Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis.
Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis.
复制标题
DOI：
10.1371/journal.pone.0177877
复制
发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Hong J
中科院分区：
文献类型：
--
作者：
Li W;Xu S;Zhang B;Zhu Y;Hua Y;Kong X;Sun L;Hong J
尿酸氧化酶是嘌呤代谢的关键酶，催化尿酸氧化为尿囊素。它用于治疗高尿酸血症和痛风，也用于诊断试剂盒。本研究采用易错聚合酶链反应和交错延伸技术从枯草芽孢杆菌中获得了一株尿酸氧化酶突变株。经过多轮诱变和筛选，获得了两个突变体6 E9和8 E279，其催化效率分别提高了2.99和3.43倍。它们的最适反应温度较低，热稳定性较高。D44 V、Q268 R和K285 Q被鉴定为通过定点诱变引入的三个最有益的氨基酸取代。通过三个突变体的随机组合获得的D44 V/Q268 R表现出最高的催化活性。与野生型尿酸氧化酶相比，D44 V/Q268 R的Km、kcat/Km和酶活分别提高了68%、83%和129%。结构建模表明，远离活性位点的突变可以对活性产生显著影响。对于其中的许多，基本的机制仍然很难从静态结构模型解释。我们还比较了同一组单点突变对野生型和最终突变体的影响。结果表明，上位性的强烈影响，这可能意味着突变影响催化通过影响蛋白质动力学，除了平衡结构。
Urate oxidase is a key enzyme in purine metabolism and catalyzes the oxidation of uric acid to allantoin. It is used to treat hyperuricemia and gout, and also in a diagnostic kit. In this study, error-prone polymerase chain reaction and staggered extension process was used to generate a mutant urate oxidase with improved enzyme activity from Bacillus subtilis. After several rounds of mutagenesis and screening, two mutants 6E9 and 8E279 were obtained which exhibited 2.99 and 3.43 times higher catalytic efficiency, respectively. They also exhibited lower optimal reaction temperature and higher thermo-stability. D44V, Q268R and K285Q were identified as the three most beneficial amino acid substitutions introduced by site-directed mutagenesis. D44V/Q268R, which was obtained through random combination of the three mutants, displayed the highest catalytic activity. The Km, kcat/Km and enzyme activity of D44V/Q268R increased by 68%, 83% and 129% respectively, compared with that of wild-type urate oxidase. Structural modeling indicated that mutations far from the active site can have significant effects on activity. For many of them, the underlying mechanisms are still difficult to explain from the static structural model. We also compared the effects of the same set of single point mutations on the wild type and on the final mutant. The results indicate strong effects of epistasis, which may imply that the mutations affect catalysis through influences on protein dynamics besides equilibrium structures.