CN1514000A - Sodium alginate catenase gene algl and its preparation method and application - Google Patents
Sodium alginate catenase gene algl and its preparation method and application Download PDFInfo
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- CN1514000A CN1514000A CNA031388248A CN03138824A CN1514000A CN 1514000 A CN1514000 A CN 1514000A CN A031388248 A CNA031388248 A CN A031388248A CN 03138824 A CN03138824 A CN 03138824A CN 1514000 A CN1514000 A CN 1514000A
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- alginate lyase
- lyase gene
- algl
- algin
- gene algl
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- 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
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Abstract
An algin lyase gene algl able to code marine pseudomonads QDA is prepared from the QDA genome DNA library by cloning, and cna be used to prepare the recombinant algin lyase. It can degradate the homopolymannuronic acid to obtain algin oligose. Said recombinant algin lyase features high stability of temp and pH value, and activity, high expression level in colibacillus 24%, and low cost.
Description
Technical field
The present invention relates to the alginate lyase gene algL of a kind of marine pseudomonas (Pseudomonas sp.) QDA.The invention also discloses the carrier that contains this gene and the production method and the application of intestinal bacteria recombinant strain and expression product thereof.
Background technology
Algin is by α-L-guluronic acid and two kinds of sugar units of beta-D-mannuronic acid continuously or the linear polysaccharide molecule that alternately is formed by connecting.In recent years, the develop rapidly along with the research of glycobiology and carbohydrate chemistry it is found that the algin oligosaccharide mixture of different polymerization degree has important pharmaceutical use, and as: reducing blood-fat, antitumor, antiviral etc., being expected to exploitation becomes marine drug of new generation.The preparation of algin oligosaccharide has several different methods, as acid degradation method, oxidation degradation method, ultrasonic degradation method and enzyme liberating method.Link to each other between the algin catenase catalysis sugar unit 1,4-glycosidic link generation hydrolysis, C4 takes place in newly-generated non-reducing end C4-hydroxyl place after fracture then, the dehydration reaction of 5-position generates C4,5 pairs of keys.The enzymic degradation of algin is better than other method, and reaction conditions gentleness, DeR are easy to control, particularly help the preparation of the single oligose of high purity, and the development and application of algin oligosaccharide medicine research is had vital role.Used the restricted substratum of sodium alginate to cultivate the biology so far from various, numerous algin catenases particularly from a large amount of marine microorganisms, have been obtained, but since enzyme active low, Substratspezifitaet is poor, can't realize industrialization production, thereby seriously hindered a large amount of preparations of algin oligosaccharide compounds and the research of structure activity relationship, and even further hindered the deep progress of application and development.Therefore, the active height of exploitation, algin catenase and genetic resources thereof that Substratspezifitaet is strong become the task of top priority.
Summary of the invention
The object of the present invention is to provide alginate lyase gene of false single-cell bacteria (Pseudomonas sp.) QDA in a kind of ocean and its production and application, it can remedy the above-mentioned deficiency of prior art.
A kind of alginate lyase gene algL is characterized in that the gene of the algin catenase of its coding marine pseudomonas QDA.
The preparation method of a kind of alginate lyase gene algL is characterized in that cloning alginate lyase gene algL with the degenerate pcr sieve method in the genome dna library of marine pseudomonas QDA.
Alginate lyase gene algL of the present invention is used to prepare escherichia coli cloning carrier pD45.
Alginate lyase gene algL of the present invention is used to prepare intestinal bacteria recombinant strain pD45/DH5 α.
Alginate lyase gene algL of the present invention is used to prepare coli expression carrier pLyase-A.
Alginate lyase gene algL of the present invention is used to prepare intestinal bacteria recombinant strain pLyase-A/Top10F '.
Alginate lyase gene algL of the present invention is used for preparation reorganization algin catenase.
The alginate lyase gene algL of the present invention algin that is used to degrade is prepared the mannuronic acid oligose.
Alginate lyase gene algL of the present invention is the gene of the algin catenase of coding marine pseudomonas QDA, reorganization algin catenase temperature and pH good stability with its production, active high, the equal polymannuronic acid of energy degraded, degraded product is mainly the oligose of polymerization degree 2-15, therefore can be used for the preparation of algin oligosaccharide.The expressed reorganization algin catenase of intestinal bacteria recombinant bacterial strain pLyase-A/Top10F ' of the present invention accounts for 24% of total protein, the expression level height, be easy to purifying, and expression amount does not have reduction after 20 generations of continuous passage, so by the present invention can mass production the reorganization algin catenase, cost is lower.
Description of drawings
Accompanying drawing 1 is the nucleotide sequence of alginate lyase gene algL.
Accompanying drawing 2 is the aminoacid sequence of inferring according to the nucleotide sequence of alginate lyase gene algL.
Embodiment
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.
The clone of embodiment 1 alginate lyase gene algL
Using degeneracy polymerase chain reaction (PCR) primer to Pseudomonas sp.) genomic library of QDA screens, clone's alginate lyase gene algL, concrete operations are: containing the NaCl concentration expressed in percentage by weight is to cultivate Pseudomonassp.QDA in 3% Luria-Bertani (LB) substratum to logarithm latter stage, extracts genomic dna.Use Sau3AI that this genomic dna is carried out enzyme and cut, agarose gel electrophoresis reclaims the segment of 5～8kb, and is connected with the BamHI complete degestion and through pBluescript II KS (+) the plasmid fragment of dephosphorylation processing.To connect product then and change in the competent cell of bacillus coli DH 5 alpha, and go up at LB solid medium (containing penbritin, X-gal, IPTG) and cultivate according to the Calcium Chloride Method of standard.Picking hickie reorganization bacterium colony, after in LB liquid nutrient medium (containing penbritin), cultivating, directly use degenerated primer that UpperA (5 ' AACCACACCGGCAARTCCAT3 ') and LowerA (5 ' GCGGCGCTT SAKYTCGTTGG3 ') are carried out bacterium liquid PCR reaction respectively, condition is: 94 ℃ of pre-sex change 4min, carry out 30 circulations with 94 ℃ of 30s, 62.6 ℃ of 30s, 72 ℃ of 30s subsequently, extend 10min at 72 ℃ at last.The PCR reaction product of each bacterial strain is through agarose gel electrophoresis, and wherein a strain shows a specific band at the 340bp place, then the recombinant bacterial strain of correspondence is cultivated in a large number, uses its plasmid DNA of standard alkaline lysis method of extracting, sequencing analysis.Final by bioinformatic analysis, determine the full nucleotide sequence (accompanying drawing 1) of alginate lyase gene algL wherein, sequence total length 1098bp encodes one and contains 365 amino acid whose protein (accompanying drawing 2).This positive transformant called after pD45/DH5 α is with its recombinant plasmid called after pD45 that contains.
The structure of embodiment 2 coli expression carrier pLyase-A
According to the alginate lyase gene algL complete sequence that obtains design upstream primer (5 ' CCGAAT TCA TCC CCC ACA GG GTT ACT AC G A3 ') and downstream primer (5 ' CCC AAGCTT GCT TTT TTG CTC TCT GCG TTC G3 '); Obtain the alginate lyase gene complete sequence with pcr amplification.The condition of this PCR is: 94 ℃ of pre-sex change 3min, carry out 30 circulations with 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 60s subsequently, and extend 10min at 72 ℃ at last.Agarose gel electrophoresis shows that there is a specific band at the 1.0kb place, and it is downcut from sepharose, cuts with Hind III and EcoR I enzyme, reclaims the dna segment of 1.0kb behind the agarose gel electrophoresis.
Coli expression carrier pBAD gIII/A is cut with Hind III and EcoR I enzyme equally, agarose electrophoresis is separated, reclaim the dna fragmentation of 4.1kb, after its dna fragmentation with above-mentioned gained 1.0kb is connected, Calcium Chloride Method according to standard changes in the bacillus coli DH 5 alpha, and screening has the transformant of amicillin resistance.Alkaline lysis method of extracting plasmid with standard, cut through HindIII and EcoR I enzyme and to obtain 1.0kb and two fragments of 4.1kb, identical with the alginate lyase gene full length fragment respectively with expression vector pBAD gIII/A size, the proof alginate lyase gene has been cloned among the expression vector pBAD gIII/A, with this recombinant plasmid called after pLyase-A.
Embodiment 3 can efficiently express the structure of the colibacillus engineering strain pLyase-A/Top10F ' of algin catenase
Expression vector pLyase-A is changed among the intestinal bacteria Top10F ' according to the Calcium Chloride Method of standard, and screening has the transformant of amicillin resistance.Alkaline lysis method of extracting plasmid with standard, cut through Hind III and EcoR I enzyme and to obtain 1.0kb and two fragments of 4.1kb, with alginate lyase gene algL full length fragment and expression vector pBAD gIII/A, prove the intestinal bacteria recombinant strain pLyase-A/Top10F ' that has obtained to efficiently express algin catenase respectively.
Embodiment 4 utilizes intestinal bacteria recombinant strain pLyase-A/Top10F ' to produce the reorganization algin catenase
Picking intestinal bacteria recombinant strain pLyase-A/Top10F ' mono-clonal is seeded in the liquid LB substratum that 2ml contains 100 μ g/ml penbritins, and 37 ℃ of 200r/min shaking culture are spent the night.By being seeded at 1: 50 in the liquid LB substratum that 100ml contains 100 μ g/ml penbritins, 37 ℃ of 200r/min shaking culture are to OD
600Be 0.6, add L-arabinose to final concentration 1mM, continue to cultivate 3h, 4 ℃ of centrifugal 30min of 5000 * g collect thalline, are resuspended in 12ml binding buffer liquid (20mM phosphate buffered saline buffer, pH7.8/0.5 M NaCl).In ultrasonication cell 15min on ice, every effect 10s suspends 10s with bacteria suspension.4 ℃ of centrifugal 30min of 12000 * g remove cell debris, and supernatant liquor is extremely used the good PreBondTMcolumn (Invitrogen) of binding buffer liquid balance through 0.22 μ m filtering with microporous membrane on the filtrate.Wash pillar to elutant OD with binding buffer liquid and dcq buffer liquid (20mM phosphate buffered saline buffer, pH7.8/0.5M NaCl)
280＜0.01, last damping fluid (20mM phosphate buffered saline buffer/0.5M NaCl) wash-out of using 5ml pH6, pH4 successively, SDS-PAGE detect albumen distribution in the elutriant, merge the elutriant that contains the single purpose band.Measure target protein concentration with the Lowry method, frozen after the packing in-80 ℃.
Embodiment 5 utilizes the reorganization algin catenase to produce mannuronic acid oligosaccharide
The reorganization algin catenase can specificity degraded polymannuronic acid molecule.During the preparation mannuronic acid oligosaccharide, polymannuronic acid is dissolved in 0.2M phosphate buffered saline buffer (pH7.5), be made into weight percent concentration and be 0.1%～0.5% solution, by weight 0.1-1: 100 add the reorganization algin catenase of gained among the embodiment 4, mix the back and bathe 3～16h 37 ℃ of temperature, the product of 70%-90% is the mannuronic acid oligosaccharide of polymerization degree 2-15.
Claims (8)
1, a kind of alginate lyase gene algL is characterized in that it is the gene of the algin catenase of coding marine pseudomonas QDA.
2, the preparation method of a kind of alginate lyase gene algL is characterized in that cloning alginate lyase gene algL with the degenerate pcr sieve method in the genome dna library of marine pseudomonas QDA.
3, the described alginate lyase gene algL of claim 1 is used to prepare escherichia coli cloning carrier pD45.
4, the described alginate lyase gene algL of claim 1 is used to prepare intestinal bacteria recombinant strain pD45/DH5 α.
5, the described alginate lyase gene algL of claim 1 is used to prepare coli expression carrier pLyase-A.
6, the described alginate lyase gene algL of claim 1 is used to prepare intestinal bacteria recombinant strain pLyase-A/Top10F '.
7, the described alginate lyase gene algL of claim 1 is used for preparation reorganization algin catenase.
8, the described alginate lyase gene algL of claim 1 is used to degrade algin prepares mannuronic acid oligosaccharide.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1920002B (en) * | 2005-08-22 | 2011-06-08 | 中国海洋大学 | Novel vibrionaceae vibrio bacterial strain and application thereof |
| CN102277340A (en) * | 2011-06-20 | 2011-12-14 | 广州立达尔生物科技股份有限公司 | Protein with seaweed polysaccharide catabolic enzyme function as well as coding genes and application thereof |
| CN102311930A (en) * | 2010-07-02 | 2012-01-11 | 中国海洋大学 | Alginate lyase produced by pseudomonas.sp.HZJ216 |
| CN105543128A (en) * | 2015-12-24 | 2016-05-04 | 山东大学 | Polar cold-adapted salt-tolerant alginate lyase and coding gene c3 and application thereof |
| CN106995811A (en) * | 2016-01-22 | 2017-08-01 | 中国科学院天津工业生物技术研究所 | A kind of algin catenase, its preparation method and application |
| CN107099545A (en) * | 2017-06-19 | 2017-08-29 | 五洲丰农业科技有限公司 | A kind of alginate lyase gene and its application |
| CN110331122A (en) * | 2019-07-24 | 2019-10-15 | 江南大学 | A kind of Escherichia coli of secreting, expressing algin catenase and its application |
| CN110951716A (en) * | 2020-01-15 | 2020-04-03 | 中国海洋大学 | Circumscribed alginate lyase VsAly7D, recombinant strain thereof and application thereof |
| CN114591936A (en) * | 2022-03-29 | 2022-06-07 | 山东大学 | Mutant type algin lyase and application thereof |
-
2003
- 2003-07-10 CN CNA031388248A patent/CN1514000A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1920002B (en) * | 2005-08-22 | 2011-06-08 | 中国海洋大学 | Novel vibrionaceae vibrio bacterial strain and application thereof |
| CN102311930A (en) * | 2010-07-02 | 2012-01-11 | 中国海洋大学 | Alginate lyase produced by pseudomonas.sp.HZJ216 |
| CN102311930B (en) * | 2010-07-02 | 2015-10-07 | 中国海洋大学 | A kind of algin catenase produced by Pseudomonas.sp.HZJ216 |
| CN102277340A (en) * | 2011-06-20 | 2011-12-14 | 广州立达尔生物科技股份有限公司 | Protein with seaweed polysaccharide catabolic enzyme function as well as coding genes and application thereof |
| CN102277340B (en) * | 2011-06-20 | 2013-01-16 | 广州立达尔生物科技股份有限公司 | Protein with seaweed polysaccharide catabolic enzyme function as well as coding genes and application thereof |
| CN105543128A (en) * | 2015-12-24 | 2016-05-04 | 山东大学 | Polar cold-adapted salt-tolerant alginate lyase and coding gene c3 and application thereof |
| CN106995811A (en) * | 2016-01-22 | 2017-08-01 | 中国科学院天津工业生物技术研究所 | A kind of algin catenase, its preparation method and application |
| CN107099545A (en) * | 2017-06-19 | 2017-08-29 | 五洲丰农业科技有限公司 | A kind of alginate lyase gene and its application |
| CN110331122A (en) * | 2019-07-24 | 2019-10-15 | 江南大学 | A kind of Escherichia coli of secreting, expressing algin catenase and its application |
| CN110951716A (en) * | 2020-01-15 | 2020-04-03 | 中国海洋大学 | Circumscribed alginate lyase VsAly7D, recombinant strain thereof and application thereof |
| CN114591936A (en) * | 2022-03-29 | 2022-06-07 | 山东大学 | Mutant type algin lyase and application thereof |
| CN114591936B (en) * | 2022-03-29 | 2023-11-28 | 山东大学 | Mutant algin lyase and application thereof |
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