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

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The production of Streptomyces griseus trypsin (SIST) 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 degrees C, and it maintained activity between pH 7.0 and 9.0 and at temperatures up to 70 degrees C. These enzymatic properties are very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.
Publisher
KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
Issue Date
2009-10
Language
English
Article Type
Article
Keywords

A-FACTOR; CHYMOTRYPSIN GENES; PROTEASE; LIVIDANS; OVEREXPRESSION; OVERPRODUCTION; PRONASE

Citation

JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, v.19, no.10, pp.1191 - 1196

ISSN
1017-7825
DOI
10.4014/jmb.0901.001
URI
http://hdl.handle.net/10203/101069
Appears in Collection
CBE-Journal Papers(저널논문)
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