| A certain amount of wheat flour was mixed with phosphate buffer solution and stirredevenly with high organization homogenate machine, after placing overnight at4℃, thesupernatant was collected by centrifugation method. The supernatant underwent the test twice ofammonium sulfate precipitation. For the first time, a certain amount of ammonium sulfatepowder (saturation is30%) was added and completely dissolved, after half an hour under4℃,the liquid supernatantand was got after centrifugal separation. For the second time, the newobtained supernatant was added to reach a70%ammonium sulfate saturation, after half anhour under4℃, the precipitate was obtained by centrifugation and dissolved by phosphatebuffer solution, after being dialyzed for2days in phosphate buffer solution, the wheatesterase enzyme solution was obtained and stored in the fridge to be used in the next step.Then the optimal conditions to determine the wheat esterase enzyme activity was fixed andthe kinetic properties of wheat esterase were also tested, such as the optimum temperature, thesuitable pH, pH stability and thermostability. The experimental results showed that the freeenzyme activity was80.26%,68.67%and10.05%of its original value after storingrespectively at40℃,50℃,60℃for8h.Two kinds of the porous polymer microspheres(GMA-AM-EGDMA) were synthesizedfrom glycidyl methacrylate(GMA) and acrylamide(AM) as monomer, ethylene glycoldimethacrylate(EGDMA) as cross-linker, methanol and distilled water as liquid porogenicagent, nano-calcium carbonate as solid porogenic agent, azodiisobutyronitrile(AIBN) asinitiator by double oil phase suspension polymerization. The ratios of the GMA, AM, thecrosslinking agent EGDMA and the nano-calcium carbonate were investigated. Under theoptimum conditions, two kinds of porous GMA-AM-EGDMA was obtained and usedseparately to immobilize the enzyme got above. During the process of the immobilization, theoptimal conditions, such as the immobilization temperature, time and the enzymeconcentration, were studied. Meanwhile, the kinetic properties of both kinds of theimmobilized enzyme were determined, such as the optimum temperature, the suitable pH, the acid and alkali stability, thermostability, operation stability and so on, the results werecompared with those of the free enzyme. The conclusion obtained indicated that: the pHstability and thermostability of both immobilized enzymes were much better than those of thefree enzyme, and both immobilized enzyme had good operational stability. The poly(GMA-AM-EGDMA) particles prepared jointly with solid-liquid porogenic reagent had abigger specific surface area, a higher activity of the immobilized enzyme and betterimmobilization effect than that prepared only with liquid porogenic agent. So, the formerporous polymer was more suitable to be used in the area of the enzyme catalyzation. |
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