An engineered strain of Pichia pastoris with high laccase production and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a laccase mutant and its application. The amino acid sequence of the laccase mutant is shown in SEQ ID No. 4. A vector containing a mutant gene is introduced into a host by means of genetic engineering to obtain a genetically engineered bacterium. The fermentation performance is verified to show that the laccase activity can reach more than 55U / mL. The optimum pH of the laccase mutant is 4.5, which is 0.5 lower than that before the mutation. The optimum temperature is 45°C. After being treated for 24 hours under the conditions of pH 4.5 and temperature 60°C, the relative activity of the enzyme still remains at about 85%. Compared with before the mutation, the heat resistance is improved, which is more conducive to application in the cellulose bioconversion industry and bioremediation.

Description

Technical field:

[0001] The invention relates to the field of biotechnology, and in particular to a Pichia pastoris engineering bacterium with high laccase production. Background technology:

[0002] Laccase (EC 1.10.3.2), belonging to the blue multi-copper enzyme family, is a copper-containing polyphenol oxidase. Laccase has a wide range of substrates and can use molecular oxygen as an electron acceptor to catalyze the redox reaction of various aromatic hydrocarbon compounds and lignin to degrade them. In addition, laccase is widely distributed in fungal secretions, higher plants, a small amount of bacteria and insects, among which the sources of fungal laccase and bacterial laccase are relatively abundant. Laccase has the functions of catalyzing the degradation of lignin, removing the toxicity of aromatic amine toxic compounds, and promoting the synthesis of fungal pigments. When oxidizing substrates, it has the advantages of substrate specificity, mild reaction conditions and harmlessness to the environment. These determine the huge potential of the application value of laccase, which has been highly valued and widely concerned in disciplines such as biology, medicine, chemistry, and environment, and has practical applications.

[0003] There is a large amount of agricultural byproduct straw in my country, but it cannot be fully utilized and can only be burned, resulting in great waste. Laccase can act on small fragments of lignin containing phenolic groups, thereby catalyzing the degradation of lignin polymers. As an important enzyme for cellulose bioconversion, combined with other related cellulase preparations, it can realize the large-scale feed and energy utilization of straw. As a cellulase additive in the production of agricultural and sideline products, straw and bran, it can improve the production performance of livestock and poultry, increase feed utilization, improve the nutritional value of feed, reduce feed costs and improve economic benefits. Therefore, further strengthening the research and development of laccase as an enzyme for cellulose bioconversion will support the development of enzyme products for cellulose bioconversion in the feed and energy industries, which has great social and economic benefits.

[0004] In addition, laccase, as a green catalyst with great application value, can oxidize a large amount of toxic (such as chlorophenol compounds and aniline substitution in chemical preparations such as pesticides, preservatives, insecticides, herbicides, and fungicides) and non-toxic compounds, and therefore plays an important role in biological decontamination. Laccase can also process organic chlorides in papermaking wastewater, and has the advantages of high catalytic efficiency, mild reaction conditions, and undemanding requirements on reaction conditions and reaction equipment. Therefore, laccase has a wide range of application value in purifying industrial wastewater, especially in sewage treatment in papermaking and textile industries. Therefore, further strengthening the research and development work of laccase as a green catalyst in biological decontamination has great social and application value in environmental protection.

[0005] There are some problems in the use of fungal laccase in the wild environment. Usually, the amount produced is small and the activity is low. It is relatively stable under normal temperature conditions, but the heat resistance is poor. Moreover, when produced by fermentation, the fermentation cycle is long, the operation is complicated, and a variety of laccase isozymes are produced, which hinders the subsequent extraction and purification. There are many influencing factors in the fermentation process. Therefore, the heterologous expression of laccase in engineering bacteria can be studied to realize the practical application of laccase. Therefore, the present invention selects a laccase-producing black Rhizopus of our company as the experimental material, mutagenesis and screening and cloning to obtain one of the laccase genes, obtains a high-yield heat-resistant laccase mutant, and by constructing an expression vector of Pichia pastoris, finally obtains an engineering bacterium expressing high laccase activity in Pichia pastoris, which meets the needs of cellulose bioconversion industrial production and bioremediation, reduces production costs, has great significance, and lays a foundation for further research and development of enzymes for cellulose bioconversion and bioremediation. Summary of the invention:

[0006] The purpose of the present invention is to provide a laccase mutant with improved enzyme activity and heat resistance and a Pichia pastoris engineered strain thereof, aiming to solve the problems of low enzyme activity of laccase in industrial production, unsatisfactory application in cellulose bioconversion and bioremediation.

[0007] The present invention is specifically implemented through the following technical solutions:

[0008] A laccase mutant with improved enzyme activity and heat resistance is firstly obtained by screening Rhizopus nigricans RN056 preserved in the applicant's laboratory through a nitrosoguanidine mutagenesis method to obtain a mutant strain, and the laccase mutant encoding gene is cloned from the mutagenic strain and a recombinant plasmid is constructed, and then expressed in Pichia pastoris GS115, thereby obtaining a Pichia pastoris engineered bacterial strain.

[0009] One of the technical solutions provided by the present invention is a laccase mutant, wherein the laccase mutant is obtained by mutating the aspartic acid at position 122 to valine, the tyrosine at position 241 to aspartic acid, the leucine at position 244 to arginine, the asparagine at position 476 to serine, and the valine at position 480 to glycine based on the wild-type laccase of RN056 shown in SEQ ID No. 2, and the amino acid sequence of the laccase mutant is shown in SEQ ID No. 4;

[0010] The present invention also provides a gene encoding the laccase mutant;

[0011] Furthermore, the nucleotide sequence of the gene encoding the laccase mutant is shown in SEQ ID No.3.

[0012] The enzymatic properties of the laccase mutant are as follows:

[0013] (1) Optimum pH: The enzyme activity is stable at pH 4.0-5.5, and the optimal action pH is 4.5;

[0014] (2) Optimum temperature: Enzyme activity is stable at 40℃-55℃, and the optimal action temperature is 45℃;

[0015] (3) Heat resistance: Under the conditions of pH 4.5 and temperature 60°C, the enzyme activity still retains more than 85% after 24 hours.

[0016] The third technical solution provided by the present invention is: a recombinant vector or a recombinant strain comprising the above-mentioned laccase mutant encoding gene, reconstructing the above-mentioned laccase mutant encoding gene into a recombinant vector, and efficiently expressing it in Pichia pastoris to obtain a recombinant strain producing high-activity laccase, and obtaining high-activity laccase by fermentation, extraction and other technologies;

[0017] Furthermore, the host cell used to express the laccase mutant is Pichia pastoris GS115;

[0018] Furthermore, the expression vector used to express the laccase mutant is a pPIC9K plasmid;

[0019] Preferably, the recombinant strain is obtained by connecting the laccase mutant encoding gene shown in SEQ ID No. 3 to the expression vector pPIC9K and expressing it in Pichia pastoris GS115.

[0020] The fourth technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the third technical solution, especially in the fermentation production of the laccase mutant shown in SEQ ID No.4.

[0021] The fifth technical solution provided by the present invention is the application of the laccase mutant shown in SEQ ID No. 4, especially in the oxidation-reduction of substrates and then hydrolysis of the substrates;

[0022] Further, it is applied in the cellulose bioconversion industry, such as removing lignin from corn stover lignocellulose hydrolysate by acting on small fragments of lignin containing phenolic groups;

[0023] Furthermore, it is used in biological decontamination or sewage treatment, such as treating organic chlorides in papermaking wastewater by oxidizing a large amount of toxic (such as chlorophenol compounds and aniline substitutes in pesticides, preservatives, insecticides, herbicides, fungicides and other chemical preparations) and non-toxic compounds.

[0024] The following definitions are used in the present invention:

[0025] 1. Nomenclature of amino acid and DNA sequences

[0026] The generally accepted IUPAC nomenclature for amino acid residues is used, either in the form of three-letter or one-letter codes. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0027] 2. Identification of Laccase Mutants

[0028] "Original amino acid + position + replaced amino acid" is used to represent the mutated amino acid in the mutant. For example, Asp122Val means that the amino acid at position 122 is replaced by Val from wild-type Asp, and the position number corresponds to the amino acid sequence number of the wild-type laccase in SEQ ID No. 2. In the present invention, RNT-1 represents wild-type laccase, and RNT-2 represents laccase mutant, and the information is shown in the following table.

[0029]

[0030] Beneficial effects:

[0031] The invention discloses a new laccase mutant, which has the characteristics of high enzyme activity and high heat resistance. The enzyme activity of the mutant fermentation liquid can reach more than 55U / mL.

[0032] The optimum reaction pH of the laccase obtained by the present invention is 4.5 and the enzyme activity is the highest. Compared with the laccase of the wild type RN056, the optimum reaction pH is reduced by 0.5, indicating that the acid resistance is improved.

[0033] The laccase obtained by the invention retains more than 85% of its enzyme activity after 24 hours under the conditions of pH 4.5 and 60°C, and has significantly improved heat resistance compared with the wild-type RN056 laccase. Description of the drawings:

[0034] Figure 1 Optimum pH curve;

[0035] Figure 2 Optimum temperature curve;

[0036] Figure 3 Heat resistance curve. Specific implementation method:

[0037] The present invention is described in more detail by specific embodiments, which are only used as examples and are not intended to limit the scope of the present invention. For those skilled in the art, improvements that can be made based on the principles of the present invention should also be considered as the scope of protection of the present invention. The molecular biology experimental methods that are not specifically described in this embodiment can be referred to in the "Molecular Cloning Experiment Guide".

[0038] The present invention provides a laccase mutant with improved enzyme activity and heat resistance. The mutant is selected from a mutated strain obtained by nitroguanidine mutagenesis of a black root mold strain (Rhizopus nigricans) RN056 preserved in the applicant's laboratory. The mutated sites obtained by gene sequencing are aspartic acid at position 122 mutated to valine, tyrosine at position 241 mutated to aspartic acid, leucine at position 244 mutated to arginine, asparagine at position 476 mutated to serine, and valine at position 480 mutated to glycine. The present invention first obtains a coding gene for the laccase mutant, connects the mutant coding gene to a pPIC9K vector to construct a recombinant plasmid, transfers it into a corresponding host bacterium GS115 for heterologous expression, and obtains the corresponding laccase of the mutant by fermentation. The laccase has high enzyme activity and is suitable for industrial production, cellulose bioconversion industry and bioremediation.

[0039] Some of the materials and methods involved in the present invention are as follows:

[0040] 1. Experimental materials and reagents:

[0041] Experimental strains and vectors: The wild-type strain is the Rhizopus nigromaculus strain RN056 preserved in the applicant's laboratory; the expression host bacteria and vectors: GS115 and pPIC9K were purchased from Novagen; host bacteria: DH5α.

[0042] Main reagents: DNA polymerase, T4 DNA ligase, DNA gel recovery kit, plasmid extraction kit, RNA extraction kit, pNPG, DNA marker, agarose, ampicillin, IPTG, X-gal: all purchased from Shanghai Bioengineering; various restriction endonucleases: purchased from NEB.

[0043] Experimental instruments: gel imager (Bio-Rad), protein electrophoresis instrument (Bio-Rad), nucleic acid electrophoresis instrument (Bio-Rad), PCR amplification instrument (Bio-Rad), high-speed centrifuge (Eppendorf).

[0044] 2. Laccase activity determination method used in the present invention:

[0045] The principle of laccase activity determination: the product of laccase decomposition of ABTS is ABTS free radicals, but the absorption coefficient of ABTS free radicals at 420nm is much greater than that of substrate ABTS. As the concentration of ABTS free radicals increases, the absorbance value increases, and the time it takes for the absorbance value to change within a certain specified range is expressed as the activity of laccase.

[0046] Definition of enzyme activity unit: Under certain conditions (temperature 30°C, pH 4.5 if not otherwise specified), one laccase unit (U) is the amount of enzyme required to convert 1 μmol ABTS in 1 min, increasing the concentration of the product ABTS free radical from 27.66 μmol / L to 36.88 μmol / L (absorbance value from 0.15 to 0.20).

[0047] Enzyme activity determination method (ABTS method): The substrate solution is preheated at a suitable temperature. During the determination, 2.9 mL of substrate solution (ABTS concentration is 0.5 mmol / L) is taken, 0.1 mL of diluted enzyme solution is added, and the time for the absorbance value to increase from 0.15 to 0.20 is recorded. Then, the enzyme activity is calculated according to the definition of enzyme activity.

[0048] The method for determining the activity of laccase at 420 nm using ABTS as substrate is as follows: pH 4.5 and temperature 30°C are selected, and the laccase is appropriately diluted so that the activity of the laccase dilution is 27.7-55.3 U / mL; during the determination, 2.9 mL of substrate solution (ABTS concentration is 0.5 mmol / L) is taken, 0.1 mL of the diluted enzyme solution is added, and the time t experienced by the laccase catalytic decomposition of 0.5 mmol / L ABTS to increase the absorbance value from 0.15 to 0.20 is determined, and the dilution multiple is changed so that the t value is within the range of 10-20 s. The activity of laccase is calculated according to the formula enzyme activity = 1000×0.1844×(0.20-0.15)×60×dilution multiple / t.

[0049] In the present invention, the amino acid sequence of wild-type laccase is shown in SEQ ID No.2: MASFLSAFAMALLLSTILLKFTIIGQRGTLFWHAHVSWLREPGKTYLLRLINAALNDELFQYPVTLHWHGIKQFKTNYADGPAHITQCPIQPNKSYIYEFKNLRSRTTLPCIFLIADDLITDFVVISPGQTMDILVQANQTEGLYYIGLNPYIAVNETLYPGDLYNCSHKETTVIPVKAGETNLLRFINAALNNELFVAIANHKMTVVATDASYTKPFTTENQEGTLWWHAHSSWLRATVYGALIIHPKLGSPYSFPMPRFPPVPPVKFDYTGNVSRGLWQPVKATKLYKLKFGAKVQIVKATESGAPPEESTAYTINGQPGFPNNCSKETTYRLQVQYGKTYLLRLVNAVMNEEMFFGIANHKLTVVAQDGAYIKPITTVGTGYGNYDETTSPKTYNLKDPPEVNTVGLPRNGWVAIRFVLPPLNDTSFATNFSNKLRSLASAQFPANVPQKVDKRFFFPKRETPILLGEWWDANPIDVTIGLGTNPCDQNNQTCQGPNFPTRPVPFNYTGTPPNNTMVRNGTKLLVIPFNTSVELIMQDTSMLGAESHPLHLHGFNFFSVIRFVANNPGVWLMHCHLDVHITWGLAMSFLVENGVSELDSLEEPPLDLPVC*。

[0050] In the present invention, the amino acid sequence of the mutant laccase is shown in SEQ ID No.4: *.

[0051] The present invention is further explained below through specific implementation modes.

[0052] Example 1 Obtaining the gene encoding the laccase mutant RNT-2

[0053] 1. Starting strain: a laccase-producing Rhizopus nigricans strain RN056 preserved in the applicant's laboratory.

[0054] 2. Culture medium:

[0055] (1) Seed culture medium: 2% glucose, 0.5% peptone, 0.15% potassium dihydrogen phosphate, 0.1% magnesium sulfate, pH 5.0, sterilized at 121°C for 20 min;

[0056] (2) YPD solid medium: 1% yeast extract, 2% peptone, 2% glucose, 2% agar powder, pH 5.0, sterilized at 121°C for 20 min;

[0057] (3) Screening medium: YPD solid medium supplemented with 1% guaiacol, sterilized at 115°C for 20 min;

[0058] (4) Shake flask rescreening culture medium: 2% glucose, 2% peptone, 1% corn steep liquor, 1% ammonium sulfate, pH 5.0, sterilized at 121°C for 20 min.

[0059] 3. Nitrosoguanidine mutagenesis:

[0060] (1) Wash the spores on the fresh slant with 10 mL of 0.1% Tween-60 and transfer them into a conical flask containing glass beads. Shake and disperse them on a shaker for 50 min. Then filter through 4 layers of lens paper and perform gradient dilution. Count the spores on a hemocytometer and adjust the spore concentration to 6 × 10 6 About pcs / mL.

[0061] (2) Preparation of nitrosoguanidine (NTG) solution: In a fume hood, weigh 25 mg of nitrosoguanidine into a brown bottle, add 2.5 mL of acetone to dissolve it, and then add 10 mL of water to prepare a 2.5 mg / mL nitrosoguanidine solution.

[0062] (3) Operation method: Rinse the prepared spore suspension with PBS (100mmol / L, pH 7.0) once, add 0.5mL PBS to make a bacterial suspension, then add 0.4mg / mL NTG solution, treat at 30℃ for 50min, and the lethality of NTG concentration is 79.5%. Centrifuge the NTG-treated bacteria, rinse with PBS three times to remove NTG residues, then suspend the bacteria in 100mL enrichment medium and culture at 30℃ for 12h. Finally, shake the bacterial suspension for 2min, dilute with sterile water to a suitable dilution, spread on the primary screening medium plate, and culture at 30℃ for 48h. After culturing, randomly pick strains with good growth from the plate for primary screening.

[0063] 4. Screening of mutant strains

[0064] (1) Primary screening: Screening was performed based on the size of the red-brown oxidation zone on the primary screening culture medium plate. The five strains with the largest red-brown oxidation zone were selected and inoculated onto YPD solid culture medium plates and continued to be cultivated until a pure culture was obtained. The five strains were named R-01, R-02, R-03, R-04, and R-05, respectively.

[0065] (2) Rescreening: The above five strains were then rescreened by fermentation shake flasks. After shake flask fermentation tests, a strain R-02 with a high laccase production was screened. The rescreening results are shown in the following table:

[0066]

[0067] R-02 obtained by mutagenesis screening was used as the starting strain. PCR primers (Forward / Rerverse) were designed according to the nucleotide sequence of the wild-type laccase RNT-1 encoding gene. The restriction site Xho I was added to the 5' end and the restriction site NotI was added to the 3' end. The encoding gene of the mutant RNT-2 was obtained by PCR. The nucleotide sequence obtained by sequencing was SEQ ID No.3, and the corresponding amino acid sequence was SEQ ID No.4.

[0068] Primer sequences:

[0069] Forward: 5'-GCC CTCGAG TCAGCAAACAGGCAAATCAAGT-3' (the underline indicates the restriction endonuclease XhoI recognition site);

[0070] Rerverse: 5'-CAT GCGGCCGC ATGGCTTCTTTCCTTAGTGCTTTTG-3' (the underline indicates the recognition site of restriction endonuclease Not I).

[0071] In the present invention, by comparing the nucleotide sequences of the wild type and the mutant, the mutation site information is obtained as shown in the following table:

[0072]

[0073] Example 2 Construction of recombinant vector pPIC9K-RNT-2

[0074] The mutant RNT-2 encoding gene (SEQ ID No. 3) and plasmid pPIC9K were digested with Xho I and Not I, respectively, and the products were recovered. The recovered RNT-2 encoding gene and pPIC9K were mixed in proportion, ligated with T4 ligase overnight at 16° C., and the ligation product was transformed into Escherichia coli DH5α competent cells. The transformation product was spread on LB (containing ampicillin) solid plates, inverted and cultured at 37° C. overnight, a single colony was picked to LB liquid culture medium, cultured at 37° C., and the target sequence was obtained by colony PCR. Sequencing comparison showed the nucleotide sequence shown in SEQ ID NO. 3, that is, the obtained recombinant vector contained the correct mutant gene, and the obtained recombinant vector was named pPIC9K-RNT-2.

[0075] Example 3 Recombinant plasmid transformation of Pichia pastoris

[0076] 1. Preparation of Pichia pastoris GS115 competent cells

[0077] (1) Pick a single colony from a Pichia pastoris plate and inoculate it into 5 mL of YPD medium at 30°C and 220 rpm overnight;

[0078] (2) Take 0.5 mL of overnight culture solution and inoculate it into 50 mL of freshly prepared YPD medium. Cultivate at 30°C and 220 rpm with shaking until the OD 600 The value reaches 1.3-1.5;

[0079] (3) Centrifuge the culture medium at 4°C, 3000 rpm for 5 min;

[0080] (4) Discard the supernatant, add 50 mL of ice-cold sterile water, and vortex to resuspend the cells;

[0081] (5) Centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant, drain the residual liquid on the tube wall, add 25 mL of ice-cold sterile water, and vortex to resuspend the cells;

[0082] (6) Centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant, drain the residual liquid on the tube wall, add 10 mL of ice-cold 1 mol / L sterile sorbitol solution, and resuspend the cells;

[0083] (7) Centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant, drain the residual liquid on the tube wall, add 1 mL of ice-cold 1 mol / L sterile sorbitol solution (pre-added with glycerol to a final concentration of 15%), and vortex to mix;

[0084] (8) Aliquot 100 μL / tube into sterile EP jars and store in a -70°C refrigerator (freshly prepared competent cells have better results).

[0085] 2. Transformation of Linearized Plasmid

[0086] The positive clones obtained in Example 2 were extracted to obtain the recombinant plasmid pPIC9K-RNT-2, which was digested with Sal I to obtain a linearized plasmid. Freshly prepared (or frozen at -70°C) GS115 competent cells were placed in an ice bath to completely thaw.

[0087] (1) Transfer 100 μL of competent cells to a new sterile EP tube, add 10 μL of linearized plasmid, blow gently to mix, and transfer to a 0.2 cm electroporation cuvette;

[0088] (2) Place the conversion cup in an ice bath for 5-10 minutes to keep it low temperature;

[0089] (3) Electroporation transformation shock conditions: 1500 V, 200 Ω, 25 μF, discharge time of about 5 ms, one shock;

[0090] (4) After electroporation, immediately add 1 mL of 1 mol / L sorbitol solution precooled at 4°C to the electroporation transformation cup, pipette and mix evenly, and place in an ice bath;

[0091] (5) Aseptically apply MD medium (1.34% YNB; 4×10 -5 % biotin; 2% glucose plate), 150 μL / plate, the coated plate was inverted and cultured at 30°C for 3-4 days;

[0092] (6) Two recombinant strains were screened on MD plates. The target sequences were obtained by colony PCR. Sequencing comparison showed that they were the nucleotide sequences shown in SEQ ID NO.3, that is, the obtained recombinant strains contained the correct mutant gene. The two recombinant strains were named Recombinant Bacteria-01 and Recombinant Bacteria-02, respectively.

[0093] Example 4 Induced expression of yeast containing recombinant plasmid pPIC9K-RNT-2

[0094] BMGY medium formula: 1.5% yeast extract, 2.5% peptone, 0.1 mol / L pH 6.0 phosphate buffer, 1.34% YNB, 4×10 -5 % biotin, 1% glycerol, and the rest water.

[0095] BMMY medium formula: 1.5% yeast extract, 2.5% peptone, 0.1 mol / L pH 6.0 phosphate buffer, 1.34% YNB, 4×10 -5 % biotin, 0.6% methanol, and the remainder water.

[0096] Recombinant strain-01, recombinant strain-02, and recombinant strain RD-01 constructed by the same method as in Example 3 with the wild-type laccase encoding gene (nucleotide sequence shown in SEQ ID NO.1) were inoculated into a conical flask filled with 30 mL of BMGY medium and cultured at 30°C and 200 r / min until OD 600 The cell density was about 10, and the cells were collected by centrifugation. The cells were resuspended in 35 mL of BMMY induction medium and cultured for 50 h at 30 °C and 200 r / min. The laccase activity in the supernatant was determined after centrifugation of the fermentation broth. The results are shown in the following table.

[0097] strain Laccase activity (U / mL) RD-01 24 Recombinant bacteria-01 35 Recombinant bacteria-02 37

[0098] Example 5 Verification of the fermentation performance of recombinant bacteria-02

[0099] The recombinant bacteria-02 obtained in Example 3 was used as the production strain for fermentation culture to produce laccase.

[0100] Seed tank medium formula: 3.9% glycerol, 1.5% ammonium dihydrogen phosphate, 0.8% potassium dihydrogen phosphate, 0.6% magnesium sulfate, 0.7% potassium sulfate, 0.05% calcium sulfate, 0.5% potassium hydroxide, the rest is water, pH 5.0;

[0101] Fermentation tank medium formula: 3.9% glycerol, 1.5% ammonium dihydrogen phosphate, 0.8% potassium dihydrogen phosphate, 0.6% magnesium sulfate, 0.7% potassium sulfate, 0.05% calcium sulfate, 0.5% potassium hydroxide, the rest is water, pH 5.0;

[0102] Carbon source: 40% glycerol;

[0103] Methanol: pure methanol;

[0104] Seed tank culture: culture temperature 30℃, initial speed 200r / min, initial air volume 2.5m 3 / h, aeration and stirring culture, pH 5.0, dissolved oxygen maintained at 20-30%, when dissolved oxygen is lower than 20%, control by increasing rotation speed and air volume, transplant when wet weight increases to 75g / L;

[0105] Fermentation tank culture: culture temperature 30℃, initial speed 200r / min, initial air volume 2.5m 3 / h, culture with ventilation and stirring, inoculation amount of 10%, pH 5.0, in the 0-20h period, 40% glycerol as a carbon source is added at a flow rate of 800g / h, the dissolved oxygen is maintained at 20-30%, when it is lower than 20%, it is controlled by increasing the rotation speed and air volume, and the bacteria are cultured to a wet weight of 200g / L; after the 20th hour period, the carbon source is stopped, the dissolved oxygen rebounds to more than 80%, and is maintained for 1.0h; then methanol is added at a rate of 200g / h, the dissolved oxygen is maintained at 20-30%, and the total fermentation period is 180h and the fermentation is completed.

[0106] The above fermentation method was used to carry out a 50L fermentation tank scale-up verification experiment with a fermentation cycle of 180h. The fermentation enzyme production of the three batches is shown in the following table. The average enzyme production level is 56.3U / mL, indicating that the recombinant strain-02 not only produces a high amount of laccase, but also has a certain stability in its fermentation performance and the enzyme activity of the laccase it produces.

[0107] Fermentation enzyme production of 3 batches of genetically engineered bacteria

[0108] batch Fermentation cycle (h) Fermentation activity (U / mL) 1 180 56 2 180 55 3 180 58

[0109] Example 6 Enzymatic properties of laccase

[0110] (1) Optimal pH

[0111] The supernatant of the fermentation broth of the recombinant bacteria-02 obtained in Example 4 was used as the mutant laccase sample, and the supernatant of the fermentation broth of RD-01 was used as the wild-type control sample. The laccase activity determination method of the present invention was used to determine the relative enzyme activities under different pH conditions of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 at 30°C, and the highest laccase activity of the two enzymes was taken as the benchmark of 100%. Figure 1 It can be seen that the laccase mutant of the present invention has stable enzyme activity in the pH range of 4.0-5.5, and the optimal pH is 4.5, which is 0.5 lower than the control. The above results show that compared with before mutation, the laccase after mutation of the present invention has higher enzyme activity under more acidic conditions, and its pH range is wider, which is more suitable for cellulose bioconversion industry and bioremediation.

[0112] (2) Optimum working temperature

[0113] The supernatant of the fermentation broth of the recombinant bacteria-02 obtained in Example 4 was used as the mutant laccase sample, and the supernatant of the fermentation broth of RD-01 was used as the wild-type control sample. The laccase activity determination method of the present invention was used to determine the enzyme activity at different temperatures of 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C under the condition of pH 4.5. The relative enzyme activity was calculated with the maximum enzyme activity of the laccase measured by each of the two enzymes as the benchmark of 100%. The results are shown in FIG. Figure 2 As shown, the laccase mutant of the present invention has stable enzyme activity at 40-55°C, and the optimum action temperature is 45°C. Compared with the wild-type laccase, the optimum reaction temperature does not change, but the optimum temperature range is improved.

[0114] (3) Heat resistance

[0115] The supernatant of the fermentation broth of the recombinant bacteria-02 obtained in Example 4 was used as the mutant laccase sample, and the supernatant of the fermentation broth of RD-01 was used as the wild-type control sample. The laccase activity of each untreated sample was taken as 100% benchmark. The two samples were heat-treated at 60°C under pH 4.5, and the enzyme activity was measured at 30°C and buffer pH 4.5 every 2 hours. The residual enzyme activity was calculated and the Figure 3 It can be seen that after 24 hours, the relative activity of the laccase mutant of the present invention still remains more than 85%. Compared with the wild-type laccase, the heat resistance of the mutant laccase is greatly improved, indicating that the laccase mutant of the present invention has good heat resistance. The above results show that compared with the pre-mutation, the improvement of heat resistance makes the mutant laccase more suitable for application in the cellulose bioconversion industry and bioremediation.

[0116] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A laccase mutant, characterized in that: The amino acid sequence of the laccase mutant is shown in SEQ ID NO.

4.

2. A gene encoding the laccase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

3.

4. A recombinant vector or recombinant strain containing the coding gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that The expression vector is pPIC9K plasmid.

6. The recombinant strain according to claim 4, characterized in that The host cell used was Pichia pastoris GS115.

7. The recombinant strain according to claim 4, characterized in that The recombinant strain is obtained by connecting the laccase mutant encoding gene shown in SEQ ID No. 3 to the expression vector pPIC9K and expressing it in Pichia pastoris GS115.

8. Use of the recombinant vector or recombinant strain according to claim 4 in producing the laccase mutant according to claim 1.

9. Use of the laccase mutant according to claim 1, characterized in that: The invention relates to the application of the laccase mutant in the hydrolysis of redox substrates, or in the bioconversion of cellulose and bioremediation.

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