CN104673814A - L-threonine aldolase from enterobacter cloacae and application thereof - Google Patents
L-threonine aldolase from enterobacter cloacae and application thereof Download PDFInfo
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- CN104673814A CN104673814A CN201510089583.4A CN201510089583A CN104673814A CN 104673814 A CN104673814 A CN 104673814A CN 201510089583 A CN201510089583 A CN 201510089583A CN 104673814 A CN104673814 A CN 104673814A
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Abstract
The invention relates to a nucleotide sequence for coding L-threonine aldolase separated from the enterobacter cloacae. The nucleotide sequence is a nucleotide sequence shown in SEQ ID NO:1, or a fragment, an analogue and a derivative of the nucleotide sequence. The nucleotide sequence for coding L-threonine aldolase is connected with an exogenous regulation sequence; and a carrier for functional expression, a cellular organism containing the carrier and a descendant of the organism are contained. A method for preparing the L-beta-hydroxy alpha-amino acid or D-beta-hydroxy alpha-amino acid from the nucleotide sequence, or polypeptide sequence, or cellular organism containing the carrier and descendant of the organism is provided.
Description
Technical field
The invention belongs to field of chemical engineering and enzyme engineering field, relating to the L-threonine zymohexase that one comes from enterobacter cloacae (Enterobacter cloacae tzyx2), is specifically nucleotide sequence and the application thereof of the L-threonine zymohexase of enterobacter cloacae.
Background technology
Zymohexase is the reversible lyase of a class, in 1934 first by the mankind cognition.Acid contracting enzyme catalysis donor (nucleophilic reagent, normally ketone) and acceptor aldehyde (electrophilic reagent) can carry out reversible aldol reaction.Most of acid contracting enzyme is very single-minded for their donor, and has larger degree of freedom for acceptor aldehyde.According to the difference of zymohexase to donor specificity, zymohexase can be divided into five large classes: di(2-ethylhexyl)phosphate pyruvic alcohol (DHAP) dependent form zymohexase, pyruvic acid and phosphoenolpyruvate dependent form zymohexase, acetaldehyde dependent form zymohexase, glycine dependent form zymohexase and dihydroxyl benzylacetone dependent form zymohexase.
Threonine aldolase (Threonine Aldolase, TA; EC4.1.2.5) belong to glycine dependent form zymohexase, the maximum feature of the type zymohexase is at cofactor pyridoxal phosphate (Pyridoxal phosphate; PLP), under existence, using amino acid as donor, after reaction, beta-hydroxy-alpha-amino acid can be generated.Threonine aldolase catalysis Threonine is cracked into glycine and acetaldehyde, and it also can catalysis glycine and acetaldehyde synthesis Threonine under certain condition.Research shows, this enzyme often demonstrates substrate tolerance very widely for its aldehyde acceptor, comprises aromatic aldehyde and the alkanoic of various replacement, therefore, threonine aldolase started to receive publicity in recent years, and people attempt to expand the application of this enzyme in synthesis, promoted the combination of chemistry and enzyme engineering.
In the beta-hydroxy-alpha-amino acid that the catalysis of threonine aldolase institute generates, comprise the key precursor of many microbiotic such as vancomycin, cyclosporin A and polyoxin D and immunosuppressor.In chemical synthesis process, the synthesis of the type beta-hydroxy-alpha-amino acid needs multi-step process to carry out separating isomers, complex process, and cost is high, and environmental pollution is serious.And utilize threonine aldolase can catalyze and synthesize the different beta-hydroxy a-amino acids of particular configuration specifically; it is without any need for protecting group, also without any need for unnecessary step, does not also produce by product; synthesis beta-hydroxy a-amino acid is the most direct beyond doubt, the most attracting mode.
According to the stereospecificity to alpha-carbon atom on acted on Threonine, this enzyme can be divided into L-type and D type two class.L-threonine zymohexase (L-TA), can be used as the resolving agent of synthesis D-β hydroxyl alpha amino acid.Can also with glycine and aldehydes for substrate one step enzyme method synthesis L-beta-hydroxy a-amino acid.
Summary of the invention
An object of the present invention is to provide the fragment of the nucleotide sequence of the encoding L-threonine zymohexase be separated from enterobacter cloacae or its nucleotide sequence, analogue or derivative.The L-threonine aldolase gene of clone from the shaft-like gram negative bacterium-enterobacter cloacae of one (Enterobacter cloacae tzyx2), this gene is connected from different expression vector, be transferred in bacterium, yeast, plant or animal, utilize its encoding L-threonine zymohexase to produce or split the methods and applications of L-beta-hydroxy a-amino acid.
Another object of the present invention is to provide L-threonine aldolase polypeptide coded by this nucleotide sequence or its fragment, analogue or derivative.
Another object of the present invention is to provide and is connected with heterologous regulatory sequence containing this gene nucleotide series, carries out the recombinant vectors of functional expression.
Another object of the present invention is to provide a kind of recombinant vectors be connected with heterologous regulatory sequence containing this gene nucleotide series or this gene nucleotide series and transforms or the host cell of transduceing and offspring thereof.
Another object of the present invention is to provide and a kind ofly transforms or the host cell of transduceing and progeny cell thereof with containing recombinant vectors that this gene nucleotide series or this gene nucleotide series be connected with heterologous regulatory sequence, or the L-threonine aldolase polypeptide coded by this nucleotide sequence produces or splits the method for L-beta-hydroxy a-amino acid.
A first aspect of the present invention, provides the fragment with the nucleotide sequence shown in SEQ ID NO:1 or this nucleotide sequence.The nucleotide sequence of the L-threonine aldolase gene be separated, it comprises a nucleotide sequence, and this nucleotide sequence is identical with a kind of nucleotide sequence being selected from lower group: (1) has the nucleotide sequence of the active polypeptide of SEQ ID NO:2 aminoacid sequence of encoding; (2) complementary with nucleotide sequence (1) nucleotide sequence, or the aforementioned nucleotide sequence having at least 65% homogeny.
In a second aspect of the present invention, provide the polypeptide coded by this nucleotide sequence of separation, this polypeptide comprises: polypeptide or its fragment with SEQ ID NO:2 aminoacid sequence.
A third aspect of the present invention, provides the plasmid expression vector containing above-mentioned nucleotide sequence, and the host cell transformed by above-mentioned nucleotide sequence or plasmid expression vector and progeny cell thereof.
A fourth aspect of the present invention, provides and a kind ofly to transform or transduce host cells and progeny cell thereof or produce with the L-threonine aldolase polypeptide coded by this nucleotide sequence or split the method for L-beta-hydroxy a-amino acid with containing plasmid expression vector that this gene nucleotide series or this gene nucleotide series be connected with heterologous regulatory sequence.
Other aspects of the present invention disclose due to this paper technology, are apparent to those skilled in the art.
As used in the present invention, " separation " refer to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).Such as, the nucleotide sequence under the native state in active somatic cell and polypeptide do not have separation and purification, but same nucleotide sequence or polypeptide as separated from native state with in other material existed, be then separation and purification.
As used herein, " nucleotide sequence of separation " refers to and is substantially free of natural other albumen relative, lipid, carbohydrate or other material.Those skilled in the art can use the DNA purification techniques of standard.
The invention provides a kind of nucleotide sequence of separation newly---the nucleotide sequence of the L-threonine zymohexase of coding enterobacter cloacae Enterobacter cloacae tzyx2, it is made up of the nucleotide sequence shown in SEQ ID NO:1 substantially, it is characterized in that: this sequence is long is 1002bp (base), is the open reading frame of encoding L-threonine zymohexase mature polypeptide SEQ ID NO:2.
The invention provides the nucleotide sequence of separation, the nucleotide sequence that this nucleotide sequence has the active polypeptide of SEQ ID NO:2 aminoacid sequence by coding forms.Particularly, nucleotide sequence of the present invention has the nucleotide sequence of SEQ ID NO:1.The nucleotide sequence of coding SEQ ID NO:2 active polypeptide comprises: the encoding sequence only having mature polypeptide; The encoding sequence of mature polypeptide and various additional coding sequence; The encoding sequence (with optional additional coding sequence) of mature polypeptide and non-coding sequence.
Polypeptide in the present invention and nucleotide sequence preferably provide with the form be separated, and are more preferably purified to homogeneous.
In the present invention, specific nucleotide sequence can obtain by multiple method.Such as, with hybridization technique isolated nucleic acid sequence well known in the art.These technology including, but not limited to: (1) hybridizes the nucleotide sequence to detect homology with probe and genome or cDNA library; (2) antibody screening of expression library is to detect the nucleotide sequence fragment of the clone with structural features.
Sequence dna fragment of the present invention also can obtain by following method: (1) is separated double chain DNA sequence from genomic dna; (2) chemical synthesising DNA sequence is to obtain the double-stranded DNA of described polypeptide.
Present invention also offers a kind of new peptide sequence, the aminoacid sequence of enterobacter cloacae L-threonine zymohexase, be made up of the aminoacid sequence shown in SEQ ID NO:2.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, improvement on synthesis, preferred recombinant polypeptide.Polypeptide of the present invention can be native purified product, or the product of chemosynthesis, or uses recombinant technology (such as, to produce in bacterium, yeast, higher plant, insect and mammalian cell from protokaryon or eucaryon host.
Can with method well-known to those having ordinary skill in the art build containing coding enterobacter cloacae L-threonine zymohexase nucleotide sequence and suitable to transcribe the/expression vector of translational control element.These methods comprise [Sambroook, et al., the Molecular Cloning such as recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology, a Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989].The nucleotide sequence of described coding enterobacter cloacae L-threonine zymohexase can be effectively connected in the appropriate promotor of expression vector, synthesizes to instruct mRNA.
The invention still further relates to the recombinant vectors of the nucleotide sequence of enterobacter cloacae L-threonine zymohexase of encoding containing the present invention or directly with the host cell that the nucleotide sequence of coding enterobacter cloacae L-threonine zymohexase produces through genetically engineered.In the present invention, the nucleotide sequence of coding enterobacter cloacae L-threonine zymohexase or the recombinant vectors containing this nucleotide sequence can be transformed or transduced into host cell, to form the genetically engineered host cell containing this nucleotide sequence or recombinant vectors.
Can be undertaken by method well-known to those having ordinary skill in the art with nucleotide sequence of the present invention or the recombinant vectors transformed host cell containing nucleotide sequence.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can collect thalline in exponential phase of growth, uses CaCl
2method process, step used is well known in the art.Also MgCl can be used
2, the methods such as electroporation are carried out.When host is eukaryote, the methods such as DNA infection protocol, microinjection, electroporation, liposome packaging can be selected.
The invention still further relates to and produce with above-mentioned genetically modified host cell, according to host cell, the method growth in common knowledge with those skilled in the art or cultivation.Such as microorganism cells is normally at 0-100 DEG C, and preferred 10-60 DEG C, also wants oxygen simultaneously.Containing carbon source in substratum, as glucose, nitrogenous source, the normally form of organonitrogen, as yeast extract, amino acid, or salt, as ammonium sulfate, trace element, as iron, magnesium salts, also has VITAMIN if necessary.The pH of substratum can keep fixing value during this period, in other words, carries out in the training period controlling or not controlling.Cultivation can batch culture, half discontinuous cultivation or cultured continuously form be carried out.After culturing, collecting cell, smashs to pieces or directly uses, and extracts L-threonine zymohexase by method well known to those skilled in the art from cell.
The invention still further relates to the method utilizing L-threonine aldolase polypeptide to produce L-beta-hydroxy a-amino acid, the method is achieved in that cultivates glycine with aldehydes together with SEQ ID NO:2.Under certain environmental conditions, be preferably temperature 10-50 DEG C, pH6-10, corresponding L-beta-hydroxy a-amino acid (as shown in Figure 1) can be catalyzed and synthesized.
The invention still further relates to the method utilizing L-threonine aldolase polypeptide to split L-beta-hydroxy a-amino acid, the method is achieved in that DL-beta-hydroxy a-amino acid is cultivated together with SEQ ID NO:2.Under certain environmental conditions, be preferably temperature 10-50 DEG C, pH6-10, correspondingly glycine and aldehydes can be split into by catalysis L-beta-hydroxy a-amino acid, and then obtain the pure D-beta-hydroxy a-amino acid (as shown in Figure 1) of mapping.
The invention still further relates to and prepare L-beta-hydroxy a-amino acid or D-beta-hydroxy a-amino acid with aforesaid method, and be applied to produce human food prods, animal-feed, makeup or pharmaceutical product use.
Classification And Nomenclature: enterobacter cloacae tzyx2
Latin literary fame: Enterobacter cloacae tzyx2
Depositary institution: China typical culture collection center
Address: Wuhan, China Wuhan University
Preservation date: on June 24th, 2012
Deposit number: CCTCC NO:M 2012240.
Accompanying drawing explanation
Fig. 1, enterobacter cloacae (Enterobacter cloacae tzyx2) L-threonine zymohexase thick Enzyme catalyzed synthesis L-beta-hydroxy a-amino acid react.
The intestinal bacteria sequencing vector pGEMT-LTA of Fig. 2, structure.
The coli expression carrier pET11-LTA of Fig. 3, structure.
Fig. 4, enterobacter cloacae (Enterobacter cloacae tzyx2) L-threonine zymohexase thick Enzyme catalyzed synthesis L-Phenserine react.
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.These embodiments should be understood only for illustration of the present invention instead of limit the scope of the invention.
Embodiment 1 is separated the nucleotide sequence of L-threonine zymohexase from enterobacter cloacae
From the cultivation enterobacter cloacae thalline of 36 hours, extract total DNA according to the method that " Molecular Cloning: A Laboratory guide " provides, getting 7 μ g is that template carries out polymerase chain reaction.According to delivered L-threonine zymohexase sequences Design, with the DNA of said extracted for template is in the enterprising performing PCR amplification of T-Gradient PCR instrument (Biometra company), reaction the primer, component and amplification condition is as follows:
Primer 1:5 '-ATGATTGATTTACGCAGTGATACCG-3 '
Primer 2: 5 '-TTAACGCTGTAAAAACGCCTGCCAG-3 '
Amplification condition: 94 DEG C of sex change 3min, then use 94 DEG C of 1min, 58 DEG C of 1min, 72 DEG C of 1min carry out 30 circulations, last 72 DEG C of 10min.Agarose gel electrophoresis detected result shows, amplification obtains the fragment that size is about 1000bp, reclaim with UNIQ-10 pillar PCR primer purification kit (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's product), recovery fragment is subcloned in sequencing vector pGEM-T (Promega Products); Plasmid construction the results are shown in accompanying drawing 2, the constructed plasmid called after pGEMT-LTA containing L-threonine aldolase gene.Connect product conversion to using CaCl
2the bacillus coli DH 5 alpha of method process, overnight incubation on the LB solid medium containing penbritin (final concentration is 100 μ g/ml); The white colony of picking grow on plates, access is containing overnight incubation in the LB liquid nutrient medium of penbritin (final concentration is 60 μ g/ml), collected by centrifugation thalline presses alkaline lysis [Sambroook, et al., 1989, Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory, New York, p19-21] extract plasmid, identify correct through NcoI and SacI double digestion and pcr amplification, order-checking (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).Sequencing result display the clip size obtained that increases be 1002bp, in Genbank database, Blast program (Basic local Alignment seatch tool) [Altschul SF et al is used by the aminoacid sequence of its coding, 1997, Nucleic Acids Res.25:3389-3402] carry out Homology search, result for retrieval shows that the homologous fragment the most similar to this fragment is L-threonine zymohexase, but and incomplete same, prove that institute's amplified fragments is new L-threonine zymohexase.
Embodiment 2: the structure of Recombinant protein expression carrier
Coding region sequence according to SEQ ID NO:1, design one pair of genes specificity amplification primer and be separated its potential open reading frame sequence:
Primer 3:5 '-GGCTCTAGA ATGATTGATTTACGCAGTGATACCG-3 ';
Primer 4:5 '-GCCGGATCC TTAACGCTGTAAAAACGCCTGCCAG-3 ';
The 5` of these two primers holds black matrix respectively containing Xba I and BamH I restriction enzyme site.Amplification condition used and reactive component the same, the display of the sequencing result of amplified production is consistent with the sequence shown in SEQ ID NO:1.Then get 50 μ l PCR primer and 1 μ l pET11a (Invitrogen company) carries out double digestion respectively, reclaim enzyme and cut large fragment, and connect 4 degree of refrigerator overnight with T4 ligase enzyme.Connect product conversion bacillus coli DH 5 alpha, by plasmid extraction and PCR screening positive clone, and carry out order-checking qualification.Plasmid construction the results are shown in accompanying drawing 3, the constructed colibacillus expression plasmid called after pET-LTA containing L-threonine aldolase gene.Connect product conversion bacillus coli DH 5 alpha, Screening and Identification goes out recombinant plasmid.
The preparation of the thick enzyme of embodiment 3:L-threonine aldolase
By the positive transformant list colony inoculation of qualification in the SOC substratum containing Amp (100 μ g/ml), 37 DEG C of shaking culture are spent the night, and bacteria concentration reaches OD
600when=1, be inoculated in the LB substratum containing Amp (100 μ g/ml) with 1%, 37 DEG C are continued to be cultured to OD
600when being in 1.2 ~ 1.5, centrifugal cell harvesting.Cell 0.1M phosphate buffer soln is resuspended, and after ultrasonication, 20000 leave the heart 1 hour, and results supernatant is the thick enzyme of L-threonine zymohexase, packing ,-20 DEG C of preservations.
Embodiment 4:L-threonine aldolase thick Enzyme catalyzed synthesis L-Phenserine (accompanying drawing 4)
Enzyme unit definition alive: under room temperature, per minute catalysis L-threonine decomposes generation 1mmol acetaldehyde, is defined as 1 Ge Meihuo unit.
Catalytic condition: damping fluid, KH
2pO
4, 50mM; PH8.0.1ml reaction system: L-TA, 77U; PLP, 13ng; Phenyl aldehyde, 10mg; Glycine, 75mg.Temperature of reaction, 25 DEG C, reaction times, 45min.
HPLC testing conditions: instrument, Agilent1260; Moving phase, 0.1% sodium heptanesulfonate: methyl alcohol=80:20; Chromatographic column, C18,5 μm, 4.6 × 150mm; Column temperature, 20 DEG C; Detector, ultraviolet; Wavelength, 225nm; Solvent, methyl alcohol.
Detected result: productive rate 80%, ee>99%.
Claims (6)
1. the nucleotide sequence of the encoding L-threonine zymohexase be separated from enterobacter cloacae, it is characterized in that it has the nucleotide sequence shown in SEQ ID NO:1, or the fragment of this nucleotide sequence, analogue or derivative, described bacillus cloacae is CCTCC NO:M 2012240 at the preserving number of China typical culture collection center.
2. according to claim 1 nucleotide sequence, it is characterized in that described nucleotide sequence is selected from:
A () coding has the nucleotide sequence of the aminoacid sequence polypeptide shown in SEQ ID NO:2;
B nucleotide sequence that () is complementary with nucleotide sequence (a), or the aforementioned nucleotide sequence having at least 65% homogeny.
3. a peptide species, is characterized in that it has the polypeptide of the aminoacid sequence shown in SEQ ID NO:2 or its amino acid variation is no more than 30%.
4. a recombinant expression vector, is characterized in that it is the recombinant vectors constructed by nucleotide sequence according to claim 1 and plasmid expression vector.
5. a genetically engineered host cell, is characterized in that it is selected from:
(a) it be the host cell transformed with nucleotide sequence according to claim 1;
(b) it be transform with recombinant expression vector according to claim 4 or the host cell of transduction.
6. any one of claim 1-5 is for the preparation of the method for L-beta-hydroxy a-amino acid or D-beta-hydroxy a-amino acid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018219107A1 (en) * | 2017-05-27 | 2018-12-06 | Enzymaster (Ningbo) Bio-Engineering Co., Ltd. | Engineered polypeptides and their applications in synthesis of beta-hydroxy-alpha-amino acids |
| CN110272856A (en) * | 2019-05-08 | 2019-09-24 | 江南大学 | A kind of recombinant bacterium that expressing D-Thr aldolase and its construction method and application |
| CN113322248A (en) * | 2021-05-12 | 2021-08-31 | 浙江工业大学 | High-temperature-resistant L-threonine aldolase and application thereof in synthesis of p-methylsulfonylphenylserine |
| CN116376989A (en) * | 2022-04-11 | 2023-07-04 | 元素驱动(杭州)生物科技有限公司 | Method for preparing keto acid and application of method in preparation of amino acid or amino acid derivative |
-
2015
- 2015-02-27 CN CN201510089583.4A patent/CN104673814B/en not_active Expired - Fee Related
Non-Patent Citations (3)
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| JI-QUAN LIU，ET AL: "Gene cloning, biochemical characterization and physiological role of a thermostable low-specificity L-threonine aldolase from Escherichia coli", 《EUR. J. BIOCHEM.》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018219107A1 (en) * | 2017-05-27 | 2018-12-06 | Enzymaster (Ningbo) Bio-Engineering Co., Ltd. | Engineered polypeptides and their applications in synthesis of beta-hydroxy-alpha-amino acids |
| US11512303B2 (en) | 2017-05-27 | 2022-11-29 | Enzymaster (Ningbo) Bio-Engineering Co., Ltd. | Engineered polypeptides and their applications in the synthesis of beta-hydroxy-alpha-amino acids |
| CN110272856A (en) * | 2019-05-08 | 2019-09-24 | 江南大学 | A kind of recombinant bacterium that expressing D-Thr aldolase and its construction method and application |
| CN113322248A (en) * | 2021-05-12 | 2021-08-31 | 浙江工业大学 | High-temperature-resistant L-threonine aldolase and application thereof in synthesis of p-methylsulfonylphenylserine |
| CN116376989A (en) * | 2022-04-11 | 2023-07-04 | 元素驱动(杭州)生物科技有限公司 | Method for preparing keto acid and application of method in preparation of amino acid or amino acid derivative |
| CN116376989B (en) * | 2022-04-11 | 2023-10-24 | 元素驱动(杭州)生物科技有限公司 | Method for preparing keto acid and application of method in preparation of amino acid or amino acid derivative |
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