High-Yield production and Kilogram-Scale preparation of 2-O-α-d-Glyceroglycoside through Whole-Cell catalysis using a novel sucrose phosphorylase

• A novel SPase with high transglycosylation activity and strict regioselectivity. • An unbuffered and salt-free whole-cell biocatalytic system. • An unprecedented αGG titer of 444 g/L achieved via one-step biocatalysis. • An integrated MF-SMB process for green and efficient product purification. • 42 kg pilot-scale product with 94.3 % purity and 91.3 % recovery setting new benchmark for 2-αGG bioproduction. 2-O-α- d -glyceroglycoside (2-αGG) is a versatile compound with significant potential for applications in cosmetic, pharmaceutical, and food industries. Achieving regio- and stereo-selective biosynthesis with high productivity and recovery continues to be a central challenge in biocatalytic manufacturing. Through bioinformatic exploration and enzymatic characterization, we identified a novel sucrose phosphorylase ( Sm SP2) from Streptococcus mutans that exhibits the highest transglycosylation activity at the glycerol 2-OH position while demonstrating low hydrolytic activity. We then developed a whole-cell biocatalytic system operating in an unbuffered, salt-free aqueous medium. Using sucrose fed-batch operation, this system achieved unprecedented αGG production of 444 g/L via one-step SPase catalysis. Furthermore, a coupled microfiltration-simulated moving bed (MF-SMB) purification process was developed to enable efficient and environmentally friendly recovery, yielding αGG with a purity of 94.3 % and a product recovery rate exceeding 91.3 %. Pilot-scale (100 L) production successfully generated 42 kg of the αGG product. This work establishes an integrated green manufacturing platform that combines enzymatic precision with industrial scalability for efficient 2-αGG production.