CN118667778A - High-temperature-resistant laccase mutant and engineering bacterium, preparation and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering of enzymes, and particularly relates to a high-temperature-resistant laccase mutant, engineering bacteria thereof, preparation and application. The invention expresses laccase gene (lac) from Bacillus pumilus in escherichia coli BL 21; the error-prone PCR technology is utilized to mutate the gene lac, and ABTS is utilized to screen the mutant, so that the mutant V347D/Y463W with improved thermal stability is selected, and the expression preparation in bacillus subtilis WB600, bacillus amyloliquefaciens CGMCC No.11218 and pichia pastoris GS115 is realized.

Description

High-temperature-resistant laccase mutant and engineering bacterium, preparation and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering of enzymes, and particularly relates to a high-temperature-resistant laccase mutant, engineering bacteria thereof, preparation and application.

Background

Agricultural industry waste, such as rice bran, wheat bran, bagasse, corn stover, peanut hull waste, is rich in lignocellulose. Currently, most waste is dumped or incinerated, which can contaminate the ecosystem or slow down the rate of decomposition. Therefore, it is important to convert these lignocellulose into commodity products that can be efficiently consumed. As renewable substrates, lignocellulose is widely studied for the production of reducing sugars and biofuels, mainly comprising hemicellulose, cellulose and lignin. In order to release cellulose and hemicellulose, a preliminary lignin removal pretreatment is required. The processes applied to the current industrial field for removing lignin are a strong alkali high-temperature pulping technology for cellulose utilization and a strong acid high-temperature xylose (or furfural) technology for hemicellulose utilization, and all the processes have the problem of prohibiting or limiting development caused by environmental pollution.

The biological enzyme method is used for replacing strong acid and alkali technology to degrade lignin, and is the most effective means for solving pollution and increasing benefit. However, due to the complexity of chemical bonds between the components of lignocellulosic straw, there are significant obstacles to degrading lignin using a single enzyme with specific properties. The lignin degradation is possible by adopting the combined action of enzyme compounding with different functions into compound enzyme under the same scene, and the technical scheme enters the industrialization stage at present, for example: patent CN 116479670B, etc.

Lignin degrading enzyme with specific functional properties, such as laccase, cannot function under conditions (such as temperature, pH, enzyme activity and the like) applied as a specific scene in the complex enzyme, and the aim is achieved through laccase mutants, genes, engineering bacteria and preparation of the laccase mutants.

Laccase (Laccase, e.c. 1.10.3.2), also known as phenolase, polyphenol oxidase, urushiol oxidase, etc., was first found in juice of urushiol, and is characterized by a metal-containing oxidase, which is one of the oldest enzymes in the history. The laccase can catalyze more than 200 substrates for oxidation, and T1Cu is a main electron acceptor in the laccase catalytic oxidation process. Electrons are transferred to trinuclear copper ion clusters (TNCs) containing T2Cu and T3Cu atoms through highly conserved His-Cys-His tripeptides, and O ₂ is reduced to H ₂ O, which catalyzes the single electron oxidation reaction of electron-rich substrates with O ₂, accompanied by the reduction of oxygen to completely non-toxic water, which is not only environmentally friendly, but also the substrate specificity is broad. In contrast to traditional methods of lignin degradation, laccase opens the phenolic ring in lignin molecules by oxidation of phenolic compounds, thereby leading to chain scission of lignin. The process makes the structure of lignin molecule more open and active, and improves the degradability and availability of lignin. Thus, the study of laccase enzymes has attracted a great deal of attention.

The sources of laccase are very wide, and are divided into plant laccase, insect laccase, fungal laccase and bacterial laccase, and the laccases from different sources have different functions. Currently, laccases are commercialized, more studied and more deeply characterized, mostly being of fungal origin, with white rot fungi laccases accounting for a relatively large number. Laccase from fungi plays an important role in lignin degradation, pigmentation, sporulation, stress defense, fungal-plant-pathogen/host interactions, etc. Research shows that laccase from plant source can promote wound healing and participate in plant

Metabolism of flavonoids, maintenance of structural integrity of cell walls, response to salt stress, degradation of environmental pollutants, and the like. Less research reports on insect-derived laccases compared to plant laccases have been reported, and the physiological processes of insect laccase tanning, hardening and pigmentation in the horny layer are essential for insect growth, development and survival. Bacterial laccase substrates oxidize very widely and perform different functions in organisms.

At present, research on fungal laccase at home and abroad is relatively extensive, and research discovers that most fungal laccase can better play a role under mild environmental conditions, the optimal temperature is usually between 30 and 55 ℃, the optimal reaction pH value is between 4 and 6, only the fungal laccase is neutral and high-temperature resistant, and the fungal laccase is usually unstable under high temperature and alkaline conditions, so that the application of the fungal laccase is limited. Compared with fungus laccase, the bacterial laccase has better heat stability and pH stability. Among them, laccase derived from bacillus has been widely studied because of its relatively good heat resistance and alkali stability. In the process of industrial degradation of straw, high temperature is often needed to assist in accelerating the decomposition and conversion of organic matters in the straw, ‌ to generate a usable fertilizer or energy. Therefore, the heat stability of the wild bacillus laccase is further improved on the basis of the wild bacillus laccase, and the application of the wild bacillus laccase in industry is facilitated.

Protein engineering refers to modifying or creating a protein with a desired function or property, such as catalytic activity, binding affinity, stability, solubility, specificity, or novelty. Two major approaches to protein engineering are rational design and irrational design (or directed evolution). Irrational design (or directed evolution) refers to the generation of large libraries of protein mutants by introducing random mutations and recombinations, followed by screening or selection to find proteins with desired properties.

The bacillus expression system has the following advantages: 1. can efficiently secrete various proteins; 2. the use of many bacillus bacteria in the fermentation industry has been quite a long history, non-pathogenic, and does not produce any endotoxin; 3. the bacillus microorganism genetic background research is quite clear, and the growth is rapid, and the bacillus microorganism genetic background research has no special requirements on nutrient substances and the like; 4. codon preference is not obvious; 5. the fermentation process is simple, bacillus belongs to aerobic bacteria, anaerobic fermentation equipment is not needed, and after the fermentation is finished, simple separation fermentation liquor and bacterial thallus can enter a separation, purification and recovery stage of target proteins; 6. has stress resistance and can be used for producing various thermostable enzyme preparations.

The pichia pastoris expression system has eukaryotic expression advantages in addition to the advantages of the bacillus expression system described above: 1. more closely to mammalian protein folding and modification, and improves protein activity. 2. High yield, and is suitable for projects with high yield requirements. 3. Is particularly suitable for proteins requiring complex post-translational modifications.

Thus, in the present invention, laccase mutants having improved thermostability are obtained by mutating laccase genes derived from Bacillus pumilus (Bacillus pumilus). And then, the high temperature resistant mutant laccase is prepared by using a bacillus and pichia pastoris expression system.

Disclosure of Invention

Based on the problem of laccase high-temperature application scene, in order to obtain laccase with improved thermal stability, the existing property of the laccase needs to be further modified, and the invention aims to provide a high-temperature-resistant laccase mutant. Expressing a laccase gene (lac) derived from Bacillus pumilus in Escherichia coli BL 21; the error-prone PCR technology is utilized to mutate the gene lac, and the ABTS is utilized to screen the mutant, so that the mutant with improved thermal stability is selected, and the expression preparation in bacillus subtilis WB600, bacillus amyloliquefaciens CGMCC No.11218 and pichia pastoris GS115 is realized.

The technical route for achieving the purpose of the invention is summarized as follows:

The laccase gene LAC from Bacillus pumilus is mutated, and the mutant V347D/Y463W and the coding gene lacm W are obtained by screening with colibacillus expression system, and the half life of the laccase gene LAC at 80 ℃ is improved to 151% of that of wild Laccase (LAC). The efficient preparation of the optimal mutant laccase V347D/Y463W is realized by using the bacillus subtilis WB600 and the pichia pastoris GS 115.

One of the technical schemes provided by the invention is a laccase mutant, wherein the laccase mutant is obtained by carrying out mutation comprising V347D and Y463W on the basis of wild laccase shown in SEQ ID NO.1, and the laccase mutant is named as V347D/Y463W mutant;

further, the V347D/Y463W mutant has an amino acid sequence shown in SEQ ID NO. 3;

Furthermore, the nucleotide sequence of the coding gene lacm of the V347D/Y463W mutant is shown as SEQ ID NO. 4.

The second technical scheme provided by the invention is a recombinant plasmid or recombinant strain containing the mutant coding gene;

Further, the adopted expression vector is pET-28a (+), and the host is escherichia coli, bacillus subtilis, bacillus amyloliquefaciens or pichia pastoris;

Still further, the host cell is E.coli BL21;

Further, the host cell is bacillus subtilis WB600, or bacillus amyloliquefaciens CGMCC No.11218, or the host cell is pichia pastoris GS115;

preferably, the recombinant strain is obtained by connecting a mutant coding gene with an expression vector pET-28a (+) and then expressing the mutant coding gene in host escherichia coli.

The third technical scheme provided by the invention is the application of the recombinant plasmid or recombinant strain in the laccase mutant in the production technical scheme I.

The fourth technical scheme provided by the invention is the application of the laccase mutant in the first technical scheme, in particular to the application in degradation of lignocellulose; further, the method is applied to degrading straw or bagasse;

further, the straw includes, but is not limited to: corn straw, rice straw or wheat straw.

The following definitions are employed in the present invention:

1. nomenclature of amino acids and DNA nucleic acid sequences

Using the accepted IUPAC nomenclature for amino acid residues, in single letter or three letter codes. The DNA nucleic acid sequence uses accepted IUPAC nomenclature.

2. Identification of laccase mutants

"Original amino acid+position+substituted amino acid" is used to denote the mutated amino acid in the LAC mutant. As in V347D, the amino acid at position 347 is replaced by Val of wild-type LAC with Asp, the numbering of the position corresponding to the amino acid sequence numbering of the mature peptide of wild-type LAC in SEQ ID NO. 1.

In the present invention, LAC represents wild-type laccase, V347D/Y463W represents laccase mutant, lower case italics LAC represents the gene encoding wild-type laccase LAC, and lower case italics lacm1 represents the gene encoding mutant V347D/Y463W.

Specific information is shown in the following table.

Advantageous effects

The invention uses error-prone PCR technology to mutate LAC wild type to obtain mutant V347D/Y463W with improved thermal stability at 80 ℃ compared with wild type, and the half life of the mutant V347D/Y463W at 80 ℃ is improved to 151% of that of wild type Laccase (LAC).

Drawings

FIG. 1 is an electrophoresis chart of PCR amplification of wild type laccase

Wherein: m is DNA MARKER, lane 1 is laccase gene lac.

FIG. 2 shows the cleavage map of pET-lac plasmid

Wherein: m is DNA MARKER, and 1 is pET-lac subjected to NcoI and XhoI double digestion.

FIG. 3 shows the enzyme activity of wild type and mutant V347D/Y463W laccase over time at 80 ℃.

Detailed Description

The technical contents of the present invention will be further described with reference to examples, but the present invention is not limited to these examples, and the scope of the present invention is not limited to the following examples.

The partial solutions and culture mediums used in the examples of the present invention are as follows:

Lysis buffer (mM): tris 20, naCl 500, dithiothreitol 1, imidazole 20.

Wash buffer (mM): tris 20, naCl 500, dithiothreitol 1, imidazole 100.

ElutionBuffer (mM): tris 20, naCl 500, dithiothreitol 1, imidazole 500.

LB medium (g/L): yeast extract 5.0, tryptone 10.0, naCl 10.0, the balance being water. 2% agar was added to the solid medium.

BMGY Medium (g/L): peptone 20.0, yeast extract 10.0, YNB 13.4, 4X 10 ^-5% biotin, 10% 1:1 mmol/L potassium phosphate buffer (pH 6.0), 0.5% glycerol.

BMMY medium (g/L): peptone 20.0, yeast extract 10.0, YNB 13.4, 4X 10 ^-5% biotin, 10% 1:1 mmol/L potassium phosphate buffer (pH 6.0), 0.5% methanol.

Fermentation medium (g/L): corn flour 64, bean cake flour 40, 2.7 amylase, 4 Na ₂HPO₄,0.3 KH₂PO₄ and the balance of water; preserving heat at 90 ℃ for 30min and sterilizing at 121 ℃ for 20min.

In the invention, the mature peptide sequence of the wild laccase LAC is shown as SEQ ID NO.1 ：MNLEKFVDELPIPEVAKPVKKNPRQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIQANRNEKVKVKWMNKLPLKHFLPVDHTIHEGHHDEPEVKTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFEREVYVYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKDDYDIPLMIMDRTFQEDGSLFYPSRPNDTPEDSDLPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRFRILNASNTRTYELHLDNDATILQIGSDGGFLPRPVRHQSFSIAPAERFDVIIDFSAYENKTITLKNTAGCGQDVNPETDANIMQFKVTRPLKGRVPKTLRLIFKPLPTLRPSQADRERTLTLSGTQDKYGRPILLLDNQFWNDPVTENPRLGSLEVWSIVNPTRGTHPIHLHLVQFRVLDRRPFDTEVYQSTGEIVYTGPNEAPPLHEQGYKDTIQAHAGEVIRIVARFVPYSGRYVWHCHILEHEDYDMMRPMDIIQ.

In the invention, the mature peptide sequence of the V347D/Y463W mutant is shown in SEQ ID NO. 3:

MNLEKFVDELPIPEVAKPVKKNPRQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIQANRNEKVKVKWMNKLPLKHFLPVDHTIHEGHHDEPEVKTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFEREVYVYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKDDYDIPLMIMDRTFQEDGSLFYPSRPNDTPEDSDLPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRFRILNASNTRTYELHLDNDATILQIGSDGGFLPRPVRHQSFSIAPAERFDVIIDFSAYENKTITLKNTAGCGQDVNPETDANIMQFKVTRPLKGRDPKTLRLIFKPLPTLRPSQADRERTLTLSGTQDKYGRPILLLDNQFWNDPVTENPRLGSLEVWSIVNPTRGTHPIHLHLVQFRVLDRRPFDTEVYQSTGEIVYTGPNEAPPLHEQGWKDTIQAHAGEVIRIVARFVPYSGRYVWHCHILEHEDYDMMRPMDIIQ.

The invention will be further illustrated by the following examples.

EXAMPLE 1 obtaining wild-type laccase Gene

1. Genomic DNA of Bacillus pumilus TCCC 11568 was extracted using a kit (OMEGA: bacterial DNA Kit) as follows:

(1) Strains were inoculated onto LB solid plates with an inoculating loop and incubated overnight at 37 ℃.

(2) Single colonies were picked from plates of cultured cells and inoculated into liquid test tube medium, shake-cultured at 37℃and 220 r/min overnight.

(3) 3 ML-5 mL bacteria solution was placed in sterilized EP tube, centrifuged at 12000 r/min for 2 min, and the supernatant was discarded.

(4) 200 Mu L of sterile water is added into an EP tube to resuspend the thalli, 50 mu L of lysozyme is added, and the mixture is blown and sucked uniformly, and the temperature is kept at 37 ℃ for 20 min.

(5) 100. Mu.L of BTL buffer and 20. Mu.L of proteinase K were added to the EP tube, mixed by vortexing, incubated at 55℃40 min, and mixed by vortexing every 20: 20 min.

(6) Add 5. Mu.L RNase and mix upside down several times and leave 10min at room temperature.

(7) Centrifuge 2 min at 12000 r/min, remove undigested fractions, transfer supernatant to a new EP tube, add 220. Mu.L BDL buffer,65℃water bath 15 min.

(8) 220 Mu L absolute ethyl alcohol is added, and the mixture is blown and sucked uniformly.

(9) Transferring the liquid in the EP pipe into a recovery column, standing for 1min, centrifuging for 1min at 12000 r/min, pouring the filtrate into the recovery column again, repeating for two times, and pouring out the waste liquid.

(10) 500. Mu.L of HBC buffer was added, and the mixture was centrifuged at 12000 r/min for 1:1 min, and the filtrate was discarded.

(11) 700 Mu L DNA wash buffer was added, left stand for 1 min, centrifuged at 12000 r/min for 1 min, and the filtrate was discarded.

(12) Adding 500 mu L DNA wash buffer, standing for 1 min, centrifuging at 12000 r/min for 1 min, and discarding the filtrate.

(13) 12000 R/min air-separation 2 min, discard the waste tube and put the recovery column into a new EP tube.

(14) The mixture is placed in a 55 ℃ metal bath for drying 10 min.

(15) 50. Mu.L of sterile water at 55℃was added, the mixture was allowed to stand at room temperature for 5 min, centrifuged at 12000 r/min for 2 min, the recovery column was discarded, and the liquid in the EP tube was the genome.

2. The genome of the extracted bacillus pumilus is used as a template, a pair of primers are designed at the upstream and downstream of an ORF frame, and restriction enzyme cutting sites NcoI and XhoI are respectively introduced, and the amplification primers of laccase gene lac of the invention are as follows:

The upstream primer P1:

5’- CATGCCATGGGCATGAACCTAGAAAAATTTGTTGACG-3’

Downstream primer P2:

5’- CCCTCGAGCTGGATGATATCCATCGGCC-3’

p1 and P2 are used as an upstream primer and a downstream primer, and the genome of the bacillus pumilus laccase is used as a template for amplification.

The reaction system for amplification is as follows:

the amplification procedure was: pre-denaturation at 98 ℃ of 30 s; denaturation at 98℃for 10 s, annealing at 54℃for 20 s, extension at 72℃for 8 s, reaction for 30 cycles; the extension is 10 min at 72 ℃. The PCR amplified product is subjected to agarose gel electrophoresis with concentration of 0.8% to obtain a band of 1530 bp (figure 1), the PCR product is recovered by using a small amount of DNA recovery kit to obtain the wild type laccase gene lac (SEQ ID NO. 2), the lac and pET-28a (+) plasmids are subjected to double digestion respectively by using restriction endonucleases NcoI and XhoI, the lac recovered by the gel digestion is connected with a carrier pET carrier to obtain a recombinant plasmid pET-lac, the digestion verification is shown in figure 2, and the recombinant plasmid pET-lac is converted into escherichia coli BL21 and is named as recombinant strain BL21/pET-lac.

Example 2LAC Activity and temperature stability determination

ABTS assay LAC enzyme activity assay method:

(1) 200 mu L of citric acid-disodium hydrogen phosphate buffer solution (50 mM and containing 5mM Cu ²⁺) with pH of 5.5 is taken in a 96-well ELISA plate, and the temperature is kept in a water bath at 80 ℃ for 1 min;

(2) Adding 10 mu L of LAC/LACM diluted to a proper concentration, uniformly mixing, and putting into 80 ℃ water bath again for heat preservation of 1 min;

(3) Then, 30 mu L of ABTS (50 mM) is added for blowing and sucking, and the mixture is uniformly mixed, and reacted in a water bath at a constant temperature of 80 ℃ for 10min, after the reaction starts and ends, OD values after the reaction starts and ends are recorded under 420 nm.

Calculating the enzyme activity:

Wherein: Δod = OD end-OD initial;

V1 represents the total volume ([ mu ] L) of the reaction system;

Δt represents the reaction time (min);

V2 adding the volume (μl) of enzyme solution;

Epsilon represents the molar absorption coefficient of the product at 420 nm, 36 mM ^-1cm^-1;

d represents the inner diameter/optical path thickness (cm) of the 96-well microplate.

Enzyme specific activity (U/mg) =enzyme activity/protein concentration.

Temperature stability measurement of LAC was placed in a buffer solution at pH 7.0 and incubated at 80℃for different times, samples were taken at regular time, residual enzyme activity was measured at pH 5.0 and 80℃after incubation was completed, and the residual enzyme activity was calculated by taking the enzyme activity without incubation as 100%, and the enzyme activity change curve was drawn to further calculate half-life.

EXAMPLE 3LAC mutant construction and screening

1. Construction of mutant libraries

Error-prone PCR: error-prone PCR was performed using the wild-type coding gene lac as a template, and the reaction system was as follows:

note that: the above reagents were obtained from Takara, takara Bio Inc.

After the system is completed, error-prone PCR reaction is carried out, and the program is set as follows:

a. pre-denaturation at 95℃5 min;

b. Denaturation: 95. 30℃ s;

c. annealing: 56. 45℃ s;

d. extension: 72. 50 ℃ s;

e.b-d reactions were carried out for 35 cycles;

f. extension at 72℃of 10 min.

After the PCR reaction is finished, the PCR amplification product is subjected to 0.8% agarose gel electrophoresis, and the PCR product is recovered by using a small amount of DNA recovery kit, so that different laccase mutant genes lacm are obtained. Recombinant plasmid pET-lacm is constructed through enzyme digestion and connection, and then the recombinant plasmid pET-lacm is transformed into escherichia coli BL21 to obtain recombinant strain BL21/pET-lacm.

2. Mutant screening

(1) Adding 2 mL sterilized LB culture medium into a 96-well plate, adding 2 [ mu ] L Kan resistance (action concentration 50 [ mu ] g/mL), adding different mutant single colonies, and culturing overnight in a shaking table at 37 ℃ with the contrast being wild type;

(2) Transferring the bacterial liquid to a new 24-well plate according to the inoculation amount of 2%, adding 1 mL sterilized LB (containing Kan resistance) into each well, carrying out shaking culture at 37 ℃ and 600 r/min for 3h (culturing until the OD600 is 0.6-0.8), adding 0.5 mM IPTG final concentration into each well, and inducing overnight culture at 16 ℃ under a shaking table;

(3) The bacterial cells were collected by using a well plate centrifuge at 5000 r/min, centrifuged at 15 min, the supernatant was removed, the mixed bacterial cells were blow-sucked with a buffer solution at pH 7.0, the bacterial cells were collected in 2mL centrifuge tubes, and cell disruption was performed by adding 80. Mu.L of lysozyme. After the crushing is completed, centrifugally collecting the crushed supernatant, dividing the crushed supernatant into two parts, wherein one part is used for measuring the enzyme activities of different mutants in a water bath kettle at 80 ℃ according to the step 1 of the example 2; after another part has been incubated at 80℃for 30 min, the residual enzyme activity is determined according to example 2, step 2;

(4) The mutant strain is obtained by selecting a mutant transformant in which the absorbance of the crushed supernatant catalytic ABTS is higher than that of the wild type strain at 420 nm and the absorbance of the catalytic ABTS is still higher than that of the wild type strain at 420 nm after the temperature is maintained at 80 ℃ for 30min, sequencing the mutant transformant to obtain a mutant gene lacm (shown as SEQ ID NO. 4), and finally, obtaining a mutant V347D/Y463W (shown as SEQ ID NO. 3) with activity and stability higher than those of the wild type strain through preliminary screening, wherein the transformant containing the mutant strain is named as recombinant strain BL21/pET-lacm1.

EXAMPLE 4 expression purification of WT and V347D/Y463W and enzyme activity assay

1. Induction expression of recombinant strains

(1) On an LB plate, respectively picking a single colony of BL21/pET-lacm1 and BL21/pET-lac, inoculating in a 5mL LB test tube (containing 50 mug/mLKan), and placing in a shaking table at 37 ℃ for shake culture for 10 h;

(2) Transferring the recombinant bacterial liquid into a 250 mL LB culture medium (final concentration is 50 mug/mL Kan), and placing the culture medium in a shaking table at 37 ℃ for shake culture for 2-2.5 h;

(3) Adding 125 mu L of IPTG (final concentration is 0.5 mmol/L), and performing induced culture on the mixture in a 16 ℃ shaking table to obtain 16-20 h;

(4) And (3) purifying the fermentation liquor to prepare the wild laccase and the laccase mutant.

2. Ni column purification of recombinant proteins

(1) Breaking thallus

The fermentation broth was collected using a centrifuge cup, 10000 rpm, centrifuged 15 min, the supernatant was removed, 20mL Lysis buffer cells were sucked by blowing and the cells were disrupted by ultrasonic waves, the cell walls were destroyed, and the intracellular proteins were released.

After the completion of the disruption, the bacterial liquid was poured into a 50 mL centrifuge tube, and centrifuged at 12000rpm at 4℃for 30 min, and the disrupted supernatant was collected.

(2) WT and V347D/Y463W binding to Nickel column

A. Before the nickel column purification process, add the appropriate amount of ddH ₂ O to the purification column and add double column volume of Lysis buffer to equilibrate the resin;

b. mixing the balanced resin and thallus supernatant, placing in magnetic stirrer, combining at speed of 80-100 r/min with 1: 1h, and maintaining low temperature (4deg.C).

(3) Protein purification

A. Adding the combined liquid into the purification column for 2-3 times in a chromatography cabinet;

b. After the binding solution is completely filtered out, 10 mL Wash buffer is added to elute the hybrid protein bound with the resin;

c. Finally, adding 10 mL pre-cooled absorption Buffer into the purification column, eluting all target proteins combined with the resin, and collecting filtrate;

d. All the eluate was transferred to an ultrafiltration centrifuge tube, and when the solution remained in the centrifuge and ultrafiltration tube by 1 mL, 50 mM of pre-chilled Tris-HCl buffer solution with pH of 7.0 was added, and the displacement was repeated twice. Purified WT and V347D/Y463W proteins were obtained.

The specific activity was determined according to example 2, step 1, and the final calculated specific activities of wild-type LAC and mutant V347D/Y463W were as follows:

The temperature stability was measured according to example 2, step 2, and thermal stability at 80℃was obtained (i.e.the enzyme activities of WT and V347D/Y463W mutants were measured according to the method of example 2, step 2, after incubation at 80℃for different times, the enzyme activity change curves were plotted, the enzyme activity change curves are shown in FIG. 3), and finally the half-lives of wild-type LAC and mutants were calculated as follows:

EXAMPLE 5 expression and preparation of laccase mutants in Bacillus subtilis

The laccase mutant V347D/Y463W coding gene lacm1 and the wild laccase coding gene lac are respectively connected with an expression plasmid pLY-3 to obtain new recombinant plasmids pLY-3-lacm1 and pLY-3-lac;

The recombinant plasmids are respectively transferred into bacillus subtilis WB600, and the mutant recombinant bacterium WB600/pLY-3-lacm1 and the wild laccase recombinant bacterium WB600/pLY-3-lac are obtained through the screening of the resistance of the kanamycin (Kan) and the enzyme digestion verification.

Recombinant strains WB600/pLY-3-lacm1 and WB600/pLY-3-lac were inoculated into fermentation medium (containing kanamycin, 50. Mu.g/mL) of 5mL, cultured overnight at 37℃at 220 r/min, transferred into fresh fermentation medium (containing kanamycin, 50. Mu.g/mL) of 50mL at an inoculum size of 2%, continuously cultured at 37℃at 220 r/min for 48 h (fermentation medium (g/L): corn meal 64, soybean meal 40), added with 2.7 amylase, na ₂HPO₄4, KH₂PO₄ 0.3.3, the balance being water, incubated at 90℃for 30min and sterilized at 121℃for 20 min.

The enzyme activity of laccase obtained by fermentation of Bacillus subtilis was determined by the ATBS method in example 2 (enzyme activity was determined by centrifuging the fermentation broth to obtain the supernatant). The wild type enzyme activity in the bacillus subtilis is 955.6U/mL, and the fermentation enzyme activity of V347D/Y463W is 1287.2U/mL.

EXAMPLE 6 expression and preparation of laccase mutant in recombinant strain of Bacillus amyloliquefaciens

The laccase mutant V347D/Y463W coding gene lacm1 and the wild laccase coding gene lac are respectively connected with an expression plasmid pLY-3 to obtain new recombinant plasmids pLY-3-lacm1 and pLY-3-lac;

The recombinant plasmid is respectively transferred into bacillus amyloliquefaciens CGMCC No.11218, and is subjected to resistance screening of kananamycin (Kan) and enzyme digestion verification to obtain mutant recombinant bacterium CGMCC No.11218/pLY-3-lacm1 and wild laccase recombinant bacterium CGMCC No.11218/pLY-3-lac.

Recombinant strains CGMCC No.11218/pLY-3-lacm1 and CGMCC No.11218/pLY-3-lac are respectively inoculated into a fermentation culture medium (containing kanamycin and 50 mu g/mL) of 5mL, cultured overnight at 37 ℃,220 r/min, transferred into a fresh fermentation culture medium (containing kanamycin and 50 mu g/mL) of 50 mL according to the inoculum size of 2%, continuously cultured at 37 ℃,220 r/min and 48 h (fermentation culture medium (g/L): corn powder 64, bean cake powder 40, 2.7 amylase, na ₂HPO₄4, KH₂PO₄ 0.3 and the balance water are added, and the temperature is kept at 90 ℃ for 30min and then sterilized at 121 ℃ for 20 min.

Laccase activity obtained by fermentation of Bacillus amyloliquefaciens was determined using the ATBS method of example 2 (enzyme activity was determined by centrifugation of the fermentation broth from supernatant). The wild type enzyme activity of the bacillus amyloliquefaciens is 1918.5U/mL, and the fermentation enzyme activity of V347D/Y463W is 2583.3U/mL.

Example 7 expression and preparation of laccase mutant in Pichia pastoris GS115 recombinant strain

The laccase mutant V347D/Y463W coding gene lacm1 and the wild laccase coding gene lac are respectively connected with an expression plasmid pPIC9K to obtain new recombinant plasmids pPIC9K-lacm1 and pPIC9K-lac;

Linearizing recombinant plasmids pPIC9K-lacm1 and pPIC9K-lac by using restriction endonuclease SalI, respectively electrically transferring the linearized recombinant plasmids into Pichia pastoris GS115, screening the resistance of the kanamicin, and performing enzyme digestion verification to obtain mutant recombinant bacteria GS115/pPIC9K-lacm1 and wild laccase recombinant bacteria GS115/pPIC9K-lac.

(1) Single colonies of recombinant expression strains GS115/pPIC9K-lacm and GS115/pPIC9K-lac were picked and inoculated into 5mL YPD tubes containing 50. Mu.g/mL Kan, cultured at 30℃and 200 r/min for 24 h;

(2) Inoculating 1 mL bacterial liquid into BMGY enrichment medium, culturing at 30deg.C and 220 r/min for 16-19 h;

(3) Collecting bacterial liquid in the BMGY culture medium by using a 50mL centrifuge tube, and centrifugally collecting bacterial bodies; adding 20mL BMMY culture medium to resuspend the thalli, centrifuging to collect the thalli, and repeating the process once; the bacteria were resuspended in 10mL BMMY medium and transferred to BMMY medium with a pipette, and methanol was added at a final concentration of 0.5% every 12: 12 h, and cultured for 6 d.

The activity of laccase obtained by fermentation of Pichia pastoris was determined using the ATBS method in example 2 (enzyme activity was determined by centrifugation of the fermentation broth from supernatant). In Pichia pastoris GS115, the fermentation activity of laccase is 1337.9U/mL, and the fermentation activity of V347D/Y463W is 1798.6U/mL.

Example 8 application of laccase mutant in degrading corn stalk and bagasse

Pre-treatment of corn stover and bagasse the corn stover and bagasse were derived from a local enterprise in Jiangsu China. Firstly, deionized water is used for cleaning to remove dirt on the surface, then the surface is placed in a constant-temperature drying box at 60 ℃ for drying so that the moisture content is below 2%, the dried raw materials are crushed by a crusher and filtered by a 40-mesh screen to ensure that the raw materials become uniform powder, and finally, the uniform powder is placed at normal temperature for subsequent use.

2. Laccase degradation lignin

The treated corn stalks and bagasse are respectively used as samples to be degraded, the wild laccase and the mutant laccase V347D/Y463W of the invention are respectively added, and a system of 20 mL is adopted: taking 1 g to-be-degraded sample in a 50 mL centrifuge tube, adding 50U of WT/mutant laccase, adding 5 mM Cu ²⁺ citric acid-disodium hydrogen phosphate buffer solution with pH of 5.0, supplementing to 20 mL, and carrying out shaking table reaction at 50 ℃ in 150 r/min water bath for 6 h.

Lignin degradation rates after laccase wild-type and mutant V347D/Y463W treatment were determined. The method comprises the following steps:

(1) Extracting the treated corn stalks and bagasse with benzene/alcohol to obtain 8 h, and drying until the corn stalks and bagasse are absolute dried for later use;

(2) 0.3 g was weighed, added to 72% sulfuric acid of 3mL, and stirred until the materials were thoroughly mixed. Mixing, and placing in 30deg.C water bath for heat preservation of 60 min;

(3) Adding 84 mL deionized water to dilute to 4%, placing into a 121 ℃ sterilizing pot, taking out after 45: 45 min, vacuum filtering by using a G3 (weighing, marked as m 0) sand core funnel, measuring the absorbance value of acidolysis solution at 205: 205 nm by using 50:50 mL filtrate, and calculating the content of acid-soluble lignin, wherein the measuring method is GB/10337-89 measurement of acid-soluble lignin in papermaking raw materials and paper pulp;

The filtered residue was washed to neutrality with hot deionized water, dried in an oven at 105 ℃, and weighed for recording (m 1). Drying, transferring into a resistance furnace, and burning at 575 ℃ to 4 h. The content of acid-insoluble lignin was calculated by taking out and cooling to room temperature and then weighing and recording (m 2). The final calculated lignin degradation rate is as follows:

1. Degradation result of lignin in corn straw

The lignin degradation rate of the mutant V347D/Y463W after treatment is 29.1%, and the lignin degradation rate of the mutant V347D/Y463W after treatment is 21.3%.

2. Degradation results of lignin in bagasse

The lignin degradation rate of the mutant V347D/Y463W after treatment is 26.5%, and the lignin degradation rate of the mutant V347D/Y463W after treatment is 19.6%.

The results show that the lignin degradation rate of the mutant on the corn straw and the bagasse is higher than that of the WT when the straw and the bagasse are treated by laccase.

The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.

Claims (8)

1. A laccase mutant is characterized in that the laccase mutant is obtained by mutating V347D and Y463W on the basis of wild laccase shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO. 3.

2. A gene encoding the laccase mutant of claim 1.

3. A recombinant plasmid or recombinant strain comprising the mutant encoding gene of claim 2.

4. Use of the recombinant plasmid or recombinant strain of claim 3 for the production of the laccase mutant of claim 1.

5. Use of the laccase mutant according to claim 1.

6. The use according to claim 5, in the degradation of lignocellulose.

7. The use of claim 6, wherein the lignocellulose includes, but is not limited to: straw and bagasse.

8. The use of claim 7, wherein the straw includes, but is not limited to, corn straw, rice straw, or wheat straw.