WO2021073011A1 - 一种生产长链二元酸的菌株及其发酵方法 - Google Patents

一种生产长链二元酸的菌株及其发酵方法 Download PDF

Info

Publication number
WO2021073011A1
WO2021073011A1 PCT/CN2020/072398 CN2020072398W WO2021073011A1 WO 2021073011 A1 WO2021073011 A1 WO 2021073011A1 CN 2020072398 W CN2020072398 W CN 2020072398W WO 2021073011 A1 WO2021073011 A1 WO 2021073011A1
Authority
WO
WIPO (PCT)
Prior art keywords
fermentation
strain
medium
saturated fatty
candida tropicalis
Prior art date
Application number
PCT/CN2020/072398
Other languages
English (en)
French (fr)
Inventor
汪江林
李葳
李秦佩
姜文晓
闵淑雅
刘修才
Original Assignee
上海凯赛生物技术股份有限公司
Cibt美国公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海凯赛生物技术股份有限公司, Cibt美国公司 filed Critical 上海凯赛生物技术股份有限公司
Priority to EP20876976.0A priority Critical patent/EP4047081A4/en
Priority to US17/769,459 priority patent/US20230203431A1/en
Publication of WO2021073011A1 publication Critical patent/WO2021073011A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • 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
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/74—Candida tropicalis

Definitions

  • the present disclosure belongs to the field of biotechnology, and specifically relates to a strain, in particular to a strain for producing long-chain dibasic acid, and a method for fermenting and producing long-chain dibasic acid by using the strain.
  • Long-chain dibasic acid is an important raw material for synthetic fragrances, nylon engineering plastics, hot melt adhesives, resins, cold-resistant plasticizers, medicines and pesticides. Its structural formula is HOOC(CH 2 ) n COOH, where n is greater than 7 Integer. Among them, ten-carbon dibasic acid is also called ten-carbon long-chain dibasic acid, sebacic acid, and its chemical formula is HOOC(CH 2 ) 8 COOH. As an important monomer raw material, it is widely used in the production of polyamide engineering plastics, such as nylon 510, nylon 1010, and nylon 610. It can also be used in high temperature resistant lubricating oil, epoxy resin curing agent, synthetic grease and artificial fragrance, cold resistant plasticizer, etc. It is one of the widely used chemical raw materials.
  • the deca-carbon dibasic acid in industrial production and application is prepared by chemical method using castor oil as raw material.
  • the main process flow is that castor oil is hydrolyzed to generate sodium ricinoleate soap, which is then further prepared to obtain ricinoleic acid.
  • the ricinoleic acid is pyrolyzed by adding alkali and heating to generate deca-carbon dibasic acid disodium salt. Further heating, adding acid, decoloring, and crystallization to obtain decadioic acid.
  • Catalytic cracking of castor oil is used to prepare deca-carbon dibasic acid.
  • the production process is complicated, and the reaction is carried out at a high temperature of 250-270°C.
  • the use of phenol or o-cresol toxic reagents pollutes the environment and severely restricts the production of deca-carbon dibasic by chemical methods.
  • the development of the acid industry is that castor oil is hydrolyzed to generate sodium ricinoleate soap, which is
  • the biological method to prepare decacarbodicarboxylic acid has the characteristics of simple production process and environmental protection. Yu Zhihua from the Institute of Microbiology, Chinese Academy of Sciences (see “Acta Microbiology” 1989 06: 0253-2654) obtained a high-yielding strain through mutagenesis and screening, and the yield of decacarbodiacid on a 16-liter fermenter reached more than 71g/L , The product of deca-carbon dibasic acid was obtained through water-phase crystallization, and the purity of the product was above 99.6%; the fermentation production of deca-carbon dibasic acid by Candida lipolytica was screened by the Institute of Forestry and Soil of the Chinese Academy of Sciences (see Journal of Microbiology, 1979, 01) The acid output is 30-40g/L.
  • decane or its analogues are often used as fermentation substrates.
  • the possible conversion pathways of such fermentation substrates in the fermentation process include: multi-step oxidation by bacteria to become target products Deca-dicarboxylic acid is consumed by the growth and maintenance of bacteria or enters the external environment after volatilization.
  • decane is toxic to microorganisms due to its own properties and inhibits the growth of microorganisms. Therefore, it is an important research direction to improve the anti-virus ability of the strain by improving the strain.
  • the first aspect of the present disclosure aims to provide a Candida tropicalis strain to solve the current problems of low cell concentration, long fermentation period, and low conversion efficiency in the process of the current Candida tropicalis strain producing decacarbodicarboxylic acid, thereby solving the problems Technical problems with high production costs.
  • the present disclosure solves the above-mentioned technical problems through the following technical solutions, and achieves the objective of the first aspect of the present disclosure.
  • the present disclosure provides a Candida tropicalis strain, which is Candida tropicalis (Am2525) Am2525, and the preservation number is CCTCC NO:M 2019419.
  • Candida tropicalis (Candida tropicalis Am2525) Am2525, which is a strain of Candida tropicalis (Candida tropicalalis) CAT N145 that oxidizes n-alkanes to produce mixed dicarboxylic acids. It was submitted to the China Center for Type Culture Collection (CCTCC) of Wuhan University on the same day, and the deposit number is CCTCC (2011192) as the starting strain, which was cultivated through combined mutagenesis and screening of atmospheric pressure and room temperature plasma ARTP and ultraviolet irradiation.
  • CTCC China Center for Type Culture Collection
  • Candida tropicalis (Candida tropicalis Am2525) Am2525 morphological characteristics: the colony is smooth and moist with milky white shiny, round and neat edges.
  • the second aspect of the present disclosure aims to propose the application of the Candida tropicalis (Am2525) Am2525 in the production of long-chain dibasic acids.
  • the present disclosure provides the application of the above-mentioned Candida tropicalis strain (Candida tropicalis Am2525) Am2525 in the production of deca dibasic acid.
  • the substrate used for fermentation conversion in the production of decacarbodibasic acid includes C10 normal alkanes, C10 linear saturated fatty acids, C10 linear saturated fatty acid esters, and C10 linear saturated fatty acids.
  • One or more of fatty acid salts include C10 normal alkanes, C10 linear saturated fatty acids, C10 linear saturated fatty acid esters, and C10 linear saturated fatty acids.
  • the third aspect of the present disclosure aims to propose a method for producing long-chain dibasic acid, especially a method for producing deca dibasic acid, so as to solve the current concentration of cells in the process of producing deca dibasic acid by Candida tropicalis strains.
  • the problems of low, long fermentation period and low conversion efficiency can solve the technical problems of high production cost.
  • the present disclosure solves the above technical problems through the following technical solutions, and achieves the third aspect of the present disclosure.
  • a method for producing long-chain dibasic acids is to prepare seed liquid by the Candida tropicalis strain (Am2525) Am2525, and/or; ferment to produce long-chain dibasic acids.
  • the long-chain dibasic acid is a C10 linear saturated dibasic acid.
  • the fermentation substrate includes one or more of C10 normal alkanes, C10 linear saturated fatty acids, C10 linear saturated fatty acid esters, and C10 linear saturated fatty acid salts.
  • the added amount of the fermentation substrate is 100-400 mL/L, and the volume ratio is the volume ratio of the substrate to the fermentation medium.
  • the temperature for preparing the seed liquid is 27-31°C; the fermentation temperature for the fermentation to produce decadioic acid is 27-31°C.
  • the method for producing long-chain dibasic acid wherein the fermentation medium for fermentation includes:
  • Nitrogen source The concentration of each nitrogen source is 0.5-10g/L
  • each inorganic salt is 0.5-12g/L.
  • the carbon source includes one or more of sucrose, glucose, maltose, molasses, fructose, rhamnose, arabinose, and sorbitol;
  • the nitrogen source includes one or more of yeast extract, corn steep liquor, urea, ammonia, ammonium sulfate, potassium nitrate and ammonium nitrate;
  • the inorganic salt includes one or more of potassium salt and sodium salt; preferably, the potassium salt includes one or more of potassium chloride, potassium nitrate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate,
  • the sodium salt includes one or more of sodium chloride, sodium nitrate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.
  • the fermentation medium includes:
  • the production method of the long-chain dibasic acid includes the following steps:
  • the culture medium for activation of the strain includes YPD medium.
  • the YPD medium includes glucose 2.0% (w/v), yeast extract 1.0% (w/v), peptone 2.0% (w/v), and the balance is water.
  • the seed culture medium includes: sucrose 10-20g/L, corn steep liquor 2-4g/L, yeast extract 3-8g/L, potassium dihydrogen phosphate 4-12g/L, urea 0.5 ⁇ 4g/L, C10 normal alkane, C10 straight chain saturated fatty acid, C10 straight chain saturated fatty acid ester and C10 straight chain saturated fatty acid salt one or more 0 ⁇ 80mL/L;
  • the culture time for preparing the seed liquid is 12 to 48 hours, and the seed maturity index is that the OD620 is 0.5 to 1.0 after being diluted 30 times with water.
  • step c) when the seed liquid is inoculated into the fermentation medium for fermentation, an inoculum of 10%-30% of the feed volume of the culture liquid is inoculated into the culture liquid.
  • step c) the fermentation is cultured at 28-31°C, the rotating speed of the shaker is 200-250 rpm, from the time when the substrate is added to the inoculation until the substrate in the fermentation broth has no significant residue, that is, the fermentation cycle is completed,
  • the fermentation period of the strain in the fermentation system is 90-200 hours.
  • the conversion efficiency of the bacterial species to the substrate in the fermentation system is more than 35%, more preferably more than 40%;
  • the present disclosure has the following remarkable features and positive effects:
  • the Candida tropicalis strain of the present disclosure has stronger resistance to the toxicity of the substrate decane than the parent, so that the bacteria can reach a higher OD during growth and fermentation, can transform more substrates, and increase productivity.
  • the production cost is reduced, the subsequent separation and purification is simple, the fermentation production process is easy to be large-scaled, and the application prospect is good.
  • Candida tropicalis (Candida tropicalis Am2525) Am2525
  • Figure 1 is a photograph of the first generation of the colony of Candida tropicalis Am2525 of the present disclosure
  • Figure 2 is a colony photograph of the sixth generation of Candida tropicalis Am2525 of the present disclosure
  • Fig. 3 is the growth curve of Candida tropicalis Am2525 in the decane seed medium containing the initial concentration of 10%, 20%, and 40% (v/v), respectively.
  • the inventor of the present application used the existing long-chain dibasic acid production strain Candida tropicalis CAT N145 (this strain has been published in the Chinese invention patent with the publication number CN 102839133A and the publication date 2012-12-26 Public, the patent procedure was submitted to the China Center for Type Culture Collection (CCTCC) at Wuhan University on June 9, 2011, and the deposit number is CCTCC M 2011192 as the starting strain, which was induced by domestication and conventional mutagenesis methods.
  • CTCC China Center for Type Culture Collection
  • Candida tropicalis (Candida tropicalis Am2525) Am2525, which can convert ten-carbon normal alkane , Linear ten-carbon saturated fatty acids, linear ten-carbon saturated fatty acid derivatives (such as linear saturated fatty acid esters, linear saturated fatty acid salts) or a mixture of two or more of them are efficiently converted into deca-carbon dibasic acid.
  • a method for producing decadicarboxylic acid which comprises the following steps:
  • the strain activation is culturing in a shaker for 1 to 2 days;
  • the culture medium for strain activation includes YPD medium, preferably, the YPD medium Including glucose 2.0% (w/v), yeast extract 1.0% (w/v), peptone 2.0% (w/v), and the balance is water.
  • the seed liquid preparation using seed culture medium is cultured at 27 ⁇ 31°C, preferably at 29°C, and the culture time is 24 ⁇ 48h.
  • the OD 620 of the obtained seed liquid is between 0.5 and 1.0 (diluted by 30 times with water), it is inoculated into a shaker flask containing a fermentation medium.
  • the seed culture medium includes: sucrose 10-20g/L, corn steep liquor 2-4g/L, yeast extract 3-8g/L, potassium dihydrogen phosphate 4-12g/L, urea 0.5-4g/L, positive ten carbon One or more of alkane, linear saturated ten-carbon fatty acid and linear saturated ten-carbon fatty acid derivative (such as linear saturated fatty acid ester, linear saturated fatty acid salt) 0-80 mL/L.
  • the temperature of the fermentation is preferably 27 to 31°C, more preferably 29°C.
  • the substrate of the fermentation includes one or more of ten-carbon normal alkanes, ten-carbon straight-chain saturated fatty acids, ten-carbon straight-chain saturated fatty acid esters, and ten-carbon straight-chain saturated fatty acid salts; preferably, the fermentation
  • the added amount of the substrate is 100-400 mL/L, and the volume ratio is the volume ratio of the substrate to the fermentation medium.
  • the fermentation medium includes:
  • Nitrogen source The concentration of each nitrogen source is 0.5-10g/L
  • each inorganic salt is 0.5-12g/L.
  • the carbon source includes one or more of sucrose, glucose, maltose, molasses, fructose, rhamnose, arabinose and sorbitol;
  • the nitrogen source includes one or more of yeast extract, corn steep liquor, urea, ammonia, ammonium sulfate, potassium nitrate and ammonium nitrate;
  • the inorganic salt includes one or more of potassium salt and sodium salt; preferably, the potassium salt includes one or more of potassium chloride, potassium nitrate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate,
  • the sodium salt includes one or more of sodium chloride, sodium nitrate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.
  • the fermentation medium includes:
  • the substrate concentration in the fermentation broth is 100-400 mL/L, and the pH of the medium is adjusted to 7.5 with NaOH solution.
  • the substrate added to the culture medium is one or more of normal alkanes, linear saturated fatty acids, and linear saturated fatty acid derivatives.
  • the substrates include C10 normal alkanes, C10 One or more of straight-chain saturated fatty acids, C10 straight-chain saturated fatty acid esters, and C10 straight-chain saturated fatty acid salts.
  • Detection of the dibasic acid content in the fermentation broth the fermentation broth is pre-treated and then detected by gas chromatography (internal standard method). Chromatographic conditions: Column: Supelco SPB-50 30m*0.53mm*0.5 ⁇ m (Cat. No. 54983). Gas chromatograph (Shimadzu, GC-2014).
  • the initial temperature is 100°C
  • the temperature is increased to 230°C at 15°C/min
  • the temperature is kept for 2min.
  • the carrier gas is hydrogen
  • the injection port temperature is 280°C
  • the FID temperature is 280°C
  • the injection volume is 4 ⁇ L.
  • the product concentration is calculated based on the ratio of the peak area of the product to the peak area of the internal standard of a known concentration.
  • V oil/V milk/V total 0.1: 0.2: 6
  • V oil 0.1 refers to the graduation height of the oil layer in the graduated test tube
  • V oil 0.2 refers to the graduation height of the emulsion layer in the graduated test tube
  • V total 6 refers to the volume of 6mL dibasic acid fermentation broth
  • the YPD medium includes glucose 2.0% (w/v), yeast extract 1.0% (w/v), peptone 2.0% (w/v), and the balance is water.
  • the Candida tropicalis Am2525 of the present disclosure has been verified by passage experiments, and after five passages, the colony morphology and the ability to produce decodicarboxylic acid have not changed significantly (see Figure 1 and Figure 2).
  • the first and sixth generation strains produced DC10 by shaking flask fermentation at 140.5g/L and 139.8g/L, respectively (adding 4.5mL nC10 as a substrate); the colony morphology: the surface of the colony is smooth, moist and milky white It is shiny, round, with neat edges. It can be seen that the passage stability of Candida tropicalis Am2525 of the present disclosure is good.
  • the concentration of decane ranges from 0 to 100 mL/L
  • the strain Candida tropicalis Am252525 presents a complete and smooth surface, indicating that the cell morphology of the strain has a strong tolerance to decane.
  • the growth of the bacteria in the volume ratio of decane and seed medium of 10%, 20%, and 40% was tested, without adding decane.
  • the seed culture medium is the blank control group, and each experiment is in triplicate.
  • Seed culture medium includes: sucrose 20g/L, corn syrup 2g/L, yeast extract 6g/L, potassium dihydrogen phosphate 8g/L, urea 2g/L, nC10 1mL; cultured at 29°C for 48h, the seed liquid is measured
  • the OD620 reached 0.7 (diluted by 30 times with water), and 3.0 mL of seed liquid was inoculated into a shake flask containing a fermentation medium (the amount of fermentation medium was 15 mL), and the fermentation medium included: sucrose 30g/L, corn syrup 5g/L, yeast extract 5g/L, potassium dihydrogen phosphate 8g/L, potassium nitrate 4g/L, sodium chloride 1.5g/L, urea 0.5g/L.
  • n-alkane nC10 added to the fermentation medium was 4.5 mL, and the fermentation medium was sterilized at 121°C for 20 minutes. Fermentation was conducted at 29°C, and the fermentation substrate in the fermentation broth was detected as 0 when the fermentation was terminated, and the cycle was 160h.
  • the content of DC10 dibasic acid in the fermentation broth was determined to be 140 g/L, the average acid production rate was 0.88 g/h ⁇ L, and the substrate mass conversion rate was 63.94%.
  • Seed culture medium includes: sucrose 20g/L, corn syrup 2g/L, yeast extract 6g/L, potassium dihydrogen phosphate 8g/L, urea 2g/L; cultured at 29°C for 48h, the measured OD620 of the seed liquid reached To 0.8 (diluted by 30 times with water), 3.0 mL of seed liquid was inoculated into a shake flask containing a fermentation medium (the amount of fermentation medium is 15 mL), the fermentation medium includes: sucrose 30g/L, corn syrup 5g/L , Yeast extract 5g/L, potassium dihydrogen phosphate 8g/L, potassium nitrate 4g/L, sodium chloride 1.5g/L, urea 0.5g/L.
  • n-alkane nC10 added to the fermentation medium was 4.5 mL, and the fermentation medium was sterilized at 121°C for 20 minutes. Fermentation was conducted at 29°C, and the fermentation substrate in the fermentation broth was detected as 0 when the fermentation was terminated, and the cycle was 180h.
  • the content of DC10 dibasic acid in the fermentation broth was determined to be 120 g/L, the average acid production rate was 0.67 g/h ⁇ L, and the substrate mass conversion rate was 54.8%.
  • Seed culture medium includes: sucrose 20g/L, corn syrup 2g/L, yeast extract 6g/L, potassium dihydrogen phosphate 8g/L, urea 2g/L, nC10 1mL; cultured at 29°C for 48h, the seed liquid is measured
  • the OD620 reached 0.7 (diluted by 30 times with water), and 3.0 mL of seed liquid was inoculated into a shake flask containing a fermentation medium (the amount of fermentation medium was 15 mL), and the fermentation medium included: sucrose 30g/L, corn syrup 5g/L, yeast extract 5g/L, potassium dihydrogen phosphate 8g/L, potassium nitrate 4g/L, sodium chloride 1.5g/L, urea 0.5g/L.
  • the normal alkane nC10 added to the fermentation medium was 1.5 mL, and the fermentation medium was sterilized at 121°C for 20 minutes. Fermentation was conducted at 29°C, and the fermentation substrate in the fermentation broth was detected as 0 when the fermentation was terminated, and the cycle was 124h.
  • the content of DC10 dibasic acid in the fermentation broth was determined to be 46 g/L, the average acid production rate was 0.37 g/h ⁇ L, and the substrate mass conversion rate was 63.1%.
  • Seed culture medium includes: sucrose 20g/L, corn syrup 2g/L, yeast extract 6g/L, potassium dihydrogen phosphate 8g/L, urea 2g/L, nC10 1mL; cultured at 29°C for 48h, the seed liquid is measured
  • the OD620 reached 0.7 (diluted by 30 times with water), and 3.0 mL of seed liquid was inoculated into a shake flask containing a fermentation medium (the amount of fermentation medium was 15 mL), and the fermentation medium included: sucrose 30g/L, corn syrup 5g/L, yeast extract 5g/L, potassium dihydrogen phosphate 8g/L, potassium nitrate 4g/L, sodium chloride 1.5g/L, urea 0.5g/L.
  • the normal alkane nC10 added to the fermentation medium was 6.0 mL, and the fermentation medium was sterilized at 121°C for 20 minutes. Fermentation was carried out at 29°C, and the fermentation substrate in the fermentation broth was detected as 0 when the fermentation was terminated, and the cycle was 192h.
  • the content of DC10 dibasic acid in the fermentation broth was determined to be 194g/L, the average acid production rate was 1.01g/h ⁇ L, and the substrate mass conversion rate was 66.46%.
  • the optical density (OD620) of the seed liquid reaches 0.5 (diluted 30 times)
  • the seed liquid is connected to the fermentation medium for fermentation transformation and inoculation
  • the amount is 10% (v/v).
  • Control the fermentation temperature to 30°C control the pH value during the fermentation process to 7.2, control the air flow rate to 0.3vvm, control the fermentation tank pressure to 0.08MPa, maintain a certain stirring speed, and maintain the dissolved oxygen above 20% during the fermentation process.
  • the bacterial cells begin to grow and multiply.
  • the fermentation substrate n-decane is added.
  • the feed rate of decane is controlled by the feeding method so that the concentration of decane in the fermentation broth during the fermentation process ranges from 4% to 6% (v/v), and the total added amount of decane, the fermentation substrate, is 1300 g.
  • the detection of the fermentation substrate in the fermentation broth is 0, the fermentation is stopped, and the fermentation is terminated when the detection of the fermentation substrate in the fermentation broth is 0, and the fermentation time is 140h.
  • the seed culture medium includes: sucrose 20g/L, corn syrup 2g/L, yeast extract 6g/L, potassium dihydrogen phosphate 8g/L, urea 2g/L, ten-carbon fatty acid methyl ester 1mL; cultivated at 29°C for 48h, It was measured that the OD620 of the seed liquid reached 0.7 (diluted by 30 times with water), and 3.0 mL of the seed liquid was inoculated into a shake flask containing a fermentation medium (the amount of fermentation medium was 15 mL), and the fermentation medium included: sucrose 30g/ L, corn steep liquor 5g/L, yeast extract 5g/L, potassium dihydrogen phosphate 8g/L, potassium nitrate 4g/L, sodium chloride 1.5g/L, and urea 0.5g/L.
  • the ten-carbon fatty acid methyl ester added to the fermentation medium was 4.5 mL, and the fermentation medium was sterilized at 121°C for 20 minutes. Fermentation was carried out at 29°C, and the fermentation substrate in the fermentation broth was detected as 0 when the fermentation was terminated, and the cycle was 162h.
  • the content of DC10 dibasic acid in the fermentation broth was determined to be 125g/L, the average acid production rate was 0.77g/h ⁇ L, and the substrate mass conversion rate was 47.66%.
  • Candida tropicalis Candida tropicalalis
  • CAT N145 glycerol tube seed into the YPD activation medium, culture for 24 hours and then add the culture solution into the seed medium (the medium is sterilized at 121°C for 20 minutes).
  • Seed culture medium includes: sucrose 20g/L, corn syrup 2g/L, yeast extract 6g/L, potassium dihydrogen phosphate 8g/L, urea 2g/L, nC10 1mL; cultured at 29°C for 48h, the seed liquid is measured OD620 reached 0.55 (diluted with water 30 times), 3.0mL seed liquid was inoculated into a shake flask containing fermentation medium (the amount of fermentation medium is 15mL), the fermentation medium includes: sucrose 30g/L, corn syrup 5g/L, yeast extract 5g/L, potassium dihydrogen phosphate 8g/L, potassium nitrate 4g/L, sodium chloride 1.5g/L, urea 0.5g/L.
  • the normal alkane nC10 added to the fermentation medium was 1.5 mL, and the fermentation medium was sterilized at 121°C for 20 minutes. Fermentation was conducted at 29°C, and the fermentation substrate in the fermentation broth was detected as 0 when the fermentation was terminated, and the fermentation period was 140h.
  • the content of DC10 dibasic acid in the fermentation broth was determined to be 30g/L, the average acid production rate was 0.21g/h ⁇ L, and the substrate mass conversion rate was 41.11%.
  • Candida tropicalis Candida tropicalalis
  • CAT N145 glycerol tube seed into the YPD activation medium, culture for 24 hours and then add the culture solution into the seed medium (the medium is sterilized at 121°C for 20 minutes).
  • Seed culture medium includes: sucrose 20g/L, corn syrup 2g/L, yeast extract 6g/L, potassium dihydrogen phosphate 8g/L, urea 2g/L, nC10 1mL; cultured at 29°C for 48h, the seed liquid is measured OD620 reached 0.55 (diluted with water 30 times), 3.0mL seed liquid was inoculated into a shake flask containing fermentation medium (the amount of fermentation medium is 15mL), the fermentation medium includes: sucrose 30g/L, corn syrup 5g/L, yeast extract 5g/L, potassium dihydrogen phosphate 8g/L, potassium nitrate 4g/L, sodium chloride 1.5g/L, urea 0.5g/L.
  • the normal alkane nC10 added to the fermentation medium was 6.0 mL, and the fermentation medium was sterilized at 121°C for 20 minutes. Fermentation was carried out at 29°C, the fermentation period was 230h, the consumption rate of the substrate was greatly reduced (from 200h, samples were taken every 10h to detect the content of the substrate), and the fermentation was stopped.
  • the content of DC10 dibasic acid in the fermentation broth was determined to be 116.8g/L, the average acid production rate was 0.51g/h ⁇ L, and the substrate mass conversion rate was 40%.
  • Candida tropicalis Candida tropicalalis
  • CAT N145 glycerol tube seed into the YPD activation medium, culture for 24 hours and then add the culture solution into the seed medium (the medium is sterilized at 121°C for 20 minutes).
  • Seed culture medium includes: sucrose 20g/L, corn syrup 2g/L, yeast extract 6g/L, potassium dihydrogen phosphate 8g/L, urea 2g/L, nC10 1mL; cultured at 29°C for 48h, the seed liquid is measured OD620 reached 0.55 (diluted with water 30 times), 3.0mL seed liquid was inoculated into a shake flask containing fermentation medium (the amount of fermentation medium is 15mL), the fermentation medium includes: sucrose 30g/L, corn syrup 5g/L, yeast extract 5g/L, potassium dihydrogen phosphate 8g/L, potassium nitrate 4g/L, sodium chloride 1.5g/L, urea 0.5g/L.
  • n-alkane nC10 added to the fermentation medium was 4.5 mL, and the fermentation medium was sterilized at 121°C for 20 minutes. Fermentation was carried out at 29°C, the fermentation period was 180h, the consumption rate of the substrate was greatly reduced (from 150h, samples were taken every 10h to detect the content of the substrate), and fermentation was stopped.
  • the content of DC10 dibasic acid in the fermentation broth was determined to be 108 g/L, the average acid production rate was 0.6 g/h ⁇ L, and the substrate mass conversion rate was 49.33%.
  • Candida tropicalis CATN145 cells in the seed culture medium at a culture temperature of 28°C.
  • the optical density (OD620) of the seed liquid cells reaches 0.5 (diluted 30 times)
  • the seed liquid is connected to the fermentation medium Fermentation and transformation are carried out in the medium, and the inoculum amount is 10% (v/v).
  • Control the fermentation temperature to 30°C control the pH value during the fermentation process to 7.2, control the air flow rate to 0.3vvm, control the fermentation tank pressure to 0.08MPa, maintain a certain stirring speed, and maintain the dissolved oxygen above 20% during the fermentation process.
  • the bacterial cells begin to grow and multiply.
  • the fermentation substrate n-decane is added.
  • the feed rate of decane is controlled by the feeding method so that the concentration of decane in the fermentation broth during the fermentation process ranges from 4% to 6% (v/v), and the total added amount of decane, the fermentation substrate, is 1300 g.
  • the fermentation time was 160h, the consumption rate of the substrate was greatly reduced (from 150h, samples were taken every 10h to detect the content of the substrate), and the fermentation was stopped.
  • the yield of decadicarboxylic acid in the fermentation broth was 144g/L
  • the mass conversion rate of the substrate was 66.4%
  • the average acid production rate was 0.9g/h ⁇ L.
  • the Candida tropicalis strain (Am2525) Am2525 used in this disclosure has better tolerance to the substrate decane than the parent Candida tropicalis CATN145.
  • the substance decane exceeds 4% to 6% (v/v) Candida tropicalis (Am2525) Am2525 can still maintain the substrate mass conversion rate above 90%, and the average acid production rate can reach 1.39g/ h ⁇ L. Therefore, the Candida tropicalis (Am2525) strain of the present disclosure, Am2525, can be used for the industrial production of deca dicarboxylic acid by enzymatic method.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Botany (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种热带假丝酵母菌株(Candida tropicalis )菌株Am2525，其保藏编号为CCTCC NO:M 2019419，以及利用该菌株发酵生产长链二元酸的方法。所述长链二元酸的生产方法包括通过所述的热带假丝酵母菌株Am2525制备种子液，和发酵生产长链二元酸。所述热带假丝酵母菌株Am2525较亲本对底物癸烷毒性的抵抗能力增强，提高了长链二元酸的产能，降低了生产成本，后续分离提纯简单，发酵生产过程易于大规模化。

Description

一种生产长链二元酸的菌株及其发酵方法 技术领域
本公开属于生物技术领域，具体涉及一种菌株，特别涉及一种生产长链二元酸的菌株，及利用该菌株发酵生产长链二元酸的方法。
背景技术
长链二元酸是合成香料、尼龙工程塑料、热熔胶、树脂、耐寒性增塑剂、医药和农药等的重要原料，其结构式为HOOC(CH 2) nCOOH，其中n为大于7的整数。其中十碳二元酸或称为十碳长链二元酸、癸二酸，化学式为HOOC(CH 2) 8COOH。其作为一种重要的单体原料，广泛用于生产聚酰胺工程塑料，如尼龙510、尼龙1010、尼龙610。也可用于耐高温润滑油、环氧树脂固化剂、合成润滑脂及人造香料、耐寒增塑剂等，是应用广泛的化工原料之一。
目前工业生产和应用的十碳二元酸是用蓖麻油为原料，通过化学法制备得到的。主要工艺流程是蓖麻油水解生成蓖麻油酸钠皂，然后进一步制备得到蓖麻油酸，蓖麻油酸在苯酚的存在下，加碱、加热进行高温裂解，生成十碳二元酸双钠盐，然后进一步加热、加酸、脱色、结晶得到十碳二元酸。采用蓖麻油催化裂解法制备十碳二元酸，生产过程复杂、在250～270℃的高温下进行反应，使用苯酚或邻甲酚有毒试剂，污染环境，严重制约着化学法生产十碳二元酸产业的发展。
生物法制备十碳二元酸具有生产工艺简单、绿色环保的特点。中国科学院微生物所的余志华(见《微生物学报》1989年06期：0253-2654)通过诱变筛选获得一株高产菌株，在16升发酵罐上获得十碳二元酸的产量达到71g/L以上，通过水相结晶得到了十碳二元酸产品，产品纯度99.6％以上；中科院林业土壤研究所(见《微生物学报》1979年01期)筛选得到的解脂假丝酵母发酵生产十碳二元酸的产量为30～40g/L。
在生物法制备十碳二元酸的过程中，癸烷或其类似物常作为发酵底物，这类发酵底物在发酵过程中可能存在的转化途径包括：被菌体多步氧化成为目的产物十碳二元酸、被菌体生长维持所消耗或者挥发后进入外部环境等。然而在发酵过程中，癸烷因其自身的性质会对微生物有毒害作用，抑制微生物的生长。因此，通过改良菌株，提高菌株的抗毒能力，是一个重要的研究方向。
发明内容
本公开的第一方面目的在于提供一种热带假丝酵母菌株，以解决目前热带假丝酵母菌株产十碳二元酸过程中菌体浓度低、发酵周期长、转化效率低的问题，进而解决生产成本高的技术问题。
本公开通过以下技术方案解决上述技术问题，达到本公开的第一方面目的。
本公开提供了一种热带假丝酵母菌株，该菌株为热带假丝酵母(Candida tropicalis Am2525)Am2525，保藏编号为CCTCC NO:M 2019419。
本公开所提供的菌种为热带假丝酵母(Candida tropicalis Am2525)Am2525，是以一株氧化正烷烃生产混合二羧酸的热带假丝酵母(Candida tropicalis)CAT N145(已于2011年6月9日提交位于武汉大学的中国典型培养物保藏中心(CCTCC)，保藏号为CCTCC M 2011192)为出发菌株，通过常压室温等离子体ARTP和紫外线照射等复合诱变、筛选培育出来的。
热带假丝酵母(Candida tropicalis Am2525)Am2525形态特征：菌落为表面光滑湿润呈乳白色有光泽、圆形、边缘整齐。
本公开的第二方面目的在于提出上述热带假丝酵母菌株(Candida tropicalis Am2525)Am2525在长链二元酸生产中的应用。
本公开提供了上述热带假丝酵母菌株(Candida tropicalis Am2525)Am2525在十碳二元酸生产中的应用。
在一优选实施方式中，所述十碳二元酸生产中用于发酵转化的底物包括C10的正烷烃、C10的直链饱和脂肪酸、C10的直链饱和脂肪酸酯和C10的直链饱和脂肪酸盐中的一种或多种。
本公开的第三方面目的在于提出一种长链二元酸的生产方法，尤其是生产十碳二元酸的方法，以解决目前热带假丝酵母菌株产十碳二元酸过程中菌体浓度低、发酵周期长、转化效率低的问题，进而解决生产成本高的技术问题。
本公开通过以下技术方案解决上述技术问题，达到本公开的第三方面目的。
一种长链二元酸的生产方法，通过所述的热带假丝酵母菌株(Candida tropicalis Am2525)Am2525制备种子液，和/或；发酵生产长链二元酸。
进一步优选地，所述长链二元酸为C10的直链饱和二元酸。
在一优选实施方式中，所述发酵的底物包括C10的正烷烃、C10的直链饱和脂肪酸、C10的直链饱和脂肪酸酯和C10的直链饱和脂肪酸盐中的一种或多种；优选地，所述发酵的底物的添加量为100～400mL/L，所述体积比为底物与发酵培养基的体积比。
在一优选实施方式中，所述制备种子液的温度为在27～31℃；所述发酵生产十碳二元酸的发酵温度为在27～31℃。
培养基的选择对于菌株发酵是非常重要的，在上述任一技术方案的基础上进一步，所述的长链二元酸的生产方法，其中，所述发酵的发酵培养基包括：
碳源      10g/L～40g/L
氮源      每种所述氮源浓度为0.5～10g/L
无机盐    每种所述无机盐浓度为0.5～12g/L。
又进一步，所述碳源包括蔗糖、葡萄糖、麦芽糖、糖蜜、果糖、鼠李糖、阿拉伯糖和山梨醇中的一种或多种；
所述氮源包括酵母膏、玉米浆、尿素、氨水、硫酸铵、硝酸钾和硝酸铵中的一种或多种；
所述无机盐包括钾盐和钠盐中的一种或多种；优选地，所述钾盐包括氯化钾、硝酸钾、磷酸二氢钾、磷酸氢二钾中的一种或多种，所述钠盐包括氯化钠、硝酸钠、磷酸二氢钠、磷酸氢二钠中的一种或多种。
优选地，所述发酵培养基包括：
Figure PCTCN2020072398-appb-000001
在上述任一技术方案的基础上进一步，所述长链二元酸的生产方法包括以下步骤：
a)菌种活化；
b)利用种子培养基制备种子液；
c)将所述种子液接种到发酵培养基中发酵。
又进一步，步骤a)中，所述菌种活化的培养基包括YPD培养基。所述YPD培养基包括葡萄糖2.0％(w/v)、酵母膏1.0％(w/v)、蛋白胨2.0％(w/v)，余量为水。
又进一步，步骤b)中，所述种子培养基包括：蔗糖10～20g/L，玉米浆2～4g/L，酵母膏3～8g/L，磷酸二氢钾4～12g/L，尿素0.5～4g/L、C10的正烷烃、C10直链饱和脂肪酸、C10直链饱和脂肪酸酯和C10直链饱和脂肪酸盐中的一种或多种0～80mL/L；
优选地，步骤b)中，制备所述种子液的培养时间12～48h，种子成熟指标为水稀释30倍后OD620为0.5～1.0。
优选地，步骤c)中，所述种子液接种到发酵培养基发酵中时，将计料体积培养液为10％～30％的接种量接种于培养液中。
优选地，步骤c)中，所述发酵为在28～31℃培养，摇床转速200～250rpm，从接种加入底物开始至发酵液中的底物无明显残留，即完成了发酵周期，所述菌种在发酵系统中的发酵周期为90～200小时。
所述菌种在发酵系统中对底物的转化效率为35％以上，进一步优选为高于40％以上；
本公开与已有技术相比具有以下显著特点和积极效果：
本公开的热带假丝酵母菌株，较亲本对底物癸烷毒性的抵抗能力增强，使得菌体在生长及发酵过程中都能达到更高的OD，能够更多地转化底物，提高产能，降低生产成本，后续分离提纯简单，发酵生产过程易于大规模化，应用前景良好。
保藏信息
菌株：热带假丝酵母(Candida tropicalis Am2525)Am2525
保藏日期：2019年6月3日
保藏单位：中国典型培养物保藏中心(地址：中国.武汉.武汉大学)
保藏编号：CCTCC NO:M 2019419
附图说明
图1是本公开的热带假丝酵母(Candida tropicalis)Am2525的第一代的菌落照片；
图2是本公开的热带假丝酵母(Candida tropicalis)Am2525的第六代的菌落照片；
图3是热带假丝酵母(Candida tropicalis)Am2525分别于含起始浓度为10％、20％、40％(v/v)的癸烷种子培养基中的生长曲线。
具体实施方式
下面结合附图，详细说明本公开。
本申请的发明人通过以现有长链二元酸生产菌株热带假丝酵母(Candida tropicalis)CAT N145(该菌株在公开号为CN 102839133A，公开日为2012-12-26的中国发明专利中已经公开，在该专利程序中已于2011年6月9日提交位于武汉大学的中国典型培养物保藏中心(CCTCC)，保藏号为CCTCC M 2011192)为出发菌株，通过驯化、常规诱变方法进行诱变育种后，例如，参照中国专利CN201110138270.5中公开的方法，筛选得到了一株新的热带假丝酵母，命名为热带假丝酵母(Candida tropicalis Am2525)Am2525，其能够将十碳的正烷烃、直链十碳饱和脂肪酸、直链十碳饱和脂肪酸衍生物(如直链饱和脂肪酸酯、直链饱和脂肪酸盐)或它们中的两种以上的混合物高效转化为十碳二元酸。
一种十碳二元酸的生产方法，其包括以下步骤：
a)菌种活化；在一个优选的具体实施方式中，所述菌种活化是摇床培养1～2天；所述菌种活化的培养基包括YPD培养基，优选地，所述YPD培养基包括葡萄糖2.0％(w/v)、酵母膏1.0％(w/v)、蛋白胨2.0％(w/v)，余量为水。
b)利用种子培养基制备种子液；在一个优选的具体实施方式中，所述利用种子培养基制备种子液，是在27～31℃培养，优选在29℃培养，培养时间24～48h，测得种子液的OD 620在0.5～1.0(水稀释30倍)时，接种到装有发酵培养基的摇瓶中。所述种子培养基包括：蔗糖10～20g/L，玉米浆2～4g/L，酵母膏3～8g/L，磷酸二氢钾4～12g/L，尿素0.5～4g/L，正十碳烷烃、直链饱和十碳脂肪酸和直链饱和十碳脂肪酸衍生物(如直链饱和脂肪酸酯、直链饱和脂肪酸盐)中的一种或多种0～80mL/L。
c)将所述种子液接种到发酵培养基中发酵，通过如上文所述的热带假丝酵母菌株(Candida tropicalis Am2525)Am2525发酵生产十碳二元酸。
步骤c)中，所述发酵的温度优选为在27～31℃，进一步优选为29℃。
所述发酵的底物包括十碳正烷烃、十碳直链饱和脂肪酸、十碳直链饱和脂肪酸酯和十碳直链饱和脂肪酸盐中的一种或多种；优选地，所述发酵的底物的添加量为100～400mL/L，所述体积比为底物与发酵培养基的体积比。
所述发酵的培养基包括：
碳源      10g/L～40g/L
氮源      每种所述氮源浓度为0.5～10g/L
无机盐    每种所述无机盐浓度为0.5～12g/L。
所述碳源包括蔗糖、葡萄糖、麦芽糖、糖蜜、果糖、鼠李糖、阿拉伯糖和山梨醇中的一种或多种；
所述氮源包括酵母膏、玉米浆、尿素、氨水、硫酸铵、硝酸钾和硝酸铵中的一种或多种；
所述无机盐包括钾盐和钠盐中的一种或多种；优选地，所述钾盐包括氯化钾、硝酸钾、磷酸二氢钾和磷酸氢二钾中的一种或多种，所述钠盐包括氯化钠、硝酸钠、磷酸二氢钠和磷酸氢二钠中的一种或多种。
在一个优选的实施例中，所述发酵的培养基包括：
Figure PCTCN2020072398-appb-000002
发酵初始时，发酵液中的底物浓度100～400mL/L，用NaOH溶液调节培养基pH至7.5。
本公开中，培养基中加入的底物，即正烷烃、直链饱和脂肪酸、直链饱和脂肪酸衍生物中的一种或多种，优选地，所述底物包括C10的正烷烃、C10的直链饱和脂肪酸、C10的直链饱和脂肪酸酯和C10的直链饱和脂肪酸盐中的一种或多种。
以下实施例中所使用的二元酸的测定方法：
发酵液中二元酸含量检测：发酵液经前处理后气相色谱检测(内标法)，色谱条件：色谱柱：Supelco SPB-50 30m*0.53mm*0.5μm(货号54983)。气相色谱仪(Shimadzu，GC-2014)。
方法：初温100℃，15℃/min升温至230℃，保持2min。载气为氢气，进样口温度280℃，FID温度280℃，进样量4μL。
根据产物峰面积和已知浓度的内标峰面积比进行产物浓度计算。
以下实施例中所残余的烷烃含量测定方法：
采用离心法测定：
(1)取6mL的二元酸发酵液(发酵液的体积为V总)，并向发酵液中加入3mL的0.1mol/L的氢氧化钠溶液，并放在80度的水浴锅中加热10min。
(2)冷却，3000转/min离心10min，离心结束后读数，油层体积V油，乳化层体积V乳；
注意：离心管必须是带刻度的。
(例如V油/V乳/V总＝0.1：0.2：6，V油0.1指油层在带刻度的试管中的刻度高度；V乳0.2指乳化层在带刻度的试管中的刻度高度；V总6指取6mL的二元酸发酵液体积)。
YPD培养基包括葡萄糖2.0％(w/v)、酵母膏1.0％(w/v)、蛋白胨2.0％(w/v)，余量为水。
实施例1
本公开的热带假丝酵母(Candida tropicalis)Am2525，经传代实验验证，经五次传代后菌落形态及生产十碳二元酸的能力均未出现明显变化(见图1、图2)。在29℃条件下，第一代和第六代菌株经摇瓶发酵产DC10分别为140.5g/L和139.8g/L(添加4.5mL nC10作为底物)；菌落形态：菌落表面光滑湿润呈乳白色有光泽、圆形、边缘整齐。可见本公开的热带假丝酵母(Candida tropicalis)Am2525的传代稳定性良好。当癸烷浓度范围为0～100mL/L时，菌株热带假丝酵母(Candida tropicalis)Am2525呈现完整和光滑的表面，说明菌株细胞形态对癸烷具有较强的耐受性。
为了进一步验证菌体在更高浓度癸烷中的耐受性，检测了菌体在癸烷和种子培养基的体积比分别为10％，20％，40％中的生长情况，不添加癸烷的种子培养基为空白对照组，每个实验平行三份。
实验结果表明，在10％，20％，40％(v/v)癸烷浓度下，热带假丝酵母(Candida tropicalis)Am2525虽然在开始时生长受到抑制，但随后生长量迅速增加，在48h时内生长量接近空白对照组。
实施例2
取1支甘油管种子接入YPD活化培养基中，培养24h后接培养液进种子培养基(培养基于121℃灭菌20分钟)。种子培养基包括：蔗糖20g/L，玉米浆2g/L，酵母膏6g/L，磷酸二氢钾8g/L，尿素2g/L，nC10 1mL；在29℃条件下培养48h，测得种子液的OD620达到了0.7(水稀释30倍)，将3.0mL种子液接种到装有发酵培养基的摇瓶中(发酵培养基的用量为15mL)，发酵培养基包括：蔗糖30g/L，玉米浆5g/L，酵母膏5g/L，磷酸二氢钾8g/L，硝酸钾4g/L，氯化钠1.5g/L，尿素0.5g/L。发酵培养基中所加的正烷烃nC10为4.5mL，发酵培养基在121℃灭菌20分钟。在29℃条件下发酵，发酵液内的发酵底物检测为0时结束发酵，周期160h。
发酵结束后，测定发酵液中的DC10二元酸的含量为140g/L，平均产酸速率为0.88g/h·L，底物质量转化率为63.94％。
实施例3
取1支甘油管种子接入YPD活化培养基中，培养24h后接培养液进种子培养基(培养基于121℃灭菌20分钟)。种子培养基包括：蔗糖20g/L，玉米浆2g/L，酵母膏6g/L，磷酸二氢钾8g/L，尿素2g/L；在29℃条件下培养48h，测得种子液的OD620达到了0.8(水稀释30倍)，将3.0mL种子液接种到装有发酵培养基的摇瓶中(发酵培养基的用量为15mL)，发酵培养基包括：蔗糖30g/L，玉米浆5g/L，酵母膏5g/L，磷酸二氢钾8g/L，硝酸钾4g/L，氯化钠1.5g/L，尿素0.5g/L。发酵培养基中所加的正烷烃nC10为4.5mL，发酵培养基在121℃灭菌20分钟。在29℃条件下发酵，发酵液内的发酵底物检测为0时结束发酵，周期180h。
发酵结束后，测定发酵液中的DC10二元酸的含量为120g/L，平均产酸速率为0.67g/h·L，底物质量转化率为54.8％。
实施例4
取1支甘油管种子接入YPD活化培养基中，培养24h后接培养液进种子培养基(培养基于121℃灭菌20分钟)。种子培养基包括：蔗糖20g/L，玉米浆2g/L，酵母膏6g/L，磷酸二氢钾8g/L，尿素2g/L，nC10 1mL；在29℃条件下培养48h，测得种子液的OD620达到了0.7(水稀释30倍)，将3.0mL种子液接种到装有发酵培养基的摇瓶中(发酵培养基的用量为15mL)，发酵培养基包括：蔗糖30g/L，玉米浆5g/L，酵母膏5g/L，磷酸二氢钾8g/L，硝酸钾4g/L，氯化钠1.5g/L，尿素0.5g/L。发酵培养基中所加的正烷烃nC10为1.5mL，发酵培养基在121℃灭菌20分钟。在29℃条件下发酵，发酵液内的发酵底物检测为0时结束发酵，周期124h。
发酵结束后，测定发酵液中的DC10二元酸的含量为46g/L，平均产酸速率为0.37g/h·L，底物质量转化率为63.1％。
实施例5
取1支甘油管种子接入YPD活化培养基中，培养24h后接培养液进种子培养基(培养基于121℃灭菌20分钟)。种子培养基包括：蔗糖20g/L，玉米浆2g/L，酵母膏6g/L，磷酸二氢钾8g/L，尿素2g/L，nC10 1mL；在29℃条件下培养48h，测得种子液的OD620达到了0.7(水稀释30倍)，将3.0mL种子液接种到装有发酵培养基的摇瓶中(发酵培养基的用量为15mL)，发酵培养基包括：蔗糖30g/L，玉米浆5g/L，酵母膏5g/L，磷酸二氢钾8g/L，硝酸钾4g/L，氯化钠1.5g/L，尿素0.5g/L。发酵培养基中所加的正烷烃nC10为6.0mL，发酵培养基在121℃灭菌20分钟。在29℃条件下发酵，发酵液内的发酵底物检测为0时结束发酵，周期192h。
发酵结束后，测定发酵液中的DC10二元酸的含量为194g/L，平均产酸速率为1.01g/h·L，底物质量转化率为66.46％。
实施例6
将1支甘油管种子接种于种子培养基，培养温度28℃，当种子液菌体光密度(OD620)达到0.5(稀释30倍)时，将种子液接入发酵培养基中进行发酵转化，接种量为10％(v/v)。控制发酵温度30℃，控制发酵过程中pH值为7.2，控制空气流量为0.3vvm，控制发酵罐压为0.08MPa，保持一定的搅拌速度，发酵过程中溶氧维持在20％以上。种子液接入发酵罐后，菌体开始生长繁殖，当发酵液中菌体光密度(OD620)达到0.5(稀释30倍)时，加入发酵底物正癸烷。采用流加方式控制癸烷的补加速率以使发酵过程中发酵液内的癸烷浓度范围为4％～6％(v/v)，发酵底物癸烷总添加量为1300g。发酵液内的发酵底物检测为0时，停止发酵，发酵液内的发酵底物检测为0时结束发酵，发酵时间140h。
检测结果：发酵结束后发酵液中十碳二元酸的产量为195g/L，底物的转化率为90％，平均产酸速率为1.39g/h·L。
实施例7
取1支甘油管种子接入YPD活化培养基中，培养24h后接培养液进种子培养基(培养基于121℃灭菌20分钟)。种子培养基包括：蔗糖20g/L，玉米浆2g/L，酵母膏6g/L，磷酸二氢钾8g/L，尿素2g/L，十碳脂肪酸甲酯1mL；在29℃条件下培养48h，测得种子液的OD620达到了0.7(水稀释30倍)，将3.0mL种子液接种到装有发酵培养基的摇瓶中(发酵培养基的用量为15mL)，发酵培养基包括：蔗糖30g/L，玉米浆5g/L，酵母膏5g/L，磷酸二氢钾8g/L，硝酸钾4g/L，氯化钠1.5g/L， 尿素0.5g/L。发酵培养基中所加的十碳脂肪酸甲酯为4.5mL，发酵培养基在121℃灭菌20分钟。在29℃条件下发酵，发酵液内的发酵底物检测为0时结束发酵，周期162h。
发酵结束后，测定发酵液中的DC10二元酸的含量为125g/L，平均产酸速率为0.77g/h·L，底物质量转化率为47.66％。
对比例1
取1支热带假丝酵母(Candida tropicalis)CAT N145甘油管种子接入YPD活化培养基中，培养24h后接培养液进种子培养基(培养基于121℃灭菌20分钟)。种子培养基包括：蔗糖20g/L，玉米浆2g/L，酵母膏6g/L，磷酸二氢钾8g/L，尿素2g/L，nC10 1mL；在29℃条件下培养48h，测得种子液的OD620达到了0.55(水稀释30倍)，将3.0mL种子液接种到装有发酵培养基的摇瓶中(发酵培养基的用量为15mL)，发酵培养基包括：蔗糖30g/L，玉米浆5g/L，酵母膏5g/L，磷酸二氢钾8g/L，硝酸钾4g/L，氯化钠1.5g/L，尿素0.5g/L。发酵培养基中所加的正烷烃nC10为1.5mL，发酵培养基在121℃灭菌20分钟。在29℃条件下发酵，发酵液内的发酵底物检测为0时结束发酵，发酵周期140h。
发酵结束后，测定发酵液中的DC10二元酸的含量为30g/L，平均产酸速率为0.21g/h·L，底物质量转化率为41.11％。
对比例2
取1支热带假丝酵母(Candida tropicalis)CAT N145甘油管种子接入YPD活化培养基中，培养24h后接培养液进种子培养基(培养基于121℃灭菌20分钟)。种子培养基包括：蔗糖20g/L，玉米浆2g/L，酵母膏6g/L，磷酸二氢钾8g/L，尿素2g/L，nC10 1mL；在29℃条件下培养48h，测得种子液的OD620达到了0.55(水稀释30倍)，将3.0mL种子液接种到装有发酵培养基的摇瓶中(发酵培养基的用量为15mL)，发酵培养基包括：蔗糖30g/L，玉米浆5g/L，酵母膏5g/L，磷酸二氢钾8g/L，硝酸钾4g/L，氯化钠1.5g/L，尿素0.5g/L。发酵培养基中所加的正烷烃nC10为6.0mL，发酵培养基在121℃灭菌20分钟。在29℃条件下发酵，发酵周期为230h，底物的消耗速率大幅降低(从200h开始每隔10h取样检测底物含量)，停止发酵。
发酵结束后，测定发酵液中的DC10二元酸的含量为116.8g/L，平均产酸速率为0.51g/h·L，底物质量转化率为40％。
对比例3
取1支热带假丝酵母(Candida tropicalis)CAT N145甘油管种子接入YPD活化培养基中，培养24h后接培养液进种子培养基(培养基于121℃灭菌20分钟)。种子培养基包括：蔗糖20g/L，玉米浆2g/L，酵母膏6g/L，磷酸二氢钾8g/L，尿素2g/L，nC10 1mL；在29℃条件下培养48h，测 得种子液的OD620达到了0.55(水稀释30倍)，将3.0mL种子液接种到装有发酵培养基的摇瓶中(发酵培养基的用量为15mL)，发酵培养基包括：蔗糖30g/L，玉米浆5g/L，酵母膏5g/L，磷酸二氢钾8g/L，硝酸钾4g/L，氯化钠1.5g/L，尿素0.5g/L。发酵培养基中所加的正烷烃nC10为4.5mL，发酵培养基在121℃灭菌20分钟。在29℃条件下发酵，发酵周期为180h，底物的消耗速率大幅降低(从150h开始每隔10h取样检测底物含量)，停止发酵。
发酵结束后，测定发酵液中的DC10二元酸的含量为108g/L，平均产酸速率为0.6g/h·L，底物质量转化率为49.33％。
对比例4
将热带假丝酵母(Candida tropicalis)CATN145菌体接种于种子培养基，培养温度28℃，当种子液菌体光密度(OD620)达到0.5(稀释30倍)时，将种子液接入发酵培养基中进行发酵转化，接种量为10％(v/v)。控制发酵温度30℃，控制发酵过程中pH值为7.2，控制空气流量为0.3vvm，控制发酵罐压为0.08MPa，保持一定的搅拌速度，发酵过程中溶氧维持在20％以上。种子液接入发酵罐后，菌体开始生长繁殖，当发酵液中菌体光密度(OD620)达到0.5(稀释30倍)时，加入发酵底物正癸烷。采用流加方式控制癸烷的补加速率以使发酵过程中发酵液内的癸烷浓度范围为4％～6％(v/v)，发酵底物癸烷总添加量为1300g。发酵时间160h，底物的消耗速率大幅降低(从150h开始每隔10h取样检测底物含量)，停止发酵。
发酵结束后发酵液中十碳二元酸的产量为144g/L，底物的质量转化率为66.4％，平均产酸速率为0.9g/h·L。
由上述实施例可知，本公开所采用的热带假丝酵母菌株(Candida tropicalis Am2525)Am2525，较亲本热带假丝酵母CATN145菌体具有更好的对底物癸烷的耐受能力，当补料底物癸烷超过4％～6％(v/v)热带假丝酵母菌株(Candida tropicalis Am2525)Am2525对底物的质量转化率仍然能够保持在90％以上，且平均产酸速率可达到1.39g/h·L。因此，本公开的热带假丝酵母菌株(Candida tropicalis Am2525)Am2525可用于酶法工业化生产十碳二元酸。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。

Claims (10)

  1. 一种热带假丝酵母菌株，其特征在于，所述菌株为热带假丝酵母(Candida tropicalis Am2525)Am2525，保藏编号为CCTCC NO:M 2019419。
  2. 一种如权利要求1所述的热带假丝酵母菌株在十碳二元酸生产中的应用。
  3. 如权利要求2所述的应用，其特征在于，用于发酵转化的底物包括C10的正烷烃、C10的直链饱和脂肪酸、C10的直链饱和脂肪酸酯和C10的直链饱和脂肪酸盐中的一种或多种。
  4. 一种长链二元酸的生产方法，其特征在于，通过如权利要求1所述的热带假丝酵母菌株(Candida tropicalis Am2525)Am2525制备种子液，和/或；发酵生产长链二元酸；优选地，所述长链二元酸为C10的直链饱和二元酸。
  5. 如权利要求4所述的方法，其特征在于，所述发酵的底物包括C10的正烷烃、C10的直链饱和脂肪酸、C10的直链饱和脂肪酸酯和C10的直链饱和脂肪酸盐中的一种或多种；优选地，所述发酵的底物的添加量为100～400mL/L。
  6. 如权利要求4所述的方法，其特征在于，所述发酵为在28～31℃培养，和/或；摇床转速200～250rpm，和/或；
    所述菌种在发酵系统中的发酵周期为90～200小时。
  7. 如权利要求4～6中任意一项所述的方法，其特征在于，所述方法包括以下步骤：
    a)菌种活化；
    b)利用种子培养基制备种子液；
    c)将所述种子液接种到发酵培养基中发酵。
  8. 如权利要求7所述的方法，其特征在于，步骤a)中，所述菌种活化的培养基包括YPD培养基；所述YPD培养基包括葡萄糖2.0％(w/v)、酵母膏1.0％(w/v)、蛋白胨2.0％(w/v)，余量为水。
  9. 如权利要求7所述的方法，其特征在于，步骤b)中，所述种子培养基包括：蔗糖10～20g/L，玉米浆2～4g/L，酵母膏3～8g/L，磷酸二氢钾4～12g/L，尿素0.5～4g/L、C10的正烷烃、C10直链饱和脂肪酸、C10直链饱和脂肪酸酯和C10直链饱和脂肪酸盐中的一种或多种0～80mL/L。
  10. 如权利要求7所述的方法，其特征在于，步骤c)中，所述种子液接种到发酵培养基发酵中时，将计料体积培养液为10％～30％的接种量接种于培养液中。
PCT/CN2020/072398 2019-10-18 2020-01-16 一种生产长链二元酸的菌株及其发酵方法 WO2021073011A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP20876976.0A EP4047081A4 (en) 2019-10-18 2020-01-16 STRAIN FOR THE PRODUCTION OF LONG-CHAIN DICARBOXYLIC ACIDS AND FERMENTATION METHOD THEREFOR
US17/769,459 US20230203431A1 (en) 2019-10-18 2020-01-16 Strain for producing long-chain dicarboxylic acids and fermentation method therefor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910992388.0 2019-10-18
CN201910992388.0A CN112680368B (zh) 2019-10-18 2019-10-18 一种生产长链二元酸的菌株及其发酵方法

Publications (1)

Publication Number Publication Date
WO2021073011A1 true WO2021073011A1 (zh) 2021-04-22

Family

ID=75444892

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/072398 WO2021073011A1 (zh) 2019-10-18 2020-01-16 一种生产长链二元酸的菌株及其发酵方法

Country Status (4)

Country Link
US (1) US20230203431A1 (zh)
EP (1) EP4047081A4 (zh)
CN (1) CN112680368B (zh)
WO (1) WO2021073011A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113943760A (zh) * 2021-12-02 2022-01-18 王婷 维斯假丝酵母发酵生产长链二元酸的方法、产品与菌种

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112680368B (zh) * 2019-10-18 2024-05-07 上海凯赛生物技术股份有限公司 一种生产长链二元酸的菌株及其发酵方法
CN112680369B (zh) * 2019-10-18 2024-05-03 上海凯赛生物技术股份有限公司 一种高产长链二元酸的菌株及其发酵方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102839133A (zh) 2011-06-21 2012-12-26 上海凯赛生物技术研发中心有限公司 一种长链二元酸生产菌株及其应用
KR20170048763A (ko) * 2015-10-27 2017-05-10 한국생명공학연구원 세바식산 생산용 미생물 및 이를 이용한 세바식산의 생산 방법
CN107326051A (zh) * 2017-08-02 2017-11-07 上海凯赛生物技术研发中心有限公司 一种微生物发酵法生产的癸二酸及其制备方法
CN109913512A (zh) * 2017-12-13 2019-06-21 上海凯赛生物技术研发中心有限公司 生物发酵生产长链二元酸的方法
CN109943599A (zh) * 2017-12-20 2019-06-28 上海凯赛生物技术研发中心有限公司 一种发酵生产长链二元酸的方法
CN109943598A (zh) * 2017-12-20 2019-06-28 上海凯赛生物技术研发中心有限公司 一种发酵生产长链二元酸的方法
CN110218746A (zh) * 2018-03-01 2019-09-10 上海凯赛生物技术研发中心有限公司 发酵生产长链二元酸的方法及发酵液、发酵处理液、污水
CN110218745A (zh) * 2018-03-01 2019-09-10 上海凯赛生物技术研发中心有限公司 发酵生产长链二元酸的方法及其得到的长链二元酸
CN110218661A (zh) * 2018-03-01 2019-09-10 上海凯赛生物技术研发中心有限公司 一种热带假丝酵母菌及其应用、以及一种长链二元酸的生产方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5001953B2 (ja) * 2005-12-30 2012-08-15 カウンシル オブ サイエンティフィック アンド インダストリアル リサーチ 長鎖ジカルボン酸の製造方法
WO2013131277A1 (en) * 2012-03-09 2013-09-12 Cathay R & D Center Co., Ltd A new candida sake strain for producing long chain dicarboxylic acids
WO2016156260A1 (fr) * 2015-03-27 2016-10-06 Fonds De Développement Des Filières Des Oléagineux Et Des Proteagineux Fidop Microorganismes et leur utilisation pour la production de diacides
CN109706192B (zh) * 2019-02-01 2022-05-10 上海凯赛生物技术股份有限公司 一种c19～c21长链二元酸的发酵生产方法
CN112680369B (zh) * 2019-10-18 2024-05-03 上海凯赛生物技术股份有限公司 一种高产长链二元酸的菌株及其发酵方法
CN112680368B (zh) * 2019-10-18 2024-05-07 上海凯赛生物技术股份有限公司 一种生产长链二元酸的菌株及其发酵方法

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102839133A (zh) 2011-06-21 2012-12-26 上海凯赛生物技术研发中心有限公司 一种长链二元酸生产菌株及其应用
KR20170048763A (ko) * 2015-10-27 2017-05-10 한국생명공학연구원 세바식산 생산용 미생물 및 이를 이용한 세바식산의 생산 방법
CN107326051A (zh) * 2017-08-02 2017-11-07 上海凯赛生物技术研发中心有限公司 一种微生物发酵法生产的癸二酸及其制备方法
CN109913512A (zh) * 2017-12-13 2019-06-21 上海凯赛生物技术研发中心有限公司 生物发酵生产长链二元酸的方法
CN109943599A (zh) * 2017-12-20 2019-06-28 上海凯赛生物技术研发中心有限公司 一种发酵生产长链二元酸的方法
CN109943598A (zh) * 2017-12-20 2019-06-28 上海凯赛生物技术研发中心有限公司 一种发酵生产长链二元酸的方法
CN110218746A (zh) * 2018-03-01 2019-09-10 上海凯赛生物技术研发中心有限公司 发酵生产长链二元酸的方法及发酵液、发酵处理液、污水
CN110218745A (zh) * 2018-03-01 2019-09-10 上海凯赛生物技术研发中心有限公司 发酵生产长链二元酸的方法及其得到的长链二元酸
CN110218661A (zh) * 2018-03-01 2019-09-10 上海凯赛生物技术研发中心有限公司 一种热带假丝酵母菌及其应用、以及一种长链二元酸的生产方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Institute of Forest Soil Science", 1979, CHINESE ACADEMY OF SCIENCES, article "Candida lipolytica"
See also references of EP4047081A4
YU ZHIHUA: "Acta Microbiologica Sinica", INSTITUTE OF MICROBIOLOGY, vol. 06, 1989, pages 0253 - 2654

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113943760A (zh) * 2021-12-02 2022-01-18 王婷 维斯假丝酵母发酵生产长链二元酸的方法、产品与菌种
CN113943760B (zh) * 2021-12-02 2023-09-12 王婷 维斯假丝酵母发酵生产长链二元酸的方法、产品与菌种

Also Published As

Publication number Publication date
CN112680368B (zh) 2024-05-07
US20230203431A1 (en) 2023-06-29
EP4047081A4 (en) 2024-04-24
EP4047081A1 (en) 2022-08-24
CN112680368A (zh) 2021-04-20

Similar Documents

Publication Publication Date Title
WO2021073011A1 (zh) 一种生产长链二元酸的菌株及其发酵方法
Canale-Parola Biology of the sugar-fermenting Sarcinae
US20240102058A1 (en) Caproate-producing bacterium with multiple substrate utilization capabilities and its applications
CN101457211B (zh) 一株肺炎克雷伯氏菌及其在制备2,3-丁二醇中的应用
CN102226159B (zh) 一株阴沟肠杆菌及其在制备2，3-丁二醇中的应用
CN102864188A (zh) 一种木质纤维素生产生物柴油的方法
CN106554931B (zh) 一株拜氏羧菌及其应用
CN102703339B (zh) 高产精氨酸脱亚胺酶菌株及用它生产l-瓜氨酸的方法
CN112239738B (zh) 一株产琥珀酸的大肠杆菌及其应用
CN102899372B (zh) 两阶段溶氧量控制发酵生产环磷酸腺苷的方法
CN103451244B (zh) 一种屎肠球菌在制备l-乳酸中的应用
CN109706192B (zh) 一种c19～c21长链二元酸的发酵生产方法
CN105801675B (zh) 一种高活性壳聚糖酶控制基因csn及利用该基因生产高活性壳聚糖酶的方法
CN111748480B (zh) 一种维斯假丝酵母及其应用
KR101245208B1 (ko) 세라믹 막 분리 배양기
CN112680369B (zh) 一种高产长链二元酸的菌株及其发酵方法
CN116426577A (zh) 一种以氢氧化钙为中和剂结合co2脉冲反馈补料发酵生产丁二酸的方法
CN111019995B (zh) 一种以丁香酚为底物发酵生成香兰素的方法
CN103725724A (zh) 一种固定化蜂房哈夫尼菌生产尸胺的方法
CN112280696A (zh) 一种通过维持超低浓度有机碳源提高酿酒酵母油脂含量的培养方法及其应用
CN115637276B (zh) 采用盐单胞菌株生产四氢嘧啶的方法
CN103937733A (zh) 一株利用蔗糖产丁二酸基因工程菌株及其发酵生产丁二酸的方法
CN114507611B (zh) α-酮戊二酸生产用耐高温酵母及发酵方法
CN115637233A (zh) 一种高产十碳二元酸的菌株及发酵方法
CN102154134A (zh) 红酵母及其产海藻糖的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20876976

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020876976

Country of ref document: EP

Effective date: 20220518