CN101481681B - Method for producing D(-)-tartaric acid or salt thereof by using gene engineering bacteria - Google Patents
Method for producing D(-)-tartaric acid or salt thereof by using gene engineering bacteria Download PDFInfo
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Abstract
The invention relates to a nucleotide sequence from microorganism for coding cis-epoxy succinate hydrase, a cloning vector containing the nucleotide sequence, a recombined host bacterium obtained by transforming the nucleotide sequence and a cis-epoxy succinate hydrase amino acid sequence coded by the recombined host bacterium, and further relates to a method of using the cis-epoxy succinate hydrase to hydrolyze cis-epoxy succinate or salts thereof to prepare D(-)-tartaric acid or salts thereof.
Description
Technical field
The present invention relates to a kind of aminoacid sequence of cis-Epoxysuccinic acid hydratase, the nucleotide sequence of the described enzyme of coding, the recombinant microorganism of the described enzyme of expression, and use described enzyme and/or recombinant microorganism to be produced the method for D (-)-tartrate or its salt by cis-form epoxy succinic acid or its salt.
Background technology
D (-)-tartrate has another name called (2S, 3S)-2,3-dihydroxy butane-Isosorbide-5-Nitrae-dicarboxylic acid is natural L (+)-tartaric isoform, seldom exist at occurring in nature, it mainly is used as chirality synthetic chiral source and resolving agent at pharmaceutical industry.D (-)-tartaric demand is just in cumulative year after year, in the urgent need to improving output, to satisfy the demand of domestic and international market at present.
Up to the present, D (-)-tartaric production method mainly contains three kinds: (1) utilizes chemical resolving agent that DL-tartrate is split as L (+)-tartrate and D (-)-tartaric chemical resolution method.Such as (Chinese J Chem Eng 2002,10 (2): 244-248) realized take L (-)-α-methylbenzyl amine (L (-)-α-methylbenzyl amine) as resolving agent the separation of DL-tartrate is made L (+)-tartrate and D (-)-tartrate such as Lola.But this class chiral resolving agent is often expensive, and optical purity and the yield of enantiomorph are all lower after once splitting, and the later separation purifying process is complicated, causes its production cost high; (2) utilize peculiar microorganism that the L (+) in the DL-tartrate-tartrate exhaustion is obtained D (-)-tartaric biological Split Method.Such as the bacterial classification that utilizes aerobacter (Aerobacter sp.) of the uncensored patent application of Japan (JP application number 24490,1975) report, (the US Patent 4904589,1990 such as Sato; EP0311835B1,1993) utilize the bacterial classification (Pseudomonas sp.) of Rhodopseudomonas, the bacterial classification (Cryptococcus sp.) of genera cryptococcus, the bacterial classification (Trichosporon sp.) of Trichosporon and the bacterial classification (Klebsiella sp.) of Klebsiella, JP-63245693 (1988) utilizes the microorganisms such as bacterial classification of Rhodopseudomonas that the L (+) in the DL-tartrate-tartrate is carried out the selectivity assimilation.But the party's ratio juris determined from raw material DL-tartrate to product D (-)-tartaric theoretical yield is the highest only has 50%, thereby make the preparation cost of the method higher; (3) biotransformation method that the cis-Epoxysuccinic acid hydratase (cis-epoxysuccinate hydrolase, ESH) that utilizes Institute of Micro-biology's secreting, expressing is D (-)-tartrate or its salt with cis-form epoxy succinic acid or its salt catalytic hydrolysis.Such as (JP-08245497,1996 such as Yamagishi; Ann N YAcad Sci 1996,799:784-785) utilize pseudomonas putida Pseudomonas putida) MCI3037, (the JP-2000-295992 such as Asai, 2000) utilize Alkaligenes bacterial classification (Alcaligenes sp.) MCI3611, (the FEMS Microbiol Lett such as Li, 2007,267:214-220) utilize bacterial classification (Bordetella sp.) 1-3 of Bordetella that cis-form epoxy succinic acid is converted into D (-)-tartrate.It is generally acknowledged, biotransformation method is produced D (-)-tartrate and is had that the enzyme stereospecificity is good, enzymatic reaction fast, optical purity of products and the characteristics such as the product yield is high, separation and purification of products is simple, not only overcome the defective that aforementioned two kinds of methods exist, and significantly reduced production cost, therefore adopted the biotransformation method of ESH enzyme to prepare the developing direction that D (-)-tartrate will become D (-)-tartrate production technology.Yet because the interior intrinsic enzyme system of microbe is very complicated, the expression amount of ESH enzyme is not high, and its activity also is subject to the multiple restriction of multiple born of the same parents' internal and external factor, causes D (-)-tartrate production efficiency low.Utilize genetic engineering technique to make up the genetic engineering bacterium with ESH enzyme coding gene, realize that efficiently expressing of ESH enzyme will become biotransformation method industrial production D (-)-tartaric key.
Summary of the invention
The present invention relates to following aspect:
(1) a kind of cis-Epoxysuccinic acid hydratase polypeptide, its origin comes from the polynucleotide encoding of Bordetella (Bordetellasp.), and its molecular weight is about 32kDa;
(2) the cis-Epoxysuccinic acid hydratase polypeptide of (1), it has the highest enzyme activity under pH 6.5,40 ℃ condition, and enzyme activity reaches more than 70% of high enzymatic activity under pH 6.5-8.5,30-50 ℃ condition;
(3) the cis-Epoxysuccinic acid hydratase polypeptide of (1), its intracellular expression by a kind of recombinant microorganism bacterial strain obtains;
(4) the cis-Epoxysuccinic acid hydratase polypeptide of (1), wherein said recombinant microorganism bacterial strain is a kind of bacterium or fungi;
(5) the cis-Epoxysuccinic acid hydratase polypeptide of (1) is characterized in that, aminoacid sequence has the homology more than 50% shown in the aminoacid sequence of described polypeptide and the SEQ ID NO:7;
(6) the cis-Epoxysuccinic acid hydratase polypeptide of (5) is characterized in that, aminoacid sequence has the homology more than 70% shown in the aminoacid sequence of described polypeptide and the SEQ ID NO:7;
(7) the cis-Epoxysuccinic acid hydratase polypeptide of (6) is characterized in that, aminoacid sequence has the homology more than 90% shown in the aminoacid sequence of described polypeptide and the SEQ ID NO:7;
(8) the cis-Epoxysuccinic acid hydratase polypeptide of (1) is characterized in that described polypeptide comprises aminoacid sequence shown in all or part of SEQ ID NO:7, and has the cis-Epoxysuccinic acid hydratase activity;
The polynucleotide of the cis-Epoxysuccinic acid hydratase polypeptide of (9) (1)-(8);
(10) polynucleotide of (9), it comprises the nucleotide sequence shown in the SEQ ID NO:6;
(11) polynucleotide of (10), it is comprised of the nucleotide sequence shown in the SEQ ID NO:6;
(12) a kind of carrier, it comprises the nucleotide sequence of (9)-(11), and optional one or more regulating and controlling sequences that derive from homology or allos microorganism;
(13) a kind of recombinant microorganism bacterial strain, the cis-Epoxysuccinic acid hydratase polypeptide of its expression (1)-(8);
(14) the recombinant microorganism bacterial strain of (13), the cis-Epoxysuccinic acid hydratase polypeptide of its expression are by the polynucleotide encoding of (9)-(11);
The recombinant microorganism bacterial strain of (15) (13)-(14), its carrier with (12) transforms;
(16) method of a kind of production D (-)-tartrate or its salt, it comprises the cis-Epoxysuccinic acid hydratase polypeptide of use (1)-(8) or by cis-Epoxysuccinic acid hydratase polypeptide and cis-form epoxy succinic acid or its reactant salt of the polynucleotide encoding of (9)-(11);
(17) method of a kind of production D (-)-tartrate or its salt, it comprises the steps:
(a) the recombinant microorganism bacterial strain of cultivation (13)-(15) in suitable medium;
(b) be fixed on the solid support or be embedded in the immobilization carrier with cell, cell extract or the cell-free extract of the recombinant microorganism bacterial strain that obtains in the step (a) or by the cis-Epoxysuccinic acid hydratase of described microorganism strains purifying;
(c) prepared product that obtains in the step (b) and cis-form epoxy succinic acid or its salt are reacted to produce D (-)-tartrate or its salt under the condition that is suitable for being hydrolyzed; With
(d) randomly reuse enzyme and/or the cell of above-mentioned fixing or embedding.
Detailed Description Of The Invention
The present invention relates to a kind of nucleotide sequence be used to a kind of cis-Epoxysuccinic acid hydratase of encoding, the Bordetella (Bordetella sp.) that it derives from separation and purification is preferably the microorganism with the common micro-organisms preservation center collection CGMCC No.2075 of China Committee for Culture Collection of Microorganisms.
The nucleotide sequence (genomic dna, cDNA or RNA) of the coding cis-Epoxysuccinic acid hydratase polypeptide that the present invention relates to, have more than 50% with nucleotide sequence shown in the SEQ ID NO:6, be preferably more than 70%, more preferably the homology more than 90%.
The nucleotide sequence of the coding cis-Epoxysuccinic acid hydratase polypeptide that the present invention relates to contains the nucleotide sequence of sequence shown in whole SEQ ID NO:6 or contains the nucleotide sequence of sequence shown in the part SEQ ID NO:6.
This sequence that refers to " nucleotide sequence that contains sequence shown in the part SEQ ID NO:6 " that the present invention relates to contains and surpasses 100 Nucleotide identical with Nucleotide in the sequence shown in the SEQ ID NO:6, the albumen that also refers to its coding have to by the similar cis-Epoxysuccinic acid hydratase vigor of the albumen (being the complete amino acid sequence of SEQ IDNO:7) of sequence encoding shown in whole SEQ ID NO:6.
" nucleotide sequence that contains sequence shown in the part SEQ ID NO:6 " of the present invention the coding albumen have to by the similar cis-Epoxysuccinic acid hydratase vigor of the albumen (being SEQ ID NO:7) of sequence encoding shown in whole SEQ ID NO:6, preferably have the above cis-Epoxysuccinic acid hydratase vigor of SEQ ID NO:780%, preferably have the cis-Epoxysuccinic acid hydratase vigor of SEQ ID NO:7100%.
The invention provides a kind of recombinant nucleotide sequence, it contains the nucleotide sequence of aforementioned coding cis-Epoxysuccinic acid hydratase polypeptide, and in abutting connection with one or more, as to derive from homology or allos microorganism regulating and controlling sequences is arranged.
Regulating and controlling sequence of the present invention, it comprises in the regulating and controlling sequences such as being selected from promoter sequence, secretory signal sequence, termination signal sequence one or more.
The present invention relates to a kind of carrier that contains the nucleotide sequence of aforementioned coding cis-Epoxysuccinic acid hydratase polypeptide, it randomly also comprises one or more regulating and controlling sequences that derive from homology or allos microorganism.
" carrier " of the present invention means that all can be used for nucleotide sequence transduction or transfection are entered the biological chemistry framework of host cell.The carrier that the present invention relates to can be one or more in plasmid, virus, phagemid, liposome, the Cationic Vesicles.Can comprise one or more aforesaid regulating and controlling sequences in the above-mentioned carrier.Carrier of the present invention is preferably plasmid, as pET is serial, pGEX is serial, pQE is serial, pP
ROEX series etc.
The host cell that the present invention relates to is preferably a kind of recombinant host cell, its by nucleotide sequence of the present invention or carrier through transforming (such as Calcium Chloride Method, sal epsom method, high voltage electric perforation method etc.).
" nucleotide sequence of the present invention or the carrier host cell through being transformed ", mean that this host itself does not have nucleotide sequence or the carrier of aforesaid coding cis-Epoxysuccinic acid hydratase polypeptide, but through after transforming, so that the host is with this nucleotide sequence or carrier.
Above-mentioned Host Strains can be expressed nucleotide sequence or the carrier of aforementioned coding cis-Epoxysuccinic acid hydratase polypeptide, and produces the aminoacid sequence of foregoing nucleotide sequence or vector encoded.The foregoing nucleotide sequence that the present invention relates to can be incorporated in selected host's the genome, or is present in the aforementioned host cell with the form of episomal vector.
For the purpose of facility, the recombinant host cell that the present invention relates to comprises microorganism, is preferably bacterium and fungi, comprises yeast.Above-mentioned recombinant host cell can the high level expression cis-Epoxysuccinic acid hydratase after transforming preferably.
Above-mentioned " high level expression " is in aforementioned recombinant host cell, realizes overexpression by adjacent regulating and controlling sequence control foregoing nucleotide sequence.
The invention still further relates to the aminoacid sequence of the cis-Epoxysuccinic acid hydratase that obtains after the separation and purification, its nucleotide coding after by aforementioned separation and purification, and (or) expressed by aforementioned recombinant host cell.
Cis-Epoxysuccinic acid hydratase of the present invention has stronger temperature stability, and 50 ℃ of insulations still remain the vigor that has more than 70% after 30 minutes, and it is at 30-50 ℃, and being preferably 35-45 ℃ has higher vigor (more than 75% of the highest enzyme work).
Cis-Epoxysuccinic acid hydratase of the present invention has stronger pH stability, at pH 4.6-9.0, be preferably pH 5.0-8.0, more preferably place between the pH 6.0-7.0 and still remain the vigor that has more than 90% after 30 minutes, it is at pH 6.0-8.5, and being preferably has higher vigor (more than 70% of the highest enzyme work) between the pH 6.0-7.0.
Cis-Epoxysuccinic acid hydratase of the present invention, its molecular weight are preferably about 32kDa (theoretical value is 32.4779kDa) at 25-35kDa.
Cis-Epoxysuccinic acid hydratase aminoacid sequence of the present invention or its polypeptide by aforementioned recombinant host cell born of the same parents outer or intracellular expression and (or) secreting, expressing.
Aminoacid sequence has more than 50% shown in cis-Epoxysuccinic acid hydratase aminoacid sequence of the present invention and the SEQ ID NO:7, is preferably more than 70%, more preferably the homology more than 90%.
Cis-Epoxysuccinic acid hydratase aminoacid sequence of the present invention has all or part of (more than 50 of aminoacid sequence shown in the SEQ ID NO:7, preferred amino acid more than 100), its cis-Epoxysuccinic acid hydratase vigor is at least more than 80% of complete amino acid sequence SEQ ID NO:7, is preferably more than 95%.That is, can insert wherein with the aminoacid sequence excalation of cis-Epoxysuccinic acid hydratase of the present invention or with partial sequence, but still can keep its vigor.The about 32kDa of the molecular weight of cis-Epoxysuccinic acid hydratase of the present invention or its partial sequence or lower or higher.
The cis-Epoxysuccinic acid hydratase that the present invention relates to can be by uncontested enzyme experimental identification, such as cis-form epoxy succinic acid or its salt hydrolysis are become D (-)-tartrate or its salt.Culturing micro-organisms and produce enzyme in suitable medium.With this culture or separate microorganism cells in this culture, and survey its enzyme activity.
In suitable cultivation, induce wild-type microorganisms or reconstitution cell of the present invention to produce cis-Epoxysuccinic acid hydratase, by method such as the column chromatography of knowing, isolate the purpose cis-Epoxysuccinic acid hydratase, detect " reactive site " of enzyme amino acid sequence, and from above-mentioned wild-type microorganisms or reconstitution cell, obtain the dna sequence dna of coding cis-Epoxysuccinic acid hydratase.
The dna sequence dna of coding cis-Epoxysuccinic acid hydratase, accession number is that the sequence of E50984 obtains among the N-end sequence by the cis-Epoxysuccinic acid hydratase that is purified into from microorganism and the GenBank.
Among the present invention, accession number is the primers of E50984 among N-end sequence by the cis-Epoxysuccinic acid hydratase that is purified into and the GenBank, through round pcr, the PCR product that about 0.9kb is contained the cis-Epoxysuccinic acid hydratase encoding sequence inserts pUC
m-T carrier, and with this plasmid called after pUC
m-T-ESH.The dna sequence dna of whole Insert Fragment detects by the sequencing technologies that the those skilled in the art knows.
Design contains the primer of Nde I and BamHI restriction endonuclease sites, pass through round pcr, obtain containing the PCR product of cis-Epoxysuccinic acid hydratase encoding sequence, and with Nde I and this PCR product of BamHI digestion with restriction enzyme, and the fragment after this enzyme cut inserts the corresponding restriction enzyme site of pET22b (+) carrier place, with this plasmid called after pET22b-ESH.The dna sequence dna of whole Insert Fragment detects by the well-known to one skilled in the art sequencing technologies of art.
The above-mentioned expression vector that builds is expressed in escherichia coli host.The above-mentioned escherichia coli host that contains the pET22b-ESH plasmid is cultivated in containing corresponding antibiotic suitable culture medium (such as the LB substratum), make being present in the host cell that the pET22b-ESH plasmid can continue, then collect above-mentioned cell and measure the cis-Epoxysuccinic acid hydratase vigor.The cis-Epoxysuccinic acid hydratase gene is not limited at expression in escherichia coli among the present invention.The dna fragmentation of coding cis-Epoxysuccinic acid hydratase can very expediently bacterium or fungi, comprise in the yeast and expressing among the present invention.For said gene can be expressed in bacterium, fungi and yeast, the dna sequence dna (such as promotor, terminator, upstream activating sequence etc.) that the dna sequence dna of coding cis-Epoxysuccinic acid hydratase can be adjacent by control is so that this gene finally is able to (excessive) in selected host express.
The dna sequence dna of coding cis-Epoxysuccinic acid hydratase can make cis-Epoxysuccinic acid hydratase secretion (extracellular expression) in the host cell substratum by appropriate modification.This modification can realize by the dna sequence dna that inserts the coding homing sequence usually.This homing sequence in selected host by mechanism of secretion so that cis-Epoxysuccinic acid hydratase be able to secreting, expressing and, in host's substratum, be able to renaturation.
The invention provides the host's who expresses cis-Epoxysuccinic acid hydratase culture condition (such as substratum, temperature etc.).
The invention provides the recombinant host cell that a kind of utilization the present invention relates to or the cis-Epoxysuccinic acid hydratase aminoacid sequence that the present invention relates to and be hydrolyzed a kind of cis-form epoxy succinic acid or its salt.Cis-form epoxy succinic acid salt involved in the present invention is the salt that cis-form epoxy succinic acid and various positively charged ion form, and positively charged ion comprises and is not limited only to ammonium ion, potassium ion, sodium ion, magnesium ion and calcium ion etc.
The enzyme that the present invention relates to can be following form use: can directly utilize dialysis or the cell of dialysis culture not; This enzyme can be used as albumen or the cell extract (the part material of the host cell that namely obtains behind the centrifugal extraction step of process one or many) of purifying.The enzyme that the present invention relates to can be with above-mentioned any form and other enzyme with above-mentioned any form combined utilization.In addition, cis-Epoxysuccinic acid hydratase is secreted into behind host cell expression in the substratum outside its born of the same parents, and in substratum renaturation.That is this zymin can be used with (or not with) one or more other enzymes associatings and with the form of crude zyme preparation or the pure zymin of part (or fully).
The cis-Epoxysuccinic acid hydratase of above-mentioned cell, cell extract, cell-free extract or purifying can be fixed on the solid support by any easy method (such as chromatography column etc.) or be embedded in (as adopting carrageenin embedding etc.) in the immobilization carrier, can continue to be hydrolyzed the substrate with epoxy group(ing), and so that above-mentioned zymin (cis-Epoxysuccinic acid hydratase of cell, cell extract, cell-free extract or purifying) can reuse.
The invention provides a kind of method of utilizing genetic engineering bacterium of the present invention to produce D (-)-tartrate or its salt.In cis-form epoxy succinic acid or its salts solution, add genetically engineered mycetocyte of the present invention, under suitable temperature, carry out enzymatic reaction, obtain D (-)-tartrate or its salt.Wherein, the volumetric molar concentration of cis-form epoxy succinic acid or its salt is 0.1～2mol/L, is preferably 0.5～1.5mol/L, more preferably 0.8～1.2mol/L; Temperature of reaction is 10～50 ℃, is preferably 25～40 ℃, more preferably 32～40 ℃; The pH value of cis-form epoxy succinic acid or its salts solution is 4.5～9.5, is preferably 6.0～8.5, more preferably 6.0～7.0.
The invention provides a kind of immobilized cell technique that utilizes and prepare genetic engineering bacterium immobilized cell of the present invention, and for the production of the method for D (-)-tartrate or its salt.Wherein, the volumetric molar concentration of cis-form epoxy succinic acid or its salt is 0.1～2mol/L, is preferably 0.5～1.5mol/L, more preferably 0.8～1.2mol/L; Temperature of reaction is 10～50 ℃, is preferably 25～40 ℃, more preferably 32～40 ℃; The pH value of cis-form epoxy succinic acid or its salts solution is 4.5～9.5, is preferably 6.0～8.5, more preferably 6.0～7.0.
Mode below by embodiment further specifies the present invention.The following examples only are for illustrative purposes, and are not to limit the scope of the invention.
Embodiment
The separation and purification of embodiment 1 cis-Epoxysuccinic acid hydratase
1. the separation and purification of cis-Epoxysuccinic acid hydratase activity
Bordetella BK-52 (Bordetella sp.BK-52) is stored in the BK-1 slant medium, and this culture medium prescription is: 0.5% glucose, and 0.5% peptone, 0.5% sodium-chlor, 0.25% yeast extract paste, 2% agar, pH 7.0.From slant medium inoculation Bordetella BK-52 to BK-2 seed culture medium, behind 30 ℃ of shaking culture 24h, transfer in the BK-3 fermention medium, induce 36h with 30 ℃ of vibrations of cis-form epoxy succinic acid salt, make it produce cis-Epoxysuccinic acid hydratase.This BK-2 seed culture based formulas is: 0.2% yeast extract, 1% glucose, 0.2% ammonium sulfate, 0.1% 3 water dipotassium hydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.001% iron vitriol, pH7.0.This BK-3 fermentative medium formula is: 1% cis-form epoxy succinic acid sodium, 0.2% yeast extract, 1% glucose, 0.2% ammonium sulfate, 0.1% 3 water dipotassium hydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.001% iron vitriol, pH7.0.Centrifugal collecting cell also saves backup in-20 ℃.
Above-mentioned freezing cell is suspended with the 0.01mol/L potassium phosphate buffer, ultrasonic 20min in ice bath, the centrifugal 30min of 8000rpm collects supernatant liquor 1.Add in the ice bath ammonium sulfate in supernatant liquor 1 to 50% saturation ratio, the centrifugal 20min of 10000rpm collects supernatant liquor 2.Then after in supernatant liquor 2, adding ammonium sulfate to 80% saturation ratio, 4 ℃ of hold over night, the centrifugal 20min of 10000rpm, collecting precipitation.Precipitation is with the dissolving of the 0.1mol/L potassium phosphate buffer of precooling, 4 ℃ of 48h that in the 0.01mol/L potassium phosphate buffer, dialyse, during change 3-4 dialyzate.Enzyme liquid after the dialysis is concentrated with PEG20000, and concentrated solution is crossed Phenyl-Sepharose post (2.6cm i.d. * 50cm), and with 0.01mol/L potassium phosphate buffer (pH8.0) wash-out.Use first the uncombined albumen of elutriant flush away of 500mL, then contain the albumen of the potassium phosphate buffer gradient elution combination of 0-0.35mol/L sodium-chlor with 1000mL, flow velocity is 1mL/min.Collect the effluent liquid of tool enzymic activity, PEG20000 is concentrated, and concentrated solution is crossed the Q-Sepharose post, and (2.6cm i.d. * 50cm), with 0.01mol/L potassium phosphate buffer (pH8.0) wash-out, flow velocity is 1mL/min.Collect the effluent liquid of tool enzymic activity, and concentrated with PEG20000, concentrated solution is crossed MonoQHR 5/5 post, and with 0.01mol/L potassium phosphate buffer (pH8.0) wash-out, flow velocity is 0.2mL/min.Collect the effluent liquid of tool enzymic activity, and concentrated with PEG20000.This process triplicate is got the collection liquid behind the part triplicate, runs 12% SDS-PAGE, and with coomassie brilliant blue R250 dyeing, observes unicity and the molecular weight thereof of protein band.
The result shows that the band in SDS-PAGE glue of the cis-Epoxysuccinic acid hydratase behind the purifying is single, and its molecular weight is about 32kDa.
2. the detection of cis-Epoxysuccinic acid hydratase activity
Get about 1.0g wet cell, physiological saline washes twice, and the cis-form epoxy succinic acid sodium (pH8.0) of using 10ml 1mol/L is that substrate suspends, behind 37 ℃ of reaction 1h, and the content of assaying reaction liquid unresolvable tartaric acid.
Enzyme unit definition alive is under above-mentioned reaction conditions, and 1.0g wet cell 1h produces the required enzyme amount of 1 μ mol tartrate.
The detection method of reaction solution unresolvable tartaric acid content is as follows: get the ammonium meta-vanadate of 2.5ml1% in the volumetric flask of a 25ml, after adding an amount of above-mentioned reaction solution, the sulfuric acid that adds again 1ml lmol/L, be settled to 25ml with distilled water, survey the light absorption value at 480nm place behind the mixing, and calculate the content of reaction solution unresolvable tartaric acid according to the typical curve of formulating.
Vigor for cis-Epoxysuccinic acid hydratase in the Quantitative detection lyoenzyme liquid, its detection method is as follows: the cis-form epoxy succinic acid sodium substrate that adds 1.0ml 1mol/L in 3.9ml 0.1mol/L potassium phosphate buffer (pH8.0), behind 37 ℃ of insulation 5min, add 0.1ml enzyme liquid and react 20min at 37 ℃, get an amount of above-mentioned reaction solution, measure wherein tartaric content.
The detection of embodiment 2 cis-Epoxysuccinic acid hydratase N-terminal aminoacid sequences
Cis-Epoxysuccinic acid hydratase N-terminal aminoacid sequence detects by method commonly used, its method is as follows: after the cis-Epoxysuccinic acid hydratase that obtains after the above-mentioned separation and purification is run SDS-PAGE, by shifting on the pvdf membrane after the Western hybridization.This film coomassie brilliant blue staining, the band that the cutting cis-Epoxysuccinic acid hydratase is corresponding also reclaims, and then uses protein sequencing instrument (Applied Biosystems Procise 492 cLC) to survey its N-terminal sequence.Sequencing result shows that its N-terminal aminoacid sequence is MTRTKLILEARI (SEQ ID NO:1).
The acquisition of embodiment 3 cis-Epoxysuccinic acid hydratase genes
Be the following primer of sequences Design of E50984: 5 '-ATGACNYGNACNAARXTN-3 ' (SEQ ID NO:2) and 5 '-CTAGTTGCTAATACCCAG-3 ' (SEQ ID NO:3) according to accession number among above-mentioned cis-Epoxysuccinic acid hydratase N-terminal sequence (SEQ ID NO:1) and the GenBank, wherein Y represents A or C, R represents A or G, X represents T or C, and N represents any one of A, C, G, four kinds of bases of T.
30 ℃ of overnight incubation of Bordetella BK-52, centrifugal collecting cell is with the genomic dna of EZ-10 pillar genome DNA extraction test kit (Bio Basic Inc.) extraction Bordetella BK-52.Take genomic dna as template, SEQ ID NO:2 and SEQ ID NO:3 are primer, obtain the fragment of one section about 0.9kb by round pcr commonly used.Its PCR program is: behind 94 ℃ of preheating 5min, and 94 ℃ of 50s, 40 ℃ of 30s, 72 ℃ of 1min, 30 circulations, last 72 ℃ are extended 10min.After the PCR product reclaims test kit (Promega) recovery purifying with Wizard PCR fragment, with pUC
m-T (Sangon) carrier connects, and according to Chung (Proc Natl Acad Sci USA 1989,86:2172-2175) etc. people's method transforms bacillus coli DH 5 alpha, screening positive clone, and order-checking (Takara Biotech), cloned plasmids called after pUC
m-T.
Embodiment 4 cis-Epoxysuccinic acid hydratase gene clonings
According to above-mentioned sequencing result design primer, be respectively 5 ' GC
CATATGATGACTCGAACCAAGTTGATACT-3 ' (SEQ ID NO:4) and 5 ' CC
GGATCCTTAGTTGCTAATACCCAGAATTT-3 ' (SEQ ID NO:5), underscore partly is respectively restriction endonuclease sites Nde I and BamHI.Take Bordetella BK-52 genomic dna as template, take SEQ ID NO:4 and SEQ ID NO:5 as primer, the open reading frame of pcr amplification cis-Epoxysuccinic acid hydratase gene.Its PCR program is: behind 94 ℃ of preheating 5min, and 94 ℃ of 50s, 57 ℃ of 30s, 72 ℃ of 1min, 30 circulations, last 72 ℃ are extended 10min.After the PCR product reclaims test kit (Promega) recovery purifying with Wizard PCR fragment, cut with Nde I and BamH I enzyme, and be connected with the appropriate site place of pET22b (+), be transformed in the e. coli bl21 according to the method among the embodiment 3, screening positive clone, the performing PCR of going forward side by side, double digestion and order-checking are identified.
Qualification result shows, the cis-Epoxysuccinic acid hydratase gene to be successfully being cloned in pET22b (+) carrier, and with this expression vector called after pET22b-ESH, this genetic engineering bacterium called after E.coliBL21-pET22b-ESH.
The feature of embodiment 5 cis-Epoxysuccinic acid hydratase genes
DNAMEN software (Lynnon Biosoft) analysis shows that it is the open reading frame of 885bp that the fragment of insertion pET22b (+) carrier has a length, is the DNA sequences encoding (SEQ ID NO:6) of cis-Epoxysuccinic acid hydratase gene.Cis-Epoxysuccinic acid hydratase gene 294 amino acid of encoding altogether, the molecular weight of prediction is 32.4779kDa, its aminoacid sequence is seen SEQ ID NO:7.The aminoacid sequence of 1-12 among the corresponding SEQ ID of sequence in the examples of implementation 2 NO:7.
Embodiment 6 cis-Epoxysuccinic acid hydratase gene Expression in Escherichia colis
The e. coli bl21 37 ℃ of shaking culture in the LB substratum that contains 100 μ g/ml penbritins that contain the pET22b-ESH plasmid are spent the night.E. coli bl21 only to contain pET22b (+) carrier without any extraneous nucleotide Insert Fragment is contrast.
According to the measuring method of cis-Epoxysuccinic acid hydratase activity among the embodiment 1, the contrast bacterium does not have the cis-Epoxysuccinic acid hydratase vigor, and the above-mentioned engineering bacteria enzyme activity that contains the cis-Epoxysuccinic acid hydratase gene is 25800U/g.
Embodiment 7 utilizes E.coli BL21-pET22b-ESH engineering bacteria of the present invention to prepare D (-)-tartrate
In the LB substratum (containing 100 μ g/ml penbritins) of E.coli BL21-pET22b-ESH engineering bacteria in 100ml embodiment 6 behind 37 ℃ of shaking culture 8h, in the access 1L LB substratum, 37 ℃ of lower aerated culture 10h.Then in nutrient solution, add 10g cis-form epoxy succinic acid disodium, after 12h shaking culture and reaction, add CaCl
2The aqueous solution filters and water washing and precipitating, obtains 13.5g four water calcium tartrates, and, crystallization refining, concentrated through sulfuric acid solution, cation and anion exchange post and oven dry obtain D (-)-tartrate 6.3g again.This D (-)-tartaric specific rotatory power is [α] by analysis
D 20=-12.1 °, content is 99.7%.
Embodiment 8 utilizes kappa-carrageenan embedding E.coli BL21-PET15b/ESH of the present invention engineering bacteria cell to prepare D (-)-tartrate
With the medium centrifugal of embodiment 7 and collect the engineering bacteria cell, with being fixed of kappa-carrageenan embedding cell.Get above-mentioned immobilized cell 10g and put into 30ml 1.0M cis-form epoxy succinic acid two sodium solutions, behind 37 ℃ of reaction 24h, the filtered and recycled immobilized cell.Add CaCl in the filtered liquid
2The aqueous solution filters after fully stirring, and washing precipitation gets 4.6g four water D (-)-calcium tartrates.With this calcium tartrate add that sulfuric acid solution, cation and anion exchange post are refining, concentrated, crystallization and oven dry obtain D (-)-tartrate 2.1g.
The immobilized cell that reclaims is put into 30ml 1.0M cis-form epoxy succinic acid two sodium solutions again, behind 37 ℃ of reaction 24h, the filtered and recycled immobilized cell.Add CaCl in the filtered liquid
2The aqueous solution filters after fully stirring, and washing precipitation gets 4.9g four water D (-)-calcium tartrates.With this calcium tartrate add that sulfuric acid solution, cation and anion exchange post are refining, concentrated, crystallization and oven dry obtain D (-)-tartrate 2.4g.
This immobilized cell can Reusability.
In summary, to have cis-form epoxy succinic acid or its salt hydrolysis be the characteristic of D (-)-tartrate or its salt to the genetic engineering bacterium that makes up of the present invention.
The feature of embodiment 9 cis-Epoxysuccinic acid hydratases
1. temperature is on the stable and active impact of cis-Epoxysuccinic acid hydratase
With the cis-Epoxysuccinic acid hydratase after the separation and purification among the embodiment 1, under differing temps, according to quick enzyme activity determination method among the embodiment 1, detected temperatures is on the impact of this enzymic activity.The result shows that cis-Epoxysuccinic acid hydratase is at 30-50 ℃, and being preferably 35-45 ℃ has higher vigor (more than 75% of the highest enzyme work).
With the cis-Epoxysuccinic acid hydratase after the separation and purification among the embodiment 1, behind the insulation 30min, according to quick enzyme activity determination method among the embodiment 1, detected temperatures is on the impact of this enzyme stability under differing temps.The result shows that cis-Epoxysuccinic acid hydratase has stronger temperature stability, surplus vigor more than 70% still behind 50 ℃ of insulation 30min.
2.pH on the stable and active impact of cis-Epoxysuccinic acid hydratase
With the cis-Epoxysuccinic acid hydratase after the separation and purification among the embodiment 1, under different pH, according to quick enzyme activity determination method among the embodiment 1, detect pH to the impact of this enzymic activity.The result shows that cis-Epoxysuccinic acid hydratase is at pH 6-8.5, and being preferably has higher vigor (more than 70% of the highest enzyme work) between the pH 6-7.
With the cis-Epoxysuccinic acid hydratase after the separation and purification among the embodiment 1, after room temperature leaves standstill 30min under the different pH, according to quick enzyme activity determination method among the embodiment 1, detect pH to the impact of this enzyme stability.The result shows that cis-Epoxysuccinic acid hydratase is preferably pH 5-8 at pH 4.6-9.0, more preferably still remains the vigor more than 90% between the pH 6-7 behind the placement 30min.
3. the dynamic characteristic of cis-Epoxysuccinic acid hydratase
Get the cis-Epoxysuccinic acid hydratase of purifying among an amount of embodiment 1, by continuous increase substrate (cis-form epoxy succinic acid sodium) concentration (12mmol/L-200mmol/L), at 37 ℃ of reactions of 0.1mol/L potassium phosphate buffer (pH6.5) 20min, calculate the K of cis-Epoxysuccinic acid hydratase by Lineweaver-Burk figure
mValue and V
MaxValue.The result shows that cis-Epoxysuccinic acid hydratase has the characteristic feature of Michaelis enzyme, and K
mValue and V
MaxValue is respectively 18.67mmol/L and 94.34mM/min/mg.
Although represented with reference to definite preferred embodiment of the present invention and described the present invention, but the one of ordinary skilled in the art will be appreciated that and can be under the prerequisite that does not deviate from the aim of the present invention that limited by appended claims and scope the present invention be carried out modification on various forms and the details.
Sequence table
Claims (4)
1. recombinant escherichia coli strain, it is characterized in that described recombinant escherichia coli strain obtains in the following way: take Bordetella BK-52 genomic dna as template, take SEQ ID NO:4 and SEQ ID NO:5 as primer, the open reading frame of pcr amplification cis-Epoxysuccinic acid hydratase gene, after the PCR product reclaims test kit recovery purifying with Wizard PCR fragment, cut with Nde I and BamHI enzyme, and be connected with the appropriate site place of pET22b (+), be transformed into afterwards in the e. coli bl21, described cis-Epoxysuccinic acid hydratase polypeptide is comprised of the aminoacid sequence shown in the SEQ ID NO:7.
2. the recombinant escherichia coli strain of claim 1, the cis-Epoxysuccinic acid hydratase polypeptide of its expression are nucleotide sequence coded by shown in the SEQ ID NO:6.
3. method of producing D (-)-tartrate or its salt, it comprises that right to use requires each described recombinant escherichia coli strain of 1-2 and cis-form epoxy succinic acid or its reactant salt.
4. method of producing D (-)-tartrate or its salt, it comprises the steps:
(a) in suitable medium, cultivate each recombinant escherichia coli strain of claim 1-2;
(b) be fixed on the solid support or be embedded in the immobilization carrier with cell, cell extract or the cell-free extract of the recombinant escherichia coli strain that obtains in the step (a) or by the cis-Epoxysuccinic acid hydratase of described recombinant escherichia coli strain purifying;
(c) prepared product that obtains in the step (b) and cis-form epoxy succinic acid or its salt are reacted to produce D (-)-tartrate or its salt under the condition that is suitable for being hydrolyzed; With
(d) randomly reuse enzyme and/or the cell of above-mentioned fixing or embedding.
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| CN102703419A (en) * | 2012-05-21 | 2012-10-03 | 中国科学院青岛生物能源与过程研究所 | Cellulose immobilized cis epoxy succinic acid hydrolase and method for preparing tartaric acid |
| CN103614398B (en) * | 2013-08-22 | 2016-01-27 | 杭州宝晶生物股份有限公司 | The encoding gene of cis-Epoxysuccinic acid hydratase, the polypeptide of its coding and related application thereof |
| CN105861463B (en) * | 2016-04-12 | 2019-08-30 | 南京工业大学 | epoxy succinic acid hydrolase and carrier and application thereof |
| CN106434713A (en) * | 2016-11-22 | 2017-02-22 | 常茂生物化学工程股份有限公司 | Epoxysuccinic acid hydrolase encoding gene and application thereof |
| CN106497844B (en) * | 2016-12-12 | 2019-05-21 | 常茂生物化学工程股份有限公司 | One plant of genetic engineering bacterium for producing L-TARTARIC ACID and its construction method and application |
| CN110468115B (en) * | 2019-07-22 | 2021-07-13 | 浙江科技学院 | Aspergillus niger cis-epoxy succinate hydrolase gene and application thereof |
| CN111088198B (en) * | 2020-01-21 | 2021-03-30 | 杭州宝晶生物股份有限公司 | Pseudomonas fuscogongensis, application thereof and method for catalytically synthesizing L (+) -tartaric acid or salt thereof |
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