CN110643583B

CN110643583B - Laccase from trichoderma unicolor as well as gene and application thereof

Info

Publication number: CN110643583B
Application number: CN201911077705.2A
Authority: CN
Inventors: 杨捷; 林沚葳; 姚炎华; 叶秀云
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2019-11-06
Filing date: 2019-11-06
Publication date: 2021-05-18
Anticipated expiration: 2039-11-06
Also published as: CN110643583A

Abstract

The invention provides laccase from trichoderma brevicompactum, a gene and application thereof, and belongs to the technical field of biology. The invention provides LacB laccase from Erysipelothrix chromophoris, the amino acid sequence of which is shown as SEQ ID NO.3, the full-length gene sequence of which is shown as SEQ ID NO.1 and the cDNA sequence of which is shown as SEQ ID NO. 2. The invention also discloses a fermentation production method of the LacB laccase, which is characterized in that the LacB laccase is produced by fermentation of the Eremotrichum, the laccase yield can reach 652U/mL after the sixth day of fermentation culture, and the method has the advantages of high yield and short enzyme production period. The LacB laccase has obvious effect in degrading environmental hormones, and has degradation rate of over 98% on diethylstilbestrol, hexestrol, dienestrol, beta-estradiol, bisphenol A and prednisolone. The LacB laccase provides a new way for treating environmental pollutants, and has good economic and social benefits.

Description

Laccase from trichoderma unicolor as well as gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to laccase from trichoderma versicolor as well as a gene and application thereof.

Background

Laccases (laccas, ec 1.10.3.2) are a class of copper-containing polyphenol oxidases, belonging to the blue multicopper oxidase family, which are widely present in fungi, plants, insects. Wherein, the white rot fungus of basidiomycota is used as the main laccase producing fungus, and has excellent capability of degrading cellulose and hemicellulose. The current research shows that laccase can efficiently and cleanly remove persistent organic pollutants (including dyes, various antibiotics and hormone-containing medicines, phenol, arylamine and derivatives thereof and the like) which cannot be effectively removed by a plurality of traditional physical and chemical methods, so that the application of the laccase in the field of environmental remediation is widely concerned.

Environmental hormone pollutants are another global significant environmental problem following ozone layer depletion, the greenhouse effect, and pollution control has received global attention. The environmental hormone is exogenous chemical substances which are released into the environment due to the production and the life of human beings and affect the normal endocrine system in human bodies and animal bodies, has the characteristics of teratogenicity, mutagenicity, carcinogenicity, reproductive toxicity and low-concentration long-term harm, is mainly derived from industrial waste water, domestic waste water, pharmaceutical industry and personal care products, and is difficult to decompose in natural environment. At present, more than 70 kinds of known environmental hormone substances exist, the laccase can degrade phenol, steroid hormones and derivatives thereof, polycyclic aromatic compounds (PAHs), chlorinated aromatic compounds, arylamines and derivatives thereof and other substances, the reaction has the characteristics of high efficiency, mild conditions, difficulty in causing secondary pollution and the like, and the laccase shows great research value and application potential in the aspect of environmental hormone treatment.

However, most fungal laccases suffer from problems of long enzyme production cycle, low enzyme production and instability, such asPanus tigrinusWhen the culture medium is cultured for 11 d in the optimized culture medium, the highest enzyme yield is 2.2U/mL,Lentinussp. the highest enzyme yield at 16 d in culture was 58U/mL,Cerrna unicolorthe highest enzyme activity of 14 d in IBB fermentation is 152U/mL, and the highest enzyme activity of another strain of tricholoma dentatum 5.1011 researched in the laboratory is 123U/mL after the culture medium is optimized in the 15 th day of fermentation, so that the application value of the strain is limited, and the strain is one of the main reasons for hindering the industrial application of laccase. Therefore, the development of enzyme sources with the characteristic of high yield, the exploration of the degradation effect and the catalytic oxidation mechanism of laccase on environmental hormone pollutants, the evaluation of the environmental effect of metabolites and the like have practical significance and good economic benefit.

Disclosure of Invention

The invention aims to provide laccase from trichoderma versicolor as well as a gene and application thereof. The novel laccase from the hirsutella chricosa has obvious effect in degrading environmental hormone pollutants and has wide application potential in the aspect of environmental pollutant treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

the LacB laccase derived from the hirsutella chromophoris has an amino acid sequence shown in SEQ ID NO. 3.

The gene of LacB laccase from Erysipelothrix lutea has a full-length sequence shown as SEQ ID NO.1 and a cDNA sequence shown as SEQ ID NO. 1.

A fermentation production method of LacB laccase from cerrena chromocor sources comprises the following steps: fermenting the hirsutella chromophoris in a fermentation medium by a liquid shake flask, centrifugally collecting fermentation liquor, and separating and purifying to obtain the laccase LacB.

The fermentation medium comprises the following components: sucrose 90 g/L, peptone 10 g/L, KH₂PO₄ 6 g/L，MgSO₄·7H₂O 4.14 g/L，CaCl₂ 0.3 g/L，NaCl 0.18 g/L，CuSO₄·5H₂O 0.0625 g/L，ZnSO₄·7H₂O 0.018 g/L，VB₁ 0.015g/L。

The LacB laccase from the hirsutella chromophoris is applied to degradation of environmental hormones.

The environmental hormones include: pregnenones: progesterone, 17 α hydroxyprogesterone acetate, medroxyprogesterone, melengestrol acetate, megestrol acetate; estrogens: diethylstilbestrol, dienestrol, beta-estradiol and bisphenol A; androgenic hormones: trenbolone, bodhitone, nandrolone, testosterone, methyltestosterone; glucocorticoids: dexamethasone, prednisolone, methylprednisolone, dexamethasone acetate, hydrocortisone, but not limited to the above environmental hormone classes.

The LacB laccase from the hirsutella chromophoris is applied to degradation of environmental hormones, and the reaction pH is 3-9; the reaction temperature is 15-55 ℃.

The LacB laccase from the trichoderma unicolor is applied to degradation of environmental hormone diethylstilbestrol, wherein the concentration of diethylstilbestrol is 10-40 mg/L, and the concentration of enzyme is 0.25-2.5U/mL.

The LacB laccase from the trichomonas chromophoris is applied to degradation of environmental hormones of estragole or dienestrol or beta-estradiol, wherein the concentration of the estragole or dienestrol or beta-estradiol is 1-40 mg/L, and the concentration of the enzyme is 0.5-2.5U/mL.

The LacB laccase from the hirsutella chricosa is applied to degradation of environmental hormone bisphenol A, wherein the concentration of the bisphenol A is 50-400 mg/L, and the enzyme concentration is 1-40U/mL.

Further, the LacB laccase from the trichomonas palustris is applied to degradation of environmental hormone diethylstilbestrol, and the reaction pH is 4-7; the reaction temperature is 15-55 ℃; the concentration of the diethylstilbestrol is 10-30 mg/L, the concentration of the enzyme is 0.25U/mL, or the concentration of the diethylstilbestrol is 30-40 mg/L, and the concentration of the enzyme is 2.0U/mL.

Further, the LacB laccase from the trichoderma brevicompactum is applied to the degradation of the environmental hormone hexanestrol, and the pH value of the LacB laccase is 5 or 6; the temperature is 25-55 ℃; the concentration of the estrene is 1-40 mg/L, and the concentration of the enzyme is 1.5U/mL.

Further, LacB laccase from trichomonas chromophila is applied to degradation of environmental hormone dienestrol, and the pH value of the LacB laccase is 5; the temperature is 15-55 ℃; the concentration of the dienestrol is 1-40 mg/L, and the concentration of the enzyme is 2.5U/mL.

Further, LacB laccase from trichomonas chromophila is applied to degradation of environmental hormone estradiol, and the pH value of the LacB laccase is 5; the temperature is 15-55 ℃; the concentration of the estradiol is 1-40 mg/L, and the concentration of the enzyme is 2.0U/mL.

Further, the LacB laccase from the trichoderma viride is applied to degradation of environmental hormone bisphenol A, and the pH value of the LacB laccase is 5; the temperature is 55 ℃; the concentration of estradiol is 50-400 mg/L, and the concentration of enzyme is 40U/mL.

The detailed technical scheme is as follows:

the invention relates to LacB laccase from Erysiphe chromophila, which is Erysiphe chromophila (C)Cerrna unicolor) 87613 strain, which is purchased from China forestry microorganism strain preservation management center and has the strain preservation number: CFCC 87613.

The invention relates to a fermentation production method of LacB laccase from Eremotrichum chrismens, which comprises the following steps: tricholoma chromophilum (A)Cerrna unicolor) 87613 inoculating the secondary seed culture of the strain to the fermentation medium at an inoculation amount of 8% (v/v), loading the fermentation medium at an amount of 60 mL/250 mL, and culturing at 30 deg.CFermenting and culturing at 200 rpm for 6 days, centrifuging, collecting fermentation liquid, separating and purifying. The enzyme yield is highest when the fermentation is carried out until the 6 th day, and the enzyme activity can reach 652.22U/mL.

The above-mentioned Erysiphe chromophoris (A), (B), (C)Cerrna unicolor) 87613 method for preparing secondary seed culture of strain:

firstly, the following steps are carried outCerrna unicolor87613 the strain was cultured on PDA plate for 4 days, 5 mycelia blocks (diameter 1 cm) of the newly grown area were inoculated into PDB seed medium (liquid volume 50 mL/100 mL), and cultured at 30 ℃ and 200 rpm for 2 days to obtain primary seed culture;

② transferring the primary seed culture into PDB culture medium (liquid loading volume 100 mL/250 mL) at a ratio of 8% (v/v), and culturing for another 2 days under the same conditions to obtain a secondary seed culture.

The separation and purification are as follows: purifying protein with AKTA equipment and anion exchange column, collecting eluate with enzyme activity, selecting high-enzyme-activity and high-purity component, and adding (NH)₄)₂SO₄Mixing uniformly to make its concentration be 1 mol/L, using AKTA system and hydrophobic chromatography column to continuously purify protein, collecting active enzyme component and storing in 4 deg.C refrigerator for later use. The purified laccase was deglycosylated using N-glycopeptidase F deglycosylation enzyme (Takara) and protein (12% acrylamide gel) was verified by SDS-PAGE. The validated samples were analyzed using a Q active Mass Spectrometer to identify the pure enzyme as LacB described above.

The present invention relates to the cloning of LacB laccase genes from C.chromogenes. Collecting mycelium of Tricholoma chromum cultured by fermentation till the 2 nd day, and extracting total RNA. And (3) synthesizing a first cDNA chain by using the total RNA of the strain as a template, and simultaneously carrying out reference-free transcriptome sequencing on the total RNA. Based on the transcriptome annotation analysis results, the cDNA sequence information of LacB was predicted. Based on the sequence information, LacB primers were designed including the complete open reading frame. Cloning cDNA of LacB laccase by using a first cDNA chain as a template and a designed LacB primer; the full-length gene sequence of LacB laccase is cloned by taking a chromous trichome genome DNA as a template and a designed LacB primer. The full-length sequence of the gene comprises a nucleotide sequence shown in SEQ ID NO. 1. The cDNA sequence comprises a nucleotide sequence shown in SEQ ID NO. 2. The DNA sequence in SEQ ID NO.1 consists of 2205 bases, comprises 11 introns, and has a cDNA sequence shown in SEQ ID NO.2 and consists of 1581 bases. The LacB laccase protein has an amino acid sequence shown as SEQ ID NO.3, and consists of 526 amino acids, and the 1 st-21 st amino acid residues from N' are signal peptide sequences; the mature enzyme is 505 amino acids.

The LacB laccase takes ABTS, guaiacol and catechol as substrates, the optimal reaction pH values are 3.5, 5.0 and 5.5 respectively, and the optimal reaction temperatures are 45, 40 and 50 ℃.

pH stability and temperature stability: mixing enzyme solution with buffer solutions (2.0-9.0) with different pH values, placing in a water bath kettle at 30 ℃ for heat preservation for 24 h and 48 h, taking ABTS as a substrate, and determining the residual enzyme activity under the optimal condition, wherein the laccase can keep 90% or more of enzyme activity in the range of pH5-9, and the enzyme activity is almost unchanged when the pH is more than 6, so that the laccase has better stability. And (2) placing the enzyme solution in a water bath kettle at 40-70 ℃ for heat preservation for 0-11 h, taking ABTS as a substrate, determining the residual enzyme activity under the optimal condition, and researching the heat stability of the laccase, wherein the laccase can be preserved for 11 h at 40 ℃ and 50 ℃, and the enzyme activity of more than 46% can be kept.

Influence of metal ions, organic solvents and inhibitors on enzyme activity: adding different metal ions, water-soluble organic solvents and enzyme inhibitors into a reaction system taking ABTS as a substrate, determining residual enzyme activity under the optimal reaction condition, and exploring the influence of the metal ions, the organic solvents and the inhibitors on the enzyme activity of the Lac B laccase by taking the enzyme activity without adding the reagents as a reference. 10 mM of Ba²⁺、Ca²⁺、Cr²⁺、Cu²⁺、K⁺、Li⁺、Na⁺Has little influence on the enzyme activity, Mg²⁺、Mn²⁺、Ni⁺And Zn²⁺The laccase has the effect of promoting the enzyme activity, when the concentration of methanol, ethanol, dimethyl sulfoxide, isopropanol, acetone and acetonitrile is 10%, the residual enzyme activity of the laccase is over 72%, the laccase has better tolerance to 1mM L-cys, 10 mM EDTA and 1mM Triton-X100, and the enzyme activity of over 75% can be kept.

The invention also relates to the application of LacB laccase in environmental hormone degradation.

Firstly, environmental hormone is treated by LacB laccase

A10 mg/L sample of environmental hormone, 10U/mL laccase, was treated at pH 6.0 (20 mM phosphate buffer), 25 ℃ for 48 h. The sample group without laccase treatment was set as blank, and the residual environmental hormone concentration was determined by high performance liquid chromatography, and each sample treatment was repeated 3 times.

The laccase has a good degradation effect on various environmental hormones, wherein the degradation effect on diethylstilbestrol, estragole, dienestrol, estradiol, bisphenol A, dexamethasone acetate and hydrocortisone is good, and the degradation rate is over 80%.

Analysis of conditions and degradation pathways affecting environmental hormone degradation

A single-factor experiment is adopted to investigate the influence of each reaction condition (including pH, temperature, enzyme concentration, substrate concentration and reaction time) on the degradation of diethylstilbestrol, dienestrol, estradiol and bisphenol A catalyzed by laccase, and a degradation product is analyzed by a high performance liquid chromatography quadrupole time of flight tandem mass spectrometer (LC-TOF MS) to conjecture a possible degradation path.

The invention has the advantages that:

(1) the invention provides a LacB laccase from Erysipelothrix. The enzyme is fermented in a fermentation medium for 6 days by a liquid shake flask, the enzyme activity can reach 652.22U/mL, the enzyme has excellent performances of high enzyme yield and short output time, and has remarkable advantages compared with the conditions that most fungal laccase enzymes have long ubiquitous periods, low enzyme yield and instability. The method has the advantages of high enzyme yield and short enzyme production period, can obviously reduce the production cost of the laccase, and provides a laccase enzyme source with high cost performance for the industrial application of the laccase.

(2) The invention also provides a fermentation medium of LacB laccase. And (3) screening out a fermentation culture medium most suitable for LacB laccase production by optimizing the types of the carbon source and the nitrogen source and the optimal addition amount of the carbon source and the nitrogen source.

(3) The LacB laccase of the invention can efficiently degrade various environmental hormones, has good degradation effect on diethylstilbestrol, dienestrol, estradiol, bisphenol A and prednisolone, and has the degradation rate of more than 98%. The LacB laccase has obvious value in the aspect of environmental hormone treatment application, provides a new way for environmental pollutant treatment, and has good economic and social benefits.

Drawings

FIG. 1 shows the enzyme production curve of Trichinella 87613 under different carbon source conditions. A: a carbon source species; b: the concentration of the carbon source.

FIG. 2 shows the enzyme production curve of Tricholoma trichotomum 87613 under different nitrogen source conditions. A: a nitrogen source species; b: the concentration of the nitrogen source.

FIG. 3 SDS-PAGE electrophoresis of LacB laccase purification results. M: protein marker; 1: fermenting the total protein of the crude enzyme liquid; 2: purifying by using a DEAE column; 3: lac B; 4: deglycosylated Lac B.

FIG. 4 enzymatic properties of LacB laccase. A: the optimum pH value; b: the optimum temperature; c: pH stability; d: and (4) temperature stability.

FIG. 5 Effect of metal ions on LacB laccase viability.

FIG. 6 effect of organic solvents on LacB laccase viability.

FIG. 7 Effect of inhibitors and surfactants on LacB laccase viability.

FIG. 8 degradation of 22 environmental hormones by laccase.

FIG. 9 affects conditions under which laccase degrades estrogen. A: pH, B: and (3) temperature.

FIG. 10 effect of enzyme concentration on laccase treatment of estrogens. A: diethylstilbestrol; b: hexylestrol; c: dienestrol; d: beta-estradiol; e: bisphenol A.

FIG. 11 is a possible way of degrading diethylstilbestrol with laccase.

Indicating the positions where the monomers may be linked, and C-C and C-O indicating that the monomers are linked by linking bonds that are C-C and C-O bonds.

FIG. 12 possible pathways for laccase degradation of hexestrol.

Indicating the location where attachment of the monomers is likely to occur.

FIG. 13 potential pathways for laccase degradation of beta-estradiol.

Indicating the location where attachment of the monomers is likely to occur.

FIG. 14 is a possible pathway for laccase degradation of bisphenol A.

Indicating the location where attachment of the monomers is likely to occur.

Detailed Description

The present invention will be fully described below with reference to specific examples, but the present invention is not limited thereto.

1. And (3) measuring the enzyme activity of the laccase: the reaction system is 2.0 mL and 1, ABTS is used as a substrate, 0.975 mL of 0.1 mol/L acetic acid-sodium acetate buffer solution (pH 3.0), 1 mL of 0.5 mmol/L ABTS and 25 mu L of appropriately diluted enzyme solution are contained, constant temperature water bath reaction is carried out at 45 ℃, and the absorbance change value of the reaction solution at the wavelength of 420 nm 5 min before the reaction is measured. Inactivated enzyme solution was used as a blank control. The enzyme activity is defined as: the enzyme amount required by catalytic oxidation of 1 mu mol of ABTS per minute is 1 enzyme activity unit (U), and the ABTS oxidation state molar absorption coefficient is 36000 L.mol^-1·cm^-1。

2. Guaiacol (Guaiacol) is used as a substrate, and 1.86 mL of 0.05 mol/L citric acid-phosphate buffer solution (pH 4.5), 100 mu L of 20 mmol/L Guaiacol solution and 40 mu L of appropriately diluted enzyme solution are contained in the substrate. Reacting in a constant-temperature water bath at 40 ℃, and measuring the absorbance change value of the reaction solution at the wavelength of 465 nm 5 min before the reaction. The inactivated enzyme solution was used as a blank. Enzyme activity is defined as: per minute catalysis 1 muThe enzyme amount required for the oxidation of the guaiacol is 1 enzyme activity unit (U), and the molar absorptivity of the guaiacol in the oxidation state is 12100 L.mol^-1·cm^-1。

3. Catechol (Catechol) was used as a substrate, and contained 1.86 mL of 0.05 mol/L citric acid-phosphate buffer (pH 4.5), 100. mu.L of 20 mmol/L guaiacol solution, and 40. mu.L of appropriately diluted enzyme solution. Reacting in a constant-temperature water bath at 40 ℃, and measuring the absorbance change value of the reaction solution at the wavelength of 400 nm 5 min before the reaction. The inactivated enzyme solution was used as a blank. Enzyme activity is defined as: the enzyme amount required for catalyzing the oxidation of 1 mu mol of guaiacol per minute is 1 enzyme activity unit (U), and the absorption coefficient of the oxidation state of catechol is 1260L & mol^-1·cm^-1。

Enzyme activity (U/mL) = NxDeltaOD xL 0³/（ξ×t）

Wherein N is the dilution multiple of the enzyme solution; Δ OD represents the value of the change in absorbance detected at the corresponding wavelength; ξ represents the substrate oxidation state molar absorption coefficient; t represents a reaction time (min).

Example 1: fermentative production of laccase

Fermentation production of LacB laccase from Erysipelothrix chromophoris: tricholoma chromophilum (A)Cerrna unicolor) 87613 the secondary seed culture of the strain is inoculated to the fermentation medium according to the inoculation amount of 8% (v/v), the liquid loading amount is 60 mL/250 mL, and the fermentation culture is carried out at 30 ℃ and 200 rpm.

Tricholoma chromophilum (A)Cerrna unicolor) 87613 method for preparing secondary seed culture of strain:

Fermentation medium: carbon source: respectively using glycerin,Glucose, lactose, sucrose, cellulose, maltodextrin, beta-dextrin and corn starch are used as carbon sources, and the addition amount of the carbon sources is 6% (w/v); nitrogen source: respectively taking ammonium nitrate, ammonium sulfate, ammonium tartrate, peptone, yeast powder and beef extract as nitrogen sources, wherein the addition amount is 1.5% (w/v); the rest components are as follows: KH (Perkin Elmer)₂PO₄ 6 g/L，MgSO₄·7H₂O 4.14 g/L，CaCl₂ 0.3 g/L，NaCl 0.18 g/L，CuSO₄·5H₂O 0.0625 g/L，ZnSO₄·7H₂O 0.018 g/L，VB₁0.015g/L。

Optimizing and screening the optimal carbon and nitrogen source types of the fermentation medium; and further optimizing the optimal addition amount of the carbon and nitrogen source on the basis.

When cane sugar is used as a carbon source, the enzyme production effect is good, the enzyme activity is high, the laccase yield is highest, the highest enzyme activity can be achieved in the sixth day of fermentation, 438.22U/mL, and the laccase yield is 73.04U/mL. d^-1(FIG. 1A). When the addition amount of the sucrose is 9 percent (w/v), the enzyme production effect is optimal and is 1.4 times of the original 6 percent (w/v) sucrose concentration, 569.78U/mL can be achieved at the 6 th day, the laccase yield is 94.96U/mL. d^-1Higher concentrations of carbon source favoured the fermentative enzyme production of this trichinosis strain (FIG. 1B). When peptone is used as a nitrogen source, the enzyme production effect is best, the laccase yield is highest, then beef extract and yeast powder are used, and generally, the strain has better laccase production capacity under the condition of a complex organic nitrogen source (figure 2A). When the concentration of peptone is 1.0%, the laccase output efficiency is highest, 108.7U/mL. d^-1On the sixth day, the enzyme production reached 652.22U/mL (FIG. 2B).

Therefore, the optimal fermentation medium for producing the LacB laccase from the Eremotrichum chromum by fermentation comprises the following components: sucrose 90 g/L, peptone 10 g/L, KH₂PO₄ 6 g/L，MgSO₄·7H₂O 4.14 g/L，CaCl₂ 0.3 g/L，NaCl 0.18 g/L，CuSO₄·5H₂O 0.0625 g/L，ZnSO₄·7H₂O 0.018 g/L，VB₁ 0.015g/L。

Example 2: acquisition of laccase Gene sequences

Extraction of total RNA: the mycelia of the trichoderma chromophilum 87613 enzyme-producing culture medium fermented to the 2 nd day are collected by suction filtration, liquid nitrogen is immediately added for grinding, and the total RNA is extracted by utilizing the operation steps of an RNeasy Plant Mini Kit. The extracted RNA was checked for RNA purity by 2% agarose gel electrophoresis and by measuring the OD260/280 ratio with NanoDrop 2000.

② transcriptome library construction and sequencing

After the total RNA sample is qualified, the reference-free transcriptome sequencing is carried out by Beijing Nuo He provenance company. mRNA was isolated by A-T base pairing using oligo (dT) -bearing magnetic beads and pIyA of mRNA. Fragmentation buffer was added to fragment the entire mRNA fragment randomly to form small fragments of about 200 bp. Under the action of reverse transcriptase, a stable double-stranded structure is formed by reverse transcription of random primers. After double-stranded cDNA is purified, end repairing is carried out, A tail is added and a sequencing joint is connected, and the size of the fragment is selected through AMPure XP beads. And performing PCR amplification to ensure that the size of most fragments is 150-200 bp, and purifying a PCR product by using an Ampure XP system to obtain a final library. Then, library detection was performed, the library was diluted to 1.5 ng/uL after quantification, and then the insert size of the library was detected using Agilent 2100. insert size was expected and the effective concentration of the library was accurately quantified using the qPCR method. And if the effective concentration of the library is more than 2 nM, the library is qualified. And (3) carrying out Illumina HiSeq sequencing on different libraries after posing according to the effective concentration and the machine data volume requirement under the target, and obtaining comprehensive gene function information by combining database analysis.

Cloning of LacB laccase Gene

And randomly breaking the complete mRNA fragments, performing computer sequencing, and splicing clean reads obtained after the sequencing is finished and quality control into different clusters. The length of splicing is 200-40000 bp. Different clusters may be annotated as the same gene, but the positions of the aligned genes may be different, and there may be overlapping portions. The results of the assembly (clusters) were therefore analyzed for classification on the principle of annotation as the same laccase gene.

Based on the analysis of the transcriptome annotation results for the classification, LacB cDNA sequence information was predicted. According to sequence information, primers including a complete development reading frame are designed, and the sequences of the primers are as follows:

LacB-F :5’-ATGGGATTGAACTCGGCTATT-3’；

LacB-R:5’-TTAAATCGCAGTTCCTTTCTTA-3’。

using the extracted total RNA as a template, first strand cDNA was synthesized using TransScript One-Step Removal and cDNA Synthesis SuperMix (TransGen Biotech Co.), and primers (LacB-F, LacB-R) were synthesized using the first strand cDNA as a template for PCR amplification.

PCR reaction (50. mu.L): 2 × EasyTaq PCR superMix 25 μ L, 75 ng/. mu.L template 1 μ L, 10 μmol/L forward and reverse primers 1 μ L, ddH₂O22. mu.L. PCR procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 2 min, and cyclic amplification for 35 times; finally, extension is carried out for 10 min at 72 ℃. After the electrophoretic detection of the PCR product, the target gene was recovered with Gel Extraction kit (OMEGA) and cloned into PMD18-T vector for sequencing analysis (Invitrogen) to obtain LacB cDNA sequence (SEQ ID NO. 2).

The PCR program is that genome DNA is taken as a template, LacB-F/LacB-R is taken as a primer, and the PCR program is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 10 min, and cyclic amplification for 35 times; finally, extension is carried out for 10 min at 72 ℃. And recovering the target gene in the gel after the electrophoresis detection of the PCR product, cloning the target gene to a PMD18-T vector, and sequencing to obtain the gene full-length sequence (SEQ ID NO. 1) of LacB.

Based on the cDNA sequence, the LacB protein is predicted to be composed of 526 amino acid residues (SEQ ID NO.3), and the 1 st to 21 st amino acid residues from the N end are a signal peptide sequence; the mature enzyme protein comprises 505 amino acid residues. The sequence of LacB coding protein is compared with that of known laccase protein by Blast software, and the comparison result is knownCerrenaLac2 (AID 59410.1) in sp, HYB07 was the highest in similarity, 80%.

Example 3: LacB laccase protein purification

Protein concentration determination: protein concentration was determined using BCA kit (Transgen). And drawing a standard curve according to the instruction method, and calculating the protein concentration of the sample to be detected according to the standard curve.

Purifying protein by using AKTA equipment and an anion exchange column, wherein liquid A is 50 mmol/L Tris-HCl buffer solution with the pH value of 8.5, liquid B is 50 mmol/L Tris-HCl buffer solution with the pH value of 8.5 and containing 1 mol/L NaCl, the anion exchange column is balanced by the liquid A and then is loaded, after the loading is finished, an elution program sets the liquid B to be 10 percent, the flow rate is 1 mL/min, eluent with enzyme activity is collected, and high-enzyme-activity components are stored in a refrigerator at the temperature of 4 ℃ for later use, after the purification is finished, the purification multiple is 3.45, the recovery rate is 54.08 percent, and the protein molecular weight is concentrated in 50.0-100.0 kD (lane 2 of figure 3).

② selecting components with higher enzyme activity and higher protein purity, adding NH₄₎₂SO₄The final concentration was 1 mol/L, and the mixture was mixed well. Further purification using AKTA System and hydrophobic chromatography column, 1 mol/L (NH)₄)₂SO₄After the solution is balanced on the column, the sample is loaded, the salt concentration is reduced, the protein is eluted, the enzyme active component is collected and stored in a refrigerator at 4 ℃ for standby, after the step is completed, the purification multiple is 8.45, the recovery rate is 14.49%, and the molecular weight of the protein is concentrated on 70.0 kD (figure 3, lane 3).

③ deglycosylation of the purified laccase using N-glycopeptidase F deglycosylation enzyme (Takara), and protein detection by SDS-PAGE (12% acrylamide gel). Under the action of deglycosylation enzyme, single laccase with molecular weight of about 60 kD is obtained (figure 3, lane 4), and the glycosylation degree is 16.7%. Identified as LacB laccase protein using Q active Mass Spectromete.

Example 4: LacB laccase enzymatic Properties

The enzyme activity of different substrates is determined as described above

Optimum temperature and pH: respectively at 20-70 deg.C and pH2.5-6.5 (citric acid-Na)₂HPO₄Buffer solution), and the influence of the temperature and the pH on the laccase activity of the trichoderma viride 87613 LacB is shown in FIGS. 4A and 4B. When the substrates are ABTS, catechol and guaiacol, the optimal reaction pH of LacB is 3.5, 5.0 and 5.5 respectively, and the optimal reaction temperature is 45, 40 and 50 ℃.

② temperature and pH stability: mixing LacB enzyme solution with citric acid-Na with pH of 2.5-8.7₂HPO₄Mixing the buffer solution 1:10 (v: v), placing in a water bath kettle at 30 ℃ for heat preservation for 24 and 48 hours respectively, measuring the residual enzyme activity by taking ABTS as a substrate, and exploring the pH stability of the laccase; the general trend of pH stability is shown in FIG. 4C, the pH stability is stable under the condition that the pH is more than 5, the temperature is kept for 48 hours, more than 90 percent of residual enzyme activity can be still kept, and the pH tolerance is strong. The enzyme activity change of LacB laccase is shown in figure 4D after heat preservation for 0-11 hours at the temperature of 40 ℃, 50 ℃, 60 ℃ and 70 ℃, LacB has better stability when heat preservation is carried out below 60 ℃, the heat preservation is carried out for 11 hours at the temperature of 40 ℃ and 50 ℃, and the residual enzyme activity of the enzyme is more than 46%.

③ Metal ions: using ABTS as substrate, respectively adding 15 kinds of metal ions (Ag) in reaction system⁺、 Ba²⁺、Ca² ⁺、Co²⁺、Cr²⁺、Cu²⁺、Fe²⁺、Hg²⁺、K⁺、Li⁺、Mg²⁺、Mn²⁺、Na⁺、Ni²⁺、Zn²⁺) The final concentration was made to be 10 mM, the enzyme activity was tested under the optimum reaction conditions, the effect of the metal ions on the LacB laccase activity was tested by calculating the relative enzyme activity with the group enzyme activity without metal ions as 100%, and the results are shown in FIG. 5, in the tested metal ions, LacB laccase on Ca²⁺、K⁺、Cr²⁺、Cu²⁺And Na⁺Has better tolerance, relatively weaker inhibition effect of the metal ions on LacB laccase and Mg²⁺、Mn²⁺、Ni⁺And Zn²⁺The addition of (A) has the effect of promoting the activity of LacB laccase, and Ag⁺、Fe²⁺And Hg²⁺On LacB laccase activityThe force has a strong inhibitory effect, followed by Co²⁺The relative enzyme activity is retained at 47%.

Organic solvent: ABTS is taken as a substrate, 6 water-soluble organic solvents (methanol, ethanol, acetone, isopropanol, acetonitrile and dimethyl sulfoxide) are respectively added into an enzyme activity test system to enable the final concentrations to be 10% and 25%, the enzyme activity is tested under the optimal reaction condition, the relative enzyme activity is calculated by taking the enzyme activity of a group without the organic solvent as 100%, the influence of the organic solvent on the enzyme activity of LacB is researched, the result is shown in figure 6, when the concentration of the organic solvent is 10%, the residual enzyme activity is more than 72%, and the dimethyl sulfoxide with the concentration of 10% has no obvious inhibition effect on the enzyme activity of LacB. When the concentration of the organic solvents is 25%, the relative enzyme activity of LacB is 16% -46%, and the inhibition effect of acetone on the laccase activity is strong.

Inhibitor: ABTS is taken as a substrate, and different kinds of inhibitors of 0.1 mM and 1mM NaN are added into an enzyme activity test system₃L-Cys, 1mM and 10 mM EDTA, 20 mM, 50 mM and 100 mM NaCl, 0.05 mM and 0.2 mM SDS, and 0.5 mM and 1mM Triton X-10, wherein the relative enzyme activity is calculated by taking the activity of the group enzyme without the inhibitor as 100%, and the influence of the inhibitor on the LacB enzyme activity is researched. The results are shown in FIG. 7, the tolerance of LacB laccase to Triton-X100 and L-cys added in the experiment is high, the relative enzyme activity of laccase is over 75%, and the inhibition effect of NaCl and SDS to LacB is obvious.

Example 5: treatment of environmental hormones with LacB laccase

And (3) degradation reaction: a10 mg/L sample of environmental hormone, 10U/mL laccase, was treated at pH 6.0 (20 mM phosphate buffer), 25 ℃ for 48 h. Samples without laccase treatment were set as blanks, and the residual environmental hormone concentration was determined by high performance liquid chromatography, with each sample treatment repeated 3 times.

The environmental hormones used included: pregnenones: progesterone, 17 α hydroxyprogesterone acetate, medroxyprogesterone, melengestrol acetate, megestrol acetate; estrogens: diethylstilbestrol, dienestrol, beta-estradiol and bisphenol A; androgenic hormones: trenbolone, bodhitone, nandrolone, testosterone, methyltestosterone; glucocorticoids: dexamethasone, prednisolone, methylprednisolone, dexamethasone acetate, hydrocortisone.

Detection conditions of high performance liquid chromatography: bisphenol A: the sample introduction amount is 10 mu L, the reversed phase C18 column (4.6 mm multiplied by 250 mm multiplied by 5 mu m), the column temperature is 30 ℃, the mobile phase is acetonitrile: water =60:40 (v/v), the flow rate is 0.8 mL/min, and the detection wavelength is 226 nm; other hormone detection conditions were as follows: the sample size was 10. mu.L, the reverse phase C18 column (4.6 mm. times.250 mm. times.3 μm), the column temperature was 30 ℃, the mobile phase was acetonitrile/water =80:20 (v/v), the flow rate was 1 mL/min, and the detection wavelength range was 200-400 nm. Extracting a chromatogram of the maximum absorption peak, and determining the hormone content in the sample by the peak-appearing time and the peak area.

The result is shown in fig. 8, the laccase has different degradation effects on different environmental hormones, has better degradation effects on diethylstilbestrol, hexestrol, dienestrol, beta-estradiol, bisphenol A and prednisolone, has a degradation rate of more than 98%, and has a degradation rate of 2.3-79.8% on the residual hormones except dexamethasone, hydrocortisone and methylprednisolone.

Example 6: influencing conditions of 5 estrogens treated with LacB laccase

Laccase is utilized to further treat 5 estrogens with better degradation effect in example 5: diethylstilbestrol, hexestrol, dienestrol and bisphenol A, and optimizing degradation conditions.

Influence of pH on the treatment Effect: 10 mg/L estrogen sample was reacted with 1U/mL laccase at 25 deg.C, pH3-9 (20 mM phosphate buffer) for 30 min, and the residual hormone content was determined by high performance liquid chromatography, the detection conditions were the same as those described in example 5, and each set of reactions was performed in triplicate. As shown in FIG. 9A, the degradation rate of 5 hormones was 87% or more at pH 5.

Influence of temperature on the treatment effect: 10 mg/L estrogen sample was reacted with 1U/mL laccase at pH6 (20 mM phosphate buffer) at 15-55 deg.C for 30 min. The results are shown in fig. 9B, the effect of temperature on estrogen treatment with laccase is relatively small in comparison with pH, and besides hexanestrol and bisphenol a, the degradation rate of laccase on other 3 estrogens is above 90% in the measured temperature range; when the reaction temperature is above 20 ℃, the degradation rate of 5 estrogens is above 81%.

Effect of enzyme concentration on treatment effect: the concentration of the diethylstilbestrol is 10, 20, 30 and 40mg/L, the concentration of the enzyme is 0.25, 0.5, 1, 1.5, 2 and 2.5U/mL; the concentrations of the estrene, the dienestrol and the beta-estradiol are 1, 5, 10, 20, 30 and 40mg/L, and the enzyme concentration is 0.5, 1, 1.5, 2 and 2.5U/mL; bisphenol A concentrations of 50, 100, 200 and 400 mg/L, enzyme concentrations of 1, 10, 20, 30, 40U/mL, at pH6 (20 mM phosphate buffer), at 25 ℃ for 30 min. The results are shown in FIGS. 10A, 10B, 10C, 10D, and 10E. For the same hormone sample, under the condition of constant enzyme concentration, the degradation efficiency is reduced along with the increase of the hormone concentration, the sample concentration is constant, and the degradation rate of the laccase on the sample is increased along with the increase of the enzyme concentration.

The laccase has good treatment effect on the diethylstilbestrol within the pH range of 3-7, and the degradation rate can reach more than 95% within the pH range of 4-7 (figure 9A); the degradation rate of the laccase to the diethylstilbestrol can be more than 90% within the range of the measured temperature of 15-55 ℃ (figure 9B); the diethylstilbestrol concentration is 10-30 mg/L, when the enzyme concentration is 0.25U/mL, the degradation rate can reach more than 91%, and the diethylstilbestrol can be almost completely degraded within 30 min along with the increase of the enzyme concentration. The concentration of the diethylstilbestrol is 30-40 mg/L, the concentration of the enzyme is increased to 2.0U/mL, and the degradation rate can reach 98% (figure 10A).

Under the conditions of pH5 and pH6, the laccase has good degradation effect on the estriol, and the degradation rate is over 80 percent (figure 9A); the removal rate of the laccase to the hexanestrol can be more than 80% when the temperature is between 25 and 55 ℃ (figure 9B); when the concentration of the estrene is 1-40 mg/L and the concentration of the enzyme is more than 1.5U/mL, the degradation rate is more than 77%, and the estrene can be almost completely degraded within 30 min along with the increase of the concentration of the enzyme (10B).

Under the conditions of pH5 and pH6, the laccase can almost completely degrade the dienestrol, and the degradation rate is over 97 percent (figure 9A); the degradation effect of laccase on dienestrol is not greatly influenced, and the removal rate of laccase on dienestrol is over 95% when the temperature is between 15 and 55 ℃ (figure 9B); when the enzyme concentration is less than 2.0U/mL, the degradation rate is reduced along with the increase of the concentration of the dienestrol substrate. When the concentration of the enzyme is 2.5U/mL, the degradation rate of the laccase to 1-40 mg/L of dienestrol reaches more than 91% (10C).

When the pH is 5, the laccase has a good degradation effect on the beta-estradiol, the degradation rate reaches 95.5%, and under the condition of the pH of 4-6, the degradation rate is 67.5% -85.3% (fig. 9A); the temperature has little influence on the degradation effect of the laccase on the beta-estradiol, and when the temperature is between 15 and 55 ℃, the removal rate of the laccase on the dienestrol is over 90 percent (figure 9B); the degradation rate of the laccase concentration of 0.25U/mL to 1-40 mg/L beta-estradiol is between 30-60%, the degradation efficiency of the laccase to the estradiol is improved along with the increase of the enzyme concentration, and the degradation rates are both 90% and above (10D) when the enzyme concentration is 2.0U/mL.

The pH has little influence on the degradation effect of the bisphenol A, the degradation rate is kept above 70 percent in the range of 3-9, and the degradation rate can reach 92 percent when the pH is 5 (figure 9A); the degradation rate of laccase to bisphenol A increases with increasing temperature in the range of 15-55 ℃, with a maximum degradation rate of 96% at 55 ℃ (FIG. 9B); the degradation effect of bisphenol A is enhanced with the increase of enzyme concentration, and when the enzyme concentration is increased to 40U/mL, the degradation rate of 50-400 mg/L bisphenol A is all above 88% (10E).

Example 7: degradation product detection

The 5 estrogen products of laccase degradation example 6 were analyzed by high performance liquid chromatography quadrupole time of flight tandem mass spectrometry (LC-TOF MS): liquid phase conditions: the sample size is 2 mu L, and other liquid phase conditions are the same as those in example 6; time-of-flight mass spectrometry conditions: ionization mode: ESI-; high resolution full scan mode: m/z is 50-1500; atomizing: nitrogen gas; flow rate of atomizing gas: 11L/min; spraying pressure: 50 psi; temperature of atomized gas: 360 ℃; capillary voltage: 3500V; taper hole voltage: 130V. When data are collected, the data are corrected in real time by using a mass reference solution two-point correction mode, and the mass-to-charge ratio (m/z) of the reference solution is 121.0509 and 922.0098. The degradation pathway of diethylstilbestrol (DES, 267.1423) by laccase is presumed to be shown in fig. 11, and 9 products with mass-to-charge ratios of 265.1256, 533.2717, 531.2577, 529.2401, 283.1366, 315.1260, 311.0945, 295.1000 and 297.1159 were identified. Dienestrol (m/z 265.1237) is structurally similar to hexestrol, presumably having the same pathway as diethylstilbestrol (pathway i, pathway ii), identifying three products with mass-to-charge ratios 529.2375, 295.0966 and 297.1124, respectively. A possible route for laccase degradation of estragol (m/z 269.1553) is shown in FIG. 12, where a product m/z 537.3010 was detected. The possible routes for degradation of beta-estradiol (m/z 271.1693) by laccases are shown in fig. 13, identifying 3 possible products with mass-to-charge ratios of 287.1640, 295.0629 and 541.3296, respectively. A possible pathway for degradation of bisphenol A (m/z 227.1084) by laccase is shown in FIG. 14, and a total of three products were analyzed, with mass-to-charge ratios of 453.2072, 679.3072 and 545.2371, respectively. From the results and the presumed degradation pathway, it can be seen that the degradation products of estrogen by laccase may be mainly oligomers.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> Fuzhou university

<120> laccase from Erysiphe versicolor, gene and application thereof

<130> 5

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 2205

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gctgcaattg ggcccgtctc cgacctgcac attgtcaaca aagaccttgc tccagatggc 120

gtacaacgtc caaccgtgct tgccggaggc acttttcctg ggacgttgat caccggtcag 180

aaagtaagga atattagttt gcgtcaaaga gccaaccaaa actaaccgcc ccgtcccata 240

gggtgacaac ttccagctca atgtcatcga tgatcttacc gacgatcgga tgttgacacc 300

aacttccatt gtgagcctat tattgtatga tttatccgaa tagtttcgca gtctgatcat 360

tggatctcta tcgctagcat tggcatggtt tcttccagaa gggaaccgct tgggccgacg 420

gtcccgcctt cgtaactcag tgccctatca tagcagataa ctccttcctg tatgacttcg 480

acgtccctga ccaggctggt actttctggt accatagtca tttatccacg cagtactgtg 540

acggtttacg tggcgccttc gttgtgtacg atcctaacga ccctcacaaa gacctgtacg 600

atgttgatga cggtgggttc caaatatttg ttctgcaaac attgtattga cggtgttcat 660

tctaatttta gagagcaccg tgattaccct tgcggattgg taccatgttc tcgcccagac 720

cgttgtcggc gctgcgtgag tagcacatac acacgctccg atacacggat actaattgtc 780

gtattgtagc actcctgatt ctaccttgat caatgggtta ggccgttcac agaccggacc 840

tgctgatgct gagctggctg tcatcagcgt tgaacataac aaacggtatg tcatctctac 900

ccgttatcct ctgtcctgct ctaattcact gtttcgtcat agataccgat tccgtttggt 960

ttcgatttcg tgcgacccca actttacctt ctccattgat ggtcataata tgactgtcat 1020

cgaagtcgac ggtgtcaaca cacaacctct gaccgtcgac tctattcaaa tcttcgccgg 1080

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tgatgtcaac agctcaatgc taaccaaccc gacgacaatt actggatccg tgctatgcca 1200

aacatcggta gaaatacaac aacactggac ggaaagaatg ccgctatcct tcgatacaag 1260

aatgcttctg tagaagagcc caagaccgtt gggggccccg ctcaatcccc attgaacgaa 1320

gcggatctgc gtccactcgt acctgctcct gtggtaggtc ttgtcccgct gttccatcgc 1380

tatttcatat taacgtttta tttttgtcaa gcctggaaac gctgttccag gtggcgcaga 1440

catcaatcac aggcttaact taactttcgt acgtacacct ggttgaaaca ttatatttcc 1500

agtctaacct ctcttgtaga gtaacggccg tttcagcatc aacaacgcct ccttcactaa 1560

tccttcggtc cccgccttat tgcaaattct gagcggcgct caaaacgctc aagatttact 1620

tccaacgggt agttacattg gccttgaact gggcaaggtc gtggagctcg ttatacctcc 1680

tctggcagtc ggaggacctc accctttcca tcttcatggc gtaagcatac cacacccctg 1740

cggccaggat gacgcagact aatcatgatt cacagcacaa tttctgggtc gtccgtagtg 1800

caggtagcga caagtataac ttcgacgatg ctatcctcag ggacgtcgta agcattggag 1860

cagggactga tgaagtcaca atccgtttcg tggtatgtct catccctcgc atttgtagac 1920

gcaagagctg atataattcc acatagacgg acaatccggg cccgtggttc ctccattgcc 1980

atattgattg gcatttggag gcaggccttg ccatcgtctt cgctgagggc atcaatcaga 2040

ccgctgcagc caaccctaca ccccgtacgt gacactgagg gtttctttat agcgctggat 2100

tactgaatcg agatttctcc agaggcatgg gatgagcttt gtcccaaata caacgggttg 2160

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gtacaacgtc caaccgtgct tgccggaggc acttttcctg ggacgttgat caccggtcag 180

aaaggtgaca acttccagct caatgtcatc gatgatctta ccgacgatcg gatgttgaca 240

ccaacttcca ttcattggca tggtttcttc cagaagggaa ccgcttgggc cgacggtccc 300

gccttcgtaa ctcagtgccc tatcatagca gataactcct tcctgtatga cttcgacgtc 360

cctgaccagg ctggtacttt ctggtaccat agtcatttat ccacgcagta ctgtgacggt 420

ttacgtggcg ccttcgttgt gtacgatcct aacgaccctc acaaagacct gtacgatgtt 480

gatgacgaga gcaccgtgat tacccttgcg gattggtacc atgttctcgc ccagaccgtt 540

gtcggcgctg ccactcctga ttctaccttg atcaatgggt taggccgttc acagaccgga 600

cctgctgatg ctgagctggc tgtcatcagc gttgaacata acaaacgata ccgattccgt 660

ttggtttcga tttcgtgcga ccccaacttt accttctcca ttgatggtca taatatgact 720

gtcatcgaag tcgacggtgt caacacacaa cctctgaccg tcgactctat tcaaatcttc 780

gccggacaga ggtattcctt tgtcctcaat gctaaccaac ccgacgacaa ttactggatc 840

cgtgctatgc caaacatcgg tagaaataca acaacactgg acggaaagaa tgccgctatc 900

cttcgataca agaatgcttc tgtagaagag cccaagaccg ttgggggccc cgctcaatcc 960

ccattgaacg aagcggatct gcgtccactc gtacctgctc ctgtgcctgg aaacgctgtt 1020

ccaggtggcg cagacatcaa tcacaggctt aacttaactt tcagtaacgg ccgtttcagc 1080

atcaacaacg cctccttcac taatccttcg gtccccgcct tattgcaaat tctgagcggc 1140

gctcaaaacg ctcaagattt acttccaacg ggtagttaca ttggccttga actgggcaag 1200

gtcgtggagc tcgttatacc tcctctggca gtcggaggac ctcacccttt ccatcttcat 1260

ggccacaatt tctgggtcgt ccgtagtgca ggtagcgaca agtataactt cgacgatgct 1320

atcctcaggg acgtcgtaag cattggagca gggactgatg aagtcacaat ccgtttcgtg 1380

acggacaatc cgggcccgtg gttcctccat tgccatattg attggcattt ggaggcaggc 1440

cttgccatcg tcttcgctga gggcatcaat cagaccgctg cagccaaccc tacaccccag 1500

gcatgggatg agctttgtcc caaatacaac gggttgagtg cgagccagaa ggtcaagcct 1560

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<210> 3

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<213> SEQ ID NO.3

<400> 3

Met Gly Leu Asn Ser Ala Ile Pro Ser Leu Ala Ile Leu Ala Leu Ser

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Val Gly Ser Tyr Ala Ala Ile Gly Pro Val Ser Asp Leu His Ile Val

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Asn Lys Asp Leu Ala Pro Asp Gly Val Gln Arg Pro Thr Val Leu Ala

35 40 45

Gly Gly Thr Phe Pro Gly Thr Leu Ile Thr Gly Gln Lys Gly Asp Asn

50 55 60

Phe Gln Leu Asn Val Ile Asp Asp Leu Thr Asp Asp Arg Met Leu Thr

65 70 75 80

Pro Thr Ser Ile His Trp His Gly Phe Phe Gln Lys Gly Thr Ala Trp

85 90 95

Ala Asp Gly Pro Ala Phe Val Thr Gln Cys Pro Ile Ile Ala Asp Asn

100 105 110

Ser Phe Leu Tyr Asp Phe Asp Val Pro Asp Gln Ala Gly Thr Phe Trp

115 120 125

Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Ala

130 135 140

Phe Val Val Tyr Asp Pro Asn Asp Pro His Lys Asp Leu Tyr Asp Val

145 150 155 160

Asp Asp Glu Ser Thr Val Ile Thr Leu Ala Asp Trp Tyr His Val Leu

165 170 175

Ala Gln Thr Val Val Gly Ala Ala Thr Pro Asp Ser Thr Leu Ile Asn

180 185 190

Gly Leu Gly Arg Ser Gln Thr Gly Pro Ala Asp Ala Glu Leu Ala Val

195 200 205

Ile Ser Val Glu His Asn Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile

210 215 220

Ser Cys Asp Pro Asn Phe Thr Phe Ser Ile Asp Gly His Asn Met Thr

225 230 235 240

Val Ile Glu Val Asp Gly Val Asn Thr Gln Pro Leu Thr Val Asp Ser

245 250 255

Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Val Leu Asn Ala Asn

260 265 270

Gln Pro Asp Asp Asn Tyr Trp Ile Arg Ala Met Pro Asn Ile Gly Arg

275 280 285

Asn Thr Thr Thr Leu Asp Gly Lys Asn Ala Ala Ile Leu Arg Tyr Lys

290 295 300

Asn Ala Ser Val Glu Glu Pro Lys Thr Val Gly Gly Pro Ala Gln Ser

305 310 315 320

Pro Leu Asn Glu Ala Asp Leu Arg Pro Leu Val Pro Ala Pro Val Pro

325 330 335

Gly Asn Ala Val Pro Gly Gly Ala Asp Ile Asn His Arg Leu Asn Leu

340 345 350

Thr Phe Ser Asn Gly Arg Phe Ser Ile Asn Asn Ala Ser Phe Thr Asn

355 360 365

Pro Ser Val Pro Ala Leu Leu Gln Ile Leu Ser Gly Ala Gln Asn Ala

370 375 380

Gln Asp Leu Leu Pro Thr Gly Ser Tyr Ile Gly Leu Glu Leu Gly Lys

385 390 395 400

Val Val Glu Leu Val Ile Pro Pro Leu Ala Val Gly Gly Pro His Pro

405 410 415

Phe His Leu His Gly His Asn Phe Trp Val Val Arg Ser Ala Gly Ser

420 425 430

Asp Lys Tyr Asn Phe Asp Asp Ala Ile Leu Arg Asp Val Val Ser Ile

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Gly Ala Gly Thr Asp Glu Val Thr Ile Arg Phe Val Thr Asp Asn Pro

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Gly Pro Trp Phe Leu His Cys His Ile Asp Trp His Leu Glu Ala Gly

465 470 475 480

Leu Ala Ile Val Phe Ala Glu Gly Ile Asn Gln Thr Ala Ala Ala Asn

485 490 495

Pro Thr Pro Gln Ala Trp Asp Glu Leu Cys Pro Lys Tyr Asn Gly Leu

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Ser Ala Ser Gln Lys Val Lys Pro Lys Lys Gly Thr Ala Ile

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<210> 4

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<213> LacB-F

<400> 4

atgggattga actcggctat t 21

<210> 5

<211> 22

<212> DNA

<213> LacB-R

<400> 5

ttaaatcgca gttcctttct ta 22

Claims

1. LacB laccase from Erysipelothrix chromophorica is characterized in that: the amino acid sequence is shown in SEQ ID NO. 3.

2. A gene of LacB laccase from Erysiphe chromocor, which is characterized in that: the full-length sequence of the gene is shown as SEQ ID NO.1, and the cDNA sequence is shown as SEQ ID NO. 2.

3. The method for the fermentative production of lac b laccase derived from trichoderma brevicompactum according to claim 1, wherein: fermenting the hirsutella chromophoris in a fermentation medium by a liquid shake flask, centrifugally collecting fermentation liquor, and separating and purifying to obtain LacB laccase; the fermentation medium comprises the following components: sucrose 90 g/L, peptone 10 g/L, KH₂PO₄ 6 g/L，MgSO₄·7H₂O 4.14 g/L，CaCl₂ 0.3 g/L，NaCl 0.18 g/L，CuSO₄·5H₂O 0.0625 g/L，ZnSO₄·7H₂O 0.018 g/L，VB₁0.015g/L；

The trichoderma viride is trichoderma viride (A)Cerrna unicolor) 87613 strain, which is purchased from China forestry microorganism strain preservation management center and has the strain preservation number:CFCC 87613。

4. the trichoderma viride-derived LacB laccase of claim 1, applied to degradation of environmental hormones;

the environmental hormones are: progesterone, medroxyprogesterone, megestrol, 17 alpha hydroxyprogesterone acetate, melengestrol acetate, medroxyprogesterone acetate, 17 alpha hydroxyprogesterone, methyltestosterone, nandrolone, testosterone, bodhilone, trenbolone, dexamethasone acetate, prednisolone, diethylstilbestrol, hexylestrol, beta-estradiol, dienestrol, bisphenol A.

5. The application of the LacB laccase derived from the trichoderma brevicompactum as claimed in claim 4 in degradation of environmental hormones is characterized in that: the LacB laccase from the hirsutella chromophoris is applied to degradation of environmental hormones, and the reaction pH is 3-9; the reaction temperature is 15-45 ℃.

6. The application of the LacB laccase derived from the trichoderma brevicompactum as claimed in claim 4 in degradation of environmental hormones is characterized in that: the LacB laccase from the trichoderma unicolor is applied to degradation of environmental hormone diethylstilbestrol, the concentration of diethylstilbestrol is 10-40 mg/L, and the enzyme concentration is 0.25-2.5U/mL.

7. The method for degrading environmental hormones by using LacB laccase derived from chaetomium globosum according to claim 6, wherein the concentration of diethylstilbestrol is 30-40 mg/L, and the enzyme concentration is 2.0-2.5U/mL.

8. The application of the LacB laccase derived from the trichoderma brevicompactum as claimed in claim 4 in degradation of environmental hormones is characterized in that: the LacB laccase from the trichomonas chromophoris is applied to degradation of environmental hormones of estragole or dienestrol or beta-estradiol, wherein the concentration of the estragole or dienestrol or beta-estradiol is 1-40 mg/L, and the concentration of the enzyme is 0.5-2.5U/mL.

9. The application of the LacB laccase derived from the trichoderma brevicompactum as claimed in claim 4 in degradation of environmental hormones is characterized in that: the LacB laccase from the hirsutella chricosa is applied to degradation of environmental hormone bisphenol A, wherein the concentration of the bisphenol A is 50-400 mg/L, and the enzyme concentration is 1-40U/mL.