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Improvement of stability and reaction behavior of enzymes in organic solutions by immobilization onto nano-porous supports

通过固定在纳米多孔载体上改善有机溶液中酶的稳定性和反应行为

基本信息

批准号：
09555256
负责人：
IMAMURA Koreyosi
金额：
$ 0.7万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

In this study, we intended to establish theoretical bases, which are required to realize enzymatic syntheses in organic solvents. We studied the synthetic reaction of aspartame precursor, Z-L-Asp-L-PheOMe, from respective amino acid derivatives (Z-Asp and PheOMe) catalyzed by thermolysin. First, we investigated various factors affecting stability of the immobilized thermolysin and analyzed its mechanism. Autolysis was found to be a main cause for inactivation in water-immiscible organic solvents and the immobilized enzyme tended to be inactivated in organic solvents with lower solubility of water. In particular, the immobilized was stable in tert-amyl alcohol probably because that aqueous phase around the enzyme immobilized is thin and as a result autolysis tended to be repressed. The method to predict the overall partition coefficient of the substrates containing acid and amine components was proposed, by taking into consideration the effect of ion-pair complexes formed between the substrates.The synthetic rate with the immobilized enzyme in an organic solvent, containing a small amount of water could be in principle estimated on the basis of the model for the biphasic reaction when the amount of enzyme loaded was not so much. Namely, in the synthetic reactions, the both components of the substrate are partitioned into the water phase inside the support, and then converted to the product by the action of the enzyme, and finally the product is transferred into the bulk organic phase.

在这项研究中，我们旨在建立在有机溶剂中实现酶促合成所需的理论基础。我们研究了在嗜热菌蛋白酶催化下，从各自的氨基酸衍生物（Z-Asp 和 PheOMe）合成阿斯巴甜前体 Z-L-Asp-L-PheOMe 的反应。首先，我们研究了影响固定化嗜热菌蛋白酶稳定性的各种因素并分析了其作用机制。发现自溶是在与水不混溶的有机溶剂中失活的主要原因，并且固定化酶在水溶解度较低的有机溶剂中倾向于失活。特别是，固定化酶在叔戊醇中稳定，这可能是因为固定化酶周围的水相很薄，因此自溶往往受到抑制。考虑底物之间形成的离子对络合物的影响，提出了预测含酸和胺成分的底物总分配系数的方法。固定化酶在含有少量水的有机溶剂中的合成速率原则上可以根据酶负载量不太多时的双相反应模型来估计。即在合成反应中，底物的两种组分均分配到载体内部的水相中，然后通过酶的作用转化为产物，最后产物转移到本体有机相中。