EstDZ3:a new esterolytic enzyme exhibiting remarkable thermostability

Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure at elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing a functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring after in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retained high levels of catalytic activity after exposure to temperatures as high as 95 oC for several hours. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modelling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold, which seems to include a subdomain insertion. This insertion is similar to the one present in its closest homologue of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a wide range of metal ions, organic solvents, and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications

EstDZ3: A New Esterolytic Enzyme Exhibiting Remarkable Thermostability

Lipolytic enzymes that retain high levels of catalytic activity when exposed to a variety of denaturing conditions are of high importance for a number of biotechnological applications. In this study, we aimed to identify new lipolytic enzymes, which are highly resistant to prolonged exposure at elevated temperatures. To achieve this, we searched for genes encoding for such proteins in the genomes of a microbial consortium residing in a hot spring located in China. After performing a functional genomic screening on a bacterium of the genus Dictyoglomus, which was isolated from this hot spring after in situ enrichment, we identified a new esterolytic enzyme, termed EstDZ3. Detailed biochemical characterization of the recombinant enzyme, revealed that it constitutes a slightly alkalophilic and highly active esterase against esters of fatty acids with short to medium chain lengths. Importantly, EstDZ3 exhibits remarkable thermostability, as it retained high levels of catalytic activity after exposure to temperatures as high as 95 oC for several hours. Interestingly, EstDZ3 was found to have very little similarity to previously characterized esterolytic enzymes. Computational modelling of the three-dimensional structure of this new enzyme predicted that it exhibits a typical α/β hydrolase fold, which seems to include a subdomain insertion. This insertion is similar to the one present in its closest homologue of known function and structure, the cinnamoyl esterase Lj0536 from Lactobacillus johnsonii. As it was found in the case of Lj0536, this structural feature is expected to be an important determinant of the catalytic properties of EstDZ3. The high levels of esterolytic activity of EstDZ3, combined with its remarkable thermostability and good stability against a wide range of metal ions, organic solvents, and other denaturing agents, render this new enzyme a candidate biocatalyst for high-temperature biotechnological applications

Discovery and characterization of a thermostable and highly halotolerant GH5 cellulase from an Icelandic hot spring isolate

Journal ArticleCopyright: © 2016 Zarafeta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.This work has been carried out in the framework of the HotZyme Project (http://hotzyme.com, grant agreement no. 265933) financed by the European Union 7th Framework Programme FP7/2007-2013, an EU FP7 Collaborative programme

ANASTASIA: An Automated Metagenomic Analysis Pipeline for Novel Enzyme Discovery Exploiting Next Generation Sequencing Data

Metagenomic analysis of environmental samples provides deep insight into the enzymatic mixture of the corresponding niches, capable of revealing peptide sequences with novel functional properties exploiting the high performance of next-generation sequencing (NGS) technologies. At the same time due to their ever increasing complexity, there is a compelling need for ever larger computational configurations to ensure proper bioinformatic analysis, and fine annotation. With the aiming to address the challenges of such an endeavor, we have developed a novel web-based application named ANASTASIA (automated nucleotide aminoacid sequences translational plAtform for systemic interpretation and analysis). ANASTASIA provides a rich environment of bioinformatic tools, either publicly available or novel, proprietary algorithms, integrated within numerous automated algorithmic workflows, and which enables versatile data processing tasks for (meta)genomic sequence datasets. ANASTASIA was initially developed in the framework of the European FP7 project HotZyme, whose aim was to perform exhaustive analysis of metagenomes derived from thermal springs around the globe and to discover new enzymes of industrial interest. ANASTASIA has evolved to become a stable and extensible environment for diversified, metagenomic, functional analyses for a range of applications overarching industrial biotechnology to biomedicine, within the frames of the ELIXIR-GR project. As a showcase, we report the successful in silico mining of a novel thermostable esterase termed “EstDZ4” from a metagenomic sample collected from a hot spring located in Krisuvik, Iceland

Discovery and characterisation of novel thermostable hydrolases through high-throughput metagenomic screening

Hydrolases are an enzyme class that catalyze the modification of a wide range ofsubstrates and can be employed in various industrial biotransformations towards theproduction of a plethora of value-added products. Their use can substitute the classicharsh chemical treatments used in industry with more eco-friendly procedures as theyrequire milder operation conditions and offer high precision transformations due to theenzyme’s highly selective nature. Even though the advantages of using enzymes inindustrial biotechnology are many, their use is restricted by the fact that commonbiocatalysts cannot function under high temperatures and other harsh conditions thatoccur in industrial environments. The cost involved in material cooling and eliminationof other denaturing conditions before adding the biocatalyst in order to protect it fromdeactivation is often prohibitive and this is the reason why enzymes do not take overthe chemical treatments very often. The employment of thermostable and overall stablebiocatalysts can alleviate the need for cooling and elimination of denaturing conditions,making enzymic biotransformations economically viable. For this reasons,biotechnologists have been focusing their efforts on the discovery of thermostablehydrolases. In nature, such enzymes can be found in the protein toolbox ofextremophiles, the organisms that have been adapted to extreme conditions throughevolutionary pressure. Due to the fact that 99% of the microbial biodiversity cannot becultivated using standard laboratory techniques, for years, the vast majority ofextremophiles and their proteins have remained unexplored. Recently, biotechnologistshave gained access to this enormous protein space through metagenomic analysis which overcomes the bottleneck of microbiological cultivation. Within this thesis, which was carried out in the framework of the multinational “Hotzyme” consortium, bioinformatic analysis and in vivo functional screening of metagenomic libraries originating from extreme environments, led to the discovery and characterisation of three novel and thermostable hydrolases. The corresponding genes coding for these enzymes were isolated from metagenomic DNA and the recombinant proteins were produced in bacterial Ε. coli cells, followed by purification and biochemical characterisation. Briefly, the first enzyme that was isolated and studied is the CelDZ1 cellulase. CelDZ1 was retrieved through bioinformatic screening of an enrichment sample originating from an Icelandic hot spring. CelDZ1 is a GH5 cellulase, with a pHoptimum at 5 and a temperature optimum at 70 oC. Interestingly, CelDZ1 is hyperhalostable and halotolerant as it is able to maintain high catalytic activity at near saturating salt concentrations. In cooperation with our colleagues, the crystal structure of CelDZ1 was studied and revealed that the new enzyme lacks the defined sugar binding subsite which is present in all homologues proteins. The second enzyme that was isolated and characterized within this study, is the EstDZ2 esterase, which was retrieved through bioinformatic screening of a Russian hot spring metagenome. The new esterase is thermostable with a half-life of more than six hours at 60 oC, and exhibits exceptional stability against high concentrations of organic solvents. Phylogenetic analysis revealed that EstDZ2 opens a new family of lipolytic enzymes termed Family XV, which bares the catalytic motif GHSAG. The study of its structural model implied that EstDZ2 is missing the largest part of the “protein lid” present in the structures of all members of the closest IV family. Finally, the third enzyme of this thesis is the EstDZ3 esterase. EstDZ3 is hyperthermostable as it retains its catalytic activity after several hours of incubation at nearboiling temperatures. The corresponding gene coding for this new enzyme was retrieved through in vivo functional screening of the genetic material of a bacterial strain isolated from an in situ enrichment of a Chinese hot spring. Apart from the exceptional thermostability, EstDZ3 also exhibits great stability against high concentrations of organic solvents. Furthermore, the study of EstDZ3’s structural model revealed that the enzyme bares a “subdomain insertion” similar to the one reported for the closest structural homologue that is expected to have a determinant role for EstDZ3’s catalytic properties. All three hydrolases, exhibit low homology to known and characterized proteins, a fact that combined with their interesting biochemical profile renders them not only candidate enzymes for biotechnological applications but also molecular models forstudying the thermostabilization mechanisms of proteins and more.Oι υδρολάσες είναι μια οικογένεια ενζύμων που καταλύουν την υδρόλυση ενόςμεγάλου εύρους υποστρωμάτων και μπορούν να χρησιμοποιηθούν στη βιομηχανικέςβιομετατροπές προς την παραγωγή πληθώρας προϊόντων υψηλής προστιθέμενης αξίας. Η χρήση τους δύναται να αντικαταστήσει τις καθαρά χημικές μεθόδους επεξεργασίαςτων υλικών καθώς απαιτεί ηπιότερες συνθήκες, και είναι αποδοτικότερη καιακριβέστερη λόγω της υψηλής εξειδίκευση των ενζύμων, οδηγώντας έτσι σε μια πιο «πράσινη» βιομηχανία. αρότι τα πλεονεκτήματα χρήσης των υδρολασών στη βιομηχανία είναι πολλά, η χρήση τους περιορίζεται από το γεγονός ότι οι συμβατικοί βιοκαταλύτες καθίστανται ανενεργοί στις υψηλές θερμοκρασίες και σε άλλες «αντίξοες» σύνηθες που συχνά επικρατούν στις βιομηχανικές διεργασίες. Το κόστος ψύξης και απομάκρυνσης των αποδιατακτικών παραγόντων πριν την προσθήκη των ενζύμων προς αποφυγή της αποδιάταξής τους, είναι τις περισσότερες φορές απαγορευτικό και αυτός είναι ο λόγος που περιορίζει την αντικατάσταση των κλασσικών χημικών κατεργασιών από ενζυμικές μετατροπές. ια λύση σε αυτό το πρόβλημα, που καθιστά τις ενζυμικές διεργασίες οικονομικά βιώσιμες, αποτελεί η χρήση θερμοσταθερών και εν γένει σταθερών υδρολασών. Για το λόγο αυτό, το ενδιαφέρον των βιοτεχνολόγων έχει στραφεί τα τελευταία χρόνια στην ανακάλυψη θερμοσταθερών βιοκαταλυτών. Στη φύση, τα ένζυμα αυτά απαντώνται στην πρωτεϊνική φαρέτρα των ακραιόφιλων οργανισμών, των οργανισμών δηλαδή που έχουν προσαρμοστεί σε ακραία περιβάλλοντα μέσω της εξελικτικής πίεσης. Επειδή όμως το 99% της μικροβιακής βιοποικιλότητας δεν μπορεί να καλλιεργηθεί στο εργαστήριο, για χρόνια, η συντριπτική πλειοψηφία των οργανισμών αυτών και των πρωτεϊνών τους παρέμεινε ανεξερεύνητη. Τα τελευταία χρόνια, μέσω της μεταγονιδιωματικής ανάλυσης που απαλείφει την ανάγκη της μικροβιολογικής καλλιέργειας, οι βιοτεχνολόγοι απέκτησαν πρόσβαση στον τεράστιο πρωτεϊνοχώρο των ακραιόφιλων οργανισμών. Σε αυτή τη διατριβή, η οποία εκπονήθηκε στο πλαίσιο της διεθνούς κοινοπραξίας “Hotzyme”, η βιοπληροφορικής και η in vivo λειτουργικής σάρωσης μεταγονιδιωματικών βιβλιοθηκών που προέκυψαν από δειγματοληψίες ακραίων ενδιαιτημάτων, οδήγησε στην ανακάλυψη τριών νέων θερμοσταθερών υδρολασών. Τα αντίστοιχα γονίδια που κωδικοποιούν για αυτές τις υδρολάσες εντοπίστηκαν και ανακτήθηκαν από τις μεταγονιδιωματικές βιβλιοθήκες, και μετά από ετερόλογη έκφραση τους σε βακτηριακά κύτταρα E. coli, τα ανασυνδυασμένη ένζυμααπμονώθηκαν και μελετήθηκαν βιοχημικά. Συνοπτικά, το πρώτο ένζυμο που απομονώθηκε είναι η θερμοανθεκτική κυτταρινάση CelDZ1. Η CelDZ1 προέκυψε από τη βιοπληροφορική σάρωση δείγματος εμπλουτισμού σλανδικής θερμοπηγής και ανήκει στη οικογένεια GH5. Η CelDZ1 είναι μια θερμοανθεκτική, οξεόφιλη κυτταρινάση με βέλτιστες συνθήκες δράσης το pH 5 και τους 70 oC. αράλληλα, η νέα κυτταρινάση παρουσιάζει εντυπωσιακή αλοανθεκτικότητα και αλοσταθερότητα αφού διατηρεί την ενεργότητα της σε συγκεντρώσεις αλάτων που τείνουν στον κορεσμό. Σε συνεργασία με τους εταίρους μας μελετήθηκε η κρυσταλλική της δομή η οποία αποκάλυψε ότι η CelDZ1 διαφέρει από τις ομόλογες πρωτεΐνες καθώς υπολείπεται της υποπεριοχής-2 πρόσδεσης των σακχαριτών. Το δεύτερο ένζυμο που απομονώθηκε και μελετήθηκε σε αυτή τη διατριβή είναι η εστεράση EstDZ2, η οποία προέκυψε μέσω βιοπληροφορικής σάρωσης του μεταγονιδιωματικού υλικού ωσικής θερμοπηγής. Η νέα εστεράση είναι θερμοσταθερή, με χρόνο ημιζωής πάνω από έξι ώρες στους 60 οC και παρουσιάζει εξαιρετική σταθερότητα σε υψηλές συγκεντρώσεις οργανικών διαλυτών. Η φυλογενετική μελέτη της νέας εστεράσης έδειξε ότι η EstDZ2 εγκαινιάζει μια νέα οικογένεια λιπολυτικών ενζύμων που φέρει το χαρακτηριστικό καταλυτικό αμινοξικό μοτίβο GHSAG, την οποία και ονομάσαμε ικογένεια XV. αράλληλα, η μελέτη του μοντέλου της τρισδιάστατης δομής της EstDZ2 έδειξε ότι πιθανότατα το νέο ένζυμο να υπολείπεται μεγάλο μέρος της χαρακτηριστικής δομής του «πρωτεϊνικού καπακιού» που συναντάται στις δομές των μελών της κοντινότερης οικογένειας V των λιπολυτικών ενζύμων.Τέλος, το τρίτο ένζυμο που απομονώθηκε και μελετήθηκε σε αυτή τη διατριβήείναι η εστεράση EstDZ3, ένα υπερ-θερμοσταθερό ένζυμο που διατηρεί τηνκαταλυτική του δράση ακόμα και μετά από πολύωρη επώαση σε θερμοκρασίες κοντά στο βρασμό. H νέα εστεράση εντοπίστηκε μετά από in vivo λειτουργική σάρωση του γενετικού υλικού βακτηριακού στελέχους που απομονώθηκε από δείγμα εμπλουτισμού κινεζικής θερμοπηγής. έρα από την εξαιρετική θερμοσταθερότητα της, η ΕstDZ3 παρουσιάζει σταθερότητα και σε υψηλές συγκεντρώσεις οργανικών διαλυτών. Παράλληλα, η μελέτη του μοντέλου της δομής της, έδειξε ότι πρόκειται για ένα ένζυμο που φέρει ένα πρωτεϊνικό ένθετο της δομής α/β, παρόμοιο με αυτό της πλησιέστερης δομικά πρωτεΐνης, που προβλέπεται να παίζει καθοριστικό ρόλο στην εκλεκτικότητα του ενζύμου. Και τα τρία νέα ένζυμα παρουσιάζουν χαμηλή ομολογία με ήδη γνωστές και χαρακτηρισμένες πρωτεΐνες κάτι που σε συνδυασμό με τα ενδιαφέροντα βιοχημικά και δομικά τους χαρακτηριστικά τα καθιστά τόσο υποψήφιους βιοκαταλύτες γιαβιοτεχνολογικές εφαρμογές όσο και μοριακά μοντέλα για τη μελέτη της θερμοσταθερότητας των πρωτεϊνών και όχι μόνο

Enzymatic esterification of cellulose substrates in non conventional media

90 σ.Ενζυμική Εστεροποίηση Κυτταρινικών Υποστρωμάτων σε μη συμβατικά Συστήματα (όπως ιοντικά υγρά, solvent free αντιδράσεις κ.λπ)Enzymatic esterification of cellulose substrates in non conventional media. Pretreated cellulose in ionic liquids and esterification in solvent free reactions.Δήμητρα Π. Ζαραφέτ

Berlin-APC: A Privacy-Friendly Dataset for Automated Passenger Counting in Public Transport

This document provides a short technical introduction to the Berlin-APC dataset. The dataset consists of two files, a HDF5 file which contains the image sequences, and a CSV file which contains the labels. The CSV file has three columns: (1) the image sequence name; (2) the number of boarding passengers in that image sequence; (3) the number of alighting passengers in that image sequence. The image sequence names also serve as keys in the HDF5 file. The HDF5 file’s datasets (indexed by the aforementioned sequence names) are float16 arrays of the shape (number of frames, 20, 25), the pixel values range between 0–1, and the frame rate is 10 frames per second

Effect of pH and temperature on the activity of CelDZ1.

<p><b>(A)</b> CelDZ1 activity was measured in the standard reaction at 40°C for 5 min at pH values ranging from 4 to 10 and <b>(B)</b> at temperatures between 40 and 90°C for 5 min in a pH 5 buffer. The reported values correspond to the mean value from three independent experiments performed in triplicate and the error bars to one standard deviation from the mean value.</p

The structure of CelDZ1.

<p><b>(A)</b> Folding of the CelDZ1α monomer is presented as a cartoon diagram and viewed from the solvent region towards the active site groove formed by the C-terminal ends of the β-strands of the (β/α)₈-barrel. The α-helices, β-strands and loops are coloured in turquoise, magenta and pink, respectively. The carbohydrate-binding module (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146454#pone.0146454.g001" target="_blank">Fig 1</a>), which contains helix α8 at the C terminus, is highlighted in green. The two catalytic residues are shown as stick models and secondary structural elements are labelled. The Met50 indicates the position of the first N-terminal residue defined in the electron density. The image was prepared using PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146454#pone.0146454.ref040" target="_blank">40</a>]. <b>(B)</b> A stereo representation of the superimposition of the monomers of CelDZ1, CelK and Cel5a displayed as grey carbon traces. The three different insertion regions are highlighted in red for CelDZ1, magenta for CelK and green for Cel5a. The cellobiose ligand bound to CelK is shown as a magenta stick model. The image was prepared using PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146454#pone.0146454.ref040" target="_blank">40</a>]. <b>(C)</b> The electrostatic potential surface of the CelDZ1 enzyme around the active site groove as viewed from the solvent region. The positive charge is shown in blue and the negative charge is shown in red. The extended active site groove, which crosses the monomer from left to right, is negatively charged disfavoring the binding of halogen ions thereby increasing halotolerance. The image was prepared with ccp4mg [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146454#pone.0146454.ref041" target="_blank">41</a>].</p