Combined engineering of substrate specificity and thermostability of Bacteroides phosphatase to promote in vitro biotransformation of d-allulose by multienzyme cascades.

Recently, in vitro multienzyme cascade biotransformation based on phosphorylation-isomerization-dephosphorylation has been extensively utilized in the industrial production of rare sugars. It exhibited distinct advantages in conversion and yield compared with the direct isomerization catalyzed by single isomerase, due to phosphorylation activating glycosyl molecules to reduce the energy barrier of isomerization. However, as a critical enzyme catalyzing the dephosphorylation to drive the equilibrium forward, phosphatases that satisfy the ideal catalytic model of 'one enzyme-one substrate' generally require high substrate specificity and catalytic activity. In this study, d-allulose 6-phosphate phosphatase (A6PP) from Bacteroides sp. CAG:462 was identified by evolutionary and structural analysis. It was subsequently modified by pocket engineering and computer-aided design to improve its substrate specificity and thermostability. The results showed that the dominant mutant M6 exhibited substantially increased substrate specificity toward d-allulose 6-phosphate with a 1.70-fold activity proportion (from 43.7 % to 74.9 %), and simultaneously obtained remarkable thermostability improvement with a 21.4-fold half-life under 50 °C. Finally, the mutants were applied to the in vitro multienzyme cascade biotransformation of d-allulose. M6 mutant produced 32.5 g/L d-allulose from 50 g/L DE 5-8 maltodextrin with conversion rate of 65.1 %, while the wild type yielded only 5.4 g/L. This study achieved high-yield biotransformation of d-allulose and provided a reference for the identification and precise engineering of specific phosphatases.