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Markošová K, Weignerová L, Rosenberg M, et al. Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris Mut(S) strain. Front Microbiol. 2015;6:1140doi: 10.3389/fmicb.2015.01140.
Markošová, K., Weignerová, L., Rosenberg, M., Křen, V., & Rebroš, M. (2015). Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris Mut(S) strain. Frontiers in microbiology, 61140. https://doi.org/10.3389/fmicb.2015.01140
Markošová, Kristína, et al. "Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris Mut(S) strain." Frontiers in microbiology vol. 6 (2015): 1140. doi: https://doi.org/10.3389/fmicb.2015.01140
Markošová K, Weignerová L, Rosenberg M, Křen V, Rebroš M. Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris Mut(S) strain. Front Microbiol. 2015 Oct 19;6:1140. doi: 10.3389/fmicb.2015.01140. eCollection 2015. PMID: 26539173; PMCID: PMC4611059.
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Front Microbiol. 2015 Oct 19;6:1140. doi: 10.3389/fmicb.2015.01140. eCollection 2015.

Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris Mut(S) strain.

Frontiers in microbiology

Kristína Markošová, Lenka Weignerová, Michal Rosenberg, Vladimír Křen, Martin Rebroš

Affiliations

  1. Department of Biochemical Technology, Slovak University of Technology Bratislava, Slovakia.
  2. Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic Prague, Czech Republic.

PMID: 26539173 PMCID: PMC4611059 DOI: 10.3389/fmicb.2015.01140

Abstract

Pichia pastoris is currently one of the most preferred microorganisms for recombinant enzyme production due to its efficient expression system. The advantages include the production of high amounts of recombinant proteins containing the appropriate posttranslational modifications and easy cultivation conditions. α-L-Rhamnosidase is a biotechnologically important enzyme in food and pharmaceutical industry, used for example in debittering of citrus fruit juices, rhamnose pruning from naringin, or enhancement of wine aromas, creating a demand for the production of an active and stable enzyme. The production of recombinant α-L-rhamnosidase cloned in the Mut(S) strain of P. pastoris KM71H was optimized. The encoding gene is located under the control of the AOX promoter, which is induced by methanol whose concentration is instrumental for these strain types. Fermentation was upscaled in bioreactors employing various media and several methanol-feeding strategies. It was found that fed batch with BSM media was more effective compared to BMMH (Buffered Methanol-complex Medium) media due to lower cost and improved biomass formation. In BSM (Basal Salt Medium) medium, the dry cell weight reached approximately 60 g/L, while in BMMH it was only 8.3 g/L, without additional glycerol, which positively influenced the amount of enzyme produced. New methanol feeding strategy, based on the level of dissolved oxygen was developed in this study. This protocol that is entirely independent on methanol monitoring was up scaled to a 19.5-L fermenter with 10-L working volume with the productivity of 13.34 mgprot/L/h and specific activity of α-L-rhamnosidase of 82 U/mg. The simplified fermentation protocol was developed for easy and effective fermentation of P. pastoris Mut(S) based on dissolved oxygen monitoring in the induction phase of an enzyme production.

Keywords: Pichia pastoris; fermentation; recombinant enzyme; upscale; α-L-rhamnosidase

References

  1. Microb Cell Fact. 2003 Jun 18;2(1):6 - PubMed
  2. Biotechnol Bioeng. 2000 Oct 5;70(1):1-8 - PubMed
  3. Biotechnol Lett. 2015 Jun;37(6):1257-64 - PubMed
  4. J Biosci Bioeng. 2002;94(4):321-5 - PubMed
  5. Biotechnol Bioeng. 2001 Jan 5;72(1):1-11 - PubMed
  6. Microb Cell Fact. 2011 Mar 03;10:14 - PubMed
  7. FEMS Microbiol Rev. 2000 Jan;24(1):45-66 - PubMed
  8. J Ind Microbiol Biotechnol. 2003 Apr;30(4):210-5 - PubMed
  9. Biotechnol Lett. 2003 Jan;25(2):173-7 - PubMed
  10. Biosci Biotechnol Biochem. 2000 Oct;64(10):2179-85 - PubMed
  11. Biotechnol Prog. 2001 May-Jun;17(3):495-502 - PubMed
  12. Arch Gerontol Geriatr. 2011 Jul-Aug;53(1):106-12 - PubMed
  13. Microb Cell Fact. 2011 Oct 27;10:85 - PubMed
  14. Biotechnol Prog. 2012 May-Jun;28(3):878-86 - PubMed
  15. Biochimie. 2003 Jan-Feb;85(1-2):109-21 - PubMed
  16. Curr Opin Biotechnol. 2002 Aug;13(4):329-32 - PubMed
  17. Lett Appl Microbiol. 2002;34(6):461-5 - PubMed
  18. Anal Biochem. 1976 May 7;72:248-54 - PubMed
  19. Chem Pharm Bull (Tokyo). 2002 Feb;50(2):175-8 - PubMed
  20. Curr Med Chem. 2001 Sep;8(11):1303-28 - PubMed
  21. Biotechnol Bioeng. 2006 Apr 5;93(5):871-9 - PubMed
  22. J Biol Chem. 2000 Mar 17;275(11):7497-504 - PubMed

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