CN110801866B - 一种制备单原子铜人工模拟过氧化物酶的方法 - Google Patents
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Abstract
本发明公开了一种制备单原子铜人工模拟过氧化物酶的方法。所述人工模拟过氧化物酶的制备直接利用廉价易得、资源丰富的人类尿液作为配体源,以单原子催化剂为模拟酶活性中心,可减少铜用量,同时提高人工过氧化物酶功能活性。单原子铜人工模拟过氧化物酶具有优良的过氧化酶活性,可催化过氧化氢高效氧化底物3,3',5,5'‑四甲基联苯胺盐酸盐,表现出类似过氧化物酶的活性特征,并且该制备方法简单,来源广泛,成本低廉,且一定程度上起到了处理人类废弃物的效果,具有较高的工业应用和环境应用潜力。
Description
技术领域
本发明涉及配位化学和酶催化技术领域,特别涉及一种制备单原子铜人工模拟过氧化物酶的方法。
背景技术
酶是一类可以进行生物催化的蛋白质。在生物反应体系中,所有代谢分子都可被看作为底物。酶几乎参与所有的细胞内反应,以提高生物系统的工作效率。酶催化生物反应的本质是降低化学反应的活化能势垒。通常来说,酶可以提高反应速率最快达到几百万倍,而自身在反应前后无任何变化,反应化学平衡也不受影响。由于酶催化具有专一性、特异性、高效性,其大批量生产具有极高的工业价值。
天然酶作为生物催化剂具有显著的优势,但由于酶的本质是一种蛋白质,难以承受苛刻的反应条件,它们可以被蛋白酶消化,在极端条件下,催化活性很容易被抑制或降低。此外,天然酶难以制备、净化和存储。为了克服天然酶的固有缺陷,研究人员一直在寻找高度稳定、生产成本低的人工模拟酶来替代天然酶。各种各样的大分子有机物,如环糊精、金属配合物、卟啉、聚合物、树状大分子与生物分子等,均已被广泛探索,用于模仿天然酶的结构与功能。
过氧化氢(H2O2)是一种强氧化剂,在生物体内生成的过氧化氢需要及时清除。过氧化物酶是氧化还原酶的一种,分布在乳汁、白细胞、血小板等体液或细胞中,该酶的辅基亦为血红素,以H2O2为电子受体,它催化H2O2直接氧化酚类或胺类化合物,具有消除H2O2和酚类胺类毒性的双重作用。如辣根过氧化物酶是催化还原过氧化氢的常用酶。但过氧化氢酶的稳定性较差,在实际应用中极易失活,因此人们多采取人工酶模拟过氧化氢酶的活性。
杨伟庆等采用微波辅助溶剂热法成功合成了铁氮共掺杂碳点(Fe-N-CDs),并利用多个技术来表征所得到的颗粒性质。铁的掺杂使整个复合物纳米颗粒具有类似天然的过氧化物酶活性,也证明了其在催化领域的潜在应用价值。赵慧晖等在研究中提到了基于BSA稳定的金纳米粒子团簇作为过氧化物酶模拟物,可被作为检测尿酸的光谱分析方法。刘艳等发现通过两亲性蛋白包覆的金纳米粒子可作为纳米酶,且具有较好的过氧化物酶的专一性和选择性。
这些开发的人工模拟过氧化物酶主要是为了解决天然酶作为一类有催化活性的蛋白质,容易受到多种物理、化学因素的影响而失活,不能广泛取代工业催化剂的缺点。然而,目前人工模拟过氧化物酶也存在如下缺陷:
(1)合成人工模拟酶的大分子价格昂贵,结构不够稳定且储存条件苛刻,不适用于大规模工业合成;
(2)人工模拟过氧化物酶的催化活性仍然显著低于生物酶,无法完全取代生物酶的位置;
(3)人工模拟过氧化物酶的功能化困难,生产时易造成污染制约了其批量生产和应用。
上述缺陷造成以现有工艺方法难以得到性质稳定、价格低廉、绿色环保且与天然酶催化活性相当的过氧化物模拟酶,限制其在生物领域的进一步应用。
发明内容
本发明是鉴于现有技术中存在的上述问题而做出的,本发明的目的在于提供一种简单、廉价、环境友好、兼具环保效应的制备单原子铜人工模拟过氧化物酶的方法。
为了实现上述目的,本发明第一技术方案,一种制备单原子铜人工模拟过氧化物酶的方法,其特征在于,包括如下的制备步骤:
(1)收集原始尿液,采用碱性阴离子交换树脂吸附尿液中的氨基酸类配体化合物;
(2)以高浓度盐溶液对吸附氨基酸配体的树脂进行解吸,同时浓缩提高氨基酸配体在水溶液中浓度;
(3)向浓缩后的氨基酸配体中加入体积比为0.5%的浓度为10g/L的硝酸铜溶液,搅拌24h,并静置沉淀2~3h,得到蓝绿色的固体沉淀物;
(4)弃去上清液,向所得沉淀物中加入等体积的冰乙酸,搅拌加热至沸腾;
(5)加入占冰乙酸体积20%的浓度为40g/L的氢氧化钠水溶液,继续搅拌5min得到蓝黑色沉淀;
(6)将反应得到的蓝黑色沉淀离心分离,用无水乙醇洗涤三次,减压干燥,即得单原子铜人工模拟过氧化物酶粉体。
本发明的第二技术方案,在第一技术方案的基础上,进一步地,限定步骤(1)中所述的原始尿液指从人体内排出,密封储存时间小于24h的新鲜尿液;
本发明的第二技术方案,在第一技术方案的基础上,进一步地,限定步骤(1)中所述的碱性阴离子交换树脂的用量为尿液重量的20%,吸附时间为24h;步骤(2)中所述的高浓度盐溶液为氯化钠、硫酸钠、氯化钾、硫酸钾、氯化铵、硫酸铵中之一种,其浓度为10g/L,等体积浸渍,解吸时间为24h。
本发明所述的合成配方中,尿液是一种廉价易得、资源丰富的人工模拟过氧化物酶配体源,人类尿液之中有91~96%为水,固体成分约占6%。成分包括含氮废物(尿素、尿酸)、氨基酸、色素、激素等。其中绝大部分可以作为金属离子,特别是过渡金属离子配体,可以和铜离子形成化学性质稳定的配合物,而这些配合物大部分具有类似人工过氧化物酶的功能活性。
铜离子是一种常见的配合物中心离子。配合物又称为络合物、复合物,包含由中心原子或离子与几个配体分子或离子以配位键相结合而形成的复杂分子或离子,通常称为“配位单元”。更特别的是,由于一个配合物结构只能具有一个中心原子,可以使用框架化学中的金属有机框架化合物(Metal Organic Framework,MOF)或共价有机框架化合物(Covalence Organic Framework,COF)作为前驱体,制备以配合物作为载体的单原子催化剂。通过将金属原子逐一分散于载体上形成单原子催化剂,可以实现金属利用的最大化,达到最高原子效率,具有潜在的、高效的人工过氧化物酶功能活性。
本发明与现有技术相比,具有如下优点:
(1)直接利用廉价易得、资源丰富的尿液为配体源,克服了传统配体价格高、来源有限的缺陷;
(2)以尿液提取物作为人工酶的资源,可以削减生活污染,变废为宝;
(3)以铜离子为配合物中心离子,最大程度提高了经济性,克服了传统纳米或纳米簇模拟酶金属用量大,催化活性低的缺陷;
(4)以单原子催化剂为模拟酶活性中心,减少铜用量,抑制生产和使用过程中的二次污染。
上述技术方案的采用,使单原子铜人工模拟过氧化物酶成为能够长期使用的经济有效的高效人工酶;其工艺简单,成本低,催化效率高、稳定性好,应用范围广,使用环境友好。
附图说明
为更清楚地说明本发明的具体实施方式,下面对具体实施方式部分描述中使用到的附图作简单说明。
图1为该单原子铜人工模拟过氧化物酶的结构示意图;
图2为该单原子铜人工模拟过氧化物酶催化过氧化氢反应的浓度-吸光度关系图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面结合附图对本发明的具体的实施方式进行详细描述。
第一实施例
采集新鲜的人尿10L,静置10h后,加入2kg碱性阴离子交换树脂,搅拌吸附尿液中的氨基酸类配体化合物,然后以高浓度盐溶液对吸附氨基酸配体的树脂进行解吸,并以浓缩法提高氨基酸配体在水溶液中浓度,向配体中加入体积比为0.5%的浓度为10g/L的硝酸铜溶液,搅拌24h,并静置沉淀2~3h,得到蓝绿色的固体沉淀物,弃去上清液,在沉淀中加入等体积的冰乙酸,搅拌加热至沸腾,然后加入占冰乙酸体积20%的浓度为40g/L的氢氧化钠水溶液,继续搅拌5min得到蓝黑色沉淀,将反应得到的蓝黑色沉淀离心分离,用无水乙醇洗涤三次,减压干燥,即得单原子铜人工模拟过氧化物酶粉体(图1为该人工模拟过氧化物酶的结构示意图)。
第二实施例
在容器内加入5mL浓度为20mmol/L的磷酸二氢钠-磷酸氢二钠缓冲液(pH 4.5),再加入0.5mL过氧化氢及0.2mL浓度为20mmol/L的3,3',5,5'-四甲基联苯胺盐酸盐,最后加入0.1mg单原子铜人工模拟过氧化物酶,剧烈搅拌,混合均匀在室温下反应10min,测定652nm波长处吸光度。过氧化氢浓度在0.02~1.0mmol/L范围内,显色产物的吸光度与过氧化氢浓度呈线性相关,此时不同浓度过氧化氢的转化率大于95%。图2为单原子铜人工模拟过氧化物酶催化不同浓度的过氧化氢反应液对应的652nm处吸光度,证明在此浓度范围内,过氧化氢的转化效率基本一致。
以上所述的具体实施方式仅用于具体说明本发明的精神,本发明的保护范围并不局限于此,对于本技术领域的技术人员来说,当然可根据本说明书中所公开的技术内容,通过变更、置换或变型的方式轻易做出其它的实施方式,这些其它的实施方式都应涵盖在本发明的保护范围之内。
Claims (3)
1.一种制备单原子铜人工模拟过氧化物酶的方法,其特征在于,制备方法如下:
(1)收集原始尿液,采用碱性阴离子交换树脂吸附尿液中的氨基酸类配体化合物;
(2)以高浓度盐溶液对吸附氨基酸配体的树脂进行解吸,同时浓缩提高氨基酸配体在水溶液中浓度;
(3)向浓缩后的氨基酸配体中加入体积比为0.5%的浓度为10g/L的硝酸铜溶液,搅拌24h,并静置沉淀2~3h,得到蓝绿色的固体沉淀物;
(4)弃去上清液,向所得沉淀物中加入等体积的冰乙酸,搅拌加热至沸腾;
(5)加入占冰乙酸体积20%的浓度为40g/L的氢氧化钠水溶液,继续搅拌5min得到蓝黑色沉淀;
(6)将反应得到的蓝黑色沉淀离心分离,用无水乙醇洗涤三次,减压干燥,即得单原子铜人工模拟过氧化物酶粉体。
2.如权利要求1中所述的一种制备单原子铜人工模拟过氧化物酶的方法,其特征在于,步骤(1)中所述的原始尿液指从人体内排出,密封储存时间小于24h的新鲜尿液;碱性阴离子交换树脂用量为尿液重量的20%,吸附时间为24h。
3.如权利要求1中所述的一种制备单原子铜人工模拟过氧化物酶的方法,其特征在于,步骤(2)中所述的高浓度盐溶液为氯化钠、硫酸钠、氯化钾、硫酸钾、氯化铵、硫酸铵中之一种,其浓度为10g/L,等体积浸渍,解吸时间为24h。
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