title: Production of bioactive γ-glutamyl transpeptidase in Escherichia coli using SUMO fusion partner and application of the recombinant enzyme to L-theanine synthesis.
abstract: The amino acid L-theanine (γ-glutamylethylamide) has potential important applications in the food and pharmaceutical industries and increased demand for this compound is expected. It is the major "umami" (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of L-theanine involving recombinant Escherichia coli γ-glutamyltranspeptidase (GGT) has been developed. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Escherichia coli γ-GGT. In order to obtain γ-GGT with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9 (consisting of glycerol and inorganic salts) and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel-nitrilotriacetic acid (Ni-NTA) resin chromatography with a yield of 115 mg per liter fermentation culture. After the SUMO/γ-GGT fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni-NTA column. Finally, about 62 mg recombinant γ-GGT was obtained from 1 l fermentation culture with no less than 95% purity. The recombinant γ-GGT showed great transpeptidase activity, with 1500 U of purified recombinant γ-GGT in a 1-l reaction system, a biosynthesis yield of 41 g of L-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC). Thus, the application of SUMO technology to the expression and purification of γ-GGT potentially could be employed for the industrial production of L-theanine.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medicine Biotechnology, College of Life Science, Nanjing Normal University, No. 1 Wenyuan Road, Nanjing 210046, Jiangsu, People's Republic of China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Cui
authors.initials: C
authors.lastname: Min
authors.forename: Fenfen
authors.initials: F
authors.lastname: Zhu
authors.forename: Yinqiang
authors.initials: Y
authors.lastname: Xin
authors.forename: Shuangquan
authors.initials: S
authors.lastname: Zhang
authors.forename: Lan
authors.initials: L
authors.lastname: Luo
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Yin
mesh_terms: D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005971:Glutamates / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D005971:Glutamates; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C026166:theanine; D005723:gamma-Glutamyltransferase