CN103243033B - Candida sake and fermentation process thereof - Google Patents
Candida sake and fermentation process thereof Download PDFInfo
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- CN103243033B CN103243033B CN201210030280.1A CN201210030280A CN103243033B CN 103243033 B CN103243033 B CN 103243033B CN 201210030280 A CN201210030280 A CN 201210030280A CN 103243033 B CN103243033 B CN 103243033B
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- 241001530515 Candida sake Species 0.000 title claims abstract description 31
- 238000000855 fermentation Methods 0.000 title description 16
- 230000004151 fermentation Effects 0.000 title description 16
- 238000000034 method Methods 0.000 claims abstract description 39
- 150000007520 diprotic acids Chemical class 0.000 claims description 29
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 239000004215 Carbon black (E152) Substances 0.000 claims description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 10
- 239000004202 carbamide Substances 0.000 claims description 10
- 229910052799 carbon Inorganic materials 0.000 claims description 10
- 229930195733 hydrocarbon Natural products 0.000 claims description 10
- 229940041514 candida albicans extract Drugs 0.000 claims description 9
- -1 n-hexadecane hydrocarbon Chemical class 0.000 claims description 9
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 9
- 239000012138 yeast extract Substances 0.000 claims description 9
- DCAYPVUWAIABOU-UHFFFAOYSA-N alpha-n-hexadecene Natural products CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 125000004432 carbon atom Chemical group C* 0.000 claims description 3
- 125000001477 organic nitrogen group Chemical group 0.000 claims description 3
- 229910002651 NO3 Inorganic materials 0.000 claims description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 2
- 235000019764 Soybean Meal Nutrition 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
- 150000003863 ammonium salts Chemical class 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
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- 239000002253 acid Substances 0.000 abstract description 25
- 238000012262 fermentative production Methods 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 16
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- 239000002609 medium Substances 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
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- 241000894006 Bacteria Species 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
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- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 239000003513 alkali Substances 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
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- 102000004169 proteins and genes Human genes 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241000222178 Candida tropicalis Species 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- 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 5
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 101150053659 POX4 gene Proteins 0.000 description 4
- 101150004239 POX5 gene Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
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- 238000000576 coating method Methods 0.000 description 4
- YCOZIPAWZNQLMR-UHFFFAOYSA-N pentadecane Chemical compound CCCCCCCCCCCCCCC YCOZIPAWZNQLMR-UHFFFAOYSA-N 0.000 description 4
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- 238000003822 preparative gas chromatography Methods 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- WGTYBPLFGIVFAS-UHFFFAOYSA-M tetramethylammonium hydroxide Chemical compound [OH-].C[N+](C)(C)C WGTYBPLFGIVFAS-UHFFFAOYSA-M 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical compound OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 3
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- BGHCVCJVXZWKCC-UHFFFAOYSA-N tetradecane Chemical compound CCCCCCCCCCCCCC BGHCVCJVXZWKCC-UHFFFAOYSA-N 0.000 description 3
- 101150033406 CYP52A12 gene Proteins 0.000 description 2
- 101100161762 Candida tropicalis POX5 gene Proteins 0.000 description 2
- 101100168396 Debaryomyces hansenii CYP52A12 gene Proteins 0.000 description 2
- 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 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
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- 239000002655 kraft paper Substances 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
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- 150000007518 monoprotic acids Chemical class 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
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- QFGCFKJIPBRJGM-UHFFFAOYSA-N 12-[(2-methylpropan-2-yl)oxy]-12-oxododecanoic acid Chemical compound CC(C)(C)OC(=O)CCCCCCCCCCC(O)=O QFGCFKJIPBRJGM-UHFFFAOYSA-N 0.000 description 1
- HCUZVMHXDRSBKX-UHFFFAOYSA-N 2-decylpropanedioic acid Chemical compound CCCCCCCCCCC(C(O)=O)C(O)=O HCUZVMHXDRSBKX-UHFFFAOYSA-N 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
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- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to the method for candida sake new strains and fermentative production long-chain biatomic acid thereof.
Description
Invention field
The present invention relates to the method for candida sake (Candidasake) and fermentative production long-chain biatomic acid thereof.
Background technology
Long-chain biatomic acid (LCDA is also referred to as long chain dicarboxylic acid and long chain diacid) comprises chemical formula HOOC (CH2)
nthe diprotic acid of COOH, wherein n>=7.LCDA is the base monomer raw material of a series of extraordinary synthetic materials.Long-chain biatomic acid and derivative monomer thereof may be used for producing extraordinary nylon, polycarbonate, powder coating, spices, hot melt adhesive, extraordinary lubricant etc., are the important source material of synthetic perfume, engineering plastics, cold resistant plasticizer, senior lubricant and the product such as polyamide hot, powder coating.
There is the chemical synthesis process of multiple long-chain biatomic acid in this area, but using method is not easy and major part obtains is the mixture of long-chain biatomic acid and short chain diacid.Therefore, need to be further purified step when producing long-chain biatomic acid by these methods.Well known microbial transformation alkane, lipid acid or its ester of utilizing produces long-chain biatomic acid.Due to the restriction of existing biological method, chemosynthesis remains the optimization approach producing long-chain biatomic acid.
This area generally uses several yeast strain to carry out fermentative production long-chain biatomic acid, and when using alkane or lipid acid to cultivate as carbon source, long-chain biatomic acid produces as by product.Yeast has three Biochemical processes when carrying out metabolism to alkane and lipid acid: alkane is oxidized to the alpha-of lipid acid, lipid acid to alpha-, omega-dicarboxylic acid omega oxidation and lipid acid to CO
2be oxidized with the degraded type beta-of water.Relative to non-biological transformation method, the bioconversion method producing diprotic acid has multiple potential advantages.Wherein mainly be use reproducible raw material as parent material and produce the ability not producing harmful chemicals by product in diprotic acid process.Another considerable advantage of biological method is used to be that this method can easily regulate to utilize identical biological catalyst and identical device to produce multiple diprotic acid.Because existing organic chemical synthesis is only suitable for production single diprotic acid, several different diprotic acid synthesis will need the exploitation of the new synthetic schemes of each diprotic acid.On the other hand, yeast bio catalyzer can utilize identical device, substratum and step to produce the diprotic acid of all lengths, and difference is only the substrate providing different carbon atom length to yeast.
Although various technology can be utilized to ferment the output of diprotic acid to improve existing yeast such as candida tropicalis (Candidatropicalis), but by providing new candida bacterial strain to produce the long-chain biatomic acid of higher output yield, dodecanedioic acid, undecane dicarboxylic acid, tetradecane diacid, pentadecane diacid and Thapsic acid can also be comprised.Therefore an object of the present invention is to provide new bacterial strain and utilize these bacterial strains to produce the method for one or more long-chain biatomic acids.Use this candiyeast can substituted chemistry synthesis method completely to produce long-chain biatomic acid, and it be high to have output, less energy-consumption, the advantage saved material.
Summary of the invention
In one embodiment of the invention, provide candida sake CATH4016CCTCCM2011488.Described bacterial classification is preserved in China typical culture collection center (Wuhan, China Wuhan University) on December 29th, 2011, and deposit number is: CCTCCM2011488.
In another embodiment of the present invention, provide the method utilizing described candida sake CATH4016CCTCCM2011488 to produce diprotic acid, said method comprising the steps of: a. cultivates candida sake CATH4016 in the substratum containing at least one nitrogenous source and at least one organic substrates; The diprotic acid cultivated from a step is reclaimed with b..
In yet another embodiment of the invention, provide the method utilizing described candida sake CATH4016CCTCCM2011488 to produce diprotic acid, said method comprising the steps of: a. cultivates candida sake CATH4016 in the substratum containing at least one nitrogenous source and at least one organic substrates, and wherein said at least one organic substrates is selected from has the alkane of 12 to 16 carbon atoms or the normal alkane of 12 to 16 carbon atoms; The diprotic acid cultivated from a step is reclaimed with b..
In the above-described embodiment, described diprotic acid is at least one at least one or positive 12 carbon to 16-dicarboxylic acid in 12 carbon to 16-dicarboxylic acid.
In yet another embodiment of the invention, provide the method utilizing described candida sake CATH4016CCTCCM2011488 to produce diprotic acid, said method comprising the steps of: a. cultivates candida sake CATH4016 in the substratum containing at least one nitrogenous source and n-hexadecane hydrocarbon; The n-hexadecane diacid cultivated from a step is reclaimed with b..
In the above-described embodiment, those skilled in the art know operable nitrogenous source.Various organonitrogen and inorganic nitrogen can be used.Organic nitrogen source includes but not limited to yeast extract paste, corn steep liquor, extractum carnis, soybean meal hydrolysate, peptone, urea, each seed amino acid and peptide etc., and preferred nitrogenous source is urea.Inorganic nitrogen-sourcedly include but not limited to nitrate, ammoniacal liquor, liquefied ammonia, ammonium salt, nitrite etc.
The purposes of candida sake H4016CCTCCM2011488, it is mainly for the production of the application of long-chain biatomic acid.
Accompanying drawing explanation
Figure 1 shows that the protein alignments figure of the POX5 gene of CATH4016.
Figure 2 shows that the POX5 gene DNA sequence comparison diagram of CATH4016.
Figure 3 shows that the protein alignments figure of the POX4 gene of CATH4016.
Figure 4 shows that the POX4 gene DNA sequence comparison diagram of CATH4016.
Figure 5 shows that the protein alignments figure of the CYTb5 gene of CATH4016.
Figure 6 shows that the CYTb5 gene DNA sequence comparison diagram of CATH4016.
Figure 7 shows that the protein alignments figure of the CYP52D2 gene of CATH4016.
Figure 8 shows that the CYP52D2 gene DNA sequence comparison diagram of CATH4016.
Figure 9 shows that the protein alignments figure of the CYP52A12 gene of CATH4016.
Figure 10 shows that the CYP52A12 gene DNA sequence comparison diagram of CATH4016.
Figure 11 shows that the protein alignments figure of the CYP52A15 gene of CATH4016.
Figure 12 shows that the CYP52A15 gene DNA sequence comparison diagram of CATH4016.
Specific embodiments
By reading following detailed description, those skilled in the art will be more readily understood these and other embodiment of the present invention, key element and advantage.Should be understood that and provide following examples to prove and to explain preferred embodiment more of the present invention and aspect further, should not be interpreted as limiting its scope.
Although to similar or suitable method is described and material can be used for practice of the present disclosure or test herein, this document describes suitable method and material.Unless otherwise defined, all technology used herein are identical with the implication that disclosure one of ordinary skill in the art understand usually with scientific terminology.If there is conflict, be as the criterion with the definition of this explanation.
When quantity, concentration or other value or parameter provide with scope or height value list, be interpreted as specificly disclose arbitrary all scopes to any high low range boundary composition, no matter whether scope discloses separately.When enumerating the scope of numerical value herein, unless otherwise indicated, this scope is intended to comprise its end points, and all integers and mark within the scope of this.When the range of definition, unintentionally scope of the present invention is limited to the particular value enumerated.
When used herein, term " comprises ", " comprising ", " having " or its other variant any, is intended to relate to non-exclusive comprising.Such as, comprise the technique of a series of key element, method, object or device and be unnecessarily limited to only those key elements, but other key element that do not list especially or that this type of technique, method, object or device are intrinsic can be comprised.
When " one " or " one " is for describing multiple key element and composition herein only for convenience of and general sense of the present disclosure is provided.This description is understood to include one or at least one and odd number also comprises plural number, unless obviously it is intended to other.
Material herein, method and example use for illustrative purposes only, unless stated otherwise, be not intended to as restriction.
embodiment
as follows to part term definition herein:
Activation medium
A kind of aqueous solution substratum that employing is called " YPD substratum ", it comprises following composition: 20g/L glucose, 10g/L yeast extract, and 20g/L peptone.Described " YPD substratum " agar containing substratum weight 2%, makes substratum at room temperature gel.PH regulator is prepared to 7.0-7.5 by water and 1N sodium hydroxide solution by described YPD substratum.By this substratum sterilizing 20min at 121 DEG C.
Shake-flask seed substratum
Another is suitable for cultivating the substratum of bacterial strain of the present invention for " seed culture medium ", and it is a kind of aqueous solution substratum, comprises following composition: 10-30g/L sucrose, 1.5-10g/L corn slurries, 1-10g/L yeast extract, 4-12g/LKH
2pO
4, 0.5-5g/L urea, and the heavy wax of 0-50ml/L.The preparation of described substratum water, and at 121 DEG C sterilizing 20min.The independent sterilizing of urea, sterilizing 15 minutes at 110 DEG C, mixes with other sterilized compositions after cooling, uses as seed culture medium.
Medium of shaking flask fermentation
Another is suitable for cultivating the substratum of bacterial strain of the present invention for " fermention medium ", and it is a kind of aqueous solution substratum, comprises following composition: 1-10g/L corn slurries, 1-10g/L yeast extract, 5-12g/LKH
2pO
4, 0-3g/L sodium-chlor, 4-12g/L saltpetre, 10-40g/L sucrose, 0.5-3g/L urea, and the single alkane of 200-300mL/L or mixed alkanes, 0-1g/L vinylformic acid, 0-0.5g/L tween 80.Described by water and 1N sodium hydroxide solution by pH regulator to 7.5-7.8.By this substratum sterilizing 20min at 121 DEG C.The independent sterilizing of urea, sterilizing 15 minutes at 110 DEG C, mixes with other sterilized compositions after cooling, uses as fermention medium.
According to the present invention, bacterial strain can be used in various zymotechnique, and comprise 500ml fermentation shake flask technique, it comprises as follows:
With transfering loop, uniform spreading on the YPD substratum of the sterilizing of cell in test tube slant is launched.By this inclined-plane stabilize at 29-30 DEG C cultivate 2 days.Then 1/3rd of this slant culture inoculations being contained in the sterilizing seed culture medium of 30ml in 500ml shaking flask and cultivating 36-48 hour at temperature is 29-30 DEG C, shaking flask speed is 200-250rpm, and the amplitude of shake is 2.5-3.5cm.Then the meat soup of seed fermentation is seeded to the fermention medium of the sterilizing be contained in 500ml fermentation flask.This culture cultivates 90-120 hour at temperature is 29-30 DEG C, and shaking flask speed is 200-240rpm, and the amplitude of shake is 2.5-3.5cm, and is maintained between 7.0-8.0 by pH by monitoring and regulating with 1N sodium hydroxide solution.Dicarboxylic acid concentration in this fermenting broth can measure after this step.
the qualification of embodiment 1 candida sake
Screen candida sake (Candidasake) from oil field, obtain the bacterial strain of the positive 16-dicarboxylic acid of high yield through chemomorphosis process.
The qualification of candida sake (Candidasake):
1. sample collecting: suburb, Accessories during Binzhou, Shandong Province Shengli Oil Field gathers, and is chocolate crude oil pollution soil sample.
2. the strain separating of collect specimen: it is 10 that all soil samples collected are made extent of dilution respectively
-3, 10
-4, 10
-5, 10
-6diluent be coated with on YPD flat board respectively.Culture condition: 30 DEG C, 48 hours.
Cultivate through separating for several times, obtain oil field saccharomycetic purifying list bacterium colony.
3. identify
With " saccharomycetic means of taxonomic research " (TheYeasts:ATaxonomicStudy) the 90th chapter mycocandida (CandidaBerkhout) 90.241 candida sake (Candidasake) comparison (P1209), determine it is candida sake.
The utilization of 3.1 Carbon and nitrogen sources
Under known microorganism identification, carry out nutrition utilize experiment.In defined medium, add following nitrogenous source or carbon source, whether yeast culture, after 2 days or 3 days, is grown to Rule of judgment, and judge negative and positive, result is as follows:
Other test: take N-acetyl-glucosamine as carbon source, nitrogenous source is (+)
At 37 DEG C be (-)
Urase is (-)
This bacterial strain is identified for candida sake (Candidasake) with this.
embodiment 2 candida sake CATH4016 mutagenesis screening step
1. a strain is separated the candida sake obtained, it is stand-by to make the preservation of glycerine pipe.
2. get the stand-by glycerine pipe bacterial strain one being preserved in cryogenic refrigerator and put thaw at RT, after thawing, access is equipped with in the 500ml triangular flask of 50mlYPD substratum, is cultivate 20-24h in the rotary shaker of 210rpm in 29 DEG C of rotating speeds.
3. in the sterile centrifugation tube of 15ml, add the YPD nutrient solution of above-mentioned 10ml, centrifugal 2 minutes of 2000rpm, removes supernatant liquor, the physiological saline centrifuge washing with 0.85% 3 times.
4. remove supernatant liquor, add the aseptic lithium chloride 10ml of 2.5%, prepare bacteria suspension.
5. the NTG mutagenic compound taking 0.08mg add in 15ml sterile centrifugation tube, and add the physiological saline of 10ml0.85%, mixing.
6. each 10ml liquid in 3 and 4 is added being placed with in the sterilized petri dishes of 15mm*3mm stirrer of 90mm, build upper cover.
7. above-mentioned flat board prepares 2 pieces again by same method process
8. this plate is put on magnetic stirring apparatus and distinguish stir process 10,15,20 minutes.
9. each process flat board processes respectively by the following method.
10. above-mentioned treatment solution is got 10ml and is added in 15ml sterile centrifugation tube, and centrifugal 2 minutes of 2000rpm, removes supernatant liquor, the physiological saline centrifuge washing with 0.85% 3 times.
11. remove supernatant liquor, and the physiological saline with 0.85% carries out gradient dilution step by step.
12. every extent of dilution are drawn 0.15ml bacterium liquid and are carried out flat board coating.
Flat board after 13. coatings puts 29 DEG C of incubators inversion cultivation 3-4 days.
14. pickings are cultivated ripe single bacterium and are transferred into 15*150mm test tube slant, put 29 DEG C of incubators and cultivate 2 days.
Getting 1/3rd inclined-plane thalline accesses after 15. slant culture maturations is equipped with in the 250ml triangular flask of 10ml fermention medium, is cultivate 4 days in the rotary shaker of 210rpm in 29 DEG C of rotating speeds.4 DEG C of refrigerator cold-storages are put on residue inclined-plane.
16. calculate the average alkali consumption of shaking flask of control strain and the alkali consumption of bacterium, determine that the consumption buck of wanted bacterium is put down, generally improve more than 10% than primary dcreening operation control strain alkali consumption.
17. according to fermentation shake flask alkali consumption number determine to carry out again the bacterial strain of multiple sieve.
The bacterial strain choosing 5 strain alkali consumptions high in 18. general 100 strain shaking flask primary dcreening operation bacterial strains carries out multiple sieve.
19. choose primary dcreening operation inclined-plane corresponding to multiple sieve bacterial strain, above-mentioned connect primary dcreening operation after remaining 2/3rds inclined-planes, then 1/5th inclined-plane thalline switching inclined-plane F2 got by shovel, put 29 DEG C of incubators and cultivate 2 days.Residue inclined-plane continues to put refrigerator cold-storage.
20. cultivate the whole access of whole ripe of inclined-plane thalline is equipped with in the 500ml triangular flask of 30ml seed culture medium, is cultivate 44-48h in the rotary shaker of 210rpm in 29 DEG C of rotating speeds.1 of control strain glycerine pipe bacterium liquid is also all accessed in 1 seed flask simultaneously.
21. cultivate the seed liquor after maturations draws 3ml and adds and be equipped with in the 500ml triangular flask of 15ml fermention medium, is cultivate 110h in the rotary shaker of 210rpm to put bottle and measure in 29 DEG C of rotating speeds.General each seed bottle correspondence connects 2 bottles of fermented liquids.
22. sieve shaking flask measuring method again: acid base titration
23. through repeatedly screening the bacterial strain obtaining high yield 16-dicarboxylic acid, called after CATH4016.
According to candida tropicalis diprotic acid pathways metabolism, selected 15 genes relevant with production diprotic acid, specific as follows: CYP52A12, CYP52A13, CYP52A14, CYP52A15, CYP52A16, CYP52A17, CYP52A18, CYP52A19, CYP52A20, CYP52D2, POX4, POX5, CPRA, CPRB, and CYTb5.Candida sake CATH4016 is delivered to specialty order-checking mechanism, adopt Illumina platform end pairing sequencing to carry out genome sequencing, and from genome sequence, extract the gene order with these 15 candida tropicalis DNA homologs.Find that candida sake CATH4016CCTCCM2011488 of the present invention has any different at the homologous gene of following gene and candida tropicalis: CYP52A12, CYP52A15, CYP52D2, POX4, POX5 and CYTb5.Fig. 1-Figure 12 is shown in gene order contrast.
embodiment 3 n-hexadecane hydrocarbon fermentation
Diprotic acid testing method in fermented product:
The preparation of diprotic acid sample: fermentation ends, in 500mL triangular flask, with 6mol/L hydrochloric acid soln adjust pH to 3.0, every bottle adds 120mL ether, shakes 100 times, place more than 30min, stratification, takes out 40mL ether extracted liquid, is added in 100mL beaker, removing ether, obtains white solid.Then the mensuration of diprotic acid is carried out.
Determine the length of diprotic acid, or whether have monoprotic acid, then need by vapor-phase chromatography GC, after product process, be separated through capillary column, the diprotic acid of different carbon chain lengths and monoprotic acid, go out peak at different time, therefore just can determine final product.Such as: chromatographic apparatus model (GC9800): capillary column FFAP30*0.53mm*0.5um, detector FID, determines that final product compares with retention time Rf and standard substance and draws, standard substance are the AR reagent that Sigma or TGI company produces.
The mensuration of diprotic acid output: will the white solid obtained be extracted, add 100mL inner mark solution (tetramethyl ammonium hydroxide solution containing 4mg/mL hexanodioic acid), when extracting after the white solid that obtains all dissolves, enter gas-chromatography, the area ratio of mark and sample in record; Take standard substance (the diprotic acid standard substance of different carbon chain lengths simultaneously, purchased from Sigma or TGI Reagent Company) about 0.4000g, add 100mL inner mark solution (tetramethyl ammonium hydroxide solution containing 4mg/mL hexanodioic acid), after standard substance all dissolve, enter gas-chromatography, the area ratio of record standard product and sample; Calculate according to the area normalization method in vapor-phase chromatography and area ratio calculating, the diacid content of different carbon chain lengths can be obtained.Obtain the standard substance of known quality and the area ratio of internal standard substance by vapor-phase chromatography, and then obtain the relation of area ratio and mass ratio.Obtained the area ratio of sample and internal standard substance again by vapor-phase chromatography, according to foregoing area ratio and the relation of mass ratio and the quality of internal standard substance, obtain the quality of sample.
The technical process of 500ml shake flask fermentation and description:
Inclined-plane seed culture-shake-flask seed cultivation-shake flask fermentation
Inclined-plane seed culture: the switching inclined-plane, freeze-drying pipe inclined-plane of 4 DEG C of Storage in refrigerator, seed flask of transferring after cultivating 48h in 29 DEG C of incubators, seed culture is between 29 DEG C of shaking tables, and 230rpm, cultivates secondary fermentation in 48 hours.The seed of fermentation shake flask access 3.0mL, after inoculation, shaking flask is put between 29 DEG C of shaking tables, puts bottle and measure product acid amount after 230rpm shaking culture 90-110h.
The substratum used in the present embodiment is specific as follows:
Inclined-plane seed culture medium:
20g/L glucose, 10g/L yeast extract and 20g/L peptone, and be YPD substratum weight 2% agar.Add agar object to be to make substratum at room temperature gel.
Shake-flask seed substratum is as follows:
| Title | Proportioning |
| Sucrose | 20g/L |
| Corn steep liquor | 3g/L |
| Yeast extract paste | 5g/L |
| KH 2PO 4 | 8g/L |
| Heavy wax | 50ml/L |
| Urea (independent sterilizing) | 3g/L |
Take various starting material according to seed culture medium proportioning table and also record the amount taken, dissolve completely with tap water starting material after taking, quantitative charger packing, 30mL/ 500mL shaking flask, packing, add the heavy wax of 1.5ml after packing toward shaking flask with 1.0mL liquid-transfering gun, 6 layers of gauze and kraft paper wrapping after at 121 DEG C sterilizing 20min, for subsequent use.
Fermention medium:
| Title | Proportioning |
| Corn steep liquor | 1.8g/L |
| Yeast extract paste | 2g/L |
| KNO 3 | 10g/L |
| NaCl | 1g/L |
| Sucrose | 30g/L |
| KH 2PO 4 | 8g/L |
| N-hexadecane hydrocarbon | 20ml/L |
| Urea (sterilizing separately) | 1g/L |
| Tween 80 | 0.5g/L |
| Vinylformic acid | 1.2 |
Take various starting material according to fermention medium proportioning and also record the amount taken, with tap water, starting material are dissolved completely after taking, regulate pH to 7.5 with alkali lye, 15mL/ 500mL shaking flask, n-hexadecane hydrocarbon is added in fermention medium, with sterilizing 20min at 6 layers of gauze and latter 121 DEG C of kraft paper wrapping, for subsequent use.
500ml shake flask fermentation result:
Single alkane 500ml shake flask fermentation result:
| Bacterial strain | Diprotic acid kind | Produce acid amount g/L |
| CAT H4016 | Positive 16 carbon | 16.77 |
embodiment 4 mixed alkanes ferments
Mixed alkanes is the mixing of n-dodecane hydrocarbon, n-tridecane hydrocarbon, n-tetradecane hydrocarbon, Pentadecane hydrocarbon and n-hexadecane hydrocarbon (nC12+nC13+nC14+nC15+nC16) equal-volume.Fermention medium is as follows:
| Title | Proportioning |
| Corn steep liquor | 1.8g/L |
| Yeast extract paste | 2g/L |
| KNO 3 | 10g/L |
| NaCl | 1g/L |
| Sucrose | 30g/L |
| KH 2PO 4 | 8g/L |
| Mixed alkanes | 20ml/L |
| Urea (sterilizing separately) | 1g/L |
| Tween 80 | 0.5g/L |
| Vinylformic acid | 1.2 |
In fermenting process, fermented product, diprotic acid kind is all identical with embodiment 3 with output test.
Mixed alkanes 500ml shake flask fermentation result:
Claims (7)
1. candida sake (
candidasake) CATH4016, its preserving number is CCTCCM2011488.
2. prepare the method for diprotic acid, said method comprising the steps of: a. cultivates candida sake CATH4016 as claimed in claim 1 in the substratum containing at least one nitrogenous source and at least one organic substrates; The diprotic acid cultivated from a step is reclaimed with b.,
Wherein said at least one organic substrates is selected from the normal alkane with 12 to 16 carbon atoms, and described diprotic acid is at least one in positive 12 carbon to positive 16-dicarboxylic acid.
3. method as claimed in claim 2, wherein said at least one organic substrates is n-hexadecane hydrocarbon.
4. the method according to any one of claim 2-3, wherein said nitrogenous source is organic nitrogen source or inorganic nitrogen-sourced.
5. method as claimed in claim 4, wherein said organic nitrogen source is one or more in yeast extract paste, corn steep liquor, extractum carnis, soybean meal hydrolysate, peptone, urea, each seed amino acid and peptide.
6. method as claimed in claim 4, wherein said inorganic nitrogen-sourced be one or more in nitrate, ammoniacal liquor, liquefied ammonia, ammonium salt.
7. candida sake is producing the application in positive 12 carbon to positive 16-dicarboxylic acid as claimed in claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1233658A (en) * | 1999-05-28 | 1999-11-03 | 清华大学 | Method for screening candida for high prodn. of long chain diacid |
| CN1570124A (en) * | 2004-05-12 | 2005-01-26 | 上海凯赛生物技术研发中心有限公司 | Long chain normal dibasic acid production method |
| CN102191187A (en) * | 2011-04-01 | 2011-09-21 | 山东华星环保集团有限公司 | Candida lipolytica strain and method for preparing long chain dicarboxylic acid by using candida lipolytica strain |
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| CN1233658A (en) * | 1999-05-28 | 1999-11-03 | 清华大学 | Method for screening candida for high prodn. of long chain diacid |
| CN1570124A (en) * | 2004-05-12 | 2005-01-26 | 上海凯赛生物技术研发中心有限公司 | Long chain normal dibasic acid production method |
| CN102191187A (en) * | 2011-04-01 | 2011-09-21 | 山东华星环保集团有限公司 | Candida lipolytica strain and method for preparing long chain dicarboxylic acid by using candida lipolytica strain |
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