Skip to main content
SpringerLink
Log in

Efficient expression and purification of recombinant alcohol oxidase in Pichia pastoris

  • Research Paper
  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

In order to improve the production of alcohol oxidase (AOX), a recombinant Pichia pastoris (P. pastoris) system was constructed by transformation of the plasmid pPIC9K-AOX into P. pastoris GS115. The effects of different expression conditions on alcohol oxidase activity in the culture supernatant were investigated in the shake flask scale. The results showed that the highest extracellular activity (562 U/L) of alcohol oxidase was obtained after 56 h induction with 4% methanol at OD600 1.0 in the medium containing 50 g/L maltose, which is about 4.2 folds higher than previously reported. High-purity functional recombinant AOX (>90%) was purified from the culture with the Ni-NTA affinity column and Sephadex G-100 chromatographical methods, with a total recovery rate of 68.9%. Further studies showed that the purified rAOX had similar enzymatic characteristics as the native enzyme, except that the thermal stability and resistance to H2O2 inhibition of rAOX were significantly greater compared to the previous report. The purified rAOX was well tolerant to various water-miscible organic solvents. This efficient expression and purification process will be promising for large-scale production of rAOX as an important diagnostic enzyme for alcohol detection in many areas.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Kumar, A. K. and P. Goswami (2008) Purification and properties of a novel broad substrate specific alcohol oxidase from Aspergillus terreus MTCC 6324. Biochim. Biophys. Acta 1784: 1552–1559.

    Article  CAS  Google Scholar 

  2. Azevedo, A., J. Cabral, D. Prazeres, T. Gibson, and L. Fonseca (2004) Thermal and operational stabilities of Hansenula polymorpha alcohol oxidase. J. Mol. Catal. B: Enzym. 27: 37–45.

    Article  CAS  Google Scholar 

  3. Ozimek, P., M. Veenhuis, and I. J. Klei (2005) Alcohol oxidase: A complex peroxisomal, oligomeric flavoprotein. FEMS Yeast Res. 5: 975–983.

    Article  CAS  Google Scholar 

  4. Visser, N. V., D. Wang, W. A. Stanley, M. R. Groves, M. Wilmanns, M. Veenhuis, and I. J. van der Klei (2007) Octameric alcohol oxidase dissociates into stable, soluble monomers upon incubation with dimethylsulfoxide. Arch. Biochem. Biophys. 459: 208–213.

    Article  CAS  Google Scholar 

  5. Lopez-Gallego, F., L. Betancor, A. Hidalgo, G. Dellamora-Ortiz, C. Mateo, R. Fernandez-Lafuente, and J. M. Guisan (2007) Stabilization of different alcohol oxidases via immobilization and post immobilization techniques. Enz. Microb. Technol. 40: 278–284.

    Article  CAS  Google Scholar 

  6. Larsson, L., J. Jimenez, P. Valero-Guillen, F. Martin-Luengo, and M. Kubin (1989) Establishment of 2-docosanol as a cellular marker compound in the identification of Mycobacterium xenopi. J. Clin. Microbiol. 27: 2388–2390.

    CAS  Google Scholar 

  7. Dhossche, D. and J. Rubinstein (1996) Drug detection in a suburban psychiatric emergency room. Ann. Clin. Psychiatry 8: 59–69.

    Article  CAS  Google Scholar 

  8. Dienys, G., S. Jarmalavicius, S. Budriene, D. Citavicius, and J. Sereikaite (2003) Alcohol oxidase from the yeast Pichia pastoris—a potential catalyst for organic synthesis. J. Mol. Catal. B: Enzym. 21: 47–49.

    Article  CAS  Google Scholar 

  9. Suye, S. (1997) Purification and properties of alcohol oxidase from Candida methanosorbosa M-2003. Curr. Microbiol. 34: 374–377.

    Article  CAS  Google Scholar 

  10. Ko, H. S., Y. Yokoyama, N. Ohno, M. Okadome, S. Amachi, H. Shinoyama, and T. Fujii (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.

    Article  CAS  Google Scholar 

  11. Kato, N., Y. Omori, Y. Tani, and K. Ogata (1976) Alcohol oxidases of Kloeckera sp. and Hansenula polymorpha. Eur. J. Biochem. 64: 341–350.

    Article  CAS  Google Scholar 

  12. Tian, G., C. D. Sobotka-Briner, J. Zysk, X. Liu, C. Birr, M. A. Sylvester, P. D. Edwards, C. D. Scott, and B. D. Greenberg (2002) Linear non-competitive inhibition of solubilized human ãsecretase by pepstatin A methylester, L685458, sulfonamides, and benzodiazepines. J. Biol. Chem. 277: 31499–31505.

    Article  CAS  Google Scholar 

  13. Cornish-Bowden, A. (1974) A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors (Short Communication). Biochem. J. 137: 143–144.

    CAS  Google Scholar 

  14. Abouseoud, M., R. Maachi, A. Amrane, S. Boudergua, and A. Nabi (2008) Evaluation of different carbon and nitrogen sources in production of biosurfactant by Pseudomonas fluorescens. Desalination 223: 143–151.

    Article  CAS  Google Scholar 

  15. Chi, Z., C. Ma, P. Wang, and H. Li (2007) Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans. Bioresour. Technol. 98: 534–538.

    Article  CAS  Google Scholar 

  16. Yildiz, H. B. and L. Toppare (2006) Biosensing approach for alcohol determination using immobilized alcohol oxidase. Biosensors Bioelectron. 21: 2306–2310.

    Article  CAS  Google Scholar 

  17. Klibanov, A. M. (1986) Enzymes that work in organic solvents. Chemtech. 16: 354–359.

    CAS  Google Scholar 

  18. Russell, A. and A. Klibanov (1988) Inhibitor-induced enzyme activation in organic solvents. J. Biol. Chem. 263: 11624–11626.

    CAS  Google Scholar 

  19. Tani, Y., T. Miya, and K. Ogata (1972) The microbial metabolism of methanol Part II. Agric. Biol. Chem. 36: 76–83.

    Article  CAS  Google Scholar 

  20. Distel, B., M. Veenhuis, and H. F. Tabak (1987) Import of alcohol oxidase into peroxisomes of Saccharomyces cerevisiae. EMBO J. 6: 3111–3116.

    CAS  Google Scholar 

  21. De Nobel, J. and J. Barnett (1991) Passage of molecules through yeast cell walls: A brief essay-review. Yeast 7: 313–323.

    Article  Google Scholar 

  22. Venturini, M., A. Morrione, P. Pisarra, E. Martegani, and M. Vanoni (1997) In Saccharomyces cerevisiae a short amino acid sequence facilitates excretion in the growth medium of periplasmic proteins. Mol. Microbiol. 23: 997–1007.

    Article  CAS  Google Scholar 

  23. Couderc, R. and J. Baratti (1980) Oxidation of methanol by the yeast, Pichia pastoris. Purification and properties of the alcohol oxidase. Agric. Biol. Chem. 44: 2279–2289.

    Article  CAS  Google Scholar 

  24. Camadro, J. M., F. Thome, N. Brouillet, and P. Labbe (1994) Purification and properties of protoporphyrinogen oxidase from the yeast Saccharomyces cerevisiae. Mitochondrial location and evidence for a precursor form of the protein. J. Biol. Chem. 269: 32085–32091.

    CAS  Google Scholar 

  25. Matsumoto, M., K. Kida, and K. Kondo (2001) Enhanced activities of lipase pretreated with organic solvents. J. Chem. Technol. Biotechnol. 76: 1070–1073.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310-027, China

    Yunping Liu, Jianfeng Pan, Jianzhong Zhu, Lei Huang, Jin Cai & Zhinan Xu

  2. Department of Biochemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310-023, China

    Peilian Wei

  3. Zhejiang Deqing Huining Biotechnology Corp. Ltd, Deqing, 313-200, China

    Jianzhong Zhu

Authors
  1. Yunping Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianfeng Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Peilian Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jianzhong Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lei Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jin Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhinan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jin Cai or Zhinan Xu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, Y., Pan, J., Wei, P. et al. Efficient expression and purification of recombinant alcohol oxidase in Pichia pastoris . Biotechnol Bioproc E 17, 693–702 (2012). https://doi.org/10.1007/s12257-011-0660-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-011-0660-z

Keyword

Search

Navigation