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Overexpression, one-step purification, and biochemical characterization of a recombinant γ-glutamyltranspeptidase from Bacillus licheniformis

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Abstract

A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant γ-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 °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-γ-glutamyl-p-nitroanilide (l-γ-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 μM when using l-γ-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 γ-glutamyl compounds.

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Acknowledgements

We are grateful to Dr. Kuo-Lung Ku for technical assistance in some of the analytical experiments, as well as Dr. Wenlung Chen for the facility support during the purification of the recombinant enzyme. This work was supported in part by a grant (NSC 94-2313-B-415-002) from National Science Council of Taiwan, Republic of China.

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  1. Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, Taiwan, 60083, Republic of China

    Long-Liu Lin, Pei-Ru Chou & Yu-Wen Hua

  2. Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan, 402, Republic of China

    Wen-Hwei Hsu

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  1. Long-Liu Lin
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Correspondence to Long-Liu Lin.

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Lin, LL., Chou, PR., Hua, YW. et al. Overexpression, one-step purification, and biochemical characterization of a recombinant γ-glutamyltranspeptidase from Bacillus licheniformis . Appl Microbiol Biotechnol 73, 103–112 (2006). https://doi.org/10.1007/s00253-006-0440-4

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