CN1351169A - Gene sequence of beta-mannase and process for preparing recombinant enzyme coded by it - Google Patents
Gene sequence of beta-mannase and process for preparing recombinant enzyme coded by it Download PDFInfo
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- CN1351169A CN1351169A CN 00130066 CN00130066A CN1351169A CN 1351169 A CN1351169 A CN 1351169A CN 00130066 CN00130066 CN 00130066 CN 00130066 A CN00130066 A CN 00130066A CN 1351169 A CN1351169 A CN 1351169A
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Abstract
A novel alkaline beta-mannanase gene obtained from the general DNA of alkaliphilic bacillus N16-5 and its preparing process is disclosed. Its prokaryotic expression plasmid is configured to transform colibacillus for expressing said beta-mannanase. Said beta-mannanase can be used to hydrolyze refined konjak starch to obtain a oligose series with high total conversion rate up to 80% or more, which can be used to prepare function food.
Description
The present invention relates to a kind of gene order of 'beta '-mannase, the preparation of the recombinase of this genes encoding and contain the goods of this enzyme.Specifically, the 'beta '-mannase dna molecular of Alkaliphilic bacillus (alkaliphilic Bacillus) N16-5 that the present invention relates to encode relates to recombinant plasmid that contains this enzyme gene and the recombinant bacterial strain of expressing corresponding enzyme.
'beta '-mannase (β-mannanase, EC 3.2.1.78) is a kind of hemicellulase, can the hydrolysis mannosans, vegetable polysaccharides such as glucomannan, polygalactomannan and gala glucomannan is (referring to Tipdon, R.S.et al.:Advances inCarbohydrate Chemistry and Biochemistry, 32:299-316, AcademicPress, New York, 1976.).Mannosans is a kind of hemicellulose resource than horn of plenty that occurring in nature exists, next in number only to Mierocrystalline cellulose.Seeds of leguminous plant, wood of coniferous tree, green coffee berry etc. all contain a large amount of mannosanss, some vegetable jelly, the mannosans of almost completely being made up of semi-lactosi or glucose and seminose as the blue or green glue in field, carob bean gum, konjaku powder etc.Utilize 'beta '-mannase that above-mentioned vegetable material is carried out deep processing and fully utilize to have very big application potential, particularly at food, have the importance using value aspect medical, because 'beta '-mannase can be degraded to mannooligo saccharide to the plant mannosans, mannooligo saccharide can promote growing (referring to Akino of human intestinal normal microflora, T.et al.:Agric.Biol.Chem., 52:773-779,1988), this is significant to the health level that improves human body.
'beta '-mannase from difference source biology separate, as genus bacillus, gas sporangium, yellow sporangium, shuttle spore bacterium, mould, wood is mould and streptomycete etc.
(7)The relevant patent and the starting point of document mainly are to handle hemicellulose in the paper industry (referring to Buchert et al.:USP5,661,021 Aug.26,1997; Ratto, M.et al.:Biotechnol.Letters, 10:661-664,1988; With Christgau et al.USP5,795,764 Aug.18,1998), a kind of beta-mannase enzyme product that mannooligo saccharide is produced that is suitable for is not arranged as yet, its subject matter is the enzymic activity complexity, the substrate level is low etc., directly influences the productive rate and the yield of mannooligo saccharide.'beta '-mannase hydrolyzing plant mannosans is reflected under the alkaline condition and carries out, and can increase the swelling of hemicellulose in water, thereby helps the effect of enzyme.Present known microorganism 'beta '-mannase optimal pH is generally at acidity or neutral range, and the facultative Alkaliphilic bacillus AM001 of Japanology, optimal pH be at 8.5-9.0, and attempts to carry out the Production by Enzymes of mannooligo saccharide, to the transformation efficiency 29% of mannooligo saccharide.In addition, the patent of mannase gene and bibliographical information more in recent years (referring to USP5,661,021 and USP5,795,764), but the report of having a liking for alkali bacterium alkaline ' beta '-mannase gene is seldom arranged.
An object of the present invention is to provide a kind of beta-mannase gene.
Another object of the present invention provides a kind of method for preparing 'beta '-mannase.
A further object of the present invention provides and contains described beta-mannase gene recombinant expression plasmid and recombinant bacterial strain, and its gene expression product is used for the High-efficient Production mannooligo saccharide.
The invention provides the dna molecular of the 'beta '-mannase of a kind of Alkaliphilic bacillus N16-5 that encodes, the aminoacid sequence of this 'beta '-mannase is SEQ ID NO:2.
According to dna molecular of the present invention, wherein, described dna molecular comprises SEQ NO:1 dna sequence dna.
The present invention also provides the recombinant expression plasmid that contains aforesaid dna sequence dna.
According to recombinant expression plasmid of the present invention, wherein, described recombinant expression plasmid is recombinant expression plasmid pMAN1 and the recombinant expression plasmid pMAN2 that contains above-described dna sequence dna.
The invention still further relates to the recombinant bacterial strain that contains aforesaid recombinant expression plasmid, comprise Alkaliphilic bacillus N16-5 bacterial strain, e. coli jm109 bacterial strain JM109MAN1 and e. coli jm109 bacterial strain JM109MAN2.
According to another aspect of the present invention, the invention provides the method that a kind of preparation has the 'beta '-mannase of enzymic activity, comprise the steps:
(1) from Alkaliphilic bacillus N16-5 bacterial strain, extracts total DNA, through the restriction enzyme portion water
Separate, obtain dna fragmentation, be connected on the pUC19 carrier and transformed into escherichia coli
JM109 obtains to contain the recombinant expression plasmid pMAN1 of beta-mannase gene and weigh
Group coli strain JM109MAN1;
(2) isolate the DNA sheet that contains beta-mannase gene from recombinant expression plasmid pMAN1
Section again through the restriction enzyme partial hydrolysis, obtains the small pieces segment DNA, is connected to pUC19
On the carrier and transformed into escherichia coli JM109, obtain to contain the weight of beta-mannase gene
Group expression plasmid pMAN2 and recombinant escherichia coli strain JM109MAN2.
Accompanying drawing is briefly described
Fig. 1 is that alkaline ' beta '-mannase gene recombination plasmid pMAN1 of the present invention makes up mode chart.
Fig. 2 is that alkaline ' beta '-mannase gene recombination plasmid pMAN2 of the present invention makes up mode chart.
Fig. 3 is the expression of alkaline ' beta '-mannase gene of the present invention in intestinal bacteria.
Fig. 4 is the 'beta '-mannase dna sequence dna from Alkaliphilic bacillus N16-5 of the present invention.
Fig. 5 is the aminoacid sequence of 'beta '-mannase of the present invention.
Describe the present invention below in conjunction with accompanying drawing.
The present invention is based on the present inventor's following discovery and finishes: Alkaliphilic bacillus N16-5 produces a large amount of alkaline ' beta '-mannases under alkali condition, this enzyme is particularly suitable for hydrolysis and plants The thing polysaccharide forms manna oligosacchride, and such as zymohydrolysis of konjaku flour etc., oligosaccharides conversion rate reaches more than 80%.
The present invention separates the gene that obtains 'beta '-mannase from Alkaliphilic bacillus N16-5, and it is The DNA of 1479bp, its dna sequence dna figure as shown in Figure 4 (SEQ ID NO:1) coding one by The protein that 493 amino acid forms, its amino acid sequence figure as shown in Figure 5 (SEQ ID NO: 2). Obtained to contain the recombinant expression plasmid of this gene by conventional method, transformed into escherichia coli makes Recombinant bacterial strain is expressed 'beta '-mannase. Therefore the present invention provides a kind of possibility simultaneously, and is namely logical Cross genetic engineering or molecular biology method, the enzyme Gene cloning that the present invention relates to is arrived basophilic gemma bar Bacterium or other acceptor bacterium are cultivated bar by Alkaliphilic bacillus N16-5 bacterial strain or other bacterial strain or at other Part produces the 'beta '-mannase that the present invention relates to. The 'beta '-mannase that the present invention relates to is special Be suitable for the enzymolysis production of manna oligosacchride, also for utilizing konjaku polysaccharide etc. to produce in a large number the sweet dew widow for raw material Sugar provides a kind of possibility.
The 'beta '-mannase that the present invention relates to is suitable for the enzymolysis production of manna oligosacchride especially, for building The vertical konjaku polysaccharide etc. of utilizing is raw material, the technology of producing in a large number manna oligosacchride, provide a kind of may.
To achieve the object of the present invention, the method for preparing 'beta '-mannase of the present invention and recombinant expression plasmid thereof and recombinant bacterial strain comprises the steps:
(1) from Alkaliphilic bacillus N16-5, extract total DNA, through the restriction enzyme partial hydrolysis,
To dna fragmentation, be connected on the pUC19 carrier and transformed into escherichia coli JM109, obtain
Restructuring plasmid pMAN1 and the recombination bacillus coli bacterium that must contain beta-mannase gene
Strain JM109MAN1;
(2) isolate the dna fragmentation that contains beta-mannase gene from restructuring plasmid pMAN1,
Through restriction enzyme hydrolysis, obtain the small pieces segment DNA again, be connected on the pUC19 carrier and turn to
Change e. coli jm109, obtain to contain the restructuring plasmid of beta-mannase gene
PMAN2 and recombination bacillus coli JM109MAN2.
Under the condition that above-mentioned beta-mannase gene is expressed, cultivate this restructuring bacterium, containing On the culture medium of beta-mannase, periphery of bacterial colonies forms transparent circle, prove this restructuring bacterium express β-Sweet dew glycan enzymatic activity. This protein has sweet dew glycan enzymatic activity, and pI is 4.3, and is the suitableeest PH9.5,70 ℃ of optimum temperatures, but effectively hydrolyzing plant sweet dew glycan produces oligosaccharides. Order-checking shows, This enzymatic structure gene is the DNA of 1479bp, the albumen that is made up of 493 amino acid of encoding Matter.
The expression product of said gene, the hydrolysis konjaku powder carries out oligosaccharides and transforms. Concentration of substrate 5-15%, reaction temperature 40-60 ℃, reaction pH 9-10, time 8-24 hour, after reaction finishes, With acetic acid, citric acid or hydrochloric acid are transferred pH to 5-6, add 0.5-2% powder or granular active carbon and take off Look. Filtration can obtain the manna oligosacchride syrup. Oligosaccharides conversion rate is more than 80%, total recovery 70% with On, wherein 2-8 sugar accounts for more than 80% of total reducing sugar, and this oligosaccharides is for the life that promotes two divergent bacillus in the body Long, raising human health level has important function.
The 'beta '-mannase that the present invention relates to is a novel 'beta '-mannase. According to enzyme Amino acid sequence similarity, at present known 'beta '-mannase belongs to glycoside hydrolase the 5th With 26 two families (referring to Ethier, N.et al.:Appl.Environ.Microbiol. 64:4428-4432,1998). By what the beta-mannase gene that the present invention relates to was derived The amino acid sequence comparative analysis, the 'beta '-mannase that the present invention relates to belongs to glycoside hydrolase the 5th A member in the family is compared with the amino acid sequence of other 'beta '-mannase of having reported, and is similar Property is less than 60%.
The performance of the 'beta '-mannase that the present invention relates to is different from known 'beta '-mannase, Its suitableeest pH and temperature are the highest in the 'beta '-mannase of finding so far. The present invention carries The structure gene that utilizes conventional method to obtain this novel 'beta '-mannase, recombinant expressed matter have been supplied Grain and restructuring thalline pattern make 'beta '-mannase of the present invention by other bacterial strain or in other cultivation Condition produces. Simultaneously, the invention provides a kind ofly take konjaku powder as raw material, enzymolysis is produced the sweet dew widow The effective ways of sugar can reach 80% to the conversion rate of manna oligosacchride.
'beta '-mannase of the present invention has following feature:
(1) produced by Alkaliphilic bacillus N16-5 or its bacterium that derives, the bacterium that derives is meant to transform this
The recombinant bacterial strain of the dna fragmentation that invention relates to;
(2) has dna sequence encoding shown in Figure 4;
(3) has aminoacid sequence shown in Figure 5;
(4) its characteristic has at least following a kind of: 1) have the mannosans enzymic activity, pI is
4.3, optimal pH 9.0,70 ℃ of optimum temperutures, molecular weight 51000 dalton; 2)
Have the mannosans enzymic activity, pI is 2.5, optimal pH 10.0, optimum temperuture 70
℃, molecular weight 38000 dalton; 3) have the mannosans enzymic activity, pI is
2.5, optimal pH 9.0,70 ℃ of optimum temperutures, molecular weight 34700 dalton;
(5) but the effectively hydrolyzing vegetable polysaccharides produces oligosaccharides.
Further, the present invention relates to a dna molecular, the 'beta '-mannase that this dna molecule encode the present invention relates to, this dna nucleotide sequence:
(1) partly forms by dna sequence dna codified shown in Figure 4;
(2) coding one protein, its aminoacid sequence aminoacid sequence as shown in the figure.
Below by embodiment the present invention is described in more detail.It should be understood that described embodiment is only used for explanation rather than restriction the present invention.The extraction of the total DNA of embodiment 1 Alkaliphilic bacillus N16-5
Adopt the therefrom isolating Alkaliphilic bacillus N16-5 of state Inner Mongol crow Du's mire alkali lake, get its fresh wet thallus 20 grams, be suspended from 10 milliliters of 50mMTris damping fluids (pH8.0), add a small amount of N,O-Diacetylmuramidase and 8 milliliters of 0.25mMEDTA (pH8.0), place 20min in 37 ℃ behind the mixing, add 2 milliliters of 10%SDS afterwards, place 5min, use equal-volume phenol respectively for 55 ℃, each extracting of chloroform is once got last supernatant solution, add 2 times of volume ethanol, reclaim DNA, respectively with 70% and dehydrated alcohol wash, precipitate and be dissolved in 0.5 milliliter of TE damping fluid (pH8.0,10mM Tris, 1mMEDTA), add 10mg/ml RNase3 μ l, 37 ℃ are incubated 1 hour, use equal-volume phenol respectively, each extracting of chloroform once, supernatant liquor adds 2 times of volume ethanol, reclaims DNA, respectively with 70% and dehydrated alcohol wash, deionized water dissolving is used in vacuum-drying.The ultraviolet spectrophotometer measurement result of dna solution is A260/A280=1.98, A260/A230=2.18.The clone of embodiment 2 beta-mannase genes
Get foregoing total dna solution 10 μ l (about 50 μ gDNA), use the limiting enzyme EcoRI partial hydrolysis,, obtain the 2-10kbDNA fragment through agarose gel electrophoresis.Get 2 μ l (5 μ g) EcoRI enzymolysis dna fragmentation and 1 μ l (1 μ g) carries out ligation through the plasmid pUC19DNA of EcoRI enzymolysis in 20 μ l linked systems, wherein contain 2 μ l (10X connects damping fluid), 1 μ lT4DNA ligase enzyme, 14 μ l water.Linked system behind the transformed competence colibacillus e. coli jm109, is applied to and contains Amp (penbritin), on the LB solid medium of IPTG and X-gal 16 ℃ of reactions 16 hours.Cultivated 16-18 hour for 37 ℃, the picking hickie is in the liquid nutrient medium that contains Amp and mannosans or contain Amp and the solid medium of mannosans, cultivated 18-20 hour for 37 ℃, can liquefy mannosans or containing on the solid medium of beta-mannase the bacterium colony that forms transparent circle is defined as the positive colony (see figure 3).Positive bacterium colony is extracted recombinant plasmid with alkaline process, with various restriction enzyme hydrolysis recombinant plasmids, confirm to have dna fragmentation to insert plasmid according to electrophoresis result, its size is about 8.0kb.The recombinant plasmid that contains this dna fragmentation is called the pMAN1 (see figure 1), and the recombination bacillus coli that contains this recombinant plasmid pMAN1 is called e. coli jm109 MAN1.
But this recombinant plasmid pMAN1 high frequency transformed into escherichia coli is expressed beta-mannase enzymic activity and anti-ammonia benzyl performance.DNA in the recombinant plasmid is inserted fragment digoxin dna marker detection kit mark, carry out Southern blot DNA hybrid experiment with the chromosomal DNA of Alkaliphilic bacillus N16-5, the dna fragmentation that inserts among the confirmation recombinant plasmid pMAN1 is from the chromosomal DNA of Alkaliphilic bacillus N16-15.The subclone and the sequence of embodiment 3 beta-mannase genes
Get insertion dna segment among the plasmid pMAN1 of 10 μ 1 in 50 μ l systems, carry out various restriction enzyme enzymolysis reactions, as: the list of AccI, HindIII, PstI, EcoRI and XbalI etc. or double digestion reaction, 37 ℃ are incubated 1.5 hours, and the agarose gel electrophoresis purifying reclaims dna fragmentation.Method as previously mentioned, single or the double enzymolysis dna fragmentation that obtains is connected to plasmid pUC19DNA or pGEM-4Z, form a series of subclone plasmids, the transformed competence colibacillus e. coli jm109, correspondingly obtain a series of recombination bacillus colis, cultivate the back and survey its enzyme and live, the result shows that containing the HincII enzyme cuts the recombination bacillus coli of dna fragmentation and have the mannosans enzymic activity, and this HincII enzyme is cut dna fragmentation and is about 3.5kb.The recombinant plasmid that contains this dna fragmentation is called the pMAN2 (see figure 2), and the recombination bacillus coli that contains this recombinant plasmid pMAN2 is called e. coli jm109 MAN2.Adopt the Sanger dideoxy method that this dna fragmentation is checked order.Sequencing result is seen Fig. 4, and the HincII enzyme is cut dna fragmentation total length 3419bp, contains the open reading frame (ORF) of a long 1479bp, is begun by the ATG initiator codon, to the ending of ATT terminator codon.Protein of forming by 492 amino acid of this complete ORF coding.The purifying and the characteristic of embodiment 4. reorganization 'beta '-mannases
The thalline of reorganization bacterium E.coli JM109MAN2 is suspended from the 10mM glycine buffer (pH9.6), utilizes the ultrasonic disruption cell, and centrifuged supernatant is the crude enzyme liquid of reorganization 'beta '-mannase.Clear enzyme solution is through DEAE-Sephadex A-25 ion-exchange chromatography on this, and hydroxyapatite adsorpting column chromatography and PAGE prepare steps such as electrophoresis and carry out purifying, and the zymin that obtains shows a band on SDS-PAGE.Utilize the standard method of known protein materialization to measure the fundamental characteristics of this reorganization 'beta '-mannase.The molecular weight of the recombinase that records with SDS-PAGE is 51000 dalton, and is close with the molecular weight of calculating in theory (54000 dalton); The iso-electric point pI of the recombinase that records with PAGEIEF is 4..3; The optimal pH of recombinase reaction is 9..5, and optimum temperuture is 70 ℃.High pressure lipuid chromatography (HPLC) records reorganization 'beta '-mannase hydrolysis Rhizoma amorphophalli powder and produces a series of oligosaccharides such as 2-8 sugar.Embodiment 5 reorganization 'beta '-mannase hydrolysis konjaku powders carry out oligosaccharides and transform
2.5L dress 1.8L water is warming up to 55 ℃ in the reactor, adds Na
2CO
34.8 gram, NaHCO
33 grams, 'beta '-mannase 1.8 * 10
4Unit, Rhizoma amorphophalli powder 180 grams.55 ℃ are incubated 16 hours.Transfer pH to 5.5-6.0 with HCl, add granular active carbon 10 grams, be warming up to 100 ℃ and kept 5 minutes, be cooled to 70 ℃ and filter (general industry filter cloth), 2-8 sugar accounts for more than 80% of total reducing sugar in the products therefrom, and the oligosaccharides transformation efficiency is more than 80%, and yield is more than 70% (table 1).
Table 1, mannooligo saccharide component change (time (h) the total reducing sugar oligosaccharides 2-6 sugar transformation efficiency 0 9.4 of gram/100ml) // 04 9.2 1.725 46% 19% 8 9.7 6.894 70% 71% 16 9.6 8.178 81% 85%
Claims (7)
1, the dna molecular of the 'beta '-mannase of coding Alkaliphilic bacillus N16-5, the aminoacid sequence of this 'beta '-mannase is SEQ ID NO:2.
2, dna molecular as claimed in claim 1 is characterized in that this molecule comprises SEQ NO:1 dna sequence dna.
3, the recombinant expression plasmid that contains the described dna sequence dna of claim 1.
4, recombinant expression plasmid as claimed in claim 3 is characterized in that this recombinant expression plasmid is the recombinant expression plasmid pMAN2 that contains the recombinant expression plasmid pMAN1 of the described dna sequence dna of claim 1 and contain 1 described dna sequence dna.
5, the recombinant bacterial strain that contains the described recombinant expression plasmid of claim 3.
6, recombinant bacterial strain as claimed in claim 4 is characterized in that described bacterial strain comprises Alkaliphilic bacillus N16-5 bacterial strain, e. coli jm109 bacterial strain JM109MAN1 and e. coli jm109 bacterial strain JM109MAN2.
7, a kind of preparation has the method for the 'beta '-mannase of enzymic activity, comprises the steps:
(1) from Alkaliphilic bacillus N16-5 bacterial strain, extracts total DNA, through the restriction enzyme partial hydrolysis, obtain dna fragmentation, be connected on the pUC19 carrier and transformed into escherichia coli JM109, obtain to contain the recombinant expression plasmid pMAN1 and the recombinant escherichia coli strain JM109MAN1 of beta-mannase gene;
(2) isolate the dna fragmentation that contains beta-mannase gene from recombinant expression plasmid pMAN1, again through the restriction enzyme partial hydrolysis, obtain the small pieces segment DNA, be connected on the pUC19 carrier and transformed into escherichia coli JM109, obtain to contain the recombinant expression plasmid pMAN2 and the recombinant escherichia coli strain JM109MAN2 of beta-mannase gene.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100410380C (en) * | 2005-07-26 | 2008-08-13 | 中国农业科学院饲料研究所 | Beta mannosidase, its coding gene and application |
| CN100420744C (en) * | 2006-04-10 | 2008-09-24 | 中国农业大学 | Beta-mannase, its expression and special engineering bacterium |
| CN102181416A (en) * | 2011-03-11 | 2011-09-14 | 中国农业科学院饲料研究所 | Alkali-resisting beta-mannase Man5A as well as gene and applications thereof |
| CN102433267A (en) * | 2010-09-29 | 2012-05-02 | 中国科学院微生物研究所 | Method for preparing beta-mannase and special strain |
| CN102533700A (en) * | 2011-12-31 | 2012-07-04 | 张珂卿 | Beta-mannase, and coding 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 |
| CN103088008A (en) * | 2011-10-31 | 2013-05-08 | 中国科学院微生物研究所 | Cytidine deaminase, its coding gene, and applications of cytidine deaminase and its coding gene |
| CN103451168A (en) * | 2012-05-30 | 2013-12-18 | 青岛蔚蓝生物集团有限公司 | Mannanases and recombinant expression bacterial strain thereof |
| CN104762243A (en) * | 2014-01-08 | 2015-07-08 | 中国科学院微生物研究所 | Recombinant strain and method for preparing alkaline beta-mannanase |
| CN105754970A (en) * | 2014-12-19 | 2016-07-13 | 中国科学院微生物研究所 | Alkaline beta-mannase, encoding genes thereof and application of encoding genes |
| CN109097348A (en) * | 2018-07-26 | 2018-12-28 | 天津科技大学 | The application of alpha-galactosidase and its complex enzyme in galactomannan degradation |
| CN110669805A (en) * | 2019-09-27 | 2020-01-10 | 天津科技大学 | Method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing locust bean gum through compounding of mannanase and galactosidase |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE528394T1 (en) * | 1998-06-10 | 2011-10-15 | Novozymes As | NOVEL MANNASES |
| US6060299A (en) * | 1998-06-10 | 2000-05-09 | Novo Nordisk A/S | Enzyme exhibiting mannase activity, cleaning compositions, and methods of use |
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2000
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100410380C (en) * | 2005-07-26 | 2008-08-13 | 中国农业科学院饲料研究所 | Beta mannosidase, its coding gene and application |
| CN100420744C (en) * | 2006-04-10 | 2008-09-24 | 中国农业大学 | Beta-mannase, its expression and special engineering bacterium |
| CN102433267B (en) * | 2010-09-29 | 2013-08-07 | 中国科学院微生物研究所 | Method for preparing beta-mannase and special strain |
| CN102433267A (en) * | 2010-09-29 | 2012-05-02 | 中国科学院微生物研究所 | Method for preparing beta-mannase and special strain |
| CN102181416A (en) * | 2011-03-11 | 2011-09-14 | 中国农业科学院饲料研究所 | Alkali-resisting beta-mannase Man5A as well as gene and applications thereof |
| CN102181416B (en) * | 2011-03-11 | 2013-06-26 | 中国农业科学院饲料研究所 | Alkali-resisting beta-mannase Man5A as well as gene and applications thereof |
| CN103088008B (en) * | 2011-10-31 | 2014-08-20 | 中国科学院微生物研究所 | Cytidine deaminase, its coding gene, and applications of cytidine deaminase and its coding gene |
| CN103088008A (en) * | 2011-10-31 | 2013-05-08 | 中国科学院微生物研究所 | Cytidine deaminase, its coding gene, and applications of cytidine deaminase and its coding gene |
| CN102533700A (en) * | 2011-12-31 | 2012-07-04 | 张珂卿 | Beta-mannase, and coding gene and application thereof |
| CN103451168A (en) * | 2012-05-30 | 2013-12-18 | 青岛蔚蓝生物集团有限公司 | Mannanases and recombinant expression bacterial strain thereof |
| CN103451168B (en) * | 2012-05-30 | 2015-03-18 | 青岛蔚蓝生物集团有限公司 | Mannanases and recombinant expression bacterial strain thereof |
| CN102994479B (en) * | 2012-11-29 | 2014-02-19 | 青岛蔚蓝生物集团有限公司 | Mannose |
| CN102994479A (en) * | 2012-11-29 | 2013-03-27 | 青岛蔚蓝生物集团有限公司 | Mannose |
| CN104762243A (en) * | 2014-01-08 | 2015-07-08 | 中国科学院微生物研究所 | Recombinant strain and method for preparing alkaline beta-mannanase |
| CN105754970A (en) * | 2014-12-19 | 2016-07-13 | 中国科学院微生物研究所 | Alkaline beta-mannase, encoding genes thereof and application of encoding genes |
| CN105754970B (en) * | 2014-12-19 | 2019-07-12 | 中国科学院微生物研究所 | A kind of application of alkaline ' beta '-mannase and its encoding gene and they |
| CN109097348A (en) * | 2018-07-26 | 2018-12-28 | 天津科技大学 | The application of alpha-galactosidase and its complex enzyme in galactomannan degradation |
| CN110669805A (en) * | 2019-09-27 | 2020-01-10 | 天津科技大学 | Method for producing galactose, mannose and mannan oligosaccharide by hydrolyzing locust bean gum through compounding of mannanase and galactosidase |
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| CN100453646C (en) | 2009-01-21 |
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Assignee: Chengdu Yong'an Pharmaceutical Co., Ltd. Assignor: Institute of Microbiology, Chinese Academy of Sciences Contract record no.: 2012990000441 Denomination of invention: Gene sequence of beta-mannase and process for preparing recombinant enzyme coded by it Granted publication date: 20090121 License type: Exclusive License Open date: 20020529 Record date: 20120625 |
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