Cloning.Expression Of Lactobacillus Plantarum Glutamate Decarboxylase And Preliminary Studying On The Enzymatic Properties

Gamma-aminobutyric acid (GABA) is one type of non-protein amino acid which is commonly found in various organisms such as plant, animal, and microorganism. GABA is an important neurotransmitter in the mammalian central nervous system and have antihypertension, sedative, anxiolytic, treatment of epilepsy, regulate hormone secretion, activation of liver and kidney, asthma control, anti-aging and other important physiological functions. So GABA can currently be used as an important functional fector used in food, medicine and feed industry.GABA is generated from L-glutamic acid under the action of catalytic decarboxylation catalyzed by decarboxylase (Glutamic acid decarboxylase, GAD) which is the only rate-limiting enzyme during this process.In this study, a high-yield GABA of Lactobacillus plantarum strain YM-4-3was isolated. The yield of GABA of this strain was up to 16.8 mM. Full length of GAD gene was achieved byPCR amplification, ligated into pMD 18-T Simple Vector, and sequenced. Nucleotide sequence analysis showed that sequence of GAD consisted of 1410 nucleotides encoding 468 amino acids, and a predicted molecular weight of 53.5 kDa. The deduced amino acid sequence was 100% similar with glutamic acid decarboxylase gene sequences from Lb. plantarum strain WCFS1 and Lb. plantarum subsp. plantarum ST-Ⅲ while it was only 28% similar with sequences from Lb. lactis.GAD gene from pMD18-T-gad recombinant plasmid were achieved by toe restriction enzyme Nde I and Eco RI. GAD gene was ligated to a pET-28a expression vector and transferred to E. coli BL21 to construct recombinant expression strain containing 6 x His tag.This recombinant strain was in logarithmic growth phase after 9h culture, in stable phase after 20h GABA concentration in the fermentation broth increases with culture and the final concentration was 30.4 mM which was 65% higher than in Lb. plantarum strain YM-4-3.The recombinant strain was induced to express GAD by IPTG The target protein was detected by SDS-PAGE protein electrophoresis. This strain could express amount of soluble protein GAD. The recombinant protein was separated and purified by Ni affinity column with specific activity of 37.6 U/mg, recovery rate of 78.4%, and the purification multiple of 10.7.The enzymatic properties of GAD purified in this study shows that, the optimum pH was 4.5; the optimal reaction temperature was 35 ℃, and the optimum PLP concentration was 150 μ mol/L. When the reaction temperature is over 65 ℃ and pH is more than 7.5, GAD completely lost activity.