WO2000018947A1 - Fermentation of clavulanic acid at a controlled level of ammonia - Google Patents
Fermentation of clavulanic acid at a controlled level of ammonia Download PDFInfo
- Publication number
- WO2000018947A1 WO2000018947A1 PCT/EP1999/007390 EP9907390W WO0018947A1 WO 2000018947 A1 WO2000018947 A1 WO 2000018947A1 EP 9907390 W EP9907390 W EP 9907390W WO 0018947 A1 WO0018947 A1 WO 0018947A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ammonium
- clavulanic acid
- concentration
- fermentation
- anyone
- Prior art date
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Classifications
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- 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
Definitions
- the present invention relates to the field of the fermentative production of clavulanic acid by fermentation of a clavulanic acid producing microorganism.
- Streptomycetes are known to produce a large variety of secondary metabolites which can be applied in the manufacturing of pharmaceuticals.
- these pharmaceuticals belong to the classes produced by the Streptomycetes and are for instance polyketides, macrolides, anthracyclines, tetracyclins, lipopeptides and ⁇ -lactams, see further Strohl ( 1 997).
- One particular example is the production of the ⁇ -lactamase inhibitor clavulanic acid, a ⁇ -lactam compound which is produced by various microbial strains belonging to the genus of Streptomycetes such as S. clavuligerus ATCC 27064, S. jumonjinensis (GB patent 1 5631 03), S. katsurahamanus IFO 1 371 6 FERM 3944 (JP patent 83009679B) and Streptomyces sp. P6621 FERM 2804 (JP patent application 551 62993A).
- Such regulations may comprise the carbon catabolite, ammonium or phosphate repression or any other kind of metabolite that represses the synthesis of the secondary metabolite of interest.
- Carbon catabolites and ammonium repress the production of cephalosporins in S. clavuligerus (Aharonowitz and Demain 1 978 and 1 979)
- nitrogen catabolites regulate the production of spiramycin in S. ambofaciens (Untrau 1 994)
- phosphate, ammonium and glutamate repress the production of clavulanic acid in S. clavuligerus (Romero et al. 1 984) .
- Various types of regulations involved in cephamycin production with various Streptomycetes are described in Omstead et al ( 1 985). High ammonium concentrations were found to repress streptonigrin biosynthesis in S. flocculus (Wallace et al. 1 990).
- a similar negative influence of ammonia on the cefaiosporin production by Streptomyces i. ⁇ o clavuligerus was already described by Aharonowitz and Demain ( 1 979) . Furthermore, as S.
- clavuligerus is urease positive, which urease is repressed by NH 4 CI (see page 2478 of Bascaran et al ( 1 989)) and ureum has been produced during the clavulanic acid production (Elson, 1 993), the production of clavulanic acid by S. clavuligerus is described to be i. 15 especially high when the concentration of ammonium is kept low (WO 96/1 8743).
- the concentration of ammonium is regulated by
- the pH during this ⁇ o fermentation has been maintained between 6.5 and 7.5 and preferably the fermentation is carried out in a fed batch, continuous or semi-continuous mode.
- the clavulanic acid production is largely improved compared to a fermentation process where the ammonium concentration is kept low ( ⁇ 50 mg/l).
- nutrients can be fed to an industrial fermentor in order to provide the optimal physiological conditions with the i 25 aim to maximize output from a given fermentation process.
- the residual ammonium concentration in a fermentation broth can be controlled at the desired concentration range by adding an ammonium source continuously or discontinously (intermittently) to the fermentor.
- ammonium, ammonium salts like ammonium chloride, ammonium nitrate, ammonium phosphate and ammonium carbonate or urea as an ammonium precursor can be added as an alkaline titrant, eventually alternating with sodium hydroxide in order to prevent excess ammonium dosage.
- the ammonium concentration can be reduced for instance by manipulation of the temperature or pH, which leads to an increased growth rate and an i. 5 increased ammonium consumption rate.
- the ammonium concentration can be controlled by adjusting the flow of the nitrogen source or by manipulation of the alkaline titrant and/or the pH.
- the ammonium concentration is maintained equal to or higher than 50 mg/l.
- the i. ⁇ o ammonium concentration must be low enough for reducing the repression of secondary metabolism and avoiding toxicity of ammonium.
- the ammonium concentration could be maintained below 2500 mg/l, preferably 1 000 mg/l, for instance 500 mg/l.
- the microorganism used for production of clavulanic acid may be i. 15 any Streptomycete, optionally improved for growth and/or clavulanic acid production by means of classical strain improvement or by recombinant DNA techniques, for instance S. clavuligerus or S. jumonjinensis.
- the production of clavulanic acid is carried out by fermentation of a Streptomycete on a suitable medium comprising various carbon and i. 20 energy sources like sugars such as glucose, fructose, sucrose, maltose, lactose, or polysaccharides like starch, maltodextrines and inuline or other fructose polymers, proteins such as flours from nuts, vegetables, seeds, cereals, grasses such as those useful in fermentation industry; soybean flour, lineseed flour, peanut flour, potato flour, sunflower, pea- or i.
- sugars such as glucose, fructose, sucrose, maltose, lactose, or polysaccharides like starch, maltodextrines and inuline or other fructose polymers
- proteins such as flours from nuts, vegetables, seeds, cereals, grasses such as those useful in fermentation industry
- soybean flour, lineseed flour peanut flour, potato flour, sunflower, pea- or i.
- nitrogen sources such as ammonium salts, ammonium, urea, nitrate, asparagine, aspartate, glutamate, lysine and from complex sources such as protein products derived from microbial source (yeast extract) or plants (corn steep liquor, soybean flour, cotton seed flour etc.) and animals (peptones) .
- Phosphor is either supplied in the form of an inorganic salt, or as a phosphor protein like casein, or bound to inositol in the form of phytate as present in many plant protein sources like soybean flour or bound in nucleotides as present in yeast extracts.
- inorganic anions such as sulphates, phosphates, chlorides, borates, molybdate, iodate and inorganic cations such as potassium, sodium, zinc, manganese, magnesium, iron, copper, cobalt, nickel may be added to the fermentation medium.
- a fermentation is started by inoculating from a preculture or inoculum fermentation at a volume of 1 to 50% of the main fermentation medium, particularly from 5 to 20%.
- the process may last from 24 to 400 hours and especially from 48 to 1 68 hours.
- the temperature will be kept between 20 and 40 °C, in particular between 25 and 35 °C, and even more particular between 25 and 30 °C.
- the pH should be maintained preferably at pH 6 to 8, more preferably between pH 6.5 and 7.5 by means of titration with an alkaline substance such as ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide, or an organic base like lysine, arginine and histidine or a combination of these alkaline substances and an acid substance, such as the inorganic acids like sulphuric acid, hydrochloric acid, phosphoric acid and nitric acid.
- an organic acid may be used such as gl ⁇ tamate, citrate, gluconate or acetate.
- the dissolved oxygen concentration is preferably controlled in the optimal range for the process by varying one or more of the following parameters: the oxygen concentration in the inlet gas, application of overpressure, modification of stirrer speed or airflow.
- the range may vary between 0 and 1 00% of air saturation.
- Carbon dioxide should be kept at non-toxic concentrations by increasing the airflow through the fermentor so that the carbon dioxide concentration in the outlet-gas is less than 5%, more particular less than 2.5%. i. 5
- the fermentation can be carried out in a batch, a fed batch, or a continuous fermentation process mode.
- the process may be carried out by controlling various non-growth limiting nutrients in their optimal concentrations.
- these growth-non-limiting nutrients i. ⁇ o may contain any relevant source of carbon, nitrogen, phosphor or sulphur or may contain oxygen.
- the recovery of the impure clavulanic acid solution as formed by the fermentative process of the present invention as well as the subsequent conversion thereof into a pharmaceutically acceptable salt i. 15 by methods known in the art do form an aspect of the present invention.
- One of the most advantageous procedures is the conversion of the impure clavulanic acid into an amino salt thereof by adding the corresponding amino salt forming compound as for instance N,N,N',N'- tetramethylethylenediamine, 1 ,3-bis(di-methylamino)-2-propanol, t- i.
- Streptomyces clavuligerus ATCC27064 was improved for clavulanic acid production by means of several rounds of classic mutation (UV, nitroso guanidine (NTG)) and selection in shake flask cultures i. 25 whereby clavulanic acid production was tested by imidazole methods as known in the art.
- the strain was conserved as vegetative mycelium grown for 48 hours in Tryptone Soytone Broth-medium (TSB-medium) at 28 °C in a shaker incubator shaken at 280 rpm and stored frozen at -80 °C. i.
- the inoculum fermentor After 72 hours of cultivation at 27 ° C, 1 00 ml of the culture is transferred to the inoculum fermentor with 70 I of steam-sterilized i. 5 medium at pH 7 containing glycerol (20-50 g/l), soybean flour (20-40 g/l), casein hydrolysate ( 1 0-50 g/l), mono potassium phosphate (2-5 g/l), a suitable trace element cocktail and synthetic antifoam ( 1 g/l).
- the inoculum fermentation medium was again grown for 72 hours at 26-30 °C keeping the dissolved oxygen concentration above 25% of air i. ⁇ o saturation by increasing airflow, agitation and backpressure if required.
- the main fermentation is inoculated by transferring 9 I of the inoculum broth to the main fermentor by pressurizing the inoculum fermentor containing 1 50 I medium sterilized by steam prior to inoculation.
- the medium contained glycerol (50-100 g/l), soybean flour (5- i. 15 20 g/l), casein hydrolysate ( 1 0-50 g/l), mono potassium phosphate (0.5-2 g/l), a suitable trace element cocktail and synthetic antifoam (0.2 - 2 g/l).
- the pH was maintained at 7 + /- 0.25 by titration with NaOH and sulphuric acid while the temperature was kept between 26 and 29 °C by pumping cooling water through the jackets of the fermentor.
- the i. 20 dissolved oxygen concentration was kept above 25% of air saturation by increasing airflow, backpressure and stirrer speed if required.
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99947466A EP1117820A1 (en) | 1998-09-29 | 1999-09-23 | Fermentation of clavulanic acid at a controlled level of ammonia |
CA002342181A CA2342181A1 (en) | 1998-09-29 | 1999-09-23 | Fermentation of clavulanic acid at a controlled level of ammonia |
AU60896/99A AU6089699A (en) | 1998-09-29 | 1999-09-23 | Fermentation of clavulanic acid at a controlled level of ammonia |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98203314 | 1998-09-29 | ||
EP98203314.4 | 1998-09-29 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000018947A1 true WO2000018947A1 (en) | 2000-04-06 |
Family
ID=8234177
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1999/007390 WO2000018947A1 (en) | 1998-09-29 | 1999-09-23 | Fermentation of clavulanic acid at a controlled level of ammonia |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP1117820A1 (en) |
CN (1) | CN1198940C (en) |
AU (1) | AU6089699A (en) |
CA (1) | CA2342181A1 (en) |
TR (1) | TR200100873T2 (en) |
WO (1) | WO2000018947A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102933717A (en) * | 2007-04-27 | 2013-02-13 | 科学与工业研究委员会 | Process for preparation of clavulanic acid employing streptomyces clavuligerus mtcc 1142 in solid state fermentation |
US9708577B2 (en) | 2013-10-02 | 2017-07-18 | Ajinomoto Co., Inc. | Apparatus for controlling ammonia and a method for controlling ammonia |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0026044A1 (en) * | 1979-08-24 | 1981-04-01 | Beecham Group Plc | Amine salt of clavulanic acid, its preparation and use |
WO1996018743A1 (en) * | 1994-12-10 | 1996-06-20 | Smithkline Beecham Plc | Process for the fermentative preparation of clavam derivatives whereby the levels of ammonia and urea in the fermentation medium are kept low |
WO1997039137A1 (en) * | 1996-04-12 | 1997-10-23 | Lek Pharmaceutical & Chemical Co. Dd | Process for the preparation of clavulanic acid |
-
1999
- 1999-09-23 WO PCT/EP1999/007390 patent/WO2000018947A1/en not_active Application Discontinuation
- 1999-09-23 TR TR2001/00873T patent/TR200100873T2/en unknown
- 1999-09-23 CA CA002342181A patent/CA2342181A1/en not_active Abandoned
- 1999-09-23 AU AU60896/99A patent/AU6089699A/en not_active Abandoned
- 1999-09-23 EP EP99947466A patent/EP1117820A1/en not_active Withdrawn
- 1999-09-23 CN CN 99811511 patent/CN1198940C/en not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0026044A1 (en) * | 1979-08-24 | 1981-04-01 | Beecham Group Plc | Amine salt of clavulanic acid, its preparation and use |
WO1996018743A1 (en) * | 1994-12-10 | 1996-06-20 | Smithkline Beecham Plc | Process for the fermentative preparation of clavam derivatives whereby the levels of ammonia and urea in the fermentation medium are kept low |
WO1997039137A1 (en) * | 1996-04-12 | 1997-10-23 | Lek Pharmaceutical & Chemical Co. Dd | Process for the preparation of clavulanic acid |
Non-Patent Citations (1)
Title |
---|
BASCARAN V ET AL: "Isolation and characterization of nitrogen-deregulated mutants of Streptomyces clavuligerus", JOURNAL OF GENERAL MICROBIOLOGY, vol. 135, no. 9, September 1989 (1989-09-01), pages 2475 - 2482, XP002086396 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102933717A (en) * | 2007-04-27 | 2013-02-13 | 科学与工业研究委员会 | Process for preparation of clavulanic acid employing streptomyces clavuligerus mtcc 1142 in solid state fermentation |
US9708577B2 (en) | 2013-10-02 | 2017-07-18 | Ajinomoto Co., Inc. | Apparatus for controlling ammonia and a method for controlling ammonia |
RU2628696C1 (en) * | 2013-10-02 | 2017-08-21 | Адзиномото Ко., Инк. | A method for producing basic amino acid (variants) |
Also Published As
Publication number | Publication date |
---|---|
TR200100873T2 (en) | 2001-07-23 |
CN1320166A (en) | 2001-10-31 |
CN1198940C (en) | 2005-04-27 |
AU6089699A (en) | 2000-04-17 |
EP1117820A1 (en) | 2001-07-25 |
CA2342181A1 (en) | 2000-04-06 |
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