阅读说明：本技术 磁性纳米粒-脂肪酶-金属有机框架复合催化材料及其制备方法和应用 (Magnetic nanoparticle-lipase-metal organic framework composite catalytic material and preparation method and application thereof ) 是由 黄和 张幸 李昺之 纪元 宋萍 于 2020-06-18 设计创作，主要内容包括：本发明公开了磁性纳米粒-脂肪酶-金属有机框架复合催化材料及其制备方法和应用,该复合催化材料以金属有机框架作为一种新型的固定化酶材料,将脂肪酶和磁性纳米粒包裹在其内部形成。本发明制备得到磁性纳米粒-脂肪酶-金属有机框架复合催化材料后,将其加入植物甾醇和酯化试剂的混合溶液中,催化酯化反应得到植物甾醇酯产品。本发明的复合催化材料能够高效催化植物甾醇的酯化反应,不仅具有良好的热稳定性和化学稳定性,还能提高酶促反应速率,并可以实现多次回收利用同时保持较高的催化活性,并且因为添加了磁性纳米粒从而简化了分离步骤。(The invention discloses a magnetic nanoparticle-lipase-metal organic framework composite catalytic material, a preparation method and application thereof. After the magnetic nanoparticle-lipase-metal organic framework composite catalytic material is prepared, the magnetic nanoparticle-lipase-metal organic framework composite catalytic material is added into a mixed solution of phytosterol and an esterification reagent, and the phytosterol ester product is obtained through catalytic esterification. The composite catalytic material can efficiently catalyze the esterification reaction of phytosterol, has good thermal stability and chemical stability, can improve the enzymatic reaction rate, can realize repeated recycling and maintain higher catalytic activity, and simplifies the separation step because of the addition of the magnetic nanoparticles.)

1. The magnetic nanoparticle-lipase-metal organic framework composite catalytic material is characterized in that a Zn-based MOFs material is used as a carrier, and the magnetic nanoparticle and lipase are wrapped inside the composite catalytic material.

2. The magnetic nanoparticle-lipase-metal organic framework composite catalytic material as claimed in claim 1, wherein the Zn-based MOFs material is ZIF-8, ZIF-90 or MAF-7.

3. The magnetic nanoparticle-Lipase-metal-organic framework composite catalytic material according to claim 1, wherein the Lipase is preferably Lipase (from Pseudomonas fluorescens), Lipozyme435 Lipase or Lipase (from Candida rugosa).

4. A preparation method of the magnetic nanoparticle-lipase-metal organic framework composite catalytic material of claim 1, characterized by comprising the following steps:

(1) preparing Magnetic Nanoparticles (MNPs) wrapped by Polydopamine (PDA): firstly Fe₃O₄Ultrasonic dispersing in chloroform, adding dopamine hydrochloride into dimethyl sulfoxide solution, and adding Fe₃O₄Stirring the chloroform solution uniformly and then carrying out oscillation reaction;

(2) after the reaction is finished, cooling to room temperature, adding n-hexane into the reaction liquid obtained in the step (1), centrifuging, taking the solid, and drying to obtain powdery PDA-MNPs;

(3) preparing a magnetic nanoparticle-lipase-metal organic framework composite catalytic material: adding the prepared PDA-MNPs powder into a dimethylformamide solution for dissolving, uniformly stirring, adding into a mixed solution of a lipase solution, a zinc nitrate solution and an imidazole solution, uniformly stirring, and reacting to obtain a reaction solution containing a composite catalytic material;

(4) and (4) carrying out centrifugal washing and air drying on the reaction liquid obtained in the step (3) to obtain the magnetic nanoparticle-lipase-metal organic framework composite catalytic material.

5. According to claim 4The preparation method is characterized in that the ultrasonic dispersion time in the step (1) is 4-6 min, and Fe₃O₄The molar ratio of the dopamine hydrochloride to the dopamine is 1: 3-1: 6, the oscillation reaction is water bath constant temperature oscillation temperature is 70-80 ℃, the rotating speed is 100-200 rpm, and the reaction time is 1-1.5 h; in the step (2), the volume ratio of n-hexane to the reaction liquid in the step (1) is 1: 1; and (3) adding 0.3-1 mg of PDA-MNPs powder into 0.6mL of dimethylformamide solution for dissolving, and stirring and reacting at 35-45 ℃ for 15-25 h to obtain a reaction solution containing the composite catalytic material.

6. The application of the magnetic nanoparticle-lipase-metal organic framework composite catalytic material of claim 1 in preparation of phytosterol ester.

7. Use according to claim 6, characterized in that it comprises the following steps:

(1) mixing phytosterol and an esterification reagent, adding an organic solvent, filling nitrogen, sealing, and oscillating and mixing;

(2) after dissolving the phytosterol, adding a magnetic nanoparticle-lipase-metal organic framework composite catalytic material and a dehydrating agent, filling nitrogen, sealing, and carrying out oscillation reaction;

(3) and (3) centrifuging the reaction liquid obtained in the step (2), taking the upper-layer oily matter, carrying out magnetic adsorption on the rest solution to collect the composite catalyst, washing for recycling, cooling the upper-layer oily matter, filtering, washing with a sodium bicarbonate solution, washing with ethanol to remove excessive esterification reagents and phytosterol, uniformly mixing with cyclohexanone, heating until the mixture is dissolved, stopping heating, naturally cooling to room temperature for crystallization, recrystallizing, filtering, and finally drying the product to obtain the phytosterol ester.

8. The use according to claim 7, wherein the phytosterol of step (1) comprises cholesterol, sitosterol, stigmasterol, or campesterol; the esterification reagent comprises pyruvic acid, conjugated linoleic acid or acetic acid; the organic solvent comprises tert-butyl alcohol, butanone, tert-amyl alcohol, cyclohexane or n-hexane, wherein the molar ratio of the phytosterol to the esterification reagent is 1: 2-1: 5.

9. The use of claim 7, wherein the sealing in step (1) is followed by shaking and mixing in a water bath at 35-40 ℃ at 100-300 rpm for 20-40 min.

10. The application of claim 7, wherein the amount of the magnetic nanoparticle-lipase-metal organic framework composite catalytic material added in step (2) is 5-10% of the mass of the substrate; the oscillation reaction is a water bath oscillation reaction at 50-60 ℃, the rotating speed is 100-300 rpm, and the reaction time is 38-45 h.

Technical Field

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

Background

Plant sterol is widely existed in plant cells and tissues as a natural functional active substance, but the low oil solubility greatly limits the application of the plant sterol in reality, while the plant sterol ester well solves the problem, and as an artificially synthesized natural substance, the plant sterol has no toxic or side effect, retains the biological activity of the plant sterol, has the functions of reducing cholesterol, diminishing inflammation, resisting cancer, regulating the level of physiological hormones in organisms and the like, can be widely applied to the industries of medicines, foods and the like as a functional ingredient, but has poor water solubility, fat solubility and low bioavailability, and greatly limits the application of the plant sterol. However, the phytosterol ester maintains the activity of the phytosterol and improves the lipid solubility of the phytosterol ester, thereby greatly improving the utilization rate of the phytosterol in human bodies.

At present, phytosterol synthesized at home and abroad is mainly long-chain saturated and unsaturated fatty acid ester, and the most commonly used phytosterol is synthesized by a chemical method and an enzymatic method. The chemical method synthesis steps are too complex, reagents are not environment-friendly, the enzymatic synthesis is more environment-friendly and green compared with the chemical method synthesis, the reaction conditions of the enzymatic synthesis are milder, the esterification rate is high, byproducts are less, the harm to the environment is small, and the like. Therefore, a novel composite catalytic material is urgently needed, and the activity of the enzyme can be maintained to a great extent on the basis of environmental protection so as to be recycled, so that the esterification cost is reduced.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a magnetic nanoparticle-lipase-metal organic framework composite catalytic material, which has the advantages of easy magnetic adsorption and separation, high porosity, large specific surface area, adjustable pore diameter, good thermal stability and chemical stability and the like, can protect the enzyme, so that the enzyme can endure certain denaturation conditions such as temperature, PH, organic solvent and the like, maintain the activity of the enzyme, realize the repeated utilization of the enzyme, solve the problem of esterification, the enzyme is easy to be dissolved in water and easy to influence the activity by the environment, is difficult to be collected for recycling, has high catalytic cost, meanwhile, due to the addition of the magnetic nanoparticles, the composite catalytic material is easier to perform subsequent magnetic adsorption separation, and the separation step of the subsequent catalytic material is simplified.

The invention also provides a preparation method and application of the magnetic nanoparticle-lipase-metal organic framework composite catalytic material, and the magnetic nanoparticle-lipase-metal organic framework composite catalytic material is used as a catalyst, so that the problems that in the process of synthesizing phytosterol ester by lipase catalysis, the existing lipase is soluble in water, the activity is easily influenced by the environment, and the lipase is difficult to collect and recycle are solved.

The technical scheme is as follows: the invention relates to a magnetic nanoparticle-lipase-metal organic framework composite catalytic material, which is formed by wrapping magnetic nanoparticles and lipase in Zn-based MOFs materials serving as carriers.

Wherein the Zn-based MOFs material is ZIF-8, ZIF-90 or MAF-7.

Preferably, the Lipase is a Lipase (from Pseudomonas fluorescens), Lipozyme435 Lipase or Lipase (Lipase from Candida rugosa).

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

(1) preparation of Polydopamine (PDA) wrapsMagnetic Nanoparticles (MNPs): firstly Fe₃O₄Ultrasonic dispersing in chloroform, adding dopamine hydrochloride into dimethyl sulfoxide solution, and adding Fe₃O₄Uniformly stirring a chloroform solution of magnetic nanoparticles, and then carrying out oscillation reaction;

(2) after the reaction is finished, cooling to room temperature, adding n-hexane into the reaction liquid obtained in the step (1), centrifuging, taking the solid, and drying to obtain powdery PDA-MNPs;

(3) preparing a magnetic nanoparticle-lipase-metal organic framework composite catalytic material: adding the prepared PDA-MNPs powder into a dimethylformamide solution for dissolving, uniformly stirring, adding into a mixed solution of a lipase solution, a zinc nitrate solution and an imidazole solution, uniformly stirring, and reacting to obtain a reaction solution containing a composite catalytic material;

(4) and (4) carrying out centrifugal washing and air drying on the reaction liquid obtained in the step (3) to obtain the magnetic nanoparticle-lipase-metal organic framework composite catalytic material.

Wherein the ultrasonic dispersion time in the step (1) is 4-6 min, and Fe₃O₄The mol ratio of the Fe-B-P-N-₃O₄Is commercial magnetic nano ferroferric oxide.

Wherein the volume ratio of the n-hexane in the step (2) to the reaction liquid in the step (1) is 1: 1; the centrifugal rotating speed is 10,000-13,000 rpm; centrifuging for 15-20 min.

And (3) adding 0.3-1 mg of PDA-MNPs powder into 0.6mL of dimethylformamide solution for dissolving, and stirring and reacting at 35-45 ℃ for 15-25 h to obtain a reaction solution containing the composite catalytic material.

Preferably, the stirring reaction is carried out at 37 ℃ and 500rpm for 24h to obtain a reaction solution containing the composite catalytic material.

Wherein the centrifugation condition in the step (4) is 8,000-12,000 rpm for 4-6 min.

The invention relates to an application of a magnetic nanoparticle-lipase-metal organic framework composite catalytic material in preparation of phytosterol ester.

The application comprises the following steps:

(1) mixing phytosterol and an esterification reagent, adding an organic solvent, filling nitrogen, sealing, and oscillating and mixing;

(2) after dissolving the phytosterol, adding a magnetic nanoparticle-lipase-metal organic framework composite catalytic material and a dehydrating agent, filling nitrogen, sealing, and carrying out oscillation reaction;

(3) and (3) centrifuging the reaction liquid obtained in the step (2), taking the upper-layer oily matter, carrying out magnetic adsorption on the rest solution to collect the composite catalyst, washing for recycling, cooling the upper-layer oily matter, filtering, washing with a sodium bicarbonate solution, washing with ethanol to remove excessive esterification reagents and phytosterol, uniformly mixing with cyclohexanone, heating until the mixture is dissolved, stopping heating, naturally cooling to room temperature for crystallization, recrystallizing, filtering, and finally drying the product to obtain the phytosterol ester.

Wherein, the phytosterol in the step (1) comprises cholesterol, sitosterol, stigmasterol or campesterol, and the esterifying reagent comprises pyruvic acid, conjugated linoleic acid or acetic acid; the organic solvent comprises tert-butyl alcohol, butanone, tert-amyl alcohol, cyclohexane or n-hexane, wherein the molar ratio of the phytosterol to the esterification reagent is 1: 2-1: 5.

Wherein, the sealed mixture obtained in the step (1) is mixed in a water bath at the temperature of 35-40 ℃ by oscillation at the rotating speed of 100-300 rpm for 20-40 min.

Wherein, the adding amount of the magnetic nanoparticle-lipase-metal organic framework composite catalytic material in the step (2) is 5-10% of the mass of the substrate (phytosterol and esterification reagent); the oscillation reaction is a water bath oscillation reaction at 50-60 ℃, the rotating speed is 100-300 rpm, and the reaction time is 38-45 h.

Further, the dehydrating agent is a molecular sieve, and the addition amount of the dehydrating agent is 40 mg/mL.

Wherein the centrifugation condition in the step (3) is 9,000-12,000 rpm for 4-6 min, the drying temperature is 70-90 ℃, and the drying time is 1-2 h.

Wherein, the nitrogen is filled to carry out inert gas protection on the reaction system.

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

the phytosterols in the present invention comprise primarily phytosterol acetate and phytosterol linoleate as oily solids.

In particular, the preparation of the present invention preferably comprises three parts:

a first part: preparation of polydopamine-coated Magnetic Nanoparticles (MNPs)

Firstly Fe₃O₄The magnetic nanoparticles are placed in a chloroform solution for ultrasonic dispersion for 4-6 min to remove the coagulation phenomenon caused by magnetic attraction and van der Waals force action. Then adding dopamine hydrochloride into dimethyl sulfoxide solution and adding Fe₃O₄Chloroform solution of magnetic nanoparticles (dopamine hydrochloride and Fe)₃O₄The mol ratio is 3: 1-6: 1), uniformly stirring, placing in a water bath, carrying out constant temperature oscillation, reacting at 70-80 ℃ and 100-200 rpm for 1-1.5 h, wherein dopamine is easily oxidized into dopaquinone in the reaction, and the dopaquinone and the residual dopamine are easily crosslinked and polymerized, so that Polydopamine (PDA) coated Fe is formed₃O₄Magnetic nanoparticles. After the reaction is finished, cooling to room temperature, adding n-hexane (the volume ratio of n-hexane to the reaction solution is 1:1), centrifuging at the rotating speed of 10,000-13,000 rpm for 15-20 min, filtering, and drying to obtain powdery PDA-MNPs;

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

1、[email protected]

Dissolving 0.3mg PDA-MNPs powder in 0.6mL DMF solution, stirring, adding Zn (NO)₃)₂·6H₂O (40mM) and Lipase (from Pseudomonas fluorescens) (0.1-0.5mg/ml), adding 2-methyl-imidazole (HmIm,640mM) solution, and adding ultrapure water to make volume. Reacting for 15-25 h at 37 ℃ and 500 rpm. After the reaction is finishedCentrifuging at 8,000-12,000 rpm for 4-6 min, and recovering the precipitate. The lipase not encapsulated by the MOFs material was then removed by washing with ultrapure water, sonication and centrifugation three times.

2、[email protected]

Dissolving 0.3mg PDA-MNPs powder in 0.6mL DMF solution, stirring, adding Zn (NO)₃)₂·6H₂O (40mM) and Lipase (from Pseudomonas fluorescens) (0.1-0.5mg/ml), and then 2-imidazole-carboxaldehyde (HICA, dissolved at high temperature, 160mM) solution was added thereto and the volume was increased by adding ultrapure water. Reacting for 15-25 h at 37 ℃ and 500 rpm. After the reaction is finished, centrifuging at 8,000-12,000 rpm for 4-6 min, and recovering the precipitate. The lipase not encapsulated by the MOFs material was then removed by washing with ultrapure water, sonication and centrifugation three times.

3、[email protected]

Dissolving 0.3mg PDA-MNPs powder in 0.6mL DMF solution, stirring, adding Zn (NO)₃)₂·6H₂O (40mM), and lipase Pseudomonas (0.1-0.5mg/ml), and 10% NH is preferably added during preparation of MNPs-Lip @ MAF-7₃·H₂O (60. mu.L) was added to a solution of 3-methyl-1,2,4-triazole (Hmtz, 120mM) and made to volume with ultrapure water. Reacting for 15-25 h at 37 ℃ and 500 rpm. After the reaction is finished, centrifuging at 8,000-12,000 rpm for 4-6 min, and recovering the precipitate. The lipase not encapsulated by the MOFs material was then removed by washing with ultrapure water, sonication and centrifugation three times.

And a third part: the magnetic nanoparticle-lipase-metal organic framework composite catalytic material (MNPs-Lip @ MOFs) catalyzes the esterification reaction of phytosterol.

Mixing phytosterol and an esterification reagent (pyruvic acid, conjugated linoleic acid and the like) in a molar ratio of 1: 2-1: 5, adding the mixture into a three-neck flask, adding a proper amount of an organic solvent, filling nitrogen, sealing, putting the mixture into a water bath oscillator at 35-40 ℃, and mixing for 20-40min at a rotating speed of 100-300 rpm. Then adding MNPs-Lip @ MOFs catalytic material and molecular sieve (40mg/mL) which are 5% -10% of the mass of the substrate as dehydrating agents, filling nitrogen, sealing, putting into a water bath oscillator at 50-60 ℃, reacting for 38-45 h at the rotating speed of 100-300 rpm, and finishing the reaction. The product was centrifuged, the upper oil was taken, and the remaining solution was magnetically adsorbed to collect the composite catalytic material and washed with ultrapure water for recycling. And cooling and filtering the upper-layer oily matter, washing for 3 times by using a 5% sodium bicarbonate solution, washing with ethanol to remove excessive esterification reagents and phytosterol, uniformly mixing with cyclohexanone, adding into a flask, heating until the materials are dissolved, stopping heating, naturally cooling to room temperature for crystallization, recrystallizing for 3 times, filtering, and finally drying at 70-90 ℃ for 1-2 hours to remove water to obtain the relatively pure phytosterol ester.

By adopting the combined process, the lipase is wrapped in the metal organic framework material, so that the influence of the environment on the lipase is reduced, the mechanical property of the lipase is enhanced, the operation stability is improved, and the magnetic nanoparticles are added, so that the catalyst and a reaction product are easier to separate, and the subsequent separation and purification steps are simplified. The composite catalytic material can efficiently catalyze the esterification reaction of phytosterol, has good thermal stability and chemical stability, can improve the enzymatic reaction rate, and can realize repeated recycling and maintain higher catalytic activity.

The invention wraps lipase and magnetic nanoparticles by a metal organic framework material to form a magnetic nanoparticle-lipase-metal organic framework composite catalytic material, esterifies phytosterol by using the composite catalytic material, and finally collects the composite catalytic material for recycling. The lipase is wrapped by the MOF material, so that the enzyme is protected from the influence of the external environment, the enzyme activity is maintained due to the change of the tolerant temperature, the pH and the organic solvent of the enzyme, the recycling rate of the enzyme is improved, the enzyme can be recycled for the esterification reaction of the phytosterol, and the esterification reaction cost of the phytosterol is reduced. In addition, the addition of the composite Magnetic Nanoparticles (MNPs) is more beneficial to simplifying the separation steps of the catalyst and reactants, improving the mechanical property of the composite catalytic material of the composite catalyst, improving the simplicity and convenience of operation, and further reducing feedback inhibition to realize the recycling of the composite catalytic material of the composite catalyst.

The invention uses Metal-Organic Frameworks (MOFs), which is a two-dimensional or three-dimensional crystal structure formed by using Metal ions as connecting points and Organic ligands as supports through self-assembly between the Metal ions and the Organic ligands. In the enzyme catalysis reaction, the enzyme is wrapped and fixed by the metal organic framework material to form the enzyme-metal organic framework material compound, which has the following advantages compared with the free enzyme: in the reaction process, the method can tolerate certain denaturation conditions, such as temperature, pH, organic solvent and the like; the porous characteristic of the metal organic framework material can promote the contact of enzyme and substrate, and improve the reaction rate. The Magnetic Nanoparticles (MNPs) are compounded on the basis of the enzyme-metal organic framework material compound, so that the separation steps of the catalyst and the reactant are simplified, the mechanical property of the composite catalytic material is improved, the simplicity and convenience of operation are improved, and the feedback inhibition is reduced to realize the reutilization of the composite catalytic material.

Has the advantages that: compared with the prior art, the magnetic nanoparticle-lipase-metal organic framework composite catalytic material prepared by the invention is a stable material which is easy to be magnetically adsorbed and separated, has high porosity and specific surface area, and has an adjustable structure, after the lipase is wrapped to form the composite catalytic material, the high-efficiency, mild and specific enzyme catalytic activity of zymogen can be greatly maintained, meanwhile, the defects of free enzyme are overcome, the storage stability of the enzyme is improved, the subsequent recovery is easy, the recovery step of the composite catalytic material is simplified, the repeated utilization rate is improved, and the reaction cost is reduced.

2. According to the invention, lipase and 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 efficiency of enzymatic reaction is accelerated. The composite catalytic material can efficiently catalyze the esterification reaction of phytosterol, has good thermal stability and chemical stability, and can simplify the subsequent separation step of the composite catalytic material due to the addition of the magnetic nanoparticles, so that the production cost can be further reduced by more simply and conveniently recycling.

3. The invention has simple preparation, convenient use and wide raw material source, and the prepared phytosterol ester maintains the activity of the phytosterol and improves the fat solubility, thereby greatly improving the utilization rate of the phytosterol in human bodies and being widely applied to industries such as medicine, food and the like.

Drawings

FIG. 1 is a schematic representation of the conversion of Lip @ ZIF-8 to phytosterol acetate catalyzed by esterification of phytosterols;

FIG. 2 is a schematic representation of the conversion of Lip @ ZIF-90 to phytosterol acetate catalyzed by esterification of phytosterols;

FIG. 3 is a graph showing the conversion of Lip @ MAF-7 to phytosterol acetate by esterification of phytosterols;

FIG. 4 is a schematic diagram of enzyme activity of a lipase-metal organic framework composite catalytic material under different environments.

Detailed Description

The invention will be better understood from the following examples. It is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.

Among them, Lipozyme435 Lipase (purchased from novicent biotechnology limited), lipolipase (lipasefms lipases) (purchased from alatin agents limited, product number L299014, abbreviated as Lipase Pseudomonas in examples), Lipase (Lipase from Candida rugosa) (sigma, L1754); fe₃O₄(purchased from Aladdin reagent Co., Ltd.; product No. 140494: 30 nm) is Fe₃O₄Magnetic nanoparticles; cholesterol, sitosterol, stigmasterol, campesterol (purchased from Aladdin reagents, Inc.), conjugated linoleic acid (purchased from national drug group chemical reagents, Inc.); molecular sieves (A), (B) and (C)From chemical agents, ltd, national drug group); dopamine hydrochloride (purchased from national pharmaceutical group chemical agents, ltd); 2-methyl-Imidazole (2-methylimidazole, available from Alatin reagent Co., Ltd.), Imidazole-2-carboxaldehyde (Imidazole-2-carbaldehyde, available from Alatin reagent Co., Ltd.), and 3-methyl-1,2,4-triazole (3-methyl-1, 2,4-triazole, available from Alatin reagent Co., Ltd.).