Expression optimization and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Escherichia coli novablue.
A truncated Escherichia coli Novablue gamma-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His(6)-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 degrees C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 degrees C and 9, respectively. The apparent K (m) and V (max) values for gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor in the transpeptidation reaction were 37.9 microM and 53.7 x 10(-3) mM min(-1), respectively. The synthesis of L -theanine was performed in a reaction mixture containing 10 mM L -Gln, 40 mM ethylamine, and 1.04 U His(6)-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.
Yao
Ya-Feng
YF
Graduate Institute of Food Science, National Chiayi University, 300 University Road, Chiayi, Taiwan.
Weng
Yih-Ming
YM
Hu
Hui-Yu
HY
Ku
Kuo-Lung
KL
Lin
Long-Liu
LL
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005971:Glutamates; D011994:Recombinant Proteins; C026166:theanine; D005723:gamma-Glutamyltransferase