High-performance biocatalyst Nicotiana attenuata α-glucan phosphorylase facilitates the robust synthesis of amylose from cellobiose

The enzymatic synthesis of amylose is a sustainable solution for the valorisation of non-food cellulose. However, the limited thermostability and catalytic efficiency of existing α -glucan phosphorylases ( α GPs) hinders its potential for industrial scalability. In this study, we identified and characterised a novel α GP from Nicotiana attenuata ( Nicα GP) via genome mining. It exhibited superior catalytic performance, with a specific activity of 82.1 U/mg under optimised conditions (40 °C and pH 5.0), and notable temperature and pH stability. Supplementation of Mg 2+ further enhanced its activity by 5 %. Kinetic analysis revealed a catalytic efficiency ( k cat / K m ) of 27.46 mM −1  s −1 for maltotetraose (G4). Furthermore, a dual-enzyme cascade system integrating Nicα GP with Clostridium thermocellum cellobiose phosphorylase achieved up to 28.4 % cellulose conversion, synthesising amylose with tunable molecular weights (6602 ± 47.13–67,154 ± 47.28 g/mol) by modulating ratios of cellobiose/G4. Molecular docking identified the conserved residues (Gly196, Arg706, Lys711, and Lys821) critical for substrate binding and pyridoxal 5′-phosphate-mediated catalysis. Overall, this study provides a new enzyme component for the artificial synthesis of amylose from non-edible biomass, facilitating the widespread application of non-edible cellulosic resources for food conversion.