Yao Ya-Feng, Weng Yih-Ming, Hu Hui-Yu, Ku Kuo-Lung, Lin Long-Liu
Graduate Institute of Food Science, National Chiayi University, 300 University Road, Chiayi, Taiwan.
Protein J. 2006 Sep;25(6):431-41. doi: 10.1007/s10930-006-9037-0.
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.
通过聚合酶链反应扩增缺少信号序列部分的前48个碱基编码序列的截短型大肠杆菌诺瓦蓝γ-谷氨酰转肽酶（EcGGT）基因，并将其克隆到表达载体pQE-30中以产生pQE-EcGGT。大肠杆菌M15（pQE-EcGGT）在20℃下用0.1 mM异丙基-β-D-硫代半乳糖苷（IPTG）诱导12小时可实现His(6)标签酶的最大产量。通过镍螯合层析将过表达的酶纯化至同质，比转肽酶活性为4.25 U/mg蛋白，最终产率为83%。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估计纯化酶亚基的分子量分别为41 kDa和21 kDa，表明即使在信号序列截短的情况下，EcGGT仍会进行翻译后切割。重组酶的最适温度和pH分别为40℃和9。在转肽反应中，以γ-谷氨酰-p-硝基苯胺作为γ-谷氨酰供体时，其表观米氏常数（K (m)）和最大反应速率（V (max)）值分别为37.9 μM和53.7×10⁻³ mM min⁻¹。L-茶氨酸的合成在含有10 mM L-谷氨酰胺、40 mM乙胺和1.04 U His(6)标签EcGGT/ml、pH 10的反应混合物中进行，转化率为45%。
