PURIFICATION AND CHARACTERIZATION OF TRYPSIN FROM AN ENTOMOPATHOGEN, NOMURAEA-RILEYI NRRL-13755

Nomuraea rileyi isolate NRRL-13755 produced a large amount of trypsin enzyme when cultured on basal salt medium containing 1% (w/v) gelatin. The trypsin was purified nearly 60-fold, with a recovery of about 13% of the initial activity from the culture supernatant. This protease e xhibited a remarkably high specific activity of nearly 370,000 IU/mg p rotein. The native molecular weight was estimated by gel permeation ch romatography to be 30 kDa, and the subunit molecular weight was determ ined to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel e lectrophoresis (SDS-PAGE). The pH and temperature optima were determin ed to be 8.5 and 35-degrees-C, respectively. With a relative trypsin a ctivity of 100%, this purified preparation showed about 10% chymoelast ase and nearly 50% chymotrypsin activity. Metal-chelating agents such as EDTA and EGTA at 2 mm inhibited the enzyme activity by 40%, whereas N-carbobenzoxy-glyCyl-L-phenylalaninamide (CBZ-gly-phe-NH2) (2 mm) an d DTT (2 mm) had no effect on activity. Trypsin inhibitor from turkey egg-white at 100 mug/ml strongly inhibited the enzyme activity.