WO2000005397A1 - Improved process for clavulanic acid production - Google Patents
Improved process for clavulanic acid production Download PDFInfo
- Publication number
- WO2000005397A1 WO2000005397A1 PCT/PT1999/000012 PT9900012W WO0005397A1 WO 2000005397 A1 WO2000005397 A1 WO 2000005397A1 PT 9900012 W PT9900012 W PT 9900012W WO 0005397 A1 WO0005397 A1 WO 0005397A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fermentation
- concentration
- broth
- culture
- clavulanic acid
- Prior art date
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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
Definitions
- This invention is related to important improvements in the clavulanic acid fermentation using strains of Streptomyces clavuligerus, low cost complex media and strategies of easy industrial implementation.
- Clavulanic acid is used in medicine in association with antibiotics that are inactivated by ⁇ - lactamases.
- Clavulanic acid (3-(2-hydroxyethylidene)-7-oxo-4-oxa-l-azabi- cyclo[3.2.0]heptane-2-carboxylic acid) is a molecule that has the structural formula
- This acid has weak antibacterial activity. However, it is a potent inhibitor of ⁇ -lactamase enzymes produced by many strains of Staphylococcus aureus, Escherichia coli, Klebsiella, Proteus, Shigella, Pseudomonas and Haemophilus influenzae.
- ⁇ -Lactamases through the hydrolysis of the ⁇ -lactam ring, inactivate several antibiotics, and make the microorganisms, which produce them, resistant to those antibiotics.
- clavulanic acid As potent inhibitor of ⁇ -lactamases, clavulanic acid is able to avoid this mechanism of resistance, widening the antibacterial activity spectrum of several antibiotics. Clavulanic acid presents good synergetic activity when associated with antibiotics such as amoxycillin, ampicillin, carbenicillin, ticarcillin, benzylpenicillin or cephaloridine, against ⁇ -lactamase-producing organisms.
- clavulanic acid production by Streptomyces clavuligerus for example (a) discontinuous fermentation using complex or chemically defined media (BR Patent 1 508 977, Beecham Group Ltd.); (b) fermentation with automatic control of pH between 6.3 and 6.7 (BR Patent 1 571 888, Glaxo Laboratories Ltd.); (c) fermentation with continuous or semicontinuous feeding of a carbon source (for example maltose or glycerol) (ES Patent 537 157, Antibioticos, S.A.); and (d) fermentation with control of soluble phosphate in the medium, at the beginning and throughout the fermentation (EP Patent 0 811 689, Antibioticos, S.A.).
- a carbon source for example maltose or glycerol
- the present invention concerns a process for clavulanic acid production by a technique of fermentation which includes the aerobic submerged culture using selected and/or culture collection strains of Streptomyces clavuligerus, or mutants thereof. Accordingly, the culture is carried out with continuous or semicontinuous feeding of one or more organic nitrogen complex sources, preferably soybean meal, so as to control the protein concentration in the filtered broth within certain limits during the time course of the fermentation.
- one or more organic nitrogen complex sources preferably soybean meal
- this invention describes new process strategies for clavulanic acid production by cultivating the producing microorganism, as for example Streptomyces clavuligerus ATCC 27064, or mutants thereof, in aerobic submerged culture using low cost complex media, in distinct conditions from those patented or reported to date, leading to important improvements in clavulanic acid fermentation.
- These conditions consist in the continuous or staggered feeding of one or more organic nitrogen complex sources, in order to control the protein concentration in the filtered broth within certain limits during the fermentation, since very high values may inhibit/repress the biosynthesis of the antibiotic, and very low values may be limiting for the desired biosynthesis.
- the organic nitrogen complex sources can be seed protein such as soybean meal, peanut meal, cottonseed meal and linseed meal, fish meal, hydrolysates and filtrates of such proteins, meat extracts and hydrolysates such as peptones, being, preferably, soybean meal.
- the amount of organic nitrogen complex source to be fed in a continuous or semicontinuous mode can be in the daily concentration of 0.1-1.5%, preferably between 0.18 and 1.0%, and/or may be such that the protein concentration in the filtered broth is between 200 and 3500 mg/L, preferably 400- 1500 mg/L throughout the fermentation.
- the initial culture medium can be composed by one or more organic nitrogen complex sources and, additionally, one or more carbon sources.
- concentration of the organic nitrogen complex source may fall preferably between 1.3 and 1.8%.
- the carbon sources may be glycerol and/or carbohydrates like starch, starch hydrolysates, dextrins and maltose. It was observed that the simultaneous use of glycerol and dextrin in the initial culture medium, preferably in the concentration ranges of, respectively, 0.9-1.3% and 1.8-2.2%, improve the production of clavulanic acid.
- one or more carbon sources may be fed continuously or semicontinuously in daily concentrations between 0.18% and 1%, and/or so that the glycerol concentration in the filtered broth is between 0.2 and 12 g/L, and/or the dextrin or maltose concentrations in the filtered broth are in the range 4-22 g/L or 2-12 g/L, respectively, during the time course of the fermentation. It was observed that the simultaneous feeding of glycerol and dextrin or maltose improve the production of clavulanic acid.
- the fermentation can be performed with continuous or semicontinuous partial discharges of fermentation broth on such way as to maintain the volume of fermentation broth between 35 and 65% of the total fermenter capacity.
- the agitator speed can be progressively increased, according to the increase in volume, broth viscosity and dry weight concentration during the fermentation, so as to improve the mixing and the dissolved oxygen levels in the culture.
- the culture can be carried out at a temperature between 26 and 29°C and the pH can be controlled between 6.5 and 6.8 by automatic addition of acid and base, such as a hydrochloric acid solution and a sodium hydroxide solution.
- acid and base such as a hydrochloric acid solution and a sodium hydroxide solution.
- Foam can be controlled by addition of antifoam, as for example, a silicone suspension.
- the fermentation vessel should be a typical tank for aerobic fermentation with agitation and aeration devices.
- the aeration volumetric flow rate per unit of broth volume may be 0.6 to 1.3 vvm.
- These tanks should be supplied with aseptic systems for continuous or semicontinuous feeding of several nutrients in the form of solution and/or suspension, with aseptic systems for continuous or semicontinuous partial discharges of fermentation broth, and possibly with variable agitator speed.
- the samples of culture broth may be vacuum filtered, preferably through filter paper previously dried in an oven at 86°C for 12 h. After the filtered broth has been collected, the filter paper with the mycelium is washed with distilled water and subsequently dried in an oven at 86°C for 24 h. The dry weight concentration of the sample is thus obtained.
- the clavulanic acid, protein, glycerol, dextrin or maltose concentrations can be measured in the filtered broth by spectrophotometric methods or, preferably, by high pressure liquid chromatographic methods, for example using the methods described in (Bird, A.E. et al. 1982, Analyst 107: 1241-1245; Foulstone, M. and Reading, C. 1982, Antimicrob. Agents Chemother. 22: 753-762), (Bradford, M.M. 1976, Anal. Biochem. 72: 248-254), (Bok, S.H. and Demain, A.L. 1977, Anal. Biochem. 81 : 18-20), (Nelson, N. 1944, J. Biol. Chem. 153: 375-380; Somogyi, M. 1952, J. Biol. Chem. 195: 19-23) respectively.
- a spore suspension of Streptomyces clavuligerus ATCC 27064 was prepared from agar slants containing hydrated dextrin 10 g, yeast extract 1 g, meat extract 1 g, bacteriological peptone 2 g, CaC0 3 2 g, agar 20 g, per litre of distilled water. The pH was corrected to 7.1 with 1M NaOH and 1M HC1.
- This spore suspension was used to inoculate various 500 mL conical flasks containing 50 mL of culture medium A (soybean meal 15 g, (87%) glycerol 10 g, hydrated dextrin 10 g, KH 2 PO 1 g, per litre of distilled water).
- culture medium A serum meal 15 g, (87%) glycerol 10 g, hydrated dextrin 10 g, KH 2 PO 1 g, per litre of distilled water.
- the pH was corrected to 7.2 with 1M NaOH and 1M HC1.
- the flasks were sterilized at 121°C for 15 min and incubated at 30°C and 110-140 rpm during 2 days. The content of 5 flasks was then mixed, giving the vegetative inoculum.
- a culture medium B composed by soybean meal 15 g, (87%) glycerol 13 g, hydrated dextrin 20 g, (50%) silicone suspension 1 g, per litre of tap water, was introduced into a 8 litre STR fermenter and sterilized for 20 min at about 120°C.
- the fermenter was inoculated with the vegetative inoculum prepared previously.
- the fermentation was carried out with automatic control of pH at 6.6 ⁇ 0,05 by addition of a 5% (v/v) HCl solution and IM NaOH solution. Foam was controlled by addition of a 50% silicone suspension. The temperature was kept between 26 and 29°C, and the aeration between 0.7 and 1.2 wm.
- the volumetric flow rate was controlled manually according to the result of the daily analysis of protein and glycerol concentrations in the filtered culture broth.
- the volumetric flow rate varied throughout the time course of the fermentation as follows:
- the volumetric flow rate was manually controlled according to the result of the daily analysis of protein, glycerol, and dextrin concentrations in the filtered broth.
- the volumetric flow rate varied in the following way throughout the time course of the fermentation :
- Partial discharges of fermentation broth were carried out (8.3% v/v) at 100 and 123 h, using a peristaltic pump with manual control, according to the increase in volume of culture broth in the fermenter.
- volume of fermentation broth volume of partial discharges of fermentation broth, dry weight concentration, clavulanic acid titre and protein, glycerol and dextrin concentrations in the filtered broth along the fermentation process were obtained:
- the culture broth At 143 h the culture broth attained a volume of 4.15 L, a clavulanic acid titre of 1374 ⁇ g/mL and a dry weight concentration of 18.7 g/L. In other words, 1839 ⁇ g of clavulanic acid and 25.0 mg of dry weight per mL of culture medium B were obtained. Considering the partial discharges, a total of 2099 ⁇ g of clavulanic acid and 28.8 mg of dry weight per mL of culture medium B were obtained.
- Culture medium B was replaced by culture medium E composed by soybean meal 15 g and (50%) silicone suspension 2 g, per litre of tap water.
- the volumetric flow rate was manually controlled according to the result of the daily analysis of the protein, glycerol and maltose concentrations in the filtered broth.
- the volumetric flow rate varied throughout the fermentation time as follows: TABLE VII
- the agitator speed and the dissolved oxygen concentration varied during the fermentation as follows:
- Partial discharges of fermentation broth were performed through a peristaltic pump of manual control, according to the increase in volume of culture broth in the fermenter.
- partial discharges of fermentation broth of 8.3%, 7.2%, 10.3%. 10.5% and 7.3% (v/v) at 96, 120, 144, 168 and 192 h, respectively, were carried out.
- volume of fermentation broth volume of partial discharges of fermentation broth, dry weight concentration, clavulanic acid titre and protein, glycerol and maltose concentrations in the filtered broth throughout the time course of the fermentation were obtained:
- the culture broth attained a volume of 4.02 L, a clavulanic acid titre of 1607 ⁇ g/mL and a dry weight concentration of 16.6 g/L.
- 2084 ⁇ g of clavulanic acid and 21.5 mg of dry weight per mL of culture medium E were obtained.
- a total of 2790 ⁇ g of clavulanic acid and 30.6 mg of dry weight per mL of culture medium E were attained.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
MXPA01000653A MXPA01000653A (en) | 1998-07-20 | 1999-07-19 | Improved process for clavulanic acid production. |
AU48088/99A AU4808899A (en) | 1998-07-20 | 1999-07-19 | Improved process for clavulanic acid production |
CA002337074A CA2337074A1 (en) | 1998-07-20 | 1999-07-19 | Improved process for clavulanic acid production |
EP99931644A EP1098989A1 (en) | 1998-07-20 | 1999-07-19 | Improved process for clavulanic acid production |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PT102181 | 1998-07-20 | ||
PT10218198A PT102181A (en) | 1998-07-20 | 1998-07-20 | IMPROVED PROCESS FOR THE PRODUCTION OF CLAVULANIC ACID |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000005397A1 true WO2000005397A1 (en) | 2000-02-03 |
Family
ID=20085781
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/PT1999/000012 WO2000005397A1 (en) | 1998-07-20 | 1999-07-19 | Improved process for clavulanic acid production |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP1098989A1 (en) |
CN (1) | CN1310766A (en) |
AU (1) | AU4808899A (en) |
CA (1) | CA2337074A1 (en) |
MX (1) | MXPA01000653A (en) |
PT (1) | PT102181A (en) |
TR (1) | TR200100167T2 (en) |
WO (1) | WO2000005397A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102277310A (en) * | 2010-06-11 | 2011-12-14 | 中国科学院上海生命科学研究院 | Vector-host system for expressing antibiotic gene clusters and application thereof |
EP2589663A1 (en) | 2011-11-04 | 2013-05-08 | LEK Pharmaceuticals d.d. | Process for production of clavulanic acid |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008132531A1 (en) * | 2007-04-27 | 2008-11-06 | Council Of Scientific And Industrial Research | Process for the preparation of clavulanic acid employing streptomyces clavuligerus mtcc 1142 in a solid state fermentation |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2604697A1 (en) * | 1975-02-07 | 1976-08-19 | Glaxo Lab Ltd | ANTIBIOTICS |
US4525353A (en) * | 1974-04-20 | 1985-06-25 | Beecham Group P.L.C. | Antibiotics |
EP0811689A1 (en) * | 1995-11-23 | 1997-12-10 | Antibioticos, S.A. | Process for the production of clavulanic acid and/or salts thereof |
-
1998
- 1998-07-20 PT PT10218198A patent/PT102181A/en not_active Application Discontinuation
-
1999
- 1999-07-19 CN CN 99808891 patent/CN1310766A/en active Pending
- 1999-07-19 EP EP99931644A patent/EP1098989A1/en not_active Withdrawn
- 1999-07-19 MX MXPA01000653A patent/MXPA01000653A/en unknown
- 1999-07-19 CA CA002337074A patent/CA2337074A1/en not_active Abandoned
- 1999-07-19 AU AU48088/99A patent/AU4808899A/en not_active Abandoned
- 1999-07-19 TR TR2001/00167T patent/TR200100167T2/en unknown
- 1999-07-19 WO PCT/PT1999/000012 patent/WO2000005397A1/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4525353A (en) * | 1974-04-20 | 1985-06-25 | Beecham Group P.L.C. | Antibiotics |
DE2604697A1 (en) * | 1975-02-07 | 1976-08-19 | Glaxo Lab Ltd | ANTIBIOTICS |
EP0811689A1 (en) * | 1995-11-23 | 1997-12-10 | Antibioticos, S.A. | Process for the production of clavulanic acid and/or salts thereof |
Non-Patent Citations (4)
Title |
---|
APPL. MICROBIOL. BIOTECHNOL. (1996), 45(1-2), 41-6 * |
CHEMICAL ABSTRACTS, vol. 122, no. 17, 24 April 1995, Columbus, Ohio, US; abstract no. 206718, PARADKAR, ASHISH S. ET AL: "Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces-- clavuligerus" XP002118367 * |
CHEMICAL ABSTRACTS, vol. 124, no. 25, 17 June 1996, Columbus, Ohio, US; abstract no. 340966, MAYER, A. F. ET AL: "Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations" XP002118366 * |
J. BACTERIOL. (1995), 177(5), 1307-14 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102277310A (en) * | 2010-06-11 | 2011-12-14 | 中国科学院上海生命科学研究院 | Vector-host system for expressing antibiotic gene clusters and application thereof |
EP2589663A1 (en) | 2011-11-04 | 2013-05-08 | LEK Pharmaceuticals d.d. | Process for production of clavulanic acid |
WO2013064687A2 (en) | 2011-11-04 | 2013-05-10 | Lek Pharmaceuticals D.D. | Process for production of clavulanic acid |
Also Published As
Publication number | Publication date |
---|---|
MXPA01000653A (en) | 2002-04-08 |
PT102181A (en) | 2000-01-31 |
TR200100167T2 (en) | 2001-05-21 |
CN1310766A (en) | 2001-08-29 |
CA2337074A1 (en) | 2000-02-03 |
AU4808899A (en) | 2000-02-14 |
EP1098989A1 (en) | 2001-05-16 |
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