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Stabilization and its Mechanism of Enzyme by Adding Sugar Containing Polymers

添加含糖聚合物对酶的稳定作用及其机理

基本信息

批准号：
08458287
负责人：
NAKAMAE Katsuhiko
金额：
$ 4.67万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08458287/
关键词：
Enzyme Polymer Stabilization Sugar Containing Polymer Activity Sugar chain Denaturation Interaction 酸素糖保存

项目摘要

Enzyme is useful in industries such as medicals, foods, fibers. However, enzyme is easy to denaturate by an environmental changes, i.e., temperature, pH and so on. Therefore, the stabilization of enzyme is important in industry. Many studies have been reported on the stabilization of enzymes ; by using stabilizers including saccharides, polyhydric alcohols, and hydrophilic polymer such as poly (ethylene glycol). This study is aimed at investigating the stabilization of enzymes in the presence of sugar containing polymers and attending an insight into the mechanisms of enzyme stabilization. trypsin, L-lactate dehydrogenase, lysozyme, and alpha-amylase were found to be stabilized upon addition of sugar containing polymers. For example, the enzymic activity of native trypsin decrease to 40% by only standing at 30"*"C for 6hr, however, that increased to 60% in the presence of sugar containing polymers. The influence of the sugar containing copolymer, especially comonomer content, on enzyme stabilization was also discussed. It was found that the enzymic activity further increased to 75% for the trypsin at the above conditions. The mechanism of stabilization could be separated into two ways in this case. Trypsin is so called protease, where trypsin molecule digest each other. The presence of polymer simply prohibits the collision of enzyme molecules. Second, the presence of sugar containing polymer enhanced the enzymic reaction and affinity between enzyme and the substrate, judging from the kinetic parameters, Vmax and Km values. Isothermal microtitlation calorimetry, proton-nuclear magnetic resonance, differential scanning calorimetry, liquid chromatography are also used to investigate the stabilization mechanism.

酶在医药、食品、纤维等工业中有着广泛的应用。然而，酶容易因环境变化而变性，即，因此，酶的稳定性在工业生产中具有重要意义。已经报道了许多关于酶的稳定化的研究;通过使用稳定剂，包括甘油、多元醇和亲水性聚合物如聚（乙二醇）。本研究旨在研究含糖聚合物对酶的稳定作用，并对酶的稳定机理进行深入探讨。发现胰蛋白酶、L-乳酸脱氢酶、溶菌酶和α-淀粉酶在加入含糖聚合物后稳定。例如，天然胰蛋白酶仅在30 ℃下静置6小时，其酶活性就下降到40%，然而，在含糖聚合物存在下，其酶活性增加到60%。讨论了含糖共聚物，特别是共聚单体含量对酶稳定性的影响。结果表明，在上述条件下，胰蛋白酶的酶活进一步提高到75%。在这种情况下，稳定化的机制可以分为两种方式。胰蛋白酶又称蛋白酶，是胰蛋白酶分子相互消化的过程。聚合物的存在只是阻止了酶分子的碰撞。其次，从动力学参数Vmax和Km值来看，含糖聚合物的存在增强了酶促反应和酶与底物之间的亲和力。利用等温微量滴定量热法、核磁共振氢谱、差示扫描量热法、液相色谱等方法研究了其稳定机理。