Spatial Orchestration of Enzyme Cascades via SpyTag/SpyCatcher Chemistry: A Tunable and High-Efficiency
Jing Zhang1, Peng He1, Zhaoyao Qian1
1School of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang, Jiangxi 330047, China.
Abstract:
Multienzyme coimmobilization technology can effectively enhance the stability and reusability of cascade enzymes and simplify the production process. In this study, SpyTag/SpyCatcher systems and site-directed mutagenesis techniques were used to construct spatially ordered coimmobilized enzyme systems. Further optimizing the preparation conditions of coimmobilized enzymes, we successfully prepared sequential coimmobilized enzymes (SiO2@β-Gal-SC-ST-LFAI) and directed coimmobilized enzymes (SiO2@β-Gal-Y69C-ST-LFAI) under mild conditions. Compared with the free enzyme system (β-Gal&LFAI), the coimmobilized enzyme systems demonstrated greater thermal stability, organic solvent tolerance, and storage stability. In addition, SiO2@β-Gal-SC-ST-LFAI and SiO2@β-Gal-Y69C-ST-LFAI retained 49.0% and 51.3% of their initial activity, respectively, after 5 cycles. Notably, the directed coimmobilization strategy significantly enhanced cascade biocatalytic efficiency, achieving a 7.75-fold improvement compared with conventional coimmobilization (SiO2@β-Gal + LFAI). The sequential and directed automated immobilization platform developed by our research provides a novel approach for the efficient immobilization of dual enzymes.
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