Purification and Characterization of Thermostable Aspartase fromBacillussp. YM55-1

A thermostable aspartase was purified from a thermophileBacillussp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75°C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. UnlikeEscherichia coliaspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, theKmandVmaxwere 28.5 mM and 700 units/mg at 30°C and 32.0 mM and 2200 units/mg at 55°C, respectively. The specific activity was four and three times higher than those ofE. coliandPseudomonas fluorescensenzymes at 30°C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55°C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.