CN102154234A - Cytochrome P450 monooxygenase with polycyclic aromatic hydrocarbon hydroxylase-like activity - Google Patents
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Abstract
The invention discloses a cytochrome P450 monooxygenase with polycyclic aromatic hydrocarbon hydroxylase-like activity, which has a nucleotide sequence shown in SEQ ID No.1. The gene is useful for preparing transgenic microorganisms or animals or plants which can produce the cytochrome P450 monooxygenase with polycyclic aromatic hydrocarbon hydroxylase-like activity and for recovering and obtaining enzymes encoded by the gene.
Description
Technical field
The present invention relates to a kind of cytopigment monooxygenase and gene thereof with polycyclic aromatic hydrocarbons hydroxylase activity; This enzyme can be applied to contain the biological restoration of polycyclic aromatic hydrocarbons wastewater treatment or soil.
Background technology
Cytochrome P450 is the monooxygenase that a class contains protoheme, and the typical mechanism of catalyzed reaction is by electron transport chain, molecular oxygen is reduced, and one of them Sauerstoffatom is added in the substrate, needs NAD (P) H as electron donor in the reaction process.P450 can the many different structure materials of catalysis reaction such as hydroxylation, epoxidation, dealkylation, deamination, dehydrogenation, dehalogenate.The different member in source has substrate spectrum widely in the P450 family, has both comprised multiple sterol and lipid in the physiological process, also comprises many xenobiontics such as dyestuff, agricultural chemicals, polycyclic aromatic hydrocarbons, polychlorobiphenyl, heterogeneous ring compound etc.Therefore, can use P450 and handle the difficult degradation compound and be converted into other biological and easily utilize material, in conjunction with conventional bioreediation means, thus more effective removal environmental pollutant.Human synthetic compound, great majority can both be as the substrate of P450, and this makes P450 have broad application prospects in the biological restoration of environmental pollution.
Hard-degraded substance in the waste water mainly is some phenyl ring classes and heterocyclic material at present, and is strong mainly due to the benzene ring substance hydrophobicity, is difficult to arrive the substrate center of most of enzyme, caused it to be difficult to be degraded by the biological treatment means of routine.Cytochrome P450 can make the c h bond fracture of compound, on the C atom, add an O atom, make the substrate hydroxylation, thereby improve the water-soluble of hydrophobic compound, make its some oxydase that can enter other, the catalytic center of peroxidase, thereby reach the purpose of thorough degraded phenyl ring and heterocyclic material.
In order to improve the application of this fermentoid in degrading polycyclic aromatic hydrocarbons class material, thereby novel cytochrome P 450 monooxygenases that can better degrading polycyclic aromatic hydrocarbons is sought in expectation.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of cytochrome P 450 monooxygenases with polycyclic aromatic hydrocarbons hydroxylase activity is provided.
The objective of the invention is to be achieved through the following technical solutions: a kind of cytochrome P 450 monooxygenases with polycyclic aromatic hydrocarbons hydroxylase activity, it has the nucleotide sequence of SEQ ID NO.1.In addition, the mutant form of nucleotide sequence shown in also comprising, described mutation type comprises: disappearance, nonsense, insertion, missense.Described nucleotide sequence coded polypeptide has the aminoacid sequence of SEQ ID NO.2.
The invention has the beneficial effects as follows, the present invention relates to
Bacillus pumilusThe separation and the expression of cytochrome P 450 monooxygenases gene with polycyclic aromatic hydrocarbons hydroxylase activity.The cytochrome P 450 monooxygenases with polycyclic aromatic hydrocarbons hydroxylase activity of transgenic microorganism or animals and plants this gene is used to produce to(for) preparation, and it is useful to reclaim the enzyme that obtains this genes encoding.In addition, the present invention also provides active amino acid sequence of polypeptide of the cytochrome P 450 monooxygenases with polycyclic aromatic hydrocarbons hydroxylase activity and functional equivalent body.Simultaneously, the present invention also provides preparation, separates, and purifying has the method for the active polypeptide of cytochrome P 450 monooxygenases of polycyclic aromatic hydrocarbons hydroxylase activity.
Embodiment
One aspect of the present invention provides a kind of energy coding to have the nucleotide sequence of the cytochrome P 450 monooxygenases polypeptide of polycyclic aromatic hydrocarbons hydroxylase activity.Said nucleotide sequence coded the have polypeptide of the aminoacid sequence among the SEQ ID NO.2 or the modified forms of described polypeptide, on this modified forms function quite or relevant with cytochrome P 450 monooxygenases.Nucleotide sequence has the polynucleotide sequence of SEQ ID NO.1 and its mutant form, and mutation type comprises: disappearance, nonsense, insertion, missense.
The present invention provides a kind of active polypeptide of cytochrome P 450 monooxygenases with polycyclic aromatic hydrocarbons hydroxylase activity on the other hand.This polypeptide has polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of the aminoacid sequence among the SEQ ID No.2.
The method that production has the cytochrome P 450 monooxygenases of polycyclic aromatic hydrocarbons hydroxylase activity is:
1, isolate the nucleotide sequence SEQ ID NO.1 of Codocyte cytochrome p 450 monooxygenase:
2, make up the expression vector that contains SEQ ID NO.1 nucleotide sequence;
3, change expression vector in the step 2 over to host cell, formation can be produced the reconstitution cell of cytochrome P 450 monooxygenases:
4, the reconstitution cell in the culturing step 3;
5, separation, purifying obtain having the cytochrome P 450 monooxygenases of polycyclic aromatic hydrocarbons hydroxylase activity.
At first, the invention provides isolatingly, coding has the polynucleotide molecule of cytochrome P 450 monooxygenases active polypeptide of polycyclic aromatic hydrocarbons hydroxylase activity, this nucleic acid molecule be from
Bacillus pumilusIn be separated to, have the nucleotide sequence of SEQ ID NO.1, its coding has 1047 amino acid whose polypeptide, infers that molecular weight is 119.21 kDa.
The invention still further relates to a kind of recombinant vectors, this carrier comprises isolating nucleic acid molecule of the present invention, and the host cell that includes recombinant vectors.Simultaneously, the present invention includes the method that makes up this recombinant vectors and host cell, and the method for producing the cytochrome P 450 monooxygenases with polycyclic aromatic hydrocarbons hydroxylase activity with the recombined engineering technology.
The present invention provides a kind of isolating cytochrome P 450 monooxygenases or polypeptide with polycyclic aromatic hydrocarbons hydroxylase activity further, it is characterized in that having SEQ ID NO.2 aminoacid sequence, or at least 70% is similar, more preferably, at least have 90%, 95%, 99% identical.
In the present invention, " isolating " DNA is meant that this DNA or segment have been arranged in its both sides under native state sequence separates, and refers to that also this DNA or segment with under the native state follow the component of nucleic acid to separate and separate with the protein of following it in cell.
In the present invention, " cytochrome P 450 monooxygenases gene " refer to the encode nucleotide sequence of cytochrome P 450 monooxygenases active polypeptide with polycyclic aromatic hydrocarbons hydroxylase activity is as nucleotide sequence and the degenerate sequence thereof of SEQ ID NO.1.This degenerate sequence be meant have one or more codons to be encoded in this sequence the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of known codon, so be low to moderate about 70% the degenerate sequence described aminoacid sequence of SEQ ID NO.2 of also encoding out with SEQ ID NO.1 nucleotide sequence homology.This term also comprises can be under the rigorous condition of moderate, more preferably under highly rigorous condition with the nucleotide sequence of the nucleotide sequence hybridization of SEQ ID NO.1.This term also comprises and SEQ ID NO.1 nucleotide sequence homology 70% at least, preferably at least 80%, more preferably at least 90%, and at least 95% nucleotide sequence best.
In the present invention, " isolating " proteic polypeptide is meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as uses column chromatography, and PAGE or HPLC method are measured the purity of polypeptide.Isolated polypeptide is substantially free of the component of following it under the native state.
In the present invention, " cytochrome P 450 monooxygenases " refers to have the active SEQ ID of the cytochrome P 450 monooxygenases NO.2 polypeptide of sequence of polycyclic aromatic hydrocarbons hydroxylase activity.This term also comprises the varient of SEQ ID NO.2 sequence, and these varients have and n cell cytochrome p 450 monooxygenase identical functions.These varients include, but is not limited to several amino acid whose disappearances, insert and/or replace, and add one or several amino acid at C latter end and/or N-terminal, also can be the difference that does not influence on the modified forms of sequence.For example, for known in the field, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C latter end and/or N-terminal and also can not change proteinic function usually.This term also comprises the active part and the reactive derivative of the cytochrome P 450 monooxygenases with polycyclic aromatic hydrocarbons hydroxylase activity.
In the present invention, can select various carrier known in the art for use, as commercially available various plasmids, clay, phage and retrovirus etc.When producing cytochrome P 450 monooxygenases of the present invention, the cytochrome P 450 monooxygenases gene order can be able to be operated the low expression regulation sequence that is connected in, thereby form the cytochrome P 450 monooxygenases expression vector.Expression vector contains replication origin and expression regulation sequence, promotor, enhanser and necessary machining information site.Expression vector also must contain alternative marker gene, as a) providing to microbiotic or other toxicant (penbritin, the protein or the b of resistance kantlex, methotrexate etc.)) complementary auxotroph protein or c) protein of the essential nutritive ingredient that does not have in the complex medium is provided.Various different hosts' appropriate flags gene be well known in the art or production firm's specification sheets famous.These expression vectors can be with well known to a person skilled in the art recombinant DNA technology preparation, as can be with reference to people such as Sambrook, and 1989 or people such as Ausubel, 1992.
Recombinant expression vector can be introduced host cell with method well known in the art, and these methods comprise: electrotransformation, Calcium Chloride Method, particle bombardment etc.The process that the external source recombinant vectors is imported host cell is called " conversion ".By cultivating host cell, induce the expression of desirable proteins, and by protein separation technology known in the art, obtain required protein as column chromatography etc.Also can adopt these protein of synthetic such as solid phase technique.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.Prokaryotic cell prokaryocyte such as intestinal bacteria commonly used, Bacillus subtilus etc.Eukaryotic cell such as yeast cell commonly used, or various animal and plant cells.
Cytochrome P 450 monooxygenases full length gene sequence of the present invention or its segment can be used the pcr amplification method usually, recombination method, or the method for synthetic obtains.For the pcr amplification method, can design primer by relevant nucleotide sequence disclosed according to the present invention, prepare with ordinary method well known by persons skilled in the art
Bacillus pumilusDNA is the mould utmost point, amplification and obtain relevant sequence.In case obtained relevant sequence, just it can be cloned into relevant carrier, change host cell again over to, from the host cell after the propagation, separate obtaining large batch of relevant sequence then by ordinary method.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the clone of cytochrome P 450 monooxygenases gene and the structure of expression vector
The cytochrome P 450 monooxygenases complete encoding sequence (SEQ ID NO.1) that obtains according to gene annotation among the embodiment, design can amplify the primer that complete coding is read frame, and on positive anti-primer, introduce restriction endonuclease sites respectively, so that construction of expression vector.With
BacilluspumilusDSM27 DNA is a template, amplifying cells cytochrome p 450 monooxygenase gene total length.Under guarantee reading the correct prerequisite of frame, recombinate to expression vector pET28a(Novagen) in, again recombinant vectors is converted into that (method for transformation is CaCl in the bacillus coli DH 5 alpha competent cell
2Method or electrotransformation), with antibiotic marker method screening (being kalamycin resistance in this example) male reorganization bacterium DH5 α-pET28a-P450.The engineering bacteria DH5 α-pET28a-P450 of picking list bacterium colony contains in the LB substratum of 30 μ g/ml kantlex jolting in 4 ml and cultivates 37 ° of C and spend the night, be converted in expression vector e. coli bl21 (DE3) competent cell after extracting recombinant plasmid, express bacterium BL21-pET28a-P450 with antibiotic marker method screening (being kalamycin resistance in this example) male recombination high efficiency.
Embodiment 2: the expression and the purifying of reconstitution cell cytochrome p 450 monooxygenase
The engineering bacteria BL21-pET28a-P450 of picking list bacterium colony contains in the LB substratum of 30 μ g/ml kantlex jolting in 4 ml and cultivates 37 ° of C and spend the night, drawing nutrient solution by the concentration of l:100 cultivated about 3 hours in new LB substratum (containing 30 μ g/ml kantlex), after reaching 0.5 to OD600, add IPTG to final concentration 0.5 mmol/L, continue at 25 ° of C and cultivate 5 respectively, 10,15,20 hours.It is centrifugal to get 1 different ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspension bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of l2000 rpm, supernatant adds electrophoresis in the 12% SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing desirable proteins.
As stated above behind the engineering bacteria of abduction delivering desirable proteins, with bacterium centrifugation, add 8 ml Binding Buffer by per 100 ml bacterium liquid, after the ultrasonication, 15000 rpm get supernatant after centrifugal 30 minutes, add 2 ml Ni-NTA resins (Invitrogen), 30 ° of C joltings were in conjunction with 30 minutes, staticly settle, abandon supernatant, after precipitation is washed 2 times with 10 ml Wash Buffer, last 2 ml chromatography columns, using Elution Buffer wash-out target protein then, is that unit is in charge of collection with 1 ml, merges enzyme the highest several pipes alive.Be stored in-80 ° of C behind the glycerine of the supernatant interpolation 50% of wash-out, and carry out the SDS-PAGE electrophoresis, detect purification effect.Protein band at 120 kDa places is cytochrome P 450 monooxygenases.
Embodiment 3: the evaluation of reconstitution cell cytochrome p 450 monooxygenase polycyclic aromatic hydrocarbons hydroxylation product
With the reconstitution cell cytochrome p 450 behind the purifying respectively at different types of polycyclic aromatic hydrocarbons room temperature condition down behind reaction 5 h, add vitriol oil termination reaction, reaction product is after the Solid-Phase Extraction purifying concentrates, utilize GC/MS that the polycyclic aromatic hydrocarbons hydroxylation product is carried out isolation identification, determine the concrete site of P450 hydroxylation polycyclic aromatic hydrocarbons.
SEQUENCE?LISTING
＜110〉Zhejiang University
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＜120〉has the cytochrome P 450 monooxygenases of polycyclic aromatic hydrocarbons hydroxylase activity
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<160> 2
?
<170> PatentIn?version?3.5
?
<210> 1
<211> 3144
<212> DNA
<213> Bacillus?pumilus
?
<400> 1
atgcaacaaa?catcaatcat?tccaaaacca?aaaacatacg?gaccttttaa?aaatattccc 60
?
catataaaaa?agggggagct?ttctcaaacc?ttttggaggc?tagcagatga?attaggaccg 120
?
atctttcagt?ttgaattttc?aaaagcaaca?agtatttttg?tttctaatca?tgaacttttc 180
?
caagaaatat?gtgatgagag?ccgttttgat?aaatacattg?ggactagtct?caataaagta 240
?
agagcatttg?caggggatgg?gttatttacg?agctggacag?aagaaccgaa?ctggagaaag 300
?
gctcaccaca?tcttgatgcc?ggcgtttagt?cagcaggcca?tgaagggcta?tcatgaaatg 360
?
atgctcgata?ttgccacaca?gcttgtacaa?aaatggcaaa?gaacaggccg?tgatgaagaa 420
?
attgaagtag?cagaggatat?gacaaagctt?actttagata?cgatcggact?ttgtggtttt 480
?
gatttcaggt?ttaacagttt?ttataaagaa?aatcagcatc?cattcatcga?aagtatggtg 540
?
aatggtttaa?gcgaagcgat?ggatcaggcg?agccggttgc?cggttgcaga?taagctaatg 600
?
atcaaaagaa?gaaaaaaatt?tgaagaaaat?gtcgatttta?tgaagcaatt?agtagatgac 660
?
attattcaag?aacggaaaaa?acaagataaa?acgggcgatg?atttactgtc?cctcatgctg 720
?
catgcaaagg?accctgaaac?aggagagcgc?ctgtcagatg?aaaatattcg?ctatcaaatt 780
?
attaccttct?taatagctgg?gcacgaaaca?acaagcgggc?tactatcctt?tgcgatttat 840
?
ttcttattaa?agaatcctga?aaaattaaag?aaagccgtcc?aagaagcaga?tgatgtactg 900
?
caaggcggac?tgccaacatt?taagcaggta?caaaaactaa?attacactcg?tatggtttta 960
?
aatgaatctc?ttcgcctctg?gccaacagcg?cctacgttct?ctctttatgc?gaaagaggac 1020
?
accgtcatcg?gagggaaata?ttcgattgaa?aagaaccaaa?gtgtctccgt?gctgctacct 1080
?
aagttacatc?gcgatcaagc?ggtatgggga?gaggatgcgg?aagaatttaa?accagagcgg 1140
?
tttctacacc?ctgaaaagat?cccgcagcat?gcctacaagc?cttttgggaa?tggacagcgt 1200
?
gcatgtattg?gcatgcaatt?cgctcttcat?gaagccacaa?tggtactggc?aatggtcctg 1260
?
cacaacctgg?aattgattga?tcacacatca?tatgaacttg?atttaaaaga?atctctgacg 1320
?
attaagccaa?acgattttaa?aatcaaagtg?cggccaagga?agcagcaact?ctttatggta 1380
?
ccaccgaaag?aagagacgaa?aaaaagcacc?acaactgatg?agtctaaagt?gaagagtcat 1440
?
ggtacaccac?tgcttgtttt?atatggatca?aatcttggca?cggcgcagca?aatcgcaaac 1500
?
gaattggctg?aagaaggaaa?agcaaaaggg?tttgatgtga?ccactgctcc?gcttgatgac 1560
?
tatacacgcc?aattaccaga?taagggtgca?gtctttatcg?tgaccgcttc?atataatgga 1620
?
catccgcctg?accatgcgaa?aaaatttgtg?gattgggtca?cgcaggagaa?agagcaggat 1680
?
ttaacaaacg?tgacgtttgc?tgtgtttgga?tgcggagatc?gaaattgggc?gagtacgtac 1740
?
cagcgtattc?ctcgtctcat?tgatgaagca?cttgaaagaa?aaggtgcgaa?gcgtgcagct 1800
?
gatttaggag?aaggtgatgc?aggcggagat?atggatgagg?ataaagaagc?atttcagaaa 1860
?
acggtcttca?agcagctcgc?aaaagaattt?cagctcacct?tccaagagaa?agggaaggaa 1920
?
aacccaaaac?tatcagttgc?ttatacaaat?gagctagtag?aacgtcctgt?ggcgaaaaca 1980
?
tatggtgcct?tttcagctgt?tgtactgaaa?aatgaagaat?tacaatctga?aaaaagtgag 2040
?
cggcaaacaa?gacatataga?gctgcaattg?cctgaaggga?aaaagtacaa?agaaggggat 2100
?
catatcggaa?ttgttccgaa?aaatagtgat?gcactcgttc?agcgggtgat?caatcgcttc 2160
?
aatctagatc?ctaagcagca?catcaagctt?tattctgaga?aaaaagcaaa?tcatttacct 2220
?
ttagatcagc?cgattcaaat?gagagaatta?cttgcgtcgc?atgttgagct?tcaagagcct 2280
?
gcaacacgta?cgcagctaag?agagctcgcc?gcatatacag?tttgtccgcc?tcaccgcgtg 2340
?
gagcttgagc?aaatggctgg?tgaagcgtat?caagaagcta?ttttaaagaa?acgagtaacc 2400
?
atgcttgact?tactagatca?atatgaagca?tgtgagctgt?catttgtgca?ctttttagca 2460
?
cttttaccag?gtttgaagcc?gcgctattat?tctatttcta?gctcaccaaa?ggtcgatgaa 2520
?
aaaagagtca?gtatcacagt?ggcggttgtg?aaagggaaag?cgtggagcgg?ccgcggagaa 2580
?
tatgccggag?tcgcatcaaa?ctatttatgt?ggtctgaagg?aaggtgaaga?agtcgcctgc 2640
?
ttcctccacg?aagcgcaggc?aggattccag?ctgccgcctt?catctgaagt?accgatgatc 2700
?
atgatcggac?cgggtacagg?aatcgctcca?tttagagggt?ttgttcaggc?aagagaagta 2760
?
tggcagaagg?aaggcaaacc?actaggcgaa?gctcacctat?attttggctg?ccgtcaccct 2820
?
catgaagatg?atctgtattt?tgaagaaatg?cagcttgcag?cgcaaaaagg?agttgtccac 2880
?
atccaccggg?cttattctcg?tcacaaagag?caaaaagtat?atgttcagca?tttgttgaaa 2940
?
gaagacggcg?gcatgttaat?caagttactt?gaccaaggtg?cgtatcttta?cgtgtgcggg 3000
?
gacggaaaag?tcatggcacc?agatgtagag?gctacactga?tcgacctcta?tcaacacgag 3060
?
aaacaatgct?cgaaggaagc?agctgaaaat?tggctgacaa?cccttgcgaa?taataataga 3120
?
tatgtaaaag?atgtatggag?ctga 3144
?
?
<210> 2
<211> 1047
<212> PRT
<213> Bacillus?pumilus
?
<400> 2
?
Met?Gln?Gln?Thr?Ser?Ile?Ile?Pro?Lys?Pro?Lys?Thr?Tyr?Gly?Pro?Phe
1 5 10 15
?
?
Lys?Asn?Ile?Pro?His?Ile?Lys?Lys?Gly?Glu?Leu?Ser?Gln?Thr?Phe?Trp
20 25 30
?
?
Arg?Leu?Ala?Asp?Glu?Leu?Gly?Pro?Ile?Phe?Gln?Phe?Glu?Phe?Ser?Lys
35 40 45
?
?
Ala?Thr?Ser?Ile?Phe?Val?Ser?Asn?His?Glu?Leu?Phe?Gln?Glu?Ile?Cys
50 55 60
?
?
Asp?Glu?Ser?Arg?Phe?Asp?Lys?Tyr?Ile?Gly?Thr?Ser?Leu?Asn?Lys?Val
65 70 75 80?
?
?
Arg?Ala?Phe?Ala?Gly?Asp?Gly?Leu?Phe?Thr?Ser?Trp?Thr?Glu?Glu?Pro
85 90 95
?
?
Asn?Trp?Arg?Lys?Ala?His?His?Ile?Leu?Met?Pro?Ala?Phe?Ser?Gln?Gln
100 105 110
?
?
Ala?Met?Lys?Gly?Tyr?His?Glu?Met?Met?Leu?Asp?Ile?Ala?Thr?Gln?Leu
115 120 125
?
?
Val?Gln?Lys?Trp?Gln?Arg?Thr?Gly?Arg?Asp?Glu?Glu?Ile?Glu?Val?Ala
130 135 140
?
?
Glu?Asp?Met?Thr?Lys?Leu?Thr?Leu?Asp?Thr?Ile?Gly?Leu?Cys?Gly?Phe
145 150 155 160
?
?
Asp?Phe?Arg?Phe?Asn?Ser?Phe?Tyr?Lys?Glu?Asn?Gln?His?Pro?Phe?Ile
165 170 175
?
?
Glu?Ser?Met?Val?Asn?Gly?Leu?Ser?Glu?Ala?Met?Asp?Gln?Ala?Ser?Arg
180 185 190
?
?
Leu?Pro?Val?Ala?Asp?Lys?Leu?Met?Ile?Lys?Arg?Arg?Lys?Lys?Phe?Glu
195 200 205
?
?
Glu?Asn?Val?Asp?Phe?Met?Lys?Gln?Leu?Val?Asp?Asp?Ile?Ile?Gln?Glu
210 215 220
?
?
Arg?Lys?Lys?Gln?Asp?Lys?Thr?Gly?Asp?Asp?Leu?Leu?Ser?Leu?Met?Leu
225 230 235 240
?
?
His?Ala?Lys?Asp?Pro?Glu?Thr?Gly?Glu?Arg?Leu?Ser?Asp?Glu?Asn?Ile
245 250 255
?
?
Arg?Tyr?Gln?Ile?Ile?Thr?Phe?Leu?Ile?Ala?Gly?His?Glu?Thr?Thr?Ser
260 265 270
?
?
Gly?Leu?Leu?Ser?Phe?Ala?Ile?Tyr?Phe?Leu?Leu?Lys?Asn?Pro?Glu?Lys
275 280 285
?
?
Leu?Lys?Lys?Ala?Val?Gln?Glu?Ala?Asp?Asp?Val?Leu?Gln?Gly?Gly?Leu
290 295 300
?
?
Pro?Thr?Phe?Lys?Gln?Val?Gln?Lys?Leu?Asn?Tyr?Thr?Arg?Met?Val?Leu
305 310 315 320
?
?
Asn?Glu?Ser?Leu?Arg?Leu?Trp?Pro?Thr?Ala?Pro?Thr?Phe?Ser?Leu?Tyr
325 330 335
?
?
Ala?Lys?Glu?Asp?Thr?Val?Ile?Gly?Gly?Lys?Tyr?Ser?Ile?Glu?Lys?Asn
340 345 350
?
?
Gln?Ser?Val?Ser?Val?Leu?Leu?Pro?Lys?Leu?His?Arg?Asp?Gln?Ala?Val
355 360 365
?
?
Trp?Gly?Glu?Asp?Ala?Glu?Glu?Phe?Lys?Pro?Glu?Arg?Phe?Leu?His?Pro
370 375 380
?
?
Glu?Lys?Ile?Pro?Gln?His?Ala?Tyr?Lys?Pro?Phe?Gly?Asn?Gly?Gln?Arg
385 390 395 400
?
?
Ala?Cys?Ile?Gly?Met?Gln?Phe?Ala?Leu?His?Glu?Ala?Thr?Met?Val?Leu
405 410 415
?
?
Ala?Met?Val?Leu?His?Asn?Leu?Glu?Leu?Ile?Asp?His?Thr?Ser?Tyr?Glu
420 425 430
?
?
Leu?Asp?Leu?Lys?Glu?Ser?Leu?Thr?Ile?Lys?Pro?Asn?Asp?Phe?Lys?Ile
435 440 445
?
?
Lys?Val?Arg?Pro?Arg?Lys?Gln?Gln?Leu?Phe?Met?Val?Pro?Pro?Lys?Glu
450 455 460
?
?
Glu?Thr?Lys?Lys?Ser?Thr?Thr?Thr?Asp?Glu?Ser?Lys?Val?Lys?Ser?His
465 470 475 480
?
?
Gly?Thr?Pro?Leu?Leu?Val?Leu?Tyr?Gly?Ser?Asn?Leu?Gly?Thr?Ala?Gln
485 490 495
?
?
Gln?Ile?Ala?Asn?Glu?Leu?Ala?Glu?Glu?Gly?Lys?Ala?Lys?Gly?Phe?Asp
500 505 510
?
?
Val?Thr?Thr?Ala?Pro?Leu?Asp?Asp?Tyr?Thr?Arg?Gln?Leu?Pro?Asp?Lys
515 520 525
?
?
Gly?Ala?Val?Phe?Ile?Val?Thr?Ala?Ser?Tyr?Asn?Gly?His?Pro?Pro?Asp
530 535 540
?
?
His?Ala?Lys?Lys?Phe?Val?Asp?Trp?Val?Thr?Gln?Glu?Lys?Glu?Gln?Asp
545 550 555 560
?
?
Leu?Thr?Asn?Val?Thr?Phe?Ala?Val?Phe?Gly?Cys?Gly?Asp?Arg?Asn?Trp
565 570 575
?
?
Ala?Ser?Thr?Tyr?Gln?Arg?Ile?Pro?Arg?Leu?Ile?Asp?Glu?Ala?Leu?Glu
580 585 590
?
?
Arg?Lys?Gly?Ala?Lys?Arg?Ala?Ala?Asp?Leu?Gly?Glu?Gly?Asp?Ala?Gly
595 600 605
?
?
Gly?Asp?Met?Asp?Glu?Asp?Lys?Glu?Ala?Phe?Gln?Lys?Thr?Val?Phe?Lys
610 615 620
?
?
Gln?Leu?Ala?Lys?Glu?Phe?Gln?Leu?Thr?Phe?Gln?Glu?Lys?Gly?Lys?Glu
625 630 635 640
?
?
Asn?Pro?Lys?Leu?Ser?Val?Ala?Tyr?Thr?Asn?Glu?Leu?Val?Glu?Arg?Pro
645 650 655
?
?
Val?Ala?Lys?Thr?Tyr?Gly?Ala?Phe?Ser?Ala?Val?Val?Leu?Lys?Asn?Glu
660 665 670
?
?
Glu?Leu?Gln?Ser?Glu?Lys?Ser?Glu?Arg?Gln?Thr?Arg?His?Ile?Glu?Leu
675 680 685
?
?
Gln?Leu?Pro?Glu?Gly?Lys?Lys?Tyr?Lys?Glu?Gly?Asp?His?Ile?Gly?Ile
690 695 700
?
?
Val?Pro?Lys?Asn?Ser?Asp?Ala?Leu?Val?Gln?Arg?Val?Ile?Asn?Arg?Phe
705 710 715 720
?
?
Asn?Leu?Asp?Pro?Lys?Gln?His?Ile?Lys?Leu?Tyr?Ser?Glu?Lys?Lys?Ala
725 730 735
?
?
Asn?His?Leu?Pro?Leu?Asp?Gln?Pro?Ile?Gln?Met?Arg?Glu?Leu?Leu?Ala
740 745 750
?
?
Ser?His?Val?Glu?Leu?Gln?Glu?Pro?Ala?Thr?Arg?Thr?Gln?Leu?Arg?Glu
755 760 765
?
?
Leu?Ala?Ala?Tyr?Thr?Val?Cys?Pro?Pro?His?Arg?Val?Glu?Leu?Glu?Gln
770 775 780
?
?
Met?Ala?Gly?Glu?Ala?Tyr?Gln?Glu?Ala?Ile?Leu?Lys?Lys?Arg?Val?Thr
785 790 795 800
?
?
Met?Leu?Asp?Leu?Leu?Asp?Gln?Tyr?Glu?Ala?Cys?Glu?Leu?Ser?Phe?Val
805 810 815
?
?
His?Phe?Leu?Ala?Leu?Leu?Pro?Gly?Leu?Lys?Pro?Arg?Tyr?Tyr?Ser?Ile
820 825 830
?
?
Ser?Ser?Ser?Pro?Lys?Val?Asp?Glu?Lys?Arg?Val?Ser?Ile?Thr?Val?Ala
835 840 845
?
?
Val?Val?Lys?Gly?Lys?Ala?Trp?Ser?Gly?Arg?Gly?Glu?Tyr?Ala?Gly?Val
850 855 860
?
?
Ala?Ser?Asn?Tyr?Leu?Cys?Gly?Leu?Lys?Glu?Gly?Glu?Glu?Val?Ala?Cys
865 870 875 880
?
?
Phe?Leu?His?Glu?Ala?Gln?Ala?Gly?Phe?Gln?Leu?Pro?Pro?Ser?Ser?Glu
885 890 895
?
?
Val?Pro?Met?Ile?Met?Ile?Gly?Pro?Gly?Thr?Gly?Ile?Ala?Pro?Phe?Arg
900 905 910
?
?
Gly?Phe?Val?Gln?Ala?Arg?Glu?Val?Trp?Gln?Lys?Glu?Gly?Lys?Pro?Leu
915 920 925
?
?
Gly?Glu?Ala?His?Leu?Tyr?Phe?Gly?Cys?Arg?His?Pro?His?Glu?Asp?Asp
930 935 940
?
?
Leu?Tyr?Phe?Glu?Glu?Met?Gln?Leu?Ala?Ala?Gln?Lys?Gly?Val?Val?His
945 950 955 960
?
?
Ile?His?Arg?Ala?Tyr?Ser?Arg?His?Lys?Glu?Gln?Lys?Val?Tyr?Val?Gln
965 970 975
?
?
His?Leu?Leu?Lys?Glu?Asp?Gly?Gly?Met?Leu?Ile?Lys?Leu?Leu?Asp?Gln
980 985 990
?
?
Gly?Ala?Tyr?Leu?Tyr?Val?Cys?Gly Asp?Gly?Lys?Val?Met Ala?Pro?Asp
995 1000 1005
?
?
Val?Glu Ala?Thr?Leu?Ile?Asp Leu?Tyr?Gln?His?Glu Lys?Gln?Cys
1010 1015 1020
?
?
Ser?Lys Glu?Ala?Ala?Glu?Asn Trp?Leu?Thr?Thr?Leu Ala?Asn?Asn
1025 1030 1035
?
?
Asn?Arg Tyr?Val?Lys?Asp?Val Trp?Ser
1040 1045
Claims (3)
1. the cytochrome P 450 monooxygenases with polycyclic aromatic hydrocarbons hydroxylase activity is characterized in that, it has the nucleotide sequence of SEQ ID NO.1.
2. according to the cytochrome P 450 monooxygenases shown in the claim 1, it is characterized in that with polycyclic aromatic hydrocarbons hydroxylase activity, the mutant form of nucleotide sequence shown in also comprising, described mutation type comprises: disappearance, nonsense, insertion, missense.
3. according to the cytochrome P 450 monooxygenases shown in the claim 1, it is characterized in that described nucleotide sequence coded polypeptide has the aminoacid sequence of SEQ ID NO.2 with polycyclic aromatic hydrocarbons hydroxylase activity.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110020493 CN102154234A (en) | 2011-01-18 | 2011-01-18 | Cytochrome P450 monooxygenase with polycyclic aromatic hydrocarbon hydroxylase-like activity |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110020493 CN102154234A (en) | 2011-01-18 | 2011-01-18 | Cytochrome P450 monooxygenase with polycyclic aromatic hydrocarbon hydroxylase-like activity |
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| Publication Number | Publication Date |
|---|---|
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509819A (en) * | 2013-09-10 | 2014-01-15 | 上海市农业科学院 | Method for improving tolerance and degradation capacity of plant to polycyclic aromatic hydrocarbon |
| CN113785051A (en) * | 2019-06-27 | 2021-12-10 | 科思创知识产权两合公司 | Modified monooxygenases for producing hydroxylated hydrocarbons |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1249779A (en) * | 1997-03-07 | 2000-04-05 | 诺瓦提斯公司 | Cytochrome P450 monooxygenases |
| CN1365393A (en) * | 1999-07-27 | 2002-08-21 | Basf公司 | Novel cytochrome p450 monooxygenases and their use for oxidizing organic compounds |
| CN1469928A (en) * | 2000-10-16 | 2004-01-21 | �����ɷ� | Cytochrome P450 monooxygenases consisting of thermophilic bacteria |
-
2011
- 2011-01-18 CN CN 201110020493 patent/CN102154234A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1249779A (en) * | 1997-03-07 | 2000-04-05 | 诺瓦提斯公司 | Cytochrome P450 monooxygenases |
| CN1365393A (en) * | 1999-07-27 | 2002-08-21 | Basf公司 | Novel cytochrome p450 monooxygenases and their use for oxidizing organic compounds |
| CN1469928A (en) * | 2000-10-16 | 2004-01-21 | �����ɷ� | Cytochrome P450 monooxygenases consisting of thermophilic bacteria |
Non-Patent Citations (1)
| Title |
|---|
| 《NCBI》 20101010 NCBI ZP_03053227 1-3 , 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509819A (en) * | 2013-09-10 | 2014-01-15 | 上海市农业科学院 | Method for improving tolerance and degradation capacity of plant to polycyclic aromatic hydrocarbon |
| CN103509819B (en) * | 2013-09-10 | 2015-08-19 | 上海市农业科学院 | A kind ofly improve the tolerance of plant to polycyclic aromatic hydrocarbons and the method for degradation capability |
| CN113785051A (en) * | 2019-06-27 | 2021-12-10 | 科思创知识产权两合公司 | Modified monooxygenases for producing hydroxylated hydrocarbons |
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Application publication date: 20110817 |