PROCESS FOR THE PREPARATION OF CLAVULANIC ACID EMPLOYING STREPTOMYCES CLAVULIGERUS MTCC 1142 IN A SOLID STATE FERMENTATION

5 Field of the Invention

The present invention relates to a solid state fermentation process for the preparation of clavulanic acid. More particularly, this invention relates to a cost effective production of the clavulanic acid under solid-state fermentation conditions using agro-industrial residues. Emergence of antibiotic resistance especially against β- 10 lactam antibiotics has been a major cause of concern recently. The drugs, which used to be our first line of defense, are rendered ineffective because of emergence of β- lactamases. Clavulanic acid is a novel potent inhibitor of bacterial β-lactamases and is produced by fermentation.

15 Background to the invention

Streptomycetes are producers of a large variety of secondary metabolites, which are useful for manufacturing of pharmaceuticals. Examples of these pharmaceuticals are for instance polyketides, macrolides, anthracyclines, tetracyclins, lipopeptides and β-lactams (Strohl W. R., Biotechnology of Industrial Antibiotics, Marcel Dekker Inc.,

20. page 1-48, 1997). Clavulanic acid (3-(2-hydroxyethylidene)-7-oxo-4-oxa-l-azabi- cyclo[3.2.0]heptane-2-carbox ylic acid) has weak antibacterial activity (Fig.l). 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. Clavulanic acid presents good synergetic activity when

25 associated with antibiotics such as amoxycillin, ampicillin, carbenicillin, ticarcillin, benzyl penicillin or cephaloridine, against β-lactamase-producing organisms. There are several microorganisms, which produce clavulanic acid, namely S. clavuligerus ATCC 27064 (US Patent 4110165, Belgian Patent 827926) S. jumonjinensis (GB patent 1563103), S. katsurahamanus IFO 13716 FERM 3944 (JP patent 83009679B) and

30 Streptomyces sp. P6621 FERM 2804 (JP patent 55162993A).

Clavulanic acid is a major β-lactam antibiotic and was discovered independently by Brown et al., (Journal of Antibiotics, 29:668-669,1976) and Napier et al., (Br.Pat.1585661,1981). It is also a potent inhibitor of bacterial β-lactamases (Mayer

& Deckwer, Applied Microbiology and Biotechnology, 20: 41-46, 1996). It appears to be active against a wide spectrum of Gram +ve and Gram -ve bacteria; however the level of observed activity is quite low, hence, it is rather co-formulated with other broad-spectrum antibiotics, which are susceptible to β-lactamases (Brown, Journal of Antimicrobials and Chemotherapy, 1 :15-48, 1981). For example, in-vitro studies indicate that minimum inhibitory concentration (MIC) of ampicillin alone exceeds 500 μg/ml when measured against a lactamase producing strain of S. aureus. On supplementation with 5 μg/ml of clavulanic acid, the MIC for combined addition was reduced to 0.1 μg/ml (Brown, Journal of Antimicrobials and Chemotherapy, 1:15-48, 1981). Following its discovery, the chemical structure of clavulanic acid was identified by Howarth et al. (Journal of Chemical Society and Chemical Commun, 266-267, 1976)

Clavulanic acid binds irreversibly with the enzyme β-lactamases to give a stable complex (Liras & Rodriguez-Garcia, Applied Microbiology and Biotechnology 54:467-475, 2000) thereby making it inactive. The β-lactamases inhibitory activity of clavulanic acid is related to its 3R, 5R stereochemistry. The combined effective action of the β-lactamase inhibition and antibacterial activity make clavulanic acid very important, both clinically and economically. Presently, Glaxo SmithKline Beecham Ltd manufactures Clavulanic acid under the trade name 'Augmentin'. In SSF, two types of process can be distinguished depending on the nature of the solid phase: In the first and the most frequently used, the solid substrate not only supplies the nutrients to the microbial culture growing in it, but also serves as an anchorage for the cells. These substrates are heterogeneous water insoluble materials from agriculture or by-products from food industry, which have an amylaceous or lignocellulosic nature like grains and grain by-products, cassava, potato, beans and sugar beet pulp. The substrate that provides all the nutrients required by the microorganism growing in it would hence be an ideal substrate. Some of the nutrients in the solid media may be available in sub-optimal concentrations, or may even be absent. In such cases, it is necessary to supplement them externally. In the second, an inert support like sugarcane bagasse, hemp, inert fibers, resins, polyurethane foam and vermiculite is impregnated with a liquid medium, which contains all the essential nutrients. Although, this strategy is not used often, but it reports some advantages. The

use of a defined liquid medium and an inert support with a homogenous physical structure improves controlling and monitoring the process, and reproducibility of fermentations.

Given the potential pharmaceutical applications of Clavulanic acid, any process ^' development, which may lead to development of a technology for cost effective fermentative production, is desirable. So far the methods used for fermentative production of clavulanic acid relied on submerged fermentation requiring costly media inputs and handling large amount of liquid fermented broth, which essentially leads to high cost of production. The present process described herein uses the solid-state fermentation method. The process also uses cheaper agro-industrial residues and clearly demonstrates that this method is a cost effective alternative to the one presently employed. The product is produced in concentrated form, hence less liquid handling is required. Given the fact that rather simple production facilities are required for fermentative production using solid-state fermentation method this method is superior to existing production method.

The nutritional requirements and environmental conditions for solid-state culture of S. clavuligerus for clavulanic acid production are not clearly demonstrated. There is only one study by Sircar et al, (Process Biochemistry, 1998, 33(3), 283-289) indicating clavulanic acid production using solid-state fermentation. However, the study has not measured the actual clavulanic acid production and medium composition also is not clearly defined. It is not clear from that study that how much of clavulanic acid was present in fermented media and how effective was the process for clavulanic acid production. The composition of the solid medium employed in the SSF included wheat rawa, soy flour, dipotassium hydrogen phosphate and sunflower oil cake. Based ^' on the above the inventors decided to go for developing this solid-state fermentation process, which is based on utilization of agro-industrial residues for fermentative production of Clavulanic acid or its salt thereof. The process resulted in good yield at a high productivity making it an alternative for existing technologies.

Objectives of the invention

The main objective of the present invention is to provide a fermentation process for the preparation of clavulanic acid using Streptomyces clavuligerus MTCCl 142.

Yet another object of the invention is to provide a process for the production of clavulanic acid using easily available carbon and nitrogen sources along with mineral salts under solid-state culture conditions.

Yet another object of the invention is to provide a media composition and environmental conditions, which can easily be attained in a fermentation laboratory setup to easily and effectively, produce the clavulanic acid. Yet another object of the invention is to utilize Streptomyces clavuligerus for the preparation of clavulanic acid under both submerged and solid-state culture conditions wherein solid-state fermentation process defined herein gives better yield.

Summary of the invention According the present invention provides a solid state fermentation process for the preparation of clavulanic acid or its salts thereof, the said process comprising the steps of: a) preparing a medium of carbon, nitrogen and mineral salt components along with surfactants and moisture contents, followed by sterilization by know method, b) inoculating the Streptomyces clavuligerus MTCCl 142 in the above said medium and growing the organism, under aerobic condition, at a temperature in the range of 25-30 ⁰ C, at a pH in the range of 6.0-7.5, for a period of 5-6 days, c) terminating the above said fermentation run and extracting the desired clavulanic acid by known extraction methods. In an embodiment of the present invention the fermentation medium used is carbon source 1.0 - 10.0 g nitrogen source (inorganic/complex) 0.05 - 1.0 % (w/w) mineral salts 0.5-10g/L @ 0.1-1% (v/w) initial moisture 65-75 % (v/w)

In yet another embodiment the source used for carbon is an agro-industrial residue selected from the group consisting of coffee husk, Rice bran, wheat bran, groundnut oil cake, sesame oil cake, olive oil cake, coconut oil cake, tamarind seed powder, soybean flakes, soybean grit and a combination thereof.

5. In yet another embodiment the sources of nitrogen used are complex organic nitrogen and inorganic nitrogen source.

In yet another embodiment the organic nitrogen complex source used is selected from the group consisting of yeast extract, beef extract, peptone, malt extract, urea, corn steep liquor, tryptone and a combination thereof. 0 In yet another embodiment the inorganic nitrogen source used is selected from the group consisting of ammonium bicarbonate, ammonium sulfate, ammonium hydrogen orthophosphates, ammonium persulfate, ammonium nitrate, ammonium chloride and a combination thereof.

In yet another embodiment the mineral salt used is selected from the group 5 consisting of KH ₂ PO ₄ , NaCl, MgSO ₄ and a combination thereof.

In yet another embodiment the surfactant used is selected from the group consisting of castor oil, Triton X-IOO, Tween 40, Tween 60, Tween 80 and silicone oil and a combination thereof.

In still another embodiment the yield of clavulanic acid used is in the range of 0 1900 μg/gds to 4700 μg/gds (gram dry substrate)

Brief description of the drawings

These and other objects, features and advantages of the present invention will become more readily apparent from a reading of the following detailed description 5 taken in conjunction with the drawings.

Fig. 1: shows the structural formula of clavulanic acid.

Fig. 2: Shows the effect of different substrates on clavulanic acid production by S. clavuligerus MTCCl 142. 0 ^' Fig. 3: Shows the effect of fermentation time on clavulanic acid production.

Fig. 4: Shows the effect of initial moisture content on clavulanic acid production.

Fig. 5: Shows the effect of inoculum size on the production of clavulanic acid in solid media.

Fig. 6: Shows the effect of inorganic nitrogen sources. Fig. 7: Shows the effect of organic nitrogen sources. Fig. 8: Shows the effect of additional carbon sources.

Fig. 9: Shows the effect of surfactants on the production of clavulanic acid in the solid media.

Detailed description of the invention

It should be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore the details disclosed herein are not to be interpreted as limiting but merely as the basis for the claims and as a basis for teaching one skilled in the art as how to make use of the invention.

A) This process is carried out in relatively simple production conditions and comprises cultivating a strain of Streptomyces clavuligerus for 5-7 days and recovering clavulanic acid or a salt thereof from the culture medium and thereafter deriving free acid. The organism is preferably S. clavuligerus MTCC 1142 or a high yielding mutant thereof, which is used in this process.

B) The cultivation of organism in presence of assimilable sources of carbon, nitrogen and mineral salt supplements takes place in a solid, semi-solid or in liquid medium. The cultivation may take place on an aerobic solid culture medium consisting of complex nutrients. The complex nutritional sources in the form of agro industrial residues are a suitable source for production.

C) The nutrient media which is useful for the cultivation of S. clavuligerus and for clavulanic acid production contains traditional agro-industrial residues such as wheat bran (WB), rice bran (RB), soybean flakes (SF), soybean grit (SG), coffee husk (CH), tamarind seed powder (TSP), coconut oil cake (COC), sesame oil cake (SOC), groundnut oil cake (GOC), olive oil cake (OOC) etc as a single substrate or in a defined combination for clavulanic acid production. D) Stock solutions of mineral salts such as KH ₂ PO ₄ , K ₂ HPO ₄ , NaCl, MgSO4 or

MgCl ₂ etc are added to fermentation media @ of 0.2-0.5 % v/w (unless otherwise specified all w/w or v/w values are with reference to carbon source)

and are usually in the range of 0.5g/l-10 g/1, preferably in the range of 0.5 -7.5 g/1 and most preferably in the range of 1 - 5 g/1.

E) The cultivation of S. clavuligerus is done usually in the temp range of 20 ⁰ C to 35 ⁰ C, preferably 22 ⁰ C to 30 ⁰ C and more preferably between 24 ⁰ C -28 ⁰ C. The pH for cultivation may be usually in the range of 5.0 to 8.5, preferably in the range of 6.0 to 7.5 and most preferably in the range of pH 6.5 to 7.2. The S. clavuligerus is cultivated in the defined media at pH and temp as described in glass conical flasks with or without shaking. The starting pH is in the range of 7.0+-0.2 and fermentation is carried out till 144 hrs. However, the maximum clavulanic acid yield is obtained preferably till 120 hrs.

F) The inorganic nitrogen source like i ammonium bi-carbonate or ammonium sulfate or ammonium hydrogen orthophosphate or ammonium persulfate or ammonium nitrate or ammonium chloride supplementation in the salt solution at a concentration of 0.01-0.2M, more preferably in the range of 0.02-0. IM and most preferably at a concentration of 0.04-0.07 M enhances the production.

G) The fermentation medium supplemented with organic nitrogen sources such as peptone or malt extract or beef extract or yeast extract or urea or corn steep liquor or corn steep solids preferably in a concentration range of 0.01% to 0.1% w/w gives enhanced production. H) The surface-active agents like castor oil or Triton X-100 or Tween 40 or Tween

60 or Tween 80 or silicone oil is added to the media preferably in the range of

0.01 % - 0.5 % for enhanced production. I) A strain of Streptomyces clavuligerus (MTCC 1142) is preferably maintained on slants of a defined medium containing yeast extract, malt extract, glucose and agar with a pH adjusted to 7.2 ± 0.2 and slants grown preferably at 25 ⁰ C-

28 ⁰ C most preferably for five days and subsequently suitably stored at 4 ⁰ C. J) The inoculum for fermentation is preferably prepared in 10 ml of distilled water containing 0.1% Tween-80 from a sporulated slant culture, preferably 5-10 days old. K) Clavulanic acid in the fermentation broth is preferably estimated by HPLC using the procedure as described by Foulstone and Reading (Antimicrob.

Agents. Chemother. 22 (1982) 753). Waters Spherisorb 5μ ODS2 column (4.6

mm x 250 mm) may preferably be used. The mobile phase is preferably methanol/(50mM) KH ₂ PO ₄ (40:60) with a pH value adjusted to pH 3.08. The flow rate is preferably maintained between 0.6-1.0 ml/min and is detected by a UV detector. L) Biomass estimation is preferably performed as per Sakurai et al, (Agriculture and Biological Chemistry, 1977, 41, 619-24). The 0.5 g fermented matter is preferably mixed with concentrated sulphuric acid (2 ml) and the reaction mixture may preferably be kept for 24 h at room temperature (25-3O ⁰ C). This mixture may be diluted with distilled water to make 1 N solution, autoclaved (15 psi for 1 h), neutralized with 1 N NaOH and made to 100 ml with distilled water. The solution (1 ml) is mixed with 1 ml acetyl acetone reagent and incubated in a boiling water bath for 20 min. After cooling, ethanol (6 ml) is added followed by the addition of 1 ml Ehrlich reagent and preferably incubated at 65 ⁰ C for 10 min. After cooling, the optical density of the reaction mixture is noted at 530 nm against the reagent blank. The result is expressed preferably as tng glucosamine per gram dry substrate (gds).

M) Moisture content is a key factor that strongly influences microbial growth and activity in SSF (Pandey A, Process Biochemistry, 27,109-17,1992; Pandey et al., Process Biochemistry, 35, 1153-1169, 2000). Filamentous fungi or Streptomycetes when cultivated on agro-industrial residues during SSF, grow best when the substrate moisture content is generally between 50 and 75% (Pandey et al., In: Solid-state fermentation in biotechnology. Asiatech Publishers New Delhi: pp. 221, 2001). The moisture content is kept preferably in the range of 45-80%, more preferably in 55-75% and most preferably 66- 73%.

N) An inoculum size of 1-5 ml (containing 8 x 10 ⁶ spores/ ml) is preferably used for optimal clavulanic acid production.

Streptomyces clavuligerus MTCCl 142

This culture is equivalent to S. clavuligerus NRRL3585. It grows aerobically at a temp. of 25 ⁰ C. The growth medium contains Yeast extract, Malt extract, glucose, agar and

distilled water. This culture is type strain for production of Clavulanic Acid, Cephalosporins and Penicillin N. This strain was a patented strain.

5 The following examples are given by the way of illustration and therefore should not be construed to limit the scope of the invention.

The medium for fermentative production of clavulanic acid is prepared as described herein. Also, unless otherwise specified all w/w of v/w values are with

10 reference to carbon.

Experiment-1

Coconut oil cake: 5 g

KH ₂ PO ₄ : 0.08 % (w/w)

15. NH ₄ NO ₃ : 0.1 % (w/w)

NaCl : 0.04 % (w/w)

MgSO ₄ : 0.04 % (w/w) pH of the medium 7.00+_ 0.2

Fermentation temp: 25 ⁰ C -28 ⁰ C

Initial moisture: 70 % (vol of liquid/total wt) Time of fermentation: 120 hrs Yield of Clavulanic Acid 1919 μg/gds

Experiment 2

Soybean oil cake 5.O g

KH ₂ PO ₄ 0.08 % (w/w)

NH ₄ NO ₃ 0.2 % (w/w)

NaCl 0.04 % (w/w)

MgSO4 0.04 % (w/w) pH of the medium 7.00+_ 0.2

Fermentation temp: 25 ⁰ C

Initial moisture: 70 % (vol of liquid/total wt)

Time of fermentation: 120 hrs

Yield of Clavulanic Acid 2586 μg/gds

Experiment 3

SOC+Soya flakes 5.O g (1:1 w/w)

Yeast Extract 0.2 % (w/w)

KH ₂ PO ₄ : 0.08 % (w/w)

NaCl: 0.04 % (w/w)

MgSO ₄ : 0.04 % (w/w) pH of the medium: 7.00+_ 0.2

Fermentation temp: 25 ⁰ C

Initial moisture: 70.58 % (vol of liquid/total wt)

Time of fermentation: 120 hrs

Yield of Clavulanic Acid 3139 μg/gds

Experiment 4

SOC 5.O g

Yeast Extract 0.2 % (w/w)

ICH ₂ PO ₄ : 0.08 % (w/w)

NaCl: 0.04 % (w/w)

MgSO ₄ : 0.04 % (w/w) pH of the medium: 7.00+_ 0.2

Fermentation temp: 25 ⁰ C

Initial moisture: 70.58 % (vol of liquid/total wt)

5 Time of fermentation: 120 hrs

Yield of Clavulanic Acid 4695 μg/gds

This invention is of potential importance for industries more specifically to Pharmaceutical industries. So far the process developed and patented by Glaxo-

10 Smithcline Beecham was used for the production of clavulanic acid. The process was a patented one, hence was not available for production to any other manufacturer. In 2002 after the expiry of clavulanic acid patent two companies started marketing of generic version of clavulanic acid. However, the cost of production was still high and needed further improvements. The present process is a novel process different from the

15 already existing processes and utilizes simple process and media inputs. The process can be readily up-scaled for industrial production purposes and will lead to clavulanic acid production at a significantly reduced price.

The foregoing description of the preferred embodiments is provided to enable any person skilled in the art to make or use the present invention. The various

20 modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. The present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Various changes and modifications

25. may be made therein without departing from spirit of the invention. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims unless they depart there from.