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Purification and Properties of Alcohol Oxidase from Pichia putida

A. R. Gvozdev1,2, I. A. Tukhvatullin2, and R. I. Gvozdev2*

1Biosensor AN Ltd., pr. Akademika Semenova 1, 142432 Chernogolovka, Moscow Region, Russia; fax: (496) 515-3588; E-mail: gari@icp.ac.ru

2Institute of Problems of Chemical Physics, Russian Academy of Sciences, pr. Akademika Semenova 1, 142432 Chernogolovka, Moscow Region, Russia; fax: (496) 522-3507; E-mail: rig@icp.ac.ru

* To whom correspondence should be addressed.

Received March 16, 2009; Revision received May 20, 2009
Alcohol oxidase (AO) was extracted from the methylotrophic yeast Pichia putida and purified using various methods. AO purified by crystallization was homogeneous based on analytical centrifugation with subsequent gel filtration and SDS-PAGE. The molecular weight of the enzyme was around 600 kDa. SDS-PAGE revealed a single protein band (74 ± 4 kDa), and 8-9 bands of native protein with similar specific AO activities and substrate specificities were identified by PAGE without SDS. Electron microscopy of a single molecule revealed eight subunits located on the top of a regular tetragon with dotted symmetry of 422 D4 providing evidence that AO consists of eight subunits. Apparently, each molecule of AO has two types of subunits with very similar molecular weights and differing from each other by the number of acidic and basic amino acid residues. Each subunit includes one molecule of FAD and 2-3 cysteine residues. The pH optimum was within 8.5-9.0. Specific activity of the enzyme varied from 10 to 50 µmol methanol/min per mg protein from batch to batch depending on separation methods and had linear relationship with protein concentration. The AO was quickly inactivated at 20°C and seemed to be stable in phosphate-citrate buffer with 30-50% (w/v) of sucrose. Different forms of 0.1-1 mm crystals of the enzyme were obtained. However the crystals did not yield X-ray reflections, apparently as a result of their molecular microheterogeneity.
KEY WORDS: alcohol oxidase, subunits, quaternary structure, crystallization

DOI: 10.1134/S000629791002015X