磁性氧化石墨烯固定化氯过氧化物酶及其在奥酸性蓝45脱色中的应用
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摘要
以氧化石墨烯和Fe_3O_4为原料制备磁性氧化石墨烯,采用吸附法将氯过氧化物酶固定在磁性氧化石墨烯上,考察了固定化体系缓冲溶液p H值、固定化时间及反应温度对固定效果的影响.以氯过氧化物酶催化氧化奥酸性蓝45染料脱色反应为模型反应,探讨了固定化氯过氧化物酶的操作稳定性.实验结果表明,p H=3.5,反应15 min、反应温度15℃为固定化氯过氧化物酶的最佳催化条件;采用共沉淀法制备载体,加入的NH_4Fe(SO_4)_2·12H_2O与氧化石墨烯(GO)质量比为10.7∶1时,得到的磁性氧化石墨烯(TMGO)的酶固载量大于二者质量比为5.35∶1时得到的磁性氧化石墨烯(FMGO),这可能与FMGO氧化石墨烯表面的Fe_3O_4含量不足有关;与游离酶相比,固定化氯过氧化物酶表现出更好的酸碱稳定性、H_2O_2稳定性、热稳定性和储存稳定性,在35~50℃,聚集或堆积的磁性氧化石墨烯(TMGO)片层打开,导致固定化酶活损失率明显小于游离酶.重复使用5次后,TMGO-氯过氧化物酶(CPO)的相对活性仍然保持在60%以上.
Magnetic graphene oxide(MGO)was prepared using graphene oxide(GO)and Fe_3O_4.Chloroperoxidase(CPO)was immobilized via adsorption method in MGO,and the immobilization conditions,such as the temperature,time and p H value of the buffer solution,were optimized.The operating stability of immobilized CPO was studied using CPO as catalyst for the degradation of acid blue 45.The activity stability of immobilized CPO was investigated from four aspects of acid base stability,H_2O_2stability,thermal stability and storage stability using CPO as catalyst for the decolorization of acid blue 45.The results show that the optimal conditions are the phosphate buffer p H=3.5,the reaction time 15 min and the temperature 15℃.TMGO{m[NH_4Fe(SO_4)_2·12H_2O]∶m(GO)=10.7∶1}isabetterimmobilizedcarrierthanFMGO{m[NH_4Fe(SO_4)_2·12H_2O]∶m(GO)=5.35∶1}.And the immobilized CPO shows a better operation stability than the free enzyme.The accumulation layer of TMGO is opened at 35—50℃,leading to the loss rate of immobilized enzyme activity significantly less than that of the free enzyme.TMGO-CPO has a good reusability that the activity is still over 60%after recycling 5 times.
Magnetic graphene oxide(MGO)was prepared using graphene oxide(GO)and Fe_3O_4.Chloroperoxidase(CPO)was immobilized via adsorption method in MGO,and the immobilization conditions,such as the temperature,time and p H value of the buffer solution,were optimized.The operating stability of immobilized CPO was studied using CPO as catalyst for the degradation of acid blue 45.The activity stability of immobilized CPO was investigated from four aspects of acid base stability,H_2O_2stability,thermal stability and storage stability using CPO as catalyst for the decolorization of acid blue 45.The results show that the optimal conditions are the phosphate buffer p H=3.5,the reaction time 15 min and the temperature 15℃.TMGO{m[NH_4Fe(SO_4)_2·12H_2O]∶m(GO)=10.7∶1}isabetterimmobilizedcarrierthanFMGO{m[NH_4Fe(SO_4)_2·12H_2O]∶m(GO)=5.35∶1}.And the immobilized CPO shows a better operation stability than the free enzyme.The accumulation layer of TMGO is opened at 35—50℃,leading to the loss rate of immobilized enzyme activity significantly less than that of the free enzyme.TMGO-CPO has a good reusability that the activity is still over 60%after recycling 5 times.
引文
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[4]Chen B.,Zhao L.L.,Sun Q.F.,Li Y.R.,Li Y.,Environmental Science&Technology,2010,33(11),50—53(谌斌,赵亮亮,孙秋芳,李燕荣,李艳.环境科学技术,2010,33(11),50—53)
[5]Xiao X.,Cui K.P.,Wang Z.,Feng Y.Y.,Technology of Water Treatment,2016,42(12),55—59(肖霄,崔康平,王郑,冯莹莹.水处理技术,2016,42(12),55—59)
[6]Wang H.L.,Zhao Y.,Ma L.K.,Fan P.H.,Xu C.B.,Jiao C.L.,Lin A.J.,Chem.J.Chinese Universities,2016,37(2),335—341(王会丽,赵越,马乐宽,范鹏浩,徐从斌,焦春磊,林爱军.高等学校化学学报,2016,37(2),335—341)
[7]Geng Z.,Yu Y.,Zhu S.Y.,Yu H.B.,Liu J.C.,Bian D.J.,Yang X.,Huo H.L.,Huo M.X.,Chem.Res.Chinese Universities,2017,33(1),36—43
[8]Fu Y.C.,Callaway Z.,Lum J.,Wang R.H.,Lin J.H.,Li Y.B.,Anal.Chem.,2014,86(4),1965—1971
[9]Palomo J.M.,Filice M.,Biotechnology Advances,2015,33(5),605—613
[10]Wu L.T.,Wu S.S.,Xu Z.,Qiu Y.B.,Li S.,Xu H.,Biosensors&Bioelectronics,2016,80,59—66
[11]Alneyadi A.H.,Shah I.,Abu Qamar S.F.,Ashraf S.S.,Biomolecules,2017,7(1),31(1—18)
[12]Morozov A.N.,Chatfield D.C.,Int.J.Mol.Sci.,2016,17(8),1297(1—12)
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[15]Rueda N.,Albuquerque T.L.,Bartolome-Cabrero R.,Lopez L.F.,Torres R.,Ortiz C.,Dos Santos J.C.S.,Barbosa O.,Lafuente R.F.,Molecules,2016,21(5),646(1—18)
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[17]Sheldon R.A.,Pelt S.,Chem.Soc.Rev.,2013,42(15),6223—6235
[18]Jiao R.J.,Tan Y.,Jiang Y.C,Hu M.C,Li S.N,Zhai Q.G.,Ind.Eng.Chem.Res.,2014,53(31),12201—12208
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