CN104478068A - Method for treating antibiotic-contaminated water through immobilized laccase and lignin mediator - Google Patents
Method for treating antibiotic-contaminated water through immobilized laccase and lignin mediator Download PDFInfo
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- CN104478068A CN104478068A CN201410604718.1A CN201410604718A CN104478068A CN 104478068 A CN104478068 A CN 104478068A CN 201410604718 A CN201410604718 A CN 201410604718A CN 104478068 A CN104478068 A CN 104478068A
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Abstract
The invention relates to a method for treating antibiotic-contaminated water through immobilized laccase and a lignin mediator. The method comprises: adding magnetic nanometer Fe3O4 particle immobilized laccase to water containing sulfanilamide and/or tetracycline antibiotic contaminants while adding a lignin mediator or lignin mediator combination, and carrying out a standing reaction for 0.1-3 h at a room temperature so as to complete catalytic degradation, such that the antibiotic contaminants in the contaminated water can be effectively removed. According to the present invention, the lignin substance is adopted as the mediator of the immobilized laccase catalysis system to promote the enzymolysis of the antibiotic in the contaminated water, such that advantages of simple treatment process, low cost and high contaminant removal rate are provided.
Description
Technical field
The invention belongs to biochemical field, be specifically related to a kind of catalyst system containing immobilized enzyme and reaction amboceptor and removing the application in microbiotic pollution.
Background technology
Along with a large amount of uses of microbiotic in farming animals fishing animal husbandry is produced, in environment, antibiotic remains is increasing, microbiotic residual in water body has higher toxicity and stability, a small amount of entered environment can jeopardize the health of the ecosystem and the Sustainable development of human society, and is difficult to be removed in the environment by self-purification.Therefore, the focus that efficient, economic residual antibiotic process new technology becomes whole world environmental worker care is researched and developed.Biodegradation technique, especially biocatalysis technology, as a kind of emerging treatment process, having that energy consumption is low, easy to operate, degradation efficiency advantages of higher, is a treatment technology had a extensive future.
Laccase (laccase, E.C.I.10.3.2) be a kind of polyphenoloxidase of cupric, there is wide in range Substratspezifitaet, can the oxidation of the multiple phenols of catalysis, amine, aromatics and heterocyclic compound, simultaneously with step four electron reaction, oxygen molecule is reduced into water environmentally friendlyly.At present, laccase obtains investigation and application widely in fields such as environmental improvement, analyzing and testing, bio-sensing and organic syntheses.Under efficient amboceptor existent condition, laccase can also be oxidized non-phenolic material, expands the range of application of laccase.But, in numerous efficient amboceptors, most amboceptor is synthetic, expensive and there is potential environmental toxicity, find efficient, cheap, come from crude substance, eco-friendly artificial amboceptor surrogate, will contribute to expand laccase water pollutant remove in application.
In addition, in laccase degradation of sewage during organic pollutant, there is easy in inactivation, reclaim and be difficult to and cannot use continuously etc. problem, limit its use in practice.One of effective measure addressed these problems by the technology of being fixed of laccase.Magnetic Nano material not only has high-specific surface area and good dispersiveness, and has paramagnetism.Adopt magnetic Nano to be the molecule space degree of freedom that carrier immobilized laccase can significantly improve the stability of laccase, repeating utilization factor and laccase, reduce inside diffusional resistance, be with a wide range of applications containing in microbiotic sewage treatment area.
Summary of the invention
The object of the present invention is to provide an immobilization laccase/lignin amboceptor catalyst system that can be used for microbiotic contaminant degradation in water body.
Another object of the present invention be to provide multiple can as the lignin amboceptor of Laccase Catalyzed microbiotic contaminant degradation or combination.
Another object of the present invention is to provide a kind of preparation method that can be used for the magnetic nano ferroferric oxide particle immobilization laccase of Degradation of Antibiotics.
The technical scheme realizing above-mentioned purpose of the present invention is:
A method for immobilization laccase/lignin mediator systems process microbiotic polluted-water, described microbiotic is sulfa antibiotics and/or tetracycline antibiotics;
Described method is in the sewage containing described microbiotic pollutent, adds magnetic nano ferroferric oxide particle immobilization laccase, and add lignin amboceptor, standing and reacting 0.1-3 hour at normal temperatures, completes catalyzed degradation simultaneously.
Wherein, described lignin amboceptor is syringyl lignin monomer derived thing and/or guaiacyl lignin monomer derivative, described syringyl lignin monomer derived thing be selected from syringic aldehyde, syringic acid, sinapinic acid and Syringylethanone one or more, described guaiacyl lignin monomer derivative be selected from Vanillin, vanillic acid, forulic acid or vanillyl methyl ketone one or more, in often liter of sewage, the addition of lignin amboceptor is 0.1-5mmol.
Preferably, described lignin amboceptor is the combination of a kind of syringyl lignin monomer derived thing and a kind of guaiacyl lignin monomer derivative, and the addition in often liter of sewage is respectively 0.1-1mmol and 0.1-1mmol.
Wherein, described magnetic nano ferroferric oxide particle immobilization laccase addition is 0.1-3g/L sewage.
Particularly, sulfa antibiotics comprises one or more in Sulphadiazine Sodium, sulfamethazine, sulfadimethoxine, sulfamethoxazole; Tetracycline antibiotics comprise in terramycin, tsiklomitsin and duomycin one or more.Usually, the antibiotic concentration polluted in water body is 0.03-3g/L.
Preferably, described magnetic nano ferroferric oxide particle immobilization laccase preparation method comprises the following steps:
(1) the magnetic nano ferroferric oxide particle of surperficial amino functional is prepared: by polyoxyethylene glycol, iron(ic) chloride, iron protochloride mixing, pass into nitrogen and be heated to 70-90 DEG C, the pH value of adjustment mixture is to 10-11, stirring reaction half an hour, be cooled to room temperature, centrifugation, gained is precipitated as the Z 250 of polyoxyethylene glycol parcel; Precipitation EtOH Sonicate is disperseed, passes into nitrogen, add silane coupling agent, at 30-50 DEG C, stirring reaction 20-30 hour, magnetic resolution, solid phase is the magnetic nano ferroferric oxide particle of amino functional;
(2) join in the glutaraldehyde solution of pH=7-9 by the magnetic nano ferroferric oxide particle of amino functional, 20-40 DEG C of stirring reaction activation 3-5 hour, washes away unreacted glutaraldehyde.Again activation magnetic nanoparticle being joined pH value is in the laccase solution of the 0.1-1mg/mL of 3-7, stirring reaction 24-48 hour at 20-40 DEG C, magnetic resolution, washes away loose laccase protein, and collecting solid phase is magnetic nano ferroferric oxide particle immobilization laccase.
Adopt method of the present invention, the activity of every gram of immobilization laccase reaches 200-500U.
Wherein preferably, stir in reaction process, the speed of stirring is 200-1000rpm.
Wherein, in step (1), described iron(ic) chloride and the mol ratio of iron protochloride are 1.7-1.8:1, and the mol ratio of silane coupling agent and magnetic nanoparticle is 3-5:1, and the consumption of polyoxyethylene glycol is 40-60g/L.
Wherein, in step (1), the polymerization degree of described polyoxyethylene glycol is 4000, adds ammoniacal liquor adjustment mixture ph, the concentration of ammoniacal liquor is 20-30% (v/v), and silane coupling agent is 3-aminopropyl triethoxysilane or 3-aminopropyl trimethoxysilane.
Wherein, in step (2), the concentration of described magnetic nanoparticle is 10mg/mL, and the concentration of glutaraldehyde is 3-5%.
Preferably, described laccase comes from glossy ganoderma, Coriolus Versicolor or flat mushroom.
Beneficial effect of the present invention is:
(1) the method for microbiotic pollutent in catalyzed degradation water body of the present invention, immobilization laccase/lignin mediator systems is utilized to dispose of sewage, lignin amboceptor can promote the catalysis of immobilization laccase, the sulfamido fast in removing water body and tetracycline antibiotics pollutent.
(2) the present invention utilizes magnetic nano ferroferric oxide particle as Laccase Immobilization carrier, has higher year enzyme amount and stability, and easily reclaim and recycling, treatment process is simple, cost is low.
Embodiment
For the ease of understanding, below will be described in detail of the present invention by specific embodiment.
The preparation of embodiment 1 magnetic nano ferroferric oxide particle immobilization laccase
Iron(ic) chloride and iron protochloride are joined in round-bottomed flask according to the mol ratio of 1.75:1, pass into nitrogen deoxygenation, add the Macrogol 4000 that concentration is 50g/L, be heated to 80 DEG C, in mixture, add ammoniacal liquor (28%, v/v) adjusted to ph is to 10-11,800rpm stirs, and reacts half an hour, is cooled to room temperature, centrifugation, gained is precipitated as the Z 250 of Macrogol 4000 parcel.
Precipitation EtOH Sonicate is disperseed, pass into nitrogen, add silane coupling agent 3-aminopropyl triethoxysilane, the mol ratio of silane coupling agent and magnetic nanoparticle is 4:1,40 DEG C, 200rpm rotating speed stirs lower reaction 20 hours, and magnetic resolution, solid phase is the magnetic nano ferroferric oxide particle of amino functional.Be 12nm through X-ray diffraction analysis median size.
The magnetic nanoparticle of amino functional is added the glutaraldehyde solution of pH=8, magnetic nanoparticle concentration 10mg/mL, glutaraldehyde concentration is 4% (v/v, the lower priming reaction of 200rpm rotating speed stirring 4 hours at 30 DEG C, magnetic resolution, washes away unreacted glutaraldehyde.(this bacterium picks up from Hubei Province's Shennongjia Nature Reserve again activation magnetic nanoparticle to be joined the ganoderma lucidum laccase that pH value is the 0.3mg/mL of 7; after separation and purification in 4 DEG C, potato slope substratum preserves) in solution; 30 DEG C; 200rpm reacts 24 hours; magnetic resolution; wash away loose laccase protein, collecting solid phase is magnetic nano ferroferric oxide particle immobilization laccase, and every gram of immobilization laccase activity is 400U.
Same preparation method, laccase is Coriolus Versicolor laccase or flat mushroom laccase, the obtained immobilized laccase of magnetic nano ferroferric oxide particle.
Embodiment 2 immobilization ganoderma lucidum laccase/lignin mediator systems removes sulfamido pollutent in water body
At l00mL, be in the water body of the sulfamethoxazole of 50mg/L containing the sulfadimethoxine that concentration is the Sulphadiazine Sodium of 50mg/L, concentration is the sulphamethazine of 50mg/L, concentration is 50mg/L or concentration respectively, add 1g magnetic nano ferroferric oxide particle immobilization ganoderma lucidum laccase (prepared by embodiment 1), and add 1mmol/L syringyl lignin monomer derived thing: syringic acid, at room temperature standing and reacting 1h.When not adding syringic acid, laccase is not degraded sulfamido pollutent, after adding syringic acid, in water body, the clearance (%) of sulfamido pollutent is as shown in table 1, table 1 result shows, immobilization ganoderma lucidum laccase/syringyl lignin amboceptoid system effectively can remove the sulfamido pollutent in water body.
Table 1 immobilization ganoderma lucidum laccase/lignin mediator systems removes sulfamido pollutent
Embodiment 3 immobilization Coriolus Versicolor laccase/lignin mediator systems removes tetracyclines pollutent in water body
At 100mL, respectively containing concentration be the tsiklomitsin of l00mg/L, concentration is the terramycin of l00mg/L, concentration is in the pollutent water body of the duomycin of l00mg/L, add 1g magnetic nano ferroferric oxide particle immobilization Coriolus Versicolor laccase (prepared by embodiment 1), and add 0.5mmol/L guaiacyl lignin monomer derivative: forulic acid, at room temperature react 0.5h.When not adding forulic acid, laccase is not degraded tetracyclines pollutent, after adding forulic acid, in water body, the clearance (%) of tsiklomitsin is as shown in table 2, table 2 result shows, under forulic acid exists, magnetic nano particle immobilized Coriolus Versicolor laccase can effectively be degraded the tetracycline antibiotics pollutent in water body.Table 2 result shows, immobilization Coriolus Versicolor laccase/guaiacyl lignin mediator systems effectively can remove the tsiklomitsin pollutent in water body.
Table 2 immobilization Coriolus Versicolor laccase/lignin mediator systems removes tetracyclines pollutent
Embodiment 4 immobilization flat mushroom laccase/lignin mediator systems removes sulfamido pollutent in water body
At l00mL, be in the water body of the sulfamethoxazole of 50mg/L containing the sulfadimethoxine that concentration is the Sulphadiazine Sodium of 50mg/L, concentration is the sulphamethazine of 50mg/L, concentration is 50mg/L and concentration respectively, add 0.8g magnetic nano ferroferric oxide grain orientation immobilization flat mushroom laccase (prepared by embodiment 1), and add 2mmol/L syringyl lignin monomer derived thing: Syringylethanone, at room temperature reacts 2h.When not adding Syringylethanone, laccase is not degraded sulfamido pollutent, after adding Syringylethanone, in water body, the clearance (%) of sulfamido pollutent is as shown in table 3, table 3 result shows, immobilization flat mushroom laccase/syringyl lignin amboceptoid system effectively can remove the sulfamido pollutent in water body.
Table 3 immobilization flat mushroom laccase/lignin mediator systems removes sulfamido pollutent
Embodiment 5 immobilization flat mushroom laccase/lignin mediator combination system removes sulfamido and tetracyclines pollutent in water body
L00mL, containing concentration be 30mg/L Sulphadiazine Sodium, 30mg/L sulphamethazine, 30mg/L tsiklomitsin and 30mg/L terramycin water body in, add 1g magnetic nano ferroferric oxide grain orientation immobilization flat mushroom laccase (prepared by embodiment 1), and add the mediator combination of 1.5mmol/L syringyl lignin monomer derived thing Syringylethanone and 1.5mmol/L guaiacyl lignin monomer derivative forulic acid respectively, at room temperature react 1h.When not adding mediator combination, laccase is not degraded microbiotic pollutent, after adding mediator combination, in water body, the clearance (%) of microbiotic pollutent is as shown in table 4, table 4 result shows, immobilization flat mushroom laccase/lignin mediator combination system effectively can remove sulfamido in water body and antibiotic contamination.
Table 4 immobilization flat mushroom laccase/lignin mediator combination system removes sulfamido pollutent
It needs to be noted, above embodiment is only to illustrate, obvious those of ordinary skill in the art according to illustrating, can make various correction and change to the present invention herein within the scope of the invention, and these are revised and change and also include in scope of the present invention.
Claims (8)
1., by a method for immobilization laccase/lignin mediator systems process microbiotic polluted-water, described microbiotic is sulfa antibiotics and/or tetracycline antibiotics;
Described method is in the sewage containing described microbiotic pollutent, adds magnetic nano ferroferric oxide particle immobilization laccase, and add lignin amboceptor, standing and reacting 0.1-3 hour at normal temperatures, completes catalyzed degradation simultaneously.
2. method according to claim 1, it is characterized in that, described lignin amboceptor is syringyl lignin monomer derived thing and/or guaiacyl lignin monomer derivative, described syringyl lignin monomer derived thing be selected from syringic aldehyde, syringic acid, sinapinic acid and Syringylethanone one or more, described guaiacyl lignin monomer derivative be selected from Vanillin, vanillic acid, forulic acid or vanillyl methyl ketone one or more, in often liter of sewage, the addition of lignin amboceptor is 0.1-5mmol.
3. method according to claim 2, it is characterized in that, described lignin amboceptor is the combination of a kind of syringyl lignin monomer derived thing and a kind of guaiacyl lignin monomer derivative, and the addition in often liter of sewage is respectively 0.1-1mmol and 0.1-1mmol.
4., according to the arbitrary described method of claim 1-3, it is characterized in that, in often liter of sewage, the addition of magnetic nano particle immobilized laccase is 0.1-3g.
5., according to the arbitrary described method of claim 1-3, it is characterized in that, described magnetic nano ferroferric oxide particle immobilization laccase preparation method comprises the following steps:
(1) the magnetic nano ferroferric oxide particle of surperficial amino functional is prepared: by polyoxyethylene glycol, iron(ic) chloride, iron protochloride mixing, pass into nitrogen and be heated to 70-90 DEG C, the pH value of adjustment mixture is to 10-11, stirring reaction half an hour, be cooled to room temperature, centrifugation, gained is precipitated as the Z 250 of polyoxyethylene glycol parcel; Precipitation EtOH Sonicate is disperseed, passes into nitrogen, add silane coupling agent, at 30-50 DEG C, stirring reaction 20-30 hour, magnetic resolution, gained solid phase is the magnetic nano ferroferric oxide particle of amino functional;
(2) the magnetic nano ferroferric oxide particle of amino functional is joined in the glutaraldehyde solution of pH=7-9,20-40 DEG C of stirring reaction activation 3-5 hour, wash away unreacted glutaraldehyde, again activation magnetic nanoparticle being joined pH value is in the laccase solution of the 0.1-1mg/mL of 3-7, stirring reaction 24-48 hour at 20-40 DEG C, magnetic resolution, washes away loose laccase protein, and collecting solid phase is magnetic nano ferroferric oxide particle immobilization laccase.
6. method according to claim 5, it is characterized in that, in described step (1), the mol ratio of iron(ic) chloride and iron protochloride is 1.7-1.8:1, the mol ratio of silane coupling agent and magnetic nanoparticle is 3-5:1, and the consumption of polyoxyethylene glycol is 40-60g/L.
7. the method according to claim 5 or 6, is characterized in that, described silane coupling agent is 3-aminopropyl triethoxysilane or 3-aminopropyl trimethoxysilane.
8. the method according to claim 5 or 6, is characterized in that, the laccase in described step (1) comes from glossy ganoderma, Coriolus Versicolor or flat mushroom.
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| CN106335994A (en) * | 2016-11-02 | 2017-01-18 | 福建省农业科学院农业质量标准与检测技术研究所 | Special aquaculture cleaning agent |
| CN106380001A (en) * | 2016-08-31 | 2017-02-08 | 天津大学 | Method for degrading sulfanilamide compounds in water environment by use of mixed bacteria |
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| CN108191072A (en) * | 2017-12-28 | 2018-06-22 | 湖南大学 | The method that antibiotic in water body is removed using hollow mesoporous carbon immobilization laccase |
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| CN109234258A (en) * | 2018-09-11 | 2019-01-18 | 新乡医学院 | Laccase natural amboceptor immobilization magnetic nano-particle and its preparation method and application |
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| CN115417507A (en) * | 2022-06-21 | 2022-12-02 | 江南大学 | Laccase for efficiently degrading veterinary drug antibiotic residues and application thereof |
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| CN115057540A (en) * | 2022-07-06 | 2022-09-16 | 吉林大学 | Phenol sewage treatment method based on nano enzyme UMP-Cu with laccase activity |
| CN116590248A (en) * | 2023-04-24 | 2023-08-15 | 青岛农业大学 | Fungus laccase gene derived from lentinus edodes, fungus laccase and application thereof in degradation of tetracycline antibiotics |
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