CN102978222A - Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof - Google Patents
Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof Download PDFInfo
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- CN102978222A CN102978222A CN2012105533234A CN201210553323A CN102978222A CN 102978222 A CN102978222 A CN 102978222A CN 2012105533234 A CN2012105533234 A CN 2012105533234A CN 201210553323 A CN201210553323 A CN 201210553323A CN 102978222 A CN102978222 A CN 102978222A
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Abstract
The invention discloses an extremely-heat-resistant beta-mannosidase gene as well as an expression protein and an application thereof, wherein the DNA (deoxyribonucleic acid) sequence of the extremely-heat-resistant beta-mannosidase gene is as shown in SEQ NO: 1, and the amino acid sequence of the extremely-heat-resistant beta-mannosidase expressed by the extremely-heat-resistant beta-mannosidase gene is as shown in SEQ NO: 2. The extremely-heat-resistant beta-mannosidase has extremely strong heat-resistant performance and the characteristic of high activity in a neutral pH condition, and the enzymatic activity is the highest and the specific enzyme activity achieves 144.6 U/mg in the conditions of 85 DEG C and a pH of 5.5; and the protease has a high enzyme activity at a temperature ranging from 70 to 95 DEG C and a pH ranging from 5.0 to 7.5. The mannosidase is extremely high in heat-resistant performance, and the residual enzyme activity is about 70% in the case that the mannosidase is heat-insulated for 2h at 80 DEG C. Via the characteristics aforementioned, the mannosidase expressed by the extremely-heat-resistant beta-mannosidase gene disclosed by the invention has more advantages than the existing mannosidase, is suitable for degradation for hemicellulose in the conditions of a high temperature of greater than 80 DEG C and a slightly acidic pH, and has a potential industrial application value.
Description
Technical field
The invention belongs to genetic engineering technique and biomass are utilized the field, be specifically related to a kind of extremely gene of anti-beta-Mannosidase the and expressing protein and application.
Background technology
Poly-grape sweet dew carbohydrate is another the important hemicellulose except polyxylose class vegetable polysaccharides.Extensively be present in the plant materialss such as coconut, Rhizoma amorphophalli powder, marine alga and softwood.Mannase and mannosidase are the most key lytic enzymes during poly-grape seminose hemicellulase is, the former is the β-1 by cutting at random mannosans, the 4-glycosidic link, mannosans is hydrolyzed to mannooligo saccharide, the latter is degraded to the seminose seminose with the mannosans of mannooligo saccharide or certain polymerization degree again, it is a kind of important saccharic nutrient substance, be distributed widely in body fluid and the tissue, playing an important role aspect the physiological regulation such as adjusting immunity system, increase wound healing, anti-inflammation effect.
Beta-Mannosidase is as a kind of toolenzyme, and it can also make the saccharification of rotted plant material material come as livestock fodder additives.Other has the research report, and beta-Mannosidase plays a significant role at biofuel and biological plastics industry.At present the beta-Mannosidase of report mainly is to derive from some fungies (such as aspergillus niger) and some normal temperature, mesophilic bacteria in the world, and the beta-Mannosidase that derives from utmost point thermophilric bacteria and ancient bacterium rarely has report.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the purpose of this invention is to provide a kind of extremely heat-resisting mannosidase gene, so that it satisfies service requirements.Another object of the present invention provides a kind of expressing protein of above-mentioned extremely heat-resisting mannosidase gene.The present invention also has a purpose to provide the application of above-mentioned a kind of extremely heat-resisting mannosidase gene.
Technical scheme: in order to realize the foregoing invention purpose, the technical solution used in the present invention is as follows:
A kind of extremely heat-resisting mannosidase gene, its dna sequence dna is shown in SEQ NO:1.
The extremely heat-resisting mannosidase of above-mentioned extremely heat-resisting mannoside enzyme gene expression, its aminoacid sequence is shown in SEQ NO:2.
A kind of extremely heat-resisting mannosidase, aminoacid sequence are replacement, the disappearance of the one or more amino-acid residues of amino acid process in the SEQ NO:2 sequence claimed in claim 2 and add the derived protein with mannoside enzymic activity that forms.
A kind of restructuring system is fastened the clone just like the dna sequence dna shown in the SEQ NO:1 at described recombinant chou; Described restructuring system comprises recombinant plasmid and recombinant bacterium.
A kind of primer for the extremely heat-resisting mannosidase gene of amplification, employed primer is to being:
man1：5’-GGAATTCCATATGGATTTCCTGCTGGGTATTAACT-3’ ；
man2：5’-CCGCTCGAGGAAGTTCAGCAGCTTATACTCTTTC-3’ 。
The above-mentioned application of extremely heat-resisting mannosidase in enzymolysis mannosans or mannooligo saccharide.The enzyme digestion reaction temperature is 70-95 ℃, pH5.0-7.5.Described mannosans derives from the mannosans of natural phant.
Above-mentioned recombinant chou ties up to the application in enzymolysis mannosans or the mannooligo saccharide.
The above-mentioned application of extremely heat-resisting mannosidase in enzymolysis caroubier mannosans.
Beneficial effect: compared with prior art, mannosidase provided by the present invention has highly active characteristic under extremely strong resistance toheat and the condition of neutral pH, and enzymic activity is the highest under 85 ℃, the condition of pH5.5, reaches 144.6U/mg than enzyme work; This proteolytic enzyme is that 70 ℃-95 ℃, pH are in the scope of 5.0-7.5 in temperature, all has higher enzyme and lives.The resistance toheat of this mannosidase is high, and in 75 ℃ reaction system, insulation 2h enzymic activity remains unchanged substantially.Above-mentioned characteristic is applicable to high temperature more than 80 ℃, second the cellulosic degraded of slant acidity pH condition so that the mannosidase that the present invention obtains has larger superiority than existing mannosidase, has potential industrial application value.This mannosidase has efficient enzymolysis ability and stronger thermostability because of it, therefore can be used as the production that a kind of efficient industrial enzyme preparation is used for seminose.
Description of drawings
Fig. 1 is the SDS-PAGE protein electrophoresis figure of mannosidase Tth manB;
Fig. 2 is the optimum temperuture figure as a result of Tth manB;
Fig. 3 is the optimal pH figure as a result of Tth manB;
Fig. 4 is the thermostability figure as a result of Tth manB;
Fig. 5 is the TLC figure of Tth manB degraded caroubier mannosans.
Embodiment
The present invention is described further below in conjunction with specific embodiment.
If employed material, reagent etc. without specified otherwise, all can obtain from commercial channels in following examples.
Can adopt the mode of following synthetic to prepare
Tth manBGene, also can with
Thermotoga thermarumTotal DNA of DSM5069 (DSMz, German) is that template obtains (the primer man1,2 that primer adopts embodiment 2) by conventional PCR method.
The mannosidase gene of present embodiment
Tth manBSynthetic by Shanghai Jierui Biology Engineering Co., Ltd, gene order is shown in SEQ NO:1.
With following primer to the mannosidase gene among the pcr amplification embodiment 1:
man1：5’-GGAATTCCATATGGATTTCCTGCTGGGTATTAACT-3’ ；
man2：5’-CCGCTCGAGGAAGTTCAGCAGCTTATACTCTTTC-3’ 。
When above-mentioned primer synthesized, man1 introduced Nde I restriction enzyme site, and man2 introduces Xho I restriction enzyme site.
PCR reaction system: 1 μ L
T.thermarumSynthetic DNA, 1 μ L man1,1 μ L man2,25 μ L Premix ExTaq, 22 μ L ultrapure waters.
PCR reaction conditions: 94 ℃ of sex change 5min; 94 ℃ of sex change 30sec, 52 ℃ of annealing 30sec, 72 ℃ are extended 3min, 30Cycles; 72 ℃ are extended 10min; 4 ℃ of insulations.
The PCR product detects output and specificity with 1% agarose gel electrophoresis, and reclaims test kit with the PCR product and carry out purifying (BIOMIGA, Shanghai).
The PCR product that purifying is crossed (embodiment 2 preparations), pET-20b(Novagen) with respectively Nde I and Xho I double digestion, agarose electrophoresis reclaims enzyme and cuts enzyme and cut PCR and carrier large fragment.Purpose fragment and carrier after rubber tapping is reclaimed, resuspended through the concentrated 8 μ L sterilized waters that add, add 1 μ L 10 * Ligase Buffer and 1 μ L Ligase, spend the night in 16 ℃ of connections.Transform intestinal bacteria pET-20b with the ligation product, then be applied to contain 100 μ g/mL Amp(penbritins) culture dish, cultivate 10-12h for 37 ℃.
From transforming the dull and stereotyped upper a plurality of single bacterium colonies of picking, adopt the plasmid of BIOMIGA to extract in a small amount test kit extraction plasmid.Check order to the plasmid double digestion checking of acquisition and to the recombinant plasmid that obtains.Sequencing result shows, has inserted the purpose fragment (length of nucleotides is 1824bp) of cloning in the pET-20b carrier, and then obtained recombinant clone, expression vector pET-20b-
Tth manB, exogenous DNA array is shown in SEQ NO:1, and the aminoacid sequence of the albumen of its expression (extremely heat-resisting mannosidase) is shown in SEQ NO:2, with this albumen called after mannosidase
Tth manB
The expression and purification of embodiment 4 restructuring mannosidase Tth manB
With recombinant clone, expression vector pET-20b-
Tth manB(embodiment 3 preparations) electricity goes to Host Strains
E. coliBL21 (DE3) (Novagen) obtains to contain the recombinant bacterium of recombinant plasmid.The recombinant bacterium of single bacterium colony is inoculated in Luria-Bertani broth (LB) substratum that 5mL contains 100 μ g/ml penbritins, and under 37 ℃ of temperature, 8-12h is cultivated in the 200rpm concussion.Get 4mL bacterium liquid and be inoculated in the 1000mL shaking flask that contains the 400mL substratum, under 37 ℃ of temperature, 200rpm shakes cultivation, as absorbancy (OD
600) when reaching 0.6-0.8, add the 1M IPTG of people 200 μ L, and under 30 ℃ of temperature, 120rpm abduction delivering 10-12h.4 ℃, with medium centrifugal 15min, collect thalline with high speed freezing centrifuge 13000rpm.With the washing of 50mL ultrapure water and under 4 ℃, with the centrifugal 15min of 13000rpm, reclaim thalline, then with 20mL 1 * Binding Buffer resuspended (0.5M NaCl, 20mM Tris-HCl, 5mM imidazole, pH7.9), in ice-water bath, with ultrasonic disruption crusher machine bacterial cell, and at 4 ℃ of centrifugal 15min of lower 13000rpm, obtain containing the crude extract of mannosidase Tth manB.
Crude extract through the thermal treatment of 70 ℃ of heat treated 30min, is removed heat labile foreign protein first, carries out purifying (method is seen His-Bind Kits, Novagen) with the Ni-NTA affinity column again, gets pure enzyme liquid.The evaluation of purifying enzyme purity and the mensuration of molecular weight adopt the SDS-PAGE method to carry out, the results are shown in Figure 1, among the figure, 1 is the Tth manB albumen in crude extract, 2 is 200 mM imidazoles wash-out purifying Tth manB albumen later, 3 is 400 mM imidazoles wash-out purifying Tth manB albumen later, and molecular weight is about 70 kDa, and is close with theoretical value.
The zymologic property analysis of embodiment 5 restructuring mannosidase Tth manB
Enzyme work is defined as: catalysis caroubier mannosans (Megazyme, Ireland) produces the required enzyme amount of 1 μ mol seminose in the 1min.
(1) optimum temperuture
Be that 6.0 0.1M imidazoles-adjacent stupid diformazan potassium hydrogen phthalate damping fluid dilution is implemented the 4 pure enzyme liquid that obtain (multiple of dilution is 30 times), carried out enzyme activity determination with the enzyme liquid after diluting with the pH value.The enzyme activity determination reaction system is 200 μ L, and by 0.5% caroubier mannosans, 100 μ L, 90 μ L 0.1M imidazoles-adjacent stupid diformazan potassium hydrogen phthalate damping fluid and 10 μ L dilution enzyme liquid form; The pH of reaction system is 6.0.Reaction system behind 60-95 ℃ of incubation 10min, is added 300 μ L DNS reagent termination reactions, measure the light absorption value of 550nm behind the boiling water bath 5min.The experiment establish 3 repetitions, the mapping of averaging, the result as shown in Figure 2, result of study shows that this mannosidase has the highest enzymic activity in the time of 85 ℃; With the live light absorption value relative reactivity 100% the most of reaction system of the enzyme under this temperature, under other temperature enzyme live reaction system light absorption value therewith the light absorption value of enzymatic activity high system as relative reactivity.Wherein, enzymic activity is higher in temperature 80-100 ℃ reaction system.
(2) optimal pH
The enzyme assay system is 200 μ L, and by 0.5% caroubier mannosans, 100 μ L, the 90 μ L 0.1M imidazoles of pH4.5-8.5-adjacent stupid diformazan potassium hydrogen phthalate damping fluid and 10 μ L dilution enzyme liquid form.Reaction system behind 85 ℃ of incubation 10min, is added 300 μ L DNS reagent termination reactions, measure the light absorption value of 550nm behind the boiling water bath 5min, repeated experiments is established in experiment 3 times, averages.The result as shown in Figure 3, result of study shows, is 5.5 o'clock at pH, mannosidase has the highest enzymic activity, is 144.6U/mg; With the enzyme under this pH value live reaction system as relative reactivity 100%, the light absorption value of the lower enzyme work of other pH value reaction system therewith the ratio of the light absorption value of enzymatic activity high system as relative reactivity.Under the condition of pH5.0-7.5, enzyme is lived all higher.
(3) recombinase thermostability
Be the pure enzyme liquid that 7.0 0.1M imidazoles-adjacent stupid diformazan potassium hydrogen phthalate damping fluid dilution embodiment 4 obtains with the pH value, carry out enzyme activity determination with the enzyme liquid after diluting.To dilute enzyme liquid at 75 ℃, 80 ℃, 85 ℃ and 90 ℃ of water-bath 30min, 60min, 90min and 120min, and measure the remaining enzyme of enzyme and live, as shown in Figure 4.PH is 5.5 in the enzyme activity determination reaction system, and measuring temperature is 85 ℃.3 repetitions are established in experiment, average, and the result as shown in Figure 4.Result of study shows, processes 2h for 75 ℃ and has no enzyme decline alive, processes 2h for 80 ℃, and remaining enzyme work is 70%, and the result shows that visible this mannosidase has stronger thermostability.
Embodiment 6 application of restructuring mannosidase Tth manB in the degraded of caroubier mannosans
Restructuring mannosidase Tth manB is to caroubier mannosans Study on degradation, and concrete steps are as described below:
(1) prepares 2% caroubier mannan solution with the 0.1M imidazoles of pH5.5-adjacent stupid diformazan potassium hydrogen phthalate damping fluid.Mixing is drawn 100 μ L mixed solutions, and the purifying enzyme that adds above-mentioned buffered soln 90 μ L and contain 10 μ L is in the reaction system of 100 μ L, and 0.5h, 1h and 2h are shaken in 80 ℃ of water-baths.
(2) collect respectively above-mentioned 0.5h, 1h and the 2h hydrolyzed solution is preserved in-20 ℃ refrigerator.
(3) developing agent and the developer of the suitable TLC of configuration said hydrolyzed liquid, propyl carbinol in the developing agent: acetic acid: water=2:1:1, developer are with 3 of 1g, the 5-orcin joins in sulfuric acid/methanol solution (2:8) of 100mL formulated.
(4) dip a small amount of 1% mannose solution and above-mentioned sample with capillary pipet, putting respectively to width is that 10cm, length are on the glass silica gel plate of 20cm, has put behind the sample with blowing that extension set dries up and silica-gel plate being positioned in the chromatography cylinder of above-mentioned developing agent.
(5) it is first air-dry to take out sheet glass behind the 5h, is positioned in 100 ℃ the loft drier to dry again, and with watering can silica-gel plate is evenly sprayed in stink cupboard, then dries in being positioned over 100 ℃ loft drier again.
The result as shown in Figure 5, M is 1% seminose reference liquid, 1,2 and 3 are respectively the hydrolyzed solution of 0.5h, 1h and 2h.Result of study shows that restructuring mannosidase Tth manB can effectively be hydrolyzed the non reducing end of caroubier mannosans fast, generates seminose.In sum, this mannosidase is suitable for the degraded of the higher mannosans of the polymerization degree, the mannooligo saccharide of effectively degrading.Therefore, this extremely heat-resisting mannosidase has potential using value in the production of seminose.
SEQUENCE LISTING
＜110〉Nanjing Forestry University
＜120〉a kind of extremely heat-resisting beta-Mannosidase gene and expressing protein and application
<130> 100
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1824
<212> DNA
<213> Thermotoga thermarum
<400> 1
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gacgagtact ttaacgaaga agttattgag aacgagatca tcgaaatgaa aaacctgggc 120
atgaacattt gtcgttcctt cctgtttctg ccgacttttt ttccgaaacc gaacaagatt 180
tctgagaagc atgttgagcg ttatctgaag ttcctgaacc tgtgcgagga gcatggcctg 240
aagaccctgc tgacctttat cgttggtcac atgagcggtg aaaactttga tccgccgttc 300
cgtaaccagc gcgacctgta catggatgaa tttatgctgc agcagcagtg tttttttgtt 360
aagtctattg ttgaaaaagt gcgttcttcc ccggcagttt acggttatat cctgtctaac 420
gaaatgccgc tgtacggtgg tactggcgaa ccggagaaag ttctgaactg ggttaagaaa 480
ctggtggaag tgatcaagag cgttgacccg acccgtccgg tgggcactgg tgatggttgt 540
tggaacgtgt ttggtggcga aaacggtttt aacctgcgtg aaatctctaa aatcgtggac 600
tatctgggtc cgcacgtgta tctgtccgag actgacgaat accgtcactc tatgatcccg 660
gaattcgtga tccgttacct gtcccagtac gacctgccga tcctgtatga agagtttggc 720
gcgtcttctg ctcaggctct ggacgagaac attgcgctgt attaccgcga agtgctgatc 780
aactgcctga ttaacggtgc tatcggtgcg ctgggttggt gtctgaacga tttcaactac 840
ccgaacatga aaccgtatct gcatcacccg tttgaactga aatttggtat cttcaaagtg 900
gacggtgcac gcaaaccggc tgcggaagag attgtgaaat tcaagaactt cgtggattct 960
ctggaaaaca ttcagtatcc gaacgcagaa gcgtgcatcc tggttccgtc ctattacaac 1020
aaacagtacc cgttctcctt tgacgatccg aaggagacct tcaaacatct gctgcaggca 1080
actgttctgt gtgcgaaagc gggtttcgtt gtggacctgg ttgaggaaga aaactataag 1140
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tgggaaaacc tgcacaagta tgttcaggct ggtggtaacc tgtatatctc ttattactgg 1260
ggcaaatatg actttcatca gggtatttgg tcccagaacc tggagtccct gatcggctgc 1320
aagctgaacc tgcgctacgg cctgaccagc tctctgccga gcaaagttaa actggaatat 1380
ggtggtctga cttggaaact gaacgtggag aagtgcaacg aatgggagaa atcctatgct 1440
ccgatcgttt ctatcctgga aaccgctgaa tctatcgagc tgggccagtc cgatctgcag 1500
ctggtgaaga acaaggtggg ctccggtaaa gtgttcttta tcaacttccc gctggagcac 1560
attctgtctg ttaacgagaa aatcaacctg tctgacctga gccatctgat ctaccgctgc 1620
atcgcgaagc aggcttctct gaacctgtgt tactgcgaca accagcgtgt tcgcgtgcgt 1680
aagatcaagt ccggtcgtaa aactctgtac ctgatccaga acatcgcatg ggacaaagag 1740
caggtgcaga ccatcttcga caactcctcc acctatcacg tgcagattga gctggacccg 1800
aaagagtata agctgctgaa cttc 1824
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<213> Thermotoga thermarum
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Claims (9)
1. extremely heat-resisting mannosidase gene, its dna sequence dna is shown in SEQ NO:1.
2. the extremely heat-resisting mannosidase of extremely heat-resisting mannoside enzyme gene expression claimed in claim 1, its aminoacid sequence is shown in SEQ NO:2.
3. extremely heat-resisting mannosidase is characterized in that: aminoacid sequence is that amino acid in the SEQ NO:2 sequence claimed in claim 2 is through replacement, the disappearance of one or more amino-acid residues and add the derived protein with mannoside enzymic activity that forms.
4. a restructuring system is characterized in that: fasten the clone just like the dna sequence dna shown in the SEQ NO:1 at described recombinant chou; Described restructuring system comprises recombinant plasmid and recombinant bacterium.
5. the primer of the extremely heat-resisting mannosidase gene claimed in claim 1 that is used for increasing, it is characterized in that: employed primer is to being:
man1：5’-GGAATTCCATATGGATTTCCTGCTGGGTATTAACT-3’ ；
man2：5’-CCGCTCGAGGAAGTTCAGCAGCTTATACTCTTTC-3’ 。
6. the application of extremely heat-resisting mannosidase claimed in claim 2 in the enzymolysis mannosans.
7. application according to claim 6 is characterized in that: the enzyme digestion reaction temperature is 70-95 ℃, pH5.0-7.5.
8. application according to claim 6 is characterized in that: described mannosans derives from caroubier.
9. recombinant chou claimed in claim 4 ties up to the application in the enzymolysis mannosans.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642779A (en) * | 2013-12-26 | 2014-03-19 | 中国农业科学院饲料研究所 | High-specific activity acidic beta-mannase Man5D as well as gene and application thereof |
| CN104004733A (en) * | 2014-05-29 | 2014-08-27 | 中国农业科学院饲料研究所 | High-temperature acid beta-mannase Man5DW1, and gene and application thereof |
| CN109385411A (en) * | 2017-08-04 | 2019-02-26 | 东莞泛亚太生物科技有限公司 | A kind of beta-Mannosidase and its application |
| CN110819610A (en) * | 2019-10-16 | 2020-02-21 | 江苏大学 | Extremely heat-resistant mannase and preparation method and application thereof |
| CN113337528A (en) * | 2021-06-29 | 2021-09-03 | 浙江农林大学 | Engineering strain of mannosidase and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110294166A1 (en) * | 2010-05-28 | 2011-12-01 | The Board Of Trustees Of The University Of Illinois | Thermostable enzymes for the hydrolysis of mannan-containing polysaccharides |
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2012
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642779A (en) * | 2013-12-26 | 2014-03-19 | 中国农业科学院饲料研究所 | High-specific activity acidic beta-mannase Man5D as well as gene and application thereof |
| CN103642779B (en) * | 2013-12-26 | 2015-09-30 | 中国农业科学院饲料研究所 | A kind of high specific activity acidic beta-mannase Man5D and gene thereof and application |
| CN104004733A (en) * | 2014-05-29 | 2014-08-27 | 中国农业科学院饲料研究所 | High-temperature acid beta-mannase Man5DW1, and gene and application thereof |
| CN104004733B (en) * | 2014-05-29 | 2016-05-04 | 中国农业科学院饲料研究所 | A kind of high-temperature acidic 'beta '-mannase Man5DW1 and gene and application |
| CN109385411A (en) * | 2017-08-04 | 2019-02-26 | 东莞泛亚太生物科技有限公司 | A kind of beta-Mannosidase and its application |
| CN110819610A (en) * | 2019-10-16 | 2020-02-21 | 江苏大学 | Extremely heat-resistant mannase and preparation method and application thereof |
| CN113337528A (en) * | 2021-06-29 | 2021-09-03 | 浙江农林大学 | Engineering strain of mannosidase and application thereof |
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