Tajabadi Naser, Baradaran Ali, Ebrahimpour Afshin, Rahim Raha A, Bakar Fatimah A, Manap Mohd Yazid A, Mohammed Abdulkarim S, Saari Nazamid
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.
Microb Biotechnol. 2015 Jul;8(4):623-32. doi: 10.1111/1751-7915.12254. Epub 2015 Mar 10.
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.
γ-氨基丁酸（GABA）是一种重要的生物活性化合物，由微生物通过谷氨酸脱羧酶（GAD）将谷氨酸脱羧生物合成。在本研究中，通过将模板脱氧核糖核酸克隆到pTZ57R/T载体中获得了全长GAD基因。GAD基因的开放阅读框显示，克隆的基因由1410个核苷酸组成，编码一个469个氨基酸的蛋白质。为了提高GABA的产量，将GAD基因克隆到pMG36e-LbGAD中，然后在植物乳杆菌Taj-Apis362细胞中表达。通过SDS-PAGE和GAD活性证实了过表达，显示出一种53 kDa的蛋白质，其酶活性比原始GAD活性增加了7倍。使用响应面法确定的GABA生产的最佳发酵条件为谷氨酸浓度497.973 mM、温度36°C、pH 5.31和时间60 h。在这些条件下，获得的最大GABA浓度（11.09 mM）与模型预测值11.23 mM相当。据我们所知，这是首次成功克隆（回克隆）并在植物乳杆菌细胞中过表达植物乳杆菌LbGAD基因的报道。重组乳杆菌可用作发酵过程中直接加入食品体系以生产富含GABA产品的发酵剂。
