A mutant of Bacillus subtilis Cot-A laccase, mutant gene and application thereof

Abstract

The present invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to a mutant of Bacillus subtilis Cot-A laccase, a mutant gene and its application. The mutant of the present invention is obtained by mutation of the 92nd amino acid of wild-type Bacillus subtilis Cot-A laccase from aspartic acid to proline; the amino acid sequence of the wild-type Bacillus subtilis Cot-A laccase is shown in the sequence with accession number H8WGE7 in UniProt. The mutant of the present invention has higher catalytic efficiency, specificity and degradation rate for polycyclic aromatic hydrocarbons including benzanthracene than the wild-type Bacillus subtilis Cot-A laccase, further improving the industrial application prospects of Bacillus subtilis Cot-A laccase.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to a mutant of Bacillus subtilis Cot-A laccase, a mutant gene and application thereof. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) are compounds composed of two or more condensed benzene rings with various structural forms. These PAHs with different structural forms are produced by natural disasters such as forest fires and volcanic eruptions or human activities such as incomplete combustion of fossil fuels, oil refining, coal gasification and waste incineration. PAHs have stable structures and low water solubility. They have carcinogenic, teratogenic and mutagenic effects, and as a persistent organic pollutant, they have attracted much attention. Although PAHs have low water solubility and low concentration in water, PAHs in the aquatic environment are widely distributed and polluted. The reason is that PAHs are easily distributed from water to sediments or organisms, enriched in sediments and organisms, destroying aquatic ecosystems, and entering the human body through the respiratory tract, skin and digestive tract, directly endangering human health. Therefore, the control of PAH pollution is imminent.

[0003] Laccase (EC1.10.3.2) is a copper-containing polyphenol oxidase that generally contains four copper ions. It can be divided into three categories based on its spectral and magnetic characteristics: one type I copper ion and one type II copper ion, which are single-electron acceptors and paramagnetic; two type III copper ions, which are di-electron acceptors and diamagnetic. These four copper ions are located in the active site of laccase and are the determining factor for the catalytic activity of laccase. The mononuclear site of type I copper atom (T1 Cu) is the substrate binding site. After accepting electrons, it is transferred to the trinuclear copper cluster site (including two type II copper atoms and one type III copper atom) through the conserved histidine-cysteine-histidine tripeptide, where molecular oxygen is reduced to water. The mechanism of laccase-catalyzed oxidation of organic matter is mainly that laccase catalyzes the single-electron oxidation of organic matter and reduces oxygen molecules to water at the same time. Laccase can then oxidize a variety of difficult-to-degrade organic matter, including benzanthracene, phenolic pollutants, polychlorinated biphenyls and their derivatives, carboxylic acids and their derivatives, aromatic amines and their derivatives, and pesticides and herbicides.

[0004] The inventors have cloned and expressed CotA laccase from the Bacillus subtilis strain CotA (B. pumilus CotA) screened by themselves. The study found that the CotA laccase has the following advantages over other types of laccases: high enzyme activity under alkaline conditions, high temperature resistance and good thermal stability, and tolerance to high concentrations of organic solvents and halogen ion environments. These excellent properties are exactly what is urgently needed for the industrial application of laccases in the field of treating polycyclic aromatic hydrocarbons. However, the wild-type CotA laccase of Bacillus subtilis CotA has a poor effect on degrading polycyclic aromatic hydrocarbons and is difficult to further industrialize. Summary of the invention

[0005] The object of the present invention is to provide a mutant of Bacillus subtilis Cot-A laccase, a mutant gene and application thereof. The mutant of Bacillus subtilis Cot-A laccase has a higher polycyclic aromatic hydrocarbon degradation rate than the wild-type Bacillus subtilis Cot-A laccase.

[0006] The present invention provides a mutant of Bacillus subtilis Cot-A laccase, wherein the 92nd amino acid of the mutant is mutated from aspartic acid to proline compared with the wild-type Bacillus subtilis Cot-A laccase;

[0007] The amino acid sequence of the wild-type Bacillus subtilis Cot-A laccase is shown in the sequence with accession number H8WGE7 in UniProt.

[0008] The present invention also provides a gene encoding a mutant of Bacillus subtilis Cot-A laccase, wherein the gene encodes the mutant as described in the above technical solution.

[0009] The present invention also provides a mutant recombinant expression vector, comprising an initial vector and the mutant gene described in the above technical solution.

[0010] Preferably, the initial vector comprises pET28a.

[0011] The present invention also provides a method for preparing the mutant recombinant expression vector described in the above technical solution, comprising the following steps: performing PCR amplification on the recombinant plasmid pET28a-Cot-A with a D92P mutant primer to obtain the mutant recombinant expression vector;

[0012] The nucleotide sequence of the forward primer of the D92P mutation primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2;

[0013] The recombinant plasmid pET28a-Cot-A is obtained by transferring the wild-type Bacillus subtilis Cot-A laccase gene into between the promoter and terminator of pET28a.

[0014] The present invention also provides a recombinant bacterium, comprising the mutant gene or recombinant expression vector described in the above technical solution.

[0015] The present invention also provides a method for preparing the mutant described in the above technical solution, comprising the following steps:

[0016] The inducing agent is used to induce the expression of the recombinant bacteria described in the above technical solution to obtain a single colony with the best ability to degrade benzanthracene, wherein the single colony includes the mutant.

[0017] Preferably, the inducing agent for inducing expression is IPTG.

[0018] The present invention also provides the use of the mutant, mutant gene, mutant recombinant expression vector described in the above technical scheme or the mutant prepared by the preparation method in the degradation of polycyclic aromatic hydrocarbons.

[0019] Preferably, the polycyclic aromatic hydrocarbons include benzanthracene.

[0020] Beneficial effects:

[0021] The present invention provides a mutant of Bacillus subtilis Cot-A laccase, wherein the 92nd amino acid of the wild-type Bacillus subtilis Cot-A laccase is mutated from aspartic acid to proline; the amino acid sequence of the wild-type Bacillus subtilis Cot-A laccase is a sequence shown in GeneBank with accession number AGO57931. The mutant of the present invention has higher catalytic efficiency, specificity and degradation rate for polycyclic aromatic hydrocarbons including benzanthracene than the wild-type Bacillus subtilis Cot-A laccase, further improving the industrial application prospect of Bacillus subtilis Cot-A laccase. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 This is the electrophoresis diagram of the wild Bacillus subtilis Cot-A mutant DNA in Example 2 of the present invention, lane 1 is the DNA Marker, and lane 2 is the electrophoresis result of the wild Bacillus subtilis Cot-A mutant DNA;

[0024] Figure 2 The predicted active sites of Bacillus subtilis Cot-A laccase and benzanthracene in Example 2 of the present invention;

[0025] Figure 3 is the kinetic curve of the wild-type Cot-A laccase in Example 4 of the present invention;

[0026] Figure 4is the kinetic curve of the mutant Cot-A laccase in Example 4 of the present invention;

[0027] Figure 5 This is a diagram showing the degradation effects of mutant Cot-A laccase and wild-type Cot-A laccase on benzanthracene as characterized by high performance liquid chromatography in Example 5 of the present invention. DETAILED DESCRIPTION

[0028] The invention provides a mutant of Bacillus subtilis Cot-A laccase, wherein the mutant is obtained by mutating the 92nd amino acid of wild-type Bacillus subtilis Cot-A laccase from aspartic acid to proline; the amino acid sequence of the wild-type Bacillus subtilis Cot-A laccase is a sequence shown in UniProt with accession number H8WGE7.

[0029] The present invention also provides a gene encoding a mutant of Bacillus subtilis Cot-A laccase, wherein the gene encodes the mutant as described in the above technical solution.

[0030] The present invention also provides a mutant recombinant expression vector, comprising an initial vector and a mutant gene and the coding gene described in the above technical solution. The initial vector of the present invention is preferably, more preferably, pET28a. The present invention preferably obtains the mutant gene by inserting it between the promoter and terminator of pET28a.

[0031] The present invention also provides a method for preparing the mutant recombinant expression vector described in the above technical scheme, comprising the following steps: performing PCR amplification on the recombinant plasmid pET28a-Cot-A with a D92P mutant primer to obtain the mutant recombinant expression vector; the nucleotide sequence of the forward primer of the D92P mutant primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; the recombinant plasmid pET28a-Cot-A is obtained by transferring the wild-type Bacillus subtilis Cot-A laccase gene into between the promoter and terminator of pET28a.

[0032] In the embodiment of the present invention, the recombinant plasmid pET28a-Cot-A was constructed by the inventors in the early stage. The recombinant plasmid pET28a-Cot-A of the present invention is preferably obtained by inserting the nucleotide sequence with the accession number KF040050 in NCBI between the promoter and terminator of pET28a.

[0033] The PCR amplification of the present invention is preferably a rolling circle PCR amplification. The nucleotide sequence shown in SEQ ID NO.1 of the present invention is specifically: 5'-CATACCATTCATCATAGCCCGAGTCAGCATGAAG AACCG-3', and the underlined nucleic acid represents the nucleotide encoding the 92nd mutant amino acid in the mutant; the nucleotide sequence shown in SEQ ID NO.2 is specifically: 5'-GCGTTCCGC CGGCGCCAGGCTAAAGCTGTTCAGTTTCAC-3'.

[0034] The present invention also provides a recombinant bacterium, comprising the coding gene of the mutant or the mutant recombinant expression vector described in the above technical solution. The original strain used to prepare the recombinant bacterium of the present invention preferably comprises Escherichia coli, more preferably BL21 competent cells.

[0035] The present invention also provides a method for preparing the mutant described in the above technical solution, comprising the following steps:

[0036] The inducing agent is used to induce the expression of the recombinant bacteria described in the above technical solution to obtain a single colony with the best ability to degrade benzanthracene, wherein the single colony includes the mutant.

[0037] After obtaining the single colony, the present invention preferably sequences the single colony to obtain the mutant; compared with the amino acid of the wild-type Bacillus subtilis Cot-A laccase, the 92nd amino acid of the mutant of the present invention is mutated from aspartic acid (Asp) to proline (Pro); the present invention has no special provisions for the sequencing method, and the conventional sequencing method in the field or the sequencing by entrusting a third party company can be used. The inducer for inducing expression in the present invention is preferably IPTG.

[0038] The method for screening the single bacterial colony that best degrades benzanthracene in the present invention preferably comprises the following steps:

[0039] Resuspend the induced expression bacteria in phosphate-citrate buffer to obtain a crude enzyme solution;

[0040] The crude enzyme solution is mixed with n-hexanol, isooctyl alcohol, rhamnolipid and benzanthracene to obtain a microemulsion system;

[0041] After the microemulsion system is subjected to microemulsion reaction under heating conditions for 20 to 24 hours, it is filtered to obtain a filtrate;

[0042] The filtrate is tested by high performance liquid chromatography, the degradation rate of benz(a)anthracene is calculated, and the single bacterial colony with the best degradation of benz(a)anthracene by the benzyl propionate is obtained;

[0043] The calculation formula of the benzanthracene degradation rate is: Degradation rate (%) = (1-A x / Ao )×100%;

[0044] A0: peak area of ​​benzanthracene degradation by wild-type Bacillus subtilis Cot-A laccase;

[0045] A x : Peak area of ​​benzanthracene degradation by crude enzyme solution.

[0046] The present invention resuspends the induced expression obtained bacteria in a phosphate-citrate buffer to obtain a crude enzyme solution. The pH value of the phosphate-citrate buffer of the present invention is preferably 4.2-4.8, more preferably 4.8; the water content is preferably 15-25%, more preferably 20%. The concentration of the crude enzyme solution of the present invention is preferably 0.4-0.8 mg / mL, more preferably 0.6 mg / mL.

[0047] After obtaining the crude enzyme solution, the present invention mixes the crude enzyme solution with n-hexanol, isooctane, rhamnolipid and benzanthracene to obtain a microemulsion system. In the microemulsion system of the present invention, the volume ratio of n-hexanol to isooctane is 1:1, the final concentration of rhamnolipid is preferably 2.8575 g / L, and the concentration of benzanthracene is preferably 5 mg / L. The mixing of the present invention is preferably ultrasonic mixing; the frequency of the ultrasonic mixing is preferably 20 to 40 Hz, more preferably 40 Hz. The present invention does not specifically limit other parameters of the ultrasonic mixing, as long as the solution after ultrasonic mixing is homogeneous and clear.

[0048] After obtaining the microemulsion system, the present invention performs a microemulsion reaction on the microemulsion system under heating conditions for 20 to 24 hours, and then filters to obtain a filtrate. The temperature of the microemulsion reaction of the present invention is preferably 20 to 35°C, more preferably 25°C; the time of the microemulsion reaction is preferably 24 hours. The microemulsion reaction of the present invention is preferably carried out under oscillation conditions, and the rotation speed of the oscillation is preferably 1000 to 1400 rpm, more preferably 1200 rpm. The present invention preferably uses an organic membrane for the filtration, and the pore size of the organic membrane for filtration is preferably 0.1 to 0.5 μm, more preferably 0.22 μm.

[0049] After obtaining the filtrate, the present invention preferably detects the filtrate by high performance liquid chromatography, calculates the degradation rate of benzanthracene, and obtains the single colony with the best degradation of benzanthracene by benzyl propionate. The present invention preferably uses a C18 chromatographic column for the high performance liquid chromatography detection; the injection volume is preferably 20 μL; the mobile phase preferably includes methanol and water, and the volume ratio of methanol to water is preferably 80:20; the flow rate of the mobile phase is preferably 1.0 mL / min. The detection wavelength of the ultraviolet detector in the high performance liquid chromatography of the present invention is preferably 254 nm.

[0050] The present invention also provides the use of the mutant, mutant gene, recombinant expression vector described in the above technical solution or the mutant prepared by the preparation method in the degradation of polycyclic aromatic hydrocarbons.

[0051] The polycyclic aromatic hydrocarbons of the present invention preferably include benzanthracene, and more preferably benzanthracene. The catalytic efficiency, specificity and degradation efficiency of polycyclic aromatic hydrocarbons of the mutants used in the present invention are improved compared with the wild-type Bacillus subtilis Cot-A laccase.

[0052] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The culture medium used in the present invention, unless otherwise specified, is generally prepared according to conventional formulations in the art or purchased through commercial channels.

[0054] Example 1

[0055] The preparation steps of wild-type Bacillus subtilis Cot-A laccase (hereinafter referred to as wild-type Cot-A laccase) are as follows:

[0056] Inoculate the recombinant expression colony Cot-A / pET28a / BL21 constructed earlier (i.e., introduce the recombinant plasmid pET28a-Cot-A into the BL21 E. coli competent state to obtain the positive recombinant expression colony) in the LB solid medium containing 0.5mM kanamycin, pick the larger monoclonal body in the LB culture medium containing 0.5mM kanamycin, culture at 37°C for 7-8h, and then inoculate the bacterial liquid into the TB culture medium containing 0.5mM kanamycin at a volume inoculation of 1%. Cultivate at 37°C for 3-4h until the OD of E. coli reaches 0.6-0.8, add 0.2mM final concentration of IPTG for induction, and continue fermentation and culture on a shaker at 18°C ​​with a speed of 200rpm for 18h, centrifuge the fermentation liquid at 4°C and 8000rpm for 2min, remove the supernatant, and collect the bacteria. The collected cells were resuspended in a phosphate-citrate buffer with a pH value of 4.8, and the liquid was centrifuged at 4°C and 8000 rpm for 2 minutes to remove the supernatant, and the cells were collected, and the operation was repeated twice. The collected cells were dissolved in a phosphate-citrate buffer with a pH value of 4.8, and the wild-type Cot-A laccase was successfully prepared.

[0057] Example 2

[0058] The preparation method of a mutant of Bacillus subtilis Cot-A laccase (hereinafter referred to as mutant Cot-A laccase) comprises the following steps:

[0059] 1) Saturation mutation

[0060] The recombinant plasmid pET28a-Cot-A containing the B. pumilus Cot-A laccase gene (GeneBank accession number KF040050) constructed in the previous period was used as a template, and the substrate benzanthracene was selected to bind to the surrounding of the laccase. (angstroms) of amino acids for confirmation of mutation positions and saturation mutagenesis;

[0061] The specific method for confirming the saturation mutation position is as follows: using Pymol software homology modeling to simulate the 3D structure of Cot-A and find the active pocket of Cot-A laccase; docking the substrate benzanthracene with laccase using Autodock vina, and using Pymol again to find the amino acid residues within 5 angstroms near the site;

[0062] Through the above operation, 19 amino acid sites that may affect the binding of substrate to laccase were obtained (such as Figure 2 As shown); Taking the coding sequence of 19 amino acid sites as the target sequence, 19 upstream primers and 1 universal downstream primer were designed (Table 1), of which S294S-R was the universal downstream primer and the remaining 19 were upstream primers. Large fragment PCR and electrophoresis detection were performed using the recombinant plasmid pET28a-Cot-A as a template to obtain a 6846bp target fragment;

[0063] Table 1 Sequences of 19 upstream primers and 1 universal downstream primer

[0064]

[0065]

[0066] The target fragment was transformed, specifically: BL21 E. coli competent cells were placed on ice to melt, 5-10 μL of the recombinant plasmid containing the target fragment was added to the BL21 E. coli competent cells, and the bacterial solution was applied to the LB solid medium containing Km antibiotics, and cultured at 37°C overnight. 92 monoclonal clones and 2 recombinant plasmid pET28a-Cot-A transformed strains were picked on the solid medium and placed in a 96-well plate, 400 μL of LB culture solution containing km antibiotics was added to each well, and after shaking the bacteria at 37°C for about 12 hours, 100 μL of bacterial solution was drawn and 50% volume of glycerol was added to build a library and preserve the seeds. 700 μL of TB medium (Terrific Broth) and IPTG inducer were added to the remaining bacterial solution in the 96-well plate to induce the bacteria to express for 18 hours at 18°C ​​and 220rpm. After the expression was completed, the bacteria were collected by centrifuge at a speed of 4000rpm and centrifuged for 10 minutes. Wash twice with disodium hydrogen phosphate and citric acid buffer adjusted to pH 4.8. Add a certain amount of disodium hydrogen phosphate-citric acid buffer with a pH of 4.8 and store at -80℃.

[0067] The Cot-A laccase expressed in the 96-well plate in the above step was subjected to a microemulsion reaction with benzanthracene, specifically: isooctane was used as the oil phase, n-hexanol was used as the cosurfactant, and the volume ratio was 1:1. Benzanthracene with a concentration of 5 mg / L was dissolved in isooctane, and rhamnolipid with a concentration of 5.8 mg / L was dissolved in n-hexanol, and ultrasonic vibration was performed until completely dissolved. The solutions were added to the 96-well plate respectively, and placed in a small blue shaker to mix the microemulsion evenly to form a microemulsion system, and reacted at 30°C for 24 hours. After the reaction, the sample was passed through a 0.22 μm organic film, and then the concentration of benzanthracene in the pretreated reaction solution was measured by HPLC / UV (Shimadzu, LC-20AT, Japan) high-performance liquid chromatograph equipped with an Inertsil ODS-P column (5 μm×250 mm×4.6 mm), with a flow rate of 1.0 mL / min, a mobile phase ratio of methanol: water = 80:20 (v / v), an ultraviolet monitor detection wavelength set to 254 nm, and an injection volume of 20 μL. The peak time of benzanthracene is 3-4 min, and the benzanthracene is quantitatively analyzed by calculating the peak area to determine which site in the 96-well plate has the best mutation effect.

[0068] The degraded sites were sequenced, and the sequencing results were compared with the original gene sequence (the gene with accession number KF040050 in GeneBank) to determine the change of the amino acid at the mutation site, that is, the ASP-92 site on Bacillus subtilis Cot-A (i.e., the 92nd amino acid of Bacillus subtilis Cot-A) was obtained as the mutation site.

[0069] 2) Preparation of recombinant bacteria

[0070] PCR reaction was performed using D92P mutant primer and recombinant plasmid pET28a-Cot-A as template.

[0071] The primers for the D92P mutation are as follows:

[0072] Forward primer: 5'-CATACCATTCATCATAGCNNKAGTCAGCATGAAGAACCG-3'

[0073] Reverse primer: 5′-GCGTTCCGCCGGCGCCAGGCTAAAGCTGTTCAGTTTCAC-3′;

[0074] The reaction system was 25 μL, and the specific components of the reaction system were: 1 μL of forward primer, 1 μL of reverse primer, 10 μL of sterile water, 0.5 μL of recombinant plasmid pET28a-Cot-A, and 12.5 μL of KODone enzyme.

[0075] The reaction conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 58°C for 5 s, and extension at 68°C for 3 min, for a total of 34 cycles; final extension at 68°C for 10 min after each cycle, and finally insulation at 12°C for 10 min.

[0076] Take 2 μL of PCR product and run it through 1% agarose gel electrophoresis. After the target product is detected, the result is as follows: Figure 2 Then, the digestion enzyme buffer is mixed with the remaining PCR product and incubated at 35-37°C for 12-14 hours to obtain the mutant recombinant expression vector; the digestion enzyme buffer is composed of 1 μL FD Buffer and 2 μL DpnI endonuclease.

[0077] Take the BL21 competent cell and put it on ice to melt, aspirate 8 μL of the mutant recombinant expression vector, put it in an ice bath for 30 minutes, heat shock it at 42°C for 90 seconds, quickly put it back on ice for 2 minutes, add 1 mL of SOC, and heat bath it at 37°C for 1 hour. Spread the transformation product on an LB plate containing 0.5 mM kanamycin, culture it at 37°C overnight, and observe the colonies. The colonies are white opaque small spots in the form of scattered single dots, and those without contamination by other bacteria are normal colonies.

[0078] Pick 96 normal single colonies from the LB plate and place them in a 96-well plate containing LB culture medium. Add 400 μL of LB culture medium containing 0.5 mM kanamycin to each well and culture on a 37°C shaker for 10 to 12 hours. Then, take 100 μL of bacterial solution and 100 μL of 50 vol% glycerol from each well for seed preservation and library construction. Add TB culture medium containing 0.2 to 0.4 mM IPTG inducer to the remaining bacterial solution. The amount of TB culture medium added is 700 μL. Culture overnight at 18 to 20°C on a shaker at 1200 rpm for 18 to 20 hours. Finally, centrifuge and collect the bacteria, which are recorded as recombinant bacteria.

[0079] 3) Expression of mutant Cot-A laccase

[0080] The preparation process of the mutant Cot-A laccase is the same as the steps in Example 1, except that the recombinant expression colony Cot-A / pET28a / BL21 in Example 1 is replaced by the recombinant bacteria obtained in step 2) of Example 2 to obtain the mutant Cot-A laccase.

[0081] Example 3

[0082] Analysis of mutant Cot-A laccase activity

[0083] 1) Definition of enzyme activity unit

[0084] When the ABTS method was used to determine the activity of laccase, the amount of enzyme required to convert 1 μmol of substrate per minute was defined as an activity unit.

[0085] 2) Enzyme activity determination steps:

[0086] Preheating: Take 2.4 mL of phosphate-citrate buffer solution with a pH value of 4.2-4.8 in a test tube, add 0.4-0.5 mL of ABTS solution (the final concentration of ABTS is 0.4-0.5 mM) to the test tube and place it in a 35-37°C water bath to preheat for 2-3 minutes;

[0087] Reaction: Add 0.1 mL of laccase solution and shake evenly.

[0088] Measurement: The shaken sample was subjected to kinetic measurement using a spectrophotometer. The change in OD value per second within 30 seconds was measured at a wavelength of 420 nm (the reaction rate was a uniform reaction) and the enzyme activity was calculated.

[0089] Enzyme activity formula:

[0090]

[0091] Where: △OD: change in absorbance OD; V 总 : total reaction volume; n: enzyme solution dilution multiple;

[0092] △t: reaction time; V0: volume of enzyme solution;

[0093] ε: molar absorption coefficient of substrate 3.6×10 4 (mol / L) -1 cm -1 .

[0094] Example 4

[0095] Determination of laccase kinetic parameters

[0096] The kinetic parameters of the enzyme were determined using ABTS of different concentrations as a substrate. The final concentration of ABTS in the 3 mL reaction system ranged from 5 to 500 uM. The 3 mL reaction system included 2 mL of phosphate-citrate buffer (pH 4.8), 0.1 mL of laccase solution, and 0.9 mL of ABTS solution (the corresponding volume of ABTS mother liquor was taken according to the final concentration and diluted to 0.9 mL with deionized water). The reaction system was placed in a 37°C water bath for a while, and the change in OD value per second within 30 seconds was measured at a wavelength of 420 nm (the reaction speed was a uniform reaction). The enzyme activity was calculated according to the enzyme activity formula in Example 3.

[0097] by Plotting a straight line, the kinetic curves of the wild-type Cot-A laccase (WT) in Example 1 and the mutant Cot-A laccase (D92P) in Example 2 are shown as follows: Figures 3-4 shown.

[0098] The slope of the straight line is Intersection with vertical axis Intersection with horizontal axis This allows us to find the kinetic parameter V max and K m After measuring the protein content, K can be calculated cat .

[0099] The kinetic parameters of the wild-type Cot-A laccase (WT) in Example 1 and the mutant Cot-A laccase (D92P) in Example 2 were calculated according to the above method, and the results are shown in Table 2:

[0100] Table 2 Kinetic parameters of wild-type Cot-A laccase (WT) and mutant Cot-A laccase (D92P)

[0101]

[0102] K m The value can determine the affinity between the enzyme and the substrate, the greater the affinity, the smaller the affinity.

[0103] At a certain enzyme concentration, the enzyme's V for a specific substrate maxis also a constant.

[0104] K cat It indicates the number of substrate molecules converted by each enzyme molecule per second when the enzyme is saturated with substrate. This constant is generally called the catalytic constant. The larger its value, the higher the catalytic efficiency of the enzyme.

[0105] K cat / K m It is the apparent second-order rate constant for the reaction between enzyme and substrate, sometimes also called the specificity constant.

[0106] It can be calculated from Table 2 that the specificity of the mutant Cot-A laccase is 1.26 times that of the wild-type Cot-A laccase, and the catalytic efficiency is 1.99 times that of the wild-type Cot-A laccase; compared with the wild-type Cot-A laccase and the mutant Cot-A laccase, the specificity and catalytic efficiency of the mutant Cot-A laccase are improved.

[0107] Example 5

[0108] A 500 μL microemulsion system was constructed, and the degradation rate of benzanthracene by the mutant Cot-A laccase was calculated with the wild-type Cot-A laccase as the control. Among them, 200 μL of n-hexanol, 200 μL of isooctane, 0.0025 mg of benzanthracene, 1.40-1.42 g of rhamnolipid, 100 μL of laccase buffer solution (0.6 mg / mL), ultrasonic (20-40 Hz) until the solution is clear, that is, the microemulsion system is successfully constructed, and it is placed in a high-speed 1200 rpm shaker and reacted at 20-25 ° C for 20-24 hours. After the reaction, the sample was passed through an organic membrane with a pore size of 0.44 μm. The sample was detected by high performance liquid chromatography at a flow rate of 1.0 mL / min. The mobile phase ratio was methanol: water = 80:20 (v / v). The detection wavelength of the ultraviolet detector was set to 254 nm. The injection volume was 20 μL. The peak time of benzanthracene was 3 to 4 min. The peak area of ​​benzanthracene degraded by wild-type Cot-A laccase was A0 = 361387, and the peak area of ​​benzanthracene degraded by mutant Cot-A laccase was A x =173466, calculate the degradation rate of benz(a)anthracene, the result is as follows Figure 5 As shown, in Figure 5 WT indicates wild-type Cot-A laccase, and D92P indicates mutant Cot-A laccase.

[0109] Degradation rate calculation formula:

[0110] Where: A0: peak area of ​​benz(a)anthracene degradation by wild-type Cot-A laccase; A x : Peak area of ​​benz(a)anthracene degradation by mutant Cot-A laccase.

[0111] Depend on Figure 5It can be concluded that the degradation rate of benz(a)anthracene by the mutant Cot-A laccase is 52%.

[0112] It can be concluded from the above examples that the mutant of the present invention has higher catalytic efficiency, specificity and degradation rate for polycyclic aromatic hydrocarbons including benzanthracene than the wild-type Bacillus subtilis Cot-A laccase.

[0113] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A mutant of Bacillus subtilis Cot-A laccase, characterized in that: The mutant is obtained by mutating the 92nd amino acid of the wild-type Bacillus subtilis Cot-A laccase from aspartic acid to proline; The amino acid sequence of the wild-type Bacillus subtilis Cot-A laccase is shown in the sequence with accession number H8WGE7 in UniProt.

2. A gene encoding a mutant of Bacillus subtilis Cot-A laccase, characterized in that: The encoding gene encodes the mutant according to claim 1.

3. A mutant recombinant expression vector, characterized in that: It comprises an initial vector and the coding gene described in claim 2.

4. The mutant recombinant expression vector according to claim 3, characterized in that: The initial vector included pET28a.

5. A recombinant bacterium, characterized in that: The invention comprises a gene encoding the mutant according to claim 2 or a mutant recombinant expression vector according to claim 3 or 4.

6. The method for preparing a mutant according to claim 1, characterized in that: The steps include: The recombinant bacteria according to claim 5 are induced to express by using an inducer to obtain a single colony that is best at degrading benzanthracene, wherein the mutant is included in the single colony.

7. The preparation method according to claim 6, characterized in that: The inducing agent is IPTG.

8. Use of the mutant according to claim 1, the mutant gene according to claim 2, the mutant recombinant expression vector according to claim 3 or 4, or the mutant prepared by the preparation method according to claim 6 or 7 in degrading polycyclic aromatic hydrocarbons.

9. The use according to claim 8, characterized in that: The polycyclic aromatic hydrocarbons include benzanthracene.

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