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CN-121991941-A - Immobilized lipase based on photo-crosslinked carboxymethyl chitosan/lignin particle composite microsphere and preparation and application thereof

CN121991941ACN 121991941 ACN121991941 ACN 121991941ACN-121991941-A

Abstract

The invention discloses immobilized lipase based on photo-crosslinked carboxymethyl chitosan/lignin particle composite microspheres and preparation and application thereof. The invention firstly carries out methacryloyl modification on carboxymethyl chitosan and propenyl modification on lignin to introduce double bonds, then takes a mixed solution of modified lignin particle dispersion liquid, modified carboxymethyl chitosan, a photoinitiator and lipase as a water phase, then adds EDC/NHS crosslinking modified carboxymethyl chitosan and lipase, adds the water phase into an oil phase to form water-in-oil emulsion, and simultaneously crosslinks the water phase into microspheres, and finally adopts ultraviolet crosslinking to further realize microsphere preparation and enzyme immobilization. The immobilized lipase has the characteristics of high enzyme carrying capacity, high catalytic activity and good mechanical property, and can be widely applied to foods, medicines and detergents as a biocatalyst.

Inventors

  • PANG YUXIA
  • FANG WUYONG
  • LOU HONGMING
  • YANG DONGJIE
  • LI ZHIXIAN
  • QIU XUEQING

Assignees

  • 华南理工大学

Dates

Publication Date
20260508
Application Date
20260212

Claims (10)

  1. 1. The preparation method of the immobilized lipase based on the photo-crosslinked carboxymethyl chitosan/lignin particle composite microsphere is characterized by comprising the following steps: (1) Dissolving lignin in alkali solution, adding allyl glycidyl ether to react to obtain propenyl lignin, and obtaining propenyl modified lignin particles through acid precipitation; (2) Dissolving carboxymethyl chitosan in deionized water, adding methacrylic anhydride to carry out double bond grafting modification, regulating pH in the reaction process, dialyzing and drying to obtain methacryloyl carboxymethyl chitosan; (3) Mixing methacryloyl carboxymethyl chitosan, propenyl modified lignin nanoparticle dispersion liquid, lipase, a photoinitiator and water to obtain an aqueous phase solution, adding EDC/NHS cross-linking agent into the aqueous phase solution, mixing the aqueous phase solution with oil phase containing surfactant, emulsifying to obtain W/O emulsion, and performing cross-linking reaction at room temperature to obtain balls; (4) And (3) carrying out photocrosslinking on the product emulsion obtained after the crosslinking reaction in the step (3) is balled, standing and layering, taking the lower layer, washing and suction filtering to obtain the immobilized lipase based on photocrosslinked carboxymethyl chitosan/lignin particle composite microspheres.
  2. 2. The preparation method according to claim 1, wherein the mass of the methacryloyl carboxymethyl chitosan in the step (3) accounts for 1.0-5.0 wt% of the aqueous phase solution, and more preferably 1.0-3.0 wt%; and/or, the concentration of the acrylated modified lignin nanoparticle dispersion liquid in the step (3) is 1.0-2.0wt%; And/or, the mass of the propenyl modified lignin nanoparticle dispersion liquid in the step (3) accounts for 50-90wt% of the aqueous phase solution; And/or the lipase in the step (3) is used in an amount of 50-500 mg/g of methacryloyl carboxymethyl chitosan, and more preferably, the lipase is used in an amount of 100-300 mg/g of methacryloyl carboxymethyl chitosan; And/or, in the step (3), the EDC addition amount is 0.5-1.0 wt% of the aqueous phase solution, the NHS addition amount is 0.2-1.0 mg/mg EDC, and further preferably, the EDC addition amount is 1.0wt% of the aqueous phase solution, and the NHS addition amount is 0.5-0.8 mg/mg EDC; And/or, the surfactant content in the oil phase containing the surfactant in the step (3) is 0.5-5.0 wt%, and more preferably, the surfactant content is 3.0-5.0 wt%; And/or, the mass ratio of the aqueous phase solution to the oil phase containing the surfactant in the step (3) is 1:2-1:10, and further preferably, the mass ratio of the aqueous phase solution to the oil phase is 1:2-1:4.
  3. 3. The preparation method according to claim 1 or 2, wherein the photocrosslinking in the step (4) is ultraviolet irradiation crosslinking, the wavelength of ultraviolet light is 365nm, the power is 180-220 w, the photocrosslinking time is 5-30 min, and further preferably, the photocrosslinking time is 5-10 min; and/or, the time of the crosslinking reaction in the step (3) is 30-90 min, and more preferably 50-60 min; and/or, the room temperature in the step (3) is 15-30 ℃.
  4. 4. The preparation method according to claim 1 or 2, wherein the mass ratio of lignin to allyl glycidyl ether in the step (1) is 1 (1.0-2.0), and further preferably the mass ratio of lignin to allyl glycidyl ether is 1 (1.0-1.4); And/or the reaction temperature in the step (1) is 50-70 ℃ and the reaction time is 8-12 h; and/or, the lignin in the step (1) is at least one of alkali lignin and enzymatic lignin.
  5. 5. The preparation method according to claim 1 or 2, wherein the mass ratio of the carboxymethyl chitosan to the methacrylic anhydride in the step (2) is 1 (1-6); and/or, the temperature of the double bond grafting modification in the step (2) is ice bath, and the time is 24-36 h.
  6. 6. The method according to claim 1 or 2, wherein the lipase in step (3) is at least one of candida rugosa lipase, aspergillus niger lipase and candida lipase; And/or the oil phase in the step (3) is at least one of liquid paraffin, ethyl acetate, dodecane, soybean oil and isopropyl myristate, more preferably at least one of liquid paraffin and soybean oil; And/or the oil phase containing the surface activity in the step (3), wherein the surface active agent is at least one of span-80, span-60, sucrose ester, tween-80, sodium dodecyl sulfate and sodium dodecyl benzene sulfonate, and more preferably at least one of span-80 and tween-80.
  7. 7. The method according to claim 1 or 2, wherein the alkali solution in step (1) is at least one of sodium hydroxide solution and potassium hydroxide solution; and/or the concentration of the alkali solution in the step (1) is 1.0-4.0 mol/L, and further preferably, the concentration of the alkali solution is 2.0-4.0 mol/L; And/or the concentration of the lignin in the alkaline solution in the step (1) is 20-60wt%, and further preferably, the concentration of the lignin in the alkaline solution is 20-50wt%; And/or, the carboxymethyl chitosan in the step (2) accounts for 0.5-2% of the deionized water; And/or, in the step (2), 0.5-2.0 mol/L sodium hydroxide aqueous solution is adopted to adjust the pH; And/or, in the step (2), the pH value is adjusted to 8-9.
  8. 8. The method of claim 1 or 2, wherein the photoinitiator of step (3) is I2959; And/or the photoinitiator in the step (3) is 0.1-2.0wt% of the aqueous phase solution, and further preferably, the photoinitiator is 0.1-0.5wt% of the aqueous phase solution; and/or, the solvent of the aqueous phase solution in the step (3) is PBS buffer solution, wherein the pH value of the PBS buffer solution is 7.0-8.0; And/or the rotational speed of the emulsification in the step (3) is 100-800 rpm, and further preferably, the rotational speed of the emulsification is 500-600 rpm; and/or, standing and layering in the step (4) for 5-30 min; And/or, in the step (4), washing for 3-6 times by adding a mixed solution of water and petroleum ether, wherein the volume ratio of the water to the petroleum ether is 1:1-5:1, and transferring the immobilized lipase of the composite microsphere into a Buchner funnel for vacuum filtration for 10-30 min.
  9. 9. The immobilized lipase based on photo-crosslinked carboxymethyl chitosan/lignin particle composite microspheres prepared by the preparation method of any one of claims 1-8.
  10. 10. The use of an immobilized lipase based on photo-crosslinked carboxymethyl chitosan/lignin particle composite microspheres according to claim 9 as a biocatalyst.

Description

Immobilized lipase based on photo-crosslinked carboxymethyl chitosan/lignin particle composite microsphere and preparation and application thereof Technical Field The invention belongs to the technical field of immobilized lipase preparation, and particularly relates to immobilized lipase based on photo-crosslinked carboxymethyl chitosan/lignin particle composite microspheres, and preparation and application thereof. Background Lipase is an important biocatalyst, and is widely applied to industries such as food processing, medicines, fine chemical engineering and the like because of the characteristics of high selectivity, mild catalytic reaction conditions, no pollution and the like. However, the problems of poor stability, easy inactivation, difficult recycling and the like of the free enzyme make it difficult to realize large-scale industrial production. Overcoming the limitations of lipase application by enzyme immobilization technology is a hotspot in research in the field of enzyme engineering. The lipase is immobilized, so that the catalytic activity of the lipase is maintained, and the stability of the lipase is improved, and the lipase can be recovered and reused. The immobilized enzyme performance 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 of immobilized enzymes. Adejanildo da S Pereira et al entrap candida lipase in chitosan microspheres (Pereira, A.d.S.; Fraga, J.L.; Souza, C.P.L.; Torres, A.G.; Amaral, P.F.F. 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), to achieve an immobilization efficiency of 51%. The immobilized enzyme can be used for catalyzing and synthesizing the beta-sitosterol oleate with high conversion rate of more than 95 percent. 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.) uses cuttlebone modified chitosan as an enzyme immobilization carrier, and covalent cross-links with trypsin through a nontoxic genipin cross-linking agent, wherein the immobilized enzyme has an enzyme activity of 390.26U/g and shows remarkably improved pH value, thermal stability and storage stability. Although chitosan has good immobilization effect on different biological enzymes, the chitosan cannot be used for immobilizing lipase due to low solubility under physiological pH, and only a chitosan carrier can be prepared first and then the lipase is loaded by an adsorption method, so that the enzyme loading amount is low. Carboxymethyl chitosan is used as a derivative of chitosan, has good water solubility at pH 3-8, and can be used for immobilizing lipase by an in-situ embedding method (lipase and carboxymethyl chitosan are simultaneously dissolved in an aqueous solution, and lipase is simultaneously embedded when carboxymethyl chitosan is solidified and molded) so as to increase enzyme carrying quantity. In addition, the most common use of immobilized lipase by an in-situ embedding method is to use glutaraldehyde crosslinking agents, so that the carboxymethyl chitosan can be crosslinked to form a carrier, and the lipase can be covalently bound on the carrier, but glutaraldehyde is toxic and has great damage to the activity of the enzyme. There is a need to develop new bio-enzyme friendly cross-linking means for preparing immobilized lipases to reduce 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 methacrylate chitin-based photocrosslinked polymer for immobilized lipase, the activity recovery rate of which is 76.0%, indicating that photocrosslinking can reduce the damage to enzyme activity in the immobilization process. However, the pure photo-crosslinking carboxymethyl chitosan microsphere has the defects of poor mechanical property and easy breakage, and is unfavorable for the repeated use of immobilized enzyme. The immobilized enzyme needs to be compounded with other reinforcing materials to improve the mechanical property of the microsphere and further improve the durability of the immobilized enzyme. Lignin is an important aromatic biopolymer which is abundantly present in nature, and lignin, as a biopolymer with a rigid structure, can significantly improve the mechanical strength of hydrogels by forming a crosslinked network with biomass-based hydrogels. Li et al （Li X H, You X Y, Wang X L, et al. Advanced lignin-based hydrogels with superior stiffness, toughness, and sensing capabilities. Advanced Functional Materials.2025.35(8)） prepare