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Purification and properties of alcohol oxidase from Pichia putida

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Abstract

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 422D4 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.

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Abbreviations

AO:

alcohol oxidase

References

  1. Janssen, F. M., Kerwin, R. M., and Ruelius, H. W. (1965) Biochem. Biophys. Res. Commun., 20, 630–636.

    Article  CAS  PubMed  Google Scholar 

  2. Janssen, F. M., and Ruelius, H. W. (1968) Biochim. Biophys. Acta, 151, 330–342.

    CAS  PubMed  Google Scholar 

  3. Kerwin, R. M., and Ruelius, H. W. (1969) Appl. Environ. Microbiol., 17, 347–351.

    CAS  Google Scholar 

  4. Van der Klei, I. J. (1991) PhD, in Alcohol Oxidase in Hansenula polymorpha, Groningen, pp. 1–146.

  5. Veenhuis, M., Salomons, F. A., and van der Klei, I. J. (2000) Microsc. Res. Techn., 51, 584–600.

    Article  CAS  Google Scholar 

  6. Van der Klei, I. J., Yurimoto, H., Sakai, Y., and Veenhuis, M. (2006) Biochim. Biophys. Acta, 1793, 1453–1462.

    Google Scholar 

  7. Fujimura, S., Nakagawa, T., Ito, T., Matsufuji, Y., Miyaji, T., and Tomizuka, N. (2007) Yeast, 24, 491–498.

    Article  CAS  PubMed  Google Scholar 

  8. Bringer, S., Sprey, B., and Sahm, H. (1979) Eur. J. Biochem., 101, 563–570.

    Article  CAS  PubMed  Google Scholar 

  9. Couderc, R., and Baratti, J. (1980) Agric. Biol. Chem., 44, 2279–2289.

    CAS  Google Scholar 

  10. Patel, R. N., Hou, C. T., Laskin, A. I., and Derelanko, P. (1981) Arch. Biochem. Biophys., 21, 481–488.

    Article  Google Scholar 

  11. Hopkins, T. R. (1984) United State Patent, N4430427.

  12. Averbach, A. Z., Pekel, N. D., Seredenko, V. I., Kulikov, A. V., Gvozdev, R. I., and Rudakova, I. P. (1995) Biochem. J., 310, 601–604.

    Google Scholar 

  13. Harrison, J. R. (1990) United State Patent, No. 4956290.

  14. Muller, F., Hopkins, T. R., Lee, J., and Bastians, P. I. H. (1992) in Chem. Biochem. Flavoenzymes (Muller, F., ed.) Vol. 3, CRC Press, Boca Baton, pp. 95–119.

    Google Scholar 

  15. Gruzman, M. B., Titorenko, V. I., Ashin, V. V., Lusta, K. A., and Trotsenko, Yu. A. (1996) Biochemistry (Moscow), 61, 1537–1544.

    Google Scholar 

  16. Suye, S. (1997) Curr. Microbiol., 34, 374–377.

    Article  CAS  PubMed  Google Scholar 

  17. Ashin, V. V., and Trotsenko, Y. A. (2000) J. Mol. Catal. B: Enzym., 10, 295–303.

    Article  CAS  Google Scholar 

  18. Ashin, V. V. (2002) Structure-Functional Characteristic of Alcohol Oxidase Isoforms from Methylotrophic Yeasts Pichia methanolica MH4: Candidate’s dissertation [in Russian], Research Institute of Biochemistry and Microbiology, Pushchino.

    Google Scholar 

  19. Ko, H.-S., Yokoyama, Y., Ohno, N., Okadome, M., Amachi, S., Shinoyama, H., and Fujii, T. (2005) J. Biosci. Bioeng., 99, 348–353.

    Article  CAS  PubMed  Google Scholar 

  20. Shleev, S. V., Shumakovich, G. P., Nikitina, O. V., Morozova, O. V., Pavlyushko, G. N., Gaida, G. Z., and Gonchar, S. V. (2006) Biochemistry (Moscow), 71, 245–250.

    CAS  PubMed  Google Scholar 

  21. Dmytruk, K. V., Smutok, O. V., Ryabova, O. B., Gayda, G. Z., Sibirny, V. A., Schuhmann, W., Gonchar, M. V., and Sibirny, A. A. (2007) BMC Biotechnol., 7, 1–7.

    Article  Google Scholar 

  22. Anthon, G. E., and Barret, D. M. (2004) J. Agric. Food. Chem., 52, 3749–3753.

    Article  CAS  PubMed  Google Scholar 

  23. De Prada, A. G., Pena, N., Mena, M. L., Reviejo, A. J., and Pingarron, J. M. (2003) Biosens. Bioelectron., 18, 127–1288.

    Google Scholar 

  24. Shkotova, L. V., Soldatkin, A. P., Gonchar, M. V., Schulman, W., and Dzyadevitch, S. V. (2006) Sci. Eng.: C, 26, 411–414.

    Article  CAS  Google Scholar 

  25. Azevedo, A. M., Prazeres, D. M. F., Cabral, J. M. S., and Fonseca, L. P. (2005) Biosens. Biotechnol., 21, 235–247.

    Article  CAS  Google Scholar 

  26. Hack, J. B., Early, J., and Brewer, K. L. (2007) Acad. Emerg. Med., 14, 1130–1134.

    Article  PubMed  Google Scholar 

  27. Guangming, W., Zhang, Y., Shuang, S., Dong, C., and Choi, M. M. F. (2007) Biosens. Bioelectron., 23, 121–129.

    Article  Google Scholar 

  28. Hattfield, G. W. (1993) United State Patent, No. 5234827

  29. Sreekrishna, Brankamp, R. G., Kropp, K. E., Blankenship, D. T., Tsay, J. T., Smith, P. L., Wierschke, J. D., Subramaniam, A., and Birkenberger, L. A. (1997) Gene, 190, 55–62.

    Article  CAS  PubMed  Google Scholar 

  30. Hollenberg, C. P., and Gellisen, G. (1997) Curr. Opin. Biotechnol., 8, 554–560.

    Article  CAS  PubMed  Google Scholar 

  31. Cregg, J. M., Cereghino, J. L., and Higgins, D. R. (2000) Mol. Biotechnol., 16, 23–52.

    Article  CAS  PubMed  Google Scholar 

  32. Van Dijk, R., Faber, K. N., Kiel, J. A., Veenhuis, M., and van der Klei, I. (2000) Enzyme Microb. Technol., 26, 793–800.

    Article  PubMed  Google Scholar 

  33. Macauley-Patrik, S., Fazenda, M. L., McNeil, B., and Harvey, L. M. (2005) Yeast, 22, 249–270.

    Article  Google Scholar 

  34. Lin-Cereghino, G. P., Godfrey, L., de la Cruz, B. J., and Johnson, S. (2006) Mol. Cell. Biol., 26, 883–897.

    Article  CAS  PubMed  Google Scholar 

  35. Hopkins, T. R. (1984) United State Patent, No. 4430427.

  36. Ornstain, L., and Davis, B. J. (1964) Ann. N. Y. Acad. Sci., 121, 404–427.

    Google Scholar 

  37. Laemmli, U. K. (1970) Nature, 227, 680–685.

    Article  CAS  PubMed  Google Scholar 

  38. Dowson, R., Elliot, D., Elliot, W., and Jones, K. (1991) Handbook in Biochemistry [Russian translation], Mir, Moscow.

    Google Scholar 

  39. Svedberg, T., and Pedersen, K. O. (1940) The Ultracentrifuge, Oxford University Press, London.

    Google Scholar 

  40. Tsuprun, V. L., Mitsova, I. Z., Blazhchuk, I. S., Gvozdev, R. I., Orlova, E. V., and Kiselev, N. A. (1985) Eur. J. Biochem., 149, 389–392.

    Article  CAS  PubMed  Google Scholar 

  41. Cregg, J. M., Madden, K. R., Barringer, K. J., Thill, G. P., and Stillman, C. A. (1989) Mol. Cell. Biol., 9, 1316–1323.

    CAS  PubMed  Google Scholar 

  42. Vonck, J., and van Bruggen, E. F. J. (1990) Biochim. Biophys. Acta, 1038, 74–79.

    CAS  PubMed  Google Scholar 

  43. Vonck, J., and van Bruggen, E. F. J. (1992) J. Bacteriol., 174, 5391–5399.

    CAS  PubMed  Google Scholar 

  44. Bystryak, L. V., Dukhuizen, L., and Harder, W. (1991) J. Gen. Microbiol., 137, 2381–2386.

    Google Scholar 

  45. Tykarska, E., Lebioda, L., Marchut, E., Steczko, J., and Stec, B. (1990) J. Prot. Chem., 9, 83–86.

    Article  CAS  Google Scholar 

  46. Boys, C. W. G., Hill, D. J., Stockley, P. G., and Woodward, J. R. (1989) J. Mol. Biol., 208, 211–212.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Biosensor AN Ltd., pr. Akademika Semenova 1, 142432, Chernogolovka, Moscow Region, Russia

    A. R. Gvozdev

  2. Institute of Problems of Chemical Physics, Russian Academy of Sciences, pr. Akademika Semenova 1, 142432, Chernogolovka, Moscow Region, Russia

    A. R. Gvozdev, I. A. Tukhvatullin & R. I. Gvozdev

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  1. A. R. Gvozdev
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  2. I. A. Tukhvatullin
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Published in Russian in Biokhimiya, 2010, Vol. 75, No. 2, pp. 297–304.

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Gvozdev, A.R., Tukhvatullin, I.A. & Gvozdev, R.I. Purification and properties of alcohol oxidase from Pichia putida . Biochemistry Moscow 75, 242–248 (2010). https://doi.org/10.1134/S000629791002015X

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