CN103695394A - Optimized acid-resistant mannase MAN26gy as well as preparation method and application thereof - Google Patents
Optimized acid-resistant mannase MAN26gy as well as preparation method and application thereof Download PDFInfo
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- CN103695394A CN103695394A CN201310700087.9A CN201310700087A CN103695394A CN 103695394 A CN103695394 A CN 103695394A CN 201310700087 A CN201310700087 A CN 201310700087A CN 103695394 A CN103695394 A CN 103695394A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
Abstract
The invention provides optimized acid-resistant mannase MAN26gy and application thereof. 85-th-site amino acid leucine (L) in an amino acid sequence of mature mannase is modified into aspartic acid (D); 103-th-site amino acid asparaginate (N) is modified into aspartic acid (D). The optimal pH value of the modified mannase is 6.0 which is improved greater in comparison with the original mannase (the optimal pH value being 7.0); the acid resistance of the modified mannase is further improved, so that over 80% of the greatest enzyme activity between the pH values of 4.0 and 7.0 can be kept, and 70% of the greatest enzyme activity also can be kept when the pH value is 3.0. According to the invention, the enzymatic properties of the modified mannase are researched, so that greater experimental data is provided for enzyme application.
Description
Technical field
The invention belongs to genetically engineered and enzyme engineering field, be specifically related to acid resistance mannase MAN26gy of a kind of optimization and its preparation method and application.
Background technology
'beta '-mannase (EC3.2.1.78) is the hemicellulase that a class can be hydrolyzed mannosans, originate more extensive, the overwhelming majority is found in the storage organs such as seed and fruit of microorganism and some plants, even in some lower animal body, also has existence.Mannase has very big-difference because of the difference character of originating.In general, in bacterium, the molecular weight of 'beta '-mannase is many between 35kD～55kD, and optimum pH is slightly acidic or neutrality, and the optimum pH of some of them Bacillus alcalophilus can reach 9.0～10.0.And the beta-mannase enzyme molecular weight of originated from fungus is about 45kD～55kD, optimum pH is 4.5～5.5.With respect to bacterium, the pH stability of originated from fungus mannase, optimum pH is all on the low side, and thermotolerance is also poor than bacterium.The mannosans enzyme molecular weight that vegetable cell produces is approximately 35kD～45kD, and optimal reaction pH is 4.5～5.5, and optimal reactive temperature and thermotolerance are all lower than bacterium and fungi.
Up to the present, have been found that nearly thousand kinds of 'beta '-mannases, the mannase of different characteristics is applied in feed, food, slurrying and oil and gas industry, wherein in feedstuff industry, is most widely used.Dregs of beans, cotton dregs, the dish dregs of rice are the most frequently used plant protein materials, but simple stomach poultry (comprising pig and chicken) only has 50-60% to the capacity usage ratio of dregs of beans.This is because the hemicellulose that contains 22.7% left and right in dregs of beans.And these non-starch polysaccharides are can not be by simple stomach animal digestion.The content of beta-mannase in dregs of beans is all higher than other common feedstuffs, as a kind of antinutritional factor beta-mannase, in the digestive tube of animal, forms gel, makes alimentary canal content have stronger stickiness, thereby affects the dietetic alimentation of animal.The interpolation Main Function of mannase is to reduce chyme viscosity, improves animal to the utilization ratio of feed etc.Due to Digestive tract such as simple stomach poultry, the properties of enzyme is had to special requirement as pH value; for mannase enzyme is better played a role in feed; conventionally the mannase that can select the pH of the suitableeest action pH and Digestive tract to be close; in recent years; along with molecular biology and engineered development; can artificially to mannase, carry out reasonable reformation, it is more suitable in fodder industry.
Summary of the invention
The object of this invention is to provide acid resistance mannase MAN26gy of a kind of optimization obtaining by rite-directed mutagenesis and its preparation method and application, the aminoacid sequence of the mannase after sudden change is as shown in SEQ ID NO:3, the present invention improves its acid resistance by rite-directed mutagenesis mannosans enzyme amino acid sequence, another object of the present invention is to provide the gene of the acid resistance mannase of coding said mutation, and its nucleotide sequence is as shown in SEQ ID NO:4.
The present invention also provides and has comprised the recombinant vectors that said mutation obtains acid resistance mannase gene MAN26gy, is preferably pET-30a-MAN26gy.The acid resistance mannase gene that sudden change is obtained is inserted between the restriction enzyme site that expression vector is suitable, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, be preferably mannase gene of the present invention is inserted between the Nde I and Not I restriction enzyme site on plasmid pET-30a, obtain recombinant expression plasmid pET-30a-MAN26gy.
The preparation method of described acid resistance mannase MAN26gy, it comprises the following steps:
1) with the encoding gene man26gy that contains mannase MAN26gy recombinant vectors transformed host cell, obtain recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induction restructuring mannosans expression of enzymes;
3) reclaim the also expressed mannase MAN26gy of purifying.
The application of high temperature resistant mannase MAN26gy described in the present invention also provides in preparing fodder additives.
Compared with prior art, advantage of the present invention and technique effect are: the present invention's technical problem first to be solved is to overcome the deficiencies in the prior art, by rite-directed mutagenesis, provides a kind of mannase that has better acid resistance, is suitable for applying in fodder industry.Due to animal digestive system, the properties of enzyme is had to special requirement as pH value, for mannase enzyme of the present invention is more suitable in fodder industry, the present invention, by rite-directed mutagenesis subtilis GH26 beta-mannase enzyme amino acid sequence, transforms this mannase acid resistance.
The present invention simulates the albumen space structure of GH26 'beta '-mannase, and carry out Multiple Sequence Alignment analysis, by overlapping pcr, carry out rite-directed mutagenesis, concrete is to be aspartic acid by the leucine of the 85th and 103 asparagine mutations, and the mannase gene after sudden change is connected to expression vector pET-30a, recombinant plasmid transformed e. coli bl21 (DE3) by after connecting, has obtained a strain recombinant bacterial strain pET-30a-MAN26gy/E.coliBL21 (DE3).Mannase MAN26gy after the sudden change obtaining has following character: improved mannase optimum pH 6.0, compared larger improvement with original mannase (optimum pH 7.0), and acid resistance further improves, between pH4.0-7.0, all can keep the more than 80% of maximum enzyme vigor, in pH3.0 situation, also can keep 40% maximum enzyme vigor.The present invention has studied the zymologic property of mannase after transformation, for the application of enzyme provides good experimental data.
In conjunction with reading after the specific embodiment of the present invention, it is clearer that the other features and advantages of the invention will become.
Accompanying drawing explanation
Fig. 1 show to recombinate after the present invention suddenlys change protein structure of mannase.
Fig. 2 show to recombinate after the present invention suddenlys change protein electrophoresis collection of illustrative plates of mannase.
Fig. 3 show to recombinate after the present invention suddenlys change optimal pH of mannase.
Fig. 4 shows the optimal pH of the original mannase of the present invention.
Fig. 5 show to recombinate after the present invention suddenlys change pH stability of mannase.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in further detail.
In following examples, do not make the experimental methods of molecular biology illustrating, all with reference to listed concrete grammar in < < molecular cloning experiment guide > > (third edition) J. Pehanorm Brooker one book, carry out, or carry out according to test kit and product description.
Embodiment 1
One, the rite-directed mutagenesis of GH26 beta-mannase gene
Mannase MAN26gy of the present invention derives from subtilis, and (deriving from commercially available subtilis all can, as be purchased from agriculture microbial strains preservation center, it is numbered ACCC01779), the aminoacid sequence of mannase MAN26gy is as following SEQ ID No:1:
LFKKHTISLLILFLLASAVLAKPIEAHTVSPVNPNAQQTTKAVMNWLAHLPNRTENRVLSGAFGGYSHDTFSMAEADRIRSATG
LSPAIYGCDYARGWLETA
NIEDSIDVSCNGDLISYWKNGGIPQISLHLANPAFQSGHFKTPITNDQYKKILDSSTAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFYPGASYVLTALNKPFAFTEVGPQTANGSFDYSLFINAIKQKYPKTIYFLAWNDEWSPAVNKGASALYHDSWTLNKGEIWNGDSLTPIVE。
The above-mentioned mannase gene man26gy that derives from subtilis that encodes, its genome sequence is as following SEQ ID No:2:
Ttgtttaagaaacatacgatctctttgctcattttatttttacttgcgtctgctgttttagcaaaaccaattgaagcgcatactgtgtcgcctgtgaatcctaatgcacagcagacaacaaaagcagtgatgaactggcttgcgcacctgccgaaccgaacggaaaacagagtcctttccggagcgttcggaggttacagtcatgacacattttctatggctgaggctgatagaatccgaagcgccaccggg
ctatcgcctgctatttatggctgcgattatgccagaggatggcttgaaacagca
aatattgaagattcaatagatgtaagctgcaacggtgatttaatttcgtattggaaaaatggcggaattccgcaaatcagtttgcacctggcgaaccctgcttttcagtcagggcattttaaaacaccgattacaaacgatcagtataaaaaaatactagattcttcaacagcagaaggaaagcggctaaatgccatgctcagcaaaattgctgacggacttcaagagctggagaaccaaggtgtgcctgttctgttcaggccactgcatgaaatgaacggcgaatggttttggtggggacttacatcatataaccaaaaggataatgaaagaatctctctatataaacagctctacaagaaaatctatcattatatgaccgacacaagaggacttgatcatttgatttgggtttactcacccgacgccaaccgagattttaaaactgatttttacccgggagcgtcttacgtgctaacagcgcttaataaaccatttgcttttacagaagtcggcccgcaaacagcaaacggcagcttcgattacagcctgttcatcaatgcaataaaacaaaaatatcctaaaaccatttactttctggcatggaatgatgaatggagcccagcagtaaacaagggtgcttcagctttatatcatgacagctggacactcaacaagggagaaatatggaatggtgattctttaacgccaatcgttgaatga。
1, the design of primer
According to the space structure of above-mentioned GH26 'beta '-mannase and in conjunction with multiple sequence compare of analysis, by the method for overlapping PCR, carry out rite-directed mutagenesis, the primer of design is as follows:
Express primer:
EXPF：5'-GGG
CATATGTTGTTTAAGAAACATACGATCTCTTTGC-3'（SEQ ID NO:5）；
EXPR：5'-GGG
GCGGCCGCTCATTCAACGATTGGCGTTAAAG-3'（SEQ ID NO:6）；
Mutant primer
L85D F：5'-CCGAAGCGCCACCGGG
GACTCGCCTGC-3'（SEQ ID NO:7）；
L85D R：5'-
GTCCCCGGTGGCGCTTCGGATTCTATCAGC-3'（SEQ ID NO:8）；
N103D F：5'-GGATGGCTTGAAACAGCA
GACATTGAAG-3'（SEQ ID NO:9）。
N103D R：5'-
GTCTGCTGTTTCAAGCCATCCTCTGGCATAATCG-3'（SEQ ID NO:10）。
2, take pET-30a-man26 as template, utilize the above-mentioned primer providing to increase, amplification condition is: 95 ℃ of 90sec; Then 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 4min30sec, repeat 30 circulations; Then 72 ℃ of 10min; Then 4 ℃ of 60min.
Amplification system is ddH
2o29 μ L, template 0.2 μ L, each 0.9 μ L of primer, FastPfu4 μ L, 5 * FastPfu Buffer10 μ L, dNTP5 μ L, amplifies the PCR product in corresponding mutational site, and utilizes sepharose to reclaim.
Through rite-directed mutagenesis, the present invention has obtained the mannase Amino Acid-Induced Site-Directed Mutation body MAN26gy in subtilis source, the 85th amino acids leucine (L) in its ripe mannosans enzyme amino acid sequence changes aspartic acid (D) into, the 103rd amino acids l-asparagine (N) changes aspartic acid (D) into, and the aminoacid sequence after sudden change is as shown in SEQ ID NO:3.
SEQ ID NO:3
LFKKHTISLLILFLLASAVLAKPIEAHTVSPVNPNAQQTTKAVMNWLAHLPNRTENRVLSGAFGGYSHDTFSMAEADRIRSATG
DSPAIYGCDYARGWLETA
DIEDSIDVSCNGDLISYWKNGGIPQISLHLANPAFQSGHFKTPITNDQYKKILDSSTAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFYPGASYVLTALNKPFAFTEVGPQTANGSFDYSLFINAIKQKYPKTIYFLAWNDEWSPAVNKGASALYHDSWTLNKGEIWNGDSLTPIVE。
The gene order man26gy of mannase after coding said mutation, this gene order is as shown in SEQ ID NO.4 particularly.
SEQ ID NO:4
ttgtttaagaaacatacgatctctttgctcattttatttttacttgcgtctgctgttttagcaaaaccaattgaagcgcatactgtgtcgcctgtgaatcctaatgcacagcagacaacaaaagcagtgatgaactggcttgcgcacctgccgaaccgaacggaaaacagagtcctttccggagcgttcggaggttacagtcatgacacattttctatggctgaggctgatagaatccgaagcgccaccggg
gactcgcctgctatttatggctgcgattatgccagaggatggcttgaaacagca
gacattgaagattcaatagatgtaagctgcaacggtgatttaatttcgtattggaaaaatggcggaattccgcaaatcagtttgcacctggcgaaccctgcttttcagtcagggcattttaaaacaccgattacaaacgatcagtataaaaaaatactagattcttcaacagcagaaggaaagcggctaaatgccatgctcagcaaaattgctgacggacttcaagagctggagaaccaaggtgtgcctgttctgttcaggccactgcatgaaatgaacggcgaatggttttggtggggacttacatcatataaccaaaaggataatgaaagaatctctctatataaacagctctacaagaaaatctatcattatatgaccgacacaagaggacttgatcatttgatttgggtttactcacccgacgccaaccgagattttaaaactgatttttacccgggagcgtcttacgtgctaacagcgcttaataaaccatttgcttttacagaagtcggcccgcaaacagcaaacggcagcttcgattacagcctgttcatcaatgcaataaaacaaaaatatcctaaaaccatttactttctggcatggaatgatgaatggagcccagcagtaaacaagggtgcttcagctttatatcatgacagctggacactcaacaagggagaaatatggaatggtgattctttaacgccaatcgttgaatga。
Two, the structure of recombinant plasmid and Transformed E .Coli BL21 (DE3)
Build recombinant expression vector L85D/N103D-pET-30a, the above-mentioned beta-mannase gene PCR product making and expression plasmid pET-30a are carried out to double digestion with Nde I and Not I simultaneously, sepharose carries out product and reclaims purifying, connects, and linked system is as follows:
16 ℃ are incubated 18 hours, get above-mentioned linked system electric shock and transform Trans-T1 competent cell, method for transformation for getting Trans-T1 competent cell to dissolving on ice from cryogenic refrigerator, the product ice bath 30min that adds 8 μ L to connect, 37 ℃ of heat shock 90sec, place 2min on ice, add 500 μ L liquid LB, 60min is cultivated in 37 ℃ of concussions, after concussion is cultivated, the centrifugal 2min of 5000rpm, discard part supernatant, the supernatant of residue approximately 100 μ L is mixed to thalline, evenly coat on the solid-state LB flat board that contains penbritin, in 37 ℃ of constant incubators, be inverted about 12h, after growing bacterium colony, choosing bacterium verifies.
Get positive colony of each mutant and cultivate, extract respectively expression plasmid, be converted into competent cell BL21 (DE3), method for transformation the same (flat board contains kantlex).After empirical tests, obtain engineering strain L85D/N103D-pET-30a/E.Coli BL21 (DE3), L85D/N103D represents two mutational sites, amino acid before the front alphabetical representative sudden change of numeral, and digital rear letter is the rear amino acid of sudden change.
Three, the purifying of recombinant bacterial strain GH26 'beta '-mannase and optimum pH analysis
Get the mutator gene engineering strain L85D/N103D-pET-30a/E.Coli BL21 (DE3) after verifying, be inoculated in respectively in 300mL LB nutrient solution, after 37 ℃ of 220rpm shaking culture 2-3h, add final concentration 0.6mMIPTG to induce 4-6h at 30 ℃.Get fermented liquid and carry out preliminary enzyme activity determination, after recording enzyme work, carry out fermented liquid and concentrate.Each fermented liquid of getting respectively after concentrating carries out purifying by Ni-NTA post, and the enzyme liquid after purifying carries out SDS-PAGE detection, and L85D/N103D-pET-30a/E.Coli BL21 (DE3) purification effect as shown in Figure 2.
The mensuration of mannosans enzymic activity: draw 2mL through the suitable enzyme liquid of dilution, join in scale test tube, then add 2mL mannan solution, concussion 3s, incubation 30min at 50 ℃.Add 5mLDNS reagent, evenly, boiling water bath heating 5min, is cooled to room temperature, adds water constant volume to 25mL, measures absorbancy at 540nm place in concussion.
The optimum pH of restructuring mannase is measured: the acetic acid-sodium acetate of the different pH value mannase liquid of purifying for (2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5) (concentration is 0.2M) damping fluid dilutes, and carries out enzymatic reaction to measure its optimal pH with the substrate of the buffer preparation mannase of above pH value.The MAN26gy mannase optimum pH that L85D/N103D-pET-30a/E.Coli BL21 (DE3) genetic engineering bacterium produces is 6.0(Fig. 3) than original mannase MAN26 optimum pH 7.0(Fig. 4) reduced by 1.0 pH values.
Four, the acid resistance analysis of recombinant bacterial strain GH26 'beta '-mannase
Draw enzyme liquid 0.5mL(liquid and directly get enzyme liquid, solid enzyme takes solid protoenzyme powder 1.00g and is dissolved in 80mL distilled water; Room temperature lower magnetic force is used 100mL volumetric flask constant volume after stirring 10min, after centrifugal, get supernatant liquor, stand-by), pH2.5, the pH3.0, pH3.5, pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH6.5, the pH7.0 acetic acid-sodium acetate buffer (0.2M) that add separately 4.5mL, after processing one hour (noting airtight) under 37 ℃ of conditions and handling, be diluted to suitable enzyme liquid to be measured, take and do not have the original enzyme liquid of processing under 37 ℃ of conditions, to process one hour as contrast, computing is residual enzyme work after one hour.Acid resistance is analyzed (Fig. 5) and is shown: MAN26gy mannase is all very stable between pH4.0-7.0, within the scope of this pH, process 1h after residual enzyme activity more than 80%.
Recombinant bacterial strain that the present invention obtains is fermented, and after fermented liquid is centrifugal, supernatant is through concentrated and purified, successfully obtain pure saltant type 'beta '-mannase, it is characterized in that by it is carried out to zymologic property analysis, compare with the optimal pH of original strain, drop to 6.0 by 7.0, make this enzyme be more suitable for applying in feed.Described feed is corn-soybean meal diet, wheat-bean pulp type daily ration, the assorted dregs of rice type daily ration of coarse cereals.
SEQUENCE LISTING
<110> Qingdao root biotechnology Group Co.,Ltd
Acid resistance mannase MAN26gy of a <120> optimization and its preparation method and application
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 342
<212> PRT
<213> Bacillus subtilis
<400> 1
Leu Phe Lys Lys His Thr Ile Ser Leu Leu Ile Leu Phe Leu Leu Ala
1 5 10 15
Ser Ala Val Leu Ala Lys Pro Ile Glu Ala His Thr Val Ser Pro Val
20 25 30
Asn Pro Asn Ala Gln Gln Thr Thr Lys Ala Val Met Asn Trp Leu Ala
35 40 45
His Leu Pro Asn Arg Thr Glu Asn Arg Val Leu Ser Gly Ala Phe Gly
50 55 60
Gly Tyr Ser His Asp Thr Phe Ser Met Ala Glu Ala Asp Arg Ile Arg
65 70 75 80
Ser Ala Thr Gly Leu Ser Pro Ala Ile Tyr Gly Cys Asp Tyr Ala Arg
85 90 95
Gly Trp Leu Glu Thr Ala Asn Ile Glu Asp Ser Ile Asp Val Ser Cys
100 105 110
Asn Gly Asp Leu Ile Ser Tyr Trp Lys Asn Gly Gly Ile Pro Gln Ile
115 120 125
Ser Leu His Leu Ala Asn Pro Ala Phe Gln Ser Gly His Phe Lys Thr
130 135 140
Pro Ile Thr Asn Asp Gln Tyr Lys Lys Ile Leu Asp Ser Ser Thr Ala
145 150 155 160
Glu Gly Lys Arg Leu Asn Ala Met Leu Ser Lys Ile Ala Asp Gly Leu
165 170 175
Gln Glu Leu Glu Asn Gln Gly Val Pro Val Leu Phe Arg Pro Leu His
180 185 190
Glu Met Asn Gly Glu Trp Phe Trp Trp Gly Leu Thr Ser Tyr Asn Gln
195 200 205
Lys Asp Asn Glu Arg Ile Ser Leu Tyr Lys Gln Leu Tyr Lys Lys Ile
210 215 220
Tyr His Tyr Met Thr Asp Thr Arg Gly Leu Asp His Leu Ile Trp Val
225 230 235 240
Tyr Ser Pro Asp Ala Asn Arg Asp Phe Lys Thr Asp Phe Tyr Pro Gly
245 250 255
Ala Ser Tyr Val Leu Thr Ala Leu Asn Lys Pro Phe Ala Phe Thr Glu
260 265 270
Val Gly Pro Gln Thr Ala Asn Gly Ser Phe Asp Tyr Ser Leu Phe Ile
275 280 285
Asn Ala Ile Lys Gln Lys Tyr Pro Lys Thr Ile Tyr Phe Leu Ala Trp
290 295 300
Asn Asp Glu Trp Ser Pro Ala Val Asn Lys Gly Ala Ser Ala Leu Tyr
305 310 315 320
His Asp Ser Trp Thr Leu Asn Lys Gly Glu Ile Trp Asn Gly Asp Ser
325 330 335
Leu Thr Pro Ile Val Glu
340
<210> 2
<211> 1029
<212> DNA
<213> Bacillus subtilis
<400> 2
ttgtttaaga aacatacgat ctctttgctc attttatttt tacttgcgtc tgctgtttta 60
gcaaaaccaa ttgaagcgca tactgtgtcg cctgtgaatc ctaatgcaca gcagacaaca 120
aaagcagtga tgaactggct tgcgcacctg ccgaaccgaa cggaaaacag agtcctttcc 180
ggagcgttcg gaggttacag tcatgacaca ttttctatgg ctgaggctga tagaatccga 240
agcgccaccg ggctatcgcc tgctatttat ggctgcgatt atgccagagg atggcttgaa 300
acagcaaata ttgaagattc aatagatgta agctgcaacg gtgatttaat ttcgtattgg 360
aaaaatggcg gaattccgca aatcagtttg cacctggcga accctgcttt tcagtcaggg 420
cattttaaaa caccgattac aaacgatcag tataaaaaaa tactagattc ttcaacagca 480
gaaggaaagc ggctaaatgc catgctcagc aaaattgctg acggacttca agagctggag 540
aaccaaggtg tgcctgttct gttcaggcca ctgcatgaaa tgaacggcga atggttttgg 600
tggggactta catcatataa ccaaaaggat aatgaaagaa tctctctata taaacagctc 660
tacaagaaaa tctatcatta tatgaccgac acaagaggac ttgatcattt gatttgggtt 720
tactcacccg acgccaaccg agattttaaa actgattttt acccgggagc gtcttacgtg 780
ctaacagcgc ttaataaacc atttgctttt acagaagtcg gcccgcaaac agcaaacggc 840
agcttcgatt acagcctgtt catcaatgca ataaaacaaa aatatcctaa aaccatttac 900
tttctggcat ggaatgatga atggagccca gcagtaaaca agggtgcttc agctttatat 960
catgacagct ggacactcaa caagggagaa atatggaatg gtgattcttt aacgccaatc 1020
gttgaatga 1029
<210> 3
<211> 342
<212> PRT
<213> Bacillus subtilis
<400> 3
Leu Phe Lys Lys His Thr Ile Ser Leu Leu Ile Leu Phe Leu Leu Ala
1 5 10 15
Ser Ala Val Leu Ala Lys Pro Ile Glu Ala His Thr Val Ser Pro Val
20 25 30
Asn Pro Asn Ala Gln Gln Thr Thr Lys Ala Val Met Asn Trp Leu Ala
35 40 45
His Leu Pro Asn Arg Thr Glu Asn Arg Val Leu Ser Gly Ala Phe Gly
50 55 60
Gly Tyr Ser His Asp Thr Phe Ser Met Ala Glu Ala Asp Arg Ile Arg
65 70 75 80
Ser Ala Thr Gly Asp Ser Pro Ala Ile Tyr Gly Cys Asp Tyr Ala Arg
85 90 95
Gly Trp Leu Glu Thr Ala Asp Ile Glu Asp Ser Ile Asp Val Ser Cys
100 105 110
Asn Gly Asp Leu Ile Ser Tyr Trp Lys Asn Gly Gly Ile Pro Gln Ile
115 120 125
Ser Leu His Leu Ala Asn Pro Ala Phe Gln Ser Gly His Phe Lys Thr
130 135 140
Pro Ile Thr Asn Asp Gln Tyr Lys Lys Ile Leu Asp Ser Ser Thr Ala
145 150 155 160
Glu Gly Lys Arg Leu Asn Ala Met Leu Ser Lys Ile Ala Asp Gly Leu
165 170 175
Gln Glu Leu Glu Asn Gln Gly Val Pro Val Leu Phe Arg Pro Leu His
180 185 190
Glu Met Asn Gly Glu Trp Phe Trp Trp Gly Leu Thr Ser Tyr Asn Gln
195 200 205
Lys Asp Asn Glu Arg Ile Ser Leu Tyr Lys Gln Leu Tyr Lys Lys Ile
210 215 220
Tyr His Tyr Met Thr Asp Thr Arg Gly Leu Asp His Leu Ile Trp Val
225 230 235 240
Tyr Ser Pro Asp Ala Asn Arg Asp Phe Lys Thr Asp Phe Tyr Pro Gly
245 250 255
Ala Ser Tyr Val Leu Thr Ala Leu Asn Lys Pro Phe Ala Phe Thr Glu
260 265 270
Val Gly Pro Gln Thr Ala Asn Gly Ser Phe Asp Tyr Ser Leu Phe Ile
275 280 285
Asn Ala Ile Lys Gln Lys Tyr Pro Lys Thr Ile Tyr Phe Leu Ala Trp
290 295 300
Asn Asp Glu Trp Ser Pro Ala Val Asn Lys Gly Ala Ser Ala Leu Tyr
305 310 315 320
His Asp Ser Trp Thr Leu Asn Lys Gly Glu Ile Trp Asn Gly Asp Ser
325 330 335
Leu Thr Pro Ile Val Glu
340
<210> 4
<211> 1029
<212> DNA
<213> Bacillus subtilis
<400> 4
ttgtttaaga aacatacgat ctctttgctc attttatttt tacttgcgtc tgctgtttta 60
gcaaaaccaa ttgaagcgca tactgtgtcg cctgtgaatc ctaatgcaca gcagacaaca 120
aaagcagtga tgaactggct tgcgcacctg ccgaaccgaa cggaaaacag agtcctttcc 180
ggagcgttcg gaggttacag tcatgacaca ttttctatgg ctgaggctga tagaatccga 240
agcgccaccg gggactcgcc tgctatttat ggctgcgatt atgccagagg atggcttgaa 300
acagcagaca ttgaagattc aatagatgta agctgcaacg gtgatttaat ttcgtattgg 360
aaaaatggcg gaattccgca aatcagtttg cacctggcga accctgcttt tcagtcaggg 420
cattttaaaa caccgattac aaacgatcag tataaaaaaa tactagattc ttcaacagca 480
gaaggaaagc ggctaaatgc catgctcagc aaaattgctg acggacttca agagctggag 540
aaccaaggtg tgcctgttct gttcaggcca ctgcatgaaa tgaacggcga atggttttgg 600
tggggactta catcatataa ccaaaaggat aatgaaagaa tctctctata taaacagctc 660
tacaagaaaa tctatcatta tatgaccgac acaagaggac ttgatcattt gatttgggtt 720
tactcacccg acgccaaccg agattttaaa actgattttt acccgggagc gtcttacgtg 780
ctaacagcgc ttaataaacc atttgctttt acagaagtcg gcccgcaaac agcaaacggc 840
agcttcgatt acagcctgtt catcaatgca ataaaacaaa aatatcctaa aaccatttac 900
tttctggcat ggaatgatga atggagccca gcagtaaaca agggtgcttc agctttatat 960
catgacagct ggacactcaa caagggagaa atatggaatg gtgattcttt aacgccaatc 1020
gttgaatga 1029
<210> 5
<211> 37
<212> DNA
<213> artificial sequence
<400> 5
gggcatatgt tgtttaagaa acatacgatc tctttgc 37
<210> 6
<211> 34
<212> DNA
<213> artificial sequence
<400> 6
ggggcggccg ctcattcaac gattggcgtt aaag 34
<210> 7
<211> 27
<212> DNA
<213> artificial sequence
<400> 7
ccgaagcgcc accggggact cgcctgc 27
<210> 8
<211> 30
<212> DNA
<213> artificial sequence
<400> 8
gtccccggtg gcgcttcgga ttctatcagc 30
<210> 9
<211> 28
<212> DNA
<213> artificial sequence
<400> 9
ggatggcttg aaacagcaga cattgaag 28
<210> 10
<211> 34
<212> DNA
<213> artificial sequence
<400> 10
gtctgctgtt tcaagccatc ctctggcata atcg 34
Claims (8)
1. an acid resistance mannase MAN26gy for optimization, is characterized in that: its aminoacid sequence is as shown in SEQ ID NO:3.
2. the acid resistance mannase MAN26gy of optimization according to claim 1, is characterized in that, the optimal ph of described mannase MAN26gy is 6.0.
3. the encoding gene of acid resistance mannase MAN26gy claimed in claim 1
man26gy, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:4.
4. comprise encoding gene described in claim 3
man26gyrecombinant vectors.
5. the encoding gene that contains according to claim 4
man26gyrecombinant vectors, it is characterized in that recombinant vectors is preferably recombinant vectors pET-30a-MAN26gy, it is by described mannase gene
man26gybe inserted on plasmid pET-30a
ndei and
notbetween I restriction enzyme site and obtain.
6. comprise encoding gene described in claim 3
man26gyrecombinant bacterial strain, it is characterized in that described bacterial strain is intestinal bacteria, yeast, genus bacillus.
7. the preparation method of acid resistance mannase MAN26gy claimed in claim 1, is characterized in that it comprises the following steps:
1) with the encoding gene that contains mannase MAN26gy
man26gyrecombinant vectors transformed host cell, obtain recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induction restructuring mannosans expression of enzymes;
3) reclaim the also expressed mannase MAN26gy of purifying.
8. the application of high temperature resistant mannase MAN26gy claimed in claim 1 in preparing fodder additives.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104774820A (en) * | 2015-04-14 | 2015-07-15 | 暨南大学 | Mannose with improved resistance to trypsin and protease and application of mannose |
| CN108085309A (en) * | 2017-12-13 | 2018-05-29 | 江南大学 | The beta-mannase enzyme mutant and its construction method that a kind of acid-resistant stability improves |
| CN110004166A (en) * | 2018-01-05 | 2019-07-12 | 中国科学院天津工业生物技术研究所 | The recombined bacillus subtilis bacterial strain and its preparation method of high efficient expression secretion 'beta '-mannase |
| CN111454974A (en) * | 2020-04-17 | 2020-07-28 | 济南爱科替维生物科技有限公司 | Endo- β -mannanohydrolase Man01929, method for mutating same into glycosyltransferase and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774820A (en) * | 2015-04-14 | 2015-07-15 | 暨南大学 | Mannose with improved resistance to trypsin and protease and application of mannose |
| CN104774820B (en) * | 2015-04-14 | 2018-01-30 | 暨南大学 | The mannase and its application that trypsase and pepsin resistance improve |
| CN108085309A (en) * | 2017-12-13 | 2018-05-29 | 江南大学 | The beta-mannase enzyme mutant and its construction method that a kind of acid-resistant stability improves |
| CN110004166A (en) * | 2018-01-05 | 2019-07-12 | 中国科学院天津工业生物技术研究所 | The recombined bacillus subtilis bacterial strain and its preparation method of high efficient expression secretion 'beta '-mannase |
| CN111454974A (en) * | 2020-04-17 | 2020-07-28 | 济南爱科替维生物科技有限公司 | Endo- β -mannanohydrolase Man01929, method for mutating same into glycosyltransferase and application thereof |
| CN111454974B (en) * | 2020-04-17 | 2021-01-19 | 济南爱科替维生物科技有限公司 | Endo-type beta-mannanohydrolase Man01929, method for mutating same into glycosyltransferase and application of endo-type beta-mannanohydrolase Man01929 |
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| Publication number | Publication date |
|---|---|
| CN103695394B (en) | 2015-07-29 |
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