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CN-122012420-A - Co-immobilized laccase-mediator system and preparation method and application thereof

CN122012420ACN 122012420 ACN122012420 ACN 122012420ACN-122012420-A

Abstract

The invention discloses a preparation method of a co-immobilized laccase-mediator system, which comprises the following steps of mixing and vibrating laccase solution and 2-methylimidazole solution, adding manganese chloride solution, vibrating and uniformly mixing, sequentially adding p-coumaric acid and 2, 2-biazal-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt ABTS (ABTS) to vibrate and react for 3-5h after the obtained mixed solution reacts for a preset time in a shaking table, adding polyethylene glycol PEG, centrifuging and washing to obtain the co-immobilized laccase-mediator system MnMOF@Lac5@M@PEG. Compared with free enzyme, the PEG modified laccase-mediator co-immobilized system improves the enzyme activity by more than 2 times, and shows excellent reusability, 100% degradation of alizarin red can be realized within 15min under the condition of 30 ℃, a green and efficient treatment technology is provided for dye degradation, and meanwhile, the immobilized laccase-mediator system has good application prospects in the fields of sewage treatment, bioremediation and the like.

Inventors

  • WANG BAOJUAN
  • YU JIAO
  • FANG ZEMIN
  • LIU SHENGLONG
  • LIU YIWEN
  • LI LINXI
  • ZHANG JIAYUAN
  • ZHANG YONG

Assignees

  • 安徽师范大学

Dates

Publication Date
20260512
Application Date
20251031

Claims (7)

  1. 1. A method for preparing a co-immobilized laccase-mediator system, comprising the steps of: (1) Uniformly mixing laccase solution and 2-methylimidazole solution, and oscillating on a vortex oscillator for 30-40s to obtain a mixed solution a; (2) Adding a manganese chloride solution into the mixed solution a obtained in the step (1), uniformly mixing, oscillating for 30-40s on a vortex oscillator, and then placing the mixed solution in a 30 ℃ horizontal shaking table for reaction for 30 min to obtain a mixed solution b; (3) Sequentially adding p-coumaric acid and ABTS with equal molar mass into the mixed solution b obtained in the step (2), uniformly mixing and stirring, reacting at the temperature of 30 ℃, adding polyethylene glycol PEG, and uniformly mixing and stirring to obtain a mixed solution C; (4) And (3) centrifuging the mixed solution C obtained in the step (3) in a centrifugal machine with the rotating speed of 10,000 rpm for 5-8 min, discarding supernatant to obtain brown precipitate, flushing the brown precipitate with deionized water for multiple times to obtain brown precipitate, and drying the brown precipitate in a 37 ℃ incubator for 10-12 h to obtain the immobilized laccase-mediator system MnMOF@Lac5@PEG@M.
  2. 2. The method of claim 1, wherein in step (2), the laccase is present in the mixed solution b at a mass concentration of 0.2-1.4 μg/mL, 2-methylimidazole is present at a molar concentration of 25-175 mM, and manganese chloride is present at a molar concentration of 50-350 mM.
  3. 3. The method of claim 1, wherein in step (3), the mixed solution c has a molar concentration of ABTS and p-coumaric acid of 0.5-12mm and a mass concentration of peg of 20 mg/mL.
  4. 4. The method of claim 1, wherein in step (3), the reaction time is 3-5h (extended range) at 30 ℃.
  5. 5. A co-immobilized laccase-mediator system prepared by the method of preparing a co-immobilized laccase-mediator system as defined in any one of claims 1 to 4.
  6. 6. Use of the co-immobilized laccase-mediator system according to any one of claims 1 to 5 in the field of dye degradation.
  7. 7. The use of co-immobilized laccase-mediator system according to claim 6 in the field of dye degradation, characterized in that alizarin red is uniformly dispersed in an immobilized lipase solution, the reaction mixture is subjected to an oscillating reaction, and after the reaction is finished, the content of alizarin red in the supernatant is determined by centrifugation.

Description

Co-immobilized laccase-mediator system and preparation method and application thereof Technical Field The invention belongs to the technical field of immobilized enzyme preparation, and particularly relates to a preparation method and application of a co-immobilized laccase-mediator system. Background Alizarin red (Alizarin Red S) is an anthraquinone dye widely used for dyeing biological tissues, calcium ion indicators and textiles such as wool, silk and the like, and is well known for its vivid color and specific chemical binding characteristics. However, alizarin red, which is a synthetic dye with potential environmental risks, is difficult to naturally degrade in the environment, has bioaccumulation property, and may cause mutability and ecotoxicity to aquatic organisms, so that residues thereof have become one of the concerns of water pollution. However, compared with the traditional physical adsorption method and chemical oxidation method, the method has the defects of limited efficiency, higher cost, possibility of generating toxic byproducts and the like, and the biodegradation method is considered to be an effective way for treating alizarin red pollution, and has the advantages of environmental friendliness, relatively simple operation, more thorough degradation and the like. Laccase (lacase) is a copper-containing polyphenol oxidase with the capability of catalyzing the oxidation of various aromatic compounds, and is widely applied to the fields of paper industry, textile decolorization, bioremediation, food processing and the like. Laccase enzymes can catalyze the oxidative degradation of dye molecules, particularly dyes containing phenolic or aromatic amine structures. Particularly, the laccase-mediator system formed in the presence of mediator molecules (such as small molecular compounds of ABTS, HBT and the like) can remarkably expand the substrate range of laccase, so that the laccase-mediator system can efficiently degrade dyes with more complex structures and higher oxidation potentials (such as anthraquinone dyes and triphenylmethane dyes), destroy chromophores of the dyes, and form degradation products with low chromaticity and even no color. However, the catalytic activity of the free laccase is often significantly affected by various environmental factors such as pH, temperature, inhibitors (such as halides) and the like, and the stability is poor in practical application, and secondly, the laccase is used as water-soluble protein, is difficult to effectively separate and recover from a complex dye wastewater system after the reaction is completed, so that the laccase cannot be recycled, the treatment cost is increased, and the application potential of the laccase in large-scale treatment of dye wastewater is limited. More importantly, the current research on the integral immobilization of laccase-mediator system is relatively few, and the research on how to immobilize laccase and mediator molecules on a carrier in a synergistic way is very necessary in the direction of intensive research, which has a key meaning for fully exerting the degradation potential of laccase-mediator system and solving the application bottleneck thereof. Therefore, aiming at laccase and a mediator system thereof, the laccase and the mediator system are immobilized on a water-insoluble carrier by an immobilization technology, so that the limitation of a free enzyme/system can be effectively overcome, the stability, reusability and operation convenience of the laccase and mediator system are obviously improved, and the efficiency of a dye degradation process is improved while the treatment cost is reduced. However, conventional support materials do not have good tunability and crystallinity, which may lead to low protein loading efficiency, instability and susceptibility of the enzyme to leaching, thereby affecting the activity of the immobilized enzyme. The Metal-organic frameworks (MOFs) are novel porous crystal hybrid materials composed of Metal ions and organic ligands through strong coordination bonds, have the advantages of adjustable pores, high specific surface area, good chemical and thermal stability and the like, and are expected to become novel excellent carriers of immobilized enzyme preparations. However, the surface charge and chemical properties of proteins or enzymes determine the ability of the immobilized enzymes to be encapsulated in MOFs, so that the enzyme activities of most immobilized enzymes are not high, and doping of different metal ions in MOFs is a way to effectively improve the activity and stability of immobilized enzymes through competitive coordination of the metal ions and organic ligands. Disclosure of Invention Aiming at the defects existing in the prior art, the invention aims to provide a novel laccase-mediator system, and a preparation method and application thereof, so as to solve the problems in the background art. In order to achieve the aim, the invention is realized by