CN102994478B - 1,4-beta-D-xylanase mutant - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a 1,4-beta-D-xylanase mutant. The genes of xylanase are mutated by using an error-prone PCR (Polymerase Chain Reaction) method and a DNA (Desoxvribose Nucleic Acid) shuffling technology; positive mutations are detected by a high-throughput screening method; parts of potential site points related to thermal stability are determined according to a half-rational design method based on a sequence alignment; and finally, the mutants related to the thermal stability are obtained by using a fixed point mutation method. Based on the building and the screening of a mutation library and the half-rational design method, the five mutants with obviously-improved thermal stability are obtained. Compared with nature mutants, the thermal inactivated half-life period of the mutants provided by the invention is improved by 2 to 52 times. Therefore, the 1,4-beta-D-xylanase mutant has potential application values in the industrial fields of preparation of paper pulp for paper marking, biological energy sources and the like.

Description

A kind of Isosorbide-5-Nitrae-β-D-xylanase mutant

Technical field

The invention belongs to genetically engineered and enzyme engineering field, particular content relates to the Isosorbide-5-Nitrae-β-D-xylanase mutant that thermotolerance improves.

Background technology

Zytase (endo-1,4-β-xylanases, EC 3.2.1.8) is with the β-1 in internal-cutting way hydrolyzed xylan molecule, 4-glycosidic link, generate xylo-oligosaccharide and wood sugar, be one of lytic enzyme of most critical in hydrolysis of hemicellulose enzyme system, industrially there is important using value.Since from the eighties in last century, zytase starts industrial application, the Application Areas of zytase constantly expands, and is used widely in the industries such as feed, pulping and paper-making, food, the energy at present.Along with the range of application of zytase is expanded further, industrial its existing performance to be had higher requirement, in long-time, keep activity stabilized, in extreme environment, keep high activity (extreme temperature or pH value etc.) or different substrates (comprising non-natural substrates) can be accepted.Wherein the thermostability of enzyme is for very important industrial application, and under hot conditions, the speed of response of enzyme is faster, can shorten reaction time, cost-saving, is also conducive to avoiding in reaction process by other microbial contamination.

Along with protein engineering and molecular biological development, the means of orthogenesis and design and rational are used to carry out artificial evolution to enzyme molecule and transform the focus having become current enzyme engineering area research.Up to the present, existing many scholars various enzyme that used this technology successfully to transform, achieves the progress (Zhao, 2007, Biotechnogy and Bioengineering 98 (2), 271-275) attracted people's attention.Wherein fallibility PCR (error-prone PCR), DNA reorganize the means that (DNA shuffling), half design and rational etc. have become conventional in the molecular modification of enzyme, greatly accelerate evolutionary process (the Lehmann and Wyss of protein, 2001 Current Opinion in Biotechnology 12 (4), 371-375).

Summary of the invention

The object of the invention is to adopt directed evolution technologies to combine half Rational design method based on sequence alignment to deriving from 1 of Geobacillus stearothermophilus simultaneously, 4-β-D-zytase XT6 carries out molecular modification, obtains the xylanase mutant that thermostability improves.

For achieving the above object, the present invention uses many wheel fallibility PCR method, DNA shuffling technology to 1,4-β-D-zytase XT6 gene suddenlys change, by high-throughput screening method, gain mutant is detected again, use the potential thermally-stabilised related locus of half Rational design method determining section simultaneously, then obtain thermally-stabilised related mutants by directed mutagenesis method.

Specific embodiment of the invention method is: deriving from G.stearothermophilus(Genbank accession number is: zytase XT6 Z29080) forms (Lapidot by 379 amino acid, Mechaly et al., 1996 Journal of Biotechnogy 51 (3), 259-264) (see SEQ IDNO.1).For obtaining rapidly XT6 complete genome sequence and improving its expression amount in intestinal bacteria, be beneficial to the transformation to XT6 enzyme molecule, according to the XT6 gene sequence information (Gat reported, Lapidot et al., 1994 Applied and Environmental Microbiology 60 (6), 1889-1896), the full genome synthetic method of PCR-based is adopted to synthesize zytase XT6 complete genome sequence.According to e. coli codon Preference, (online software DNAWorks is optimized to its DNA sequence dna simultaneously, http://helixweb.nih.gov/dnaworks/, optimum synthesis zytase XT6 gene order be SEQ ID NO.2), normally functioning zymoprotein is overexpression Zhang in e. coli bl21-DE3, Peiet al., 2009 Chinese Journal of Applied and Environmental Biology 15 (2), 271-275).Employing fallibility PCR method, DNA shuffling technology carry out random mutation to it, with the efficient mutation library of pET 28a (+) vector construction, again the plasmid containing mutator gene is proceeded in expressive host E.coli BL21-DE3, select mono-clonal expressing protein in 96 orifice plates.Centrifugal resuspended after, get part bacterium liquid add 1% concentration xylan substrate reaction appropriate time after, with DNS method termination reaction, measure enzymic activity.Meanwhile, separately get part bacterium liquid thermal treatment certain hour, measure residual activity.Residual activity is transferred in 96 new well culture plates higher than the bacterial strain of contrast, carries out repeating screening.Residual activity screening obtained, higher than the bacterial strain of contrast, serves the order-checking of Hai Ying fine horse Bioisystech Co., Ltd, obtains mutant DNA sequence dna information.Detailed protocol is shown in embodiment 2.

Adopt half Rational design method based on sequence alignment to obtain heat stable mutants ((Lehmann, Loch et al., 2002 Proteinengineering 15 (5), 403-411) simultaneously.Specific practice is, first 17 xylanase amino acid sequence of XT6 aminoacid sequence and other different sourcess are compared, tentatively define the site that may affect enzyme stability, the single-point mutants in each site is obtained by rite-directed mutagenesis, measure its heat inactivation transformation period, and compare with wild-type, obtain the mutant F360L of enzyme heat stability.Detailed protocol is shown in embodiment 6.

Screen and half Rational design method through the above-mentioned mutated library to building, obtain the mutant that 20 thermostabilitys improve, be respectively 10E11,05D03,09D05,08A07,05F03,04D08,04F06,04H09,08G01,06B01,04D03,03B11,04H12,02E04,06H07, F360L, FAM, FAMG, FTAMG and FC06T, sequence information is shown in SEQ ID NO.3-NO.22, and its feature is as follows:

10E11: the phenylalanine of the 53rd of this enzyme sports tyrosine (DNA sequence dna becomes TAT from TTT).

05D03: the alanine mutation of the 257th of this enzyme is α-amino-isovaleric acid (GCA becomes GAT), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA).

09D05: the Threonine of the 121st of this enzyme sports Isoleucine (ACC becomes ATC), the glycine mutation of the 364th is aspartic acid (GGC becomes GAC).

08A07: the methionine(Met) of the 271st of this enzyme sports Isoleucine (ATG becomes ATA).

05F03: the alanine mutation of 257 of this enzyme is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA), the glycine mutation of the 364th is aspartic acid (GGC becomes GAC).

04D08: the phenylalanine of the 53rd of this enzyme sports tyrosine (TTT becomes TAT), the lysine mutation of the 73rd is Isoleucine (AAA becomes ATA), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA).

04F06: the phenylalanine of the 53rd of this enzyme sports tyrosine (TTT becomes TAT), the glycine mutation of the 364th is aspartic acid (GGC becomes GAC), the lysine mutation of the 380th is Threonine (AAA becomes ACA).

04H09: the phenylalanine of the 53rd of this enzyme sports tyrosine (TTT becomes TAT), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA), the lysine mutation of the 380th is Isoleucine (AAA becomes ATA).

08G01: the phenylalanine of the 53rd of this enzyme sports tyrosine (TTT becomes TAT), the glutamic acid mutation of the 113rd is aspartic acid (GAA becomes GAT), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA).

06B01: the Threonine of the 213rd of this enzyme sports Isoleucine (ACT becomes ATT), the alanine mutation of the 257th is leucine (GCA becomes TTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA), the glycine mutation of the 364th is aspartic acid (GGC becomes GAC).

04D03: the lysine mutation of the 26th of this enzyme is Isoleucine (AAA becomes ATA), the phenylalanine of the 53rd sports tyrosine (TTT becomes TAT), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA), the glycine mutation of the 364th is aspartic acid (GGC becomes GAC).

03B11: the glutamic acid mutation of the 138th of this enzyme is aspartic acid (GAA becomes GAC), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine, (ATG becomes ATA), the glycine mutation of the 364th are aspartic acid (GGC becomes GAC).

04H12: the phenylalanine of the 53rd of this enzyme sports tyrosine (TTT becomes TAT), the proline(Pro) of the 116th sports Threonine (CCG becomes ACG), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA).

02E04: the asparagine mutation of the 17th of this enzyme is Methionin (AAC becomes AAA), the phenylalanine of the 53rd sports tyrosine (TTT becomes TAT), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA).

06H07: the phenylalanine of the 53rd of this enzyme sports tyrosine (TTT becomes TAT), the lysine mutation of the 73rd is Isoleucine (AAA becomes ATA), the Threonine of the 121st sports Isoleucine (ACC becomes ATC), the valine mutation of the 219th is L-Ala (GAT becomes GCT), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA), the glycine mutation of the 364th is aspartic acid (GGC becomes GAC), the lysine mutation of the 380th is l-asparagine (AAA becomes AAC).

F360L: the phenylalanine of the 361st of this enzyme sports leucine (TTT becomes TTA).

FAM: the phenylalanine of the 53rd of this enzyme sports tyrosine (TTT becomes TAT), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA).

FAMG: the phenylalanine of the 53rd of this enzyme sports tyrosine (TTT becomes TAT), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA), the glycine mutation of the 364th is aspartic acid (GGC becomes GAC).

FTAMG: the phenylalanine of the 53rd of this enzyme sports tyrosine (TTT becomes TAT), the Threonine of the 121st sports Isoleucine (ACC becomes ATC), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA), the glycine mutation of the 364th is aspartic acid (GGC becomes GAC).

FC06T: the asparagine mutation of the 17th of this enzyme is Methionin (AAC becomes AAA), the lysine mutation of the 26th is Isoleucine (AAA becomes ATA), the phenylalanine of the 53rd sports tyrosine (TTT becomes TAT), the lysine mutation of the 73rd is Isoleucine (AAA becomes ATA), the glutamic acid mutation of the 113rd is aspartic acid (GAA becomes GAT), the Threonine of the 121st sports Isoleucine (ACC becomes ATC), the glutamic acid mutation of the 138th is aspartic acid (GAA becomes GAC), the valine mutation of the 219th is L-Ala (GAT becomes GCT), the alanine mutation of the 257th is α-amino-isovaleric acid (GCA becomes GTA), the methionine(Met) of the 271st sports Isoleucine (ATG becomes ATA), the phenylalanine of the 361st sports leucine (TTT becomes TTA), the glycine mutation of the 364th is aspartic acid (GGC becomes GAC), the lysine mutation of the 380th is l-asparagine (AAA becomes AAC).

The gene of said mutation body is connected into carrier pET 28a (+), and expresses in host cell E. coli Escherichiacoli BL21-DE3, obtain mutant zymoprotein.

Compare wild-type 1,4-β-D-zytase XT6,20 mutant thermostabilitys of the present invention significantly improve, improve 2.4,2.5,2.1,3.1,5.1,6.7,8,8.8,10.4,9.2,19.9,13.7,8.5,10.4,23,2.3,7.3,15,19 and 52 times respectively in the heat inactivation transformation period (t1/2) of 75 ° of C, demonstrate at paper-making pulping, the industrial potential using value such as bioenergy.

Accompanying drawing explanation

Fig. 1 is the mensuration of remnant enzyme activity under 7 the mutant differing tempss comprising all thermally-stabilised relevant mutational sites found.

Fig. 2 is that the speed of reaction of 7 mutant enzyme at different temperatures comprising all thermally-stabilised relevant mutational sites found measures.

SEQ ID NO.1 for deriving from Geobacillus stearothermophilus(Genebank accession number is: zytase XT6 aminoacid sequence Z29080), and SEQ ID NO.2 is the xylanase gene XT6 nucleotide sequence that the present invention optimizes.SEQ ID NO.3 is the aminoacid sequence of mutant 10E11.SEQ ID NO.4 is the aminoacid sequence of mutant 05D03.

SEQ ID NO.5 is the aminoacid sequence of mutant 09D05.SEQ ID NO.6 is the aminoacid sequence of mutant 08A07.SEQ IDNO.7 is the aminoacid sequence of 05F03.SEQ ID NO.8 is the aminoacid sequence of 04D08.SEQ ID NO.9 is the aminoacid sequence of 04F06.SEQ ID NO.10 is the aminoacid sequence of 04H09.SEQ ID NO.11 is the aminoacid sequence of 08G01.SEQ ID NO.12 is the aminoacid sequence of 06B01.SEQ ID NO.13 is the aminoacid sequence of 04D03.SEQ ID NO.14 is the aminoacid sequence of 03B11.SEQ IDNO.15 is the aminoacid sequence of 04H12.SEQ ID NO.16 is the aminoacid sequence of 02E04.SEQ ID NO.17 is the aminoacid sequence of 06H07.SEQ ID NO.18 is the aminoacid sequence of F360L.SEQ ID NO.19 is the aminoacid sequence of FAM.SEQ ID NO.20 is the aminoacid sequence of FAMG.SEQ ID NO.21 is the aminoacid sequence of FTAMG.SEQ ID NO.22 is the aminoacid sequence of FC06T.

Embodiment

Below in conjunction with embodiment, the present invention will be further described, it is pointed out that the present embodiment only for explaining the present invention, but not limitation of the scope of the invention.

Embodiment 1 fallibility PCR (error – prone PCR) method builds zytase XT6 mutated library

Adopt with the XT6 (see SEQ ID NO.2) after optimization for template amplification Isosorbide-5-Nitrae-β-D-zytase XT6 gene, introduce sudden change at random.

The primer is: 5 '-TAGGAGGTCATATGAAAAATGCGGACAGCTATGCG-3 ',

5′-ATACGCGGATCCCTATTTGTGATCAATGATCGCCCAATACGCCGGCTT-3′

Reaction conditions is: 94 ° of C denaturation 10min, 94 ° of C sex change 30s, and 60 ° of C annealing 60s and 72 ° of C extends 2min, totally 25 circulations, 0.8% agarose electrophoresis, and test kit reclaims goal gene fragment.

According to the method that NEB company 2007-2008 products catalogue specification sheets describes, after double digested with Nde I and BamH III, ligation is carried out with pET 28a (+) carrier (kalamycin resistance gene) cut through same enzyme, reaction conditions is: carrier and fragment in molar ratio 1:3 ratio mixing, add the T4 ligase enzyme of 400 units, 16 ° of C spend the night.Electric shocking method proceeds to intestinal bacteria DH10B, obtains the mutant library more than 105 clones.

The screening of embodiment 2 zytase XT6 mutant library

Extract plasmid by embodiment 1 after mutation library clone collection, proceed to E. coli expression strains BL21-DE3, the LB of coating containing kantlex is dull and stereotyped, cultivates 12h.Picking mono-clonal is in 96 orifice plates, and 150 μ L TB substratum (receiving mycin containing 50 μ g/mL cards, 1mMIPTG) are contained in every hole, and 37 ° of C, 245rpm, 36h is cultivated in concussion.It is dull and stereotyped in LB solid medium that 96 orifice plate reproducers copy each mono-clonal, after 37 ° of C cultivate 12h, and 4 ° of C Refrigerator stores.With the cell culture in the volley of rifle fire gently each hole of sucking-off 96 orifice plate, be allocated in 96 orifice plates of A plate and B plate by corresponding position, the culture in each hole of every block 96 orifice plate is 70 μ L.4000rpm, 4 ° of C, centrifugal 10min, supernatant discarded, somatic cells 30 μ L in each hole, the sodium phosphate buffer of 50mM, pH 7.6 is resuspended.A plate directly adds 50 μ L, 1% xylan substrate of the sodium phosphate buffer preparation of 50mM, pH 7.6,37 ° of C, 245rpm, and reaction 1.5h, adds 120 μ LDNS solution, and fully mixing is placed on baking oven, colour developing, and screening enzymic activity is higher than the mutant of wild type control; B plate preservative film is sealed, and be placed in 85 ° of C baking ovens, be placed on rapidly-20 ° of C after 2h process, temperature is down to room temperature fast.Add 50 μ L 50mM, the 1%xylan substrate of the sodium phosphate buffer preparation of pH 7.6,37 ° of C, 245rpm, reaction 3h, adds 120 μ L DNS solution, heating colour developing.Measure mutant residual activity, get the residual activity bacterial strain higher than wild-type XT6 in 96 new well culture plates, carry out repeating screening.Screen 4 mutant, be respectively 10E11,05D03,09D05,08A07, residual activity is 2-3 times of wild type control, and picking mono-clonal serves the order-checking of Hai Ying fine horse biotech company respectively.

The preparation of embodiment 3 mutant and wild-type hot zymoprotein runic thing and the mensuration of thermostability

3.1 crude enzyme liquid preparation and enzyme activity determination methods

The mutant mono-clonal that picking screens is in 20mL TB(50mg/mL kantlex, 1.0mM IPTG), 37 ° of C cultivate and after inducing 36h, 6000rpm, centrifugal 10min collects thalline.Supernatant discarded, the resuspended thalline of 12mL sodium phosphate buffer (50mM, pH7.6), ultrasonic disruption cell, extracts zymoprotein crude extract.

Enzyme activity determination: in 10mL centrifuge tube, reaction system is: the crude enzyme liquid (diluting 50 times with phosphate buffered saline buffer) of 0.2mL dilution, 1.8mL substrate solution (in advance at 50 DEG C of temperature bath 30min), 50 DEG C of water-baths, 5min, then add 3mLDNS solution termination reaction, after mixing, boil 5min in boiling water bath, be cooled to room temperature immediately with cold water, 540nm place measures light absorption value.Three, each sample repetition (Bailey, Bielyet al., 1992 Journal of biotechnolgy 23 (3), 257-270).Enzyme activity unit defines: at pH 7.6, under 50 DEG C of conditions, and per minute produces 1mmol reducing sugar or the enzyme amount required for its equivalent is a unit of activity (U).

The mensuration of 3.2 XT6 enzyme mutant thermostabilitys

The crude enzyme liquid 40mL of protein concentration 0.1mg/mL is placed in the PCR pipe of capacity 250mL, the repetition of 3, each sample, by PCR instrument in 75 ° of C process different time, then sample hose is positioned over cooled on ice, 4 DEG C, 12000rpm, centrifugal 25min, the enzyme liquid got after appropriate process measures residual activity, and measuring method is shown in 3.1.With the enzyme liquid without pyroprocessing for reference, obtain remnant enzyme activity per-cent.Take treatment time as X-axis, the natural logarithm of remnant enzyme activity per-cent is Y-axis, scatter diagram is done with origin75 software, add Trendline, obtain the linear equation of enzyme heat treatment time and residual activity, natural logarithm with remaining 50% is 3.912023 to calculate the corresponding time, and this time is the heat inactivation transformation period t1/2 of mutant.Each mutant is at pH 7.6, and the heat inactivation transformation period under 75 ° of C conditions is in table 1.Mutant 10E11,05D03,09D05 and 08A07 are respectively 2.4,2.5,2.1 and 3.1 times of wild-type in the heat inactivation transformation period of 75 ° of C.

The crude enzyme liquid 300mL of protein concentration 0.1mg/ml is placed in 1.5mL centrifuge tube, and the repetition of 3, each sample, at pH 7.6, processes 20min under differing temps, rapid placement cooled on ice, 4 ° of C, 12000rpm, centrifugal 25min, gets appropriate enzyme liquid and measures residual activity, with reference to case study on implementation 3.1.With the enzyme liquid without pyroprocessing for reference, obtain remnant enzyme activity per-cent, the results are shown in Figure of description 2, compare with wild-type, mutant 10E11,05D03, the thermostability of 09D05 and 08A07 is significantly increased, and wild-type xylanase, after 72 ° of C process 20min, is only left the activity of 10%, and 4 mutant under the same conditions, the residual activity of 50% still can be kept.

3.3 temperature are on the impact of enzyme mutant speed of reaction

Under pH 7.5 condition, measure zytase wild-type and saltant type maximum speed of reaction at different temperatures, the concentration of enzyme is 0.1mg/mL, and temperature of reaction is respectively 60 ° of C, 65 ° of C, 70 ° of C, 75 ° of C, 80 ° of C, 85 ° of C, 90 ° of C, 95 ° of C.Measuring method is with 3.1.With activity during maximum speed of reaction for 100%, it is ordinate zou that the enzyme measured at other temperature is lived with its ratio per-cent, and temperature of reaction is X-coordinate, draws the curve that enzyme work changes with temperature of reaction.The results are shown in Figure of description 2.Mutant 10E11,05D03,09D05 and 08A07 have maximum speed of reaction at 82 ° of C, improve 5 ° of C than wild-type.

Embodiment 4 second takes turns structure and the screening of fallibility PCR mutated library

The structure of 4.1 mutated library

Three mutant 10E11,05D03,09D05 extraction plasmids works second screened divided by first round fallibility PCR are taken turns outside the template of fallibility PCR, and in the building process in random mutant storehouse, the selection of primer, PCR reaction conditions, the structure in library is with embodiment 1.Result obtains the random mutant storehouse more than 105 clones.

The screening of 4.2 mutated library

E. coli expression strains BL21-DE3 is proceeded to by building the mutant obtained in step 4.1, screening activity/thermally-stabilised gain mutant time with 10E11,05D03,09D05 for reference, heat treatment time is 150min, all the other operations are with embodiment 2, get activity/residual activity than saltant type 10E11,05D03,09D05, high bacterial strain in 96 new well culture plates, carry out repeating screening.Screen the enzyme mutant that 10 thermostabilitys improve, be respectively 05F03,04D08,04F06,04H09,08G01,06B01,04D03,03B11,04H12,02E04.The thermally-stabilised mutant of picking send and checks order in Shanghai Ying Jun Bioisystech Co., Ltd.

The mensuration of 4.3 mutant thermostabilitys

Measuring method is with embodiment 3.2, result (table 1) shows, mutant 05F03,04D08,04F06,04H09,08G01,06B01,04D03,03B11,04H12,02E04 are 5.1,6.7,8,8.8,10.4,9.2,19.9,13.7,8.5 and 10.4 times of wild-type in the heat inactivation transformation period of 75 ° of C.

The structure in embodiment 5XT6 mutant gene DNA shuffling mutations storehouse and screening

The structure in 5.1DNA shuffling mutations storehouse

With zytase XT6 mutant 10E11,05D03,09D05,08A07,05F03,04D08,04F06,04H09,08G01,06B01,04D03,03B11,04H12,02E04 of screening for template, pair of primers:

5′-GTGAGCGGATAACAATTCCC-3′,

5′-CCTCAAGACCCGTTTAGAGG-3′

Increase each mutant gene respectively, and reaction conditions is: 94 ° of C denaturation 10min, 94 ° of C sex change 30s, and 60 ° of C annealing 30s and 72 ° of C extends 2min, totally 25 circulations, 0.8% agarose electrophoresis, and test kit reclaims purifying goal gene.According to the method for Stemmer, the DNA goal gene fragment process appropriate time that DNase I will obtain, reclaim the small segment (Stemmer of 50 ~ 150bp, 1994 Proceedings of theNational Academy of Science of the United States of America 91 (22), 10747-10751).Pass through without primer PCR and have primer PCR, the primer is:

5′-TAGGAGGTCATATGAAAAATGCGGACAGCTATGCG-3′,

5′-ATACGCGGATCCCTATTTGTGATCAATGATCGCCCAATACGCCGGCTT-3′

Obtain the total length goal gene after recombinating.The construction process in DNA shuffling mutations library, with embodiment 1, obtains the mutant library more than 105 clones.

The screening in 5.2 DNA shuffling mutations storehouses

Screening gain mutant takes turns the highest mutant 04D03 of the thermostability that obtains in fallibility PCR for reference with second, heat treatment time is 240min, all the other operations, with embodiment 2, get the residual activity bacterial strain higher than saltant type 04D03 in 96 new well culture plates, carry out repeating screening.Screen mutant 06H07.Send and check order in Shanghai Ying Jun Bioisystech Co., Ltd.

The mensuration of 5.3 mutant thermostabilitys

Measuring method is with embodiment 3.2, and result (table 1) shows, mutant 06H07 thermostability is compared with wild-type and improved a lot, and the heat inactivation transformation period under 75 ° of C conditions is 23 times of wild-type.

Embodiment 6 is based on half design and rational of sequence alignment

Xylanase amino acid sequence closer for F10 family 17 sibships known in zytase XT6 aminoacid sequence and protein library (SwissPort) is compared (table 2).In the xylanase amino acid sequence of these 17 different sourcess, 9 zytases derive from thermophile bacteria, and 8 from mesophilic bacteria.Their aminoacid sequence is compared with zytase XT6, and amino acid similarity degree is between 40%-75%.Sequence alignment software used is Clustal X.

Comparison result finds, Phe95, Ile152, Tyr200 in zytase XT6 aminoacid sequence, the amino acid that the amino acid of Asn262, Phe361 and Ile376 position occupies the majority in its homology xylanase amino acid sequence corresponding position is respectively Gly, Val, Phe, Ile, Leu and Val.Directed mutagenesis method changes these sites, the primer:

XYL94-F：5′-GGCATGGATATTCGTGGTCACACCCTGGTGTGG-3′

XYL94-R：5′-CCACACCAGGGTGTGACCACGAATATCCATGCC-3′

XYL151-F：5′-GAACGTTATAAGGATGATGTAAAGTACTGGGATGTAG-3′

XYL151-R：5′-CTACATCCCAGTACTTTACATCATCCTTATAACGTTC-3′

XYL199-F：5′-GGCGATAACATTAAGTTATTCATGAATGACTACAATACCG-3′

XYL199-R：5′-CGGTATTGTAGTCATTCATGAATAACTTAATGTTATCGCC-3′

XYL261-F：5′-GCACTGGGCCTGGATATCCAAATTACCGAACTGG-3′

XYL261-R：5′-CCAGTTCGGTAATTTGGATATCCAGGCCCAGTGC-3′

360-F：5′-GCAAAGATGCGCCGCTTGTGTTTGGCCCGG-3′

360-R：5′-CCGGGCCAAACACAAGCGGCGCATCTTTGC-3′

XYL375-F：5′-CCGGCGTATTGGGCGGTCATTGATCACAAATAG-3′

XYL375-R：5′-CTATTTGTGATCAATGACCGCCCAATACGCCGG-3′

PCR condition is: 10 × Buffer 5mL, primer (10mM) each 6mL, dNTP(2.5mM) 6mL, pfu(2.5U/mL) 1mL, plasmid 10ng, ultrapure water supplies 50mL, condition: 95 ° of C denaturation 30s, 95 ° of C sex change 30s, 55 ° of C annealing 1min, 68 ° of C extend 7min, totally 12 circulations.PCR primer 1mL DpnI process, 37 ° of C process 1h.PCR primer 10mL chemical method proceeds to E.coli DH5a.Send and check order in Shanghai Ying Jun Bioisystech Co., Ltd.After order-checking is correct, extracts plasmid and proceed to expression strain E.coliBL21-DE3.Extract mutant zymoprotein, measure its thermostability under 75 ° of C.Measuring method is with embodiment 3.2.Result obtains the mutant F360L that thermostability improves, and being 10.5min in the heat inactivation transformation period of 75 ° of C, is 2.3 times (table 3) of wild-type.

The combination of embodiment 7 mutational site builds XT6 new mutant

The structure of 7.1 mutant FAM

The phenylalanine of 53 of mutant 05D03 is sported tyrosine by directed mutagenesis method, and build mutant FAM, primer used is as follows:

52-F:5′-GATGCTGAAGCGTCATTATAACTCAATTGTGGCGG-3′，

52-R:5′-CCGCCACAATTGAGTTATAATGACGCTTCAGCATC-3′

PCR condition and operation, with embodiment 6, obtain new mutant FAM.

The structure of 7.2 mutant FAMG

The glycine mutation of 364 of mutant FAM is aspartic acid by fixed-point mutation method, builds mutant FAMG.Primer used is as follows:

363-F:5′-GCGCCGTTTGTGTTTGACCCGGATTACAAAGTGAAGC-3′，

363-R:5′-GCTTCACTTTGTAATCCGGGTCAAACACAAACGGCGC-3′

PCR condition and subsequent operations, with embodiment 6, obtain mutant FAMG.

The structure of 7.3 mutant FTAMG

The Threonine of 121 of mutant FAMG is sported Isoleucine by fixed-point mutation method, and build mutant FTAMG, primer used is as follows:

120-F:5′-CCGATGGTGAACGAGATCGATCCGGTGAAACGTG-3′，

120-R:5′-CACGTTTCACCGGATCGATCTCGTTCACCATCGG-3′

PCR condition and subsequent operations, with embodiment 6, obtain mutant FTAMG.

The structure of 7.4 mutant FC06T

The asparagine mutation of 17 of mutant 06H07 is Methionin by fixed-point mutation method, the lysine mutation of 26 is Isoleucine, the proline(Pro) of 113 sports Threonine, the glutamic acid mutation of 138 is aspartic acid, the phenylalanine of 361 sports leucine, build mutant FC06T, primer used is as follows:

16-F：5′-GCATATTAGCGCCCTGAAAGCGCCACAGCTGG-3′

16-R：5′-CCAGCTGTGGCGCTTTCAGGGCGCTAATATGC-3′

25-F：5′-CAGCTGGACCAACGGTACATAAATGAATTTACTATTGGTGCG-3′

25-R：5′-CGCACCAATAGTAAATTCATTTATGTACCGTTGGTCCAGCTG-3′

112-F：5′-GGTTCTTCCTTGACAAAGATGGAAAACCGATGGTG-3′

112-R：5′-CACCATCGGTTTTCCATCTTTGTCAAGGAAGAACC-3′

137-F：5′-CTGCTGAAACGCTTGGACACCCACATCAAAACC-3′

137-R：5′-GGTTTTGATGTGGGTGTCCAAGCGTTTCAGCAG-3′

360-F：5′-GCAAAGATGCGCCGCTTGTGTTTGGCCCGG-3′

360-R：5′-CCGGGCCAAACACAAGCGGCGCATCTTTGC-3′

PCR condition and subsequent operations, with embodiment 6, obtain mutant FC06T.

7.5XT6 mutant thermal stability determination

Xylanase mutant FAM, FAMG, FTAMG, FC06T thermal stability determination method is with 3.2, and measurement result shows: FAM, FAMG, FTAMG, the FC06T heat inactivation transformation period under 75 ° of C conditions is 7.3,14.8,19.4 and 52 times (table 1) of wild-type respectively; FTAMG, FC06T measures residual activity after 20min thermal treatment, during 78 ° of C, wild-type residual activity is lower than 1%, and mutant FTAMG still can keep 20% activity, FC06T still can keep the activity of 50%, when temperature reaches 80 ° of C, the residual activity of FTAMG is lower than 5%, and the residual activity of mutant FC06T is 30%.Temperature measures with 3.3 the impact of enzyme mutant speed of reaction, and mutant FTAMG, FC06T have maximum speed of reaction at 87 ° of C, improve 10 ° of C than wild-type.The results are shown in Figure of description 2.

Table 1 mutant is 75 ° of C heat inactivation transformation period

The F10 family zytase feature of table 2 different sources

The table 3 single-point mutants heat inactivation transformation period

SEQ?ID?NO.1

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHFNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAALGLDNQITELDVSMYGWPPRA

YPTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNA

PYAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.2：

ATGAAAAATGCGGACAGCTATGCGAAAAAACCGCATATTAGCGCCCTGAACGCGCCACAGCTGGACC

AACGGTACAAAAATGAATTTACTATTGGTGCGGCGGTGGAACCCTATCAGCTGCAGAATGAGAAAGAT

GTGCAGATGCTGAAGCGTCATTTTAACTCAATTGTGGCGGAAAATGTGATGAAGCCTATTAGCATTCA

GCCGGAAGAAGGGAAATTCAATTTCGAACAGGCGGATCGTATTGTGAAATTTGCGAAAGCGAATGGC

ATGGATATTCGTTTTCACACCCTGGTGTGGCATAGCCAAGTGCCGCAGTGGTTCTTCCTTGACAAAGA

AGGAAAACCGATGGTGAACGAGACCGATCCGGTGAAACGTGAGCAGAATAAACAGTTGCTGCTGAA

ACGCTTGGAAACCCACATCAAAACCATTGTTGAACGTTATAAGGATGATATAAAGTACTGGGATGTAG

TGAATGAGGTAGTTGGCGATGATGGCAAACTGCGTAATAGCCCGTGGTATCAGATTGCGGGCATTGATT

ACATTAAAGTGGCGTTCCAGGCGGCACGTAAATATGGTGGCGATAACATTAAGTTATACATGAATGACT

ACAATACCGAAGTAGAACCGAAACGCACTGCGCTGTATAATCTGGTAAAACAGCTGAAAGAAGAGGG

CGTGCCGATTGACGGCATTGGGCATCAGAGCCACATTCAAATTGGCTGGCCGAGCGAAGCGGAAATA

GAAAAGACCATCAATATGTTTGCAGCACTGGGCCTGGATAACCAAATTACCGAACTGGATGTAAGCAT

GTACGGATGGCCACCGCGTGCGTATCCGACCTATGATGCGATTCCGAAACAGAAATTCCTGGATCAAG

CAGCTCGGTATGACCGGCTGTTCAAACTGTATGAAAAGCTGAGCGACAAAATTAGCAATGTGACCTTT

TGGGGAATTGCGGATAATCACACCTGGCTGGATAGCCGTGCGGATGTGTATTACGATGCCAATGGCAA

TGTTGTGGTCGATCCGAATGCGCCCTACGCGAAAGTGGAAAAAGGCAAGGGCAAAGATGCGCCGTTT

GTGTTTGGCCCGGATTACAAAGTGAAGCCGGCGTATTGGGCGATCATTGATCACAAATAG

SEQ?ID?NO.3：(10E11)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAALGLDNQITELDVSMYGWPPRA

YPTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNA

PYAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.4：(05D03)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHFNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.5：(09D05)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHFNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNEIDPVKREQNKQLLLKRLET

HIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAALGLDNQITELDVSMYGWPPRA

YPTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNA

PYAKVEKGKGKDAPFVFDPDYKVKPAYWAIIDHK

SEQ?ID?NO.6：(08A07)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHFNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAALGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.7：(05F03)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHFNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFDPDYKVKPAYWAIIDHK

SEQ?ID?NO.8：(04D08)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGIFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLET

HIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.9：(04F06)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAALGLDNQITELDVSMYGWPPRA

YPTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNA

PYAKVEKGKGKDAPFVFDPDYKVKPAYWAIIDHT

SEQ?ID?NO.10：(04H09)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHI

SEQ?ID?NO.11：(08G01)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKDGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.12：(06B01)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHFNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRIALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFALLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFDPDYKVKPAYWAIIDHK

SEQ?ID?NO.13：(04D03)

MKNADSYAKKPHISALNAPQLDQRYINEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFDPDYKVKPAYWAIIDHK

SEQ?ID?NO.14：(03B11)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHFNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLD

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFDPDYKVKPAYWAIIDHK

SEQ?ID?NO.15：(04H12)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKTMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.16：(02E04)

MKNADSYAKKPHISALKAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.17：(06H07)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGIFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNEIDPVKREQNKQLLLKRLET

HIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLAKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHN

SEQ?ID?NO.18：(F360L)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHFNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAALGLDNQITELDVSMYGWPPRA

YPTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNA

PYAKVEKGKGKDAPLVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.19：(FAM)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFGPDYKVKPAYWAIIDHK

SEQ?ID?NO.20：(FAMG)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNETDPVKREQNKQLLLKRLE

THIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFDPDYKVKPAYWAIIDHK

SEQ?ID?NO.21：(FTAMG)

MKNADSYAKKPHISALNAPQLDQRYKNEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGKFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKEGKPMVNEIDPVKREQNKQLLLKRLET

HIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLVKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPFVFDPDYKVKPAYWAIIDHK

SEQ?ID?NO.22：(FC06T)

MKNADSYAKKPHISALKAPQLDQRYINEFTIGAAVEPYQLQNEKDVQMLKRHYNSIVAENVMKPISIQPE

EGIFNFEQADRIVKFAKANGMDIRFHTLVWHSQVPQWFFLDKDGKPMVNEIDPVKREQNKQLLLKRLDT

HIKTIVERYKDDIKYWDVVNEVVGDDGKLRNSPWYQIAGIDYIKVAFQAARKYGGDNIKLYMNDYNTE

VEPKRTALYNLAKQLKEEGVPIDGIGHQSHIQIGWPSEAEIEKTINMFAVLGLDNQITELDVSIYGWPPRAY

PTYDAIPKQKFLDQAARYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRADVYYDANGNVVVDPNAP

YAKVEKGKGKDAPLVFDPDYKVKPAYWAIIDHN

Claims (6)

1. an Isosorbide-5-Nitrae-β-D-xylanase mutant for thermostability raising, is characterized in that: based on the Isosorbide-5-Nitrae-β-D-zytase XT6 aminoacid sequence shown in SEQ ID NO.1, its methionine(Met) of the 271st is sported Isoleucine.

2. Isosorbide-5-Nitrae-β-D-the xylanase mutant that improves of thermostability, is characterized in that: be α-amino-isovaleric acid by the alanine mutation of the 257th of mutant according to claim 1 the.

3. Isosorbide-5-Nitrae-β-D-the xylanase mutant that improves of thermostability, is characterized in that: the alanine mutation of the 257th of mutant according to claim 1 the is α-amino-isovaleric acid and the glycine mutation of the 364th is aspartic acid.

4. a thermostability improve 1,4-β-D-xylanase mutant, is characterized in that: the Threonine of the 213rd of mutant according to claim 1 the is sported Isoleucine, the alanine mutation of the 257th is leucine and the glycine mutation of 364 is aspartic acid.

5. a thermostability improve 1,4-β-D-xylanase mutant, is characterized in that: be aspartic acid by the glutamic acid mutation of the 138th of mutant according to claim 1 the, the alanine mutation of the 257th is α-amino-isovaleric acid and the glycine mutation of the 364th is aspartic acid.

6. 1 of a kind of thermostability raising as described in claim 1 or 2 or 3 or 4 or 5,4-β-D-xylanase mutant, it is characterized in that, the gene of mutant is connected into pET28a (+) carrier, and express in host cell E. coli Escherichia coli BL21-DE3, obtain mutant zymoprotein.