Highly thermostable neutral protease from Bacillus stearothermophilus

Abstract

Bacillus stearothermophilus MK232, which produced a highly thermostable neutral protease, was isolated from a natural environment. By several steps of mutagenesis, a hyper-producing mutant strain, YG185, was obtained. The enzyme productivity was twice as much as that of the original strain. This extracellular neutral protease was purified and crystallized. The molecular weight of the enzyme was 34,000 by SDS-polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the enzyme activity were 7.5 and 70°C, respectively, and the enzyme was stable at pH 5–10 and below 70°C. The thermostability and specific activity of the new protease are around 10% and 40% higher than those of thermolysin (the neutral protease from Bacillus thermoproteolyticus), respectively. The enzyme was inactivated by EDTA, but not by phenylmethylsulfonyl fluoride. These results indicate that the enzyme is a highly thermostable neutral-(metallo)protease.