Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Overproduction of Bacterial Trypsin in Streptomyces - Optimization for Streptomyces griseus Trypsin Production by Recombinant Streptomyces

미생물을 이용한 트립신 과대 생산 연구 - Streptomyces용 숙주-벡터계를 이용한 트립신 유전자의 대량발현 최적화 -

  • Kim, Jong-Hee (Department of Food and Nutrition, Seoil College) ;
  • Hong, Soon-Kwang (Department of Biological Science & Institute of Bioscience and Biotechnology, Myongji University)
  • 김종희 (서일대학 식품영양학과) ;
  • 홍순광 (명지대학교 생명과학정보학부.생명공학연구소)
  • Published : 2008.03.28
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Abstract

The expression vector (pWHM3-TR1R2) for sprT gene encoding Streptomyces griseus trypsin (SGT) followed by two regulatory genes, sgtR1 and sgtR2, was introduced into Streptomyces lividans TK24 and Streptomyces griseus IFO 13350. Various media with different compositions were used to maximize the productivity of SGT in the recombinant trains. he SGT productivity was best when the transformant of S. lividans TK24 was cultivated in R2YE medium (0.74 unit/mL) at 5 days of cultivation. C5/L (0.66 unit/mL) medium also gave a good productivity, but Livid (0.08 unit/mL) and NDSK (0.06 unit/mL) yielded poor productivities. S. griseus IFO 13350/pWHM3-TR1R2 produced SGT by 1.518 unit/mL (C5/L), 1.284unit/mL (R2YE),0.932 unit/mL (NDSK), and 0.295 unit/mL (Livid) at 7 days of cultivation, which was much higher than those from S. lividans TK24/TR1R2. The SGT protein was purified from the culture broth of S. griseus IFO 13350/pWHM3-TR1R2 in C5/L to homogeneity via ammonium sulfate fractionation, and CM-sepharose and SP-sepharose column chromatographies. The specific activity of purified SGT was 69,252 unit/mg, and the final purification fold and recovery yield were 6.5 and 1.4%, respectively.

Streptomyces griseus trypsin (SGT)을 코드하는 sprT 유전자와 그 하류에 존재하는 두 개의 조절 유전자 rsgtR1 및 sgtR2를 동시에 갖고 있는 재조합 벡터 pWHM3-TR1R2를 S. lividans TK24 및 S. griseus IFO 13350에 도입하여, 트립신의 생산성을 더욱 증대시킬 수 있는 배지를 조사하였다. S. lividans TK24/pWHM3-TR1R2의 경우 배양 5일을 기준으로 R2YE에서 가장 높은 생산성(0.74 unit/mL)을 나타냈고, C5/L. (0.66 unit/mL), Livid (0.08 unit/mL), NDSK(0.06 unit/mL) 순으로 나타났다 S. griseus IFO 13350/pWHM3-TR1R2의 경우에는 전반적으로 배양 7일에 트립신 활성이 가장 높았으며, C5/L (1.518 unit/mL), R2YE(1.284 unit/mL), NDSK (0.932 unit/mL), Livid (0.295 unit/mL) 순으로 나타났다. S. griseus IFO 13350/pWHM3-TR1R2를 C5/L 배지에서 7일간 배양한 배양액으로부터 $25%{\sim}60%$ ammonium sulfate 침전, CM-sepharose 및 Sp-sepharose column chromatography를 통하여 트립신을 고순도로 정제할 수 있었다. 최종 purification fold는 6.5배, 순수 정제된 트립신의 specific activity는 69,252 unit/mg, 회수율은 1.4%이었다.

Keywords

References

  1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  2. Chi, W.-J., J.-M. Kim, S.-S. Choi, D.-K. Kang, and S.-K. Hong. 2001. Overexpression of SGPA and SGT induces morphological changes in Streptomyces lividans. J. Microbiol. Biotehnol. 11: 1077-1086
  3. Chi, W.-J., M.-S. Kim, J.-H. Kim, D.-K. Kang, and S.-K. Hong. 2005. Molecular cloning and analysis of the genes in the vicinity of Streptomyces griseus trypsin (SGT) gene from Streptomyces griseus ATCC 10137. Kor. J. Microbiol. 41: 255-261
  4. Chi, W.-J., Y.-H. Kim, J.-H. Kim, D.-K. Kang, S.-S. Kang, J.-W. Suh, and S.-K. Hong. 2003. Streptomyces griseus Trypsin (SGT) has gelatinase activity and its proteolytic activity is enhanced by manganese. J. Microbiol. Biotechnol. 41: 289-294
  5. Choi, S.-S., J. H. Kim, J.-H. Kim, D.-K. Kang, S.-S. Kang, and S.-K. Hong. Functional anaylsis of sprD gene encoding Streptomyces griseus protease D (SGPD) in Stretomyces griseus. J. Microbiol. Biotechnol. 16: 312-317
  6. Choi, E.-Y., E.-A. Oh, J.-H. Kim, D.-K. Kang and S.-K. Hong. 2007. Distinct regulation of the sprC gene encoding Streptomyces griseus protease C from other chymotrypsin genes in Streptomyces griseus IFO13350. J. Microbiol. Biotechnol. 17: 81-88
  7. Choi, S.-S., W.-J. Chi, J.-H. Lee, S.-S. Kang, B. C. Jeong, and S.-K. Hong. 2001. Overexpression of the sprD gene encoding Streptomyces griseus protease D stimulates actinorhodin production in Streptomyces lividans. J. Microbiol. 39: 305-313
  8. Gregory, D. G., D. E. Ordaz, and W. R. Strohl. 1989. Overexpression of extracellular protease activity by Streptomyces C5-A13 in fed-batch fermentation. Appl. Microbiol. Biotechnol. 31: 119-124 https://doi.org/10.1007/BF00262447
  9. Hong, S.-K., A. Matsumoto, S. Horinouchi, and T. Beppu. 1993. Effects of protein kinase inhibitors on in vitro protein phosphorylation and cellular differentiation of Streptomyces griseus. Mol. Gen. Genet. 236: 347-354 https://doi.org/10.1007/BF00277132
  10. Horinouchi, S. 2002. A microbial hormone, A-factor, as a master switch for morphological differentiation and secondary metabolism in Streptomyces griseus. Front Biosci. 7: 2045-2057 https://doi.org/10.2741/horinouc
  11. Jayant, S. A. and W. R. Strohl. 1993. Purification and properties of an extracellular aminopeptidase from Streptomyces lividans 1326. J. Gen. Microbiol. 139: 417-424. https://doi.org/10.1099/00221287-139-3-417
  12. Kato, J., W.-J. Chi, Y. Ohnishi, S.-K. Hong, and S. Horinouchi. 2005. Transcriptional control by a-factor of two trypsin genes in Streptomyces griseus. J. Bacteriol. 187: 286-295 https://doi.org/10.1128/JB.187.1.286-295.2005
  13. Kieser, H., M. J. Bibb, M. J. Buttner, F. K. Chater, and D. A. Hopwood. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK
  14. Kim, J.C., S.H. Cha, S.T. Jeong, S.K. Oh, and S.M. Byun. 1991. Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene. Biochem. Biophys. Res. Commun. 181: 707-713 https://doi.org/10.1016/0006-291X(91)91248-B
  15. Kim, Y.-H., S.-S. Choi, D.-K. Kang, S.-S. Kang, B. C. Jeong and S.-K. Hong. 2004. Overexpression of sprA and sprB genes is tightly regulated in Streptomyces griseus. J. Microbiol. Biotechnol. 14: 1350-1355
  16. Koo, B.-J., J.M. Kim, S.-M. Byun, and S.-K. Hong. 1999. Optimal production conditions of Streptomyces griseus trypsin (SGT) in Streptomyces lividans. J. Biochem. Mol. Biol. 32: 86-91
  17. Koo, B.J., K.H. Bae, S.M. Byun, and S.-K. Hong. 1998. Purification and characterization of Streptomyces griseus trypsin overexpressed in Streptomyces lividans. J. Microbiol. Biotechnol. 8: 333-340
  18. Oh, E. A., M.-S. Kim, W.-J. Chi, J.-H. Kim and S.-K. Hong. 2007. Characterization of the sgtR1 and sgtR2 genes and their role in regulating expression of the sprT gene encoding Streptomyces griseus trypsin. FEMS Microbiol. Lett. 276: 75-82 https://doi.org/10.1111/j.1574-6968.2007.00907.x
  19. Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  20. Tomono A., Y. Tasi, Y. Ohnishi, and S. Horinouchi. 2005. Three chymotrypsin genes are members of the AdpA regulon in the A-factor regulatory cascade in Streptomyces griseus. J. Bacteriol. 187: 6341-6353 https://doi.org/10.1128/JB.187.18.6341-6353.2005
  21. Trop, M. and Y. Birk. 1970. The specificity of proteases from Streptomyces griseus (Pronase). J. Biochem. 116: 19-25 https://doi.org/10.1042/bj1160019
  22. Yang, H.-Y., S.-S. Choi, W.-J. Chi, J.-H. Kim, D.-K. Kang, J. Chun, S.-S. Kang, and S.-K. Hong. 2005. Identification of the sprU gene encoding an additional sprT homologous trypsin-type protease in Streptomyces griseus. J. Microbiol. Biotechnol. 14: 1125-1129