Preparation And Application Of Immobilized Enzyme Using Alginate/Graphene Oxide As The Carrier

Sodium alginate(SA)is a natural polysaccharide extracted from brown seaweeds.Due to its some unique properties such as non-toxicity,hydrophilicity,good biocompatibility and relatively low cost,SA has been usually used as immobilization support.However,neat SA exhibits many disadvantages,such as strong hydrophilic character,poor mechanical properties and low thermal stability which greatly restrict its applications.One significant strategy is to fill inorganic or organic nanoparticles into SA matrix to overcome its disadvantages.Graphene oxide(GO)has been attracted a great deal of attention in recent years owning to its low-cost,high surface area and mechanical properties.GO has numerous and various oxygen-containing functional groups(hydroxyl,epoxy,carbonyl and carboxyl groups,etc.).GO has also been widely used as a support for enzyme immobilization.Complexation of GO with SA can not only enhance the thermal stability and mechanical properties of the material,but alao improve the immobilization effect of enzyme.In the present work,SA/GO beads were prepared by gel method,then SA/GO beads were further activated by N,N'-dicyclohexylcarbodiimide(DCC)/N-hydroxysuccinimide(NHS).The DCC/NHS/SA/GO beads were used as the support of immobilized pectinase(I-P)and immobilized pectinase-glucoamylase(I-PG).The conditions and parameters of I-P and I-PG preparation were optimized by employing single factor tests and response surface method(RSM)/orthogonal experiment.Finally,the I-PG was used in the preparation of pumpkin-haw juices.The main contents and results of the present study are as follows:1.The SA/GO beads were activated by glutaraldehyde,epoxy chloropropan,DCC/NHS respectively.The results showed that DCC/NHS is the optimal activating agents for the immobilization of pectinase onto SA/GO(4%wtGO).The immobilization conditions of pectinase,optimized by employing single factor tests and RSM,are ascertained as:the concentration of pectinase 10.24 mg/mL,immobilization pH 3.0,immobilization temperature 30 ?,immobilization time 30 min.The verification experiment showed that the catalytic activity of I-P prepared under the optimal conditions reached 1236.86±40.21 U/g.2.In the present work,Fourier Transform Infra Red Spectroscopy(FT-IR)and Scanning electron microscope(SEM)analyzed the changes in the molecular-structure of SA/GO beads and the microstructure of SA/GO beads during the preparation of I-P demonstrated the good compatibility of the SA and GO.Pectinase was immobilized onto SA/GO beads via covalent bonds(amide linkage).Thermogravimetric analysis indicated that SA/GO has a superior thermal stability to that of SA.The kinetic parameters of I-P were measured using pectin as a substrate,which followed Michaelis-Menten equation.The Km1 of I-P was determined to be 7.71 mg/mL which was higher than that of free one(6.73 mg/mL).The optimal pH of free pectinase was 4.5,while that of I-P shifted to pH 4.0.The optimal temperature of I-P was improved to 60 ?,10 ? higher than that of free form.Furthermore,The I-P had a higher thermal stability than free pectinase.Reusability study indicated that the activity of I-P even retained over 70%of initial state after 6 times cycles.3.The immobilization conditions of I-PG were optimized by using single factor tests and the orthogonal experiments:immobilization pH 4.0,immobilization temperature 40 ?,immobilization time 35 min.The kinetic parameters of I-PG were measured as follows:The optimal pH of I-PG shifted to more acidic condition(pH 4.5)as compared to that of free enzyme.The optimal temperature of I-PG was improved to 60 ?.Furthermore,The I-PG had a higher thermal stability than free enzyme.The activity of I-PG even retained over 60%of initial state after 6 times reusing.4.The optimal conditions for the enzymolysis of pumpkin-hawthorn juices by I-PG enzymolysis,taking the total soluble solid(TSS),light transmittance,reducing sugar content as indexs,were determined by employing the single factor experiments and the orthogonal experiments.The optimized enzymatic conditions are:enzyme concentration 0.5 g/100mL,reaction temperature 60 ?,reaction time 100 min.The verification experiment showed that TSS,light transmittance,reducing sugar content of the pumpkin-hawthorn juices prepared under optimal conditions were measured to be 5.4±0.1%,94.3±0.36%,15.6±0.31 mg/mL,respectively.Analyses on the changes of the main components in pumpkin-hawthorn juices before and after treatment with I-PG indicated that after treatment with I-PG,the pectin content and the soluble protein were reduced,the light transmittance was increased,which can improve the clarity of fruit juice and avoid the secondary turbidity of fruit juice.In addition,the reducing sugar content which was more easily absorbed by the body was increased.