Cloning And Expression Of Glutamate Decarboxylase Gene From Lactobacillus Brevis

Glutamate decarboxylase (GAD, EC 4.1.1.15) is one kind of pyridoxal 5'-phosphate (PLP) dependent enzyme widely distributed in nature, can catalyze a-decarboxylation of L-glutamate or its salts into y-aminobutyric acid (GABA). GABA is a four-carbon non-protein amino acid found in living cells of various organisms and acts as a major cerebral neurotransmitter in the central nervous system of animals. Recently, much attention has been paid to its use as a functional bioactive ingredient in foods and pharmaceuticals, as well as its industrial production by biosynthesis with microorganism or GAD.Among different GABA producing microorganisms, lactic acid bacteria is a more promising biocatalyst for the preparation of GABA because of its high yields of GABA and reliable safety proved by its application in food. Currently, some Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus sakei have been isolated as candidates for preparation of GABA. We have also isolated a GABA-producing strain Lactobacillus brevis CGMCC 1306 which GABA-production capability was higher compared with other reported lactic acid bacteria. In this study, we try to clone, express its glutamate decarboxylase in recombinant E. coli and study its enzyme characters.Firstly, we studied the systematic bacteriology information of this high GABA-producing strain through its 16s rDNA sequence. Then three new glutamate decarboxylase (GAD) genes from Lactobacillus brevis CGMCC 1306 was cloned using homology-based degenerate PCR, which were designed based on bioinformatics analysis of existing GAD genes from Lactobacillus brevis. They were inserted into T-A cloning vector pMD 19-T Simple Vector and sequenced, the open reading frame of the cloned GAD genes comprised 1407,1440 and 1881 nucleotides.Secondly, these three GAD genes were cut from those T-A cloning vectors using restriction Enzymes and inserted into expression vector pET-28a(+), then three His×6 tagged recombinant GAD was expressed in E.coli BL21 (DE3). Determined with SDS-PAGE, their molecular weight were 53kDa (GAD1407),58kDa (GAD1440) and 66kDa (GAD 1881) respectively. GAD 1407 and GAD 1440 have found expressed as a soluble protein in the cell, but the activity can be detected only in GAD 1407 now. Then the expression conditions of GAD 1407 had been optimized to improve its production.Finally, GAD1407 was purified with Ni-NTA-agarose chromatography. The biochemical characterization of this GAD were studied, including its optimistic temperature, optimistic pH and its stability of temperature. Different from other reported GAD of Lactobacillus brevis, its activity is not dependent on activation by ammonium sulfate or addition of pyridoxal 5'-phosphate (PLP).In brief, this study provided a successful example of cloning GAD gene with degenerate primers directly from genomic DNA of Lactobacillus brevis. The expressed GAD displayed unique characteristics and activity of 67.62 U/ml enzyme solution, which may be useful for preparation of GABA as well as research on GAD structure-function relationship.