First Example of the Extracellular Surface Expression of Intrinsically Periplasmic Escherichia coli γ-Glutamyltranspeptidase, a Member of the N-Terminal Nucleophile Hydrolase Superfamily, and the Use of Cells as a Catalyst for γ-Glutamylvalylglycine Production.

Although the purified Escherichia coli γ-glutamyltranspeptidase has much higher transpeptidation activity than hydrolysis activity, almost all γ-glutamyltranspeptidase activity is hydrolysis activity in vivo, that is when measured using the whole cells. By using the Met1 to Arg232 fragment of E. coli YiaT or the CapA of Bacillus subtilis subsp. Natto as an anchor protein, we succeeded in expressing E. coli γ-glutamyltranspeptidase on the extracellular surface of the cells, and these cells showed higher transpeptidation activity than hydrolysis activity in the presence of NaCl. Furthermore, E. coli cells overexpressing γ-glutamyltranspeptidase without an anchor from the T5 promoter maintained γ-glutamyltranspeptidase on the extracellular surface of the cells immediately after being harvested from the culture medium, but the enzyme was released from the extracellular surface of the cells subsequently in the absence of NaCl. Using these cells expressing γ-glutamyltranspeptidase on the extracellular surface, γ-Glu-Val-Gly, a kokumi compound, was successfully produced.

Agmatine production by Escherichia coli cells expressing SpeA on the extracellular surface.

A plasmid was constructed to express the CapA-SpeA fusion protein from the capA gene, which encodes one of the subunits of capsular poly-γ-glutamate synthetase of Bacillus subtilis subsp. natto, and the speA gene, encoding biosynthetic arginine decarboxylase (EC 4.1.1.19) of Escherichia coli, under the control of the T5 promoter. The expression of SpeA on the extracellular surface of cells was confirmed by confocal microscopy with the anti-SpeAE. coli antibody and anti-rabbit IgG L & H conjugated with Alexa Fluor 488. The constructed strain SH2290 produced 200 mM agmatine from 200 mM arginine, 20 mM MgSO4, 0.9 % NaCl, and 0.02 mg/mL pyridoxal 5'-phosphate (initial pH 5.3) by adjusting pH of the reaction mixture to 6.8 with HCl after each sampling during the reaction. The addition of pyridoxal 5'-phosphate to the reaction mixture was required for the maximum agmatine production. The present results demonstrate that the expression of enzymes on the extracellular surface of cells is a very powerful method for enzymatic conversion.