The Immobilization Of β-galactosidase Using Covalent Binding And Its Application

In this thesis, β-galactosidase was immobilized using expoxy-modified D152cationexchange r esin by c ovalent bi nding. Compared with traditional covalent method, t hismethod have a lot of advantage including mild immobilization condition, simple technicalprocess and strong intergration between enzyme and carrier, simultaneously, immobilizedenzyme has good stability and repeatability. In the paper, the conditions of immobilization,the properties of the immobilized enzyme and the lactose hydrolysis kinetics catalyzed byimmobilized enzyme were researched.1. The conditions of immobilizationThe ef fects of （NH₄）₂SO₄concentration, enzyme a ddition quantity, pH value,temperature and time on β-galactosidase immobilization by modified-expoxy D152cationexchange resin were investigated, moreover, the structure of immobilized enzyme wascharacterized by i nfrared spectrum（IR） a nd scanning e lectron m icroscope（SEM）. Theresults indicated that under t he c onditions of （NH₄）₂SO₄concentration1.75mol·L^-1,enzyme addition quantity1.54U·g^-1, pH6.5, temperature35℃and time4h, the specificactivity and activity recovery of immobilized β-galactosidase is0.127U·g^-1and8.25%,respectively.2. The properties of immobilized enzyme（1）Kinetics: The ef fects of substrate conc entration, time, pH va lue, temperature,activators a nd i nhibitors on O NPG hydr olysis r eaction rate catalyzed by immobilizedβ-galactosidase were i nvestigated. Results a s f ollows: the opt imum p H va lue a ndtemperature f or thi s immobilized e nzyme to c atalyse O NPG hydr olsis ar e6.5⁷.5and35℃⁴5℃, respectively; the K_mfor ONPG is equal to245.652μmol·L^-1; Ca²⁺、Mn²⁺andFe²⁺perform activation at <10^-3mol·L^-1; Mg²⁺perform activation at <10^-5mol·L^-1, butinhibition at>10^-4mol·L^-1; Zn²⁺perform activation at <10^-5mol·L^-1or>10^-4mol·L^-1;Cu2+perform inhibition at <10^-5mol·L^-1or>10^-4mol·L^-1.（2）Stability: The effects of pH value, temperature, time and operation on the stabilityof immobilized β-galactosidase were investigated. Results as follows: This immobilizedenzyme is suited to be saved at pH7.5and lower than20℃; when it is saved in pH7.5environment at5℃and20℃, its half-life is32d and28d, respectively; its batch operatingstability is better and its residual activity exceeds85%after ten consecutive reactions. 3. The kinetics of lactose hydrolysis which catalyzed by immobilized enzymeThe effects of lactose concentration, time, pH value, temperature on lactose hydrolysisreaction rate catalyzed by i mmobilized β-galactosidase were i nvestigated. Results asfollows: The optimum pH value and temperature for this immobilized enzyme to catalyzelactose hydrolysis is7.0and35℃, respectively, the K_mfor lactose is equal to649.314μmol·L^-1; enzyme activity reduce to1/25of that for ONPG hydrolsis approximately; thereis significant positive linear correlation between lactose hydrolyzing rate and reaction time.