基于聚乙烯亚胺改性纳米磁球的木瓜蛋白酶固定化  

Immobilization of Papain Based on Polyethyleneimine Modified Nano-magnetic Spheres

作　　者：梁杰 林国荣 邹汉勋 周凤超 郭天章 曾捷 刘涛 杨磊 蔡力锋 LIANG Jie;LIN Guorong;ZOU Hanxun;ZHOU Fengchao;GUO Tianzhang;ZENG Jie;LIU Tao;YANG Lei;CAI Lifeng(College of Environmental and Biological Engineering,Putian University,Putian 351100,China;Key Laboratory of Ecology-Toxicological Effects&Rubber Research Institute,Putian 351100,China;Key Laboratory of Ecological Environment and Information Atlas,Fujian Provincial University,Putian 351100,China;Fujian Province Sailor Foods Co.,Ltd.,Putian 351100,China)

机构地区：[1]莆田学院环境与生物工程学院,福建莆田351100 [2]福建省新型污染物生态毒理效应与控制重点实验室,福建莆田351100 [3]生态环境及其信息图谱福建省高校重点实验室,福建莆田351100 [4]福建省水手食品有限公司,福建莆田351100

出　　处：《食品工业科技》2024年第23期151-160,共10页Science and Technology of Food Industry

基　　金：福建省自然科学基金(2021J011107,2022J011160,2022J011164);福建省科学技术协会福建省科技经济融合服务平台(FJKX-2024XRH08)。

摘　　要：为实现木瓜蛋白酶(Papain,PAP)的高效固定化,本文构建了易于磁分离的亲水性三维立体结构的固定化载体,采用单因素和响应面法优化了固定条件。在磁性Fe_(3)O_(4)表面包覆聚甲基丙烯酸甲酯(PMMA)制备核-壳结构的纳米磁球Fe_(3)O_(4)@PMMA,接枝不同分子量聚乙烯亚胺(Polyethyleneimide,PEI),将PAP物理吸附在PEI上,以戊二醛(Glutaraldehyde,GA)为交联剂,构筑易于磁分离的亲水性三维立体结构的固定化载体Fe_(3)O_(4)@PMMA-PEI,对PAP交联封装并对其微观结构和理化性质进行表征。以纳米磁球的固定化能力为评价指标,探究其固定PAP的最佳交联时间、戊二醛浓度、交联温度、交联转速等。结果表明,磁球表面PMMA酯解后形成的羧基含量为0.95 mmol/g,接枝不同分子量PEI后,磁球表面氨基含量为0.16~0.48 mmol/g;纳米磁球对PAP的固载量可通过接枝PEI的分子量进行调控,当PEI分子量为1800时,固载量达到120.71 mg/g,选择PEI1800为接枝空间臂。纳米磁球微观形貌较规整,单体分散性良好,粒径约150~200 nm。通过响应面试验优化得到最佳条件:当交联时间为32 min、戊二醛浓度为0.02%、交联温度为25℃、交联转速为90 r/min时,Fe_(3)O_(4)@PMMA-PEI 1800对PAP的固载量达139.80 mg/g。Fe_(3)O_(4)@PMMA-PEI 1800-PAP在重复使用5次之后,相对酶活力能保持80.35%。本研究设计合成纳米结构的磁性微球,实现PAP在纳米磁球表面的高效固定,为固定化PAP的研究和应用提供实验依据。To achieve efficient immobilization of papain(PAP),this study constructed a hydrophilic three-dimensional matrix for easy magnetic separation of the immobilized enzyme.The immobilization conditions were optimized using single-factor and response methodology.A core-shell structured nano-magnetic bead Fe_(3)O_(4)@PMMA was prepared by coating the magnetic Fe_(3)O_(4) with poly(methyl methacrylate)(PMMA),followed by grafting polyethyleneimine(PEI)of different molecular weights.PAP was physically adsorbed onto the PEI,and then cross-linked using glutaraldehyde(GA)to construct an easily magnetically separable hydrophilic three-dimensional immobilization matrix Fe_(3)O_(4)@PMMA-PEI.The cross-linked encapsulation of PAP and its microstructure and physicochemical properties were characterized.Using the immobilization capacity of the nano-magnetic beads as the evaluation criterion,the optimal cross-linking time,glutaraldehyde concentration,cross-linking temperature,and cross-linking speed for immobilizing PAP were investigated.The results indicated that the carboxyl group content formed after the ester hydrolysis of the PMMA on the surface of the magnetic beads was 0.95 mmol/g.Following the grafting of PEI with different molecular weights,the amino group content on the surface of the magnetic beads was 0.16~0.48 mmol/g.The loading capacity of PAP on the nano-magnetic beads could be modulated by the molecular weight of the grafted PEI.When the molecular weight of PEI was 1800,the loading capacity reached 120.71 mg/g.PEI 1800 was selected as the grafting spacer arms.The nanomagnetic spheres exhibited well-defined morphology,uniform particle size distribution in the range of approximately 150~200 nm.Through response surface experiments,the best conditions were optimized:Cross-linking time of 32 minutes,glutaraldehyde concentration of 0.02%,cross-linking temperature of 25℃,and cross-linking speed of 90 revolutions per minute.Under these conditions,Fe_(3)O_(4)@PMMA-PEI 1800 achieved a PAP loading capacity of 139.80 mg/g.Impor

关 键 词：纳米磁球 木瓜蛋白酶(PAP) 聚乙烯亚胺(PEI) 固定化 固载量 

分 类 号：TS201.25[轻工技术与工程—食品科学]