Abstract
Alanine racemase [EC 5. 1. 1. 1], which catalyzes the interconversion of D-, L-alanine, was partially purified from the muscle of the black tiger prawn Penaeus monodon using DEAE-cellulose, DEAE-Toyopearl, hydroxyapatite, Phenyl- and Butyl-Toyopearl, and Gel-Toyopearl HW column chro-matographies. The final enzyme preparation was not homogeneous but the purification was as high as about 17, 700-fold with a final yield of 2.5%.
Apparent molecular weight of the enzyme in its native form was 85, 000. The maximal activity was attained at 35-40°C and at around pH 8. 5. The alanine racemase was inactivated between 40 and 60°C and was also rather unstable during low temperature storage. The enzyme acts specifically on D-, L-alanine as substrates, but not on the other amino acids in the present assay conditions. The enzyme did not require pyridoxal 5'-phosphate or FAD as a cofactor. The enzyme was inhibited strongly with pyruvate and L-alanine, which are metabolites from D-alanine.
