Self‐assembled multienzyme complex facilitates synthesis of glucosylglycerol from maltodextrin and glycerol

Abstract BACKGROUND Compound 2‐ O ‐α‐ d ‐glucosylglycerol (2αGG) naturally serves as a compatible osmolyte in acclimation to environmental stresses, such as high osmolarity, dryness, and extreme temperature. It presents several bioactivities and has been used in the food, agriculture, and cosmetics areas. RESULTS In the present study, we attempted to synthesize the 2αGG from low‐cost maltodextrin and glycerol by constructing an in vitro multi‐enzyme system. The system contained two core enzymes, namely glucan phosphorylases (GPs) and glucosylglycerol phosphorylases (GGPs), and two auxiliary enzymes, namely isoamylase and 4‐α‐glucanotransferase. Several new GGPs from different organisms were characterized with the function of converting α‐G1P and glycerol to sole stereo‐configuration product 2αGG. Then, polypeptide SpyTag‐SpyCatcher was employed to construct a self‐assembled multienzyme complex, and different combinations between enzymes and peptides were constructed and tested. The best self‐assembled multienzyme complex exhibited three‐fold higher productivity compared to that of free enzyme. This reaction system also produced 240 m m (61 g L −1 ) 2αGG under high substrate concentration, with a conversion yield of 86%. CONCLUSION The present study provides an efficient approach for producing 2αGG. It also demonstrates that the SpyTag‐SpyCatcher system could be applied to construct other multienzyme complexes for increased productivity and product titer. © 2023 Society of Chemical Industry.