Purification and characterization of an enzyme that has dihydroxyacetone-reducing activity from methanol-grown Hansenula ofunaensis

• Published in: Journal of bioscience and bioengineering 1999, Vol.88 (2), p.148-152
• Main Authors: Yamada-Onodera, Keiko, Ono, Kenji, Tani, Yoshiki
• Format: Article
• Language: English
• Subjects: alcohol dehydrogenase; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; dihydroxyacetone reductase; Fundamental and applied biological sciences. Psychology; glycerol dehydrogenase; Hansenula ofunaensis; Miscellaneous; Mission oriented research
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/S1389-1723(99)80193-5

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°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.