CN110468051B - K252A fermentation medium and preparation method thereof - Google Patents
K252A fermentation medium and preparation method thereof Download PDFInfo
- Publication number
- CN110468051B CN110468051B CN201910698830.9A CN201910698830A CN110468051B CN 110468051 B CN110468051 B CN 110468051B CN 201910698830 A CN201910698830 A CN 201910698830A CN 110468051 B CN110468051 B CN 110468051B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- percent
- seed culture
- culture medium
- solution
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
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
- C12N1/20—Bacteria; 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a K252A fermentation medium and a preparation method thereof, the fermentation medium can provide nutrition for growth and metabolism of Nocardiopsis strains through optimized component collocation and addition of functional factors such as choline chloride, disodium succinate, methionine and the like, and can also provide an intermediate for biosynthesis of K252A after metabolic conversion, thereby finally obtaining a fermentation liquid with high yield of K252A. When the fermentation medium provided by the invention is used for shake flask fermentation for 9 days, the K252A titer is higher than 2.60 g/L. By controlling two key process conditions of pH and dissolved oxygen and simultaneously optimizing a feeding control strategy of glucose and functional factors, the culture is carried out for about 264 hours, the K252A titer can reach 5.79g/L, the level is obviously improved compared with the level reported in the literature, and the method has the basis of industrial production.
Description
Technical Field
The invention relates to the technical field of industrial microbial fermentation, in particular to a K252A fermentation medium and a method for preparing K252A from the fermentation medium.
Background
K252A, CAS No. 99533-80-9, was a metabolite isolated from Nocardiopsis culture by HIROSHI KASE et al in 1986, and was then structurally characterized to finally confirm that it is an analog of staurosporine, a potent inhibitor of protein kinase C and tyrosine kinase, IC 50 The value was 32.9 nM. At present, a large number of documents report that antibiotic K252A has potent antitumor activity, but in vitro studies have shown no antimicrobial activity or rodent toxicity in vivo. More extensive studies have demonstrated that antibiotic K252A is a potent Ca 2+ Calponin kinase II inhibitors which also inhibit other kinase activities, in particular myosin light chain kinase, cyclic adenosine monophosphate dependent Protein Kinase (PKA), Protein Kinase C (PKC) and cGMP dependent Protein Kinase (PKG).
Nocardiopsis species (Nocardiopsis sp.) are known to produce K252A, and HIROSHI KASE was briefly reported in 1986, but only about 110. mu.g/ml of K252A was obtained in The fermentation broth (Hiroshi Kase, Kazuyuki Iwahashi, Yuzuru Matsuda. K-252a, a flux Inhibitor of Protein Kinase C m Microbiological Origin [ J ]. The Journal of Antibiotics,1986,39(8): 1059-. Mitsutaka Kino reported that K252A could be fermented at a level of 2g/L in a fermenter, but it has not been disclosed in the following documents (Mitsutaka Kino, Kenzo Shono, Tetsuo Nishinura, et al.practical Preparation of K-252a from a Fermentation Solution [ J ]. biosci.Biotechnol.biochem.,1998,62(8): 1627) 1629.). Sanjay R.Chemburgkar et al report that the K252A fermentation level is 1-4 g/L in patent US20090239271A1, and protect the separation method of K252A, but do not disclose the specific fermentation medium formula and fermentation culture method.
Disclosure of Invention
In order to improve the yield of K252A, the invention takes Nocardiopsis sp as a production strain, researches and optimizes a fermentation medium and a fermentation method, and finally obtains the fermentation medium and the method for preparing K252A from the fermentation medium. The culture medium and the method can provide sufficient nutrition for growth of Nocardiopsis species, can promote the metabolic rate of Nocardiopsis species, provide an intermediate for biosynthesis of K252A through metabolic conversion of functional factors, and finally greatly improve the fermentation yield of K252A.
A fermentation medium of K252A, which comprises the following components in percentage by weight:
wherein the functional factors are:
the composition comprises choline chloride, disodium succinate and methionine, wherein the content of the choline chloride is 0.01-0.05% of the weight of the fermentation medium, the content of the disodium succinate is 0.08-0.4% of the weight of the fermentation medium, and the content of the methionine is 0.26-1.3% of the weight of the fermentation medium.
Preferably, the carbon source is one or a combination of two or more of the following: glycerol, dextrin, soluble starch, maltose and methyl oleate. The carbon source comprises a quick-acting carbon source and a slow-acting carbon source, and is mainly used for providing energy required for growth and reproduction of thalli and carbon components required for synthesizing the thalli at different periods, stimulating hypha growth and simultaneously providing a carbon skeleton for product synthesis.
As a further preference, wherein the carbon source is preferably methyl oleate or preferably a combination comprising methyl oleate. The methyl oleate has multiple effects, is easy to foam to the top tank in a disinfection stage, and can reduce the dosage of a substrate defoamer due to the defoaming effect, so that the inhibition effect of the defoamer on the growth of thalli is reduced; in the culture stage, the carbon source can be slowly utilized by the thalli as a basic carbon source, and the diffusion of products from the inside of the cell to the outside of the cell can be accelerated, so that the feedback inhibition effect is reduced.
Preferably, the nitrogen source is one or a combination of two or more of the following: soybean cake powder, cottonseed cake powder, corn flour, yeast extract and corn steep liquor dry powder. The nitrogen source comprises quick-acting and slow-acting nitrogen sources, and is mainly used for constructing cell substances such as thallus amino acid, protein, nucleic acid and the like at different periods and a nitrogen skeleton for product synthesis.
Preferably, the inorganic salt is one or a combination of two or more of the following: ammonium salt, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, sulfate, nitrate, preferably a combination of ammonium nitrate, magnesium sulfate, dipotassium hydrogen phosphate, and calcium carbonate. The inorganic salts can constitute the cellular components of the bacterial cells, and can be used as important components of microbial enzymes, and can also be used as activators or inhibitors of certain enzymes so as to regulate the pathway and efficiency of product synthesis. The inorganic salt can also buffer and regulate the pH value of the culture medium, construct a proper buffer system, possibly maintain proper osmotic pressure, oxidation-reduction potential and the like, and participate in the electron transfer in the biological metabolic process.
Preferably, the defoamer is a THIX-298 defoamer.
Preferably, the fermentation medium comprises the following components in percentage by weight:
the pH value of the fermentation medium before disinfection is 6.4-8.0.
The fermentation medium not only contains a carbon source, but also contains components such as a nitrogen source, inorganic salts and functional factors, so that sufficient nutrition can be provided for Nocardiopsis sp, the metabolic rate of Nocardiopsis sp can be promoted, an intermediate is provided for K252A biosynthesis, and the fermentation yield of K252A is greatly increased finally.
The invention also provides a method for preparing K252A by using the fermentation medium, which comprises the following steps:
the shaking flask fermentation method comprises the following steps: inoculating the shake flask seed culture solution into the fermentation culture medium according to the volume ratio of 5-15%, culturing at 26-32 ℃ under the condition of 150-500 rpm for 220-240 h to obtain fermentation liquor containing K252A;
or
The tank fermentation method comprises the following steps: inoculating the tank seed culture solution into the fermentation culture medium according to the proportion of 5-10% of the volume of the fermentation culture medium, culturing at 26-32 ℃, and keeping dissolved oxygen at 15-30% all the time through stirring and ventilation linkage control. After inoculation for 24h, starting to culture, and controlling the pH value to be 6.8 +/-0.2 by using ammonia water; and starting after 36h of inoculation, feeding the glucose solution and the functional factor mixed solution in the period, stopping feeding until 1 day before the end of culture, and performing the culture period for 220-264 h to obtain the fermentation liquor containing K252A.
Preferably, the control strategy of feeding the glucose solution in the tank fermentation method is to maintain the concentration of glucose in the fermentation liquid within the range of 1-5 g/L, and the concentration of the fed-in glucose solution is 500 g/L.
Preferably, the functional factor mixed liquor fed-batch in the tank fermentation method comprises the following components: 5g/L of choline chloride, 40g/L of disodium succinate, 130g/L of methionine and the balance of water, wherein the feeding speed of the functional factor mixed solution is controlled to be 0.05-0.1% of the volume of the initial fermentation medium per hour.
Preferably, the shake flask seed culture fluid is obtained by the following steps:
1) screening Nocardiopsis strains: selecting a bacterial colony with a large starch hydrolysis ring by adopting a solid plate culture medium;
2) preparing an effective inoculation source: selecting excellent bacterial colonies, inoculating the bacterial colonies on a solid slant culture medium, performing static culture for 5 days at the temperature of 26-32 ℃ and the relative humidity of 55-65%, and collecting fresh and mature bacterial lawn serving as an effective inoculation source;
3) and (3) seed culture in a shaking flask: inoculating the seed source obtained in the step 2) into a seed culture medium, and culturing for 24-72 h at the temperature of 26-32 ℃ and the rpm of 150-330 to obtain a shake flask seed culture solution;
the tank seed culture solution is obtained by the following steps: inoculating the shake flask seed culture solution into a seed culture medium in a seed tank, and carrying out amplification culture for 18-96 h under the conditions of 26-32 ℃, 50-300 rpm and 0.35vvm to obtain a tank seed culture solution.
Further preferably, the solid plate culture medium in the step 1) and the solid slant culture medium in the step 2) comprise the following components in percentage by weight:
the pH value of the solid culture medium before disinfection is 6.5-7.5, corn starch is used as a carbon source in the step 1) for strain screening, the metabolic capability of the strain can be visually judged according to the size of a starch hydrolysis ring by a bacterial colony, the strain with relatively poor metabolic capability is eliminated, the diameter of the bacterial colony cultured by the culture medium is larger than or equal to 5mm, the bacterial colony is tall and full of lawn.
Preferably, the seed culture medium in step 3) comprises the following components in percentage by weight:
the pH value of the seed culture medium before disinfection is 6.5-7.5, and the seed culture medium can stimulate the growth of hyphae and provide sufficient carbon and nitrogen source nutrition, so that the hyphae are divergent, strong and vigorous, and are not easy to age.
By using the fermentation medium and the method for preparing K252A, the K252A titer is higher than 2.60g/L and is 5.26 times of that of the fermentation medium without functional factors after shaking flask fermentation is carried out for 9 days. Through the fermentation tank process condition and feeding control, the K252A titer can reach 5.79g/L at most after the culture is carried out for about 264 hours, and the level is obviously improved compared with the level reported in the current literature.
Compared with the prior art, the invention has the following advantages:
1. the fermentation medium provided by the invention can provide nutrition for growth and metabolism of Nocardiopsis strains, and the K252A titer is higher than 2.6g/L by adding choline chloride, disodium succinate and methionine combined functional factors.
2. In the tank fermentation culture, the feed supplement control strategy of glucose and functional factors is optimized by controlling two key process conditions of pH and dissolved oxygen, so that the content of K252A in the fermentation liquid is further greatly improved to be more than 5.7g/L, and the level is obviously improved compared with the level reported in the literature.
3. The K252A fermentation medium and the preparation method thereof are verified by carrying out process amplification in a 500L scale fermentation tank, the content of K252A in the fermentation liquid can reach more than 5.7g/L, and the K252A fermentation medium and the preparation method thereof have the basis of industrial production.
Detailed Description
In order to explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is made with reference to specific embodiments. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The media components used in the following examples are as follows:
solid plate medium composition: 1% of corn starch, 0.2% of hydrolyzed casein, 0.2% of beef extract, 0.2% of yeast extract, 2% of agar and the balance of water, wherein the pH value of the culture medium before disinfection is 6.5-7.5.
Solid slant culture medium composition: 1% of corn starch, 0.2% of hydrolyzed casein, 0.2% of beef extract, 0.2% of yeast extract, 2% of agar and the balance of water, wherein the pH value of the culture medium before disinfection is 6.5-7.5.
The seed culture medium comprises the following components: 0.5% of glycerol, 3% of corn starch, 2% of soybean cake powder, 0.5% of yeast extract, 0.5% of corn steep liquor dry powder, 0.3% of calcium carbonate and the balance of water, wherein the pH value of the seed culture medium before disinfection is 6.5-7.5.
The strain sources in the invention are as follows: purchased from the American agricultural research culture Collection, Nocardiaopsis sp.K-252, NRRL 15532.
Comparative example 1:
1) screening Nocardiopsis strains: taking a working strain freezing tube, separating and dibbling the working strain onto a solid plate culture medium, and selecting a bacterial colony with a large starch hydrolysis ring;
2) preparing an effective inoculation source: 1 excellent colony is selected and inoculated on a solid slant culture medium, standing culture is carried out for 5 days at the temperature of 28 ℃ and the relative humidity of 60 percent, and fresh and mature lawn is immediately collected to be used as an effective inoculation source;
3) and (3) seed culture in a shaking flask: the solid inclined surface lawn of the step 2) is about 1cm 2 Inoculating into 50ml seed culture medium, and culturing at 28 deg.C and 200rpm for 72h to obtain shake flask seed culture solution;
4) and (3) shake flask fermentation culture: inoculating the seed culture solution obtained in the step 3) into a fermentation culture medium without methyl oleate and functional factors according to the volume ratio of 5%, and culturing for 9 days at the temperature of 28 ℃ and at the speed of 200rpm to obtain a fermentation liquid containing K252A;
the fermentation medium formula (without functional factors) comprises: 0.4% of glycerol, 1% of maltose, 9% of maltodextrin, 4% of soluble starch, 0.2% of soybean cake powder, 1% of corn flour, 1% of cottonseed cake powder, 3% of yeast extract, 0.5% of corn steep liquor dry powder, 0.1% of ammonium nitrate, 0.5% of magnesium sulfate, 0.02% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and the balance of water, wherein the pH value of the culture medium before disinfection is 6.5.
5) Sample treatment and detection: adding 5ml of anhydrous methanol into 1ml of fermentation liquor, uniformly mixing, performing ultrasonic treatment for 1 hour, uniformly mixing again, centrifuging or filtering, taking supernatant, and analyzing and determining by HPLC, wherein the content of K252A in the fermentation liquor is 0.504 g/L.
Example 1:
1) screening Nocardiopsis strains: taking a working strain freezing tube, separating and dibbling the working strain onto a solid plate culture medium, and selecting a bacterial colony with a large starch hydrolysis ring;
2) preparing an effective inoculation source: 1 excellent colony is picked and inoculated on a solid slant culture medium, standing culture is carried out for 5 days at the temperature of 28 ℃ and the relative humidity of 60 percent, and fresh and mature lawn is immediately collected to be used as an effective inoculation source;
3) and (3) seed culture in a shaking flask: the solid inclined surface lawn of the step 2) is about 1cm 2 Inoculating into 50ml seed culture medium, and culturing at 28 deg.C and 200rpm for 72h to obtain shake flask seed culture solution;
4) and (3) shake flask fermentation culture: inoculating the seed culture solution obtained in the step 3) into a methyl oleate-free fermentation culture medium added with functional factors according to the volume ratio of 5%, and culturing for 9 days at the temperature of 28 ℃ and the speed of 200rpm to obtain a fermentation liquid containing K252A;
the fermentation medium formula (containing functional factors) comprises the following components: 0.4% of glycerol, 1% of maltose, 9% of maltodextrin, 4% of soluble starch, 0.2% of soybean cake powder, 1% of corn flour, 1% of cottonseed cake powder, 3% of yeast extract, 0.5% of corn steep liquor dry powder, 0.1% of ammonium nitrate, 0.5% of magnesium sulfate, 0.02% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate, 0.05% of choline chloride, 0.4% of disodium succinate, 1.3% of methionine and the balance of water, wherein the pH value of the culture medium before disinfection is 6.5.
5) Sample treatment and detection: adding 5ml of anhydrous methanol into 1ml of fermentation liquor, uniformly mixing, performing ultrasonic treatment for 1h, uniformly mixing again, centrifuging or filtering, taking supernatant, and analyzing and determining by HPLC (high performance liquid chromatography), wherein the content of K252A in the fermentation liquor is 2.651 g/L.
Example 2:
1) screening Nocardiopsis strains: taking a working strain freezing tube, separating and dibbling the working strain onto a solid plate culture medium, and selecting a bacterial colony with a large starch hydrolysis ring;
2) preparing an effective inoculation source: 1 excellent colony is selected and inoculated on a solid slant culture medium, standing culture is carried out for 5 days at the temperature of 28 ℃ and the relative humidity of 60 percent, and fresh and mature lawn is immediately collected to be used as an effective inoculation source;
3) and (3) shake flask seed culture: the solid slant lawn in the step 2) is about 1cm 2 Inoculating into 50ml seed culture medium, and culturing at 28 deg.C and 200rpm for 72h to obtain shake flask seed culture solution;
4) and (3) shake flask fermentation culture: inoculating the seed culture solution obtained in the step 3) into a fermentation culture medium which contains methyl oleate and is added with functional factors according to the volume ratio of 5%, and culturing for 9 days at the temperature of 28 ℃ and the speed of 200rpm to obtain a fermentation liquid containing K252A;
the fermentation medium formula (containing functional factors and methyl oleate) comprises the following components: 1.5% of methyl oleate, 0.4% of glycerol, 1% of maltose, 9% of maltodextrin, 4% of soluble starch, 0.2% of soybean cake powder, 1% of corn flour, 1% of cottonseed cake powder, 3% of yeast extract, 0.5% of corn steep liquor dry powder, 0.1% of ammonium nitrate, 0.5% of magnesium sulfate, 0.02% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate, 0.05% of choline chloride, 0.4% of disodium succinate, 1.3% of methionine and the balance of water, wherein the pH value of the culture medium before sterilization is 6.5.
5) Sample treatment and detection: adding 5ml of anhydrous methanol into 1ml of fermentation liquor, uniformly mixing, performing ultrasonic treatment for 1 hour, uniformly mixing again, centrifuging or filtering, taking supernatant, and analyzing and determining by HPLC, wherein the content of K252A in the fermentation liquor is 2.773 g/L.
Comparative example 2:
1) screening Nocardiopsis strains: taking a working strain cryopreservation tube, separating and dibbling the working strain to a solid plate culture medium, and selecting a bacterial colony with a large starch hydrolysis ring;
2) preparing an effective inoculation source: 1 excellent colony is selected and inoculated into a solid slant culture medium, standing culture is carried out for 5 days at the temperature of 28 ℃ and the relative humidity of 60 percent, and fresh and mature lawn is immediately collected to be used as an effective inoculation source;
3) and (3) seed culture in a shaking flask: the solid inclined surface lawn of the step 2) is about 1cm 2 Inoculating into 50ml seed culture medium, culturing at 28 deg.C and 200rpm for 68 hr to obtain shake flask seed culture solution;
4) tank fermentation culture: inoculating the shake flask seed culture solution obtained in the step 3) into 10L of seed culture medium according to the proportion of 0.25% of the volume of the culture medium, and performing amplification culture for 48h under the conditions of 29 ℃, 150rpm and 0.35vvm to obtain a tank seed culture solution; inoculating the tank seed culture solution into a fermentation culture medium without containing methyl oleate according to the proportion of 8% of the volume of the fermentation culture medium, culturing under the conditions of 28 ℃, 150-500 rpm and 0.35vvm without pH and dissolved oxygen control and material supplementation for 256 hours to obtain a fermentation liquid containing K252A;
the fermentation medium formula (containing functional factors) comprises the following components: 0.4% of glycerol, 1% of maltose, 5% of maltodextrin, 3% of soluble starch, 0.6% of soybean cake powder, 1.5% of corn flour, 1.5% of cottonseed cake powder, 2.5% of yeast extract, 0.75% of corn steep liquor dry powder, 0.1% of ammonium nitrate, 0.5% of magnesium sulfate, 0.02% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate, 0.05% of choline chloride, 0.4% of disodium succinate, 1.3% of methionine and the balance of water, wherein the pH value of the culture medium before sterilization is 6.5.
5) Sample treatment and detection: adding 5ml of anhydrous methanol into 1ml of fermentation liquor, uniformly mixing, performing ultrasonic treatment for 1h, uniformly mixing again, centrifuging or filtering, taking supernatant, and analyzing and determining by HPLC (high performance liquid chromatography), wherein the content of K252A in the fermentation liquor is 2.746 g/L.
Comparative example 3:
1) screening Nocardiopsis strains: taking a working strain freezing tube, separating and dibbling the working strain onto a solid plate culture medium, and selecting a bacterial colony with a large starch hydrolysis ring;
2) preparing an effective inoculation source: 1 excellent colony is selected and inoculated to a solid slant culture medium, standing culture is carried out for 5 days at the temperature of 28 ℃ and the relative humidity of 60 percent, and fresh and mature lawn is immediately collected to be used as an effective inoculation source;
3) and (3) seed culture in a shaking flask: the solid inclined surface lawn of the step 2) is about 1cm 2 Inoculating into 50ml seed culture medium, culturing at 28 deg.C and 200rpm for 68 hr to obtain shake flask seed culture solution;
4) tank fermentation culture: inoculating the shake flask seed culture solution obtained in the step 3) into 10L of seed culture medium according to the proportion of 0.25% of the volume of the culture medium, and performing amplification culture for 48h under the conditions of 29 ℃, 150rpm and 0.35vvm to obtain a tank seed culture solution; inoculating the tank seed culture solution into a fermentation culture medium containing 1.5% of methyl oleate according to the proportion of 8% of the volume of the fermentation culture medium, culturing at 28 ℃, 150-500 rpm and 0.35vvm without pH and dissolved oxygen control and feeding, and culturing for 260 hours to obtain a fermentation liquid containing K252A;
the fermentation medium formula (containing functional factors and methyl oleate) comprises the following components: 1.5% of methyl oleate, 0.4% of glycerol, 1% of maltose, 5% of maltodextrin, 3% of soluble starch, 0.6% of soybean cake powder, 1.5% of corn flour, 1.5% of cottonseed cake powder, 2.5% of yeast extract, 0.75% of corn steep liquor dry powder, 0.1% of ammonium nitrate, 0.5% of magnesium sulfate, 0.02% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate, 0.05% of choline chloride, 0.4% of disodium succinate, 1.3% of methionine and the balance of water, wherein the pH value of the culture medium before disinfection is 6.5.
5) Sample treatment and detection: taking 1ml of fermentation liquor, adding 5ml of anhydrous methanol, uniformly mixing, carrying out ultrasonic treatment for 1h, uniformly mixing again, centrifuging or filtering, taking supernatant, and analyzing and determining by HPLC, wherein the content of K252A in the fermentation liquor is 2.918 g/L.
Example 3:
1) screening Nocardiopsis strains: taking a working strain cryopreservation tube, separating and dibbling the working strain to a solid plate culture medium, and selecting a bacterial colony with a large starch hydrolysis ring;
2) preparing an effective inoculation source: 1 excellent colony is selected and inoculated into a solid slant culture medium, standing culture is carried out for 5 days at the temperature of 28 ℃ and the relative humidity of 60 percent, and fresh and mature lawn is immediately collected to be used as an effective inoculation source;
3) and (3) seed culture in a shaking flask: the solid inclined surface lawn of the step 2) is about 1cm 2 Inoculating into 50ml seed culture medium, culturing at 28 deg.C and 200rpm for 68 hr to obtain shake flask seed culture solution;
4) tank fermentation culture: inoculating the shake flask seed culture solution obtained in the step 3) into 10L of seed culture medium according to the proportion of 0.25% of the volume of the culture medium, and carrying out amplification culture for 48h under the conditions of 29 ℃, 150rpm and 0.35vvm to obtain a tank seed culture solution; and inoculating the tank seed culture solution into a fermentation culture medium containing 3% of methyl oleate and low-concentration functional factors according to the proportion of 8% of the volume of the fermentation culture medium, and maintaining dissolved oxygen by 15-30% under the condition of 28 ℃ through stirring and ventilation linkage control. After inoculation for 24h, the pH was automatically controlled to 6.8. + -. 0.2 with ammonia until the end of the culture. After inoculation for 36 hours, feeding 500g/L of glucose solution and functional factor mixed solution, maintaining the concentration of glucose in the fermentation broth to be 1-5 g/L, stopping feeding the glucose solution and the functional factor mixed solution until 240 hours, and continuously culturing for 264 hours to obtain fermentation broth containing K252A;
the fermentation medium formula (containing functional factors and methyl oleate) comprises the following components: 0.4% of glycerol, 0.5% of maltose, 5% of maltodextrin, 3% of soluble starch, 3% of methyl oleate, 0.6% of soybean cake powder, 1.5% of corn flour, 1.5% of cottonseed cake powder, 2.5% of yeast extract, 0.75% of corn steep liquor dry powder, 0.1% of ammonium nitrate, 0.5% of magnesium sulfate, 0.02% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate, 0.01% of choline chloride, 0.08% of disodium succinate, 0.26% of methionine and the balance of water, wherein the pH value of the culture medium before sterilization is 6.5.
The functional factor mixed solution comprises: 5g/L of choline chloride, 40g/L of disodium succinate, 130g/L of methionine and the balance of water;
5) sample treatment and detection: and (3) adding 9ml of anhydrous methanol into 1ml of fermentation liquor, uniformly mixing, performing ultrasonic treatment for 1 hour, uniformly mixing again, centrifuging or filtering, taking supernatant, and analyzing and determining by using HPLC (high performance liquid chromatography), wherein the content of K252A in the fermentation liquor is 5.702 g/L.
Example 4:
1) screening Nocardiopsis strains: taking a working strain freezing tube, separating and dibbling the working strain onto a solid plate culture medium, and selecting a bacterial colony with a large starch hydrolysis ring;
2) preparing an effective inoculation source: 1 excellent colony is selected and inoculated into a solid slant culture medium, standing culture is carried out for 5 days at the temperature of 28 ℃ and the relative humidity of 60 percent, and fresh and mature lawn is immediately collected to be used as an effective inoculation source;
3) and (3) seed culture in a shaking flask: the solid slant lawn in the step 2) is about 1cm 2 Inoculating into 50ml seed culture medium, culturing at 28 deg.C and 200rpm for 68 hr to obtain shake flask seed culture solution;
4) tank fermentation culture: inoculating the shake flask seed culture solution obtained in the step 3) into 30L of seed culture medium according to the proportion of 0.25% of the volume of the culture medium, and culturing for 52h under the conditions of 29 ℃, 150rpm and 0.35vvm to obtain a tank seed culture solution; and inoculating the tank seed culture solution into a fermentation culture medium containing 3% of methyl oleate and low-concentration functional factors according to the proportion of 6% of the volume of the fermentation culture medium, and maintaining dissolved oxygen by 15-30% under the condition of 28 ℃ through stirring and ventilation linkage control. After inoculation is carried out for 24 hours, the pH is automatically controlled within the range of 6.8 +/-0.2 by using ammonia water, after inoculation is carried out for 36 hours, 500g/L of glucose solution and functional factor mixed liquor are fed, the concentration of glucose in the fermentation liquor is maintained at 1-5 g/L, the feeding of the glucose solution and the functional factor mixed liquor is stopped until 240 hours, and the fermentation liquor containing K252A is obtained after continuous culture for 264 hours;
the fermentation medium formula (containing functional factors and methyl oleate) comprises the following components: 3% of methyl oleate, 0.4% of glycerol, 0.5% of maltose, 5% of maltodextrin, 3% of soluble starch, 0.6% of soybean cake powder, 1.5% of corn flour, 1.5% of cottonseed cake powder, 2.5% of yeast extract, 0.75% of corn steep liquor dry powder, 0.1% of ammonium nitrate, 0.5% of magnesium sulfate, 0.02% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate, 0.01% of choline chloride, 0.08% of disodium succinate, 0.26% of methionine and the balance of water, wherein the pH value of the culture medium before disinfection is 6.5.
The functional factor mixed solution comprises: 5g/L of choline chloride, 40g/L of disodium succinate, 130g/L of methionine and the balance of water;
5) sample treatment and detection: and (3) adding 9ml of anhydrous methanol into 1ml of fermentation liquor, uniformly mixing, performing ultrasonic treatment for 1 hour, uniformly mixing, centrifuging or filtering, taking supernatant, and analyzing and determining by using HPLC (high performance liquid chromatography), wherein the content of K252A in the fermentation liquor is 5.790 g/L.
From the comparative data of comparative example 1 and example 1, it can be seen that the potency of K252A is significantly improved by adding the choline chloride, disodium succinate and methionine combined functional factor, and reaches 2.6g/L, which is about 5.26 times that of the non-added functional factor.
From the comparison of example 1 with example 2 and the comparison of comparative example 2 with comparative example 3, it can be seen that the addition of methyl oleate to the fermentation medium of the invention, in particular to the tank fermentation medium, is advantageous for the increase in the yield of K252A.
Compared with the comparative data of the comparative example 3 and the examples 3-4, the invention has the advantages that the two key process conditions of pH and dissolved oxygen are controlled, the feeding control strategy of the mixed solution of glucose and functional factors is optimized, the content of K252A in the fermentation liquid is further greatly improved, the content can still reach 5.79g/L after pilot plant test amplification, the level is obviously improved compared with that reported in the literature, and the industrial production basis is provided.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by changing and modifying the embodiments described herein or by using the equivalent structures or equivalent processes of the present invention, and are included in the scope of the present invention.
Claims (5)
1. A fermentation medium for preparing K252A is characterized by comprising the following components in percentage by weight:
0.2-0.5% of glycerin
0.5 to 1 percent of maltose
5-9% of maltodextrin
3 to 4 percent of soluble starch
0 to 3 percent of methyl oleate
0.2-0.6% of soybean cake powder
1-1.5% of corn flour
1-1.5% of cottonseed cake powder
2 to 3 percent of yeast extract
0.5 to 1 percent of corn steep liquor dry powder
0.1 to 0.5 percent of ammonium nitrate
0.5 to 0.8 percent of magnesium sulfate
Dipotassium hydrogen phosphate 0.01-0.02%
0.1 to 0.3 percent of calcium carbonate
0.01 to 0.05 percent of choline chloride
0.08-0.4% of disodium succinate
0.26 to 1.3 percent of methionine
The balance of water,
the pH value of the fermentation medium before disinfection is 6.4-8.0.
2. Method for the preparation of K252A from a fermentation medium according to claim 1, characterized in that: which comprises the steps of preparing a mixture of a plurality of raw materials,
the shake flask fermentation method comprises the following steps: inoculating the shake flask seed culture solution into the fermentation culture medium according to the volume ratio of 5-15%, culturing at 26-32 ℃ and 150-500 rpm for 200-240 h to obtain a fermentation solution containing K252A; or alternatively
The tank fermentation method comprises the following steps: inoculating a tank seed culture solution into the fermentation culture medium in the claim 1 according to the proportion of 5-10% of the volume of the fermentation culture medium, culturing at 26-32 ℃, and keeping dissolved oxygen at 15-30% all the time through stirring and aeration linkage control; after inoculation for 24 hours, starting to culture, and adjusting the pH value to be 6.8 +/-0.2 by using ammonia water; after inoculation for 36h, feeding the glucose solution and the functional factor mixed solution in the period, stopping feeding until 1 day before the culture is finished, and obtaining fermentation liquor containing K252A after the culture period is 220-264 h; wherein,
the shake flask seed culture solution is obtained through the following steps: will be provided withNocardiopsissp.K-252 and NRRL 15532 are inoculated into a seed culture medium and cultured for 24-72 h at the temperature of 26-32 ℃ and the rpm of 150-330 to obtain a shake flask seed culture solution;
the tank seed culture solution is obtained by the following steps: inoculating the shake flask seed culture solution into a seed culture medium in a seed tank, and carrying out amplification culture for 18-96 h at 26-32 ℃ and 50-300 rpm to obtain a tank seed culture solution.
3. The method of claim 2 for preparing K252A, wherein: the control strategy of fed-batch glucose solution in the tank fermentation method is to maintain the concentration of glucose in fermentation liquor within the range of 1-5 g/L, and the concentration of the fed-batch glucose solution is 500 g/L.
4. The method of claim 2 for preparing K252A, wherein: the functional factor mixed liquid fed-batch in the tank fermentation method comprises the following components: 5g/L of choline chloride, 40g/L of disodium succinate, 130g/L of methionine and the balance of water; the speed of feeding the functional factor mixed liquor is controlled to be 0.05-0.1% of the volume of the initial fermentation culture medium per hour.
5. The method of claim 2 for preparing K252A, wherein: the seed culture medium comprises the following components in percentage by weight:
0.5 percent of glycerin
3 percent of corn starch
2 percent of soybean cake powder
0.5% of yeast extract
0.5 percent of corn steep liquor dry powder
0.3 percent of calcium carbonate
The balance of water;
the pH value of the seed culture medium before disinfection is 6.5-7.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910698830.9A CN110468051B (en) | 2019-07-31 | 2019-07-31 | K252A fermentation medium and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910698830.9A CN110468051B (en) | 2019-07-31 | 2019-07-31 | K252A fermentation medium and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110468051A CN110468051A (en) | 2019-11-19 |
| CN110468051B true CN110468051B (en) | 2022-08-23 |
Family
ID=68509306
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910698830.9A Active CN110468051B (en) | 2019-07-31 | 2019-07-31 | K252A fermentation medium and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110468051B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113122591B (en) * | 2019-12-30 | 2022-11-22 | 上海医药工业研究院 | A method for producing staurosporine by fermentation |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09275993A (en) * | 1996-04-16 | 1997-10-28 | Kyowa Hakko Kogyo Co Ltd | Purification of k-252a |
| CN105238709A (en) * | 2015-09-09 | 2016-01-13 | 中国热带农业科学院热带生物技术研究所 | Marine actinomycete strain with meloidogyne insecticidal activity and application of marine actinomycete strain |
| CN105802895A (en) * | 2016-05-17 | 2016-07-27 | 中国极地研究中心 | Fermentation medium and fermentation method of Streptomyces sp. |
| CN108060193A (en) * | 2018-01-31 | 2018-05-22 | 天俱时工程科技集团有限公司 | The production technology of avermectin |
| CN108949845A (en) * | 2018-08-08 | 2018-12-07 | 福建康鸿生物科技有限公司 | A kind of fermentation medium and the method that mupirocin is prepared by fermentation medium |
| CN109576196A (en) * | 2019-01-25 | 2019-04-05 | 北大方正集团有限公司 | A kind of production method of the fermentation medium for producing doractin and doractin |
-
2019
- 2019-07-31 CN CN201910698830.9A patent/CN110468051B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09275993A (en) * | 1996-04-16 | 1997-10-28 | Kyowa Hakko Kogyo Co Ltd | Purification of k-252a |
| CN105238709A (en) * | 2015-09-09 | 2016-01-13 | 中国热带农业科学院热带生物技术研究所 | Marine actinomycete strain with meloidogyne insecticidal activity and application of marine actinomycete strain |
| CN105802895A (en) * | 2016-05-17 | 2016-07-27 | 中国极地研究中心 | Fermentation medium and fermentation method of Streptomyces sp. |
| CN108060193A (en) * | 2018-01-31 | 2018-05-22 | 天俱时工程科技集团有限公司 | The production technology of avermectin |
| CN108949845A (en) * | 2018-08-08 | 2018-12-07 | 福建康鸿生物科技有限公司 | A kind of fermentation medium and the method that mupirocin is prepared by fermentation medium |
| CN109576196A (en) * | 2019-01-25 | 2019-04-05 | 北大方正集团有限公司 | A kind of production method of the fermentation medium for producing doractin and doractin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110468051A (en) | 2019-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111304106B (en) | A strain of Bacillus clausii and method for producing tetrahydropyrimidine using the same | |
| CN105112476B (en) | A kind of method for fermentation production of lipopeptide biosurfactant | |
| CN108034599B (en) | One plant of Lactobacillus brevis for efficiently synthesizing γ-aminobutyric acid from brewed spirit system | |
| CN102559552B (en) | Production method and application of high-yield gamma-aminobutyric acid | |
| CN101245362B (en) | Method for producing polypeptide antibiotic enramycin by fermentation method | |
| CN100430471C (en) | Engineering strain producing riboflavin and method for producing riboflavin | |
| CN112795607A (en) | Method for improving adenosine fermentation yield | |
| SK287293B6 (en) | A method for fermentation of polymyxin B by means of productive microorganism Bacillus polymyxa | |
| CN110468051B (en) | K252A fermentation medium and preparation method thereof | |
| CN114606276B (en) | Method for improving fermentation yield of L-threonine | |
| CN111363708B (en) | Method for improving butyric acid fermentation level | |
| Elisashvili et al. | Extracellular polysaccharide production by culinary-medicinal Shiitake mushroom Lentinus edodes (Berk.) Singer and Pleurotus (Fr.) P. Karst. species depending on carbon and nitrogen source | |
| CN101709318B (en) | Method for preparing glutathione by fed-batch fermentation of Candida utilis | |
| TWI627278B (en) | Mutant of corynebacterium glutamicum producing l-histidine and method for producing l-histidine using the same | |
| JPS583678B2 (en) | Continuous fermentation production method for L-tryptophan | |
| CN114276937A (en) | Method for fermenting paecilomyces hepiali by using Chinese yam as carbon source | |
| CN114015607A (en) | Bacillus amyloliquefaciens for high yield of 5-methyltetrahydrofolic acid and application thereof | |
| Nguyen et al. | Citric acid production by Aspergillus niger using media containing low concentrations of glucose or corn starch | |
| CN100365129C (en) | An improved method for preparing thioadenosylmethionine | |
| US20060270004A1 (en) | Fermentation processes with low concentrations of carbon-and nitrogen-containing nutrients | |
| JPH05244973A (en) | Actinomadura fibrosa sp. nov. nrrl18348 and production of polyether-based antibiotic from this strain | |
| RU2103350C1 (en) | Yeast strain rhodotorula glutinis - a producer of carotinoids | |
| Rao et al. | Effect of tweens on the production of ergot alkaloids by Aspergillus fumigatus | |
| KR0169061B1 (en) | A process for preparing xylitol by controlling fermenting condition | |
| CN116855562A (en) | Method for producing adenosine by fermentation of nucleoside metabolic factor culture medium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |