【摘要】 为解决现有D-阿洛酮糖3-差向异构酶（DAEase）热稳定性差的产业问题，本文采用系统发育指导的大数据挖掘、合理修饰和祖先序列重建策略（ASR），重建了具有不同催化结构域DAEase的祖先序列，构建了表达载体，通过重组表达与分子对接筛选出了DAEase A13并进行酶学性质表征，此外，还基于结构分析与分子动力学模拟揭示了DAEase A13热稳定性增强的分子机制。结果表明，基于ASR策略所构建的A13 70℃时半衰期可达8.4 h，其热稳定性较野生（WT）酶显著增强，最大转化率为31%，催化活性也略高于WT酶。立体结构模拟与分子动力学模拟揭示了ASR A13中大量氢键和疏水作用的增加维持了高温下酶分子结构的稳定性，是其热稳定性增强的主要因素。研究结果证实了ASR策略可以改造DAEase使其稳定性、活性和混杂性增强，可以为D-阿洛酮糖工业生产提供良好的生物催化剂。更多还原

【Abstract】 To solve the problem of poor thermal stability of the current D-allulose 3-epimerase(DAEase), the ancestor sequences of DAEase with different catalytic domains were reconstructed by big data mining, reasonable modification and ancestor sequence reconstruction(ASR) strategy under the guidance of phylogenetic information. The expression vectors of the ancestor sequences were constructed, and DAEase A13 with significantly enhanced thermal stability was screened by recombinant expression and molecular docking, and its enzymatic properties were characterized. In addition, the molecular mechanism of thermal stability enhancement of DAEase A13 was revealed based on structural analysis and molecular dynamics. The results showed that the half-life of A13 constructed based on ASR strategy could reach 8.4 h at 70 ℃,indicating that its thermal stability was significantly enhanced compared with that of wild-type(WT) enzyme. The maximum conversion rate of A13 reached 31%, indicating that the catalytic activity of A13 was slightly higher than that of WT enzyme. The structural and molecular dynamics analysis revealed that the increase in hydrogen bonding and hydrophobic interaction in ASR A13 was the main factor responsible for maintaining the stability of the enzyme’s molecular structure at high temperatures. The results showed that ASR strategy could modify DAEases to enhance the stability,activity or hybridity, which could provide superior biocatalyst sources for various industrial applications of functional sugars.更多还原