Synthesis Of D-psicose From D-glucose Catalysedby Immobilized Enzymes On Yeast Spores

D-psicose is a rare sugar which is particularly uncommon in nature. As an ideal sweetener, D-psicose has many good properties, such as low-energy, poor metabolic capacity and suppression of hepatic lipogenic enzyme activity. Therefore, D-psicose has many potential applications in food and pharmaceutical fields. D-psicose is a C3 epimer of D-fructose and D-psicose-3-epimerase(DPEase) catalyses the interconversion between each other. Xylose isomerase(XI) catalyses the reversible isomerization between D-glucose and D-fructose. In this study, XI from Thermus thermophilus HB8 and DPEase from Agrobacterium tumefaciens were immobilized on yeast spores based on the structure of spore by biological and chemical methods, respectively. The biosynthesis of D-psicose from D-glucose was realized catalyzed by immobilized enzymes on spore wall.According to the biological characteristics of Saccharomyces cerevisiae, soluble secreted proteins could be encapsulated in spore wall during the process of sporulation. The spore wall has a multi-layer structure which consists of mannan, β-glucan, chitosan and dityrosine from the innermost layer to outmost layer. Previous study has shown that the outmost dityrosine layer of spore wall could improve the resistance of spore against ether and protect target enzymes from degradation. Although dit1Δ spores without dityrosine layer could promote the interaction between enzymes and substrates, the resistances against environmental pressure were decrease. For osw2Δ spores, the existence of loose dityrosine layer could not only resist environmental pressure, but also increase the permeability of substrates. In this study, XI from T. thermophilus was expressed and immobilized in yeast spores(wild type, osw2Δ and dit1Δ spores). The results of RFP localization, western blot analysis and activity assay showed that XI was successfully expressed and immobilized on yeast spores. Moreover, osw2Δ spores showed good properties, such as good stability, substrate permeability and reusability. In addition, XI showed the highest activity at 85 oC, which avoided the germination of spore in the presence of D-glucose.In industry, heterologous enzymes from prokaryotes were usually expressed in eukaryotes like S. cerevisiae. However, the differences of protein modification such as glycosylation during the process of protein maturation may lead to inactivity of enzymes. In this study, when DPEase from A. tumefaciens was expressed in S. cerevisiae, no activity was detected. To address this problem, the potential glycosylation sites were mutated to preclude the glycosylation effect. Unfortunately, no activity was still observed. According to our previous study, DPEase was successfully immobilized on dit1Δ spores to function as “chitosan beads” after activated with cross-linking agent glutaraldhyde. Moreover, immobilized DPEase showed many good properties, such as p H and thermal stabilities and reusabilities.The synthesis of D-psicose was performed using immobilized enzymes via one-step and two-step methods. Due to the big difference of optimal reaction temperatures between XI and DPEase, the conversion rate was only 4 % via one-step method. What’s worse, the immobilized DPEase showed poor reusability at a high reaction temperature. Therefore, a two-step method was employed and both steps were performed under its optimal temperature. Although the conversion rate of D-psicose was about 5 % via two-step method, the highest conversion yield could reach 12 % after 3 cycles by repeating the two-step process.