Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis.
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 Mg(2+), K(+), and Na(+) can activate BLrGGT, whereas that of Pb(2+) 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 microM 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.
Lin
Long-Liu
LL
Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, Taiwan, 60083, Republic of China. llin@mail.ncyu.edu.tw
Chou
Pei-Ru
PR
Hua
Yu-Wen
YW
Hsu
Wen-Hwei
WH
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005973:Glutamine / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007854:Lead / Q000494:pharmacology; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D005973:Glutamine; D007854:Lead; D012965:Sodium Chloride; C029891:lead chloride; D011189:Potassium Chloride; C001300:gamma-glutamine-4-nitroanilide; D005723:gamma-Glutamyltransferase