Multiple Molecular Forms of Alcohol Oxidase from the Methylotrophic Yeast Pichia methanolica

M. B. Gruzman,¹ V. I. Titorenko,¹ V. V. Ashin,¹ K. A. Lusta,¹ and Yu. A. Trotsenko^1,2

¹Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (7-095) 923-36-02; E-mail: trotsenko@ibpm.serpukhov.su

²To whom correspondence should be addressed.

Submitted June 10, 1996; revision submitted July 10, 1996.

Alcohol oxidase (AO) isolated from Pichia methanolica MH4 exists in nine molecular forms. A combination of ion-exchange high-performance liquid chromatography (HPLC) and preparative electrophoresis under nondenaturing conditions resulted in isolation, purification, and partial characterization of individual AO forms displaying equal molecular weights but different pI (5.2-4.2), the K_m for methanol (0.5-6.5 mM), and the contents of mFAD (78.7-18.1%). The ratio of relative levels of these isoforms was an important feature that consistently changed in consecutive phases of the culture growth and upon changes in the growth rate in the chemostat mode. The isoform displaying the highest electrophoretic mobility dominated at the beginning of exponential growth during periodic cultivation and at high growth rates during cultivation in a chemostat, whereas the low-mobility isoform was mainly found in the late stationary phase and at low growth rates during chemostat cultivation. Mechanisms that can mediate the formation of multiple forms of AO and their functional roles are discussed.

KEY WORDS: methylotrophic yeasts, alcohol oxidase, isoenzymes.

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