尿酸酶是诊断工具酶、防治肿瘤溶解综合征药物、针对难治性痛风唯一有效药物，应用要求其在pH7.4活性高且稳定性好。苛求芽孢杆菌尿酸酶(BFU)在pH7.4稳定性好且溶解度高、活性表达易；其双突变体V144A/Y319R在pH9.2时Vm接近50kU/g，生理条件下热失活半衰期接近120天，但在pH7.4时仅余20%活性。季也蒙毕赤酵母尿酸酶(MGU)热稳定性较好、Vm接近12kU/g、最适pH接近7.4，但溶解度低、重组表达收率低。本项目基于三维构象分析，比较BFU和MGU决定其最适pH的相互作用网络差异；将MGU最适pH决定因素移植到BFU使BFU突变体最适pH接近7.4，再突变Y319为R提高热稳定性；对此突变体催化残基附近区域定向进化，基于序列活性关系合理设计，筛选在pH7.4更高活性突变体；评价高活性稳定BFU突变体在尿酸测定中的应用和清除血浆尿酸药效，为其应用奠定基础。

Uricase is a crucial enzyme for in vitro diagnosis, a drug to treat tumor-lysis-syndrom and the sole effective drug to treat refractory gout, whose higher activity and stronger thermostability at pH 7.4 are preferred for application. The uricase from Bacillus fastidious ATCC29604 (BFU) exhibited excellent thermostability at pH 7.4 and ease for active expression; its double mutant V144A/Y319R had the maximum activity of about 50 kU/g at pH 9.2, a half-life time of about 120 days under physiological conditions, but reserved less than 20% activity at pH 7.4. The uricase from Meyerozyma guilliermondii (CGMCC 2.1008; MGU) showed good thermostability at pH 7.4, the maximum activity of about 13 kU/g and a wonderful optimum pH close to pH 7.0, but tolerated lower solubility and challenges on recombinant expression. This project aims to (1) reveal the differences in the interaction networks determining optimum pH of BFU and MGU through the analyses of three-dimensional conformation and experimental verification, (2) transplant such interaction networks determining optimum pH of MGU into BFU to yield BFU mutants bearing optimum pH close to pH 7.4, whose Y319 was then mutated into R for stronger thremostability so that these mutants can serve as the starting enzymes for subsequent engineering, (3) perform directed evolution of the starting enzymes focused on the regions close to catalytic residues and rational design of their mutants based on sequence-activity relationship, to obtain stable mutants of much higher activity at pH 7.4, (4) evaluate the application of the potent stable mutants in both serum uric acid assay and the elimination of plasma uric acid. These researches will promote the applications of BFU mutants of neutral optimum pH and much higher activity.