d-Allulose production via a simplified in vitro multienzyme cascade strategy: Biosynthesis and crystallization

d- Allulose is a rare sugar with multiple physiological functions whose application has been restricted by low conversion rate, weak thermostability of the used enzymes, and complex production. In this study, a simplified multienzyme cascade strategy was designed for biosynthesis of d- allulose from d- glucose, and a detailed investigation of the crucial enzyme allulose 6-phosphate phosphatase was conducted. Under optimized conditions, the yield of d- allulose reached 4.12 ± 0.14 and 80.20 ± 3.20 mmol/L using 10.0 and 200.0 mmol/L d- glucose as substrate, respectively. Furthermore, the influence of various factors on the cooling crystallization of d- allulose was investigated, and the crystallization process was optimized. Consequently, a crystallization recovery rate of 77.43% ± 2.50% and a purity of 98.70% ± 0.05% were achieved. Moreover, the obtained crystals of d -allulose were characterized by XRD, TGA-DSC, and optical microscopy.