Skip to main content
SpringerLink
Log in

A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors

  • Original Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1 % (w/v) and galactose 0.5 % (w/v), while in the feed step the culture medium was constituted by 5 % (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59 %) and cyprosin B activity (91 %) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6 × 104 U·mg−1, corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4 %. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Lund BM, Knox MR, Sims AP (1984) The effect of oxygen and redox potential on growth of Clostridium botulinum type E from a spore inoculum. Food Microbiol 1:277–287

    Article  Google Scholar 

  2. Lee J, Lee SY, Park S, Middelberg AP (1999) Control of fed-batch fermentations. Biotechnol Adv 17:29–48

    Article  CAS  Google Scholar 

  3. Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8:423–488. doi:10.1002/yea.320080602

    Article  CAS  Google Scholar 

  4. Sampaio PN, Calado CRC, Sousa L, Bressler DC, Pais MS, Fonseca LP (2010) Optimization of the culture medium composition using response surface methodology for new recombinant cyprosin B production in bioreactor for cheese production. Eur Food Res Technol 231:339–346. doi:10.1007/s00217-010-1281-z

    Article  CAS  Google Scholar 

  5. Sampaio PN, Sousa L, Calado CRC, Pais MS, Fonseca LP (2011) Use of chemometrics in the selection of a Saccharomyces cerevisiae expression system for recombinant cyprosin B production. Biotechnol Lett 33:2111–2119. doi:10.1007/s10529-011-0678-5

    Article  CAS  Google Scholar 

  6. Cordeiro MC, Xue ZT, Pietrzak M, Pais MS, Brodelius PE (1994) Isolation and characterization of a cDNA from flowers of Cynara cardunculus encoding cyprosin (an aspartic proteinase) and its use to study the organ-specific expression of cyprosin. Plant Mol Biol 24:733–741

    Article  CAS  Google Scholar 

  7. Heimgartner U, Pietrzak M, Geertsen R, Brodelius P, Silva Figueiredo AC, Pais MS (1990) Purification and partial characterization of milk clotting proteases from flowers of Cynara cardunculus. Phytochemistry 29:1405–1410

    Article  CAS  Google Scholar 

  8. Verissimo P, Esteves C, Faro CJ, Pires EV (1995) The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities. Biotechnol Lett 17:621–626

    Article  CAS  Google Scholar 

  9. Ramalho-Santos M, Pissara J, Veríssimo P, Pereira S, Salema R, Pires E, Faro C (1997) Cardosin A, an abundant aspartic proteinase, accumulates in protein storage vacuoles in the stigmatic papillae of Cynara cardunculus L. Planta 203:204–212

    Article  CAS  Google Scholar 

  10. Barros R, Ferreira C, Silva S, Malcata F (2001) Quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulphate. Enzyme Microb Technol 29:541–547

    Article  CAS  Google Scholar 

  11. Twining SS (1984) Fluorescein isothiocyanate–labelled casein assay for proteolytic enzymes. Anal Biochem 143:30–34

    Article  CAS  Google Scholar 

  12. Laemmli UK (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227:680–686

    Article  CAS  Google Scholar 

  13. Berridge NJ (1952) Some observations on the determination of the activity of rennet. Analyst London 77:57–62

    Article  CAS  Google Scholar 

  14. Kapat A, Jung JK, Park YH (2000) Effect of continuous feeding of galactose on the production of recombinant glucose oxidase using S. cerevisiae. Bioprocess Eng 23:37–40

    Article  CAS  Google Scholar 

  15. Caunt P, Impoolsup A, Greenfield PF (1989) The effect of oxygen limitation on stability of a recombinant plasmid in S. cerevisiae. Biotechnol Lett 11:5–10

    Article  CAS  Google Scholar 

  16. Lu-Chau TA, Guillan A, Nuñez MJ, Roca E, Lema JM (2004) Anaerobic and aerobic continuous culture of S. cerevisiae: comparison of plasmid stability and EXG1 gene expression. Bioprocess Biosyst Eng 26:159–163

    Article  CAS  Google Scholar 

  17. Konz JO, King J, Cooney CL (1998) Effects of oxygen on recombinant protein expression. Biotechnol Prog 14:393–409

    Article  CAS  Google Scholar 

  18. Käpelli O (1986) Regulation of carbon metabolism in Saccharomyces cerevisiae and related yeasts. Adv Microb Physiol 28:181–208

    Article  Google Scholar 

  19. Salmon JM (1998) Determination of oxygen utilization pathways in an industrial strain of Saccharomyces cerevisiae during enological fermentation. J Ferment Bioeng 86:154–163

    Article  CAS  Google Scholar 

  20. Ferreira BS, Calado CRC, van Keulen F, Fonseca LP, Cabral JMS, Fonseca MR (2004) Recombinant Saccharomyces cerevisiae strain triggers acetate production to fuel biosynthetic pathways. J Biotech 109:159–167

    Article  CAS  Google Scholar 

  21. Oosttergardt K, Larsson C, Bill RM, Stahlberg A, Boles E, Hohmann S, Gustafsson L (2004) Switching the mode of metabolism in the yeast Saccharomyces cerevisiae. EMBO Rep 5:532–537

    Article  Google Scholar 

  22. Krause M, Ukkonen K, Haataja T, Ruottinen M, Glumoff T, Neubauer A, Neubauer P, Vasala A (2010) A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb Cell Fact 9:1–11

    Article  Google Scholar 

  23. Sampaio PN, Fortes AM, Cabral JMS, Pais MS, Fonseca LP (2008) Production and characterization of recombinant cyprosin B in Saccharomyces cerevisiae W303-1A strain. J Biosc and Bioeng 105:305–312. doi:10.1263/jbb.105.305

    Article  CAS  Google Scholar 

  24. Calado CRC, Almeida C, Cabral JMS, Fonseca LP (2003) Development of fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant S. cerevisiae SU50 strain. J Biosc Bioeng 96:141–148

    Article  CAS  Google Scholar 

  25. Jin SY, Shimizu K (1995) Metabolic pathway analysis of recombinant S. cerevisiae with a galactose-inducible promoter based on a signal flow modelling approach. J Ferment Bioeng 80:541–551

    Article  CAS  Google Scholar 

  26. Mendoza-Vega O, Hebert C et al (1994) Production of recombinant hirudin by high cell density fed-batch cultivations of a Saccharomyces cerevisiae strain: physiological considerations during the bioprocess design. J Biotechnol 32:249–259

    Article  CAS  Google Scholar 

  27. Calado CRC, Taipa MA, Cabral JMS, Fonseca LP (2002) Optimization of culture conditions and characterization of cutinase produced by recombinant Saccharomyces cerevisiae. Enzym Microb Technol 31:161–170

    Article  CAS  Google Scholar 

  28. Zabriski DW, Arcuri EJ (1986) Factors influencing productivity of cultivations employing recombinant microorganisms. Enzym Microb Technol 8:706–717

    Article  Google Scholar 

  29. Gagnon P, Grund E, Lindback T (1995) Media selection and rapid development of binding conditions for high performance, hydrophobic interaction chromatography (HIC) LC-GC. Int 8:502–510

    Google Scholar 

  30. White PC, Cordeiro MC, Arnold D, Brodelius PE, Kay J (1999) Processing, activity and inhibition of recombinant cyprosin, and aspartic proteinase from cardoon (Cynara cardunculus). J Biol Chem 274:16685–16693

    Article  CAS  Google Scholar 

  31. Faro C, Ramalho-Santos M, Vieira M, Mendes A, Simões I, Andrade R, Veríssimo P, Lin X, Tang J, Pires E (1999) Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase. J Biol Chem 274:28724–28729

    Article  CAS  Google Scholar 

  32. Ramalho-Santos M, Pissarra J, Pires E, Faro C (1998) Cardosinogen A: the precursor form of the major aspartic proteinase from cardoon. In: James Michel NG (ed) Aspartic proteinases: retroviral and cellular enzymes. Plenum Press, New York, pp 253–258

    Chapter  Google Scholar 

Download references

Acknowledgments

We thank Dr. Filomena Calixto for providing S. cerevisiae BJ1991strain transformed with CYPRO11 gene.

Author information

Authors and Affiliations

  1. BioFIG, Unit of Molecular Biology and Plant Biotechnology, Plant Systems Biology Laboratory, Institute of Applied Science and Technology, Faculty of Sciences of University of Lisbon, Campo Grande, 1749-016, Lisbon, Portugal

    P. N. Sampaio & M. S. Pais

  2. Department of Bioengineering, Instituto Superior Técnico (IST), University of Lisbon, Av Rovisco Pais, 1049-001, Lisbon, Portugal

    P. N. Sampaio & L. P. Fonseca

  3. Institute for Biotechnology and Bioengineering (IBB), Centre for Biological and Chemical Engineering, IST, UL, Lisbon, Portugal

    L. P. Fonseca

Authors
  1. P. N. Sampaio
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. S. Pais
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L. P. Fonseca
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P. N. Sampaio.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sampaio, P.N., Pais, M.S. & Fonseca, L.P. A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors. Bioprocess Biosyst Eng 37, 2515–2527 (2014). https://doi.org/10.1007/s00449-014-1229-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-014-1229-y

Keywords

Search

Navigation