A metal-organic framework material for co-immobilizing multiple enzymes, its preparation method and application

Abstract

This invention belongs to the field of biocatalysis technology, specifically relating to a method for preparing and applying a multi-enzyme co-immobilized with a metal-organic framework (MOF). The method involves mixing an organic ligand solution with β-glucosidase, then adding a metal salt solution, reacting, centrifuging, and washing to obtain MOF@β-glucosidase. Glutaraldehyde is added to the MOF@β-glucosidase for activation, followed by reaction, centrifugation, and washing. Cellulase solution is then added, reacted, centrifuged, and washed to obtain the multi-enzyme co-immobilized with the MOF. This invention uses MOFs as a carrier to achieve partitioned co-immobilization of multiple enzymes, providing protection for enzyme molecules while maintaining the high stability of the material itself, and enhancing enzyme stability at different temperatures and pH levels. This material enables cascade catalysis of the substrate, improving hydrolysis efficiency, and has good recyclability. In actual production, it can achieve rapid separation from the substrate, reducing production costs.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a multi-enzyme co-immobilized in a metal-organic framework material, its preparation method, and its application. Background Technology

[0002] Cellulose is the most abundant forest plant resource and the largest and most widely distributed renewable biomass resource. Through appropriate physicochemical methods, it can be converted into high-quality fine chemicals, biomass energy, and industrial products, and is now widely used in chemical, energy, food, and medical fields. As a polysaccharide, cellulose can be chemically degraded into monosaccharides or oligosaccharides. Enzymatic hydrolysis, as a high-conversion-rate, low-energy-consumption, and environmentally friendly method, has great potential in the cellulose hydrolysis industry. However, current cellulase (Cx) enzyme systems suffer from a low β-glucosidase (BG) content, which prevents endoglucanases and exoglucanases from reaching their maximum hydrolytic activity. This leads to the accumulation of cellobiose in the hydrolysate, reducing the amount of sugar available for subsequent fermentation. Therefore, supplementing the cellulase system with additional BG can effectively accelerate the conversion of cellobiose to glucose, thereby increasing the cellulose hydrolysis rate and glucose yield. However, BG has unstable biological activity and is prone to inactivation under industrial production conditions. Furthermore, its water solubility makes it difficult to recycle and reuse, which greatly increases the cost of hydrolysis.

[0003] Immobilized enzymes are a technique that uses physical or chemical methods to convert water-soluble enzymes into water-insoluble ones or immobilize them on a solid support while retaining their activity. Immobilization enhances enzyme stability, improves their ability to adapt to the environment, and reduces the inhibitory effects of compounds present in the reaction medium. Furthermore, the recyclability of immobilized enzymes improves enzyme utilization efficiency, reduces production costs, and allows for better control of reaction conditions and processes, thus facilitating industrial and continuous production. Currently, research using metal-organic frameworks (MOFs) as enzyme immobilization carriers has been reported, but this mainly involves preparing immobilized enzyme materials based on the principle of physical adsorption. This method requires selecting or designing MOFs with specific pore sizes according to the desired enzyme molecule size and characteristics to achieve enzyme immobilization. This method is cumbersome, prone to enzyme detachment, and typically only suitable for single-enzyme immobilization studies, presenting difficulties when applied to the co-immobilization of multiple enzymes with different molecular sizes and characteristics. Therefore, achieving multi-enzyme immobilization using MOFs as carriers under mild conditions, while improving enzyme stability and recyclability, and enabling cascade catalysis of substrates remains a challenge. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying a metal-organic framework material co-immobilized multi-enzyme. Specifically, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing multi-enzymes co-immobilized on metal-organic framework materials, comprising the following steps: The organic ligand solution was mixed with β-glucosidase, then a metal salt solution was added, the reaction was carried out, centrifuged, and washed to obtain MOF@β-glucosidase. Glutaraldehyde was added to the MOF@β-glucosidase for activation, followed by reaction, centrifugation, washing, and then cellulase solution was added for reaction. After the reaction was completed, centrifugation and washing were performed to finally obtain the metal-organic framework material co-immobilized multi-enzyme.

[0005] This invention utilizes a biomimetic mineralization method and a glutaraldehyde cross-linking method to prepare MOF@β-glucosidase / cellulase materials, achieving the partitioned co-immobilization of β-glucosidase and cellulase on MOF materials under mild conditions. This material enables the two enzymes to exchange substances through the pores on the carrier. Cellulose is hydrolyzed into small molecules of glucose and cellobiose by the cellulase cross-linked on the outside of the material. The cellobiose then enters the carrier through the MOF pores, where it is further hydrolyzed into glucose by β-glucosidase and transported out of the carrier through the pores. This cascade catalysis of the two enzymes avoids the accumulation of transition products and substrate competitive inhibition during hydrolysis, thus significantly improving catalytic efficiency.

[0006] This invention utilizes immobilized enzyme technology to co-immobilize BG and Cx in a certain ratio into a specific carrier, which can improve the stability and recyclability of the enzyme. By constructing a cascade catalysis of enzymes, it achieves efficient degradation of cellulose, which is of great significance to the development of the green cellulose hydrolysis industry.

[0007] As a further preferred embodiment, the organic ligand solution is prepared by mixing the organic ligand with an acetate-sodium acetate buffer solution at a pH of 4.5–6.5 and a concentration of 50 mM; wherein the concentration of the prepared organic ligand solution is 2.5 mM–15 mM. More preferably, the organic ligand is 2-aminoterephthalic acid. The organic ligand solution is prepared using an acetate-sodium acetate buffer solution to maintain the pH at this level throughout the subsequent reaction process, which facilitates the final preparation of multi-enzyme co-immobilized in the metal-organic framework material.

[0008] As a further preferred embodiment, the metal salt solution is an iron salt solution or an aluminum salt solution, and the concentration of the metal salt solution is 2.5 mM to 10 mM.

[0009] As a further preferred embodiment, the mass concentration of glutaraldehyde is 2.5%. If the glutaraldehyde concentration is too low, cross-linking between the MOF and the enzyme cannot be achieved, while if the concentration is too high, the enzyme will be inactivated.

[0010] As a further preferred embodiment, the ratio of organic ligand solution, β-glucosidase, metal salt solution, glutaraldehyde and cellulase is 5 mL: 2 mg: 5 mL: 10 mL~20 mL: 1 mg~5 mg.

[0011] As a further preferred embodiment, the reaction time is 0.5 h to 8 h.

[0012] In a second aspect, the present invention also provides a multi-enzyme co-immobilized in a metal-organic framework material prepared by the above-described preparation method.

[0013] In a third aspect, the present invention also provides the application of the above-mentioned metal-organic framework materials for co-immobilization of multiple enzymes in cellulose hydrolysis.

[0014] A fourth aspect of the present invention also provides a method for hydrolyzing cellulose, comprising the following steps: The aforementioned metal-organic framework material was used to co-immobilize multiple enzymes, which were uniformly dispersed in a substrate solution. The mixture was then incubated with shaking and centrifuged to obtain glucose. The substrate solution was a mixture of carboxymethyl cellulose and cellulose.

[0015] The beneficial effects of this invention are as follows: (1) This invention prepared MOF@β-glucosidase / cellulase material by using biomimetic mineralization method and glutaraldehyde crosslinking method, realizing the dual enzyme immobilization of β-glucosidase and cellulase by MOF material under mild conditions, and constructed a multi-enzyme regional co-immobilized MOF material, realizing multi-enzyme cascade catalysis of cellulose, avoiding the accumulation of transition products and substrate competitive inhibition, significantly improving catalytic efficiency, and at the same time, improving the existing problems of reduced cellulase hydrolysis efficiency, low enzyme utilization, high cost, poor enzyme stability and harsh use conditions; (2) The present invention uses MOFs as carrier materials, which have good physical and chemical stability and can protect enzyme molecules while maintaining the high stability of the material itself, thereby enhancing the stability of enzymes at different temperatures and pH. (3) The MOF@β-glucosidase / cellulase material prepared by the present invention has good recycling performance. In actual production, it can achieve rapid separation from the substrate, realize recycling in production, and reduce production costs. Attached Figure Description

[0016] Figure 1 The figure shown is a comparison of the thermal stability of NH2-MIL-53(Fe)@β-glucosidase and free β-glucosidase. Figure 2The image shows the recycling performance of NH2-MIL-53(Fe)@β-glucosidase; Figure 3 The image shown is a scanning electron microscope image of NH2-MIL-53(Fe)@β-glucosidase / cellulase. Figure 4 The image shows the recycling performance of NH2-MIL-53(Fe)@β-glucosidase / cellulase; Figure 5 The figure shown is a standard curve of bovine serum albumin. Detailed Implementation

[0017] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0018] In the following examples, the method for calculating the encapsulation efficiency of the immobilized enzyme is as follows: E(%)=(1-C x / C0)×100%(1) In the formula, E represents the encapsulation efficiency (%); C0 is the protein concentration of the free enzyme obtained by substituting the absorbance value into the standard curve (mg / mL); C x Substitute the absorbance values ​​into the bovine serum albumin (BSA) standard curve (see...). Figure 5 The protein concentration (mg / mL) of the immobilized enzyme supernatant obtained.

[0019] Example 1 A method for preparing multi-enzymes co-immobilized in a metal-organic framework material, specifically comprising the following steps: Step 1: Add 2 mg of β-glucosidase to 5 mL of 10 mM 2-aminoterephthalic acid solution to obtain an organic ligand solution containing β-glucosidase. Then, slowly add 5 mL of 5 mM Fe(NO3)3·9H2O aqueous solution. Stir at 500 rpm for 2 h to obtain a mixed solution. Centrifuge the prepared mixture at 10000 rpm for 10 min and wash three times with deionized water to obtain NH2-MIL-53(Fe)@β-glucosidase material.

[0020] Step 2: Weigh 25 mg of NH2-MIL-53(Fe)@β-glucosidase, add 20 mL of 2.5% glutaraldehyde, stir at room temperature for 3 h. After the reaction is complete, centrifuge at 10000 rpm for 10 min to separate the solid, wash three times with deionized water, add 5 mg of cellulase, stir at room temperature for 8 h, centrifuge at 10000 rpm for 10 min to separate the solid, wash three times with deionized water, and vacuum dry to obtain NH2-MIL-53(Fe)@β-glucosidase / cellulase solid.

[0021] The preparation process of the above 2-aminoterephthalic acid solution is as follows: Preparation of acetate-sodium acetate buffer: Weigh 2.86 g of anhydrous sodium acetate solid and dissolve it in a small amount of pure water. Pipette 0.87 mL of glacial acetic acid into the sodium acetate solution and dilute to a 1000 mL volumetric flask. Adjust the pH of the buffer to 4.5–6.5 using a small amount of 50 mM sodium acetate solution and 50 mM acetic acid solution.

[0022] Preparation of aminoterephthalic acid solution: Weigh 0.036 g of 2-aminoterephthalic acid solid into a 50 mL centrifuge tube, add 10 mL of acetate-sodium acetate buffer, and shake well. Since the ligand has low solubility in the buffer, it needs to be deprotonated. Add small amounts of ammonia water several times and shake well until the solid is completely dissolved and the solution is a clear purplish-brown color. Then add an appropriate amount of citric acid solution (1 mM) to adjust the pH of the solution. Finally, add buffer to bring the solution volume to 20 mL and store at room temperature for later use.

[0023] The following describes the performance determination and characterization of the materials prepared above: (1) The content of unencapsulated protein in the supernatant of the NH2-MIL-53(Fe)@β-glucosidase material obtained in step 1 was determined by Coomassie brilliant blue method at a wavelength of 595 nm, and the encapsulation rate of β-glucosidase was calculated to be 88.49%.

[0024] The stability test of NH2-MIL-53(Fe)@β-glucosidase samples was conducted as follows: Figure 1 In step a, NH2-MIL-53(Fe)@β-glucosidase and free enzyme (free β-glucosidase) were stored at 20℃, 30℃, 40℃, and 50℃ for 1 h, respectively, and then hydrolyzed using p-nitrophenol as a substrate. The relative enzyme activities of the two were compared. Figure 1In the figures, b represents the storage of NH2-MIL-53(Fe)@β-glucosidase and free enzyme at 50℃ for 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, respectively, followed by hydrolysis with p-nitrophenol as a substrate. The relative enzyme activities of the two enzymes were compared, and the results are as follows: Figure 1 (b), and by Figure 1 It is known that immobilized enzymes have better thermal stability than free enzymes.

[0025] Cyclic hydrolysis experiments were conducted on NH2-MIL-53(Fe)@β-glucosidase samples using p-nitrophenol as the substrate. After the reaction, the samples were centrifuged, and the supernatant was collected for later use. The solid samples were washed three times with deionized water by centrifugation, and then fresh substrate solution was added to continue the hydrolysis reaction. This process was repeated four times. The product concentration in the supernatant was measured after each reaction, and the relative enzyme activity of all experimental groups was calculated with the first measurement result set as 100%. The results are as follows: Figure 2 As shown, after 5 cycles of hydrolysis, the relative activity of the immobilized enzyme remained at 64%.

[0026] (2) The NH2-MIL-53(Fe)@β-glucosidase / cellulase solid obtained in step 2 was characterized by scanning electron microscopy, and the results are as follows: Figure 3 As shown, by Figure 3 It can be seen that the sample is irregularly shaped like gravel, and honeycomb-like channels of 20-50 nm can be observed on the cross-section of the material at 200 nm.

[0027] The NH2-MIL-53(Fe)@β-glucosidase / cellulase sample was applied to the hydrolysis of carboxymethyl cellulose. Using carboxymethyl cellulose as a substrate, the NH2-MIL-53(Fe)@β-glucosidase / cellulase sample and an equal amount of cellulase were hydrolyzed at pH 5.0 and 50℃. Compared to pure cellulase, the hydrolysis efficiency was increased by 22%. Furthermore, the NH2-MIL-53(Fe)@β-glucosidase / cellulase exhibited good recyclability, such as… Figure 4 As shown, the material retains 61% of its relative enzyme activity after being hydrolyzed five times.

[0028] Example 2 A method for preparing multi-enzymes co-immobilized in a metal-organic framework material, specifically comprising the following steps: Step 1: Add 2 mg of β-glucosidase to 5 mL of 10 mM 2-aminoterephthalic acid solution to obtain an organic ligand solution containing β-glucosidase. Then, slowly add 5 mL of 10 mM AlCl3·6H2O solution and stir at 500 rpm for 2 h to obtain a mixed solution. Centrifuge the prepared mixture at 10000 rpm for 10 min and wash three times with deionized water to obtain NH2-MIL-53(Al)@β-glucosidase material.

[0029] Step 2: Weigh 25 mg of NH2-MIL-53(Al)@β-glucosidase, add 20 mL of 2.5% glutaraldehyde, stir at room temperature for 3 h. After the reaction is complete, centrifuge at 10000 rpm for 10 min to separate the solid, wash three times with deionized water, add 5 mg of cellulase, stir at room temperature for 8 h, centrifuge at 10000 rpm for 10 min to separate the solid, wash three times with deionized water, and vacuum dry to obtain NH2-MIL-53(Al)@β-glucosidase / cellulase solid.

[0030] The following describes the performance determination and characterization of the materials prepared above: (1) The content of unencapsulated protein in the supernatant of the NH2-MIL-53(Al)@β-glucosidase material obtained in step 1 was determined by Coomassie brilliant blue method at a wavelength of 595 nm, and the encapsulation rate of β-glucosidase was calculated to be 87.75%.

[0031] (2) When the NH2-MIL-53(Al)@β-glucosidase / cellulase sample was applied to the hydrolysis of carboxymethyl cellulose, its hydrolysis efficiency was increased by 27% compared with pure cellulase.

[0032] Although the description of the invention has been quite detailed and particularly of several described embodiments, it is not intended to limit it to any of these details or embodiments or any particular embodiment, but should be considered as providing a broad possible interpretation of the claims by referring to the appended claims and taking into account the prior art, thereby effectively covering the intended scope of the invention. Furthermore, the invention has been described above with respect to embodiments foreseeable by the inventors in order to provide a useful description, and non-substantial modifications to the invention that have not yet been foreseen may still represent equivalent modifications.

Claims

1. A method for preparing multi-enzymes co-immobilized in a metal-organic framework material, characterized in that, Includes the following steps: The organic ligand solution was mixed with β-glucosidase, then a metal salt solution was added, the reaction was carried out, centrifuged, and washed to obtain MOF@β-glucosidase; the organic ligand was 2-aminoterephthalic acid; the metal salt solution was an iron salt solution or an aluminum salt solution. The organic ligand solution was prepared by mixing an acetate-sodium acetate buffer solution with a pH of 4.5–6.5 and a concentration of 50 mM with the organic ligand. Glutaraldehyde was added to the MOF@β-glucosidase for activation, followed by reaction, centrifugation, washing, and then cellulase solution was added for reaction. After the reaction was completed, centrifugation and washing were performed to finally obtain the metal-organic framework material co-immobilized multi-enzyme. The ratio of organic ligand solution, β-glucosidase, metal salt solution, glutaraldehyde, and cellulase is 5 mL: 2 mg: 5 mL: 10 mL~20 mL: 1 mg~5 mg; The concentration of the organic ligand solution is 2.5 mM to 15 mM; The concentration of the metal salt solution is 2.5 mM to 10 mM; The mass concentration of glutaraldehyde is 2.5%.

2. The preparation method according to claim 1, characterized in that, The reaction time is 0.5 h to 8 h.

3. A metal-organic framework material for co-immobilizing multiple enzymes, characterized in that, It is prepared by the preparation method according to any one of claims 1-2.

4. The application of the metal-organic framework material co-immobilized with multiple enzymes as described in claim 3 in cellulose hydrolysis.

5. A method for hydrolyzing cellulose, characterized in that, Includes the following steps: The multi-enzyme co-immobilized by the metal-organic framework material of claim 1 is uniformly dispersed in the substrate solution, then shaken and incubated, and centrifuged to obtain glucose; The substrate solution is carboxymethyl cellulose or a cellulose solution.

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