10.6092/issn.2531-7342/16067
L-Asparaginase has a greater demand due to its potential application in the food industry as well as in the pharmaceutical industry. To meet this demand, optimization of nutritional as well as physical conditions is critical for scaling up the production of L-Asparaginase. This study aims to achieve enhanced L-Asparaginase production, a therapeutic enzyme from the endophytic fungus Fusarium sp. LCJ324 isolated from Glycosmis mauritiana (Lam.) Tanaka belonging to the Rutaceae family. Fusarium sp. LCJ324 exhibited L-Asparaginase activity of 8.75 ± 0.55 U mL-1 in modified Czapek Dox broth, the highest among all the seven different media tested by submerged fermentation. Different sources and concentrations of carbon, nitrogen and amino acid inducers (nutritional factors) as well as pH and temperature (physical parameters) were optimized to achieve enhanced L-Asparaginase production. Maximum L-Asparaginase activity of 19.94 ± 0.35 U mL-1 was obtained at 30º C with dextrose (3 g L-1), ammonium sulphate (30 g L-1), and L-Asparagine (20 g L-1) at pH 6. Response Surface Methodology was employed for statistical optimization of minimum and maximum levels of the selected parameters. The levels of parameters suggested by the response surface methodology for maximum production of L-Asparaginase were similar to the conventional optimization. A 2.29 fold increase in L-Asparaginase production was obtained through conventional and statistical optimization.
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Submerged fermentation
The nutritional and cultural conditions in shake flask culture for improved production of L-Asparaginase from endophytic fungus <em>Fusarium</em> sp. LCJ324: A sequential statistical method
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John Joel Gnanadoss
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Microbial and Environmental Biotechnology Research Unit, Department of Plant Biology and Biotechnology, Loyola College, Chennai-600034, Tamil Nadu, India
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Yap LS, Lee WL, Ting ASY (2021) Optimization of L-Asparaginase production from endophytic Fusarium proliferatum using OFAT and RSM and its cytotoxic evaluation. Journal of Microbiological Methods 191:106358. https://doi.org/10.1016/j.mimet.2021.106358
The nutritional and cultural conditions in shake flask culture for improved production of L-Asparaginase from endophytic fungus <em>Fusarium</em> sp. LCJ324: A sequential statistical method
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                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 52 (2023): 62-75
                Journal homepage: https://italianmycology.unibo.it/

Research article

  The nutritional and cultural conditions in shake flask culture for
 improved production of L-Asparaginase from endophytic fungus
      Fusarium sp. LCJ324: A sequential statistical method

               Elangovan Udayan, Anbarasu Kathiravan, John Joel Gnanadoss
Microbial and Environmental Biotechnology Research Unit, Department of Plant Biology and Biotechnology, Loyola
College, Chennai-600034, Tamil Nadu, India

Corresponding author e-mail: joelgna@gmail.com


Article info
Received 20/12/2022; accepted 17/2/2023
https://doi.org/10.6092/issn.2531-7342/16067

Abstract
L-Asparaginase has a greater demand due to its potential application in the food industry as well as in the
pharmaceutical industry. To meet this demand, optimization of nutritional as well as physical conditions is
critical for scaling up the production of L-Asparaginase. This study aims to achieve enhanced L-Asparaginase
production, a therapeutic enzyme from the endophytic fungus Fusarium sp. LCJ324 isolated from Glycosmis
mauritiana (Lam.) Tanaka belonging to the Rutaceae family. Fusarium sp. LCJ324 exhibited L-Asparaginase
activity of 8.75 ± 0.55 U mL-1 in modified Czapek Dox broth, the highest among all the seven different
media tested by submerged fermentation. Different sources and concentrations of carbon, nitrogen and amino
acid inducers (nutritional factors) as well as pH and temperature (physical parameters) were optimized to
achieve enhanced L-Asparaginase production. Maximum L-Asparaginase activity of 19.94 ± 0.35 U mL-1
was obtained at 30º C with dextrose (3 g L-1), ammonium sulphate (30 g L-1), and L-Asparagine (20 g L-1) at
pH 6. Response Surface Methodology was employed for statistical optimization of minimum and maximum
levels of the selected parameters. The levels of parameters suggested by the response surface methodology for
maximum production of L-Asparaginase were similar to the conventional optimization. A 2.29 fold increase in
L-Asparaginase production was obtained through conventional and statistical optimization.

Keywords
Modified Czapek Dox broth, L-Asparagine, one factor at a time, response surface methodology, submerged
fermentation


Introduction

The enzyme L-Asparaginase (EC 3.5.1.1) catalyses the hydrolysis of L-asparagine to L-aspartic
acid and ammonia. Cancerous cells typically require asparagine because they have lost the ability to
express the gene encoding asparagine synthetase (Ekpenyong et al., 2021). Thus, these cells depend
on external sources of L-asparagine for their persistence. Therefore, the usage of L-Asparaginase as an
intravenous injection will reduce L-Asparagine levels and in turn prevents the growth of cancerous cells.
As a result, in the treatment of children with acute lymphoblastic leukaemia (ALL), L-Asparaginase
is considered as the most effective drug (Nunes et al., 2020). Furthermore, L-Asparaginase is widely

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and maintained by ABIS-AlmaDL. © 2023 The Authors. This work is licensed under CC BY 4.0
Udayan et al.                                                    Italian Journal of Mycology 52 (2023): 62-75

https://doi.org/10.6092/issn.2531-7342/16067



used to treat other types of cancer such as lymphosarcoma, Hodgkin’s disease, reticulosarcoma as
well as different types of leukaemia (El-Naggar et al., 2018). L-Asparaginase finds application in
food industry namely to reduce the development of acrylamide (carcinogenic substance formed in
foods that have undergone heat processing) in starchy foods. L-Asparaginase based biosensors that
can quantify L-Asparagine levels finds application in both pharmaceutical as well as food industry
(Nunes et al., 2020).
    The sources of L-Asparaginase are plants (Bano and Sivaramakrishnan, 1980; Oza et al., 2011;
Mohamed et al., 2015) and microbes. The complicated process of obtaining L-Asparaginase from
plants coupled with the low yield restricts the plants as a go to source for L-Asparaginase production.
Microbial source of L-Asparaginase includes bacteria, actinomycetes and fungi. L-Asparaginase from
bacteria Acetinobacter soli was reported by Jiao et al. (2020). Alrumman et al. (2019) has reported
that Bacillus licheniformis was capable of yielding 8.1 U mL-1 of L-Asparaginase. There are two
clinically approved formulations of bacterial L-Asparaginases (Erwinia chrysanthemi and E. coli)
that are in use for ALL treatments. The bacterial derived (prokaryotic sources) L-Asparaginase has
high glutaminase activity and long-term therapeutic use causes unpleasant side effects due to normal
cell toxicity (bacterial toxins in the enzyme) and the dissociation of the enzyme from the bloodstream,
limits its usage (Ramya et al., 2012; El-Naggar et al., 2014).
    L-Asparaginase from fungal sources can have fewer adverse effects as they belong to the
eukaryotic type of organisms (Ali et al., 1994; Meghavarnam et al., 2022). Endophytic fungi are
capable of producing L-Asparaginase but are less explored. Endophytic fungi isolated from medicinal
plants have huge potential for the production of numerous metabolites and enzymes. Production
of L-Asparaginase using endophytic fungi can be done by solid state or submerged fermentation.
In submerged fermentation, the utilization of substrate by fungi is rapid with the added advantage
of having minimal chances for contamination. This method is well-suited for the extraction of
extracellular metabolites from microbes (Couto and Sanromán, 2006). The endophytic fungus
Fusarium sp. LCJ324 produced significant L-Asparaginase of 8.84 U mL-1 in submerged fermentation
using modified Czapek Dox broth (Udayan and Gnanadoss, 2023). However, to deal with low yields
of the enzyme, optimization of the nutritional parameters and physical conditions of the medium is
necessary in order to improve the yields. Optimization of the bioprocess can be done by One Factor
at a Time (OFAT) method which is a classical method of optimization. OFAT method of optimization
is tedious and expensive. However, the development of contemporary mathematical and statistical
techniques can assist in media optimization. Response Surface Methodology (RSM) is one such
statistical tool that can be used in analyzing different levels of variables, interaction and their impact
on product formation. RSM generates an experimental design that reduces the number of experiments
and increases the efficiency of the optimization process (Li et al., 2015; Abdollahzadeh et al., 2020).
The study was aimed to formulate and optimize the medium for enhanced L-Asparaginase production
by the endophytic fungus Fusarium sp. LCJ324 using OFAT and RSM methods.

Materials and methods

Isolation and characterization of endophytic fungal culture
The endophytic fungal culture LCJ324 was isolated from Glycosmis mauritiana (Lam.) Tanaka
belonging to the Rutaceae family (Udayan and Gnanadoss, 2023). LCJ324 exhibited high
L-Asparaginase enzyme activity and was subjected to molecular characterization using ITS1
(5’TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5’TCCTCCGCTTATTGATATGC-3′) primer
pair. The endophytic fungal culture LCJ324 was identified as Fusarium sp. The pure culture was
maintained on PDA slants at 4°C.

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Selection of medium for L-Asparaginase production
The endophytic fungus Fusarium sp. LCJ324 was cultured in seven different media (glucose asparagine
broth, asparagine dextrose salts broth, glycerol asparagine broth, modified M9 broth, modified M2
broth, modified Czapek Dox broth and inorganic salt starch asparagine broth) to identify the medium
that best supports production of L-Asparaginase. The shake flask method of culturing was followed
in this experiment to identify the medium that supports maximum production of L-Asparaginase. A
hundred mL of each media was prepared in a separate 250 mL conical flask, sterilized and supplemented
with streptomycin (100 µg mL-1). Three mycelial discs (5 mm) of four days old Fusarium sp. LCJ324
maintained in PDA were inoculated into the flasks under aseptic conditions and incubated at 120 rpm
in an orbital shaker (REMI RIS-24BL) for 8 days. The L-Asparaginase activity was evaluated every
24 h using the supernatant obtained by centrifuging 0.5 mL of crude culture filtrate.

L-Asparaginase assay
L-Asparaginase assay was done by Nesslerization method described by Imada et al. (1973). Culture
filtrate was pipetted out daily to make a reaction mixture (2.5 mL) that contained 500 µl of each of
the following culture filtrate, 0.5 M Tris buffer, 0.04 M L-asparagine and distilled water. The reaction
mixture was incubated for about 30 min followed by the addition of 500 µl of Trichloroacetic acid.
The reaction mixture was then centrifuged at 10,000 rpm for 5 min. A hundred µl of supernatant was
pipetted out from the reaction solution to which distilled water (3700 µl) and Nesseler’s reagent (200
µl) was added and incubated for 20 min. The brownish yellow colour developed was read at 450 nm
using an UV visible spectrophotometer (Elico). At 37 ± 2 °C, one unit of asparaginase is defined as
the enzyme quantity required to catalyze the creation of 1 µmol of NH4/min.

     Units mL-1 enzyme = (μM of ammonia liberated) (Volume of reaction solution)
                       (Volume of supernatant) (Incubation Time) (Volume of crude enzyme)

Volume of reaction solution - 2.5 mL
Volume of supernatant - 0.1 mL
Incubation Time - 30 min
Volume of crude enzyme - 0.5 mL

Studies for optimizing production parameters of L-Asparaginase enzyme
Optimization studies are necessary to evaluate the various factors and conditions to improve enzyme
production. The following factors such as carbon source, nitrogen source, amino acid inducer and
culture conditions like pH and temperature were optimized by following one factor at a time method.
Optimization was carried out by varying any one factor or condition while keeping other variables
as constant. Modified Czapek Dox broth medium (g L-1): [Glucose (2), L-Asparagine (10), KH2PO4
(1.52), MgSO4.7H2O (0.52), KCL (0.52), Cu(NO3).3H2O (0.001), ZnSO4.7H2O (0.001), FeSO4.7H2O
(0.001)] (Jenila and Gnanadoss, 2018) was used for the optimization of L-Asparaginase production
using Fusarium sp. LCJ324. The optimized parameter was used in the subsequent studies.

Optimization of carbon source and its concentration
Effect of addition of different carbon sources such as lactose, sucrose, dextrose, maltose and galactose
at 2 g L-1 on L-Asparaginase production by Fusarium sp. LCJ324 was studied by substituting the
carbon source in the modified Czapek Dox broth medium. The standard medium was used as a
control. The best carbon source identified was further studied to find the optimum concentration for
L-Asparaginase production. The concentration of the best carbon source was varied from 1 to 6 g L-1.
L-Asparaginase activity and protein activity were determined every day.
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Optimization of nitrogen source and its concentration
Effect of different nitrogen sources on L-Asparaginase production by Fusarium sp. LCJ324 was
studied. Peptone, potassium nitrate, urea, ammonium sulphate and yeast extract are the different
nitrogen sources used in this study at a concentration of 10 g L-1. The standard medium was used as
a control. The best nitrogen source was further scrutinized to find the optimum concentration. The
concentration of the nitrogen source was varied from 5 to 30 g L-1.

Optimization of amino acid inducer sources and its concentration
Effect of different amino acid sources such as L-Asparagine, L-Arginine, L-Tryptophan, L-Tyrosine
and L-Glutamic acid on L-Asparaginase production by Fusarium sp. LCJ324 was studied at a
concentration of 10 g L-1. After the selection of the best amino acid source, the optimal concentration for
the enhanced L-Asparaginase production was studied by varying the concentrations of L-Asparagine
from 5 to 30 g L-1.

Optimization of initial pH
Effect of initial pH of the medium on the production of L-Asparaginase by Fusarium sp. LCJ324 was
studied by varying the pH of the modified Czapek Dox broth in the range of 4 to 9. The pH of the
medium was varied using 0.1N HCL and 0.1N NaOH.

Optimization of temperature
The effect of different temperatures on L-Asparaginase production was studied. The inoculated flasks
were incubated in varying temperatures ranging from 28 ºC to 36 ºC.

Optimization of L-Asparaginase production using RSM
RSM is a statistical tool that is employed to optimize the output when more than two factors are
involved. Central composite design (CCD) is the widely used tool in RSM to design experiments.
After the selection of major parameters and their ranges, levels were coded for the study using Face
Centre Central Composite Design (FCCCD). The parameters such as dextrose, ammonium sulphate,
L-Asparagine and pH were studied. Three different levels (-1, 0, 1) of the four parameters were coded
in the software Design Expert (version 13, Stat-Ease, Inc., Minneapolis, MN).
    Thirty experiments were generated by varying the concentrations of each of the selected four
parameters. The experiments were carried out and their response (enzyme activity) was noted.
A polynomial equation was generated that is in accordance with the data as well as related to all
the variables in the experiment. The polynomial equation generated using the four factors for
L-Asparaginase production is:
    Y= β0 + β1X1+ β2X2+ β3X3+ β4X4+ β11X12+ β22X22+ β33X32+ + β44X42+ β12X1X2+ β13X1X3+ β14X1X4+
β23X2X3+ β24X2X4+ β34X3X4

   In the above equation, Y represents the L-Asparaginase (predicted response) of the model; β0
represents intercept; β1, β2, β3, β4 represent linear coefficient; β11, β22, β33, β44 represent squared
coefficients; β12, β13, β14, β23, β24, β34 represents interaction coefficients. Analysis of variance (ANOVA)
was used as the statistical method to look into the significance and suitability of the model. The
Design Expert 13.0 software was employed in handling all of the three-dimensional and counter
plotting, as well as the statistical analysis.




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Comparison of L-Asparaginase production using original and optimized medium
L-Asparginase production by Fusarium sp. LCJ324 using the original and optimized medium was
studied. Fusarium sp. LCJ324 was inoculated and cultured for 8 days in separate flasks containing the
original and optimized medium. The L-Asparaginase activity was determined every day.

Results and Discussion

Isolation and Characterization of Endophytic fungal culture
The endophytic fungal LCJ324 isolated from Glycosmis mauritiana (Lam.) Tanaka was identified
as Fusarium sp. LCJ324 using ITS sequence. Fusarium sp. LCJ324 showed high L-Asparaginase
activity in screening medium modified Czapek Dox. The obtained ITS sequence was submitted to
Genbank and the accession number MZ646108 was obtained. Similarly, Hatamzadeh et al. (2020)
performed screening for L-Asparaginase enzyme from endophytic fungal isolates in modified Czapek
Dox medium.

Selection of medium for L-Asparaginase production
The L-Asparaginase production was tested using the seven different media for 8 days and the
production varied in all the media. Modified Czapek Dox broth medium showed the highest
L-Asparaginase activity of 8.70 ± 0.52 U mL-1 on the 5th day when compared to other media. The
lowest L-Asparaginase activity of 3.89 ± 0.25 U mL-1 was noted in inorganic salt starch asparagine
broth. Intermediate L-Asparaginase activity was observed in modified M9 broth (8.12 ± 0.35 U mL-1),
glycerol asparagine broth (7.48 ± 0.44 U mL-1), asparagine dextrose salts broth (7.14 ± 0.23 U mL-1),
modified M2 broth (6.63 ± 0.29 U mL-1) and glucose asparagine broth (6.73 ± 0.35 U mL-1).
   Submerged fermentation for L-Asparaginase production was investigated by Elshafei and El-
Ghonemy (2015) from Trichoderma viridae Pers and was found to be 14.23 ± 0.87 U mL-1. Jenila
and Gnanadoss (2018) studied L-Asparaginase production from five different liquid media. Among
them modified Czapek Dox broth showed significant L-Asparaginase activity and was subsequently
subjected to conventional optimization. L-Asparaginase from Fusarium equiseti (Corda) Sacc.
under submerged fermentation produced 40.78 U mL-1 of enzyme after conventional optimization
of modified Czapek Dox broth (El-Gendy et al., 2021). L-Asparaginase production from endophytic
Lasiodiplodia theobromae (Pat.) Grifton & Maubl. was studied in modified Czapek Dox broth
(Balbool and Azeem, 2020).

Optimization of carbon source and its concentration
The effect of different carbon sources on L-Asparaginase production such as lactose, sucrose,
dextrose, maltose and galactose was studied. Dextrose yielded higher L-Asparaginase activity of
11.90 ± 0.57 U mL-1 when compared to other carbon sources on the 5th day (Table 1). The effect
of different concentrations of dextrose on L-Asparaginase activity was studied, 3 g L-1 of dextrose
increased L-Asparaginase activity to 13.20 ± 0.57 U mL-1 (Fig. 1).




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Table 1 - Effect of different nutritional parameters on the production of L-Asparaginase.
Source                                                      L-Asparaginase activity U mL-1
Carbon source (2 g L )  -1

Lactose                                                     9.81 ± 0.52
Sucrose                                                     9.36 ± 0.29
Dextrose                                                    11.90 ± 0.57
Maltose                                                     9.02 ± 0.52
Galactose                                                   9.07 ± 0.52
Nitrogen source (10 g L )  -1

Peptone                                                     5.64 ± 0.29
Potassium nitrate                                           3.58 ± 0.17
Urea                                                        5.83 ± 0.40
Ammonium sulphate                                           14.70 ± 0.52
Yeast extract                                               5.95 ± 0.52
Amino acid inducer (10 g L-1)
L-Asparagine                                                18.80 ± 0.58
L-Arginine                                                  16.00 ± 0.58
L-Trytophan                                                 17.30 ± 0.64
L-Tyrosine                                                  16.50 ± 0.58
L-Glutamic acid                                             15.00 ± 0.69
Values are mean ±SEM
Values differ (p<0.05) as analyzed by one-way ANOVA

    The primary structural and functional element in microbial cells is carbon. Although fungi may
use a wide range of carbon molecules, most of them prefer simple sugars (Uzma et al., 2016).
Among 9 different carbon sources tested, sucrose and molasses increased L-Asparaginase activity for
Aspergillus candidus P. Micheli ex Haller (Ekpenyong et al., 2021). Two g L-1 of glucose was used
as the carbon source for enhanced production of L-Asparaginase (6.21 U) using Aspergillus terreus
Thom (Costa-silva et al., 2019). Two g L-1 of sucrose was found to be best carbon souce for enhancing
L-Asparaginase production (0.166 U mL-1) by Fusarium solani (Mart.) Sacc. (Uzma et al., 2016).
Study on optimization of nutritional parameters revealed that 2 g L-1 of glucose as best carbon source
for increasing L-Asparaginase production by Fusarium proliferatum (Matsuch.) Nirenberg (Yap et
al., 2021).




                                                                             Fig. 1 - Effect of dextrose concentrations
                                                                             on the production of L-Asparaginase by
                                                                             Fusarium sp. LCJ324 on the 5th day.


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Optimization of nitrogen source and its concentration
The effect of different nitrogen sources on L-Asparaginase production in modified Czapek Dox
broth was studied. Ammonium sulphate as nitrogen source yielded better L-Asparaginase production
by Fusarium sp. LCJ324 (14.70 ± 0.52 U mL-1) than other nitrogen sources (Table 1). Different
concentration of ammonium sulphate was tested for optimum production of L-Asparaginase and 30 g
L-1 of ammonium sulphate resulted in enhancing the L-Asparaginase activity to 16.30 ± 0.64 U mL-1
(Fig. 2).




                                                                  Fig. 2 - Effect of ammonium sulphate
                                                                  concentrations on the L-Asparaginase
                                                                  production by Fusarium sp. LCJ324 on
                                                                  the 5th day.

   Nitrogen as a limiting nutrient is crucial for the fungi’s metabolism as well as for the formation
of L-Asparaginase (El-Hadi et al., 2017). Ammonium sulphate as nitrogen source for Fusarium sp.
at a concentration of 20 g L-1 increased the L-Asparaginase activity to 13.69 U mL-1 (Jenila and
Gnanadoss, 2018). Yeast extract, proline, ammonium chloride, asparagine and sodium nitrate were
examined as different nitrogen sources to enhance L-Asparaginase activity. Ammonium chloride as
nitrogen source for Fereydounia khargensis significantly increased the L-Asparaginase activity to
51.7 U mL-1 from 13.6 U mL-1 (Fazeli et al., 2021). Ammonium nitrate and sodium nitrate as nitrogen
source demonstrated increased L-Asparaginase activity for Fusarium solani (Uzma et al., 2016).

Optimization of amino acid inducer source and its concentration
The effect of different amino acid inducer sources on L-Asparaginase production was studied. The
addition of L-Asparagine as amino acid inducer yielded better L-Asparaginase activity of 18.80 ±
0.58 U mL-1 (Table 1). Subsequently, the effect of different concentrations of L-Asparagine was
studied. Maximum L-Asparaginase activity of 19.10 ± 0.69 U mL-1 was observed at 20 g L-1 of
L-Asparagine (Fig. 3).
   One g L-1 of L-Asparagine was used as an inducer for increasing L-Asparaginase activity of
Trichoderma viridae to 223 U g-1 while other inducers such as L-aspartic acid and L-glutamic acid
resulted in reduced L-Asparaginase activity (Elshafei and El-Ghonemy, 2015). The final composition
of the optimized medium for increased production of L-Asparaginase using Talaromyces pinophilus
(Hedge.) Samson, Yilmaz, Frisrad & Seifert devised by Krishnapura and Belur (2020) had 17.2 g L-1
of L-Asparagine as an inducer.




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                                                                Fig. 3 - Effect of L-Asparagine
                                                                concentrations on the L-Asparaginase
                                                                production by Fusarium sp. LCJ324 on
                                                                the 5th day.


Optimization of initial pH
The effect of different initial pH in the medium for L-Asparaginase production was studied since
pH is an important physical parameter for increased enzyme production. In the present study, the
optimum L-Asparaginase activity of 19.30 ± 0.23 U mL-1 was achieved at initial pH 6 (Fig. 4).
   Similarly, maximum L-Asparaginase activity at pH 7 was reported by Hosamani and Kaliwal
(2011), Chandrasekhar (2012), and Uzma et al. (2016). pH 6.5 was found to be favourable for
L-Asparaginase enzyme production for Talaromyces pinophilus. An increase in pH led to a decrease
in enzyme activity (Krishnapura and Belur, 2020). Maximum L-Asparaginase enzyme yield was
obtained from Aspergillus niger P. Micheli ex Haller at pH 6.5 (Mishra, 2006). pH 7 was identified
as the optimum pH for increased L-Asparaginase production (32.25 U mL-1) by Aspergillus terreus
(Kalyanasundaram et al., 2015). In another study Aspergillus terreus, a marine derived endophytic
fungus yielded maximum L-Asparaginase activity at pH 6 (Farag et al., 2015). pH of the production
medium plays a vital role in affecting the enzyme production both positively and negatively.




                                                               Fig. 4 - Effect of pH on L-Asparaginase
                                                               production by Fusarium sp. LCJ324 on
                                                               the 5th day.




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Optimization of temperature
The suitable incubation temperature of Fusarium sp. LCJ324 was studied for enhancing
L-Asparaginase production. A temperature of 30°C was found to be the optimum temperature for
increased L-Asparaginase activity of 19.94 ± 0.346 U mL-1 (Fig. 5). Higher temperatures resulted in
a decrease in enzyme production.




                                                                 Fig. 5 - Effect of temperature on
                                                                 L-Asparaginase production by Fusarium
                                                                 sp. LCJ324 on the 5th day.
   An incubation temperature of 30°C was reported as the optimum temperature for the L-Asparaginase
production from Aspergillus sp. (Pundir et al., 2020; Priya and Subhashini, 2022) and Fusarium
proliferatum (Yap et al., 2021). An incubation temperature of 29 °C was found to be the best condition
for L-Asparaginase production using Aspergillus terreus (Da Rocha et al., 2019). Incubation
temperature is a crucial factor for enzyme production as it influences both growth of the organisms as
well as the rate of the chemical reaction in the medium (Shah et al., 2014).

Optimization of L-Asparaginase production using RSM
RSM was employed in finding the influence of the four major parameters (dextrose, ammonium
sulphate, L-Asparagine and pH) on the L-Asparaginase production. Using FCCCD 30 trials were
generated. The trial runs were conducted in triplicates and the results were analyzed using RSM.
Table 2 displays the design matrix and the accompanying outcomes of the observed and predicted
responses for L-Asparaginase production.
   The analysis of variance (ANOVA) of the model yielded P-value of <0.0001 which indicates
that the model is significant. The R2 value of 0.9594 was obtained from this study indicating a good
connection between the observed and predicted responses. Statistical optimization for enhancing
L-Asparaginase production from Aspergillus candidus was done using RSM and R2 value of 0.9874
was reported (Ekpenyong et al., 2021). Similarly, R2 values of 0.9488 and 0.9679 were reported
for RSM mediated statistical optimization of L-Asparaginase production from Penicillium lilacinum
Thom and Cladosporium tenuissimum Cooke respectively (Hamed et al., 2021; Vimal and Kumar,
2022). The F and P value of lack of fit was found to be 2.44 and 0.1682 respectively. Indicating lack
of fit was insignificant and the model is very effective.
   The obtained polynomial equation for L-Asparaginase production is as follows:
   Y= 19.34 + 0.5244X1 + 0.5517X2 + 0.4872X3 – 0.0478X4 - 0.0319X1X2 + 0.0169X1X3 + 0.3906X1X4
+ 0.1106X2X3 + 0.4194X2X4 + 0.0731X3X4 - 0.7011X12 - 0.5161X22 - 0.7061X32 – 1.68 X42
   where, Y represents the L-Asparaginase activity, X1, X2, X3 and X4 represents dextrose, ammonium
sulphate, L-Asparagine and pH respectively.

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Udayan et al.                                                         Italian Journal of Mycology 52 (2023): 62-75

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Table 2 - Observed and predicted values with factors in coded values for L-Asparaginase production using FCCCD.
   Run          Dextrose   Ammonium sulphate       L-Asparagine        pH        L-Asparaginase activity U mL-1
                                                                                 Actual value Predicted value
     1              0                 0                   1              0           18.84            19.12
     2              0                 1                   0              0           19.18            19.38
     3             -1                 1                  -1             -1           14.70            15.31
     4              1                -1                   1              1           16.88            16.13
     5             -1                 0                   0              0           17.77            18.11
     6              0                 0                   0              0           19.96            19.34
     7              1                -1                  -1              1           14.86            15.20
     8             -1                -1                  -1              1           13.20            13.34
     9              0                 0                   0              0           19.40            19.34
    10              1                 1                   1             -1           16.84            16.56
    11             -1                -1                   1             -1           15.69            15.77
    12              1                 1                  -1             -1           15.46            15.48
    13              1                 0                   0              0           18.96            19.16
    14             -1                 1                   1              1           16.35            16.43
    15              0                 0                   0              0           19.46            19.34
    16              1                 1                   1              1           17.80            18.23
    17             -1                -1                  -1             -1           15.62            15.20
    18              0                 0                   0              0           19.99            19.34
    19              0                 0                  -1              0           17.88            18.15
    20              0                -1                   0              0           17.92            18.27
    21              0                 0                   0              1           17.12            17.61
    22              0                 0                   0             -1           17.65            17.71
    23             -1                -1                   1              1           14.21            14.20
    24             -1                 1                  -1              1           15.61            15.12
    25              0                 0                   0              0           19.82            19.34
    26              1                -1                   1             -1           15.64            16.14
    27              1                 1                  -1              1           17.08            16.85
    28              1                -1                  -1             -1           15.72            15.50
    29              0                0                   0               0           19.05            19.34
    30             -1                1                   1              -1           16.65            16.32

   Four components were combined into pairs for the construction of three-dimensional response plots
to predict the L-Asparaginase production for various values of the investigated variables and examine
the interaction between them (Fig. 6). Since the observed and predicted values of L-Asparaginase
production were found to be in close agreement, the model was validated successfully.
   The results of the present study indicate that both OFAT and RSM evaluation yielded the same
optimum condition for increased L-Asparaginase production. The results coincide with the results
reported by Yap et al. (2021). Optimization of culture conditions for better production of L-Asparaginase
has been reported using Fusarium sp. (Jenila and Gnanadoss, 2018), Fusarium solani (As-Suhbani
and Bhosale, 2020), Talaromyces pinophilus (Krishnapura and Belur, 2020) and Aspergillus candidus
(Ekpenyong et al., 2021).


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Udayan et al.                                                     Italian Journal of Mycology 52 (2023): 62-75

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Fig. 6 - Three dimensional plots from RSM showing the interaction between variables a) Dextrose and ammonium
sulphate b) Dextrose and pH c) Ammonium sulphate and L-Asparagine.


Comparison of L-Asparaginase production using original and optimized medium
Comparative study of L-Asparaginase production by Fusarium sp. LCJ324 in the original and
optimized medium was done. L-Asparaginase activity for Fusarium sp. LCJ324 was found to
be 8.78 ± 0.52 U mL-1 in original medium and 19.95 ± 0.59 U mL-1 in optimized medium (g L-1;
Dextrose (3), Ammonium sulphate (30), L-Asparagine (2), KH2PO4 (1.52), MgSO4.7H2O (0.52), KCL
(0.52), Cu(NO3).3H2O (0.001), ZnSO4.7H2O (0.001), FeSO4.7H2O (0.001). A 2.29-fold increase in
L-Asparaginase production was obtained in optimized medium in comparison to the original medium.

Conclusion

This study has revealed that Fusarium sp. LCJ324, an endophytic fungus isolated from Glycosmis
mauritiana was capable of producing L-Asparaginase, a highly intriguing enzyme with significant
applications in the food and pharmaceutical industries. To increase the yield of L-Asparaginase,
the best medium was formulated by comparing seven different media. Modified Czapek Dox broth
medium formulation was selected and taken up for optimization to achieve maximum production
of L-Asparaginase. The main components that were optimized are carbon, nitrogen, amino acid
inducer sources, pH and temperature. By combining the application of conventional and statistical
methods of optimization, the selection of suitable medium components at correct levels maximized
the L-Asparaginase production. This resulted in 2.29-fold increase in the L-Asparaginase yield from
8.7 to 19.99 U mL-1. To conclude, given the paucity of studies on L-Asparaginase production from
endophytic fungi, the findings of the current study open up fresh avenues for designing novel methods
for the synthesis of this enzyme, with the potential to yield L-Asparaginase with superior properties
to L-Asparaginase produced by bacteria that are available on the market currently.

Acknowledgement
We the authors are thankful to the management of Loyola College, Chennai for providing necessary
support and encouragement throughout the study.




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https://doi.org/10.6092/issn.2531-7342/16067



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