季也蒙毕赤酵母尿酸酶和苛求芽孢杆菌尿酸酶最适pH影响因素的比较和应用

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.

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。

尿酸酶是体外诊断工具酶、防治肿瘤溶解综合征药物、针对难治性痛风的唯一有效药物，这些应用要求尿酸酶在中性pH活性高且热稳定性好。此前研究发现苛求芽孢杆菌尿酸酶(BFU)突变体V144A/Y319R有超强热稳定性、高催化效力但最适pH偏碱，而季也蒙毕赤酵母尿酸酶(MGU)最适pH接近中性、最大比活性较低，本项目已按计划完成：(1)通过三维构象比较分析解析决定MGU最适pH接近中性的催化残基所参与静电-氢键网络、BFU高催化效力相关的催化网络独特组成，发现MGU催化中心有独特的静电-氢键网络、BFU的活性中心有特殊的催化残基；将BFU所具有的特殊催化相关残基移植到MGU的对等空间位置获得MGU突变体I260R，发现其在pH7.2最大比活性约55kU/g(与BFU突变体V144A/Y319R在pH9.2最大比活性无统计差异)，且接近野生型MGU的1.6倍、超过旭化成及Sigma在2015年后推广诊断尿酸酶pH7.2比活性的3倍(测定血清尿酸所用双试剂法要求最适pH接近7.2的尿酸酶)，热稳定性提高到满足课题组所建立双试剂法试剂盒专利配方的要求，但水溶性低故纯化成本高；分析I260R活性中心组成发现，其亚基间界面存在因I260R突变所产生的新的静电作用；(2)建立用微量蛋白样品定量测定蛋白溶解度的快速、简便且通用新方法；(3)对MGU阳性突变体I260R，参照BFU表面离子对结构设计其表面多位点突变生成与BFU表面对应的离子对，发现此类突变都显著提升MGU突变体的溶解度，特别是三位点协同突变体溶解度提升近5倍而达到与BFU相当水平，且其最适pH仍接近中性，但最大比活性却显著下降；(4)尝试用表面经小分子兼性离子对高密度修饰的离子交换介质纯化MGU，发现其纯化效力达到常规离子交换剂的3倍且废液量显著减少；这可能是解决低溶解性MGU突变体纯化的方向，也是后续的研究任务。