CN110272878A - Laccase TvLac and its encoding gene and application - Google Patents
Laccase TvLac and its encoding gene and application Download PDFInfo
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Abstract
The invention belongs to field of agricultural biotechnology, and in particular to originated from fungus laccase TvLac and its encoding gene and application.The present invention provides a kind of laccase TvLac in Trametes versicolor source.Laccase of the invention can effectively degrade the mycotoxin of different types of structure under mediator assistance, can be widely used for food and feed mycotoxin detoxification field.
Description
Technical field
The invention belongs to agricultural biological technical fields, and in particular to laccase TvLac and its encoding gene and application.
Background technique
Mycotoxin is fungal secondary metabolite, harmful to the health of domestic animal, poultry and the mankind.Common mycotoxin
Including aflatoxin, zearalenone, vomitoxin (deoxynivalenol), citrinin, ochratoxin, volt
Horse rhzomorph, patulin and single-ended spore mycotoxin can be divided into tool cyclic structure and not have two toxoids of cyclic structure.Greatly
Most mycotoxins belong in the subgroup of tool cyclic structure such as aflatoxin and zearalenone, usually poly- by fungi
Ketone approach is synthesized.For example, aflatoxin (aflatoxin) B1 is generated by aspergillus flavus (Aspergillus flavus),
The five yuan of carbocyclic rings and two dihydrofuran rings arranged side by side of coumarin ring and two sides with core are that very strong liver causes
Cancer object.In addition, the structure of zearalenone is m-dihydroxy-benzoic acid phenol lactone.
Physical absorption (or inactivation) and bioconversion are main by two kinds of the mycotoxin detoxification in food and feed
Mode.In addition to this, using microorganism, especially by the enzyme that microorganism generates, it is a kind of new for carrying out the detoxification of mycotoxin
Emerging detoxification means.Pantoic acid lactone hydrolase used in existing enzyme process degrading mold toxin technology and some still cannot
The enzyme of classification can only can specifically degrade single kind toxin by oxidation or Hydrolytic Mechanism degradative fungi toxin,
In practical applications due to it is at high cost the disadvantages of and be extremely restricted.
Summary of the invention
The purpose of the present invention is to provide a kind of laccase TvLac.
A further object of the present invention is to provide the encoding genes of above-mentioned laccase TvLac.
A further object of the present invention is to provide the recombinant expression carriers for containing above-mentioned encoding gene.
A further object of the present invention is to provide the recombinant bacterial strains for containing above-mentioned encoding gene.
A further object of the present invention is to provide the preparation methods of above-mentioned laccase TvLac.
A further object of the present invention is to provide the applications of above-mentioned laccase TvLac.
A further object of the present invention is to provide the mycotoxin degradation agents for containing above-mentioned laccase TvLac.
Laccase TvLac of the invention, amino acid sequence is as shown in SEQ ID No.1:
MGKFHSFVNVVALSLSLSGRVFGAIGPVTDLTISNADVTPDGITRAAVLAGGVFPGPLITGNKGDEFQI
NVIDNLTNETMLKSTTIHWHGIFQAGTNWADGAAFVNQCPIATGNSFLYDFTVPDQAGTFWYHSHLSTQYCDGLRGP
LVVYDPDDANASLYDVDDDTTVITLADWYHTAAKLGPAFPAGPDSVLINGLGRFSGDGGGATNLTVITVTQGKRYRF
RLVSISCDPNFTFSIDGHNMTIIEVGGVNHEALDVDSIQIFAGQRYSFILNANQSIDNYWIRAIPNTGTTDTTGGVN
SAILRYDTAEEIEPTTNATTSVIPLTETDLVPLDNPAAPGDPQVGGVDLAMSLDFSFNGSNFFINNETFVPPTVPVL
LQILSGAQDAASLLPNGSVYTLPSNSTIEISFPIITTDGALNAPGAPHPFHLHGHTFSVVRSAGSSTFNYANPVRRD
TVSTGNSGDNVTIRFTTDNPGPWFLHCHIDFHLDAGFAIVFAEDTADTASANPVPTAWSDLCPTYDALDSSDL
Wherein, which includes 527 amino acid, and 23 amino acid of N-terminal are its signal peptide sequence, signal peptide sequence such as SEQ
Shown in ID No:2:
MGKFHSFVNVVALSLSLSGRVFG
The theoretical molecular weight of mature laccase TvLac is 53.7kDa, and amino acid sequence is as shown in SEQ ID NO.3:
AIGPVTDLTISNADVTPDGITRAAVLAGGVFPGPLITGNKGDEFQINVIDNLTNETMLKSTTIHWHGIF
QAGTNWADGAAFVNQCPIATGNSFLYDFTVPDQAGTFWYHSHLSTQYCDGLRGPLVVYDPDDANASLYDVDDDTTVI
TLADWYHTAAKLGPAFPAGPDSVLINGLGRFSGDGGGATNLTVITVTQGKRYRFRLVSISCDPNFTFSIDGHNMTII
EVGGVNHEALDVDSIQIFAGQRYSFILNANQSIDNYWIRAIPNTGTTDTTGGVNSAILRYDTAEEIEPTTNATTSVI
PLTETDLVPLDNPAAPGDPQVGGVDLAMSLDFSFNGSNFFINNETFVPPTVPVLLQILSGAQDAASLLPNGSVYTLP
SNSTIEISFPIITTDGALNAPGAPHPFHLHGHTFSVVRSAGSSTFNYANPVRRDTVSTGNSGDNVTIRFTTDNPGPW
FLHCHIDFHLDAGFAIVFAEDTADTASANPVPTAWSDLCPTYDALDSSDL
The present invention also provides the gene order for encoding above-mentioned laccase TvLac, genome sequence such as SEQ ID No.4 institutes
Show:
Atgggcaagtttcactcttttgtgaacgtcgtcgcccttagtctttctttgagcggtcgtgtgttcgg
cgccattgggcccgtcaccgacttgactatctctaacgccgatgttacgcctgacggcattactcgtgctgctgtc
ctcgcgggcggcgtcttccccgggcccctcattaccggcaacaagggggatgaattccagatcaatgtcatcgaca
acctgaccaacgagaccatgttgaagtcgaccacaatccactggcatggtatcttccaggccggcaccaactgggc
agacggcgcggccttcgtgaaccagtgccctatcgccacgggaaactcgttcttgtacgacttcaccgttcctgat
caagccggcaccttctggtaccacagccacctgtccacccagtactgtgacggcctgcgcggtcctcttgtggtct
acgaccccgacgatgccaacgcgtctctttacgacgtcgatgacgatactacggttattacgcttgcggactggta
ccacactgcggcgaagctgggccctgccttccccgcgggtccggatagcgtcttgatcaacggtcttggtcggttc
tccggcgatggtggaggagcgacaaacctcaccgtgatcaccgtcacgcaaggcaaacggtaccgcttccgccttg
tgtcgatctcgtgcgaccccaacttcacgttctcgatcgacgggcacaacatgaccatcatagaggtgggcggcgt
caaccacgaggccttggacgtcgactccattcagatttttgcggggcagcggtactccttcatcctcaacgccaac
cagtccatcgacaactactggatccgcgcgatccccaacaccggtaccaccgacaccacgggcggcgtgaactctg
ccattctgcgctacgacaccgccgaagagatcgagcctacgaccaacgcgaccacctccgtcatccctcttaccga
gacggatttggtcccacttgacaaccctgcggctcccggcgacccccaggttggcggtgttgacctggctatgagt
ctcgacttctcctttaacggttccaacttctttatcaacaacgagaccttcgtcccgcccacagtgcccgtactcc
tgcagattttgagcggtgcgcaggacgcggcgagcctgctccccaacggcagtgtctacacactcccttcgaactc
gaccattgagatctcgttccctatcatcaccaccgacggtgctctgaacgcacccggtgctccgcaccccttccat
ctccacggtcacactttctcggtggtgcgcagcgccgggagctcgaccttcaactacgccaacccggtccgacggg
acaccgtcagtactggtaactctggcgacaacgtcactatccgcttcacgaccgacaacccgggcccgtggttcct
ccactgtcacatcgacttccacctggacgcgggcttcgccatcgtctttgcggaggacactgcggacaccgcgtcc
gcgaatcccgttcctacggcgtggagcgatttgtgccccacttacgatgctttggactcgtccgacctctaa
Laccase TvLac coding gene sequence overall length 1584bp.Wherein, the base sequence of signal peptide such as SEQ ID No.5 institute
Show:
Atgggcaagtttcactcttttgtgaacgtcgtcgcccttagtctttctttgagcggtcgtgtgttcggc
CDNA (the removing signal peptide) sequence of mature laccase TvLac is as shown in SEQ ID No.6:
Gccattgggcccgtcaccgacttgactatctctaacgccgatgttacgcctgacggcattactcgtgc
tgctgtcctcgcgggcggcgtcttccccgggcccctcattaccggcaacaagggggatgaattccagatcaatgtc
atcgacaacctgaccaacgagaccatgttgaagtcgaccacaatccactggcatggtatcttccaggccggcacca
actgggcagacggcgcggccttcgtgaaccagtgccctatcgccacgggaaactcgttcttgtacgacttcaccgt
tcctgatcaagccggcaccttctggtaccacagccacctgtccacccagtactgtgacggcctgcgcggtcctctt
gtggtctacgaccccgacgatgccaacgcgtctctttacgacgtcgatgacgatactacggttattacgcttgcgg
actggtaccacactgcggcgaagctgggccctgccttccccgcgggtccggatagcgtcttgatcaacggtcttgg
tcggttctccggcgatggtggaggagcgacaaacctcaccgtgatcaccgtcacgcaaggcaaacggtaccgcttc
cgccttgtgtcgatctcgtgcgaccccaacttcacgttctcgatcgacgggcacaacatgaccatcatagaggtgg
gcggcgtcaaccacgaggccttggacgtcgactccattcagatttttgcggggcagcggtactccttcatcctcaa
cgccaaccagtccatcgacaactactggatccgcgcgatccccaacaccggtaccaccgacaccacgggcggcgtg
aactctgccattctgcgctacgacaccgccgaagagatcgagcctacgaccaacgcgaccacctccgtcatccctc
ttaccgagacggatttggtcccacttgacaaccctgcggctcccggcgacccccaggttggcggtgttgacctggc
tatgagtctcgacttctcctttaacggttccaacttctttatcaacaacgagaccttcgtcccgcccacagtgccc
gtactcctgcagattttgagcggtgcgcaggacgcggcgagcctgctccccaacggcagtgtctacacactccctt
cgaactcgaccattgagatctcgttccctatcatcaccaccgacggtgctctgaacgcacccggtgctccgcaccc
cttccatctccacggtcacactttctcggtggtgcgcagcgccgggagctcgaccttcaactacgccaacccggtc
cgacgggacaccgtcagtactggtaactctggcgacaacgtcactatccgcttcacgaccgacaacccgggcccgt
ggttcctccactgtcacatcgacttccacctggacgcgggcttcgccatcgtctttgcggaggacactgcggacac
cgcgtccgcgaatcccgttcctacggcgtggagcgatttgtgccccacttacgatgctttggactcgtccgacctc
taa
The present invention also provides the recombinant vector comprising above-mentioned laccase TvLac, preferably pPIC9-TvLac.It will be of the invention
Laccase gene be inserted between suitable restriction enzyme cleavage sites of the expression vector, make its nucleotide sequence it is operable with expression
Regulating and controlling sequence is connected.As the most preferred embodiment of the invention, laccase TvLac of the invention is inserted into plasmid
Between EcoRI-NotI restriction enzyme site on pPIC9, the nucleotide sequence is made to be located at the downstream of AOX promoter and by it
Regulation, obtains restructured Pichia pastoris in expression plasmid pPIC9-TvLac.
The present invention also provides the recombinant bacterial strains comprising above-mentioned laccase TvLac, preferably recombinant pichia yeast strain
GS115/TvLac。
The present invention also provides a kind of methods for preparing laccase TvLac, comprising the following steps:
(1) host cell is converted with the recombinant expression carrier comprising encoding laccases TvLac gene, obtains recombinant bacterial strain；
(2) recombinant bacterial strain, induction laccase TvLac expression are cultivated；
(3) laccase TvLac after purification is obtained using anion-exchange chromatography.
The present invention also provides the applications of above-mentioned laccase TvLac, especially in terms of detoxicating mycotoxin, can effectively drop
Solve aflatoxin B1, zearalenone.
The present invention also provides a kind of mycotoxin degradation agents comprising laccase TvLac and mediator, mediator are NSC 611398
Or other can assist the compound of laccase degradative fungi toxin, such as synthesize mediator (- two (3- ethyl-benzothiazole-of 2,2- hydrazine
6- sulfonic acid) (ABTS), murexide (VIO) etc.), natural amboceptor (syringaldehyde, acetosyringone, p-Coumaric Acid etc.) or multi-metal oxygen
Hydrochlorate.
Beneficial effects of the present invention:
The present invention provides a kind of laccase TvLac, have preferable degradation energy to aflatoxin B1, zearalenone
Power.
Detailed description of the invention
Degradation rate of Fig. 1 display recombination laccase TvLac to aflatoxin B1, zearalenone；
The HPLC of Fig. 2 display recombination laccase TvLac degrading aflatoxin B 1 analyzes result；
The HPLC of Fig. 3 display recombination laccase TvLac degrading zearalenone analyzes result.
Specific embodiment
Test material and reagent
1, gene and carrier: yeast expression vector pPIC9 and bacterial strain Pichia pastoris GS115；
2, enzyme and other biochemical reagents: restriction endonuclease, recombinase, aflatoxin B1, zearalenone, syringic acid first
Ester；Trifluoroacetic acid aqueous solution, trifluoroacetic acid, Tris；
3, culture medium: culture medium of pichia pastoris YPD (2% glucose, 2% peptone, 1% yeast extract), BMGY
(1% glycerol, 2% peptone, 1% yeast extract, 10%YNB, 1 ‰ Biotin), BMMY (0.5% methanol, 2% peptone,
1% yeast extract, 10%YNB, 1 ‰ Biotin).
The clone of 1 laccase TvLac encoding gene of embodiment
Laccase TvLac gene source designs specific primer, goes forward side by side in Trametes versicolor (Trametes versicolor)
Row PCR amplification, primer sequence are as follows:
TvLac-F:5'-GCTGAAGCTTACGTAGAATTCGCCATTGGGCCCGTCACCGA-3'；
TvLac-R:5'-AAGGCGAATTAATTCGCGGCCGCGAGGTCGGACGAGTCCA-3'。
PCR product is separated and recovered from purifying with 1% agarose gel electrophoresis, is analyzed its nucleotide sequence such as SEQ ID
Shown in No.4.
Double digestion is carried out to PCR amplification gene products therefrom and pPIC9 carrier using restriction enzyme EcoRI, NotI,
Digestion products are separated and recovered from purifying with 1% agarose gel electrophoresis.Using T4 DNA ligase by after digestion carrier and
Genetic fragment is attached and converts Pichia pastoris cloning host, obtains restructured Pichia pastoris in expression plasmid pPIC9-TvLac.
The preparation of the recombination of embodiment 2 laccase TvLac
The restructured Pichia pastoris in expression plasmid pPIC9-TvLac containing laccase TvLac of acquisition is converted into Pichia pastoris
GS115 obtains recombinant yeast pichia pastoris GS115/TvLac.
The Pichia pastoris GS115 engineered strain containing target gene TvLac is taken, is inoculated in 50mL YPD culture solution, 30
DEG C, after 220rpm shaken cultivation 48h, in the switching of 2% ratio in 300mL BMGY culture medium, 30 DEG C of 220rpm shaken cultivations
48h is replaced BMMY culture medium (containing 0.2mM CuSO4), is expressed using methanol induction external source laccase protein.It is every to supplement for 24 hours
0.5% methanol, coinduction 72h.12000rpm centrifugation removal thallus, is concentrated into 5ml for supernatant using film packet, is placed in 10mM phosphoric acid
Dialysed overnight in disodium hydrogen-citrate buffer solution (pH 6.5).It is purified using Anionic column chromatography, collects electrophoretically pure wash
De- component is simultaneously dialysed to 10mM disodium hydrogen phosphate-citrate buffer solution (pH 6.5) preservation.
3 TvLac- NSC 611398 mediator systems degrading aflatoxin B 1 of embodiment
Aflatoxin B1 is dissolved into the mother liquor that 50mg/L is configured in dimethyl sulfoxide, by following reaction system:
50mM Tris-HCl (pH 7.0), 20 μ L aflatoxin B1 solution (50mg/L), 20 μ L NSC 611398s (10mM), 20 μ L
TvLac(300U/L).The system of laccase TvLac not to be added as control, reaction system sets 3 repetitions.Reaction is at 30 DEG C
It carries out, the acetonitrile that three times volume is added after 10h terminates reaction, using high performance liquid chromatography (HPLC) analysis aflatoxin B1
Degradation rate.Liquid chromatogram is Shimadzu Nexera UHPLC efficient liquid phase chromatographic analysis system, and chromatography column is Zorbax SB-
C18 (4.6 × 250mm, 5 μm), mobile phase A (water of 0.06%TFA), Mobile phase B (acetonitrile of 0.05%TFA)；Gradient elution
Condition 0%B is eluted 4 minutes, and 0%-100%B is eluted 15 minutes, and 100%B is eluted 6 minutes；Detection wavelength 365nm.
As a result as shown in Figure 1 and Figure 2, part aflatoxin B1 has been degraded, degradation rate 69.0%.
4 TvLac- NSC 611398 mediator systems degrading zearalenone of embodiment
Zearalenone is dissolved into the mother liquor that 50mg/L is configured in dimethyl sulfoxide, by following reaction system:
50mM Tris-HCl (pH 7.0), 20 μ L aflatoxin B1 solution (50mg/L), 20 μ L NSC 611398s (10mM), 20 μ L
TvLac(300U/L).The system of laccase TvLac not to be added as control, reaction system sets 3 repetitions.Reaction is at 30 DEG C
It carries out, the acetonitrile that three times volume is added after 10h terminates reaction, using high performance liquid chromatography (HPLC) analysis zearalenone
Degradation rate.Liquid chromatogram is Shimadzu Nexera UHPLC efficient liquid phase chromatographic analysis system, and chromatography column is Zorbax SB-
C18 (4.6 × 250mm, 5 μm), mobile phase A (water of 0.06%TFA), Mobile phase B (acetonitrile of 0.05%TFA)；Gradient elution
Condition 0%B is eluted 4 minutes, and 0%-100%B is eluted 15 minutes, and 100%B is eluted 6 minutes；Detection wavelength 316nm.
As a result as shown in Figure 1, Figure 3, part zearalenone has been degraded, degradation rate 100%.
Laccase TvLac respectively reaches 69.0% He to the degradation rate of aflatoxin B1 and zearalenone in the present invention
100%, which is above the data of existing laccase, if the laccase in the source SCC0152 is to the degradation rate of aflatoxin B1
The laccase in the source about 52.4%, T.versicolor is 81.7% to the degradation rate of zearalenone.
Sequence table
<110>Institute of Feeds,China Academy of Agriculture Sciences
<120>laccase TvLac and its encoding gene and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 527
<212> PRT
<213>Trametes versicolor (Trametes versicolor)
<400> 1
Met Gly Lys Phe His Ser Phe Val Asn Val Val Ala Leu Ser Leu Ser
1 5 10 15
Leu Ser Gly Arg Val Phe Gly Ala Ile Gly Pro Val Thr Asp Leu Thr
20 25 30
Ile Ser Asn Ala Asp Val Thr Pro Asp Gly Ile Thr Arg Ala Ala Val
35 40 45
Leu Ala Gly Gly Val Phe Pro Gly Pro Leu Ile Thr Gly Asn Lys Gly
50 55 60
Asp Glu Phe Gln Ile Asn Val Ile Asp Asn Leu Thr Asn Glu Thr Met
65 70 75 80
Leu Lys Ser Thr Thr Ile His Trp His Gly Ile Phe Gln Ala Gly Thr
85 90 95
Asn Trp Ala Asp Gly Ala Ala Phe Val Asn Gln Cys Pro Ile Ala Thr
100 105 110
Gly Asn Ser Phe Leu Tyr Asp Phe Thr Val Pro Asp Gln Ala Gly Thr
115 120 125
Phe Trp Tyr His Ser His Leu Ser Thr Gln Tyr Cys Asp Gly Leu Arg
130 135 140
Gly Pro Leu Val Val Tyr Asp Pro Asp Asp Ala Asn Ala Ser Leu Tyr
145 150 155 160
Asp Val Asp Asp Asp Thr Thr Val Ile Thr Leu Ala Asp Trp Tyr His
165 170 175
Thr Ala Ala Lys Leu Gly Pro Ala Phe Pro Ala Gly Pro Asp Ser Val
180 185 190
Leu Ile Asn Gly Leu Gly Arg Phe Ser Gly Asp Gly Gly Gly Ala Thr
195 200 205
Asn Leu Thr Val Ile Thr Val Thr Gln Gly Lys Arg Tyr Arg Phe Arg
210 215 220
Leu Val Ser Ile Ser Cys Asp Pro Asn Phe Thr Phe Ser Ile Asp Gly
225 230 235 240
His Asn Met Thr Ile Ile Glu Val Gly Gly Val Asn His Glu Ala Leu
245 250 255
Asp Val Asp Ser Ile Gln Ile Phe Ala Gly Gln Arg Tyr Ser Phe Ile
260 265 270
Leu Asn Ala Asn Gln Ser Ile Asp Asn Tyr Trp Ile Arg Ala Ile Pro
275 280 285
Asn Thr Gly Thr Thr Asp Thr Thr Gly Gly Val Asn Ser Ala Ile Leu
290 295 300
Arg Tyr Asp Thr Ala Glu Glu Ile Glu Pro Thr Thr Asn Ala Thr Thr
305 310 315 320
Ser Val Ile Pro Leu Thr Glu Thr Asp Leu Val Pro Leu Asp Asn Pro
325 330 335
Ala Ala Pro Gly Asp Pro Gln Val Gly Gly Val Asp Leu Ala Met Ser
340 345 350
Leu Asp Phe Ser Phe Asn Gly Ser Asn Phe Phe Ile Asn Asn Glu Thr
355 360 365
Phe Val Pro Pro Thr Val Pro Val Leu Leu Gln Ile Leu Ser Gly Ala
370 375 380
Gln Asp Ala Ala Ser Leu Leu Pro Asn Gly Ser Val Tyr Thr Leu Pro
385 390 395 400
Ser Asn Ser Thr Ile Glu Ile Ser Phe Pro Ile Ile Thr Thr Asp Gly
405 410 415
Ala Leu Asn Ala Pro Gly Ala Pro His Pro Phe His Leu His Gly His
420 425 430
Thr Phe Ser Val Val Arg Ser Ala Gly Ser Ser Thr Phe Asn Tyr Ala
435 440 445
Asn Pro Val Arg Arg Asp Thr Val Ser Thr Gly Asn Ser Gly Asp Asn
450 455 460
Val Thr Ile Arg Phe Thr Thr Asp Asn Pro Gly Pro Trp Phe Leu His
465 470 475 480
Cys His Ile Asp Phe His Leu Asp Ala Gly Phe Ala Ile Val Phe Ala
485 490 495
Glu Asp Thr Ala Asp Thr Ala Ser Ala Asn Pro Val Pro Thr Ala Trp
500 505 510
Ser Asp Leu Cys Pro Thr Tyr Asp Ala Leu Asp Ser Ser Asp Leu
515 520 525
<210> 2
<211> 23
<212> PRT
<213>Trametes versicolor (Trametes versicolor)
<400> 2
Met Gly Lys Phe His Ser Phe Val Asn Val Val Ala Leu Ser Leu Ser
1 5 10 15
Leu Ser Gly Arg Val Phe Gly
20
<210> 3
<211> 504
<212> PRT
<213>Trametes versicolor (Trametes versicolor)
<400> 3
Ala Ile Gly Pro Val Thr Asp Leu Thr Ile Ser Asn Ala Asp Val Thr
1 5 10 15
Pro Asp Gly Ile Thr Arg Ala Ala Val Leu Ala Gly Gly Val Phe Pro
20 25 30
Gly Pro Leu Ile Thr Gly Asn Lys Gly Asp Glu Phe Gln Ile Asn Val
35 40 45
Ile Asp Asn Leu Thr Asn Glu Thr Met Leu Lys Ser Thr Thr Ile His
50 55 60
Trp His Gly Ile Phe Gln Ala Gly Thr Asn Trp Ala Asp Gly Ala Ala
65 70 75 80
Phe Val Asn Gln Cys Pro Ile Ala Thr Gly Asn Ser Phe Leu Tyr Asp
85 90 95
Phe Thr Val Pro Asp Gln Ala Gly Thr Phe Trp Tyr His Ser His Leu
100 105 110
Ser Thr Gln Tyr Cys Asp Gly Leu Arg Gly Pro Leu Val Val Tyr Asp
115 120 125
Pro Asp Asp Ala Asn Ala Ser Leu Tyr Asp Val Asp Asp Asp Thr Thr
130 135 140
Val Ile Thr Leu Ala Asp Trp Tyr His Thr Ala Ala Lys Leu Gly Pro
145 150 155 160
Ala Phe Pro Ala Gly Pro Asp Ser Val Leu Ile Asn Gly Leu Gly Arg
165 170 175
Phe Ser Gly Asp Gly Gly Gly Ala Thr Asn Leu Thr Val Ile Thr Val
180 185 190
Thr Gln Gly Lys Arg Tyr Arg Phe Arg Leu Val Ser Ile Ser Cys Asp
195 200 205
Pro Asn Phe Thr Phe Ser Ile Asp Gly His Asn Met Thr Ile Ile Glu
210 215 220
Val Gly Gly Val Asn His Glu Ala Leu Asp Val Asp Ser Ile Gln Ile
225 230 235 240
Phe Ala Gly Gln Arg Tyr Ser Phe Ile Leu Asn Ala Asn Gln Ser Ile
245 250 255
Asp Asn Tyr Trp Ile Arg Ala Ile Pro Asn Thr Gly Thr Thr Asp Thr
260 265 270
Thr Gly Gly Val Asn Ser Ala Ile Leu Arg Tyr Asp Thr Ala Glu Glu
275 280 285
Ile Glu Pro Thr Thr Asn Ala Thr Thr Ser Val Ile Pro Leu Thr Glu
290 295 300
Thr Asp Leu Val Pro Leu Asp Asn Pro Ala Ala Pro Gly Asp Pro Gln
305 310 315 320
Val Gly Gly Val Asp Leu Ala Met Ser Leu Asp Phe Ser Phe Asn Gly
325 330 335
Ser Asn Phe Phe Ile Asn Asn Glu Thr Phe Val Pro Pro Thr Val Pro
340 345 350
Val Leu Leu Gln Ile Leu Ser Gly Ala Gln Asp Ala Ala Ser Leu Leu
355 360 365
Pro Asn Gly Ser Val Tyr Thr Leu Pro Ser Asn Ser Thr Ile Glu Ile
370 375 380
Ser Phe Pro Ile Ile Thr Thr Asp Gly Ala Leu Asn Ala Pro Gly Ala
385 390 395 400
Pro His Pro Phe His Leu His Gly His Thr Phe Ser Val Val Arg Ser
405 410 415
Ala Gly Ser Ser Thr Phe Asn Tyr Ala Asn Pro Val Arg Arg Asp Thr
420 425 430
Val Ser Thr Gly Asn Ser Gly Asp Asn Val Thr Ile Arg Phe Thr Thr
435 440 445
Asp Asn Pro Gly Pro Trp Phe Leu His Cys His Ile Asp Phe His Leu
450 455 460
Asp Ala Gly Phe Ala Ile Val Phe Ala Glu Asp Thr Ala Asp Thr Ala
465 470 475 480
Ser Ala Asn Pro Val Pro Thr Ala Trp Ser Asp Leu Cys Pro Thr Tyr
485 490 495
Asp Ala Leu Asp Ser Ser Asp Leu
500
<210> 4
<211> 1584
<212> DNA
<213>Trametes versicolor (Trametes versicolor)
<400> 4
atgggcaagt ttcactcttt tgtgaacgtc gtcgccctta gtctttcttt gagcggtcgt 60
gtgttcggcg ccattgggcc cgtcaccgac ttgactatct ctaacgccga tgttacgcct 120
gacggcatta ctcgtgctgc tgtcctcgcg ggcggcgtct tccccgggcc cctcattacc 180
ggcaacaagg gggatgaatt ccagatcaat gtcatcgaca acctgaccaa cgagaccatg 240
ttgaagtcga ccacaatcca ctggcatggt atcttccagg ccggcaccaa ctgggcagac 300
ggcgcggcct tcgtgaacca gtgccctatc gccacgggaa actcgttctt gtacgacttc 360
accgttcctg atcaagccgg caccttctgg taccacagcc acctgtccac ccagtactgt 420
gacggcctgc gcggtcctct tgtggtctac gaccccgacg atgccaacgc gtctctttac 480
gacgtcgatg acgatactac ggttattacg cttgcggact ggtaccacac tgcggcgaag 540
ctgggccctg ccttccccgc gggtccggat agcgtcttga tcaacggtct tggtcggttc 600
tccggcgatg gtggaggagc gacaaacctc accgtgatca ccgtcacgca aggcaaacgg 660
taccgcttcc gccttgtgtc gatctcgtgc gaccccaact tcacgttctc gatcgacggg 720
cacaacatga ccatcataga ggtgggcggc gtcaaccacg aggccttgga cgtcgactcc 780
attcagattt ttgcggggca gcggtactcc ttcatcctca acgccaacca gtccatcgac 840
aactactgga tccgcgcgat ccccaacacc ggtaccaccg acaccacggg cggcgtgaac 900
tctgccattc tgcgctacga caccgccgaa gagatcgagc ctacgaccaa cgcgaccacc 960
tccgtcatcc ctcttaccga gacggatttg gtcccacttg acaaccctgc ggctcccggc 1020
gacccccagg ttggcggtgt tgacctggct atgagtctcg acttctcctt taacggttcc 1080
aacttcttta tcaacaacga gaccttcgtc ccgcccacag tgcccgtact cctgcagatt 1140
ttgagcggtg cgcaggacgc ggcgagcctg ctccccaacg gcagtgtcta cacactccct 1200
tcgaactcga ccattgagat ctcgttccct atcatcacca ccgacggtgc tctgaacgca 1260
cccggtgctc cgcacccctt ccatctccac ggtcacactt tctcggtggt gcgcagcgcc 1320
gggagctcga ccttcaacta cgccaacccg gtccgacggg acaccgtcag tactggtaac 1380
tctggcgaca acgtcactat ccgcttcacg accgacaacc cgggcccgtg gttcctccac 1440
tgtcacatcg acttccacct ggacgcgggc ttcgccatcg tctttgcgga ggacactgcg 1500
gacaccgcgt ccgcgaatcc cgttcctacg gcgtggagcg atttgtgccc cacttacgat 1560
gctttggact cgtccgacct ctaa 1584
<210> 5
<211> 69
<212> DNA
<213>Trametes versicolor (Trametes versicolor)
<400> 5
atgggcaagt ttcactcttt tgtgaacgtc gtcgccctta gtctttcttt gagcggtcgt 60
gtgttcggc 69
<210> 6
<211> 1515
<212> DNA
<213>Trametes versicolor (Trametes versicolor)
<400> 6
gccattgggc ccgtcaccga cttgactatc tctaacgccg atgttacgcc tgacggcatt 60
actcgtgctg ctgtcctcgc gggcggcgtc ttccccgggc ccctcattac cggcaacaag 120
ggggatgaat tccagatcaa tgtcatcgac aacctgacca acgagaccat gttgaagtcg 180
accacaatcc actggcatgg tatcttccag gccggcacca actgggcaga cggcgcggcc 240
ttcgtgaacc agtgccctat cgccacggga aactcgttct tgtacgactt caccgttcct 300
gatcaagccg gcaccttctg gtaccacagc cacctgtcca cccagtactg tgacggcctg 360
cgcggtcctc ttgtggtcta cgaccccgac gatgccaacg cgtctcttta cgacgtcgat 420
gacgatacta cggttattac gcttgcggac tggtaccaca ctgcggcgaa gctgggccct 480
gccttccccg cgggtccgga tagcgtcttg atcaacggtc ttggtcggtt ctccggcgat 540
ggtggaggag cgacaaacct caccgtgatc accgtcacgc aaggcaaacg gtaccgcttc 600
cgccttgtgt cgatctcgtg cgaccccaac ttcacgttct cgatcgacgg gcacaacatg 660
accatcatag aggtgggcgg cgtcaaccac gaggccttgg acgtcgactc cattcagatt 720
tttgcggggc agcggtactc cttcatcctc aacgccaacc agtccatcga caactactgg 780
atccgcgcga tccccaacac cggtaccacc gacaccacgg gcggcgtgaa ctctgccatt 840
ctgcgctacg acaccgccga agagatcgag cctacgacca acgcgaccac ctccgtcatc 900
cctcttaccg agacggattt ggtcccactt gacaaccctg cggctcccgg cgacccccag 960
gttggcggtg ttgacctggc tatgagtctc gacttctcct ttaacggttc caacttcttt 1020
atcaacaacg agaccttcgt cccgcccaca gtgcccgtac tcctgcagat tttgagcggt 1080
gcgcaggacg cggcgagcct gctccccaac ggcagtgtct acacactccc ttcgaactcg 1140
accattgaga tctcgttccc tatcatcacc accgacggtg ctctgaacgc acccggtgct 1200
ccgcacccct tccatctcca cggtcacact ttctcggtgg tgcgcagcgc cgggagctcg 1260
accttcaact acgccaaccc ggtccgacgg gacaccgtca gtactggtaa ctctggcgac 1320
aacgtcacta tccgcttcac gaccgacaac ccgggcccgt ggttcctcca ctgtcacatc 1380
gacttccacc tggacgcggg cttcgccatc gtctttgcgg aggacactgc ggacaccgcg 1440
tccgcgaatc ccgttcctac ggcgtggagc gatttgtgcc ccacttacga tgctttggac 1500
tcgtccgacc tctaa 1515
Claims (10)
1. laccase TvLac, which is characterized in that its amino acid sequence is as shown in SEQ ID No.1 or SEQ ID No.3.
2. laccase TvLac gene, which is characterized in that it encodes laccase TvLac described in claim 1.
3. laccase TvLac gene according to claim 2, which is characterized in that its nucleotide sequence such as SEQ ID No.4 or
Shown in SEQ ID No.6.
4. including the recombinant expression carrier of laccase TvLac gene as claimed in claim 2.
5. including the recombinant bacterial strain of laccase TvLac gene as claimed in claim 2.
6. the method for preparing laccase TvLac described in claim 1, which is characterized in that the described method comprises the following steps:
(1) host cell is converted with the recombinant expression carrier comprising encoding laccases TvLac gene, obtains recombinant bacterial strain；
(2) recombinant bacterial strain, induction laccase TvLac expression are cultivated；
(3) the laccase TvLac is purified.
7. the application of laccase TvLac described in claim 1.
8. application of the laccase TvLac described in claim 1 in terms of detoxicating mycotoxin.
9. mycotoxin degradation agent, which is characterized in that the mycotoxin degradation agent includes laccase described in claim 1
TvLac and mediator.
10. mycotoxin degradation agent according to claim 9, which is characterized in that the mediator is NSC 611398.
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| CN114480365A (en) * | 2022-01-18 | 2022-05-13 | 南昌大学 | Macromolecule-enzyme-inorganic hybrid nano flower, preparation method thereof and application thereof in degrading mycotoxin in edible oil |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111690622A (en) * | 2020-07-29 | 2020-09-22 | 中国农业科学院北京畜牧兽医研究所 | Laccase g2589 derived from fungi as well as gene and application thereof |
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| CN114480365A (en) * | 2022-01-18 | 2022-05-13 | 南昌大学 | Macromolecule-enzyme-inorganic hybrid nano flower, preparation method thereof and application thereof in degrading mycotoxin in edible oil |
| CN114480365B (en) * | 2022-01-18 | 2024-05-28 | 南昌大学 | Polymer-enzyme-inorganic hybrid nanoflower, preparation method thereof and application thereof in degrading mycotoxin in edible oil |
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