CN1418959A - High temp. resistant pyruvate carboxylase gene and coded polypeptide and preparation process thereof - Google Patents

Abstract

The present invention relates to coding separated DNA with activity or its functional identical variant and polypeptide with high-temp. resisting pyruvate carboxylase activity which is produced by utilziing recombinant DNA technology and the described separated DNA or its functional identical various. By using tengchong thermophilic anaerobe whole genome sequencing and analysis as basis said invention clones and separates the high-temp resisting pyruvate carboxylase gene. Said gene is useful for preparing transgenic microbe or animal and plant to produce high-temp. resisting pyruvate carboxylase and for covering and obtaining the enzyme coded by said gene. Besides, said invention also provides amino acid sequence of polypeptide with high-temp. resisting pyruvate carboxylase activity and functional identical body.

Description

High temperature resistant pyruvate carboxylase gene and encoded polypeptides thereof and preparation method

Technical field

The present invention relates to sudden change or genetic engineering, relate in particular to a kind of high temperature resistant pyruvate carboxylase gene sequence and encoded polypeptides and preparation method.

Background technology

Play a very important role in pyruvate carboxylase (Pruvate carboxylase) the organism intermediate product metabolic process, its catalysis forms oxaloacetic acid by carboxyl donor and pyruvic acid, follows the hydrolysis of ATP simultaneously.This reaction forms lipid acid for tricarboxylic acid cycle, and amino acid and neurotransmitter etc. provides important intermediate product---oxaloacetic acid.Pyruvate carboxylase has the conservative property of height, and on nearly all biology such as bacterium, fungi is all found the existence of this enzyme in higher plant and the animal, is a member in the vitamin H dependent enzyme family.The vitamin H prothetic group combines with the polypeptide chain covalency of this enzyme.Under normal circumstances this enzyme constitutes the tetramer by four identical polypeptide chains.The catalytic entire reaction of pyruvate carboxylase comprises two-step reaction, and occurs in isolated two different loci on the interior space of cell, the carrier that covalently bound with it vitamin H is then rolled into a ball as carboxyl.The first step reaction is that vitamin H is carboxylated under ATP energy supply and the effect of carboxyl donor, and the while is as the substrate of next step reaction.The second step reaction is that the carboxyl with carboxybiotin is transferred to pyruvic acid.Some amino-acid residues role in reaction of the mechanism of part chemical reaction and this enzyme is illustrated, compare by homology the aminoacid sequence between the enzyme with identity function, the functional domain of yeast pyruvate carboxylase has been pushed to be measured, research to the quaternary structure of this enzyme is carried out, known it form a tetrahedral structure by four subunits, the main attemperator of enzymic activity is an acetyl-CoA, it at first activates the ATP cracking in the first step reaction, follow the variation of tetramer structure conformation simultaneously, the biotin carboxylase territory that ATP relies on makes that to be connected in the attached group of vitamin H that vitamin H/carboxyl carries on the special lysine residue in territory (BCCP) carboxylated.Acetyl-CoA activates this reaction by the cleavage rate that increases ATP.In the reaction of second step, the BCCP territory offers pyruvic acid with carboxyl, by the transfer carboxylase active zone catalysis of this enzyme.Past is very restricted the mechanism of action and the Regulation Mechanism of this enzyme of research owing to lack the information of this enzymatic structure, and the research of other vitamin H dependence enzyme too.Along with the clone's success of this enzyme and the research of three-dimensional structure in various organism deepen continuously, believe that the research to the reaction mechanism of this enzyme will obtain bigger progress.Though the high-resolution X ray crystalline tomograph of this enzyme is not also arranged, existing electron microscope observation, the cloning and sequencing analysis of limited proteolysis analysis and gene etc. at present.Known this enzyme constitutes tetrahedral structure by four identical subunits, and each subunit has three functional domains: biotin carboxylase territory, carboxyl transferring field and biotin carboxylase territory.

Tengchong thermophilc anaerobe (Thermoanaerobacter tangcongensis), it is a kind of microorganism that lives in the hot spring of Yunnan Province of China province Tengchong County, it is a kind of thermophilic eubacterium (eubacteria), optimum growth temperature is 75 degrees centigrade, anaerobic growth, the gramstaining reaction is positive.It is at first found by Microbe Inst., Chinese Academy of Sciences and has carried out the analysis on the taxonomy.Bacterial classification is kept at Chinese microorganism and preserves center MB4 ^T(Chinese collection of microorganisms AS 1.2430 ^T=JCM 11007 ^T).This thermophilc anaerobe is the distinctive species of China, and the high temperature resistant pyruvate carboxylase that is had in its body also has own its specific structure.

Summary of the invention

One of purpose of the present invention provides a kind of isolating, and coding has the nucleotide sequence of the active polypeptide of high temperature resistant pyruvate carboxylase.

Two of purpose of the present invention provides a kind of isolating, has high temperature resistant pyruvate carboxylase active polypeptide.

Purpose of the present invention also provide thermophilc anaerobe the pyruvate carboxylase recombinant vectors, contain the host cell of recombinant vectors, and produce proteic method.

One aspect of the present invention provides a kind of can the coding to have the nucleotide sequence of the active polypeptide of high temperature resistant pyruvate carboxylase.Said nucleotide sequence coded the have polypeptide of the aminoacid sequence among the SEQ ID NO.2 or the modified forms of described polypeptide, on this modified forms function quite or relevant with pyruvate carboxylase.Nucleotide sequence has the polynucleotide sequence of SEQ ID NO.1 and its mutant form, and mutation type comprises: disappearance, nonsense, insertion, missense.

The present invention provides a kind of high temperature resistant pyruvate carboxylase active polypeptide on the other hand.This polypeptide has polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of the aminoacid sequence among the SEQ ID No.2.

The method of producing high temperature resistant pyruvate carboxylase is:

1) isolate the coding high temperature resistant pyruvate carboxylase nucleotide sequence SEQ ID NO.1;

2) make up the expression vector that contains SEQ ID NO.1 nucleotide sequence;

3) with step 2) in expression vector change host cell over to, form the reconstitution cell can produce high temperature resistant pyruvate carboxylase;

4) culturing step 3) in reconstitution cell;

5) separation, purifying obtain high temperature resistant pyruvate carboxylase.

The present invention relates to the separation and the expression of the high temperature resistant pyruvate carboxylase gene of thermophilc anaerobe.Based on Tengchong thermophilc anaerobe genome sequencing and analysis, clone and separate high temperature resistant pyruvate carboxylase gene.The high temperature resistant pyruvate carboxylase of transgenic microorganism or animals and plants this gene is used to produce to(for) preparation, and it is useful to reclaim the enzyme that obtains this genes encoding.In addition, the present invention also provides and has had active amino acid sequence of polypeptide of high temperature resistant pyruvate carboxylase and functional equivalent body.Simultaneously, the present invention also provides preparation, separates, and purifying has the method for the active polypeptide of high temperature resistant pyruvate carboxylase.

Description of drawings

Fig. 1 is an order-checking library construction flow chart of steps;

Fig. 2 is order-checking and data analysis schema;

Fig. 3 is that forward and reverse sequencing result is analyzed synoptic diagram;

Fig. 4 Part of Co smid end sequencing result schematic diagram;

Embodiment

At first, the invention provides isolating, the encode polynucleotide molecule of the active polypeptide of high temperature resistant pyruvate carboxylase, this nucleic acid molecule is by obtaining Tengchong thermophilc anaerobe genome sequencing and analysis, nucleotide sequence with SEQ.ID NO.1, its coding has the polypeptide that 465 amino acid are read frame, infers that molecular weight is 52875 dalton.

The invention still further relates to a kind of recombinant vectors, this carrier comprises isolating nucleic acid molecule of the present invention, and the host cell that includes recombinant vectors.Simultaneously, the present invention includes the method that makes up this recombinant vectors and host cell, and the method for producing high temperature resistant pyruvate carboxylase with the recombined engineering technology.

The present invention provides a kind of isolating high temperature resistant pyruvate carboxylase or polypeptide further, it is characterized in that having SEQ.ID NO.2 aminoacid sequence, or at least 70% is similar, more preferably, have at least 90%, 95%, 99% identical.

In the present invention, " isolating " DNA is meant that this DNA or segment have been arranged in its both sides under native state sequence separates, refer to that also this DNA or segment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, " high temperature resistant pyruvate carboxylase gene " refers to encode and has the nucleotide sequence of the active polypeptide of high temperature resistant pyruvate carboxylase, as nucleotide sequence and the degenerate sequence thereof of SEQ.ID NO.1.This degenerate sequence be meant have one or more codons to be encoded in this sequence the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of known codon, so be low to moderate about 70% the degenerate sequence described aminoacid sequence of SEQ ID NO.2 of also encoding out with SEQ ID NO.1 nucleotide sequence homology.This term also comprises can be under the rigorous condition of moderate, more preferably under highly rigorous condition with the nucleotide sequence of the nucleotide sequence hybridization of SEQ ID NO.1.This term also comprises and SEQ ID NO.1 nucleotide sequence homology 70% at least, preferably at least 80%, more preferably at least 90%, and at least 95% nucleotide sequence best.

In the present invention, " isolating " proteic polypeptide is meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as uses column chromatography, and PAGE or HPLC method are measured the purity of polypeptide.Isolated polypeptide is substantially free of the component of following it under the native state.

In the present invention, " high temperature resistant pyruvate carboxylase " refers to have the active SEQID NO.2 of high temperature resistant pyruvate carboxylase polypeptide of sequence.This term also comprises the varient of SEQ ID NO.2 sequence, and these varients have and natural high temperature resistant pyruvate carboxylase identical functions.These varients include, but is not limited to several amino acid whose disappearances, insert and/or replace, and add one or several amino acid at C latter end and/or N-terminal, also can be the difference that does not influence on the modified forms of sequence.For example, for known in the field, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C latter end and/or N-terminal and also can not change proteinic function usually.This term also comprises the active part and the reactive derivative of high temperature resistant pyruvate carboxylase.

In the present invention, can select various carrier known in the art for use, as commercially available various plasmids, clay, phage and retrovirus etc.When producing high temperature resistant pyruvate carboxylase of the present invention, high temperature resistant pyruvate carboxylase gene sequence can be able to be operated and lowly be connected in expression regulation sequence, thereby form high temperature resistant pyruvate carboxylase expression vector.Expression vector contains replication origin and expression regulation sequence, promotor, enhanser and necessary machining information site.Expression vector also must contain alternative marker gene, as a) providing to microbiotic or other toxicant (penbritin, the protein or the b of resistance kantlex, methotrexate etc.)) complementary auxotroph protein or c) protein of the essential nutritive ingredient that does not have in the complex medium is provided.Various different hosts' appropriate flags gene be well known in the art or production firm's specification sheets famous.These expression vectors can be with well known to a person skilled in the art recombinant DNA technology preparation, as can be with reference to people such as Sambrook, and 1989 or people such as Ausubel, 1992.

Recombinant expression vector can be introduced host cell with method well known in the art, and these methods comprise: electrotransformation, Calcium Chloride Method, particle bombardment etc.The process that the external source recombinant vectors is imported host cell is called " conversion ".By cultivating host cell, induce the expression of desirable proteins, and by protein separation technology known in the art, obtain required protein as column chromatography etc.Also can adopt these protein of synthetic such as solid phase technique.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.Prokaryotic cell prokaryocyte such as intestinal bacteria commonly used, Bacillus subtilus etc.Eukaryotic cell such as yeast cell commonly used, or various animal and plant cells.

High temperature resistant pyruvate carboxylase gene full length sequence of the present invention or its segment can be used the pcr amplification method usually, recombination method, or the method for synthetic obtains.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence design primer, is template with the thermophilc anaerobe complete genome DNA of ordinary method preparation well known by persons skilled in the art, increases and obtains relevant sequence.In case obtained relevant sequence, just it can be cloned into relevant carrier, change host cell again over to, from the host cell after the propagation, separate obtaining large batch of relevant sequence then by ordinary method.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1: make up the order-checking library

The structure in order-checking library adopts full genome shotgun approach (shotgun) to carry out.At first cultivate the Tengchong thermophilc anaerobe, cultural method is pressed Marmur (1961) method and is collected bacterium by (Yanfen Xue, 2000) improved MB substratum (Balch et al., 1979), extracts total DNA.For the randomness of the library construction that guarantees to check order, farthest avoid producing the problem of breakage hot spot, that adopts several different methods, different condition builds the storehouse principle.。Adopt earlier physics cutting method (comprise supersonic method and shear with Hydroshear Machine), next is selected for use AluI to carry out the random partial enzyme according to this bacterium genome signature and cuts.Adopt varying strength to handle sample when physics is sheared, handle sample by enzyme amount gradient is set when enzyme is cut.Sample after the processing adopts electrophoresis fraction collection 1.5-4kb dna fragmentation after flat terminal the processing, be connected with the dephosphorylized pUC18 that cuts through the SmaI enzyme, connects product has made up random sequencing by electric Transformed E .coli DH5 α library.Simultaneously, (cut genomic dna in the order-checking library that has also made up long insertion fragment (about 10kb) for the ease of the later overlap joint of lengthy motion picture disconnected (contig) with Sau3AI random partial enzyme, electrophoresis is collected the fragment about 10kb, is connected, makes up the library with the dephosphorylized pUC18 that cuts through the BamHI enzyme).The order-checking of these two ends in library can obtain the relation between the contig in the process of finishing figure (finishing), and can solve the difficulty that bigger gap causes filling-up hole.Build storehouse flow process such as (see figure 1).

Embodiment 2: gene order-checking

When finishing the genomic order-checking of Tengchong thermophilc anaerobe, two kinds of full-automatic sequenator: ABI377 and MegaBACE 1000 have mainly been used.These two kinds of sequenators all are to utilize the principle of electrophoresis (see figure 2) that checks order, and can finish 96 samples at every turn.ABI377 is the product of PE company, is a kind of of ABI series.It belongs to the plate gel electrophoresis sequenator.MegaBACE 1000 is products of Pharmacia Corp, belongs to the capillary gel electrophoresis sequenator.

Embodiment 3:Basecalling and sequencing quality monitoring

So-called Basecalling is meant the process that obtains correct base sequence from the raw data file that sequenator obtains.Because that obtain on the sequenator is A, T, G, the light intensity variation track (trace) of the different wave length that four kinds of base pairs of C are answered need take certain algorithm therefrom correctly to identify the base of different track correspondences with computer.What we used is Phred software (Ewing B, Hillier L, 1998), and reason is that its result is more reliable, and other programs that its result exports in the same software package of being more convenient for are further analyzed.

Phred carries out the algorithm principle of Basecalling, is the shape according to each peak in the track, and spacing, and factor such as signal to noise ratio are judged the base type, simultaneously this base are provided reliability information, i.e. the sequencing quality of base.In large scale sequencing, the monitoring of sequencing quality is crucial, and it directly influences the decision-making to order-checking, comprises the structure in library, the size of fraction of coverage.Can in time feed back the error that may occur in the order-checking experiment simultaneously.

Embodiment 4: sequence assembly

So-called sequence assembly is exactly full genome shotgun approach, and the sample sequence that claims the shotgun random sequencing to obtain again is assembled into successive lengthy motion picture disconnected (contig), mainly utilizes the overlap between them for referencial use.Consider the influence that has carrier in the order-checking, need earlier sample sequence to be unloaded body and handle.Here used software cross_match and the back used software Phrap of splicing are the software (Gordon D, Abajian C, 1998) of U.S. Washington university, and its ultimate principle is Swith-Waterman algorithm (WatermanMS, 1990).This is a kind of dynamic algorithm, after having considered the comparison between the sequence in twos, can obtain the publicly-owned sequence (consensus sequence) of one group of sequence.Sample sequence behind the removal carrier splices with Phrap again.In when splicing, the sequencing quality of base also has been considered, and the confidence level of resulting publicly-owned each base of sequence is calculated by the sequencing quality of the sample of forming this publicly-owned sequence.

Embodiment 5: gene annotation

After obtaining genomic most of sequence (frame diagram of finishing the work) substantially, just need carry out note to genome, comprise out frame frame (Open Reading Frame, prediction ORF), the prediction of gene function, and the pulsating analysis of special RNA etc.

The first step adopts the GLIMMER2.0 (Delcher of default parameter, A.L., Harmon, D.1999) and ORPHEUS (Frishman, D.1998) software prediction gene coded sequence, open frame and the non-coding region (intergenic region) of all predictions all use BLAST software (Altschul, S.F.et al.1997) and the irredundant albumen database (non-redundant protein database) of NCBI relatively to find the gene that may miss then.When judging the starting point of a gene, will be with reference to various relevant informations, as sequence homology, ribosome bind site, possible signal peptide sequence and promoter sequence etc.If open in the frame when a plurality of promotor occurring at one, generally adopt the starting point of first promotor as gene.(Ermolaeva M.D.2000) predicts the transcription terminator that does not rely on Rho (ρ) factor at non-coding region to adopt TransTerm software.If this terminator be positioned at a gene the catchment too at a distance, then may hint a minigene lose or the mistake that checks order has shortened this gene artificially, can be used as the reference of further analysis.When determining to move frame sudden change and point mutation, main basis is judged with the proteinic similarity in the database.If protein is corresponding to the situation of two encoding sequences adjacent one another are, then be considered to a non-activity gene (pseudogene pseudogenes), produce the abort phenomenon because this illustrates between these two encoding sequences owing to suddenling change, and then gene is lost activity.All analytical resultss use Artemissequence viewer software (Rutherford, K.et al.2000) to carry out manual analysis again.Some are obviously shown eclipsed with other code sequence and open frame, and length does not have homology and wherein do not have tangible promotor or termination is regional opens frame and will be removed less than 150 base pairs and in the data with existing storehouse.

Proteinic functional fragment (motif) and functional area (domain) employing and Pfam, PRINTS, PROSITE, ProDom and SMART database respectively compare, the result uses InterPro database (Apweiler, R.et al.2001) to carry out Macro or mass analysis again.According to the COGs database (Tatusov, R.L.et al.2001) of NCBI and with reference to other result of querying database determine protein in the COGs classification functional classification and possible pathways metabolism.Confirm membranin, abc transport albumen and stride the film functional domain with TMHMM software (Krogh, A.et al.2001).The employing Gram-negative bacteria is a parameter, with SIGNALP2.0 software (Nielsen, H.et al.1999) analytical signal peptide zone.(4) filling-up hole

After finishing genomic work frame chart, will carry out the filling-up hole work of difficulty more, promptly finish the order-checking of whole genome 100%, obtain an annular genome.Groundwork is exactly that the contig that obtains is previously coupled together.This is a very concrete and numerous and diverse job.Main method comprises:

A. utilize forward and reverse order-checking sample message in the order-checking in the order-checking process, we have carried out two-way order-checking to some sample intentionally, check order simultaneously promptly that certain inserts pulsating two ends, institute's calling sequence are spliced with other sequences again.Because the relation of this a pair of sequence on genome is certain, distance between it is roughly known, according to this information, one can confirm whether certain section contig is reliable, the 2nd, when this a pair of sequence lays respectively on the different contig, can determine direction relations and the position relation of these two contig, for further contrived experiment provides with reference to (see figure 3).

B. long insertion segment and Cosmid end sequencing are based on same principle, and we can make up the insertion segment library of different lengths, and only to its two ends order-checking, its particular location is analyzed in splicing then.These libraries comprise that length is the long Cosmid library of inserting about segment storehouse and 20-40Kb of 9-12Kb.Specific analytical method is same as above.Figure 4 shows that Part of Co smid end sequencing result.

C.PCR and the terminal Walking of extension test

According to contig direction and position relation that A and B provided, further Biochemistry Experiment just can have been carried out.As design a pair of primer and carry out pcr amplification, or carry out end extension (Walking) with a certain contig end sequence synthetic primer and come filling-up hole etc.

Embodiment 6: the preparation of pyruvate carboxylase and purification

According to the pyruvate carboxylase complete encoding sequence (SEQ ID NO.1) that gene annotation among the embodiment obtains, design can amplify the primer that complete coding is read frame, and introduces restriction endonuclease sites respectively on positive anti-primer, so that construction of expression vector.Plasmid DNA with the order-checking library that obtains among the embodiment 1 is a template, behind pcr amplification, guarantee to read recombinate under the correct prerequisite of frame to the pGEX-2T carrier (Pharmacia, Piscataway, NJ).Again recombinant vectors is transformed into that (method for transformation is CaCL in the bacillus coli DH 5 alpha ₂Method or electrotransformation).Screening and Identification to the engineering bacteria DH5 α-pGEX-2T-PycA that contains expression vector.

The engineering bacteria DH5 α-pGEX-2T-PycA of picking list bacterium colony contains in the LB substratum of 100 μ g/ml penbritins jolting in 3ml and cultivates 37 ℃ and spend the night, draw nutrient solution by 1: 100 concentration and in new LB substratum (containing 100 μ g/ml penbritins), cultivated about 3 hours, to OD ₆₀₀After reaching 0.5, add IPTG, continue at 37 ℃ and cultivated respectively 0,1,2,3 hours to final concentration 1mmol/L.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing desirable proteins.

As stated above behind the engineering bacteria of abduction delivering desirable proteins, with bacterium centrifugation, add 50% saturated Triptide Sepharose 4B of 20mlPBS by every 400ml bacterium, 37 ℃ of joltings were in conjunction with 30 minutes, 10000rpm precipitated the Triptide Sepharose 4B that combines desirable proteins in centrifugal 10 minutes, abandoned supernatant.Add 100 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid gained precipitation, room temperature was put 10 minutes, and supernatant is the albumen of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored in-80 ℃, and carries out the SDS-PAGE electrophoresis, detects purification effect.Protein band at 52875 dalton place is pyruvate carboxylase.

1.SEQ ID NO.1 ( 1 ) ：a.：1398b.：DNAc.：d.： ( 2 ) ： ( 3 ) ：atggaaaggagagttaaaataacagaaacagttctaagagatgcccatcagtctttgatggctactcgcatgactacagaggaaatgctgcctattgcagaaaagcttgataaggtgggatatcattctcttgaggtttggggaggagctacctttgatgcttgtctcaggtttttaaatgaagacccgtgggagaggctgcgggaattaaaaaagaggataaaaaatacacccctgcaaatgcttttaagagggcaaaatctggtaggatacagacattatccggatgacattgtagaaaaatttatagaaaaggctgtagctaatggaatagacataataaggatctttgatgctttgaatgatgtgagaaatttggaagtttccataaaggcgacaaaaagggtaggagcccatgcgcaagggactcttgtctatactataagccctgtccacactatagaccactacataagagtggcaaaagaactggtagaattaggtgtagattctatctgcataaaggacatgtcgggcatattgagcccttatgtagcctatgaactggtgaagaggctcaaagagacagttaatgttcccatacagctgcacggccattacacgagtggaatggcttccatgacttatttaaaagccatagaagcaggggtggatgcgatagatacagcaatttctcctttagctttgggcacttctcagcctgctacagagaccatggtggcagctttgaagggaactcaatatgatacagggcttgaccttgaacttctctcagaaattgcttcatattttaaggaagtaaagcgaaatcactataaagaaacagatttctcaatggtaatgggtatagatactgatgtgcttgtctaccaggttccgggaggaatgctttcaaatctcattgcacagctaaaagagcaaaaagcgcttgacaagtataaagaagttctagaagagataccgagggtgagagaggacctaggatatccgcctctagttactccaatgagtcaaatggtaggtacacaggcggttttaaatgtgataactggtgagaggtataaaatggttccaaaagagataaaggattatgtcaagggactgtacggcaggcctcctgcaccaatttctgatgagataaagaggaaaataataggtgatgaagaggtaattgagataaggccggcggacctcttaaaaccacagtttgaagaggtaaaagaagaaataaaagagtattacgaacaggaagaggatgtactgacatatgctctttttccgcaagtggctaaaaagttttttgagtacagaagggcgaaaaagtattttatagattctagtatggttggtgagggaaacagagtgtatcctgtctaa2.SEQ ID NO.2 ( 1 ) ：a.：465b.：c.：d.： ( 2 ) ： ( 3 ) MERRVKITETVLRDAHQSLMATRMTTEEMLPIAEKLDKVGYHSLEVWGGATFDACLRFLNEDPWERLRELKKRIKNTPLQMLLRGQNLVGYRHYPDDIVEKFIEKAVANGIDIIRIFDALNDVRNLEVSIKATKRVGAHAQGTLVYTISPVHTIDHYIRVAKELVELGVDSICIKDMSGILSPYVAYELVKRLKETVNVPIQLHGHYTSGMASMTYLKAIEAGVDAIDTAISPLALGTSQPATETMVAALKGTQYDTGLDLELLSEIASYFKEVKRNHYKETDFSMVMGIDTDVLVYQVPGGMLSNLIAQLKEQKALDKYKEVLEEIPRVREDLGYPPLVTPMSQMVGTQAVLNVITGERYKMVPKEIKDYVKGLYGRPPAPISDEIKRKIIGDEEVIEIRPADLLKPQFEEVKEEIKEYYEQEEDVLTYALFPQVAKKFFEYRRAKKYFIDSSMVGEGNRVYPV

Claims (8)

1. isolated DNA molecule is characterized in that: it is the nucleotide sequence that coding has the polypeptide of high temperature resistant pyruvate carboxylase protein-active.

2. dna molecular as claimed in claim 1, it is characterized in that: the polypeptide of the aminoacid sequence among the said nucleotide sequence coded SEQ.ID of the having NO.2 or the modified forms of described polypeptide, on this modified forms function quite or relevant with high temperature resistant pyruvate carboxylase.

3. dna molecular as claimed in claim 1 is characterized in that: said nucleotide sequence has the polynucleotide sequence of SEQID NO.1 and its mutant form, and mutation type comprises: disappearance, nonsense, insertion, missense.

4. polypeptide separated, it is characterized in that: it has high temperature resistant pyruvate carboxylase activity.

5. polypeptide as claimed in claim 4 is characterized in that: it has polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative of the aminoacid sequence among the SEQ ID No.2.

6. carrier, it is characterized in that: it contains the DNA in the claim 1.

7. host cell is characterized in that: it is prokaryotic cell prokaryocyte or the eukaryotic cell that transforms with the described carrier of claim 6.

8. method for preparing high temperature resistant pyruvate carboxylase is characterized in that this method comprises:

1) isolate the coding high temperature resistant pyruvate carboxylase gene nucleotide sequence SEQ ID NO.1;

2) make up the expression vector that contains SEQ ID NO.1 nucleotide sequence;

3) with step 2) in expression vector change host cell over to, form the reconstitution cell can produce high temperature resistant pyruvate carboxylase;

4) culturing step 3) in reconstitution cell;

5) separation, purifying obtain high temperature resistant pyruvate carboxylase.

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