CN101381735A - Thermophilic long chain alkanol alcohol dehydrogenase and encode gene and application thereof - Google Patents

Thermophilic long chain alkanol alcohol dehydrogenase and encode gene and application thereof Download PDF

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CN101381735A
CN101381735A CNA2007101495406A CN200710149540A CN101381735A CN 101381735 A CN101381735 A CN 101381735A CN A2007101495406 A CNA2007101495406 A CN A2007101495406A CN 200710149540 A CN200710149540 A CN 200710149540A CN 101381735 A CN101381735 A CN 101381735A
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alcoholdehydrogenase
long chain
chain alkanol
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王磊
刘雪倩
冯露
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Nankai University
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01192—Long-chain-alcohol dehydrogenase (1.1.1.192)

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Abstract

The invention relates to an enzyme and an encoding gene thereof, in particular to alcohol dehydrogenase capable of degrading alphanol and an encoding gene thereof. The invention discloses the gene which can encode the alcohol dehydrogenase, wherein the alcohol dehydrogenase can degrade the alcohol dehydrogenase of the alphanol. Moreover, the invention provides a recombinant plasmid capable of expressing the alcohol dehydrogenase and a recombinant bacterium capable of generating the alcohol dehydrogenase. The enzyme has the ability of degrading the alphanol the carbon content of which his more than 16, and can be widely acted on alphanol between C12 and C30; and the optimum substrate is ethanol.

Description

Thermophilic long chain alkanol alcoholdehydrogenase and encoding gene thereof and application
Technical field
The present invention relates to a kind of enzyme and encoding gene thereof, particularly a kind of alcoholdehydrogenase and encoding gene thereof of the long chain alkanol of can degrading.
Background technology
Alcoholdehydrogenase (Alcohol Dehydrogenase, ADH, EC 1.1.1.1) is one of redox catalyst important in the organism, and a lot of alcohols metabolism are all finished by alcoholdehydrogenase catalysis.This enzyme all has comparatively widely at biocatalysis, biomedical sector to be used.In vivo, a lot of alcohols metabolism are all finished by alcoholdehydrogenase catalysis.In animal, plant, microorganism, eukaryote and protokaryon bacterium, all find alcoholdehydrogenase, had at least the enzyme of 25 EC numberings to be called as alcoholdehydrogenase.
The alcoholdehydrogenase wide material sources, of a great variety.Alcoholdehydrogenase in the degradable alkane bacterium of having reported at present mainly contains following three major types: I type, Zn 2+The long-chain alcohol desaturase that relies on, about 350 amino-acid residues of each subunit; The II type, short-chain alcohol dehydrogenase, each subunit contain 250 amino-acid residues, as Bt-ADH; The III type, Fe 2+The alcoholdehydrogenase that relies on, and about 38kDa size (TomohisaKato etc., Journal of Bioscience and Bioengineering, 2001.vol.91, No.1,100-102).The substrate kind that alcoholdehydrogenase can act on is various, comprises Fatty Alcohol(C12-C14 and C12-C18), aromatic alcohol and the styryl carbinol etc. of ethanol.
Aspect alcoholdehydrogenase research, the bacterial classification that relates to has: thermophilic anaerobic ethanol bacterium (Thermoanaerobacter ethanolicus JW200), thermophilic anaerobic ethanol bacterium (Thermoanaerobacter ethanolicus ATCC 31550), extreme thermophile bacteria (Thermotoga hypogea sp.Nov), cow mycobacteria (Mycobacterium bovis BCG), clostridium butyricum (Clostridium butyricum VPI1718); But research is the clearest to be yeast.Alcohol Dehydrogenase from Yeast is the tetramer, and single subunit peptide chain contains 347 amino-acid residues, molecular weight subunit 35000.Alcohol Dehydrogenase from Yeast is strong to Substratspezifitaet, higher to ethanol and other straight chain primary alcohol activity, only minority secondary alcohol and side chain alkanol are had 143～283 of active amino acid residue sequence and be the coenzyme calmodulin binding domain CaM, 1～142 and 284～336 of amino acid residue sequence is the catalytic activity zone.Coenzyme and enzyme in conjunction with the time enzyme conformation change, catalyst structure domain turns to the coenzyme binding domains after rotating about 10 °, the latter rotates about 1.5 °.Form the crack between two structural domains, a chamber bag is formed in two structural domains and crack, and substrate and coenzyme just are combined in the dark chamber bag of intermediate formation, and the side chain of chamber bag one side is hydrophobic, and the opposite side side chain is hydrophilic, comprises the Zn of catalytic activity 2+And ligand (F.Mark Dickinson etc., Chemico-Biological Interactions130-132 (2001) 417-423; Ueda M etc., Methods Enzymol.1990; 188:171-5).
About alcohol dehydrogenase gene up-to-date patent and bibliographical information.A kind of ethanol dehydrogenase and gene (No. 01126435.7, date of application 2001.08.09) thereof have been reported as hair people such as make people rich; People such as P.Hildebrandt have reported the alcoholdehydrogenase (Appl Microbiol Biotechnol, 59:483-487,2002) of Pseudomonas fluorescens (Pseudomonas fluorescensDSM50106); Peter J.Holt etc. has reported the alcoholdehydrogenase (FEMS Microbiology Letters, 190:57-62,2000) of thermophilic anaerobic ethanol bacterium (Thermoanaerobacter ethanolicusJW200); People such as Jean-Marc Wilkin have reported the alcoholdehydrogenase (Eur.J.Biochem, 262:299-307,1999) in the cow mycobacteria (Mycobacterium bovis BCG).
At present microorganism is carried out the function of alkane degradation and tens kinds of bacterial strains (P.Hildebrandt etc. that Mechanism Study mainly concentrates on Rhodopseudomonas (Pseudomonas), acinetobacter (Acinetobacter), Blaw Bordetella (Prauserella), Rhod seven kinds such as (Rhodococcus), Appl Microbiol Biotechnol, 2002,59:483-487; Peter J.Holt etc., MicrobiologyLetters, 2000,190:57-62; Ce line Raynaud etc., PNAS 2003, April 29:5010-5015; Jean-Marc Wilkinl etc., Eur.J.Biochem, 1999,262:299-307; RyokoIwamoto etc., Archives of Biochemistryand Biophysics, 2002,398:203-212), the gene of each the step reaction in its alkane degradation approach is dispersed in genomic different positions mostly, or cluster is arranged (as Pseudomonas putida OCT plasmid) in plasmid.Though the research report to the alkanol desaturase is more, the substrate of its effect still is confined to short-and-medium alkanol, especially ethanol and butanols, with the alkanol more than the 16C is that the alcoholdehydrogenase of substrate yet there are no report (Akio Tani etc., Appl.Environ.Microbiol, Dec.2000, p.5231-5235; Ishige, T. etc., Appl.Environ.Microbiol.2000,66:3481-348; M E Singer, J Bacteriol.1985 December; 164 (3): 1017-1024; T Kotani etc., J Bacteriol, Dec.2003, p:7120-7128; Ching T.Hou etc., Applied And Environmental Microbiology, July 1983, p.98-105; KesenMa etc., Journal Of Bacteriology, Feb.1999, p.1163-1170).
In the petroleum hydrocarbon degradation approach, the long chain alkanol desaturase is crucial key enzyme, research to this enzyme provides stronger evidence for the mechanism of furtheing investigate thermophilic denitrifying bacillocin NG80-2 degraded long chain alkane pathways metabolism, also provides fundamental basis for the pathways metabolism of exploring other alkane degradation bacterial.Simultaneously, the ability of the thermophilic of this enzyme and degraded long chain alkanol has important industrial application value, can be applied to the treating processes of petroleum pollution and petroleum wastewater and improve the microorganism oil recovery.Therefore await the long-chain alcohol desaturase is further studied.
Summary of the invention
An object of the present invention is to provide a kind of gene, its a kind of thermophilic long chain alkanol alcoholdehydrogenase of encoding;
Another object of the present invention provides a kind of thermophilic long chain alkanol alcoholdehydrogenase of its long chain alkanol (more than the 16C) of can degrading;
Another object of the present invention provides a kind of recombinant plasmid that can express the thermophilic long chain alkanol alcoholdehydrogenase;
A further object of the present invention provides a kind of reorganization bacterium that can produce the thermophilic long chain alkanol alcoholdehydrogenase.
To achieve these goals, the present invention adopts following technical scheme:
The present invention proposes a kind of gene of the thermophilic long chain alkanol alcoholdehydrogenase of encoding, its have be selected from a), b) or nucleotide sequence c):
A) nucleotide sequence shown in the SEQ ID NO:1;
B) because the degeneracy of genetic code is different from SEQ ID NO:1 but the amino acid sequence coded aminoacid sequence identical nucleotide sequence coded with SEQ ID NO:1;
C) under stringent hybridization condition with above-mentioned a) or b) in sequence hybridization, and coding has the nucleotide sequence of active alcoholdehydrogenase.
In one embodiment of the invention, the gene of above-mentioned coding thermophilic long chain alkanol alcoholdehydrogenase has the nucleotide sequence shown in the SEQ ID NO:1.
The present invention proposes a kind of thermophilic long chain alkanol alcoholdehydrogenase, its have be selected from following d), e) or aminoacid sequence f):
D) above-mentioned a), b) or c) described nucleotide sequence coded aminoacid sequence;
E) aminoacid sequence shown in the SEQ ID NO:2;
F) disappearance above-mentioned e), replace or insert the aminoacid sequence behind one or more amino acid, and the protein with this sequence there is the activity of alcoholdehydrogenase.
In one embodiment of the invention, above-mentioned thermophilic long chain alkanol alcoholdehydrogenase has the aminoacid sequence shown in the SEQ IDNO:2.
The present invention proposes a kind of recombinant plasmid of expressing the thermophilic long chain alkanol alcoholdehydrogenase, and it comprises the gene of above-mentioned coding alcoholdehydrogenase at least.
In one embodiment of the invention, the carrier of above-mentioned recombinant plasmid is pET-28a (+).
The present invention proposes a kind of reorganization bacterium that produces the thermophilic long chain alkanol alcoholdehydrogenase, has imported the gene of above-mentioned coding alcoholdehydrogenase in this bacterium.
In one embodiment of the invention, above-mentioned reorganization bacterium is intestinal bacteria, is preferably e. coli strain bl21.
The present invention also provides a kind of application of thermophilic long chain alkanol alcoholdehydrogenase, it is characterized in that this enzyme is used for the treating processes of petroleum pollution and petroleum wastewater.
The present invention also provides a kind of application of thermophilic long chain alkanol alcoholdehydrogenase, it is characterized in that this enzyme is used for the process in oil production, to improve the microorganism oil recovery.
Should be pointed out that the above-mentioned term of mentioning " stringent condition " implication in this manual is meant has formed so-called specific hybridization and has not formed non-specific hybridization under this condition.For example, this stringent condition can be, homology each other is not less than can hybridize between 70% the DNA and be lower than between the DNA of above-mentioned numerical value and can not hybridize, and preferably homology is no less than between 90% the DNA and can hybridizes.For common wash conditions in the Southern hybridization, can for example be following hybridization conditions: with Hybond membrane place prehybridization solution (the 0.25mol/L sodium phosphate buffer, pH7.0,7%SDS) in, 50 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.0,7%SDS, isotope-labeled nucleotide fragments), 50 ℃ of hybridization 12hr; Abandon hybridization solution, add film washing liquid I (2 * SSC and 0.1%SDS), wash film 2 times for 50 ℃, each 30min; Add film washing liquid II (0.5 * SSC and 0.1%SDS), wash film 30min for 50 ℃.
The person of ordinary skill in the field will be appreciated that, the dna sequence dna of coding alcoholdehydrogenase of the present invention comprises that also coding carries out the proteinic nucleotide sequence that one or more amino acid are replaced, inserted or disappearance also still has this enzymic activity to the aminoacid sequence of the expressed enzyme molecule of nucleotide sequence shown in the SEQ ID NO:1.
In addition, the amino acid of the expressed enzyme molecule of alcohol dehydrogenase gene of the present invention is carried out one or more amino acid replace, insert or lack resulting protein, also can reach purpose of the present invention.Thereby the present invention also comprises with the aminoacid sequence shown in the SEQ ID NO:2 having at least 70% homology, preferably has at least 90% homology, but has the active protein of alcoholdehydrogenase simultaneously.The term that uses above " a plurality of " can be the number less than 100, is preferably the number less than 10.
Compare with known alcoholdehydrogenase, the alcoholdehydrogenase that the present invention proposes is a kind of novel alcoholdehydrogenase, by the alcohol dehydrogenase gene deduced amino acid comparative analysis that the present invention is proposed, alcoholdehydrogenase of the present invention is compared with the aminoacid sequence of the enzyme that other has been reported, and most similaritys are all less than 45%.Similarity is higher than 45% alcoholdehydrogenase all not to be done function and identifies, is mainly derived from bacillus acidocldarius (Bacillus kaustophilus HTA426) (YP_148778.1); Bacillus licheniformis (Bacillus licheniformis ATCC 14580) (YP_080422.1); Bacillus cereus (Bacillus cereus G9241) (ZP_00239930); Bacillus thuringiensis (Bacillusthuringiensis str.Al Hakam) (YP_896094); Bacillus thuringiensis (Bacillusthuringiensis serovar konkukian str.97-27) (YP_037785.1); Bacillus cereus (Bacillus cereus ATCC 10987) (NP_980044.1); Bacillus cereus (Bacilluscereus E33L) (YP_085061.1).
This shows, alcoholdehydrogenase of the present invention is with after the aminoacid sequence of other alcoholdehydrogenase of having reported is compared, similarity is greater than in 45% the bacterial strain of discovery, basically all carried out the research of sequence alignment, the pertinent literature that does not have discovery that the function of the alcohol dehydrogenase gene of corresponding bacterial strain is identified.By the amino acid sequence similarity of enzyme, the alcoholdehydrogenase of this invention belongs to III type, Fe 2+The alcoholdehydrogenase that relies on.
Compared with prior art, the present invention adopts the beneficial effect of technique scheme to be:
In addition, alcoholdehydrogenase of the present invention is different from the alcoholdehydrogenase of having reported, and enzyme activity detects when different substrate, proves that this enzyme has the ability of high temperature resistant degraded long chain alkanol (greater than 16 carbon).Though the suitableeest substrate of this enzyme is an octanol, have certain degradation capability for lauryl alcohol to the long-chain alcohol between the triacontanol, optimal reactive temperature is 60 ℃, has very high thermostability, this is the performance of its thermophilic just.
The degraded of long chain alkane is to be long chain alkanol by paraffin hydrocarbon mono oxygenase with the alkane hydroxylation, be the long-chain alkanal by the further dehydrogenation of alkanol desaturase again, continue dehydrogenation by full desaturase again and generate the long-chain alkanoic acid, entering the final complete oxidation of tricarboxylic acid cycle after generating long-chain alkanoic acid coenzyme A under the effect of long-chain alkanoic acid CoA ligase is carbonic acid gas and water.In the petroleum hydrocarbon degradation approach, long chain alkanol desaturase among the thermophilic denitrifying bacillocin NG80-2 is crucial key enzyme, it can high temperature resistant and efficiently utilize the catalysate long chain alkanol of paraffin hydrocarbon mono oxygenase, makes its further dehydrogenation, improves the catalytic efficiency in the alkane degradation approach; On the other hand, it has than long chain paraffin hydrocarbon mono oxygenase substrate scope widely, not only can the catalysis long chain alkanol, also short chain alcohol there is very high catalytic capability.Therefore, the mechanism that the research of long chain alkanol desaturase is not only helped understanding in depth thermophilic denitrifying bacillocin NG80-2 degraded long chain alkane approach, for the pathways metabolism of exploring other alkane degradation bacterial is provided fundamental basis, this enzyme structure efficient expression engineering strain and large-scale purification are had more wide prospect in industrial application and economic worth.
Based on the above-mentioned characteristic and the function of long chain alkanol alcoholdehydrogenase of the present invention, this enzyme can be in the processing of petroleum pollution and petroleum wastewater and widespread use in improving the microorganism oil recovery.On the one hand, this enzyme long chain alkanol of at high temperature degrading, thereby strengthen the reaction of long chain alkane through aforementioned degradation pathway, improve light paraffins and heavy paraffin hydrocarbon ratio, improve characteristic such as crude oil fluidity, help crude oil production, the raising oil recovery; On the other hand, this enzyme is at high temperature to the degradation characteristic of long chain alkane, and the processing of the high temperature petroleum wastewater that brought by oilfield exploitation, the race leakage of oil transportation operation, the caused environment petroleum pollution of overflow and petrochemical complex is also had a good application prospect.
Description of drawings
Fig. 1 is the alcohol dehydrogenase gene construction of recombinant plasmid mode chart in the embodiment of the invention.Be updated to clear chart
Fig. 2 represents alcoholdehydrogenase SDS-PAGE electrophorogram of the present invention
Fig. 3 represents the ratio vigor of alcoholdehydrogenase of the present invention to different substrates
Fig. 4 represents the ratio vigor of alcoholdehydrogenase of the present invention under differing temps.Chart uses black lines
Fig. 5 represents the ratio vigor of alcoholdehydrogenase of the present invention under different pH values.Chart uses black lines
Fig. 6 A and Fig. 6 B represent alcoholdehydrogenase vigor vapor detection figure of the present invention
Embodiment
Also the present invention is described in further detail in conjunction with the accompanying drawings below by specific embodiment.Below each embodiment be only used for the explanation rather than the restriction the present invention.
Embodiment one
1. the extraction of the total DNA of thermophilic denitrifying bacillocin NG80-2 (CGMCC No.1228) studies have shown that, can be isolated the gene of coding alcoholdehydrogenase by thermophilic denitrifying bacillocin (Geobacillus thermodenitrificans) NG80-2 genome.Therefore, in the present embodiment, (its preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.1228 to the thermophilic denitrifying bacillocin NG80-2 that employing obtains from Chinese Tianjin Dagang Oilfield official 69-8 block oil-well strata water sepn, preservation date is on October 09th, 2004, patent of invention and obtaining the authorization in the applicant country, denomination of invention: thermophilic denitrifying bacillocin and screening thereof and application, the patent No.: ZL200410072759.7), get the fresh culture thing 3ml of its incubated overnight, centrifugal collection thalline, thalline is suspended from the 250 microlitre 50mMTris damping fluids (pH8.0), add 10 microlitre 0.4M EDTA (pH8.0), 37 ℃ of insulation 20min behind the mixing, add 30 microlitre 20mg/ml N,O-Diacetylmuramidases afterwards, 37 ℃ are incubated 20min again behind the mixing, add 5 microlitre 20mg/ml Proteinase Ks again, behind the gentle mixing, add 20 microlitre 10%SDS again, 50 ℃ are incubated to solution and clarify, use equal-volume phenol respectively: chloroform: primary isoamyl alcohol extracting twice, chloroform: the primary isoamyl alcohol extracting once, last supernatant solution, the dehydrated alcohol that adds 2.5 times of volume precoolings, reclaim DNA, wash with 70% ethanol, precipitation is dissolved in 100 microlitre TE damping fluid (pH8.0,10mMTris, 1mMEDTA), add 10mg/ml RNase 2 microlitres, 65 ℃ of insulation 30min, use phenol respectively: chloroform: primary isoamyl alcohol, chloroform: each extracting of primary isoamyl alcohol once, supernatant liquor adds the dehydrated alcohol of 2.5 times of volume precoolings, reclaims DNA, washes with 70% ethanol, vacuum-drying, precipitation is dissolved in 50 microlitre TE damping fluids.The ultraviolet spectrophotometer measurement result of dna solution is A260/A280=1.95, A260=0.73.
2. the clone of alcohol dehydrogenase gene of the present invention and screening
Get foregoing total dna solution 0.5 microlitre (about 10ng) as template, as primer, and carry out 20 cycle P CR by the PCR loop parameter of following setting with following oligonucleotide sequence.
The PCR loop parameter of setting is as follows:
95℃，5min；95℃，30s；50℃，45s；72℃，2min；72℃，5min；4℃，2hr
Primer sequence is as follows:
Upstream primer: 5 '-CGGAATTCATGCAAAATTTTACGTTTCGCA-3 ';
Downstream primer: 5 '-ACGCGTCGACTTATAAAGACGCACGCAAAATG-3 ',
Above-mentioned PCR product EcoRI and SalI double digestion, the agarose gel electrophoresis through 0.8% is cut glue recovery 1.1kb enzyme and is cut the product segment.With through same restricted type restriction endonuclease enzymolysis and cut the plasmid pET-28a (+) that glue reclaims and be connected, behind the transformed competence colibacillus bacillus coli DH 5 alpha, be applied on the LB solid medium that contains 50 μ g/mlKan (kantlex).Cultivated 12 hours for 37 ℃, the picking mono-clonal is transferred in the 20ml LB substratum cultivates (50 μ g/ml Kan), cultivate after 12 hours for 37 ℃, the extraction plasmid is identified, pET-28a (+) plasmid that is inserted with the dna sequence dna of SEQ ID NO:1 is a recombinant plasmid pLW1168 (see figure 1), (this bacterial strain can be ordered to the thick general biotechnology in development area, Tianjin development corporation, Ltd. to change recombinant plasmid pLW1168 over to the expressive host e. coli bl21, article No. is 69387-3) in, the recombinant bacterial strain that obtains containing this recombinant plasmid is H1454, but this recombinant plasmid pLW1168 high frequency transformed into escherichia coli BL21 expresses the active and anti-kantlex performance of alcoholdehydrogenase.
Adopt the Sanger method that this dna fragmentation is checked order, sequencing result shows that the insertion fragment contains the open reading frame of a long 1161bp, the protein of being made up of 387 amino acid of encoding.
Embodiment twoThe purifying of alcoholdehydrogenase and characteristic
Above-mentioned reorganization bacterium H1454 mono-clonal is inserted 20ml to be contained in the LB substratum of 50 μ g/ml Kan, 37 ℃, 180rpm/min cultivated 12 hours, then culture is inserted the LB substratum (totally 10 are shaken bottle) that 200ml contains 50 μ g/ml Kan by 1% (v/v) inoculum size, 37 ℃, it is 0.6 o'clock that 220rpm/min cultivates OD600, add IPTG to final concentration be 0.1mM, 45 ℃, 180rpm/min induced 2.5 hours.Centrifugal collection thalline is suspended from and contains in 50mM Tris-HCl (pH8.0) damping fluid, utilizes the ultrasonic disruption cell, and centrifuged supernatant is the crude extract of alcoholdehydrogenase.This supernatant liquor is through chelating sepharose (Chelating Sepharose) nickel affinity column chromatography purification, and the zymin that obtains shows a band (see figure 2) on SDS-PAGE.Utilize the standard method of known protein materialization to measure the fundamental characteristics of this alcoholdehydrogenase.The molecular weight of the recombinase that records with SDS-PAGE is 45000 dalton, and is similar to the molecular weight of calculating in theory (42800 dalton); The iso-electric point pI of the recombinase that isoelectric point precipitation records is 5.18.This alcoholdehydrogenase all has effect to the long-chain alcohol of C15～C30.
Embodiment three
1. the ratio vigor when measuring the different substrate of alcoholdehydrogenase of the present invention
The alcoholdehydrogenase that makes in the foregoing description two is carried out the mensuration of different substrates, and concrete grammar is: prepare 200 μ l reaction systems, coenzyme NAD +Final concentration be 1mM, add the substrate in 1mM methyl alcohol, ethanol, butanols, hexanol, octanol, decyl alcohol, lauryl alcohol, tetradecyl alcohol, hexadecanol, stearyl alcohol, eicosanol, tetracosanol, octacosanol, the triacontanol respectively, certain density alcoholdehydrogenase uses the Tris-HCl of 50mM pH8.0 to mend to 200 μ l.Mixing, reaction is 10 minutes in 60 ℃ of water-baths, after reaction finishes, ice bath 5 minutes, termination reaction.With the chloroform extraction of 1:1, measure photoabsorption then at the 340nm place.Generate the required enzyme amount of 1 μ molNADH with per minute and be defined as 1 enzyme activity unit.Measurement result is referring to Fig. 3, and as can be seen, the suitableeest substrate of this alcoholdehydrogenase is an octanol from this table.
2. measure the ratio vigor of alcoholdehydrogenase of the present invention under differing temps
The alcoholdehydrogenase that makes in the foregoing description two is carried out the mensuration of optimal reactive temperature, concrete grammar is: prepare 200 μ l reaction systems, the final concentration of substrate octanol is 100mM, coenzyme NAD+final concentration be 1mM, certain density alcoholdehydrogenase uses the Tris-HCl of 50mM pH8.0 to mend to 200 μ l.Mixing, respectively at reaction in 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, the 75 ℃ water-baths 10 minutes, after reaction finishes, ice bath 5 minutes, termination reaction.With the chloroform extraction of 1:1, measure photoabsorption then at the 340nm place.Measurement result is referring to Fig. 4.As can be seen from the figure, the optimal reactive temperature of this alcoholdehydrogenase is about 60 ℃.
3. measure the ratio vigor of alcoholdehydrogenase of the present invention under different pH
The alcoholdehydrogenase that makes in the foregoing description two is carried out the mensuration of optimum pH, concrete grammar is: prepare 200 μ l reaction systems, the final concentration of substrate octanol is 100mM, coenzyme NAD+final concentration be 1mM, certain density alcoholdehydrogenase uses the damping fluid (prescription sees Table 1) of pH6.0, pH6.5, pH7.0, pH7.5, pH8.0, pH8.5, pH9.0 to mend to 200 μ l respectively.Mixing, reaction is 10 minutes in 60 ℃ of water-baths, after reaction finishes, ice bath 5 minutes, termination reaction.With the chloroform extraction of 1:1, measure photoabsorption then at the 340nm place.Measurement result is referring to Fig. 5, and as can be seen from the figure, the optimum pH of this alcoholdehydrogenase is about 8.0.
Used damping fluid in the table 1 alcoholdehydrogenase enzyme activity determination
The pH scope Damping fluid 1 Damping fluid 2
pH5.8-8.0 0.1MKH 2PO 4 0.1M?NaOH
pH8.6-10.6 0.1M glycine 0.1M?NaOH
4. measure the influence that alcoholdehydrogenase of the present invention is subjected to different ions
To the mensuration that the alcoholdehydrogenase that makes in the foregoing description two carries out different ions influence, concrete grammar is: prepare 200 μ l reaction systems, the final concentration of substrate octanol is 100mM, coenzyme NAD+final concentration be 1mM, add FeCl respectively 2, FeSO 4, CuSO 4, MgCl 2, CoCl 2, ZnSO 4, MnCl 2, AlCl 3, CaCl 2, NiCl 2, NaCl, KCl, EDTA to 1mM or SDS to 0.05%, certain density alcoholdehydrogenase is mended to 200 μ l with the Tris-HCl of 50mM pH8.0.Mixing, reaction is 10 minutes in 60 ℃ of water-baths, after reaction finishes, ice bath 5 minutes, termination reaction.With the chloroform extraction of 1:1, measure photoabsorption then at the 340nm place.Generate the required enzyme amount of 1 μ molNADH with per minute and be defined as 1 enzyme activity unit, measurement result is referring to table 2.
Table 2 different ions is to the influence of this alcoholdehydrogenase vigor
Ion Concentration Relative reactivity (%)
Do not have 1mM 100
FeCl 2 1mM 63.69
FeSO 4 1mM 69.01
CuSO 4 1mM 10.63
MgCl 2 1mM 0.66
CoCl 2 1mM 58.37
ZnSO 4 1mM 15.15
MnCl 2 1mM 23.37
AlCl 3 1mM 1.86
CaCl 2 1mM 0
NiCl 2 1mM 19.54
NaCl 1mM 136.96
KCl 1mM 73.40
EDTA 1mM 73.40
SDS 0.05％ 0
5. the vapor detection of alcoholdehydrogenase vigor of the present invention
The alcoholdehydrogenase that makes in the foregoing description two is carried out gas phase measure, concrete grammar is: preparation 1ml reaction system, and the final concentration of substrate hexadecanol is 0.5mM, tensio-active agent is 6% with the w/w ratio of substrate.Coenzyme NAD+final concentration be 1mM, certain density alcoholdehydrogenase is mended to 1ml with the Tris-HCl of 50mMpH8.0.Mixing, 60 ℃ are reacted the regular hour down.After reaction finishes, ice bath 5 minutes, termination reaction.Add and contain pentadecane, with the minimizing of agilent6820 gas chromatograph systems measurement reaction substrate as interior target n-hexane extraction.Measurement result is referring to Fig. 6 A and Fig. 6 B, and as can be seen from the figure, with the reaction times prolongation, substrate constantly reduces.
Though the present invention discloses as above with preferred embodiment, so it is not that any person of ordinary skill in the field not breaking away from change and the improvement that the spirit and scope of the present invention are done, will fall into protection scope of the present invention in order to qualification the present invention.
Sequence table
＜110〉Nankai University
＜120〉thermophilic long chain alkanol alcoholdehydrogenase and encoding gene thereof and application
<130>7P13005-CN
<160>1
<170>PatentIn?version?3.3
<210>1
<211>1164
<212>DNA
＜213〉nucleotide sequence of coding alcoholdehydrogenase
<400>1
Figure A200710149540D00161
<210>2
<211>387
<212>PRT
＜213〉aminoacid sequence of alcoholdehydrogenase
<400>2
Figure A200710149540D00172
Figure A200710149540D00181

Claims (14)

1, the gene of coding thermophilic long chain alkanol alcoholdehydrogenase is characterized in that, this gene have be selected from a), b) or nucleotide sequence c):
A) nucleotide sequence shown in the SEQ ID NO:1;
B) be different from SEQ ID NO:1 but the amino acid sequence coded aminoacid sequence identical nucleotide sequence coded with SEQ ID NO:1;
C) under stringent hybridization condition with above-mentioned a) or b) in sequence hybridization, and coding has the nucleotide sequence of active alcoholdehydrogenase.
2, thermophilic long chain alkanol alcoholdehydrogenase according to claim 1 is characterized in that, has to be selected from following d), e) or aminoacid sequence f):
D) above-mentioned a), b) or c) described nucleotide sequence coded aminoacid sequence;
E) aminoacid sequence shown in the SEQ ID NO:2;
F) disappearance above-mentioned e), replace or insert the aminoacid sequence behind one or more amino acid, and the protein with this sequence there is the activity of alcoholdehydrogenase.
According to the described thermophilic long chain alkanol alcoholdehydrogenase of claim 2, it is characterized in that 3, described alcoholdehydrogenase has the homology with the aminoacid sequence at least 70% shown in the SEQ ID NO:2, the protein that has this sequence simultaneously has the activity of alcoholdehydrogenase.
According to the described thermophilic long chain alkanol alcoholdehydrogenase of claim 3, it is characterized in that 4, described alcoholdehydrogenase has the homology with the aminoacid sequence at least 90% shown in the SEQ ID NO:2, the protein that has this sequence simultaneously has the activity of alcoholdehydrogenase.
According to the described thermophilic long chain alkanol alcoholdehydrogenase of claim 2, it is characterized in that 5, a plurality of aminoacid sequences of disappearance, replacement or insertion are 2-100 in the aminoacid sequence shown in the described SEQ of the having ID NO:2.
According to the described thermophilic long chain alkanol alcoholdehydrogenase of claim 5, it is characterized in that 6, a plurality of aminoacid sequences of disappearance, replacement or insertion are 2-10 in the aminoacid sequence shown in the described SEQ of the having ID NO:2.
7, according to the described thermophilic long chain alkanol alcoholdehydrogenase of claim 2, it is characterized in that described alcoholdehydrogenase belongs to III type, Fe 2+The alcoholdehydrogenase that relies on.
8, a kind of recombinant plasmid of expressing the thermophilic long chain alkanol alcoholdehydrogenase is characterized in that, it comprises the gene of the described coding alcoholdehydrogenase of claim 1 at least.
9, a kind of recombinant plasmid of expressing the thermophilic long chain alkanol alcoholdehydrogenase according to claim 8 is characterized in that, the carrier of described recombinant plasmid is pET-28a (+).
10, a kind of reorganization bacterium that produces the described thermophilic long chain alkanol alcoholdehydrogenase of claim 2 is characterized in that, has imported the gene of the described coding alcoholdehydrogenase of claim 1 in this reorganization bacterium.
11, a kind of reorganization bacterium that produces the thermophilic long chain alkanol alcoholdehydrogenase according to claim 10 is characterized in that, described reorganization bacterium is intestinal bacteria.
12, a kind of reorganization bacterium that produces the thermophilic long chain alkanol alcoholdehydrogenase according to claim 11 is characterized in that described intestinal bacteria are e. coli strain bl21.
13. the application of each described thermophilic long chain alkanol alcoholdehydrogenase of claim 2 to 7 is characterized in that this enzyme is used for the treating processes of petroleum pollution and petroleum wastewater.
14. the application of each described thermophilic long chain alkanol alcoholdehydrogenase of claim 2 to 7 is characterized in that this enzyme is used for the process in oil production, to improve the microorganism oil recovery.
CNA2007101495406A 2007-09-06 2007-09-06 Thermophilic long chain alkanol alcohol dehydrogenase and encode gene and application thereof Pending CN101381735A (en)

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* Cited by examiner, † Cited by third party
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CN102936586A (en) * 2012-12-10 2013-02-20 南开大学 Thermophilic alphanol alcohol dehydrogenase and crystal structure thereof
CN103184154A (en) * 2013-04-02 2013-07-03 南开大学 Biotechnology capable of producing No. 380 admiralty fuel oil and application thereof
CN110777126A (en) * 2019-11-25 2020-02-11 南开大学 High-temperature anaerobic long-chain alkanol alcohol dehydrogenase and application thereof
CN114539366A (en) * 2022-03-22 2022-05-27 南开大学 GntR family regulatory protein and application thereof in constructing efficient alkane degrading bacteria

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TWI275645B (en) * 2000-02-16 2007-03-11 Daicel Chemical Industries Ltd. (R)-2-octanol dehydrogenases, methods for producing the enzymes, DNA encoding the enzymes, and methods for producing alcohols using the enzymes
CA2510657A1 (en) * 2005-07-18 2007-01-18 Institut Francais Du Petrole Polypeptides with an activity in the mtbe degradation pathway and their use

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102936586A (en) * 2012-12-10 2013-02-20 南开大学 Thermophilic alphanol alcohol dehydrogenase and crystal structure thereof
CN103184154A (en) * 2013-04-02 2013-07-03 南开大学 Biotechnology capable of producing No. 380 admiralty fuel oil and application thereof
CN110777126A (en) * 2019-11-25 2020-02-11 南开大学 High-temperature anaerobic long-chain alkanol alcohol dehydrogenase and application thereof
CN114539366A (en) * 2022-03-22 2022-05-27 南开大学 GntR family regulatory protein and application thereof in constructing efficient alkane degrading bacteria
CN114539366B (en) * 2022-03-22 2023-09-08 南开大学 GntR family regulatory protein and application thereof in construction of efficient alkane degrading bacteria

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