Cloning, expression of glutaminase from Pseudomonas nitroreducens and application to theanine synthesis

It has been reported that glutaminase (GLS) from Pseudomonas nitroreducens can be used in a new method for producing L-theanine. In this study, a pair of degenerate primers was designed to obtain the glutaminase gene from P. nitroreducens DSM 14399. The glutaminase gene (PnGLS gene) was 909 bps long encoding a protein of 302 amino acids, containing several key catalytic residues of GLSs (Ser61, Lys64, Asn111, Lys193 and Ser194). The optimum conditions for the production of theanine were 0.3 M L-glutamine, 1.5 M ethylamine and 1.5 U/5 ml recombinant PnGLS, pH 10.0 (100 mM borate buffer), 5 h incubation at 37 degrees C. L-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC) at a concentration of 21.8 g/L reaction mixture; a yield of about 40%. Using recombinant PnGLS is a feasible route for the future industrial production of L-theanine.