A method for preparing a nanofiber composite carrier and its application

Abstract

This invention discloses a method for preparing a nanofiber composite carrier and its application. The invention employs a blending method to prepare a nanofiber membrane by blending polyvinyl alcohol and aqueous polyurethane. Simultaneously, electrostatic spraying introduces a crosslinking agent to further enhance the mechanical properties of the membrane, resulting in a nanofiber composite carrier with good biocompatibility. Furthermore, an immobilized enzyme is prepared using an adsorption method. This immobilized enzyme exhibits good catalytic activity and manipulability. For example, after repeated use, the immobilized hemicellulase retained 80.3% of its initial enzyme activity on the eighth use. Similarly, the immobilized cellulase retained 86.1% of its initial enzyme activity on the eighth use. The composite carrier disclosed in this invention shows good application results in the field of immobilized enzymes, enabling the reuse of waste liquid from viscose fiber production processes, and has promising industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme technology, and relates to a method for preparing an electrospun nanofiber composite carrier and its application in immobilized enzymes. Background Technology

[0002] Xylanases are a class of enzymes that degrade xylan. They hydrolyze xylan into xylo-oligosaccharides, including xylobiose, xylotriose, and other xylooligosaccharides, as well as a small amount of arabinose, by hydrolyzing the β-1,4-glycosidic bonds of xylose molecules. Hemicellulases are complex enzyme systems, mainly including xylanases and mannanases, which also degrade xylan. Cellulose can promote the dissolution of plant cell walls, and by hydrolyzing cellulose, it promotes the release of hemicellulose, which is beneficial for subsequent hydrolysis to prepare xylooligosaccharides. Xylanases, hemicellulases, and cellulases are often used to hydrolyze lignocellulose to produce high-value-added products and achieve the green conversion of lignocellulose. However, free enzymes have disadvantages such as difficulty in recycling and poor stability, which increases the cost of use. Immobilized enzymes, on the other hand, have the characteristics of easy recycling, reusability, and good stability, which make up for the shortcomings of free enzymes.

[0003] Among various materials for immobilized enzymes, nanomaterials have attracted widespread attention from researchers due to their advantages such as high specific surface area, multiple attachment sites, and good biocompatibility. However, powdered or granular nanomaterials are difficult to separate and recover in reaction systems, leading to the formation of new impurities and limiting the application of immobilized enzymes. Nanofiber membranes not only possess microscopic nanostructures but also macroscopic continuity. Electrospun nanofibers, as an excellent immobilization carrier, have become a research hotspot for immobilized enzymes, showing promising application prospects in the field.

[0004] In recent years, numerous processes for immobilizing enzymes using nanofibers have been developed both domestically and internationally. For example, Niu et al. disclosed a method for immobilizing laccase using polylactic acid electrospun fibers. Polylactic acid fibers exhibit good biocompatibility, are easily degraded, and do not generate secondary pollution. Liu et al. disclosed a method for preparing composite fibers, where peptides are prepared into nanofibers and grafted onto poly(glycidyl methacrylate-co-methyl acrylate) electrospun fibers, improving enzyme immobilization performance and film-forming properties. These studies demonstrate that the unique structure of electrospun nanofibers enables them to achieve good results in the field of enzyme immobilization. This application utilizes a blend of polyvinyl alcohol and waterborne polyurethane to prepare composite materials, achieving beneficial effects in immobilizing hydrolytic enzymes. Zong et al. prepared an elastic three-dimensional fiber sponge with noise reduction function by simultaneously electrospinning and electrospraying polysulfone and polyurethane wrapped around graphene oxide nanosheets, verifying the feasibility and unique effect of combining electrospinning and electrospraying. This application, while preparing composite fibers through electrospinning, uses electrospraying to form micro-droplets that adhere to the fibers, resulting in a material with excellent hydrophilicity, biocompatibility, and stability. Luo et al. disclosed a method for preparing immobilized enzymes using magnetic chitosan microspheres. The immobilized proteases and amylases showed beneficial effects in sludge hydrolysis. Fan et al. disclosed a method for immobilizing naringinase using polyurethane nanofiber membranes. The prepared immobilized enzymes can be reused multiple times and can effectively hydrolyze naringin, removing bitter substances. Related studies have shown that immobilized hydrolytic enzymes can effectively improve enzyme stability while maintaining enzyme hydrolytic activity, exhibiting good storage stability and showing promising application prospects in the food processing field.

[0005] Existing research has shown that nanofiber-immobilized enzymes prepared using insoluble polymers have exhibited excellent performance. However, the application of electrospun fibers immobilized with polyvinyl alcohol (PVA)-based water-soluble polymers remains limited. The poor stability and high water solubility of nanofibers need to be addressed. Immersing the fibers in a crosslinking agent solution can reduce solubility, but the fiber membrane structure is still damaged. Therefore, researching a novel multi-nozzle blending process, in which the crosslinking agent is added to the electrospun fibers via electrostatic spraying during electrospinning, completes crosslinking without damaging the fiber structure. Rapidly preparing nanofiber-immobilized enzymes with high stability, good biocompatibility, and high catalytic activity is a key technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] This invention provides a method for preparing a nanofiber composite carrier and its application. The reagents used in this invention are sourced from a reagent company.

[0007] The technical solution adopted in this invention is as follows: A method for preparing a nanofiber composite carrier, comprising the following steps: (1) Mix polyvinyl alcohol and distilled water in a certain proportion, heat in a boiling water bath and stir continuously until fully dissolved, filter with gauze and cool to room temperature to obtain a polyvinyl alcohol aqueous solution; (2) Take a certain volume of the polyvinyl alcohol solution in (1), add a certain amount of surfactant, dilute and dissolve with water, stir for 4-8 h, sonicate at room temperature for 10-30 min and let stand to obtain a polyvinyl alcohol spinning solution; (3) Take a certain mass of the polyvinyl alcohol solution in (1), add a certain amount of waterborne polyurethane, dilute with water and stir for 4-8 h to obtain a polyvinyl alcohol-waterborne polyurethane spinning solution; (4) Take a certain mass of crosslinking agent and dissolve in water, add a certain mass of polyvinylpyrrolidone, stir and dissolve to obtain a crosslinking solution; (5) Transfer the prepared spinning solution and crosslinking solution to a 2.5-10 mL syringe with a needle inner diameter of 0.63-0.90 mm, place horizontally in a four-piece push pump, electrospin at a temperature of 20-30℃ and a humidity of 40-70%, and dry in an oven at 40-50℃ for 12-24 hours. h; (6) Place the nanofiber membrane in glutaraldehyde vapor for 4-8 h and wash it repeatedly with distilled water to obtain the nanofiber composite carrier.

[0008] Furthermore, in (2), the concentration of the diluted polyvinyl alcohol is 5-13%, and the surfactant is preferably one or more of hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, and Tween-20, and the ratio of the surfactant to polyvinyl alcohol is 0.2 / 1 to 0.8 / 1 g / g.

[0009] Furthermore, the ratio of waterborne polyurethane to polyvinyl alcohol in (3) is 1.25 / 1 to 1.5 / 1 g / g.

[0010] Furthermore, in (4), the crosslinking agent is preferably at least one of borax, boric acid, glyoxal, and glutaraldehyde, and the ratio of the crosslinking agent to polyvinylpyrrolidone is 0.7 / 1 to 1.8 / 1 g / g.

[0011] Furthermore, the spinning process parameters in (5) are: positive high voltage 13~19 kV, negative high voltage 0~-2 kV, pushing speed 0.03~0.05 mm / min, translation speed 80~100 mm / min, needle distance from receiving device is 10~15 cm, receiving device is receiving roller wrapped with release paper, receiving speed of receiving roller is 40~60 r / m.

[0012] Furthermore, the glutaraldehyde vapor in (6) is preferably saturated vapor.

[0013] Furthermore, in the nanofiber composite carrier, the electrospun fibers have a diameter of several hundred nanometers, and the waterborne polyurethane is distributed on the fiber surface.

[0014] The present invention also discloses the application of a composite carrier prepared by the above method in immobilized enzymes.

[0015] Furthermore, the composite carrier was immersed in an enzyme buffer solution, placed on a shaker and shaken for 30-50 minutes, then removed and washed with buffer solution to obtain nanofiber immobilized enzyme.

[0016] Furthermore, in the enzyme buffer solution, the buffer is preferably at least one of phosphate buffer, acetate buffer, and citrate buffer, the pH value of the buffer is preferably 4-6, the buffer concentration is preferably 0.01-0.20 mol / L, the enzyme concentration is 1-10 mg / mL, and the enzyme is preferably at least one of hemicellulase, xylanase, and cellulase.

[0017] Furthermore, the substrates of nanofiber immobilized xylanase and nanofiber immobilized hemicellulase are one or more of corn cob xylan, straw xylan, coconut shell xylan and viscose fiber xylan; the substrate of nanofiber immobilized cellulase is cellulose.

[0018] Furthermore, the preparation process of the viscose fiber xylan is as follows: take 50-100 mL of hemicellulose extract, add three times the volume of 95% ethanol, let stand overnight, filter and take the solid, wash the solid with 95% ethanol and dry it to obtain the alcohol precipitation product xylan, and recover the ethanol by rotary evaporation of the filtrate.

[0019] Furthermore, the method for using nanofiber immobilized enzyme is as follows: Dissolve the substrate in phosphate buffer at pH 5-6, with a substrate concentration preferably of 0.5%-5%. Add the immobilized enzyme, incubate at 50°C with shaking for 0.5-4 h, remove the immobilized enzyme, collect the supernatant, and set aside for later use.

[0020] Furthermore, the method for determining reducing sugar content (DNS method) is as follows: Take 2 mL of the test solution, add 2 mL of DNS solution, boil in a water bath for 2 min, dilute 10 times, and measure the absorbance value at 540 nm. Use glucose as a standard to prepare a standard curve.

[0021] Furthermore, enzyme activity is defined as follows: the number of micromoles of reducing sugar released per minute by 1 g of immobilized enzyme from the substrate solution at 50°C. The calculation formula is as follows: (1) In the formula: V -- Solution volume, mL; C0 -- concentration of reducing sugar in the solution, mg / mL; M -- Molar mass of reducing sugar, g / mol; t -- Enzymatic hydrolysis reaction time, min; m -- Immobilized enzyme mass, g.

[0022] The advantages of this invention are: A four-component blended spinning method was employed, simultaneously collecting composite fibers composed of three types of fibers: polyvinyl alcohol (PVA) to form a fiber membrane; 4% PVA-waterborne polyurethane, where the waterborne polyurethane fibers act as adhesives; and saturated boric acid-polyvinylpyrrolidone (PVP), which forms a fine spray in an electric field, causing the PVA fibers to crosslink and resulting in a nanofiber mat of a certain thickness. The nanofibers prepared by this invention have small diameters, large specific surface areas, good hydrophilicity, and excellent mechanical strength. The immobilized enzymes prepared exhibit high activity and stability, enabling the reuse of waste liquid from viscose fiber production processes. Attached Figure Description

[0023] Figure 1 A statistical chart showing the enzyme activity retention rate after multiple uses of immobilized hemicellulase.

[0024] Figure 2 A statistical chart showing the enzyme activity retention rate after multiple uses of immobilized cellulase. Detailed Implementation

[0025] The specific embodiments of the present invention will be further described in detail below with reference to examples. These examples are used to further illustrate the content of the present invention, but do not limit the scope of protection of the present invention. All related content and modifications involved in the present invention fall within the scope of protection of the present invention.

[0026] Example 1: Preparation of nanofiber composite carrier and application of immobilized xylanase (1) Mix 7-8 g of polyvinyl alcohol with distilled water, heat in a boiling water bath and stir continuously until fully dissolved, filter with gauze and cool to room temperature to obtain an 8-13% polyvinyl alcohol aqueous solution; (2) Take the polyvinyl alcohol solution from step (1), add hexadecyltrimethylammonium chloride, dilute and dissolve with water, stir for 4-6 h, sonicate at room temperature for 20-30 min and let stand to obtain a polyvinyl alcohol spinning solution containing 4-6% hexadecyltrimethylammonium chloride; (3) Take the polyvinyl alcohol solution from step (1), add aqueous polyurethane, dilute and stir with water for 4-6 h to obtain a 3% polyvinyl alcohol-4% aqueous polyurethane spinning solution; (4) Take 0.9-1.0 g of boric acid and dissolve it in 10 mL of water, add 1.0 g of polyvinylpyrrolidone, stir and dissolve to obtain a crosslinking solution; (5) Transfer the prepared spinning solution and crosslinking solution to a 2.5-5 mL syringe with a needle inner diameter of 0.63-0.80 mm, place it horizontally in a four-piece push pump, and adjust the positive high pressure to 15-17. kV, negative high voltage -2 kV, propulsion speed 0.03~0.05 mm / min, translation speed 80~100 mm / min, distance between needle tip and receiving device is 10~12 cm, receiving device is receiving roller wrapped with release paper, receiving speed of receiving roller is 50~60 r / m, electrospinning in an environment with temperature of 20~30℃ and humidity of 40~70%, and drying in an oven at 45~50℃ for 12~15 h; (6) crosslink the dried nanofiber membrane in saturated glutaraldehyde vapor for 6~8 h, wash with distilled water to obtain nanofiber composite carrier.

[0027] The composite carrier was immersed in a xylanase buffer solution with a concentration of 8-10 mg / mL. The buffer solution was a phosphate buffer solution with a pH of 4-6 (0.01-0.1 mol / L). The solution was shaken on a shaker for 30-50 min. After removal, the carrier was washed with the buffer solution to obtain nanofiber immobilized xylanase.

[0028] Xylan from viscose cellulose was dissolved in a phosphate buffer solution with a pH of 5-6 to prepare a 0.5-1% xylan solution. Immobilized xylanase was added, and the solution was incubated in a water bath at 50°C for 30-60 min. The immobilized enzyme was removed, and the reducing sugar content of the enzymatic digest supernatant was determined.

[0029] The yield, expressed as the total amount of reducing sugars (mg) obtained from the enzymatic hydrolysis of each gram of precipitated alcohol solid (g), was 231.72 mg / g.

[0030] Example 2: Preparation of nanofiber composite carrier and application of immobilized hemicellulase Mix 10-12 g of polyvinyl alcohol with distilled water, heat in a boiling water bath and stir continuously until fully dissolved, filter with gauze and cool to room temperature to obtain an 8-13% polyvinyl alcohol aqueous solution; (2) Take the polyvinyl alcohol solution from step (1), add hexadecyltrimethylammonium bromide, dilute and dissolve with water, stir for 6-8 h, sonicate at room temperature for 20-30 min and let stand to obtain a polyvinyl alcohol spinning solution containing 4-6% hexadecyltrimethylammonium bromide; (3) Take the polyvinyl alcohol solution from step (1), add aqueous polyurethane, dilute and stir with water for 4-6 h to obtain a 4% polyvinyl alcohol-5% aqueous polyurethane spinning solution; (4) Take 0.9-1.2 g of boric acid and dissolve it in 10 mL of water, add 1.2 g of polyvinylpyrrolidone, stir and dissolve to obtain a crosslinking solution; (5) Transfer the prepared spinning solution and crosslinking solution to a 2.5-10 mL syringe with a needle inner diameter of 0.70-0.80 mm, place it horizontally in a four-piece push pump, and adjust the positive high pressure to 17. kV, negative high voltage -2 kV, propulsion speed 0.04~0.05 mm / min, translation speed 80~100 mm / min, distance between needle tip and receiving device is 10~14 cm, receiving device is receiving roller wrapped with release paper, receiving speed of receiving roller is 50~60 r / m, electrospinning in an environment with temperature of 20~30℃ and humidity of 40~70%, and drying in an oven at 40~50℃ for 12~15 h; (6) crosslink the dried nanofiber membrane in saturated glutaraldehyde vapor for 6~8 h, wash with distilled water to obtain nanofiber composite carrier.

[0031] The composite carrier was immersed in a hemicellulase buffer solution with a concentration of 8-10 mg / mL. The buffer solution was a phosphate buffer solution with a pH of 4-60.1-0.15 mol / L. The solution was shaken on a shaker for 30-50 min. After removal, the carrier was washed with the buffer solution to obtain nanofiber immobilized hemicellulase.

[0032] Corn cob xylan was dissolved in a phosphate buffer solution with a pH of 5-6 to prepare a 0.5-1% xylan solution. Immobilized hemicellulase was added, and the solution was incubated in a 50°C water bath for 30-60 min. The immobilized hemicellulase was then removed, and its activity was measured. This process was repeated. After 8 reuses of the nanofiber-immobilized hemicellulase, the enzyme activity retention rate was 80.3%. (See details...) Figure 1 .

[0033] Example 3: Preparation of nanofiber composite carrier and application of immobilized cellulase (1) Mix 10-12 g of polyvinyl alcohol with distilled water, heat in a boiling water bath and stir continuously until fully dissolved, filter with gauze and cool to room temperature to obtain an 8-13% polyvinyl alcohol aqueous solution; (2) Take the polyvinyl alcohol solution from step (1), add hexadecyltrimethylammonium bromide, dilute and dissolve with water, stir for 6-8 h, sonicate at room temperature for 20-30 min and let stand to obtain a polyvinyl alcohol spinning solution containing 4-6% hexadecyltrimethylammonium bromide; (3) Take the polyvinyl alcohol solution from step (1), add aqueous polyurethane, dilute with water and stir for 4-6 h to obtain a 4% polyvinyl alcohol-5% aqueous polyurethane spinning solution; (4) Dissolve 0.9-1.2 g of boric acid in 10 mL of water, add 1.2 g of polyvinylpyrrolidone, stir and dissolve to obtain a crosslinking solution; (5) Transfer the prepared spinning solution and crosslinking solution to a 2.5-10 mL syringe with a needle inner diameter of 0.70-0.80 mm. In a syringe of mm, it is placed horizontally in a four-piece propulsion pump. The positive high voltage is adjusted to 17 kV, the negative high voltage to -2 kV, the propulsion speed is 0.04~0.05 mm / min, the translation speed is 80~100 mm / min, the distance between the needle and the receiving device is 10~14 cm, the receiving device is a receiving roller wrapped with release paper, the receiving speed of the receiving roller is 50~60 r / m, electrospinning is carried out in an environment with a temperature of 20~30℃ and a humidity of 40~70%, and then dried in an oven at 40~50℃ for 12~15 h; (6) the dried nanofiber membrane is placed in saturated glutaraldehyde vapor for crosslinking for 6~8 h, and washed with distilled water to obtain the nanofiber composite carrier.

[0034] The composite carrier was immersed in a cellulase buffer solution with a concentration of 8-10 mg / mL. The buffer solution was a phosphate buffer solution with a pH of 4-60.1-0.15 mol / L. The solution was shaken on a shaker for 30-50 min. After removal, the carrier was washed with the buffer solution to obtain nanofiber immobilized cellulase.

[0035] Cellulose was added to a phosphate buffer solution with a pH of 5-6 to prepare a substrate solution with a concentration of 0.5-1%. Immobilized cellulase was then added, and the solution was incubated in a 50°C water bath for 30-60 min. The immobilized cellulase was then removed, and its activity was measured. This process was repeated. After 8 reuses of the nanofiber-immobilized cellulase, the enzyme activity retention rate was 86.1%. (See details...) Figure 2 .

Claims

1. A method for preparing a nanofiber composite carrier, characterized in that, Includes the following steps: (1) Mix polyvinyl alcohol and distilled water in a certain proportion, heat in a boiling water bath and stir continuously until fully dissolved, filter with gauze and cool to room temperature to obtain a polyvinyl alcohol aqueous solution; (2) Take a certain volume of the polyvinyl alcohol aqueous solution in (1), add a certain amount of surfactant, dilute and dissolve with water, stir for 4~8 h, sonicate at room temperature for 10~30 min and let stand to obtain a polyvinyl alcohol spinning solution; (3) Take a certain mass of the polyvinyl alcohol aqueous solution in (1), add a certain amount of waterborne polyurethane, dilute with water and stir for 4~8 h to obtain a polyvinyl alcohol-waterborne polyurethane spinning solution; (4) Take a certain mass of crosslinking agent dissolved in water, add a certain mass of polyvinylpyrrolidone, stir and dissolve to obtain a crosslinking solution; (5) Transfer the prepared polyvinyl alcohol, polyvinyl alcohol-waterborne polyurethane spinning solution and crosslinking solution to 2.5~10 mL of needle with an inner diameter of 0.63~0.90 mm. In a syringe of mm, it is placed horizontally in a four-piece propulsion pump and electrospun in an environment with a temperature of 20~30℃ and a humidity of 40~70%. The crosslinking solution is added to the electrospun fiber in the form of electrostatic spray and dried in an oven at 40~50℃ for 12~24 h; (6) The nanofiber membrane is placed in glutaraldehyde vapor for 4~8 h and washed multiple times with distilled water to obtain a nanofiber composite carrier; the crosslinking agent in (4) is at least one of borax, boric acid, glyoxal, and glutaraldehyde, and the ratio of crosslinking agent to polyvinylpyrrolidone is 0.7 / 1~1.8 / 1 g / g.

2. The method for preparing a nanofiber composite carrier according to claim 1, characterized in that, The concentration of polyvinyl alcohol after dilution in (2) is 5-13%, and the surfactant is one or more of hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, and Tween-20. The ratio of surfactant to polyvinyl alcohol is 0.2 / 1 to 0.8 / 1 g / g.

3. The method for preparing a nanofiber composite carrier according to claim 1, characterized in that, The ratio of waterborne polyurethane to polyvinyl alcohol in (3) is 1.25 / 1 to 1.5 / 1 g / g.

4. The method for preparing a nanofiber composite carrier according to claim 1, characterized in that, The spinning process parameters in (5) are: positive high voltage 13~19 kV, negative high voltage 0~-2 kV, pushing speed 0.03~0.05 mm / min, translation speed 80~100 mm / min, needle distance from receiving device is 10~15 cm, receiving device is receiving roller wrapped with release paper, receiving speed of receiving roller is 40~60 r / m.

5. The method for preparing a nanofiber composite carrier according to claim 1, characterized in that, In the nanofiber composite carrier, the electrospun fibers have a diameter of several hundred nanometers, and the waterborne polyurethane is distributed on the surface of the fibers obtained from the polyvinyl alcohol-waterborne polyurethane spinning solution.

6. The application of the composite carrier obtained by the preparation method according to claim 1 in immobilized enzymes.

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