CN106755225B - Fermentation method of pneumocandin B0 - Google Patents
Fermentation method of pneumocandin B0 Download PDFInfo
- Publication number
- CN106755225B CN106755225B CN201710048503.XA CN201710048503A CN106755225B CN 106755225 B CN106755225 B CN 106755225B CN 201710048503 A CN201710048503 A CN 201710048503A CN 106755225 B CN106755225 B CN 106755225B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- dissolved oxygen
- percent
- controlling
- pneumocandin
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a novel fermentation method of pneumocandin B0, which can reduce the content of pneumocandin C0 in the fermentation process and greatly reduce the cost of a downstream extraction process. The strain used in the method is Glarea lozoyensis, vitamin b5 is added in the whole fermentation process, the pH is strictly controlled, and the content of pneumocandin C0 in the fermentation liquor obtained by the method can be reduced from 6% to 1.5%.
Description
Technical Field
The invention belongs to the technical field of bioengineering, relates to microbial fermentation of antifungal medicines, and particularly relates to a microbial fermentation method of pneumocandin B0.
Background
Pneumocandin B0 is a secondary metabolite synthesized by fermentation of the mold Glarea lozoyensis. The Glaralozoyensis fermentation product has 12 analogues such as A0, B1, B2, C0, D0, E0 and the like besides a target product B0, wherein the structure of C0 is the closest to that of B0, the structural difference between the two is trans-3-hydroxyproline in B0, and trans-4-hydroxyproline in C0.
Pneumocandin B0 Cas: 135575-42-7
Pneumocandin C0 Cas: 144074-96-4
In the prior art, a resin column process is mainly adopted for purifying the pneumocandin B0, and when the column chromatography is applied to industrial production, time is consumed, the use amount of a solvent is large, the environmental pollution is easy to cause, the loss of raw materials is large, and the production cost is greatly improved. C0 is an isomer of pneumocandin B0, differing in structure by a single hydroxyl position; the C0 impurity could not be separated from pneumocandin B0 in reverse phase chromatography, only by normal phase chromatography. The C0 impurity in the fermentation broth is typically 10% and if not effectively controlled, a mixture of pneumocandin B0 and C0 is obtained. The C0 impurity can participate in the subsequent reaction, and seriously influences the quality of the caspofungin acetate.
Disclosure of Invention
In order to solve the problems, the inventor develops a new fermentation method of pneumocandin B0 through exploration, and the method can reduce the content of pneumocandin C0 in the fermentation process and greatly reduce the cost of a downstream extraction process.
The strain used in the process is Glarea lozoyensis, and the fermentation conditions are as follows:
fermentation medium (wt): 3.0% of lactose, 1.0% of threonine, 1.0% of yeast powder, 1.2% of proline and KH2PO40.15%, magnesium sulfate heptahydrate 0.05%, MES buffer salt 1.5%, pH 5.3.
The temperature is 24-26 ℃ in the fermentation process, the initial ventilation volume is 0.5-1.0 VVM, and the tank pressure is 0.04-0.06 MPa. The ventilation and the rotating speed are gradually increased in the fermentation process to ensure that the dissolved oxygen is not less than 20 percent, the rotating speed is maximum 600 r/min, and the ventilation is maximum 1.2 VVM.
After fermentation for 48 hours, vitamin b5 is added, and the addition amount is 20-40 mg/l according to the volume of the culture medium.
After fermentation for 72h (the ventilation volume and the rotating speed are adjusted to the upper limit), the pH control range is adjusted according to the change of the dissolved oxygen volume until the fermentation is finished: when the dissolved oxygen is not less than 45%, controlling the pH value to be 5.0-5.4; controlling the pH value to be 5.4-5.8 when the dissolved oxygen is 35% -45%; controlling the pH value to be 5.8-6.2 when the dissolved oxygen content is 25% -35%; when the dissolved oxygen is less than 25%, controlling the pH value to be 6.2-6.6.
The content of the pneumocandin C0 in the fermentation liquid obtained by fermentation can be reduced from 6 percent to 1.5 percent.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Example 1
Fermentation strain: glarea lozoyensis
Fermentation medium (wt): 3.0% of lactose, 1.0% of threonine, 1.0% of yeast powder, 1.2% of proline and KH2PO40.15%, magnesium sulfate heptahydrate 0.05%, MES buffer salt 1.5%, pH 5.3.
Culturing in 50L fermenter, sterilizing at 121 deg.C for 30 min with 30L culture medium. The amount of the fermentation broth is 1.5L, the culture temperature of the fermentation broth is 25 ℃, the initial aeration rate is 0.9VVM, 200 revolutions and the tank pressure is 0.05Mpa, the aeration rate and the rotation speed are gradually increased in the fermentation process to ensure that the dissolved oxygen is not lower than 20 percent, the rotation speed is 600 revolutions per minute at maximum, and the aeration rate is 1.2VVM at maximum.
After fermentation for 48h, vitamin b5 was added in an amount of 30 mg/l. After fermentation for 72h (at the moment, the ventilation volume and the rotating speed are adjusted to the upper limit), the pH control range is adjusted according to the dissolved oxygen change until the fermentation is finished: when the dissolved oxygen content is not lower than 45%, controlling the pH to be 5.0-5.4, controlling the dissolved oxygen content at 96h to be 35-45%, controlling the pH to be 5.4-5.8, controlling the dissolved oxygen content at 110h to be 25-35%, controlling the pH to be 5.8-6.2, controlling the dissolved oxygen content at 130h to be lower than 25%, controlling the pH to be 6.2-6.6, controlling the dissolved oxygen content at 144h to be 25%, controlling the pH to be 5.8-6.2, controlling the dissolved oxygen content at 168h to be 35%, maintaining the dissolved oxygen content at 35-45%, controlling the pH to be 5.4-5.8, finishing fermentation at 240h, discharging a tank, and determining the fermentation unit of the pneumocandin B0 to be 1048mg/l and the content of the pneumocandin C0 to be 1.5%.
Example 2 (comparative example without addition of vitamin b 5)
Fermentation strain: glarea lozoyensis
Fermentation medium (wt): 3.0% of lactose, 1.0% of threonine, 1.0% of yeast powder, 1.2% of proline and KH2PO40.15%, magnesium sulfate heptahydrate 0.05%, MES buffer salt 1.5%, pH 5.3.
Culturing in 50L fermenter, sterilizing at 121 deg.C for 30 min with 30L culture medium. The amount of the fermentation broth is 1.5L, the culture temperature of the fermentation broth is 25 ℃, the initial aeration rate is 0.9VVM, 200 revolutions and the tank pressure is 0.05Mpa, the aeration rate and the rotation speed are gradually increased in the fermentation process to ensure that the dissolved oxygen is not lower than 20 percent, the rotation speed is 600 revolutions per minute at maximum, and the aeration rate is 1.2VVM at maximum.
After fermentation for 72h (at the moment, the ventilation volume and the rotating speed are adjusted to the upper limit), the pH control range is adjusted according to the dissolved oxygen change until the fermentation is finished: when the dissolved oxygen content is not lower than 45%, controlling the pH to be 5.0-5.4, controlling the dissolved oxygen content at 96h to be 35-45%, controlling the pH to be 5.4-5.8, controlling the dissolved oxygen content at 110h to be 25-35%, controlling the pH to be 5.8-6.2, controlling the dissolved oxygen content at 130h to be lower than 25%, controlling the pH to be 6.2-6.6, controlling the dissolved oxygen content at 144h to be increased back to 25%, controlling the pH to be 5.8-6.2, controlling the dissolved oxygen content at 168h to be increased back to 35%, maintaining the dissolved oxygen content at 35-45%, controlling the pH to be 5.4-5.8, finishing fermentation at 240h, putting a tank, and determining the fermentation unit of the pneumocandin B0 to be 1025mg/l and the content of the pneumocandin C0 to.
Claims (1)
1. A fermentation method of pneumocandin B0, wherein the strain used is Glarea lozoyensis, and the fermentation conditions of the method are as follows:
fermentation medium: 3.0 percent of lactose, 1.0 percent of threonine, 1.0 percent of yeast powder, 1.2 percent of proline and KH2PO4The mass fraction of the MES buffer salt is 0.15 percent, the mass fraction of magnesium sulfate heptahydrate is 0.05 percent, the mass fraction of MES buffer salt is 1.5 percent, and the pH value is 5.3;
the temperature is 25 ℃, the initial ventilation volume is 0.9VVM, the tank pressure is 0.05Mpa in the fermentation process, the ventilation volume and the rotating speed are gradually increased in the fermentation process to ensure that the dissolved oxygen is not less than 20 percent, the rotating speed is 600 r/min at the maximum, and the ventilation volume is 1.2VVM at the maximum;
after fermentation for 48h, vitamin b5 is added, the addition amount is 30mg/l according to the volume of the culture medium,
after fermentation is carried out for 72h, the ventilation volume and the rotating speed are adjusted to the upper limit, the pH control range is adjusted according to the change of the dissolved oxygen volume until the fermentation is finished: when the dissolved oxygen is not less than 45%, controlling the pH value to be 5.0-5.4; when the dissolved oxygen amount is 35-45%, controlling the pH value to be 5.4-5.8; when the dissolved oxygen amount is 25-35%, controlling the pH value to be 5.8-6.2; when the dissolved oxygen is less than 25%, controlling the pH value to be 6.2-6.6;
the fermentation culture period is 10 days, and the fermentation culture period is 10 days later.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710048503.XA CN106755225B (en) | 2017-01-20 | 2017-01-20 | Fermentation method of pneumocandin B0 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710048503.XA CN106755225B (en) | 2017-01-20 | 2017-01-20 | Fermentation method of pneumocandin B0 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106755225A CN106755225A (en) | 2017-05-31 |
| CN106755225B true CN106755225B (en) | 2020-06-26 |
Family
ID=58942145
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710048503.XA Active CN106755225B (en) | 2017-01-20 | 2017-01-20 | Fermentation method of pneumocandin B0 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106755225B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115747283A (en) * | 2021-09-03 | 2023-03-07 | 杭州中美华东制药有限公司 | Method for preparing pneumocandin B0 through fermentation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103087928A (en) * | 2013-01-25 | 2013-05-08 | 杭州华东医药集团生物工程研究所有限公司 | Fungus Glarea lozoyensis and application thereof for controlling microbial metabolite Pneumocandin Kangding B0 |
| CN103289900A (en) * | 2012-02-22 | 2013-09-11 | 上海来益生物药物研究开发中心有限责任公司 | High-yield strain for pneumocandin B0 and application for same |
| CN104145021A (en) * | 2012-01-13 | 2014-11-12 | 中化帝斯曼制药有限公司荷兰公司 | Cyclopeptide fermentation at increased metal ion concentration |
-
2017
- 2017-01-20 CN CN201710048503.XA patent/CN106755225B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104145021A (en) * | 2012-01-13 | 2014-11-12 | 中化帝斯曼制药有限公司荷兰公司 | Cyclopeptide fermentation at increased metal ion concentration |
| CN103289900A (en) * | 2012-02-22 | 2013-09-11 | 上海来益生物药物研究开发中心有限责任公司 | High-yield strain for pneumocandin B0 and application for same |
| CN103087928A (en) * | 2013-01-25 | 2013-05-08 | 杭州华东医药集团生物工程研究所有限公司 | Fungus Glarea lozoyensis and application thereof for controlling microbial metabolite Pneumocandin Kangding B0 |
Non-Patent Citations (3)
| Title |
|---|
| Improvement in the titer of echinocandin-type antibiotics: a;Jan S.Tkacz等;《Journal of Industrial Microbiology》;19930228;第11卷(第2期);第97页左栏第4段、第100页表5及图3和第102页右栏第2段 * |
| Novel proline hydroxylase activities in the pneumocandin-producing;L.Petersen等;《Applied Microbiology and Biotechnology》;20030306;第62卷(第2期);全文 * |
| Pilot-scale process sensitivity studies for the scaleup of a fungal fermentation for the production of pneumocandins;Pollard DJ等;《Biotechnology and Bioengineering》;20020505;第78卷(第3期);第275页图5及第277页表1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106755225A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fernández-Moreno et al. | The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces | |
| Liu et al. | MpigE, a gene involved in pigment biosynthesis in Monascus ruber M7 | |
| Prasad et al. | Hyaluronic acid production is enhanced by the additional co-expression of UDP-glucose pyrophosphorylase in Lactococcus lactis | |
| Udeh et al. | Role of magnesium ions on yeast performance during very high gravity fermentation | |
| CN106755225B (en) | Fermentation method of pneumocandin B0 | |
| WO2009081276A3 (en) | Fermentation processes for cultivating streptococci and purification processes for obtaining cps therefrom | |
| NZ714121A (en) | Fermented malt beverage and production method therefor | |
| CN107141774A (en) | A kind of high transparency polyether-type bimirror face TPU film | |
| Joshi et al. | Exopolysaccharide production by a lactic acid bacteria, Leuconostoc lactis isolated from ethnically fermented beverage | |
| CN108129831A (en) | Smooth TPU film in a kind of food grade polyester type bimirror face and preparation method thereof | |
| Goldbeck et al. | Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum | |
| Matsushika et al. | Evaluation of Saccharomyces cerevisiae GAS1 with respect to its involvement in tolerance to low pH and salt stress | |
| CN104250660A (en) | Antibacterial peptide fermentation culture medium formula | |
| WO2009124966A2 (en) | Conversion of stilbenoids | |
| CN109680032B (en) | Method for synthesizing 7, 21-dihydroxy-20-methylpregna-4-en-3-one by using microorganisms | |
| CN102559813A (en) | Low-impurity acarbose and preparation method thereof | |
| CN106755224B (en) | The fermentation process of Caspofungin fermentation intermediate | |
| CN106868079B (en) | Culture medium for fermenting polymyxin B sulfate and method for producing polymyxin B sulfate through fermentation | |
| EP2182007B1 (en) | Process for preparing human G-CSF | |
| PARK et al. | Doxorubicin productivity improvement by the recombinant Streptomyces peucetius with high-copy regulatory genes cultured in the optimized media composition | |
| CN105671112A (en) | Method of preparing A82846B through fermentation | |
| CN106978409B (en) | Efficient preparation method of alpha-glucosidase | |
| CN108753878A (en) | The fermentation process of micafen sodium intermediate | |
| CN109112151B (en) | Gene cassette for finely regulating and controlling composition ratio of 4-hydroxybutyric acid in copolymer and application thereof | |
| CN104561168A (en) | Fermentation culture medium for fermentation production of lovastatin and method for replenishing water in fermentation process |
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 | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: C2218, c2218 building, Suzhou Xinghu Industrial Park, Jiangsu Province Patentee after: Borui Pharmaceutical (Suzhou) Co., Ltd Address before: 215123 building C27, bio nano Park, 218 Xinghu street, Suzhou Industrial Park, Jiangsu Province Patentee before: XINTAI PHARMACEUTICAL (SUZHOU) Co.,Ltd. |