Journal of Microbiology and Biotechnology

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

DOI QR Code

Medium Optimization and Application of Affinity Column Chromatography for Trypsin Production from Recombinant Streptomyces griseus

  • Chi, Won-Jae (Department of Biological Science, Myongji University) ;
  • Song, Ju-Hyun (Department of Biological Science, Myongji University) ;
  • Oh, Eun-A. (Department of Biological Science, Myongji University) ;
  • Park, Seong-Whan (R&D Center, DyneBio Inc.) ;
  • Chang, Yong-Keun (Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology) ;
  • Kim, Eung-Soo (Department of Biological Engineering, Inha University) ;
  • Hong, Soon-Kwang (Department of Biological Science, Myongji University)
  • Published : 2009.10.31
Download PDF
⟨ Previous Next ⟩

Abstract

The production of Streptomyces griseus trypsin (SGT) by S. griseus IFO13350 transformed with the expression vector pWHM3-TR1R2, containing sprT encoding SGT and the two positive regulatory genes sgtR1 and sgtR2, was investigated in various media. Cultivation in Ferm-0 gave 1.4 times more trypsin activity than in C5/L medium. In addition, replacement of 2% glucose and 1% skim milk in Ferm-0 with 2% dextrin and 1% tryptone (designated Ferm-II) enhanced trypsin activity 4.1-fold. To simplify the purification process, the supernatant from the S. griseus transformant cultured in Ferm-II medium was fractionated with ammonium sulfate (25-55%), then subjected to Hitrap Benzamidine FF affinity column chromatography. The specific activity of SGT purified by one-step chromatography was 69,550 unit/mg protein and the overall purification yield was above 8%, indicating that this method is more effective than those previously reported. Purified SGT was most active at pH 8.0 and $50^{\circ}C$, and it maintained activity between pH 7.0 and 9.0 and at temperatures up to $70^{\circ}C$. These enzymatic properties are very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.

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. Biotechnol. 11:1077-1086
  3. 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
  4. Choi, S.-S., J. H. Kim, J.-H. Kim, D.-K. Kang, S.-S. Kang, and S.-K. Hong. 2006. Functional analysis of sprD gene encoding Streptomyces griseus protease D (SGPD) in Streptomyces griseus. J. Microbiol. Biotechnol. 16: 312-317
  5. 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
  6. 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
  7. Kato, J. Y., 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
  8. Khokhlov, A. S., I. I. Tovarova, L. N. Borisova, S. A. Pliner, L. A. Schevchenko, E. Y. Kornitskaya, N. S. Ivkina, and I. A. Rapoport. 1976. The A-factor, responsible for the biosynthesis of streptomycin by a mutant strain of Actinomyces streptomycini. Dokl. Akad. Nauk SSSR 177: 232-235
  9. Kieser, H., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, United Kingdom
  10. Kim, J.-H. and S.-K. Hong. 2008. Overproduction of bacterial trypsin in Streptomyces - optimization for Streptomyces griseus trypsin production by recombinant Streptomyces. Kor. J. Appl. Microbiol. Biotechnol. 36: 28-33
  11. 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. Comm. 181: 707-713 https://doi.org/10.1016/0006-291X(91)91248-B
  12. 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
  13. Koo, B.-J., K. H. Bae, S.-M. Byun, and S.-K. Hong. 1998. Purification and characterization of Streptomcyes griseus trypsin overexpressed in Streptomyces lividans. J. Microbiol. Biotechnol. 8: 333-340
  14. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 https://doi.org/10.1038/227680a0
  15. Narahashi, Y., K. Shibuya, and M. Yanagita. 1968. Studies on proteolytic enzymes (pronase) of Streptomyces griseus K-1. II. Separation of exo-and endopeptidases of pronase. J. Biochem. 64: 427-437
  16. 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
  17. Olfason, R. W. and L. B. Smillie. 1975. Enzymatic and physicochemical properties of Streptomyces griseus trypsin. Biochemistry 14: 1161-1167 https://doi.org/10.1021/bi00677a010
  18. Tomono, A., Y. Tsai, 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
  19. Trop, M. and Y. Birk. 1970. The specificity of proteases from Streptomyces grisseus (pronase). Biochem. J. 116: 19-25
  20. 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. 15:1125-1129

Cited by

  1. Enhancement of Protein Secretion by TatAC Overexpression in Streptomyces griseus vol.16, pp.1, 2009, https://doi.org/10.1007/s12257-010-0382-7
  2. Functional expression of trypsin from Streptomyces griseus by Pichia pastoris vol.39, pp.11, 2009, https://doi.org/10.1007/s10295-012-1172-3
  3. Streptomyces flavogriseus HS1: Isolation and Characterization of Extracellular Proteases and Their Compatibility with Laundry Detergents vol.2014, pp.None, 2009, https://doi.org/10.1155/2014/345980
  4. Improved Production of Active Streptomyces griseus Trypsin with a Novel Auto-Catalyzed Strategy vol.6, pp.None, 2009, https://doi.org/10.1038/srep23158