The present dissertation seeks to deepen our understanding of hydrogen transfers and especially C-H bond activations in enzymes. Hydrogen transfers are ubiquitous in chemistry and biology and a thorough understanding of how they occur and what factors influence them will facilitate developments in biomimetic catalysis, rational drug design, and other fields. A particular difficulty with H-transfers is the importance of nuclear quantum effects to the reaction, particularly tunneling. The overall scope of the work here aims to examine how experimental kinetic isotope effects (KIEs) can be interpreted with a particular type of tunneling model, referred to as Marcus-like models, to yield a semi-quantitative picture of the physical mechanisms of H-transfers. Previous work had used this kind of model to qualitatively interpret experimental data using a combination of intuition and generalized theories. The work here examines these theories in quantitative detail, testing and calibrating our intuition in the context of several experimental systems. The first chapter of research (ch. II) focusses on the temperature dependence of primary KIEs and how these experiments can be quantitatively interpreted as a probe for certain kinds of enzyme or solvent dynamics. The subsequent chapters (ch. III-VI) focus on the use of secondary KIEs to determine the detailed structures of tunneling ready states (TRSs) and how the dynamics of H-tunneling affect those structures. These chapters focus primarily on the TRS of the enzyme alcohol dehydrogenase, but by examining an uncatalyzed analogue to that reaction (ch. VI), the work gains some insight about similarities and differences between catalyzed and uncatalyzed reactions. In summary, the work uncovers some principles of catalysis, not just the mechanism of a catalyzed reaction. The mechanism of C-H activation presented here provides an elegant solution to problems that have been vexing to accommodate within traditional models. This work constitutes some initial steps in making Marcus-like models quantitatively useful as a supplement or even replacement for traditional models of reactivity.

Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R)- and 2-(S)-hydroxypropyl-CoM (HPC) dehydrogenases (DH)) of the epoxide carboxylation pathway in Xanthobacter autotrophicus strain Py2. The main goal of this dissertation is to kinetically, mechanistically and structurally characterize S-HPCDH and through the comparison studies with R-HPCDH reveal the basis for high degree of stereoselectivity exhibited by both enzymes. Analysis of the molecular structure of R-HPCDH and the homology model of S-HPCDH suggests a mechanism of substrate specificity in which the binding of the substrate sulfonate moiety at distinct sites on each stereoselective enzyme directs the orientation of the appropriate substrate enantiomer for the hydride abstraction. The positively charged residues responsible for binding the CoM moiety of the substrate were identified in R-HPCDH (Arg152 and Arg196), and in S-HPCDH (Arg211 and Lys214). Site-directed mutagenesis confirmed their importance in binding and orienting physiological substrates, but not the substrates lacking the CoM moiety. Extensive kinetic and mechanistic characterization of S-HPCDH reveals its key catalytic features similar to those of R-HPCDH, but also points out a few important differences. Furthermore, the role of the methionine residues flanking the substrate in the active site of both dehydrogenases was investigated. Substitution of these residues to alanine resulted in enzymes with significantly altered catalytic parameters and suggested their importance in binding and catalysis. Additionally, the X-ray crystal structures of the Met187Ala and Met192Ala mutants of R-HPCDH have revealed their role as "gate keepers," protecting the active site from the surrounding solvent. Kinetic analysis of Met187Leu and Met192Leu mutants implied a structural, rather than catalytic function of the methionines. It is proposed that steric clashes of the terminal methyl group of the HPC substrates with the nicotinamide ring of NAD+ are a major determinant of the enantioselectivity in S-HPCDH. This research provides the first side-by-side characterization of a pair of short-chain dehydrogenase/reductase (SDR) enzymes expressed simultaneously to act on two enantiomers of the same alcohol produced in a metabolic pathway. The R-HPCDH and S-HPCDH enzymes are distinguished from all other known members of the SDR family in using the novel sulfonate functional group of coenzyme M as a handle for chiral discrimination. These results provide a standard for examining the molecular basis of stereoselectivity in other such enzyme pairs.

Alkylierte polyzyklische aromatische Kohlenwasserstoffe (alk-PAK) kommen zusammen mit rein aromatischen polyzyklischen Kohlenwasserstoffen u.a. im Zigarettenrauch, Dieselabgasen sowie einigen Lebensmitteln (z.B. Freilandgemüse, planzliche Öle und Fette) vor. Benzylische Hydroxylierung und nachfolgende Sulfokonjugation ist ein wichtiger Bioaktivierungsweg für einige alk-PAK. Oxidation der benzylischen Alkohole durch Alkoholdehydrogenasen (ADH) und Aldehyddehydrogenasen (ALDH) zur Carbonsäure könnte einen wichtigen Detoxifizierungsweg in Konkurrenz zur Aktivierung durch Sulfotransferasen (SULT) darstellen, was für 1-Hydroxymethylpyren in der Ratte bereits gezeigt wurde (Ma, L., Kuhlow, A. & Glatt, H. (2002). Polycyclic Aromat Compnds 22, 933-946). Durch Hemmung der ADH und/oder ALDH ist eine verstärkte Aktivierung zu erwarten, wie in der besagten Studie ebenfalls nachgewiesen wurde. Insbesondere Ethanol kommt in diesem Zusammenhang eine Rolle als möglicher Risikofaktor für alk-PAK induzierte Kanzerogenese zu. Menschen konsumieren häufig große Mengen Ethanol und oft besteht eine Koexposition mit alk-PAK (z.B. durch Rauchen). Ähnliches gilt für 5-(Hydroxymethyl)-2-furfural (HMF), einem Pyrolyseprodukt reduzierender Zucker, dem gegenüber Menschen in recht hohen Mengen exponiert sind. Auch bei HMF steht der ADH- und ALDH-vermittelte oxidative Metabolismus in Konkurrenz zu einer Aktivierung durch Sulfokonjugation. Um die Bedeutung humaner ADH und ALDH im Metabolismus von alk-PAK und von HMF aufzuklären, wurden alle bekannten humanen ADH sowie die humanen ALDH2 und 3A1 (aus theoretischen Überlegungen heraus die vielversprechendsten Formen) für kinetische Analysen in Bakterien exprimiert. Als Enzymquelle dienten zytosolische Präparationen und durch Anionenaustauschchromatographie partiell gereinigte Enzyme. In der vorliegenden Arbeit wurde nachgewiesen, dass primäre benzylische Alkohole von Methyl- und Dimethylpyrenen gute Substrate humaner ADH sind. Sekundäre benzylische Alkohole und benzylische Alkohole von alk-PAK mit größerem Kohlenwasserstoffgrundgerüst erwiesen sich dagegen als schlechte Substrate. Vier Formen (ADH1C, 2, 3 und 4) wurden näher analysiert. Dazu wurden sie partiell gereinigt, primär um die störende endogene Bakterien-ADH zu eliminieren. Alle untersuchten ADH waren in der Lage Pyrenylmethanole zu oxidieren. Insbesondere ADH2 katalysierte die Oxidation der Pyrenylmethanole effizient, aber auch für ADH1C und 4 waren die Pyrenylmethanole gute Substrate. ADH3 oxidierte die Pyrenylmethanole mit geringer katalytischer Effizienz. Die Reduktion der entsprechenden Pyrenaldehyde durch ADH1C, 2 und 4 wurde mit noch höherer Effizienz katalysiert als die Oxidation der Pyrenylmethanole, was die Bedeutung von ALDH für die effiziente Detoxifizierung dieser Verbindungen unterstreicht. In einer an diese Arbeit angelehnten Diplomarbeit (Rost, K. (2007). Universität Potsdam, Mathematisch-Naturwissenschaftliche Fakultät) wurde auch tatsächlich gezeigt, dass humane ALDH2 aber auch ALDH3A1 in der Lage sind, die Pyrenaldehyde zu Pyrenylcarbonsäuren zu oxidieren. Die bestimmten kinetischen Parameter legen nahe, dass insbesondere ALDH2 von Bedeutung für die Detoxifizierung von Methyl- und Dimethylpyrenen ist. Schon allein auf Grund der an der Detoxifizierung beteiligten Enzyme ist Ethanolaufnahme bei Koexposition mit Pyrenderivaten als Risiokofaktor anzusehen. Es ist wahrscheinlich, dass Ethanol und, nach dessen Oxidation, Acetaldehyd als konkurrierende Substrate die ADH- und ALDH-katalysierte Oxidation von Pyrenylmethanolen bzw. Pyrenaldehyden inhibieren und somit zu einer verstärkten SULT-vermittelten Aktivierung der Pyrenylmethanole führen. In der Tat wurde eine effiziente Inhibition der ADH2-katalysierten Oxidation von 1-Hydroxymethylpyren und von 1-(Hydroxymethyl)-8-methylpyren durch physiologisch relevante Ethanolkonzentrationen nachgewiesen. Drei humane ADH (4, 2 und 3), die HMF effizient zum 2,5-Diformylfuran oxidieren können, wurden identifiziert. Durch ALDH-katalysierte Weiteroxidation dieser Substanz entsteht schließlich 2,5-Furandicarbonsäure, die nach HMF-Exposition auch tatsächlich im menschlichen Urin gefunden wurde (Jellum, E., Børresen, H. C. & Eldjarn, L. (1973). Clin Chim Acta 47, 191-201). Weiter wurde gezeigt, dass ALDH3A1, aber auch ALDH2 HMF effizient zur 5-(Hydroxymethyl)-2-furancarbonsäure (HMFA) oxidieren können, ein weiterer nachgewiesener HMF Metabolit in vivo. Dass die ADH-katalysierte Oxidation von HMFA und nachfolgende ALDH-katalysierte Oxidation zur Bildung von 2,5-Furandicarbonsäure einen nennenswerten Anteil beträgt, kann aufgrund der kinetischen Daten für HMFA als Substrat humaner ADH ausgeschlossen werden. Die beobachteten Enzymaktivitäten lassen den Schluss zu, dass Ethanolaufnahme zu einer Reduktion des oxidativen HMF Metabolismus führt und somit eine Aktivierung von HMF durch Sulfokonjugation begünstigt. / Alkylated polycyclic aromatic hydrocabons (alk-PAH), together with purely aromatic PAH, are present e.g. in tobacco smoke, diesel exhausts and also in some foods (e.g. outdoor vegetables, vegetable oils). Benzylic hydroxylation and subsequent sulfo conjugation is an important metabolic activation pathway for some of these compounds. Nevertheless, oxidation of the benzylic alcohols by alcohol dehydrogenases (ADH) and subsequently by aldehyde dehydrogenases (ALDH) can compete with the sulfo conjugation. Therefore, this pathway is probably important in the detoxification as could be shown for the representative compound 1-hydroxymethylpyrene in the rat (Ma, L., Kuhlow, A. & Glatt, H. (2002). Polycyclic Aromat Compnds 22, 933-946). Inhibition of ADH and/or ALDH should increase bioactivation as indeed was shown for 1-hydroxymethylpyrene in this study. Particularly ethanol, a competing ADH substrate, is of high interest in this context. Humans often consume large quantities of ethanol and often they are coexposed to alk-PAH (e.g. due to tobacco smoking). Similar relationships can be considered for 5-(hydroxymethyl)-2-furfural (HMF), a common pyrolysate of reducing sugars with high exposure to humans. Oxidative metabolism of HMF by ADH and ALDH also competes with its bioactivation by sulfotransferases (SULT). To clarify the importance of human ADH and ALDH in the metabolism of alk-PAH and HMF, all known human ADH as well as human ALDH2 and 3A1 (the most promising forms according to theoretical considerations) were expressed in bacteria for kinetic anlalyses. Cytosolic preparations or enzymes partially purified by anion exchange chromatography were used as enzyme source. In the present study it was shown that primary benzylic alcohols of methyl- and dimethylpyrenes were good substrates for human ADH. However, secondary benzylic alcohols and benzylic alcohols derived from alk-PAH with a bulkier hydrocarbon skeletal were poor substrates for human ADH. The most promising forms (ADH1C, 2, 3 and 4) were partially purified and further analysed. The purification step was necessary to eliminate the bacterial ADH. Particularly ADH2 was efficient for oxidation of pyrenylmethanols, although ADH1C and 4 were relatively efficient too. ADH3 was also capable of oxidising the tested pyrenylmethanols but with low catalytic efficiency. The reduction of the corresponding pyrene aldehydes was catalysed by ADH1C, 2 and 4 even with higher efficiency than the oxidation of the pyrenylmethanols emphasising the importance of ALDH for the detoxification of these compounds. In a diploma work related to the present study (Rost, K. (2007). University of Potsdam, Mathematisch-Naturwissenschaftliche Fakultät) it was shown that human ALDH2, but also ALDH3A1, can oxidise pyrene aldehydes to pyrenylcarboxylic acids. Particularly ALDH2 efficiently catalyse these reactions and, therefore, is probably of importance for the detoxification of methyl- and dimethylpyrenes. Due to the enzymes involved ethanol consumption could be a risk factor for methyl- and dimethylpyrene induced damage in the case of coexposure to methyl- and dimethylpyrenes. It is probable that ethanol and, after its oxidation, acetaldehyde will inhibit the ADH- and ALDH-catalysed oxidation of pyrenylmethanols and pyrenealdehydes. Indeed, it was shown that ADH2 catalysed oxidation of 1-hydroxymethylpyrene and of 1-(hydroxymethyl)-8-methylpyrene was efficiently inhibited by physiologically attainable concentrations of ethanol. Three human ADHs (4, 2 and 3) that efficiently oxidise HMF to 2,5-diformylfuran were identified. Further oxidation by ALDH leads to 2,5-furandicarboxylic acid, which was found in human urine after exposure to HMF (Jellum, E., Børresen, H. C. & Eldjarn, L. (1973). Clin Chim Acta 47, 191-201). Moreover, it was shown that human ALDH3A1 and also ALDH2 efficiently oxidise HMF to 5-(hydroxymethyl)-2-furancarboxylic acid (HMFA), which was also found in human urine. That 2,5-furandicarboxylic acid can be formed in significant amounts by ADH-catalysed oxidation of HMFA and subsequent oxidation by ALDH could be ruled out due to the kinetic data with HMFA as a substrate for human ADH. Due to the enzymes involved it is probable that ethanol consumption will inhibit the oxidative metabolism of HMF and, therefore, will increase the sulfo conjugation of HMF.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 96-104).

Zinc is a metal involved in structure of many enzymes, in the growth and differentiation of plants. Wheat is one of the most consumed cereals. Some wheat cultivars can&amp / #8217 / t deal with zinc deficiency and this situation not only reduces grain yield but also weakens the resistance of cereals to diseases and impairs the nutritional quality of the grain. Some wheat cultivars are not affected by zinc deficiency. In this study, &amp / #8216 / differential display&amp / #8217 / , used for determination differentially expressed genes between two samples, was performed. The most zinc efficient bread wheat cultivar Kira&ccedil / -66 was grown in hydroponics medium and samples were taken at different time periods. RNA isolations were done and differential display technique was performed. After examining the results, differentially expressed bands were selected and sequenced. DNA sequence analysis were done in available databases which showed that three of the bands were fragments of putative zinc transporters. In this study we have found threee putative gene fragments using differential display technique on zinc efficient plants grown under differeing zinc concentrations. These fragments showed homology with zinc transporter, ABC transporter and ADH (Alcohol Dehydrogenase). It is known that all of these three genes are involved in zinc efficiency mechanism. Further studies will be conducted on these gene fragments.

Thesis (Ph. D.)--University of Washington, 1989. / Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 7 uppsatser.

Thesis (Ph.D. in Toxicology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 137-149).

Made available in DSpace on 2014-06-11T19:23:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-07Bitstream added on 2014-06-13T19:09:26Z : No. of bitstreams: 1 reis_jpz_me_arafcf.pdf: 425057 bytes, checksum: 37bfdbfbb7c3c607942b9445950e32e0 (MD5) / Universidade Estadual Paulista (UNESP) / 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)... / 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)

Membrane-bound NAD-independent alcohol dehydrogenase (NAD-independent ADH) from Acetobacter and Gluconobacter species has shown appreciable enantioselectivity on the oxidation of chiral primary and secondary alcohols. A NADIndependent alcohol dehydrogenase from mesophilic Acetobacter sp was isolated and characterized as a quinohaemprotein alcohol dehydrogenase QH-ADH. QHADH showed enantiopreference for the oxidation of (S)-enantiomer during the kinetic resolution of racemic secondary alcohols. Due to the growing interest for thermotolerant enzymes in the biocatalysis area, this study was performed to isolate acetic acid bacteria from vinegar factories in the northeast region of Brazil aiming to obtain a thermotolerant QH-ADH. Among the strains isolated, two showed characteristics of the Acetobacter genus (LBVE1 e LBVE4). Biomass production of the two strains were conduced on shaker flasks assays with three different ethanol concentrations as carbon source (10, 15 e 20 g/L) incubated in three different temperatures (30 35 e 37ºC). The highest biomass production for the two strains was obtained on the assays with 20 g/L ethanol, 30 ºC. However, both strains presented different growth conductions for the production of biomass highly active in NADindependent ADH. LBVE1 biomass produced on 10 g/L ethanol, 30ºC, showed the highest specific activity, 10.44 U/mg Proteín while the highest specific activity for LBVE4, 8.04 U/mg Proteín, was obtained on the biomass produced in assays with 15 g/L ethanol, 30 ºC. The results of the substrate specificity of the NAD-independent ADH present in the crude extract of both strains for selected primary and secondary alcohols. For primary alcohols, LBVE1 crude extract showed a decrease in the activity values with the rise of the carbon chain. Kinetic assays were performed to calculate apparent kinetics constants, using ethanol as substrate, for the NADindependent ADH present in the crude extract of both strains. LBVE1 crude extracts from cells grown in 10 and 20 g/L ethanol showed comparable values for Kmapp and VMax app. However, only extract from LBVE4 grown in 20 g/L ethanol had Kmapp value comparable to the one found for LBVE1 extracts analyzed. / Fundação de Amparo a Pesquisa do Estado de Alagoas / Álcool desidrogenase NAD-independente, enzima ligada a membrana, das espécies Acetobacter e Gluconobacter mostram apreciável enantioseletividade na oxidação de álcoois quirais primários e secundários. Em estudos anteriores, esta enzima foi isolada e caracterizada de espécies mesofílicas Acetobacter sp como uma quinohemoproteína álcool desidrogenase (QH-ADH). QH-ADH mostrou enantiopreferência pela oxidação do (S)-enantiômero durante a resolução cinética de álcoois secundários racêmicos. Devido ao crescente interesse por enzimas termotolerantes na área de biocatálise, este estudo foi conduzido com intuito de isolar bactérias termotolerantes do ácido acético de fábricas de vinagre na região nordeste do Brasil, almejando obter uma QH-ADH termotolerante. Dentre as espécies isoladas, duas mostraram características do gênero Acetobacter (LBVE1 e LBVE4). A produção de biomassa foi conduzida em agitador sob aeração de 200 rpm com três diferentes concentrações de etanol (10, 15 e 20 g/L) e incubadas em três temperaturas (30 35 e 37ºC). Os dois isolados apresentaram maior produção de biomassa nas condições quando cultivadas a 20 g/L etanol na 30ºC. Todavia, as condições para produção de uma biomassa ativa em álcool desidrogenase NADindependente variaram para as duas cepas. Cultivos de LBVE1 com 10 g/L de etanol, 30ºC, apresentou maior atividade específica cerca de 10,44 U/mgProteína. Enquanto que cultivos de LBVE4 mostrou atividade específica mais alta, 8,04 U/mgP nos cultivos a 15 g/L de etanol, 30ºC. As análises para investigar os perfis de especificidades para o substrato nos extratos brutos de LBVE1 e LBVE4, contendo ADH NAD-independente foram realizadas usando álcoois racêmicos com diferentes tamanhos de cadeia carbônica, dentre eles o rincoforol que é utilizado como feromônio. Também foram medidos os parâmetros cinéticos aparentes nos extratos brutos de ambas as cepas contendo atividade em ADH NAD-independente. Os extratos brutos células de LBVE1 cultivados 10 g/L e 20 g/L etanol apresentaram valores de Kmapp e VMax app comparáveis. Contudo, somente os extratos brutos de células de LBVE4 cultivados a 20 g/L etanol tiveram valores de Kmapp semelhantes aos encontrados para os extratos de células de LBVE1 estudados.