Kazuhito FUJII

Analysis of enzymic characteristic of recombinant aspartate racemase variant from blood shell Scapharca broughtonii

Ryo-hei YAMADA, Yoshiki KERA, Shouji TAKAHASHI

Aspartate racemase (AspR) from the blood shell Scapharca broughtonii (SbAspR) is the only pyridoxal 5�L-phosphate(PLP)-dependent AspR purified and characterized so far, and its activity is modulated by certain nucleotides. We successfully cloned the gene and characterized the recombinant enzyme expressed in and purified from Escherichia coli. The recombinant enzyme was, however, distinguished from the native enzyme from the shell by its certain biochemical properties, indicating the possibility of the presence of its variant in the shell. In this study, we newly isolated the gene encoding a possible AspR variant and expressed it and analyzed the product.
The AspR variant gene was isolated by PCR with total cDNA from the shell as a template. The variant gene encodes an SbAspR containing several different amio acid residues from the recombinant enzyme analysed previously. The variant was purified from E.coli crude extract by Blue Sepharose and Sephacryl S-100 chromatography. The purified variant had a specific activity of 9.89 U/mg protein and similar properties to those of the previous recombinant enzyme.