Cloning, sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain CGMCC 1306

In previous work, our research group isolated and bred a gamma-aminobutyric acid (GABA)-producing microorganism, CGMCC 1306, from fresh unpasteurized milk and found that the strain possessed high glutamate decarboxylase (GAD) activity. However, the cells of the strain grew poorly during submerged fermentation, with the highest dry cell weight only 2.78 g/L. To enhance the efficiency of GABA biosynthesis and achieve GAD production in high cell-density fermentations, the gene from CGMCC 1306 was cloned using degenerate PCR. Nucleotide sequencing analysis showed that the cloned gene comprised 1407 bp and encoded a 468-amino acid protein. The gene with a hexa-His tag was inserted into pET-28a(+) and expressed in BL21. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the recombinant protein product had a molecular weight of approximately 53 kDa, which corresponded to the predicted size of the deduced protein (53.47 kDa). High-performance liquid chromatography analysis showed that the purified recombinant protein was capable of catalyzing alpha-decarboxylation of -sodium glutamate into GABA, which confirmed that the protein was derived from a gene. GAD activity was the highest at pH 4.8 and 48A degrees C. Using Lineweaver-Burk plots, the and of the recombinant enzyme were 10.26 mM and 8.86 U/mg, respectively. Its activity was not dependent on the addition of pyridoxal 5'-phosphate. These results strongly suggest that cloning of the gene is not only of interest to researchers in terms of affecting GAD production by protein engineering strategies, but that it may increase GAD production by high cell-density fermentation and enhance the efficiency of GABA biosynthesis.