Two-step biosynthesis of d-allulose via a multienzyme cascade for the bioconversion of fruit juices.

d-Allulose, a low-calorie rare sugar with potential as sucrose substitute for diabetics, can be produced using d-allulose 3-epimerase (DAE). Here, we characterized a putative thermostable DAE from Pirellula sp. SH-Sr6A (PsDAE), with a half-life of 6 h at 60 °C. Bioconversion of 500 g/L d-fructose using immobilized PsDAE on epoxy support yielded 152.7 g/L d-allulose, which maintained 80% of the initial activity after 11 reuse cycles. A multienzyme cascade system was developed to convert sucrose to d-allulose comprising sucrose invertase, d-glucose isomerase and PsDAE. Fruit juices were treated using this system to convert the high-calorie sugars, such as sucrose, d-glucose, and d-fructose, into d-allulose. The content of d-allulose among total monosaccharides in the treated fruit juice remained between 16 and 19% during 15 reaction cycles. This study provides an efficient strategy for the development of functional fruit juices containing d-allulose for diabetics and other special consumer categories.