CN109234258B - 漆酶天然介体固定化磁性纳米粒子及其制备方法和应用 - Google Patents

漆酶天然介体固定化磁性纳米粒子及其制备方法和应用 Download PDF

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CN109234258B
CN109234258B CN201811058692.XA CN201811058692A CN109234258B CN 109234258 B CN109234258 B CN 109234258B CN 201811058692 A CN201811058692 A CN 201811058692A CN 109234258 B CN109234258 B CN 109234258B
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黄娟
杨赟
张坤
王耀坤
刘友勋
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Xinxiang Medical University
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Abstract

本发明提供了一种漆酶天然介体固定化磁性纳米粒子及其制备方法和应用。该漆酶天然介体固定化磁性纳米粒子的制备方法，包括如下步骤：将表面氨基化的磁性纳米粒子与带有羧基的漆酶天然介体进行酰胺化反应。本发明首次将漆酶的天然介体有效地固定于磁性纳米载体上并表现出高活性，该天然介体修饰磁性纳米粒子制备工艺简单、成本低廉。本发明得到的漆酶天然介体修饰的磁纳米粒子可以有效地作为漆酶介体使用，可以重复使用达8次以上。

Description

漆酶天然介体固定化磁性纳米粒子及其制备方法和应用
技术领域
本发明涉及环境治理技术领域，更具体地，涉及一种漆酶天然介体固定化磁性纳米粒子及其制备方法和应用。
背景技术
漆酶(EC 1.10.3.2，苯二酚氧化酶)是一种氧化酶，有很广泛底物范围，在许多工业和生物技术中得到应用，是一类非常重要的酶。研究报道表明，在单独应用漆酶时，其对氧化还原电位较高的底物，不能够发生完全的催化作用。但是，在加入一些被称为漆酶介体的小分子物质后，如1-羟基苯并***(HBT)、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)、芥子酸、阿魏酸、4-羟基肉桂酸、丁香醛等，能够显著促进漆酶对非酚类或非酶类底物的催化作用。这些介体作为电子载体，能使电子在酶和非酚类底物之间转移。漆酶介体的发现，进一步促进了漆酶的广泛使用，在漆酶-介体***在造纸工业、有机合成、生物传感器和环境处理等多个领域有广泛应用。目前，使用较多的漆酶介体都属于人工合成，但这些合成介体的使用存在一些问题：其一，其具有一定的毒性，使用后废弃会造成环境污染；其二，这些介体的活化后会反作用于漆酶蛋白的氨基酸从而使漆酶活性降低或失活；其三，人工和成的介体成本高，导致漆酶介体***在实际应用中成本高，难以推广应用。部分研究者考虑用漆酶的天然介体代替合成介体，因为天然介体本身是一种漆酶底物，且能够稳定漆酶构象，保持漆酶的高活性，更重要的是天然介体对环境无毒性。但天然介体使用后，也存在随着废液流失难以回收，不能根本地解决漆酶介体在应用中的成本问题。
将磁性纳米材料应用于各种小分子和生物大分子的固定化已经成为研究的热点。由于磁性纳米载体材料本身具备的磁响应和粒子表面积大、粒径小的特点，给固定化的分子带来了优势，如易分离、不易被反应环境中的酸碱等腐蚀、稳定性强、回收率高、成本低等。目前为止，将漆酶固定化和合成介体固定化均有相关的研究报道，但还没有发现将漆酶天然介体固定化的报道。
在染料生产和纺织的过程中会使用到大量的染料，而很多染料具有良好的水溶性，生产过程和洗涤过程中产生的染料废水，对于水体会造成明显的污染。大部分染料具有一定的毒性，难以被环境吸收和生物降解，影响水生生物的生存和人体健康，造成严重的环境污染问题，因此当今面临的一个重大环境污染问题是如何除去污水中的染料。寻找高效且低成本的除去工业废水中染料的方法是一个重大的挑战，现已应用的方法有吸附、混凝、生物降解、化学降解和光降解等，但这些方法普遍存在效率低和成本高等缺陷。生物降解技术，尤其是生物催化技术，作为一种新兴的处理方法，具有能耗低、易操作、降解效率高等优点，是一项前景广阔的处理技术。采用磁性纳米为载体固定漆酶天然介体能够明显提高漆酶的稳定性和介体重复利用率，从而降低介体使用成本，在含染料污水处理领域中具有广泛的应用前景。
发明内容
本发明的第一目的在于提供一种漆酶天然介体固定化磁性纳米粒子的制备方法。该制备方法包括如下步骤：将表面氨基化的磁性纳米粒子与带有羧基的漆酶天然介体进行酰胺化反应。
本发明拟通过对表面氨基化的磁性纳米粒子与带有羧基的漆酶天然介体进行酰胺化反应，利用羧基和氨基反应生成稳定的酰胺键，从而使带有羧基的漆酶天然介体能共价连接到磁性纳米粒子上，该方法可以将漆酶天然介体有效地固定在磁性纳米载体上，得到的漆酶天然介体固定化磁性纳米粒子具有很高的活性。
在本发明一个优选实施方式中，所述磁性纳米粒子为磁性四氧化三铁纳米粒子、磁性铁酸锌纳米粒子或磁性铁酸锰纳米粒子；优选为磁性四氧化三铁纳米粒子。
其中，所述表面氨基化的磁性纳米粒子可以由3-氨丙基三乙氧基硅烷(APTES)或者3-氨丙基三甲氧基硅烷(APTS)与磁性纳米粒子发生硅烷化反应制得。但用APTES硅烷化的磁性纳米粒子所含的氨基量比APTS硅烷化的磁性纳米粒子所含的氨基量要高，这有利于后续反应中连接更多的漆酶天然介体。
在本发明一个优选实施方式中，所述带有羧基的漆酶天然介体为芥子酸、阿魏酸或4-羟基肉桂酸，优选为芥子酸。上述几种介体相比较，介子酸更容易被连接到氨基化的磁性纳米粒子表面。
在本发明一个优选实施方式中，所述表面氨基化的磁性纳米粒子与带有羧基的漆酶天然介体的质量比为1:2～4:1，优选为1.6:1。
在本发明一个优选实施方式中，上述制备方法中酰胺化反应体系中优选缩合剂、催化剂、缚酸剂和溶剂。即上述制备方法具体包括：
将表面氨基化的磁性纳米粒子、带有羧基的漆酶天然介体、缩合剂、催化剂和缚酸剂在溶剂中混合，进行酰胺化反应。
其中，所述缩合剂优选为二环己基碳二亚胺、二异丙基碳二亚胺和1-(3-二甲胺基丙基)-3-乙基碳二亚胺(EDC)中的一种，进一步优选为EDC。上述几种缩合剂相比较，EDC使用效果更好，能大幅提高介体固定量。
其中，所述催化剂优选为4-N,N-二甲基吡啶或4-吡咯烷基吡啶(4-PPY)，进一步优选为4-PPY。上述几种催化剂相比较，4-PPY使用效果更好，能大幅提高介体固定量。
其中，所述缚酸剂为三乙胺或1,8-二氮杂二环十一碳-7-烯(DBU)，进一步优选为DBU。上述几种缚酸剂相比较，DBU使用效果更好，能大幅提高介体固定量。
其中，所述溶剂优选为N,N-二甲基甲酰胺或二甲基亚砜。
在本发明一个优选实施方式中，所述带有羧基的漆酶天然介体：缩合剂：缚酸剂：催化剂＝(1～3):(1～3):(1～3):(1～3):(1～3)。
在本发明一个优选实施方式中，上述制备方法具体包括：
将所述带有羧基的漆酶天然介体、缩合剂和溶剂在-4～10℃下活化1～30min，在向其中加入表面氨基化的磁性纳米粒子、催化剂和缚酸剂，在氮气存在下，10～50℃下酰胺化反应5～48h，优选为至少20h。
在本发明一个优选实施方式中，上述制备方法具体包括：
将芥子酸、EDC和溶剂在-4～10℃下活化1～30min，在向其中加入表面氨基化的四氧化三铁磁性纳米粒子、4-PPY和DBU，在氮气存在下，10～50℃下酰胺化反应5～48h。
在上述方案中，活化的温度优选为0～5℃，时间优选为15～20min。酰胺化的温度优选为20～25℃，时间优选为至少20h。
在本发明中，通常将上述酰胺化得到的产物进行洗涤，即得到纯的漆酶天然介体固定化磁性纳米粒子。优选使用DMF和水对产物分别洗涤。
由上述制备方法制得的漆酶天然介体固定化磁性纳米粒子可以有效地去除有机染料污染物。
即本发明的另一目的在于提供由上述的制备方法制得的漆酶天然介体固定化磁性纳米粒子。
本发明的再一目的在于提供上述制备方法或由上述制备方法得到的漆酶天然介体固定化磁性纳米粒子在污染物降解、造纸工业或木质素降解中的应用。
其中，优选在污染物降解中的应用。其中，该污染物优选为靛胭脂(Acid Blue74)。
溶液的脱色测试结果表明，本发明得到的漆酶天然介体修饰的磁纳米粒子可以有效地作为漆酶介体使用，能完成对靛胭脂的催化降解作用，同时使用后的漆酶天然介体固定化磁性纳米粒子方便回收重复使用，可以重复使用达8次以上。
与现有技术相比，本发明的有益效果是：
(1)本发明首次将漆酶的天然介体有效地固定于磁性纳米载体上并表现出高活性，该天然介体修饰磁性纳米粒子制备工艺简单、成本低廉。
(2)本发明提供的漆酶天然介体固定化磁性纳米粒子本身无毒对环境友好，能稳定漆酶活性，提高酶的利用效率。可以通过外部磁场对固定化天然介体简便回收，反复使用，从而提高介体的利用率，降低生产成本，有利于漆酶-介体***的推广使用。
(3)本发明提供的漆酶天然介体固定化磁性纳米粒子还可作为多种氧化酶的介体而广泛的应用于污染物降解、造纸工业、木质素降解及有机合成等环境治理和能源与化学等相关领域。
附图说明
图1是本发明实施例1制得修饰APTES的磁纳米粒子前后的红外图；
图2是本发明实施例1制得修饰介子酸(SA)的磁性纳米粒子前后的红外图；
图3是本发明实验例1染料溶液脱色前后的紫外-可见光扫描图谱；
图4是本发明实验例1漆酶、漆酶+SA、漆酶+固定化SA等不同体系脱色效率比较图；
图5是本发明实验例1制得的芥子酸固定化的磁纳米粒子作为漆酶介体重复使用数据图；
图6是本发明实验例1芥子酸固定化的磁纳米粒子重复使用前后的红外图；
图7是本发明实验例1介子酸固定化的磁纳米粒子在外磁场影响前(a)后(b)的照片。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1
本实施例提供了一种漆酶天然介体介子酸固定化磁纳米粒子Fe3O4(SA@APTES@Fe3O4纳米粒子)，其制备方法包括如下步骤：
(1)称取3.34gFeSO4·7H2O和4.86gFeCl3·6H2O加入圆底烧瓶中，再加入40ml的蒸馏水，搅拌溶解并升温至80℃，然后迅速加入12ml浓氨水，在80℃下持续搅拌反应2h，所制备的磁纳米(Fe3O4)用磁铁分离、反复用乙醇和蒸馏水洗涤至中性。将制备好的纳米粒子分散于蒸馏水中配置为1.0g L-1Fe3O4分散液，备用。
(2)称量750mg上述(1)制备的Fe3O4纳米进行活化，加入50ml去离子水后超声分散10min.维持60℃水浴加热，机械搅拌，N2保护下反应，加入一定量的稀盐酸(0.001mol/L)将反应体系pH调节到4，然后每隔0.5h测定反应体系的pH，及时补加稀盐酸使反应体系的pH维持在4，直到pH不再变化。
(3)称取上述(2)步骤活化后的Fe3O4纳米粒子加入100ml乙醇水溶液(50％)，用冰醋酸调节pH至4，室温下超声分散10min(40KHz)，然后加入APTES，反应用N2保护，60℃水浴加热，机械搅拌反应10h。反应完毕后依次用去离子水、无水乙醇和无水***洗至无油状悬浮物，真空干燥，即得表面氨基化的磁性纳米(APTES@Fe3O4)。
图1对比了磁性纳米Fe3O4修饰前后的红外图谱，发现修饰后的磁性纳米在3480cm-1有较强的氨基吸收峰，在1030cm-1有较强的Si-O键吸收峰，表明磁性纳米粒子表面已成功修饰上APTES。
(4)称取0.25g芥子酸(SA)溶解于10ml DMF溶剂并加入到三颈瓶中，称取0.28g的EDC溶解于1ml DMF中，加入三颈瓶并使其反应体系保持0℃活20min。随后加入0.4g APTES@Fe3O4、0.21ml DBU，机械搅拌5min后，再称取0.184g 4-PPY溶于1ml的DMF中并缓慢加入三颈瓶中，反应体系在25℃，N2保护下搅拌20h，回收纳米粒子并用DMF和蒸馏水分别洗涤5次，最终得到芥子酸固定化的磁性纳米粒子SA@APTES@Fe3O4。
根据图2的红外谱图发现SA@APTES@Fe3O4纳米粒子在1630、3450cm-1有较强的酰胺键吸收峰。和SA的红外谱型比较，修饰后纳米粒子上包含有很多SA的特征峰，由此可知芥子酸已成功的通过酰胺化反应修饰到纳米粒子表面。
实验例1
实施例1中得到的芥子酸固定化的SA@APTES@Fe3O4作为漆酶介体对染料的去除应用。
(1)取漆酶介体SA@APTES@Fe3O4纳米粒子2mg，加入到1mL的pH＝5.0的柠檬酸-柠檬酸钠缓冲液(0.1mol/L)，再加入0.5U的漆酶，然后加入0.01mg染料靛胭脂，振荡反应至完全，30min后蓝色溶液变成无色。
图3紫外-可见光扫描图谱显示了染料在610nm的特征峰消失，表明其被催化降解。
图4比较了漆酶、漆酶+SA、漆酶+SA@APTES@Fe3O4等不同体系对AB染料的脱色效率，结果表明芥子酸固定化的SA@APTES
@Fe3O4能作为漆酶介体使用，并且其和没有固定化的SA达到相同的效果。
(2)通过磁力回收SA@APTES@Fe3O4纳米粒子，随后用蒸馏水洗涤5次，回收得到的SA@APTES@Fe3O4纳米粒子重复上述循环使用。
其对靛胭脂去除效果和重复使用变化见图5，由图5可知循环使用8次后，仍然保持对AB有较高的去除作用效果。
图6显示了SA@APTES@Fe3O4纳米粒子重复使用前后的红外谱型没有任何改变，表明重复使用多次后漆酶天然介体SA仍能稳定连接在纳米颗粒表面。
纳米粒子的磁性可以很方便地通过外加磁场的方法加以验证。
图7是定性表明经漆酶天然介体修饰的Fe3O4纳米粒子在外磁场的作用前后的照片，表明经介体修饰后SA@APTES@Fe3O4纳米粒子依然有很强的磁性，完全能在外磁场的作用下定向移动。
使用固定漆酶天然介体的SA@APTES@Fe3O4纳米粒子和漆酶混合物来去除有机染料靛胭脂溶液的测试结果表明，SA@APTES@Fe3O4可以有效地作为漆酶介体使用，能完成对有机染料靛胭脂的催化降解作用，同时使用后的SA@APTES@Fe3O4纳米粒子能重复使用，并能保持很好的稳定性。
最后，本发明的方法仅为较佳的实施方案，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (4)

1.一种漆酶天然介体固定化磁性纳米粒子的制备方法，其特征在于，包括如下步骤：
将带有羧基的漆酶天然介体、缩合剂和溶剂在-4～10℃下活化1～30min，在向其中加入表面氨基化的磁性纳米粒子、催化剂和缚酸剂，在氮气存在下，10～50℃下酰胺化至少反应20h；所述表面氨基化的磁性纳米粒子与带有羧基的漆酶天然介体的质量比为1.6:1；
所述磁性纳米粒子为磁性四氧化三铁纳米粒子；
所述带有羧基的漆酶天然介体为芥子酸；
所述缩合剂为1-(3-二甲胺基丙基)-3-乙基碳二亚胺；
所述催化剂为4-吡咯烷基吡啶；
所述缚酸剂为4-吡咯烷基吡啶；
所述溶剂为N,N-二甲基甲酰胺或二甲基亚砜；
所述带有羧基的漆酶天然介体：缩合剂：缚酸剂：催化剂＝(1～3):(1～3):(1～3):(1～3):(1～3)。
2.根据权利要求1所述的制备方法，其特征在于，所述表面氨基化的磁性纳米粒子由3-氨丙基三乙氧基硅烷与磁性纳米粒子发生硅烷化反应制得。
3.权利要求1或2所述的制备方法制得的漆酶天然介体固定化磁性纳米粒子。
4.权利要求1或2所述的制备方法或权利要求3所述的漆酶天然介体固定化磁性纳米粒子在污染物降解、造纸工业或木质素降解中的应用。
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