CN113930401A - Method for improving laccase catalytic activity, mutant Lcc9-M1, gene and application - Google Patents
Method for improving laccase catalytic activity, mutant Lcc9-M1, gene and application Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, in particular to a method for improving laccase catalytic activity, a mutant Lcc9-M1, a gene and application. The mutant with improved catalytic activity is obtained by site-directed mutagenesis of wild laccase Lcc 9. The laccase mutant provided by the invention has good enzymatic properties, and can be applied to industries such as feed, food, sewage treatment, medicine and the like.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a method for improving the catalytic activity of laccase, a mutant Lcc9-M1, a gene and application.
Background
Laccase (EC 1.10.3.2) is a copper-containing polyphenol oxidase enzyme, widely found in higher plants, fungi, bacteria, insects, and lichen. Wherein, the laccase from fungus has the characteristics of most extensive distribution, highest oxidation-reduction potential, wide substrate spectrum, simple separation and purification and simple identification. Laccase has considerable application prospect in industrial biocatalysis, more than 200 catalytic substrates are known at present, and the catalytic substrates mainly comprise 6 types of phenols (mainly catechol, hydroquinone and other polyphenols and derivatives), arylamines and derivatives, carboxylic acid and derivatives, steroid hormones, biological pigments, ferrocene compounds and derivatives. In the presence of redox mediators, laccase can further catalyze more non-phenolic substrates, such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, azo dyes, organophosphorus pesticides and lignin macromolecular compounds. At present, laccase has important applications in green chemistry such as environmental remediation, biological monitoring, food processing, fiber modification, prevention of dyeing, pharmacy, and organic synthesis.
The catalytic activity has been widely paid attention as an important index for measuring the industrial application value of the enzyme. Besides obtaining natural enzymes with high catalytic activity by mass screening, the improvement of enzyme molecules by means of protein engineering is also a research hotspot in the field of enzyme engineering at present.
Disclosure of Invention
To further optimize derived fromCoprinopsis cinereaThe enzymatic properties of laccase Lcc9, and the present invention was proposed and completed.
The invention aims to provide laccase mutants with improved catalytic activity.
The invention also aims to provide a coding gene of the laccase mutant.
The invention further aims to provide a recombinant vector containing the laccase mutant coding gene.
It is still another object of the present invention to provide a recombinant strain comprising the gene encoding the laccase mutant described above.
It is a further object of the invention to provide a method for the preparation of a laccase with improved catalytic activity.
The invention further aims to provide application of the laccase mutant.
The invention mutates wild laccase Lcc9 to obtain laccase mutant with improved catalytic activity, wherein the amino acid sequence of the mother wild laccase Lcc9 is shown in SEQ ID NO. 1.
The amino acid sequence of the mature wild type laccase Lcc9 after the signal peptide is removed is shown in SEQ ID NO. 2.
According to the specific embodiment of the invention, the amino acid at position 165 of the wild-type laccase Lcc9 shown in the amino acid sequence SEQ ID NO:1 is mutated from Asp to Glu, the amino acid at position 253 is mutated from Met to Ile, the amino acid at position 268 is mutated from Val to Ile, the amino acid at position 372 is mutated from Ser to Leu, and the amino acid at position 447 is mutated from Asp to Asn, so that the laccase mutant Lcc9-M1 is obtained.
According to the specific embodiment of the invention, the amino acid sequence of the laccase mutant Lcc9-M1 is shown as SEQ ID NO. 3.
The amino acid sequence of the mature laccase mutant Lcc9-M1 after the signal peptide is removed is shown as SEQ ID NO. 4.
The invention provides a gene for coding the laccase mutant Lcc 9-M1.
According to the specific embodiment of the invention, the gene sequence of the wild-type laccase Lcc9 is shown in SEQ ID NO: 5, respectively.
The gene sequence of the mature wild type laccase Lcc9 after the signal peptide is removed is shown in SEQ ID NO. 6.
According to the specific embodiment of the invention, the sequence of the encoding gene of the laccase mutant Lcc9-M1 is shown as SEQ ID NO. 7.
The encoding gene sequence of the mature laccase mutant Lcc9-M1 after the signal peptide is removed is shown as SEQ ID NO. 8.
The method for improving the catalytic activity of laccase according to the invention comprises the following steps:
the method comprises the steps of mutating the 165 th amino acid of the wild-type laccase Lcc9 from Asp to Glu, the 253 th amino acid from Met to Ile, the 268 th amino acid from Val to Ile, the 372 nd amino acid from Ser to Leu, and the 447 th amino acid from Asp to Asn, or mutating the 145 th amino acid of the wild-type laccase Lcc9 from Asp to Glu, the 233 th amino acid from Met to Ile, the 248 th amino acid from Val to Ile, the 352 nd amino acid from Ser to Leu, and the 427 th amino acid from Asp to Asn, wherein the amino acid sequence is shown in SEQ ID NO. 1.
The invention provides a recombinant vector containing the encoding gene of the laccase mutant Lcc 9-M1.
The invention also provides a recombinant strain containing the encoding gene of the laccase mutant Lcc9-M1, the preferred strain is Pichia pastoris GS115, and the recombinant strain containing the laccase mutant gene is recombinant gibberellin GS 115-Lcc9-M1。
According to an embodiment of the invention, the method for preparing a laccase with improved catalytic activity is as follows:
(1) transforming host cells by using a recombinant vector containing the encoding gene of the laccase mutant Lcc9-M1 to obtain a recombinant strain;
(2) culturing the recombinant strain, and inducing laccase expression;
(3) recovering and purifying the expressed laccase.
The invention has the beneficial effects that:
the invention mutates wild laccase Lcc9, and the specific activity of laccase mutant Lcc9-M1 is improved by 36% compared with that of wild laccase, thus confirming that the laccase mutant provided by the invention has higher catalytic activity, providing important clues for researching the catalytic activity improvement of Lcc9, and simultaneously providing reference for improving the molecular improvement of other laccases of the family. The catalytic activity of the laccase mutant Lcc9-M1 is greatly improved, and the laccase mutant Lcc9-M1 has good enzymological properties, can be applied to industries such as feed, food, sewage treatment and medicine, and has wide application prospects.
Drawings
FIG. 1 shows SDS-PAGE analysis of recombinant laccase mutants and wild-type expressed in Pichia pastoris;
FIG. 2 shows the pH optimum of laccase mutants with improved catalytic activity compared to wild type;
FIG. 3 shows the temperature optima of laccase mutants with improved catalytic activity with the wild type;
FIG. 4 shows a graph comparing specific activity of laccase mutants with wild type with improved catalytic activity.
Detailed Description
Test materials and reagents
1. Bacterial strain and carrier: expression hostE.coliBL21(DE3) was purchased from Novovozap Biotechnology, Inc.;PichiapastorisGS115 is stored in the laboratory, and expression plasmid vectors pET28a and pPIC9r are stored in the laboratory.
2. Biochemical reagents: restriction enzymes were purchased from NEB; fastpfdna polymerase was purchased from novispan biotechnology limited; purchase of DNA recovery kit from OMEGA; a plasmid miniprep medium-volume kit was purchased from Tiangen Biotechnology Ltd. GeneMorph II Random Mutagenesis Kit was purchased from Aglient. Chaperonin plasmids were purchased from TAKARA. The reagents are all domestic analytical pure reagents (all of which can be purchased from common biochemical reagents).
3. Culture medium:
(1) LB culture medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;
(2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose;
(3) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;
(4) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34% YNB, 0.00004% Biotin;
(5) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (V/V);
(6) ampicillin: 50 mg/mL, 0.22 μ M filter membrane filter out;
(7) 50 × TAE: 57.1 mL/L glacial acetic acid, 242 g/L Tris alkali and 50M EDTA are adjusted to pH8.0;
(8) chloramphenicol: 20 mg/mL, 0.22 μ M filter membrane filter out;
(9) lysozyme: 10 mg/mL, dissolved in 10 mM phosphate buffer (pH 7.0).
Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.
Example 1 error-prone PCR and library construction
To include the Lcc9 genepET28a-Lcc9Plasmid (40 ng/. mu.L) was used as a template, diluted 10-fold, 50-fold, 100-fold respectively to determine the mutation frequency, and PCR was performed using GeneMorph II Random Mutagenesis Kit with the sequence of 40bp at the junction of the Lcc9 gene and the vector as a primer (forward primer: SEQ ID NO: 9; reverse primer SEQ ID NO: 10). The PCR product was recovered and ligated by homologous recombinationNcoI/XhoI on the double digested pET28a vector. By heat shock transformationThe top10 E.coli was transformed competent, single clones were picked and the mutation frequency was verified from the sequencing. Extracting the mixed plasmid, and co-transforming the mixed plasmid and pGro7 into a BL21 Escherichia coli expression vector.
Example 2 cloning selection and preliminary screening of mutants
(1) The first day, 800. mu.L of liquid LB medium (mixed with 50. mu.g/mL kanamycin sulfate (Kan) and 20 ug/mL chloramphenicol (Cm)) was added to each well of the 96-well plate;
(2) single clones were picked with the QPix 420 automated clone selection system and plated into 96-well plates with wild-type and empty-carrier controls at the periphery and in the middle. Placing in a high-speed oscillator at 37 deg.C for 16-18h at 750 r/min;
(3) the next day 200 ul LB (5 mM CuSO) was added to 96-well plates using a line gun40.5 mM IPTG,20 mg/ml L-Arabinose), placing in a high-speed oscillator for culturing at 16 ℃ and 750r/min for 22-24 h;
(4) preserving strains on a plate on the third day, centrifuging a 96-well plate to collect thalli, freezing at the temperature of minus 80 ℃ for 2-3 h after discarding supernatant, taking out the frozen thalli to melt at room temperature, adding 300 mu L of 10mg/m lysozyme, and putting the thalli into a high-speed oscillator to break the walls at 750r/min for 1 h;
(5) the 96-well plate with the wall broken is centrifuged at 2500r/min for 10min, 100. mu.L of the supernatant and 100. mu.L of substrate with 2 mM ABTS concentration are mixed uniformly in another 96-well plate one by one, and then the color reaction is observed. And recording the suspected mutant with relatively dark color, and further re-screening and verifying. The laccase mutant Lcc9-M1 is obtained, wherein the 165 th amino acid of the wild-type laccase Lcc9 shown in SEQ ID NO. 1 is mutated from Asp to Glu, the 253 th amino acid is mutated from Met to Ile, the 268 th amino acid is mutated from Val to Ile, the 372 nd amino acid is mutated from Ser to Leu, and the 447 th amino acid is mutated from Asp to Asn.
EXAMPLE 3 preparation of laccase mutants with improved catalytic Activity
(1) Laccase mutant recombinant vectorpPIC9r-Lcc9-M1Preparation of
Cloning laccase mutant sequence fragment (removing signal peptide) with improved enzyme activity to expression vector pPIC-9r, and naming aspPIC9r-Lcc9-M1。
(2) Large-scale expression of laccase mutant Lcc9-M1 with improved catalytic activity in shake flask level in pichia pastoris
The obtained mutant gene with improved catalytic activityLcc9-M1The recombinant plasmid of (1)pPIC9r-Lcc9-M1Transforming Pichia pastoris GS115 to obtain recombinant yeast strain GS115/Lcc9-M1. Taking a GS115 strain containing the recombinant plasmid, inoculating the strain into a 1L triangular flask of 300 mL BMGY medium, and placing the strain at 30 ℃ and shaking the strain at 220 rpm for 48 h; after this time, the culture broth was centrifuged at 3000 g for 5 min, the supernatant was discarded, and the pellet was resuspended in 200 mL BMMY medium containing 0.5% methanol and again placed at 30 ℃ at 220 rpm for induction culture. 0.5 mL of methanol is added every 12 h, so that the concentration of the methanol in the bacterial liquid is kept at 0.5%, and meanwhile, the supernatant is taken for enzyme activity detection.
(3) Purification of recombinant laccase
The shake flask-expressed recombinant protease supernatant was collected, concentrated by passing through a 10kDa membrane pack while medium therein was replaced with a low-salt buffer, and then further concentrated using a 10kDa ultrafiltration tube. And (3) concentrating the recombinant laccase Lcc9-M1 which can be diluted to a certain multiple, and purifying by ion exchange chromatography. Specifically, 5.0 mL of laccase Lcc9 and mutant Lcc9-M1 concentrated solution are taken to pass through a HiTrap Q Sepharose XL anion column which is balanced by 10 mmol/L phosphate buffer solution (pH 6.0) in advance, then linear gradient elution is carried out by 1 mol/L NaCl, and the eluate collected in the step is subjected to enzyme activity detection and protein concentration determination. Protein electrophoresis is carried out on the purified protein, and dyeing is carried out by using Coomassie brilliant blue dyeing liquid, and the result shows that the recombinant laccase is expressed in the pichia pastoris and is electrophoretically pure after purification (figure 1). In the same way, a recombinant vector containing a mutant sequence of the signal peptide is constructed, and pichia pastoris GS115 is transformed to obtain a recombinant strain.
Example 4 Activity analysis of laccase mutants and wild type with improved recombinant catalytic Activity
The laccase of the invention is subjected to activity analysis by using ABTS as a substrate. The specific method comprises the following steps: laccase enzyme activity was determined by measuring laccase activity against 1mM ABTS (. epsilon.) in 50 mM Tris-NaOH buffer (pH 2.5)420=36000 M-1.cm-1) The oxidation rate of (2). The reaction is carried out at 30 DEG CAnd 3 min, and detecting the wavelength at 420 nm. Laccase activity unit definition: under certain conditions, the amount of enzyme required to produce one μmol of oxidation product per minute.
(1) Comparison of optimum pH analysis
The purified (example 3) expressed laccase Lcc9 and mutant Lcc9-M1 were subjected to enzymatic reactions at different pH to determine their pH optima. The buffer solution is a Tris-NaOH buffer system with the pH value of 2.0-4.0. The optimal pH values of the purified laccase Lcc9 and the mutant Lcc9-M1 are measured in buffer systems with different pH values at 30 ℃, as shown in FIG. 2, and the results show that the optimal pH values of the Lcc9 and the mutant Lcc9-M1 are both 2.5.
(2) Comparison of optimum temperature analysis
The purified laccase is tested for enzyme activity under different temperatures (30-70 ℃) under respective optimum pH conditions, as shown in FIG. 3, the analysis and experiment results show that the optimum temperatures of Lcc9 and mutant Lcc9-M1 are both 60 ℃, and the overall change trends of the Lcc9 and the mutant Lcc9-M1 are similar.
(3) Comparison of specific Activity
The laccase wild type Lcc9 after purification (example 3) was enzymatically reacted with mutant Lcc9-M1 at pH 2.560 ℃ to determine its enzymatic activity.
The specific activity measurement result is shown in FIG. 4, the specific activity of the wild type Lcc9 is 315U/mg, and the specific activity of the mutant Lcc9-M1 is 429U/mg, which is improved by about 36% compared with the wild type.
The above description is provided for the purpose of illustrating the present invention and is not intended to limit the scope of the present invention.
Sequence listing
<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences
<120> method for improving laccase catalytic activity, mutant Lcc9-M1, gene and application
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Gly Pro Trp Phe Phe His Cys His Ile Glu Phe His Leu Val Leu Gly
465 470 475 480
Leu Ala Met Val Phe Met Glu Ala Pro Ser Asp Ile Pro Ser Thr Ser
485 490 495
Pro Pro Pro Pro Ser Trp Ser Glu Leu Cys Pro Lys Phe Glu Ser Leu
500 505 510
Pro Ala Ser Ala Thr Ser Ile Gln Ile Val Pro Thr Pro
515 520 525
<210> 4
<211> 505
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gln Ile Leu Gly Pro Thr Ser Thr Met Thr Val Ser Asn Ile Asp Ala
1 5 10 15
Ser Pro Asp Gly Phe Asn Arg Pro Val Val Ala Val Asn Gly Gln His
20 25 30
Pro Gly Pro Leu Val Arg Ala Asn Lys Gly Asp Asn Phe Arg Ile Asn
35 40 45
Val Val Asn Asp Leu Asn Asp Pro Thr Met Leu Arg Gln Thr Ser Val
50 55 60
His Trp His Gly Val Phe Gln His Gly Thr Ala Trp Ala Asp Gly Pro
65 70 75 80
Asp Gly Val Thr Gln Cys Pro Ile Ala Gln Asn Gly Glu Ser Phe Glu
85 90 95
Tyr Arg Phe Asn Ala Gly Asn Glu Ala Gly Thr Phe Trp Tyr His Ser
100 105 110
His Phe Gly Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Leu Val Ile
115 120 125
Tyr Asp Pro Asn Asp Pro His Arg Asn Leu Tyr Asp Val Asp Asn Ala
130 135 140
Glu Thr Val Ile Thr Leu Val Asp Trp Tyr His Leu Gln Ala Pro Ser
145 150 155 160
Ile Glu Gly Pro Ala Leu Ser Asp Ala Thr Leu Ile Asn Gly Lys Gly
165 170 175
Arg Arg Pro Gly Gly Pro Glu Thr Asp Ile Ala Ile Val Asn Val Gln
180 185 190
Arg Asn Arg Arg Tyr Arg Phe Arg Leu Val Ser Met Ser Cys Asp Pro
195 200 205
Asn Tyr Lys Phe Ser Ile Asp Gly His Lys Leu Thr Val Ile Glu Ala
210 215 220
Asp Gly Gln Leu Thr Glu Pro Leu Ile Val Asp Glu Ile Gln Ile Phe
225 230 235 240
Ala Gly Gln Arg Tyr Ser Phe Ile Leu Ser Ala Asn Arg Pro Val Gly
245 250 255
Asn Tyr Trp Ile Arg Ala Ile Pro Asn Val Gly Ser Asn Asn Leu Pro
260 265 270
Asn Phe Ser Ser Gly Gly Ile Asn Ser Ala Ile Leu Arg Tyr Ala Gly
275 280 285
Ala Pro Asn Ala Asn Pro Thr Ser Thr Pro Val Thr Asn Pro Val Ala
290 295 300
Leu His Glu Ser Asn Leu His Ala Leu Leu Asn Pro Gly Ala Pro Gly
305 310 315 320
Gly Ser Gly Pro Ala Asp Glu Asn Ile Val Leu Gln Met Gly Leu Gly
325 330 335
Pro Ala Gly Phe Glu Ile Asn Gly Val Thr Trp Ala Asn Pro Asp Leu
340 345 350
Pro Val Met Val Gln Ile Met Asn Gly Val Pro Pro Ala Asp Ile Val
355 360 365
Pro Ser Gly Ala Ile His Thr Leu Pro Arg Asn Arg Val Val Glu Val
370 375 380
Ser Ile Pro Gly Phe Glu Leu Ala Gly Pro His Pro Phe His Leu His
385 390 395 400
Gly His Ala Phe Ser Val Val Arg Ser Ala Gly Ser Ser Thr Tyr Asn
405 410 415
Tyr Glu Asn Pro Val Arg Arg Asp Val Val Asn Val Gly Gly Ala Ser
420 425 430
Asp Asn Val Thr Ile Arg Phe Thr Thr Asp Asn Pro Gly Pro Trp Phe
435 440 445
Phe His Cys His Ile Glu Phe His Leu Val Leu Gly Leu Ala Met Val
450 455 460
Phe Met Glu Ala Pro Ser Asp Ile Pro Ser Thr Ser Pro Pro Pro Pro
465 470 475 480
Ser Trp Ser Glu Leu Cys Pro Lys Phe Glu Ser Leu Pro Ala Ser Ala
485 490 495
Thr Ser Ile Gln Ile Val Pro Thr Pro
500 505
<210> 5
<211> 1581
<212> DNA
<213> Coprinopsicinerea griseus (Coprinopsicinerea)
<400> 5
atgtcaagaa agctcttctc gctggcatat cttgcagttg ttcttgtttc agtcgcgggc 60
cagatactag gaccaaccag caccatgact gtttcaaaca tcgatgcatc acctgatgga 120
tttaatcgtc cagtagtcgc tgttaacgga caacatcctg gcccactagt acgtgcgaac 180
aaaggagata actttagaat caacgttgtt aacgatctta acgatcctac aatgcttaga 240
caaacatcag ttcattggca tggagtgttc cagcacggaa cagcatgggc agatggacct 300
gatggagtta cacaatgtcc tatcgcacag aatggcgaat catttgaata tagatttaac 360
gcaggaaacg aagcaggaac attctggtac cattcacatt tcggcacgca atattgtgat 420
ggacttagag ggccattggt catttacgat cctaacgatc ctcatagaaa cctttatgat 480
gttgataacg cagatacagt tatcacactt gttgattggt atcatcttca agcaccttca 540
atcgaaggac ctgcactttc agatgcaaca cttatcaacg gaaagggtcg cagacctgga 600
ggacctgaaa cagatatcgc aatcgttaac gttcaaagaa acagaagata tagatttaga 660
cttgtttcaa tgtcatgtga tcctaactat aaattctcga ttgatggaca taaacttaca 720
gttatcgaag cagatggaca acttacagaa cctcttatgg ttgatgaaat ccaaatcttc 780
gccggccaga ggtactcatt tgttctttca gcaaatcggc cagtaggtaa ttattggatc 840
agagcaatcc ctaacgttgg atcaaacaac cttcctaact tcagtagcgg aggaatcaac 900
tcagcaatcc ttagatatgc aggagcacct aacgcaaacc cgacgtctac gcctgtcacg 960
aaccctgttg cacttcatga atcaaacctt catgcacttc ttaaccctgg agcacctgga 1020
ggatcaggac ctgcagatga gaatatagtt cttcaaatgg gacttggacc tgcaggattt 1080
gaaatcaacg gagttacatg ggcaaaccct gattcacctg ttatggttca aatcatgaac 1140
ggagttcctc ctgcagatat cgttccttca ggagcaatcc atacacttcc tagaaacaga 1200
gttgttgaag tttcaatccc tggatttgaa cttgcaggac ctcatccttt ccacctgcat 1260
ggacatgcat tcagcgtggt tagatcagca ggatcatcaa catataacta tgagaatccc 1320
gttagaagag atgttgttga tgttggagga gcatcagata acgttacaat cagatttaca 1380
acagataacc ctggaccttg gttcttccac tgccatatcg aatttcatct tgttcttgga 1440
cttgcaatgg tattcatgga ggcaccttca gatatccctt caacatcacc tcctcctcct 1500
tcatggtcag aactttgtcc taaatttgaa tcacttcctg catcagcaac atcaatccaa 1560
atcgttccta caccttgatg a 1581
<210> 6
<211> 1521
<212> DNA
<213> Coprinopsicinerea griseus (Coprinopsicinerea)
<400> 6
cagatactag gaccaaccag caccatgact gtttcaaaca tcgatgcatc acctgatgga 60
tttaatcgtc cagtagtcgc tgttaacgga caacatcctg gcccactagt acgtgcgaac 120
aaaggagata actttagaat caacgttgtt aacgatctta acgatcctac aatgcttaga 180
caaacatcag ttcattggca tggagtgttc cagcacggaa cagcatgggc agatggacct 240
gatggagtta cacaatgtcc tatcgcacag aatggcgaat catttgaata tagatttaac 300
gcaggaaacg aagcaggaac attctggtac cattcacatt tcggcacgca atattgtgat 360
ggacttagag ggccattggt catttacgat cctaacgatc ctcatagaaa cctttatgat 420
gttgataacg cagatacagt tatcacactt gttgattggt atcatcttca agcaccttca 480
atcgaaggac ctgcactttc agatgcaaca cttatcaacg gaaagggtcg cagacctgga 540
ggacctgaaa cagatatcgc aatcgttaac gttcaaagaa acagaagata tagatttaga 600
cttgtttcaa tgtcatgtga tcctaactat aaattctcga ttgatggaca taaacttaca 660
gttatcgaag cagatggaca acttacagaa cctcttatgg ttgatgaaat ccaaatcttc 720
gccggccaga ggtactcatt tgttctttca gcaaatcggc cagtaggtaa ttattggatc 780
agagcaatcc ctaacgttgg atcaaacaac cttcctaact tcagtagcgg aggaatcaac 840
tcagcaatcc ttagatatgc aggagcacct aacgcaaacc cgacgtctac gcctgtcacg 900
aaccctgttg cacttcatga atcaaacctt catgcacttc ttaaccctgg agcacctgga 960
ggatcaggac ctgcagatga gaatatagtt cttcaaatgg gacttggacc tgcaggattt 1020
gaaatcaacg gagttacatg ggcaaaccct gattcacctg ttatggttca aatcatgaac 1080
ggagttcctc ctgcagatat cgttccttca ggagcaatcc atacacttcc tagaaacaga 1140
gttgttgaag tttcaatccc tggatttgaa cttgcaggac ctcatccttt ccacctgcat 1200
ggacatgcat tcagcgtggt tagatcagca ggatcatcaa catataacta tgagaatccc 1260
gttagaagag atgttgttga tgttggagga gcatcagata acgttacaat cagatttaca 1320
acagataacc ctggaccttg gttcttccac tgccatatcg aatttcatct tgttcttgga 1380
cttgcaatgg tattcatgga ggcaccttca gatatccctt caacatcacc tcctcctcct 1440
tcatggtcag aactttgtcc taaatttgaa tcacttcctg catcagcaac atcaatccaa 1500
atcgttccta caccttgatg a 1521
<210> 7
<211> 1581
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atgtcaagaa agctcttctc gctggcatat cttgcagttg ttcttgtttc agtcgcgggc 60
cagatactag gaccaaccag caccatgact gtttcaaaca tcgatgcatc acctgatgga 120
tttaatcgtc cagtagtcgc tgttaacgga caacatcctg gcccactagt acgtgcgaac 180
aaaggagata actttagaat caacgttgtt aacgatctta acgatcctac aatgcttaga 240
caaacatcag ttcattggca tggagtgttc cagcacggaa cagcatgggc agatggacct 300
gatggagtta cacaatgtcc tatcgcacag aatggcgaat catttgaata tagatttaac 360
gcaggaaacg aagcaggaac attctggtac cattcacatt tcggcacgca atattgtgat 420
ggacttagag ggccattggt catttacgat cctaacgatc ctcatagaaa cctttatgat 480
gttgataacg cagaaacagt tatcacactt gttgattggt atcatcttca agcaccttca 540
atcgaaggac ctgcactttc agatgcaaca cttatcaacg gaaagggtcg cagacctgga 600
ggacctgaaa cagatatcgc aatcgttaac gttcaaagaa acagaagata tagatttaga 660
cttgtttcaa tgtcatgtga tcctaactat aaattctcga ttgatggaca taaacttaca 720
gttatcgaag cagatggaca acttacagaa cctcttatcg ttgatgaaat ccaaatcttc 780
gccggccaga ggtactcatt tattctttca gcaaatcggc cagtaggtaa ttattggatc 840
agagcaatcc ctaacgttgg atcaaacaac cttcctaact tcagtagcgg aggaatcaac 900
tcagcaatcc ttagatatgc aggagcacct aacgcaaacc cgacgtctac gcctgtcacg 960
aaccctgttg cacttcatga atcaaacctt catgcacttc ttaaccctgg agcacctgga 1020
ggatcaggac ctgcagatga gaatatagtt cttcaaatgg gacttggacc tgcaggattt 1080
gaaatcaacg gagttacatg ggcaaaccct gatttacctg ttatggttca aatcatgaac 1140
ggagttcctc ctgcagatat cgttccttca ggagcaatcc atacacttcc tagaaacaga 1200
gttgttgaag tttcaatccc tggatttgaa cttgcaggac ctcatccttt ccacctgcat 1260
ggacatgcat tcagcgtggt tagatcagca ggatcatcaa catataacta tgagaatccc 1320
gttagaagag atgttgttaa tgttggagga gcatcagata acgttacaat cagatttaca 1380
acagataacc ctggaccttg gttcttccac tgccatatcg aatttcatct tgttcttgga 1440
cttgcaatgg tattcatgga ggcaccttca gatatccctt caacatcacc tcctcctcct 1500
tcatggtcag aactttgtcc taaatttgaa tcacttcctg catcagcaac atcaatccaa 1560
atcgttccta caccttgatg a 1581
<210> 8
<211> 1521
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cagatactag gaccaaccag caccatgact gtttcaaaca tcgatgcatc acctgatgga 60
tttaatcgtc cagtagtcgc tgttaacgga caacatcctg gcccactagt acgtgcgaac 120
aaaggagata actttagaat caacgttgtt aacgatctta acgatcctac aatgcttaga 180
caaacatcag ttcattggca tggagtgttc cagcacggaa cagcatgggc agatggacct 240
gatggagtta cacaatgtcc tatcgcacag aatggcgaat catttgaata tagatttaac 300
gcaggaaacg aagcaggaac attctggtac cattcacatt tcggcacgca atattgtgat 360
ggacttagag ggccattggt catttacgat cctaacgatc ctcatagaaa cctttatgat 420
gttgataacg cagaaacagt tatcacactt gttgattggt atcatcttca agcaccttca 480
atcgaaggac ctgcactttc agatgcaaca cttatcaacg gaaagggtcg cagacctgga 540
ggacctgaaa cagatatcgc aatcgttaac gttcaaagaa acagaagata tagatttaga 600
cttgtttcaa tgtcatgtga tcctaactat aaattctcga ttgatggaca taaacttaca 660
gttatcgaag cagatggaca acttacagaa cctcttatcg ttgatgaaat ccaaatcttc 720
gccggccaga ggtactcatt tattctttca gcaaatcggc cagtaggtaa ttattggatc 780
agagcaatcc ctaacgttgg atcaaacaac cttcctaact tcagtagcgg aggaatcaac 840
tcagcaatcc ttagatatgc aggagcacct aacgcaaacc cgacgtctac gcctgtcacg 900
aaccctgttg cacttcatga atcaaacctt catgcacttc ttaaccctgg agcacctgga 960
ggatcaggac ctgcagatga gaatatagtt cttcaaatgg gacttggacc tgcaggattt 1020
gaaatcaacg gagttacatg ggcaaaccct gatttacctg ttatggttca aatcatgaac 1080
ggagttcctc ctgcagatat cgttccttca ggagcaatcc atacacttcc tagaaacaga 1140
gttgttgaag tttcaatccc tggatttgaa cttgcaggac ctcatccttt ccacctgcat 1200
ggacatgcat tcagcgtggt tagatcagca ggatcatcaa catataacta tgagaatccc 1260
gttagaagag atgttgttaa tgttggagga gcatcagata acgttacaat cagatttaca 1320
acagataacc ctggaccttg gttcttccac tgccatatcg aatttcatct tgttcttgga 1380
cttgcaatgg tattcatgga ggcaccttca gatatccctt caacatcacc tcctcctcct 1440
tcatggtcag aactttgtcc taaatttgaa tcacttcctg catcagcaac atcaatccaa 1500
atcgttccta caccttgatg a 1521
<210> 9
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ctttaagaag gagatatacc atgcagatac taggaccaac cagcac 46
<210> 10
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cagtggtggt ggtggtggtg ctcgagaggt gtaggaacga tttggattg 49
Claims (8)
1. The laccase mutant Lcc9-M1 with improved catalytic activity is characterized in that the amino acid sequence of the laccase mutant is shown as SEQ ID NO. 3 or NO. 4.
2. A method of increasing the catalytic activity of a laccase enzyme, the method comprising the steps of:
the 165 th amino acid of the wild laccase Lcc9 with the amino acid sequence shown as SEQ ID NO. 1 is mutated into Glu from Asp, the 253 th amino acid is mutated into Ile from Met, the 268 th amino acid is mutated into Val, the 372 nd amino acid is mutated into Leu from Ser, the 447 th amino acid is mutated into Asn from Asp,
or the 145 th amino acid of the wild-type laccase Lcc9 with the amino acid sequence shown as SEQ ID NO. 2 is mutated from Asp to Glu, the 233 th amino acid is mutated from Met to Ile, the 248 th amino acid is mutated from Val to Ile, the 352 nd amino acid is mutated from Ser to Leu, and the 427 th amino acid is mutated from Asp to Asn.
3. Laccase mutant gene, characterized in that it encodes the laccase mutant Lcc9-M1 with improved catalytic activity according to claim 1.
4. The laccase mutant gene according to claim 3, wherein the nucleotide sequence is shown in SEQ ID NO. 7 or NO. 8.
5. A recombinant vector comprising the laccase mutant gene of claim 3.
6. A recombinant strain comprising the laccase mutant gene of claim 3.
7. A method for producing a laccase with improved catalytic activity, comprising the steps of:
(1) transforming a host cell with a recombinant vector comprising the encoding gene of the laccase mutant Lcc9-M1 with improved catalytic activity of claim 1 to obtain a recombinant strain;
(2) culturing the recombinant strain, and inducing and expressing laccase;
(3) recovering and purifying the expressed laccase.
8. The use of the laccase mutant Lcc9-M1 with improved catalytic activity as claimed in claim 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703212A (en) * | 2022-03-01 | 2022-07-05 | 东华大学 | Method for modifying laccase by using random mutation method of specific section and laccase strain LAC123 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8410336B2 (en) * | 2007-12-12 | 2013-04-02 | Monsanto Technology Llc | Transgenic plants with enhanced agronomic traits |
| CN109295017B (en) * | 2018-07-24 | 2021-08-31 | 安徽大学 | Fungal laccase mutant PIE5, and expression strain and application thereof |
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2021
- 2021-12-21 CN CN202111566938.6A patent/CN113930401B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8410336B2 (en) * | 2007-12-12 | 2013-04-02 | Monsanto Technology Llc | Transgenic plants with enhanced agronomic traits |
| CN109295017B (en) * | 2018-07-24 | 2021-08-31 | 安徽大学 | Fungal laccase mutant PIE5, and expression strain and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| KILARU,S., HOEGGER,P.J. AND KUES,U.: "TPA_exp: laccase 9 [Coprinopsis cinerea okayama7#130]", 《GENBANK: DAA04514.1》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114703212A (en) * | 2022-03-01 | 2022-07-05 | 东华大学 | Method for modifying laccase by using random mutation method of specific section and laccase strain LAC123 |
| CN114703212B (en) * | 2022-03-01 | 2024-03-29 | 东华大学 | Method for modifying laccase by using specific segment random mutation method and laccase strain LAC123 |
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