Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha

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 a...

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Published in:Biochemistry (Moscow) 2006-03, Vol.71 (3), p.245-250
Main Authors: Shleev, S V, Shumakovich, G P, Nikitina, O V, Morozova, O V, Pavlishko, H M, Gayda, G Z, Gonchar, M V
Format: Article
Language:English
Subjects:
Alcohol Oxidoreductases - genetics
Alcohol Oxidoreductases - isolation & purification
Alcohol Oxidoreductases - metabolism
Ethanol - metabolism
Formaldehyde - metabolism
Fungal Proteins - genetics
Fungal Proteins - isolation & purification
Fungal Proteins - metabolism
Hansenula polymorpha
Methanol - metabolism
Molecular Weight
Pichia - enzymology
Protein Structure, Secondary
Substrate Specificity
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Summary: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 (approximately 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.
ISSN:0006-2979
1608-3040
0320-9725
DOI:10.1134/s0006297906030035