CN102071174A - (2R, 3R)-2,3-butanediol dehydrogenase and coding gene and application thereof - Google Patents
(2R, 3R)-2,3-butanediol dehydrogenase and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses (2R, 3R)-2,3-butanediol dehydrogenase from paenibacillus polymyxa ATCC12321 and a coding gene and application thereof. Zymology verifies that the dehydrogenase has new characteristics: (2R, 3R)-2,3-butanediol is catalyzed to generate (R)-acetoin and meso-2,3-butanediol is catalyzed to generate (S)-acetoin by taking NAD+ as coenzyme, and the (R)-acetoin is catalyzed to generate (2R, 3R)-2,3-butanediol, the (S)-acetoin is catalyzed to generate meso-2,3-butanediol, and butanedione is catalyzed to generate (R)-acetoin by taking NADH as coenzyme. Moreover, the dehydrogenase substrate has high specificity and wide industrial application prospect.
Description
Technical field
The present invention relates to enzyme and encoding gene thereof and application, particularly relate to one derive from Paenibacillus polymyxa (Paenibacillus polymyxa ATCC12321) (2R, 3R)-2,3-butanediol dehydrogenation enzyme and encoding gene thereof and application.
Background technology
2, (2,3-butanediol BD) is widely used in fields such as chemical industry, food, aerospace fuel to the 3-butyleneglycol.Its dewatered product methylethylketone can be made the solvent of resin, paint etc.; Dewatered product 1,3-butadiene after the esterification can be used for synthetic rubber, polyester and polyurethane; Calorific value higher (27,200kJ/kg) can be used as fuel dope; Form octane isomer with the methylethylketone dehydrogenation and can produce senior aviation with oily; BD also can prepare printing ink, perfume, fumigant, moistening agent, tenderizer, softening agent, explosive and medicine chiral support etc.BD chemosynthesis cost height and process are loaded down with trivial details, and suitability for industrialized production is difficulty.It is to avoid the difficulty of chemosynthesis that biological process prepares the BD purpose, realizes that by traditional petroleum refining be the biorefinery transition of raw material to renewable resources.
And as chiral intermediate (2R, 3R)-2, the 3-butyleneglycol is mainly used in fine chemicals such as the pharmaceutical intermediate of high added value and liquid crystal material, its price is a meso-form 2, more than 1000 times of 3-butyleneglycol, though its market requirement is little, profit is surprising.With respect to meso-form 2, the 3-butyleneglycol, (2R, 3R)-2, the research of 3-butyleneglycol is also fewer, and is rare especially to the research of its gene and zymetology for chiral purity.(the 2R that existing unique report is crossed, 3R)-2,3-butanediol dehydrogenation enzyme only has the bacterial strain that separates acquisition from yeast saccharomyces cerevisiae, but this bacterial strain is not suitable for the industrial production butyleneglycol, output extremely low (about 1mM), this enzyme only are that to keep yeast self physiological function required, do not have practical application in industry to be worth [J.Bio.Chem., 275 (46), 35876-35885 (2000)].
Summary of the invention
The invention provides a kind of industrial application value higher (2R, 3R)-2,3-butanediol dehydrogenation enzyme.
(2R provided by the present invention, 3R)-2,3-butanediol dehydrogenation enzyme source available from U.S. Biological resources preservation center (http://www.atcc.org/), is one of following amino acid residue sequences in Paenibacillus polymyxa (Paenibacillus polymyxa ATCC12321):
1) the SEQ ID NO:1 in the sequence table;
2) amino acid residue sequence with SEQ ID NO:1 in the sequence table passes through replacement, disappearance or the interpolation of amino-acid residue and has (2R, 3R)-2, the protein of 3-butanediol dehydrogenation enzyme or the effect of acetoin reductase enzyme, new protein and SEQ ID NO:1 homology reach 80% or higher.。
SEQ ID NO:1 in the sequence table is made up of 350 amino-acid residues.
Encode above-mentioned (2R, 3R)-2, the gene of 3-butanediol dehydrogenation enzyme is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID NO:2 in the sequence table;
2) dna sequence dna of SEQ ID NO:1 in the code sequence tabulation;
3) coded sequence 80% or above with come from the sequence table SEQ ID NO:1 and have (2R, 3R)-2, the nucleotide sequence of 3-butanediol dehydrogenation enzyme or the effect of acetoin reductase enzyme;
4) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the SEQ ID NO:2 in the sequence table.
The rigorous condition of described height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.
SEQ ID NO:2 in the sequence table is by 1050 based compositions, and its encoding sequence is from 5 ' end 1-1050 bit base, has the protein of the amino acid residue sequence of SEQ ID NO:1 in the sequence table.
Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.
(2R, 3R)-2, arbitrary segmental primer is to also within protection scope of the present invention in the 3-butanediol dehydrogenation enzyme gene in amplification.
Another object of the present invention provide a kind of expression above-mentioned (2R, 3R)-2, the method for 3-butanediol dehydrogenation enzyme.
Expression provided by the present invention above-mentioned (2R, 3R)-2, the method for 3-butanediol dehydrogenation enzyme, be above-mentioned with containing (2R, 3R)-2, the recombinant expression vector of 3-butanediol dehydrogenation enzyme gene imports host cell, express and obtain (2R, 3R)-2,3-butanediol dehydrogenation enzyme.
But described host is the protokaryon or the eukaryotic cell of arbitrary expression alien gene.
Described prokaryotic cell prokaryocyte can be colibacillus, as E.coli BL21, E.coli M15, E.coli JM109, E.coliDH5 α or E.coli LG90 etc.; Also can be subtilis Bacillus subtilis or Paenibacillus polymyxa Paenibacillus polymyxa.
Described eukaryotic cell can be mammalian cell, yeast cell and vegetable cell etc.Comprise COS-7, CHO, BHK-21, NIH3T3, Pichia pastoris or TBY2 etc.
Be used for making up and contain that (2R, 3R)-2, the carrier that sets out of the expression of recombinant e. coli carrier of 3-butanediol dehydrogenation enzyme gene can be pET-22b, pET28, pET32, pQE-30, pGEX-4T-2, pBR322 or pUC18 etc.
With pET-22b be set out containing of vector construction described (2R, 3R)-2, the intestinal bacteria secretion expression carrier called after pET22b-2R3R of 3-butanediol dehydrogenation enzyme gene.
Above-mentioned recombinant expression vector all can make up according to ordinary method.
When described host is colibacillus, need to add IPTG and carry out abduction delivering, add IPTG concentration be 0.01mM-5mM, be preferably 1mM.
That cultivation contains is of the present invention, and (2R, 3R)-2, the substratum and the culture condition of the host cell of 3-butanediol dehydrogenation enzyme gene all can be substratum and the culture condition of cultivating the host that sets out.
Application of the present invention, comprise direct utilization (2R, 3R)-2,3-butanediol dehydrogenation enzyme, perhaps utilize (2R, 3R)-2,3-butanediol dehydrogenation enzyme gene host cell carry out (2R, 3R)-2, the production of 3-butyleneglycol or the production of acetoin.
Utilization provided by the present invention contain (2R, 3R)-2, the cells produce of 3-butanediol dehydrogenation enzyme gene (2R, 3R)-2, the method for 3-butyleneglycol specifically may further comprise the steps:
1) with (2R 3R)-2, after the recombinant expression vector pET22b-2R3R of 3-butanediol dehydrogenation enzyme gene imports intestinal bacteria E.coli BL21 (DE3) pLysS, obtains recombination bacillus coli E.coli RBDH;
2) the single bacterium colony of E.coli RBDH is transferred in the LB liquid nutrient medium, inducible protein is expressed under 37 ℃, 1mM IPTG condition, obtain describedly having (2R, 3R)-2, the cell of 3-butanediol dehydrogenation enzyme activity is as biological catalyst;
3) utilize step 2) biological catalyst that obtains, add (3R)-acetoin as substrate, 37 ℃ of reactions 12 hours down, obtain target product (2R, 3R)-2, the 3-butyleneglycol.When the substrate addition is 1000mM, detects and obtain target product 871mM.
At above-mentioned production (2R, 3R)-2, in the 3-butyleneglycol method, described step 2) has (2R in, 3R)-2, the expression method of the cell of 3-butanediol dehydrogenation enzyme activity is specially: the single bacterium colony of E.coli RBDH of random choose is transferred to 5mL and contains the LB liquid nutrient medium of 100 μ g/mL Ampicillin Trihydrates and 34 μ g/mL paraxin from flat board, cultivated 12 hours for 37 ℃, be forwarded in the fresh LB liquid nutrient medium that contains 100 μ g/mL Ampicillin Trihydrates and 34 μ g/mL paraxin in 1% ratio then, 37 ℃ are cultured to thalline OD
600Value reaches 0.6, and the IPTG inducible protein that adds 1mM was then expressed 5 hours, collects thalline and is resuspended in the phosphate buffered saline buffer of 200mM, regulates thalline OD
600Value is 20, obtain describedly to have (2R, 3R)-2, the cell of 3-butanediol dehydrogenation enzyme activity.
Utilization provided by the present invention (2R, 3R)-2,3-butanediol dehydrogenation enzyme is produced (2R, 3R)-2, the method for 3-butyleneglycol is to utilize described (2R, 3R)-2,3-butanediol dehydrogenation enzyme adds (3R)-acetoin as substrate and coenzyme NAD H, in the phosphate buffered liquid system of pH 8.0,70 ℃ were reacted 4 hours, obtain (2R, 3R)-2, the 3-butyleneglycol.
The present invention (2R, 3R)-2, the application of 3-butanediol dehydrogenation enzyme also further comprises:
A) with NAD+ be coenzyme, with (2R, 3R)-2,3-butanediol dehydrogenation enzyme is so that (2R, 3R)-2, the 3-butyleneglycol is 70 ℃ of substrates, pH 11.0 preparation (R)-acetoins.
B) with NADH be coenzyme, with (2R, 3R)-2,3-butanediol dehydrogenation enzyme is 70 ℃ of substrates with the dimethyl diketone, pH 11.0 preparation (R)-acetoins.
C) with NAD+ be coenzyme, with (2R, 3R)-2,3-butanediol dehydrogenation enzyme, with racemization type meso-2, the 3-butyleneglycol is 70 ℃ of substrates, pH 11.0 preparation (S)-acetoins;
D) with NADH be coenzyme, with (2R, 3R)-2,3-butanediol dehydrogenation enzyme is 70 ℃ of substrates with meso-form (S)-acetoin, pH 8.0 preparation meso-form meso-2,3-butyleneglycol.
Concrete, the invention provides the method for production (3R)-acetoin, with described (2R, 3R)-2,3-butanediol dehydrogenation enzyme carries out one of following operation:
A) in phosphate buffered saline buffer (pH 11.0) system of 100mM, the NADH that adds 2mM is a coenzyme, and the 100mM dimethyl diketone is as substrate, and 70 ℃ were reacted 4 hours, and obtained (3R)-acetoin (3R)-acetoin;
B) in phosphate buffered saline buffer (pH 11.0) system of 100mM, the NAD+ that adds 4mM is a coenzyme, and 100mM (2R, 3R)-2, the 3-butyleneglycol is a substrate, 70 ℃ were reacted 4 hours, and obtained (3R)-acetoin (3R)-acetoin.
Concrete, the invention provides the method for production (3S)-acetoin, utilize described (2R, 3R)-2,3-butanediol dehydrogenation enzyme, in phosphate buffered saline buffer (pH 11.0) system of 100mM, the NAD+ that adds 4mM is a coenzyme, the meso-2 of 100mM, and the 3-butyleneglycol is as substrate, 70 ℃ were reacted 4 hours, and obtained (3S)-acetoin.
In whole application of the present invention, (2R, 3R)-2, the optimal reactive temperature of 3-butanediol dehydrogenation enzyme is 70 ℃; Described (2R, 3R)-2, the optimum pH that 3-butanediol dehydrogenation enzyme participates in the glycol oxidation reaction is 11.0, the optimum pH that participates in the ketone reduction reaction is 8.0.
The invention provides one derive from Paenibacillus polymyxa (Paenibacillus polymyxa ATCC12321) (2R, 3R)-2,3-butanediol dehydrogenation enzyme and encoding gene thereof.By the zymetology checking, confirm that this enzyme has brand-new characteristic, with NAD+ coenzyme, catalysis (2R, 3R)-2, the 3-butyleneglycol generates (R)-acetoin, catalysis meso-form meso-2, the 3-butyleneglycol generates (S)-acetoin; With NADH is coenzyme, and catalysis (R)-acetoin generation (2R, 3R)-2, the 3-butyleneglycol, catalysis (S)-acetoin generates the meso-2 of meso-form, the 3-butyleneglycol, the catalysis dimethyl diketone only generates (R)-acetoin.In addition, this enzyme substrates specificity height, prospects for commercial application is wide.
Below in conjunction with specific embodiment the present invention is described in further details.
Description of drawings
Fig. 1 is the molecular weight size that SDS-PAGE (A) and native-PAGE (B) detect purifying protein
Fig. 2 be (2R, 3R)-2,3-butanediol dehydrogenation enzyme optimal reaction pH pH-value determination pH result
Embodiment
Embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Method therefor is ordinary method if no special instructions among the following embodiment.
(2R from Paenibacillus polymyxa (Paenibacillus polymyxa ATCC12321), 3R)-2,3-butanediol dehydrogenation enzyme gene is after analyzing by genome sequencing, note, to obtain from Paenibacilluspolymyxa ATCC12321 genome amplification by the PCR means.Wherein Paenibacillus polymyxa ATCC12321 bacterial strain is available from U.S. Biological resources preservation center (http://www.atcc.org/), and the public can directly freely buy from U.S. Biological resources preservation center.
The clone's process and the construction of prokaryotic expression vector process thereof of this gene may further comprise the steps:
One, (2R, 3R)-2, the clone of 3-butanediol dehydrogenation enzyme gene
Through genome sequencing, and behind the note genomic information, obtained the gene of the butanediol dehydrogenation enzyme of may encoding, design following primer: P1:5 '-CGCATATGCAAGCATTGAGATGGCATGG-3 ' (restriction enzyme site of design is NdeI) and P2:5 '-ATCTCGAGGGCTTTCGGAGATACCAGGAT-3 ' (restriction enzyme site of design is XhoI) according to gene order, the total gene of Paenibacillus polymyxa ATCC12321 with extraction is a template, with P1 and P2 is primer, and the mode by PCR increases and obtains this gene fragment.(2R to the clone, 3R)-2,3-butanediol dehydrogenation enzyme gene carries out nucleotide sequencing, the sequencing result shows that this gene has the nucleotide sequence of SEQ IDNO:2 in the sequence table, by 1050 based compositions, the protein of the amino acid residue sequence of SEQ ID NO:1 in the tabulation of 5 ' end 1-the 1050th bit base code sequence.On amino acid levels, carried out sequence relatively, be somebody's turn to do (2R, 3R)-2,3-butanediol dehydrogenation enzyme with from 2 of a bacillus subtilis (Bacillus subtilis), 3 butanediol dehydrogenation enzyme [Appl.Environ.Microbiol., 74 (22), 6832-6838 (2008)] amino acid similarity level only 67%, with (2R from yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), 3R)-2,3 butanediol dehydrogenation enzyme [J.Bio.Chem., 275 (46), 35876-35885 (2000)] the amino acid similarity degree has only 35%, be lower than 22% with amino acid similarity level from Thermoanaerobacter brockii and Clostridium beijerinckii alcoholdehydrogenase [Org.Biomol.Chem., 7,3914-3917 (2009)], with from 2 of klebsiella (Klebsiella pneumoniae), 3-butanediol dehydrogenation enzyme [J.Ferment.Bioeng.83:32-37 (1997)] does not have homology.
Two, (2R, 3R)-2, the structure of 3-butanediol dehydrogenation enzyme gene prokaryotic carrier
With step 1 after gene fragment that PCR obtains is with NdeI and XhoI double digestion, be connected in the expression vector pET22b that same enzyme double digestion is handled, to contain (2R, 3R)-2, connection product transformed into escherichia coli E.coli BL21 (DE3) pLysS of 3-butanediol dehydrogenation enzyme gene, after cutting evaluation, PCR and enzyme will contain (2R, 3R)-2, the positive colony plasmid called after pET22b-2R3R of 3-butanediol dehydrogenation enzyme gene, available its carries out (2R, 3R)-2, the expression of 3-butanediol dehydrogenation enzyme.
(the 2R that contains with embodiment 1 acquisition, 3R)-2,3-butanediol dehydrogenation enzyme Prokaryotic Expression carrier pET22b-2R3R transformed into escherichia coli E.coli BL21 (DE3) pLysS, selecting positive monoclonal is shaking under 37 ℃ after bacterium is 0.6 to OD600, adding 1mM IPTG induces, centrifugal collection thalline after 5 hours, be resuspended in ultrasonic damping fluid behind the multigelation and (contain the 20mM phosphate buffered saline buffer, 300mM NaCl, the 10mM imidazoles, pH 7.4) in, the ultrasonication cell is collected supernatant and precipitation respectively, carry out the SDS-PAGE electrophoretic analysis, the result shows that the prokaryotic expression carrier that carries goal gene obtains great expression in intestinal bacteria E.coli BL21 (DE3) pLysS, and single molecular weight subunit of the recombinant protein of expression is 38kDa, conforms to expected results.
Carry out protein purification by the His-tag label, concrete grammar is: obtain supernatant liquor through ultrasonic disruption, combine with the Ni-NTA gel, the albumen that contains the His-tag label will be attached on the Ni-NTA gel, use cleaning buffer solution (20mM phosphate buffered saline buffer then, 300mM NaCl, the 50mM imidazoles, pH 7.4) wash the foreign protein of non-specific binding, use elution buffer (20mM phosphate buffered saline buffer at last, 300mM NaCl, the 500mM imidazoles, pH 7.4) with the target protein wash-out, promptly obtain (the 2R behind the purifying, 3R)-2,3-butanediol dehydrogenation enzyme uses molecular sieve gel post exchange buffering liquid to be 200mM phosphate buffered saline buffer (pH 8.0) then, and obtaining enzyme liquid is final (2R, 3R)-2,3-butanediol dehydrogenation enzyme.(swimming lane M is the molecular weight of albumen standard to purification result shown in Figure 1A, swimming lane 1 is a purifying protein), the result of SDS-PAGE shows that it is 38kDa that purifying obtains albumen list molecular weight subunit, further detect purifying protein molecular weight size (Figure 1B under active condition by Native-PAGE, shown in the swimming lane 3), the molecular weight size of this albumen under active condition is 76kDa, shows that this proteic active condition is a dimer protein.
Carrying out enzymatic property with following reaction identifies:
This reaction is for reversible reaction, with the variation of the NADH foundation (it is coenzyme that the recombinant protein of acquisition can only utilize NAD+/NADH, can not utilize NADP+/NADPH to be coenzyme) as enzyme activity determination.
With NAD+ is coenzyme, and catalysis (2R, 3R)-2, the 3-butyleneglycol generates (R)-acetoin, and (under 70 ℃, pH 11.0 conditions, with 100mM (2R, 3R)-2, the 3-butyleneglycol is a substrate, the NAD+ of 4mM is a coenzyme), the result only generates (R)-acetoin; Catalysis meso-form meso-2,3-butyleneglycol generate (S)-acetoin (under 70 ℃, pH 11.0 conditions, with the meso-form meso-2 of 100mM, the 3-butyleneglycol is a substrate, and the NAD+ of 4mM is a coenzyme), and the result only generates (S)-acetoin.
With NADH is coenzyme, catalysis (R)-acetoin generate (2R, 3R)-2,3-butyleneglycol (under 70 ℃, pH 8.0 conditions, be substrate with (R)-acetoin of 10mM, the NADH of 0.2mM is a coenzyme), the result only generate (2R, 3R)-2, the 3-butyleneglycol; Catalysis (S)-acetoin generates the meso-2 of meso-form, 3-butyleneglycol (under 70 ℃, pH 8.0 conditions, be substrate with (S)-acetoin of 10mM, the NADH of 0.2mM is a coenzyme), and the result only generates meso-2, the 3-butyleneglycol; Catalysis dimethyl diketone (under 70 ℃, pH 8.0 conditions, be substrate with the dimethyl diketone of 10mM, the NADH of 0.2mM is a coenzyme), the result only generates (R)-acetoin.
This albumen to (2S, 3S)-2, the 3-butyleneglycol without any active (under 70 ℃, pH 11.0 conditions, with (2R, 3R)-2, the 3-butyleneglycol is a substrate, the NAD+ of 4mM is a coenzyme, the result does not have spawn and detects, and does not have the generation of NADH yet) of 100mM.
The target protein that above-mentioned test-results shows expression really for (2R, 3R)-2,3-butanediol dehydrogenation enzyme.
Test one, (2R, 3R)-2, the optimal reaction pH value of 3-butanediol dehydrogenation enzyme is definite
In the ketone reduction reaction, promptly generate 2 from acetoin, 3-butyleneglycol the Direction of Reaction under 25 ℃ of conditions, is a substrate with 10mM (R/S)-acetoin, the NADH of 0.2mM is a coenzyme, has measured this enzyme in the scope of pH4.0 to 9.0, the changing conditions of enzymic activity.The result shows (as Fig. 2 (a) width of cloth), and the optimal pH of the ketone reduction reaction of this enzyme is 8.0.Buffer system: the phosphate buffer (pH 5 to 8) of 100mM citric acid buffer system (pH 4 to 6), 100mM and the Tris-HCl buffer system (pH 8 to 9) of 100mM.
In the glycol oxidation reaction, promptly from 2, the 3-butyleneglycol is to acetoin the Direction of Reaction, under 25 ℃ of conditions, with 100mM (2R, 3R)-2, the 3-butyleneglycol is a substrate, the NAD+ of 4mM is a coenzyme, has measured this enzyme in the scope of pH6.0 to 12.0, the changing conditions of enzymic activity.The result shows (as Fig. 2 (b) width of cloth), and the glycol oxidation reaction optimal pH of this enzyme is 11.0.The Tris-HCl buffer system (pH 8 to 9) of the phosphate buffer of buffer system: 100mM (pH 6 to 8), 100mM, the sodium bicarbonate buffer system (pH 9 to 11) of 100mM and glycine-sodium hydroxide buffer system (pH 11-12) of 100mM.
Test two, (2R, 3R)-2, the optimal reactive temperature of 3-butanediol dehydrogenation enzyme is determined
The measuring method that enzyme is lived is the variation of measuring NADH under the 340nm ultraviolet wavelength, and the enzyme work of a unit is defined as the NADH of per minute oxidation 1 μ mol.(2R, 3R)-2, the condition determination of 3-butanediol dehydrogenation enzyme optimal reactive temperature is as follows: in ketone reduction reaction system, (R/S)-acetoin of 10mM is a substrate, and the NADH of 0.2mM is a coenzyme, the phosphate buffered saline buffer of 100mM (pH 8.0) system; The glycol oxidation reaction system, with 100mM (2R, 3R)-2, the 3-butyleneglycol is a substrate, the NAD+ of 4mM is a coenzyme, the sodium bicarbonate buffer liquid of 100mM (pH 11.0) system.Ketone reduction reaction and glycol oxidation reaction have all been measured respectively from 40 ℃ to 80 ℃ enzyme activity.The result shows that in ketone reduction reaction and glycol oxidation reaction, the optimal reactive temperature of this enzyme all is 70 ℃.
Test three, (2R, 3R)-2, the substrate specificity of 3-butanediol dehydrogenation enzyme mensuration
Under the optimum reaction conditions of this enzyme, measured the substrate specificity of this enzyme in glycol oxidation reaction and ketone reduction reaction respectively.Concrete grammar is as follows:
1, in the glycol oxidation reaction, it is 100mM that substrate adds concentration, and 4mM NAD+ is a coenzyme, 100mM sodium bicarbonate buffer system (pH 11.0), 70 ℃ of temperature of reaction.To (2R, 3R)-2, the vigor of 3-butyleneglycol is set at 100%, and the relative energy value of other compound is as shown in table 1.
In the reaction of table 1 glycol oxidation (2R, 3R)-2, the substrate specificity measurement result of 3-butanediol dehydrogenation enzyme
" ± " represents the standard difference of three parallel tests
Above-mentioned test-results shows, in the glycol oxidation reaction optimum response substrate of this enzyme be (2R, 3R)-2, secondly the 3-butyleneglycol is meso-form meso-2, the 3-butyleneglycol is to (2S, 3s)-2, the 3-butyleneglycol is without any vigor.
2, in the ketone reduction reaction, it is 10mM that substrate adds concentration, and 0.2mM NADH is a coenzyme, 100mM phosphate buffer (pH 8.0), 70 ℃ of temperature of reaction.Vigor to (3R/3S)-acetoin is set at 100%, and the relative energy value of other test chemical is as shown in table 2.
In the table 2 ketone reduction reaction (2R, 3R)-2, the substrate specificity measurement result of 3-butanediol dehydrogenation enzyme
" ± " represents the standard difference of three parallel tests
Above-mentioned test-results shows that the best substrate of this enzyme is (3R/3S)-acetoin in the ketone reduction reaction, secondly is dimethyl diketone, and the di(2-ethylhexyl)phosphate pyruvic alcohol is not had vigor.
Embodiment 4, utilize the present invention (2R, 3R)-2,3-butanediol dehydrogenation enzyme produce (2R, 3R)-2, the 3-butyleneglycol
By the method for embodiment 2 obtain behind the purifying (2R, 3R)-2,3-butanediol dehydrogenation enzyme uses molecular sieve gel post exchange buffering liquid to be 200mM phosphate buffered saline buffer (pH 8.0) then, obtain (2R, 3R)-2,3-butanediol dehydrogenation enzyme enzyme liquid.
Obtain (2R above utilizing, 3R)-2,3-butanediol dehydrogenation enzyme, (the 3R)-acetoin of interpolation 100mM and the NADH of 100mM are as substrate, and in phosphate buffered saline buffer (pH 8.0) system of 100mM, 70 ℃ were reacted 4 hours, detection obtains 69mM's (2R, 3R)-2, the 3-butyleneglycol is far above the output of the 1mM of yeast report.
Embodiment 5, utilize contain the present invention (2R, 3R)-2, the cells produce of 3-butanediol dehydrogenation enzyme (2R, 3R)-2, the 3-butyleneglycol
To carry (2R, 3R)-2, behind 3-butanediol dehydrogenation enzyme Prokaryotic Expression carrier pET22b-2R3R transformed into escherichia coli E.coli BL21 (DE3) pLysS, obtain recombination bacillus coli E.coli RBDH, through PCR and expression checking, prove this bacterial strain have (2R, 3R)-2,3-butanediol dehydrogenation enzyme activity.
The single bacterium colony of E.coli RBDH of random choose is transferred to 5mL and contains the LB liquid nutrient medium of 100 μ g/mL Ampicillin Trihydrates and 34 μ g/mL paraxin from flat board, cultivated 12 hours for 37 ℃, 1% be forwarded in the fresh LB liquid nutrient medium that contains 100 μ g/mL Ampicillin Trihydrates and 34 μ g/mL paraxin then, 37 ℃ are cultured to thalline OD600 value and reach 0.6, the IPTG inducible protein that adds 1mM was then expressed 5 hours, collect thalline and be resuspended in the phosphate buffered saline buffer of 200mM, regulate thalline OD
600Value is 20, obtains to have that (2R, 3R)-2, the cell of 3-butanediol dehydrogenation enzyme activity is as biological catalyst.
The biological catalyst that utilization obtains above, (the 3R)-acetoin by adding 1000mM is as substrate, reaction is 12 hours under 37 ℃ of conditions, can detect obtain 871mM (2R, 3R)-2, the 3-butyleneglycol is far above the output of the 1mM of yeast report.
Embodiment 6, utilize (2R, 3R)-2,3-butanediol dehydrogenation enzyme production acetoin
With the method identical with embodiment 2 obtain (2R, 3R)-2,3-butanediol dehydrogenation enzyme, utilize this enzyme to carry out following reaction then:
A) in phosphate buffered saline buffer (pH 11.0) system of 100mM, the NADH that adds 2mM is a coenzyme, and the 100mM dimethyl diketone is as substrate, and 70 ℃ were reacted 4 hours, and obtained (3R)-acetoin (3R)-acetoin 83mM.
B) in phosphate buffered saline buffer (pH 11.0) system of 100mM, the NAD+ that adds 4mM is a coenzyme, and 100mM (2R, 3R)-2, the 3-butyleneglycol is a substrate, 70 ℃ were reacted 4 hours, and obtained (3R)-acetoin (3R)-acetoin 76mM.
C) in phosphate buffered saline buffer (pH 11.0) system of 100mM, the NAD+ that adds 4mM is a coenzyme, and the meso-2 of 100mM, 3-butyleneglycol are as substrate, and 70 ℃ were reacted 4 hours, and obtained (3S)-acetoin 56mM.
Claims (10)
1. (2R, 3R)-2,3-butanediol dehydrogenation enzyme is one of following amino acid residue sequences:
1) the SEQ ID NO:1 in the sequence table;
2) amino acid residue sequence with SEQ ID NO:1 in the sequence table passes through replacement, disappearance or the interpolation of amino-acid residue and has (2R, 3R)-2, the protein of 3-butanediol dehydrogenation enzyme or the effect of acetoin reductase enzyme, the homology of this protein and SEQ ID NO:1 reaches 80% or higher.
The coding claim 1 described (2R, 3R)-2, the gene of 3-butanediol dehydrogenation enzyme is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID NO:2 in the sequence table;
2) dna sequence dna of SEQ ID NO:1 in the code sequence tabulation;
3) protein sequence that SEQ ID NO:1 limits in coded protein sequence and the sequence table have 80% above homology and have (2R, 3R)-2, the nucleotide sequence of 3-butanediol dehydrogenation enzyme or the effect of acetoin reductase enzyme;
4) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with the SEQ ID NO:2 in the sequence table.
3. contain the described expression carrier of claim 2, transgenic cell line and host bacterium.
One kind express claim 1 described (2R, 3R)-2, the method for 3-butanediol dehydrogenation enzyme, be will contain claim 2 described (2R, 3R)-2, the recombinant expression vector of 3-butanediol dehydrogenation enzyme gene imports host cell, expression obtain (2R, 3R)-2,3-butanediol dehydrogenation enzyme.
5. expression method according to claim 4, it is characterized in that: be used for making up and contain the described (2R of claim 2,3R)-2, the carrier that sets out of the expression of recombinant e. coli carrier of 3-butanediol dehydrogenation enzyme gene is pET-22b, pET28, pET32, pQE-30, pGEX-4T-2, pBR322 or pUC18; Be the carrier that sets out with pET-22b, containing of structure is described, and (2R, 3R)-2, the coli expression carrier of 3-butanediol dehydrogenation enzyme gene is pET22b-2R3R.
A 6. production (2R, 3R)-2, the method for 3-butyleneglycol specifically may further comprise the steps:
1) described (2R 3R)-2, after the recombinant expression vector pET22b-2R3R of 3-butanediol dehydrogenation enzyme gene imports intestinal bacteria E.coli BL21 (DE3) pLysS, obtains recombination bacillus coli E.coliRBDH with claim 5;
2) the single bacterium colony of E.coli RBDH is transferred in the LB liquid nutrient medium, inducible protein is expressed under 37 ℃, 1mM IPTG condition, had (2R, 3R)-2, the cell of 3-butanediol dehydrogenation enzyme activity is as biological catalyst;
3) utilize step 2) biological catalyst that obtains, add (3R)-acetoin as substrate, 37 ℃ of reactions 12 hours down, obtain (2R, 3R)-2, the 3-butyleneglycol.
7. produce (2R for one kind, 3R)-2, the method for 3-butyleneglycol specifically comprises: utilize claim 1 or 4 described (2R, 3R)-2,3-butanediol dehydrogenation enzyme adds (3R)-acetoin as substrate and coenzyme NAD H, in the phosphate buffered liquid system of pH 8.0,70 ℃ were reacted 4 hours, obtain (2R, 3R)-2, the 3-butyleneglycol.
8. (2R, 3R)-2, the application of 3-butanediol dehydrogenation enzyme is one of following reaction:
A) with NAD+ be coenzyme, with (2R, 3R)-2,700 ℃ of 3-butanediol dehydrogenation enzymes, 11.0 times catalysis of pH (2R, 3R)-2,3-butyleneglycol preparation (R)-acetoin; Reaction conditions:
B) with NADH be coenzyme, with (2R, 3R)-2,70 ℃ of 3-butanediol dehydrogenation enzymes, pH 11.0 catalysis dimethyl diketone preparation (R)-acetoin;
C) with NAD+ be coenzyme, with (2R, 3R)-2,70 ℃ of 3-butanediol dehydrogenation enzymes, pH 11.0 catalysis meso-form meso-2,3-butyleneglycol preparation (S)-acetoin;
D) with NADH be coenzyme, with (2R, 3R)-2,70 ℃ of 3-butanediol dehydrogenation enzymes, pH 8.0 catalysis meso-form (S) acetoins prepare meso-form meso-2, the 3-butyleneglycol.
9. produce the method for (3R)-acetoin, it is characterized in that, utilize claim 1 or 4 described (2R, 3R)-2,3-butanediol dehydrogenation enzyme carries out one of following operation:
A) in phosphate buffered saline buffer (pH 11.0) system of 100mM, the NADH that adds 2mM is a coenzyme, and the 100mM dimethyl diketone is as substrate, and 70 ℃ were reacted 4 hours, and obtained (3R)-acetoin (3R)-acetoin;
B) in phosphate buffered saline buffer (pH 11.0) system of 100mM, the NAD+ that adds 4mM is a coenzyme, and 100mM (2R, 3R)-2, the 3-butyleneglycol is a substrate, 70 ℃ were reacted 4 hours, and obtained (3R)-acetoin (3R)-acetoin.
10. produce the method for (35)-acetoin, it is characterized in that, utilize claim 1 or 4 described (2R, 3R)-2,3-butanediol dehydrogenation enzyme, in phosphate buffered saline buffer (pH 11.0) system of 100mM, the NAD+ that adds 4mM is a coenzyme, the meso-2 of 100mM, and the 3-butyleneglycol is as substrate, 70 ℃ were reacted 4 hours, and obtained (3S)-acetoin.
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