Screening of secreted proteases of Paenibacillus larvae by using substrate-SDS-polyacrylamide gel electrophoresis

Abstract

SummaryThis study is focused on a primary proteolytic screening of the secreted products of P. larvae cultures by means of substrate SDS-PAGE and using specific inhibitors for the determination of protease classes. Two- and five-day cultures (cultivation medium: MYPGP broth) were used for primary screening. Proteolytic activity was detected only in gels containing gelatin in the area of 87, 74 and 42 kDa in two-day culture and in the area of 87,74,42 and 40 kDa in five-day culture. By using various protease inhibitors, it was considered that the 87 and 74 kDa stable proteases (most active at pH 7) presented in two-day and five-day culture can be metalloproteases, by virtue of their sensitivity to EDTA, 1, 10-phenantroline and EGTA (metal chelators) in standard concentrations, and by the lack of sensitivity to inhibitors of the other classes of protease. The 40 kDa protease in two-day culture was inhibited also by standard concentration of metal chelators, and was most active at pH 6. The 40 and 42 kDa proteases in five-day culture (most active also at pH 6) were inhibited by 1, 10- phenantroline in 2 mM concentration; the EDTA partially inhibited them at 8 mM concentration. All proteases were partially inhibited by 5 mM DTT in incubation buffers. On the other hand, the 2-mercaptoethanol in sample buffer did not alter the proteolytic activity. The inhibited 87 and 74 kDa proteases (by means of EDTA) can be reactivated by Ca2+ and partial by Fe2+ ions. The 40 kDa proteases in twoday culture can be reactivated by Zn2+ ions. No proteolytic activity was detected on the casein substrate gels.