Biochemical Characteristics of an Alanine Racemase from Aeromonas hydrophil HBNUAh01

摘要

We reveal that the genome of Aeromonas hydrophila HBNUAhOl has an alanine racemase gene (alr-2); this gene encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain MB2795. The gene alr-2 was cloned and expressed in E. coli BL21 (DE3). The gene has an open reading frame (ORF) of 1230 bp encoding a protein of 369 amino acids with a calculated molecular mass of 40.39 kD. The amino acid sequence deduced revealed similarity of 98, 84 and 68% with alanine racemases from A. hydrophila ATCC7966, Aeromonas caviae_Ae398， Aeromonas vickers_b565， respectively. The optimal temperature and pH for enzyme activity was 37°C and pH 10. The enzyme had broad substrate specificity. The alanine racemase was shown to belong to the pyridoxal 5'-phosphate (PLP)-dependent family of enzymes that require a certain concentration of PLP for activity. The kinetic parameters K_m and V_(max) at 40°C of alanine racemase were 35.9 mM, 2898 units/mg for L-alanine and 15.36 mM， 1209 units/mg for D-alanine, respectively.