Please use this identifier to cite or link to this item:
|標題:||Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis|
|期刊/報告no：:||Applied Microbiology and Biotechnology, Volume 73, Issue 1, Page(s) 103-112.|
|摘要:||A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant gamma-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6-8 and 40 degrees C, respectively. The chloride salt of metal ions Mg2+, K+, and Na+ can activate BLrGGT, whereas that of Pb2+ dramatically inhibited it. The substrate specificity study showed that L-gamma-glutamyl-p-nitroanilide (L-gamma-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k (cat) of 105 s(-1) and a K (m) of 21 mu M when using L-gamma-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important gamma-glutamyl compounds.|
|Appears in Collections:||期刊論文|
Show full item record
TAIR Related Article
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.