CN107177612A - A kind of circumscribed-type algin catenase, gene and its application - Google Patents
A kind of circumscribed-type algin catenase, gene and its application Download PDFInfo
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- CN107177612A CN107177612A CN201710367707.XA CN201710367707A CN107177612A CN 107177612 A CN107177612 A CN 107177612A CN 201710367707 A CN201710367707 A CN 201710367707A CN 107177612 A CN107177612 A CN 107177612A
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- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02003—Poly(beta-D-mannuronate) lyase (4.2.2.3)
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Abstract
The invention discloses a kind of circumscribed-type algin catenase, gene and its application.Present invention clone from marine bacteria microvesicle bacterium ALW1 obtains new alginate lyase gene algl17, the bp of size 2220, encodes 739 amino acid, protein classification is classified as the families of PL 17.The gene is expressed and purified in E.coli, obtains recombinating AlgL17, molecular size range is about 85 kDa.The enzyme is to sodium alginate, D polymannuronates（polyM）It is active with algin oligosaccharide, and to L guluronic acids（polyG）With compared with low activity, showing that the recombinase is the few algin catenase of polyM specificity.Restructuring AlgL17 may act on sodium alginate, produce the hexose uronic acid of 4 deoxidation L erythros 5（DEH）, it is circumscribed-type algin catenase to show the enzyme.The recombinase can directly degrade sea-tangle, produce reduced sugar.The present invention successfully constructs the recombinant vector containing above-mentioned new alginate lyase gene, realizes heterogenous expression, and the industrialized production and application for the enzyme provide good basis.
Description
Technical field
The present invention relates to genetic engineering and the technical field of enzyme engineering, more particularly to a kind of circumscribed-type algin catenase,
Gene and its application.
Background technology
China's marine algae resource enriches.Brown alga is a kind of important economical alga, and representing plant includes sea-tangle, pelvetia silquosa and horse
Waste dish etc..Brown alga contains the polysaccharide such as algin, laminaran and fucoidin, accounts for the 40%~80% of degreasing brown alga dry weight, its
Middle algin content is enriched the most.Algin is by beta-D-mannuronic acid and its C5 epimer α-L- guluronic acids
Two kinds of alditol acid monomers connect the linear polymeric polymer to be formed by β-Isosorbide-5-Nitrae glycosidic bond, be widely used in food, medicine,
In the fields such as chemical industry.Algin can be synthesized largely in the cell membrane and intercellular matrix of brown alga, can also be by some
Specific bacteria is produced.In recent years, ethanol is produced by raw material of brown alga turns into the focus that algae bio fuel is studied.
Algin catenase cracks the glycosidic bond of algin by beta-elimination reaction.The enzyme can be cut many as endo-type
Sugar, also can cut algin or algin oligosaccharide end as circumscribed-type.Algin oligosaccharide is produced using algin catenase,
Have the advantages that efficient, degradation condition is gentle, hydrolysate is difficult to be destroyed, recovery process is simple, free from environmental pollution, is brown
Research of the algae oligosaccharides in terms of functional food, multifunctional cosmetics, new product medicine provides new means with application.
Except for preparing algin oligosaccharide, algin catenase is also applied to treatment fibrosis tumour, prepares protoplast, from brown
Extracted in algae in bioactive substance, analysis brown alga plastic structure, production bioenergy etc..
Algin catenase is mainly derived from the bodies such as multiple-microorganism, marine algae, sea mollusk and echinoderm
It is interior.The present inventor studies and devised a circumscribed-type brown alga for coming from microvesicle bacterium ALW1 (Microbulbifer sp.ALW1)
Thus glue lyases, gene and its application, this case produce.
The content of the invention
It is an object of the invention to provide a kind of circumscribed-type algin catenase, gene and its application, the enzyme can be used for producing
Raw monose and oligosaccharides, can be applied in the fields such as food, medicine, chemical industry, the energy.
To achieve these goals, the technical scheme adopted by the invention to solve the technical problem is that：
A kind of gene for encoding circumscribed-type algin catenase AlgL17, its nucleotide sequence is as shown in SEQ ID NO.1.
Gene size is 2220 bp.
A kind of circumscribed-type algin catenase AlgL17, its amino acid sequence is as shown in SEQ ID NO.2.The encoding histone
739 amino acid residues.
A kind of algin catenase expression vector, the expression containing described circumscribed-type algin catenase AlgL17 genes
Carrier pET-28a-algl17.
A kind of preparation method of algin catenase, including carried using the expression containing alginate lyase gene
Body converts host cell, cultivates transformant, restructuring algin catenase is obtained from culture.Certainly, algin can also be split
Solution enzyme AlgL17 genes are transfected into suitable eucaryote host, including yeast and mammalian cell etc..
As the preferred embodiment of embodiment, described host cell is Escherichia coli.
As the preferred embodiment of embodiment, including using the expression vector conversion containing alginate lyase gene
To host cell e. coli bl21 (DE3), induced through IPTG, obtain the restructuring algin catenase of solubility expression.
As the preferred embodiment of embodiment, the final concentration of 0.1mmol/L of described IPTG, inducing temperature is 18 DEG C.
One kind restructuring algin catenase, including using the expression vector conversion containing alginate lyase gene
Host cell, cultivates transformant, recombinase is obtained from culture.
As the preferred embodiment of embodiment, using sodium alginate as substrate, the optimal reaction temperature of the restructuring algin catenase
Spend for 35 DEG C, optimal reaction pH is 8.0.Recombinate AlgL17 stable at 25 DEG C, it is unstable at 30 DEG C and 35 DEG C.The enzyme exists
PH 5.0-8.0 have good stability.By the Na for studying various concentrations+Influence to enzymatic activity, as a result shows, Na+It is right
Restructuring enzyme activity has activation, and 0.7mol/L NaCl activation is most strong.Inhibitor EDTA, β-ME and DTT can suppress
The activity of recombinase.The enzyme has good resistance to Triton X-100, Tween 20 and Tween 80.On the contrary, SDS and
The activity of CTAB inhibitory enzymes significantly.
Recombinate AlgL17 active to sodium alginate, polyM and algin oligosaccharide, and have polyG compared with low activity,
It is the few algin catenase of polyM specificity to show the recombinase.Recombinase may act on sodium alginate and produce DEH, show this
Enzyme is circumscribed-type algin catenase.The sea-tangle in addition, recombinase can directly degrade, produces reduced sugar.
Present invention clone from microvesicle bacterium ALW1 (Microbulbifer sp.ALW1) obtains a new algin and split
Solve enzyme AlgL17 genes, it was found that the zymoprotein of the gene code has in the fields such as food, medicine, chemical industry, the energy
Application potential.Meanwhile, this invention, which is also cloned, have been obtained the algin catenase can be achieved with the engineering bacteria of great expression
Large-scale production, good basis is provided for follow-up industrial applications.
Brief description of the drawings
Fig. 1 is algin catenase AlgL17 induced expression and its SDS-PAGE figures of purification assays；Wherein, 1：Molecule
Measure standard protein； 2：Containing pET-28a (+) negative bacterium, IPTG inductions；3：Positive bacteria containing pET-28a-algl17, is not induced；4：
Positive bacteria containing pET-28a-algl17, IPTG inductions；5：Recombinant protein after purification；
Fig. 2 is temperature to restructuring algin catenase AlgL17 activity and the influence curve figure of stability；A：Temperature pair
Recombinate the influence of enzymatic activity；B：Influence of the temperature to restructuring enzyme stability；
Fig. 3 is pH to restructuring algin catenase AlgL17 activity and the influence curve figure of stability；
Fig. 4 is influence curve figure of the metal ion to restructuring algin catenase AlgL17 activity；A：Different metal ions
Influence to recombinating enzymatic activity；B：Na+Influence to recombinating enzymatic activity；
Fig. 5 is restructuring algin catenase AlgL17 substrate specificity analysis chart；
Fig. 6 acts on the enzymolysis product analysis chart of sodium alginate for restructuring algin catenase AlgL17；A：Sodium alginate
Liquid-phase chromatographic analysis after enzymolysis processing；B：Sodium alginate liquid-phase chromatographic analysis without enzymolysis processing；C：5.28min main peak
The product mass spectra analysis at place；D：Product mass spectra analysis at 6.94 min main peaks；
Fig. 7 acts on the analysis chart of Kelp Powder for restructuring algin catenase AlgL17.
Embodiment
Embodiment 1：The recombinant expression plasmid of algin catenase AlgL17 genes and the structure of recombinant bacterial strain
Microvesicle bacterium ALW1 (Microbulbifer sp.ALW1) is extracted using bacterial genomes extracts kit (TaKaRa)
Genome, utilize the enzyme gene specific primer, expand algin catenase AlgL17 genes：
Sense primer (SEQ ID NO.3)：5'-CGCGGATCCGTTGAACATCCCAACCTGGTGATCG-3'；
Anti-sense primer (SEQ ID NO.4)：5'-CCGCTCGAGGTTAGTAATTTGGAAGAGTTTTGCG-3'；
PCR reaction conditions are：95℃5min；94 DEG C of 45sec, 50 DEG C of 45sec, 72 DEG C, 2min, 30 circulations；72℃
10min.After the detection of PCR product utilizations agarose gel electrophoresis, target gene, product and pET-28a (+) carrier are reclaimed in rubber tapping
It is attached, target gene is inserted in pET-28a (+) carrier, gained construct is named as after BamHI and XhoI digestions
pET-28a-algl17.Once confirming gene order by being sequenced, recombinant plasmid is imported into e. coli bl21 (DE3).
Embodiment 2：Utilize recombinant strains expression and purification of Recombinant algin catenase
After recombinant strains incubated overnight, by volume 1：100, which are transferred to 250mL LB fluid nutrient mediums, (contains 50 μ g/
ML kanamycins) in, 37 DEG C, 180r/min cultivate to OD600 and reach 0.8, add final concentration of 0.1mmol/L IPTG, 18
DEG C, 180r/min culture 20h.Thalline is collected by centrifuging 10min in 6,000 × g.Cell is resuspended in combination buffer
(50mmol/L NaH2PO4, 300mmol/L sodium chloride, 15mmol/L imidazoles, pH 8.0) in, ultrasonic disruption is carried out on ice
Processing.18 at 4 DEG C, 000 × g centrifugation 20min collect supernatant, Ni-NTA affinity chromatographys are then carried out, by lavation buffer solution
(50mmol/L NaH2PO4, 300mmol/L sodium chloride, 30mmol/L imidazoles, pH 8.0) washing after, utilize elution buffer
(50mmol/L NaH2PO4, 300mmol/L sodium chloride, 250mmol/L imidazoles, pH 8.0) and elution, collect eluent.It will obtain
Restructuring algin catenase AlgL17 with 50mmol/L sodium phosphate buffers (pH 8.0) dialyse after, detected through SDS-PAGE,
The molecular weight of recombinant protein is 85kDa, as a result as shown in Figure 1.Determined using BCA protein determination kits (Pierce, USA)
Protein concentration, the concentration of recombinase is about 0.6mg/mL.
Embodiment 3：The viability examination of algin catenase
The amount of the reduced sugar discharged is determined by using 3,5- dinitrosalicylic acids (DNS) method to detect that algin is cracked
The activity of enzyme.Concrete operations：10 μ L enzyme liquids (0.6mg/mL) are added to 390 μ L and contain 2.5mg/mL sodium alginates
In 50mmol/L sodium phosphate buffers (pH 8.0), incubated at 35 DEG C after 10min, add 400 μ L DNS reagents, and 100
Boiling water bath heating 10min carrys out terminating reaction at DEG C.It is cooled to after room temperature, uses Epoch2T spectrophotometers (BioTeK, USA)
The burst size of reduced sugar is monitored at 540nm.The algin catenase activity definition of one unit is every point under condition determination
Clock discharges the enzyme amount needed for 1 μm of oL reduced sugar.
Embodiment 4：The influence of temperature and pH to restructuring algin catenase AlgL17
The optimal reactive temperature of algin catenase is determined in the range of 25 DEG C -60 DEG C, concrete operations：Take institute above
System, reacts 10min 25, under the conditions of 30,35,40,45,50,55 and 60 DEG C respectively, determines 540nm light absorption value.
Measurement result (Fig. 2A) shows that algin catenase AlgL17 optimal reactive temperatures are 35 DEG C, and in 25-45 DEG C of temperature
In the range of exhibit more than 60% maximum activity, 60 DEG C almost without activity.The thermal stability analysis of enzyme is 25,30 and 35
It is measured at DEG C.Concrete operations are：Enzyme liquid after purification is handled into 10-60min at different temperatures.By sample after heat treatment
Product are placed on to be cooled down immediately on ice.The residual activity of enzyme is measured by described standard method, is defined with undressed enzymatic activity
For 100%.Measurement result (Fig. 2 B) shows that the enzyme is stablized relatively at 25 DEG C.30 DEG C handle 30min make its 84% it is relative
Loss of enzyme activity.The enzyme is almost no active after being heat-treated at 35 DEG C.These results show that the recombinase has thermally labile
Property.
The optimal reaction pH of algin catenase is determined in the range of 4.0-11.0.Determining the buffer solution used is：
50mmol/L citrate buffer solutions (pH 4.0-6.0), 50mmol/L sodium phosphate buffers (pH 7.0-8.0), 50mmol/L bars
Than appropriate sodium-HCl (pH 8.5-9.0) and 50 mmol/L glycine-NaOH (pH is 9.5-11.0).Measurement result (Fig. 3) shows,
Algin catenase AlgL17 optimal reaction pH is 8.0, and the enzyme exhibits more than 30% in the range of 7.5-9.0 pH
Maximum activity.The pH stability of algin catenase is measured in the range of 4.0-11.0.Concrete operations are：Enzyme is placed in
In the buffer solution of different pH value, 25 DEG C incubate after 1h, determine the residual activity of enzyme, are defined as with undressed enzymatic activity
100%.Measurement result (Fig. 3) shows that recombinase its stability when pH scopes are 5.0-8.0 is maintained at initial activity
More than 50%, in the residual vigor of 9.5 times holdings 18% of pH.
Embodiment 5：Influence of the additive to restructuring algin catenase AlgL17 activity
Add final concentration of 1mmol/L or 10mmol/L different metal salt ion respectively in algin catenase, wrap
Include NaCl, KCl, CaCl2、MgCl2、FeCl2、CuCl2、CoCl2、MnCl2、BaCl2、CdCl2And ZnCl2.It is placed in 25 DEG C of decentralizations
Put after 1h, the residual activity of enzyme is determined, to be not added with the enzyme activity of metal ion as 100%.Measurement result (Fig. 4 A) display,
Fe2+、Cu2+、 Co2+、Mn2+、Ba2+、Cd2+And Zn2+The activity of meeting inhibitory enzyme, Cd2+And Zn2+Influence it is notable.1mmol/L K+、Ca2+And Mg2+Activity on enzyme does not influence, and inhibited under the conditions of 10mmol/L.1mmol/L and 10mmol/L
Na+The activity of enzyme can be promoted.Further study the Na of various concentrations+Influence to enzymatic activity, as a result (Fig. 4 B) show,
0.7mol/L NaCl activation highest, with 170% relative activity, but the enzymatic activity quilt under 1.0mol/L NaCl
Suppress.These results show that restructuring algin catenase is Na+The enzyme of activation.
Added respectively in algin catenase final concentration of 1mmol/L or 10mmol/L inhibitor (including EDTA, β-
ME and DTT) and 0.1% (w/v or v/v) or 1% (w/v or v/v) detergent (including SDS, Triton X-100, Tween
20th, Tween 80 and CTAB).It is placed at 25 DEG C and places after 1h, the residual activity of enzyme is determined, to be not added with inhibitor or de-sludging
The enzyme activity of agent is 100%.As shown in table 1, EDTA, β-ME and DTT are shown measurement result under the two kinds of concentration tested
Go out the inhibitory action to enzymatic activity.The enzyme has good resistance to Triton X-100, Tween 20 and Tween 80.Phase
Instead, the activity of SDS and CTAB inhibitory enzyme significantly.Under the conditions of concentration is 1% SDS and CTAB, enzyme almost completely loses work
Property.
The influence of the inhibitor of table 1 and detergent to restructuring algin catenase activity
Embodiment 6：Recombinate algin catenase AlgL17 substrate specificity
Using 2mg/mL sodium alginates, polyM, polyG and algin oligosaccharide (3.3kDa) as substrate, pass through analysis
Light absorption value size at 235nm determines the activity of algin catenase, the substrate specificity of studying enzyme.Concrete operations are：
Added respectively into the different substrate solutions of 8mL at 100 μ L enzyme solutions (0.6mg/mL), 35 DEG C and react 90min.By using UV-
5200 spectrophotometers (Shanghai Yuan Xi Instrument Ltd.) determine light absorption value of the reactant mixture in 235nm, what reflection was produced
The amount of unsaturated uronic acid.As a result (Fig. 5) is shown, restructuring algin catenase is to polyM, sodium alginate and algin oligosaccharide
It is active, but low-down activity is shown to polyG.These results show that recombinase is the few algin of polyM specificity
Lyases.
Embodiment 7：Restructuring algin catenase AlgL17 acts on the enzymolysis product analysis of sodium alginate
Take algin catenase (6U) to be added in 30mL 5.0mg/mL sodium alginate substrate solutions, carried out at 35 DEG C
Reaction.Every 1 h samplings once, the amount of the reduced sugar of release is determined by DNS methods.React after 8h, addition 3U algin splits
Enzyme is solved into reactant mixture, continues to react 9h at the same temperature, the amount of the reduced sugar now discharged does not change.Pass through
Mixture 10min is heated in boiling water, then carrys out terminating reaction in cooled on ice 5min.18,000 × g centrifuges 20min at 4 DEG C
Afterwards, collect supernatant and be used as sample of hydrolysate.Using equipped with ACQUITY BEH C18 posts (2.1 × 150mm, 1.7 μm)
ACQUITY HPLC/MALDI SYNAPT Q-TOF MS (Waters, USA) identification enzymolysis product.Two mobile phase difference
It is solution A：0.1% (v/v) aqueous formic acid, solution B：Acetonitrile, gradient flow velocity 0.3mL/min.Use SYNAPT Q-TOF matter
Amount detector (Waters) obtains mass spectrogram.As a result (Fig. 6) shows, the liquid phase color of restructuring AlgL17 degraded sodium alginate products
Spectrum occurs two main peaks (Fig. 6 A) in retention time 5.28min and 6.94min, and the sodium alginate not digested does not occur
These peaks (Fig. 6 B).The product in above-mentioned main peak, in the negative ion mode, the polymerization of product mass-to-charge ratio (m/z) are determined using MS
Spend the quasi-molecular ions [DPx-H] that (DPs) occurs—(x=1,2,3,4,5) it is respectively 1 (175m/z), 2 (351 m/z), 3 (527m/
Z), 4 (703m/z) and 5 (879m/z).Mass spectral results (Fig. 6 C-D) show that the sodium alginate degraded of restructuring AlgL17 mediations is produced
Thing is DEH, disaccharides, trisaccharide and tetrose.
Embodiment 8：Restructuring algin catenase AlgL17 acts on Kelp Powder
Cleaned in distilled water after sea-tangle piece, by dry sample grind into powder.Powder is added to 50mmol/L phosphorus
In sour sodium buffer solution (pH 8.0), 5mg/mL Kelp Powder substrate is prepared.6U algin catenases are added in 30mL substrates, 35
Enzymatic reaction is carried out at DEG C, every 1h samplings.The sample boiling water bath heating 5min terminating reactions taken, then in cooled on ice
5min.18 at 4 DEG C, after 000 × g centrifugations 20min, the amount of the reduced sugar discharged in supernatant is determined using DNS methods.React 6h
Afterwards, 3U algin catenases are added into reactant mixture, continue to react 14h, now reduced sugar release at the same temperature
Amount does not change.Negative control will be used as without the substrate for recombinating ferment treatment.As a result (Fig. 7) is shown, with prolonging for processing time
It is long, the concentration increase of reduced sugar.Recombinate after AlgL17 processing substrate (150mg) 20h, the reduced sugar of release is stable, and its amount reaches
About 16mg.These results show that restructuring algin catenase AlgL17 can act on sea-tangle and produce reduced sugar.
In summary, present invention clone from Microbulbifer sp.ALW1 obtains new algin catenase
AlgL17 gene, it was found that the degradable sodium alginate of zymoprotein of the gene code produces monose and oligosaccharides, and can degrade sea-tangle
Powder produces reduced sugar, can be applied in the fields such as food, medicine, chemical industry, the energy.Meanwhile, this invention, which is also cloned, to be obtained
The large-scale production of the algin catenase can be achieved, is follow-up industrial applications are carried with the engineering bacteria of great expression
Good basis is supplied.
All deformations that one of ordinary skill in the art directly can export or associate from the disclosure of invention, all should
It is considered protection scope of the present invention.
SEQUENCE LISTING
<110>Collects The American University
<120>A kind of circumscribed-type algin catenase, gene and its application
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 2220
<212> DNA
<213>Microballoon stem bacterium（Microbulbifer sp.）
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atgtttggta aatttttctt taaccagcgt aaagctctgt ccgccgccgt cgcggtgctg 60
gcaatggctc cattgtcggg acttgccgtt gaacatccca acctggtgat cgacgccgca 120
gacgtggatg ccatgcaagg cgcggtagca aaacccggtc gctttcgcag cgcatttctc 180
gcgtccaaat cagcggtaga ccatgcgctg caagtgccac tggcggtacc agtgcccacc 240
gacgcgggtg gaggctatac ccacgagcag cacaaaaaga actatcagct gatgtacaac 300
gctggcgtgc tttatcagat taccgaagat cccaaatatg cggaacgcgt gcgggatatg 360
ctgctggctt atgccgatct gtaccctacc ctgccgctcc atcccaagcg tcgcccgggg 420
gcagaaaatc ccggcaagct gttctggcag agcctgaatg aagcggtttg gctggtgtac 480
accatccagg cctacgatct gattcgccct tcattgtcca atgcagaagc agaaaaaatc 540
gaacagggtg cgctgcgccc ggtggctaaa tttctttccg tagaatcccc ggccaccttc 600
aacaaggtgc ataaccacgg cacctggttg accgccggag tgggtatggc gggctatgta 660
ttggacgagc cagagtgggt tgagcaggcg ctcctcgacc tggacaagtc cggcaagggt 720
ggcttccttc gccagctgaa caccctgttc tcaccggatg gctactacaa cgaaggtccc 780
tactaccagc gctacgcgct gatgccattt gtcaccttcg ccaaagcgat cgaaaacaac 840
gagccggaac ggggcatttt caagtaccgc gatggcattg tgatgaaggc catcgatacc 900
accatccagc tcagctacaa caatctgttt ttcccgatca acgatgccat caagagtaaa 960
ggcatcgaca cctcggagct ggtgctgggc gtcaccattg cctatggcga gtccggcaat 1020
ccacaactgc tggatatcgc cgacagacag catcagattc tgttgtccgg tgacggcctc 1080
aaggttgcgc agggactcga cgccggcgca ctacagcctt acccgttcaa gtctttcgcc 1140
ttccgcgatg gaaaggacgg cgacgaaggc gcactggttg tgctgcgcca gcaaaccgac 1200
ggcgaccagg cgttagtgtt caagccagcc gctcagggca tgggccacgg tcactttgac 1260
cgcttcctga atgtggaagc caaaaatggc ggccgctatc tgcaagaaaa cgaaacctgg 1380
gccaagcaga ccattgcgca caacacggtg gtggtggacg agacctccca cttcgacaac 1440
aacctgaaaa tcgcaaacag gaaccatcct gagttgctgt tcttccacgc ggatgaccag 1500
gtcaaaatca gtgcggcaga aatcgattcc gcctatcccg gtgtcagctt gaaacgcact 1560
ctggcgctcg ttaacaaccc ggagagcggc aatagcttcg ccatcgatgt gttcggtgtg 1620
gaatccagcc agaaacacca gttggacctt cctttgcact acaacggcca gctggtggat 1680
accaatttca ggttgcaggg ctttaccgac agtctcaagg cactgggtac caacaatggc 1740
taccagcacc tttggctcaa agcccgcggc aaaccggatt ccggactggc acaggtcacc 1800
tggctgaacg acaacggccg tttctatacc caatccagcc tggtagacgg caaaaccgag 1860
ctgttgttca ccgagctggg cgccaacgat cccaacttca acctgcgctc cgaaaaaggc 1920
tttatcgctc gccgcaatgg tgcgcgttca catacctttg taagcgtgtt ggagccccat 1980
ggcgagtaca acccgtccaa ggaatttacc ctggaggcgg aaagccaagt gcaggcgcta 2040
cagcatcggc aggccggtga cctcgaactg atcgccatcg gcatcaagaa cggtgccaca 2100
cagctgcttg cgtacaaccg cagtagcaat gtgccagaag aactcgaaaa catttttgaa 2160
tatgacggcc gtaagtacca gttcactggc cgcgcaaaac tcttccaaat tactaactga 2220
<210> 2
<211> 739
<212> PRT
<213>Microballoon stem bacterium（Microbulbifer sp.）
<400> 2
Met Phe Gly Lys Phe Phe Phe Asn Gln Arg Lys Ala Leu Ser Ala Ala
1 5 10 15
Val Ala Val Leu Ala Met Ala Pro Leu Ser Gly Leu Ala Val Glu His
20 25 30
Pro Asn Leu Val Ile Asp Ala Ala Asp Val Asp Ala Met Gln Gly Ala
35 40 45
Val Ala Lys Pro Gly Arg Phe Arg Ser Ala Phe Leu Ala Ser Lys Ser
50 55 60
Ala Val Asp His Ala Leu Gln Val Pro Leu Ala Val Pro Val Pro Thr
65 70 75 80
Asp Ala Gly Gly Gly Tyr Thr His Glu Gln His Lys Lys Asn Tyr Gln
85 90 95
Leu Met Tyr Asn Ala Gly Val Leu Tyr Gln Ile Thr Glu Asp Pro Lys
100 105 110
Tyr Ala Glu Arg Val Arg Asp Met Leu Leu Ala Tyr Ala Asp Leu Tyr
115 120 125
Pro Thr Leu Pro Leu His Pro Lys Arg Arg Pro Gly Ala Glu Asn Pro
130 135 140
Gly Lys Leu Phe Trp Gln Ser Leu Asn Glu Ala Val Trp Leu Val Tyr
145 150 155 160
Thr Ile Gln Ala Tyr Asp Leu Ile Arg Pro Ser Leu Ser Asn Ala Glu
165 170 175
Ala Glu Lys Ile Glu Gln Gly Ala Leu Arg Pro Val Ala Lys Phe Leu
180 185 190
Ser Val Glu Ser Pro Ala Thr Phe Asn Lys Val His Asn His Gly Thr
195 200 205
Trp Leu Thr Ala Gly Val Gly Met Ala Gly Tyr Val Leu Asp Glu Pro
210 215 220
Glu Trp Val Glu Gln Ala Leu Leu Asp Leu Asp Lys Ser Gly Lys Gly
225 230 235 240
Gly Phe Leu Arg Gln Leu Asn Thr Leu Phe Ser Pro Asp Gly Tyr Tyr
245 250 255
Asn Glu Gly Pro Tyr Tyr Gln Arg Tyr Ala Leu Met Pro Phe Val Thr
260 265 270
Phe Ala Lys Ala Ile Glu Asn Asn Glu Pro Glu Arg Gly Ile Phe Lys
275 280 285
Tyr Arg Asp Gly Ile Val Met Lys Ala Ile Asp Thr Thr Ile Gln Leu
290 295 300
Ser Tyr Asn Asn Leu Phe Phe Pro Ile Asn Asp Ala Ile Lys Ser Lys
305 310 315 320
Gly Ile Asp Thr Ser Glu Leu Val Leu Gly Val Thr Ile Ala Tyr Gly
325 330 335
Glu Ser Gly Asn Pro Gln Leu Leu Asp Ile Ala Asp Arg Gln His Gln
340 345 350
Ile Leu Leu Ser Gly Asp Gly Leu Lys Val Ala Gln Gly Leu Asp Ala
355 360 365
Gly Ala Leu Gln Pro Tyr Pro Phe Lys Ser Phe Ala Phe Arg Asp Gly
370 375 380
Lys Asp Gly Asp Glu Gly Ala Leu Val Val Leu Arg Gln Gln Thr Asp
385 390 395 400
Gly Asp Gln Ala Leu Val Phe Lys Pro Ala Ala Gln Gly Met Gly His
405 410 415
Gly His Phe Asp Lys Leu Thr Trp Gln Phe Tyr Asp Arg Gly Glu Glu
420 425 430
Ile Val Thr Asp Tyr Gly Ala Ala Arg Phe Leu Asn Val Glu Ala Lys
435 440 445
Asn Gly Gly Arg Tyr Leu Gln Glu Asn Glu Thr Trp Ala Lys Gln Thr
450 455 460
Ile Ala His Asn Thr Val Val Val Asp Glu Thr Ser His Phe Asp Asn
465 470 475 480
Asn Leu Lys Ile Ala Asn Arg Asn His Pro Glu Leu Leu Phe Phe His
485 490 495
Ala Asp Asp Gln Val Lys Ile Ser Ala Ala Glu Ile Asp Ser Ala Tyr
500 505 510
Pro Gly Val Ser Leu Lys Arg Thr Leu Ala Leu Val Asn Asn Pro Glu
515 520 525
Ser Gly Asn Ser Phe Ala Ile Asp Val Phe Gly Val Glu Ser Ser Gln
530 535 540
Lys His Gln Leu Asp Leu Pro Leu His Tyr Asn Gly Gln Leu Val Asp
545 550 555 560
Thr Asn Phe Arg Leu Gln Gly Phe Thr Asp Ser Leu Lys Ala Leu Gly
565 570 575
Thr Asn Asn Gly Tyr Gln His Leu Trp Leu Lys Ala Arg Gly Lys Pro
580 585 590
Asp Ser Gly Leu Ala Gln Val Thr Trp Leu Asn Asp Asn Gly Arg Phe
595 600 605
Tyr Thr Gln Ser Ser Leu Val Asp Gly Lys Thr Glu Leu Leu Phe Thr
610 615 620
Glu Leu Gly Ala Asn Asp Pro Asn Phe Asn Leu Arg Ser Glu Lys Gly
625 630 635 640
Phe Ile Ala Arg Arg Asn Gly Ala Arg Ser His Thr Phe Val Ser Val
645 650 655
Leu Glu Pro His Gly Glu Tyr Asn Pro Ser Lys Glu Phe Thr Leu Glu
660 665 670
Ala Glu Ser Gln Val Gln Ala Leu Gln His Arg Gln Ala Gly Asp Leu
675 680 685
Glu Leu Ile Ala Ile Gly Ile Lys Asn Gly Ala Thr Gln Leu Leu Ala
690 695 700
Tyr Asn Arg Ser Ser Asn Val Pro Glu Glu Leu Glu Asn Ile Phe Glu
705 710 715 720
Tyr Asp Gly Arg Lys Tyr Gln Phe Thr Gly Arg Ala Lys Leu Phe Gln
725 730 735
Ile Thr Asn
<210> 3
<211> 34
<212> DNA
<213>Artificial sequence
<400> 3
cgcggatccgttgaacatcccaacctggtgatcg 34
<210> 4
<211> 34
<212> DNA
<213>Artificial sequence
<400> 4
ccgctcgaggttagtaatttggaagagttttgcg 34
Claims (10)
1. a kind of gene for encoding circumscribed-type algin catenase AlgL17, it is characterised in that：Its nucleotide sequence such as SEQ ID
Shown in NO.1.
2. a kind of circumscribed-type algin catenase AlgL17, it is characterised in that：Its amino acid sequence is as shown in SEQ ID NO.2.
3. a kind of algin catenase expression vector, it is characterised in that：Contain algin catenase as claimed in claim 1
The expression vector pET-28a-algl17 of AlgL17 genes.
4. a kind of preparation method of algin catenase, it is characterised in that：Including containing algin using described in claim 3
The expression vector conversion host cell of lyases AlgL17 genes, cultivates transformant, and restructuring algin is obtained from culture and is split
Solve enzyme.
5. a kind of preparation method of algin catenase according to claim 4, it is characterised in that：Described host cell
For Escherichia coli.
6. a kind of preparation method of algin catenase according to claim 5, it is characterised in that：Including using described
Expression vector containing algin catenase AlgL17 genes is converted to host cell e. coli bl21（DE3）, lured through IPTG
Lead, obtain the restructuring algin catenase of solubility expression.
7. a kind of preparation method of algin catenase according to claim 6, it is characterised in that：Described IPTG is dense eventually
Spend for 0.1 mmol/L, inducing temperature is 18oC.
8. one kind restructuring algin catenase, it is characterised in that：Including containing algin catenase using described in claim 3
The expression vector conversion host cell of AlgL17 genes, cultivates transformant, restructuring algin catenase is obtained from culture.
9. a kind of restructuring algin catenase according to claim 8, it is characterised in that：The restructuring algin catenase is made
Optimal reactive temperature and optimal reaction pH for sodium alginate substrate are respectively 35oC and 8.0；The recombinase is stable in 25oC,
Unstable under 30oC and 35oC, pH stability range is 5.0-8.0；Sodium ion has activation to restructuring enzyme activity.
10. a kind of application for recombinating algin catenase, it is characterised in that：The restructuring algin catenase may act on alginic acid
Sodium produces DEH, or the restructuring algin catenase can directly degrade sea-tangle, produces reduced sugar.
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