基于非天然氨基酸改造的可控固定化酶制备及催化拆分应用研究

To avoid the unwanted chemical linkage and to prevent the active site of enzyme from being buried and destroyed, which results from the redundant dependence on the amino group of natural amino acids in side chain and the random chemical linkage in traditional covalent immobilization (Figure 1A), an idea for controllable and covalent immobilization of enzyme (Figure 1B) is proposed based on the reconstruction of enzyme using unnatural amino acid (UAA). In this method, lipase (43KDa) are used as model enzymes for their low molecular weight and simple structure. Immobilization site could be chose by making use of terminator and selecting the inserted site of unnatural amino acid (UAA) based on the site-specific mutagenesis. The structure of unnatural amino acid (UAA) is very similar with that of corresponding amino acid in the enzyme peptide chain. Under the orientation of UAA for the site-specific chemical reaction of its side chain group, the recombinant targetted enzyme will be covalently immobilized on the support facilely using cu-free clicking chemistry. Effect of the structure, physical-chemical properties and location of UAA in enzyme structure on the biocatalytic property and thermo-stability would be carefully examined. Relation between covalent linkage, linkage amount and immobilization region with biocatalytic property and thermo-stability would also be investigated. When this immobilized enzyme are used in the enzymatic resolution of intermediate of moxifloxacin strengthened using microwave irradiation,effects of some factors such as the power for microwave irradiation on the enzymatic catalysis will be examined carefuully and the mechanism for the process will be explored depending on the experiments research. By changing the UAA insert site, selectivity of covalent immobilization site will be enhanced and random chemical linkage in conventional covalent immobilization will be avoided. Resolution of the intermediate of moxifloxacin will be strenthened and accelerated under microwave irradiation.For the favorable conclusion drew from the previous research, respectively, further research will be carried out to enrich the enzyme immobilization and catalysis study.

针对传统酶固定中共价连接依赖侧链氨基且随机进行而引起的错误连接，以及由此产生的固定位点选择被动、活性中心易遭掩埋和破坏等问题（图1A），提出基于非天然氨基酸改造的酶可控共价固定化设想（图1B），并依据“死码活用”原理结合定点突变方法，对结构相对简单和应用较广的脂肪酶开展此研究。首先，以对应氨基酸结构类似的非天然氨基酸改造酶蛋白，借助非天然氨基酸定位及生物正交化学连接实现固定, 建立基于非天然氨基酸改造的酶固定化方法；其次，在酶结构分析基础上调整改造过程以主动选择固定位点和连接方式，考察非天然氨基酸结构及位置等对固定化酶性能影响；最后，借此固定化酶催化拆分大位阻环仲胺化合物并辅以微波强化，考察微波辐射诸因素对催化过程影响并探索其强化机制。在稳定酶结构前提下凭借非天然氨基酸作用克服传统共价固定诸多弊端，以微波强化提高环仲胺拆分速率，从而在前期探索良好基础上，进一步深入并丰富酶固定化和催化研究。

本项目主要针对传统酶固定中共价连接依赖侧链氨基且随机进行而引起的错误连接，以及由此产生的固定位点选择被动、活性中心易遭掩埋和破坏等问题，将非天然氨基酸改造以及点击化学引入酶的可控共价固定化，对结构相对简单和应用较广的脂肪酶（43KDa）、醛酮还原酶(31KDa）、脱氢酶(29KDa）等开展此研究。. 首先，基于单点插入修饰的酶固定化。以对应氨基酸(如：酪氨酸)结构类似的非天然氨基酸（如：对叠氮苯丙氨酸）改造酶蛋白，借助非天然氨基酸定位及生物正交化学连接实现固定, 制备了一步纯化与固定化的固定化脂肪酶，相对活力最高达到137%，初步建立基于非天然氨基酸改造的酶固定化方法，并将此方法推广到醛酮还原酶、醇脱氢酶的研究中；同时发现在醛酮还原酶和脱氢酶的改造中，通过基因组改造的宿主MG1655表达，可以大大提高修饰酶的表达量，单点突变并插入非天然氨基酸后表达量能达到野生型水平，这为项目设计的新型固定化方法进一步完善和应用推广奠定了基础。. 其次，基于多点插入修饰的酶固定化。在酶结构分析基础上调整改造过程以主动选择固定位点，考察非天然氨基酸结构及位置等对固定化酶性能影响，当经过酶蛋白的生物信息学研究和选择位点，即便是多点插入修饰也未见酶催化活性的显著降低；且插入非天然氨基酸修饰制备的固定化酶热稳定性随非天然氨基酸插入数目增多明显增强。. 最后，新型固定化酶的催化应用。主要研究了固定化醛酮还原酶和脱氢酶的催化制备抗癌药物克唑替尼中间体(S)-2,6-二氯-3-氟苯乙醇。在适宜的条件下对于合成手性乙醇具有最高的催化活性，催化产率高达90.8％，最终产物(S)-2,6-二氯-3-氟苯乙醇显示出高与99.98%的ee值。初步的实验室小量实验批次催化6个循环周期（12 h一个循环）后仍保持约初始活性的80%，这为后期计划的放大实验奠定了较好的基础。. 项目中在前期基础上我们还拓展了相关的研究，如：新型固定化方法的拓展与应用以及酶蛋白改造应用的研究等，进一步丰富了固定化酶制备中的科学问题探索和应用催化研究。