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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- 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
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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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110468115A (en) * | 2019-07-22 | 2019-11-19 | 浙江科技学院 | A kind of aspergillus niger cis-Epoxysuccinate hydrolase gene and its application |
| 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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| CN110468115A (en) * | 2019-07-22 | 2019-11-19 | 浙江科技学院 | A kind of aspergillus niger cis-Epoxysuccinate hydrolase gene and its application |
| CN110468115B (en) * | 2019-07-22 | 2021-07-13 | 浙江科技学院 | Aspergillus niger cis-epoxy succinate hydrolase gene and application thereof |
| CN111778165A (en) * | 2020-08-11 | 2020-10-16 | 哈尔滨工业大学 | Aspergillus niger DFY1 and application thereof |
| 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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