CN1513998A - Sodium alginate lyase gene and its preparation method and application - Google Patents
Sodium alginate lyase gene and its preparation method and application Download PDFInfo
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- CN1513998A CN1513998A CNA031124143A CN03112414A CN1513998A CN 1513998 A CN1513998 A CN 1513998A CN A031124143 A CNA031124143 A CN A031124143A CN 03112414 A CN03112414 A CN 03112414A CN 1513998 A CN1513998 A CN 1513998A
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- alginate lyase
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Abstract
An algin lyase gene aly VI able to code (Vibro sp.) QY101 is disclosed. It can be used to prepare the recombinant algin lyase, which has high stability of temp and pH value and activity and can be used to degradate algin, polymannuronic acid and polyguluronic acid to obtain alginic oligose. Its advantages are high expression level in colibacillus, easy purifying, high stability for 20 generations and low cost.
Description
Technical field
The present invention relates to a kind of alginate lyase gene that derives from vibrio marinopraesens QY101.The invention also discloses the carrier that contains this gene and the production and the application of coli strain and expression product thereof.
Background technology
Algin is the linear anionic polysaccharide that is connected to form by β-1,4 glycosidic link by beta-D-mannuronic acid (M) and α-L-guluronic acid (G).Two kinds of uronic acids can be homogeneous or alternately arrange, and replace block thereby form equal polymannuronic acid section (polyM), homopolymerization guluronic acid section (polyG) and MG.Brown alga and some bacterium all can produce algin, and the algin that wherein derives from brown alga has been widely used in industry such as food, medicine, weaving, oil.Recent studies show that, therefore antitumor, antiviral, anti-oxidant, the anti-ageing multiple biological activity of waiting for a long time that brown alga oligose has attracts much attention gradually.Acid-hydrolysis method is adopted in the preparation of brown alga oligose at present more, and this method is difficult to the control degradation degree, the product heterogeneity, and to the equipment requirements height, environmental pollution is serious.Recently people attempt producing oligosaccharides with alginate lyase degraded algin, and this method is easy to control, product specificity height, and free from environmental pollution.In addition, alginate lyase and microbiotic share and can effectively treat pulmonary cystic fibrosis.Therefore increasing to the demand of alginate lyase.But existing alginate lyase is produced bacterial strain and is all had the difficulty that production of enzyme is low, be difficult to separation and purification, causes production cost high, has therefore seriously hindered the exploitation and the application of brown alga oligose.
Summary of the invention
The object of the present invention is to provide a kind of vibrios (Vibrio sp.) QY101 alginate lyase gene alyVI and its production and application, it can remedy the above-mentioned deficiency of prior art.
A kind of alginate lyase gene alyVI is characterized in that it is the gene of the alginate lyase of coding vibrios (Vibriosp.) QY101.
The preparation method of a kind of alginate lyase gene alyVI, it is characterized in that the total DNA with vibrios (Vibrio sp.) QY101 is a template, obtain the complete sequence of alginate lyase gene alyVI through the method for degenerate pcr and inverse PCR, the method by PCR obtains this alginate lyase gene alyVI again.
Alginate lyase gene alyVI of the present invention is used to prepare coli expression carrier pET24-alyVI.
Alginate lyase gene alyVI of the present invention is used to prepare intestinal bacteria recombinant strain BL21-pET24-alyVI.
Alginate lyase gene alyVI of the present invention is used for preparation reorganization alginate lyase.
The alginate lyase gene alyVI of the present invention algin that is used to degrade is prepared brown alga oligose.
Alginate lyase gene alyVI of the present invention is the gene of coding vibrios (Vibrio sp.) QY101 alginate lyase, reorganization alginate lyase temperature and pH good stability with its production, active high, can degrade algin, polymannuronic acid section and poly-guluronic acid section, degraded product is mainly the brown alga oligose of polymerization degree 4-16, therefore can be used for the preparation of brown alga oligose.The expressed reorganization alginate lyase of intestinal bacteria recombinant strain BL21-pET24-alyVI of the present invention accounts for 35% 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 alginate lyase, cost is lower.
(1) description of drawings
Accompanying drawing 1 is the nucleotide sequence of alginate lyase gene aly
Accompanying drawing 2 is the aminoacid sequence of inferring according to the nucleotide sequence of alginate lyase gene aly
(2) 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 alyVI and the structure of coli expression carrier pET24-alyVI
In containing Luria-Bertani (LB) substratum of 3% (concentration expressed in percentage by weight) NaCl, cultivate vibrios QY101 to logarithm latter stage, extract bacteria total DNA.By the analysis revealed to known alginate lyase aminoacid sequence, the N of alginate lyase end and C end have one section conservative region respectively.According to two degenerated primer Paly1 of this two sections conservative regions design (5 '-CGBTCDGARCTBCGBGMRATG-3 ') and Paly2 (5 '-RTARTTRCCBGCYTTRAARTA-3 '), total DNA with vibrios QY101 is a template, utilizes pcr amplification to obtain alginate lyase portion gene fragment.The condition of this PCR is: 94 ℃ of pre-sex change 3min, carry out 30 circulations with 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 60s subsequently, and extend 10min at 72 ℃ at last.Agarose electrophoresis shows that there is a specific band at the 0.51kb place, it is downcut from sepharose reclaim rear clone, check order and analyze.
According to acquired alginate lyase Gene Partial sequences Design reverse primer Paly3 (5 '-TATTTTCACCATCCCACTGC-3 ') and Paly4 (5 '-AGCCATTCAGTGTCAACCTA-3 ').Total DNA of vibrios QY101 is carried out after with Bgl II complete degestion from connecting, carry out the inverse PCR amplification as template, obtain alginate lyase gene known array both sides unknown nucleotide sequence with the connection product of gained.The PCR condition is: 94 ℃ of pre-sex change 3min, carry out 30 circulations with 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 60s subsequently, and extend 10min at 72 ℃ at last.Agarose electrophoresis shows that there is a specific band at the 4.5kb place, it is downcut from sepharose reclaim rear clone, check order and analyze, thereby obtain alginate lyase gene aly complete sequence (accompanying drawing 1).Wherein the coding region is 197-1211bp, 338 the amino acid whose protein (accompanying drawing 2) of encoding.
According to alginate lyase gene aly complete sequence design primer Paly7 (5 '-GGAATTCCATATGAAAACCTCATGGTTTATC-3 ') and Paly8 (5 '-CCGCTCGAGTCTATGATCAATATTGATCTTC-3 '), total DNA with vibrios QY101 is a template, utilizes pcr amplification to obtain alginate lyase gene complete sequence.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 electrophoresis shows that there is a specific band at the 1.0kb place, after it is reclaimed from the sepharose cutting-out, cuts with NdeI and XhoI enzyme, separates through agarose electrophoresis again, reclaims the dna fragmentation of 1.0kb.Coli expression carrier pET-24a (+) is cut with NdeI and XhoI enzyme equally, agarose electrophoresis is separated, reclaim the dna fragmentation of 5.3kb, after its dna fragmentation with above-mentioned gained 1.0kb is connected, Calcium Chloride Method according to standard changes among the intestinal bacteria E.coli DH5 α, and screening has the transformant of amicillin resistance.Alkaline lysis method of extracting plasmid with standard, cut through NdeI and XhoI enzyme and to obtain 1.0kb and two fragments of 5.3kb, identical with alginate lyase gene aly full length fragment respectively with expression vector pET-24a (+) size, proof alginate lyase full length sequence has been cloned among the expression vector pET-24a (+), with this recombinant plasmid called after pET24-alyVI.
Embodiment 2 can efficiently express the structure of the intestinal bacteria recombinant strain BL21-pET24-alyVI of alginate lyase
The expression vector pET24-alyVI of gained among the embodiment 1 is changed in the e. coli bl21 (DE3) 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 NdeI and XhoI enzyme and to obtain 1.0kb and two fragments of 5.3kb, identical with alginate lyase gene aly full length fragment respectively with expression vector pET-24a (+) size, prove the intestinal bacteria recombinant strain BL21-pET24-alyVI that has obtained to efficiently express alginate lyase.
Embodiment 3 utilizes intestinal bacteria recombinant strain BL21-pET24-alyVI to produce the reorganization alginate lyase
The mono-clonal of the intestinal bacteria recombinant strain BL21-pET24-alyVI of gained among the embodiment 2 is seeded in the liquid LB substratum that 3ml contains 50 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 50 μ g/ml penbritins, 37 ℃ of 200r/min shaking culture are to OD
600Be 0.5, add IPTG, continue to cultivate 3h to final concentration 1mM, 4 ℃ of centrifugal 30min of 5000 * g collect thalline, are resuspended in binding buffer liquid (20mM phosphate buffered saline buffer, pH7.8/0.5M NaCl), add N,O-Diacetylmuramidase to final concentration 0.5mg/ml, room temperature effect 30min.In ultrasonication cell 30min 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 PreBond of binding buffer liquid balance through 0.22 μ m filtering with microporous membrane on the filtrate
TMColumn (Invitrogen).Use binding buffer liquid and dcq buffer liquid (20mM phosphate buffered saline buffer, pH6.0/0.5M NaCl) flushing pillar to elutant OD successively
280＜0.01, last elution buffer (20mM phosphate buffered saline buffer, pH6.0/0.5M NaCl) (imidazole concentration is followed successively by 50mM, 200mM, 350mM, 500mM) the washing pillar that contains the different concns imidazoles successively with 5ml is collected elutriant.SDS-PAGE detects albumen distribution in the elutriant, merges and contains single purpose band elutriant, and imidazoles is removed in dialysis to 20mM phosphate buffered saline buffer (pH7.8), measures target protein concentration with the Lowry method, and is frozen in-80 ℃ after the packing.
Embodiment 4 utilizes the reorganization alginate lyase to prepare brown alga oligose
Reorganization alginate lyase can degrade sleeve phycocolloid, polymannuronic acid section, poly-guluronic acid section.During the preparation brown alga oligose, (molecular-weight average 20KDa) is dissolved in 0.1M phosphate buffered saline buffer (pH7.5) with algin, be made into weight percent concentration and be 0.1%～1.0% solution, by weight 0.1-1: 100 add the reorganization alginate lyase of gained among the embodiment 3, mix the back and bathe 1～24h 30 ℃ of temperature, the product of 60%-80% is the brown alga oligose of polymerization degree 4-16.
Claims (6)
1, a kind of alginate lyase gene alyVI is characterized in that it is the gene of coding vibrios (Vibriosp.) QY101 alginate lyase.
2, the preparation method of a kind of alginate lyase gene alyVI, it is characterized in that the total DNA with vibrios (Vibrio sp.) QY101 is a template, obtain the complete sequence of alginate lyase gene alyVI through the method for degenerate pcr and inverse PCR, the method by PCR obtains this alginate lyase gene alyVI again.
3, the described alginate lyase gene of claim 1 alyVI is used to prepare coli expression carrier pET24-alyVI.
4, the described alginate lyase gene of claim 1 alyVI is used to prepare intestinal bacteria recombinant strain BL21-pET24-alyVI.
5, the described alginate lyase gene of claim 1 alyVI is used for preparation reorganization alginate lyase.
6, the described alginate lyase gene of claim 1 alyVI is used to degrade algin prepares brown alga oligose.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009086685A1 (en) * | 2007-12-29 | 2009-07-16 | Dalian Yaweite Bioengineering Co., Ltd. | An alginic acid with low molecular weight, its salts, uses, preparative methods, pharmaceutical compositions and foods |
| CN102864136A (en) * | 2012-10-19 | 2013-01-09 | 上海海洋大学 | Method for utilizing high-density culture gene engineering bacteria to prepare alginate lyase |
| CN104830880A (en) * | 2015-04-13 | 2015-08-12 | 昆明理工大学 | Alginate lyase SHA-I gene and expression vector thereof |
| CN105154458A (en) * | 2015-10-13 | 2015-12-16 | 滨州医学院 | Gene of novel alginate endolyase, engineering bacterium and application |
| CN108753642A (en) * | 2018-05-10 | 2018-11-06 | 江南大学 | One plant of production algin catenase bacterial strain Yue Shi Flavobacterium |
-
2003
- 2003-05-29 CN CNA031124143A patent/CN1513998A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009086685A1 (en) * | 2007-12-29 | 2009-07-16 | Dalian Yaweite Bioengineering Co., Ltd. | An alginic acid with low molecular weight, its salts, uses, preparative methods, pharmaceutical compositions and foods |
| US8686053B2 (en) | 2007-12-29 | 2014-04-01 | Chuanxing YU | Alginic acid with low molecular weight, its salts, uses, preparative methods, pharmaceutical compositions and foods |
| CN102864136A (en) * | 2012-10-19 | 2013-01-09 | 上海海洋大学 | Method for utilizing high-density culture gene engineering bacteria to prepare alginate lyase |
| CN104830880A (en) * | 2015-04-13 | 2015-08-12 | 昆明理工大学 | Alginate lyase SHA-I gene and expression vector thereof |
| CN104830880B (en) * | 2015-04-13 | 2018-03-06 | 昆明理工大学 | A kind of alginate lyase SHA I genes and its expression vector |
| CN105154458A (en) * | 2015-10-13 | 2015-12-16 | 滨州医学院 | Gene of novel alginate endolyase, engineering bacterium and application |
| CN105154458B (en) * | 2015-10-13 | 2018-03-23 | 滨州医学院 | A kind of new endo-type alginate lyase gene and engineering bacteria and application |
| CN108753642A (en) * | 2018-05-10 | 2018-11-06 | 江南大学 | One plant of production algin catenase bacterial strain Yue Shi Flavobacterium |
| CN108753642B (en) * | 2018-05-10 | 2020-02-11 | 江南大学 | Flavobacterium johnsonii strain for producing alginate lyase |
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