In vitro assembly of trehalose bi-enzyme complex with artificial scaffold protein

Trehalose is an important rare sugar with stable properties, which protects biomolecules from the environment. We herein introduce a dual enzyme cascade strategy that a scaffold protein-mediated assembly method for trehalose bi-enzyme complex was used to cascade a variety of functionally related enzymes to continuously and efficiently catalyze the substrate and enhance the overall catalytic effect. Based on the specific interaction of cohesin-dockerin, we implement the assembly, discuss the assembly efficiency, and expound on how to improve the utilization of enzymes by constructing a bi-enzyme complex under the best conditions in vitro. Moreover, the amount of trehalose produced by the bi-enzyme complex reached 1.5 times that of the free enzyme mixture at 40 h and maintained an increasing trend. With the model of scaffold protein-mediated bi-enzyme complex, the cascade reaction and catalytic effect can be improved compared with free enzyme mixtures, which has a far-reaching prospect not only for the preparation of trehalose but also for future industrial applications.