CN114085827B - 一种腈水合酶-无机杂化纳米花催化剂及其应用 - Google Patents
一种腈水合酶-无机杂化纳米花催化剂及其应用 Download PDFInfo
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims abstract description 8
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Abstract
本发明公开了一种腈水合酶‑无机杂化纳米花催化剂的制备方法及应用。将腈水合酶溶液与磷酸钠、羧甲基纤维素钠CMC混合，向上述混合液中加入硫酸亚铁溶液，孵育一段时间后，得到腈水合酶‑无机杂化纳米花催化剂。本发明得到的复合催化剂用于催化水解脂肪族二腈(尤其是己二腈)生成更具经济价值的5‑氰基戊酰胺等酰胺类化合物，相比较游离的腈水合酶，固定化酶在高温、偏酸的反应条件下催化效果有明显提高，同时表现出显著的底物耐受性。
Description
技术领域
本发明属于绿色化学领域，涉及一种腈水合酶-无机杂化纳米花催化剂制备方法及其应用。
背景技术
腈水合酶(EC 4.2.1.84,nitrile hydratase,简称NHase)是一类催化腈类化合物生成酰胺类化合物的金属酶，例如：在全细胞或者游离的腈水合酶的催化作用下，丙烯腈可以转化为丙烯酰胺；3-氰基吡啶可以转化为烟酰胺；己二腈可以转化为5-氰基戊酰胺。酰胺类物质在有机合成、医药、农药等方面有着十分重要的应用。自然界中产腈水合酶的微生物大多来源于红球菌属，假单胞菌属，短杆菌属等。
工业上大多采用传统的化学方法将腈类物质转化成酰胺类物质，但其反应条件苛刻需要在高温高压下进行，而且伴随大量副产物和污染物生成，产率低，产物难分离，三废处理也会造成环境的严重污染。而采用微生物法，以腈水合酶为催化剂将腈类物质水合生成酰胺，因为它具有严格的化学、区域和对映选择性及底物专一性，作用条件温和，使其产物纯度高、副产物少，并且对设备腐蚀小。因此，采用腈水合酶催化符合经济环保的绿色环境发展方向，适应当今社会可持续发展的要求。但是，由于游离酶的温度、pH和操作稳定性低，难以回收利用等，阻碍了其在工业生产上的应用。因此将游离酶进行固定化可以在一定程度上提高酶的耐受性，并且能够循环使用，方便下游过程的操作。
目前应用较多的固定化酶载体有磁性纳米多孔材料，金属有机框架，共价有机框架，有机无机杂化纳米材料等。其中有机-无机杂化纳米花(NF)是一类由金属离子和生物分子(蛋白质、DNA、小有机分子等)通过自组装形成的花状纳米材料。相比于其他材料，杂化纳米花具有独特的优点：①纳米花具有较大的比表面积，且传质阻力较小；②合成条件温和，也不需添加任何有毒物质，从而避免了对其活性的影响；③有些金属离子对酶活性的提高具有促进作用；④纳米花独特的分层纳米结构，增强了酶活性和稳定性。
发明内容
本发明提供了一种腈水合酶-无机杂化纳米花的方法，调节固定化酶的微环境，使其在反应条件苛刻下依旧保持较高的催化活性。
本发明的技术方案为：
一种腈水合酶-无机杂化纳米花催化剂，所述催化剂具有纳米花状形貌，以Fe3(PO4)2纳米花为载体，负载腈水合酶。并对合成的腈水合酶-无机杂化纳米花进行SEM表征，确定其形貌粒径以及元素分布。
上述腈水合酶-无机杂化纳米花催化剂的制备方法，包括以下步骤：
(1)向腈水合酶溶液中加入羧甲基纤维素钠，混合均匀后，在连续搅拌下加入一定量的磷酸三钠溶液；其中，腈水合酶溶液浓度1mg/ml，CMC的质量浓度为0.5％，磷酸三钠浓度为12.5mmol/L；
(2)搅拌15min后，向上述混合液中逐滴加入硫酸亚铁溶液，整个操作在氮气下进行；其中，硫酸亚铁溶液浓度为100mmol/L，含腈水合酶的CMC：磷酸三钠：硫酸亚铁体积比为2:1:1；
(3)继续搅拌，将得到的混合液孵育处理后，离心收集沉淀，磷酸盐缓冲液洗涤三次，得到腈水合酶-无机杂化纳米花；其中，孵育温度为4℃，孵育时间为12h，离心条件为8000rpm，2min，磷酸盐缓冲液浓度为50mmol/L，pH为7.4。
上述腈水合酶-无机杂化纳米花催化剂催化底物脂肪族二腈(己二腈)的应用，与游离腈水合酶对比其催化性能，包括以下步骤，
(1)向腈水合酶-无机杂化纳米花催化剂和游离腈水合酶溶液中加入同等量的底物己二腈，在不同反应温度，反应pH下进行反应，确定二者最佳反应条件；其中，加入腈水合酶的质量为0.05mg，己二腈的反应终浓度为20mmol/L；
(2)向腈水合酶-无机杂化纳米花催化剂和游离腈水合酶溶液中加入不同浓度的底物己二腈，在最佳反应条件下，对比催化性能。其中，己二腈反应终浓度分别为50mmol/L，100mmol/L，150mmol/L，200mmol/L，反应温度为35℃，反应pH为7.5。
本发明的有益效果是：
(1)腈水合酶-无机杂化纳米花催化剂的制备方法简单，能够很好的保存酶活性，与游离腈水合酶相比，其对底物耐受性有明显提高；
(2)纳米花作为催化剂在反应完成后分离操作简单，可通过简单的离心直接从反应液中去除；
(3)该方法成本低廉，反应产物和过程绿色环保，符合经济环保的绿色环境发展方向，适应当今社会可持续发展的要求。
附图说明
图1A是腈水合酶-无机杂化纳米花催化剂的扫描电镜图。
图1B是腈水合酶-无机杂化纳米花催化剂的X射线能谱(EDS)分析图。
图2A是游离腈水合酶和腈水合酶-无机杂化纳米花最适pH比较图。
图2B是游离腈水合酶和腈水合酶-无机杂化纳米花最适温度比较图。
图3A是游离腈水合酶与腈水合酶-无机杂化纳米花催化剂底物耐受性比较图，其中的己二腈浓度50mmol/L。
图3B是游离腈水合酶与腈水合酶-无机杂化纳米花催化剂底物耐受性比较图，其中的己二腈浓度100mmol/L。
图3C是游离腈水合酶与腈水合酶-无机杂化纳米花催化剂底物耐受性比较图，其中的己二腈浓度150mmol/L。
图3D是游离腈水合酶与腈水合酶-无机杂化纳米花催化剂底物耐受性比较图，其中的己二腈浓度200mmol/L。
具体实施方式
实施例1：制备一种腈水合酶-无机杂化纳米花催化剂
在连续搅拌下，将0.5％CMC(含1mg/mL腈水合酶)的20mL中加入10mL磷酸三钠水溶液(12.5mmol/L)，15min后，向上述混合物中滴加10mL(100mmol/L)硫酸亚铁水溶液，继续搅拌。将得到的混合溶液在4℃下孵育12h。以8000rpm离心收集沉淀物，用磷酸盐缓冲液(pH7.4)洗涤三次。所有操作都在N2气氛下进行。
本实施例制备的腈水合酶-无机杂化纳米花的SEM图如图1所示，从图1中可以看出制备的杂化纳米花呈现花苞状，粒径在3μm左右，具有较高的比较面积；其X射线能谱分析图中可观察到Fe、P、O、C、N五种元素，其中C、N元素来源于腈水合酶，Fe、P、O元素来源于载体Fe3(PO4)2，这一结果表明腈水合酶被成功固定在载体Fe3(PO4)2纳米花上。
实施例2：游离腈水合酶和腈水合酶-无机杂化纳米花催化剂的最佳反应条件
取100μL等质量浓度的游离腈水合酶和腈水合酶-无机杂化纳米花，加入350μL磷酸盐缓冲液中，最后加入50μL，200mmol/L己二腈使其反应终浓度为20mmol/L，在35℃，250rpm下振动反应5min，用500μL甲醇终止反应，采用高效液相色谱对目标产物进行检测。改变反应温度，在20-60℃之间检测酶活；改变磷酸盐缓冲液pH值，在5.0-12.0之间检测酶活。
本实施例测定的游离腈水合酶和腈水合酶-无机杂化纳米花催化剂的最佳反应条件如图2所示，从图2中可以看出二者最适反应温度均为35℃，最适反应pH均为7.5。值得注意的是，固定化酶在高温，极端pH环境下耐受性更强，相比游离酶更稳定。
实施例3：游离腈水合酶和腈水合酶-无机杂化纳米花催化剂的底物耐受性测定
取等质量浓度的游离腈水合酶和腈水合酶-无机杂化纳米花置于磷酸盐缓冲液中，加入己二腈，使其反应终浓度分别为50mmol/L，100mmol/L，150mmol/L，200mmol/L，分别反应2min，5min，10min，15min，20min，40min，60min后甲醇终止反应，以上述实施例2的方法测定其酶活，计算目标产物5-氰基戊酰胺的产率。
本实例测定的底物耐受性如图3所示，从图3中可以观察到，当游离腈水合酶催化底物己二腈浓度大于100mmol/L时，5-氰基戊酰胺的产率急剧下降，由50mmol/L100％的产率降为几乎失活状态，但腈水合酶-无机杂化纳米花的底物耐受性有显著提高，随着反应时间的延长，可以在15min左右将100mmol/L及150mmol/L的己二腈完全转化，且在己二腈浓度为200mmol/L下目标产物5-氰基戊酰胺依旧保持70％左右的产率。
Claims (6)
1.一种腈水合酶-无机杂化纳米花催化剂，其特征在于，所述催化剂具有纳米花状形貌，以Fe3(PO4)2纳米花为载体，负载腈水合酶；其采用以下步骤制备得到：
(1)在连续搅拌下，将磷酸三钠Na3PO4与含腈水合酶的羧甲基纤维素钠CMC溶液混合；腈水合酶浓度为1 mg/ml，磷酸三钠浓度为12.5 mmol/L，CMC质量浓度为0.5%；
(2)搅拌15 min后，向上述溶液中滴加浓度为100 mmol/L的硫酸亚铁，所有操作都在氮气环境中进行；含腈水合酶的CMC：磷酸三钠：硫酸亚铁体积比为2：1：1；
(3)继续搅拌，将得到的混合液孵育处理后，离心收集沉淀，磷酸盐缓冲液洗涤三次，得到腈水合酶-无机杂化纳米花。
2.根据权利要求1所述的腈水合酶-无机杂化纳米花催化剂，其特征在于，步骤(3)所述孵育温度为4 ℃，孵育时间为12 h；所述磷酸盐缓冲液浓度为50 mmol/L，pH为7.4；离心处理的速率为8000 rpm，离心处理的时间为2 min，离心处理的温度为4 ℃。
3.权利要求1或2所述的腈水合酶-无机杂化纳米花催化剂在催化己二腈的应用。
4.根据权利要求3所述的应用，其特征在于，包括以下步骤：将腈水合酶-无机杂化纳米花催化剂加入到己二腈溶液中，调节反应pH，反应温度和己二腈浓度。
5.根据权利要求3所述的应用，其特征在于，腈水合酶-无机杂化纳米花催化剂的添加浓度均为0.5 mg/ml。
6.根据权利要求3所述的应用，其特征在于，其特征在于，所述己二腈浓度为50-200mmol/L，反应温度为20-60 ℃，反应pH为5.0-12.0。
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| CN113106137A (zh) * | 2021-03-05 | 2021-07-13 | 华南理工大学 | 一种蛋白质-无机杂化纳米花及其制备方法 |
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