Screening Of Gamma-glutamylmethylamide Synthetase-producing Strain

γ-glutamylmethylamide synthetase(γ-GMAS) (EC 6.3.4.12) is a ligase, which can catalytic glutamate and methylamine synthesisγ-glutamylmethylamide in the presence of ATP and Mn2+.γ-glutamylmethylamide and theanine (γ-ethylamino-L-glutamic acid)own a very similar structure, soγ-glutamylmethylamide synthetase can still catalytic glutamate and ethylamine synthesis theanine under certain conditions,this provides a new idea for theanine production.γ-glutamylmethylamide synthetase, in foreign countries, was reported mainly in the bacterial bodys, including Hyphomicrobium vulgare, Pseudomonas, and some methyl nutrition marines with methylamine as the sole carbon and nitrogen sources.In recent years,lots of research onγ-glutamylmethylamide synthetase forming theanine are reported in abroad.But we have not yet seen relevant reports about liquid fermentation of Pseudomonas.sp producingγ-glutamylmethylamide synthetase, in view of this, the paper presented a preliminary study on theγ-glutamylmethylamide synthetase production of Pseudomonas.sp by submerged fermentation.Firstly, we use UV-induced Pseudomonas.sp to select mutant strain with higherγ-glutamylmethylamide synthetase.then Plackett-Burman and RSM Response Surface was designed to optimize fermentation conditions, and improve enzyme production; Finally,γ-glutamylmethylamide synthetase was cruded and part of its enzymatic properties were studied in order to provide further research for theoretical foundation and scientific basis.The mainly research results are as follows:First, the initial strain Pseudomonas.sp was treatmented by UV mutagenesis, we received a good mutations strain UV-19 with highγ-glutamylmethylamide synthetase activity and high genetic stability after screening and rescreening.Secondly, the obtained good mutations strain for the experiments,γ-glutamylmethylamide synthetase activity by fermentation as index,screening out the optimum enzyme production carbon and nitrogen sources, oxygen speed, culture temperature, incubation time,substrate-induced, and the main factors influencing fermentation were screening through Plackett-Burman (PB) design,they are glucose (p=0.0003), peptone (p=0.0244), initial pH value (p=0.0033) and liquid volume (p=0.0122). Again, based on the PB experiments,γ-glutamylmethylamide synthetase activity is for evaluation index,the main factors were further optimized through response surface method (Response Surface Methodology, RSM) central composite experimental design,the optimum fermentation conditions for enzyme production was determined:glucose 15.0g/L, peptone 12.0g/L, NaCl 5.0g/L, MgSO₄ 0.2g/L, KH₂PO₄ 0.5g/L, methylamine hydrochloride 1.0g/L, initial pH6.5, liquid volume 72mL/250mL, culture time 42h, culture temperature 30℃, speed 160r/min.Finally,γ-glutamylmethylamide synthetase of dominant mutant was crude, part of the crude enzyme properties (optimum temperature and temperature stability, optimum pH and pH stability, the impact of metal ions, etc.) were studied,the results indict that the optimal enzyme and substrate reaction time is 40min, the optimal reaction temperature is 40℃, temperature stability range is 10-30℃,optimum pH is 7.5,pH stability range is pH5.5-6.5,metal ions Mn²⁺,Mg²⁺could activeγ-glutamylmethylamide synthetase.