One pot cascade biosynthesis of D-allulose from D-glucose and its kinetic modelling

One pot cascade of enzymatic catalysis was applied for D-allulose synthesis from D-glucose by the combination of D-glucose isomerase (GI) from Escherichia coli MG1655 and D-allulose 3-epimerase (DAE) from Agrobacterium tumefaciens C58. The synthesis process was investigated by characterizing the initial substrate concentration, molar ratios of GI and DAE, the enzyme loading, and contents of cobalt and magnesium ions. The increase of enzyme loading and the molar ratios of GI vs DAE caused the raising of D-allulose yield from 4% to 9%, verifying that the generation rate of D-fructose was an essential factor for rare sugar conversion improvement. The optimal enzyme loadings required for reactions with substrate concentrations of 100 g L⁻¹ and 200 g L⁻¹ were 60 U and 200 U. Kinetics stimulation was conducted to reveal the underlying relationship within the three sugars converted by dual-enzyme, with the fitting curve validated the reliability of the model.