CN1702173A - Coded high temperature acid alpha-amylase gene and its synthesis method, cloning and expression - Google Patents
Coded high temperature acid alpha-amylase gene and its synthesis method, cloning and expression Download PDFInfo
- Publication number
- CN1702173A CN1702173A CN 200510081647 CN200510081647A CN1702173A CN 1702173 A CN1702173 A CN 1702173A CN 200510081647 CN200510081647 CN 200510081647 CN 200510081647 A CN200510081647 A CN 200510081647A CN 1702173 A CN1702173 A CN 1702173A
- Authority
- CN
- China
- Prior art keywords
- amyf
- oligonucleotide
- high temperature
- gene
- acid alpha
- 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)
Abstract
It is a coding high-temperature acidic alpha-amylase gene and the preparation, clone, and expression, belonging to the field of microbiological gene engineering. The invention provides a synthesized high-temperature acidic alpha-amylase gene amyF and the method for preparation, the nucleotide sequence of amyF, the clone carrier pSK-amyF containing amyF and secretory expression carrier pBL-amyF for bacillus, and the construction of recombination bacillus, the preparation of recombinase and the further analysis of enzymatic nature with pBL-amyF. It can express effectively and prepare recombinant high-temperature acidic alpha-amylase in bacillus, and realizes the quick hydrolysis of starch in high-temperature acidic condition.
Description
Technical field
A kind of coded high temperature acid alpha-amylase gene and synthetic, clone thereof and express belong to the microbiological genetic engineering field.Specifically overlap the oligonucleotide of encoding novel high temperature acid alpha-amylase by engineered means design one, use the gene total synthesis method to obtain encoding novel high temperature acid alpha-amylase gene amyF again, be used in and realize efficiently expressing and preparing recombinant protein in the genus bacillus.
Technical background
Starch is a family macromolecule material that is formed by glucose polymerisation.Starch is made up of amylose starch and amylopectin two portions.Starch small grain is generally circular or irregular shape, and size is 1-100 μ m, keeps its structure with inner hydrogen bond.Starch granules inside is complicated crystal structure, and the composition that is positioned at particle surface owing to reasons such as moisture losses forms a kind of hard-packed adventitia, and external force is had stronger resistibility.Therefore, the water insoluble and organic solvent of starch granules.Starch is the industrial main raw material of producing glucose and dextrin.Traditional method is hydrolyzed into micromolecular dextrin and glucose with acid with starch, and this method is replaced by enzymatic hydrolysis at present.
Starch is as the application of raw material in industry, and its topmost mode is that starch is hydrolyzed into glucose, maltose or oligosaccharides syrup.These syrup are used as raw material subsequently and are used for as industrial production such as alcohol, organic acid, amino acid or directly are used for other industrial circles as product.The starch hydrolysis process process usually comprises following two steps: liquefaction and saccharification.In the starch liquefacation process, starch granules forms emulsion in cold water, after being heated to 60～700 ℃, the starch granules water-swelling, volume increases sharply, and crystalline structure is destroyed, the particle adventitia splits, and forms a kind of thick liquid (this process is called gelatinization) of pasty state.During the higher starch fluid gelatinization of concentration, liquefaction rapidly, otherwise the viscosity of starch fluid sharply rising equipment can't be moved, the starch of gelatinization can take place aging after temperature reduces and can't liquefy in addition.Therefore, must to tolerate starch pasting at least temperature required for the amylase that is used for starch liquefacation.After the starch pasting, molecular chain is directly exposed under the effect of enzyme molecule, and molecular chain is cut off rapidly, shortens, and finally forms low-molecular-weight dextrin, and liquid viscosity sharply descends, and this process is called as liquefaction.Main flow technology in the existing heavy industrialization starch hydrolysis process is moment high-temperature injection liquefaction, and the internal temperature of starch wine with dregs reaches more than 1050 ℃ during liquefaction.In addition, it is tactful all to adopt waste liquid to reflux in sugaring that the present overwhelming majority is raw material with starch and the zymotechnique, and the pH value of starch wine with dregs can so be reduced to about 4.5-5.0.Therefore, the high reactivity of α-Dian Fenmei under high temperature and acidic conditions is extremely important to the optimization and the simplification of starch liquefacation technology.
The mode of acquisition new type high temperature acid alpha-amylase is summarized two kinds.A kind of is on the basis of existing high temperature resistant α-Dian Fenmei, by relevant means such as gene clone, protein engineerings protein molecule is transformed, and obtains high temperature resistant, acid-resistant alpha-amylase mutant with this.The existing many successful reports of the work of this respect.Another kind is to seek high temperature acid alpha-amylase novel molecular (Jorgenson etc. 1997, ApplEnviron Microbiol) from occurring in nature, as the α-Dian Fenmei of Pyrococcus sp., but produces enzyme level less than 2U/mL.But no matter by any mode, the highly active α-Dian Fenmei molecule that has under the high-temperature acidic condition that is obtained must be through efficiently just having the industrial application meaning with a large amount of cheap preparations.
Genus bacillus is a leather Lan Shi positive bacteria, in industrial production, use for many years as subtilis, bacillus megaterium, bacstearothermophilus, Bacillus licheniformis etc., be proved safe and reliable and have the ability of efficient, a large amount of secretory proteins.With the genus bacillus is that the host cell efficiently expressing exogenous gene also has some successful reports.
Summary of the invention
The purpose of this invention is to provide a kind of coded high temperature acid alpha-amylase gene and synthetic, clone thereof and express, adopt the external combination technique of chemosynthesis and molecule to seek a kind of high temperature acid alpha-amylase gene that in genus bacillus, efficiently expresses.
Technical solution of the present invention:, analyze the structure, the functional zone composition characteristic that obtain alpha-amylase family with self-built α-Dian Fenmei protein families database.With self-built tRNA analysis software subtilis 168 and Bacillus licheniformis ATCC14580 genome, obtain tRNA encoding gene feature and codon usage frequency parameter.Classify the basis as with the nucleotides sequence of Pyrococcus sp.DSM3638 α-Dian Fenmei encoding gene again, use DNAMAN software, design 78 and be used for new gene synthetic oligonucleotide, oligonucleotide is adjacent on same chain, and with its complementary chain some bases are arranged are eclipsed, and the melting temperature(Tm) of overlapping region is controlled at 45-50 ℃.Carry out the synthetic of full gene with two external splicings of step and amplification technique, obtain full-length gene amyF.Gene amyF is cloned in the EcoR V site of pBlueScript II SK (-), obtains recombinant plasmid pSK-amyF.Recombinant plasmid pSK-amyF is used for the sequencing of amyF and subsequent experimental amyF is provided.SmaI and EcoRI enzyme are cut pSK-amyF, and rubber tapping is reclaimed and obtained total length amyF.AmyF is cloned into SmaI and the EcoRI site of secreted expression carrier pBL-WZX, obtains recombinant plasmid pBL-amyF.Transform Bacillus licheniformis with recombinant plasmid pBL-amyF, make up the reorganization bacterium and the preparation recombinase that contain gene amyF.
The nucleotide sequence of gene amyF is as follows:
gcaaaatact?tggaacttga?agaaggagga?gttatcatgc?aagcatttta?ttgggatgtt 60
ccgggcggag?gaatttggtg?ggatcatatc?agatcgaaaa?ttccggaatg?gtatgaagct 120
ggaatctcag?caatctggct?gccgccgccg?agcaaaggca?tgagcggagg?atattcaatg 180
ggctacgatc?cgtatgatta?ctttgatctg?ggcgaatact?accagaaagg?aacggtcgaa 240
acgcgctttg?gatcaaaaga?agaactggtg?agattgatcc?aaacggccca?tgcctatgga 300
atcaaagtca?tcgccgatgt?cgttatcaac?catagggctg?gcggcgacct?ggaatggaac 360
ccgttcgttg?gagattacac?atggacagac?ttttctaaag?ttgcctcagg?caaatataca 420
gctaactatc?tggacttcca?tccgaacgaa?cttcattgct?gcgacgaagg?aacgtttgga 480
ggatttccgg?atatctgcca?tcataaagaa?tgggatcagt?actggctgtg?gaaaagcaat 540
gaaagctatg?ctgcttatct?gagaagcatc?ggatttgatg?gctggagatt?tgactatgtt 600
aaaggctatg?gagcttgggt?tgtcagagac?tggcttaatt?ggtggggagg?ctgggcagtt 660
ggagaatact?gggacacaaa?tgtcgatgca?ctgctgagct?gggcatatga?aagcggcgca 720
aaagtctttg?acttcccgct?gtactataaa?atggatgaag?catttgacaa?taacaacatt 780
ccggcactgg?tctatgccct?gcaaaacgga?caaacggtcg?tttcgagaga?tccgtttaaa 840
gcagtcacgt?tcgttgccaa?tcatgacaca?gatatcatct?ggaacaaata?tccggcatat 900
gcgttcatct?tgacatatga?aggacagccg?gtcatcttct?acagggactt?tgaagaatgg 960
ctgaacaaag?ataaactgat?taacctgatt?tggatccatg?atcatttggc?aggaggaagc 1020
acaacaattg?tctactacga?caacgatgaa?ctgatctttg?tgagaaatgg?agattctaga 1080
aggccgggcc?ttatcacgta?cattaacttg?agcccgaact?gggttggcag?gtgggtctac 1140
gttccgaaat?ttgcaggcgc?ttgcattcat?gaatacacgg?gaaacctggg?aggatgggtc 1200
gataaaagag?tcgatagcag?cggatgggtc?tacctggaag?caccgccgca?tgatccggct 1260
aacggctact?atggctactc?cgtctggagc?tattgcggcg?ttggc 1305
Beneficial effect of the present invention: the invention provides a kind of from the alpha-amylase gene constructional feature, in conjunction with the genus bacillus codon usage frequency, obtain the new type high temperature acid alpha-amylase gene sequence amyF that can efficiently express genus bacillus by full gene synthetic method and molecular evolution method, the preparation method of new type high temperature acid alpha-amylase gene is provided, cloning vector pSK-amyF that contains high temperature acid alpha-amylase gene and the expression vector pBL-amyF that expresses in genus bacillus are provided.Utilize achievement of the present invention, can be used for the structure and the mass preparation of the genetic engineering bacterium of high temperature acid alpha-amylase suitability for industrialized production, improve the starch hydrolysis efficiency and simplify starch liquefacation and saccharification operation.The gene synthesis strategy that is provided can also be used for the molecular modification purpose of other genes.
1, high temperature acid alpha-amylase gene amyF of the present invention can realize in genus bacillus that efficient secretory expression, expression level are that Pyrococcus sp.DSM3638 produces more than 225 times of enzyme level.
2, the optimum temperature of the expression product of gene amyF of the present invention is 95-98 ℃, and the suitableeest action pH scope is 4.8-6.5, and has good thermostability.Quick hydrolyzed starch under the high-temperature acidic condition.
3, full gene synthesis technology of the present invention can be used for the complete synthesis of other gene.
Description of drawings
The building-up process of Fig. 1 gene amyF.
The physical map of Fig. 2 recombinant plasmid pBL-amyF.
The optimal reactive temperature of Fig. 3 recombinase.
The relative vigor of Fig. 4 recombinase under condition of different pH.
Embodiment
Embodiment 1: high temperature acid alpha-amylase gene is complete synthesis with the design of oligonucleotide sequence and the chemosynthesis of oligonucleotide.
According to α-Dian Fenmei protein families analysis of biological information and genus bacillus tRNA genomic constitution and codon use characteristic, nucleotides sequence with Pyrococcus sp.DSM3638 α-Dian Fenmei encoding gene is classified the basis as, the nucleotide sequence of the high temperature acid alpha-amylase encoding gene that Computer Design makes new advances, the one group of oligonucleotide formed by 78 sequences that used the DNAMAN software design.Wherein among F0 and the R38 underscore mark be respectively EcoRI and SmaI restriction enzyme site.The utilization dna synthesizer obtains this group oligonucleotide through chemosynthesis, and the nucleotides sequence of 78 oligonucleotide is classified as:
F0 gagagaaa
ga?attctacttg?gaacttgaag?aaggagga
R0 aaaatgcttg catgataact cctccttctt?caagtt
F1 gttatcatgc aagcatttta ttgggatgtt?ccgg
R1 accaaattcc tccgcccgga acatcccaat
F2 gcggaggaat ttggtgggat catatcagat?cga
R2 cataccattc cggaattttc gatctgatat?gatccc
F3 aaattccgga atggtatgaa gctggaatct?cagc
R3 gcggcagcca gattgctgag attccagctt
F4 aatctggctg ccgccgccga gcaaaggc
R4 gaatatcctc cgctcatgcc tttgctcggc?g
F5 atgagcggag gatattcaat gggctacgat?cc
R5 cccagatcaa agtaatcata cggatcgtag?cccatt
F6 gtatgattac tttgatctgg gcgaatacta?ccagaaagga
R6 cgcgtttcga ccgttccttt ctggtagtat?tcg
F7 acggtcgaaa cgcgctttgg atcaaaagaa?gaac
R7 ttggatcaat ctcaccagtt cttcttttga?tccaaag
F8 tggtgagatt gatccaaacg gcccatgcct?a
R8 gcgatgactt tgattccata ggcatgggcc?gt
F9 tggaatcaaa gtcatcgccg atgtcgttat?caacc
R9 gccgccagcc?ctatggttga?taacgacatc?g
F10 atagggctgg?cggcgacctg?gaatggaacc
R10 gtaatctcca?acgaacgggt?tccattccag?gtc
F11 cgttcgttgg?agattacaca?tggacagact?tttct
R11 gcctgaggca?actttagaaa?agtctgtcca?tgt
F12 aaagttgcct?caggcaaata?tacagctaac?tatctgg
R12 cgttcggatg?gaagtccaga?tagttagctg?tatattt
F13 acttccatcc?gaacgaactt?cattgctgcg?a
R13 tccaaacgtt?ccttcgtcgc?agcaatgaag?tt
F14 cgaaggaacg?tttggaggat?ttccggatat?ctgc
R14 tgatcccatt?ctttatgatg?gcagatatcc?ggaaatcc
F15 catcataaag?aatgggatca?gtactggctg?tggaa
R15 gcatagcttt?cattgctttt?ccacagccag?tac
F16 aagcaatgaa?agctatgctg?cttatctgag?aagca
R16 cagccatcaa?atccgatgct?tctcagataa?gca
F17 tcggatttga?ggctggagat?ttgactatgt?taaagg
R17 aacccaagct?ccatagcctt?taacatagtc?aaatctc
F18 ctatggagct?tgggttgtca?gagactggct?taa
R18 agcctcccca?ccaattaagc?cagtctctga?c
F19 ttggtgggga?ggctgggcag?ttggagaat
R19 acatttgtgt?cccagtattc?tccaactgcc?c
F20 actgggacac?aaatgtcgat?gcactgctga
R20 ctttcatatg?cccagctcag?cagtgcatcg
F21 gctgggcata?tgaaagcggc?gcaaaagtct
R21 cagcgggaag?tcaaagactt?ttgcgccg
F22 ttgacttccc?gctgtactat?aaaatggatg?aagca
R22 ggaatgttgt?tattgtcaaa?tgcttcatcc?attttatagt?a
F23 tttgacaata?acaacattcc?ggcactggtc?tatgc
R23 tgtccgtttt?gcagggcata?gaccagtgcc
F24 cctgcaaaac?ggacaaacgg?tcgtttcgag
R24 actgctttaa?acggatctct?cgaaacgacc?gtt
F25 agatccgttt aaagcagtca?cgttcgttgc?ca
R25 gatgatatct gtgtcatgat?tggcaacgaa?cgtg
F26 atcatgacac agatatcatc?tggaacaaat?atccggc
R26 gtcaagatga acgcatatgc?cggatatttg?ttcca
F27 atatgcgttc atcttgacat?atgaaggaca?gccg
R27 tccctgtaga agatgaccgg?ctgtccttca?tat
F28 gtcatcttct acagggactt?tgaagaatgg?ctgaa
R28 caggttaatc agtttatctt?tgttcagcca?ttcttcaaag
F29 caaagataaa ctgattaacc?tgatttggat?ccatgatcat?t
R29 gcttcctcct gccaaatgat?catggatcca?aat
F30 tggcaggagg aagcacaaca?attgtctact?acg
R30 agatcagttc atcgttgtcg?tagtagacaa?ttgttgt
F31 acaacgatga actgatcttt?gtgagaaatg?gagattc
R31 ggcccggcct tctagaatct?ccatttctca?caa
F32 tagaaggccg ggccttatca?cgtacattaa?cttga
R32 aacccagttc gggctcaagt?taatgtacgt?gataa
F33 gcccgaactg ggttggcagg?tgggtcta
R33 tgcaaatttc ggaacgtaga?cccacctgcc
F34 cgttccgaaa tttgcaggcg?cttgcattca
R34 ggtttcccgt gtattcatga?atgcaagcgc?c
F35 tgaatacacg ggaaacctgg?gaggatgggt?c
R35 gctgctatcg actcttttat?cgacccatcc?tccca
F36 gataaaagag tcgatagcag?cggatgggtc?tacctgg
R36 atgcggcggt gcttccaggt?agacccatcc
F37 aagcaccgcc gcatgatccg?gctaacgg
R37 acggagtagc catagtagcc?gt?tagccgga?tc
F38 ctactatggc tactccgtct?ggagctattg?cggc
R38 aaa
cccgggctagccaacgc?cgcaatagct?ccag
Embodiment 2. high temperature acid alpha-amylase amyF's is complete synthesis
The complete synthesis employing oligonucleotide in vitro combination technique of high temperature acid alpha-amylase amyF is carried out in two steps.The first step in 50 μ l reaction systems, adds 200mmol/L dNTPs, whole 78 oligonucleotide of 0.1mmol/L and the PfuDNA polymerase of 0.5U.Oligonucleotide splicing pcr amplification condition is 1 * (95 ℃ of 2min); 35 * (94 ℃ of 30s, 52 ℃ of 30s, 70 ℃ of 2min30s); 1 * (70 ℃ of 10min).In second step, in 50 μ l reaction systems, add above-mentioned oligonucleotide splicing product 2 μ l, 200mmol/L dNTPs, 1mmol/L primers F 0, the Pfu DNA polymerase of 1mmol/L primer R38 and 0.5U.The pcr amplification condition is 1 * (95 ℃ of 2min); 35 * (95 ℃ of 30s, 52 ℃ of 30s, 70 ℃ of 2min30s); 1 * (70 ℃ of 10min).The synthetic gene is introduced EcoRI and SmaI restriction enzyme site at the two ends of gene respectively by primers F 0 and primer R38.Behind the synthetic gene purifying that obtains, cut and be cloned into EcoRI and SmaI enzyme in the EcoR V site of pBlueScript II SK (-), obtain cloning vector pSK-amyF, sequencing confirms that gene synthesizes successfully (Fig. 1).
Embodiment 3: the structure of recombinant plasmid expression vector pBL-amyF
With EcoRI and SmaI enzyme cutting clone carrier pSK-amyF, full-length gene amyF is reclaimed in rubber tapping.Gene amyF is cloned among the EcoRI and SmaI restriction enzyme site of the integrated expression vector pBL-WZX of genus bacillus, obtains recombinant plasmid pBL-amyF (Fig. 2).In recombinant plasmid pBL-amyF, gene amyF is respectively at P
AmyLTranscribe and translate under the control, in the secretion of the peptide-mediated amyF gene product of amyL encoded signals.
The expression of embodiment 4:amyF in genus bacillus
Behind the recombinant plasmid pBL-amyF purifying, transform Bacillus licheniformis by electric method for transformation.Transformation mixture is at the LB of the sulphuric acid kanamycin of 5 μ g/mL plate screening positive transformant.In the LB substratum, behind the cultivation 48h, the centrifuging and taking supernatant liquor is surveyed enzyme and is lived with resulting recon direct inoculation.The high yield enzyme level of reorganization bacterium reaches 4.5mg/ml, and expression level is 225 times that Pyrococcus sp.DSM3638 produces enzyme level.Choose wherein strain reorganization bacterium and be used for the preparation of recombinase and the further analysis of zymologic property.The result shows optimum temperuture 95-98 ℃ (Fig. 3) of recombinase, optimal pH 4.8-6.5 (Fig. 4).
Claims (4)
1, a kind of high temperature acid alpha-amylase gene amyF of synthetic, its nucleotides sequence is classified as:
gcaaaatact?tggaacttga?agaaggagga?gttatcatgc?aagcatttta?ttgggatgtt 60
ccgggcggag?gaatttggtg?ggatcatatc?agatcgaaaa?ttccggaatg?gtatgaagct 120
ggaatctcag?caatctggct?gccgccgccg?agcaaaggca?tgagcggagg?atattcaatg 180
ggctacgatc?cgtatgatta?ctttgatctg?ggcgaatact?accagaaagg?aacggtcgaa 240
acgcgctttg?gatcaaaaga?agaactggtg?agattgatcc?aaacggccca?tgcctatgga 300
atcaaagtca?tcgccgatgt?cgttatcaac?catagggctg?gcggcgacct?ggaatggaac 360
ccgttcgttg?gagattacac?atggacagac?ttttctaaag?ttgcctcagg?caaatataca 420
gctaactatc?tggacttcca?tccgaacgaa?cttcattgct?gcgacgaagg?aacgtttgga 480
ggatttccgg?atatctgcca?tcataaagaa?tgggatcagt?actggctgtg?gaaaagcaat 540
gaaagctatg?ctgcttatct?gagaagcatc?ggatttgatg?gctggagatt?tgactatgtt 600
aaaggctatg?gagcttgggt?tgtcagagac?tggcttaatt?ggtggggagg?ctgggcagtt 660
ggagaatact?gggacacaaa?tgtcgatgca?ctgctgagct?gggcatatga?aagcggcgca 720
aaagtctttg?acttcccgct?gtactataaa?atggatgaag?catttgacaa?taacaacatt 780
ccggcactgg?tctatgccct?gcaaaacgga?caaacggtcg?tttcgagaga?tccgtttaaa 840
gcagtcacgt?tcgttgccaa?tcatgacaca?gatatcatct?ggaacaaata?tccggcatat 900
gcgttcatct?tgacatatga?aggacagccg?gtcatcttct?acagggactt?tgaagaatgg 960
ctgaacaaag?ataaactgat?taacctgatt?tggatccatg?atcatttggc?aggaggaagc 1020
acaacaattg?tctactacga?caacgatgaa?ctgatctttg?tgagaaatgg?agattctaga 1080
aggccgggcc?ttatcacgta?cattaacttg?agcccgaact?gggttggcag?gtgggtctac 1140
gttccgaaat?ttgcaggcgc?ttgcattcat?gaatacacgg?gaaacctggg?aggatgggtc 1200
gataaaagag?tcgatagcag?cggatgggtc?tacctggaag?caccgccgca?tgatccggct 1260
aacggctact?atggctactc?cgtctggagc?tattgcggcg?ttggc 1305
2. synthetic, the clone of high temperature acid alpha-amylase gene amyF and expressing according to claim 1 is characterized in that
(A) amyF's is synthetic: the nucleotides sequence with Pyrococcus sp.DSM3638 α-Dian Fenmei encoding gene is classified the basis as, use DNAMAN software, designed one group of 78 oligonucleotide, the utilization dna synthesizer obtains this group oligonucleotide through chemosynthesis, and the nucleotides sequence of 78 oligonucleotide is classified as:
F0 gagagaaa
ga?attctacttg?gaacttgaag?aaggagga
R0 aaaatgcttg?catgataact?cctccttctt?caagtt
F1 gttatcatgc?aagcatttta?ttgggatgtt?ccgg
R1 accaaattcc?tccgcccgga?acatcccaat
F2 gcggaggaat?ttggtgggat?catatcagat?cga
R2 cataccattc?cggaattttc?gatctgatat?gatccc
F3 aaattccgga?atggtatgaa?gctggaatct?cagc
R3 gcggcagcca?gattgctgag?attccagctt
F4 aatctggctg?ccgccgccga?gcaaaggc
R4 gaatatcctc?cgctcatgcc?tttgctcggc?g
F5 atgagcggag?gatattcaat?gggctacgat?cc
R5 cccagatcaa?agtaatcata?cggatcgtag?cccatt
F6 gtatgattac?tttgatctgg?gcgaatacta?ccagaaagga
R6 cgcgtttcga?ccgttccttt?ctggtagtat?tcg
F7 acggtcgaaa?cgcgctttgg?atcaaaagaa?gaac
R7 ttggatcaat?ctcaccagtt?cttcttttga?tccaaag
F8 tggtgagatt?gatccaaacg?gcccatgcct?a
R8 gcgatgactt?tgattccata?ggcatgggcc?gt
F9 tggaatcaaa?gtcatcgccg?atgtcgttat?caacc
R9 gccgccagcc?ctatggttga?taacgacatc?g
F10 atagggctgg?cggcgacctg?gaatggaacc
R10 gtaatctcca?acgaacgggt?tccattccag?gtc
F11 cgttcgttgg?agattacaca?tggacagact?tttct
R11 gcctgaggca?actttagaaa?agtctgtcca?tgt
F12 aaagttgcct?caggcaaata?tacagctaac?tatctgg
R12 cgttcggatg?gaagtccaga?tagttagctg?tatattt
F13 acttccatcc?gaacgaactt?cattgctgcg?a
R13 tccaaacgtt?ccttcgtcgc?agcaatgaag?tt
F14 cgaaggaacg?tttggaggat?ttccggatat?ctgc
R14 tgatcccatt?ctttatgatg?gcagatatcc?ggaaatcc
F15 catcataaag?aatgggatca?gtactggctg?tggaa
R15 gcatagcttt?cattgctttt?ccacagccag?tac
F16 aagcaatgaa?agctatgctg?cttatctgag?aagca
R16 cagccatcaa?atccgatgct?tctcagataa?gca
F17 tcggatttga?ggctggagat?ttgactatgt?taaagg
R17 aacccaagct?ccatagcctt?taacatagtc?aaatctc
F18 ctatggagct?tgggttgtca?gagactggct?taa
R18 agcctcccca?ccaattaagc?cagtctctga?c
F19 ttggtgggga?ggctgggcag?ttggagaat
R19 acatttgtgt?cccagtattc?tccaactgcc?c
F20 actgggacac?aaatgtcgat?gcactgctga
R20 ctttcatatg?cccagctcag?cagtgcatcg
F21 gctgggcata?tgaaagcggc?gcaaaagtct
R21 cagcgggaag?tcaaagactt?ttgcgccg
F22 ttgacttccc?gctgtactat?aaaatggatg?aagca
R22 ggaatgttgt?tattgtcaaa?tgcttcatcc?attttatagt?a
F23 tttgacaata?acaacattcc?ggcactggtc?tatgc
R23 tgtccgtttt?gcagggcata?gaccagtgcc
F24 cctgcaaaac?ggacaaacgg?tcgtttcgag
R24 actgctttaa?acggatctct?cgaaacgacc?gtt
F25 agatccgttt?aaagcagtca?cgttcgttgc?ca
R25 gatgatatct?gtgtcatgat?tggcaacgaa?cgtg
F26 atcatgacac?agatatcatc?tggaacaaat?atccggc
R26 gtcaagatga?acgcatatgc?cggatatttg?ttcca
F27 atatgcgttc?atcttgacat?atgaaggaca?gccg
R27 tccctgtaga?agatgaccgg?ctgtccttca?tat
F28 gtcatcttct?acagggactt?tgaagaatgg?ctgaa
R28 caggttaatc?agtttatctt?tgttcagcca?ttcttcaaag
F29 caaagataaa?ctgattaacc?tgatttggat?ccatgatcat?t
R29 gcttcctcct?gccaaatgat?catggatcca?aat
F30 tggcaggagg?aagcacaaca?attgtctact?acg
R30 agatcagttc?atcgttgtcg?tagtagacaa?ttgttgt
F31 acaacgatga?actgatcttt?gtgagaaatg?gagattc
R31 ggcccggcct?tctagaatct?ccatttctca?caa
F32 tagaaggccg?ggccttatca?cgtacattaa?cttga
R32 aacccagttc?gggctcaagt?taatgtacgt?gataa
F33 gcccgaactg?ggttggcagg?tgggtcta
R33 tgcaaatttc?ggaacgtaga?cccacctgcc
F34 cgttccgaaa?tttgcaggcg?cttgcattca
R34 ggtttcccgt?gtattcatga?atgcaagcgc?c
F35 tgaatacacg?ggaaacctgg?gaggatgggt?c
R35 gctgctatcg?actcttttat?cgacccatcc?tccca
F36 gataaaagag?tcgatagcag?cggatgggtc?tacctgg
R36 atgcggcggt?gcttccaggt?agacccatcc
F37 aagcaccgcc?gcatgatccg?gctaacgg
R37 acggagtagc?catagtagcc?gttagccgga?tc
F38 ctactatggc?tactccgtct?ggagctattg?cggc
R38 aaa
cccgggc?tagccaacgc?cgcaatagct?ccag
Adopt the oligonucleotide in vitro combination technique to carry out the synthetic of amyF, the synthetic of amyF carries out in two steps: the first step in 50 μ l reaction systems, adds 200mmol/L dNTPs, 0.1mmol/L whole 78 oligonucleotide and the Pfu DNA polymerase of 0.5U, the pcr amplification condition is 95 ℃ of 2min; 94 ℃ of 30s, 52 ℃ of 30s, 35 circulations of 70 ℃ of 2min 30s; 70 ℃ of 10min; Second step in 50 μ l reaction systems, added above-mentioned oligonucleotide splicing product 2 μ l, 200mmol/L dNTPs, and 1mmol/L primers F 0, the Pfu DNA polymerase of 1mmol/L primer R38 and 0.5U, the pcr amplification condition is 95 ℃ of 2min; 95 ℃ of 30s, 52 ℃ of 30s, 35 circulations of 70 ℃ of 2min 30s; 70 ℃ of 10min; The underscore of primers F 0 sequence partly is manually-injected EcoR I restriction enzyme site, and the underscore of primer R38 sequence partly is manually-injected Sma I restriction enzyme site;
(B) clone of amyF: EcoR I and Sma I restriction enzyme site at synthetic amyF two ends, cut by EcoR I and Sma I enzyme, amyF is cloned into obtains cloning vector pSK-amyF in the EcoRV site of pBlueScript II SK (-);
(C) expression of amyF: Sma I and EcoR I enzyme are cut pSK-amyF, rubber tapping is reclaimed and is obtained total length amyF, amyF is cloned into Sma I and the EcoR I site of expression vector pBL-WZX, obtains the secreted from bacillus expression vector pBL-amyF of high temperature acid alpha-amylase gene amyF.
3, synthesizing according to the described high temperature acid alpha-amylase gene amyF of claim 2, it is characterized in that design oligonucleotides, codon is a genus bacillus preference codon, and oligonucleotide length is that 28-41 base do not wait, and the complementary sequence of 15-25 base is contained at the two ends of adjacent oligonucleotide.
4, as the application of the expression product pBL-amyF of high temperature acid alpha-amylase gene amyF as described in the claim 2, it is characterized in that under the high-temperature acidic condition, having the ability of fast liquefying starch, optimal reactive temperature is 95-98 ℃, and optimal reaction pH value is 4.8-6.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510081647 CN1702173A (en) | 2005-06-27 | 2005-06-27 | Coded high temperature acid alpha-amylase gene and its synthesis method, cloning and expression |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510081647 CN1702173A (en) | 2005-06-27 | 2005-06-27 | Coded high temperature acid alpha-amylase gene and its synthesis method, cloning and expression |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1702173A true CN1702173A (en) | 2005-11-30 |
Family
ID=35632136
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510081647 Pending CN1702173A (en) | 2005-06-27 | 2005-06-27 | Coded high temperature acid alpha-amylase gene and its synthesis method, cloning and expression |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1702173A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514327B (en) * | 2008-02-19 | 2011-01-12 | 无锡德冠生物科技有限公司 | Acid-proof high-temperature amylase strain and method for producing acid-proof high-temperature amylase |
| CN102477437A (en) * | 2010-11-30 | 2012-05-30 | 苏州普瑞信生物技术有限公司 | Acid alpha-amylase with recombination genes and application thereof |
| CN102618518A (en) * | 2012-03-22 | 2012-08-01 | 无锡德冠生物科技有限公司 | Wide-temperature amylase production method |
-
2005
- 2005-06-27 CN CN 200510081647 patent/CN1702173A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514327B (en) * | 2008-02-19 | 2011-01-12 | 无锡德冠生物科技有限公司 | Acid-proof high-temperature amylase strain and method for producing acid-proof high-temperature amylase |
| CN102477437A (en) * | 2010-11-30 | 2012-05-30 | 苏州普瑞信生物技术有限公司 | Acid alpha-amylase with recombination genes and application thereof |
| CN102618518A (en) * | 2012-03-22 | 2012-08-01 | 无锡德冠生物科技有限公司 | Wide-temperature amylase production method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1249784A (en) | In vitro method for construction of DNA library | |
| CN1276082C (en) | Method for synthesizing C DNA | |
| RU2011115100A (en) | METHOD FOR PRODUCING AN ADDITIVE FOR ENZYMIC DIVISION OF MYCOTOXINS, AND ALSO THE ADDITIVE AND ITS USE | |
| US20150050658A1 (en) | Gene shuffling methods | |
| JP2007074939A5 (en) | ||
| CN111100835B (en) | PET degradation biocatalyst and application thereof | |
| KR102183558B1 (en) | Gene encoding L-alanyl-L-glutamine biosynthetic enzyme and use thereof | |
| CN1702173A (en) | Coded high temperature acid alpha-amylase gene and its synthesis method, cloning and expression | |
| WO2021178934A1 (en) | Class ii, type v crispr systems | |
| JP6988000B2 (en) | Ketoreductase variants and their applications | |
| CN1763178A (en) | Recombinant bacterium with granular methane monooxygenase activity and application thereof | |
| CN1080306C (en) | Adjusting and controlling factor for expression of nitrile solution deenzyme and gene thereof | |
| CN101052713A (en) | Single protein production in living cells facilitated by a messenger RNA interferase | |
| CN1884501A (en) | Glutamine synthetase and its dedicated expression engineered bacteria and uses | |
| CN1590549A (en) | Method of constructing T carrier | |
| CN1908159A (en) | D-amino acid preparation strain and construction method thereof | |
| CN1740322A (en) | Para-gene sequence for exogenous insertion vector of corn strain MON863 | |
| CN1223676C (en) | High temperaturealpha- amylase and coding gene thereof | |
| CN1796548A (en) | Reducing enzyme of nitryl, phenylnitryl, encoding gene and application | |
| CN111363709A (en) | Genetic engineering bacterium for improving isoprene yield and construction method and application thereof | |
| CN1802436A (en) | Plasmid having a function of T-vector and expression vector, and expression of the target gene using the same | |
| CN1763204A (en) | Constitutive-expression vector and its construction method and application | |
| CN114774384B (en) | Taq DNA polymerase mutant and screening method thereof | |
| CN114958886A (en) | Simple gene synthesis method | |
| CN117844665B (en) | Recombinant yeast engineering strain, preparation thereof and application thereof in degradation of PET plastic |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |