Science in China Series C: Life Sciences

, Volume 51, Issue 1, pp 52–59 | Cite as

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis

  • Zhang Min 
  • Zhao Cong 
  • Du LianXiang 
  • Lu FuPing 
  • Gao Chen 
Article

Abstract

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.8-fold, with a specific activity of 16530 U mg−1. As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65°C and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 °C.

Keywords

Bacillus subtilis thermophilic neutral protease expression purification characterization 

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References

  1. 1.
    Nascimento W C A, Martins M L L. Production and properties of an extracellular protease from thermophilic Bacillus sp. Braz J Microbiol, 2004, 35: 1–2CrossRefGoogle Scholar
  2. 2.
    Pastor M D, Lorda G S, Balatti A. Protease obtention using Bacillus subtilis 3411 and amaranth seed meal medium at different aeration rates. Braz J Microbiol, 2001, 32: 1–8CrossRefGoogle Scholar
  3. 3.
    Ward O P. Proteolytic Enzymes. Oxford: Pergamon Press, 1985, 789–818Google Scholar
  4. 4.
    Sookkheo B, Sinchaikul S, Phutrakul S, et al. Purification and characterization of the highly thermostable proteases from Bacillus stearothermophilus TLS33. Protein Expr Purif, 2000, 20: 142–151PubMedCrossRefGoogle Scholar
  5. 5.
    Oledzka G., Dabrowski S, Kur J. High-level expression, secretion, and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris. Protein Expr Purif, 2003, 29: 223–229PubMedGoogle Scholar
  6. 6.
    Ferrero M A, Castro G R, Abate C M, et al. Thermostable alkaline protease of Bacillus licheniformis MIR29: isolation, production and characterization. Appl Microbiol Biotechnol, 1996, 45: 327–332CrossRefGoogle Scholar
  7. 7.
    Kumar C G. Purification and characterization of a thermostable alkaline protease from alkalophilic Bacillus pumilus. Lett Appl Microbiol, 2002, 34: 13–17PubMedCrossRefGoogle Scholar
  8. 8.
    Hyenung J J, Byoung C K, Yu R P, et al. A novel subtilisn-like serine protease from Thermoanerobacter yonseiensis KB-1: Its cloning, expression, and biochemical properties. Extremophiles, 2002, 6: 233–243CrossRefGoogle Scholar
  9. 9.
    Helmann J D. Compilation and analysis of Bacillus subtilis σA-dependent promoter sequences: Evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res, 1995, 23: 2351–2360PubMedCrossRefGoogle Scholar
  10. 10.
    Van den Burg B, Eijsink V G H, Veltman O R, et al. Evolution reversed: Engineering an enzyme to resist boiling. Proc Natl Acad Sci USA, 1998, 95: 2056–2060PubMedCrossRefGoogle Scholar
  11. 11.
    Rao M B, Tankasale A M, Ghatge M S, et al. Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev, 1998, 62: 597–634PubMedGoogle Scholar
  12. 12.
    Fujii M, Takagi M, Imanaka T, et al. Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis. J Bacteriol, 1983, 154: 831–837PubMedGoogle Scholar
  13. 13.
    Huang G R, Ying T J, Huo P, et al. Purification and characterization of a protease from Thermophilic bacillus strain HS08. Afr J Biotechnol, 2006, 5(24): 2433–2438Google Scholar
  14. 14.
    Doi R H, Wong S L, Kawamura F. Potential use of Bacillus subtilis for secretion and production of foreign proteins. Trends Biotechnol, 1986, 4: 232–235CrossRefGoogle Scholar
  15. 15.
    Goeddle D V. Expression in B. subtilis. Meth Enzymol, 1990, 85: 199–228Google Scholar
  16. 16.
    Behnke D, Gerlach D. Cloning and expression in Escherichia coli, Bacillus subtilis, and Streptococcus sanguis of a gene for staphylokinase, a bacterial plasminogen activator. Mol Gen Genet, 1987, 210: 528–534PubMedCrossRefGoogle Scholar
  17. 17.
    Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. New York: Cold Spring Harbor Laboratory, 1989Google Scholar
  18. 18.
    Chang S, Cohen S N. High-frequency transformation of Bacillus subtilis protoplast by plasmid DNA. Mol Gen Genet, 1979, 168: 111–115PubMedCrossRefGoogle Scholar
  19. 19.
    Ledoux M, Lamy F. Determination of proteins and sulfobetaine with the folin-phenol reagent. Anal Biochem, 1986, 157: 28–31PubMedCrossRefGoogle Scholar
  20. 20.
    Kubo M, Imanaka T. Cloning and nucleotide sequence of the highly thermostable neutral protease gene from Bacillus stearothermophilus. J Gen Microbiol, 1988, 134(7): 1883–1892PubMedGoogle Scholar
  21. 21.
    Priest F G. Extracellular enzyme synthesis in the genus Bacillus. J Bacteriol, 1977, 41: 711–753Google Scholar
  22. 22.
    Ayusawa D, Yoneda Y, Yamane K, et al. Pleiotropic phenomena in autolytic enzyme(s) content, flagellation, and simultaneous hyperproduction of extracellular α-amylase and protease in a Bacillus subtilis mutant. Bacteriol, 1975, 124: 459–469Google Scholar
  23. 23.
    Jacobs M F. Expression of the subtilisin Carlsberg-encoding gene in Bacillus licheniformis and Bacillus subtilis. Gene, 1995, 152: 69–74PubMedCrossRefGoogle Scholar
  24. 24.
    Pan X F. Effects of degU32(Hy), degQa and degR pleiotropic regulatory genes on the growth and protease fermentation of Bacillus Subtilis Ki-2-132. Acta Genet Sin, 2006, 33: 373–380PubMedCrossRefGoogle Scholar

Copyright information

© Science in China Press 2008

Authors and Affiliations

  • Zhang Min 
    • 1
    • 2
  • Zhao Cong 
    • 2
  • Du LianXiang 
    • 2
  • Lu FuPing 
    • 2
  • Gao Chen 
    • 3
  1. 1.College of EngineeringShenyang Agricultural UniversityShenyangChina
  2. 2.College of BiotechnologyTianjin University of Science and TechnologyTianjinChina
  3. 3.College of Life ScienceNankai UniversityTianjinChina

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