CN113862252B - Method for immobilizing laccase by titanate nanotube composite material - Google Patents

Method for immobilizing laccase by titanate nanotube composite material Download PDF

Info

Publication number
CN113862252B
CN113862252B CN202111262130.9A CN202111262130A CN113862252B CN 113862252 B CN113862252 B CN 113862252B CN 202111262130 A CN202111262130 A CN 202111262130A CN 113862252 B CN113862252 B CN 113862252B
Authority
CN
China
Prior art keywords
laccase
titanate
composite material
nanotube composite
nanotubes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111262130.9A
Other languages
Chinese (zh)
Other versions
CN113862252A (en
Inventor
张慧君
王俊峰
潘建泽
张鹏鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Yinong Biotechnology Co ltd
Original Assignee
Jiangsu Yinong Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Yinong Biotechnology Co ltd filed Critical Jiangsu Yinong Biotechnology Co ltd
Priority to CN202111262130.9A priority Critical patent/CN113862252B/en
Publication of CN113862252A publication Critical patent/CN113862252A/en
Application granted granted Critical
Publication of CN113862252B publication Critical patent/CN113862252B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14Enzymes or microbial cells immobilised on or in an inorganic carrier
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/0203Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of metals not provided for in B01J20/04
    • B01J20/0211Compounds of Ti, Zr, Hf
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/348Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
    • C12N9/0055Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061Laccase (1.10.3.2)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y110/00Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002Laccase (1.10.3.2)
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00Nature of the contaminant
    • C02F2101/30Organic compounds
    • C02F2101/34Organic compounds containing oxygen
    • C02F2101/345Phenols
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00Nature of the contaminant
    • C02F2101/30Organic compounds
    • C02F2101/36Organic compounds containing halogen

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Water Supply & Treatment (AREA)
  • Environmental & Geological Engineering (AREA)
  • Hydrology & Water Resources (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Inorganic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

The invention discloses a method for immobilizing laccase by using a titanate nanotube composite material, which comprises the steps of firstly preparing titanate nanotubes, then modifying the titanate nanotubes by using mixed molten salt, and finally grafting chitosan on the modified titanate nanotubes, wherein the obtained material can realize the high-efficiency degradation of 2, 4-dichlorophenol after being used for immobilizing the laccase, the degradation rate is close to 99 percent at most, and the material has good acid-base stability, good thermal stability and good application prospect.

Description

Method for immobilizing laccase by titanate nanotube composite material
Technical Field
The invention belongs to the technical field of laccase immobilization, and particularly relates to a method for immobilizing laccase by using a titanate nanotube composite material.
Background
Laccase is a copper-containing polyphenol oxidase, and is widely present in various organisms. Because of the better stability and specificity of the laccase, the laccase has wide application in the fields of wastewater treatment, biosensor construction, food industry and the like. However, due to the constitution of laccase proteins, free laccases are very sensitive to the external environment and are very prone to inactivation. Accordingly, laccase immobilization technology has been developed. In the laccase immobilization technology, an immobilization carrier plays a key role, and common laccase immobilization carriers comprise inorganic materials, natural polymer materials, synthetic polymer materials and the like.
Among inorganic materials, graphene and carbon nanotubes are widely used, and patent documents CN105647902A, CN107988196A, CN108018281A, CN109912048A, etc. all report related technical solutions. However, due to the defects of hydrophobicity and easy aggregation of the carbon nanotubes, the carbon nanotubes usually need to be treated under special conditions such as strong acid, and potential chemical residues can affect the activity of laccase. In contrast, the carbonate nanotubes have the potential to be excellent carriers of laccase due to the advantages of hydrophilicity, easy modification and the like. Based on the above, the invention provides a method for immobilizing laccase by using a titanate nanotube composite material.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for immobilizing laccase by using a titanate nanotube composite material.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for immobilizing laccase by using titanate nanotube composite materials comprises the following steps:
(1) preparing a laccase immobilized carrier;
(2) immobilizing laccase;
wherein, the preparation of the laccase immobilized carrier comprises the following steps:
s1, preparing titanate nanotubes;
s2, modification of titanate nanotubes: mixing the titanate nanotubes obtained in the step S1 with mixed salt, then carrying out high-temperature reaction in a tubular furnace, and stirring and washing until the pH value is neutral after the reaction is finished, so as to obtain modified titanate nanotubes;
s3, preparing a titanate nanotube composite material: dispersing the modified titanate nanotubes obtained in the step S2 in an acetic acid solution, and uniformly dispersing by ultrasonic; and then adding the mixed solution a containing chitosan, stirring and mixing, and centrifuging, washing and drying after stirring and mixing to obtain the titanate nanotube composite material.
Preferably, the method for preparing titanate nanotubes in step S1 is as follows: adding titanium dioxide nano powder into a sodium hydroxide solution, performing ultrasonic dispersion uniformly, performing hydrothermal reaction at 120-150 ℃ for 1-5 days, washing with deionized water after the reaction is finished, soaking the product in hydrochloric acid, and washing with deionized water again after the soaking is finished to obtain the titanate nanotube.
Preferably, the mixed salt in step S2 is a mixture of sodium nitrate and sodium carbonate or a mixture of potassium nitrate and potassium hydroxide.
Preferably, the mass ratio of the sodium nitrate to the sodium carbonate is 1:1, and the mass ratio of the potassium nitrate to the potassium hydroxide is 1: 1.
Preferably, the mass ratio of the mixed salt to the titanate nanotubes in step S2 is 10-50: 1.
Preferably, the high-temperature reaction temperature in the step S2 is 340-350 ℃, the high-temperature reaction time is 1-4 h, and the reaction temperature rise rate is 12 ℃/min.
Preferably, the chitosan-containing mixed solution a in step S3 is prepared by mixing 100mg of chitosan and 100mL of 0.1mol/L acetic acid solution.
Preferably, the amount of the modified titanate nanotubes is 0.4-0.8 g, and the volume ratio of the acetic acid solution to the mixed solution a is 1: 1.
preferably, in the step S3, the stirring and mixing temperature is room temperature, the time is 12-36 h, and the stirring speed is 350 r/min.
Preferably, the laccase immobilization in step (2) comprises the steps of: at room temperature, taking 2mL of 1% wt glutaraldehyde solution, adding 0.4g titanate nanotube composite material, oscillating at the rotating speed of 180r/min, and then carrying out centrifugal separation to obtain a carrier; adding the obtained carrier into 1mL of acetic acid-sodium acetate buffer solution containing laccase with the pH value of 5.5, oscillating for 4h at room temperature, and performing suction filtration and washing to obtain immobilized laccase;
wherein the concentration of the laccase is 0.8 g/L.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, titanate nanotubes are prepared, mixed molten salt is used for modifying the titanate nanotubes, chitosan is grafted on the modified titanate nanotubes, the obtained material can realize efficient degradation of 2, 4-dichlorophenol after being used for immobilizing laccase, the highest degradation can reach about 99%, and the immobilized laccase has good acid-base stability and good thermal stability.
(2) The method creatively uses the molten salt method commonly used for carbon nanotube modification for modification of titanate nanotubes, can selectively corrode the titanate nanotubes, and can increase the roughness of the titanate surface by ionizing metal molten salt and violently colliding with the titanate surface at high temperature to form irregular surface defects; therefore, more active adsorption sites are provided for the subsequent self-assembly of the chitosan, and the fixation amount of the laccase is further improved.
(3) According to the invention, chitosan is grafted on the surface of the modified titanate nanotube, rich charges of the chitosan are utilized to be self-assembled and combined on the surface of the modified titanate nanotube, the modified titanate nanotube has rich binding sites, and the condition that the stability of the immobilized laccase is poor due to easy separation of the chitosan through direct self-assembly is avoided.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly illustrated, the present invention will be further explained in detail with reference to the embodiments.
It is particularly emphasized that laccases are commercially available from Aldrich; the titanate is rutile titanium dioxide, available from aladdin corporation. Other materials or reagents of the present invention are also commercially available, unless otherwise specified.
Example 1
A method for immobilizing laccase by using titanate nanotube composite materials comprises the following steps:
(1) preparing a laccase immobilized carrier:
(2) at room temperature, taking 2mL of 1% wt glutaraldehyde solution, adding 0.4g laccase immobilized carrier, oscillating at the rotating speed of 180r/min, and then carrying out centrifugal separation to obtain a cross-linked carrier; adding the obtained cross-linked carrier into 1mL of laccase-containing acetic acid-sodium acetate buffer solution with pH of 5.5, oscillating for 4h at room temperature, performing suction filtration, and washing to obtain immobilized laccase;
wherein the concentration of the laccase is 0.8 g/L;
wherein, the preparation of the laccase immobilized carrier comprises the following steps:
s1, preparing titanate nanotubes: adding 3g of titanium dioxide nano powder into 100mL of 10mol/L sodium hydroxide solution, performing ultrasonic dispersion uniformly, performing hydrothermal reaction at 125 ℃ for 2.5d, washing with deionized water after the reaction is finished, soaking the product in hydrochloric acid for 12h, and cleaning with deionized water after the soaking is finished to obtain a titanate nanotube;
s2, modification of titanate nanotubes: mixing 2g of titanate nanotube obtained in the step S1 with 30g of mixed salt (formed by mixing sodium nitrate and sodium carbonate in a mass ratio of 1: 1), then reacting for 2 hours in a tubular furnace at 340 ℃, wherein the reaction temperature rise rate is 12 ℃/min, and stirring and washing until the pH value is neutral after the reaction is finished, so as to obtain the modified titanate nanotube;
s3, preparing a titanate nanotube composite material: dispersing 0.5g of the modified titanate nanotube obtained in the step S2 in 100mL of 0.1mol/L acetic acid solution, and uniformly dispersing by ultrasonic; and then adding 100mL of chitosan-containing mixed solution a (formed by mixing 100mg of chitosan and 100mL of 0.1mol/L acetic acid solution), stirring and mixing for 12h at room temperature, wherein the stirring speed is 350r/min, and after stirring and mixing, centrifuging, washing and drying to obtain the titanate nanotube composite material.
Example 2
A method for immobilizing laccase by using titanate nanotube composite materials comprises the following steps:
(1) preparation of laccase immobilized carrier:
(2) at room temperature, taking 2mL of 1% wt glutaraldehyde solution, adding 0.4g laccase immobilized carrier, oscillating at the rotating speed of 180r/min, and then carrying out centrifugal separation to obtain a cross-linked carrier; adding the obtained cross-linked carrier into 1mL of laccase-containing acetic acid-sodium acetate buffer solution with pH of 5.5, oscillating for 4h at room temperature, performing suction filtration, and washing to obtain immobilized laccase;
wherein the concentration of the laccase is 0.8 g/L;
wherein, the preparation of the laccase immobilized carrier comprises the following steps:
s1, preparing titanate nanotubes: adding 2.5g of titanium dioxide nano powder into 100mL of 10mol/L sodium hydroxide solution, performing ultrasonic dispersion uniformly, performing hydrothermal reaction at 130 ℃ for 3d, washing with deionized water after the reaction is finished, soaking the product in hydrochloric acid for 16h, and cleaning with deionized water after the soaking is finished to obtain a titanate nano tube;
s2, modification of titanate nanotubes: mixing 2.5g of titanate nanotube obtained in the step S1 with 30g of mixed salt (formed by mixing sodium nitrate and sodium carbonate in a mass ratio of 1: 1), then reacting in a tube furnace at 342 ℃ for 2.5h, wherein the reaction temperature rise rate is 12 ℃/min, and stirring and washing after the reaction is finished until the pH value is neutral to obtain the modified titanate nanotube;
s3, preparing a titanate nanotube composite material: dispersing 0.5g of the modified titanate nanotube obtained in the step S2 in 100mL of 0.1mol/L acetic acid solution, and uniformly dispersing by ultrasonic; and then adding 100mL of chitosan-containing mixed solution a (formed by mixing 100mg of chitosan and 100mL of 0.1mol/L acetic acid solution), stirring and mixing at room temperature for 16h, wherein the stirring speed is 350r/min, and after stirring and mixing are finished, centrifuging, washing and drying to obtain the titanate nanotube composite material.
Example 3
A method for immobilizing laccase by using titanate nanotube composite materials comprises the following steps:
(1) preparation of laccase immobilized carrier:
(2) at room temperature, taking 2mL of 1% wt glutaraldehyde solution, adding 0.4g laccase immobilized carrier, oscillating at the rotating speed of 180r/min, and then carrying out centrifugal separation to obtain a cross-linked carrier; adding the obtained cross-linked carrier into 1mL of laccase-containing acetic acid-sodium acetate buffer solution with pH of 5.5, oscillating for 4h at room temperature, performing suction filtration, and washing to obtain immobilized laccase;
wherein the concentration of the laccase is 0.8 g/L;
wherein, the preparation of the laccase immobilized carrier comprises the following steps:
s1, preparing titanate nanotubes: adding 2g of titanium dioxide nano powder into 100mL of 10mol/L sodium hydroxide solution, performing ultrasonic dispersion uniformly, performing hydrothermal reaction at 130 ℃ for 3d, washing with deionized water after the reaction is finished, soaking the product in hydrochloric acid for 16h, and cleaning with deionized water after the soaking is finished to obtain a titanate nanotube;
s2, modification of titanate nanotubes: mixing 3g of titanate nanotube obtained in the step S1 with 30g of mixed salt (formed by mixing sodium nitrate and sodium carbonate in a mass ratio of 1: 1), then reacting in a tubular furnace at 345 ℃ for 3h, wherein the reaction temperature rise rate is 12 ℃/min, and stirring and washing after the reaction is finished until the pH value is neutral to obtain the modified titanate nanotube;
s3, preparing a titanate nanotube composite material: dispersing 0.5g of the modified titanate nanotube obtained in the step S2 in 100mL of 0.1mol/L acetic acid solution, and uniformly dispersing by ultrasonic; and then adding 100mL of chitosan-containing mixed solution a (formed by mixing 100mg of chitosan and 100mL of 0.1mol/L acetic acid solution), stirring and mixing for 24 hours at room temperature, wherein the stirring speed is 350r/min, and after stirring and mixing, centrifuging, washing and drying to obtain the titanate nanotube composite material.
Comparative example 1
A method for immobilizing laccase by using titanate nanotube composite materials comprises the following steps:
(1) preparation of laccase immobilized carrier:
(2) at room temperature, taking 2mL of 1% wt glutaraldehyde solution, adding 0.4g laccase immobilized carrier, oscillating at the rotating speed of 180r/min, and then carrying out centrifugal separation to obtain a cross-linked carrier; adding the obtained cross-linked carrier into 1mL of laccase-containing acetic acid-sodium acetate buffer solution with pH of 5.5, oscillating for 4h at room temperature, performing suction filtration, and washing to obtain immobilized laccase;
wherein the concentration of the laccase is 0.8 g/L;
wherein, the preparation of the laccase immobilized carrier comprises the following steps:
s1, preparing titanate nanotubes: adding 2.5g of titanium dioxide nano powder into 100mL of 10mol/L sodium hydroxide solution, performing ultrasonic dispersion uniformly, performing hydrothermal reaction at 130 ℃ for 3d, washing with deionized water after the reaction is finished, soaking the product in hydrochloric acid for 16h, and cleaning with deionized water after the soaking is finished to obtain titanate nanotubes;
s2, preparing a titanate nanotube composite material: dispersing 0.5g of titanate nanotubes obtained in the step S1 in 100mL of 0.1mol/L acetic acid solution, and uniformly dispersing by ultrasonic; and then adding 100mL of chitosan-containing mixed solution a (formed by mixing 100mg of chitosan and 100mL of 0.1mol/L acetic acid solution), stirring and mixing for 16h at room temperature, wherein the stirring speed is 350r/min, and after stirring and mixing, centrifuging, washing and drying to obtain the titanate nanotube composite material.
The immobilized laccase prepared in examples 1 to 3 and comparative example 1 is subjected to enzyme activity test, immobilized laccase amount test, degradation adsorption performance test and thermal stability test. The specific test method is as follows:
(1) enzyme Activity assay
Adding 2mL of 1mg/mL free laccase or 2 mg of obtained immobilized laccase into 1mmol/L ABTS solution, carrying out oscillation reaction at 25 ℃ for 15min, placing in an ice bath to terminate the reaction, carrying out centrifugal separation, and measuring the absorbance change of the supernatant at 420nm by using an ultraviolet-visible spectrophotometer to calculate the corresponding enzyme activity.
In this example, the model of the uv-vis spectrophotometer is new century T6.
(2) Immobilized laccase quantity test
a) Drawing a protein standard curve: adding 1000 mug/mL of standard protein solution and distilled water according to different amounts, then sequentially adding 5mL of Coomassie brilliant blue G-250 reagent, and after dyeing for 5min, measuring the absorbance of the reagent, and drawing a protein standard curve by taking the protein content as the abscissa and the absorbance as the ordinate.
b) Determination of the amount of immobilized laccase: testing the laccase amount by adopting a Coomassie brilliant blue method; wherein, the enzyme amount calculation formula is as follows:
Q(%)=(C o -C)V/W*100;
in the above formula, Q represents the enzyme adsorption amount, C o Concentration of laccase in solution before immobilization; c is the concentration of laccase in the immobilized solution, V is the total volume of the immobilized solution, and W is the mass of the carrier. In the present case, C o =0.8 g/L,V=1mL=1*10 -3 L, W =0.4 g. The specific test and calculation results are shown in table 1.
TABLE 1 immobilized laccase quantity test results
Amount of immobilized laccase/(mg/g)
Example 1 184.1
Example 2 185.6
Example 3 184.9
Comparative example 1 152.4
As can be seen from Table 1, the immobilization amount of laccase in each example of the invention exceeds 180 mg/g, and the immobilization efficiency is high.
(3) Degradation adsorption of 2, 4-dichlorophenol
Mixing 0.15g of immobilized laccase with 15mL of 10 mg/L2, 4-dichlorophenol, reacting for 5h at room temperature, centrifuging, diluting 10mL of supernatant, and sequentially adding 1.25mL of 0.5mol/L NH 3 ·H 2 O, 0.5mL of 2 percent 4-aminoantipyrine, 0.5mL of 80g/L potassium ferricyanide solution, stirring and reacting for 15min, measuring the absorbance change at the wavelength of 510nm by an ultraviolet spectrophotometer, and calculating to obtain the immobilized laccase p-2, 4-dichlorobenzeneThe adsorption degradation rate of phenol.
In this example, HPLC is Shimadzu LC-20A.
TABLE 22, 4-dichlorophenol degradation adsorption test results
Degradation adsorption rate/%
Example 1 97.8
Example 2 98.9
Example 3 98.2
Comparative example 1 84.27
From the above table, it can be seen that the immobilized laccase prepared in the examples of the present application has a good adsorption degradation rate for 2, 4-dichlorophenol.
(4) Stability test
And (3) testing thermal stability: the immobilized laccase (100 mg) prepared in examples 1-3 and comparative example 1 and the free enzyme (100 mg) are stored for 1.5h at different temperatures, and the residual activity of the enzyme is calculated according to the enzyme activity test method.
Test results show that the residual activity of the immobilized enzyme obtained in the embodiments 1 to 3 is still maintained to be more than 77 percent after the immobilized enzyme is stored for 2 hours at the temperature of 60 ℃; the remaining activity of the enzyme in comparative example 1 was 68%; whereas the residual activity of the free laccase was only 58.6%.
Testing acid-base stability: and (3) storing the immobilized laccase (100 mg) prepared in the examples 1-3 and the free enzyme (100 mg) prepared in the comparative example 1 in 20mL of PBS buffer solution with the pH value of 3-8 for 12h at room temperature, then adding 2mL of 1.00mmol/L ABTS solution for mixing, stirring for 1h at room temperature, stopping the reaction in an ice bath, taking supernate, and calculating the residual activity of the enzyme according to the enzyme activity test method. The enzyme residual activity of the immobilized laccase obtained in the examples 1-3 is higher in the range of pH value 3-8 compared with that of the immobilized laccase obtained in the comparative example 1, and the enzyme residual activity of the immobilized laccase obtained in the examples 1-3 is more than 80% in the range of pH value 3-6.
The above description describes a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention as claimed. Any modification, equivalent replacement and improvement without departing from the principle and spirit of the present invention shall be considered to be within the protection scope of the present claims.

Claims (5)

1. A method for immobilizing laccase by using titanate nanotube composite material is characterized by comprising the following steps:
(1) preparing a laccase immobilized carrier;
(2) immobilizing laccase;
wherein, the preparation of the laccase immobilized carrier comprises the following steps:
s1, preparing titanate nanotubes;
s2, modification of titanate nanotubes: mixing the titanate nanotubes obtained in the step S1 with mixed salt, then carrying out high-temperature reaction in a tubular furnace, and stirring and washing until the pH value is neutral after the reaction is finished, so as to obtain modified titanate nanotubes;
s3, preparing a titanate nanotube composite material: dispersing the modified titanate nanotubes obtained in the step S2 in an acetic acid solution, and uniformly dispersing by ultrasonic; then adding mixed solution a containing chitosan, then starting stirring and mixing, and after stirring and mixing are finished, centrifuging, washing and drying to obtain the titanate nanotube composite material;
wherein, the mixed salt in the step S2 is a mixture of sodium nitrate and sodium carbonate;
wherein the mass ratio of the sodium nitrate to the sodium carbonate is 1: 1;
wherein the mass ratio of the mixed salt to the titanate nanotubes in the step S2 is 10-50: 1;
wherein the high-temperature reaction temperature in the step S2 is 340-350 ℃, the high-temperature reaction time is 1-4 h, and the reaction temperature rise rate is 12 ℃/min;
wherein, the laccase immobilization in the step (2) comprises the following steps: at room temperature, taking 2mL of 1% wt glutaraldehyde solution, adding 0.4g titanate nanotube composite material, oscillating at the rotating speed of 180r/min, and then carrying out centrifugal separation to obtain a carrier; adding the obtained carrier into 1mL of acetic acid-sodium acetate buffer solution containing laccase with the pH value of 5.5, oscillating for 4 hours at room temperature, and performing suction filtration and washing to obtain immobilized laccase;
wherein the concentration of the laccase is 0.8 g/L.
2. The method of immobilizing laccase enzyme by titanate nanotube composite material according to claim 1, wherein the method for preparing titanate nanotubes in step S1 is as follows: adding titanium dioxide nano powder into a sodium hydroxide solution, performing ultrasonic dispersion uniformly, performing hydrothermal reaction at 120-150 ℃ for 1-5 days, washing with deionized water after the reaction is finished, soaking the product in hydrochloric acid, and washing with deionized water again after the soaking is finished to obtain the titanate nanotube.
3. The method of claim 1, wherein step S3 is performed by mixing chitosan-containing mixed solution a with 100mg chitosan and 100mL of 0.1mol/L acetic acid solution.
4. The method of immobilizing laccase by using titanate nanotube composite material as claimed in claim 3, wherein the amount of modified titanate nanotubes is 0.4-0.8 g, and the volume ratio of acetic acid solution to mixed solution a is 1: 1.
5. the method of immobilizing laccase according to claim 3, wherein the step S3 is performed at room temperature for 12-36 h, and the stirring speed is 350 r/min.
CN202111262130.9A 2021-10-28 2021-10-28 Method for immobilizing laccase by titanate nanotube composite material Active CN113862252B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111262130.9A CN113862252B (en) 2021-10-28 2021-10-28 Method for immobilizing laccase by titanate nanotube composite material

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111262130.9A CN113862252B (en) 2021-10-28 2021-10-28 Method for immobilizing laccase by titanate nanotube composite material

Publications (2)

Publication Number Publication Date
CN113862252A CN113862252A (en) 2021-12-31
CN113862252B true CN113862252B (en) 2022-09-27

Family

ID=78998055

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111262130.9A Active CN113862252B (en) 2021-10-28 2021-10-28 Method for immobilizing laccase by titanate nanotube composite material

Country Status (1)

Country Link
CN (1) CN113862252B (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100455517C (en) * 2006-01-17 2009-01-28 河南大学 Method for preparing Nano tube of titanate
CN101502785B (en) * 2008-11-25 2011-01-05 中国科学院合肥物质科学研究院 Method for preparing nano titanates for removing surface modification of heavy metal ion

Also Published As

Publication number Publication date
CN113862252A (en) 2021-12-31

Similar Documents

Publication Publication Date Title
Liu et al. Biocompatible magnetic cellulose–chitosan hybrid gel microspheres reconstituted from ionic liquids for enzyme immobilization
SU1215624A3 (en) Method of producing polymer solution for extracting petroleum
CN109232953B (en) Polyvinimidyl chloramine type antibacterial cellulose membrane, preparation method and application
CN108384767A (en) Transaminase mutant and its application
Yang et al. Robust glucose oxidase with a Fe 3 O 4@ C-silica nanohybrid structure
Sahin et al. Determination of optimum conditions for glucose-6-phosphate dehydrogenase immobilization on chitosan-coated magnetic nanoparticles and its characterization
CN1702782A (en) Water-based magnetic liquid and method for making same
CN107418950A (en) Multi-arm magnetic composite microsphere immobilized HRP and its preparation method and application
CN108850779B (en) PEG-ACS/M-siRNA nano-composite and application thereof, and method for reducing histamine content in fish meal storage
Payne et al. Comparison of cross-linked branched and linear poly (ethylene imine) microgel microstructures and their impact in antimicrobial behavior, copper chelation, and carbon dioxide capture
CN108849974A (en) Graphene oxide-chitosan hydrochloride composite antibacterial material preparation method and application
El‐Ghaffar et al. Immobilization of α‐amylase onto chitosan and its amino acid condensation adducts
CN113862252B (en) Method for immobilizing laccase by titanate nanotube composite material
Bazazi et al. Polysaccharide-based C-dots and polysaccharide/C-dot nanocomposites: Fabrication strategies and applications
Ozyilmaz et al. Preparation of regenerable magnetic nanoparticles for cellulase immobilization: Improvement of enzymatic activity and stability
CN104437394B (en) Dual-layer high-amino density plant fiber-based adsorption material and preparation method and application thereof
Ein Ali Afjeh et al. Characteristics of glucose oxidase immobilized on Magnetic Chitosan Nanoparticles
Haroun et al. Functionalized multi-walled carbon nanotubes as emerging carrier for biological applications
WO2024037662A1 (en) Preparation method for and use of bismuth ion filter membrane
CN107338238B (en) Immobilized laccase and preparation method thereof
Cheng et al. Studies on the properties and co-immobilization of manganese peroxidase
CN109836522B (en) Zwitterionic polymer grafted nano-medium with weak-hydrophobicity side chain and preparation and immobilized enzyme method thereof
KR101717818B1 (en) Fe2O3 yolk shell nano structure and enzyme immobilization using the same
CN113951280A (en) Preparation method of lysozyme-polysaccharide nano-composite with antibacterial property
CN106591274B (en) A kind of immobilized nucleic acids enzyme P1 and preparation method thereof is applied with it

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant