Extraction, purification and characterization of a peptidoglycan hydrolase from vegetative cells of Clostridium perfringens type A.

Abstract

A peptidoglycan hydrolase (PGH), which causes the germination and lysis of the coat-stripped spores of Clostridium perfringens, was extracted from perfringens vegetative cells. The enzyme was partially purified and showed an MW of 125,000 and a pI of 7.6. It was sensitive to a variety of monovalent and divalent cations and was inhibited by sulfhydryl-active agents. The sites of PGH action, the cell wall and the spore-cortex, were determined by the release of free amino groups from cell wall fragments and the release of soluble fluorescent products from fluorescamine-labelled cortical fragments of the C. perfringens spores. The enzyme appears distinct from the extracellular initiation protein produced by C. perfringens. PGH may be an autolysin with corticolytic activity.Clostridium perfringens is a common cause of human food poisoning in Western countries. Illness results from the ingestion of large numbers of vegetative cells which sporulate andd produce an enterotoxin in the intestine (30). The organism is commonly found in raw meat and poultry, frequently in the spore state. Temperature abuse of food allows germination and subsequent multiplication of vegetative cells.In previous papers we have described the extraction and isolation of a spore-lytic enzyme (SLE) from whole spores of C. perfringens (15, 16). And we have previously isolated and characterized an extracellular spore-lytic (germination) enzyme from vegetative cells of this organism (12,25,27,34,35). The latter enzyme causes the germination of coatless spores and hydrolysis of cortical fragments of this organism. The enzyme was termed initiation protein (IP) by Cassier and Sebald (6) who first detected it by its ability to promote recovery of heat-injured spores of this organism, presumably by replacing the inactivated, native, spore-germination enzyme.

Purification and characterization of an extracellular alpha-amylase from Clostridium perfringens type A

“…Thin-layer chromatography was employed for identification of oligosaccharides in starch hydrolysates (14). Isoelectrofocusing was performed as described by Labbé et al (18).…”

Sporulation-promoting ability of Clostridium perfringens culture fluids

“…These substances may result in a decrease in the guanosine triphosphate pool, which triggers sporulation. Several enzyme systems that are associated with sporulation (proteases, peptidoglycan hydrolase, and amylase) in C. perfringens have been studied (11,13,21,22). Here we report a sporulation-inducing factor produced by enterotoxin-positive (Ent ϩ ) and Ent Ϫ strains of C. perfringens.…”