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Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization

  • Biotechnology Methods
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca2+ and inhibited by Zn2+, Mn2+, Al3+, and Cu2+. The K m and k cat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system.

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References

  1. Andrews PC, Minth CD, Dixon JE (1982) Immunochemical characterization of a proline endopeptidase from rat brain. Its relationship to proline endopeptidase from other tissues and from other species. J Biol Chem 257:5861–5865

    CAS  PubMed  Google Scholar 

  2. Arentz-Hansen H, McAdam SN, Molberg O, Fleckenstein B, Lundin KEA (2002) Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues. Gastroenterology 123:803–809

    Article  PubMed  Google Scholar 

  3. Aron PM, Shellhammer TH (2010) A discussion of polyphenols in beer physical and flavour stability. J Inst Brew 116:369–380

    Article  CAS  Google Scholar 

  4. Capiralla H, Hiroi T, Hirokawa T, Maeda S (2002) Purification and characterization of a hydrophobic amino acid -specific endopeptidase from Halobacterium halobium S9 with potential application in debittering of protein hydrolysates. Process Biochem 38:571–579

    Article  CAS  Google Scholar 

  5. Chevallier S, Goeltz P, Thibault P, Banville D, Gagnon J (1992) Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of the active-site serine. J Biol Chem 267:8192–8199

    CAS  PubMed  Google Scholar 

  6. Edens L, Dekker P, Van der Hoeven R, Deen F, De Roos A, Floris R (2005) Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates. J Agric Food Chem 53:7950–7957

    Article  CAS  PubMed  Google Scholar 

  7. Esparza Y, Huaiquil A, Neira L, Leyton A, Rubilar M, Salazar L, Shene C (2011) Optimization of process conditions for the production of a prolyl endopeptidase by Aspergillus niger ATCC 11414 in solid state fermentation. Food Sci Biotechnol 20:1323–1330

    Article  CAS  Google Scholar 

  8. Gass J, Ehren J, Strohmeier G, Isaacs I, Khosla C (2005) Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy. Biotechnol Bioeng 92:674–684

    Article  CAS  PubMed  Google Scholar 

  9. Goertges S (1982) Problematik der Eiweissstabilisierung (Problems with protein stabilization in winemaking). Weinwirtschaft 118:931–935

    CAS  Google Scholar 

  10. Goldman BS, Nierman WC, Kaiser D, Slater SC, Durkin AS, Eisen J, Ronning CM et al (2006) Evolution of sensory complexity recorded in a myxobacterial genome. Proc Natl Acad Sci USA 103:15200–15205

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Goptar IA, Lysogorskaya EN, Filippova IY, Vinokurov KS, Zhuzhikov DP, Elpidina EN (2005) Prolyl endopeptidases from the midgut of the yellow mealworm Tenebrio molitor. FEBS J 272:154–155

    Google Scholar 

  12. Hough JSBD, Stevens R, Young TW (1982) Malting and brewing science, 2nd edn. Chapman & Hall, London, 2

  13. Kabashima T, Fujii M, Meng Y, Ito K, Yoshimoto T (1998) Prolyl endopeptidase from Sphingomonas capsulata: isolation and characterization of the enzyme and nucleotide sequence of the gene. Arch Biochem Biophys 358:141–148

    Article  CAS  PubMed  Google Scholar 

  14. Kanatani A, Yoshimoto T, Kitazono A, Kokubo T, Tsuru D (1993) Prolyl endopeptidase from Aeromonas hydrophila: cloning, sequencing, and expression of the enzyme gene, and characterization of the expressed enzyme. J Biochem 113:790–796

    CAS  PubMed  Google Scholar 

  15. Kang C, Yu XW, Xu Y (2013) Gene cloning and enzymatic characterization of an endoprotease Endo-Pro-Aspergillus niger. J Ind Microbiol Biotechnol 40:855–864

    Article  CAS  PubMed  Google Scholar 

  16. Kang C, Yu XW, Xu Y (2014) Purification and characterization of a prolyl endopeptidase isolated from Aspergillus oryzae. J Ind Microbiol Biotechnol 41:49–55

    Article  CAS  PubMed  Google Scholar 

  17. Siebert KJ (1999) Effects of protein-polyphenol interactions on beverage haze, stabilization, and analysis. J Agric Food Chem 47:353–362

    Article  CAS  PubMed  Google Scholar 

  18. Kubota K, Tanokura M, Takahashi K (2005) Purification and characterization of a novel prolyl endopeptidase from Aspergillus niger. Proc Jpn Acad B Phys 81:447–453

    Article  CAS  Google Scholar 

  19. Kuwabara T (1992) Characterization of a prolyl endopeptidase from spinach thylakoids. FEBS Lett 300:127–130

    Article  CAS  PubMed  Google Scholar 

  20. Lopez M, Edens L (2005) Effective prevention of chill-haze in beer using an acid proline-specific endoprotease from Aspergillus niger. J Agric Food Chem 53:7944–7949

    Article  CAS  PubMed  Google Scholar 

  21. Mary Booth WJD, Fhaoláin IN, Jennings PV, O’Cuinn G (1990) Proline-specific peptidases of Streptococcus cremoris AM2. J Dairy Res 57:79–88

    Article  Google Scholar 

  22. Mentlein R (1988) Proline residues in the maturation and degradation of peptide hormones and neuropeptides. FEBS Lett 234:251–256

    Article  CAS  PubMed  Google Scholar 

  23. Nadzeyka A, Altenhofen U, Zahn H (1979) The significance of beer proteins in relationship to cold break and age-related haze formation. Brauwissenschaft 32:167–172

    CAS  Google Scholar 

  24. Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS et al (2005) Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438:1151–1156

    Article  CAS  PubMed  Google Scholar 

  25. O’Leary RM, Gallagher SP, O’Connor B (1996) Purification and characterization of a novel membrane-bound form of prolyl endopeptidase from bovine brain. Int J Biochem Cell Biol 28:441–449

    Article  PubMed  Google Scholar 

  26. O’Leary RM, O’Connor B (1995) Identification and localisation of a synaptosomal membrane prolyl endopeptidase from bovine brain. Eur J Biochem 227:277–283

    Article  PubMed  Google Scholar 

  27. Oyama H, Aoki H, Amano M, Mizuki E, Yoshimoto T, Tsuru D, Murao S (1997) Purification and characterization of a prolyl endopeptidase from Pseudomonas sp. KU-22. J Ferment Bioeng 84:538–542

    Article  CAS  Google Scholar 

  28. Riggle HM, Fisher MA (2009) Purification of a prolyl endopeptidase from Aspergillus oryzae and evaluation of its ability to digest gluten. Abstracts of Papers of the American Chemical Society 237

  29. Sattar AK, Yamamoto N, Yoshimoto T, Tsuru D (1990) Purification and characterization of an extracellular prolyl endopeptidase from Agaricus bisporus. J Biochem 107:256–261

    CAS  PubMed  Google Scholar 

  30. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucl Acid Res 31:3381–3385

    Article  CAS  Google Scholar 

  31. Shan L, Marti T, Sollid LM, Gray GM, Khosla C (2004) Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue. Biochem J 383:311–318

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Shen GX, Shi JP (1999) Cloning and nucleotide sequencing of prolyl endopeptidase gene from Aeromonas punctata subsp. punctata. Acta Biochim Biophys Sin 31:567–571

    CAS  PubMed  Google Scholar 

  33. Soisson SM, Patel SB, Abeywickrema PD, Byrne NJ, Diehl RE et al (2010) Structural definition and substrate specificity of the S28 protease family: the crystal structure of human prolylcarboxypeptidase. BMC Struct Biol 10:16

    Article  PubMed Central  PubMed  Google Scholar 

  34. Sridhar VR, Hughes JE, Welker DL, Broadbent JR, Steele JL (2005) Identification of endopeptidase genes from the genomic sequence of Lactobacillus helveticus CNRZ32 and the role of these genes in hydrolysis of model bitter peptides. Appl Environ Microb 71:3025–3032

    Article  CAS  Google Scholar 

  35. Szwajcer-Dey E, Rasmussen J, Meldal M, Breddam K (1992) Proline-specific endopeptidases from microbial sources: isolation of an enzyme from a Xanthomonas sp. J Bacteriol 174:2454–2459

    CAS  PubMed Central  PubMed  Google Scholar 

  36. Yoshimoto T, Walter R, Tsuru D (1980) Proline-specific endopeptidase from Flavobacterium. Purification and properties. J Biol Chem 255:4786–4792

    CAS  PubMed  Google Scholar 

  37. Yoshimoto T, Kanatani A, Shimoda T, Inaoka T, Kokubo T, Tsuru D (1991) Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene. J Biochem 110:873–878

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Financial support from the National High Technology Research and Development Program of China (863 Program) (No. 2012AA022207), the National Key Basic Research and Development Program of China (973 Program) (No. 2011CB710800), the High-end Foreign Experts Recruitment Program (GDW20123200113) and the 111 Project (111-2-06) are greatly appreciated.

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China

    Chao Kang, Xiao-Wei Yu & Yan Xu

  2. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China

    Chao Kang, Xiao-Wei Yu & Yan Xu

Authors
  1. Chao Kang
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  2. Xiao-Wei Yu
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  3. Yan Xu
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Correspondence to Xiao-Wei Yu or Yan Xu.

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Kang, C., Yu, XW. & Xu, Y. Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization. J Ind Microbiol Biotechnol 42, 263–272 (2015). https://doi.org/10.1007/s10295-014-1571-8

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  • DOI: https://doi.org/10.1007/s10295-014-1571-8

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