Immobilized lipase based on photo-crosslinked carboxymethyl chitosan / lignin particle composite microspheres as well as preparation and application of immobilized lipase

Abstract

The invention discloses immobilized lipase based on photo-crosslinked carboxymethyl chitosan / lignin particle composite microspheres as well as preparation and application of the immobilized lipase. The preparation method comprises the following steps: firstly, carrying out methacrylation modification on carboxymethyl chitosan and carrying out propenylation modification on lignin to introduce double bonds; then taking a mixed solution of the modified lignin particle dispersion liquid, the modified carboxymethyl chitosan, a photoinitiator and lipase as a water phase, then adding EDC / NHS to crosslink the modified carboxymethyl chitosan and the lipase, adding the water phase into the oil phase to form a water-in-oil emulsion, and simultaneously crosslinking to form microspheres; and finally, further realizing microsphere preparation and enzyme immobilization by adopting ultraviolet crosslinking. The immobilized lipase has the characteristics of high enzyme loading amount, high catalytic activity and good mechanical property, and can be widely applied to food, preparation of medicines and detergents as a biocatalyst.

Description

Technical Field

[0001] This invention belongs to the field of immobilized lipase preparation technology, specifically relating to immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres and its preparation and application. Background Technology

[0002] Lipases are an important class of biocatalysts, widely used in food processing, pharmaceuticals, and fine chemicals due to their high selectivity, mild catalytic reaction conditions, and lack of pollution. However, the poor stability, easy inactivation, and difficulty in recycling of free enzymes hinder their large-scale industrial production. Overcoming these limitations through enzyme immobilization technology has become a hot research topic in enzyme engineering. Immobilizing lipases preserves their catalytic activity while improving their stability, allowing for recovery and repeated use.

[0003] The performance of immobilized enzymes is related to the type of immobilized enzyme carrier. Chitosan and its derivatives have good biodegradability, biocompatibility, antibacterial activity, and adhesiveness, and are often used as carriers for immobilized enzymes. Adejanildo da S. Pereira et al. encapsulated Candida rugosa lipase in chitosan microspheres (Pereira, AdS; Fraga, JL; Souza, CPL; Torres, AG; Amaral, PFF β-Sitosterol Oleate Synthesis by Candida rugosa Lipase in a Solvent-Free Mini Reactor System: Free and Immobilized on Chitosan-Alginate Beads. Catalysts 2023, 13, 780), achieving an immobilization efficiency of 51%. The immobilized enzyme-catalyzed synthesis of β-sitosterol oleate can achieve a high conversion rate of over 95%. Li et al. (Li, H., Liu, Z., Shi, R., Yang, C., & Li, D. Cuttlebone modified chitosan used as immobilized trypsin carrier and degradation of bovine hemoglobin. Preparative Biochemistry & Biotechnology, 2025, 1–8.) used cuttlebone-modified chitosan as an enzyme immobilization carrier. Through covalent cross-linking with trypsin using a non-toxic genipin cross-linking agent, the immobilized enzyme activity reached 390.26 U / g, and it exhibited significantly improved pH, thermal stability, and storage stability. Although chitosan has good immobilization effects on various biological enzymes, its low solubility at physiological pH limits its use for immobilizing lipases. In situ embedding is not possible; a chitosan carrier must be prepared first, followed by adsorption to load the lipase, resulting in a lower enzyme loading capacity.

[0004] Carboxymethyl chitosan, as a derivative of chitosan, exhibits good water solubility in the pH range of 3-8. It can be used to immobilize lipases via in-situ embedding (lipase and carboxymethyl chitosan are simultaneously dissolved in an aqueous solution, and the lipase is simultaneously embedded during the solidification process) to increase enzyme loading. Furthermore, the most common method for in-situ immobilization of lipases using carboxymethyl chitosan employs glutaraldehyde as a cross-linking agent. This agent can cross-link carboxymethyl chitosan to form a carrier and covalently bind the lipase to the carrier. However, glutaraldehyde is toxic and significantly damages enzyme activity. Therefore, it is necessary to develop new bio-enzyme-friendly cross-linking methods to prepare immobilized lipases to minimize the damage to enzyme activity during the immobilization process. Didem Omay et al. (Omay, D. Immobilization of lipase onto a photo-crosslinked polymer network: Characterization and polymerization applications. Biocatalysis and Biotransformation, 2014, 32(2), 132-140.) prepared a methacrylated chitin-based photocrosslinked polymer for immobilizing lipase, achieving a lipase activity recovery rate of 76.0%, indicating that photocrosslinking can reduce enzyme activity damage during immobilization. However, pure photocrosslinked carboxymethyl chitosan microspheres suffer from poor mechanical properties and are easily broken, hindering the reusability of immobilized enzymes. Therefore, it is necessary to combine them with other reinforcing materials to improve the mechanical properties of the microspheres, thereby enhancing the durability of the immobilized enzymes.

[0005] Lignin is an important aromatic biopolymer abundant in nature. As a biopolymer with a rigid structure, the cross-linking network formed by lignin and biomass-based hydrogels can significantly improve the mechanical strength of the hydrogels. Li et al. (Li XH, You XY, Wang XL, et al. Advanced lignin-based hydrogels with superior stiffness, toughness, and sensing capabilities. Advanced Functional Materials. 2025.35(8)) prepared lignin / poly(N,N-dimethylacrylamide) (PDMA) and sodium alginate / calcium ion (SA / Ca) hydrogels using a solvent exchange method. 2+A dual-network hydrogel was prepared, and it was found that the prepared DL / S0.1Ca0.5 hydrogel had a multi-level energy dissipation structure, exhibiting high stiffness and toughness, with a tensile stress of 3.7 MPa, a tensile strain of 1100%, and a tensile modulus of 8.7 MPa. Gu et al. (Gu, Y., Xu, C., Wang, Y. et al. Compressible, anti-fatigue, extreme environment adaptable, and biocompatible supramolecular organohydrogel enabled by lignosulfonate triggered noncovalent network. Nat Commun, 2025, 16, 160) added lignosulfonate nanoparticles to chitosan / gelatin hydrogel, which resulted in a hydrogel with high compressive strength (54 MPa) and toughness (3.54 MJ / m). 3 It is 100 times and 70 times higher than pure chitosan / gelatin hydrogel, and also has excellent self-healing and fatigue resistance properties.

[0006] Furthermore, lignin, formed by the dehydrogenation polymerization of three main phenylpropane units, is hydrophobic. Studies have also found that lignin, as a hydrophobic support, significantly enhances the catalytic activity of lipases. Based on the catalytic mechanism and interfacial activation of lipases, lipases immobilized on hydrophobic supports exhibit higher activity. This is because the surface properties of the hydrophobic support allow the hydrophobic oligopeptides covering the active sites of the lipase to adsorb onto the surface and open through hydrophobic interactions. This not only effectively immobilizes and stabilizes the lipase on the hydrophobic support but also maintains the lipase in a stable open state through the interaction between the active site and the support surface, thus improving its catalytic efficiency. Immobilizing lipases through interfacial adsorption between the lipase and the hydrophobic support is also a common strategy for preparing immobilized lipases. Neto et al. (Girão Neto, CAC, Prasilde, ICM Enzymatic synthesis of citronellyl butyrate by lipase B from Candida antarctica immobilized on magnetic cashew apple bagasse lignin. Process Biochemistry, 2023, 131, 244-255.) immobilized citronellal butyrate by combining lignin with magnetic nanoparticles. The results showed that lignin can promote lipase activity. The immobilized lipase achieved a 97% yield of citronellal butyrate at 50°C, compared to only 55% for the free enzyme. Furthermore, the immobilized lipase retained approximately 95% of its catalytic activity after 10 cycles. Therefore, introducing lignin into a carboxymethyl chitosan-based support for lipase immobilization helps improve the mechanical strength of the hydrogel and also activates the lipase.

[0007] Using lignin combined with natural polymer materials for enzyme immobilization, although combining material properties, results in the lignin component and the polymer matrix being mainly bound by weak physical forces. Under repeated use or harsh reaction environments, there are problems of structural loosening and enzyme leakage. Summary of the Invention

[0008] To address the shortcomings and deficiencies of existing technologies, the primary objective of this invention is to provide a method for preparing immobilized lipases based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0009] This invention proposes to first modify carboxymethyl chitosan by methacrylation to impart photocrosslinking properties, and simultaneously modify lignin by allylation to impart photocrosslinking properties, and then prepare lignin nanoparticles by acid precipitation self-assembly; then, the modified carboxymethyl chitosan and the modified lignin nanoparticles are combined with lipase to prepare immobilized lipase.

[0010] Specifically, this invention utilizes EDC / NHS (EDC being 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and NHS being N-hydroxysuccinimide) and photocrosslinking to prepare immobilized enzymes. First, double bonds are introduced by methacrylylation modification of carboxymethyl chitosan and propylene modification of lignin. Then, a mixture of modified lignin particle dispersion, modified carboxymethyl chitosan, photoinitiator, and lipase is used as the aqueous phase. Next, EDC / NHS crosslinked modified carboxymethyl chitosan and lipase are added, and the aqueous phase is added to the oil phase to form a water-in-oil emulsion, simultaneously crosslinking into microspheres. Finally, ultraviolet light crosslinking is used to further achieve microsphere preparation and enzyme immobilization. The double bonds introduced by the modified lignin can undergo photocrosslinking with the double bonds of the modified carboxymethyl chitosan under ultraviolet light to form strong covalent bonds. At the same time, the presence of lignin particles enhances the carboxymethyl chitosan hydrogel network, and its hydrophobic benzene rings are beneficial to stimulating the catalytic activity of lipase. The EDC / NHS crosslinking agent can form crosslinks between the carboxyl and amino groups on the modified carboxymethyl chitosan, as well as between carboxymethyl chitosan and lipase, thereby reducing leakage of the final immobilized enzyme during use and improving recycling performance.

[0011] Another objective of this invention is to provide an immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres prepared by the above-described method, which has the characteristics of high enzyme loading capacity and catalytic activity, as well as good mechanical properties.

[0012] Another object of the present invention is to provide the application of the above-mentioned immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0013] To achieve this objective, the present invention adopts the following technical solution:

[0014] In a first aspect, the present invention provides a method for preparing immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres, comprising the following steps:

[0015] (1) Dissolve lignin in an alkaline solution, add allyl glycidyl ether to react and obtain propylene-modified lignin; then obtain propylene-modified lignin particles by acid precipitation;

[0016] (2) Dissolve carboxymethyl chitosan in deionized water, add methacrylic anhydride for double bond grafting modification, adjust pH during reaction, dialyze, and dry to obtain methacrylated carboxymethyl chitosan.

[0017] (3) A aqueous solution was obtained by mixing methacrylated carboxymethyl chitosan, propylene-modified lignin nanoparticle dispersion, lipase, photoinitiator and water; EDC / NHS crosslinking agent was added to the aqueous solution, and then it was emulsified with an oil phase containing surfactant to obtain W / O emulsion, which was crosslinked into spheres at room temperature.

[0018] (4) The product emulsion after the cross-linking reaction in step (3) is photocrosslinked, allowed to stand and separate into layers, the lower layer is removed, washed and filtered to obtain the immobilized lipase based on the carboxymethyl chitosan / lignin particle composite microspheres.

[0019] Preferably, the lignin in step (1) is at least one of alkali lignin and enzymatically hydrolyzed lignin; most preferably, it is alkali lignin.

[0020] Preferably, the alkaline solution in step (1) is at least one of sodium hydroxide solution and potassium hydroxide solution, and the concentration of the alkaline solution is 1.0 to 4.0 mol / L; the concentration of lignin in the alkaline solution is 20 to 60 wt%; more preferably, the concentration of the alkaline solution is 2.0 to 4.0 mol / L; and the concentration of lignin in the alkaline solution is 20 to 50 wt%.

[0021] Preferably, the mass ratio of lignin to allyl glycidyl ether in step (1) is 1:(1.0 to 2.0); more preferably, the mass ratio of lignin to allyl glycidyl ether is 1:(1.0 to 1.4).

[0022] Preferably, the reaction temperature in step (1) is 50–70 °C and the reaction time is 8–12 h; more preferably, the reaction temperature is 60 °C and the reaction time is 10 h.

[0023] Preferably, the acid precipitation in step (1) refers to dialysis in deionized water at room temperature using a 1000 Da dialysis bag until the pH of the dialysis solution is 5-6.

[0024] Preferably, the carboxymethyl chitosan in step (2) accounts for 0.5-2% of the mass of deionized water, more preferably 1%.

[0025] Preferably, the mass ratio of carboxymethyl chitosan to methacrylic anhydride in step (2) is 1:(1-6).

[0026] Preferably, the double bond grafting modification in step (2) is performed in an ice bath for 24–36 hours. The temperature of the ice bath is 0–5°C.

[0027] Preferably, in step (2), the pH is adjusted using a 0.5–2.0 mol / L sodium hydroxide aqueous solution.

[0028] Preferably, the pH range adjusted in step (2) is 8 to 9.

[0029] Preferably, the dialysis in step (2) refers to dialysis for 5 days at room temperature using an 8000-10000 Da dialysis bag.

[0030] Preferably, the drying in step (2) is freeze drying.

[0031] Preferably, the mass of the methacrylamide carboxymethyl chitosan in step (3) accounts for 1.0 to 5.0 wt% of the aqueous solution, and more preferably 1.0 to 3.0 wt%.

[0032] Preferably, the concentration of the propylene-modified lignin nanoparticle dispersion in step (3) is 1.0 to 2.0 wt%; more preferably 1.0 wt%.

[0033] Preferably, the mass of the propylene-modified lignin nanoparticle dispersion in step (3) accounts for 50 to 90 wt% of the aqueous solution.

[0034] Preferably, the lipase in step (3) is at least one of Candida pleuropsis lipase, Aspergillus niger lipase and Candida lipase.

[0035] Preferably, the amount of lipase used in step (3) is 50-500 mg / g methacrylamide carboxymethyl chitosan; more preferably, the amount of lipase used is 100-300 mg / g methacrylamide carboxymethyl chitosan.

[0036] Preferably, the photoinitiator in step (3) is I2959; the amount of the photoinitiator is 0.1 to 2.0 wt% of the aqueous solution; more preferably, the amount of the photoinitiator is 0.1 to 0.5 wt% of the aqueous solution.

[0037] Preferably, the solvent of the aqueous phase solution in step (3) is PBS buffer; the pH of the PBS buffer is 7.0 to 8.0.

[0038] Preferably, in the surface-active oil phase of step (3), the surfactant content is 0.5 to 5.0 wt%; more preferably, the surfactant content is 3.0 to 5.0 wt%.

[0039] Preferably, the oil phase in step (3) is at least one of liquid paraffin, ethyl acetate, dodecane, soybean oil, and isopropyl myristate; more preferably, it is at least one of liquid paraffin and soybean oil.

[0040] Preferably, in the surface-active oil phase of step (3), the surfactant is at least one of Span-80, Span-60, sucrose ester, Tween-80, sodium dodecyl sulfate, and sodium dodecylbenzene sulfonate; more preferably, it is at least one of Span-80 and Tween-80.

[0041] Preferably, the mass ratio of the aqueous solution and the oil phase containing the surfactant in step (3) is 1:2 to 1:10; more preferably, the mass ratio of the aqueous solution and the oil phase is 1:2 to 1:4.

[0042] Preferably, the emulsification speed in step (3) is 100-800 rpm; more preferably, the emulsification speed is 500-600 rpm.

[0043] Preferably, in step (3), the amount of EDC added is 0.5 to 1.0 wt% of the aqueous solution, and the amount of NHS is 0.2 to 1.0 mg / mg EDC; more preferably, the amount of EDC added is 1.0 wt% of the aqueous solution, and the amount of NHS is 0.5 to 0.8 mg / mg EDC.

[0044] Preferably, the crosslinking reaction time in step (3) is 30 to 90 min; more preferably 50 to 60 min.

[0045] Preferably, the room temperature in step (3) is 15 to 30°C.

[0046] Preferably, the photocrosslinking in step (4) is ultraviolet light irradiation crosslinking, the ultraviolet light wavelength is 365nm and the power is 180~220W; the photocrosslinking time is 5~30 min; more preferably, the photocrosslinking time is 5~10 min.

[0047] Preferably, the settling time in step (4) is 5 to 30 minutes; more preferably 15 minutes.

[0048] Preferably, the washing and filtration in step (4) are as follows: wash 3 to 6 times with a mixture of water and petroleum ether, wherein the volume ratio of water to petroleum ether is 1:1 to 5:1, and then transfer the composite microsphere immobilized lipase to a Buchner funnel for vacuum filtration for 10 to 30 minutes.

[0049] Secondly, the present invention provides an immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres prepared by the above preparation method.

[0050] The immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres prepared in this invention has a particle size of 400–800 μm.

[0051] Thirdly, the present invention provides the application of the above-mentioned immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres as a biocatalyst.

[0052] Preferably, the immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres is used as a biocatalyst in food, pharmaceutical preparation, and detergents.

[0053] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0054] 1. This invention crosslinks lipase, modified carboxymethyl chitosan, and modified lignin to form composite microspheres, while simultaneously embedding the lipase in situ within the composite microspheres. The resulting composite microspheres have a high enzyme loading capacity. Using photocrosslinking and EDC / NHS crosslinking instead of traditional glutaraldehyde crosslinking to prepare immobilized lipase reduces damage to enzyme activity during immobilization. Furthermore, the lipase is firmly bound to the carrier, exhibits good stability, and is not easily detached from the microspheres.

[0055] 2. The microspheres prepared by this invention have excellent mechanical properties. By grafting double bonds onto lignin and introducing double bonds into carboxymethyl chitosan through amidation, both can undergo free radical reactions under ultraviolet light. Furthermore, the carboxymethyl chitosan forms a double-crosslinked network structure through EDC / NHS crosslinking, enhancing the mechanical properties of the composite microspheres and improving the reusability of the immobilized lipase microspheres.

[0056] 3. The enzyme-carrying composite microspheres prepared by this invention have good catalytic performance. The hydrophobicity of the lignin particles helps to open the "lid" covering the active center of the lipase, which has a positive impact on improving the catalytic performance of the lipase. Attached Figure Description

[0057] Figure 1 The infrared spectra of carboxymethyl chitosan and methacryloxylated carboxymethyl chitosan prepared in Example 1 are shown.

[0058] Figure 2 The images show the 1H NMR spectra of carboxymethyl chitosan and methacryloxylated carboxymethyl chitosan prepared in Example 1.

[0059] Figure 3 The infrared spectra of alkali lignin and propylene-modified alkali lignin prepared in Example 1 are shown.

[0060] Figure 4 This is an optical microscope image of the immobilized lipase on the photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres prepared in Example 1.

[0061] Figure 5The image shows a laser confocal image of the immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres prepared in Example 1 (left image shows green fluorescence of lipase, right image shows blue fluorescence of lignin).

[0062] Figure 6 The mechanical properties and enzyme activity of the immobilized lipases obtained in Examples 1-7 and Comparative Examples 1-3 are shown.

[0063] Figure 7 The image shows the sphericity of the immobilized lipase in the photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres prepared in Example 1 and the immobilized lipases in Comparative Examples 1-3.

[0064] Figure 8 This is a diagram showing the enzyme leakage of the immobilized lipase from photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres prepared in Example 1 and the immobilized lipase from pure photocrosslinked carboxymethyl chitosan microspheres in Comparative Example 1. Detailed Implementation

[0065] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0066] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. All raw materials and reagents used, unless otherwise specified, are commercially available conventional products.

[0067] Example 1

[0068] (1) Preparation of propylene-modified lignin: 2.0 g of alkali lignin was dissolved in 2.0 mol / L sodium hydroxide solution to prepare 10 g 20 wt% alkali lignin solution; 2.08 mL of allyl glycidyl ether was added dropwise to the alkali lignin solution under stirring and reacted at 60 °C for 10 h; the solution after reaction was poured into a dialysis bag (1000 Da) and dialyzed in deionized water to remove unreacted reactants. Dialysis was continued until the pH of the dialysate was 5-6 to obtain a dispersion of propylene-modified lignin nanoparticles.

[0069] (2) Preparation of modified carboxymethyl chitosan: 1.0 g of carboxymethyl chitosan was dissolved in 100 mL of deionized water, and 2.5 mL of methacrylic anhydride was added dropwise. The reaction was carried out under ice bath conditions for 24 h. During this period, the pH was adjusted to 8-9 with 1 mol / L sodium hydroxide aqueous solution. The solution after the reaction was poured into a dialysis bag (8000-10000 Da) and dialyzed for 5 days. The solution was then freeze-dried to obtain methacrylated carboxymethyl chitosan.

[0070] (3) Preparation of W / O emulsion: 5 g of modified lignin nanoparticle dispersion (1.0 wt%) obtained in step (1), 0.3 g of methacrylamide carboxymethyl chitosan obtained in step (2), 0.09 g of Candida lipolytica lipase, 0.01 g of photoinitiator I2959 and PBS buffer (pH 8.0) were mixed to prepare 10 g of aqueous solution; 0.1 g of EDC and 0.05 g of NHS were added to the aqueous solution, and then the aqueous solution was added to 20 g of liquid paraffin containing 5 wt% Span-80. The mixture was emulsified and crosslinked at 500 rpm for 60 min by mechanical stirring to obtain crosslinked W / O solution.

[0071] (4) Preparation of immobilized lipase: The cross-linked W / O solution obtained in step (3) was placed under a UV lamp (wavelength 365nm, power 200W) for 5 min of photocrosslinking. After the reaction was completed, it was allowed to stand for 15 min to separate into layers, and the upper oil phase was poured off. The mixture was washed three times with 200 ml of a mixture containing 100 ml of deionized water and 100 ml of petroleum ether, and then transferred to a Buchner funnel for vacuum filtration for 20 min to obtain immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0072] Example 2

[0073] (1) Preparation of propylene-modified lignin: 2.0 g of enzymatically hydrolyzed lignin was dissolved in 1.0 mol / L potassium hydroxide solution to prepare 10 g 20 wt% enzymatically hydrolyzed lignin solution; 2.91 mL of allyl glycidyl ether was added dropwise to the enzymatically hydrolyzed lignin solution under stirring and reacted at 60 °C for 10 h; the solution after reaction was poured into a dialysis bag (1000 Da) and dialyzed in deionized water to remove unreacted reactants. Dialysis was continued until the pH of the dialysate was 5-6 to obtain a dispersion of propylene-modified lignin nanoparticles.

[0074] (2) Preparation of modified carboxymethyl chitosan: 1.0 g of carboxymethyl chitosan was dissolved in 100 mL of deionized water, and 1.0 mL of methacrylic anhydride was added dropwise. The reaction was carried out under ice bath conditions for 24 h. During this period, the pH was adjusted to 8-9 with 1 mol / L sodium hydroxide aqueous solution. The solution after the reaction was poured into a dialysis bag (8000-10000 Da) and dialyzed for 5 days. The solution was then freeze-dried to obtain methacrylated carboxymethyl chitosan.

[0075] (3) Preparation of W / O emulsion: 6 g of modified lignin nanoparticle dispersion (1.0 wt%) obtained in step (1), 0.2 g of methacrylated carboxymethyl chitosan obtained in step (2), 0.06 g of Aspergillus niger lipase, 0.02 g of photoinitiator I2959 and PBS buffer (pH 8.0) were mixed to prepare 10 g of aqueous solution; 0.05 g of EDC and 0.05 g of NHS were added to the aqueous solution, and then the aqueous solution was added to 30 g of ethyl acetate containing 3.5 wt% Span-60. The mixture was emulsified at 600 rpm for 50 min by mechanical stirring to obtain cross-linked W / O solution.

[0076] (4) Preparation of immobilized lipase: The cross-linked W / O solution obtained in step (3) was placed under a UV lamp (wavelength 365nm, power 200W) for 10 min of photocrosslinking. After the reaction was completed, it was allowed to stand for 15 min to separate into layers, and the upper oil phase was poured off. The mixture was washed three times with 200 ml of a mixture containing 150 ml of deionized water and 50 ml of petroleum ether, and then transferred to a Buchner funnel for vacuum filtration for 30 min to obtain immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0077] Example 3

[0078] (1) Preparation of modified lignin: 3.0 g of enzymatically hydrolyzed lignin was dissolved in 3.0 mol / L potassium hydroxide solution to prepare 10 g 30 wt% enzymatically hydrolyzed lignin solution; 3.43 mL of allyl glycidyl ether was added dropwise to the enzymatically hydrolyzed lignin solution under stirring and reacted at 60 °C for 10 h; the solution after reaction was poured into a dialysis bag (1000 Da) and dialyzed in deionized water to remove unreacted reactants. Dialysis was continued until the pH of the dialysate was 5-6 to obtain a dispersion of propylene-modified lignin nanoparticles.

[0079] (2) Preparation of modified carboxymethyl chitosan: 1.0 g of carboxymethyl chitosan was dissolved in 100 mL of deionized water, and 5.0 mL of methacrylic anhydride was added dropwise. The reaction was carried out under ice bath conditions for 24 h. During the reaction, the pH was adjusted to 8-9 with 1 mol / L sodium hydroxide aqueous solution. The solution after the reaction was poured into a dialysis bag (8000-10000 Da) and dialyzed for 5 days. The solution was then freeze-dried to obtain methacrylated carboxymethyl chitosan.

[0080] (3) Preparation of W / O emulsion: 7 g of modified lignin nanoparticle dispersion (1.0 wt%) obtained in step (1), 0.3 g of methacrylamide carboxymethyl chitosan obtained in step (2), 0.09 g of Candida lipase, 0.05 g of photoinitiator I2959 and PBS buffer (pH 8.0) were used to prepare 10 g of aqueous solution; 0.075 g of EDC and 0.0375 g of NHS were added to the aqueous solution, and then the aqueous solution was added to 20 g of ethyl acetate containing 5.0 wt% sodium dodecylbenzene sulfate. The mixture was emulsified at 500 rpm for 60 min by mechanical stirring to obtain cross-linked W / O solution.

[0081] (4) Preparation of immobilized lipase: The cross-linked W / O solution obtained in step (3) was placed under a UV lamp (wavelength 365nm, power 200W) for 5 min of photocrosslinking. After the reaction was completed, it was allowed to stand for 15 min to separate into layers, and the upper oil phase was poured off. The mixture was washed three times with 200 ml of a mixture containing 100 ml of deionized water and 100 ml of petroleum ether, and then transferred to a Buchner funnel for vacuum filtration for 10 min to obtain immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0082] Example 4

[0083] (1) Preparation of propylene-modified lignin nanoparticles: 3.5 g of alkali lignin was dissolved in 3.0 mol / L sodium hydroxide solution to prepare 10 g 35 wt% alkali lignin solution; 4.37 mL of allyl glycidyl ether was added dropwise to the alkali lignin solution under stirring and reacted at 60 °C for 10 h; the solution after reaction was poured into a dialysis bag (1000 Da) and dialyzed in deionized water to remove unreacted reactants. Dialysis was continued until the pH of the dialysate was 5-6 to obtain a dispersion of propylene-modified lignin nanoparticles.

[0084] (2) Preparation of modified carboxymethyl chitosan: 1.0 g of carboxymethyl chitosan was dissolved in 100 mL of deionized water, and 2.5 mL of methacrylic anhydride was added dropwise. The reaction was carried out under ice bath conditions for 24 h. During the reaction, the pH was adjusted to 8-9 with 1 mol / L sodium hydroxide aqueous solution. The solution after the reaction was poured into a dialysis bag (8000-10000 Da) and dialyzed for 5 days. The solution was then freeze-dried to obtain methacrylated carboxymethyl chitosan.

[0085] (3) Preparation of W / O emulsion: 8 g of modified lignin nanoparticle dispersion (1.0 wt%) obtained in step (1), 0.3 g of methacrylamide carboxymethyl chitosan obtained in step (2), 0.09 g of Aspergillus niger lipase, 0.03 g of photoinitiator I2959 and PBS buffer (pH 8.0) were used to prepare 10 g of aqueous solution; 0.1 g of EDC and 0.05 g of NHS were added to the aqueous solution, and then the aqueous solution was added to 30 g of liquid paraffin containing 5.0 wt% sodium dodecylbenzene sulfate. The mixture was emulsified at 500 rpm for 50 min by mechanical stirring to obtain cross-linked W / O solution.

[0086] (4) Preparation of immobilized lipase: The cross-linked W / O solution obtained in step (3) was placed under a UV lamp (wavelength 365nm, power 200W) for 5 min of photocrosslinking. After the reaction was completed, it was allowed to stand for 15 min to separate into layers, and the upper oil phase was poured off. The mixture was washed three times with 200 ml of a mixture containing 100 ml of deionized water and 100 ml of petroleum ether, and then transferred to a Buchner funnel for vacuum filtration for 10 min to obtain immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0087] Example 5

[0088] (1) Preparation of modified lignin: 4.0 g of alkali lignin was dissolved in 4.0 mol / L sodium hydroxide solution to prepare 10 g 40 wt% alkali lignin solution; 5.41 mL of allyl glycidyl ether was added dropwise to the alkali lignin solution under stirring and reacted at 60℃ for 10 h; the solution after reaction was poured into a dialysis bag (1000 Da) and dialyzed in deionized water to remove unreacted reactants. Dialysis was continued until the pH of the dialysate was 5-6 to obtain a dispersion of propylene-modified lignin nanoparticles.

[0089] (2) Preparation of modified carboxymethyl chitosan: 1.0 g of carboxymethyl chitosan was dissolved in 100 mL of deionized water, and 4.0 mL of methacrylic anhydride was added dropwise. The reaction was carried out under ice bath conditions for 24 h. During the reaction, the pH was adjusted to 8-9 with 1 mol / L sodium hydroxide aqueous solution. The solution after the reaction was poured into a dialysis bag (8000-10000 Da) and dialyzed for 5 days. The solution was then freeze-dried to obtain methacrylated carboxymethyl chitosan.

[0090] (3) Preparation of W / O emulsion: 9 g of modified lignin nanoparticle dispersion (1.0 wt%) obtained in step (1), 0.3 g of methacrylamide carboxymethyl chitosan obtained in step (2), 0.08 g of Candida lipase, 0.05 g of photoinitiator I2959 and PBS buffer (pH 8.0) were mixed to prepare 10 g of aqueous solution; 0.1 g of EDC and 0.075 g of NHS were added to the aqueous solution, and then the aqueous solution was added to 20 g of liquid paraffin containing 5 wt% Span-80. The mixture was emulsified at 500 rpm for 60 min by mechanical stirring to obtain cross-linked W / O solution.

[0091] (4) Preparation of immobilized lipase: The cross-linked W / O solution obtained in step (3) was placed under a UV lamp (wavelength 365nm, power 200W) for 5 min of photocrosslinking. After the reaction was completed, it was allowed to stand for 15 min to separate into layers, and the upper oil phase was poured off. The mixture was washed three times with 200 ml of a mixture containing 100 ml of deionized water and 100 ml of petroleum ether, and then transferred to a Buchner funnel for vacuum filtration for 10 min to obtain immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0092] Example 6

[0093] (1) Preparation of modified lignin: 4.5 g of enzymatically hydrolyzed lignin was dissolved in 3.0 mol / L sodium hydroxide solution to prepare 10 g 45 wt% enzymatically hydrolyzed lignin solution; 6.32 mL of allyl glycidyl ether was added dropwise to the enzymatically hydrolyzed lignin solution under stirring and reacted at 60 °C for 10 h; the solution after reaction was poured into a dialysis bag (1000 Da) and dialyzed in deionized water to remove unreacted reactants. Dialysis was continued until the pH of the dialysate was 5-6 to obtain a dispersion of propylene-modified lignin nanoparticles.

[0094] (2) Preparation of modified carboxymethyl chitosan: 1.0 g of carboxymethyl chitosan was dissolved in 100 mL of deionized water, and 4.0 mL of methacrylic anhydride was added dropwise. The reaction was carried out under ice bath conditions for 24 h. During the reaction, the pH was adjusted to 8-9 with 1 mol / L sodium hydroxide aqueous solution. The solution after the reaction was poured into a dialysis bag (8000-10000 Da) and dialyzed for 5 days. The solution was then freeze-dried to obtain methacrylated carboxymethyl chitosan.

[0095] (3) Preparation of W / O emulsion: 5 g of modified lignin nanoparticle dispersion (1.0 wt%) obtained in step (1), 0.2 g of methacrylamide carboxymethyl chitosan obtained in step (2), 0.06 g of Candida lipase, 0.03 g of photoinitiator I2959 and PBS buffer (pH 8.0) were used to prepare 10 g of aqueous solution; 0.075 g of EDC and 0.075 g of NHS were added to the aqueous solution, and then the aqueous solution was added to 20 g of liquid paraffin containing 5 wt% sodium dodecylbenzene sulfate. The mixture was emulsified at 500 rpm for 50 min by mechanical stirring to obtain cross-linked W / O solution.

[0096] (4) Preparation of immobilized lipase: The cross-linked W / O solution obtained in step (3) was placed under a UV lamp (wavelength 365nm, power 200W) for 10 min of photocrosslinking. After the reaction was completed, it was allowed to stand for 15 min to separate into layers, and the upper oil phase was poured off. The mixture of 200 ml containing 100 ml of deionized water and 100 ml of petroleum ether was poured in and washed three times. Then it was transferred to a Buchner funnel and vacuum filtered for 10 min to obtain immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0097] Example 7

[0098] (1) Preparation of modified lignin: 5.0 g of alkali lignin was dissolved in 4.0 mol / L sodium hydroxide solution to prepare 10 g 50 wt% alkali lignin solution; 7.28 mL of allyl glycidyl ether was added dropwise to the alkali lignin solution under stirring and reacted at 60℃ for 10 h; the solution after reaction was poured into a dialysis bag (1000 Da) and dialyzed in deionized water to remove unreacted reactants. Dialysis was continued until the pH of the dialysate was 5-6 to obtain a dispersion of propylene-modified lignin nanoparticles.

[0099] (2) Preparation of modified carboxymethyl chitosan: 1.0 g of carboxymethyl chitosan was dissolved in 100 mL of deionized water, and 2.5 mL of methacrylic anhydride was added dropwise. The reaction was carried out under ice bath conditions for 24 h. During the reaction, the pH was adjusted to 8-9 with 1 mol / L sodium hydroxide aqueous solution. The solution after the reaction was poured into a dialysis bag (8000-10000 Da) and dialyzed for 5 days. The solution was then freeze-dried to obtain methacrylated carboxymethyl chitosan.

[0100] (3) Preparation of W / O emulsion: 5 g of modified lignin nanoparticle dispersion (1.0 wt%) obtained in step (1), 0.3 g of methacrylamide carboxymethyl chitosan obtained in step (2), 0.07 g of Candida lipase, 0.01 g of photoinitiator I2959 and PBS buffer (pH 8.0) were mixed to prepare 10 g of aqueous solution; 0.1 g of EDC and 0.05 g of NHS were added to the aqueous solution, and then the aqueous solution was added to 30 g of liquid paraffin containing 3.0 wt% sucrose ester. The mixture was emulsified at 600 rpm for 60 min by mechanical stirring to obtain cross-linked W / O solution.

[0101] (4) Preparation of immobilized lipase: The cross-linked W / O solution obtained in step (3) was placed under a UV lamp (wavelength 365nm, power 200W) for 5 min of photocrosslinking. After the reaction was completed, it was allowed to stand for 15 min to separate into layers, and the upper oil phase was poured off. The mixture was washed three times with 200 ml of a mixture containing 100 ml of deionized water and 100 ml of petroleum ether, and then transferred to a Buchner funnel for vacuum filtration for 10 min to obtain immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0102] Comparative Example 1

[0103] To compare the effects of lignin on the hydrophobic properties, mechanical properties, and enzyme activity of immobilized enzymes, an immobilized lipase based on pure methacryloyl carboxymethyl chitosan microspheres was prepared without the addition of lignin, referring to Example 1.

[0104] (1) Dissolve 1.0 g of carboxymethyl chitosan in 100 mL of deionized water, add 2.5 mL of methacrylic anhydride dropwise, and react under ice bath conditions for 24 h. During this time, adjust the pH to 8-9 with 1 mol / L sodium hydroxide aqueous solution. Pour the solution after reaction into a dialysis bag (8000-10000 Da) and dialyze for 5 days. Freeze dry to obtain methacrylated carboxymethyl chitosan.

[0105] (2) Prepare a 10 g aqueous solution by mixing 0.3 g of methacrylated carboxymethyl chitosan, 0.09 g of Candida pleuropsis lipase, 0.01 g of photoinitiator I2959 with PBS buffer (pH 8.0); add 0.1 g of EDC and 0.05 g of NHS to the aqueous solution, and then add the aqueous solution to 20 g of liquid paraffin containing 5 wt% Span-80. Emulsify and crosslink at 500 rpm for 60 min using mechanical stirring to obtain a crosslinked W / O solution.

[0106] (3) Place the cross-linked W / O solution under a UV lamp (wavelength 365nm, power 200W) for 5 min of light cross-linking. After the reaction is complete, let it stand for 15 min to allow it to separate into layers, pour off the upper oil phase, and wash it three times with a mixture of 200 ml containing 100 ml deionized water and 100 ml petroleum ether. Then transfer it to a Buchner funnel and vacuum filter for 20 min to obtain immobilized lipase based on photocross-linked carboxymethyl chitosan microspheres.

[0107] Comparative Example 2

[0108] To compare the effects of modified lignin on the mechanical properties and enzyme activity of immobilized enzymes, an immobilized lipase based on carboxymethyl chitosan / alkali lignin composite microspheres was prepared without modification of alkali lignin, referring to Example 1.

[0109] (1) Dissolve 1.0 g of carboxymethyl chitosan in 100 mL of deionized water, add 2.5 mL of methacrylic anhydride dropwise, and react under ice bath conditions for 24 h. During this time, adjust the pH to 8-9 with 1 mol / L sodium hydroxide aqueous solution. Pour the solution after reaction into a dialysis bag (8000-10000 Da) and dialyze for 5 days. Freeze dry to obtain methacrylated carboxymethyl chitosan.

[0110] (2) Prepare a 10 g aqueous solution by mixing 0.3 g of methacrylated carboxymethyl chitosan, 5 g of alkali lignin dispersion (1.0 wt%), 0.09 g of Candida pleuropsis lipase, 0.01 g of photoinitiator I2959 with PBS buffer (pH 8.0); add 0.1 g of EDC and 0.05 g of NHS to the aqueous solution, and then add the aqueous solution to 20 g of liquid paraffin containing 5 wt% Span-80. Emulsify and crosslink at 500 rpm for 60 min using mechanical stirring to obtain a crosslinked W / O solution.

[0111] (3) Place the cross-linked W / O solution under a UV lamp (wavelength 365nm, power 200W) for 5 min of light cross-linking. After the reaction is complete, let it stand for 15 min to allow it to separate into layers, pour off the upper oil phase, and wash it three times with a mixture of 200 ml containing 100 ml deionized water and 100 ml petroleum ether. Then transfer it to a Buchner funnel and vacuum filter for 20 min to obtain the immobilized lipase based on photocross-linked carboxymethyl chitosan / lignin composite microspheres.

[0112] Comparative Example 3

[0113] To compare the effects of photocrosslinking on the mechanical properties and activity of immobilized enzymes, immobilized enzyme microspheres were prepared using only EDC / NHS crosslinking (without UV photocrosslinking and curing). Immobilized lipase based on carboxymethyl chitosan / lignin nanoparticle composite microspheres was prepared in accordance with Example 1.

[0114] (1) Dissolve 2.0 g of alkali lignin in 2.0 mol / L sodium hydroxide solution to prepare 10 g 20 wt% alkali lignin solution; add 2.08 mL of allyl glycidyl ether dropwise to the alkali lignin solution under stirring and react at 60 °C for 10 h; pour the solution after reaction into a dialysis bag (1000 Da) and dialyze it in deionized water to remove unreacted reactants. Dialyze until the pH of the dialysate is 5-6 to obtain a dispersion of propylene-modified lignin nanoparticles.

[0115] (2) Dissolve 1.0 g of carboxymethyl chitosan in 100 mL of deionized water, add 2.5 mL of methacrylic anhydride dropwise, and react under ice bath conditions for 24 h. During this time, adjust the pH to 8-9 with 1 mol / L sodium hydroxide aqueous solution. Pour the solution after reaction into a dialysis bag (8000-10000 Da) and dialyze for 5 days. Freeze dry to obtain methacrylated carboxymethyl chitosan.

[0116] (3) Prepare a 10g aqueous solution by mixing 5g of modified lignin nanoparticle dispersion (1.0wt%), 0.3g of methacrylamide carboxymethyl chitosan, 0.09g of Candida pleuropsis lipase, 0.01g of photoinitiator I2959 with PBS buffer (pH 8.0); add 0.1g of EDC and 0.05g of NHS to the aqueous solution, and then add the aqueous solution to 20g of liquid paraffin containing 5wt% Span-80. Emulsify and crosslink at 500rpm for 60min using mechanical stirring to obtain a crosslinked W / O solution. After the reaction is completed, let it stand for 15min to separate the layers, pour off the upper oil phase, and wash it three times with a mixture of 200ml of 100ml deionized water and 100ml petroleum ether. Then transfer it to a Buchner funnel and vacuum filter for 20min to obtain immobilized lipase based on EDC / NHS crosslinked carboxymethyl chitosan / lignin particle composite microspheres.

[0117] Comparative Example 4

[0118] To compare the effect of the timing of EDC / NHS addition on the mechanical properties and enzyme activity of the immobilized enzyme, an immobilized lipase based on carboxymethyl chitosan / lignin nanoparticle composite microspheres was prepared by emulsifying into a W / O emulsion and then adding the EDC / NHS crosslinking agent, referring to Example 1.

[0119] (1) Dissolve 2.0 g of alkali lignin in 2.0 mol / L sodium hydroxide solution to prepare 10 g 20 wt% alkali lignin solution; add 2.08 mL of allyl glycidyl ether dropwise to the alkali lignin solution under stirring and react at 60 °C for 10 h; pour the solution after reaction into a dialysis bag (1000 Da) and dialyze it in deionized water to remove unreacted reactants. Dialyze until the pH of the dialysate is 5-6 to obtain a dispersion of propylene-modified lignin nanoparticles.

[0120] (2) Dissolve 1.0 g of carboxymethyl chitosan in 100 mL of deionized water, add 2.5 mL of methacrylic anhydride dropwise, and react under ice bath conditions for 24 h. During this time, adjust the pH to 8-9 with 1 mol / L sodium hydroxide aqueous solution. Pour the solution after reaction into a dialysis bag (8000-10000 Da) and dialyze for 5 days. Freeze dry to obtain methacrylated carboxymethyl chitosan.

[0121] (3) Prepare a 10g aqueous solution by mixing 5g of modified lignin nanoparticle dispersion (1.0wt%), 0.3g of methacrylamide carboxymethyl chitosan, 0.09g of Candida pleuropsis lipase, 0.01g of photoinitiator I2959 with PBS buffer (pH 8.0); add the aqueous phase to 20g of liquid paraffin containing 5 wt% Span-80 and emulsify at 500rpm for 30min with mechanical stirring; add 2.0g of crosslinking agent solution containing 50mg EDC and 25mg NHS dropwise to the obtained W / O solution and stir crosslinking at room temperature for 60min to obtain a crosslinked W / O solution.

[0122] (4) Place the cross-linked W / O solution under a UV lamp (wavelength 365nm, power 200W) for 5 min of light cross-linking. After the reaction is complete, let it stand for 15 min to allow it to separate into layers, pour off the upper oil phase, and wash it three times with a mixture of 200 ml containing 100 ml deionized water and 100 ml petroleum ether. Then transfer it to a Buchner funnel and vacuum filter for 20 min to obtain the immobilized lipase based on photocross-linked carboxymethyl chitosan / lignin composite microspheres.

[0123] Example effect description:

[0124] The effects are mainly illustrated using Example 1 as an example.

[0125] Methacrylamide-modified carboxymethyl chitosan with photocrosslinking properties was prepared by amidation reaction of methacrylic anhydride with the amino groups on carboxymethyl chitosan. Infrared spectrum. Figure 1 The infrared spectrum shows that at 3400 cm⁻¹ -1 The broad absorption peak at 2900 cm⁻¹ is an overlap of the hydroxyl stretching vibration peak and the imine stretching vibration peak. -1The absorption peak at 1427 cm⁻¹ is attributed to the methylene group. -1 and 1624 cm -1 The absorption peak at 1710 cm⁻¹ is due to the bending vibration of the amino group and the stretching vibration of the carbonyl group on carboxymethyl chitosan. -1 The double bond stretching vibration band at 1427 cm⁻¹, and the discovery of amino groups at 1427 cm⁻¹ -1 The weakening of the bending vibration band at the point due to the grafting of methacrylic anhydride indicates the successful synthesis of methacrylated carboxymethyl chitosan.

[0126] Methacrylamide-modified carboxymethyl chitosan was characterized by nuclear magnetic resonance. Figure 2 The figure shows the 1H NMR spectra of carboxymethyl chitosan and methacryloxycarboxymethyl chitosan. The results are shown in the figure. Characteristic chemical shifts were observed at 5.6, 6.1 and 1.7 ppm, which are proton signals of C(CH3)=CH2-, indicating that the double bond was successfully attached to the main chain of carboxymethyl chitosan.

[0127] Figure 3 The infrared spectrum of propylene-modified lignin is shown in the following figures. Figure 3 The results show that propylene-based alkali lignin is present at 1641 cm⁻¹. -1 It exhibits a strong C=C vibrational absorption peak, especially at 1100 cm⁻¹. -1 and 952cm -1 There are strong vibrational absorption peaks at this point, which are attributed to the vibrational absorption peaks of COC and CH (-C=CH2) in the allylated alkali lignin molecule, respectively. These peaks appear at 3445 cm⁻¹ in both allylated alkali lignin and alkali lignin. -1 Both exhibit strong absorption peaks, which are the vibrational absorption peaks of the phenolic hydroxyl groups in lignin and the alcoholic hydroxyl groups in the allylated alkali lignin molecules, respectively. Comparing the infrared spectra of the two demonstrates that the double bond was successfully incorporated into the alkali lignin molecule.

[0128] from Figure 4 An optical microscope image of the immobilized lipase based on carboxymethyl chitosan / lignin-based cross-linked microspheres prepared in Example 1 shows that the immobilized enzyme is a regular spherical shape with a smooth surface.

[0129] To investigate the distribution of lignin and lipase in the carboxymethyl chitosan / lignin double crosslinked composite microspheres prepared in Example 1, and whether the lipase was successfully loaded, lignin and lipase were labeled with fluorescein isothiocyanate (FITC) to prepare composite microspheres, and laser confocal microscopy was performed at excitation wavelengths of 405 and 490 nm, respectively. Figure 5 This is a laser confocal microscope image of the immobilized lipase composite microspheres. It can be seen that the blue fluorescent lignin and the green fluorescent lipase are distributed on the composite microspheres, indicating that the lipase has been successfully loaded onto the composite microspheres.

[0130] The force required for the immobilized lipase microspheres prepared in the examples and comparative examples to break apart was determined using a texture analyzer. A higher force indicates better mechanical properties of the microspheres. Texture analyzer test parameters: A 5 mm diameter test probe was used, with the probe initially positioned 5 cm from the origin of the platform. The probe was lowered at a rate of 2 mm / min. Pressure was applied upon contact with the microsphere, and the test was stopped when the deformation reached 60%. The recorded pressure at the stopping point was the breaking force (N) of the microsphere. Each sample was measured at least five times, and the average value was taken.

[0131] The catalytic performance of the immobilized lipases prepared in the examples and comparative examples was determined using the p-nitrophenyl laurate hydrolysis method. The concentration of the product p-nitrophenol was calculated by measuring the absorbance at 410 nm using a microplate reader. Specific steps: ① Dissolve p-nitrophenyl laurate in isopropanol to prepare a substrate solution with a concentration of 3.33 mg / mL. ② Disperse 10 mg of the immobilized enzyme in 900 μL of 50 mmol / L Tris-HCl buffer (pH 8.0), preheat at 40 °C, add 50 μL of the substrate solution, mix thoroughly, and react in a 40 °C water bath for 20 min. ③ After the reaction, add 200 μL of the liquid to each well of a 96-well plate, and measure the absorbance at 410 nm using a microplate reader. Each sample was measured in triplicate. One enzyme activity unit (U) represents the amount of enzyme required to catalyze the hydrolysis to produce 1 μmol of p-nitrophenol per minute. The enzyme activity was calculated using the following formula.

[0132]

[0133] In the formula, E is the enzyme activity, U / mg; C is the p-NP concentration, μmol / L; V is the reaction volume, L; T is the reaction time, min; and M is the enzyme dosage, mg.

[0134] Carboxymethyl chitosan methacrylate modification involves an amidation reaction between methacrylic anhydride and the amino groups on carboxymethyl chitosan, resulting in a reduced amino content. EDC / NHS crosslinking, on the other hand, involves crosslinking the amino and carboxyl groups on carboxymethyl chitosan. In Comparative Example 4, after emulsification to form a W / O solution, EDC / NHS was added. The crosslinking agent had to diffuse from the external oil phase into each tiny water droplet for crosslinking. Due to the reduced amino content after carboxymethyl chitosan modification and the fact that EDC / NHS is a water-soluble crosslinking agent, the crosslinking efficiency was relatively low, resulting in a low microsphere formation rate and poor immobilized enzyme spheroidization. Therefore, adding EDC / NHS crosslinking agent to the aqueous phase before emulsification can improve crosslinking efficiency, resulting in better immobilized enzyme spheroidization and more regular shapes.

[0135] Figure 6The mechanical properties and enzyme activity of the immobilized lipases obtained in Examples 1-7 and Comparative Examples 1-3 are shown. The higher breaking force and enzyme activity of Comparative Example 2 compared to Comparative Example 1 indicate that the addition of lignin, with its rigid polycyclic aromatic hydrocarbon structure and hydrophobic properties, not only improves the support strength but also enhances the catalytic activity of the lipase. The enzyme activities of Examples 1-7 were 1.54-1.90 U / mg, and the breaking force was 5.4-7.5 N. The enzyme activity and breaking force of Example 1 were both higher than those of Comparative Examples 1-3. The fact that the breaking force and enzyme activity of Example 1 were higher than those of Comparative Example 2 demonstrates that propylene modification of alkali lignin can improve the enzyme activity and mechanical properties of the immobilized enzyme. This is because after lignin is propylene-modified, the hydrophobicity of the modified lignin particles is conducive to improving lipase activity. Moreover, it can undergo free radical polymerization and crosslinking with methacryloxycarboxymethyl chitosan through photocrosslinking, further improving the degree of crosslinking of the carrier and enhancing the mechanical properties of the microspheres. The fact that the breaking force and enzyme activity of Example 1 are higher than those of Comparative Example 3 indicates that the introduction of double bonds through propylene-modification of alkali lignin can form another crosslinking network with methacryloxycarboxymethyl chitosan under ultraviolet light. This forms a double crosslinking network with the EDC / NHS crosslinked carboxymethyl chitosan hydrogel network, further increasing the degree of crosslinking of the carrier, improving mechanical properties, and making it less prone to leakage of the loaded lipase.

[0136] Twenty immobilized lipase particles from the photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres of Example 1 and 20 immobilized lipase particles from Comparative Examples 1-3 were randomly selected and placed in vials. 20 mL of deionized water and a magnetic stir bar were added. The mixture was stirred at 500 rpm for 6 hours at room temperature, and the number of intact microsphere particles was observed. Multiple measurements were taken, and the average value was used to calculate the microsphere integrity rate (%). Figure 7 The spheric integrity rates of the microspheres based on carboxymethyl chitosan / lignin double crosslinked composites in Example 1 and the immobilized lipases in Comparative Examples 1-3 are shown. The spheric integrity rate of the immobilized enzyme microspheres in Example 1 was 89%, which was higher than that in Comparative Examples 1-3. This demonstrates that the introduction of double bonds through propylene modification of lignin, combined with the photoinitiated free radical polymerization of methacryloxylated carboxymethyl chitosan, and the fact that the methacryloxylated carboxymethyl chitosan itself is crosslinked via EDC / NHS, further enhances the mechanical properties of the microspheres through the double crosslinked network.

[0137] To further investigate the lipase shedding of the two immobilized enzymes in Example 1 and Comparative Example 1, immobilized enzymes with the same enzyme content were dispersed in deionized water and stirred. The lipase concentration of the supernatant was measured, and the amount of lipase leakage was calculated.

[0138] Figure 8The enzyme leakage amounts of the immobilized lipase prepared in Example 1 and the immobilized lipase on pure photocrosslinked carboxymethyl chitosan microspheres in Comparative Example 1 are compared. Over time, the leakage amount of lipase on both types of immobilized enzyme microspheres showed a gradual increasing trend. However, the enzyme leakage amount in Example 1 was consistently lower than that in Comparative Example 1, indicating that incorporating propylene-modified lignin into the carrier can provide more immobilization sites for the lipase and reduce enzyme leakage. Simultaneously, the propylene-modified lignin and methacryloxylated carboxymethyl chitosan formed a double crosslinked network. This dense double crosslinked network structure can prevent lipase leakage, thereby enabling the immobilized enzyme to maintain better recyclability.

[0139] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres, characterized in that, Includes the following steps: (1) Dissolve lignin in an alkaline solution, add allyl glycidyl ether to react and obtain propylene-modified lignin; then obtain propylene-modified lignin particles by acid precipitation; (2) Dissolve carboxymethyl chitosan in deionized water, add methacrylic anhydride for double bond grafting modification, adjust pH during reaction, dialyze, and dry to obtain methacrylated carboxymethyl chitosan. (3) A aqueous solution was obtained by mixing methacrylated carboxymethyl chitosan, propylene-modified lignin nanoparticle dispersion, lipase, photoinitiator and water; EDC / NHS crosslinking agent was added to the aqueous solution, and then it was emulsified with an oil phase containing surfactant to obtain W / O emulsion, which was crosslinked into spheres at room temperature. (4) The product emulsion after the cross-linking reaction in step (3) is photocrosslinked, allowed to stand and separate into layers, the lower layer is removed, washed and filtered to obtain the immobilized lipase based on the carboxymethyl chitosan / lignin particle composite microspheres.

2. The preparation method according to claim 1, characterized in that, In step (3), the mass of the methacrylamide carboxymethyl chitosan in the aqueous solution is 1.0–5.0 wt%, more preferably 1.0–3.0 wt%. And / or, the concentration of the propylene-modified lignin nanoparticle dispersion in step (3) is 1.0–2.0 wt%; And / or, the mass of the propylene-modified lignin nanoparticle dispersion in step (3) accounts for 50-90 wt% of the aqueous solution; And / or, the amount of lipase used in step (3) is 50-500 mg / g methacrylamide carboxymethyl chitosan; more preferably, the amount of lipase used is 100-300 mg / g methacrylamide carboxymethyl chitosan. And / or, in step (3), the amount of EDC added is 0.5 to 1.0 wt% of the aqueous solution, and the amount of NHS is 0.2 to 1.0 mg / mg EDC; more preferably, the amount of EDC added is 1.0 wt% of the aqueous solution, and the amount of NHS is 0.5 to 0.8 mg / mg EDC; And / or, in the surface-active oil phase of step (3), the surfactant content is 0.5–5.0 wt%; more preferably, the surfactant content is 3.0–5.0 wt%. And / or, the mass ratio of the aqueous solution and the oil phase containing the surfactant in step (3) is 1:2 to 1:10; more preferably, the mass ratio of the aqueous solution and the oil phase is 1:2 to 1:

4.

3. The preparation method according to claim 1 or 2, characterized in that, The photocrosslinking in step (4) is ultraviolet light irradiation crosslinking, the ultraviolet light wavelength is 365nm, the power is 180~220W; the photocrosslinking time is 5~30 min; more preferably, the photocrosslinking time is 5~10 min; And / or, the crosslinking reaction time in step (3) is 30 to 90 min; more preferably 50 to 60 min; And / or, the room temperature in step (3) is 15 to 30°C.

4. The preparation method according to claim 1 or 2, characterized in that, In step (1), the mass ratio of lignin to allyl glycidyl ether is 1:(1.0-2.0); more preferably, the mass ratio of lignin to allyl glycidyl ether is 1:(1.0-1.4). And / or, the temperature of the reaction in step (1) is 50–70 °C and the reaction time is 8–12 h; And / or, the lignin in step (1) is at least one of alkali lignin and enzymatically hydrolyzed lignin.

5. The preparation method according to claim 1 or 2, characterized in that, The mass ratio of carboxymethyl chitosan to methacrylic anhydride in step (2) is 1:(1-6); And / or, the temperature for double bond grafting modification in step (2) is an ice bath, and the time is 24 to 36 h.

6. The preparation method according to claim 1 or 2, characterized in that, The lipase mentioned in step (3) is at least one of Candida pleuropsis lipase, Aspergillus niger lipase and Candida lipase; And / or, in step (3), the oil phase is at least one of liquid paraffin, ethyl acetate, dodecane, soybean oil, and isopropyl myristate; more preferably, it is at least one of liquid paraffin and soybean oil. And / or, in the surface-active oil phase of step (3), the surfactant is at least one of Span-80, Span-60, sucrose ester, Tween-80, sodium dodecyl sulfate and sodium dodecylbenzene sulfonate; more preferably at least one of Span-80 and Tween-80.

7. The preparation method according to claim 1 or 2, characterized in that, The alkaline solution in step (1) is at least one of sodium hydroxide solution and potassium hydroxide solution; And / or, the concentration of the alkaline solution in step (1) is 1.0 to 4.0 mol / L; more preferably, the concentration of the alkaline solution is 2.0 to 4.0 mol / L; And / or, the concentration of lignin in the alkaline solution in step (1) is 20–60 wt%; more preferably, the concentration of lignin in the alkaline solution is 20–50 wt%. And / or, the carboxymethyl chitosan in step (2) accounts for 0.5% to 2% of the mass of deionized water; And / or, in step (2), the pH is adjusted using a 0.5–2.0 mol / L sodium hydroxide aqueous solution; And / or, the pH range adjusted in step (2) is 8 to 9.

8. The preparation method according to claim 1 or 2, characterized in that, The photoinitiator in step (3) is I2959; And / or, the amount of photoinitiator used in step (3) is 0.1 to 2.0 wt% of the aqueous solution; more preferably, the amount of photoinitiator used is 0.1 to 0.5 wt% of the aqueous solution; And / or, the solvent of the aqueous phase solution in step (3) is PBS buffer; the pH of the PBS buffer is 7.0 to 8.0; And / or, the emulsification speed in step (3) is 100 to 800 rpm; more preferably, the emulsification speed is 500 to 600 rpm; And / or, the settling time for the stratification in step (4) is 5 to 30 minutes; And / or, the washing and filtration in step (4) are as follows: wash 3 to 6 times with a mixture of water and petroleum ether, wherein the volume ratio of water to petroleum ether is 1:1 to 5:1, and then transfer the composite microsphere immobilized lipase to a Buchner funnel for vacuum filtration for 10 to 30 minutes.

9. An immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the immobilized lipase based on photocrosslinked carboxymethyl chitosan / lignin particle composite microspheres as a biocatalyst according to claim 9.

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