Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 56-75).

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical sciences Candidate: Bc. Monika Šmídlová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Effect of selected cytostatics for the treatment of leukemia on the activity of human carbonyl reducing enzymes Key words: reductases, leukemia, cytostatics, inhibition Anthracycline antibiotics, especially daunorubicin, are widely used for the treatment of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Although the efficacy of these drugs is high, treatment is still limited due to cardiotoxicity and tumor cell resistance to anthracyclines. Mechanisms that contribute to the formation of anthracycline resistance include metabolic biotransformation (reduction) to less efficient secondary alcohols. The reduction is calatyzed by carbonyl reducing enzymes belonging to aldo-keto reductase (AKR) and short chain dehydrogenase/reductase (SDR) superfamilies. The discovery of AKR and SDR inhibitors could help to overcome anthracycline resistance and also reduce cardiotoxicity caused by these drugs. The aim of the diploma thesis was to find out whether all-trans-retinoic acid, cyclophosphamide, cytarabine, cladribine and prednisolone are able to inhibit anthracycline reductases AKR1A1, AKR1B10, AKR1C3, AKR7A2...

As revisões sistemáticas são consideradas os desenhos experimentais capazes de guiar os cuidados em saúde. Contudo, as revisões narrativas ainda são largamente publicadas até o presente momento. No âmbito do tratamento das doenças periodontais, diversos estudos têm reportado que o uso adjuvante de estatinas à terapia periodontal mecânica pode acarretar melhorias nos parâmetros clínicos periodontais, como adicionais reduções de profundidade de sondagem e ganhos de inserção clínica. O objetivo deste trabalho é contextualizar a informação advinda de uma revisão sistemática realizada sobre um tema clínico significativo e compreender qual o seu papel como suporte da atenção ao paciente periodontal, a partir da compreensão obtida no estudo bibliométrico de revisões de literatura. No estudo bibliométrico, uma amostra representativa das revisões de literatura, publicadas na base Scopus, foi selecionada. Tipo de revisão, número de citações, ano de publicação, temática do estudo e outras variáveis foram coletadas. Nesse estudo, observou-se que o número de revisões sistemática tem aumentado significativamente ao longo dos anos quando comparados com o número de revisões narrativas. Apesar disso, o número ajustado de citações das revisões sistemáticas não difere significativamente das recebidas nas revisões narrativas. Já na revisão sistemática de uso adjuvante de estatinas, uma estratégia de busca nas bases Pubmed, Scopus e Embase foi realizada para identificar todos os ensaios clínicos que tenham utilizado estatinas como adjuvantes ao tratamento periodontal mecânico em comparação à terapia periodontal mecânica isolada ou associada a placebo. Quinze estudos foram selecionados. Observou-se que, na maioria dos estudos, o uso adjuvante de estatina apresentou adicionais reduções de profundidade de sondagem e ganhos de inserção clínica quando comparado com seus respectivos grupos controles. Contudo, a alta heterogeneidade desse resultado e o grande número de estudos ser executado por um mesmo grupo de pesquisa são fatores limitadores dessa revisão sistemática. Dessa maneira, pode-se concluir que o número de revisões sistemáticas vem aumentando a longo do tempo, porém o seu número de citações parece não acompanhar as mesmas tendências. Além disso, o uso adjuvante de estatina na terapia periodontal ainda não deve ser recomendado até a execução de outros estudos com melhor qualidade. / Systematic review is considered the experimental design capable of guiding the health care. However, narrative reviews are broadly published nowadays. Regarding periodontal diseases treatment, several studies reported that the adjuvant use of statins to mechanical periodontal treatment may promote additional improvements in clinical periodontal parameters, such as additional reduction in probing depth and clinical attachment gain. This study aimed to contextualize the information obtained in a systematic review about a significant clinical thematic and comprehend is its role in the periodontal care, through information gathered in a bibliometric study of literature review studies in Dentistry. In the bibliometric study, a representative sample of literature reviews studies, published in Scopus database, was selected. Type of review, number of citations, year of publication, study thematic, and other variables were collected. It was showed that the number of systematic reviews increased throughout the years in comparison to narrative reviews. Additionally, the Dentistry clinical fields presented the highest number of published systematic reviews. Despite of that, the mean adjusted number of citations granted for systematic reviews did not differ from the narrative ones. Regarding the performed systematic review, a search strategy was conducted on Pubmed, Scopus, and Embase databases to identify all clinical trials that used statins as adjuvant to mechanical periodontal treatment in comparison to mechanical periodontal treatment alone or in association with placebo. Fifteen studies were included. It was showed that most of the included studies presented additional reduction in probing depth and clinical attachment gain in comparison to their control groups. However, the high heterogeneity among the studies and the high number of studies conducted by the same research group are limitations of the present systematic review. It was concluded that the number of systematic review are increasing dramatically throughout the years, but this trend is not followed by the number of citations granted to this type of study. Furthermore, the adjuvant use of statin in the mechanical periodontal therapy may not be recommended until further well-designed studies have been published.

As revisões sistemáticas são consideradas os desenhos experimentais capazes de guiar os cuidados em saúde. Contudo, as revisões narrativas ainda são largamente publicadas até o presente momento. No âmbito do tratamento das doenças periodontais, diversos estudos têm reportado que o uso adjuvante de estatinas à terapia periodontal mecânica pode acarretar melhorias nos parâmetros clínicos periodontais, como adicionais reduções de profundidade de sondagem e ganhos de inserção clínica. O objetivo deste trabalho é contextualizar a informação advinda de uma revisão sistemática realizada sobre um tema clínico significativo e compreender qual o seu papel como suporte da atenção ao paciente periodontal, a partir da compreensão obtida no estudo bibliométrico de revisões de literatura. No estudo bibliométrico, uma amostra representativa das revisões de literatura, publicadas na base Scopus, foi selecionada. Tipo de revisão, número de citações, ano de publicação, temática do estudo e outras variáveis foram coletadas. Nesse estudo, observou-se que o número de revisões sistemática tem aumentado significativamente ao longo dos anos quando comparados com o número de revisões narrativas. Apesar disso, o número ajustado de citações das revisões sistemáticas não difere significativamente das recebidas nas revisões narrativas. Já na revisão sistemática de uso adjuvante de estatinas, uma estratégia de busca nas bases Pubmed, Scopus e Embase foi realizada para identificar todos os ensaios clínicos que tenham utilizado estatinas como adjuvantes ao tratamento periodontal mecânico em comparação à terapia periodontal mecânica isolada ou associada a placebo. Quinze estudos foram selecionados. Observou-se que, na maioria dos estudos, o uso adjuvante de estatina apresentou adicionais reduções de profundidade de sondagem e ganhos de inserção clínica quando comparado com seus respectivos grupos controles. Contudo, a alta heterogeneidade desse resultado e o grande número de estudos ser executado por um mesmo grupo de pesquisa são fatores limitadores dessa revisão sistemática. Dessa maneira, pode-se concluir que o número de revisões sistemáticas vem aumentando a longo do tempo, porém o seu número de citações parece não acompanhar as mesmas tendências. Além disso, o uso adjuvante de estatina na terapia periodontal ainda não deve ser recomendado até a execução de outros estudos com melhor qualidade. / Systematic review is considered the experimental design capable of guiding the health care. However, narrative reviews are broadly published nowadays. Regarding periodontal diseases treatment, several studies reported that the adjuvant use of statins to mechanical periodontal treatment may promote additional improvements in clinical periodontal parameters, such as additional reduction in probing depth and clinical attachment gain. This study aimed to contextualize the information obtained in a systematic review about a significant clinical thematic and comprehend is its role in the periodontal care, through information gathered in a bibliometric study of literature review studies in Dentistry. In the bibliometric study, a representative sample of literature reviews studies, published in Scopus database, was selected. Type of review, number of citations, year of publication, study thematic, and other variables were collected. It was showed that the number of systematic reviews increased throughout the years in comparison to narrative reviews. Additionally, the Dentistry clinical fields presented the highest number of published systematic reviews. Despite of that, the mean adjusted number of citations granted for systematic reviews did not differ from the narrative ones. Regarding the performed systematic review, a search strategy was conducted on Pubmed, Scopus, and Embase databases to identify all clinical trials that used statins as adjuvant to mechanical periodontal treatment in comparison to mechanical periodontal treatment alone or in association with placebo. Fifteen studies were included. It was showed that most of the included studies presented additional reduction in probing depth and clinical attachment gain in comparison to their control groups. However, the high heterogeneity among the studies and the high number of studies conducted by the same research group are limitations of the present systematic review. It was concluded that the number of systematic review are increasing dramatically throughout the years, but this trend is not followed by the number of citations granted to this type of study. Furthermore, the adjuvant use of statin in the mechanical periodontal therapy may not be recommended until further well-designed studies have been published.

nÃo hà / O aumento nos Ãltimos anos de indivÃduos imunocomprometidos, como portadores da SÃndrome da ImunodeficiÃncia Adquirida, de doenÃas malignas, transplantados e outros usuÃrios de terapias imunossupressoras, favorece o surgimento de infecÃÃes oportunistas, principalmente as de teor fÃngico, como a candidÃase e a criptococose. Apesar de a terapia antifÃngica atual ser eficiente na maioria dos casos, algumas vezes fazem-se necessÃrias novas drogas que atuem como alternativa ou como coadjuvantes no tratamento para potencializar o efeito dos antifÃngicos utilizados. As estatinas sÃo fÃrmacos hipolipemiantes mais prescritos mundialmente para doenÃas cardiovasculares. Entretanto, recentemente, tem sido descritos outros efeitos benÃficos destas drogas, como, por exemplo, o controle de infecÃÃes. Este trabalho teve como objetivo determinar a atividade antifÃngica in vitro das estatinas ante 51 cepas de Candida, sendo 16 de C. albicans, 11 de C. krusei, 12 de C. tropicalis e 12 de C. parapsilosis, e 25 cepas de Cryptococcus, sendo 12 de C. gattii e 13 de C. neoformans, por meio de testes de microdiluiÃÃo em caldo, segundo documento M27-A3 padronizado pelo CLSI. O intervalo de concentraÃÃo testado para pravastatina foi de 50 a 0,0977 mg/mL, para sinvastatina, 1 a 0,0020 mg/mL e para atorvastatina, 10 a 0,0200 mg/mL. Pravastatina inibiu 37 leveduras do gÃnero Candida apresentando concentraÃÃo inibitÃria mÃnima (CIM) na faixa de 1,56 a 6,25 mg /mL e as cepas restantes nÃo foram inibidas mesmo na maior concentraÃÃo testada (50 mg /mL), enquanto que sinvastatina e atorvastatina apresentaram atividade antifÃngica sobre todas as 51 cepas avaliadas, apresentando CIMs de 0,02 a 1 mg / mL e 0,04 a 5,00 mg / mL, respectivamente. Para o gÃnero Cryptococcus, apenas 4 cepas foram inibidas ante a pravastatina (CIM = 25 mg / mL), por outro lado, sinvastatina inibiu todas as 25 cepas (CIM = 0,06 a 1 mg / mL), e atorvastatina apenas 8 cepas (CIM = 0,62 a 2,5 mg / mL), sendo que as 17 restantes nÃo foram inibidas mesmo na maior concentraÃÃo testada ( ≥ 10 mg / mL). Foi determinada concentraÃÃo fungicida mÃnima (CFM) de pravastatina sobre 15 cepas do gÃnero Candida (CFM = 3,12 a 25 mg / mL), de sinvastatina sobre 34 cepas (CFM = 0,03 a 1 mg / mL), e de atorvastatina sobre 16 cepas (CFM = 0,04 a 0,31 mg / mL). Para o gÃnero Cryptococcus, das 25 cepas testadas, pravastatina exibiu CFM sobre apenas 3 cepas (CFM = 50 mg / mL), sinvastatina sobre 21 cepas (CFM = 0,12 a 1 mg / mL), e atorvastatina sobre 1 cepa (CFM = 1 mg / mL). Esta atividade inibitÃria in vitro de estatinas sobre espÃcies de Candida e Cryptococcus, abre uma perspectiva importante para a investigaÃÃo do possÃvel uso destas drogas com finalidade antifÃngica in vivo. / In the past years, fungal opportunistic infections, especially, candidiasis and cryptococcosis, have become more frequent because of the increase in the number of immunocompromised individuals, such as AIDS, transplant and cancer patients and those that are on immunosuppressive therapy. In spite of being effective, sometimes it is necessary to use new drugs as alternatives or as adjuvants in order to potentiate the effect of the classical antifungal therapy. Statins are the most prescribed hypolipemiant drugs worldwide for preventing cardiovascular diseases. However, other benefic effects for these drugs have been described, such as the control of infections. This work aimed at determining the antifungal activity of statins against Candida spp. and Cryptococcus spp. The minimum inhibitory concentrations (MICs) for three different statins (pravastatin, simvastatin and atorvastatin) were determined against 51 strains of Candida spp. (16 C. albicans, 11 C.krusei, 12 C. tropicalis and 12 C. parapsilosis) and 25 strains of Cryptococcus spp. (12 C. gattii and 13 C. neoformans), through broth microdilution assay, according to the Clinical Laboratory Standards Institute (CLSI - Document M27-A3). The concentration tested for pravastatin ranged from 50 to 0.0977 mg/mL, for simvastatin, it ranged from 1 to 0.0020 mg/mL and, for atorvastatin, it varied from 10 to 0.0200 mg/mL. Pravastatin inhibited 37 Candida strains, with MICs varying from 1.56 to 6.25 mg/mL and the remaining strains were not inhibited, even at the highest concentration tested (50 mg/mL). Simvastatin and atorvastatin, on the other hand, inhibited all 51 Candida strains evaluated, presenting MICs ranging from 0.02 to 1 mg/mL and from 0.04 to 5 mg/mL, respectively. Concerning Cryptococcus spp., only four strains were inhibited by pravastatin (MIC=25 mg/mL), while all 25 strains were inhibited by simvastatin (0.06≤MIC≤1 mg/mL) and eight were inhibited by atorvastatin (0.62≤MIC≤2.5 mg/mL) and the remaining 17 were not susceptible to the highest atorvastatin concentration tested (10 mg/mL). The minimum fungicidal concentrations (MFCs) for the tested statins were also determined. The MFC for pravastatin against Candida spp. was determined against 15 strains (3.12≤MIC≤25 mg/mL). The MFC values for simvastatin were determined for 34 strains of Candida spp. (0.03≤MFC≤1 mg/mL), while those for atorvastatin were determined against 16 strains (0.04≤MFC≤0,31 mg/mL). Concerning Cryptococcus spp., the 25 strains tested, MFC values for pravastatin were found against three strains (MFC=50 mg/mL), while those for simvastatin were determined against 21 strains (0.12≤MFC≤1 mg/mL) and those for atorvastatin were determined against one single strain (MFC=1 mg/mL). This in vitro inhibitory activity of statins against Candida spp. and Cryptococcus spp. creates an important perspective for the use of these drugs in vivo in order to control fungal infections.

Mycobacterium avium is human and animal opportunistic pathogen responsible for disseminated disease in immunocompromised patients. Mycobacteria have a capacity to adapt to the environmental conditions by inducing enzyme activities and altering their metabolism. M. avium A5 cells were grown in a defined minimal medium (Nitrogen Test Medium) with glutamine, nitrite, nitrate, or ammonia as sole nitrogen source at a concentration of 2 mM at 370C aerobically. The strain grew well on all the nitrogen sources except nitrite. It grew slowly on nitrite with a generation time of 6 days and cultures were not viable after 4 weeks of storage. These data confirm that M. avium can utilize a single nitrogen source in a defined minimal medium as documented by McCarthy (1987). M. avium genome has been sequenced and contains genes sharing sequence similarities to respiratory nitrate reductase and dissimilatory nitrite reductases. Because, M. avium can use nitrate or nitrite as sole nitrogen source for growth (McCarthy, 1987), it must have assimilatory nitrate- and nitrite-reductases. Nitrate- and nitrite-reductase activities of M. avium cells growing aerobically or undergoing anaerobic shift in the presence of ammonia, nitrate or ammonia and nitrate in combination were measured. M. avium produced nitrate- as well as nitrite-reductase activity. Nitrite- and nitrate-reductases used either NADH or NADPH as an electron donor. Nitrite reductase activity was greater than nitrate reductase activity. This observation supports the rapid reduction of nitrite and slow reduction of nitrate in M. avium as documented by McCarthy (1987) and explained why M. avium gives a negative result by the standard nitrate reductase test. In addition to assimilatory enzyme activity, M. avium A5 also produced dissimilatory nitrate- and nitrite-reductase activities. / Master of Science

Les aldose réductases (AKR1B) sont des oxydoréductases dépendantes du NADPH initialement décrites pour leurs fonctions de détoxication cellulaire et de réduction du glucose. La découverte de l’expression d’Akr1b7 dans le tissu adipeux murin ainsi que l’activité prostaglandine F2α synthase (PGFS) spécifique de certaines isoformes suggèrent des rôles biologiques inédits pour ces enzymes. La prostaglandine F2α (PGF2α) inhibant l’adipogenèse, cette fonction PGFS met en avant l’implication des AKR1B dans la physiologie du tissu adipeux blanc (TAB). L’objectif de ces travaux était de caractériser l’expression de l’ensemble des AKR1B au sein des TAB murins et humains et de comprendre leur impact sur l’homéostasie du tissu adipeux et en particulier sur l’adipogenèse et la lipolyse. Nous avons montré que l’ensemble des AKR1B était exprimé dans le TAB murin. Akr1b3, Akr1b8 et Akr1b16 sont exprimées à la fois dans les fractions stroma‑vasculaires (contenant des cellules immunitaires, vasculaires, progénitrices…) et adipocytaires. A l’inverse, Akr1b7 n’est pas exprimé par les adipocytes. Les analyses réalisées in vitro indiquent qu’à l’exception d’Akr1b16, les isoformes murines des AKR1B voient leur expression augmenter précocement et transitoirement au cours de l’adipogenèse. Chez l’homme, l’isoforme AKR1B1 est exprimée dans le TAB sous‑cutané de patients obèses alors qu’AKR1B10 est difficilement détectable (western blot, RT‑qPCR). In vitro, l’expression d’AKR1B1 augmente tout au long de la différenciation adipocytaire contrairement à AKR1B10 qui est préférentiellement exprimé dans les cellules indifférenciées. L’utilisation d’un inhibiteur spécifique des AKR1B montre que l’activité PGFS d’AKR1B1 constitue un frein à l’adipogenèse. Nous montrons aussi que les mécanismes régulant l’action de la PGF2α diffèrent en fonction des espèces. Chez l’homme, l’expression du récepteur FP est régulée dans le temps alors que dans les cellules murines, c’est l’expression des PGFS et donc la synthèse de PGF2α qui définit, au cours de l’adipogenèse, la fenêtre d’action de cette prostaglandine. Les souris invalidées pour la PGFS Akr1b7 présentent une diminution des quantités intra‑tissulaires en PGF2α associée à une expansion accrue de leurs tissus adipeux due à une augmentation de l’adipogenèse et à une hypertrophie adipocytaire sans modification de l’expression des enzymes impliquées dans la lipogenèse (Volat et al., 2012). Ces données en accord avec le rôle anti‑adipogénique de la PGF2α suggèrent aussi une action sur la lipolyse. Nous démontrons ici que la perte d’Akr1b7 entraîne une diminution de l’activité lipolytique du TAB. L’utilisation de cellules murines (3T3‑L1) et humaines (hMADS) différenciées en adipocytes, nous a permis de montrer que la stimulation de l’activité lipolytique suite à l’activation du récepteur FP résultait en partie d’une augmentation de la phosphorylation de HSL (forme active) et de l’accumulation de la lipase ATGL. Le troisième volet de ce travail de thèse a consisté à caractériser un modèle de souris transgénique surexprimant AKR1B1 dans le TAB (souris aP2‑AKR1B1) afin d’étudier le rôle biologique de cette isoforme humaine. / Aldose reductases are NADPH-dependent oxydoreductases described for their involvement in cellular detoxification and glucose reduction. The discovery of Akr1b7 expression in murine adipose tissue together with the prostaglandin F2α Synthase (PGFS) activity of some isoforms suggest unreleased biological roles for these enzymes. Prostaglandin F2α (PGF2α) inhibiting adipogenesis, this PGFS function highlights AKR1B potential involvement in white adipose tissue (WAT) physiology. This work aimed at characterising the expression of all AKR1B in both murine and human WAT and understanding their impact on adipose tissue homeostasis and especially on adipogenesis and lipolysis. We showed that all AKR1B were expressed in murine WAT. Akr1b3, Akr1b8 and Akr1b16 were both expressed in the stromal vascular fraction (containing immune cells, vascular cells, progenitors…) and in the adipose fraction. In contrast, Akr1b7 was not expressed in adipocytes. In vitro analyses indicated that, except for Akr1b16, murine AKR1B isoform expression increased early and transiently during adipogenesis. In human, AKR1B1 was expressed in human subcutaneous WAT from obese patients whereas AKR1B10 was hardly detectable (western blot, RT‑qPCR). In vitro, AKR1B1 expression increased throughout adipocyte differentiation unlike AKR1B10, which was preferentially expressed in undifferentiated cells. Using an AKR1B specific inhibitor, we demonstrated that AKR1B1 PGFS activity was a dampen to adipogenesis. We also showed that mechanisms regulating PGF2α action differed according to the species. In human cells, the expression of FP receptor was time-regulated whereas, in murine cells, PGFS expression and thus, PGF2α synthesis, limited PGF2α activity during adipogenesis. Akr1b7 knockout mice have decreased PGF2α intratissular levels associated with an expansion of adipose tissue resulting from an increase of adipogenesis and an adipocyte hypertrophia without any modification of lipogenic enzymes expression (Volat et al., 2012). These data, in agreement with PGF2α anti-adipogenic action, suggest an impact on lipolysis. We demonstrated that loss of Akr1b7 led to a decrease of WAT lipolytic activity. The use of murine (3T3‑L1) and human (hMADS) differentiated cells allowed us to show that the stimulation of lipolysis in response to FP activation was, in part, due to an increase of HSL phosphorylation (active form) and an increase of ATGL accumulation. The third part of this work consisted in characterizing the phenotype of transgenic mice overexpressing AKR1B1 in WAT (aP2‑AKR1B1 mice) in order to study the biological role of this human isoform.

Novel biocatalysts for the enantioselective reduction of imines and reductive amination of a broad range of carbonyl compounds have been developed. Unlike other imine reductases (IREDs), the IRED from Amycolaptosis orientalis (AoIRED) features an aprotic "catalytic" residue Asn171 and as such became an interesting candidate for detailed mechanistic, specificity and stereoselectivity studies. AoIRED has been shown to be an efficient catalyst for the enantioselective reduction of imines and iminium ions to yield the corresponding chiral amines in high conversions and good to excellent enantioselectivity. The enzyme exhibits unusual stereoselective properties, displaying a selectivity switch for structurally similar substrates and in certain cases for the same substrate depending on the age of the enzyme. Mutagenesis studies have highlighted important residues that may play key roles in the substrate specificity and stereoselectivity of the enzyme. The reductive aminase from Aspergillus oryzae (AspRedAm) is a multifunctional catalyst that efficiently catalyses i) the reductive coupling of carbonyl compounds and amine nucleophiles, ii) the enantioselective reduction of prochiral cyclic and preformed imines or iii) the oxidative deamination of amines towards kinetic resolution of racemic amines. Detailed kinetic studies have led to the construction of a kinetic model/mechanism and based on structure guided investigation of conserved active site residues, a putative catalytic mechanism has been proposed. It has also been possible to engineer wild-type AspRedAm for improved stereoselectivity as well as to invert the enzyme's enantioselectivity towards a range of substrates. Using AspRedAm as a catalyst, efficient systems have been developed that allow the kinetic resolution of several racemic amines. This thesis has been organised into separate chapters each addressing a specific theme. Chapter 1 gives an overview of recent advances in the field of amine biocatalysis with emphasis on biocatalytic imine reduction and reductive amination; it also outlines the objectives of this project. Chapter 2 describes methods and materials used in these studies while Chapters 3-7 present and discuss results from different projects that constitute the work in this thesis. Initial discovery and characterisation studies of IREDs are described in Chapter 3. Chapter 4 describes detailed characterisation of AoIRED with particular emphasis on stereoselectivity and synthetic applicability while Chapter 5 presents and discusses results from the study of the reductive aminase (AspRedAm) from Aspergillus oryzae. Chapters 6 and 7 respectively describe the engineering of AoIRED and AspRedAm, and the application of AspRedAm in kinetic resolution of racemic amines. The results from these chapters have been summarised and discussed in Chapter 8 and recommendations for future directions in this field have been offered.

Asymmetric synthesis with biocatalyst has become an increasingly interesting and cost effective manufacturing process in fine chemicals, pharmaceuticals, and agrochemical intermediates. Enoate reductases from the Old Yellow Enzyme family offer high substrate efficiency, region, stereo-, and enantioselectivity in the catalyzed biotransformations. Asymmetric reduction of activated C=C bond is one of the most widely applied synthetic tools for the potential to generate up to two stereogenic centers in one step reaction. The thesis contributed to the development and characterization of the Old Yellow Enzyme family members including NRSal from Salmonella typhimurium, YersER from Yersinia bercoviei, KYE1 from Kluyveromyces lactis, and XenA from Pseudomonas putida. We explored the possible new chemistry, gathered further understanding of enzymes functionality and biochemistry, evaluated parameters such as enzyme stability, productivity, and selectivity, and improved enzyme specificity through computational guided protein engineering method. In overall, the increasing knowledge about this Old Yellow Enzyme family together with recent advances in biotechnology renders the enoate reductases a tool of choice for industrial applications.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 5 uppsatser.