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

Abstract

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°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.

Keywords::

• Glutaminase
• Pseudomonas nitroreducens
• γ-glutamyl transfer reaction
• L-theanine

Acknowledgements

We thank Professor Jian He of NJAU for providing the bacterial P. nitroreducens DSM 14399 (ATCC 33634). We also wish to thank Zilie Liu, associate professor, for performing the HPLC analysis.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by the special funding from NJNU for talent faculty and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, No. 164320H106).