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

publication date: Sep 30, 2007
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Journal of Apicultural Research
Vol. 46 (3) pp. 160-164
DOI
Date
September 2007
 
Article Title
 
Screening of secreted proteases of Paenibacillus larvae by using substrate-SDS-polyacrylamide gel electrophoresis
Author(s)
Jaroslav Hrabák and Karel Martínek
Abstract
This 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 metal loproteases, 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.
Keywords
Paenibacillus larvae, American foulbrood, honey bee disease, Apis mellifera, proteolytic enzymes, protease, proteinase, metalloprotease, substrate electrophoresis.
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