Medium optimization and application of an affinity column chromatography for streptomyces griseus trypsin production from the recombinant Streptomyces griseus.
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 50 degrees C, and maintained their activities between pH 7.0 and pH 9.0, and up to 70 degrees C. These enzymatic properties were very similar to those of authentic eukaryotic trypsin purified from bovine pancreas.
Chi
Won-Jae
WJ
Department of Biological Science, Myongji University, Yongin, Korea.
Song
Ju-Hyun
JH
Oh
Eun A
EA
Park
Seong-Whan
SW
Chang
Yong Keun
YK
Kim
Eung-Soo
ES
Hong
Soon-Kwang
SK
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D003470:Culture Media / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D005818:Genetic Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013305:Streptomyces griseus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D014357:Trypsin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D014357:Trypsin