CN114958788B

CN114958788B - High-temperature-resistant laccase as well as gene, strain and application thereof

Info

Publication number: CN114958788B
Application number: CN202210726253.1A
Authority: CN
Inventors: 杨江科; 乐琛; 张俊雄; 雷磊
Original assignee: Wuhan Polytechnic University
Current assignee: Wuhan Polytechnic University
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2023-04-25
Anticipated expiration: 2042-06-24
Also published as: CN114958788A

Abstract

The invention belongs to the technical field of genetic engineering, and relates to a high-temperature-resistant laccase, a gene, a strain and application. The amino acid sequence of the high temperature resistant laccase CotAGold is shown in SEQ ID NO:1, the high-temperature resistance and the activity are obviously improved after redesign and transformation. According to the invention, laccase CotAGold with high temperature resistance and high enzyme activity is obtained by modifying laccase wild laccase CotA. The laccase CotAGold obtained by the invention lays a foundation for the application of laccase in the industrial fields of pulp decolorization, jeans rinsing and the like.

Description

High-temperature-resistant laccase as well as gene, strain and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a high-temperature-resistant laccase and encoding genes, a recombinant expression vector, a recombinant expression strain, and a preparation method and application thereof. The enzyme can be used in a plurality of industrial fields such as degradation of aflatoxin B1, decolorization of dye such as indigo and the like.

Background

Laccase is a polyphenol oxidase, and has stronger oxidizing ability and wide substrate. Thus, laccase enzymes have very wide application. For example, in food products, laccase enzymes can be used to degrade aflatoxins in the food or feed, thereby detoxify the food and extend its shelf life. Can also be used for hydrolyzing undesirable phenols in beer, improving taste, and prolonging storage time; in the textile sector, it can be used to degrade organic dyes, the product of which is clean water; in the papermaking field, can be used for lignin decolorization and wastewater treatment; in the field of biosensors, as a biosensor, morphine, codeine and catecholamine can be detected, and phenol or other enzymes in fruit juice and flavonoid compounds in plants can be judged.

Aflatoxin B1 is a common mycotoxin in corn, peanuts and other cereals, and is a toxic secondary metabolite produced by aspergillus flavus and aspergillus parasiticus. It has strong toxicity, carcinogenicity and teratogenicity, pollutes various economic crops, causes the safety problems of food, feed and the like, can cause diseases such as liver cancer, slow growth, damage of reproductive system and the like, and forms a great threat to the health of people and livestock. Due to the contamination of mycotoxins in food and feed, there is an urgent need to develop a multifunctional enzyme to degrade a variety of mycotoxins including aflatoxin B1.

Current detoxification methods mainly include physical, chemical and biological methods. Physical methods include radiation, heat treatment and adsorption. The radiation method itself has the problem of radioactive pollution, and the radiated food products have some safety problems. The high temperature during the heat treatment process can destroy the quality and flavor of the grains, and the detoxification effect is incomplete. The adsorption method has the problems of large adsorbent dosage, limited adsorption toxin types and the like. The chemical method adopts alkali treatment and oxidation treatment to break the molecular structure so as to achieve the purpose of detoxification, but the method also breaks the nutrient components of the grains and introduces new chemical reagents. The safety of the degradation products is not yet clear. Biodegradation refers to detoxification by biocatalysis using microorganisms or their enzymes and agents. The detoxification condition is mild and effective. Currently, a great deal of research is focused mainly on the single detoxification of toxins degraded by microorganisms and their metabolites. The detoxification method has poor universality, strong pertinence and poor integrity. The method for degrading two toxins by using various probiotics simultaneously is low in efficiency, complex in experiment and narrow in application range, and is not suitable for natural toxic foods and grains.

Industrial application of laccase requires laccase not only high activity but also good thermal stability. For example, in pulp rinsing, high temperatures are required for pulp rinsing and drying. Common thermolabile laccases tend to lose activity at high temperatures. In the field of clothing manufacturing, laccase is used in jeans decolorization process requiring high temperature environment. The decolorization efficiency of the pulp in a high temperature environment is also to be improved. Therefore, the high-temperature-resistant laccase has wide industrial application prospect. However, the industrial application of laccase in the present stage is to be solved, including but not limited to the following points: 1) Lack of high temperature resistant laccase variety; 2) Limited activity and enzyme yield; 3) The economic cost is high.

For the problems, the genetic engineering method is adopted to modify laccase, so that the method can not only improve the enzyme characteristics and obtain high-temperature-resistant laccase, but also improve the enzyme yield, greatly reduce the economic cost and meet the requirements of industrial application.

Disclosure of Invention

Aiming at improving the defects or shortcomings of the laccase at present, the invention aims to provide high-temperature-resistant laccase cotagld and a coding gene cotAgold and application thereof, wherein the problems of poor heat resistance, low yield and the like of the laccase are overcome by improving the amino acid sequence of the key original laccase cotA from bacillus, the sequence for coding the laccase gene cotA and the like. The invention can realize the application of the laccase CotAGold in industry, can obtain the laccase production strain with good application prospect, and is very beneficial to the popularization and application of the laccase CotAGold. The laccase CotAGold obtained by the invention can be applied to various fields, is a novel high-temperature-resistant laccase which can be widely applied to the industrial fields of dye decolorization, papermaking, food, feed, textile and the like, and can be especially applied to degradation of aflatoxin and decolorization of dyes such as indigo, lignin and the like, thereby greatly reducing the cost required by production and improving the efficiency.

The first aspect of the invention provides a high temperature resistant laccase cotagld, the amino acid sequence of the laccase cotagld is shown in SEQ ID NO: 1.

In a second aspect the invention provides a method for obtaining said laccase cotagld comprising engineering a wild type laccase CotA: aspartic acid D at position 113 was mutated to proline P, aspartic acid D at position 187 was mutated to glycine G, and isoleucine I at position 255 was mutated to leucine L.

The third aspect of the invention provides a high temperature resistant laccase gene cotAgold for encoding the laccase, wherein the base sequence of the laccase gene cotAgold is shown in SEQ ID NO: 2.

Compared with the prior art, the technical scheme of the invention can effectively solve the problems of weak heat resistance, low catalytic efficiency and the like of the existing laccase due to the improvement of the amino acid sequence of the original laccase CotA, the sequence for encoding the laccase gene CotA and the like.

In a fourth aspect, the invention provides a recombinant expression vector comprising said laccase gene cotAgold.

The fifth aspect of the present invention provides a method for constructing the recombinant expression vector, comprising the steps of: respectively introducing restriction enzyme NdeI and HindIII cleavage sites at two ends of the laccase gene cotAgold; the cotAgold gene cut by double digestion of NdeI and HindIII is inserted into an escherichia coli expression vector pET21a cut by double digestion of NdeI and HindIII, and a laccase recombinant expression vector pET21a-cotAgold is obtained.

In a sixth aspect, the present invention provides a recombinant expression strain, comprising the recombinant expression vector, wherein the host cell of the recombinant expression strain is escherichia coli.

According to one embodiment of the present invention, the method for preparing the recombinant expression strain comprises the steps of: linearizing the recombinant expression vector pET21a-cotAgold with the restriction enzyme BamHI; the linearized recombinant expression vector pET21a-cotAgold is introduced into escherichia coli by means of electrotransformation, and positive clones are screened on an LB plate containing ampicillin resistance to obtain the recombinant expression strain.

The seventh aspect of the present invention provides a method for producing the recombinant expression strain, comprising the steps of: and (3) introducing the laccase recombinant expression vector into a host cell to obtain a recombinant expression strain.

According to an eighth aspect of the present invention, there is provided a method for producing the high temperature resistant laccase, culturing the recombinant expression strain, and obtaining the laccase from the culture.

The method specifically comprises the following steps:

(1) Transforming host cells with the recombinant vector to obtain recombinant strains;

(2) Fermenting the recombinant strain to obtain expressed laccase cotagld;

(3) The expressed laccase cotagld was collected and purified.

According to a specific embodiment of the present invention, the method for preparing laccase by recombinant expression strain comprises the following steps: single colonies of the recombinant expression strain were picked up and inoculated into 5mL of LB liquid medium, cultured at 37℃and 170rpm overnight as seed solution, and 2mL of the seed solution was inoculated into a shake flask containing 0.1 mg/mL. Culturing at 37deg.C and 170rpm for 2-3 hr, adding 0.1mM IPTG and 1mM copper ion, culturing for 3 hr, centrifuging, collecting bacteria, and storing in refrigerator at-10deg.C. The fermentation supernatant was taken to determine laccase activity and protein content was determined using BCA method.

In a ninth aspect the invention provides the use of the laccase in contact with aflatoxin B1 for degradation thereof.

In a tenth aspect the invention provides another use of the laccase, which is contacted with a dye, preferably for indigo degradation.

The invention improves the high temperature resistant property and activity of the laccase CotA by modifying the amino acid sequence of the key laccase CotA and the gene cotA sequence for encoding the laccase CotA. In the preparation process of laccase, the preparation process is simple, the yield is high, and the production cost of laccase is reduced. The produced laccase has outstanding high-temperature resistance, can be used for degrading aflatoxin B1, has strong degradation capability on dye indigo, and can be used for rinsing jeans, paper pulp and the like.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 is a gel diagram of laccase production and protein analysis of wild laccase CotA recombinant strain and modified laccase CotAgold recombinant strain in shake flask according to the invention.

FIG. 2 is a graph showing the laccase catalytic ability change of the high temperature resistant laccase CotAgold and the wild type laccase CotA in the examples of the invention after 5min treatment at 45-85deg.C.

FIG. 3 is a graph showing the comparison of the degradation rate of aflatoxin at 70℃for the high temperature resistant laccase CotAgold and the wild laccase CotA in the examples of the invention.

FIG. 4 is a qualitative analysis of the indigo-breaking capacity of the modified laccase CotAgold compared to the indigo-breaking capacity of the wild-type laccase in the examples of the invention.

FIG. 5 is a graph showing the comparison of the indigo degradation rate at 70℃for the high temperature resistant laccase CotAgold and the wild type laccase CotA in the examples of the invention.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein.

The specific conditions not specified in the examples were either conventional or manufacturer-recommended. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

This example is intended to illustrate the modification of wild-type laccase CotA to a thermostable laccase CotAGold by site-directed mutagenesis. The concrete transformation process comprises the following steps:

site-directed mutagenesis was performed on the wild-type laccase CotA by analysis of the three-dimensional structure of the laccase, resulting in a total of 3 mutation sites (D113P, D187G, I L), which were analyzed using PyMol. After D113P (proline is used for replacing aspartic acid), the rigidity of a loop region of the protein can be improved, and the stability of the protein is obviously improved; D187G (glycine to aspartic acid) was used for this point mutation due to analysis of the secondary structure of the protein, where the beta-turn was found to be too stiff, resulting in difficulty in folding, affecting the activity of the protein, and increasing the flexibility of the region after mutation; I255L, this mutation occurs in the core region of the protein, which results in improved hydrophobic interactions within the fold, and thus in improved stability and activity of the protein.

The site-directed mutagenesis technique is a routine procedure in the art, and the specific procedures are well known to those skilled in the art. The amino acid sequence of the modified laccase CotAGold is shown as SEQ ID NO: 1. The amino acid sequence of the wild laccase CotA is shown as SEQ ID NO: 3.

Example 2

The embodiment is used for explaining the sequence characteristics of the modified high temperature resistant laccase gene. The present example redesigns the nucleotide sequence of laccase gene CotAGold based on the amino acid sequence of mutated laccase CotAGold. Specifically including replacing codons of low frequency with codons of high frequency; the complexity and the minimum free energy of the secondary structure of the mRNA encoded by the gene are reduced. Balancing the GC content and distribution in the gene, and removing the repetitive sequences and cis-acting elements in the gene. The laccase gene sequence is designed, and laccase gene fragments are obtained by an artificial synthesis method.

According to the embodiment, a brand new laccase gene cotA sequence is artificially designed according to the amino acid sequence of the wild laccase cotA, and the laccase gene fragment is obtained by an artificial synthesis method. The techniques for artificially synthesizing genes are conventional in the art, and the specific procedures are well known to those skilled in the art. The base sequence of the laccase gene cotAgold is shown in SEQ ID NO:2, the base sequence of the laccase gene cotA is shown as SEQ ID NO: 4.

Example 3

This example is to illustrate the wild laccase CotA and the engineered high temperature resistant laccase cotagld recombinant expression vector.

(1) Adding enzyme cutting sites Nde I and Hind III at two ends of a designed high-temperature-resistant laccase gene cotAgold, and carrying out enzyme cutting on the gene by the Nde I and Hind III and collecting enzyme cutting products; vector pET21a was digested simultaneously with Nde I and Hind iii and the digested products were collected. Wherein, the enzyme digestion system is as follows: mu.L of DNA, 1. Mu.L of Nde I, 1. Mu.L of HindIII, 10. Mu.L of Buffer, and water were added to fill up to a volume of 100. Mu.L, and the mixture was digested at 37℃for 4 hours.

(2) The cleavage product of the laccase gene cotAgold and the cleavage product of the vector pET21a were ligated overnight at 16℃with T4 DNA ligase. The connection system is as follows: 7. Mu.L of the gene fragment, 1. Mu.L of pET21a fragment, 1. Mu.L of T4 ligase buffer, 0.5. Mu.L of T4 DNA ligase, and water was added to make up to 10. Mu.L. After ligation, the recombinant expression vector pET21a-cotAgold was obtained.

(3) The method is adopted to construct a wild laccase cotA recombinant vector pET21a-cotA.

The embodiment constructs laccase wild laccase gene cotA and the modified laccase gene cotAGold recombinant expression plasmid.

Example 4

The example is used to illustrate the acquisition and expression of recombinant strains of laccase wild laccase CotA and modified high temperature resistant laccase cotagld.

(1) The recombinant expression vector pET21a-cotAgold obtained in example 3 was transformed into E.coli BL21 (DE 3), and the electrotransformation method was as follows: (i) 1. Mu.L of the linearized recombinant expression plasmid was mixed with 90. Mu.L of fresh E.coli competent cells and placed on an ice box for 5min in an ice bath. (ii) The electrotometer is turned on, the electric shock parameters are regulated, the voltage is 1600V, the resistance is 200 omega, and the capacitance is 25 mu F. The linearization product of the ice bath and competent cell mixture were transferred to an electric rotating cup on ice for electric shock when it was cold. (iii) 1mL of AMP, which had been preheated at 28℃was rapidly added to the electric beaker, transferred to a 1.5mL sterile centrifuge tube, and allowed to stand in an incubator at 37℃for 2 hours. (iv) 100. Mu.L of the bacterial liquid was spread on an ampicillin-resistant LB plate, and the plate was allowed to stand for 3 days in an incubator at 37 ℃. After single bacterial colony is grown, the colibacillus recombinant strain containing the cotAgold gene is obtained.

(2) Constructing a laccase wild-type laccase CotA recombinant strain according to the method of the step (1).

(3) The recombinant strain was inoculated into 5mL of LB medium and cultured overnight in a constant temperature shaker at 37 ℃. Inoculating to 250mL LB medium, culturing at 37deg.C for 3 hr, transferring to 16deg.C, culturing, adding IPTG with final concentration of 0.1mM and CuSO with final concentration of 1mM ₄ And (5) performing induction culture for 12-16h. Then, the cultured bacterial liquid is subjected to pressure crushing, and ultrasonic crushing is performed after the bacterial liquid is subjected to pressure crushing. And then breakingThe bacterial liquid was centrifuged at a low temperature refrigerated centrifuge for 20min at 8000r/min, and one fraction was collected as a total sample. Then, ni-NTA column separation was performed, the prepared column was transferred to a 50mL centrifuge tube, the centrifuge tube was capped, sealed with a sealing film, and then handled with a cradle for 1 hour. And then adding the centrifuged bacterial liquid into a shaking column, flowing out, and collecting FT. After the sample flows out, pouring the lysis buffer into a 50mL centrifuge tube for loading bacteria liquid after centrifugation, shaking uniformly, pouring into a column for draining, and repeating for three times. And adding a wash buffer into the column to drain out, and collecting a wash sample. Then adding an filtration buffer into the column, draining, and collecting an effluent sample. Proteins were then collected, 12 tubes per protein, about 1mL per tube. And coomassie brilliant blue identification was performed on the 12 samples collected. And then adding the identified sample into an ultrafiltration tube, centrifuging at 5000r/min until about 1.5mL of the upper part of the ultrafiltration tube is left, and respectively carrying out Coomassie brilliant blue identification on the upper part and the lower part of the ultrafiltration tube. Finally, the ultrafiltered sample is dialyzed. And (5) sub-packaging after treatment. The result was verified by running the gel as shown in FIG. 1. FIG. 1 shows a gel diagram of laccase production and protein analysis of wild laccase CotA recombinant strain and modified laccase CotAgold recombinant strain in shake flasks.

(4) The protein content is determined by BCA method, and the specific operation steps are as follows: firstly preparing BCA working solution, wherein the preparation method is to add 50 volumes of A solution and 1 volume of B solution, and fully and uniformly mixing to obtain BCA working solution. And drawing a standard curve, adding 0-20 mu L of protein solution into a reaction system to ensure that the protein content in the system is 0-200 mu g/mL, adding 200 mu L of BCA working solution into the reaction system, reacting for 30min in a baking oven at 60 ℃, and finally testing the absorbance of A562 to draw the standard curve. Adding a sample with the same volume into a sample hole of a 96-well plate, adding water to 20 mu L, adding 200 mu L of BCA working solution into a reaction system, reacting for 30min in an oven at 60 ℃, testing the absorbance of A562, and finally calculating the protein content.

(5) Determination of laccase Activity: adding 500 mu L of BR Buffer with pH of 5.5 into a 2mL EP tube, adding 10 mu L of 0.1M ABTS, mixing uniformly by vortex, placing all control groups of the experimental groups on a 70 ℃ metal bath for preheating for 2 minutes, then sequentially adding 2 mu L of purified enzyme solution into the experimental groups, wherein the interval time is the same, the two are 15s, then reacting on the metal bath for 5min, sequentially adding 500 mu L of methanol according to the same interval time for stopping reaction, mixing uniformly by vortex, adding 500 mu L of methanol into the control groups for stopping, mixing uniformly by vortex, taking 200 mu L of the reaction solution after stopping, adding the 200 mu L of the reaction solution into a 96-well plate, reading the value of OD420 on an enzyme label instrument, and calculating the enzyme activity according to the OD420 values of the experimental groups and the control groups.

The enzyme activity calculation formula is

Δod: absorbance change before and after reaction

ξ：36000M ^-1 ·cm ^-1

l: measured by using an enzyme-labeled instrument, is 0.58cm

T: time unit: minute (min)

[E] The method comprises the following steps Protein content μg/mL

V _Enzymes : volume of enzyme dosage

V _{Reaction system} : volume of the whole reaction system

According to the procedure described in this example, the present invention obtained wild-type laccase CotA and the redesigned recombinant strain of laccase CotAgold, and subjected to enzyme-producing fermentation in shake flasks.

Example 5

This example was used to analyze the heat resistance of wild-type laccase CotA and redesigned laccase CotAgold.

(1) And (3) respectively placing the laccase wild laccase CotA and the laccase CotAgold after optimization at 45-85 ℃ for 30min, carrying out enzyme activity measurement on the treated sample, setting the enzyme activity at the highest temperature of the enzyme activity as 100%, and calculating the enzyme activities of the samples treated at different temperatures.

(2) According to the above method, the present invention determines the heat resistance of the wild-type laccase CotA and the optimized laccase CotAgold. The results are shown in FIG. 2 (average enzyme activity of CotAgold on left and average enzyme activity of CotA on right), and the high temperature resistance of the modified laccase CotAgold is significantly better than that of the wild-type laccase CotA. The enzyme activity of the wild laccase CotA is 18.7% when the wild laccase CotA is not subjected to the 81.1 ℃ standing treatment for 30min, and the enzyme activity retention rate of the optimized laccase CotAgold is 23.3%.

Example 6

This example was used to analyze the degradation capacity of wild-type laccase CotA and redesigned laccase cotagld for aflatoxin.

(1) The laccase wild-type laccase CotA and the optimized laccase cotagld are placed at 70 ℃ to be respectively contacted with 50 mug/mL of aflatoxin B1, and are degraded, the content of Huang Qumei toxin is measured every 0.33h, and meanwhile, enzymes with different concentrations are used for degradation, and the concentrations are shown in table 1 below.

TABLE 1 enzyme concentration used for degradation of aflatoxin B1 by wild laccase and modified laccase

(2) According to the method, the degradation efficiency of the wild laccase CotA and the optimized laccase CotAGold on the aflatoxin B1 is measured. The results are shown in FIG. 3 (time min on the horizontal axis, degradation efficiency of CotA on the left side, and degradation efficiency of CotAgold on the right side), and the ability of the modified laccase CotAgold to degrade aflatoxin B1 was significantly better than that of the wild-type laccase CotA. The wild laccase CotA requires 4 hours to completely degrade aflatoxin B1 in the system, while the modified laccase cotagld requires only 1.67 hours to completely degrade Huang Qumei toxin B1 in the system.

Example 7

This example was used to analyze the degradation capacity of wild-type laccase CotA and redesigned laccase cotagld for indigo dye.

(1) Placing laccase wild laccase CotA and optimized laccase CotAGold at 70deg.C, respectively contacting with 1ng/mL indigo, degrading, and measuring indigo content at intervals. The qualitative results after 12h of reaction are shown in FIG. 4.

(2) According to the method, the degradation efficiency of the wild laccase CotA and the optimized laccase cotagld on indigo is measured. The results are shown in FIG. 4 (from left to right: degradation with laccase CotAGold, degradation with CotA, no enzymatic degradation, respectively), the ability of the modified laccase CotAGold to degrade indigo is significantly better than the wild-type laccase CotA. As shown in FIG. 5 (the left column is the degradation efficiency of CotA and the right column is the degradation efficiency of CotAgold), the wild-type laccase CotA takes 6 hours to reach the peak of degradation, the modified laccase CotAGold only takes 2 hours to reach the peak, the modified laccase CotAGold degradation rate can reach approximately 80%, and the wild-type laccase CotA degradation rate can only reach about 30%.

The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described.

Sequence listing

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Met Thr Leu Glu Lys Phe Val Asp Ala Leu Pro Ile Pro Asp Thr Leu

1 5 10 15

Lys Pro Val Gln Gln Ser Lys Glu Lys Thr Tyr Tyr Glu Val Thr Met

20 25 30

Glu Glu Cys Thr His Gln Leu His Arg Asp Leu Pro Pro Thr Arg Leu

35 40 45

Trp Gly Tyr Asn Gly Leu Phe Pro Gly Pro Thr Ile Glu Val Lys Arg

50 55 60

Asn Glu Asn Val Tyr Val Lys Trp Met Asn Asn Leu Pro Ser Thr His

65 70 75 80

Phe Leu Pro Ile Asp His Thr Ile His His Ser Asp Ser Gln His Glu

85 90 95

Glu Pro Glu Val Lys Thr Val Val His Leu His Gly Gly Val Thr Pro

100 105 110

Asp Asp Ser Asp Gly Tyr Pro Glu Ala Trp Phe Ser Lys Asp Phe Glu

115 120 125

Gln Thr Gly Pro Tyr Phe Lys Arg Glu Val Tyr His Tyr Pro Asn Gln

130 135 140

Gln Arg Gly Ala Ile Leu Trp Tyr His Asp His Ala Met Ala Leu Thr

145 150 155 160

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

165 170 175

Pro Lys Glu Lys Arg Leu Lys Leu Pro Ser Asp Glu Tyr Asp Val Pro

180 185 190

Leu Leu Ile Thr Asp Arg Thr Ile Asn Glu Asp Gly Ser Leu Phe Tyr

195 200 205

Pro Ser Ala Pro Glu Asn Pro Ser Pro Ser Leu Pro Asn Pro Ser Ile

210 215 220

Val Pro Ala Phe Cys Gly Glu Thr Ile Leu Val Asn Gly Lys Val Trp

225 230 235 240

Pro Tyr Leu Glu Val Glu Pro Arg Lys Tyr Arg Phe Arg Val Ile Asn

245 250 255

Ala Ser Asn Thr Arg Thr Tyr Asn Leu Ser Leu Asp Asn Gly Gly Asp

260 265 270

Phe Ile Gln Ile Gly Ser Asp Gly Gly Leu Leu Pro Arg Ser Val Lys

275 280 285

Leu Asn Ser Phe Ser Leu Ala Pro Ala Glu Arg Tyr Asp Ile Ile Ile

290 295 300

Asp Phe Thr Ala Tyr Glu Gly Glu Ser Ile Ile Leu Ala Asn Ser Ala

305 310 315 320

Gly Cys Gly Gly Asp Val Asn Pro Glu Thr Asp Ala Asn Ile Met Gln

325 330 335

Phe Arg Val Thr Lys Pro Leu Ala Gln Lys Asp Glu Ser Arg Lys Pro

340 345 350

Lys Tyr Leu Ala Ser Tyr Pro Ser Val Gln His Glu Arg Ile Gln Asn

355 360 365

Ile Arg Thr Leu Lys Leu Ala Gly Thr Gln Asp Glu Tyr Gly Arg Pro

370 375 380

Val Leu Leu Leu Asn Asn Lys Arg Trp His Asp Pro Val Thr Glu Thr

385 390 395 400

Pro Lys Val Gly Thr Thr Glu Ile Trp Ser Ile Ile Asn Pro Thr Arg

405 410 415

Gly Thr His Pro Ile His Leu His Leu Val Ser Phe Arg Val Leu Asp

420 425 430

Arg Arg Pro Phe Asp Ile Ala Arg Tyr Gln Glu Ser Gly Glu Leu Ser

435 440 445

Tyr Thr Gly Pro Ala Val Pro Pro Pro Pro Ser Glu Lys Gly Trp Lys

450 455 460

Asp Thr Ile Gln Ala His Ala Gly Glu Val Leu Arg Ile Ala Ala Thr

465 470 475 480

Phe Gly Pro Tyr Ser Gly Arg Tyr Val Trp His Cys His Ile Leu Glu

485 490 495

His Glu Asp Tyr Asp Met Met Arg Pro Met Asp Ile Thr Asp Pro His

500 505 510

Lys

<210> 4

<211> 1539

<212> DNA

<213> LACCASE (LACCASE)

<400> 4

atgacacttg aaaaatttgt ggatgctctc ccaatcccag atacactaaa gccagtacag 60

caatcaaaag aaaaaacata ctacgaagtc accatggagg aatgcactca tcagctccat 120

cgcgatctcc ctccaacccg cctgtggggc tacaacggct tatttccggg accgaccatt 180

gaggttaaaa gaaatgaaaa cgtatatgta aaatggatga ataaccttcc ttccacgcat 240

ttccttccga ttgatcacac cattcatcac agtgacagcc agcatgaaga gcccgaggta 300

aagactgttg ttcatttaca cggcggcgtc acgccagatg atagtgacgg gtatccggag 360

gcttggtttt ccaaagactt tgaacaaaca ggaccttatt tcaaaagaga ggtttatcat 420

tatccaaacc agcagcgcgg ggctatattg tggtatcacg atcacgccat ggcgctcacc 480

aggctaaatg tctatgccgg acttgtcggt gcatatatca ttcatgaccc aaaggaaaaa 540

cgcttaaaac tgccttcaga cgaatacgat gtgccgcttc ttatcacaga ccgcacgatc 600

aatgaggatg gttctttgtt ttatccgagc gcaccggaaa acccttctcc gtcactgcct 660

aatccttcaa tcgttccggc tttttgcgga gaaaccatac tcgtcaacgg gaaggtatgg 720

ccatacttgg aagtcgagcc aaggaaatac cgattccgtg tcatcaacgc ctccaataca 780

agaacctata acctgtcact cgataatggc ggagatttta ttcagattgg ttcagatgga 840

gggctcctgc cgcgatctgt taaactgaat tctttcagcc ttgcgcctgc tgaacgttac 900

gatatcatca ttgacttcac agcatatgaa ggagaatcga tcattttggc aaacagcgcg 960

ggctgcggcg gtgacgtcaa tcctgaaaca gatgcgaata tcatgcaatt cagagtcaca 1020

aaaccattgg cacaaaaaga cgaaagcaga aagccgaagt acctcgcctc atacccttcg 1080

gtacagcatg aaagaataca aaacatcaga acgttaaaac tggcaggcac ccaggacgaa 1140

tacggcagac ccgtccttct gcttaataac aaacgctggc acgatcccgt cacagaaaca 1200

ccaaaagtcg gcacaactga aatatggtcc attatcaacc cgacacgcgg aacacatccg 1260

atccacctgc atctagtctc cttccgtgta ttagaccggc ggccgtttga tatcgcccgt 1320

tatcaagaaa gcggggaatt gtcctatacc ggtccggctg tcccgccgcc gccaagtgaa 1380

aagggctgga aagacaccat tcaagcgcat gcaggtgaag tcctgagaat cgcggcgaca 1440

ttcggtccgt acagcggacg atacgtatgg cattgccata ttctagagca tgaagactat 1500

gacatgatga gaccgatgga tataactgat ccccataaa 1539

Claims

1. The high temperature resistant laccase cotagld is characterized in that the amino acid sequence of the laccase cotagld is shown in SEQ ID NO: 1.

2. The method of obtaining laccase cotagld of claim 1, having the amino acid sequence of SEQ ID NO:3, modifying a wild laccase CotA shown in the formula 3: aspartic acid D at position 113 was mutated to proline P, aspartic acid D at position 187 was mutated to glycine G, and isoleucine I at position 255 was mutated to leucine L.

3. High-temperature-resistant laccase genecotAgoldCharacterized in that the laccase genecotAgoldThe base sequence of (2) is shown as SEQ ID NO: 2.

4. A recombinant expression vector comprising the laccase gene of claim 3cotAgold。

5. The recombinant meter of claim 4The construction method of the vector is characterized by comprising the following steps: the laccase gene of claim 3cotAgoldRespectively introducing restriction enzymes at both ends of (a)Nde I andHind III, enzyme cutting site; warp knitting machineNdeI andHindIII double enzyme digestioncotAgoldGene insertion is likewise carried outNdeI andHindIII double-enzyme-cut colibacillus expression vector pET21a to obtain laccase recombinant expression vector pET21a-cotAgold。

6. A recombinant expression strain, comprising the recombinant expression vector of claim 4, wherein the host cell of the recombinant expression vector is E.coli.

7. The method for preparing the recombinant expression strain according to claim 6, comprising the steps of: and (3) introducing the laccase recombinant expression vector into a host cell to obtain a recombinant expression strain.

8. The method for producing a high temperature resistant laccase as claimed in claim 1, characterized in that the recombinant expression strain as claimed in claim 6 is cultivated and the laccase is obtained from the culture.

9. Use of the laccase according to claim 1, characterized in that the laccase according to claim 1 is contacted with aflatoxin B1 for its degradation.

10. Use of the laccase according to claim 1, characterized in that the laccase according to claim 1 is contacted with a dye for dye indigo degradation.