Identification and Purification of a hemolysin from Bacillus group by Zymography method

Hemolysin is an extracellular protein which is secreted by many pathogenic and non-pathogenic bacteria. This protein is mostly active on plasma membrane of red blood cells. The objective of this study was to extract hemolysin from bacillus strains producing it, measure the molecular weight, and identify the functional characteristics of this enzyme. At first, hemolysin-producing bacillus strains were identified and then the strongest one was isolated. The hemolysin produced by this bacterium was extracted using Zymography and the effect of lysing it on blood agar medium was studied. Finally, SDS-PAGE method was used to measure the molecular weight of hemolysin. A new method was applied for purification and analysis of properties of the produced hemolysin. Among the 8 studied strains, M3, with the access number of KC577596.1, was the best and strongest strain degrading blood on blood agar medium. The hemolysin produced by this strain was extracted and its lysis properties were studied. SDS-PAGE method showed that this strain of bacillus is able to produce two types of hemolysin with molecular weights of 13 and 23 kDa. According to the results, the studied strain, which was firstly isolated at Microbiology laboratories of University of Isfahan, contains a strong type of hemolysin with a high lysis power. Given the high power of lysis of this hemolysin and as M3 strain of bacillus is non-pathogenic, various applications of this protein can be used in industrial and medical sectors.