CN113930416A - Preparation method of biochar immobilized laccase based on compound modification of sodium hydroxide and ferroferric oxide crystals - Google Patents
Preparation method of biochar immobilized laccase based on compound modification of sodium hydroxide and ferroferric oxide crystals Download PDFInfo
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 title claims abstract description 114
- 108010029541 Laccase Proteins 0.000 title claims abstract description 110
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 title claims abstract description 45
- 239000013078 crystal Substances 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 238000012986 modification Methods 0.000 title claims description 16
- 230000004048 modification Effects 0.000 title claims description 16
- 150000001875 compounds Chemical class 0.000 title claims description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 38
- 102000004190 Enzymes Human genes 0.000 claims abstract description 38
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000002131 composite material Substances 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000011068 loading method Methods 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 10
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 8
- 239000003513 alkali Substances 0.000 claims abstract description 7
- 238000002474 experimental method Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims abstract description 6
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims abstract description 4
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 40
- 230000000694 effects Effects 0.000 claims description 38
- 239000010902 straw Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 241000209140 Triticum Species 0.000 claims description 16
- 235000021307 Triticum Nutrition 0.000 claims description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 14
- 238000002835 absorbance Methods 0.000 claims description 14
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 14
- 239000010431 corundum Substances 0.000 claims description 13
- 229910052593 corundum Inorganic materials 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 13
- 239000010453 quartz Substances 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- 239000003610 charcoal Substances 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims description 4
- 230000007613 environmental effect Effects 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 238000000197 pyrolysis Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 229910021577 Iron(II) chloride Inorganic materials 0.000 claims description 2
- 230000008033 biological extinction Effects 0.000 claims description 2
- 230000008859 change Effects 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000000967 suction filtration Methods 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 abstract description 3
- 229960002089 ferrous chloride Drugs 0.000 abstract 1
- 239000011148 porous material Substances 0.000 description 11
- 238000001179 sorption measurement Methods 0.000 description 11
- 239000000463 material Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 6
- 239000003344 environmental pollutant Substances 0.000 description 6
- 231100000719 pollutant Toxicity 0.000 description 6
- 108010093096 Immobilized Enzymes Proteins 0.000 description 4
- 238000004132 cross linking Methods 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000005389 magnetism Effects 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 241000235349 Ascomycota Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 241000208225 Rhus Species 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
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- 125000000524 functional group Chemical group 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
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- 239000003607 modifier Substances 0.000 description 1
- 239000002052 molecular layer Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
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- 238000002336 sorption--desorption measurement Methods 0.000 description 1
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- 239000012085 test solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B32/00—Carbon; Compounds thereof
- C01B32/05—Preparation or purification of carbon not covered by groups C01B32/15, C01B32/20, C01B32/25, C01B32/30
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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Abstract
The invention discloses a preparation method of immobilized laccase based on sodium hydroxide and ferroferric oxide crystal composite modified biochar. Then, fully mixing the alkali modified biochar with ferric chloride and ferrous chloride hydrate in an ethanol solution, and pyrolyzing the mixture in a tubular furnace at 500 ℃ to obtain magnetic porous biochar SBC @ Fe3O4Dispersing the SBC @ Fe in a glutaraldehyde solution to obtain SBC @ Fe3O4The magnetically modified glutaraldehyde of @ GA activates biochar. Finally, the carrier is subjected to a three-factor five-horizontal orthogonal experimental method according to different enzyme-carbon ratiosAdding laccase solutions with different concentration gradients, and stirring for several hours by using a magnetic stirrer to obtain SBC @ Fe3O4The product of @ GA @ Lac, and the optimal immobilization method is selected according to the loading capacity of laccase. The invention has the advantages of low cost, suitability for large-scale preparation, less enzyme loss and the like.
Description
Technical Field
The invention belongs to the field of laccase immobilization, relates to an immobilized laccase and a preparation method thereof, and particularly relates to a preparation method of a composite modified biochar immobilized laccase based on sodium hydroxide and ferroferric oxide crystals.
Background
Laccase (EC1.10.3.2) is a copper-rich oxidoreductase that degrades phenolic and non-phenolic complex compounds, often as catalysts for various aromatic compounds. The origin of laccases can be divided into four families, plant, fungus, bacteria and insect. Laccases were first found in the secretions of the plant sumac and co-exist in the cell walls of countless higher plants. However, the plant laccase is abundant, but the research on the fungal laccase is complete. Within a broad family of fungi there are numerous fungi that can produce laccases, such as ascomycetes, basidiomycetes and deuteromycetes. With the enhancement of awareness of human to environmental protection and the increase of the demand for resource utilization, laccase has wide application in aspects such as organic synthesis, dye decolorization, biosensor preparation, wastewater treatment, soil remediation and the like due to its high efficiency and specificity. However, free laccase still has obvious defects in the aspects of thermal stability, reusability, pollutant degradation efficiency and the like. Therefore, the immobilization by the combination of the immobilized laccase and a loading material, including adsorption, crosslinking, embedding, covalent bonding and the like, is one of the main strategies for improving the stability and reusability of the laccase nowadays. Meanwhile, the dual-functionality of adsorbing or degrading pollutants by laccase and materials can be realized by properly selecting the load materials, so that the removal efficiency of the pollutants is greatly improved.
Biochar is a carbon-containing solid substance prepared by biomass under the condition of limited oxygen, and becomes one of main immobilized support materials due to high porosity, large specific surface area and strong adsorption capacity to heavy metals and organic pollutants. However, the traditional biochar has low adsorption capacity to pollutants and is difficult to recover from the environment after application, so the pore structure of the traditional biochar can be further changed through modification, and the functional groups on the surface of the traditional biochar are increased, so that the traditional biochar has stronger adsorption and fixation capacity to pollutants. The alkali modification can reduce the ash content of the biochar and increase the number of hydroxyl and carboxyl. Magnetic modification can introduce magnetic substances into the biochar, so that the biochar is more convenient to separate and recycle from the environment, and the treatment cost is reduced. Therefore, the modified biochar is used as a load material of the immobilization technology, and has profound environmental significance for maintaining the activity of the enzyme, improving the stability of the immobilized enzyme and improving the reusability.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a preparation method of a composite modified biochar immobilized laccase based on sodium hydroxide and ferroferric oxide crystals, which has low cost, is convenient to recycle from the environment and improves the storage stability and the operation stability of the enzyme. The invention aims to solve the defects that the existing immobilized enzymes are prepared by a plurality of methods, but a single immobilization technology is mainly used, such as embedding, adsorption, crosslinking and the like, and the methods are not favorable for maintaining the activity of the enzymes and have insufficient enzyme loading capacity. And the loss of enzyme activity is reduced, the loading capacity of the enzyme is increased, the recovery rate of the immobilized product is improved, and meanwhile, the immobilized product can be produced and applied in a large scale by selecting a low-cost loading material.
The purpose of the invention is realized by the following technical scheme: a preparation method of biochar immobilized laccase based on compound modification of sodium hydroxide and ferroferric oxide crystals comprises the following steps:
(1) heating the dried wheat straw powder in a muffle furnace at 500 ℃ for 2.5 hours, and pyrolyzing to prepare biochar;
the average pore diameter of the wheat straw biochar is 3nm-16 nm;
the pore size of the wheat straw biochar is 0.001cm3/g-0.008cm3/g；
The specific surface area of the wheat straw biochar is 5.991m2/g。
(2) Preparing sodium hydroxide modified biochar:
and (2) sufficiently mixing 20g of the biochar (WBC) obtained in the step (1) with 4mol/L of NaOH solution according to a mass ratio of 1 to 3, stirring the mixture for two hours at room temperature by using a magnetic stirrer, and then drying the mixture in an oven at 105 ℃. And (3) placing the dried sample in a 250ml corundum boat, introducing 80-100 ml/min nitrogen, heating to 500 ℃ at 10 ℃/min in a tube furnace, heating to 800 ℃ at 5 ℃/min, keeping the temperature for 90min, and then cooling to room temperature. Soaking the prepared biochar in 1.5mol/L hydrochloric acid for two hours, then washing the biochar with deionized water for a plurality of times, drying the biochar in an oven, and homogenizing the biochar with a 100-mesh sieve to obtain sodium hydroxide modified biochar (SBC) which is stored in a glass drying dish for later use.
The average pore diameter of the sodium hydroxide modified biochar is 2nm-21 nm;
the pore size of the sodium hydroxide modified biochar is 0.01cm3/g-0.04cm3/g；
The specific surface area of the sodium hydroxide modified biochar is 41.285m2/g。
(3) Preparing magnetic modified ferroferric oxide crystal biochar:
mixing the alkali modified biochar obtained in the step (2) with 0.02mol FeCl3·6H2O and 0.01mol FeCl2·4H2O was mixed well in 200ml ethanol solution. And putting the mixture into a 250ml corundum boat, covering the corundum boat with tinfoil in a tube furnace, heating the corundum boat to 500 ℃ at the speed of 10 ℃/min, carrying out pyrolysis and heat preservation for 110min, introducing nitrogen at the speed of about 80-100 ml/min for protection, and cooling the corundum boat to room temperature. Then washing with deionized water for several times to prepare the magnetic porous biochar SBC @ Fe3O4。
The average pore diameter of the magnetic porous biochar is 2nm-6 nm;
the pore size of the magnetic porous biochar is 0.01cm3/g-0.06cm3/g；
The specific surface area of the magnetic porous biochar is 78.262m2/g。
(4) Preparing immobilized laccase: 1g of the magnetically modified biochar obtained in step (3) was dispersed in 25ml of glutaraldehyde solution for 2.5 hours. Subsequently, the activated support was washed several times with deionized water and citric acid-disodium hydrogen phosphate buffer pH 5, and dried in an oven at 105 deg.C to obtainTo SBC @ Fe3O4The magnetically modified glutaraldehyde of @ GA activates biochar. Adding laccase solutions with different concentration gradients into the carrier according to different enzyme-carbon ratios by a three-factor five-horizontal orthogonal experimental method, and stirring for several hours by using a magnetic stirrer to obtain SBC @ Fe3O4The product of @ GA @ Lac, and the optimal immobilization method is selected according to the loading amount of the laccase. The product was washed several times with citric acid-disodium hydrogen phosphate buffer at pH 5, filtered with a magnet and stored in a refrigerator at-20 deg.C for further use.
The oven temperature in the selected step (1) is 105 ℃, and the heating time is 220 min.
The preparation conditions of the sodium hydroxide solution in the step (2) are as follows: 80g of NaOH powder was dissolved in 500ml of distilled water to prepare a 4mol/L NaOH solution.
The temperature of the oven in the step (2) is 105-120 ℃.
The ethanol solution in the step (3) is analytically pure ethanol.
The glutaraldehyde solution in the step (4) is a glutaraldehyde solution with the volume ratio of 2.5%.
The citric acid-disodium hydrogen phosphate buffer solution in the step (4) is prepared by mixing 0.1mol/L citric acid solution and 0.2mol/L disodium hydrogen phosphate solution.
And (4) adopting a 0.22 mu m organic nylon filter membrane as the suction filtration membrane in the step (4).
The three factors of the orthogonal experiment in the step (4) are enzyme-to-carbon ratio (mass ratio), concentration of laccase solution (U/mL) and immobilization time (h).
Wherein, the five level variables of the charcoal enzyme ratio are respectively 1:2, 1:3, 1:4, 1:5 and 1:6, the five level variables of the concentration of the laccase solution are respectively 2, 4, 6, 8 and 10, and the five level variables of the immobilization time are respectively 8, 12, 16, 20 and 24.
The technical progress of the invention is represented as follows: the method can realize the immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal. Thereby overcoming the defect of short maintenance time of laccase activity.
1. The invention adopts the wheat straw as the biomass, and the wheat straw biochar as the raw material can not only relieve the pollution problem caused by straw burning, but also be used as an environmental modifier to be depended on the environment for a long time, and can synergistically adsorb and degrade pollutants with laccase.
2. The invention adopts the adsorption crosslinking composite immobilization technology to immobilize the laccase, has good immobilization effect, less laccase loss, reusability and improved thermal stability.
3. The invention adopts the magnetically modified biochar as the load material, has strong magnetism, can realize the recycling in the environment, has simple operation and low preparation cost, is suitable for large-scale production, and has obvious economic benefit and industrial value.
Drawings
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
FIG. 1 is SEM images of magnetically modified ferriferrous oxide crystal biochar, magnetically modified glutaraldehyde activated biochar and immobilized laccase prepared by the invention. Wherein, (a) is magnetic modified ferroferric oxide crystal biochar, (b) is magnetic modified glutaraldehyde activated biochar, and (c) is immobilized laccase.
FIG. 2 is an X-ray diffraction diagram of magnetically modified ferroferric oxide crystal biochar, magnetically modified glutaraldehyde activated biochar and immobilized laccase prepared by the method.
FIG. 3 is N of the wheat straw biochar, the sodium hydroxide modified biochar and the magnetic modified ferroferric oxide crystal biochar prepared by the invention2Adsorption-desorption curves and pore size distribution plots.
FIG. 4 is a graph of the hysteresis regression of magnetically modified glutaraldehyde-activated biochar and immobilized laccase prepared according to the present invention.
FIG. 5 is a graph comparing the pH stability of free laccase and immobilized laccase.
FIG. 6 is a graph comparing the temperature stability of free laccase and immobilized laccase.
FIG. 7 is a graph of the operational stability of immobilized laccase.
Detailed Description
The invention is further described below with reference to the following figures and examples.
The laccase was selected in the examples of the present invention and purchased from a source leaf organism (80498-15-3) with a specification of 120U/g.
The equipment used in the method mainly comprises a muffle furnace, a tube furnace, a magnetic stirrer, a vacuum suction filter, an ultraviolet spectrophotometer, a field emission scanning electron microscope, a vibration sample magnetometer, an X-ray diffractometer, a full-automatic specific surface area and porosity analyzer and the like.
The invention will be further described with reference to the drawings and specific preferred embodiments without thereby departing from the scope of the invention.
Example 1:
according to the immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal, laccase is immobilized on the modified porous charcoal through an adsorption crosslinking composite technology.
The preparation method of the biochar immobilized laccase based on the compound modification of the sodium hydroxide and the ferroferric oxide crystals in the embodiment comprises the following steps:
(1) preparation of wheat straw biochar
60g of natural wheat straw powder is taken, dried in an oven at 105 ℃, then pyrolyzed in a muffle furnace at 500 ℃ to obtain the wheat straw biochar, and the wheat straw biochar is placed in a glass drying dish for storage and standby.
(2) Preparation of sodium hydroxide modified biochar
And (2) fully mixing 20g of biochar (WBC) obtained in the step (1) with 4mol/L of NaOH solution according to the mass ratio of 1:3, stirring for two hours at room temperature by using a magnetic stirrer, and then drying in an oven at 105-120 ℃. The dried sample was placed in a 250ml corundum boat and charged with 80ml/min nitrogen, heated to 500 ℃ at 10 ℃/min in a tube furnace, heated to 800 ℃ at 5 ℃/min and held for 90min, and then cooled to room temperature. Soaking the prepared biochar in 1.5mol/L hydrochloric acid for two hours, then washing the biochar with deionized water for a plurality of times, drying the biochar in an oven, and homogenizing the biochar with a 100-mesh sieve to obtain sodium hydroxide modified biochar (SBC) which is stored in a glass drying dish for later use.
(3) Preparation of magnetic modified ferroferric oxide crystal biochar
Mixing SBC obtained in the step (2) with 0.02mol FeCl3·6H2O，0.01molFeCl2·4H2O was mixed well in 200ml of an analytically pure ethanol solution. Placing the mixture in a 250ml corundum boat, covering the corundum boat in a tube furnace by using tinfoil, heating the corundum boat to 500 ℃ at the speed of 10 ℃/min, carrying out pyrolysis and keeping the temperature for 110min, introducing nitrogen at the speed of about 80ml/min for protection, and cooling the corundum boat to room temperature. Then washing with deionized water for several times to prepare the magnetic porous biochar SBC @ Fe3O4And storing in a glass drying dish for later use.
(4) Preparation of immobilized laccase
1g of the magnetically modified biochar obtained in step (3) was dispersed in 25ml of glutaraldehyde solution for 2.5 hours. Subsequently, the activated support was washed several times with deionized water and citric acid-disodium hydrogen phosphate buffer at pH 5, and dried in an oven to obtain SBC @ Fe3O4The magnetically modified glutaraldehyde of @ GA activates biochar. Adding laccase solutions with different concentration gradients into the carrier according to different enzyme-carbon ratios by a three-factor five-horizontal orthogonal experimental method, and stirring for several hours by using a magnetic stirrer to obtain SBC @ Fe3O4The product of @ GA @ Lac, and the optimal immobilization method is selected according to the loading amount of the laccase. The product was washed several times with citric acid-disodium hydrogen phosphate buffer at pH 5, filtered with a magnet and stored in a refrigerator at-20 deg.C for further use.
And (3) measuring the load of the immobilized enzyme:
weighing 0.05g of immobilized enzyme, adding 1mL of buffer solution, centrifuging for 10min (the temperature is 4 ℃, and the centrifugal force is 8000g), and taking supernatant as the liquid to be detected. Respectively adding 500 microliters of distilled water and 500 microliters of Coomassie brilliant blue indicator into a quartz cuvette as blanks, then respectively adding 500 microliters of solution to be detected and 500 microliters of Coomassie brilliant blue indicator into the quartz cuvette, measuring absorbance at 620nm, and obtaining the loading capacity (mg/g) of laccase by calculation, wherein the calculation formula is as follows:
protein concentration C of the initial laccasei(mg/mL)＝0.07×(ΔA+0.0007)
Protein concentration of the immobilized laccaseCf(mg/mL)＝0.07×(ΔA+0.0007)
Where Δ A represents the change in absorbance before and after the measurement.
Wherein, V1Representing the amount of free laccase solution (mL) added during the initial laccase protein concentration determination, V2Representing the amount of the test solution (mL) added during the determination of the concentration of the immobilized laccase protein.
And (3) enzyme activity determination:
taking about 15mg of free laccase or immobilized laccase in a 15ml centrifuge tube, sequentially adding 2ml of citric acid-disodium hydrogen phosphate buffer solution with the optimal pH value and 2ml of 0.5mmoL/LABTS solution, immediately centrifuging (the centrifugal force is 8000g and the temperature is 4 ℃) for 10min, taking 1ml of supernatant in a quartz slit cuvette, measuring the absorbance at 420nm, fully oscillating, carrying out water bath at the optimal temperature for 3min, centrifuging again (the centrifugal force is 8000g and the temperature is 4 ℃) for 10min, taking 1ml of supernatant in the quartz slit cuvette, and measuring the absorbance again at 420 nm. The enzyme activity was calculated using the ABTS equation as follows:
wherein Δ A represents the increase in absorbance of ABTS, VtRepresents the volume (L) of the reaction system, and ε represents the molar extinction coefficient (36000 L.M) of ABTS-1·cm-1) DeltaT represents the reaction time (min) and m represents the mass of the enzyme (g).
Example 2:
an orthogonal experimental assay study for the preparation of immobilized laccase in example 1:
according to the orthogonal experimental arrangement, 25 groups of experiments are carried out by changing different factors, and the measurement results are shown in the following table 1:
TABLE 1
The results of the range analysis are shown in the following table 2:
TABLE 2
| Level of | Carbon to enzyme ratio (g/g) | Enzyme concentration (U/mL) | Immobilization time (h) |
| 1 | 0.3692 | 0.3864 | 0.2320 |
| 2 | 0.3137 | 0.3107 | 0.2301 |
| 3 | 0.3323 | 0.3162 | 0.5150 |
| 4 | 0.3628 | 0.3338 | 0.2526 |
| 5 | 0.3443 | 0.3753 | 0.4926 |
| Delta | 0.0554 | 0.0756 | 0.2849 |
| Rank of |
3 | 2 | 1 |
According to the results of range analysis, the optimal immobilization conditions of the immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal are as follows: the ratio of charcoal to enzyme is 1:2, the concentration of laccase is 2U/mL, and the immobilization time is 16 h. And the priority order of the three factors is immobilization time, laccase concentration and carbon-enzyme ratio.
Example 3:
the immobilized laccase was prepared according to the optimal immobilization conditions found in example 2 and subjected to performance characterization and testing:
the magnetic modified ferriferrous oxide crystal biochar, the magnetic modified glutaraldehyde activated biochar and the immobilized laccase in example 1 were subjected to field emission Scanning Electron Microscope (SEM) detection and analysis, and the results are shown in fig. 1. Wherein (a) is magnetic modified ferroferric oxide crystal biochar, (b) is magnetic modified glutaraldehyde activated biochar, and (c) is immobilized laccase. As can be seen from the graph (a), the pores of the biochar after the ferroferric oxide crystal is magnetically modified become more uniform, and as can be seen from the graph (b), the biochar is activated by the magnetically modified glutaraldehyde to enlarge part of the pores of the porous biochar, and as can be seen from the graph (c), the biochar activated by the magnetically modified glutaraldehyde is used as a carrier, the laccase is successfully immobilized on the carrier material, and the structure of the immobilized laccase obtained after the laccase is immobilized is unchanged.
The magnetic modified ferroferric oxide crystal biochar, the magnetic modified glutaraldehyde activated biochar and the immobilized laccase in example 1 were subjected to X-ray diffraction (XRD) analysis, wherein (a) is the magnetic modified ferroferric oxide crystal biochar, (b) is the magnetic modified glutaraldehyde activated biochar, and (c) is the immobilized laccase. As can be seen from FIG. 2, two diffraction peaks appeared at 30.1 ℃ and 35.6 ℃ in the pattern, which was designated as Fe according to PDF (89-0691)3O4Crystal, indicating that the magnetic modified charcoal is loaded with Fe3O4And (4) crystals.
The wheat straw biochar, the sodium hydroxide modified biochar and the magnetic modified ferroferric oxide crystal biochar in the example 1 are subjected to full-automatic specific surface area and porosity analysis (BET). As can be seen from FIG. 3, the adsorption isotherms from wheat straw biochar to sodium hydroxide modified biochar to magnetic modified ferroferric oxide crystal biochar transition from type I to type II, the type II adsorption isotherm is mostly a non-porous or typical multi-molecular-layer physical adsorption process on a macroporous adsorbent, and the combination of the pore size distribution diagram shows that the mesoporous and macroporous ratios of the sodium hydroxide modified biochar and the magnetic modified ferroferric oxide crystal biochar are increased.
The magnetically modified glutaraldehyde-activated biochar and immobilized laccase from example 1 were subjected to vibro-sample magnetic intensity analysis (VSM). As can be seen from FIG. 4, the saturation magnetic field intensity of the biochar activated by the magnetically modified glutaraldehyde serving as the load material is about 7.3emu/g, while the saturation magnetic field intensity of the immobilized laccase can be increased to about 17emu/g, which is increased by 1.33 times and belongs to strong magnetism. The loading of the laccase on the magnetic material is shown to not only not weaken the magnetism, but also enhance the magnetism, and is more beneficial to the adsorption effect and the recovery function of the immobilized laccase in the environment.
Example 4:
the immobilized laccase was prepared according to the optimal immobilization conditions obtained in example 2 and subjected to stability tests, including pH stability, temperature stability and handling stability, with the following steps and results:
(1) stability of pH
In the process of measuring the activities of free laccase and immobilized laccase, the pH value of a citric acid-disodium hydrogen phosphate buffer solution is changed to measure the enzyme activity under different pH environment gradients, so that the optimum pH value of the laccase activity is obtained, wherein the pH gradient is set to be 2.5, 3, 4, 5, 6 and 7. The specific method comprises the following steps: taking about 15mg of free laccase or immobilized laccase in a 15ml centrifuge tube, sequentially adding 2ml of citric acid-disodium hydrogen phosphate buffer solution with different pH gradients and 2ml of 0.5mmoL/LABTS solution, immediately centrifuging (the centrifugal force is 8000g,4 ℃) for 10min, taking 1ml of supernatant in a quartz slit cuvette, measuring the absorbance at 420nm, fully oscillating, reacting at room temperature for 3min, centrifuging again (the centrifugal force is 8000g,4 ℃) for 10min, taking 1ml of supernatant in the quartz slit cuvette, and measuring the absorbance again at 420 nm. Calculating enzyme activity by using an ABTS method formula, and taking the ratio of the enzyme activity under different pH gradients to the highest enzyme activity as relative enzyme activity, and recording the relative enzyme activity in percentage.
As can be seen from FIG. 5, the optimum pH range of the immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal is wider than 2.5-4, and the optimum pH values of 5 and 3 are respectively selected as the optimum pH values for determining the activities of the free laccase and the immobilized laccase.
(2) Temperature stability
In the process of measuring the activities of free laccase and immobilized laccase, the enzyme activities under different temperature environment gradients are measured by changing the reaction temperature of a water bath, so that the optimal temperature of the laccase activity is obtained, wherein the temperature gradients are set to be 20, 30, 40, 50, 60 and 70 ℃. The specific method comprises the following steps: and (2) adding about 15mg of free laccase or immobilized laccase into a 15ml centrifuge tube, sequentially adding 2ml of citric acid-disodium hydrogen phosphate buffer solution with the optimum pH value measured in the step (2) and 2ml of 0.5mmoL/LABTS solution, immediately centrifuging (the centrifugal force is 8000g and 4 ℃) for 10min, taking 1ml of supernatant into a quartz slit cuvette, measuring the absorbance at 420nm, fully oscillating, reacting for 3min under different water bath temperature gradients, centrifuging again (the centrifugal force is 8000g and 4 ℃) for 10min, taking 1ml of supernatant into the quartz slit cuvette, and measuring the absorbance again at 420 nm. Calculating enzyme activity by using an ABTS method formula, and taking the ratio of the enzyme activity under different temperature gradients to the highest enzyme activity as relative enzyme activity, and recording the relative enzyme activity in percentage.
As can be seen from FIG. 6, the temperatures of 50 ℃ and 60 ℃ were selected as the optimum temperatures for determining the activities of the free laccase and the immobilized laccase, respectively.
(3) Stability of operation
Adsorbing the immobilized laccase after the enzyme activity is determined on the outer side of a centrifugal tube by using a magnet, pouring out the added buffer solution and an ABTS substrate solution, washing the immobilized laccase and the ABTS substrate solution by using deionized water for three times, continuously determining the activity of the immobilized laccase after the cyclic recovery according to the optimal temperature of the immobilized laccase obtained in the step (2) by using the method for determining the activity in the step (2), repeating the cycle for ten times, and taking the ratio of the enzyme activity determined each time to the first enzyme activity as the relative enzyme activity, and recording the relative enzyme activity in percentage.
As can be seen from FIG. 7, the enzyme activity was hardly decreased after the first three operations, and was maintained at about 90% until the fourth operation. The enzyme activity after the fifth time is reduced to about 60%, and the enzyme activity is stably maintained at about 30% from the sixth time to the tenth time. The immobilized laccase based on the sodium hydroxide and ferroferric oxide crystal composite modified charcoal can be stably used for about four to five times.
Claims (8)
1. A preparation method of biochar immobilized laccase based on compound modification of sodium hydroxide and ferroferric oxide crystals is characterized by comprising the following steps:
(1) preparing the wheat straw biochar: taking natural wheat straw powder, drying in an oven, and then pyrolyzing in a muffle furnace to obtain wheat straw biochar WBC;
(2) preparing alkali modified charcoal: fully mixing the biochar WBC obtained in the step (1) with NaOH solution according to the mass ratio of 1:3, stirring for two hours at room temperature by using a magnetic stirrer, then placing the mixture in an oven for drying, placing a dried sample in a corundum boat, carrying out pyrolysis in a tubular furnace by using nitrogen as an environmental atmosphere, cooling to room temperature, soaking the prepared biochar for two hours by using 1.5mol/L hydrochloric acid, then washing for a plurality of times by using deionized water, drying in the oven, homogenizing by using a 100-mesh sieve to obtain alkali modified biochar SBC, and storing the alkali modified biochar SBC in a glass drying dish for later use;
(3) preparing magnetic modified biochar: mixing the alkali modified biochar obtained in the step (2) with 0.02mol FeCl36H2O and 0.01mol FeCl2·4H2Fully mixing O in ethanol solution, putting the mixture into a corundum boat, covering the corundum boat with tinfoil, pyrolyzing the mixture in a tube furnace, cooling the mixture to room temperature, and then washing the mixture for a plurality of times by using deionized water to prepare the magnetic porous biochar SBC @ Fe3O4；
(4) Preparing immobilized laccase: dispersing the magnetically modified biochar obtained in the step (3) in a glutaraldehyde solution, washing the activated carrier with deionized water and a citric acid-disodium hydrogen phosphate buffer solution for several times, and drying in an oven to obtain SBC @ Fe3O4The magnetic modification glutaraldehyde of @ GA activates biochar, laccase solutions with different concentration gradients are added into the carrier according to different enzyme-carbon ratios through a three-factor five-level orthogonal experimental method, and the mixture is stirred for a plurality of hours by a magnetic stirrer to obtain SBC @ Fe3O4The product of @ GA @ Lac is chosen according to the loading capacity of laccase, the product is washed for several times by using citric acid-disodium hydrogen phosphate buffer solution, and is subjected to suction filtration and magnet collection and storage for later use.
2. The preparation method of the biochar immobilized laccase based on sodium hydroxide and ferroferric oxide crystal composite modification according to claim 1, which is characterized in that,
the temperature of the oven in the step (1) is 105 ℃, and the heat preservation time is 220 min;
in the step (1), the heating temperature of the muffle furnace is 500 ℃, and the heating time is 2.5 h;
the concentration of the sodium hydroxide solution in the step (2) is 4 mol/L;
the temperature of the oven in the step (2) is 105-120 ℃;
the flow rate of nitrogen of the tubular furnace in the step (2) is 80-100 ml/min, the heating rate is 10 ℃/min, the heating temperature is 500 ℃, the heating temperature is 800 ℃ at the heating rate of 5 ℃/min, and the heat preservation time is 90 min;
the flow rate of nitrogen of the tubular furnace in the step (3) is 80-100 ml/min, the heating temperature is 500 ℃, the heating rate is 10 ℃/min, and the heat preservation time is 110 min;
the temperature of the oven in the step (4) is 105 ℃;
the pH value of the citric acid-disodium hydrogen phosphate buffer solution in the step (4) is 5, the citric acid-disodium hydrogen phosphate buffer solution is prepared by mixing 0.1mol/L citric acid solution and 0.2mol/L disodium hydrogen phosphate solution,
the volume ratio of the glutaraldehyde solution in the selected step (4) is 2.5%.
3. The preparation method of the biochar-immobilized laccase based on composite modification of sodium hydroxide and ferroferric oxide crystals according to claim 1, wherein the three factors set by the orthogonal experimental method in the step (4) are as follows: the charcoal enzyme ratio is mg/mg, the concentration of the laccase solution is U/mL, and the immobilization time is h.
4. The preparation method of the biochar-immobilized laccase based on composite modification of sodium hydroxide and ferroferric oxide crystals according to claim 1, wherein five proportional gradients of enzyme-to-carbon ratio mg/mg in the step (4) are 1:2,1: 3,1: 4,1: 5,1: 6.
5. the preparation method of the biochar-immobilized laccase based on sodium hydroxide and ferroferric oxide crystal composite modification according to claim 1, characterized in that five proportional gradients of the concentration U/mL of the laccase solution in the step (4) are 2, 4, 6, 8 and 10U/mL.
6. The preparation method of the biochar immobilized laccase based on sodium hydroxide and ferroferric oxide crystal composite modification according to claim 1, wherein five proportional gradients of the immobilization time h in the step (4) are 8, 12, 16, 20 and 24.
7. The preparation method of the biochar-immobilized laccase based on sodium hydroxide and ferroferric oxide crystal composite modification according to claim 1, wherein the determination method of laccase load in the step (4) is as follows: weighing 0.05g of immobilized product, adding 1mL of buffer solution, centrifuging for 10min at the temperature of 4 ℃ and the centrifugal force of 8000g, taking supernatant as a solution to be detected, respectively adding 500 microliters of distilled water and 500 microliters of Coomassie brilliant blue indicator into a quartz cuvette as a blank, respectively adding 500 microliters of solution to be detected and 500 microliters of Coomassie brilliant blue indicator into the quartz cuvette, detecting absorbance at 620nm, and calculating to obtain the loading mg/g of laccase;
the formula is as follows:
protein concentration C of the initial laccasei(mg/mL)＝0.07×(ΔA+0.0007)
Protein concentration C of the immobilized laccasef(mg/mL)＝0.07×(ΔA+0.0007)
Wherein Δ A represents the change in absorbance before and after the measurement,
wherein, V1Representing the amount of free laccase solution mL, V added during the initial laccase protein concentration determination2And the amount of the liquid to be measured mL added in the process of measuring the concentration of the immobilized laccase protein is represented.
8. The preparation method of the biochar-immobilized laccase based on sodium hydroxide and ferroferric oxide crystal composite modification according to claim 1, characterized in that the determination method of the enzyme activity is as follows: taking about 15mg of free laccase or immobilized laccase in a 15ml centrifuge tube, sequentially adding 2ml of citric acid-disodium hydrogen phosphate buffer solution with the optimal pH value and 2ml of 0.5mmoL/LABTS solution, immediately centrifuging for 10min at the centrifugal force of 8000g and 4 ℃, taking 1ml of supernatant in a quartz slit cuvette, measuring the absorbance at 420nm, fully oscillating, carrying out water bath for 3min at the optimal temperature, centrifuging again for 10min at the centrifugal force of 8000g and 4 ℃, taking 1ml of supernatant in the quartz slit cuvette, measuring the absorbance again at 420nm, and calculating the enzyme activity by using an ABTS method formula;
the formula is as follows:
wherein Δ A represents the increase in absorbance of ABTS, VtThe volume L of the reaction system, ε the molar extinction coefficient of ABTS,. DELTA.T the reaction time min and m the mass g of the enzyme.
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