Purification and characterization of alanine racemase from hepatopancreas of black-tiger prawn, Penaeus monodon

Abstract

Alanine racemase has been purified to homogeneity from the hepatopancreas of the black tiger prawn, Panaeus mondon. The enzyme depends on pyridoxal 5′-phosphate and consists of two subunits with an identical molecular weight of 41,000. V max and K m values for l-alanine are 460 μmol/min/mg and 50 mM, and those for d-alanine are 94 μmol/min/mg and 24 mM, respectively. The enzyme is highly specific toward alanine. Among other amino acids examined, only serine served as a substrate: l-serine was racemized at a rate of approximately 0.5% of that of l-alanine. The prawn enzyme is immunochemically distinguishable from the enzymes of Bacillus stearothermophilus and Schizosaccharomyces pombe, which resemble each other. The prawn enzyme is activated and stabilized by the presence of monovalent anions including chloride. This is consistent with the previous hypothesis (e.g. E. Fujita, E. Okuma, H. Abe, Comp. Biochem. Physiol. 116A (1997) 83–87) that d-alanine serves as an osmoregulator in marine and euryhaline animals.