Enzymatic synthesis of neotrehalose: Deciphering the β-1 catalytic promiscuity of transglycolytic starch-active enzymes.

Neotrehalose is a naturally occurring, non-reducing, rare disaccharide with promising nutritional, medical, and synthetic applications, but its low natural abundance and complex chemical synthesis limit commercial production. This study evaluated starch-active enzymes as a sustainable route for its synthesis from renewable substrates. A one-step biocatalytic process using starch and/or maltose as glucosyl donors was developed with the hyperthermophilic α-amylase from Thermotoga maritima. Optimized conditions yielded up to 23.2 g neotrehalose/L (11.60 g neotrehalose/100 g initial maltose), among the most efficient enzymatic methods reported. Combining maltose and starch resulted in an additive effect, increasing productivity and reducing reaction time. Mechanistic analysis showed that neotrehalose is formed through β-anomer-specific transglycosylation at the reducing end of maltose or maltooligosaccharides, generating neotrehalose-core oligosaccharide intermediates. Structural studies with inositol scaffolds examined how the presence and orientation of hydroxyl groups in β-glucosyl acceptors influence enzyme-substrate recognition and the capacity of AmyA to synthesize neotrehalose.