CN117511891A

CN117511891A - Pichia pastoris engineering strain for high laccase production and application thereof

Info

Publication number: CN117511891A
Application number: CN202311249480.0A
Authority: CN
Inventors: 张辉; 曹世源; 张法玲; 王克芬; 王兴吉; 佟新伟
Original assignee: Shandong Lonct Enzymes Co ltd
Current assignee: Shandong Lonct Enzymes Co ltd
Priority date: 2023-09-25
Filing date: 2023-09-25
Publication date: 2024-02-06

Abstract

The invention belongs to the technical field of biology, and particularly relates to a laccase mutant and application thereof. The amino acid sequence of the laccase mutant is shown as SEQ ID No.4, the vector containing the mutant gene is introduced into a host by means of genetic engineering to obtain genetically engineered bacteria, and the laccase enzyme activity can reach more than 55U/mL through fermentation performance verification. The laccase mutant has the optimal pH value of 4.5, which is reduced by 0.5 compared with the pH value before mutation, the optimal temperature is 45 ℃, the treatment is carried out for 24 hours under the conditions of the pH value of 4.5 and the temperature of 60 ℃, the relative activity of the enzyme is still about 85 percent, and compared with the pH value before mutation, the heat resistance is improved, so that the laccase mutant is more beneficial to being applied to the cellulose bioconversion industry and biological decontamination.

Description

Pichia pastoris engineering strain for high laccase production and application thereof

Technical field:

the invention relates to the technical field of biology, in particular to a Pichia pastoris engineering strain for high laccase production.

The background technology is as follows:

laccase (EC 1.10.3.2), belonging to the family of blue multicopper enzymes, is a copper-containing polyphenol oxidase, has a wider substrate for laccase action, and can utilize molecular oxygen as an electron acceptor to catalyze oxidation-reduction reaction of various aromatic hydrocarbon compounds and lignin to degrade the aromatic hydrocarbon compounds. In addition, laccase is widely distributed in fungal secretions, higher plants, a small amount of bacteria and insects, wherein the sources of the fungal laccase and the bacterial laccase are relatively abundant. Laccase has the functions of catalyzing degradation of lignin, removing toxicity of aromatic amine toxic compounds, promoting synthesis of fungal pigment and the like, and has the advantages of substrate specificity, mild reaction conditions, environmental friendliness and the like when oxidizing a substrate. The great potential of laccase application value is determined, and the laccase is highly valued and widely focused in the disciplines of biology, medicine, chemistry, environment and the like, and has practical application.

The straw which is a large amount of agricultural byproducts in China can not be fully utilized and can only be burnt, so that great waste is caused. Laccase can act on small fragments of lignin containing phenolic groups, thereby catalyzing degradation of lignin polymers. As an important enzyme for cellulose bioconversion, the enzyme can be combined with other relevant cellulase preparations to realize large-scale fodder and energy utilization of straws. As a cellulase additive for producing agricultural and sideline products, such as scraps, straws and chaff, the production performance of livestock and poultry can be improved, the feed utilization rate can be improved, the nutritive value of the feed can be improved, the feed cost can be reduced, and the economic benefit can be improved. Therefore, the laccase is further enhanced to serve as research and development work of the enzyme for cellulose bioconversion, the development of enzyme products for cellulose bioconversion in the feed and energy industries is supported, and great social and economic benefits are achieved.

In addition, laccase is used as a green catalyst with extremely wide application value, and can oxidize a large number of toxic (such as chlorophenol compounds and aniline substitutes in chemical preparations such as pesticides, preservatives, insecticides, herbicides, bactericides and the like) and nontoxic compounds, so that the laccase plays an important role in biological decontamination. The laccase can also treat organic chloride in the papermaking waste liquid, and has the advantages of high catalytic efficiency, mild reaction conditions, no harsh requirements on the reaction conditions and the reaction equipment, and the like. Therefore, laccase has wide application value in purifying industrial wastewater, especially in sewage treatment in paper making and textile industries. Therefore, the laccase is further enhanced to serve as a green catalyst for research and development of biological decontamination, and has great social and application values in the aspect of environmental protection.

The fungus laccase in the wild environment has some problems in use, is small in quantity and low in activity, is relatively stable under normal temperature conditions, is relatively poor in heat resistance, has long fermentation period and complex operation when being produced by fermentation, and can produce various laccase isoenzymes, so that the follow-up extraction and purification are hindered, and the influence factors are more in the fermentation process, so that the heterologous expression of the laccase in engineering bacteria can be studied to realize the practical application of the laccase. Therefore, the invention selects the rhizopus nigricans which produces laccase from the company as an experimental material, one laccase gene is obtained by mutagenesis and screening and cloning, a high-yield heat-resistant laccase mutant is obtained, and the engineering bacteria with higher laccase activity in pichia pastoris is finally obtained by constructing an expression vector of the pichia pastoris, thereby meeting the requirements of cellulose bioconversion industrial production and biological decontamination, reducing the production cost, having great significance and laying the foundation for further research and development work of enzymes for cellulose bioconversion and biological decontamination.

The invention comprises the following steps:

the invention aims to provide a laccase mutant with improved enzyme activity and heat resistance and a pichia pastoris engineering strain thereof. Aims to solve the problems of low enzyme activity, biotransformation of cellulose and non-ideal biological decontamination application of laccase in industrial production.

The invention is realized by the following technical scheme:

a laccase mutant with improved enzyme activity and heat resistance is prepared by screening Rhizopus nigricans (Rhizopus nigricans) RN056 preserved in the laboratory of the applicant through nitrosoguanidine mutagenesis to obtain a mutant strain, cloning laccase mutant encoding genes from the mutant strain, constructing recombinant plasmids, and expressing in Pichia pastoris GS115 to obtain Pichia pastoris engineering strain.

One of the technical schemes provided by the invention is as follows: the laccase mutant is obtained by mutating 122 th aspartic acid into valine, 241 th tyrosine into aspartic acid, 244 th leucine into arginine, 476 th asparagine into serine and 480 th valine into glycine on the basis of RN056 wild laccase shown in SEQ ID No.2, and the amino acid sequence of the laccase mutant is shown in SEQ ID No. 4;

the invention also provides a coding gene of the laccase mutant;

further, the nucleotide sequence of the encoding gene of the laccase mutant is shown as SEQ ID No. 3.

The enzymatic properties of the laccase mutants are as follows:

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

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

(3) Heat resistance: the enzyme has the activity of more than 85% after 24 hours under the conditions of pH4.5 and temperature of 60 ℃.

The third technical scheme provided by the invention is as follows: the recombinant vector or recombinant strain containing the laccase mutant encoding gene is used for reconstructing the recombinant vector from the laccase mutant encoding gene and efficiently expressing the laccase mutant encoding gene in pichia pastoris to obtain the recombinant strain for producing the laccase with high activity, and the laccase with high activity is obtained through fermentation, extraction and other technologies;

further, the host cell for expressing the laccase mutant is pichia pastoris GS115;

further, the expression vector for expressing the laccase mutant is pPIC9K plasmid;

preferably, the recombinant strain is obtained by connecting a laccase mutant coding gene shown in SEQ ID No.3 with an expression vector pPIC9K and expressing in Pichia pastoris GS115.

The invention provides a technical scheme that: the recombinant vector or the recombinant strain is applied to the technical scheme III, in particular to the application in the fermentation production of laccase mutants shown in SEQ ID No.4.

The technical scheme provided by the invention is as follows: the laccase mutant shown in SEQ ID No.4 is applied to oxidation-reduction of a substrate and further hydrolysis of the substrate;

further, it is an application in the cellulose bioconversion industry, such as removal of lignin from corn straw lignocellulosic hydrolysate by acting on small fragments of lignin containing phenolic groups;

further, the method is applied to biological decontamination or sewage treatment, such as treatment of organic chloride in papermaking waste liquid by oxidizing a large amount of toxic compounds (such as chlorophenol compounds and aniline substitutes in chemical preparations such as pesticides, preservatives, pesticides, herbicides, bactericides and the like) and nontoxic compounds.

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, three-letter or single-letter codes are used. 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 mutant. If Asp122Val indicates that the amino acid at position 122 is replaced by Val from the wild-type Asp, the numbering of the position corresponds to the amino acid sequence numbering of the wild-type laccase in SEQ ID No. 2. In the present invention, RNT-1 represents a wild-type laccase and RNT-2 represents a laccase mutant, with the information given in the following Table.

The beneficial effects are that:

the invention discloses a novel laccase mutant, which has the characteristics of high enzyme activity and high heat resistance, and the enzyme activity of the mutant fermentation liquor can reach more than 55U/mL.

The pH of the optimum reaction of the laccase obtained by the invention is at 4.5, the enzyme activity is highest, and compared with the laccase of wild RN056, the pH of the optimum reaction is reduced by 0.5, which indicates that the acid resistance is improved.

The laccase obtained by the invention has the enzyme activity remaining more than 85% after 24 hours under the conditions of pH4.5 and 60 ℃, and has obviously improved heat resistance compared with the laccase of wild RN056.

Description of the drawings:

FIG. 1 is an optimum pH curve;

FIG. 2 is an optimum temperature profile;

FIG. 3 is a heat resistance curve.

The specific embodiment is as follows:

the invention will now be described in more detail by way of specific examples, which are given by way of illustration only and are not intended to limit the scope of the invention. Modifications which would occur to those skilled in the art based on the principles of this invention are also considered to be within the scope of this invention. The experimental method of molecular biology, which is not specifically described in this example, can be referred to the "guidelines for molecular cloning experiments".

The invention provides a laccase mutant with improved enzyme activity and heat resistance, which is selected from a mutant strain obtained by nitrosoguanidine mutagenesis of a rhizopus nigricans strain (Rhizopus nigricans) RN056 preserved in the laboratory of the applicant, wherein mutation sites obtained by gene sequencing are mutation of 122 th aspartic acid into valine, 241 th tyrosine into aspartic acid, 244 th leucine into arginine, 476 th asparagine into serine and 480 th valine. The invention firstly obtains the encoding gene of laccase mutant, connects the encoding gene of the mutant with pPIC9K carrier to construct recombinant plasmid, transfers the recombinant plasmid into corresponding host bacteria GS115 for heterologous expression, and ferments to obtain laccase corresponding to the mutant. The laccase has higher enzyme activity, and is suitable for industrial production, cellulose bioconversion industry and biological decontamination.

The invention relates to a part of materials and a method as follows:

1. experimental materials and reagents:

experimental strains and vectors: the wild strain is rhizopus nigricans strain RN056 preserved in the laboratory of the applicant; expression host bacteria and vectors: GS115 and pPIC9K were purchased from Novagen; host bacteria: DH5 alpha.

The main reagent comprises: DNA polymerase, T4 DNA ligase, DNA gel recovery kit, plasmid extraction kit, RNA extraction kit, pNPG, DNAmarker, agarose, ampicillin, IPTG, X-gal: all purchased from Shanghai; various restriction enzymes: purchased from NEB corporation.

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

2. The laccase activity determination method adopted by the invention comprises the following steps:

principle of measurement of laccase enzyme activity: the product of laccase decomposition of ABTS is ABTS radical, however the absorbance coefficient of ABTS radical is much greater than substrate ABTS at 420 nm. As ABTS radical concentration increases, absorbance values become greater and the time elapsed when absorbance values vary within a certain specified range is expressed as laccase activity.

Definition of enzyme activity unit: under certain conditions (which are not specified, but refer to a temperature of 30 ℃ C. And a pH of 4.5), one laccase unit (U) is converted into 1. Mu. Mol ABTS in 1min, so that the concentration of the product ABTS free radicals is increased from 27.66. Mu. Mol/L to 36.88. Mu. Mol/L (the absorbance is increased from 0.15 to 0.20).

Enzyme activity measurement method (ABTS method): the substrate solution was preheated at a suitable temperature in advance, 2.9mL of the substrate solution (ABTS concentration: 0.5 mmol/L) was measured, 0.1mL of the diluted enzyme solution was added, the time for increasing the absorbance value from 0.15 to 0.20 was recorded, and then the enzyme activity was calculated according to the enzyme activity definition.

The method for measuring laccase activity under 420nm by using ABTS as a substrate comprises the following steps: the pH value is selected to be 4.5, the temperature is 30 ℃, and the laccase is properly diluted, so that the activity of laccase diluent is 27.7-55.3U/mL; 2.9mL of substrate solution (ABTS concentration is 0.5 mmol/L) is taken during measurement, 0.1mL of diluted enzyme solution is added, the time t for the laccase to catalyze and decompose 0.5mmol/L ABTS to increase the absorbance value from 0.15 to 0.20 is measured, and the dilution multiple is changed to ensure that the t value is in the range of 10-20 s. Laccase activity was calculated according to the formula enzyme activity = 1000 x 0.1844 x (0.20-0.15) x 60 x dilution/t.

In the invention, the amino acid sequence of the wild laccase is shown as SEQ ID No. 2: MASFLSAFAMALLLSTILLKFTIIGQRGTLFWHAHVSWLREPGKTYLLRLINAALNDELFQYPVTLHWHGIKQFKTNYADGPAHITQCPIQPNKSYIYEFKNLRSRTTLPCIFLIADDLITDFVVISPGQTMDILVQANQTEGLYYIGLNPYIAVNETLYPGDLYNCSHKETTVIPVKAGETNLLRFINAALNNELFVAIANHKMTVVATDASYTKPFTTENQEGTLWWHAHSSWLRATVYGALIIHPKLGSPYSFPMPRFPPVPPVKFDYTGNVSRGLWQPVKATKLYKLKFGAKVQIVKATESGAPPEESTAYTINGQPGFPNNCSKETTYRLQVQYGKTYLLRLVNAVMNEEMFFGIANHKLTVVAQDGAYIKPITTVGTGYGNYDETTSPKTYNLKDPPEVNTVGLPRNGWVAIRFVLPPLNDTSFATNFSNKLRSLASAQFPANVPQKVDKRFFFPKRETPILLGEWWDANPIDVTIGLGTNPCDQNNQTCQGPNFPTRPVPFNYTGTPPNNTMVRNGTKLLVIPFNTSVELIMQDTSMLGAESHPLHLHGFNFFSVIRFVANNPGVWLMHCHLDVHITWGLAMSFLVENGVSELDSLEEPPLDLPVC.

In the invention, the amino acid sequence of the mutant laccase is shown as SEQ ID No. 4: MASFLSAFAMALLLSTILLKFTIIGQRGTLFWHAHVSWLREPGKTYLLRLINAALNDELFQYPVTLHWHGIKQFKTNYADGPAHITQCPIQPNKSYIYEFKNLRSRTTLPCIFLIADDLITVFVVISPGQTMDILVQANQTEGLYYIGLNPYIAVNETLYPGDLYNCSHKETTVIPVKAGETNLLRFINAALNNELFVAIANHKMTVVATDASYTKPFTTENQEGTLWWHAHSSWLRATVDGARIIHPKLGSPYSFPMPRFPPVPPVKFDYTGNVSRGLWQPVKATKLYKLKFGAKVQIVKATESGAPPEESTAYTINGQPGFPNNCSKETTYRLQVQYGKTYLLRLVNAVMNEEMFFGIANHKLTVVAQDGAYIKPITTVGTGYGNYDETTSPKTYNLKDPPEVNTVGLPRNGWVAIRFVLPPLNDTSFATNFSNKLRSLASAQFPANVPQKVDKRFFFPKRETPILLGEWWDASPIDGTIGLGTNPCDQNNQTCQGPNFPTRPVPFNYTGTPPNNTMVRNGTKLLVIPFNTSVELIMQDTSMLGAESHPLHLHGFNFFSVIRFVANNPGVWLMHCHLDVHITWGLAMSFLVENGVSELDSLEEPPLDLPVC.

The invention is further illustrated by the following detailed description.

EXAMPLE 1 acquisition of laccase mutant RNT-2 coding Gene

1. Starting strain: a laccase-producing Rhizopus niveus strain (Rhizopus nigricans) RN056 deposited in the applicant's laboratory.

2. Culture medium:

(1) Seed culture medium: glucose 2%, peptone 0.5%, potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.1%, pH 5.0, sterilizing at 121deg.C for 20min;

(2) YPD solid medium: 1% of yeast extract, 2% of peptone, 2% of glucose, 2% of agar powder, pH 5.0 and sterilizing at 121 ℃ for 20min;

(3) Primary screening of the culture medium: adding 1% guaiacol into YPD solid culture medium, and sterilizing at 115 deg.C for 20min;

(4) Shake flask rescreening medium: 2% of glucose, 2% of peptone, 1% of corn steep liquor, 1% of ammonium sulfate, pH 5.0 and sterilization at 121 ℃ for 20min.

3. Nitrosoguanidine mutagenesis:

(1) Taking 10mL of 0.1% Tween-60 to wash spores on fresh inclined planes, transferring the spores into a triangular flask containing glass beads, vibrating and scattering the spores on a shaking table for 50min, filtering the spores with 4 layers of mirror cleaning paper, performing gradient dilution, counting the spores on a blood cell counting plate and adjusting the concentration of the spores to be 6 multiplied by 10 ⁶ About one/mL.

(2) Nitrosoguanidine (NTG) solution formulation: 25mg of nitrosoguanidine was weighed into a brown bottle in a fume hood, dissolved in 2.5mL of acetone, and 10mL of water was added after dissolution to prepare a 2.5mg/mL nitrosoguanidine solution.

(3) The operation method comprises the following steps: the spore suspension was rinsed 1 time with PBS (100 mmol/L, pH 7.0.0), 0.5mL of PBS was added to prepare a bacterial suspension, and then 0.4mg/mL of NTG solution was added thereto, and the solution was treated at 30℃for 50 minutes, with a mortality rate of 79.5% in NTG concentration. The NTG-treated cells were centrifuged, washed 3 times with PBS to remove NTG residues, then the cells were suspended in 100mL of enrichment medium and cultured at 30℃for 12 hours, finally the bacterial suspension was shaken for 2 minutes, diluted with sterile water to a suitable dilution, spread on a plate of a preliminary screening medium, and cultured at 30℃for 48 hours. After the culture, randomly picking the strain with good growth vigor from the flat plate, and performing primary screening.

4. Mutant screening

(1) And (3) primary screening: screening according to the size of reddish brown oxidation rings on a primary screening culture medium plate, selecting 5 strains with the maximum reddish brown oxidation rings, inoculating the strains to a YPD solid culture medium plate, and continuously culturing until pure cultures are obtained, wherein the 5 strains are named R-01, R-02, R-03, R-04 and R-05 in sequence.

(2) And (3) re-screening: and then carrying out fermentation shaking re-screening on the 5 strains, and screening to obtain 1 strain R-02 with higher laccase yield through shaking fermentation test, wherein the re-screening result is shown in the following table:

the R-02 obtained by mutagenesis screening is used as an original strain, a PCR primer (Forward/reverse) is designed according to the nucleotide sequence of a wild laccase RNT-1 coding gene, an enzyme cutting site Xho I is added at the 5 'end, an enzyme cutting site Not I is added at the 3' end, the coding gene of mutant RNT-2 is obtained through PCR, and the nucleotide sequence of the coding gene is SEQ ID No.3 and the corresponding amino acid sequence is SEQ ID No.4.

Primer sequence:

Forward：5’-GCCCTCGAGTCAGCAAACAGGCAAATCAAGT-3' (underlined as restriction enzyme Xho I recognition site);

Rerverse：5’-CATGCGGCCGCATGGCTTCTTTCCTTAGTGCTTTTG-3' (underlined as restriction endonuclease Not I recognition site).

In the present invention, mutation site information is obtained by comparing nucleotide sequences of the wild type and the mutant as follows:

EXAMPLE 2 construction of recombinant vector pPIC9K-RNT-2

The mutant RNT-2 coding gene (SEQ ID No. 3) and plasmid pPIC9K are respectively subjected to XhoI and NotI digestion, products are recovered, the recovered RNT-2 coding gene and pPIC9K are mixed in proportion, the mixture is connected with T4 ligase at 16 ℃ overnight, the connection products are used for transforming E.coli DH5 alpha competent cells, the transformation products are coated on LB (ampicillin-containing) solid plates, the inversion culture is carried out at 37 ℃ overnight, single colony is picked up to LB liquid culture medium, the colony PCR is carried out at 37 ℃ to obtain target sequences, the sequencing comparison shows that the nucleotide sequences shown as SEQ ID No.3 are obtained, the obtained recombinant vector contains correct mutant genes, and the obtained recombinant vector is named pPIC9K-RNT-2.

EXAMPLE 3 transformation of Pichia pastoris with recombinant plasmid

1. Preparation of pichia pastoris GS115 competent cells

(1) Picking single colony of Pichia pastoris flat plate, inoculating to 5mL YPD culture medium, and oscillating at 30deg.C and 220r/min overnight;

(2) Inoculating 0.5mL of the overnight cultured bacterial liquid into 50mL of freshly prepared YPD medium, and shaking culturing at 30 ℃ for 220r/min to make OD ₆₀₀ The value reaches 1.3-1.5;

(3) Taking the culture solution, and centrifuging at 4 ℃ and 3000r/min for 5min;

(4) Removing the supernatant, adding 50mL of sterile water precooled on ice, and shaking to resuspend thalli;

(5) Centrifuging at 4deg.C for 5min at 3000r/min, removing supernatant, sucking residual liquid on the wall of the dry tube, adding 25mL of sterile water precooled on ice, and shaking to resuspension thallus;

(6) Centrifuging at 4deg.C for 5min at 3000r/min, removing supernatant, sucking residual liquid on the wall of the dry tube, adding 10mL of pre-cooled 1mol/L sterile sorbitol solution on ice, and re-suspending thallus;

(7) Centrifuging at 4deg.C for 5min at 3000r/min, removing supernatant, sucking residual liquid on the wall of the tube, adding 1mL of pre-cooled 1mol/L sterile sorbitol solution (glycerol is added in advance to a final concentration of 15%), and shaking and mixing;

(8) 100. Mu.L/tube was dispensed into sterile EP jars and stored frozen in a refrigerator at-70℃with better results for freshly prepared competent cells.

2. Transformation of linearized plasmids

The positive clone obtained in example 2 was extracted to obtain a recombinant plasmid pPIC9K-RNT-2, and the recombinant plasmid was subjected to single cleavage with Sal I to obtain a linearized plasmid. Freshly prepared (or frozen at-70 ℃) GS115 competent cells were placed in an ice bath and allowed to thaw completely.

(1) 100. Mu.L of competent cells were removed to a new sterile EP tube, 10. Mu.L of linearized plasmid was added, gently mixed, aspirated and transferred to a 0.2cm electroporation cuvette;

(2) Placing the conversion cup in ice bath for 5-10min, and maintaining low temperature;

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

(4) After electric shock, 1mL of 1mol/L sorbitol solution precooled at 4 ℃ is added into an electric shock conversion cup immediately, and the mixture is blown uniformly by a pipetting gun and placed into an ice bath;

(5) MD medium (1.34% YNB; 4X 10) was applied aseptically on an ultra clean bench ^-5 % biotin; 2% glucose plate), 150. Mu.L/plate, and the coated plate was incubated upside down at 30℃for 3-4 days;

(6) Screening two recombinant strains on an MD (machine direction) plate, obtaining a target sequence through colony PCR (polymerase chain reaction), and sequencing and comparing the target sequence with the nucleotide sequence shown as SEQ ID NO.3, wherein the obtained recombinant strains contain correct mutant genes, and the two recombinant strains are respectively named as recombinant strain-01 and recombinant strain-02.

EXAMPLE 4 Induction of expression by yeasts containing recombinant plasmid pPIC9K-RNT-2

BMGY medium formula: 1.5% Yeast extract, 2.5% peptone, 0.1mol/L phosphate buffer pH 6.0, 1.34% YNB, 4X 10) ^-5 % biotin, 1% glycerol and the balance water.

BMMY medium formula: 1.5% Yeast extract, 2.5% peptone, 0.1mol/L phosphate buffer pH 6.0, 1.34% YNB, 4X 10) ^-5 % biotin, 0.6% methanol, the balance water.

Recombinant strain RD-01 constructed by recombinant strain-01, recombinant strain-02 and wild laccase coding gene (nucleotide sequence shown as SEQ ID NO. 1) by the same method as in example 3 is respectively inoculated into triangular flask filled with 30mL BMGY culture medium, and cultured at 30deg.C and 200r/min to OD ₆₀₀ About 10, cells were collected by centrifugation, resuspended in 35mL of BMMY-induced medium, and cultured at 30℃for 50 hours under 200r/min, and the laccase enzyme activity in the supernatant was measured after centrifugation of the fermentation broth, and the results are shown in the following Table.

Strain	Laccase enzyme activity (U/mL)
		RD-01	24
Recombinant bacterium-01	35
		Recombinant bacterium-02	37

EXAMPLE 5 fermentation Performance verification of recombinant bacterium-02

The recombinant bacterium-02 obtained in example 3 was used for producing laccase by fermentation culture of the production strain.

The formula of the seed tank culture medium comprises: 3.9% of glycerol, 1.5% of monoammonium phosphate, 0.8% of monopotassium phosphate, 0.6% of magnesium sulfate, 0.7% of potassium sulfate, 0.05% of calcium sulfate, 0.5% of potassium hydroxide, and the balance of water, wherein the pH value is 5.0;

the formula of the fermentation tank culture medium comprises: 3.9% of glycerol, 1.5% of monoammonium phosphate, 0.8% of monopotassium phosphate, 0.6% of magnesium sulfate, 0.7% of potassium sulfate, 0.05% of calcium sulfate, 0.5% of potassium hydroxide, and the balance of water, wherein the pH value is 5.0;

carbon source: 40% glycerol;

methanol: pure methanol;

seed pot culture: the culture temperature is 30 ℃, the initial rotating speed is 200r/min, and the initial air quantity is 2.5m ³ And (3) carrying out ventilation stirring culture, wherein the pH value is 5.0, the dissolved oxygen is kept at 20-30%, when the dissolved oxygen is lower than 20%, the rotation speed and the air quantity are increased to control, and when the wet weight is increased to 75g/L, the seeds are transplanted;

culturing in a fermentation tank: the culture temperature is 30 ℃, the initial rotating speed is 200r/min, and the initial air quantity is 2.5m ³ And (3) carrying out ventilation stirring culture, wherein the inoculum size is 10%, the pH is 5.0, the period is 0-20h, 40% of glycerol as a carbon source is fed at the flow rate of 800g/h, the dissolved oxygen is kept at 20-30%, when the dissolved oxygen is lower than 20%, the rotation speed and the air quantity are increased to control, and the thallus is cultured until the wet weight is 200g/L; stopping supplementing the carbon source after the 20 th period, and keeping the dissolved oxygen rebounded to more than 80% for 1.0h; and then methanol is fed at the speed of 200g/h, dissolved oxygen is kept at 20-30%, and the fermentation is finished until the total fermentation period is 180 h.

The 50L fermentation tank amplification verification experiment is carried out by adopting the fermentation method, the fermentation period is 180 hours, the fermentation and enzyme production conditions of 3 batches are shown in the following table, the average enzyme production level is 56.3U/mL, and the strain recombinant bacterium-02 not only has high laccase production, but also has certain stability in fermentation performance and enzyme activity of the laccase produced by the strain recombinant bacterium-02.

3 fermentation enzyme production conditions of batch of genetically engineered bacteria

Batch of	Fermentation period (h)	Fermentation activity (U/mL)
			1	180	56
2	180	55
			3	180	58

EXAMPLE 6 enzymatic Properties of laccase

(1) pH optimum action

The recombinant bacterium-02 fermentation broth supernatant obtained in example 4 is taken as a laccase sample after mutation, the RD-01 fermentation broth supernatant is taken as a wild type control sample, and the laccase activity determination method provided by the invention is adopted to determine the relative enzyme activities under different conditions of pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 at 30 ℃, and the highest enzyme activities of laccase measured by the two enzymes are taken as 100% of the reference. As can be seen from FIG. 1, the laccase mutants of the invention have stable enzymatic activity in the pH range of 4.0-5.5, and an optimal pH of 4.5, which is reduced by 0.5 compared with the control. The results show that compared with the laccase before mutation, the laccase after mutation has higher enzyme activity under higher acid condition, has wider pH range of action, and is more suitable for the cellulose bioconversion industry and biological decontamination.

(2) Optimum operating temperature

The laccase activity determination method of the invention is adopted by taking the supernatant of the recombinant bacterium-02 fermentation broth obtained in the example 4 as a mutated laccase sample and the supernatant of the RD-01 fermentation broth as a wild type control sample, and under the condition of pH4.5, the enzyme activities at different temperatures of 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ are determined, the relative enzyme activities are calculated by taking the highest enzyme activities of the laccase measured by the two enzymes as 100% as the reference, and the result is shown in figure 2, the laccase mutant of the invention has stable enzyme activity at 40-55 ℃, the optimal action temperature is 45 ℃, and the optimal reaction temperature is unchanged compared with the wild type laccase, but the optimal temperature range is improved.

(3) Heat resistance

The recombinant bacterium-02 fermentation broth supernatant obtained in the example 4 is taken as a laccase sample after mutation, RD-01 fermentation broth supernatant is taken as a wild type control sample, and the two samples are respectively subjected to heat preservation treatment at 60 ℃ under the condition of pH4.5 on the basis of 100% of laccase enzyme activity which is not treated, and the enzyme activity is measured at the temperature of 30 ℃ and the buffer pH4.5 every 2 hours, so that the residual enzyme activity is calculated, and as can be seen from FIG. 3, after 24 hours, the laccase mutant of the invention has the relative activity which is still more than 85%, and compared with the wild type laccase, the heat resistance of the laccase mutant is greatly improved, so that the laccase mutant has good heat resistance. The above results indicate that the improved heat resistance makes the mutated laccase more suitable for application in the cellulose bioconversion industry and bioremediation than before mutation.

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

1. The laccase mutant is characterized by having an amino acid sequence shown in SEQ ID NO.4.

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

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

4. A recombinant vector or recombinant strain comprising the coding gene of claim 2.

5. The recombinant vector of claim 4, wherein the expression vector is a pPIC9K plasmid.

6. The recombinant strain of claim 4, wherein the host cell is pichia pastoris GS115.

7. The recombinant strain of claim 4, wherein the recombinant strain is obtained by ligating a laccase mutant encoding gene shown in SEQ ID No.3 with an expression vector pPIC9K and expressing the gene in Pichia pastoris GS115.

8. Use of the recombinant vector or recombinant strain of claim 4 for the production of the laccase mutant of claim 1.

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

10. The use of a laccase mutant according to claim 9, characterized in that the laccase mutant is used in the hydrolysis of redox substrates or in the bioconversion of cellulose, bioremediation.