A novel thermophilic α-glucan phosphorylase enables molecular weight-tunable amylose biosynthesis from cellobiose via a dual-enzyme cascade system

Non-food cellulose bioconversion to amylose offers a promising avenue for sustainable biomanufacturing while maximizing non-food biomass utilization. Nevertheless, current α -glucan phosphorylases ( α GPs) exhibit insufficient thermostability and suboptimal catalytic performance, thereby limiting their feasibility for industrial implementation. In this study, a novel thermostable α GP from Dictyoglomus sp. NZ13-RE01 ( Dspα GP) was identified and characterized through computational genome mining. The specific enzyme activity of Dspα GP was 36.3 ± 2.1 U/mg under the optimal conditions of temperature 70 °C and pH 5.0. The enzyme thermal melting temperature ( T m ) was 82.26 °C, and the residual activity retained 55.5 % after stored at 70 °C for 12 h. These results demonstrated the enzyme an exceptional catalytic efficiency and remarkable thermal stability. Kinetic analysis revealed superior substrate specificity toward maltotriose and maltotetraose with k ca t / K m values of 1.96 and 1.83 mM −1 s −1 , respectively. Integration of Dspα GP with Clostridium thermocellum cellobiose phosphorylase established a dual-enzyme cascade system capable of synthesizing amylose with tunable molecular weight (7392–16,632 g/mol) by adjusting the cellobiose/G4 ratio, a conversion efficiency of 35.67 % was achieved, significantly surpassing existing systems. Site-directed mutagenesis combined with molecular docking revealed that conserved residues Gly32, Arg364, Lys370, Tyr432, and Lys464 play critical roles in substrate binding and catalytic processes. This study provides enhanced process efficiency and operational stability for sustainable carbohydrate bioconversion applications and promoting valorization of non-edible cellulose resources.