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

• A novel d -allulose 3-epimerase was characterized with a half-life of 6 h at 60 °C. • Immobilized d -allulose 3-epimerase shows well-reusability during 11 reuse cycles. • A multienzyme cascade system was developed to convert sucrose to d -allulose. • The content of d -allulose in treated fruit juice remained about 17% during 15 cycles. 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.