The Immobilization Of Laccase Using Covalent Binding And Its Application

Laccase is one kind of polyphenol oxidases, which catalyzes phenolic compounds andaromatic compounds to oxidize. Advantage of the immobilized enzyme is that it can berecycled. Therefore, it has great significance to develop a good carrier and immobilizedenzyme immobilization method for the study of enzyme engineering applications. In thisthesis, using the epoxy-modified cation exchange resin D152as the carrier by the methodof covalent connecting to immobilize laccase.Compared with traditional methods, thismethod has advantages of fixed mild conditions, simple operation process and firmconnection between enzymes and solid carrier, etc.and the resulting immobilized enzymehas good stability and reusability. This article has a systemic study on immobilizationconditions to laccase, nature of immobilized enzyme and degradation conditions ofimmobilized laccase to2,4-dichlorophen.1.Study on Immobilized ConditionsStudying the influences of (NH4)2SO4concentration, enzyme dosage, reaction pH,reaction temperature and reaction time to modified cation exchange resin immobilizedlaccase. And infrared spectroscopy to characterize the structure of the immobilized laccase.The results show that: under the conditions of1.5mol/L of (NH4)2SO4concentration, theamount of enzyme0.2U/g, pH6.5, temperature of35℃and time of3h, immobilizedlaccase has highest specific activity and activity recovery, respectively0.0264U/g and3.67%.2.Study on enzymatic properties of immobilized laccaseStudying on enzymatic properties of immobilized laccase, the optimal reaction pH ofimmobilized laccase was4.5, compared with the free laccase, optimal PH value shift to thealkaline pH; optimum temperature immobilized of laccase was50℃; Km value of laccasewas0.962mmol/L, compared with the free laccase, Km value of laccase increased,indicating the immobilized laccase reduced affinity with the substrate; the relative activityof immobilized laccase was40%at55℃, the relative activity of free laccase was10%at55℃; After repeating use of immobilized laccase for10times, the relative activity ofenzyme was still10%.3. Degradation of immobilized laccase to2,4-dichlorophenolUsing obtained immobilized laccase to degrade2,4-dichlorophen, studied the influence of substrate concentration, pH of the reaction and the reaction temperature to4-dichlorophen degradation process, degradating effect was the best at15mg/L ofconcentration to2,4-dichlorophen, degradation rate reached at35%, the removal rate at80%. The optimum reaction pH was5, the optimum temperature was40℃, After repeatinguse of immobilized laccase for5times, the removal rate was still before10%.