CN101603044B - Alcohol dehydrogenase and gene and recombinase thereof - Google Patents

Abstract

The invention discloses an alcohol dehydrogenase and gene thereof. The gene is one of following the nucleotide sequences: 1) base sequence shown in SEQ ID No.1 of a sequence table; 2) protein consisting of amino acid sequence shown in SEQ ID No.2 of the sequence table. The invention discloses recombinant expression vector comprising the gene and expression transformant, and recombinant alcohol dehydrogenase and preparation method and application thereof. By separating new alcohol dehydrogenase gene from Lactobacilluskefir DSM20587 gene group, the invention expands resources of alcohol dehydrogenase gene, simultaneously provides scientific proof for metabolic engineering research of Lactobacilluskefir DSM20587 and gene engineering establishment, and provides excellent alcohol dehydrogenase for stereoselectivly transforming ketone compound with large side-chain to corresponding alcohol in production of bio-organic synthesis.

Description

A kind of ethanol dehydrogenase and gene thereof and recombinase

Technical field

The invention belongs to and belong to technical field of bioengineering, particularly a kind of ethanol dehydrogenase and gene thereof, and contain the recombinant expression vector of this gene, recombinant expressed transformant, and a kind of recombinant alcohol dehydrogenase and preparation method thereof and using.

Background technology

Ethanol dehydrogenase (E.C.1.1.1.1.) is called alcohol dehydrogenase or keto-reductase abroad, promptly ethanol dehydrogenase (alcoholdehydrogenase, ADH) or keto reductase, the domestic acetaldehyde ethanol dehydrogenase that also cries.Ethanol dehydrogenase has the ability of stereospecificity ground reduction prochirality carbonyl compound, is very important in a redox enzymes classification.Ethanol dehydrogenase can be used for efficient synthetic optically active alcohol that has, and latter's industry that becomes more meticulous is often produced and used the key structure unit.See from angle of practical application, utilize NADH especially to have significant application value as the ethanol dehydrogenase of cofactor, reason is to set up a kind of effective formate/hydrogenlyase system aspect the NADH regeneration; And the ethanol dehydrogenase that relies on for NADPH by contrast, the validity of its existing cofactor regeneration circulating system is just much lower.

Existing existing report is found the multiple ethanol dehydrogenase that can be used for organic synthesis, and wherein using maximum is to buy the three kinds of ethanol dehydrogenases from yeast, horse liver and Thermoanaerobium brockii that obtain from commercial sources.The Substratspezifitaet of these three kinds of enzymes and stereoselectivity all have remarkable difference, but they can both reduce keto compounds widely.These three kinds of enzymes also all have some limitations, and for example the stability of life-time service substrate not enough or that accept is limited, and these have limited their application technically.

Up to the present, can reducing methyl phenyl ketone for example and this type of verivate thereof in the ethanol dehydrogenase of having reported, to have the ketone compounds of huge side chain few.Hummel etc. are separated to the ethanol dehydrogenase that a kind of NADPH relies on from bacillus caucasicus Lacobacillus kefir DSM 20587 when screening new biological catalyst; Can methyl phenyl ketone and verivate thereof be changed into (R)-alcohol (Hummel W.New alcoholdehydrogenases for the synthesis of chiral compounds.Adv Biochem EngBiotechnol .1997; 58:145-84); In addition, in Rhodococcus Rhodococcuserythropolis, find the ethanol dehydrogenase (RE-ADH) that a kind of new NADH relies on.Compare with the ethanol dehydrogenase that the NADPH of L.kefir relies on, the similar ketone of this alcohol dehydrogenase endonuclease capable catalytic reduction is corresponding (S)-alcohol.Hummel etc. have studied the purifying and the biochemical property of the alcoholdehydrogenase of this NADH dependence, and it is successfully applied to biological organic synthesis.

In sum, seeking the alcohol dehydrogenase gene that clone also has potential using value, is all significant from enlarging the genetic resources or the angle of production application with NADH as the ethanol dehydrogenase of cofactor especially.

Bacillus caucasicus Lactobacillus kefir DSM 20587 is that unique strain of report at present can be through self entrained S)-ADH with (R)-ADH link coupled two goes on foot reduction reaction; Efficiently, the catalysis of high enantioselectivity ground generates 6-chloro-(3R; 5S)-bacterial strain (Amidjojo M, Franco-Lara E, the Nowak A of dihydroxyl hecanoic acid t-butyl ester; Link H; Weuster-Botz D.Asymmetric synthesis oftert-butyl (3R, 5S) 6-chloro-dihydroxyhexanoate with Lactobacillus kefir.ApplMicrobiol Biotechnol., 2005; 69 (1): 9-15), the latter is the important chiral building block that medicine industry is produced anti-cholesterol drugs HMG-CoA reductase inhibitor.Have only its (R)-ADH gene to obtain the clone at present; Still have nothing to do in correlative study report (Pfruender H, Amidjojo M, the Hang F of (S)-ADH gene and enzyme; Weuster-Botz D.Production of Lactobacillus kefir cells forasymmetric synthesis of a 3; 5-dihydroxycarboxylate.Appl Microbiol Biotechnol., 2005,67 (5): 619-22.).

Summary of the invention

The technical problem that the present invention will solve is exactly the limitation that the substrate that in biological organic synthesis chipal compounds, all exists to existing ethanol dehydrogenase and coenzyme rely on type; A kind of new good ethanol dehydrogenase and gene thereof are provided; The recombinant expression vector, the transformant that contain this gene; And the recombinase of this ethanol dehydrogenase and its production and application, this new alcohol dehydrogenase endonuclease capable relies on NADH, and the ketone compounds that catalytic reduction has huge side chain is corresponding alcohol.

The present invention solves the problems of the technologies described above one of technical scheme of being adopted: a kind of alcohol dehydrogenase gene is characterized in that be one of following nucleotide sequences: 1) it has the base sequence shown in the SEQ ID No.1 in the sequence table; The protein of 2) encoding and forming by the aminoacid sequence shown in the SEQ ID No.2 in the sequence table.

Two of technical scheme of the present invention provides a kind of ethanol dehydrogenase, and its aminoacid sequence is shown in SEQ ID No.2 in the sequence table.This preferable ethanol dehydrogenase is the ethanol dehydrogenase that derives from the NADH dependence of Lactobacillus kefir DSM20587 bacterial strain.

According to the present invention; Described alcohol dehydrogenase gene adh is can biocatalysis producing 6-chloro-(3R; 5S)-and the total DNA of bacillus caucasicus Lactobacillus kefir DSM 20587 bacterial strains of dihydroxyl hecanoic acid t-butyl ester is a template, obtains through pcr amplification.Specifically can comprise the steps:

(1) the existing known bacterium alcohol dehydrogenase enzyme amino acid sequence of compare of analysis is found out the conservative aminoacid sequence district (BlockS) of 2 tile heights, and the zine ion combination territory and the cofactor of corresponding ethanol dehydrogenase high conservative combine the territory to two conservative regions respectively.Convert the protein sequence of these two conservative regions into the degenerate core nucleotide sequence, and calculate degeneracy, according to this degenerate core nucleotide sequence design pair of degenerate primers.Total DNA is a template with Lactobacillus kefir DSM 20587 bacterial strains, carries out pcr amplification with this degenerated primer and obtains the section of DNA sequence.Measure the dna sequence dna of this PCR product.

5 ' end of the dna sequence dna that (2) in step (1), records designs 3 nested special primer SP1, SP2, SP3 and SP1 ' separately with 3 ' end; SP2 ', SP3 ' matches with 13 kinds of degenerated primers (AP1-AP13) respectively; Total DNA is a template with Lactobacillus kefir DSM 20587 bacterial strains; Carry out TAIL-PCR amplification, and product is carried out dna sequencing, the flanking sequence at the dna sequence dna two ends that obtain recording in the step (1).

(3) the two ends flank sequence assembly that each the TAIL-PCR amplification that records in dna sequence dna that records in the step (1) and the step (2) is got obtains the full length gene sequence that has initiator codon ATG and terminator codon TAG shown in SEQ ID No.1 in the sequence table.

According to the present invention, described alcohol dehydrogenase gene adh total length 1044bp, wherein A, C, G, four kinds of Nucleotide numbers of T and per-cent are respectively 273 (26.1%), 254 (24.3%), 260 (24.9%), 257 (24.6%).Initiator codon is ATG, and terminator codon is TAG.This sequence intronless has complete opening code-reading frame, 347 amino acid of encoding.Coded product, promptly the aminoacid sequence of ethanol dehydrogenase of the present invention is shown in SEQ ID No.2 in the sequence table, and molecular weight is 37.07kD, and iso-electric point pI is 5.70.Protein sequence compare of analysis through the U.S.'s state-run biotechnology information center sequence similarity search utility (NCBIBlast); Show with registered ethanol dehydrogenase homology and be up to 72%; Wherein the ethanol dehydrogenase amino acid sequence homology with Lu Te Shi lactobacillus spp Lactobacillus reuteri 100-23 and Lactobacillusreuteri F275 is the highest, is 72%.Proved also that thus alcohol dehydrogenase gene of the present invention is a new alcohol dehydrogenase gene.

Certainly, as is known to the person skilled in the art, alcohol dehydrogenase gene of the present invention also can be proteinic other any nucleotide sequence that coding is made up of the aminoacid sequence shown in the SEQ ID No.2 in the sequence table.And ethanol dehydrogenase of the present invention is not limited only to the aminoacid sequence shown in the SEQ ID No.2 in the sequence table; Can also be in the aminoacid sequence shown in the SEQ ID No.2 through replacement, lack or add one or several amino-acid residue and have identical alcohol dehydrogenase activity by the proteinic nucleotide sequence of sequence 2 deutero-; The situation of the replacement of these amino-acid residues, disappearance or interpolation comprises: add one or several amino acid at C-terminal and/or N-terminal, merge, do not influence situation such as the pro forma difference of sequence modification like the amino acid with vector encoded.Correspondingly, alcohol dehydrogenase gene of the present invention also can be the nucleotide sequence of these alcohol dehydrogenase enzyme derivatives of the present invention of coding or analogue.

Three of technical scheme of the present invention provides a kind of recombinant expression vector, comprises the nucleotide sequence of above-mentioned alcohol dehydrogenase gene of the present invention.The preferred prokaryotic expression carrier of the carrier framework of this recombinant expression vector, more preferably pET-28a (+).

Four of technical scheme of the present invention provides a kind of recombinant expressed transformant, comprises above-mentioned nucleotide sequence any of the present invention or recombinant expression vector.This recombinant expressed transformant preferred bacterium, more preferably intestinal bacteria E.coli BL21.DE3.

Five of technical scheme of the present invention provides a kind of preparation method of recombinant alcohol dehydrogenase, cultivates the recombinant expressed transformant of the invention described above, obtains the ethanol dehydrogenase of reorganization.The recombinant expressed transformant of intestinal bacteria E.coli BL21.DE3 of the present invention gives expression to the target protein of about 44kD after IPTG induces, confirmed that alcohol dehydrogenase gene of the present invention is a gene with expressive function.

Six of technical scheme of the present invention provide said method prepared and recombinant alcohol dehydrogenase.

Seven of technical scheme of the present invention provides described ethanol dehydrogenase or recombinant alcohol dehydrogenase are converted into carbonyl compound corresponding alcohol in biological organic synthesis application.Preferable, this enzyme transforms aldehyde compound or ketone compounds such as methyl phenyl ketone and the verivate thereof such as the parachloroacetophenone that have a bigger side-chain radical in biological organic synthesis and is reduced to corresponding (S)-alcohol.Described aldehyde compound is preferable is selected from positive propionic aldehyde, butyraldehyde-n, isobutyric aldehyde, valeraldehyde, n-hexyl aldehyde, enanthaldehyde, phenyl aldehyde, phenylacetic aldehyde, salicylic aldehyde, o-chlorobenzaldehyde, itral and phenylacrolein.Described ketone compounds is preferable is selected from ketopentamethylene, propione, 4-hydroxy-4-methyl-2 pentanone, methyl phenyl ketone and parachloroacetophenone.This enzyme can also transform ester compound in biological organic synthesis and be reduced to corresponding (S)-alcohol.Described ester compound is methyl aceto acetate preferably.

Alcohol dehydrogenase gene provided by the invention also can be used for the biological metabolism approach of studying or makes up genetic engineering bacterium.Described biology is preferably Lactobacillus kefir DSM 20587 bacterial strains.

Than prior art; Beneficial effect of the present invention is following: the present invention has isolated new alcohol dehydrogenase gene from Lactobacillus kefirDSM 20587 genomes; Enlarged the resource of alcohol dehydrogenase gene, metabolic engineering research and the structure genetic engineering bacterium for bacillus caucasicus Lactobacillus kefir DSM 20587 provides scientific basis simultaneously.Separation of the present invention can high enantioselectivity ground conversion methyl phenyl ketone be (S)-phenylethyl alcohol from the alcoholdehydrogenase (LK-ADH) of Lactobacillus kefir DSM20587; Enantiomeric excess value reaches 99.4%; The type of coenzyme dependence simultaneously is the NADH type, shows that this enzyme is that a kind of NADH relies on (S)-alcoholdehydrogenase.Therefore the present invention produces the keto compounds that neutral body optionally with carbonyl compound, particularly has a huge side chain for biological organic synthesis and is converted into optically pure alcohol, like (S)-phenylethyl alcohol good alcoholdehydrogenase is provided.

Description of drawings

Below in conjunction with description of drawings feature and advantage of the present invention.

Fig. 1 is the increase TAIL-PCR reacting flow chart of the segmental two ends of alcohol dehydrogenase gene part flanking sequence of the present invention.

Fig. 2 is the splicing and the TAIL-PCR schema of alcohol dehydrogenase gene adh full length sequence of the present invention.

Fig. 3 is ethanol dehydrogenase inclusion body dialysis renaturation purifying electrophoretic analysis figure of the present invention; Wherein 1,2 swimming lanes are eluting fractions of inclusion body washings I and II; What 3 swimming lanes showed is the white component of solubilization of inclusion bodies liquid eggs; 4 swimming lanes promptly are the recombinase liquid through the dialysis renaturation, and what the 5th swimming lane showed is the albumen in the supernatant that obtains behind the broken wall.

Fig. 4 is pET-28a of the present invention (+)-adh plasmid construction figure.

Fig. 5 is that the GC of LK-ADH asymmetric reduction methyl phenyl ketone product analyzes.A: (S)-phenylethyl alcohol with (R)-phenylethyl alcohol standard substance (Sigma-Aldrich); B: reaction solution sample. methyl phenyl ketone, (R)-phenylethyl alcohol and (S)-phenylethyl alcohol RT is respectively 5.623,7.634, and 7.884min.

Fig. 6-1 is the influence of pH to the oxidizing reaction enzyme activity.

Fig. 6-2 is the influence of pH to the reduction reaction enzyme activity.

Fig. 7-1 is the influence of temperature to the oxidizing reaction enzyme activity.

Fig. 7-2 is the influence of temperature to the reduction reaction enzyme activity.

Fig. 8-1 is the double reciprocal curve of methyl phenyl ketone concentration and speed of response.

Fig. 8-2 is the double reciprocal curve of isopropyl alcohol concentration and speed of response.

Fig. 9-1 is the stability of recombinase under-20 ℃.

Fig. 9-2 is the stability under 25 ℃ of the recombinases.

Fig. 9-3 is the thermostability of recombinase.

Embodiment

Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

The design of embodiment 1 PCR degenerated primer

According to the bacterium ethanol dehydrogenase protein sequence of including among the GenBank; Concrete bacterial classification is seen table 1; AlignX multisequencing comparison programanalysis through Vector NTI Advance 10 software packages; Find out the conservative aminoacid sequence district (BlockS) of 2 tile heights, these two conservative regions zine ion of corresponding ethanol dehydrogenase high conservative respectively combine territory and cofactor to combine the territory.Convert the protein sequence of these two conservative regions into the degenerate core nucleotide sequence, and calculate degeneracy, according to this degenerate core nucleotide sequence design pair of degenerate primers, the sequence of this primer is referring to SEQ ID No.3 and 4 in table 2 and the sequence table.

The proteic bacterial classification of ethanol dehydrogenase of table 1. design alcohol dehydrogenase gene degenerated primer institute reference

The degenerated primer of table 2. amplification ethanol dehydrogenase

Annotate: Y=C or T; W=A or T; S=C or G; K=G or T; R=A or G; M=A or C; B=G or C or T; H=A or C or T; D=A or G or T; V=A or G or C.

The extraction of the total DNA of embodiment 2 Lactobacillus kefir DSM, 20587 strain gene groups

1) cultivates Lactobacillus kefir DSM 20587 cell 300ml (available from ANS DSMZ,

Number:35411).

2) 3, the centrifugal 10min of 000g is resuspended in 5ml SET solution.Add N,O-Diacetylmuramidase (final concentration 1mg/ml), cultivated 1 hour for 37 ℃.

3) add 1/10 volume 10% (w/v) SDS and Proteinase K (final concentration 0.5mg/ml), cultivate 2 hours (or put upside down mixing) for 55 ℃.Add 1/3 volume 5M NaCl, 1 times of volume chloroform, incubated at room temperature 0.5 hour is often put upside down mixing.

4) 4, the centrifugal 15min of 500g moves into new pipe mutually with upper water.

5) add 1 times of volume Virahol, put upside down the pipe mixing gently, centrifugal back DNA deposition is used 70% washing with alcohol, and vacuum-drying is dissolved in TE solution.

The segmental pcr amplification of embodiment 3 alcohol dehydrogenase gene parts

The total DNA of Lactobacillus kefir DSM 20587 strain gene groups with embodiment 2 gained is a template; With 1 designed primer of embodiment (synthetic) by Invitrogen Shanghai Ying Jun Bioisystech Co., Ltd; Carry out pcr amplification (amplified reaction uses the DR001A PCR test kit available from the precious biotechnology in TaKaRa Dalian ltd) reaction system and see table 3, response procedures is following:

Table 3.PCR reaction system is formed

The PCR response procedures:

1 95 ℃ of preparatory sex change 3min of Step,

2 95 ℃ of sex change 30sec of Step,

3 60 ℃ of annealing of Step 30sec,

Step extends 30sec for 4 72 ℃,

Step 5 repeats Step2 to Step4, and circular flow is 30 times altogether,

Step extends 10min for 6 72 ℃,

74 ℃ of maintenances of Step.

The order-checking of embodiment 4 PCR products

Reclaim the PCR product of embodiment 3 gained, follow these steps to operation:

(1) gets the PCR reaction solution 5 μ l of embodiment 3 gained, add 1 μ l, 6 * loading buffer, use 1% sepharose to carry out electrophoresis.

(2) the DNA running gel is placed under the long-wave ultra violet lamp, cut out the gel that contains the dna fragmentation that amplifies, the plastic centrifuge tube of weighing in advance (available from Eppendorf company) of packing into weighs up the weight of gel.

(3) the 6mol/L NaI (the 0.1g gel adds 300 μ l) of three times of gel weight of adding melts in 45 ℃ of water bath heat preservation to gels fully.

(4) add 5 μ l glass milk, (this glass milk is prepared by following steps: 10g silica gel (Sigma S5631) is suspended in the 100ml PBS damping fluid vortex mixer mixing, leaves standstill the 2h hypsokinesis and removes suspended substance; Centrifugal (Beckman Avanti J-25; Rotor JA17,2000g, 2min) abandoning supernatant; The deposition be suspended in again in the 3mol/L NaI solution, with after this solution packing 4 ℃ of preservations).

(5) place ice bath to keep 10min, and whenever at a distance from centrifuge tube of 1～2min vibration.

(6) centrifugal (desk centrifuge, 13,000r/min, 30sec) deposition glass milk, abandoning supernatant.

(7) the New Wash elution buffer (NaCl50mmol/L of the adding freezing preservations of 200 μ l (20 ℃); EDTA (pH7.5) 2.5mmol/L; Tris (pH7.5) 10mmol/L; Ethanol 50%), in vortex mixer suspension glass milk, centrifugal (desk centrifuge, 13,000r/min, 30sec) washing.This process needs triplicate, and last washing should be blotted as far as possible and remained in the pipe elution buffer at the end.

(8) add 5 μ l sterile distilled waters, vibration suspends glass milk, places 45 ℃ of water bath heat preservation 10min, and the DNA that makes absorption is by desorb, centrifugal (desk centrifuge, 13,000r/min, 30sec), with new centrifuge tube of supernatant immigration.This process needs triplicate, merges supernatant.

(9) the glass milk that possibly bring into of centrifugal removal (desk centrifuge, 13,000r/min 30sec), moves into a new centrifuge tube with supernatant, is the recovery product.

Reclaim product and be connected, connect and carry out to specifications with the pMD19-T cloning vector.The pMD19-T that adopts is a kind of cloning vector commonly used, and size is 2692bp, has the Amp resistant gene available from the precious biotechnology in TaKaRa Dalian ltd.

Connect product Transformed E coli DH5 α competent cell.Carry out blue hickie screening on the LB screening flat board of penbritin, X-gal and IPTG containing, and extract hickie bacterium colony plasmid and carry out bacterium colony PCR to identify positive recombinant.Wherein the preparation process of E coli DH5 α competent cell is following:

(1) with the freezing preservation of glycerine bacterium liquid E coli DH5 α (giving birth to worker biotech company) inoculation 2ml LB liquid nutrient medium (Tryptones 1% available from Shanghai; NaCl 0.5%; Yeast extract 1%; PH7.4), spend the night in 37 ℃ of shaking culture.

(2) inoculate the 250ml triangular flask that 20ml LB liquid nutrient medium is housed with 1% inoculum size, in about 37 ℃ of shaking culture 2h, control OD ₆₀₀0.3～0.5.

(3) with the aseptic plastic centrifuge tube (available from Beckman company) of bacterium liquid immigration 50ml, in ice bath, place 10min.

(4) centrifugal 6,000r/min, 4 ℃, 10min reclaims thalline.

(5) nutrient solution that inclines, and centrifuge tube is inverted so that the trace nutrient solution of final residual flows to end.

(6) bacterial sediment is suspended in the ice-cold 0.1mol/L CaCl of 10ml ₂In the solution, thalline is centrifugal recovery after washing.

(7) thalline is suspended in the ice-cold 0.1mol/L CaCl of 10ml again ₂In the solution, in ice bath, keep 30min.

(8) centrifugal recovery somatic cells, the upper solution of inclining also is inverted centrifuge tube so that the solution of final residual flows to end.

(9) thalline is suspended in the ice-cold 0.1mol/L CaCl of 800 μ l ₂In the solution, place 4 ℃ or-70 ℃ of refrigerators preservations, be competent cell.

Use CaCl ₂Method Transformed E coli DH5 α competent cell, step is following:

(1) with the DNA of the above-mentioned pMD19-T of being connected to cloning vector connect product with the aseptic suction nozzle adding of refrigerative contain above-mentioned preparation 100 μ l competent cells, place the plastic centrifuge tube of ice bath (available from Eppendorf company) in advance; With finger tapping centrifuge tube bottom; Careful mixing keeps 30min in ice bath.

(2) will contain centrifuge tube heat shock 90sec in 42 ℃ of waters bath with thermostatic control that DNA connects product and competent cell, not shake centrifuge tube therebetween.

(3) place ice bath to keep 2min centrifuge tube fast.

(4) add 800 μ l LB liquid nutrient mediums, put upside down mixing, 37 ℃, 120r/min vibration incubation 1h makes cell proliferation.

(5) cell transformed is coated the agar plate surface that contains penbritin, X-gal and IPTG.

(6) place room temperature to be absorbed flat board until liquid.

(7) be inverted flat board, cultivate 16h in 37 ℃.

The screening of positive recombinant and evaluation:

(1) blue hickie reaction screening positive recombinant: according to the principle of α-Hu Bu reaction; The white colony that on the agar plate of additional penbritin, X-gal and IPTG, produces is the bacterium colony that has recombinant plasmid; Blue colonies is the bacterium colony that has the recirculation carrier, but so positive recon of white colony preliminary evaluation.

(2) the bacterium colony PCR of positive recombinant identifies: picking hickie bacterium colony, utilize universal primer to carry out PCR.The PCR response procedures is the same constant, carries out 0.8% agarose gel electrophoresis, and electrophoresis showed goes out and target DNA band of the same size, proves that the plasmid reorganization is correct, is positive recombinant.

Determined dna sequence and analysis:

Positive recombinant carries out sequencing by the handsome Bioisystech Co., Ltd in Shanghai; Promptly get the segmental dna sequence dna of alcohol dehydrogenase gene part of Lactobacillus kefir DSM 20587 bacterial strains; Be the dna sequence dna between two conservative regions of alcohol dehydrogenase gene, these two conservative regions zine ion of corresponding ethanol dehydrogenase high conservatives respectively combine territory and cofactor combination territory.

Embodiment 5 adopts the TAIL-PCR segmental two ends of the above-mentioned alcohol dehydrogenase gene part flanking sequence that increases

According to the TAIL-PCR principle; Be the basis with the embodiment 4 last dna sequence dnas of measuring; Design 3 nested special primer SP1, SP2, SP3 and SP1 ', SP2 ', SP3 ' at its 5 ' end separately with 3 ' end; With 13 kinds of degenerated primers (AP1-AP13) pairing, obtain the flanking sequence at its two ends through pcr amplification respectively.Concrete primer sequence is seen SEQ ID No.5～23 in table 4 and the sequence table.

The flanking sequence the primer at table 4.TAIL-PCR amplification alcohol dehydrogenase gene part fragment two ends

Annotate: N=A or G or C or T; W=A or T; S=C or G.

TAIL-PCR principle wherein; Primer design principle and the concrete experimental implementation step of TAIL-PCR used in the table 4 of the present invention are following:

1. TAIL-PCR principle: the TAIL-PCR full name is that hot asymmetric interlaced PCR (ThermalAsymmetric Interlaced PCR) is a kind of Protocols in Molecular Biology that is used for separating with the contiguous unknown dna sequence dna of known array.The TAIL-PCR ultimate principle is 3 nested Auele Specific Primers of known array design (special prime, abbreviation SP1, the SP2 that utilizes target sequence other; SP3; About 20bp), degenerated primer (Arbitrarydegenerate prime is called for short AP) is combined at random with (about the 14bp) of a plurality of weak points with low Tm value respectively with them, with genomic dna as template; Length and specific difference according to primer design asymmetric temperature cycle, carry out specific amplification through fractional order reaction.Generally have at least a kind of degenerated primer can and Auele Specific Primer between utilize the difference of annealing temperature to carry out hot asymmetric PCR reaction, can obtain the flanking sequence of known array through three nest-type PRCs reactions.The concrete reaction process of TAIL-PCR is as shown in Figure 1.

2. TAIL-PCR Auele Specific Primer of the present invention reaches degenerated primer principle of design at random: the Auele Specific Primer design direction is for needing the zone of ignorance direction of amplification, and the position of SP2 is in the inboard of SP1, and SP3 is positioned at the inboard of SP2.Distance between per two primers generally is advisable with 60-100bp.The design of primers principle: the length of primer is 22-26nt, GC content 45-55%, 60-70 ℃ of Tm value.Degenerated primer is according to the ubiquitous proteinic conserved amino acid sequence design of species at random, and length is about 14bp, and the Tm value is 30～48 ℃.In order to increase annealed possibility between degenerated primer and target sequence, except 3 of 3 ' end the base, the base of other position comprises the degenerate core thuja acid.

3. the concrete experimental implementation step of TAIL-PCR of the present invention is following:

1) genomic dna obtains.

The extraction of the total DNA of Lactobacillus kefir DSM 20587 strain gene groups is with embodiment 2.

2) affirmation of known array.

The dna sequence dna that before carrying out the PCR experiment, embodiment 4 is recorded carries out 3 order-checkings, to guarantee the safety of sequencing result.

3) 1st PCR reaction.

Genomic dna is got in right amount as template after OD measures accurately quantitatively, and as upstream primer, SP1 or SP1 ' primer are downstream primer, carry out 1st PCR reaction with AP primer (any one in 13 kinds, below be example with the AP1 primer).Amplified reaction uses the DRR002A PCR test kit available from the precious biotechnology in TaKaRa Dalian ltd.

● by following component preparation 1st PCR reaction solution.

● 1st PCR reaction conditions is following:

94℃ 1min

98℃ 1min

94℃ 30sec；25℃ 3min；72℃2～4min

72℃ 10min

4) 2nd PCR reaction.

After 50 times of 1st PCR reaction solution dilutions, get the template of 1 μ l as 2nd PCR reaction, be upstream primer with the AP1 primer, SP2 or SP2 ' primer are downstream primer, carry out 2nd PCR reaction.

● by following component preparation 2nd PCR reaction solution.

● 2nd PCR reaction conditions is following:

72℃ 10min

5) 3rd PCR reaction.

After 50 times of 2nd PCR reaction solution dilutions, get the template of 1 μ l as 3rd PCR reaction, be upstream primer with the AP1 primer, SP3 or SP3 ' primer are downstream primer, carry out 3rd PCR reaction.

● by following component preparation 3rd PCR reaction solution.

● 3rd PCR reaction conditions is following:

72℃ 10min

6) get 1st, 2nd, each 5 μ l of 3rd PCR reaction solution, the sepharose of use 1% carries out electrophoresis.

7) cutting glue and reclaim electrophoretic band clearly, is that primer carries out dna sequencing to the PCR product with SP3 and SP3 '.

Splicing, pcr amplification, clone and the order-checking of embodiment 6 alcohol dehydrogenase gene full length sequences

Each TAIL-PCR amplification that records among dna sequence dna that in embodiment 4, records and the embodiment 5 and the basis of two ends flanking sequence on; Analyze in conjunction with Blastn, Primer Premier5.0 software and VectorNTI ORF, splice the full length gene sequence (see figure 2) that to have initiator codon ATG and terminator codon TAG.According to sequences Design total length checking primer ADH-forward and ADH-reverse (seeing SEQ ID No.24 and 25 in table 5 and the sequence table) that splicing gets, total DNA is a template amplification total length alcohol dehydrogenase gene with Lactobacillus kefir DSM 20587 strain gene groups.The PCR reaction conditions is 95 ℃ of 3min, 95 ℃ of 30sec, and 56 ℃ of 1min, 72 ℃ of 1min30sec, totally 30 circulations, last 72 ℃ are extended 10min.The PCR product cloning is carried out the sequence checking to the pMD19-T carrier.Through measuring the sequence of this PCR product, show that both nucleotide sequences are in full accord after comparing with the sequence of splicing gained.

Table 5. amplification alcohol dehydrogenase gene full length sequence the primer

The nucleotide sequence of the gene of above-mentioned gained is shown in SEQ ID No.1 in the sequence table.Full length gene 1044bp, wherein A, C, G, four kinds of Nucleotide numbers of T and per-cent are respectively 273 (26.1%), 254 (24.3%), 260 (24.9%), 257 (24.6%).This sequence intronless has complete opening code-reading frame, 347 amino acid of encoding.The aminoacid sequence of coded product is shown in SEQ ID No.2 in the sequence table, and molecular weight is 37.07kD, and iso-electric point pI is 5.70.

The sequence of the gene of gained uploaded on the http://www.ncbi.nlm.gov website with blast program carry out sequence homology relatively and marking.Through NCBI Blastn and Blastp nucleic acid and protein sequence compare of analysis, can not find identical sequence at the GenBank lane database.In registered Protein Data Bank, the homology of LK-ADH and all sequences all is no more than 78%, and wherein zinc dependence alcoholdehydrogenase (LB-ADH) homology with short lactobacillus L.brevis ATCC 367 codings is 78% (GenBank accession number YP_795041); The homology that contains zinc alcoholdehydrogenase (LR-ADH) of encoding with Lu Te Shi lactobacillus spp Lactobacillus reuteri 100-23 is 72% (GenBank accession number ZP_01273263).Two kinds of proteic sequences that LK-ADH and above-mentioned homology are the highest demonstrate homology highly in conservative position, territory (calmodulin binding domain CaM of zine ion binding site and cofactor), only a residue position difference are arranged.Proved also that thus alcohol dehydrogenase gene of the present invention is a new alcohol dehydrogenase gene.

The structure of embodiment 7 expression vectors and transformant

MCS sequence according to pET-28a (+) expression vector; Initiator codon ATG place at alcohol dehydrogenase gene adh introduces the BamHI restriction enzyme site; Introduce the HindIII restriction enzyme site at terminator codon TAG place, design and synthesize primer (seeing SEQ ID No.26 and 27 in table 6 and the sequence table).Total DNA is a template with Lactobacillus kefir DSM 20587 strain gene groups, the PCR reaction conditions: 95 ℃ of preparatory sex change 3min, 95 ℃ of sex change 30sec; 58 ℃ of annealing 30sec; 72 ℃ are extended 1min 10sec, and circular flow is 30 times altogether, and last 72 ℃ are extended 10min.Reclaim the PCR product; Through BamHI and HindIII double digestion, reclaim through glue again, be connected 16h with same pET-28a (+) plasmid (purchasing company) in 16 ℃ in Novagen with BamHI and HindIII double digestion and glue recovery; Make recombinant plasmid pET-28a (+)-adh, plasmid construction figure sees Fig. 4.Should connect product Transformed E coli DH5 α competent cell.Connect product and use CaCl ₂Be applied on the kalamycin resistance flat board that contains 50 μ g/mL behind the method Transformed E .coli DH5 α competent cell.Extract the plasmid (concrete steps see below) of the transformant that grows on the resistant panel, carry out enzyme with BamHI and HindIII and cut checking.

Table 6. alcohol dehydrogenase gene is expressed and is used primer

The underscore place is restriction enzyme site BamHI (forward) and HindIII (oppositely).

The extraction step of the sub-plasmid of recombinant conversion:

1) choose the test tube that single colony inoculation contains the kantlex of 5ml LB and 50 μ g/ml from the conversion flat board of grow overnight, 37 ℃ of shaking culture are spent the night;

2) draw 1ml bacterium liquid in the eppendorf centrifuge tube, centrifugal (Eppendorf 5415R type, 13000r/min, 1min, down together), abandon supernatant, vacuum blots residual liquid;

3) will precipitate and be suspended in 100 μ l Solution I solution again, vibration disperses thalline on the vortex mixer, places ice bath;

4) add the freshly prepared Solution II of 200 μ l solution, cover tight lid after, put upside down pipe Solution II solution fully contacted with thalline so that lysis, ice bath 3-5min;

5) add the 150 μ l Solution III solution of ice bath in advance, it is fully mixed that the high vibration pipe makes it, and places ice bath 3～5min;

6) centrifugal 10min moves to another centrifuge tube with supernatant, adds 2 times of volume ethanol, places 30min for-20 ℃;

7) centrifugal 10min abandons supernatant.Deposition with 70% washing with alcohol once, and is dry in vacuum drier, adds 25 μ l TE damping fluid dissolution precipitations and obtains the sub-plasmid of recombinant conversion.

Solution I solution (mmol/L): glucose 50, EDTA 10, and Tris-Cl 25, and pH 8.0.

Solution II solution: NaOH 0.2mol/L, SDS 1 (w/v) %.

Solution III solution: 5mol/L Potassium ethanoate 60ml, glacial acetic acid 11.5ml, TE damping fluid (mmol/L): Tris-HCl 10, and EDTA 1, and pH 8.0.

The transformant plasmid that empirical tests is correct adopts CaCl again ₂Method Transformed E .coli BL21.DE3 (U.S. Novagen company) competent cell; Get positive colony and carry out the restriction analysis evaluation through the resistance screening of medium; Proof contains correct insertion fragment, obtains being used for conversion bacterial strain E.coliBL21 (DE3)/pET-28a (+)-adh (preparation of above-mentioned competent escherichia coli cell and the CaCl of genetic expression ₂The concrete step that method transforms is referring to embodiment 4).

The expression of embodiment 8 recombinant alcohol dehydrogenase (extraction of recombinant alcohol dehydrogenase crude enzyme liquid)

(1) engineering strain E.coli BL21 (DE3)/pET-28a (+)-adh of making of inoculation embodiment 7 cultivates 16h for 37 ℃ in containing the antibiotic LB liquid nutrient medium of kantlex (50 μ g/mL).With the fresh LB liquid nutrient medium of 1% inoculum size switching, 37 ℃ are cultured to OD with nutrient solution ₆₀₀After being 0.6, adding IPTG is that 1mM carries out abduction delivering 4h to final concentration.

(2) the bacterium liquid after drawing 1ml and inducing is in 4 ℃ of eppendorf centrifuge tubes, and 13,200rpm is centrifugal, and 10min abandons supernatant;

(3) add 0.5ml pH6.0 sodium phosphate buffer and (contain MgCl ₂1mM) resuspended, add 0.5ml pH6.0 sodium phosphate buffer once more after repeating step (2) washs once to thalline and (contain MgCl ₂1mM) resuspended;

(4) place ice bath supersonic wave wall breaking 3min (250W, work 5s, intermittently 5s).4 ℃ of cytoclasis liquid, 13, the centrifugal 20min of 200rpm.

(5) deposition that obtains behind the supersonic wave wall breaking adds 1ml inclusion body washings I; Repeatedly behind the pressure-vaccum in 16; 4 ℃ of centrifugal 20min of 100g (Eppendorf5415R small frozen supercentrifuge) go to add behind the supernatant 1ml inclusion body cleaning solution II and carry out second time with method and wash.

(6) deposition of the inclusion body after the washing adds 5ml solubilization of inclusion bodies liquid, and room temperature is placed about 2h and stirred frequently and helps dissolving to dissolve until complete.4 ℃ 16, the centrifugal 20min of 100g, sucking-off supernatant obtain purer inclusion body protein liquid.

(7) pack into the dialysis tubing handled of inclusion body protein liquid is dialysed in 4 ℃ step by step, and dialyzate contains urea 4M respectively, 2M, 1M and 0M.Dialysis at different levels continue 8～16h respectively.Last is taken turns and is not urea-containing dialysis, and dialyzate is selected the damping fluid of different pH values as required.

The inclusion body washings:

Washings I:

Tris pH8.0 50mmol/L EDTA 1mmol/L

Triton X-100 0.5% urea 2mol/L

Cleaning solution II:

Tris pH8.0 50mmol/L EDTA 0.5mmol/L

Urea 1mol/L

Solubilization of inclusion bodies liquid:

Potassium phosphate buffer

PH6.0 0.1mol/L urea 8mol/L

MgCl ₂ 1mmol/L

Analyzing discovery through SDS-PAGE only needs through the dialysis renaturation, and target protein has just reached enough purity.The protein sample of each step of separation and purification is carried out the SDS-PAGE analytical results shown in Fig. 4-4.What the 5th swimming lane showed is the last white protein that broken wall obtains, and 1,2 swimming lanes are eluting fractions of inclusion body washings I and II, and what 3 swimming lanes showed is the white component of solubilization of inclusion bodies liquid eggs, and 4 swimming lanes promptly are the recombinase liquid through the dialysis renaturation.Recombinant protein behind the purifying is a band on the SDS-PAGE running gel, gray scale scanning shows that purity more than 95%, records concentration 0.84mg/ml with the Bradford method.Used enzyme liquid all is this enzyme liquid among the embodiment below the present invention.

Embodiment 9 coenzyme rely on the mensuration of type

Method: with the methyl phenyl ketone is substrate, and it is active that spectrophotometer 340nm (ε 340=6.22mmol/L-1cm-1) detects the alcoholdehydrogenase catalytic reduction reaction down.Active testing conditions is: 30 ℃, reaction system TV 1ml contains: the 10mmol/L methyl phenyl ketone; The potassium phosphate buffer of 100mmol/L pH6.0; 0.25mmol/L NADH (or NADPH); And the enzyme liquid of 0.2ml.Add enzyme liquid afterreaction and pick up counting, measure the variation of 340nm light absorption value in 1min.

The enzyme activity definition: under 30 ℃, the enzyme amount of the NADH (or NADPH) of PM catalyzed conversion 1 μ mol is defined as an enzyme activity unit (U).

The calculation formula that enzyme is lived is: enzyme (U)=EW * V/6.22 alive

(EW is the variation of 340nm absorbancy in the 1min; V is the volume of reaction solution, ml; 6.22, molar extinction coefficient)

Result:, be that to record LK-ADH according to the method described above respectively be 7.23 * 10 for the activity of methyl phenyl ketone to coenzyme with NADH and NADPH respectively for the coenzyme of confirming alcoholdehydrogenase LK-ADH relies on type ^-2U/ml enzyme liquid and 0.90 * 10 ^-2U/ml enzyme liquid, the former is 8 times of the latter.This result shows: the LK-ADH of the bacillus caucasicus L.kefir DSM 20587 of being cloned into is a kind of NADH dependent form alcoholdehydrogenase.

What measure enzyme employing alive among the embodiment below the present invention all is the method among the embodiment 9.

The mensuration of embodiment 10 enantioselectivities

Method: with the methyl phenyl ketone is that substrate is measured the enantioselectivity of alcoholdehydrogenase LK-ADH in asymmetric reduction reaction.

Reaction system TV 1ml contains: 0.20mmol/L NADH; 7.7 μ l Virahol (0.1mol/L); 1.2 μ l methyl phenyl ketone (10mmol/L); The potassium phosphate buffer of 176 μ l 100mmol/L pH6.0 (contains 1mmol/L MgCl ₂); The alcoholdehydrogenase LK-ADH (5.6U/ml) of 800 μ l purifying.Behind the mixing in 30 ℃ of oscillatory reaction 4h, 4, the centrifugal 20min of 000rpm gets supernatant with the extracting of 1 times of volume chloroform.The chloroform layer extraction liquid that obtains is used for gas chromatographic analysis, according to calculated by peak area product optical purity (enantiomeric excess value, e.e value), and confirms the enantioselectivity of alcoholdehydrogenase LK-ADH thus.

Gas chromatography analysis method:

Gas chromatograph: Agilent 6890 chromatographic working stations

Chiral chromatographic column: CP-Chirasil-DEX CB column (25m; Diameter:25 μ m)

Column temperature program: 60 ℃ of 5min at, 195 ℃ of 5 ℃/min to

Flow rate of carrier gas: 1.3ml/min

Carrier gas: helium

Sample size: 1 μ l

Detector FID

According to the method described above, alcoholdehydrogenase LK-ADH gas chromatographic analysis result sees Fig. 5.

Behind the reaction 4h, methyl phenyl ketone (RT: 5.623min) peak area=23.88; (R)-phenylethyl alcohol (Sigma-Aldrich) (RT: 7.634min) peak area=1.42; (S)-phenylethyl alcohol (Sigma-Aldrich) (RT: 7.884min): peak area=462.28.Reorganization alcoholdehydrogenase LK-ADH optics enantioselectivity in the reaction of asymmetric reduction methyl phenyl ketone is the S-configuration.Wherein:

Enantiomeric excess value (e.e value)=(462.28-1.42)/(462.28+1.42) * 100%=99.4%.

The test of embodiment 11 substrate specificities

Method:, test the oxidation activity of alcoholdehydrogenase LK-ADH and a series of alcohol compounds behind the purifying and active respectively with the reduction reaction of a series of ketone, aldehyde compound according to following method.

Oxidizing reaction is 100% calculating relative reactivity with the enzyme work to Virahol; Reduction reaction is 100% calculating relative reactivity with the enzyme work to methyl phenyl ketone.All substrate final concentrations are 10mmol/L.

1. oxidizing reaction

With the Virahol is substrate, and spectrophotometer 340nm (ε 340=6.22mmol/L-1cm-1) detects the alcoholdehydrogenase oxidation activity down.Active testing conditions is: 30 ℃, reaction system TV 1ml contains: the 10mmol/L Virahol; The potassium phosphate buffer of 100mmol/L pH6.0; 0.25mmol/L NAD+; And the enzyme liquid of 0.2ml.Add enzyme liquid afterreaction and pick up counting, measure the variation of 340nm light absorption value in 1min

2. reduction reaction

With the methyl phenyl ketone is substrate, and it is active that spectrophotometer 340nm (ε 340=6.22mmol/L-1cm-1) detects the alcoholdehydrogenase reduction reaction down.Active testing conditions is: 30 ℃, reaction system TV 1ml contains: the 10mmol/L methyl phenyl ketone; The potassium phosphate buffer of 100mmol/L pH6.0; 0.25mmol/L NADH; And the enzyme liquid of 0.2ml.Add enzyme liquid afterreaction and pick up counting, measure the variation of 340nm light absorption value in 1min

The result: according to the method described above, measured the substrate specificity of alcoholdehydrogenase LK-ADH in oxidizing reaction and reduction reaction respectively with a series of alcohol compounds and ketone, aldehyde compound, the result sees table 7.

The substrate specificity of table 7. alcoholdehydrogenase LK-ADH

Mensuration through substrate specificity finds that alcoholdehydrogenase LK-ADH generally shows higher relatively activity to surveys aldehyde compound, and ketone compounds such as methyl phenyl ketone and the verivate thereof such as the parachloroacetophenone that have bigger side-chain radical in addition also can be reduced to corresponding alcohol by the effective conversion of this enzyme.

The optimal reaction pH value of embodiment 12 enzymes

Method:

(1) enzyme liquid is mixed with different pH systems, is respectively: 3.6,4.0,4.6,5.0,5.6,6.0,6.6,7.0,7.6 and 8.0.Wherein the pH value is the citric acid-sodium citrate damping fluid preparation with 100mmol/L of 3.6 to 5.6 enzyme liquid, and pH value is that 6.0 to 8.0 enzyme liquid is prepared with the potassium phosphate buffer of 100mmol/L.

(2) measure purified recombinant enzyme in oxidizing reaction and the reduction reaction respectively in the corresponding down residual enzyme vigor of condition of different pH.

(3) be 100% with the highest enzyme work, calculate and draw the vigor change curve of enzyme under different pH values, the ph optimum of research enzymatic reaction.

The result: the experiment of the optimal reaction pH value of enzyme when carrying out oxidizing reaction and reduction reaction according to the method described above, the result is shown in Fig. 6-1 and 6-2.The optimal reaction pH value of this enzyme is about 5.6 when experimental data can be found out oxidizing reaction, and when reaction pH is reduced to below 4.0 or is adjusted to more than 6.6, enzyme activity descends rapidly.The optimal reaction pH value of enzyme is about 5.0 during reduction reaction, equally is reduced to 4.0 below or is adjusted to neutrality more than 7.0 as reaction pH, and enzyme activity descends rapidly.

The optimal reactive temperature of embodiment 13 enzymes

Method:

(1) uses 4 ℃ of ice baths and different bath temperatures: 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃.

(2) enzyme liquid is placed above-mentioned differing temps insulation 10min, its corresponding residual enzyme vigor in the sampling and measuring reduction reaction in the citric acid-sodium citrate damping fluid (pH5.0) of 100mmol/L.

(3) enzyme liquid is placed above-mentioned differing temps insulation 10min, its corresponding residual enzyme vigor in the sampling and measuring oxidizing reaction in the citric acid-sodium citrate damping fluid (pH5.6) of 100mmol/L.

(4) be 100% with the highest enzyme value alive, calculate and also draw the vigor change curve of enzyme under differing temps, investigate the optimal reactive temperature of enzyme.

The result: carry out the experiment of the optimal reactive temperature of enzyme according to the method described above, the result is shown in Fig. 7-1,7-2.The vigor that this enzyme all showed about 35 ℃ when experimental data showed oxidizing reaction and reduction reaction is the highest.In 30 ℃～50 ℃ scopes, the activity of LK-ADH is all more stable in oxidizing reaction or reduction reaction, and temperature surpasses 50 ℃ of enzymic activitys and significantly reduces.

Embodiment 14 dissimilar additives are to the influence of enzyme activity

Method: measured the influence of several kinds of common metal ions, metal ion chelation agent, tensio-active agent, protein denaturant, sulfydryl reductive agent respectively to enzymic activity.Test condition: according to the method for embodiment 9; In the 100mmol/L of pH5.0 citric acid-sodium citrate damping fluid reaction system, add additive and the enzyme liquid that final concentration is 10mmol/L and behind 35 ℃ of insulation 10min, add the substrate methyl phenyl ketone, investigate of the influence of different substance enzyme activity.With the reaction system that does not contain additive is that relative reactivity is calculated in the contrast alive of 100% enzyme.Institute's test substances is:

(1) metals ion is to enzyme influence alive

K ⁺、Na ⁺、Mg ²⁺、Ni ²⁺、Ca ²⁺、Mn ²⁺(1mmol/L)、Fe ³⁺、Zn ²⁺，Cu ²⁺(10μmol/L)。

(2) other additives are to the influence of enzyme activity

Urea, tween-80 (Tween-80), methyl-sulphoxide (DMSO), YD 30 (EDTA), beta-mercaptoethanol, WR 34678 (DTT), sodium laurylsulfonate (SDS).

The result: measured the influence to enzymic activity of several metal ion species, metal ion chelation agent, tensio-active agent, protein denaturant, sulfydryl reductive agent according to the method described above, the result sees table 8.

The dissimilar additives of table 8. are to the influence of enzyme activity

Embodiment 15 kinetic constant K _mMensuration

Method: in the 100mmol/L of pH5.0 citric acid-sodium citrate buffer solution system,, measure the initial velocity of enzyme reduction reaction with the substrate methyl phenyl ketone of enzyme liquid and different concns (1,2,3,4,5,6,7,8,9,10mmol/L) effect; In the 100mmol/L of pH5.6 citric acid-sodium citrate buffer solution system; With the substrate Virahol of enzyme liquid and different concns (0.2,0.5,1,2,5,10mmol/L) effect; Measure the initial velocity of oxydasis reaction, map with Lineweaver-Burk double-reciprocal plot method respectively.

Get equation: Y=BX+C is B=K wherein _m/ V _MaxC=1/V _Max

Obtaining with the methyl phenyl ketone with this is the reduction reaction K of substrate _mValue and be the oxidizing reaction K of substrate with the Virahol _mValue.

The result:

(1) reduction reaction K _m

With 1,2,3,4,5,6,7,8,9, the 10mmol/L methyl phenyl ketone is as substrate; Measure substrate reactions speed (table 9-1); With of the inverse mapping (see Fig. 8-1) of Lineweaver-Burk double-reciprocal plot method, get equation: y=80.49x+1.4537 with the inverse and the concentration of substrate of maximum response speed; Calculate reduction reaction: K _m=55.37mmol/L.

Table 9-1. measures reduction reaction K _mThe maximum reaction velocity of value

Concentration of substrate (mmol/L)	1	2	3	4	5	6	7	8	9	10
											V max (×10 -2U/mg.min)	1.210	2.493	3.720	4.255	5.780	6.406	7.194	9.191	9.708	10.905

(2) oxidizing reaction Km

With 0.2,0.5,1,2,5, the 10mmol/L Virahol is as substrate; Measure substrate reactions speed (table 9-2); With of the inverse mapping (see Fig. 8-2) of Lineweaver-Burk double-reciprocal plot method, get equation: y=103.18x+1.1646 with the inverse and the concentration of substrate of maximum response speed; Calculate oxidizing reaction: Km=88.60mmol/L

Table 9-2. measures oxidizing reaction K _mThe maximum reaction velocity of value

Concentration of substrate (mmol/L)	0.2	0.5	1	2	5	10
							V max(×10 -3U/mg.min)	1.923	5.319	7.353	25.641	50.012	83.333

The stability of embodiment 16 enzymes

Method:

(1)-20 the variation of ℃ freezing enzyme activity

Enzyme liquid is placed-20 ℃ of refrigerator freezings, and sampling once in 35 ℃, is measured enzyme activity in the 100mmol/LpH5.0 citric acid-sodium citrate buffer solution system weekly.Soprano alive is 100% with enzyme, investigates the stability of enzyme under-20 ℃.

(2) 25 ℃ of normal temperature are incubated the influence to enzyme activity

Place 25 ℃ of water-baths to be incubated enzyme liquid, at set intervals the sampling and measuring enzyme activity.Soprano alive is 100% with enzyme, investigates the stability of enzyme in the time of 25 ℃.

(3) thermostability of enzyme under differing temps

Enzyme liquid is incubated 1h, the residue vigor of sampling and measuring enzyme at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 50 ℃, 60 ℃ respectively in 100mmol/L citric acid-sodium citrate damping fluid (pH5.0).Soprano alive is 100% with enzyme, investigates the thermostability of enzyme.

The result:

(1)-20 the variation of ℃ freezing enzyme activity

Enzyme is lived over time under ℃ freezing according to the method described above-20, and the result is shown in Fig. 9-1.Can keep more than 90% in the activity in preservation 2 time-of-weeks of this enzyme under the refrigerated situation, about 70% vigor of preservation 4 week back residue, preservation still remains 50% vigor after 5 weeks, explain this enzyme can be under freezing situation short-term storage.

(2) 25 ℃ of normal temperature are incubated the influence to enzyme activity

Carry out the experiment of 25 ℃ of insulations to the enzyme activity influence according to the method described above, the result is shown in Fig. 9-2.The active decline in the preceding 4h of 25 ℃ of insulations of this enzyme is less, the vigor of residue 96% during 4h; Behind 24h, enzyme activity still has 90% residue, and behind the insulation 2d, the residue vigor of enzyme is reduced to original about 50%, and enzyme is lived and continued to descend afterwards.

(3) thermostability of enzyme under differing temps

The reorganization alcoholdehydrogenase is investigated in the thermostability under the differing temps, and the result is shown in Fig. 9-3.Enzyme vigor behind 20,25,30,35 ℃ of insulation 1h all reduces less than tangible; The residue vigor is original 95% behind 35 ℃ of insulation 1h; It is original 11% that temperature reaches after 45 ℃ the rapid drawdown of living of insulation 1h enzyme, shows that 35～40 ℃ is the limit range of this enzyme tolerable temperature.

Sequence table

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<220>

<221>misc_feature

<222>(1，4，6，7，8，10，13，14，15，16)

< 223>v=a or g or c; R=a or g; S=c or g; K=g or t; Y=c or t; M=a or c

<400>4

vccrassscr ccrkymcc 18

<210>5

<211>23

<212>DNA

< 213>artificial sequence

<220>

< 223>primer SP1

<400>5

ttcggcttga taaccagcac taa 23

<210>6

<211>26

<212>DNA

< 213>artificial sequence

<220>

< 223>primer SP2

<400>6

aaccagcact aaagttgtcg ctatga 26

<210>7

<211>23

<212>DNA

< 213>artificial sequence

<220>

< 223>primer SP3

<400>7

tggtgcgatt acaaagtctc ccg 23

<210>8

<211>20

<212>DNA

< 213>artificial sequence

<220>

< 223>primer SP1 '

<400>8

cgttaccagc atgccgagtg 20

<210>9

<211>23

<212>DNA

< 213>artificial sequence

<220>

< 223>primer SP2 '

<400>9

catgccgagt ggtcactggt taa 23

<210>10

<211>20

<212>DNA

< 213>artificial sequence

<220>

< 223>primer SP3 '

<400>10

atgctgcccg tgtcgccaac 20

<210>11

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP1

<220>

<221>misc_feature

<222>(1，7，8，11，13)

< 223>n=a or g or c or t; S=c or g; W=a or t

<400>11

ngtcgaswga nawgaa 16

<210>12

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP2

<220>

<221>misc_feature

<222>(3，5，8，10，13)

< 223>w=a or t; N=a or g or c or t; S=c or g

<400>12

tgwgnagwan casaga 16

<210>13

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP3

<220>

<221>misc_feature

<222>(3，5，8，10，13)

< 223>w=a or t; N=a or g or c or t

<400>13

agwgnagwan cawagg 16

<210>14

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP4

<220>

<221>misc_feature

<222>(1，5，8，10，13)

< 223>s=c or g; N=a or g or c or t

<400>14

sttgntastn ctntgc 16

<210>15

<211>15

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP5

<220>

<221>misc_feature

<222>(1，6，8，10，12)

< 223>n=a or g or c or t; S=c or g; W=a or t

<400>15

ntcgastwts gwgtt 15

<210>16

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP6

<220>

<221>misc_feature

<222>(1，5，8，10，13)

< 223>w=a or t; N=a or g or c or t

<400>16

wgtgnagwan canaga 16

<210>17

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP7

<220>

<221>misc_feature

<222>(3，6，8，11，13)

< 223>w=a or t; N=a or g or c or t; S=c or g

<400>17

cawcgncnga nasgaa 16

<210>18

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP8

<220>

<221>misc_feature

<222>(3，5，8，11，13)

< 223>n=a or g or c or t; W=a or t; S=c or g

<400>18

tcstncgnac ntwgga 16

<210>19

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP9

<220>

<221>misc_feature

<222>(1，5，8，10，13)

< 223>w=a or t; N=a or g or c or t

<400>19

wcagntgwtn gtnctg 16

<210>20

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP10

<220>

<221>misc_feature

<222>(5，8，11，13)

< 223>n=a or g or c or t

<400>20

tcttncgnac ntngga 16

<210>21

<211>15

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP11

<220>

<221>misc_feature

<222>(4，7，10，12)

< 223>n=a or g or c or t

<400>21

ttgnagnacn anagg 15

<210>22

<211>16

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP12

<220>

<221>misc_feature

<222>(3，7，8，10，13)

< 223>n=a or g or c or t; S=c or g; W=a or t

<400>22

gtncgaswca nawgtt 16

<210>23

<211>15

<212>DNA

< 213>artificial sequence

<220>

< 223>primer AP13

<220>

<221>misc_feature

<222>(1，6，8，10，12，14)

< 223>n=a or g or c or t; S=c or g; W=a or t

<400>23

ntcagstwts gwgwt 15

<210>24

<211>26

<212>DNA

< 213>artificial sequence

<220>

< 223>ADH-forward primer

<400>24

atgaaatcaa ccatttttgt aaaacc 26

<210>25

<211>22

<212>DNA

< 213>artificial sequence

<220>

< 223>ADH-reverse primer

<400>25

ctatttttga gcgacaacca ac 22

<210>26

<211>29

<212>DNA

< 213>artificial sequence

<220>

< 223>pET-28a-adh-forward primer

<400>26

gcgggatcca tgaaatcaac catttttgt 29

<210>27

<211>28

<212>DNA

< 213>artificial sequence

<220>

< 223>pET-28a-adh-reverse primer

<400>27

gcgaagcttc tatttttgag cgacaacc 28

Claims (10)

1. an alcohol dehydrogenase gene is characterized in that, is one of following nucleotide sequences:

1) it is the base sequence shown in the SEQ ID No.1 in the sequence table;

The protein of 2) encoding and forming by the aminoacid sequence shown in the SEQ ID No.2 in the sequence table.

2. an ethanol dehydrogenase is characterized in that, its aminoacid sequence is shown in SEQ ID No.2 in the sequence table.

3. ethanol dehydrogenase according to claim 2 is characterized in that, the ethanol dehydrogenase that it relies on for the NADH that derives from bacillus caucasicus (Lactobacillus kefir) DSM 20587 bacterial strains.

4. a recombinant expression vector is characterized in that, comprises the nucleotide sequence of the described alcohol dehydrogenase gene of claim 1.

5. recombinant expression vector according to claim 4 is characterized in that, carrier framework is pET-28a (+).

6. a recombinant expressed transformant is characterized in that, comprises described any nucleotide sequence of claim 1 or claim 4 or 5 described recombinant expression vectors.

7. recombinant expressed transformant according to claim 6 is characterized in that, it is intestinal bacteria E.coli BL21.DE3.

8. the preparation method of a recombinant alcohol dehydrogenase is characterized in that, comprises cultivating claim 6 or 7 described recombinant expressed transformant, obtains the ethanol dehydrogenase of reorganization.

9. prepared recombinant alcohol dehydrogenase of the preparation method by claim 8.

10. application that in biological organic synthesis, carbonyl compound is converted into corresponding alcohol like claim 2 or 3 described ethanol dehydrogenases or the described recombinant alcohol dehydrogenase of claim 9.

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