Resin Immobilized Laccase Optimization And Mathematical Model

Of enzyme to maintain its efficiency, specificity and characteristics of mild enzymatic reaction, while there are high stability, easy separation and recovery, can be repeatedly used, continuous and controlled operation, simple process and a series of advantages. Therefore, the development of a good carrier for immobilized and immobilized enzyme engineering methods important to study. In this paper, application of such a pore size of macroporous resin D380 with a larger surface area than the polymer material, using glutaraldehyde as crosslinking agent on the immobilized laccase:the resin immobilized laccase conditions were optimized, immobilization conditions established mathematical model of free and immobilized laccase enzymatic properties of the various comparative analysis with immobilized laccase degradation of dye had a preliminary trial.Some effects of enzyme immobilization of 9 single-factor study, optimization of the best single factor laccase immobilized good condition:the crosslinker concentration of 10%, the time of 2 hours cross-linking, cross-linking optimum temperature 40℃, the immobilized pH is 6.0, the enzyme dilution of 500, the enzyme was 10 ml, the PEB ion concentration of 0.2 mol/ L, the t fixed time of 6 hours, the fixed temperature of 30℃.By orthogonal test to optimize the immobilization conditions, using SPSS software and the SAS V8.2 software to optimize the response surface analysis and constructed a mathematical model of the conclusion that the optimum conditions:the best cross-linking agent concentration of 10%, the best time of 2 hours cross-linking, cross-linking optimum temperature 31℃, the best immobilized pH 7.2, the best enzyme dilution of 900, the best to the enzyme was 10ml, the best concentration of PEB is 0.2 mol/L, the best fixed time of 6 hours, the best constant temperature of 30℃. The test results under the optimum conditions are:the average results of immobilized enzyme activity was 15.53 U/g, the experimental value (15.53 U/g) and the model of the theoretical value (15.95 U/g) only the difference between the theoretical value of 2.63%, and further shows that the model is feasible.Immobilized and free laccase optimum pH, optimum temperature, thermal stability, Michaelis constant and enzymatic properties were compared:the free enzyme's optimum temperature is 50℃, the optimum temperature of immobilized enzyme were is 40℃, the immobilized laccase can be conducted at higher temperature reaction; free enzyme and immobilized laccase optimum pH were pH= 3 office, but when pH＞4 enzyme activity in higher fixed at the time, Description of the immobilized enzyme is more acid; immobilized laccase in pH 3-5 environment preservation 1h, the relative activity remained at 80%. The free laccase in pH 3-5 environment is not stable relative activity to 70% of the lowest, indicating the pH of immobilized laccase was more stable than free enzyme stability; fixed enzyme in 20℃-40℃heating for 2 hours after enzyme Activity remained at 75%, while the free enzyme activity dropped to 50% or less, laccase immobilized through its thermal stability has been significantly improved; immobilized enzyme remained after 30 days about 60% of activity, while activity of free enzyme activity in 30 days only 20% remaining, indicating good storage stability of the fixed enzyme; fixed enzyme in recycled 10 times, can still maintain 40% of the activity, indicating that a fixed Enzymes can be used repeatedly.Free laccase, laccase immobilized on the dye Congo red studies show,12 h degradation rates were 23.66%,38.77%, immobilized laccase decolorization better than free laccase, indicating immobilized enzyme for the enzyme laccase response provides a good micro-environment, the degradation process in the long run, the immobilized enzyme by less impact on the environment, on the pH and temperature have a larger scope, and ultimately achieving a higher degradation.