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Heterologous expression and enzymatic characterization of γ-glutamyltranspeptidase from Bacillus amyloliquefaciens

  • Microbial Physiology and Biochemistry
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Abstract

γ-Glutamyltranspeptidase (GGT) catalyzes the cleavage of γ-glutamyl compounds and the transfer of γ-glutamyl moiety to water or to amino acid/peptide acceptors. GGT can be utilized for the generation of γ-glutamyl peptides or glutamic acid, which are used as food taste enhancers. In the present study, Bacillus amyloliquefaciens SMB469 with high GGT activity was isolated from Doenjang, a traditional fermented soy food of Korea. The gene encoding GGT from B. amyloliquefaciens SMB469 (BaGGT469) was cloned from the isolate, and heterologously expressed in E. coli and B. subtilis. For comparison, three additional GGT genes were cloned from B. subtilis 168, B. licheniformis DSM 13, and B. amyloliquefaciens FZB42. The BaGGT469 protein was composed of 591 amino acids. The final protein comprises two separate polypeptide chains of 45.7 and 19.7 kDa, generated via autocatalytic cleavage. The specific activity of BaGGT469 was determined to be 17.8 U/mg with γ-L-glutamyl-p-nitroanilide as the substrate and diglycine as the acceptor. GGTs from B. amyloliquefaciens showed 1.4- and 1.7-fold higher transpeptidase activities than those from B. subtilis and B. licheniformis, respectively. Especially, recombinant B. subtilis expressing BaGGT469 demonstrated 11- and 23-fold higher GGT activity than recombinant E. coli and the native B. amyloliquefaciens, respectively, did. These results suggest that BaGGT469 can be utilized for the enzymatic production of various γ-glutamyl compounds.

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References

  • Abe, K., Ito, Y., Ohmachi, T., and Asada, Y. 1997. Purification and properties of two isozymes of γ-glutamyltranspeptidase from Bacillus subtilis TAM-4. Biosci. Biotechnol. Biochem. 61, 1621–1625.

    Article  CAS  PubMed  Google Scholar 

  • Castellano, I., Di Salle, A., Merlino, A., Rossi, M., and La Cara, F. 2011. Gene cloning and protein expression of γ-glutamyltranspeptidases from Thermus thermophilus and Deinococcus radiodurans: Comparison of molecular and structural properties with mesophilic counterparts. Extremophiles 15, 259–270.

    Article  CAS  PubMed  Google Scholar 

  • Castellano, I. and Merlino, A. 2012. γ-Glutamyltranspeptidases: Sequence, structure, biochemical properties, and biotechnological applications. Cell. Mol. Life Sci. 69, 3381–3394.

    Article  CAS  PubMed  Google Scholar 

  • Castellano, I., Merlino, A., Rossi, M., and La Cara, F. 2010. Biochemical and structural properties of γ-glutamyl transpeptidase from Geobacillus thermodenitrificans: An enzyme specialized in hydrolase activity. Biochimie 92, 464–474.

    Article  CAS  PubMed  Google Scholar 

  • Chen, X., Su, L., Wu, D., and Wu, J. 2014. Application of recombinant Bacillus subtilis γ-glutamyltranspeptidase to the production of L-theanine. Process Biochem. 49, 1429–1439.

    Article  CAS  Google Scholar 

  • Chevalier, C., Thiberge, J.M., Ferrero, R.L., and Labigne, A. 1999. Essential role of Helicobacter pylori γ-glutamyltranspeptidase for the colonization of the gastric mucosa of mice. Mol. Microbiol. 31, 1359–1372.

    Article  CAS  PubMed  Google Scholar 

  • Choi, C.H., Kim, S.H., Jang, J.H., Park, J.T., Shim, J.H., Kim, Y.W., and Park, K.H. 2010. Enzymatic synthesis of glycosylated puerarin using maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis. J. Sci. Food Agric. 90, 1179–1184.

    Article  CAS  PubMed  Google Scholar 

  • Di Stefano, A., Sozio, P., and Cerasa, L.S. 2008. Antiparkinson prodrugs. Molecules 13, 46–68.

    Article  PubMed  Google Scholar 

  • Imaoka, M., Yano, S., Okumura, M., Hibi, T., and Wakayama, M. 2010. Molecular cloning and characterization of γ-glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694. Biosci. Biotechnol. Biochem. 74, 1936–1939.

    Article  CAS  PubMed  Google Scholar 

  • Kijima, K. and Suzuki, H. 2007. Improving the umami taste of soy sauce by the addition of bacterial γ-glutamyltranspeptidase as a glutaminase to the fermentation mixture. Enzyme Microb. Technol. 41, 80–84.

    Article  CAS  Google Scholar 

  • Kim, J., Jang, S., Kim, A., Su, H., Gunawardhana, N., Jeon, Y.E., Bak, E.J., Kim, J.H., and Cha, J.H. 2016. Role of bacterial γ-glutamyltranspeptidase as a novel virulence factor in bone-resorbing pathogenesis. J. Microbiol. 54, 396–402.

    Article  CAS  PubMed  Google Scholar 

  • Lin, L.L., Chou, P.R., Hua, Y.W., and Hsu, W.H. 2006. Overexpression, one-step purification, and biochemical characterization of a recombinant γ-glutamyltranspeptidase from Bacillus licheniformis. Appl. Microbiol. Biotechnol. 73, 103–112.

    Article  CAS  PubMed  Google Scholar 

  • Minami, H., Suzuki, H., and Kumagai, H. 2003. Salt-tolerant γ-glutamyltranspeptidase from Bacillus subtilis 168 with glutaminase activity. Enzyme Microb. Technol. 32, 431–438.

    Article  CAS  Google Scholar 

  • Nakayama, R., Kumagai, H., and Tochikura, T. 1984. Purification and properties of γ-glutamyltranspeptidase from Proteus mirabilis. J. Bacteriol. 160, 341–346.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ogawa, Y., Hosoyama, H., Hamano, M., and Motai, H. 1991. Purification and properties of γ-glutamyltranspeptidase from Bacillus subtilis (natto). Agric. Biol. Chem. 55, 2971–2977.

    CAS  PubMed  Google Scholar 

  • Oinonen, C. and Rouvinen, J. 2000. Structural comparison of Ntnhydrolases. Protein Sci. 9, 2329–2337.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Orlowski, M. and Meister, A. 1963. γ-Glutamyl-p-nitroanilide: A new convenient substrate for determination and study of L-and D-γ-glutamyltranspeptidase activities. Biochim. Biophys. Acta 73, 679–681.

    Article  CAS  PubMed  Google Scholar 

  • Penninckx, M.J. and Jaspers, C.J. 1985. Characterization of γ-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae. Biochimie 67, 999–1006.

    Article  CAS  PubMed  Google Scholar 

  • Rajpert-de Meyts, E., Heisterkamp, N., and Groffen, J. 1988. Cloning and nucleotide sequence of human γ-glutamyl transpeptidase. Proc. Natl. Acad. Sci. USA 85, 8840–8844.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sadaie, Y. and Kada, T. 1983. Formation of competent Bacillus subtilis cells. J. Bacteriol. 153, 813–821.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shruti, B. and Rani, G. 2014. Theanine synthesis using γ-glutamyl transpeptidase from Bacillus licheniformis ER-15. J. Agric. Food Chem. 62, 9151–9159.

    Article  Google Scholar 

  • Shuai, Y., Zhang, T., Mu, W., and Jiang, B. 2011. Purification and characterization of γ-glutamyltranspeptidase from Bacillus subtilis SK11.004. J. Agric. Food Chem. 59, 6233–6238.

    Article  CAS  PubMed  Google Scholar 

  • Suzuki, H., Izuka, S., Miyakawa, N., and Kumagai, H. 2002a. Enzymatic production of theanine, an “umami” component of tea, from glutamine and ethylamine with bacterial γ-glutamyltranspeptidase. Enzyme Microb. Technol. 31, 884–889.

    Article  CAS  Google Scholar 

  • Suzuki, H., Kajimoto, Y., and Kumagai, H. 2002b. Improvement of the bitter taste of amino acids through the transpeptidation reaction of bacterial γ-glutamyltranspeptidase. J. Agric. Food Chem. 50, 313–318.

    Article  CAS  PubMed  Google Scholar 

  • Suzuki, H., Kumagai, H., and Tochikura, T. 1986. γ-Glutamyltranspeptidase from Escherichia coli K-12: Purification and properties. J. Bacteriol. 168, 1325–1331.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suzuki, H., Yamada, C., and Kato, K. 2007. γ-Glutamyl compounds and their enzymatic production using bacterial γ-glutamyltranspeptidase. Amino Acids 32, 333–340.

    Article  CAS  PubMed  Google Scholar 

  • Toelstede, S. and Hofmann, T. 2009. Kokumi-active glutamyl peptides in cheeses and their biogeneration by Penicillium roquefortii. J. Agric. Food Chem. 57, 3738–3748.

    Article  CAS  PubMed  Google Scholar 

  • Xu, K. and Strauch, M.A. 1996. Identification, sequence, and expression of the gene encoding γ-glutamyltranspeptidase in Bacillus subtilis. J. Bacteriol. 178, 4319–4322.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University, Cheongju, 28644, Republic of Korea

    Jung-Min Lee, Jaejung Lee, Gyeong-Hwa Nam, Byung-Sam Son, Myoung-Uoon Jang & Tae-Jip Kim

  2. Sempio Fermentation Research Center, Sempio Foods Company, Cheongju, 28156, Republic of Korea

    Jaejung Lee, So-Won Lee & Byung-Serk Hurh

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  1. Jung-Min Lee
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  2. Jaejung Lee
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Correspondence to Tae-Jip Kim.

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Lee, JM., Lee, J., Nam, GH. et al. Heterologous expression and enzymatic characterization of γ-glutamyltranspeptidase from Bacillus amyloliquefaciens . J Microbiol. 55, 147–152 (2017). https://doi.org/10.1007/s12275-017-6638-6

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  • DOI: https://doi.org/10.1007/s12275-017-6638-6

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