Purification and characterization of clostridium perfringense type A alpha toxin

Introduction and Objectives: Clostridium perfringens is a gram positive, rod shape anaerobic, spore forming pathogenic bacterium with the ability to produce different bacterial exotoxins. The alpha toxin from clostridium perfringens type A is a metallophospholipase that causes food born disease marked by diarrhea and abdominal discomfort or even death. Materials and Methods: The bacterium source was taken from Mashhad Razi vaccine and serum researches institute. For obtaining maximum bacterial growth and toxin production, different media were formulated and applied as growth media. The sources of nitrogen, carbon, minerals, and vitamins were subjected in the process. in addition, time and temperature conditions were optimized. During growth optimization, the amount of toxin was monitored with hemolysis and lecithinase assay and protein quantification. After optimization, the purification was performed with salting out, gel filtration and ion exchange chromatography and in each step the yield of toxin and its purity was controlled with SDS-PAGE and hemolysin activity. The zymography method was used for activity and structure of the protein. The selected medium with the highest toxin production yield consists of 0.5% carbon source, 4% nitrogen source, 0.2% trace elements and 0.25%phosphate buffer. The maximum activity of enzyme was in the presence of zinc and magnesium as metal sources. The SDS-PAGE results were shown that enzyme was successfully purified as a single band with estimated molecular weight 43 KD. Results: According to the results, the optimized culture medium and growth conditions for alpha toxin production were applicable for vaccine production. Also, the purified toxin can be used in diagnostic application and vaccine researches.

Keywords:

Clostridium perfringens , alpha toxin , enzyme , optimized , purification , characterization chromatography.