Immobilization Of Trehalose Synthase

Trehalose is a non-reducing disaccharide, has good physical and chemical properties and special biological characteristics, plays an increasingly important role in many fields. Trehalose synthase is an intramolecular glycosyltransferase, can converts maltose into trehalose by transglycosylation in one-step, the process is simple, easy to be controled and low cost, is an important way to product trehalose. Enzymes as biocatalysts, is efficient, loyal catalytic properties, widely used in many fields. But the free enzyme is easy to be inactive, difficult to purified and can't be re-used etc, which increase the difficulty and cost of industrial application. Immobilized enzyme has the characteristics of good enzyme stability, reusable, easy to separate from substrate etc, provides a wider range of applications for biological enzyme. Immobilized trehalose synthase, combined with trehalose synthase's simple technology and good features of immobilization technology, provides a new way for the industrial production of trehalose.This paper aims to find an immobilization methods of immobilized enzyme with easy preparation and good enzymatic properties, providing effective and workable solutions for the production of trehalose. The thesis mainly researched the suitable immobilized buffer and carrier, the carrier preparation, immobilization conditions, immobilized enzyme properties. Conclusions are as folows:(1) Porous alumina (Al2O3), titania (TiO2), attapulgite and polyacrylonitrile (PAN) membrane as carrier respectively.Na2HPO4-C6H8O7, KH2PO4-NaOH, Na2HPO4-NaH2PO4, Na2HPO4-KH2PO4 solution as the buffer system respectively, using adsorption immobilized trehalose synthase. Results showed that Na2HPO4-C6HgO7 solution as the enzyme preparation buffer, four Carriers showed better immobilized enzyme activity and specific activity; PAN membrane as the carrier, still retained 78% of the initial activity after repeated ten times, the other three carriers were almost inactivated. Therefore, selected Na2HPO4-C6H8O7 solution as the buffer, PAN membrane as the carrier.(2) PAN membrane were prepared by phase inversion, analyzed the impact of immobilization effect and mechanical properties of the membrane by changing PAN concentration, evaporation time, coagulation bath temperature, coagulation bath concentration the groove depth. The results showed that the immobilized position, evaporation time, coagulation bath concentration had little effect on immobilization. The optimum membrane preparation process:PAN mass fraction was 15%, evaporation time was 90 s, temperature of coagulation bath was 25?, glass groove depth was 0.1 mm. PAN membrane had good mechanical properties and immobilization effect under the process.(3) PAN membrane as carrier, Na2HPO4-C6H8O7 solution as the buffer, immobilized trehalose synthase by adsorption, optimized immobilized conditions. The results showed that: the optimal immobilization conditions were pH 7.6, temperature 35?, enzyme dosage 0.1 mg·cm-2 (free enzyme 1.85 mg·mL-1, specific enzyme activity:1.19 U·mg-1,), time 3 h, immobilized coupling rate 70.9%, activity recovered rate 50.21%. Immobilized enzyme optimum reaction temperature is 40?, increased 10? than the free enzyme; the optimum pH and initial substrate concentration were 7.4 and 200 g·L-1, remained good enzyme activity within a range of pH 5.4?8.0 and 20?50?; Ca2+, Zn2+, Fe2+, Mn2+ had little activation; Cu2+ had a slight inhibitory effect, Co2+, Al3+, Ba2+, Mg2+, Ni2+ were almost no effect, showed higher tolerance to metal irons than the free enzyme; the highest trehalose content was 50.9%, by-product of glucose content was 9.1%, which reduced by 6 percentage points compared with the free enzyme; the reaction of 12 h under optimum conditions as a batch reaction, retained 64.4% of the initial activity after repeated six times, showed good operational stability.Immobilized trehalose synthase on PAN membrane, has the following special features: ?For carrier, preparation process is simple, low cost and reusable; ?The enzyme used for immobilization is crude enzyme without purification, reduced the cost of enzyme greatly; immobilized enzyme preparation only need adsorption of 3 h; ?The membrane carrier's shape is adjustable, reactor design is flexible;?The byproducts of glucose content is 9.1%,which is the lowest content in the current reported studies.