CN1367251A - High-temperature-resistant spermine succinic lyase gene and polypeptide coded by same and preparation method - Google Patents

Abstract

The invention discloses a high-temperature resistant spermine succinic lyase gene, a polypeptide coded by the same and a preparation method thereof. It relates to a separated DNA with high-temperature resistant spermine succinic acid lyase activity or its functional equivalent variant and uses recombinant DNA technique to produce polypeptide with high-temperature resistant spermine succinic acid lyase activity or its functional equivalent variant with the separated DNA. The invention clones and separates the high temperature resistant argininosuccinate lyase gene based on sequencing and analysis of Tengchong thermophilic anaerobic bacteria whole genome. The gene is used to produce transgenic microbe or transgenic animal and plant of high temperature resistant argininosuccinate lyase and the enzyme coded by the gene is recovered. In addition, the invention also provides an amino acid sequence and a functional equivalent of the polypeptide with the high-temperature-resistant argininosuccinate lyase activity. Meanwhile, the invention also provides a method for preparing, separating and purifying the polypeptide with the high-temperature-resistant argininosuccinase activity.

Description

High temperature resistant spermine succsinic acid lyase gene and encoded polypeptides and preparation method

Technical field

The present invention relates to field of genetic engineering, particularly, relate to a kind of high temperature resistant spermine succsinic acid lyase gene and encoded polypeptides thereof and prepare the method for this polypeptide.

Background technology

Spermine succsinic acid lyase (argininosuccinate lyase) is with regard to one of enzyme required in amino acid transportation and the metabolic process.Its effect is that the catalysis argininosuccinic acid generates arginine and fumarate.

Spermine succsinic acid lyase is a kind of delta-crystallin (delta-Crystallin), is the synthetic and metabolic enzyme of a seed amino acid, also is a kind of urine cyclophorase.Its aminoacid sequence has 439 amino acid, infers that molecular weight is 50497 dalton.

Spermine base succsinic acid lyase is important a kind of enzyme in the urine circulation, and its effect is a cracked ammonium.Its disappearance can cause the huge injury to body.Spermine base succsinic acid lyase disappearance disease produces by heredity, and is autosomal recessive inheritance (the 7th on the karyomit(e)), and this sick symptom generally occurs in the later stage of patient's age, but but shows as very high mortality ratio for baby due.Clinical symptom comprise ewborn infant in just birth stage blood because the many ammonia of blood of fainting of too much producing of ammonia, all property, backwardness, hepatomegaly, erythema fash, hair fragility etc.Result of laboratory test has shown that the hyperammonemia that the disappearance of spermine succsinic acid lyase causes is secondary respiratory alkalosis.The toxic action of the maximum of hyperammonemia is for central nervous system, and it may cause cerebral edema.

Spermine base succsinic acid lyase is indispensable a kind of enzyme in the process of the intravital nitrogen of body discharges.If abnormal words can cause the accumulation of intravital nitrogen, cause the injury huge to body.

Therefore the synthetic of spermine base succsinic acid lyase can be applied to pharmaceutical sector, and fields such as agricultural, food-processing industry are with a wide range of applications.

The Tengchong thermophilc anaerobe that the present invention relates to (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 to be found and carried out the analysis on the taxonomy by Microbe Inst., Chinese Academy of Sciences.Bacterial classification is kept at Chinese microorganism and preserves center MB4 ^T(Chinese collection of microorganisms AS1.2430 ^T＝JCM11007 ^T)。This thermophilc anaerobe is the distinctive species of China, and the high temperature resistant spermine succsinic acid lyase 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 spermine succsinic acid lyase.

Two of purpose of the present invention provides a kind of isolating, has high temperature resistant spermine succsinic acid lyase active polypeptide.

Purpose of the present invention also provide the DNA that contains the high temperature resistant spermine succsinic acid lyase of encoding recombinant vectors, contain the host cell of aforementioned recombinant vectors, and this proteic method of preparation.

A first aspect of the present invention provides a kind of coding to have the nucleotide sequence of the active polypeptide of high temperature resistant spermine succsinic acid lyase.This nucleotide sequence can encode the polypeptide with 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 spermine succsinic acid lyase.Nucleotide sequence has the polynucleotide sequence of SEQ ID NO.1 and its mutant form, and mutation type comprises: disappearance, nonsense, insertion, missense.

A second aspect of the present invention provides a kind of high temperature resistant spermine succsinic acid lyase active polypeptide.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 present invention also provides the method for preparing high temperature resistant spermine succsinic acid lyase, and this method comprises the steps:

1) isolate the coding high temperature resistant spermine succsinic acid lyase gene nucleotide sequence such as SEQ ID NO.1;

2) structure contains the expression vector of the nucleotide sequence of SEQ ID NO.1;

3) with step 2) in expression vector change host cell over to, form the reconstitution cell can produce high temperature resistant spermine succsinic acid lyase;

4) culturing step 3) in reconstitution cell;

5) separation, purifying obtain high temperature resistant spermine succsinic acid lyase.

Description of drawings

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

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

Embodiment

The invention provides isolating, the encode nucleic acid molecule of the active polypeptide of high temperature resistant spermine succsinic acid lyase, this nucleic acid molecule has the nucleotide sequence shown in the SEQ ID NO.1 by Tengchong thermophilc anaerobe genome sequencing and analysis are obtained.It has been encoded and has had that high temperature resistant spermine succsinic acid lyase is active 439 amino acid whose polypeptide, and the supposition molecular weight of this polypeptide is 50497 dalton.

The present invention also provides a kind of recombinant vectors, and 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 utilize the recombined engineering technology to produce the method for high temperature resistant spermine succsinic acid lyase.

The present invention provides a kind of isolating high temperature resistant spermine succsinic acid lyase or polypeptide further, and it has the aminoacid sequence shown in the SEQ ID NO.2, 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 fragment have been arranged in its both sides under native state sequence separates, refer to that also this DNA or fragment 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 spermine succsinic acid lyase gene " refers to encode and has the nucleotide sequence of the active polypeptide of high temperature resistant spermine succsinic acid lyase, 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 IDNO.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 spermine succsinic acid lyase " refers to have the active SEQ ID of high temperature resistant spermine succsinic acid lyase NO.2 polypeptide of sequence.This term also comprises the varient of SEQ ID NO.2 sequence, and these varients have and natural high temperature resistant spermine succsinic acid lyase 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 fragments and the reactive derivative of high temperature resistant spermine succsinic acid lyase.

In the present invention, can select various carrier known in the art for use, as various plasmids, clay, phage and the retrovirus of selling on the market etc.When producing high temperature resistant spermine succsinic acid lyase of the present invention, high temperature resistant spermine succsinic acid lyase gene sequence operationally can be connected in expression regulation sequence, thereby form high temperature resistant spermine succsinic acid lyase expression vector.This 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 is well known in the art or production firm's specification sheets indicates.These expression vectors can be with recombinant DNA technology known in those skilled in the art preparation, as can be with reference to people's such as Sambrook way (1989), or people's such as Ausubel way (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 spermine succsinic acid lyase gene full length sequence of the present invention or its fragment can be used PCR (polymerase chain reaction) TRAP 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 by (Yanfen Xue, 2000) improved MB substratum (Balch et al., 1979), press Marmur (1961) method and collect bacterium, extract 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 after flat terminal the processing.The 5-4kb dna fragmentation is connected with the dephosphorylized pUC18 that cuts through the SmaI enzyme, connects product by electric Transformed E.Coli DH5 α has made up the library of random sequencing.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 contig (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 (contig) in structure is finished the process of figure (finishing), and can solve the difficulty that cause filling-up hole in bigger hole (gap).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 ABI 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: data analysis

1) 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.

2) sequence assembly

So-called sequence assembly, the sample sequence that full genome shotgun approach (claiming shotgun again) random sequencing is obtained is assembled into successive contig (contig) exactly, 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.

3): gene annotation

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

The first step adopts GLIMMER2.0 (Delcher, the A of default parameter.L。，Harmon，D。1999) and ORPHEUS (Frishman, D.1998) software prediction gene coded sequence, then all predictions open frame and non-coding region (intergenic region) is all used BLAST software (Altschul, S.F。etal。1997) relatively find the gene that to miss with the irredundant albumen database (non-redundantprotein database) of NCBI (American National biotechnology information center).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 when in an open reading frame, a plurality of promotor occurring, generally adopt the starting point of first promotor as gene.Adopt TransTerm software (Ermolaeva, M.D。2000) predict the transcription terminator that does not rely on Rho (ρ) factor at non-coding region.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 seudogenes), 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 are used Artemis sequenceviewer software (Rutherford, K again.et?al。2000) carry out manual analysis.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 open reading frame that termination is regional will be removed less than 150 base pairs and in the data with existing storehouse.

Proteinic function fragment (motif) and functional area (domain) employing and Pfam, PRINTS, PROSITE, ProDom and SMART database respectively compare, and the result uses InterPro database (Apweiler, R.et al again.2001) carry out Macro or mass analysis.COGs database (Tatusov, R according to NCBI.L。et?al。2001) and with reference to other result of querying database determine protein in COGs classification functional classification and possible pathways metabolism.With TMHMM software (Krogh, A.et?al。2001) confirm membranin, abc transport albumen and stride the film functional domain.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.Main method comprises:

A. utilize the 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 segmental 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 them 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 reference.

B. long fragment and equipment type carrier clay (Cosmid) end sequencing of inserting

Based on same principle, we can make up the insertion fragment 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 sheet phase library and 20-40Kb of 9-12Kb.Specific analytical method is same as above.

C.PCR and terminal extend (Walking) test

The direction of the contig that is provided according to above-mentioned steps A and B and position relation, 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 4: the preparation of spermine succsinic acid lyase and spermine succsinic acid lyase gene complete encoding sequence (the SEQ IDNO that purifies and obtain according to gene annotation among the embodiment.1), designs and to amplify the primer that complete coding is read frame, and on positive anti-primer, introduce restriction endonuclease sites respectively, 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).The engineering bacteria DH5 α-pGEX-2T-ArgH that contains expression vector that Screening and Identification obtains.

The engineering bacteria DH5 α-pGEX-2T-ArgH 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 20ml PBS 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 SDS-PAGE SEQ ID NO.1 electrophoresis, detects purification effect.Protein band at 50497 dalton place is spermine succsinic acid lyase.

1、EQ ID NO.1 ( 1 ) ：a.：1320b.：DNAc.：d.： ( 2 ) ： ( 3 ) ：atgaagctgtggggaggtaggtttaaaaaggacttggacaaactgatggaggatttcaattcctccatttcttttgatataaggcttttaaagtatgatataatgggctctattgcccatgcaaagggtctttacaaggctggcgttttaacagaagaggagcttaatttgatagaaaaa agggcttaaagaaatacttgaggaatccgagttaaaagaaatacctcaggatgaagatgttcattcctatgtagaaaggcttcttgtagagaaaataggcgatgtagggcgtaaacttcacacaggaagaagcagaaacgaccaagttgcgacggatgaaaggctttatttaagagatgagatagacaaaataaaagaggatttaataaagcttatagatactctaaaagaaatggctgaaacttataaagaaacaattatgccgggatatacgcatttacagagggcacagcctgtgacttttggacattatcttcttgcctatgttgaaatgtttaaaagagatttgtcaaggcttgatgatatgatgaaaagagtcaatgtaatgcctttgggttctggtgctcttgcaggtactacctttaatatcgacagatattatgtcgcttctcttttagggttcgatgatataaccttaaacagtatggacggggttagtgatagagattttgtgatagaatttttaagctttgcttctataacaatgatgcatttgagcagattctgtgaggaattaattttgtggtctacaaaggaatttgactttattgaaatggacgatagattttcaacaggaagcagtatgatgccgcaaaagaaaaatcctgatgcagcagaattaataaggggcaaaaccggcagggtttacggcgatttgattacaatacttaccgtgatgaaaggactccctcttgcatataacaaagacatgcaggaggataaagaggccttgtttgacggaatagacactttaaaaatgtctttgaaagtatttacagaaatgataaagacaattaaagtaaaaacagacaacatggaaaaagctgcaaaatatggttatatgaatgctacagattttgcagactatcttgtacaaaaaggtattccttttaggaccgcccatgaaattgcaggcaaagttgtcttatatgcgatagaacgaaacttaccaattgaagagttatcacttgaagaacttaaaaaattcagcgatgtaatagaaaaagatgtatatgaggcgatagatataaaaaatattttaaaaaagagaaagacaataggttcaccgaggatattctaa2.SEQ ID NO.2 ( 1 ) ：a.：439b.：c.：d.： ( 2 ) ： ( 3 ) MKLWGGRFKKDLDKLMEDFNSSISFDIRLLKYDIMGSIAHAKGLYKAGVLTEEELNLIEKGLKEILEESELKEIPQDEDVHSYVERLLVEKIGDVGRKLHTGRSRNDQVATDERLYLRDEIDKIKEDLIKLIDTLKEMAETYKETIMPGYTHLQRAQPVTFGHYLLAYVEMFKRDLSRLDDMMKRVNVMPLGSGALAGTTFNIDRYYVASLLGFDDITLNSMDGVSDRDFVIEFLSFASITMMHLSRFCEELILWSTKEFDFIEMDDRFSTGSSMMPQKKNPDAAELIRGKTGRVYGDLITILTVMKGLPLAYNKDMQEDKEALFDGIDTLKMSLKVFTEMIKTIKVKTDNMEKAAKYGYMNATDFADYLVQKGIPFRTAHEIAGKVVLYAIERNLPIEELSLEELKKFSDVIEKDVYEAIDIKNILKKRKTIGSPRIF

Claims (8)

1. an isolated DNA molecule is characterized in that: be the nucleotide sequence that coding has the polypeptide of high temperature resistant spermine succsinic acid lyase 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 of the having ID NO.2 or the modified forms of this polypeptide, on this modified forms function quite or relevant with high temperature resistant spermine succsinic acid lyase.

3. dna molecular as claimed in claim 1 is characterized in that: said nucleotide sequence has the polynucleotide sequence of SEQ ID 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 spermine succsinic acid lyase 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. a method for preparing high temperature resistant spermine succsinic acid lyase is characterized in that this method comprises the steps:

1) isolate the coding high temperature resistant spermine succsinic acid lyase gene nucleotide sequence such as SEQ ID NO.1;

2) structure contains the expression vector of the nucleotide sequence of SEQ ID NO.1;

3) with step 2) in expression vector change host cell over to, form the reconstitution cell can produce high temperature resistant spermine succsinic acid lyase;

4) culturing step 3) in reconstitution cell;

5) separation, purifying obtain high temperature resistant spermine succsinic acid lyase.

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