CN104845955B - A kind of alkalescent xylanase mutant and its application - Google Patents

A kind of alkalescent xylanase mutant and its application Download PDF

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CN104845955B
CN104845955B CN201510207695.5A CN201510207695A CN104845955B CN 104845955 B CN104845955 B CN 104845955B CN 201510207695 A CN201510207695 A CN 201510207695A CN 104845955 B CN104845955 B CN 104845955B
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gln
ser
gly
ala
xyn
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CN104845955A (en
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苏瑞荣
苏琳
吴福彪
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Heze University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases

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Abstract

The present invention relates to a kind of alkalescent xylanase mutant and its application.The alkalescent xylanase mutant, described mutant are that amino acid sequence is SEQ ID NO:176th, 210,253 position amino acid mutations of 2 alkalescent xylanase are Ala, Gly and Met.The present invention is expanded using degenerate primer from soil genome obtains a kind of xylanase gene Xyn, and then has obtained Xyn mutant Xyn 1 using fallibility PCR method；Xyn 1 does not have significant changes relative to Xyn optimal reactive temperatures and highest enzyme activity, but, its optimal reaction pH changes into 9 11, optimal reaction pH is more biased towards in alkalescence, and there is good pH stability, Xyn 1 can be used for the degraded of cellulosic material in the basic conditions, and broader scope is provided for the application of zytase.

Description

A kind of alkalescent xylanase mutant and its application
（One）Technical field
The invention belongs to bioengineering field, and in particular to a kind of alkalescent xylanase mutant and its application, the wood are poly- Carbohydrase mutant is partial to alkalescence relative to parent xylanase, its optimal pH.
（Two）Background technology
Xylan（xylan）It is a kind of poly pentose, is the Main Ingredients and Appearance of plant hemicellulose, is that nature relaying is fine The abundant fertile absorber resource xylan of content second after dimension element, and renewable resource the abundantest in nature it One.The main chain of xylan is connected into by β-D- xylopyranoses residues through β-Isosorbide-5-Nitrae-glycosidic bond, can there is different side chain substituents.
The complete hydrolysis of xylan needs various enzymes in xylan hydrolysis enzyme system to cooperate with completion, including β -1 between each other, 4- endo-xylanases, xylobiase, α-L-arabinose glycosides enzyme, α-D- glucuronidases, acetyl group xylan esterase and Phenolic acid (forulic acid and p- coumaric acids) esterase, wherein zytase is most critical.
Zytase belongs to hydrolase, be it is a kind of can be by xylan degrading into xylo-oligosaccharide or the complex enzyme of xylose System, including a variety of restriction endonucleases and excision enzyme.Zytase is divided into family 10 and family 11 in glycoside hydrolase；Family 10 Xylan molecular weight it is high, complicated, hydrolysate generally produces the smaller oligosaccharide of molecular weight；Family 11 xylan Molecular weight is smaller, there is very high specificity to xylan.The molecular structure of zytase and physicochemical property it is different with its source and There is bigger difference, some zytases comprise only single catalyst structure domain, and other then have catalysis a variety of with other simultaneously On-catalytic domain.
With deepening continuously for research, zytase is increasingly shown in industries such as pulping and paper-making, food, feeds Its huge applications；For the zytase of different biological sources, molecular weight ranges 8-145kD, pI scope are pH 3- 10.5, the reaction optimal pH scope of most of enzymes is 4-7, and optimum temperature is 40-60 DEG C；According to actual conditions, it is necessary to develop resistance to Sour, alkaline-resisting characteristic zytase type, to increase the application of zytase.
（Three）The content of the invention
The present invention needs to solve the problems, such as to be to be directed to prior art, and the invention provides a kind of alkalescent xylanase mutant And its application, the xylanase mutant Xyn-1 that obtains is relative to parent xylanase by the present invention, optimal reaction pH be more biased towards in Alkalescence, broader scope is provided for the application of zytase.
The present invention is achieved through the following technical solutions：
A kind of alkalescent xylanase mutant, it is characterized in that：Described mutant is that amino acid sequence is SEQ ID NO:176th, 210,253 position amino acid mutations of 2 alkalescent xylanase are Ala, Gly and Met.
The alkalescent xylanase mutant of the present invention, the amino acid sequence of alkalescent xylanase is SEQ ID NO:4, it is described The optimal reaction pH of alkalescent xylanase is 9-11.
A kind of gene, encode the gene of the alkalescent xylanase mutant.Its nucleotides sequence is classified as SEQ ID NO:3.
A kind of recombinant vector, the recombinant vector are the carrier for carrying coding alkalescent xylanase mutant gene.
A kind of host cell, include the recombinant vector.
A kind of application in degradation of fibers cellulosic material in the basic conditions of alkalescent xylanase mutant.
A kind of application in degradation of fibers cellulosic material in the basic conditions of alkalescent xylanase mutant.
The cellulosic material is xylan, and the pH of the alkalescence condition is 9-11.
The present invention is expanded using degenerate primer from pedotheque obtains a kind of zytase Xyn, its nucleotide sequence such as SEQ ID NO:Shown in 1, amino acid sequence such as SEQ ID NO:Shown in 2, its optimal reactive temperature is 35-45 DEG C, and optimal reaction pH is 4-6；Afterwards, the present invention carries out gene mutation to Xyn using the method for fallibility PCR amplifications, and screening has obtained optimal reaction pH hairs The raw mutant enzyme Xyn-1 changed.Xyn-1 has amino acid sites at three to undergo mutation, optimal reactive temperature is relative to Xyn 35-45 DEG C, optimal reaction pH is 9-11.
Beneficial effects of the present invention：The present invention is expanded using degenerate primer from soil genome obtains a kind of zytase Gene Xyn, and then obtained Xyn mutant Xyn-1 using fallibility PCR method；Xyn-1 is relative to Xyn optimal reaction temperature Degree and highest enzyme activity do not have significant changes, and still, its optimal reaction pH changes into 9-11, and optimal reaction pH is more biased towards in alkali Property, and there is good pH stability, Xyn-1 can be used for the degraded of cellulosic material in the basic conditions, be xylan The application of enzyme provides broader scope.
（Four）Brief description of the drawings
Fig. 1：Zytase Xyn enzyme activity change curves under different pH；
Fig. 2：Zytase Xyn enzyme activity change curves at different temperatures；
Fig. 3：Xylanase mutant Xyn-1 enzyme activity change curves under different pH；
Fig. 4：Xylanase mutant Xyn-1 enzyme activity change curves at different temperatures；
Fig. 5：Xyn and Xyn-1 pH stability test figures, wherein, Xyn is tested under conditions of pH=5, Xyn-1 It is to be tested under conditions of pH=10.
（Five）Embodiment
With reference to embodiment, the present invention will be further described, and the experimental method of unreceipted actual conditions can in embodiment Routinely technology is operated.Those skilled in the art can be better understood from the present invention by embodiment；It is but of the invention Protection domain is not limited to provided embodiment.
Embodiment 1：Zytase Xyn acquisition
Soil genomic DNA, agarose gel electrophoresis detection are extracted using PowerMax kits (MOBIO, USA) DNA mass.DNA mean concentrations after purification reach 90ng/ μ l.
Conserved regions design degenerate primer (the 5'- of zytase according to disclosed in NCBI GGAATTCCWYAYTAYGCATWGHCC-3', 5'-CATGCCTGHTWYCCRAAGCA-3'), reacted using touchdown PCR from soil Zytase is obtained in genomic DNA, PCR programs are as follows： 95 °C 5 min；95 °C 30 s, 65 °C-50 °C 30 S, 72 °C of 70 s, totally 15 circulations；95 °C of 30 s, 50 °C of 30 s, 72 °C of 70 s, totally 30 circulations；Last 72 ° C extends 10 min.
Amplified fragments are connected with pMD18-T carriers, and sequencing obtains zytase Xyn, its nucleotide sequence such as SEQ ID NO:1 It is shown, the amino acid sequence such as SEQ ID NO of coding:Shown in 2.
Embodiment 2：Xyn heterogenous expression and purifying
Xyn genes are connected with vector plasmid pET-22b (+) and are transformed into E. coli BL21 (DE3) expressive host In cell.Using IPTG induced expression Xyn, and the Xyn albumen purified is obtained using Ni-NTA affinity chromatographys.
Embodiment 3：Xyn Enzyme activity assay
The enzyme activity determination of zytase uses DNS methods, and concrete operations are as follows：
DNS is prepared：10 grams of NaOH are weighed, are dissolved in 400ml distilled waters, then weigh 10g dinitrosalicylic acids, 2g benzene Phenol, 0.5g anhydrous sodium sulfites, 200g Rochelle salts, are dissolved in 300ml distilled waters, two kinds of solution mixing, Constant volume is kept in dark place to 1 liter.
In 100 μ l reaction systems, final concentration of 1% (w/w) birch xylan is added, final concentration of 100mM's pH6.0 Na2HPO4/NaH2PO4Buffer solution, then adds the appropriate enzyme liquid that certain dilution factor is diluted to the buffer solution, 40 DEG C Reaction 10 minutes, adding 100 μ l DNS terminating reactions, (control is first to be added in above-mentioned reaction system after 100 μ l DNS again Enzyme-added liquid), react in boiling water bath 5 minutes and develop the color, survey 540nm absorbance values with ELIASA, sample measurements subtract profit after control Enzyme-activity unit (U) is calculated with standard curve.
Enzyme-activity unit (U) is defined as the enzyme amount needed for 1 μm of ol reduced sugar of catalyzing hydrolysis xylan generation per minute.
Xyn optimal pHs are detected in the range of 40 DEG C, pH 3-12；Optimum temperature is under the conditions of optimal pH, 20-70 DEG C of model Enclose interior detection.
From Fig. 1 and Fig. 2, Xyn optimal reaction pH is 4-6, and optimal reactive temperature is 35-45 DEG C.
Embodiment 4：Zytase Xyn-1 acquisition and Activity determination
According to Xyn nucleotide sequence, xylanase mutant Xyn-1 is obtained using fallibility PCR method, sequencing result shows Show Xyn-1 nucleotide sequence such as SEQ ID NO:Shown in 3, the amino acid sequence such as SEQ ID NO of coding:Shown in 4.Xyn-1 phases For Xyn, the amino acid of Ser176, Asn210 and Asp253 position is undergone mutation, and sports Ala, Gly and Met respectively.
Fallibility pcr amplification reaction system is：10 × PCR buffer10.0 μ L, 25mmol/L the μ L of MnCl2 2.0, 25mmol/L MgCl2 22.0 μ L, 100mmol/L dATP 0.2 μ L, 100mmol/L dGTP 0.2 μ L, 100mmol/L DCTP 1.0 μ L, 100mmol/L dTTP 1.0 μ L, 50mmol/L each 2.0 μ L of upstream and downstream primer, DNA profiling 1 μ L, Lc Taq3.0U, it is 100 μ L to add ultra-pure water to reaction cumulative volume；Fallibility PCR amplification conditions are：95 DEG C of 3min that unwind, 94 DEG C of denaturation 30sec；42 DEG C of annealing 30sec；72 DEG C of extension 3min；30 circular responses are carried out altogether；Then continue to extend 10min in 72 DEG C.
By the mutant gene Xyn-1 that fallibility PCR is obtained according to embodiment 2-3 method carry out heterogenous expression, purifying with And Activity determination, from Fig. 3-4, Xyn-1 optimal reaction pH is 9-11, and alkalescence is more biased towards compared with Xyn；Xyn-1 is most Suitable reaction temperature is 35-45 DEG C, and compared with Xyn, optimal reactive temperature does not change.
In addition, Xyn-1 highest enzyme activity, compared with Xyn, difference is not notable, and the stability under optimal reaction pH Also do not weaken（Referring to Fig. 5）；This shows that the Xyn position amino acid sites of the 176th, 210 and 253 rise to its optimal reaction pH Key effect, what mutant Xyn-1 can stablize in the basic conditions plays a role；The xylanase mutant of the present invention Xyn-1 can be used for degradation of xylan in the basic conditions and other cellulosic materials.
Specification nucleotides and amino acid sequence table
SEQ ID NO:1
atggcaggga tcttccttgc tatcgtatca agccagtcag ctcttgcaac tggcacgagc 60
aagttgcttg gaaacatcat cccatcaagt gttccacgaa actgggatac atatcggaac 120
cagcctacag cagcaaatgg atgcaaatgg ggatgtgttc agaattcgca agcatcattc 180
attggagaga ttgtgactca gcattcagtc atgcaaacac agcagggatc acatttaagt 240
tccatccatc agtttggaga agccaagaac caggatggat tggatccttg tcaggcaatg 300
actcaacaac aggcagttgt tagttggatc gaagcagcag gaaaacatat tcaagcgcac 360
aaggagttga tgttgtcaat gaagcactac atgcaccagc aagctgcaga aactcacttg 420
ctggatcagg aagctcagga tgggattgga ttgtctggtc attcaagcag gcaaaacaat 480
tcacttccaa gttcaaaact cttgatcaat caatatggta ttatcagtga tccaagtgaa 540
gctcgtcaat atgttgaaat tattgatatt ttgaagagca atagcttaac acatggtatc 600
ggaatccttg gccatcaact caatgtcaac acagtatcag catcaacagc tcaatcagtt 660
cttgacacac gtggagcgac aggtgtttca aaatatgttt cagaacttga tgcaaatgga 720
aacacagaac aagagcaaca agctatctat gagagagatt tccgagttct ttggacacac 780
aaatcagtgc aaggaatcac acattggggc tatatcacag gccagacatg gaacgatgga 840
acctattga 849
SEQ ID NO:2
Met Ala Gly Ile Phe Leu Ala Ile Val Ser Ser Gln Ser Ala Leu Ala
1 5 10 15
Thr Gly Thr Ser Lys Leu Leu Gly Asn Ile Ile Pro Ser Ser Val Pro
20 25 30
Arg Asn Trp Asp Thr Tyr Arg Asn Gln Pro Thr Ala Ala Asn Gly Cys
35 40 45
Lys Trp Gly Cys Val Gln Asn Ser Gln Ala Ser Phe Ile Gly Glu Ile
50 55 60
Val Thr Gln His Ser Val Met Gln Thr Gln Gln Gly Ser His Leu Ser
65 70 75 80
Ser Ile His Gln Phe Gly Glu Ala Lys Asn Gln Asp Gly Leu Asp Pro
85 90 95
Cys Gln Ala Met Thr Gln Gln Gln Ala Val Val Ser Trp Ile Glu Ala
100 105 110
Ala Gly Lys His Ile Gln Ala His Lys Glu Leu Met Leu Ser Met Lys
115 120 125
His Tyr Met His Gln Gln Ala Ala Glu Thr His Leu Leu Asp Gln Glu
130 135 140
Ala Gln Asp Gly Ile Gly Leu Ser Gly His Ser Ser Arg Gln Asn Asn
145 150 155 160
Ser Leu Pro Ser Ser Lys Leu Leu Ile Asn Gln Tyr Gly Ile Ile Ser
165 170 175
Asp Pro Ser Glu Ala Arg Gln Tyr Val Glu Ile Ile Asp Ile Leu Lys
180 185 190
Ser Asn Ser Leu Thr His Gly Ile Gly Ile Leu Gly His Gln Leu Asn
195 200 205
Val Asn Thr Val Ser Ala Ser Thr Ala Gln Ser Val Leu Asp Thr Arg
210 215 220
Gly Ala Thr Gly Val Ser Lys Tyr Val Ser Glu Leu Asp Ala Asn Gly
225 230 235 240
Asn Thr Glu Gln Glu Gln Gln Ala Ile Tyr Glu Arg Asp Phe Arg Val
245 250 255
Leu Trp Thr His Lys Ser Val Gln Gly Ile Thr His Trp Gly Tyr Ile
260 265 270
Thr Gly Gln Thr Trp Asn Asp Gly Thr Tyr
275 280
SEQ ID NO:3
atggcaggga tcttccttgc tatcgtatca agccagtcag ctcttgcaac tggcacgagc 60
aagttgcttg gaaacatcat cccatcaagt gttccacgaa actgggatac atatcggaac 120
cagcctacag cagcaaatgg atgcaaatgg ggatgtgttc agaattcgca agcatcattc 180
attggagaga ttgtgactca gcattcagtc atgcaaacac agcagggatc acatttaagt 240
tccatccatc agtttggaga agccaagaac caggatggat tggatccttg tcaggcaatg 300
actcaacaac aggcagttgt tagttggatc gaagcagcag gaaaacatat tcaagcgcac 360
aaggagttga tgttgtcaat gaagcactac atgcaccagc aagctgcaga aactcacttg 420
ctggatcagg aagctcagga tgggattgga ttgtctggtc attcaagcag gcaaaacaat 480
tcacttccaa gttcaaaact cttgatcaat caatatggta ttatcgcaga tccaagtgaa 540
gctcgtcaat atgttgaaat tattgatatt ttgaagagca atagcttaac acatggtatc 600
ggaatccttg gccatcaact caatgtcggg acagtatcag catcaacagc tcaatcagtt 660
cttgacacac gtggagcgac aggtgtttca aaatatgttt cagaacttga tgcaaatgga 720
aacacagaac aagagcaaca agctatctat gagagaatgt tccgagttct ttggacacac 780
aaatcagtgc aaggaatcac acattggggc tatatcacag gccagacatg gaacgatgga 840
acctattga 849
SEQ ID NO:4
Met Ala Gly Ile Phe Leu Ala Ile Val Ser Ser Gln Ser Ala Leu Ala
1 5 10 15
Thr Gly Thr Ser Lys Leu Leu Gly Asn Ile Ile Pro Ser Ser Val Pro
20 25 30
Arg Asn Trp Asp Thr Tyr Arg Asn Gln Pro Thr Ala Ala Asn Gly Cys
35 40 45
Lys Trp Gly Cys Val Gln Asn Ser Gln Ala Ser Phe Ile Gly Glu Ile
50 55 60
Val Thr Gln His Ser Val Met Gln Thr Gln Gln Gly Ser His Leu Ser
65 70 75 80
Ser Ile His Gln Phe Gly Glu Ala Lys Asn Gln Asp Gly Leu Asp Pro
85 90 95
Cys Gln Ala Met Thr Gln Gln Gln Ala Val Val Ser Trp Ile Glu Ala
100 105 110
Ala Gly Lys His Ile Gln Ala His Lys Glu Leu Met Leu Ser Met Lys
115 120 125
His Tyr Met His Gln Gln Ala Ala Glu Thr His Leu Leu Asp Gln Glu
130 135 140
Ala Gln Asp Gly Ile Gly Leu Ser Gly His Ser Ser Arg Gln Asn Asn
145 150 155 160
Ser Leu Pro Ser Ser Lys Leu Leu Ile Asn Gln Tyr Gly Ile Ile Ala
165 170 175
Asp Pro Ser Glu Ala Arg Gln Tyr Val Glu Ile Ile Asp Ile Leu Lys
180 185 190
Ser Asn Ser Leu Thr His Gly Ile Gly Ile Leu Gly His Gln Leu Asn
195 200 205
Val Gly Thr Val Ser Ala Ser Thr Ala Gln Ser Val Leu Asp Thr Arg
210 215 220
Gly Ala Thr Gly Val Ser Lys Tyr Val Ser Glu Leu Asp Ala Asn Gly
225 230 235 240
Asn Thr Glu Gln Glu Gln Gln Ala Ile Tyr Glu Arg Met Phe Arg Val
245 250 255
Leu Trp Thr His Lys Ser Val Gln Gly Ile Thr His Trp Gly Tyr Ile
260 265 270
Thr Gly Gln Thr Trp Asn Asp Gly Thr Tyr 。
275 280
SEQUENCE LISTING
<170> PatentIn version 3.5
<210> 1
<211> 849
<212> DNA
<213> 1
SEQ ID NO:1
atggcaggga tcttccttgc tatcgtatca agccagtcag ctcttgcaac tggcacgagc 60
aagttgcttg gaaacatcat cccatcaagt gttccacgaa actgggatac atatcggaac 120
cagcctacag cagcaaatgg atgcaaatgg ggatgtgttc agaattcgca agcatcattc 180
attggagaga ttgtgactca gcattcagtc atgcaaacac agcagggatc acatttaagt 240
tccatccatc agtttggaga agccaagaac caggatggat tggatccttg tcaggcaatg 300
actcaacaac aggcagttgt tagttggatc gaagcagcag gaaaacatat tcaagcgcac 360
aaggagttga tgttgtcaat gaagcactac atgcaccagc aagctgcaga aactcacttg 420
ctggatcagg aagctcagga tgggattgga ttgtctggtc attcaagcag gcaaaacaat 480
tcacttccaa gttcaaaact cttgatcaat caatatggta ttatcagtga tccaagtgaa 540
gctcgtcaat atgttgaaat tattgatatt ttgaagagca atagcttaac acatggtatc 600
ggaatccttg gccatcaact caatgtcaac acagtatcag catcaacagc tcaatcagtt 660
cttgacacac gtggagcgac aggtgtttca aaatatgttt cagaacttga tgcaaatgga 720
aacacagaac aagagcaaca agctatctat gagagagatt tccgagttct ttggacacac 780
aaatcagtgc aaggaatcac acattggggc tatatcacag gccagacatg gaacgatgga 840
acctattga 849
SEQ ID NO:2
Met Ala Gly Ile Phe Leu Ala Ile Val Ser Ser Gln Ser Ala Leu Ala
1 5 10 15
Thr Gly Thr Ser Lys Leu Leu Gly Asn Ile Ile Pro Ser Ser Val Pro
20 25 30
Arg Asn Trp Asp Thr Tyr Arg Asn Gln Pro Thr Ala Ala Asn Gly Cys
35 40 45
Lys Trp Gly Cys Val Gln Asn Ser Gln Ala Ser Phe Ile Gly Glu Ile
50 55 60
Val Thr Gln His Ser Val Met Gln Thr Gln Gln Gly Ser His Leu Ser
65 70 75 80
Ser Ile His Gln Phe Gly Glu Ala Lys Asn Gln Asp Gly Leu Asp Pro
85 90 95
Cys Gln Ala Met Thr Gln Gln Gln Ala Val Val Ser Trp Ile Glu Ala
100 105 110
Ala Gly Lys His Ile Gln Ala His Lys Glu Leu Met Leu Ser Met Lys
115 120 125
His Tyr Met His Gln Gln Ala Ala Glu Thr His Leu Leu Asp Gln Glu
130 135 140
Ala Gln Asp Gly Ile Gly Leu Ser Gly His Ser Ser Arg Gln Asn Asn
145 150 155 160
Ser Leu Pro Ser Ser Lys Leu Leu Ile Asn Gln Tyr Gly Ile Ile Ser
165 170 175
Asp Pro Ser Glu Ala Arg Gln Tyr Val Glu Ile Ile Asp Ile Leu Lys
180 185 190
Ser Asn Ser Leu Thr His Gly Ile Gly Ile Leu Gly His Gln Leu Asn
195 200 205
Val Asn Thr Val Ser Ala Ser Thr Ala Gln Ser Val Leu Asp Thr Arg
210 215 220
Gly Ala Thr Gly Val Ser Lys Tyr Val Ser Glu Leu Asp Ala Asn Gly
225 230 235 240
Asn Thr Glu Gln Glu Gln Gln Ala Ile Tyr Glu Arg Asp Phe Arg Val
245 250 255
Leu Trp Thr His Lys Ser Val Gln Gly Ile Thr His Trp Gly Tyr Ile
260 265 270
Thr Gly Gln Thr Trp Asn Asp Gly Thr Tyr
275 280
SEQ ID NO:3
atggcaggga tcttccttgc tatcgtatca agccagtcag ctcttgcaac tggcacgagc 60
aagttgcttg gaaacatcat cccatcaagt gttccacgaa actgggatac atatcggaac 120
cagcctacag cagcaaatgg atgcaaatgg ggatgtgttc agaattcgca agcatcattc 180
attggagaga ttgtgactca gcattcagtc atgcaaacac agcagggatc acatttaagt 240
tccatccatc agtttggaga agccaagaac caggatggat tggatccttg tcaggcaatg 300
actcaacaac aggcagttgt tagttggatc gaagcagcag gaaaacatat tcaagcgcac 360
aaggagttga tgttgtcaat gaagcactac atgcaccagc aagctgcaga aactcacttg 420
ctggatcagg aagctcagga tgggattgga ttgtctggtc attcaagcag gcaaaacaat 480
tcacttccaa gttcaaaact cttgatcaat caatatggta ttatcgcaga tccaagtgaa 540
gctcgtcaat atgttgaaat tattgatatt ttgaagagca atagcttaac acatggtatc 600
ggaatccttg gccatcaact caatgtcggg acagtatcag catcaacagc tcaatcagtt 660
cttgacacac gtggagcgac aggtgtttca aaatatgttt cagaacttga tgcaaatgga 720
aacacagaac aagagcaaca agctatctat gagagaatgt tccgagttct ttggacacac 780
aaatcagtgc aaggaatcac acattggggc tatatcacag gccagacatg gaacgatgga 840
acctattga 849
SEQ ID NO:4
Met Ala Gly Ile Phe Leu Ala Ile Val Ser Ser Gln Ser Ala Leu Ala
1 5 10 15
Thr Gly Thr Ser Lys Leu Leu Gly Asn Ile Ile Pro Ser Ser Val Pro
20 25 30
Arg Asn Trp Asp Thr Tyr Arg Asn Gln Pro Thr Ala Ala Asn Gly Cys
35 40 45
Lys Trp Gly Cys Val Gln Asn Ser Gln Ala Ser Phe Ile Gly Glu Ile
50 55 60
Val Thr Gln His Ser Val Met Gln Thr Gln Gln Gly Ser His Leu Ser
65 70 75 80
Ser Ile His Gln Phe Gly Glu Ala Lys Asn Gln Asp Gly Leu Asp Pro
85 90 95
Cys Gln Ala Met Thr Gln Gln Gln Ala Val Val Ser Trp Ile Glu Ala
100 105 110
Ala Gly Lys His Ile Gln Ala His Lys Glu Leu Met Leu Ser Met Lys
115 120 125
His Tyr Met His Gln Gln Ala Ala Glu Thr His Leu Leu Asp Gln Glu
130 135 140
Ala Gln Asp Gly Ile Gly Leu Ser Gly His Ser Ser Arg Gln Asn Asn
145 150 155 160
Ser Leu Pro Ser Ser Lys Leu Leu Ile Asn Gln Tyr Gly Ile Ile Ala
165 170 175
Asp Pro Ser Glu Ala Arg Gln Tyr Val Glu Ile Ile Asp Ile Leu Lys
180 185 190
Ser Asn Ser Leu Thr His Gly Ile Gly Ile Leu Gly His Gln Leu Asn
195 200 205
Val Gly Thr Val Ser Ala Ser Thr Ala Gln Ser Val Leu Asp Thr Arg
210 215 220
Gly Ala Thr Gly Val Ser Lys Tyr Val Ser Glu Leu Asp Ala Asn Gly
225 230 235 240
Asn Thr Glu Gln Glu Gln Gln Ala Ile Tyr Glu Arg Met Phe Arg Val
245 250 255
Leu Trp Thr His Lys Ser Val Gln Gly Ile Thr His Trp Gly Tyr Ile
260 265 270
Thr Gly Gln Thr Trp Asn Asp Gly Thr Tyr
275 280

Claims (7)

  1. A kind of 1. alkalescent xylanase mutant, it is characterised in that：The amino acid sequence of the mutant is SEQ ID NO:4.
  2. A kind of 2. gene, it is characterised in that：The gene is the base of alkalescent xylanase mutant described in coding claim 1 Cause.
  3. 3. gene according to claim 2, it is characterised in that：The nucleotides sequence of the gene is classified as SEQ ID NO:3.
  4. A kind of 4. recombinant vector, it is characterised in that：The recombinant vector includes the gene described in Claims 2 or 3.
  5. A kind of 5. host cell, it is characterised in that：Include the recombinant vector described in claim 4.
  6. 6. alkalescent xylanase mutant according to claim 1 application in degradation of fibers cellulosic material in the basic conditions.
  7. 7. application according to claim 6, it is characterised in that：The cellulosic material is xylan, the alkalescence condition PH be 9-11.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103045624A (en) * 2012-11-20 2013-04-17 天津科技大学 High-temperature resistant and high-alkali resistant xylanase as well as gene, engineering bacterium and preparation method of xylanase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103045624A (en) * 2012-11-20 2013-04-17 天津科技大学 High-temperature resistant and high-alkali resistant xylanase as well as gene, engineering bacterium and preparation method of xylanase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Directed evolution of Streptomyces lividans xylanase B toward enhanced thermal and alkaline pH stability";Tao Xia 等;《World Journal of Microbiology and Biotechnology》;20090131;第25卷(第1期);第93-100页 *
"Expression of an alkalo-tolerant fungal xylanase enhanced by directed evolution in Pichia pastoris and Escherichia coli";Nokuthula Peace Mchunu 等;《Journal of Biotechnology》;20090420;第141卷(第1-2期);摘要 *

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