CN105713889A - Alginate lyase Alga and coding gene and application thereof - Google Patents

Abstract

The present invention discloses an alginate lyase Alga genetic sequence and a preparation method and application thereof. Alginate lyase Alga is derived from Vibrio alginolyticus 40B. The present invention provides a process for preparing the novel alginate lyase Alga, a genetic engineering method is used, an alginate lyase Alga coding gene is cloned into an E. coli expression vector to obtain a recombinant E. coli strain capable of heterologous expression of the alginate lyase Alga, and the alginate lyase Alga prepared by heterologous expression of the recombinant E. coli strain has the function of degrading sodium alginate to prepare sodium alginate oligosaccharide. The alginate lyase Alga can be widely used in chemical industry, agriculture, food, feed additives, pharmaceuticals and algae genetic engineering and other fields.

Description

A kind of algin catenase Alga and encoding gene thereof and application

Technical field

The present invention designs a kind of alginate lyase gene alga.Present invention also offers containing this gene Carrier and E. coli recombinant stain and the production method of expression product thereof and application.

Background technology

Algin is by α-L-guluronic acid (G) and two kinds of monosaccharide of beta-D-mannuronic acid (M) The line style acidic polysaccharose of aldehydic acid composition is thin universally present in the kelp class plant such as Thallus Laminariae (Thallus Eckloniae), Thallus Laminariae In cell wall.Along with what glycobiology and carbohydrate chemistry were studied progressively gos deep into, many marine oligosaccharides, the most brown Catabolite-the algin oligosaccharide of algin has various biological activity.Such as, the Algin of low polymerization degree Oligosaccharide has the biologic activity such as blood fat reducing, antiviral, antitumor and antioxidation, the exploitation having For medicine.Prepare algin oligosaccharide with Algin for raw material and have a multiple method: acid hydrolyzation, oxidation degradation method, Sonication and enzymatic degradation method.

Wherein, acid hydrolyzation and oxidation degradation method degradation condition are difficult to control to, and operate complex, the longest. Sonication is typically used in conjunction with other edman degradation Edmans, and the catabolite degree of polymerization is high, is difficult to prepare oligosaccharide； It is gentle that enzymic degradation has degradation condition relative to other biodegrading process, and reaction condition is easily controllable, product High specificity, yield advantages of higher, therefore become the prefered method preparing algin oligosaccharide.

Prepare the enzyme i.e. algin catenase that algin oligosaccharide is used, belong to polysaceharide lyase family Member, it is possible to the Isosorbide-5-Nitrae glycosidic bond between catalysis uronic acid unit hydrolyzes, and be newly formed after fracture is non- Reducing end generates double bond.According to the difference of its substrate specificity, algin catenase can be divided three classes: The polymannuronate lyases (EC4.2.2.3) of specific degradation polymannuronate, specific degradation The guluronic acid lyases (EC4.2.2.11) of guluronic acid and above two fragment of degrading Difunctional algin catenase.Owing to it has Substratspezifitaet, algin catenase is mainly used in system For aspects such as the algin oligosaccharide of specificity structure, the chemical constitutions of research Algin；Additionally, Algin Lyases can also be applied to treat lung cyst fibrosis, the protoplast of the kelps such as preparation Thallus Laminariae (Thallus Eckloniae) Deng.

Algin catenase is mainly derived from marine animal and plant (including sea mollusk, marine algae) With multiple-microorganism (including marine bacteria, soil bacteria and fungus).It is different according to its substrate specificity, Two big classes can be divided into: the lyases (EC4.2.2.3) of specific degradation polymannuronate and specificity drop Solve the lyases (EC4.2.2.11) of guluronic acid；In addition with some above two of can degrading The difunctional lyases of fragment, arises primarily at pseudoalteromonas NO272 The algin catenase Aly of (Pseudoalteromonas sp.NO272)；From pseudoalteromonas The AlySJ-02 of isolated in Pseudoaltermonas sp.SM0524；From Isoptericola Halotolerans isolated algin catenase and from Fructus Hordei Germinatus Stenotrophomonas (Stenotrophomsa maltophilia KJ-2) clones the algin catenase obtained；Above four kinds Lyases is respectively provided with the ability of degraded PM and PG, and catabolite mostly is the oligosaccharide of DP2-6.Bifunctional Algin catenase due to its widely substrate specificity and there is in commercial production important application valency Value, is mainly used in the production of algin oligosaccharide.Algin catenase involved in the present invention has degraded two The ability of kind of fragment, the product degree of polymerization simultaneously for different substrates is more single, is all main with trisaccharide Product, has important industrial application value.

Summary of the invention

First purpose of the present invention is to provide a kind of novel algin catenase Alga and encoding gene thereof.

Second object of the present invention is to provide a kind of method preparing novel algin catenase Alga.

Third object of the present invention is to provide novel algin catenase Alga DNA recombinant expression plasmid With recombination engineered strain.

Fourth object of the present invention is to provide novel algin catenase Alga in Algin is degraded Application.

Algin catenase Alga provided by the present invention, derives from cholerae strain Vibrio Alginolyticus 40B, its aminoacid sequence has one of following feature:

1) the SEQ ID NO.2 in sequence table is from 1-521 or the 18-521 position ammonia that aminoterminal starts Base acid residue sequence, wherein 1-17 position is signal peptide sequence, and 18-521 position is that activated Algin splits Solve the aminoacid of enzyme Alga.

2) the 1-521 position SEQ ID NO.2 in sequence table started from aminoterminal or 18-521 position Amino acid residue sequence has degraded Brown algae through the replacement of amino acid residue and/or disappearance and/or after adding The protein of glue activity.

The invention provides the encoding gene (named alga) of novel algin catenase Alga, tool There is one of following nucleotide sequence feature:

1) deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in sequence table；

2) DNA (deoxyribonucleic acid) (DNA) sequence of SEQ ID NO.1 aminoacid sequence in polynucleotide；

The aminoacid sequence of the novel algin catenase Alga of the present invention and nucleotide coding sequence thereof are also Can obtain according to the aminoacid sequence of the Alga of prediction and nucleotide sequence synthetic thereof.

The method preparing recombinase Alga, is that encoding gene alga is cloned into recombinant expression carrier, leads Enter host cell, it is thus achieved that recombinant expressed algin catenase.

The expression vector of described recombinant expressed inscribe algin catenase Alga can be escherichia coli table Reach carrier, Yeast expression carrier, bacillus subtilis expression vector, lactic acid bacteria expression vectors, streptomycete expression Carrier, phage vector, filamentous fungi expression vector, plant expression vector, insect expression vector or Mammalian cell expression vector etc..

For recombinant bacterium or the transgenic cell line of recombinant expressed inscribe algin catenase Alga, permissible Be e. coli host cell (as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α etc.), yeast host cells is (such as Saccharomyces Cerevisiae, Pichia pastoris, Kluyveromyces lactis etc.), bacillus subtilis place Chief cell (such as Bacillus subtilis R25, Bacillus subtilis 9920 etc.), lactic acid Bacterium host cell (such as Lactic acid bacteria COCC101 etc.), actinomycetes host cell (as Streptomyces spp. etc.), filamentous fungal host cell (such as Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans etc.), elder brother Worm cell (such as Bombyx mori, Antharaea eucalypti etc.) or mammalian cell are (in as State hamster ovary cell CHO, baby hamster kidney cell BHK, CHL cells CHL etc.).

The novel algin catenase Alga of the present invention derives from vibrio bacterial strain Vibrio Alginolyticus 40B, by genome digging technology, analyzes Vibrio alginolyticus40B Alginate lyase gene possible in genome, according to homologous sequence by design specific primer, gram The grand algin catenase Alga total length encoding gene that obtains, this gene code head of district 1566bp, encode 521 Individual aminoacid, molecular weight is about 56.72kDa, belongs to polysaceharide lyase family 7.Escherichia coli expression obtains Recombinase Alga, during with Algin for substrate, 30 DEG C, there is high enzymatic activity under the conditions of pH7.0, Rate activity reaches 65.97U/mg.

Accompanying drawing explanation

Fig. 1: the protein three-dimensional structure model of novel algin catenase Alga: overall structure be glove-like-β- Sandwich structure (glove-like-β-sandwich), wherein comprise 3 minor spiral structures (H1: 22-26；H2:76-79；And 2 antiparallel beta-pleated sheet A and B, A H3:185-189) Comprising 8 β-strand structures, B comprises 7 β-strand structures.

Fig. 2: novel algin catenase Alga expresses and the polyacrylamide gel electrophoresis figure of purification (SDS-PAGE).Each swimming lane add sample respectively: M: protein standard molecular weight (marker), Band order from top to bottom is: 170kDa, 130kDa, 110kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa；Swimming lane 1: supernatant after restructuring bacterial cell disruption, applied sample amount is 10 μ l, swimming lane 2: recombinant bacterium breaks Broken rear supernatant is through the effluent of ni-sepharose purification, applied sample amount 10 μ l；Swimming lane 3:10mmol imidazole elution, Applied sample amount 10 μ l；Swimming lane 4:20mmol imidazole elution, applied sample amount 10 μ l；Swimming lane 5:50mmol Imidazole elution, applied sample amount 10 μ l；Swimming lane 6:250mmol imidazole elution, applied sample amount 10 μ l.

Fig. 3: temperature is for the impact of algin catenase Alga activity.

Fig. 4: pH for impact active for algin catenase Alga.

Fig. 5: temperature is for the impact of the stability of algin catenase Alga.

Fig. 6: pH for the impact of the stability of algin catenase Alga.

The substrate Preference curve of Fig. 7: algin catenase Alga.

Fig. 8: algin catenase is for the catabolite ESI-MS collection of illustrative plates of three kinds of substrates.A: Algin substrate The ESI-MS spectrogram of catabolite, the ESI-MS spectrogram of the catabolite of B:polyM substrate, C: The ESI-MS spectrogram of the catabolite of polyG substrate.

Detailed description of the invention

Embodiment 1

The cultivation of Vibrio alginolyticus 40B and strain gene group DNA extract

The strain used is vibrio W13, and the monoclonal colony inoculation of picking Vibrio sp.W13 bacterial strain arrives In 50ml fluid medium, be subsequently placed into temperature be 30 DEG C, revolution be that the shaking table of 220rpmin is cultivated After 24 hours, medium centrifugal is collected thalline and abandons supernatant culture medium.

The liquid culture based formulas used is (g/L): Carnis Bovis seu Bubali cream 5g, glucose 15g, yeast leaching powder 1.0g, NaCl 5.0g, MgSO4 7H2O 0.5g, CaCl2 0.2g, KH2PO4 1.0g, FeSO4 7H2O 0.02g, PH value is 7.0.

Taking the most cultured bacterium solution of 4mL, be placed in 2mLtube pipe, 5000g, 10min are abundant Centrifugal.Supernatant discarded, uses bacterial genomes to extract test kit (Thermal Scientific, USA) Carry out genome extraction, operate as follows: will with 180 μ L Digestive systems (Digestion Solution) Thalline is resuspended, adds 20 μ L E.C. 3.4.21.64s, fully after mixing, and 56 DEG C of incubation 30min.Add After 20 μ L RnaseA, fully mixing, room temperature places 10min.Add 200 μ L lysate (Lysis Solution), quickly mixing, process does not exceeds 15s.Add 400 μ L 50% (v/v) ethanol Solution, fully mixes.Above-mentioned solution is transferred in adsorption column, stands 1min, be centrifuged 6000g, 1min, Abandon waste liquid, adsorption column is transferred in a new 2mL tube pipe.Add 500 μ L wash buffer I, 8000g, 1min are centrifugal, abandon waste liquid.Add 500 μ L wash buffer II, 8000g, 1min is centrifuged, and abandons waste liquid.It is repeated once.Suction attached column 12000g, 3min is centrifugal, by post Residual liquid fully dries.200 μ L eluent (Elution are added in adsorption column matrix membrane centre position Buffer) standing 1min, 8000g, 1min are centrifugal, obtain genome.

The clone of embodiment 2 algin catenase Alga encoding gene and qualification

According to the alginate lyase gene design possible with Vibrio alginolyticus40B genome Following amplimer: forward primer (5 '-CGCATGAAGCATATTTTCTTCAA-3 ') and downstream primer (5’-CCGGCCTTGGTACTTACCAAAAG-3’).With extract strain gene group as template, carry out PCR amplification obtains algin catenase Alga full length gene sequence.PCR condition is: 94 DEG C of denaturations 3min, carries out 30 circulations, finally at 72 DEG C with 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min subsequently Extend 10min.Agarose gel electrophoresis shows a specific band between 1.0kb and 2.0kb, It is cut from agarose gel, uses DNA gel to reclaim test kit and be purified.

The DNA fragmentation of purification is connected on cloning vehicle pMD19-T, converts bacillus coli DH 5 alpha impression In state cell, at LB solid medium (containing 100 μ g/ml ampicillin, 24 μ g/ml X-Gal With 40 μ g/ml IPTG) in cultivate after, picking white colony use amplimer carry out PCR checking. PCR condition is 94 DEG C of denaturations 3min, carries out with 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min subsequently 30 circulations, finally extend 10min at 72 DEG C.Will appear from the recombinant bacterial strain corresponding to specific band big Amount is cultivated, and uses plasmid extraction kit to carry out plasmid extraction, carries out sequencing analysis.Result shows Brown algae Full nucleotide sequence total length 1566bp of glue lyase gene, nucleotide sequence is as shown in SEQ ID NO.1； Encoding 521 aminoacid, aminoacid sequence is as shown in SEQ ID NO.2, and albumen theoretical molecular is 56.72 kDa.By the structural information of SignalP4.1server software analysis algin catenase Alga, result It is signal peptide sequence that display N end starts to the 17th aminoacid, and 18-521 amino acids belongs to brown Algin lyase family 2；With PHYRE2 homology Modeling Server (http://www.sbg.bio.ic.ac.uk/servers/phyre2/html/page.cgi？Id=inde X) protein three-dimensional structure of algin catenase Alga is carried out homology modeling.The Alga finally given Protein three-dimensional structure model as shown in Figure 1.

Sequence table

SEQ ID NO.1

Alga encoding gene, open reading frame (ORF) total length 1566bp, wherein 1-51bp regional code Signal peptide sequence, 52-1566bp encodes algin catenase maturase sequence.

ATGAAGCATATTTTCTTCAAAAGCTTGTTAGCTTCTTCTATTTTACTTGCTGTGGGCTGTAA TAGTACGGCGACCATGGTAGATCCATTTCCAAACAACCAAGAAACTGGTGCTGATATTCTGACTCC CGTTACGATAACGGCGAGTAGTCATGATGGTAATGTTCCAGAGCAGTTGTTTGACCAAGACATTAT GACACGTTGGTCAGCAAATGGTGATGGTGAGTGGGCGATGTTGGATTACGGCTCGGTTTATGAGTT CGACGCAATTCAAGCCTCGTTCAGCAAAGGTAACGAACGCGTCAGCAAATTTGATGTTCAGTTTAG TACTGATGGTGAAAACTGGGTGACGGTTATTGAGGGTGCGCAAAGCTCTGGTCGCGCACTTGGCTT AGAGCGCTTCCAGTTTGAGCCTGCAGTAAAAGCACGTTATGTTCGTTACGTTGGCCACGGTAATAG CAAAAACCAATGGAACTCAGTTACTGAGTTAGCTGCAGTTAATTGCGGAATTAATGCCTGTCCGGC AAGTCACATCATTACTGATGACGTCGTTAACGCTGAAGCGGCTATGATTGCGCAAATGAAAGCGGA GCAAAAGGCACAAAAGGATTTACTCAAAAAGAATCGTAAAGGCGACTTTGGAGCACCAATCGTCCG TCCTTGTGAAACGACTGTGACGTGTGATCTTTCTAAAGCGATGCCTTACCCTACGTTACCGAAAGA GCCACTCGCTACAAATGCACCGGGTGAAAACTTTGACTTAACACGTTGGAAACTGACGACACCTTT CGACCATGACAAGGATGGTCGTGCTGATGATATTGATGAGTGGGACTTGGCTAACGGCTTCCAACA CTCTGATATATTCTACACAGCAGATGACGGCGGAATGGTATTCAAGAGCTATGTGAAAGGCGCTCG AACTTCTGAAAACACTAAATACGCGCGAACTGAGTTACGTACCATGCTTCGTGCTGGTGATAAGTC TTACAGCACAAAAGGTGTAAATCCTAATAACTGGGTATTCAGTTCTGCACCAGTTGAAGATCAAAA AGAAGCGGGTGGTGTAGACGGTACGCTTGAAGCTACTCTGAAGATTGACCACGCAACCACAACTGG TCAGTCGCATGAAGTTGGCCGCTTTATTATCGGCCAGATTCATGACAAAGATGATGAGCCAATTCG TCTTTATTATCGTAAGCTACCAGACCAACCAACAGGTACAGTTTACTTTGCTCACGAAAGAACCAA AACTGGAACAGAGGATTACTACAGCTTAGTGGGTGATATGACTGGTGAAATCGGTGACGATGGCAT CGCGCTAGGTGAAAAATTCAGCTACATCATTGATGTGAAAGGCAACACAATGACAGTTACGGTAAA ACGTGATGGTAAAGATGATGTTGTTCAAGTCGTAGATATGAGTGACAGTGGCTATGACGAAGGTGG CAGGTATATGTACTTTAAAGCCGGTGTCTATAACCAGAATATGTACGGTAATCCAGATGATTACGC ACAAGCTACTTTCTATAAATTAAAACAATCTTTTGGTAAGTACCAAGGCTAG

SEQ ID NO.2

Algin catenase Alga N end start to the 17th aminoacid be signal peptide sequence, and 18-521 Amino acids belongs to algin catenase family 2 (Alginate Lyase 2fami ly).

MKHIFFKSLLASSILLAVGCNSTATMVDPFPNNQETGADILTPVTITASSHDGNVPEQLFDQ DIMTRWSANGDGEWAMLDYGSVYEFDAIQASFSKGNERVSKFDVQFSTDGENWVTVIEGAQSSGRA LGLERFQFEPAVKARYVRYVGHGNSKNQWNSVTELAAVNCGINACPASHIITDDVVNAEAAMIAQM KAEQKAQKDLLKKNRKGDFGAPIVRPCETTVTCDLSKAMPYPTLPKEPLATNAPGENFDLTRWKLT TPFDHDKDGRADDIDEWDLANGFQHSDIFYTADDGGMVFKSYVKGARTSENTKYARTELRTMLRAG DKSYSTKGVNPNNWVFSSAPVEDQKEAGGVDGTLEATLKIDHATTTGQSHEVGRFIIGQIHDKDDE PIRLYYRKLPDQPTGTVYFAHERTKTGTEDYYSLVGDMTGEIGDDGIALGEKFSYIIDVKGNTMTV TVKRDGKDDVVQVVDMSDSGYDEGGRYMYFKAGVYNQNMYGNPDDYAQATFYKLKQSFGKYQG

Embodiment 3

Algin catenase Alga recombinant expression carrier builds

According to alginate lyase gene complete sequence design (removal signal peptide) forward primer (5 '- CGCGGATCCGTGGGCTGTAATAGTACGGC-3 ') and downstream primer (5 '- CCGCTCGAGGCCTTGGTACTTACCAAAAG-3 '), carry out PCR amplification and obtain algin catenase Alga full length gene sequence.PCR condition is: 94 DEG C of denaturations 3min, subsequently with 94 DEG C of 30s, 55 DEG C 30s, 72 DEG C of 2min carry out 30 circulations, finally extend 10min at 72 DEG C.PCR primer Bam HI Reclaim after carrying out enzyme action with Xho I；By coli expression carrier pET21a (+) use Bam HI equally Reclaim after carrying out enzyme action with Xho I, after it being connected with above-mentioned gained genes of interest, convert escherichia coli In DH5 α, screening has the transformant of amicillin resistance.By the plasmid extraction kit extracting positive Transformant plasmid, uses Bam HI and Xho I to carry out digestion verification, obtains length and be respectively 5.5kb With two fragments of about 1.5kb, it was demonstrated that alginate lyase gene has been cloned into expression vector pET21a On (+), by named for this recombiant plasmid pET21a-alga.

Prepared by the embodiment 4 algin catenase Alga gene recombinant expressed and purification in escherichia coli

Recombinant expression plasmid pET21a-alga is converted coli strain BL21 (DE3) (purchased from the U.S. Novagen company), then according to the operating procedure that the said firm provides carries out algin catenase Alga and lures Lead expression and purification.The purification situation of algin catenase Alga is detected by polyacrylamide gel electrophoresis, Result is as in figure 2 it is shown, Alga after purification presents single band on running gel, and position and prediction Molecular weight matches.

The characterization analysis of embodiment 5 algin catenase Alga

(1) pH and the temperature impact on enzymatic activity

It is 1% sodium alginate substrate, the Alga enzyme liquid of purification and the 20mM of different pH value by mass concentration Tris-HCl, phosphate, acetate buffer (pH scope is 2.0-11.0) are by 9:1:10 (volume Than) ratio mixing after, 30 DEG C react 30 minutes, by dinitrosalicylic acid system survey enzyme activity.Knot Fruit display Alga reaches maximum vigor when pH 7.0, show the optimal reaction pH of Alga be 7.0 (as Fig. 3)

Under optimum pH, it is 1% sodium alginate substrate, the Alga enzyme liquid of purification and 20mM by mass concentration Tris-HCl buffer (pH7.0) is mixed in the ratio of 9:1:10 (volume ratio), respectively in not equality of temperature Degree (20 DEG C-70 DEG C) reacts 30 minutes, surveys enzyme activity by dinitrosalicylic acid system.Result shows Alga reaches maximum vigor when 30 DEG C, shows that the optimal reactive temperature of Alga is 30 DEG C (such as Fig. 4).

It is 1% sodium alginate substrate, the Alga enzyme liquid of purification and 50mM Tris-HCl by mass concentration After buffer is mixed in the ratio of 9:1:10 (volume ratio), under optimum temperature and optimum pH, react 30 Minute, survey enzyme activity by dinitrosalicylic acid system, then with the quantification of protein examination being purchased from green skies company Agent box measures the protein content of Alga enzyme liquid, and result shows that the ratio work of sodium alginate is by restructuring Alga 65.97U/mg。

(2) pH and the temperature impact on enzyme stability

Will under different temperatures (20 DEG C-80 DEG C) Alga enzyme liquid after heat treatment 30min and mass concentration Being 1% sodium alginate substrate solution is mixed in the ratio of 1:9 (volume ratio), then at optimum temperature and Measure residual enzyme under suitable pH to live, be defined as 100% relative activity to live without the enzyme liquid enzyme of heat treatment (relativie activity), result shows that Alga has preferably at a temperature of less than 40 DEG C Heat stability (such as Fig. 5).

Will be at 30 DEG C, the different Alga enzyme liquid after pH (pH4-10) preincubate 24h with mass concentration is 1% sodium alginate substrate solution is mixed in the ratio of 1:9 (volume ratio), then at optimum temperature and the suitableeest Measuring residual enzyme under pH to live, living with the enzyme liquid enzyme processed without pH is defined as 100% relative activity (relativie activity), result shows in the range of pH6～10, and Alga enzyme is lived and still kept More than 60%, show that Alga is relatively wide (such as Fig. 6) to pH value tolerance range.

The substrate Preference of Alga

By the Alga of purification respectively with the sodium alginate that mass concentration is 0.1%, polymannuronate fragment (polyM) press 1:9's (volume ratio) with poly-three kinds of different substrates of guluronic acid fragment (polyG) Ratio mixes, and then at 30 DEG C, reacts under the conditions of pH7.0.The product taking differential responses time point surveys it 235nm ultraviolet absorption value, finds that 235nm absorption value is gradually increased, according to purple along with enzymolysis time extends Outer method survey algin catenase principle (Qing-Da An et al.Process Biochemistry, 2008, 43:842 847), it was demonstrated that Alga is lyases.It addition, as it is shown in fig. 7, Alga is to poly-ancient Lip river The degraded of alduronic acid fragment, sodium alginate and polymannuronate fragment is respectively provided with preferable degrading activity, Show that Alga is a difunctional algin catenase.

The impact on Alga activity of embodiment 6 metal ion

It is that 1% sodium alginate substrate, the Alga enzyme liquid of purification and 50mM Tris-HCl delay by mass concentration Rush liquid (pH7.0) to mix in the ratio of 8:2:10 (volume ratio), then add not in reaction system Same metal ion, final concentration of 5mM or 10mM of ion of interpolation, then reacts 30 points at 30 DEG C Clock, surveys enzyme activity by aforesaid dinitrosalicylic acid system.Alga when matched group is for being not added with any metal ion Activity (being set as 100%), result is as shown in the table.Experimental result shows, Ca2+, Ba2+, Co2+ Can increase Alga activity, Mn2+, Fe2+, Ni2+ are active substantially without impact, Cu2+, Zn2+ on Alga Inhibitory action is presented Deng the work of other ions enzymes；Concentration is that the NaCl of 300-500mM splits for Algin The activity solving enzyme Alga has significant facilitation.

The impact on Alga activity of table 1 metal ion

Table 2NaCl concentration is for algin catenase Alga activity influence

The ESI-MS of the catabolite of three kinds of substrates is analyzed by embodiment 7 algin catenase Alga

It is 0.1% substrate (sodium alginate, polyM and polyG), the Alga enzyme of purification by mass concentration After liquid and 50mM Tris-HCl buffer are mixed in the ratio of 9:1:10 (volume ratio), at pH7.0, React under the conditions of 30 DEG C, the product after enzymolysis 72 hours is carried out Electrospray Ionization Mass Spectrometry.Electron spray matter Spectral condition is for using positive ion mode, ion source voltage: 4.5kV；Sheath gas velocity: 30arb；Capillary tube Temperature: 275～300 DEG C；Pipe mirror voltage: 250V；Sweep limits: 150-2000.

As shown in Figure 8, Alga for the product of three kinds of different substrates all with trisaccharide as primary product.Therefore, Algin catenase Alga can be used for the preparation of sodium alginate oligosaccharide and degrades relevant to sodium alginate Field, add including chemical industry, agricultural, food, feedstuff, medicine and Sargassum genetic engineering etc..

Claims (7)

1. algin catenase Alga, have that in sequence table, SEQ ID NO.2 starts from aminoterminal 1-521 or 18-521 amino acids residue sequence.

2. an encoding gene of algin catenase Alga described in claim 1, has in sequence table DNA (deoxyribonucleic acid) (DNA) sequence of SEQ ID NO.1.

3. the preparation method of the algin catenase described in a claim 1, it is characterised in that: be by Alginate lyase gene described in claim 2 is cloned into recombinant expression carrier, imports host cell, Obtain recombinant expressed algin catenase；

The expression vector of described recombinant expressed algin catenase, refers to coli expression carrier, ferment Female expression vector, bacillus subtilis expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phagocytosis Body carrier, filamentous fungi expression vector, plant expression vector, insect expression vector or mammal are thin Cellular expression carrier；

For recombinant bacterium or the transgenic cell line of recombinant expressed algin catenase, refer to escherichia coli place Escherichia coli BL21 in chief cell, Escherichia coli JM109 or Escherichia Saccharomyces cerevisiae, Pichia in coli DH5 α, yeast host cells Bacillus in pastoris or Kluyveromyces lactis, bacillus subtilis host cell Lactic acid in subtilis R25 or Bacillus subtilis 9920, lactic acid bacteria host cell Streptomyces spp. in bacteria COCC101, actinomycetes host cell, filamentous fungi place Trichoderma viride, Trichoderma reesei, Aspergillus niger in chief cell Or Bombyx mori or Antharaea in Aspergillus nidulans, insect cell Eucalypti, the Chinese hamster ovary cell CHO in mammalian cell, baby hamster kidney cell BHK Or the one in CHL cells CHL.

4. the application in alginate is degraded of the algin catenase described in a claim 1.

Apply the most as claimed in claim 4, it is characterised in that: described algin catenase has One of following purposes or more than two kinds；

1) be used for rupturing the glycosidic bond of alginate (sodium alginate), it is thus achieved that alginate oligosaccharide；

2) for degrading alginate (sodium alginate) component in red algae or Brown algae cell wall, extracting Protoplast, studies for food and algae；

Or 3) be used for destroying pathogenic bacterium Pseudomonas aeruginosa (Pseudomonas aeruginosa) institute shape Sodium alginate composition in the Mycoderma become.

Apply the most as claimed in claim 4, it is characterised in that: described algin catenase Alga with After the mixing of other Algin digestive enzymes, for collaborative fracture alginate glycosidic bond.

Apply the most as claimed in claim 4, it is characterised in that: described algin catenase for Sodium alginate be the raw material production degree of polymerization be the sodium alginate oligosaccharide of 3.