Pickering emulsion microcapsule of interface immobilized lipase as well as preparation method and application of Pickering emulsion microcapsule

Abstract

The invention belongs to the technical field of biochemical engineering, and particularly relates to a Pickering emulsion microcapsule of interface immobilized lipase as well as a preparation method and application of the Pickering emulsion microcapsule. The preparation method of the Pickering emulsion microcapsule comprises the following steps: ultrasonically dispersing silicon oxide nanoparticles which have an emulsifying function and can adsorb enzyme into an organic solvent to serve as an oil phase, dissolving lipase into a buffer solution to serve as a water phase, mixing the water phase and the oil phase, and carrying out ultrasonic dispersion to obtain the Pickering emulsion microcapsule. And then adding a silane coupling agent, organic amine and an organic solvent, and carrying out interface gel-sol treatment to obtain the Pickering emulsion microcapsule. The Pickering emulsion microcapsule disclosed by the invention has better high temperature resistance and pressure resistance, and can realize efficient preparation of epoxide in a fixed bed continuous enzyme catalysis olefin epoxidation reaction.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to Pickering emulsion microcapsules of interface immobilized lipase and a preparation method and application thereof. Background Art

[0002] Due to the increasingly serious problems of environmental pollution and resource scarcity, the development of new chemical synthesis technology guided by green chemistry requirements has broad development prospects. Olefins are an important chemical raw material with a large market demand and a wide range of applications. The unsaturated carbon-carbon double bonds they have can be chemically modified to generate high-value products, such as epoxidation reactions. Olefin epoxides are an important product intermediate that can be used in the subsequent production of chemical products such as drugs, perfumes, agricultural chemicals, and food additives. At present, the olefin epoxidation methods are mainly chemical and biological methods. The chemical synthesis method requires the use of metal catalysts and strong acids. The catalyst has poor thermal stability during the production process and will produce a large number of by-products, which is very unfriendly to the environment. The biological method uses enzyme-catalyzed epoxidation reactions, and the reaction conditions are mild and green. However, the enzyme has low solubility in organic solvents, limited tolerance to oxidants and polar reagents, and wild enzymes are difficult to express in Escherichia coli, with poor selectivity, all of which limit the industrial development of enzymatic epoxidation of olefins. Therefore, how to further improve the stability of the immobilized enzyme system and realize continuous flow catalytic olefin epoxidation is still an important and challenging task. Summary of the invention

[0003] Aiming at the shortcomings of the prior art bioenzymatic olefin epoxidation process, such as low reaction conversion rate, high amount of oxidant added and poor stability of enzyme catalyst, a Pickering emulsion microcapsule of interface immobilized lipase is provided, which can realize the efficient preparation of epoxides in the fixed-bed continuous enzyme-catalyzed olefin epoxidation reaction.

[0004] To achieve the above object, the technical solution of the present invention is as follows:

[0005] In a first aspect, the present invention provides a method for preparing Pickering emulsion microcapsules of interfacially immobilized lipase, comprising the following steps:

[0006] Step 1: Ultrasonic dispersion of silica nanoparticles with emulsification function and capable of adsorbing enzymes in an organic solvent as an oil phase, dissolving lipase in a buffer solution as an aqueous phase, and mixing the aqueous phase with the oil phase to obtain an oil-in-water Pickering emulsion for interfacial enzyme adsorption;

[0007] Step 3: Add silane coupling agent, organic amine and organic solvent to the water-in-oil Pickering emulsion, and obtain the interface-immobilized lipase Pickering emulsion microcapsules through interfacial gel-sol.

[0008] Furthermore, the silica nanoparticles having an emulsifying function and capable of adsorbing enzymes in step 1 are positively charged silica nanoparticles obtained by modifying silica with hydrophobic silane alone or with hydrophobic silane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride, wherein the hydrophobic silane is any one of methyltrimethoxysilane, dichlorodimethylsilane, tetramethylsilane, triethoxy(octyl)silane, tris(trimethylsilyl)silane, n-octadecyltriethoxysilane, and octyltrimethoxysilane.

[0009] Furthermore, when hydrophobic silane modifies silica alone, the mass ratio of silica to hydrophobic silane is 1:0.05 to 1:2; when hydrophobic silane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride are used to modify silica together, the mass ratio of silica, hydrophobic silane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride is 1:0.05:0.05 to 1:2:2.

[0010] Furthermore, in step 1, the lipase is any one of Candida antarctica enzyme A, Candida antarctica enzyme B, and Candida plicata lipase; and the organic solvent is any one of toluene, n-octane, cyclohexane, benzene, n-dodecane, and cyclopentyl methyl ether.

[0011] Furthermore, the buffer solution in step 1 includes any one of a phosphate buffer, a phthalic acid-hydrochloric acid buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium citrate buffer; the mass ratio of the buffer solution to the silicon oxide nanoparticles is 1:0.01 to 1:5, and the volume ratio of the buffer solution to the organic solvent is 1:0.3 to 1:20.

[0012] Furthermore, polyethylene glycol, glycerol or a low eutectic solvent is added to the buffer solution to increase the stability of the lipase.

[0013] Furthermore, in step 2, the silane coupling agent is any one of methyltrimethoxysilane, dichlorodimethylsilane, tetramethylsilane, triethoxy(octyl)silane, tri(trimethylsilyl)silane, n-octadecyltriethoxysilane and octyltrimethoxysilane; the organic amine is n-hexylamine or triethylamine; the temperature of the interfacial gel-sol is 30-80°C, and the time is 6-72h.

[0014] In a second aspect, the present invention provides Pickering emulsion microcapsules with interfacially immobilized lipase prepared by the preparation method described in the first aspect, wherein the Pickering emulsion microcapsules have interfacially immobilized lipase and form a hydrophobic silicon oxide shell layer at the Pickering emulsion interface.

[0015] In a third aspect, the present invention provides the use of the Pickering emulsion microcapsules of the interface-immobilized lipase described in the second aspect for fixed-bed continuous enzyme-catalyzed olefin epoxidation reaction.

[0016] Further, the reaction conditions are as follows: the reaction temperature is 25 to 100° C., the reactant flow rate is 0.2 to 20.0 mL / h, the reactants include olefins, hydrogen peroxide or urea peroxide and organic carboxylic acid (ester), wherein the organic carboxylic acid (ester) is C2-C 20 Long chain carboxylic acids (esters) or isomeric acids (esters).

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention, based on the Pickering emulsion, utilizes the hydrophobicity of silicon oxide at the emulsion interface or the modified charged method to adsorb the enzyme to the emulsion interface, and grows a porous solid shell layer at the emulsion interface through an interfacial sol-gel method to immobilize the enzyme to the interface of the emulsion. At the same time, the different internal compositions can adjust the microenvironment where the enzyme is located, thereby improving the stability of the enzyme. Moreover, the formed shell layer has high stability and can be directly filled in a fixed bed reactor for continuous reaction. By regulating the hydrophobicity and charged properties of the shell layer, the toxic and side effects of the external environment on the internal enzyme are effectively avoided, and the problem of the substrate bringing out water during the reaction, resulting in reduced enzyme activity, is avoided.

[0019] 2. The traditional Pickering emulsion has very poor high temperature resistance. At high temperature, bubbles will appear in the water phase, causing the emulsion to break and collapse, and the internal water phase will be lost. The present invention prepares microcapsules by growing a shell layer at the interface of the Pickering emulsion, and refluxes the organic phase at 110°C. When heated, the emulsion has no obvious change and no bubbles are generated. Electron microscopy observation shows that the Pickering emulsion droplet microcapsules still maintain a uniform morphology and no obvious change in structure, indicating that the microcapsules have good high temperature resistance.

[0020] 3. In the continuous flow process of traditional Pickering emulsion, the substrate filling height and concentration are too high, which may cause the emulsion to break, so the conversion of higher substrate concentration cannot be achieved. On the basis of retaining the advantages of Pickering emulsion, the present invention grows a porous shell layer on the interface of emulsion droplets, which can not only be used for the conversion of higher concentration substrates, but also avoid the problems of emulsion breaking caused by the reaction.

[0021] 4. In the continuous flow process of traditional Pickering emulsion, the emulsion is easily demulsified in polar solvents due to its poor resistance to polar solvents, which limits the selection of continuous phase. The Pickering emulsion droplet microcapsules of the present invention can withstand polar solvents such as ethanol, ethyl acetate, N,N-dimethylformamide, etc., which expands the selection range of the continuous phase and increases the range of substrates applicable to this method.

[0022] 5. In the continuous flow process of the traditional Pickering emulsion method, the pressure resistance of the emulsion is poor. The present invention uses a cross-linked Pickering emulsion to prepare microcapsules, and controls the flow rate and pressure of the organic solvent in the continuous flow system of the microcapsules to 50 mL / h and 1 MPa respectively by a pump. It is found that the microcapsules can be maintained without deformation for 48 hours, indicating that the microcapsules have good pressure resistance.

[0023] 6. The present invention only needs to select appropriate solid particles and silane coupling agents to prepare Pickering emulsion microcapsules through a sol-gel method. The particle size, mechanical strength and permeability can be regulated by changing the amount of solid particles, the type and amount of coupling agents, and the length of cross-linking time, and the controllability is strong. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is an optical microscope image of the water-in-oil Pickering emulsion prepared in Example 1 of the present invention;

[0025] Figure 2 This is a scanning electron microscope image of the Pickering emulsion microcapsules of the interfacially immobilized lipase prepared in Example 1 of the present invention;

[0026] Figure 3 This is a curve showing the change in conversion rate over time of the microcapsules of the interface-immobilized lipase prepared in Example 1 of the present invention when applied to the cyclooctene epoxidation reaction. DETAILED DESCRIPTION

[0027] The examples described below are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures, but do not limit the protection scope of the patent of the present invention. Any technical solution obtained in the form of equivalent replacement or equivalent transformation should fall within the protection scope of the present invention.

[0028] Example 1

[0029] Preparation of Pickering emulsion microcapsules of interfacially immobilized lipase

[0030] Step 1: Disperse 2.0 g of dried 20 nm silica into 70 mL of toluene, add 0.77 g of dichlorodimethylsilane and 1.2 g of n-hexylamine, stir at 60 °C for 4 h, then centrifuge, wash, and dry to obtain dichlorodimethylsilane-modified silica nanoparticles;

[0031] Step 2: Weigh 0.04 g of dichlorodimethylsilane-modified silica nanoparticles and ultrasonically disperse them in 4 mL of n-octane as the oil phase system, take 300 μL of Antarctic Candida enzyme B and dissolve it in 2 mL of phosphate buffer as the water phase system, mix the oil and water phases, and use a high-speed homogenizer to shear at 3500 rpm to obtain an oil-in-water Pickering emulsion (morphology as shown in FIG. Figure 1 shown);

[0032] Step 3: Add 10 mL of n-octane, 0.6 g of methyltrimethoxysilane, and 0.3 g of n-hexylamine to the water-in-oil Pickering emulsion, and obtain the Pickering emulsion microcapsules of the interfacial immobilized lipase (morphology as shown in FIG. Figure 2 shown).

[0033] Application of Pickering emulsion microcapsules for interfacial immobilization of lipase

[0034] The Pickering emulsion microcapsules of the above-mentioned interface-immobilized lipase were filled in a fixed bed reactor, the reaction temperature was set at 35°C, and an ethyl acetate solution containing 0.75 mol / L hydrogen peroxide and 0.5 mol / L cyclooctene was introduced at a flow rate of 1 mL / h for reaction. The reaction was continued for 800 h, and the yield was stabilized at 99% after 20 h (e.g. Figure 3 shown).

[0035] Example 2

[0036] Preparation of Pickering emulsion microcapsules of interfacially immobilized lipase

[0037] Step 1: Disperse 1.0 g of dried 20 nm silica into 30 mL of toluene, add 0.70 g of octyltrimethoxysilane, 0.20 g of N-methoxysilylpropyl-N,N,N-trimethylammonium chloride and 0.6 g of n-hexylamine, stir at 120 ° C for 4 h, then centrifuge, wash and dry to obtain silica nanoparticles modified with octyltrimethoxysilane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride;

[0038] Step 2: Weigh 0.04 g of silica nanoparticles modified with octyltrimethoxysilane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride and ultrasonically disperse them in 4 mL of n-octane as the oil phase system, take 300 μL of Antarctic Candida enzyme B and dissolve it in 2 mL of phthalic acid-hydrochloric acid buffer as the water phase system, mix the oil and water phases, and use a high-speed homogenizer to shear at 3500 revolutions per minute to obtain an oil-in-water Pickering emulsion;

[0039] Step 3: Add 20 mL of n-octane, 0.6 g of methyltrimethoxysilane, and 0.3 g of n-hexylamine to the water-in-oil Pickering emulsion, and obtain the Pickering emulsion microcapsules of the interfacially immobilized lipase through interfacial gel-sol.

[0040] Application of Pickering emulsion microcapsules for interfacial immobilization of lipase

[0041] The Pickering emulsion microcapsules of the above-mentioned interface-immobilized lipase were filled in a fixed bed reactor, the reaction temperature was set at 35°C, and an ethyl acetate solution containing 0.75 mol / L hydrogen peroxide and 0.5 mol / L cyclohexene was introduced at a flow rate of 1 mL / h for reaction. The reaction was continued for 400 h, and the yield was stabilized at 99%.

[0042] Example 3

[0043] Preparation of Pickering emulsion microcapsules of interfacially immobilized lipase

[0044] Step 1: Disperse 1.0 g of dried 20 nm silica into 30 mL of toluene, add 1.20 g of n-octadecyltriethoxysilane and 0.6 g of n-hexylamine, stir at 120 ° C for 4 h, then centrifuge, wash, and dry, and then centrifuge, wash, and dry to obtain n-octadecyltriethoxysilane-modified silica nanoparticles;

[0045] Step 2: Weigh 0.08 g of silicon oxide nanoparticles modified with n-octadecyltriethoxysilane and ultrasonically disperse them in 8 mL of n-octane as the oil phase system, take 12.6 mg of Antarctic Candida enzyme B and dissolve it in 4 mL of phosphate buffer as the water phase system, mix the oil and water phases, and use a high-speed homogenizer to shear at 3500 revolutions per minute to obtain an oil-in-water Pickering emulsion;

[0046] Step 3: Add 20 mL of n-octane, 1.2 g of dichlorodimethylsilane and 0.6 g of n-hexylamine to the water-in-oil Pickering emulsion, and obtain the Pickering emulsion microcapsules of the interfacially immobilized lipase through interfacial gel-sol.

[0047] Application of Pickering emulsion microcapsules for interfacial immobilization of lipase

[0048] The Pickering emulsion microcapsules of the above-mentioned interface-immobilized lipase were filled in a fixed bed reactor, the reaction temperature was set at 35°C, and an ethyl acetate solution containing 0.75 mol / L hydrogen peroxide and 0.5 mol / L styrene was introduced at a flow rate of 1 mL / h for reaction. The reaction was carried out continuously for 400 h, and the yield was stabilized at more than 85%.

[0049] Example 4

[0050] Preparation of Pickering emulsion microcapsules of interfacially immobilized lipase

[0051] Step 1: Disperse 1.0 g of dried 20 nm silica in 30 mL of toluene, add 0.80 g of tris(trimethylsilyl)silane and 0.4 g of triethylamine, stir at 120 ° C for 4 h, then centrifuge, wash, and dry, and then centrifuge, wash, and dry to obtain tris(trimethylsilyl)silane-modified silica nanoparticles;

[0052] Step 2: Weigh 0.16 g of tri(trimethylsilyl)silane-modified silica nanoparticles and ultrasonically disperse them in 16 mL of n-octane as the oil phase system, take 25.2 mg of Antarctic Candida enzyme B and dissolve it in 8 mL of phosphate buffer and 2 mL of glycerol as the water phase system, mix the oil and water phases, and use a high-speed homogenizer to shear at 3500 revolutions per minute to obtain an oil-in-water Pickering emulsion;

[0053] Step 3: Add 40 mL of toluene, 1.8 g of methyltrimethoxysilane and 1.2 g of triethylamine to the water-in-oil Pickering emulsion, and obtain the Pickering emulsion microcapsules of the interfacially immobilized lipase through interfacial gel-sol.

[0054] Application of Pickering emulsion microcapsules for interfacial immobilization of lipase

[0055] The Pickering emulsion microcapsules of the above-mentioned interface-immobilized lipase were filled in a fixed bed reactor, the reaction temperature was set at 35°C, and an ethyl acetate solution containing 0.75 mol / L hydrogen peroxide and 0.5 mol / L cyclohexanone was introduced at a flow rate of 1 mL / h for reaction. The reaction was continued for 400 h, and the yield was stabilized at 91%.

[0056] Example 5

[0057] Preparation of Pickering emulsion microcapsules of interfacially immobilized lipase

[0058] Step 1: Disperse 1.0 g of dried 20 nm silica in 30 mL of toluene, add 0.80 g of n-octadecyltriethoxysilane, 0.20 g of N-methoxysilylpropyl-N,N,N-trimethylammonium chloride and 0.4 g of triethylamine, stir at 120 ° C for 4 h, then centrifuge, wash, dry, and then centrifuge, wash, and dry to obtain silicon oxide nanoparticles modified by n-octadecyltriethoxysilane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride;

[0059] Step 2: Weigh 0.16 g of silicon oxide nanoparticles modified by n-octadecyltriethoxysilane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride and ultrasonically disperse them in 16 mL of n-octane as the oil phase system, take 25.2 mg of Antarctic Candida enzyme B and dissolve it in 8 mL of citric acid-sodium citrate solution and 3 mL of low eutectic solvent as the water phase system, mix the oil and water phases, and use a high-speed homogenizer to shear at 3500 revolutions per minute to obtain an oil-in-water Pickering emulsion;

[0060] Step 3: Add 40 mL of toluene, 2.4 g of methyltrimethoxysilane and 1.8 g of triethylamine to the water-in-oil Pickering emulsion, and obtain the interface-immobilized lipase Pickering emulsion microcapsules through interfacial gel-sol.

[0061] Application of Pickering emulsion microcapsules for interfacial immobilization of lipase

[0062] The Pickering emulsion microcapsules of the above-mentioned interface-immobilized lipase were filled in a fixed bed reactor, the reaction temperature was set at 35°C, and an ethyl acetate solution containing 0.75 mol / L hydrogen peroxide and 0.5 mol / L 4-heptanone was introduced at a flow rate of 1 mL / h for reaction. The reaction was continued for 400 h, and the yield was stabilized at 85%.

[0063] The contents not described in detail in the specification of the present invention belong to the prior art known to the professional and technical personnel in the field. Although the illustrative specific embodiments of the present invention are described above to facilitate the understanding of the present invention by the technical personnel in the field, it should be clear that the present invention is not limited to the scope of the specific embodiments. For the ordinary technical personnel in the field, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

Claims

1. A method for preparing Pickering emulsion microcapsules of interfacially immobilized lipase, characterized in that: The following steps are involved: Step 1: Ultrasonic dispersion of silica nanoparticles with emulsification function and capable of adsorbing enzymes in an organic solvent as an oil phase, dissolving lipase in a buffer solution as an aqueous phase, and mixing the aqueous phase with the oil phase to obtain an oil-in-water Pickering emulsion for interfacial enzyme adsorption; Step 2: Add silane coupling agent, organic amine and organic solvent to the water-in-oil Pickering emulsion, and obtain the Pickering emulsion microcapsules of interfacial immobilized lipase through interfacial gel-sol.

2. The method for preparing the Pickering emulsion microcapsules of interfacially immobilized lipase according to claim 1, characterized in that: The silica nanoparticles having an emulsifying function and capable of adsorbing enzymes in step 1 are positively charged silica nanoparticles obtained by modifying silica with hydrophobic silane alone or with hydrophobic silane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride, wherein the hydrophobic silane is any one of methyltrimethoxysilane, dichlorodimethylsilane, tetramethylsilane, triethoxy(octyl)silane, tris(trimethylsilyl)silane, n-octadecyltriethoxysilane, and octyltrimethoxysilane.

3. The method for preparing the Pickering emulsion microcapsules of interfacially immobilized lipase according to claim 2, characterized in that: When hydrophobic silane modifies silica alone, the mass ratio of silica to hydrophobic silane is 1:0.05 to 1:2; when hydrophobic silane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride are used to modify silica together, the mass ratio of silica, hydrophobic silane and N-methoxysilylpropyl-N,N,N-trimethylammonium chloride is 1:0.05:0.05 to 1:2:

2.

4. The method for preparing the Pickering emulsion microcapsules of interfacially immobilized lipase according to claim 1, characterized in that: In the step 1, the lipase is any one of Candida antarctica enzyme A, Candida antarctica enzyme B, and Candida plicata lipase; and the organic solvent is any one of toluene, n-octane, cyclohexane, benzene, n-dodecane, and cyclopentyl methyl ether.

5. The method for preparing the Pickering emulsion microcapsules of interfacially immobilized lipase according to claim 1, characterized in that: The buffer solution in step 1 includes any one of a phosphate buffer, a phthalic acid-hydrochloric acid buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium citrate buffer; the mass ratio of the buffer solution to the silicon oxide nanoparticles is 1:0.01 to 1:5, and the volume ratio of the buffer solution to the organic solvent is 1:0.3 to 1:

20.

6. The method for preparing the Pickering emulsion microcapsules of interfacially immobilized lipase according to claim 5, characterized in that: Polyethylene glycol, glycerol or a low eutectic solvent is also added to the buffer solution to increase the stability of the lipase.

7. The method for preparing the Pickering emulsion microcapsules of interfacially immobilized lipase according to claim 1, characterized in that: In step 2, the silane coupling agent is any one of methyltrimethoxysilane, dichlorodimethylsilane, tetramethylsilane, triethoxy(octyl)silane, tri(trimethylsilyl)silane, n-octadecyltriethoxysilane and octyltrimethoxysilane; the organic amine is n-hexylamine or triethylamine; the temperature of the interface gel-sol is 30-80°C, and the time is 6-72h.

8. The Pickering emulsion microcapsules of interfacially immobilized lipase prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The Pickering emulsion microcapsule interface immobilizes lipase and forms a hydrophobic silicon oxide shell layer at the Pickering emulsion interface.

9. The use of the Pickering emulsion microcapsules of interfacially immobilized lipase according to claim 8, characterized in that: Used for fixed-bed continuous enzyme-catalyzed olefin epoxidation reaction.

10. The use of the Pickering emulsion microcapsules for interfacial immobilized lipase according to claim 9, characterized in that: The reaction conditions are as follows: the reaction temperature is 25 to 100° C., the flow rate of the reactants is 0.2 to 20.0 mL / h, the reactants include olefins, hydrogen peroxide or urea peroxide and organic carboxylic acids (esters), wherein the organic carboxylic acids (esters) are C2-C 20 Long chain carboxylic acids (esters) or isomeric acids (esters).

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