| Purpose: This study carried out the immoblization of lipase by using HPD 750 macroporous absorbent resin of medium polarity and LX-120 poxy resin as the carrier and epoxy agent Poly(ethylene glycol)diglycidyl ether as cross-linker.By preparing two immobilized lipases,the application value of these two resins and this crosslinker for the production of industrial immobilized enzymes was explored.Methods:(1)By comparing the enzymatic activity of the immobilized enzyme and the recovery rate of the enzyme activity,the suitable immobilized macroporous resin and epoxy resin were determined.(2)By comparing the enzymatic activity of the immobilized enzyme,an appropriate immobilization cross-linker was determined.(3)By comparing the enzymatic activity of the immobilized enzyme,the optimum temperature,time,p H,and amount of enzyme for the adsorption immobilization and the covalent immobilization of the epoxy resin were determined,and the optimum conditions were optimized by the response surface experiment.(4)By comparing the enzymatic activity of the immobilized enzyme,the optimum temperature,time,and cross-linker concentration of the macroporous resin for cross-linking immobilization were determined,and the optimum conditions were optimized by the response surface experiment.(5)By comparing the enzymatic activity of the immobilized enzyme,the thermal stability,acid-base stability,operational stability and storage stability of the immobilized enzyme prepared were investigated.Results:(1)The best carrier for lipase adsorption-crosslinking immobilization was styrene-based medium-polar macroporous resin HPD 750;the best cross-linker was novel epoxy crosslinker polyethylene glycol diglycidyl ether;the best carrier for the covalent immobilization was epoxy resin LX-120.(2)Response surface experiments to optimize the macroporous resin adsorption-crosslinked immobilized conditions: adsorption temperature being 45 ?,enzyme loading of lipase being 60 mg per gram carrier,the concentration of cross-linker being 0.7 %,cross-linking time being 12.5 h,the cross-linking temperature being 30 ? and p H value being 6.36.(3)Response surface experiments to optimize the epoxy resin covalently bonded binding immobilized conditions: p H 6.27,adsorption time 2 h,adsorption temperature 26 °C,to the amount of enzyme 18.5 mg/g.(4)Under those optimized conditions,the activity of adsorption-crosslinking immobilized enzyme was 565.31 U/g,and the enzyme activity recovery rate was 32.16 %;the activity of the covalently immobilized enzyme was 544.28 U/g,the enzyme activity recovery was 73.67 %.(5)The optimum reaction temperature for adsorption-crosslinking immobilized enzyme on macroporous resin and free enzyme was 60 °C.When the temperature increased from 60 °C to 100 °C,the activity of immobilized enzyme and free enzyme decreased,but the activity of free enzyme decreased rapidly.At 100 °C,the activity of immobilized enzyme still remained above 71.2 %,but the free enzyme activity was only 38.75 %.At 60 °C for 7 h,the activity of immobilized enzyme remained approximately 72.94 %,while the free enzyme activity retained 55.47 %.In the environment of p H 5 to p H 8,the activity retention rate of immobilized enzyme was higher than that of free enzyme.After 10 consecutive operations,the enzymatic activity of the immobilized enzyme was retained at 34.86 %.Stored at 4 °C for 30 days,the immobilized enzyme activity retained 64.81 %.In summary,when compared to the free enzyme,the prepared immobilized enzyme had significantly improved in thermal and acid-base stability,and had a better stability in operation and storage.(6)The optimal reaction temperature for the covalently immobilized enzyme and free enzyme of the epoxy resin was 60 °C.After incubation at 60 °C for 7 h,the activity of immobilized enzyme was about 66.21 %,while that of free enzyme was retained at 55.47 %.Under the environment of p H 5 to p H 10,the enzyme activity retention rate of immobilized enzyme was significantly higher than that of free enzyme.After 10 consecutive operations,the immobilized enzyme activity retained 64.21 %.Stored at 4 °C for 30 days,the immobilized enzyme activity retained 78.14 %.Therefore,when compared to the free enzyme,thermal and acid-base stability,operation stability and storage stability of the prepared covalent immobilized enzyme was significantly improved.,Conclusion:(1)Domestic macroporous resin HPD 750 and epoxy resin LX-120 can be used as carriers for lipase immobilization;(2)Polyethylene glycol diglycidyl ether can be cross-linked;(3)adsorption-crosslinked immobilized and covalently bound immobilized lipases was successfully established;(4)Compared to adsorption-crosslinking immobilized enzyme,covalently binds immobilized enzyme on epoxy resin has better acid-base stability,operational stability and storage stability. |
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