Cloning, expression and characterization of an aspartate aminotransferase gene from Lactobacillus brevis CGMCC 1306

• Main Authors: Sheng Hu, Xiang Zhang, Yi Lu, Yue-Cheng Lin, Dong-Fang Xie, Hui Fang, Jun Huang, Le-He Mei
• Format: Article
• Language: English
• Published: Taylor & Francis Group 2017-05-01
• Series: Biotechnology & Biotechnological Equipment
• Subjects: Aspartate aminotransferase; psychrophilic enzyme; phase diagram; phylogenetic analysis; Lactobacillus brevis
• Online Access: http://dx.doi.org/10.1080/13102818.2017.1304181
• ISSN: 1310-2818; 1314-3530

An aspartate aminotransferase (AATase) gene from Lactobacillus brevis CGMCC 1306 was cloned, which contains a 1182-bp open reading frame coding for 393 amino acids (41.43 kDa). When expressed in Escherichia coli BL21 (DE3), the recombinant AATase was purified and subsequently characterized. The recombinant AATase can catalyse the conversion of L-Asp to L-Glu, and the kcat/Km was determined to be 25.5 (mmol/L)−1 s−1 for L-Asp and 207.8 m(mol/L)−1 s−1 for α-ketoglutarate. With optimum temperature as 25 ˚C, the AATase may be a novel and special psychrophilic enzyme which exhibited a good thermal stability below 55 ˚C. The conserved active site residue of AATase was identified as Lys237 by phylogenetic analysis. Secondary structure of the enzyme includes α-helix (39.2%), β-sheet (5.5%), β-turn (8.8%), and random coil (36.5%) by circular dichroism spectral analysis. Phase diagram for the fluorescence data analysis showed that guanidinium chloride-induced unfolding of AATase involved at least one intermediate.