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.

From Tea Leaves to Factories: A Review of Research Progress in l-Theanine Biosynthesis and Production

“…Since E. coli GGT is directed into the periplasm, free 43 and immobilized 66 whole E. coli cells possessing GGT activity have been applied to produce L-theanine from GAME with conversion rates of 95% and 69.3%, respectively. Several fusion technologies, such as thioredoxin (Trx), 68 glutathione Stransferase (GST), 69 and small ubiquitin-related modifier (SUMO) 42 tag fusion, were also conducted to enhance recombinant expression of E. coli GGT. Ultimately, using the purified E. coli GGT of the SUMO fusion system, 41 g/L of Ltheanine was produced from 0.267 M L-glutamine and 2 M ethylamine after 24 h reaction.…”

Application of Cell-Free Protein Synthesis System for the Biosynthesis of l-Theanine

“…In addition, the CFPS reaction is fast (hours), whereas in vivo production needs days for cell cultivation and l -theanine formation. Although the yield of l -theanine in our current CFPS system is not as high as in previous reports, − we believe that rational strategies are available to increase the yield in the future. For example, using different cell-free systems like Bacillus subtilis - or Pseudomonas putida -based , CFPS to express BsGS and PtGS, respectively, might be beneficial to the expression level and solubility of related enzymes and the resulting l -theanine yield.…”

“…So far, l -theanine biosynthesis has been reported using either purified enzymes or engineered microbial host cells with titers ranging from tens to hundreds of millimoles (mM) of product. − However, these strategies have their own limitations. For example, the enzyme purification process is time-consuming and laborious.…”

Recombinant Scorpine Produced Using SUMO Fusion Partner in Escherichia coli Has the Activities against Clinically Isolated Bacteria and Inhibits the Plasmodium falciparum Parasitemia In Vitro

“…Human SUMO-1, a 101 amino acid polypeptide, shares 50% sequence identity with human SUMO-2/ SUMO-3 [31], which are close homologues. It is known that SUMO, fused at the N-terminus with other proteins, can fold and protect the protein by its chaperoning properties, making it a useful tag for heterologous expression [12,32]. All SUMO genes encode precursor proteins with a short C-terminal sequence that extends from the conserved C-terminal Gly-Gly motif.…”

“…It is known that the SUMO, fused at the N-terminus with other proteins, can fold and protect the protein by its chaperoning properties, making it a useful tag for heterologous expression. These advantages include the manner in which protein expression is enhanced, proteolytic degradation of the target protein is decreased, protein folding and solubility are increased, and purification and detection are simplified [10][11][12].…”