| Immobilized enzyme technology is a hot issue in the field of modern biocatalysis,and to improve the stability of enzyme is also the prerequisite and basis of its industrial application.When immobilized,enzyme can significantly improve its resistance to adverse environmental conditions.With the wide application of biocatalyst in industrial production,the traditional single forms of enzyme immobilization methods such as adsorption,embedding,covalent bonding and cross-linking immobilization are unable to meet the needs of industrial development.Therefore,studies on the immobilization enzyme technology have been turned to the double or multiple compound immobilization and non-traditional enzyme immobilization direction.These new methods provide a new idea for the development of enzyme immobilization,which can effectively solve the shortcomings of traditional enzyme immobilization technology and make an immobilized enzyme system more suitable for practical application.In this paper,almond ?-glucosidase was used as the research object.Based on the aqueous in situ polymerization technology,several different scale and morphological structures of immobilized ?-glucosidase were designed and prepared to improve the stability of the enzyme.The morphology and structure of the prepared ?-glucosidase nanogel,cross-linked ?-glucosidase aggregates(CLEAs)gel and almond powder gel were characterized,and then the author further made a study and discussion on the preparation conditions,enzymatic properties and catalytic properties of the system,which provided the theoretical basis for its industrial application.The major research contents are presented as follows:(1)The ?-glucosidase nanogel was prepared by means of the aqueous two-step in situ polymerization.Through the optimization of preparation conditions,the activity recovery of ?-glucosidase nanogel could reach up to 81.4%.?-Glucosidase nanogel was characterized by infrared spectroscopy(FT-IR),polyacrylamide gel electrophoresis(SDS-PAGE),dynamic light scattering(DLS),scanning electron microscopy(SEM)and fluorescence spectroscopy,the results showed that the prepared P-glucosidase nanogel was single-protein enzyme nanogel;the particle size was mainly distributed between 15nm to 40nm;the thickness of nanogel layer was about 3.5nm and maintained its native enzyme protein structure.The results of enzymatic properties studies showed that the Km value of ?-glucosidase nanogel was 3.61mmol/L and the Vmax was 2.52?mol/(min mg),and the affinity for the substrate was only slightly lower than that of the native enzyme.Compared with the native enzyme,the ?-glucosidase nanogel was significantly improved in pH stability,thermal stability,organic solvents tolerance and storage stability.(2)With substrates such as glucose and tyrosol,the ?-glucosidase nanogel was used as a catalyst to synthesize salidroside in a non-aqueous system.The effects of different organic solvents,water content,enzyme dosage,pH,temperature and the substrate concentration on the synthesis of salidroside were investigated.The results showed that the t-butanol was the best organic solvent;the water content of system was 5%;the enzyme dosage was 4.0U/mL;the pH value was 6.0;the reaction temperature was 60 ?;the concentrations of glucose and tyrosol were respectively 300mmol/L and 900mmol/L.Under these conditions,the concentration of salidroside could reach up to 23.7%after reaction for 96h.(3)The ?-glucosidase was purified from crude extract of bitter almond by means of the three-phase partitioning.Different parameters,such as different organic solvents and its concentration,ammonium sulfate saturation,pH and temperature were optimized to get highest purity fold and yield.The results showed that the t-butanol was the best organic solvent;the ratio of the volume of crude extract to t-butanol was 1.0:1.5;ammonium sulfate saturation was 50%;the pH of system was 5.0 and the optimum temperature was 25?.Under the optimized conditions,the purification factor of P-glucosidase was 5.97 and the activity recovery was 85.7%.(4)The almond P-glucosidase prepared by the three-phase partitioning was theresearch object,the CLEAs gel was prepared by combining the cross-linked enzyme aggregates with the aqueous in situ polymerization.Through optimization ofpreparation conditions,the activity recovery of CLEAs gel was 62.7%.Themorphological and structural characterization results showed that the average particlesize of the prepared CLEAs gel was about 50.390?m and the thickness of the polymershell was about 700nm.The results of enzymatic properties studies showed that the pH stability,thermal stability and organic solvents tolerance of CLEAs gel were significantly improved compared with free enzyme and CLEAs.In the continuous process of salidroside synthesis,CLEAs gel could still maintain high catalytic synthesis efficiency,and its catalytic effect was far better than that of free enzyme and CLEAs.(5)The native almond powder was used as the carrier of ?-glucosidase,based on the aqueous in situ polymerization technology,the almond powder gel was successfully prepared,in which almond powder gel retained 85%of the catalytic activity of its native counterpart.The morphological and structural characterization results showed that the average particle size of the prepared almond powder gel was about 45.437?m and the thickness of the surface polymer shell was only 460nm.The results of enzymatic properties studies showed that the pH stability,thermal stability,organic solvents tolerance and operational stability of almond powder gel were significantly improved compared with native almond powder.In the synthesis of salidroside,almond powder gel was continuously used for five batches,the average yield of salidroside was 16.1%,and the average catalytic synthesis capacity was3.63g/(L·U).Hence,the almond powder gel was manifested prominent advantages insalidroside production,which had wide applications on industry. |
PDF Full Text Request