CN102994478A - 1,4-beta-D-xylanase mutant - Google Patents
1,4-beta-D-xylanase mutant Download PDFInfo
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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
Technical field
The invention belongs to genetically engineered and enzyme engineering field, particular content relates to Isosorbide-5-Nitrae-β that thermotolerance improves-D-xylanase mutant.
Background technology
Zytase (endo-1,4-β-xylanases, EC 3.2.1.8) is with the β-1 in the internal-cutting way hydrolyzed xylan molecule, the 4-glycosidic link, generating xylo-oligosaccharide and wood sugar, is one of lytic enzyme of most critical in the hydrolysis of hemicellulose enzyme system, has an important using value industrial.Since the eighties in last century, zytase began industrial application, the Application Areas of zytase constantly enlarged, and was 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 further expanded, industrial its existing performance is had higher requirement, as keeping activity stabilized in long-time, in extreme environment, keep high activity (extreme temperature or pH value etc.) or can accept different substrate (comprising non-natural substrates).Wherein Thermostability is very important for industrial application, and under the hot conditions, the speed of response of enzyme is faster, can shorten reaction time, saves cost, also is conducive to avoid in the reaction process by other microbial contamination.
Along with protein engineering and molecular biological development, use the means of orthogenesis and design and rational that the enzyme molecule is carried out the artificial evolution and transforms the focus that has become current enzyme engineering area research.Up to the present, existing many scholars use this technology success transformation various enzymes, obtained the progress that attracts people's attention (Zhao, 2007, Biotechnogy and Bioengineering 98 (2), 271-275).Wherein fallibility PCR (error-prone PCR), DNA reorganization (DNA shuffling), half design and rational etc. have become the means of commonly using in the molecular modification of enzyme, greatly accelerated 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 the orthogenesis technology simultaneously in conjunction with based on half Rational design method of sequence alignment to deriving from 1 of Geobacillus stearothermophilus, 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 simultaneously the potential thermally-stabilised related locus of half Rational design method determining section, obtain thermally-stabilised related mutants by the rite-directed mutagenesis method again.
Specific implementation method of the present invention is: deriving from the 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 the XT6 complete genome sequence and improving its Expression in Escherichia coli amount, be beneficial to the transformation to XT6 enzyme molecule, according to the XT6 gene order information (Gat that has reported, Lapidot et al., 1994 Applied and Environmental Microbiology 60 (6), 1889-1896), adopt the full method for synthesizing gene of PCR-based to synthesize zytase XT6 complete genome sequence.According to the e. coli codon Preference its dna sequence dna is optimized (online software DNAWorks simultaneously, http://helixweb.nih.gov/dnaworks/, optimizing synthetic zytase XT6 gene order is 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).Adopt fallibility PCR method, DNA shuffling technology that it is carried out random mutation, with the efficient mutation library of pET 28a (+) vector construction, the plasmid that will contain again mutator gene changes among the expressive host E.coli BL21-DE3, selects mono-clonal expressing protein in 96 orifice plates.Centrifugal resuspended after, after getting part bacterium liquid and adding 1% concentration xylan substrate reactions appropriate time, with DNS method termination reaction, measure enzymic activity.Simultaneously, other gets part bacterium liquid thermal treatment certain hour, measures residual activity.The bacterial strain that residual activity is higher than contrast is transferred in the 96 new well culture plates, repeats screening.The residual activity that screening is obtained is higher than the bacterial strain of contrast, serves the order-checking of extra large Ying Jun Bioisystech Co., Ltd, obtains mutant dna sequence dna information.Detailed protocol is seen embodiment 2.
Adopt simultaneously based on half Rational design method of sequence alignment obtain heat stable mutants ((Lehmann, Loch et al., 2002 Proteinengineering 15 (5), 403-411).Specific practice is, at first 17 zytase aminoacid sequences of XT6 aminoacid sequence and other different sourcess are compared, tentatively define the site that may affect enzyme stability, obtain the simple point mutation body in each site by rite-directed mutagenesis, measure its heat inactivation transformation period, and with wild-type relatively, obtain the mutant F360L of enzyme heat stability.Detailed protocol is seen embodiment 6.
Through above-mentioned sudden change library screening and half Rational design method to making up, 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 seen SEQ ID NO.3-NO.22, and its feature is as follows:
10E11: the 53rd phenylalanine of this enzyme sports tyrosine (dna sequence dna becomes TAT by TTT).
05D03: the 257th alanine mutation of this enzyme is that α-amino-isovaleric acid (GCA becomes GAT), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA).
09D05: the 121st Threonine of this enzyme sports Isoleucine (ACC becomes ATC), the 364th glycine mutation is aspartic acid (GGC becomes GAC).
08A07: the 271st methionine(Met) of this enzyme sports Isoleucine (ATG becomes ATA).
05F03: 257 alanine mutation of this enzyme is that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA), the 364th glycine mutation is aspartic acid (GGC becomes GAC).
04D08: it is that Isoleucine (AAA becomes ATA), the 257th alanine mutation are that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA) that the 53rd phenylalanine of this enzyme sports tyrosine (TTT becomes TAT), the 73rd lysine mutation.
04F06: it is that aspartic acid (GGC becomes GAC), the 380th lysine mutation are Threonine (AAA becomes ACA) that the 53rd phenylalanine of this enzyme sports tyrosine (TTT becomes TAT), the 364th glycine mutation.
04H09: it is that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA), the 380th lysine mutation is Isoleucine (AAA becomes ATA) that the 53rd phenylalanine of this enzyme sports tyrosine (TTT becomes TAT), the 257th alanine mutation.
08G01: the 53rd phenylalanine of this enzyme sports tyrosine (TTT becomes TAT), the 113rd L-glutamic acid, and to sport aspartic acid (GAA becomes GAT), the 257th alanine mutation be that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA).
06B01: it is that leucine (GCA becomes TTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA), the 364th glycine mutation is aspartic acid (GGC becomes GAC) that the 213rd Threonine of this enzyme sports Isoleucine (ACT becomes ATT), the 257th alanine mutation.
04D03: the 26th lysine mutation of this enzyme is that to sport tyrosine (TTT becomes TAT), the 257th alanine mutation be that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA), the 364th glycine mutation is aspartic acid (GGC becomes GAC) for Isoleucine (AAA becomes ATA), the 53rd phenylalanine.
03B11: it is that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine that the 138th L-glutamic acid of this enzyme sports aspartic acid (GAA becomes GAC), the 257th alanine mutation, and (ATG becomes ATA), the 364th glycine mutation are aspartic acid (GGC becomes GAC).
04H12: the 53rd phenylalanine of this enzyme sports tyrosine (TTT becomes TAT), the 116th proline(Pro), and to sport Threonine (CCG becomes ACG), the 257th alanine mutation be that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA).
02E04: the 17th asparagine mutation of this enzyme is that to sport tyrosine (TTT becomes TAT), the 257th alanine mutation be that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA) for Methionin (AAC becomes AAA), the 53rd phenylalanine.
06H07: the 53rd phenylalanine of this enzyme sports tyrosine (TTT becomes TAT), the 73rd lysine mutation is Isoleucine (AAA becomes ATA), the 121st Threonine sports Isoleucine (ACC becomes ATC), the 219th α-amino-isovaleric acid sports L-Ala (GAT becomes GCT), the 257th alanine mutation is α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sports Isoleucine (ATG becomes ATA), the 364th glycine mutation is aspartic acid (GGC becomes GAC), the 380th lysine mutation is l-asparagine (AAA becomes AAC).
F360L: the 361st phenylalanine of this enzyme sports leucine (TTT becomes TTA).
FAM: it is that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA) that the 53rd phenylalanine of this enzyme sports tyrosine (TTT becomes TAT), the 257th alanine mutation.
FAMG: it is that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA), the 364th glycine mutation is aspartic acid (GGC becomes GAC) that the 53rd phenylalanine of this enzyme sports tyrosine (TTT becomes TAT), the 257th alanine mutation.
FTAMG: the 53rd phenylalanine of this enzyme sports tyrosine (TTT becomes TAT), the 121st Threonine, and to sport Isoleucine (ACC becomes ATC), the 257th alanine mutation be that α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sport Isoleucine (ATG becomes ATA), the 364th glycine mutation is aspartic acid (GGC becomes GAC).
FC06T: the 17th asparagine mutation of this enzyme is Methionin (AAC becomes AAA), the 26th lysine mutation is Isoleucine (AAA becomes ATA), the 53rd phenylalanine sports tyrosine (TTT becomes TAT), the 73rd lysine mutation is Isoleucine (AAA becomes ATA), the 113rd L-glutamic acid sports aspartic acid (GAA becomes GAT), the 121st Threonine sports Isoleucine (ACC becomes ATC), the 138th L-glutamic acid sports aspartic acid (GAA becomes GAC), the 219th α-amino-isovaleric acid sports L-Ala (GAT becomes GCT), the 257th alanine mutation is α-amino-isovaleric acid (GCA becomes GTA), the 271st methionine(Met) sports Isoleucine (ATG becomes ATA), the 361st phenylalanine sports leucine (TTT becomes TTA), the 364th glycine mutation is aspartic acid (GGC becomes GAC), the 380th lysine mutation is l-asparagine (AAA becomes AAC).
The gene of said mutation body is connected into carrier pET 28a (+), and in host cell intestinal bacteria Escherichiacoli BL21-DE3, expresses, obtain mutant enzyme albumen.
Compare wild-type 1,4-β-D-zytase XT6,20 mutant thermostabilitys of the present invention obviously 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 have been improved respectively in the heat inactivation transformation period of 75 ° of C (t1/2), demonstrate at paper-making pulping the industrial potential using value such as bioenergy.
Description of drawings
Fig. 1 is the mensuration that comprises remnant enzyme activity under 7 mutant differing tempss of all thermally-stabilised relevant mutational sites of having found.
Fig. 2 is that the speed of reaction that comprises 7 mutant enzyme under differing temps of all thermally-stabilised relevant mutational sites of having found is measured.
SEQ ID NO.1 for deriving from Geobacillus stearothermophilus(Genebank accession number is: zytase XT6 aminoacid sequence Z29080), SEQ ID NO.2 are 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
The present invention will be further described below in conjunction with embodiment, it is pointed out that present embodiment only is used for explaining the present invention, but not limitation of the scope of the invention.
Embodiment 1 fallibility PCR (error – prone PCR) method makes up zytase XT6 sudden change library
Adopt
XT6 (seeing SEQ ID NO.2) after to optimize introduces at random and suddenlys change as template amplification Isosorbide-5-Nitrae-β-D-zytase XT6 gene.
The primer is: 5 '-TAGGAGGTCATATGAAAAATGCGGACAGCTATGCG-3 ',
5′-ATACGCGGATCCCTATTTGTGATCAATGATCGCCCAATACGCCGGCTT-3′
Reaction conditions is: 94 ° of C denaturation 10min, and 94 ° of C sex change 30s, 60 ° of C annealing 60s and 72 ° of C extend 2min, totally 25 circulations, 0.8% agarose electrophoresis, test kit reclaims the goal gene fragment.
Method according to the description of the 2007-2008 of NEB company products catalogue specification sheets, after Nde I and the digestion of BamH III double digestion, carry out ligation with pET 28a (+) carrier (kalamycin resistance gene) of cutting through same enzyme, reaction conditions is: carrier and the fragment in molar ratio ratio of 1:3 are mixed, add the T4 ligase enzyme of 400 units, 16 ° of C spend the night.Electric shocking method changes intestinal bacteria DH10B over to, obtains surpassing 105 clones' mutant library.
The screening of embodiment 2 zytase XT6 mutant libraries
Extract plasmid after the mutation library clone collects among the embodiment 1, change escherichia coli expression bacterial strain BL21-DE3 over to, it is dull and stereotyped to be coated with the LB that contains kantlex, cultivates 12h.The picking mono-clonal is in 96 orifice plates, every hole contain 150 μ L TB substratum (contain 50 μ g/mL cards and receive mycin, 1mMIPTG), 37 ° of C, 245rpm, 36h is cultivated in concussion.It is dull and stereotyped in the 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 volley of rifle fire cell culture in each hole of sucking-off 96 orifice plates gently, be allocated in by the corresponding position in 96 orifice plates of A plate and B plate, the culture in every each hole of 96 orifice plates is 70 μ L.4000rpm, 4 ° of C, centrifugal 10min, supernatant discarded, in each hole somatic cells with 30 μ L, 50mM, the sodium phosphate buffer of pH 7.6 is resuspended.The A plate directly adds 50 μ L, 50mM, and 1% xylan substrate of the sodium phosphate buffer preparation of pH 7.6,37 ° of C, 245rpm, reaction 1.5h adds 120 μ LDNS solution, and fully mixing is placed on baking oven, colour developing, the screening enzymic activity is higher than the mutant of wild-type contrast; The B plate is sealed with preservative film, places 85 ° of C baking ovens, and 2h is placed on rapidly-20 ° of C after processing, and 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, the heating colour developing.Measure the mutant residual activity, get residual activity than the high bacterial strain of wild-type XT6 in 96 new well culture plates, repeat screening.Screen 4 mutant, be respectively 10E11,05D03,09D05,08A07, residual activity be wild-type contrast 2-3 doubly, the picking mono-clonal is served the order-checking of extra large Ying Jun biotech company respectively.
The preparation of embodiment 3 mutant and the hot zymoprotein runic of wild-type thing and the mensuration of thermostability
3.1 crude enzyme liquid preparation and enzyme activity determination method
The mutant mono-clonal that picking screens is in 20mL TB(50mg/mL kantlex, 1.0mM IPTG), 37 ° of C cultivate and induce 36h after, 6000rpm, centrifugal 10min collects thalline.Supernatant discarded, the resuspended thalline of 12mL sodium phosphate buffer (50mM, pH7.6), the ultrasonic disruption cell extracts the zymoprotein crude extract.
Enzyme activity determination: in the 10mL centrifuge tube, reaction system is: the crude enzyme liquid of 0.2mL dilution (with 50 times of phosphate buffered saline buffer dilutions), 1.8mL substrate solution (bathing 30min 50 ℃ of temperature in advance), 50 ℃ of water-baths, 5min adds 3mLDNS solution termination reaction again, behind the mixing, boil 5min in the boiling water bath, be cooled to room temperature with cold water immediately, the 540nm place measures light absorption value.Three repetitions of each sample (Bailey, Bielyet al., 1992 Journal of biotechnolgy 23 (3), 257-270).The enzyme activity unit definition: under 7.6,50 ℃ of conditions of pH, per minute produces the 1mmol reducing sugar or the needed enzyme amount of its equivalent is a unit of activity (U).
3.2 the mensuration of XT6 enzyme mutant thermostability
The crude enzyme liquid 40mL of protein concentration 0.1mg/mL is placed the PCR pipe of capacity 250mL, 3 repetitions of each sample, process the different time with the PCR instrument at 75 ° of C, then sample hose is positioned over cooled on ice, 4 ℃, 12000rpm, centrifugal 25min, the enzyme liquid of getting after an amount of processing is measured residual activity, and measuring method sees 3.1.Take without the enzyme liquid of crossing pyroprocessing as reference, obtain remnant enzyme activity per-cent.Take the treatment time as X-axis, the natural logarithm of remnant enzyme activity per-cent is Y-axis, do scatter diagram with origin75 software, add Trendline, obtain the linear equation of enzyme heat treatment time and residual activity, natural logarithm take remaining 50% calculates the corresponding time as 3.912023, and this time is the heat inactivation transformation period t1/2 of mutant.The heat inactivation transformation period of each mutant under 7.6,75 ° of C conditions of pH sees 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 the 1.5mL centrifuge tube, and 3 repetitions of each sample at pH 7.6, are processed 20min under the differing temps, place rapidly cooled on ice, 4 ° of C, 12000rpm, centrifugal 25min gets an amount of enzyme liquid and measures residual activity, with reference to case study on implementation 3.1.Take without the enzyme liquid of crossing pyroprocessing as reference, obtain remnant enzyme activity per-cent, the results are shown in Figure of description 2, compare mutant 10E11 with wild-type, 05D03, the thermostability of 09D05 and 08A07 is significantly increased, and the wild-type zytase only is left 10% activity after 72 ° of C process 20min, and 4 mutant under the same conditions, still can keep 50% residual activity.
3.3 temperature is on the impact of enzyme mutant speed of reaction
Under pH 7.5 conditions, measure zytase wild-type and the maximum speed of reaction of mutant under differing temps, the concentration of enzyme is 0.1mg/mL, 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.Activity during take maximum speed of reaction is 100%, and the enzyme of measuring under other temperature is lived and its ratio per-cent is ordinate zou, 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 in fallibility PCR sudden change library
4.1 the structure in sudden change library
Do second and take turns outside the template of fallibility PCR divided by three mutant 10E11,05D03,09D05 extraction plasmid that first round fallibility PCR screens, in the building process in random mutation body storehouse, the selection of primer, the PCR reaction conditions, the structure in library is with embodiment 1.The result obtains surpassing 105 clones' random mutation body storehouse.
4.2 the screening in sudden change library
Change escherichia coli expression bacterial strain BL21-DE3 over to making up the mutant that obtains in the step 4.1, screening during activity/thermally-stabilised gain mutant take 10E11,05D03,09D05 as reference, heat treatment time is 150min, all the other operations are with embodiment 2, get activity/residual activity than mutant 10E11,05D03,09D05, high bacterial strain in 96 new well culture plates, repeat 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 in Shanghai Ying Jun Bioisystech Co., Ltd and checks order.
4.3 the mensuration of mutant thermostability
Measuring method is with embodiment 3.2, result's (table 1) shows that 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.
Structure and the screening of embodiment 5XT6 mutant gene DNA reorganization mutation library
5.1DNA the structure of reorganization mutation library
Take the zytase XT6 mutant 10E11 that screens, 05D03,09D05,08A07,05F03,04D08,04F06,04H09,08G01,06B01,04D03,03B11,04H12,02E04 as template, pair of primers:
5′-GTGAGCGGATAACAATTCCC-3′,
5′-CCTCAAGACCCGTTTAGAGG-3′
Each mutant gene that increases respectively, reaction conditions is: 94 ° of C denaturation 10min, 94 ° of C sex change 30s, 60 ° of C annealing 30s and 72 ° of C extend 2min, totally 25 circulations, 0.8% agarose electrophoresis, test kit reclaims the purifying goal gene.Method according to Stemmer, DNase I processes appropriate time with the DNA goal gene fragment that obtains, 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).Process is without primer PCR and primer PCR is arranged, and the primer is:
5′-TAGGAGGTCATATGAAAAATGCGGACAGCTATGCG-3′,
5′-ATACGCGGATCCCTATTTGTGATCAATGATCGCCCAATACGCCGGCTT-3′
Total length goal gene after obtaining recombinating.The construction process in DNA reorganization sudden change library obtains to surpass 105 clones' mutant library with embodiment 1.
5.2 the screening of DNA reorganization mutation library
The screening gain mutant is taken turns the highest mutant 04D03 of the thermostability that obtains among the fallibility PCR as reference take second, heat treatment time is 240min, all the other operations are with embodiment 2, get residual activity than the high bacterial strain of mutant 04D03 in 96 new well culture plates, repeat screening.Screen mutant 06H07.Send in Shanghai Ying Jun Bioisystech Co., Ltd and check order.
5.3 the mensuration of mutant thermostability
Measuring method is with embodiment 3.2, and result's (table 1) shows that 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
With the closer zytase aminoacid sequence of 17 sibships of F10 family known in zytase XT6 aminoacid sequence and the protein library (SwissPort) compare (table 2).In the zytase aminoacid 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 the amino acid similarity degree is between 40%-75%.The used software of sequence alignment is Clustal X.
Comparison result is found, Phe95 in the zytase XT6 aminoacid sequence, Ile152, Tyr200, Asn262, the amino acid of Phe361 and Ile376 position is respectively Gly, Val at the amino acid that its homology zytase aminoacid sequence corresponding position occupies the majority, Phe, Ile, Leu and Val.The rite-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′
The PCR condition is: 10 * Buffer 5mL, primer (10mM) each 6mL, dNTP(2.5mM) 6mL, pfu(2.5U/mL) 1mL, plasmid 10ng, ultrapure water is supplied 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.The PCR product is processed with 1mL DpnI, and 37 ° of C process 1h.PCR product 10mL chemical method changes E.coli DH5a over to.Send in Shanghai Ying Jun Bioisystech Co., Ltd and check order.After order-checking is correct, extracts plasmid and change expression strain E.coliBL21-DE3 over to.Extract mutant enzyme albumen, measure its thermostability under 75 ° of C.Measuring method is with embodiment 3.2.The result obtains the mutant F360L that thermostability improves, and is 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 sites makes up XT6 new mutant body
7.1 the structure of mutant FAM
The rite-directed mutagenesis method sports tyrosine with 53 the phenylalanine of mutant 05D03, makes up mutant FAM, and used primer is as follows:
52-F:5′-GATGCTGAAGCGTCATTATAACTCAATTGTGGCGG-3′，
52-R:5′-CCGCCACAATTGAGTTATAATGACGCTTCAGCATC-3′
PCR condition and operation obtain new mutant body FAM with embodiment 6.
7.2 the structure of mutant FAMG
Fixed-point mutation method is aspartic acid with 364 the glycine mutation of mutant FAM, makes up mutant FAMG.Used primer is as follows:
363-F:5′-GCGCCGTTTGTGTTTGACCCGGATTACAAAGTGAAGC-3′，
363-R:5′-GCTTCACTTTGTAATCCGGGTCAAACACAAACGGCGC-3′
PCR condition and subsequent operations obtain mutant FAMG with embodiment 6.
7.3 the structure of mutant FTAMG
Fixed-point mutation method sports Isoleucine with 121 the Threonine of mutant FAMG, makes up mutant FTAMG, and used primer is as follows:
120-F:5′-CCGATGGTGAACGAGATCGATCCGGTGAAACGTG-3′，
120-R:5′-CACGTTTCACCGGATCGATCTCGTTCACCATCGG-3′
PCR condition and subsequent operations obtain mutant FTAMG with embodiment 6.
7.4 the structure of mutant FC06T
Fixed-point mutation method is Methionin with 17 the asparagine mutation of mutant 06H07,26 lysine mutation is Isoleucine, 113 proline(Pro) sports Threonine, 138 L-glutamic acid sports aspartic acid, 361 phenylalanine sports leucine, make up mutant FC06T, used primer 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 obtain mutant FC06T with embodiment 6.
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 heat inactivation transformation period of FC06T under 75 ° of C conditions is respectively 7.3,14.8,19.4 and 52 times (table 1) of wild-type; FTAMG, FC06T measures residual activity after 20min thermal treatment, during 78 ° of C, the wild-type residual activity is lower than 1%, and mutant FTAMG still can keep 20% activity, and FC06T still can keep 50% activity, 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 is measured with 3.3 the impact of enzyme mutant speed of reaction, mutant FTAMG, and FC06T has maximum speed of reaction at 87 ° of C, improves 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 simple point mutation body heat inactivation transformation period
[0055
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. one kind 1,4-β-D-xylanase mutant, it is characterized in that: take the Isosorbide-5-Nitrae-β shown in the SEQ ID NO.1-D-zytase XT6 aminoacid sequence as the basis, after sudden change, have the aminoacid sequence of following Characteristics of Mutation: its methionine(Met) of the 271st is sported Isoleucine.
2. Isosorbide-5-Nitrae-β-D-xylanase mutant is characterized in that: be α-amino-isovaleric acid with the 257th alanine mutation of mutant claimed in claim 1.
3. Isosorbide-5-Nitrae-β-D-xylanase mutant is characterized in that: be that α-amino-isovaleric acid and the 364th 's glycine mutation is aspartic acid with the 257th alanine mutation of mutant claimed in claim 1.
4. Isosorbide-5-Nitrae-β-D-xylanase mutant is characterized in that: it is that leucine and 364 s' glycine mutation is aspartic acid that the 213rd Threonine of mutant claimed in claim 1 is sported Isoleucine, the 257th alanine mutation.
5. Isosorbide-5-Nitrae-β-D-xylanase mutant is characterized in that: it is that α-amino-isovaleric acid and the 364th 's glycine mutation is aspartic acid that the 138th L-glutamic acid of mutant claimed in claim 1 is sported aspartic acid, the 257th alanine mutation.
6. such as claim 1 or 2 or 3 or 4 or 5 described a kind of 1,4-β-D-xylanase mutant is characterized in that, the gene of mutant is connected into pET 28a (+) carrier, and in host cell intestinal bacteria Escherichiacoli BL21-DE3, express, obtain mutant enzyme albumen.
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Cited By (8)
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| CN103993025A (en) * | 2013-12-24 | 2014-08-20 | 北京伟嘉人生物技术有限公司 | Xylanase coding gene with excellent heat resistance and specific activity and application thereof |
| WO2015007033A1 (en) * | 2013-07-16 | 2015-01-22 | 中国农业科学院饲料研究所 | Mutant of xylanase xynas9-m with improved thermal stability and gene and use thereof |
| CN107338241A (en) * | 2016-05-03 | 2017-11-10 | 中国科学院深圳先进技术研究院 | A kind of method that evolution is oriented to gene promoter |
| CN111690632A (en) * | 2020-07-14 | 2020-09-22 | 云南师范大学 | Endo-xylanase mutant S23E11, and preparation method and application thereof |
| CN111748542A (en) * | 2020-07-14 | 2020-10-09 | 云南师范大学 | Endo-xylanase mutant S07A11, and preparation method and application thereof |
| CN111849943A (en) * | 2020-07-14 | 2020-10-30 | 云南师范大学 | Endo-xylanase mutant S06H03, and preparation method and application thereof |
| CN111849942A (en) * | 2020-07-14 | 2020-10-30 | 云南师范大学 | Endo-xylanase mutant S44A09, and preparation method and application thereof |
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