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 α-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 ∼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.