MXPA01000653A

MXPA01000653A - Improved process for clavulanic acid production.

Info

Publication number: MXPA01000653A
Application number: MXPA01000653A
Authority: MX
Inventors: Almeida Nabais Ana Maria De
Original assignee: Dsm Nv
Priority date: 1998-07-20
Filing date: 1999-07-19
Publication date: 2002-04-08
Also published as: PT102181A; TR200100167T2; CN1310766A; CA2337074A1; AU4808899A; EP1098989A1; WO2000005397A1

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

IMPROVED PROCESS FOR THE PRODUCTION OF CLAVULAN ACID Scope of the Invention This invention relates to important improvements in the fermentation of clavulanic acid using strains of Streptomyces clavuligerus, less complex and low cost strategies of easy industrial implementation. Clavulanic acid is used in medicine in association with antibiotics that are inactivated by ß-lactamases. BACKGROUND OF THE INVENTION Clavulanic acid (3- (2-hydroxyethylidene) -7-oxo-4-oxa-l-azabicyclo [3.2.0] heptan-2-carboxylic acid) is a molecule having the structural formula 20 This acid has weak antibacterial activity. However, it is a potent inhibitor of the ß-lactamase enzymes produced by many strains of Staphylococcus aureus, Escherichia coli, Klebsiella, Proteus, Shigella, Pseudomonas and Haemophilus influenzae-β-lactamases, through 25 Ref: 126575 of the hydrolysis of the ß-lactam ring, inactivates several antibiotics, and makes the microorganisms that produce them resistant to those antibiotics. As a potent inhibitor of ß-lactamases, clavulanic acid is able to avoid this mechanism of resistance, extending the spectrum of antibacterial activity of several antibiotics. Clavulanic acid has good synergistic activity when it is associated with antibiotics such as amoxilicilina, ampicillin, carbenicillin, ticarcillin, benzylpenicillin or cephaloridin, against organisms that produce ß-lactamase. There are several microorganisms which produce clavulanic acid, namely Streptomyces clavuligerus, Streptomyces jumonj inensis (Patent ES 543 854) and Streptomyces ka tsurahamanus (Patent JP 5 ^ 3-104796, Takeda Chemical Industries, Ltd.). There are several processes described for the production of clavulanic acid by Streptomyces clavuligerus, for example (a) discontinuous fermentation using complex or chemically defined media (patent BR 1 508 977, Beecham Group Ltd.); (b) fermentation with automatic pH control between 6.3 and 6.7 (Patent BR 1 571 888, Glaxo Laboratories Ltd.); (c) fermentation with continuous or semi-continuous feeding of a carbon source (for example maltose or glycerol) (Patent ES 537 157, Antibiotics, S.A.); and (d) fermentation with control of the soluble phosphate in the medium, at the beginning and through the fermentation (EP 0 811 689, Antibiotics, S.A.). In the discontinuous fermentation of Streptomyces clavuligerus using soluble media, it is generally observed that the time course of the title of clavulanic acid is closely related to the concentration in dry weight until a maximum value is reached, followed by a decay of both. The decay of the concentration in dry weight is attributed to the sporulation and / or lysis of the mycelium. The decay of the title of clavulanic acid may be due to the fact that its rate of degradation is greater than its production rate.

Description of the Invention The present invention relates to a process for the production of clavulanic acid by a fermentation technique which includes aerobic submerged culture using selected strains and / or culture collection of Streptomyces clavul igerus, or mutants thereof. Accordingly, the culture is carried out with continuous or semi-continuous feeding of one or more complex sources of organic nitrogen, preferably soybean meal, to control the concentration of protein in the filtered broth within a certain limit during the time course The conditions described in this invention successfully lead to the following important improvements in the fermentation of clavulanic acid: (a) a significant increase in the production of clavulanic acid, (b) the prevention of lysis of the mycelium through fermentation; (c) a continuous increase in the title of clavulanic acid and in the concentration in dry weight through fermentation (without partial discharges of the fermentation broth); and / or (d) a continuous increase in the title of the clavulanic acid, maintaining the concentration in dry weight approximately constant from a certain point in time (with partial discharges of the fermentation broth). Therefore, this invention describes new process strategies for the production of clavulanic acid during the culture of the microorganism that produces it, as for example Streptomyces clavuligerus ATCC 27064, or mutants thereof, in anaerobic submerged culture using low cost complex media , in different conditions to those patented or reported to date, which lead to significant improvements in the fermentation of clavulanic acid. These conditions consist of the continuous or staged feeding of one or more complex sources of organic nitrogen, to control the concentration of protein in the filtered broth within certain limits during fermentation, since very high values can inhibit / repress the biosynthesis of the antibiotic, and very low values can limit the desired biosynthesis. Complex sources of organic nitrogen may be seeded proteins such as soybean meal, peanut flour, cottonseed meal and linseed meal, fishmeal, hydrolysates and filtrates of such proteins, meat extracts or hydrolysates such as peptones, preferably, soybean meal. The amount of complex source of organic nitrogen to be fed in a continuous or semi-continuous mode may be in the daily concentration of 0.1-1.5%, preferably between 0.18 and 1.0%, and / or may be such that the concentration of protein in the filtered broth is between 200 and 3500 mg / L, preferably 4Q0-1500 mg / L through the fermentation. According to this process, the initial culture medium can be composed of one or more complex sources of organic nitrogen and, additionally, one or more carbon sources. The concentration of the complex source of organic nitrogen may preferably fall between 1.3 and 1.8%. The carbon source can be glycerol and / or carbohydrates such as 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% ', improves the production of clavulanic acid. Additionally, one or more carbon sources may be fed continuously or semicontinuously at daily concentrations of between 0.18% and 1%, and / or such that the concentration of glycerol in the filtered broth is between 0.2 and 12 g / L, and / or the concentrations of dextrin or maltose in the filtered broth are in the range of 4-22 g / L or 2-12 g / L, respectively, during the temporary course of fermentation. It will be noted that the simultaneous feeding of glycerol and dextrin or maltose improves the production of clavulanic acid. The fermentation can be carried out with continuous or semi-continuous partial discharges of the fermentation broth, in such a way that the volume of the fermentation broth is maintained between 35 and 65% of the total capacity of the fermenter. The speed of the agitator can be increased progressively, according to the increase of the volume, the viscosity of the broth and the concentration in dry weight during the fermentation, to improve the mixing and the oxygen levels dissolved in the culture. The cultivation can be carried out at a temperature of between 26 and 29 ° C and the pH can be controlled between 6.5 and 6.8 by the automatic addition of acid and base, such as a solution of hydrochloric acid and a solution of sodium hydroxide. The foam can be controlled by the addition of an antifoam, such as a silicone suspension. The fermentation vessel should be a typical tank for aerobic fermentation with agitation and aeration devices. The volumetric flow rate of aeration per unit volume of broth can be from 0.6 to 1.3 vvm. These tanks must be equipped with aseptic systems for the continuous or semi-continuous feeding of several nutrients in the form of solution and / or suspension., with aseptic systems for continuous or semi-continuous partial discharges of fermentation broth, and possibly with a variable agitator speed. To carry out the analysis, the samples of culture broth can be filtered under vacuum, 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 then dried in an oven at 86 ° C for 24 h. In this way the concentration in dry weight of the sample is obtained. The concentrations of clavulanic acid, protein, glycerol, dextrin or maltose can be measured in the filtered broth by spectrophotometric methods or, preferably, by chromatographic methods of high pressure liquids, for example using the methods described in (Bird, AE et al., 1982, Analyst 107: 1241-1245; Foulstone, M. and Reading, C. 1982, Atimicrob. Agents Chemother. 22: 753-762), (Bradford, MM 1976, Anal. Biochem. 72: 248-254), (Bok, SH and Demain, AL 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 following examples.

EXAMPLE 1 A suspension of Streptomyces clavuligerus ATCC 27064 spores of agar slices containing 10 g of hydrated dextrin, 10 g of yeast extract, 1 g of meat extract, 2 g of bacteriological peptone, 2 g of CaCO3, was prepared. 20 g of agar, per liter of distilled water. The pH was corrected to 7.1 with 1M NaOH and 1M HCl. This spore suspension was used to inoculate several 500 mL conical flasks containing 50 mL of culture medium A (15 g of soy flour, 10 g of glycerol (875%), 10 g of hydrated dextrin, 1 g of KH2P04 , per liter of distilled water). The pH was corrected to 7.2 with 1M NaOH and 1M HCl. The containers were sterilized at 121 ° C for 15 minutes and incubated at 30 ° C and 110-140 rpm for 2 days. The content of 5 bottles was then mixed giving the vegetative inoculum. Separately, 3.1 mL of a culture medium B compound was introduced by 15 g of soybean meal, 13 g of glycerol (87%), 20 g of hydrated dextrin, 1 g of silicone suspension (50%), by liter of tap water, in an 8-liter STR fermentor and sterilized for 20 minutes at approximately 20 ° C. The fermenter was inoculated with the vegetative inoculum prepared above. The fermentation was carried out with automatic pH control at 6.610.05 by the addition of a 5% (volume / volume) HCl solution and a 1M NaOH solution. The foam was controlled by the addition of a 50% silicone suspension. The temperature was maintained between 26 and 29 ° C, and the aeration between 0.7 and 1.2 vvm. At 24 h, the staged feeding of a culture medium C composed of 65 g of soy flour and 100 g of glycerol (87%), per liter of tap water, was started using a variable speed peristaltic pump. The volumetric flow rate was manually controlled according to the result of the daily protein analysis and the glycerol concentrations in the filtered culture broth. From this mode, the volumetric flow rate varied through the time course of the fermentation as follows: TABLE II The following volume values of the fermentation broth, concentration in dry weight, clavulanic acid titre and concentrations of protein, glycerol and dextrin in the filtered broth were obtained through the temporary course of the fermentation: TABLE I Since the volume, the viscosity of the broth and the concentration in dry weight were increased through the course of the fermentation, the speed of the agitator was increased manually, in such a way that the mixing and dissolved oxygen levels in the culture were improved. . In this way, the speed of the agitator and the concentration of the dissolved oxygen varied in the following way during the fermentation: TABLE III Sporulation was observed under the microscope during fermentation, but no mycium lysis was detected. At 166 h, the culture broth reached a volume of 4.92 L, a multiple of clavulanic acid 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 below. The stepwise or gradual feeding of a culture medium D composed of 65 g of soy flour, 56.3 g of glycerol (87%), 86.6 g of hydrated dextrin and 2 g of silicone suspension (50%), per liter of water from the tap, was carried out from 24 h from the fermentation time until the end of the fermentation. The velocity of the volumetric flow was controlled manually according to the result of the daily analysis of the concentrations of protein, glycerol and dextrin in the filtered broth. In this way the volumetric flow rate varied as follows through the time course of the fermentation: TABLE IV The agitator speed and the concentration of dissolved oxygen vary throughout the fermentation time as follows.

TABLE V Partial discharges of the fermentation broth (8.3% volume / volume) were carried out at 100 and 123 h, using a peristaltic pump with manual control, according to the increase in the volume of the culture broth in the fermenter. The following values were obtained for the volume of the fermentation broth, volume of the partial discharges of the fermentation broth, concentration in dry weight, clavulanic acid titre and concentrations of protein, glycerol and dextrin in the filtered broth throughout the fermentation process. : Table VI Sporulation was observed through fermentation, and at the beginning of mycelium lysis at the end of fermentation (at 143 h), under a microscope. At 143 h, the culture medium reached a volume of 4.15 Lj. a clavulanic acid titer 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 were obtained B.

EXAMPLE 3 The fermentation was carried out according to example 1, except for the changes described below. Culture medium B was replaced by culture medium E composed of 15 g of soy flour and 2 g of silicone suspension (50%), per liter of tap water. The stepwise or gradual feeding of a culture medium F composed of 65 g of soybean meal, 70 g of glycerol (87%) and 65 g of monohydrated maltose, per liter of tap water, took place from the 0 h of the fermentation time. The volumetric flow rate was controlled manually according to the result of the daily analysis of the concentrations of protein, glycerol and maltose in the filtered broth. In this way, the volumetric flow rate varied through the fermentation time as follows: TABLE VII The agitator speed and dissolved oxygen concentration varied during fermentation as follows.

TABLE VIII Partial discharges of the fermentation broth were made through a manual control peristaltic, according to the increase of the volume of the culture broth in the fermenter. In this way, partial discharges of fermentation broth of 8.3%, 7.2%, 10.3%, 10.5% and 7.3% (volume / volume) were carried out at 96, 120, 144, 168 and 192 h, respectively. The following volume values of the fermentation broth, volume of the partial discharges of the fermentation broth, concentration in dry weight, clavulanic acid titre and concentrations of protein, glycerol and maltose in the filtered broth were obtained throughout the fermentation process. : Table IX Sporulation was observed under the microscope through fermentation, but no mycium lysis was detected. At 216 h the culture broth reached a volume of 4.02 L, a clavulanic acid titer 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 E culture medium were obtained. Taking into account the partial discharges, a total of 2790 μg of clavulanic acid and 30.6 mg of dry weight per mL were obtained. of culture medium E. It is noted that in relation to this date, the best method by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims

CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A process for the production of clavulanic acid by fermentation, characterized in that it comprises the aerobic immersed culture of a strain of Streptomyces clavuligerus, or mutants thereof, where the cultivation is carried out via fermentation using continuous or semi-continuous feeding in a medium of cultivation of one or more complex sources of organic nitrogen, thereby controlling the concentration of protein in the filtered broth within certain limits during fermentation. 2. The process according to claim 1, characterized in that one or more complex sources of organic nitrogen are selected from the group consisting of seed protein, fish meal, hydrolysates and filtrates of such proteins, meat extracts and hydrolysates such as peptones, where the seed protein is selected from the group consisting of soybean meal, peanut meal, cottonseed meal and rapeseed meal. 3. The process according to claim 1 or claim 2, characterized in that one or more complex sources of organic nitrogen are fed in a continuous or semi-continuous mode at a daily concentration of 0.1-1.5%, preferably between 0.18 and 1.0%, so that the protein concentration of the filtered broth is between 200 and 3500 mg / L, preferably 400-1500 mg / L during the fermentation. . The process according to claims 1 to 3, characterized in that initially the culture medium comprises one or more complex sources of organic nitrogen in a concentration between 1.3 and 1.8%, glycerol at a concentration ranging between 0.9-1.3% and dextrin at a concentration that fluctuates between 1.8-2.2%. 5. The process according to any of claims 1 to 4, characterized in that one or more carbon sources are fed continuously or semicontinuously at a daily concentration of between 0.18% and 1%, so that the concentration of glycerol in the filtered broth is in the range of 0.2 and 12 g / L, and dextrin is present simultaneously 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 through fermentation. 6. The process according to any of claims 1 to 5, characterized in that the process also comprises effecting unloading continuous or semi-continuous partitions of the fermentation broth, thus maintaining the volume of the fermentation broth between 35 and 65% of the capacity total of the fermentor. The process according to any of the preceding claims, characterized in that the process also comprises stirring the culture medium with a stirring device, where the speed of the stirring device increases progressively according to the increases in volume, viscosity of the broth and dry weight concentration during the fermentation, thereby improving the mixing, as well as improving the dissolved oxygen levels in the culture medium.