【作者】 刘倩；

【导师】 倪良；

【作者基本信息】 江苏大学 ， 化学工程， 2017， 硕士

【摘要】 作为一种天然催化剂,酶在催化过程中具有的催化效率高,反应条件温和,高度专一的特点,同时也有易失活和难回收等阻碍酶工业应用的特点。为了利用酶优良催化性能的同时克服上述弱点,固定化酶成为专家学者们的研究重点。而固定化酶的性能主要取决于固定化方法和所使用的载体材料。本论文中,我们将制备三种不同的复合材料作为载体,以共价结合的方式,分别固定化纤维素酶或辣根过氧化物酶,具体内容如下:(1)设计合成了磁性复合微球Fe3O4@SiO2@P(GMA-co-NIPAM),并对其性能和形貌进行表征。该微球呈现直径约为350 nm的核-壳-壳结构,同时具有超顺磁性。将其应用到纤维素酶的固定化过程中,最佳固定化条件下得到纤维素酶固载量约为233.00 mg g-1。对固定化酶的酶学性能进行了测定,相对于游离酶,固定化酶的催化温度域拓宽;催化pH值没有太大影响;温度稳定性,贮藏稳定性均有提升。利用固定化酶反复水解催化羧甲基纤维素6次后,相对酶活为65.6%,具有较高的重复操作性。此外,固定化纤维素酶具有一定的热变性复性能力。(2)设计合成磁性多臂复合微球Fe3O4@PAA-6-arm-PEG-NH2,并对性能和形貌进行表征。利用戊二醛连接辣根过氧化物酶和材料表面的氨基,得到共价固定化辣根过氧化物酶。其次,探讨了固定化过程(戊二醛浓度,材料量,时间,温度)对固定化结果的影响,得到酶固载量约为139.82 mg g-1,远远高于其他学者的研究。固定化辣根过氧化物酶的酶学性能都有了一定程度的提升。其中,在贮存60天后,固定化辣根过氧化物酶的相对酶活高达71.05%;催化反应8次后,相对酶活为61.06%。最后,探讨了该固定化辣根过氧化物酶降解水溶液中苯酚的能力,优化降解过程,如苯酚浓度、过氧化氢量、固定化酶用量、反应时间。在此基础上,对含有苯酚的水溶液进行降解,对比载体材料和游离辣根过氧化物酶,固定化酶的降解速率明显高于游离酶和载体材料。(3)设计合成了磁性氧化石墨烯复合材料GO-Fe3O4-6-arm-PEG-NH2,并对其形貌结构进行表征。该材料呈现近乎透明的薄膜状,具有超顺磁性,饱和磁化强度为30.80 emu g-1。利用GO-Fe3O4-6-arm-PEG-NH2固定化辣根过氧化物酶,最佳酶固载量约为186.34 mg g-1。相比游离酶,固定化辣根过氧化物酶对催化环境的条件耐受性略有增强。温度稳定性,贮存稳定性和操作稳定性有所提升。利用固定化辣根过氧化物酶进行苯酚降解实验,由于氧化石墨烯材料具有一定类酶催化性能,固定化辣根过氧化物酶对苯酚的降解效果远远高于游离酶和载体材料。更多还原

【Abstract】 As a natural catalyst,enzymes have characteristics of high catalytic efficiency,mild reaction condition and high specificity in the catalytic process,but they also have characteristics of easy deactivation and difficult recovery,which inhibit their industrial application.In order to using the excellent catalytic performance of enzyme,overcoming the weakness,immobilized enzyme became the focus field for experts and scholars.And the performance of the immobilized enzyme mainly depends on the immobilization methods and carrier materials.In this work,we prepared three kinds of composites as carriers and immobilized cellulase or horseradish peroxidase in covalent bonding.The specific works are listed as follows:(1)The magnetic composite microspheres Fe3O4@SiO2@P(GMA-co-NIPAM)was synthesized and characterized.The microspheres exhibited a core-shell-shell structure with a diameter of about 350 nm and were superparamagnetic.They were applied to the cellulase immobilization process,and the cellulase loading amount was about 233.00 mg g-1 under the optimum immobilization conditions.The enzymatic properties of the immobilized enzyme were determined.Compared with the free enzyme,the catalytic temperature range of the immobilized enzyme was widened,and the catalytic pH value was not greatly affected.The thermal stability and and storage stability were improved.After repeated hydrolysis of carboxymethyl cellulose for 6 times,the relative activity was 65.6%.In addition,immobilized cellulase has thermal denaturation and refolding ability.(2)The magnetic multi-arm composite microspheres Fe3O4 @ PAA @6-arm-PEG-NH2 were designed and synthetized,and their properties and morphology were characterized.The glutaraldehyde was used to connect the horseradish peroxidase and the amino groups on the surface of the material to obtain covalently immobilized horseradish peroxidase.Secondly,the effects of immobilization process(glutaraldehyde concentration,amount of material,time and temperature)on the immobilization results were discussed.The enzyme loading amount was about 139.82 mg g-1,which was much higher than that of other scholars.The enzymatic properties of immobilized horseradish peroxidase have been improved.The relative activity of immobilized horseradish peroxidase was 71.05%after storage for 60 days.The catalytic activity was 61.06%after 8 times of catalytic reaction.Finally,the ability of the immobilized horseradish peroxidase degrading phenol in aqueous solution was discussed.The degradation process,such as phenol concentration,hydrogen peroxide content,immobilized enzyme dosage and reaction time,were optimized.On the basis of this,the degradation rate of the immobilized enzyme was significantly higher than that of the free enzyme and the carrier material.(3)The magnetic graphene composites GO-Fe3O4-6-arm-PEG-NH2 were designed and synthesized,and its morphological structure was characterized.The material exhibits a nearly transparent film,with superparamagnetism and a saturation magnetization of 30.80 emu g-1.The optimal enzyme loading amount was about 186.34 mg g-1 by using GO-Fe3O4-6-arm-PEG-NH2 immobilized horseradish peroxidase.Compared to free enzymes,immobilized horseradish peroxidase has slightly enhanced tolerance to the catalytic environment.Thermal stability,storage stability and operational stability have been improved.The efficiency of immobilized horseradish peroxidase on the removal of phenol was much higher than that of free enzyme and carrier material,because of the enzyme-like catalytic performance of the graphene material.更多还原