Purification and characterization of an alkaline proteinase produced by Pimelobacter sp. Z-483

An extracellular alkaline proteinase was purified from the culture filtrate of Pimelobacter sp. Z-483. The molecular mass of the purified enzyme was estimated to be approximately 23 kDa by SDS-PAGE and 27 kDa by gel filtration. The optimum pH and temperature for the hydrolysis of casein were approximately 9 and 50°C, respectively. The enzyme activity was strongly inhibited by 1,10-phenanthroline and mercury chloride, but not by EDTA, phosphoramidon and Zincov. The amino terminal sequence of the enzyme was similar to that of an alkaline elastase (MAP1) from Myxococcus xanthus. However, the specificity of the alkaline proteinase in the cleavage of the oxidized insulin B-chain was different from those of the Myxococcus enzyme and animal elastase.