CA2337074A1 - Improved process for clavulanic acid production - Google Patents
Improved process for clavulanic acid production Download PDFInfo
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- CA2337074A1 CA2337074A1 CA002337074A CA2337074A CA2337074A1 CA 2337074 A1 CA2337074 A1 CA 2337074A1 CA 002337074 A CA002337074 A CA 002337074A CA 2337074 A CA2337074 A CA 2337074A CA 2337074 A1 CA2337074 A1 CA 2337074A1
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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
Abstract
The present invention concerns a process for clavulanic acid production by aerobic fermentation 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. The described conditions led to significant improvements in the clavulanic acid fermentation.
Description
WO 00/05397 PCT/PT99/OUOI2 _ DESCRIPTION
"IMPROVED PROCESS FOR
CLAVULANIC ACID PRODUCTION"
Scope of the Invention 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 (3-lactamases.
Background to the Invention Clavulanic acid (3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabi-cyclo[3.2.0]heptane-2-carboxylic acid) is a molecule that has the structural formula CHzC~H
H I
H~
'C-C~O\C%C~
H'~ I I I H
,C-N C.
CC!OH
H
This acid has weak antibacterial activity. However, it is a potent inhibitor of ~3-lactamase enzymes produced by many strains of Staphylococcus aureus, Escherichia coli, Klebsiella, Proteus, Shigella, Pseudomonas and Haemophilus influenzae. ~3-Lactamases, through the hydrolysis of the (3-lactam ring, inactivate several antibiotics, and make the microorganisms, which produce them, resistant to those antibiotics.
As potent inhibitor of ~i-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 (3-lactamase-producing organisms.
There are several microorganisms which produce clavulanic acid, namely Streptomyces clavuligerus, Streptomyces jumonjinensis (ES Patent 543 854) and Streptomyces katsurahamanus (JP Patent 53-104796, Takeda Chemical Industries, Ltd.). There are various processes described for 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.).
In discontinuous fermentations of Streptomyces clavuligerus using soluble media, it is generally observed that the time course of clavulanic acid titre is closely related to the dry weight concentration until a maximum value is reached, followed by a decay of both. Decay of dry weight concentration is ascribed to sporulation and/or mycelium lysis. Decay of clavulanic acid titre can be due to its degradation rate being higher than its production rate.
WO 00!05397 PCT/PT99/00012 _ Description of the Invention 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.
The conditions described in this invention successfully led to the following important improvements in clavulanic acid fermentation: (a) a significant increase in the clavulanic acid production; (b) the prevention of mycelium lysis throughout the fermentation; (c) a continuous increase in the clavulanic acid titre and in the dry weight concentration throughout the fermentation (without partial discharges of fermentation broth); and/or (d) a continuous increase in the clavulanic acid titre, maintaining the dry weight concentration approximately constant from a certain point in time (with partial discharges of fermentation broth).
Therefore, 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 WO 00/05397 PCT/PT99/00012 _ 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 1500 mg/L throughout the fermentation.
According to this process, the initial culture medium can be composed by one or more organic nitrogen complex sources and, additionally, one or more carbon sources. The 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.
Additionally, 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 WO 00/05397 PCT/PT99/00012 _ -$-semicontinuous partial discharges of fermentation broth on such way as to maintain the volume of fermentation broth between 3$ and 6$% 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. 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.
To carry out the analysis, 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 summary of the results obtained is presented through the various examples that follow.
EXAMPLE I
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, CaC03 2 g, agar 20 g, per litre of distilled water. The pH was corrected to 7.1 with 1 M NaOH and 1 M HCI.
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, KH2P04 1 g, per litre of distilled water). The pH was corrected to 7.2 with 1 M NaOH and 1 M HCI. 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.
Separately, 3.1 L of 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 _7_ 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 6.60,05 by addition of a 5% (v/v) HCl solution and 1M NaOH solution. Foam was controlled by addition of a SO% silicone suspension. The temperature was kept between 26 and 29°C, and the aeration between 0.7 and 1.2 vvm.
At 24 h the staggered feeding of a culture medium C composed by soybean meal 65 g and (87%) glycerol 100 g, per litre of tap water, was started, using a peristaltic pump of variable rate. 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. Thus, the volumetric flow rate varied throughout the time course of the fermentation as follows:
TABLEI
Time Flow h mL/h Since the volume, broth viscosity and dry weight concentration increased throughout the course of the fermentation, the agitator speed was manually increased, on such way as to improve the mixing and the dissolved oxygen levels in the culture. Thus, the agitator speed and the dissolved oxygen concentration varied in the following way during the fermentation:
WO 00!05397 PCT/PT99/Off012 _ _$-TABLE II
Time Agitator Speed Dissolved Oxygen (h m 0-24 500 100.0-75.6 24-56 600 89.6-52.5 56-72 700 56.8-48.5 72-79 800 51.3-49.9 79-96 1000 48.5-29.7 96-1 O 1100 41.7-34.3 1 O 1-1171200 36.9-39.2 117-166 1300 45.9-21.6 The following values of volume of fermentation broth, dry weight concentration, clavulanic acid titre and protein, glycerol and dextrin concentrations in the filtered broth, throughout the time course of the fermentation, were obtained:
TABLE III
Time Volume ClavulanicDry WeightProteinGlycerolDextrin (h) (L) Acid (g/L) (mg/L) (g/L) (g/L) /mL
0 3.34 0 5.3 3064 10.9 21.9 24 3.34 90 7.0 860 9.0 19.3 48 3.50 441 9.6 511 8.6 13.4 72 3.67 698 11.2 494 7.0 8.4 96 3.83 857 12.2 674 3.9 7.7 120 4.20 1021 13.8 854 4.4 7.4 144 4.65 1096 14.7 911 4.5 5.9 166 4.92 1224 15.6 1154 2.8 7.2 Sporulation was observed under the microscope throughout the fermentation, but mycelium lysis was not detected.
At 166 h the culture broth attained a volume of 4.92 L, a clavulanic acid titre of 1224 ~g/mL and a dry weight concentration of 15.6 g/L. In other words, 1943 ~,g of clavulanic acid and 24.8 mg of dry weight per mL of culture medium B were obtained.
The fermentation was carried out as described in example 1, except for the changes described next.
The staggered feeding of a culture medium D composed by soybean meal 65 g, (87%) glycerol 56.3 g, hydrated dextrin 86.6 g and (50%) silicone suspension 2 g, per litre of tap water, was carried out from 24 h fermentation time until the end of the fermentation. 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. Thus, the volumetric flow rate varied in the following way throughout the time course of the fermentation TABLE IV
Time Flow h mL/h The agitator speed and the dissolved oxygen concentration varied throughout the fermentation time as follows:
TABLE V
Time Agitator Speed Dissolved Oxygen h m 0-24 S00 99.4-71.9 24-5 S 600 7 8.9-54.0 55-75 700 66.6-53.2 75-107 800 59.0-44.4 107-131 900 51.4-21.0 131-143 1000 29.2-3 5.6 Partial discharges of fermentation broth were carned 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.
The following values of 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:
TABLE VI
Time Volume Partial ClavulanicDry WeightProteinGlycerolDextrin (h) (L) DischargeAcid (g/L) (mg/L) (g/L) (g/L) Volume (~,g/mL) L
0 3.24 - 0 5.5 1673 11.4 20.8 24 3.26 - 91 6.8 718 9.4 18.3 48 3.56 - 426 10.6 634 8.2 19.0 75 3.88 - 801 14.2 318 7.8 16.1 100 4.20 0.35 1035 16.2 464 4.2 17.5 123 4.21 0.35 1264 17.3 674 2.2 24.6 143 4.15 - 1374 18.7 647 0.6 22.8 Sporulation throughout the fermentation, and the beginning of mycelium lysis at the end of the fermentation (at 143 h) were observed under the microscope.
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 elavulanic 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.
The fermentation was carried out according to example 1, except for the changes described next.
Culture medium B was replaced by culture medium E composed by soybean meal 1 S g and (50%) silicone suspension 2 g, per litre of tap water.
The staggered feeding of a culture medium F composed by soybean meal 65 g, (87%) glycerol 70 g and monohydrated maltose 65 g, per litre of tap water, took place from 0 h of fermentation time. 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. Thus, the volumetric flow rate varied throughout the fermentation time as follows:
WO 00/05397 PCT/PT99/OObl2 TABLE VII
Time Flow h mL/h The agitator speed and the dissolved oxygen concentration varied during the fermentation as follows:
TABLE VIII
Time Agitator Speed Dissolved Oxygen h m 0-24 500 98.6-68.9 24-48 600 74.4-79.9 48-72 700 82.0-73.1 72-146 800 80.6-51.7 146-216 900 54.4-37.9 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. Thus, 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.
The following values of 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 WO 00/05397 PCT/PT99/Oti012 _ throughout the time course of the fermentation were obtained:
TABLE IX
Time Volume Partial ClavulanicDry WeightProteinGlycerolMaltose (h) (L) DischargeAcid (g/L) (mg/L) (g/L) (g/L) Volume (~tg/mL) L
0 3.14 - 0 5.4 3137 0.3 2.5 24 3.32 - 123 7.0 962 0.7 2.7 48 3.62 - 496 10.2 742 2.0 3.8 72 3.93 - 759 11.9 625 2.3 4.6 96 4.20 0.3 5 904 13 .2 770 1.9 5.3 120 4.16 0.30 1074 13.9 862 1.2 4.7 144 4.35 0.45 1164 15.7 850 3.6 7.8 168 4.28 0.45 1295 16.1 853 5.1 8.2 192 4.10 0.30 1475 16.3 810 6.0 7.9 216 4.02 - 1607 16.6 936 6.4 8.2 Sporulation was observed under the microscope throughout the fermentation, but no mycelium lysis was detected.
At 216 h 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. In other words, 2084 ~,g of clavulanic acid and 21.5 mg of dry weight per mL of culture medium E were obtained. Taking into consideration the partial discharges, a total of 2790 ~g of clavulanic acid and 30.6 mg of dry weight per mL of culture medium E were attained.
"IMPROVED PROCESS FOR
CLAVULANIC ACID PRODUCTION"
Scope of the Invention 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 (3-lactamases.
Background to the Invention Clavulanic acid (3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabi-cyclo[3.2.0]heptane-2-carboxylic acid) is a molecule that has the structural formula CHzC~H
H I
H~
'C-C~O\C%C~
H'~ I I I H
,C-N C.
CC!OH
H
This acid has weak antibacterial activity. However, it is a potent inhibitor of ~3-lactamase enzymes produced by many strains of Staphylococcus aureus, Escherichia coli, Klebsiella, Proteus, Shigella, Pseudomonas and Haemophilus influenzae. ~3-Lactamases, through the hydrolysis of the (3-lactam ring, inactivate several antibiotics, and make the microorganisms, which produce them, resistant to those antibiotics.
As potent inhibitor of ~i-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 (3-lactamase-producing organisms.
There are several microorganisms which produce clavulanic acid, namely Streptomyces clavuligerus, Streptomyces jumonjinensis (ES Patent 543 854) and Streptomyces katsurahamanus (JP Patent 53-104796, Takeda Chemical Industries, Ltd.). There are various processes described for 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.).
In discontinuous fermentations of Streptomyces clavuligerus using soluble media, it is generally observed that the time course of clavulanic acid titre is closely related to the dry weight concentration until a maximum value is reached, followed by a decay of both. Decay of dry weight concentration is ascribed to sporulation and/or mycelium lysis. Decay of clavulanic acid titre can be due to its degradation rate being higher than its production rate.
WO 00!05397 PCT/PT99/00012 _ Description of the Invention 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.
The conditions described in this invention successfully led to the following important improvements in clavulanic acid fermentation: (a) a significant increase in the clavulanic acid production; (b) the prevention of mycelium lysis throughout the fermentation; (c) a continuous increase in the clavulanic acid titre and in the dry weight concentration throughout the fermentation (without partial discharges of fermentation broth); and/or (d) a continuous increase in the clavulanic acid titre, maintaining the dry weight concentration approximately constant from a certain point in time (with partial discharges of fermentation broth).
Therefore, 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 WO 00/05397 PCT/PT99/00012 _ 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 1500 mg/L throughout the fermentation.
According to this process, the initial culture medium can be composed by one or more organic nitrogen complex sources and, additionally, one or more carbon sources. The 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.
Additionally, 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 WO 00/05397 PCT/PT99/00012 _ -$-semicontinuous partial discharges of fermentation broth on such way as to maintain the volume of fermentation broth between 3$ and 6$% 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. 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.
To carry out the analysis, 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 summary of the results obtained is presented through the various examples that follow.
EXAMPLE I
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, CaC03 2 g, agar 20 g, per litre of distilled water. The pH was corrected to 7.1 with 1 M NaOH and 1 M HCI.
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, KH2P04 1 g, per litre of distilled water). The pH was corrected to 7.2 with 1 M NaOH and 1 M HCI. 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.
Separately, 3.1 L of 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 _7_ 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 6.60,05 by addition of a 5% (v/v) HCl solution and 1M NaOH solution. Foam was controlled by addition of a SO% silicone suspension. The temperature was kept between 26 and 29°C, and the aeration between 0.7 and 1.2 vvm.
At 24 h the staggered feeding of a culture medium C composed by soybean meal 65 g and (87%) glycerol 100 g, per litre of tap water, was started, using a peristaltic pump of variable rate. 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. Thus, the volumetric flow rate varied throughout the time course of the fermentation as follows:
TABLEI
Time Flow h mL/h Since the volume, broth viscosity and dry weight concentration increased throughout the course of the fermentation, the agitator speed was manually increased, on such way as to improve the mixing and the dissolved oxygen levels in the culture. Thus, the agitator speed and the dissolved oxygen concentration varied in the following way during the fermentation:
WO 00!05397 PCT/PT99/Off012 _ _$-TABLE II
Time Agitator Speed Dissolved Oxygen (h m 0-24 500 100.0-75.6 24-56 600 89.6-52.5 56-72 700 56.8-48.5 72-79 800 51.3-49.9 79-96 1000 48.5-29.7 96-1 O 1100 41.7-34.3 1 O 1-1171200 36.9-39.2 117-166 1300 45.9-21.6 The following values of volume of fermentation broth, dry weight concentration, clavulanic acid titre and protein, glycerol and dextrin concentrations in the filtered broth, throughout the time course of the fermentation, were obtained:
TABLE III
Time Volume ClavulanicDry WeightProteinGlycerolDextrin (h) (L) Acid (g/L) (mg/L) (g/L) (g/L) /mL
0 3.34 0 5.3 3064 10.9 21.9 24 3.34 90 7.0 860 9.0 19.3 48 3.50 441 9.6 511 8.6 13.4 72 3.67 698 11.2 494 7.0 8.4 96 3.83 857 12.2 674 3.9 7.7 120 4.20 1021 13.8 854 4.4 7.4 144 4.65 1096 14.7 911 4.5 5.9 166 4.92 1224 15.6 1154 2.8 7.2 Sporulation was observed under the microscope throughout the fermentation, but mycelium lysis was not detected.
At 166 h the culture broth attained a volume of 4.92 L, a clavulanic acid titre of 1224 ~g/mL and a dry weight concentration of 15.6 g/L. In other words, 1943 ~,g of clavulanic acid and 24.8 mg of dry weight per mL of culture medium B were obtained.
The fermentation was carried out as described in example 1, except for the changes described next.
The staggered feeding of a culture medium D composed by soybean meal 65 g, (87%) glycerol 56.3 g, hydrated dextrin 86.6 g and (50%) silicone suspension 2 g, per litre of tap water, was carried out from 24 h fermentation time until the end of the fermentation. 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. Thus, the volumetric flow rate varied in the following way throughout the time course of the fermentation TABLE IV
Time Flow h mL/h The agitator speed and the dissolved oxygen concentration varied throughout the fermentation time as follows:
TABLE V
Time Agitator Speed Dissolved Oxygen h m 0-24 S00 99.4-71.9 24-5 S 600 7 8.9-54.0 55-75 700 66.6-53.2 75-107 800 59.0-44.4 107-131 900 51.4-21.0 131-143 1000 29.2-3 5.6 Partial discharges of fermentation broth were carned 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.
The following values of 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:
TABLE VI
Time Volume Partial ClavulanicDry WeightProteinGlycerolDextrin (h) (L) DischargeAcid (g/L) (mg/L) (g/L) (g/L) Volume (~,g/mL) L
0 3.24 - 0 5.5 1673 11.4 20.8 24 3.26 - 91 6.8 718 9.4 18.3 48 3.56 - 426 10.6 634 8.2 19.0 75 3.88 - 801 14.2 318 7.8 16.1 100 4.20 0.35 1035 16.2 464 4.2 17.5 123 4.21 0.35 1264 17.3 674 2.2 24.6 143 4.15 - 1374 18.7 647 0.6 22.8 Sporulation throughout the fermentation, and the beginning of mycelium lysis at the end of the fermentation (at 143 h) were observed under the microscope.
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 elavulanic 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.
The fermentation was carried out according to example 1, except for the changes described next.
Culture medium B was replaced by culture medium E composed by soybean meal 1 S g and (50%) silicone suspension 2 g, per litre of tap water.
The staggered feeding of a culture medium F composed by soybean meal 65 g, (87%) glycerol 70 g and monohydrated maltose 65 g, per litre of tap water, took place from 0 h of fermentation time. 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. Thus, the volumetric flow rate varied throughout the fermentation time as follows:
WO 00/05397 PCT/PT99/OObl2 TABLE VII
Time Flow h mL/h The agitator speed and the dissolved oxygen concentration varied during the fermentation as follows:
TABLE VIII
Time Agitator Speed Dissolved Oxygen h m 0-24 500 98.6-68.9 24-48 600 74.4-79.9 48-72 700 82.0-73.1 72-146 800 80.6-51.7 146-216 900 54.4-37.9 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. Thus, 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.
The following values of 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 WO 00/05397 PCT/PT99/Oti012 _ throughout the time course of the fermentation were obtained:
TABLE IX
Time Volume Partial ClavulanicDry WeightProteinGlycerolMaltose (h) (L) DischargeAcid (g/L) (mg/L) (g/L) (g/L) Volume (~tg/mL) L
0 3.14 - 0 5.4 3137 0.3 2.5 24 3.32 - 123 7.0 962 0.7 2.7 48 3.62 - 496 10.2 742 2.0 3.8 72 3.93 - 759 11.9 625 2.3 4.6 96 4.20 0.3 5 904 13 .2 770 1.9 5.3 120 4.16 0.30 1074 13.9 862 1.2 4.7 144 4.35 0.45 1164 15.7 850 3.6 7.8 168 4.28 0.45 1295 16.1 853 5.1 8.2 192 4.10 0.30 1475 16.3 810 6.0 7.9 216 4.02 - 1607 16.6 936 6.4 8.2 Sporulation was observed under the microscope throughout the fermentation, but no mycelium lysis was detected.
At 216 h 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. In other words, 2084 ~,g of clavulanic acid and 21.5 mg of dry weight per mL of culture medium E were obtained. Taking into consideration the partial discharges, a total of 2790 ~g of clavulanic acid and 30.6 mg of dry weight per mL of culture medium E were attained.
Claims (7)
1. Process for clavulanic acid production by fermentation comprising aerobic submerged culturing of a strain of Streptomyces clavuligerus, or mutants thereof, wherein the culturing is carried out via fermentation using continuous or semicontinuous feeding into a culture medium of one or more organic nitrogen complex sources, thereby controlling protein concentration of filtered broth within certain limits during fermentation.
2. Process according to claim 1, wherein the one or more organic nitrogen complex sources are selected from the group consisting of seed protein, fish meal, hydrolysates and filtrates of such proteins, meat extracts and hydrolysates such as peptones, wherein the seed protein is selected from the group consisting of soybean meal, peanut meal, cottonseed meal and linseed meal.
3. Process according to claim 1 or claim 2, wherein the one or more organic nitrogen complex sources are fed in a continuous or semicontinuous mode at a daily concentration of 0.1-1.5%, preferably between 0.18 and 1.0%, such that the protein concentration of the filtered broth is between 200 and 3500 mg/L, preferably 400-1500 mg/L during fermentation.
4. Process according to claims 1 to 3, wherein initially the culture medium comprises one or more organic nitrogen complex sources in a concentration between 1.3 and 1.8%, glycerol at a concentration ranging between 0.9-1.3% and dextrin at a concentration ranging between 1.8-2.2%.
5. Process according to any of claims 1 to 4, wherein one or more carbon sources are fed continuously or semicontinuously at a daily concentration between 0.18 and 1 %, such that the glycerol concentration in the filtered broth is in the range of 0.2-12 g/L, and simultaneously dextrin is present in the filtered broth at a concentration of 4-22 g/L, or maltose is present in the filtered broth at a concentration of 2-12 g/L throughout the fermentation.
6. Process according to any of the claims 1 to 5, wherein the process also comprises performing continuous or semicontinuous partial discharges of fermentation broth, thereby maintaining the volume of the fermentation broth between 35 and 65% of the total fermenter capacity.
7. Process according to any of the preceding claims, wherein the process also comprises agitating the culture medium with an agitation device, wherein speed of the agitation device is progressively increased according to increases in volume, broth viscosity and dry weight concentration during fermentation, thereby improving mixing, as well as improving dissolved oxygen levels in the culture medium.
Applications Claiming Priority (3)
| 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 |
| PCT/PT1999/000012 WO2000005397A1 (en) | 1998-07-20 | 1999-07-19 | Improved process for clavulanic acid production |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2337074A1 true CA2337074A1 (en) | 2000-02-03 |
Family
ID=20085781
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002337074A Abandoned CA2337074A1 (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) |
Families Citing this family (3)
| 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 |
| CN102277310B (en) * | 2010-06-11 | 2013-04-10 | 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 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IE41110B1 (en) * | 1974-04-20 | 1979-10-24 | Beecham Group Ltd | Esters of clavulanic acid |
| DK141099B (en) * | 1975-02-07 | 1980-01-14 | Glaxo Lab Ltd | Process for purifying clavulanic acid or a salt thereof with a base. |
| ES2101658B1 (en) * | 1995-11-23 | 1998-03-01 | Antibioticos Sa | NEW PROCEDURE FOR THE PRODUCTION OF CLAVULANIC ACID AND ITS SALTS. |
-
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
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 |
| AU4808899A (en) | 2000-02-14 |
| EP1098989A1 (en) | 2001-05-16 |
| WO2000005397A1 (en) | 2000-02-03 |
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