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.