Skip to main content
SpringerLink
Log in

Enhancing functional expression of β-glucosidase in Pichia pastoris by co-expressing protein disulfide isomerase

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

Abstract

The expression of heterologous proteins may exert severe stress on the host cells at different levels. Protein folding and disulfide bond formation were identified as rate-limited steps in recombinant protein secretion in yeast cells. For the production of β-glucosidase in Pichia pastoris, final β-glucosidase activity reached 1,749 U/mL after fermentation optimization in a 3 L bioreactor, while the specific activity decreased from 620 to 467 U/mg, indicating a potential protein misfolding. To solve this problem, protein disulfide isomerase, a chaperone protein which may effectively regulate disulfide bond formation and protein folding, was co-expressed with β-glucosidase. In the co-expression system, a β-glucosidase production level of 2,553 U/mL was achieved and the specific activity of the enzyme reached 721 U/mg, which is 1.54 fold that of the control.

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. Chantasingh, D. (2006) Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris. Protein Expr. Purif. 46: 143–149.

    Article  CAS  Google Scholar 

  2. Cereghino, J. L. and J. M. Cregg (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24: 45–66.

    Article  CAS  Google Scholar 

  3. Chantasingh, D. (2006) Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris. Protein Expr. Purif. 46: 143–149.

    Article  CAS  Google Scholar 

  4. Zhang, W. (2006) Enhanced secretion of heterologous proteins in Pichia pastoris following overexpression of Saccharomyces cerevisiae chaperone proteins. Biotechnol. Prog. 22: 1090–1095.

    Article  CAS  Google Scholar 

  5. Hohenblum, H. (2004) Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol. Bioeng. 85: 367–375.

    Article  CAS  Google Scholar 

  6. Agashe, V. R. and F. U. Hartl (2000) Roles of molecular chaperones in cytoplasmic protein folding. Semin. Cell Dev. Biol. 11: 15–25.

    Article  CAS  Google Scholar 

  7. Qu Lin, L. J. -Y. and Z. Qi (2010) Effect of coexpressing protein disulfide isomerase on expression of IFNβ-HSA fusion protein in Pichia pastoris. Chin. J. Biologicals. 23: 346–349.

    Google Scholar 

  8. Xu, P. (2005) Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding. Metab. Eng. 7: 269–279.

    Article  CAS  Google Scholar 

  9. Powers, S. L. and A. S. Robinson (2007) PDI improves secretion of redox-inactive beta-glucosidase. Biotechnol. Prog. 23: 364–369.

    Article  CAS  Google Scholar 

  10. Sinnott, M. L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. American Chem. Society 90: 1171–1202.

    CAS  Google Scholar 

  11. Duff, S. J. B. and W. D. Murray. (1996) Bioconversion of forest products industry waste cellulosics to fuel ethanol: A review. Bioresource Technol. 55: l–33.

    Article  Google Scholar 

  12. Sheehan, J. and M. Himmel (1999) Enzymes, energy, and the environment: A strategic perspective on the US Department of Energy’s research and development activities for bioethanol. Biotechnol. Prog. 15: 817–827.

    Article  CAS  Google Scholar 

  13. Vulfson, E. N., R. Patel, and B. Law (1990) Alkyl glucoside synthesis in a water-organic two-phase system. Biotechnol.Lett. 12: 397–402.

    Article  CAS  Google Scholar 

  14. Heather, F. and R. E. H. Seidle (2005) Transglusidic reactions of the Aspergillus niger Family 3 β-glucosidase Qualitative and quantitative analyses and evidence that the transglucosidic rate is independent of pH. Arch. Biochem. Biophys. 436: 254–264.

    Article  Google Scholar 

  15. Lingling Liu, S. Z. (2009) Production of gentiooligosaccharide by recombinant β-glucosidase. Acta Mcrobiol. Sinica. 49: 597–602.

    Google Scholar 

  16. Joseph Sambrook, D. W. R (2001) Molecular cloning: A laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

    Google Scholar 

  17. Jason, D., B. C. T. Smith, and A. S. Robinson (2003) Protein disulfide isomerase. But not binding protein, overexpression enhances secretion of a Non-disulfide-bonded protein in yeast. Biotechnol. Bioeng. 85: 340–350.

    Google Scholar 

  18. Shu, W. R. (2007) Method of producing recombinant Aspergillus niger β-glucosidase and an aroma spreading plant. US Patent 7,223,902.

  19. Salmon, J. -M. (2006) Interactions between yeast, oxygen and polyphenols during alcoholic fermentations: Practical implications. Food Sci. Technol. 39: 959–965.

    CAS  Google Scholar 

  20. Perrone, G. G., S. X. Tan, and I. W. Dawes (2008) Reactive oxygen species and yeast apoptosis. Biochim. Biophys. Acta 1783: 1354–1368.

    Article  CAS  Google Scholar 

  21. Wang, Z. (2009) Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation. Bioresour. Technol. 101: 1318–1323.

    Article  Google Scholar 

  22. Joan Lin Cereghino, J. M. C. (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24: 45–66.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214-122, China

    Jian-hong Zhang, Dan Wu, Jian Chen & Jing Wu

  2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214-122, China

    Jian-hong Zhang, Dan Wu, Jian Chen & Jing Wu

Authors
  1. Jian-hong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dan Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jian Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jing Wu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, Jh., Wu, D., Chen, J. et al. Enhancing functional expression of β-glucosidase in Pichia pastoris by co-expressing protein disulfide isomerase. Biotechnol Bioproc E 16, 1196–1200 (2011). https://doi.org/10.1007/s12257-011-0136-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12257-011-0136-1

Keywords

Search

Navigation