Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarumTaj-Apis362 for high gamma-aminobutyric acid production

• Published in: Microbial biotechnology Vol. 8; no. 4; pp. 623 - 632
• Main Authors: Tajabadi, Naser, Baradaran, Ali, Ebrahimpour, Afshin, Rahim, Raha A, Bakar, Fatimah A, Manap, Mohd Yazid A, Mohammed, Abdulkarim S, Saari, Nazamid
• Format: Journal Article
• Language: English
• Published: 01.07.2015
• Subjects: Lactobacillus; Lactobacillus plantarum
• ISSN: 1751-7915; 1751-7915
• DOI: 10.1111/1751-7915.12254

Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarumTaj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973mM, temperature 36 degree C, pH 5.31 and time 60h. Under the conditions, maximum GABA concentration obtained (11.09mM) was comparable with the predicted value by the model at 11.23mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L.plantarum to L.plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products. A full-length GAD gene was obtained by cloning the template DNA to pTZ57R/T vector. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The over-expression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by 7 folds compared to the original GAD activity.The optimal fermentation conditions for GABA production established using response surface methodology