CN106434579A - Laccase from Klebsiella pneumoniae, as well as recombinant strain and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering of enzymes, and relates to a novel laccase derived from Klebsiella pneumoniae and a preparation method thereof. A strain of Klebsiella pneumoniae capable of producing laccase was amplified by PCR technology to amplify the gene of the new laccase, and then the new laccase gene was carried out in the Bacillus subtilis expression system and the Pichia pastoris expression system respectively. The recombinant strains of high-stable laccase of Bacillus subtilis and the recombinant strains of high-stable laccase free expression of Pichia pastoris were respectively obtained, so as to realize the preparation of new laccases. At the same time, the new bacterial laccase has good decolorization effect on azo and anthraquinone dyes.

Description

Laccase derived from Klebsiella pneumoniae and its recombinant strain and preparation

Technical field:

The present invention relates to a novel laccase derived from Klebsiella pneumoniae and its gene, engineering bacteria and preparation method, in particular to obtaining a recombinant expression strain expressing the novel laccase by means of genetic engineering technology and molecular biology, and The application of the bacterial laccase protein in the dye decolorization industry belongs to the technical field of enzyme genetic engineering.

Background technique:

Laccase (Laccase, E.C.1.10.3.2), also known as urushiol oxidase and multi-copper oxidase, is a copper-containing polyphenol oxidase, which is compatible with ascorbate oxidase in plants and ceruloplasmin in mammals. , Cytochrome C oxidase, and bilirubin oxidase are homologous and belong to the blue multi-copper oxidase family. It can catalyze the oxidation of various phenolic and non-phenolic compounds to generate corresponding quinones, and at the same time reduce molecular oxygen to water with the transfer of electrons, and no other by-products are formed during the reaction.

Laccase is recognized as an environmentally friendly "green enzyme" and is widely distributed in nature. Laccases can be divided into four categories according to their sources: plant laccases, animal laccases, fungal laccases and bacterial laccases. Laccase was first discovered in the juice components of Japanese sumac. In subsequent studies, researchers found laccase from various plants, but plant laccase contains a large number of other oxidases during the extraction process, making the process of purifying laccase more complicated. It was later confirmed that laccase exists in animals, fungi and bacteria in addition to plants.

Fungal laccases mainly exist in higher fungi (especially basidiomycetes). Currently, industrial commercial laccases are mainly derived from fungi. However, fungal laccases are easily affected by hydroxyl ions, and their activity is very low or almost non-existent under alkaline conditions. , thermal stability is also poor, and the growth cycle of filamentous fungi is long, and the medium requirements are high. High-yielding strains express themselves or use eukaryotic expression systems for recombinant expression, which has disadvantages such as long production cycle, high cost, and low yield, which seriously affects the industrial application of fungal laccase.

Bacterial laccases include CotA protein of Bacillus, PpoA protein of Halomonas, CueO protein of Escherichia coli, etc. Compared with fungal laccase, bacterial laccase can overcome the above-mentioned shortcomings of fungal laccase, and under alkaline conditions It has good catalytic activity and high stability. Bacterial laccase has some unique advantages, such as: no need for glycosylation modification, wide optimum pH range of enzyme, good temperature stability, Cu ²⁺ resistance, etc. These properties are exactly what is needed for the current industrial application of laccases. Therefore, the study of bacterial laccase is of great significance to the expansion of the application field of laccase. At present, there are few studies on bacterial laccases, and more bacterial laccase genes with high activity and high stability need to be further explored and studied.

Since laccases come from different sources, and laccases from different sources have certain differences in structure and properties, laccases from different sources will show certain differences in their catalytic properties. Laccase has a wide range of substrates and can catalyze the oxidation and polymerization of more than 250 organic substances including polyphenols, diamines, aromatic amines, and carboxylic acids. Because of its special catalytic performance and wide range of substrates, laccase has a wide range of applications, including pollutant degradation, dye decolorization and detoxification, pulp and textile bleaching, oral health tooth whitening agent, bleaching laundry detergent, food flavor Improvement, feed nutrition improvement, bioelectronic research and development, etc. For many applications, the oxidative capacity of laccases can be enhanced through the use of mediators. Currently known mediators include: HBT (1-hydroxybenzotriazole), ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, syringic acid Methyl ester, NHA (N-hydroxyacetanilide), NEIAA (N-acetyl-N-phenylhydroxylamine), HBTO (3-hydroxyl,2,3-benzotriazin-4(3H)one), VIO (violet uric acid).

Bacillus subtilis is a Gram-positive bacteria. The Bacillus subtilis expression system has the following advantages: 1. It can efficiently secrete various proteins; 2. Many Bacillus subtilis have been used in the fermentation industry for a long time, and they are non-pathogenic and do not produce any endotoxins; 3. 1. The genetic background of Bacillus is very clear, and it grows rapidly, and has no special requirements for nutrients; 4. The codon preference is not obvious; 5. The fermentation process is simple, Bacillus subtilis is an aerobic bacteria, no need Anaerobic fermentation equipment, after the fermentation, simply separate the fermentation broth and bacterial cells, and then enter the stage of separation, purification and recovery of the target protein; 6. It has stress resistance and can produce a variety of heat-resistant enzyme preparations.

Pichia pastoris is a single-celled lower eukaryote with ordinary culture conditions and rapid growth and reproduction. When the Pichia pastoris system is used to express genetic engineering products, it can be produced on a large scale, effectively reducing production costs. The Pichia pastoris expression system has many advantages when expressing foreign target proteins, has broad prospects, and is suitable for industrialization and large-scale production. The carrier is expressed by the promoter of the alcohol oxidase gene, and the expression level of the exogenous target protein is very high, and the expression level of P. ) is 10 times or even 100 times higher. 2. High stability: the expression vector of the Pichia pastoris expression system exists through integration on the yeast chromosome, and replicates with the replication of the chromosome, not in the form of plasmid self-replication, so the stability of the recombinant strain is very high. 3. High secretion expression: The leader sequence of factor a in the expression vector of the Pichia pastoris expression system is a secretion signal sequence with good secretion effect, which can secrete the expression product out of the cell through a certain way, and at the same time relieve the host cell The metabolic load is conducive to the continuous growth of cells. 4. Post-translational processing and modification of target protein: Pichia pastoris has a complete subcellular structure of eukaryotic organisms, and can carry out post-translational processing and modification of eukaryotic proteins (such as glycosylation, lipid phthalation, phosphorylation, protein Cleavage, folding and formation of disulfide bonds, etc.), so that the structure of the secreted protein is closer to the natural protein, and the degree of glycosylation is appropriate, which is suitable for clinical use. 5. The genetic background research is relatively clear, and the high-level expression of the target protein can be achieved through the regulation mechanism of gene expression. 6. It is easy to carry out industrial production. Pichia pastoris is a single-celled microorganism, which has the advantages of low production cost, simple nutritional requirements (the carbon source of yeast is generally glycerol, glucose and methanol, etc.), simple fermentation process, and high-density fermentation. Yeast has the potential to produce exogenous proteins by large-scale fermentation, and the yeast expression system self-secretes very few miscellaneous proteins, which is beneficial to the purification of target proteins. The Pichia pastoris expression system has become the most important tool and model for modern molecular biology research, and it is an ideal tool for expressing foreign genes.

In the present invention, the novel laccase encoding gene is derived from Klebsiella pneumoniae, which belongs to bacteria, and the novel laccase encoding gene in the Klebsiella pneumoniae genome is cloned, and the novel laccase gene is expressed in the Bacillus subtilis expression system and Pichia pastoris expression system to obtain recombinant strains with high stability of Bacillus subtilis laccase and recombinant strains with free expression of high stability laccase of Pichia pastoris respectively. After the recombinant strains are fermented and treated accordingly, high stability laccase catalyst, and the new recombinant laccase can decolorize anthraquinone and azo dyes.

Invention content:

The purpose of the present invention is to overcome and avoid the deficiencies in the current industrial production of laccase, and provide a novel bacterial laccase coding gene derived from Klebsiella pneumoniae and an engineering process for expressing the novel bacterial laccase gene strain.

The technical route for realizing the purpose of the present invention is as follows: using the genome of Klebsiella pneumoniae as a template, and according to the reported Klebsiella pneumoniae laccase mature peptide gene, analyze its conserved sequence, and design the laccase mature peptide gene of the present invention Peptide gene amplification primers P1 and P2, P3 and P4, wherein the upstream primer P1 and downstream primer P2 are used to amplify the target gene expressed in Bacillus subtilis, and the upstream and downstream primers introduce restriction enzyme sites respectively BamH I, Hind III; upstream primer P3 and downstream primer P4 are used to amplify the target gene expressed in Pichia pastoris GS115, and the upstream and downstream primers introduce restriction enzyme sites EcoR I and Not I respectively. The laccase gene Lac of Klebsiella pneumoniae was obtained by PCR cloning, and the recombinant plasmids pBSA43-Lac and pPIC9K-Lac were constructed after connecting it with the pBSA43 vector and the pPIC9K vector respectively; the recombinant strain JM109/pBSA43 was obtained by transforming Escherichia coli JM109 -Lac and JM109/pPIC9K-Lac. Once again, the correct recombinant plasmids pBSA43-Lac and pPIC9K-Lac were successfully expressed in Bacillus subtilis WB600 and Pichia pastoris GS115, respectively, to obtain recombinant strains producing a new type of laccase with high stability, and further optimize the fermentation process to obtain high-yield A novel laccase with high stability.

In order to achieve the above object, one of the technical solutions provided by the present invention is: a novel bacterial laccase derived from a strain of Klebsiella pneumoniae screened by the inventor, whose amino acid sequence is as shown in the sequence listing Shown in SEQ ID No: 2;

When the laccase uses ABTS as a substrate to determine the enzymatic properties of the new laccase, the optimum action temperature is 35°C, and the optimum action pH is 4; when DMP is used as the substrate to determine the enzymatic properties of the new laccase, the optimum action temperature is The temperature is 70°C, and the optimum pH is 8;

At the same time, the pH stability and thermal stability of the new laccase were measured with DMP as the substrate. The results showed that the new bacterial laccase had good stability in the range of pH 5-9, and the specific performance was that it was incubated at pH 5, pH 6 and pH 9. After 35 hours, the residual enzyme activity is above 60%. After 35 hours of incubation at pH7 and pH8, the residual enzyme activity is above 85%. It has good thermal stability at 30-60°C. The specific performance is that the remaining enzyme activity is More than 95%, when incubated at 50-60°C for 4 hours, the residual enzyme activity is more than 50%. Compared with the reported laccase derived from Klebsiella pneumoniae, the pH stability of the novel laccase to be protected by this patent and better temperature stability;

The coding gene of the laccase is Lac, and the base sequence is shown in SEQ ID No: 1 in the sequence listing;

In order to achieve the above purpose, the second technical solution provided by the present invention is: reconstruct the recombinant vector of the above gene, and express it efficiently in Bacillus subtilis WB600 and Pichia pastoris GS115 to obtain a recombinant strain producing a new type of laccase with high stability , and further obtain a high-yield and high-stability novel laccase through optimization of the fermentation process;

The host cell used to express the novel bacterial laccase is Bacillus subtilis WB600, and the expression vector is pBSA43;

The host cell used to express the novel bacterial laccase is Pichia pastoris GS115, and the expression vector is pPIC9K;

Experimental procedure of the present invention is summarized as follows:

1. A novel laccase gene derived from Klebsiella pneumoniae, constructing a recombinant bacterial strain expressing the novel laccase (Bacillus subtilis WB600/pBSA43-Lac) and the preparation process of the novel bacterial laccase include the following steps :

(1) Obtain a bacterial strain capable of producing laccase through high-throughput strain screening, that is, Klebsiella pneumoniae;

(2) Using the genome of Klebsiella pneumoniae as a template, according to the reported Klebsiella pneumoniae laccase mature peptide gene, analyze its conserved sequence, and design the amplification primer P1 and the laccase mature peptide gene of the present invention P2, the laccase gene Lac of Klebsiella pneumoniae was obtained by PCR cloning, and after it was connected with the Escherichia coli-Bacillus subtilis shuttle plasmid pBSA43, the recombinant plasmid pBSA43-Lac was constructed and transformed into E. pBSA43-Lac; obtain Klebsiella pneumoniae novel laccase coding gene;

(3) Transform the recombinant plasmid pBSA43-Lac into Bacillus subtilis WB600, and construct the recombinant strain WB600/pBSA43-Lac;

(4) Fermenting the recombinant bacterial strain to prepare a novel bacterial laccase with high stability;

(5) Preparation of a novel laccase with high stability.

2. A novel laccase gene derived from Klebsiella pneumoniae, constructing a recombinant strain (Pichia GS115/pPIC9K-Lac) free to express the novel laccase and the preparation process of the novel bacterial laccase include the following step:

(1) A strain capable of producing laccase, Klebsiella pneumoniae, was obtained through high-throughput strain screening.

(2) Using the genome of Klebsiella pneumoniae as a template, according to the reported Klebsiella pneumoniae laccase mature peptide gene, analyze its conserved sequence, and design the amplification primers P3 and P3 of the laccase mature peptide gene of the present invention P4, the laccase gene Lac of Klebsiella pneumoniae was obtained by PCR cloning, and after it was connected with the Escherichia coli-Pichia pastoris shuttle plasmid pPIC9K, the recombinant plasmid pPIC9K-Lac was constructed and transformed into E. pPIC9K-Lac; get Klebsiella pneumoniae novel laccase coding gene;

(3) Transform the recombinant plasmid pPIC9K-Lac into Pichia pastoris GS115, and construct the recombinant strain GS115/pPIC9K-Lac;

(4) Screening the obtained recombinant strains with geneticin and measuring the enzyme activity of laccase to obtain a recombinant strain producing highly stable laccase;

(5) Fermenting the high-yield bacterial strain to prepare a highly stable novel bacterial laccase;

(6) Preparation of novel laccase with high stability.

Beneficial effect:

1. The present invention obtains a bacterial strain capable of producing laccase through screening of specific high-throughput bacterial strains capable of producing laccase, that is, Klebsiella pneumoniae, and the laccase gene of the bacterial strain obtained by PCR amplification, It is sequenced as a new base sequence.

2. In the present invention, the novel recombinant laccase prepared by fermentation of the novel laccase recombinant strain expressed by Bacillus subtilis and the free expression of the novel laccase recombinant strain by Pichia pastoris has the following advantages in enzymatic properties: ABTS is used as a substrate to determine the novel laccase For the enzymatic properties of laccase, the optimum action temperature is 35°C, and the optimum action pH is 4; when DMP is used as the substrate to determine the enzymatic properties of new laccase, the optimum action temperature is 70°C, and the optimum action pH is 8 At the same time, the novel bacterial laccase is stable in the pH range of 5-9, and has good thermal stability at 30-60°C.

3. The new bacterial recombinant laccase has good decolorization effect on azo and anthraquinone dyes. Therefore, the recombinant laccase has great application potential in practical applications.

Description of drawings

Fig. 1 is the PCR amplification electrophoresis figure of novel laccase mature peptide gene of the present invention;

Among them: M is DNA Marker, 1 and 2 are laccase mature peptide gene Lac respectively;

Fig. 2 is the verification diagram of restriction enzyme digestion of recombinant plasmid pBSA43-Lac of the present invention;

Among them: M is the DNA Marker, 1 is the BamHI single-digestion map of the recombinant plasmid pBSA43-Lac, and 2 is the BamHI and Hind III double-digestion map of the recombinant plasmid pBSA43-Lac;

Fig. 3 is the verification diagram of restriction enzyme digestion of the recombinant plasmid pPIC9K-Lac of the present invention;

Among them: M is the DNA Marker, 1 is the EcoR I single-digestion map of the recombinant plasmid pPIC9K-Lac, and 2 is the EcoR I and Not I double-digestion map of the recombinant plasmid pPIC9K-Lac.

detailed description

The technical content of the present invention will be further described below in conjunction with examples, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited with the following embodiments.

Example 1: Acquisition of Klebsiella pneumoniae Novel Laccase Mature Peptide Gene

1. The new laccase mature peptide gene is derived from Klebsiella pneumoniae screened in our laboratory, and its genomic DNA is extracted. The extraction steps of Klebsiella pneumoniae genomic DNA are as follows:

(1) Inoculate and streak on LB solid plates from glycerol tubes, and culture at 37°C for 12 hours;

(2) Pick a single colony from the culture plate and inoculate it in 5mL liquid LB medium, and culture it at 220r/min, 37°C for 12h;

(3) Dispense the bacterial liquid into sterilized 1.5mL microcentrifuge tubes, centrifuge at 12000r/min for 1min to collect the bacterial cells, and discard the supernatant;

(4) Resuspend the precipitate in 200 μL of pre-cooled solution I and repeatedly pipette and mix with a pipette tip, then add 50 μL of 50 mg/mL lysozyme, and bathe in water at 37°C for 1 hour;

(5) Add 20 μL of 10% SDS and 10 μL of proteinase K, and bathe in water at 65°C for 2-3 hours;

(6) Add 250 μL of newly prepared solution II, cover the mouth of the tube tightly, and gently turn the 1.5 mL EP tube up and down 6 to 8 times to fully lyse the bacteria in the EP tube;

(7) Add 350 μL of pre-cooled solution III, and immediately and gently turn the 1.5 mL EP tube up and down 6 to 8 times, at this time, white flocculent precipitates will appear in the EP tube;

(8) Centrifuge at 12000r/min for 10min, transfer the supernatant to another EP tube, add an equal volume of Tris saturated phenol/chloroform (1:1) mixed solution, mix well, centrifuge at 12000r/min for 10min, and then Transfer the supernatant to another EP tube;

(9) Repeated extraction 2 times, then extracted 1 time with equal volume of chloroform to remove trace phenol;

(10) Add 2 times the volume of absolute ethanol, mix well, and place at -20°C for 30 minutes. Centrifuge at 12000r/min for 10min to collect the precipitate;

(11) Wash the precipitate with 70% ethanol for 2 to 3 times, and discard the raffinate;

(12) Dry in air for 20-30min, and dissolve the precipitate with 30 μL sterilized ddH2O;

2. Search through the NCBI gene bank, analyze its conserved sequence according to the reported Klebsiella pneumoniae laccase mature peptide gene, and design the amplification primers for the laccase mature peptide coding gene of the present invention as follows:

Upstream primer P1 (SEQ ID NO.3): 5'—CGCGGATCCTCAACGTCGAGACTTCCTCA—3'

Downstream primer P2 (SEQ ID NO.4): 5'—AAAACTGCAGTTAAACCGTGAACCCCAAC—3'

Upstream primer P3 (SEQ ID NO.5): 5'—CCGGAATTCCAACGTCGAGACTTCCTCAA—3'

Downstream primer P4 (SEQ ID NO.6): 5'—ATAAGAATGCGGCCGCTTAAACCGTGAACCCCAAC—3'

Wherein, the upstream primer P1 and the downstream primer P2 are used to amplify the target gene expressed in Bacillus subtilis, and the upstream and downstream primers introduce restriction enzyme sites BamH I and Hind III respectively; the upstream primer P3 and the downstream primer P4 are Used to amplify the target gene expressed in Pichia pastoris GS115, the upstream and downstream primers were respectively introduced into the restriction enzyme cutting sites EcoR I, Not I.

The amplification template is Klebsiella pneumoniae genomic DNA, and the reaction conditions for its amplification are:

The amplification conditions were: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 1 min and 40 s for 30 cycles; extension at 72°C for 10 min. The PCR amplified product is through 0.8% agarose gel electrophoresis, obtains the band (Fig. 1) of 1600bp, reclaims PCR product with a small amount of DNA gel recovery kit, and carries out double enzyme cutting and purifying recovery, obtains pneumoniae clone of the present invention See sequence 1 for the gene Lac encoding a novel laccase mature peptide of Lebsiella.

Example 2: Construction of a novel laccase recombinant bacterium with high stability of Bacillus subtilis

1. Construction of expression vector pBSA43

pBSA43 is obtained by using the E. coli-Bacillus subtilis shuttle cloning vector pBE2 as the backbone, cloning into a strong Bacillus constitutive promoter P43, and the fructan sucrase signal sequence sacB that can directly secrete the recombinant protein into the medium . It has the Amp ^r gene, which can use ampicillin resistance as a selection marker in Escherichia coli; it also has the Km ^r gene, which can use kanamycin resistance as a selection marker in Bacillus subtilis and Bacillus licheniformis.

2. Construction of a novel laccase expression vector pBSA43-Lac

The novel laccase gene (Lac) amplified by PCR and recovered after double digestion with BamH I and Hind III was ligated with the same double digestion Bacillus subtilis expression vector pBSA43, and the ligated product was transformed into Escherichia coli JM109 In the competent cells, after Amp resistance screening, positive transformants were selected, the transformant plasmids were extracted, and single- and double-enzyme digestion verification and sequencing were performed to confirm that the correct recombinant strain JM109/pBSA43-Lac was constructed.

3. Transformation of Bacillus subtilis WB600 with recombinant expression vector pBSA43-Lac

Add 1 μL (50ng/μL) of the pBSA43-Lac recombinant plasmid to 50 μL of Bacillus subtilis WB600 competent cells and mix well, then transfer to a pre-cooled electroporation cup (1mm), and ice-bath for 1-1.5min, Electric shock once (25uF, 200Ω, 4.5-5.0ms). Immediately after the electric shock, 1 mL recovery medium (LB+0.5mol/L sorbitol+0.38mol/L mannitol) was added. After shaking and culturing at 37°C for 3 hours, spread the resuscitated material on an LB plate containing kanamycin, culture at 37°C for 12-24 hours, pick out positive transformants, and perform single and double enzyme digestion verification to confirm that subtilis Bacillus recombinant strain WB600/pBSA43-Lac.

Example 3: Construction of recombinant strains for the free expression of novel laccase from Pichia pastoris

1. Construction of a novel laccase expression vector pPIC9K-Lac

The novel laccase gene (Lac) amplified by PCR and recovered after EcoR I and Not I double-digestion was ligated with the same double-digestion Pichia expression vector pPIC9K with ligase; the ligated product was transformed into Escherichia coli JM109 In the competent cells, select positive transformants through Amp resistance screening; extract positive transformant plasmids, extract plasmids after shaking tube culture at 37°C, and conduct preliminary verification of single and double enzyme digestion, and verify the correct enzyme digestion The recombinant plasmid was named pPIC9K-Lac; the positive clones verified by enzyme digestion were sent to Beijing Huada Gene Technology Co., Ltd. for sequencing to further ensure the correctness of the target gene, and finally confirm the construction of the correct recombinant strain JM109/pPIC9K-Lac .

2. Construction of new laccase recombinant strains and screening of new laccase high expression recombinant strains

(1) Preparation of linearized recombinant expression plasmid pPIC9K-Lac

Before the recombinant plasmid is electrotransformed into Pichia pastoris GS115, the constructed recombinant expression plasmid pPIC9K-Lac should be linearized to improve the integration efficiency of the recombinant plasmid on the Pichia pastoris chromosome. Each transformation requires 15 μg of linearized plasmid DNA, and the purer the plasmid, the higher the transformation efficiency. Linearization was performed with two restriction enzymes, SacI and SalI. After digestion, use a small DNA recovery kit to recover the linearized digestion products.

(2) Electrotransformation of Pichia pastoris GS115 with linearized plasmid pPIC9K-Lac, identification of positive transformants and screening of new strains with high expression of laccase

a. Add the recombinant plasmid DNA (15 μg) recovered by linearization to 100 μL Pichia pastoris GS115 competent cells prepared in advance, mix well, and after 20 minutes in ice bath, add the mixed reaction solution into the pre-ice bath In the electric spinner cup.

b. Ice-bath the transformation cup filled with the transformation solution for 5 minutes, and perform electrotransformation of Pichia pastoris according to the parameters recommended by the electroporation device (voltage 2500V, electroporation time 5ms).

c. Immediately after the pulse, add 1 mL of pre-cooled 1 mol/L sorbitol solution to the electro-cup, and transfer the transformation solution to a new 1.5 mL centrifuge tube.

d. Cultivate statically at 30°C for 1-2 hours, pipette 200 μL of Pichia GS115 electrotransfer solution and spread it on the MD medium plate.

e. Static culture at 30°C until transformants appear.

f. Pick a single transformant colony and dissolve it in 10 μL of deionized water, take 2 μL of the bacterial liquid, add Lyticase wall-breaking enzyme, react at 30°C for 10 minutes, and immediately put the reaction solution in a -80°C refrigerator for 10 minutes to fully lyse the yeast cell wall and release The genome was used as a template for PCR. Pichia pastoris GS115/pPIC9K transformed into empty plasmid pPIC9K was used as a control to determine positive transformants.

g. On the basis of determining the positive transformants, use resistance plates containing different concentrations of geneticin to screen the transformants with high geneticin resistance, and then measure the new paints of these transformants with high geneticin resistance respectively. The enzymatic activity of the enzyme was used to obtain a novel laccase-producing strain GS115/pPIC9K-Lac.

Embodiment 4: the mensuration of laccase enzymatic activity

Laccases from different sources (plants, animals, fungi, bacteria) have different affinities for substrate reactions, and the reaction substrates, reaction conditions and enzyme activity unit definition used in the enzyme activity assay method have a great influence on the assay results of laccase activity. influences. p-phenylenediamine, guaiacol, o-toluidine, catechol, urushiol, syringaldazine, 2,6-dimethoxyphenol, 2,2'-azinobis(3-ethyl Benzothiazoline-6-sulfonic acid) diammonium salts can be used as substrates for the determination of laccase activity. Among them, o-toluidine has poor water solubility; when using guaiacol as a substrate, the reaction is very stable, but the reaction time is long and the measured laccase activity value is low, so it is rarely used; when using cloves When aldazine is used as substrate to measure laccase activity, although the laccase activity value is higher, the reaction is unstable when the amount of substrate and enzyme solution is higher. If the method of reducing the amount of substrate and enzyme solution is adopted, it can be compared Overcome the defect that the reaction is not easy to terminate to the greatest extent. When ABTS is used as the substrate to determine the enzyme activity of laccase, it has the characteristics of sensitive response, good stability and good reproducibility.

The methods for determining the enzyme activity of laccase include HPLC method, oxygen measurement method, spectrophotometry method, spectrum method and so on. Determination of laccase activity with ABTS as substrate is one of the most common methods in foreign countries. It has the following advantages and disadvantages: (1) ABTS is easily soluble in water and is still relatively stable at room temperature for 6 months. (2) Its reaction has only one step, that is, from ABTS to its cationic radical. The cationic radical of ABTS is light blue-green in aqueous solution, and is relatively stable, usually within several hours or several days. (3) Under the action of laccase, the molar absorptivity coefficient of ABTS colored solution is higher, indicating that the sensitivity of the determination using it as a substrate is higher. (4) So far, there has been no report that ABTS has carcinogenicity, mutagenesis or strong toxicity. (5) Quantitative analysis of laccase with ABTS as substrate, usually at 420nm to measure the change of absorbance within 3min. Quantitative analysis with this substrate has the disadvantage that the absorbance of a solution of ABTS cationic radicals is easily affected by the concentration of unreacted ABTS in solution. This has just produced a problem: the molar absorptivity coefficient 36000L/(mol cm) under 420nm is obtained under the condition of pure ABTS cationic free radical, and in the actual measurement reaction, ABTS is excessive, and this just causes people Enzyme activity is underestimated. This effect cannot be ignored, because when the reaction is terminated, there must be at least 1 mmol/L of ABTS in the solution. Despite the above-mentioned problems when using ABTS as a substrate, it is still one of the best substrates for the determination of laccase activity.

1. Using ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) as the substrate to determine the enzyme activity of laccase

(1) Principle of measuring laccase activity with ABTS as substrate

Laccase decomposes ABTS into ABTS cationic radicals, but the absorption coefficient of ABTS cationic radicals at 420nm is much larger than the substrate ABTS. Therefore, with the continuous reaction of laccase and substrate ABTS, the concentration of ABTS free radicals increases gradually, and its absorbance value also increases gradually.

Definition of enzyme activity: under certain conditions, the amount of enzyme required to oxidize 1 μmol of ABTS per minute is defined as an enzyme activity unit.

(2) Method and steps for measuring laccase enzyme activity with ABTS as substrate

a. Take 200 ^μL of 0.1 mol/L citric acid-disodium hydrogen phosphate buffer (pH=2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8) or 200 μL of 0.05 mol/L glycine-sodium hydroxide buffer (pH=8.5, 9, 9.5, 10, 10.5, 11) containing 4 mmol/LCu ²⁺ in a 96-well plate, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90°C for 1 min.

b. Add 10 μL of enzyme solution diluted by an appropriate multiple, then add 30 μL of substrate (50 mmol/L ABTS), mix well, and record the initial OD value of the reaction and the OD value after 3 minutes of reaction.

2. Using 2,6-dimethoxyphenol (2,6-dimethoxyphenol, DMP) as the substrate to determine the enzyme activity of laccase

(1) Principle of Determination of Laccase Activity Using DMP as Substrate

Laccase can oxidize DMP to 3,5,3',5'-tetramethoxydiphenylquinone, which has a maximum absorption peak at 468nm, and its molar absorptivity coefficient is 49.6mM ^-1 cm ^-1 . The concentration of the product within the concentration range has a linear relationship with its absorbance value, and the formation rate of the calculated product is determined by measuring the change in absorbance.

Definition of enzyme activity: Under certain conditions, the amount of enzyme required to oxidize 1 μmol of DMP per minute is defined as an enzyme activity unit.

(2) Method and steps for measuring laccase enzyme activity with DMP as substrate

a. Take 200 ^μL of 0.1 mol/L citric acid-disodium hydrogen phosphate buffer (pH=2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8) or 200 μL of 0.05 mol/L glycine-sodium hydroxide buffer (pH=8.5, 9, 9.5, 10, 10.5, 11) containing 4 mmol/LCu ²⁺ in a 96-well plate, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90°C for 1 min.

b. Add 10 μL of enzyme solution diluted to an appropriate multiple, then add 30 μL of substrate (8 mmol/L DMP), mix well, and record the initial OD value of the reaction and the OD value after 3 minutes of reaction.

3. Calculation of enzyme activity

Enzyme activity

In the formula: △OD represents the change in absorbance from the beginning to the end of the reaction.

V ₁ represents the total volume of the reaction system.

Δt represents the time taken from the beginning to the end of the reaction.

V ₂ represents the volume of enzyme solution in the reaction system.

ε represents when ABTS is used as the substrate, the molar absorptivity of its product at 420nm is 36mM ^-1 cm ^-1 .

ε represents when DMP is used as the substrate, the molar absorptivity coefficient of its product at 468nm is 49.6mM ^-1 cm ^-1 .

d represents the inner diameter of the light absorbing cup or the thickness of the optical path (cm).

The enzymatic properties of the novel laccase are determined by the above method, and the enzymatic properties of the novel laccase are as follows:

When using ABTS as the substrate to determine the enzymatic properties of the new laccase, the optimum action temperature is 35°C, and the optimum action pH is 4; when using DMP as the substrate to determine the enzymatic properties of the new laccase, the optimum action temperature is 70°C , the optimum pH is 8;

The pH stability and thermal stability of the new laccase were measured with DMP as the substrate. The results showed that the new bacterial laccase had good stability in the range of pH 5-9. Measured at pH 8 and 70°C, the residual enzyme activity was above 60%, incubated at pH 7 and pH 8 for 35 hours, and measured at pH 8 and 70°C, the residual enzyme activity was above 85%;

It has good thermal stability at 30-60°C, and it is specifically shown that after incubation at 30-40°C for 5 hours, the residual enzyme activity measured at pH 8 and 70°C is more than 95%, and at 50-60°C for 4 hours, at pH 8 and 70°C The measured residual enzyme activity is more than 50%. Compared with the reported laccase derived from Klebsiella pneumoniae, the novel laccase to be protected in this patent has better pH stability and temperature stability.

Embodiment 5: Expression and preparation of novel laccase in recombinant strain of Bacillus subtilis

Inoculate the recombinant strain of Bacillus subtilis WB600/pBSA43-Lac in 5 mL of LB liquid medium (containing kanamycin, 50 μg/mL), culture overnight at 37 ° C, 220 r/min, transfer to 50 mL according to 2% inoculum In the fresh LB medium, continue to cultivate at 37°C and 220r/min for 48h, and a highly stable novel laccase crude enzyme solution can be prepared, and the enzyme activity of the new laccase crude enzyme solution is determined with ABTS as the substrate (pH4, 35°C conditions), the laccase activity of the recombinant strain expressing the new laccase from Bacillus subtilis could reach 16.8 U/mL after fermentation, and the enzyme activity of the crude enzyme solution of the new laccase (pH8, 70°C) was determined with DMP as the substrate. Subtilis subtilis The activity of laccase can reach 19.7U/mL after fermentation by the recombinant strain of Bacillus expressing the new laccase; then the new laccase is precipitated by the fractional salting-out method, the protein precipitate is collected, dissolved, dialyzed to remove salt, and then subjected to ion exchange chromatography, After gel chromatography, the novel laccase pure enzyme powder was prepared by freeze-drying.

Example 7: Expression and preparation of novel laccase in Pichia pastoris free expression recombinant strain

Inoculate the Pichia pastoris free-expressing novel laccase recombinant strain GS115/pPIC9K-Lac cultured on the YPD solid plate into the YPD liquid medium, and culture at 30°C and 220r/min for 24h. Transfer to fresh BMGY medium with 2% inoculum, continue culturing at 30°C, 220r/min for 24h, then centrifuge at 6000rpm/min for 10min to collect the bacteria, and transfer the bacteria to BMMY medium. Then cultivate at 30°C, 220r/min, and add methanol every 12h to keep the final concentration at 0.5% V/V. After culturing for 120h, a crude enzyme solution of a new type of laccase can be obtained, which is determined by using ABTS as a substrate. The enzyme activity of the novel laccase crude enzyme solution (at pH 4, 35°C), the laccase enzyme activity of the recombinant strain expressing the new laccase free from Pichia pastoris after fermentation can reach 15.3U/mL, and the crude enzyme of the new laccase was determined with DMP as the substrate Enzyme activity of the enzyme solution (at pH 8, 70°C), the laccase activity of the recombinant strain expressing the new laccase free from Pichia pastoris can reach 18.6U/mL after fermentation; then the new laccase is precipitated by the fractional salting-out method, and the protein precipitate is collected After dissolving, dialyze to remove salt, then go through ion-exchange chromatography and gel chromatography, and then freeze-dry to obtain a new type of laccase pure enzyme powder.

Embodiment 8: Calculation of decolorization treatment and decolorization rate of novel laccase to dyestuff

To decolorize anthraquinone and azo dyes with new laccase enzyme solution, it is necessary to consider factors in the catalytic reaction system: reaction temperature, reaction pH value, dye type and concentration, mediator type and concentration, enzyme solution dosage, decolorization time and other factors . The dyes used in this study include: Reactive Brilliant Blue X-BR, Reactive Brilliant Blue K-GR, Reactive Brilliant Blue KN-R, Reactive Deep Blue M-2GE, Congo Red, Cotton Blue, Malachite Green, Azo Fluorescent Pink, Bromophenol blue, acid medium black PV.

(1) Method and steps for decolorizing dyestuff by novel laccase

The laccase-catalyzed dye decolorization treatment uses a 300 μL reaction system:

a. Take 275 μL of 0.1 mol/L citric acid-disodium hydrogen phosphate buffer (pH=4) containing 4 mmol/L Cu ²⁺ or 0.05 mol/L glycine-hydroxide containing 4 mmol/L Cu ²⁺ Sodium buffer solution (pH=8), in a 96-well plate, incubated at 70° C. for 1 min.

b. Add 15 μL of the above dye with a concentration of 2000 mg/L.

c. Add 10 μL of laccase enzyme solution (corresponding to an enzyme activity of 0.05 U/mL) diluted by an appropriate multiple (or under the same experimental conditions, add an equal amount of enzyme solution that was inactivated at 100°C for 15 minutes as a blank control), and measure Its absorbance A ₀ .

d. Decolorize at 70°C for 60 minutes, and measure the absorbance as A ₁ .

Each of the above decolorization treatments was repeated 3 times, and the results were averaged.

(2) Calculation of decolorization rate:

After the obtained two strains of recombinant bacteria were fermented, the above-mentioned dyes were decolorized by using the prepared novel laccase enzyme powder according to the above-mentioned decolorization steps, and the decolorization rates reached more than 85% in 60 minutes.

Claims (7)

1. a kind of novel bacterial laccase from Klebsiella pneumonia is it is characterised in that the aminoacid of described bacterial laccase SEQ ID No in sequence such as sequence table：Shown in 2.

2. a kind of novel bacterial laccase from Klebsiella pneumonia according to claim 1 is it is characterised in that compile The gene of the described bacterial laccase of code is Lac, its base sequence such as sequence table SEQ ID No：Shown in 1.

3. a kind of novel bacterial laccase from Klebsiella pneumonia according to claim 1 is it is characterised in that institute The preparation method stating bacterial laccase comprises the steps：

(1) gene described in claim 2 is carried out enzyme action, be connected with expression vector and obtained new recombinant vector；

(2) recombinant vector is transformed in host cell, obtains recombinant bacterial strain, afterwards recombinant bacterial strain ferments, obtain high stable Property bacterial laccase.

4. the cloning vehicle containing the novel laccase enzyme gene described in claim 2, expression vector and host cell.

5. the cloning vehicle containing novel laccase enzyme gene according to claim 4, expression vector and host cell, its It is characterised by, described cloning vehicle is pBSA43 carrier, described expression vector is pBSA43, described host cell is withered Careless bacilluss WB600.

6. the cloning vehicle of novel laccase enzyme gene according to claim 4, expression vector and host cell, its feature It is, described cloning vehicle is pPIC9K carrier, described expression vector is pPIC9K, described host cell is to finish red ferment Female GS115.

7. a kind of novel bacterial laccase from Klebsiella pneumonia according to claim 1 is in azo and Anthraquinones The application of dye decolored aspect.