Yoshinori Muroki

Purification and characterization of bivalve mollusk Scapharca broughtonii aspartate racemase expressed in E. coli

Ryo-hei YAMADA, Yoshio KERA, Shouji TAKAHASHI

Aspartate racemase (AspR) from blood shell Scapharca broughtonii (SbAspR) is the only pyridoxal 5'-phosphate-dependent AspR purified and characterized so far. The enzyme activity is markedly modulated by some nucleotides. Recently, the SbAspR gene was cloned and expressed in Escherichia coli. In the present study, we purified and characterized the recombinant enzyme expressed in E. coli.
The recombinant aspartate racemase was purified by Blue Sepharose and Sephacryl S-100 chromatography. The purified recombinant enzyme exhibited similar properties to those of the enzyme from S. broughtonii as follows: The recombinant enzyme is a homodimer as indicated by its behavior on gel filtration; its absorption spectra and inhibitors show that it is a PLP-dependent enzyme; its optimum pH and temperature are pH 8.5 and 35��C, respectively; it is strictly specific for aspartate; its activity is modulated by nucleotides. However, it exhibited some different properties from those of the native enzyme: The activity of recombinant enzyme is enhanced in a simple saturating manner with increasing AMP concentration, which, in contrast, becomes progressively less effective above 6 mM for the native enzyme; the affinity of the recombinant enzyme for ATP was much higher than that for the native enzyme, and inhibition of the recombinant enzyme by 10 ��M ATP was comparative to that of the native enzyme by 1 mM ATP.