Abstract
Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l−1, respectively.
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References
Azevedo AM, Prazeres DM, Cabral JM, Fonseca LP (2005) Ethanol biosensors based on alcohol oxidase. Biosens Bioelectron 21:235–247
Barzana E, Klibanov AM, Karel M (1989) A colorimetric method for the enzymatic analysis of gases: the determination of ethanol and formaldehyde vapors using solid alcohol oxidase. Anal Biochem 182:109–115
Belghith H, Romette JL, Thomas D (1987) An enzyme electrode for on-line determination of ethanol and methanol. Biotech Bioeng 30:1001–1005
Bormann C, Sahm H (1978) Degradation of microbodies in relation to activities of alcohol oxidase and catalase in Candida boidinii. Arch Microbiol 117:67–72
Bringer S, Sprey B, Sahm H (1979) Purification and properties of alcohol oxidase from Poria contigua. Eur J Biochem 101:563–570
Bystrykh LV, Dijkhuizen L, Harder W (1991) Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha. J Gen Microbiol 137:2381–2386
Chen RLC, Matsumoto K (1995) Sequential enzymatic monitoring of glucose, ethanol and glutamate in bioreactor fermentation broth containing a high salt concentration by a multi-channel flow-injection analysis method. Anal Chim Acta 308:145–151
Couderc R, Baratti J (1980) Oxidation of methanol by the yeast, Pichia pastoris. Purification and properties of the alcohol oxidase. Agric Biol Chem 44:2279–2289
Egli T, van Dijken JP, Veenhuis M, Harder W, Fiechter A (1980) Methanol metabolism in yeasts: regulation of the synthesis of catabolic enzymes. Arch Microbiol 124:115–121
Fujii T, Tonomura K (1972) Oxidation of methanol, formaldehyde and formate by a Candida species. Agric Biol Chem 36:2297–2306
Holzmann K, Schreiner E, Schwab H (2002) A Penicillium chrysogenum gene (aox) identified by specific induction upon shifting pH encodes for a protein which shows high homology to fungal alcohol oxidases. Curr Genet 40:339–344
Janssen FW, Kerwin RM, Ruelius HW (1975) Alcohol oxidase from Basidiomycetes. Methods Enzymol 41:364–369
Kato N, Omori Y, Tani Y, Ogata K (1976) Alcohol oxidases of Kloeckera sp. and Hansenula polymorpha. Catalytic properties and subunit structures. Eur J Biochem 64:341–350
Kato N, Miyawaki N, Sakazawa C (1982) Oxidation of formaldehyde by resistant yeasts Debaryomyces vanriji and Trichosporon penicillatum. Agric Biol Chem 46:655–661
Kerwin RM, Ruelius HW (1969) Production of alcohol oxidase by several Basidiomycetes. Appl Microbiol 17:347–351
Ko HS, Yokoyama Y, Ohno N, Okadome M, Amachi S, Shinoyama H, Fujii T (2005) Purification and characterization of intracellular and extracellular, thermostable and alkali-tolerant alcohol oxidases produced by a thermophilic fungus, Thermoascus aurantiacus NBRC 31693. J Biosci Bioeng 99:348–353
Kondo T, Morikawa Y, Hayashi N, Kitamoto N (2002) Purification and characterization of formate oxidase from a formaldehyde-resistant fungus. FEMS Microbiol Lett 214:137–142
Koutz P, Davis GR, Stillman C, Barringer K, Cregg J, Thill G (1989) Structural comparison of the Pichia pastoris alcohol oxidase genes. Yeast 5:167–177
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Ledeboer AM, Edens L, Maat J, Visser C, Bos JW, Verrips CT, Janowicz Z, Eckart M, Roggenkamp R, Hollenberg CP (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13:3063–3082
Liden H, Vijayakumar AR, Gorton L, Marko-Varga G (1998) Rapid alcohol determination in plasma and urine by column liquid chromatography with biosensor detection. J Pharm Biomed Anal 17:1111–1128
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Nash T (1953) The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem J 55:416–421
Ozimek P, Veenhuis M, van der Klei IJ (2005) Alcohol oxidase: a complex peroxisomal, oligomeric flavoprotein. FEMS Yeast Res 5:975–983
Patel RN, Hou CT, Laskin AI, Derelanko P (1981) Microbial oxidation of methanol: properties of crystallized alcohol oxidase from a yeast, Pichia sp. Arch Biochem Biophys 210:481–488
Sahm H (1975) Oxidation of formaldehyde by alcohol oxidase of Candida boidinii. Arch Microbiol 105:179–181
Sakai Y, Tani Y (1992) Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast, Candida boidinii S2. Gene 114:67–73
Segers G, Bradshaw N, Archer D, Blissett K, Oliver RP (2001) Alcohol oxidase is a novel pathogenicity factor for Cladosporium fulvum, but aldehyde dehydrogenase is dispensable. Mol Plant Microbe Interact 14:367–377
Soldevila AI, Ghabrial SA (2001) A novel alcohol oxidase/RNA-binding protein with affinity for mycovirus double-stranded RNA from the filamentous fungus Helminthosporium (Cochliobolus) victoriae. Molecular and functional characterization. J Biol Chem 276:4652–4661
Tani Y, Miya T, Nishikawa H, Ogata K (1972a) The microbial metabolism of methanol. Part I. Formation and crystallization of methanol-oxidizing enzyme in a methanol-utilizing yeast, Kloeckera sp. No. 2201. Agric Biol Chem 36:68–75
Tani Y, Miya T, Ogata K (1972b) The microbial metabolism of methanol. Part II. Properties of crystalline alcohol oxidase from Kloeckera sp. No. 2201. Agric Biol Chem 36:76–83
Udagawa S (1978) Paecilomyces variotii Bain. In ed. Udagawa S, Tsubaki K, Horie Y, Miura K, Minoura K, Yamazaki M, Yokoyama T, Watanabe S (eds) Kinrui Zukan, Vol. 2, 3rd edn. Kodansha (in Japanese), Tokyo, pp 1074–1075
van Dijken JP, Otto R, Harder W (1976) Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism. Arch Microbiol 111:137–144
Veenhuis M, Zwart K, Harder W (1978) Degradation of peroxisomes after transfer of methanol-grown Hansenula polymorpha into glucose-containing media. FEMS Microbiol Lett 3:21–28
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This work was performed through Special Coordination Funds for Promoting Science and Technology (Leading Research Utilizing Potential of Regional Science and Technology) of the Science and Technology Agency of the Japanese Government.
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Kondo, T., Morikawa, Y. & Hayashi, N. Purification and characterization of alcohol oxidase from Paecilomyces variotii isolated as a formaldehyde-resistant fungus. Appl Microbiol Biotechnol 77, 995–1002 (2008). https://doi.org/10.1007/s00253-007-1237-9
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DOI: https://doi.org/10.1007/s00253-007-1237-9