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Expression Optimization and Biochemical Characterization of a Recombinant γ-Glutamyltranspeptidase from Escherichia coli Novablue

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Abstract

A truncated Escherichia coli Novablue γ-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 His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °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 °C and 9, respectively. The apparent K m and V max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 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 His6-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.

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Abbreviations

GGT:

γ-glutamyltranspeptidase

EcGGT:

Escherichia coli GGT

X-gal:

5–bromo-4-chloro-3-indolyl-β-D-galactopyranoside

IPTG:

isopropyl-β-D-thiogalactopyranoside

PAGE:

polyacrylamide gel electrophoresis

SDS-PAGE:

sodium dodecyl sulfate-PAGE

L-γ-Glu-p-NA:

L-γ-glutamyl-p-nitroanilide.

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Acknowledgments

This work was supported by a grant (NSC-94-2313-B-241-002) from National Science Council of the Republic of China.

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Authors and Affiliations

  1. Graduate Institute of Food Science, National Chiayi University, 300 University Road, 60083, Chiayi, Taiwan

    Ya-Feng Yao & Yih-Ming Weng

  2. Department of Food and Nutrition, Hungkuang University, Taichung, 433, Taiwan

    Hui-Yu Hu

  3. Department of Applied Chemistry, National Chiayi University, 300 University Road, 60083, Chiayi, Taiwan

    Kuo-Lung Ku & Long-Liu Lin

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  1. Ya-Feng Yao
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Correspondence to Long-Liu Lin.

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Yao, YF., Weng, YM., Hu, HY. et al. Expression Optimization and Biochemical Characterization of a Recombinant γ-Glutamyltranspeptidase from Escherichia coli Novablue. Protein J 25, 431–441 (2006). https://doi.org/10.1007/s10930-006-9037-0

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