阅读说明：本技术 一种降解玉米秸秆的方法 (Method for degrading corn straw ) 是由 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 于 2019-11-29 设计创作，主要内容包括：一种降解玉米秸秆的方法,其特征在于：具体是将玉米秸秆机械粉碎、过筛、烘干后,用以Cu<Sup>2+</Sup>修饰的Fe<Sub>3</Sub>O4-NH<Sub>2</Sub>-PEI磁性纳米粒子作为载体的固定化漆酶对玉米秸秆中的木质素进行降解,然后回收固定化漆酶,将降解后的玉米秸秆煮沸后加入NaOH进行洗涤,再加入纤维素酶进一步降解纤维素。本发明方法增强了固定化漆酶与底物亲和力,提高了漆酶在应用过程中的稳定性以及活性,从而提高降解效率,对木质素的降解率达到40.76%以上。消除了固定化漆酶以及降解产生的酚类物质等降解产物对纤维素酶活性的抑制,从而保证了纤维素酶活性,提高了纤维素的转化率,其转化率达到46.85%。(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 used 2+ Modified Fe 3 O4‑NH 2 Degrading 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. Eliminates the degradation of immobilized laccase and phenols generated by degradationThe hydrolysate can inhibit the activity of cellulase, so that the activity of cellulase is ensured, the conversion rate of cellulose is improved, and the conversion rate reaches 46.85%.)

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 the 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, adding NaOH for washing, and then adding cellulase to further degrade cellulose.

2. The method for degrading corn stover according to claim 1, wherein the step of: the method for degrading the lignin in the corn straws by using the immobilized laccase specifically comprises the step of carrying out oscillation treatment on the immobilized laccase, the corn straws and a NaAC-HAC buffer solution at 35 ℃ for 12-72 hours.

3. The method for degrading corn stover according to claim 1, wherein the step of: the oscillation rate of the oscillation process is 160 revolutions/min.

4. The method for degrading corn stover according to claim 1, wherein the step of: the addition amount of the immobilized laccase is 10-60U g^-1。

5. The method for degrading corn stover according to claim 1, wherein the step of: the mass-volume ratio of the corn straws to the NaAC-HAC buffer solution with the pH value of 4.6 is 1 g: 200 μ L.

6. The method for degrading corn stover according to claim 1, wherein the step of: and boiling the corn straws for 20-30min, cooling, and adding a 0.1% NaOH solution for washing.

7. The method for degrading corn stover according to claim 1, wherein the step of: the immobilized laccase is Cu^{2 +}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.

8. The method for degrading corn stover according to claim 1, wherein the step of: the buffer solution containing laccase 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, concentration of laccaseIs 0.5-4 mg/mL.

9. 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^-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.

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^-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 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 utilized_mA value of 5.91, close to freeThe affinity of the laccase and the substrate improves the degradation capability of the laccase.

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 laccase with 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; the white rot fungus laccase is provided by key laboratories for straw bio-utilization of education department; 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)₃O₄nanoparticles withimmobilizedCu²⁺for high purity functional proteins adsorption ", Yuepan Guan et al, gels and Surfaces A: physical chemical and Engineering applications, Vol.443: 552-559, 2014).