CN101805726A - Heat-resistance neutral xylanase, coding gene and application thereof - Google Patents
Heat-resistance neutral xylanase, coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses heat-resistance neutral xylanase, a coding gene and application thereof. The heat-resistance neutral xylanase is the protein shown as (a) or (b): (a) the protein formed by amino acid sequences shown as a first sequence in a sequence table; or (b) the protein which is obtained through substitution and/or deletion and/or addition of one or a plurality of amino acid residues on an amino acid sequence shown as (a), has the xylanase activity and is derived from (a) or (b). The xylanase provided by the invention has the characteristics of heat resistance and high enzymatic activity under the condition of neutral pH. Experiments show that the invention has the highest enzymatic activity which is 92.5 U/mg dry protein powder under the conditions of the temperature of 70 DEG C and the pH value of 7.0, the xylanase has higher enzymatic activity when the temperature is in a range between 55 DEG C and 75 DEG C, and the enzymatic activity is also higher when the pH value is in a range between 6.0 and 8.0. When the xylanase is maintained for 30 minutes under the condition of 60 DEG C, the enzymatic activity is not reduced, and the result shows that the xylanase has high heat resistance. Because of the characteristics, the xylanase of the invention is more applicable to the conditions of high temperature and neutral pH than the ordinary enzyme.
Description
Technical field
The present invention relates to a kind of heat-resistance neutral xylanase and encoding gene thereof and application.
Background technology
Xylan is a kind of abundant renewable resource of occurring in nature, is the most representative hemicellulose, accounts for 1/3～1/2 of hemicellulose, is except that Mierocrystalline cellulose, the polysaccharide that occurring in nature is the abundantest.Compare the easier conversion that is degraded by microorganisms of hemicellulose with Mierocrystalline cellulose.
Zytase is the lytic enzyme of most critical in the xylan hydrolysis enzyme system, and by hydrolysis wood sugar molecule β-1, the 4-glycosidic link is low xylans such as wood oligose and xylo-bioses with xylan hydrolysis, and a small amount of wood sugar and pectinose.Handle sulfate pulping with zytase and can reduce the consumption that following bleaching is handled chemical agent.Because can being used for the pre-bleaching, fodder additives of paper pulp, zytase improves the Energy value of feed and livestock and poultry to the specific absorption of feed, the bifidus bacillus in the propagation enteron aisle, be used for aspects such as food improver and wine brewing, so as a kind of industrial enzymes, the zytase tool is using value widely.
Summary of the invention
An object of the present invention is to provide a kind of protein and encoding gene thereof.
Protein provided by the present invention, be following (a) or (b) shown in protein:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence shown in (a) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have xylanase activity by (a) or (b) deutero-protein.
Described encoding gene is following 1) or 2) or 3) dna molecular:
1) nucleotide sequence is the dna molecular shown in the sequence 2 in the sequence table;
2) under stringent condition with 1) the dna sequence dna hybridization that limits and coding proteic dna molecular with xylanase activity;
3) with 1) dna sequence dna that limits has 90% above homology, and encode and have the proteic dna molecular of xylanase activity.
Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
In order to make the albumen in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (a) but in the albumen synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Proteic encoding gene in above-mentioned (a) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence table 1, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain above-mentioned arbitrary described encoding gene also belong to protection scope of the present invention.
The recombinant expression vector that described recombinant vectors obtains for the multiple clone site that above-mentioned arbitrary described encoding gene is inserted the carrier pHBM905A that sets out; Described reorganization bacterium is the recombinant expression vector that the multiple clone site that above-mentioned arbitrary described encoding gene inserts the carrier pHBM905A that sets out obtains to be transformed Pichia yeast GS115 obtain.
Available existing expression vector establishment contains the recombinant expression vector of described gene.When using described gene constructed recombinant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be used in combination with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
The total length of above-mentioned arbitrary described encoding gene of increasing and any segmental primer thereof are to also belonging to protection scope of the present invention.
A primer sequence of described primer centering is shown in sequence in the sequence table 3, and another primer sequence is shown in sequence in the sequence table 4.
The application of above-mentioned arbitrary described protein in degradation of xylan also belongs to protection scope of the present invention.
The application of above-mentioned arbitrary described encoding gene in degradation of xylan also belongs to protection scope of the present invention.
The application of above-mentioned arbitrary described recombinant vectors in degradation of xylan also belongs to protection scope of the present invention.
The application of above-mentioned arbitrary described reorganization bacterium in degradation of xylan also belongs to protection scope of the present invention.
The application of above-mentioned arbitrary described expression cassette in degradation of xylan also belongs to protection scope of the present invention.
The application that above-mentioned arbitrary described transgenic cell ties up in the degradation of xylan also belongs to protection scope of the present invention.
In above-mentioned arbitrary described application, the condition of described degraded comprises: the temperature of reaction system is 55 ℃-75 ℃, is preferably 70 ℃, and the pH value of reaction system is 6.0-8.0, is preferably 7.0.
In above-mentioned arbitrary described application, described xylan is birch xylan, oat xylan or beechwood xylan.
Zytase provided by the present invention has heat-resistant quality and active high characteristic under condition of neutral pH.Experiment showed, that zytase of the present invention is to have the highest enzymic activity under 7.0 the condition at 70 ℃, pH, is 92.5U/mg albumen dry powder; This enzyme is being in 55 ℃-75 ℃ the scope in temperature, and enzyme is lived all higher; In the pH value was the scope of 6.0-8.0, enzyme was lived also all higher.This enzyme kept 30 minutes under 60 ℃ of conditions, and enzymic activity does not descend, and proved this enzyme thermotolerance height.Above-mentioned characteristic makes enzyme of the present invention more be applicable to high temperature, pH neutral condition than general zytase.
Description of drawings
Fig. 1 is the proteic SDS-PAGE electrophorogram of xy10.
Fig. 2 is the variation of heat resistant xylanase activity with temperature.
Fig. 3 is the variation of heat resistant xylanase activity with pH.
Fig. 4 is the thermostability variation diagram of heat resistant xylanase.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The preparation of embodiment 1, albumen and gene and function
One, gene and proteic discovery
1, the extraction of the total DNA of bacillus sp.N16-5: adopt bacillus sp.N16-5, get its fresh wet thallus 20 grams, be suspended from 10 milliliters of 50mM Tris damping fluids (pH8.0), add N,O-Diacetylmuramidase and 8 milliliters of 0.25mM EDTA (pH8.0), place 20min for 37 ℃ behind the mixing; Add 2 milliliters of 10%SDS afterwards, place 5min, use equal-volume phenol, each extracting of chloroform respectively once for 55 ℃; Get last supernatant solution, add 2 times of volume ethanol, reclaim DNA, respectively with 70% and dehydrated alcohol wash; Precipitation is dissolved in 0.5 milliliter of TE damping fluid, and (pH8.0,10mM Tris 1mMEDTA), add 10mg/ml RNase 3 μ l, and 37 ℃ are incubated 1 hour, use equal-volume phenol, each extracting of chloroform respectively once; Supernatant solution adds 2 times of volume ethanol, reclaims DNA, respectively with 70% and dehydrated alcohol wash, deionized water dissolving is used in vacuum-drying.
2, the discovery of neutral xylanase
1) get total dna solution 10 μ l (about 50 μ g DNA), partially digested with restriction enzyme Sau3AI, through agarose gel electrophoresis, reclaim the 3-9kb dna fragmentation.
2) get total dna solution 10 μ l (about 50 μ g DNA), partially digested with restriction enzyme Sau3AI, through agarose gel electrophoresis, reclaim the 2-8kb dna fragmentation.
3) ligation is 16 hours:
Linked system (20 μ l): 2 μ l (5 μ g) Sau3AI enzymolysis dna fragmentation;
1 μ l (1 μ g) is through BamHI enzymolysis and dephosphorylized plasmid pUC18 DNA;
2 μ l 10x connect damping fluid;
1 μ l T4DNA ligase enzyme;
14 μ l water.
4) with ligation product transformed competence colibacillus bacillus coli DH 5 alpha, be applied to then on the solid medium that contains 50ug/ml Amp (penbritin), 1% crosslinked xylan, cultivated 16-18 hour for 37 ℃, periphery of bacterial colonies has the positive colony that is of transparent circle.
3, positive colony in the Amp-LB substratum 37 ℃ cultivated 16-18 hour, have xylanase activity through active testing.
4, the recombinant plasmid in the positive colony is checked order.Sequencing result shows, in the recombinant plasmid, at pUC18 (available from TAKARA, catalog number is 3218) inserted a dna fragmentation in the skeleton, this dna fragmentation contains the open reading frame (ORF) of a long 1230bp, and the ORF nucleotide sequence is shown in the sequence 2 of sequence table, and its amino acid sequence coded is shown in sequence in the sequence table 1, this albumen note is made xy10 albumen, this gene note is made the xy10 gene.
Two, gene and proteic preparation
Bacillus sp.N16-5 CGMCC No.0369 is available from China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC).
Plasmid pHBM905A is at document (Molecular cloning and heterologous expression of a newxylanase gene from Plectosphaerella cucumerina in Pichia pastoris.AppliedMicrobial Biotechnology, 2007, disclosed in 74:339-346).
Pichia yeast GS115 is available from Invitrogen company, and catalog number is K171001.
(1) preparation of xy10 gene
Can be prepared as follows the xy10 gene; Also can adopt the method for synthetic to obtain the xy10 gene.
Total DNA with bacillus sp.N16-5 is a template, with following primer to carrying out pcr amplification.
Primer is to as follows:
Forward primer: 5 '-
GtcaGtgtcgtttaggtcatactttttaatttactc-3 ' (sequence 3);
Reverse primer: 5 '-
GgccaTtaatcaataattctccagtaagcaggc-3 ' (sequence 4);
The underscore of forward primer partly is the joint of restriction enzyme site CpoI, and the underscore of reverse primer partly is the joint of restriction enzyme site NotI.
The PCR reaction system:
10 * damping fluid, 5 μ l
dNTP 4μl
ExTaq archaeal dna polymerase 0.5 μ l
Template 0.5 μ l
Water 38 μ l.
The PCR reaction conditions: 94 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change-58 ℃ of annealing in 30 seconds 30 seconds were extended 1 minute 30 seconds for-72 ℃ then, 30 circulations, last 72 ℃ were extended 10 minutes.
The PCR product detects output and specificity with 1% agarose gel electrophoresis, and with DNA purification kit (ultrathin centrifugal column type, day root company production) purifying.
(2) structure of recombinant expression vector
1, the PCR product behind the purifying is handled (method of processing: PCR product, dNTP, T4 DNA polymerase are mixed 20min at 12 ℃) with T4 DNA polymerase, obtain the sticky end of NotI and CpoI, agarose electrophoresis recycling product.
2, with plasmid pHBM905A NotI and CpoI double digestion, agarose electrophoresis reclaims enzyme and cuts the big fragment of carrier.
3, the enzyme of the recovery product of step 1 and step 2 is cut the big fragment of carrier and be connected, coat the LB flat board that contains 50 μ g/ml penbritins, 37 ℃ of incubated overnight, picking mono-clonal after connecting product electric shock transformed into escherichia coli DH5 α; Mono-clonal is inserted the liquid LB culture medium culturing that contains 50 μ g/ml penbritins, extract plasmid; Plasmid is carried out bacterium colony PCR checking with forward primer and reverse primer, and the result obtains the correct amplified production of size, and the recombinant plasmid that preliminary proof makes up is correct; Further carry out the plasmid order-checking, the result shows, inserted the xy10 gene shown in the sequence 2 in the sequence table between the NotI of pHBM905A and CpoI restriction enzyme site, and direction of insertion is correct, further the recombinant plasmid of proof structure is correct, with this recombinant plasmid called after pHBM905A-xy10.
(3) preparation of engineering bacteria
Plasmid pHBM905A-xy10 is coated the MD flat board through electric shock after the SalI linearizing after transforming Pichia yeast GS115, and 30 ℃, 48h cultivates, and obtains containing the engineering bacteria of plasmid pHBM905A-xy10, and note is made GS115/pHBM905A-xy10.
Replace pHBM905A-xy10 with pHBM905A, transform Pichia yeast GS115, obtain containing the reorganization bacterium of pHBM905A, in contrast bacterium.The positive reorganization bacterium note that changes pHBM905A over to is made GS115/pHBM905A.
The composition of MD flat board: by YNB (Yeast Nitrogen Base), vitamin H (biotin), D-glucose (dextrose), (NH
4)
2SO
4, agar (agar) and water forms; The concentration of each material in the MD flat board is: (YNB) 3.4g/L, vitamin H 4x10
-5%, glucose 20g/L, (NH
4)
2SO
410g/L, agar 15g/L.
YNB is available from Difco company, and catalog number is 0919-07.
(4) preparation of neutral xylanase and purifying
Gel-filtration prepacked column GE Superdex 75 is available from Amersham Biosciences, and catalog number is 17-5174-01.
The BMGY substratum is formed: be made up of yeast extract, peptone, potassium primary phosphate, YNB, G ﹠ W; The concentration of each composition in substratum is: yeast extract (yeast extract) 10g/L, peptone (peptone) 20g/L, potassium primary phosphate (potassium phosphate) 100mM, pH 6.0, YNB 3.4g/L, glycerine (glycerol) 10g/L.
The BMMY substratum is formed: be made up of yeast extract, peptone, potassium primary phosphate, YNB and water; The concentration of each composition in substratum is: yeast extract (yeast extract) 10g/L, peptone (peptone) 20g/L, potassium primary phosphate (potassium phosphate) 100mM, pH 6.0, YNB 3.4g/L.
The positive bacterium GS115/pHBM905A-xy10 that recombinates is inoculated in the BMGY substratum, cultivates 48h for 30 ℃, this moment culture system OD
600=15 o'clock, go to the BMMY substratum and continue for 25 ℃ to cultivate 72h, every 12 hours, add 1.5% methyl alcohol (even the volumetric concentration of methyl alcohol in culture system is 1.5%), the note of all substances in the culture vessel is made fermented liquid;
With the centrifugal collection supernatant of fermented liquid 5000rpm, 10min, supernatant is concentrated into 400 μ L by Millipore ultrafiltration pipe (molecular weight cut-off of ultrafiltration pipe is 10000); Carry out molecular sieve purification with gel-filtration prepacked column GE Superdex 75 then, remove foreign protein and pigment, purification step is: elder generation is 7.5 Na with the pH value
2HPO
4-NaH
2PO
4Damping fluid balance pillar is gone up sample again, collects maximum peak, is target protein; The elutriant note of collecting is made pure enzyme liquid; After pure enzyme liquid concentrated by Millipore ultrafiltration pipe, vacuum lyophilization obtained zymoprotein dry powder.
GS115/pHBM905A adopts identical step to cultivate and purifying with the contrast bacterium, and the solution that obtains is enzyme liquid in contrast.
The pure enzyme liquid of experimental group is carried out the SDS-PAGE electrophoresis, and the result shows that the proteic molecular weight of Xy10 of experimental group purifying is about 48kDa shown in swimming lane among Fig. 12, and the 45kDa that infers than theory is big, may be glycosylated result.
Glycosylase endoH is available from Biolab company, and catalog number is P0703S.Product carried when Denature buffer and G5 buffer all bought enzyme.
Pure enzyme liquid is handled with deglycosylating enzyme endoH, treatment process is: 2 μ l Denature buffer and the pure enzyme liquid of 18 μ l were handled 10 minutes for 100 ℃, add 2.5 μ l G5 buffer and 1.5 μ l endoH enzymes then and handled 1 hour for 37 ℃, the enzyme liquid after the processing carries out the SDS-PAGE electrophoresis again; The protein band that the result obtains diminishing is about 45kDa.Electrophoretic effects as shown in Figure 1, among Fig. 1, swimming lane 3 expression molecular weight of albumen standards (100,75,50,35,25kDa), the Xy10 albumen of swimming lane 2 expressions after molecular sieve purification, the Xy10 albumen after swimming lane 1 expression is handled through deglycosylating enzyme.
Three, proteic zymologic property analysis
Enzyme unit definition alive is: the catalysis birch xylan produces the required enzyme amount of 1 μ mol wood sugar in the 1min.
Birch xylan is available from sigma, and catalog number is X0502.
(1) optimum temperuture
With the pH value 7.0 Na
2HPO
4-NaH
2PO
4Pure enzyme liquid in the damping fluid dilution experiment two carries out enzyme activity determination with the enzyme liquid after the dilution.Enzyme liquid after dilution note is made dilution enzyme liquid.
Experimental group: the enzyme activity determination reaction system is 0.5ml, is made up of 0.48ml solution A and 0.02ml dilution enzyme liquid; The pH value of reaction system is 7.0; Reaction system behind specified temp incubation 10min, is added 0.5ml dinitrosalicylic acid solution (DNS) termination reaction, measure the light absorption value of 520nm then behind the boiling water bath 5min.
The preparation of every 100ml solution A: with the Na of the pH7.0 of 90ml 100mM
2HPO
4-NaH
2PO
4Damping fluid, 1g birch xylan and 10mi water mix, and handle 3-5 minute to the birch xylan dissolving, obtain the 100ml solution A for 100 ℃.
The result as shown in Figure 2.Show that in the time of 70 ℃, zytase has the highest enzymic activity, be 92.5U/mg albumen dry powder; Enzyme under this temperature is lived the light absorption value of reaction system as relative reactivity 100%, and enzyme is lived the ratio of light absorption value of the therewith enzymatic activity high system of light absorption value of reaction system as relative reactivity under other temperature.Enzyme when temperature is 55 ℃ is lived and is that it is 74.4U/mg that 66.56U/mg, the enzyme in the time of 60 ℃ live, and the enzyme in the time of 75 ℃ is lived and is 46.3U/mg.
The albumen (note compares enzyme liquid) that obtains with contrast bacterium GS115/pHBM905A carries out above-mentioned experiment, and the result is no matter under which temperature condition, and control enzyme liquid does not all have the activity of degradation of xylan.
Repetition, unanimity are as a result established in experiment 3 times.
(2) optimal pH
Dilution enzyme liquid in following each group all is to obtain with the pure enzyme liquid in the damping fluid dilution experiment two in each group.
Experimental group: the determination of activity system is 0.5ml, is made up of 0.48ml solution B (B1, B2, B3, B4, B5, B6 or B7) and 0.02ml dilution enzyme liquid;
The preparation of every 100ml solution B 1: with the Na of 90ml 50mM
2HPO
4-citrate buffer solution, 1g birch xylan mix, and regulate pH value to 4.0, add 10ml water again, obtain 100ml solution B 1.
The preparation of every 100ml solution B 2: identical with solution B 1, the pH value of solution B 2 that different is is 5.0.
The preparation of every 100ml solution B 3: with the NaH of 90ml 50mM
2PO
4-Na
2HPO
4Damping fluid, 1g birch xylan mix, and regulate pH value to 6.0, add 10ml water again, obtain 100ml solution B 3.
The preparation of every 100ml solution B 4: identical with solution B 3, the pH value of solution B 4 that different is is 7.0.
The preparation of every 100ml solution B 5: Tris-HCl damping fluid, the 1g birch xylan of 90ml 50mM are mixed, regulate pH value to 8.0, add 10ml water again, obtain 100ml solution B 5.
The preparation of every 100ml solution B 6: Glycine-NaOH damping fluid, the 1g birch xylan of 90ml 50mM are mixed, regulate pH value to 9.0, add 10ml water again, obtain 100ml solution B 6.
The preparation of every 100ml solution B 7: identical with solution B 6, the pH value of solution B 7 that different is is 10.0.
Above solution all need be handled at 100 ℃ and dissolve to birch xylan in 3-5 minute.
Reaction system at 70 ℃, behind the incubation 10min, is added 0.5ml dinitrosalicylic acid solution (DNS) termination reaction, measure the light absorption value of 520nm then behind the boiling water bath 5min.
3 repetitions are established in experiment.
The result as shown in Figure 3.Showing, is 7.0 o'clock in the pH value, and zytase has the highest enzymic activity, is 92.5U/mg albumen dry powder; Enzyme under this pH value is lived the light absorption value of reaction system as relative reactivity 100%, other pH value down the ratio of the light absorption value of the therewith enzymatic activity high system of light absorption value of enzyme work reaction system as relative reactivity.In the pH value is that 6.0 o'clock enzyme is lived and to be that 63.5U/mg, pH value are that 6.5 o'clock enzyme is lived and is that 88U/mg, pH value are that 7.5 o'clock enzyme is lived and is that 74U/mg, pH value are 8.0 o'clock the enzyme 64.8U/mg of being alive.
The albumen (note compares enzyme liquid) that obtains with contrast bacterium GS115/pHBM905A carries out above-mentioned experiment, and the result is no matter under which pH value condition, and control enzyme liquid does not all have the activity of degradation of xylan.
(3) enzyme heat stability
With the pH value 7.0 Na
2HPO
4-NaH
2PO
4Pure enzyme liquid in the damping fluid dilution experiment two carries out enzyme activity determination with the enzyme liquid after the dilution.Enzyme liquid after dilution note is made dilution enzyme liquid.
To dilute enzyme liquid and place 15 and 30 minutes 60 ℃, 70 ℃, 80 ℃ water-baths respectively, measure the residual activity of enzyme.The pH value is 7.0, measures temperature to be 70 ℃ in the enzyme activity determination reaction system, and all the other conditions and step are with identical described in the experiment ().3 repetitions are established in experiment.
Result such as Fig. 4 show, handle for 60 ℃ and do not see enzyme decline alive in 30 minutes; Show that this enzyme thermotolerance under 60 ℃ of conditions is fine.
Sequence table
＜110〉Institute of Microorganism, Academia Sinica
＜120〉a kind of heat-resistance neutral xylanase and encoding gene thereof and application
<160>4
<210>1
<211>392
<212>PRT
＜213〉genus bacillus (Bacillus sp.)
<400>1
Met?Leu?Lys?Ser?Phe?Lys?Val?Phe?Cys?Val?Ala?Gly?Leu?Ser?Ile?Pro
1 5 10 15
Leu?Leu?Val?Gly?Gly?Gly?Leu?Ser?Ser?Val?Val?Thr?Ala?Lys?Glu?Gly
20 25 30
Pro?Glu?Ala?Gly?Val?Asn?Gly?Gln?Val?Asn?Asn?Ser?Pro?Phe?Ala?Trp
35 40 45
Glu?Val?Ala?Ser?Leu?Ala?Glu?Arg?Tyr?Asp?Gly?Gln?Phe?Asp?Ile?Gly
50 55 60
Ala?Ala?Val?Glu?Pro?Glu?His?Leu?Glu?Gly?Arg?Arg?Ala?Gln?Ile?Leu
65 70 75 80
Glu?His?His?Tyr?Asn?Ser?Leu?Val?Ala?Glu?Asn?Ala?Met?Lys?Pro?Val
85 90 95
Ser?Leu?Gln?Pro?Arg?Glu?Gly?Glu?Trp?Asn?Trp?Gly?Gly?Ala?Asp?Arg
100 105 110
Ile?Val?Asn?Phe?Ala?Arg?Gln?His?Asn?Met?Glu?Leu?Arg?Phe?His?Thr
115 120 125
Leu?Val?Trp?His?Ser?Gln?Val?Pro?Glu?Trp?Phe?Phe?Ile?Asp?Lys?Asp
130 135 140
Gly?Asn?Arg?Met?Val?Asp?Glu?Thr?Asn?Pro?Ala?Lys?Arg?Glu?Ala?Asn
145 150 155 160
Lys?Gln?Leu?Leu?Leu?Glu?Arg?Met?Glu?Thr?His?Ile?Lys?Thr?Val?Val
165 170 175
Glu?Arg?Tyr?Lys?Asp?Asp?Val?Thr?Ser?Trp?Asp?Val?Val?Asn?Glu?Val
180 185 190
Ile?Asp?Asp?Gly?Gly?Gly?Leu?Arg?Asn?Ser?Glu?Trp?Tyr?Gln?Ile?Thr
195 200 205
Gly?Thr?Asp?Tyr?Ile?Lys?Val?Ala?Phe?Glu?Thr?Ala?Arg?Lys?Tyr?Ala
210 215 220
Gly?Glu?Asp?Ala?Lys?Leu?Tyr?Ile?Asn?Asp?Tyr?Asn?Thr?Glu?Ile?Pro
225 230 235 240
Ser?Lys?Arg?Asp?Asp?Leu?Tyr?Asn?Leu?Val?Lys?Asp?Leu?Leu?Asp?Gln
245 250 255
Gly?Val?Pro?Ile?Asp?Gly?Val?Gly?His?Gln?Ala?His?Ile?Gln?Ile?Gly
260 265 270
Trp?Pro?Ser?Leu?Glu?Asp?Thr?Arg?Ala?Ser?Phe?Asp?Lys?Phe?Thr?Ser
275 280 285
Leu?Gly?Leu?Asp?Asn?Gln?Val?Thr?Glu?Leu?Asp?Met?Ser?Leu?Tyr?Gly
290 295 300
Trp?Pro?Pro?Thr?Gly?Ala?Tyr?Thr?Ser?Tyr?Asp?Asp?Ile?Pro?Glu?Tyr
305 310 315 320
Leu?Leu?Gln?Ala?Gln?Ala?Asp?Arg?Tyr?Asp?Gln?Leu?Phe?Gln?Leu?Tyr
325 330 335
Glu?Glu?Leu?Asp?Ala?Asp?Ile?Ser?Ser?Val?Thr?Phe?Trp?Gly?Ile?Ala
340 345 350
Asp?Asn?His?Thr?Trp?Leu?Asp?Asp?Arg?Ala?Arg?Gln?Tyr?Asn?Asn?Gly
355 360 365
Val?Gly?Val?Asp?Ala?Pro?Phe?Val?Phe?Asp?His?Asn?Tyr?Arg?Val?Lys
370 375 380
Pro?Ala?Tyr?Trp?Arg?Ile?Ile?Asp
385 390
<210>2
<211>1179
<212>DNA
＜213〉genus bacillus (Bacillus sp.)
<400>2
atgttaaaat?cttttaaggt?gttttgtgtg?gcgggtttgt?cgatcccttt?attagttgga 60
ggtggcttga?gtagtgtcgt?aacagctaag?gaaggaccgg?aagcaggtgt?gaatggacaa 120
gtcaataata?gcccttttgc?gtgggaagtt?gcttctcttg?ctgagcgata?tgatggccaa 180
tttgatattg?gagcagcagt?tgaaccagag?cacttagagg?gaagaagagc?acagatttta 240
gagcatcatt?ataatagcct?tgtggcggaa?aatgcgatga?aacctgtatc?cctccaaccg 300
agagaaggtg?agtggaattg?gggaggcgct?gacagaatcg?tgaattttgc?ccgacaacat 360
aacatggagc?ttcgttttca?cacactcgtt?tggcatagcc?aagtacctga?atggtttttc 420
atcgataaag?acggtaatcg?tatggtggat?gaaacaaatc?cagcgaaacg?tgaggctaat 480
aaacagcttt?tattagagcg?gatggaaaca?catatcaaaa?cggttgtgga?acgttataaa 540
gatgatgtaa?catcatggga?tgtggtgaat?gaagtcattg?atgatggcgg?cggacttcgt 600
aattcggaat?ggtatcaaat?tacggggaca?gactatatta?aggttgcttt?tgaaacagca 660
agaaaatatg?ctggtgaaga?tgcaaagctg?tacattaatg?attataacac?cgaaattcca 720
tcaaagagag?atgaccttta?caaccttgtc?aaagacttat?tagaccaggg?agtaccaatt 780
gacggggtag?gacaccaggc?gcatattcaa?attggctggc?cttcacttga?agatacaaga 840
gcctcttttg?acaagtttac?tagcttaggg?ttggacaacc?aggtaactga?gttggacatg 900
agcctttatg?gctggccacc?aacaggggca?tacacatctt?atgatgatat?tccagaatac 960
cttcttcaag?ctcaagcaga?ccgatatgat?cagctattcc?agctatatga?agaattagac 1020
gctgatatta?gcagtgtgac?attctgggga?attgctgata?atcatacttg?gcttgatgac 1080
cgtgcaagac?agtacaataa?tggtgtaggt?gtagatgcac?catttgtttt?tgatcataat 1140
tatcgcgtca?agcctgctta?ctggagaatt?attgattaa 1179
<210>3
<211>32
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
gtcaatgtta?aaatctttta?aggtgttttg?tg 32
<210>4
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
ggccattaat?caataattct?ccagtaagca?ggc 33
Claims (9)
1. protein, be following (a) or (b) shown in protein:
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence shown in (a) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have xylanase activity by (a) or (b) deutero-protein.
2. the described proteinic encoding gene of claim 1.
3. encoding gene according to claim 2 is characterized in that: described encoding gene is following 1) or 2) or 3) dna molecular:
1) nucleotide sequence is the dna molecular shown in the sequence 2 in the sequence table;
2) under stringent condition with 1) the dna sequence dna hybridization that limits and coding proteic dna molecular with xylanase activity;
3) with 1) dna sequence dna that limits has 90% above homology, and encode and have the proteic dna molecular of xylanase activity.
4. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 described encoding genes.
5. total length and any segmental primer thereof of amplification claim 2 or 3 described encoding genes are right.
6. primer according to claim 5 is right, it is characterized in that: a primer sequence of described primer centering is shown in sequence in the sequence table 3, and another primer sequence is shown in sequence in the sequence table 4.
7. the application of the described protein of claim 1 in degradation of xylan; Claim 2 or 3 application of described encoding gene in degradation of xylan; The application of the described recombinant vectors of claim 4 in degradation of xylan; The application of claim 4 described reorganization bacterium in degradation of xylan; The application of the described expression cassette of claim 4 in degradation of xylan; Or the described transgenic cell of claim 4 ties up to the application in the degradation of xylan.
8. application according to claim 7 is characterized in that: the condition of described degraded comprises: the temperature of reaction system is 55 ℃-75 ℃, is preferably 70 ℃, and the pH value of reaction system is 6.0-8.0, is preferably 7.0.
9. according to claim 7 or 8 described application, it is characterized in that: described xylan is birch xylan, oat xylan or beechwood xylan.
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| CN2010101528276A CN101805726B (en) | 2010-04-19 | 2010-04-19 | Heat-resistance neutral xylanase, coding gene and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002471A (en) * | 2010-11-05 | 2011-04-06 | 广东溢多利生物科技股份有限公司 | Novel xylanase V-XYL as well as gene, high-efficiency expression method and application thereof |
| CN102586207A (en) * | 2012-03-01 | 2012-07-18 | 上海戴迪实业发展有限公司 | Method for preparing neutral bleaching xylanase |
| CN103060290A (en) * | 2011-10-18 | 2013-04-24 | 中国科学院微生物研究所 | Alkaline xylanase, its coding gene and application |
| CN103060289A (en) * | 2011-10-18 | 2013-04-24 | 中国科学院微生物研究所 | Salt-tolerant xylanase, its coding gene and application |
| CN105483098A (en) * | 2016-01-26 | 2016-04-13 | 深圳大学 | Neutral endo-xylanase and encoding gene and application thereof |
| CN107129976A (en) * | 2017-06-02 | 2017-09-05 | 中国农业科学院饲料研究所 | A kind of neutral high-temperature xylanase and its encoding gene and its application |
| CN107502602A (en) * | 2017-09-01 | 2017-12-22 | 上海市农业科学院 | The preparation and its application for the hot rod bacterium xylanase mutant that two heat resistances improve |
| CN109652393A (en) * | 2017-10-12 | 2019-04-19 | 中国科学院微生物研究所 | A kind of zytase xylanase-m and its encoding gene and application with high thermal stability |
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| CN1405304A (en) * | 2001-09-14 | 2003-03-26 | 中国农业科学院饲料研究所 | Heat-resisting, antiproteinase acidic-neutral xylanase and its gene |
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| US5871730A (en) * | 1994-07-29 | 1999-02-16 | Universite De Sherbrooke | Thermostable xylanase DNA, protein and methods of use |
| CN1405304A (en) * | 2001-09-14 | 2003-03-26 | 中国农业科学院饲料研究所 | Heat-resisting, antiproteinase acidic-neutral xylanase and its gene |
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| 《微生物学报》 20040229 曾艳等 嗜碱菌（Bacillus sp.）ZBAW6的木聚糖酶的分离纯化及其性质 第75-78页 1-9 第44卷, 第1期 2 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102002471A (en) * | 2010-11-05 | 2011-04-06 | 广东溢多利生物科技股份有限公司 | Novel xylanase V-XYL as well as gene, high-efficiency expression method and application thereof |
| CN102002471B (en) * | 2010-11-05 | 2012-07-04 | 广东溢多利生物科技股份有限公司 | Novel xylanase V-XYL as well as gene, high-efficiency expression method and application thereof |
| CN103060290A (en) * | 2011-10-18 | 2013-04-24 | 中国科学院微生物研究所 | Alkaline xylanase, its coding gene and application |
| CN103060289A (en) * | 2011-10-18 | 2013-04-24 | 中国科学院微生物研究所 | Salt-tolerant xylanase, its coding gene and application |
| CN103060290B (en) * | 2011-10-18 | 2014-05-28 | 中国科学院微生物研究所 | Alkaline xylanase, its coding gene and application |
| CN103060289B (en) * | 2011-10-18 | 2014-05-28 | 中国科学院微生物研究所 | Salt-tolerant xylanase, its coding gene and application |
| CN102586207A (en) * | 2012-03-01 | 2012-07-18 | 上海戴迪实业发展有限公司 | Method for preparing neutral bleaching xylanase |
| CN105483098A (en) * | 2016-01-26 | 2016-04-13 | 深圳大学 | Neutral endo-xylanase and encoding gene and application thereof |
| CN107129976A (en) * | 2017-06-02 | 2017-09-05 | 中国农业科学院饲料研究所 | A kind of neutral high-temperature xylanase and its encoding gene and its application |
| CN107129976B (en) * | 2017-06-02 | 2020-07-14 | 中国农业科学院饲料研究所 | Xylanase, coding gene thereof and application thereof |
| CN107502602A (en) * | 2017-09-01 | 2017-12-22 | 上海市农业科学院 | The preparation and its application for the hot rod bacterium xylanase mutant that two heat resistances improve |
| CN109652393A (en) * | 2017-10-12 | 2019-04-19 | 中国科学院微生物研究所 | A kind of zytase xylanase-m and its encoding gene and application with high thermal stability |
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| CN101805726B (en) | 2012-05-02 |
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