Abstract：
Protein stability is crucial in enzymatic catalysis. To improve the efficiency in the searching for thermostablizing mutations, we applied a sequence consensus approach focusing on dimeric interface residues of ketoreductase ChKRED20. The strategy returned a success rate of 43%, revealing 9 beneficial mutations from 21 candidates with improved kinetic or thermodynamic stability. Several combinatorial mutants were then constructed, and mutant M8K displayed the highest thermostability, with a melting temperature (Tm) of 89 °C and a half-inactivation temperature (T50) of 93.4 °C, both of over 35 °C increase compared to the wild-type. M8K could remain stable for at least 7 days at its optimal reaction temperature of 55 °C. Its inactivation half-life (t1/2) was 110 min at 90 °C, while the wild-type was 18.6 min at 60 °C. The results were interpreted in the context of structural and molecular dynamic simulation analysis, which revealed the addition of intramolecular interactions, decreased conformational flexibility and increased compactness, all in agreement with the observed effect.
摘要：
蛋白质稳定性在酶催化中至关重要。为了提高寻找恒温突变的效率，我们应用了一种序列共有方法，重点是酮还原酶ChKRED20的二聚体界面残基。该策略的成功率为43%，从21个候选物中发现9个有益的突变，具有改善的动力学或热力学稳定性。然后构建了几个组合突变体，突变体M8K表现出最高的热稳定性，熔化温度（Tm）为89°C，半失活温度（T50）为93.4°C，与野生型相比，两者均超过35°C。M8K在55°C的最佳反应温度下可以保持稳定至少7天。它的失活半衰期(t1/2)在90℃为110分钟，而野生型在60℃为18.6分钟。在结构和分子动力学模拟分析的背景下解释了结果，这揭示了分子内相互作用的加入，降低构象的灵活性和增加的紧密度，都与观察到的效果一致。
