Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Characterization of Protease Produced by Bacillus amyloliquefaciens HTP-8 Isolated from Korean Fermented Anchovy Sauce.

멸치 어간장으로부터 분리한 Bacillus amyloliquefaciens HTP-8 이 생산하는 단백질 분해효소의 특성

  • 임형택 (경남대학교 생명과학부) ;
  • 정순경 (양산대학 식품가공제과제빵과) ;
  • 김기남 (경남대학교 생명과학부) ;
  • 하정욱 (경남대학교 생명과학부) ;
  • 백현동 (경남대학교 생명과학부)
  • Published : 2002.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

For commercial production of Korean fermented anchovy sauce through rapid fermentation, a bacterial strain which showed the high protease activity was isolated from a commercially fermented anchovy sauce. The isolate was Bacillus amyloliquefaciens, and named as B. amyloliquefaciens HTP-8. The incubation temperature, initial pH, and cultivation time for optimal production of protease by B. amyloliquefaciens HTP-8 were $30^{\circ}C$, 7.0, and 3 days, respectively. In jar fermenter, B. amyloliquefaciens HTP-8 showed higher protease activity when grown at pH 7.0. The protease was partially purified by 80% ammonium sulfate precipitation and CM-Sephadex C-50 ion exchange chromatography. The partially purified enzyme had specific activity of 103.3 units/mg, yield of 0.4%, and purification fold of 43.0. The optimal pH and temperature for the protease activity were 10.0 and $50^{\circ}C$, respectively. The protease was relatively stable at the pH range of 7.0~12.0 and at the temperatures below 4$0^{\circ}C$. The activity of the enzyme was inhibited by $Ag^{＋}$ /, $Ba^{2＋}$ and selectively inhibited by PMSF, suggesting that it is a serine protease.

저염상태에서 속성 발효를 통한 멸치 어간장의 대량 생산을 위하여 시중 어간장에서 단백질 분해효소 생산이 우수한 균주를 분리 동정하여, B. amyloliquefaciens HTP-8로 명명하였다. 효소생산을 위한 배양 최적온도, 초기 pH, 그리고 배양시간은 각각 $30^{\circ}C$, pH 7.0과 3일이었다. Jar fermenter 배양시 pH를 7.0으로. 조절한 경우가 조절하지 않은 경우에 비해 효소활성이 최대에 이르는 시간이 단축되었다. 효소의 부분정제는 조효소액을 80% ammonium sulfate에 의한 염석과 CM-Sephadex C-50을 이용하여 정제한 결과, 수율이 0.4%, 정제배수가 43.0배였다. 정제효소의 최적 pH와 온도는 pH 10.0과 $50^{\circ}C$였으며, pH 7.0~l2.0의 pH 범위와 $40^{\circ}$ 이하에서 안정하였다. 금속이온 중 $Ag^{＋}$ /, $Ba^{2＋}$ /에 의하여 효소활성이 저해되었다. 한편, 본 효소는 PMSF에 의하여 선택적으로 활성이 억제됨으로써 활성부위에 serine을 가진 serine protease에 속하는 것으로 판단되었다.

Keywords

References

  1. Hagihara, B., H. Matrubara, M. Nakai, and K. Okunuki. 1958. Crystalline bacterial proteinase. I. Preparation of crystalline proteinase of Bacillus subtilis. J Biochem. 45: 185194. https://doi.org/10.1093/oxfordjournals.jbchem.a126856
  2. Horikoshi, K. and T. Akiba. 1982. Alkalophilic Microorganisms. pp 93-101. Scientific Societies Press, Tokyo.
  3. Hwang, S. Y 1995. Purification and characterization of an extracellular serine protease from Bacillus sp. strain KUN17. Kor. J Appl. Microbio!. Biotechnol. 23: 53-59.
  4. Johnvesly, B. and R. N. Gajanan. 2001. Production of bleach-stable and halo-tolerant alkaline protease by an alkalophilic Bacillus pumilus ill 05 isolated from cement industry effluents. J Microbiol. Biotechnol. 11: 558-563.
  5. Sakka, K., K. Shimada, and K. I. Matsushima. 1985. Purification and some properties of serine proteinase from a mutant of Aspergillus niger. J Ferment. Technol. 63: 479483.
  6. Kim, S. J., D. B. Shin, and B. H. Ryu. 1993. Continuous rapid fermentation of sardine soy sauce by using column type reactor packed immobilized yeast cells. Korean J. Food Sci. Technol. 25: 154-159.
  7. Lee, E. G., E. H. Park, and H. H. Hyun. 2000. Purification and characterization of two alkaline proteases produced by Pseudomonas sp. BK7. J Microbiol. Biotechnol. 10: 677684.
  8. Lee, E. H., C. B. Ahn, J. S. Kim, C. W. Lim, S. W. Lee, and Y. A. Choi. 1988. Processing and taste compounds of fish sauces from filefish scrap. J Korean Soc. Food Nutr. 17: 326-335.
  9. Lee, E. H., H. S. Park, C. B. Ahn, and G. C. Hwang. 1986. Preparation of fish sauce from mackerel scrap. J Korean Soc. Food Nutr. 15: 201-206.
  10. Lee, E. H., T. H. Lee, J. S. Kim, and C. B. Ahn, 1989. Processing and taste compounds of the fish sauce from skipjack scrap. Bull. Korean Fish. Soc. 22: 25-35.
  11. Lim, S. I. 2000. Purification and characterization of protease produced by Aspergillus wentti isolated from Korean traditional meju. Korean J. Food Sci. Techno!. 32: 161-167.
  12. Lim, S. I., H. K. Kim, and J. Y. Yoo. 2000. Characteristics of protease produced by Bacillus subtilis PCA 20-3 isolated from Korean traditional meju. Korean J Food Sci. Technol. 32: 154-160.
  13. Lim, S. I. and J. Y Yoo. 1999. Purification and characteristics of protease produced by Syncephalastrum racemosum PDA 132-2 from Korean traditional meju. J Korean Soc. Food Sci. Nutr. 28: 1010-1016.
  14. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J Bioi. Chem. 193: 265-275.
  15. Ok, M., M. S. Kim, W. S. Seo, J. Y. Cha, and Y. S. Cho. 2000. Characterization of extracellular protease of Bacillus sp. WRD-1 isolated from soil. Kor. J Appl. Microbiol. Biotechnol. 28: 329-333.
  16. Ryu, B. H., S. J. Kim, and D. B. Shin. 1992. Lactic acid, ethylalcohol and 4-ethylguaiacol contents of rapid fermentation of sardine soy sauce prepared by using immobilized whole cells. KoreanJ Food Sci. Technol. 24: 456-462.
  17. Kobayashi, T., A. Ogasawara, S. Ito, and M. Saitoh. 1985. Purification and some properties of alkaline proteinase produced by Pseudomonas maltophilia. Agric. Bioi. Chem. 49: 693-698.
  18. Yeum, D. M., T. G. Lee, J. R. Do, O. K Kim, Y. B. Park, S. B. Kim, and Y. H. Park. 1993. Characteristics of angiotensinI converting enzyme inhibitors derived from fermented fish product. 2. Characteristics of angiotensin-I converting enzyme inhibitors of fish sauce prepared from sardine, Sardinops melanosticta. Bull Korean Fish. Soc. 26: 416-423.
  19. Yun, S. w., K P. Lee, J. H. Yu, C. S. Shin, and D. H. Oh. 1989. Purification and properties of alkaline protease from Streptomyces sp, YSA-130. Kor. J Appl. Microbiol. Biotechnol. 17: 358-364.