Programming an Orthogonal Self-Assembling Protein Cascade Based on Reactive Peptide-Protein Pairs for In Vitro Enzymatic Trehalose Production

: Trehalose is an important rare sugar that protects biomolecules against environmental stress. We herein introduce a dual enzyme cascade strategy that regulates the proportion of cargos and scaffolds, to maximize the benefits of enzyme immobilization. Based upon the self-assembling properties of the shell protein (EutM) from the ethanolamine utilization (Eut) bacterial microcompartment, we implemented the catalytic synthesis of trehalose from soluble starch with the coimmobilization of alpha amylase and trehalose synthase. This strategy improved enzymatic cascade activity and operational stability. The cascade system enabled the efficient production of trehalose with a yield of similar to 3.44 g/(L U), 1.5 times that of the free system. Moreover, its activity was maintained over 12 h, while the free system was almost completely inactivated after 4 h, demonstrating significantly enhanced thermostability. In conclusion, an attractive self-assembly coimmobilization platform was developed, which provides an effective biological process for the enzymatic synthesis of trehalose in vitro