한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제30권1호
  • /
  • Pages.26-32
  • /
  • 2002
  • /
  • 1598-642X(pISSN)
  • /
  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

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

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

  • 임형택 (경남대학교 생명과학부) ;
  • 정순경 (양산대학 식품가공제과제빵과) ;
  • 김기남 (경남대학교 생명과학부) ;
  • 하정욱 (경남대학교 생명과학부) ;
  • 백현동 (경남대학교 생명과학부)
  • 발행 : 2002.03.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

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.

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.

키워드

참고문헌

  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.