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World Journal of Microbiology and Biotechnology

, Volume 28, Issue 8, pp 2651–2659 | Cite as

Hydrolytic activity of Virgibacillus sp. SK37, a starter culture of fish sauce fermentation, and its cell-bound proteinases

  • Sornchai Sinsuwan
  • Sureelak Rodtong
  • Jirawat YongsawatdigulEmail author
Original Paper

Abstract

Fish sauce production relies on a natural fermentation process requiring 12–18 months for process completion. Virgibacillus sp. SK37 has been shown to be a potential strain for fish sauce acceleration. However, hydrolytic activity of proteinases bound at cell surface of this strain has not been well elucidated. Addition of 0.2 % CaCl2 (w/w) in conjunction with starter cultures of Virgibacillus sp. SK 37 increased protein hydrolysis as measured by α-amino group content throughout fermentation (P < 0.05). Cell-bound proteinases from Virgibacillus sp. SK 37 were extracted into a free form by incubating the washed cells in Ca2+-free buffer at 37 °C for 2 h. Cell-bound proteinases revealed molecular mass of 19, 20, 22, 32, 34, and 44 kDa based on a synthetic peptide zymogram. The proteinases showed subtilisin-like serine characteristics with the highest activity at 50 °C and pH 8 and 11. Activity of the extracted proteinases increased ~4 times at ≥100 mM CaCl2. In addition, CaCl2 enhanced thermal stability of the extracted proteinases. Enzymes showed proteolytic activity in either the absence or presence of 10 and 25 % NaCl toward fish muscle, soy protein isolate, and casein substrates. Cell-bound proteinases were likely to play an important role in protein hydrolysis during fish sauce fermentation.

Keywords

Virgibacillus sp. Moderately halophilic bacterium Cell-bound proteinase Fish sauce fermentation 

Notes

Acknowledgments

This work was supported by a research grant from the National Science and Technology Development Agency (NSTDA), Thailand under research agreement No. BT-B-01-FT-19-5014 and Suranaree University of Technology under a Food Protein Research Unit Grant Agreement.

References

  1. Adler-Nissen J (1979) Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid. J Agric Food Chem 27:1256–1262CrossRefGoogle Scholar
  2. Barrett AJ, Kirschke H (1981) Cathepsin B, cathepsin H, and cathepsin L. In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol 80. Academic Press, New York, pp 535–561Google Scholar
  3. Chu N-M, Chao Y, Bi R-C (1995) The 2 Å crystal structure of subtilisin E with PMSF inhibitor. Protein Eng 8:211–215CrossRefGoogle Scholar
  4. Espeche Turbay MB, Savoy de Giori G, Hebert EM (2009) Release of the cell-envelope-associated proteinase of Lactobacillus delbrueckii subspecies lactis CRL 581 is dependent upon pH and temperatue. J Agric Food Chem 57:8607–8611CrossRefGoogle Scholar
  5. Exterkate FA (2000) Structural changes and interactions involved in the Ca2+-triggered stabilization of the cell-bound cell envelope proteinase in Lactococcus lactis subsp. cremoris SK11. Appl Environ Microbiol 66:2021–2028CrossRefGoogle Scholar
  6. Fernández de Palencia P, Peláez C, Martín-Hernández MC (1997) Specificity of the bound and free forms of the cell-envelope proteinase of Lactobacillus casei subsp. IFPL 731 towards the αs1-casein-(1-23)-fragment. Lett Appl Microbiol 25:388–392CrossRefGoogle Scholar
  7. Fernandez-Espla MD, Garault P, Monnet V, Rul F (2000) Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization. Appl Environ Microbiol 66:4772–4778CrossRefGoogle Scholar
  8. Fricke B, Parchmann O, Aurich H (1993) Membrane-bound proteinases of Halobacterium halobium. J Basic Microbiol 1:9–18CrossRefGoogle Scholar
  9. Fricke B, Buchmann T, Friebe S (1995) Unusual chromatographic behavior and one-step purification of a novel membrane proteinase from Bacillus cereus. J Chromatogr 715:247–258CrossRefGoogle Scholar
  10. García-Carreño FL, Dimes LE, Haard NF (1993) Substrate-gel electrophoresis for composition and molecular weight of proteinase or proteinaceous proteinase inhibitors. Anal Biochem 214:65–69CrossRefGoogle Scholar
  11. Gilbert C, Atlan D, Blanc B, Portailer R, Germond JE, Lapierre L, Mollet B (1996) A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbrueckii subsp. bulgaricus. J Bacteriol 178:3059–3065Google Scholar
  12. Graycer TP, Ballinger MD, Wells JA (2004) Subtilisin. In: Barrett AJ, Rawlings ND, Woessner JF (eds) Handbook of proteolytic enzymes, 2nd edn. Elsevier Academic Press, Amsterdam, pp 1786–1792Google Scholar
  13. Laan H, Konings WN (1989) Mechanism of proteinase release from Lactococcus lactis subsp. cremoris Wg2. Appl Environ Microbiol 55:3101–3106Google Scholar
  14. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  16. Phrommao E, Rodtong S, Yongsawatdigul J (2010) Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37. J Appl Microbiol 110:191–201CrossRefGoogle Scholar
  17. Shahbal S, Hemme D, Renault P (1993) Characterization of a cell envelope-associated proteinase activity from Streptococcus thermophilus H-strains. Appl Environ Microbiol 59:177–182Google Scholar
  18. Siezen RJ (1999) Multi-domain, cell-envelope proteinases of lactic acid bacteria. Antonie Van Leeuwenhoek 76:139–155CrossRefGoogle Scholar
  19. Sinsuwan S, Rodtong S, Yongsawatdigul J (2007) NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation. J Food Sci 72:264–269CrossRefGoogle Scholar
  20. Sinsuwan S, Rodtong S, Yongsawatdigul J (2008a) Production and characterization of NaCl-activated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation. Process Biochem 43:185–192CrossRefGoogle Scholar
  21. Sinsuwan S, Rodtong S, Yongsawatdigul J (2008b) Characterization of Ca2+-activated cell-bound proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation. LWT Food Sci Technol 41:2166–2174CrossRefGoogle Scholar
  22. Sinsuwan S, Rodtong S, Yongsawatdigul J (2010a) Purification and characterization of a salt-activated and organic solvent-stable heterotrimer proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce. J Agric Food Chem 58:248–256CrossRefGoogle Scholar
  23. Sinsuwan S, Rodtong S, Yongsawatdigul J (2010b) A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce. Food Chem 119:573–579CrossRefGoogle Scholar
  24. Sinsuwan S, Rodtong S, Yongsawatdigul J (2011) Evidence of cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation. J Food Sci 76:413–419CrossRefGoogle Scholar
  25. Tsakalidou E, Anastasiou R, Vandenberghe I, van Beeumen J, Kalantzopoulos G (1999) Cell-wall-bound proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: characterization and specificity for β-casein. Appl Environ Microbiol 65:2035–2040Google Scholar
  26. Yasothornsrikul S, Hook VY (2000) Detection of proteolytic activity by fluorescent zymogram in-gel assays. Biotechniques 28:1166–1173Google Scholar
  27. Yongsawatdigul J, Rodtong S, Raksakulthai N (2007) Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures. J Food Sci 72:382–390CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Sornchai Sinsuwan
    • 1
    • 3
  • Sureelak Rodtong
    • 2
    • 3
  • Jirawat Yongsawatdigul
    • 1
    • 3
    Email author
  1. 1.School of Food Technology, Institute of Agricultural TechnologySuranaree University of TechnologyNakhon RatchasimaThailand
  2. 2.School of Microbiology, Institute of ScienceSuranaree University of TechnologyNakhon RatchasimaThailand
  3. 3.Microbial Cultures Research Center for Food and Bioplastics ProductionSuranaree University of TechnologyNakhon RatchasimaThailand

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