Purification and Characterization of an Enzyme That Has Dihydroxyacetone-Reducing Activity from Methanol-Grown Hansenula ofunaensis
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概要
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An intracellular enzyme having reduction activity towards dihydroxyacetone(DHA), and that was induced by DHA, was purified and characterized from a methanol-grown yeast, Hansenula ofunaensis. After harvesting cells grown in a 1% methanol medium until the early stationary phase, the enzyme was purified through ammonium sulfate fractionation and a series of ion-exchange, hydrophobic, and gel-filtration column chromatographies. SDS-PAGE and HPLC showed the enzyme to be a homo dimer composed of two identical subunits, each with a molecular mass of 38 kDa. The optimum pHs for DHA reduction and glycerol oxidation were 6.0 and 7.0, respectively. The optimum temperature for enzyme activity was 55℃. The enzyme reduced several other compounds, including acetaldehyde, acetol, 2-butanone and 3-methyl-2-butanone, more effectively than it did DHA, while its oxidation activity was higher towards ethanol, 2-propanol, 1, 2-propanediol, 2, 3-butanediol and 1, 3-butanediol than towards glycerol. The K_m values for DHA in reduction and glycerol in oxidation were 430 mM and 4M, respectively. The purified enzyme had high K_m values for glycerol and DHA and low K_m values for 2-butanol and butanone, although physiologically it had a role in DHA metabolism. There were similarities between the purified enzyme and sec-alcohol dehydrogenases reported previously in their behavior towards inhibitors and metal ions, as well as in their K_m values for 2-butanol and 2-butanone, but differences in their subunit molecular masses and activities for ethanol. At pH 9.8, the oxidative activity of the purified enzyme for L-2-butanol was about eleven times higher than that for D-2-butanol.
- 公益社団法人日本生物工学会の論文
- 1999-08-25
著者
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Tani Yoshiki
Graduate School Of Biological Sciences Nara Institute Of Science And Technology
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Yamada‐onodera K
Nara Inst. Sci. And Technol. Nara Jpn
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Yamada-onodera Keiko
Graduate School Of Biological Sciences Nara Institute Of Science And Technology
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ONO KENJI
Faculty of Agriculture, Kyoto University
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Tani Yoshiki
Graduate School Of Biological Sciences Advanced Institute Of Science And Technology
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Ono Kenji
Faculty Of Agriculture Kyoto University
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Yamada-onodera Keiko
Graduate School Of Biological Sciences Nara Inst. Of Sci. And Technol.
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