Purification and Characterization of Amino Acid Racemase with Very Broad Substrate Specificity from Aeromonas caviae

Abstract

A bacterium which grows in a medium containing d-serine as a sole nitrogen source has been isolated from soil and identified as Aeromonas caviae. The bacterium had a high enzyme activity catalyzing racemization of various amino acids. The enzyme, purified to homogeneity by polyacrylamide gel electrophoresis and ultracentrifugation, has a molecular weight of about 76,000, and is composed of two subunits identical in molecular weight (40,000). The results of enantiomeric analysis of the reaction product and kinetic examination of the reaction indicate that the enzyme catalyzes the complete racemization of substrates. The enzyme has absorption maxima at 280 and 420 nm, and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. The holoenzyme is resolved to the apoenzyme by treatment with hydroxylamine, and reconstituted by the addition of pyridoxal 5′-phosphate. The very broad substrate specificity of the A. caviae amino acid racemase is comparable to that of the Pseudomonas striata enzyme. However, they share no common antigenic determinants.