Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Enzyme Production of A Protease-producing Strain, Bacillus sp SH-8 Isolated from Insect-eating Plant

식충식물로부터 Protease를 생산하는 Bacillus sp. SH-8의 분리와 효소 생산성

  • 윤기홍 (우송대학교 식품영양.식품과학부) ;
  • 이미성 (우송대학교 식품영양.식품과학부) ;
  • 박병완 (우송대학교 식품영양.식품과학부) ;
  • 박용하 (영남대학교 응용미생물학과) ;
  • 김홍익 ((주)프로바이오닉) ;
  • 김정현 (대구과학고등학교) ;
  • 김문숙 (대구과학고등학교)
  • Published : 2006.12.28
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Abstract

A bacterium producing the extracellular protease was isolated from insect-eating plant and has been identified as a member of the genus Bacillus based on partial 165 rRNA sequences. In order to develop the medium composition, effects of ingredients including nitrogen sources, carbon source, metal ions and phosphate were examined for protease production of the isolate, SH-8. Soluble starch increased the protease productivity, while glucose repressed it. Yeast extract was effective nitrogen source for enzyme production, but the pretense production of Bacillus sp. SH-8 was reduced by large amount of yeast extract. The calcium was found to induce pretense activity as well as protease productivity. However, cell growth and enzyme production was completely inhibited by divalent ions such as $Zn^{2+}$, $Cu^{2+}$, $Co^{2+}$ and $Mn^{2+}$. The maximum protease productivity was reached 435 unit/ml in the optimized medium consisting of soluble starch (2%), yeast extract (0.3%), $CaCl_2$ (0.3%), $K_2HPO_4$ (0.01%) and $KH_2PO_4$ (0.01%). The pretense activity of culture filtrate was dramatically decreased after incubation for 26 h.

식충식물에서 채취된 시료로부터 protease치 생산균으로 분리된 SH-8은 그람 양성간균으로 16S rRNA외 부분 염기서열에 근거하여 Bacillus속 균주로 확인되었다. Protease 생산을 위한 배지를 제조하기 위해 질소원, 탄소원, 인, 금속이온의 성분을 변화시키면서 균의 성장과 효소 생산성을 비교하였다. 포도당을 탄소원으로 사용하였을 때는 균의 성장은 정상적으로 일어나지만, pretense생산이 완전히 억제되는 것으로 나타났으며, 가용성 전분을 탄소원으로 사용하였을 때 효소 생산성이 가장 높았다. 질소원으로는 yeast extact가 효소 생산에 가장 적합하였으며, 농도가 높으면 효소 생산성이 저하되었다. 한편 2가 금속이온중 $Zn^{2+}$, $Cu^{2+}$, $Co^{2+}$, $Mn^{2+}$을 배지에 첨가하였을 때는 균의 성장이 심하게 저해되었으며, 효소 생산도 되지 않았다. $CaCl_2$를 첨가한 배지에서는 효소 생산성이 증가되었으며, 효소 반응에 $CaCl_2$를 첨가하였을 때도 효소 활성이 증가하였다. 이로보아 $CaCl_2$는 Bacillus sp. SH-8의 protease 생산성과 활성을 모두 증가시키는 것으로 판단된다. 가용성 전분(2%), yeast extract(0.3%), $CaCl_2$(0.3%), $CaCl_2$(0.01%)와 $KH_2PO_4$(0.01%)를 포함하는 것으로 구성된 최적화 배지에서 최대효소 생산성은 435 U/ml로 나타났으며 26시간이 되었을 때 배양액 내 효소활성은 급격히 감소하였다.

Keywords

References

  1. Banerjee, U. C., R. K. Sani, W. Azmi, and R. Sani. 1999. Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochem. 35: 213-219 https://doi.org/10.1016/S0032-9592(99)00053-9
  2. Banik, R. M. and M. Prakash. 2004. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res. 19: 135-140
  3. Dhandapani, R. and R. Vijayaragvan. 1994. Production of thermophilic, extracellular alkaline proteases by B. stearothermophilus AP-4. Wor. J. Microbiol. Biotechnol. 10: 33-35 https://doi.org/10.1007/BF00357559
  4. Donovan, W. P., Y. Tin, and A. C. Slaney. 1997. Cloning of the nprA gene for neutral protease A of Bacillus thuringiensis and effect of in vivo deletion of nprA on insecticidal crystal proteins. Appl. Environ. Microbiol. 63: 2311-2317
  5. Drucker, H. 1972. Regulation of exocellular protease in Neurospora crassa: induction and repression of enzyme synthesis. J. Bacteriol. 110: 1041-1049
  6. Feder, J. and J. M. Schuck 1970. Studies on the Bacillus subtilis neutral-protease- and Bacillus thermoproteolyticus thermolysin-catalyzed hydrolysis of dipeptide substrates. Biochem. 9: 2784-2791 https://doi.org/10.1021/bi00816a005
  7. Ferrero, M. A., G. R. Castro, C. M. Abate, M. D. Baigori, and F. Sineriz. 1996. Thermostable alkaline protease Bacillus licheniformis MIR 29: isolation, production and characterization. Appl. Microbiol. Biotechnol. 45: 327-332 https://doi.org/10.1007/s002530050691
  8. Fricke, B., K. Drossler, I. Willhardt, A. Schierhorn, S. Menge, and P. Rucknagel. 2001. The cell envelope-bound metalloprotease(camelysin) from Bacillus cereus is a possible pathogenic factor. Biochim. Biophys. Acta 1537: 132-146
  9. Fukushima, Y., H. Itoh, T. Fukasa, and H. Motai. 1989. Continuous protease production in a carbon-limited chemostat culture by salt tolerant Aspergillus oryzae. Appl. Microbiol. Biotechnol. 30: 604-608
  10. Ghorbel-Frikha, B., A. Sellami-Kamoun, N. Fakhfakh, A. Haddar, L. Manni, and M. Nasri. 2005. Production and purification of a calcium-dependent protease from Bacillus cereus BG1. J. Ind. Microbiol. Biotechnol. 32: 186-194 https://doi.org/10.1007/s10295-005-0228-z
  11. Giesecke, U. E. G. Bierbaum, H. Rudde, U. Spohn, and C. Wandrey. 1991. Production of alkaline protease with Bacillus licheniformis in a controlled fed-batch process. Appl. Microbiol. Biotechnol. 35: 720-724
  12. Gupta, R., O. K. Beg, and P. Lorenz. 2002. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol Biotechnol. 59: 13-32
  13. Hayano, K., M. Takeuchi, and E. Ichishima. 1987. Characterization of a metalloprotease component extracted from soil. Biol. Fertil. Soils 4: 179-183
  14. Holmes, M. A. and B. W. Matthews. 1982. Structure of thermolysin refined at 1.6 ${\AA}$ resolution. J. Mol. Biol. 160: 623-639 https://doi.org/10.1016/0022-2836(82)90319-9
  15. Khannous, L., N. Souissi, B. Ghorbel, R. Jarboui, M. Kallel, M. Nasri, and N. Gharsallah. 2003. Treatment of saline wastewaters from marine-products processing factories by activated sludge reactor. Environ. Technol. 24: 1261-1268 https://doi.org/10.1080/09593330309385668
  16. Kim, S. S., Y. J. Kim, and I. K. Rhee. 2001. Purification and characterization of a novel extracellular protease from Bacillus cereus KCTC 3674. Arch. Microbiol. 175: 458-461 https://doi.org/10.1007/s002030100282
  17. Lee, E. -H., C. -J. Kim, and K. -H. Yoon. 2005. Characterization and xylanase productivity of Streptomyces sp. WL2. Kor. J. Microbiol. Biotechnol. 33: 178-183
  18. Mehrotra, S., P. K. Pandey, R. Guar, and N. S. Darmwal. 1999. The production of alkaline protease by a Bacillus species isolate. Bioresour. Technol. 67: 201-203 https://doi.org/10.1016/S0960-8524(98)00107-2
  19. Nilegaonkar, S. S., V. P. Zambare, P. P. Kanekar, P. K. Dhakephalkar, and S. S. Sarnaik. 2007. Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour. Technol. (in press)
  20. Nishiya, Y. and T. Imanaka. 1990. Cloning and nucleotide sequences of the Bacillus stearothermophilus neutral protease gene and its transcriptional activator gene. J. Bacteriol. 172: 4861-4869
  21. Priest, F. G. 1977. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol. Rev. 41: 711-753
  22. Stark, W., R. A. Pauptit, K. S. Wilson, and J. N. Jansonius. 1992. The structure of neutral protease from Bacillus cereus at 0.2-nm resolution. Eur. J. Biochem. 207: 781-791 https://doi.org/10.1111/j.1432-1033.1992.tb17109.x
  23. Tran, L., X. -C. Wu, and S. -L. Wong. 1991. Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis. J. Bacteriol. 173: 6364-6372
  24. Vasantha, N., L. Thompson, C. Rhodes, C. Banner, J. Nagle, and D. Filpula. 1984. Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. J. Bacteriol. 159: 811-819
  25. Veltman, O. R., G. Vriend, H. J. C. Berendsen, B. van den Burg, G. Venema, and V. G. H. Eijsink. 1998. A single calcium binding site is crucial for the calcium-dependent thermal stability of thermolysin-like proteases. Biochemistry 37: 5312-5319 https://doi.org/10.1021/bi9725879
  26. Watanabe, K. and K. Hayano. 1993. Source of soli protease in paddy field. Can. J. Microbiol. 39: 1035-1040
  27. Wetmore, D. R., S. L. Wong, and R. S. Roche. 1992. The role of the prosequence in the processing and secretion of the thermolysin-like protease from Bacillus cereus. Mol. Microbiol. 6: 1593-1604 https://doi.org/10.1111/j.1365-2958.1992.tb00884.x
  28. Yang, M., E. Ferrari, and D. Henner. 1984. Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-deletion mutation. J. Bacteriol. 160: 15-21