CN102154357B - Organic solvent responded expression vector and application thereof in biological catalysis - Google Patents
Organic solvent responded expression vector and application thereof in biological catalysis Download PDFInfo
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Abstract
The invention relates to an organic solvent responded expression vector and application thereof in biological catalysis, belonging to the field of biotechnology; the organic solvent response broad host constitutive expression vector comprises a vector pMMS and a vector pMMRS, and consists of a DNA element, a replicor RSF1010, a selection marker gene bla, multiple cloning sites, an intergenic sequence and an organic solvent response promoter Psrp; and the expression vector is characterized in that the nucleotide sequence of the pMMS is shown by SEQ ID No. 1, and the nucleotide sequence of the pMMRS is shown by SEQ ID No. 2. The invention has advantages in biological catalysis, especially in double-liquid-phase biological catalysis. The invention is better in economy and effectiveness, and ensures engineering bacteria constructed by organic solvent induction vector to be better than genetic engineering bacteria constructed by common isopropyl beta D thiogalactopy ranoside (IPTG) induction vector under the condition of high-concentration organic solvent exists.
Description
Technical field
What the present invention relates to is a kind of organic solvent expression vector and application thereof of biological technical field, is specially a kind ofly to reply promotor P with organic solvent
SrpThe expression vector that the organic solvent that makes up is replied and the application in biocatalysis thereof.
Background technology
Biocatalysis has application potential widely at synthetic fine chemicals and biological restoration field., environmental friendliness energy-conservation as a clock, safety and effective means, whole-cell catalytic is attracting increasing concern.In biocatalysis, usually to touch organic solvent.Such as in the biocatalysis process in order to increase dissolubility of reactants or to avoid the reasons such as decomposition of product usually will introduce the biliquid phase reaction that contains organic solvent; The Polluted area that carries out biological restoration at many needs also is flooded with the organic solvent of the various concentration that reach capacity, such as benzene,toluene,xylene etc.For common micro-organisms, organic solvent is poisonous, because organic solvent can destroy the structure of biomacromolecule, thereby and can upset the ordered structure of phospholipid bilayer in the cytolemma accumulation, and then destroy the integrity of the structure and function of cytolemma.The toxicity of organic solvent is that biocatalysis is at a big obstacle of environment and the application of fine chemicals production field.
Since the first strain organic solvent-resistant microorganism in 1989 was separated, people had been separated to a large amount of organic solvent-resistant microorganisms from various ecotopes, were many with pseudomonas putida wherein.These microorganisms are a kind of of extreme microorganism, and they are rugged by cytolemma, the murder by poisoning that methods such as expression organic solvent efflux pump are resisted organic solvent.The organic solvent-resistant microorganism all has the important use potentiality at the numerous areas of biocatalysis, and such as biological restoration, fine chemicals is synthetic etc.Pseudomonas putida has the surprised characteristics of many people of allowing, and has powerful metabolic capacity such as pseudomonas putida, and aromatic series and the fatty compounds of the arbitrary form of almost can degrading comprise the compound that some are hypertoxic.Pseudomonas putida still is the main monoid in the soil microorganisms, is a kind of relatively bacterial strain of safety, thereby, bigger development potentiality is arranged in recombinant DNA technology and environmental applications.Of particular concern is that some bacterial strain of pseudomonas putida has the ability of tolerance organic solvent, this makes them become very useful bacterial strain in Industrial Catalysis and environmental area.This wherein pseudomonas putida (Pseudomonas putida) S12 be an outstanding representative (Ruijssenaars H, 2007, J.Biotechnol.131:S205-S206), this strain bacterium is being developed to produce the platform bacterial strain of aromatic compound with saccharide compound.Yet, these organic solvent-resistant microorganisms often do not possess the catalytic activity that people want, therefore to utilize organic solvent tolerance bacterium carry out catalysis or environment remediation just the engineered means of needs use these microorganisms are transformed, to give its unique catalytic capability, expansion application of microorganism scope can enoughly be suitable for various industry and environmental demand.
Heterologous expression system is to transform microorganism, and the strong instrument of expansion microorganism strains range of application is widely used in biocatalysis, such as fine chemicals production, biological restoration and biological desulphurization.Suitable heterologous expression system not only can make the bacterial strain transformation of microorganism or structure become easily, and can give the genetic engineering bacterium excellent characteristic, makes that the scale operation that utilizes biological catalyst to carry out is more economical feasible.At present, only there is a spot of genetic manipulation instrument in the pseudomonas putida of organic solvent tolerance, to use, and existing operational tool mostly is based on the intestinal bacteria lactose operons, need use the expensive IPTG as inductor when inducing, this has limited them in the application of industry and environmental area.
Promotor P
SrpIt is the promotor of the organic solvent efflux pump gene of organic solvent tolerance bacterial strain pseudomonas putida (P.putida) S12, this promotor can be induced transcribing of promotor gene down at multiple cheap organic solvent, such as can start transcribing of downstream gene under the inducing of aromatic hydrocarbon, alkane and alcohols.And this promotor is not replied such as pH, temperature, salt concn or organic acid for common environmental stress, and these characteristics will make that its downstream gene is easier and be controlled by the people.Especially it should be noted that these organic solvents also are simultaneously solvents commonly used in the industrial biocatalysis, and in contaminate environment, also exist often.This promotor is as the gene expression regulation element of expression vector, to have very big advantage, not only can substitute expensive IPTG with cheap organic solvent, reduce the derived cost of biocatalysis, can also directly utilize the organic solvent that exists in microbiological industry and the environment that expression of gene is induced easily, the save operation step reduces cost.Therefore, this promotor is fallen in biocatalysis especially biliquid phase reaction and environmental organism reparation field and is had very big application potential.
Summary of the invention
The present invention is directed to deficiency of the prior art, proposed expression vector that a kind of organic solvent replys and the application in biocatalysis thereof, the present invention replys promotor P with organic solvent
SrpMake up, showed better economic and validity, be much better than to induce with common IPTG the genetic engineering bacterium of vector construction.
The present invention is achieved by the following technical solutions:
The present invention relates to the expression vector that a kind of organic solvent is replied, be specially wide host's constitutive expression carrier that organic solvent is replied.
Described wide host's constitutive expression carrier comprises carrier pMMS and carrier pMMRS, replys promotor P by DNA element, replicon RSF1010, selection markers gene bla, multiple clone site, intergenic sequence and organic solvent
SrpForm, it is characterized in that the nucleotide sequence of described carrier pMMS is shown in SEQ ID No.1, the nucleotide sequence of described carrier pMMRS is shown in SEQ ID No.2.
The host of described carrier is pseudomonas (containing Pseudomonas stutzeri, pseudomonas putida or Pseudomonas fluorescens) and intestinal bacteria.
Described DNA element, wherein promotor P
SrpBe derived from pseudomonas putida (P.putida) S12 with regulatory gene srpRS, other elements come from carrier pMMB66EH.
Described carrier pMMS, namely bacillus coli DH 5 alpha/pMMS (Escherichia coli DH5 α/pMMS) be deposited in Chinese typical culture collection center, preserving number is CCTCC M 2010319, the preservation time is: on November 29th, 2010.
Described carrier pMMRS, namely bacillus coli DH 5 alpha/pMMRS (Escherichia coli DH5 α/pMMRS) be deposited in Chinese typical culture collection center, preserving number is CCTCC M 2010320, the preservation time is: on November 29th, 2010.
Described pseudomonas putida, i.e. pseudomonas putida DS23 (Psudomonas putida DS23), this bacterial strain has been deposited in Chinese typical culture collection center, and preserving number is CCTCC M 2010321, and the preservation time is: on November 29th, 2010.
Described pseudomonas putida, i.e. pseudomonas putida DT23 (Psudomonas putida DT23), this bacterial strain has been deposited in Chinese typical culture collection center, and preserving number is CCTCC M 2010322, and the preservation time is: on November 29th, 2010.
The typical culture collection centre address of China is: Wuhan City Wuhan University, loujia hill belongs, postcode: 430072.
The present invention also studies the application of expression vector in biocatalysis that above-mentioned organic solvent is replied, described carrier pMMS and pMMRS, with an organic solvent compounds is as inductor, can make organic solvent and replying, can be used for making up the biocatalysis engineering bacteria, and be applicable to comprise that compound is synthetic, the biocatalysis of biological restoration, bio-transformation.
Application in biocatalysis, carrier pMMS and pMMRS are used for making up engineering bacteria, and constructed engineering bacteria is used for biocatalysis.
The present invention is with promotor P
SrpBe the basis, utilize the existing wide host range carrier pMMB66EH in laboratory, made up can be in intestinal bacteria and pseudomonas self-replicating, and carrier pMMS and the pMMRS that can be induced by multiple organic solvent compounds, and operation report gene (chloramphenicol acetyl transferasegene, cat) study the operability of constructed carrier, studied the application of carrier construction in biocatalysis especially biliquid phase biocatalysis.
The carrier pMMS that the present invention is constructed and pMMRS can be in all kinds of pseudomonad strain the expression of promotor gene, wherein pMMRS shows as inducible expression vector in all bacterial strains, pMMS is induction type in pseudomonas putida (P.putida) S12, shows as constitutive expression carrier in other bacterial strains.
The present invention is application example with the biological desulphurization reaction of dibenzothiophene simultaneously, has investigated the advantage of constructed expression vector in biocatalysis especially biliquid phase biocatalysis.The engineering bacteria DS23 that at first used vector construction of the present invention has made up the reorganization bacterium with general carrier (IPTG induces) and has made up engineering bacteria DT23.Carried out a series of biliquid phase reaction with this two strains bacterium then.Find that DS23 can be by the vigor of multiple organic solvent abduction delivering desulfurization, DS23 is better than the DT23 vigor of inducing with IPTG in biliquid phase reaction system, has showed better economic and validity.And under high levels of organic solvents (50% normal hexane) existence condition, DS23 can make the dibenzothiophene of 1mM be degraded 67% in 24h, and the average response speed of initial 6h is 2.41mmolg (stem cell is heavy)
-1h
-1, this is much better than to induce with common IPTG the genetic engineering bacterium of vector construction.
Description of drawings
The structure of Fig. 1 carrier pMMS
Fig. 2 PCR detects carrier pMMS-cat, M1, M2: molecular weight marker thing; C1: contrast; S1: sample
The structure of Fig. 3 carrier pMMRS
Fig. 4 PCR detects the structure of carrier pMMRS-cat, M3, M4:DNA molecular weight marker thing; S3: be template with carrier pMMRS-cat, PlacI.f (MluI) and PlacI.r (MluI) carry out the result of pcr amplification for primer; S4: be template with carrier pMMRS-cat, PlacI.f (MluI) and srpR.r (ApaI) carry out the result of pcr amplification for primer; Molecular weight unit is bp
The structure of Fig. 5 carrier pMMS-ABCD
The structure of Fig. 6 PCR checking pMMS-ABCD, M1, M2: molecular weight marker thing; Swimming lane 1-5: be template with the recombinant vectors, the temperature dszABCD gene that PCR obtains of rising progressively; Lane C: be the contrast PCR of template with the pMMS carrier
The desulfurization situation of bacterial strain when Fig. 7 different organic solvents is done inductor and reaction medium
The comparison of Fig. 8 DT23 and DS23 desulfurization vigor
The sweetening effectiveness of DS23 when the organic solvent of Fig. 9 high density 50% (volume ratio) exists,
Dibenzothiophene concentration; (▲) 2-xenol (dibenzothiophene degraded product) concentration
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used for explanation the present invention, limit the scope of the invention and be not used in.The experimental technique of unreceipted actual conditions among the following embodiment, according to ordinary method, specifically referring to " molecular cloning laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to reagent and test kit manufacturer.All DNA elements of using of the present invention and microorganism strains all come from has published documents and materials, and itself all can be obtained its sequence information and DNA by the public, sees table 1 and table 2 for details; Other various materials all are that the present invention makes up, and can construct with method of the present invention again; Wherein carrier pMMABCD makes up in document before this seminar and the patent that (Tao et al.2006, Appl.Environ.Microbiol.72:4604-4609), the present invention uses it as the reference of the operability of research and establishment carrier.
The bacterial strain tabulation that present embodiment relates to:
Table 1
The carrier tabulation that present embodiment relates to:
Table 2
The primer tabulation that present embodiment relates to:
Table 3
| The primer title | Sequence |
| cat.f(HindIII) | 5’-CTGT AAGCTTGCTAAGGAAGCTAAAATGG-3’ |
| cat.r(HindIII) | 5’-ATCG AAGCTTTAAAAAAATTACGCCCCG-3’ |
| Psrp.f(MluI) | 5’-TCG ACGCGTTCGACGCTGCTCTGG-3’ |
| Psrp.r(EroRI) | 5’-AGAC GAATTCTGGCTCCATCTCTC-3’ |
| dszD.f(EcoRI) | 5’-GAG GAATTCATGTCTGACAAGCCGAATGCC-3’ |
| dszC.r(HindIII) | 5’-GATCA AAGCTTCAGATCCTCAGGAGGTGAAGC-3’ |
| srpS.f(MluI) | 5’-AAT ACGCGTGAGCTCTACAGTGGC-3 |
| srpR.r(ApaI) | 5’-ATT GGGCCCGTCGACTATAGCG-3’ |
| PlacI.f(MluI) | 5’-GCT ACGCGTTGCAGTCGATAAG-3’ |
| PlacI.r(MluI) | 5’-AT ACGCGTCCAGTTGTTGTGCC-3’ |
Organic solvent is replied the structure of expression vector pMMS and pMMRS
Promotor P
SrpIn bacterial strain S12, instruct the expression of organic solvent tolerance gene, be a kind of promotor that can be induced by multiple organic solvent compounds, these compounds are mostly relatively more cheap, and wherein have much industrial have widely use, such as normal hexane, hexanaphthene and toluene.Be that the heterologous expression system of fundamental construction will be the good alternative system of lac promoter expression system undoubtedly with this promotor.The above-mentioned P of present embodiment
SrpPromotor has been replaced the former promotor of pMMB66EH, has made up the expression vector pMMS (Fig. 1) that can be induced by organic solvent.For this reason, at first on the multiple clone site of wide host range carrier pMMB66EH, insert a reporter gene, chloramphenicol resistance gene cat makes up pMMB66EH-cat.Carry out double digestion (MluI+EcoRI) then, insert the promoter fragment that is primer PCR expands from the S12 genome with Psrp.f (MluI) and Psrp.r (EcoRI), obtain pMMS-cat.Again this carrier is carried out enzyme with HindIII and cut, reclaim big fragment and carry out the carrier cyclisation from connecting, can obtain purpose carrier pMMS (Fig. 1).
In the vector construction process with chlorampenicol resistant as the activity index whether chloramphenicol resistance gene inserts, whether the carrier that builds provides the molecular biology evidence proof correct with the method for pcr amplification.Fig. 2 is the result who carrier pMMS-cat is verified with PCR method, can not amplify band with primer Psrp.f (MluI) and Psrp.r (EcoRI) when using pMMB66EH-cat as template (contrast) as seen from the figure, can amplify the band of 200～300bp during as template with pMMS-cat, this and clone's promotor P
SrpSize conform to, the structure that carrier pMMS-cat is described is successful.
Further the method for order-checking is adopted in checking.Excise chloramphenicol resistance gene with HindIII, cyclisation uses the method for dna sequencing finally to verify Zi the ultimate aim carrier pMMS that gets continuously, and sequencing result shows the in full accord of the sequence of measured carrier construction and expectation.
The pMMS of above-mentioned structure itself does not contain regulatory gene (repressor gene), and therefore, its expression is composing type, is not subjected to the regulation and control (except in P.putida S12) of inductor, and present embodiment has made up the carrier pMMRS that contains repressor gene again for this reason.At first from the S12 genome, clone repressor gene srpSR, be connected into then and obtain pMMSx on the pMMS-cat, and then the weak constitutive promoter of clone's lacI gene obtains carrier pMMRS-cat before placing srpSR, cuts at enzyme and obtain final objective carrier pMMRS (Fig. 3) behind concatemerization.
That Fig. 4 shows is the result of the PCR checking of carrier pMMRS-cat, as can be seen from the figure uses primer PlacI.f (MluI) and PlacI.r (MluI) to amplify a dna fragmentation about 200～300bp, the P of this fragment and expectation from pMMRS-cat
LacISize consistent.Use primer PlacI.f (MluI) and srpR.r (ApaI) can amplify one greater than the dna fragmentation of 2kb simultaneously, the P that this fragment and expectation obtain
LacIThe size of-srpS-srpR fragment is consistent.The carrier pMMRS-cat that the result shows successful structure.The same, enzyme cuts except reporter gene cat, and cyclisation also is finally to confirm with the method for order-checking from the pMMRS that finally obtains, and its result shows the sequence of gained and estimates that the destination carrier sequence that obtains is in full accord, illustrates finally successfully to have obtained carrier pMMRS.
Carrier operability and host range
Exactness host range for further checking carrier structure, also the carrier of above-mentioned structure has been imported in the table 1 in each pseudomonas in the present embodiment, checked inductor (organic solvent) when existing carrier can give the host bacterium with chlorampenicol resistant, the result shows and contains the pMMS-cat pseudomonas, when inductor exists, can grow at the LB substratum that contains 5g/l paraxin, the carrier that this explanation makes up contains correct target start, and has the ability that starts downstream gene expression under the solvent-induced condition having.
Table 4 inductor (organic solvent) when existing carrier pMMS-cat and pMMRS-cat to the influence of each bacterial strain chloramphenicol sensitivity
| P.putida S12 | P.putida Idaho | P.putida KT2440 | P.fluorescens ACB | |
| When not containing carrier | Responsive | Responsive | Responsive | Responsive |
| When containing pMMS-cat | Insensitive | Insensitive | Insensitive | Insensitive |
| When containing pMMRS-cat | Insensitive | Insensitive | Insensitive | Insensitive |
Result in the table 4 shows that carrier pMMRS-cat also can give various bacterial strain chlorampenicol resistants, and resistance need be added organic solvent as inductor, and this induces carrier character identical with the organic solvent that expection makes up.The bacterial strain that carrier pMMS-cat can give each test equally is with chlorampenicol resistant, but owing to do not contain regulatory gene srpRS, the expression of its chloramphenicol resistance gene in the bacterial strain except S12 is composing type, does not need to induce.This is because the S12 bacterial strain itself contains regulatory gene, can suppress P under the situation of no inductor
SrpPromotor, and do not contain regulatory gene srpSR in other the bacterial strain, thereby can't suppress promotor P
SrpCarrier pMMS and pMMRS that these presentation of results present embodiments make up have the potentiality of using in multiple pseudomonas.Find also in the test that carrier pMMRS-cat can cause serious solvent shock when using in bacterial strain S12, growth has very long lag period behind the organic solvent having added namely to cause S12.This phenomenon be by the expression inhibiting of the solvent efflux pump promotor repressor gene srpSR among the carrier pMMRS-cat on the S12 karyomit(e) efflux pump expression of gene cause.This shows that it is inappropriate using pMMRS in bacterial strain S12, but can use carrier pMMS.
The dibenzothiophene desulfurization bacterium makes up
Biological desulphurization is a kind of desulfurization of fossil fuel method of environmental protection and energy saving, is a kind of replacement scheme of potential chemical desulfurization.The general profit two-phase system that uses of biological desulphurization that carries out the fuel oil of oil and petroleum refining carries out, and is a kind of of two-phase biocatalysis.Fuel oil is multiple organic solvent compounds, such as the mixture of alkane aromatic hydrocarbon etc., thereby, in biological desulphurization, exist the same problem of biphasic reaction, namely organic solvent is to the toxicity of cell.Made up the desulfurization bacterial strain A4 that solvent tolerant is arranged of strain reorganization with pMMB66EH before, wherein contain carrier pMMABCD, specifically be recorded in implementation of China example number of patent application 200610044410.1, publication number CN1861785, in open day on November 15th, 2006, present embodiment will be the characteristics that the organic solvent inducible expression carrier of example investigation structure is used in biocatalysis with the biological desulphurization.At first carrier pMMS-cat is carried out enzyme and cut, from carrier pMMABCD, with the fragment of PCR method amplification dszABCD, carry out enzyme and cut then, be connected to enzyme and cut on the pMMS-cat of processing, obtain recombinant vectors pMMS-ABCD.After the structure, the transformant that screening is obtained carries out the PCR checking.Extraction carrier wherein uses primer dszD.f (EcoRI) and dszC.r (HindIII) to carry out PCR as template, result (Fig. 6) as shown below, and this shows that the gained transformant is correct transformant, wherein contains target recombinant vectors pMMS-ABCD.Extraction carrier electricity consumption method for transformation wherein changes among the organic solvent-resistant bacterial strain pseudomonas putida S12 and goes, measure desulphurizing activated then, finishing screen is selected activated transformant, finally obtained active better conversion of a strain through screening, comprise pseudomonas putida (P.putida) DS23, this bacterial strain has been stored in Chinese typical culture collection center (CCTCC M 2010321).Simultaneously carrier pMMABCD is changed over to pseudomonas putida S12, with the contrast as research DS23, obtain active better conversion through screening and comprise pseudomonas putida (P.putida) DT23, this bacterial strain has been deposited in Chinese typical culture collection center (CCTCC M 2010322).
Dibenzothiophene desulfurization in the different organic solvents
Contain diversified organic solvent in the fuel oil, these organic solvents have different toxicity to cell, and they are for P
SrpInducibility also be not quite similar, therefore, be necessary to inquire into the operability of carrier when using different organic solvents of structure.Present embodiment has selected different organic solvent (normal hexane, normal heptane, n-decane, hexanaphthene and n-Heptyl alcohol) to carry out this research.With an organic solvent induce the desulfurization genetic expression of bacterium at first respectively, use corresponding organic solvent to carry out the biliquid phase reaction then, measure the degree of desulfurization at last with HPLC.Biliquid phase reaction water uses the M9 substratum, and organic phase is used the corresponding organic solvent of 20% (v/v).The result is (Fig. 7) as shown in the figure, after the reaction of 12h, use various organic solvents to be respectively as the dibenzothiophene degradation rate of inductor and reaction medium: 57.2% (normal hexane), 49.8% (normal heptane), 51.5% (n-decane), 27.4% (hexanaphthene) and 27.7% (n-Heptyl alcohol).For the organic solvent that all relate to, the carrier of structure can be induced, and has embodied the activity of desulfurization in biphasic reaction, and obviously normal hexane is best in the organic solvent of all uses.Normal hexane occupies bigger component in fuel oil, and this organic solvent is comparatively more common than other several application in catalysis, and this has reflected from a side that also the carrier that makes up the present embodiment has wider range of application in biocatalysis.
Reply the comparison that carrier and common IPTG induce the dibenzothiophene desulfurization bacterium of vector construction with solvent
Induce carrier for the organic solvent of further research and establishment, relatively use different culture condition to cultivate bacterial strain DT23 and bacterial strain DS23, be used for the biliquid phase reaction then.At first this two strains bacterium is carried out different cultivations, DT23 uses LB+Ap+IPTG (AB) and LB+Ap+IPTG+ normal hexane (EF) respectively, DS23 cultivates (CD) with the LB+Ap+ normal hexane, is that reaction medium (AEC) and M9+30% normal hexane are that reaction medium (BDF) carries out the biological desulphurization reaction with M9 then.With the palliating degradation degree of HPLC mensuration dibenzothiophene, the result is (Fig. 8) as shown in the figure.All above-mentioned situation cultured cells all have very strong sweetening power in M9, can be starting point concentration the dibenzothiophene degraded 99% of 0.5mM at 12h, and major portion can be finished in preceding two hours.Inducing of this explanation normal hexane is the same with IPTG effective, induced desulfurization expression of gene preferably, in the diphasic system that contains 30% normal hexane, the DT23 that LB+Ap+IPTG cultivates does not have the vigor of desulfurization, this is because do not add organic solvent in the substratum in advance, thereby do not have to induce the organic solvent tolerance of bacterial strain, when carrying out the biliquid phase reaction, can produce the solvent shock and cause most necrocytosis, thereby show as the vigor that does not have desulfurization.The DS23 cell that the DT23 that the LB+Ap+IPTG+ normal hexane is cultivated and LB+Ap+ normal hexane are cultivated has been degraded the desulfurization vigor of 26% and 56%, DS23 in the dibenzothiophene of 0.5mM respectively than DT23 high twice nearly in the desulphurization reaction of 12h.In diphasic system, use the organic solvent of present embodiment structure to induce carrier on cost or desulfuration efficiency, all to have advantage than the carrier that IPTG induces no matter the result shows.
Embodiment 6
The desulfurization of dibenzothiophene in the high density normal hexane
Water oil ratio low (less than 1: 9) is the important factor that influences desulphurization cost always in the biological desulphurization process, and improving water oil ratio is urgent problem.Therefore present embodiment has carried out the trial of desulfurization under the high water oil ratio condition.At first use LB+Ap+ normal hexane culturing cell, the desulfurization that carries out the biliquid phase reaction with the M9 that contains 50% normal hexane then, the result is (Fig. 9) as shown.In 24 hours biliquid phase reaction process, DS23 is with the dibenzothiophene degraded 67% of 1mM, and wherein 38% is preceding 6h degraded, and the average response speed of initial 6h is 2.41mmol g (dry cell weight)
-1h
-1This result than the result who uses P.putida A4 (utilizing the organic solvent-resistant desulfurization bacterium of the expression vector establishment that IPTG induces) to obtain good (Tao et al.2006, Appl.Environ.Microbiol.72:4604-4609).The various biocatalysis of using microorganism to carry out in order to prevent microbial contamination, all inevitably will be carried out disinfecting action, and this is an operation that will expend a large amount of energy.The carrier that present embodiment is made up and organic solvent-resistant microorganism are in conjunction with use, can avoid this operation, because seldom there is microorganism under the condition that organic solvent exists, to survive, this has just been avoided microbiological contamination, also reduced the generation of proteolytic enzyme simultaneously like this, be conducive to biological catalyst and in follow-up reaction process, keep stable (Sardessai and Bhosle 2004, Biotechnol Prog.20:655-660).Therefore, except the advantage of above-mentioned proof, the organic solvent inducible expression carrier that present embodiment makes up also has the advantage of large-scale application, because it can be so that open-sky technique becomes possibility.
Claims (2)
1. the expression vector that organic solvent is replied is characterized in that, this carrier is carrier pMMRS, and by the DNA element, replicon RSF1010, selection markers gene bla, multiple clone site, intergenic sequence and organic solvent are replied promotor P
SrpForm;
The nucleotide sequence of described carrier pMMRS is shown in SEQ ID No.2.
2. the application of expression vector in biocatalysis that organic solvent according to claim 1 is replied, it is characterized in that, described carrier pMMRS with an organic solvent compounds makes organic solvent as inductor and replying, and is used for making up the biocatalysis engineering bacteria.
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Non-Patent Citations (2)
| Title |
|---|
| Jasper kieboom等.Active efflux of orgnic solvents by pseudomonas putida S12 is induced by solvents.《Journal of bacteriology》.1998,第180卷(第24期),6769-6771. * |
| Jens p.Furste等.Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector.《GENE》.1986,第48卷119-131. * |
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