History teaches us that novel materials, such as chlorofluorocarbon and asbestos, can have dire unintended consequences to human and environmental health. The exponential growth of the field of nanotechnology and the products developed along the way provide the opportunity for a new paradigm of design thinking, in which human and environmental impacts are considered early on in product development. In particular, nanocellulose is touted as a promising green nanomaterial, as it is sourced from an effectively inexhaustible feedstock of wood-based cellulose and is assumed to be harmless to the environment since it is derived from a natural material and assumed to be biodegradable. The various forms of nanocellulose possess an impressive diversity of properties, making it suitable for a wide variety of applications such as drug delivery, reinforcement, food additives, and iridescent make-up. However, as nanomaterials can have different properties relative to their bulk form, it is questionable whether they are truly environmentally friendly, particularly in terms of their biodegradability and potential impacts to receiving environments. Given the projected mass-scale application of nanocellulose and the inevitability of its subsequent release into environment, the purpose of this study was to determine the biodegradability of nanocellulose and the response of environmentally-relevant microbial communities. Specifically, it was hypothesized that cellulose in the nano size range would display distinct biodegradation patterns and rates, relative to larger forms of cellulose. Further, it was hypothesized that modification of nanocellulose, in terms of morphology and surface properties (e.g., charge), would further influence its biodegradability. Wetlands and anaerobic digesters were selected as two environmentally-relevant receiving environments that also play critical roles in global carbon turnover. To examine the biodegradability of nanocellulose, two distinct microbial consortia were enriched from wetland (W) and anaerobic digester (AD) inocula and applied in parallel experiments. The consortia were grown under anaerobic conditions with microcrystalline cellulose as the sole carbon substrate over a period of 246 days before being aliquoted to microcosms for subsequent biodegradation assays. Various forms of nanocellulose were spiked into the microcosms and compared with microcrystalline cellulose as a non nano reference. Microcosms were sacrificed in triplicate with time to monitor cellulose degradation as well as various measures of microbial community response. Microbial communities were characterized in terms of gene markers for total bacteria (16S rRNA genes) and anaerobic cellulose degraders (glycoside hydrolase family 48 genes, i.e., cel48) as well as high throughput amplicon sequencing of 16S rRNA genes (V4 region). A series of three studies examined: 1) the effect of nanocrystalline versus microcrystalline cellulose; 2) the effects of nanocellulose morphology (crystalline rod versus filament) and surface functionalization (cationic and anionic); and 3) metagenomic characterization of cellulose degrading communities using next-generation DNA sequencing. It was found that the nano- size range did not hinder cellulose degradation, in fact, nanocrystalline cellulose degraded slightly faster than microcrystalline cellulose according to 1st order kinetics (1st order decay constants: 0.62±0.08 wk-1 for anionic nanocrystalline cellulose versus 0.39±0.05 wk-1 for microcrystalline cellulose exposed to AD culture; 0.69±0.04 wk-1 for anionic nanocrystalline cellulose versus 0.58±0.05 wk-1 for microcrystalline cellulose exposed to W). Experiments comparing the effects of surface functionalization indicated that anionic nanocellulose degraded faster than cationic cellulose (1st order decay constants for cationic nanocrystalline cellulose: 0.48±0.06 wk-1 and 0.58±0.07 wk-1 on exposure to AD and W cultures respectively). Measurements of 16S rRNA and cel48 genes were consistent with this trend of greater biological growth and cellulose-degrading potential in the anionic nanocellulose condition, suggesting that surface properties can influence biodegradation patterns. Taxonomic characterization of 16S rRNA gene amplicons suggested that taxa known to contain anaerobic cellulose degraders were enriched in both W and AD consortia, which shifted in a distinct manner in response to exposure to the different cellulosic materials. This suggests that distinct groups of microbes may drive the biodegradation of different forms of cellulose. Further, metagenomic investigation provided new insight into taxonomic and functional aspects of anaerobic cellulose degradation, including identification of enzymatic families associated with degradation of the various forms of cellulose. Overall, the findings of this study advance understanding of anaerobic cellulose degradation and indicate that nanocellulose is likely to readily degrade in receiving environments and not pose an environmental concern. / Ph. D.

N-methylputrescine oxidase (MPO) is a member of the diamine oxidase (DAO) class of enzymes believed to be responsible for synthesis of the alkaloid nicotine in the roots of Nicotiana tabacum (Mizusaki et al., 1972). A purportedly pure MPO protein from tobacco root culture extracts was used to generate immune antiserum in rabbits (McLauchlan et al., 1993). In an attempt to clone a cDNA encoding MPO, we used this antiserum to screen a tobacco cDNA expression library. Unexpectedly, two previously unreported genes with strong homology to members of a gene family encoding S-adenosylhomocysteine hydrolase (SAHH) in N. sylvestris and a gene encoding SAHH in N. tabacum were cloned instead. SAHH is an enzyme of the S-adenosylmethionine (SAM) recycling pathway, which also includes SAM synthetase (SAMS) and methionine synthase (MS). These results led to the hypothesis of a multi-enzyme complex, or metabolon, of at least one member of the nicotine biosynthesis pathway, i.e., MPO, and at least one member of the SAM recycling pathway, i.e., SAHH, during nicotine biosynthesis. Metabolons are stable noncovalent complexes in cells that ensure sufficient passage of the product of one enzyme reaction to the next enzyme in the pathway via a "channel" without equilibrating with the bulk solution (Ovádi, 1991). My research employed co-immunoprecipitation studies to determine if other SAM recycling enzymes are associated in a complex with MPO and SAHH, as well as Northern and Western blot analyses to determine if the genes encoding SAM recycling pathway enzymes are coordinately regulated during nicotine biosynthesis. Our results indicate that nicotine biosynthesis-inducing conditions result in differential mRNA accumulation patterns of the three enzymes of the SAM recycling pathway, although to different extents. However, protein levels of SAM recycling pathway members do not appear to reflect the differential mRNA accumulation patterns. We have firmly established an association of SAHH and an enzyme with DAO activity, purportedly MPO. If the enzyme is proven to be MPO, then our data would constitute the first documentation of an alkaloid metabolon. Finally, using a degenerate primer PCR approach, we have cloned a 986-bp gene fragment with homology to copper amine oxidases, the class to which MPO belongs. / Master of Science

Yes / The crotonases comprise a widely distributed enzyme superfamily that has multiple roles in both primary and secondary metabolism. Many crotonases employ oxyanion hole-mediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) thioester substrates (e.g., malonyl-CoA, α,β-unsaturated CoA esters) both with nucleophiles and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved fold formed from a central β-sheet core surrounded by α-helices, which typically oligomerizes to form a trimer or dimer of trimers. The presence of a common structural platform and mechanisms involving intermediates with diverse reactivity implies that crotonases have considerable potential for biocatalysis and synthetic biology, as supported by pioneering protein engineering studies on them. In this Perspective, we give an overview of crotonase diversity and structural biology and then illustrate the scope of crotonase catalysis and potential for biocatalysis. / Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Wellcome Trust

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Dans ce projet de thèse, une variété de techniques a été employée pour étudier l’alpha-L-arabinofuranosidase de Thermobacillus xylanilyticus (TxAbf), notamment en ce qui concerne les relations structure/fonctions et son activité de transglycosylation. Nos travaux ont eu pour objectif d’apporter un éclairage quant au rôle de la dynamique dans l’activité catalytique de la TxAbf, en se focalisant sur le mouvement de la boucle bêta2alpha2, et d’explorer la spécificité du sous-site [+1], un élément du site actif qui est particulièrement pertinent pour l’activité de transglycosylation. Enfin, nous avons entrepris des travaux d’ingénierie visant la création de transarabinofuranosylases performantes. Nos résultats confirment le rôle important de la boucle bêta2alpha2 et suggèrent que le mouvement de celle-ci permet de relocaliser les résidus His98 et Trp99 de manière à créer un site actif opérationnel. Le résidu Trp99 apparaît comme un élément clé du sous-site [-1] de la TxAbf, alors que le résidu His98, qui n’est pas conservé dans l’ensemble des enzymes de la famille GH51, participerait à la formation d’un sous-site [+2’]. Concernant le sous-site [+1], nos résultats confirment la large spécificité celui-ci et montrent clairement que l’encombrement stérique à la position C-5 des glycosides accepteurs est défavorable à la réaction de transglycosylation. Par ailleurs, nous avons pu réaliser pour la première fois la synthèse de trisaccharides, utilisant comme accepteur l’alpha-D-xylobioside de benzyle et comme donneur le β-D-galactofuranoside de para-nitrophényle. Enfin, nos travaux de mutagenèse aléatoire et le criblage de banques a permis d’identifier deux mutations Phe26Leu et Trp178Arg, qui se situent au niveau des sous-sites [-1] et [+1], respectivement. Selon nos premières analyses, les mutants correspondants rendraient moins favorable la déglycosylation de l’intermédiaire glycosyl-enzyme par une molécule d’eau, réduisant ainsi l'hydrolyse secondaire et stabilisant par la même occasion le produit de synthèse. En employant une deuxième méthode de criblage plus sophistiquée, impliquant l’utilisation d’accepteurs xylo-oligosaccharidiques, nous avons pu obtenir des enzymes mutées qui (i) catalysent des réactions de transglycosylation en présence de xylobiose (l’enzyme sauvage ne catalysant que très faiblement cette réaction) (ii) se caractérisent par une absence quasi-totale d’hydrolyse secondaire et (iii) comportent des mutations situées à différentes positions (e.g. au niveau des sous-sites [-1], [+1] et [+2’]) et qui semblent moduler le ratio Transglycosylation/Hydrolyse en faveur de la synthèse / In this investigation, a variety of techniques to study the Thermobacillus xylanilyticus alpha-L-arabinofuranosidase (TxAbf) have been employed, especially with regard to structure-functions relations and the enzyme’s ability to catalyze transglycosylation reactions. The aim of our work was to better understand the dynamic role of the bêta2alpha2 loop and to explore the substrate specificity of the subsite [+1], an important active site element with respect to transglycosylation. Finally, this work has focused on the creation of new transarabinofuranosylases using random engineering and screening approaches.Our results confirm the important role of the bêta2alpha2 loop and suggest that its movement during catalysis relocalizes residues His98 and Trp99 and thus permits the formation of a catalytically-viable active site configuration. Trp99 is relocalized from a solvent exposed position into a buried position and forms a critical element of subsite [-1], whereas His98, a residue that is not conserved in all GH51 members, appears to form a part of subsite [+2’]. Regarding subsite [+1], our results confirm its wide specificity and indicate that steric bulkiness at the C-5 position of glycoside acceptors leads to reduced transglycosylation. In this work, we have also demonstrated for the first time the synthesis by TxAbf of trisaccharides, using benzyl alpha-D-xylobioside as the acceptor and para-nitrophenyl β-D-galactofuranoside as the donor. Finally, random mutagenesis and screening has led to the identification of two mutations Phe26Leu and Trp178Arg, which are located in sub-sites [-1] and [+1] respectively, that appear to reduce the water-mediated deglycosylation of the glycosyl-enzyme intermediate. Consequently, the corresponding mutants reduce secondary hydrolysis and favourably affect the operational stability of synthetic products. Using a second more sophisticated screening method that involves the use of xylo-oligosaccharide acceptors, it has been possible to isolate mutant enzymes that (i) catalyze transglycosylation reactions in the presence of xylobioside (a reaction that is poorly catalyzed by wild type TxAbf), (ii) show almost no secondary hydrolysis, (iii) display point mutations at several key locations (e.g. in sub-sites [-1], [+1] and [+2’]) that seem to modulate the Transglycosylation/Hydrolysis ratio in favour of synthesis

<p>Epoxides are three-membered cyclic ethers formed in the metabolism of foreign substances and as endogenous metabolites. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to yield the corresponding diols. EHs have been implicated in diverse functions such as detoxification of various toxic epoxides, as well as regulation of signal substance levels.</p><p>The main goal of this thesis was to investigate and characterize the α/β hydrolase fold EH. The first part concerns the identifictaion of an EH in <i>Saccharomyces cerevisiae</i>. The second part involves detailed mechanistic and structural studies of a plant EH from potato, StEH1. </p><p>Despite the important function of EH, no EH has previously been established in <i>S. cerevisiae</i>. By sequence analysis, we have identified a new subclass of EH present in yeast and in a wide range of microorganisms. The <i>S. cerevisiae</i> protein was produced recombinantly and was shown to display low catalytic activity with tested epoxide substrates. </p><p>In plants, EHs are involved in the general defence system, both in the metabolism of the cutin layer and in stress response to pathogens. The catalytic mechanism of recombinantly expressed wild type and mutant potato EH were investigated in detail using the two enantiomers of <i>trans</i>-stilbene oxide (TSO). The proposed catalytic residues of StEH1 were confirmed. StEH1 is slightly enantioselective for the <i>S,S</i>-enantiomer of<i> trans</i>-stilbene oxide. Furthermore, distinct pH dependence of the two enantiomers probably reflects differences in the microscopic rate constants of the substrates. The detailed function of the two catalytic tyrosines was also studied. The behavior of the tyrosine pair resembles that of a bidentate Lewis acid and we conclude that these tyrosines function as Lewis acids rather then proton donors.</p><p>The three dimensional structure of StEH1 was solved, representing the first structure of a plant EH. The structure provided information about the substrate specificity of StEH1.</p>

Cellulosabaserade material är världens rikligast förekommande förnyelsebara råvara. Växters cellväggar är naturliga kompositmaterial där den kristallina cellulosan är inbäddad i en väv av hemicellulosa, strukturproteiner och lignin. Xyloglukaner är en viktig hemicellulosagrupp som omger och korslänkar den kristallina cellulosan i cellväggarna. I denna avhandling undersöks undersöks sambanden mellan struktur och funktion hos olika xyloglukan-aktiva enzymer. En modell för effektiv enzymatisk omvandling av biomassa ges av cellulosomen hos den anaeroba prokaryota organismen Clostridium thermocellum. Cellulosomen är ett proteinkomplex med hög molmassa och flera olika enzymaktiviteter, bl.a. det inverterande xyloglukan-endohydrolaset CtXGH74A. Proteinstrukturen för CtXGH74A har lösts i komplex med xyloglukanoligosackarider, som stabliliserar vissa loopar/slingor som är oordnade i apostrukturen. Ytterligare detaljerade kinetiska och produktananalyser har genomförts för att entydigt visa att CtXGH74A är ett endoxyloglukanas vars slutliga nedbrytningsprodukt är Glc4-baserade xyloglukanoligosackarider. Som jämförelse innehåller glykosidhydrolasfamilj 16 (GH16) såväl hydrolytiska endoxyloglukanaser som xyloglukantransglykosylaser (XETs) från växter. För att utreda vad som bestämmer förhållandet mellan transglykosylering och hydrolys i xyloglukanaktiva enzymer från familj GH 16 jämfördes struktur och kinetik hos ett strikt transglykosylas, PttXET16-34 från hybridasp, med ett nära besläktat hydrolytiskt enzym, NXG1 från krasse. I NXG1 identifierades en viktig förlängningsloop, som vid trunkering gav ett muterat enzym med högre transglykosyleringshastighet och minskad hydrolytisk aktivitet. Kinetikstudierna genomfördes med hjälp av nyutvecklade känsliga provmetoder med väldefinerade XGO:er och ett antal kromogena XGO-arylglykosider. En detaljerad förståelse av enzymologin inom GH16 möjliggjorde utvecklingen av en ny kemoenzymatisk metod för biomimetisk fiberytmodifiering med hjälp av PttXET16-34s translgykosyleringsaktivitet. Aminoalditolderivat av xyloglukanoligosackarider användes som nyckelintermediärer för att introducera ny kemisk funktionalitet hos xyloglukan, såsom kromoforer, reaktiva grupper, proteinligander och initiatorer för polymeriseringsreaktioner. Tekniken innebär ett nytt och mångsidigt verktyg för fiberytmodifiering. / Zellulosehaltige Materialien sind die häufigsten erneuerbaren Rohmaterialien auf der Welt. Pflanzenzellwände sind natürliche Kompositmaterialien, sie enthalten kristalline Zellulose, die in einer Matrix aus Hemizellulosen, Proteinen und Lignin eingebettet sind. Xyloglukane sind eine wichtige Gruppe der Hemizellulosen, sie ummanteln und verbinden Zellulose in der pflanzlichen Zellwand. In dieser Abhandlung werden Strukturen von drei Xyloglukanaktiven Enzymen in Beziehung zu ihrer Funktion untersucht. Ein Paradigma für effizienter Nutzung von Biomasse ist das Cellulosom des anaerob lebenden Bakteriums Clostridium thermocellum. Das Cellulosom ist ein hochmolekularer Komplex von Proteinen mit vielen verschiedenen Aktivitäten, darunter ist auch die invertierende Xyloglukan Endohydrolase CtXGH74A. Die Proteinstruktur von CtXGH74A wurde im Komplex mit Xyloglukanoligosacchariden (XGO) gelöst, welche ungeordnete Loops der apo-Struktur stabilisierten. Durch weitere detaillierte Analyse der Kinetik und Reaktionsprodukte konnte schlüssig gezeigt werden, daß CtXGH74A eine Endoglukanase ist, die Glc4-basierte XGO produziert. Im Vergleich dazu enthält die retentierende Glykosidhydrolasefamilie 16 (GH16) sowohl hydrolytische Endoxyloglukanasen als auch Transglykosidasen von Pflanzen. Um zu erklären welche Faktoren das Verhältnis zwischen Transglykosidase und Hydrolase Aktivität bei GH16 Xyloglukanaktiven Enzymen bestimmen wurde eine reine Transglykosidase PttXET16-34 von Hybridaspen mit einem nah verwandten hydrolytischen Enzym NXG1 von Kapuzinerkresse strukturell und kinetisch verglichen. Als Schlüsselstelle wurde eine Verlängerung eines Loops in NXG1 identifiziert, Verkürzung des Loops führte zu einer Mutante mit erhöhter Transglykosylierungsrate bei verminderter hydrolytischer Aktivität. Kinetische Studien wurden erleichtert durch neu entwickelte hochempfindliche Methoden für Aktivitätsmessung, die auf XGO oder chromogene Aryl-XGO als definierte Substrate zurückgreifen. Detailliertes Verständnis von GH16 Enzymologie hat den Weg für die Entwicklung für eine neuartige Methode für biomimetische Oberflächenmodifikation von Zellulosefibern geebnet, dafür wurde die transglykosylierende Aktivität von PttXET16-34 angewendet. Aminoalditol-derivate von XGO wurden als wichtigste Zwischenprodukte angewendet, um neue chemische Funktionalitäten in Xyloglukan einzuführen, darunter waren Chromophore, reaktive Gruppen, Proteinliganden und Initiatoren für Polymerisationsreaktionen. Die modifizierten Xyloglukane wurden an eine Reihe von verschiedenen Zellulosematerialien gebunden und veränderten die Oberflächeneigenschaften dramatisch. Diese Methode ist ein neues wertvolles Werkzeug für Oberflächenmodifikation von Zellulosen. / Cellulosic materials are the most abundant renewable resource in the world; plant cell walls are natural composite materials containing crystalline cellulose embedded in a matrix of hemicelluloses, structural proteins, and lignin. Xyloglucans are an important group of hemicelluloses, which coat and cross-link crystalline cellulose in the plant cell wall. In this thesis, structure-function relationships of a range of xyloglucan-active enzymes were examined. A paradigm for efficient enzymatic biomass utilization is the cellulosome of the anaerobic bacterium Clostridium thermocellum. The cellulosome is a high molecular weight complex of proteins with diverse enzyme activities, including the inverting xyloglucan endo-hydrolase CtXGH74A. The protein structure of CtXGH74A was solved in complex with xyloglucan oligosaccharides (XGOs) which stabilized disordered loops of the apo-structure. Further detailed kinetic and product analyses were used to conclusively demonstrate that CtXGH74A is an endo-xyloglucase that produces Glc4-based XGOs as limit digestion products. In comparison, the retaining glycoside hydrolase family 16 (GH16) contains hydrolytic endo-xyloglucanases as well as xyloglucan transglycosylases (XETs) from plants. To elucidate the determinants of the transglycosylase/hydrolysis ratio in GH16 xyloglucan-active enzymes, a strict transglycosylase, PttXET16-34 from hybrid aspen, was compared structurally and kinetically with the closely related hydrolytic enzyme NXG1 from nasturtium. A key loop extension was identified in NXG1, truncation of which yielded a mutant enzyme that exhibited an increased transglycosylase rate and reduced hydrolytic activity. Kinetic studies were facilitated by the development of new, sensitive assays using well-defined XGOs and a series of chromogenic XGO aryl-glycosides. A detailed understanding of GH16 xyloglucan enzymology has paved the way for the development of a novel chemo-enzymatic approach for biomimetic fiber surface modification, in which the transglycosylating activity of PttXET16-34 was employed. Aminoalditol derivates of XGOs were used as key intermediates to incorporate novel chemical functionality into xyloglucan, including chromophores, reactive groups, protein ligands, and initiators for polymerization reactions. The resulting modified xyloglucans were subsequently bound to a range of cellulose materials to radically alter surface properties. As such, the technology provides a novel, versatile toolkit for fiber surface modification. / QC 20100624

Epoxides are three-membered cyclic ethers formed in the metabolism of foreign substances and as endogenous metabolites. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to yield the corresponding diols. EHs have been implicated in diverse functions such as detoxification of various toxic epoxides, as well as regulation of signal substance levels. The main goal of this thesis was to investigate and characterize the α/β hydrolase fold EH. The first part concerns the identifictaion of an EH in Saccharomyces cerevisiae. The second part involves detailed mechanistic and structural studies of a plant EH from potato, StEH1. Despite the important function of EH, no EH has previously been established in S. cerevisiae. By sequence analysis, we have identified a new subclass of EH present in yeast and in a wide range of microorganisms. The S. cerevisiae protein was produced recombinantly and was shown to display low catalytic activity with tested epoxide substrates. In plants, EHs are involved in the general defence system, both in the metabolism of the cutin layer and in stress response to pathogens. The catalytic mechanism of recombinantly expressed wild type and mutant potato EH were investigated in detail using the two enantiomers of trans-stilbene oxide (TSO). The proposed catalytic residues of StEH1 were confirmed. StEH1 is slightly enantioselective for the S,S-enantiomer of trans-stilbene oxide. Furthermore, distinct pH dependence of the two enantiomers probably reflects differences in the microscopic rate constants of the substrates. The detailed function of the two catalytic tyrosines was also studied. The behavior of the tyrosine pair resembles that of a bidentate Lewis acid and we conclude that these tyrosines function as Lewis acids rather then proton donors. The three dimensional structure of StEH1 was solved, representing the first structure of a plant EH. The structure provided information about the substrate specificity of StEH1.

Der Actinomycet T. fusca KW3, isoliert aus Kompost, bildete während der Kultivierung im Mineralsalz-Spurenelement-Vitamin-Minimalmedium nach Zusatz von PET-Fasern eine 52 kDa Carboxylesterase (TfCa), welche effizient zyklische PET Trimere (CTR) hydrolysiert. Die TfCa besitzt einen pI von 4,8, eine Substratspezifität gegenüber kurzkettigen p-Nitrophenyl-Estern und wird durch Phenylmethylsulfonylfluorid (PMSF) und Tosyl-L-Phenylalanin-Chloromethylketon (TPCK) in der Aktivität gehemmt. Die Carboxylesterase hydrolysiert kein Cutin oder Poly-ε-caprolacton (PCL). CTR hingegen wurden durch die TfCa mit einem Km von 0,5 mM und einer Vmax von 9,3 μmol/min/mg bei optimalen Bedingungen (60°C, pH 6) hydrolysiert. Das aktive Zentrum der Carboxylesterase besteht aus den Aminosäuren Ser185, Glu319 und His415, wobei das Serin in das katalytische Motiv G-E-S-A-G eingebettet ist. Während der Reaktion setzte die TfCa auch Hydrolyseprodukte aus PET-Fasern und -Filmen frei. Der Nachweis der Hydrolyse erfolgte durch Umkehrphasen-Hochleistungsflüssigkeitschromatographie der Abbauprodukte und bei den PET-Filmen zusätzlich mittels Rasterelektronenmikroskopie. Dabei zeigte die Carboxylesterase verglichen mit anderen PET-Hydrolasen eine geringere Effizienz, was durch die Lage des aktiven Zentrums in einer Bindungstasche und der daraus folgenden schlechten Zugänglichkeit für polymere Substrate begründet werden kann. Bei der Hydrolyse der viel kleineren CTR war die TfCa deutlich effektiver, was auf eine höhere Spezifität gegenüber kurzkettigen PET Substraten hinweist.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal