Use of novel assays to indicate that O-esters and S-esters are produced by the same enzyme in brewing yeast |
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Authors: | Bamforth C W Kanauchi M |
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Affiliation: | Department of Food Science and Technology, University of California, Davis, CA 95616-8598, USA. cwbamforth@ucdavis.edu |
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Abstract: | Ethanol- and methanethiol-dependent removal of acetyl-CoA by crude extracts of ale yeast has been monitored using a decrease in OD232. Activity has also been detected in these extracts after fractionation on polyacrylamide gels, in this case using a novel assay in which the coenzyme A produced in the reaction is linked via DCPIP reduction to color formation from nitroblue tetrazolium. Ethanol- and methanethiol-dependent activities migrate identically on such gels, and only one band of color formation was observed. Furthermore they displayed closely similar sensitivity to heating at 40 degrees C and 60 degrees C and pH optima, with activity maximal at pH 7.5. It is likely that a single enzyme is responsible for the formation of O-esters and S-esters in yeast. Initial kinetic studies indicate that methanethiol has higher affinity for the enzyme than has ethanol and a higher maximum velocity. However, the enzyme has a much lower Km for acetyl-CoA, suggesting that the alcohol or thiol substrate is the more likely substrate to be limiting. |
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Keywords: | Ethanol Methanethiol Ethyl acetate Methyl thioacetate Acetyl-CoA Alcohol acetyl transferase |
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