Purification and Characterization of a Thermostable Neutral Metalloprotease I from Chloroflexus aurantiacus J-10-fl

Abstract

Chloroflexus aurantiacus J-10-fl was found to contain two types (protease I and protease II) of thermostable proteases which were separated by Butyl-Toyopearl 650M chromatography. Protease I was purified to electrophoretic homogeneity from the culture broth of C. aurantiacus J-10-fl. The molecular mass of protease I was estimated to be approximately 66 kDa by SDS-PAGE, and the value of approximately 66kDa was also obtained by the Hedrick-Smith method, indicating that protease I was a monomer. The isoelectric point was 6.2. Protease I activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, and o-phenanthroline. The optimum pH for the activity of protease I was around 8.0. Addition of Ca²⁺ increased the pH and heat stabilities of protease I. The activity was stable between pH 4.0–11.0 and up to 75°C, and the maximum activity was observed at 70°C in the presence of 2mM CaCl₂. Protease I was resistant to the treatment by denaturing reagents (8 M urea or 1% SDS) at pH 8.0 and 20°C for 24 h. The sites of cleavage. in oxidized insulin B chain by protease I were similar to those by other microbial neutral metalloproteases. Elastase activity of protease I was not detected.