CN105543128A - Polar cold-adapted salt-tolerant alginate lyase and coding gene c3 and application thereof - Google Patents
Polar cold-adapted salt-tolerant alginate lyase and coding gene c3 and application thereof Download PDFInfo
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Abstract
The invention relates to polar cold-adapted salt-tolerant alginate lyase, a coding gene c3 of the polar cold-adapted salt-tolerant alginate lyase and application of the polar cold-adapted salt-tolerant alginate lyase, belonging to the technical field of biotechnology. A cold-adapted salt-tolerant alginate lyase gene c3 disclosed by the invention is obtained by means of cloning a genome DNA of Psychromonas sp.C-3 separated from a kelp sample from the Arctic Yellow River Station. The lyase disclosed by the invention belongs to the seventh polysaccharide lyase family, has the effects of excellent cold adaptability and excellent NaCl tolerance, and has large application potentiality in the fields of food and pharmaceuticals.
Description
Technical field
The present invention relates to a kind of polar region and fit cold salt tolerant alginate lyase and encoding gene c3 thereof and application, belong to technical field of biotechnology.
Background technology
Alginic acid (alginate, alginicacid) has another name called algin, seaweed gel, is a kind of acidic polysaccharose being mainly derived from marine products brown alga.Alginic acid is the non-branched polysaccharide be formed by connecting by Isosorbide-5-Nitrae glycosidic link by beta-D-mannuronic acid (β-D-mannuronate, M) and its epimer α-L-guluronic acid (α-L-guluronate, G) two kinds of monomers.Current commercial alginic acid exists mainly with the form of alginate.Alginate has good wetting ability, becomes gel, toughness and biocompatibility, is widely used in multiple fields such as food, medicine and chemical industry.And alginic acid degraded product-alginate oligosaccharide/brown alga oligose has the active and pharmaceutical use of the various biological such as antitumor, growth promoting effects, enhancing immunocompetence, anti-oxidant, anti-inflammatory, paid close attention to widely in recent years, become new study hotspot.
Alginate lyase can the alignic degraded of catalysis, by β-elimination machining function in the C-O glycosidic link containing gulose and seminose, at the non-reducing end C4 of product, between 5, generate unsaturated uronic acid 4-deoxy-L-erythro-hex-4-enopyranosyluronicacid.This enzyme can be divided into β-D-1 according to the difference of substrate specificity, 4-mannuronic acid lyase (EC4.2.2.3, PM type) and α-L-1,4-guluronic acid lyase (EC4.2.2.11, PG type).Polymannuronic acid and poly-golonic acid acid fragment are specific to alginic acid molecule, and impart alginate oligosaccharide and the various biologic activity of brown alga oligose, have wide development prospect in medicine, field of health care products, its demand is also increasing.Relative to traditional acidolysis mode of production, with alginate lyase enzyme process prepare alginate oligosaccharide/oligosaccharides have reaction conditions gentleness, can handling strong, productive rate advantages of higher, thus become the instrument favourable by force of the high-valued application of brown alga biological resources.
Alginate lyase extensively distributes at occurring in nature, is mainly derived from microorganism, marine algae, sea mollusk and echinoderms etc.The alginate lyase kind of bacterial origin is many, and source is wide, is convenient to produce, is convenient to the features such as heterogenous expression and has good application potential.The alginate lyase that microorganism produces is middle temperature enzyme mostly, and low temperature and suitable cold salt tolerant alginate lyase then rarely have report.Suitable cold salt tolerant alginate lyase has very high activity under low temperature height salt condition, has very large application potential in food and medicine field.The discovery of novel suitable cold salt tolerant alginate lyase all has great importance for the Study and appliance of this enzyme.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of marine brown acid cleavage enzyme and encoding gene c3 thereof and application are provided.
One strain cold Zymomonas mobilis (Psychromonassp.) C-3, on December 14th, 2015 is preserved in China typical culture collection center, Bayi Road Luo Jia Shan, Wuchang District, Wuhan City, Hubei Province, address, preserving number CCTCCM2015748.
A kind of marine brown lyase gene c3, nucleotide sequence is as shown in SEQIDNO.1.
The marine brown acid cleavage enzyme C3 of said gene coding, aminoacid sequence is as shown in SEQIDNO.2.
A kind of recombinant expression vector, this expression vector includes the function fragment of nucleotide sequence as shown in SEQIDNO.1.
A kind of reconstitution cell, this Host Strains includes above-mentioned recombinant expression vector or expresses above-mentioned marine brown acid cleavage enzyme C3.
Above-mentioned marine brown acid cleavage enzyme C3 and/or the application of above-mentioned marine brown lyase gene c3 in alginic acid cracking.
Alginate lyase gene c3 clones from the genomic dna of the Psychromonassp.C3 of Arctic Yellow River Station sea-tangle sample from separation to obtain.To this bacterial strain full genome order-checking possessing alginic acid degradation capability, obtain encoding the gene c3 of alginate lyase, its signal peptide length is predicted in signal peptide prediction website, and according to its sequences Design Auele Specific Primer, the gene c3 of the coding alginate lyase that utilized round pcr to clone from the genomic dna of Psychromonassp.C3, constructs containing the expression vector of alginate lyase gene c3 and measures containing the nucleotide sequence of intestinal bacteria reconstitution cell to this gene of this expression vector.Sequencing result shows that alginate lyase gene c3 is an open reading frame containing 861 Nucleotide, and this open reading frame is encoded 286 amino acid altogether.The signal peptide of alginate lyase C3 is made up of 20 amino acid to use online signal peptide prediction website SignalP to predict, therefore alginate lyase C3 be one containing 20 amino acid whose signal peptides, 286 amino acid whose polypeptide of encoding altogether.Sequential analysis shows, alginate lyase C3 belongs to polysaceharide lyase the 7th family.Property testing is carried out to the alginate lyase C3 of the purifying cutting signal peptide.Result shows that this enzyme reveals selective degradation to mannuronic acid segment table.Optimal pH is 8.0, and in the scope of pH6.0 ~ 10.0 stable existence.The suitableeest enzyme temperature alive is 20 DEG C, and still remains above the vigor of 40% at 0 DEG C, shows good suitable to cold.C3 also shows good tolerance to the NaCl of high density.
Beneficial effect:
1, alginate lyase C3 of the present invention has high enzyme active at low temperatures, unstable under middle high temperature, thus ensure that its security used;
2, alginate lyase C3 of the present invention shows the highly active feature of remarkable high salt, and the enzymic activity in 3MNaCl is still more than 100%;
3, only degrade this characteristic of PM of alginate lyase C3 of the present invention can be used for preparing mannuronic acid oligosaccharide from sodium alginate.
Accompanying drawing explanation
Fig. 1, the electrophorogram of the gene fragment of coding polar region alginate lyase C3 of being cloned by pcr amplification
Wherein swimming lane 2 is the DNA fragmentation of amplification
Fig. 2, the alginic acid lyase C3 electrophorogram that heterogenous expression purifying obtains in intestinal bacteria;
The enzyme temperature curve alive of Fig. 3, polar region alginate lyase C3;
Wherein: (A) temperature is on the impact of enzymic activity, and (B) temperature is on the impact of enzyme stability;
The NaCl of Fig. 4, different concns is to the influence curve of polar region alginate lyase C3 enzymic activity;
The enzyme pH curve alive of Fig. 5, polar region alginate lyase C3;
The substrate specificity analysis of Fig. 6, polar region alginate lyase C3.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described, but institute of the present invention protection domain is not limited thereto.
Substratum:
Enrichment medium: 0.5wt% peptone, 0.1wt% yeast powder, 0.5wt% sodium alginate, artificial seawater is prepared, pH6.5.
Isolation medium: 0.5wt% peptone, 0.1wt% yeast powder, 0.5wt% sodium alginate, 1.5wt% agar, artificial seawater is prepared, pH6.5.
LB liquid nutrient medium: 1wt% peptone, 0.5wt% yeast powder, 1wt%NaCl, distilled water is prepared.
LB solid plate: 1wt% peptone, 0.5wt% yeast powder, 1wt%NaCl, 1.5wt% agar, distilled water is prepared.
Embodiment 1: the acquisition of polar region alginate lyase C3 coding gene sequence and sequential analysis thereof
Bacterial classification is originated: bacterial strain Psychromonassp.C3 is separated the sea-tangle sample gathered from Arctic Yellow River Station.The cold Zymomonas mobilis Psychromonassp.C-3 of one strain, on December 14th, 2015 is preserved in China typical culture collection center, Bayi Road Luo Jia Shan, Wuchang District, Wuhan City, Hubei Province, address, preserving number CCTCCM2015748.
Concrete steps are as follows:
The screening of 1.1 bacterial strains
Sea-tangle sample is put into enrichment medium cultivate, then coat isolation medium flat board with sterilizing seawater gradient dilution.Choose well-grown bacterium colony and carry out pure culture.Get pure culture bacterial strain, dibbling, on isolation medium flat board, is cultivated 48h for 15 DEG C, is measured the diameter of bacterial strain.Then the cetylpyridinium chloride solution that 5mL mass concentration is 10% is added, Rotating Plates makes dissolution homogeneity be paved with whole plate gently, and standing 10min fully dyes, and then rinses media surface with clear water and removes cetylpyridinium chloride solution, measure the diameter of transparent circle, and calculate the diameter ratio of transparent circle and bacterium colony.
The determination of 1.2 polar region alginate lyase C3 gene orders
Choose bacterial strain C3 solid plate producing transparent degraded circle, a large amount of its genomic dna of extraction:
(1) cultivate 100ml bacterial cultures to state of saturation, mass concentration is that 3%NaCl solution washes thalline 2 times;
(2) throw out adds 5ml mass concentration is 3%NaCl solution, repeatedly blows and beats make it resuspended with suction pipe;
(3) add 10mlTE, gently shake mixing;
(4) add N,O-Diacetylmuramidase, make its final concentration be 1mg/ml, 37 DEG C of effect 1 ~ 2h;
(5) add mass concentration be 20%SDS and 20mg/mL Proteinase K to final concentration 0.1mg/ml, 55 DEG C of incubation 3h;
(6) in thalline, the CTAB/NaCl solution 4ml of 65 DEG C of preheatings is added, 65 DEG C of incubation 20min;
(7) add after equal-volume phenol/chloroform/primary isoamyl alcohol (25:24:1) shakes 10min centrifugal, extracting 1 time, supernatant liquor uses equal-volume chloroform 1 time again;
(8) in supernatant, add the dehydrated alcohol of 2 times of volume precoolings, with glass stick, DNA is stirred silk;
(9) DNA mass concentration is that 70% ethanol and dehydrated alcohol respectively wash 1 time, is dissolved in appropriate 0.1 × SSC after slightly dry, is placed on 4 DEG C of Refrigerator stores (can preserve for a long time in-20 DEG C) by ultraviolet spectrophotometer mensuration DNA concentration and purity.
By to its gene annotation after full genome order-checking, the gene c3 of the alginate lyase that obtains encoding.This gene 861bp, the open reading frame wherein containing a 861bp, its polar region alginate lyase C3 that encodes, initiator codon is positioned at 1bp, and terminator codon is positioned at 859bp, 286 amino acid whose albumen of encoding altogether.The nucleotide sequence of the gene c3 obtained is as shown in SEQIDNO.1.The aminoacid sequence of the albumen of its coding is as shown in SEQIDNO.2.
Embodiment 2: the clone of alginate lyase C3, heterogenous expression and separation and purification
2.1 utilize PCR to increase to polar region alginate lyase c3 gene order
(1) two Auele Specific Primers are designed according to polar region alginate lyase c3 gene order:
C3F:GGAATTC
cATATGaATGTTCAGTTTTCTAATCAAGATG (SEQIDNO.3), what mark with underscore is NdeI restriction enzyme site; SEQIDNO.3
C3R:CCG
cTCGAGtTATTTAGTTTCTAACGCTTTATATTC (SEQIDNO.4), what mark with underscore is XhoI restriction enzyme site; SEQIDNO.4
Primer is synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.
(2) with C3F and C3R for primer, with the DNA of bacterial strain C3 for template, by FastPfuDNA polysaccharase (purchased from Transgen company) amplifying target genes fragment;
PCR reaction conditions is: 95 DEG C of denaturation 2min; Then 95 DEG C of sex change 30sec, 50 DEG C of annealing 30sec, 72 DEG C extend 1min, after 30 circulations; 72 DEG C extend 10min.
(3) carry out 1wt% agarose gel electrophoresis to pcr amplification product, result shows the DNA fragmentation (as Fig. 1) of acquisition one treaty 800bp.Then reclaim test kit with the DNA of Omega company, according to it, recovery amplification of DNA fragments is described.
(4) with restriction enzyme NdeI and XhoI, respectively double digestion reaction is carried out to recovery fragment and plasmid pET22b.After enzyme cuts end, 1wt% agarose gel electrophoresis is carried out to digestion products, then reclaim test kit with the DNA of Omega company, according to it, recovery amplification of DNA fragments and carrier segments are described.C3 gene fragment through double digestion is connected on pET22b carrier, 16 DEG C of connections of spending the night.
(5) bacillus coli DH 5 alpha competence is prepared by method E. coli competent preparing by " Molecular Cloning: A Laboratory guide ".
(6) by the heat-shock transformed method on " Molecular Cloning: A Laboratory guide ", the restructuring pET22b carrier connected is gone to bacillus coli DH 5 alpha competence.
(7) bacillus coli DH 5 alpha transformed coats the LB solid medium containing 100 μ g/ml penbritins, 37 DEG C of incubated overnight.Select positive colony, be forwarded in LB liquid nutrient medium and cultivate, extract plasmid, carry out NdeI/XhoI double digestion, send Beijing Hua Da genome company to check order by the plasmid that digestion verification is correct.
Above-mentioned LB solid medium component is as follows:
1wt% peptone, 0.5wt% yeast powder, 1wt%NaCl, 1.5wt% agar, distilled water is prepared.
Above-mentioned LB liquid nutrient medium component is as follows:
1wt% peptone, 0.5wt% yeast powder, 1wt%NaCl, distilled water is prepared.
2.2 recombinant expression vector pET22b-c3 are transformed in e. coli bl21 (DE3)
(1) e. coli bl21 competence is prepared by method E. coli competent preparing by " Molecular Cloning: A Laboratory guide ";
(2) by the heat-shock transformed method on " Molecular Cloning: A Laboratory guide ", the correct recombinant vectors pET22b-c3 of order-checking is gone to e. coli bl21 competence;
(3) e. coli bl21 of conversion is applied in the LB substratum containing 100 μ g/ml penbritins, 37 DEG C of incubated overnight.
2.3 polar region alginate lyase c3 genes abduction delivering and purifying in intestinal bacteria
(1) the large stretch of lawn of scraping on flat board, is connected to 100ml containing in the LB liquid nutrient medium of 100 μ g/ml penbritins, cultivates 2 ~ 3h for 37 DEG C;
(2) be transferred to 1,000ml containing in the LB liquid nutrient medium of 100 μ g/ml penbritins by 1% (v/v) inoculum size, 37 DEG C are cultured to OD
600be 0.6, adding IPTG to final concentration is 0.4mM, continues to cultivate 18h at 15 DEG C of shaking tables;
(3) collect the LB nutrient solution through IPTG abduction delivering, the centrifugal 5min of 9,000rpm, collect thalline;
(4) with 50mMTris-HCl damping fluid (pH8.0) the suspension thalline containing 100mMNaCl;
(5) the bacterium liquid of Eddy diffusion is carried out pressure breaking;
(6) by the centrifugal 1h of bacterium liquid 12,000rpm after fragmentation, supernatant liquor is collected;
(7) affinity chromatography is carried out in supernatant liquor requirement to specifications;
(8) the sample SDS-PAGE collected after chromatography detects purity, proves the pure enzyme of electrophoresis (as Fig. 2) obtaining alginate lyase C3.Remove imidazoles by sieve chromatography, be finally placed in-20 DEG C and save backup, obtained polar region alginate lyase C3.
Embodiment 3: the property testing of polar region alginate lyase C3
3.1 optimum temperutures and temperature stability analysis
Standard reaction is:
Sodium alginate (available from Sigma) substrate of 80 μ l5mg/l and 100 μ l concentration are that 50mMTris-HCl (pH8.0) mixed solution is after 20 DEG C of preheating 5min, add polar region alginate lyase C3 enzyme liquid that concentration that 20 μ l have diluted is 1mg/ml and in 20 DEG C of reaction 30min, boiling water bath 5min termination reactions.Measure OD
235value, using the reaction of not enzyme-added liquid as blank.Enzyme activity unit (U) is defined as: under certain temperature, per minute produces the enzyme amount of absorption value increase required for 0.1 that product makes 235nm place.
The mensuration of optimal reactive temperature: take sodium alginate as substrate, in 25mMTris-HCl (pH8.0) damping fluid, detects the enzyme of polar region alginate lyase C3 at 0 DEG C, 10 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C respectively and lives.The highest enzyme is lived and is defined as 100%.
Result shows that the suitableeest enzyme of this enzyme temperature alive is 20 DEG C (as Fig. 3 A).
Temperature stability is analyzed: enzyme liquid is incubation at 20 DEG C, 30 DEG C and 35 DEG C respectively, gets the different time detecting alginate lyase C3 of identical enzyme amount incubation at 20 DEG C, remaining vigor in 25mMTris-HCl (pH8.0) damping fluid.0 DEG C of enzyme is lived and is defined as 100%.
Result shows, lower than under 30 DEG C of conditions, alginate lyase C3 energy stable existence, under 35 DEG C of conditions, become unstable, after 35 DEG C of incubation 30min, residual enzyme activity is less than 40% (as Fig. 3 B).
The tolerance analysis of 3.2 couples of NaCl
The NaCl of different concns is added respectively to reaction system, final concentration is respectively 0,0.5M, 1.0M, 1.5M, 2.0M, 2.5M, 3.0M, the enzyme measured respectively under different N aCl concentration conditions is lived, and not add the enzyme work of NaCl as blank, is lived by the highest enzyme and is defined as 100%.
Result shows that this enzyme is salt tolerant enzyme.C3 enzyme is lived by the activation of NaCl; When salt concn is up to 3.0M, C3 enzyme is lived still higher than without the enzyme activity (as Fig. 4) during NaCl.
3.3 optimal pH analyses
Secure ph in 5.0 ~ 11.0 scopes, the Britton-Robinson damping fluid of 1 the pH unit in interval.Measure alginate lyase C3 20 DEG C, enzyme under condition of different pH lives, the highest enzyme is lived and is defined as 100%.
Result shows that alginate lyase C3 optimal pH is 8.0 (as Fig. 5).
3.4 substrate specificity analyses
With three kinds of substrate sodium alginates, poly-guluronic acid and polymannuronic acid in 20 DEG C, react respectively with alginate lyase C3 in 25mMTris-HCl (pH8.0) damping fluid, relatively enzyme is to the active size of different substrate, the degradation of substrates Preference of enzyme analysis.The highest enzyme is lived and is defined as 100%.
Result shows that this enzyme is PM specificity.
4. result
CTAB/NaCl method is utilized to be extracted the genomic dna of Psychromonassp.C3.To be checked order by full genome and gene annotation analysis obtains the nucleotide sequence of the gene c3 of the open reading frame coding alginate lyase C3 of a 861bp, and obtained the DNA fragmentation (Fig. 1) of this gene by pcr amplification.Gene c3 is carried out heterogenous expression and purifying in intestinal bacteria, obtains the activated alginate lyase C3 (Fig. 2) belonging to polysaceharide lyase the 7th family.The suitableeest enzyme of this enzyme temperature alive is 20 DEG C, 0 DEG C time, the enzyme of more than 30% still can be kept to live (Fig. 3), have significant salt activator and the feature of salt tolerant (Fig. 4), optimal pH is 8.0 (Fig. 5), has selective degradation (Fig. 6) to mannuronic acid section.Therefore, the alginate lyase C3 that gene c3 encodes is the alginate lyase of a novel suitable cold salt tolerant.
Claims (6)
1. a strain cold Zymomonas mobilis (Psychromonassp.) C-3, on December 14th, 2015 is preserved in China typical culture collection center, Bayi Road Luo Jia Shan, Wuchang District, Wuhan City, Hubei Province, address, preserving number CCTCCM2015748.
2., by a Zymomonas mobilis Psychromonassp.C-3 alginate lyase gene c3 cold described in claim 1, nucleotide sequence is as shown in SEQIDNO.1.
3. the alginate lyase C3 of alginate lyase gene c3 coding described in claim 2, aminoacid sequence is as shown in SEQIDNO.2.
4. a recombinant expression vector, this expression vector includes the function fragment of nucleotide sequence as shown in SEQIDNO.1.
5. a reconstitution cell, this Host Strains includes recombinant expression vector described in claim 4 or expresses alginate lyase C3 described in claim 3.
6. the application of alginate lyase gene c3 in food and medicine field described in alginate lyase C3 described in claim 3 and/or claim 2.
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| CN110982831A (en) * | 2019-09-10 | 2020-04-10 | 集美大学 | Application of gene AlgL23 with cold adaptability |
| CN112831436A (en) * | 2021-01-20 | 2021-05-25 | 山东大学 | Polar region bacillus 1,3/1, 4-xylan degrading bacteria and culture method and application thereof |
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Cited By (6)
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| CN109295043A (en) * | 2018-10-19 | 2019-02-01 | 中国科学院天津工业生物技术研究所 | A kind of novel algin catenase, preparation method and application |
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| CN110982831A (en) * | 2019-09-10 | 2020-04-10 | 集美大学 | Application of gene AlgL23 with cold adaptability |
| CN110982831B (en) * | 2019-09-10 | 2022-07-26 | 集美大学 | Application of gene AlgL23 with cold adaptability |
| CN112831436A (en) * | 2021-01-20 | 2021-05-25 | 山东大学 | Polar region bacillus 1,3/1, 4-xylan degrading bacteria and culture method and application thereof |
| CN117821432A (en) * | 2023-12-19 | 2024-04-05 | 江南大学 | Method for improving thermal stability of algin lyase and application thereof |
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