CN113856761B

CN113856761B - Preparation method and application of defective metal organic framework composite catalytic material

Info

Publication number: CN113856761B
Application number: CN202111235409.8A
Authority: CN
Inventors: 黄和; 张幸; 高婉柠; 纪元; 张鹏; 吴正章
Original assignee: Nanjing Normal University
Current assignee: Nanjing Normal University
Filing date: 2021-10-22
Publication date: 2024-07-02
Anticipated expiration: 2041-10-22

Abstract

The preparation method and application of the 'defective' metal organic framework composite catalytic material are characterized in that the 'defective' porous crystal material is used as a carrier, magnetic nanoparticles and lipase are wrapped in the carrier to form the 'defective' metal organic framework composite catalytic material, and the 'defective' porous crystal material is formed by acid etching of Zn base and 2-formaldehyde imidazole. The invention takes a metal organic framework as a novel immobilized enzyme material, wraps lipase and magnetic nano particles in the immobilized enzyme material to prepare a composite catalytic material, and then etches the composite catalytic material by acid, and applies the 'defective' composite catalytic material to the esterification reaction of plant sterol in a solvent-free system to catalyze the esterification reaction to obtain a plant sterol ester product. The composite catalytic material can not only efficiently catalyze the esterification reaction of the phytosterol in a solvent-free system, but also improve the thermal stability and chemical stability of lipase, and can realize repeated recycling and simultaneously maintain higher catalytic activity.

Description

Preparation method and application of defective metal organic framework composite catalytic material

Technical Field

The invention relates to the technical field of catalysts, in particular to a defective magnetic nanoparticle-lipase-metal organic framework composite catalytic material, and a preparation method and application thereof.

Background

Phytosterols are plant-derived natural compounds that are widely distributed in the tissues of roots, stems, leaves, and seeds of plants. Phytosterol has important physiological functions of cholesterol reduction, cancer resistance, inflammation resistance, oxidation resistance and the like, but the phytosterol cannot be synthesized from the head in a human body, and must be obtained from diet, and free phytosterol has the defects of poor solubility, high melting point, easy oxidation, poor absorption in the human body and the like. In order to overcome these problems of phytosterols and to make them reasonably applicable to medicine, the food industry is often improved by esterification.

The esterification reagent, such as conjugated linoleic acid used in the experiment, is an unsaturated fatty acid and has various physiological functions of reducing blood cholesterol, preventing atherosclerosis, resisting cancer and oxidization, improving organism immunity, promoting growth and the like, but the conjugated linoleic acid is unstable and is easy to oxidize. The method can be used for esterifying the conjugated linoleic acid with the phytosterol, so that the fat solubility and bioavailability of the phytosterol can be effectively improved, the oxidation stability of the conjugated linoleic acid can be improved, the physiological effectiveness of the conjugated linoleic acid can be fully exerted, and the application of the conjugated linoleic acid and the phytosterol in functional foods can be greatly promoted. The esterification catalytic processes currently used industrially add organic solvents to increase the solubility of the substrate, but this affects the enzymatic activity to a large extent. For a solvent-free system, only the substrate and the catalyst are present in the system, so that the loss of an esterification product is further reduced while the enzyme activity is maintained, and the esterification cost is reduced.

At present, the synthesis of the plant sterol ester is carried out by a chemical method and an enzymatic method at home and abroad, the synthesis steps of the chemical method are too complex, the used reagents are not environment-friendly, and the treatment method is too complex when the product is recycled. Compared with a chemical synthesis method, the enzymatic synthesis method has the characteristics of environmental protection, environment friendliness, milder reaction conditions, high esterification rate, few byproducts, small environmental hazard and the like. However, when free enzyme is used for catalysis, the free enzyme has the defects of easy influence of surrounding environment on enzyme activity, low recycling rate, high use cost and the like. At present, the immobilization of free enzyme by using MOF material becomes a popular research, MOF material is a novel crystalline porous material formed by combining metal ions and organic molecules, and can improve the thermal stability, pH stability and reusability of enzyme, but at the same time, the MOF material has small pore diameter, and mass transfer resistance can be generated in the process of catalytic reaction, so that the catalytic efficiency of enzyme is affected. Therefore, a novel composite catalytic material and an esterification method are urgently needed, the activity and repeatability of enzyme are maintained to a great extent on the basis of environmental protection, the esterification cost is reduced, and the step of collecting downstream products is reduced.

Disclosure of Invention

The invention aims to: aiming at the problems existing in the prior art, the invention provides a 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material, which has the advantages of easy magnetic adsorption and separation, large specific surface area, good thermal stability and chemical stability and the like; compared with the common MOF composite catalytic material, the defective composite catalytic material has larger catalytic activity due to larger frame pore diameter. The enzyme is protected on one hand, so that the enzyme can resist a certain degree of denaturation conditions such as temperature, PH, organic solvents and the like, the activity of the enzyme is maintained, the enzyme is recycled for a plurality of times, and on the other hand, the catalytic efficiency of the composite catalytic material is improved. The problems that in the esterification reaction, enzyme is easy to dissolve in water and is easy to influence the activity of the enzyme by environment, the enzyme is difficult to collect for recycling and the catalysis cost is high are effectively solved, and meanwhile, the addition of the magnetic nanoparticles can enable the composite catalytic material to be easier for subsequent magnetic adsorption separation, so that the separation steps of the subsequent catalytic material are simplified.

The invention also provides a preparation method of the defective magnetic nanoparticle-lipase-metal organic framework composite catalytic material. According to the preparation method disclosed by the invention, the aperture of the MOF material is enlarged through specific acid and etching time, so that the mass transfer resistance is reduced, and the catalytic efficiency of the enzyme is improved.

The third purpose of the invention is to provide the application of the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material to the esterification reaction of plant sterols in a solvent-free system, and the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material is used as a catalyst to solve the problems that lipase is dissolved in water and the activity is easily affected by the environment in the process of synthesizing plant sterol ester by lipase catalysis, and is difficult to collect for repeated use; by using the esterification method of the solvent-free system plant sterol, the loss of enzyme activity is reduced, the steps of collecting and separating downstream products are further reduced, and the yield of plant sterol ester is improved, so that the reaction cost is reduced. The esterification mode of the solvent-free system provided by the invention further reduces the loss of enzyme activity, and simplifies the downstream separation step, thereby reducing the industrial production cost.

The technical scheme is as follows: in order to achieve the above purpose, the invention provides a 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material, which takes a 'defective' porous crystal Material (MOFs) as a carrier, and wraps magnetic nanoparticles and lipase in the composite catalytic material to form, wherein the 'defective' porous crystal material is formed by acid etching Zn-based and 2-formaldehyde imidazole.

Preferably, the MOFs material synthesized by Zn base and 2-formaldehyde imidazole is ZIF-90, and the ZIF-90 is subjected to acid etching to form 'defective' ZIF-90.

Wherein the lipase is a lipase (CRL, lipase from Candida rugosa) produced by candida.

Wherein the acid is Tri-HCl, tannic acid or citric acid with pH of 3-7.

The preparation method of the defective magnetic nanoparticle-lipase-metal organic framework composite catalytic material comprises the following steps:

(1) Preparation of Magnetic Nanoparticles (MNPs): firstly, adding ferric chloride and ferrous chloride powder into a container containing water, adding magnetons into the container, heating and stirring the mixture in inert gas for reaction, and cooling the mixture to room temperature after the reaction is finished;

(2) Adding ammonia water into the reaction solution obtained in the step (1), adjusting pH, heating and stirring for reaction;

(3) Cooling the solution obtained in the step (2) to room temperature, recovering the magnetic nanoparticles under the magnetic field condition, and adding water to prepare MNP solution;

(4) Preparing a magnetic nanoparticle-lipase-metal organic framework composite catalytic material: adding the prepared MNP solution into a mixed solution of lipase solution, zinc nitrate solution and 2-formaldehyde imidazole solution, uniformly stirring, and reacting to obtain a reaction solution containing a composite catalytic material;

(5) And (3) centrifugally washing the reaction liquid in the step (4), adding the solid precipitate into acid for etching, and centrifugally washing the reaction liquid to form the defective metal organic framework composite catalytic material.

Wherein, in the step (1), the molar ratio of the ferric chloride to the ferrous chloride is 1:1-1:4, the temperature of the heating and stirring reaction is 70-80 ℃, the rotating speed is 200-400 rpm, and the reaction time is 1-3 h.

Wherein the volume ratio of the ammonia water in the step (2) to the reaction solution in the step (1) is 1:1-1:3, the pH is adjusted to 9-11, the heating and stirring reaction is carried out at 75-90 ℃, the rotating speed is 200-400 rpm, and the reaction is carried out for 0.5-2 h.

Wherein the concentration of the MNP solution prepared in the step (3) is 0.5-2 mg/mL

Wherein, step (4) adding 0.1-0.6 mL MNPs solution into mixed solution containing lipase solution, zinc nitrate solution and imidazole solution, wherein the molar ratio of zinc to 2-formaldehyde imidazole is 1:1-1:6, the concentration of lipase is 0.48mM, stirring the reaction at 35-45 ℃ for reacting for 10-20 h to obtain reaction solution containing composite catalytic material; the volume ratio of the MNPs solution, the lipase solution, the zinc nitrate solution and the imidazole solution is 4:5:8.

Preferably, the stirring reaction is carried out at 37℃and 500rpm for 12 hours to obtain a reaction solution containing the composite catalytic material.

Wherein, the centrifugation condition in the step (5) is 8,000-12,000 rpm for 2-6 min.

Wherein, in the step (5), the solid precipitate is put into acid for etching for 3-15 min. And compared to the activity of the solid without acid etching.

The invention relates to an application of a defective metal organic framework composite catalytic material in esterification reaction for efficiently catalyzing and synthesizing plant sterol ester.

Preferably, the application process is as follows:

(1) Adding a defective magnetic nanoparticle-lipase-metal organic framework composite catalytic material into an esterifying agent, putting a magneton, sealing, heating and stirring for reaction;

(2) After the esterifying agent and the defect type composite catalytic material are uniformly mixed, adding the phytosterol, sealing, heating and stirring for reaction;

(3) Centrifuging the reaction solution obtained in the step (2), taking an upper oily substance, magnetically adsorbing the rest solution to collect the composite catalyst, washing for recycling, and cooling the upper oily substance to obtain the phytosterol ester. After the reaction, the reaction product may be analyzed for its composition using ISQ mass spectrometry and micro-gas chromatography-ultra-gas chromatography equipped with capillary columns. The sample was injected into the gas chromatograph with helium as the carrier gas, and the flow rate, oven temperature, injector temperature, transmission line temperature, ion source temperature, and mass spectrometer scan range were set. And obtaining peak area values of all substances in the product by using a peak area normalization method, so as to calculate the esterification yield.

Wherein, the esterification reagent in the step (1) is conjugated linoleic acid, linolenic acid or oleic acid; the plant sterol in the step (2) is stigmasterol, B-sitosterol or campesterol; the mass ratio of the phytosterol to the esterifying reagent is 5-12%; the reaction systems of the steps (1) and (2) are solvent-free systems;

after the step (1) is sealed, oscillating and mixing in a water bath at 40-60 ℃ for 5-20 min, wherein the rotating speed is 100-300 rpm; the addition amount of the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material is 1% -10% of the mass of a substrate (plant sterol and esterification reagent); wherein the reaction in the step (2) is a water bath oscillation reaction at 45-65 ℃, the rotating speed is 200-400 rpm, and the reaction time is 42-60 h.

Wherein the centrifugation condition in the step (3) is 9,000-12,000 rpm, 2-6 min, and the drying temperature is 70-90 ℃ for 1-2 h.

Wherein, the catalytic reaction route of the enzyme composite catalytic material in the steps (1) and (2) is as follows:

Wherein the capillary column in the step (3) is 30m multiplied by 0.25mm multiplied by 0.25 mu m, DB-5, agilent; the sample injected into the chromatographic column is 1 mu L, and the split ratio is 1:8-1:12; the flow rate of helium is 1.2-1.5 mL/min; heating the oven at 40-60 ℃ for 1-4 min, then raising the temperature to 300-350 ℃ at 10-18 ℃/min and keeping the temperature for 2-4 min; the temperature of the injector is 240-260 ℃; the temperature of the transmission line is 250 ℃; the ion source temperature was 240 ℃.

Specifically, the preparation of the present invention preferably comprises three parts:

a first part: preparation of Magnetic Nanoparticles (MNPs)

Firstly, adding ferric chloride and ferrous chloride into a double-neck flask (the mol of the ferric chloride and the ferrous chloride is 1:1-1:4), adding a magneton, introducing nitrogen, discharging oxygen so as to enable a reaction system to be carried out under the anaerobic condition, reducing oxidation of reactants, placing the reaction system into a water bath constant temperature stirrer, and reacting for 1-3 hours at the temperature of 70-80 ℃ at 200-400 rpm. After the solution is cooled to room temperature, ammonia water is added into the reaction solution, the pH value of the solution is adjusted to 9-11 (the volume ratio of the ammonia water to the reaction solution is 1:1-1:4), the solution is placed into a water bath constant temperature stirrer, and the reaction is carried out for 0.5-2 h at the temperature of 75-90 ℃ at 200-400 rpm. And (3) after the solution is cooled to room temperature, recovering Magnetic Nanoparticles (MNPs) under the magnetic field condition, and adding water to prepare MNP solution with the concentration of 0.5-2 mg/mL.

A second part: preparation of defective magnetic nanoparticle-lipase-metal organic framework composite catalytic material

1、CRL/MNP@ZIF-90

Adding 0.3-0.6 mL of prepared MNP solution into lipase solution (0.5 mL,0.48 mM), zinc nitrate solution and imidazole solution mixed solution (the molar ratio of the zinc nitrate solution to the imidazole solution is 1:1-1:6, the volume of the mixed solution is 0.8-1.5 mL), and stirring uniformly by using ultrapure water, wherein the stirring reaction is carried out at 35-45 ℃ at the rotating speed of 300r/min, and the reaction solution containing the composite catalytic material is obtained after the reaction for 10-20 h.

2. "Defective" CRL/MNP@ZIF-90

And (3) centrifugally washing the reaction solution in the step (1) (8,000-12,000 rpm, 2-6 min) to obtain the magnetic nanoparticle-lipase-metal organic framework composite catalytic material. The composite catalytic material is subjected to acid etching for different time (3-15 min) by using Tri-HCl, tannic acid and citric acid with different pH values, and the activity of the composite catalytic material is compared with that of a solid which is not subjected to acid etching. And (3) centrifugally washing the obtained solid to obtain the defect type magnetic nanoparticle-lipase-metal organic framework composite catalytic material. After the reaction, the mixture is centrifuged at 8,000 to 12,000rpm for 4 to 6 minutes, and the precipitate is recovered. Then washed with ultrapure water, sonicated and centrifuged three times to remove lipase not encapsulated by MOFs material.

Third section: the "defective" magnetic nanoparticle-lipase-metal organic framework composite catalytic material ("defective" CRL/mnp@zif-90) catalyzes the esterification reaction of phytosterols.

Adding the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material with the mass of 8% of the substrate into an esterifying agent, putting a magneton into the esterifying agent, putting the esterifying agent into a water bath constant temperature magnetic stirrer (40-60 ℃, 100-300 rpm), heating, stirring and sealing for reaction for 5-20 min. After the esterifying agent and the defective composite catalytic material are uniformly mixed, adding the plant sterol (the mass ratio of the plant sterol to the esterifying agent is 5-12%), placing the mixture in a water bath constant temperature magnetic stirrer (45-65 ℃ and 200-400 rpm), heating, stirring and sealing for reaction for 42-60 h. After the reaction was completed, the solution was cooled to room temperature, the reaction solution was centrifuged (9,000 to 12,000rpm, 2 to 6 minutes), the upper oily substance was taken, and the remaining solution was magnetically adsorbed to collect the composite catalyst and washed with ultrapure water for reuse. After cooling the upper oil, the phytosterol ester was obtained. The reaction product was analyzed for its composition using ISQ mass spectrometry and micro-gas chromatography (Thermo Fisher) equipped with capillary columns (30 m 0.25mm 0.25 μm, DB-5, agilent). 1 mu L of sample is injected into gas chromatography, and the split ratio is 1:8-1: 12. helium is used as a carrier gas, and the flow rate is set to 1.2-1.5 mL/min. The operation conditions are set as follows, the temperature of the oven is heated to 40-60 ℃ for 1-4 min, then the temperature is increased to 300-350 ℃ at 10-18 ℃/min and kept at constant temperature for 2-4 min; the temperature of the injector is 240-260 ℃; the temperature of the transmission line is 250 ℃; the ion source temperature was 240 ℃. And obtaining peak area values of all substances in the product by using a peak area normalization method, so as to calculate the esterification yield.

The preparation process disclosed by the invention has the advantages that the lipase is wrapped in the metal organic framework material, so that the influence of the environment on the enzyme is reduced, the mechanical property of the enzyme is enhanced, the operation stability is improved, the separation of the catalyst and a reaction product is easier due to the added magnetic nanoparticles, and the subsequent separation and purification steps are simplified.

The composite catalytic material can efficiently catalyze the esterification reaction of the phytosterol in a solvent-free system, has good thermal stability and chemical stability, can improve the enzymatic reaction rate, realizes repeated recycling, simultaneously keeps higher catalytic activity, has high esterification rate, causes little pollution to the environment, reduces the esterification cost and reduces the downstream separation and recovery steps.

The MOF material is used for wrapping lipase, so that the enzyme activity is reduced or even inactivated due to the influence of external environment, the tolerance of the enzyme to temperature, PH and organic solvents is improved, the recycling rate of the enzyme is improved, and the enzyme can be recycled for the esterification reaction of plant sterols, so that the esterification reaction cost of the plant sterols is greatly reduced. In addition, the addition of the composite Magnetic Nanoparticles (MNPs) is more beneficial to the downstream separation and recovery steps, improves the mechanical properties of the composite catalytic material of the composite catalyst, and improves the operation simplicity. The aperture of the MOF material can be enlarged by a certain degree of acid etching, and the mass transfer resistance of the MOF material during enzyme catalysis is reduced, so that the catalysis efficiency of the enzyme is improved. However, in the etching process, the pore diameters of different MOF materials, the acids used for etching and the etching time are greatly affected. According to the invention, through specific acid and specific etching conditions, efficient catalysis of immobilized lipase under a solvent-free system is realized.

The innovation point of the invention is that 1) the lipase is immobilized through ZIF-90, and the ZIF-90 is etched through acid to form a larger metal frame aperture, so that the mass transfer resistance in the enzyme catalytic reaction is reduced, and the catalytic efficiency of the enzyme is improved. 2) The stable ZIF-90 which is suitable for effectively wrapping enzyme is found from a plurality of MOF materials, the synthesis condition of the ZIF-90 is mild, high temperature and high pressure are not needed, the cost of raw materials is low, and the synthesis process is simple. 3) The material is etched in different time by using different pH values and different acids, so that the most suitable etching conditions are found, and the etching conditions are mild, the method is simple and the effect is excellent by using the pH 5Tris-HCl to etch for 9 min. 4) The material is suitable for immobilizing lipase through a relatively stable ZIF-90 material, so that the stability and the recycling property of the enzyme under extreme conditions are improved. 5) The 'defective' composite catalytic material after acid etching realizes the synthesis of the plant sterol ester. 6) The synthesis of the plant sterol ester in a solvent-free system is realized, the esterification yield is improved while the enzyme activity is maintained, and the production cost is reduced. In the reaction of lipase esterification synthesis in the prior art, an organic solvent system is generally needed, which is to increase the solubility of a substrate and facilitate the catalysis of lipase, but the organic solvent can reduce the activity of lipase, and the specific 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material and the esterification method thereof can perform good catalytic reaction even in a solvent-free system. 7) The free enzyme can be catalyzed in a solvent-free system, so that the catalytic property of the enzyme is kept as much as possible, and the esterification catalytic reaction can be completed in the solvent-free system, which is not completed by a plurality of immobilized lipases; 8) The addition of the composite Magnetic Nanoparticles (MNPs) is more beneficial to simplifying the separation step of the catalyst and reactants, improving the mechanical property of the composite catalytic material of the composite catalyst, improving the operation simplicity, and further reducing the feedback inhibition to realize the recycling of the composite catalytic material of the composite catalyst.

The mechanism is as follows: the invention uses Metal-organic frame materials (Metal-Organic Frameworks, MOFs), which are two-dimensional or three-dimensional crystal structures formed by self-assembly between Metal ions and organic ligands by taking the Metal ions as connection points and organic ligands as supports. In the enzyme catalytic reaction, enzymes are wrapped and fixed by metal organic framework materials, and the formation of the enzyme-metal organic framework material compound has the following advantages compared with free enzymes: in the reaction process, the method can resist a certain degree of denaturation conditions such as temperature, pH, organic solvents and the like; the porous nature of the metal organic framework material may promote contact of the enzyme with the substrate, increasing the reaction rate. The composite Magnetic Nanoparticles (MNPs) are combined on the basis of an enzyme-metal organic framework material compound, so that the separation step of a catalyst and reactants is simplified, the mechanical property of the composite catalytic material is improved, the operation simplicity is improved, and the feedback inhibition is reduced to realize the recycling of the composite catalytic material. The metal organic framework can lead to material disintegration under acidic condition and collapse, and by utilizing the characteristic, the composite catalytic material is placed in acid with different acidity to etch for a certain time, namely, the defect can be utilized, and the surface aperture of the composite catalytic material is enlarged on the basis of not collapsing, so that the mass transfer resistance is reduced, the catalytic efficiency is improved, and the catalytic effect of immobilized enzyme is further improved. The invention utilizes the synthesized 'defect type' magnetic nanoparticle-lipase-metal organic framework composite catalytic material with mesopores to catalyze the synthesis of the phytosterol ester under the condition of no solvent system, and improves the esterification yield. Thereby reducing the production cost to a certain extent, and at the same time, exhibiting environmental friendliness.

MOF material (metal organic frame) can improve enzyme stability, improve recovery utilization ratio, and the magnetic nanoparticles added can reduce downstream separation recovery steps, thereby improving recovery efficiency. On the basis, the MOF material is etched by using acid, and the etching objects (1:1, 1:6) are screened in the etching acid and etching time, so that the aperture is enlarged, and the catalytic efficiency of the composite catalytic material can be improved. In addition, the composite catalytic material synthesized by the method catalyzes and synthesizes the phytosterol ester in a solvent-free system. The solvent-free system reduces the damage of organic solvent to enzyme, and simultaneously, the solvent-free system can also reduce the downstream recovery step and loss, and various plant sterols can be used in the invention.

In addition, the invention adopts a specific method to control the enzyme immobilization time and dosage, the temperature and time in the reaction process and the rotating speed of the magnetic stirrer, and finally realizes the efficient catalytic reaction under a solvent-free system.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

1. the prepared 'defective type' magnetic nanoparticle-lipase-metal organic framework composite catalytic material is a stable material which is easy to magnetically adsorb and separate, has high porosity and high specific surface area and adjustable structure, can maintain the original high-efficiency, mild and specific enzyme catalytic activity of enzyme to a great extent after wrapping lipase to form the composite catalytic material, overcomes the defect of free enzyme, improves the storage stability of enzyme, is easy to recover subsequently, simplifies the recovery step of the composite catalytic material, improves the recycling rate, and reduces the reaction cost. Compared with the common composite catalytic material, the defective composite catalytic material has better catalytic activity.

2. According to the invention, the lipase and the magnetic nanoparticles are wrapped in the metal organic framework material, so that the influence of the environment on the enzyme activity is reduced, the enzyme activity is maintained, the mechanical property of the enzyme is enhanced, the operation stability is improved, and the enzymatic reaction efficiency is accelerated. The composite catalytic material can efficiently catalyze the esterification reaction of the phytosterol in a solvent-free system, has good thermal stability and chemical stability, and is environment-friendly while the esterification conversion rate is increased because of the addition of the defective magnetic nanoparticles and the solvent-free system, and the subsequent separation step of the composite catalytic material can be simplified, so that the composite catalytic material can be more simply recycled and the production cost can be further reduced.

3. The invention has simple preparation, convenient use and wide raw material sources, and the prepared plant sterol ester has environmental friendliness while retaining the activity of the plant sterol, reduces the steps of downstream recovery, and improves the fat-soluble and esterification conversion rate, thereby greatly improving the utilization rate of the plant sterol in human bodies, and can be widely applied to industries such as medicines, foods and the like.

Drawings

FIG. 1 is a schematic diagram of conversion rates of stigmasterol linoleate produced by catalyzing esterification of phytosterol with different ratios of CRL/MNP@ZIF-90;

FIG. 2 is a schematic diagram showing the conversion rate of stigmasterol linoleate produced by catalyzing esterification of phytosterol after different acid etching with a metal framework ratio Zn: ICA (2-formaldehyde imidazole) of 1:6 and CRL/MNP@ZIF-90;

FIG. 3 is a schematic diagram showing the conversion rate of stigmasterol linoleate produced by catalyzing esterification of phytosterol after etching with Tris-HCl at different pH values of CRL/MNP@ZIF-90 with a metal framework ratio of Zn: ICA 1:6;

FIG. 4 is a schematic diagram showing the conversion rate of stigmasterol linoleate produced by catalyzing the esterification of phytosterol after etching with pH 5Tris-HCl for different times with a metal frame ratio Zn: ICA of 1:6 and CRL/MNP@ZIF-90;

FIG. 5 is a schematic illustration of recycling of a metal organic framework composite catalytic material;

FIG. 6 is a schematic diagram of enzyme activity at different temperatures of a metal organic framework composite catalytic material;

FIG. 7 is a schematic diagram of enzyme activity at different pH of a metal organic framework composite catalytic material.

Detailed Description

The invention will be better understood from the following examples. It will be readily understood by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.

Wherein the lipase (CRL, lipase from Candida rugosa) (available from sigma) is a lipase produced by candida; ferric chloride, ferrous chloride, ammonia, stigmasterol, B-sitosterol, campesterol (purchased from ala Ding Shiji limited), conjugated linoleic acid, linolenic acid, oleic acid (purchased from national pharmaceutical systems chemical company); 2-Imidazole-carboxaldehyde (2-formaldehyde-imidazole, ICA, purchased from ala Ding Shiji limited), zinc nitrate hexahydrate (zinc nitrate hexahydrate, purchased from ala Ding Shiji limited).

PH 3, 4, 5, 6, 7, 100mg/mL Tri-HCl, tannic acid, citric acid were formulated.

Example 1

The preparation method of the Magnetic Nanoparticles (MNPs) comprises the following steps:

Firstly, adding ferric chloride and ferrous chloride (total mass is 4 g) powder into a double-neck flask containing 60mL of water (the mole of ferric chloride and ferrous chloride is 1:2), adding magnetons, introducing nitrogen, discharging oxygen so as to enable a reaction system to be carried out under the anaerobic condition, reducing oxidation of reactants, putting into a water bath constant temperature stirrer, and reacting for 1h at 70 ℃ and 350 rpm. After the solution cooled to room temperature, ammonia water was added to the reaction solution, the pH of the solution was adjusted to 9, and the solution was placed in a water bath thermostatic stirrer and reacted at 75℃and 300rpm for 1 hour. And (3) after the solution is cooled to room temperature, recovering Magnetic Nanoparticles (MNPs) under the magnetic field condition, and adding water to prepare MNP solution with the concentration of 1 mg/mL.

Example 2

The preparation method of the magnetic nanoparticle-lipase-metal organic framework composite catalytic material comprises the following steps:

CRL/MNP@ZIF-90

Adding 0.4mL of MNP solution prepared in example 1 into a mixed solution (prepared into metal organic frame compounds with different proportions) of CRL lipase solution (0.5 mL,0.48 mM), zinc nitrate solution and 2-formaldehyde Imidazole (ICA), preparing a series of metal organic frame compounds (such as 1:1 to 1:6) with the molar ratio of zinc to imidazole, wherein the volume of the mixed solution is 0.8mL, and the solution is fixed to 1.8mL by ultrapure water, stirring uniformly, and reacting for 15 hours at 35 ℃ at the rotating speed of 300r/min to obtain a reaction solution containing the composite catalytic material.

Example 3

The preparation method of the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material (the 'defective' CRL/MNP@ZIF-90) comprises the following steps:

1. Acid etching is carried out on the composite material by utilizing Tri-HCl to form the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material

The reaction solution in example 2 was subjected to centrifugal washing (10,000 rpm,2 min) to obtain a solid precipitated magnetic nanoparticle-lipase-metal-organic framework composite catalytic material (non "defective"). The solid precipitate was added to a specific acid solution, using Tri-HCl (100 mg/mL) at pH 5, for Zn: the composite catalytic material with ICA ratio of 1:6 was subjected to acid etching at room temperature for 9min, and after the reaction was completed, centrifuged at 10,000rpm for 4min, and the precipitate was recovered. And then washing with ultrapure water, carrying out ultrasonic treatment and centrifuging for three times to remove lipase which is not wrapped by MOFs material, thus obtaining the 'defect' magnetic nanoparticle-lipase-metal organic framework composite catalytic material.

2. Acid etching is carried out on the composite material by using tannic acid to form the defective magnetic nanoparticle-lipase-metal organic framework composite catalytic material

Step 2 adopts the same preparation method as step 1, except that the etched acid is replaced by Tri-HCl with pH 5 to tannic acid with pH 5, the etching time is 9min, and after the reaction is finished, the reaction is centrifuged at 10,000rpm for 4min, and the precipitate is recovered. And then washing with ultrapure water, carrying out ultrasonic treatment and centrifuging for three times to remove lipase which is not wrapped by MOFs material, thus obtaining the 'defect' magnetic nanoparticle-lipase-metal organic framework composite catalytic material.

3. Acid etching is carried out on the composite material by utilizing citric acid to form the defective magnetic nanoparticle-lipase-metal organic framework composite catalytic material

Step 3 adopts the same preparation method as step 1, except that the etched acid is replaced by Tri-HCl with pH 5 to citric acid with pH 5, the etching time is 9min, and after the reaction is finished, the reaction is centrifuged at 10,000rpm for 4min, and the precipitate is recovered. And then washing with ultrapure water, carrying out ultrasonic treatment and centrifuging for three times to remove lipase which is not wrapped by MOFs material, thus obtaining the 'defect' magnetic nanoparticle-lipase-metal organic framework composite catalytic material.

4. Acid etching is carried out on the composite material by utilizing Tri-HCl with different pH values to form the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material

Step 4 adopts the same preparation method as step 1, except that the etched acid is replaced by Tri-HCl with pH 5 and 3-7, the etching time is 9min, and after the reaction is finished, the reaction is centrifuged at 10,000rpm for 4min, and the precipitate is recovered. And then washing with ultrapure water, carrying out ultrasonic treatment and centrifuging for three times to remove lipase which is not wrapped by MOFs material, thus obtaining the 'defect' magnetic nanoparticle-lipase-metal organic framework composite catalytic material.

5. Acid etching is carried out on the composite material for different time by utilizing pH 5Tri-HCl to form the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material

Step 5 adopts the same preparation method as in step 1, except that the etching time is replaced by 0-15 min from 9min, the etched acid is replaced by Tri-HCl with pH 5 and Tri-HCl with pH 3-7, and after the reaction is finished, the reaction is centrifuged at 10,000rpm for 4min, and the precipitate is recovered. And then washing with ultrapure water, carrying out ultrasonic treatment and centrifuging for three times to remove lipase which is not wrapped by MOFs material, thus obtaining the 'defect' magnetic nanoparticle-lipase-metal organic framework composite catalytic material.

Example 4

The preparation method of the stigmasterol linoleate by catalyzing esterification of plant sterols by using 'defective' CRL/MNP@ZIF-90 comprises the following steps:

The "defective" CRL/MNP@ZIF-90 accounting for 8% of the mass of the substrate was added to conjugated linoleic acid (200 mg), the mixture was placed in a magnetic stirrer at constant temperature in a water bath (40 ℃ C., 200 rpm), and the mixture was heated, stirred and sealed for reaction for 5min. After the esterifying agent and the defect type composite catalytic material are uniformly mixed, stigmasterol (20 mg) is added, and the mixture is placed in a water bath constant temperature magnetic stirrer (60 ℃ C., 300 rpm), heated, stirred and sealed for reaction for 50 hours. After the reaction was completed, the solution was cooled to room temperature, and the reaction mixture was centrifuged (10,000 rpm,2 min) to obtain an upper oily substance, namely, a phytosterol ester. The remaining solution was magnetically adsorbed to collect the composite catalyst and washed with ultrapure water for reuse.

The phytosterin esters were quantitatively analyzed by gas chromatography mass spectrometry using ISQ mass spectrometry and micro gas chromatography ultra gas chromatography (Thermo Fisher) equipped with capillary columns (30 m x 0.25mm x 0.25 μm, DB-5, agilent). 1 μl of sample was injected into the gas chromatograph with a split ratio of 1:10. Helium was used as the carrier gas, and the flow rate was set to 1.2mL/min. The operating conditions were set as follows: heating the oven to 50 ℃ for 2min, then heating to 300 ℃ at 15 ℃/min and keeping the temperature for 4min; injector temperature 240 ℃; the temperature of the transmission line is 250 ℃; the ion source temperature was 240 ℃. And obtaining peak area values of all substances in the product by using a peak area normalization method, so as to calculate the esterification yield. The esterification conversion rate can reach 92% through calculation. By using the same esterification method, the repeated utilization rate of the defective CRL/MNP@ZIF-90 composite catalytic material is evaluated, and the conversion rate can still reach more than 63.7% when the material is repeatedly utilized for 5 times.

After the esterification reaction is finished, centrifuging a product, collecting an upper oily substance, adding a magnet into the collected solution, adsorbing a composite catalytic material, taking down adsorbed particles, washing with ultrapure water, drying, repeatedly carrying out the phytosterol esterification reaction, calculating the esterification conversion rate, and adding magnetic nanoparticles, so that the subsequent separation step of the composite catalytic material can be simplified, further the production cost can be reduced more simply and conveniently by recycling, and the esterification conversion rate can be improved, the environmental pollution can be reduced, and the downstream separation and recovery steps can be reduced because the catalytic reaction is carried out without a solvent system.

Example 5

Example 5 the same preparation as in example 4 was used, except that stigmasterol was replaced with sitosterol, the mass ratio of sitosterol to conjugated linoleic acid was 5%, and the catalytic material was prepared in a solvent-free system using the "defective" CRL/mnp@zif-90 prepared in example 3 to catalyze esterification of phytosterol to sitosterol linoleate. After sealing, mixing in 60 ℃ water bath with shaking at 100rpm for 5min; the addition amount of the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material is 1% of the mass of a substrate, the reaction is a water bath oscillation reaction at 65 ℃, the rotating speed is 200rpm, and the reaction time is 60 hours.

Example 6

Example 6 the same preparation as in example 4 was used, except that stigmasterol was replaced with campesterol, the mass ratio of campesterol to conjugated linoleic acid was 12%, and the catalytic material was prepared in a solvent-free system using the "defective" CRL/mnp@zif-90 prepared in example 3 to catalyze esterification of phytosterol to campesterol linoleate. After sealing, mixing in a water bath at 50 ℃ with shaking, wherein the rotating speed is 300rpm, and the time is 5min; the addition amount of the 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material is 10% of the mass of a substrate, the reaction is a 45 ℃ water bath oscillation reaction, the rotating speed is 400rpm, and the reaction time is 42h.

Example 7

Example 7 the same preparation as in example 4 was used, except that conjugated linoleic acid was replaced with linolenic acid and the mass ratio of stigmasterol to linolenic acid was 10%, and the catalytic material was prepared in a solvent-free system using the "defective" CRL/mnp@zif-90 prepared in example 3 to catalyze esterification of phytosterol to produce stigmasterol linolenate.

Example 8

Example 8 the same preparation as in example 4 was used, except that conjugated linoleic acid was replaced with oleic acid and the mass ratio of stigmasterol to oleic acid was 10%, and the catalytic material was prepared in a solvent-free system using the "defective" CRL/mnp@zif-90 prepared in example 3 to catalyze the esterification of phytosterols to stigmasterol oleate.

Example 9

The enzyme activity of the metal organic framework composite catalytic material of the different proportions in example 2 of the present invention was tested by using the esterification catalytic reaction of example 4, and the enzyme activity of free lipase not coated with MOFs material. The results are shown in FIG. 1 (free lipase and ZIF-90-encapsulated lipase in different proportions, in the order from left to right in the figure, with enzyme activity expressed as ester conversion).

Example 10

1:1 Preparation method of 'defective' magnetic nanoparticle-lipase-metal organic framework composite catalytic material: example 10 the same preparation as in example 3 was used, except that the metal frame consisted of Zn: ICA ratio 1:6 was replaced with Zn: ICA ratio is 1:1, etching time of etched acid is 9min for pH 5 of Tri-HCl, and the 1:1 'defect type' magnetic nanoparticle-lipase-metal organic framework composite catalytic material is synthesized. The same preparation method as in example 4 was used for the activity detection method.

Example 11

Reusability search

The 1:1 magnetic nanoparticle-lipase-metal organic framework composite catalytic material synthesized in example 2, example 3 and example 10, the 1:6 magnetic nanoparticle-lipase-metal organic framework composite catalytic material, the 1:6 'defect type' magnetic nanoparticle-lipase-metal organic framework composite catalytic material, the 1:1 'defect type' magnetic nanoparticle-lipase-metal organic framework composite catalytic material, free lipase and the like are utilized to carry out 5-round repeated utilization experiments, distilled water is used for cleaning the materials three times after each experiment is ended, and then the materials are put into the next catalytic reaction, and the activity detection method adopts the same preparation method as that of example 4.

Example 12

Investigation of Activity under extreme conditions

Activity tests were performed under extreme conditions (pH, temperature) using the 1:1 magnetic nanoparticle-lipase-metal organic framework composite catalytic materials synthesized in example 2, example 3, example 10, 1:6 magnetic nanoparticle-lipase-metal organic framework composite catalytic materials, 1:6 "defective" magnetic nanoparticle-lipase-metal organic framework composite catalytic materials, 1:1 "defective" magnetic nanoparticle-lipase-metal organic framework composite catalytic materials, free lipase, and the like. The same preparation method as in example 4 was used for the activity detection method. The esterification conversion of the enzyme activity of the composite catalytic material treated with different acids, different pH and different etching times in example 3 with free lipase not coated with MOFs material was tested by using the esterification catalytic reaction of example 4. The results are shown in FIGS. 2, 3 and 4.

The composite catalytic materials formed under the optimal conditions in example 2 and example 3 (Zn: ICA ratio of 1:1, and composite catalytic material having Zn: ICA ratio of 1:6 were etched in Tris-HCl of pH 5 for 9 min) and free enzyme were treated in solutions of different temperatures, organic solvents and different pH, respectively, for 2 hours (example 12). The composite catalytic material and the free enzyme (the input amount of the free enzyme is consistent with that of the enzyme in the composite material) obtained after treatment are used for the esterification reaction of the plant sterol (the method of the example 4), and then the plant sterol ester is quantitatively analyzed by a gas chromatography mass spectrometer, so that the conversion rate of the esterification reaction (the conversion rate is the ratio of the residual plant sterol after the reaction to the plant sterol before the reaction) is calculated.

As can be seen from the analysis of the experimental results of FIG. 1, the esterification conversion rate of the free lipase is the best, but the free lipase is easy to deactivate in the organic solvent at the polar pH and temperature, the recovery efficiency is low, and the activity of the recovered enzyme is also reduced, while the esterification catalytic efficiency of the immobilized enzyme of the invention can not reach the free lipase, but the immobilized enzyme has good stability, high recycling rate and more cost reduction effect.

The specific acid used in the present invention as in example 3 was etched with Tris-HCl being the best and significantly superior to other acids commonly found in enzymatic reactions such as tannic acid and citric acid, and other acids such as malic acid, succinic acid, and the like. Although Tris-HCl is a common acid in enzymatic reactions, it is very specific and prominent in the present invention that the esterification conversion of immobilized lipases can be increased.

In the embodiment of the invention, under the condition of not being subjected to acid etching, the esterification catalytic efficiency reaches 91.2% when the Zn: ICA is 1:1, and the catalytic effect of Zn: ICA is only 82.4% at worst; however, according to the experimental results of FIGS. 2, 3 and 4, it can be found that the catalytic activity of the defect type composite catalytic material obtained by acid etching with pH 5Tris-HCl for 9min is highest in different acid etching times when Zn: ICA is 1:6, the esterification catalytic efficiency is up to 95.2%, and is higher than that of the composite catalytic material which is not acid etched when Zn: ICA is 1:1 when Zn: ICA is 1:6; the invention shows that under the specific acid treatment, the non-defective type material with the worst catalytic effect Zn: ICA of 1:6 forms a defective type composite material with the best effect instead, and the effect is superior to the non-defective type material with the best effect (4 percent is obviously improved in esterification conversion rate).

The results of fig. 5 were obtained using the procedure of example 11, and the results of fig. 6 and 7 were obtained using the procedure of example 12. From the analysis of the experimental results of FIGS. 5, 6 and 7, it can be seen that the enzyme activity of the free lipase is best under the optimal reaction conditions of the lipase, but the enzyme activity of the free lipase under the extreme conditions is very low (50-80 ℃ C., pH 3-6), and the reusability of the free lipase is very poor, so that the production cost is greatly increased when the free lipase is applied to industrial production; compared with free enzyme, the immobilized lipase has a good protection effect on the enzyme under the polar condition, and the recycling rate is improved; comparing the 1:1 composite catalytic material, the 1:1 'defective' composite catalytic material and the 1:6 'defective' composite catalytic material, wherein the 1:6 'defective' composite catalytic material has the best catalytic efficiency under the optimal reaction condition (pH 7, 30-40 ℃) of lipase; compared with the free lipase, the 1:6 'defective' composite catalytic material has better stability and recycling property under extreme conditions. In conclusion, the defective composite catalytic material with the ratio of 1:6 has better industrial production value.

Example 13

Example 13 was prepared in the same manner as in example 1, except that: the molar ratio of the ferric chloride to the ferrous chloride is 1:1, the temperature is 80 ℃, the rotating speed is 200rpm, and the reaction time is 3 hours; adjusting the pH of ammonia water to 9, heating and stirring to react at 90 ℃ at 200rpm for 2 hours; the concentration of the MNP solution prepared in the step (3) is 2mg/mL.

Example 14

Example 14 was prepared in the same manner as example 1, except that: in the step (1), the molar ratio of the ferric chloride to the ferrous chloride is 1:4.

Claims

1. The application of a 'defective' metal organic framework composite catalytic material in catalyzing esterification reaction of plant sterol in a solvent-free system is characterized in that the composite catalytic material takes a 'defective' porous crystal material as a carrier, magnetic nanoparticles and lipase are wrapped in the carrier to form the composite catalytic material, and the 'defective' porous crystal material is formed by acid etching of MOFs (metal organic frameworks) material synthesized by zinc nitrate and 2-formaldehyde imidazole; the lipase is candida lipase; the acid is Tris-HCl with pH of 3-7.

2. The use according to claim 1, characterized in that the process for the preparation of the "defective" metal-organic framework composite catalytic material comprises the following steps:

(1) Preparing magnetic nanoparticles: firstly, adding ferric chloride and ferrous chloride powder into a container containing water, adding magnetons into the container, heating and stirring the mixture in inert gas for reaction, and cooling the mixture to room temperature after the reaction is finished;

(5) And (3) centrifugally washing the reaction liquid in the step (4), and adding the solid precipitate into acid to etch to form the defective metal organic framework composite catalytic material.

3. The use according to claim 2, wherein in step (1) the molar ratio of ferric chloride to ferrous chloride is 1:1-1:4, the heating and stirring reaction is carried out at a temperature of 70-80 ℃, the rotating speed is 200-400 rpm, and the reaction time is 1-3 h; the pH value of the step (2) is adjusted to 9-11, the temperature of the heating and stirring reaction is 75-90 ℃, the rotating speed is 200-400 rpm, and the reaction lasts for 0.5-2 hours; the concentration of the MNP solution prepared in the step (3) is 0.5-2 mg/mL.

4. The use according to claim 2, wherein in the step (4), 0.1-0.6 mL MNPs solution is added into a mixed solution containing lipase solution, zinc nitrate solution and 2-formaldehyde imidazole solution, the molar ratio of zinc to 2-formaldehyde imidazole is 1:1-1:6, and the reaction is stirred at 35-45 ℃ for reaction 10-20: 20 h to obtain a reaction solution containing the composite catalytic material.

5. The use according to claim 2, wherein the solid precipitate is placed in an acid for an etching time of 3-15 min in step (5).

6. The application according to claim 1, wherein the process of the application is:

(3) Centrifuging the reaction solution obtained in the step (2), taking an upper oily substance, magnetically adsorbing the rest solution to collect the composite catalyst, washing for recycling, and cooling the upper oily substance to obtain the phytosterol ester.

7. The use according to claim 6, wherein the esterifying reagent in step (1) is conjugated linoleic acid, linolenic acid or oleic acid; the plant sterol in the step (2) is stigmasterol, B-sitosterol or campesterol; the mass ratio of the phytosterol to the esterifying reagent is 5-12%; the reaction systems of the steps (1) and (2) are solvent-free systems; after the sealing in the step (1), oscillating and mixing in a water bath at the temperature of 40-60 ℃ for 5-20 min, wherein the rotating speed is 100-300 rpm; the addition amount of the defective magnetic nanoparticle-lipase-metal organic framework composite catalytic material in the step (1) is 1% -10% of the mass of a substrate; the reaction in the step (2) is a water bath oscillation reaction at 45-65 ℃, the rotating speed is 200-400 rpm, and the reaction time is 42-60 h.