CN102174494A - Marine cryophilic endo beta-xylanase XynB as well as expressing gene xynB and application thereof - Google Patents

Abstract

The invention relates to a marine cold-adapted endo-beta-xylanase and its expression gene xynB and its application, belonging to the technical field of biotechnology. A marine cold-adapted endo-beta-xylanase XynB, the amino acid sequence of which is shown in SEQ ID NO.1. As well as the gene xynB encoding the marine cold-adapted endo-β-xylanase XynB, the nucleotide sequence is shown in SEQ ID NO.2. The β-xylanase XynB of the present invention has relatively high catalytic efficiency at 0-35°C, and can be used in pulp bleaching, xylooligosaccharide production, flour modification, etc. at room temperature, which can save the traditional process. heating step, thereby saving energy.

Description

The suitable cold inscribe beta-xylanase XynB in a kind of ocean and expressing gene xynB and application

Technical field

The present invention relates to the suitable cold inscribe beta-xylanase in a kind of ocean and expressing gene xynB and application, belong to technical field of biotechnology.

Background technology

Beta-xylanase be by bacterium and fungus secretion can degradation of xylan glycoside hydrolase, the overwhelming majority belongs to glycoside hydrolase the 10th family and the 11st family, finds in recent years that the minority beta-xylanase belonged to the 5th, 7,8 and 43 families.Wherein the 8th family's major part is a cellulase, also comprise a part of chitinase and zytase, at present the 8th family's zytase through property research has only 2, is respectively from the pXyl of Pseudoalteromonas.haloplanktis TAH3a with from the Xyn8 of environment genomic library.According to the crystal structure analysis result to pXyl, the 8th family's zytase may be folded into (a/a) of distortion ₆Barrel-like structure is centered on by outer and each 6 a spiral of internal layer and to form, and this topological framework is found in the 15th family's amylase and the 65th family's maltose phosphorylase also once in the 9th family's endoglucanase.Similar with the 11st family's zytase, the 8th family's zytase contains a bigger substrate in conjunction with the crack, can hold at least 6 xylose residues, the amino-acid residue that plays katalysis is L-glutamic acid and aspartic acid, both close proximity and be positioned at the fissured middle position of three-dimensional structure.Different with the retaining catalyst mechanism of the 10th and 11 family's zytases, the 8th family's zytase is taked the mode of opposite inverting.According to existing result of study, the Substratspezifitaet of the 8th family zytase effect is more intense, and general only have hydrolytic action to xylan, to the not effect of other glycan classes such as Mierocrystalline cellulose.

Beta-xylanase has a wide range of applications in industries such as papermaking, food and feed.In the pulping and paper-making industry, the use of beta-xylanase can reduce the consumption of acid bleaching agent in the pulp bleaching process (as chlorine, dioxide peroxide), reduces and pollutes.In food service industry, beta-xylanase can decompose from natural hemicellulose such as cotton seed hulls, bagasse and corn cob and obtains xylo-oligosaccharide, and this sugar is the current remarkable a kind of functional oligose that is subjected to most.Its sugariness is 40% of a sucrose, can not cause that glucose level rises significantly in the blood plasma after edible.Can be made into the food of different sugarinesses with xylo-oligosaccharide, can replace glucose, as diabetics's dietetic food.The use of beta-xylanase can be converted into nutritional substance with the non-starch polysaccharide in the feed in feedstuff industry, eliminates its anti-oxidant action, thereby improves the transformation efficiency of feed.Beta-xylanase is applied to China grass degumming, can reduces environmental pollution, reduce the damage of degumming technology fiber.

Because the extensive use of beta-xylanase is all paid attention to by the countries in the world scientist the research and development of beta-xylanase always.The novel beta-xylanase that exploitation has new purposes has very important theory significance and using value.Since the seventies in 20th century, along with gene recombination technology and development of molecular biology, the encoding gene of many beta-xylanases and primary structure are studied in great detail, and the minority enzyme are arranged by crystallization.

In beta-xylanase, fit cold zytase owing to have very high activity at low temperatures, therefore at association with pulp bleaching, wood oligose production, warm zytase has more superiority in industries such as flour modification and the feed ratio, has been subjected to investigator's extensive concern.Studies show that in recent years, polar region exist a large amount of microorganisms, and these microorganism long term growth have formed cryogenic special construction of adaptation and special mechanism under cryogenic extreme environment, and therefore, the polar region microorganism is a good material of finding novel suitable cold enzyme.But because difficulties such as sampling and cultivation are at present relatively less to the research of polar region microorganism.

Summary of the invention

The present invention is directed to the deficiencies in the prior art, the suitable cold inscribe beta-xylanase XynB in a kind of ocean and expressing gene xynB and application are provided.

The technical term that the present invention uses:

1.CTAB/NaCl method: be meant at containing the more bacterium of polysaccharose substance, extracting on the bacterial genomes DNA ordinary method basis, add CTAB (bromination n-Hexadecane trimethyl ammonium) and sodium-chlor and remove polysaccharose substance, thereby reach a kind of method of extraction effect preferably.

2.PCR technology: the chain polymerization enzyme reaction, utilize archaeal dna polymerase, template, primer and dNTP carry out the technology of amplification in vitro specific DNA fragments.

3.CDD analysis software: a kind of software that is used for inferring and analyzing the structural domain information of specific amino acids sequence formation that provides on the NCBI website.

Cold inscribe beta-xylanase XynB is fitted in a kind of ocean, and aminoacid sequence is shown in SEQ ID N0.1.

The encode gene xynB of the suitable cold inscribe beta-xylanase XynB in above-mentioned ocean, nucleotide sequence is shown in SEQ ID NO.2.

A kind of expression vector that contains nucleotide sequence shown in the SEQ ID No.2.

The reconstitution cell that contains nucleotide sequence shown in the above-mentioned SEQ ID No.2 or above-mentioned expression vector.

The suitable cold inscribe beta-xylanase XynB in above-mentioned ocean and/or said gene xynB application aspect hydrolysis oat xylan, beech wood glycan, the birch xylan in association with pulp bleaching, wood oligose production, flour modification or fodder production industry.

The gene xynB of the suitable cold inscribe beta-xylanase in ocean of the present invention is from being stored in German microbial strains preservation center, and culture presevation number is the genomic dna that 15026 bacterium Glaciecola mesophila KMM241 extracts.Glaciecolamesophila KMM241 separates in the seawater invertebrates body of the arctic and obtains.According to the genome of the highest strain bacterium Pseudoalteromonas atlantica T6c of the 16S rDNA sequence similarity of Glaciecola mesophilaKMM241 in one the 8th family's xylanase gene sequences Design primer, utilize round pcr from the genomic dna of Glaciecola mesophilaKMM241, to clone the gene of the suitable cold inscribe beta-xylanase xynB in coding ocean.Nucleotide sequence to this gene is measured.Sequencing result shows in the dna fragmentation of acquisition and contains an open reading frame with 2739 Nucleotide that this open reading frame promptly is the gene xynB of coding zytase XynB, 912 amino acid of encoding altogether.Therefore, zytase XynB is one and contains 912 amino acid whose polypeptide.The structure of the precursor of zytase XynB comprises signal peptide and maturing enzyme two portions.Maturing enzyme contains 869 amino acid, is a zytase of not reporting as yet with new sequence in glycoside hydrolase the 8th family.

Beneficial effect:

1, beta-xylanase XynB of the present invention has than higher catalytic efficiency at 0-35 ℃, is applied to association with pulp bleaching, wood oligose production, and flour modification etc. can at room temperature be carried out, and can save the heating step of traditional technology, thus save energy;

2, the beta-xylanase XynB of the present invention restriction endonuclease that is a kind of strictness, hydrolyzed xylan can produce oligosaccharides such as xylotriose, Xylotetrose, can be widely used in the production process of wood oligose;

3, beta-xylanase XynB of the present invention has the high enzyme activity at low temperatures, and extremely unstable under the middle high temperature, middle temperature just can make its complete deactivation, thereby has guaranteed the security of its use.

Description of drawings

The electrophorogram of the genomic dna of the Glaciecola mesophila KMM241 of Fig. 1, extraction;

Wherein: 1, the genomic dna of Glaciecola mesophila KMM241,2, dna molecular amount mark (marker);

The electrophorogram of the gene fragment of Fig. 2, the coding XynB by pcr amplification clone;

Wherein: 1, dna molecular amount mark (marker), 2, the amplification dna fragmentation;

Fig. 3, in intestinal bacteria, carry out the suitable cold beta-xylanase XynB electrophorogram of heterogenous expression and purifying;

M, molecular weight protein marker (marker); 1, the supernatant liquor electrophorogram of e. coli bl21 behind IPTG abduction delivering thalline ultrasonic disruption that contains expression plasmid; 2, supernatant liquor is through the electrophorogram behind the anion-exchange chromatography; 3, the pure beta-xylanase XynB electrophorogram behind the sample process nickel post affinitive layer purification;

Fig. 4, be the enzyme of the suitable cold beta-xylanase XynB temperature curve of living;

Fig. 5, be the enzyme of the suitable cold beta-xylanase XynB pH curve of living;

Embodiment

The present invention will be further described below in conjunction with drawings and Examples, but institute of the present invention protection domain is not limited thereto.

Embodiment 1:

Cold inscribe beta-xylanase XynB is fitted in a kind of ocean, and aminoacid sequence is shown in SEQ ID NO.1.The encode gene xynB of the suitable cold inscribe beta-xylanase XynB in above-mentioned ocean, nucleotide sequence is shown in SEQ ID NO.2.

Gene xynB is 2739bp altogether, wherein contains the suitable cold endo-xylanase XynB of open reading frame coding of a 2739bp, and initiator codon is positioned at 1bp, and terminator codon is positioned at 2737bp, 912 amino acid of encoding altogether.

Embodiment 2: the cloning process of gene xynB

Bacterial classification source: Glaciecola mesophila KMM241 is available from German microbial strains preservation center (DSM), and culture presevation number is: 15026.

Concrete steps are as follows:

1. fit the clone of cold endo-xylanase XynB encoding gene

1.1 the extraction of Glaciecola mesophila KMM241 genomic dna reference " fine works molecular biology experiment guide ":

(1) cultivates the 50ml bacterial cultures to state of saturation, get the centrifugal 5min of culture 12000g of 15ml;

(2) throw out adds 5670 μ l TE, blows and beats repeatedly with suction pipe and makes it resuspended, then, adds 300 μ l 10%SDS and 30 μ l 20mg/mL Proteinase Ks, mixing, 37 ℃ of incubation 2.5-3h;

(3) add 1000 μ l 5M NaCl, fully mixing adds 800 μ l CTAB/NaCl solution again, and mixing is in 65 ℃ of incubation 30-60min;

(4) add isopyknic mixed solution (this mixed solution is by 24: 1 by volume mixed getting of chloroform/primary isoamyl alcohol), mixing, 4 ℃, the centrifugal 20min of 12000g changes supernatant liquor in the new pipe over to;

(5) add isopyknic mixed solution (chloroform/primary isoamyl alcohol mixed in 24: 1 by volume), mixing, 4 ℃, the centrifugal 30min of 14000g forwards supernatant in the new pipe to;

(6) add isopyknic mixed solution (chloroform/primary isoamyl alcohol mixed in 24: 1 by volume), mixing, 4 ℃, the centrifugal 30min of 16000g forwards supernatant in the new pipe to;

(7) add 0.6 times of volume Virahol, mix (3min slowly turns upside down) gently, place 4 ℃ of refrigerators to keep 10min, the flocks ditch is gone out, change in the centrifuge tube of a 5ml with a thin curved glass rod;

(8) 70% washing with alcohol of usefulness 3ml is 2 times, uses absolute ethanol washing again 2 times, and the centrifugal 2min of each 12000g abandons supernatant, and precipitation is DNA, and DNA is air-dry on Bechtop;

(9) add 100-200 μ l TE damping fluid, 4 ℃ of dissolving DNAs that spend the night;

(10) adding 10mg/ml RNase is 50 μ g/mL to final concentration, 37 ℃ of incubation 1h;

(11) add equal-volume phenol/chloroform/primary isoamyl alcohol (mixing in 1: 1: 1 by volume) extracting 1 time, supernatant liquor is used chloroform/primary isoamyl alcohol (mixing in 1: 1: 1 by volume) extracting 1 time again;

(12) dehydrated alcohol of adding 1/10 volume 3M NaAc (pH5.2) and 2 times of volume precoolings in supernatant is inverted and is mixed;

(13) supernatant discarded gently after centrifugal, the DNA precipitation is respectively washed 1 time with 70% ethanol and dehydrated alcohol, is dissolved among an amount of TE buffer after dried slightly, measures DNA concentration and purity with ultraviolet spectrophotometer and is placed on 4 ℃ of refrigerators preservations (can in-20 ℃ of prolonged preservation).

1.2 design of primers is with synthetic

Beta-xylanase gene order design two Auele Specific Primer F8X1:ATGAGCACTTTATCAGTCAG (SEQ ID NO.3) according to existing Pseudoalteromonas atlantica T6c bacterium in the database; F8X4:TTATTCAGGCTCGTTTTC (SEQ ID NO.4), this primer give birth to worker biotech company by Shanghai and synthesize;

1.3 utilize PCR to carry out the gene order amplification

(1) being primer with F8X1 and F8X2, is template with the genomic dna, does pcr amplification.The PCR reaction conditions is: 95 ℃, and 5 minutes; 95 ℃ then, 1 minute; 50 ℃, 1 minute; 72 ℃, 4 minutes, after 32 circulations, 72 ℃, extended 10 minutes.

(2) pcr amplification product is carried out 1% agarose gel electrophoresis, the result shows the dna fragmentation that obtains a treaty 2700bp.DNA with Omega company reclaims test kit according to its explanation recovery amplification of DNA fragments then.

2. gene sequencing

(1) dna fragmentation is connected with cloning vector

Be connected on the pGEMT carrier of Promega company reclaiming dna fragmentation.The ligation system:

Ligase enzyme Buffer 1 μ l

Carrier pGEMT 1 μ l

Exogenous dna fragment 3 μ l

T ₄Ligase enzyme 1 μ l

Add ddH ₂O to 10 μ l

Cover tight lid, finger flicks centrifuge tube, and the mixing sample changeed 2 seconds on whizzer, and sample is concentrated on the pipe end, and 16 ℃ of connections are spent the night.

(2) go up the competent method of preparation intestinal bacteria by " molecular cloning experiment guide " and prepare the bacillus coli DH 5 alpha competence.

(3) the reorganization pGEMT carrier that will connect by the heat shock method for transformation on " molecular cloning experiment guide " goes to the bacillus coli DH 5 alpha competence.

(4) bacillus coli DH 5 alpha of Zhuan Huaing is applied to the Mai Kangkai substratum that contains 100 μ g/L penbritins, 37 ℃ of incubated overnight, select white transformant as template, as primer, verify by bacterium colony PCR whether plasmid in the white transformant contains the dna fragmentation of pcr amplification with F8X1 and F8X2.

(5) transformant that plasmid is contained the dna fragmentation of pcr amplification is served the order-checking of sea living worker Bioisystech Co., Ltd.Therefore, obtain the sequence 2739bp of zytase XynB encoding gene xynB.Sequence is shown in SEQ ID NO.1.The open reading frame that wherein contains the coding zytase XynB of a 2739bp by the blastX software analysis discovery of NCBI website.Initiator codon is positioned at 1bp, and terminator codon is positioned at 1270bp, 912 amino acid of encoding altogether.

Embodiment 3: the heterogenous expression of zytase XynB and purifying

3. the expression of gene xynB in intestinal bacteria

3.1 the structure of expression vector pet-22b-xynB

(1) utilize low dose of plasmid extraction kit, step is to specifications extracted recombinant plasmid pGEMT-xynB. from the transformant that contains reorganization pGEMT plasmid

(2) according to two Auele Specific Primers of sequencing result design

XYB1:CGAC GGATCCWhat GAGCACTTTATCAGTCAG (SEQ ID NO.5) marked with underscore is the BamHI restriction enzyme site.XYB2:CGACG CTCGAGWhat TTCAGGCTCGTTTTC (SEQ ID NO.6) marked with underscore is the XhoI restriction enzyme site.Above-mentioned primer is given birth to worker biotech company by Shanghai and is synthesized.

(3) utilizing PCR to carry out the gene order amplification, is primer with XYB1 and XYB2, is template with reorganization pGEMT plasmid, does pcr amplification.The PCR reaction conditions is: 95 ℃, and 5 minutes; 95 ℃ then, 1 minute; 50 ℃, 1 minute; 72 ℃, 4 minutes, after 32 circulations, 72 ℃, extended 10 minutes.

(4) pcr amplification product is carried out 1% agarose gel electrophoresis, the result shows the dna fragmentation that obtains a treaty 2700bp.DNA with Omega company reclaims test kit according to its explanation recovery amplification of DNA fragments then.

(5) carry out double digestion with restriction enzyme BamHI and XhoI to reclaiming fragment and plasmid pet-22b.The endonuclease reaction system is as follows:

Enzyme cutting buffering liquid 2 μ l enzyme cutting buffering liquids 2 μ l

Restriction endonuclease BamHI 1 μ l restriction endonuclease BamHI 1 μ l

Restriction endonuclease XhoI 1 μ l restriction endonuclease XhoI 1 μ l

Reclaim dna fragmentation 10 μ l plasmid pet-22b 6 μ l

Add ddH ₂O to 20 μ l adds ddH ₂O to 20 μ l

Be placed in 37 ℃ of water-baths and reacted 3 hours.Enzyme is cut product carry out 1% agarose gel electrophoresis, the DNA with Omega company reclaims test kit according to its explanation recovery amplification of DNA fragments then.

(6) will be connected on the pet-22b carrier through the xynB gene fragment of double digestion.The ligation system:

Ligase enzyme Buffer 1 μ l

Carrier pet-22b 1 μ l

XynA gene fragment 3 μ l

T ₄Ligase enzyme 1 μ l

Add ddH ₂O to 10 μ l

Cover tight lid, finger flicks centrifuge tube, and the mixing sample changeed 2 seconds on whizzer, and sample is concentrated on the pipe end, connects in 16 ℃ of shaking tables and spends the night.

(7) go up the competent method of preparation intestinal bacteria by " molecular cloning experiment guide " and prepare the bacillus coli DH 5 alpha competence;

(8) the reorganization pet-22b carrier that will connect by the heat shock method for transformation on " molecular cloning experiment guide " goes to the bacillus coli DH 5 alpha competence;

(9) bacillus coli DH 5 alpha after will transforming is applied in the LB substratum that contains 100 μ g/mL penbritins 37 ℃ of incubated overnight;

(10) picking list bacterium colony on flat board is connected to 5mL and contains in the LB liquid nutrient medium of 100 μ g/mL penbritins 37 ℃ of incubated overnight;

(11) collect 3mL bacterium liquid, the centrifugal 2min of 12000g obtains thalline, extracts recombinant plasmid pet-22b-xynB with test kit.Place-20 ℃ of preservations.

3.2 expression vector pet-22b-xynB is transformed in the e. coli bl21 (DE3)

(1) goes up the competent method of preparation intestinal bacteria by " molecular cloning experiment guide " and prepare the e. coli bl21 competence;

(2) the reorganization pet-22b carrier that will connect by the heat shock method for transformation on " molecular cloning experiment guide " goes to the e. coli bl21 competence;

(3) e. coli bl21 that transforms is applied in the LB substratum that contains 100 μ g/mL penbritins 37 ℃ of incubated overnight.

3.3 gene xynB is abduction delivering and purifying in intestinal bacteria

(1) picking list bacterium colony on flat board is connected to 20mL and contains in the LB liquid nutrient medium of 100 μ g/mL penbritins 37 ℃ of incubated overnight;

(2) 1% inoculum sizes are transferred in the fresh LB substratum that contains 100 μ g/mL penbritins, and 37 ℃ are cultured to bacteria concentration OD0.8, and adding IPTG is 0.5mmol/L to final concentration, and continuation is cultivated 20h at 15 ℃ of shaking tables;

(3) collection is through the 500mL LB nutrient solution of IPTG abduction delivering, and the centrifugal 10min of 12000g collects thalline;

(4) the Binding damping fluid suspension thalline of usefulness 20mL;

The bacterium liquid that (5) will suspend again carry out ultrasonic disruption (400W, 10min);

(6), collect supernatant liquor with the centrifugal 10min of bacterium liquid 12000g after the fragmentation;

(7) nickel post affinity chromatography is carried out in supernatant liquor requirement to specifications;

(8) sample of collecting behind the chromatography detects purity with SDS-PAGE, proves the pure enzyme of the electrophoresis that obtains zytase XynB (Fig. 3).Tris-HCl (pH7.5) damping fluid with 20mmol/L is dialysed 3-4 time.Place-20 ℃ of preservations standby at last.

Embodiment 4: the property testing of zytase XynB

4.1 substrate specificity

(1) with 50mmol/L sodium phosphate buffer (pH7.0) compound concentration is 1% different substrate solutions, comprises the oat xylan, beech wood glycan, birch xylan, cellulose acetate, Sargassum polysaccharides, mannosans, chitin, starch;

(2) in the quantity tube of 20mL, add the different substrate solution of 0.9mL, place 30 ℃ of water-bath preheating 5min, add the pure enzyme liquid of 0.1mL, reaction 10min, add 1.5mLDNS solution termination reaction then, place boiling water bath to heat 5min quantity tube, add the distilled water constant volume at last to 15mL;

(3) characterize hydrolysis degree by measuring the amount that 550nm place light absorption value calculates the reducing sugar of generation.This enzyme is as shown in table 1 to the hydrolytic activity of different substrates.

Table 1

Substrate	Relative enzyme (%) alive
		The beech wood glycan	100

Birch xylan	90.6
		The oat xylan	75.5
Carboxymethyl cellulose	0
		Sargassum polysaccharides	0
Mannosans	0
		Chitin	0
Starch	0

The temperature 4.2 the suitableeest enzyme is lived

Under 0,5,10,15,20,25,30,35,40,45,50,55,60 ℃ of condition, measure the enzyme of beta-xylanase XynB respectively and live, to determine the optimal reactive temperature of enzyme.Measuring enzyme concrete grammar alive is:

The beech xylan solution of 90 μ L 1% adds the pure enzyme liquid of 10 μ L, reacts 10min under the differing temps, adds 150 μ LDNS liquid termination reactions, boils 5min.Add 1.25mL distilled water mixing, measure light absorption value in the 550nm place.Calculate the amount of the reducing sugar of generation according to the wood sugar typical curve.The result shows the suitableeest enzyme of this enzyme 35 ℃ of temperature (as Fig. 4) alive.

4.3 optimal pH

Under pH4.0,5.0,5.5,6.0,7.0,8.0,9.0,10.0,11.0 conditions, measure the enzyme of zytase respectively and live, to determine the optimal pH of this enzyme.Buffer system (50mmol/L): pH 4.0-6.0, citrate buffer solution; PH 6.0-8.0, phosphoric acid buffer; PH 8.0-9.0, the Tris-hydrochloride buffer; PH 9.0-11.0, glycine-NaOH damping fluid.

Concrete grammar is 1% a beech xylan solution of preparing different pH with above-mentioned damping fluid, measures the enzyme of beta-xylanase XynB under condition of different pH according to the method in 5.3 then and lives.The result shows that the optimal pH of this enzyme is 7.0 (as Fig. 5).

5. result

Utilize the CTAB/NaCl method to extract the genomic dna (Fig. 1) of Glaciecola mesophila KMM241.Obtain the nucleotide sequence of the gene xynB of coding beta-xylanase XynB through order-checking by pcr amplification obtain the encoding gene DNA fragment (Fig. 2) of beta-xylanase XynB.Gene xynB contains the suitable cold endo-xylanase XynB of open reading frame coding of a 2739bp, and initiator codon is positioned at 1bp, and terminator codon is positioned at 2737bp, 912 amino acid of encoding altogether.Utilize the structure of the CDD software analysis beta-xylanase XynB of NCBI website, the structure of the precursor of beta-xylanase XynB comprises signal peptide and catalyst structure domain and N-end unknown function structural domain three parts.Gene xynB is carried out heterogenous expression and purifying in intestinal bacteria, obtain ripe activated beta-xylanase XynB (Fig. 3).Maturing enzyme beta-xylanase XynB contains 869 amino acid whose catalyst structure domains, is an enzyme of not reporting as yet with new sequence in lytic enzyme the 8th family.The suitableeest enzyme temperature alive is 35 ℃ (Fig. 4), and optimal pH is 6.0-7.0 (Fig. 5).Therefore, the beta-xylanase XynB of gene xynB coding is the suitable cold endo-xylanase of a neutrality.

Claims (5)

1. cold inscribe beta-xylanase XynB is fitted in an ocean, and aminoacid sequence is shown in SEQ ID NO.1.

2. the gene xynB of cold inscribe beta-xylanase XynB is fitted in the described ocean of coding claim 1, and nucleotide sequence is shown in SEQ ID NO.2.

3. expression vector that contains nucleotide sequence shown in the SEQ ID No.2.

4. the reconstitution cell that contains the described SEQ ID of claim 2 No.2 nucleotide sequence or the described expression vector of claim 3.

5. the suitable cold inscribe beta-xylanase XynB in the described ocean of claim 1 and/or the described gene xynB of claim 2 application aspect hydrolysis oat xylan, beech wood glycan, the birch xylan in association with pulp bleaching, wood oligose production, flour modification or fodder production industry.

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