Programmable amylose architectures via α-glucan phosphorylase coupling: From process variables to structure–property map

A predictive linkage between operating variables and amylose architecture is needed because current phosphorylase-based syntheses from non-food cellulose, though valuable, lack a process–structure–property map for design and manufacturing. The dual-enzyme cascade reaction for amylose synthesis was investigated by systematic variation of temperature, pH, enzyme stoichiometry and addition timing, and the cellobiose/maltotetraose primer ratio, coupled with time-resolved monitoring and multiscale characterization. Under the optimized conditions (pH 5.0, 50 °C), varying the cellobiose/maltotetraose ratio from 1:0.125 to 1:32 enabled the Ct CBP– Nicα GP cascade to produce amylose spanning 1821 ± 20.06–69,252 ± 30.02 g/mol, with a maximum yield of 35.84%. The concurrent delineation of quantitative distribution curves for cellobiose, glucose-1-phosphate, glucose, and inorganic phosphate revealed the synthesis reaction to be influenced by glucose threshold and delineated into three stages. The availability of primer was found to be the primary variable in programming chain length, polymorph selection ( V -B → V-C → A-V), morphology (granules → aggregates → films), and thermal degradation and gelatinisation properties. Overall, this study provides a predictive framework for synthesising amylose with controllable structure and properties, while also outlining the pathway and theoretical basis for the large-scale conversion of non-food cellulose into high-value carbohydrates.