CN106148222B - A kind of bacterium and its application in production 16-dicarboxylic acid - Google Patents
A kind of bacterium and its application in production 16-dicarboxylic acid Download PDFInfo
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- CN106148222B CN106148222B CN201610137025.5A CN201610137025A CN106148222B CN 106148222 B CN106148222 B CN 106148222B CN 201610137025 A CN201610137025 A CN 201610137025A CN 106148222 B CN106148222 B CN 106148222B
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- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
Abstract
The present invention provides a kind of novel bacterias, areAcinetobacterBelong to, belongs to microorganisms technical field, simultaneously, provide its application in production 16-dicarboxylic acid, bacterium provided by the invention is able to produce 16-dicarboxylic acid, produces 16-dicarboxylic acid using bacterium provided by the invention, avoids and produce DC using Candida tropicalis in the prior art16The harm of bacterial strain, novel bacteria fermenting and producing DC provided by the invention16Yield also greater than yield prepared by other biological method in the prior art.
Description
Technical field
The present invention relates to efficient production DC16Bacterium, while be related to its prepare DC16Method, and in particular to using culture
Method generates DC from oil-polluted soils, to energy metabolism hexadecane hydrocarbon16Bacterium be enriched with, screened, separated and identified, and
The DC of production is metabolized to it16It separated, purified and is measured.
Background technique
DC16It is a kind of important synthesis material, musk ambrette ketone can be used to, instead of natural musk, for preparing in a variety of
Patent medicine.DC16It is not present in nature, and chemical industry method is also difficult to synthesize, therefore, biological metabolism method becomes production DC16Most
Effective method.Up to the present, bioanalysis produces DC16Be using Candida tropicalis (Candida cloacae) come
It completes, it has not been found that producing DC using bacterium16Report.With Candida tropicalis (Candida cloacae) fermentation method
Prepare DC16Report appear in the seventies earliest, as in Japan tail et al. with cloaca Candida (Candida cloacae)
MR-12 produces DC16.Later, Chen Yuantong using candida tropicalis (Candida cloacae) mutant strain UH-3-9 production
DC16, by adding acrylic acid into reaction system, to inhibit the beta oxidation of dicarboxylic acids, reduce candida tropicalis
(Candida cloacae) to having generated DC16Decomposition, improve DC16Yield, DC when fermenting 3 days16Yield reach
54 g/L are above the yield of 40 g/L of Gao Zhongxiang (1990) and plant village (1987) report or so.The public affairs such as Cao Wubo (2011)
Opened using candida tropicalis (Candida cloacae) mutant strain ly-6 fermentation hexadecane production DC16Patent of invention,
After adding hexadecane and fermenting 165 hours, DC16Yield be 170.5 g/L, conversion ratio 88.1%.Candida tropicalis
Bacterium (Candida cloacae), i.e., Candida tropicalis, category Cryptococeales, Cryptococcaceae are a kind of fungies, can cause urgency
Property, subacute or chronic infection, are the most common nosomycosises, and can lead to skin, mucous membrane, internal organ and be damaged.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of production DC16Bacterium, and provide its and specific make
Preparation Method.The present invention, which is not only separated to one plant, can efficiently be metabolized production DC16Novel species bacterium, enrich microbial resources library, and
DC16Fermentation production rate be higher than before research, while avoid using Candida tropicalis ferment harm.
Bacterium number provided by the present invention is STKD-1, belongs to acinetobacter calcoaceticusAcinetobacter sp.Belong to, bacterium
Strain deposit number is CGMCCNo.11839, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, ground
Location is in BeiChen West Road, Chaoyang District, BeiJing City No.1 institute 3, and the deposit date is on December 9th, 2015.
Specific bacterium provided by the invention is to prepare the preparation step in 16-dicarboxylic acid as follows:
(1) culture medium is prepared
Fermentation medium II:
Hexadecane 1.5g/L, peptone 4.0g/L, (NH4)2SO40.15g/L, KH2PO40.8g/L, NaCl
0.1g/L, deionized water 1000mL, adjusting pH is 7.2, is dispensed into 500 mL triangular flasks, and 121 DEG C of 20min sterilizings are stand-by；
Liquid screening medium:
K2HPO41.5g/L, MgSO4 ·7H2O 0.5g/L, NH4NO3 1.5g/L, FeCl30.025g/L, it is anhydrous
CaCl20.01g/L, 1.5 g/L of hexadecane adjust pH to 7.0 with the NaOH of 2M, and 121 DEG C of 20 min sterilizes, to
With；
(2) it ferments
The preculture process of hexadecane zymogenous bacteria: by strain inoculated into liquid screening medium, 28 DEG C, 150
3 d of rpm/min condition shaken cultivation, obtains pre-culture solution；
Fermentation process: taking pre-culture solution 20mL to be inoculated into 3L fermentation medium II, and 28 DEG C, 150 rpm/min oscillation training
It supports, adjusts fermentation liquid pH to 7.2 every 24 h, 6 mol/L NaOH solutions, incubation time is 20 d, takes hair after fermentation
The extraction and measurement of zymotic fluid progress 16-dicarboxylic acid；
(3) extraction of 16-dicarboxylic acid
Fermentation liquid is heated to 85 DEG C, is while stirring 11.0 with NaOH adjustment pH, carries out film suction filtration, filter used while hot
Film is nitrocellulose filter, and aperture is 0.22 μm, collects smoke filtrate；Smoke filtrate is stood into 12 h under the conditions of 4 DEG C, takes suction filtration
Dilute sulfuric acid is added in liquid upper liquid, and adjustment pH stands 12 h under the conditions of being 2.0,4 DEG C, collects bottom precipitation；Take smoke filtrate lower part
Deionized water dissolving is added in precipitating, adjusts pH value of solution to 12 h are stood under the conditions of 2.0,4 DEG C with dilute sulfuric acid, collects bottom precipitation；
Merge two parts precipitating, low-temperature vacuum drying is to get total 16-dicarboxylic acid；
(4) gas chromatography mass spectrometry method measures 16-dicarboxylic acid content.
The present invention provides a kind of energy metabolisms to generate DC16Novel bacteria, avoid in the prior art using Candida tropicalis
Produce DC16The harm of bacterial strain, meanwhile, novel bacteria fermenting and producing DC provided by the invention16Yield be greater than in the prior art other
The yield of bioanalysis.
Detailed description of the invention
Fig. 1 is that gas chromatography mass spectrometry (GC-MS) measures D16As a result；
Fig. 2 is that high-efficiency fermenting produces DC16The phylogenetic tree of bacterial strain STDK-1.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated.
Embodiment 1
1, the enrichment of bacterium
Take being put into beaker near petrochemical industry gas station by 10 g of soil of oil pollution in the Huangdao District of Qingdao, then to its
The middle deionized water that 90 mL are added is impregnated and is stirred, and 30 min are stood, and 5 mL of leachate is taken to be added to the enrichment training of 100mL liquid
It supports in base, shaken cultivation (150 rpm/min) 5 d, obtain enrichment culture liquid under the conditions of 28 DEG C；Enriched medium forms (/L):
Beef extract 3.0g, peptone 10.0g, NaCl 5.0g are that 7.6,121 DEG C of 20 min sterilizings are stand-by with NaOH tune pH；
2, the screening and purifying of hexadecane zymogenous bacteria
Screening and culturing medium forms (/L): K2HPO41.5g, MgSO4 ·7H2O 0.5g, NH4NO31.5g, FeCl3
0.025g, anhydrous CaCl20.01g, hexadecane 1.5g, agar 20g adjust pH to 7.0 with the NaOH of 2M, and 121 DEG C
20 min sterilize, while hot inverted plate, and up to solid plate culture medium after cooling, agar 4g/L is added in semisolid screening culture medium,
Other compositions and content are constant, and agar is not added in liquid screening medium；Separation process: above-mentioned 20 μ L of enrichment culture liquid is taken to be added dropwise
It onto solid plate culture medium, then smears uniformly, 3 d of stationary culture under the conditions of 28 DEG C, 5 fast single colonies of picking growth
Carry out streak plate culture respectively again, plating medium and condition of culture used are the same, and 5 groups of plates are obtained.It sees respectively
5 groups of flat-plate bacterial colony features are examined, if colony characteristics are inconsistent, then continue to repeat culture of crossing, until colony characteristics are consistent, put down at this time
Plate bacterium colony is single bacterium (pure bacterium).It is cultivated by above-mentioned multiple scribing line, isolates 5 plants of pure bacterium, be denoted as STKD-1, STKD-2,
STKD-3, STKD-4 and STKD-5 will be stored in semisolid screening culture medium, for use under the conditions of this 5 plants 4 DEG C of pure bacterium respectively；
3, the fermentation of bacterium generates DC16Used medium composition
(1) fermentation medium I forms (/L): hexadecane 2g, NaCl 0.5g, (NH4)2SO40.15g, MgSO4·
7H2O 0.03g, NaNO3 0.1 g, NaH2PO40.5g, FeCl30.04 g, deionized water are prepared, and are 7.2 with NaOH tune pH,
It is dispensed into 500 mL triangular flasks, 121 DEG C of 20min sterilizings are stand-by；
(2) fermentation medium II forms (/L): hexadecane 1.5g, peptone 4.0g, (NH4)2SO40.15g,
KH2PO40.8g, NaCl 0.1g, deionized water 1000mL, adjusting pH is 7.2, is dispensed into 500 mL triangular flasks, 121 DEG C
20min sterilizing is stand-by；
(3) fermentation medium III forms (/L): hexadecane 2.5 g, KH2PO41.5g, K2HPO42.0g, NH4
NO30.5g, NaCl 0.5g, MgSO4·7H2O 0.01g, anhydrous CaCl20.01g, FeSO40.002g, deionized water
1000mL, adjusting pH is 7.2, is dispensed into 500 mL triangular flasks, and 121 DEG C of 20min sterilizings are stand-by；
4, specific fermentation process:
(1) the preculture process of hexadecane zymogenous bacteria: by bacterial strain STKD-1, STKD-2, STKD-3, STKD-4 and
STKD-5 is inoculated into liquid screening medium respectively, and 28 DEG C of condition oscillations (150 rpm/min) cultivate 3 d, obtains preculture
Liquid；
(2) fermentation process: pre-culture solution 20mL points of STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5 are taken respectively
It is not inoculated into 3L fermentation medium I, fermentation medium II and fermentation medium III, 28 DEG C of oscillation (150 rpm/min) trainings
It supports, adjusts fermentation liquid pH to 7.2 every 24 h, 6 mol/L NaOH solutions, incubation time is 20 d, takes hair after fermentation
Zymotic fluid carries out DC16Extraction and measurement；
5、DC16Extraction process
Above-mentioned fermentation liquid is heated to 85 DEG C, is while stirring 11.0 with NaOH adjustment pH, carries out film suction filtration, institute while hot
It is nitrocellulose filter with filter membrane, aperture is 0.22 μm, collects smoke filtrate, is at this time soluble 16-dicarboxylic acid sodium in filtrate
(under the conditions of temperature is 85 DEG C, 16-dicarboxylic acid sodium is solubilised state).Then smoke filtrate is stood into 12 h under the conditions of 4 DEG C,
Solubility reduces and is deposited in bottom under 16-dicarboxylic acid sodium low temperature, further locates to upper liquid and bottom precipitation respectively
Reason, upper liquid suspense make liquid (I), and bottom precipitation suspense precipitates (I)；
Note: remain a small amount of 16-dicarboxylic acid sodium in liquid (I)；It is processed for 16-dicarboxylic acid sodium liquid (I) to precipitate (I)
Journey: to dilute sulfuric acid is added in liquid (I), adjustment pH is 2.0, remains in the 16-dicarboxylic acid sodium in liquid (I) at this time in acid item
DC is formed under part16, 12 h, DC are stood under the conditions of 4 DEG C16Solubility reduces under cryogenic conditions, is deposited to container with crystal form
Precipitating is collected in bottom, is denoted as precipitating (II)；
Precipitate (I) treatment process: to deionized water dissolving is added in precipitating (I), then with the pH of dilute sulfuric acid adjustment solution
12 h, DC are stood under the conditions of to 2.0,4 DEG C16Crystallization is denoted as precipitating (III) in bottom of bottle under cryogenic conditions；
Merge precipitating (II) and precipitating (III), low-temperature vacuum drying is to get to total DC16, because utilizing separation in the present invention
5 kinds of bacterial strains (STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5) being purified to are respectively to three kinds of fermentation mediums (I, II
And III) carry out hexadecane hydrocarbon fermenting and producing DC16, so 15 groups of total DC are obtained16；
6, gas chromatography mass spectrometry (GC-MS) method measures DC16
(1) gas chromatography mass spectrometry (GC-MS) measures DC16Operating parameter
GC-MS parameter setting: the post case inner equilibrium time is 0.25 min, sets maximum temperature as 325 DEG C, column temperature heats up
Program is 0 min up to 150 DEG C, and then 6 DEG C/min is warming up to 300 DEG C, and total run time is 35 min；Sample volume is 1 μ L,
Sample clean pumping velocity is 300 μ L/min；
(2) gas chromatography mass spectrometry (GC-MS) measures DC16Result
5 bacterial strains (STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5) with above-mentioned three kinds of fermentation mediums (I, II and
III fermentation) is carried out respectively generates DC16, fermentation liquid is handled to obtain 15 groups of fermentation crystal by crystallization, low temperature drying, uses methanol
15 groups of crystal are dissolved, lysate is used as sample introduction, and using the material composition of GC-MS measurement crystalline solid, operating parameter is shown in 6 (1).It surveys
The result shows that, 15 groups of crystalline solid appearance times are consistent calmly, compared using database, determine that 15 groups of crystalline solid occur at 7.5min
Peak be DC16, such as Fig. 1；
7、DC16The calculating of yield and the determination of optimum operating condition
DC16Fermentation production rate calculated according to weight.To 5 bacterium of STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5
Strain is fermented in the fluid nutrient medium of 3L 20 d respectively, by crystallization and low temperature drying, by the DC of acquisition16It weighs, according to
Fermentating liquid volume calculates separately DC16Fermentation production rate (table 1).As can be seen from Table 1, three kinds of different fermentation mediums (I,
II and III) in, bacterial strain STKD-1 produces DC to hexadecane hydrocarbon fermentation16Yield be maximum, respectively 145.3g/L,
176.2 g/L and 114.7 g/L.And compare three kinds of different fermentation mediums (I, II and III) to DC16Fermentation production rate come
It sees, DC of the fermentation medium II to 5 bacterial strains16Fermentation production rate be maximum, so fermenting and producing DC16Optimal combination be fermentation
Medium ii and bacterial strain STKD-1.According to the above results, bacterial strain STKD-1 is chosen in subsequent experimental and is further analyzed for representative；
D of 15 bacterial strain of table in different fermentations culture medium (I, II and III) when 20 d of fermentation16Fermentation production rate (unit: g/
L)
8, the taxology identification of STKD-1
5 bacterial strains (STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5) fermenting and producing DC16The result shows that, bacterial strain
STKD-1 is maximum to the fermentation production rate of hexadecane hydrocarbon, therefore, chooses bacterial strain STKD-1, solves to its systematics position
Analysis；
(1) bacterial strain STKD-1DNA is extracted
The extraction of bacterial strain STKD-1 DNA is completed using kit-DP303, and extraction step in kit referring to illustrating
Book；
(2) PCR expands bacterial strain STKD-1 DNA
Utilize 5 '-AGA GTTTGA TCC TGG CTC AG-3 ' of forward primer PF and 5 '-GGY TAC of reverse primer PR
CTT GTT ACG ACT T-3 ' carries out PCR amplification to bacterial strain STKD-1 16S rDNA.PCR reaction system (50 μ L) includes: 1 μ
L Taq archaeal dna polymerase, 0.5 μ L template DNA, 5 μ L 10 × PCR buffers, 3.5 μ L MgCl2, 0.5 μ L PF and PR, 2 μ L
DNTP, 37 μ L ultrapure waters.PCR reaction condition: 95 DEG C of 3 min of initial denaturation, 95 DEG C of denaturation 45S, 55 DEG C of annealing 45S, 72 DEG C extend
90S, 32 circulations, then 72 DEG C of 10 min of extension；
(3) DNA sequencing
PCR product is sequenced with DNA sequencer (model: Applied Biosystems 3730XL), by what is measured
Sequence is sent to DDBJ (DNA Data Bank of Japan), and the accession number for obtaining bacterial strain STKD-1 is LC094963.
Particular sequence is as follows:
aacacatgca agtcgagcgg agagaggtag cttgctaccg atcttagcgg cggacgggtg
agtaatgctt aggaatctgc ctattagtgg gggacaacat ttcgaaagga atgctaatac
cgcatacgtc ctacgggaga aagcagggga tcttcggacc ttgcgctaat agatgagcct
aagtcggatt agctagttgg tggggtaaag gcctaccaag gcgacgatct gtagcgggtc
tgagaggatg atccgccaca ctgggactga gacacggccc agactcctac gggaggcagc
agtggggaat attggacaat gggcgcaagc ctgatccagc catgccgcgt gtgtgaagaa
ggccttatgg ttgtaaagca ctttaagcga ggaggaggct actttagtta atacctagag
atagtggacg ttactcgcag aataagcacc ggctaactct gtgccagcag ccgcggtaat
acagagggtg caagcgttaa tcggatttac tgggcgtaaa gcgcgcgtag gcggctaatt
aagtcaaatg tgaaatcccc gagcttaact tgggaattgc attcgatact ggttagctag
agtgtgggag aggatggtag aattccaggt gtagcggtga aatgcgtaga gatctggagg
aataccgatg gcgaaggcag ccatctggcc taacactgac gctgaggtgc gaaagcatgg
ggagcaaaca ggattagata ccctggtagt ccatgccgta aacgatgtct actagccgtt
ggggcctttg aggctttagt gccgcattta acgcgataag tagaccgcct ggggagtacg
gtcgcaagac taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg
tttaattcga tgcaacgcga agaaccttac ctggccttga catagtaaga actttccaga
gatggattgg tgccttcggg aacttacata caggtgctgc atggctgtcg tcagctcgtg
tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttttccttat ttgccagcga
gtaatgtcgg gaactttaag gatactgcca gtgacaaact ggaggaaggc ggggacgacg
tcaagtcatc atggccctta cggccagggc tacacacgtg ctacaatggt cggtacaaag
ggttgctacc tagcgatagg atgctaatct caaaaagccg atcgtagtcc ggattggagt
ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg cggatcagaa tgccgcggtg
aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtttg ttgcaccaga
Agtagctagc ctaactgcaa agagggcggt (SEQ ID NO:1)；
(4) creation of genealogical tree
Phylogenetic tree wound is carried out using 16S rDNA sequence of the nearest neighbour method to the higher bacterial strain of its homology of bacterial strain STDK-1
It builds, software used is CustalX2.1 and Mega5.From genealogical tree (Fig. 2) as can be seen that bacterial strain STDK-1 belongs toAcinetobacterBelong to, and withAcinetobacter calcoaceticus(X81661) 16S rDNA sequence similarity
For highest, it is 98%, is a kind of novel species bacterium, is named asAcinetobactersp.STDK-1(LC094963)。
The present invention isolates energy metabolism from soil and generates DC16Novel bacteria, then in the culture medium of different compositions plus
Enter sole carbon source of the hexadecane hydrocarbon as bacterium, to its shaker fermentation culture, fermentation liquid is obtained, to DC in fermentation liquid16It carries out
It isolates and purifies, and measurement DC after 20 d fermentation in 3L fermentor16Yield.In addition, generating DC to energy metabolism16It is thin
The DNA of bacterium extracts, be sequenced and phyletic evolution position is also analyzed.The result shows that in 5 bacterial strains isolated and purified
In (STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5), when bacterial strain STKD-1 ferments in fermentation medium II, DC16
Yield it is maximum, be 176.2 g/L, generate DC using Candida tropicalis metabolism than what is reported before16Yield it is all high.It is logical
Cross Phylogenetic analysis, bacterial strain STKD-1 withAcinetobacter calcoaceticus(X81661) 16S rDNA sequence
Column similarity is highest (98%), is a kind of novel species bacterium, is named asAcinetobacterSp.STDK-1, the bacterial strain log in serial number
For LC094963.
<120>a kind of bacterium and its application in production 16-dicarboxylic acid
<160>1
<210>1
<211>1410
<212>DNA
<213>acinetobacter calcoaceticus (Acinetobacter) is derived from
<222>(1)..(1410)
<400>1
aacacatgca agtcgagcgg agagaggtag cttgctaccg atcttagcgg cggacgggtg 60
agtaatgctt aggaatctgc ctattagtgg gggacaacat ttcgaaagga atgctaatac 120
cgcatacgtc ctacgggaga aagcagggga tcttcggacc ttgcgctaat agatgagcct 180
aagtcggatt agctagttgg tggggtaaag gcctaccaag gcgacgatct gtagcgggtc 240
tgagaggatg atccgccaca ctgggactga gacacggccc agactcctac gggaggcagc 300
agtggggaat attggacaat gggcgcaagc ctgatccagc catgccgcgt gtgtgaagaa 360
ggccttatgg ttgtaaagca ctttaagcga ggaggaggct actttagtta atacctagag 420
atagtggacg ttactcgcag aataagcacc ggctaactct gtgccagcag ccgcggtaat 480
acagagggtg caagcgttaa tcggatttac tgggcgtaaa gcgcgcgtag gcggctaatt 540
aagtcaaatg tgaaatcccc gagcttaact tgggaattgc attcgatact ggttagctag 600
agtgtgggag aggatggtag aattccaggt gtagcggtga aatgcgtaga gatctggagg 660
aataccgatg gcgaaggcag ccatctggcc taacactgac gctgaggtgc gaaagcatgg 720
ggagcaaaca ggattagata ccctggtagt ccatgccgta aacgatgtct actagccgtt 780
ggggcctttg aggctttagt gccgcattta acgcgataag tagaccgcct ggggagtacg 840
gtcgcaagac taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg 900
tttaattcga tgcaacgcga agaaccttac ctggccttga catagtaaga actttccaga 960
gatggattgg tgccttcggg aacttacata caggtgctgc atggctgtcg tcagctcgtg 1020
tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttttccttat ttgccagcga 1080
gtaatgtcgg gaactttaag gatactgcca gtgacaaact ggaggaaggc ggggacgacg 1140
tcaagtcatc atggccctta cggccagggc tacacacgtg ctacaatggt cggtacaaag 1200
ggttgctacc tagcgatagg atgctaatct caaaaagccg atcgtagtcc ggattggagt 1260
ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg cggatcagaa tgccgcggtg 1320
aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtttg ttgcaccaga 1380
agtagctagc ctaactgcaa agagggcggt 1410
Claims (1)
1. deposit number CGMCCNo.11839'sAcinetobacterBelong to bacterial strain and preparing the application in 16-dicarboxylic acid, specifically
Steps are as follows:
(1) culture medium is prepared
Fermentation medium II:
Hexadecane 1.5g/L, peptone 4.0g/L, (NH4)2SO40.15g/L, KH2PO40.8g/L, NaCl 0.1g/L,
Deionized water 1000mL, adjusting pH is 7.2, is dispensed into 500 mL triangular flasks, and 121 DEG C of 20min sterilizings are stand-by；
Liquid screening medium:
K2HPO41.5g/L, MgSO4 ·7H2O 0.5g/L, NH4NO3 1.5g/L, FeCl30.025g/L, anhydrous CaCl2
0.01g/L, 1.5 g/L of hexadecane adjust pH to 7.0 with the NaOH of 2M, and 121 DEG C of 20 min sterilizes, for use；
(2) it ferments
The preculture process of hexadecane zymogenous bacteria: by strain inoculated into liquid screening medium, 28 DEG C, 150 rpm/
3 d of min condition shaken cultivation, obtains pre-culture solution；
Fermentation process: taking pre-culture solution 20mL to be inoculated into 3L fermentation medium II, and 28 DEG C, 150 rpm/min shaken cultivations,
Fermentation liquid pH to 7.2 is adjusted every 24 h, 6 mol/L NaOH solutions, incubation time is 20 d, takes fermentation liquid after fermentation
Carry out the extraction and measurement of 16-dicarboxylic acid；
(3) extraction of 16-dicarboxylic acid
Fermentation liquid is heated to 85 DEG C, is while stirring 11.0 with NaOH adjustment pH, carries out film suction filtration while hot, filter membrane used is
Nitrocellulose filter, aperture are 0.22 μm, collect smoke filtrate；Smoke filtrate is stood into 12 h under the conditions of 4 DEG C, is taken in smoke filtrate
Dilute sulfuric acid is added in portion's liquid, and adjustment pH stands 12 h under the conditions of being 2.0,4 DEG C, collects bottom precipitation；Smoke filtrate lower part is taken to precipitate
Deionized water dissolving is added, adjusts pH value of solution to 12 h are stood under the conditions of 2.0,4 DEG C with dilute sulfuric acid, collects bottom precipitation；Merge
Two parts precipitating, low-temperature vacuum drying is to get total 16-dicarboxylic acid；
(4) gas chromatography mass spectrometry method measures 16-dicarboxylic acid content.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610137025.5A CN106148222B (en) | 2016-03-11 | 2016-03-11 | A kind of bacterium and its application in production 16-dicarboxylic acid |
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| CN102115768A (en) * | 2009-12-30 | 2011-07-06 | 曹务波 | Method for producing hexadecanedioic acid by synchronously fermenting n-hexadecane with microbe |
| CN102839133A (en) * | 2011-06-21 | 2012-12-26 | 上海凯赛生物技术研发中心有限公司 | Strain producing long chain dibasic acid, and application thereof |
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| CN102115768A (en) * | 2009-12-30 | 2011-07-06 | 曹务波 | Method for producing hexadecanedioic acid by synchronously fermenting n-hexadecane with microbe |
| CN102839133A (en) * | 2011-06-21 | 2012-12-26 | 上海凯赛生物技术研发中心有限公司 | Strain producing long chain dibasic acid, and application thereof |
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| LC094963.1;Hongsheng,L., 等;《Genbank》;20151114;第1-2页 * |
| 发酵法生产长链二元酸研究进展;徐成勇 等;《生物工程进展》;20021231;第22卷(第2期);第66-69页 * |
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