Study on the activation mechanism of the spore-lytic enzyme (s) during germination of Bacillus cereus spores.

1.鉴定了蜡状芽孢杆菌和枯草芽孢杆菌孢子萌发过程中分解孢子皮质的酶(Sleb)，并克隆了相应的基因(Sleb)。蜡样芽孢杆菌野生型和突变型Slib在大肠杆菌中的表达表明，其C末端独特的半胱氨酸残基对其酶活性具有重要的功能。Slib基因的缺失显著影响了L-丙氨酸介导的枯草杆菌孢子萌发过程，表明该酶与通用发芽剂L-丙氨酸相对应。从蜡样芽胞杆菌孢子中分离纯化出一种新的类枯草杆菌蛋白水解酶，并对其性质进行了研究。该酶以静电的方式结合在孢子表面，但孢子中的酶损失并不影响孢子萌发，说明该酶不参与孢子萌发过程。然而，该酶具有较高的比活力和对HgCl2等SH试剂的敏感性。对该酶编码基因的克隆表明，该酶属于枯草杆菌蛋白亚家族。考察了蔗糖单烷基酸酯(SE)的抗菌活性。SE结合在蜡样芽孢杆菌孢子表面，结合的数量似乎与SE的抑菌活性有关。孢子萌发过程中SE的抑菌活性丧失是由于SE被孢子释放的酯酶分解所致。

1. Enzymes (SleB) that hydrolyze spore cortex during germination of Bacillus cereus and Bacillus subtilis spores were identified and corresponding genes (sleB) were cloned.2. Expression of wild-type and mutant SleB of B.cereus in Escherichia coli revealed that the unique cysteine residue at C-terminal portion SleB is functionally important for its enzymatic activity.3. Deletion of sleB significantly affected the L-alanine-mediated germination process of B.subtilis spores, indicating the enzyme corresponds to the universal germinant, L-alanine.4. Novel subtilisin-like protease was purified from B.cereus spores and characterized. The protease binds to the surface of spores electrostatically, but loss of enzyme from spores did not affect spore germination, indicating that the protease is not involved in the germination process. However, the protease is unique in its high specific activity and sensitivity to SH-reagents, such as HgCl_2. Cloning of the gene encoding the protease revealed the enzyme is categorized into subfamily of subtilisin.5. Antimicrobial activities of sucrose monoalkylates (sugar ester, SE) were examined. SEs bind to the surface of B.cereus spores, and numbers of binding seems to relate to the antimicrobial activity of SE.Loss of the antimicrobial activity of SE during spore germination was revealed to be caused through the lysis of SE by esterase released from spores.