Hyperthermophiles grow optimally at 80 ??C and above, and many of them have the ability to utilize various carbohydrates as carbon source and produce ethanol as an end product. Alcohol dehydrogenase (ADH) is a key enzyme responsible for alcohol production, catalyzing interconversions between alcohols and corresponding ketones or aldehydes. ADHs from hyperthermophiles are of great interests due to their thermostability, high activity and enantioselectivity. The gene encoding ADH from hyperthermophilic archaeon Thermococcus guaymasensis was cloned, sequenced and over-expressed. DNA fragments of the genes encoding the ADHs were amplified directly from the corresponding genomic DNA by combining the use of conventional and inverse PCRs. The entire gene was detected to be 1092 bp and the deduced amino acid sequence had a total of 364 amino acids with a calculated molecular mass of 39463 Dalton. The enzyme belonged to the family of zinc-containing ADHs with catalytic zinc only. It was verified that the enzyme had binding motifs of catalytic zinc only (GHEX2GX5GX2V, residues 62-76) and coenzyme NADP (GXGX2G, residues 183-188). The tertiary structural modeling showed two typical domains, one catalytic domain close to amino-terminal (N-terminal) end and one coenzyme-binding domain close to carboxy-terminal (C-terminal) end. Since its codon usage pattern seemed to be different from that of Escherichia coli, the enzyme was over-expressed in the E. coli codon plus strain using pET-30a vector. The recombinant enzyme was detected to be soluble and active (1073 U/mg), which was virtually the same to the native enzyme (1049 U/mg). The recombinant ADH possessed almost identical properties with the native enzyme. The optimal pHs for ethanol oxidation and acetaldehyde reduction were 10.5 and 7.5 respectively, while the activity for alcohol oxidation was much higher than that of aldehyde reduction. The enzyme activity was inhibited in the presence of 100 ??M Zn2+ in the assay mixture and it has a half-life of 6 hours after exposure to air. Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90 ??C. The gene encoding an alcohol dehydrogenase from T. hypogea was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained successfully by sequencing all the DNA fragments amplified from PCR. The deduced amino acid sequence was found to have high degrees of identity (~72%) to iron-containing ADHs from Thermotoga species and harbored typical iron and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. The structural modeling showed that N-terminal domain of ThADH contained ??/??-dinucleotide-binding motif and its C-terminal domain was ??-helix-rich region including iron-binding motif. The gene encoding T. hypogea ADH was functionally expressed in E. coli using the vector pET-30a. The recombinant protein was expressed optimally in E. coli grown in the presence of 1 mM ferrous and induced by 0.4-0.6 mM IPTG. The recombinant enzyme was found to be soluble, active and thermostable, and had a subunit size of 43 kDa revealed by SDS-PAGE analyses. The native ADH from T. hypogea was purified to homogeneity for comparative analysis using a three-step liquid chromatography while the recombinant ADH over-expressed in E. coli was isolated by a simpler procedure including one-hour heat treatment. The activity of the purified recombinant enzyme was 69 U/mg and presented almost identical properties with the native enzyme. The optimal pHs for ethanol oxidation and acetaldehyde reduction were 11.0 and 8.0 respectively, while activity for alcohol oxidation were higher than that of aldehyde reduction. The enzyme was oxygen sensitive and it had a half-life (t1/2) of 20 minutes after exposed to air. The enzyme remained 50% activity after incubation at 70 ??C for 2 hours. Successful high-level expression of T. hypogea ADH in E. coli will significantly facilitate further study on the catalytic mechanism of iron-containing ADHs. In summary, both zinc- and iron-containing ADHs from two hyperthermophiles were successfully cloned, sequenced and overexpressed in mesophilic host E. coli, and such a high-level expression of ADH genes provides possibilities for three dimensional structural analysis by X-ray crystallography and enzyme modification by mutagenesis, which will help further explore mechanisms of catalysis and protein thermostability of iron and zinc-containing ADHs and their potential applications in biotechnology.

Diante da biodiversidade de micro-organismos existentes na natureza e da necessidade de descobrir novos biocatalisadores para a síntese de bloco de construções quirais e de produtos químicos de alto valor agregado, o presente trabalho teve como objetivo realizar a bioprospeção de micro-organismos para aplicação em reações de oxidação enantiosseletiva de álcoois. Os micro-organismos foram obtidos pelo processo de isolamento induzido de amostras de solo/sedimento coletadas no Parque Estadual Turístico do Alto Ribeira (PETAR) e na Antártica. A partir das amostras coletadas (43 de solo e 13 de sedimento) foram isolados 130 microorganimos mesofílicos (isolados de amostras de solo do PETAR) e 232 microorganimos psicrofílicos/psicrotróficos (isolados de amostras de solo/sedimento da Antártica). Realizamos também a avaliação do espectro de atividade enzimática dos micro-organismos em reações de oxidação enantiosseletiva, e para isso empregamos derivados para substituídos do (R,S)-1-(fenil)etanol. Dentre os micro-organismos estudados, 15 psicrofílicos/psicrotróficos e 11 micro-organismos mesofílicos apresentaram álcool desidrogenases que catalisaram a oxidação do enantiômero (S) do álcool racêmico à sua correspondente cetona. Nesses casos, as linhagens possuem atividade de álcool desidrogenase em que o rendimento de obtenção da cetona foi > 10 % e excesso enantiomérico > 60 %. Por outro lado 6 micro-organismos mesofílicos apresentaram álcool desidrogenases que catalisam a oxidação do enantiômero (R) do álcool racêmico à sua correspondente cetona. Foi realizada a caracterização taxonômica para alguns micro-organismos através do sequenciamento do 16S rDNA. Dentre os micro-organismos caracterizados, destacaram-se as bactérias Flavobacterium sp., Arthrobacter sp., Acinetobacter sp., e o Bacillus sp. por apresentar excelente atividade enzimática. Flavobacterium sp. e a Arthrobacter sp. foram selecionadas para o estudo de otimização da reação de oxidação do (R,S)-1-(4- metilfenil)etanol. Nesse processo foram utilizadas as células ressuspensas em tampão fosfato em diferentes temperaturas (5-30 ºC) e tempos reacionais (24-72 horas). A partir desse estudo, observou-se que a Flavobacterium sp. possui uma excelente atividade enzimática a 10 ºC e a Arthrobacter sp. a 25 ºC. Foram determinadas curvas de crescimentos destas bactérias em 15, 20 e 25 ºC, em que ambas apresentaram crescimento ótimo a 25 ºC, indicando que estas bactérias são psicrotróficas / In the view of the microorganisms biodiversity in nature and the necessity to discover new biocatalysts for chiral synthesis of building blocks and high value-added chemical products, the present work was aimed at making the bioprospection of microorganisms for application in enantioselective oxidation reactions of alcohols. Microorganisms were isolated by enrichment technique from soil/sediment samples collected from the State Park of Alto Ribeira (PETAR) and Antarctic. 130 Mesophilic microorganisms were isolated from soil samples of PETAR, and 232 psychrophilic/psychrotrophic microorganisms from soil/sediment samples of Antarctic. We also evaluated the enzymatic activity of the microorganisms in enantioselective oxidation reactions, by using derivatives of substituted para (RS)-1- phenylethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotrophic and 11 mesophilic strains contain alcohol dehydrogenases that catalyze the (S)-enantiomer oxidation of racemic alcohols to its corresponding ketone. In these cases, the strains showed alcohol dehydrogenase activity in which the ketone yields were higher than 10 % and the enantiomeric excess >60 %. Moreover, 6 mesophilic microorganisms showed alcohol dehydrogenases that catalyze the R)-enantiomer oxidation of racemic alcohols to its corresponding ketone. It was performed taxonomic characterization for some microorganisms by sequencing the 16S rDNA. Among the characterized microorganisms, Flavobacterium sp., Arthrobacter sp., Acinetobacter sp. and Bacillus sp. showed excellent enzymatic activity. The Flavobacterium sp. and Arthrobacter sp. were selected for optimization study of oxidation of the (R,S)-1-(4-methyl-phenyl)ethanol. In this process, bacterial cells were resuspended in phosphate buffer at different temperatures (5-30 °C) and reaction times (24-72 h). From these studies, it was observed that the Flavobacterium sp. has an excellent enzymatic activity at 10 ºC and Arthrobacter sp. at 25 ºC. We determined the growth curves of these bacteria in 15, 20 and 25 ° C. Both strains showed optimum growth at 25 ° C, indicating that these bacteria are psychrotrophics.

Neste trabalho, foi avaliada a reatividade de compostos orgânicos contendo boro frente a reações catalisadas por enzimas. Diferentes exemplos de álcoois secundários quirais contendo boro foram acetilados enantiosseletivamente pela lipase de Candida antarctica (CALB). Todas as reações ocorreram com excelente enantiosseletividade (E >200) e ambos os produtos acetilados, bem como os álcoois remanescentes foram obtidos com alto excesso enantiomérico (ee >99%). Além da resolução cinética enzimática, foi avaliado também o processo de resolução cinética dinâmica quimio-enzimática, empregando complexos de rutênio como agentes de racemização. Após estabelecer as condições adequadas para a reação, foi possível obter o produto acetilado de configuração-(R) com 83% de conversão, mantendo a enantiosseletividade observada na reação de resolução cinética enzimática. Como extensão ao trabalho realizado com os álcoois contendo boro, foi estudada a resolução cinética de aminas primárias quirais contendo boro. Foram avaliadas diversas condições reacionais para alcançar a máxima resolução cinética destas aminas via acilação enantiosseletiva catalisada pela lipase CALB. Excelente enantiosseletividade (E >200) e altos excessos enantioméricos (até >99%) foram obtidos utilizando acetato de etila tanto como reagente doador de acila como solvente da reação. Adicionalmente, foi investigada a reação de biorredução de cetonas pró-quirais contendo boro. Os melhores resultados foram obtidos com as enzimas álcool desidrogenases (ADHs) purificadas dos microorganismos Rhodococcus ruber e Lactobacillus brevis. A ADH de R. ruber (ADH-A) promoveu a biorredução das cetonas aos respectivos álcoois de configuração-(R) com excelente enantiosseletividade (ee >99%), enquanto a ADH de L. brevis (ADH-LB) catalisou a redução de alguns exemplos de cetonas aos respectivos álcoois de configuração-(S), também com excelente enantiosseletividade. / In this work, was evaluated the reactivity of boron-containing organic compounds in enzyme-catalyzed reactions. Different examples of boron-containing chiral secondary alcohols were resolved by enantioselective acetylation mediated by lipase from Candida. antarctica (CALB). All reactions showed excellent enantioselectivities (E >200) and both remaining substrates and acetylated product were obtained in high enantiomeric excesses (up to >99%). Besides the enzymatic kinetic resolution, the chemoenzymatic dynamic kinetic resolution was also evaluated, using ruthenium complexes as racemization agents. After establishing the best conditions, the acetylated (R)-product was obtained with 83% conversion, maintaining the high enantioselectivity observed in the enzymatic kinetic resolution. As an expansion of work with boron-containing alcohols, the kinetic resolution of boron-containing chiral amines was studied. Several reaction conditions were studied to achieve the kinetic resolution of boron-containing amines via enantioselective acylation mediated by CAL-B. Excellent enantioselectivity (E >200) and high enantiomeric excess (up to >99%) of both the remaining amines and amides were obtained using ethyl acetate as both acyl donor and the reaction solvent. Additionally, the bioreduction of pro-chiral boron-containing ketones was investigated. The best results were obtained with purified alcohol dehydrogenases (ADH) from Rhodococcus ruber and Lactobacillus brevis. The ADH from R. ruber (ADH-A) mediated the bioreduction of ketones to their respective (R)-alcohols with excellent enantioselectivity (ee >99%), while the ADH from L. brevis (ADH-LB) catalyzed the reduction of some ketones to their respective (S)-alcohols, also with excellent enantioselectivity (ee >99%).

Neste trabalho, foi avaliada a reatividade de compostos orgânicos contendo boro frente a reações catalisadas por enzimas. Diferentes exemplos de álcoois secundários quirais contendo boro foram acetilados enantiosseletivamente pela lipase de Candida antarctica (CALB). Todas as reações ocorreram com excelente enantiosseletividade (E >200) e ambos os produtos acetilados, bem como os álcoois remanescentes foram obtidos com alto excesso enantiomérico (ee >99%). Além da resolução cinética enzimática, foi avaliado também o processo de resolução cinética dinâmica quimio-enzimática, empregando complexos de rutênio como agentes de racemização. Após estabelecer as condições adequadas para a reação, foi possível obter o produto acetilado de configuração-(R) com 83% de conversão, mantendo a enantiosseletividade observada na reação de resolução cinética enzimática. Como extensão ao trabalho realizado com os álcoois contendo boro, foi estudada a resolução cinética de aminas primárias quirais contendo boro. Foram avaliadas diversas condições reacionais para alcançar a máxima resolução cinética destas aminas via acilação enantiosseletiva catalisada pela lipase CALB. Excelente enantiosseletividade (E >200) e altos excessos enantioméricos (até >99%) foram obtidos utilizando acetato de etila tanto como reagente doador de acila como solvente da reação. Adicionalmente, foi investigada a reação de biorredução de cetonas pró-quirais contendo boro. Os melhores resultados foram obtidos com as enzimas álcool desidrogenases (ADHs) purificadas dos microorganismos Rhodococcus ruber e Lactobacillus brevis. A ADH de R. ruber (ADH-A) promoveu a biorredução das cetonas aos respectivos álcoois de configuração-(R) com excelente enantiosseletividade (ee >99%), enquanto a ADH de L. brevis (ADH-LB) catalisou a redução de alguns exemplos de cetonas aos respectivos álcoois de configuração-(S), também com excelente enantiosseletividade. / In this work, was evaluated the reactivity of boron-containing organic compounds in enzyme-catalyzed reactions. Different examples of boron-containing chiral secondary alcohols were resolved by enantioselective acetylation mediated by lipase from Candida. antarctica (CALB). All reactions showed excellent enantioselectivities (E >200) and both remaining substrates and acetylated product were obtained in high enantiomeric excesses (up to >99%). Besides the enzymatic kinetic resolution, the chemoenzymatic dynamic kinetic resolution was also evaluated, using ruthenium complexes as racemization agents. After establishing the best conditions, the acetylated (R)-product was obtained with 83% conversion, maintaining the high enantioselectivity observed in the enzymatic kinetic resolution. As an expansion of work with boron-containing alcohols, the kinetic resolution of boron-containing chiral amines was studied. Several reaction conditions were studied to achieve the kinetic resolution of boron-containing amines via enantioselective acylation mediated by CAL-B. Excellent enantioselectivity (E >200) and high enantiomeric excess (up to >99%) of both the remaining amines and amides were obtained using ethyl acetate as both acyl donor and the reaction solvent. Additionally, the bioreduction of pro-chiral boron-containing ketones was investigated. The best results were obtained with purified alcohol dehydrogenases (ADH) from Rhodococcus ruber and Lactobacillus brevis. The ADH from R. ruber (ADH-A) mediated the bioreduction of ketones to their respective (R)-alcohols with excellent enantioselectivity (ee >99%), while the ADH from L. brevis (ADH-LB) catalyzed the reduction of some ketones to their respective (S)-alcohols, also with excellent enantioselectivity (ee >99%).

Diante da biodiversidade de micro-organismos existentes na natureza e da necessidade de descobrir novos biocatalisadores para a síntese de bloco de construções quirais e de produtos químicos de alto valor agregado, o presente trabalho teve como objetivo realizar a bioprospeção de micro-organismos para aplicação em reações de oxidação enantiosseletiva de álcoois. Os micro-organismos foram obtidos pelo processo de isolamento induzido de amostras de solo/sedimento coletadas no Parque Estadual Turístico do Alto Ribeira (PETAR) e na Antártica. A partir das amostras coletadas (43 de solo e 13 de sedimento) foram isolados 130 microorganimos mesofílicos (isolados de amostras de solo do PETAR) e 232 microorganimos psicrofílicos/psicrotróficos (isolados de amostras de solo/sedimento da Antártica). Realizamos também a avaliação do espectro de atividade enzimática dos micro-organismos em reações de oxidação enantiosseletiva, e para isso empregamos derivados para substituídos do (R,S)-1-(fenil)etanol. Dentre os micro-organismos estudados, 15 psicrofílicos/psicrotróficos e 11 micro-organismos mesofílicos apresentaram álcool desidrogenases que catalisaram a oxidação do enantiômero (S) do álcool racêmico à sua correspondente cetona. Nesses casos, as linhagens possuem atividade de álcool desidrogenase em que o rendimento de obtenção da cetona foi > 10 % e excesso enantiomérico > 60 %. Por outro lado 6 micro-organismos mesofílicos apresentaram álcool desidrogenases que catalisam a oxidação do enantiômero (R) do álcool racêmico à sua correspondente cetona. Foi realizada a caracterização taxonômica para alguns micro-organismos através do sequenciamento do 16S rDNA. Dentre os micro-organismos caracterizados, destacaram-se as bactérias Flavobacterium sp., Arthrobacter sp., Acinetobacter sp., e o Bacillus sp. por apresentar excelente atividade enzimática. Flavobacterium sp. e a Arthrobacter sp. foram selecionadas para o estudo de otimização da reação de oxidação do (R,S)-1-(4- metilfenil)etanol. Nesse processo foram utilizadas as células ressuspensas em tampão fosfato em diferentes temperaturas (5-30 ºC) e tempos reacionais (24-72 horas). A partir desse estudo, observou-se que a Flavobacterium sp. possui uma excelente atividade enzimática a 10 ºC e a Arthrobacter sp. a 25 ºC. Foram determinadas curvas de crescimentos destas bactérias em 15, 20 e 25 ºC, em que ambas apresentaram crescimento ótimo a 25 ºC, indicando que estas bactérias são psicrotróficas / In the view of the microorganisms biodiversity in nature and the necessity to discover new biocatalysts for chiral synthesis of building blocks and high value-added chemical products, the present work was aimed at making the bioprospection of microorganisms for application in enantioselective oxidation reactions of alcohols. Microorganisms were isolated by enrichment technique from soil/sediment samples collected from the State Park of Alto Ribeira (PETAR) and Antarctic. 130 Mesophilic microorganisms were isolated from soil samples of PETAR, and 232 psychrophilic/psychrotrophic microorganisms from soil/sediment samples of Antarctic. We also evaluated the enzymatic activity of the microorganisms in enantioselective oxidation reactions, by using derivatives of substituted para (RS)-1- phenylethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotrophic and 11 mesophilic strains contain alcohol dehydrogenases that catalyze the (S)-enantiomer oxidation of racemic alcohols to its corresponding ketone. In these cases, the strains showed alcohol dehydrogenase activity in which the ketone yields were higher than 10 % and the enantiomeric excess >60 %. Moreover, 6 mesophilic microorganisms showed alcohol dehydrogenases that catalyze the R)-enantiomer oxidation of racemic alcohols to its corresponding ketone. It was performed taxonomic characterization for some microorganisms by sequencing the 16S rDNA. Among the characterized microorganisms, Flavobacterium sp., Arthrobacter sp., Acinetobacter sp. and Bacillus sp. showed excellent enzymatic activity. The Flavobacterium sp. and Arthrobacter sp. were selected for optimization study of oxidation of the (R,S)-1-(4-methyl-phenyl)ethanol. In this process, bacterial cells were resuspended in phosphate buffer at different temperatures (5-30 °C) and reaction times (24-72 h). From these studies, it was observed that the Flavobacterium sp. has an excellent enzymatic activity at 10 ºC and Arthrobacter sp. at 25 ºC. We determined the growth curves of these bacteria in 15, 20 and 25 ° C. Both strains showed optimum growth at 25 ° C, indicating that these bacteria are psychrotrophics.

Ph. D. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Hydrocarbon pollution is becoming a growing environmental concern in South Africa and globally. This inadvertently supports the need to identify enzymes for their targeted degradation. The search for novel biocatalysts such as monooxygenases, alcohol dehydrogenases and aldehyde dehydrogenases, have relied on conventional culture-based techniques but this allows sourcing of the biomolecules from only 1-10 % of the microbial population leaving the majority of the biomolecules unaccounted for in 90-99 % of the microbial community. The implementation of a metagenomics approach, a culture-independent technique, ensures that more or less than 100 % of the microbial community is assessed. This increases the chance of finding novel enzymes with superior physico-chemical and catalytic traits. Hydrocarbon polluted soils present a rich environment with an adapted microbial diversity. It was thus extrapolated that it could be a potential source of novel monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH) involved in hydrocarbon degradation pathways. Therefore, the aim of the study was to extract metagenomic DNA from hydrocarbon contaminated soils and construct a metagenomic fosmid library and screen the library for monooxygenases, alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH). Accordingly, the fosmid library was constructed from metagenome of hydrocarbon-contaminated soil. Then the library was functionally screened using hexadecane, octadecene and cyclohexane as substrates and fifteen positive clones were selected. The fosmid constructs of the positive clones were sequenced using PacBio next generation sequencing platform. The sequences were de novo assembled and analysed using CLC Genomic Workbench. The open reading frames (ORF) of the contigs were identified by blasting the contigs against uniport database. Accordingly, four novel genes namely amo-vut1, aol-vut3, dhy-sc-vut5 and dhy-g-vut7 that showed close similarity with our target enzymes were further analysed in silico and codon-optimized as per Escherichia coli codon preference. The codon adjusted sequences were synthesised and cloned into pET30a(+) expression vector. However, it is worth noting that expression of amo-vut1 was not successful since it was later identified to be a multi-pass member protein, which made it insoluble despite the use of detergent to the effect. There is a need to meticulously genetically engineer amo-vut1 to remove the signal and other membrane-bound peptides while maintaining its activity. Yet the other three constructs were successfully transformed and expressed in E. coli BL21 (DE3). The enzymes were purified and characterized and cocktail for hydrolysis of hexanol was succesfully engineered based on AOL-VUT3, DHY-SC-VUT5 and DHY-G-VUT7. Therefore, novel enzymes were mined from metagenome of fossil-oil contaminated soil and effective hydrocarbon-degrading enzyme cocktails containing their combination were successfully engineered.