CN100368535C - Dismutase of hydroxyamino-benzene, its coded gene, and application - Google Patents
Dismutase of hydroxyamino-benzene, its coded gene, and application Download PDFInfo
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- CN100368535C CN100368535C CNB2004101028369A CN200410102836A CN100368535C CN 100368535 C CN100368535 C CN 100368535C CN B2004101028369 A CNB2004101028369 A CN B2004101028369A CN 200410102836 A CN200410102836 A CN 200410102836A CN 100368535 C CN100368535 C CN 100368535C
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The present invention relates to hydroxylamine benzene dismutase, a coding gene thereof and an application thereof. The hydroxylamine benzene dismutase of the present invention is protein with the amino acid residue sequence of SEQ ID No. 1 in the sequence list or protein which has the functions of oxyammonia benzene dismutase and is formed by that one to ten amino acid residues of the amino acid residue sequence of SEQ ID No. 1 in the sequence list are substituted, deleted or added. The hydroxylamine benzene dismutase and the coding gene thereof can play an important role in the production of o-aminophenol aromatic compounds and relevant derived compounds thereof.
Description
Technical field
The present invention relates to a kind of oxyammonia benzene fork position enzyme and encoding gene and application in microorganism biological technology and the gene engineering technology field.
Background technology
O-aminophenol is an intermediate of producing dyestuff, medicine, spices, is mainly used in and makes Naphthol AS-PH, is widely used in dye industry, also is used for medical technology and fragrance industry, as: ortho vanillin etc.Therefore, along with the development of dyeing industry, perfumery and medical circle, market also can continue to increase the demand of the intermediate of synthetic these products.In addition, aromatics has amino and hydroxyl simultaneously on position adjacent, can make this type of aromatics aggregate into macromolecular compound, forms the molecular material that some have new physico-chemical property.
At present, technologies such as iron powder reducing method or liquid phase catalytic hydrogenation method are adopted in the production of o-aminophenol and adjacent oxyammonia benzene quasi-aromatic compound, cost height (needing pure hydrogen) not only, and pollute (producing a large amount of iron mud that has the machine poisonous substance) to environment easily.Utilizing the biological processing of enzymatic reaction and transforming is emerging in recent years biotechnology, can under normal temperature and pressure conditions, transform processing and obtain the high-quality product of high purity with very economical, high-efficiency method, be applied at present, as the production of chiral drug and the production of acrylamide at field of biological pharmacy, biological chemical field.Oxyammonia benzene fork position enzyme or the discovery with zymoprotein of similar oxyammonia benzene fork position enzyme catalysis function make the bio-transformation of adjacent oxyammonia benzene quasi-aromatic compound become possibility.
Summary of the invention
The purpose of this invention is to provide a kind of oxyammonia benzene fork position enzyme and encoding gene thereof.
Oxyammonia benzene fork provided by the present invention position enzyme derives from Comamonas testosteroni (Comamonastestosteroni) CNB1 CGMCC No.1028 bacterial strain, is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 1;
2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and the protein with oxyammonia benzene fork position enzyme function.
Wherein, the sequence in the sequence table 1 is made up of 150 amino-acid residues.
The encoding gene of above-mentioned oxyammonia benzene fork position enzyme also belongs to protection scope of the present invention.It can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 1 protein sequence;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
Wherein, the SEQ ID № in the sequence table: 2 by 453 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to the 453rd bit base.
Contain expression carrier of the present invention, clone and host bacterium and all belong to protection scope of the present invention.
Oxyammonia benzene fork of the present invention position enzyme can be expressed as follows: the recombinant expression vector that will contain above-mentioned oxyammonia benzene fork position enzyme coding gene imports host cell and obtains transformant, cultivates this transformant, expresses obtaining oxyammonia benzene fork position enzyme.
The carrier that sets out that is used for making up the recombinant expression vector that contains above-mentioned oxyammonia benzene fork position enzyme coding gene can be carriers such as the plasmid expression vector in genetically engineered field, virus expression carrier, Yeast expression carrier, as pGEX serial carrier, pET serial carrier, Cosmid carrier, pPIC9K.The host that the host selects and above-mentioned expression vector adapts.The culture condition of described transformant is identical with its host's that sets out culture condition, needs the transformant of abduction delivering foreign gene, adopts corresponding inductor abduction delivering oxyammonia benzene fork of the present invention position enzyme.When being the host with the bacterium, after above-mentioned oxyammonia benzene fork position enzyme coding gene can being connected to carrier S uperCos 1, pack with Gigapack III XL packaging protein, maybe this gene is connected to other expression vectors, then packaged carrier or connection carrier are transformed into competence intestinal bacteria or other bacteriums, through LB substratum or other culture medium culturing, contain the bacterium colony that connects gene fragment and just be transformant, with SuperCos 1 is that the transformant of carrier does not need just to induce and can express enzymic activity, is that the transformant of carrier then needs the corresponding inductor abduction delivering of carrier enzymic activity with other expression vector.The production that these transformants can be used to produce oxyammonia benzene fork position enzyme of the present invention or be directly used in adjacent amino (benzene) phenols aromatics and relevant derivative compound thereof.
The expression amount of oxyammonia benzene fork position enzyme of the present invention in intestinal bacteria can reach 800-1000mg/L.It is that the specific activity of substrate can reach 98Umg with oxyammonia benzene
-1Min
-1The enzymatic experiment shows that oxyammonia benzene fork of the present invention position enzyme can oxyammonia benzene be a substrate not only, and can be with 2-oxyammonia chlorinated benzene, 3-oxyammonia chlorinated benzene, 4-oxyammonia chlorinated benzene and 2, and 4-two oxyammonia chlorinated benzene are that substrate carries out the position catalysis of hydroxylamino fork.The substrate wide range of this enzyme, hydroxyl that can not only catalysis oxyammonia benzene and amino disproportionation reaction, and can be substrate with the multiple aromatics that contains hydroxylamino, catalysis forms corresponding adjacent hydroxyl amino aromatics.
The present invention provides a kind of new oxyammonia benzene fork position enzyme for biological production o-aminophenol compounds and relevant derivative compound thereof, and this enzyme can carry out the position reaction of hydroxylamino fork by catalysis oxyammonia benzene-like compounds, forms o-aminophenol and relevant derivative compound thereof.Oxyammonia benzene fork position enzyme of the present invention and encoding gene thereof will play a significant role in the production of adjacent amino (benzene) phenols aromatics and relevant derivative compound thereof.
Embodiment
Experimental technique in following examples is ordinary method if no special instructions.
The acquisition of embodiment 1, oxyammonia benzene of the present invention fork position enzyme gene
The single bacterium colony of Comamonas testosteroni (Comamonas testosteroni) CNB1 CGMCC No.1028 bacterial strain on the LB flat board chosen in the LB nutrient solution, 30 ℃ of shaking culture, collect thalline, extract genomic dna, with the partially digested segment to 30kb of Mbo I.(available from StratageneCompany, USA), after carrier was cut with Xba I enzyme earlier, dephosphorylation was cut into two fragments of 6.8kb and 1.1kb again with BamH I enzyme as carrier with SuperCos 1.Carrier after fragment that genome is partially digested and the processing is used T at 16 ℃
4Dna ligase connects.The fragment that connects (available from Stratagene Company, USA) is packed with Gigapack III XL packaging protein.Transfection to intestinal bacteria XL 1-blue MR (available from Stratagene Company, USA) in, set up Cosmid gene library.Screen the library with the 2-amino-phenol as substrate, cultivated two days for 30 ℃ in the 2-amino-phenol aqueous solution, solution is the colourless positive clone of clone, screens 3 positive colonies from gene library.One of them clone's plasmid is extracted order-checking.Sequencing result is carried out ORF to be analyzed, on NCBI, carry out the blastx comparison, from sequence, analyzing the gene order of coding oxyammonia benzene fork position enzyme, its amino acid sequence coded and hydroxylaminobenzene mutase[Pseudomonas pseudoalcaligenes JS45] sequence similarity of (accession number is AAB94123) is 41%; With hydroxylaminobenzene mutase[Pseudomonasputida HS12] sequence similarity of (accession number is AAK26516) is 40%.The nucleotide sequence of this gene and its amino acid sequence coded are respectively shown in sequence in the sequence table 2 and sequence 1.
The expression and the Function Identification thereof of embodiment 2, oxyammonia benzene of the present invention fork position enzyme
1, the expression of an oxyammonia benzene fork enzyme of the present invention
Extracting the genomic dna of Comamonas testosteroni (Comamonas restosteroni) CNB1 CGMCC No.1028 bacterial strain, is template with it, at primer 1:5 '-GTCC
GAATTCAAGGAGACCCCTTCATGCC-3 ' (base of band underscore is an EcoR I recognition site, and runic is an initiator codon) and primer 2: 5 '-GTCA
AAGCTTUnder the guiding of TGCGGGAAGTCTGATGGT-3 ' (base of band underscore is a Hind III recognition site, and runic is a terminator codon), pcr amplification oxyammonia benzene fork of the present invention position enzyme gene.Wherein, 50 μ l PCR reaction systems are: template (60ng/ μ l) 0.5 μ l; DNTP (every kind of 10mM) 1 μ l; Primer (each 25 μ M) 1 μ l; 10 * damping fluid, 5 μ l; DdH
2O 42.1 μ l; Taq (5U/ μ l) 0.4 μ l.Wherein, 10 * damping fluid comes from TaKaRa Taq test kit (TaKaRa company, Code No.:DR100A).The PCR temperature condition is 94 ℃ of 2min of elder generation; 94 ℃ of 30sec then, 58 ℃ of 45sec, 72 ℃ of 45sec, totally 30 circulations; 72 ℃ of 5min again.Pcr amplification product is carried out 1% agarose gel electrophoresis, and the result shows the band that increases about a 450bp, the size with predict the outcome consistent.Reclaim the fragment of the about 450bp of size, be cloned into the pGEM-T carrier, carry out gene sequencing, the result shows that an oxyammonia benzene fork enzyme gene that amplification obtains has the nucleotide sequence of SEQ ID NO:2.Utilize restriction enzyme EcoRI and HindIII that this pcr amplification product is cloned into pET-21a (+) (available from Novagen company, USA) the corresponding site in, obtain recombinant plasmid, recombinant plasmid transformed is arrived in the competence e. coli bl21 (DE3), then at the enterprising row filter of resistance LB substratum (penbritin 100 μ g/ml); Under the guiding of primer 1 and primer 2, the positive colony (transformant) that screens with the method validation of bacterium colony PCR; Transformant is placed 37 ℃ of LB liquid nutrient mediums, 150rpm shaking culture, to nectar degree OD
600When being 0.8 left and right sides, the IPTG that adds 1mM induced 1.5 hours, expressed oxyammonia benzene fork position enzyme.(USA), carry out according to product manual for HisBind Res in Chromatography, Novagen company, and its expression amount is 1000mg/L by the concrete operations step by HisTag pillar purifying for oxyammonia benzene fork position enzyme.
2, the mensuration of an oxyammonia benzene fork enzymic activity of the present invention
(1) preparation of oil of mirbane nitroreductase
Extracting the genomic dna of Comamonas testosteroni (Comamonas testosteroni) CNB1 CGMCC No.1028 bacterial strain, is template with it, at primer 3:5 '-GACGTTT
CATATGCCGACCAGCCCGTTC-3 ' (base of band underscore is the NdeI recognition site, and runic is an initiator codon) and primer 4:5 '-TG
GGATCCUnder the guiding of TAATTCGTGGACGAAGGTGG-3 ' (base of band underscore is the BamHI restriction enzyme site, and runic is a terminator codon), pcr amplification oil of mirbane nitroreductase gene.Wherein, 50 μ l PCR reaction systems are: template (60ng/ μ l) 0.5 μ l; DNTP (every kind of 10mM) 1 μ l; Primer (each 25 μ M) 1 μ l; 10 * damping fluid, 5 μ l; DdH
2O 42.1 μ l; Taq (5U/ μ l) 0.4 μ l.Wherein, 10 * damping fluid comes from TaKaRa Taq test kit (TaKaRa company, Code No.:DR100A).The PCR temperature condition is 94 ℃ of 2min of elder generation; 94 ℃ of 30sec then, 40 ℃ of 45sec, 72 ℃ of 45sec, totally 30 circulations; 72 ℃ of 5min again.Pcr amplification product is carried out 1% agarose gel electrophoresis, and the result shows the band that increases about a 700bp, the size with predict the outcome consistent.Reclaim the fragment of the about 700bp of size, be cloned into the pGEM-T carrier, carry out gene sequencing, the result shows that the oil of mirbane nitroreductase gene that obtains of amplification has the nucleotide sequence of SEQ ID NO:3 in the sequence table, and coding has the oil of mirbane nitroreductase of the amino acid residue sequence of sequence 4 in the sequence table.Utilize restriction enzyme NdeI and BamHI that this pcr amplification product is cloned into pET-21a (+) (available from Novagen company, USA) the corresponding site in, obtain recombinant plasmid, recombinant plasmid transformed in competence e. coli bl21 (DE3), is carried out the positive colony screening then on resistance LB substratum (penbritin 100 μ g/ml); And under the guiding by primer 3 and primer 4, use of the checking of the method for bacterium colony PCR to the positive colony (transformant) of screening; Transformant is placed 37 ℃ of LB liquid nutrient mediums, 150rpm shaking culture, to nectar degree OD
600When being 0.8 left and right sides, the IPTG that adds 1mM induced 1.5 hours, obtained the oil of mirbane nitroreductase.(USA), carry out according to product manual for HisBind Resin Chromatography, Novagen company by the concrete operations step by HisTag pillar purifying for the oil of mirbane nitroreductase.
(2) preparation of oxyammonia benzene
Be substrate with oil of mirbane and be coenzyme with NADPH, with oil of mirbane nitroreductase catalytic preparation, concrete grammar is as follows: 3 μ mol oil of mirbane, 10 μ mol NADPH, 50mmol/L phosphate buffered saline buffer (pH8.0) 20mL contains the pure protein solution of 3～9mg oil of mirbane nitroreductase, adds to 30mL with sterilized water, react 5～10min under the room temperature, then contain the oxyammonia benzene (0.7～1.0mg/L) of higher concentration in the reaction system.
(3) mensuration of an oxyammonia benzene fork enzymic activity of the present invention
With oxyammonia benzene is example, illustrates the measuring method of enzymic activity.The product that oxyammonia benzene forms under the katalysis of oxyammonia benzene fork position enzyme is an o-aminophenol, and it has maximum absorption band at the 235nm place, the increasing amount of the absorbance value at 235nm place in the ultraviolet spectrophotometer analytical unit time, and the ratio that can record oxyammonia benzene fork position enzyme is lived.Reaction system comprises 0.3 μ mol oxyammonia benzene, and 50mmol/L phosphate buffered saline buffer (pH8.0) 1mL contains the pure protein solution of 0.3～0.9mg oxyammonia benzene fork position enzyme, and the enzymatic reaction cumulative volume is 3mL, reacts 5min under the room temperature.The enzyme activity of 1 unit is defined as per minute and generates the required enzyme amount of 1 μ mol o-aminophenol.The result shows that this oxyammonia benzene fork position specific enzyme activity is 98Umg
-1Min
-1
3, the Function Identification of oxyammonia benzene fork position enzyme
Do the enzymic catalytic reaction experiment with the oxyammonia benzene fork position enzyme that step 1 obtains, respectively with oxyammonia benzene, 2-oxyammonia chlorinated benzene, 3-oxyammonia chlorinated benzene, 4-oxyammonia chlorinated benzene and 2,4-two oxyammonia chlorinated benzene detect the generation of its product as the substrate of this enzyme at 235nm.Reaction conditions is as follows: 0.3 μ mol substrate (as oxyammonia benzene), 50mmol/L phosphate buffered saline buffer (pH8.0) 1mL contains the pure protein solution of 0.3～0.9mg oxyammonia benzene fork position enzyme, and the enzymatic reaction cumulative volume is 3mL, react 5min under the room temperature, in the rate of change of 235nm place check absorbance value.The result is as shown in table 1, shows that this enzyme all has hydroxylamino fork position catalytic activity to these the five kinds aromatics that contain hydroxylamino.
The substrate scope of table 1 oxyammonia benzene fork of the present invention position enzyme
| Substrate | Enzyme is than (the Umg that lives -1·min -1) |
| Oxyammonia benzene 2-oxyammonia chlorinated benzene 3-oxyammonia chlorinated benzene 4-oxyammonia chlorinated benzene 2,4-two oxyammonia chlorinated benzene | 98 54 67 88 35 |
* the preparation of substrate is with the preparation of above-mentioned oxyammonia benzene, with oil of mirbane replace with adjacent chloronitrobenzene, a chloro oil of mirbane respectively, to chloronitrobenzene and 2, the 4-dinitro-chloro-benzene.
4, the evaluation of oxyammonia benzene fork position enzyme catalysis product
The enzymatic reaction cumulative volume is 3mL, contain 0.3 μ mol oxyammonia benzene, 50mmol/L phosphate buffered saline buffer (pH8.0) 1mL contains the pure protein solution of 0.5mg oxyammonia benzene fork position enzyme, after reacting 5min under the room temperature, add isopyknic ethyl acetate, oscillation extraction 10min, 5000rpm is centrifugal, get upper organic phase, carefully ethyl acetate is dried up with nitrogen, residue with water dissolution (product I), is carried out next step analysis.With product I is 2-amino phenol 1,6-dioxygenase (2-amino phenol 1, the 6-dioxygenase is 200310118812.8 according to application number, name is called the method preparation of describing in the application for a patent for invention of " 2-amino phenol 1; 6-dioxygenase, its gene and purposes ") the effect substrate carry out enzymatic reaction, the ultraviolet absorption collection of illustrative plates of the product that obtains be 2-amino phenol 1 with the standard substance of o-aminophenol, the effect substrate of 6-dioxygenase and the ultraviolet absorption collection of illustrative plates unanimity of the product that obtains.Product I is carried out HPLC analyze, analysis condition is as follows: HP1050 type liquid chromatograph; The C-18 reversed-phase column (4.6mm, 250mm, Agilent, USA); Moving phase is methyl alcohol: water=60: 40 (v/v); Flow rate of mobile phase is 1ml/min; Detecting wavelength is 210nm and 280nm.Analytical results shows that the time of holding back of the standard substance of product I and o-aminophenol is 2.766min.Be o-aminophenol by above two provable product I of test.
Sequence table
<160>4
<210>1
<211>150
<212>PRT
＜213〉Comamonas testosteroni (Comamonas testosteroni)
<400>1
Met Pro Ala Gln Pro Asn Val Ala Tyr Phe Thr His Arg Leu Leu Gln
1 5 10 15
Ala Gly Phe Val Leu Phe Leu Leu Gly Leu Leu Thr Gly Ile Val Ile
20 25 30
Pro Ala Ala Gln Leu Pro Arg Met Ala Leu Ser Ser His Leu Gln Gly
35 40 45
Val Met Asn Gly Ser Phe Leu Ile Ala Leu Gly Leu Cys Trp Lys His
50 55 60
Leu Val Leu Pro Ser Trp Ala Glu Arg Met Ala Phe Leu Ser Ala Ile
65 70 75 80
Thr Gly Thr Tyr Ala Asn Trp Ala Ala Thr Leu Leu Ser Ala Phe Thr
85 90 95
Gly Ala Ala Pro Met Met Pro Ile Ala Gly Gly Gly Ser Val Gly Thr
100 105 110
Pro Phe His Glu Met Leu Val Ser Gly Leu Leu Leu Phe Leu Ile Leu
115 120 125
Ala Met Ile Leu Thr Cys Val Leu Val Leu Trp Gly Leu His Arg Arg
130 135 140
Ser Gly Val Pro Ala Pro
145 150
<210>2
<211>453
<212>DNA
＜213〉Comamonas testosteroni (Comamonas testosteroni)
<400>2
atgcctgccc agccgaatgt cgcttatttc acccatcgcc ttctacaggc agggttcgtg 60
cttttccttc ttgggctcct gacgggtatt gtcattccag ccgcccaact tccgcgcatg 120
gcactgtcca gccatctcca gggcgtcatg aatggctcct tcctcatcgc tctcggactt 180
tgctggaagc atctcgttct tccgtcctgg gccgagagga tggcgttcct ttcggccatt 240
acggggacat atgcgaactg ggcggcgacg ctgctttcag ctttcaccgg tgctgccccg 300
atgatgccca ttgccggtgg cggaagcgtt ggtactccct ttcacgaaat gctggtgtcc 360
ggcctcctgc tcttcctgat cctggccatg atcctcacct gcgtgcttgt gctgtggggg 420
cttcaccgtc ggtcgggtgt cccagcacca tga 453
<210>3
<211>684
<212>DNA
＜213〉Comamonas testosteroni (Comamonas testosteroni)
<400>3
atgccgacca gcccgttcat tgatgatctg atacgcgacc gtcgcacgaa gcgtggtttt 60
ctggatcagc cagtgccgat agaaatggtg aaggacatcc tttcagtggc aaagtatacg 120
cctagttcga gtaacaccca gccgtggcgc tgttatgttt taaccggtga ggcgcgcgag 180
cgcgttacta cggccgcggt ggaggcgtac cgcggagcac ccgaaggcct gaagccggag 240
tattcatact ttcctgaacc actgcatgaa ccctatgcga cccgtttcaa ttcgtttcgc 300
ggacaactgg gcgacgctga aggatgctgc cgaagtgata taaccgggcg gcgtcgatac 360
gtcgagcgcc agttccgttt tttcgacgcg ccggtaggcc tcattttcac gatggatcgc 420
cgtctggaat gggccagttt catttgctat ggctgctttc tccagaacat catgctggca 480
gccaagggtc gtggtctcga tacttgccct caaggactat ggtccctgca gcacccggtg 540
ctgcgcactg aactcaacct tcctgatgat caaatggtgg tagcggggat gtcgctgggc 600
tgggccgaca atagcatggc agtgaaccag atgagtatgt ccagggtgga actggaagag 660
ttcaccacct tcgtccacga atga 684
<210>4
<211>227
<212>PRT
＜213〉Comamonas testosteroni (Comamonas testosteroni)
<400>4
Met Pro Thr Ser Pro Phe Ile Asp Asp Leu Ile Arg Asp Arg Arg Thr
1 5 10 15
Lys Arg Gly Phe Leu Asp Gln Pro Val Pro Ile Glu Met Val Lys Asp
20 25 30
Ile Leu Ser Val Ala Lys Tyr Thr Pro Ser Ser Ser Asn Thr Gln Pro
35 40 45
Trp Arg Cys Tyr Val Leu Thr Gly Glu Ala Arg Glu Arg Val Thr Thr
50 55 60
Ala Ala Val Glu Ala Tyr Arg Gly Ala Pro Glu Gly Leu Lys Pro Glu
65 70 75 80
Tyr Ser Tyr Phe Pro Glu Pro Leu His Glu Pro Tyr Ala Thr Arg Phe
85 90 95
Asn Ser Phe Arg Gly Gln Leu Gly Asp Ala Glu Gly Cys Cys Arg Ser
100 105 110
Asp Ile Thr Gly Arg Arg Arg Tyr Val Glu Arg Gln Phe Arg Phe Phe
115 120 125
Asp Ala Pro Val Gly Leu Ile Phe Thr Met Asp Arg Arg Leu Glu Trp
130 135 140
Ala Ser Phe Ile Cys Tyr Gly Cys Phe Leu Gln Asn Ile Met Leu Ala
145 150 155 160
Ala Lys Gly Arg Gly Leu Asp Thr Cys Pro Gln Gly Leu Trp Ser Leu
165 170 175
Gln His Pro Val Leu Arg Thr Glu Leu Asn Leu Pro Asp Asp Gln Met
180 185 190
Val Val Ala Gly Met Ser Leu Gly Trp Ala Asp Asn Ser Met Ala Val
195 200 205
Asn Gln Met Ser Met Ser Arg Val Glu Leu Glu Glu Phe Thr Thr Phe
210 215 220
Val His Glu
225
Claims (8)
1. oxyammonia benzene fork position enzyme, its amino acid residue sequence is shown in SEQ ID NO:1.
2. the encoding gene of the described oxyammonia benzene of claim 1 fork position enzyme.
3. gene according to claim 2 is characterized in that: the nucleotide sequence of described oxyammonia benzene fork position enzyme coding gene is shown in SEQ ID NO:2.
4. the expression vector that contains claim 2 or 3 described oxyammonia benzene fork position enzyme coding genes.
5. the clone that contains claim 2 or 3 described oxyammonia benzene fork position enzyme coding genes.
6. the host bacterium that contains claim 2 or 3 described oxyammonia benzene fork position enzyme coding genes.
7. the application of the described oxyammonia benzene fork of claim 1 position enzyme in the o-aminophenol quasi-aromatic compound is produced.
8. claim 2 or 3 application of described oxyammonia benzene fork position enzyme coding gene in the o-aminophenol quasi-aromatic compound is produced.
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| CN102286508A (en) * | 2011-06-23 | 2011-12-21 | 浙江大学 | Recombinant plasmid for degrading nitrobenzene, gene engineering bacteria and preparation method thereof |
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| CN101100654B (en) * | 2007-06-19 | 2011-07-27 | 中国科学院微生物研究所 | Method for producing 4,6-diaminoresorcinol and special-purpose engineering bacterium for the same |
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| CN102286508A (en) * | 2011-06-23 | 2011-12-21 | 浙江大学 | Recombinant plasmid for degrading nitrobenzene, gene engineering bacteria and preparation method thereof |
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