阅读说明：本技术 一种基于半胱氨酸-组氨酸二肽与铜离子复合物纳米酶的制备方法及其应用 (Preparation method and application of nano-enzyme based on cysteine-histidine dipeptide and copper ion compound ) 是由 齐崴 黄仁亮 王晶辉 苏荣欣 于 2019-05-05 设计创作，主要内容包括：本发明公开了一种制备半胱氨酸-组氨酸二肽与铜离子配位复合物纳米酶(CH-Cu)的方法,包括：1)称取半胱氨酸-组氨酸二肽粉末20-120mg,溶解于0.5-6mL去离子水中,室温下超声10-60min；2)称取可溶性铜盐10-120mg,溶解于0.5-5mL去离子水中,室温下超声10-60min；3)将步骤1)得到的半胱氨酸-组氨酸二肽溶液与步骤2)得到的铜盐溶液与2-8mL N,N-二甲基甲酰胺(DMF)混合,超声10-60min；4)将步骤3)得到的混合溶液转移至耐压管或水热釜中,在80-140℃下反应2-8h,之后离心收集,用N,N-二甲基甲酰胺和去离子水分别清洗三次,得到粒径为30～200nm的半胱氨酸-组氨酸二肽与铜离子配位复合物纳米酶颗粒。本发明还公开了所制得的纳米酶在降解2,4-二氯苯酚、对苯二酚和肾上腺素方面的应用。(The invention discloses a method for preparing cysteine-histidine dipeptide and copper ion coordination complex nano enzyme (CH-Cu), which comprises the following steps: 1) weighing 20-120mg of cysteine-histidine dipeptide powder, dissolving in 0.5-6mL of deionized water, and performing ultrasonic treatment at room temperature for 10-60 min; 2) weighing 10-120mg of soluble copper salt, dissolving in 0.5-5mL of deionized water, and performing ultrasonic treatment at room temperature for 10-60 min; 3) mixing the cysteine-histidine dipeptide solution obtained in the step 1) and the copper salt solution obtained in the step 2) with 2-8mL of N, N-Dimethylformamide (DMF), and performing ultrasonic treatment for 10-60 min; 4) transferring the mixed solution obtained in the step 3) into a pressure-resistant pipe or a hydrothermal kettle, reacting for 2-8h at 80-140 ℃, then centrifugally collecting, and respectively washing with N, N-dimethylformamide and deionized water for three times to obtain the cysteine-histidine dipeptide and copper ion coordination complex nano-enzyme particles with the particle size of 30-200 nm. The invention also discloses application of the prepared nano enzyme in degrading 2, 4-dichlorophenol, hydroquinone and adrenalin.)

1. A preparation method of cysteine-histidine dipeptide and copper ion coordination complex nano-enzyme is characterized by comprising the following steps:

1) weighing 20-120mg of cysteine-histidine dipeptide powder, dissolving in 0.5-6mL of deionized water, and performing ultrasonic treatment at room temperature for 10-60 min;

2) weighing 10-120mg of soluble copper salt, dissolving in 0.5-5mL of deionized water, and performing ultrasonic treatment at room temperature for 10-60 min;

3) mixing the cysteine-histidine dipeptide solution obtained in the step 1) and the copper salt solution obtained in the step 2) with 2-8mL of N, N-dimethylformamide, and carrying out ultrasonic treatment for 10-60 min;

4) transferring the mixed solution obtained in the step 3) into a pressure-resistant pipe or a hydrothermal kettle, reacting for 2-8h at 80-140 ℃, then centrifugally collecting, and respectively washing with N, N-dimethylformamide and deionized water for three times to obtain the cysteine-histidine dipeptide and copper ion coordination complex nano-enzyme particles with the particle size of 30-200 nm.

2. The method of claim 1, wherein: the soluble copper salt is one of copper chloride, copper sulfate and copper nitrate or two or three of the copper chloride, the copper sulfate and the copper nitrate in any proportion.

3. The method of claim 1, wherein: in step 1), cysteine-histidine dipeptide powder, having a mass of 62mg, was dissolved in 2mL of deionized water.

4. The method of claim 1, wherein: in step 2), the mass of the soluble copper salt was 41mg, dissolved in 1mL of deionized water.

5. The method of claim 1, wherein: in the step 3), the amount of N, N-dimethylformamide is 4 mL.

6. The method of claim 1, wherein: the reaction temperature of the step 4) is 140 ℃, and the reaction time is 4.5 h.

7. The application of the cysteine-histidine dipeptide and copper ion coordination complex nano-enzyme prepared by the preparation method of any one of claims 1 to 6 in the degradation and removal of phenolic pollutants.

8. Use according to claim 7, characterized in that: the phenolic pollutants are 2, 4-dichlorophenol and hydroquinone.

9. The use of the cysteine-histidine dipeptide and copper ion coordination complex nanoenzyme prepared by the preparation method of any one of claims 1 to 6 for degrading and removing epinephrine.

Technical Field

The invention relates to the field of nano enzyme biomaterials, in particular to a preparation method and application of a nano enzyme based on a cysteine-histidine dipeptide and copper ion compound.

Background

The wide application of phenolic substances in the fields of wood preservation, insecticides, disinfectants and the like makes the phenolic substances become common environmental pollutants. The phenolic substance is used as a hormone interfering substance, has carcinogenicity, reproduction, neurotoxicity and the like, and seriously threatens the health of human beings.

The laccase is a copper-rich oxidase which takes oxygen as an oxidant to oxidize various phenols or amine organic matters, and can effectively remove phenol pollutants. Because the final product of the reaction is water, laccase is often used as a green catalyst in the fields of water treatment or soil bioremediation and the like. However, the practical application of laccases is limited by the low stability and low reuse of laccases in complex environments. In order to enhance the catalytic stability and recyclability of laccases, enzyme immobilization techniques are widely used. Although the development of enzyme immobilization technology has improved the reusability of enzymes, the enzyme immobilization technology does not fundamentally solve the disadvantage of poor enzyme catalytic stability due to the variability of enzyme molecular structure in complex catalytic systems.

Nanoenzymes are a class of nanomaterials with enzymatic activity, and have been extensively studied due to their low cost, high stability, and other advantages. At present, the nano enzyme is mainly based on nano materials such as noble metals, metal oxides, carbon and the like, and is widely applied to the fields of virus detection, degradation of organic phosphorus toxicants, sterilization and the like. Compared with natural enzyme, the nano enzyme is a nano material, so that the nano enzyme has better stability and durability in a catalytic process.

In order to obtain the high-stability biocatalyst, the nanoenzyme is constructed by an organic-inorganic compound formed by coordination of metal ions and organic ligands, and the method has the advantages of flexible synthetic method, controllable scale, high biocompatibility and the like. However, due to the structural complexity of laccase catalytic centers, nano-enzymes with laccase activity have been reported only rarely.

Disclosure of Invention

The invention aims to provide a preparation method of cysteine-histidine dipeptide and copper ion complex nano enzyme (CH-Cu), and the prepared nano enzyme has high catalytic activity, reusability and catalytic stability.

The invention also aims to provide the application of the cysteine-histidine dipeptide and copper ion complex nano enzyme (CH-Cu) in the degradation and removal of phenolic pollutants, so that chlorophenol and hydroquinone can be better degraded and detection is facilitated.

Therefore, the technical scheme of the invention is as follows:

a preparation method of cysteine-histidine dipeptide and copper ion coordination complex nano-enzyme (CH-Cu) prepares organic-inorganic composite nano-particles by a hydrothermal synthesis method, and comprises the following steps:

1) weighing 20-120mg of cysteine-histidine dipeptide powder, dissolving in 0.5-6mL of deionized water, and performing ultrasonic treatment at room temperature for 10-60 min;

2) weighing 10-120mg of soluble copper salt, dissolving in 0.5-5mL of deionized water, and performing ultrasonic treatment at room temperature for 10-60 min;

3) mixing the cysteine-histidine dipeptide solution obtained in the step 1) and the copper salt solution obtained in the step 2) with 2-8mLN, N-Dimethylformamide (DMF), and performing ultrasonic treatment for 10-60 min;

4) transferring the mixed solution obtained in the step 3) into a pressure-resistant pipe or a hydrothermal kettle, reacting for 2-8h at 80-140 ℃, then centrifugally collecting, and respectively washing with N, N-dimethylformamide and deionized water for three times to obtain the cysteine-histidine dipeptide and copper ion coordination complex nano-enzyme particles with the particle size of 30-200 nm.

Wherein the soluble copper salt is one of copper chloride, copper sulfate and copper nitrate or two or three of the copper chloride, the copper sulfate and the copper nitrate in any proportion.

In the above step 1), the cysteine-histidine dipeptide powder was dissolved in 2mL of deionized water at a mass of 62 mg.

In the step 2), the mass of the soluble copper salt is 41mg, and the soluble copper salt is dissolved in 1mL of deionized water.

In the step 3), the amount of N, N-dimethylformamide is 4 mL.

The reaction temperature in the step 4) is 140 ℃, and the reaction time is 4.5 h.

The invention also provides application of the cysteine-histidine dipeptide and copper ion coordination complex (CH-Cu) prepared by the method in degradation and removal of phenolic pollutants, wherein the phenolic pollutants are 2, 4-dichlorophenol and hydroquinone.

The invention also provides application of the cysteine-histidine dipeptide and copper ion coordination complex (CH-Cu) prepared by the method in degrading and removing epinephrine.

The invention has the following beneficial effects:

(1) compared with the traditional nano enzyme, the cysteine-histidine dipeptide and copper ion coordination complex nano enzyme (CH-Cu) prepared by the invention has the advantages that the used materials are natural, non-toxic, green and environment-friendly, the preparation process is safe and the conditions are mild.

(2) The nano enzyme (CH-Cu) prepared by the method has high catalytic activity and reusability, and shows excellent catalytic stability under the conditions of extreme pH, high temperature, long-time storage, high salt content and the like.

(3) Compared with laccase, the nano enzyme (CH-Cu) prepared by the invention has higher capability of degrading chlorophenol and hydroquinone.

(4) Compared with laccase, the nano-enzyme (CH-Cu) prepared by the invention has a lower detection limit for detection of the phenolic substances (epinephrine), and can be used for more accurately determining the concentration of the phenolic substances (epinephrine).

Drawings

FIG. 1 is a scanning electron micrograph of nanoenzyme (CH-Cu) prepared in example 1;

FIG. 2 shows the catalytic activity of nanoenzymes (CH-Cu) prepared in example 3 under different pH conditions;

FIG. 3a is the data of catalytic stability of nanoenzyme (CH-Cu) prepared in example 3 at different pH;

FIG. 3b is the data of catalytic stability of nanoenzyme (CH-Cu) prepared in example 3 at different temperatures;

FIG. 3c is the data of catalytic stability of nanoenzyme (CH-Cu) prepared in example 3 at different storage times;

FIG. 3d is the catalytic stability data of nanoenzyme (CH-Cu) prepared in example 3 at different NaCl concentrations;

FIG. 3e is the catalytic stability data of nanoenzyme (CH-Cu) prepared in example 3 at different ethanol concentrations;

FIG. 3f is the recycling data of nanoenzyme (CH-Cu) prepared in example 3;

fig. 4 is a standard curve and a standard colorimetric card for portable detection of epinephrine by using a nano-enzyme (CH-Cu) smart phone.

Detailed Description