Les lipases/acyltransférases homologues à CpLIP2 de Candida parapsilosis forment un groupe phylogénétique marqué (au moins 56% d’identité entre les séquences protéiques) . Elles partagent le phénotype d’une activité significative d’acyltransfert, et ce, même dans un milieu aqueux avec une forte activité thermodynamique de l’eau (aW > 0.95), mais diffèrent dans leurs spécificités de substrats. L’identification et la caractérisation de nouvelles lipases/acyltransférases, CalLAc8 et CalLAc5 de Candida albicans et CduLAc de Candida dublininensis, ont apporté de nouveaux éclaircissements sur les relations structure/fonction au sein de cette famille particulière. Dans un premier temps, une définition claire et une méthodologie simple pour évaluer la capacité des enzymes lipolytiques à catalyser l’acyltransfert ont été élaborées. Puis, une stratégie d’ingénierie des protéines, basée sur une analyse comparative des structures 3D et de la mutagénèse dirigée, a été appliquée dans le but d’identifier les déterminants structuraux impliqués dans l’activité d’acyltransfert et la spécificité de substrat des lipases/acyltransférases. Il a été démontré que le caractère hydrophobe d’une cavité située sous le site actif était déterminant pour l’activité de transfert en favorisant les nucléophiles moins polaires que l’eau dans l’étape de désacylation du mécanisme catalytique. Ainsi, des mutants améliorés de plusieurs enzymes sauvages ont pu être élaborés. En parallèle, des enzymes chimériques ont été construites sur la base d’échanges rationnels de sous-domaines (corps principal, chapeau et volet C-terminal). Leur caractérisation a confirmé le rôle du chapeau dans la spécificité de substrat et le rôle principal de « l’acyltransfer pocket » dans la capacité d’acyltransfert. Une potentielle protéine ancestrale de la famille PaleoLAc a également été conçue pour trouver de nouveaux résidus clés et donner un aperçu de l’histoire évolutive de la spécificité de substrats. / Lipases/acyltransferases homologous to CpLIP2 from Candida parapsilosis constitute a consistent phylogenetic subgroup with at least 56% identity. They share the phenotype of a significant acyltransfer activity, even in aqueous media with a high thermodynamic activity of water (aW > 0.95), but are divergent in their substrate specificities. The identification and the characterization of new lipases/acyltransferases, CalLAc8 and CalLAc5 from Candida albicans and CduLAc from Candida dublininensis, brought new enlightenments to the structure/function relationships in this peculiar family. After the elaboration of a clear definition and a simple methodology to assess the acyltransferase character of lipolytic enzymes, a rational design strategy, based on comparative 3D structure analysis and site-directed mutagenesis, was applied to find structural determinants of the acyltransfer ability and the substrate specificities of lipases/acyltransferases. It was evidenced that the hydrophobicity of a cavity located under the active site was determinant for the acyltransfer activity. This allowed the improvement of the acyltransfer activity of several natural enzymes. In parallel, chimeric enzymes with rational exchanges of protein subdomains (main core, cap and C-term flap) were designed, and their characterization confirmed the role of the cap in the substrates specificity and the main role of the acyltransfer pocket in the acyltransfer ability. A putative ancestral protein of the family PaleoLAc was also designed to find new key residues and to give insights on the evolutionary history of the substrate specificities.

Le développement des technologies de recombinaison en biologie moléculaire fut un point tournant pour les sciences biologiques. Depuis cette découverte, diverses avancées extraordinaires qui ont un impact direct sur les humains ont pu être accomplies dans les domaines de recherches qui découlent de cette technologie. L’étude des enzymes produites en utilisant cette technique est le fondement de leurs applications éventuellement accessibles. À cet effet, la biocatalyse est un sous-domaine de l’enzymologie en développement continuel. Les chimistes et ingénieurs utilisent les composantes de systèmes biologiques ou même des systèmes complets afin de complémenter ou remplacer des méthodologies existantes. Cette thèse étudie la famille d’enzymes transglutaminase (TGase) comme biocatalyseur afin d’explorer et d’étendre l’ubiquité et les innovations rendues possibles grâce aux enzymes. Les TGases sont des enzymes versatiles. Leur homologue bactérien, la transglutaminase bactérienne (MTG), est couramment utilisé à l’échelle industrielle pour la transformation alimentaire. Depuis une dizaines d’années, de nombreux efforts ont été faits afin de trouver de nouvelles applications des TGases. En premier lieu, une revue des accomplissements, progrès et défis reliés au développement des TGases sera décrite. Les TGases sont intrinsèquement des catalyseurs de la formation de lien isopeptidiques entre une glutamine et une lysine. Par ce fait, elles ont été initialement testées dans cette thèse pour la synthèse de peptides. Une forme de l’enzyme TGase de mammifères fut en mesure de générer les composés dipeptidiques Gly-Xaa et D-Ala-Gly avec une faible conversion. La MTG possède plusieurs caractéristiques qui font de cette enzyme un candidat intéressant pour le développement de biotechnologies. Elle est stable, non dépendante d’un cofacteur et connait peu de compétition pour sa réaction catalytique inverse. La majeure partie de cette thèse porte exclusivement sur l’utilisation de la MTG. Nous avons développé et caractérisé une réaction chimio-enzymatique en un seul pot pour la conjugaison de peptides et protéines. La présence de glutathion en quantité suffisante permet de contourner l’incompatibilité de la MTG avec le cuivre et ouvre la porte à l’utilisation de la réaction de cycloaddition entre un alcyne et un azoture catalysée par le cuivre, afin d’effectuer le marquage fluorescent de protéines. L’utilisation d’autres méthodes de chimie « click » sans métaux fut aussi étudiée afin d’incorporer divers substrats protéiques. Le marquage de protéines avec la MTG fut investigué de manière combinatoire. Précisément, la ligation de Staudinger, la cycloaddition azoture-alcyne promue par la tension de cycle, ainsi que la ligation de tetrazine (TL) ont été testées. Différents niveaux de conversion ont été atteints, le plus prometteur étant celui obtenu avec la TL. Une étude par cristallographie a été effectuée afin d’élucider comment les substrats contenant une glutamine interagissent avec la MTG. Une méthode de purification alternative de la MTG a été développée afin d’atteindre ce but. Une discussion sur les stratégies et défis est présentée. Finalement, la conjugaison entre un système contenant la MTG comme biocatalyseur de marquage, le domaine B1 de la protéine G (GB1) comme substrat et d’un fluorophore contenant une amine comme sonde fut étudié. Comme deux des constituants de ce système sont des protéines, l’ingénierie d’enzyme peut être entreprise afin d’améliorer leurs propriétés. Une banque de 24 variantes de GB1 fut construite grâce à une approche semi-rationnelle afin d’investiguer quels facteurs sont déterminants pour la sélectivité de la MTG envers la glutamine. Chaque variante étudiée comportait une seule glutamine à une position variable afin d’évaluer l’impact des éléments de structure secondaire où se retrouve la glutamine. L’efficacité pour le marquage a pu être améliorée d’au moins un ordre de grandeur pour huit des substitutions étudiées. Comme chacune des structures secondaires fut marquée, il fut démontré que la MTG n’en préfère pas une en particulier. De plus, la réactivité de la MTG envers la variante I6Q-GB1 fut augmentée en créant des mutations dans son site actif. Ces résultats permettent de comprendre d’avantage la sélectivité de la MTG envers la glutamine, tout en démontrant le potentiel de cette enzyme à être modifiée afin d’être améliorée. / The development of recombinant molecular biology technologies was a turning point for the biological sciences, which has since evolved into dozens upon dozens of different subfields and contributed to extraordinary advances for humans. At the core of many of these advances are the enzymes produced by these techniques, with efforts to understand their form and function laying the groundwork for their application. One of these continuously advancing subfields rooted in enzymology is biocatalysis, in which chemists and engineers embrace biological components and systems to complement, or even replace, existing methodologies. This thesis seeks to further contribute to the advancement and ubiquity of enzymes to be incorporated into future innovations. To this end, transglutaminase (TGase) is the biocatalyst selected for study. TGases are versatile enzymes, with the bacterial homolog, microbial transglutaminase (MTG) being readily used in industrial processes for years, particularly for food processing. An abundance of efforts seeking to apply TGases to other processes have been made within the last decade. We commence by reviewing the accomplishments, progress, and challenges to developing TGase towards new goals. TGase naturally catalyzes the formation of isopeptide bonds utilizing a glutamine and lysine substrates, and one of its first unconventional applications we investigated was for peptide synthesis. We determined the ability and specificity of one form of TGase for various amino acid-derived substrates, observing the formation of Gly-Xaa and D-Ala-Gly dipeptide products, albeit at a low conversion. MTG exhibits several characteristics that make it an appealing candidate for biotechnological development, such as its independence from a cofactor, little competition for its reverse catalytic reaction, and increased stability relative to mammalian TGases. Therefore, the remainder of this thesis pertains exclusively to MTG. We developed and extensively characterized a one-pot chemoenzymatic peptide and protein conjugation scheme. The presence of sufficient glutathione circumvents the incompatibility of the copper-catalyzed azide-alkyne cycloaddition with MTG owing to the presence of copper. We ultimately utilized this chemoenzymatic conjugation scheme for fluorescent protein labeling. We continue to expand upon combinatorial methods to undertake protein labeling by investigating to what extent metal-free click chemistries can be utilized in combination with MTG. Specifically, the Staudinger ligation, strain-promoted azide-alkyne cycloaddition, and tetrazine ligation (TL) were assayed on protein substrates to reveal varying levels of effective conjugation, with the TL being the most promising of the three. The details surrounding the manner in which MTG interacts with its glutamine-containing substrate remains unclear. To address this knowledge gap, we sought to pursue crystallography studies, which required the development a modified purification strategy. We discuss the strategies we investigated and the challenges surrounding such efforts. Finally, we present a conjugation system consisting of MTG as the labeling biocatalyst, the B1 domain of Protein G (GB1) as a substrate, and a small-molecule amine belonging to a recently developed class of fluorophores as a probe. As two components of this system are proteins, enzyme engineering can be applied to further improve their properties. A semi-rational approach was used to generate a 24-member GB1 library to probe the structural determinants of MTG’s glutamine selectivity. Each variant contained a single glutamine at varying positions covering all secondary structure elements, and assayed for reactivity. Eight substitutions resulting in an increased labeling efficiency of at least an order of magnitude were distributed throughout all secondary structure elements, indicating that MTG does not favor one preferentially. In addition, introducing point mutations within MTG’s active site also resulted in increased reactivity towards variant I6Q-GB1. Our results contribute further to understanding the nature of MTG’s glutamine selectivity, while simultaneously demonstrating the potential enzyme engineering has to improve and adjust this system.

This thesis describes the rational design of a novel enzyme, a thiolester hydrolase, derived from human glutathione transferase (GST) A1-1 by the introduction of a single histidine residue. The first section of the thesis describes the design and the determination of the reaction mechanism. The design was based on the crystal structure of human GST A1-1 complexed with S-benzylglutathione. The resulting enzyme, A216H, catalyzed the hydrolysis of the non-natural substrate GSB, a thiolester of glutathione and benzoic acid. The reaction followed saturation kinetics with a kcat of 0.00078 min-1 and KM of 5 μM. The rate constant ratio, (kcat/KM)/kuncat, was found to be more than 107 M-1. The introduction of a single His residue in position 216 opened up a novel reaction pathway in human GST A1-1 and is a nice example of catalytic promiscuity. The substrate requirements were investigated and A216H was found to be selective since only two out of 18 GS-thiolesters tested were substrates for A216H. The reaction mechanism of the A216H-catalyzed hydrolysis of GSB was determined and found to proceed via an acyl intermediate at Y9. The hydrolysis was catalyzed by H216 that acts as a general base and the deacylation was found to be the rate-determining step. The Y9-intermediate could be selectively trapped by oxygen nucleophiles and primary alcohols, in particular 1-propanol and trifluoroethanol, were the most efficient. In addition, saturation kinetics was obtained in the acyl transfer reaction with 1-propanol indicating the presence of a second binding site in A216H. The second section of this thesis describes the site-specific covalent modification of human GST A1-1. The addition of GSB to the wild-type protein results in a site-specific benzoylation of only one tyrosine residue, Y9, out of ten present in the protein (one out of totally 51 nucleophiles). The reaction was tested with five GST classes (Alpha, Mu, Pi, Theta and Omega) and found to be specific for the Alpha class isoenzymes. The covalent modification reaction was further refined to target a single lysine residue, K216, providing a more stable linkage in the form of an amide bond. The reaction was found to be versatile and approximately 50% of the GS-thiolesters tested acylated K216, including a fluorophore. / <p>On the day of the public defence the status of article II was: Submitted and article IV was: In press.</p>

For a sustainable future, there is a call to increase the market share of bio-based technologies and materials. Microbial-based technologies have the potential and the ability to contribute substantively on many levels to global efforts to achieve sustainability. Development and utilization of microbial technologies is, however, an extensive process involving numerous steps, including the discovery of novel technologies and the development of industrially viable production systems. In the presented thesis, individual steps of microbial biotechnology development were addressed. In the first part of the study, a variety of methodological approaches were employed in order to study the effect of the anthropogenic activity (i.e., decades lasting production of penicillin G) on the structure of soil microbial communities. Moreover, both cultivable and non-cultivable fractions of populations were subjected to functional screening in order to unravel the biotechnological potential of the microorganisms in terms of production of enzymes involved in biotransformation of beta-lactam antibiotics: penicillin G acylase (PGA) and alpha amino acid ester hydrolase (AEH). Our results indicated that the impacted communities harbour a microbial community with increased diversity and richness. However, on the...

Elevated reaction temperatures are crucial for the efficient enzymatic degradation of polyethylene terephthalate (PET). A disulfide bridge was introduced to the polyester hydrolase TfCut2 to substitute its calcium binding site. The melting point of the resulting variant increased to 94.7°C (wild-type TfCut2: 69.8 °C) and its half-inactivation temperature to 84.6 °C (TfCut2: 67.3 °C). The variant D204C-E253C-D174R obtained by introducing further mutations at vicinal residues showed a temperature optimum between 75 and 80 °C compared to 65 and 70 °C of the wild-type enzyme. The variant caused a weight loss of PET films of 25.0 +/- 0.8% (TfCut2: 0.3 +/-0.1%) at 70 °C after a reaction time of 48 h. The results demonstrate that a highly efficient and calcium-independent thermostable polyester hydrolase can be obtained by replacing its calcium binding site with a disulfide bridge.

Many different chemicals are used in the everyday life, like detergents and pharmaceuticals. However, their production has a big impact on health and environment as much of the raw materials are not renewable and the standard ways of production in many cases includes toxic and environmentally hazardous components. As the population and as the life standard increases all over the planet, the demand for different important chemicals, like pharmaceuticals, will increase. A way to handle this is to apply the concept of Green chemistry, where biocatalysis, in the form of enzymes, is a very good alternative. Enzymes do not normally function in industrial processes and needs modifications through protein engineering to cope in such conditions. To be able to efficiently improve an enzyme, there is a need to understand the mechanism and characteristics of that enzyme. Acyloins (α-hydroxy ketones) are important building blocks in the synthesis of pharmaceuticals. In this thesis, the enzyme alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber has been in focus, as it has been shown to display a wide substrate scope, also accepting aryl-substituted alcohols. The aim has been to study the usefulness of ADH-A as a biocatalyst towards production of acyloins and its activity with aryl-substituted vicinal diols and to study substrate-, regio-, and enantioselectivity of this enzyme. This thesis is based on four different papers where the focus of the first has been to biochemically characterize ADH-A and determine its mechanism, kinetics and its substrate-, regio-, and enantioselectivity. The second and third paper aims towards deeper understanding of some aspects of selectivity of ADH-A. Non-productive binding and its importance for enantioselectivity is studied in the second paper by evolving ADH-A towards increased activity with the least favored enantiomer through protein engineering. In the third paper, regioselectivity is in focus, where an evolved variant displaying reversed regioselectivity is studied. In the fourth and last paper ADH-A is studied towards the possibility to increase its activity towards aryl-substituted vicinal diols, with R-1-phenyl ethane-1,2-diol as the model substrate, and the possibility to link ADH-A with an epoxide hydrolase to produce acyloins from racemic epoxides.

La transglutaminase microbienne (Microbial transglutaminase : MTG) est fortement exploitée dans l’industrie textile et alimentaire afin de modifier l’apparence et la texture de divers produits. Elle catalyse la formation de liaisons iso-peptidiques entre des protéines par l’entremise d’une réaction de transfert d’acyle entre le groupement γ-carboxamide d’une glutamine provenant d’un substrat donneur d’acyle, et le groupement ε-amino d’une lysine provenant d’un substrat accepteur d’acyle. La MTG est tolérante à un large éventail de conditions réactionnelles, ce qui rend propice le développement de cette enzyme en tant que biocatalyseur. Ayant pour but le développement de la MTG en tant qu’alternative plus soutenable à la synthèse d’amides, nous avons étudié la réactivité d’une gamme de substrats donneurs et accepteurs non-naturels. Des composés chimiquement diversifiés, de faible masse moléculaire, ont été testés en tant que substrats accepteurs alternatifs. Il fut démontré que la MTG accepte une large gamme de composés à cet effet. Nous avons démontré, pour la première fois, que des acides aminés non-ramifiés et courts, tels la glycine, peuvent servir de substrat accepteur. Les α-acides aminés estérifiés Thr, Ser, Cys et Trp, mais pas Ile, sont également réactifs. En étendant la recherche à des composés non-naturels, il fut observé qu’un cycle aromatique est bénéfique pour la réactivité, bien que les substituants réduisent l’activité. Fait notable, des amines de faible masse moléculaire, portant les groupements de forte densité électronique azidure ou alcyne, sont très réactives. La MTG catalyse donc efficacement la modification de peptides qui pourront ensuite être modifiés ou marqués par la chimie ‘click’. Ainsi, la MTG accepte une variété de substrats accepteurs naturels et non-naturels, élargissant la portée de modification des peptides contenant la glutamine. Afin de sonder le potentiel biocatalytique de la MTG par rapport aux substrats donneurs, des analogues plus petits du peptide modèle Z-Gln-Gly furent testés; aucun n’a réagi. Nous avons toutefois démontré, pour la première fois, la faible réactivité d’esters en tant que substrats donneurs de la MTG. L’éventuelle amélioration de cette réactivité permettrait de faire de la MTG un biocatalyseur plus général pour la synthèse d’amides. Mots clés: Lien amide, biocatalyse, biotransformation, transglutaminase, arrimage moléculaire, criblage de substrats, ingénierie de substrats. / Microbial transglutaminase (MTG) is used extensively in the food and textile industry to alter the appearance and texture of products. MTG catalyses the formation of isopeptide linkages between proteins by an acyl transfer reaction between the γ-carboxamide group of a glutamine ‘acyl-donor’ substrate, and the ε-amino group of a lysine ‘acyl-acceptor’ substrate. MTG is tolerant to a broad range of reaction conditions and is therefore suitable for further development as a biocatalyst. Toward developing MTG as a “green” alternative for amide synthesis, we have investigated a range of non-native donor and acceptor substrates to probe the scope of MTG reactivity. Small, chemically varied compounds were tested as alternative acyl-acceptor substrates. We observed a broad acceptor specificity. We show, for the first time, that very short-chain alkyl-based amino acids such as glycine can serve as acceptor substrates. The esterified α-amino acids Thr, Ser, Cys and Trp – but not Ile – also show reactivity. Extending the search to non-natural compounds, an aromatic ring was observed to be beneficial for reactivity, although ring substituents reduced reactivity. Overall, bonding of the amine to a less hindered carbon increases reactivity. Importantly, very small amines carrying either the electron-rich azide or the alkyne groups required for click chemistry were highly reactive as acceptor substrates, providing a ready route to minimally modified, ‘clickable’ peptides. These results demonstrate that MTG is tolerant to a variety of chemically varied natural and non-natural acceptor substrates, which broadens the scope for modification of glutamine-containing peptides. To further probe the biocatalytic potential of MTG in terms of the donor substrate, smaller analogues of the model substrate Z-Gln-Gly were tested. We did not find product formation with substrates smaller than the model substrate. We observed, for the first time, trace esterase activity with MTG. Future improvement of this activity would render MTG a more attractive, general biocatalyst for amide bond formation.

L'activation de molécules inertes telles que les alcanes constitue l'un des défis les plus difficiles en catalyse, du fait de la grande stabilité de la liaison C-H. Pour répondre aux principes de la chimie verte, les méthodes de fonctionnalisation doivent respecter un certain nombre d'exigences, telles que l'utilisation de solvants et de réactifs non toxiques, la réduction des apports énergétiques, en association avec une activité élevée. Afin de satisfaire ces conditions, nous nous sommes dirigés vers l'utilisation d'un système enzymatique. En effet, les liaisons C-H non activées peuvent être fonctionnalisées en conditions douces par des monooxygénases, telles que les cytochromes P450, mais leur activité est relativement faible. Dans le but de disposer de cytochromes P450 plus actifs sur les alcanes, nous décrivons la fusion entre un membre de la famille des CYP153 et un partenaire donneur d'électrons. Cette protéine de fusion a été caractérisée, et ses propriétés catalytiques étudiées. Nous avons montré que la fusion augmente de manière considérable l'activité alcane hydroxylase. Nous avons, dans un second temps, continué d'exploiter le fort potentiel de ce biocatalyseur en tentant de réduire le volume de son site actif par mutagénèse dirigée, en vue de l'hydroxylation des alcanes gazeux, notamment le méthane. Enfin, différentes modifications des conditions réactionnelles nous ont permis d'atteindre une activité non égalée pour l'hydroxylation terminale de l'octane. / Activation of inert molecules such as alkanes is considered as one of the most difficult challenges in catalysis, due to the high stability of the C-H bond. To comply with the principles of green chemistry, functionalization methods must respect multiple requirements, such as the use of non-toxic solvents and reagents, in addition to reducing energy usage whilst maintaining maximal activity. To satisfy these conditions, we decided to focus on the use of an enzymatic system. Indeed, unactivated C-H bonds can be functionalized under mild conditions by monooxygenases, such as cytochrome P450s, but their activity is relatively limited. In order to have cytochrome P450s more active on alkanes, we describe the fusion between a member of the CYP153 family and an electron donor partner. This fusion protein has been characterized and its catalytic properties studied. We have shown that the fusion increases significantly the alkane hydroxylase activity. Our second step was to continue to exploit the potential of this biocatalyst by attempting to reduce the volume of its active site using site-directed mutagenesis for the hydroxylation of gaseous alkanes, including methane. Finally, various modifications of the reaction conditions allowed us to achieve the terminal hydroxylation of octane with a previously unequalled activity.

The enzymatic degradation of polyethylene terephthalate (PET) occurs at mild reaction conditions and may find applications in environmentally friendly plastic waste recycling processes. The hydrolytic activity of the homologous polyester hydrolases LC cutinase (LCC) from a compost metagenome and TfCut2 from Thermobifida fusca KW3 against PET films was strongly influenced by the reaction medium buffers tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis rate of PET films in 0.2 M Tris, while the rate of TfCut2 was 2.1-fold lower at this buffer concentration. At a Tris concentration of 1 M, the hydrolysis rate of LCC decreased by more than 90% and of TfCut2 by about 80%. In 0.2 M MOPS or sodium phosphate buffer, no significant differences in the maximum initial hydrolysis rates of PET films by both enzymes were detected. When the concentration of MOPS was increased to 1 M, the hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2 remained low compared to the increasing hydrolysis rates observed at higher concentrations of sodium phosphate buffer. In contrast, the activity of LCC did not change at different concentrations of this buffer. An inhibition study suggested a competitive inhibition of TfCut2 and LCC by Tris and MOPS. Molecular docking showed that Tris and MOPS interfered with the binding of the polymeric substrate in a groove located at the protein surface. A comparison of the Ki values and the average binding energies indicated MOPS as the stronger inhibitor of the both enzymes.

Nitrilases are enzymes which catalyze the hydrolysis of a nitrile into the corresponding carboxylic acid and ammonia. These enzymes are potentially applicable in biocatalysis and bioremediation because of their advantages over the conventional (chemical) methods of nitrile hydrolysis (lower demand for energy, safety, simplicity, high yields, selectivity). In this work, genome mining was used to search for the sequences of hypothetical nitrilases from filamentous fungi. The amino acid sequences of previously characterized fungal nitrilases were used as the templates. Then the new synthetic genes together with other genes from our nitrilase library were expressed in E. coli and the substrate specificities of the enzymes thus produced were compared. Significant attention was focused on the relationships between the sequence of the enzyme and its substrate specificity. The arylacetonitrilases from Arthroderma benhamiae (NitAb) and Nectria haematococca (NitNh) were purified and characterized. Their substrate specificities, kinetic parameters, pH and temperature profiles and subunit and holoenzyme size were assessed. NitAb and NitNh together with other recombinant fungal nitrilases were employed in the hydrolysis of high concentrations of (R,S)-mandelonitrile in a batch or fed-batch mode. Nitrilase from...