Bioproduction of Rare d-Allulose from d-Glucose via Borate-Assisted Isomerization

d -Allulose is a low-calorie functional rare sugar with excellent processing suitability and unique physiological efficacy. d -Allulose is primarily produced from d -fructose through enzymatic epimerization, facing the constraints of a low conversion yield and high production cost. In this study, a double-enzyme cascade system with tetraborate-assisted isomerization was constructed for the efficient production of d -allulose from inexpensive d -glucose. With the introduction of sodium tetraborate (STB), capable of forming complexes with diol-bearing sugars, the conversion yield of d -allulose from d -glucose substantially escalated from the initial 17.37% to 44.97%. Furthermore, d -allulose was found to exhibit the most pronounced binding affinity for STB with an association constant of 1980.51 M –1, notably surpassing that of d -fructose (183.31 M –1 ) and d -glucose (35.37 M –1 ). Additionally, the structural analysis of the sugar–STB complexes demonstrated that d -allulose reacted with STB via the cis 2,3-hydroxyl groups in the α-furanose form. Finally, the mechanism underlying STB-assisted isomerization was proposed, emphasizing the preferential formation of an allulose–STB complex that effectively shifts the isomerization equilibrium to the allulose side, thereby resulting in high yield of d -allulose. Such an STB-facilitated isomerization system would also provide a guidance for the cost-effective synthesis of other rare sugars.