CN1026129C - Process for producing long-chain alpha, omega-dicarboxylic acid from orthoalkanes by microbe fermentation - Google Patents
Process for producing long-chain alpha, omega-dicarboxylic acid from orthoalkanes by microbe fermentation Download PDFInfo
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- CN1026129C CN1026129C CN 89102548 CN89102548A CN1026129C CN 1026129 C CN1026129 C CN 1026129C CN 89102548 CN89102548 CN 89102548 CN 89102548 A CN89102548 A CN 89102548A CN 1026129 C CN1026129 C CN 1026129C
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Abstract
The present invention relates to a method for using microorganism fermentation n-alkane to produce alpha. Omega-dicarboxylic acid, particularly to a method for highly producing heptadeca-carbon dicarboxylic acid. Seed liquid cultivated by a mutant NP-260 of a strain of candida tropicalis is transferred to the mixed liquor of the n-alkane whose pH value is from 5.0 to 8.5 and a fermentation medium with the density of 90 to 60%(V/V), wherein the n-alkane comprises 12 to 20 carbon atoms with the density of 10 to 40%(V/V). The fermentation medium is composed of alkali metal phosphates with the density of 5 to 14 grams per liter, sodium chloride with the density of 0.5 to 2.0 grams per liter, urea with the density of 0.5 to 2.0 grams per liter, yeast cream with the density of 0.5 to 4 grams per liter, corn steep liquor with the density of 0.5 to 2 grams per liter, acrylic acid with the density of 0.2 to 3 ml per liter and vitamins with the density of 10 to 100 milligrams per liter. The mixture is maintained for 48 to 170 hours at the temperature of 24 to 32 DEG C, and then, the generated alpha. Omega-dicarboxylic acid is separated and purified.
Description
The invention relates to microbial fermentation from normal alkane production long-chain alpha alpha, omega-dicarboxylic acid, particularly from the method for n-heptadecane fermentative production 17 carbon dicarboxylic acid.
17 carbon dicarboxylic acid are important source material of the famous and precious spices civet of synthetic.Since relatively more difficult with the long chain dicarboxylic acid that chemical process is produced more than 12 carbon, therefore adopt microbial fermentation to produce the research topic that long chain dicarboxylic acid has become people.As everyone knows, along with the increase of carbonatoms, the solubleness of long-chain normal alkane and the dicarboxylic acid corresponding with it can be more and more littler.In utilizing the method for microbial fermentation, because littler as n-heptadecane and product 17 carbon dicarboxylic acid solubleness in water of substrate, therefore bigger than the long chain dicarboxylic acid difficulty of producing 12-15 carbon.Above normal alkane and the dicarboxylic acid of the easiest oxidation 16 carbon of microorganism in general, thereby to accumulate the above dicarboxylic acid of 16 carbon, improve its output and be still a difficult problem that awaits solving.
All disclose the method for utilizing microbial fermentation to produce long chain dicarboxylic acid in USP4339536 and USP4624920, wherein the former produces long chain dicarboxylic acid with candida tropicalis assimilation straight chain hydrocarbon.As can be seen, the long chain dicarboxylic acid of the highest carbon number of producing with this method is 13 carbon dicarboxylic acid from example, and output reaches 45 grams per liters.The latter then be with Mycobacterium spKSM-B-33 from 6-22 carbon normal alkane, lipid acid and derivative thereof under 40-50 ℃ of higher temperatures and aeration condition, the method for fermentative preparation 6-22 carbon dicarboxylic acid in the developing medium that nitrogenous source and carbon source are arranged.In the example, only provide at 50 milliliters or 500 milliliters and shaken in the test tube, with the method as carbon source system 10-16 carbon dicarboxylic acid such as sec.-propyl palmitic acid, methyl palmitic acid, n-hexadecane, wherein the production peak of 14 carbon dicarboxylic acid is 16 mg/litre nutrient solutions, and the output of 16 carbon dicarboxylic acid only is 3 mg/litre nutrient solutions.
The dicarboxylic acid of the men's strain candida tropicalis mutant strain M2030 in the South Sea, village from various single alkane production respective chain length planted by Japan, wherein from n-heptadecane fermentative production 17 carbon dicarboxylic acid, output is 70 grams per liters on 3 liters of fermentor tanks, the n-heptadecane input amount is 30%(V/V), the n-heptadecane transformation efficiency is less than 30%.
When Shanghai Shen Yong waits resting cell (dense 15 * 10 °/milliliter of bacterium) the conversion n-heptadecane of human candida tropicalis N-15 high density to become 17 carbon dicarboxylic acid by force, reach 101 grams per liters, transformation efficiency 63.1%(" plant physiology journal " 1980, Vol.6, No.1).Institute of Micro-biology of the Chinese Academy of Sciences once used candida tropicalis mutant strain U in addition
2-21Generate 10-18 carbon dicarboxylic acid with grown cell and resting cell from various normal alkane fermentations of 10-18 carbon and conversion, wherein the output of 17 carbon dicarboxylic acid is respectively 41.8 grams per liters and 52.5 grams per liters.From above prior art as can be seen, the microbial fermentation generation long chain dicarboxylic acid particularly output of 17 carbon dicarboxylic acid awaits further raising, could adapt to need of industrial production.Once the application number of submitting to before the present invention has been for providing the fermentation process of fermentation strain that can high yield 16 carbon α alpha, omega-dicarboxylic acids in 87105445 the application, thereby the purpose of this invention is to provide a kind of two ends oxidation long-chain normal alkane strong and the output that new candida tropicalis mutant strain that the β-Yang Hua ability is weak and corresponding fermentation process have further improved 17 carbon dicarboxylic acid of n-heptadecane ability particularly that is more suitable for suitability for industrialized production.
The used bacterial strain of the present invention is candida tropicalis mutant strain (Candida tropicatism utant NP-260), be to generate the stronger candida tropicalis (referring to " microorganism journal " 20(1) of long chain dicarboxylic acid ability: 88-93 with a strain two ends oxidation normal alkane, 1980) be starting strain, by Sodium Nitrite and ultraviolet repeatedly mutagenic obtained.The used bacterial strain of the present invention is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, is numbered: CGMCC No.0139.
Seed culture medium of the present invention has: the wort of (1) 10 Bahrain adds the solid inclined-plane that 2% agar is made; (2) the alkane seed culture medium comprises KH
2PO
4The 6-10 grams per liter, corn steep liquor 2-4 grams per liter, sucrose 2-5 grams per liter, yeast extract paste 3-6 grams per liter, urea 2-5 grams per liter, heavy wax 40-60 milliliter/liter, tap water preparation, pH value about 5.
The process of cultivating seed is: get a transfering loop NP-260 yeast thalline, be coated on the wheat tooth juice solid inclined-plane (15 * 180 test tubes, every liquid amount 6-7 milliliter is put into the inclined-plane) and cultivated 40 hours in 28-30 ℃.Getting an above-mentioned cultured NP-260 bacterial classification all scrapes in the alkane seed culture medium of 30ml/250ml triangular flask, cultivated 36-44 hour on 220 rev/mins rotary shaker in 28-30 ℃, as first order seed, again cultured first order seed 20-25 milliliter is inserted in the alkane seed culture medium of 300ml/3000ml triangular flask or 500ml/5000ml triangular flask, on 28-30 ℃ of 180 rev/mins of rotary shakers, cultivated 2 days, as the ferment-seeded of fermentor tank.
With mutant strain candida tropicalis NP-260 production long-chain alpha alpha, omega-dicarboxylic acid of the present invention, particularly the concrete grammar of 17 carbon dicarboxylic acid is: ferment-seeded is inserted the 10-40%(V/V that contains that pH5.0-8.5 is preferably 7.0-7.6) normal alkane and the 90-60%(V/V of a 12-20 carbon atom) in the mixed solution of fermention medium.Consisting of of fermention medium: alkali metal phosphate 5-14 grams per liter is preferably the 6-10 grams per liter, sodium-chlor 0.5-2.0 grams per liter, urea 0.5-2.0 grams per liter, be preferably the 0.8-1.3 grams per liter, yeast extract paste 0.5-4 grams per liter is preferably the 2-3 grams per liter, corn steep liquor 0.5-2 grams per liter, vinylformic acid 0.2-3 milliliter/liter, be preferably the 0.5-2 milliliter/liter, VITAMIN 10-100 mg/litre, be preferably the 30-60 mg/litre, and some other known nutrition source.At 24-32 ℃, temperature preferably is to keep under 28-30 ℃ 48-170 hour with said mixture, then the α alpha, omega-dicarboxylic acid that produces is separated purification.The content of positive hydrocarbon is 10-20%(V/V in when beginning fermentation mixed solution), add normal alkane between in due course then, be as the criterion all the time 〉=5%(V/V) with the normal alkane concentration of fermented liquid.Alkali metal phosphate can be from KH
2PO
4NaH
2PO
4, K
2HPO
4, Na
2HPO
4Among select a kind of.VITAMIN can be selected vitamin A or B for use.
After the fermentation ends, fermented liquid is heated to about 85 ℃, adds NaOH and regulate about pH value to 10, remove thalline while hot.Clear liquid is separated out a large amount of 17 carbon dicarboxylic acid sodium salt crystals after placing cooling, and inclining crystalline mother solution (at 16-18 ℃, 17 carbon dicarboxylic acid content are about 1%).Sodium salt crystal cleans with a small amount of tap water, drains.Use behind the crystalline mother solution concentrating under reduced pressure with method and handle collection 17 carbon dicarboxylic acid sodium salts, above gained sodium salt is dissolved in the boiling water, regulate the pH value and decolour suction filtration for 10-12; Filtrate thin up post-heating adds sulfuric acid acidation to 90-100 ℃, and the pH value is 2-3, and leave standstill crystallisation by cooling and spend the night, suction filtration washing then, oven dry obtains white plates 17 carbon dicarboxylic acid crystallizates.
With candida tropicalis mutant strain NP-260 of the present invention and fermentation process, can obtain output higher 12-20 carbon α alpha, omega-dicarboxylic acid, particularly 17 carbon α alpha, omega-dicarboxylic acids.With present method fermentation 6 days, 17 carbon dicarboxylic acid output reached as high as 133 grams per liters, and transformation efficiency is more than 60%.
Example one
(1) gets the NP-260 of a transfering loop, be coated on 15 * 180 Boiling tube wort solid inclined-planes, cultivated 2 days at 28 ℃;
(2) get one of above-mentioned bacterial classification, insert in 250 milliliters of triangular flasks that 30 milliliters of alkane seed culture mediums are housed and on 220 rev/mins rotary shaker, cultivated 40 hours in 28-30 ℃.KH in the alkane seed culture medium
2PO
48 grams per liters, yeast extract paste 5 grams per liters, corn steep liquor 3 grams per liters, sucrose 3 grams per liters, urine is 3 grams per liters not, 50 milliliters/liter in heavy wax, tap water preparation, pH value 5.0.
(3) in 500 milliliters of triangular flasks of 15 milliliters of fermention mediums are housed, insert 4 milliliters of above-mentioned seed liquor, 200 rev/mins of rotary shaker top fermentations 4 days, transferred a pH value in 7.5-8.0 with sodium hydroxide in per 24 hours.Fermention medium contains NaH
2PO
410 grams per liters, yeast extract paste 1.5 grams per liters, corn steep liquor 1 grams per liter, sodium-chlor 1 grams per liter, urea 1 grams per liter, 150 milliliters/liter of n-heptadecane, the tap water preparation, regulating the pH value is 7.2.Sterilized 30 minutes down at 110 ℃.After the fermentation ends, to 2-3,, obtain 17 carbon dicarboxylic acid white crystals with boiling off ether after 100 milliliters of ether extraction with the salt acid for adjusting pH value.Use the standard caustic soda solution titration, calculate that 17 carbon dicarboxylic acid content are 83.3 grams per liters in the fermented liquid.
Example two
Seed culture based component and cultural method are with example 1.KH in the fermention medium
2PO
48 grams per liters, sodium-chlor 1 grams per liter, yeast extract paste 2 grams per liters, corn steep liquor 1 grams per liter, urea 1 grams per liter, 200 milliliters/liter of n-heptadecane.Insert 4 milliliters of seed liquor, fermented 4 days.Per 24 hours transfer a pH value is 7.5-8.0.Fermentation ends with salt acid for adjusting pH value to 3, boils off ether then with 100 milliliters of ether extraction and obtains 17 carbon dicarboxylic acid white crystals.Use the standard caustic soda solution titration, calculate that 17 carbon dicarboxylic acid content are 87.7 grams per liters in the fermented liquid.
Example three
(1) gets a transfering loop NP-260 thalline, be coated on 15 * 180 the Boiling tube wort solid inclined-plane, totally 6, cultivated 2 days for 28 ℃; (2) a big slant strains access of 2 days of above-mentioned cultivation is equipped with in 250 milliliters of triangular flasks of 30 milliliters of malt extract mediums, totally 6 bottles, on 28 ℃ of following rotary shakers of 220 rev/mins, cultivated 2 days; (3) wort kind liquid 1 bottle graft of cultivating in (2) is gone into to be equipped with in 3000 milliliters of triangular flasks of 210 milliliters of alkane seed culture mediums (seed culture based component: KH
2PO
46 grams per liters, yeast extract paste 3 grams per liters, corn steep liquor 3 grams per liters, sucrose 3 grams per liters, heavy wax 50 grams per liters, urea 3 grams per liters), totally 6 bottles at 28 ℃, cultivated about 40 hours on 180 rev/mins the rotary shaker; (4) one of the kind liquid access of cultivating in (3) is equipped with in the automatic controlling tank of 16 liters of 8 liters of fermention mediums, wherein contains KH
2PO
464 grams, sodium-chlor 8 grams, yeast extract paste 16 grams, corn steep liquor 8 grams, 8 milliliters in vinylformic acid, vitamins B
12150 milligrams, urea 8 grams, defoamer 8 grams are dissolved in 5 liters of tap water, regulate pH value to 7.5 with sodium hydroxide, and 1.2 liters of n-heptadecane,, inoculate in the time of 29-30 ℃ after 30 minutes 121 ℃ of sterilizations; (5) after the inoculation, at 30 ℃ of stirring velocity 500-700 rev/mins, air flow 1: 1-1: 1.5, tank pressure 10-15 pound/centimetre
2Condition bottom fermentation 140 hours, wherein at third and fourth, respectively added in five days into 400 milliliters of n-heptadecane.Ferment after 24 hours with sodium hydroxide adjust pH to 7.6.From 48 hours, the pH value was controlled at 7.5 automatically until fermentation ends.(6) add the heating of 12 premium on currency after the fermentation ends, be warming up to 80 ℃, adding sodium hydroxide regulates after the pH value to 12, centrifugal remaining n-heptadecane and the thalline told, clear liquid concentrates, and crystallisation by cooling filters, collect the sodium salt crystal of 17 carbon dicarboxylic acid, the refining then sodium salt crystal that obtains white 17 carbon dicarboxylic acid.Recording 17 carbon dicarboxylic acid is 133 grams per liters, transformation efficiency 61.8%, refining total recovery 77.6%.
With vapor-phase chromatography 17 carbon dicarboxylic acid are carried out qualitative analysis (instrument model: day the island proper Tianjin GC7A), as Fig. 1, table 1, shown in Figure 2.1-6 is respectively the peak of known 11-16 carbon dicarboxylic acid among Fig. 1, makes Fig. 2 of table 1 and expression carbon number law according to this spectrogram, can judge thus among Fig. 1 that 7 is 17 carbon dicarboxylic acid.Fig. 3 is 17 carbon dicarboxylic acid purity check color atlass (the instrument model is the same), and recording its purity is 95.4%.
Table 1
Standard dicarboxylic acid first
Ester component and unknown sample C
11C
12C
13C
14C
15C
16C
Unknown
The reservation of each component
Time Vt(branch) 1.50 1.88 2.39 3.09 4.12 5.34 7.10
Vt-V
0(branch) 1.20 1.58 2.09 2.79 3.82 5.04 6.80
LgVt-V
00.08 0.20 0.32 0.45 0.58 0.70 0.83
Annotate: dead time V
0It is 0.30 minute
Claims (7)
1, a kind of method of utilizing microbial fermentation production long-chain alpha alpha, omega-dicarboxylic acid is characterized in that:
(1) using candida tropicalis mutant strain NP-260 (Candida tropicalis mutant NP-260, the seed liquor of CGMCC NO.0139) cultivating inserts the pH value and contains in the mixed solution of the normal alkane of 12-20 carbon atom of 10-40% (V/V) and 90-60% (V/V) fermention medium for 5.0-8.5, consisting of of fermention medium: alkali metal phosphate 5-14 grams per liter, sodium-chlor 0.5-2.0 grams per liter, urea 0.5-2.0 grams per liter, yeast extract paste 0.5-4 grams per liter, corn steep liquor 0.5-2 grams per liter, vinylformic acid 0.2-3 milliliter/liter, vitamin B group 10-100 mg/litre.
(2) said mixture was kept under 24-32 ℃ 48-170 hour.
(3) the α alpha, omega-dicarboxylic acid that produces is separated purification.
2, method according to claim 1, when it is characterized in that beginning to ferment in the mixed solution content of normal alkane be 10-20%(V/V), add normal alkane then during the fermentation, the normal alkane concentration that makes fermented liquid all the time 〉=5%(V/V).
3, method according to claim 1 is characterized in that in the composition of fermention medium: alkali metal phosphate 6-10 grams per liter, and urea 0.8-1.3 grams per liter, yeast extract paste 2-3 grams per liter, vinylformic acid 0.5-2 milliliter/liter, vitamin B group 30-60 mg/litre.
4, according to claim 1,2 method is characterized in that said normal alkane contains 15-17 carbon atom.
5, according to claim 1,2 method is characterized in that normal alkane contains 17 carbon atoms.
6, according to claim 1,3 described methods, wherein alkali metal phosphate is KH
2PO
4Or NaH
2PO
4Or K
2HPO
4Or Na
2HPO
4
7, method according to claim 1 is characterized in that the temperature of fermenting is controlled at 28-30 ℃, and the pH value of fermented liquid is controlled at 7.0-7.6.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1049688C (en) * | 1995-10-05 | 2000-02-23 | 中国石油化工总公司 | Method for treatment of alpha, omega dibasic acid fermentation liquor |
| CN1048754C (en) * | 1995-11-09 | 2000-01-26 | 中国科学院微生物研究所 | Process for producing long-chain alpha, omega-dicarboxylic acid by synchronous fermentation of microbe |
| CN1067725C (en) * | 1998-12-16 | 2001-06-27 | 中国石油化工集团公司 | Process for producing alpha, omega-long chain binary acid by using microorganism fermentation |
| ATE332389T1 (en) * | 1999-09-30 | 2006-07-15 | Cognis Ip Man Gmbh | IMPROVED FERMENTATION PROCESS |
| CN102115765B (en) * | 2009-12-30 | 2013-07-03 | 张艾琳 | Method for producing heptadecanedioic acid by fermenting and converting n-heptadecane |
| CN111349664B (en) * | 2018-12-24 | 2022-05-31 | 中国科学院微生物研究所 | Method for producing long-chain dicarboxylic acid by using saturated fatty acid as substrate |
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