Molecole bioreattive: un contributo innovativo delle biotecnologie per il restauro dei Beni Culturali
10.6092/issn.1973-9494/4191
SummaryOne of the first and most important steps during the conservative restoration of an artifact, is the action of cleaning, which enables the removal of organic layers such as animal glue or other protein mixtures that cover the original surface. Cleaning must be performed carefully and selectively, making a distinction between the different areas, so as not to cause irreversible damage to the surface of the object, and remove the deposits without acting directly on the original surface materials. In this study hydrolases isolated from marine organisms, whose peculiarity is to be active in a range of temperature between 4° C and 37° C, are proposed as an alternative to classic cleaning methods. RiassuntoLa pulitura, azione che permette la rimozione di strati soprammessi dalla superficie di un manufatto, è una delle prime e più importanti fasi attuate in interventi di restauro conservativo. Questa deve essere eseguita in modo da non causare danni irreversibili, quindi effettuata in modo selettivo, differenziando l’azione tra le diverse zone e rimuovendo in successione gli strati di deposito, senza interagire in modo diretto con i materiali originali della superficie. In questo studio sono state utilizzate nuove idrolasi isolate da organismi marini, la cui attività proteasica è stata saggiata in laboratorio, presentano la rilevante peculiarità è di essere attivi in un range di temperature compreso tra 4°C e 37°C e sono proposte come valida alternativa ai classici metodi di pulitura.RésuméLe nettoyage est l’action qui permet d’enlever les couches accumulées sur la surface d’un ouvrage, et c’est l’une des premières phases les plus importantes mises en œuvre dans les interventions de restauration-conservation. Le nettoyage doit faire en sorte de ne pas provoquer de dommages irréversibles : il est donc effectué de manière sélective en différenciant l’action suivant les zones, et en enlevant en succession les couches de dépôt sans interagir directement avec les matériaux originaux de la surface. Dans cette étude, on a utilisé de nouvelles hydrolases isolées d’organismes marins, dont l’activité protéasique a été testée en laboratoire. Elles présentent la particularité importante d’être actives dans un intervalle de températures compris entre4°Cet37°C. Elles sont proposées comme une alternative valable aux méthodes classiques de nettoyage. ZusammenfassungDie Säuberung, d.h. das Abtragen von abgelagerten Schichten auf einem Gegenstand, ist eine der ersten und wichtigsten Phasen bei konservativen Restaurierungsmaßnahmen. Sie muss so erfolgen, dass keine nicht wieder gutzumachenden Schäden entstehen, d.h. sie muss selektiv vorgenommen werden, durch unterschiedliche Bearbeitung der verschiedenen Zonen und aufeinanderfolgendes Abtragen der abgelagerten Schichten ohne direkt auf die originalen Materialien der Oberfläche einzuwirken. In dieser Studie wurden neue, von Meeresorganismen isolierte Hydrolasen eingesetzt, deren eiweißspaltende Wirkung im Labor getestet wurde. Diese haben die relevante Eigenschaft, dass sie in einem Temperaturbereich zwischen 4 und 37°C aktiv sind und eine gute Alternative zur den herkömmlichen Säuberungsmethoden bieten. ResumenLa limpieza, acción que permite remover capas superpuestas de la superficie de una obra, es una de las primeras y más importantes fases llevadas a cabo en los trabajos de restauración conservativa. Dicha fase ha de ser realizada sin provocar daños irreversibles, por lo tanto es efectuada de forma selectiva, diferenciando el tipo de acción en las distintas zonas y quitando sucesivamente las capas de depósito, sin interaccionar directamente con los materiales originales de la superficie. En este estudio se han utilizado nuevas hidrolasas aisladas de organismos marinos, cuya actividad proteasa ha sido probada en laboratorio, con la importante peculiaridad de que permanecen activas en una franja de temperatura situada entre 4°C y 37°C y se proponen como una alternativa apropiada ante métodos clásicos de limpieza. РезюмеОчистка, действие, которое позволяет удалить накопленные слои с поверхности артефакта, является одним из первых и наиболее важных шагов, применяемых при консервативной реставрации. Она должна производиться при недопущении необратимых повреждений, следовательно,  ее проведение должно быть выборочным, при дифференциации вмешательств в различных зонах и, удаляя в последовательности слои накопления, не соприкасаясь напрямую с оригинальными материалами поверхности. В этом исследовании были использованы новые гидролазы, выделенные из морских организмов, протеазная активность которых была проанализирована в лаборатории, и которые имеют важную особенность, состоящую в том, что они могут быть активными в диапазоне температур от 4 ° С до 37 ° С и могут являться эффективной альтернативой традиционным методам очистки. 摘要清洁，即去除物品表面层层覆盖物的操作，是分阶段修复措施中最首先和最重要的一步。这必须在不会造成不可弥补伤害的前提下进行，所以应该用选择性的清洁，在不同区域要用不同的操作方式并且相继除去覆盖层，切忌直接与表面原始材料相互影响。这项研究中从海洋生物内提取了新的水解酶，其蛋白酶活性已通过实验室检测，它表现出的相关特性是在4°C ~37°C的温度范围内活跃并被提议为很好的替代传统清洁的新方法。
application/pdf
1973-9494
4191
en
Franco Palla
Conservation Science in Cultural Heritage
conservative restoration
Bioactive molecules: innovative contributions of biotechnology to the restoration of cultural heritage
Text.Serial.Journal
https://conservation-science.unibo.it/
13
Articles
2013-12-30
2014-02-09
2013-12-30
2015-11-02
369-373
CSCH
Franco Palla
2013/12/30
10.6092/issn.1973-9494/4191
369
1973-9494
conservative restoration
en
373
PALLA F., CAMMARATA M., TRAPANI M.R., GHERSI G., SALAMONE M., SEBATIANELLI M., 2013, Novel proteases from marine organisms with potential interest in restoration procedure, in Science and Technology for the Conservation of Cultural Heritage CRC Press, Taylor & Francis Group, (October 2013, in press).
Bioactive molecules: innovative contributions of biotechnology to the restoration of cultural heritage
https://conservation-science.unibo.it/article/download/4191/3641
13
B    IOACTIVE MOLECULES: INNOVATIVE CONTRIBUTIONS OF
     BIOTECHNOLOGY TO THE RESTORATION OF CULTURAL HERITAGE



   Franco Palla●1
   Laboratorio di Biologia e Biotecnologie per i Beni Culturali,
   Dipartimento di Scienze e Tecnologie Biologiche,
   Chimiche e Farmaceutiche, Università degli Studi di Palermo



   Keywords: biocleaning, proteases, removal of aged layers, conservative restoration

   1. Introduction

    Since the beginning of the twenty-first century, biotechnology has provided innova-
tive techniques useful in the field of diagnosing biodeterioration in cultural heritage and
in defining conservation/restoration strategies. From the point of view of diagnosis,
molecular biology technology allows sample amounts to be minimised and information
in diagnosing microbial contamination of cultural assets to be optimised, thus reveal-
ing unknown species that have never been cultured [1-4]. This approach, based on
genomic DNA analysis, has also proven to be very useful in revealing and identifying
microbial particles in the bioaerosol of indoor environments that may be related to po-
tential health problems in visitors/professional operators [5-7].
    From another point of view, science and technology have provided and continue to
provide innovative protocols for the biological cleaning (biocleaning) of the surfaces of




                                                                                                    CONSERVATION SCIENCE IN CULTURAL HERITAGE
works of art. It is well known that the action of cleaning represents one of the first and
most important steps in a conservative restoration project. It must be performed care-
fully and in a selective way, making a distinction between different areas, with the aim
of preventing irreversible damage to the surface of the object, and removing deposits
without acting directly on the original materials of the same surface.
    So far many scientific publications have referred that biocleaning, performed by
purified enzymes or living bacteria, seems to meet these standards of accuracy. In par-
ticular, several bacterial strains, with specific cellular activities, have long been used
effectively for remediating polluted environments; in addition, bioreactive molecules
(enzymatic protein or enzymes) isolated from different biological systems may be ap-
plied to remove overlapping organic layers (proteins, oils, starches) from the surface
of historic and artstic works.
    The first biocleaning attempts with enzymes date back to 1970 and were initially
perfomed on paper-based artifacts and polychrome canvases. However, they were
frequently isolated experiences. Wendelbo, reported the enzymatic treatment (trypsin
in phosphate buffer at pH 8.0, for 10 minutes at 40° C) on some pages of a book
pasted together with animal glue [8]. Segal and Cooper [9] applied a double enzymatic
treatment, to remove adhesives composed of starch and protein, using both amylase


● Corresponding author: franco.palla@unipa.it

                                                                                              369
                                                                                                                     (in phosphate buffer pH 7.0, incubating at 38 °C up to 60 minutes ) and protease (in
                                                                                                                     buffer phosphate at pH 7.5 and incubated at 40° C). Makes, in 1982, reported the first
                                                                                                                     application of an amylase/protease mixture to remove glue paste from the back of an
                                                                                                                     oil painting [10] and later, in 1988, the use of a mixture of enzymes to remove a protein/
                                                                                                                     oily binder from a painted surface [11]. Lipases have been applied in different restora-
                                                                                                                     tion procedures regarding the removal of aged acrylic resins (Paraloid B72) used as a
                                                                                                                     coating [12] or oil-based overpaints, by immobilizing the enzymes in aqueous gel [13].
                                                                                                                     Enzymatic decoloration of pigmented spots on marble surfaces produced by Serratia
F. Palla - Bioactive molecules: innovative contributions of biotechnology to the restoration of cultural heritage




                                                                                                                     bacteria, was performed by the fungal enzyme laccase [14].
                                                                                                                         An alternative to cleaning with purified enzymes is the use of live bacterial cells, a
                                                                                                                     potential application first perceived by Atlas in 1988, and later implemented by Sorlini,
                                                                                                                     Ranalli and Cappitelli [15-17]. In particular, the application of bacteria Desulfovibrio
                                                                                                                     desulfuricans (supported by Carbogel) allowed the removal of black crusts, reducing
                                                                                                                     sulphates to hydrogen sulphide. The same research group performed the bio-cleaning
                                                                                                                     of one of the frescoes in the monumental cemetery of Pisa, using Pseudomonas viable
                                                                                                                     bacterial cells (P. stutzeri, A29 strain) able to degrade glue layers, minimizing the risk
                                                                                                                     of pigment alteration, reaching an efficiency of 80-90%. The cleaning was performed
                                                                                                                     on different zones of the fresco, keeping the bacteria on the surface for a time between
                                                                                                                     8-12 hours at 28-30° C. Most of the organic layer was degraded by means of the
                                                                                                                     metabolic activity of the bacteria, while the residual glue was removed by protease
                                                                                                                     (TypeXIX) enzyme solution [18]. In these studies the destructive activity  resulting from
                                                                                                                     the metabolism of the bacterial colonies, was successfully used to reduce sulphate in
                                                                                                                     the black crust or hydrolyze animal glue layers. Bioremoval of animal glue from mural
                                                                                                                     paintings, by P. stutzeri viable bacteria, has recently been reported by Bosch [19].


                                                                                                                       2. Potentially useful new enzymes for biocleaning surfaces of works of art

                                                                                                                         Currently, hydrolases (amylase, protease, esterase) that are commercially available
                                                                                                                     and used for biocleaning  surfaces of works of art have been isolated and purified from
                                                                                                                     terrestrial biosystems and applied at temperatures > 30° C.
                                                                                                                         It is possible to isolate amylase from animal tissues or microbial sources (Bacil-
                                                                                                                     lus sp. or Aspergillus sp.); lipase derived from animal tissues (pancreas), from plant
                                                                                                                     tissues (seeds of oats and wheat) or from microbial species (Bacillus, Aspergillus,
                                                                                                                     Penicillium). Pepsin, trypsin and protease can be extracted from both animal tissues
                                                                                                                     (pancreas, stomach) and microbial cells (Bacillus, Aspergillus).
                                                                                                                         The rational use of a purified enzyme solution or a mixture of them, requires infor-
                                                                                                                     mation on: i) the nature of the material to be removed (proteins, starches, oils, fats,
                                                                                                                     waxes); ii) the hydrolytic activity and specificity of the enzyme action; iii) pH and salt
                                                                                                                     concentrations; iv) temperature. Reaction temperature represents a crucial point in
                                                                                                                     biocleaning protocols since it is neither advisable to use preheated (>30°C) enzyme
                                                                                                                     solutions nor to heat the artifact surface to which it must be applied.
                                                                                                                         In order to avoid undesirable effects on works of art, related to the reaction tem-
                                                                                                                     perature of the enzyme, in our laboratory we are developing biocleaning protocols
                                                                                                                     involving the use of new hydrolases isolated from marine organisms. Marine organ-
                                                                                                                     isms are a potentially rich source of different enzymes with unique properties and with
                                                                                                                     a wide range of interesting applications in the field of scientific research and industry.
                                                                                                                     Two batches of hydrolase, with collagenolytic-proteolytic activity were isolated from


                                                                                                               370
vertebrate and invertebrate marine organisms, and the protease activity tested with
SDS-PAGE or with the Kembhavi method [20, 21].
    The most interesting feature of these enzymes is that they are active in a range
of temperatures between 4° C and 37°C.
    We have recently performed the removal of protein layers (mainly animal glue) both
from ad hoc arranged test specimens (laboratory test) and from polychrome wooden
furniture (Museo Diocesano Palermo), selectively and for short lengths of time (5-10
minutes), performed at a temperature of 22+3° C; a range of “room temperature” (19
to 25.5 °C), which coincided with restoration laboratories or storage environments.
    The cleaning tests showed reliable results, without the heating of the enzyme solu-
tions or of the surfaces on which they were applied [22, 23]. This particular feature
makes these proteases more appropriate than others, usually active at higher tem-
peratures.

      3. Conclusions

    Our hypothesis is that these enzymes will implement the knowledge and the ef-
ficiency of bio-cleaning protocols and can be used to hydrolyze and easily remove
animal glue layers that alter the painted surface of artistic works. This feature consti-
tutes an innovation compared to products currently available and is in accordance
with modern criteria for restoration: compatibility with the original constituent materials;
minimal intervention; actual applicability of the product with a reduction in costs and
operating times.
    The advantages of using enzymes (or living bacteria) as an alternative to traditional
methods lies in the fact that the first generally induce more specific and highly selec-
tive processes and are therefore non-invasive. Typically they act only on target com-
pounds, without attacking molecules other than those for which they are used.




                                                                                                     CONSERVATION SCIENCE IN CULTURAL HERITAGE
    Further research, however, is in progress to complete purification and to determine
the properties of the single components isolated from invertebrate marine organisms.
Moreover, we have also partially purified enzymes with lipolytic activity.
    As well as for proteases, lipases represent a valid alternative to conventional meth-
ods for removing aged siccative oil, as they represent safer working conditions both as
regards the operator’s health and the integrity of the artwork.
    Moreover, purified enzymes which are already active at temperatures below 30°
C, such as those that can be isolated from marine organisms are extremely useful in
projects designed for the restoration of historic-artistic works of art.

      Acknowledgements

    The author wishes to thank all collaborators involved in the isolation and analysis
of enzymes from invertebrate marine organisms used in this work. This study was
developed in the project It@cha, ‘Ricerca e Competitività 2007 - 2013’, PON 01_0625
(UNIPA).

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  Biographical notes

     Franco Palla, is Associate professor of Environmental and Applied Botanical Sci-
ences at the University of Palermo, Italy. He is involved in the Academic Five-Year De-
gree Course in Conservation and Restoration of Cultural Heritage, both as teacher and
Vice-Coordinator. He is a Member of the National Order of Biologists of the Commis-




                                                                                                   CONSERVATION SCIENCE IN CULTURAL HERITAGE
sion for the Protection of Cultural Heritage; Scientific coordinator of UNIPA Research
Unit - Project PON01_00625, IT@CHA (Italian Technology for Advanced Applications
in Cultural Assets). He is one of the members of the working groups of experts in the
Italy-Cambodia Cooperation Project “Training of Cultural Heritage Experts”, University
of Palermo - Royal University of Fine Art and Ministry for Culture and Fine Arts, Ang-
kor, Cambodia. He is a Scientific Consultant in the field of Biological deterioration, in
restoration projects regarding archaeological sites, works of art and historical- artistic
artifacts. Professor Palla is also a member of the Advisory Committee of the Journal
“Conservation Science in Cultural Heritage”. Currently Professor Palla is the scien-
tific coordinator of the Laboratory of Biology and Biotechnology for Cultural Heritage
at the Department of Biological, Chemical, Pharmaceutical Sciences and Technology
(STEBICEF) at the University of Palermo.




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