Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts
---
paper_title: Enzymes for Technical Applications

paper_content:

Introduction ::: Historical Outline ::: Enzymes for the Detergent Industry ::: Introduction ::: History ::: Overview of Enzymes ::: Latest Innovations ::: Future Perspectives ::: ::: ::: Enzymes for the Starch Industry ::: Introduction ::: History ::: Enzymes ::: Latest Innovations ::: Perspectives ::: ::: ::: Enzymes for Biofuel ::: Introduction ::: History ::: Enzymes and Latest Innovations ::: Future Perspectives ::: ::: ::: Enzymes for the Textile Industry ::: Introduction ::: History ::: Enzymes ::: Latest Innovations ::: ::: ::: Enzymes for the Pulp and Paper Industry ::: Introduction ::: Overview of Selected Applications ::: Enzymes ::: Future Perspectives ::: ::: ::: Enzymes for Organic Synthesis ::: Introduction ::: History ::: Enzymes ::: Latest Innovations ::: Perspectives ::: ::: ::: Enzymes for Processing of Fats and Oils ::: Introduction ::: History ::: Enzymes ::: Latest Innovations ::: Perspectives ::: ::: ::: Key Technologies for the Discovery of Industrial Enzymes ::: Exploring Nature's Diversity ::: Protein Optimization ::: Conclusion ::: ::: ::: Acknowledgments ::: ::: ::: Keywords: ::: ::: industrial enzymes; ::: starch; ::: textile; ::: detergent; ::: fuel; ::: fats; ::: oil; ::: organic synthesis; ::: pulp and paper; ::: diversity; ::: screening; ::: assay technology; ::: molecular biology; ::: protein engineering; ::: rational protein design; ::: directed evolution
---
paper_title: Industrial enzyme applications.

paper_content:

The effective catalytic properties of enzymes have already promoted their introduction into several industrial products and processes. Recent developments in biotechnology, particularly in areas such as protein engineering and directed evolution, have provided important tools for the efficient development of new enzymes. This has resulted in the development of enzymes with improved properties for established technical applications and in the production of new enzymes tailor-made for entirely new areas of application where enzymes have not previously been used.
---
paper_title: Industrial enzymes in bioindustrial sector development: An Indian perspective

paper_content:

The bioindustrial and information technology sectors have been the two most-decisive factors for the rising Indian economy over the last 15 years. India is the second fastest growing economy in the world, with a gross domestic product (GDP) of 9.2 per cent in 2006–2007. Industrial enzymes production and marketing predominantly cover the developments incurred in the bioindustrial sector. In 2006–2007, the bioindustrial sector made an impressive turnover of Rs. 3950m, with a growth rate of 5.33 per cent. India imports about 70 per cent of the total enzyme consumption. Pharmaceutical enzymes are the representatives of industrial enzymes' demand in India and cover almost 50 per cent of the total enzyme demand, followed by detergent enzymes (20 per cent) and textile enzymes (20 per cent). The global market for enzymes was estimated to be about $ 2bn in 2004 in tune with an average annual growth rate of 3.3 per cent. This article touches upon the importance of major industrial enzymes in the development of the bioindustrial sector in India. Also, applications of enzymes, manufacturing industries and major suppliers or distributors of enzymes have been discussed.
---
paper_title: Building sustainable competitive advantage from knowledge in the region: The industrial enzymes industry

paper_content:

This paper analyses the foundations of regional knowledge and its long-term impact on the region's companies' and how a particular knowledge has developed an ability to stay competitive within a specific technological field. The case illustrates how the Copenhagen region has been able to develop a dominating position in the global market for industrial enzymes from 1870--2004. The case of industrial enzymes shows how a region has been able to build sustainable competitive advantages from its distinctive competencies. This is done through a mixture of outsourcing and in sourcing of competencies, knowledge and technologies from other regions in a ramified set of interacting networks.
---
paper_title: Industrial microbial enzymes: their discovery by screening and use in large-scale production of useful chemicals in Japan.

paper_content:

The application of microbial enzymes to large-scale organic synthesis is currently attracting much attention, and has been uniquely developed especially in Japan. The discovery of new microbial enzymes through extensive and persistent screening has brought about many new and simple routes for synthetic processes. The application of these enzymes in so-called 'hybrid processes' of enzymatic and chemical reactions, provide one possible way to solve environmental problems.
---
paper_title: History of Industrial Biotransformations – Dreams and Realities

paper_content:

This user-friendly and application-oriented overview draws on extensive literature and patent research, and is unique in its systematic structure for easy comparison. For this second edition every chapter has been rewritten, with each process updated and over 30 new ones added.
---
paper_title: Applications of cold-adapted proteases in the food industry.

paper_content:

Publisher Summary This chapter discusses that enzyme technology has been an integral part of food processing and improvement of food quality for a long time and advances in enzyme technology will be important for the production of better and safer foods. The utilization of by-products from the fishing and agricultural industries for added value largely depends on a thorough understanding of enzyme reactions. Relatively few enzymes are used in a wide variety of food processes, for the production of corn syrup and speciality fats, and to facilitate juice extraction from fruits, in addition to their use as antimicrobial preservatives. The most common food processing enzymes are hydrolases, including the proteases, glucosidases, and lipases. Proteolytic cold-adapted “superactive” enzymes from marine sources offer advantages for the food industry, although their purification and structure determination are often problematic. The tremendous knowledge gathered on enzyme structures and function in recent years has opened up a new era for food processing applications with enzymes. A new phase of enzyme technology may revolutionize the ways foods are prepared and processed in the near future. It is becoming easier to obtain relatively large numbers of naturally rare proteins, crystallography requires smaller amounts of purified proteins and smaller crystals are easier to work with than ever before.
---
paper_title: Enzymes for Technical Applications

paper_content:

Introduction ::: Historical Outline ::: Enzymes for the Detergent Industry ::: Introduction ::: History ::: Overview of Enzymes ::: Latest Innovations ::: Future Perspectives ::: ::: ::: Enzymes for the Starch Industry ::: Introduction ::: History ::: Enzymes ::: Latest Innovations ::: Perspectives ::: ::: ::: Enzymes for Biofuel ::: Introduction ::: History ::: Enzymes and Latest Innovations ::: Future Perspectives ::: ::: ::: Enzymes for the Textile Industry ::: Introduction ::: History ::: Enzymes ::: Latest Innovations ::: ::: ::: Enzymes for the Pulp and Paper Industry ::: Introduction ::: Overview of Selected Applications ::: Enzymes ::: Future Perspectives ::: ::: ::: Enzymes for Organic Synthesis ::: Introduction ::: History ::: Enzymes ::: Latest Innovations ::: Perspectives ::: ::: ::: Enzymes for Processing of Fats and Oils ::: Introduction ::: History ::: Enzymes ::: Latest Innovations ::: Perspectives ::: ::: ::: Key Technologies for the Discovery of Industrial Enzymes ::: Exploring Nature's Diversity ::: Protein Optimization ::: Conclusion ::: ::: ::: Acknowledgments ::: ::: ::: Keywords: ::: ::: industrial enzymes; ::: starch; ::: textile; ::: detergent; ::: fuel; ::: fats; ::: oil; ::: organic synthesis; ::: pulp and paper; ::: diversity; ::: screening; ::: assay technology; ::: molecular biology; ::: protein engineering; ::: rational protein design; ::: directed evolution
---
paper_title: Marine glycosyl hydrolases in the hydrolysis and synthesis of oligosaccharides.

paper_content:

The marine ecosystem can be considered a rather unexplored source of biological material (e.g. natural substances with therapeutic activity) and can also be a surprising source of enzymes carrying new and interesting catalytic activities to be applied in biocatalysis. The use of glycosyl hydrolases from marine environments dates back to the end of the 1960s and was mainly focused on the development of sensitive and reliable hydrolytic methods for the analysis of sugar chains. As a result not all the benefits of a particular enzymatic activity have been investigated, especially regarding the transglycosylation potential of these enzymes for the synthesis of glycosidic bonds. In this review, the potential of marine sources will be demonstrated reporting on the few examples found in literature for the synthesis and hydrolysis of biologically relevant oligosaccharides catalyzed by glycosyl hydrolases of marine origin. Particular emphasis is given to the synthesis of glycosidic bonds, which is easy by the use of glycosyl hydrolases. Further aspects considered in this review are applications of these biocatalysts for vegetal waste treatment in recovering useful materials, for structural identification and for preparation of target materials from new purified polysaccharides, for the synthesis or modification of food-related compounds and for glycobiology related studies.
---
paper_title: Transglycosylation reactions using glycosyl hydrolases from Thermotoga neapolitana, a marine hydrogen-producing bacterium

paper_content:

The use of the extremophilic marine bacterium Thermotoga neapolitana (DSM 4359) for the bioproduction of hydrogen using complex carbohydrates as feedstock has been recently described in a patent. In this growth condition glycosyl hydrolases can be well expressed. An important issue to plan economical feasibility of biological hydrogen production comprises techniques for recovering useful materials such as physiologically active biomolecules from biomasses grown in large quantities. ::: ::: The present paper describes a series of enzymatic transglycosylation reactions performed using the crude homogenate of T. neapolitana. The study is focused on synthetic features of the transglycosylating enzymes. Xylosidase/xylanase activity seems to be the most abundant leading to convenient syntheses of interesting series of pure (β-1,4)-xylooligosaccharides of different aglycones such as 1-hexanol (producing promising candidates for new surfactants), 9-fluorene methanol (obtaining anti-HSV agents), 1,4-butanediol (for the synthesis of new glycolipids), and geraniol (producing aroma compounds). Furthermore, the regioselectivity during galactose, fucose, glucose, and mannose enzymatic transfers is also investigated. The knowledge of synthetic characteristics of all these enzymes will be useful in the feasibility evaluation of large-scale processes of the biohydrogen production based on T. neapolitana.
---
paper_title: The Biocatalytic Potential of Extremophiles and Extremozymes

paper_content:

Summary Extremophiles are bizarre microorganisms that can grow and thrive in extreme environments, which were formerly considered too hostile to support life. The extreme conditions may be high or low temperature, high or low pH, high salinity, high metal concentrations, very low nutrient content, very low water activity, high radiation, high pressure and low oxygen tension. Some extremophiles are subject to multiple stress conditions. Extremophiles are structurally adapted at the molecular level to withstand these harsh conditions. The biocatalysts, called extremozymes, produced by these microorganisms, are proteins that function under extreme conditions. Due to their extreme stability, extremozymes offer new opportunities for biocatalysis and biotransformation. Examples of extremozymes include cellulases, amylases, xylanases, proteases, pectinases, keratinases, lipases, esterases, catalases, peroxidases and phytases, which have great potential for application in various biotechnological processes. Currently, only 1–2 % of the microorganisms on the Earth have been commercially exploited and amongst these there are only a few examples of extremophiles. However, the renewed interest that is currently emerging as a result of new developments in the cultivation and production of extremophiles and success in the cloning and expression of their genes in mesophilic hosts will increase the biocatalytic applications of extremozymes.
---
paper_title: Purification and Partial Characterization of Esterase from Marine Vibrio fischeri

paper_content:

Lipolytic enzymes from marine microbes have been the focus of intense and growing research. The bioluminescence bacterium Vibrio fischeri was produced esterase enzyme when the medium contained specific substrate. The esterase was purified from the concentrated culture supernatant. The most active fractions were obtained using the technique of precipitation with 1N HCl. The precipitated fraction was purified by ion exchange chromatography (DEAE-Cellulose) and gel filteration chromatography (Sephadex G200). The purified active fraction exhibiting final specific activity of 300U/mg and characterized; the optimum pH was 7.5, the optimum temperature was 30°C. The enzyme was very stable at the temperature 30°C and at wide range of pH. The enzyme was monomeric protein having molecular mass of 37 KDa estimated by native PAGE assay.
---
paper_title: Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

paper_content:

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of > 80 degrees C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.
---
paper_title: Two Extremely Thermostable Xylanases of the Hyperthermophilic Bacterium Thermotoga maritima MSB8.

paper_content:

During growth with xylose or xylan as the source of carbon, xylanase production by Thermotoga maritima MSB8 was enhanced about 10-fold compared with growth with glucose or starch. Two extremely thermostable endoxylanases (1,4-(beta)-d-xylan-xylanohydrolase, EC 3.2.1.8), designated XynA and XynB, were identified and purified from cells of this organism. XynA and XynB occurred as proteins with apparent molecular masses of about 120 and 40 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum activity at the optimal pH (pH 6.2 and pH 5.4 for XynA and XynB, respectively) was measured at about 92(deg)C for XynA (10-min assay) and at about 105(deg)C for XynB (5-min assay). XynB activity was stimulated twofold by the addition of 500 mM NaCl, while XynA displayed maximum activity without the addition of salt. Both xylanases were tolerant of relatively high salt concentrations. At 2 M (about 12% wt/vol) NaCl, XynA and XynB retained 49 and 65%, respectively, of their maximum activities. In contrast to XynB, XynA was able to adsorb to microcrystalline cellulose. Antibodies raised against a recombinant truncated XynA protein cross-reacted with XynB, indicating that the enzymes may have sequence or structural similarities. Part of the xylanase activity appeared to be associated with the outer membrane of T. maritima cells, since more than 40% of the total xylanase activity present in the crude cellular extract was found in the membrane fraction after high-speed centrifugation. Most of the membrane-bound activity appeared to be due to the 120-kDa xylanase XynA.
---
paper_title: Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus.

paper_content:

Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a beta-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native beta-glucosidase has a molecular mass of 230 +/- 20 kDa, composed of 58 +/- 2-kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102-105 degrees C. From Lineweaver-Burk plots, Vmax values of 470 U/mg and 700 U/mg were found for cellobiose (Km = 20 mM) and p-nitrophenyl-beta-D-glucopyranoside (Km = 0.15 mM), respectively. The purified enzyme also exhibits high beta-galactosidase activity and beta-xylosidase activity, but shows no activity towards alpha-linked disaccharides or beta-linked polymers, like cellulose. The purified beta-glucosidase shows a remarkable thermostability with a half life of 85 h at 100 degrees C and 13 h at 110 degrees C.
---
paper_title: Inulinase production by the marine yeast Cryptococcus aureus G7a and inulin hydrolysis by the crude inulinase

paper_content:

Abstract The marine yeast strain G7a isolated from sediment of China South Sea was found to secrete a large amount of inulinase into the medium. This marine yeast strain was identified to be a strain of Cryptococcus aureus according to the results of routine yeast identification and molecular methods. The crude inulinase produced by this marine yeast showed the highest activity at pH 5.0 and 50 °C. The optimal medium for inulinase production was artificial seawater containing inulin 4.0% (w/v), K 2 HPO 4 0.3% (w/v), yeast extract 0.5% (w/v), KCl 0.5% (w/v), CaCl 2 0.12% (w/v), NaCl 4.0% (w/v) and MgCl 2 ·6H 2 O 0.6% (w/v), while the optimal cultivation conditions for inulinase production were pH 5.0, a temperature of 28 °C and a shaking speed of 170 rpm. Under the optimal conditions, over 85.0 U/ml of inulinase activity was produced within 42 h of fermentation at shake flask level. This is very high level of inulinase activity produced by yeasts. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.
---
paper_title: Recent Advances in Microbial Raw Starch Degrading Enzymes

paper_content:

Raw starch degrading enzymes (RSDE) refer to enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. These promising enzymes can significantly reduce energy and simplify the process in starch industry. RSDE are ubiquitous and produced by plants, animals, and microorganisms. However, microbial sources are the most preferred one for large-scale production. During the past few decades, RSDE have been studied extensively. This paper reviews the recent development in the production, purification, properties, and application of microbial RSDE. This is the first review on microbial RSDE to date.
---
paper_title: Characterization of Amylolytic Enzyme Activities Associated with the Hyperthermophilic Archaebacterium Pyrococcus furiosus

paper_content:

The hyperthermophilic archaebacterium Pyrococcus furiosus produces several amylolytic enzymes in response to the presence of complex carbohydrates in the growth medium. These enzyme activities, {alpha}-glucosidase, pullulanase, and {alpha}-amylase, were detected in both cell extracts and culture supernatants. All activities were characterized by temperature optima of at least 100{degree}C as well as a high degree of thermostability. The existence of this collection of activities in P. furiosus suggests that polysaccharide availability in its growth environment is a significant aspect of the niche from which it was isolated.
---
paper_title: Production of a high maltose-forming, hyperthermostable and Ca 2+ - independent amylopullulanase by an extreme thermophile Geobacillus thermoleovorans in submerged fermentation

paper_content:

Production of a hyperthermostable, high maltose-forming and Ca 2+ -independent amylopullulanase by an extreme thermophile Geobacillus thermoleovorans was studied in submerged fermentation. Parametric optimization led to secretion of amylopullulanase (2850 U L -1 of α-amylase, 840 U L -1 of pullulanase) in a starch-yeast extract medium (2% starch, 0.1% ammonium sulphate, 0.3% yeast extract, 0.1% K2HPO4, 0.1% NaCl, 0.01% MgSO4.7H2O and 0.1% Maltose) using 2% inoculum (12 h) at 70°C and 200 rpm in 20 h. The addition of trace elements, detergents, surfactants and additives did not stimulate growth or enzyme production. The hydrolysis of pullulan and starch yielded maltotriose, and maltose, maltotriose and maltotetraose as the major end products, respectively. The cultivation of the organism in a laboratory fermenter showed (i) abolition of the lag phase, (ii) a 2-fold increase in enzyme production (5880 U L -1 of α-amylase, 1900 U L -1 of pullulanase) and (iii) a reduction in fermentation time to 7-8 h.
---
paper_title: "Enzyme Test Bench," a high-throughput enzyme characterization technique including the long-term stability.

paper_content:

A new high throughput technique for enzyme characterization with specific attention to the long term stability, called "Enzyme Test Bench," is presented. The concept of the Enzyme Test Bench consists of short term enzyme tests in 96-well microtiter plates under partly extreme conditions to predict the enzyme long term stability under moderate conditions. The technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles in sequential experiments by optimal non-linear experimental design, the long term deactivation effects can be purposefully accelerated and detected within hours. During the experiment the enzyme activity is measured online to estimate the model parameters from the obtained data. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The engineered instrumentation provides for simultaneous automated assaying by fluorescent measurements, mixing and homogenous temperature control in the range of 10-85 +/- 0.5 degrees C. A universal fluorescent assay for online acquisition of ester hydrolysis reactions by pH-shift is developed and established. The developed instrumentation and assay are applied to characterize two esterases. The results of the characterization, carried out in microtiter plates applying short term experiments of hours, are in good agreement with the results of long term experiments at different temperatures in 1 L stirred tank reactors of a week. Thus, the new technique allows for both: the enzyme screening with regard to the long term stability and the choice of the optimal process temperature regarding such process parameters as turn over number, space time yield or optimal process duration. The comparison of the temperature dependent behavior of both characterized enzymes clearly demonstrates that the frequently applied estimation of long term stability at moderate temperatures by simple activity measurements after exposing the enzymes to elevated temperatures may lead to suboptimal enzyme selection. Thus, temperature dependent enzyme characterization is essential in primary screening to predict its long term behavior.
---
paper_title: Characterization of hyperthermostable α-amylase from Geobacillus sp. IIPTN

paper_content:

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable α-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60°C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80°C and pH 5.5–8.5. Maximum enzyme production was in exponential phase with activity 135 U ml−1 at 60°C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml−1 at pH 5.0 and 80°C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg−1 protein. The molecular mass of the purified enzyme was 97 KDa. The values of K m and V max were 36 mg ml−1 and 222 µmol mg−1 protein min−1, respectively. The amylase was stable over a broad range of temperature from 40°C to 120°C and pH ranges from 5 to 10. The enzyme was stimulated with Mn2+, whereas it was inhibited by Hg2+, Cu2+, Zn2+, Mg2+, and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.
---
paper_title: Dispelling the Myths--Biocatalysis in Industrial Synthesis

paper_content:

Biocatalysis has emerged as an important tool in the industrial synthesis of bulk chemicals, pharmaceutical and agrochemical intermediates, active pharmaceuticals, and food ingredients. However, the number and diversity of the applications are modest, perhaps in part because of perceived or real limitations of biocatalysts, such as limited enzyme availability, substrate scope, and operational stability. Recent scientific breakthroughs in genomics, directed enzyme evolution, and the exploitation of biodiversity should help to overcome these limitations. As a result, we expect many new industrial applications of biocatalysis to be realized, from single-step enzymatic conversions to customized multistep microbial synthesis by means of metabolic pathway engineering.
---
paper_title: Novel approaches in microbial enzyme production

paper_content:

Abstract The rapid developments in food biotechnology with an increase in novel applications of enzyme technology create new demands for the availability of food grade enzymes. Relatively few enzymes are today available in large commercial quantities, and new applications require enzymes with new characteristics. Enzymes for food and feed applications are obtained from animal, plant, and microbial sources, although most are of microbial origin. The majority of microbial industrial enzymes are extracellular hydrolases, but others such as glucose isomerase and glucose oxidase are cell bound. Such intracellular enzymes are used either as whole cell preparations or they are recovered and purified to a varying degree. Microbial enzymes are usually produced by batch aerobic submerged fermentation, although continuous culture technology is also applied. Certain fungal enzymes are produced by solid‐state fermentation, and novel methods such as aqueous two‐phase systems and immobilized viable cell techniques have ...
---
paper_title: Biophysical and biochemical characterization of a hyperthermostable and Ca2+ -independent alpha-Amylase of an extreme thermophile Geobacillus thermoleovorans.

paper_content:

alpha-Amylases reported from various microbial sources have been shown to be moderately thermostable and Ca2+ dependent. The bacterial strain used in this investigation is an extremely thermophilic bacterium Geobacillus thermoleovorans that produces a novel alpha-amylase (26 kDa; alpha-amylase gt), which is hyperthermostable (Topt 100 degrees C) and does not require Ca2+ for its activity/stability. These special features of alpha-amylase gt make it applicable in starch saccharification process. The structural aspects of alpha-amylase gt are, therefore, of significant interest to understand its structure-function relationship. The circular dichroism spectroscopic data revealed the native alpha-amylase gt to contain 25% alpha-helix, 21% beta-sheet, and 54% random coils. The addition of urea, at high concentration (8 M), appeared to expose the buried Trp residues of the native alpha-amylase gt to the aqueous environment and thus showed low fluorophore. Fluorescence-quenching experiments using KI, CsCl, N-bromosuccinimide, and acrylamide revealed interesting features of the tryptophan microenvironment. Analysis of Ksv and fa values of KI, CsCl, and acrylamide suggested the overall Trp microenvironment in alpha-amylase to be slightly electropositive. Fluorescence-quenching studies with acrylamide revealed the occurrence of both collisional as well as static quenching processes. There was no change in the alpha-helix content or the enzyme activity with an increase in temperature (60-100 degrees C) that suggested a critical role of the alpha-helix content in maintaining the catalytic activity.
---
paper_title: A Ca-independent α-amylase that is active and stable at low pH from the Bacillus sp. KR-8104

paper_content:

Abstract Bacillus sp. KR-8104 was selected from a set of 18 bacteria strains isolated from soil samples and screened for production of amylase. The maximum productivity obtained at pH 5–6 and 60–65 h after cultivation in production medium. New extracellular Ca-independent α-amylase was highly purified using ion exchange and hydrophobic interaction chromatography, which showed a single band with an apparent molecular weight of 59 kDa by SDS-PAGE. This enzyme is active in a wide pH range with its maximum activity at low pH values (4.0–6.0) and has the 90% of its maximum activity at pH 3.5. The α-amylase is optimally active at 75–80 °C. The presence or absence of Ca 2+ and EDTA did not affect enzyme activity and thermal stability.
---
paper_title: A thermostable glucose isomerase having a relatively low optimum pH: study of activity and molecular cloning of the corresponding gene

paper_content:

A thermostable glucose isomerase from a newly isolated thermophilic Streptomyces sp. SK strain, had a wide pH range with an optimum of 6 at 60°C and 6.4 at 90°C. It was optimally active at 95°C and completely stable at 80°C for at least 5.5 h with a half-life of 5 h at 90°C. Using E.coli as a host strain and an internal fragment of xylA of S. olivochromogenes as a probe, a 6.5 kb DNA fragment from Streptomyces sp. SK was cloned. This fragment carries the entire xylA gene since the recombinant plasmid complements the E.coli xyl-5 mutant strain HB101. © Rapid Science Ltd. 1998
---
paper_title: Purification and characterization of thermoalkalophilic xylanase isolated from the Enterobacter sp. MTCC 5112.

paper_content:

Thermoalkalophilic Enterobacter sp. MTCC 5112 was isolated from a sediment sample collected from the Mandovi estuary on the west coast of India. This culture produced extracellular xylanase. The xylanase enzyme was isolated by ammonium sulfate (80%) fractionation and purified to homogeneity using size exclusion and ion exchange chromatography. The molecular mass of the xylanase was approximately 43 kDa. The optimal pH of the xylanase activity was 9, and at room temperature it showed 100% stability at pH 7, 8 and 9 for 3 h. The optimal temperature for the enzyme activity was 100 degrees C at pH 9.0. At 80 degrees C and pH 9, 90% of the enzyme activity was retained after 40 min. At 70 and 60 degrees C, the enzyme retained 64% and 85% of its activity after 18 h, respectively, while at 50 degrees C and pH 9 the enzyme remained stable for days. For xylan, the enzyme gave a K(m) value of 3.3 mg ml(-1) and a V(max) value of 5,000 micromol min(-1) mg(-1) when the reaction was carried out at 100 degrees C and pH 9. In the presence of metal ions such as Co(2+), Zn(2+), Fe(2+), Cu(2+), Mg(2+) and Ca(2+) the activity of the enzyme increased, whereas strong inhibition of enzyme activity was observed in the presence of Hg(2+) and EDTA. To the best of our knowledge, this is the first report on the production of xylanase by this bacterium.
---
paper_title: Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

paper_content:

Abstract A thermostable alkaline α-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8 mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS–PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 °C with an optimum of 60 °C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 °C. The enzyme was highly active (95%) in alkaline range of pH (10.0–11.5), and it was almost completely active up to 100 °C with 96% of the original activity remaining after heat treatment at 100 °C for 30 min. Enzyme activity was enhanced in the presence of 5 mM CaCl 2 (130%) and inhibition with 5 mM by ZnCl 2 , NaCl, Na–sulphide, EDTA, PMSF (3 mM), Urea (8 M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0–11.0 and 60 °C for 24 h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.
---
paper_title: Characterization of Amylolytic Enzymes, Having Both α-1,4 and α-1,6 Hydrolytic Activity, from the Thermophilic Archaea Pyrococcus furiosus and Thermococcus litoralis

paper_content:

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea Thermococcus litoralis (optimal growth temperature, 90°C) and Pyrococcus furiosus (optimal growth temperature, 98°C). The molecular mass of the T. litoralis enzyme was estimated at 119,000 Da by electrophoresis, while the P. furiosus enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca2+, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140°C. The addition of Ca2+ also affected substrate binding, as evidenced by a decrease in Km for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the α-1,6 linkages in pullulan, α-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller α-1,4-linked oligosaccharides. The enzymes from T. litoralis and P. furiosus appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.
---
paper_title: Sources, Properties and Suitability of New Thermostable Enzymes in Food Processing

paper_content:

Investigations concerning recombinant α-amylases from Pyrococcus woesei and thermostable α-glucosidase from Thermus thermophilus indicate their suitability for starch processing. Furthermore, the study of recombinant β-galactosidase from Pyrococcus woesei suitable for purpose of low lactose milk and whey production are also presented. The activity of this enzyme in a wide pH range of 4.3–6.6 and high thermostability suggests that it can be used for processing of dairy products at temperatures which restrict microbial growth during a long operating time of continuous-flow reactor with an immobilized enzyme system. Preparation of recombinant α-amylase and β-galactosidase was facilitated by cloning and expression of genes from Pyrococcus woesei in Escherichia coli host. Satisfactory level of recombinant enzymes purification was achieved by thermal precipitation of native proteins originated from Escherichia coli. The obtained α-amylase has maximal activity at pH 5.6 and 93°C. The half-life of this preparation (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120°C. An advantageous attribute of recombinant α -amylase is independence of its activity and stability on calcium salt. α-Glucosidase from Thermus thermophilus also not require metal ions for stability and retained about 80% of maximal activity at pH range 5.8–6.9. Thus, this enzyme can be used together with recombinant α-amylase.
---
paper_title: Production of Industrial Enzymes (Amylase, Carboxymethylcellulase and Protease) by Bacteria Isolated from Marine Sedentary Organisms

paper_content:

The abilities of bacteria isolated from eight marine sedentary organisms, six marine sponges (Spirastrella sp., Phyllospongia sp., Ircinia sp., Aaptos sp., Azorica sp. and Axinella sp.), one soft coral (Lobophytum sp.) and one alga (Sargassum sp.) to produce industrial enzymes (amylase, carboxymethylcellulase and protease) were examined. The mean total viable counts of the bacterial isolates ranged from 8.7 × 10 4 to 8.4 x 10 5 cfu/g wet weight of the organism. All eight organisms harboured amylase (0.05-0.5 IU/ml), carboxymethylcellulase (0.05-0.5 IU/ml) and protease (0.1-0.5 IU/ml) producing bacteria. Of 56 bacterial strains tested, as many as 60 to 83% of the strains produced at least one of the three enzymes, and 47% of strains were able to produce all three enzymes. High activities (> 0.5 IU/ml) of the three enzymes were recorded in bacterial strains belonging to the genera Alcaligenes and Bacillus. From the results of this study, it appears that bacteria associated with marine sedentary organisms are the novel source of industrial enzymes for possible commercial applications and may play an important role in enzyme-catalysed organic matter cycling in marine environments.
---
paper_title: Purification and biochemical characterization of a native invertase from the hydrogen-producing Thermotoga neapolitana (DSM 4359)

paper_content:

This is the first report describing the purification and enzymatic properties of a native invertase (beta-D-fructosidase) in Thermotogales. The invertase of the hydrogen-producing thermophilic bacterium Thermotoga neapolitana DSM 4359 (hereby named Tni) was a monomer of about 47 kDa having an amino acid sequence quite different from other invertases studied up to now. Its properties and substrates specificity let us classify this protein as a solute-binding protein with invertase activity. Tni was specific for the fructose moiety and the enzyme released fructose from sucrose and raffinose and the fructose polymer inulin was hydrolyzed in an endo-type fashion. Tni had an optimum temperature of 85 degrees C at pH 6.0. At temperatures of 80-85 degrees C, the enzyme retained at least 50% of its initial activity during a 6 h preincubation period. Tni had a K(m) and k(cat)/K(m) values (at 85 degrees C and pH 6.0) of about 14 mM and 5.2 x 10(8) M(-1) s(-1), respectively.
---
paper_title: A novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization.

paper_content:

This study reports the purification and characterization of a novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1. Maximum alpha-amylase activity (53 U mL(-1)) was obtained at 45 degrees C after 44 h of incubation. The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography, and showed a molecular weight of 56 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH 5.0, performed stability over a broad range of pH 4.5-11.0, and was optimally active at 40-50 degrees C. The enzyme preparation had a strong digesting ability towards various raw starches and efficiently hydrolyzed raw corn starch at a concentration of 20% and pH 5.0, which were normally used in the starch industries, in a period of 12h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel alpha-amylase.
---
paper_title: Starch-hydrolyzing enzymes from thermophilic archaea and bacteria.

paper_content:

Extremophlic microorganisms have developed a variety of molecular strategies in order to survive in harsh conditions. For the utilization of natural polymeric substrates such as starch, a number of extremophiles, belonging to different taxonomic groups, produce amylolytic enzymes. This class of enzyme is important not only for the study of biocatalysis and protein stability at extreme conditions but also for the many biotechnological opportunities they offer. In this review, we report on the different molecular properties of thermostable archaeal and bacterial enzymes including alpha-amylase, alpha-glucosidase, glucoamylase, pullulanase, and cyclodextrin glycosyltransferase. Comparison of the primary sequence of the pyrococcal pullulanase with other members of the glucosyl hydrolase family revealed that significant differences are responsible for the mode of action of these enzymes.
---
paper_title: Atypical Ca2+-independent, raw-starch hydrolysing α-amylase from Bacillus sp. GRE1: characterization and gene isolation

paper_content:

Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca2+-independent thermostable α-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental conditions was 2,360 U l−1. Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca2+ produced similar temperature optima of 65–70°C. The optimum pH was in the range of 5.5–6.0. The enzyme maintained 50% of its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0–9.0. The V max and K m values for soluble starch were 42 mg reducing sugar min−1 and 4.98 mg starch ml−1, respectively. Strong inhibitors of enzyme activity included Cu2+, Zn2+ and Fe2+. The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species α-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization temperatures. Rather than the higher oligosaccharides normally produced by Bacillus α-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme.
---
paper_title: Properties of thermostable extracellular FOS-producing fructofuranosidase from Cryptococcus sp.

paper_content:

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, showing a substrate inhibitory model. The K m, K i and v max values were shown to be 64 mM, 3 M and 159.6 μmol mL−1 min−1 for FA and 131 mM, 1.6 M and 377.8 μmol mL−1 min−1 for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 °C, while the best stability was achieved at pH 4.5 and temperatures below 60 °C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).
---
paper_title: Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

paper_content:

Abstract A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg 2+ ions. Ca 2+ , dithiothreitol, and Mn 2+ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V max and K m values of 154 U mg −1 and 1.3 mg ml −1 . The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.
---
paper_title: Purification and characterization of heat-stable exo-inulinase from Streptomyces sp.

paper_content:

Abstract The extracellular exo-inulinase from Streptomyces sp. was purified from the culture broth by ammonium sulphate precipitation, followed by successive chromatographies on DEAE-Sephacel and ConA-CL Agarose columns. The enzyme was purified 18-folds with 4.8% activity yield from the starting culture broth. The purified enzyme gave a single band on gel electrophoresis and its molecular weight was estimated to be 45 kDa. The optimum temperature and pH for enzyme activity were 70 °C and 6.0, respectively. All metal salts except NaNO3, EDTA and HgCl2 were tolerated and did not adversely affect inulinase activity. The effect of thermal stabilizers on inulinase activity was studied. Consequently, glycerol and mannitol have been shown to have a protective effect on enzyme activity. As compared to sucrose and raffinose the purified enzyme had a lower Km (1.63 mM) and higher Vmax (450 mM) for inulin. All these conditions make Streptomyces sp., a potential candidate for industrial enzymatic production of high fructose syrup and in other large-scale biotechnological processes.
---
paper_title: Thermostable inulinases secreted by yeast and yeast-like strains from the Brazilian semi-arid region.

paper_content:

The use of inulinases provides an alternative to the chemical process of inulin hydrolysis to obtain fructose syrup, and can reduce processing steps, time, and costs in the food industry. The objective of this work was to screen the thermostable inulinases produced by yeast and yeast-like strains isolated from the Brazilian semi-arid region. Thermostability was studied at different temperatures (60 degrees C, 70 degrees C, 80 degrees C and 90 degrees C) and for increasing periods of time (0-50 min). Thirty-three microorganisms were tested, and 27 showed inulinase activity with specific activities ranging from 0.98 to 73.79 micromol/mg protein/min. Three strains (CCMB 300, CCMB327 and CCMB328) showed the desired combination of high specific activity and a small reduction in residual activity when submitted to heat treatment (>or=60 degrees C). Our results indicate that the inulinases produced by these three yeast strains from the Brazilian semi-arid region have great potential to be used for inulin hydrolysis in the food industry.
---
paper_title: A novel raw starch digesting thermostable α-amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch

paper_content:

Abstract A new strain of Bacillus sp. I-3, isolated from natural soil samples, showed a high raw starch digesting activity towards potato starch. Upon optimization of various environmental and cultural conditions, the yield of α-amylase reached 642 U/mL. The kinetic characterization of partially purified enzyme exhibited the maximum activity at 70 °C, pH 7.0 and revealed a high thermostability in the presence of 10 mM CaCl 2 ·2H 2 O where it could retain more than 90% residual activity at 70 °C after 3.5 h. At 80, 90 and 100 °C, the enzyme retained 80, 59 and 26% of its maximum activity after 2.5, 0.5 and 0.5 h, respectively. The enzyme preparation had a strong affinity towards raw potato starch granules and was almost completely adsorbed onto it. It also hydrolyzed raw potato starch at a concentration of 12.5% significantly in a short period of time of 12 h.
---
paper_title: Glucose-to-fructose conversion at high temperatures with xylose (glucose) isomerases fromStreptomyces murinus and two hyperthermophilicThermotoga species

paper_content:

The conversion of glucose to fructose at elevated temperatures, as catalyzed by soluble and immobilized xylose (glucose) isomerases from the hyperthermophiles Thermotoga maritima (TMGI) and Thermotoga neapolitana 5068 (TNGI) and from the mesophile Streptomyces murinus (SMGI), was examined. At pH 7.0 in the presence of Mg(2+), the temperature optima for the three soluble enzymes were 85 degrees C (SMGI), 95 degrees to 100 degrees C (TNGI), and >100 degrees C (TMGI). Under certain conditions, soluble forms of the three enzymes exhibited an unusual, multiphasic inactivation behavior in which the decay rate slowed considerably after an initial rapid decline. However, the inactivation of the enzymes covalently immobilized to glass beads, monophasic in most cases, was characterized by a first-order decay rate intermediate between those of the initial rapid and slower phases for the soluble enzymes. Enzyme productivities for the three immobilized GIs were determined experimentally in the presence of Mg(2+). The highest productivities measured were 750 and 760 kg fructose per kilogram SMGI at 60 degrees C and 70 degrees C, respectively. The highest productivity for both TMGI and TNGI in the presence of Mg(2+) occurred at 70 degrees C, pH 7.0, with approximately 230 and 200 kg fructose per kilogram enzyme for TNGI and TMGI, respectively. At 80 degrees C and in the presence of Mg(2+), productivities for the three enzymes ranged from 31 to 273. A simple mathematical model, which accounted for thermal effects on kinetics, glucose-fructose equilibrium, and enzyme inactivation, was used to examine the potential for high-fructose corn syrup (HFCS) production at 80 degrees C and above using TNGI and SMGI under optimal conditions, which included the presence of both Co(2+) and Mg(2+). In the presence of both cations, these enzymes showed the potential to catalyze glucose-to-fructose conversion at 80 degrees C with estimated lifetime productivities on the order of 2000 kg fructose per kilogram enzyme, a value competitive with enzymes currently used at 55 degrees to 65 degrees C, but with the additional advantage of higher fructose concentrations. At 90 degrees C, the estimated productivity for SMGI dropped to 200, whereas, for TNGI, lifetime productivities on the order of 1000 were estimated. Assuming that the most favorable biocatalytic and thermostability features of these enzymes can be captured in immobilized form and the chemical lability of substrates and products can be minimized, HFCS production at high temperatures could be used to achieve higher fructose concentrations as well as create alternative processing strategies.
---
paper_title: A Review of Proteotlytic Enzymes from Marine Organisms and Their Application in the Food Industry

paper_content:

Proteolytic enzyme are widely used as food processing aids. In recent years, proteolytic enzymes from marine organisms have been used as process aids. Other possible uses of enzymes form marine animals in the food industry are based on the unique properties of these enzymes. Examples of unique properties include ease of heat denaturation, high molecular activity at low temperatures and ability to catalyze hydrolysis of native proteins. The possible advantage of using an enzyme form a marine organism in specific applications rather than a conventional plant, animal or microbial source is discussed.
---
paper_title: Comparative analysis of thermostability of extracellular inulinase activity from Aspergillus fumigatus with commercially available (Novozyme) inulinase

paper_content:

Thermostable inulinase activity was identified in the extracellular extract of Aspergillus fumigatus. At its optimum temperature of 60 °C, the ammonium sulphate fraction retained approximately 70% of its maximum activity after 72 h incubation in the absence of inulin. The two isoforms of A. fumigatus inulinase were purified and their thermostability was studied. In the presence of inulin, isoform II was more thermostable when compared to other two fractions and retained approximately 54% activity after 3 h at 60 °C. The higher thermostability of inulinase of A. fumigatus makes it a potential candidate for commercial production of fructose.
---
paper_title: Enzymes from fish and aquatic invertebrates and their application in the food industry

paper_content:

Abstract Enzymatic methods have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. Aquatic environment contains the largest pool of diversified genetic material and, hence represents an enormous potential for different sources of enzymes. In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates has been achieved and some interesting and novel applications related to marine enzymes in food processing have emerged. This review summarizes information related to digestive and muscular enzymes in fish and aquatic invertebrates. In addition, important developments in food applications of marine enzymes are reported.
---
paper_title: Marine biotechnology for production of food ingredients.

paper_content:

The marine world represents a largely untapped reservoir of bioactive ingredients that can be applied to numerous aspects of food processing, storage, and fortification. Due to the wide range of environments they survive in, marine organisms have developed unique properties and bioactive compounds that, in some cases, are unparalleled by their terrestrial counterparts. Enzymes extracted from fish and marine microorganisms can provide numerous advantages over traditional enzymes used in food processing due to their ability to function at extremes of temperature and pH. Fish proteins such as collagens and their gelatin derivatives operate at relatively low temperatures and can be used in heat-sensitive processes such as gelling and clarifying. Polysaccharides derived from algae, including algins, carrageenans, and agar, are widely used for their ability to form gels and act as thickeners and stabilizers in a variety of foods. Besides applications in food processing, a number of marine-derived compounds, such as omega-3 polyunsaturated fatty acids and photosynthetic pigments, are important to the nutraceutical industry. These bioactive ingredients provide a myriad of health benefits, including reduction of coronary heart disease, anticarcinogenic and anti-inflammatory activity. Despite the vast possibilities for the use of marine organisms in the food industry, tools of biotechnology are required for successful cultivation and isolation of these unique bioactive compounds. In this chapter, recent developments and upcoming areas of research that utilize advances in biotechnology in the production of food ingredients from marine sources are introduced and discussed.
---
paper_title: Analysis of the gene for β-fructosidase (invertase, inulinase) of the hyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed in Escherichia coli

paper_content:

This is the first report describing the gene structure and the enzymatic properties of a β-fructosidase of a hyperthermophilic organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other β-fructosidases. On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the β-anomeric configuration of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis of sucrose and inulin with kcat/Km values (at 75 °C, pH 5.5) of about 4.1 × 104 M−1s−1 and 3.1 × 104 M−1s−1 respectively. BfrA had an optimum temperature of 90–95 °C (10-min assay) and was extremely insensitive to thermo-inactivation. During 5 h at temperatures up to 80 °C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the most thermostable β-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-β-d-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability recommend it for use in biotechnology.
---
paper_title: Commercial scale biocatalysis: myths and realities

paper_content:

The unique ways in which enzymes are differentiated from other catalysts translate into special advantages. Understanding these advantages is the key toward better matching of biocatalysts needs in industrial chemistry. Specific cases where enzymes and biotransformations have been used successfully at the production scale are examined, permitting the realities of using biocatalysts to be separated from the misconceptions and myths. Five such misconceptions will be examined in the context of examples of some commercially-successful biocatalytic processes.
---
paper_title: A Novel Cold-Active and Alkali-Stable β-Glucosidase Gene Isolated from the Marine Bacterium Martelella mediterranea

paper_content:

A β-glucosidase gene designated gluc3m was cloned through construction of a genomic library of Martelella mediterranea 2928. The gluc3m consisted of 2,496 bp and encoded a peptide of 832 amino acids that shared the greatest amino acid similarity (59%) with a β-glucosidase of family 3 glycoside hydrolase from Agrobacterium radiobacter K84. The optimum reaction temperature and pH of Gluc3M were 45 °C and 8.0, respectively. The K (m) and V (max) for p-nitrophenyl-β-D: -glucopyranoside were 0.18 mg/ml and 196.08 µmol/min/mg enzyme, respectively. Gluc3M was found to be highly alkali stable, retaining 80% of its maximum enzymatic activity after treatment with pH 11.0 buffers for 24 h. Furthermore, the activity of Gluc3M improved remarkably in the presence of univalent metal ions, whereas it was inhibited in the presence of divalent ions. Gluc3M also exhibited significant activities toward various substrates including pNPGlu, pNPGal, salicin, and konjac powder. It is important to note that Gluc3M is a cold-active enzyme that showed over 50% of the maximum enzymatic activity at 4 °C. SWISS-MODEL revealed that the amino acids near the conserved domain SDW of Gluc3M contributed to the cold-active ability. Based on these characteristics, Gluc3M has the potential for use in additional studies and for industrial applications.
---
paper_title: A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable alpha-amylase.

paper_content:

High throughput screening of microbial DNA libraries was used to identify alpha-amylases with phenotypic characteristics compatible with large scale corn wet milling process conditions. Single and multiorganism DNA libraries originating from various environments were targeted for activity and sequence-based screening approaches. After initial screening, 15 clones were designated as primary hits based upon activity at pH 4.5 or 95 degrees C without addition of endogenous Ca(2+). After further characterization, three enzyme candidates were chosen each with an exceptional expression of one or more aspects of the necessary phenotype: temperature stability, pH optimum, lowered reliance on Ca(2+) and/or enzyme rate. To combine the best aspects of the three phenotypes to optimize process compatibility, the natural gene homologues were used as a parental sequence set for gene reassembly. Approximately 21,000 chimeric daughter sequences were generated and subsets screened using a process-specific, high throughput activity assay. Gene reassembly resulted in numerous improved mutants with combined optimal phenotypes of expression, temperature stability, and pH optimum. After biochemical and process-specific characterization of these gene products, one alpha-amylase with exceptional process compatibility and economics was identified. This paper describes the synergistic approach of combining environmental discovery and laboratory evolution for identification and optimization of industrially important biocatalysts.
---
paper_title: Safety regulations of food enzymes

paper_content:

Summary The majority of industrial enzymes available at present is used in food industry. Safety regulations of food enzymes differ among countries, including fundamental aspects, whether a pre-market approval is needed and on the level of details, e.g. what particular information manufacturers have to provide in the course of safety evaluation. Occupational safety concerns focus on allergenic properties as it is well established that enzymes are potent inhalative sensitizers and can cause allergic reactions including asthma. Otherwise toxic substances including bacterial toxins and mycotoxins might also be present in enzyme isolates and might thus constitute a safety risk to consumers. Safety evaluation procedures seem to be appropriate as no incidents have been reported so far, resulting in suggestion for reduced test packages. Safety precaution and monitoring measures established by industry have also reduced but not entirely eradicated occupational risks. Challenges to regulators and industry arise from unresolved issues, e.g. whether enzymes might be contact sensitizers, and from the lack of harmonisation of both legislation and safety evaluation. In the EU, most food enzymes are not covered by food safety regulations neither on Community nor on national level. On top of this the availability of enzymes with new and unusual properties raises questions of safety. In the EU there seems to be a chance that these challenges will be tackled in the course of establishing a harmonised legislation on food enzymes.
---
paper_title: A new cold-adapted β-D-galactosidase from the Antarctic Arthrobacter sp. 32c – gene cloning, overexpression, purification and properties

paper_content:

BackgroundThe development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production.ResultsIn this article, we present a new β-D-galactosidase as a candidate to be applied in the above mentioned biotechnological processes. The gene encoding this β-D-galactosidase has been isolated from the genomic DNA library of Antarctic bacterium Arthrobacter sp. 32c, sequenced, cloned, expressed in Escherichia coli and Pichia pastoris, purified and characterized. 27 mg of β-D-galactosidase was purified from 1 L of culture with the use of an intracellular E. coli expression system. The protein was also produced extracellularly by P. pastoris in high amounts giving approximately 137 mg and 97 mg of purified enzyme from 1 L of P. pastoris culture for the AOX1 and a constitutive system, respectively. The enzyme was purified to electrophoretic homogeneity by using either one step- or a fast two step- procedure including protein precipitation and affinity chromatography. The enzyme was found to be active as a homotrimeric protein consisting of 695 amino acid residues in each monomer. Although, the maximum activity of the enzyme was determined at pH 6.5 and 50°C, 60% of the maximum activity of the enzyme was determined at 25°C and 15% of the maximum activity was detected at 0°C.ConclusionThe properties of Arthrobacter sp. 32cβ-D-galactosidase suggest that this enzyme could be useful for low-cost, industrial conversion of lactose into galactose and glucose in milk products and could be an interesting alternative for the production of ethanol from lactose-based feedstock.
---
paper_title: Food-processing enzymes from recombinant microorganisms--a review.

paper_content:

Enzymes are commonly used in food processing and in the production of food ingredients. Enzymes traditionally isolated from culturable microorganisms, plants, and mammalian tissues are often not well-adapted to the conditions used in modern food production methods. The use of recombinant DNA technology has made it possible to manufacture novel enzymes suitable for specific food-processing conditions. Such enzymes may be discovered by screening microorganisms sampled from diverse environments or developed by modification of known enzymes using modern methods of protein engineering or molecular evolution. As a result, several important food-processing enzymes such as amylases and lipases with properties tailored to particular food applications have become available. Another important achievement is improvement of microbial production strains. For example, several microbial strains recently developed for enzyme production have been engineered to increase enzyme yield by deleting native genes encoding extracellular proteases. Moreover, certain fungal production strains have been modified to reduce or eliminate their potential for production of toxic secondary metabolites. In this article, we discuss the safety of microorganisms used as hosts for enzyme-encoding genes, the construction of recombinant production strains, and methods of improving enzyme properties. We also briefly describe the manufacture and safety assessment of enzyme preparations and summarize options for submitting information on enzyme preparations to the US Food and Drug Administration.
---
paper_title: Extremophiles: developments of their special functions and potential resources.

paper_content:

Extremophilles are valuable resources in biotechnology. Enzymes from extremophiles are expected to fill the gap between biological and chemical processes due to their unusual properties. Especially enzymes from hyperthermophiles that can grow at above 90°C were devoted owing to its extraordinary thermostability and denaturant tolerance. Screening trials of hyperthermophilic microorganisms were performed by a number of microbiologists and various unique strains were isolated from natural environments. One of the most successful uses of thermostable enzymes was DNA polymerase in the polymerase chain reaction (PCR). Thermostable enzymes are used in the chemical, food, pharmaceutical, paper and textile industries. Recombinant forms of thermostable enzymes that have been expressed in Escherichia coli are commonly utilized in industrial applications however their enzymatic characteristics and tertiary structure are different from the native ones produced in the original strains. In vitro heat treatment induces a structural conversion of the recombinant protein to its natural form. High temperature itself plays an important role in determining the specific characteristics and tertiary structure of the enzyme. Recent studies have revealed that hyperthermophiles can grow under numerous conditions not only in geothermal or deep-sea thermal environments. Technological advances have allowed DNA to be isolated from natural environments. Now genes could be isolated from microorganisms that have not been cultured. In this review, innovative approaches to hunt genes from natural environments without pure culturing of microorganisms are also discussed.
---
paper_title: Cold-active acid β-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans

paper_content:

Summary In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans . The isolated strains were able to grow on lactose at below 5 °C, and showed cold-active acid β -galactosidase activity even at 0 °C and pH 4.0 in the extracellular fractions. Moreover, K m of β -galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 °C, and the activity could hydrolyze lactose in milk at 10 °C. The findings in this study indicate the possibility that the isolated strains produce novel acid β -galactosidases that are able to hydrolyze lactose at low temperature.
---
paper_title: Isolation and characterization of psychrophilic yeasts producing cold-adapted pectinolytic enzymes.

paper_content:

AIMS ::: The present study was conducted to screen for psychrophilic yeasts that are able to degrade pectin compounds at low temperature, and to examine the cold-active pectinolytic enzymes produced by the isolated psychrophilic yeasts. ::: ::: ::: METHODS AND RESULTS ::: Psychrophilic yeasts, which grow on pectin as a sole carbon source, pectinolytic-psychrophilic yeast (PPY) strains PPY-3, 4, 5 and 6, were isolated from soil from Abashiri (Hokkaido, Japan). The sequences of 28S rDNA D1/D2 of strains PPY-3 and 4 indicated a taxonomic affiliation to Cryptococcus cylindricus and Mrakia frigida, respectively, strains PPY-5 and 6 belonged to Cystofilobasidium capitatum. The isolated strains were able to grow on pectin at below 5 degrees C, and showed the activities of several cold-active pectinolytic enzymes. ::: ::: ::: CONCLUSION ::: The findings of this study indicate the possibility that the isolated strains produce novel pectinolytic enzymes that are able to degrade pectin compounds at low temperature. ::: ::: ::: SIGNIFICANCE AND IMPACT OF THE STUDY ::: It is possible that the cold-active pectinolytic enzymes from the isolated strains can be applied to the food industry, e.g. the clarification of fruit juice below 5 degrees C.
---
paper_title: Enzymes from fish and aquatic invertebrates and their application in the food industry

paper_content:

Abstract Enzymatic methods have become an important and indispensable part of the processes used by the modern food and feed industry to produce a large and diversified range of products for human and animal consumption. Aquatic environment contains the largest pool of diversified genetic material and, hence represents an enormous potential for different sources of enzymes. In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates has been achieved and some interesting and novel applications related to marine enzymes in food processing have emerged. This review summarizes information related to digestive and muscular enzymes in fish and aquatic invertebrates. In addition, important developments in food applications of marine enzymes are reported.
---
paper_title: Protein thermostability in Archaea and Eubacteria

paper_content:

In order to survive at high temperatures, thermophilic prokaryotes (Archaea and Eubacteria) adopt different strategies. Among several important contributing factors for stability of proteins are CG- rich codons, the ratio of charged amino acids compared to uncharged amino acids, ionic interactions, amino acid preferences and their distri- bution, post-translational modifications, and solute accumulation. How- ever, these factors may differ from taxon to taxon, both within and be- tween species depending upon the composition of proteins found in these organisms. This is exemplified in the case of differences in strategies adopted by soluble proteins and membrane proteins. Therefore, it ap- pears that no single factor or combination of factors together can be universally attributed to the provision of thermal stability in proteins.
---
paper_title: Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

paper_content:

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of > 80 degrees C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.
---
paper_title: Directed evolution of enzyme stability

paper_content:

Abstract Modern enzyme development relies to an increasing extent on strategies based on diversity generation followed by screening for variants with optimised properties. In principle, these directed evolution strategies might be used for optimising any enzyme property, which can be screened for in an economically feasible way, even if the molecular basis of that property is not known. Stability is an interesting property of enzymes because (1) it is of great industrial importance, (2) it is relatively easy to screen for, and (3) the molecular basis of stability relates closely to contemporary issues in protein science such as the protein folding problem and protein folding diseases. Thus, engineering enzyme stability is of both commercial and scientific interest. Here, we review how directed evolution has contributed to the development of stable enzymes and to new insight into the principles of protein stability. Several recent examples are described. These examples show that directed evolution is an effective strategy to obtain stable enzymes, especially when used in combination with rational or semi-rational engineering strategies. With respect to the principles of protein stability, some important lessons to learn from recent efforts in directed evolution are (1) that there are many structural ways to stabilize a protein, which are not always easy to rationalize, (2) that proteins may very well be stabilized by optimizing their surfaces, and (3) that high thermal stability may be obtained without forfeiture of catalytic performance at low temperatures.
---
paper_title: Assembly of mutations for improving thermostability of Escherichia coli AppA2 phytase

paper_content:

We previously identified a number of mutations in Escherichia coli AppA2 phytase for enhancing its thermostability. The objective of the present study was to determine if these mutations (K46E, K65E, G103S, D112N, D144N, S209G, V227A, and G344D) could be sequentially added to further improve the thermostability of AppA2. Compared with the wild-type enzyme, two variants (D144N/V227A and D144N/V227A/G344D) out of the eight resulting mutants showed 15% enhancement in thermostability (as measured by residual activity after being heated at 80°C for 10 min) and 4 to 5°C increases in the melting temperatures (Tm). Based on the structural predictions with a highly homologous AppA phytase, the substitution D144N introduces a side-chain–side-chain hydrogen bond, thereby stabilizing the loop region (Gln137–Asn144), and the V227A substitution might eliminate structural hindrance between Val222 and Val227 that face each other in the β-hairpin structure. In addition, overall catalytic efficiency (kcat/Km) of the two mutants was also improved (P < 0.05) compared to the wild type. However, no further improvement in thermostability was observed by adding other mutations to D144N/V227A/G344D, which might result from unfavorable electrostatic interactions or structural perturbation. In conclusion, our results underscore the potential as well as difficulty of predicting synergistic effects of multiple mutations on thermostability within phytase.
---
paper_title: Combinatorial engineering to enhance thermostability of amylosucrase.

paper_content:

Amylosucrase is a transglucosidase that catalyzes amylose-like polymer synthesis from sucrose substrate. About 60,000 amylosucrase variants from two libraries generated by the MutaGen random mutagenesis method were submitted to an in vivo selection procedure leading to the isolation of more than 7000 active variants. These clones were then screened for increased thermostability using an automated screening process. This experiment yielded three improved variants (two double mutants and one single mutant) showing 3.5- to 10-fold increased half-lives at 50 degrees C compared to the wild-type enzyme. Structural analysis revealed that the main differences between wild-type amylosucrase and the most improved variant (R20C/A451T) might reside in the reorganization of salt bridges involving the surface residue R20 and the introduction of a hydrogen-bonding interaction between T451 of the B' domain and D488 of flexible loop 8. This double mutant is the most thermostable amylosucrase known to date and the only one usable at 50 degrees C. At this temperature, amylose synthesis by this variant using high sucrose concentration (600 mM) led to the production of amylose chains twice as long as those obtained by the wild-type enzyme at 30 degrees C.
---
paper_title: Strategies for Improving Enzymes for Efficient Biocatalysis

paper_content:

An electromechanical brake tightening device for a rail vehicle, includes a brake actuator to apply and release brakes. The actuator has a power converter to convert energy supplied by the brake actuator to a brake application movement. The power converter includes a shear force measuring bolt and/or a brake lever, arranged in a power transmission path of the converter to measure braking power.
---
paper_title: Advances in laboratory evolution of enzymes.

paper_content:

We address recent developments in the area of laboratory, or directed evolution, with a focus on enzymes and on new methodologies of generic potential. We survey three main areas: (i) library making techniques, including the application of computational and rational methods for library design; (ii) screening and selection techniques, including recent applications of enzyme screening by FACS (fluorescence activated cell sorter); (iii) new approaches for performing directed evolution, and in particular, the application of 'neutral drifts' (libraries generated by rounds of mutation and selection for the enzyme's original function) and of consensus mutations to generate highly evolvable starting points for directed evolution.
---
paper_title: Recent Advances in Biocatalysis by Directed Enzyme Evolution

paper_content:

Naturally occurring enzymes are remarkable biocatalysts with numerous potential applications in industry and medicine. However, many of their catalyst properties often need to be further tailored to meet the specific requirements of a given application. Within this context, directed evolution has emerged over the past decade as a powerful tool for engineering enzymes with new or improved functions. This review summarizes recent advances in applying directed evolution approaches to alter various enzyme properties such as activity, selectivity (enantio- and regio-), substrate specificity, stability, and solubility. Special attention will be paid to the creation of novel enzyme activities and products by directed evolution.
---
paper_title: Structural features of thermozymes

paper_content:

Enzymes synthesized by thermophiles and hyperthermophiles are known as thermozymes. These enzymes are typically thermostable, or resistant to irreversible inactivation at high temperatures, and thermophilic, i.e. optimally active at elevated temperatures between 60 and 125 °C. Enzyme thermostability encompasses thermodynamic stability and kinetic stability. Thermodynamic stability is defined by the enzyme's free energy of stabilization (ΔGstab) and by its melting temperature (Tm). An enzyme's kinetic stability is often expressed as its halflife (t1/2) at defined temperature. ΔGstab of thermophilic proteins is 5–20 kcal/mol higher than that of mesophilic proteins. The thermostability mechanisms for thermozymes are varied and depend on the enzyme; nevertheless, some common features can be identified as contributing to stability. These features include more interactions (i.e. hydrogen bonds, electrostatic interactions, hydrophobic interactions, disulfide bonds, metal binding) than in less stable enzymes and superior conformational structure (i.e. more rigid, higher packing efficiency, reduced entropy of unfolding, conformational strain release and stability of α-helix). Understanding of the stabilizing features will greatly facilitate reengineering of some of the mesozymes to more stable thermozymes.
---
paper_title: Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution.

paper_content:

Abstract With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational ‘consensus concept’ are compared with those of rational design and directed evolution approaches.
---
paper_title: Directed evolution drives the next generation of biocatalysts.

paper_content:

Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technology. Directed evolution involves repeated rounds of (i) random gene library generation, (ii) expression of genes in a suitable host and (iii) screening of libraries of variant enzymes for the property of interest. Both in vitro screening-based methods and in vivo selection-based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodology, applications in synthetic transformations and strategies for the generation of new enzyme function.
---
paper_title: Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR

paper_content:

Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60–80°C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80°C for 10 min), and 6–7°C increases in melting temperatures (Tm). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (kcat / Km) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.
---
paper_title: Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH

paper_content:

The Thermotoga neapolitana xylose isomerase (TNXI) is extremely thermostable and optimally active at 95 degrees C. Its derivative, TNXI Val185Thr (V185T), is the most active type II xylose isomerase reported, with a catalytic efficiency of 25.1 s(-1) mM(-1) toward glucose at 80 degrees C (pH 7.0). To further optimize TNXI's potential industrial utility, two rounds of random mutagenesis and low temperature/low pH activity screening were performed using the TNXI V185T-encoding gene as the template. Two highly active mutants were obtained, 3A2 (V185T/L282P) and 1F1 (V185T/L282P/F186S). 1F1 was more active than 3A2, which in turn was more active than TNXI V185T at all temperatures and pH values tested. 3A2 and 1F1's high activities at low temperatures were due to significantly lower activation energies (57 and 44 kJ/mol, respectively) than that of TNXI and V185T (87 kJ/mol). Mutation L282P introduced a kink in helix alpha7 of 3A2's (alpha/beta)8 barrel. Surprisingly, this mutation kinetically destabilized 3A2 only at pH 5.5. 1F1 displayed kinetic stability slightly above that of TNXI V185T. In 1F1, mutation F186S creates a cavity that disrupts a four-residue network of aromatic interactions. How the conformation of the neighboring residues is affected by this cavity and how these conformational changes increase 1F1's stability still remain unclear.
---
paper_title: Developments in Directed Evolution for Improving Enzyme Functions

paper_content:

The engineering of enzymes with altered activity, specificity, and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. In vitro recombination techniques such as DNA shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), iterative truncation for the creation of hybrid enzymes (ITCHY), recombined extension on truncated templates (RETT), and so on have been developed to mimic and accelerate nature's recombination strategy. This review discusses gradual advances in the techniques and strategies used for the directed evolution of biocatalytic enzymes aimed at improving the quality and potential of enzyme libraries, their advantages, and disadvantages.
---
paper_title: Directed evolution of enzymes for product chemistry

paper_content:

Covering: up to April 2004 ::: This review discusses the achievements of directed evolution of enzymes relevant to natural and unnatural product chemistry focusing on the period 1998–2004, citing 156 references. A few selected papers published before 1998 are also cited for their fundamental relevance, together with additional reviews covering other aspects of directed evolution applied to closely related topics. An overview of the latest methods applied in directed evolution is given in the introduction, together with a range of approaches for the selection of variants of enzymes with desired catalytic properties. All classes of enzymes are systematically covered.
---
paper_title: Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance

paper_content:

The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75 degrees C, which was 15 degrees C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9 degrees C. The half-life of ThMA-DM was 172 min at 80 degrees C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.
---
paper_title: Directed enzyme evolution: climbing fitness peaks one amino acid at a time.

paper_content:

Directed evolution can generate a remarkable range of new enzyme properties. Alternate substrate specificities and reaction selectivities are readily accessible in enzymes from families that are naturally functionally diverse. Activities on new substrates can be obtained by improving variants with broadened specificities or by step-wise evolution through a sequence of more and more challenging substrates. Evolution of highly specific enzymes has been demonstrated, even with positive selection alone. It is apparent that many solutions exist for any given problem, and there are often many paths that lead uphill, one step at a time.
---
paper_title: Molecular and industrial aspects of glucose isomerase.

paper_content:

Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the presence of two signature sequences, VXW(GP)GREG(YSTAE)E and (LIVM)EPKPX(EQ)P. The use of an inexpensive inducer in the fermentation medium devoid of Co2+ and redesigning of a tailor-made GI with increased thermostability, higher affinity for glucose, and lower pH optimum will contribute significantly to the development of an economically feasible commercial process for enzymatic isomerization of glucose to fructose. Manipulation of the GI gene by site-directed mutagenesis holds promise that a GI suitable for biotechnological applications will be produced in the foreseeable future.
---
paper_title: Engineering Enzymes for Biocatalysis

paper_content:

Protein engineering techniques have been available for over two decades beginning with the development of methods for genetic engineering. Since that time, the engineering of enzymes has advanced rapidly along with a revolution in the range and efficiency of new techniques and strategies for designing and evolving proteins in the laboratory. While recent advances in high-throughput screening techniques are permitting larger libraries of enzymes to be screened more rapidly, a combination of new genetic tools and computational methods are enabling the efficient application of random mutagenesis targeted to areas of enzyme structures that are more likely to elicit the desired enhancement of their biocatalytic properties. Meanwhile the rational design of enzyme properties, in particular, by computational design is showing great potential. This review article summarises recent and important patents relating to the engineering of enzymes for biocatalysis.
---
paper_title: History of Industrial Biotransformations – Dreams and Realities

paper_content:

This user-friendly and application-oriented overview draws on extensive literature and patent research, and is unique in its systematic structure for easy comparison. For this second edition every chapter has been rewritten, with each process updated and over 30 new ones added.
---
paper_title: Thermal Stabilization of Bacillus subtilis Family-11 Xylanase by Directed Evolution

paper_content:

Abstract We used directed evolution to enhance the thermostability of glycosyl hydrolase family-11 xylanase from Bacillus subtilis. By combining random point mutagenesis, saturation mutagenesis, and DNA shuffling, a thermostable variant, Xylst, was identified which contained three amino acid substitutions: Q7H, N8F, and S179C. The half-inactivation temperature (the midpoint of the melting curves) for the Xylst variant compared with the wild-type enzyme after incubation for 10 min was elevated from 58 to 68 °C. At 60 °C the wild-type enzyme was inactivated within 5 min, but Xylst retained full activity for at least 2 h. The stabilization was accompanied by evidence of thermophilicity; that is, an increase in the optimal reaction temperature from 55 to 65 °C and lower activity at low temperatures and higher activity at higher temperatures relative to wild type. To elucidate the mechanism of thermal stabilization, three-dimensional structures were determined for the wild-type and Xylst enzymes. A cavity was identified around Gln-7/Asn-8 in wild type that was filled with bulky, hydrophobic residues in Xylst. This site was not identified by previous approaches, but directed evolution identified the region as a weak point. Formation of an intermolecular disulfide bridge via Cys-179 was observed between monomers in Xylst. However, the stability was essentially the same in the presence and absence of a reducing agent, indicating that the increased hydrophobicity around the Cys-179 accounted for the stability.
---
paper_title: Engineering of Phytase for Improved Activity at Low pH

paper_content:

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.
---
paper_title: Modification of optimal pH in L-arabinose isomerase from Geobacillus stearothermophilus for D-galactose isomerization

paper_content:

Abstract l -Arabinose isomerase from Geobacillus stearothermophilus (GSAI; EC 5.3.1.4) has been genetically evolved to increase the reaction rate toward d -galactose, which is not a natural substrate. To change the optimal pH of GSAI for d -galactose isomerization (pH optimum at 8.5), we investigated the single point mutations influencing the activity based on the sequences of the previously evolved enzymes. Among the seven point mutations found in the evolved enzymes, mutations at Val 408 and Asn 475 were determined to be highly influential mutation points for d -galactose isomerization activity. A random mutation was introduced into sites Val 408 and Asn 475 (X408V and X475N), and candidates were screened based on non-optimal pH conditions. Among the mutations of X408V and X475N, mutations of Q408V and R408V were selected. The optimal pH of the both mutations Q408V and R408V was shifted to pH 7.5. At the shifted optimal pH, the d -galactose isomerization activities of Q408V and R408V were 60 and 30% higher than that of the wild type at pH 8.5, respectively.
---
paper_title: Applications of cold-adapted proteases in the food industry.

paper_content:

Publisher Summary This chapter discusses that enzyme technology has been an integral part of food processing and improvement of food quality for a long time and advances in enzyme technology will be important for the production of better and safer foods. The utilization of by-products from the fishing and agricultural industries for added value largely depends on a thorough understanding of enzyme reactions. Relatively few enzymes are used in a wide variety of food processes, for the production of corn syrup and speciality fats, and to facilitate juice extraction from fruits, in addition to their use as antimicrobial preservatives. The most common food processing enzymes are hydrolases, including the proteases, glucosidases, and lipases. Proteolytic cold-adapted “superactive” enzymes from marine sources offer advantages for the food industry, although their purification and structure determination are often problematic. The tremendous knowledge gathered on enzyme structures and function in recent years has opened up a new era for food processing applications with enzymes. A new phase of enzyme technology may revolutionize the ways foods are prepared and processed in the near future. It is becoming easier to obtain relatively large numbers of naturally rare proteins, crystallography requires smaller amounts of purified proteins and smaller crystals are easier to work with than ever before.
---
paper_title: Transglycosylation reactions using glycosyl hydrolases from Thermotoga neapolitana, a marine hydrogen-producing bacterium

paper_content:

The use of the extremophilic marine bacterium Thermotoga neapolitana (DSM 4359) for the bioproduction of hydrogen using complex carbohydrates as feedstock has been recently described in a patent. In this growth condition glycosyl hydrolases can be well expressed. An important issue to plan economical feasibility of biological hydrogen production comprises techniques for recovering useful materials such as physiologically active biomolecules from biomasses grown in large quantities. ::: ::: The present paper describes a series of enzymatic transglycosylation reactions performed using the crude homogenate of T. neapolitana. The study is focused on synthetic features of the transglycosylating enzymes. Xylosidase/xylanase activity seems to be the most abundant leading to convenient syntheses of interesting series of pure (β-1,4)-xylooligosaccharides of different aglycones such as 1-hexanol (producing promising candidates for new surfactants), 9-fluorene methanol (obtaining anti-HSV agents), 1,4-butanediol (for the synthesis of new glycolipids), and geraniol (producing aroma compounds). Furthermore, the regioselectivity during galactose, fucose, glucose, and mannose enzymatic transfers is also investigated. The knowledge of synthetic characteristics of all these enzymes will be useful in the feasibility evaluation of large-scale processes of the biohydrogen production based on T. neapolitana.
---
paper_title: Current studies on biological tagatose production using l-arabinose isomerase: a review and future perspective

paper_content:

d-Tagatose is a hexoketose monosaccharide sweetener, which is an isomer of d-galactose and is rarely found in nature. Recently, there has been industrial interest in d-tagatose as a low-calorie sugar-substituting sweetener. This article describes the properties and metabolism of tagatose as well as its commercial importance. The comparison between the biological tagatose production and the chemical production was reviewed based on the example of the glucose isomerization into fructose. The industrial problems facing its commercial application is described and evolving potential solutions are suggested.
---
paper_title: Molecular and biotechnological aspects of xylanases.

paper_content:

Hemicellulolytic microorganisms play a significant role in nature by recycling hemicellulose, one of the main components of plant polysaccharides. Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. Recently cellulase-free xylanases have received great attention in the development of environmentally friendly technologies in the paper and pulp industry. In microorganisms that produce xylanases low molecular mass fragments of xylan and their positional isomers play a key role in regulating its biosynthesis. Xylanase and cellulase production appear to be regulated separately, although the pleiotropy of mutations, which causes the elimination of both genes, suggests some linkage in the synthesis of the two enzymes. Xylanases are found in a cornucopia of organisms and the genes encoding them have been cloned in homologous and heterologous hosts with the objectives of overproducing the enzyme and altering its properties to suit commercial applications. Sequence analyses of xylanases have revealed distinct catalytic and cellulose binding domains, with a separate non-catalytic domain that has been reported to confer enhanced thermostability in some xylanases. Analyses of three-dimensional structures and the properties of mutants have revealed the involvement of specific tyrosine and tryptophan residues in the substrate binding site and of glutamate and aspartate residues in the catalytic mechanism. Many lines of evidence suggest that xylanases operate via a double displacement mechanism in which the anomeric configuration is retained, although some of the enzymes catalyze single displacement reactions with inversion of configuration. Based on a dendrogram obtained from amino acid sequence similarities the evolutionary relationship between xylanases is assessed. In addition the properties of xylanases from extremophilic organisms have been evaluated in terms of biotechnological applications.
---
paper_title: Starch and α-glucan acting enzymes, modulating their properties by directed evolution

paper_content:

Starch is the major food reserve in plants and forms a large part of the daily calorie intake in the human diet. Industrially, starch has become a major raw material in the production of various products including bio-ethanol, coating and anti-staling agents. The complexity and diversity of these starch based industries and the demand for high quality end products through extensive starch processing, can only be met through the use of a broad range of starch and alpha-glucan modifying enzymes. The economic importance of these enzymes is such that the starch industry has grown to be the largest market for enzymes after the detergent industry. However, as the starch based industries expand and develop the demand for more efficient enzymes leading to lower production cost and higher quality products increases. This in turn stimulates interest in modifying the properties of existing starch and alpha-glucan acting enzymes through a variety of molecular evolution strategies. Within this review we examine and discuss the directed evolution strategies applied in the modulation of specific properties of starch and alpha-glucan acting enzymes and highlight the recent developments in the field of directed evolution techniques which are likely to be implemented in the future engineering of these enzymes.
---
paper_title: Protein engineering to improve the thermostability of glucoamylase from Aspergillus awamori based on molecular dynamics simulations

paper_content:

Twelve mutations were constructed to improve the thermostability of glucoamylase from Aspergillus awamori based on the results of molecular dynamics simulations. The thermal unfolding of the catalytic domain followed a putative hierarchical behavior. In addition, the unfolding of the 13 alpha-helices obeyed the random ordered mechanism, in which the alpha-helices 8, 1 and 11 unfolded more rapidly than the others. The catalytic center was well protected by the (alpha/alpha)(6)-barrel at simulation temperatures up to 600 K, whereas the catalytic base, E400, migrated from its original interior pocket to the surface of the catalytic domain by surmounting the hydrophobic barrier provided by alpha-helices 12 and 13 at 800 K. The disulfide bonds engineered to 'lock' the alpha-helix 11 on the surface of the catalytic domain dramatically increased the thermostability. Substituting G396 and G407 with Ala residues slightly increased the thermostability, whereas their specific activity and catalytic efficiency were reduced. This indicates that the introduced residues with higher hydrophobicity were favorable in the loop between alpha-helices 12 and 13, whereas they partially destroyed the hydrogen bond and salt linkage network in the catalytic center. Alpha-helices 12 and 13 can be stabilized by introducing residues with higher hydrophobicity, except for the H391M mutation.
---
paper_title: Assembly of mutations for improving thermostability of Escherichia coli AppA2 phytase

paper_content:

We previously identified a number of mutations in Escherichia coli AppA2 phytase for enhancing its thermostability. The objective of the present study was to determine if these mutations (K46E, K65E, G103S, D112N, D144N, S209G, V227A, and G344D) could be sequentially added to further improve the thermostability of AppA2. Compared with the wild-type enzyme, two variants (D144N/V227A and D144N/V227A/G344D) out of the eight resulting mutants showed 15% enhancement in thermostability (as measured by residual activity after being heated at 80°C for 10 min) and 4 to 5°C increases in the melting temperatures (Tm). Based on the structural predictions with a highly homologous AppA phytase, the substitution D144N introduces a side-chain–side-chain hydrogen bond, thereby stabilizing the loop region (Gln137–Asn144), and the V227A substitution might eliminate structural hindrance between Val222 and Val227 that face each other in the β-hairpin structure. In addition, overall catalytic efficiency (kcat/Km) of the two mutants was also improved (P < 0.05) compared to the wild type. However, no further improvement in thermostability was observed by adding other mutations to D144N/V227A/G344D, which might result from unfavorable electrostatic interactions or structural perturbation. In conclusion, our results underscore the potential as well as difficulty of predicting synergistic effects of multiple mutations on thermostability within phytase.
---
paper_title: Mutations in two amino acids in phyI1s from Aspergillus niger 113 improve its phytase activity

paper_content:

We applied in vitro mutagenesis and colony screening, using the wild type phyI1s gene from Aspergillus niger 113 as the template, and obtained two mutant phyI1s (gene products) after one round of screening. The two mutants had mutations at two nucleic acid sites, resulting in changes in two amino acids: K41E, E121F. None of the amino acid substitutions in the two mutants was in a position reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two mutants indicated that the substitutions gave rise to 2.5- and 3.1-fold increased specific activity, and a 1.78- and 3.24-fold reduced affinity for sodium phytate. In addition, the overall catalytic efficiency (kcat/Km) of the two mutants was changed by 0.52-fold and 0.68-fold compared to that of the wild type. Such mutants will be instrumental for the structure–function study of the enzyme and for industrial application.
---
paper_title: Enzymatic conversion of d-galactose to d-tagatose: heterologous expression and characterisation of a thermostable l-arabinose isomerase from Thermoanaerobacter mathranii

paper_content:

The ability to convert d-galactose into d-tagatose was compared among a number of bacterial l-arabinose isomerases (araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis–Menten constants of the enzyme determined with l-arabinose, d-galactose and d-fucose also indicated that this enzyme is an unusual, versatile l-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of d-tagatose at 65 °C. Starting from a 30% solution of d-galactose, the yield of d-tagatose was 42% and no sugars other than d-tagatose and d-galactose were detected. Direct conversion of lactose to d-tagatose in a single reactor was demonstrated using a thermostable β-galactosidase together with the thermostable l-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.
---
paper_title: Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity

paper_content:

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme™, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.
---
paper_title: Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR

paper_content:

Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60–80°C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80°C for 10 min), and 6–7°C increases in melting temperatures (Tm). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (kcat / Km) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.
---
paper_title: Co-expression of l-arabinose isomerase and d-glucose isomerase in E. coli and development of an efficient process producing simultaneously d-tagatose and d-fructose

paper_content:

Abstract To develop a feasible enzymatic process for the concomitant d -tagatose and d -fructose production, the thermostable l -arabinose isomerase of Bacillus stearothermophilus US100 ( l -AI US100) and the mutant d -glucose isomerase obtained from that of Streptomyces SK (SKGI-A103G) were successfully co-expressed in Escherichia coli HB101 strain. The recombinant cells were immobilized in alginate beads and showed, similarly to the free cells, optimal temperatures for d -galactose and d -glucose isomerisation of 80 and 85 °C, respectively. The two isomerases were optimally active at pH 7.5. Cell entrapment significantly enhanced the acidotolerance of the two isomerases, as well as their stability at high temperatures. To perform simultaneous isomerisation of d -galactose and d -glucose at 65 °C and pH 7.5 in packed-bed bioreactor, cells concentration, dilution rate, productivity and bioconversion rate were optimized to be 32 g/l, 2.6 h −1 , 3 g/l h and 30%, respectively.
---
paper_title: Selected mutations in Bacillus subtilis levansucrase semi-conserved regions affecting its biochemical properties

paper_content:

Levansucrases (LS) are fructosyltransferases (FTFs) belonging to family 68 of glycoside hydrolases (GH68) using sucrose as substrate to synthesize levan, a fructose polymer. From a multiple sequence analysis of GH68 family proteins, nine residues were selected and their role in acceptor and product specificity, as well as in biochemical Bacillus subtilis LS properties, was investigated. A product specificity modification was obtained with mutants Y429N and R433A that no longer produce levan but exclusively oligosaccharides. An effect of the mutation S164A was observed on enzyme stability and kinetic behavior; this mutation also induces a levan activation effect that enhances the reaction rate. We report the crystallographic structure of this mutant and found that S164 is an important residue to maintain the nucleophile position in the active site. We also found evidence of the important role of Y429 in acceptor specificity: this is a key residue coordinating the sucrose position in the catalytic domain-binding pocket. Some of these mutations resulted in LS with a broad range of specificities and new biochemical properties.
---
paper_title: Cumulative improvements of thermostability and pH-activity profile of Aspergillus niger PhyA phytase by site-directed mutagenesis

paper_content:

Aspergillus niger phytase (PhyA) has been used as a feed supplement to reduce manure phosphorus excretion of swine and poultry but lacks sufficient thermostability for feed pelleting and appropriate pH-activity profile for phytate hydrolysis in the stomach of animals. Previously, a thermostable mutant PhyA18 and two pH-activity profile-improved mutants E228K and K300E were developed. In this study, the mutations were combined to determine if both improvements were cumulative. Four substitutions (S149P, F131L, K112R, and K195R) identified from random mutagenesis were added sequentially to the combined mutants to further improve their thermostability. Mutant E228K shifted the optimum pH of the parent one from 5.5 to 4.0 and increased (P < 0.05) its specific activity at pH 3.5, whereas mutant K300E eliminated the activity dip at pH 3.5 shown in the wild type. Mutant S149P further improved thermostability over PhyA18. Our results illustrate the feasibility and structural basis to improve thermostability and pH-activity profile of PhyA phytase by assembling mutations derived from rational design and random mutagenesis.
---
paper_title: Use of Random and Saturation Mutageneses To Improve the Properties of Thermus aquaticus Amylomaltase for Efficient Production of Cycloamyloses

paper_content:

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of α-1,4 glucans to produce cyclic α-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.
---
paper_title: Hyperthermostabilization of Bacillus licheniformis α-amylase and modulation of its stability over a 50°C temperature range

paper_content:

Bacillus licheniformis α-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories. For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability. This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA. Remarkably, the Q264S–N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function. The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23°C higher than the wild-type enzyme. In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25°C in the half-inactivation temperature of the double mutant enzyme compared with wild-type. These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant. Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50°C through a few point mutations.
---
paper_title: Single amino acid residue changes in subsite ) 1 of inulosucrase from Lactobacillus reuteri 121 strongly influence the size of products synthesized

paper_content:

Bacterial fructansucrase enzymes belong to glycoside hydrolase family 68 and catalyze transglycosylation reactions with sucrose, resulting in the synthesis of fructooligosaccharides and/or a fructan polymer. Significant differences in fructansucrase enzyme product specificities can be observed, i.e. in the type of polymer (levan or inulin) synthesized, and in the ratio of polymer versus fructooligosaccharide synthesis. The Lactobacillus reuteri 121 inulosucrase enzyme produces a diverse range of fructooligosaccharide molecules and a minor amount of inulin polymer [with β(2-1) linkages]. The three-dimensional structure of levansucrase (SacB) of Bacillus subtilis revealed eight amino acid residues interacting with sucrose. Sequence alignments showed that six of these eight amino acid residues, including the catalytic triad (D272, E523 and D424, inulosucrase numbering), are completely conserved in glycoside hydrolase family 68. The other three completely conserved residues are located at the - 1 subsite (W271, W340 and R423). Our aim was to investigate the roles of these conserved amino acid residues in inulosucrase mutant proteins with regard to activity and product profile. Inulosucrase mutants W340N and R423H were virtually inactive, confirming the essential role of these residues in the inulosucrase active site. Inulosucrase mutants R423K and W271N were less strongly affected in activity, and displayed an altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer. Our data show that the - 1 subsite is not only important for substrate recognition and catalysis, but also plays an important role in determining the size of the products synthesized. © 2006 The Authors.
---
paper_title: Computational tools for designing and engineering biocatalysts.

paper_content:

Current computational tools to assist experimentalists for the design and engineering of proteins with desired catalytic properties are reviewed. The applications of these tools for de novo design of protein active sites, optimization of substrate access and product exit pathways, redesign of protein-protein interfaces, identification of neutral/advantageous/deleterious mutations in the libraries from directed evolution and stabilization of protein structures are described. Remarkable progress is seen in de novo design of enzymes catalyzing a chemical reaction for which a natural biocatalyst does not exist. Yet, constructed biocatalysts do not match natural enzymes in their efficiency, suggesting that more research is needed to capture all the important features of natural biocatalysts in theoretical designs.
---
paper_title: Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production.

paper_content:

L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.
---
paper_title: Mutations at subsite −3 in cyclodextrin glycosyltransferase from Paenibacillus macerans enhancing α-cyclodextrin specificity

paper_content:

A major disadvantage of cyclodextrin production is the limited cyclodextrin product specificity of cyclodextrin glycosyltransferase (CGTase). Here, we described mutations of Asp372 and Tyr89 at subsite −3 in the CGTase from Paenibacillus macerans strain JFB05-01. The results showed that Asp372 and Tyr89 played important roles in cyclodextrin product specificity of CGTase. The replacement of Asp372 by lysine and Tyr89 by aspartic acid, asparagine, lysine, and arginine resulted in a shift in specificity towards the production of α-cyclodextrin, which was most apparent for the mutants D372K and Y89R. Furthermore, the changes in cyclodextrin product specificity for the single mutants D372K and Y89R could be combined in the double mutant D372K/Y89R, which displayed a 1.5-fold increase in the production of α-cyclodextrin, with a concomitant 43% decrease in the production of β-cyclodextrin when compared to the wild-type CGTase. Thus, the D372K and Y89R single and double mutants were much more suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The enhanced α-cyclodextrin specificity of these mutants might be a result of stabilizing the bent conformation of the intermediate in the cyclization reaction.
---
paper_title: Increasing the thermostability of d-xylose isomerase by introduction of a proline into the turn of a random coil

paper_content:

Thermostability can be increased by introducing prolines at suitable sites in target proteins. Two single (G138P, G247D) mutants and one double (G138P/G247D) mutant of xylose isomerase from Streptomyces diastaticus No.7, strain M1033 have been constructed by site-directed mutagenesis. With respect to the wild-type enzyme, G138P showed about a 100% increase in thermostability, and G247D showed an increased catalytic activity. Significantly, the double mutant, G138P/G247D displayed even higher activity than G247D and better heat stability than G138P. Its half life was about 2.5-fold greater than the wild-type enzyme, using xylose as a substrate. Molecular modelling suggested that the introduction of a proline residue in the turn of a random coil may cause the surrounding conformation to be tightened by reducing the backbone flexibility. The change in thermostability can, therefore, be explained based on changes in the molecular rigidity. Furthermore, the improvements in the properties of the double mutant indicated that the advantages of two single mutants can be combined effectively.
---
paper_title: Thermozymes: Identifying molecular determinants of protein structural and functional stability

paper_content:

Thermozymes are thermostable enzymes that function optimally between 60°C and 125°C. These extremozymes are a ‘hot' research topic because they are remarkable tools for studying protein stability, as well as for developing industrial and specialty biotechnologies. Most protein-stabilization mechanisms (i.e. hydrophobic interactions, packing efficiency, salt-bridges, hydrogen bonds, reduction of conformational strain, reduction of the entropy of unfolding, α-helix stabilization, loop stabilization and resistance to covalent destruction) have been identified by stability studies using mesophilic models. Recent structural comparisons between mesophilic enzymes and thermozymes validate these mechanisms.
---
paper_title: Engineering of cyclodextrin glycosyltransferase reaction and product specificity

paper_content:

Many plants produce starch, a high molecular weight polymer of glucose, for storage as a carbon and energy source. These starch molecules are mostly found in seeds (e.g., wheats) or roots (e.g., potato) in the form of granules consisting of two types of glucan polymers: highly branched amylopectin and linear amylose. Many bacteria are able to use starch as a carbon and energy source for growth. For this purpose these micro-organisms convert starch molecules extracellularly into molecules suitable for uptake and further conversion by the cells. A whole range of starch-degrading enzymes with diierent reaction speci¢cities has evolved in these organisms yielding a wide variety of products. A number of these enzymes ¢nd application in the industrial processing of starch, either for modi¢cation of starch molecules or for the production of speci¢c degradation products. These enzymes are therefore studied extensively, leading to increasing knowledge of their reaction mechanisms and factors determining substrate and product speci¢city. A particularly interesting enzyme is cyclodextrin glycosyltransferase (CGTase), which has the unique capability of forming cyclodextrins from starch. This enzyme is a member of the K-amylase family (family 13) of glycosyl hydrolases. Current insights in the catalytic mechanism employed by these enzymes is discussed. Emphasis in this review is on structural and mechanistic features of CGTase determining cyclodextrin product speci¢city.
---
paper_title: Engineering Enzymes for Biocatalysis

paper_content:

Protein engineering techniques have been available for over two decades beginning with the development of methods for genetic engineering. Since that time, the engineering of enzymes has advanced rapidly along with a revolution in the range and efficiency of new techniques and strategies for designing and evolving proteins in the laboratory. While recent advances in high-throughput screening techniques are permitting larger libraries of enzymes to be screened more rapidly, a combination of new genetic tools and computational methods are enabling the efficient application of random mutagenesis targeted to areas of enzyme structures that are more likely to elicit the desired enhancement of their biocatalytic properties. Meanwhile the rational design of enzyme properties, in particular, by computational design is showing great potential. This review article summarises recent and important patents relating to the engineering of enzymes for biocatalysis.
---
paper_title: γ-Cyclodextrin: a review on enzymatic production and applications

paper_content:

Cyclodextrins are cyclic α-1,4-glucans that are produced from starch or starch derivates using cyclodextrin glycosyltransferase (CGTase). The most common forms are α-, β-, and γ-cyclodextrins. This mini-review focuses on the enzymatic production, unique properties, and applications of γ-cyclodextrin as well as its difference with α- and β-cyclodextrins. As all known wild-type CGTases produce a mixture of α-, β-, and γ-cyclodextrins, the obtaining of a CGTase predominantly producing γ-cyclodextrin is discussed. Recently, more economic production processes for γ-cyclodextrin have been developed using improved γ-CGTases and appropriate complexing agents. Compared with α- and β-cyclodextrins, γ-cyclodextrin has a larger internal cavity, higher water solubility, and more bioavailability, so it has wider applications in many industries, especially in the food and pharmaceutical industries.
---
paper_title: Tagatose: properties, applications, and biotechnological processes

paper_content:

d-Tagatose has attracted a great deal of attention in recent years due to its health benefits and similar properties to sucrose. d-Tagatose can be used as a low-calorie sweetener, as an intermediate for synthesis of other optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulation. Biotransformation of d-tagatose has been produced using several biocatalyst sources. Among the biocatalysts, l-arabinose isomerase has been mostly applied for d-tagatose production because of the industrial feasibility for the use of d-galactose as a substrate. In this article, the characterization of many l-arabinose isomerases and their d-tagatose production is compared. Protein engineering and immobilization of the enzyme for increasing the conversion rate of d-galactose to d-tagatose are also reviewed.
---
paper_title: Glutamic acid 219 is critical for the thermostability of a truncated α-amylase from alkaliphilic and thermophilic Bacillus sp. strain TS-23

paper_content:

The functional and structural significance of glutamic acid 219 of a N- and C-terminally truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was explored by the approach of site-directed saturation mutagenesis. The expressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular mass was determined to be approximately 54 kDa by SDS/PAGE. Except E219F, E219P, and E219W, all other mutant enzymes exhibited a lower shift in their optimum temperatures with respect to the wild-type enzyme. A decreased thermostability was also found in all of the mutant enzymes when compared with the wild-type form of BACΔNC. Except E219F, E219P, and E219W mutant enzymes, greater than 2-fold decrease in k cat and a similar substrate affinity relative to the wild-type BACΔNC were observed for the rest mutant enzymes. Based on these observations, it is suggested that Glu-219 apparently plays an important role in the thermostability of BACΔNC.
---
paper_title: Thermal Stabilization of Bacillus subtilis Family-11 Xylanase by Directed Evolution

paper_content:

Abstract We used directed evolution to enhance the thermostability of glycosyl hydrolase family-11 xylanase from Bacillus subtilis. By combining random point mutagenesis, saturation mutagenesis, and DNA shuffling, a thermostable variant, Xylst, was identified which contained three amino acid substitutions: Q7H, N8F, and S179C. The half-inactivation temperature (the midpoint of the melting curves) for the Xylst variant compared with the wild-type enzyme after incubation for 10 min was elevated from 58 to 68 °C. At 60 °C the wild-type enzyme was inactivated within 5 min, but Xylst retained full activity for at least 2 h. The stabilization was accompanied by evidence of thermophilicity; that is, an increase in the optimal reaction temperature from 55 to 65 °C and lower activity at low temperatures and higher activity at higher temperatures relative to wild type. To elucidate the mechanism of thermal stabilization, three-dimensional structures were determined for the wild-type and Xylst enzymes. A cavity was identified around Gln-7/Asn-8 in wild type that was filled with bulky, hydrophobic residues in Xylst. This site was not identified by previous approaches, but directed evolution identified the region as a weak point. Formation of an intermolecular disulfide bridge via Cys-179 was observed between monomers in Xylst. However, the stability was essentially the same in the presence and absence of a reducing agent, indicating that the increased hydrophobicity around the Cys-179 accounted for the stability.
---
paper_title: Perspectives and applications of immobilised β-galactosidase in food industry – a review

paper_content:

β-Galactosidase is an important industrial enzyme in the hydrolysis of milk and whey lactose. The enzymatic hydrolysis of lactose allows to avoid health and environmental problems posed by this disaccharide. In addition, this enzyme catalyses the formation of galacto-oligosaccharides, which are prebiotic additives for the so-called "healthy foods". β-Galactosidase is one of the relatively few enzymes that have been used in large-scale processes in both free and immobilised forms. This article presents a review of recent trends in immobilisation of β-galactosidase and their application in food industry.
---
paper_title: Application of encapsulated enzymes to accelerate cheese ripening

paper_content:

Abstract The suitability of gellan, κ-carrageenan and a high-melting-fat-fraction of milk fat (HMFF) to encapsulate protease enzymes (Flavourzyme) and impact in accelerating Cheddar cheese ripening were studied. The rates of enzyme entrapment were 48.2%, 55.6%, and 38.9% for gellan, κ-carrageenan and HMFF, respectively. The enzyme capsules were incorporated into milk during cheese manufacture. The moisture content of cheeses with added gum capsules was higher than control cheeses. Casein (β) degradation was monitored by High-Performance Capillary Electrophoresis. All cheeses treated with encapsulated enzyme showed higher rates of proteolysis than the control cheese throughout the ripening period. The rate of proteolysis was greater with cheeses made incorporating κ-carrageenan capsules containing protease. Cheese texture and sensory quality were not significantly influenced by the type of encapsulating material (gum or milk fat). Differences in textural and sensory quality between treated and control cheeses were consistent with release of protease enzymes from capsules.
---
paper_title: Impact of liposome-encapsulated enzyme cocktails on cheddar cheese ripening

paper_content:

Abstract Cheddar cheese proteolysis and lipolysis were accelerated using liposome-encapsulated enzymatic cocktails. Flavourzyme, neutral bacterial protease, acid fungal protease and lipase (Palatase M) were individually entrapped in liposomes and added to cheese milk prior to renneting. Flavourzyme was tested alone at three concentrations (Z1, Z2 and Z3 cheeses). Enzyme cocktails consisted of lipase and bacterial protease (BP cheeses), lipase and fungal protease (FP cheeses) or lipase and Flavourzyme (ZP cheeses). The resulting cheeses were chemically, rheologically and organoleptically evaluated during 3 months of ripening at 8 °C. Levels of free fatty acids and appearance of bitter and astringent peptides were measured. Certain enzyme treatments (BP and ZP) resulted in cheeses with more mature texture and higher flavor intensity in a shorter time compared with control cheeses. No bitter defect was detected except in 90-day-old FP cheese. A full aged Cheddar flavor was developed in Z3 and ZP cheeses, while treatment BP led to strong typical Cheddar flavor by the second month and did not exhibit any off-flavor when ripening was extended for a further month.
---
paper_title: Microencapsulation of enzymes for potential application in acceleration of cheese ripening

paper_content:

Abstract An encapsulation efficiency of 70% was achieved when flavourzyme was encapsulated in selected polymers and gelled in 0.1 m CaCl 2 containing 0.1% (w/v) chitosan. Enzyme retention in capsules did not increase with alginate concentration or with coating of capsules with 0.15% (w/v) alginate or 0.05% (w/v) poly l -lysine. Gelling duration of 10 min was found to be optimum, while 2.0% trisodium citrate was necessary for efficient release of encapsulated flavourzyme. During 10-week storage after freeze-drying and freezing, about 80% retention of enzyme activity was noted in flavourzyme capsules with or without poly l -lysine coating, while air-dried capsules retained about 53% activity and 32% on coating with poly l -lysine over 10 weeks. Flavourzyme leakage from capsules increased with an increase in the duration of the simulated cheese press from 4 to 16 h. Capsule addition before rennetting resulted in an even distribution compared with capsule addition before salting.
---
paper_title: Immobilization of Bacillus subtilis α-amylase and characterization of its enzymatic properties

paper_content:

Abstract Bacillus subtilis I (α-amylase was immobilized on different carriers by different methods of immobilization. The immobilized enzymes were prepared by physical adsorption on aminoalkylsilane-alumina (AS-alumina), ionic binding onto DEAE-cellulose, covalent binding on chitin, and entrapment in polyacrylamide had the highest activities. The specific activity of the immobilized enzymes, calculated on bound protein basis, were 13.5–50% of the original specific activity exhibited by the free enzyme. The optimal pH of the immobilized enzymes was shifted to lower values than for the free enzyme. The optimum reaction temperature was determined to be 45 °C for the free amylase, whereas that for the immobilized enzymes was shifted to 60-65°C. In all cases, K m values for the hydrolysis of starch of the immobilized enzymes were higher than that of the native enzyme. Compared to the free form, the immobilized enzymes exhibited improved thermal stability and higher values of activation energy of denaturation.
---
paper_title: Hydrolysis of sucrose by invertase entrapped in polyvinyl alcohol hydrogel capsules

paper_content:

Invertase was immobilised into polyvinyl alcohol capsules (LentiKats®). The enzyme retained 86% of its original activity after the immobilisation process. Kinetic constants, pH and temperature profiles, and stability were determined, for both free and immobilised enzyme. Entrapped invertase was used for 45 repeated batch hydrolyses and no decrease in enzymatic activity was observed. The immobilised enzyme retained 78% of its initial activity after 8 months of storage.
---
paper_title: A simple entrapment of glucoamylase into LentiKats® as an efficient catalyst for maltodextrin hydrolysis

paper_content:

The glucoamylase from Aspergillus niger was immobilized into a poly(vinylalcohol) hydrogel lens-shaped capsules LentiKats ® . Immobilization broadened the pH optimum of the enzyme. The KM of glucoamylase increased after immobilization (approximately 1.5 times on maltose), which reflected the decrease in affinity of enzyme for its substrate. Immobilized enzyme retained excellent long-term operational stability for both, batch and continuous mode of hydrolysis. Specific enzymatic activity of the immobilized enzyme (0.67 g g −1 h −1 ) was constant for 63 days of continuous operation at 45 ◦ C.
---
paper_title: Synthesis of galacto-oligosaccharide from lactose using ?-galactosidase fromKluyveromyces lactis: Studies on batch and continuous UF membrane-fitted bioreactors

paper_content:

A study of galacto-oligosaccharides (GOS) synthesis from lactose with β-galactosidase from Kluyveromyces lactis (Maxilact® L2000) was carried out. The synthesis was performed using various initial lactose concentrations ranging from 220 to 400 mg/mL and enzyme concentrations ranging from 3 to 9 U/mL, and was investigated at 40°C and pH 7, in a stirred-tank reactor. In the experimental range examined, the results showed the amount of GOS formed depended on lactose concentration but not on enzyme concentration. Galactose was a competitive inhibitor, while glucose was a non-competitive inhibitor. In a further study, a laboratory-scale reactor system, fitted with a 10-kDa NMWCO composite regenerated cellulose membrane, was used in a continuous process. The reactor was operated in cross-flow mode. The effect of operating pressures on flux and productivity was investigated by applying different transmembrane pressures to the system. The continuous process showed better production performance compared to the batch synthesis with the same lactose and enzyme concentrations at 40°C, pH 7. Comparison of product structures from batch and continuous processes, analyzed by HPAE-PAD and methylation analysis, showed similarities but differed from the structures found in a commercial GOS product (Vivinal®GOS). © 2004 Wiley Periodicals, Inc.
---
paper_title: Immobilization of malto-oligosaccharide forming α-amylase from Bacillus subtilis KCC103: properties and application in starch hydrolysis

paper_content:

BACKGROUND: ::: ::: ::: ::: A malto-oligosaccharide forming α-amylase from Bacillus subtilis KCC103 immobilized in calcium alginate beads was repeatedly used in batch processes of starch hydrolysis. The degree of starch degradation and operational stability of the immobilized system were optimized by varying the physical characteristics and composition of the beads. The products formed from hydrolysis of various starches by α-amylase immobilized in different supports were analyzed. ::: ::: ::: ::: RESULTS: ::: ::: ::: ::: Immobilized beads prepared from 3% (w/v) alginate and 4% (w/v) CaCl2 were suitable for up to 10 repeated uses, losing only 25% of their efficiency. On addition of 1% silica gel to alginate prior to gelation, the operational stability of the immobilized enzyme was enhanced to 20 cycles of operation, retaining > 90% of the initial efficiency. Distribution of malto-oligosaccharides in the starch hydrolyzate depended on the type of starch, reaction time and mode of immobilization. Soluble starch and potato starch formed a wide range of malto-oligosaccharides (G1–G5). Starches from wheat, rice and corn formed a narrow range of smaller oligosaccharides (G1–G3) as the major products. ::: ::: ::: ::: CONCLUSION: ::: ::: ::: ::: The immobilized beads of α-amylase from KCC103 prepared from alginate plus silica gel showed high efficiency and operational stability for hydrolysis of starch. This immobilized system is useful for production of malto-oligosaccharides applied in the food and pharmaceutical industries. Since this KCC103 amylase can be produced at low cost utilizing agro-residues in a short time and immobilized enzyme can be recycled, the overall cost of malto-oligosaccharide production would be economical for industrial application. Copyright © 2008 Society of Chemical Industry
---
paper_title: A new innovative process to produce lactose-reduced skim milk.

paper_content:

The research field for applications for lactose hydrolysis has been investigated for some decades. Lactose intolerance, improvement for technical processing of solutions containing lactose and utilisation of lactose in whey are main topics in development of biotechnological processes. In this article, the establishment of a hollow fiber membrane reactor process for enzymatic lactose hydrolysis is reported. Mesophilic beta-galactosidases were circulated abluminally during luminal flow of skim milk. The main problem, microorganisms growth in the enzyme solution, was minimised by sterile filtration and UV irradiation. In order to characterise the process parameters, such as skim milk concentration, enzyme activity and flow rates were varied. In comparison to a batch process, enzyme activity could be used longer and enzyme rest into the product should not occur. Furthermore, the three-dimensional separation of the substrate from the enzyme solution minimise blocking and washing out effects, which restrict processes with immobilised enzymes. A conversion rate of 78.11% was achieved at a skim milk flow rate of 9.9l h(-1), enzyme activity of 120 Uml(-1) and a temperature of 23+/-2 degrees C in a hollow fiber reactor with a membrane area of 4.9 m2.
---
paper_title: Leaching studies over immobilized a-amylase. importance of the nature of enzyme attachment

paper_content:

The enzyme α-amylase was immobilized on montmorillonite K-10 via adsorption and covalent bonding. These two modes of attachment play a vital role in the leaching of enzyme. Covalent bonding retains most of the enzyme even at high loadings. The significance of temperature has also been established. Higher temperatures lead to severe leaching in case of adsorbed enzyme. Covalent linkage, being of higher strength, does not yield to enzyme leaching even at high temperatures.
---
paper_title: Development of dried liposomes containing beta-galactosidase for the digestion of lactose in milk.

paper_content:

The hydrolyzed-lactose milk for lactase-deficient subjects has a sweeter taste than whole milk, and some subjects dislike its taste. In order to cope with this shortcoming, we examined whether beta-galactosidase, which hydrolyzes lactose, added to the whole milk in the form of dried liposomes, would be able to digest lactose in milk following the lysis of liposomes in the presence of bile salts. Dried liposomes containing beta-galactosidase were prepared in the presence of trehalose by the dehydration-rehydration vesicle method to overcome the instability of the conventional liposome suspension. The stability of liposomal membranes was evaluated by measuring the activity of entrapped beta-galactosidase under various storage conditions. By treating liposomes with trehalose, which was found to prevent the fusion of liposomes and the leakage of entrapped drug, the entrapping efficiency increased up to fourfold. Over 95% of dried liposomes which had been stored at 17 degrees C for 60 days were reconstituted to liposomes upon rehydration process. From the stability study, dried liposomes were found to retain 87% of beta-galactosidase activity at 17 degrees C after 60 days and to be more stable than the multilamellar vesicle suspension prepared without trehalose. The lysis study showed that dried liposomes were hardly lyzed in the simulated gastric fluid with pepsin, but lyzed immediately more than 90% in 0.01 M deoxycholic acid. Lactose hydrolysis in the presence of deoxycholic acid after the addition of dried liposome-entrapped beta-galactosidase to whole milk was proportional to the quantity of entrapped beta-galactosidase and the amount of dried liposomes added. These results demonstrate that beta-galactosidase entrapped in liposome is stable and reconstituted mostly upon rehydration, and can digest lactose in milk after the efficient lysis of liposomes in the presence of bile salts. This study implies that beta-galactosidase entrapped in liposome may be applied to whole milk for lactase-deficient subjects.
---
paper_title: Encapsulation in LentiKats of Dextransucrase from Leuconostoc mesenteroides NRRL B-1299, and its Effect on Product Selectivity

paper_content:

Insoluble (cell-bound) dextransucrase from Leuconostoc mesenteroides B-1299 was encapsulated in highly elastic and stable hydrogels formed by polyvinyl alcohol. The gelation was carried out by controlled partial drying at room temperature, resulting in lens-shaped particles, called LentiKats. A similar recovery of activity (approximately 55%) was achieved when compared with entrapment in calcium alginate gels. Under reaction conditions, the protein leakage in LentiKats was reduced from 18% to 4% by pre-treatment of the dextransucrase with glutaraldehyde. The immobilized dextransucrases were tested in the acceptor reaction with methyl α-D-glucopyranoside. The conversion to oligosaccharides using Lentikat-dextransucrase was higher than that obtained for alginate-dextransucrase, probably due to the reduction of diffusional limitations derived from its lenticular shape. In addition, a shift of selectivity towards the synthesis of oligosaccharides containing α(1→2) bonds was observed for the Lentikat-biocataly...
---
paper_title: Use of Immobilized Enzymes in the Food Industry

paper_content:

Most of the literature on immobilized enzymes has resulted from research and development during the past 40 years. A number of reviews have appeared (1–11) that discuss the preparation, characteristics, and uses of immobilized enzymes. Actual commercialization of immobilized enzyme bioprocesses has not occurred at a rapid pace in the food industry, perhaps owing to a natural reluctance to replace existing soluble enzyme processes and the fact that most of the industry involves low value-added products with small profit margins. Nevertheless, immobilized enzyme technology has gained acceptance in several areas. The most successful example is the production of high-fructose corn syrup using immobilized glucose isomerase, which is a unique product produced only with the immobilized enzyme.
---
paper_title: Advances in enzyme immobilisation

paper_content:

Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment. New commercial resins (Sepabeads) exhibit improved protein binding capacity. Novel methods of enzyme self-immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment. Apart from retention, recovery and stabilisation, other advantages to enzyme immobilisation have emerged, such as enhanced enzyme activity, modification of substrate selectivity and enantioselectivity, and multi-enzyme reactions. These advances promise to enhance the roles of immobilisation enzymes in industry, while opening the door for novel applications.
---
paper_title: Enzymes inside lipid vesicles: preparation, reactivity and applications.

paper_content:

There are a number of methods that can be used for the preparation of enzyme-containing lipid vesicles (liposomes) which are lipid dispersions that contain water-soluble enzymes in the trapped aqueous space. This has been shown by many investigations carried out with a variety of enzymes. A review of these studies is given and some of the main results are summarized. With respect to the vesicle-forming amphiphiles used, most preparations are based on phosphatidylcholine, either the natural mixtures obtained from soybean or egg yolk, or chemically defined compounds, such as DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Charged enzyme-containing lipid vesicles are often prepared by adding a certain amount of a negatively charged amphiphile (typically dicetylphosphate) or a positively charged lipid (usually stearylamine). The presence of charges in the vesicle membrane may lead to an adsorption of the enzyme onto the interior or exterior site of the vesicle bilayers. If (i) the high enzyme encapsulation efficiencies; (ii) avoidance of the use of organic solvents during the entrapment procedure; (iii) relatively monodisperse spherical vesicles of about 100 nm diameter; and (iv) a high degree of unilamellarity are required, then the use of the so-called ‘dehydration–rehydration method’, followed by the ‘extrusion technique’ has shown to be superior over other procedures. In addition to many investigations in the field of cheese production—there are several studies on the (potential) medical and biomedical applications of enzyme-containing lipid vesicles (e.g. in the enzyme-replacement therapy or for immunoassays)—including a few in vivo studies. In many cases, the enzyme molecules are expected to be released from the vesicles at the target site, and the vesicles in these cases serve as the carrier system. For (potential) medical applications as enzyme carriers in the blood circulation, the preparation of sterically stabilized lipid vesicles has proven to be advantageous. Regarding the use of enzyme-containing vesicles as submicrometer-sized nanoreactors, substrates are added to the bulk phase. Upon permeation across the vesicle bilayer(s), the trapped enzymes inside the vesicles catalyze the conversion of the substrate molecules into products. Using physical (e.g. microwave irradiation) or chemical methods (e.g. addition of micelle-forming amphiphiles at sublytic concentration), the bilayer permeability can be controlled to a certain extent. A detailed molecular understanding of these (usually) submicrometer-sized bioreactor systems is still not there. There are only a few approaches towards a deeper understanding and modeling of the catalytic activity of the entrapped enzyme molecules upon externally added substrates. Using micrometer-sized vesicles (so-called ‘giant vesicles’) as simple models for the lipidic matrix of biological cells, enzyme molecules can be microinjected inside individual target vesicles, and the corresponding enzymatic reaction can be monitored by fluorescence microscopy using appropriate fluorogenic substrate molecules.
---
paper_title: IMMOBILIZATION OF GLUCOSE ISOMERASE IN SURFACE-MODIFIED ALGINATE GEL BEADS

paper_content:

In this study, glucose isomerase enzyme was entrapped into modified and nonmodified calcium alginate gel beads. Various characteristics of free and immobilized enzymes such as the optimum pH, temperature and dependence of activity on storage and operational stability were evaluated. The optimum pH and temperature of free and immobilized glucose isomerase were found to be the same values as 7.5 and 60C, respectively. For free and immobilized enzymes, kinetic parameters were calculated as 1.79 × 10−2 and 8.27 × 10−3 mol/L for Km, and 2.39 × 10−3 and 6.03 × 10−3 mol/L min for Vmax, respectively. After 42 days of storage at 4C, free enzyme retained 56% of its initial activity, while for the immobilized enzyme, this value was observed as 86%. The immobilized samples were used repeatedly 22 times by retaining more than 85% of their initial activity. ::: ::: ::: ::: PRACTICAL APPLICATIONS ::: ::: Glucose isomerase (GI) serves as an interesting model for studying structure–function relationships by advanced biochemical and genetic engineering techniques. Besides its academic importance, it has received increased attention by industries for its use in producing high-fructose corn syrup (HFCS) and for its potential application in the production of ethanol from hemicelluloses. The use of GI is expensive because it is an intracellular enzyme, and large quantities are needed to compensate for the high Km for glucose. Therefore, it is important to immobilize GI for its industrial applications. Recently, several methods for immobilizing GI have been developed. However, only a few are economical and yield enzyme preparations with properties that are suitable for commercial production of HFCS and ethanol.
---
paper_title: Stability and catalytic kinetics of microencapsulated β-galactosidase in liposomes prepared by the dehydration–rehydration method

paper_content:

Lipid vesicles prepared by the dehydration–rehydration method were used as carrier for the microencapsulation of β-galactosidase in order to permit the hydrolysis of the milk lactose following the lysis of liposomes in the presence of gastric fluid. Some characteristics of the liposomal and free β-galactosidase were compared. The kinetic behaviour of the enzyme was altered substantially in presence of lipid vesicles. The kinetic study indicated an decrease in both substrate affinity and maximum velocity when β-galactosidase was associated with phospholipid vesicles. Differences in the activity of free and liposomal β-galactosidase as a function of pH and temperature were found, although the optimum incubation temperature of free and entrapped enzymes remained similar. However, the optimum pH for liposomal enzyme was more acid than that for free enzyme. First-order kinetic analysis of thermal inactivation of enzymes showed that the activation energy with free enzyme was smaller than that with liposomal enzyme. In relation to the ΔH* and ΔS*, their values were greater than that those with free enzymes. These results confirm that β-galactosidase entrapped in liposomes showed superior thermal stability at all temperatures evaluated. Moreover, the proteolytic stability of the β-galactosidase was enhanced by encapsulation in liposomes.
---
paper_title: Immobilized enzyme technology for food applications.

paper_content:

Publisher Summary This chapter describes that there are very few examples of commercial processes that utilize immobilized enzymes for food constituent modifications. In order for the immobilized process to be more economical or more useful than the soluble enzyme, either the cost per unit of product must be less or the product formed can only be produced with an immobilized enzyme. The two most successful examples of the use of immobilized enzymes are the production of high-fructose corn syrup and trans-free oils. The use of enzymes for food constituent modification has several advantages over the use of chemicals. The reactions are specific with generally fewer side reactions. Numerous publications over the past few years have focused on the development of immobilized enzymes for future commercial use, yet currently there are few successful examples of immobilized enzymes for food processing despite the advantages associated with immobilized enzyme processing. The immobilized form of glucose isomerase is used for the production of high-fructose corn syrup and immobilized lipases are used for production of diacylglycerols and trans-free fats, and oils. Despite the numerous benefits associated with immobilized enzymes, the economics of the system outweighs most other benefits.
---
paper_title: Entrapment of β-galactosidase in polyvinylalcohol hydrogel

paper_content:

β-Galactosidase isolated from Aspergillus oryzae was immobilized in lens-shaped polyvinylalcohol capsules (with activity 25 U g−1) giving 32% of its original activity. Immobilization did not change the pH optimum (4.5) of lactose hydrolysis. The relative enzyme activity during product inhibition testing was, in average, 10% higher for immobilized enzyme. No decrease of activity was observed after 35 repeated batch runs and during 530 h of continuous hydrolysis of lactose (10%, w/v) at 45°C. The immobilized enzyme was stable for 14 months without any change of activity during the storage at 4°C and pH 4.5.
---
paper_title: Immobilization of inulinase for sucrose hydrolysis

paper_content:

A commercial inulinase preparation immobilized on various supports was used for sucrose hydrolysis. Entrapment and encapsulation in Ca-alginate and entrapment in an alginate–silicate sol–gel matrix were evaluated. Best results were obtained with Ca-alginate beads. The influence of sodium-alginate concentration on the immobilization yield was assessed. Inulinase entrapped in Ca-alginate beads displayed high activity in the range 50–60 °C, whereas the optimum for the free enzyme was 60 °C. The optimum pH of the immobilized enzyme was slightly more acidic (4.0) than the one observed for the free form (4.5). The apparent KM for sucrose of the immobilized inulinase was 184 mM, as compared to 82 mM for free inulinase, as a result of diffusion resistances.
---
paper_title: Improvement of enzyme activity, stability and selectivity via immobilization techniques

paper_content:

Abstract In spite of their excellent catalytic properties, enzyme properties have to be usually improved before their implementation at industrial scale (where many cycles of high yield processes are desired). Generally, soluble enzymes have to be immobilized to be reused for long times in industrial reactors and, in addition to that, some other critical enzyme properties have to be improved like stability, activity, inhibition by reaction products, selectivity towards non-natural substrates. Some strategies to improve these enzyme properties during the performance of tailor-made enzyme immobilization protocols are here reviewed. In this way, immobilized enzymes may also exhibit much better functional properties than the corresponding soluble enzymes by very simple immobilization protocols. For example, multipoint and multisubunit covalent immobilization improve the stability of monomeric or multimeric enzymes. Moreover, enantioselectivity of different enzymes (e.g., lipases) may be also dramatically improved (from E = 1 to >100) by performing different immobilization protocols of the same enzyme. In all cases, enzyme engineering via immobilization techniques is perfectly compatible with other chemical or biological approaches to improve enzyme functions and the final success depend on the availability of a wide battery of immobilization protocols.
---
paper_title: History of Industrial Biotransformations – Dreams and Realities

paper_content:

This user-friendly and application-oriented overview draws on extensive literature and patent research, and is unique in its systematic structure for easy comparison. For this second edition every chapter has been rewritten, with each process updated and over 30 new ones added.
---
paper_title: A novel approach to develop β-galactosidase entrapped in liposomes in order to prevent an immediate hydrolysis of lactose in milk

paper_content:

Abstract A fractional factorial screening design and a response surface methodology were applied to optimize the entrapment of β -galactosidase in liposomes by dehydration-rehydration vesicle method. The effect of lipid (phosphatidylcholine and cholesterol), enzyme concentration, buffer pH, type and time of sonication, and sucrose concentration were the parameters selected for this study. The cholesterol:phosphatidylcholine (Ch:PC) ratio was the most important factor followed by type of sonicator and enzyme:lipid (E:L) ratio. The buffer pH, sucrose concentration and sonication time did not play an important role in the response. Two of the factors (Ch:PC and E:L ratios) were further studied in a 3 2 central composite design. The optimized liposomal formulation with an entrapment efficiency of 28% was obtained with Ch:PC and E:L ratios of 0.53 and 13.76, respectively, at pH 6 and employing a probe type sonicator.
---
paper_title: A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities

paper_content:

BackgroundThe use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities.ResultsComplete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity.ConclusionA single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.
---
paper_title: Immobilization of pectinex ultra sp-l pectinase and its application to production of fructooligosaccharides

paper_content:

Pectinex Ultra SP-L, a commercial pectinase with fructosyl-transferase (FTF) activity, is able to catalyze the production of short chain fructooligosaccharides (FOS). It was immobilized onto an anion exchange resin by a combined method. The optimal biocatalyst/matrix ratio (16.7 g enzyme solution/g dry support) as well as the most favourable conditions of the immobilization: the concentration of cross-linking agent (0.125%) and the time of chemical fixation (15 min) have been determined. The temperature and pH optima of the solid-phase biocatalyst have been found 53 °C and 5.6, respectively. It showed almost no decrease in its activity during 12 reaction cycles. Based on these results, lab-scale synthesis experiments have been carried out for fructooligosaccharide production under the determined optimal operational conditions.
---
paper_title: Hyperstabilization of a thermophilic esterase by multipoint covalent attachment

paper_content:

Abstract A strategy for the hyperstabilization of a thermophilic esterase from Bacillus stearothermophilus by multipoint covalent attachment of the enzyme through amino groups to glyoxyl agarose gels has been developed. All immobilized esterase derivatives, whether multipoint or one-point linkage, showed improved operational stability under conditions in which the free enzyme was inactivated by interprotein interactions (e.g., aggregation at the isoelectric point) or by interaction with surfaces (adsorption at hydrophobic surfaces, denaturation by liquid-liquid interfaces, etc.). However, only via optimization of the major variables that control the extent of the enzyme-support interaction (contact time between the immobilized enzyme and the activated support, temperature, etc.) was it possible to obtain enzyme derivatives with a significantly increased stability with respect to denaturing reagents. The optimized multipoint covalent immobilization process increased the stability of the esterase preparations by factors of up to 600-fold (depending on the pH) compared to single-point covalent derivatives, with a retention of 65% of the initial activity after immobilization. Apparent stabilization factors of up to 30,000-fold were obtained when comparing the stability of the multipoint immobilized esterase with the soluble enzyme. A clear correlation between thermostabilization and enhanced stability in the presence of organic cosolvents was observed. Multipoint covalently linked esterase derivatives retained more than 70% of the initial activity after 1 week in 50% DMF or DMS at 30°C.
---
paper_title: Immobilization and enzymatic activity of β-glucosidase on mesoporous SBA-15 silica

paper_content:

The mesoporous silicate SBA-15 has shown to be a good support for the immobilization of β-glucosidase from almonds, an enzyme with high molecular weight (ca. 130 kDa for the dimer). An enzyme loading of 430 mg per gram of support (3.2 ± 0.2 μmol g−1 of SBA-15) was achieved at 7 h. The optimum pH for the immobilization was 3.5. The electrostatic interactions between the surface of SBA-15 and the enzyme molecules were the driving force of the adsorption process. The immobilized β-glucosidase presented enzymatic activity on the hydrolysis of the 4-nitrophenyl-β-d-glucopyranoside at 3.5 pH. The catalytic activity was similar to the free enzyme for reaction time of 30 min. When the reaction pH was higher (5.5 pH) the enzyme was desorbed.
---
paper_title: Enhanced stability of glucoamylase through chemical crosslinking

paper_content:

A series of bifunctional chemical modification reagents, presenting variations in both the chemistry of the functional groups and in the length of the spacer between the two reactive groups, have been evaluated as agents for enhancing the thermal stability of purifiedAspergillus niger amyloglucosidase by means of intramolecular cross-linking. Several chemical modifiers (e.g., diimidoesters) were identified that more than double the half-life of this industrially important enzyme during incubation at 65°C in the absence of substrate. The increased stability of the modified enzymes has been correlated with changes in the fluorescence-monitored thermal denaturation curves of the modified enzymes, relative to that of the native enzyme.
---
paper_title: Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions

paper_content:

BackgroundIncreasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts.ResultsTwo types of procedures were used for the preparation of biocatalysts from purified wild type LS (WT LS) B. subtilis and the R360K and Y429N LS mutants: purified enzymes aggregated with glutaraldehyde (cross-linked enzyme aggregates: CLEAs), and covalently immobilized enzymes in Eupergit C®. The biocatalysts were characterized and used for fructoside synthesis using xylose as an acceptor model. CLEAs were able to catalyze the synthesis of fructosides as efficiently as soluble enzymes. The specific activity of CLEAs prepared from wild type LS (44.9 U/mg of CLEA), R360K (56.5 U/mg of CLEA) and Y429N (1.2 U/mg of CLEA) mutants were approximately 70, 40 and 200-fold higher, respectively, than equivalent Eupergit C® immobilized enzyme preparations (U/mg of Eupergit), where units refer to global LS activity. In contrast, the specific activity of the free enzymes was 160, 171.2 and 1.5 U/mg of protein, respectively. Moreover, all CLEAs had higher thermal stability than corresponding soluble enzymes. In the long term, the operational stability was affected by levan synthesis.ConclusionThis is the first report of cross-linked transglycosidases aggregates. CLEAs prepared from purified LS and mutants have the highest specific activity for immobilized fructosyltransferases (FTFs) reported in the literature. CLEAs from R360K and Y429N LS mutants were particularly suitable for fructosyl-xyloside synthesis as the absence of levan synthesis decreases diffusion limitation and increases operational stability.
---
paper_title: Immobilisation of Bacillus subtilis NRC33a levansucrase and some studies on its properties

paper_content:

Bacillus subtilis NRC33a levansucrase was immobilised on different carriers using different immobilisation methods including physical adsorption, covalent binding, ionic binding and entrapment. The immobilised enzyme prepared by covalent binding on chitosan through 3% gluteraldehyde had the highest immobilization yield (81.51%). Therefore, it was used as a typical example for Bacillus subtilis NRC33a immobilised levansucrase and its properties were investigated. The time of the reaction and substrate concentration revealed that the activity of the immobilised enzyme was relatively lower than the free enzyme. The immobilised levansucrase showed a slight increase in activity compared with the free enzyme above 35°C. The activation energies were 6.62 and 9.27 kcal mol-1 for free and immobilised enzyme respectively. Although the thermal stability of the immobilised levansucrase was significantly improved in comparison to the free form, the deactivation energy of the immobilised enzyme was lower than that of the free enzyme. The half life of the immobilised and free levansucrase was also determined. The effect of different pH values reported that at acidic pH the activity of the immobilised levansucrase was higher than that of the free enzyme. The study of pH stability of free and immobilised levansucrase showed that the immobilisation process protected the enzyme from alkaline and severe acidic media. The effect of various metal ions showed that the free levansucrase was more sensitive to the inhibitory effect of the investigated substances. Immobilised levansucrase retained 51.13% after 14 repeated uses.
---
paper_title: Immobilization and stabilization of a cyclodextrin glycosyltransferase by covalent attachment on highly activated glyoxyl-agarose supports.

paper_content:

Covalent immobilization of cyclodextrin glycosyltransferase on glyoxyl-agarose beads promotes a very high stabilization of the enzyme against any distorting agent (temperature, pH, organic solvents). For example, the optimized immobilized preparation preserves 90% of initial activity when incubated for 22 h in 30% ethanol at pH 7 and 40 °C. Other immobilized preparations (obtained via other immobilization protocols) exhibit less than 10% of activity after incubation under similar conditions. Optimized glyoxyl-agarose immobilized preparation expressed a high percentage of catalytic activity (70%). Immobilization using any technique prevents enzyme inactivation by air bubbles during strong stirring of the enzyme. Stabilization of the enzyme immobilized on glyoxyl-agarose is higher when using the highest activation degree (75 μmol of glyoxyl per milliliter of support) as well as when performing long enzyme-support incubation times (4 h) at room temperature. Multipoint covalent immobilization seems to be responsible for this very high stabilization associated to the immobilization process on highly activated glyoxyl-agarose. The stabilization of the enzyme against the inactivation by ethanol seems to be interesting to improve cyclodextrin production: ethanol strongly inhibits the enzymatic degradation of cyclodextrin while hardly affecting the cyclodextrin production rate of the immobilized-stabilized preparation.
---
paper_title: Immobilization of the recombinant invertase INVB from Zymomonas mobilis on Nylon-6

paper_content:

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 °C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 °C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 °C. The values for kinetic parameters were determined as: 984 and 98 mM for \( K_{m}^{\text{app}} \) of immobilized and free re-INVB, respectively. \( K_{\text{cat}}^{\text{app}} \) values for immobilized and free enzymes were 6.1 × 102 and 1.2 × 104 s−1, respectively, and immobilized re-INVB showed \( V_{\max }^{\text{app}} \) of 158.73 μmol h min−1 mg−1. Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 °C and 70% at 40 °C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.
---
paper_title: Immobilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F on Eupergit C supports.

paper_content:

Dextransucrase from Leuconostoc mesenteroides B-512F was immobilized on epoxy-activated acrylic polymers with different textural properties (Eupergit C and Eupergit C 250L). Prior to immobilization, dextransucrase was treated with dextranase to remove the dextran layer covering the enzyme surface, thus increasing the accessibility of its reactive groups to the epoxide centers of the support. Elimination of 99% of the initial carbohydrate content was determined by the anthrone method. To prevent enzyme inactivation, the immobilization was carried out at pH 5.4, at which the coupling to the support took place through the carboxylic groups of the enzyme. The effects of the amount (mg) of dextransucrase added per gram of support (from 0.2:1 to 30:1), temperature and contact time were studied. Maximum activity recovery of 22% was achieved using Eupergit C 250L. Using this macroporous support, the maximum specific activity (710 U/g biocatalyst) was significantly higher than that obtained with the less porous Eupergit C (226 U/g biocatalyst). The dextransucrase immobilized on Eupergit C 250L showed similar optimal temperature (30 degrees C) and pH (5-6) compared with the native enzyme. In contrast, a notable stabilization effect at 30 degrees C was observed as a consequence of immobilization. After a fast partial inactivation, the dextransucrase immobilized on Eupergit C 250L maintained more than 40% of the initial activity over the following 2 days. The features of this immobilized system are very attractive for its application in batch and fixed-bed bioreactors.
---
paper_title: Galacto-oligosaccharide synthesis by immobilized Aspergillus oryzae β-galactosidase

paper_content:

Abstract Aspergillus oryzae β-galactosidase was immobilized by three different techniques, namely adsorption on celite, covalent coupling to chitosan and aggregation by cross-linking. These techniques were compared in terms of the yield of immobilized preparation, enzymatic characteristics, stability and efficiency in oligosaccharide synthesis. Immobilization led to increase in K m in each case. Immobilization on chitosan gave maximum enzyme yield and oligosaccharide synthesis. At 60 °C, the chitosan-immobilized enzyme was stabilized (by 1.6-fold) due to protection effect of the matrix. However, at 65 °C, the t 1/2 of cross-linked enzyme aggregates (CLEA) of β-galactosidase was 1.07 h as compared to 0.79 h in the case of free enzyme. Both chitosan-immobilized enzyme and CLEA were used for oligosaccharide synthesis. Using 20% (w/v) lactose, the chitosan-immobilized enzyme gave maximum oligosaccharide yield (17.3% of the total sugar) as compared to free enzyme (10.0%) in 2 h at 40 °C. CLEA were instead found effective in lactose hydrolysis yielding 78% monosaccharide in 12 h.
---
paper_title: Xylitol purification by cross-linked glucose isomerase crystals

paper_content:

Xylitol is specifically bound by active, cross-linked glucose isomerase crystals (CLGI). CLGI can be used to purify xylitol or concentrate it from dilute and impure solutions. Bound xylitol can be eluted from CLGI by Ca2 and the material reactivated by Mg2. The binding capacity is 1 mg xylitol per 525 mg CLGI which equals one molecule per active center. CLGI can further be used to purify xylitol and sorbitol from impure mixtures of arabinitol, mannitol, ribitol and monosaccharides. © Rapid Science Ltd. 1998
---
paper_title: Production of low methoxyl pectin using immobilized pectinesterase bioreactors

paper_content:

Abstract Soluble pectinesterase (EC 3.1.1.11) from germinating seeds of Vigna sinensis has been immobilized on egg shell as well as in gelatin matrices. The egg shell-immobilized enzyme showed higher specific activity and purification fold than that immobilized in gelatin. There is a shift in the optimum pH without distortion of the profile on immobilization of the enzyme on egg shell and in gelatin as well. The immobilized enzymes showed higher optimum temperature than the soluble enzyme and also a marked increase in thermal stability. Pectinesterase immobilized on egg shell without glutaraldehyde was reusable 32 times at 30°C, compared to 24 times for enzyme immobilized on egg shell with glutaraldehyde treatment and 18 times for that immobilized in gelatin. Three types of reactors: fixed bed, fluidized bed and stirred tank have been prepared using hen egg shell-bound pectinesterase. Fluidized bed reactor (2×10 cm), operated at an optimum flow rate of 5 ml/h and temperature 35°C, showed better results for production of low methoxyl pectin.
---
paper_title: Hydrolysis of sucrose by invertase immobilized on nylon-6 microbeads

paper_content:

Abstract A commercial extracellular invertase (EC 3.2.1.26) from Saccharomyces cerevisiae has been inmobilized by covalent bonding on novel microbeads of nylon-6 using glutaraldehyde. The enzyme was strongly bound on the support, immobilized with an efficiency factor of 0.93. The biocatalyst showed a maximum enzyme activity when immobilized at pH 5.0, but optimum pH activity for both immobilized and free invertases was 5.5. The optimum temperatures for immobilized and free enzymes were 60 and 65 °C, respectively. Kinetic parameters were determined for immobilized and free invertases: V max values were 1.37 and 1.06 mmol min −1 mg −1 , respectively. The K m and K i values were 0.029 and 0.71 M for immobilized invertase and 0.024 and 0.69 M for free invertase. It was found that the thermal stability of the immobilized invertase with regard to the free one increased by 25% at 50 °C, 38% at 60 °C and 750% at 70 °C. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous sucrose hydrolysis. The effects of two different sugar concentrations and three flow rates on the productivity of the reactor and on the specific productivity of the biocatalyst were studied. The system demonstrated a very good productivity up to 2.0 M sugar concentration, with conversion factors of 0.95 and 0.97, depending on sucrose concentration in the feeding. This approach may serve as a simple technique and can be a feasible alternative to continuous sucrose hydrolysis in a fixed bed reactor for the preparation of fructose-rich syrup.
---
paper_title: Reversible immobilization of a thermophilic β-galactosidase via ionic adsorption on PEI-coated Sepabeads

paper_content:

Abstract The immobilization of the enzyme β-galactosidase from Thermus sp. T2 was performed via ionic adsorption onto two different supports: a new anionic exchanger resin, based on the coating of Sepabeads internal surfaces with polyethylenimine (PEI) polymers ( M w 25,000), and conventional DEAE-agarose. Immobilization proceeded very rapidly in both cases, but the adsorption strength was much higher in the case of PEI-Sepabeads than in DEAE-supports at both pH 5 and 7 (e.g. at pH 7 and 0.4 M NaCl, less than 5% of enzyme was eluted from PEI-support while more than 70% protein was eluted from DEAE-agarose). Interestingly, the PEI-derivatives remained almost fully active at pH 5 and 7 after several weeks of incubation at 50 °C, conditions that allows the hydrolysis of lactose in milk coupled with the antimicrobial treatment usually performed.
---
paper_title: Glucoamylase immobilized on montmorillonite: influence of nature of binding on surface properties of clay-support and activity of enzyme

paper_content:

Glucoamylase was immobilized on acid activated montmorillonite clay via two different procedures namely adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N2 adsorption measurements and the activity of immobilized glucoamylase for starch hydrolysis was determined in a batch reactor. XRD shows intercalation of enzyme into the clay matrix during both immobilization procedures. Intercalation occurs via the side chains of the amino acid residues, the entire polypeptide backbone being situated at the periphery of the clay matrix. 27Al NMR studies revealed the different nature of interaction of enzyme with the support for both immobilization techniques. N2 adsorption measurements indicated a sharp drop in surface area and pore volume for the covalently bound glucoamylase that suggested severe pore blockage. Activity studies were performed in a batch reactor. The adsorbed and covalently bound glucoamylase retained 49% and 66% activity of the free enzyme respectively. They showed enhanced pH and thermal stabilities. The immobilized enzymes also followed Michaelis–Menten kinetics. K m was greater than the free enzyme that was attributed to an effect of immobilization. The immobilized preparations demonstrated increased reusability as well as storage stability.
---
paper_title: A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin

paper_content:

Abstract A novel method was developed for the immobilization of glucoamylase from Aspergillus niger . The enzyme was immobilized onto polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin, resulting in 85% immobilization yield. The immobilized enzyme has been fully active for 30 days. In addition, the immobilized enzyme retained 90 and 75% of its activity in 60 and 90 days, respectively. The enzyme optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4 and 65 °C, respectively. The kinetic parameters for the hydrolysis of maltodextrin by free and immobilized glucoamylase were also determined. The K m values for free and immobilized enzyme were 7.5 and 10.1 g maltodextrin/l, respectively. The V max values for free and immobilized enzyme were estimated as 20 and 16 μmol glucose/(min μl enzyme), respectively. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes.
---
paper_title: COVALENT IMMOBILIZATION OF INVERTASE ON CHEMICALLY ACTIVATED POLY (STYRENE-2-HYDROXYETHYL METHACRYLATE) MICROBEADS

paper_content:

Properties of invertase immobilized on poly(2-hydroxyethyl methacrylate) microbeads activated by epichlorohydrin or cyanuric chloride were studied. After 20 repeated uses for 3 days, the activity of the immobilized enzyme was 92–93%.
---
paper_title: Covalent immobilization of invertase on polyurethane, plast-film and ferromagnetic Dacron.

paper_content:

Invertase was covalently immobilized on polyurethane (PU), inox plate covered with plast-film layer and ferromagnetic azide-Dacron. The immobilization processes, physico-chemical parameters and a model for coupling reactions were studied. The preliminary studies for selection of the support showed that the best activity was obtained for PU treated with HCl, polyethylenimine and glutaraldehyde (156.7+/-4.9 U/g support). All plast-film-invertase derivatives did not show activity and the Dacron-invertase derivative showed an activity of 105.39 U/g support. The invertase immobilized in presence of substrate (10% w/v sucrose) was the most efficient (832.74+/-1.48 U/g support). The optimal pH was shifted from 4.5 (free enzyme) to 5.0 (immobilized derivative) and optimal temperature was not affected. Activation energy values of free enzyme, Dacron-invertase and PU-invertase were 32.4+/-0.34 kJ/mol, 33.4+/-0.36 kJ/mol and 44.0+/-0.67 kJ/mol, respectively. The PU-invertase could be used over 2 months without considerable activity loss (68.5% activity retention) and retained 12.6% (287.97+/-27.9U/g support) of the activity after five cycles.
---
paper_title: Immobilization of Bacillus macerans cyclodextrin glycosyltransferase fused with poly-lysine using cation exchanger

paper_content:

Immobilized enzymes have provided tremendous advantages for the efficient production of biomaterials. There is increasing demand on simple and convenient protein immobilization methods because protein microarray is emerging as a cutting-edge technology for the proteome analysis and diagnosis. It has been shown that a poly-lysine tag facilitates protein purification and refolding processes. This study demonstrates that the same poly-lysine tag can be employed for the immobilization of enzyme on a solid support without deterioration of its enzymatic characteristics. Cyclodextrin glycosyltransferase (CGTase) derived from Bacillus macerans was fused to consecutive 10 lysine residues (CGTK10ase) and electrostatically immobilized on a cation exchanger. Analyses on the binding characteristics, effects of pH and temperature on enzyme stability and operational stability indicate that the poly-lysine tag is also effective for non-covalent immobilization of CGTase. Though the poly-lysine-mediated immobilization is reversible, binding force is strong enough to block protein leakage from the solid support at neutral and basic pH.
---
paper_title: Covalent immobilisation of invertase onto a reactive film composed of 2-hydroxyethyl methacrylate and glycidyl methacrylate: properties and application in a continuous flow system

paper_content:

Abstract Invertase was covalently immobilised on the poly(hydroxyethyl methacrylate-co-glycidyl methacrylate) (poly(HEMA–GMA)) film. The invertase immobilisation capacity of the films was increased as the GMA ratio increased in the film structure. The immobilised invertase on the poly(HEMA–GMA-3) composition exhibited an activity of 32.7 U cm −2 film. The optimum temperature of the immobilised invertase increased by 5 °C, and the optimal pH values for the free and the immobilised enzymes were determined as 5.0. The retained activity of the immobilised invertase was between 53 and 85%. Kinetic parameters were determined for immobilised invertase as well as for the free enzyme. The values of the Michael’s constant K m of invertase were significantly larger, ca. 2.7 times upon immobilisation, indicating decreased affinity by the enzyme for its substrate, whereas V max was smaller for immobilised invertase. Activity of the immobilised invertase was quite stable with respect to free counterpart. After 168 h reaction, only 8% of immobilised invertase activity was lost. The operational inactivation rate constant ( k opi ) of the immobilised invertase at 35 °C with 200 mM sucrose was 8.23×10 −6 min −1 .
---
paper_title: Fixed bed reactor performance of invertase immobilized on montmorillonite

paper_content:

Invertase was immobilized on acid activated montmorillonite via two independent procedures, adsorption and covalent binding. The immobilized enzymes were characterized by XRD, NMR and N2 adsorption measurements and their activity was tested in a fixed bed reactor. XRD revealed that the enzyme was situated on the periphery of the clay and the side chains of different amino acid residues were involved in intercalation with the clay matrix. NMR demonstrated that tetrahedral Al was linked to the enzyme during adsorption and the octahedral Al was involved during covalent binding. Secondary interaction of the enzyme with Al was also observed. N2 adsorption studies showed that covalent binding of enzymes caused pore blockage since the highly polymeric species were located at the pore entrance. The fixed bed reactor proved to be efficient for the immobilized invertase. The optimum pH and pH stability improved upon immobilization. The kinetic parameters calculated also showed an enhanced efficiency of the immobilized systems. They could be used continuously for long period. Covalently bound invertase demonstrated greater operational stability.
---
paper_title: Immobilization of α-amylase on zirconia: a heterogeneous biocatalyst for starch hydrolysis

paper_content:

Abstract α-Amylase was immobilized on zirconia via adsorption. The support and the immobilized enzymes were characterized using XRD, IR spectra and N 2 adsorption studies. The efficiency of immobilized enzymes for starch hydrolysis was tested in a batch reactor. The effect of calcination temperatures on properties of the support as well as upon immobilization was studied. From XRD, IR and N 2 adsorption studies it was confirmed that the enzyme was adsorbed on the external surface of the support. pH, buffer concentration and substrate concentration had a significant influence on the activity of immobilized enzyme. Immobilization improved the pH stability of the enzyme. The Michaelis–Menten kinetic constants were calculated from Hanes–Woolf plot. K m for immobilized systems was higher than the free enzyme indicating a decreased affinity by the enzyme for its substrate, which may be due to interparticle diffusional mass transfer restrictions.
---
paper_title: Immobilization of β-galactosidase onto magnetic poly(GMA–MMA) beads for hydrolysis of lactose in bed reactor

paper_content:

Abstract In the present study, novel magnetic beads were prepared from glycidylmethacrylate and methylmethacrylate via suspension polymerization in the presence of a cross-linker (i.e. ethylenedimethylmethacrylate). The magnetic poly(GMA–MMA) beads were characterized with scanning electron microscope, FT-IR and ESR spectrophotometers. The reactive character of the epoxy groups allowed the attachment of the amino groups. The aminated magnetic beads were used for the covalent immobilization of β-galactosidase via glutaric dialdehyde activation. The maximum amount of immobilized β-galactosidase on the magnetic poly(GMA–MMA) beads was 9.87 mg/g support. The values of Michaelis constants K m for immobilized β-galactosidase was significant larger, indicating decreased affinity by the enzyme for its substrate, whereas V max values were smaller for the immobilized β-galactosidase. However, the β-galactosidase immobilized on the magnetic poly(GMA–MMA) beads resulted in an increase in enzyme stability with time. Optimum operational temperature for immobilized enzyme was 5 °C higher than that of the free enzyme and was significantly broader. Finally, a bed reactor with β-galactosidase immobilized was used for hydrolysis of lactose. The enzyme reactor operated continuously at 35 °C for 60 h and the immobilized enzyme lost about 12% of its initial activity after this period.
---
paper_title: Carrier-bound Immobilized Enzymes : Principles , Application and Design

paper_content:

Foreword. 1. Introduction: Immobilized Enzymes: Past, Present and Prospects. 2. Adsorption-based Immobilization. 3. Covalent Enzyme Immobilization. 4. Enzyme Entrapment. 5. Enzyme Encapsulation. 6. Unconventional Enzyme Immobilization. Subject Index.
---
paper_title: Optimization of invertase immobilization by adsorption in ionic exchange resin for sucrose hydrolysis

paper_content:

Abstract This work presents as a main objective to study the immobilization process of yeast invertase by adsorption in the ion exchanging resin Duolite A-568 for invert sugar production. Initially, a kinetic study of the soluble form of the enzyme was carried out. At the sequence was studied the immobilization process of yeast invertase in the weakly exchanging anionic resin Duolite A-568. The influences of the pH, enzyme concentration and temperature in the enzyme immobilization were analyzed through a central composite design (CCD). The results indicated that the retention of the catalytic activity in immobilization was strongly dependent of these variables, being maximum in a pH value of 5.0, with an enzyme concentration of 12.5 g/L (1.875 g of protein per liter) and temperature of 30 °C. The simultaneous influence of pH and temperature on the free and immobilized invertase activity was also studied through a CCD.
---
paper_title: Immobilization of enzymes in microtiter plate scale

paper_content:

Different immobilization methods were adapted to the 96-well microtiter plate scale using esterases as model enzymes. The methods tested were based on adsorption, coprecipitation, aggregation and covalent bonding. The protein covered microcrystals proved to be the best method in terms of yield and expressed activity for the test reaction, which was the alcoholysis of p-nitrophenyl acetate with 1-propanol under anhydrous conditions.
---
paper_title: Immobilization of glucoamylase via its carbohydrate moiety on macroporous poly(GMA-co-EGDMA)

paper_content:

We succeeded in the immobilization of 190 mg of periodate oxidized glucoamylase per gram of macroporous polymer. The covalently immobilized enzyme had a specific activity of 1100 U/g. The temperature and pH optimum as well as kinetic parameters were determined. The immobilized enzyme was tested in different types of reactors for hydrolysis of concentrated maltose and starch hydrolysate syrups. The DE value of 98.6, obtained the immobilized enzyme, was slightly higher than that for the soluble form, when acting on 20% substrates.During continuous use in a packed bed reactor over a period of 4 weeks the immobilized enzyme produced 1300 kg of glucose per 1 L of reactor volume without any decrease in its activity.
---
paper_title: Epoxy-amino groups: a new tool for improved immobilization of proteins by the epoxy method.

paper_content:

The properties of a new commercially available amino-epoxy support (amino-epoxy-Sepabeads) for immobilizing enzymes have been compared to those of conventional epoxy supports. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. Thus, this support has a great anionic exchanger power and a high number of epoxy groups. We have found a number of advantages to this new heterofunctional support. Immobilization proceeds at low ionic strength using amino epoxy Sepabeads while requiring high ionic strength using conventional monofunctional epoxy supports. Immobilization is much more rapid using amino-epoxy supports than employing conventional epoxy supports. The possibility of achieving immobilized preparations in which the enzyme orientation may be different to that obtained using the traditional hydrophobic supports (with likely effects in terms of activity or stability). Stability of the immobilized enzyme has been found to be much higher using the new support than in preparations using the conventional ones in many cases. Here we show some examples of these advantages using different enzymes (beta-galactosidases, lipase, glutaryl acylase, invertase, and glucoamylase).
---
paper_title: Covalent immobilization of a glucoamylase to bagasse dialdehyde cellulose

paper_content:

Cellulose fibres from bagasse were oxidized by sodium periodate in sulphuric acid media at positions 2 and 3 of the anhydroglucose unit to produce dialdehyde cellulose. The aldehyde groups of the dialdehyde cellulose were able to react with amino groups of a glucoamylase to form covalent bonds and result in a dialdehyde cellulose immobilized enzyme. The optimum pH of this immobilized enzyme and free enzyme were in the range of 3.0–5.0 and 3.5–5.0, respectively. The optimum temperature for both the free and immobilized enzymes was 60–65 °C. The relative remaining activity of the immobilized enzyme was 36% and its stability was very good, since it could be reused for over 30 cycles. Its activity decreased from the first to the seventh reuse cycles, due to the slow detachment of non-covalently bound enzyme. However, activity tended to stabilize after the seventh cycle of reuse, indicating very stable covalent binding between the enzyme and dialdehyde cellulose.
---
paper_title: Immobilization of glucose isomerase on Indion 48-R

paper_content:

Abstract The glucose isomerase from Streptomyces sp. NCIM 2730 was produced using wheat bran or bagasse hydrolyzate as an inducer and immobilized on an inexpensive anion exchange resin Indion 48-R. The adsorption capacity and efficiency of the resin were found to be 25 U g −1 and 60%, respectively. The properties of soluble and immobilized glucose isomerase were compared. The stability of the enzyme at higher temperatures and acidic pH is increased due to immobilization. Concentrated glucose syrup (25%) was efficiently converted to fructose by the immobilized enzyme. After the seventh reuse, the conversion capacity of immobilized glucose isomerase was reduced to 61%, with a 35% loss in enzyme activity.
---
paper_title: Method for immobilizing invertase by adsorption on Dowex® anionic exchange resin

paper_content:

This communication describes a method for adsorbing the invertase (EC.3.2.1.26) on DOWEX® anion exchange resin. Among the types of DOWEX® resins studied (1x8:50-400; 1x4:50-400 and 1x2:100-400), 1X4-200 was the most suitable, because it adsorbed the invertase molecules completely and the complex 1X4-200/invertase retained 100% of the catalytic activity. Moreover, no leakage of enzyme from the support was noted at the end of the sucrose hydrolysis.
---
paper_title: Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

paper_content:

Abstract The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant ( K m ) and the maximum rate of reaction ( V max ) were calculated from the Lineweaver–Burk plot. The K m and V max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.
---
paper_title: Advances in the design of new epoxy supports for enzyme immobilization–stabilization

paper_content:

Multipoint covalent immobilization of enzymes (through very short spacer arms) on support surfaces promotes a very interesting ‘rigidification’ of protein molecules. In this case, the relative positions of each residue of the enzyme involved in the immobilization process have to be preserved unchanged during any conformational change induced on the immobilized enzyme by any distorting agent (heat, organic solvents etc.). In this way, multipoint covalent immobilization should induce a very strong stabilization of immobilized enzymes. Epoxy-activated supports are able to chemically react with all nucleophile groups placed on the protein surface: lysine, histidine, cysteine, tyrosine etc. Besides, epoxy groups are very stable. This allows the performance of very long enzyme–support reactions, enabling us to get very intense multipoint covalent attachment. In this way, these epoxy supports seem to be very suitable to stabilize industrial enzymes by multipoint covalent attachment. However, epoxy groups exhibit a low intermolecular reactivity towards nucleophiles and hence the enzymes are not able to directly react with the epoxy supports. Thus a rapid physical adsorption of enzymes on the supports becomes a first step, followed by an additional rapid ‘intramolecular’ reaction between the already adsorbed enzyme and the activated support. In this situation, a suitable first orientation of the enzyme on the support (e.g. through regions that are very rich in nucleophiles) is obviously necessary to get a very intense additional multipoint covalent immobilization. The preparation of different ‘generations’ of epoxy supports and the design of different protocols to fully control the first interaction between enzymes and epoxy supports will be reviewed in this paper. Finally, the possibilities of a directed immobilization of mutated enzymes (change of an amino acid by cysteine on specific points of the protein surface) on tailor-made disulfide-epoxy supports will be discussed as an almost-ideal procedure to achieve very intense and very efficient rigidification of a desired region of industrial enzymes.
---
paper_title: Carrier-bound Immobilized Enzymes : Principles , Application and Design

paper_content:

Foreword. 1. Introduction: Immobilized Enzymes: Past, Present and Prospects. 2. Adsorption-based Immobilization. 3. Covalent Enzyme Immobilization. 4. Enzyme Entrapment. 5. Enzyme Encapsulation. 6. Unconventional Enzyme Immobilization. Subject Index.
---

Title: Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts

Section 1: Introduction
Description 1: Brief introduction to the historical and modern applications of enzymes in food processing.

Section 2: General Aspects and the Screening Approach
Description 2: Discuss the various classes of enzymes used in food processing, their characteristics, and how they are screened and improved for better performance.

Section 3: Improving Biocatalysts: Beyond Screening
Description 3: Detailed overview of methods such as protein engineering, directed evolution, and computational tools used to enhance enzyme stability and effectiveness.

Section 4: Immobilization
Description 4: Discussion on the various techniques and benefits of immobilizing enzymes to improve their stability, activity, and reusability.

Section 5: Typical Bioreactors
Description 5: Description of the common bioreactor configurations used in continuous enzymatic processes and their operational considerations.

Section 6: Conclusions and Future Perspectives
Description 6: Summary of the current trends, challenges, and future directions in the application of enzymes in food processing.