Preparation method of mesoporous MOFs (Metal-Organic Frameworks) material with water-acid dual stability and application of mesoporous MOFs material in high-value conversion of grease

Abstract

The invention provides a preparation method of a mesoporous MOFs (Metal-Organic Frameworks) material with water-acid dual stability and application of the mesoporous MOFs material in high-value conversion of grease. The preparation method comprises the following steps: firstly, preparing a mesoporous MOFs material of which the average pore size is greater than 10nm by a lauryl sodium sulfate mediated soft template method, and then dissociating an outer layer by using sodium citrate to increase the outer specific surface area of the outer layer, so as to obtain the mesoporous MOFs material (Meso-MIL-88A) with water-acid dual stability. The adsorption isotherm of the Meso-MIL-88A accords with a Langmuir model, the adsorption of macromolecular lipase belongs to single-molecule adsorption, and the defect that the specific enzyme activity is reduced due to enzyme accumulation caused by the fact that a macroporous material serves as an enzyme immobilization carrier is fundamentally avoided. The Meso-MIL-88A can be used for efficiently immobilizing lipase and synthesizing structural phospholipids, and has a good application prospect in the field of lipase-mediated grease high-valued treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological chemical industry, in particular, to a preparation method of mesoporous MOFs material with water-acid dual stability and application thereof in oil high-value conversion. BACKGROUND

[0002] Metal-organic frameworks (MOFs) are three-dimensional ordered porous materials formed by organic ligands and metal ions through coordination bonds. Due to its special physical and chemical properties, such as controllable pore size, high specific surface area, easy modification and modification, MOFs have great application potential in the fields of gas storage, separation, catalysis, molecular recognition, biomedicine and enzyme immobilization. However, the pore size of the current MOFs is mainly microporous (less than 2 nm), and this microporous structure often hinders the diffusion of macromolecules inside the material, making it difficult to be used for the immobilization of macromolecular lipase (lipase molecule size is often greater than 5 nm). Macroporous MOFs are too large in pore size (greater than 100 nm), which easily leads to enzyme molecule accumulation during the immobilization of lipase, seriously affecting the efficiency of enzyme catalysis. Therefore, the development of mesoporous MOFs has become a promising strategy to cope with these challenges, and has become the focus of research. In addition to the influence of pore size on the immobilization of macromolecular lipase, the stability of the material itself also seriously affects its use. Although MOFs as a new type of immobilized enzyme carrier have good development and application prospects, its stability greatly limits its application in practical systems. Among the reported MOFs, some have acid stability, some have water stability, and there is no report on MOFs with acid-water dual stability. The high-value conversion of oil catalyzed by lipase needs to occur at the oil-water interface, and the MOF used as the immobilized carrier of lipase needs to have water-acid dual stability. SUMMARY

[0003] The purpose of the present application is to provide a preparation method of mesoporous MOFs material with water-acid dual stability and application thereof in oil high-value conversion.

[0004] In order to achieve the purpose of the present application, in the first aspect, the present application provides a preparation method of mesoporous MOFs material with water-acid dual stability, which is prepared by soft template method, comprising the following steps: (1) Dissolve 3 mmol of fumaric acid (FA) and 2.50 g of sodium dodecyl sulfate (SDS) in 140 mL of deionized water, preheat at 55℃ with 250 rpm stirring for 1 hour to form a stable micellar system, denoted as solution A; (2) Dissolve 1 mmol of FeCl3·6H2O in 10 mL of deionized water and add to the A solution, react at 55°C for 24 hours under 250 rpm stirring, after the reaction, collect the light red-brown precipitate by centrifugation, and then clean the light red-brown precipitate by immersing in 150 mL of ethanol for about 6 hours to remove SDS and any unreacted precursor solution, and then vacuum dry at 60°C to obtain the dried mesoporous material; (3) Disperse 100 mg of the dried mesoporous material obtained in step (2) in 50 mL of deionized water, and add 10 mL of 0.02-0.04 mol / L sodium citrate solution, and keep the mixture at 30-50°C for 1-2 hours to dissociate part of the external MOFs and expose the internal pores. The product obtained after dissociation is a mesoporous MOFs material with water-acid dual stability, denoted as Meso-MIL-88A (i.e. MIL-88A).

[0005] In a second aspect, the present application provides a mesoporous MOFs material prepared according to the method.

[0006] The average pore size of the mesoporous MOFs material is greater than 10 nm.

[0007] In a third aspect, the present application provides any one of the following applications of the mesoporous MOFs material: 1) for enzyme immobilization field; 2) for oil and fat high-value conversion.

[0008] In a fourth aspect, the present application provides an immobilized lipase prepared by using the mesoporous MOFs material.

[0009] In a fifth aspect, the present application provides a method for oil and fat high-value conversion by using the mesoporous MOFs material to immobilize lipase, the method comprising: 1) disperse the mesoporous MOFs material in deionized water, mix with a lipase solution, then collect the immobilized lipase by centrifugation, clean with water, and then freeze-dry; 2) add the immobilized lipase to a primary or multi-stage enzyme reactor to carry out enzymatic conversion reaction of oil and fat.

[0010] Further, in 2), 200-2000 units of standard enzyme activity of immobilized lipase per unit of oil mass is added.

[0011] Further, 1) specifically: 50 mg of the mesoporous MOFs material is added to 800 μL of deionized water and sonicated for 15 minutes to disperse it evenly. Then, 200 μL of lipase solution with a concentration of 10-30% is added to the system. The resulting mixture is placed in a constant temperature shaker at 30-50°C (preferably 40°C) and 200 rpm for 3-6 hours (preferably 4 hours). Then, it is centrifuged at 8000 rpm for 3 minutes to collect the immobilized lipase, washed once with water, and finally freeze-dried overnight.

[0012] In this invention, the lipase includes, but is not limited to, Candida antarcticus (…). Candida antarctica ), thermophilic fungi ( Thermomyces lanuginosus Aspergillus niger (), Aspergillus niger Aspergillus oryzae ( ) Aspergillus oryzae ), Rhizopus oryzae ( Rhizomucor miehei ), Rhizopus oryzae ( Rhizopus oryzae Lipase.

[0013] In a sixth aspect, the present invention provides the application of the method in the preparation of structured phospholipids (such as phosphatidyl-DHA, phosphatidyl-EPA, etc.).

[0014] This invention, based on the hard-soft acid-base (HSAB) theory, screened out MOFs with acid-water stability, specifically MIL-88A, and for the first time synthesized mesoporous MIL-88A (Meso-MIL-88A) with an average pore size greater than 10 nm via a sodium dodecyl sulfate (SDS)-mediated soft template method. Furthermore, its outer layer was dissociated using sodium citrate to increase its external specific surface area. Meso-MIL-88A exhibits excellent performance in lipase immobilization and oil repurposing processes.

[0015] The mesoporous MIL-88A prepared by this invention has dual stability in water and acid, which breaks through the limitations of existing MOFs carriers in terms of stability and pore size, and has a very good application prospect in the fields of macromolecular lipase immobilization and high-value conversion of oils. Attached Figure Description

[0016] Figure 1 In a preferred embodiment of the present invention, Meso-MIL-88A was synthesized using a soft template strategy. A: SEM and TEM images of MIL-88A with different amounts of SDS. 1-4 are SEM images of C-MIL-88A (without SDS), MIL-88A with 50 mg SDS, 250 mg SDS, and 500 mg SDS in a 15 mL synthesis system, respectively. 5-6 are TEM images of the above four materials. B: (1) SEM and (2) Fe 3+ TEM images of SDS-coordinated with Fe. C: Adding different SDS and Fe...3+ SEM and TEM images of MIL-88A. 1-3 are SEM images of MIL-88A synthesized under the conditions of 15 mL of synthesis system, 50 mg of SDS and 0.05 mmol of Fe 3+ , 250 mg of SDS and 0.1 mmol of Fe 3 + , 500 mg of SDS and 0.4 mmol of Fe 3+ SEM images of MIL-88A synthesized under the conditions of 15 mL of synthesis system, 50 mg of SDS and 0.05 mmol of Fe

[0017] Figure 2 are SEM images of MIL-88A added with different sodium citrate solutions; B: FTIR spectra of C-MIL-88A, Meso-MIL-88A and Meso-MIL-88A; C: XRD patterns of C-MIL-88A, Meso-MIL-88A and Meso-MIL-88A.

[0018] Figure 3 are immobilization performance of C-MIL-88A and Meso-MIL-88A in the preferred embodiments of the present application. A: loading and loading efficiency of ET 2.0@C-MIL-88A and ET 2.0@Meso-MIL-88A; B: specific activity and recovery rate of ET 2.0@C-MIL-88A and ET 2.0@Meso-MIL-88A; C and D are CalB@C-MIL-88A and CalB@Meso-MIL-88A, respectively. DETAILED DESCRIPTION

[0019] The present application provides a kind of mesoporous MOFs immobilized carrier preparation with water-acid double stability and is used for lipase immobilization and oil high-value conversion technology.

[0020] The present application adopts the following technical scheme: First, mesoporous MOFs material with average pore size greater than 10 nm is prepared by sodium dodecyl sulfate mediated soft template method, and then the outer layer is dissociated by sodium citrate to increase the external specific surface area, to obtain mesoporous MOFs material (Meso-MIL-88A) with water-acid double stability.

[0021] The preparation method of mesoporous MOFs material MIL-88A (Meso-MIL-88A) is soft template method.

[0022] Preferably, the soft template method is sodium dodecyl sulfate mediated.

[0023] The preferred process for the preparation of Meso-MIL-88A is: 3 mmol fumaric acid (FA) and 2.50 g sodium dodecyl sulfate (SDS) are dissolved in 140 mL deionized water, preheated at 55 °C with stirring at 250 rpm for 1 h to form a stable micellar system (denoted as solution A). Subsequently, 1 mmol FeCl3-6H2O is dissolved in 10 mL deionized water and added to solution A and reacted (55 °C, 250 rpm) for 24 h. The light red-brown precipitate is then collected by centrifugation and immersed in 150 mL ethanol by sonication for about 6 h to remove SDS and any unreacted precursor solution, to obtain mesoporous MIL-88A, which is further dried under vacuum at 60 °C to obtain dried mesoporous MIL-88A.

[0024] The preferred process for the dissociation of citric acid is: the dried mesoporous MIL-88A (100 mg) prepared above is dispersed in 50 mL deionized water, and 10 mL 0.02 mol / L sodium citrate solution is added to the system. The mixture is kept at 30 °C for 1 h to dissociate part of the external MOFs and expose the internal pores. The product obtained after dissociation is Meso-MIL-88A with an average pore size of greater than 10 nm.

[0025] Further, the Meso-MIL-88A immobilized lipase is used for the preparation of high-value products of oils and fats such as structured phospholipids.

[0026] The lipase includes but is not limited to lipase derived from Candida antarctica (Novozyme 435® Candida antarctica ), Thermomyces (Lipex® Thermomyces lanuginosus ), Aspergillus niger (Lipase AFS® Aspergillus niger ), Aspergillus oryzae (Lipase Amano® Aspergillus oryzae ), Rhizomucor miehei (Lipase Lipozyme® Rhizomucor miehei ), Rhizopus oryzae (Lipase Lipex® Rhizopus oryzae ).

[0027] Further, the Meso-MIL-88A immobilized lipase is used for the preparation of high-value products of oils and fats such as structured phospholipids.

[0028] The adsorption isotherm of the prepared Meso-MIL-88A conforms to the Langmuir model, and the adsorption of macromolecular lipase belongs to monomolecular adsorption, which fundamentally avoids the disadvantage of enzyme accumulation caused by using macroporous materials as enzyme immobilization carriers, thereby reducing the specific enzyme activity. The Meso-MIL-88A can efficiently immobilize lipase and be used for the synthesis of structural phospholipids, and has a good application prospect in the field of lipase-mediated oil high-value.

[0029] The following examples are intended to illustrate the present application but not to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are the conventional means known to those skilled in the art, and the raw materials used are commercially available.

[0030] Example 1 Synthesis of microporous C-MIL-88A and mesoporous Meso-MIL-88A materials with water-acid dual stability

[0031] Synthesis of microporous C-MIL-88A: 10 mmol of FeCl3·6H2O and 30 mmol of fumaric acid were dissolved in 75 mL of deionized water, respectively, then the FeCl3·6H2O solution and the fumaric acid suspension were mixed in a flask, and reacted in a shaker (55℃, 250 rpm) for 24 hours, and the unreacted precursor solution was removed by centrifugation at 8000 rpm for 10 minutes, the obtained solid was washed with ethanol several times, and the obtained solid was separated by centrifugation (8000 rpm, 10 minutes) and dried at 60℃ under vacuum overnight to obtain C-MIL-88A.

[0032] Synthesis of mesoporous Meso-MIL-88A: synthesized by a soft template method as shown in Figure 1 The optimal conditions are as follows: 3 mmol of fumaric acid (FA) and 2.50 g of sodium dodecyl sulfate (SDS) are dissolved in 140 mL of deionized water, preheated at 55℃ with stirring at 250 rpm for 1 hour to form a stable micellar system. Then, 1 mmol of FeCl3·6H2O is dissolved in 10 mL of deionized water and added to the above mixed solution for reaction. The reaction is carried out at 55℃ with stirring at 250 rpm for 24 hours. Then, the light red-brown precipitate is collected by centrifugation, and the precipitate is immersed in 150 mL of ethanol by ultrasonic treatment for about 6 hours to remove SDS and any unreacted precursor solution. After thorough washing, the obtained MOFs are dried at 60℃ under vacuum overnight. Then, 100 mg of MOFs powder is dispersed in 50 mL of deionized water, and 10 mL of 0.02 mol / L sodium citrate solution is added. The mixture is kept at 30℃ for 1 hour to dissociate part of the external MOFs and expose the internal pores. The product obtained after dissociation is denoted as Meso-MIL-88A. The relevant characterization of the Meso-MIL-88A carrier obtained by citric acid dissociation is as follows: Figure 2The results are shown in Figure 1. The presence of mesopores in the obtained material is known, and the external specific surface area of Meso-MIL-88A is significantly higher than that of microporous C-MIL-88A and the material not subjected to citric acid dissociation. Moreover, in combination with electron microscopy observation, it was found that the mesopores of Meso-MIL-88A have a relatively wide pore distribution of about 10 nm. Figure 2 The results are shown in Figure 1. The presence of mesopores in the obtained material is known, and the external specific surface area of Meso-MIL-88A is significantly higher than that of microporous C-MIL-88A and the material not subjected to citric acid dissociation. Moreover, in combination with electron microscopy observation, it was found that the mesopores of Meso-MIL-88A have a relatively wide pore distribution of about 10 nm.

[0033] Example 2 Meso-MIL-88 and C-MIL-88A immobilized lipase

[0034] 50 mg of the carrier was added to 800 μL of deionized water. The carrier was uniformly dispersed by ultrasonic treatment for 15 minutes. Subsequently, 200 μL of a lipase (lipase concentration of 30%, for example, derived from Candida antarctica , Aspergillus oryzae , Rhizomucor miehei Lipase) solution was added to the mixture, and the obtained mixture was placed in a constant temperature shaker at 40°C, 200 rpm for 4 hours. Then, the immobilized lipase was collected by centrifugation at 8000 rpm for 3 minutes. The collected immobilized lipase was washed once with water, and then freeze-dried overnight. Regardless of the immobilization amount, the enzyme activity recovery rate or specific enzyme activity, the Meso-MIL-88A immobilized lipase was significantly higher than that of the C-MIL-88A immobilized lipase (P < 0.05). Figure 3

[0035] Example 3 Meso-MIL-88 immobilized lipase catalyzed synthesis of structured phospholipids

[0036] Example 1

[0037] DHA-ethyl ester and soybean phosphatidylcholine (PC) in a molar ratio of 10:1 were placed in an enzyme reactor, and 200 units of standard enzyme activity of Meso-MIL-88A immobilized lipase (derived from Candida antarctica ) per unit of oil mass was added, and the reaction was carried out at 55°C, 250 rpm for 24 hours. The DHA insertion rate on the structured phospholipid Sn-1 was 80%.

[0038] Example 2

[0039] DHA-ethyl ester and soybean phosphatidylcholine (PC) in a molar ratio of 20:1 were placed in an enzyme reactor, and 400 units of standard enzyme activity of Meso-MIL-88A immobilized lipase (derived from Aspergillus oryzae ) per unit of oil mass and 300 units of standard enzyme activity of Meso-MIL-88A immobilized lipase (derived from Candida antarctica ) per unit of oil mass were added, and the reaction was carried out at 60°C, 250 rpm for 24 hours. The DHA insertion rate on the structured phospholipid Sn-1 was 85%. ​

[0040] Example 3

[0041] EPA-ethyl ester in a molar ratio of 30:1 with soybean phosphatidylcholine (PC) was placed in an enzyme reactor, Meso-MIL-88A immobilized lipase (derived from Rhizomucor miehei ) was added at 2000 units of enzyme activity per mass of oil, 45°C, 250 rpm, for 24 hours, and the incorporation rate of EPA on the structural phospholipid Sn-1 was 90%.

[0042] While the present application has been described in detail with particular references to illustrative embodiments thereof, it will be understood that modifications and improvements can be made to the disclosed embodiments without departing from the spirit and scope of the application. Accordingly, no limitation is intended by the description set forth herein, and all such possible modifications and improvements are intended to be included within the scope of the present application as defined by the following claims.

Claims

1. A method for preparing mesoporous MOFs material with water-acid dual stability, characterized in that, The mesoporous MOFs material with water-acid dual stability is prepared by a soft template method, and the method comprises the following steps: (1) 3 mmol of fumaric acid and 2.50 g of sodium dodecyl sulfate are dissolved in 140 mL of deionized water, and the solution is preheated at 55 °C under stirring at 250 rpm for 1 hour to form a stable micellar system, which is recorded as solution A; (2) 1 mmol of FeCl3·6H2O is dissolved in 10 mL of deionized water and added to the solution A, and the mixture is reacted at 55 °C under stirring at 250 rpm for 24 hours; after the reaction, a light red-brown precipitate is collected by centrifugation, and the light red-brown precipitate is immersed in 150 mL of ethanol by ultrasonic treatment for 6 hours for cleaning, and then vacuum dried to obtain a mesoporous material; (3) 100 mg of the dried mesoporous material obtained in step (2) is dispersed in 50 mL of deionized water, and 10 mL of 0.02-0.04 mol / L sodium citrate solution is added; the mixture is kept at 30-50 °C for 1-2 hours to obtain a mesoporous MOFs material with water-acid dual stability, which is recorded as Meso-MIL-88A.

2. The mesoporous MOFs material prepared by the method according to claim 1.

3. The mesoporous MOFs material of claim 2, wherein, The average pore size is greater than 10 nm.

4. Any one of the following applications of the mesoporous MOFs material according to claim 2 or 3: 1) in the field of enzyme immobilization; 2) in the high-value conversion of oil and fat.

5. A method for high value conversion of oils and fats using the mesoporous MOFs material immobilized lipase according to claim 2 or 3, characterized in that, The method comprises: 1) dispersing the mesoporous MOFs material in deionized water, mixing with a lipase solution, then collecting the immobilized lipase by centrifugation, washing with water, and then freeze-drying; 2) adding the immobilized lipase to a primary or multi-stage enzyme reactor for enzymatic conversion reaction of oil and fat.

6. The method of claim 5, wherein, 2) 200-2000 units of standard enzyme activity of immobilized lipase per unit of oil mass are added.

7. The method of claim 5, wherein, 1) Specifically, 50 mg of the mesoporous MOFs material is added to 800 μL of deionized water, and ultrasonic treatment is performed for 15 minutes to uniformly disperse the mesoporous MOFs material; then 200 μL of a lipase solution with a concentration of 10-30% is added to the system, and the obtained mixture is placed in a constant temperature shaking bed at 30-50 °C and 200 rpm for 3-6 hours; then the immobilized lipase is collected by centrifugation at 8000 rpm for 3 minutes, washed once with water, and finally freeze-dried overnight.

8. The method according to any one of claims 5-7, characterized in that, The lipase includes a lipase derived from Candida antarctica ( Candida antarctica ), Thermomyces ( Thermomyces lanuginosus ), Aspergillus niger ( Aspergillus niger ), Aspergillus oryzae ( Aspergillus oryzae ), Rhizomucor miehei ( Rhizomucor miehei ), Rhizopus oryzae ( Rhizopus oryzae ).

9. The method according to any one of claims 1-8 for preparing structural phospholipids.

10. Use according to claim 9, characterized in that, The structural phospholipids include phosphatidyl-DHA and phosphatidyl-EPA.

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