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

本研究旨在为在有机溶剂中实现酶促合成奠定理论基础。研究了热裂解酶催化下从氨基酸衍生物Z-Asp和PheOMe合成阿斯巴甜前体Z-L-Asp-L-PheOMe的反应。首先，考察了影响固定化热裂解酶稳定性的各种因素，并对其作用机理进行了分析。在不溶于水的有机溶剂中，自溶是导致酶失活的主要原因，而在水的溶解度较低的有机溶剂中，固定化酶往往失活。特别是，固定化的酶在叔戊醇中是稳定的，可能是因为固定化的酶周围的水相很薄，因此自溶倾向于被抑制。通过考虑底物间形成的离子对络合物的影响，提出了一种预测含酸胺类底物总分配系数的方法，在酶负载量不大的情况下，根据两相反应模型，原则上可以估算固定化酶在含少量水的有机溶剂中的合成速率。也就是说，在合成反应中，底物的两个组分被分配到载体内部的水相中，然后在酶的作用下转化为产物，最后产物转移到主体有机相中。

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.