CN102994407B

CN102994407B - Flavobacterium strain and incision alginate lyase coding gene, preparation and application

Info

Publication number: CN102994407B
Application number: CN201110424529.2A
Authority: CN
Inventors: 杜昱光; 黄李淑馨; 李曙光; 赵小明; 曹海龙; 刘航
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2011-12-16
Filing date: 2011-12-16
Publication date: 2014-10-29
Anticipated expiration: 2031-12-16
Also published as: CN102994407A; WO2013087043A1

Abstract

The invention discloses a gen sequence of incision alginate lyase Alg2A, as well as a preparation method and application thereof. The incision alginate lyase Alg2A is from soil new separated strain Flavobacterium sp.S20. The invention also provides a preparation method of the novel alginate lyase, namely a technical method by utilizing genetic engineering. According to the method, the gene of the novel alginate lyase is cloned to an escherichia coli expression vector to obtain an escherichia coli recombined strain capable of heterologously expressing the alginate lyase, and the alginate lyase Alg2A prepared by heterologous expression of the strain has the function of preparing sodium alginateoligosaccharide by degrading sodium alginate. The alginate lyase Alg2A provided by the invention can be widely applied to the fields of chemical industry, agriculture, food, feed additive, medicine, algae genetic engineering and the like.

Description

Flavobacterium bacterial strain and inscribe algin catenase encoding gene and preparation and application

Technical field

The present invention relates to gene order of a kind of inscribe algin catenase Alg2A and its preparation method and application.The present invention also provides recombinant plasmid and the recombination engineering strain of this inscribe algin catenase.Inscribe algin catenase Alg2A of the present invention can be widely used in the fields such as chemical industry, agricultural, food, feed interpolation, medicine and marine alga genetic engineering.

Background technology

The wide ocean marine algae resource that is richly stored with, and wherein mainly formed by blue-green algae, green alga, red algae and the large class of brown alga four.Algin (algin) is a kind of straight chain acidic polysaccharose, under native state, algin main existence form in brown alga cell walls is the alkaline metal salts such as water-soluble sodium alginate (sodium alginate), potassium and water-insoluble alginic acid (alginic acid) and the alginic acid salt (alginates) of being combined with the above metal ion of divalent thereof.Sodium alginate (commodity are called sodium alginate) or other alginates are in the market mainly to obtain from brown alga.Research finds that the oligosaccharides that degraded sodium alginate obtains has multiple biological activity, such as immunomodulatory, growth promotion, inducing plant resistance and raising protein stability etc., thereby can be widely used in chemical industry, agricultural, food, feed interpolation and medicine and other fields (Dou Yong, Guangxi light industry, 2009,10:12-13).Sodium alginate can be degraded by several different methods, comprises chemical degradation method, mechanical degradation method and enzyme liberating method.Chemical degradation method is taking acid degradation as main, but the method degradation condition is difficult to control, operate more complicated, length consuming time.Mechanical degradation method comprises radiation method and ultrasonic method etc., generally uses together with other edman degradation Edmans, and the limit molecular mass of degraded product is 50ku left and right, is difficult for making oligosaccharides.And there is degradation condition gentleness with algin catenase degraded sodium alginate, yield advantages of higher, and because the Substratspezifitaet of enzyme can provide information for follow-up study oligosaccharides chemical structure, so algin catenase progressively becomes the method for preferential degraded sodium alginate.In addition, algin catenase can also be applied to research (the Wong TY et al.Annual Review of Microbiology in the fields such as treatment, seaweed fodder processing and the marine alga genetic engineering of pulmonary cyst fibrosis disease, 2000,54:289-340).

Alginate lyase derives from marine animal and plant and multiple-microorganism (comprising marine bacteria, soil bacteria and fungi).Sodium alginate lyase can be divided into two large classes by its Substratspezifitaet: Isosorbide-5-Nitrae-beta-D-mannuronic acid fragment lyase (Ec 4.2.2.3) and Isosorbide-5-Nitrae-a-L-guluronic acid fragment lyase (EC 4.2.2.11).So far, the production of algin catenase mostly relies on original product enzyme animals and plants or microorganism and obtains zymoprotein, although this kind of method can effectively obtain a certain amount of zymoprotein, output is limited, and cost is higher, more difficultly meets practical application request.Along with the development of biotechnology, provide the technological method that utilizes genetically engineered High-efficient Production algin catenase.Dong Eun Kim etc. are according to the similar sequences design PCR primer of known correlation function gene, from Streptomyces sp.ALG-5 bacterial strain, be cloned into an alginate lyase gene, and in Escherichia coli BL21 (DE3) successful expression (Kim et al.Marine Biotechnology.2009.11:10-16).Adopt this strategy to have certain understanding could design PCR primer to genes involved sequence, and that find is the recruit in a certain class formation or functional similarity protein, the gene that more difficult discovery is brand-new.

Bacterium producing multi enzyme preparation is built to its genomic library, can break through the restriction of PCR clone gene, fully excavate and utilize the genetic resources of microorganism, then the method for combined function screening, brand-new gene can be found.Caswell etc. are cloned into the alginate lyase gene to the degraded of guluronic acid fragments specific from the genomic library of Klebsiella pneumoniae PG1 bacterial strain, the molecular weight of this enzyme is 28kDa (Caswell et al.Gene, 1989,75:127-134).The people such as Maki and Kraiwattanapong has successively been cloned into two alginate lyase genes from the genomic library of Pseudomonas sp.OS-ALG-9 bacterial strain, one is the aly to the degraded of polymannuronic acid fragments specific, this genes encoding head of district 1365bp, 398 amino acid of having encoded, the about 50kDa of molecular weight (Maki et al.Journal of General Microbiology, 1993,139:987-993).Another is alyII, this genes encoding head of district 2141bp, 713 amino acid of having encoded, the about 79kDa of molecular weight (Kraiwattanapong et al.Bioscience Biotechnology and Biochemistry, 1997,61 (11): 1853-1857).Up to now, producing algin catenase for Flavobacterium studies less.Two kinds of algin catenases the crude enzyme liquid that the people such as Takeuchi produce from Flavobacterium multivolum K-11 with column chromatography, are purified to, a kind of is that molecular weight is the guluronic acid lyase (EC 4.2.2.11) (Takeuchi et al.Food Science and Technology International.1997.3 (1): 22-26) of 43kDa, another kind is that molecular weight is 32kDa, the algin catenase (Takeuchi et al.Food Science and Technology International.1997.3 (4): 388-392) identical to these two kinds of fragment degradation of substrates speed of guluronic acid fragment and polymannuronic acid fragment.The people such as An are newly separated to the Flavobacterium sp.LXA bacterial strain of a strain product algin catenase, they from the fermentation culture of this bacterial strain with ammonium sulfate precipitation method partial purification algin catenase, and by enzyme production and the activity research (An et al.Journal of Applied Microbiology.2009.106:161-170) for algin oligosaccharide.The alginate lyase gene that derives from present Flavobacterium there is not yet report.

Summary of the invention

First object of the present invention is to provide a kind of new bacterial strain of Flavobacterium that produces algin catenase, and it is Flavobacterium S20, Classification And Nomenclature: Flavobacterium Flavobacterium sp., deposit number: CGMCC NO.5026.

Second object of the present invention is to provide a kind of new and effective inscribe algin catenase Alg2A and encoding gene thereof.

The 3rd object of the present invention is to provide a kind of method of preparing new and effective inscribe algin catenase Alg2A.

The 4th object of the present invention is to provide and contains described inscribe algin catenase Alg2A DNA recombinant expression plasmid and recombination engineering strain.

Another object of the present invention is to provide the application of new and effective inscribe algin catenase Alg2A in sodium alginate degraded.

Inscribe algin catenase Alg2A provided by the present invention, derives from the new bacterial strain Flavobacterium of the Flavobacterium of separation and purification sp.S20 in soil, and its aminoacid sequence has one of following feature:

1) the SEQ ID NO.2 in sequence table starts from aminoterminal 1-288 or 23-288 amino acids residue sequence, wherein 1-22 position is signal peptide, 23-288 position is the amino acid of activated algin catenase Alg2A.

2) 1-288 SEQ ID NO.2 in sequence table being started from aminoterminal or 23-288 amino acids residue sequence are through the replacement of amino-acid residue and/or disappearance and/or after adding, have the protein of degraded sodium alginate activity.

3) with sequence table in the homology of the aminoacid sequence that limits of SEQ ID NO.2 reach 80% and above and there is the protein of degraded alginate activity.

The present invention also provides the encoding gene (called after alg2A) of inscribe algin catenase Alg2A, has one of following nucleotide sequence feature:

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

2) thymus nucleic acid (DNA) sequence of SEQ ID NO.2 aminoacid sequence in code sequence list;

3) homology of thymus nucleic acid (DNA) sequence limiting with SEQ ID NO.1 reach 80% and more than, and thymus nucleic acid (DNA) sequence of alginate protein of degrading of encoding.

The aminoacid sequence of algin catenase Alg2A of the present invention and nucleotide coding sequence thereof also can obtain according to the aminoacid sequence of the Alg2A of prediction and nucleotide coding sequence synthetic thereof.

Preparing the method for recombinase Alg2A, is that encoding gene alg2A is cloned into recombinant expression vector, imports host cell, obtains recombinant expressed inscribe algin catenase.

The expression vector of described recombinant expressed inscribe algin catenase Alg2A can be coli expression carrier, Yeast expression carrier, Bacillus subtilus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungus 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 Alg2A, can be that e. coli host cell is (as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α etc.), yeast host cell is (as Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis etc.), Bacillus subtilus host cell is (as Bacillus subtilis R25, Bacillus subtilis 9920 etc.), milk-acid bacteria host cell (as Lactic acid bacteria COCC101 etc.), actinomycetes host cell (as Streptomyces spp. etc.), filamentous fungal host cell (as Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans etc.), insect cell (as Bombyxmori, Antharaea eucalypti etc.) or mammalian cell (as Chinese hamster ovary cell CHO, immature hamster kidney cell BHK, Chinese hamster pneumonocyte CHL etc.).

Alg2A of the present invention derives from the new bacterial strain Flavobacterium of the Flavobacterium of separation and purification sp.S20 in soil, by the screening active ingredients to Flavobacterium sp.S20 genome dna library, can the degrade positive colony of sodium alginate of acquisition, further obtain the complete encoding sequence of inscribe algin catenase Alg2A by the method for order-checking, homology comparison, this genes encoding head of district 867bp, 288 amino acid of having encoded, the about 33kD of molecular weight, belongs to algin catenase family 2.The Alg2A that Recombinant protein expression obtains during taking sodium alginate as substrate, has enzymatic activity high under 45 DEG C, the condition of pH8.5, and specific activity reaches 300U/mg.With respect to sodium alginate and these two kinds of substrates of polymannuronic acid fragment (polyM), Alg2A has higher degrading activity to poly-guluronic acid fragment (polyG).

The fields such as inscribe algin catenase Alg2A of the present invention can widespread use chemical industry, agricultural, food, feed interpolation, medicine and marine alga genetic engineering.

Brief description of the drawings

Fig. 1: the protein three-dimensional structure model of inscribe algin catenase Alg2A

Fig. 2: the polyacrylamide gel electrophoresis figure (SDS-PAGE) of recombined endo algin catenase Alg2A Expression and purification.The sample that each swimming lane adds is respectively: M: Protein biomarker (marker), and band from top to bottom size is 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD; Swimming lane 1: supernatant after recombinant bacterium broken wall, applied sample amount 10 μ l, swimming lane 2: after recombinant bacterium fragmentation supernatant through the effluent liquid of ni-sepharose purification, applied sample amount 10 μ l, the imidazoles wash-out of swimming lane 3:100mmol is collected liquid, applied sample amount 10 μ l.

Fig. 3: the activity influence curve of pH value to inscribe algin catenase Alg2A.

Fig. 4: the activity influence curve of temperature to inscribe algin catenase Alg2A.

Fig. 5: the stability influence curve of temperature to inscribe algin catenase Alg2A.

Fig. 6: the stability influence curve of pH value to inscribe algin catenase Alg2A.

Fig. 7: the substrate Preference curve of inscribe algin catenase Alg2A.

Fig. 8: electrospray ionization mass spectrum (ESI-MS) analysis chart of inscribe algin catenase Alg2A degraded sodium alginate products therefrom.

The bacterial strain of Flavobacterium, it is Flavobacterium S20, Classification And Nomenclature: Flavobacterium Flavobacterium sp., be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number: CGMCC NO.5026, preservation date on 07 05th, 2011.

Embodiment

The cultivation of the new bacterial strain Flavobacterium of embodiment 1 Flavobacterium sp.S20 and product algin catenase

The bacterial classification adopting is Flavobacterium S20, the mono-clonal of picking Flavobacterium sp.S20 bacterial strain (CGMCC NO.5026) is inoculated in 50ml liquid nutrient medium, then the shaking table that to put into temperature and be 30 DEG C, revolution be 220rpmin was cultivated after 48 hours, and medium centrifugal is collected to Flavobacterium sp.S20 thalline and retained supernatant substratum.By carrying out ultrasonic bacteria breaking after the resuspended thalline of 5ml phosphate buffered saline buffer (20mM, pH7.0).

The liquid culture based formulas using is (g/L): extractum carnis 5g, glucose 15g, yeast soak powder 1.0g, NaCl 5.0g, MgSO ₄7H ₂o 0.5g, CaCl ₂0.2g, KH ₂pO ₄1.0g, FeSO ₄7H ₂o 0.02g, pH value is 7.0.

Enzyme activity unit (U) definition: per minute catalysis sodium alginate produces the needed enzyme amount of 1 μ mol reducing sugar.Survey algin according to dinitrosalicylic acid and split method (the Qing-Da An et al.Process Biochemistry that enzyme enzyme is lived, 2008,43:842-847), recording enzyme in the liquid nutrient medium of Flavobacterium sp.S20 bacterial strain lives as 0.37U/ml, and this cellular lysate liquid enzyme is lived as 1.34U/ml, thus with the total algin catenase enzyme of the nutrient solution work that 50ml liquid nutrient medium cultivation Flavobacterium sp.S20 bacterial strain 48h obtains be 1.71U/ml.

The extraction of embodiment 2 Flavobacterium sp.S20 strain gene group DNAs

Get the fresh bacterium liquid of Flavobacterium S20 of 2ml incubated overnight, centrifugal (12,000rmp, 3min) collect thalline.Wash thalline three times with phosphate buffered saline buffer (20mM, pH7.0), add 650 μ l DNA extraction buffer (100mM Tris-HCl; 100mM Na ₂eDTA; 100mM Na ₃pO ₄; 1.5M NaCl; 1% (w/v) cetyl trimethylammonium bromide; PH8.0), after mixing, put-80 DEG C freezing, be then placed in 65 DEG C of water-baths and melt, so multigelation three times.After cooling, add 4 μ l N,O-Diacetylmuramidases (100mg/L) level in shaking table to vibrate (37 DEG C, 225rpm) 30min, then add the follow-up persistent oscillation 30min of 3 μ l Proteinase K (20mg/mL), finally add 50 μ l 20% (w/v) sodium lauryl sulphate, after mixing, 65 DEG C of insulation 2h (mixing every the 20min centrifuge tube that turns upside down).12, the centrifugal 10min of 000rpm room temperature, collect supernatant liquor, add the saturated phenol of 500 μ l: chloroform: primary isoamyl alcohol (volume ratio is 25: 24: 1) is by carrying twice, then add 250 μ l chloroforms: primary isoamyl alcohol (volume ratio is 24: 1) extracting once adds the Virahol of 0.6 times of volume afterwards, room temperature is placed after 20min, the centrifugal 15min of 12,000rpm.The ethanol rinsing that precipitation is 70% by volumetric concentration, dry rear with 60 μ l TE damping fluid (10mM Tris-HCl, 1mM Na ₂eDTA, pH8.0) dissolve, put into-20 DEG C of preservations after adding 1 μ l rnase (remove RNA).

The structure in embodiment 3 Flavobacterium sp.S20 bacterial strain total genomic dna libraries and the clone that algin catenase activity is expressed in screening from library

With Sau3A I enzyme, the Flavobacterium S20 strain gene group DNA extracting is carried out to random enzyme and cut, agarose gel electrophoresis separation enzyme is cut the DNA fragmentation of product acquisition 3-10kb.These fragments are cut with through BamHI enzyme and remove 5 ' with alkaline phosphatase and hold the pGEM11z carrier of phosphate to be connected, connect product and transform intestinal bacteria Top10 bacterial strain, converted product is coated and is contained X-gal (the chloro-3-indoles-β-D-of the bromo-4-of 5-galactoside, 40 μ g/ml), IPTG (sec.-propyl-β-D-sulfo-galactopyranoside, 40 μ g/ml), Amp (Ampicillin Trihydrate, 50 μ g/ml) Luria-Bertani substratum solid plate on, 37 DEG C cultivate 12～16h.By the white intestinal bacteria transformant picking growing (200 μ l LB substratum/hole) in 96 well culture plate holes, after 37 DEG C of cultivation 12～16h, in each cultivation plate hole, adding 150 μ l volumetric concentrations is 50% glycerine, then be stored in-80 DEG C, be stored in institute's adularescent intestinal bacteria transformant in 96 well culture plate holes and be the genome dna library clone of Flavobacterium S20 bacterial strain.Sau3A I enzyme, BamHI enzyme and alkaline phosphatase are all purchased from precious biotech firm, the system of enzyme-to-substrate reaction, the description of product operation that temperature and time all provides according to company.

The genomic library clone of Flavobacterium sp.S20 bacterial strain is inoculated in 96 well culture plates, and 200 μ l Luria-Bertani substratum (containing Ampicillin Trihydrate 50 μ g/ml) are equipped with in each hole, centrifugal collection thalline after 37 DEG C of cultivation 12h.Every hole adds after 20 μ l phosphoric acid buffers (20mM, pH7.0), by freezing-80 DEG C of thalline placements, is then placed in 37 DEG C of thawings, so repeats freeze thawing and three times thalline is broken.Whether the cellular lysate liquid that detects all library clones by foregoing dinitrosalicylic acid method has enzyme to live, result shows the library clone expression algin catenase activity that is numbered VII 2H, by recombinant plasmid called after pGEM11z-VII 2H contained this library clone.

On embodiment 4 recombinant plasmid pGEM11z-VII 2H, express order-checking and the sequential analysis thereof of the gene of algin catenase activity

The active recombinant plasmid pGEM11z-VII of expression algin catenase 2H is sent to Invitrogen Corp.'s order-checking.Sequencing result is analyzed with the software on NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/).NCBI analysis software used is Open Reading Frame Finder (ORF Finder, http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi).

On NCBI analytical results demonstration recombinant plasmid pGEM11z-VII 2H, carry an alginate lyase gene (called after alg2A), this genes encoding head of district 867bp, its nucleotide sequence is as shown in SEQ ID NO 1.520 Nucleotide in the whole genome sequence (NCBI number of registration: CP002825.1) of alg2A and Lacinutrix sp.5H-3-7-4 have 71% homology.

The algin catenase Alg2A of alg2A coding is made up of 288 amino acid, and its aminoacid sequence is as shown in SEQ ID NO 2, and the theoretical molecular of protein is about 33kD.With Simple Modular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de/) analyze the structural information of algin catenase Alg2A, result shows that N end beginning to the 22nd amino acid is signal peptide sequence, and 23-288 amino acids belongs to algin catenase family 2.The protein three-dimensional structure of algin catenase Alg2A is carried out to homology modeling with SWISS-MODEL homology modeling service device (http://swissmodel.expasy.org), the Alg2A protein three-dimensional structure model finally obtaining as shown in Figure 1.

Sequence table

SEQ?ID?NO.1

ATGAGCATACAATTTTCAAAAATCTTATTACTAACGGTTCTA

GCAACTGCTACAATTAGTAATGCACAGGATAAAAAATCAAA

AAGCAAAACTGCTAAAATTGATTGGTCTCATTGGACGGTTAC

TGTGCCTGAGGAGAATCCAGATAAACCAGGTAAGCCGTACT

CTTTAGGGTATCCTGAAATACTAAATTATGCTGAGGATAAAA

TCGCATCCAAGTACATGTACGATGACCCAAAAGACAAGTCT

GTTGTTTTTTATGCCTTTCCTTCGGGAGTGACCACGGCTAATA

CGCATTATTCTCGTTCTGAGCTAAGAGAGACAATGGAAACTG

GTAGCAATAAGGTCAACTGGACATTTGCAAAAGGCGGTAAA

ATGAGAGGTACGTATGCTATTGACGACATTTCAAAAGAGCC

AGATGGCAAATACAGCCGCGTTATTATTGCGCAAATTCACGG

TGTATTAACGGATGAACAACGTGATTTAATTGGTCAAAAAG

ACAACAATGCACCGCCTATTTTGAAAGTGTATTGGGATAAAG

GAAAAATTCGTGTGAAAACAAAAGTACTTAAAGATTTGAAC

GCGCCTTATAAAGAAATGCTTTTAGAACATGCTTGGGGTGAT

GATGAAGGTCGAAATTTTAAAGAGAAAATCGATTTAAACAC

AAGGTTTACTCTAGAAGTGAAAGTTTCGGATGGGCGAATGG

AAGTGATTTTAAATGATACAGAATCATTGGTTTACGATGATA

TTCACATGAAAAAATGGGGGATATTCGAAAATTATTTTAAAG

CAGGGAATTATTTTCAATCTAAAACACCAGGTACCTTTGCAA

AGGTAAAAATATATTCTTTACAAGTTACTCATTAG

SEQ?ID?NO.2

MSIQFSKILLLTVLATATISNAQDKKSKSKTAKIDWSHWTVTVP

EENPDKPGKPYSLGYPEILNYAEDKIASKYMYDDPKDKSVVFY

AFPSGVTTANTHYSRSELRETMETGSNKVNWTFAKGGKMRGT

YAIDDISKEPDGKYSRVIIAQIHGVLTDEQRDLIGQKDNNAPPIL

KVYWDKGKIRVKTKVLKDLNAPYKEMLLEHAWGDDEGRNFK

EKIDLNTRFTLEVKVSDGRMEVILNDTESLVYDDIHMKKWGIFE

NYFKAGNYFQSKTPGTFAKVKIYSLQVTH

Recombinant expressed in intestinal bacteria of embodiment 5 alg2A genes

Taking above-mentioned recombinant plasmid pGEM11z-VII 2H as template, by following primer to carrying out pcr amplification.Primer pair is as follows: forward primer Alg2A-F:

( CATATGCAGGATAAA

AAATCAAAAAGCAAAACTG), reverse primer Alg2A-R:

( CTCGAGATGAG

TAACTTGTAAAGAATAT), what forward primer underscore marked is restriction enzyme Ned I site, and what reverse primer underscore marked is restriction enzyme Xho I site.Taq archaeal dna polymerase is purchased from precious biotech firm, and the description of product that PCR reaction system provides according to company operates.PCR reaction conditions: 94 DEG C of denaturations 5 minutes, then-72 DEG C of 94 DEG C of sex change-50 DEG C of annealing in 30 seconds 30 seconds are extended 1 minute, 30 circulations, last 72 DEG C are extended 10 minutes.By Ned I and Xho I double digestion for PCR product, agarose gel electrophoresis reclaims the PCR product that enzyme is cut.To be purchased from product pET-21a carrier Ned I and the Xho I double digestion of Novagen company of the U.S., agarose gel electrophoresis reclaims enzyme and cuts carrier large fragment.Ned I and Xho I are all purchased from precious biotech firm, the system of enzyme-to-substrate reaction, the description of product operation that temperature and time all provides according to company.

PCR product through double digestion is connected through double digestion pET-21a carrier with same, connect after product transforms intestinal bacteria Top10 bacterial strain and coat on the Luria-Bertani substratum solid plate that contains 50 μ g/ml Ampicillin Trihydrates, cultivate 14h, picking mono-clonal for 37 DEG C; Mono-clonal is accessed in the liquid Luria-Bertani substratum that contains 50 μ g/ml Ampicillin Trihydrates and cultivated, extract plasmid; Plasmid is carried out to bacterium colony PCR checking with forward primer Alg2A-F and reverse primer Alg2A-R, and result obtains the amplified production that size is correct, and the recombinant plasmid that preliminary proof builds is correct; Then this recombinant plasmid is sent to Invitrogen Corp.'s order-checking, result shows, between the Ned of pET-21a I and Xho I restriction enzyme site, insert the alg2A gene shown in SEQ ID NO 1, and direction of insertion is correct, so further prove that the recombinant plasmid building is correct, by this recombinant plasmid called after pET21a-Alg2A.

PET21a-Alg2A is transformed to coli strain BL21 (DE3) (purchased from Novagen company of the U.S.), and the operation steps then providing according to the said firm is carried out algin catenase Alg2A abduction delivering and purifying.The purifying situation that detects algin catenase Alg2A with polyacrylamide gel electrophoresis, as shown in Figure 2, the algin catenase Alg2A after purifying is single band to result on running gel, and the molecular weight of position and prediction matches.The zymologic property analysis of embodiment 6 algin catenase Alg2A

(1) pH and the temperature impact on enzymic activity

Be after the Alg2A enzyme liquid of 1% sodium alginate substrate, purifying and the 20mM Tris-HCl damping fluid of different pH values (pH scope is 6.0-10.0) mix in the ratio of 8: 2: 10 (volume ratio) by mass concentration, 40 DEG C of reactions 10 minutes, survey enzyme activity by aforesaid dinitrosalicylic acid method.Result shows that Alg2A reaches maximum vigor in the time of pH 8.5, and the optimal reaction pH that shows Alg2A is 8.5 (as Fig. 3)

Under optimal pH, the Alg2A enzyme liquid and the 20mM Tris-HCl damping fluid (pH8.5) that are 1% sodium alginate substrate, purifying by mass concentration mix in the ratio of 8: 2: 10 (volume ratio), differing temps (20 DEG C-70 DEG C) reaction 10 minutes, survey enzyme activity by aforesaid dinitrosalicylic acid method respectively.Result shows that Alg2A reaches maximum vigor in the time of 45 DEG C, and the optimal reactive temperature that shows Alg2A is 45 DEG C (as Fig. 4).

Be after the Alg2A enzyme liquid of 1% sodium alginate substrate, purifying and 50mM Tris-HCl damping fluid mix in the ratio of 8: 2: 10 (volume ratio) by mass concentration, under optimum temperuture and optimal pH, react 10 minutes, survey enzyme activity by aforesaid dinitrosalicylic acid method, then with the protein content of quantification of protein kit measurement Alg2A enzyme liquid that is purchased from betting office, the result Alg2A that shows to recombinate is 300U/mg to the specific activity of sodium alginate.

(2) pH and the temperature impact on enzyme stability

Be that 1% sodium alginate substrate solution mixes in the ratio of 2: 8 (volume ratio) by the Alg2A enzyme liquid after the lower thermal treatment 1h of differing temps (20 DEG C-50 DEG C) with mass concentration, then under optimum temperuture and optimal pH, measuring residual enzyme lives, to be defined as 100% relative activity (relativie activity) without the work of heat treated enzyme liquid enzyme, result shows that Alg2A has good thermostability (as Fig. 5) at the temperature lower than 40 DEG C.

Will be at 30 DEG C, Alg2A enzyme liquid after different pH (pH4-10) preincubate 24h is that 1% sodium alginate substrate solution mixes in the ratio of 2: 8 (volume ratio) with mass concentration, then under optimum temperuture and optimal pH, measuring residual enzyme lives, be defined as 100% relative activity (relativie activity) with the enzyme liquid enzyme work of processing without pH, result is presented in the scope of pH5～10, Alg2A enzyme is lived and is still kept more than 60%, shows that Alg2A is to wide (as Fig. 6) of pH value tolerance range.

(3) the substrate Preference of Alg2A

The sodium alginate that is 1% from mass concentration respectively by the Alg2A of purifying, polymannuronic acid fragment (polyM) and three kinds of different substrates of poly-guluronic acid fragment (polyG) mix in the ratio of 2: 8 (volume ratio), then at 30 DEG C, under pH8.5 condition, react.Get the product of differential responses time point and survey its 235nm ultraviolet absorption value, find along with enzymolysis time extends, 235nm absorption value increases gradually, survey principle (the Qing-Da An et al.Process Biochemistry of algin catenase according to ultraviolet method, 2008,43:842-847), confirm that Alg2A is lyase.In addition, as shown in Figure 7, Alg2A faster than the degraded to sodium alginate and polymannuronic acid fragment, shows the poly-guluronic acid fragment of Alg2A preference degraded to poly-guluronic acid segment degradation speed.

The impact of embodiment 7 metal ions on Alg2A activity

The Alg2A enzyme liquid and the 50mM Tris-HCl damping fluid (pH8.5) that are 1% sodium alginate substrate, purifying by mass concentration mix in the ratio of 8: 2: 10 (volume ratio), then in reaction system, add different metal ions, the ion final concentration adding is 5mM or 10mM, then 45 DEG C of reactions 10 minutes, survey enzyme activity by aforesaid dinitrosalicylic acid method.Control group is the activity (being set as 100%) of Alg2A while not adding any metal ion, and result is as shown in the table.Experimental result shows, K ⁺, Na ⁺can increase Alg2A activity, Li ⁺, Mg ²⁺active substantially without impact, Ca on Alg2A ²⁺, Fe ²⁺present restraining effect Deng other ions enzyme work.

Table 1: the impact of metal ion on Alg2A activity

The electrospray ionization mass spectrum (ESI-MS) of embodiment 8 Alg2A degraded sodium alginate products therefroms is analyzed

Be after the Alg2A enzyme liquid of 1% sodium alginate substrate, purifying and 50mM Tris-HCl damping fluid mix in the ratio of 8: 2: 10 (volume ratio) by mass concentration, at pH8.5, under 45 DEG C of conditions, react, the product of choosing different enzymolysis times (0.5,1,1.5,2 hour) carries out Electrospray Ionization Mass Spectrometry.Electrospray ionization mass spectrum condition is for adopting positive ion mode, ion source voltage: 4.5kV; Sheath gas velocity: 30arb; Capillary temperature: 275～300 DEG C; Pipe mirror voltage: 250V; Sweep limit: 150-2000.

Wherein the enzymolysis electrospray ionization mass spectrum figure of 2 hours as shown in Figure 8.On mass spectrum, show the sodium alginate oligosaccharides that has a series of different polymerization degrees in the Alg2A degraded sodium alginate product of 2 hours, this result has fully proved that Alg2A belongs to inscribe algin catenase.Therefore, inscribe algin catenase Alg2A can be used to the preparation of sodium alginate oligosaccharides and to the sodium alginate relevant field of degrading, comprise chemical industry, agricultural, food, feed interpolation, medicine and marine alga genetic engineering etc.

Claims

1. the bacterial strain of Flavobacterium, it is Flavobacterium S20, Classification And Nomenclature: Flavobacterium Flavobacterium sp., be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number: CGMCC NO.5026, preservation date on 07 05th, 2011.

2. an inscribe alginate lyase gene Alg2A who derives from bacterial strain (Flavobacterium sp.S20) described in claim 1, its nucleotide sequence has one of following feature:

1) be thymus nucleic acid (DNA) sequence of SEQ ID NO.1 in sequence table;

2) thymus nucleic acid (DNA) sequence of coding SEQ ID NO.2 aminoacid sequence.

3. according to the inscribe algin catenase of inscribe alginate lyase gene coding claimed in claim 2, the aminoacid sequence of its coding is 1-288 or the 23-288 amino acids residue sequence that in sequence table, SEQ ID NO.2 starts from aminoterminal.

4. prepare the method for inscribe algin catenase as claimed in claim 3 for one kind, it is characterized in that: be that inscribe alginate lyase gene claimed in claim 2 is cloned into recombinant expression vector, import host cell, obtain recombinant expressed inscribe algin catenase;

The expression vector of described recombinant expressed inscribe algin catenase, refers to coli expression carrier, Yeast expression carrier, Bacillus subtilus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungus expression vector, plant expression vector, insect expression vector or mammalian cell expression vector.

5. in accordance with the method for claim 4, it is characterized in that: for recombinant bacterium or the transgenic cell line of recombinant expressed inscribe algin catenase, refer to e. coli host cell, yeast host cell, Bacillus subtilus host cell, milk-acid bacteria host cell, actinomycetes host cell, filamentous fungal host cell, insect cell, the one in mammalian cell.

6. the application of inscribe algin catenase as claimed in claim 3 in alginate degraded, is characterized in that: have one of following purposes or more than two kinds;

1) for the glycosidic link of rupture alginate or algal polysaccharide, obtain alginate oligosaccharides;

2) for the alginate component of degrade red algae or brown alga cell walls, extracting protoplastis, protoplastis is for food and algae research.

7. application as claimed in claim 6, is characterized in that: after described inscribe algin catenase Alg2A mixes with other algin degrading enzymes, for the application of collaborative fracture alginate glycosidic link aspect.