IMPROVED PROCESS FOR CLAVULANIC ACID PRODUCTION

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: 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 β- lactamases. Background to the Invention 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 H This acid has weak antibacterial activity. However, it is a potent inhibitor of β-lactamase enzymes produced by many strains of Staphylococcus aureus, Escherichia coli, Klebsiella, Proteus, Shigella, Pseudomonas and Haemophilus influenzae. β-Lactamases, through the hydrolysis of the β-lactam ring, inactivate several antibiotics, and make the microorganisms, which produce them, resistant to those antibiotics. As potent inhibitor of β-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 β-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. 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 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. 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 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. 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 1 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). 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. 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 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. 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: 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: TABLE II 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 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 μg/mL and a dry weight concentration of 15.6 g/L. In other words, 1943 μg of clavulanic acid and 24.8 mg of dry weight per mL of culture medium B were obtained. EXAMPLE 2 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 The agitator speed and the dissolved oxygen concentration varied throughout the fermentation time as follows: TABLE V 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. 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 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 μg/mL and a dry weight concentration of 18.7 g/L. In other words, 1839 μg 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 μg of clavulanic acid and 28.8 mg of dry weight per mL of culture medium B were obtained. EXAMPLE 3 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 15 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: TABLE VII The agitator speed and the dissolved oxygen concentration varied during the fermentation as follows: TABLE VIII 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 throughout the time course of the fermentation were obtained: TABLE IX 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 μg/mL and a dry weight concentration of 16.6 g/L. In other words, 2084 μg 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 μg of clavulanic acid and 30.6 mg of dry weight per mL of culture medium E were attained.

Claims: CLAIMS 1. 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, wherein the culture is carried out with continuous or semicontinuous feeding of one or more organic nitrogen complex sources, preferably soybean meal, in order to control the protein concentration in the filtered broth within certain limits during the time course of the fermentation. 2. Process according to claim 1, wherein the organic nitrogen complex sources are 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. 3. Process according to claim 1 or 2, wherein the amount of organic nitrogen complex source to be fed in a continuous or semicontinuous mode is in the daily concentration of 0.1-1.5%, preferably between 0.18 and 1.0%, and/or is such that the protein concentration in the filtered broth is between 200 and 3500 mg/L, preferably 400-1500 mg/L, during the fermentation. 4. Process according to any of the claims 1 to 3, wherein the initial culture medium is constituted preferably by one or more organic nitrogen complex sources in a concentration between 1.3 and 1.8%, glycerol and dextrin in the concentration range 0.9-1.3% and 1.8-2.2%, respectively. 5. Process according to any of the claims 1 to 4, wherein glycerol and dextrin or maltose are additionally fed, continuous or semicontinuously, at a daily concentration between 0.18 and 1%, and/or so that the glycerol concentration in the filtered broth is in the range 0.2-12 g/L, and so that the dextrin or maltose concentration in the filtered broth is 4-22 g/L or 2-12 g/L, respectively, throughout the fermentation. 6. Process according to any of the claims 1 to 5, wherein continuous or semicontinuous partial discharges of fermentation broth are performed on such way as to maintain the volume of fermentation broth between 35 and 65% of the total fermenter capacity. 7. Process according to any of the preceding claims, wherein the agitator speed is progressively increased according to the increase in volume, broth viscosity and dry weight concentration during the fermentation, on such way as to improve the mixing, as well as the dissolved oxygen levels in the culture.

Inventors: DE ALMEIDA NABAIS ANA MARIA, REGALO DA FONSECA MARIA MANUEL, DE ALMEIDA NABAIS, ANA MARIA, REGALO DA FONSECA, MARIA, MANUELA
Applicants: ALMEIDA NABAIS ANA MARIA DE, DSM NV, REGALO DA FONSECA MARIA MANUEL, DE ALMEIDA NABAIS, ANA MARIA, DSM N.V., REGALO DA FONSECA, MARIA, MANUELA
Application year: 1999
Main classification: C12P17
IPC codes: C12P17