|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Bacterial spore cortex-lytic enzymes (SCLEs), which act as N-acetylmuramyl-L-alanine amidase, were purified in an active form from the exudate of fully germinated spores of Bacillus cereus and Clostridium perfringens. The enzymes hydrolysed decoated spores and caused phase-darkening of the spores, but had minimal activity on isolated peptidoglycan substrates. They shared similar sensitivities to temperature, pH,ionic strenght, and a variety of chemicals. Both SCLEs were localized on the exterior of cortex layrs in the spore. However, analyzes of their genes showed a diversity of structures of germination-specific amidases. There was no homology in their amino acid alignments, except for a motif involved in binding of the enzymes to spore peptidoglycan. The C.perfringens amidase was synthesized in a form comprising N-terminal prepro-region, N-terminal pro-region, mature form and C-terminal pro-region, and an inactive precursor with N-terminal pro-region is precessed to produce an active mature enzyme. Such an activation process lacks in the B.cereus enzyme, and the enzyme was produced as a precursor posessing a cleavable signal sequence. Such a signal sequence was not found in the sequence of C.perfringens amidase. This indicates that there is species-spcific mechanism on translocation of the enzymes to reach final destination on spores. Thus, evolutionary divergence might lead to many cortex-lytic enzymes differing in the specificities and modes of action and in the mechanism of activation during germination. Furthermore, the fact that the enzymes are heat sensitive only when removed from intact spores indicate the presence of some heat protective mechanism for enzymes in rather peripheral location in the dormant spore, which is not dependent on the core environment of spores.