A protease from the marine sponge Callyspongia schulzi

Aqueous extracts of 25 marine sponge species (from coral reefs of Papua New Guinea) were screened for proteolytic activity. Only one sponge, Callyspongia schulzi, showed remarkable activity. A protease hydrolyzing casein as well as the synthetic substrate alpha-N-benzoyl-L-arginine ethyl ester was isolated from the sponge extract by gel filtration, ion-exchange and HPLC absorption chromatography. The enzyme was homogenous in SDS-PAGE exhibiting an apparent molecular weight of 80 kDa. Its pH optimum was in the range of 9-11, it was remarkably heat-stable and was not inhibited by phenylmethane sulfonylfluoride, soybean trypsin inhibitor, aprotinin or alpha1-antitrypsin, but by EDTA and 1,10-phenanthroline suggesting properties of a metalloprotease. The protease hydrolyzed the oxidized insulin B-chain between Arg22-Gly23 and Lys29-Ala30, similar to trypsin.