Summary auto-generated
This study describes the purification and characterization of a spore-lytic enzyme released from Bacillus cereus spores during germination. The enzyme, a 24 kDa protein with an amidase or peptidase function, was isolated from germination exudate and purified through multiple chromatographic steps. The enzyme caused loss of optical density and phase-darkening in coat-stripped spores, characteristic changes of spore germination, but showed minimal activity on isolated peptidoglycan. The purified enzyme had an N-terminal sequence of 19 amino acids (FSNQVIQRGASGEKVIELQ) and required relatively high ionic strength and non-ionic surfactants for stability. In its bound spore form, the enzyme was heat-resistant, but once solubilized, it became heat-sensitive and readily inactivated by thiol-reactive reagents. The enzyme's activity depended on salt concentration, with optimal activity around 30 mM NaCl, and it functioned optimally at neutral pH. Analysis of enzyme-degraded spores showed release of free amino groups but not reducing sugars, indicating peptide bond cleavage without polysaccharide degradation.
Key findings
- A 24 kDa spore-lytic enzyme is released into germination exudate during Bacillus cereus spore germination and causes characteristic refractility changes in coat-stripped spores
- The enzyme appears to be an amidase or peptidase that cleaves peptide bonds in spore cortex but does not degrade polysaccharides
- The solubilized enzyme is heat-sensitive and readily inactivated by thiol reagents, requiring high ionic strength and non-ionic surfactants for stability
- Enzyme activity depends on salt concentration (optimal ~30 mM), neutral pH, and involves thiol-sensitive sites critical for both enzyme function and spore germination
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Abstract
The exudate of fully germinated spores of Bacillus cereus IFO 13597 in 0.25 M sodium phosphate buffer, pH 7.0, was found to contain a spore- lytic enzyme. This enzyme was found to cause loss of absorbance in coat- stripped spore suspensions and phase-darkening of the spores but had minimal activity on isolated peptidoglycan substrates. The enzyme was purified in an active form and identified as a 24 kDa protein which is either an amidase or a peptidase. The amino-terminal 19 residues had the following sequence: FSNQVIQRGASGEKVIELQ. The spore-lytic enzyme retained its activity in a medium of a relatively high ionic strength containing a non-ionic surfactant such as nonaethyleneglycol n-dodecyl ether. This activity was optimum at a salt concentration of about 30 mM in assay buffer at neutral pH. In contrast to the enzyme in a spore- bound form, the enzyme in solution was shown to be heat-sensitive and was readily inactivated by thiol reagents.