新型低共熔离子液体对生物还原反应过程的强化及机理研究

Biocatalysis based on deep eutectic solvents (DESs) is an emerging research area. In the present proposal, aimed to promote bioreduction process and coenzyme regeneration, we will design and synthesize a task-specific DES by rational design strategy based on bioreduction characteristics and demand of green chemistry, which is fully derived from biomaterials, and suitable for biocatalytic process of poorly soluble substrate and bioreduction involving coenzyme regeneration. The bioreduction of prochiral ketones to their corresponding chiral alcohols catalyzed by recombinant-whole-cell overexpressing carbonyl reductase of LXCAR was used as model reaction. The functionalized amino acid or oligopeptide-based deep eutectic ionic liquids will be synthesized based on rational design strategy by combining the organic salt (quaternary ammonium salt) with benign hydrogen bond donor (amino acid or oligopeptide) in order to improve mass transfer and strengthen coenzyme regeneration in the bioreduction. A novel reaction system containing amino acid-based or oligopeptide-based deep eutectic ionic liquid as co-solvent will be constructed based on structure-activity relationship between the biocompatibility of amino acid or oligopeptide-based deep eutectic ionic liquids to recombinant-whole-cell, and the component and physicochemical properties of designed task-specific DESs. The fundamental law of efficient preparation of chiral alcohols conducted in novel deep eutectic ionic liquid-containing system by microbial whole-cell will be illustrated. By the study on the mechanism of process promotion and cofacter regeneration strengthing in this reaction medium, a newly regulatory strategy for bioreduction mediated by amino acid-based or oligopeptide-based deep eutectic ionic liquids-containing medium will be established. The implementation of the present project has great scientific significance in the development of green alternative solvents for biocatalytic reaction, and will broaden the function and application of designed task-specific and environmentally friendly deep eutectic solvents.

低共熔离子液体应用于生物催化是近年来介质工程研究的新兴领域。项目以生物反应过程强化和绿色化为目的，依据绿色化学发展要求和生物还原反应特性（涉及辅酶再生），提出强化过程传质的有机盐中性分子偶联促进辅酶再生的氢键供体的设计新策略，以生物基原料制备系列新型环境友好的季铵盐-氨基酸/短肽类功能化低共熔离子液体，并应用于生物催化新介质体系。通过考察低共熔离子液体组成-生物相容性构效关系，研究其对重组工程菌LXCAR催化制备系列手性取代芳基醇反应的影响规律和反应机理，探明新型低共熔离子液体对生物还原过程中辅酶再生和过程强化的影响机制，建立新型调控生物催化还原效率的介质工程新方法，拓展低共熔离子液体促进辅酶再生的新特性，为适用于生物催化的新型绿色替代溶剂开发提供新思路，为强化生物还原反应效率和环境友好性开辟一条新途径。

低共熔离子液体应用于生物催化是近年来的研究热点。项目以生物反应过程强化和绿色化为目的，依据绿色化学发展要求和生物还原反应过程涉及辅酶再生的特性，基于低共熔离子液体结构的可设计性，从天然生物原料出发制备得到了系列环境友好的新型氯化胆碱/氨基酸（短肽）类低共熔离子液体，以及生物相容性更好的基于醋酸胆碱的系列生物基低共熔溶剂，并成功应用于以半乳糖地霉 (Galactomyces geotrichum) ZJPH1810、棘孢木霉(Trichoderma asperellum)ZJPH0810、红串红球菌(Rhodococcus erythropolis) XS1012、热带假丝酵母(Candida tropicalis)104和重组E. coli等不同微生物细胞为催化剂，以阿瑞匹坦、依替米贝、西他列汀等抗肿瘤和降糖、降脂药物关键手性中间体制备为模型反应的生物还原过程，取得了明显优于常用的缓冲液介质体系的催化结果。项目主要研究内容和取得的成果如下：①制备得到了系列基于氯化胆碱的低共熔离子液体，发现氯化胆碱/氨基酸类和氯化胆碱/谷胱甘肽低共熔离子液体可明显提高生物还原产率，且具有促进反应中辅酶再生作用；②基于低共熔离子液体结构的可设计性，通过改变构成低共熔溶剂的氢键供体（HBD）和氢键受体（HBA）种类，考察低共熔离子液体组成以及与微生物细胞生物相容性的构效关系，获得了较传统的基于氯化胆碱类低共熔溶剂生物相容性更好，更利于生物还原反应的基于醋酸胆碱和甜菜碱类低共熔离子液体，其中氢键供体（HBD）对还原反应的影响规律为：醇类 < 糖类 < 氨基酸类，酸性氨基酸 <中性氨基酸 < 碱性氨基酸，以氨基酸和糖类为氢键供体的低共熔离子液体具有促进生物还原过程中辅酶再生的作用；③研究发现氧环境对生物还原反应具有明显的影响，控制低氧浓度可有效提高真菌细胞介导的生物还原产率；④通过改变反应介质体系中L-肉碱:赖氨酸低共熔离子液体的浓度，可使还原产物的构型发生反转。通过研究，丰富了可用于生物催化过程的低共熔离子液体种类，解决了传统咪唑类和吡啶类离子液体存在的毒性大、生物降解性差的问题，为适用于生物催化的新型绿色替代溶剂开发提供了新思路，为强化生物还原反应效率和环境友好性开辟了新途径。