La lipase B de Candida antarctica (CALB) est un enzyme présentant des propriétés énantiosélectives très intéressantes pour l'obtention de molécules énantio pures par dédoublement cinétique de mélanges racémiques,molécules utilisées comme synthons dans l'industrie pharmaceutique. En effet, le principe actif de nombreux médicaments est efficace sous une forme énantio pure, l'autre forme chirale pouvant se révéler délétère pour l'organisme.Les travaux de la thèse s'intéressent à mieux comprendre l'origine de l'énantiosélectivité de la lipase B de Candida antarctica, en particulier pour la résolution d'alcools secondaires par des réactions de transestérification.Nous utilisons pour la première fois la méthode de la perturbation de l'énergie libre pour estimer la différence d'énergie libre entre les intermédiaires tétraédriques obtenus avec les formes R et S d'alcools énantiomères pour une série d'alcools secondaires, dans le but de prédire in silico l'énantiosélectivité de la CALB. Les paramètres cinétiques apparents d'une réaction avec deux alcools substrats énantiopurs sont expérimentalement déterminés et permettent de définir la contribution respective du Km et du kcat de chaque énantiomère pour la définition de l'énantiosélectivité. L'étude expérimentale de l'effet d'empreinte par des molécules co-substrats est réalisée,ainsi qu'une étude par modélisation moléculaire de l'effet d'empreinte par le premier ester substrat de la réaction qui pourrait modifier la conformation du site actif de la CALB. La troisième partie porte sur l'étude de la CALB et de trois variants (T42V, S47A et T42V/S47A) chez lesquels les acides aminés dans la poche stéréospécifiques ont mutés. T42V et S47A permettent d'obtenir une augmentation de l'énantiosélectivité. L'étude propose une étude détaillée de la conformation du site actif à partir de simulations de trajectoires de dynamique moléculaire

<p>This thesis describes strategies for and examples ofcellulose fiber modification.The ability of an engineered biocatalyst, acellulose-binding module fused to the<i>Candida antarctica</i>lipase B, to catalyze ring-openingpolymerization of e-caprolactone in close proximity tocellulose fiber surfaces was explored. The water content in thesystem was found to regulate the polymer molecular weight,whereas the temperature primarily influenced the reaction rate.The hydrophobicity of the cellulose sample increased as aresult of the presence of surface-deposited polyester.</p><p>A two-step enzymatic method was also investigated. Here,Candida antarctica lipase B catalyzed the acylation ofxyloglucan oligosaccharides.The modified carbohydrates werethen incorporated into longer xyloglucan molecules through theaction of a xyloglucan endotransglycosylase. The modifiedxyloglucan chains were finally deposited on a cellulosesubstrate.</p><p>The action of<i>Candida antarctica</i>lipase B was further investigated inthe copolymerization of e-caprolactone and D,L-lactide.Copolymerizations with different e-caprolactone-to-D,L-lactideratios were carried out. Initially, the polymerization wasslowed by the presence of D,L-lactide. During this stage,D,L-lactide was consumed more rapidly than ε-caprolactoneand the incorporation occurred dimer-wise with regard to thelactic acid units.</p><p>Morphological studies on wood fibers were conducted using asol-gel mineralization method. The replicas produced werestudied, without additional sample preparation, by electronmicroscopy and nitrogen adsorption. Information concerning thestructure and accessibility of the porous fiber wall wasobtained. Studies of never-dried kraft pulp casts revealedmicro-cavities and cellulose fibrils with mean widths of 4.7(±2) and 3.6 (±1) nm, respectively.</p><p>Finally, cationic catalysis by simple carboxylic acids wasstudied. L-Lactic acid was shown to catalyze the ring-openingpolymerization of ε-caprolactone in bulk at 120 °C.The reaction was initiated with methylß-D-glucopyranoside, sucrose or raffinose, which resultedin carbohydrate-functionalized polyesters. The regioselectivityof the acylation was well in agreement with the correspondinglipase-catalyzed reaction. The polymerization was alsoinitiated with a hexahydroxy-functional compound, whichresulted in a dendrimer-like star polymer. The L-lactic acidwas readily recycled, which made consecutive reactions usingthe same catalyst possible.</p><p><b>Keywords:</b><i>Candida antarctica</i>lipase B, cationic catalysis,cellulose-binding module, dendrimer, enzymatic polymerization,fiber modification, silica-cast replica, sol-gelmineralization, organocatalysis, xyloglucanendotransglycosylase</p>

A chemoselective catalyst is preferred in a chemical reaction where protecting groups otherwise are needed. The two lipases Candida antarctica lipase B and Rhizomucor miehei lipase showed large chemoselectivity ratios, defined as (kcat/KM)OH / (kcat/KM)SH, in a transacylation reaction with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptor (paper I). The chemoselectivity ratio of the uncatalyzed reaction was 120 in favour of the alcohol. Compared to the uncatalyzed reaction, the chemoselectivity was 730 times higher for Candida antarctica lipase B and ten times higher for Rhizomucor miehei lipase. The KM towards the thiol was more than two orders of magnitude higher than the KM towards the corresponding alcohol. This was the dominating contribution to the high chemoselectivity displayed by the two lipases. In a novel approach, Candida antarctica lipase B was used as catalyst for enzymatic synthesis of thiol-functionalized polyesters in a one-pot reaction without using protecting groups (paper II). Poly(e-caprolactone) with a free thiol at one of the ends was synthesized in an enzymatic ring-opening polymerization initiated with mercaptoethanol or terminated with either 3-mercaptopropionic acid or g-thiobutyrolactone.

La lipase B de Candida antarctica (CALB) est un enzyme présentant des propriétés énantiosélectives très intéressantes pour l’obtention de molécules énantio pures par dédoublement cinétique de mélanges racémiques,molécules utilisées comme synthons dans l’industrie pharmaceutique. En effet, le principe actif de nombreux médicaments est efficace sous une forme énantio pure, l’autre forme chirale pouvant se révéler délétère pour l’organisme.Les travaux de la thèse s’intéressent à mieux comprendre l’origine de l’énantiosélectivité de la lipase B de Candida antarctica, en particulier pour la résolution d’alcools secondaires par des réactions de transestérification.Nous utilisons pour la première fois la méthode de la perturbation de l’énergie libre pour estimer la différence d’énergie libre entre les intermédiaires tétraédriques obtenus avec les formes R et S d’alcools énantiomères pour une série d’alcools secondaires, dans le but de prédire in silico l’énantiosélectivité de la CALB. Les paramètres cinétiques apparents d’une réaction avec deux alcools substrats énantiopurs sont expérimentalement déterminés et permettent de définir la contribution respective du Km et du kcat de chaque énantiomère pour la définition de l’énantiosélectivité. L’étude expérimentale de l’effet d’empreinte par des molécules co-substrats est réalisée,ainsi qu’une étude par modélisation moléculaire de l’effet d’empreinte par le premier ester substrat de la réaction qui pourrait modifier la conformation du site actif de la CALB. La troisième partie porte sur l’étude de la CALB et de trois variants (T42V, S47A et T42V/S47A) chez lesquels les acides aminés dans la poche stéréospécifiques ont mutés. T42V et S47A permettent d’obtenir une augmentation de l’énantiosélectivité. L’étude propose une étude détaillée de la conformation du site actif à partir de simulations de trajectoires de dynamique moléculaire / The lipase B from Candida antarctica is an enzyme displaying enantioselective properties which are interesting to obtain enantio pure compounds by kinetic resolution of racemic mixtures, which are used as pharmaceutical intermediates and fine chemicals. Indeed, for most of the drugs, only one of the two chiral formsis efficient as bioactive compound, whereas the other chiral form may display deleterious effects. Present work concerns the understanding of the origin of Candida antarctica lipase B enantioselectivity, and more especially in case of the resolution of secondary alcohols by transesterification. We used, for the first time, the free energy perturbation method to evaluate the free energy difference between tetrahedral intermediates with R and S alcohol enantiomers for a series of secondary alcohols in order to predict in silico enantiomeric ratio of CALB-catalyzed reactions. The apparent kinetic parameters were experimentally determined for two enantio pure substrates and allow to evalute the relative contribution of both Km and kcat for R and S enantiomers in the enantiomeric ratio of CALB-catalyzed reactions. Experimental study of imprinting effect hypothesis by co-substrate molecules was done. Molecular modeling studies of imprinting effect hypothesis were performed, in which the first substrate ester of the reaction could mould the active site. At least, the third part of this thesis concerns the study of wild-type CALB and three different variants (T42V and S47A which allow to increase enantioselectivity and T42/S47A) of CALB by molecular modeling. A detailed study of the conformation of the stereo specificity pocket in the active site is presented, based on molecular dynamics simulations.

<p>Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods.</p><p>In <strong>paper I </strong>a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.</p><p>In paper<strong> II </strong>and <strong>III</strong>, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase,<em> Candida antarctica</em> lipase<strong> </strong>B<strong> </strong>and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD⁺. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance.</p>

<p>The potential use of enzymes in industrial synthesis of epoxidized soybean oil has been limited through the high cost of the enzyme catalyst, in this work we evaluate the effectiveness of chemo enzymatic epoxidation of high oleic soybean oil (HOSBO) using lipase B from <i>Candida antarctica </i>(CALB) on immobilization support Immobead 150 and H₂O₂in a solvent-free system. Additionally, we evaluated the production decay rates for hydrolytic activity and epoxide product formation over consecutive batches to determine half-life of the enzyme catalyst. </p> <p> Batch epoxidation of HOSBO using CALB on 4wt% loading shows yields higher than 90% after 12 hrs. of reaction, and with a correlation to the consumption of double bonds suggesting that the reaction is selective and limiting side product reactions. Non-selective hydrolysis of oil was not found beyond the initial hydrolysis degree of raw HOSBO. Evaluations of decay given by epoxide product formation and released free fatty acids shows a half-life of the enzyme catalyst on these activities is of 22 ad 25 hrs. respectively. Finally, we evaluated the physical parameters influencing this decay, and found that H₂O₂ presence is the most important parameter of enzyme inactivation with no significant effect from its slowed addition. We propose a new reactor configuration for the analysis of the specific steps on epoxide formation through peracid intermediates. </p>

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Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods. In paper I a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage. In paper II and III, three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance. / QC 20101108