Prokaryotic Expression And Emzymatic Characterization Of Glutamate Decarboxylase From Lactobacillus Plantarum

?-aminobutyric acid?GABA? is an important neurotransmitter of inhibition,which has a series of important physiological functions.GABA is a functional food hygiene factor, and the preparation and application of GABA has attracted great attention for long time. Glutamate decarboxylase?GAD? is a key speed limited enzyme for production of GABA using the method of biocatalysis.Fermentation of GABA in traditional way has a low yield, and is not good way for large-scale production, while it is an economic way to produce GABA catalyzed by GAD in vitro. In order to gain enough GAD for GABA production, a recombined engineering bacteria E.coli BL21-p GEX-4T-gad B was constructed, high catalysis activity GAD had been got through process of induction expression and purification, and the enzyme characterition of recombined GAD had been studied in our research. The major experiment results were as follows:1. According to the gad B gene sequence of L.plantarum QL-14, a pair of specific primers were designed and synthesized, then the gene gad B of glutamate decarboxylase was amplified by PCR, recombinant plasmid p GEX-4T-gad B was constructed after target gad B connected into fusion expression vector p GEX-4T-3, and E. coli prokaryotic expression system was successfully built, after plasmid p GEX-4T-gad B transformed into E.coli BL21. The sequencing results showed that glutamate decarboxylase coding gene gad had the most similarty?99.6%? with that of L.plantarum WCFS1 published in Gene Bank.2. By bioinformatics analysis of cloned GAD, we found that ORF of the gene gad B is 1410 bp, encoding 469 amino acids. The molecular formula of recombined GAD was C₂₄₁₆H₃₆₅₈N₆₄₈O₆₉₁S₂₃ and the theoretically relative predicted molecular weight and isoelectric point was 53574.9u and 5.58 respectively. GAD was hydrophilic protein,and it had a structure domain of pyridoxal phosphate?PLP?-dependent aspartate aminotransferase superfamily. There was no signal peptides bind at the N-terminal of GAD which could transfer it outside a cell. Secondary structure showed that ?-helix 52.67%, extended strand 31.56%, beta-turn 7.25%, random coil 8.53% respectively. Based on molecular phylogenetic tree, we found that cloned GAD had the closest evolutionary relationship?99%? with Lactobacillus plantarum subsp.plantarum NC8.3. Exogenous gene gad B was induced and expressed by IPTG. Expression conditions was optimized, while the optimal concentration of IPTG was 1.0m M, and it was induced at a temperature of 16? over night so that most recombinant proteins were in the form of soluble protein. In addition,recombinant GAD was purified by means of affinity chromatography method. A Berthelot colorimetric method of determining purified GAD activity assay with hight sensitivity was proposed based on the principle of Berthelot. We had eliminated interruptions of substract and reagent?Tris, GSH?. The optimal concentration of substract was 0.02 M,and concentration of purified GAD was 0.626mg/m L of which had a specific activity of 9.9U/mg.4. The enzymatic properties showed that: recombined GAD had the highest activity when p H was 4.8 and temperature was about 40?, it still had a 80% activity when GAD was conducted 30 min at a temperature of 70?, it had almost lost none of its activity, after preserverd at 0? for a month. The optimal addition amount of coenzyme PLP was 0.005 m M. Fe²⁺, Ca²⁺, Mg²⁺, Cu²⁺ had increased the enzyme activity. Tween 20 acted as nonionic surfactant had increased activity of GAD mostly, whrereas it had an opposite effect for SDS acted as LAS. Isoamylol was a motivating organic solvent for recombined GAD. The michaelis constant of GAD showed that Km was 23.33 m M, Vmax was 1.133?M/min.