Purification and characterization of an enzyme that has dihydroxyacetone-reducing activity from methanol-grown Hansenula ofunaensis.
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 fractination 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 degrees C. 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 4 M, respectively. The K(m) values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, 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.
Yamada-Onodera
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Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 Japan.
Ono
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Tani
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