Hyperthermophiles are microorganisms that can grow at temperatures close to the boiling point of water or above. They are potential resources of thermostable enzymes including alcohol dehydrogenases (ADHs). Both Thermococcus guaymasensis and Thermotoga hypogea produce ethanol as an end product using glucose as substrate. However, the metabolic pathway and enzymes involved in alcohol production by these hyperthermophiles were not clear. ADH is a key enzyme responsible for alcohol metabolism, and the enzyme has been purified and characterized. T. hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90??C. The NADP+-dependent ADH from T. hypogea was purified to homogeneity and a homodimeric protein with a subunit size of 40 ?? 1 kDa. A part of its encoding gene was cloned and sequenced, from which a major part of the amino acid sequence of the enzyme was deduced and found to have high similarities to iron-containing ADHs from other Thermotoga species and harbored typical iron and NADP+-binding motifs. The conserved domain search showed that T. hypogea ADH was a member of the family of uncharacterized iron-containing ADHs. The iron content of the enzyme was determined to be 1.02 ?? 0.06 g-atoms per subunit. It is the first characterized iron-containing ADH from hyperthermophilic bacteria. Similar to known iron-containing ADHs, T. hypogea ADH was oxygen sensitive; however, the loss of enzyme activity upon exposure to oxygen could be recovered by incubation with dithiothreitol and Fe2+. The enzyme was thermostable with a half-life of about 10 h at 70??C, and its catalytic activity increased along with the rise of temperatures up to 95??C. Optimal pH values for the production and oxidation of alcohol were determined to be 8.0 and 11.0, respectively. The enzyme had a broad specificity in utilizing primary alcohols and aldehydes as substrates. Apparent Km values for ethanol and 1-butanol were much higher than that for acetaldehyde and butyraldehyde and thus the enzyme was likely to catalyze the reduction of aldehydes to alcohols in vivo. T. guaymasensis is a hyperthermophilic anaerobic archaeon capable of catalyzing the starch degradation and producing ethanol and acetoin as end-products. The purified T. guaymasensis ADH was an NADP+-dependent homotetramer with a subunit of 40 ?? 1 kDa. The enzyme was a primary-secondary ADH, but it exhibited substrate preference on secondary alcohols and corresponding ketones. In particular, it catalyzed the reduction of diacetyl to 2, 3-butanediol via acetoin in which the reduction from diacetyl to acetoin was irreversible. For the oxidation of 2, 3-butanediol, the enzyme exhibited higher activities on (2R, 3R)-(-)-2, 3-butanediol and meso-2, 3-butanediol than (2S, 3S)-(+)-2, 3-butanediol while the stereoselective reduction of racemic (R/S)-acetoin produced (2R, 3R)-(-)-2, 3-butanediol and meso-butanediol but not (2S, 3S)-(+)-2, 3-butanediol. The optimal pHs for the oxidation and formation of alcohols were determined to be 10.5 and 7.5, respectively. The enzyme activity increased along with the rise of temperatures up to 95??C, and it was highly stable with a half-life of 24 hours at 95??C. The enzyme was resistant to 30% (v/v) methanol (retaining 40% of its full activity). NADPH for the ketone reduction was efficiently regenerated using isopropanol as a substrate. The apparent Km value for NADPH was 40 times lower than that of NADP+, and the specificity constant with NADPH were 5 times higher than that of NADP+. Therefore, the physiological role of the enzyme was likely to be responsible for the reactions involving the NADPH oxidation???coupled formation of ethanol and/or acetoin. The fully active T. guaymasensis ADH contained 0.9 ?? 0.03 g atom zinc per subunit determined by inductively coupled plasma mass spectrometry (ICP-MS) and was the first characterized zinc-containing ADH from Thermococcus species. The gene encoding this enzyme was cloned and sequenced, and the deduced amino acid sequence contained 364 amino acids showing high similarities (85%) to those ADHs from Thermoanaerobacter species which have only the catalytic zinc atom. The motif analyses also indicated the enzyme lacked of the structural zinc-binding motif; thus, zinc might play a catalytic role in the enzyme. Further analyses showed the presence of the conserved domains of L-threonine dehydrogenases; however, the enzyme could not oxidize L-threonine or L-serine. Distinct from most of zinc-containing ADHs, the enzyme activity was almost fully inhibited by 100 ??M Zn2+ in the assay mixture. Moreover, it was sensitive to oxygen. An NADP+-dependent ADH was purified from the hyperthermophilic anaerobic archaeon Thermococcus strain ES1, an ethanol producer. The recombinant enzyme over-expressed in Escherichia coli was purified using a two-step procedure including heat treatment, and characterized in comparison with the native enzyme. The purified recombinant enzyme exhibited a specific activity of 52.8 U mg-1, close to that of the native enzyme (57 U mg-1). Both native and recombinant enzymes were homotetramers with a subunit size of 45 ?? 1 kDa. Their optimal pHs for the ethanol oxidation and acetaldehyde reduction were determined to be 10.5 and 7.0, respectively. Both enzymes were able to oxidize a series of primary alcohols and diols. Metal contents of the fully active recombinant enzyme were determined by ICP-MS to be 1.0 ?? 0.04 g atom iron per subunit, and both iron-containing enzymes were oxygen sensitive. Their kinetic parameters showed lower Km-values of acetaldehyde and NADPH than those of ethanol and NADP+, suggesting the native enzyme could be involved in ethanol formation in vivo. The recombinant and native enzymes had almost identical characteristics and thus its encoding gene was successfully over-expressed in E. coli. The deduced amino acid sequence of the ADH from Thermococcus strain ES1 was a 406 amino acid polypeptide. Its amino acid sequence showed high identities (80%) to iron-containing ADHs from the related archaea Thermococcus zilligii and Thermococcus hydrothermalis. The conserved domain search revealed it belonged to the family of iron-containing ADHs. Moreover, the sequence of the enzyme had catalytic metal and dinucleotide-binding motifs typical for iron-containing ADHs. In conclusion, the results indicate that iron- and zinc-containing ADHs from hyperthermophiles have significant differences in terms of biophysical, biochemical and molecular properties. The hyperthermophilic bacterial and archaeal iron-containing ADHs are divergent while the zinc-containing ADH from T. guaymasensis has significant similarity to thermophilic bacterial ones.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

This is an investigation into alcohol dehydrogenases (ADHs) from Geobacillus thermoglucosidasius. Eighteen ADHs have been studied, with seven taken for closer inspection. Characterisation was carried out to determine the industrial significance of these enzymes, starting with the substrate scope of the ADHs. The key results obtained are as follows: ADH A is the alcohol dehydrogenase domain of the bifunctional ADHE enzyme (Extance, 2012; Extance et al., 2013). It has been determined that the substrate scope, whilst restricted to linear aliphatic aldehydes, extends at least to dodecanal. Also, with a specificity constant of 167 mM-1 min-1 it appears that ADH A could prefer butanal to shorter-chain aldehydes such as ethanal and propanal with specificity constants of 38 mM-1 min-1 and 35 mM-1 min-1, respectively. Thus ADH A may have a preference for longer aldehydes than previously believed due to its native role in the production of ethanol from acetyl-coA. ADH B was previously investigated for its potential role in the production of butanol. Here it was confirmed as an NADH-dependent ADH, with a substrate scope limited to five carbon length substrates and smaller, with residual activity with C6 substrates. ADH B demonstrated activity with ethyl 4-chloroacetoacetate, an intermediate in the production of statins. Further, an estimated half-life whilst stored at 4°C of 770 days; retention of 86% activity with 10vol% ethyl acetate and 92% activity with 10vol% acetonitrile; and a specific activity of 27 U mg-1 with 3M 2-butanone are all indications that ADH B is a potentially useful enzyme for industry. The last enzyme to be previously investigated was ADH C, which in this work was confirmed to be an acetoin reductase with a very small substrate scope exclusively based around the acetoin motif, and therefore no further work was conducted. ADH D and ADH F both have broad substrate scopes including the industrially-relevant substrates, 5-norbornene-2-carboxaldehyde, 1-phenyl-1,2-propanedione, ethyl 4-chloroacetoacetate and ethyl-2-oxo-4-phenylbutyrate. ADH D is an NADPH-dependent enzyme whereas ADH F can utilise both NADH and NADPH. Both enzymes are annotated as aldo-keto reductases, which is further indicated by multiple sequence alignment with the most similar available protein sequences and crystal structures. Thus, these two enzymes are the first aldo-keto reductases to be examined from moderate thermophiles, and are tentatively assigned in the AKR family as AKR6D1 and AKR5G4 respectively. ADH D has a very low KM (≤0.1 μM) with NADPH, giving a specificity constant of 2,800,000 mM-1 min-1, substantially higher than any other noted. ADH D showed >80% activity from pH 5.0 - 8.0. The enzyme was resistant to solvents DMSO (at 5 vol%) and ethyl acetate, acetonitrile and cyclopentyl methyl ether (at 20vol%). ADH F had the broadest substrate scope of any ADH tested, with 1-phenyl-1,2-propanedione the most preferred substrate with a KM of 0.010 mM and a specificity constant of 54,000 mM-1 min-1. It greatly preferred sodium phosphate at pH 7.0, as almost any deviation resulted in a substantial loss of activity. Activity of ≥70% was recorded in 5vol% DMSO, ethyl acetate, acetonitrile, cyclopentyl methyl ether and 50vol% hexane . Both ADH D and F have optimal activities at 70 °C and both may have application in the biotechnology industry for the production of pharmaceutical intermediates and other high value chemicals. ADH E acts solely as an aldehyde reductase, with Vmax using NADH of 74, 331, 320 and 281 U mg-1 for methanal, ethanal, propanal and butanal, respectively. Activity with NADPH was limited (< 1% compared with NADH). Activity was also noted with higher aldehydes such as octanal and furfural. ADH G is an NADPH-dependent ADH utilizing aldehydes only. It has an optimal temperature of 60°C with a half-life of under two hours at that temperature. In conclusion, this thesis reports a feasibility study into the potential industrial use of specific enzymes for a variety of purposes ranging from the production of pharmaceutical intermediates to bioremediation. ADHs D and F are most likely to have use in the biotechnology industry, and ADHs B and E may be suitable for cofactor regeneration. ADH E may additionally be useful in the bioremediation industry. In addition, the anticipated biological significance of these enzymes is described.

by Lau King-Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 119-142). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / ABBREVIATIONS --- p.IV / TABLE OF CONTENTS --- p.V / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- PURIFICATION OF ADH-I & ADH-C --- p.25 / Chapter CHAPTER 3 --- "PURIFICATION & IDENTIFICATION OF ""ADH-ACTIVATING"" PROTEASE" --- p.60 / Chapter CHAPTER 4 --- ACTIVATION OF ADH-I BY COMMERCIAL PROTEASE & BY ACETIMIDYLATION --- p.90 / Chapter CHAPTER 5 --- CONCLUSION --- p.114 / REFERENCES --- p.118

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2004. / Härtill 5 uppsatser.

Thesis (Ph.D.) - University of St Andrews, May 2008.

Resumo: O presente trabalho descreve e compara duas metodologias enzimáticas de dosagem de etanol (métodos UV e colorimétrico), que utilizam desidrogenase alcoólica (álcool: NAD+: oxidoredutase EC 1.1.1.1) de fermento de panificação (Mauri Brasil Ind. Comp. e Imp. Ltda), adquirida no comércio na forma desidratada. A atividade da álcool desidrogenase (ADH) presente no extrato bruto de levedura de panificação, da ordem de 5,66 U/mL, foi utilizada nos ensaios colorimétricos, enquanto que nos ensaios no ultravioleta (UV), atividades ao redor de 30 U/mL foram obtidas através da otimização das condições de extração e purificação parcial da ADH. A estabilidade da ADH foi mantida durante 2 meses, na forma liofilizada a 4oC (retenção de 96,2% de sua atividade), na presença de 1 mM de azida de sódio. A mesma preparação enzimática, reconstituída em PEG 15% e armazenada durante 12 meses em freezer (-18oC), apresentou retenção de 50% de sua atividade até 2 meses. O método ultravioleta de dosagem de etanol (detecção na faixa de 2,3 x 10-4 g/L a 6,91 x 10-3 g/L ou 5 æM a 150 æM) baseia-se na conversão enzimática do etanol a acetaldeído, através de reação de óxido-redução, tendo o NAD+ como aceptor de elétrons. O NADH formado pela reação é quantificado com leituras espectrofométricas a 340 nm, conforme descrito por Gattás (2002). Uma preparação enzimática parcialmente purificada e diluída foi utilizada na quantificação de etanol em diferentes bebidas, mostrando desvios dos teores alcoólicos de no máximo 2,1% quando comparados às especificações do produtor. O ensaio de etanol em vinho foi realizado com recuperação da ordem de 99,25% em amostra contendo, originalmente, 249,65 g/L de etanol. O método colorimétrico de dosagem de etanol (detecção na faixa de 4,6 x 10-2 g/L a 23,0 x 10-2 g/L ou 1000 æM a 5000 æM)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present work describes and compares two enzymatic methodology of ethanol dosage (UV and colorimetric methods), that use alcohol dehydrogenase (alcohol: NAD+: oxidoredutase EC 1.1.1.1) of baker’s yeast (Mauri Brasil Ind. Comp. e Imp. Ltda) acquired in the commerce in the dry form. The activity of the alcohol dehydrogenase (ADH) at around 5.66 U/mL present in the crude extract of baker’s yeast was used in the colorimetric assay, whereas activities at around of 30 U/mL were obtained through the optimization of the extraction conditions and partial purification of ADH in the ultraviolet assay (UV). The stability of liophilized ADH at 4ºC was maintained for 2 months (retention of 96.2% of activity) in the presence of 1 mM sodium azide. The same enzymatic preparation reconstituted in PEG 15% and stored for 12 months in freezer (-18ºC) presented retention of 50% of your activity up to 2 months. The ultraviolet method of ethanol dosage (detection range of 2.3 x10-4 g/L to 6.91 x 10-3 g/L or 5 æM to 150 æM) is based on the enzymatic conversion of ethanol into acetaldehyde, through oxido-reduction reaction with NAD+ as the aceptor of electrons. The NADH formed by the reaction was quantified spectrophotometrically at 340 nm, as described by Gattás (2002). A partially purified and diluted enzymatic preparation was used for ethanol quantification in different beverages, showing alcoholic contents deviations up to 2.1% when compared to the specifications of the manufacturer. The ethanol assay in wine was accomplished with a recovery at around 99.25% in sample originally containing 249.65 g/L of ethanol. The colorimetric method of ethanol dosage (detection range of 4.6 x 10-2 g/L to 23.0 x 10-2 g/L or 1000 æM to 5000 æM) uses the color reagents system MTT/PMS dissolved in saline phosphate buffer (PBS), determined spectrophotometrically at 570-655 nm... (Complete abstract, click electronic address below) / Orientador: Edwil Aparecida de Lucca Gattás / Coorientador: Maristela de Freitas Sanches Peres / Banca: Rubens Monti / Banca: Luis Henrique Souza Guimarães / Mestre

Gracias a la resurrección de varias enzimas de nuestros antepasados primates se han identificado varias mutaciones que ocurrieron hace, aproximadamente, 10 millones de años, las cuales le confirieron a nuestros antepasados una capacidad mucho mayor para metabolizar etanol. Este episodio de evolución enzimática coincidió con un cambio climático global asociado con la reducción de los bosques africanos y la transición de nuestros antepasados de un estilo de vida arbórea a un estilo de vida terrestre en el cual la fruta altamente fermentada era más común. Estos estudios sugieren que la evolución de las enzimas de nuestros antepasados pueden haberles permitido explotar una fuente alternativa de alimento cuando la comida era escasa. / We have resurrected ancient enzymes from our primate ancestors and identified several mutations occurring approximately 10 million years ago that endowed our ancestors with an enhanced capacity to metabolize ethanol.  This episode of enzyme evolution coincided with a global climate change associated with shrinking African forests and our ancestor’s transition from an arboreal lifestyle to a terrestrial lifestyle where highly fermented fruit is more common. These studies suggest that the evolution of our ancestor’s enzymes may have enabled our ancestors to exploit an alternative source of nourishment during a time when food was scarce.

Orientadores: Fumio Yokoya, Wilma Aparecida Spinosa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-12T23:38:20Z (GMT). No. of bitstreams: 1 SantosJunior_Vitoriodos_D.pdf: 1117180 bytes, checksum: 0701207136023e01100a6834c8566c3b (MD5) Previous issue date: 2009 / Resumo: As bacterias aceticas sao utilizadas na producao de vinagre. O requerimento nutricional das bacterias aceticas e pouco conhecido e a reproducao desses microrganismos e muito dificil. Os reatores de fermentacao acetica submersa promovem condicoes de estresse fisiologico: acidez (acima de 10% p/v), teor alcoolico (2% p/v) e oxigenacao (0,4 vvm). Esses parametros sao considerados extremos e indicam a necessidade de ativacao de rotas metabolicas especificas, para a manutencao da capacidade de conversao alcool-acido acetico e divisao celular. Este estudo tem como objetivo contribuir na avaliacao qualitativa e quantitativa do efeito de minerais sobre a produtividade de acido acetico, que e definida como a porcentagem de acido acetico produzido em 24 horas. Os experimentos foram conduzidos com linhagem de Acetobacter sp., proveniente de industria produtora de vinagre. As etapas do trabalho foram: ativacao do reator com nutriente padrao; teste de efeitos dos minerais, empregando como ferramenta estatistica um delineamento fatorial incompleto (2(5-1)), isolamento e identificação dos microrganismos, utilizando provas bioquimicas classicas e analise molecular da expressao de RNA mensageiro, por meio da tecnica do DDRT/PCR, com utilizacao de primers da enzima alcool desidrogenase (ADH). Com os resultados obtidos a partir de um ensaio fatorial incompleto para a linhagem estudada, foram selecionados como minerais que aumentam a produtividade, sao eles: o ferro, o molibdenio e o manganes e, ainda, como minerais que diminuem a produtividade, o zinco e o boro. A linhagem isolada do reator apresentou todas as características bioquimicas do genero Acetobacter e a analise molecular evidenciou a expressão do gene para a enzima alcool desidrogenase em todos os experimentos / Abstract: The acetical bacteria are used in vinegar production. The nutritional requirement of acetical bacteria is poorly known, and the maintenance of these microorganisms is too difficult. The reactors of submerged acetical fermentation promote conditions of physiologic stress: acidity (up to 10% p/v), alcohol (2%p/v) and oxygenation (0,4 vvm).These parameters are considerate extremes and indicate the necessity of the activation of specifics metabolic to the maintenance of alcohol-acetic acid transformation capacity and cell division. This research has in view to contribute to the qualitatively and quantitatively evaluation of minerals effects on the productivity of acetic acid, which is defined as the acetic acid percentage produced until 24 hours. These experiments were conducted with of Acetobacter sp. strain, proceeding from vinegar industries. The work steps were: activation of the reactor with standard nutrient; minerals effects tests using the fractional factorial design (25-1) as statistic tool; isolation and identification of the microorganisms, employing classical biochemistry proofs and molecular analysis of the RNA messenger expression, through DDRT/PCR techniques, applying alcohol dehydrogenase enzyme primers. With the results obtained from the fractional factorial design for the strains studied, were selected minerals as the iron, the molybdenum and the manganese that increase productivity and, yet, minerals as zinc and boron that decrease it. The reactor isolated strain presented all biochemical characteristics of Acetobacter genus and the molecular analysis evidenced the expression of the gene for the enzyme alcohol dehidrogenase in all experiments / Doutorado / Doutor em Ciência de Alimentos