CN103114056B - Aldehyde dehydrogenase and preparation method thereof - Google Patents
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Abstract
The invention discloses an aldehyde dehydrogenase and a preparation method thereof, and relates to aldehyde dehydrogenases. Strains for producing the aldehyde dehydrogenase Fwaldh are Flammeovirga Pacifica H2. The preparation method comprises the following steps of: cloning an aldehyde dehydrogenase gene fwaldh; inserting the aldehyde dehydrogenase gene fwaldh into an expression vector, and constructing a recombinant expression vector carrying the aldehyde dehydrogenase gene fwaldh; transforming the recombinant expression vector into E.coil BL21; selecting positive clones from transformed E.coil BL21 and carrying out fermentation culture on the positive clones in a culture medium; collecting fermented E.coil BL21 cells by centrifugation, re-suspending the E.coil BL21 cells in a cracking buffer so as to be subjected to cracking; carrying out centrifugation on cracked suspension and collecting supernatant; and uniformly mixing the supernatant with Ni-NTA Agarose, and purifying according to the instructions of a purification kit, thereby obtaining the aldehyde dehydrogenase Fwaldh.
Description
Technical field
The present invention relates to a kind of aldehyde dehydrogenase, especially relate to the Novel aldehydes dehydrogenase gene of Pacific Ocean heat color bacillus (FlammeovirgaPacifica) H2 deriving from deep-sea, and recombinant expressed obtained aldehyde dehydrogenase and preparation method thereof.
Background technology
Aldehyde dehydrogenase (Aldehyde dehydrogenase) (EC1.21.3) is can the enzyme superfamily of oxidation of aldehydes NAD (the P)+dependent form of catalyzing endogenous and external source.Up to now, 19 kinds of aldehyde dehydrogenase genes are found from the mankind, the metabolism (Crabb, Matsumoto, Chang, & You, 2004) of life and external acetaldehyde in these genes participate in.Investigator finds, aldehyde dehydrogenase also has important effect (Huang et al., 2008 plant and microorganism environment resistant pressure (arid, high salt) aspect; Li, Gao, Yu, Wang, & An, 2003), (Yang such as Yang, Zhang, Wang, Wood, & Zhang, 2012) be transformed in intestinal bacteria by the aldehyde dehydrogenase gene in red for tooth rib moss, transformed bacteria obtains the ability that recombinant bacterium also has drought-resistant and high salt.(the Kato such as Tomohisa kato, Miyanaga, Kanaya, & Morikawa, 2010) obtain the aldehyde dehydrogenase gene of long chain alkane of can degrading from Geobacillusthermoleovorans B23, Jyumpei Kokayashi etc. can improve the aldehyde dehydrogenase of hydrogen output low-concentration acetic acid salt from Rhodobacter sphaeroides.In patent, the aldehyde dehydrogenase gene of different sources is cloned, checks order and expresses, and is mainly used in and utilizes L-sorbosone to produce vitamins C or 2-KGA, the healthcare products etc. relieved the effect of alcohol.
Summary of the invention
The first object of the present invention is to provide a kind of Novel aldehydes dehydrogenase gene fwaldh.
The second object of the present invention is to provide a kind of aldehyde dehydrogenase Fwaldh encoded by described aldehyde dehydrogenase gene fwaldh.
The third object of the present invention is to provide the preparation method of aldehyde dehydrogenase Fwaldh.
The production bacterial strain of described aldehyde dehydrogenase Fwaldh is Pacific Ocean heat color bacillus (Flammeovirga Pacifica) H2, this bacterial strain is preserved in China typical culture collection center on June 13rd, 2012, deposit number is: CCTCC NO:M2012229, address is Wuhan, Wuhan University, postcode 430072.
The sequence (SEQ ID No.1) of described Novel aldehydes dehydrogenase gene fwaldh is as follows:
The information of SEQ ID No.1:
1) title: Flammeovirga Pacifica aldehyde dehydrogenase gene;
2) molecule type: DNA;
3) sequence signature:
A) length: 1440bp;
B) type: nucleic acid;
C) chain: double-strand;
D) topological framework: linear.
The aminoacid sequence (SEQ ID No.2) of the aldehyde dehydrogenase Fwaldh that described aldehyde dehydrogenase gene fwaldh encodes is as follows:
The information of SEQ ID No.2:
1) title: Flammeovirga Pacifica aldehyde dehydrogenase;
2) molecule type: protein;
3) sequence signature:
A) length: 479aa;
B) type: amino acid.
The preparation method of described aldehyde dehydrogenase Fwaldh comprises the following steps:
1) described aldehyde dehydrogenase gene fwaldh is cloned;
2) described aldehyde dehydrogenase gene fwaldh is inserted expression vector, build the recombinant expression vector carrying described aldehyde dehydrogenase gene fwaldh;
3) described recombinant expression vector is transformed in E.coli BL21;
4) choose the positive colony transformed in rear E.coli BL21 and carry out fermentation culture in substratum;
5) the E.coli BL21 cell after collected by centrifugation fermentation, resuspended described E.coli BL21 cell carries out cracking in lysis buffer;
6) suspension after cracking in step 5) is carried out centrifugal, collect supernatant liquor;
7) supernatant liquor and Ni-NTA Agarose are mixed, carry out purifying according to purification kit specification sheets, obtain described aldehyde dehydrogenase Fwaldh.
In step 2) in, described expression vector can be pET-His carrier.
In step 4), described substratum can be the LB substratum containing 100 μ g/mL penbritins, and add 100 μ g/mL penbritins during induction, described fermentation condition can be: at 37 DEG C, shaking culture is to A
600=0.6, adding isopropylthio-β-D-galactoside (IPTG) to final concentration is 50 μ g/mL, and adds 0.2% glucose, is then induced to A in 28 DEG C
600=0.8, then be changed to and be induced to A at 16 DEG C
600=1.2.
In step 5), described lysis buffer formula can be: 0.3mol/L NaCl, 10mmol/L imidazoles, 50mmol/LNaH
2pO
4, pH8.0.
In step 6), described centrifugal condition can be 18000g, centrifugal 20min.
In the present invention, the outstanding feature of Novel aldehydes desaturase shows as:
1. novel gene order, does not have similar complete ORF gene order in ncbi database.
2. Novel aldehydes desaturase Fwaldh can in intestinal bacteria stably express, add glucose in preparation method's step 4) and significantly can reduce and local express and the stability of plasmid in intestinal bacteria can be maintained.Add 100 μ g/mL penbritins during induction to supplement by the degraded of the β-lactamase of expression-secretion to penbritin, thus prevent the loss of recombinant plasmid.
3. the aldehyde dehydrogenase vigor after purifying is very high, and stability is strong under cryogenic, has high reactivity in the basic conditions, can overcome existing aldehyde dehydrogenase vigor low, easily spontaneous oxidation occur, and enzyme purification process is loaded down with trivial details waits deficiency.
4. recombinant expressed aldehyde dehydrogenase can efficient catalytic formaldehyde, acetaldehyde degradation be formic acid, and acetic acid, can be widely used in the degraded and cell acetaldehyde metabolic detoxification etc. of aldehyde pollutants in environment.
The present invention clones and obtains Novel aldehydes dehydrogenase gene fwaldh from Deep-Sea Microorganisms Flammeovirga Pacifica genome, is transformed in intestinal bacteria and carries out stable a large amount of heterogenous expression, obtain a kind of aldehyde dehydrogenase Fwaldh.This enzyme energy efficient catalytic acetaldehyde, propionic aldehyde, butyraldehyde and valeraldehyde generate acetic acid, propionic acid, butyric acid, positive valeric acid respectively, at food, chemical field, play an important role in the metabolism of cell acetaldehyde and biological environment resistant pressure.The invention provides a kind of method of aldehyde dehydrogenase gene clonal expression, albumen fast purifying.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE collection of illustrative plates of the recombinant expressed purifying of aldehyde dehydrogenase Fwaldh, M is albumen Marker, 1 is that BL21pET-his is unloaded, and 2 is the expression of recombinant vectors pET-Fwaldh in bacterial strain BL21 (induction), and 3 is the abduction delivering of recombinant vectors pET-Fwaldh in bacterial strain BL21.
Fig. 2 is the SDS-PAGE after restructuring aldehyde dehydrogenase Fwaldh purifying, and M is albumen Marker, and 1 is the target protein Fwaldh(55kDa through Ni-NTA purifying gained).
Fig. 3 is the optimal pH of aldehyde dehydrogenase Fwaldh recombinant protein.In figure 3, X-coordinate is pH value, and ordinate zou is enzyme activity (%).
Fig. 4 is aldehyde dehydrogenase Fwaldh recombinant protein optimal reactive temperature.In the diagram, X-coordinate is temperature (DEG C), and ordinate zou is enzymic activity (%).
Embodiment
Below in conjunction with specific embodiments and the drawings, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as Molecular Cloning: A Laboratory room handbook (New York:Cold Spring Harbor Laboratory Press, 2001) experiment condition described in, or according to the condition that reagent or instrument manufacturer facility business advise.
The extraction of 1.Flammeovirga Pacifica genomic dna
With the Flammeovirga sp.wpaga001 bacterial classification that transfering loop picking-80 DEG C is preserved, 2216E culture medium flat plate (a kind of bacteria culture medium, containing the Tryptones of 1%, the yeast extract of 0.2%, 1.5% agar powder, seawater 100mL) the single bacterium colony of upper line separation.37 DEG C of overnight incubation put by flat board after line, (a kind of bacteria culture medium in picking list colony inoculation to the 2216E nutrient solution pipe of 5mL, containing the Tryptones of 1%, the yeast extract of 0.2%, seawater 100mL), 37 DEG C of wave and culture spend the night, and collect bacterium, supernatant discarded after the centrifugal 8min of 6000g, precipitation Eddy diffusion is in 567 μ L TE damping fluid (10mMTris-HCl, 1mM EDTA, pH8.0), add 30 μ L 10%SDS, shake up, add 3 μ L Proteinase Ks (20mg/mL) again, mix gently, 37 DEG C of water-bath 1h.Add 100 μ L 5mol/L NaCl, fully mix, then add 80 μ L CTAB(cetyltriethylammonium bromides)/NaCl, softly mix, 65 DEG C of water-bath 10min.With phenol/centrifugal 10min of chloroform/primary isoamyl alcohol (25: 24: 1) extracting twice, 15000g, supernatant is carefully transferred in another centrifuge tube.With chloroform/primary isoamyl alcohol (24: 1) extracting once, the centrifugal 10min of 15000g, uses isopropanol precipitating DNA, then uses washing with alcohol twice, precipitation be dissolved in redistilled water ,-20 DEG C of preservations.
2. the pcr amplification of aldehyde dehydrogenase fwaldh gene and sequential analysis
According to the gene annotation result after the order-checking of Flammeovirga Pacifica strain gene group, the primers F of design aldehyde dehydrogenase: 5 '-GCGC
gGATCCaTGGAAAATGTAATTATTACTTC-3 ' (being scribed ss BamHI restriction enzyme site) (SEQ ID No.3), R:5 '-CCGGAA
gCTAGCtCAAAGCTTTGTAATG-3 ' (being scribed ss NheI restriction enzyme site) (SEQ ID No.4).With the Flammeovirga Pacifica genomic dna extracted for template, the fwaldh gene of the total length that increases with primers F and R.PCR reaction conditions is: contain in the reaction system of 50 μ L, 100ng template, 400nmo/L primers F, 400nmo/L primer R, 200 μm of o/L dNTP, 2.5mmo/L Mg
2+, 5U Primer Star HSTaq enzyme (purchased from TaKaRa company), 5 μ L10 × PCR reaction buffers; Reaction conditions: 94 DEG C of 5min; 98 DEG C of 10s, 52 DEG C of 45s, 72 DEG C of 1.5min(30cycles); 72 DEG C of 10min; 4 DEG C of preservations.PCR primer after purifying is carried out enzyme and is cut, and the pET-His carrier after cutting with enzyme enzyme of the same race is connected, CaCl
2method is transformed in intestinal bacteria Top10, chooses positive colony and checks order.
Derive the aminoacid sequence of aldehyde dehydrogenase Fwaldh according to the nucleotide sequence obtained, totally 479 amino-acid residues, its aminoacid sequence refers to SEQ ID No.2.
The information of SEQ ID No.3:
1) molecule type: oligonucleotide;
2) sequence signature:
A) length: 33bp;
B) type: nucleic acid;
C) chain: strand;
D) topological framework: linear.
The information of SEQ ID No.4:
1) molecule type: oligonucleotide;
2) sequence signature:
A) length: 28bp;
B) type: nucleic acid;
C) chain: strand;
D) topological framework: linear.
3. the expression and purification of restructuring Fwaldh
Clone's aldehyde dehydrogenase gene fwaldh, is inserted in expression vector pET-His, builds the recombinant expression vector carrying aldehyde dehydrogenase gene fwaldh.To the recombinant expression vector pET-Fwaldh Transformed E .coli BL21 of fwaldh gene be carried, choose positive colony containing 100 μ g/mL penbritins LB substratum in 37 DEG C shake training to A
600when=0.6, add isopropylthio-β-D-galactoside (IPTG) to final concentration 50 μm of ol/L, add 100 μ g/mL penbritins before induction, 28 DEG C are induced to A
600=0.8,16 DEG C are induced to A
600=1.2, after bacterium liquid is collected in the centrifuge tube of 200mL, 5000g centrifugation bacterial cell.By bacterial cell Eddy diffusion, at the lysis buffer of 20mL, (lysis buffer formula is: 0.3mol/L NaCl, 10mmol/L imidazoles, 50mmol/L NaH
2pO
4, pH8.0) in, ultrasonication becomes translucent to bacterium liquid, the centrifugal 20min of 18000g, supernatant with mix with the Ni-NTA Agarose that lysis buffer balances in advance, 4 DEG C in conjunction with 1h, purge process illustrates according to purification kit (purchased from Qiagen company) carries out.The albumen of purifying through 12% SDS-PAGE electrophoretic analysis, its molecular weight is about 55kDa, and purity reaches more than 95% (result is see Fig. 1).
4. the zymologic property of restructuring aldehyde dehydrogenase Fwaldh
The measuring method of 4.1 restructuring aldehyde dehydrogenase Fwaldh enzyme activities is as follows:
Fwaldh(150 μ g/mL after dilution purifying), 2mM coenzyme NAD
+, 2mM acetaldehyde, propionic aldehyde, butyraldehyde or valeraldehyde, 1mM DTT(dithiothreitol (DTT)), 50mmol/L Glycine-NaOH damping fluid (pH9.0), reaction cumulative volume 1mL.After reacting 15min at 35 DEG C, 340nm place surveys light absorption value.It is 1U that enzymic activity is defined as the enzyme amount that per minute produces 1 μm of ol NADH.By Enzyme assay, with acetaldehyde, propionic aldehyde, butyraldehyde, valeraldehyde is the V that substrate calculates restructuring aldehyde dehydrogenase Fwaldh
maxbe respectively 1.8U/mg/min, 2.0U/mg/min, 5.3U/mg/min, 5.2U/mg/min.
The optimal pH measuring method of 4.2 restructuring aldehyde dehydrogenase Fwaldh is as follows:
Purified restructuring Fwaldh is that substrate carries out enzymatic reaction to measure its optimal pH under different pH value with butyraldehyde.Restructuring Fwaldh carries out restructuring Fwadh enzyme activity determination at 35 DEG C of temperature in the damping fluid of different pH.Result (Fig. 2) shows, the optimal pH of restructuring Fwaldh is 9.0.
Optimum temperuture and the temperature stability measuring method of 4.3 restructuring aldehyde dehydrogenase Fwaldh are as follows:
The optimum temperuture of restructuring Fwaldh to be determined as with butyraldehyde be that substrate carries out enzymatic reaction under Glycine-NaOH damping fluid (pH9.0) system and differing temps.Thermal stability determination, for restructuring Fwaldh is at 50 DEG C, 55 DEG C, processes different time respectively, then carry out enzyme assay at 35 DEG C at 60 DEG C.Enzymatic reaction optimum temperuture measurement result (Fig. 3) shows, its optimum temperuture is 35 DEG C, and this enzyme has higher activity at low temperatures, 20 DEG C have 63% activity, 10 DEG C still have 30% activity, this enzyme, after room temperature places one week, still has the enzyme activity of 60%.The heat stability test of enzyme shows, restructuring Fwaldh is thermolability enzyme, and after 50 DEG C of process 1h, residual enzyme is lived and also had about 10%; 60 DEG C, after 70 DEG C of process 1h, enzyme work is 0% substantially.
Claims (9)
1. aldehyde dehydrogenase gene fwaldh, is characterized in that its DNA sequence dna (SEQ ID No.1) is as follows:
atggaaaatg taattattac ttctgatcag aacgttcaaa atattgaaga tatattcatt 60
catttaagaa agaatacaca agcaattaaa gacactaatg caaagcaaag gattaaaaaa 120
ttaaagagct tagagaagta tattttagat catagatcag aaattcagag tgctatacac 180
cacgatttca agaaaaatcc aactgaaact gatgccacag aaatactatc aacattaggt 240
ttacttagac atactaaaaa gaatttaaag aaatggatga agccaacaaa agtaggtaca 300
ccattaagta tgcttggcac ttcttccaaa gttattgagg aacctaaagg agtttgttta 360
attatatctc cttggaatta tcctttttat ttagctattg gtccgcttat ttatgctatt 420
gctgcaggga atacagttgc tttaaaacct tcagaacttt cttcaaacac ctctcttttt 480
atagaaaaga tggtcaaaga gttatttcca cctgatgaga tcaacgttat tttgggtggt 540
gtggaaactt ctacaaaact acttgagaaa aagtttgatc atattttctt tacaggtagc 600
cctcaagtag gtaaagtgat tatgaatgcc gcagctaaac acctcacttc tgttactttg 660
gaactaggag ggaaatctcc tacagtactt ttaaatgata cagacttaaa agatgctgca 720
gacaaaatta catggggtaa atttatcaat gccggtcaga catgtattgc tcctgattat 780
tgcttaattc caaaagaact ggaaactcaa tttattgatc agatgcaaaa taccatcaat 840
gagttttact ctcctgatgg tgaaattatt gagctttcta aagattttgc aagaatcatt 900
tctaaaaaac atttcttgag acttcaacat ttaattcatg atgctgcaga aaaaggagcg 960
actgtaatga tgaatggaga aatgcatgaa gattctaaat atattcctcc aacaattatt 1020
agcaacgttt ctgacgatat gctcatcatg caagaagaaa tttttggtcc aattttacca 1080
atcaaaacct acacacaaat agaggaggct ttgtcttata tttcttctaa agaaaaacca 1140
ttagcattat atatcttcgg acaaaagaag aaagatataa atcatgtatt gaaaaatact 1200
acagcaggtg gtacttcaat taatgaaact gtcatacata tcaataaccc tgaattacct 1260
tttggtgggg ttaataatag tggcataggg aaatctcatg gtatatatgg ttttaaagga 1320
tttagtaatc aaagagctat tttgaaaaat agagtaggca tgacgggcat caaacctgtc 1380
tatcctcctt atggtaattt ggctacaaaa cttttagatt ttatcattac aaagctttga 1440
The information of SEQ ID No.1:
1) title: Flammeovirga Pacifica aldehyde dehydrogenase gene;
2) molecule type: DNA;
3) sequence signature:
A) length: 1440bp;
B) type: nucleic acid;
C) chain: double-strand;
D) topological framework: linear.
2. the aldehyde dehydrogenase Fwaldh as claimed in claim 1 coded by gene fwaldh, is characterized in that its aminoacid sequence (SEQID No.2) is as follows:
MENVIITSDQ NVQNIEDIFI HLRKNTQAIK DTNAKQRIKK LKSLEKYILD HRSEIQSAIH 60
HDFKKNPTET DATEILSTLG LLRHTKKNLK KWMKPTKVGT PLSMLGTSSK VIEEPKGVCL 120
IISPWNYPFY LAIGPLIYAI AAGNTVALKP SELSSNTSLF IEKMVKELFP PDEINVILGG 180
VETSTKLLEK KFDHIFFTGS PQVGKVIMNA AAKHLTSVTL ELGGKSPTVL LNDTDLKDAA 240
DKITWGKFIN AGQTCIAPDY CLIPKELETQ FIDQMQNTIN EFYSPDGEII ELSKDFARII 300
SKKHFLRLQH LIHDAAEKGA TVMMNGEMHE DSKYIPPTII SNVSDDMLIM QEEIFGPILP 360
IKTYTQIEEA LSYISSKEKP LALYIFGQKK KDINHVLKNT TAGGTSINET VIHINNPELP 420
FGGVNNSGIG KSHGIYGFKG FSNQRAILKN RVGMTGIKPV YPPYGNLATK LLDFIITKL 479
The information of SEQ ID No.2:
1) title: Flammeovirga Pacifica aldehyde dehydrogenase;
2) molecule type: protein;
3) sequence signature:
A) length: 479aa;
B) type: amino acid.
3. the preparation method of aldehyde dehydrogenase Fwaldh as claimed in claim 2, is characterized in that comprising the following steps:
1) aldehyde dehydrogenase gene fwaldh described in claim 1 is cloned;
2) described aldehyde dehydrogenase gene fwaldh is inserted expression vector, build the recombinant expression vector carrying described aldehyde dehydrogenase gene fwaldh;
3) described recombinant expression vector is transformed in E.coli BL21;
4) choose the positive colony transformed in rear E.coli BL21 and carry out fermentation culture in substratum;
5) the E.coli BL21 cell after collected by centrifugation fermentation, resuspended described E.coli BL21 cell carries out cracking in lysis buffer;
6) by step 5) in suspension after cracking carry out centrifugal, collect supernatant liquor;
7) supernatant liquor and Ni-NTA Agarose are mixed, carry out purifying according to purification kit specification sheets, obtain described aldehyde dehydrogenase Fwaldh.
4. the preparation method of aldehyde dehydrogenase Fwaldh as claimed in claim 3, is characterized in that in step 2) in, described expression vector is pET-His carrier.
5. the preparation method of aldehyde dehydrogenase Fwaldh as claimed in claim 3, is characterized in that in step 4) in, described substratum is the LB substratum containing 100 μ g/mL penbritins.
6. the preparation method of aldehyde dehydrogenase Fwaldh as claimed in claim 5, is characterized in that described substratum, adds 100 μ g/mL penbritins when inducing.
7. the preparation method of aldehyde dehydrogenase Fwaldh as claimed in claim 3, is characterized in that in step 4) in, described fermentation condition is: at 37 DEG C, shaking culture is to A
600=0.6, adding isopropylthio-β-D-galactoside to final concentration is 50 μ g/mL, and adds 0.2% glucose, is then induced to A in 28 DEG C
600=0.8, then be changed to and be induced to A at 16 DEG C
600=1.2.
8. the preparation method of aldehyde dehydrogenase Fwaldh as claimed in claim 3, is characterized in that in step 5) in, described lysis buffer formula is: 0.3mol/L NaCl, 10mmol/L imidazoles, 50mmol/L NaH
2pO
4, pH8.0.
9. the preparation method of aldehyde dehydrogenase Fwaldh as claimed in claim 3, is characterized in that in step 6) in, described centrifugal condition is 18000g, centrifugal 20min.
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