High Efficiency Of L-Glutamine Production By Coupling Genetic Engineered Bacterial Glutamine Synthetase With Yeast Alcoholic Fermentation System

Glutamine is an important conditionally necessary amino acid in human body. In this paper, high efficiency of L-glutamine production by coupling genetic engineered bacterial glutamine synthetase with yeast alcoholic fermentation system was studied.Glutamine Synthetase gene(glnA) was amplified from Bacillus subtilis genomic DNA with primers designed according to sequences reported in EMBL data bank, then it was inserted into expression vector pET28b, the sequence of glnA was proved to be the same as that reported in the data bank by DNA sequencing. After transformation of this recombinant plasmid pET28b-glnA into BL-21 (DE3) strain, Lactose and IPTG were used to induce gene expression at 37 C separately. Both of them can induce GS expression efficiently. The induced protein is proved to be soluble and occupies about 88% of the total proteins by SDS-PAGE analysis. The soluble GS was purified by Ni2+ chelating sepharose column. After purification, the purified enzyme was proved active. ResuLts reveal that the optmum temperature of this enzyme is 60C and optmum pH is 6.5 in biosynthetic reaction. After induction, the enzyme activity in crude extract of BL-21/pET28b-glnA is 83 times higher than that of original BL-21 extract. Mn2+ can obviously increase the activity and stability of this enzyme. Experiments show that the transformation efficiency of glutamate to glutamine is more than 95% by using glutamate, ammonium choloride and ATP as substrates.Because of the high cost from ATP, a system coupling glutamine synthetase with yeast for ATP regenaration was established. In this system, GS utilizes ATP released by yeast fermentation to synthesize glutamine. The good efficiency was achieved by treating yeast with 2% toluence to increase its permeability and with the presence of 250 mM glucose and 200 mM phosphate , the transformation efficiency of glutamate to glutamine by this system is more than 80%, the average yield of glutamine is 22g/L.