[Construction of a recombinant Escherichia coli BL21/ pET-28a-lpgad and the optimization of transformation conditions for the efficient production of gamma-aminobutyric acid].
In order to enhance gamma-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 showed 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 degrees C, respectively. At the same time, we found that Ca2+ and Mg2+ could increase the activity significantly. Based on this, we investigated gamma-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions. Accordingly, the yield of gamma-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 gamma-aminobutyric acid was improved by 42.5% compared with that under the unoptimized transformation conditions. This paved a way for the gamma-aminobutyric acid construction of the industrial applications.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Lingzhi
L
Tian
Meijuan
M
Xu
Zhiming
Z
Rao
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D005968:Glutamate Decarboxylase / Q000096:biosynthesis* / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism*; D048191:Lactiplantibacillus plantarum / Q000201:enzymology / Q000235:genetics; D011995:Recombination, Genetic; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis*
D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase