阅读说明：本技术 一种HMFO@MOFs复合物材料及其制备方法和应用 (HMFO @ MOFs composite material and preparation method and application thereof ) 是由 尹恒 巩凤芹 于 2019-04-23 设计创作，主要内容包括：本发明涉及无机有机纳米催化剂及其应用领域,具体的说是2-甲基咪唑、锌离子和5-羟甲基糠醛氧化酶(5-hydroxymethylfurfural oxidase,HMFO)制备一种新型扇形多层纳米花状结构的复合材料,该复合材料结构不同于ZIF-8经典的菱形十二面体,而由多片层组合成扇形纳米花结构,更有利于小分子扩散及底物的传递,有利于提高催化效率。本发明采用的技术方案采用水溶液作为溶剂,不含有机溶剂,绿色环保。反应只需在室温下进行,能耗小,简单方便。本发明所述的HMFO@MOFs复合物保留了HMFO的催化活性,可催化HMF生成一系列高附加值产物,具有应用价值。(The invention relates to an inorganic organic nano catalyst and the application field thereof, in particular to a novel composite material with a fan-shaped multilayer nano flower-shaped structure prepared from 2-methylimidazole, zinc ions and 5-hydroxymethyl furfural oxidase (HMFO), the structure of the composite material is different from that of a ZIF-8 classic rhombic dodecahedron, and a fan-shaped nano flower structure is formed by combining a plurality of layers, so that the small molecule diffusion and the substrate transfer are facilitated, and the catalytic efficiency is improved. The technical scheme adopted by the invention adopts the aqueous solution as the solvent, does not contain an organic solvent, and is green and environment-friendly. The reaction is carried out only at room temperature, so that the energy consumption is low, and the method is simple and convenient. The HMFO @ MOFs compound disclosed by the invention reserves the catalytic activity of HMFO, can catalyze HMF to generate a series of high value-added products, and has application value.)

1. An HMFO @ MOFs composite characterized by: the composite material is obtained by mixing 5-hydroxymethylfurfural oxidase, 2-methylimidazole and a zinc ion precursor; the zinc ion precursor is zinc acetate or zinc nitrate.

2. The HMFO @ MOFs composite according to claim 1, wherein the mixing temperature is 25-40 ℃ and the mixing time is 10-60 min.

3. The HMFO @ MOFs composite according to claim 1, wherein the HMFO @ MOFs composite morphology is fanned nanoflower composed of sheet-like structures.

4. A method of making the HMFO @ MOFs composite of claim 1, characterized in that: the method comprises the following steps: mixing a 5-hydroxymethylfurfural oxidase solution, a 2-methylimidazole solution and a zinc ion precursor solution, stirring at 25-40 ℃ for 10-60min, standing for 12-24h, and washing to obtain the HMFO @ MOFs composite material.

5. The method of making HMFO @ MOFs composite according to claim 4, characterized in that: the concentration of the 2-methylimidazole solution is 120-200 mM; the concentration of the zinc precursor solution is 30-50 mM; the concentration of the 5-hydroxymethylfurfural oxidase solution is 0.5-2.0 mg/mL; the volume ratio of the 2-methylimidazole solution to the zinc precursor solution to the 5-hydroxymethylfurfural oxidase solution is 0.5-1: 0.25; the specific activity of the 5-hydroxymethylfurfural oxidase is 0.005-0.01U/mg.

6. The process for the preparation of HMFO @ MOFs composite according to claim 4 or 5, characterized in that: the solvent of the 2-methylimidazole solution and the zinc precursor solution is water, and the solvent of the 5-hydroxymethylfurfural oxidase solution is a phosphate buffer solution; the pH value of the phosphate buffer solution is 7.2, and the concentration is 0.1 mol/L.

7. The method of making the HMFO @ MOFs composite according to claim 4, wherein the concentration of the 5-hydroxymethylfurfural oxidase solution is 1.0 mg/mL; the specific activity of the 5-hydroxymethylfurfural oxidase is 0.01U/mg; the stirring time is 30 min; the stirring temperature is 25 ℃; the standing time is 18 h.

8. Use of the HMFO @ MOFs composite according to any one of claims 1 to 3 or the HMFO @ MOFs composite prepared by the preparation process according to any one of claims 4 to 7, characterized in that: the HMFO @ MOFs composite catalyzes the oxidation of 5-Hydroxymethylfurfural (HMF) to 2, 5-Diformylfuran (DFF) and 5-hydroxymethyl-2-furancarboxylic acid (FFA) under oxygen and/or air conditions.

9. Use according to claim 8, characterized in that: dissolving 5-Hydroxymethylfurfural (HMF) in a phosphate buffer solution to form an HMF solution, adding the HMFO @ MOFs composite material, and reacting at 25-30 ℃ for 24-85 h; the pH value of the phosphate buffer solution is 7.9, and the concentration of the phosphate buffer solution is 50 mmol/L; the concentration of the HMF solution is 0.5-2.0 mg/mL; the mass ratio (mg/mg) of the added HMFO @ MOFs to the HMF in the HMF solution is 10-100: 1.

10. Use according to claim 9, wherein the temperature of the catalytic reaction is 25-30 ℃ and the concentration of the HMF solution is 0.5 mg/mL.

Technical Field

The invention relates to the field of inorganic and organic nano-catalysts and application thereof, in particular to a MOFs (metal-organic frameworks) immobilized 5-hydroxymethylfurfural oxidase composite material, a preparation method thereof and application of the MOFs immobilized 5-hydroxymethylfurfural oxidase composite material in catalyzing 5-hydroxymethylfurfural to generate a series of high value-added compounds.

Technical Field

The free enzyme has the characteristics of unique high selectivity, high catalytic activity, mild condition and environmental protection, and is widely applied to the fields of chemical industry, pharmacy, food and the like. However, the stability of the free enzyme is poor and recovery is difficult, thereby limiting its industrial application. In order to solve the problems of free enzymes, the technology of immobilized enzymes is developed and developed. At present, research on novel material immobilized enzymes mainly focuses on more model enzymes, including glucose oxidase, horseradish peroxidase, lipase and the like, and the research types and the research ranges of the enzymes are relatively narrow. Because the selection of the immobilized carrier needs to comprehensively consider the molecular size, amino acid composition, structural characteristics and physicochemical properties of the target enzyme, no general immobilization method aiming at a certain class of enzymes exists so far, and the research of wider structural properties and mechanisms is still lacked. Therefore, the research range of the immobilized enzyme of the novel material is expanded, and the method has very important significance for the basic and application research of the enzyme-novel material composite structure.

Currently, HMF mostly catalyzes fructose or glucose production by a chemical catalyst, and the product contains many byproducts. In 2015, Jin et al (Chemical Engineering Science,2015,124:170-178.) realized efficient adsorption separation of 5-hydroxymethylfurfural in mixed solution by preparing a series of Metal-organic frameworks (MOFs), and compared the specific adsorption effects of ZIF-8, ZIF-90 and ZIF-93 on HMF, the result showed that the adsorption amount of ZIF-8 was the highest. 5-hydroxymethylfurfural oxidase (HMFO) as a glucose-methanol-choline oxidase can effectively catalyze 5-Hydroxymethylfurfural (HMF) to generate a series of high value-added compounds. In view of the characteristic that ZIF-8 specifically adsorbs HMF, the catalytic effect of the immobilized HMFO on HMF can be theoretically improved by immobilizing the HMFO through the ZIF-8.

Disclosure of Invention

The invention aims to provide a novel composite HMFO @ MOFs composite structure with a fan-shaped multilayer nanoflower-like structure, a preparation method and application thereof, and the composite structure has the function of catalyzing 5-hydroxymethylfurfural to generate a series of high value-added compounds. According to the invention, the raw materials 2-methylimidazole and zinc ions of ZIF-8 are adopted to realize immobilization on HMFO for the first time, and the nano biocatalyst HMFO @ MOFs with a novel structure is prepared, wherein the structure is different from a rhombic dodecahedron of the ZIF-8 classic, and is in a fan-shaped multilayer nanoflower shape. Has higher enzyme loading capacity, and the immobilized enzyme has higher stability and can be recycled. The composite structure can efficiently and continuously oxidize and catalyze the 5-hydroxymethylfurfural to generate a series of compounds with high added values. The patent expands the research range of nano biocatalysts and realizes the green preparation process of 2, 5-diformylfuran and 5-hydroxymethyl-2-furancarboxylic acid.

In order to achieve the above object, the invention adopts the following technical scheme:

the invention provides an HMFO @ MOFs composite material, which is prepared by mixing 2-methylimidazole, zinc ions and 5-hydroxymethylfurfural oxidase; the 5-hydroxymethylfurfural oxidase is disclosed in the patent application document CN 108118064.

The specific preparation process of the 5-hydroxymethylfurfural oxidase, which is shown in SEQ ID NO.1 in the gene sequence table of the 5-hydroxymethylfurfural oxidase, is as follows:

optimally synthesizing a 5-hydroxymethylfurfural oxidase gene (SEQ ID NO.1 of a sequence table), connecting an HMF oxidase gene to a pPICZa-A expression vector by using restriction endonucleases XhoI and XbaI, converting the HMF oxidase gene to escherichia coli Top10, screening and identifying positive clones by colony PCR and enzyme digestion, amplifying recombinant plasmids in the escherichia coli, recovering plasmids by using a plasmid purification kit, performing sequencing analysis, and transferring the correctly sequenced plasmids into escherichia coli E.coli BL21(DE3) in a calcium transfer mode. E.coli positive clone transformants were picked from the plates, inoculated into 5.0ml LA medium, cultured overnight at 37 ℃ at 220 r/min. The next day, 1.0mL of overnight strain was inoculated into 100.0mL of new LA medium, OD600 was about 0.6-0.8 at 37 ℃ and 220r/min, 0.1mmol/L of IPTG was added, culture was performed at 16 ℃ and 220r/min for 20h, the strain was collected by centrifugation, 30.0mL of NaCl (0.5mol/L) solution was added to resuspend the strain, and centrifugation was performed again. 10.0ml of the liquid suspension of the cell culture was resuspended in a buffer and the cell wall was broken by sonication on ice. Centrifuging and collecting supernatant to obtain crude enzyme solution. And purifying the crude enzyme solution by a Ni-NTA affinity chromatography column to obtain pure 5-hydroxymethylfurfural oxidase solution.

Based on the technical scheme, the mixing temperature is preferably 25-40 ℃, and the mixing time is preferably 10-60 min.

Based on the technical scheme, the HMFO @ MOFs composite material is preferably in a fan-shaped nanometer flower formed by a sheet structure, the composite material is formed by mixing 2-methylimidazole and zinc ions with 5-hydroxymethylfurfural oxidase, and a ZIF-8 structure formed by inducing the combination of the 2-methylimidazole and the zinc ions by the 5-hydroxymethylfurfural oxidase is in a fan-shaped nanometer flower shape instead of a traditional rhombic dodecahedron.

The invention also provides a preparation method of the HMFO @ MOFs composite material, which is characterized by comprising the following steps of: the method comprises the following steps: mixing a 5-hydroxymethylfurfural oxidase solution, a 2-methylimidazole solution and a zinc precursor solution, stirring at 25-30 ℃ for 10-60min, standing for 12-24h, and washing to obtain the HMFO @ MOFs composite material.

Based on the technical scheme, the concentration of the 2-methylimidazole solution is preferably 120-200 mM; the concentration of the zinc precursor solution is 30-50 mM; the concentration of the 5-hydroxymethylfurfural oxidase solution is 0.5-2.0 mg/mL; the volume ratio of the 2-methylimidazole solution to the zinc precursor solution to the 5-hydroxymethylfurfural oxidase solution is (0.5-1): 0.25; the specific activity of the 5-hydroxymethylfurfural oxidase is 0.005-0.01U/mg. Based on the technical scheme, preferably, the solvent of the 2-methylimidazole solution and the zinc precursor solution is water, and the solvent of the 5-hydroxymethylfurfural oxidase solution is a phosphate buffer solution; the pH value of the phosphoric acid buffer solution is 7.2, and the concentration is 0.1 mol/L; the zinc precursor is zinc acetate and zinc nitrate.

Based on the technical scheme, preferably, the concentration of the 5-hydroxymethylfurfural oxidase solution is 1.0 mg/mL; the specific activity of the 5-hydroxymethylfurfural oxidase is 0.01U/mg; the stirring time is 30 min; the stirring temperature is 25 ℃; the standing time is 18 h.

In a further aspect, the present invention provides a use of the above HMFO @ MOFs composite or the HMFO @ MOFs composite prepared by the above preparation method, wherein the reaction is carried out under aerobic conditions, i.e., in the presence of oxygen and/or air, and the HMFO @ MOFs composite catalyzes the oxidation of 5-Hydroxymethylfurfural (HMF) to 2, 5-Diformylfuran (DFF) and 5-hydroxymethyl-2-furancarboxylic acid (FFA).

Based on the technical scheme, preferably taking a 5-Hydroxymethylfurfural (HMF) solution, adding the HMFO @ MOFs composite material, and reacting at 30 ℃ for 24-85 h; the reverse reaction; the solution of the HMF is a phosphate buffer solution, the pH value of the phosphate buffer solution is 7.9, and the concentration of the phosphate buffer solution is 50 mmol/L; the concentration of the HMF solution is 0.5-1.0 mg/mL; the mass ratio (mg/mg) of the added HMFO @ MOFs to the added HMF is (10:1) - (100: 1).

Based on the technical scheme, the temperature of the catalytic reaction is 30 ℃; the concentration of the HMF solution was 0.5 mg/mL.

Advantageous effects

1. The technical scheme of the invention adopts aqueous solution as solvent, does not contain organic solvent, and is green and environment-friendly.

2. The technical scheme of the invention only needs to be carried out at room temperature, and has the advantages of low energy consumption, simplicity and convenience.

3. The HMFO @ MOFs compound structure is different from a ZIF-8 classic rhombic dodecahedron, and a plurality of layers are combined to form a fan-shaped nanoflower structure, so that the compound structure has high enzyme loading capacity, is more favorable for small molecule diffusion and substrate transfer, and is favorable for increasing the catalytic efficiency.

4. The HMFO @ MOFs compound disclosed by the invention reserves the catalytic activity of HMFO, can catalyze HMF to generate a series of high value-added products, and has application value.

5. The immobilized HMFO @ MOFs has better stability and reusability.

Drawings

FIG. 1 is an SDS-PAGE pattern of HMFO in example 1.

FIG. 2 is an SDS-PAGE analysis of HMFO @ MOFs prepared in example 1.

FIG. 3 is a scanning electron micrograph of HMFO @ MOFs prepared in example 1.

FIG. 4 is a SDS-PAGE analysis of HMFO @ MOFs prepared in example 2.

FIG. 5 is a scanning electron micrograph of HMFO @ MOFs prepared in example 2.

FIG. 6 is a SDS-PAGE analysis of HMFO @ MOFs prepared in example 3.

FIG. 7 is a scanning electron micrograph of HMFO @ MOFs prepared in example 3.

FIG. 8 is a SDS-PAGE analysis of HMFO @ MOFs prepared in example 4.

FIG. 9 is a scanning electron micrograph of HMFO @ MOFs prepared in example 4.

Detailed Description

The invention is further illustrated by the following examples: