α-酮己二酰-7-氨基头孢烷酸酰化酶的定向进化研究

Single-pot conversion of cephalosporin C to 7-aminocephalosporanic acid (7-ACA) in absence of hydrogen peroxide could be carried out by a tri-enzymatic system, which would be the new focus of international research. In this tri-enzymatic system, the operation stability of the biocatalysts could be improved remarkably, and hence the producing cost of 7-ACA was effectively reduced. The α-ketoadipyl-7-ACA (AKA-7-ACA) acylase with high enzymatic activity was the key for the large-scale production of 7-ACA in a single pot by this tri-enzymatic system. In this study, the molecular structure of AKA-7-ACA acylase from the Pseudomonas species will be investigated. The directed evolution in vitro of the key amino acids in the enzyme active center will be performed by using error-prone PCR technology. The recombinant E.coli JM105 will be constructed. The high throughput screening for AKA-7-ACA acylase will be designed by synthesizing analogs of AKA-7-ACA and using the Bacillus cereus ATCC 13061 as the directed strain. The mutants expressing AKA-7-ACA acylase with high enzymatic activity will be screened from the mutant library. This project will contribute to the solution for further cost reduction of 7-ACA production and enhancement of its international competitiveness.

"三酶一步法"制备7-氨基头孢烷酸（7-ACA）因避免了反应体系中存在过氧化氢而降低酶催化剂的使用寿命、造成生产成本提高等问题，将成为国际研究新热点。高活力α-酮己二酰-7-氨基头孢烷酸（AKA-7-ACA）酰化酶的获得是解决"三酶一步法"实现工业化生产7-ACA的一个瓶颈。本项目拟对假单胞菌来源的AKA-7-ACA酰化酶分子结构进行研究，运用基因重组技术对酶活性中心氨基酸序列进行基因体外定向进化，构建重组大肠杆菌E.coli JM105，通过制备AKA-7-ACA结构类似物，以蜡样芽孢杆菌Bacillus cereus ATCC 13061作为指示菌，设计一个AKA-7-ACA酰化酶的高通量筛选系统，从定向进化获得的突变文库中，筛选出高活力的AKA-7-ACA酰化酶产生菌。本项目研究对进一步降低7-ACA生产成本、提高其国际竞争力具有重要的理论指导与现实意义。

“三酶一步法”工艺制备7-氨基头孢烷酸（7-ACA）因反应体系中不存在过氧化氢而避免化合物被氧化降解和酶催化剂被氧化失活等问题，成为酶工程领域关注热点。戊二酰-7-ACA酰化酶（GLA）对底物α-酮己二酰-7-氨基头孢烷酸（AKA-7-ACA）的特异性酶活力低是三酶一步法工艺实现工业化生产7-ACA的一个瓶颈。本课题以假单胞菌NK703来源的GLA基因为模板，构建了重组菌pET-28b/BL21。通过同源比对与同源建模确定酶活性中心八个关键位点（N21β，P22β，H23β，T69β，V70β，N244β，K374β，S377β），利用分子对接技术确定突变位点替代氨基酸，通过设计引物，利用PCR技术体外扩增，产物重组到含kan抗性标记的质粒pET-28b，并转入到宿主BL21，经过含LB+Kan液体培养液进行初筛获得17个单倍体突变子。经发酵与酶活力测定发现突变子V70βF来源酰化酶对底物AKA-7-ACA有较高的特异性活力（Km为原来的48%；酶活力为GLA的55%）。突变子V70βF通过发酵罐发酵表达获得6732U/L的GLA酶活力（3702 U/L的AKA-7-ACA酶活力）。另外，我们也进行细胞液预处理、酶纯化与纳米材料石墨烯固定化及酶学研究。三酶系统包括透性化毕赤酵母细胞D-氨基酸氧化酶和过氧化氢酶以及氧化石墨烯固定化GLA一步法转化头孢菌素C，反应体系无过氧化氢条件下可获得85.5% 7-ACA产率。本研究结果对指导GLA定向进化和研究进一步降低7-ACA生产成本、提高其国际竞争力具有理论与现实意义。