title: Heterologous expression and enzymatic characterization of γ-glutamyltranspeptidase from Bacillus amyloliquefaciens.
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.
authors.affiliation: Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University, Cheongju, 28644, Republic of Korea.
authors.forename: Jung-Min
authors.initials: JM
authors.lastname: Lee
authors.affiliation: Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University, Cheongju, 28644, Republic of Korea.|Sempio Fermentation Research Center, Sempio Foods Company, Cheongju, 28156, Republic of Korea.
authors.forename: Jaejung
authors.initials: J
authors.lastname: Lee
authors.affiliation: Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University, Cheongju, 28644, Republic of Korea.
authors.forename: Gyeong-Hwa
authors.initials: GH
authors.lastname: Nam
authors.affiliation: Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University, Cheongju, 28644, Republic of Korea.
authors.forename: Byung-Sam
authors.initials: BS
authors.lastname: Son
authors.affiliation: Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University, Cheongju, 28644, Republic of Korea.
authors.forename: Myoung-Uoon
authors.initials: MU
authors.lastname: Jang
authors.affiliation: Sempio Fermentation Research Center, Sempio Foods Company, Cheongju, 28156, Republic of Korea.
authors.forename: So-Won
authors.initials: SW
authors.lastname: Lee
authors.affiliation: Sempio Fermentation Research Center, Sempio Foods Company, Cheongju, 28156, Republic of Korea.
authors.forename: Byung-Serk
authors.initials: BS
authors.lastname: Hurh
authors.affiliation: Division of Animal, Horticultural and Food Sciences, Graduate School of Chungbuk National University, Cheongju, 28644, Republic of Korea. tjkim@cbnu.ac.kr.
authors.forename: Tae-Jip
authors.initials: TJ
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D000069977:Bacillus amyloliquefaciens / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D006033:Glycylglycine; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D056910:Republic of Korea; D045730:Soy Foods / Q000382:microbiology; D013379:Substrate Specificity; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D006033:Glycylglycine; D005723:gamma-Glutamyltransferase
keywords: Bacillus amyloliquefaciens; enzymatic characterization; heterologous expression; γ-glutamyltranpeptidase