Alkaline protease from Neurospora crassa. Purification and partial characterization.

A simple purification procedure has been developed for the extracellular alkaline protease from Neurospora crassa. Key steps in the purification were: 1) the choice of gelatin as the protein inducer, which induces optimally at a much lower concentration than other commonly employed protein inducers; 2) heat treatment, during which the inducer is digested by the protease; and 3) a concentration step that eliminates the usual precipitation procedures and removes much of the digested protein inducer. These procedures were followed by routine ion exchange chromatography and gel filtration. The preparation was homogeneous, as determined by gel electrophoresis and ultracentrifugal analyses. A molecular weight of approximately 30,500 was determined by amino acid analysis, gel electrophoresis, and sedimentation equilibrium. The protease has 100% activity from pH 6.0 to 10.0, is heat labile above 45 degrees C, and susceptible to autodigestion. Hydrolysis of the beta chain from insulin indicates a preferential cleavage on the carboxyl group side of neutral and aromatic amino acids.