智能多液相界面酶催化体系的构建及其微观调控机制研究

Interfacial enzymes have attracted widespread attention as powerful tools for the synthesis of bio-chemicals using two immiscible reactants. However, its industrial application has been limited due to the low catalytic efficiency in the conventional O/W two-phase reaction system, in which the catalytic activity of the enzymes can be dramatically affected by the low interfacial area and the product inhibition occurring in both the phases. Recently, although a great deal of studies have been attempted to develop a novel interfacial enzyme reaction system with high efficiency, but none of these have appeared good enough for large-scale applications. A multi-liquid phase system (MLPS) was designed particularly for carrying out interfacial enzyme reactions therein, which has appeared advantageous due to the high catalytic efficiency, facile recovery/reuse of the catalysts and convenient separation/isolation of the products. However, the enzyme catalytic rates in the system are too fast, which limits its application because of the difficulty of controlling the reaction in some complex reactions. In our recent study, in some specific phase compositions in some special MLPSs, a turning point of the phase distribution could be discovered. In the turning point, the phase distribution can be turned over and the catalytic efficiency of the enzyme can be dramatic changed with small changes in some controllable factors, such as pH and temperature of MLPS. As a result, at the turning point of MLPS, the reaction can be “intelligent” controlled by changing the controllable factors. Therefore, the study on MLPS has important theoretical significance and application value. However, its application was limited because almost no study on the basic theory was done. Enzymatic resolution has received extensive attention due to the huge demand in the medical field, but its application was restricted due to some problems, such as low efficiency and difficult control in the production process. For the project, the chiral resolution of mandelic acid (an important chiral drug intermediate) was used as model reaction to study the catalytic mechanisms, regulative rule and molecular mechanism of enzymes of MLPS. This study can provide theoretical support for the application of the novel enzymatic reaction system in the field of nonaqueous enzymology.

界面酶具有催化非互溶底物反应的独特特性，获得了广泛的关注。但该类酶仅能在界面表现活力，且同时受到两相中产物的抑制，导致传统双相体系催化效率低，严重制约其应用。多液相体系是申请人首次开发的一种新型催化体系，既可大幅提高反应速率，又可同步实现产物分离和酶的回用，具有重要的应用价值。但是该体系过快的反应速度，导致在某些复杂反应中，反应难于控制的难题，限制了其应用范围。近期我们发现，某些特殊体系，存在相分布高敏性受控于pH值等因素而发生突然改变的“拐点”，此时，酶的催化特性也会出现相应的“受制拐点”，形成新型“智能多液相体系”。通过该体系有望对反应进行智能调控，具有重要的理论意义和应用价值。酶法手性拆分生产手性药物受到了广泛重视，但产物抑制导致的效率低，难于控制等问题制约了其发展。本项目拟以酶法手性拆分扁桃酸为模型，考察该体系的催化特性、分子机制及调控规律，为非水相酶学的新型催化体系提供理论支撑。

界面酶具有催化非互溶底物反应的独特特性，获得了广泛的关注。但该类酶仅能在界面表现活力，且同时受到两相中产物的抑制，导致传统双相体系催化效率低，严重制约其应用。多液相体系既可大幅提高反应速率，又可同步实现产物分离和酶的回用，具有重要的应用价值。但是该体系存在某些复杂反应中反应难于控制以及选择性提高量难于满足工业要求等难题，限制了其应用范围。本课题首先构建和筛选了多个高效率的多液相酶催化体系。并验证了其在脂肪酶水解拆分扁桃酸、萘普生、苯乙醇等多个手性醇或酸以及磷脂酶选择性水解去除油中磷脂等多种酶催化反应都有较好的适用性，研究表明在该类体系中，最优条件下，绝大部分酶（大于90%）均可富集于富酶相并便于重复利用，并且酶的催化效率和选择性均可大幅提升。在此基础上，构建了筛选和构建了以离子液体[BMIM]BF4/柠檬酸钠体系和异丙醇/硫酸铵体系为代表的两相可发生突然反转的智能多液相体系，并结合成相物质含量、搅拌转速等动力学因素对智能多液相酶催化体系的影响，进一步利用“相反转”智能调控间歇反应过程，不但大幅提升了脱胶反应速率，而且可将有害副产物脂肪酸的生成减少85%以上。针对多液相手性拆分体系中，存在的单次拆分的选择性虽有大幅提升，但其拆分效率依然难于满足工业需求的难题，考虑将酯化拆分和多液相酶水解拆分相偶联等方式，利用多液相酶催化体系能够大幅减少手性副产物生成的优势，成功制备了高纯度的手性产物，手性产物R-扁桃酸e.e.值可达到98.06%。对其微观结构的深入研究，我们发现多液相体系会形成O/W/W乳液体系或双乳液体系两种微观结构。在智能多液相体系中，双乳液体系聚集存在较大差异，该现象与 “拐点”两侧酶催化效率突变的主要影响因素。最后，初步尝试及构建了基于智能多液相体系的连续装置，实现了高效、可控化运转，其可连续运转16小时以上，反应效率没有明显的变化，为界面酶的工业应用提供了新的工程范例和理论支撑。