Improving trehalose production through the design and optimization of linker peptides in scaffold protein

Trehalose is an excellent bioprotective agent with substantial applications in biomedicine, cosmetics, and food. Currently, trehalose production utilizes a bi-enzyme method involving free maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase). The spatial distance between the active sites of the two enzymes can be shortened by assembling a bi-enzyme complex using a scaffold protein to improve catalytic efficiency. In this study, previously constructed MTSase-CcDoc and CtDoc-MTHase fusion enzymes were combined with a scaffold protein to form a bi-enzyme complex via Cohesin-Dockerin interaction to catalyze trehalose production using maltodextrin as a substrate. By designing the structural length and rigidity of the 12 linker peptides to replace the native linker peptide in the scaffold protein, trehalose production was increased by 1.24 and 1.17 times compared with that using free enzymes and the native linker peptides, respectively. Predictive models of enzyme binding sites, along with structural and docking analyses, further confirmed the enhanced catalytic activity and structural integrity of the bi-enzyme complex, providing a foundation for developing efficient trehalose synthesis pathways.