Exploring the substrate spectrum of phylogenetically distinct bacterial polyesterases

The rapid escalation of plastic waste accumulation presents a significant threat of the modern world, demanding an immediate solution. Over the last years, utilization of the enzymatic machinery of various microorganisms has emerged as an environmentally friendly asset in tackling this pressing global challenge. Thus, various hydrolases have been demonstrated to effectively degrade polyesters. Plastic waste streams often consist of a variety of different polyesters, as impurities, mainly due to wrong disposal practices, rendering recycling process challenging. The elucidation of the selective degradation of polyesters by hydrolases could offer a proper solution to this problem, enhancing the recyclability performance. Towards this, our study focused on the investigation of four bacterial polyesterases, including DaPUase, IsPETase, PfPHOase, and Se1JFR, a novel PETase-like lipase. The enzymes, which were biochemically characterized and structurally analyzed, demonstrated degradation ability of synthetic plastics. While a consistent pattern of polyesters’ degradation was observed across all enzymes, Se1JFR stood out in the degradation of PBS, PLA, and polyether PU. Additionally, it exhibited comparable results to IsPETase, a benchmark mesophilic PETase, in the degradation of PCL and semi-crystalline PET. Our results point out the wide substrate spectrum of bacterial hydrolases and underscore the significant potential of PETase-like enzymes in polyesters degradation

Discovery and Biochemical Characterization of a Novel Polyesterase for the Degradation of Synthetic Plastics

Plastic waste poses an enormous environmental problem as a result of soil and ocean contamination, causing the release of microplastics that end up in humans through the food web. Enzymatic degradation of plastics has emerged as an alternative to traditional recycling processes. In the present work, we used bioinfomatics tools to discover a gene coding for a putative polyester degrading enzyme (polyesterase). The gene was heterologously expressed, purified and biochemically characterized. Furthermore, its ability to degrade polyethylene terephthalate (PET) model substrates and synthetic plastics was assessed

Identification and Characterization of New Laccase Biocatalysts from Pseudomonas Species Suitable for Degradation of Synthetic Textile Dyes

Laccases are multicopper-oxidases with variety of biotechnological applications. While predominantly used, fungal laccases have limitations such as narrow pH and temperature range and their production via heterologous protein expression is more complex due to posttranslational modifications. In comparison, bacterial enzymes, including laccases, usually possess higher thermal and pH stability, and are more suitable for expression and genetic manipulations in bacterial expression hosts. Therefore, the aim of this study was to identify, recombinantly express, and characterize novel laccases from Pseudomonas spp. A combination of approaches including DNA sequence analysis, N-terminal protein sequencing, and genome sequencing data analysis for laccase amplification, cloning, and overexpression have been used. Four active recombinant laccases were obtained, one each from P. putida KT2440 and P. putida CA-3, and two from P. putida F6. The new laccases exhibited broad temperature and pH range and high thermal stability, as well as the potential to degrade selection of synthetic textile dyes. The best performing laccase was CopA from P. putida F6 which degraded five out of seven tested dyes, including Amido Black 10B, Brom Cresol Purple, Evans Blue, Reactive Black 5, and Remazol Brilliant Blue. This work highlighted species of Pseudomonas genus as still being good sources of biocatalytically relevant enzymes

Progressing Plastics Circularity: A Review of Mechano-Biocatalytic Approaches for Waste Plastic (Re)valorization

Inspirational concepts, and the transfer of analogs from natural biology to science and engineering, has produced many excellent technologies to date, spanning vaccines to modern architectural feats. This review highlights that answers to the pressing global petroleum-based plastic waste challenges, can be found within the mechanics and mechanisms natural ecosystems. Here, a suite of technological and engineering approaches, which can be implemented to operate in tandem with nature's prescription for regenerative material circularity, is presented as a route to plastics sustainability. A number of mechanical/green chemical (pre)treatment methodologies, which simulate natural weathering and arthropodal dismantling activities are reviewed, including: mechanical milling, reactive extrusion, ultrasonic-, UV- and degradation using supercritical CO2. Akin to natural mechanical degradation, the purpose of the pretreatments is to render the plastic materials more amenable to microbial and biocatalytic activities, to yield effective depolymerization and (re)valorization. While biotechnological based degradation and depolymerization of both recalcitrant and bioplastics are at a relatively early stage of development, the potential for acceleration and expedition of valuable output monomers and oligomers yields is considerable. To date a limited number of independent mechano-green chemical approaches and a considerable and growing number of standalone enzymatic and microbial degradation studies have been reported. A convergent strategy, one which forges mechano-green chemical treatments together with the enzymatic and microbial actions, is largely lacking at this time. An overview of the reported microbial and enzymatic degradations of petroleum-based synthetic polymer plastics, specifically: low-density polyethylene (LDPE), high-density polyethylene (HDPE), polystyrene (PS), polyethylene terephthalate (PET), polyurethanes (PU) and polycaprolactone (PCL) and selected prevalent bio-based or bio-polymers [polylactic acid (PLA), polyhydroxyalkanoates (PHAs) and polybutylene succinate (PBS)], is detailed. The harvesting of depolymerization products to produce new materials and higher-value products is also a key endeavor in effectively completing the circle for plastics. Our challenge is now to effectively combine and conjugate the requisite cross disciplinary approaches and progress the essential science and engineering technologies to categorically complete the life-cycle for plastics

The use of chitosan in protecting wooden artifacts from damage by mold fungi

Background: Many buildings in Egypt e.g. museums, mosques and churches, do not possess controlled environments for minimizing the risks of damage of wooden artifacts due to the growth of fungi. Fungal damage usually appears as change in wood color, appearance of stains, and sometimes deformation of wooden surfaces. In this study we focused on the effect that some fungi exert on the properties of wooden artifacts and evaluated the effectiveness of different concentrations of chitosan on their protection against damage by mold fungi. Results: Samples were collected from different monuments and environments, and fungi growing on them were isolated and identified. The isolated Penicillium chrysogenum , Aspergillus flavus and Aspergillus niger strains were used for the infestation of new pitch pine samples. The results revealed that the lightness of samples infected with any of the tested fungi decreased with increasing incubation times. XRD analysis showed that the crystallinity of incubated samples treated individually with the different concentrations of chitosan was lower than the crystallinity of infected samples. The crystallinity index measured by the first and the second method decreased after the first and second months but increased after the third and fourth months. This may due to the reducing of amorphous part by enzymes or acids produced by fungi in wooden samples. Conclusions: The growth of fungi on the treated wood samples decreased with increasing the concentration of chitosan. Hence, it was demonstrated that chitosan prevented fungal growth, and its use could be recommended for the protection of archeological wooden artifacts

Supplementary data for the article: Djapovic, M.; Milivojevic, D.; Ilic-Tomic, T.; Lješević, M.; Nikolaivits, E.; Topakas, E.; Maslak, V.; Nikodinovic-Runic, J. Synthesis and Characterization of Polyethylene Terephthalate (PET) Precursors and Potential Degradation Products: Toxicity Study and Application in Discovery of Novel PETases. Chemosphere 2021, 275, 130005. https://doi.org/10.1016/j.chemosphere.2021.130005.

Supplementary material for: [https://doi.org/10.1016/j.chemosphere.2021.130005]Related to published version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1415]Related to accepted version: [https://imagine.imgge.bg.ac.rs/handle/123456789/1732

Supplementary data for: "Synthesis and characterization of polyethylene terephthalate (PET) precursors and potential degradation products: Toxicity study and application in discovery of novel PETases"

Supplementary material for: Djapovic, M.; Milivojevic, D.; Ilic-Tomic, T.; Lješević, M.; Nikolaivits, E.; Topakas, E.; Maslak, V.; Nikodinovic-Runic, J. Synthesis and Characterization of Polyethylene Terephthalate (PET) Precursors and Potential Degradation Products: Toxicity Study and Application in Discovery of Novel PETases. Chemosphere 2021, 275, 130005. [https://doi.org/10.1016/j.chemosphere.2021.130005]Published version of the article: [https://cer.ihtm.bg.ac.rs/handle/123456789/4658]Accepted version of the article: [https://cer.ihtm.bg.ac.rs/handle/123456789/4659

Discovery of novel fungal biocatalysts and their application in bioremediation of chlorinated aromatic pollutants

The present thesis aimed to the discovery and modification of biocatalysts to be applied in the bioremediation of chlorinated aromatic pollutants. Two methods were followed in order to achieve this objective. One is based on the exploitation of microorganisms from poorly studied ecosystems and the study of the mechanisms by which they detoxify xenobiotics. The second method is based on the in silico search and discovery of protein sequences with potential activity on aromatic pollutants and modification of these enzymes by protein engineering tools in order to improve their properties. This research falls into both fields of Environmental Biotechnology and Biocatalysis. In the first part of the thesis, the potential of 104 fungal strains for the biotransformation of two chlorinated aromatic pollutants, namely 2,4-dichlorophenol (2,4-DCP) and 2,4,5-trichlorodiphenyl (PCB29) was studied. These fungi were isolated as symbionts of marine invertebrates, which were collected from different regions of the world and at different depths. Nine of these strains showed high potential for the removal of 1 mM 2,4-DCP at yields above 55%. These strains belonged to the genera Penicillium, Aspergillus, Chrysosporium, Cladosporium and Tritirachium. Metabolite analysis of these strains revealed many compounds, some of which are reported for the first time in the literature, such as the conjugates of 2,4-DCP with glutamine and cysteine, as well as dichlorocatechol palmitate and sulfated chlorophenol. In every case, metabolites were less toxic than the original compound. Most strains were able to partially or completely dechlorinate the pollutant leading to hydroxyquinol. Furthermore, Tritirachium strain was able to cleave the benzene ring by expressing an enzyme with catechol 1,2-dioxygenase (C12O) activity, producing 2-hydroxy-muconic acid, effectively assimilating the pollutant. The enzyme with C12O activity that was isolated from the extracellular culture medium of Tritirachium sp., was biochemically characterized, presenting very interesting features. Upon proteomic analysis, it was revealed that this enzyme is in fact a catalase with C12O activity. This is the first time in the literature that such double catalytic action is mentioned. Additionally, as a C12O, this enzyme could cleave 4-chlorocatechol in addition to catechol and hydroxyquinol, which makes it very attractive in bioremediation applications. The isolated marine-derived fungi were further investigated regarding their ability to remove PCB29 in solutions saturated with the pollutant. Out of the 104 strains tested, 8 were able to remove the pollutant almost entirely (>98.5%) and belonged to the genera Penicillium, Aspergillus, Purpureocillium, Cladosporium and Alternaria. During the removal of PCB29 the studied strains did not express dihydroxyphenyl 2,3-dioxygenase, catechol dioxygenase or alkane dehalogenase activities. However, one Cladosporium strain expressed laccase activity at much higher levels than the rest of the strains. Two laccase isozymes were isolated from the extracellular liquid of this strain after cultivation in a 12-L batch bioreactor. These enzymes exhibited similar biochemical characteristics, in terms of their optimum activity conditions and substrate scope. One of the two, however, showed the ability of PCB29 removal by 71% in the presence of a mediator. In the second part of the thesis, the in silico discovery, cloning and expression of a polyphenoloxidase (PPO) gene from the fungus Thermothelomyces thermophila in Pichia pastoris heterologous expression system was performed. After extensive biochemical characterization of the isolated enzyme, its use in the removal of various mono- and dichlorophenols was tested. In order to study both the effect of specific amino acid residues on the enzyme's stereoselectivity and to increase its catalytic activity in dichlorophenols, its modification was performed by point mutations. Of the five variants that were constructed, one showed 5fold increase of oxidation activity on 3,5-DCP, with only two point mutations.Η παρούσα διδακτορική διατριβή στόχευσε στην ανακάλυψη και τροποποίηση βιοκαταλυτών με εφαρμογή στη βιοεξυγίανση χλωριωμένων αρωματικών ρύπων. Για την επίτευξη του στόχου αυτού ακολουθήθηκαν δύο μέθοδοι. Η μία βασίζεται στην εκμετάλλευση μικροοργανισμών που προέρχονται από ελάχιστα μελετημένα οικοσυστήματα και στη μελέτη των μηχανισμών με τους οποίους αυτοί αποτοξικοποιούν ξενοβιοτικά. Η δεύτερη μέθοδος βασίζεται στην in silico αναζήτηση και ανακάλυψη πρωτεϊνικών αλληλουχιών με πιθανότητα δράσης σε αρωματικούς ρύπους και η τροποποίηση των ενζύμων αυτών με εργαλεία πρωτεϊνικής μηχανικής, για τη βελτίωση των ιδιοτήτων τους. Η έρευνα αυτή εμπίπτει τόσο στο πεδίο της Περιβαλλοντικής Βιοτεχνολογίας και στο πεδίο της Βιοκατάλυσης. Στο πρώτο κομμάτι της διατριβής μελετήθηκε το δυναμικό 104 μυκητιακών στελεχών για τη βιομετατροπή δύο χλωριωμένων αρωματικών ρύπων, της 2,4-διχλωροφαινόλης (2,4-DCP) και του 2,4,5-τριχλωροδιφαινυλίου (PCB29). Οι μύκητες αυτοί είχαν απομονωθεί ως συμβιώτες θαλάσσιων ασπόνδυλων, τα οποία είχαν συλλεχθεί από διαφορετικές περιοχές του κόσμου και διαφορετικά βάθη. Από τα στελέχη αυτά, 9 έδειξαν υψηλή δυνατότητα απομάκρυνσης 1 mM 2,4-DCP, σε ποσοστά υψηλότερα του 55%. Τα στελέχη αυτά ανήκαν στα γένη Penicillium, Aspergillus, Chrysosporium, Cladosporium και Tritirachium. Η ανάλυση μεταβολιτών των στελεχών αυτών αποκάλυψε πολλούς μεταβολίτες, κάποιοι εκ των οποίων αναφέρονται για πρώτη φορά στη βιβλιογραφία, όπως τα συζεύγματα της 2,4-DCP με γλουταμίνη και κυστεΐνη, καθώς επίσης και ο παλμιτικός εστέρας της διχλωροκατεχόλης και η θειωμένη χλωροφαινόλη. Σε κάθε περίπτωση όλοι οι μεταβολίτες ήταν λιγότερο τοξικοί από την αρχική ένωση. Τα περισσότερα στελέχη μπόρεσαν να αποχλωριώσουν μερικώς ή πλήρως το ρύπο παράγοντας υδροξυκινόλη. Μάλιστα, το στέλεχος Tritirachium είχε τη δυνατότητα να πραγματοποιεί διάνοιξη του βενζολικoύ δακτυλίου, με την έκφραση ενός ενζύμου με δράση 1,2-διοξυγενάσης της κατεχόλης (C12O), παράγοντας υδροξυ-μουκονικό οξύ, αφομοιώνοντας πρακτικά το ρύπο.Το ένζυμο με ενεργότητα C12O απομονώθηκε από το εξωκυτταρικό υγρό του μύκητα και χαρακτηρίστηκε βιοχημικά, παρουσιάζοντας πολύ ενδιαφέροντα χαρακτηριστικά. Κατά την πρωτεομική ανάλυση, αποκαλύφθηκε ότι το ένζυμο αυτό είναι στην πραγματικότητα καταλάση με δράση C12O. Η διπλή αυτή καταλυτική δράση αναφέρεται για πρώτη φορά στη παγκόσμια βιβλιογραφία. Επίσης, ως C12O, το ένζυμο αυτό μπορούσε να διασπάσει και την 4-χλωροκατεχόλη εκτός από την κατεχόλη και την υδροξυκινόλη, γεγονός που το κάνει πολύ ελκυστικό σε εφαρμογές βιοεξυγίανσης. Στη συνέχεια της έρευνας αναφορικά με τους θαλάσσια προερχόμενους μύκητες, μελετήθηκε η δυνατότητά τους να απομακρύνουν το PCB29 σε διαλύματα κορεσμένα με το ρύπο. Από τα 104 στελέχη, 8 κατάφεραν να απομακρύνουν το ρύπο σχεδόν εξολοκλήρου (>98.5%), τα οποία ανήκαν στα γένη Penicillium, Aspergillus, Purpureocillium, Cladosporium και Alternaria. Κατά τη διαδικασία απομάκρυνσης του ρύπου, κανένα από τα μελετούμενα στελέχη δεν εξέφραζε ενεργότητες διοξυγενασών του 2,3-διυδροξυ-διφαινυλίου, διοξυγενασών της κατεχόλης ή αφαλογoνάσης των αλκανίων. Ένα, όμως, στέλεχος Cladosporium εξέφραζε ενεργότητα λακκάσης σε επίπεδα πολύ υψηλότερα από τα υπόλοιπα στελέχη. Από το εξωκυτταρικό υγρό του στελέχους αυτού, πραγματοποιήθηκε απομόνωση δύο ισοενζύμων λακκάσης, ύστερα από καλλιέργεια σε βιοαντιδραστήρα διαλείποντος έργου 12 L. Τα ένζυμα αυτά παρουσίασαν παρόμοια βιοχημικά χαρακτηριστικά, αναφορικά με τις βέλτιστες συνθήκες δράσης τους και το εύρος των υποστρωμάτων που μπορούν να οξειδώσουν. Το ένα από τα δύο όμως παρουσίασε τη δυνατότητα απομάκρυνσης του PCB29 σε ποσοστό 71% σε αντιδράσεις παρουσία μεσολαβητή. Στο δεύτερο τμήμα της διατριβής πραγματοποιήθηκε η in silico ανακάλυψη, κλωνοποίηση και έκφραση ενός γονιδίου πολυφαινολοξειδάσης (PPO) από τoν μύκητα Thermothelomyces thermophila στο ετερόλογο σύστημα Pichia pastoris. Ύστερα από εκτενή βιοχημικό χαρακτηρισμό του απομονωμένου ενζύμου, δοκιμάστηκε η χρήση του στην απομάκρυνση διαφόρων μονο- και δι-χλωροφαινολών. Με στόχο τόσο τη μελέτη της επίδρασης συγκεκριμένων αμινοξέων στην στερεοεκλεκτικότητα του ενζύμου, όσο και την αύξηση της καταλυτικής του ενεργότητας σε διχλωροφαινόλες, πραγματοποιήθηκε η τροποποίησή του με σημειακές μεταλλάξεις. Από τα πέντε τροποποιημένα ένζυμα που κατασκευάστηκαν, στο ένα η ενεργότητα στην οξείδωση της 3,5-DCP πενταπλασιάστηκε με μόνο δύο μεταλλάξεις

Engineering of the heterologous expression and characterization of a cutinase from the fungus Fusarium oxysporum

129 σ.Σκοπός της παρούσας διπλωματικής εργασίας ήταν η ετερόλογη έκφραση μιας κουτινάσης (FoCut16606) του μεσόφιλου μύκητα Fusarium oxysporum σε τρεις διαφορετικούς ξενιστές, με στόχο τη βελτίωση της σταθερότητας και αποδοτικότητας του ανασυνδυασμένου βιοκαταλύτη. Αρχικά, η FoCut16606 εκφράσθηκε στο βακτήριο Escherichia coli BL21, το οποίο είχε μετασχηματισθεί με τον πλασμιδιακό φορέα pET22b(+) που έφερε το αντίστοιχο γονίδιο (foxg_16606.3). Η ανασυνδυασμένη πρωτεΐνη παρουσίασε πολύ μικρή θερμοσταθερότητα ακόμα και σε χαμηλές θερμοκρασίες επώασης. Με στόχο τη βελτίωση της σταθερότητας, η αναδίπλωση του ενζύμου πραγματοποιήθηκε στον περιπλασμικό χώρο των E. coli BL21 χρησιμοποιώντας τη σηματοδοτική αλληλουχία pelB. Η περιπλασμική έκφραση δίνει τη δυνατότητα δημιουργίας δισουλφιδικών δεσμών, που κρίνονται απαραίτητοι για την αύξηση της σταθερότητας. Παρόλο που η ανασυνδυασμένη κουτινάση παρουσίασε βελτιωμένες ιδιότητες, δεν θεωρήθηκε ικανοποιητική για εφαρμογές στη Βιομηχανία. Για την δημιουργία δισουλφιδικών δεσμών στο κυτταροπλασματικό περιβάλλον, χρησιμοποιήθηκε ως ξενιστής το βακτηριακό στέλεχος E. coli Origami2. Παρότι δοκιμάστηκε και η περιπλασμική έκφραση του ενζύμου, χρησιμοποιώντας τη σηματοδοτική αλληλουχία pelB, το ένζυμο που παρήχθη ενδοκυτταρικά βρέθηκε σταθερότερο. Δεν υφίστατο απώλειες ενεργότητας στους 20 οC, ενώ διατήρησε λιγότερο από το 20% της αρχικής του ενεργότητας ύστερα από 4 h στους 40 οC. Ο εντοπισμός ενεργότητας κουτινάσης σε όλα τα προαναφερθέντα συστήματα έκφρασης μελετήθηκε τόσο στον εξωκυτταρικό χώρο, όσο και στον εσωκυτταρικό ως διαλυτή πρωτεΐνη ή έγκλειστα σωμάτια (inclusion bodies), σε δύο διαφορετικές θερμοκρασίες επαγωγής (37 και 16 οC). Στις παραγωγές που πραγματοποιήθηκαν στους 37 οC η πλειοψηφία της ενεργότητας κουτινάσης εντοπίστηκε στα inclusion bodies, ενώ στους 16 οC στο κυτταρόπλασμα. Ύστερα από απομόνωση, τα ανασυνδυασμένα ένζυμα ( ̴23 kDa) χρησιμοποιήθηκαν για κινητικές μελέτες σε τρεις εστέρες της παρα-νιτροφαινόλης με διαφορετικό μήκος αλυσίδας – C2, C4 και C12. Με κριτήριο την κινητική σταθερά Km, τα ανασυνδυασμένα ένζυμα έδειξαν μεγαλύτερη συγγένεια με τον εστέρα με τα 4 άτομα άνθρακα. Η θερμοσταθερότητα της FoCut16606 αυξήθηκε σημαντικά, όταν αυτή εκφράσθηκε σε ευκαρυωτικό ξενιστή, τη μεθυλότροφη ζύμη Pichia pastoris η οποία δίνει τη δυνατότητα μετα-μεταφραστικών τροποποιήσεων, όπως σχηματισμό δισουλφιδικών δεσμών και γλυκοζυλίωση. Τα κύτταρα της P. pastoris X33 μετασχηματίσθηκαν με τον πλασμιδιακό φορέα pPICZαC, ο οποίος έφερε το γονίδιο foxg_16606.3 ρυθμιζόμενο από τον υποκινητή της αλκοολικής οξειδάσης (AOX1), καθώς και την αλληλουχία α-factor (προέλευση από τη ζύμη Saccharomyces cerevisiae) για την έκκριση της ανασυνδυασμένης πρωτεΐνης εξωκυτταρικά. Το ανασυνδυασμένο αυτό ένζυμο παρέμενε σταθερό στους 30 οC, ενώ έχανε το 40% τις αρχικής του ενεργότητας στους 55 οC και το 80% στους 70 οC, ύστερα από 4 h επώαση. Στη συνέχεια πραγματοποιήθηκε έρευνα της επίπτωσης διαφόρων οργανικών διαλυτών στην ενζυμική ενεργότητα των ανασυνδυασμένων πρωτεϊνών. Όλες έδειξαν αντοχή στην επώαση στους διαλύτες και περισσότερο το προϊόν της περιπλασμικής έκφρασης των BL21. Αυτή η ιδιότητα καθιστά την FoCut16606 κατάλληλη για εφαρμογές σε μη συμβατικά συστήματα.The purpose of the present diploma thesis was the heterologous expression of a cutinase (FoCut16606) from the mesophilic fungus Fusarium oxysporum in three different hosts, in order to improve the stability and efficiency of the recombinant biocatalyst. Initially, FoCut16606 was expressed in Escherichia coli BL21, which was transformed with the plasmid vector pET22b(+) carrying the corresponding gene (foxg_16606.3). The recombinant protein showed very little thermal stability even at low incubation temperatures. To overcome temperature sensitivity, the expression took place in the perisplasmic space of E. coli BL21 using the signal peptide pelB. Periplasmic expression allows the formation of disulfide bonds which are deemed necessary to increase stability. Although the recombinant cutinase displayed improved properties, it was not considered satisfactory for industrial applications. For the creation of disulfide bonds in the cytosolic environment, bacterial strain E. coli Origami2 was subsequently used as the host. Although periplasmic expression was also tested, using pelB signal peptide, the enzyme produced intracellularly was more stable. It suffered no activity losses at 20 οC and retained less than 20% of its initial activity after a 4-hour incubation at 40 οC. The occurrence of cutinase activity in all the aforementioned expression systems was examined both in the extracellular and intracellular space, as soluble protein or inclusion bodies, in two different temperatures (37 και 16 οC). Most of the cutinase activity was detected in inclsion bodies at 37 οC expression, while at 16 οC it was mainly found in the cytosol. After the purification of the recombinant enzymes ( ̴23 kDa) kinetic studies were performed using three synthetic esters with different carbon chain length – C2, C4 και C12. Judging by the values of Km kinetic constant, the enzymes showed higher affinity with the C4 ester. The thermostability of FoCut16606 increased significantly when it was expressed in an eukaryotic host, the methylotrophic yeast Pichia pastoris. The latter is capable of performing post-translational modifications such as disulfide bond formation and glycosylation. P. pastoris X33 cells were transformed by the vector pPICZαC carrying foxg_16606.3 gene, regulated by the alcohol oxidase promoter (AOX1) and the a-factor sequence (from the yeast Saccharomyces cerevisiae) for extracellular secretion of the recombinant protein. This enzyme remained stable during incubation at 30 οC and lost 40% kai 80% of its initial activity after a 4-hour incubation in 55 οC and 70 οC respectively. Finally, the impact of various organic solvents on the enzymatic activity of recombinant proteins was investigated. All enzymes showed high tolerance in the solvents used, but the product of the periplasmic expression in BL21 seemed to be the most tolerant. This property of FoCut16606 makes it suitable for application in non-conventional media.Ευστράτιος Λ. Νικολάιβιτ

A Middle-Aged Enzyme Still in Its Prime: Recent Advances in the Field of Cutinases

Cutinases are α/β hydrolases, and their role in nature is the degradation of cutin. Such enzymes are usually produced by phytopathogenic microorganisms in order to penetrate their hosts. The first focused studies on cutinases started around 50 years ago. Since then, numerous cutinases have been isolated and characterized, aiming at the elucidation of their structure⁻function relations. Our deeper understanding of cutinases determines the applications by which they could be utilized; from food processing and detergents, to ester synthesis and polymerizations. However, cutinases are mainly efficient in the degradation of polyesters, a natural function. Therefore, these enzymes have been successfully applied for the biodegradation of plastics, as well as for the delicate superficial hydrolysis of polymeric materials prior to their functionalization. Even though research on this family of enzymes essentially began five decades ago, they are still involved in many reports; novel enzymes are being discovered, and new fields of applications arise, leading to numerous related publications per year. Perhaps the future of cutinases lies in their evolved descendants, such as polyesterases, and particularly PETases. The present article reviews the biochemical and structural characteristics of cutinases and cutinase-like hydrolases, and their applications in the field of bioremediation and biocatalysis