Purification and Characterization of Alpha Toxin Clostridium perfringens Type A

Zahra Heshmati,Mohsen Fathi Najafi,Gholam Ronaghi

doi:10.21608/eajbsc.2020.115949

Abstract

Clostridium perfringens is a toxin-producing anaerobe with the ability to cause multiple diseases in humans and animals. One of the toxins produced by all strains of this bacterium is an alpha toxin (phospholipase C). This study aimed to purify and characterize alpha-toxin. C. perfringens type A was grown in an optimized medium, the culture supernatant was separated, and ammonium sulfate precipitated protein was subjected to ion exchange and gel filtration chromatography methods. The purification steps were monitored by SDS-PAGE and hemolysin activity for each sample and performed by the hemolysin assay method. The purified alpha-toxin appeared in a single band in 43 KDa. The enzymatic efficiency yield was about 88% with a specific activity of 69170 and 654 fold of purification. According to the results, alpha-toxin is Zn and Mg metal-enzyme dependent on the temperature and pH optimum of 40 °C and 6, respectively. The enzyme Km and Vmax values of 3.8 mg/ml and 0.19 were calculated as the enzyme in the presence of lecithin. The partially purified alpha toxin with specific characters can be used for different applications such as antibody production and determination of the anti-toxin rate in animal serum.

Highlights

Clostridium perfringens is an anaerobe residing in water, sewage, soil, and intestinal contents of humans and animals
All types of C. perfringens produce alpha toxin, but the highest production is reported in C. perfringens type A (Zückert et al, 1998)
Clostridium perfringens type A, was grown in optimized medium containing 3% (w/v) meat peptone, 0.5%(w/v) maltose, 0.5%(w/v) glucose,1%(w/v) yeast extract, 0.5% (w/v) sodium chloride, 0.25% (w/v) disodium phosphate in pH 7 under anaerobic condition at 37°C for 25 hours

Summary

Introduction

Clostridium perfringens is an anaerobe residing in water, sewage, soil, and intestinal contents of humans and animals. The bacterium has five strains according to the production of different toxins. All types of C. perfringens produce alpha toxin, but the highest production is reported in C. perfringens type A (Zückert et al, 1998). Alpha toxin is dangerous to human and animal health. This bacterium causes gas gangrene, food born poisoning, necrosis, and enterotoxaemia. The gas gangrene, caused by alpha toxin leads to bradycardia, shock, and eventually death(Cavalcanti et al, 2004)

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