51ºÚÁϳԹÏÍø

Abstract

Γ-glutamyl transpeptidase (GGT) is a well known enzyme for the synthesis of various γ-glutmayl compounds, and theanine is one of the most important neutraceutical among them. GGT gene from Bacillus licheniformis ER-15 was cloned and expressed in E. coli BL21 harboring pET 51b vector. Protein was purified by Ni2+-NTA resin and biochemically characterized with pH and temperature optima to be pH 9 and 60⁰C respectively. Enzyme showed three times transpeptidase activity with respect to hydrolysis in case of glycylglycine as an acceptor while 1.5 times with ethylamine at 37⁰C. For the enzymatic synthesis of theanine, acetone precipitated enzyme was used which can be stored at 4⁰C with a shelf-life of more than 3 months. Various parameters for theanine synthesis viz. pH, donor and acceptor concentrations, enzyme concentration, time and temperature were optimized to be pH 9, 20mM glutamine, 200mM ethylamine, 0.75U/mL, 4h and 37⁰C respectively, in a 10ml reaction volume. Theanine was estimated using a novel RP-HPLC method without employing derivatization of theanine at 203nm. Above 80% conversion was obtained using above parameters. The process was then scaled up to 1L reaction volume and theanine was batch purified using Dowex 50W X 8 hydrogen form resin and eluted using ammonia water with a recovery of around 85%. Further optimization of theanine production was done by employing fed-batch method as the enzyme is stable at 37⁰C for more than 24h.

Biography