Purification and Characterization of Alcohol Oxidase from a Genetically Constructed Over-producing Strain of the Methylotrophic Yeast Hansenula polymorpha

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S. V. Shleev¹, G. P. Shumakovich¹, O. V. Nikitina¹, O. V. Morozova¹, H. M. Pavlishko², G. Z. Gayda², and M. V. Gonchar^2,3*

¹Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, 119071 Moscow, Russia; fax: (7-495) 954-2732; E-mail: shleev@inbi.ras.ru

²Department of Cell Regulatory Systems, Institute of Cell Biology, ul. Drahomanov 14/16, 79005 Lviv, Ukraine; fax: +380-322-742-144; E-mail: gonchar@biochem.lviv.ua

³Institute of Biotechnology, University of Rzeszow, Rejtana 16C, 35310 Rzeszow, Poland

^* To whom correspondence should be addressed.

Received March 24, 2005; Revision received September 12, 2005

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Alcohol oxidase (AOX) has been purified 8-fold from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha C-105 (gcr1 catX) with impaired glucose-induced catabolite repression and completely devoid of catalase. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and HPLC. Some physicochemical and biochemical properties of AOX were studied in detail: molecular weight (~620 kD), isoelectric point (pI 6.1), and UV-VIS, circular dichroism (CD), and fluorescence spectra. The content of different secondary structure motifs of the enzyme has been calculated from the CD spectra using a computer program. It was found that the native protein contains about 50% alpha-helix, 25% beta-sheet, and about 20% random structures. The kinetic parameters for different substrates, such as methanol, ethanol, and formaldehyde, were measured using a Clark oxygen electrode. The rate of enzymatic oxidation of formaldehyde by alcohol oxidase from H. polymorpha is only twice lower compared to the best substrate of the enzyme, methanol.

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KEY WORDS: alcohol oxidase, over-producing strain, secondary structure, spectral and kinetic properties

DOI: 10.1134/S0006297906030035

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