CN116870899A - Nanometer enzyme synthesis method with laccase-like activity based on manganese compound - Google Patents
Nanometer enzyme synthesis method with laccase-like activity based on manganese compound Download PDFInfo
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Abstract
Description
技术领域Technical field
本发明涉及锰化合物和纳米酶领域，具体涉及基于锰化合物的具有类漆酶活性的纳米酶合成方法。The present invention relates to the field of manganese compounds and nanozymes, and specifically relates to a method for synthesizing nanozymes with laccase-like activity based on manganese compounds.
背景技术Background technique
漆酶是一种含四个铜离子的多酚氧化酶，发生反应后生成水，因此其本质上是一种环保型酵素。因此，漆酶可作为绿色催化剂应用于水处理、土壤生物修复等方面。然而，由于成本高，在复杂环境中的稳定性差，回收难，严重阻碍了漆酶的实际应用。纳米酶相较于传统生物酶，具有更好的环境稳定性，成本低，可回收，易于加工。本发明专利的内容涉及基于锰化合物的具有类漆酶活性的纳米酶合成方法。Laccase is a polyphenol oxidase containing four copper ions, which reacts to produce water, so it is essentially an environmentally friendly enzyme. Therefore, laccase can be used as a green catalyst in water treatment, soil bioremediation, etc. However, the high cost, poor stability in complex environments, and difficulty in recycling have seriously hindered the practical application of laccase. Compared with traditional biological enzymes, nanozymes have better environmental stability, low cost, recyclability, and easy processing. The content of the patent of the present invention relates to the synthesis method of nanozymes with laccase-like activity based on manganese compounds.
发明内容Contents of the invention
本发明专利的内容涉及基于锰化合物的具有类漆酶活性的纳米酶合成方法。具有类漆酶活性的纳米酶，本发明简称为漆酶纳米酶。本方法具有操作简易、程序简单等优点。The content of the patent of the present invention relates to the synthesis method of nanozymes with laccase-like activity based on manganese compounds. Nanozymes with laccase-like activity are referred to as laccase nanozymes in the present invention. This method has the advantages of easy operation and simple procedures.
进一步地，本专利所述基于锰化合物的具有类漆酶活性的纳米酶，也称为基于锰化合物的漆酶纳米酶；Further, the nanozyme with laccase-like activity based on manganese compounds described in this patent is also called a laccase nanozyme based on manganese compounds;
进一步地，本专利所述的锰化合物，包括但不限于Mn3O4,MnCaO2，MnO2,和其他含锰和氧的化合物；Further, the manganese compounds described in this patent include but are not limited to Mn 3 O 4 , MnCaO 2 , MnO 2 , and other compounds containing manganese and oxygen;
进一步地，本专利所述的锰化合物，包括但不限于由Mn3O4,MnCaO2，MnO2,和其他含锰和氧的化合物中的单一化合物；Further, the manganese compounds described in this patent include, but are not limited to, single compounds consisting of Mn 3 O 4 , MnCaO 2 , MnO 2 , and other compounds containing manganese and oxygen;
进一步地，本专利所述的锰化合物，包括但不限于由MnO2, Mn3O4,MnCaO2，和其他含锰和氧的化合物中的两种或两种以上化合物构成的混合锰化合物；Further, the manganese compounds described in this patent include but are not limited to mixed manganese compounds composed of two or more compounds among MnO 2 , Mn 3 O 4 , MnCaO 2 , and other compounds containing manganese and oxygen;
进一步地，本专利所述的基于锰化合物的具有类漆酶活性的纳米酶，包括但不限于由MnO2, Mn3O4,MnCaO2，和其他含锰和氧的化合物中的单一化合物，或其中的两种或两种以上的混合锰化合物；Furthermore, the nanozymes with laccase-like activity based on manganese compounds described in this patent include but are not limited to single compounds composed of MnO 2 , Mn 3 O 4 , MnCaO 2 , and other compounds containing manganese and oxygen. or a mixture of two or more manganese compounds thereof;
进一步地，本专利所述的基于锰化合物的具有类漆酶活性的纳米酶，包括但不限于由MnO2, Mn3O4,MnCaO2，和其他含锰和氧的化合物中的单一化合物，或其中的两种或两种以上的混合锰化合物与其他材料构成的复合材料；Furthermore, the nanozymes with laccase-like activity based on manganese compounds described in this patent include but are not limited to single compounds composed of MnO 2 , Mn 3 O 4 , MnCaO 2 , and other compounds containing manganese and oxygen. Or a composite material composed of two or more mixed manganese compounds and other materials;
进一步地，本专利所述的锰化合物或混合锰化合物与其他材料构成复合材料，其中的其他材料包括但不限于石墨烯及其衍生材料，碳纳米管及其衍生材料，或其他可以作为锰化合物或混合锰化合物载体材料的材料。Furthermore, the manganese compound or mixed manganese compound described in this patent forms a composite material with other materials. The other materials include but are not limited to graphene and its derivative materials, carbon nanotubes and its derivative materials, or other materials that can be used as manganese compounds. Or materials mixed with manganese compound carrier materials.
进一步地，本专利所述的锰化合物或混合锰化合物与其他材料构成复合材料，是锰化合物或混合锰化合物与其他材料通过共价，吸附，氢键，亲水作用，憎水作用，或范德华力，或这些作用的部分或全部的综合作用的相互结合或连接起来的材料。Furthermore, the manganese compound or mixed manganese compound and other materials described in this patent constitute a composite material. The manganese compound or mixed manganese compound and other materials form a composite material through covalence, adsorption, hydrogen bonding, hydrophilic interaction, hydrophobic interaction, or van der Waals. Materials that combine or connect forces, or the combined effects of some or all of these effects.
附图说明Description of the drawings
图1. 三种不同方法验证两种基于锰化合物的具有类漆酶活性的纳米酶 (Mn3O4/rGO和MnCaO2) 的活性。Figure 1. Three different methods to verify the activity of two manganese compound-based nanozymes with laccase-like activity (Mn 3 O 4 /rGO and MnCaO 2 ).
（A）ABTS方法。漆酶能够分解底物ABTS产生ABTS自由基（ABTS*），产物在420 nm处具有特征吸收峰，通过吸光值变化表征漆酶的活性。[ABTS]: 0.93 mM; [Mn3O4/rGO] 和[MnCaO2]: 0.33 mg/mL。 ABTS = 2,2'-Azinobis-(3-ethylbenzthiazoline-6-),2 ,2-联氮-二(3-乙基-/>-6-/>)二铵盐。(A) ABTS method. Laccase can decompose the substrate ABTS to produce ABTS free radicals (ABTS*). The product has a characteristic absorption peak at 420 nm. The activity of laccase is characterized by changes in absorbance value. [ABTS]: 0.93 mM; [Mn 3 O 4 /rGO] and [MnCaO 2 ]: 0.33 mg/mL. ABTS = 2,2'-Azinobis-(3-ethylbenzthiazoline-6- ),2,2-azino-bis(3-ethyl-/> -6-/> ) diammonium salt.
（B）双酚A-4-氨基安替比林显色法；双酚A （Bisphenol ， BPA）与4-氨基安替比林（4-Aminoantipyrine，4-AP），在漆酶作用下，发生典型显色反应在510 nm出现明显吸收峰。[BPA]: 0.32 mM; [4-AP]: 8 mM。(B) Bisphenol A-4-aminoantipyrine chromogenic method; bisphenol A (Bisphenol, BPA) and 4-Aminoantipyrine (4-AP), under the action of laccase, A typical color reaction occurs and an obvious absorption peak appears at 510 nm. [BPA]: 0.32 mM; [4-AP]: 8 mM.
（C）丁香醛连氮法 。通过改进Merck公司对漆酶活性的测试方法来表征漆酶的活性。具体为，在漆酶作用下，与丁香醛连氮 （Syringaldazine）反应，在530 nm处，测试反应10 min后溶液吸光度的变化。[Syringaldazine]: 0.22 mM; [Mn3O4/rGO] 和 [MnCaO2]:0.33 mg/mL。(C) Syringaldazine method. The activity of laccase was characterized by improving Merck's testing method for laccase activity. Specifically, under the action of laccase, react with syringaldazine, and test the change in absorbance of the solution after 10 minutes of reaction at 530 nm. [Syringaldazine]: 0.22 mM; [Mn 3 O 4 /rGO] and [MnCaO 2 ]: 0.33 mg/mL.
具体实施方式Detailed ways
下面结合两个基于锰化合物的漆酶纳米酶的合成展示本发明的具体实施例。Specific embodiments of the present invention are demonstrated below in conjunction with the synthesis of two laccase nanozymes based on manganese compounds.
(1)Mn3O4/rGO作为漆酶纳米酶的制备：具体是将一定量的氧化石墨烯用乙醇充分分散，一定量的醋酸锰用水溶解，然后将醋酸锰水溶液、氨水、水混合后，加入氧化石墨烯分散液，将混合物在60-120℃下剧烈搅拌10小时，将混合物移到反应釜，100-180℃反应3小时，用水和乙醇清洗产物，于60℃真空干燥得到Mn3O4/rGO纳米酶。(1) Preparation of Mn 3 O 4 /rGO as laccase nanozyme: Specifically, a certain amount of graphene oxide is fully dispersed in ethanol, a certain amount of manganese acetate is dissolved in water, and then the manganese acetate aqueous solution, ammonia water, and water are mixed. , add graphene oxide dispersion, stir the mixture vigorously at 60-120°C for 10 hours, move the mixture to the reaction kettle, react at 100-180°C for 3 hours, wash the product with water and ethanol, and dry it under vacuum at 60°C to obtain Mn 3 O 4 /rGO nanozyme.
(2)MnCaO2作为漆酶纳米酶的制备：具体是将一定量的氯化钙和醋酸锰分别溶解在水中，混合得到溶液A，将高锰酸钾和氢氧化钾混合得到热的溶液B，溶液A、B混合，剧烈搅拌生成深色沉淀物，继续搅拌2小时，得到悬浮液，离心、洗涤，50-80℃下干燥12小时，再在300-600℃下煅烧10小时，最终MnCaO2纳米酶。(2) Preparation of MnCaO 2 as laccase nanozyme: Specifically, a certain amount of calcium chloride and manganese acetate are dissolved in water respectively, and mixed to obtain solution A, and potassium permanganate and potassium hydroxide are mixed to obtain hot solution B. , mix solutions A and B, stir vigorously to form a dark precipitate, continue stirring for 2 hours to obtain a suspension, centrifuge, wash, dry at 50-80°C for 12 hours, and then calcine at 300-600°C for 10 hours, finally MnCaO 2 nanozymes.
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