CN115090329B - 一种Cu-二硫苏糖醇纳米仿生漆酶及其降解污染物和检测肾上腺素的应用 - Google Patents
一种Cu-二硫苏糖醇纳米仿生漆酶及其降解污染物和检测肾上腺素的应用 Download PDFInfo
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Abstract
本发明公开了一种Cu‑二硫苏糖醇纳米仿生漆酶及其降解污染物和检测肾上腺素的应用，模仿天然酶的催化中心结构，将Cu2+和二硫苏糖醇(DTT)利用“一锅法”合成了具有类漆酶活性的Cu‑DTT纳米粒子，它具有易合成、条件温和、价格低廉等优势。与天然漆酶相比，Cu‑DTT纳米粒子具有更高的Vmax和更低的Km，表现出良好的催化活性和底物普适性，且Cu‑DTT纳米粒子在不同温度、长期贮藏和高盐浓度下表现出更强的稳定性，可以降解不同的酚类污染物。此外，基于Cu‑DTT纳米粒子建立了一种快速定量检测肾上腺素的方法，在环境催化和快速检测方面有很好的应用前景。
Description
技术领域
本发明属于类漆酶材料技术领域，具体涉及一种具有类漆酶活性的纳米材料及其降解酚类污染物和检测肾上腺素的应用。
背景技术
随着近现代工业的迅速发展，以及早期人们缺少对保护环境的意识，因此导致全球的生态环境日益恶化。其中酚类污染物是当前重要的环境污染物之一。酚类污染物，如氯酚、双酚等，是一种毒性很强的有机污染物，广泛存在于石油化工、印染、塑料、纺织、农药等行业，对生态环境造成了极大的危害。酚类污染物是一种毒性很强的有机化合物，具有高毒性、持久性、流动性等特点。这些酚类污染物会侵入人体细胞原浆，在内分泌系统、生殖系统、神经系统、免疫系统以及癌变等方面对人体健康造成严重的威胁。因此，检测降解酚类污染物具有十分重要的现实意义。
漆酶(Laccase)是一种含有四个铜离子(Cu2+)的多酚氧化酶，是多铜氧化酶(MCOs)中的一种，以单体糖蛋白的形式存在，可以催化多种有机底物的氧化。由于漆酶具有良好的底物亲和力，同时有着种类丰富的特异性底物，且反应后的唯一副产物是水(H2O)，是一种性能良好的绿色催化剂。因此被广泛应用于脱色加工、降解木质素、纸浆漂白、生物燃料、食品安全、绿色有机物合成、降解环境中有毒物质等方面。虽然漆酶在工业生产以及污染物降解中被广泛应用，然而天然漆酶在复杂的反应环境中难以保存，稳定性较差，难以对其进行回收进而反复利用，且投入成本较高，这些问题都严重阻碍了漆酶在实际生产生活中的应用。因此，亟需寻找一种催化效率高、稳定性好、易于合成的纳米酶来解决这一问题。
目前为止，纳米酶作为新一代的人工模拟酶，兼具纳米材料的物理化学性质和酶催化功能。与天然酶相比，纳米酶具有成本低、可批量合成、适用于工业生产、可调节催化活性、稳定性高等优点，人们已经将它广泛应用于肿瘤诊断与治疗、血糖和尿酸的检测、免疫检测、农药和神经毒剂的监测等方面。漆酶能够催化氧化2,4-二氯苯酚(2,4-DP)和4-氨基安替吡啉(4-AP)反应产生红色物质的性质来检测其类漆酶活性，并测定纳米酶动力学参数米氏常数(Km)和Vmax。肾上腺素(EP)是一种由肾上腺髓质或者神经系统所分泌的儿茶酚胺，可以加快人体的血液循环流动以及心跳速率，在临床中常被用作治疗支气管哮喘、心律失常以及过敏性休克等症状的药物。肾上腺素的氧化产物可作为肾上腺素的检测方法，肾上腺素的定量检测对于疾病的诊断和药物分析至关重要。
发明内容
本发明的目的是提供一种催化效率高、稳定性好、易于储存、价格低廉的Cu-二硫苏糖醇纳米仿生漆酶，并为该仿生漆酶提供新的应用。
针对上述目的，本发明采用的Cu-二硫苏糖醇纳米仿生漆酶是铜离子与二硫苏糖醇(DTT)络合形成的纳米粒子，其呈颗粒状，大小均匀，尺寸为30～50nm。
本发明Cu-二硫苏糖醇纳米仿生漆酶的制备方法为：将DTT与氯化铜按摩尔比为1:2～2:1加入去离子水与N,N-二甲基甲酰胺的混合溶剂中，所得混合溶液转移至高压反应釜中，在密封条件下130～150℃静置反应4～5h，反应完后离心分离，沉淀依次用无水乙醇、超纯水洗涤后，冷冻干燥，得到Cu-二硫苏糖醇纳米仿生漆酶。
本发明Cu-二硫苏糖醇纳米仿生漆酶可用于降解酚类污染物，如2,4-二氯苯酚、对苯二酚、邻苯二酚、对碘苯酚等。
本发明Cu-二硫苏糖醇纳米仿生漆酶还可用于检测肾上腺素。
本发明的有益效果如下：
本发明通过二硫苏糖醇(DTT)与铜离子的络合反应，合成了一种新型的类漆酶，它具有易合成、条件温和、价格低廉等优势。与天然漆酶相比，本发明的Cu-二硫苏糖醇纳米仿生漆酶具有更高的Vmax和更低的Km，表现出良好的催化活性和底物普适性。与天然漆酶相比，Cu-二硫苏糖醇纳米仿生漆酶在不同温度、长期贮藏和高盐浓度下表现出更强的稳定性，可以降解不同的酚类污染物，且催化降解能力要强于天然漆酶。此外，本发明基于Cu-二硫苏糖醇纳米仿生漆酶建立了一种快速定量检测肾上腺素的方法，在环境催化和快速检测方面有很好的应用前景。因此，本发明将Cu-二硫苏糖醇纳米仿生漆酶用于肾上腺素的定量检测，在环境催化和快速检测等领域有非常重要的应用前景。
附图说明
图1是Cu-DTT纳米粒子的SEM表征图。
图2是Cu-DTT纳米粒子的FTIR图谱。
图3是Cu-DTT纳米粒子催化2,4-DP、4-AP的紫外-可见光吸收光谱(A)和漆酶与Cu-DTT NPs催化氧化2,4-DP与4-AP反应紫外-可见吸收光谱(B)图。
图4是Cu-DTT纳米粒子与天然漆酶在不同温度下的催化稳定性。
图5是Cu-DTT纳米粒子与天然漆酶在不同浓度NaCl中的催化稳定性。
图6是Cu-DTT纳米粒子与天然漆酶在室温下储存不同时间的催化稳定性。
图7是Cu-DTT纳米粒子和天然漆酶催化降解不同酚类污染物在λ＝510nm处的吸光度值。
图8是Cu-DTT纳米粒子与天然漆酶催化氧化不同浓度肾上腺素的反应显色图和在λ＝485nm处的吸光度值。
具体实施方式
下面结合附图和实施例对本发明进一步详细说明，但本发明的保护范围不仅限于这些实施例。
实施例1
将10mL 0.12mol/L DTT水溶液加入到5mL 0.24mol/L CuCl2水溶液中，并加入20mL N,N-二甲基甲酰胺，将上述混合溶液转移至高压反应釜中，置于电热恒温鼓风干燥箱中，在140℃下反应4.5h。反应冷却后，使用高速冷冻离心机以10000r/min的转速进行离心，沉淀用无水乙醇洗涤3次，并用超纯水洗涤3次后，进行冷冻干燥，得到Cu-DTT纳米粒子，即Cu-二硫苏糖醇纳米仿生漆酶，密封保存于4℃冰箱上层。
利用场发射扫描电镜(SEM)、傅里叶变换红外光谱仪(FTIR)对Cu-DTT纳米粒子进行了表征。从图1可以看出，Cu-DTT纳米粒子呈颗粒状，大小均匀，尺寸约为30～50nm。图2中，波长在2553cm-1和3340cm-1左右是DTT中S-H和O-H的伸缩振动，Cu-DTT纳米粒子红外光谱图中2553cm-1的S-H键消失以及946cm-1出现了新的特征峰，说明DTT与Cu2+离子相互作用形成金属-有机硫醇键，即Cu-DTT纳米粒子成功合成。
对上述合成的Cu-DTT纳米粒子进行各种性能测试，具体如下：
1、类漆酶活性测定
将Cu-DTT纳米粒子放入研钵中研磨成粉末状，取1mg粉末溶解于1mL超纯水中，配制成1mg/mL Cu-DTT溶液。将2,4-DP和4-AP分别溶解在30mM pH 7.0的MES缓冲溶液中，分别配制成浓度为1mg/mL的溶液。取80μL 1mg/mL 2,4-DP溶液和80μL 1mg/mL 4-AP溶液于离心管中，加入40μL 1mg/mL Cu-DTT溶液，振荡摇匀，在25℃下反应60min。测定反应后溶液在λ＝510nm处的紫外可见吸光度值，并对溶液的颜色进行拍照。取1mg/mL的漆酶溶液(0.5U/mg)代替Cu-DTT溶液催化2,4-DP和4-AP，在相同条件下反应后测定其在λ＝510nm处的吸光度值，并对溶液的颜色进行拍照。由图3的对比结果可以看出，只有当Cu-DTT纳米粒子与2,4-DP、4-AP同时存在时，才能在λ＝510nm处产生吸收峰，说明Cu-DTT纳米粒子具有类漆酶活性，能够催化氧化2,4-DP和4-AP的反应，使溶液颜色呈红色(图3A)。将Cu-DTT NPs与天然漆酶进行活性对比，Cu-DTT NPs在λ＝510nm处的吸光度值明显高于天然漆酶，而且显示出的红色也明显比天然漆酶深(图3B)，说明Cu-DTT纳米粒子具有类漆酶活性，且活性较天然漆酶更强。
2、类漆酶动力学参数测定
(1)用30mM pH为7.0的MES缓冲溶液分别配制浓度为10μg/mL、20μg/mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL的2,4-DP溶液，分别取80μL不同浓度的2,4-DP溶液与80μL1mg/mL 4-AP溶液、40μL 1mg/mL Cu-DTT溶液混合，在25℃下每隔1min测定其在λ＝510nm处的紫外可见吸光光度值。通过v-c图可知不同浓度2,4-DP下的反应速率Vmax，通过1/V-1/S图可知Cu-DTT对应的米氏常数Km。
(2)分别取80μL不同浓度的2,4-DP溶液与80μL 4-AP溶液、40μL 1mg/mL天然漆酶溶液混合，在40℃下每隔1min测定其在λ＝510nm处的紫外可见吸光光度值。通过v-c图可知不同浓度2,4-DP下的反应速率Vmax，通过1/V-1/S图可知天然漆酶对应的米氏常数Km。得到的Vmax与Km数值如表1所示。Cu-DTT纳米粒子的Vmax高于漆酶，Km低于天然漆酶，说明Cu-DTT纳米粒子比天然的漆酶活性高。
表1是Cu-DTT纳米粒子和漆酶的动力学参数
3、催化稳定性测定
(1)测定温度对催化稳定性的影响
取80μL1 mg/mL 2,4-DP溶液、80μL 1mg/mL 4-AP溶液于离心管中，并分别加入20μL 1mg/mL Cu-DTT溶液和20μL 1mg/mL天然漆酶溶液，振荡均匀，分别放置于4℃、20℃、40℃、60℃、80℃以及100℃的环境中，反应60min测定其上清液在λ＝510nm处的吸光度值。以20℃下的催化活性为参考值，分别测定Cu-DTT纳米粒子和漆酶在4～100℃下的相对活性(见图4)。从图4中可以看出，在60℃之前，Cu-DTT纳米粒子与天然漆酶的酶活性都随着温度的升高而升高。当温度高于60℃时，天然漆酶的酶活性骤降且几乎失活，此时Cu-DTT纳米粒子依旧具有较好的酶活性。由此说明，Cu-DTT纳米粒子较之天然漆酶具有更好的酶活性，表现出良好的热稳定性。
(2)测定盐类对催化稳定性的影响
取80μL1 mg/mL 2,4-DP溶液、80μL 1mg/mL 4-AP溶液于离心管中，并分别加入20μL 1mg/mL Cu-DTT溶液和20μL 1mg/mL天然漆酶溶液，接下来分别加入不同浓度的NaCl水溶液(0、100、200、300、400、500mM)，振荡均匀，置于室温下反应60min，测定其上清液在λ＝510nm处的吸光度值。如图5所示，随着NaCl浓度的增加，Cu-DTT纳米粒子的酶活性在一定程度上也有所提高，其催化活性甚至提高到178％。当Cl-浓度超过100mM时，Cu-DTT纳米粒子仍维持较高的催化活性。而Cl-的加入使得天然漆酶的酶活性迅速下降，当Cl-浓度超过100mM时，天然漆酶几乎失活。由此可以得出结论，Cu-DTT纳米粒子较之天然漆酶具有更好的酶活性，且当Cl-存在时更加稳定。
(3)测定储存时间对催化稳定性的影响
分别将1mg/mL Cu-DTT溶液和1mg/mL天然漆酶溶液置于室温条件下进行保存，每隔2天按下述方法进行测定：取80μL 1mg/mL 2,4-DP溶液、80μL 1mg/mL4-AP溶液于离心管中，并分别加入20μL 1mg/mL Cu-DTT溶液和20μL 1mg/mL天然漆酶溶液，震荡均匀，置于室温下反应60min进行显色，测定其上清液在λ＝510nm处的吸光度值。如图6所示，Cu-DTT纳米粒子和天然漆酶均在室温下进行储存，随着时间的推移，两者的催化活性均有所下降。贮藏10天后，天然漆酶的催化活性几乎完全丧失。相同贮存期后，Cu-DTT纳米粒子的催化活性仍保持在82％。由此说明，Cu-DTT纳米粒子与天然漆酶相比更易于储存。
实施例2
实施例1的Cu-DTT纳米粒子降解酚类污染物的应用
用超纯水配制质量浓度均为1mg/mL的Cu-DTT溶液和天然漆酶溶液，并将2,4-DP、对苯二酚、邻苯二酚、对碘苯酚分别溶解在30mM pH 6.8的MES缓冲溶液中，配制成质量浓度为1mg/mL的溶液。取100μL 1mg/mL 2,4-DP(对苯二酚、邻苯二酚、对碘苯酚)溶液、80μL1mg/mL 4-AP溶液于离心管中，分别加入20μL 1mg/mL Cu-DTT溶液天然漆酶溶液，震荡均匀，置于室温下反应60min进行显色，测定其上清液在λ＝510nm处的吸光度值。如图7所示，Cu-DTT纳米粒子和天然漆酶对这四种酚类污染物均有催化降解的作用，而Cu-DTT纳米粒子对不同酚类污染物的降解能力都强于天然漆酶。由此可以得出结论，漆酶可以降解不同的酚类污染物，具有良好的底物普遍性，且Cu-DTT纳米粒子的催化降解能力要强于天然漆酶。
实施例3
实施例1的Cu-DTT纳米粒子检测肾上腺素的应用
用30mM pH 6.8的MES缓冲溶液分别配制浓度为10μg/mL、20μg/mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL、120μg/mL的肾上腺素溶液。取180μL不同浓度的肾上腺素溶液，并分别加入20μL 1mg/mL Cu-DTT纳米粒子溶液和20μL 1mg/mL天然漆酶溶液，震荡均匀，室温下反应60min，测定其上清液在λ＝485nm处的吸光度值，结果如图8所示。从图8的结果可以看出，随着肾上腺素浓度的增加，Cu-DTT纳米粒子和天然漆酶催化底物的溶液颜色也随之变深。肾上腺素的氧化产物在485nm处的吸光度值随肾上腺素浓度的增加而增加，Cu-DTT纳米粒子催化氧化肾上腺素的吸光度值始终大于天然漆酶吸光度值，并且拟合后曲线都呈现出良好的线性关系，由此说明Cu-DTT纳米粒子可用于定量检测肾上腺素且效果优于天然漆酶。
Claims (3)
1.一种Cu-二硫苏糖醇纳米仿生漆酶，其特征在于：所述仿生漆酶是铜离子与二硫苏糖醇络合形成的纳米粒子，其呈颗粒状，大小均匀，尺寸为30～50 nm；
所述纳米仿生漆酶的制备方法为：将二硫苏糖醇与氯化铜按摩尔比为1:2～2:1加入去离子水与N,N-二甲基甲酰胺的混合溶剂中，所得混合溶液转移至高压反应釜中，在密封条件下130～150 ℃静置反应4～5 h，反应完后离心分离，沉淀依次用无水乙醇、超纯水洗涤后，冷冻干燥，得到Cu-二硫苏糖醇纳米仿生漆酶。
2.权利要求1所述的Cu-二硫苏糖醇纳米仿生漆酶在降解酚类污染物中的应用。
3.权利要求1所述的Cu-二硫苏糖醇纳米仿生漆酶在检测肾上腺素中的应用。
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