Expression And Immobilization Of D-psicose 3-epimerase And Its Application In The Production Of D-psicose

D-Psicose, a rarel ketohexose, is an isomer of D-Fructose. As a novel low-calorie functional factor, it has a great potential for use in a variety of applications, such as food, medicine and health care products. D-psicose 3-epimerase is an intracellular enzyme that possesses the catalytic activity for the conversion of D-fructose to D-psicose. Considering that D-psicose is mainly used in food industry, the food-grade Bacillus subtilis was used as expression system of D-psicose 3-epimerase. In this study, recombinants Bacillus subtilis with the DPE gene from Clostridium cellulolyticum H10 (CCDPE) and the DPE gene from Ruminococcus sp. (RDPE) were constructed, respectively. The CCDPE gene expressed in Bacillus subtilis was purified and characterized in detail. The immobilization of the curde enzyme of CCDPE and RDPE was studied, respectively, and the physicochemical property of this two immobilized enzymes were also compared.The length of DPE gene from Clostridium cellulolyticum H10 was 882 bp, and the molecular mass of the enzyme was estimated to be approximately 33 kDa. The Bacillus subtilis cells harbouring CCDPE gene were cultivated in LB medium containing 50μg kanamycin/ml at 37℃ and 200 rpm. The cells were harvested by centrifugation and disrupted by sonication. The supernatant was obtained by centrifugation. The crude extract was purified using Ni-NTA affinity column and Source 15Q anion-exchange chromatography column. It was showed that the purity of recombinant CCDPE was purified to more than 90% from the SDS-PAGE results. Moreover, the vast majority of the recombinant protein is soluble. The purified enzyme was optimally active at 50℃ and pH 8.0. The enzyme activity was not with metal ion exist dependency relationship, but Co2+ could accelerate the activity of CCDPE. The recombinanted CCDPE has higher catalytic efficiency for D-psicose.Various materials and methods were used to immobilize CCDPE. It has been found that the enzyme recovery was higher when using chitosan as the carrier. The optimal ratio of enzyme to carrier was 15:1 (U/g), and the optimal time for immobilization was 3 h. After immobilized under this optimal condition, the CCDPE showed the highest activity recovery, which was 45%. In addition, the crude enzyme of RDPE was also immobilized with the same method. The effects of pH, temperature and metal ions on the activity of free and immobilized CCDPE and RDPE were investigated, respectively. For CCDPE, the optimum temperature and pH of the immobilized enzyme were 55℃ and 8.5,respectively,which was higher than those of the free enzyme (50℃,8.0). For RDPE, the optimum temperature of the immobilized and free enzyme were the same, but the optimum pH of the immobilized enzyme (9.0) higher than that of the free enzyme (8.0). The activity of free and immobilized CCDPE and RDPE were inhibited in the presence of Ni2+, Cu2+ and Zn2+, but the activity of free CCDPE was enhanced in the presence of Co2+ and Mn2+. Furthermore, Mn2+ and Fe3+ can strengthen the activity of free RDPE.The continuous enzymatic conversion of D-fructose to D-psicose with an immobilized CCDPE in packed-bed reactor was studied. The productivity of 6.7 g·L-1·h-1 was achieved with the yield of 19% at the optimal flow rate of 2.5 mL/min.Continuous production was performed at 40℃, after 168 h the reaction yield was maintained above 13%.