Activities of the enzymes of the Ehrlich pathway and formation of branched-chain alcohols in Saccharomyces cerevisiae and Candida utilis grown in continuous culture on valine or ammonium as sole nitrogen source (original) (raw)
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Yeast, 2014
Higher alcohol formation by yeast is of great interest in the field of fermented beverages. Among them, medium-chain alcohols impact greatly the final flavour profile of alcoholic beverages, even at low concentrations. It is widely accepted that amino acid metabolism in yeasts directly influences higher alcohol formation, especially the catabolism of aromatic and branched-chain amino acids. However, it is not clear how the availability of oxygen and glucose metabolism influence the final higher alcohol levels in fermented beverages. Here, using an industrial Brazilian cachaça strain of Saccharomyces cerevisiae, we investigated the effect of oxygen limitation and glucose pulse on the accumulation of higher alcohol compounds in batch cultures, with glucose (20 g/l) and leucine (9.8 g/l) as the carbon and nitrogen sources, respectively. Fermentative metabolites and CO 2 /O 2 balance were analysed in order to correlate the results with physiological data. Our results show that the accumulation of isoamyl alcohol by yeast is independent of oxygen availability in the medium, depending mainly on leucine, α-keto-acids and/or NADH pools. Highavailability leucine experiments showed a novel and unexpected accumulation of isobutanol, active amyl alcohol and 2-phenylethanol, which could be attributed to de novo biosynthesis of valine, isoleucine and phenylalanine and subsequent outflow of these pathways. In carbon-exhausted conditions, our results also describe, for the first time, the metabolization of isoamyl alcohol, isobutanol, active amyl alcohol but not of 2-phenylethanol, by yeast strains in stationary phase, suggesting a
The alcohol dehydrogenases ofSaccharomyces cerevisiae: a comprehensive review
FEMS Yeast Research, 2008
Alcohol dehydrogenases (ADHs) constitute a large family of enzymes responsible for the reversible oxidation of alcohols to aldehydes with the concomitant reduction of NAD 1 or NADP 1. These enzymes have been identified not only in yeasts, but also in several other eukaryotes and even prokaryotes. The ADHs of Saccharomyces cerevisiae have been studied intensively for over half a century. With the ever-evolving techniques available for scientific analysis and since the completion of the Yeast Genome Project, a vast amount of new information has been generated during the past 10 years. This review attempts to provide a brief summary of the wealth of knowledge gained from earlier studies as well as more recent work. Relevant aspects regarding the primary and secondary structure, kinetic characteristics, function and molecular regulation of the ADHs in S. cerevisiae are discussed in detail. A brief outlook also contemplates possible future research opportunities.
Structure and function of yeast alcohol dehydrogenase
Journal of the Serbian Chemical Society, 2000
1. Introduction 2. Isoenzymes of YADH 3. Substrate specificity 4. Kinetic mechanism 5. Primary structure 6. The active site 7. Mutations in the yeast enzyme 8. Chemical mechanism 9. Binding of coenzymes 10. Hydride transfer This article has been corrected. Link to the correction 10.2298/JSC0008609E
Activity of yeast alcohol dehydrogenases on benzyl alcohols and benzaldehydes
Chemico-Biological Interactions, 2009
The substrate specificities of yeast alcohol dehydrogenases I and II from Saccharomyces cerevisiae (SceADH1 and SceADH2) and Saccharomyces carlsbergensis (ScbADH1) were studied. For this work, the gene for the S. carlsbergensis ADH1 was cloned, sequenced and expressed. The amino acid sequence of ScbADH1 differs at four positions as compared to SceADH1, including substitutions of two glutamine residues with glutamic acid residues, and has the same sequence as the commercial yeast enzyme, which apparently is prepared from S. carlsbergensis. The electrophoretic mobilities of ScbADH1, SceADH2 and commercial ADH are similar. The kinetics and specificities of ScbADH1 and SceADH1 acting on branched, long-chain and benzyl alcohols are very similar, but the catalytic efficiency of SceADH2 is about 10 to 100-fold higher on these substrates. A three dimensional structure of SceADH1 shows that the substrate binding pocket has Met-270, whereas SceADH2 has Leu-270, which allows larger substrates to bind. The reduction of a series of p-substituted benzaldehydes catalyzed by SceADH2 is significantly enhanced by electronwithdrawing groups, whereas the oxidation of p-substituted aromatic alcohols may be only slightly affected by the substituents. The substituent effects on catalysis generally reflect the effects on the equilibrium constant for the reaction, where electron-withdrawing substituents favor alcohol. The results are consistent with a transition state that is electronically similar to the alcohol, supporting previous results obtained with commercial yeast ADH.
World Journal of Microbiology and Biotechnology, 2005
The effect of glucose and dissolved oxygen in a synthetic medium simulating the standard composition of grape juice on the production of ethyl acetate and isoamyl acetate by a Saccharomyces cerevisiae strain during alcoholic fermentation was studied. The specific in vitro activity of alcohol acetyltransferase (AATase, EC 2.3.1.84) and esterases (ESase, EC 3.1.1.1; hydrolysis and synthesis of esters) in cell-free extracts was also examined. The specific activity of AATase for ethyl acetate was found to peak at the beginning of the exponential growth phase and that for isoamyl acetate at its end. While the glucose concentration only affected the maximum specific activity of AATase, and only slightly, oxygen inhibited such activity, to a greater extent for isoamyl acetate than for ethyl acetate. On the other hand, esterases were found to catalyse the synthesis of ethyl acetate only at a low or medium glucose concentration (50 or 100 g l)1 , respectively), and to reach their maximum hydrolytic activity on isoamyl acetate during the stationary growth phase. The highest ethyl acetate and isoamyl acetate concentrations in the medium were obtained with a glucose concentration of 250 g l)1 and semianaerobic conditions.
FEMS Yeast Research, 2006
In Saccharomyces cerevisiae, branched-chain amino acid transaminases (BCAAT ases) are encoded by the BAT1 and BAT2 genes. BCAATases catalyse the transfer of amino groups between those amino acids and a-keto-acids. a-Keto-acids are precursors for the biosynthesis of higher alcohols, which significantly influence the aroma and flavour of yeast-derived fermentation products. The objective of this study was to investigate the influence of BAT-gene expression on general yeast physiology, on aroma and flavour compound formation and on the sensory characteristics of wines and distillates. For this purpose, the genes were overexpressed and deleted in a laboratory strain, BY4742, and overexpressed in an industrial wine yeast strain, VIN13. The data show that, with the exception of a slow growth phenotype observed for the BAT1 deletion strain, the fermentation behaviour of the strains was unaffected by the modifications. The chemical and sensory analysis of fermentation products revealed a strong correction between BAT gene expression and the formation of many aroma compounds. The data suggest that the adjustment of BAT gene expression could play an important role in assisting winemakers in their endeavour to produce wines with specific flavour profiles.
Novel substrates of yeast alcohol dehydrogenase-4. Allyl alcohol and ethylene glycol
Iubmb Life, 1999
In the present work, we have determined the steady-state kinetic constants for yeast alcohol dehydrogenase-catalyzed oxidation of allyl alcohol (H2C=CH.CH2OH) and ethylene glycol (HOCHg.CH2OH) with NAD § at pH 8.0; also, a kinetic mechanism for the former reaction was determined at the same pH. In addition, it was found that acrolein is a potent inhibitor of yeast alcohol dehydrogenase.
Aldehyde dismutase activity of yeast alcohol dehydrogenase
1999
Yeast alcohol dehydrogenase (EC 1.1.1.1) is able to catalyze the oxidation of acetaldehyde by NAD + with a concomitant formation of ethanol, at pH 8.8 and pH 7.1; the stoichiometry of aldehyde oxidation vs. ethanol formation is 2:1. This enzymatic reaction obeys the Michaelis-Menten kinetics and was characterized by a high K M for acetaldehyde (68 mM) and a low k cat (2.3 s −1), at pH 8.8, 22 • C. There is no visible burst of NADH during the reaction, from pH 7.1-10.1. Therefore, we have concluded that the enzyme catalyzes an apparent dismutation of two molecules of acetaldehyde into a molecule of acetic acid and a molecule of ethanol.
The Enzymatic Approach to Making of Alcoholic Beverages
International Journal Bioautomation, 2011
Immobilized yeast invertase was applied for treatment of alcoholic beverages with the aim of transformation of higher alcohols into alkylfructosides. Gas-liquid chromatography of treated water-alcoholic medium containing 3.0 mg/l isoamyl alcohol and 4% saccharose by immobilized invertase had shown the convertion of 40% isoamyl alcohol, which amounts to 1.8 mg/l absolute alcohol. Other parameters remained at the previous level. The high level of enzyme activity was observed when the initial concentration of sucrose in the reaction mixture attained 4.0-12.5%. Tasting of treated samples indicated the improvement of quality and degustational properties of beverages, they had softer and more harmonious taste and aroma in comparison with control sample and finished Vodka, which completed the cycle of technological processing.