CN101597597B - A kind of 4-nitrophenol-4-monooxygenase gene and its preparation method and use - Google Patents
A kind of 4-nitrophenol-4-monooxygenase gene and its preparation method and use Download PDFInfo
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- CN101597597B CN101597597B CN2009100628819A CN200910062881A CN101597597B CN 101597597 B CN101597597 B CN 101597597B CN 2009100628819 A CN2009100628819 A CN 2009100628819A CN 200910062881 A CN200910062881 A CN 200910062881A CN 101597597 B CN101597597 B CN 101597597B
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Abstract
本发明公开了一种4-硝基酚-4-单加氧酶基因及制备方法和用途，一种分离的多肽，序列为SEQ ID NO：1所示的核苷酸序列。其步骤是：首先以恶臭假单胞菌NyZ402菌株的基因组为模板，利用PCR的方法扩增获得一段对苯二酚双加氧酶基因片段；其次是通过染色体步移的方法克隆获得参与4-硝基酚代谢的基因簇，获得pnpC基因附近的基因；第三是对获得的基因簇序列进行比对分析，获取4-硝基酚-4-单加氧酶基因，在国际基因数据库中，与所涉及的4-硝基酚-4-单加氧酶蛋白质序列无明显相似性。4-硝基酚-4-单加氧酶基因在芳香烃环境污染物降解中的应用。该方法易行，操作简便，能够快速的获得目的基因片段，尤其是长片段。为解决硝基酚环境污染问题具有着重要的理论和应用价值。The invention discloses a 4-nitrophenol-4-monooxygenase gene, its preparation method and use, and an isolated polypeptide whose sequence is the nucleotide sequence shown in SEQ ID NO:1. The steps are as follows: firstly, using the genome of Pseudomonas putida NyZ402 strain as a template, amplify and obtain a segment of hydroquinone dioxygenase gene fragment by PCR method; secondly, obtain a fragment of the gene involved in 4- The gene clusters of nitrophenol metabolism, to obtain the genes near the pnpC gene; the third is to compare and analyze the sequence of the obtained gene clusters, to obtain the 4-nitrophenol-4-monooxygenase gene, in the international gene database, No apparent similarity to the involved 4-nitrophenol-4-monooxygenase protein sequence. Application of 4-nitrophenol-4-monooxygenase gene in the degradation of aromatic hydrocarbon environmental pollutants. The method is easy to operate and easy to operate, and can quickly obtain target gene fragments, especially long fragments. It has important theoretical and practical value to solve the problem of nitrophenol environmental pollution.
Description
Technical field
The present invention relates to a kind of 4-nitrophenol-4-monooxygenase gene, the preparation method who also relates to this gene simultaneously, the endonuclease capable catalytic environment pollutent 4-nitrophenol of this genes encoding and the degraded of 4-nitro-pyrocatechol, toxic pollutant 4-nitrophenol and 4-nitro-pyrocatechol in can the katabolism environment.The invention still further relates to a kind of method of utilizing the activated 4-nitrophenol of this genetic expression-4-monooxygenase, and the application of 4-nitrophenol-4-monooxygenase in the metabolism of environmental pollutant microbiological degradation.
Background technology
Nitrophenol is one of the most widely used arene compound, also is the medelling compound of research aromatic hydrocarbon microbial metabolism approach and mechanism.4-nitrophenol (para-nitrophenol wherein, 4-nitrophenol, PNP) be synthetic precursors such as a kind of important chemical material, Chang Zuowei medicine, dyestuff, agricultural chemicals, in using the organic phosphorous insecticide environment, also can cause the accumulation of nitrophenol simultaneously owing to the hydrolysis of organophosphorus.This compounds be everlasting produce and use in be released in the environment, they are difficult to degrade, can be residual in the environment midium or long term.The 4-nitrophenol can influence the substance metabolism of human body, and enrichment and scale effect are arranged in food chain, has danger such as teratogenesis, mutagenesis, cause cancer, serious threat human beings'health and existence.Therefore classified as preferential environmental pollutant (PriorityPollutant List) (http://oaspub.epa.gov/wqsdatabase/) by EPA.
Enter nitrophenol and other organic pollutant in the environment, originally occurring in nature does not exist microorganism and the enzyme system that utilizes them, but through evolution of long period of time, the mutation of the spontaneous mutation Cheng Xin that microorganism has, what have passes through the generation inducible enzyme to adapt to new envrionment conditions, thereby can utilize these pollutents to grow, make pollutent be degraded or transform simultaneously as carbon source, nitrogenous source or the energy.The metabolism research of 4-nitrophenol is subjected to people's attention always, and many strains report of the pure cultures of bacteria of metabolism nitrophenol has fully been arranged.With respect to other nitryl aromatic hydrocarbon compound, the pathways metabolism of 4-nitrophenol is the most various.The oxidative metabolism approach of 4-nitrophenol is the topmost pathways metabolism of reporting at present, according to the difference of intermediate product, can be divided into Resorcinol approach and benzenetriol approach.
The Resorcinol pathways metabolism of 4-nitrophenol extensively is present in the Gram-negative bacteria, in this approach, the 4-nitrophenol at first is oxidized to benzoquinone (p-benzoquinone), and the benzoquinone is reduced to Resorcinol (hydroquinone) under the reductase enzyme effect, enter the open loop approach then.Resorcinol open loop under the effect of dioxygenase generate γ-hydroxymuconic acid semialdehyde (γ-hydroxymuconicsemialdehyde, HMSA), the latter under the desaturase effect, be converted into maleoyl acetate (maleylacetate, MA).MA under the effect of MA reductase enzyme, generate β-ketoadipic acid (β-ketoadipate) and finally enter tricarboxylic acid cycle (Tricarboxylic acid cycle, TCA), [Spain JC﹠amp; Gibson DT (1991) Pathway for biodegradation of p-nitrophenol in aMoraxella sp.Appl.Environ.Microbiol.57:812-819.].
The 4-nitrophenol pathways metabolism of another classics is benzenetriol approach, and this pathways metabolism extensively exists in gram-positive microorganism.The 4-nitrophenol forms benzenetriol at 2-and 3-position hydroxylation (1,2,4-benzenetriol hydroxyquinol), has the generation of intermediate product 4-nitro-pyrocatechol (4-nitrocatechol) in the metabolic process in this approach.Benzenetriol is generated maleoyl acetate by open loop under the effect of dioxygenase, be reduced into β-ketoadipic acid [Jain RK, Dreisbach JH﹠amp then; Spain JC (1994) Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp.Appl.Environ.Microbiol.60:3030-3032.].
Though the separation of 4-nitrophenol metabolism bacterial strain and the research of pathways metabolism have been carried out half a century, seldom about the research of 4-nitrophenol pathways metabolism on the molecular biology level.At present, reported metabolic gene of participation 4-nitrophenol and operon from two bacterial strains, this two strains bacterium is gram-positive microorganism, all is through oxyhydroquinone approach degraded 4-nitrophenol.Respectively from Rhodococcus opacus SAO 101[Kitagawa W, Kimura N﹠amp; Kamagata Y (2004) Anovel p-nitrophenol degradation gene cluster from a Gram-positive bacterium, Rhodococcus opacus SAO101.J.Bacteriol.186:4894-4902.] and Arthrobacter sp.JS443 bacterial strain [Perry LL﹠amp; Zylstra GJ (2007) Cloning of a gene cluster involved in thecatabolism of p-nitrophenol by Arthrobacter sp.strain JS443and characterization ofthep-nitrophenol monooxygenase.J.Bacteriol.189:7563-7572.].The enzyme of the most critical in its metabolic process--4-nitrophenol monooxygenase has also obtained the clone respectively and has identified.These two kinds of 4-nitrophenol monooxygenases all belong to the flavine monooxygenase of two-pack, comprise a reductase enzyme component and a monooxygenase component that relies on flavine that relies on NAD (P) H, and its protein sequence have higher consistence (72%).Participate in the metabolic gene cluster of 4-nitrophenol and be respectively EF052871 and AY131335 in the sequence accession number in international gene database (GenBank).
The 4-nitrophenol does not appear in the newspapers through the metabolic molecular biology research of Resorcinol approach always, does not have the report of corresponding gene order in the GenBank database yet.
Summary of the invention
The objective of the invention is to be to provide the gene order of a kind of 4-nitro-4-monooxygenase, this sequence is from the 4-degrading mononitrophenol bacterium in the environment, and other gene in this gene and the database does not have the obvious sequence similarity, is a kind of brand-new gene.The present invention is for the diversity that makes full use of microbial metabolism pollutent ability, have important theory and using value for solving the nitrophenol problem of environmental pollution.
Another object of the present invention is the gene order preparation method who has been to provide a kind of 4-nitro-4-monooxygenase, this is easy to implement the method, and is easy and simple to handle, and this method adopts round pcr and chromosome walking technology, can obtain target gene fragment, especially long segment fast.
A further object of the present invention is to be to provide the application of a kind of 4-nitrophenol-4-monooxygenase gene in the degraded of aromatic hydrocarbon environmental pollutant.4-nitro-4-monooxygenase can katabolism 4-nitrophenol etc. environmental pollutant, in the environmental treatment of nitryl aromatic hydrocarbon pollutant, have research and using value.
One of technical essential of the present invention is separation and the evaluation of 4-degrading mononitrophenol microorganism.
The method of a kind of isolation identification 4-degrading mononitrophenol bacterium is provided in one embodiment of the invention.This bacterial strain is to separate to obtain from the sample that 4-nitrophenol pollution history is arranged, screening method is " a selectivity cultivation concentration method ", promptly in minimum medium, only add the 4-nitrophenol as sole carbon source, nitrogenous source and the energy, only can utilize the 4-nitrophenol could breed, reach the purpose of enrichment and purifying with this as the bacterium of carbon source, nitrogenous source and the energy.By single bacterium colony isolation identification, obtain a strain pure cultures of bacteria at last, through being accredited as pseudomonas putida, called after NyZ402 (Pseudomonas putidaNyZ402).This bacterium can utilize the 4-nitrophenol to grow as carbon source, nitrogenous source and the energy, can transform fully, degradation of contaminant 4-nitrophenol and 4-nitro-pyrocatechol, realizes the complete detoxification of toxic pollutant.
Microorganism strains involved in the present invention includes but are not limited to pseudomonas putida and belongs to, because the extensive existence of Horizontal Gene Transfer between microorganism, bacterial population in all environment, comprise educable and can not cultured microorganism, the aromatic hydrocarbon metabolic gene of can obtain, heredity is involved in the present invention.
Two of technical essential of the present invention be the clone to participate in 4-nitrophenol metabolic gene cluster sequence, the especially gene of 4-nitrophenol-4-monooxygenase (bunch) sequence.
As described in the background art, also do not report in international nucleic acid database (GenBank), there is not the login of correlated series through the metabolic open source literature of Resorcinol approach at present about the 4-nitrophenol yet.And the 4-nitrophenol is different fully with the benzenetriol pathways metabolism through the Resorcinol pathways metabolism, the gene order of its coding control, and especially the gene order of 4-nitrophenol monooxygenase is also inequality.Therefore, be difficult to based on homologous sequence, by the method for PCR or Southern hybridization obtain participation 4-nitrophenol through the gene of the metabolic 4-nitrophenol of Resorcinol approach-4-monooxygenase (bunch) sequence.
In one embodiment of the invention, a kind of clone gene order that obtains 4-nitrophenol-4-monooxygenase and the gene cluster sequence method that participates in the Resorcinol pathways metabolism of 4-nitrophenol are provided, the gene order preparation method of a kind of 4-nitro-4-monooxygenase the steps include:
(1) amplification of Resorcinol dioxygenase gene.At first (from soil, separate with pseudomonas putida NyZ402 bacterial strain, method such as embodiment 1) genome be template, utilize the method amplification of PCR to obtain one section Resorcinol dioxygenase gene (pnpC) fragment, the open loop of the Resorcinol dioxygenase possibility catalysis Resorcinol of this genes encoding, the downstream pathways metabolism of participation 4-nitrophenol.
(2) clone obtains to participate in the metabolic gene cluster of 4-nitrophenol.By the method (GenomeWalker of BD company test kit) of chromosome walking, just can obtain near other gene cluster sequence of pnpC gene.Because it is chain often to participate in the metabolic gene cluster of aromatic hydrocarbon, is compact arranged in genome.Therefore, may just comprise the enzyme of catalysis 4-nitrophenol in the upstream and downstream gene order of pnpC gene through the metabolic most critical of Resorcinol--the gene order of 4-nitrophenol-4-monooxygenase.
(3) 4-nitrophenol-4-monooxygenase gene is obtained in sequential analysis.The gene cluster sequence that obtains is compared, the result shows, in international gene database (GenBank), to 4-nitrophenol-4-monooxygenase protein sequence involved in the present invention the most similar be the sequence (accession number is XP_754665) that derives from the monooxygenase of Aspergillusfumigatus Af293, but similarity only is 38%, this just illustrates that the designed gene of the present invention is a brand-new gene, a kind of brand-new metabolic enzyme of participation 4-nitrophenol--4-nitrophenol-4-monooxygenase of can encoding.A kind of isolated polypeptide, its sequence are the nucleotide sequence shown in the SEQ ID NO:1.A kind of isolated polypeptide, its sequence are the aminoacid sequence shown in the SEQ ID NO:2.
4-nitrophenol-4-monooxygenase involved in the present invention is made up of 1233 Nucleotide, and wherein the 1231-1233 position is terminator codon TAG.
4-nitrophenol-4-monooxygenase nucleotide sequence (SEQ ID NO:1)
ATGGAAACAATTGATGGCGTAGTGGTTGTCGGCGGAGGCCCGGTTGGGTTGCTTACAG
CATTGAAACTTGGCAAGGCCGGTGTGCGGGTAGTTGTCCTGGAGTCCGAATCGGGCGT
TTCACCCTCACCGCGGGCGGTTGCCTATATGCCACCCACTGCTGCGGCACTGGACCGTT
TCGGGTTGCTCGACGACATTCGCAAACGTGCTGTGTGGTGCCCTGATTTCGCTTACCGT
CACGGTAATGGCGAACTGATCGCGAAAATGGACTGGGCTGTTCTGGCACAGGACACTG
ATTACCCCTATATGCTGTTGCTGGCGCAAAACCATGTGTCCAATGTAATCGTGGAGCATC
TTCGCAAAC fourth CTCCAATGTCGAAATTCGCTGGAATCACGCGGTCGAAGACATTGAGCA
GGACGACGACTATGTGACCATGGAAACCAGCGGCCCTGCCGGAAAAGCACGTTTGCG
GGCGAAATGGGTTGCTGCCACCGACGGTGCACGCAGTACCGTACGAGGAAAAATCGG
GCTGTCCTTTGATGGTATTACTTGGTCGGAACGCCTGGTTGCGACAAACGTGTTCTATG
ACTTTTCCCTGCACGGCTACTCTCGGGCCAACTTCGTTCACGACCCGGTAGACTGGGC
CGTCGTCGTTCAGCTCGACAAAACCGGCCTGTGGCGTGTGTGCTATGGCGAAGATCCT
GAGATCTCCGAGGCTGAAGTCCGCCGCCGCTTGCCGGAGCGTTTCAAGCGCCTGCTGC
CGGGAGCGCCGACGCCCGATCAGTATCGGGTCGACTACCTCAATCCTTACCGTGTCCA
CCAGCGTTGCGCCGCCGAGTTCCGCCGTGGACGGGTGATCCTGGCCGGAGATGCTGCC
CATGCGACCAACCCGATGGGGGGGCTGGGACTGTCGGGGGGCGTACTTGATGCCGAG
CATTTGGCCGAGGCGTTGATTGCGGTGATCAAAGAAGGCGCTTCTACCAAGGTGCTGG
ATGAGTACTCTATCGATCGGCGCAAAGTCTTCCTCGAGTTCACGTCCCCGACCGCGACT
GCCAACTTCACCTGGATGAAAGAAAGTGACCCTGCCCAGCGTGCTCGTGATAACGCAA
TGTTTGATCATGCGGGCAAGGACTTGAAAGTCATGCGTGAAATTCTTCTGGACTTCGA
GAAGCTCAACGGCCGGCGCGTGATCGCTCCACGACAATATGCGCAAGCGCTTGAAAGT
ATGGGCGCATAG
The protein amino acid sequence of this genes encoding is as follows.
The aminoacid sequence of 4-nitrophenol-4-monooxygenase (SEQ ID NO:2)
METIDGVVVVGGGPVGLLTALKLGKAGVRVVVLESESGVSPSPRAVAYMPPTAAALDRFG
LLDDIRKRAVWCPDFAYRHGNGELIAKMDWAVLAQDTDYPYMLLLAQNHVSNVIVEHLR
KLSNVEIRWNHAVEDIEQDDDYVTMETSGPAGKARLRAKWVAATDGARSTVRGKIGLSF
DGITWSERLVATNVFYDFSLHGYSRANFVHDPVDWAVVVQLDKTGLWRVCYGEDPEISEA
EVRRRLPERFKRLLPGAPTPDQYRVDYLNPYRVHQRCAAEFRRGRVILAGDAAHATNPM
GGLGLSGGVLDAEHLAEALIAVIKEGASTKVLDEYSIDRRKVFLEFTSPTATANFTWMKES
DPAQRARDNAMFDHAGKDLKVMREILLDFEKLNGRRVIAPRQYAQALESMGA.
The fine 4-nitrophenol-4-monooxygenase that the present invention relates to is made of 410 amino acid, and molecular weight is 45.5kD, and iso-electric point is 6.5.
The acquisition methods of 4-nitro involved in the present invention-4-monooxygenase gene sequence includes but are not limited to aforesaid method.4-nitro-4-the monooxygenase gene that the present invention relates to can also obtain by the following method: (1) subclone: with gene fragment involved in the present invention is template, be template perhaps with the recombinant plasmid that contains gene fragment involved in the present invention, be template perhaps with the engineering strain DNA that contains gene fragment involved in the present invention, by conventional molecular biology working method (as PCR), subclone obtains corresponding gene fragment, these fragments can comprise gene order involved in the present invention whole or part, these fragments can be on bacterium, yeast, animal and plant cells or other eucaryon, express in the prokaryotic organism, obtain protein or polypeptide fragment, these protein or polypeptide fragment have identical biological function with protein involved in the present invention; (2) synthetic: according to amino acid involved in the present invention or nucleotide sequence, can be by the method synthetic DNA fragment of chemosynthesis, these fragments can comprise gene order involved in the present invention whole or part, can be cloned in the corresponding carrier and express, expressed protein or polypeptide have identical biological function with protein involved in the present invention.
Gene fragment involved in the present invention can be modified, and such as some nucleotide sequence of mutator gene, but does not influence its encoded protein matter aminoacid sequence; Can increase, lack amino acid involved in the present invention or nucleotide sequence, these are modified can not influence proteinic biochemical characteristic, higher structure and proteinic biological function.Such as: can add secreting signal peptide at protein or polypeptide N-end or C-end, can also add that suitable joint (as 6 Histidines) is convenient to proteins extraction, purifying.
Three of technical essential of the present invention is functional verifications of 4-nitrophenol-4-monooxygenase gene.
As described in the background art, 4-nitrophenol-4-monooxygenase gene involved in the present invention does not have disclosed report as yet, and the function of verifying gene involved in the present invention is the prerequisite that further develops this gene.
The authentication method of a kind of 4-of evaluation nitrophenol-4-monooxygenase gene and proteins encoded function thereof is provided in one embodiment of the present of invention (embodiment 4).This method comprises: the pcr amplification of (1) 4-nitrophenol-4-monooxygenase gene.Concrete grammar is, extracts the genome of pseudomonas putida NyZ402 bacterial strain, is template pcr amplification 4-nitrophenol-4-monooxygenase gene with it, and the primer is P1:5 ' ATT
GAA TTC ATA TGG GCC GTG ATA GGA GA3 ' (base of band underscore is the recognition site of EcoRI and NdeI, and primer has comprised the SD sequence) and P2:5 ' AGT T
GA ATT CAC TCC TAC GAC GAA AGA TCG3 ' (base of band underscore is the recognition site of EcoRI).The PCR reaction system is: template (30ng/ μ l) 1 μ l; DNTP (every kind of 10mM) 2 μ l; Primer (each 10 μ M) 2 μ l; 10 * damping fluid, 5 μ l; DdH
2O 39.5 μ l; Pyrobest DNA Polymerase 0.5 μ l.Wherein 10 * damping fluid and Pyrobest DNAPolymerase are all from Takaka company.PCR reaction conditions: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s of 30 round-robin, 57 ℃ of annealing 45s, 72 ℃ are extended 1.5min; 72 ℃ are extended 7min again.(2) recovery of target gene fragment, be cloned into carrier.Concrete grammar is, with the PCR product with 1% (weightmeasurement ratio) agarose gel electrophoresis, obtain the fragment that a size is about 1300bp, reclaim fragment, cut and be cloned in pUC19 (Takaka company) carrier with the EcoRI enzyme, sequential analysis is also carried out in order-checking, and the result proves that the sequence that amplification obtains is consistent with the 4-nitrophenol-4-monooxygenase gene of expection, does not have the sudden change generation in the PCR process.(3) in intestinal bacteria (E.coli), express 4-nitrophenol-4-monooxygenase, concrete grammar is, utilize restriction enzyme EcoRI and NdeI with in PCR product cloning pET5a (Novagen company) expression vector that extremely the corresponding enzyme of process is cut, screening positive clone on the LB flat board that contains penbritin (100 μ g/ml).Positive transformant is placed the 100ml LB that contains penbritin (final concentration 100 μ g/ml), 37 ℃, 200rpm (rev/min) cultivate OD under the condition
600Value is about 0.6, adds 0.1mM IPTG (Isopropyl-β-D-thiogalactopyranoside) continue to induce 3-4h in nutrient solution.The thalline 12 that abduction delivering is good, the centrifugal collection of 000g, ultrasonic disruption is 10 minutes in ice bath, and 4 ℃, 200, centrifugal 1 hour of 000g, the gained supernatant is crude enzyme liquid and is used for follow-up research.(4) activity of the 4-nitrophenol-4-monooxygenase by this gene coded protein product of determined by ultraviolet spectrophotometry.Reaction is initial to add substrate, and (the 420nm molar extinction coefficient is 7 to substrate 4-nitrophenol, 000M
-1Cm
-1) consumption determine by the reduction of 400nm.Reaction system comprises substrate 4-nitrophenol 0.05mM, NADPH 0.2mM, FAD 0.04mM, albumen crude enzyme liquid 2 μ g.Be reflected in the 20mM phosphoric acid buffer and carry out under (pH 7.0) room temperature (identical below 20-25 ℃), (λ max=340nm, molar extinction coefficient are 6 to mensuration, 220M with 340nm place coenzyme NADP 11 charateristic avsorption band than living
-1Cm
-1) minimizing measure.The enzyme unit that lives is defined as under the room temperature (identical below 20-25 ℃), the required enzyme amount of increase of the minimizing of per minute 1 μ mol substrate or product, and the ratio of enzyme is lived and is what of every gram protease activity unit.(5) evaluation of 4-nitrophenol-4-monooxygenase catalysate.Concrete grammar is, the transformant that will contain 4-nitrophenol-4-monooxygenase gene places the 100ml LB that contains penbritin (final concentration 100 μ g/ml), 37 ℃, 200rpm (rev/min) cultivate OD under the condition
600Value is about 0.6, adds 0.1mM IPTG and continue to induce 3-4h in nutrient solution.The culture 12 that abduction delivering is good, the centrifugal collection of 000g, after thalline cleans twice with 20mM phosphoric acid buffer (pH7.0), be resuspended in the identical damping fluid, the 4-nitrophenol that adds 0.5mM, 30 ℃, 200rpm temperature are bathed sampling in 10 minutes and are placed and directly carry out HPLC (high performance liquid chromatography) analysis on ice.Testing conditions is: Gilson chromatographic instrument Discover chromatograph (SupelcoTM HPLC Columns, USA) C-18 (250x 4.6mm), each sample size is 2 μ l, moving phase is 30% methyl alcohol and 70% water (volume ratio), flow velocity 1ml/min, detect product at the 290nm place with the 119UV/VIS detector, detected result shows has Resorcinol to generate (retention time is 4.0min) in the reaction solution, illustrate after the 4-nitrophenol is through 4-nitrophenol-4-monooxygenase catalysis to detect the product Resorcinol.
Gene-expression system involved in the present invention includes but are not limited to the E.coli-pET5a system, is applied to clone of the present invention, expression vector includes but are not limited to following carrier: pUC serial carrier (Takaka company), pGEM-T (Progema company), pET serial carrier (Novagen company)." the molecular cloning laboratory manual " that carrier feature and cloning process can be write referring to cold spring harbor laboratory.--carrier system includes but are not limited to following system: bacterium--the phage system that is applied to host of the present invention, bacterium--plasmid vector system, yeast--yeast vector system, Mammals--viral system (vaccinia virus, adenovirus etc.), insect cell--rhabdovirus system.Gene involved in the present invention with can be cloned among the corresponding host after carrier is connected so that the preservation of gene, amplification and expression, the biological degradation that is applied to environmental pollutant such as 4-nitrophenol that can also be direct or indirect is handled.
The function verification method of 4-nitrophenol-4-monooxygenase gene involved in the present invention includes but are not limited to ultraviolet spectrophotometry, gas-chromatography (GC) or high pressure liquid chromatography (HPLC) analytical method, and other ordinary method that can prove the enzymology of this protein-active can be used and the present invention.
Four of technical essential of the present invention is the application in the biological degradation of 4-nitrophenol environmental pollutant of 4-nitrophenol-4-monooxygenase.
In one embodiment of the invention, method--the engineering strain biodegradation method that provides a proof 4-nitrophenol-4-monooxygenase can degrade environmental pollutant 4-nitrophenol.This method is to utilize the E.coli bacterial strain (Novagen company) of expressing 4-nitrophenol-4-monooxygenase, the 4-nitrophenol of degrading fully in the may command environment of laboratory.
4-nitrophenol-4-monooxygenase application method in the environmental pollutant biological degradation involved in the present invention includes but are not limited to the engineering strain biological degradation method, using gene involved in the present invention carries out the method that environmental pollutant handle and includes but are not limited to: will gene involved in the present invention direct or be cloned into various host-vector systems, be discharged in the contaminate environment then; Utilize gene constructed engineering strain involved in the present invention, be directly released in the contaminate environment; Utilization contains the activated albumen of the engineering strain expression of gene involved in the present invention, directly applies to the processing of environmental pollutant then.
The present invention compared with prior art, have the following advantages and effect: (1) 4-nitrophenol-4-monooxygenase gene involved in the present invention is a kind of brand-new 4-degrading mononitrophenol gene, can encode and produce 4-nitrophenol-4-monooxygenase, the degraded of this endonuclease capable catalysis 4-nitrophenol and 4-nitro-pyrocatechol, realize the detoxification treatment of this pollutant, treating processes is nontoxic, can not produce " secondary pollution ", leftover problem is few, in the biological degradation of environmental pollutant great application prospect is arranged.(2) in the degradation process of pollutent, do not need to add any auxiliary material, do not destroy the required edatope of plant-growth, reduce of the influence of Pollutant Treatment process to greatest extent environment.(3) easy and simple to handle, cost is low, is beneficial to and promotes the use of.(4) contaminant degradation speed is fast, surrounding environment is disturbed lacked, and in one embodiment of the invention, provides a kind of method of the 4-of degraded nitrophenol, through just finishing the removal fully of pollutent in 14 hours.
Description of drawings
Fig. 1 .4-nitrophenol-4-monooxygenase Degradation and Transformation 4-nitrophenol
The inoculation (initial concentration 160 μ M) in 4-nitrophenol pollutent that will contain 4-nitrophenol-4-monooxygenase gene, per hour sampling, the degraded situation of detection 4-nitrophenol.X-coordinate is time (unit: hour), and ordinate is a 4-nitrophenol concentration (unit: μ M).
Fig. 2 bacterial strain NyZ402 katabolism pollutent 4-nitrophenol
4-nitrophenol starting point concentration is 0.5mM, and the inoculation thalline is that LB cleans cell.(■) culture light absorption value (OD
600); (●) 4-NP concentration; (▲) nitrite ion concentration.The release of the metabolic simultaneous nitrite ion of 4-nitrophenol.
Embodiment
Substratum among all embodiment and molecular biology working method are familiar with by these those skilled in the art, can be with reference to " molecular cloning " (laboratory manuals such as Sambrook, the cold spring port, 1989) reach " fine works molecular biology experiment guide " (work such as U.S./F. Ao Sibai, Yan Ziying etc. translate, Beijing, Science Press, 1998).The invention will be further described below in conjunction with drawings and Examples, but not as the restriction to interest field of the present invention.
Separation and the evaluation of embodiment 1. pseudomonas putida NyZ402
From the sedimentation basin of sewage disposal of the chemical plant that produces compounds such as nitrophenol (any compound factory), gather the active sludge sample.Get the 1g sample and add (minimum medium (10g peptone, 10g yeast powder, 10g sodium-chlor in the 100ml enrichment medium.121 ℃ of sterilizations, 20min).Add the 4-nitrophenol as sole carbon, nitrogenous source), 30 ℃ of enrichment culture 3 days, continuous then 3 switching enrichment mediums improve 4-nitrophenol concentration in the substratum gradually, and 4-nitrophenol concentration is brought up to 2mM in substratum.Separate single bacterium colony with the line of solid enrichment medium then, the single bacterium colony of selecting to grow, go down to posterity stable, that degradation capability is good of a strain is studied.16S rDNA sequencing result shows that this bacterial strain belongs to pseudomonas putida and belongs to called after NyZ402 (Pseudomonasputida NyZ402).
The acquisition of embodiment 2.4-nitrophenol-4-monooxygenase gene
1) amplification of Resorcinol dioxygenase gene
With the pseudomonas putida NyZ402 inoculation that is separated in the LB liquid nutrient medium, 30 ℃ of shaking table overnight incubation.Centrifugal collection bacterial cell extracts genomic dna.
According to two primers of the conserved sequence of the benzenetriol dioxygenase gene among GenBank design, be the benzenetriol dioxygenase gene of this bacterial strain of template pcr amplification with the genomic dna of NyZ402 bacterial strain.Primer sequence is: upstream, GTC GAC ATA TGA CCG ATC AAG ACAAAG CCA TC, downstream: AAG CTT GGA TTC ATG ACT CAC TCT GCC TCCATG ACG A.50 μ l PCR reaction systems contain: template (30ng/ μ l) 1 μ l; DNTP (every kind of 10mM) 2 μ l; Primer (each 10 μ M) 2 μ l; 10 * damping fluid, 5 μ l; DdH
2O 39.5 μ l; RTaq0.5 μ l.Wherein 10 * damping fluid and rTaq are all from Takaka company.PCR reaction conditions: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s of 30 round-robin, 53 ℃ of annealing 45s, 72 ℃ are extended 1min; 72 ℃ are extended 7min again.Pcr amplification obtains the band that size is 800bp, reclaim this fragment and order-checking, the result shows that the benzenetriol dioxygenase gene among this fragment and the GenBank has higher similarity (consensus dna sequence 90%), shows that this fragment may be for participating in the 4-metabolic Resorcinol ring-opening enzyme of nitrophenol (dioxygenase) gene (pnpC) fragment in the NyZ402 bacterial strain.
2) clone obtains to participate in the metabolic gene cluster of 4-nitrophenol
With this fragment is starting point, can obtain the gene order of this fragment upstream and downstream with the method for chromosome walking (genomic walking), method is carried out [Siebert PD referring to document, et al., (1995) An improved PCR method for walking in uncloned genomic DNA.Nucl.Acids Res.23:1087-1088.].Move by 3 successive step, obtain to comprise the fragment of the 15kb of pnpC gene altogether.
3) 4-nitrophenol-4-monooxygenase gene is obtained in sequential analysis
With DNAstar software (DNAstar company product) sequence that obtains is analyzed, inferred possible open reading frame (ORF), be translated into amino acid sequence corresponding, and compare with the sequence in the GenBank database.The result shows that this fragment has comprised the full gene that may participate in 4-nitrophenol pathways metabolism.The metabolic key enzyme of catalysis 4-nitrophenol--4-nitrophenol-4-monooxygenase gene (pnpA) wherein, with sequence (accession number the is XP_754665) similarity of the monooxygenase that derives from Aspergillus fumigatus Af293 be 38%; Monooxygenase sequence (accession number the is YP_001507373) similarity that relies on the FAD that derives from Frankiasp.EAN1pec is 33%.This shows that also the known gene of function does not have the obvious sequence similarity among this gene and the GenBank, is a brand-new gene, a kind of new albumen of encoding--4-nitrophenol-4-monooxygenase.By the sequence alignment analytical proof, cloned and obtained the metabolic gene cluster sequence of participation 4-nitrophenol, comprise 4-nitrophenol-4-monooxygenase.
The expression of embodiment 3.4-nitrophenol-4-monooxygenase in intestinal bacteria (E.coli)
Extracting the genome of pseudomonas putida NyZ402 bacterial strain, is template pcr amplification 4-nitrophenol-4-monooxygenase gene with it, and the primer is P1:5 ' ATT
GAA TTC ATA TGGGCC GTG ATA GGA GA3 ' (base of band underscore is the recognition site of EcoRI and NdeI, and primer has comprised the SD sequence) and P2:5 ' AGT T
GA ATT CAC TCC TAC GACGAA AGA TCG3 ' (base of band underscore is the recognition site of EcoRI).The PCR reaction system is: template (30ng/ μ l) 1 μ l; DNTP (every kind of 10mM) 2 μ l; Primer (each 10 μ M) 2 μ l; 10 * damping fluid, 5 μ l; DdH
2O 39.5 μ l; Pyrobest DNA Polymerase 0.5 μ l.Wherein 10 * damping fluid and Pyrobest DNA Polymerase are all from Takaka company.PCR reaction conditions: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 45s of 30 round-robin, 57 ℃ of annealing 45s, 72 ℃ are extended 1.5min; 72 ℃ are extended 7min again.With the PCR product with 1% (weightmeasurement ratio) agarose gel electrophoresis, obtain the fragment that a size is about 1300bp, reclaim fragment, cut and be cloned in pUC19 (Takaka company) carrier with the EcoRI enzyme, order-checking is also carried out sequential analysis, the result proves that the sequence that obtains of amplification is consistent with the 4-nitrophenol-4-monooxygenase gene of expection, does not have the sudden change generation in the PCR process.
Utilize restriction enzyme EcoRI and NdeI with in PCR product cloning pET5a (Novagen company) expression vector that extremely the corresponding enzyme of process is cut, the recombinant plasmid pZWWQ001 that obtains is converted among the E.coli BL21 (DE3) (Novagen company), screening positive clone on the LB flat board that contains penbritin (100 μ g/ml) is verified the positive colony that screens with primer P1 and P2.Positive transformant is placed the 100mlLB that contains penbritin (final concentration 100 μ g/ml), 37 ℃, 200rpm (rev/min) cultivate OD under the condition
600Value is about 0.6, adds 0.1mM IPTG (Isopropyl-β-D-thiogalactopyranoside) continue to induce 3-4h in nutrient solution.The thalline 12 that abduction delivering is good, the centrifugal collection of 000g, thalline is resuspended in the identical damping fluid after using the 20mM phosphoric acid buffer (pH 7.0) of ice bath to clean twice.Ultrasonic disruption is 10 minutes in ice bath, and 4 ℃, 200, centrifugal 1 hour of 000g, the gained supernatant is crude enzyme liquid and is used for follow-up research.
The Function Identification of embodiment 4.4-nitrophenol-4-monooxygenase gene
1) 4-nitrophenol-4-monooxygenase enzyme activity determination
Do the enzymic catalytic reaction experiment in intestinal bacteria (E.coli) (Novagen company), to express the 4-nitrophenol-4-monooxygenase crude enzyme liquid that obtains.The determination of activity of 4-nitrophenol-4-monooxygenase detects by ultraviolet spectrophotometry.Reaction is initial to add substrate, and (the 420nm molar extinction coefficient is 7 to substrate 4-nitrophenol, 000M
-1Cm
-1) consumption determine by the reduction of 400nm.Reaction system comprises substrate 4-nitrophenol 0.05mM, NADPH (DPNH I) 0.2mM, FAD (flavin adenine dinucleotide) 0.04mM, albumen crude enzyme liquid 2 μ g.Be reflected in the 20mM phosphoric acid buffer and carry out under (pH 7.0) room temperature, (λ max=340nm, molar extinction coefficient are 6 to mensuration, 220M with 340nm place coenzyme NADP 11 charateristic avsorption band than living
-1Cm
-1) minimizing measure.The enzyme unit that lives is defined as under the room temperature (identical below 20-25 ℃), the required enzyme amount of increase of the minimizing of per minute 1 μ mol substrate or product, and the ratio of enzyme is lived and is what of every gram protease activity unit.Measurement result is 49.6U/g for this 4-nitrophenol-4-monooxygenase specific activity.
2) evaluation of 4-nitrophenol-4-monooxygenase catalysate
Method by bio-transformation is identified 4-nitrophenol-4-monooxygenase catalysis 4-nitrophenol product, specific practice is as follows: the transformant that will contain 4-nitrophenol-4-monooxygenase gene places the 100ml LB that contains penbritin (final concentration 100 μ g/ml), 37 ℃, 200rpm (rev/min) cultivate OD under the condition
600Value is about 0.6, adds 0.1mM IPTG and continue to induce 3-4h in nutrient solution.The culture 12 that abduction delivering is good, the centrifugal collection of 000g, after thalline cleans twice with 20mM phosphoric acid buffer (pH 7.0), be resuspended in the identical damping fluid, the 4-nitrophenol that adds 0.5mM, 30 ℃, 200rpm temperature are bathed sampling in 10 minutes and are placed and directly carry out HPLC (high performance liquid chromatography) analysis on ice.Testing conditions is: Gilson chromatographic instrument Discover chromatograph (SupelcoTM HPLC Columns, USA) C-18 (250x 4.6mm), each sample size is 2 μ l, moving phase is 30% methyl alcohol and 70% water (volume ratio), flow velocity 1ml/min, detect product at the 290nm place with the 119UV/VIS detector, detected result shows has Resorcinol to generate (retention time is 4.0min) in the reaction solution, illustrate after the 4-nitrophenol is through 4-nitrophenol-4-monooxygenase catalysis to detect the product Resorcinol.
3) 4-nitrophenol-4-monooxygenase enzymatic property research
Be the enzyme activity determination that substrate carries out 4-nitrophenol-4-monooxygenase with 4-nitrophenol, 2-nitrophenol, 3-nitrophenol, 4-nitro-pyrocatechol respectively.The result shows, 4-nitrophenol-4-monooxygenase is only to 4-nitrophenol and the effect of 4-nitro-pyrocatechol, have stronger Substratspezifitaet, and 4-nitrophenol-4-monooxygenase to the catalysis speed of its natural substrate 4-nitrophenol obviously greater than the 4-nitro-pyrocatechol, as shown in the table.The endonuclease capable katabolism 4-nitrophenol and the 4-nitro-pyrocatechol of final certification 4-nitrophenol-4-monooxygenase gene coding.
4-nitrophenol-4-monooxygenase substrate scope is lived with relative enzyme
The embodiment 5.4-nitrophenol-application of 4-monooxygenase in contaminant degradation
Intestinal bacteria (the E.coli that will contain 4-nitrophenol-4-monooxygenase gene, Takara company product) engineering strain (as making up among the embodiment 3) is inoculated among the 100ml LB that contains penbritin (final concentration 100 μ g/ml), 37 ℃, 200rpm (rev/min) cultivate OD under the condition
600Value is about 0.6, adds 0.1mM IPTG and continue to induce 3-4h in nutrient solution.The culture 12 that abduction delivering is good, the centrifugal collection of 000g, be resuspended in the 20mM phosphoric acid buffer (pH 7.0), the 4-nitrophenol that adds 160 μ M, per hour sampling detects the degradation of substrates situation with HPLC, the result shows, contain the engineering bacteria of 4-nitrophenol-4-monooxygenase gene can be in 14 hours Degradation and Transformation 4-nitrophenol fully, degradation rate reaches 99%, as shown in Figure 1.
The application of embodiment 6. bacterial strain NyZ402 in 4-nitrophenol contaminant degradation
Will be in liquid LB the pseudomonas putida NyZ402 bacterial strain of grow overnight clean the back with minimum medium (MM) and inoculate in the MM substratum of 50ml into the inoculum size of 1% (weightmeasurement ratio), add 0.5mM substrate 4-nitrophenol and supply strain growth as unique carbon, nitrogenous source, timing sampling is measured concentration of substrate and cell concentration (OD
600) variation.Shown in the accompanying drawing 2, bacterial strain NyZ402 can be with the 4-nitrophenol as sole carbon, nitrogenous source growth as a result, and the 4-nitrophenol reduces the release of simultaneous nitrite ion, and this shows that bacterial strain NyZ402 is by oxidative pathway metabolism 4-nitrophenol.With single colony inoculation thalline to the MM substratum, be 0.5mM with concentration gradient respectively, 1mM, 1.5mM, the 4-nitrophenol of 2mM and 2.5mM is grown as sole carbon, the nitrogenous source of bacterial strain NyZ402 growth, reached and showed that NyZ402 can reach 2mM to the tolerance concentration of 4-nitrophenol, when 4-nitrophenol concentration is higher than 2mM, not regrowth of NyZ402.In addition, NyZ402 can utilize substrate 4-nitrophenol, pyrocatechol and Resorcinol growth, but can not utilize substrate 4-nitro-pyrocatechol, 2-nitrophenol, 3-nitrophenol, hydroxyquinol, 2,6-dinitrophenol, p-nitrobenzoic acid, oil of mirbane, Meta-dinitrobenzene, 2,4-dinitrotoluene (DNT), 2,4, the growth of 6-picric acid.
SEQUENCE?LISTING
＜110〉Wuhan Virology Institute,Chinan academy of Sciences
＜120〉a kind of 4-nitrophenol-4-monooxygenase gene and preparation method and purposes
＜130〉a kind of 4-nitrophenol-4-monooxygenase gene and preparation method and purposes
<160>1
<170>PatentIn?version?3.3
<210>1
<211>1233
<212>Type：DNA
<213>Pseudomonas?putida
<400>1
atggaaacaa?ttgatggcgt?agtggttgtc?ggcggaggcc?cggttgggtt?gcttacagca 60
ttgaaacttg?gcaaggccgg?tgtgcgggta?gttgtcctgg?agtccgaatc?gggcgtttca 120
ccctcaccgc?gggcggttgc?ctatatgcca?cccactgctg?cggcactgga?ccgtttcggg 180
ttgctcgacg?acattcgcaa?acgtgctgtg?tggtgccctg?atttcgctta?ccgtcacggt 240
aatggcgaac?tgatcgcgaa?aatggactgg?gctgttctgg?cacaggacac?tgattacccc 300
tatatgctgt?tgctggcgca?aaaccatgtg?tccaatgtaa?tcgtggagca?tcttcgcaaa 360
ctctccaatg?tcgaaattcg?ctggaatcac?gcggtcgaag?acattgagca?ggacgacgac 420
tatgtgacca?tggaaaccag?cggccctgcc?ggaaaagcac?gtttgcgggc?gaaatgggtt 480
gctgccaccg?acggtgcacg?cagtaccgta?cgaggaaaaa?tcgggctgtc?ctttgatggt 540
attacttggt?cggaacgcct?ggttgcgaca?aacgtgttct?atgacttttc?cctgcacggc 600
tactctcggg?ccaacttcgt?tcacgacccg?gtagactggg?ccgtcgtcgt?tcagctcgac 660
aaaaccggcc?tgtggcgtgt?gtgctatggc?gaagatcctg?agatctccga?ggctgaagtc 720
cgccgccgct?tgccggagcg?tttcaagcgc?ctgctgccgg?gagcgccgac?gcccgatcag 780
tatcgggtcg?actacctcaa?tccttaccgt?gtccaccagc?gttgcgccgc?cgagttccgc 840
cgtggacggg?tgatcctggc?cggagatgct?gcccatgcga?ccaacccgat?gggggggctg 900
ggactgtcgg?ggggcgtact?tgatgccgag?catttggccg?aggcgttgat?tgcggtgatc 960
aaagaaggcg?cttctaccaa?ggtgctggat?gagtactcta?tcgatcggcg?caaagtcttc 1020
ctcgagttca?cgtccccgac?cgcgactgcc?aacttcacct?ggatgaaaga?aagtgaccct 1080
gcccagcgtg?ctcgtgataa?cgcaatgttt?gatcatgcgg?gcaaggactt?gaaagtcatg 1140
cgtgaaattc?ttctggactt?cgagaagctc?aacggccggc?gcgtgatcgc?tccacgacaa 1200
tatgcgcaag?cgcttgaaag?tatgggcgca?tag 1233
Claims (3)
1. isolated polypeptide, its nucleotide sequence coding is shown in the SEQ ID NO:1.
2. isolated polypeptide, its sequence is the aminoacid sequence shown in the SEQ ID NO:2.
3. the application of the described a kind of 4-nitrophenol of claim 1-4-monooxygenase gene in the degraded of 4-nitrophenol environmental pollutant.
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