The Production Of SUMO Glutaminase Gene And Its Application In Glutamine Synthesis

L-glutamine(L-Gln)is a L-glutamate r-hydroxyl acylation amino acid,which is one of the basic amino acids used to biosynthesize protein.In the human body,L-Gln accounts for more than half of free amino acids and is the most abundant plus important amino acid in the human organism.L-Gln can be used widely,especially in medical field.Clinical research progress in recent years indicates that L-Gln clinical value and usage would probably exceed vitamin C in the future.According to L-Gln sales trends from European and America market,to minimum extent,L-Gln raw materials demand in China is up to 5000 tons per year.At present,China is the largest producer and consumer of L-glutamic acid in the world.However,research on fermentation of L-Gln,a kind of Glutamic acid analogues,was reported until the 1980's,and there is still a huge gap of L-Gln production technology between China and other developed countries.Therefore,medical grade L-Gln has being always imported from foreign countries.L-Gln was sold at RMB1,000,000/ton in the world market.Its production cost is 3-4 times of L-glutamate.Along with the improvement of human health,the accumulation of wealth and the deepening of the study on the mechanism of L-Gln,function of L-Gln will be developed further.As mentioned above,L-Gln is an important essential amino acid in human body and potential demand in food or medicine industry is expected.We first developed the application of small ubiquitin-related modifier(SUMO)fusion technology to the expression and purification of recombinant Bacillus subtilis GS.SUMO modification is to help correct protein folding and help improve recombinant glutamine synthetase activity and solubility.Thus,through our laboratory established coupling genetic engineered bacterial glutamine synthetase(GS)with yeast alcoholic fermentation system,we have solved the problem of high cost and low efficiency in traditional process of enzymatic production of glutamine,and have developed a way of the high efficient L-Gln production by GS.In order to obtain GS with high L-Gln forming activity,safety and low cost for food and pharmaceutics industry,0.1%(w/v)lactose was selected as inducer.The fusion protein was expressed in totally soluble form in E.coli,and the expression was verified by SDS-PAGE and Western blot analysis.The fusion protein was purified to 90%purity by nickel nitrilo-triacetic acid(Ni-NTA)resin chromatography with a yield of 625 mg per liter fermentation culture.After the SUMO/GS fusion protein was cleaved by the SUMO protease,the cleaved sample was reapplied to a Ni-NTA.Finally,about 121 mg recombinant GS was obtained from 1 L fermentation culture with no less than 96%purity.The recombinant purified GS showed great transferase activity(23 U/mg).The biosynthesis yield of L-Gln was high of 27.5 g/1 detected by high pressure liquid chromatography(HPLC)or thin-layer chromatography in 50 ml reaction system with 25 U recombinant GS.Thus,the SUMO-mediated GS expression and purification system potentially could be employed for the industrial production of L-Gln.