CN113430182B - Bacterial laccase from Astrospiraceae of elephant intestinal tract and gene thereof - Google Patents

Abstract

The invention discloses a bacterial laccase derived from the Asian elephant intestinal Lachnospiraceae and its gene. The amino acid sequence of the bacterial laccase is shown in SEQ ID NO.1, and the coding gene is shown in SEQ ID NO.2. The genome of an uncultured Lachnospiraceae NK4A136 bacterium was assembled by binning technology, and the laccase gene containing copper polyphenol oxidoreductase was annotated by InterPro and Pfam databases, and the bacterial laccase was prepared by genetic engineering technology. Compared with fungal-derived laccase, the bacterial laccase has better pH stability and temperature stability.

Description

A Bacterial Laccase and Its Gene Derived from the Lachnospiraceae Gut of Asian Elephants

technical field

The invention belongs to the technical field of genetic engineering of enzymes, and relates to a bacterial laccase derived from the Asian elephant intestinal Lachnospiraceae and its gene, and the recombination expressing the novel laccase is obtained through the metagenomic sequencing analysis technology and molecular biology means expression strain.

Background technique

Laccase (Laccase, EC 1.10.3.2), also known as phenolase and polyphenol oxidase, belongs to a class of multi-copper oxidases, which can oxidize a variety of aromatic compounds, and finally reduce molecular oxygen to generate water. Therefore, laccase is a kind of green catalyst with important application value.

Laccase is widely distributed in fungi, bacteria, insects, animals and plants. Compared with animal and plant source laccase, the source of microbial laccase is more abundant. Laccase derived from fungi has high activity at pH 4-6 and 30-55°C, but industrial applications often require high temperature and strong alkali environment, which limits the application and high cost of laccase. Moreover, the long growth cycle of the fungus has harsh requirements on the medium, making the fermenter vulnerable to mechanical damage. These problems have seriously affected the industrial application of fungal laccase. Bacterial laccase does not require glycosylation modification, usually exists in the form of monomeric protein bodies, has a wide range of pH, good thermal stability, and Cu ²⁺ resistance. These have important implications for the wider application of laccases. At present, there are few excavation of bacterial laccase gene resources, and new bacterial laccases with high activity and high stability need to be further discovered.

With the development of second-generation sequencing technology, combined with metagenomics analysis methods, in the mining of microbial functional enzyme genes, it can successfully avoid the traditional technical problems of pure microbial culture, and can quickly discover a large number of functional enzyme genes in a short time , increased functional enzyme gene

The extensiveness and effectiveness of screening have greatly promoted the improvement of the cloning efficiency of functional enzyme genes, and provided a new research strategy for finding and discovering new laccase genes.

Contents of the invention

The purpose of the present invention is to overcome and avoid the deficiencies of laccase in industrial production and application, and provide a new type of bacterial laccase encoding gene derived from the intestinal tract of Asian elephants from the uncultivated Lachnospiraceae and a method for expressing the new type of bacterial laccase gene Engineering strains.

In the present invention, the coding gene of the novel laccase is derived from the Asian elephant intestinal microbial metagenome, and the genome of an uncultivated Lachnospiraceae NK4A136 bacterium is assembled by using Binning technology (completeness is 96.44%, The pollution rate is 0), and the polyphenol-containing polyphenol oxidoreductase laccase gene (IPR003730 and PF02578.16) is annotated by InterPro and Pfam databases. The amino acid sequence of the laccase gene is compared with the existing laccase amino acid sequence in the NR database The highest agreement is about 52%. The novel laccase-encoding gene was cloned and expressed in the Bacillus subtilis expression system to obtain a highly stable laccase recombinant strain of Bacillus subtilis.

In order to achieve the above-mentioned technical purpose, the present invention is specifically realized through the following technical solutions:

A bacterial laccase derived from the intestinal Lachnospiraceae of Asian elephants, the amino acid sequence of the bacterial laccase is shown in SEQ ID NO.1.

When the bacterial laccase uses the substrate 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) to measure the enzymatic properties of the new laccase, the best effect The temperature is 70°C and the optimum pH is 5.

The pH stability and thermal stability of the new laccase were measured with the substrate ABTS, and the results showed that it had good stability in the range of pH 5-6 at 60°C. Novel laccases from bacteria with better pH and thermostability than laccases of fungal origin.

The gene encoding the bacterial laccase is also prepared within the protection scope of the present invention, and its nucleotide sequence is shown in SEQ ID NO.2.

In another aspect of the present invention, the gene encoding the above-mentioned bacterial laccase is inserted into an expression vector, and its nucleotide sequence is connected with an expression control sequence. A recombinant vector comprising the encoding gene is provided.

As a preferred embodiment of the present invention, the gene encoding the bacterial laccase described above is connected with the expression vector pBE-S to obtain the recombinant Escherichia coli expression plasmid pBE-S-Laac2.

More preferably, the host cell used to express the bacterial laccase is E. coli DH5α.

In another aspect of the present invention, a recombinant strain comprising the gene encoding the bacterial laccase is also provided.

The preferred recombinant strain is Bacillus subtilis, more preferred is the recombinant strain WB600/pBE-S-Lacc2.

In another aspect of the present invention, the preparation method of above-mentioned novel bacterial laccase is provided, comprising the following steps:

1) Using the intestinal metagenome DNA of the Asian elephant as a template, and according to the metagenomic component box technology to assemble the gene encoding laccase in the genome of the uncultured Lachnospiraceae bacteria, analyze its conserved sequence, and design the gene encoding the laccase The amplification primers are Pf and Pr, and the target laccase coding gene lacc2 is obtained by PCR amplification;

Upstream primer Pf: CAATTCATGAATGATATGACAGCAG,

Downstream primer Pr: GTCGACCTATCTAGCAAGCATGATAAC;

2) The laccase-encoding gene lacc2 and the expression vector pBE-S were double digested with EcoRI and SalI, ligated with T4 ligase, and then transformed into the cloning host E.coli DH5α. After colony PCR, enzyme digestion and sequencing verified the correctness, the recombinant was obtained Plasmid pBE-S-laac2;

3) The recombinant plasmid pBE-S-laac2 was transformed into the host Bacillus subtilis WB600 and successfully expressed to obtain a recombinant strain producing a novel laccase with high stability;

4) A novel laccase Lacc2 with high yield and high stability was obtained by optimizing the fermentation process.

In another aspect of the present invention, the application of the laccase Lacc2 in the decolorization of crystal violet, alizarin red, isatin and reactive black 5 is provided.

The decolorization rates of the new laccase Lacc2 to crystal violet, alizarin red, isatin and reactive black 5 were 80.8%, 81.6%, 68.5% and 45.85% within 6 hours without mediator; After syringone, the decolorization rate of the new laccase Lacc2 to Reactive Black 5 increased to 84.95% within 6 hours.

The beneficial effects of the present invention are:

The present invention assembles and obtains the genome of an uncultivated Lachnospiraceae NK4A136 bacterium (the completeness is 96.44%, and the pollution rate is 0) through the assembly of the macrogene component box technology, which is annotated by the InterPro and Pfam databases to annotate the redox of polyphenols containing copper The enzyme laccase gene is prepared by genetic engineering technology to obtain bacterial laccase Lacc2. Compared with the laccase derived from fungi, the bacterial laccase Lacc2 has good stability in the range of pH 5-6 at 70° C., and its pH stability and temperature stability are better.

Description of drawings

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

Fig. 2 is the restriction electrophoresis diagram of recombinant plasmid pBE-S-laac2 of the present invention;

Fig. 3 is novel laccase Lacc2 optimal action temperature curve of the present invention;

Fig. 4 is the optimal action pH curve of novel laccase Lacc2 of the present invention;

Fig. 5 is the decolorization effect of novel laccase Lacc2 of the present invention to 4 kinds of dyes, A: the decolorization effect of novel laccase Lacc2 to 4 kinds of dyes under the condition of no mediator, B: novel laccase Lacc2 is paired 4 kinds under the condition of mediator Decolorization effect of dyes.

detailed description

The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The original data of Asian elephant intestinal metagenomic sequencing used in the present invention has been deposited in the Genome Sequence Archives of the Big Data Center of the Chinese Academy of Sciences, and the accession number is CRA003369.

Example 1 Acquisition of Lachnospiraceae Novel Bacterial Laccase Gene from Uncultivated Asian Elephant Gut

1. The new laccase-encoding gene is derived from the metagenome of Asian elephant fresh feces preserved in our laboratory, and its metagenomic DNA was extracted using a kit (QIAamp DNA Stool Mini Kit). The extraction steps of the metagenome DNA of fresh Asian elephant feces are as follows:

1) Weigh about 0.2g of Asian elephant feces into a sterilized centrifuge tube;

2) Add 1.4mL buffer ASL, vortex continuously for several minutes until fully mixed, incubate at 70°C for 5min, and then vortex mix for 15s;

3) Centrifuge at 20,000g for 1min to remove fecal impurities, and transfer the supernatant to a new centrifuge tube;

4) Immediately mix for about 1 min after adding 1 inhibitEX Tablet, then place at room temperature for 1 min to allow the inhibitor to be adsorbed on the inhibitEX matrix, and centrifuge at 20,000g for 3 min;

5) Take the supernatant to a new centrifuge tube, and centrifuge once more as in 4);

6) Pipette the supernatant into a new centrifuge tube to which 15 μL proteinase K has been added, add 200 μL buffer AL, mix thoroughly and incubate at 70°C for 10 min;

7) Add 200 μL of absolute ethanol, mix thoroughly, transfer to the QIAamp adsorption column, and centrifuge at 20,000g for 1 min (if the liquid does not pass through the column, repeat the centrifugation once);

8) Wash the column with 500 μL of buffer AW1 and AW2 respectively, and centrifuge for 3 minutes as in 7), then transfer the column to a new collection tube;

9) Add an appropriate amount of buffer AE to the center of the adsorption column, place at room temperature for 1 min, and centrifuge for 1 min to elute. The obtained metagenomic DNA can be used for downstream molecular biology experiments or stored at -20°C for future use.

2. Through the Binning assembly technology of metagenomics analysis, the genome of an uncultivated Lachnospiraceae NK4A136 bacteria (completeness 99.73%, pollution rate 0) was obtained, which was annotated by InterPro and Pfam databases Contains a polycopper polyphenol oxidoreductase laccase gene (IPR003730 and PF02578.16), the result of NR database blast shows that the highest amino acid identity with the NR database laccase is about 52%, and the conservation of this new laccase is analyzed Sequence, design the amplification primer of laccase coding gene of the present invention as follows:

Upstream primer Pf: CAATTCATGAATGATATGACAGCAG,

Downstream primer Pr: GTCGACCTATCTAGCAAGCATGATAAC;

The upper and lower primers Pf and Pr are used to amplify the laccase gene expressed in Bacillus subtilis, and the restriction sites EcoRI and SalI are respectively introduced into the upper and lower primers.

The amplification template is Asian elephant intestinal metagenomic DNA. The PCR amplification reaction system is: EX Taq 1.25 μL, 10XBuffer 10 μL, dNTP mix 5 μL, Pf and Pr 2.5 μL each, DNA 5 μL, ddH ₂ 0 73.75 μL, total volume 100 μL .

The amplification reaction conditions are: 94°C, 5min; 94°C, 30s; 55-61°C, 30s; 72°C, 1min (2-4, 30 cycles); 72°C, 10min; 4°C, 10min.

The PCR product was verified by 0.8% agarose gel electrophoresis to obtain a 795bp band (Fig. 1). After the PCR product was recovered from the gel, it was digested and purified to obtain the new type of uncultured Lachnospiraceae in the intestinal tract of Asian elephants of the present invention. The bacterial laccase encoding gene lacc2 is shown in the sequence SEQ ID NO.2.

Example 2 Construction of a novel bacterial laccase recombinant plasmid derived from uncultured Lachnospiraceae in the intestinal tract of Asian elephants

1) Digest the expression plasmid pBE-S with restriction endonucleases EcoRI and SalI, and then use T4 ligase to digest the purified product of the pBE-S plasmid and the purified product of the target gene at 16°C overnight Ligation, the ligation product was chemically transformed into E.coli DH5α.

2) After colony PCR identification, double enzyme digestion identification (Figure 2) and correct sequencing, the recombinant plasmid pBE-S-lacc2 was obtained;

3) The cloned strain E. coli DH5α/pBE-S-lacc2 of the verified correct recombinant plasmid pBE-S-lacc2 was stored at -80°C with 15% glycerol.

Example 3 Construction of Bacillus subtilis High Stability Novel Laccase Recombinant Bacteria

1) Add 1 μL (50ng/μL) of pBE-S-lacc2 recombinant plasmid to Bacillus subtilis WB600 competent cells, and mix gently;

2) After mixing, transfer to a pre-cooled electric cup, ice bath for 1-1.5min, and then electric shock once (3.0kV, 3.0-4.5ms), after electric shock, immediately add 1mL recovery medium (LB+0.5mol /L sorbitol+0.38mol/L mannitol);

3) After shaking culture at 37°C for 3 hours, spread the bacterial solution on the LB plate containing Kan resistance, and incubate at 37°C for about 16 hours;

4) The positive transformants were picked and verified by double enzyme digestion to confirm that the recombinant Bacillus subtilis strain WB600/pBE-S-lacc2 expressing lacc2 was obtained.

The mensuration of embodiment 4 laccase enzymatic activity

The determination of enzyme activity was performed in three parallel experiments, and the results were averaged.

Enzyme activity unit (U): the amount of laccase required to oxidize 1 μmol ABTS in 1 min.

Mix 480 μL of disodium hydrogen phosphate-citrate buffer solution and 100 μL of 3 mmol/L ABTS thoroughly in advance, and place in a water bath at 50°C for 5 minutes. Quickly add 20 µL of Lacc1 laccase solution. Read the OD value at a wavelength of 420nm.

The calculation formula of laccase activity is as follows:

Enzyme activity (U/L)=(Δ _OD ×V ₁ ×n)/(Δ _t ×V ₂ ×ε×10 ^-6 )

In the formula, Δ _OD is the absorbance difference between the beginning and the end; V ₁ is the total volume of the reaction system (mL); n is the dilution factor of the enzyme solution; Δ _t is the reaction time (min) _; The volume of the enzyme solution (mL); ε is the molar digestion coefficient (L/(mol·cm)).

Optimum pH of laccase: at 50°C, place the reaction system in buffers with different pH (2.0-11.0), measure the enzyme activity, take the highest enzyme activity as 100%, and compare to obtain the optimum reaction pH.

Optimum temperature: Under the optimal reaction pH condition, test the enzyme activity of the reaction system at different temperatures (30, 40, 50, 60, 70, 80, 90, 100°C), and take the highest enzyme activity as 100 %, to get the optimum reaction temperature of laccase.

pH stability: place the enzyme solution in buffer solutions of different pH, put it in the room at 4°C for 5 hours, take the highest enzyme activity as 100%, and measure the remaining enzyme activity.

Determination of thermal stability: under the optimal reaction pH condition, the laccase was incubated under different temperature gradients (30-90° C.) for 2 hours, and the remaining enzyme activity of Lacc1 laccase was determined with the highest enzyme activity as 100%.

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 substrate to determine the enzymatic properties of the new laccase, the optimum temperature is 70°C (Figure 3), and the optimum pH is 5 (Figure 4);

The pH stability and thermal stability of the new laccase were measured with ABTS as substrate, and the results showed that the new bacterial laccase had good stability in the range of pH 5-6 at 60°C. When stored at 4°C and pH=5 for 4 days, the residual enzyme activity reached about 300%, and when stored at 4°C and pH=6.5 for 4 days, the residual enzyme activity reached about 200%; Days, the residual enzyme activity reaches about 200%, and when stored at 4°C and pH=6.5 for 10 days, the residual enzyme activity reaches about 200% (the above enzyme activities are all measured at 70°C and pH5, and the initial enzyme activity is used as 100%).

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

1) Pick a single colony of the recombinant Bacillus subtilis strain WB600/pBE-S-lacc2 and inoculate it into LB liquid medium containing kanamycin, and cultivate overnight at 37°C with shaking;

2) Inoculate the seeds obtained in step 1) into 50 mL of LB liquid medium in an amount of 2%, and culture with shaking at 37° C. for about 48 hours;

3) Centrifuge the fermented liquid obtained in step 2), collect the supernatant, and obtain a high-stability novel laccase crude enzyme liquid;

4) Using ABTS as a substrate to measure the enzyme activity of the new laccase crude enzyme solution (at pH 5, 70°C), the laccase activity of the recombinant strain expressing the new laccase in Bacillus subtilis can reach 300U/mL after fermentation, and then use the grading Precipitate the new laccase by salting out method, collect the protein precipitate, dissolve, dialyze to remove salt, and then go through ion exchange chromatography, gel chromatography, and freeze-dry to obtain the pure enzyme powder of the new laccase.

Example 6 Calculation of decolorization treatment and decolorization rate of 4 kinds of dyestuffs by novel laccase Lacc2

The initial concentration of alizarin is 1,000mg/L, the final concentration is 100mg/L, and the maximum absorption wavelength is 520nm; the initial concentration of reactive black 5 is 400mg/L, the final concentration is 40mg/L, and the maximum absorption wavelength is 597nm; The concentration is 250mg/L, the final concentration is 25mg/L, and the maximum absorption wavelength is 610nm; the initial concentration of crystal violet is 50mg/L, the final concentration is 5mg/L, and the maximum absorption wavelength is 583nm. The total volume of the mediator-free decolorization reaction system is 12 mL, including 1.2 mL of dye mother solution, 100 μL of new laccase Lacc2 crude enzyme solution and 10.7 mL of 0.2 mol/L Na ₂ HPO ₄ -NaHPO ₄ buffer solution at pH 5. The total volume of the decolorization reaction system with the mediator is 12 mL, including 12 μL of 0.1 mol/L acetosyringone, 1.2 mL of the dye mother solution, 100 μL of the new laccase Lacc2 crude enzyme solution and 0.2 mol/L Na ₂ HPO ₄ -NaHPO at pH 5 ₄ Buffer 10.688 mL. Decolorization was carried out in a shaker at 37°C and 200r/min, and the optical density values of different dyes at the maximum absorption wavelength were measured at 2h, 4h and 6h, and the decolorization rate of various dyes by the new laccase Lacc2 was calculated. The formula for calculating the decolorization rate is: R=(A ₀ -A)/A ₀ ×100%, where A is the optical density value of the dye during regular sampling, and A0 is the optical density value of the initial dye, repeated 3 times.

The new laccase Lacc2 decolorized 80.8%, 81.6%, 68.5% and 45.85% of crystal violet, alizarin red, isatin and reactive black 5 within 6 hours without mediator, and crystal violet and alizarin The decolorization effect of red is better; after the mediator acetosyringone is added to the system, the decolorization rate of the new laccase Lacc2 to reactive black 5 increases to 84.95% within 6 hours (Figure 5).

Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variants, the scope of the invention is defined by the appended claims and their equivalents.

sequence listing

<110> Yunnan Normal University

<120> A Bacterial Laccase and Its Gene Derived from the Lachnospiraceae in the Gut of Asian Elephants

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acaagtaaaa tcgcgaggcc tgcatatgga cctggctgga tggcagaggc cgatggctat 300

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Claims (5)

1. A bacterial laccase derived from Asian elephant intestinal metagenome is characterized in that the amino acid sequence of the bacterial laccase is shown in SEQ ID NO. 1.

2. The gene encoding the bacterial laccase of claim 1, wherein the nucleotide sequence is shown in SEQ ID No.2.

3. A recombinant plasmid comprising the coding gene of claim 2.

4. A recombinant bacterium comprising the coding gene according to claim 2.

5. Use of the bacterial laccase enzyme of claim 1 for the decolorization of crystal violet, alizarin red, isatin and active black 5.

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