Immobilization Of β-Galactosidase On Carboxymethyl Cellulose-Chitosan Compound

The main reason of causing lactose malabsorption is the lack of lactase. To make use of immobilizedβ-Galactosi'dase to hydrolyze lactose in milk can settle lactose malabsorption effectively. In this work, carboxymethyl cellulose-chitosan polyelectrolyte complex (CMC-CS), dialdehyde carboxymethyl cellulose-chitosan compound (dialdehyde CMC-CS) and dialdehyde carboxymethyl cellulose ester (dialdehyde CMC ester) were prepared by carboxymethyl cellulose (CMC) and chitosan (CS).β-Galactosidase was immobilized on mentioned above products. The immobilized condition was optimized and the properties of immobilized enzyme were studied. The film forming ability of CMC-CS was investigated. The adsorption capacity of metallic ion and the adsorption mechanism were discussed. The research was supported by grants from Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) (No.20060225008) and Graduate Innovation Projects of Northeast Forest University.CMC-CS compound were prepared by co-solution method in the acid medium.The intermolecular mechanism of action was discussed by FTIR,X-RD,SEM and DSC. The results showed that CMC and CS generated polyelectrolyte complex.They existed electrostatic and hydrogen bonds interaction, which weren't a simple mixing. CMC-CS compound membranes with different weight ratios(l:2,1:1,2:1) were prepared. The results indicated that the electrostatic and hydrogen bonds between CS and CMC were the best, when the CMC-CS membrane with a weight ratio was 1:1. Tension strength of the CMC-CS membranes reached 34.44 MPa, which was more than CS membranes'64.47%. The maximal acidity increased from pH value 5 to 4 and the tension strength of CMC-CS membranes was 25.71 MPa after dunking in pH value 6. The absorption rate of CMC-CS membranes could be effectivlly reduced. The intermoleculars of CMC-CS membranes were degration between 170℃and 330℃. The ionic bonds were fractured at.217.4℃and the electrostatic interaction was disappeared. The adsorption capacity of Pb2+ was 213.76μg/cm2.The static and dynamic filtrate-chelating capacity for Cu2+ per unit area of CMC-CS compound membrane were 231.25μg/cm2 and 24.05μg/cm2, respectively.β-Galactosidase was immobilized on CMC-CS polyelectrolyte complex with glutaraldehyde by crosslinking reaction. The immobilization conditions and the properties of the immobilized enzyme were studied. The results indicated that the max ratio activity was 0.0235 U/g, when the concentrantion of glutaraldehyde was 0.5%, the adding amountβ-galactosidase was 1 mg/mL per 0.5 g CMC-CS, immobilization and crosslink time were 9 and 3 h, respectively. The optimal reaction temperature and pH value of the immobilized enzyme were 50℃and 7; and of free enzyme were 30℃and 9.The immobilized enzyme showed lower thermal stability than that of the free enzyme, because of carrier and enzyme occurred irreversible reaction, but pH value stability of the immobilized enzyme were better than that of the free enzyme. The immobilizedβ-galactosidase showed higher operation stability when it was used repeatedly for three times. The Michaelis constant Km of the immobilized enzyme was 0.7051μmol/mL, which was lower than that of the free enzyme. It indicated that the affinity of immobilized enzyme and substrate increased and it was beneficial to the reaction.Dialdehyde CMC-CS compound was prepared from CMC with NaIO4 as oxidant. The structures of CMC-CS was characterized by FTIR and DSC. The results showed that C2 and C3 bit of hydroxy groups were oxidized to aldehyde groups. CMC and CS generated polyelectrolyte complex. They existed electrostatic bonds interaction.β-Galactosidase was immobilized on dialdehyde CMC-CS compound.The max ratio activity was 0.1156 U/g. The immobilized enzyme showed good thermal stability at a low temperature, but less thermal stability at a high temperature. The ratio activity of immobilized enzyme was first increases and then decreases with the increase of pH value. The ratio activity of immobilized enzyme was almost unchanged after using repeatedly for six times. The Michaelis constant of the immobilized enzyme lower than that of the free enzyme, which was beneficial to the reaction.Dialdehyde CMC ester was prepared by selective oxidation of CMC with epoxy chloropropane as cross linker and periodate sodium as oxidant. Dialdehyde CMC ester was characterized by FTIR, X-RD, DSC and measuring the aldehyde group.The properties of immobilized enzyme onto dialdehyde CMC ester were studied. The results showed that the optimal synthetical condition was that the ration of NaIO4 to esterification CMC was 2 to 1 by weight, the reaction temperature was 40℃, the reaction time was 2 hours and pH was 2,with aldehyde content of 80.67% under this condition. Carboxy groups of CMC were transformed into ester groups after esterification reaction. There were aldehyde groups in the products after oxidation reaction. Solubility property analysis showed that esterification CMC and dialdehyde CMC ester were not dissolved in common organic solvents. The max ratio activity was 0.0771 U/g, when the adding amountβ-galactosidase was 1 mg/mL, immobilization time was 6 h. The immobilized enzyme showed higher thermal stability than that of the free enzyme. The pH value stability of the immobilized enzyme onto dialdehyde CMC ester were greatly improved, whose pH alue stability was between onto CMC-CS's and dialdehyde CMC-CS's. The reusability was good. But the Michaelis constant of the immobilized enzyme showed that the affinity of immobilized enzyme and substrate decreased.