Preparation And Characterization Of Immobilization α-amylase Base On P(DMAA-co-AM) Hydrogel

α-amylase is an important biological enzyme,free enzyme is great environmentalsensitivity,unstable,easy to inactivation,and can’t realize the continuous catalyticreaction and automation,its wide application is limited seriously,so,the research ofα-amylase immobilization is very important.As hydrogel with good mechanicalperformance, stable physical and chemical properties, swelling andundissolve,etc.hydrogel has vast potential for future development in manyfields,includeing materials seperation and purification,drug delivery system andenzyme immobilization,etc. This paper studyed α-amylase immobilized by hydrogeland its performance.The activity of immobilization enzyme are closely related to the carrier materialsand the immobilized methods,In this paper, taking P（DMAA-co-AM） hydrogel withgood biocompatibility as carrier immobilized α-amylase by situ polymerization andphysical adsorption,studyed optimal conditions of preparation immobilizationα-amylase and performance of immobiization α-amylase. Base on this,introducesodium alginate（SA） to P（DMAA-co-AM） hydrogel to improve the performance ofP（DMAA-co-AM） hydrogel,study the performance of immobilized α-amylase byP（DMAA-co-AM） hydrogel modified by SA.The main contents of this paper asfollows:1.Taking P（DMAA-co-AM） hydrogel as carrier,studyed separately optimalconditions of preparation immobilization α-amylase by situ polymerization andphysical adsorption.The results showed that:under the condition ofn（DMAA）:n（AM）=2:1,the amount of BMA is0.3%, the amount of APS is0.6%,theconcentration of α-amylase is1.0mg/mL,immobilization α-amylase by situpolymerization appeared the maximum activity.Under this condition,fixed rated up to95.16%;while,n（DMAA）:n（AM）=1:1,the amount of BMA is0.2%, the amount ofAPS is0.6%,the concentration of α-amylase is2.5mg/mL,immobilization α-amylaseby physical adsorption appeared the maximum activity.Under this condition,fixedrated is63.51%. 2.Taking P（DMAA-co-AM） hydrogel as carrier, immobilization α-amylase bysitu polymerization and physical adsorption,study the influence of temperature andpH on immobilization enzyme and free enzyme.The results showed that:the optimumtemperatures and pH of free enzyme are50℃and6.0,immobilization α-amylase bysitu polymerization shifted to60℃and6.6, immobilization α-amylase by physicaladsorption has the same optimum temperatures and pH to free enzyme.Study thestability of the immobilization α-amylase by the two methods,the results showedthat:immobilization α-amylase by situ polymerization has wider activity temperatureand pH range,in addition,temperature stability,operation stability, storage stability andfixed rate are satisfying;While,physical adsorption has more simple operation processand doesn’t change molecules structure of enzyme in immobilization process.3.Taking P（DMAA-co-AM）/SA hydrogel as carrier,study the optimum conditionbase on immobilized α-amylase by P（DMAA-co-AM） hydrogel.The results showedthat:the amount of SA is2%， P（DMAA-co-AM） hydrogel modified by SAimmobilization α-amylase has the maximum activity,under this condition,fixedrated is58.23%.The optimum temperatures and pH of immobilization α-amylase is50℃and6.6, the use of pH range of immobilization enzyme is broaden,at the sametime, its stability is improved obviously.4.The enzymatic reaction kinetics of free and immobilization enzyme showedthat：immobilization α-amylase has better substrate affinity than free enzyme.The Kmvalues and V_maxvalues of free enzyme are5.16mg/mL,2.56mg/min;the Kmvaluesand V_maxvalues are1.33mg/mL,0.52mg/min and1.44mg/mL,0.58mg/min for bothimmobilization α-amylase by situ polymerization and physical adsorption.The Kmvalues and V_maxvalues of immobilization α-amylase modified by SA are1.07mg/mL,0.54mg/min,and improved the substrate affinity after modified by SA.