Laboratory of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China; College of Food Science and Technology, Nanchang University, Nanchang 214122, China.
Laboratory of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China.
Int J Biol Macromol. 2020 Nov 1;162:445-453. doi: 10.1016/j.ijbiomac.2020.06.141. Epub 2020 Jun 18.
Artificial multienzyme biocatalysts have played a crucial role in biosynthesis because they allow for conducting complex reactions. Here, we reorted a facile approach to fabricate multienzyme nanodevices with high catalytic activity and stability based on protein assembly and chemical crosslinking. The self-assembled partner SpyCatcher and SpyTag were genetically fused with 2,3-butanediol hydrogenase and formate hydrogenase to produce KgBDH-SC (SpyCatcher-fused 2,3-butanediol hydrogenase) and FDH-ST (SpyTag-fused formate hydrogenase), respectively. After assembling the two fusion proteins, the complexes were then immobilized on the functionalized silicon dioxide nanoparticles by glutaraldehyde, forming KgBDH-SC-ST-FDH-SiO with the capability of reducing 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol with cofactor regeneration. Under the optimal conditions, the created co-immobilized enzymes performed 49% activity recovery compared with the mixture of free enzymes as well as showed 2.9-fold higher catalytic activity than the traditional random co-immobilized enzymes. Moreover, KgBDH-SC-ST-FDH-SiO showed better pH stability and organic solvents stability than the free enzymes, and remained 52.5% overall catalytic activity after 8 cycles. Finally, the co-immobilized enzymes can reduce 60 mM HAP for fabrication of (R)-PED with cofactor regeneration in the phosphate buffer reaction system, affording 83.9% yield and above 99% optical purity.
人工多酶生物催化剂在生物合成中发挥了关键作用，因为它们可以进行复杂的反应。在这里，我们报告了一种基于蛋白质组装和化学交联的简便方法来制备具有高催化活性和稳定性的多酶纳米器件。自组装伴侣 SpyCatcher 和 SpyTag 分别与 2,3-丁二醇氢酶和甲酸氢酶基因融合，产生 KgBDH-SC（SpyCatcher 融合的 2,3-丁二醇氢酶）和 FDH-ST（SpyTag 融合的甲酸氢酶）。将两种融合蛋白组装后，用戊二醛将复合物固定在功能化的二氧化硅纳米粒子上，形成具有辅酶再生能力的 KgBDH-SC-ST-FDH-SiO，可将 2-羟基苯乙酮还原为（R）-1-苯基-1,2-乙二醇。在最佳条件下，与游离酶混合物相比，所创建的共固定化酶的活性恢复了 49%，并且比传统的随机共固定化酶的催化活性高 2.9 倍。此外，KgBDH-SC-ST-FDH-SiO 比游离酶具有更好的 pH 稳定性和有机溶剂稳定性，经过 8 个循环后，其总催化活性仍保持在 52.5%。最后，在磷酸盐缓冲反应体系中，共固定化酶可以还原 60mM HAP 以制备（R）-PED，同时进行辅酶再生，产物收率为 83.9%，光学纯度高于 99%。
