干酪乳杆菌自溶酶AclB的性质、功能及调控机理研究

Autolysins are a species of cell wall hydrolases leading to bacterial lysis. Timely autolysis of Lactobacillus casei was in favor of acceleration of cheese ripening during its manufacture. The process could be interpreted by the release of intracellular esterases and peptidases, and subsequently the flavor development caused by amino acid metabolism..Currently, there were very few studies concerning the identification and characterization of lactobacillus autolysins, and moreover, their controlling mechanisms remained unexplored. Given the context, a novel autolysin AclB, as well as a putative regulatory component WalK’/WalR’, was identified from Lb. casei BL23 strain. It was then reasonable to hypothesize that the expression of AclB was strictly regulated by WalK’/WalR’. .The systems involving controllable autolysis and membrane protein expression had been established during our previous studies, which were supposed to provide the essential technical tools and thoughts with the current project. Therefore, to test the aforementioned hypothesis and also investigate the enzymatic properties, physiological functions and application potential of AclB, a series of studies including inactivation and complementation, specificity determination, construction and application of controlled lysis vector, overexpression and purification of WalR’/WalK’ as well as in vivo and in vitro binding assays needed to be carried out. .These investigations should be expected to shed new light on the regulatory mechanisms of lactobacillus autolysins, further paving the way for their potential application in cheese manufacture.

自溶酶是细菌释放出的一种细胞壁水解酶，作用于细胞壁导致细胞破裂死亡。奶酪生产中的后成熟即是利用发酵菌株自溶后释放出胞内酶，从而将大分子物质水解形成风味物质。干酪乳杆菌是奶酪发酵的重要菌株，比较基因组发现干酪乳杆菌BL23能编码产生一种自溶酶AclB及一种潜在的调控元件WalK’/WalR’，但该菌株如何调控其自溶酶表达并通过其自溶对奶酪成熟施加影响，此研究目前尚未见诸报道。本项目拟以干酪乳杆菌BL23为材料，利用基因敲除、酶谱分析探究自溶酶AclB的性质、功能及用于奶酪模型实验的可行性；利用乳酸乳球菌为宿主，构建膜蛋白表达载体，大量制备调控元件WalK’/WalR’；在此基础上，利用免疫共沉淀、凝胶阻滞分析等技术，体内外分析WalK’/WalR’蛋白与AclB启动子的结合能力，揭示WalK’/WalR’对AclB的调控机制，为实时控制干酪乳杆菌裂解并提高奶酪生产效率提供理论依据。

自溶酶是细菌释放出的一种细胞壁水解酶，作用于细胞壁导致细胞破裂死亡。奶酪生产中的后成熟即是利用发酵菌株自溶后释放出胞内水解酶，从而将大分子物质水解形成风味物质。干酪乳杆菌是奶酪发酵的重要菌株，比较基因组发现干酪乳杆菌BL23能编码产生一种自溶酶AclB及一种潜在的调控元件WalK’/WalR’，但该菌株自溶酶的特性和功能尚未得到研究。此外，干酪乳杆菌BL23作为一种原宿主和定殖终点均在胃肠道的益生菌，其是否可通过对肠道菌群的重塑而影响机体正常的生理活动是我们感兴趣的课题。同样，上述交互作用是否为自溶酶等细胞表面蛋白所介导同样具有重要的研究价值。.本研究以干酪乳杆菌BL23及其他代表性的乳酸菌株为材料，利用基因敲除、酶谱分析探究自溶酶AclB的性质、功能及用于奶酪模型实验的可行性；利用乳酸乳球菌为宿主，构建膜蛋白表达载体，大量制备调控元件WalK’/WalR’；以干酪乳杆菌BL23为肠道定殖益生菌进行动物实验，探究该菌定殖对学习记忆损伤的修复效应及该效应对自溶酶AclB的依赖性。.研究结果表明：AclB是干酪乳杆菌BL23的主自溶酶，具有显著的肽聚糖水解活性，且最适作用pH为5.0；AclB突变菌呈现出长链状，指示出细胞分裂后分离缺陷的特征，表明AclB的自溶性质在干酪乳杆菌细胞分离过程中起到重要的介导作用；以AclB为关键元件构建的可控裂解表达系统可有效促进干酪乳杆菌适时裂解及奶酪生产中的胞内酶释放；利用Mistic膜蛋白过表达载体在乳酸乳球菌中大量表达膜蛋白WalK，并对WalK/R完成了分离纯化；WalK/R的条件突变未对自溶酶AclB的表达产生明显影响；干酪乳杆菌BL23的肠道定殖能够显著挽救SD大鼠的学习记忆损伤，该过程伴随着明显的肠道菌群重塑过程，并与脑中海马区EZH2分子的表达水平密切相关；上述益生菌的有益效应由AclB蛋白所介导，因其特异突变菌无法完成同样的修复过程。.本课题全面研究了干酪乳杆菌主自溶酶AclB的性质、功能以及在奶酪生产和益生菌定殖方面的作用，为实时控制干酪乳杆菌裂解以及提高奶酪生产效率奠定理论基础。