CN104004733B - A kind of high-temperature acidic 'beta '-mannase Man5DW1 and gene and application - Google Patents
A kind of high-temperature acidic 'beta '-mannase Man5DW1 and gene and application Download PDFInfo
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Abstract
The present invention relates to genetic engineering field. Particularly, the present invention relates to a kind of high-temperature acidic 'beta '-mannase Man5DW1 and gene and application, is its amino acid sequence as SEQ? ID? NO.1 or SEQ? ID? shown in NO.2. The invention provides a new mannase gene, the mannase of its coding has acidity, high temperature, good heat resistance and anti-metal ion ability, can be applied to the industry such as feed, food, medicine. Just can realize according to technical scheme of the present invention the mannase that utilizes the good applicable commercial Application of genetic engineering means nature of production.
Description
Technical field
The present invention relates to genetic engineering field. Particularly, the present invention relates to a kind of high-temperature acidic 'beta '-mannaseMan5DW1 and gene thereof and application.
Background technology
The materials such as plant cell wall three major polymers: cellulose, hemicellulose and lignin form. Mannosan is that plant half is fineTieing up plain important component, is the wire polymer being formed by connecting by β-Isosorbide-5-Nitrae-D-MANNOSE, on the side chain of polysaccharide, mainly contains PortugalThe substituted radicals such as grape glycosyl, acetyl group and galactosyl. 'beta '-mannase (β-mannanaseEC3.2.1.78) is a kind ofThe inscribe hydrolase of hydrolysis mannosan, with internal-cutting way degraded mannose backbone β-l, 4 glycosidic bonds, discharge short β-l, 4Manna oligosacchride.
In recent years, along with the discovery of manna oligosacchride physiological function, the increasing of the rise of green feed and people's environmental consciousnessBy force, the regeneration research of the energy, research and the utilization of people to 'beta '-mannase entered a new stage. β-GanReveal dextranase and be widely used in food, medicine, feed, papermaking, textile printing and dyeing, oil exploitation, fine chemistry industry and biological skillThe numerous areas such as art, are a kind of novel industrial enzymes, have very large potential using value.
'beta '-mannase is extensively present in the biologies such as bacterium, actinomyces, fungi, plant, animal. Bacterial origin sweetRevealing dextranase is mainly the mannase of sour partial neutral. Its molecular weight is many between 35kDa～55kDa, optimal reaction effectTemperature is 50 DEG C～70 DEG C. Most study is bacillus at present, except the suitableeest action pH of Bacillus alcalophilus reachesMore than pH9.0, optimal reaction pH is between 5.5～8.0 mostly. It is acid that the 'beta '-mannase of fungi is generally, and molecular weight is largeAbout 45kDa～55kDa, the suitableeest action pH is 4.0～6.0, and optimum temperature is 55 DEG C～75 DEG C. Bacterium relatively,The 'beta '-mannase optimal reaction pH value of originated from fungus, pH stability is all on the low side, and heat resistance is poorer than bacterium.
Mannase in bacterium generally has good heat resistance. and 50～70 DEG C of optimal reaction temperatures, fungi producesEnzyme optimal reactive temperature be 55～75 DEG C. But heat resistance is poor compared with bacillus. The thermophilic of mannase of plant sourceDegree approaches normal temperature, and heat resistance is all poorer than bacterium and fungal source. Heat-resisting mannase has improved its answering in industryBy value.
Although being cloned, many 'beta '-mannases separate and property testing the character spy of these enzymes at present both at home and abroad,Levy, all have some defects, for example, pH sphere of action is improper, poor heat stability, and expression is low etc., all can not meet realityThe needs of application. Therefore people wish to find a kind of new 'beta '-mannase that can meet practical application request, fromApply in the industries such as feed, food, medicine and can further promote this 'beta '-mannase.
In the Alternariasp. bacterial strain that the present invention separates from saussurea involucrata soil, obtain a new 'beta '-mannaseGene, the mannase of its coding has following advantage: acidity, high temperature, good heat endurance, strong metal ionResistance, easy fermenting and producing. All these advantages all mean that neoteric 'beta '-mannase is at feed, food, medicine etc.In industry, will more there is using value than former reported 'beta '-mannase.
Summary of the invention
The object of this invention is to provide the 'beta '-mannase of a kind of acidity, high temperature, good heat endurance.
A further object of the present invention is to provide the gene of above-mentioned 'beta '-mannase.
A further object of the present invention is to provide the recombinant vector that comprises above-mentioned 'beta '-mannase.
A further object of the present invention is to provide the recombinant bacterial strain that comprises above-mentioned beta-mannase gene.
A further object of the present invention is to provide a kind of method of preparing 'beta '-mannase.
A further object of the present invention is to provide the application of above-mentioned 'beta '-mannase.
The present invention's technical problem first to be solved is to overcome the deficiencies in the prior art, provide a kind of character good,Be suitable for the new high temperature 'beta '-mannase of applying in the industries such as feed, food, medicine. This 'beta '-mannaseMan5DW1, its amino acid sequence is as SEQIDNO.1:
Wherein, 374 amino acid of this enzyme total length, 15 amino acid of N end are signal peptide sequence " MKLLSILSLCATAAA ".
Therefore, the theoretical molecular of ripe 'beta '-mannase Man5DW1 is 39.5kDa, and its amino acid sequence is as SEQIDNO.2：
This 'beta '-mannase Man5DW1 has the feature such as acidity, high temperature simultaneously. Optimal pH is 5.0, at pH3.5-Within the scope of pH9.0, this enzyme can maintain its more than 40% enzyme activity; 70 DEG C of optimum temperatures, within the scope of 40 DEG C-80 DEG C, toolHave more than 40% enzyme activity, this enzyme belongs to high temperature enzyme, at 60 DEG C, processes 60min, and enzyme is lived and substantially do not lost, even this enzymeAt 65 DEG C, process 20min, still can keep 30% enzyme activity, there is good stability; There is fabulous anti-metalThe ability of ion and chemical reagent; High density fermentation enzymatic activity is high simultaneously, is easy to suitability for industrialized production.
The present invention also provides the gene of the above-mentioned 'beta '-mannase of encoding. The complete genome sequence of this enzyme is as SEQIDShown in NO.3:
The present invention is separated and has been cloned this beta-mannase gene man5DW1 by the method for PCR, and DNA complete sequence dividesAnalyse result and show, 'beta '-mannase Man5DW1 structural gene total length 1170bp, contains 1 introne ,+131～178bp, forIts intron sequences, the long 1122bp of cDNA, its cDNA sequence is as shown in SEQIDNO.4:
Wherein, the base sequence of signal peptide is:
“atgaagctactttcaatcctctcgctatgcgcaactgcggcggca”
Therefore, the coded sequence of ripe gene is
Shown in SEQIDNO.5:
Maturation protein theoretical molecular is 39.5kDa, and this enzyme belongs to glycosyl hydrolase the 5th family. By 'beta '-mannaseGene man5DW1cDNA sequence and the amino acid sequence of deriving carry out BLAST comparison in GenBank to be found, determinesMan5DW1 is a kind of new mannase.
The present invention also provides the recombinant vector that comprises above-mentioned beta-mannase gene, is preferably pPIC9-man5DW1.Beta-mannase gene of the present invention is inserted between the restriction enzyme site that expression vector is suitable, makes its nucleotides sequenceBe listed as and be exercisablely connected with expression regulation sequence. As the most preferred embodiment of the present invention, be preferably β-GanReveal xylanase gene and be inserted between the EcoRI and NotI restriction enzyme site on plasmid pPIC9, make this nucleotide sequenceBe positioned at the downstream of AOXl promoter and regulated and controled by it, obtaining expression of recombinant yeast plasmid pPIC9-man5DW1.
The present invention also provides the recombinant bacterial strain that comprises above-mentioned beta-mannase gene, is preferably recombinant bacterial strain GS115/man5DW1。
The present invention also provides a kind of method of preparing acidity, high temperature 'beta '-mannase, comprises the following steps:
1) with above-mentioned recombinant vector transformed host cell, obtain recombinant bacterial strain;
2) cultivate recombinant bacterial strain, the expression of induction restructuring 'beta '-mannase; And
3) reclaim the also expressed 'beta '-mannase of purifying.
Wherein, preferred described host cell is Pichia pastoris (Pichiapastoris) cell, brewer's yeast(Saccharomycescerevisiae) cell or Hansenula polymorpha (Hansenulapolymorpha) cell, preferably willExpression of recombinant yeast plasmid transforms Pichia pastoris (Pichicpastoris) GS115, obtains recombinant bacterial strain GS115/man5DW1。
The present invention also provides the application of above-mentioned 'beta '-mannase. Use genetic engineering means to carry out industrialization and produce high temperatureThe mannosan enzyme product of acid high specific activity have not been reported.
The invention provides a new mannase gene, the mannase of its coding has acidity, high temperature,Good heat resistance and anti-metal ion ability, can be applied to the industry such as feed, food, medicine. According to technical side of the present inventionCase just can realize the mannase that utilizes the good applicable commercial Application of genetic engineering means nature of production.
Brief description of the drawings
The SDS-PAGE of the 'beta '-mannase that Fig. 1 man5DW1 expresses in Pichia pastoris analyzes, l, molecular weight standard;2, the restructuring 'beta '-mannase of purifying.
The recombinate optimum pH of 'beta '-mannase of Fig. 2 the present invention.
The pH stability of Fig. 3 'beta '-mannase of the present invention.
Fig. 4 'beta '-mannase optimal reactive temperature of the present invention.
Fig. 5 beta-mannase enzyme heat stability of the present invention.
Detailed description of the invention
Test material and reagent
1, bacterial strain and carrier: Pichia pastoris (PichiapastorisGS115) is for preserving in this laboratory; Pichia pastoris tableReach carrier pPIC9 and bacterial strain GS115 purchased from Invitrogen company.
2, enzyme and other biochemical reagents: restriction endonuclease is purchased from TaKaRa company, and ligase is purchased from Invitrogen company. SwallowWheat xylan is purchased from Sigma company, and other is all domestic reagent (all can buy and obtain from common biochemical reagents company).
3, culture medium:
(I) culture medium: 30g/L wheat bran, 30g/L maize cob meal, 30g/L dregs of beans, 5g/L konjaku flour, 5g/L (NH4)SO4，1g/LKH2PO4，0.5g/LMgSO4·7H2O，0.01g/LFeSO4·7H2O，0.2g/LCaCl2In 1L deionized waterIn, 121 DEG C, sterilization treatment 20min under 15 pounds of conditions
(2) Escherichia coli culture medium LB (126 peptones, 0.5% yeast extract, 126NaCI, pH7.O).
(3) BMGY culture medium; 1% yeast extract, 2% peptone, 1.34%YNB, 0.000049 < Biotin, 1% is sweetOil (v/v).
(4) BMMY culture medium: replace glycerine divided by 0.5% methyl alcohol, all the other compositions are all identical with BMGY, pH4.0.
Illustrate: the experimental methods of molecular biology that in following examples, work illustrates, all with reference to " molecular cloning experimentGuide " listed concrete grammar carries out in (third edition) J. Pehanorm Brooker one book, or according to kit and product descriptionCarry out.
The clone of embodiment 1 beta-mannase coding gene man5DW1
Extract genomic DNA
By the Liquid Culture bacterium of 3 days, the centrifugal 10min of 12,000rpm, the mycelium of collection adds the mortar of high-temperature sterilizationIn, be ground to rapidly powder with liquid nitrogen, then ground thalline is transferred to one new, 15mlCTAB lysate is housedIn 50mL centrifuge tube, soft turned upside down mixes, and is placed in 70 DEG C of water-bath insulation 3h, and every 20min, turned upside down softly mixesOnce, so that abundant cracking thalline. 4 DEG C, 12, the centrifugal 10min of 000rpm, draws supernatant in new centrifuge tube, the body such as addsLong-pending chloroform extracting, room temperature is placed 5min. 4 DEG C, 12, the centrifugal 10min of 000rpm. Get supernatant and add again isopyknic phenol/chloroformExtracting, room temperature is placed 5min. 4 DEG C, 12, the centrifugal 10min of 000rpm. So that as far as possible except foreigh protein removing, then get supernatant and the body such as addLong-pending isopropyl alcohol, leaves standstill after 5min in room temperature, the centrifugal l0min of l0000rpm at 4 DEG C. Abandon supernatant, precipitation is washed with 70% ethanolTwice, vacuum drying, adds appropriate TE to dissolve, be placed in-20 DEG C for subsequent use.
Degenerate primer P1, P2 (in table 1) have been synthesized in the beta-mannase gene conserved sequence design of delivering according to oneself. WithTotal DNA is that template is carried out pcr amplification. PCR response parameter is: 95 DEG C of 5min; 94 DEG C of 30sec, 50～45 DEG C of 30sec, 72 DEG C30sec, 12 circulations (wherein after each circulation, renaturation temperature declines 1 DEG C); 94 DEG C of 30min, 45 DEG C of 30sec, 72 DEG C of 30sec,30 circulations; 72 DEG C of 10min. Obtain an about 180bp fragment, this fragment is reclaimed to Hou Songruibo Bioisystech Co., Ltd and surveyOrder.
The nucleotide sequence design TAIL-PCR primer uspl obtaining according to order-checking, usp2, usp3; Dspl, dsp2, dsp3(in table 1). Obtain the flanking sequence of known sequence by TAIL-PCR, amplification obtains after product reclaims sending farsighted rich biological skillThe order-checking of art Co., Ltd. After the sheet cracked ends splicing of checking order correct, obtain full-length gene.
The primer that this experiment of table 1 is required
The acquisition of embodiment 2 'beta '-mannase cDNA
Extract total RNA, utilize Oligo (dT)20Obtain a chain of cDNA with reverse transcriptase, then design amplification is open readsFrame primers F and R (in table 1), this strand cDNA that increases, obtains the cDNA sequence of mannase, amplification obtains productReclaim the order-checking of Hou Songruibo Bioisystech Co., Ltd.
Contain 1 and include by finding after the genome sequence to mannase and cDNA sequence alignment that this gene hasSon, the long 1122bp of cDNA, encode 373 amino acid and a terminator codon, 15 amino acid of N end are its signal peptide sequence,The gene that proves the coding mannase that separation clone obtains from Alternariasp. through comparison is new gene.
The structure of embodiment 3 'beta '-mannase engineered strains
(1) structure of expression vector and the expression at yeast
Taking the cDNA that checks order mannase Man5DW1 correct as template, design has been synthesized with EcoRI and NotIThe primers F of restriction enzyme site and R (in table 1), increase in the code area of the maturation protein to Man5DW1. And utilizeEcoRI and NotI enzyme are cut PCR product, connect and enter expression vector pPIC9 (Invitrogen, SanDiego), and β-sweet dew is poly-The sequence of carbohydrase Man5DW1 maturation protein is inserted into the downstream of the signal peptide sequence of above-mentioned expression vector, just forms with signal peptideTrue reading frame, is built into Yeast expression carrier pPIC9-man5DW1, transforms competent escherichia coli cell JM109. PositiveTransformant carries out DNA sequencing, and order-checking shows that transformant that sequence is correct is for preparing in a large number recombinant plasmid. Use restriction enzymeBglII carries out linearisation expression plasmid carrier DNA, and electric shock transformed yeast GS115 competent cell, coats histidine defectProperty RDB flat board, cultivate 2-3 days for 30 DEG C, the transformant that picking is grown on RDB flat board carries out further expressing experiment, toolGymnastics please refer to Pichia anomala expression operation manual.
Build in the same way the expression vector containing the cDNA of Man5DW1 signal peptide sequence, and transform.
(2) screening of high mannosan enzymatic activity transformant
With sterilized toothpick picking list bacterium colony from the long RDB plate that has transformant, first put MM according to numbering upper, then pointTo the MD flat board of corresponding numbering, on each flat board, put 100 single bacterium colonies, amount to 200 transformants; Point is had to transformantMM, MD flat board are placed in 30 DEG C of incubators and cultivate 1～2 day, grow to bacterium colony. Picking transformant inoculation from MD flat board by numberIn the centrifuge tube of 3mLBMGY culture medium is housed, 30 DEG C, 220rpm shaking table cultivation 48h; Shaking table is cultivated to the bacterium liquid 3 of 48h,The centrifugal 15min of 000 × g, removes supernatant, the BMMY culture medium that adds again 1mL to contain 0.5% methyl alcohol in centrifuge tube, 30 DEG C,220rpm induces cultivation; Induction is cultivated after 48h, and the centrifugal 5min of 3,000 × g, gets supernatant and detect for enzymatic activity, therefrom filters outThe transformant of high mannosan enzymatic activity, concrete operations please refer to Pichia anomala expression operation manual.
The recombinate preparation of 'beta '-mannase of embodiment 4
(1) great expression of beta-mannase gene Man5DW1 shaking flask level in Pichia pastoris
Filter out enzyme higher transformant alive, be inoculated in the 1L triangular flask of 300mLBMGY fluid nutrient medium, 30 DEG C,220rpm shaking table shaken cultivation 48h; The centrifugal 5min of 5,000rpm, softly abandons supernatant, then adds 100mL to contain 0.5% to thallineThe BMMY fluid nutrient medium of methyl alcohol, 30 DEG C, 72h is cultivated in 220rpm induction. Between induction culture period, interval 24h adds methyl alcohol one timeSolution, with the loss of compensation methyl alcohol, makes methanol concentration remain on 0.5% left and right; (3) 12, the centrifugal 10min of 000 × g, collects supernatantZymotic fluid, detects enzymatic activity and carries out the analysis of SDS-PAGE protein electrophoresis.
(2) purifying of restructuring 'beta '-mannase
Collect the restructuring 'beta '-mannase supernatant that shaking flask is expressed, concentrate by 10kDa film bag, use less salt simultaneouslyBuffer solution is replaced culture medium wherein, then further concentrated with 10kDa super filter tube. The concentrated weight that can be diluted to certain multipleGroup Man5DW1, carries out purifying by ion-exchange chromatography. Particularly, get Man5DW1 concentrate 2.0mL and use 20mM through in advanceThe HiTrapQSepharoseXL anion column that Tris-HCl (pH7.5) balance is crossed, then carries out with the NaCl of 0-1mol/LLinear gradient elution, the eluent that substep is collected detects enzymatic activity and carries out the mensuration of protein concentration, utilizes SDS-PAGE electricityThe purity (Fig. 1) of swimming analyzing proteins.
The embodiment 5 'beta '-mannase some properties analysis of recombinating
Adopt DNS method to carry out activity analysis to mannase of the present invention. Concrete grammar is as follows: at pH4.5, and 90 DEG C of barsUnder part, the reaction system of 1mL comprises the dilution enzyme liquid that l00 μ L is suitable, 900 μ L substrates, and reaction l0rnin, adds 1.5mLDNSCessation reaction, boiling water boiling 5mn. Cooling rear 540nm measures OD value. Mannosan unit of enzyme activity definition: under certain condition, everyMinute decomposing mannosan, to generate l μ mol reduced sugar required enzyme amount be 1 active unit (U).
(1) optimal pH of mannase Man5DW1 and pH stability
The mannase Man5DW1 that purified embodiment 3 expresses carries out enzymatic reaction to measure under different pHIts optimal pH. Buffer solution used is that citric acid one sodium hydrogen phosphate series buffer solution and the pH9.0～l0.0 of pH2.2～8.0 is sweetPropylhomoserin-NaOH series buffer solution. The mannase Man5DW1 of purifying measures at buffer system .50 DEG C of different pHPH adaptive result (Fig. 2) show: the optimal pH of Man5DW1 is 5.0, and within the scope of pH3.5-pH9.0, this enzyme can maintain itMore than 40% enzyme activity. Enzyme liquid is processed to 60min in the buffer solution of different pH values at 37 DEG C, then measure enzymatic activity to grindStudy carefully the pH stability of enzyme. Result shows (Fig. 3), and analysis result shows can maintain more than 80% enzyme between pH4.0-pH9.0Vigor, illustrates that this enzyme has good pH stability.
(2) mannase Man5DW1 reaction optimum temperature and heat endurance
The mannase of purifying, under pH5.0 condition, is measured the enzymatic activity under different temperatures (30-90 DEG C), analyzes realTest result and show to show, the optimal reactive temperature of this enzyme is 70 DEG C, within the scope of 40 DEG C-80 DEG C, still has more than 40% enzymeVigor, this enzyme belongs to high temperature enzyme (Fig. 4). Its optimum temperature is 70 DEG C. Temperature tolerance is determined as mannase under different temperaturesProcess different time, then at 70 DEG C, carry out enzyme assay. Heat endurance experiment shows: man5-DW1 processes at 60 DEG C60min, residual enzyme work is more than 95%, even if this enzyme is processed 20min at 65 DEG C, still can keep 30% enzyme activity,This shows that this enzyme has good stability (Fig. 5).
The mensuration of the kinetic parameter of embodiment 6 mannase Man5DW1
With carob (the 0.25 – 5mgml of variable concentrations-1) be substrate, at citric acid-sodium hydrogen phosphate buffer solution(pH5.0) in buffer solution system, measure enzymatic activity at 70 DEG C, calculate its KmValue. After measured, the K during taking carob as substratemValue and Vmax are respectively 1.58mgml–1With 1,302 μ molmin–1mg–1. Be 619U/mg than vigor.
The impact of embodiment 7 different chemical reagent mannase Man5DW1 enzymatic activitys.
In enzymatic reaction system, add different chemical reagent (final concentration is respectively 1mmol/L and 5mmol/L), researchThe impact of different chemical reagent on enzymatic activity. Result shows: Mn2+，Co2+, β-Mercaptoethanol is right at 1mM and 5mMEnzyme work has facilitation, and wherein β-Mercaptoethanol effect is the strongest; And SDS, Ag+In the time of 1mM, enzyme is lived and substantially do not had shadowRing, but strong inhibition enzyme is lived in the time of 5mM; Zn2+，Pb+，Cu2+Inhibitory enzyme is lived slightly.
The impact of the various chemical reagent of table 2 on mannase Man5DW1 vigor
Claims (9)
1. a high-temperature acidic 'beta '-mannase Man5DW1, is characterized in that, its amino acid sequence as SEQIDNO.1 orShown in SEQIDNO.2.
2. a high-temperature acidic beta-mannase gene, is characterized in that, a kind of high-temperature acidic claimed in claim 1 of encoding'beta '-mannase Man5DW1.
3. high-temperature acidic beta-mannase gene according to claim 2, is characterized in that, its nucleotide sequence asShown in SEQIDNO.3, SEQIDNO.4 or SEQIDNO.5.
4. comprise the recombinant expression carrier of high-temperature acidic beta-mannase gene described in claim 2.
5. comprise the recombinant expression carrier pPIC9-man5DW1 of high-temperature acidic beta-mannase gene described in claim 2, itsIn, the high-temperature acidic beta-mannase gene by nucleotide sequence as shown in SEQIDNO.5 is with EcoRI and NotI enzymeCut, connect and enter expression vector pPIC9, be built into recombinant expression carrier pPIC9-man5DW1.
6. comprise the recombinant bacterial strain of high-temperature acidic beta-mannase gene described in claim 2.
7. comprise the recombinant bacterial strain GS115/man5DW1 of high-temperature acidic beta-mannase gene described in claim 2, wherein,By express the high-temperature acidic beta-mannase of nucleotide sequence as shown in SEQIDNO.5 in Pichia pastoris GS115Enzyme gene obtains described recombinant bacterial strain GS115/man5DW1.
8. a method of preparing high-temperature acidic 'beta '-mannase Man5DW1, is characterized in that, comprises the following steps:
(1) with recombinant expression carrier transformed host cell claimed in claim 4;
(2) cultivate host cell;
(3) separation and purification obtains high-temperature acidic 'beta '-mannase Man5DW1.
Described in claim 1 high-temperature acidic 'beta '-mannase Man5DW1 for being hydrolyzed the application of mannosan.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392241A (en) * | 2007-09-18 | 2009-03-25 | 中国科学院微生物研究所 | Beta-mannanase, gene, preparation method, vector and host cell |
| CN101724614A (en) * | 2010-01-12 | 2010-06-09 | 中南大学 | Acid beta-mannase, genes, engineering bacteria and structure thereof |
| CN102392036A (en) * | 2011-12-12 | 2012-03-28 | 江南大学 | Cloning of beta-1,4-endomannase (Tvi Man5A) gene and preparation of recombinase |
| CN102628056A (en) * | 2012-04-24 | 2012-08-08 | 中南林业科技大学 | Acid-resistant and high temperature-resistant beta-mannase gene and application thereof |
| CN102676477A (en) * | 2011-08-19 | 2012-09-19 | 济南诺能生物工程有限公司 | Transformation of acidic beta-mannase gene and construction of engineering bacteria of acidic beta-mannase gene |
| CN102978187A (en) * | 2011-09-06 | 2013-03-20 | 北京卫诺恩生物科技有限公司 | High-activity beta-mannanase MAN5A with pH value within range of 2.5-6.5, and gene and application thereof |
| CN102978222A (en) * | 2012-12-19 | 2013-03-20 | 南京林业大学 | Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof |
| CN102994479A (en) * | 2012-11-29 | 2013-03-27 | 青岛蔚蓝生物集团有限公司 | Mannose |
-
2014
- 2014-05-29 CN CN201410234352.3A patent/CN104004733B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392241A (en) * | 2007-09-18 | 2009-03-25 | 中国科学院微生物研究所 | Beta-mannanase, gene, preparation method, vector and host cell |
| CN101724614A (en) * | 2010-01-12 | 2010-06-09 | 中南大学 | Acid beta-mannase, genes, engineering bacteria and structure thereof |
| CN102676477A (en) * | 2011-08-19 | 2012-09-19 | 济南诺能生物工程有限公司 | Transformation of acidic beta-mannase gene and construction of engineering bacteria of acidic beta-mannase gene |
| CN102978187A (en) * | 2011-09-06 | 2013-03-20 | 北京卫诺恩生物科技有限公司 | High-activity beta-mannanase MAN5A with pH value within range of 2.5-6.5, and gene and application thereof |
| CN102392036A (en) * | 2011-12-12 | 2012-03-28 | 江南大学 | Cloning of beta-1,4-endomannase (Tvi Man5A) gene and preparation of recombinase |
| CN102628056A (en) * | 2012-04-24 | 2012-08-08 | 中南林业科技大学 | Acid-resistant and high temperature-resistant beta-mannase gene and application thereof |
| CN102994479A (en) * | 2012-11-29 | 2013-03-27 | 青岛蔚蓝生物集团有限公司 | Mannose |
| CN102978222A (en) * | 2012-12-19 | 2013-03-20 | 南京林业大学 | Extremely-heat-resistant beta-mannosidase gene as well as expression protein and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Expression of Bacillus subtilis MA139 β-mannanase in Pichia pastoris and the Enzyme Characterization;Jiayun Qiao et al.;《Appl Biochem Biotechnol》;20101231;第160卷;1362-1370 * |
| β-甘露聚糖酶基因的克隆表达及酶学性质;毛绍名 等;《中南林学院学报》;20061231;第26卷(第6期);17-21 * |
| β-甘露聚糖酶研究进展;崔栩 等;《中国畜牧杂志》;20101231;第46卷(第15期);69-72 * |
| 微生物β-甘露聚糖酶的制备与应用研究进展;许牡丹 等;《动物医学进展》;20061231;第27卷(第9期);31-34 * |
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