Construction of a recombinant Escherichia coli BL21/ pET-28a-lpgad and the optimization of transformation conditions for the efficient production of γ-aminobutyric acid

Abstract:

In order to enhance γ-aminobutyric acid production from L-glutamate efficiently, we amplified the key enzyme glutamate decarboxylase (GAD) encoding gene lpgad from the strain Lactobacillus plantarum GB 01-21 which was obtained by way of multi-mutagenesis and overexpressed it in E. coli BL21. Then we purified GAD by Ni-NTA affinity chromatography and characterized the enzyme to optimize the conditions of the whole-cell transformation. The results show that the recombinant E. coli BL21 (pET-28a-lpgad) produced 8.53 U/mg GAD, which was increased by 3.24 fold compared with the GAD activity in L. plantarum. The optimum pH and temperature of the enzyme were pH 4.8 and 37 °C, respectively. At the same time, we found that Ca2+ and Mg2+ could increase the activity significantly. Based on this, we investigated γ-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions. Accordingly, the yield of γ-aminobutyric acid was 204.5 g/L at 24 h when the 600 g L-glutamate was added and the mole conversion rate had reached 97.92%. The production of γ-aminobutyric acid was improved by 42.5% compared with that under the unoptimized transformation conditions. This paved a way for the γ-aminobutyric acid construction of the industrial applications.