One-pot synthesis of γ-cyclodextrin of high purity from non-food cellulose via an in vitro ATP-free synthetic enzymatic biosystem

γ-Cyclodextrin (γ-CD) is a cyclic oligosaccharide composed of eight glucose molecules linked together via α-1,4-glycosidic bonds. It has a wide range of applications in the pharmaceutical, food, and chemical industries. Two pathways were designed for the synthesis of γ-CD from the non-food feedstock cellulose via an in vitro adenosine triphosphate (ATP) -free synthetic enzymatic biosystem. Cellulose was employed as the substrate for producing cellobiose, which was subsequently converted to γ-CD via a cascade reaction utilizing five enzymes. A stoichiometric conversion of cellulose to γ-CD was achieved by adding the synthesis module for glucose-1-phosphate (G-1-P) and optimizing the reaction conditions. The productivity of γ-CD obtained via pathway II-condition III was as high as 517 g/m³·h from cellobiose, representing a 16-fold increase compared to pathway II-condition I. A process for producing γ-CD from cellulose was established in this study, which yielded γ-CD of >90 % purity. This study presents a novel process that could be employed in next-generation biorefineries and a strategy for improving the economic considerations associated with cellulose utilization.