Purification and characterization of isoamyl acetate-hydrolyzing esterase encoded by the IAH1 gene of Saccharomyces cerevisiae from a recombinant Escherichia coli |
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| Authors: | K. Fukuda Y. Kiyokawa T. Yanagiuchi Y. Wakai K. Kitamoto Y. Inoue A. Kimura |
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| Affiliation: | (1) Kizakura Sake Brewing Co., Ltd., 223, Shioya-machi, Fushimi-ku, Kyoto 612-8046, Japan e-mail: fukudakz@mbox.kyoto-inet.or.jp Tel.: +81-75-611-4101 ex. 560 Fax: +81-75-622-3510, JP;(2) Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, JP;(3) Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan, JP |
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| Abstract: | The IAH1 gene of Saccharomyces cerevisiae encodes an esterase that preferentially acts on isoamyl acetate; however, the enzyme has not yet been completely purified from the yeast S. cerevisiae. We constructed the IAH1 gene expression system in Escherichia coli, and purified the IAH1 gene product (Iah1p). The amount of Iah1p produced by recombinant E. coli was more than 40% of total cellular proteins. The molecular size of Iah1p was 28 kDa by SDS-polyacrylamide gel electrophoresis. Judging from the molecular weight estimation by gel filtration of purified Iah1p, the enzyme was thought to be a homodimer. The K m values for isoamyl acetate and isobutyl acetate were 40.3 mM and 15.3 mM, respectively. The enzyme activity was inhibited by Hg2+, p-chloromercuribenzoate, and diisopropylfluorophosphate. Received: 23 May 1999 / Received revision: 27 October 1999 / Accepted: 5 November 1999 |
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