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

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 k(cat)/K-m was determined to be 25.5 (mmol/L)(-1) s(-1) for L-Asp and 207.8 m(mol/L)(-1) s(-1) for alpha-ketoglutarate. With optimum temperature as 25 degrees C, the AATase may be a novel and special psychrophilic enzyme which exhibited a good thermal stability below 55 degrees C. The conserved active site residue of AATase was identified as Lys237 by phylogenetic analysis. Secondary structure of the enzyme includes alpha-helix (39.2%), beta-sheet (5.5%), beta-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.