Abstract：
D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+, and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization. The half-life of the best variant E268R-EKL16 at 50 °C was dramatically increased 30-fold to 135.3 h, and it maintained 90 % of its activity after 13 consecutive reaction cycles. Additionally, we identified that metal ions played a key role in stabilizing the tetramer structure of Rum55, and the dependence on metal ions for E268R-EKL16 was significantly reduced. This study provides a useful route for improving the thermostability of DAEs, opening up new possibilities for the industrial production of D-allulose.
摘要：
D-阿洛酮糖,一种非常理想的糖替代品，主要使用D-阿洛酮糖3-差向异构酶(DAE)产生。然而,可用的DAE酶的可用性是有限的。在这项研究中,我们发现并改造了一种新的DAERum55，它来自人类肠道细菌Ruminococussp。CAG55.Rum55的活性严格依赖于Co2+的存在，在50℃下，平衡转化率为30.6%，半衰期为4.5h。为了提高其性能，我们设计了Rum55的界面相互作用来稳定它的四聚体结构，然后将最佳变体E268R与自组装肽连接以形成活性酶聚集体作为无载体固定化。最佳变体E268R-EKL16在50°C的半衰期急剧增加30倍，达到135.3小时，在连续13个反应循环后，它保持了90%的活性。此外,我们发现金属离子在稳定Rum55的四聚体结构中起着关键作用，并且E268R-EKL16对金属离子的依赖性显着降低。本研究为提高DAEs的热稳定性提供了一条有用的途径。为D-阿洛酮糖的工业生产开辟了新的可能性。
