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

• Published in: Journal of microbiology and biotechnology Vol. 19; no. 10; pp. 1191 - 1196
• Main Authors: Chi, W.J., Myongji University, Yongin, Republic of Korea, Song, J.H., Myongji University, Yongin, Republic of Korea, Oh, E.A., Myongji University, Yongin, Republic of Korea, Park, S.W., DyneBio Inc., Seongnam, Republic of Korea, Chang, Y.K., Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea, Kim, E.S., Inha University, Incheon, Republic of Korea, Hong, S.K., Myongji University, Yongin, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Seoul Korean Society for Applied Microbiology 01.10.2009; 한국미생물·생명공학회
• Subjects: affinity column chromatography; Biological and medical sciences; Biotechnology; Fundamental and applied biological sciences. Psychology; medium optimization; STREPTOMYCES GRISEUS; Streptomyces griseus trypsin (SGT); 생물학; medium optimization; Streptomycetaceae; Serine endopeptidases; Enzyme; Streptomyces griseus trypsin (SGT); Optimization; Actinomycetales; Peptidases; Trypsin; Affinity chromatography; Production; Bacteria; Hydrolases; Actinomycetes; Streptomyces griseus; Application; affinity column chromatography
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.0901.001

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℃, and it maintained activity between pH 7.0 and 9.0 and at temperatures up to 70℃. These enzymatic properties are very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.