CN110846357B - Method for degrading corn straw - Google Patents

Method for degrading corn straw Download PDF

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CN110846357B
CN110846357B CN201911201549.6A CN201911201549A CN110846357B CN 110846357 B CN110846357 B CN 110846357B CN 201911201549 A CN201911201549 A CN 201911201549A CN 110846357 B CN110846357 B CN 110846357B
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laccase
corn
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immobilized
pei
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CN110846357A (en
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孙旸
陈�光
苟泽昌
孙春玉
苏瑛杰
雷志鹏
张斯童
吴卓夫
王撼宇
卢芳
邓青青
顾剑锋
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Jilin Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • 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)
    • 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)
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis

Abstract

A method for degrading corn straws is characterized by comprising the following steps: the corn stalk is mechanically crushed, sieved and dried, and then Cu is used2+Modified Fe3O4‑NH2Degrading lignin in the corn straws by using immobilized laccase with PEI magnetic nanoparticles as carriers, then recovering the immobilized laccase, boiling the degraded corn straws, adding NaOH for washing, and then adding cellulase to further degrade cellulose. The method enhances the affinity of the immobilized laccase with the substrate, and improves the stability and the activity of the laccase in the application process, thereby improving the degradation efficiency, and the degradation rate of the laccase to lignin reaches more than 40.76 percent. The inhibition of the immobilized laccase and degradation products such as phenolic substances generated by degradation on the activity of the cellulase is eliminated, so that the activity of the cellulase is ensured, the conversion rate of the cellulose is improved, and the conversion rate reaches 46.85%.

Description

Method for degrading corn straw
Technical Field
The invention belongs to the field of straw degradation, and particularly relates to a method for degrading corn straws by using immobilized laccase.
Background
Corn stover is a very abundant renewable resource, and its content of lignocellulose is the most valuable biological energy source. The lignocellulose of different plants has different compositions and structures, and the lignocellulose of the corn straws mainly comprises cellulose, hemicellulose and lignin. However, cellulose, hemicellulose and lignin in the corn stalks are interwoven to form a cell wall structure, the interwoven structure determines that the degradation of any component is bound by other components, particularly the lignin and the hemicellulose are combined in a covalent bond mode to wrap the cellulose, a natural barrier is formed to prevent the cellulase from contacting cellulose molecules, so that the decomposition of the cellulose is limited, the efficiency of degrading the stalks by cellulolytic enzymes is low, and the effective utilization of lignocellulose of the corn stalks is prevented. Therefore, the effective pretreatment of the straws is the most important link in the resource utilization of the straws, and the key point of the effective utilization of the lignocellulose is whether the lignin can be degraded or not.
Laccase is a promising mild and clean lignin degradation path as a lignin degradation enzyme, and plays an important role in the lignin degradation process. Laccase destroys the structure of lignin by changing the hydrophobicity and porosity of the lignin, and improves the combination of cellulase and cellulose, thereby improving the utilization rate of lignocellulose. However, laccase is not widely used in industry because of its high production cost, poor stability, easy deformation and inactivation due to environmental change during use.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for efficiently degrading corn straws.
The purpose of the invention is realized by the following technical scheme:
a method for degrading corn straws is characterized by comprising the following steps: the corn stalk is mechanically crushed, sieved and dried, and then Cu is used2+Modified Fe3O4-NH2Degrading lignin in the corn straws by using immobilized laccase with PEI magnetic nanoparticles as carriers, then recovering the immobilized laccase, boiling the degraded corn straws, adding NaOH for washing, and then adding cellulase to further degrade cellulose.
Further, the step of degrading the lignin in the corn straws by using the immobilized laccase is to perform oscillation treatment on the immobilized laccase, the corn straws and the NaAC-HAC buffer solution at 35 ℃ for 12-72 hours.
Further, the oscillation rate was 160 rpm.
Further, the pH of the NaAC-HAC buffer was 4.6.
Further, the addition amount of the immobilized laccase is 10-60U g-1。
Further, the mass-volume ratio of the corn straws to the NaAC-HAC buffer solution is 1 g: 200 μ L.
Further, the corn straw is boiled for 20-30min, and is washed by adding 0.1% NaOH solution after being cooled.
Further, the mass-volume ratio of the corn straw to the immobilized laccase is 1 g: 50 mL.
Further, the immobilized laccase is Cu2+Modified Fe3O4-NH2PEI magnetic nano-particles as a carrier, in particular Cu2+Modified Fe3O4-NH2-PEI magnetic nanoparticles are dissolved in a buffer solution containing laccase and subjected to oscillation treatment at a speed of 150 rpm for 40-70min at 25 ℃; after reaction, the prepared Cu2+Modified Fe3O4-NH2-separating PEI laccase magnetic nanoparticles from the supernatant; using ultrapure water to Cu2+Modified Fe3O4-NH2-successive washings of PEI laccase magnetic nanoparticles until no laccase activity was detected in the detergent.
Further, the laccase is white rot fungus laccase.
Laccase inactivation is easily caused in the laccase immobilization process, and the immobilized laccase is easily desorbed.
The inventor finds that laccase contains rich amino acid residues, can form a special covalent bond with transition metal, adsorbs laccase in a metal affinity manner to form immobilized laccase, effectively ensures the activity of the laccase in the process of degrading corn straw lignin, can specifically immobilize the laccase in the use process, and effectively reduces the probability that the laccase is desorbed and turns into a free state.
Further, the laccase-containing buffer solution is NaAC-HAC buffer solution with pH of 4.6, Cu2+Modified Fe3O4-NH2-the mass-to-volume ratio of the PEI magnetic nanoparticles to the NaAC-HAC buffer is 1 mg: 10mL, and the concentration of the laccase is 0.5-4 mg/mL.
Preferably, the shaking treatment time is 50min, and the concentration of the laccase is 2.5 mg/mL.
A method for degrading corn stalks is characterized by comprising the following steps:
(1) mechanically pulverizing corn stalks, sieving with a 40-mesh sieve, and drying at 105 deg.C;
(2) immobilization of laccase in Cu2+Modified Fe3O4-NH2Forming immobilized laccase on PEI magnetic nanoparticles, specifically Cu2+Modified Fe3O4-NH2-PEI magnetic nanoparticles are dissolved in NaAC-HAC buffer solution containing laccase, and the solution is subjected to oscillation treatment at the speed of 150 rpm at 25 ℃ for 40-70 min; after reaction, the prepared Cu2+Modified Fe3O4-NH2-separating PEI laccase magnetic nanoparticles from the supernatant; using ultrapure water to Cu2+Modified Fe3O4-NH2-successive washing of the PEI laccase magnetic nanoparticles until no laccase activity is detected in the detergent; wherein the pH of the NaAC-HAC buffer solution is 4.6, the concentration of the laccase is 0.5-4mg/mL, and the concentration of the laccase is Cu2+Modified Fe3O4-NH2Mass-to-volume ratio of PEI magnetic nanoparticles to NaAC-HAC buffer solution was 1 mg: 10 mL;
(3) carrying out oscillation treatment on the immobilized laccase prepared in the step (2), the corn straw treated in the step (1) and a NaAC-HAC buffer solution at 35 ℃ at a speed of 160 r/min for 12-72 h; laccase addition amount is 10-60Ug-1Maize strawThe mass-to-volume ratio of the buffer solution to NaAC-HAC buffer solution with pH of 4.6 is 1 g: 200 mu L;
(4) and (3) adsorbing and recovering the immobilized laccase by using a magnet after treatment, washing the corn straws after the immobilized laccase is recovered, boiling the corn straws with clear water for 20-30min, cooling the corn straws, washing the corn straws with 0.1% NaOH solution, wherein the mass-volume ratio of the corn straws to the NaOH is 1 g: 50 mL;
(5) and (4) adding cellulase into the corn straws treated in the step (4), and oscillating for 72 hours at the temperature of 50 ℃ at the speed of 140 revolutions per minute, wherein the adding amount of the cellulase is 30fpu per gram.
Because laccase is immobilized, the flexibility of laccase movement in a system is reduced, the binding rate of laccase and a substrate is reduced, and the utilization of active sites of laccase is hindered; therefore, the laccase is immobilized to greatly restrict the degradation capability of the laccase to lignin, the technical problem is effectively solved, the immobilized laccase and the substrate have good affinity, the immobilized laccase and the substrate are effectively combined, the active site of the laccase is fully utilized, and the Mie constant K of the affinity of the immobilized laccase and the substrate is fully utilizedmThe value is 5.91, the affinity of the free laccase and the substrate is close, and the degradation capability of the laccase is improved.
In addition, the inventor finds that laccase exists in a system and has an inhibiting effect on the activity of cellulase in the process of degrading lignin by laccase, and degradation products such as phenolic substances and the like generated in the process of degrading lignin by laccase also have an inhibiting effect on the activity of cellulase, so that the conversion rate of cellulose is low. According to the invention, the immobilized laccase is used for degrading and pretreating lignin in the corn straws, after the immobilized laccase is recovered, the inhibition effect of a large amount of laccase on the cellulase is eliminated, and then the inhibition effect of a small amount of laccase falling from a carrier into a system in the degradation process and degradation products such as phenols generated by degrading the lignin by the laccase on the cellulase are also eliminated through the combination of the steps of boiling the corn straws after the lignin is degraded and washing the corn straws by NaOH, so that the hydrolysis capability of the subsequent cellulase on the cellulase in the corn straws is improved.
The invention has the following technical effects:
(1) by passingLaccase enzyme and Cu2+Modified Fe3O4-NH2The metal affinity adsorption of PEI magnetic nano particles can recycle laccase and reduce the inactivation rate of laccase, the activity of the laccase is about 50% after 6 cycles of the recycling, and the industrial application cost of the laccase is greatly reduced.
(2) The affinity of the immobilized laccase and lignin is increased, and the activity and stability of the laccase in the application process are improved, so that the degradation efficiency is improved, and the degradation rate of the lignin is over 40.76%.
(3) The inhibition effect of degradation products such as phenolic substances and the like and the immobilized laccase on the activity of the cellulase is eliminated, so that the activity of the cellulase is ensured, the conversion rate of the cellulose is improved, and the conversion rate reaches 46.85%.
Drawings
FIG. 1: scanning electron micrographs of the corn stalks before and after degradation.
FIG. 2: FTIR patterns and XRD patterns before and after the degradation of the corn stalks in the invention.
FIG. 3: temperature and pH influence curves on the stability of laccase before and after immobilization.
FIG. 4: stability of immobilized laccase and free laccase to ABTS.
FIG. 5: the influence of the immobilized laccase on lignin degradation is avoided;
wherein, a is a concentration-efficiency curve chart and b is a time-efficiency curve chart.
FIG. 6: cellulose conversion efficiency profiles for different pretreatments.
Detailed Description
The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-mentioned disclosure.
The raw materials used in the invention are as follows:
corn stalks; biological utilization of white rot fungus laccase from straws of education departmentProviding by a point laboratory; cellulase, polyethylenimine (PEI, branched, Mw 10000g/mol) purchased from Sigma Aldrich (usa); ferric chloride hexahydrate (FeCl)3·6H2O), Ethylenediamine (EDA), glacial acetic acid (HAC), sodium acetate (NaAC), copper sulfate (CuSO)4·5H2O), sodium hydroxide (NaOH), Ethylene Glycol (EG), and disodium Ethylenediaminetetraacetate (EDTA), available from pharmaceutical chemicals ltd; glutaraldehyde (GA, 50% aqueous solution), sodium Chloroacetate (CH)2ClCOONa) was provided by tianjin zhiyuan reagent ltd; sodium borohydride (NaBH)4)。
Fe3O4-NH2Preparation: 4.3g of FeCl3·6H2O is added with 0.67g of ethylenediamine, mixed and then added with 1.34g of sodium acetate and 20mL of ethylene glycol, and reacted for 8h at 200 ℃. Fe3O4-NH2Also available from Beijing Gega nanotechnology, Inc.
Cu in the invention2+Modified Fe3O4-NH2Preparation of PEI magnetic nanoparticles according to the prior art ("Synthesis of polyethylene modified Fe)3O4 nanoparticles with immobilizedCu2+for high purity functional proteins adsorption ", Yuepan Guan et al, gels and Surfaces A: physical chemical and Engineering applications, Vol.443: 552-559, 2014).
Example 1:
a method for degrading corn stalks is characterized by comprising the following steps:
(1) mechanically pulverizing corn stalks, sieving with a 40-mesh sieve, and drying at 105 deg.C;
(2) mixing Cu2+Modified Fe3O4-NH2-PEI magnetic nanoparticles are dissolved in NaAC-HAC buffer solution containing laccase, and subjected to oscillation treatment at a speed of 150 rpm for 40min at 25 ℃; after reaction, the prepared Cu2+Modified Fe3O4-NH2-separating PEI laccase magnetic nanoparticles from the supernatant; using ultrapure water to Cu2+Modified Fe3O4-NH2Continuous washing of the PEI laccase magnetic nanoparticles until the detergent is finishedUntil laccase activity is not detected; wherein the pH of the NaAC-HAC buffer solution is 4.6, the concentration of the laccase is 0.5mg/mL, and the Cu content is2+Modified Fe3O4-NH2Mass-to-volume ratio of PEI magnetic nanoparticles to NaAC-HAC buffer solution was 1 mg: 10 mL;
(3) carrying out oscillation treatment on the immobilized laccase prepared in the step (2), the corn straw treated in the step (1) and a NaAC buffer solution at 35 ℃ at a speed of 160 rpm for 12 hours; laccase addition amount is 10Ug-1The mass-to-volume ratio of the corn straws to the NaAC buffer solution with the pH value of 4.6 is 1 g: 200 mu L;
(4) and (3) adsorbing and recovering the immobilized laccase by using a magnet after treatment, washing the corn straws recovered with the immobilized laccase, boiling the washed corn straws in clear water for 30min, cooling the corn straws, washing the corn straws by using a 0.1% NaOH solution, wherein the mass-volume ratio of the corn straws to the NaOH is 1 g: 50 mL;
(5) and (4) adding cellulase into the corn straws treated in the step (4), and oscillating for 72 hours at the temperature of 50 ℃ at the speed of 140 revolutions per minute, wherein the adding amount of the cellulase is 30fpu per gram.
Example 2:
a method for degrading corn stalks is characterized by comprising the following steps:
(1) mechanically pulverizing corn stalks, sieving with a 40-mesh sieve, and drying at 105 deg.C;
(2) immobilization of laccase in Cu2+Modified Fe3O4-NH2Forming immobilized laccase on PEI magnetic nanoparticles, specifically Cu2+Modified Fe3O4-NH2-PEI magnetic nanoparticles are dissolved in NaAC-HAC buffer solution containing laccase, and subjected to oscillation treatment at the speed of 150 rpm at 25 ℃ for 70 min; after reaction, the prepared Cu2+Modified Fe3O4-NH2-separating PEI laccase magnetic nanoparticles from the supernatant; using ultrapure water to Cu2+Modified Fe3O4-NH2-successive washing of the PEI laccase magnetic nanoparticles until no laccase activity is detected in the detergent; wherein the pH of the NaAC-HAC buffer solution is 4.6, the concentration of the laccase is 4mg/mL, and the concentration of Cu is2+Modified Fe3O4-NH2-PEI magnetic nanoparticlesThe mass-to-volume ratio of the particles to the NaAC-HAC buffer solution was 1 mg: 10 mL;
(3) carrying out oscillation treatment on the immobilized laccase prepared in the step (2), the corn straw treated in the step (1) and a NaAC buffer solution at 35 ℃ at a speed of 160 rpm for 72 hours; laccase addition amount is 60Ug-1The mass-to-volume ratio of the corn straws to the NaAC buffer solution with the pH value of 4.6 is 1 g: 200 mu L;
(4) and (3) adsorbing and recovering the immobilized laccase by using a magnet after treatment, washing the corn straws recovered with the immobilized laccase, boiling the washed corn straws in clear water for 20min, cooling the corn straws, washing the corn straws by using a 0.1% NaOH solution, wherein the mass-volume ratio of the corn straws to the NaOH is 1 g: 50 mL;
(5) and (4) adding cellulase into the corn straws treated in the step (4), and oscillating for 72 hours at the temperature of 50 ℃ at the speed of 140 revolutions per minute, wherein the adding amount of the cellulase is 30fpu per gram.
Example 3:
a method for degrading corn stalks is characterized by comprising the following steps:
(1) mechanically pulverizing corn stalks, sieving with a 40-mesh sieve, and drying at 105 deg.C;
(2) immobilization of laccase in Cu2+Modified Fe3O4-NH2Forming immobilized laccase on PEI magnetic nanoparticles, specifically Cu2+Modified Fe3O4-NH2-PEI magnetic nanoparticles are dissolved in NaAC-HAC buffer solution containing laccase, and subjected to oscillation treatment at the speed of 150 rpm for 50min at 25 ℃; after reaction, the prepared Cu2+Modified Fe3O4-NH2-separating PEI laccase magnetic nanoparticles from the supernatant; using ultrapure water to Cu2+Modified Fe3O4-NH2-successive washing of the PEI laccase magnetic nanoparticles until no laccase activity is detected in the detergent; wherein the pH of the NaAC-HAC buffer solution is 4.6, the concentration of the laccase is 20.5mg/mL, and the Cu content is2+Modified Fe3O4-NH2Mass-to-volume ratio of PEI magnetic nanoparticles to NaAC-HAC buffer solution was 1 mg: 10 mL;
(3) the immobilized laccase prepared in the step (2) and the corn straw treated in the step (1)Carrying out oscillation treatment on the stalks and the NaAC buffer solution at 35 ℃ at the speed of 160 rpm for 40 h; laccase addition amount is 50Ug-1The mass-volume ratio of the corn straws to the NaAC-HAC buffer solution with the pH value of 4.6 is 1 g: 200 mu L;
(4) and (3) adsorbing and recovering the immobilized laccase by using a magnet after treatment, washing the corn straws after the immobilized laccase is recovered, boiling the corn straws with clear water for 25min, cooling the corn straws, washing the corn straws with 0.1% NaOH solution, wherein the mass volume ratio of the corn straws to the NaOH is 1 g: 50 mL;
(5) and (4) adding cellulase into the corn straws treated in the step (4), and oscillating for 45 hours at the temperature of 50 ℃ at the speed of 140 revolutions per minute, wherein the addition amount of the cellulase is 30fpu per gram.
The immobilized laccase prepared in the step (2) has an immobilization rate of 87.4% and an immobilization efficiency of 85.2%.
The free laccase and immobilized laccase were the highest in activity at pH 4.6 and 35 ℃ as shown in FIGS. 3a and 3 b. Due to Cu2+Modified Fe3O4-NH2The PEI magnetic nanoparticles and the laccase have strong combination effect, and special covalent bonds are formed between amino groups and transition metal ions, so that the immobilized laccase has a larger application range to temperature and pH value and has better stability.
The reaction rates of NaAC-HAC buffer (pH 4.6) laccase and ABTS were measured at 30 ℃ and Lineweaver Burk curves were used to estimate the kinetic properties (Mie constant (K) for free laccase and immobilized laccasem) And maximum reaction rate (V)max)). Km and VmaxThe values were calculated according to the Lineweaver-burk plot double reciprocal model as described in equations (1) and (2) below:
V＝(Vmax[S])/([S]+Km) (1)
1/V＝[(Km/Vmax)(1/[S])]+1/Vmax) (2)
km reflects the affinity of the enzyme for the substrate, KmThe smaller the value, the higher the affinity of the enzyme for the substrate. K for the oxidation reaction was calculated using the slope and intercept of the Lineweaver Burk plot, respectivelymAnd Vmax. Kinetic measurements of free laccase and immobilized laccase are shown in FIG. 4Shown in the figure. Substituting the formula to calculate the K of the free laccasemValue 5.86, K of immobilized laccasemThe value was 5.91. It is known that the affinity of immobilized laccase for the substrate approaches that of free laccase.
SEM analysis shows that the change of the surface structure of the corn straw before and after degradation of the immobilized laccase is respectively shown in figure 1a and figure 1b, and the observation result of a scanning electron microscope shows that the surface of the corn straw becomes rough, porous and disordered after degradation.
In studying the effect of laccase concentration and treatment time on lignin degradation, the lignin degradation rate gradually increased when the concentration of immobilized laccase increased, and when the concentration of laccase exceeded 50U g-1At this time, the degradation rate of lignin slowly increased, as shown in fig. 5 a. When the concentration of the immobilized laccase is 50U g-1And when the treatment time is 72 hours, the lignin degradation rate can reach 40.76 percent, as shown in figure 5 b.
Different pretreatment methods have different cellulose conversion rates. After the corn straws are pretreated by using the free laccase, the inactivated laccase and the immobilized laccase to degrade lignin, the cellulose conversion rates are 14.35%, 14.51% and 27.33% respectively. After the maize straws are degraded by using the inactivated laccase and the free laccase, compared with the maize straws which are not degraded by using the laccase, the cellulose conversion rate is obviously reduced. Compared with the free laccase pretreatment and the inactivated laccase pretreatment, the cellulose conversion rate of the immobilized laccase pretreatment corn stalks is respectively improved by 12.98% and 12.82%, while the cellulose conversion rate is only improved by 2.42% compared with the corn stalks which are not subjected to the lignin degradation pretreatment, as shown in fig. 6 a. The immobilized laccase drops a small amount of laccase from the carrier in the corn straw pretreatment process, and degradation products such as phenolic compounds generated by lignin degradation inhibit the activity of the cellulase. In order to completely eliminate the inhibition effect of laccase, corn straws treated by immobilized laccase are treated by a method of boiling and then adding 0.1% NaOH for washing, so that the inhibition effect of degradation products such as a small amount of laccase and phenolic substances dropped from a carrier on the activity of cellulase is eliminated. The results show that the cellulose conversion rate of the corn stalks treated with the immobilized laccase reaches 46.85%, which is 23.9% higher than that of the corn stalks which are not washed with laccase and boiled and 0.1% NaOH, as shown in FIG. 6 b.

Claims (4)

1. A method for degrading corn straws is characterized by comprising the following steps: the corn stalk is mechanically crushed, sieved and dried, and then Cu is used2+Modified Fe3O4-NH2Degrading lignin in corn straws by using immobilized laccase with PEI magnetic nanoparticles as carriers, then recovering the immobilized laccase, boiling the corn straws after degrading the lignin for 20-30min, adding 0.1% NaOH for washing, then adding cellulase for further degrading cellulose, specifically, carrying out oscillation treatment on the lignin in the corn straws by using the immobilized laccase, the corn straws and NaAC-HAC buffer solution with the pH of 4.6 at 35 ℃ at the oscillation rate of 160 r/min for 12-72 h, wherein the addition amount of the immobilized laccase is 10-60U g-1The mass-volume ratio of the corn straws to the NaAC-HAC buffer solution is 1 g: 200 μ L.
2. The method for degrading corn stover according to claim 1, wherein the step of: the immobilized laccase is Cu2 +Modified Fe3O4-NH2PEI magnetic nano-particles as a carrier, in particular Cu2+Modified Fe3O4-NH2-PEI magnetic nanoparticles are dissolved in a buffer solution containing laccase and subjected to oscillation treatment at a speed of 150 rpm for 40-70min at 25 ℃; after reaction, the prepared Cu2+Modified Fe3O4-NH2-separating PEI laccase magnetic nanoparticles from the supernatant; using ultrapure water to Cu2+Modified Fe3O4-NH2-successive washings of PEI laccase magnetic nanoparticles until no laccase activity was detected in the detergent.
3. The method for degrading corn stover according to claim 2, wherein the step of: the laccase-containing buffer solution is NaAC-HA with pH of 4.6C buffer solution, Cu2+Modified Fe3O4-NH2-the mass-to-volume ratio of the PEI magnetic nanoparticles to the NaAC-HAC buffer is 1 mg: 10mL, and the concentration of the laccase is 0.5-4 mg/mL.
4. The method for degrading corn stover according to claim 1, comprising the steps of:
(1) mechanically pulverizing corn stalks, sieving with a 40-mesh sieve, and drying at 105 deg.C;
(2) immobilization of laccase in Cu2+Modified Fe3O4-NH2Forming immobilized laccase on PEI magnetic nanoparticles, specifically Cu2+Modified Fe3O4-NH2-PEI magnetic nanoparticles are dissolved in NaAC-HAC buffer solution containing laccase, and the solution is subjected to oscillation treatment at the speed of 150 rpm at 25 ℃ for 40-70 min; after reaction, the prepared Cu2+Modified Fe3O4-NH2-separating PEI laccase magnetic nanoparticles from the supernatant; using ultrapure water to Cu2+Modified Fe3O4-NH2-successive washing of the PEI laccase magnetic nanoparticles until no laccase activity is detected in the detergent; wherein the pH of the NaAC-HAC buffer solution is 4.6, the concentration of the laccase is 0.5-4mg/mL, and the concentration of the laccase is Cu2+Modified Fe3O4-NH2Mass-to-volume ratio of PEI magnetic nanoparticles to NaAC-HAC buffer solution was 1 mg: 10 mL;
(3) carrying out oscillation treatment on the immobilized laccase prepared in the step (2), the corn straw treated in the step (1) and a NaAC-HAC buffer solution at 35 ℃ at a speed of 160 r/min for 12-72 h; laccase addition amount is 10-60Ug-1The mass-volume ratio of the corn straws to the NaAC-HAC buffer solution is 1 g: 200 mu L;
(4) and (3) adsorbing and recovering the immobilized laccase by using a magnet after treatment, washing the corn straws recovered with the immobilized laccase, boiling the washed corn straws in clear water for 20-30min, cooling the corn straws, washing the corn straws by using 0.1% NaOH solution, wherein the mass-volume ratio of the corn straws to the NaOH is 1 g: 50 mL;
(5) and (4) adding cellulase into the corn straws treated in the step (4), and oscillating for 72 hours at the temperature of 50 ℃ at the speed of 140 revolutions per minute, wherein the adding amount of the cellulase is 30fpu per gram.
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CN102206624A (en) * 2011-04-15 2011-10-05 北京师范大学 Magnetic composite microsphere immobilized laccase and preparation method thereof
CN106480130A (en) * 2016-12-09 2017-03-08 厦门庚能新材料技术有限公司 A kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw
CN108079978A (en) * 2017-12-15 2018-05-29 吉林化工学院 A kind of Fe3O4-NH2The preparation method and applications of-PEI magnetic Nano materials

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CN102206624A (en) * 2011-04-15 2011-10-05 北京师范大学 Magnetic composite microsphere immobilized laccase and preparation method thereof
CN106480130A (en) * 2016-12-09 2017-03-08 厦门庚能新材料技术有限公司 A kind of method applying recyclable magnetic Nano immobilized enzyme hydrolyzing straw
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