Cloning,Expression,and Immobilization Of Sucrose Phosphorylase

Sucrose phosphorylase is a specificity enzyme which catalyzes transglycosylation. It could transfer alpha-glucosyl residues from donor, such as sucrose and a-D-glucose 1-phosphate, to various acceptor molecules with hydroxyl and carboxyl. It has vast application to improve the water-solubility, stability and biocompatibility of some commercial compounds.A sucrose phosphorylase gene (named BLSpase) was firstly cloned from Bifidobacterium longum, expressed in Escherichia coli and the product was purified. The purity of BLSpase was higher than 95% and its molecular weight was about 57 kDa. The specific activity of BLSpase was 2.143U/mg at 30℃, pH 7.0. The optimum temperature and pH of BLSpase were 45℃ and 6.0, respectively. Co2+ could improve its catalysis activity. Then the preparation of cross-linked enzyme aggregates by glutaraldehyde (GA) was explored. After adding bovine serum albumin (BSA), the activity recovery was about 76% at this time. The optimal temperature of the co-aggregation of BLSpase and BSA (named BSA-BLSpase-CLEAs) was 5℃ higher than the free enzyme. BSA-BLSpase-CLEAs were still stable after incubation at 60℃ for 1 hour with the relative residual activity about 85%. After BSA-BLSpase-CLEAs were stored at 4℃ for 15 days, the relative residual activity was 80%. Mn2+, Cu2+ and Co2+ were activators of the BSA-BLSpase-CLEAs. At last, BLSpase was immobilized on chitosan microsphere with GA and tris(hydroxymethyl)phosphine (THP) as crosslinker, respectively. After optimization, the activity of the immobilized enzyme using GA as the crosslinking agent (named GA-BLSpase) was 217.2 U/g, with the optimum temperature was 15℃ higher than the free enzyme; the activity of the immobilized enzyme using THP as the crosslinking agent (named THP-BLSpase) was 346.2 U/g, with the optimum temperature was 20℃ higher than the free enzyme, and its optimal pH was changed 3 units to acidic direction than the free enzyme. THP-BLSpase was stable after incubation in pH 4.0 for 30 minutes with the relative residual activity about 100%. Li+, Cu2+ and Mg2+ were activators of GA-BLSpase. And Mn2+ could make the activity of THP-BLSpase increase 1.2-fold.In summary, the optimum temperature and thermostability of the immobilized BLSpase were greater than the free enzyme. The two kinds of immobilized enzyme with carriers (GA-BLSpase and THP-BLSpase) were more suitable for the reaction with high temperature among them. They have good stability in operation which could improve the utilization, and can be applied to the industrial production of the higher reaction temperature. BSA-BLSpase-CLEAs have long storage stability, and were suitable for long-term preservation of transportation needs. THP-BLSpase showed a high activity and stability under acidic conditions, and could catalyze transglycosylation reaction to the acceptor with carboxyl group. This convenient procedure thus allows us to simultaneously improve stability and specificity, and could find widespread application in the field of sucrose phosphorylase engineering.