Hybrid metal organic framework material immobilized enzyme as well as preparation method and application thereof

Abstract

The invention discloses a hybrid metal organic framework material immobilized enzyme which is prepared by the following steps: adding a mesoporous silicon material loaded with metal ions, transaminase and an organic ligand into an HEPES buffer solution or ultrapure water to obtain a preparation reaction system, sequentially reacting under stirring and ultrasonic conditions, centrifuging and washing. The invention also discloses application of the hybrid metal organic framework material in catalyzing aldehyde or ketone and amine to synthesize organic amine compounds. A one-pot synthesis method is adopted, transaminase serves as an immobilization object, meso-porous silicon loaded with metal ions serves as a core to physically adsorb the transaminase, a shell adopts organic carboxylic acid sodium salt and the metal ions to form a metal organic framework material, and the hybrid metal organic framework material immobilized enzyme of a core-shell structure is prepared. Compared with a free enzyme, the immobilized enzyme can effectively improve the activity and stability of the enzyme, has good stability under various severe conditions such as high temperature, extreme pH, organic reagents and the like, and is high in repeated utilization rate.

Description

Technical Field

[0001] The present invention belongs to the field of bio-enzyme catalysts and materials, and relates to a hybrid metal organic framework material immobilized enzyme, a preparation method and application thereof, and specifically to a hybrid metal organic framework material immobilized transaminase, a preparation method and application thereof in the synthesis of amine compounds. Background Art

[0002] Organic amines are an important class of compounds, often appearing as key synthetic building blocks in the chemical structures of natural products, pharmaceuticals, agrochemicals, and polymers. Most agrochemicals and over 75% of the top 200 best-selling pharmaceuticals contain organic amine structures. However, chemical amination reactions are typically carried out under harsh reaction conditions, resulting in low product yields, poor optical purity, and high levels of waste, waste, and other environmental impacts. Transaminases (TAs) have great potential for large-scale amine synthesis due to their high enantioselectivity and broad substrate adaptability. Among them, ω-transaminases (ω-TAs), as pyridoxal-5'-phosphate (PLP)-dependent enzymes, can directly convert aldehydes or ketones into amines, making ω-TAs more versatile in biocatalytic conversions and making the synthesis of chiral amines more feasible and efficient.

[0003] Because free enzymes are susceptible to inactivation due to external conditions, immobilization is often an effective method to enhance their stability and reusability. Metal-organic frameworks (MOFs) are porous materials composed of orderly coordinated metal ions and organic ligand units. They offer advantages such as high porosity, large surface area, and tunable pore size, making them ideal carriers for immobilized enzymes. They can effectively improve enzymes' tolerance to harsh conditions such as high temperatures, extreme pH values, and organic solvents, enhancing their reusability.

[0004] Currently, there have been reports of using MOFs as carriers for enzyme immobilization, with immobilization methods primarily categorized as physical adsorption and in situ enzyme encapsulation. The physical adsorption method involves first synthesizing the complete material and then mixing it with an enzyme solution to achieve enzyme immobilization. This method places high demands on the material's specific surface area, pore size, and other properties. Furthermore, the enzymes loaded into the material are prone to detachment during application, resulting in poor reusability. Furthermore, the preparation of many materials involves high temperatures and the use of organic solvents, which contravenes sustainable development goals. The in situ enzyme encapsulation method, another common method for enzyme immobilization, involves incorporating the enzyme during material synthesis. This method is simple to operate, but requires mild synthesis conditions that do not damage enzyme activity. However, patent CN113856761A places the immobilized enzyme in acid for etching, and this method is not suitable for acid-intolerant enzymes; patent CN118581076A uses a slightly acidic ligand solution and organic reagents such as glutaraldehyde, which is only suitable for glucosidase and has a limited range of applications; patent CN118599822A uses a metal organic framework and graphene hybrid material, but has a low enzyme activity recovery rate of only 48.7%; patent CN117467653A uses sodium alginate hydrogel beads cross-linked with MOFs, which has poor stability and only exhibits good enzyme activity at an appropriate pH and temperature.

[0005] In summary, the use of MOFs as carriers for enzyme immobilization has the following drawbacks: low enzyme reuse rates, the need for high temperatures, organic solvents, and acidic conditions, which are overly harsh or complex in their preparation, hindering the preservation of enzyme activity, and failing to meet the immobilization requirements of most enzymes. If these issues are not addressed, the practical application of such enzyme immobilization methods will be significantly reduced. Furthermore, metal-organic frameworks (MOFs) also suffer from low atom economy, poor reusability, and stability, significantly hindering their practical application. To address this issue, the effective combination of MOFs with other materials to prepare hybrid MOF composites has been extensively studied. Summary of the Invention

[0006] The purpose of the present invention is to solve the problems of the prior art and to provide a hybrid metal organic framework material immobilized enzyme and a preparation method and application thereof.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A hybrid metal organic framework material immobilized enzyme is prepared by adding a mesoporous silicon material loaded with metal ions, a transaminase and an organic ligand into a HEPES buffer or ultrapure water to obtain a reaction system, reacting under stirring and ultrasonic conditions in sequence, and then centrifuging and washing.

[0009] A method for preparing the hybrid metal-organic framework material immobilized enzyme comprises:

[0010] S1: ultrasonically dispersing the metal ion-loaded mesoporous silica material in HEPES buffer or ultrapure water, adding the transaminase solution and the organic ligand solution under stirring to obtain a preparation reaction system;

[0011] S2: Prepare the reaction system, react under stirring and ultrasonic conditions, and centrifuge to obtain the product;

[0012] S3: washing the product with HEPES buffer or ultrapure water to obtain the hybrid metal-organic framework material immobilized enzyme.

[0013] In S1, the metal ion-loaded mesoporous silicon material is prepared by high-temperature reaction of MCM-41 mesoporous silicon and metal salt using MCM-41 mesoporous silicon as a carrier.

[0014] The metal ions are calcium ions, zinc ions, iron ions, nickel ions or copper ions.

[0015] The metal salt is selected from nitrates, hydrochlorides and sulfates of calcium, zinc, iron, nickel and copper.

[0016] The usage ratio of the MCM-41 mesoporous silicon and the metal salt is (0.10-1.50):(5-50) g / mmol, preferably 1:35-1:40 g / mmol.

[0017] The metal ion-loaded mesoporous silicon material is prepared by the following method: MCM-41 mesoporous silicon is uniformly dispersed in a DMF solution of a metal salt, first ultrasonicated for 10 to 100 minutes, and then stirred at room temperature for 10 to 200 minutes; then, it is transferred to a high-pressure reactor, reacted at 100 to 250° C. for 12 to 48 hours, cooled to room temperature, centrifuged, washed with methanol three times, and vacuum dried at 40 to 80° C. overnight to obtain the metal ion-loaded mesoporous silicon material.

[0018] The centrifugal speed is 6000-12000 rpm, and the centrifugal time is 5-30 minutes.

[0019] The transaminase is S-ω-transaminase, specifically S-ω-transaminase from Bacillus megaterium (BmSTA, Uniprot ID: A0A1C7D190).

[0020] Specifically, the transaminase is obtained by the following method: the gene sequence of S-ω-transaminase (BmSTA, Uniprot ID: A0A1C7D190) from Bacillus megaterium is selected as the target gene sequence; the gene sequence is cloned into the pET-28a(+) vector using BamHI and HindIII as restriction sites to obtain a recombinant plasmid; the recombinant plasmid is transformed into Escherichia coli BL21(DE3) host cells, and after fermentation and expression in TB medium, BmSTA recombinant E. coli cells are obtained. The target protease is obtained after ultrasonic disruption and purification. Those skilled in the art can implement this technical solution through routine operations.

[0021] The transaminase solution is a HEPES buffer solution of transaminase.

[0022] The concentration of the transaminase in the preparation reaction system is 1-10 mg / mL, preferably 4-6 mg / mL.

[0023] The mass ratio of the metal ion-loaded mesoporous silicon material to the transaminase is 1:1 to 1:2, preferably 1:1.4.

[0024] The organic ligand is selected from sodium salt ligands such as trimesic acid sodium salt, 2-aminoterephthalic acid sodium salt, and side-chain extended aryl tricarboxylate sodium ligands; the side-chain extended aryl tricarboxylate sodium ligand is selected from 4,4',4"-[1,3,5-benzenetriyltris(carbonylimino)]triphenylacetate sodium (BTAA-Na), 4,4',4"-[1,3,5-benzenetriyltris(carbonylimino)]tribenzoate sodium (BTFA-Na), and 6,6',6"-[1,3,5-benzenetriyltris(carbonylimino)]trinaphthoate sodium (BTNA-Na).

[0025] The side chain extended aryl tricarboxylate sodium ligand is prepared by the following method: 5-10 mmol of p-aminophenylacetic acid, p-aminobenzoic acid or 6-amino-2-naphthoic acid is dissolved in 10-50 mL of N,N-dimethylacetamide (DMA), and 0.10-1.50 mmol of 4-dimethylaminopyridine, react at 50-90°C for 10-80 minutes, add dropwise 1-10 mL of a 1-20% (m / V) 1,3,5-benzenetricarboxylic acid chloride DMA solution, react at 10-40°C for 1-10 hours, add 10-200 mL of water to terminate the reaction, precipitate a white solid, filter with suction, wash the filter cake with water, and dry to obtain a white solid; disperse 0.5-5 g of the white solid in 3-15 mL of ultrapure water, slowly add dropwise 1-5 mL of a 5-30% by mass NaOH solution under ice-water bath, stir magnetically for 5-60 minutes, slowly add 50-200 mL of ethanol, precipitate a white solid, let stand, filter with suction, discard the filtrate, wash the filter cake with anhydrous ethanol, and dry to obtain a sodium aryl tricarboxylate ligand with extended side chains.

[0026] The organic ligand solution is a HEPES buffer solution or an aqueous solution of the organic ligand.

[0027] The usage ratio of the metal ion-loaded mesoporous silicon material and the organic ligand is 1:1 to 1:2 mg / μmol.

[0028] Specifically, the mesoporous silica material loaded with metal ions is ultrasonically dispersed in HEPES buffer or ultrapure water, and the transaminase solution is added under stirring. After the addition is completed, magnetic stirring is performed for 1 to 10 minutes, and then the organic ligand solution is added under stirring to obtain a preparation reaction system.

[0029] The stirring may be magnetic stirring.

[0030] In S2, the stirring may be magnetic stirring.

[0031] The stirring temperature is 4°C to room temperature; the stirring speed is 500 to 1500 rpm, and the stirring time is 12 minutes to 3 hours.

[0032] The ultrasonic power is 300-600W, and the ultrasonic time is 10-40 minutes.

[0033] The separation is centrifugal separation; the rotation speed of the centrifugal separation is 6000-12000 rpm.

[0034] Another object of the present invention is to provide the use of the hybrid metal organic framework material in catalyzing the synthesis of organic amine compounds from aldehydes or ketones and amines.

[0035] Furthermore, the application is: using aldehyde or ketone, amine as substrate, mixing the hybrid metal organic framework material immobilized enzyme with aldehyde or ketone, amine, coenzyme, and reaction solvent, and incubating with shaking to obtain an amine compound.

[0036] The mass ratio of the hybrid metal organic framework material immobilized enzyme to the aldehyde or ketone is 1:100 to 1:200.

[0037] The aldehydes are 1,4-difluorobenzaldehyde, 3-pyridinecarboxaldehyde, 4-pyridinecarboxaldehyde, 2-pyridinecarboxaldehyde, 4-fluorobenzaldehyde, furfural, etc.; the amines are isopropylamine, benzylamine, α-phenylethylamine; and the coenzyme is PLP.

[0038] The molar ratio of the aldehyde or ketone to the amine is 1:1 to 1:10, preferably 1:2 to 1:2.5.

[0039] The molar ratio of the aldehyde or ketone to the coenzyme is 10:1 to 50:1, preferably 10:1 to 15:1.

[0040] The reaction solvent is HEPES buffer or water; the pH of the reaction system is 6-8.

[0041] The incubation temperature is 20-50° C., the incubation time is 2-48 hours, and the oscillation speed is 100-200 rpm.

[0042] Beneficial effects of the present invention:

[0043] (1) The present invention can realize the catalytic synthesis of amine compounds and the simple and rapid recovery of immobilized enzymes through enzymes and hybrid metal-organic framework materials.

[0044] (2) The present invention adopts sodium salt ligands to solve the problem that some ligands have poor solubility in the aqueous phase system, resulting in low binding efficiency with the metal source. The sodium salt ligands can react with the metal source at room temperature, with a fast binding rate and little damage to the enzyme, which is conducive to maintaining its activity.

[0045] (3) Through the ligand extension strategy, a one-step chemical method is used to synthesize a side chain extended ligand. The ligand can be used to prepare a new core-shell structure hybrid mesoporous silica MOF immobilized enzyme biocomposite material through simple operation under a mild method. This material can further improve the catalytic activity of the enzyme.

[0046] (5) Mesoporous silica has the characteristics of high stability, large pore size and the ability to form strong functional group interaction. The present invention adopts a one-pot synthesis method. Under mild conditions, transaminase is immobilized as the target, and the mesoporous silica loaded with metal ions is used as the core to physically adsorb the transaminase. The outer shell uses organic carboxylic acid sodium salt as a ligand to form a metal-organic framework material with metal ions. Finally, a new core-shell structure hybrid metal-organic framework material immobilized enzyme biocomposite material is prepared, which effectively retains the excellent catalytic activity of the enzyme. The immobilized enzyme of the present invention is suitable for immobilizing any protease catalyst due to its mild preparation conditions.

[0047] Free enzyme catalytic reactions can only be used once and cannot be recycled. They are also intolerant to harsh conditions, which limits the application of enzymes. Compared with free enzymes, the hybrid metal organic framework material immobilized enzyme of the present invention can effectively improve the activity and stability of the enzyme. The immobilized enzyme catalyzes the synthesis of chiral amine compounds and has good stability in various harsh conditions such as high temperature, extreme pH (such as pH 2, pH 10, pH 12, etc.), organic reagents, etc., is easy to separate from the reaction solution, and has a high reuse rate. At the same time, the immobilized enzyme material is used to achieve efficient synthesis of amine compounds, providing a green and efficient means for enzyme-catalyzed asymmetric synthesis of key chiral compounds.

[0048] (5) The use of MOFs as carriers to immobilize enzymes through physical adsorption or in situ synthesis has the following problems: low reuse rate of immobilized enzymes, and the preparation of immobilized enzymes involves harsh or complex conditions such as high temperature, organic solvents, and acidic conditions, which are not conducive to maintaining the original activity of the enzymes. In addition, the use of metal-organic framework materials alone to immobilize enzymes also has problems such as low atom economy, reusability, and poor stability. The effective combination of MOFs with structurally stable mesoporous silica materials to prepare hybrid MOF composite materials can effectively solve the above problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a transmission electron microscope image of M&B@NFF prepared in Example 1.

[0050] Figure 2 This is a comparison of the catalytic activities of the M&B@NFF prepared in Example 1 and the free enzyme after being treated with different pH solvents.

[0051] Figure 3 This is a comparison of the catalytic activities of the M&B@NFF prepared in Example 1 and the free enzyme after being treated with different organic solvents.

[0052] Figure 4 Comparison of the catalytic activity of M&B@NFF prepared in Example 1 and free enzyme after treatment at different temperatures

[0053] Figure 5This is a graph showing the reusability of the M&B@NFF prepared in Example 1. DETAILED DESCRIPTION

[0054] The following are specific embodiments of the present invention, which are used to illustrate the technical solutions and features of the present invention and are intended to assist those skilled in the art in understanding the present invention and implementing it accordingly. However, the implementation of the technical solutions of the present invention is not limited thereto. Any equivalent transformations or modifications made according to the spirit of the present invention shall fall within the scope of protection of the present invention.

[0055] MCM-41 Mesoporous Silica: Mesoporous Silica Material-41 (Mobil Oil Company).

[0056] BmSTA: S-ω-aminotransferase from Bacillus megaterium (Uniprot ID: A0A1C7D190).

[0057] BTAA-Na: sodium 4,4',4"-[1,3,5-benzenetriyltris(carbonylimino)]triphenylacetate. The synthesis process is as follows:

[0058] p-Aminophenylacetic acid (0.90 g, 6.47 mmol) was dissolved in 20 mL of N,N-dimethylacetamide (DMA), and 4-dimethylaminopyridine (25.0 mg, 0.21 mmol) was added. The mixture was reacted at 80°C for 30 min, and then 10 mL of 1,3,5-benzenetricarboxylic acid chloride solution in DMA (20%, m / V) was added dropwise. The mixture was reacted at 40°C for 5 h. 100 mL of water was added to terminate the reaction. A white solid was precipitated and filtered. The filter cake was washed with water (20 mL × 3). The solid was dried in vacuo at 50°C for 12 h to obtain a white solid (1.01 g, 92%).

[0059] Salt formation: Weigh 1.01 g of the above white solid and disperse it in 5 mL of ultrapure water. In an ice-water bath, slowly add 3 mL of 10% NaOH solution dropwise. Stir magnetically for 20 min. Slowly add 80 mL of anhydrous ethanol to precipitate a white solid. Let it stand for 2 h, filter, discard the filtrate, wash the filter cake with anhydrous ethanol, and dry the resulting solid in a vacuum at 40°C overnight to obtain sodium 4,4',4"-[1,3,5-benzenetriyltris(carbonylimino)]triphenylacetate, denoted as BTAA-Na. 1 H NMR (400MHz, DMSO-d6) δ11.09(s,3H),8.84(s,3H),7.73-7.52(m,6H),7.25-7.05(m,6H),3.22(s,6H).

[0060] BTFA-Na: sodium 4,4',4"-[1,3,5-benzenetriyltris(carbonylimino)]tribenzoate. BTFA-Na is prepared by referring to the synthesis method of BTAA-Na, except that p-aminophenylacetic acid is replaced with an equal amount of p-aminobenzoic acid. 1 H NMR (400 MHz, heavy water, deuterium oxide) δ 8.29 (d, J = 2.7 Hz, 3H), 7.77-7.61 (m, 6H), 7.51-7.37 (m, 6H).

[0061] BTNA-Na: sodium 6,6',6"-[1,3,5-benzenetriyltris(carbonylimino)]trinaphthoate. BTNA-Na was prepared by referring to the synthesis method of BTAA-Na, except that p-aminophenylacetic acid was replaced with an equal amount of 6-amino-2-naphthoic acid. 1H NMR (400 MHz, DMSO-d6) δ 10.94 (d, J = 5.9 Hz, 3H), 8.82 (s, 3H), 8.57 (s, 3H), 8.54 (s, 4H), 8.11 (t, J = 8.0 Hz, 4H), 7.94 (d, J = 4.6 Hz, 11H).

[0062] Trimellitic acid sodium salt: prepared using trimesic acid and sodium hydroxide as raw materials according to the salt-forming conditions of BTAA-Na.

[0063] 2-Aminoterephthalic acid sodium salt: prepared using 2-aminoterephthalic acid and sodium hydroxide as raw materials according to the salt-forming conditions of BTAA-Na.

[0064] PLP: pyridoxal phosphate;

[0065] mM:mmol / L.

[0066] Example 1

[0067] S1: Take 0.25g of MCM-41 mesoporous silica and disperse it in a DMF solution of Ni(NO3)2·6H2O (613mM, 15mL), first ultrasonicate for 30min, and then stir at room temperature for 1.5h; then transfer it to a high-pressure reactor, react at 150℃ for 24h, cool to room temperature, and centrifuge (10000rpm, 10min). The product is washed three times with methanol and vacuum-dried at 40℃ overnight to obtain a mesoporous silica material loaded with metal ions, which is recorded as MCM-41-Ni.

[0068] The DMF solution of Ni(NO3)2·6H2O was replaced with DMF solution or aqueous solution of Zn(NO3)2·6H2O, Ca(NO3)2·6H2O, Cu(NO3)2·2.5H2O, and Fe(NO3)3·9H2O with equal molar concentrations. The obtained mesoporous silicon materials loaded with metal ions were named MCM-41-Zn, MCM-41-Cu, MCM-41-Fe, and MCM-41-Ca, respectively.

[0069] S2: Weigh 2 mg of MCM-41-Ni and disperse it in 100 μL of HEPES buffer. Ultrasonic dispersion is performed for 10 minutes. 400 μL of 7 mg / mL BmSTA in HEPES buffer is added under magnetic stirring. After the addition is complete, magnetic stirring is performed for 1 minute. Then, 64 μL of 36 mM BTFA-Na aqueous solution is added dropwise under magnetic stirring to obtain a preparative reaction system.

[0070] In order to uniformly disperse the hybrid MOF-immobilized enzyme material, the preparation reaction system was first magnetically stirred (speed 1000 rpm) at room temperature for 32 min, then ultrasonicated (ultrasonic power 500 W) for 16 min, centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the precipitate was taken;

[0071] S3: The precipitate was washed three times with HEPES buffer to remove unreacted ligands, enzyme proteins, etc., and freeze-dried. The resulting solid was the hybrid metal-organic framework material immobilized enzyme, denoted as M&B@NFF.

[0072] Figure 1 This is a scanning electron microscope image of M&B@NFF, such as Figure 1 As shown, the material presents a clear structure with mesoporous silica MCM-41 as the core and enzyme-loaded MOF as the shell, and its particle size is about 150 to 200 nm.

[0073] M&B@NFF is used to synthesize amine compounds. The specific operation is as follows:

[0074] A substrate solution containing furfural, isopropylamine and PLP was prepared using HEPES buffer. In the substrate solution, the concentration of furfural was 12 mM, the concentration of isopropylamine was 28 mM, and the concentration of PLP was 1 mM.

[0075] 3mg of M&B@NFF was added to 400μL of a substrate solution containing furfural, isopropylamine, and PLP to produce a mixture. The mixture was incubated in a constant temperature shaking incubator at 37°C and 200 rpm for 3 hours. After the reaction, the reaction solution was collected by centrifugation and the product furfural was detected by high-performance liquid chromatography.

[0076] M&B@NFF was replaced with the free enzyme BmSTA with an equal enzyme amount, and the other reaction conditions remained unchanged. The product furfural amine catalyzed by the free enzyme was detected by high-performance liquid chromatography.

[0077] The concentration of furfural amine in the reaction solution of M&B@NFF and equal amount of free enzyme was measured, which represented the activity of M&B@NFF or free enzyme. The enzyme activity recovery (A) was calculated according to formula (1). R ) to evaluate the enzyme activity retention ability of M&B@NFF.

[0078]

[0079] Among them, A i and A f represent the activities of immobilized enzyme and free enzyme, respectively.

[0080] Compared with the free enzyme with the same enzyme amount, the enzyme activity retention rate of M&B@NFF is R The value is 144.4%.

[0081] Example 2

[0082] S1: Take 0.25g of MCM-41 mesoporous silica and disperse it in a DMF solution of Fe(NO3)3·9H2O (613mM, 15mL), first ultrasonicate for 30min, and then stir at room temperature for 1.5h; then, transfer it to a high-pressure reactor, react at 150℃ for 24h, cool to room temperature, and centrifuge (10000rpm, 10min). The product is washed three times with methanol and dried in vacuum at 40℃ overnight to obtain a mesoporous silica material loaded with metal ions, which is recorded as MCM-41-Fe.

[0083] Weigh 2 mg of MCM-41-Fe and disperse it in 100 μL of HEPES buffer. Ultrasonic dispersion is performed for 10 minutes. Then, 400 μL of a 7 mg / mL solution of BmSTA in HEPES buffer is added under magnetic stirring. After the addition is complete, magnetic stirring is performed for 1 minute. Then, 64 μL of a 36 mM aqueous solution of BTAA-Na is added dropwise under magnetic stirring to obtain a preparative reaction system.

[0084] S2: The reaction system was first magnetically stirred (speed 1000 rpm) at room temperature for 32 min, then ultrasonicated (ultrasonic power 500 W) for 16 min, and centrifuged at 8000 rpm for 5 min. The supernatant was discarded and the precipitate was collected.

[0085] S3: The precipitate was washed three times with HEPES. The resulting solid was the hybrid metal-organic framework immobilized enzyme, denoted as M&B@FAF.

[0086] M&B@FAF is used to synthesize amine compounds. The specific operation is as follows:

[0087] A substrate solution containing furfural, isopropylamine and PLP was prepared using HEPES buffer. In the substrate solution, the concentration of furfural was 12 mM, the concentration of isopropylamine was 28 mM, and the concentration of PLP was 1 mM.

[0088] 3mg of M&B@FAF was added to 400μL of a substrate solution containing furfural, isopropylamine, and PLP to produce a mixture. The mixture was incubated in a constant-temperature shaking incubator at 37°C and 200rpm for 3 hours. After the reaction, the reaction solution was centrifuged and collected. The product, furfural, was detected by high-performance liquid chromatography. Compared to an equal amount of free enzyme, the enzyme activity retention rate was 108.5%.

[0089] Example 3

[0090] S1: Take 0.25g of MCM-41 mesoporous silica and disperse it in a DMF solution of Zn(NO3)2·6H2O (613mM, 15mL), first ultrasonicate it for 30min, and then stir it at room temperature for 1.5h; then transfer it to a high-pressure reactor, react at 150℃ for 24h, cool to room temperature, and centrifuge it (10000rpm, 10min); wash it with methanol three times and vacuum dry it at 40℃ overnight to obtain a mesoporous silica material loaded with metal ions, which is recorded as MCM-41-Zn.

[0091] 2 mg of MCM-41-Zn was weighed and dispersed in 100 μL of HEPES buffer. Ultrasonic dispersion was performed for 10 minutes. 400 μL of a 7 mg / mL solution of BmSTA in HEPES buffer was then added under magnetic stirring. After the addition was complete, magnetic stirring was performed for 1 minute. Then, 64 μL of a 36 mM aqueous solution of BTNA-Na was added dropwise under magnetic stirring to obtain a preparative reaction system.

[0092] S2: The reaction system was first magnetically stirred (speed 1000 rpm) at room temperature for 32 min, then ultrasonicated (ultrasonic power 500 W) for 16 min, and centrifuged at 8000 rpm for 5 min. The supernatant was discarded and the precipitate was collected.

[0093] S3: The precipitate was washed three times with HEPES. The resulting solid was the hybrid metal-organic framework immobilized enzyme, denoted as M&B@ZNF.

[0094] M&B@ZNF is used to synthesize amine compounds. The specific operation is as follows:

[0095] A substrate solution containing furfural, isopropylamine and PLP was prepared using HEPES buffer. In the substrate solution, the concentration of furfural was 12 mM, the concentration of isopropylamine was 28 mM, and the concentration of PLP was 1 mM.

[0096] 3mg of M&B@ZNF was added to 400μL of a substrate solution containing furfural, isopropylamine, and PLP to produce a mixture. The mixture was incubated in a constant-temperature shaking incubator at 37°C and 200rpm for 3 hours. After the reaction, the reaction solution was centrifuged and collected. High-performance liquid chromatography (HPLC) was used to analyze the product furfural, revealing an enzyme activity retention rate of 113.6% compared to an equal amount of free enzyme.

[0097] Example 4

[0098] The following test experiment was carried out using the hybrid metal organic framework material immobilized enzyme M&B@NFF synthesized in Example 1.

[0099] Test Example 1: pH tolerance test

[0100] A substrate solution containing furfural, isopropylamine and PLP was prepared using HEPES buffer. In the substrate solution, the concentration of furfural was 12 mM, the concentration of isopropylamine was 28 mM, and the concentration of PLP was 1 mM.

[0101] 3 mg of M&B@NFF or an equivalent amount of BmSTA was dispersed in 200 μL of an aqueous solution at pH 2, 4, 6, 7, 8, 10, or 12 (pH adjusted with 2 M hydrochloric acid or 10% (m / v) sodium hydroxide). The mixture was incubated at 37°C with shaking for 2 hours. The mixture was then added to 400 μL of a substrate solution containing furfural, isopropylamine, and PLP. The mixture was incubated in a constant temperature shaking incubator at 37°C and 200 rpm for 3 hours. After completion of the reaction, the reaction solution was centrifuged and collected. The relative activities of the free BmSTA and immobilized M&B@NFF enzymes were determined by high-performance liquid chromatography.

[0102] The calculation of relative activity is shown as follows:

[0103]

[0104] Among them, A v Represents the enzyme activity measured under various variable conditions, A f It indicates the enzymatic activity of the free enzyme under optimal conditions (pH 7, 37°C, HEPES buffer as solvent).

[0105] Comparison of the catalytic activities of M&B@NFF and free enzyme BmSTA after treatment with different pH values ​​is shown in Figure 2 Compared with the free enzyme, the catalytic activity of the immobilized enzyme M&B@NFF was improved, especially with higher tolerance to extreme pH (such as pH 2, pH 10, pH 12, etc.).

[0106] Test Example 2: Organic solvent tolerance test

[0107] A substrate solution containing furfural, isopropylamine and PLP was prepared using HEPES buffer. In the substrate solution, the concentration of furfural was 12 mM, the concentration of isopropylamine was 28 mM, and the concentration of PLP was 1 mM.

[0108] 3 mg of M&B@NFF or an equivalent amount of BmSTA was dispersed in 200 μL of HEPES buffer and HEPES buffer containing 20% ​​(volume fraction) MeOH, EtOH, DMSO, MeCN, THF, or DMSO, respectively. The mixture was incubated at 37°C with shaking for 2 hours. The mixture was then added to 400 μL of a substrate solution containing furfural, isopropylamine, and PLP. The mixture was incubated in a constant temperature shaking incubator at 37°C and 200 rpm for 3 hours. After completion of the reaction, the reaction solution was centrifuged and collected. The relative activities of the free and immobilized enzymes were determined by high-performance liquid chromatography.

[0109] Figure 3 This is a comparison of the catalytic activities of M&B@NFF and the free enzyme BmSTA after treatment with different organic solvents. Compared with the free enzyme, the immobilized enzyme M&B@NFF has a higher tolerance to organic solvents.

[0110] Test Example 3: High Temperature Tolerance Test

[0111] A substrate solution containing furfural, isopropylamine and PLP was prepared using HEPES buffer. In the substrate solution, the concentration of furfural was 12 mM, the concentration of isopropylamine was 28 mM, and the concentration of PLP was 1 mM.

[0112] 3 mg of M&B@NFF or an equivalent amount of BmSTA was dispersed in 200 μL of HEPES buffer and incubated in a water bath at 25°C, 37°C, 44°C, 50°C, 60°C, 70°C, and 80°C for 2 hours. The mixture was then added to 400 μL of a substrate solution containing furfural, isopropylamine, and PLP. The mixture was incubated in a constant-temperature shaking incubator at 37°C and 200 rpm for 3 hours. After completion of the reaction, the reaction solution was centrifuged and collected. The relative activities of the free and immobilized enzymes were determined by high-performance liquid chromatography.

[0113] Figure 4 This is a comparison of the catalytic activities of M&B@NFF and the free enzyme BmSTA after treatment under different temperature conditions. Compared with the free enzyme, the immobilized enzyme composite material has a higher tolerance to high temperature (40-80°C).

[0114] Example 5

[0115] Reusability test

[0116] A substrate solution containing furfural, isopropylamine and PLP was prepared using HEPES buffer. In the substrate solution, the concentration of furfural was 12 mM, the concentration of isopropylamine was 28 mM, and the concentration of PLP was 1 mM.

[0117] 3mg of M&B@NFF was added to 400μL of a substrate solution containing furfural, isopropylamine, and PLP to form a mixture. The mixture was incubated in a constant-temperature shaking incubator at 37°C and 200 rpm for 3 hours. After the reaction, the reaction solution was centrifuged and collected. The relative activities of the free and immobilized enzymes were determined by high-performance liquid chromatography. The solid was washed once with HEPES buffer and resuspended in the same substrate solution for the next round of catalytic reaction. This cycle was repeated 10 times, and the initial enzyme activity of M&B@NFF was used as a control to evaluate its reusability.

[0118] like Figure 5 As shown in the figure, after 10 cycles, M&B@NF showed no obvious activity decline.

Claims

1. A hybrid metal-organic framework material immobilized enzyme, characterized by: The method comprises adding a mesoporous silicon material loaded with metal ions, a transaminase and an organic ligand into a HEPES buffer or ultrapure water to obtain a preparation reaction system, reacting the system under stirring and ultrasonic conditions, and then centrifuging and washing the system.

2. The hybrid metal-organic framework immobilized enzyme according to claim 1, characterized in that: The metal ion-loaded mesoporous silicon material is prepared by high-temperature reaction of MCM-41 mesoporous silicon and a metal salt using MCM-41 mesoporous silicon as a carrier; wherein the metal ion is calcium ion, zinc ion, iron ion, nickel ion or copper ion; the metal salt is selected from nitrate, hydrochloride or sulfate of calcium, zinc, iron, nickel or copper; and the dosage ratio of the MCM-41 mesoporous silicon and the metal salt is (0.10-1.50):(5-50) g / mmol.

3. The hybrid metal-organic framework immobilized enzyme according to claim 2, characterized in that: The usage ratio of the MCM-41 mesoporous silicon and the metal salt is 1:35-1:40 g / mmol.

4. The hybrid metal-organic framework immobilized enzyme according to claim 1 or 2, characterized in that: The metal ion-loaded mesoporous silicon material is prepared by the following method: MCM-41 mesoporous silicon is uniformly dispersed in a DMF solution of a metal salt, first ultrasonicated for 10 to 100 minutes, and then stirred at room temperature for 10 to 200 minutes; then, it is transferred to a high-pressure reactor, reacted at 100 to 250° C. for 12 to 48 hours, cooled to room temperature, centrifuged, washed with methanol three times, and vacuum dried at 40 to 80° C. overnight to obtain the metal ion-loaded mesoporous silicon material.

5. The hybrid metal-organic framework immobilized enzyme according to claim 1, characterized in that: The transaminase is S-ω-transaminase; and the mass ratio of the metal ion-loaded mesoporous silicon material to the transaminase is 1:1 to 1:

2.

6. The hybrid metal-organic framework material immobilized enzyme according to claim 1, characterized in that: The organic ligand is selected from sodium salt of trimesic acid, sodium salt of 2-aminoterephthalic acid, and sodium aryl tricarboxylate ligand with extended side chain; the sodium aryl tricarboxylate ligand with extended side chain is selected from sodium 4,4',4"-[1,3,5-benzenetriyltris(carbonylimino)]triphenylacetate, sodium 4,4',4"-[1,3,5-benzenetriyltris(carbonylimino)]tribenzoate, and sodium 6,6',6"-[1,3,5-benzenetriyltris(carbonylimino)]trinaphthoate; the usage ratio of the metal ion-loaded mesoporous silicon material and the organic ligand is 1:1 to 1:2 mg / μmol.

7. A method for preparing an enzyme immobilized on a hybrid metal-organic framework according to claim 1, characterized in that: include: S1: ultrasonically dispersing the metal ion-loaded mesoporous silica material in HEPES buffer or ultrapure water, adding the transaminase solution and the organic ligand solution under stirring to obtain a preparation reaction system; S2: Prepare the reaction system, react under stirring and ultrasonic conditions, and centrifuge to obtain the product; S3: washing the product with HEPES buffer or ultrapure water to obtain the hybrid metal-organic framework material immobilized enzyme.

8. The method for preparing an enzyme immobilized on a hybrid metal-organic framework according to claim 7, characterized in that: In S2, the stirring temperature is 4°C to room temperature; the stirring speed is 500 to 1500 rpm, and the stirring time is 12 minutes to 3 hours; the ultrasonic power is 300 to 600 W, and the ultrasonic time is 10 to 40 minutes.

9. Use of the hybrid metal organic framework material according to claim 1 in catalyzing the synthesis of organic amine compounds from aldehydes or ketones and amines.

10. The use according to claim 9, characterized in that: Using aldehyde or ketone, amine as substrate, the hybrid metal organic framework material immobilized enzyme according to claim 1 is mixed with aldehyde or ketone, amine, coenzyme, and reaction solvent, and incubated with shaking to obtain an amine compound; Wherein, the mass ratio of the hybrid metal organic framework material immobilized enzyme to the aldehyde or ketone is 1:100 to 1:200; The aldehydes are 1,4-difluorobenzaldehyde, 3-pyridinecarboxaldehyde, 4-pyridinecarboxaldehyde, 2-pyridinecarboxaldehyde, 4-fluorobenzaldehyde, furfural, etc. The amine is isopropylamine, benzylamine, or α-phenylethylamine; The coenzyme is PLP; The molar ratio of the aldehyde or ketone to the amine is 1:1 to 1:10, preferably 1:2 to 1:2.5; The molar ratio of the aldehyde or ketone to the coenzyme is 10:1 to 50:1, preferably 10:1 to 15:1; The reaction solvent is HEPES buffer or water; the pH of the reaction system is 6-8; The incubation temperature is 20-50° C., the incubation time is 2-48 hours, and the oscillation speed is 100-200 rpm.

Images