Pseudozyma (Candida) antarctica lipase B (PalB) was expressed in Escherichia coli facilitating protein engineering. The lack of glycosylation was evaluated for a deeper understanding of the difficulties in expressing PalB in E. coli. Different systems were tested: periplasmic expression in Rosetta (DE3), cytosolic expression in Rosetta-gami 2(DE3), Origami 2(DE3), and coexpression of groES and groEL. Periplasmic expression resulted 5.2 mg/L active PalB at 16 °C in shake flasks. This expression level was improved by using the EnBase technology, enabling fed-batch cultivation in 24-deep well scale. The feed rate was titrated with the addition of α-amylase, which slowly releases glucose as energy source. Different media were evaluated where the EnBase mineral salt medium resulted in 7.0 mg/L of active PalB. Protein secreted directly into the media was obtained using the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for screening and production of PalB in P. pastoris. A protease sensitive fusion protein CBM-PalB (cellulose-binding module) was used as a model system. When optimised, the expression system resulted in 46 mg/L lipase in 72 hours in shake flask, 37 mg/L lipase in 28 hours in 96-deep-well plate format, and 2.9 g PalB per 10 L bioreactor cultivation. The E. coli expression system was used to express a small focused library of PalB variants, designed to prevent water from entering the active site through a hypothesised tunnel. Screening of the library was performed with a developed assay, allowing for simultaneous detection of both transacylation and hydrolytic activity. From the library a mutant S47L, in which the inner part of the tunnel was blocked, was found to catalyse transacylation of vinyl butyrate in 20 mM butanol 14 times faster than hydrolysis. Water tunnels, assisting water in reaching the active sites, were furthermore found by molecular modelling in many hydrolases. Molecular modelling showed a specific water tunnel in PalB. This was supported by experimental data, where the double mutant Q46A S47L catalysed transacylation faster than hydrolysis compared to the wild type PalB. / <p>QC 20100818</p>

Neste trabalho, duas preparações de lipase imobilizada (EC 3.1.1.3), tipo B (CALB) de Candida antarctica, foram comparadas como biocatalisadores na síntese de ésteres metílicos. CALB imobilizada comercialmente (Novozym 435 - CALB-435) e CALB imobilizada em esferas de estireno-divinilbenzeno (CALB-MCI) foram testadas para as reações de transesterificação. Um delineamento composto central rotacional (DDCR) e metodologia de superfície de resposta (MSR) foram utilizados para otimizar a relação razão molar álcool:óleo, teor de enzimas, e a água adicionada nas reações. As duas preparações de enzimas mostraram diferentes condições ótimas para a produção de ésteres metílicos, com a taxa inicial da reação de 51,47 mmol L-1 h-1 para CALB-435, e 57 mmol L-1 h-1 para CALB-MCI. O estudo do tempo de reação indicou que em 72 h foi possível obter conversões próximas a 100 % para ambos os derivados. Um alto rendimento de conversão foi obtido sob as condições otimizadas, indicando que a RSM pode ser usada para descrever adequadamente a relação entre os parâmetros de reação e da resposta (teor de ésteres metílicos). Sobre a estabilidade operacional durante as experiências de reutilização, ambos preparados enzimáticos mantiveram 70 % de sua atividade inicial após oito bateladas, sugerindo sua aplicabilidade na produção de biodiesel. / In this work two preparations of immobilized lipase (EC 3.1.1.3), type B (CALB) from Candida antarctica, were compared as biocatalysts in the synthesis of esters. Commercial Novozym 435 (CALB-435) and CALB immobilized on styrene-divinylbenzene beads (CALB-MCI) were tested for the transesterification reactions. Central composite design rotational (DCCR) and response surface methodology (RSM) were used to optimize the substrate molar ratio, enzyme content, and the added water. The two enzyme preparations have shown different optimal conditions for the production of methyl esters, with initial rates of reaction 51.47 mmol L-1 h-1 for CALB-435, and 57 mmol L-1 h-1 for CALB-MCI. The study of reaction time indicated that in 72 h it was possible to obtain conversions close to 100 % for both derivatives. A high yield conversion was obtained under the optimized conditions, indicating that RSM can be used to adequately describe the relationship between the reaction parameters and the response (yield conversion) in lipase-catalyzed biodiesel synthesis. Over the operational stability during experiments of batch reuse, both prepared enzymatic maintained 70 % of their initial activity after eight batches, suggesting their potential for economical application on biodiesel production.

Neste trabalho, duas preparações de lipase imobilizada (EC 3.1.1.3), tipo B (CALB) de Candida antarctica, foram comparadas como biocatalisadores na síntese de ésteres metílicos. CALB imobilizada comercialmente (Novozym 435 - CALB-435) e CALB imobilizada em esferas de estireno-divinilbenzeno (CALB-MCI) foram testadas para as reações de transesterificação. Um delineamento composto central rotacional (DDCR) e metodologia de superfície de resposta (MSR) foram utilizados para otimizar a relação razão molar álcool:óleo, teor de enzimas, e a água adicionada nas reações. As duas preparações de enzimas mostraram diferentes condições ótimas para a produção de ésteres metílicos, com a taxa inicial da reação de 51,47 mmol L-1 h-1 para CALB-435, e 57 mmol L-1 h-1 para CALB-MCI. O estudo do tempo de reação indicou que em 72 h foi possível obter conversões próximas a 100 % para ambos os derivados. Um alto rendimento de conversão foi obtido sob as condições otimizadas, indicando que a RSM pode ser usada para descrever adequadamente a relação entre os parâmetros de reação e da resposta (teor de ésteres metílicos). Sobre a estabilidade operacional durante as experiências de reutilização, ambos preparados enzimáticos mantiveram 70 % de sua atividade inicial após oito bateladas, sugerindo sua aplicabilidade na produção de biodiesel. / In this work two preparations of immobilized lipase (EC 3.1.1.3), type B (CALB) from Candida antarctica, were compared as biocatalysts in the synthesis of esters. Commercial Novozym 435 (CALB-435) and CALB immobilized on styrene-divinylbenzene beads (CALB-MCI) were tested for the transesterification reactions. Central composite design rotational (DCCR) and response surface methodology (RSM) were used to optimize the substrate molar ratio, enzyme content, and the added water. The two enzyme preparations have shown different optimal conditions for the production of methyl esters, with initial rates of reaction 51.47 mmol L-1 h-1 for CALB-435, and 57 mmol L-1 h-1 for CALB-MCI. The study of reaction time indicated that in 72 h it was possible to obtain conversions close to 100 % for both derivatives. A high yield conversion was obtained under the optimized conditions, indicating that RSM can be used to adequately describe the relationship between the reaction parameters and the response (yield conversion) in lipase-catalyzed biodiesel synthesis. Over the operational stability during experiments of batch reuse, both prepared enzymatic maintained 70 % of their initial activity after eight batches, suggesting their potential for economical application on biodiesel production.

Neste trabalho, duas preparações de lipase imobilizada (EC 3.1.1.3), tipo B (CALB) de Candida antarctica, foram comparadas como biocatalisadores na síntese de ésteres metílicos. CALB imobilizada comercialmente (Novozym 435 - CALB-435) e CALB imobilizada em esferas de estireno-divinilbenzeno (CALB-MCI) foram testadas para as reações de transesterificação. Um delineamento composto central rotacional (DDCR) e metodologia de superfície de resposta (MSR) foram utilizados para otimizar a relação razão molar álcool:óleo, teor de enzimas, e a água adicionada nas reações. As duas preparações de enzimas mostraram diferentes condições ótimas para a produção de ésteres metílicos, com a taxa inicial da reação de 51,47 mmol L-1 h-1 para CALB-435, e 57 mmol L-1 h-1 para CALB-MCI. O estudo do tempo de reação indicou que em 72 h foi possível obter conversões próximas a 100 % para ambos os derivados. Um alto rendimento de conversão foi obtido sob as condições otimizadas, indicando que a RSM pode ser usada para descrever adequadamente a relação entre os parâmetros de reação e da resposta (teor de ésteres metílicos). Sobre a estabilidade operacional durante as experiências de reutilização, ambos preparados enzimáticos mantiveram 70 % de sua atividade inicial após oito bateladas, sugerindo sua aplicabilidade na produção de biodiesel. / In this work two preparations of immobilized lipase (EC 3.1.1.3), type B (CALB) from Candida antarctica, were compared as biocatalysts in the synthesis of esters. Commercial Novozym 435 (CALB-435) and CALB immobilized on styrene-divinylbenzene beads (CALB-MCI) were tested for the transesterification reactions. Central composite design rotational (DCCR) and response surface methodology (RSM) were used to optimize the substrate molar ratio, enzyme content, and the added water. The two enzyme preparations have shown different optimal conditions for the production of methyl esters, with initial rates of reaction 51.47 mmol L-1 h-1 for CALB-435, and 57 mmol L-1 h-1 for CALB-MCI. The study of reaction time indicated that in 72 h it was possible to obtain conversions close to 100 % for both derivatives. A high yield conversion was obtained under the optimized conditions, indicating that RSM can be used to adequately describe the relationship between the reaction parameters and the response (yield conversion) in lipase-catalyzed biodiesel synthesis. Over the operational stability during experiments of batch reuse, both prepared enzymatic maintained 70 % of their initial activity after eight batches, suggesting their potential for economical application on biodiesel production.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-02-26T13:58:25Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO DE MESTRADO -DARTAGNAN PIRES - OK!! (1).pdf: 6019641 bytes, checksum: 2024556a4ff3ddf948edf6eeeb9dbd55 (MD5) / Made available in DSpace on 2016-02-26T13:58:25Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO DE MESTRADO -DARTAGNAN PIRES - OK!! (1).pdf: 6019641 bytes, checksum: 2024556a4ff3ddf948edf6eeeb9dbd55 (MD5) Previous issue date: 2013-12-13 / CNPq / Derivados de furano e tiofeno 2-substituídos são compostos bastante difundidos no meio ambiente e desempenham papel importante em processos bioquímicos. Além disso, estes compostos podem ser utilizados como building blocks na preparação de diversas moléculas bioativas. No entanto, reagentes prejudiciais ao meio ambiente e muitas etapas reacionais, são geralmente atribuídos às metodologias sintéticas que empregam estes derivados. Atualmente, poucos trabalhos têm reportado a utilização de enzimas para este tipo de sistema. Dessa forma, nosso grupo demonstrou interesse em propor uma metodologia quimioenzimática para preparação de derivados de furano e tiofeno 2-substituídos em suas formas enantiomericamente pura, e aplicá-los na preparação de moléculas bioativas enantiodependentes. A metodologia proposta emprega um número reduzido de etapas sintéticas e utiliza a enzima CAL-B na etapa chave para obtenção dos respectivos enantiômeros separadamente. Inicialmente, uma série de alcoóis secundários racêmicos contendo grupos heterocíclicos foi sintetizada em rendimentos que variaram de moderados a excelentes (40-98%) e submetidos à resolução cinética enzimática catalisada por lipase (RCE). Adicionalmente, um derivado dibromo vinilfurano 2-substituído foi empregado em estudos visando à síntese total da modiolida A, uma macrolactona de origem natural contendo 10 membros em seu anel. Este fragmento foi utilizado em uma reação do tipo Corey-Fuchs para obtenção de um intermediário avançado na síntese desta molécula. / Furan and Tiophene derivatives are widespread compounds in the environment and they also play important role on biochemical process. In addition these compounds can be employed as building blocks in the synthesis of bioactive molecules. However, environmentally harmful reagents and many reaction steps are generally assigned to synthetic methodologies that employ it. Nowadays, few works have been reported enzyme applications in this type of system. Here, we propose a chemoenzymatic methodology to prepare enantioenriched secondary alcohols containing furan and tiophene heterocyclic group and apply them in the preparation of bioactive enantiodependent molecules. The proposed methodology employs a reduced number of synthetic steps and CAL-B enzyme at the key step of deracemization. Initially, the racemic secundary alcohols containing heterocyclic groups were synthesized from moderate to good yields (40-98%) and each of them was submitted to lipase-catalyzed enzymatic kinetic resolution (EKR). Furthermore, a dibromovinylfuran 2-substituted has been employed in studies forward the total synthesis of Modiolide A, a 10-membered ring of natural lactone. This fragment has been used on Corey-Fuchs reaction in order to prepare an advanced intermediate to obtain the Modiolida A.

Orientador: Telma Teixeira Franco / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-07T15:01:29Z (GMT). No. of bitstreams: 1 Tsukamoto_Junko_D.pdf: 1866337 bytes, checksum: 5cac22304ad7fddc116a61d4e57a941f (MD5) Previous issue date: 2006 / Resumo: Reações enzimáticas em meio orgânico têm despertado grande interesse industrial devido às vantagens de obtenção de produtos puros pela maior especificidade das enzimas, pela redução de gastos energéticos e pela possibilidade de desenvolvimento de condições menos agressivas de processo. O uso de enzimas em síntese é uma alternativa viável à síntese orgânica e pode ser compreendida como processo relacionado ao novo conceito de ¿biotecnologia industrial¿ (white biotechnology), devido às preocupações ambientais e da sustentabilidade dos processos químicos. Processos biotecnológicos permitem a exploração racional de materiais renováveis (açúcares) para obtenção de produtos industriais, diminuindo a degradação ambiental. Uma rota enzimática é aqui proposta para obtenção de acrilatos de açúcares, monômero para confecção de polímeros biodegradáveis. O uso de enzimas em síntese orgânica oferece vantagens devido a especificidades na formação de produto, onde os custos de separação, purificação e tratamento de resíduos são minimizados. As lipases são responsáveis por 20% das biotransformações realizadas atualmente. Ésteres derivados de carboidratos e de ácidos graxos vêm sendo produzidos para a indústria alimentícia e farmacêutica, entretanto, mas não se encontram ainda ésteres tais como acrilatos de carboidratos no mercado. Esse projeto visou um estudo da produção de acrilatos de carboidratos pela reação direta via enzimática pela lipase comercial CalB. A esterificação de três monossacarídeos (D-glicose, D-frutose e D-xilose) e dois dissacarídeos (sacarose e maltose) com ácido acrílico pela CalB no solvente terc-butanol foi estudada. O efeito dos três seguintes parâmetros foi avaliado: temperatura, razão molar entre os reagentes (açúcar: ácido acrílico) e quantidade de enzima sobre a conversão a ésteres foi avaliada. A conversão da reação foi acompanhada por cromatografia líquida (CLAE-IR) e por MALDI TOF MS / Abstract: Industrial interest in enzymatic reactions in organic media is due to the possibility of obtain pure compounds with the use of highly specific enzyme, the decrease in energy costs and the possibility of using mild process conditions. The development of new enzymatic processes for chemical synthesis as a viable alternative to the organic synthesis is related to the new concept of "industrial biotechnology" (white biotechnology), with higher concerns with the environment and the sustainability of chemical processes. Biotechnological processes that make a more rational use of renewed resources such as sugars to produce chemicals decrease, in most cases, the environmental impact. In this work an enzymatic route is proposed for the production of sugar acrylates, a biodegradable monomer. The use of enzymes in organic media has the advantage of high specificity to the desired product, lower separation and purification costs, and minimization of residue formation. Currently, lipases are responsible for 20% of all biotransformations. Esters derived from fatty acid and carbohydrates are produced for the food and pharmaceutical industries. However, acrylate esters of carbohydrate are still not found in the market. This aim of this work was to study the production of acrylates of carbohydrates by the direct enzymatic reaction of sugars with acrylic acid on the commercial lipase CalB. The esterification of three monosaccharide (D-glucose, D-fructose and xylose) and two disaccharides (sucrose and maltose) with acrylic acid on CalB in tert-butanol was studied. The effects of temperature, molar ratio of the reactants (sugar to acrylic acid) and amount of enzyme on the conversion to esters were studied. The esterification reaction was monitored by HPLC and MALDI TOF MS / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

Les céramides sont des lipides de la classe des sphingolipides issus de la N-acylation d’une base sphingoide par un acide gras. Ces lipides et leurs analogues suscitent un grand intérêt comme composants actifs dans les industries pharmaceutique et cosmétique. Parmi les biocatalyseurs capables de réaliser la synthèse de ce type de lipide, la lipase B de Candida antarctica semble être l’enzyme la plus adaptée à la production de « pseudo-céramides » à partir d’amino-polyols. Dans ce contexte, nous avons abordé l’étude de l’acylation de composés de type « amino-alcool »catalysée par la lipase B de Candida antarctica, en gardant à l'esprit une approche fondamentale afin d’élargir les connaissances actuelles sur ce sujet. La première partie de notre travail a ainsi traité de l’étude cinétique de l’acylation de composés monofonctionnels afin de déterminer les mécanismes réactionnels et l’énantio sélectivité de la lipase B de Candida antarctica pour les réactions de N-acylation et de O-acylation. Les parties suivantes de notre travail ont porté sur une étude structure-réactivité du substrat accepteur d’acyle et sur l’étude de l’influence du solvant utilisé (solvant organique ou liquide ionique) afin de déterminer les facteurs clés influençant la chimio sélectivité et la régio sélectivité de la lipase B de Candida antarctica lors de l’acylation de composés multifonctionnels de type « amino-alcool ». Finalement, à partir des connaissances acquises dans les différentes parties, nous avons développé et optimisé un procédé de synthèse enzymatique de « pseudo-céramides » (O,N-diacyl aminopropanediols) mis en oeuvre en réacteur continu à « lit fixe ». / Ceramides are lipids from the sphingolipide class derived from the N-acylation of a sphingoid base from a fatty acid. These lipids and their analogs are compounds of interest used as active components in pharmaceutical and cosmetic industries. Among biocatalysts able to synthesize this type of lipids, Candida antarctica lipase B appears to be the most appropriate enzyme for the production of "pseudo-ceramides" derived from amino-polyols. In this context, we have studied the acylation of amino-alcohol-like compounds catalyzed by Candida antarctica lipase B, keeping inmind a fundamental approach to expand the current knowledge on this subject. The first part of our work aimed to determine the reaction mechanisms and the enantio selectivity exhibited by Candida antarctica lipase B during O-acylation and N-acylation reactions, by conducting a kinetic study of mono functional compound acylation. In the following parts of our work, we performed a structurere activity study of the acyl acceptor substrate and evaluated the effect of the solvent used (organic solvent or ionic liquid) to determine the key factors influencing the chemo selectivity and the regio selectivity of the Candida antarctica lipase B-catalyzed acylation of polyfunctional amino alcohol compounds. Finally, starting from the knowledge acquired in the previous parts, we have developed and optimized an enzymatic process of “pseudo-ceramide” (O,N-diacylaminopropanediol) synthesis performed in a continuous packed-bed reactor.

L’objectif de cette thèse était de mieux comprendre les paramètres structuraux et environnementaux gouvernant l’énantiosélectivité de la lipase B de Candida antarctica (CALB), lors de la résolution des alcools secondaires linéaires chiraux. Ces composés sont notamment utilisés comme synthons chiraux dans l’industrie pharmaceutique. Dans un premier temps, une étude systématique des orientations du butan-2-ol et du pentan-2-ol au sein du site actif a été réalisée par modélisation moléculaire. Les résultats suggèrent l’existence de modes d’arrimages supplémentaires à ceux mentionnés dans la littérature. La comparaison selon l’énergie potentielle du substrat des structures les plus stables, combinée à la prise en compte de modes de fixation non productifs, nous a permis d’expliquer qualitativement l’énantiopréférence de la CALB pour la forme R. A l’aide du réacteur solide/gaz, nous avons montré que les résidus hydrophobes formant le canal d’accès du substrat au site actif (Ile189, Leu278 et Ala282), jouaient un rôle significatif dans l’énantiosélectivité de la CALB vis-à-vis des alcools secondaires. L’influence de la partie alkoxy du donneur d’acyle sur le ratio énantiomérique a également pu être mise en évidence. Par ailleurs, nous avons montré que des mutations isostériques de la poche stéréosélective conduisaient à des variations des paramètres thermodynamiques d’activation de l’acylation du pentan-2-ol, probablement suite à des modifications du réseau de liaisons hydrogène formé entre les résidus de la poche. Les études d’adsorption de l'eau et des substrats sur l’enzyme immobilisée nous ont permis de relier l'état d'hydratation de la CALB avec les effets particuliers de l'eau sur l’activité et la sélectivité de l’enzyme. / The aim of this thesis was to understand more precisely the structural and environmental parameters governing the enantioselectivity of lipase B from Candida antarctica (CALB), involved in the discrimination of chiral secondary alcohols enantiomers. These compounds are used in particular for the synthesis of enantiomerically pure pharmaceutical molecules. Initially a systematic study of the orientation of butan-2-ol and pentan-2-ol in the active site was performed by molecular modeling. The results suggest the existence of additional binding modes to those mentioned in the literature. The potential energy comparison of the most stable conformations of the substrate, combined with the existence of non productive binding modes, allowed us to explain qualitatively the enantiopreference of CALB for the R form. Using the solid/gas reactor, we have shown that hydrophobic residues forming the access channel of substrate to the active site (Ile189, Leu278 and Ala282), played a significant role in the enantioselectivity of CALB towards secondary alcohols. The influence of the alkoxy part of the acyl donor on the enatiomeric ratio E has also been highlighted. Furthermore, we showed that isosteric mutations of the stereoselective pocket led to changes in pentan-2-ol activation thermodynamic parameters of acylation, probably due to changes in the hydrogen bonds network formed between residues of the pocket. Studies of water and substrates adsorption on the immobilized enzyme allowed us to relate the hydration status of CALB with specific effects of water on the enzyme activity and selectivity.

Dans un contexte du développement durable, de nouvelles architectures macromoléculaires biosourcées ont été synthétisées à partir de synthons (diacides et diols) pouvant être obtenus par voies fermentaires à partir de sources carbonées issues de la biomasse. Dans un premier temps, différents copolyesters aliphatiques ont été synthétisés en masse, à l’aide d’un catalyseur organométallique à base de titane, à partir de diacides (acides succinique et adipique) et de diols (1,3-propanediol, 1,4-butanediol et 2,3-butanediol) courts. Dans un deuxième temps, des architectures macromoléculaires similaires ont été obtenues par catalyse enzymatique en solution à l’aide de la lipase B de Candida antarctica. L’influence de la longueur et de la structure des monomères sur leur réactivité en présence de la lipase a été particulièrement étudiée. Dans un troisième et dernier temps, des architectures macromoléculaires à base d’oligomères hydroxytéléchéliques d’un polyester bactérien : le poly((R)-3-hydroxybutyrate) (PHB)tels que des poly(ester-éther-uréthane)s et des copolyesters ont été obtenues soit par couplage de chaîne à l’aide d’un diisocyanate, ou par transestérification organométallique et enzymatique. Ces études ont permis d’analyser en détail l’effet de l’addition des synthons biosourcés dans les architectures macromoléculaires et notamment sur la structure cristalline, la stabilité thermique et les propriétés thermiques et optiques de ces polymères. De plus, le grand potentiel de la catalyse enzymatique pour la synthèse de polyesters et celui de l’utilisation d’oligomères de PHB pour l’élaboration de nouveaux matériaux performants ont pu être largement démontrés. / In the context of sustainable development, new biobased and aliphatic macromolecular architectures were synthesized from building blocks that can be obtained by fermentation routes using carbon sources from the biomass. First, several aliphatic copolyesters were synthesized in bulk from short dicarboxylic acids (such as succinic and adipic acids) and diols (such as 1,3-propanediol, 1,4-butanediol and 2,3-butanediol) by organometallic catalysis using an effective titanium-based catalyst. In a second time, similar macromolecular architectures were synthesized by an enzymatic process in solution using Candida antarctica lipase B as catalyst. The influence of the alkyl chain length and the structure of monomers on their reactivity toward the lipase were particularly discussed. In the third and last part, new macromolecular architectures based on hydroxytelechelic oligomers of a bacterial polyester: poly((R)-3-hydroxybutyrate) (PHB), such as poly(ester-ether-urethane)s and copolyesters, were obtained by either chain-coupling using a diisocyanate, or organometallic and enzymatic transesterification, respectively.These studies permitted to determine a close relationship between the effect of the building blocks structure integrated in the final macromolecular architectures and the intrinsic properties, such as the crystalline structure, the thermal stability and the thermal and optical properties, of these polymers. In addition, the great potential of the lipase-catalyzed synthesis of polyesters and the use of PHB oligomers for developing new high performance materials has been clearly established.

<p> </p><p>A chemoselective catalyst is preferred in a chemical reaction where protecting groups otherwise are needed. The two lipases <em>Candida antarctica </em>lipase B and <em>Rhizomucor miehei</em> lipase showed large chemoselectivity ratios, defined as (<em>k_cat</em>/<em>K</em>_M)_OH/ (<em>k_cat</em>/<em>K</em>_M)_SH, in a transacylation reaction with ethyl octanoate as acyl donor and hexanol or hexanethiol as acyl acceptor (<strong>paper I</strong>). 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 <em>Candida antarctica </em>lipase B and ten times higher for <em>Rhizomucor miehei</em> lipase. The <em>K</em>_M towards the thiol was more than two orders of magnitude higher than the <em>K</em>_M towards the corresponding alcohol. This was the dominating contribution to the high chemoselectivity displayed by the two lipases. In a novel approach, <em>Candida antarctica </em>lipase B was used as catalyst for enzymatic synthesis of thiol-functionalized polyesters in a one-pot reaction without using protecting groups (<strong>paper II</strong>). 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.</p><p> </p>