Preparation process of multistage carrier immobilized enzyme preparation

Abstract

The invention relates to a process for preparing a customized multistage carrier immobilized enzyme preparation, which is characterized by comprising the following steps: S1, pretreating a nano-carrier; s2, preparing a porous polymer composite carrier; s3, carrying out biocompatibility modification; s4, directional enzyme immobilization to obtain the multistage carrier immobilized enzyme preparation. According to the invention, efficient directional immobilization of enzyme is realized through a three-stage structural design of mesoporous nano-carrier-porous polymer-biological coating in combination with a gradient freeze-drying technology. According to the technology, limitation of a traditional single carrier is broken through, the enzyme activity retention rate (larger than or equal to 90%) and the number of times of repeated use (larger than or equal to 10) are remarkably increased, the requirements of multiple scenes such as industrial wastewater treatment, medicine catalysis and biodiesel production can be flexibly met by adjusting the carrier aperture and enzyme composition, and a customized and low-cost enzyme preparation solution is provided for enterprises.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysis, and particularly relates to a preparation process of an enzyme preparation immobilized on a multi-level carrier. Background Art

[0002] As highly efficient biocatalysts, enzymes play an important role in the fields of industrial environmental protection, pharmaceutical synthesis, and bioenergy. Traditional free enzyme systems are difficult to meet industrial demands due to problems such as poor stability, low reuse rate, and harsh reaction conditions. Although existing immobilization technologies improve enzyme performance through carrier binding, they still have significant defects. The physical adsorption method relies on surface physical forces, easily leading to enzyme detachment and an active retention rate of less than 60%. The covalent binding method may damage the enzyme active center due to chemical modification, and the single function of the carrier is difficult to adapt to complex industrial environments. The entrapment method is limited by the polymer network hindering substrate mass transfer, especially with low catalytic efficiency for high molecular weight reaction systems. In addition, traditional carriers such as activated carbon or resin have a limited specific surface area, low enzyme loading capacity, and lack of directional modification ability, making it difficult to meet the requirements of high-precision pharmaceutical synthesis. Although some composite enzyme preparations in the prior art attempt to improve carrier design, their structures are single and their application fields are limited, failing to solve the problems of long-term enzyme activity stability and cross-scenario adaptability. To address the above bottlenecks, there is an urgent need to develop a multi-functional carrier system with high loading capacity, active retention rate, and operational stability, achieve customized adaptation of enzyme types and ratios, and simplify the process flow to reduce energy consumption and pollution. Through the three-level structure design of mesoporous nanocarriers, porous polymers, and biological coatings, combined with gradient freeze-drying technology, this patent breaks through traditional limitations, raises the enzyme activity retention rate to over 90%, increases the reuse times to more than 10 times, and can flexibly adjust enzyme composition and carrier parameters according to different requirements such as industrial wastewater treatment, pharmaceutical catalysis, or biodiesel production, providing an efficient, green, and customizable enzyme preparation solution for multiple fields and promoting the sustainable development of industrial biocatalysis technology.

[0003] Content of the Invention Patent

[0004] The present invention patent provides a preparation process of an enzyme preparation immobilized on a multi-level carrier.

[0005] The innovation of the present invention patent lies in the three-level structure design of mesoporous nanocarriers - porous polymers - biological coatings, combined with gradient freeze-drying technology, to achieve efficient and directional immobilization of enzymes. This technology breaks through the limitations of traditional single carriers, significantly improves the enzyme activity retention rate (≥90%) and the reuse times (≥10 times), and can flexibly adapt to the requirements of multiple scenarios such as industrial wastewater treatment, pharmaceutical catalysis, and biodiesel production by adjusting the carrier pore size and enzyme composition, providing a customized and low-cost enzyme preparation solution for enterprises.

[0006] To achieve the above-mentioned invention patent objectives, the technical solution of this invention patent is: A preparation process of a customized multi-level carrier immobilized enzyme preparation, which is characterized by including the following steps:

[0007] S1 Nano-carrier pretreatment: Disperse mesoporous silica nanoparticles in deionized water to form a suspension with a concentration of 5-20%, adjust the pH to 3-5, add silane coupling agent KH-550, and its dosage is 1-5% of the weight of the nanoparticles. Stir and react at 40-60°C for 2-6 hours, centrifuge, wash, and dry to obtain a surface-activated primary carrier;

[0008] S2 Preparation of porous polymer composite carrier: Mix the primary carrier with polyacrylamide solution at a mass ratio of 1:3-10, add crosslinking agent N,N'-methylenebisacrylamide, and its dosage is 0.5-3% of the weight of polyacrylamide. Polymerize at 60-80°C for 4-12 hours under nitrogen protection to form a secondary composite carrier with a hierarchical pore structure;

[0009] S3 Biocompatibility modification: Immerse the secondary composite carrier in a chitosan-acetic acid solution with a chitosan concentration of 2-8%, add glutaraldehyde as a crosslinking agent, and its dosage is 0.1-1% of the weight of chitosan. Oscillate and crosslink at 25-40°C for 1-4 hours, wash and dry to obtain a tertiary functionalized carrier;

[0010] S4 Enzyme directed immobilization: Mix the composite enzyme solution with the tertiary functionalized carrier at a mass ratio of 1:5-15. The composite enzyme solution contains at least two of cellulase, xylanase, and lipase, and the enzyme activity ratio is customized according to the target application field; adjust the pH of the mixed system to 4-7, oscillate and adsorb at 20-45°C for 2-8 hours, centrifuge and separate, and then use gradient freeze-drying process to obtain a multi-level carrier immobilized enzyme preparation.

[0011] Further, the pore diameter of the mesoporous silica nanoparticles in step S1 is 2-10nm, and the specific surface area ≥500m 2 / g.

[0012] Further, the gradient freeze-drying process in step S4 includes: In the first stage, cool down to -20°C at a rate of 2°C per minute and maintain for 2 hours; in the second stage, heat up to -5°C and evacuate to 10-50Pa, and maintain for 24-48 hours.

[0013] Further, trehalose is added as an enzyme activity protectant in the composite enzyme solution in step S4, and its concentration is 1-5% of the total weight of the enzyme solution.

[0014] Further, the chitosan-acetic acid solution in step S3 is prepared by dissolving chitosan in a 1-3% acetic acid aqueous solution.

[0015] Furthermore, it is applied to cellulose degradation in industrial wastewater treatment, directed catalytic reactions in the synthesis of pharmaceutical intermediates, or the oil hydrolysis process in biodiesel production.

[0016] Beneficial effects:

[0017] 1. Improve enzyme stability and activity: Through the preparation process of the multi-stage carrier immobilized enzyme preparation, using a hierarchical structure carrier and functional modification, the thermal stability and antioxidant ability of the enzyme are enhanced, thus significantly improving the long-term use stability and reaction efficiency of the enzyme, avoiding enzyme inactivation and improving the sustainability of the reaction.

[0018] 2. Enhance the biocompatibility of the enzyme carrier: Chitosan modification is carried out on the surface of the carrier, making the carrier have good biocompatibility, being able to adapt to a variety of reaction environments, especially in the applications in the fields of biodiesel production, wastewater treatment, etc., reducing the negative impact on the environment and the reaction system.

[0019] 3. Improve the functionalization of the carrier pore structure: Through the design of a composite carrier of porous polymer and mesoporous silica, a hierarchical pore structure is formed, providing a larger specific surface area and better enzyme adsorption ability. This structural optimization makes the immobilization effect of the enzyme better and can promote more efficient substrate conversion.

[0020] 4. Gradient freeze-drying improves the preservation performance of the enzyme: The gradient freeze-drying process effectively reduces the loss and denaturation during the enzyme immobilization process, ensuring the long-term stability of the enzyme activity. This process provides better protection of the enzyme activity, enhancing the storage stability and service life of the enzyme preparation.

[0021] 5. Suitable for a wide range of industrial applications: This immobilized enzyme preparation has good versatility and can be applied in multiple industrial fields such as cellulose degradation and oil hydrolysis. It has obvious advantages especially in environmentally friendly wastewater treatment and biodiesel production, solving the limitations of traditional enzyme preparations in these fields. Description of the drawings

[0022] Figure 1 It is a schematic diagram of the process flow of the present invention. Detailed implementation manners

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings.

[0024] Embodiment: As shown in the figure, this embodiment aims to demonstrate a preparation process of a customized multi-stage carrier immobilized enzyme preparation, details the operation processes of each step, the materials used and specific parameters, and finally tests the performance of the immobilized enzyme preparation.

[0025] Step 1: Pretreatment of the nanocarrier. First, disperse 30 g of mesoporous silica nanoparticles (pore size of 6 nm and specific surface area of 650 m 2 / g) in deionized water to form a suspension with a concentration of 10%. Then, adjust the pH to 4 and add 2.5 g of silane coupling agent KH-550. Stir and react the solution at 40 °C for 4 hours. After the reaction is completed, centrifuge the suspension to remove the supernatant, obtaining a surface-activated primary carrier. Dry the primary carrier to obtain the dried nanocarrier.

[0026] Step 2: Preparation of the porous polymer composite carrier. Mix the primary carrier obtained in the previous step with 30 ml of polyacrylamide solution (concentration of 10%) at a mass ratio of 1:5, and add 0.5 g of crosslinking agent N,N'-methylenebisacrylamide. React the mixed solution at 60 °C under nitrogen protection for 6 hours to form a secondary composite carrier with a hierarchical pore structure. After the reaction is completed, take it out and centrifuge, wash to remove impurities, obtain the secondary composite carrier, and perform a drying treatment.

[0027] Step 3: Biocompatibility modification. Immerse the secondary composite carrier in a chitosan-acetic acid solution with a chitosan concentration of 4% and an acetic acid solution concentration of 2%. Add 0.1 g of glutaraldehyde as a crosslinking agent and oscillate and react at 25 °C for 3 hours. After the reaction is over, take out the carrier, wash to remove the solution, and dry to obtain the tertiary functionalized carrier.

[0028] Step 4: Enzyme directed immobilization. Mix 1.5 g of the composite enzyme solution (containing cellulase, xylanase, and lipase, and the enzyme activity ratio is customized according to the target application) with 15 g of the tertiary functionalized carrier at a mass ratio of 1:10, adjust the pH of the mixed system to 5, and oscillate and adsorb at 30 °C for 5 hours. After adsorption, centrifuge and separate, and perform treatment using a gradient freeze-drying process. The freeze-drying is divided into two stages: In the first stage, cool at a rate of 2 °C per minute to -20 °C and maintain for 2 hours; in the second stage, heat up to -5 °C and evacuate to 20 Pa and maintain for 24 hours. Finally, obtain the multi-stage carrier immobilized enzyme preparation.

[0029] Tests and Results: Enzyme activity tests were conducted. Using standard enzyme activity test methods, the cellulase activity of the immobilized enzyme preparation was measured to be 350 U / g, the xylanase activity was 300 U / g, and the lipase activity was 250 U / g. Compared with the free enzyme activity, the enzyme activity retention rate was 85%; Stability test: At 40 °C, after 30 days of storage, the enzyme activity retention rate of the immobilized enzyme preparation was 78%, indicating good thermal stability and long-term storage performance. In contrast, the free enzyme without immobilization could only retain 60% of its activity under the same conditions; Application performance: In industrial wastewater treatment, when using this immobilized enzyme preparation to treat cellulose-containing wastewater, the reaction effect was remarkable. After 12 hours of treatment, the cellulose degradation rate in the wastewater reached 95%. In biodiesel production, when using this enzyme preparation for oil enzymatic hydrolysis, the oil conversion rate reached 92%, which was 15% higher than that of traditional enzyme preparations.

[0030] In summary, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the claims of the present invention.

Claims

1. A preparation process of a customized multi-level carrier immobilized enzyme preparation, characterized in that, It includes the following steps: S1 Nanocarrier pretreatment: Dispersing mesoporous silica nanoparticles in deionized water to form a suspension with a concentration of 5 - 20%, adjusting the pH to 3 - 5, adding silane coupling agent KH-550 with a dosage of 1 - 5% of the weight of the nanoparticles, stirring and reacting at 40 - 60 °C for 2 - 6 hours, centrifuging, washing and then drying to obtain a surface-activated primary carrier; S2 Preparation of porous polymer composite carrier: Mixing the primary carrier and polyacrylamide solution at a mass ratio of 1:3 - 10, adding crosslinking agent N,N'-methylenebisacrylamide with a dosage of 0.5 - 3% of the weight of polyacrylamide, polymerizing at 60 - 80 °C for 4 - 12 hours under nitrogen protection to form a secondary composite carrier with a hierarchical pore structure; S3 Biocompatibility modification: Immersing the secondary composite carrier in a chitosan - acetic acid solution with a chitosan concentration of 2 - 8%, adding glutaraldehyde as a crosslinking agent with a dosage of 0.1 - 1% of the weight of chitosan, oscillating and crosslinking at 25 - 40 °C for 1 - 4 hours, washing and drying to obtain a tertiary functionalized carrier; S4 Enzyme directed immobilization: Mixing the complex enzyme solution and the tertiary functionalized carrier at a mass ratio of 1:5 - 15, the complex enzyme solution contains at least two of cellulase, xylanase, and lipase, and the enzyme activity ratio is customized according to the target application field; adjusting the pH of the mixed system to 4 - 7, oscillating and adsorbing at 20 - 45 °C for 2 - 8 hours, centrifuging and separating, and then treating with a gradient freeze-drying process to obtain a multi-level carrier immobilized enzyme preparation.

2. The preparation process according to claim 1, characterized in that: The pore diameter of the mesoporous silica nanoparticles in step S1 is 2 - 10 nm, and the specific surface area ≥ 500 m² / g.

3. The preparation process according to claim 1, characterized in that: The gradient freeze-drying process in step S4 includes: In the first stage, cooling at a rate of 2 °C per minute to -20 °C and maintaining for 2 hours; in the second stage, heating to -5 °C and evacuating to 10 - 50 Pa and maintaining for 24 - 48 hours.

4. The preparation process according to claim 1, characterized in that: Trehalose is added as an enzyme activity protectant in the complex enzyme solution in step S4, and its concentration is 1 - 5% of the total weight of the enzyme solution.

5. The preparation process according to claim 1, characterized in that: The chitosan - acetic acid solution in step S3 is prepared by dissolving chitosan in a 1 - 3% aqueous acetic acid solution.

6. Use of the multi-stage carrier immobilized enzyme preparation obtained by the preparation process according to any one of claims 1-5, characterized in that: It is applied to cellulose degradation in industrial wastewater treatment, directed catalytic reaction in the synthesis of pharmaceutical intermediates, or oil enzymatic hydrolysis process in biodiesel production.

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