CN101974543A - Extracellular pullulanase encoding gene derived from bacilli and application thereof - Google Patents
Extracellular pullulanase encoding gene derived from bacilli and application thereof Download PDFInfo
- Publication number
- CN101974543A CN101974543A CN2010102562876A CN201010256287A CN101974543A CN 101974543 A CN101974543 A CN 101974543A CN 2010102562876 A CN2010102562876 A CN 2010102562876A CN 201010256287 A CN201010256287 A CN 201010256287A CN 101974543 A CN101974543 A CN 101974543A
- Authority
- CN
- China
- Prior art keywords
- pullulanase
- pula042
- gene
- starch
- bacillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The invention relates to an extracellular pullulanase encoding gene derived from bacilli and application thereof, which belong to the technical field of microorganism and enzyme engineering. A bacillus sp.042 with pullulan decomposing capacity is screened, the complete sequence of the encoding area of a pullulanase encoding gene pulA042 is cloned by utilizing an inverse PCR (Polymerase Chain Reaction) technology, sequence analysis shows that the whole length of the encoding area of the gene is 2571 bases, a polypeptide chain with 856 amino acid residues is encoded, and a typical signal peptide is formed by 32 amino acid residues at the N end of the polypeptide chain, so that an extracellular pullulanase is encoded by the gene pulA042. The expression product of the pullulanase gene pulA042 of the bacillus sp.042 has the activity of decomposing pullulan and red pullulan, can decompose an alpha-1,6 glycosidic bond in starch, can not decompose an alpha-1,4 glycosidic bond and is an I-type pullulanase. A recombinant enzyme produced by utilizing the gene pulA042 is used for decomposing the alpha-1,6 glycosidic bond in starch or dextrin molecules in starch processing.
Description
Technical field
The present invention relates to adopt a kind of Pullulanase encoding gene and the application thereof of genetic engineering means acquisition, belong to microorganism, technical field of enzyme engineering.
Background technology
Starchy material is one of industrial raw material of widespread use the most so far, starch is the mixture that is made of amylose starch and amylopectin, wherein amylose starch passes through α-1 by glucose, the 4-glycosidic link is connected to form, and amylopectin also contains branched chain, that connect bifurcation is α-1, the 6-glycosidic link.Amylolytic enzyme is the class of enzymes that can be hydrolyzed and utilize starchy material, and the most commonly used with α-Dian Fenmei and saccharifying enzyme, these two kinds of enzymes all act on the α-1 of starch molecule, 4-glycosidic link.Because the starch of industrial use generally all contains a certain amount of side chain, therefore the thorough hydrolysis of starch molecule be needed to use a class specificity to cut amylopectin tapping point α-1, the enzyme of 6-glycosidic link, this kind of enzyme is commonly referred to as separates an enzyme, the most commonly used with Pullulanase and isoamylase, both α-1 in can both the hydrolysis amylopectin, 6-glycosidic link, and have only the former can hydrolysis α-1 in the Propiram molecule, the 6-glycosidic link.Pullulanase and α-Dian Fenmei and saccharifying enzyme are used and can make starch molecule thoroughly be hydrolyzed to glucose, improve the starchy material utilization ratio, therefore in industries such as starch refine dsugar, Production of Fuel Ethanol important purposes and good market outlook are arranged.Pullulanase prepares at amylose starch in addition, also is widely used in the production of high fructose syrup, high maltose syrup etc.The present invention screens the genus bacillus that a strain has the Propiram degradation capability from occurring in nature, and it is numbered Bacillus sp.042.By Southern Yangtze University's China's colleges and universities' industrial microorganism resource and information center (http://www.cicim-cu.Jiangnan.edu.cn) collection, its collection is numbered Bacillus sp.CICIM B4312 to this bacterium.Utilize genetic engineering technique to clone a Pullulanase encoding gene pulA042 from Bacillus sp.042 genomic dna, Function Identification confirms pulA042 coding I type Pullulanase.
Summary of the invention
The technical problem to be solved in the present invention is: mainly utilize gene recombination technology to produce the bacterium genomic dna the outer Pullulanase encoding gene of clone born of the same parents and confirm its function from Pullulanase, provide genetic resources for utilizing gene recombination technology production Pullulanase.
Technical scheme of the present invention: a kind of outer Pullulanase encoding gene of born of the same parents that derives from bacillus sp.042, called after pulA042, its nucleotides sequence is classified SEQ ID NO:1 as, the aminoacid sequence of the Pullulanase protoenzyme of its coding is SEQ ID NO:2, the intestinal bacteria transformant of the recombinant plasmid pMD-PA that gene pulA042 and cloning vector pMD18-T Simple form, classification called after JM109/pMD-PA, be deposited in Chinese typical culture collection center, deposit number CCTCC NO:M 2010200.
The preparation method of described Pullulanase encoding gene pulA042 is as follows:
(1) genus bacillus that produces Pullulanase from the nature screening: gather soil sample from nature, adopt pyroprocessing to kill the nourishing body cell, the enrichment genus bacillus.The Pullulanase bacterium is produced in enrichment on the substratum that with the Propiram is sole carbon source.Further survey the bacterium that the outer Pullulanase of born of the same parents is produced in screening by shake-flask culture and the biopsy of Pullulanase enzyme.Confirm that with 16s rDNA gene sequencing the microorganism of screening belongs to bacillus.This takes turns the set out bacterium of the selected bacillus sp.042 of work for Pullulanase gene clone research.
(2) clone of Bacillus sp.042 Pullulanase gene:
The first step:, be that template PCR obtains Pullulanase Gene Partial fragment with Bacillus sp.042 chromosomal DNA according to bacterium Pullulanase gene conserved regions design primer.Sequential analysis obtains this fragment nucleotide sequence, and designs a pair of inverse PCR primer according to obtaining dna sequence dna.
Second step: utilize the inverse PCR technology to obtain the complete sequence of Pullulanase encoding gene.
Utilize a pair of inverse PCR primer of previous step design, obtain Pullulanase encoding gene coding region global DNA fragment by PCR, this fragment is connected with cloning vector pMD18-T Simple, and transformed into escherichia coli JM109 screens transformant containing on the LB flat board of penbritin.Extract the plasmid order-checking from the transformant that obtains, obtain Pullulanase encoding gene coding region complete sequence, the Pullulanase encoding gene called after pulA042 that obtains, its nucleotides sequence classify SEQ ID NO:1 as.
The 3rd step: obtain Bacillus sp.042 Pullulanase encoding gene
Design a pair of primer again with amplification Bacillus sp.042 Pullulanase coding region complete genome according to SEQ ID NO:1, with Bacillus sp.042 chromosomal DNA is template, pcr amplification obtains the dna fragmentation about a 2.5kb, be connected with cloning vector pMD18-T Simple behind the amplified fragments purifying, transformed into escherichia coli JM109 screens transformant containing on the LB flat board of penbritin.The aminoacid sequence of the Pullulanase protoenzyme of its coding is SEQ ID NO:2.The intestinal bacteria transformant of the recombinant plasmid pMD-PA that gene pulA042 and cloning vector pMD18-T Simple form, its classification called after JM109/pMD-PA, be deposited in Chinese typical culture collection center, be called for short CCTCC, deposit number CCTCC NO:M 2010200.
The application of Pullulanase encoding gene pulA042
Pullulanase encoding gene pulA042 can be used for carrying out heterogenous expression at different host cells, and expression product is used for starch-splitting and dextrin molecule α-1,6 glycosidic link.
Material and method
Common molecular biology method:
Unless mention, DNA operation and the molecular biology method that transforms according to standard carry out (1989 molecular cloning laboratory manuals such as Sambrook).
Unless otherwise mentioned, PCR manipulates standard method and the PCR response data carries out, can be referring to (1989 molecular cloning laboratory manuals such as Sambrook).
DNA operates the specification sheets use of employed enzyme according to supplier.
Primer is inventor herein's design and gives birth to worker's biotechnology company limited by Shanghai and synthesize.
Cloning vector pMD18-T Simple is a Dalian precious biotechnology company limited product, and production code member is D506A.
Inverse PCR technology reference literature carries out [Ochman H, Gerber AS and Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction.Genetics 120:621-623].
E. coli jm109, e. coli bl21 (DE3), coli expression carrier pET28a provides by Chinese colleges and universities industrial microorganism resource and information center (http://www.cicim-cu.jiangnan.edu.cn).
Enzyme that DNA manipulates and test kit
Unless otherwise mentioned, the enzyme that all DNA manipulate, restriction enzyme for example, ligase enzyme etc. are Dalian precious biotechnology company limited product.The archaeal dna polymerase that the PCR reaction is used is the precious biotechnology product E x Taq of company limited in Dalian, and production code member is DRR006A.The enzyme that all DNA manipulate employed damping fluid when reaction is attached giving when buying enzyme, and buffer concentration is 10 times of reaction desired concn.The test kit that the pillar dna fragmentation reclaims test kit and reclaim dna fragmentation from running gel is Dalian precious biotechnology company limited product, and production code member is respectively DV807A and DV805A.
Other reagent
Propiram is a Sigma company product, and red Propiram is a Megazyme company product, and glutinous rice starch and W-Gum are water chestnut lake, Zhejiang starch factory product, and the yeast nitrogen base is the O/O of a Difco company type yeast nitrogen base, and this product is carbonaceous sources and amino acid not.Red Propiram, glutinous rice starch and corn starch solution all adopt the phosphoric acid buffer preparation of 10mM, pH 6.0, and phosphoric acid buffer uses the distilled water preparation.Induce IPTG that bacillus coli gene express to use to be precious biotechnology company limited product, production code member is D9030A.
Substratum
LB describes in " 1989 molecular cloning laboratory manuals such as Sambrook ".With the Propiram is the solid medium of sole carbon source: yeast nitrogen base 1g/L, and Propiram 2g/L, agar powder 15g/L prepares with distilled water; Fermention medium: yeast nitrogen base 1g/L, Propiram 2g/L, (NH
3)
2SO
40.1g/L, prepare with distilled water.Shake-flask culture all adopts the 500mL triangular flask, shakes in bottle cabinet at constant temperature by the 220r/min condition under 37 ℃ of conditions and carries out.
Iodine solution
Press document [Jiang Xirui etc. newly organized zymin practical technique handbook .2002, Beijing: light industry press] in the 398th page " rare iodine liquid " preparation.
The 16s rDNA gene sequencing of bacterium
By document carry out [Chen Yuanyuan, Niu Dandan, Wang Zhengxiang. the method for a kind of Rapid identification bacterium. food and fermentation industries, 2007,33 (5): 29-31].
The Pullulanase vitality test
The Pullulanase enzyme activity determination carries out [Bibel M by the method for bibliographical information, Brett C, Gosslar U, et al.Isolation and analysis of amylolytic enzymes of the hyperthermophilic bacteriumThermotoga maritime.FEMS Microbiol Lett, 1998,158:9～15]
Beneficial effect of the present invention: the Pullulanase of the Pullulanase encoding gene coding that obtains after the invention process has α in starch-splitting and the dextrin-1 in starch processing, the function of 6 glycosidic links, the Pullulanase encoding gene that utilizes the present invention to obtain makes up the high efficiency production that recombinant microorganism can be realized Pullulanase.
Biological material specimens preservation: the intestinal bacteria transformant of the recombinant plasmid pMD-PA that gene pulA042 and cloning vector pMD18-T Simple form, classification called after JM109/pMD-PA, be deposited in Chinese typical culture collection center, preservation date: on August 11st, 2010, deposit number CCTCC NO:M2010200.
Description of drawings
Fig. 1 pulA042 gene expression product is to the degraded of red Propiram.
Reaction solution supernatant liquor behind alcohol precipitation of broken liquid of A:BL21 among Fig. 1 (DE3)/pET28a and red Propiram is colourless, shows that red Propiram is not decomposed.The supernatant liquor of reaction solution behind alcohol precipitation of broken liquid of B:BL21 among Fig. 1 (DE3)/pET-PA and red Propiram is red, shows the red Propiram of part and is degraded.
The reaction of Fig. 2 pulA042 gene expression product and glutinous rice starch.
The mixed solution of the broken liquid of A:BL21 among Fig. 2 (DE3)/pET-PA and glutinous rice starch changes mazarine with the color of Iod R into by red-purple after reaction, show that the side chain of glutinous rice starch is cut off, the branching content decline of starch.The mixed solution of the broken liquid of B:BL21 among Fig. 2 (DE3)/pET28a and glutinous rice starch still is a red-purple with the color of Iod R after reaction, and the side chain of demonstration glutinous rice starch is not cut off.
The reaction of Fig. 3 pulA042 gene expression product and W-Gum.
A among Fig. 3: the mixed solution of the reorganization broken liquid of bacterium BL21 (DE3)/pET28a and W-Gum still is a mazarine with the color of Iod R after reaction; B: the mixed solution of the broken liquid of reorganization bacterium BL21 (DE3)/pET-PA and W-Gum equally still is a mazarine with the color of Iod R after reaction, and considerable change does not take place the demonstration W-Gum.
Embodiment
The invention will be further described by embodiment, and embodiment is with the scope that does not limit the present invention in any way.
Embodiment 1: the acquisition of producing Pullulanase bacillus sp.042
Genus bacillus from nature screening product Pullulanase: from gathering the acid soil sample of pH about 4 near the cured good fortune hot spring in Tengchong County, Yunnan Province, normal temperature is preserved in freshness protection package.In the laboratory, above-mentioned soil sample is put into beaker, add 5 times of distilled waters, be incubated 1 hour at 100 ℃ behind the mixing.The above-mentioned mixed solution coating that takes a morsel is the solid medium flat board of sole carbon source with the Propiram, cultivates three days for 37 ℃, obtains 308 bacterium colonies altogether, and microscopic examination determines that wherein 187 strains are rod-shaped bacterium.Above-mentioned bacillar bacterium colony is rule the separation back respectively with single colony inoculation fermention medium, and shake-flask culture is got supernatant liquor detection Pullulanase enzyme and is lived after 2 days, wherein detect tangible Pullulanase activity in the 3 strain bacteria culture fluids.Carry out the 16s rDNA gene sequencing of bacterium respectively, the 16s rDNA Gene Partial sequence that wherein is numbered 042 bacterium is SEQ ID NO:3, utilize BLAST software with SEQ ID NO:3 and the American National biotechnology (www.ncbi.nlm.nih.gov of information center, abbreviation NCBI) gene order of including is compared, the result shows that (the NCBI accession number: HM449698.1) approaching, similarity is 96% for the 16s rDNA gene order of SEQID NO:3 and cured shape genus bacillus PCSBB.According to above experimental result, 042 bacterium should belong to bacillus, called after Bacillus sp.042.Bacillus sp.042 is used to next step experiment.
The clone of embodiment 2:Bacillus sp.042 Pullulanase gene:
The first step: the partial nucleotide sequence that obtains Bacillus sp.042 Pullulanase gene
According to bacterium Pullulanase gene conserved regions design pair of degenerate primers:
5’-TGGGG(A，T，C，G)TA(T，C)AA(T，C)CC-3’(SEQ?ID?NO：4)
5’-GG(A，G)TT(A，G)TA(A，T，G，C)CCCCA-3’(SEQ?ID?NO：5)
With Bacillus sp.042 chromosomal DNA is that template is carried out pcr amplification and obtained amplified fragments about a 0.8kb.To be connected with cloning vector pMD18-T Simple behind the amplified production purifying, the recombinant plasmid that obtains carries out sequential analysis, the result shows the PCR product total length 860bp that obtains, the partial sequence similarity of the Pullulanase gene in its nucleotide sequence and Bacillus acidopullulyticus source is up to 50%, this result shows that the fragment that above-mentioned PCR obtains is the part of Pullulanase encoding gene.
Second step: the complete sequence that obtains Bacillus sp.042 Pullulanase encoding gene
Obtain Bacillus sp.042 Pullulanase encoding gene part fragment nucleotide sequence according to the first step and design a pair of inverse PCR primer:
5’-CTGAAAGTGGCATTAAACAG-3’(SEQ?ID?NO：6)
5’-TAATTTATCAAACTCTTCGC-3’(SEQ?ID?NO：7)
Utilize inverse PCR technology clone Bacillus sp.042 Pullulanase gene complete coding region.
Get the about 10 μ g of Bacillus sp.042 chromosomal DNA, be dissolved in the 100 μ L distilled waters.Add 11 μ L enzyme cutting buffering liquids and 1 μ L restriction enzyme Hind III in above-mentioned dna solution, 37 ℃ of enzymes are cut after 2 hours and are reclaimed the dna fragmentation that the test kit recovery is cut through enzyme with the pillar dna fragmentation.Get the solution that the contains dna fragmentation 44 μ L of above-mentioned recovery, add ligation damping fluid 5 μ L, ligase enzyme 1 μ L, 16 ℃ of connections are spent the night.With above-mentioned connection product is template, utilize the inverse PCR primer to carry out pcr amplification, electrophoresis result shows, inverse PCR obtains the amplified fragments about a 3kb, above-mentioned amplified fragments is separated the back be connected with cloning vector pMD-18T Simple, recombinant plasmid carries out sequential analysis, and with obtaining sequence and the fragment comparison that obtains sequence, obtain the complete sequence of Pullulanase encoding gene, i.e. SEQ ID NO:1.
The 3rd step: obtain Bacillus sp.042 Pullulanase encoding gene
Design a pair of primer again with amplification Bacillus sp.042 Pullulanase coding region complete genome according to SEQ ID NO:1 sequence, primer sequence is as follows:
5’-GTGCAAATTACAAAAAAATTAAT-3’(SEQ?ID?NO：8)
5’-TTATTTAATCGGTTTCTCTGTT-3’(SEQ?ID?NO：9)
With Bacillus sp.042 chromosomal DNA is template, pcr amplification obtains the dna fragmentation about a 2.5kb, be connected with cloning vector pMD18-T Simple behind the amplified fragments purifying, transformed into escherichia coli JM109 screens transformant containing on the LB flat board of penbritin.Extract the plasmid order-checking from the transformant that obtains, obtain Pullulanase encoding gene coding region complete sequence, this unnamed gene is pulA042, and its nucleotides sequence is classified SEQ ID NO:1 as, and the aminoacid sequence of the Pullulanase protoenzyme of its coding is SEQ ID NO:2.The intestinal bacteria transformant of the recombinant plasmid pMD-PA that gene pulA042 and cloning vector pMD18-T Simple form, its classification called after JM109/pMD-PA has been deposited in Chinese typical culture collection center, deposit number CCTCC NO:M 2010200.
Embodiment 3: Pullulanase expression of gene and recombinase functional analysis
With the recombinant plasmid pMD-PA that contains the pulA042 gene is template, uses primer:
5’-AATGAATCGTTTCAAATTCAAAAACAAC-3’(SEQ?ID?NO：10)
5’-AATAAGCTTTTATTTAATCGGTTTCTCTG-3’(SEQ?ID?NO：11)
Carry out pcr amplification, obtain the mature peptide coding region of pulA042 gene, cutting the back with restriction endonuclease EcoRI with Hind III enzyme is connected with the coli expression carrier pET28a that cuts through same enzyme, construction recombination plasmid pET-PA, recombinant plasmid transformed e. coli bl21 (DE3) obtains recombination bacillus coli BL21 (DE3)/pET-PA.Recombination bacillus coli BL21 (DE3)/pET-PA shake-flask culture to nutrient solution opacity in the LB liquid nutrient medium is OD
600Adding IPTG at=0.8 o'clock is 0.5mg/mL to final concentration, and nutrient solution ultrasonication smudge cells after 4 hours is cultivated in continuation.In contrast, reorganization bacterium BL21 (the DE3)/pET28a that obtains with empty plasmid pET28a transformed into escherichia coli BL21 (DE3) cultivates too and carries out cytoclasis.The broken liquid of pair cell detects the Pullulanase vigor, and the result shows that the cytoclasis liquid of bacterium BL21 (the DE3)/pET-PA that recombinates has tangible Pullulanase vigor, and enzyme work is about 6U/mL.And the cytoclasis liquid Pullulanase vigor of reorganization bacterium BL21 (DE3)/pET28a is 0.As seen, the expression product of gene pulA042 mature peptide encoding gene has the Propiram degrading activity.
For further verifying the function of pulA042 mature peptide encoding gene expression product, reorganization bacterium BL21 (the DE3)/pET-PA of preparation in the last joint and the cytoclasis liquid of BL21 (DE3)/pET28a are reacted with red Propiram, glutinous rice starch, W-Gum respectively.
The reaction result of cytoclasis liquid and red Propiram as shown in Figure 1.
The cytoclasis liquid of getting 0.1mL reorganization bacterium BL21 (DE3)/pET-PA and BL21 (DE3)/pET28a mixes with the red Propiram solution of 0.3mL 0.5% respectively, 37 ℃ of reactions adding 1mL alcohol after about 1 hour, and behind the mixing 8, the centrifugal 10min of 000r/min.Because red Propiram is a macromolecular compound, mix generating precipitation with alcohol.If red Propiram is degraded, then form small molecules, do not mix to form precipitation with alcohol.By A among Fig. 1 as seen, reaction solution supernatant liquor behind alcohol precipitation of broken liquid of BL21 (DE3)/pET28a and red Propiram is colourless, and red Propiram precipitation concentrates on the centrifuge tube bottom, shows that red Propiram is not decomposed.Among Fig. 1 B as seen, the broken liquid of BL21 (DE3)/pET-PA and the reaction solution of red Propiram through alcohol precipitation and centrifugal after supernatant liquor still be red, and the precipitation of centrifuge tube bottom is seldom, shows that most of red Propiram is degraded to small molecules.As seen from Figure 1, pulA042 mature peptide encoding gene expression product has the Propiram degradation capability.
Glutinous rice starch is the higher starch of a class branching content, because the branching content height, and glutinous rice starch and Iod R displaing amaranth rather than based on the mazarine of the starch of straight chain.The enzyme of α-1,6 glycosidic link contact in glutinous rice starch and the single-minded starch-splitting, its side chain will be cut off, and mix with iodine again, and reaction solution shows as mazarine.The reaction result of cytoclasis liquid and glutinous rice starch as shown in Figure 2.
The recombinate cytoclasis liquid of bacterium BL21 (DE3)/pET-PA and BL21 (DE3)/pET28a of 0.2mL is mixed with the glutinous rice starch solution of 1mL 0.3% respectively, and 37 ℃ of reactions add the equal-volume iodine solution after 1 hour, mix.By A among Fig. 2 as seen: the mixed solution of the broken liquid of BL21 (DE3)/pET-PA and glutinous rice starch changes mazarine with the color of Iod R into by red-purple after reaction, show that the side chain of glutinous rice starch is cut off, the branching content decline of starch.By B among Fig. 2 as seen: the broken liquid of BL21 (DE3)/pET28a and glutinous rice starch still are red-purple with the color of Iod R after reaction, and the side chain of demonstration glutinous rice starch is not cut off.As seen from Figure 2, pulA042 mature peptide encoding gene expression product has the function of decomposing amylopectin α-1,6 glycosidic link.
W-Gum is the starch of a class based on straight chain, shows mazarine with Iod R.The α of W-Gum-1,4 glycosidic link is decomposed, and forms dextrin, then shows as redness or red-purple with Iod R again.The reaction result of cytoclasis liquid and W-Gum as shown in Figure 3.
The recombinate cytoclasis liquid of bacterium BL21 (DE3)/pET-PA and BL21 (DE3)/pET28a of 0.2mL is mixed with the corn starch solution of 1mL 0.3% respectively, and 37 ℃ of reactions add the equal-volume iodine solution after about 6 hours, mix.As seen from Figure 3: the broken liquid of reorganization bacterium BL21 (DE3)/pET28a and BL21 (DE3)/pET-PA and the mixed solution of W-Gum equally all are mazarine with the color of Iod R after reaction, and considerable change does not take place the demonstration W-Gum.As seen from Figure 3, pulA042 mature peptide encoding gene expression product does not have the function of decomposing alpha-1,4 glycosidic link.
Comprehensive above-mentioned experiment as seen, pulA042 mature peptide encoding gene expression product has the ability of decomposing α-1,6 glycosidic link in Propiram and the amylopectin, decomposing alpha-1,4 glycosidic link not meets the characteristics of I type Pullulanase.This class Pullulanase can be used for the decomposition of α-1,6 glycosidic link in starch or the dextrin molecule in starch processing.Present embodiment adopts escherichia coli expression pulA042 mature peptide encoding gene, and this is a kind of but pa042 expression of gene mode obviously is not limited to.
Claims (4)
1. outer Pullulanase encoding gene of the born of the same parents that derive from bacillus Bacillus sp.042, called after pulA042, its nucleotides sequence classify SEQ ID NO:1 as.
2. the aminoacid sequence of the Pullulanase protoenzyme of the described gene pulA042 coding of claim 1 is SEQID NO:2.
3. the intestinal bacteria transformant of the recombinant plasmid pMD-PA that forms of described gene pulA042 of claim 1 and cloning vector pMD18-T Simple, classification called after JM109/pMD-PA, be deposited in Chinese typical culture collection center, deposit number CCTCC NO:M 2010200.
4. the application of the described gene pulA042 of claim 1 is characterized in that:
The reorganization Pullulanase that the recombinant microorganisms express of utilizing Pullulanase encoding gene pulA042 to make up produces has the ability of decomposing Propiram, α-1 in the energy degraded starch, 6 glycosidic links, α-1 does not degrade, 4 glycosidic links, be a kind of I type Pullulanase, the reorganization Pullulanase that utilizes gene pulA042 to produce is used for α-1,6 glycosidic link of starch-splitting or dextrin molecule in starch processing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102562876A CN101974543B (en) | 2010-08-18 | 2010-08-18 | Extracellular pullulanase encoding gene derived from bacilli and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102562876A CN101974543B (en) | 2010-08-18 | 2010-08-18 | Extracellular pullulanase encoding gene derived from bacilli and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101974543A true CN101974543A (en) | 2011-02-16 |
| CN101974543B CN101974543B (en) | 2012-05-23 |
Family
ID=43574451
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102562876A Active CN101974543B (en) | 2010-08-18 | 2010-08-18 | Extracellular pullulanase encoding gene derived from bacilli and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101974543B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533818A (en) * | 2012-01-12 | 2012-07-04 | 广西大学 | Bacillus cereus-derived pullulanase gene and applications thereof |
| CN102676557A (en) * | 2012-05-11 | 2012-09-19 | 复旦大学 | Encoding gene of type I pullulanase as well as recombinant expression and application thereof |
| CN102676480A (en) * | 2012-06-08 | 2012-09-19 | 江南大学 | Method for producing extracellular pullulanase by applying auto-induction culture medium and dual-temperature control strategy |
| CN102676437A (en) * | 2012-06-08 | 2012-09-19 | 江南大学 | Producing method of extracellular pullulanase and special microorganism thereof |
| CN102965361A (en) * | 2012-12-04 | 2013-03-13 | 昆明爱科特生物科技有限公司 | Pullulanase XWPu2 and gene thereof |
| CN104946561A (en) * | 2015-05-27 | 2015-09-30 | 孙会忠 | Pullulanase active strain in caltrop endophyte and culture method of pullulanase active strain |
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5147795A (en) * | 1989-08-31 | 1992-09-15 | Kao Corporation | Alkaline pullulanase from bacillus sp. ferm p-10887 |
| CN1609188A (en) * | 2004-11-16 | 2005-04-27 | 江南大学 | Prussian blue producing strain and prussian blue producing process with the strain |
-
2010
- 2010-08-18 CN CN2010102562876A patent/CN101974543B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5147795A (en) * | 1989-08-31 | 1992-09-15 | Kao Corporation | Alkaline pullulanase from bacillus sp. ferm p-10887 |
| CN1609188A (en) * | 2004-11-16 | 2005-04-27 | 江南大学 | Prussian blue producing strain and prussian blue producing process with the strain |
Non-Patent Citations (2)
| Title |
|---|
| 《生物学杂志》 20090430 张明焱等 地衣芽孢杆菌普鲁兰酶编码基因的鉴定 第8-10页 1-4 第26卷, 第2期 2 * |
| 《食品研究与开发》 20071231 杨华第等 普鲁兰酶产生菌的分离与鉴定 第4-6页 1-4 第28卷, 第9期 2 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| CN102533818A (en) * | 2012-01-12 | 2012-07-04 | 广西大学 | Bacillus cereus-derived pullulanase gene and applications thereof |
| CN102676557A (en) * | 2012-05-11 | 2012-09-19 | 复旦大学 | Encoding gene of type I pullulanase as well as recombinant expression and application thereof |
| CN102676480A (en) * | 2012-06-08 | 2012-09-19 | 江南大学 | Method for producing extracellular pullulanase by applying auto-induction culture medium and dual-temperature control strategy |
| CN102676437A (en) * | 2012-06-08 | 2012-09-19 | 江南大学 | Producing method of extracellular pullulanase and special microorganism thereof |
| CN102965361A (en) * | 2012-12-04 | 2013-03-13 | 昆明爱科特生物科技有限公司 | Pullulanase XWPu2 and gene thereof |
| CN102965361B (en) * | 2012-12-04 | 2014-05-28 | 昆明爱科特生物科技有限公司 | Pullulanase XWPu2 and gene thereof |
| CN104946561A (en) * | 2015-05-27 | 2015-09-30 | 孙会忠 | Pullulanase active strain in caltrop endophyte and culture method of pullulanase active strain |
| CN104946561B (en) * | 2015-05-27 | 2017-11-14 | 河南科技大学 | Pullulanase active bacterial strain and its cultural method in puncture vine endophyte |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
| US11878079B2 (en) | 2017-04-14 | 2024-01-23 | Capsugel Belgium Nv | Pullulan capsules |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101974543B (en) | 2012-05-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101974543B (en) | Extracellular pullulanase encoding gene derived from bacilli and application thereof | |
| Sorokin et al. | Halo (natrono) archaea isolated from hypersaline lakes utilize cellulose and chitin as growth substrates | |
| CN112481240B (en) | GH16 family heat-resistant glucanase mutant and construction method and application thereof | |
| CN103060353B (en) | Alginate lyase biosynthesis gene cluster | |
| CN106414728A (en) | Agarooligosaccharide hydrolase and method for producing 3,6-anhydro-l-galactose and galactose from agarose by using same | |
| CN102676557B (en) | Encoding gene of type I pullulanase as well as recombinant expression and application thereof | |
| CN112725319B (en) | Alginate lyase FaAly7 with polyG substrate specificity and application thereof | |
| CN105950640A (en) | Preparation method of marine bacteria-derived kappa-carrageenase gene and recombinase | |
| Zhang et al. | Identification of chitinolytic enzymes in Chitinolyticbacter meiyuanensis and mechanism of efficiently hydrolyzing chitin to N-acetyl glucosamine | |
| CN102286443A (en) | Method for producing mixture of recombinant isoamylase, alpha-amylase and glucamylase | |
| CN109929861B (en) | Encoding gene of glucomannanase, enzyme, preparation and application thereof | |
| CN110452919B (en) | Truncated alginate lyase Aly7B-CDII gene and application thereof | |
| CN112899257B (en) | Thermophilic chitinase Chi304 mutant and application thereof in degradation of chitin | |
| CN112941089B (en) | Alginate lyase mutant gene, alginate lyase mutant, engineering bacterium containing mutant, construction method and application | |
| CN102154190B (en) | Engineering escherichia coli capable of efficiently producing hyaluronic acid and preparation method thereof | |
| CN105886487A (en) | Chitin histone deacetylase mutant | |
| CN102382790A (en) | Recombinant bacillus subtilis for high yield of catalase (CAT), and construction method and application thereof | |
| Abdalla et al. | Genetic and biochemical characterization of thermophilic β‐cyclodextrin glucanotransferase from Gracilibacillus alcaliphilus SK51. 001 | |
| Hegazy et al. | Improvement of cellulose degradation by cloning of endo-β-1, 3-1, 4 glucanase (bgls) gene from Bacillus subtilis BTN7A strain | |
| CN101736023A (en) | Cellulose hydrolytic enzyme beta-1,4 glucose incision enzyme gene | |
| KR20100040438A (en) | A novel agarase and an enzymatic production method of agarooligosaccharide from agarose using the same | |
| Yavari-Bafghi et al. | Isolation, screening and identification of haloarchaea with chitinolytic activity from hypersaline lakes of Iran | |
| CN105543128A (en) | Polar cold-adapted salt-tolerant alginate lyase and coding gene c3 and application thereof | |
| CN104087604A (en) | Genetic expression sequence of inulin fructotransferase | |
| Brumm et al. | Identification, cloning and characterization of Dictyoglomus turgidum CelA, an endoglucanase with cellulase and mannanase activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20201116 Address after: 226500 No. 6 Haiyang South Road, Chengnan Street, Rugao City, Nantong City, Jiangsu Province Patentee after: LAIDING ELECTRONIC MATERIAL TECHNOLOGY Co.,Ltd. Address before: 214122 Jiangsu Province, Wuxi City Lake Road No. 1800, Jiangnan University Institute of biological engineering Patentee before: Jiangnan University |