CN109609388A - A kind of aspergillus niger and its application - Google Patents
A kind of aspergillus niger and its application Download PDFInfo
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- CN109609388A CN109609388A CN201811591691.1A CN201811591691A CN109609388A CN 109609388 A CN109609388 A CN 109609388A CN 201811591691 A CN201811591691 A CN 201811591691A CN 109609388 A CN109609388 A CN 109609388A
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- aspergillus niger
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- 241000228245 Aspergillus niger Species 0.000 title claims abstract description 66
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims abstract description 30
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims abstract description 29
- 239000001358 L(+)-tartaric acid Substances 0.000 claims abstract description 19
- 235000011002 L(+)-tartaric acid Nutrition 0.000 claims abstract description 19
- FEWJPZIEWOKRBE-LWMBPPNESA-N L-(+)-Tartaric acid Natural products OC(=O)[C@@H](O)[C@H](O)C(O)=O FEWJPZIEWOKRBE-LWMBPPNESA-N 0.000 claims abstract description 19
- 150000003839 salts Chemical class 0.000 claims abstract description 18
- DCEMCPAKSGRHCN-UHFFFAOYSA-N oxirane-2,3-dicarboxylic acid Chemical compound OC(=O)C1OC1C(O)=O DCEMCPAKSGRHCN-UHFFFAOYSA-N 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 27
- 239000001963 growth medium Substances 0.000 claims description 26
- 238000000855 fermentation Methods 0.000 claims description 15
- 230000004151 fermentation Effects 0.000 claims description 15
- GUPPESBEIQALOS-UHFFFAOYSA-L calcium tartrate Chemical compound [Ca+2].[O-]C(=O)C(O)C(O)C([O-])=O GUPPESBEIQALOS-UHFFFAOYSA-L 0.000 claims description 11
- 239000001427 calcium tartrate Substances 0.000 claims description 11
- 235000011035 calcium tartrate Nutrition 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 8
- 239000012530 fluid Substances 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 6
- 239000011591 potassium Substances 0.000 claims description 5
- 229910052700 potassium Inorganic materials 0.000 claims description 5
- 239000004593 Epoxy Substances 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 238000005903 acid hydrolysis reaction Methods 0.000 claims description 4
- 230000010307 cell transformation Effects 0.000 claims description 4
- 238000002425 crystallisation Methods 0.000 claims description 4
- 230000008025 crystallization Effects 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 238000009938 salting Methods 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims 1
- 238000005349 anion exchange Methods 0.000 claims 1
- 238000005341 cation exchange Methods 0.000 claims 1
- 150000001768 cations Chemical class 0.000 claims 1
- 239000002244 precipitate Substances 0.000 claims 1
- 239000004575 stone Substances 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 9
- 238000000034 method Methods 0.000 abstract description 9
- 244000005700 microbiome Species 0.000 abstract description 7
- 238000004321 preservation Methods 0.000 abstract description 5
- 241000894006 Bacteria Species 0.000 description 9
- 229960001367 tartaric acid Drugs 0.000 description 9
- 239000012265 solid product Substances 0.000 description 8
- 235000002906 tartaric acid Nutrition 0.000 description 8
- 239000011975 tartaric acid Substances 0.000 description 8
- 244000061456 Solanum tuberosum Species 0.000 description 6
- 235000002595 Solanum tuberosum Nutrition 0.000 description 6
- 239000000706 filtrate Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- XONPDZSGENTBNJ-UHFFFAOYSA-N molecular hydrogen;sodium Chemical compound [Na].[H][H] XONPDZSGENTBNJ-UHFFFAOYSA-N 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 238000004566 IR spectroscopy Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 241000233866 Fungi Species 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 230000002538 fungal effect Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 241000589220 Acetobacter Species 0.000 description 2
- 241000590020 Achromobacter Species 0.000 description 2
- 241000589291 Acinetobacter Species 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000589158 Agrobacterium Species 0.000 description 2
- 241000588986 Alcaligenes Species 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000588748 Klebsiella Species 0.000 description 2
- -1 L (+)-winestone Acid Chemical compound 0.000 description 2
- 241000187654 Nocardia Species 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 241000589180 Rhizobium Species 0.000 description 2
- 244000061458 Solanum melongena Species 0.000 description 2
- 235000002597 Solanum melongena Nutrition 0.000 description 2
- 235000009754 Vitis X bourquina Nutrition 0.000 description 2
- 235000012333 Vitis X labruscana Nutrition 0.000 description 2
- 240000006365 Vitis vinifera Species 0.000 description 2
- 235000014787 Vitis vinifera Nutrition 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108700005088 Fungal Genes Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 108091023242 Internal transcribed spacer Proteins 0.000 description 1
- 241001185603 Labrys Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 101100412856 Mus musculus Rhod gene Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 1
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae 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
- 101100242191 Tetraodon nigroviridis rho gene Proteins 0.000 description 1
- 208000035199 Tetraploidy Diseases 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- UNTBPXHCXVWYOI-UHFFFAOYSA-O azanium;oxido(dioxo)vanadium Chemical compound [NH4+].[O-][V](=O)=O UNTBPXHCXVWYOI-UHFFFAOYSA-O 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000001175 calcium sulphate Substances 0.000 description 1
- 235000011132 calcium sulphate Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 description 1
- CYUUZGXOQDCCGH-UHFFFAOYSA-N dodecyl dodecanoate Chemical compound CCCCCCCCCCCCOC(=O)CCCCCCCCCCC CYUUZGXOQDCCGH-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006735 epoxidation reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 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
- 230000007774 longterm Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- FWFGVMYFCODZRD-UHFFFAOYSA-N oxidanium;hydrogen sulfate Chemical compound O.OS(O)(=O)=O FWFGVMYFCODZRD-UHFFFAOYSA-N 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000011044 succinic acid Nutrition 0.000 description 1
- 150000003444 succinic acids Chemical class 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Botany (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
A kind of aspergillus niger and its application, belong to technical field of bioengineering.One aspect of the present invention provide a kind of aspergillus niger (Aspergillus niger) WH-2, for the bacterial strain on December 03rd, 2018 in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number is CGMCC No.16799, depositary institution address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3;Another aspect of the present invention provides the aspergillus niger and is preparing the application in L (+)-tartaric acid or its salt, provides new method to obtain L (+)-tartaric acid or its salt.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of aspergillus niger and its application.
Background technique
Tartaric acid, a kind of α-carboxylic acid also known as 2,3- dihydroxysuccinic acid or 2,3- dyhydrobutanedioic acid.1769, Sweden
Chemist Carl Wilhelm Scheele is most early in having found that L (+)-tartaric acid is deposited in the leftover bits and pieces arcilla of grape wine
Therefore named " tartaric acid ".L (+)-tartaric acid is widely present in nature, therefore be otherwise known as " Threaric acid ", especially sieve
Content is higher in shop sign in the form of a streamer fruit and grape, is important food emulsifying agent, beverage acid, medical resolving agent, calcium sulphate retarder, print
Resist agent, photographic developer, metal polish are contaminated, is widely used in food industry, medication chemistry and construction industry.Microorganism turns
Change method is current L (+)-tartaric acid industrialized production main stream approach, i.e., using cis-butenedioic anhydride as raw material, passes through hydrolysis and epoxidation reaction
Become cis-form epoxy succinic acid or its salt, then utilize the microorganism containing cis-Epoxysuccinate hydrolase, biocatalysis is suitable
Formula Epoxysuccinic acid or its salt generate L (+)-tartaric acid or its salt.
The strain reported at present for producing L (+)-tartaric acid has: Nocardia (Nocardia), Corynebacterium
(Coryncbacterium), Rhod (Rhodococcus), rhizobium (Rhizobium), pseudomonas
(Pseudomonas), achromobacter (Achromobacter), acetobacter (Acetobacter), Agrobacterium
(Agrobacterium), alcaligenes (Alcaligenes), acinetobacter (Acinetobacter), Klebsiella
Belong to (Klebsiella) and double end bacterium (Labrys).The strain of these reported production L (+)-tartaric acid is bacterium, is had no
Utilize the report of fungi production L (+)-tartaric acid.
Summary of the invention
In view of the problems of the existing technology, it is an object of the invention to design to provide the skill of a kind of aspergillus niger and its application
Art scheme.
A kind of described aspergillus niger (Aspergillus niger) WH-2, the bacterial strain is on December 03rd, 2018 in China
Microbiological Culture Collection administration committee common micro-organisms center preservation, deposit number be CGMCC No.16799, it is proposed that classification
Name are as follows: aspergillus niger Aspergillus niger, depositary institution address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
A kind of aspergillus niger WH-2 is preparing the application in L (+)-tartaric acid or its salt.
The application, it is characterised in that the following steps are included:
1) aspergillus niger WH-2 is cultivated in slant medium, 28 DEG C are cultivated 3~5 days to dense spore is grown, and spore is added
Suspension scrapes spore, washes down spore sufficiently, be filtered to remove mycelium with three layers of sterile lens wiping paper;
2) filtered above-mentioned spore liquid presses 3 × 106A/mL concentration is seeded in seed culture medium, 28 DEG C of shaken cultivations
24~36h obtains Aspergillus niger strain seed liquor;
3) above-mentioned Aspergillus niger strain seed liquor is taken to be forwarded in culture medium, 28 DEG C shaken cultivation 2~3 days, obtain phase
Then obtained aspergillus niger cell and fermentation liquid are added to cis-form epoxy succinic acid respectively by the aspergillus niger cell and fermentation liquid answered
Or in its salting liquid, 30 DEG C of shaken cultivations convert 3 days, obtain cell transformation liquid and fermentation liquid conversion fluid;
4) excessive CaCl is separately added into two kinds of conversion fluids2, Calcium Tartrate is obtained, filters and is rushed with distilled water
Calcium Tartrate is washed, then with sulfuric acid solution Calcium Tartrate, the acid hydrolysis solution obtained after then filtering is handed over by zwitterion
L (+)-tartaric acid or the finished product of its salt are obtained after changing purification, concentration, crystallization, drying.
The application, it is characterised in that the spore suspension: weighing the NaCl of the Tween80 and 1.8g of 0.2g,
200ml distilled water is added, is uniformly mixed, it is spare to put 4 DEG C of refrigerators by 121 DEG C of high pressure sterilization 20min;The slant medium
For PDA culture medium;The seed culture medium is PDB culture medium;The culture medium is PDB minimal medium.
The application, it is characterised in that the cis-form epoxy succinic acid salt selects sodium hydrogen cis-epoxysuccinate or cis-
Epoxysuccinic acid potassium.
The present inventor is by long-term and in-depth study, and separation obtains a kind of new microorganism from rural area soil
Bacterial strain, the new bacterial strain are identified as aspergillus niger (Aspergillus niger).Aspergillus niger strain and its fermentation liquid of the invention
L (+)-tartaric acid or its salt can be generated by hydrolysis cis-form epoxy succinic acid or its salt.To for obtain L (+)-tartaric acid or its
Salt provides new method.
Specific embodiment
The present invention is further described in conjunction with the embodiments.
In the examples below, spore suspension used are as follows: weigh the NaCl of the Tween80 and 1.8g of 0.2g, then plus
Enter 200ml distilled water, is uniformly mixed, it is spare to put 4 DEG C of refrigerators by 121 DEG C of high pressure sterilization 20min.
The composition of various culture mediums used is as follows:
(1) configuration method of plate screening culture medium are as follows: weigh 3g sodium nitrate, 1g dipotassium hydrogen phosphate, seven water sulfuric acid of 0.5g
Magnesium, 0.5g potassium chloride, 0.01g ferrous sulfate, 30g sucrose, 5g sodium hydrogen cis-epoxysuccinate, 20g agar, with distilled water mend to
1000ml, 121 DEG C of high pressure sterilization 20min.
(2) group of slant medium becomes self-control PDA culture medium, preparation method are as follows: the fresh potato of 200g peeling is weighed,
Potato is cut into small pieces and is put into pot, water 1000ml is added, is heated to boiling, keeps 30min.Again with double gauze while hot in measuring cup
Upper filtering, leaves filtrate.The agar of 20g glucose and 15~20g is added, and filtrate is supplemented to 1000ml, 121 DEG C of high pressures are gone out
Bacterium 20min.After the completion of sterilizing, sterilized eggplant-shape bottle is poured on the super-clean bench that ultraviolet disinfection is crossed, to inclined-plane in eggplant-shape bottle
After culture medium cooled and solidified, it is sealed against and a night is put in indoor inversion.If there is no long bacterium on slant medium, 4 DEG C are put it into
Refrigerator is spare.
(3) group of seed culture medium becomes self-control PDB culture medium, preparation method are as follows: the fresh potato of 200g peeling is weighed,
Potato is cut into small pieces and is put into pot, water 1000ml is added, is heated to boiling, keeps 30min.Again with double gauze while hot in measuring cup
Upper filtering, leaves filtrate.10g glucose is added, and filtrate is supplemented to 1000ml, 121 DEG C of high pressure sterilization 20min.
(4) group of culture medium becomes self-control PDB minimal medium, preparation method are as follows: weighs the new of 200g peeling
Fresh potato, potato is cut into small pieces and is put into pot, adds water 1000ml, is heated to boiling, and keeps 30min.It is taken advantage of again with double gauze
Heat filters on measuring cup, leaves filtrate.Addition 10g glucose, 1.4g ammonium nitrate, 20g yeast powder, tri- water dipotassium hydrogen phosphate of 1g,
PH6.0, and filtrate is supplemented to 1000ml, 121 DEG C of high pressure sterilization 20min.
Embodiment 1: the screening of aspergillus niger (Aspergillus niger) WH-2
The rural area soil that acquisition has fruits and vegetables rotten in Hangzhou, Zhejiang province city, weighs 1g soil sample, pours into rapidly equipped with bead
In sterile saline triangular flask, mixes, be then diluted to 10 respectively with pipette-2、10-3、10-4、10-5Times.It draws respectively
10-2、10-3、10-4、10-5Times dilution is injected on plate screening culture medium, is inverted culture dish after even spread, in 28 DEG C
In constant incubator, cultivate 2~5 days.The single colonie grown on picking plate is inoculated in the 250ml equipped with 50ml culture medium
In triangular flask, 28 DEG C, 180rpm shaken cultivation 2~3 days collect thallus and fermentation liquid respectively.In the cell and fermentation liquid of collection
In be separately added into the suspension of 10ml1M pH8.0 sodium hydrogen cis-epoxysuccinate, 30 DEG C after oscillating reactions 3 days, with ammonium metavanadate development process
(Liu Yeqing, Yan Wenkang, Zhou Wenlong wait the colorimetric method industrial microorganism of tartaric acid, 1983,13:32-37.) identification is anti-
Answer in liquid that whether there is or not tartaric acid generations.
The present invention has obtained one plant to have cis-form epoxy succinic acid or its salt hydrolysis being L after largely screen
The bacterial strain of (+)-tartaric acid or its salt characteristic, is stored in slant medium, is survived through CGMCC proof.The bacterial strain preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation registration number CGMCC
No.16799 is named as aspergillus niger (Aspergillus niger) WH-2, preservation day: on December 03rd, 2018, depositary institution
Location: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica (postcode: 100101).
Embodiment 2: the identification of aspergillus niger (Aspergillus niger) WH-2
Step 1: the Morphological Identification of bacterium
To isolated bacterial strain, according to " Fungal identification handbook " (Wei Jingchao is edited, Shanghai science tech publishing house,
1979) method observes the structures such as bacterium colony, mycelia, spore using PDA plate, microscope and scanning electron microscope.As a result it shows
Show, bacterial strain is cultivated 3 days for 30 DEG C in PDA culture medium, and bacterium colony is rounded, and surface texture is in granular form, and spore is black or dark brown
Color, conidium are spherical shape, and mycelia is flourishing.Referring to " Fungal identification handbook ", bacterium is primarily determined as aspergillus niger
(Aspergillus niger)。
Step 2: the molecular biology identification of bacterium
Aspergillus niger (Aspergillus is extracted using fungal gene group reagent box (being purchased from Takara, Code No.9765)
Niger) the genomic DNA of WH-2CGMCC No.16799.
Using Fungi Identification PCR Kit (being purchased from Takara, Code No.RR178), kit is utilized
The conserved sequence of included rDNA be primer, the internal transcribed spacer ribose rDNA (region ITS) of the unknown fungi of PCR amplification,
Tetraploid rice is carried out with sequence known in GenBank after sequencing, by unknown Fungal identification to category or kind.
PCR reaction system are as follows: step 2 resulting 50~100ng of template DNA, PCR Premix 25 μ l, Forward
Primer (20pmol/ μ l) 0.5 μ l, Reverse primer (20pmol/ μ l) 0.5 μ l, dH2O supplies 50 μ l of total volume.It uses
1% agarose gel electrophoresis carries out the verifying of PCR product, general ITS section length be about 300~1000bp (base-pair) no
Deng.
PCR reaction condition is as follows:
Use Ago-Gel DNA QIAquick Gel Extraction Kit (being purchased from Sangon company, Code No.518131) gel extraction mesh
Segment, and using Seq Reverse Primer and Seq Forward Primer as primer carry out DNA sequencing (Sangon be public
Department).Sequencing result shows that the ITS nucleotide sequence of aspergillus niger (Aspergillus niger) WH-2CGMCC No.16799 is such as
Shown in SEQ ID NO:1, i.e., aspergillus niger of the invention has ITS sequence shown in SEQ ID NO:1.
To there is ITS nucleotide sequence shown in SEQ ID NO:1 obtained in above-described embodiment 1, existed with blast program
It is compared in ncbi database, comparison result is as shown in table 1, belongs to aspergillus niger (Aspergillus niger).
According to the qualification result of above-mentioned morphology and molecular biology, the bacterial strain tested belongs to aspergillus niger
(Aspergillus niger)。
The BLAST of the ITS sequence of 1 aspergillus niger of table (Aspergillus niger) WH-2CGMCC No.16799 compares knot
Fruit
Embodiment 3: cis-form epoxy succinic acid potassium and aspergillus niger (Aspergillus niger) WH-2CGMCC are utilized
No.16799 produces L (+)-tartaric acid
First with slant medium culture aspergillus niger (Aspergillus niger) WH-2CGMCC in eggplant bottle
No.16799,28 DEG C are cultivated 3~5 days to dense spore is grown, and 5ml spore suspension is added, and are shoveled scraping spore with inoculation, are made spore
Son is sufficiently washed down, is filtered to remove mycelium with three layers of sterile lens wiping paper.Filtered above-mentioned spore liquid uses blood after diluting
Ball count plate counts, by 3x106A mL-1Concentration inoculating spores into the 250ml conical flask for the seed culture medium that 50ml is housed,
28 DEG C of 180rpm 24~36h of shaken cultivation obtain the Aspergillus niger strain seed liquor.Take the seed liquor of above-mentioned 20ml aspergillus niger
Inoculation equipped in the 1000ml conical flask of 200ml culture medium, 28 DEG C 180rpm shaken cultivation 2~3 days, obtain corresponding black
Aspergillus cell and fermentation liquid, the cis- epoxy amber for being respectively then 1M by the concentration that obtained aspergillus niger cell is added to 200ml
In amber potassium, 30 DEG C, 180rpm shaken cultivation converts 3 days, obtains cell transformation liquid and fermentation liquid conversion fluid.Again respectively to two kinds
Excessive CaCl is added in conversion fluid2, obtain Calcium Tartrate, filter and with distilled water flushing respectively obtain 37.6g and
37.4g Calcium Tartrate, then with sulfuric acid solution Calcium Tartrate, the acid hydrolysis solution obtained after filtering again passes through zwitterion
22.8g and 22.7g solid product is respectively obtained after exchange purification, concentration, crystallization, drying.Through infrared spectroscopy, ultraviolet spectra, core
Magnetic resonance spectrum, Mass Spectrometer Method determine that the solid product is tartaric acid.
In the present embodiment, the infrared spectroscopy of sample is examined with Nicolet-Nexus670 Fourier transform formula infrared spectrometer
It surveys;Nuclear magnetic resonance spectroscopy and the carbon spectrum of sample are detected with Bruker Avance DMX500 Nuclear Magnetic Resonance;The mass spectrum of sample is used
Bruker Esquire 3000plusMass spectrograph detection;The optical activity of sample is detected with WZZ-2B polarimeter.Specific rotatory power detection,
The specific rotatory power of the solid product isProve that the solid product is dextroform tartaric acid, i.e. L (+)-winestone
Acid, and purity is 99.9%.
Embodiment 4: sodium hydrogen cis-epoxysuccinate and aspergillus niger (Aspergillus niger) WH-2CGMCC are utilized
No.16799 produces L (+)-tartaric acid
First with slant medium culture aspergillus niger (Aspergillus niger) WH-2CGMCC in eggplant bottle
No.16799,28 DEG C are cultivated 3~5 days to dense spore is grown, and 5ml spore suspension is added, and are shoveled scraping spore with inoculation, are made spore
Son is sufficiently washed down, is filtered to remove mycelium with three layers of sterile lens wiping paper.Filtered above-mentioned spore liquid uses blood after diluting
Ball count plate counts, by 3x106A mL-1Concentration inoculating spores into the 250ml conical flask for the seed culture medium that 50ml is housed,
28 DEG C of 180rpm 24~36h of shaken cultivation obtain the Aspergillus niger strain seed liquor.Take the seed liquor of above-mentioned 20ml aspergillus niger
Inoculation equipped in the 1000ml conical flask of 200ml culture medium, 28 DEG C 180rpm shaken cultivation 2~3 days, obtain respectively corresponding
Aspergillus niger cell and fermentation liquid, then respectively by obtained aspergillus niger cell and fermentation liquid be added to 200ml concentration be 1M
Cis- epoxy succinic sodium in, 30 DEG C, 180rpm shaken cultivation, convert 3 days, obtain cell transformation liquid and fermentation liquid conversion fluid.
Excessive CaCl is added into conversion fluid again2, Calcium Tartrate is obtained, filters and respectively obtains 38.5g with distilled water flushing
With 38.3g Calcium Tartrate, then with sulfuric acid solution Calcium Tartrate, the acid hydrolysis solution that is obtained after filtering again by yin-yang from
23.5g and 23.4g solid product is respectively obtained after son exchange purification, concentration, crystallization, drying.
Through infrared spectroscopy, ultraviolet spectra, nuclear magnetic resoance spectrum, Mass Spectrometer Method, determine that the solid product is tartaric acid.This implementation
In example, the infrared spectroscopy of sample is detected with Nicolet-Nexus670 Fourier transform formula infrared spectrometer;The nuclear-magnetism of sample is total
Hydrogen spectrum of shaking and carbon spectrum are detected with Bruker Avance DMX500 Nuclear Magnetic Resonance;The mass spectrum of sample Bruker Esquire
3000plusMass spectrograph detection;The optical activity of sample is detected with WZZ-2B polarimeter.Specific rotatory power detection, the ratio of the solid product
Optical activity isProve that the solid product is dextroform tartaric acid, i.e. L (+)-tartaric acid, and purity is
99.9%.
In summary, aspergillus niger of the invention (Aspergillus niger) WH-2CGMCC No.16799 and its fermentation
It is L (+)-tartaric acid or the characteristic of its salt that liquid, which has cis-form epoxy succinic acid or its salt hydrolysis,.
It should be noted that cis-form epoxy succinic acid salt of the present invention can be above-mentioned cis-form epoxy succinic acid potassium
Or sodium hydrogen cis-epoxysuccinate, it can also be the salt of other cis-form epoxy succinic acids such as cis-form epoxy succinic acid calcium.
Sequence table
<110>Scientific and Technological Institutes Of Zhejiang
<120>a kind of aspergillus niger and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 586
<212> DNA
<213>aspergillus niger (Aspergillus niger)
<400> 1
tgcggaagga tcattaccga gtgcgggtcc tttgggccca acctcccatc cgtgtctatt 60
ataccctgtt gcttcggcgg gcccgccgct tgtcggccgc cgggggggcg cctttgcccc 120
ccgggcccgt gcccgccgga gaccccaaca cgaacactgt ctgaaagcgt gcagtctgag 180
ttgattgaat gcaatcagtt aaaactttca acaatggatc tcttggttcc ggcatcgatg 240
aagaacgcag cgaaatgcga taactaatgt gaattgcaga attcagtgaa tcatcgagtc 300
tttgaacgca cattgcgccc cctggtattc cggggggcat gcctgtccga gcgtcattgc 360
tgccctcaag cccggcttgt gtgttgggtc gccgtccccc tctccggggg gacgggcccg 420
aaaggcagcg gcggcaccgc gtccgatcct cgagcgtatg gggctttgtc acatgctctg 480
taggattggc cggcgcctgc cgacgttttc caaccatttt ttccaggttg acctcggatc 540
aggtagggat acccgctgaa cttaagcata tcaataaagg cggagg 586
Claims (5)
1. a kind of aspergillus niger (Aspergillus niger) WH-2, deposit number are as follows: CGMCC No.16799.
2. a kind of aspergillus niger WH-2 as described in claim 1 is preparing the application in L (+)-tartaric acid or its salt.
3. application as claimed in claim 2, it is characterised in that the following steps are included:
1) aspergillus niger WH-2 is cultivated in slant medium, 28 DEG C are cultivated 3~5 days to dense spore is grown, and spore suspension is added
Liquid scrapes spore, washes down spore sufficiently, be filtered to remove mycelium with three layers of sterile lens wiping paper;
2) filtered above-mentioned spore liquid presses 3 × 106A/mL concentration is seeded in seed culture medium, and 28 DEG C of shaken cultivations 24~
36h obtains Aspergillus niger strain seed liquor;
3) above-mentioned Aspergillus niger strain seed liquor is taken to be forwarded in culture medium, 28 DEG C shaken cultivation 2~3 days, obtain corresponding
Aspergillus niger cell and fermentation liquid, then respectively by obtained aspergillus niger cell and fermentation liquid be added to cis-form epoxy succinic acid or its
In salting liquid, 30 DEG C of shaken cultivations convert 3 days, obtain cell transformation liquid and fermentation liquid conversion fluid;
4) excessive CaCl is separately added into two kinds of conversion fluids2, obtain Calcium Tartrate, filter and with distilled water flushing wine
Stone acid calcium precipitate, then with sulfuric acid solution Calcium Tartrate, the acid hydrolysis solution obtained after then filtering is by cation and anion exchange essence
L (+)-tartaric acid or the finished product of its salt are obtained after system, concentration, crystallization, drying.
4. application as claimed in claim 3, it is characterised in that the spore suspension: weigh 0.2g Tween80 and
The NaCl of 1.8g adds 200ml distilled water, is uniformly mixed, it is spare to put 4 DEG C of refrigerators by 121 DEG C of high pressure sterilization 20min;Described
Slant medium is PDA culture medium;The seed culture medium is PDB culture medium;The culture medium is PDB inorganic salts
Culture medium.
5. application as claimed in claim 3, it is characterised in that the cis-form epoxy succinic acid salt selects cis- epoxy succinic
Sour sodium or cis-form epoxy succinic acid potassium.
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CN111778165A (en) * | 2020-08-11 | 2020-10-16 | 哈尔滨工业大学 | Aspergillus niger DFY1 and application thereof |
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CN111778165B (en) * | 2020-08-11 | 2022-10-25 | 哈尔滨工业大学 | Aspergillus niger DFY1 and application thereof |
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