SALIVARY AMYLASE--MICROBIAL INTERACTIONS AND HYDROXYAPATITE BINDING

唾液的成分可以在定居和繁殖过程中发挥重要作用 口腔细菌的代谢。一种丰富的唾液成分，具有 最近被证明与口腔绿色杆菌相互作用的链球菌是唾液 淀粉酶。事实上，淀粉酶现在被认为至少拥有三个 生物功能：1)复合碳水化合物的水解性；2)结合 3)与羟基磷灰石(HAP)结合。的目标是 目前的应用是为了理解三者的结构基础 口腔中淀粉酶的功能。这将通过以下方式实现 从唾液腺总mRNA中扩增人唾液淀粉酶基因 使用聚合酶链式反应(聚合酶链式反应)。扩增的淀粉酶cDNA会 在大肠杆菌系统中克隆和表达，并获得具有生物活性的重组 人唾液淀粉酶的纯化及其与天然淀粉酶的比较 酶活性、链球菌结合、HAP结合和继发性 用圆二色谱(CD)分析了其结构。这是经过优化的 然后使用表达系统来表达其中的淀粉酶 突变是使用特定残基的定点突变产生的 与酶功能有关。这些突变的淀粉酶将被比较 对野生型蛋白的酶结合和链球菌结合 活动。基于已发表文献的唾液淀粉酶分子模型 其他淀粉酶的氨基酸序列和可利用的晶体结构数据 将被构建并用于解释现场定向的效果 生物功能上的突变。最后，的HAP结合域 唾液淀粉酶将使用淀粉酶的选择性碎片化来定位 固定在HAP上。一旦确定，重组淀粉酶cDNA包含 该区域的突变将按照描述的那样构建和表达 上面。然后将对这些突变的淀粉酶进行测试，以确定它们是否具有 促进细菌对羟基磷灰石的黏附以及酶活性和 链球菌结合。对淀粉酶功能的了解可能会使 具有增强的酶和细菌结合的淀粉酶类似物的设计 活性，但结合HAP的能力减弱。这样的构造可能会 在促进口腔细菌清除的同时 抑制细菌对牙齿的黏附。这样的代理人的可用性 作为生物相容和有效的成分，可能被证明是有用的 抗菌斑剂或人工唾液。

Components of saliva can play an important role in the colonization and metabolism of oral bacteria. One abundant salivary component that has recently been shown to interact with oral viridans streptococci is salivary amylase. In fact, amylase is now thought to possess at least three biological functions: 1) hydrolysis of complex carbohydrates; 2) binding to bacteria; and 3) binding to hydroxyapatite (HAP). The goal of the present application is to understand the structural basis of the three functions of amylase in the oral cavity. This will be accomplished by amplification of human salivary amylase cDNA from total salivary gland mRNA using the polymerase chain reaction (PCR). the amplified amylase cDNA will be cloned and expressed in an E. coli system and the bioactive recombinant human salivary amylase purified and compared to native amylase for enzymatic activity, streptococcal binding, HAP binding and secondary structure by circular dichroism (CD) spectroscopy. This optimized expression system will then be used to express amylases in which point mutations are produced using site-directed mutagenesis of specific residues implicated in enzymatic function. These mutant amylases will be compared to the wild type protein for enzymatic and streptococcal binding activities. A molecular model of salivary amylase based on the published amino acid sequence and available crystal structure data of other amylases will be constructed and used to interpret effects of the site-directed mutations on the biological functions. Finally, the HAP binding domain of salivary amylase will be mapped using selective fragmentation of amylase immobilized on HAP. Once identified, recombinant amylase cDNAs containing mutations in this region will be constructed and expressed as described above. These mutant amylases will then be tested for their ability to promote bacterial adhesion to HAP a well as for enzymatic activity and streptococcal binding. An understanding of amylase function may allow the design of amylase analogs having enhanced enzymatic and bacterial binding activities but diminished ability to bind HAP. Such a construct might promote bacterial clearance from the oral cavity while simultaneously inhibiting bacterial adhesion to teeth. The availability of such an agent may prove useful as a component of biologically compatible and efficacious anti-plaque agents or artificial salivas.