Purification and characterization of thermostable aspartase from Bacillus sp YM55-1

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55- 1 and characterized in terms of activity and stability. The enzyme was isol ated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-excha nge and AF-Red Toyopearl chromatographies. The native molecular weight of a spartase determined by gel filtration was about 200,000, and this enzyme wa s composed of four identical monomers with molecular weights of 51,000 dete rmined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence o f magnesium ion at alkaline pH. At the optimum pH, the Ii, and V-max were 2 8.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times hi gher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degree s C, respectively. Eighty percent of the activity was retained after a 60-m in incubation at 55 degrees C, and the enzyme was also resistant to chemica l denaturants; 80% of the initial specific activity was detected in assay m ixtures containing 1.0 M guanidine hydrochloride. The purified enzyme share d a high sequence homology in the N-terminal region with aspartases from Ot her organisms, (C) 1999 Academic Press.