CN110004070A - One plant of production zytase aspergillus niger and its construction method and application - Google Patents
One plant of production zytase aspergillus niger and its construction method and application Download PDFInfo
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- CN110004070A CN110004070A CN201910284360.1A CN201910284360A CN110004070A CN 110004070 A CN110004070 A CN 110004070A CN 201910284360 A CN201910284360 A CN 201910284360A CN 110004070 A CN110004070 A CN 110004070A
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- China
- Prior art keywords
- aspergillus niger
- gene
- msb2
- xylan
- zytase
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- Granted
Links
- 241000228245 Aspergillus niger Species 0.000 title claims abstract description 48
- 238000010276 construction Methods 0.000 title claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 title claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 33
- 238000000855 fermentation Methods 0.000 claims abstract description 31
- 230000004151 fermentation Effects 0.000 claims abstract description 31
- 150000004823 xylans Chemical class 0.000 claims abstract description 30
- 229920001221 xylan Polymers 0.000 claims abstract description 29
- 241000894006 Bacteria Species 0.000 claims abstract description 16
- 230000001580 bacterial effect Effects 0.000 claims abstract description 11
- 239000002773 nucleotide Substances 0.000 claims abstract description 11
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 11
- 230000002779 inactivation Effects 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 241000196324 Embryophyta Species 0.000 claims description 12
- 235000015099 wheat brans Nutrition 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 239000007836 KH2PO4 Substances 0.000 claims description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 210000001938 protoplast Anatomy 0.000 claims description 7
- 238000003209 gene knockout Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 4
- 229920000053 polysorbate 80 Polymers 0.000 claims description 4
- 229920002635 polyurethane Polymers 0.000 claims description 4
- 239000004814 polyurethane Substances 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 241000228212 Aspergillus Species 0.000 claims description 3
- 229910052564 epsomite Inorganic materials 0.000 claims description 3
- 230000006801 homologous recombination Effects 0.000 claims description 3
- 238000002744 homologous recombination Methods 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000002657 fibrous material Substances 0.000 claims 1
- 238000007654 immersion Methods 0.000 claims 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 16
- 108090000790 Enzymes Proteins 0.000 description 14
- 102000004190 Enzymes Human genes 0.000 description 14
- 229940088598 enzyme Drugs 0.000 description 14
- 239000001963 growth medium Substances 0.000 description 14
- 239000007788 liquid Substances 0.000 description 12
- 239000002609 medium Substances 0.000 description 9
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- 239000001965 potato dextrose agar Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 230000009182 swimming Effects 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 229960003487 xylose Drugs 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000003892 spreading Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004061 bleaching Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000004804 polysaccharides Chemical class 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- 241001370055 Aspergillus niger CBS 513.88 Species 0.000 description 1
- 108010001682 Dextranase Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241001655322 Streptomycetales Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000002154 agricultural waste Substances 0.000 description 1
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 description 1
- 230000000433 anti-nutritional effect Effects 0.000 description 1
- 238000004500 asepsis Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 108010081495 driselase Proteins 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019256 formaldehyde Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 108010087005 glusulase Proteins 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
Abstract
The invention discloses the aspergillus niger that one plant produces xylan, Msb2 gene is inactivated in the bacterial strain；The nucleotide sequence of the Msb2 gene is as shown in SEQ ID NO.1, and the nucleotide sequence after the Msb2 gene inactivation is as shown in SEQ ID NO.2.The invention also discloses the construction methods of said gene engineering bacteria.The present invention further discloses application of the genetic engineering bacterium in fermenting and producing zytase.The present invention reduces aspergillus niger biomembrane yield during fermentation produces zytase, and adhesion dies down, and improves the yield of zytase, shortens fermentation period by the aspergillus niger of building Msb2 gene.
Description
Technical field
The invention belongs to biological gene engineering fields, and in particular to one plant production xylan aspergillus niger and its
Construction method and application.
Background technique
Plant fiber is that storage capacity is maximum, most abundant in nature, and most cheap a kind of renewable resource, wherein half
Cellulose accounts for about 15%~30%.As the main constituents of hemicellulose, xylan is distributed widely in the thin of higher plant
It is the polysaccharide that amount of storage is only second to cellulose in nature in cell wall.Zytase is that one kind can be by xylan degrading at low
The hydrolase of glycan and xylose is mainly made of β-Isosorbide-5-Nitrae-endo-xylanase and xylobiase, and function is cut respectively
Xylose glycosidic bond in disconnected xylan backbone and oligosaccharide is further degraded to monomeric wood sugar.
Zytase has in numerous areas and is widely applied as a kind of industrial enzyme preparation simultaneously.In plant feed,
Addition zytase can degrade the anti-nutritional factors non-starch polysaccharide in feed, to improve utilization of the poultry to nutriment
Rate.In food service industry, the higher agricultural wastes of hemicellulose level can be degraded into xylo-oligosaccharide by zytase.In life
Object energy conversion field, zytase can act synergistically with cellulase system, and plant fiber is degraded as pentose and hexose, and with
It further passes through micro-organisms ethyl alcohol as fermentation substrate.In bio-pulping bleaching field, zytase can be used as life
Object bleaching assistant is applied to paper industry.
Currently, zytase relies primarily on microbe fermentation method acquisition, it is now known to the microorganism of synthesis zytase
Relate generally to fungi, streptomycete, bacterium and yeast etc..Aspergillus bacterial strain is the strain excellent for producing zytase, becomes domestic
The emphasis of outer zytase PRODUCTION TRAITS.
Mycelium is fixed on paddy stalk or some other inert material, or using the embedding of the carrier materials such as sodium alginate
Method is reused, both reduces fermentation broth viscosity, conducive to the transmitting of oxygen and nutriment, improve cell utilization rate and
Biological reactions rates, while the post-processings such as separation and Extraction for being also convenient for product are processed.In recent years, the immobilization fermentation of filamentous fungi
Production has obtained more and more concerns.
Summary of the invention
Goal of the invention: low output, fermentation period are long when in order to solve fermentation of Aspergillus niger production zytase in the prior art
The problem of.
The present invention also technical problems to be solved are to provide the construction method of above-mentioned aspergillus niger.
The last technical problems to be solved of the present invention are to provide above-mentioned aspergillus niger and prepare xylan in fermentation
Application in enzyme.
Summary of the invention:
The black-koji mould of one plant of production xylan, classification naming are black-koji mould (Aspergillus niger), bacterial strain number
For SJ1, it is preserved in China typical culture collection center, the preservation time is on February 28th, 2019, deposit number CCTCC
NO:M201911, preservation address are Wuhan, China Wuhan University, postcode 430072.The bacterial strain is by dilution soil, with base
Plinth xylan screening and culturing medium screening gained, culture medium prescription are xylan 10g/L, KH2PO4、KH2PO4、MgSO4、NH4NO3、
NaCl、MnS04、ZnSO4、FeSO4, agar consumption equal 2%.It is aspergillus niger by strain idenfication, systematic evolution tree is as shown in Figure 1.
The black-koji mould of above-mentioned production xylan is preparing the application in xylan within protection scope of the present invention.
The aspergillus niger of one plant of production xylan, Msb2 gene inactivation in the bacterial strain.The inactivation refers to this
Msb2 gene cannot express to obtain active protein in bacterial strain, and the inactivation, which can be, is inserted into one in Msb2 gene
Section nucleotide sequence perhaps lacks the partial sequence of the Msb2 gene in aspergillus niger or is replaced with other nucleotide sequences
The complete sequence of Msb2 gene.
Wherein, core of the nucleotide sequence of the Msb2 gene as shown in SEQ ID NO.1, after the Msb2 gene inactivation
Nucleotide sequence is as shown in SEQ ID NO.2.Msb2 gene be aspergillus niger CBS513.88 signal mucin Msb2 (Gene ID:
4986934) homologous protein, 90% similarity.Msb2 overall length 2955bp hygromycin is substituted for by the method for homologous recombination to resist
Property Expression element 2740bp.
Wherein, the aspergillus niger is aspergillus niger SJ1, is preserved in China typical culture collection center, deposit number CCTCC
NO:M201911。
The construction method of the aspergillus niger of above-mentioned production xylan, includes the following steps:
(1) aspergillus niger SJ1 genome is extracted；
(2) gene knockout segment △ Msb2, the nucleotide sequence of the gene knockout segment such as SEQ ID NO.3 institute are constructed
Show；
(3) aspergillus niger protoplast is prepared；
(4) gene knockout segment is imported in protoplast and carries out homologous recombination, obtain Msb2 gene inactivation aspergillus niger base
Because of engineering bacteria.
The aspergillus niger of above-mentioned production xylan is preparing applying in protection model of the invention in zytase
Within enclosing.
Specific application method is as follows: to produce the aspergillus niger of xylan as fermenting agent, porous fibre material
Material is fixation support, and fermentation prepares zytase, and the fixation support is polyurethane.
The fixation support is pre-processed by the following method:
The fixation support NaOH of 0.5~2M is impregnated into 30min~2h, being rinsed with water to pH is neutrality, in 0.5~2M
30min-2h is impregnated in HCl, being rinsed with water to pH is neutrality, and drying is obtained by pretreated fixation support；
As preferred: the fixation support NaOH of 1M being impregnated 1h, being rinsed with water to pH is neutrality, is soaked in 1M HCl
1h is steeped, being rinsed with water to pH is neutrality, and drying is obtained by pretreated fixation support.
The condition of the fermentation is as follows:
Fermentation medium: 10~70g/L of xylan, NaNO32~6g/L, KH2PO40.3~2g/L, MgSO4·7H2O
0.1~0.8g/L, 0.1~0.8g/L of Tween-80, with the preparation of wheat bran leachate；It is preferred that xylan 30g/L, NaNO3 4.2g/
L、KH2PO4 1.0g/L、MgSO4·7H2O 0.5g/L, Tween-80 0.5g/L；
The wheat bran leachate is prepared as follows: weighing the wheat bran of 15~60g/L, 800mL water is added, boils
It after 30min-2h, filters to get filtrate, is settled to 1L again with water；It is preferred that weighing the wheat bran of 40g/L, 800mL water is added, boils 1h
Afterwards, it filters to get filtrate, is settled to 1L again with water.
Fermentation condition is as follows: 28~32 DEG C, 150~300r/min culture 3~6d, preferably 30 DEG C, 220r/min culture 4d.
The utility model has the advantages that
The present invention reduces the formation of biomembrane, and then improve by knocking out to biofilm formation correlation Msb2 gene
The yield of zytase improves thallus to the Utilization ability of substrate, shortens fermentation period.The present invention is carried by immobilization of polyurethane
Body realizes shake flask fermentation and produces zytase, and enzyme activity is stablized, and enzyme activity is improved by 1515.34U/mL to 1633.27U/mL, and yield mentions
High by 7.8%, the period foreshortens to 3.5d by 4d.The present invention is pure cleaned medium using culture medium, and fermentation ends obtain on fermentation liquid
It is clearly xylan enzyme solution, can be directly used for subsequent experimental.
Detailed description of the invention
Fig. 1 is the systematic evolution tree of aspergillus niger original bacteria.
Fig. 2 is the electrophoretogram of aspergillus niger SJ1 genome, and in Fig. 1,1,2 swimming lanes are aspergillus niger SJ1 genome, and M is
10000bp DNA marker。
Fig. 3 is PAN7-1 plasmid map；
Fig. 4 is the electrophoretogram of Msb2 upstream region of gene homology arm, lower homology arm and resistance gene fragment, and M is 2000bp DNA
Marker, swimming lane 1 are the upstream Msb2 homology arm, and swimming lane 2 is resistance gene fragment, and swimming lane 3 is the downstream Msb2 homology arm.
Fig. 5 is the electrophoretogram that gene △ Msb2 knocks out segment, and swimming lane 1 is that △ Msb2 knocks out segment, and M is 5000bp DNA
marker；
Fig. 6 is violet staining figure；
Fig. 7 is violet staining OD value disparity map；
Fig. 8 is the fermentation results of original aspergillus niger and aspergillus niger；
Fig. 9 is carrier bacterium amount attachment figure after aspergillus niger original bacteria and Msb2 knock-out bacterial strain immobilization fermentation 4d.
Specific embodiment
Embodiment 1: aspergillus niger SJ1 genome is extracted.
Aspergillus niger spore is accessed into YPD culture medium, 30 DEG C, 220rpm culture 12h.12000rpm, 5min is centrifuged at 4 DEG C,
Abandon supernatant.It is washed with PBS (8g/L NaCl, 0.2g/L KCl, 1.44g/L Na2HPO4,0.24g/L KH2PO4, pH 7.4)
Twice.(4 DEG C, 12000rpm, 2min) are centrifuged immediately after, abandon supernatant.By thallus after liquid nitrogen grinding powdering, according to
TaKaRa MiniBEST Plant Genomic DNA Extraction Kit kit specification is operated.Pass through agar
Sugared gel electrophoresis measurement aspergillus niger genome concentration is as shown in Figure 2.
Embodiment 2: building gene △ Msb2 knocks out segment.
(1) design primer: PCR obtains Msb2 upstream region of gene homology arm and downstream homology arm, the Msb2 upstream region of gene are same
The nucleotide sequence of source arm is as shown in SEQ ID NO.4, the nucleotide sequence of the Msb2 downstream of gene homology arm such as SEQ ID
Shown in NO.5, go out resistance expression's element, pAN7-1 plasmid map such as Fig. 3, on Msb2 by template PCR amplifications of pAN7-1 plasmid
It swims homology arm, downstream homology arm and resistance fragments and carries out glue recovery purifying, use 0.8% (0.8g/100mL) Ago-Gel pair
The upstream Msb2 homology arm, downstream homology arm, the resistance element of purifying carry out electrophoresis detection, as a result such as Fig. 4.Then homologous to swim
Arm, downstream homology arm, resistance element construct the knockout segment △ Msb2 of Msb2 using the method for overlap pcr, to △
Msb2 knocks out segment glue recovery purifying, agarose gel electrophoresis and sequence verification.△ Msb2 knocks out segment result such as Fig. 5.Primer
It is as follows:
1 △ Msb2 of table knocks out segment and constructs primer
PCR system is as follows:
Glue recycling obtains Msb2 or more homology arm, and resistance fragments and △ Msb2 knock out segment:
Use takara kit (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit
Ver.4.0) kits.
Embodiment 3: the preparation and conversion of aspergillus niger protoplast.
1. aspergillus niger is inoculated into 3 PDA plates, spore is covered with, for use.
(0.02% tween80,115 DEG C of sterilizing 20min are added in 0.9%NaCl 2. adding and scraping spore buffer in right amount.)
Into plate, spore is scraped with spreading rod, obtains spore liquid, blood counting chamber counts.
3. inoculation 107For a spore to 50ml YPD culture medium, 30 ° of 220rpm cultivate 10h.
4. preparing enzyme solution, (Yatalase, lysing enzyme, driselase, each 0.1g/10ml of glusulase, dextranase
50 μ l/10ml, 400 μ l/10ml of cellulase), then in triangular flask with 20ml asepsis injector filtration sterilization to sterilizing.
5. after spore germination, being filtered with Miracloth, mycelia is left, weighs, takes 2g mycelia into enzyme solution.30°
3h is digested under 200rpm.
6. being filtered after the completion of enzymatic hydrolysis with Miracloth, filtrate is taken, 4 ° of 5000rpm are centrifuged 10min.Supernatant is removed, 1ml is added
1M sorbierite is mixed with rifle pressure-vaccum, adds 15ml sorbierite, is centrifuged, is removed supernatant.Then it repeats primary.Remove supernatant, adds
Entering 1ml buffer, (100ml contains KCl 4.47g, and CaCl2 0.735g, MOP 0.2093g, KOH tune pH are blown to 6.0) with rifle
Persorption is even, and it is stand-by then to put 4 ° of refrigerators.
9. drawing △ Msb2 knocks out segment into 1.5ml sterile centrifugation tube
10. ready-made protoplast is mixed several times with the soft pressure-vaccum of 1ml liquid-transfering gun and (is then not necessarily to mix if it is what is now done before
It is even or need to only turn upside down several times just)
11. inhaling the protoplast of 100 μ l into 1.5ml centrifuge tube, segment is knocked out with 10ul △ Msb2 and is mixed, adds 50
The buffer solution A (100ml contains PEG8000 25g, CaCl2 1.47g, KCl 4.47g, 10mM Tris pH7.5) of μ l mixes,
It is placed on ice and timing 20min.
12. after 20min, 1ml buffer solution A being added, turns upside down several times, it is placed on room temperature and timing 20min.
After 20min, draws 200,300,500 μ l and be coated on the PDA culture medium that hygromycin concentration is 150mmol/L, just set
Culture.
Embodiment 4: bacterium colony PCR verifying.
1. aspergillus niger is inoculated into 3 PDA plates, spore is covered with, for use.
2. adding and scraping spore buffer into plate in right amount, spore is scraped with spreading rod, obtains spore liquid, blood counting chamber meter
Number.
3. take 2ul spore to be added in buffer (100ml contains 0.01M Tris, 10Mm EDTA, 1M KCl, PH9.5),
95 DEG C water-bath 10 minutes, take out ice bath 5 minutes, be template, with verify primer △ Msbcheck1 (sequence:
CGCTCTGTCCATTCATTTCAGC it) carries out PCR with △ Msbcheck2 (sequence: ATGGCAACATCAACAACGCT) primer and tests
Card, pcr obtain segment and carry out sequence verification, obtain the knock-out bacterial strain for knocking out Msb2.
Embodiment 5: violet staining experiment
It by original aspergillus niger and knocks out bacterium and is inoculated into PDA plate respectively, spore to be covered with adds 3mL to scrape spore buffer to putting down
In plate, spore is scraped with spreading rod, is transferred in the 5mL centrifuge tube of sterilizing, with spore buffer constant volume is scraped to 2mL, obtains spore
Sub- liquid.It is quantified with blood counting chamber and is diluted to 106A/mL, continues thereafter with and is diluted to 105, 104, 103。
It is previously added the aspergillus minimal medium of 1ml in 24 orifice plates, then takes 2uL to be inoculated with the spore liquid of various concentration
Into culture medium.Stationary culture 48h at 30 DEG C makes aspergillus niger form a film in orifice plate bottom.Culture medium is outwelled later, rinses 2 with PBS
After, 0.1% violet staining 15min is added.Crystal violet is outwelled later, after being rinsed 2 times with PBS, glacial acetic acid is added and is shaking
It swings and places 30min in instrument, crystal violet is made to decolourize.Then observation and microplate reader detection OD570 are carried out.Violet staining hole is hardened
Fruit is as shown in fig. 6, OD value disparity map is as shown in Figure 7.
Embodiment 6: fermentation verifying.
1, carrier is polyurethane material, and preprocess method: impregnating 1h for the carrier Na (OH) of 1M, after being cleaned with pure water,
1h is impregnated in 1M HCl, be with pure water rinsing to pH later it is neutral, place into 65 DEG C of baking ovens that drying to constant weight.Phase will be cut into
Carrier with size is added in culture medium in right amount, 115 DEG C of sterilizing 20min together with culture medium, cooling stand-by.
2, wheat bran leachate: weighing the wheat bran of 40g/L, and 800mL water is added, after boiling 1h, must be filtered with 4 layers of filtered through gauze
Liquid is settled to 1L with water again.
3, culture medium prescription: slant medium: potato dextrose agar (PDA) culture medium.Seed culture medium sucrose
10g/L, with the preparation of wheat bran leachate.Fermentation medium (g/L): xylan 30, NaNO3 4.2, KH2PO4 1.0, MgSO4
7H2O 0.5, Tween-80 0.5, with the preparation of wheat bran leachate.The above culture medium 121 DEG C, 105Pa condition sterilize 15min, to
With.
4. preservation of bacteria strain is inoculated in PDA slant medium, after being placed in 30 DEG C of constant incubator culture 3d, access is equipped with
In the 500mL shaking flask of 100mL seed culture medium, 30 DEG C, 220r/min culture for 24 hours.
5. cultured seed is seeded to fermentation medium, 30 DEG C, 220r/min culture 4d with 5% (volume fraction).
6. prepared by crude enzyme liquid: fermentation liquid refrigerated centrifuge 5000rpm is centrifuged 10min, i.e., crude enzyme liquid.
7. xylanase activity measures: taking appropriate diluted 50 μ L of crude enzyme liquid, the citric acid of the 0.05mol/L of 0.45mL is added
The xylan substrate of buffer (pH 4.8) and 1mL, 1.2% (mass fraction) reacts 30min under the conditions of 50 DEG C.Xylose is raw
It is carried out at the measurement reference literature of amount.Enzyme activity defines (U): reacting 30min under the conditions of 50 DEG C, substrate of degrading per minute generates 1 μ
Enzyme amount needed for mol xylose.
8. in the above way, aspergillus niger SJ1 and Msb2 knock-out bacterial strain to be inoculated into the 100mL seed culture for adding 0.1g carrier
Base culture for 24 hours afterwards switching carrier to fermentation medium, 30 DEG C, 220r/min cultivate 4d, different time takes fermentation liquid to detect enzyme activity,
Enzyme-activity data is as shown in Figure 8.Carrier after immobilization fermentation 4d is taken out, rinses 3 times mycelia for washing off absorption with PBS.It obtains
Adhere to the carrier of mycelia, as shown in figure 9, the bacterium amount of original bacteria is more, the bacterium amount for knocking out bacterium is moderate.
Sequence table
<110>Nanjing University of Technology
<120>one plants of production zytase aspergillus nigers and its construction method and application
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2955
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgatcgcgc ccacgacttc attgttggcc gcgttcctgt ccttgggcgg cctcgagctc 60
gtcgctgccc gggaggcagg acagatccgg gacttgggag cgcgtgaggt tagctccgcc 120
gttcctggtt cctcccagcc cgttacgcct gtcgagttga caaccaggtc cgaaacgagt 180
acgcatgatt cagcgccgac ttcggacaat gaacaaacaa gcccgatcgt tgacccttca 240
cctgctgtta tcgttgttcc tgtaaccatt tccgtggacg ccaatggtgt tacgcatacc 300
attgtgggca cggcacccaa cgttcccggt gcttcaacga caactagctc cacccttgag 360
cgtgcagaga gtaccccggc ggcagctgct gctacttcga cggcgagcgc gactacatcg 420
gacaaggatt caactaccag tgactcggaa aagcagaaat ctacatccga atccgagaaa 480
cacacttccg cgtctgaatc gtcagcgaaa ccagaggcag cttcttcgtc agcagcagca 540
acgacggcag agacagcttc ttcgtcggcc gcagcggcag cagcagcagc tactacatct 600
agccagagtt cgttggctga ctcctgggag agcttcattt caagtatctt ggggtcctct 660
accagcacca gcgcaacctc cacggccagc cctaccgcat cgccagccga gtccagctct 720
gcaacccccg ctcccgcttc aagctctgct ggcgtcattg tagatgtcac ctcgagcaag 780
agttctacaa gcgatagctc gacgacttcg gagagctcta cggctaagga ttccaccact 840
cagagctctt ccagcccagc atcctcgacc cctgcttcca ctacggagtc ttcggccgag 900
gcttcgcaat cggaatctgc cacttctagc tcgaaggcag agacttcgag tcaacttatc 960
actagccaga gcgcatctgc ccagagttct tccgctcagg tccctctggc tgagtctact 1020
tcgaagtccg agtcttcaac ccagagctcc tcgagtcagg ttacatctgt tcaggccacc 1080
tcatcaggcg cagcctctca gactactggg tcagcatcct cgagcctcat cccgtctgca 1140
tctgaagatg tgacctcttc ttcttccacc ccctcatcat caggaggtgt cctcggcttc 1200
ctctcggacc tcttccctac taccaccact tccagcggat ctgcctctgc tactagcaag 1260
agtgcaagcg agtctgccga ctccagtgct ttccctctga gcctccctac catctccgtt 1320
agtgttcctg tggttacgcc tgcctcatct tctggatcgg tcaatattgt ttctgcccta 1380
cttggcacat cctcaggcgc ttccttggaa ccttcggcca ttagtctggg tatctcggcc 1440
acttccacgg ctcttatccc cggagcttcc agcggaaccc agagctcctc agcgggagcc 1500
tcgactccga tcatcagcgg tggcattgtc atcatgccca cttcgactga atctgcttct 1560
gcttccgtct caggatctgc ctcgacgggt ctttttggaa ccacttcaga atcagcatct 1620
tcattggctt cgggatccgc ctctggatct gcgggcgtta ttcctacatc ggtgtctggg 1680
tctgtatctg gatctgcagg actgttccct acgtcggcgt ctggatccgt ttcgggatct 1740
gcaggtgtta ttcctacatc ggctcctggc tctgcatctg gatctgtgtc gggatctgct 1800
tctggatccg tttcaggctc tgccattggc accagctctg catccaagtt tacctccatt 1860
tctggccctg cctcttcgat tcctttgatc acttccacca aggagtcctc gggtctggta 1920
tctgcgacgc cttcctatgc cccgtccact accagcaagg aaacgcccac gcctatcgca 1980
caacccacca cgacgaccac ctcggccgct gagacgactg tcgcccccaa accgaccacg 2040
accacctccg attctgacaa ctacctgccg tcaagcattc tcgtggttga gcccacaact 2100
actacgtcac tgagcacggg cacagctacc cattcgagta ccagcactgc tgctgctcag 2160
cttcctggat cgatctctcc ttctggtggt cttcccgagc agccggccaa cgataccttg 2220
gtccaaatcg gattcaatgg caaactgcgc tggagctttg tggccacaac gcccttgtct 2280
tcctcccaga tcttcatgta tgttcccgag ggtatgggct acgcgttgca gctcaatgac 2340
tctgacgtgg tcatgtacga cctccaaccc tacgacaact cggcaagcac tggctacatt 2400
gctactgttg cgctgatgta catcccttcg gatgacgttg acaccctgag aaagatgctc 2460
cacaacccta tttcgaggct ctacgagcag cccaacgaga cggtcaagac attgatggcc 2520
atgatcgacc cttccatccc tcttctggtg ggcgactcgg gttcttccag tggcagctcg 2580
tccaacagcg gtggatcggg tagcagcagc agcaacggcg atggcagtga cggtggctac 2640
gaccaaagcg atgctggtac cagctcatct ggaaccacca aggctagtgc cgttggtata 2700
ggatgtggtg ctgtggccgg tgctgcagca tatggtgctg gtatgttctg ggttgcgcgt 2760
cggtaccgga agaagcgcca gttgcaccag cgctccccct cgagcgtcga ccagatgagc 2820
gaaggccgcg gcgctgggtc cgtcttcggc gcaggtggcc gtctctcccg caacagccag 2880
aacagccggg gcactggccg cacccagatg atcagcgcgc cggtgatggc cgagaactcg 2940
ctcggctgga actaa 2955
<210> 2
<211> 2798
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gctattgtta cccaaccatt cgaatcaatt aaaatccgcc gcctccacca tttgtagaaa 60
aatgtgacga actcgtgagc tctgtacagt gaccggtgac tctttctggc atgcggagag 120
acggacggac gcagagagaa gggctgagta ataagccact ggccagacag ctctggcggc 180
tctgaggtgc agtggatgat tattaatccg ggaccggccg cccctccgcc ccgaagtgga 240
aaggctggtg tgcccctcgt tgaccaagaa tctattgcat catcggagaa tatggagctt 300
catcgaatca ccggcagtaa gcgaaggaga atgtgaagcc aggggtgtat agccgtcggc 360
gaaatagcat gccattaacc taggtacaga agtccaattg cttccgatct ggtaaaagat 420
tcacgagata gtaccttctc cgaagtaggt agagcgagta cccggcgcgt aagctcccta 480
attggcccat ccggcatctg tagggcgtcc aaatatcgtg cctctcctgc tttgcccggt 540
gtatgaaacc ggaaaggccg ctcaggagct ggccagcggc gcagaccggg aacacaagct 600
ggcagtcgac ccatccggtg ctctgcactc gacctgctga ggtccctcag tccctggtag 660
gcagctttgc cccgtctgtc cgcccggtgt gtcggcgggg ttgacaaggt cgttgcgtca 720
gtccaacatt tgttgccata ttttcctgct ctccccacca gctgctcttt tcttttctct 780
ttcttttccc atcttcagta tattcatctt cccatccaag aacctttatt tcccctaagt 840
aagtactttg ctacatccat actccatcct tcccatccct tattcctttg aacctttcag 900
ttcgagcttt cccacttcat cgcagcttga ctaacagcta ccccgcttga gcagacatca 960
ccatgcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg 1020
tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag 1080
gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt 1140
atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg 1200
aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag 1260
acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga 1320
tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg 1380
gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact 1440
ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga 1500
tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca 1560
acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt 1620
tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta 1680
tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc 1740
tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca 1800
atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg 1860
ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg 1920
tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat 1980
agagtagatg ccgaccgcgg gatccactta acgttactga aatcatcaaa cagcttgacg 2040
aatctggata taagatcgtt ggtgtcgatg tcagctccgg agttgagaca aatggtgttc 2100
aggatctcga taagatacgt tcatttgtcc aagcagcaaa gagtgccttc tagtgattta 2160
atagctccat gtcaacaaga ataaaacgcg ttttcgggtt tacctcttcc agatacagct 2220
catctgcaat gcattaatgc attgactgca acctagtaac gccttncagg ctccggcgaa 2280
gagaagaata gcttagcaga gctattttca ttttcgggag acgagatcaa gcagatcaac 2340
ggtcgtcaag agacctacga gactgaggaa tccgctcttg gctccacgcg actatatatt 2400
tgtctctaat tgtactttga catgctcctc ttctttactc tgatagcttg actatgaaaa 2460
ttccgtcacc agcncctggg ttcgcaaaga taattgcatg tttcttcctt gaactctcaa 2520
gcctacagga cacacattca tcgtaggtat aaacctcgaa atcanttcct actaagatgg 2580
tatacaatag taaccatgca tggttgccta gtgaatgctc cgtaacaccc aatacgccgg 2640
ccgaaacttt tttacaactc tcctatgagt cgtttaccca gaatgcacag gtacacttgt 2700
ttagaggtaa tccttctttc tagaagtcct cgtgtactgt gtaagcgccc actccacatc 2760
tccactcgag cccatcagta tttctcctat aaaccatg 2798
<210> 3
<211> 6740
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ttctccgtat actattgatc gtttggcatc tagtgtacct tttgatgggg ggttcgacca 60
ggacggcatg cgtacatgcc ttctccattg ggacgttcct ccggctagac acgcacatac 120
tgtccgttca cactatccat tcattcattc ggccattagt aaatacactc atacatatta 180
gccgggatgt aatgacttgt agtgcatgag gtgtaggcag agagtccaga accggtgaca 240
cgcaacgaca gcaacgaaag caactccagt atcaaatagc aaagacctag aacatgctca 300
accccaggtt gttgcgggca tttgtcgtca gtgagtgagc aaatgtgcaa ttgggctaat 360
gaggatgcga ggtgaagata cggcaaccaa ggtacaatac cgtacggcaa gaggtgggac 420
aaagtggaag ggggcccacc tctacccggt gcgtgtagtt tatggccttg tcatggggtg 480
gttggcattg tagccacggc cttttccttt gcttttcccg cgtgcttcct gccttttccc 540
ccctctgctt tggtgtgcct cgtttttccc ctgccgtgcc tttttccctt ccttccctcc 600
tcccccccct tttttccagt ggtaccaaac cacaactccc acagcccaac ccggaaatcc 660
aagtggtggt ggtaagagct aagtaaagta aagtaaagtc aaaaagtaaa agccgggccc 720
caaaataatc aaatctacca aaaaaaggcg atgacacgga ccggcaaacc aactaagcca 780
aactaatcca aaccgccagc cagaagtaat ctccccttcc ctctcactct cctttcattc 840
attttagtgc tgttgttatt attactgttc ctaattacta ccaccatttt tttgattttg 900
atttttcccg tccttccttt ggtattatta ttattattct tctgcttctt cactttgcat 960
cgcctatctt tcttttcatt ctttactatc atcctttccc tcttcctcct ctctcttcct 1020
ctctctttcc ccccctcttc gccccgtttt tgcttcgcca gcgccggtgc ttcgttgacg 1080
tagcccacct gcgaacacct tttctacgtc aacgagccgt tgggacttgc caattcatcc 1140
tcagcctttc tctccttgca tcccatcccc ctcccacggt cgcgcgccct cttcaggtac 1200
taccaccacc tccaaccgcc gttcaccata acaacccacc cccctcctcc actaccatta 1260
ccacctccac ctcctccctc cctctccacg gccgtttttc tcacttaaca tccctttctt 1320
agatcttgtc gctcgctcct taccgcctct acattcgtta ttgttttgtt tgctggtagt 1380
ttttcgtgag gggttactgt tggtttctcg tcgaccacac cccctgccgc tctttgaacc 1440
cgctttgttt ataccgaact cccgccgttg gtgcgcgcac atccatccgt cagctgtcag 1500
tgcactatta gagcttgtcg ctttcgatac ttttgattcg gagatttttt tggtgatata 1560
gttttgtgat atctgcaaac tttctggttg tttgttcgac tcaatatacc aggaagctta 1620
ccgaccgttg tcatcgcatc cccagccgtc gaccgattcg ggatatcgcc atcgctcgcg 1680
ccatccttcg attgagatag gttcgcttca agtggttcaa atcgtggaga ggtggatata 1740
cgaaaacgcc gggaatagga catctatata gcccgggggt tagactattc gaatttccaa 1800
ccaccgaccc catgcatcag caatcttaac gactggcttt gatccttgac gtttgaatac 1860
tccgtgccta gggatatttg gttttggagg ttcccatctg agccttcgct ctgtccattc 1920
atttcagcat tgttacggtc ttttcgacca cctgaccact ctttatcgcc tgctattgtt 1980
acccaaccat tcgaatcaat taaaatccgc cgcctccacc atttgtagaa aaatgtgacg 2040
aactcgtgag ctctgtacag tgaccggtga ctctttctgg catgcggaga gacggacgga 2100
cgcagagaga agggctgagt aataagccac tggccagaca gctctggcgg ctctgaggtg 2160
cagtggatga ttattaatcc gggaccggcc gcccctccgc cccgaagtgg aaaggctggt 2220
gtgcccctcg ttgaccaaga atctattgca tcatcggaga atatggagct tcatcgaatc 2280
accggcagta agcgaaggag aatgtgaagc caggggtgta tagccgtcgg cgaaatagca 2340
tgccattaac ctaggtacag aagtccaatt gcttccgatc tggtaaaaga ttcacgagat 2400
agtaccttct ccgaagtagg tagagcgagt acccggcgcg taagctccct aattggccca 2460
tccggcatct gtagggcgtc caaatatcgt gcctctcctg ctttgcccgg tgtatgaaac 2520
cggaaaggcc gctcaggagc tggccagcgg cgcagaccgg gaacacaagc tggcagtcga 2580
cccatccggt gctctgcact cgacctgctg aggtccctca gtccctggta ggcagctttg 2640
ccccgtctgt ccgcccggtg tgtcggcggg gttgacaagg tcgttgcgtc agtccaacat 2700
ttgttgccat attttcctgc tctccccacc agctgctctt ttcttttctc tttcttttcc 2760
catcttcagt atattcatct tcccatccaa gaacctttat ttcccctaag taagtacttt 2820
gctacatcca tactccatcc ttcccatccc ttattccttt gaacctttca gttcgagctt 2880
tcccacttca tcgcagcttg actaacagct accccgcttg agcagacatc accatgcctg 2940
aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc 3000
tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg 3060
gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc 3120
ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg 3180
agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg 3240
aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg 3300
ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca 3360
ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg 3420
tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg 3480
ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc 3540
tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt 3600
cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc 3660
agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt 3720
atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg 3780
atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg 3840
ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac 3900
tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagagtagat 3960
gccgaccgcg ggatccactt aacgttactg aaatcatcaa acagcttgac gaatctggat 4020
ataagatcgt tggtgtcgat gtcagctccg gagttgagac aaatggtgtt caggatctcg 4080
ataagatacg ttcatttgtc caagcagcaa agagtgcctt ctagtgattt aatagctcca 4140
tgtcaacaag aataaaacgc gttttcgggt ttacctcttc cagatacagc tcatctgcaa 4200
tgcattaatg cattgactgc aacctagtaa cgccttncag gctccggcga agagaagaat 4260
agcttagcag agctattttc attttcggga gacgagatca agcagatcaa cggtcgtcaa 4320
gagacctacg agactgagga atccgctctt ggctccacgc gactatatat ttgtctctaa 4380
ttgtactttg acatgctcct cttctttact ctgatagctt gactatgaaa attccgtcac 4440
cagcncctgg gttcgcaaag ataattgcat gtttcttcct tgaactctca agcctacagg 4500
acacacattc atcgtaggta taaacctcga aatcanttcc tactaagatg gtatacaata 4560
gtaaccatgc atggttgcct agtgaatgct ccgtaacacc caatacgccg gccgaaactt 4620
ttttacaact ctcctatgag tcgtttaccc agaatgcaca ggtacacttg tttagaggta 4680
atccttcttt ctagaagtcc tcgtgtactg tgtaagcgcc cactccacat ctccactcga 4740
gcccatcagt atttctccta taaaccatgg atcgtgtact ttgttcaata ctagacaggg 4800
ccaaaatata agccgaagcc atgacgcaac gctctagaga ggttgttggg ggcgttgggc 4860
ccctaatctg tgtggaatac ataccaagcg ttgttgatgt tgccatttgt tcccaactgt 4920
attcacattt tatcttgata tgccatcgcg tcctcatttc cctggcattt gtttgattat 4980
atcctttttc tttttctttt ctactcttct tattccccac acgttttgtg ccattttcca 5040
ctaccttttc atccattcca ttcttactct ccttatggtg acacagaaaa agacctgcca 5100
ccgcaatcac ttttttactc ttcttctatc ccattttcta tatcttcttt ccatcctctc 5160
catatgacct atcccatctt ccctcagcca tggatctacc tatccctcct gactgaactc 5220
accccatccc ctacgctttc ttaatgattg ggtggtaggt atctgctgtg acgagtgaac 5280
gccgtgctat actctcctac cccgcgtttt gttttctacc tctttgtccg tcctttttta 5340
tctcgctatg tgtacagatg tatatttctc ttttcttatt tttcaagtag atcgaaatgc 5400
aagcatgcaa agctagctat ttagcttagc ccagctagtt ggattaaatc tgactcggtt 5460
gggttgggct gaatgaaaat gttcaaatgt ggttgattct tttgtagaga tgtaggagta 5520
gttctctgat tgttttgatg tgtgaaggta tttttgaacc gggacctagg tagggattgt 5580
ttctgttgat aggcaaatgc ggaggaccga tgagtagcag gtaatgtgtt tgatctggtc 5640
cttgaatgat gtggttcgtg gtaatgatga tgactgatga tgtttggctg aaaggagagc 5700
agaaaaacgc taagagacaa ttattatgta cagtacaaga atgcagcgga gagagaggcg 5760
aatgataaga aagtcagaag aaccatttcg tccttccatc cgtccatccc tccatcgcca 5820
atgacataag aaaaccccgg gcaagagaaa aagaagagtt aaaatgaaga gatactggaa 5880
cgaaagtgta ggaagggatc aatcaatcac tcaatcactc attcttctct ttcgaattcg 5940
acccgaaacc caacaacgcc aacggacccc tctcgccgcg ctgctcacgc tcctgcatgc 6000
gctgctcctt cgctcgacga cgttcctcct tatacttctc ccaggagacg ttcgcatcgt 6060
agcccatgac ctcgccatta ttcttctcag cctggaattt aaggaagtgg cttagagagg 6120
tctcctcgtc atgaggcgtc ctcacggcct gaggtaacgg acggacggca tcttcaccgg 6180
catacgcacg gagtccggta tggaaggccc attcgacggc ctcttcgacg ggcttgtcga 6240
ataggtaggg gaggaaaggc acgacggaga gaccgagcta gcaagtagta atttgcatgt 6300
gttagcggaa taattctata ccgcaagcgg agaaacagaa ctcaccccga taggcgccca 6360
agtacggaaa aagacactcc gggaattctt caacatgcgc ccagagtact tgaccaccga 6420
gtggatggta aaggccggaa gacccatact ggcgataccc tggaacacgg cgcgctgggc 6480
catgacggtg cggtagtctt cagctagggg gacgtgagtc gtgggccatg gcatcagcgt 6540
atcgctgctg ctactgcctg ttaacgagcc ggcgatgttg ccggttacca tgcccttggc 6600
gacttgctcg tgggtaaggt cgctggcatc tttgtaggcc tcgcagggag gcgctaggac 6660
gcggcgattg cggaggtagg ctttgtagcc ttcgtgggcg acatcgccga ttagataggt 6720
ccaggaaatg ccgtaggcgg 6740
<210> 4
<211> 1797
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gagagtccag aaccggtgac acgcaacgac agcaacgaaa gcaactccag tatcaaatag 60
caaagaccta gaacatgctc aaccccaggt tgttgcgggc atttgtcgtc agtgagtgag 120
caaatgtgca attgggctaa tgaggatgcg aggtgaagat acggcaacca aggtacaata 180
ccgtacggca agaggtggga caaagtggaa gggggcccac ctctacccgg tgcgtgtagt 240
ttatggcctt gtcatggggt ggttggcatt gtagccacgg ccttttcctt tgcttttccc 300
gcgtgcttcc tgccttttcc cccctctgct ttggtgtgcc tcgtttttcc cctgccgtgc 360
ctttttccct tccttccctc ctcccccccc ttttttccag tggtaccaaa ccacaactcc 420
cacagcccaa cccggaaatc caagtggtgg tggtaagagc taagtaaagt aaagtaaagt 480
caaaaagtaa aagccgggcc ccaaaataat caaatctacc aaaaaaaggc gatgacacgg 540
accggcaaac caactaagcc aaactaatcc aaaccgccag ccagaagtaa tctccccttc 600
cctctcactc tcctttcatt cattttagtg ctgttgttat tattactgtt cctaattact 660
accaccattt ttttgatttt gatttttccc gtccttcctt tggtattatt attattattc 720
ttctgcttct tcactttgca tcgcctatct ttcttttcat tctttactat catcctttcc 780
ctcttcctcc tctctcttcc tctctctttc cccccctctt cgccccgttt ttgcttcgcc 840
agcgccggtg cttcgttgac gtagcccacc tgcgaacacc ttttctacgt caacgagccg 900
ttgggacttg ccaattcatc ctcagccttt ctctccttgc atcccatccc cctcccacgg 960
tcgcgcgccc tcttcaggta ctaccaccac ctccaaccgc cgttcaccat aacaacccac 1020
ccccctcctc cactaccatt accacctcca cctcctccct ccctctccac ggccgttttt 1080
ctcacttaac atccctttct tagatcttgt cgctcgctcc ttaccgcctc tacattcgtt 1140
attgttttgt ttgctggtag tttttcgtga ggggttactg ttggtttctc gtcgaccaca 1200
ccccctgccg ctctttgaac ccgctttgtt tataccgaac tcccgccgtt ggtgcgcgca 1260
catccatccg tcagctgtca gtgcactatt agagcttgtc gctttcgata cttttgattc 1320
ggagattttt ttggtgatat agttttgtga tatctgcaaa ctttctggtt gtttgttcga 1380
ctcaatatac caggaagctt accgaccgtt gtcatcgcat ccccagccgt cgaccgattc 1440
gggatatcgc catcgctcgc gccatccttc gattgagata ggttcgcttc aagtggttca 1500
aatcgtggag aggtggatat acgaaaacgc cgggaatagg acatctatat agcccggggg 1560
ttagactatt cgaatttcca accaccgacc ccatgcatca gcaatcttaa cgactggctt 1620
tgatccttga cgtttgaata ctccgtgcct agggatattt ggttttggag gttcccatct 1680
gagccttcgc tctgtccatt catttcagca ttgttacggt cttttcgacc acctgaccac 1740
tctttatcgc ctgctattgt tacccaacca ttcgaatcaa ttaaaatccg ccgcctc 1797
<210> 5
<211> 1930
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gcccactcca catctccact cgagcccatc agtatttctc ctataaacca tggatcgtgt 60
actttgttca atactagaca gggccaaaat ataagccgaa gccatgacgc aacgctctag 120
agaggttgtt gggggcgttg ggcccctaat ctgtgtggaa tacataccaa gcgttgttga 180
tgttgccatt tgttcccaac tgtattcaca ttttatcttg atatgccatc gcgtcctcat 240
ttccctggca tttgtttgat tatatccttt ttctttttct tttctactct tcttattccc 300
cacacgtttt gtgccatttt ccactacctt ttcatccatt ccattcttac tctccttatg 360
gtgacacaga aaaagacctg ccaccgcaat cactttttta ctcttcttct atcccatttt 420
ctatatcttc tttccatcct ctccatatga cctatcccat cttccctcag ccatggatct 480
acctatccct cctgactgaa ctcaccccat cccctacgct ttcttaatga ttgggtggta 540
ggtatctgct gtgacgagtg aacgccgtgc tatactctcc taccccgcgt tttgttttct 600
acctctttgt ccgtcctttt ttatctcgct atgtgtacag atgtatattt ctcttttctt 660
atttttcaag tagatcgaaa tgcaagcatg caaagctagc tatttagctt agcccagcta 720
gttggattaa atctgactcg gttgggttgg gctgaatgaa aatgttcaaa tgtggttgat 780
tcttttgtag agatgtagga gtagttctct gattgttttg atgtgtgaag gtatttttga 840
accgggacct aggtagggat tgtttctgtt gataggcaaa tgcggaggac cgatgagtag 900
caggtaatgt gtttgatctg gtccttgaat gatgtggttc gtggtaatga tgatgactga 960
tgatgtttgg ctgaaaggag agcagaaaaa cgctaagaga caattattat gtacagtaca 1020
agaatgcagc ggagagagag gcgaatgata agaaagtcag aagaaccatt tcgtccttcc 1080
atccgtccat ccctccatcg ccaatgacat aagaaaaccc cgggcaagag aaaaagaaga 1140
gttaaaatga agagatactg gaacgaaagt gtaggaaggg atcaatcaat cactcaatca 1200
ctcattcttc tctttcgaat tcgacccgaa acccaacaac gccaacggac ccctctcgcc 1260
gcgctgctca cgctcctgca tgcgctgctc cttcgctcga cgacgttcct ccttatactt 1320
ctcccaggag acgttcgcat cgtagcccat gacctcgcca ttattcttct cagcctggaa 1380
tttaaggaag tggcttagag aggtctcctc gtcatgaggc gtcctcacgg cctgaggtaa 1440
cggacggacg gcatcttcac cggcatacgc acggagtccg gtatggaagg cccattcgac 1500
ggcctcttcg acgggcttgt cgaataggta ggggaggaaa ggcacgacgg agagaccgag 1560
ctagcaagta gtaatttgca tgtgttagcg gaataattct ataccgcaag cggagaaaca 1620
gaactcaccc cgataggcgc ccaagtacgg aaaaagacac tccgggaatt cttcaacatg 1680
cgcccagagt acttgaccac cgagtggatg gtaaaggccg gaagacccat actggcgata 1740
ccctggaaca cggcgcgctg ggccatgacg gtgcggtagt cttcagctag ggggacgtga 1800
gtcgtgggcc atggcatcag cgtatcgctg ctgctactgc ctgttaacga gccggcgatg 1860
ttgccggtta ccatgccctt ggcgacttgc tcgtgggtaa ggtcgctggc atctttgtag 1920
gcctcgcagg 1930
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gagagtccag aaccggtgac 20
<210> 7
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gaggcggcgg attttaattg attcgaatgg ttgggtaaca atagc 45
<210> 8
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gctattgtta cccaaccatt cgaatcaatt aaaatccgcc gcctc 45
<210> 9
<211> 52
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
catggtttat aggagaaata ctgatgggct cgagtggaga tgtggagtgg gc 52
<210> 10
<211> 52
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gcccactcca catctccact cgagcccatc agtatttctc ctataaacca tg 52
<210> 11
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
cctgcgaggc ctacaaagat 20
<210> 12
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cgctctgtcc attcatttca gc 22
<210> 13
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
atggcaacat caacaacgct 20
Claims (10)
1. the black-koji mould of one plant of production xylan, classification naming are black-koji mould (Aspergillus niger), bacterial strain number is
SJ1 has been preserved in China typical culture collection center, and the preservation time is on February 28th, 2019, deposit number CCTCC
NO:M201911。
2. the black-koji mould for producing xylan described in claim 1 is preparing the application in xylan.
3. the aspergillus niger of one plant of production xylan, which is characterized in that Msb2 gene inactivates in the bacterial strain.
4. the aspergillus niger according to claim 2 for producing xylan, which is characterized in that the Msb2 gene
Nucleotide sequence is as shown in SEQ ID NO.1, and the nucleotide sequence after the Msb2 gene inactivation is as shown in SEQ ID NO.2.
5. the aspergillus niger according to claim 1 for producing xylan, which is characterized in that the aspergillus niger is black
Aspergillus SJ1, is preserved in China typical culture collection center, and deposit number is CCTCC NO:M201911.
6. the construction method of any aspergillus niger for producing xylan of claim 3~5, which is characterized in that packet
Include following steps:
(1) aspergillus niger SJ1 genome is extracted；
(2) gene knockout segment △ Msb2 is constructed, the nucleotide sequence of the gene knockout segment is as shown in SEQ ID NO.3；
(3) aspergillus niger protoplast is prepared；
(4) gene knockout segment is imported in protoplast and carries out homologous recombination, obtain Msb2 gene inactivation aspergillus niger gene work
Journey bacterium.
7. any aspergillus niger for producing xylan of claim 3~5 is preparing the application in zytase.
8. application according to claim 7, which comprises the steps of: to produce the aspergillus niger gene of xylan
Engineering bacteria is fermenting agent, and porous fibrous material is fixation support, and fermentation prepares zytase, and the fixation support is
Polyurethane.
9. application according to claim 8, which is characterized in that the fixation support is located in advance by the following method
Reason:
The fixation support NaOH of 0.5~2M is impregnated into 30min~2h, being rinsed with water to pH is neutrality, in 0.5~2M HCl
Middle immersion 30min~2h, being rinsed with water to pH is neutrality, and drying is obtained by pretreated fixation support.
10. application according to claim 8, which is characterized in that the fermentation, fermentation condition are as follows:
Fermentation medium: 10~70g/L of xylan, NaNO32~6g/L, KH2PO40.3~2g/L, MgSO4·7H2O 0.1~
0.8g/L, 0.1~0.8g/L of Tween-80, with the preparation of wheat bran leachate；
The wheat bran leachate is prepared as follows: weighing the wheat bran of 15~60g/L, 800mL water is added, boils
It after 30min~2h, filters to get filtrate, is settled to 1L again with water；
Fermentation condition is as follows: 28~32 DEG C, 150~300r/min culture, 3~6d.
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Cited By (2)
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| CN111088173A (en) * | 2019-12-26 | 2020-05-01 | 南京高新工大生物技术研究院有限公司 | Aspergillus niger genetically engineered bacterium and construction method and application thereof |
| CN112708567A (en) * | 2021-01-26 | 2021-04-27 | 天津科技大学 | Fructosyltransferase and high-yield strain thereof |
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| CN112708567A (en) * | 2021-01-26 | 2021-04-27 | 天津科技大学 | Fructosyltransferase and high-yield strain thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110004070A8 (en) | 2020-07-31 |
| CN110004070B (en) | 2020-11-03 |
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Correction item: Biological Conservation Information Correct: CCTCC|NO:M2019115|2019.02.28 Number: 28-02 Page: The title page Volume: 35 Correction item: Biological Conservation Information Number: 28-02 Volume: 35 |
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Address after: 210000, 5 new model street, Gulou District, Jiangsu, Nanjing Applicant after: NANJING TECH University Address before: 210000 Nanjing University of Technology, Nanjing, Pukou, Jiangsu Province, 30 Zhujiang South Road Applicant before: NANJING TECH University |
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