CN107129976A

CN107129976A - A kind of neutral high-temperature xylanase and its encoding gene and its application

Info

Publication number: CN107129976A
Application number: CN201710409349.4A
Authority: CN
Inventors: 姚斌; 马锐; 柏映国; 罗会颖; 黄火清; 苏小运; 王苑; 涂涛; 王亚茹; 孟昆
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Animal Science of CAAS
Priority date: 2017-06-02
Filing date: 2017-06-02
Publication date: 2017-09-05
Anticipated expiration: 2037-06-02
Also published as: CN107129976B

Abstract

The invention provides a kind of neutral high-temperature xylanase CtXyn10A and its gene and application.The neutral high-temperature xylanase CtXyn10A that the present invention is provided is the protein with the amino acid sequence shown in the SEQ ID № .3 and/or SEQ ID № .5 in sequence table, and/or the substitution by the amino acid residue sequence shown in the SEQ ID № .3 and/or SEQ ID № .5 in sequence table by one or several amino acid residues and/or missing and/or addition and the protein of amino acid sequence protein derived as shown in the SEQ ID № .3 and/or SEQ ID № .5 in sequence table with xylanase activity.The zytase CtXyn10A optimal pHs 6.0 7.0 of the present invention, 70 DEG C of optimum temperature, in the enzyme activity of 90 DEG C and pH9.0 holdings more than 20%, than living for 461U/mg；The zytase CtXyn10A of the present invention has the activity of zytase, dextranase, cellulase, and is easy to industrial fermentation production, as a kind of new wide spectrum enzyme preparation, can be widely used for food, papermaking, energy industry etc..

Description

A kind of neutral high-temperature xylanase and its encoding gene and its application

Technical field

The invention belongs to genetic engineering field, and in particular to a kind of neutral high-temperature xylanase and its encoding gene should with it With.

Background technology

Xylan (xylan) be the second abundant plant biomass that content in nature is only second to cellulose it is main into Point (Lynd et al.Microbiology and Molecular Biology Review, 2002,66:506-577), in life There is potential value in terms of the thing energy.Existing biotechnology with the cellulose components in efficient degradation plant and can be changed into Fermentable glucose, the latter generates bio-ethanol under yeast effect.The xylan of high component not only limits cellulase With the association reaction of cellulose, suppress catalytic reaction, and as byproduct be discharged into nature can cause nutrition accumulationization so as to Destroy the ecosystem (Polizeli et al.Applied Microbiology and Biotechnology, 2005,67: 577-591).Therefore in biomass bioconversion procedure, zytase is generally added, the physics of xylan is on the one hand removed Barrier, promotes combination (the Hu et al.Biotechnology for Biofuels, 2011,4 of cellulase and cellulose: 14；Qing and Wyman Biotechnology for Biofuels,2011,4:18) wood oligose, on the other hand generated Fermentable xylose can also be further degraded into, 10-25% raw material (Jin et are provided for the production of bio-ethanol al.Applied and Environmental Microbiology,2004,70:6816-6825)。

Structure based on amino acid sequence and catalytic domain, most of zytase (EC 3.2.1.8) is divided in glycoside hydrolysis Enzyme the 10th and 11 families.Compared with the 11st family's zytase of single-minded degradation of xylan, the zytase of the 10th family has Wide in range substrate specificity, in addition to acting on xylan, can also a variety of substrates such as catalytic cellulose, glucan degraded, because This has wider application prospect in industrial circle.

Existing bio-ethanol processing technology includes the acid-base pretreatment of lignocellulosic, long-time high temperature enzyme reaction, from And substantial amounts of energy and soda acid chemical reagent are consumed, cause the significantly lifting and serious environmental pollution of cost.Therefore develop The lignocellulolytic enzymes of neutral high temperature tie up to the saving energy, environmental protection, are easy in terms of catalytic reaction with important Meaning (Ji et al.Biotechnology for Biofuels, 2014,7:130).

The content of the invention

It is an object of the present invention to provide a kind of albumen, entitled Ctxyn10A, from Tianshan Mountains cladosporium Cladosporium tianshanense SL-14。

Albumen of the present invention for it is following 1), 2) or 3) described in albumen：

1) there is the protein of the amino acid sequence shown in the SEQ ID № .3 in sequence table；

2) there is the protein of the amino acid sequence shown in the SEQ ID № .5 in sequence table；

3) amino acid residue sequence shown in the SEQ ID № .3 and/or SEQ ID № .5 in sequence table is passed through one Or the substitution of several amino acid residues and/or missing and/or addition and with xylanase activity by 1) and/or 2) derivative Protein.

Amino acid sequence in sequence table shown in SEQ ID № .3 is made up of 352 amino acid residues；Wherein, N-terminal 18 Amino acid is the signal peptide sequence of prediction, with SEQ ID № in sequence table：Amino acid sequence shown in 4.

Amino acid sequence in sequence table shown in SEQ ID № .5 is made up of 334 amino acid residues；That is Ctxyn10A into Soft-boiled eggs are white.

It is above-mentioned 1), 2) He 3) in Ctxyn10A albumen can be artificial synthesized, also can first synthesize its encoding gene, then given birth to Thing expression is obtained.It is above-mentioned 1), 2) He 3) in the encoding gene of Ctxyn10A albumen can be by by SEQ in sequence table in sequence table ID №：1、SEQ ID №：2, and/or SEQ ID №：DNA sequences shown in 2 the 55th -1059 shown nucleotide sequences Lieque is obtained after losing the codon of one or several amino acid residues, and/or the missense mutation of one or several base-pairs of progress.

The nucleic acid molecules for encoding the Ctxyn10A albumen fall within protection scope of the present invention.

The nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also be RNA, such as mRNA, hnRNA or tRNA.

The present invention's a further object is a kind of encoding gene of offer, it is characterised in that the encoding gene has following One of nucleotide sequence：

1) polynucleotide sequence of albumen of the present invention is encoded；

2) SEQ ID № in sequence table：1、SEQ ID №：2, and/or SEQ ID №：Shown in the 55th -1059 of 2 Nucleotide sequence；

3) SEQ ID № in polynucleotide：The polynucleotide sequence of 3 and/or SEQ ID № .5 protein sequences；

4) nucleotide sequence that can hybridize under high high stringency conditions with the DNA sequence dna of 1), 2), and/or 3) any restriction；

5) there is more than 90% homology with the DNA sequence dna of 1), 2), 3), and/or 4) any restriction, and encodes identical work( Can protein DNA sequence.

Specifically, the homology is more than 95%；Specific again is more than 96%；Specific again is more than 97%；Again Specific is more than 98%；Specific again is more than 99%.

Above-mentioned high high stringency conditions can be that with 6 × SSC, 0.5%SDS solution hybridizes at 65 DEG C, then with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Wherein, SEQ ID № in sequence table：Nucleotide sequence shown in 1, common 1202bp is respectively containing 2 length 59bp and 84bp intron sequences, 2 intron sequences have SEQ ID № in sequence table respectively：Shown in 1 141-199 nucleotide sequences and the 512nd-595 nucleotide sequences；Remove the cDNA after 2 intron sequences Long 1059bp, with SEQ ID № in sequence table：SEQ ID № in 2 nucleotide sequence, polynucleotide：Ammonia shown in 3 Base acid sequence and a terminator codon, i.e., Ctxyn10A albumen of the present invention；With SEQ ID № in sequence table：The of 2 SEQ ID № in the nucleic acid fragment polynucleotide of 55-1059 nucleotide sequences：Amino acid sequence shown in 5, i.e. this hair The bright Ctxyn10A maturation proteins.

The present invention's a further object is a kind of carrier of offer, expression cassette, transgenic cell line, recombinant bacterium, and/or micro- life Thing, the carrier, expression cassette, transgenic cell line, recombinant bacterium, and/or microorganism contain described encoding gene.

Specifically, the carrier includes recombinant cloning vector, recombinant expression carrier；Specifically include pPIC- again Ctxyn10A；The bacterium bag of setting out of the recombinant bacterium includes Escherichia coli, yeast, bacillus, Bacillus acidi lactici, aspergillus, and/or wood It is mould；Again specifically, the bacterium bag of setting out of the recombinant bacterium includes Pichia pastoris, brewer's yeast, and/or many types of inferior yeast；Again specifically, The bacterium germination that goes out of the recombinant bacterium is Pichia pastoris GS115；Again specifically, the recombinant bacterium is specially GS115/Ctxyn10A；Institute Stating microorganism includes Tianshan Mountains cladosporium Cladosporium tianshanense SL-14；

The recombinant expression carrier can use existing expression vector establishment.The expression vector can also include foreign gene 3 ' ends untranslated region, i.e., the DNA fragmentation comprising polyadenylation signals and any other participation mRNA processing or gene expression.Institute State the 3 ' ends that the bootable polyadenylic acid of polyadenylation signals is added to mRNA precursor.Use the gene constructed recombinant expression carrier When, any enhanced, composing type, organizing specific type or inducible promoter can be added before its transcription initiation nucleotides, They can be used alone or are used in combination with other promoters；In addition, using the gene constructed recombinant expression carrier of the present invention When, it is also possible to use enhancer, including translational enhancer or transcriptional enhancer.For the ease of entering to transgenic plant cells or plant Row identification and screening, can be processed to plant expression vector used, and color change can be produced by such as adding the expression in microorganism Enzyme or luminophor gene (gus gene, GFP genes, luciferase genes etc.), resistant antibiotic marker (gentamicin label, kanamycins label etc.) or anti-chemical reagent marker gene (such as anti-herbicide gene).From The security consideration of genetically modified organism, can be not added with any selected marker, directly screen transformant with adverse circumstance.

The present invention's a further object is a kind of primer pair of offer, the amplifiable encoding gene total length of primer pair or Its any fragment.

A further object is for the present invention provides a kind of preparation method of albumen, and methods described includes：

1) encoding gene of the present invention is prepared；

2) prepare comprising step 1) recombinant expression carrier of the encoding gene；

3) step 2 is made) expression vector expression to be to obtain destination protein.

Specifically, methods described also includes the deglycosylation of destination protein；It is described to make step 2) the expression vector expression Specific steps include by the expression vector import microorganism in, express it；Again specifically, the microorganism includes large intestine Bacillus, yeast, bacillus, Bacillus acidi lactici, aspergillus, and/or trichoderma；Again specifically, the microorganism includes Pichia pastoris, beer Brewer yeast, and/or many types of inferior yeast；Again specifically, the microorganism is Pichia pastoris GS115；Specifically, the recombination expression Carrier includes pPIC-Ctxyn10A.

The albumen that the protein preparation method is prepared falls within the scope of protection of the invention；Specifically, the albumen Including Ctxyn10A albumen of the present invention and modification structure；Specifically, the modification structure includes glycosylation structure.

It is also another object of the present invention to provide albumen of the present invention, encoding gene, recombinant vector, expression cassette, turn The albumen that gene cell system, recombinant bacterium, and/or microorganism, primer pair, protein preparation method, the preparation method are prepared Application.

Specifically, the application is included in following 1) -8) it is at least one in application：

1) zytase is being prepared and/or containing the application in zytase Related product；

2) xylanase mutant is being prepared and/or containing the application in xylanase mutant Related product；

3) in Prepare restructuring zytase and/or the application in recombined xylanase Related product is contained；

4) prepare wood oligose, xylobiose, the sugar of wood one, containing wood oligose, containing xylobiose, and/or containing the sugared phase of wood one Close the application in product；

5) have as cellulase, dextranase and/or preparation in cellulase, dextranase enzymatic activity Related product Application；

6) application in degradation of xylan, cellulose, and/or glucan；

7) in application and preparation in industry, the processing of agricultural, food, medicine, feed, the energy, waste and/or field of environment protection In product in application；

8) application in industry, the processing of agricultural, food, medicine, feed, the energy, waste and/or field of environment protection.

Specifically, the xylan include the poly- Arabic wood of beech xylan, solvable wheat, it is birch xylan, insoluble Wheat Arabinoxylan；The cellulose includes carboxymethyl cellulose, lichenin；The glucan is poly- including barley Portugal Sugar.

Specifically, the zytase includes albumen of the present invention and/or prepared by protein preparation method of the present invention Obtained albumen；The xylanase mutant is included albumen of the present invention and/or protein preparation method of the present invention The albumen that the albumen prepared is obtained after point mutation；The recombined xylanase include by albumen of the present invention and/ Or the albumen that the albumen for preparing of protein preparation method of the present invention is obtained after homologous recombination.

Specifically, the application be included in it is following it is 1), 2), 3), and/or 4) described under the conditions of application：

1) pH includes 4.0-12.0；

2) temperature includes 0 DEG C -90 DEG C；

3) environment of metal ion, and/or chemical reagent is included；

4) substrate includes xylan, cellulose, and/or glucan.

It is preferred that, the pH includes 5.0-8.0；Further preferably, the pH includes 6.0-7.0；Most preferably, the pH is 6.5；It is preferred that, the temperature includes 50 DEG C -80 DEG C；It is preferred that, the temperature includes 50 DEG C -75 DEG C；It is preferred that, the temperature Including 60 DEG C -75 DEG C；Most preferably, the temperature is 70 DEG C；The metal ion, and/or chemical reagent include Ni²⁺, Co²⁺, Na⁺, Cr³⁺, Mg²⁺, Mn²⁺, K⁺, Cu²⁺, Ca²⁺, Pb²⁺, Zn²⁺, Fe³⁺, beta -mercaptoethanol, EDTA, SDS；The metal ion and/ Or the concentration of chemical reagent includes 5mmol/L；The xylan includes the Arabic wooden poly-, birch of beech xylan, solvable wheat Xylan, insoluble Wheat Arabinoxylan；The cellulose includes carboxymethyl cellulose, lichenin；The glucan Including barley.

It is also another object of the present invention to provide a kind of preparation method of zytase, methods described includes：From Zytase is extracted in Cladosporium tianshanense SL-14.

The zytase CtXyn10A that the present invention is provided is in the range of neutral (pH 5.0-8.0) and high temperature (50 DEG C -75 DEG C) It is respectively provided with the characteristic of high activity.The present invention screens Tianshan Mountains cladosporium Cladosporium tianshanense SL-14 (China Agriculture DSMZ ACCC 32710) produced by zytase CtXyn10A, its optimum pH be 6.0-7.0, The enzymatic activity of maintenance more than 80% in the range of pH5.0-8.0；Optimum temperature be 70 DEG C, be respectively provided between 60-75 DEG C 70% with On enzyme activity；There is 10% enzyme activity under the conditions of 0 DEG C；And there is degradation to a variety of xylans, glucan, cellulose. The also unprecedented report of the neutral high-temperature xylanase of this property.

The present invention overcomes the deficiencies in the prior art, and obtaining the fungal resources of high-temperature xylanase, there is provided a kind of property It is excellent, be suitable in food, papermaking, energy industry using new neutral high-temperature xylanase CtXyn10A.The present invention's Zytase CtXyn10A optimal pHs are 6.0-7.0, there is higher enzymatic activity in pH5.0~8.0；PH stability is good；Than work Power is 461U/mg；With wide in range substrate specificity.The characteristics of its neutral high temperature, it is possible to decrease zytase is in industrial production Energy cost.PH wide adaptation ranges, can improve aquatic feeds digestible energy and metabolic energy, reduce formulation cost, reduce environment dirty Dye.Its wide in range substrate specificity can improve the nutritive value of grain processing co-product, lift feed product quality.Xylan Enzyme CtXyn10A can be applied to brewing industry, reduce material viscosity, and diastatic action can be conducive in stratum granulosum, starch is improved Utilization rate, increases the yield of alcohol.Can also be under normal temperature condition, by the xylan in paper industry waste material and agricultural wastes It can be converted into wood oligose, and wood oligose can be by bacterium, yeast and fungal transformation into valuable fuel.Therefore, xylan Applications of the enzyme CtXyn10A in energy industry also shows that its huge potentiality.Zytase CtXyn10A hydrolysates (wood Sugar and xylo-oligosaccharide) food service industry is can be applicable to, it is used as thickener, fatty sub, prebiotic oligosaccharides and freeze proof food additives； Xylan is used in combination with other materials in pharmaceuticals industry, can delay the release of drug ingedient.Zytase and its hydrolysis Product can also be further converted to liquid fuel, solvent and low calorie sweetener.

Brief description of the drawings

Fig. 1 is the recombined xylanase CtXyn10A expressed in recombinant bacterium GS115/Ctxyn10A SDS-PAGE analyses,

Wherein, swimming lane 1 is deglycosylated purification of Recombinant zytase；Swimming lane 2 is purification of Recombinant zytase；Swimming lane 3 For low molecular weight protein Marker.

Fig. 2 is recombined xylanase CtXyn10A optimal pH measurement result figure.

Fig. 3 is recombined xylanase CtXyn10A pH Stability Determination result figures.

Fig. 4 is recombined xylanase CtXyn10A optimum temperature measurement result figure.

Fig. 5 is recombined xylanase CtXyn10A thermal stability determination result figure.

Embodiment

Explanation：

Experimental method used in following embodiments is conventional method unless otherwise specified.

Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiments《Molecular Cloning:A Laboratory guide》 Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or is carried out according to kit and product description.

Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.

Test material and reagent：

1st, bacterial strain and carrier：Tianshan Mountains cladosporium Cladosporium tianshanense SL- used in following embodiments 14, it is stored in agriculture DSMZ (No.12 ,zhongguancun south street,Haidian District, Beijing, Chinese agriculture of the Chinese Academy of Agricultural Sciences Agricultural DSMZ of the academy of sciences, 100081), its preserving number is：ACCC32710, the public can be commercially available this from the center Bacterial strain.Yeast expression vector pPIC9 and bacterial strain GS115 is purchased from Invitrogen companies.

2nd, enzyme and other biochemical reagents：Restriction endonuclease is purchased from TaKaRa companies, and ligase is purchased from Invitrogen companies.Beech Wooden xylan is purchased from Sigma companies, and other is all domestic reagent (can be commercially available from common biochemical Reagent Company).

3rd, culture medium：

(1) Cladosporium tianshanense SL-14 culture mediums are potato juice culture medium：1000mL potatos Juice, 10g glucose, 25g agar, pH5.0.

(2) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).

(3) BMGY culture mediums：1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% is sweet Oily (V/V).

(4) BMMY culture mediums：Divided by 0.5% methanol (V/V) replace glycerine, remaining composition is identical with BMGY, pH4.0.

The clone of embodiment 1, neutral high-temperature xylanase CtXyn10A genomic DNA

Extract Tianshan Mountains cladosporium Cladosporium tianshanense SL-14 genomic DNAs：

The filtering of the Liquid Culture mycelium aseptic filter paper of 3 days is put into mortar, 2mL extract solutions are added, 5min is ground, Then lapping liquid is placed in 50mL centrifuge tubes, 65 DEG C of water-baths crack 20min, are mixed once every 10min, at 4 DEG C 10000rpm centrifuges 5min.Supernatant extrct foreigh protein removing in phenol/chloroform is taken, then takes supernatant to add isometric isopropanol, in It is stored at room temperature after 5min, 10000rpm centrifuges 10min at 4 DEG C.Supernatant is abandoned, precipitation is washed twice with 70% ethanol, and vacuum is done It is dry, add appropriate TE dissolving, be placed in -20 DEG C it is standby.

According to the sequence analysis of Cladosporium tianshanense SL-14 genome frame diagrams, design synthesis is special Different primer Ctxyn10A-F/Ctxyn10A-R：

Ctxyn10A-F：5'-ATGCGTTTCACTGAGGTCTTCACTGCTC-3'；

Ctxyn10A-R：5'-TCACTTCTTGCCACCCTTGATGCCC-3'；

Using Tianshan Mountains cladosporium Cladosporium tianshanense SL-14 STb genes as template, with above-mentioned Ctxyn10A-F/Ctxyn10A-R is special primer, enters performing PCR amplification.

PCR response parameters are：94 DEG C of denaturation 5min；Then 94 DEG C are denatured 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extensions 60sec, 72 DEG C of insulation 10min after 30 circulations.

Above-mentioned PCR reactions obtain the nucleic acid fragment of an about 1200bp, are connected after the fragment is reclaimed with pEASY-T3 carriers Send three rich Bioisystech Co., Ltd's sequencings.

Sequencing result shows that the sequence for the nucleic acid fragment that above-mentioned PCR amplifications are obtained has SEQ ID № in sequence table：1 institute The nucleotide sequence shown, common 1202bp.This had into SEQ ID № in sequence table：The nucleic acid fragment of nucleotide sequence shown in 1 It is named as Ctxyn10A.

The acquisition of the neutral high-temperature xylanase CtXyn10A genes of embodiment 2, encoding mature

Tianshan Mountains cladosporium Cladosporium tianshanense SL-14 total serum IgE is extracted, is obtained using reverse transcriptase A cDNA chain, then expands the single-stranded cDNA with primer Ctxyn10A-F/Ctxyn10A-R, and amplification is obtained after product recovery Send three rich Bioisystech Co., Ltd's sequencings.

Ctxyn10A-F：5'-GGGGAATTCCACCCTTCGGCTCCCAAGGAC-3'；

Ctxyn10A-R：5'-GGGGCGGCCGCTCACTTCTTGCCACCCTTGATGCC-3'；

After the genome sequence for the nucleic acid fragment surveyed by comparing embodiment 1 and above-mentioned pcr amplification product sequencing result, It was found that the gene has 2 intrones, the long 1059bp of cDNA, with SEQ ID № in sequence table：2 nucleotide sequence, code sequence SEQ ID № in list：Amino acid sequence and a terminator codon shown in 3.With SEQ ID № in sequence table：Shown in 3 Amino acid sequence albumen, totally 352 amino acid residues, 18 amino acid of N-terminal for prediction signal peptide sequence, with sequence SEQ ID № in table：Amino acid sequence shown in 4.SEQ ID № in sequence table：1 the 141st -199 nucleotide sequences It is respectively 59bp and 84bp intron sequences with the 512nd -595 nucleotide sequences

Above-mentioned pcr amplification product sequencing result shows that above-mentioned PCR amplifications, which obtain nucleic acid fragment, includes the double enzymes of EcoRI+NotI It is with SEQ ID № in sequence table in the middle of enzyme site, above-mentioned two restriction enzyme site：2 the 55th -1059 nucleotide sequences Nucleic acid fragment.

With SEQ ID № in sequence table：The nucleic acid fragment of 2 the 55th -1059 nucleotide sequence is Ctxyn10A SEQ ID № in the nucleic acid fragment of gene encoding mature protein part, polynucleotide：Amino acid sequence shown in 5；Will Xylanase sequence on the nucleic acid fragment and GenBank of Ctxyn10A gene encoding mature protein parts carries out homologous ratio Compared with highest uniformity is 74%, and amino acid sequence highest uniformity is 72%, it was demonstrated that from Tianshan Mountains cladosporium Cladosporium The gene for the encoding xylanase that separation clone obtains is new gene in tianshanense SL-14.

The preparation of embodiment 3, neutral high-temperature xylanase CtXyn10A

Yeast expression vector pPIC9 is subjected to double digestion (EcoRI+NotI), while above-described embodiment 2 is prepared into The nucleic acid fragment double digestion (EcoRI+NotI) of the encoding mature albumen arrived, cuts out the genetic fragment and double enzymes of encoding mature albumen Expression vector pPIC9 connections after cutting.

The correctness of sequence verification sequence.The sequence of the foreign gene inserted in gained recombinant plasmid is SEQ ID №：2 55-1059 nucleotides, pPIC-Ctxyn10A is named as by the recombinant plasmid.

Above-mentioned recombinant plasmid pPIC-Ctxyn10A is converted into Pichia pastoris GS115；Positive restructuring bacterium is extracted into plasmid order-checking The correctness of sequence is verified, the correct recombinant pichia yeast strain containing recombinant plasmid pPIC-Ctxyn10A will be sequenced and names For GS115/Ctxyn10A.

Recombinant bacterium GS115/Ctxyn10A bacterial strains are taken, are inoculated in 300mL BMGY nutrient solutions, 30 DEG C of 250rpm vibration trainings Support after 48h, thalline is collected by centrifugation.Then it is resuspended in 100mL BMMY culture mediums, 30 DEG C of 250rpm shaken cultivations.Induce after 72h, Supernatant is collected by centrifugation, purifying protein determines zytase CtXyn10A vigor.

SDS-PAGE results are as shown in figure 1, Fig. 1 results show, zytase CtXyn10A is in Pichia pastoris recombinant bacterium Expressed in GS115/Ctxyn10A.Expressed zytase CtXyn10A is after purifying, its target protein Content reach more than the 90% of total protein, reach that electrophoresis is pure.After ENDO-H de-glycosylations, the molecular weight and reason of purifying protein It is consistent by value, it is 37.4kDa.

The enzyme activity determination of embodiment 4, neutral high-temperature xylanase CtXyn10A

The vigor for the zytase CtXyn10A that above-described embodiment 3 is prepared is determined using DNS methods, specific method is such as Under：Under the conditions of pH6.5,70 DEG C, 1mL reaction system includes the dilution enzyme liquid 100 μ L suitably, 900 μ L substrates, reaction 10min, adds 1.5mL DNS terminating reactions, boiling water boiling 5min.540nm determines OD values after cooling.1 enzyme-activity unit (U) definition For the enzyme amount per minute for discharging 1 μm of ol reduced sugar under given conditions.After measured, the neutrality that embodiment 3 is prepared is high Warm zytase CtXyn10A enzyme activity is 160U/mL.

Embodiment 5, neutral high-temperature xylanase CtXyn10A property are determined

1st, recombined xylanase CtXyn10A optimal pH and the assay method of pH stability are as follows：

The recombined xylanase that embodiment 3 is purified carries out enzymatic reaction to determine its optimal pH under different pH.Will The different pH of substrate xylan 0.1mol/L citrate-phosphate disodium hydrogen buffer solutions, carry out xylanase activity at 30 DEG C Power is determined.As a result as shown in Fig. 2 zytase CtXyn10A optimal pH is 6.0-7.0, in the range of pH5.0~8.0, Enzymatic activity is kept at more than the 80% of maximum enzyme activity.Zytase 37 DEG C of processing in above-mentioned various different pH buffer solution 60min, then enzymatic activity is determined at 70 DEG C in pH6.5 buffer solution systems, with the pH patience of studying enzyme.As a result as shown in figure 3, wood Dextranase CtXyn10A is very stable between pH 4.0-12.0, and remaining enzymatic activity exists after processing 60min in the range of this pH More than 80%, this illustrates that this enzyme has preferable pH stability.

2nd, the optimum temperature of zytase and thermal stability determination method are as follows：

The optimum temperature of zytase is determined as in citrate-phosphate disodium hydrogen buffer solution (pH6.5) buffer solution system And enzymatic reaction is carried out under different temperatures.Temperature tolerance is determined as zytase and handles different time at different temperatures, then PH6.5, carry out enzyme assay at 70 DEG C.Enzyme reaction optimum temperature measurement result is as shown in figure 4, zytase CtXyn10A Optimum temperature is 70 DEG C, than living for 461U/mg；There is greater activity in 50 DEG C -75 DEG C of high temperature range；Between 60 DEG C -75 DEG C It is respectively provided with more than 70% enzyme activity；There is 10% enzyme activity under the conditions of 0 DEG C；The heat stabilization test result of enzyme such as Fig. 5 (Fig. 5 In result shown in 80 DEG C to have the substrate reactions time 10 minutes；80 DEG C in Fig. 4, result and to be expressed in no 80 DEG C of warm bath of substrate certain Enzyme activity is determined after time) shown in, zytase CtXyn10A stability at 60 DEG C is very good, 60min is incubated at 70 DEG C, completely Lose enzyme activity.

In addition, also measured were enzyme activity of the zytase CtXyn10A at 90 DEG C and pH9.0, as a result show that it is maintained More than 20% enzyme activity.

It is above-mentioned test result indicates that, zytase CtXyn10A is neutral high temperature enzyme, the characteristics of with neutral high temperature.

3rd, the Km values determination methods of zytase are as follows：

It is substrate with the beech xylan of various concentrations, in citrate-phosphate disodium hydrogen buffer solution (pH6.5) buffering liquid In system, enzymatic activity is determined at 70 DEG C, its Km, Vmax, kcat and kcat/Km value at 70 DEG C is calculated.After measured, this wood is poly- Km values of the carbohydrase CtXyn10A by substrate of beech xylan at 70 DEG C is 0.64mg/mL, and maximum reaction velocity Vmax is 450 μm ol/minmg, kcat values are that 281/s and kcat/Km values are 434ml/mgs., should test result indicates that CtXyn10A with The affinity of substrate is stronger, also has higher catalytic efficiency under the high temperature conditions, it is similar that its zymologic property is significantly better than sequence Fungal xylanases FoXyn10A (optimal pH 7.0,45 DEG C of optimum temperature, Km 0.8mg/mL, Vmax1.22 μm of ol/min mg；Christakopoulos et al.Carbohydrate Research,1997,302:191-195)

4th, influence of the different metal ions chemical reagent to zytase CtXyn10A enzyme activity is determined as follows：

The different metal ions and chemical reagent of various concentrations are added in enzymatic reaction system, various chemistry examinations are studied Influence of the agent to zytase CtXyn10A vigor, the various final concentration of 5mmol/L of material.Determined under the conditions of 70 DEG C, pH6.5 Enzymatic activity.As a result as shown in table 1, most of ions and chemical reagent are when concentration is 5mmol to recombined xylanase CtXyn10A vigor has not significant impact, and SDS has partial inhibition.Mn²⁺Can increase to restructuring enzyme activity in 5mmol Originally 1.32 times.Should be test result indicates that CtXyn10A has preferable chemoresistance, a small amount of Mn of appropriate addition²⁺Can be with Increase enzyme activity, so as to reduce application cost.

5th, zytase CtXyn10A substrate specificity

Different substrates, research zytase CtXyn10A substrate specificity are added in enzymatic reaction system.70 DEG C, determine enzymatic activity under the conditions of pH6.5.As a result as shown in table 2, zytase CtXyn10A has zytase, fiber simultaneously The activity of plain enzyme and dextranase.

Should test result indicates that, zytase CtXyn10A has wide in range substrate specificity, except acting on xylan Outside, can also a variety of substrates such as catalytic cellulose, glucan degraded, therefore have in industrial circle before widely application Scape.

Table 1

Table 2

Substrate	With respect to enzyme activity (%)
		Beech xylan (1%)	100.0
Solvable Wheat Arabinoxylan (1%)	130.6
		Birch xylan (1%)	88.1
Insoluble Wheat Arabinoxylan (1%)	49.0
		CMC (1%)	31.7
Lichenin (1%)	24.6
		Barley (0.5%)	8.9

6th, degrade beech xylan products of zytase CtXyn10A are analyzed as follows：

Added in 500 μ L 1% xylan under the enzyme liquid of 100 μ L purifying, optimum temperature and be incubated 12h.Use absolute ethyl alcohol Zymoprotein is precipitated, 2500 chromatographs of supernatant, examined using High Performance Anion Exchange Chromatography Coupled with Pulsed Amperometric (HPAEC-PAD) Survey method, carries out the analysis of sugar type in product.Analysis result shows：The production of zytase CtXyn10A degraded beech xylans If owner's xylobiose and the sugar of wood one.Xylobiose content is 68% in product, and a wooden sugared content is 24%.The experimental result table Bright, zytase CtXyn10A can be used for the production Related product such as xylobiose and a wooden sugar.

Sequence table

<110>Feed institute of the Chinese Academy of Agricultural Sciences

<120>A kind of neutral high-temperature xylanase and its encoding gene and its application

<160>5

<210>1

<211>1202

<212>DNA

<213>Tianshan Mountains cladosporium Cladosporium tianshanense SL-14

<400>1

atgcgtttca ctgaggtctt cactgctctt acgctggccg cttcggcggt tgcccaccct 60

tcggctccca aggacaagaa gggtttggcc actgctatga aggctcgtgg aagggagttt 120

atcggcacag cccttacact gtacgtggtt gattgatctc tcaaggattt cgactttttt 180

gctgacttca agattccagt cgcggcaacg agaccgaaga agccattgct cgcaacaacg 240

ccgacttcaa ctctttcacg ccagagaatg caatgaagtg ggaggccatt gagcctaacc 300

gcaacaactt caccttcagc gacgccgacc gctaccgcga ctgggccaag gccaataaga 360

aggaaatcca ctgccacact cttgtctggc actctcagct ccctccttgg gtcgctgctg 420

gcaactacga caacaagacc ctgataggga tcatggaaaa ccacatcaag aacgtggctg 480

gccgctacaa ggacgtgtgc acccgctggg agtaagtgga cccgactttt ttttctcgac 540

tttcgacgaa cttttttccg gacttgcaac gaaccatcaa ctaacaccat aacagcgtag 600

tcaacgaggc gttggaggag gacggtacct accgctcctc acccttctac gacaccatcg 660

gcgaagcttt catcccaatc gccttcaaat tcgccaagaa gtacagcccc aagtccgagc 720

tcttctacaa cgactacaac ctcgagtaca atggcaacaa gaccctcggc gccaagcgca 780

tcgtcaagct ggtccagagc tacggcgtgc acatcgacgg cgtgggtctg caagcccact 840

tggcccagga agtcaccccg accgccggcg ccctgcccga ccaagccact ctcgagaccg 900

ttctcagagg cttcacttcc ctggacgtcg atgttgttta caccgagatt gatatccgca 960

tgaacacccc cagcaccccg gccaagctca agacacaggc caaggctttc gagactgttg 1020

ctcgcagctg tcttgctgtc aagaggtgca ttggaatgac cgtttggggt atttcagacg 1080

ctttctcgtg gatccctggt gtattccctg gtgagggcgc tgcgcttctt tgggacgaga 1140

accttaagaa gaagccggct tacgatggct tctacaaggg catcaagggt ggcaagaagt 1200

ga 1202

<210>2

<211>1059

<212>DNA

<213>Tianshan Mountains cladosporium Cladosporium tianshanense SL-14

<400>2

atgcgtttca ctgaggtctt cactgctctt acgctggccg cttcggcggt tgcccaccct 60

tcggctccca aggacaagaa gggtttggcc actgctatga aggctcgtgg aagggagttt 120

atcggcacag cccttacact tcgcggcaac gagaccgaag aagccattgc tcgcaacaac 180

gccgacttca actctttcac gccagagaat gcaatgaagt gggaggccat tgagcctaac 240

cgcaacaact tcaccttcag cgacgccgac cgctaccgcg actgggccaa ggccaataag 300

aaggaaatcc actgccacac tcttgtctgg cactctcagc tccctccttg ggtcgctgct 360

ggcaactacg acaacaagac cctgataggg atcatggaaa accacatcaa gaacgtggct 420

ggccgctaca aggacgtgtg cacccgctgg gacgtagtca acgaggcgtt ggaggaggac 480

ggtacctacc gctcctcacc cttctacgac accatcggcg aagctttcat cccaatcgcc 540

ttcaaattcg ccaagaagta cagccccaag tccgagctct tctacaacga ctacaacctc 600

gagtacaatg gcaacaagac cctcggcgcc aagcgcatcg tcaagctggt ccagagctac 660

ggcgtgcaca tcgacggcgt gggtctgcaa gcccacttgg cccaggaagt caccccgacc 720

gccggcgccc tgcccgacca agccactctc gagaccgttc tcagaggctt cacttccctg 780

gacgtcgatg ttgtttacac cgagattgat atccgcatga acacccccag caccccggcc 840

aagctcaaga cacaggccaa ggctttcgag actgttgctc gcagctgtct tgctgtcaag 900

aggtgcattg gaatgaccgt ttggggtatt tcagacgctt tctcgtggat ccctggtgta 960

ttccctggtg agggcgctgc gcttctttgg gacgagaacc ttaagaagaa gccggcttac 1020

gatggcttct acaagggcat caagggtggc aagaagtga 1059

<210>3

<211>352

<212> PRT

<213>Tianshan Mountains cladosporium Cladosporium tianshanense SL-14

<400>3

Met Arg Phe Thr Glu Val Phe Thr Ala Leu Thr Leu Ala Ala Ser Ala

1 5 10 15

Val Ala His Pro Ser Ala Pro Lys Asp Lys Lys Gly Leu Ala Thr Ala

20 25 30

Met Lys Ala Arg Gly Arg Glu Phe Ile Gly Thr Ala Leu Thr Leu Arg

35 40 45

Gly Asn Glu Thr Glu Glu Ala Ile Ala Arg Asn Asn Ala Asp Phe Asn

50 55 60

Ser Phe Thr Pro Glu Asn Ala Met Lys Trp Glu Ala Ile Glu Pro Asn

65 70 75 80

Arg Asn Asn Phe Thr Phe Ser Asp Ala Asp Arg Tyr Arg Asp Trp Ala

85 90 95

Lys Ala Asn Lys Lys Glu Ile His Cys His Thr Leu Val Trp His Ser

100 105 110

Gln Leu Pro Pro Trp Val Ala Ala Gly Asn Tyr Asp Asn Lys Thr Leu

115 120 125

Ile Gly Ile Met Glu Asn His Ile Lys Asn Val Ala Gly Arg Tyr Lys

130 135 140

Asp Val Cys Thr Arg Trp Asp Val Val Asn Glu Ala Leu Glu Glu Asp

145 150 155 160

Gly Thr Tyr Arg Ser Ser Pro Phe Tyr Asp Thr Ile Gly Glu Ala Phe

165 170 175

Ile Pro Ile Ala Phe Lys Phe Ala Lys Lys Tyr Ser Pro Lys Ser Glu

180 185 190

Leu Phe Tyr Asn Asp Tyr Asn Leu Glu Tyr Asn Gly Asn Lys Thr Leu

195 200 205

Gly Ala Lys Arg Ile Val Lys Leu Val Gln Ser Tyr Gly Val His Ile

210 215 220

Asp Gly Val Gly Leu Gln Ala His Leu Ala Gln Glu Val Thr Pro Thr

225 230 235 240

Ala Gly Ala Leu Pro Asp Gln Ala Thr Leu Glu Thr Val Leu Arg Gly

245 250 255

Phe Thr Ser Leu Asp Val Asp Val Val Tyr Thr Glu Ile Asp Ile Arg

260 265 270

Met Asn Thr Pro Ser Thr Pro Ala Lys Leu Lys Thr Gln Ala Lys Ala

275 280 285

Phe Glu Thr Val Ala Arg Ser Cys Leu Ala Val Lys Arg Cys Ile Gly

290 295 300

Met Thr Val Trp Gly Ile Ser Asp Ala Phe Ser Trp Ile Pro Gly Val

305 310 315 320

Phe Pro Gly Glu Gly Ala Ala Leu Leu Trp Asp Glu Asn Leu Lys Lys

325 330 335

Lys Pro Ala Tyr Asp Gly Phe Tyr Lys Gly Ile Lys Gly Gly Lys Lys

340 345 350

<210>4

<211>18

<212> PRT

<213>Tianshan Mountains cladosporium Cladosporium tianshanense SL-14

<400>4

Met Arg Phe Thr Glu Val Phe Thr Ala Leu Thr Leu Ala Ala Ser Ala

1 5 10 15

Val Ala

<210>5

<211>334

<212> PRT

<213>Tianshan Mountains cladosporium Cladosporium tianshanense SL-14

<400>5

His Pro Ser Ala Pro Lys Asp Lys Lys Gly Leu Ala Thr Ala Met Lys

1 5 10 15

Ala Arg Gly Arg Glu Phe Ile Gly Thr Ala Leu Thr Leu Arg Gly Asn

20 25 30

Glu Thr Glu Glu Ala Ile Ala Arg Asn Asn Ala Asp Phe Asn Ser Phe

35 40 45

Thr Pro Glu Asn Ala Met Lys Trp Glu Ala Ile Glu Pro Asn Arg Asn

50 55 60

Asn Phe Thr Phe Ser Asp Ala Asp Arg Tyr Arg Asp Trp Ala Lys Ala

65 70 75 80

Asn Lys Lys Glu Ile His Cys His Thr Leu Val Trp His Ser Gln Leu

85 90 95

Pro Pro Trp Val Ala Ala Gly Asn Tyr Asp Asn Lys Thr Leu Ile Gly

100 105 110

Ile Met Glu Asn His Ile Lys Asn Val Ala Gly Arg Tyr Lys Asp Val

115 120 125

Cys Thr Arg Trp Asp Val Val Asn Glu Ala Leu Glu Glu Asp Gly Thr

130 135 140

Tyr Arg Ser Ser Pro Phe Tyr Asp Thr Ile Gly Glu Ala Phe Ile Pro

145 150 155 160

Ile Ala Phe Lys Phe Ala Lys Lys Tyr Ser Pro Lys Ser Glu Leu Phe

165 170 175

Tyr Asn Asp Tyr Asn Leu Glu Tyr Asn Gly Asn Lys Thr Leu Gly Ala

180 185 190

Lys Arg Ile Val Lys Leu Val Gln Ser Tyr Gly Val His Ile Asp Gly

195 200 205

Val Gly Leu Gln Ala His Leu Ala Gln Glu Val Thr Pro Thr Ala Gly

210 215 220

Ala Leu Pro Asp Gln Ala Thr Leu Glu Thr Val Leu Arg Gly Phe Thr

225 230 235 240

Ser Leu Asp Val Asp Val Val Tyr Thr Glu Ile Asp Ile Arg Met Asn

245 250 255

Thr Pro Ser Thr Pro Ala Lys Leu Lys Thr Gln Ala Lys Ala Phe Glu

260 265 270

Thr Val Ala Arg Ser Cys Leu Ala Val Lys Arg Cys Ile Gly Met Thr

275 280 285

Val Trp Gly Ile Ser Asp Ala Phe Ser Trp Ile Pro Gly Val Phe Pro

290 295 300

Gly Glu Gly Ala Ala Leu Leu Trp Asp Glu Asn Leu Lys Lys Lys Pro

305 310 315 320

Ala Tyr Asp Gly Phe Tyr Lys Gly Ile Lys Gly Gly Lys Lys

325 330

Claims

1. a kind of albumen, it is characterised in that the albumen for it is following 1), 2) or 3) described in albumen：

3) amino acid residue sequence shown in the SEQ ID № .3 and/or SEQ ID № .5 in sequence table is passed through one or several The substitution of individual amino acid residue and/or missing and/or addition and with xylanase activity 1) and/or 2) as derived from albumen Matter.

2. a kind of encoding gene, it is characterised in that the encoding gene has one of following nucleotide sequence：

1) polynucleotide sequence of albumen described in coding claim 1；

2) SEQ ID № in sequence table：1、SEQ ID №：2, and/or SEQ ID №：2 the 55th -1059 shown nucleosides Acid sequence；

5) there is more than 90% homology, and coding identical function egg with the DNA sequence dna of 1), 2), 3), and/or 4) any restriction The DNA sequence dna of white matter.

3. a kind of carrier, expression cassette, transgenic cell line, recombinant bacterium, and/or microorganism, it is characterised in that the carrier, table Contain the encoding gene described in claim 2 up to box, transgenic cell line, recombinant bacterium, and/or microorganism.

4. a kind of primer pair, it is characterised in that encoding gene total length described in the amplifiable claim 2 of primer pair or its is any Fragment.

5. the albumen that the preparation method and methods described of a kind of albumen are prepared, it is characterised in that methods described includes：

1) encoding gene described in claim 2 is prepared；

6. the encoding gene described in albumen, claim 2, the recombinant vector described in claim 3, table described in claim 1 Up to preparation side described in primer pair described in box, transgenic cell line, recombinant bacterium, and/or microorganism, claim 4, claim 5 The application for the albumen that method and the preparation method are prepared.

7. application according to claim 6, it is characterised in that the application is included in following 1) -8) it is at least one in Using：

4) prepare wood oligose, xylobiose, the sugar of wood one, containing wood oligose, containing xylobiose, and/or containing the related production of the sugar of wood one Application in product；

5) as cellulase, dextranase and/or preparation with answering in cellulase, dextranase enzymatic activity Related product With；

6) application in degradation of xylan, cellulose, and/or glucan；

7) in application and preparation in industry, the processing of agricultural, food, medicine, feed, the energy, waste and/or field of environment protection Application in product；

8. application according to claim 7, it is characterised in that the zytase includes claim 1 or claim 5 The albumen；The xylanase mutant includes being obtained albumen described in claim 1 or claim 5 after point mutation Albumen；The recombined xylanase includes the egg for obtaining albumen described in claim 1 or claim 5 after homologous recombination In vain.

9. according to any described application in claim 6,7, and/or 8, it is characterised in that the application be included in it is following 1), 2) application under the conditions of described in, 3), and/or 4)：

1) pH includes 4.0-12.0；

2) temperature includes 0 DEG C -90 DEG C；

3) environment of metal ion, and/or chemical reagent is included；

4) substrate includes xylan, cellulose, and/or glucan.

10. a kind of preparation method of zytase, it is characterised in that methods described includes：From Cladosporium Zytase is extracted in tianshanense SL-14.