Journal of Zhejiang University SCIENCE B

, Volume 10, Issue 10, pp 769–776 | Cite as

Effects of proteinase a on cultivation and viability characteristics of industrial Saccharomyces cerevisiae WZ65

  • Hong-bo Zhang
  • Hai-feng Zhang
  • Qi-he Chen
  • Hui Ruan
  • Ming-liang Fu
  • Guo-qing He


Proteinase A (PrA), encoded by PEP4 gene, is a key enzyme in the vacuoles of Saccharomyces cerevisiae. We characterized the effects of PrA on cell growth and glucose metabolism in the industrial S. cerevisiae WZ65. It was observed that the lag phase of cell growth of partial PEP4 gene deletion mutant (36 h) and PrA-negative mutant (48 h) was significantly extended, compared with the wild type strain (24 h) (P<0.05), but PrA had no effect on glucose metabolism either under shaking or steady state cultivations. The logistic model was chosen to evaluate the effect of PrA on S. cerevisiae cell growth, and PrA was found to promote cell growth against insufficient oxygen condition in steady state cultivation, but had no effect in shaking cultivation. The effects of glucose starvation on cell growth of partial PEP4 gene deletion strain and PrA-negative mutant were also evaluated. The results show that PrA partial deficiency increased the adaption of S. cerevisiae to unfavorable nutrient environment, but had no effect on glucose metabolism under the stress of low glucose. During heat shock test, at 60 °C the reduced cell viability rate (RCVR) was 10% for the wild type S. cerevisiae and 90% for both mutant strains (P<0.01), suggesting that PrA was a negative factor for S. cerevisiae cells to survive under heat shock. As temperatures rose from 60 °C to 70 °C, the wild type S. cerevisiae had significantly lower relative glucose consumption rate (RGCR) (61.0% and 80.0%) than the partial mutant (78.0% and 98.5%) and the complete mutant (80.0% and 98.0%) (P<0.05), suggesting that, in coping with heat shock, cells of the PrA mutants increased their glucose consumption to survive. The present study may provide meaningful information for brewing industry; however, the role of PrA in industrial S. cerevisiae physiology is complex and needs to be further investigated.

Key words

Proteinase A (PrA) PEP4 gene Saccharomyces cerevisiae WZ65 Cell metabolism Viability 

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  1. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72(1–2):248–254. [doi:10.1016/0003-2697(76)90527-3]PubMedCrossRefGoogle Scholar
  2. Brandberg, T., Gustafsson, L., Franzén, C.J., 2007. The impact of severe nitrogen limitation and microaerobic conditions on extended continuous cultivations of Saccharomyces cerevisiae with cell recirculation. Enzyme Microb. Technol., 40(4):585–593. [doi:10.1016/j.enzmictec.2006.05.032]CrossRefGoogle Scholar
  3. Cooper, D.J., Stewart, G.G., Bryce, J.H., 2000. Yeast proteolytic activity during high and low gravity wort fermentations and its effect on head retention. J. Inst. Brew., 1066(4):197–201.Google Scholar
  4. Davidson, J.F., Schiestl, R.H., 2001a. Cytotoxic and genotoxic consequences of heat stress are dependent on the presence of oxygen in Saccharomyces cerevisiae. J. Bacteriol., 183(15):4580–4587. [doi:10.1128/JB.183.15.4580-4587.2001]PubMedCrossRefGoogle Scholar
  5. Davidson, J.F., Schiestl, R.H., 2001b. Mitochondrial respiratory electron carriers are involved in oxidative stress during heat stress in Saccharomyces cerevisiae. Mol. Cell. Biol., 21(24):8483–8489. [doi:10.1128/MCB.21.24.8483-8489.2001]PubMedCrossRefGoogle Scholar
  6. Fernandes, P.M.B., Domitrovic, T., Kao, C.M., Kurtenach, E., 2004. Genomic expression pattern in Saccharomyces cerevisiae cells in response to high hydrostatic pressure. FEMS Lett., 556(1):153–160. [doi:10.1016/S0014-5793(03)01396-6]Google Scholar
  7. He, G.Q., Wang, Z.Y., Liu, Z.S., Chen, Q.H., Ruan, H., Schwarz, P.B., 2006. Relationship of proteinase activity, foam proteins, and head retention in unpasteurized beer. J. Am. Soc. Brew. Chem., 64(1):33–38.Google Scholar
  8. Jones, E.W., 1991. Three proteolytic systems in the yeast Saccharomyces cerevisiae. J. Biol. Chem., 266(13):7963–7966.PubMedGoogle Scholar
  9. Kim, I.S., Moon, H.Y., Yun, H.S., Jin, I., 2006. Heat shock causes oxidative stress and induces a variety of cell rescue proteins in Saccharomyces cerevisiae KNU5377. J. Microb., 44(5):492–501.Google Scholar
  10. Li, S.C., Kane, P.M., 2009. The yeast lysosome-like vacuole: Endpoint and crossroads. Biochim. Biophys. Acta, 1793(4):650–663. [doi:10.1016/j.bbamcr.2008.08.003]PubMedCrossRefGoogle Scholar
  11. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 31(3):426–427. [doi:10.1021/ac60147a030]CrossRefGoogle Scholar
  12. Parr, C.L., Keates, R.A.B., Bryksa, B.C., Ogawa, M., Yada, R.Y., 2007. The structure and function of Saccharomyces cerevisiae proteinase A. Yeast, 24(6):467–480. [doi:10.1002/yea.1485]PubMedCrossRefGoogle Scholar
  13. Sambrook, J., Fritsch, E.F., Maniatis, T., 2002. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p.1–1859.Google Scholar
  14. Simões, I., Faro, C., 2004. Structure and function of plant aspartic proteinases. Eur. J. Biochem., 271(11):2067–2075. [doi:10.1111/j.1432-1033.2004.04136.x]PubMedCrossRefGoogle Scholar
  15. Stevens, T.H., Esmon, B., Schekman, R., 1982. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell, 30(2):439–448. [doi:10.1016/0092-8674(82)90241-0]PubMedCrossRefGoogle Scholar
  16. Tang, Q.Y., Feng, M.G., 1997. Practical Statistics and DPS Data Processing System. China Agricultural Press, Beijing, p.1–292 (in Chinese).Google Scholar
  17. Teichert, U., Mechlere, B., Muller, H., Wolf, D.H., 1989. Lysosomal (vacuolar) proteinases of yeast are essential catalysts for protein degradation, differentiation, and cell survival. J. Biol. Chem., 264(27):16037–16045.PubMedGoogle Scholar
  18. van den Hazel, H.B., Kielland-Brandt, M.C., Winther, J.R., 2005. Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens. Eur. J. Biochem., 207(1):277–283. [doi:10.1111/j.1432-1033.1992.tb17048.x]CrossRefGoogle Scholar
  19. Wang, Z.Y., He, G.Q., Liu, Z.S., Ruan, H., Chen, Q.H., Xiong, H.P., 2005. Purification of yeast proteinase A from fresh beer and its specificity on foam proteins. Int. J. Food Sci. Technol., 40(8):835–840. [doi:10.1111/j.1365-2621.2005.01000.x]CrossRefGoogle Scholar
  20. Wang, Z.Y., He, G.Q., Ruan, H., Liu, Z.S., Yang, L.F., Zhang, B.R., 2007. Construction of proteinase A deficient transformant of industrial brewing yeast. Eur. Food Res. Technol., 225(5–6):831–835. [doi:10.1007/s00217-006-0488-5]CrossRefGoogle Scholar
  21. Zaman, S., Lippman, S.I., Zhao, X., Broach, J.R., 2008. How Saccharomyces responds to nutrients. Annu. Rev. Genet., 42(1):27–81. [doi:10.1146/annurev.genet.41.110306.130206]PubMedCrossRefGoogle Scholar
  22. Zhang, Q., Chen, Q.H., Fu, M.L., Wang, J.L., Zhang, H.B., He, G.Q., 2008. Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination. J. Zhejiang Univ. Sci. B, 9(7):527–535. [doi:10.1631/jzus.B0820019]PubMedCrossRefGoogle Scholar
  23. Zubenko, G.S., Park, F.J., Jones, E.W., 1983. Mutations in PEP4 locus of Saccharomyces cerevisiae block final step in maturation of two vacuolar hydrolases. Proc. Natl. Acad. Sci. USA, 80(2):510–514. [doi:10.1073/pnas.80.2.510]PubMedCrossRefGoogle Scholar

Copyright information

© Zhejiang University and Springer Berlin Heidelberg 2009

Authors and Affiliations

  1. 1.Department of Food Science and NutritionZhejiang UniversityHangzhouChina

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