High-throughput screening transformation and preparation method of efficient lignin peroxidase and degradation effect of straw lignin

Abstract

The invention belongs to the field of biotechnology and environmental management, and particularly relates to a high-throughput screening transformation and preparation method of efficient lignin peroxidase and a degradation effect of straw lignin. An efficient screening platform is established on the basis of an escherichia coli exocrine system, rapid quantitative detection of the enzyme activity is achieved in combination with an ABTS chromogenic reaction, and the screening efficiency can be improved. Key mutation sites (M52I, A188E and 308I) are determined through a directed evolution technology, the mutant D3 is obtained through combination mutation and a bacillus subtilis surface display technology, the enzyme activity of the mutant D3 is improved by 2.35 times, the degradation efficiency of alkali lignin is improved by 50%, and the mutant D3 is easy to operate and high in stability and economic benefit. The method can be widely applied to efficient degradation of lignin in agricultural wastes (such as straws) or environment-friendly treatment of industrial wastewater (such as papermaking wastewater), and a novel enzyme preparation solution is provided for biomass resource utilization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, straw feed and environmental governance, and particularly relates to a high-efficiency lignin peroxidase high-throughput screening modification and preparation method and straw lignin degradation. BACKGROUND

[0002] Lignin peroxidase (EC 1.11.1.14, LiP) is an important heme-containing oxidase in the superfamily of peroxidases, which mainly participates in the efficient degradation of lignin. As a key enzyme secreted by white rot fungi, it has a very high redox potential and can catalyze the cleavage of C-C and C-O bonds in complex lignin macromolecules. Due to its high oxidative capacity for refractory organic pollutants, lignin peroxidase has broad application prospects in the fields of bioenergy production (such as lignocellulosic biomass conversion), paper industry (lignin removal and bleaching), environmental pollution control (degradation of persistent organic pollutants), agricultural waste treatment, and biocatalytic synthesis. Its unique catalytic mechanism and environmental friendly characteristics make it an important tool enzyme in the field of green biotechnology.

[0003] However, the activity of lignin peroxidase produced by nature and expressed heterologously or homologously is low, which is the main limiting factor for its application in the field of biotechnology. In bioenergy production, low-activity LiP degrades lignin slowly, and the residual lignin fragments inhibit the activity of cellulase, which not only prolongs the production cycle of bioethanol but also increases the cost due to the need for additional enzymatic steps. In the paper industry, low-activity LiP cannot efficiently replace traditional chlorine bleaching agents, and its environmental friendly advantage is offset by low process efficiency, making it difficult for the industry to smoothly realize green transformation. When facing high concentrations of organic pollutants (such as pesticides and polycyclic aromatic hydrocarbons), a large amount of enzyme preparation is needed for several days or even weeks of reaction to completely treat them, which is too low in economy and timeliness, making it difficult to meet the needs of large-scale application. Therefore, combining enzyme molecular engineering with immobilization technology can improve the activity of enzymes.

[0004] Directed evolution is a protein engineering strategy based on the principle of simulating natural evolution. By error-prone PCR or DNA shuffling on the coding gene of target enzyme, random mutations are introduced, and then the mutant gene is introduced into the host expression system to generate a library containing millions of mutants. Finally, high-quality mutants with target properties such as catalytic activity or stability are screened from the library. However, the traditional screening method has low throughput and high cost, and it is difficult to cover the diversity of the library, so it is gradually eliminated. Therefore, high-throughput screening technology must be used to achieve rapid screening. To break through the traditional screening method, a color substrate can be selected, and a microplate spectrophotometer can be used for rapid quantitative detection, which facilitates the establishment of a high-throughput screening platform. The high-throughput screening platform constructed by color reaction can cover the diversity of the library. By using the secretion technology of Escherichia coli, the recombinant protein is secreted into the periplasmic space, which not only establishes the mutual relationship between genotype and phenotype, but also realizes the effect of single cell sorting. The high-activity enzyme preparation obtained is immobilized by Bacillus subtilis surface display technology, which is expected to become an important biological manufacturing tool to solve the major challenges of lignin degradation and pollution control. SUMMARY

[0005] In view of the wide industrial application prospect of lignin peroxidase, in view of the shortcomings of existing enzyme preparation production technology and directed evolution screening technology, the present application adopts the following technical solutions: ① A recombinant lignin peroxidase engineering bacterium is constructed based on the outer secretion of Escherichia coli for the first time, which is used to prepare LiP enzyme preparation; ② Based on ① and combined with directed evolution technology, a lignin peroxidase mutant library is constructed, and single cell sorting is realized by secreting recombinant PcLiP into the periplasmic space of Escherichia coli; ③ Based on the absorbance change and color change of the free radical cation (ABTS + ) generated by the oxidation of ABTS, a 96-well plate-based high-throughput colorimetric screening method for lignin peroxidase is established; ④ Based on ② and ③, five recombinant lignin peroxidase mutant strains with improved catalytic activity are screened; ⑤ Based on ④, three mutant sites (M52I, A188E, S308I) with significantly improved activity are selected for combination, and an engineering strain containing immobilized enzyme mutant D3 (M52I-A188E-S308I) is obtained; ⑥ A treatment method for degrading alkali lignin and wheat straw lignin by using immobilized lignin peroxidase is provided.

[0006] The application provides a high-throughput screening and modification method of high-efficiency lignin peroxidase mutants, the lignin peroxidase mutants are obtained through preliminary screening of mutants based on an E. coli exosporulation high-throughput screening scheme, and the lignin peroxidase mutants are obtained through combination mutation, that is, the methionine (Met) at the 52nd position of the lignin peroxidase PcLiP shown in the amino acid sequence SEQ ID NO. 2 is mutated into isoleucine (Ile), the alanine (Ala) at the 188th position is mutated into glutamic acid (Glu), and the serine (Ser) at the 308th position is mutated into isoleucine (Ile).

[0007] To achieve the above-mentioned application purposes, the technical solutions adopted by the application are as follows:

[0008] (I), first, a soluble recombinant lignin peroxidase preparation is prepared, and the steps are as follows:

[0009] 1. The lignin peroxidase (Lignin peroxidase, PcLiP) gene sequence (GenBank: X15599) derived from Phanerochaete chrysosporium is optimized according to the codon bias of E. coli and Bacillus subtilis, a gene encoding PcLiP is obtained, the nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0010] 2. The coding PcLiP gene in step 1 is used as a template, and PCR amplification is carried out using P1 primers; the PcLiP gene fragment obtained through amplification is double-digested using restriction endonucleases NcoI and XhoI, and is connected with a linearized fragment of the expression vector pET-22b of E. coli subjected to the same enzyme digestion, the obtained connection product is transformed into E. coli DH5a competent cells; after the plasmid is extracted, the recombinant plasmid DH5a-pET-22b-PcLiP is obtained. Subsequently, the recombinant plasmid DH5a-pET-22b-PcLiP is transformed into E. coli BL21 (DE3) competent cells, and the recombinant lignin peroxidase engineering bacteria that can be used for periplasmic space expression are obtained, and are recorded as E. coli BL21 (DE3)-pET-22b-PcLiP.

[0011] Preferably, the P1 primers include P1-F: TGGCCATGGCACCGAATCTGGAT and P1-R: GTGCTCGAGATTCGGTGACGGC.

[0012] 3. The recombinant lignin peroxidase engineered E. coli BL21(DE3)-pET-22b-PcLiP obtained in step 2 is inoculated into LB medium containing ampicillin sodium, and after first cultivation, a culture solution is obtained. The culture solution is inoculated into LB liquid medium containing ampicillin sodium for secondary cultivation. When OD600 is 0.6-0.8, soluble inducer is added for continued induction cultivation. After the induction cultivation is completed, the bacterial cell precipitate is collected by centrifugation, resuspended with a lysozyme-containing crushing buffer to obtain a resuspension solution. After incubation, the supernatant is collected by centrifugation again, and the lignin peroxidase enzyme preparation is obtained, which is recorded as E-PcLiP.

[0013] Preferably, the inoculation amount of the recombinant lignin peroxidase engineered bacteria in step 3 is 1-3% into LB medium containing ampicillin sodium, and the concentration of ampicillin sodium in the LB medium is 0.1-0.2 mg / mL. The inoculation amount of the culture solution into LB liquid medium containing ampicillin sodium is 1-3% (v / v). The components of the lysozyme-containing crushing buffer are: 0.5 mg / mL lysozyme, 200 mM Tris, 500 mM sucrose, 1 mM EDTA, pH 8.0. The first cultivation conditions are 30-40°C, 160-200 rpm for 10-16 h. The second cultivation conditions are 30-40°C, 160-200 rpm, 2-6 h. The addition amount of the soluble inducer is 1-5% of the volume of the secondary culture solution. After the soluble inducer is added, the induction cultivation conditions are 12-20°C, 8-16 h. The composition of the soluble inducer is: 2.5% Triton X-100, 2.5% Tween 80, 10% ethanol, 5% glycerol, 50 mM CaCl2, 3 mM IPTG.

[0014] The bacterial cell precipitate is resuspended in the lysozyme-containing crushing buffer, and the use amount ratio is: 0.5-1 g: 2.5-5 mL. The incubation conditions are: 30-40°C, 30-45 min.

[0015] (II) Preparation of high-catalytic-activity mutants based on high-throughput screening method, the steps are as follows:

[0016] 4. The recombinant plasmid DH5α-pET-22b-PcLiP obtained in step 2 is used as a template for error-prone PCR (eq-PCR) amplification using P1 primer. 2+ By reducing the template specificity of DNA polymerase, Mg 2+ By stabilizing non-complementary base pairs, the two synergistic effects can significantly increase the mutation frequency. Therefore, by adjusting the concentrations of Mn 2+ and Mg 2+ , the mutation rate can be controlled.

[0017] Easy Taq PCR reaction system: template 1 ng; forward and reverse primers (10 mM) 1 μL each; 10 x Easy Taq Buffer: 5 μL; dNTPs (2.5 mM): 4 μL; Easy Taq DNA polymerase: 0.5 μL; Mg 2+ Final concentration 3-5 mM and Mn 2 + Final concentration 0.6-1 mM, and finally add sterile ddH2O to make up 50 μL reaction system; based on the reaction system to amplify PcLiP eq gene fragment.

[0018] 5. Use restriction endonuclease NcoI and XhoI to double digest the PcLiP eq gene fragment amplified in step 4 and recombinant plasmid DH5α-pET-22b-PcLiP, to obtain double-digested PcLiP eq fragment and pET-22b linearized vector; recover the double-digested PcLiP eq fragment and pET-22b linearized vector, then perform ligation, and the obtained ligation product is transformed into E. coli DH5α competent cells. Extract the plasmid together to obtain a recombinant mutant plasmid library (DH5α-pET-22b-PcLiP eq ), which is transformed into E. coli BL21(DE3) competent cells to obtain a recombinant lignin peroxidase mutant strain library, denoted as E. coli(DE3) BL21-pET-22b-PcLiP eq .

[0019] 6. Perform high-throughput screening of E. coli secretion expression of the recombinant lignin peroxidase mutant strain library E. coli(DE3) BL21-pET-22b-PcLiP eq in step 5, the specific steps are as follows:

[0020] In a sterile clean bench, transform the recombinant engineering bacteria E. coli(DE3) BL21-pET-22b-PcLiP eqThe single clone mutant strain on the mutation plate is inoculated into a 96-deep well plate containing LB liquid medium with ampicillin sodium, recorded as plate A, and cultured in plate A for a period of time, then transferred to another 96-deep well plate containing LB liquid medium with ampicillin sodium, recorded as plate B; after culturing in plate B for a period of time, N positions are randomly selected in plate B, N is a positive integer, then the bacterial liquid is taken from the N positions respectively to detect OD600, to judge the growth condition of each mutant; when the OD600 of each well is within the range of 0.6-0.8 and the OD600 values differ by no more than 0.1, then the soluble inducer is added to plate B for induction culture;

[0021] After the induction culture is completed, the supernatant is discarded by centrifugation and the precipitate is collected, the lysing buffer containing lysozyme is added to the precipitate in each well to resuspend the precipitate to obtain a suspension, and the mutant recombinant protein suspension is obtained after incubation; after centrifugation of the mutant recombinant protein suspension, the supernatant is transferred to a new 96-shallow well plate, recorded as plate C, for enzyme activity determination of the mutant, and the obtained supernatant is the mutant recombinant protein of the mutant; after enzyme activity determination, high-activity mutants are screened, respectively recorded as H2(L180A), B3(M290L), D8(M52I), E9(A188E) and A10(S308I), and the encoded nucleotide sequences are shown in SEQ ID NO. 3-7;

[0022] The final concentration of ampicillin sodium in plate A and plate B in step 6 is 0.1-0.2 mg / mL, and the culture conditions in plate A and plate B are both 30-40°C, 160-200 rpm constant temperature shaking incubation in an incubator; N≥3, preferably 8; the induction culture conditions are 12-20°C, 160-200 rpm in a low-temperature constant temperature shaking incubation in an incubator for 8-16 h;

[0023] The inoculation amount from plate A to plate B is 1%-5% (v / v), and the addition amount of the soluble inducer is 1-5% of the volume of the culture medium in plate B;

[0024] The centrifugation condition is 4,000 rpm for 15-20 min, the concentration of lysozyme in the lysozyme-containing lysis buffer is 0.5 mg / mL, and the composition of the lysis buffer is: 200 mM Tris, 500 mM sucrose, 1 mM EDTA, pH 8.0; the incubation is carried out in a constant temperature water bath, the temperature is 30-40°C, and the incubation time is 30-45 min.

[0025] (Three) Construction of Bacillus subtilis surface-displayed lignin peroxidase:

[0026] 7. The recombinant plasmid DH5α-pET-22b-PcLiP obtained in step 2 was used as a template, and P2 primers containing Spe I and Hind III restriction sites were used to amplify the PcLiP fragment with Spe I and Hind III restriction sites at both ends. The amplified PcLiP fragment and the DH5α-pHS-CotB plasmid were double-digested with restriction endonucleases Spe I and Hind III, respectively, and the double-digested PcLiP fragment and linearized pHS-CotB vector fragment were purified by gel recovery, respectively, followed by a ligation reaction of the recovered fragments. The ligation product was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the recombinant plasmid DH5α-pHS-CotB-PcLiP.

[0027] The recombinant plasmid DH5α-pHS-CotB-PcLiP was further transformed into B. subtilis WB800N competent cells to obtain a recombinant lignin peroxidase engineering strain that can be used for surface display of B. subtilis, designated as B. subtilis WB800N-pHS-CotB-PcLiP.

[0028] The preferred P2 primers include: P2-F: GGTTCAACTAGTATGGCACCGAATCTG, and P2-R: TAGAAAGCTTTCAGTGGTGGTGGTG.

[0029] 8. The recombinant lignin peroxidase engineering strain B. subtilis WB800N-pHS-CotB-PcLiP obtained in step 7 was inoculated into LB medium containing kanamycin and chloramphenicol, and a culture solution was obtained after a first cultivation. Then, the first culture solution was inoculated into Difco sporulation medium (DSM) for a second cultivation to obtain a spore culture. The bacterial cells were collected by centrifugation, resuspended in GTE buffer, and then incubated and centrifuged again. The precipitate was collected and washed with PBS buffer, and finally, the washed precipitate was resuspended in PBS buffer to obtain a pure spore solution, which was an enzyme preparation, designated as CotB-PcLiP.

[0030] Preferably, the inoculation amount of the recombinant lignin peroxidase engineering bacteria B. subtilis WB800N-pHS-CotB-PcLiP in step 7 is 1-3% in the LB medium containing kanamycin and chloramphenicol, wherein the concentration of chloramphenicol in the LB liquid medium is 5-10 μg / mL, and the concentration of kanamycin is 5-10 μg / mL. The inoculation amount of the first obtained culture solution in the Difco spore formation medium is 1-3% (v / v). The first culture condition is 30-40°C, 160-200 rpm for 10-16 h; the second culture condition is 30-40°C, 160-200 rpm for 30-48 h. The GTE buffer composition: 50 mM glucose, 20 mM Tris-HCl, 10 mM EDTA, 2 mg / mL, pH 7.5. The precipitate is resuspended in GTE buffer, and the volume ratio of the precipitate to GTE buffer is 1:10-20 (v / v). The incubation condition is 30-40°C for 30-60 min; the OD600 of the pure spore solution is 1-2.

[0031] (IV) Construction of B. subtilis surface-displayed lignin peroxidase mutants:

[0032] 9. Based on the 5 mutants screened in step 6, it is found that the enzyme activities of mutants D8 (M52I), E9 (A188E), and A10 (S308) are all increased by more than 70% compared with E-PcLiP. Therefore, three mutation sites M52I, A188E, and S308I are selected for combined mutation, and a combined mutant plasmid DH5α-pHS-CotB-PcLiP-D3 is constructed by three rounds of whole plasmid PCR. The nucleotide sequence of the three-point combined mutant PcLiP-D3 is shown in SEQ ID NO. 8, and the amino acid sequence is shown in SEQ ID NO. 9.

[0033] The specific steps of constructing the combined mutant plasmid by three rounds of whole plasmid PCR are as follows:

[0034] (1) Using DH5α-pHS-CotB-PcLiP plasmid as the template, using mutation primers M52I-F and M52I-R, whole plasmid PCR site-directed mutation is carried out to mutate the catalytic residue Met52 to Ile, and the whole plasmid PCR product containing M52I mutation is amplified. Then the above PCR product is treated with restriction endonuclease DpnI, the original template plasmid is selectively digested, and the DpnI-treated (containing mutation) PCR product is transformed into E. coli DH5α competent cells, the plasmid is extracted, and the mutant plasmid DH5α-pHS-CotB-PcLiP-M52I is obtained.

[0035] Preferably, the primers for full-plasmid PCR site-directed mutation of M52I are the primers at both ends of M52I, and the sequences are as follows: M52I-F: AAGCGCATGAAGCGCTGCGTATCGTTTTTCATGATAGC, and M52I-R: ATACGCAGCGCTTCATGCGCTTCCGCACCACACTG.

[0036] (2) Using the DH5a-pHS-CotB-PcLiP-M52I plasmid obtained in step (1) as a template, and using the mutation primers A188E-F and A188E-R, full-plasmid PCR is performed to obtain a PCR product containing the A188E mutation. The PCR product is treated with the restriction endonuclease DpnI to selectively digest the original template plasmid. The DpnI-treated (containing mutation) PCR product is transformed into E. coli DH5a competent cells, and plasmid extraction is performed to obtain the mutant plasmid DH5a-pHS-CotB-PcLiP-M52I-A188E.

[0037] Preferably, the primers for full-plasmid PCR site-directed mutation of A188E are the primers at both ends of A188E, and the sequences are as follows: A188E-F: CGCACATTCAATTGCGGAGGCAAATGATGTGGAC, and A188E-R: CGCAATTGAATGTGCGCTCAGCAGCCAAACT.

[0038] (3) Using the mutant plasmid DH5a-pHS-CotB-PcLiP-M52I-A188E in step (2) as a template, and using the S308I mutation primers, a PCR product containing the S308I mutation is obtained. The PCR product is treated with the restriction endonuclease DpnI to selectively digest the original template plasmid; the DpnI-treated (containing mutation) PCR product is transformed into E. coli DH5a competent cells, and plasmid extraction is performed to obtain the mutant plasmid DH5a-pHS-CotB-PcLiP-M52I-A188E-S308I, which is denoted as DH5a-pHS-CotB-PcLiP-D3; the mutant gene fragment of the lignin peroxidase carried by the plasmid is denoted as PcLiP-D3;

[0039] The nucleotide sequence of the PcLiP-D3 gene is shown in SEQ ID NO. 8, and the amino acid sequence is shown in SEQ ID NO. 9.

[0040] Preferably, the primers for full-plasmid PCR site-directed mutation of S308I are the primers at both ends of S308I, and the sequences are as follows: S308I-F: CTGTTAATTTTGGCCCGATCTTTTTTCCG, and S308I-R: TCGGGCCAAAATTAACAGGTTTCGGTGCC.

[0041] The amplification system of the whole plasmid PCR in steps (1)-(3) is as follows: 10 μM of both end primers, 1 μL each; 2 x PrimeSTARMax Premix 10 μL; 1 μL of template plasmid, with a concentration of 0.1-1 ng / μL; and ddH2O is added to make up to 20 μL.

[0042] The whole plasmid PCR amplification procedure is as follows: 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 55°C annealing for 15 s, 72°C extension for 10 s, for a total of 30 cycles; and 72°C final extension for 10 min.

[0043] (4) The DH5α-pHS-CotB-PcLiP-D3 plasmid in step (3) is transformed into B. subtilis WB800N competent cells to obtain a recombinant lignin peroxidase mutant engineering bacterium, which is denoted as B. subtilis WB800N-pHS-CotB-PcLiP-D3.

[0044] (5) The recombinant lignin peroxidase mutant engineering bacterium B. subtilis WB800N-pHS-CotB-PcLiP-D3 in step (4) is inoculated into LB culture medium containing kanamycin and chloramphenicol, and after the first cultivation, a culture solution is obtained. Then, the culture solution is inoculated into Difco spore formation culture medium (DSM) for secondary cultivation to obtain a spore culture. The bacterial body precipitate is collected by centrifugation, and the precipitate is resuspended using GTE buffer to obtain a resuspension solution. After incubation, the precipitate is collected again by centrifugation and washed with PBS buffer. Finally, the washed precipitate is resuspended in PBS buffer to obtain a spore suspension, which is the enzyme preparation, denoted as CotB-PcLiP-D3.

[0045] Preferably, the recombination lignin peroxidase mutant engineering bacteria B. subtilis WB800N-pHS-CotB-PcLiP-D3 in step (5) is inoculated into LB medium containing kanamycin and chloramphenicol at an inoculation amount of 1-3%, wherein the concentration of chloramphenicol in the LB liquid medium is 5-10 μg / mL, and the concentration of kanamycin is 5-10 μg / mL. The inoculation amount of the culture solution into the Difco spore formation medium is 1-3% (v / v); the first culture condition is 30-40°C, 160-200 rpm for 10-16 h. The second culture condition is 30-40°C, 160-200 rpm, 30-48 h. The composition of the GTE buffer is: 50 mM glucose, 20 mM Tris-HCl, 10 mM EDTA, 2 mg / mL, pH 7.5. The precipitate is resuspended in GTE buffer, and the volume ratio of the precipitate to GTE buffer is 1:10-20. The incubation condition is: 30-40°C for 30-60 min; the OD600 of the spore suspension is 1-2.

[0046] (V) Application of the enzyme preparation:

[0047] S1, use of mutant enzyme preparation for degrading alkali lignin

[0048] The application provides application of a recombination lignin peroxidase immobilized enzyme preparation CotB-PcLiP-D3 in degrading alkali lignin, and the specific method is as follows:

[0049] Taking alkali lignin as the substrate, the buffer system is Britton-Robinson (BR) buffer, and the cofactor is H2O2.

[0050] The above components are mixed, that is, alkali lignin is used as raw material, mutant enzyme preparation CotB-PcLiP-D3 is added, BR buffer and H2O2 are added, a reaction system is formed, and alkali lignin can be degraded after reaction.

[0051] The pH of the BR buffer is 2.5-4.5; in the reaction system, the concentration of alkali lignin is 0.8-1.2 g / L, the concentration of BR buffer is 25-100 mM, and the concentration of H2O2 is 2-4 mM; the OD600 of the mutant enzyme preparation is 1.0, and the addition amount is 15-30% (v / v) of the reaction system.

[0052] S2, use of mutant enzyme preparation for degrading straw lignin

[0053] Specifically, the application relates to application of mutant enzyme preparation CotB-PcLiP-D3 in degrading wheat straw lignin.

[0054] The steps are as follows: using crushed wheat straw as raw material, combined with mutant enzyme preparation, and synergistic mixed enzymes to form a mixed system, controlling the moisture content of the mixed system, and reacting under certain temperature conditions, the lignin in the wheat straw can be degraded; the mixed enzyme is composed of laccase, glucose oxidase, glucanase, xylanase, acid cellulase, cellobiase and mannosease.

[0055] Preferably, based on the mass of wheat straw, the OD600 of the mutant enzyme preparation CotB-PcLiP-D3 in the steps is 2.0, and the addition amount is 5%-40% (ml / g, v / m), i.e., 5-40ml:100g; the addition amount of laccase is 1-3%; the addition amount of glucose oxidase is 1-3%; the addition amount of glucanase, xylanase, acid cellulase, cellobiase, and mannanase is 0.1% each; the water content of the mixed system is controlled at 40-60%, the temperature is 30-45℃, and the reaction time is 70-96h.

[0056] The advantages and technical effects of this invention are:

[0057] (1) The present invention provides a method for producing a soluble lignin peroxidase enzyme preparation, which uses an Escherichia coli secretion expression system to direct the enzyme to the periplasmic space, thereby achieving a simple and efficient enzyme preparation preparation.

[0058] (2) This invention provides a directed evolution scheme for lignin peroxidase. It employs error-prone PCR technology (with the addition of Mn). 2+ and Mg 2+ (To improve the mutation rate) Construct a random mutant library; Establish a mutant library based on the E. coli periplasmic secretion system; Establish a 96-well plate colorimetric high-throughput screening platform: Utilize lignin peroxidase to catalyze the oxidation of ABTS to generate free radical cations (ABTS). + By detecting ABTS + Rapidly screen for highly active mutants by measuring absorbance changes at 420 nm.

[0059] (3) This invention combines high-quality mutation sites (positions 52, 188, and 308) obtained through high-throughput screening and utilizes Bacillus subtilis surface display technology to easily and rapidly obtain large quantities of immobilized lignin peroxidase preparations. The resulting CotB-PcLiP-D3 immobilized enzyme has high activity, strong stability, and recyclability, enabling the industrial production of lignin peroxidase.

[0060] (4) The immobilized enzyme preparation CotB-PcLiP-D3 prepared in this invention can efficiently degrade alkali lignin and wheat straw lignin. Pretreatment experiments show that it significantly improves the degradation rate of wheat straw lignin.

[0061] (5) The present application integrates three major technical innovations: the establishment of a high-throughput screening platform by E. coli periplasmic secretion, the acquisition of high-quality mutants by multi-site combinatorial mutation, and the immobilized production of enzyme preparations by B. subtilis surface display; this can provide a reference for solving the technical problem of large-scale industrial production of lignin peroxidase. The immobilized enzyme obtained has high activity and can be recycled, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 Comparison chart of lignin peroxidase original sequence and optimized sequence.

[0063] Figure 2 PCR amplification of PcLiP gene fragment.

[0064] Figure 3 Physical map of recombinant plasmid pET-22b-PcLiP.

[0065] Figure 4 Protein expression identification of PcLiP.

[0066] Figure 5 Qualitative enzyme activity detection of PcLiP.

[0067] Figure 6 Error-prone PCR electrophoresis map.

[0068] Figure 7 Double enzyme digestion of error-prone PcLiP fragment and pET-22b vector.

[0069] Figure 8 Verification of positive rate of mutant library.

[0070] Figure 9 Establishment of high-throughput screening scheme.

[0071] Figure 10 Screening template.

[0072] Figure 11 Enzyme activity detection of high-quality mutants.

[0073] Figure 12 PCR specific verification of 5 strains of mutants.

[0074] Figure 13 Expression identification of immobilized enzyme wild type.

[0075] Figure 14 Expression identification of immobilized enzyme mutant.

[0076] Figure 15 Activity detection of immobilized enzyme, wherein WT refers to wild type CotB-PcLiP, and D3 refers to mutant CotB-PcLiP-D3.

[0077] Figure 16 For immobilized enzyme degradation of alkali lignin.

[0078] Figure 17 For the reusability of immobilized enzyme. DETAILED DESCRIPTION

[0079] The following examples are used to further illustrate the present application. Based on the foregoing description and examples, those skilled in the art can understand the core features of the present application, and can make various modifications and adjustments to the present application without departing from the spirit and scope of the present application, to adapt to different application scenarios and conditions. Unless otherwise specified, the experimental materials used in the following examples can be obtained by commercial means; the technical methods used in the present application are the existing technologies in the art.

[0080] Reagent material description:

[0081] Laccase, glucose oxidase, dextranase, acid cellulase, xylanase, cellobiase are commercially available products, purchased from Xiasheng (Ningxia) Biotechnology Development Co., Ltd.;

[0082] ABTS was purchased from Shanghai Maier Company, and alkali lignin was purchased from Macklin Biotech Co., Ltd.

[0083] Receptor bacteria E. coli DH5a, BL21 are chemically transformed competent cells prepared using E. coli DH5a, BL21 strains, purchased from Shanghai Sunway Biotech Co., Ltd.

[0084] The preparation method of Bacillus subtilis WB800N competent cells is described in the reference (Zhang Tianxi. Expression identification of thermophilic esterase of Clostridium thermocellum and spore surface display of Bacillus subtilis, [Master's thesis]. Zhenjiang, Jiangsu: Jiangsu University, 2016.).

[0085] LB medium: per L, formula is 0.5% yeast extract powder, 1% tryptone, 0.5% sodium chloride;

[0086] DSM medium: per L, 0.8% nutrient broth, 0.1 KCl, 0.025% MgSO4.7H2O, 10 μM MnCl2, 1 mM Ca(NO3)2·4H2O, 1 μM FeSO4.

[0087] Example 1: Preparation method of soluble expression of recombinant lignin peroxidase;

[0088] I. Construction of recombinant lignin peroxidase engineering bacteria:

[0089] The lignin peroxidase (PcLiP) gene sequence (GenBank: X15599) from *Phanerochaete chrysosporium* was optimized according to the codon preference of *Escherichia coli* and *Bacillus subtilis* to obtain the gene encoding PcLiP. The nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence is shown in SEQ ID NO.2. Figure 1 This is a sequence alignment diagram of the original sequence and the codon-optimized sequence of the lignin peroxidase (PcLiP) gene.

[0090] Design primers containing NcoI and XhoI restriction sites (P1) to amplify the PcLiP gene sequence. The forward primer sequence P1-F is: TGGCCATGGCACCGAATCTGGAT, and the reverse primer sequence P1-R is: GTGCTCGAGATTCGGTGACGGC. PCR amplification of the target gene fragment PcLiP is as follows: Figure 2 Lane 1 is shown. The PCR reaction conditions were: 98℃ pre-denaturation for 3 min; 30 cycles of denaturation at 98℃ for 10 s, annealing at 55℃ for 15 s, and extension at 72℃ for 12 s; and a final extension at 72℃ for 10 min. Subsequently, the amplified PcLiP gene fragment was double-digested with restriction endonucleases NcoI and XhoI, and ligated to a linearized fragment of the *E. coli* expression vector pET-22b digested with the same restriction enzymes. The ligation product was transformed into *E. coli* DH5α competent cells, and the plasmid was extracted to obtain the recombinant plasmid DH5α-pET-22b-PcLiP. The physical map of the recombinant plasmid DH5α-pET-22b-PcLiP is shown below. Figure 3 As shown.

[0091] The recombinant plasmid DH5α-pET-22b-PcLiP obtained above was transformed into E. coli BL21(DE3) competent cells to obtain the recombinant lignin peroxidase engineered strain E. coli BL21(DE3)-pET-22b-PcLiP that can be used for periplasmic spatial expression.

[0092] II. Expression and Identification of Recombinant Engineered Bacterium E. coli BL21(DE3)-pET-22b-PcLiP

[0093] The recombinant engineering bacteria E. coli BL21(DE3)-pET-22b-PcLiP was inoculated into 5 mL LB medium containing ampicillin sodium (final concentration of 0.1 mg / mL) at an inoculation amount of 1%, and cultured at 37°C and 180 rpm overnight. The overnight cultured bacteria were inoculated into 100 mL LB medium containing ampicillin sodium (final concentration of 0.1 mg / mL) at an inoculation amount of 1%, and cultured at 37°C and 180 rpm until the OD600 was 0.8 to obtain a secondary culture. Then, 1% of the volume of the secondary culture was added with a soluble inducer (soluble inducer components: 2.5% Triton X-100, 2.5% Tween 80, 10% ethanol, 5% glycerol, 50 mM CaCl2, 3 mM IPTG) in a clean bench, and induced and cultured at 16°C and 180 rpm for 12 h to obtain the culture.

[0094] The lysozyme method was used to direct release of the lignin peroxidase preparation: 100 mL of the culture was centrifuged at 10,000 rpm for 20 min, and the precipitate was collected. 1 g of the precipitate was resuspended in 5 mL of a lysis buffer solution containing lysozyme (lysis buffer solution components: 0.5 mg / mL lysozyme, 200 mM Tris 500 mM sucrose, 1 mM EDTA, pH 8.0) to obtain a suspension. After the suspension was incubated at 37°C for 40 min, it was centrifuged at 10,000 rpm for 15 min, and the obtained supernatant was the lignin peroxidase enzyme preparation, denoted as E-PcLiP.

[0095] 20 μL of the E-PcLiP enzyme preparation was mixed with 5 μL of 5×Loading buffer, treated in a 100°C metal bath for 10 min, and then subjected to Western Blot analysis verification. The primary antibody used was Rabbit Anti-6×His Tag Mouse Monoclonal Antibody (used at a concentration of 1:2000), and the secondary antibody was Anti-mouse-IgG-FITC conjugates with Fluorescein Isothiocyanate (at a concentration of 1:5000), both of which were purchased from Shanghai Biotechnology Co., Ltd. Finally, Omni-ECL TM The basic type of chemiluminescence kit (Shanghai Yaenzyme) was used for color development. The Western Blot verification results are shown in Figure 4As shown: a single specific band can be seen in lane 4 of E-PcLiP protein at around 40 kDa, while no specific band can be seen in lane 3 of the precipitate of the engineered bacteria, lane 2 of the periplasmic space extract of the strain carrying empty vector pET-22b and lane 1 of the precipitate of the strain carrying empty vector pET-22b, which is consistent with the expectation. The results show that the recombinant lignin peroxidase preparation E-PcLiP is successfully secreted into the periplasmic space of E. coli.

[0096] III. Qualitative detection of the recombinant lignin peroxidase enzyme preparation

[0097] Since the dye Azure B cannot react with laccase and manganese peroxidase, it is also not easy to be decolorized under biological conditions. Lignin peroxidase can catalyze the oxidation of Azure B in the presence of hydrogen peroxide, destroy its structure, and cause the color to disappear. By observing the color change of the Azure B solution, the degradation activity of lignin peroxidase can be qualitatively detected. Therefore, Azure B dye is used as a qualitative test reagent to preliminarily identify whether the recombinant lignin peroxidase has activity.

[0098] In a 96-well plate, 50 μL of 0.1M BR buffer (Britton-Robinson buffer, pH 3.0), 50 μL of 0.16 mM Azure B solution, 100 μL of lignin peroxidase preparation (E-PcLiP), 50 μL of 5 mM H2O2 solution were added. Inactivated E-PcLiP and periplasmic space extract of the strain carrying empty vector pET-22b were used as controls, and they were left at room temperature. If the reaction system changes from blue to colorless, it indicates that the recombinant lignin peroxidase has activity, otherwise the enzyme has no activity. The results of the qualitative detection are shown in Figure 5 As shown, it is found that inactivated E-PcLiP (pET-22b-PcLiP inactivated) and periplasmic space extract of the strain carrying empty vector pET-22b (pET-22b) cannot decolorize Azure B, only the experimental group containing active E-PcLiP (pET-22b-PcLiP) can decolorize Azure B. This result excludes the influence of non-enzyme factors on the degradation of Azure B, and also proves that the obtained E-PcLiP has the activity of lignin peroxidase.

[0099] Example 2: Directed evolution and high-throughput screening method of recombinant lignin peroxidase;

[0100] (1) Construction of recombinant engineered bacteria E. coli BL21(DE3)-pET-22b-PcLiP eq Mutant library

[0101] The recombinant plasmid DH5α-pET-22b-PcLiP was used as a template, and P1 primer was used in the PCR reaction system with the addition of Mg 2+ The Mn 2+ The Mn 2+ and Mg 2+ can significantly increase the frequency of errors introduced by DNA polymerase during replication: Mn 2+ by reducing the specificity of the polymerase to the template, and Mg 2+ by increasing the stability of the reaction system and promoting the stability of non-complementary base pairs, both of which synergistically increase the mutation frequency. Therefore, by reasonably adjusting the concentrations of Mn 2+ and Mg 2+ , the mutation rate of error-prone PCR can be effectively controlled.

[0102] The error-prone PCR reaction system (50 μL) includes: template 1 ng, forward and reverse primers (10 μM) 1 μL each, 10×Easy Taq Buffer 5 μL, dNTPs (2.5 mM) 4 μL, Easy Taq DNA polymerase 0.5 μL, and the addition of Mg 2+ (4 mM final concentration) and Mn 2+ (1 mM final concentration), respectively, and finally supplemented with sterile ddH2O to make up the system. The amplified PcLiP eq gene fragment has an electrophoresis map as shown in lanes 1-5 of Figure 6 , which is consistent with the expected results.

[0103] The PcLiP eq gene fragment amplified by error-prone PCR was recovered using a DNA gel recovery kit. The PcLiP eq gene fragment and the recombinant plasmid DH5α-pET-22b-PcLiP were double-digested with restriction endonucleases NcoI and XhoI, respectively, at 37°C for 40 min, and the electrophoresis results are shown in lanes 1-4 of Figure 7 . Figure 7 . eq The bands in lanes 1-4 of

[0104] Subsequently, the double-digested linearized vector pET-22b and the PcLiP eq gene fragment were ligated using T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells, and after the growth of the transformants, the plasmid was extracted to obtain the recombinant mutant plasmid library DH5α-pET-22b-PcLiP eqThe plasmid library was transformed into E. coli BL21(DE3) competent cells and plated on solid LB agar plates containing ampicillin sodium (0.1 mg / mL) and cultured overnight. A subset of transformants were picked and verified by PCR using P3 primers (P3-F: TAATACGACTCACTATAGGG, P3-R: GCTAGTATTGCTCAGCGG).

[0105] The results of 1% agarose gel electrophoresis to detect the positive rate of mutants are as follows: Figure 8 As shown, all transformants in lanes 1-16 were positive, indicating successful transformation and successful construction of the recombinant lignin peroxidase mutant strain library E. coli BL21(DE3)-pET-22b-PcLiP. eq .

[0106] (2) Obtaining mutant recombinant protein

[0107] The recombinant lignin peroxidase mutant strain library E. coli BL21(DE3)-pET-22b-PcLiP eq The specific steps for high-throughput screening of E. coli secretory expression are as follows:

[0108] In a sterile laminar flow hood, single-clonal strains from the mutant library plates were picked and transferred to 96-well plates (plate A, each well containing 800 μL of LB liquid medium containing 0.1 mg / mL ampicillin sodium). The plates were incubated at 37°C with shaking at 180 rpm for 12 h to obtain the culture medium. This culture was then transferred at a 2% (v / v) inoculation rate to another 96-well plate (plate B, each well containing 800 μL of LB liquid medium containing 0.1 mg / mL ampicillin sodium). The plates were incubated at 37°C with shaking at 180 rpm for 6 h. Eight wells were randomly selected, and 200 μL of the bacterial culture was collected to measure OD600 and assess the growth status of each mutant. If the OD600 values ​​of each well are essentially the same (essentially the same is defined as an OD600 value difference ≤ 0.1 and OD600 ≈ 0.8), add 1% soluble inducer to plate B and induce culture for 12 h at 16℃ and 180 rpm in a low-temperature constant-temperature shaking incubator. Add 15% glycerol to the remaining culture medium in plate A and store the mutant library at -80℃.

[0109] After the induction culture, the bacteria were collected by centrifugation at 4000 rpm for 20 min using a desktop low-speed centrifuge (2x2x96-hole rotor), and the supernatant was discarded. 200 μL of lysis buffer (200 mM Tris, 500 mM sucrose, 1 mM EDTA, pH 8.0) containing 0.5 mg / mL lysozyme was added to each well of the precipitate, and the precipitate was resuspended to obtain a suspension. The suspension was incubated in a 37°C constant temperature water bath for 40 min. After incubation, centrifugation was performed, and 100 μL of supernatant was aspirated using a syringe and transferred to a new shallow 96-well plate (C plate). The supernatant was the crude enzyme solution of the mutant, which was used for subsequent enzyme activity determination to screen high-activity mutants.

[0110] (3) Establishing a high-throughput screening method for recombinant lignin peroxidase mutant strains (colorimetric method)

[0111] A high-throughput screening method based on the colorimetric method was established for screening high-quality mutants from a mutant strain library. The scheme of high-throughput screening is shown in Figure 9 .

[0112] Lignin peroxidase can rapidly oxidize ABTS through a single electron transfer mechanism to generate a stable blue-green cation radical (ABTS + ). The absorbance change of the radical cation (ABTS + ) generated by the oxidation of ABTS was detected at 420 nm using a microplate reader. The increase in absorbance reflects the degree of oxidation of ABTS and is directly proportional to the depth of the solution color (blue-green intensity). The more the absorbance increases, the darker the color, indicating that the enzyme catalytic reaction is more complete and the enzyme activity is higher. Therefore, ABTS is selected as the reaction substrate for high-throughput screening by the colorimetric method, which can quickly and sensitively screen high-activity mutants.

[0113] The enzyme activity calculation formula is:

[0114] ΔA: Absorbance change of ABTS oxidation product in the reaction system (usually detected at 420 nm).

[0115] ε: Molar extinction coefficient of ABTS cation radical (ABTS + ) (unit: L·mol -1 ·cm -1 ), commonly used value: ε 420 nm=3.6x10 4 L·mol -1 ·cm -1

[0116] Vtotal: Total volume of the reaction system (unit: L).

[0117] t: Reaction time (unit: min).

[0118] V enzyme: enzyme liquid volume added (unit: L).

[0119] D: enzyme liquid dilution multiple (if the original enzyme liquid is diluted for use)

[0120] Enzyme activity determination method: 25mM BR buffer and 1.25mM ABTS were added to each well of plate C (containing mutant crude enzyme liquid), and finally 2mM H2O2 solution was added to start the reaction. Immediately use the enzyme marker to detect the absorbance change value at 420nm within 10min. The activity of E-PcLiP was used as a control to screen mutants with higher enzyme activity than the control. The more the absorbance increases, the higher the enzyme activity.

[0121] The first round of high-throughput screening detected a total of 1800 mutants, and 15 mutants with high enzyme activity were further screened by colorimetric method. The screening result sample is shown in Figure 10 .

[0122] (4) Mutant rescreening and identification

[0123] The 15 strains with high enzyme activity obtained by preliminary screening were inoculated according to their corresponding 96-well plate positions, and the original strains were inoculated together with the recombinant engineering bacteria E. coli BL21(DE3)-pET-22b-PcLiP for amplification culture. Respectively inoculated into 10mL LB liquid medium containing 0.1mg / mL ampicillin sodium, cultured at 37℃, 180rpm overnight, and the culture solution was obtained. Then the culture solution was transferred to 100mL LB liquid medium containing 0.1mg / mL ampicillin sodium at an inoculation amount of 1%(v / v), and cultured at 37℃, 180rpm until OD600 was 0.8. In the clean bench, add soluble inducer (soluble inducer components: 2.5% Triton X-100, 2.5% Tween 80, 10% ethanol, 5% glycerol, 50mM CaCl2 and 3mM IPTG) corresponding to 1% of the culture solution volume, and induce culture at 16℃, 180rpm for 12h. Centrifuge 100mL culture at 10000rpm for 20min to collect bacterial precipitate. Take 1g bacterial precipitate and resuspend in 5mL lysis buffer solution containing lysozyme (lysis buffer solution components: 0.5mg / mL lysozyme, 200mM Tris 500mM sucrose, 1mM EDTA, pH8.0) to obtain a suspension. The suspension was incubated at 37℃ for 40min, then centrifuged at 10000rpm for 15min, and the supernatant was collected. Based on the 15 groups of supernatants corresponding to the 15 strains, 15 mutant proteins were obtained. The enzyme activity of E-PcLiP was used as a control to detect the enzyme activity of the mutants.

[0124] The E-PcLiP and 15 mutant protein solutions were respectively taken 100 μL, 25 mM BR buffer (pH 3.0) was added, 1.25 mM ABTS solution was added, and finally 2 mM H2O2 was added to start the reaction. The change in absorbance at 420 nm was detected by an enzyme marker. The enzyme activity of each mutant was evaluated based on the enzyme activity of E-PcLiP. The enzyme activity of E-PcLiP and 15 mutant proteins was determined by repeated measurement. The relative enzyme activity of each mutant was calculated based on the enzyme activity of unmutated E-PcLiP as 100%. The activity of 5 mutants (H2, B3, D8, E9, A10) was significantly higher than that of the E-PcLiP control group. The corresponding amino acid site mutations of the 5 mutants were: H2 (Leu180Ala), B3 (Met290Leu), D8 (Met52Ile), E9 (Ala188Glu), and A10 (Ser308Ile). The encoded nucleotide sequences are shown in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7, respectively.

[0125] As shown in Figure 11 , the enzyme activity of mutant A10 was the highest, reaching (228±4.8)%, followed by D8, which was (190%±6.3)%, and E9, which was (174%±6.2)%.

[0126] The 5 mutants (H2, B3, D8, E9, A10) screened were subjected to specific verification by PCR using P3 primers. The results are shown in Figure 12 , and the size of the amplified band was as expected (about 1300 bp). The target band was excised and sequenced, and the sequencing results were compared with the original PcLiP gene sequence. It was finally confirmed that the enzyme activity of the 5 mutants was improved compared with E-PcLiP.

[0127] The sequencing alignment results clearly showed the mutation sites: mutant H2 was the mutation of leucine (Leu, L) at position 180 to alanine (Ala, A); mutant B3 was the mutation of methionine (Met, M) at position 290 to leucine (Leu, L); mutant D8 was the mutation of methionine (Met, M) at position 52 to isoleucine (Ile, I); mutant E9 was the mutation of alanine (Ala, A) at position 188 to glutamic acid (Glu, E); and mutant A10 was the mutation of serine (Ser, S) at position 308 to isoleucine (Ile, I).

[0128] Example 3: Preparation of a fixed enzyme preparation of Bacillus subtilis surface display

[0129] (1) Construction of B. subtilis WB800N-pHS-CotB-PcLiP

[0130] The recombinant plasmid DH5α-pET-22b-PcLiP was used as a template, and P2 primers (P2-F: GGTTCAACTAGTATGGCACCGAATCTG, P2-R: TAGAAAGCTTTCAGTGGTGGTGGTG) containing Spe I and Hind III restriction sites were used to amplify the PcLiP fragment with Spe I and Hind III restriction sites at both ends by PCR.

[0131] The amplified PcLiP fragment and the DH5α-pHS-CotB plasmid were double-digested with restriction endonucleases Spe I and Hind III, respectively. The double-digested PcLiP fragment and the linearized pHS-CotB vector fragment were gel-purified, respectively. The recovered fragments were subjected to a ligation reaction, and the ligation product was transformed into E. coli DH5α competent cells. The plasmid was extracted to obtain the recombinant plasmid DH5α-pHS-CotB-PcLiP. The recombinant plasmid DH5α-pHS-CotB-PcLiP was transformed into B. subtilis WB800N competent cells to obtain a recombinant lignin peroxidase engineering strain that can be used for B. subtilis surface display, which was named B. subtilis WB800N-pHS-CotB-PcLiP.

[0132] (2) Induction of recombinant PcLiP spores

[0133] B. subtilis WB800N-pHS-CotB-PcLiP was inoculated into LB liquid medium containing 7.5 μg / mL chloramphenicol and 10 μg / mL kanamycin at an inoculation amount of 1%, and cultured at 37°C and 180 rpm for 12 h to obtain a culture solution. Then, the first obtained culture solution was inoculated into Difco spore formation medium (DSM) for secondary culture, and incubated at 37°C and 180 rpm for 36 h to obtain a spore culture. Due to nutrient depletion, the recombinant B. subtilis existed in the form of spores after being cultured in the DSM for 36 h.

[0134] The spore culture was centrifuged at 4°C at 8,000 rpm for 10 min, and the spore precipitate was collected and resuspended in GTE buffer (50 mM glucose, 20 mM Tris-HCl, 10 mM EDTA, 2 mg / mL lysozyme, pH 7.5) to obtain a resuspension. The volume ratio of the precipitate to the GTE buffer was 1:10. The resuspension was incubated at 37°C for 45 min, and then centrifuged at 4°C at 8,000 rpm for 10 min to collect the precipitate. The precipitate was washed twice with 1 M NaCl, 1 M KCl, and sterile phosphate buffered saline (PBS, pH 7.4). Finally, the purified spores were resuspended in PBS, and the OD600 was adjusted to 1.0. The obtained spore suspension was denoted as enzyme preparation CotB-PcLiP.

[0135] (3) Western Blotting and immunofluorescence verification of spore surface display of PcLiP

[0136] S1, Western Blotting verification:

[0137] The purified spore suspension stored at -20°C was taken for verification. The spore suspension was centrifuged to collect the precipitate, which was resuspended in an equal volume of SDS-DTT extraction buffer (containing 1.5% SDS and 50 mM DTT) and treated in a 70°C metal bath for 2 h to extract the CotB-PcLiP fusion protein. The extracted CotB-PcLiP fusion protein and the control CotB protein were subjected to Western blotting analysis. After incubation with the primary antibody (Rabbit Anti-6xHis Tag Mouse Monoclonal Antibody, 1:2000) and the secondary antibody (Anti-mouse-IgG-FITC Conjugates, 1:5000), color development was performed using the Omni-ECL TM Basic Chemiluminescence Kit (Shanghai Yezhe) and exposed. The verification results are shown in Figure 13 (a) The figure shows that the recombinant fusion protein CotB-PcLiP (corresponding to lane 2) displayed on the surface presents a single clear band at 81 kDa, while the control CotB protein (corresponding to lane 1) does not appear the corresponding band. This result shows that the recombinant lignin peroxidase is successfully displayed on the surface of Bacillus subtilis spores.

[0138] S2, immunofluorescence verification:

[0139] First, the glass slides were cleaned with alcohol, soaked in 0.1% (w / v) gelatin solution for 5 min, and then dried vertically overnight. 10 μL of appropriately diluted CotB-PcLiP spore suspension was dropped onto the center of the treated glass slide, and then air-dried at room temperature. The glass slide was rinsed with PBS buffer for 3 times, 5 min each time. The PBS buffer containing 5% skim milk was used for blocking at room temperature for 30 min. The glass slide was rinsed with PBS buffer for 5 times, 5 min each time. The primary antibody (Rabbit Anti-6xHis Tag Mouse Monoclonal Antibody, 1:2000) was added, and then incubated at 4°C overnight. The glass slide was washed with PBS buffer at room temperature for 3 times, 5 min each time. The fluorescent secondary antibody (Goat Anti-Mouse IgG / FITC, diluted 1:100) was added, and then incubated at room temperature for 2 h in the dark. The glass slide was washed with PBS buffer for 3 times, 5 min each time. The spore samples of control CotB were treated synchronously. The observation was performed under an inverted fluorescence microscope. The results are shown in Fig. 6(b), wherein the spores (No. 4) of the fusion protein CotB-PcLiP showed green fluorescence, while no fluorescence was detected in the spores (No. 3) of the control CotB. This result further confirmed that the lignin peroxidase was successfully displayed on the surface of Bacillus subtilis. Figure 13

[0140] (4) Construction of mutant B. subtilis WB800N-pHS-CotB-PcLiP-D3

[0141] The five mutants obtained by high-throughput screening were analyzed, and it was found that the enzyme activities of mutants D8 (M52I), E9 (A188E), and A10 (S308) were all increased by more than 70% compared with E-PcLiP, ranking in the top three. Therefore, the three mutant sites M52I, A188E, and S308I were further selected for combined mutation, and a combined mutant plasmid DH5α-pHS-CotB-PcLiP-D3 was constructed through three rounds of whole plasmid PCR. The nucleotide sequence of the three-point combined mutant PcLiP-D3 is shown in SEQ ID NO. 8, and the amino acid sequence is shown in SEQ ID NO. 9.

[0142] Specific steps for constructing the combined mutant plasmid:

[0143] ​S1, Constructing M52I single-point mutant plasmid. Using DH5α-pHS-CotB-PcLiP plasmid as template, using mutation primers M52I-F and M52I-R, full plasmid PCR site-directed mutation was carried out to mutate catalytic residue Met52 to Ile, and full plasmid PCR product containing M52I mutation was amplified. Then the above PCR product was treated with restriction endonuclease DpnI, and the original template plasmid was selectively digested by DpnI treatment. The DpnI-treated (containing mutation) PCR product was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the mutant plasmid DH5α-pHS-CotB-PcLiP-M52I.

[0144] Full plasmid PCR was used to amplify site-directed mutation. The concentration of primers was 10 μM, 1 μL of primers at both ends of M52I (M52I-F: AAGCGCATGAAGCGCTGCGTATCGTTTTTCATGATAGC, M52I-R: ATACGCAGCGCTTCATGCGCTTCCGCACCACACTG); 10 μL of 2×PrimeSTAR Max Premix was added; 1 μL of DH5α-pHS-CotB-PcLiP plasmid with a concentration of 0.5 ng / μL; 7 μL of ddH2O was added to make up 20 μL of PCR reaction system.

[0145] The amplification program of full plasmid PCR was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 70 s, for a total of 30 cycles; 72℃ final extension for 10 min.

[0146] S2, Constructing A188E double-point mutant plasmid based on M52I. The DH5α-pHS-CotB-PcLiP-M52I plasmid obtained in the above step was used as a template, and mutation primers A188E-F and A188E-R were used for full plasmid PCR to amplify PCR products containing A188E mutation. The PCR product containing A188E mutation was treated with restriction endonuclease DpnI to selectively digest the original template plasmid. The DpnI-treated (containing mutation) PCR product was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the mutant plasmid DH5α-pHS-CotB-PcLiP-M52I-A188E.

[0147] The amplification system of PCR includes:

[0148] A188E-F (CGCACATTCAATTGCGGAGGCAAATGATGTGGAC) and A188E-R (CGCAATTGAATGTGCGCTCAGCAGCCAAACT) 1 μL each, 1 μL of DH5α-pHS-cotB-PcLiP-M52I mutant plasmid with a concentration of 0.5 ng / μL, 10 μL of 2×PrimeSTAR Max Premix, and ddH2O to make up 20 μL of PCR reaction system.

[0149] The amplification procedure of the whole plasmid PCR was as follows: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 s, 55 °C annealing for 15 s, 72 °C extension for 70 s, for a total of 30 cycles; and 72 °C terminal extension for 10 min.

[0150] S3, S308I three-point mutant plasmid (DH5α-pHS-CotB-PcLiP-D3) was constructed based on M52I-A188E. The S308I mutant primer was used to amplify the PCR product containing the S308I mutation using the mutant plasmid DH5α-pHS-CotB-PcLiP-M52I-A188E as the template. The PCR product containing the S308I mutation was treated with restriction endonuclease DpnI to selectively digest the original template plasmid. The PCR product containing the S308I mutation treated with DpnI was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the mutant plasmid DH5α-pHS-CotB-PcLiP-M52I-A188E-S308I, which was designated as DH5α-pHS-CotB-PcLiP-D3. The mutant gene fragment of the lignin peroxidase carried by the plasmid was designated as PcLiP-D3. The nucleotide sequence of the PcLiP-D3 gene is shown in SEQ ID NO. 8, and the amino acid sequence is shown in SEQ ID NO. 9.

[0151] The amplification system of PCR included: S308I-F (CTGTTAATTTTGGCCCGATCTTTTTTCCGG) and S308I-R (TCGGGCCAAAATTAACAGGTTTCGGTGCC) 1 μL each, 1 μL of DH5α-pHS-cotB-PcLiP-M52I-A188E mutant plasmid with a concentration of 0.5 ng / μL, 10 μL of 2×PrimeSTAR Max Premix, and ddH2O to make up 20 μL of PCR reaction system. The amplification procedure of the whole plasmid PCR was as follows: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 s, 55 °C annealing for 15 s, 72 °C extension for 70 s, for a total of 30 cycles; and 72 °C terminal extension for 10 min.

[0152] S4, constructing a B. subtilis surface display engineering bacteria and spore preparation;

[0153] The recombinant plasmid DH5a-pHS-CotB-PcLiP-D3 was transformed into B. subtilis WB800N competent cells to obtain the recombinant engineering bacteria B. subtilis WB800N-pHS-CotB-PcLiP-D3. The recombinant engineering bacteria B. subtilis WB800N-pHS-CotB-PcLiP-D3 was inoculated into LB liquid medium containing kanamycin (7.5 pg / mL) and chloramphenicol (10 pg / mL) at an inoculation amount of 1% (g / ml, i.e. 1 g: 100 ml), and cultured at 37°C, 180 rpm for 12 h to obtain a culture solution. The culture solution was transferred to Difco spore formation medium (DSM) at an inoculation amount of 1% (v / v), and incubated at 37°C, 180 rpm for 36 h to obtain a spore culture. The spore culture was collected by centrifugation at 4°C, 8000 rpm for 10 min, and the precipitate was resuspended in a lysozyme-containing GTE buffer (50 mM glucose, 20 mM Tris-HCl, 10 mM EDTA, 2 mg / mL lysozyme, pH 7.5) to obtain a resuspension; wherein the volume ratio of the precipitate to the GTE buffer was 1:10. The resuspension was incubated at 37°C for 45 min, and the precipitate was collected by centrifugation and washed with PBS buffer (8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4, pH 7.4 per 1 L), and finally the washed precipitate was resuspended in PBS buffer, and the OD600 was adjusted to 1.0 to obtain a spore suspension, which was denoted as CotB-PcLiP-D3.

[0154] The spore suspension was centrifuged to collect the precipitate, which was resuspended in an equal volume of SDS-DTT extraction buffer (containing 1.5% SDS and 50 mM DTT), and treated in a 70°C metal bath for 2 h to extract the CotB-PcLiP-D3 fusion protein. The extracted CotB-PcLiP-D3 fusion protein and the control CotB protein were subjected to Western blotting analysis. After incubation with the primary antibody (Rabbit Anti-6xHis Tag Mouse Monoclonal Antibody, 1:2000) and the secondary antibody (Anti-mouse-IgG-FITC Conjugates, 1:5000), color development was performed using Omni-ECL TM The verification results are shown in FIG. 6. Figure 14As shown in Figure (a), the recombinant fusion protein CotB-PcLiP-D3 (corresponding to lane 1) exhibits a single, clear band at 81 kDa, while the control CotB protein (corresponding to lane 2) does not show a corresponding band. This result indicates that the recombinant lignin peroxidase D3 mutant was successfully displayed on the surface of Bacillus subtilis spores.

[0155] First, clean the glass slides with alcohol, soak them in 0.1% (w / v) gelatin solution for 5 min, remove them, and dry them upright overnight. Add 10 μL of appropriately diluted CotB-PcLiP-D3 spore suspension to the center of the treated slides and air dry at room temperature. Rinse the slides three times with PBS buffer, 5 min each time. Block with PBS buffer containing 5% skim milk powder at room temperature for 30 min. Rinse five times with PBS buffer, 5 min each time. Add primary antibody (RabbitAnti-6×His Tag MouseMonoclonalAntibody, 1:2000) and incubate overnight at 4°C. Rinse three times with PBS buffer at room temperature, 5 min each time. Add fluorescent secondary antibody (GoatAnti-Mouse IgG / FITC, diluted 1:100) and incubate at room temperature in the dark for 2 h. Rinse the slides three times with PBS buffer, 5 min each time. Simultaneously treat control CotB spore samples. Observe under an inverted fluorescence microscope. The verification results are as follows Figure 13 As shown in (b): the spores displaying the fusion protein CotB-PcLiP-D3 (number 4) exhibited green fluorescence, while the spores of the control group CotB (number 3) showed no fluorescence. This result further confirms that the lignin peroxidase mutant was successfully displayed on the surface of Bacillus subtilis.

[0156] Enzyme activity reaction system: 25 mM BR buffer (pH 5.0), 1.25 mM MABTS, 100 μL of CotB-PcLiP or CotB-PcLiP-D3 spore suspension with OD600 of 1.0, and finally 2 mM H2O2 to start the reaction. Enzyme activity was quantified by measuring absorbance changes at 420 nm over 10 min using a microplate reader.

[0157] The enzyme activity of CotB-PcLiP was 100%. The results are as follows... Figure 15 As shown, the enzyme activity of the CotB-PcLiP-D3 mutant (abbreviated as D3 in the figure) was increased to 335% of that of CotB-PcLiP (abbreviated as WT in the figure), which is 2.35 times higher.

[0158] Example 4: Lignin peroxidase immobilized enzyme preparation for alkali lignin degradation;

[0159] (1) Preparation and performance evaluation of recombinant lignin peroxidase immobilized enzyme preparation

[0160] The degradation experiments were carried out with the recombinant lignin peroxidase immobilized enzyme preparations CotB-PcLiP and CotB-PcLiP-D3 prepared in Example 3.

[0161] Lignin peroxidase has high oxidation capacity and adaptability to complex structures, and can degrade alkali lignin. The enzyme has the highest activity in acidic conditions, and can form highly active free radicals (such as lignin radicals), thereby initiating the degradation of alkali lignin.

[0162] (2) Alkali lignin degradation capacity determination

[0163] The alkali lignin degradation capacity of the immobilized enzyme preparation CotB-PcLiP or CotB-PcLiP-D3 was determined using alkali lignin as the substrate.

[0164] The reaction system was composed as follows: addition of immobilized enzyme preparation CotB-PcLiP or CotB-PcLiP-D3 (OD600 adjusted to 1.0), 1 g / L alkali lignin, 25 mM BR buffer (pH 3.0), 2 mM H2O2. The addition amount of immobilized enzyme preparation was 25% (v / v) of the reaction system.

[0165] The reaction system was reacted at 37°C for 24 h, and the supernatant was taken as the sample to be tested after centrifugation. The absorbance (OD value) was determined at a wavelength of 280 nm, and the alkali lignin degradation rate was calculated. The results are shown in Figure 16 The degradation rate of the wild-type CotB-PcLiP (control, abbreviated as WT) was 100%, and the degradation rate of the mutant CotB-PcLiP-D3 (abbreviated as D3) was 150% of the wild-type, i.e. the degradation efficiency was increased by 0.5 times, indicating that the degradation capacity of the mutant was significantly better than that of the wild-type.

[0166] (3) Evaluation of the operational stability of the immobilized enzyme

[0167] To evaluate the operational stability of the mutant CotB-PcLiP-D3, alkali lignin was used as the substrate for 5 rounds of continuous catalytic cycle tests.

[0168] The reaction system was as follows: 25% (volume of immobilized enzyme preparation: volume of reaction system) of immobilized enzyme preparation CotB-PcLiP-D3 (OD600 adjusted to 1.0), 1 g / L alkali lignin, 25 mM BR buffer (pH 3.0), 2 mM H2O2. The reaction system was reacted at 37°C for 24 h, and the supernatant was taken as the sample to be tested after centrifugation. The absorbance was measured at 280 nm, and the alkali lignin degradation rate was calculated. The reaction conditions were the same as above, and after the reaction was completed, the immobilized enzyme was recovered by high-speed centrifugation (10,000 rpm, 10 min), and was reused after being washed twice with PBS. Taking the initial degradation rate as 100%, the recovery performance was quantified by the relative degradation rate. The results are shown in Figure 17 Fig. 2, where the abscissa (1)-(5) represents the number of cycles. After the first three cycles, the alkali lignin degradation rate of mutant CotB-PcLiP-D3 could be maintained above 70%. After the fifth cycle, the degradation rate could still reach 30%. This indicates that the immobilized enzyme CotB-PcLiP-D3 has good operational stability, providing a reference for the industrial application of LiP in the future. The LiP immobilized enzyme produced based on the spore surface display technology is expected to provide new possibilities for environmentally friendly industrial production.

[0169] Example 5: Use of immobilized enzyme preparation for wheat straw pretreatment

[0170] The immobilized enzyme preparation CotB-PcLiP or CotB-PcLiP-D3 prepared in Example 3 was used, with the difference being that the OD600 was adjusted to 2.0.

[0171] The wheat straw was crushed and passed through a 40-mesh sieve, and was placed in a dry environment for standby.

[0172] Wheat straw was used as raw material, and the mixed system was composed of ①-⑧, and the water content of the mixed system was controlled at 50%. After stirring uniformly, it was placed in a constant temperature and humidity incubator with a humidity of 70%, and was degraded at 37°C for 96 h. After the degradation was completed, the lignin content in the product was determined by the filter bag method, and the lignin degradation rate of wheat straw was used as an index to evaluate the pretreatment effect of the immobilized enzyme preparation on wheat straw. The results are shown in the following table.

[0173] 5 g of wheat straw was weighed for each part, and the group was set as follows according to the mass of wheat straw:

[0174] ① Mixed enzyme; ② Mixed enzyme + 10% CotB; ③ Mixed enzyme + 10% CotB-PcLiP; ④ Mixed enzyme + 10% CotB-PcLiP-D3; ⑤ Mixed enzyme + 5% CotB-PcLiP-D3;

[0175] V. Mixed enzymes + 15% CotB-PcLiP-D3; VI. Mixed enzymes + 20% CotB-PcLiP-D3; VII. Mixed enzymes + 25% CotB-PcLiP-D3.

[0176] Note: Mixed enzymes composition: 2% laccase (enzyme activity 10,000 U / g), 2% glucose oxidase (enzyme activity 10,000 U / g), 0.1% glucanase (enzyme activity 50,000 U / g), 0.1% xylanase (enzyme activity 30,000 U / g), 0.1% acid cellulase (enzyme activity 10,000 U / g), 0.1% cellobiose (enzyme activity 10,000 U / g), 0.1% mannase (enzyme activity 50,000 U / g).

[0177] Group Lignin degradation rate ① 1.2％ ② 8.96％ ③ 10.25％ ④ 16.29％ ⑤ 13.57％ ⑥ 20.57％ ⑦ 24.16％ ⑧ 24.23％

[0178] Description: The above examples are only used to illustrate the present application and not limit the technical solutions described in the present application; therefore, although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present application can still be modified or replaced by equivalents; and all technical solutions and improvements that do not deviate from the spirit and scope of the present application should be covered within the scope of the claims of the present application.

[0179] Sequence Listing:

[0180] SEQ ID NO. 1

[0181]

[0182] SEQ ID NO. 2:

[0183] APNLDKRVACPDGVHTASNAACCAWFPVLDDIQQNLFHGGQCGAEAHEALRMVFHDSIAISPKLQSQGKFGGGGADGSIITFSSIETTYHPNIGLDEVVAIQKPFIAKHGVTRGDFIAFAGAVGVSNCPGAPQMQFFLGRPEATQAAPDGLVPEPFHTIDQVLARMLDAGGFDEIETVWLLSAHSIAAANDVDPTISGLPFDSTPGQFDSQFFVETQLRGTAFPGKTGIQGTVMSPLKGEMRLQTDHLFARDSRTACEWQSFVNNQTKLQEDFQFIFTALSTLGHDMNAMTDCSEVIPAPKPVNFGPSFFPAGKTHADIEQACASTPFPTLITAPGPSASVARIPPPPSPN

[0184] SEQ ID NO. 3: H2(L180A)

[0185]

[0186] SEQ ID NO. 4: B3 (M290L)

[0187]

[0188] SEQ ID NO. 5: D8 (M52I)

[0189]

[0190] SEQ ID NO. 6: E9 (A188E)

[0191]

[0192] SEQ ID NO. 7: A10 (S308I)

[0193]

[0194] SEQ ID NO. 8: D3 (M52I-A188E-S308I)

[0195]

[0196] SEQ ID NO. 9: D3 (M52I-A188E-S308I)

[0197] APNLDKRVACPDGVHTASNAACCAWFPVLDDIQQNLFHGGQCGAEAHEALRIVFHDSIAISPKLQSQGKFGGGGADGSIITFSSIETTYHPNIGLDEVVAIQKPFIAKHGVTRGDFIAFAGAVGVSNCPGAPQMQFFLGRPEATQAAPDGLVPEPFHTIDQVLARMLDAGGFDEIETVWLLSAHSIAEANDVDPTISGLPFDSTPGQFDSQFFVETQLRGTAFPGKTGIQGTVMSPLKGEMRLQTDHLFARDSRTACEWQSFVNNQTKLQEDFQFIFTALSTLGHDMNAMTDCSEVIPAPKPVNFGPIFFPAGKTHADIEQACASTPFPTLITAPGPSASVARIPPPPSPN

Claims

1. A high-throughput screening engineering method of high-efficiency lignin peroxidase mutants, characterized in that, The lignin peroxidase mutant is obtained by screening the primary mutant obtained based on the high-throughput screening scheme of the E. coli exosporulation and combining mutations to obtain the lignin peroxidase mutant PcLiP-D3, i.e. the methionine (Met) at position 52 of the lignin peroxidase PcLiP shown in the amino acid sequence SEQ ID NO. 2 is mutated into isoleucine (Ile), the alanine (Ala) at position 188 is mutated into glutamic acid (Glu), and the serine (Ser) at position 308 is mutated into isoleucine (Ile); wherein the nucleotide sequence of the lignin peroxidase PcLiP is shown in SEQ ID NO. 1; The preparation method of the lignin peroxidase mutant is as follows: (1) The lignin peroxidase gene sequence (GenBank: X15599) derived from P. chrysosporium is optimized according to the codon bias of E. coli and B. subtilis to obtain a gene encoding PcLiP, and the nucleotide sequence is shown in SEQ ID NO. 1 and the amino acid sequence is shown in SEQ ID NO. 2; (2) The P1 primer is used for PCR amplification with the PcLiP-encoding gene in step (1) as the template to obtain the amplified PcLiP gene fragment; the amplified PcLiP gene fragment is double-digested with restriction endonucleases NcoI and XhoI, and then connected with the linearized fragment of the E. coli expression vector pET-22b digested with the same enzymes, and the obtained connection product is transformed into E. coli DH5α competent cells, and after extracting the plasmid, the recombinant plasmid DH5α-pET-22b-PcLiP is obtained; then, the recombinant plasmid DH5α-pET-22b-PcLiP is transformed into E. coli BL21 (DE3) competent cells to obtain a recombinant lignin peroxidase engineering bacterium that can be expressed in the periplasmic space, which is denoted as E. coli BL21 (DE3)-pET-22b-PcLiP; (3) Using the recombinant plasmid DH5α-pET-22b-PcLiP obtained in step (2) as a template, error-prone PCR amplification was performed using P1 primer to obtain PcLiP eq gene fragment; (4) The amplified PcLiP gene fragment in step (3) was digested with restriction endonucleases Ncol and Xhol, respectively, to obtain the digested PcLiP gene fragment eq and the recombinant plasmid DH5α-pET-22b-PcLiP eq The digested PcLiP gene fragment and the linearized pET-22b vector were recovered. eq After the digested PcLiP gene fragment and the linearized pET-22b vector were ligated, the ligation product was transformed into E. coli DH5α competent cells, plasmids were extracted, and a recombinant mutant plasmid library was obtained, which was recorded as DH5α-pET-22b-PcLiP eq The recombinant mutant plasmid library was transformed into E. coli BL21(DE3) competent cells, and a recombinant lignin peroxidase mutant strain library was obtained, which was recorded as E. coli(DE3) BL21-pET-22b-PcLiP eq ; (5) The recombinant lignin peroxidase mutant strain library E. coli(DE3)BL21-pET-22b-PcLiP from step (4) is used. eq The specific steps for high-throughput screening of the periplasmic secretion pathway in Escherichia coli are as follows: In a sterile laminar flow hood, the recombinant engineered bacteria E. coli BL21(DE3)-pET-22b-PcLiP was... eq The monoclonal mutant strain on the mutant plate was inoculated into a 96-well plate containing ampicillin sodium in LB liquid medium, denoted as plate A. After being cultured in plate A for a period of time, it was transferred to another 96-well plate containing ampicillin sodium in LB liquid medium, denoted as plate B. After being cultured in plate B for a period of time, N positions were randomly selected in plate B, where N is a positive integer. Then, bacterial culture was taken from each of the N positions to detect OD600 and determine the growth status of each mutant. When the OD600 of each well was in the range of 0.6 to 0.8 and the difference in OD600 values ​​was within 0.1, a soluble inducer was added to plate B for induction culture. After the induction culture is completed, the supernatant is discarded by centrifugation, and the lysing buffer containing lysozyme is added to each well of the precipitate to resuspend the precipitate to obtain a suspension, and after incubation, a suspension of mutant recombinant protein is obtained; after centrifugation of the mutant recombinant protein suspension, the supernatant is transferred to a new shallow 96-well plate, denoted as plate C, and the mutant enzyme activity is determined, and the obtained supernatant is the mutant recombinant protein of the mutant; after enzyme activity determination, high-activity mutants are screened, which are denoted as H2 (L180A), B3 (M290L), D8 (M52I), E9 (A188E) and A10 (S308I), and the encoded nucleotide sequences are shown in SEQ ID NO. 3-7, respectively; (6) Based on the 5 mutants screened in step (5), the enzyme activity of mutants D8 (M52I), E9 (A188E), and A10 (S308) is all improved by more than 70% compared with E-PcLiP, ranking in the top three; therefore, three mutant sites M52I, A188E, and S308I are further selected for combined mutation, and a combined mutant plasmid DH5α-pHS-CotB-PcLiP-D3 is constructed through three rounds of whole plasmid PCR; wherein the three-point combined mutant is PcLiP-D3, the nucleotide sequence is shown as SEQ ID NO. 8, and the amino acid sequence is shown as SEQ ID NO.

9.

2. The high-throughput screening and modification method for efficient lignin peroxidase mutants according to claim 1, characterized in that, The three rounds of whole plasmid PCR for constructing the combined mutant plasmid DH5α-pHS-CotB-PcLiP-D3 in step (6) are as follows: ① Using the recombinant plasmid DH5α-pET-22b-PcLiP as a template, a PcLiP fragment with SpeI and HindIII restriction sites at both ends is amplified using P2 primers containing SpeI and HindIII restriction sites; the amplified PcLiP fragment and the DH5α-pHS-CotB plasmid are double-digested using restriction endonucleases SpeI and HindIII, respectively; the double-digested PcLiP fragment and the linearized pHS-CotB vector fragment are gel-purified, respectively; and the recovered fragments are subjected to a ligation reaction to obtain a ligation product which is transformed into E. coli DH5α competent cells, and the plasmid is extracted to obtain the recombinant plasmid DH5α-pHS-CotB-PcLiP; ② Using the recombinant plasmid DH5α-pHS-CotB-PcLiP of step ① as a template, a full plasmid PCR product containing the M52I mutation is amplified by one-step full plasmid PCR by mutating the 52nd methionine (Met52) to isoleucine (Ile52); then the full plasmid PCR product containing the M52I mutation is treated with restriction endonuclease DpnI, the original template plasmid is selectively digested, and the DpnI-treated full plasmid PCR product containing the M52I mutation is transformed into E. coli DH5α competent cells, and the plasmid is extracted to obtain the mutant plasmid DH5α-pHS-CotB-PcLiP-M52I; ③ Using the mutant plasmid DH5α-pHS-CotB-PcLiP-M52I obtained in step ② as a template, a PCR product containing the A188E mutation is amplified using A188E mutant primers; the PCR product containing the A188E mutation is treated with restriction endonuclease DpnI to selectively digest the original template plasmid; the DpnI-treated PCR product containing the A188E mutation is transformed into E. coli DH5α competent cells, and the plasmid is extracted to obtain the mutant plasmid DH5α-pHS-CotB-PcLiP-M52I-A188E; (4) using the mutant plasmid DH5α-pHS-CotB-PcLiP-M52I-A188E in step (3) as a template, a PCR product containing the S308I mutation was obtained by using a S308I mutation primer; the PCR product containing the S308I mutation was treated with DpnI to selectively digest the original template plasmid; and the PCR product containing the S308I mutation treated with DpnI was transformed into E. coli DH5α competent cells, and plasmid was extracted to obtain a mutant plasmid DH5α-pHS-CotB-PcLiP-M52I-A188E-S308I, denoted as DH5α-pHS-CotB-PcLiP-D3; wherein the mutant plasmid carries a lignin peroxidase mutant gene fragment denoted as PcLiP-D3.

3. The method of claim 1, wherein the high-throughput screening of a high efficiency lignin peroxidase mutant is characterized by, The error-prone PCR amplification reaction system in step (3) is as follows: 1 ng of template; 1 μL each of forward and reverse primers, both at 10 μM; 5 μL of 10×Easy Taq Buffer; 4 μL of dNTPs at 2.5 mM; 0.5 μL of Easy Taq DNA polymerase; 1 μL of Mg 2+ Final concentration 3-5 mM and Mn 2+ Final concentration 0.6-1 mM, and sterile ddH2O was added to make up the 50 μL reaction system; In step (5), the final concentration of ampicillin sodium in the A plate and the B plate is 0.1-0.2 mg / mL, and the culture conditions in the A plate and the B plate are 30-40 °C, 160-200 rpm in a constant temperature shaking incubator for 8-16 h; N≥3, preferably 8; the induction culture conditions are 12-20 °C, 160-200 rpm in a low-temperature constant temperature shaking incubator for 8-16 h; The inoculation amount from the A plate to the B plate is 1%-5% (v / v), and the addition amount of the soluble inducer is 1-5% of the volume of the culture medium in the B plate; The centrifugation condition is 4000 rpm for 15-20 min, the concentration of lysozyme in the lysozyme-containing breaking buffer is 0.5 mg / mL, and the composition of the breaking buffer is: 200 mM Tris, 500 mM sucrose, 1 mM EDTA, pH 8.0; the incubation is carried out in a constant temperature water bath, the temperature is 30-40 °C, and the incubation time is 30-45 min.

4. A gene encoding the mutant lignin peroxidase of claim 1, which is capable of efficiently degrading lignin, characterized by, The nucleotide sequence of the lignin peroxidase mutant gene encoding the lignin-degrading enzyme is shown in SEQ ID NO. 8, and the encoded amino acid sequence is shown in SEQ ID NO.

9.

5. An engineered bacterium expressing the high-efficiency lignin-degrading lignin peroxidase mutant of claim 1, characterized in that, The lignin peroxidase mutant engineering bacteria comprise an expression vector pHS-CotB-PcLiP-D3; the expression vector pHS-CotB-PcLiP-D3 comprises the lignin peroxidase mutant gene encoding the lignin-degrading enzyme according to claim 4; The lignin peroxidase mutant engineering bacteria are obtained by transforming the expression vector pHS-CotB-PcLiP-D3 into host cells.

6. The engineered bacteria of the lignin peroxidase mutant degrading lignin efficiently according to claim 5, characterized by, The obtaining step of the engineering bacteria is as follows: the plasmid pHS-CotB-PcLiP-D3 is transformed into E. coli DH5a competent cells, and a mutant plasmid DH5a-pHS-CotB-PcLiP-D3 is obtained by extraction; Bacillus subtilis WB800N is used as a host bacteria, and the mutant plasmid DH5a-pHS-CotB-PcLiP-D3 is introduced into the host bacteria by a chemical transformation method to construct a recombinant engineering bacteria B. subtilis WB800N-pHS-CotB-PcLiP-D3.

7. A method for preparing a high-efficiency lignin-degrading lignin peroxidase mutant enzyme preparation, characterized by, Based on the recombinant engineering bacteria B. subtilis WB800N-pHS-CotB-PcLiP-D3 of claim 6, the recombinant spore suspension of the surface-displayed lignin peroxidase is obtained by inoculating the bacteria into a culture medium and centrifugation; or the spore suspension is freeze-dried to prepare a solid powder; and the recombinant spore suspension or the solid powder is a lignin peroxidase mutant enzyme preparation for efficiently degrading lignin, and is denoted as CotB-PcLiP-D3.

8. The method for preparing the lignin peroxidase mutant enzyme preparation according to claim 7, characterized in that, The recombinant engineering bacteria B. subtilis WB800N-pHS-CotB-PcLiP-D3 is inoculated into an LB culture medium containing kanamycin and chloramphenicol, and a culture solution is obtained after a first culture; then the culture solution is inoculated into a DSM culture medium, and a bacterial body precipitate is collected by centrifugation after a second culture; the precipitate is resuspended using a GTE buffer containing lysozyme to obtain a resuspension; the precipitate is collected by centrifugation after incubation, and is washed using a PBS buffer; the precipitate is collected after washing, and is resuspended in the PBS buffer again to obtain a recombinant spore protein suspension displaying lignin peroxidase on the surface; or the recombinant spore protein suspension is freeze-dried to obtain a solid powder; and the recombinant spore protein suspension or the solid powder is a lignin peroxidase mutant enzyme preparation for efficiently degrading lignin, and is denoted as CotB-PcLiP-D3; The concentration of the lysozyme in the GTE buffer containing the lysozyme is 2 mg / mL, and the components of the GTE buffer are as follows: 10 mM EDTA, 20 mM Tris-HCl with pH = 7.5, and 50 mM glucose. The recombination engineering bacteria B. subtilis WB800N-pHS-CotB-PcLiP-D3 is inoculated into LB medium containing kanamycin and chloramphenicol at an inoculation amount of 1-3%, wherein the concentration of chloramphenicol in the LB liquid medium is 5-10 μg / mL, and the concentration of kanamycin is 5-10 μg / mL; the culture liquid is inoculated into DSM medium at an inoculation amount of 1-3% (v / v); the first culture is carried out at 30-40°C, 160-200 rpm for 10-16 h; the second culture is carried out at 30-40°C, 160-200 rpm for 30-48 h; the GTE buffer is composed of 50 mM glucose, 20 mM Tris-HCl, 10 mM EDTA, 2 mg / mL, pH 7.5; the volume ratio of the precipitate to the GTE buffer is 1:10-20; the incubation is carried out at 30-40°C for 30-60 min; and the OD600 of the recombinant spore protein suspension is 1-2.

9. Use of a lignin peroxidase mutant enzyme preparation prepared according to the method of claim 7 or 8 for degrading alkali lignin or straw lignin.

10. Use according to claim 9, characterized in that, The steps are as follows: (1) When degrading alkali lignin, alkali lignin is used as raw material, lignin peroxidase mutant enzyme preparation CotB-PcLiP-D3 is added, and BR buffer and H2O2 are added simultaneously to form a reaction system, and alkali lignin can be degraded after reaction; The pH of the BR buffer is 2.5-4.5; in the reaction system, the concentration of alkali lignin is 0.8-1.2 g / L, the concentration of BR buffer is 25-100 mM, and the concentration of H2O2 is 2-4 mM; the OD600 of the CotB-PcLiP-D3 is 1.0, and the addition amount is 15-30% (v / v) of the reaction system; (2) When degrading straw lignin, wheat straw is used as raw material, lignin peroxidase mutant enzyme preparation CotB-PcLiP-D3 is combined, and mixed enzymes are added simultaneously to form a mixed system, and the water content of the mixed system is controlled, and lignin in the wheat straw can be degraded after reaction under certain temperature conditions; the mixed enzymes are composed of laccase, glucose oxidase, glucanase, xylanase, acid cellulase, cellobiose and mannanase; In terms of the mass of the wheat straw, the OD600 of CotB-PcLiP-D3 in the step is 2.0, and the addition amount is 5%-40% (ml / g, v / m), i.e. 5-40 ml: 100 g; the addition amount of laccase is 1-3%; the addition amount of glucose oxidase is 1-3%; the addition amounts of glucanase, xylanase, acid cellulase, cellobiose and mannanase are all 0.1%; the water content of the mixed system is controlled to be 40-60%, the temperature is controlled to be 30-45°C, and the reaction time is 70-96 h.

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