title: Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.
abstract: 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 plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa 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 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. 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.
authors.affiliation: Department of Food Science, Faculty of Food Science and Technology, University Putra Malaysia, Serdang, Selangor, 43400, Malaysia.|Department of Honey Bee, Animal Science Research Institute of Iran (ASRI), Karaj, Iran.
authors.forename: Naser
authors.initials: N
authors.lastname: Tajabadi
authors.affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, Serdang, Selangor, 43400, Malaysia.
authors.forename: Ali
authors.initials: A
authors.lastname: Baradaran
authors.affiliation: Department of Food Science, Faculty of Food Science and Technology, University Putra Malaysia, Serdang, Selangor, 43400, Malaysia.
authors.forename: Afshin
authors.initials: A
authors.lastname: Ebrahimpour
authors.affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, Serdang, Selangor, 43400, Malaysia.
authors.forename: Raha A
authors.initials: RA
authors.lastname: Rahim
authors.affiliation: Department of Food Science, Faculty of Food Science and Technology, University Putra Malaysia, Serdang, Selangor, 43400, Malaysia.
authors.forename: Fatimah A
authors.initials: FA
authors.lastname: Bakar
authors.affiliation: Department of Food Science, Faculty of Food Science and Technology, University Putra Malaysia, Serdang, Selangor, 43400, Malaysia.
authors.forename: Mohd Yazid A
authors.initials: MY
authors.lastname: Manap
authors.affiliation: Department of Food Science, Faculty of Food Science and Technology, University Putra Malaysia, Serdang, Selangor, 43400, Malaysia.
authors.forename: Abdulkarim S
authors.initials: AS
authors.lastname: Mohammed
authors.affiliation: Department of Food Science, Faculty of Food Science and Technology, University Putra Malaysia, Serdang, Selangor, 43400, Malaysia.
authors.forename: Nazamid
authors.initials: N
authors.lastname: Saari
mesh_terms: D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D048191:Lactiplantibacillus plantarum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids; D013696:Temperature; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase