Expression, Purification, And Characterization Of Recombinant Glutamine Synthetase

For the growth of plants,the nitrogen assimilation is a very important physiological process.Firstly,inorganic nitrogen must be assimilated into glutamic acid and glutamine organic nitrogen for plant to absorption and utilization.Glutamine synthetase (GS) is a key enzyme involved in ammonia assimilation of this process.In addition,GS is also a best mode for the gene specific expression and expression regulation in molecular biology reserch.GS expression not only have tissue organ specificity,but also effected by the environment (for instance,light,nitrogen,carbon dioxide) and growth development factor. So,higher plants glutamine synthetase become one of the hot research topic to people interested in this year.Glutamine synthetase has a very wide range of application prospect. Its unique nutritional and medicinal value has drawn wide attention from all over the world.According to relevant data,domestic consumption of glutamine in 2000 is 350t,but the domestic production of glutamine is 180t,the output of such far cannot satisfy the needs of the domestic market.In recent years,our country national economy rapid development,people’s living standards greatly improved,public health consciousness constantly reinforced,the market demand of glutamine is also growing rapidly..Our country is a large country with a population of 1.3 billion,Cranial nerve functional disease and digestive tract ulcer are frequently-occurring disease.So the demand for medicinal amino acid is also very large.There is a huge potential for glutamine in the pharmaceutical market.However,for a long time,due to the enzyme activity is not high,the practical application is inhibited by the factors of low specific activity and enzyme production.Although the glutamine synthetase widely exist in plants,the enzyme production efficiency is not high,it is hard to get a lot of products.In this paper, a new glutamine synthetase gene from Vitis vinifera was synthesized chemically according to the Escherichia coli bias codon and was synthesised by PTDS.For further research of grape enzymology characteristics of glutamine synthetase and its related functions,we constructed prokaryotic expression vector transformed recombinant plasmid into secretory expression of E.coli.Through enzyme activity identification,SDS-PAGE,we got the grapes of glutamine synthetase expression positive strains. In this paper,wu used transferase method and biosynthetase method to study the basic enzyme characteristics and the affect of the restraint of PPT to the recombinant.Results indicate that,The subunit molecular mass of GSI was 40 kDa. Optimal condition for y-glutamyl transferase activity was found to be 42℃ at PH 6.5 with 1_mM Mn2+ and bio synthetic activity was found to be 45℃ at PH7.0 with characteristics20 mM Mg2+.Kinetic analysis reveled Km for glutamate,ammonium and ATP to be 3.5,2.02.2.34mM,respectively. We also found that the synthesis of γ-glutamylhydroxamate in the absence of ADP wan observed with Mn2+.Lys and Gly has certain inhibitory effect on VvGSl.The results can provide tbackground knowledge to the industrial application of glutamine synthetase,and provide related basis to improve the production effciency of medicinal glutamine.