CN1417339A - High temperature-resisting cellulase gene sequence and its encoded polypeptide and prepn process - Google Patents
High temperature-resisting cellulase gene sequence and its encoded polypeptide and prepn process Download PDFInfo
- Publication number
- CN1417339A CN1417339A CN 01132116 CN01132116A CN1417339A CN 1417339 A CN1417339 A CN 1417339A CN 01132116 CN01132116 CN 01132116 CN 01132116 A CN01132116 A CN 01132116A CN 1417339 A CN1417339 A CN 1417339A
- Authority
- CN
- China
- Prior art keywords
- high temperature
- polypeptide
- sequence
- gene
- cellulase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a technology to code separate DNA separating with activity and functional isomutant and to recombinant DNA as well as the production of polypeptide with high temperature resistant cellulase activity or its functional isomutant by the said separate DNA. Based on the sequencing and analysis of Tengchong thermophilic anaerobe whole genome, high temperature resistant cellulase gene is cloned and separated. The gene is useful in preparing corresponding transgenic microbe, plant and animal and recovering the gene encoded enzyme. In addition, the present invention also provides the amino acid sequence of the polypeptide with high temperature resistant cellulase activity and its functional identity. The present invention also provides the method of preparing, separating and purifying the polypeptide with high temperature resistant cellulase activity.
Description
Technical field
The present invention relates to the microorganism hereditary field, be specifically related to belong to a kind of isolating high temperature-resisting cellulase gene sequence and the encoded polypeptides and the preparation method of thermophilc anaerobe.High temperature-resisting cellulase can be widely used in technical fields such as gene clone, gene diagnosis and gene therapy.
Background technology
The complicated enzyme that cellulase is made up of multiple lytic enzyme is mainly to come from fungi and bacterium.According to the difference of each enzyme function wherein, be divided into following three major types: (1) endoglucanase (1.4-β-D-glucan giucanohydrolase or endo-1.4-β-D-glucanase E.C 3.2.1.4,), this fermentoid general action is in the noncrystalline domain of Mierocrystalline cellulose inside, random hydrolysis β-1.4-glycosidic link, with the brachymemma of long chain cellulose molecule, produce the small molecules Mierocrystalline cellulose of a large amount of band non reducing ends.(2) dextran excision enzyme (1.4-β-D-glucan cellobiohydrolase or exo-1.4-β-D-glucanase, E.C3.2.1.91,), this fermentoid acts on Mierocrystalline cellulose linear molecule end, hydrolysis β-1.4-glycosidic link, cut next cellobiose molecule, so be called cellobiohydrolase (cellobiohydrolase) again at every turn.(3) beta-glucan glycosides enzyme (β-1.4-glucosidase, E.C3.2.1.21), this fermentoid generally is hydrolyzed into glucose molecule with cellobiose.
Focusing on of current people's research improves on production of enzyme, vigor and the stability.Wang Yi just waits (1981) that natural wild strain is screened, and has obtained highly active desirable strain.Usefulness NTG such as Tao Shuxing and UV etc. carry out mutagenic treatment to the funnel-form oyster mushroom strain, obtain the vigor mutants which had HCA15 higher 1.86 times than former bacterial strain of avicelase.Yu Fengming etc. are by Sn-9106 bacterial strain enzyme activity maximum in the solid medium of 10% wheat bran content, and along with the increase of wheat bran content, cellulase activity reduces gradually.The cellulase that Chahal report Trichodermareesei is produced by solid state fermentation is lower than the productive rate height and the cost of liquid state fermentation.Zhang Yongliangs etc. (1992) are used two ring ethyl carbonyl diimine activation sodium alginates, connect with cellulase then, cellulase after modifying like this has enhancing largely to pH value, thermostability, and (Hg2+, Cu2+, Ag2+, Ca2+) has stronger resistibility to some inhibitor.Sparson (1989) all utilizes immobilization technologies such as carrier combination and embedding that cellulase is carried out stabilization treatment, has obtained effect preferably.In addition, adopt biotechnology (transformation and the design of gene clone, genetic expression, cellulase protein molecule) to obtain desired high cellulase-producing.At present, people are considering to adopt manual simulation's enzyme to further investigate cellulase.
Because thermophile bacteria can be used for production plurality of enzymes preparation, for example cellulase, proteolytic enzyme, amylase, lipase, inulinase etc., have Heat stability is good, rate of catalysis reaction height by the zymin that produces in these microorganisms, be easy at room temperature preserve, in recent years, the thermophile bacteria enzyme becomes the research focus.Shimizus has reported the clone of thermostability cellulose enzyme gene.Saito (1990) has reported the effect of the plain enzyme gene of thermophilic anaerobic heat resistant fibre in yeast saccharomyces cerevisiae again.
In sum, the application prospect of cellulase is very wide, is that deep discussion still need be done in aspects such as composition, thermotolerance, operative temperature, pH value and protease inhibitor hydrolysis ability to enzyme.
Cellulase has application prospect in following several respects:
1, the application in petroleum fracturing liquid: oil may be split derivatived cellulose (CMC) and the fracturing liquid hydrolysis of vegetable jelly class in the liquid and generate non-toxic substances such as glucose.
2, the application on textile industry: may become a kind of specific enzyme that is used for the super gentle thing arrangement of natural fabric.
3, the application in veterinary drug: may become a kind of new drug of the big domestic animal gastroenteropathy of treatment, assert efficient the reaching more than 98% of treatment through tests such as military veterinary institutes.
4, the application in brewery industry: in soy sauce, vinegar production, use the utilization ratio that can improve protein, starch and fruit juice.
5, the application of cellulase on Animal nutrition: decompose abundant renewable resources-Mierocrystalline cellulose, making it exploitation becomes feedstuff raw material.
Tengchong thermophilc anaerobe (Thermoanaerobacter tangcongensis) 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 ofmicroorganisms AS 1.2430
T=JCM 11007
T).This thermophilc anaerobe is the distinctive species of China, and the high temperature-resisting cellulase that is had in its body also has own its specific structure.
Summary of the invention
The technical problem to be solved in the present invention is: provide a kind of very practical, well behaved high temperature-resisting cellulase gene sequence and encoded polypeptides and preparation method.The present invention utilizes predictive genes software to obtain one section sequence that contains 1041 base pairs by to Tengchong thermophilc anaerobe genome sequencing, with this gene of pcr amplification, is cloned into then among the expression vector pBV220, and obtains high expression level in intestinal bacteria.Studies show that by gene sequencing research and protein function this gene is the gene order of a coding high temperature-resisting cellulase.The high temperature-resisting cellulase 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 amino acid sequence of polypeptide and the functional equivalent body with high temperature resistant DNA polymerase activity.Simultaneously, the present invention also provides preparation, separates, and purifying has the method for the polypeptide of high temperature resistant DNA polymerase activity.
The technical solution adopted in the present invention is: the present invention relates to a kind of separated DNA, its can the encode nucleotide sequence of polypeptide with high temperature resistant DNA polymerase activity.
Above-mentioned separated DNA, it also has the nucleotide sequence of the modified forms of the polypeptide of the aminoacid sequence among the coding SEQ ID NO:2 or described polypeptide, on this modified forms function quite or relevant with high temperature-resisting cellulase.
Above-mentioned separated DNA, it also has the polynucleotide sequence of SEQ ID NO:1 and its mutant form, and mutation type comprises disappearance, nonsense, insertion, missense.
The invention still further relates to a kind of polypeptide separated, it has high temperature resistant DNA polymerase activity.
Above-mentioned polypeptide separated, it has the polypeptide of the aminoacid sequence among the SEQ ID NO:2, or its conservative property variation polypeptide or its active fragments or its reactive derivative.
The invention still further relates to a kind of carrier, it contains the separated DNA of the nucleotide sequence of the polypeptide with high temperature resistant DNA polymerase activity of encoding.
The invention still further relates to the above-mentioned carrier transformed host cells of a kind of usefulness, comprise prokaryotic cell prokaryocyte or eukaryotic cell.
The invention still further relates to a kind of method of producing high temperature-resisting cellulase, comprising:
1) isolates nucleotide sequence SEQ.ID NO.1 with coding high temperature-resisting cellulase polypeptide;
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, formation can be produced the reconstitution cell of high temperature-resisting cellulase polypeptide;
4) culturing step 3) in reconstitution cell;
5) polypeptide that separate, purifying obtains high temperature resistant DNA polymerase activity.
The invention has the beneficial effects as follows: the high temperature-resisting cellulase of transgenic microorganism or animals and plants the present invention is used to produce to(for) preparation, and it is useful to reclaim the enzyme that obtains this genes encoding.
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-resisting cellulase gene " refer to the encode nucleotide sequence of polypeptide with high temperature resistant DNA polymerase activity is 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-resisting cellulase albumen " refers to have the SEQ ID NO.2 polypeptide of sequence of high temperature resistant DNA polymerase activity.This term also comprises the varient of SEQ ID NO.2 sequence, and these varients have and natural high temperature-resisting cellulase 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-resisting cellulase.
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-resisting cellulase of the present invention, high temperature-resisting cellulase gene sequence operationally can be connected in expression regulation sequence, thereby form the high temperature-resisting cellulase 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 (penbritins, 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-resisting cellulase full length gene 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.
Description of drawings
Fig. 1 is an embodiment of the invention order-checking library construction block diagram.
Fig. 2 is embodiment of the invention order-checking and data analysis schema.
Embodiment
To be described in detail the present invention by embodiment below:
1) makes 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 the storehouse flow process as shown in Figure 1.
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 principle of electrophoresis to check 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.
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 here 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.
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 (Waterman MS, 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.
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 (intergenicregion) 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 Artemis sequence 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.
6. the preparation of high temperature-resisting cellulase and purification
According to the high temperature-resisting cellulase complete encoding sequence that arrives (SEQ ID NO.1) of gene annotation among the embodiment, 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.According to the predictive genes result,,, increased Xbal I site at upstream primer for ease of the clone at this upstream region of gene and downstream design primer.
For obtaining to have the gene mutation body of part base deletion, design primer again in the both sides of required disappearance part.All primers are responsible for synthetic by Beijing AudioCodes biotech firm.
With the 1st) plasmid DNA in the order-checking library that obtains in the part is 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-that contains expression vector?
The engineering bacteria DH5 α-pGEX-2T-of picking list bacterium colony? containing in the LB substratum of 100gg/ml penbritin jolting in 3ml cultivates 37 ℃ and spends 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.? the protein band at Kda place is high temperature-resisting cellulase.
Sum up
The present invention has introduced a kind of high temperature-resisting cellulase or fragment and gene thereof of purifying.It has the activity of Tengchong thermophilc anaerobe (T.tengcongensis) high temperature-resisting cellulase.Is the molecular weight of pure enzyme under SDS-PAGE approximately among the present invention? high temperature-resisting cellulase not only has the activity of cellulase but also have higher thermostability, has broad application prospects in industrial production, petrochemical complex, medicine production and feed processing and other fields.
Sequence table
1.SEQ?ID?NO.1
(1) sequence signature:
A. length: 1041 base pairs
B. type: DNA/RNA
C. chain: two strands
D. geometry: linearity
(2) molecule type: Nucleotide
( 3 ) ：atggattataaggagctgttaaaaaagcttagtgaaagtcacggggtctccggacatgaaagaggaatttatcagcttttgaaaaaagaatttgaagagatttccgatgaagttctagaggacaattttggaaatttaatttttaaaaagaagggtttaaaaggcaagtataaggtgatgctggcagcccatttggatgaaatagggcttatggtgaaagatattgatgagaaaggatttataaaatttacaccggttggaggagtggaccaaagaacccttccttctcaagaagtcatagtgcacggtaaaaaagaattattgggagttatagggagtaaaccgcctcatctcttatcctccgaagatatggagaaggcaataaaaatagacgatatgtacgtagacgtagggcttccgaaagaggaagtagagaagctggtaagcattggagatattataactgtgaaaagggagtttagagaacttttgaatgataatgtttcaggaaaggctttagacgatagggcaggggttgtggtgatggcagtatgccttgatgaactaaggaagatgtaccattatcacgacgtgtatgctgtagctactcttcaggaagaggtgggggtaagaggagctataacttcttcttacaatattgaacctgacatagctatagcgattgatgtgactcatgcgaaagcgaggggagttagccgcgacatagagatagggaaagggcctgcgattgggaaggggcctaatattcatcctgctgtatataaagggcttgtagatatagcgaaaaaatacaatataaattatcagatagagccgcttcctgggcattcaggcactgatgcatgggcaatccaggtgtcaaaaaaaggagttcctacagggcttgtgtcaatacctttgaagtacatgcatacttctgtggaaactgctaatatgaaagacataatagaaagcggcagactgctggcacactatattgccaatctgcctgaggaattggagggacatttatgttattaa
2.SEQ?ID?NO.2
(1) sequence signature:
A. length: 346 amino acid
B. type: polypeptide
C. chain: strand
D. geometry: solid
(2) molecule type: protein
(3) sequence description MDYKELLKKLSESHGVSGHERGIYQLLKKEFEEISDEVLEDNFGNLIFKKKGLKGK YKVMLAAHLDEIGLMVKDIDEKGFIKFTPVGGVDQRTLPSQEVIVHGKKELLGVIG SKPPHLLSSEDMEKAIKIDDMYVDVGLPKEEVEKLVSIGDIITVKREFRELLNDNV SGKALDDRAGVVVMAVCLDELRKMYHYHDVYAVATLQEEVGVRGAITSSYNIEPDI AIAIDVTHAKARGVSRDIEIGKGPAIGKGPNIHPAVYKGLVDIAKKYNINYQIEPL PGHSGTDAWAIQVSKKGVPTGLVSIPLKYMHTSVETANMKDIIESGRLLAHYIANL PEELEGHLCY
Claims (8)
1. separated DNA is characterized in that: it is the nucleotide sequence that coding has the polypeptide of high temperature resistant DNA polymerase activity.
2. separated DNA according to claim 1, it is characterized in that: its coding has the nucleotide sequence of the modified forms of the polypeptide of the aminoacid sequence among the SEQ ID NO:2 or described polypeptide, on this modified forms function quite or relevant with high temperature-resisting cellulase.
3. separated DNA according to claim 1 is characterized in that: it 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 DNA polymerase activity.
5. polypeptide separated according to claim 4 is characterized in that: it has the polypeptide of the aminoacid sequence among the SEQ ID NO:2, or its conservative property variation polypeptide or its active fragments or its reactive derivative.
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. one kind prepares the proteic method of high temperature-resisting cellulase, it is characterized in that this method comprises:
1) isolates the proteic nucleotide sequence SEQ of coding high temperature-resisting cellulase IDNO.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, formation can be produced the proteic reconstitution cell of high temperature-resisting cellulase;
4) culturing step 3) in reconstitution cell;
5) separation, purifying obtain high temperature-resisting cellulase albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01132116 CN1417339A (en) | 2001-11-06 | 2001-11-06 | High temperature-resisting cellulase gene sequence and its encoded polypeptide and prepn process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01132116 CN1417339A (en) | 2001-11-06 | 2001-11-06 | High temperature-resisting cellulase gene sequence and its encoded polypeptide and prepn process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1417339A true CN1417339A (en) | 2003-05-14 |
Family
ID=4671159
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01132116 Pending CN1417339A (en) | 2001-11-06 | 2001-11-06 | High temperature-resisting cellulase gene sequence and its encoded polypeptide and prepn process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1417339A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102771630A (en) * | 2012-06-07 | 2012-11-14 | 宁夏伊品生物科技股份有限公司 | Fermentation and coating of lysine and cellulose |
-
2001
- 2001-11-06 CN CN 01132116 patent/CN1417339A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102771630A (en) * | 2012-06-07 | 2012-11-14 | 宁夏伊品生物科技股份有限公司 | Fermentation and coating of lysine and cellulose |
| CN102771630B (en) * | 2012-06-07 | 2013-10-16 | 宁夏伊品生物科技股份有限公司 | Fermentation and coating of lysine and cellulose |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1417339A (en) | High temperature-resisting cellulase gene sequence and its encoded polypeptide and prepn process | |
| CN1500872A (en) | High temperaturebeta- glucosaccharase, coding gene and uses thereof | |
| CN1417338A (en) | High temperature-resisting DNA polymerase gene sequence and its encoded polypeptide and prepn process | |
| CN1418954A (en) | High temp. resistant HSP 70 molecular chaperone and coded polypeptide and preparation process thereof | |
| CN1164750C (en) | High-temperature resistant transaldolase gene, polypeptide coded by same and preparation method of polypeptide | |
| CN1371996A (en) | High-temperature resistant spermidine synthase gene sequence, polypeptide coded by same and method for preparing polypeptide | |
| CN1198928C (en) | High-temperature resistant ribosomal protein L15 gene and its coded polypeptide and preparing process | |
| CN1379103A (en) | High-temperature resistant glucan phosphorylase gene and polypeptide coded by same and preparation method | |
| CN1420172A (en) | High-temp resistant CTP synthetase gene, polypeptide coded therewith and preparing method thereof | |
| CN1390943A (en) | High-temp. resistant 6-phosphoglucose isomerase gene and its coded polypeptide and preparing process | |
| CN1366059A (en) | Refractory phosphomannose isomerase gene and its polypeptide coded by it and preparing process | |
| CN1379101A (en) | Refractory thymidine phosphorylase gene and its polypeptide coded by it and preparing process | |
| CN1379098A (en) | Refractory histidinol dehydrogenase gene and its polypeptide coded by it and preparing process | |
| CN1379094A (en) | High-temperature-resistant tyrosyl tRNA synthetase gene, polypeptide coded by same and preparation method | |
| CN1379099A (en) | High-temperature-resistant 6-phosphogluconate dehydrogenase gene and polypeptide coded by same and preparation method | |
| CN1367249A (en) | High-temp. resistant chorismate synthase gene and its coded polypeptide and preparation method | |
| CN1379108A (en) | Refractory phosphoheptose isomerase gene and its polypeptide coded by it and preparing process | |
| CN1379100A (en) | High-temp. resistant dihydroorotate synthase gene and its coded polypeptide and preparing method | |
| CN1390941A (en) | Refractory cytidylic acid kinase gene and its polypeptide coded by it and preparing process | |
| CN1379107A (en) | High-temp. resistant cysteine synthase gene and its coded polypeptide and preparing method | |
| CN1364908A (en) | High-temperature resistant enolase gene and its coded polypeptide and preparation method | |
| CN1420179A (en) | High-temp. resistant reverse gyrase gene, polypeptide coded therewith and preparing method thereof | |
| CN1366058A (en) | Refractory triosephosphate isomerase gene and its polypeptide coded by it and preparing process | |
| CN1367250A (en) | High-temperature-resistant isocitrate dehydrogenase gene, polypeptide coded by same and preparation method of polypeptide | |
| CN1382804A (en) | High-temperature-resistant phosphoribosyl anthranilate isomerase gene, polypeptide coded by same and preparation method of polypeptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |