Purification and characterization of a novel extracellular protease from Beauveria bassiana

Abstract

An extracellular protease designated BBP (Beauveria bassiana protease) was purified from a B. bassiana isolate and subsequently characterized. It was produced in the late stages of growth in a gelatin medium, and was purified using a combination of ionexchange chromatography and preparative gel electrophoresis. Inhibition by phenylmethylsulphonyl fluoride and chymostatin indicates that BBP is a serine type with chymotrypsin activity. Substrate specificity indicates that the protease has elastase activity as well. The protease was stable at 25 °C and had an alkaline pH optimum (7.5–9.5). Another protease was purified from the same isolate that produced BBP, and was identified as Pr1 based on its isoelectric point, amino terminal sequence, substrate specificity and cuticle degrading ability. A comparison between the two showed that BBP had a lower isoelectric point (pl 7.5) than Pr1 (pl ≥ 10) and was 0.5 kDa smaller. The proteases also exhibited differences in substrate specificity with Pr1 being most active against Suc-Ala-Ala-Pro-Phe-pNA, and BBP being most active against MeOSuc-Ala-Ala-Pro-Met-pNA. The two proteases had equal cuticle-degrading activity and both were expressed in cuticle media, although the timing of their expression differed.