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J. Microbiol. Biotechnol. 2009; 19(10): 1191-1196

Published online October 28, 2009 https://doi.org/10.4014/jmb.0901.001

Copyright © The Korean Society for Microbiology and Biotechnology.

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

Won-Jae Chi 1, Ju-Hyun Song 1, Eun A. Oh 1, Seong-Whan Park 1, Yong Keun Chang 3, Eung-Soo Kim 4 and Soon-Kwang Hong 1*

Department of Biological Science, Myongji University, Yongin 449-728, Korea, 1R&D Center, DyneBio Inc., Seongnam 460-120, Korea, 2Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Taejon 305-701,Korea, 3Department of Biological Engineering, Inha University, Incheon 402-751, Korea

Abstract

The expression vector pWHM3-TR1R2, which contains
sprT encoding Streptomyces griseus trypsin (SGT) and two
positive regulatory genes (sgtR1 and sgtR2), was introduced
into S. griseus IFO13350 and the productivity of SGT by
the transformant was investigated in various media. Among
the tested media, Ferm-0 gave 1.4 times more trypsin
activity than C5/L medium. In addition, replacement of
2% glucose and 1% skim milk in Ferm-0 medium with
2% dextrin and 1% tryptone (named as Ferm-II medium)
yielded significantly enhanced trypsin activity, by 4.1-fold,
than that of Ferm-0. For simplifying the purification
process, the cultural supernatant of S. griseus transformant
in Ferm-II medium was fractionated with ammonium
sulfate (25%-55%), and then applied to Hitrap benzamidine
FF affinity column chromatography. The specific activity
of the purified SGT by one-step column chromatography
was 69,550 unit/mg protein, and the overall purification
yield was above 8%, which was more effective than the
methods of previous reports. The trypsin activity of the
purified SGT was most active at pH 8.0 and 50oC, and
maintained their activities between pH 7.0 and pH 9.0, and
up to 70oC. These enzymatic properties were very similar to
those of authentic eukaryotic trypsin purified from bovine
pancreas.

Keywords: Streptomyces griseus Trypsin(SGT), medium optimization, affinity column chromatography