CN1224709C - Beta-agaropectionase gene aga B, its preparation method and application - Google Patents
Beta-agaropectionase gene aga B, its preparation method and application Download PDFInfo
- Publication number
- CN1224709C CN1224709C CNB031125182A CN03112518A CN1224709C CN 1224709 C CN1224709 C CN 1224709C CN B031125182 A CNB031125182 A CN B031125182A CN 03112518 A CN03112518 A CN 03112518A CN 1224709 C CN1224709 C CN 1224709C
- Authority
- CN
- China
- Prior art keywords
- agarase
- agab
- beta
- gene
- agar
- 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.)
- Expired - Fee Related
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 7
- 238000002360 preparation method Methods 0.000 title abstract description 11
- 101710110830 Beta-agarase Proteins 0.000 claims abstract description 34
- 101150092006 agaB gene Proteins 0.000 claims abstract description 27
- 241000894006 Bacteria Species 0.000 claims description 17
- 239000010977 jade Substances 0.000 claims description 11
- 229920001542 oligosaccharide Polymers 0.000 claims description 11
- 150000002482 oligosaccharides Chemical class 0.000 claims description 11
- 230000000968 intestinal effect Effects 0.000 claims description 10
- 241000588724 Escherichia coli Species 0.000 claims description 7
- 241000588902 Zymomonas mobilis Species 0.000 claims description 7
- 238000010367 cloning Methods 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 229920000936 Agarose Polymers 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 abstract description 13
- 238000006116 polymerization reaction Methods 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 241000519582 Pseudoalteromonas sp. Species 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 239000008272 agar Substances 0.000 abstract 4
- 239000007857 degradation product Substances 0.000 abstract 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N Glycolaldehyde Chemical compound OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 11
- 239000007788 liquid Substances 0.000 description 10
- 108010045649 agarase Proteins 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- 241000206672 Gelidium Species 0.000 description 6
- 235000010419 agar Nutrition 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 102000003960 Ligases Human genes 0.000 description 5
- 108090000364 Ligases Proteins 0.000 description 5
- 239000012634 fragment Substances 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000195493 Cryptophyta Species 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 101100008047 Caenorhabditis elegans cut-3 gene Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 206010047400 Vibrio infections Diseases 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 150000003538 tetroses Chemical class 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a beta-agarase gene agaB which is characterized in that the gene is used for coding the beta-agarase of pseudoalteromonas sp. CY24. The beta-agarase coded by the beta-agarase gene of the present invention can specifically degrade agar to produce new agar oligose whose polymerization degree is from 8 to 14, and the content of each main new agar oligose is greater than 20% in degradation products; thus, an effective method is provided for the large-scale preparation of novel agar oligose whose polymerization degree is eight, ten, twelve and fourteen, and the defect of low polymerization degree (from diose to hexaose in general) of final degradation products of beta-agarase in the prior art is overcome. The recombinant beta-agarase expressed by a colibacillus recombinant strain BL21-pET24-agaB accounts for 25% of total protein and has high expression level and easy purification, and thus, the present invention can realize the mass production of the recombinant beta-agarase and is low in cost.
Description
Technical field
The present invention relates to a kind of β-agarase gene agaB, the preparation and the application of particularly a kind of β-agarase gene agaB that derives from false other Zymomonas mobilis (Pseudoalteromonas sp.) CY24 and clone thereof, expression, expression product.
Background technology
Agarase is mainly derived from marine microorganism, at present from marine pseudomonas, vibrios separation and purification go out multiple agarase.According to the mode of action difference of agarase, can be divided into two classes to it: α-agarase and β-agarase.α-1,3 glycosidic link of α-agarase cracking agarose, generating with β-D-semi-lactosi is non reducing end and with 3,6-inner ether-α-L-semi-lactosi is the fine jade oligosaccharides series of reducing end under neutral; β-1,4 glycosidic link of β-agarase cracking agarose, generating with β-D-semi-lactosi is reducing end under neutral and with 3,6-inner ether-α-L-semi-lactosi is the new fine jade oligosaccharides series of non reducing end.Agarase is with a wide range of applications, and it is used to the preparation of algae protoplastis and the fundamental research of algae physiological and biochemical property the earliest.In addition, it also is used for the separation of high-molecular-weight DNA and is used for the preparation of agaropectin oligose as toolenzyme.Particularly, it is found that the agaropectin oligose of different polymerization degree has important pharmaceutical use, as antitumor, anti-ageing, anti-infective etc. in recent years along with the develop rapidly of glycobiology and chemical research.But, the preparation of oligosaccharides is still a global technical barrier, the method reaction conditions of traditional acid degradation agar-agar is more violent, specificity is poor, hydrolysate is destroyed easily, is unfavorable for the analysis and the recovery of product, and the enzyme digestion reaction condition is easy to control, help a large amount of preparations of specific oligosaccharides, so enzymolysis process replacing acid solution has become trend.The β of Fa Xianing-agarase degraded product is new fine jade disaccharides, new fine jade tetrose and Xin Qiong six sugar mostly up to now, and therefore present market has only the standard substance of two, four, six sugar, does not satisfy the needs of production and scientific research far away.
Summary of the invention
The purpose of this invention is to provide β-agarase gene agaB of the other Zymomonas mobilis CY24 of a kind of vacation and its production and application, it can remedy the above-mentioned deficiency of prior art.
A kind of β-agarase gene agaB is characterized in that it is the gene agaB of β-agarase of the false other Zymomonas mobilis CY24 of coding.
The preparation method of a kind of β-agarase gene agaB is characterized in that the gene agaB with β-agarase of the false other Zymomonas mobilis CY24 of shotgun cloning.
β of the present invention-agarase gene agaB is used to prepare escherichia coli cloning carrier pBS-agaB.
β of the present invention-agarase gene agaB is used to prepare intestinal bacteria recombinant strain DH5 α-pBS-agaB.
β of the present invention-agarase gene agaB is used to prepare coli expression carrier pET24-agaB.
β of the present invention-agarase gene agaB is used to prepare intestinal bacteria recombinant strain BL21 (DE3)-pET24-agaB.
β of the present invention-agarase gene agaB is used to prepare recombinant beta-agarase.
β of the present invention-agarase gene agaB agar-agar that is used to degrade is prepared agaropectin oligose.
β-the agarase of β of the present invention-agarase gene agaB coding can be degraded specifically, and to produce the polymerization degree be the new fine jade oligosaccharides of 8-14 to agar-agar, every kind of main new fine jade oligosaccharides content in degraded product is greater than 20%, therefore for the polymerization degree is that a large amount of preparations of eight, ten, 12 and 14 new fine jade oligosaccharides provide a kind of strong instrument, and remedied the shortcoming of β-agarase degraded end product polymerization degree low (being generally 2-6 sugar) in the past.Expressed recombinant beta-the agarase of intestinal bacteria recombinant strain BL21-pET24-agaB of the present invention accounts for 25% of total protein, and the expression level height is easy to purifying, thus can mass production recombinant beta-agarase by the present invention, and cost is lower.
Description of drawings and embodiment
Accompanying drawing 2 is the aminoacid sequence of inferring according to the nucleotide sequence of β-agarase agaB gene.
Following examples will help those of ordinary skill in the art further to understand the present invention, but not limit the present invention in any form.
Embodiment 1: the preparation of β-agarase gene agaB
Adopt β-agarase gene agaB of the false other Zymomonas mobilis CY24 of shotgun cloning, concrete operations are to utilize agar-agar to select degraded active clone of agar-agar and order-checking for the flat screen of sole carbon source from the false other Zymomonas mobilis genomic library that this laboratory has made up.Utilize biological software Primer5.0 to analyze single open reading frame and proper restriction site, single open reading frame is carried out enzyme respectively cut subclone further with the activated purpose clone of agar-agar plate screening.According to sequencing result design upstream primer (5 '-CATATGTTAAAGCGCCACCAAGC-3 ') and downstream primer (5 '-CTCGAGTTGGCAAGTATAACCT-3 '), with the genomic dna is template, carry out the PCR reaction, PCR reacts (10 μ l reaction system) composed as follows: the upstream and downstream primer of 0.8 μ l dNTP, 1 μ l, 10 * PCR damping fluid, 0.05 μ l (5U/ μ l) Taq enzyme, 1 μ l dna profiling, each 0.1 μ l.Reaction conditions is: 94 ℃ of pre-sex change 2min, 94 ℃ of 30s, 56.7 ℃ of 30s, 72 ℃ of 90s then, totally 30 circulations, 72 ℃ were continued to extend 15min when reaction finished, then the PCR product is reclaimed and obtain β-agarase gene agaB, its concrete nucleotide sequence is seen accompanying drawing 1.
Embodiment 2: the structure of escherichia coli cloning carrier pBS-agaB
β-agarase agaB gene is connected with carrier pBS-T's, 20 μ l reaction system: pBS-T, 0.5 μ l, the PCR product 10 μ l that embodiment 1 obtains, T
4Dna ligase 1.0 μ l, 10 * T
4Dna ligase damping fluid 2.0 μ l, ddH
216 ℃ of connections of O 6.5 μ l resultant connection liquid that spends the night can obtain escherichia coli cloning carrier pBS-agaB, is used for transformed into escherichia coli DH5 α.
Embodiment 3: the structure of intestinal bacteria recombinant strain DH5 α-pBS-agaB
Add competence E.coli DH5 α that 100 μ l thaw and be connected liquid to the 1.5mlEppendorf pipe with 10 μ l embodiment 2 obtain, ice bath 30min, 42 ℃ of 90s, ice bath 3min adds LB substratum 900 μ l, 37 ℃ of shaking culture 1 hour.Bacterium liquid with coat on the LB flat board that contains 50 μ g/ml penbritins 37 ℃ of overnight incubation after 4 μ l IPTG and 40 μ l X-gal mix.The single bacterium colony of picking white is PCR and is detected, and presents the positive conversion bacterium colony of the special band person of 1.4kb in the agarose gel electrophoresis, promptly contains bacillus coli DH 5 alpha-pBS-agaB of pBS-agaB.Utilize alkaline lysis from DH5 α-pBS-agaB, to extract plasmid, measure the nucleotide sequence of external source insertion sequence agaB with the order-checking universal primer of pBluescript II KS (+).
Embodiment 4: the structure of coli expression carrier pET24-agaB
Cloning vector pBS-agaB and expression vector PET-24a cut with NdeI and XhoI enzyme respectively, and the enzyme tangent condition is as follows:
Reaction system 1:ddH
2O 67 μ l, 10 * H Buffer, 10 μ l, pBS-agaB plasmid DNA 20 μ l, NdeI 1.5 μ l, XhoI 1.5 μ l
Reaction system 2:ddH
2O 66 μ l, 10 * H Buffer, 10 μ l, PET-24a plasmid DNA 20 μ l, NdeI 2 μ l, XhoI 2 μ l
37 ℃ of following enzymes were cut 3 hours, reclaimed two purpose fragment 1.4kb and 5.3kb behind the electrophoresis respectively, and the two is identical with expression vector pET-24a (+) clip size with β-agarase agaB full length gene fragment respectively, uses T
4Dna ligase connects, and condition of contact is as follows: ddH
2O 11 μ l, 10 * T
4Dna ligase damping fluid 2.0 μ l, 5.3kb fragment 4 μ l, 1.4kb fragment 2 μ l, T
4Dna ligase 1.0 μ l, 16 ℃ of connections are spent the night and can be obtained coli expression carrier pET24-agaB, are used for transformed into escherichia coli BL21 (DE3).
Embodiment 5: the structure of intestinal bacteria recombinant strain BL21 (DE3)-pET24-agaB
Add competence BL21 (DE3) that 100 μ l thaw and be connected liquid to the 1.5mlEppendorf pipe with 10 μ l embodiment 4 obtain, ice bath 30min, 42 ℃ of 90s, ice bath 3min, add LB substratum 900 μ l, 37 ℃ of shaking culture 1 hour are coated bacterium liquid on the LB flat board that contains 30 μ g/ml kantlex 37 ℃ of overnight incubation.The single bacterium colony of picking white is PCR and is detected, and presents the positive conversion bacterium colony of the special band person of 1.4kb in the agarose gel electrophoresis, promptly contains intestinal bacteria recombinant strain BL21 (DE3)-pET24-agaB of pET24-agaB.
Embodiment 6: the preparation of recombinant beta-agarase
Picking intestinal bacteria recombinant strain BL21 (DE3)-pET24-agaB mono-clonal is in containing the liquid nutrient medium that concentration is 30 μ g/ml kantlex LB, and 37 ℃ of shaking culture are spent the night.Be seeded to 1L by 1:50 and contain in the liquid LB substratum that concentration is 30 μ g/ml kantlex, 37 ℃ of 200r/min shaking culture are to OD
600Be 0.6, adding final concentration is 1m M IPTG and 0.01% (weight ratio) Sodium desoxycholate, 25 ℃ of abduction delivering 5h.Induce 4 ℃ of centrifugal 10min of 10000g of bacterium liquid, collect supernatant liquor, supernatant liquor successively concentrates through ultrafiltration and embedding, extremely uses binding buffer liquid (the Native Binding Buffer pH7.8) PreBond that balance is good on the concentrated solution
TMResin column (Invitrogen) is respectively 6.0 and 5.5 lavation buffer solution (Native Wash Buffer) washing affinity column to elutant OD with binding buffer liquid and pH value successively
280＜0.01, be 4.0 elutriant (Native Elution Buffer) wash-out affinity column at last with pH, collect elutriant.SDS-PAGE detects albumen distribution in the elutriant, merges the elutriant that contains the single purpose band and can obtain recombinant beta-agarase, and its speculating acid sequence is seen accompanying drawing 2.Measure target protein concentration with the Lowry method, after the packing with albumen frozen in-80 ℃ with standby.
Embodiment 7: expression product is used to prepare new fine jade oligosaccharides
When preparing new fine jade oligosaccharides, agarose is dissolved in 0.1M phosphate buffered saline buffer (pH7.5), be made into weight percent concentration and be 0.1%～1.0% solution, by weight 0.1-1: 100 add the recombinant beta-agarase of gained among the embodiment 6, after mixing, 30 ℃ of temperature are bathed 5-24h, and 80-90% is that the polymerization degree is the new fine jade oligosaccharides of 8-14.
Claims (7)
1, a kind of β-agarase gene agaB is characterized in that it is the gene of β-agarase of the false other Zymomonas mobilis CY24 of coding, and has
ATGTTAAAGCGCCACCAAGCTTCAAGGAAGGTTTCATCATCATTAGTGGCTTTGTTAAAGCCT
TCGGCCATTGCGGTTCTAGCAACCGTTGCTTTGGCTAGCGGCGCAAATGCCGCTAACTATACG
GCCAGCAATGCCAGCCAGCTAAGTGCTCGTTTGCAAGATGCAGCCAACAACGGCACCGGCGTA
GATGTTATTACCATTCAAGGCAGCATATTTACCGACCAGCAAATTGATATTCAAACTCCGGTA
ACTATTCAAGGTGCAGCTGGGTTTCGCGTAAGCCGTATCATTCGTACTTCTGATGATGGTTTT
CAACCTTTGTTTAATATTCAAAGTTCGAATGTGACTATTCGTAATTTGCTGCTAATTGATGAG
AAAGGCCAAAATACTAATACCCAAGTGGCCAGTGAAGCAGGGAACGACCACTCAAATGCGCGC
TTAATTAACATTCCTTACGAAGATGCTTATCAGCAAATTGCTAACATCACCATCGAAAACAAT
ACCTTTGAAAATACCGCTGTTGGTGTGGCGTCTTCTGGTTTAATTCCGCGTAATTTGTCGATT
ACCAATAATGACTTTATTAAAGTGAATCGCAGCGTAGAGCTGCTACGAGATGTTGGCCGGGTA
TACAACGTGTGGAATGTAAGTGCTAACAACGTAGTGCTTAACGGCGGAACACTAAACATTTCA
AATAACCGTATTCGTGGTAATCGAGTGCGTTTAGGGATCTCTGTAGATGCGGGCAACGACGGG
GTTTACGTGCCGCCAAGCTTTACCAACATTCCGTTTTTTGATGCAGCAGCCCGTGCTCAGTTT
AGTGATAAACCGGTCGTGTTTGCCAATGGCTCGCAAGTTAACTCAAACACTGTAGAGGGGGCC
AACGAGTTTGGCATTGCTTTGGCTACTGTGGCTAACGTAACTGTTGCGGGTAACACAGTGAGT
ACAACAGAAGATGACATTAACTCGTCTGACGATATCGAGAACAACTTCACAGCGGGCATCAAT
GTTGAACATAACAGCCGCGATATTGTGGTAGATAGCAATACTATTACCGTGGGTGCTTCAGGC
AACTTTGCTACGGGTATTAACGTATTGGCCTTTCAAGATCACCACGCGCCGTTAAATCATGCT
CAAGCTAGCAGCAACATAACTTTGGTTAGAAACATCTTTAAAGGAACTGGCGAGAACACCATT
TTAGCCTTTGGCTTCTCTAACTTAGTGGTTGAAGACAACAATGCCTCGCAGTTTACAACGCGT
AACCCTTACCAAGTAACGGCGTCTTTTTACAACGTGCCTTGTGGTTTAAGTACTAGCACTGCA
CGCGGTACTAACAACAATATTCGTTACAACCAAAGCTCGTTTAACGGCACCGGCAATGCGCCG
Nucleotide sequence shown in the CAGTACTACGACAAAAACGGTAACGTTGTGTCAGGTTATACTTGCCAATAG.
2, escherichia coli cloning carrier pBS-agaB, it contains the described β of claim 1-agarase gene agaB.
3, intestinal bacteria recombinant strain DH5 α-pBS-agaB, it contains the described escherichia coli cloning carrier of claim 2 pBS-agaB.
4, coli expression carrier pET24-agaB, it contains the described β of claim 1-agarase gene agaB.
5, intestinal bacteria recombinant strain BL21 (DE3)-pET24-agaB, it contains the described coli expression carrier pET24-agaB of claim 4.
6, recombinant beta-agarase, it is by the described intestinal bacteria recombinant strain of claim 5 BL21 (DE3)-pET24-agaB generation of fermenting.
7, the described recombinant beta-agarase of claim 6 prepares application in the new fine jade oligosaccharides at the degraded agarose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031125182A CN1224709C (en) | 2003-06-06 | 2003-06-06 | Beta-agaropectionase gene aga B, its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031125182A CN1224709C (en) | 2003-06-06 | 2003-06-06 | Beta-agaropectionase gene aga B, its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1460717A CN1460717A (en) | 2003-12-10 |
| CN1224709C true CN1224709C (en) | 2005-10-26 |
Family
ID=29591020
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031125182A Expired - Fee Related CN1224709C (en) | 2003-06-06 | 2003-06-06 | Beta-agaropectionase gene aga B, its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1224709C (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103194435B (en) * | 2013-04-09 | 2014-07-09 | 中国海洋大学 | Beta-agarase and applications thereof |
| KR101521711B1 (en) * | 2013-10-14 | 2015-05-19 | 고려대학교 산학협력단 | Novel β-agarooligosaccharide hydrolase and enzymatic production method of 3,6-anhydro-L-galactose and galactose from agarose by using the same |
| CN105420142B (en) * | 2015-10-13 | 2018-11-09 | 中国海洋大学 | A method of producing agarase from the bacterium Pseudoalteromonas.sp.QJ97 of marine organisms and with it |
| CN105506032A (en) * | 2016-02-25 | 2016-04-20 | 福建省绿麒食品胶体有限公司 | Preparation method and application of Gracilaria agar oligosaccharide promoting growth of Lactobacillus bulgaricus |
| CN106834379A (en) * | 2016-09-26 | 2017-06-13 | 中国海洋大学 | A kind of preparation method and applications of new fine jade tetrose |
| CN112370368A (en) * | 2019-12-27 | 2021-02-19 | 青岛博智汇力生物科技有限公司 | Moisturizing cream containing neoagaro-oligosaccharide |
-
2003
- 2003-06-06 CN CNB031125182A patent/CN1224709C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1460717A (en) | 2003-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Albers et al. | Glucose transport in the extremely thermoacidophilic Sulfolobus solfataricus involves a high-affinity membrane-integrated binding protein | |
| CN109295043B (en) | Alginate lyase, and preparation method and application thereof | |
| Sorokin | Halo (natrono) archaea isolated from hypersaline lakes utilize cellulose and chitin as growth substrates | |
| Park et al. | Cloning and characterization of a novel oligoalginate lyase from a newly isolated bacterium Sphingomonas sp. MJ-3 | |
| Zhou et al. | Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3′-phosphoadenosine-5′-phosphosulfate | |
| Singhania et al. | Genetic modification for enhancing bacterial cellulose production and its applications | |
| V. Gomes et al. | Heterologous hyaluronic acid production in Kluyveromyces lactis | |
| US11884954B2 (en) | Protein complex based on DNA enzymes of E family of Escherichia coli and application thereof in artificial protein scaffolds | |
| CN110452919B (en) | Truncated alginate lyase Aly7B-CDII gene and application thereof | |
| CN106414728A (en) | Agarooligosaccharide hydrolase and method for producing 3,6-anhydro-l-galactose and galactose from agarose by using same | |
| CN112708609B (en) | Chitosanase OUC-CsnPa and application thereof | |
| Yan et al. | Genes encoding the production of extracellular polysaccharide bioflocculant are clustered on a 30-kb DNA segment in Bacillus licheniformis | |
| CN1224709C (en) | Beta-agaropectionase gene aga B, its preparation method and application | |
| CN106995811A (en) | A kind of algin catenase, its preparation method and application | |
| Sorokin et al. | Halococcoides cellulosivorans gen. nov., sp. nov., an extremely halophilic cellulose-utilizing haloarchaeon from hypersaline lakes | |
| CN112725319A (en) | Alginate lyase FaAly7 with polyG substrate specificity and application thereof | |
| Sun et al. | Heterologous expression and purification of a marine alginate lyase in Escherichia coli | |
| CN104004732B (en) | A kind of chitosanase and its preparation method and application | |
| Jiang et al. | Enzymatic verification and comparative analysis of carrageenan metabolism pathways in marine bacterium Flavobacterium algicola | |
| CN110643622A (en) | Alginate lyase gene and application thereof | |
| Thakur et al. | Genomic characterization provides genetic evidence for bacterial cellulose synthesis by Acetobacter pasteurianus RSV-4 strain | |
| Xu et al. | Purification and characterization of cold-adapted and salt-tolerant dextranase from Cellulosimicrobium sp. THN1 and its potential application for treatment of dental plaque | |
| Abaramak et al. | Functional and molecular characterization of the Halomicrobium sp. IBSBa Inulosucrase | |
| CN1239708C (en) | Beta-agaropectinase gene aga, its preparation method and application | |
| Nair et al. | Identification of an additional minor pilin essential for piliation in the archaeon Methanococcus maripaludis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: QINGDAO OCEAN UNIVERSITY OF CHINA HOLDINGS CO., LT Free format text: FORMER OWNER: OCEAN UNIVERSITY OF CHINA Effective date: 20130507 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 266003 QINGDAO, SHANDONG PROVINCE TO: 266071 QINGDAO, SHANDONG PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20130507 Address after: 266071 Hongkong East Road, Laoshan District, Shandong, China, No. 23, No. Patentee after: Qingdao Ocean University of China Holdings Ltd. Address before: 266003 No. 5 fish Hill Road, Shandong, Qingdao Patentee before: Ocean University of China |
|
| ASS | Succession or assignment of patent right |
Owner name: QINGDAO HAIDA HAITANG BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: QINGDAO OCEAN UNIVERSITY OF CHINA HOLDINGS CO., LTD. Effective date: 20131010 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 266071 QINGDAO, SHANDONG PROVINCE TO: 266061 QINGDAO, SHANDONG PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20131010 Address after: 1704 room 182-6, Fortune Tower, No. 266061 Haier Road, Laoshan District, Shandong, Qingdao Patentee after: QINGDAO HAIDA HAITANG BIOTECH CO.,LTD. Address before: 266071 Hongkong East Road, Laoshan District, Shandong, China, No. 23, No. Patentee before: Qingdao Ocean University of China Holdings Ltd. |
|
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20031210 Assignee: QINGDAO BZ OLIGO BIOTECH Co.,Ltd. Assignor: QINGDAO HAIDA HAITANG BIOTECH CO.,LTD. Contract record no.: 2014370000031 Denomination of invention: Beta-agaropectionase gene aga B, its preparation method and application Granted publication date: 20051026 License type: Exclusive License Record date: 20140324 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210527 Address after: 266000 No. 23 Hong Kong East Road, Laoshan District, Qingdao City, Shandong Province Patentee after: Qingdao Ocean University of China Holdings Ltd. Address before: 266061 room 1704, Fortune Building, 182-6 Haier Road, Laoshan District, Qingdao City, Shandong Province Patentee before: QINGDAO HAIDA HAITANG BIOTECH Co.,Ltd. |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20051026 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |