BIOCHEMICAL-PROPERTIES OF A NOVEL METALLOPROTEASE FROM STAPHYLOCOCCUS-HYICUS SUBSP HYICUS INVOLVED IN EXTRACELLULAR LIPASE PROCESSING

Two extracellular proteases from Staphylococcus hyicus subsp. hyicus, ShpI and ShpII, have been characterized. ShpI is a neutral metalloprot ease with broad substrate specificity; the gene has been cloned and se quenced. ShpII, characterized here, is mainly produced in the late log arithmic growth phase in contrast to ShpI, which is mainly produced in the late stationary growth phase. ShpII was purified from culture med ium of S. hyicus by ammonium sulfate precipitation and DEAE-Sepharose chromatography. The molecular mass, estimated by sodium dodecyl sulfat e-polyacrylamide gel electrophoresis, was 34 kDa. The temperature opti mum of ShplI was 55 degrees C, and the pH optimum was 7.4. ShpII, a ne utral metalloprotease, was strongly inhibited by zinc and calcium chel ators. The amino-terminal sequence of the active enzyme was similar to the corresponding region of a Staphylococcus epidermidis metalloprote ase. The substrate specificity of ShpII was similar to that of thermol ysin-like proteases, with the exception that ShpII also recognized aro matic amino acids. We demonstrated in vitro that ShpII, but not ShpI, cleaved the 86-kDa S. hyicus subsp. hyicus prolipase between Thr-245 a nd Val-246 to generate the mature 46-kDa lipase. Results of additional in vivo experiments supported the model that ShpII is necessary for t he extracellular processing and maturation of S. hyicus subsp. hyicus lipase.