A process for the production of actinomycin d

Abstract

The present invention describes a fermentation process with specifically designed medium for improved production of pharmaceutically important drug (actinomycin D) using actinobacterial strain Streptomyces marokkonensis MTCC-25511, isolated from cold deserts of NW Himalayas (Barchey) India. The process involves 12 days fermentation under specific fermentation conditions i.e. temperature 28ºC±2ºC, pH7.0±2.0 with 10% inoculum size followed by downstream processing involving purification of actinomycin D by conventional solvent as well as single step purification method. The direct single step purification involved binding of the metabolites of fermented broth with macroporous adsorbent resin and elution of pure compound Actinomycin D using methanol / water / acetone. The present invention provided high productivity i.e. ~16.7mg / L / h of actinomycin D with >95% purity.

Description

[0001]PT / 2025 / 10079 A PROCESS FOR THE PRODUCTION OF ACTINOMYCIN D FIELD OF THE INVENTION: The present invention relates to a fermentation based process for production and purification 5 of a pharmaceutically important drug (actinomycin D) using actinobacterium isolated from cold deserts of NW Himalayas. BACKGROUND OF THE INVENTION 10 The actinomycins are a family of bicyclic chromopeptide lactones, that shares the chromophoric phenoxazinone dicarboxylic acid, to which two non-ribosomal pentapeptide lactones are attached. The class is represented by actinomycin D, however, more than 41 actinomycins, mainly with variations in the peptide chain of the structures have been reported so far. Actinomycin was isolated for the first time by Waksman and Woodruff in 1940 from 15 Streptomyces antibioticus (Science 118, no.3062 (1953): 259-266). Although it has not been successful as an antibiotic, however, it has huge demand as a chemotherapeutic agent. Actinomycin D acts as a transcription inhibitor, which binds to the DNA duplex at the transcription initiation step, thus prevents the elongation of RNA polymerase. Conformation of the molecule is extremely well adapted for intercalation into a right-handed DNA helix, 20 favoring the establishment of hydrophobic interactions that stabilize the DNA / antibiotic complex. A number of species of Streptomyces have been explored for the production of actinomycin D (Antimicrobial Agents and Chemotherapy 11, no.2 (1977): 281-290..; Journal of Industrial Microbiology and Biotechnology 29, no. 6 (2002): 299-302.; Letters in Applied25 Microbiology 49, no. 4 (2009): 450-455.; Medicinal Chemistry Research 17 (2008): 114- 122.Brazilian Journal of Chemical Engineering 19 (2002): 277-285 disclosed the bioprocess development of actinomycin D using three species of Streptomyces i.e. S. regensis DAUFPE 30 3053, S. felleus DAUFPE 3079, S. parvulus with yield of 1.53g / L. Patent no. CN112680387A reported 1.38 mg / L yield of actinomycin D with media composition 2-3% soluble starch, 3-4% soyabean flour, 0.1-1% peptone, 0.5-1.5% corn flour, 0.01-0.1% dipotassium hydrogen phosphate with pH 7.0-7.2. PT / 2025 / 10079 Applied microbiology and biotechnology 95 (2012): 919-927 discloses yield of actinomycin D i.e.1.770 g / L using strain of S. sp. MS449 in MPG medium (glucose 1.0 %, millet meal 2.0 %, cotton seed gluten meal 2.0 %, and MOPS 2.0 %, pH 7.0 5 Patent no. BR102015005953A2 has disclosed the production of actinomycin D and its analogues using Streptomyces longwoodensis with yield 1.184 g / L. Medicinal Chemistry Research 17 (2008): 114-122 disclosed the production of actinomycin D with the yield of 850 mg / L using the mutant strain of Streptomyces sindenensis. 10 Journal of Chemical and Pharmaceutical Research (2011), 3(5):281-289 discloses the production of actinomycin D with the concentration of ~2g / L at shake flask have been reported till date Several isolation strategies have been employed for the purification of actinomycin D. Sousa 15 et. al. claims the use of toluene-acetone system (7:3) for the purification of actinomycin D (Brazilian Journal of Chemical Engineering 19 (2002): 277-285). Patent no. CN102391967A describes the use of acetone to obtain crude extract, then subjected to the HP20 macroporous adsorptive resins, acetone washout followed by HPLC 20 for isolation of actinomycin D. Patent no. CN112010942A discloses the purification of actinomycin D using ethyl acetate as solvent to obtain crude extract, followed by normal phase silica gel column, elution with cyclohexane-ethyl acetate and then dried fraction was subjected to Supercritical fluid 25 chromatography. Journal of Pharmaceutical and Biomedical Analysis 195 (2021): 113835 describes the only method that denies the use of organic solvents for the purification of actinomycin D and its analogs, the method involves the use of Supercritical fluid chromatography to fractionate the 30 pure actinomycin D and its analogs. Microorganisms (2020): 8(3) 351 discloses that actinomycin D could also be used as biocontrol agent against phytopathogens. PT / 2025 / 10079 The following Table 1 highlights the strains reported for the production of actinomycin D: Table 1: Strains reported in literature for actinomycin D and their yields S.no. Strain Yield No. of References / Patent no. days 1. Streptomyces. regensis DAUFPE 3053 12 mg / L 8 Sousa et.al.2002 (250 mL shake https: / / doi.org / 10.1590 / S010 flask) 4-66322002000300002 2. Streptomyces felleus DAUFPE 3079 20 mg / L 8 Sousa et.al.2002 (250 mL shake https: / / doi.org / 10.1590 / S010 flask) 4-66322002000300002 3. Streptomyces parvulus 1.530g / L 8 Sousa et.al.2002 (14-liter https: / / doi.org / 10.1590 / S010 bioreactor) 4-66322002000300002 4. Streptomyces rubiginosus FIM-ZI9-12 1.383g / L 3-6 CN112680387A (mutant) (fermentation tank) 5. Streptomyces sindenensis 850 mg / L 7 Praveen et.al.2008 (3L stirred tank https: / / doi.org / 10.1007 / BF bioreactor) 03179453 6. Streptomyces halstedii MTCC 6817 512 mg / L 7 Praveen et.al.2008 (shake flask) https: / / doi.org / 10.1007 / s0004 4-007-9042-7 7. Streptomyces anulatus MTCC 6818 620 mg / L 7 Praveen et.al.2008 (shake flask) https: / / doi.org / 10.1007 / s0004 4-007-9042-7 8. Streptomyces griseoruber 210 mg / L 6 Praveen et.al.2009 (shake flask) https: / / doi.org / 10.1111 / j.147 2-765X.2009.02689.x 9. Streptomyces sindenensis-M-46 1.26 to ~2 gm / L 5 Tripathi et.al.2011 (shake flask) Journal of Chemical and Pharmaceutical Research2011, 3(5):281-289 10. Streptomyces sp. MS449 (S. 1.77g / L 15 Chen et.al.2012 avermitilis) (250-ml shake https: / / doi.org / 10.1007 / s0 flask) 0253-012-4079-z 11. 467mg / L 10 Toumatia et.al.2015 Streptomyces sp. IA1 (500-ml shake flask) https: / / doi.org / 10.1002 / job m.201400202 12. Streptomyces hydrogenans IB310 13.99 mg / L 5 Kulkarni et.al.2017 (shake flask) https: / / doi.org / 10.1016 / j .bcab.2017.02.009 PT / 2025 / 10079 Streptomyces tauricus 2.58mg / L 10 Rather et. al.2017 (shake flask) https: / / doi.org / 10.1007 / s0 0044-017-1842-9 960 mg / L 7-10 Wei et.al.2017 Streptomyces flavogriseus NJ-4 (250-ml shake https: / / doi.org / 10.7717 / flask) peerj.3601 / supp-2 Streptomyces smyrnaeus UKAQ_23 561.3mg / kg 7 Qureshi et.al.2021 (solid state https: / / doi.org / 10.1038 / s4 fermentation) 1598-021-93285-7 Streptomyces longwoodensis 1.184 g / L 7 BR102015005953A2 (2L shake flask) Streptomyces marokkonensis 4.0 g / L 10 Present invention (MTCC-25511) (500L bioreactor) Comparison with prior art reveals a significant improvement over the prior art, with the enhanced yields of 4.0 g / L of Actinomycin D in 10 days. The yields are ~ two fold, which is far better than the reported literature having production of ~2.0gm / L at shake flask fermentation. The present invention also involves the single step purification of Actinomycin D (>95% purity), without involving solvent extraction step with higher productivity and purity. OBJECTIVES OF THE INVENTION Main objective of the present invention was to provide an improved and efficient process for the production of Actinomycin D and its analogues, an improved and efficient process for the downstream and purification of Actinomycin D and its analogues. Yet another objective of the invention was to develop one step downstream processing that allows the purification of Actinomycin D. Yet another objective of the invention was to develop a cost effective process with improved productivity and minimal use of solvents to achieve high purity (>90%) actinomycin D. SUMMARY OF THE INVENTION The present invention relates to the field of fermentation based process development of pharmaceutically important drug (actinomycin D) using actinobacterial strain, isolated from cold deserts of NW Himalayas. PT / 2025 / 10079 In a preferred embodiment of the present invention, a fermentation process with specifically designed medium was developed for the production of actinomycin D with enhanced yields. In still another embodiment of the invention, the downstream process involves purification of 5 actinomycin D by conventional solvent and single step purification method. In yet another embodiment of the invention, the downstream process involves purification by direct single step, which involves binding of the metabolites of fermented broth with macroporous adsorbent resin and elution of pure compound Actinomycin D using 10 methanol / water / acetone. In still another embodiment of the invention, the process provided high productivity i.e. ~16.7 mg / L / h and >95% purity of actinomycin D. 15 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: A) TLC profile of Fraction.2: B) analytical HPLC analyses of Fraction 2 Figure 2: Semi-preparative Separation of Fraction 2. Figure 3: The Purity of Actinomycin D obtained by (a) solvent extraction method and (b) single step purification using macroporous adsorbent resin 20 DETAILED DESCRIPTION OF THE INVENTION Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings 25 In an embodiment, the present invention specifically related to the process development for the production of actinomycin D directly from fermented broth with higher yield ranging from 250 mg / L to 4.0 g / L or even higher under specified physicochemical conditions e.g. in presence of certain elicitors / precursors i.e. Valine, Arginine, Tyrosine, Asparagine, Sodium acetate and Glycerol as compared to literature value, thus supporting the idea of green 30 technology In an embodiment, the present invention describes the exploration of an isolate Streptomyces marokkonensis (MTCC-25511) from cold deserts for the production of bioactive (Actinomycin D) and its analogues. PT / 2025 / 10079 The term bioactive herein describes the compound that affects a live creature, tissue, or cell. After isolation the pure culture was maintained and preserved on same medium. The morphological characteristics were investigated on International Streptomyces Project - 2 and 5 Starch Casein Agar media, respectively at 28oC±2oC. The cultural characteristics of isolate grown on SCA were observed as powdery white colony with circular and smooth ends. Molecular characterization has been done by 16S rRNA partial gene sequencing using -10 ACTT- was purified by Gel elution / SAP. In an embodiment of the invention the purified PCR product was sequenced by Sanger sequencing herein the Sanger sequencing describes one of the DNA sequencing method by 15 Dideoxynucleotide based termination to generate family of fragments of DNA molecules which are fluorescent tagged and analyzed to generate DNA sequence and the partial sequence was assembled and submitted to NCBI with Accession number OM540372. The culture has been submitted to Microbial Type Culture Collection, India with Accession no. MTCC- 25511. 20 One embodiment of the invention is the process for the production of actinomycins involves solid state as well as submerged fermentation, wherein consistent extracellular as well as intracellular production takes place in various fermentation media at shake flask level as well as in bioreactor upto 500L size, under various fermentation conditions (either alone or in 25 combination). In an embodiment of the invention the optimization of different media herein refers to a medium for cultivation of a wide variety of microbes species including or which is liquid at room temperature. 30 In another embodiment of the invention Media may comprise carbon sources both simple and complex, nitrogen sources (one or multiple) along with trace elements such as Calcium carbonate, Magnesium sulphate, Di-potassium hydrogen phosphate, Ferrous sulphate, Potassium chloride, Sodium chloride, Ammonium sulphate / nitrate etc. PT / 2025 / 10079 In another embodiment the carbon sources used were selected from the list of Glucose, Fructose, Sucrose, Mannitol, Glycerol, Pectin, Lactose, Maltose, Mannose, Gluconic acid, Chitosan, Dextrin, Starch, Xylose, Molasses, Corn steep liquor, dextrin, Inositol, Chitin 5 Sorbitol where in the preferred carbon sources are glucose, starch, dextrin, chitosan, molasses, gluconic acid and glycerol either alone or combinations of these nutrients. In an another embodiment, in addition to carbon sources, different nitrogen sources used were soyabean meal, yeast extract, peptone, sodium / potassium nitrate, casein, casein 10 hydrolysate and ammonium nitrate, urea, arginine, asparagine, ammonium phosphate, ammonium sulphate, beef extract; yeast extract, soyabean meal, peptone, N / Z amine A, N / Z amine B, Casein hydrolysate, Peptone, Sodium nitrate, Valine, Urea, Arginine, Potassium nitrate, Ammonium sulphate support Actinomycin D and its analogues production; wherein preferred nitrogen sources are soybean meal, yeast extract, peptone, sodium / potassium 15 nitrate, casein, casein hydrolysate and ammonium nitrate or combinations of these nutrients. Meat solubles, peptone, Beef extract, corn meal, enzyme-hydrolyzed casein, and nitrate salts are also useful for the production of Actinomycin D and its analogues. In an embodiment, trace elements salts like Calcium carbonate, Magnesium sulphate, Di- 20 potassium hydrogen phosphate, Ferrous sulphate, Potassium chloride, Sodium chloride, Ammonium sulphate / nitrate etc. are helpful in combination for the growth of the organism and production of Actinomycin D and its analogues In an embodiment the present invention involves the use of modified production medium. 25 The term production medium involves the medium used for the production of bioactive compounds and the term modified production medium involves the medium having same composition as production medium but with different concentration. 30 The different media PM-1, PM-2 , PM-3, PM-4, PM-5, PM-6, PM-7, PM-8, PM-9, PM-10, PM-11 & PM-12 were optimized to enhance the yield of actinomycinD. The composition of the media from PM-1to PM-12 are shown in the Table-3. PT / 2025 / 10079 In another embodiment, the present invention involves the use of modified production medium. The term production medium involves the suitable nutritional medium required by the microorganism for the production of bioactive compounds during fermentation process at bioreactor level. The modified production medium involves the optimized constituent concentrations in the production medium. The modified medium was designed using Design of Experiments for the production of actinomycin D and further validated by performing experiments in triplicate for better accuracy and precision. In an another embodiment of the invention the optimization of different physico-chemical parameters where in the different temperature ranges i.e.10oC, 20oC, 30oC, 40oC with maximum yield at temperature 28oC, pH range i.e.5.0, 6.0, 7.0, 8.0, 9.0 with maximum yield at pH 7.0 and 10.0, inoculum size of 5%,10%,15%,20% with maximum yield at 10% inoculum size Another embodiment of the invention is the purification of actinomycin D directly from fermented broth and through convention method as reported in literature. The fermented broth was homogenized followed by separation of cell biomass by filtration and filtrate was used for direct binding with macroporous adsorbent resin (Diaion HP20). Diaion HP20 column (3 cm x 60 cm, 150 mL) was prepared by soaking Diaion HP20 resin (Sigma aldrich, USA) in Milli-Q water. Finally, the column was eluted with methanol at a flow rate of 2 mL / min, collecting 30 mL fractions. Each fraction was checked for its compositions by TLC using 100% EtOAc for TLC development. Anisaldehyde stain was used for detection of the fractions and all the fractions with actinomycin D were combined and concentrated to dryness to give orange red color powder (actinomycin D). In an embodiment the purity and recovery of pure compound was compared with the conventional solvent extraction method to validate the results. The structure of the Actinomycin D was confirmed based on1H,13C NMR in Pyridine-D5 on Bruker-Advance DPX FT-NMR 400 MHz instruments (100 MHz for13C NMR). and further their comparison with literature values. Experiments were performed on a Waters 1525 HPLC system (Waters, Milford, MA, USA) equipped with a binary gradient pump, auto sampler, column oven, photodiode array detector, PT / 2025 / 10079 and data acquirement and processing was operated by the Waters Empower 3 software (Waters, Milford, MA, USA). One embodiment of the invention is to minimize the use of solvents for the purification of 5 actinomycin D where in the fermented broth has been directly bound to macroporous adsorbent resin followed by elution step to get the purified Actinomycin D. Thus, present process involves higher production of actinomycin D under optimized physico-chemical conditions along with single step downstream process for the purification, without using solvent extraction step. 10 EXAMPLES The following examples are given by way of illustration of the working of the invention in actual practice and should not be construed to limit the scope of the present invention. 15 Example 1: Isolation and characterization of Streptomyces marokkonensis (MTCC- 25511). The actinobacteria strain Streptomyces marokkonensis (MTCC-25511) was isolated from soil sample collected from cold deserts of North Western Himalayas 34.57450N, 76.23200E, (Barchey) India on Starch-Casein agar (SCA) medium. The pure culture was maintained and 20 preserved on same medium. The morphological characteristics were investigated on ISP2 and SCA, respectively at 28oC±2oC. The cultural characteristics of isolate grown on SCA were observed as powdery white colony with circular and smooth ends. Aerial colonies appear white during early growth and yellow pigmentation starts appearing with time. 25 Molecular characterization was performed by 16S rRNA partial gene sequencing using - -ald Amp GT master mix. The 1500bp ampliconwas purified by Gel elution / SAP. The purified product was sequenced by Sanger sequencing and the partial sequence was assembled and submitted to NCBI with Accession number 30 OM540372. The culture has been submitted to Microbial Type Culture Collection, India with Accession no. MTCC- 25511. Example 2: Maintenance of culture PT / 2025 / 10079 The culture Streptomyces marokkonensis MTCC-25511) was maintained on glass test tubes of 18×150 mm2size having starch casein agar slants (Table 2), by transferring a loopful of culture from matured slant and kept for incubation at 28oC±2oC for 4-6 days with subsequent sub-culturing after two weeks. The glycerol stocks and lyophilized cultures were kept for long term storage of the culture. Table 2: Components of media for preservation of cultures Streptomyces marokkonensis MTCC-25511. Ingredients Composition (g / L) Starch 10.0 KNO32.0 K2HPO4 2.0 Casein 0.3 MgSO4.7H2O 0.05 NaCl 2.0 CaCO3 0.02 FeSO4.7H2O 0.01 pH 7.0 Agar 20.0 Example 3: Preparation of culture inoculum Pre-inoculum was prepared in a narrow mouth 500 mL Erlenmeyer flask containing 100 mL medium (Table 3) by transferring loopful of culture from freshly grown culture plate. The inoculated medium was incubated at about 28oC±2oC for about 48-72 hours on a rotating shaker at 250 RPM. Table 3: Components of media used for inoculum preparation Ingredients Composition (g / L) Soluble starch 25.0 Soyabean meal 15.0 Yeast extract 2.0 CaCO3 4.0 pH 7.0 PT / 2025 / 10079 Preferably, 5-10% of pre-inoculum was transferred to 1000 mL Erlenmeyer flasks containing 300 mL seed medium. The seed inoculum was prepared using same medium components as of pre-inoculum. The inoculum was prepared by incubating the flasks at 28±2oC for about 48-72 hours on a rotating shaker at 200 RPM. Example 4: Production of actinomycin D and its analogues Different production media were used for actinomycin D production as shown in Table 4. Out of these PM-11 was observed to give maximum yield of actinomycin D. Further PM-11 was modified (Table 5) to enhance the yield. The production medium was designed after optimizing concentration of medium components using DoE and further validated by the experiments carried out in triplicate with the modified medium for better accuracy and precision. After optimization the concentration of both carbon sources (24 g / L Soluble starch and 46 g / L dextrin) remained same, however concentration of nitrogen sources i.e. peptone and yeast extract were modified to 2 g / L and 7.5 g / L, respectively. Similarly, in case of CaCO3 and K2HPO4 the concentrations were modified as 1.5 g / L after optimization for better production of actinomycin D. Table 4: Components used for production medium S. No. Medium Composition in g / L 1 PM-1 Glycerol-20.0; Soyabean meal-20; Sodium chloride-3.0 2 PM-2 Starch-25.0; Soyabean meal-15.0; Yeast extract-2.0; CaCO3 -4.0 3 PM-3 Dextrin-1.0; Glucose-1.0; Soyabean meal-5.0; Yeast extract-2.0; CaCO3 -1.50 4 PM-4 Glucose-50.0; Glycerol-5.0; Peptone-10.0; Malt extract-1.50; Soyabean meal-10.0; MgSO4.7H2O-1.0; CaCO3 -5.0; Beef extract-5.0 5 PM-5 Glucose-2.0; Soyabean meal-10.0; Starch-30.0; Corn Steep liquor-1.0; Peptone-3.0; CaCO3-5.0 6 PM-6 Starch-10.0; KNO3-2.0; Casein-0.30; K2HPo4-2.0; MgSO4-0.05, NaCl-2.0 7 PM-7 Starch-10.0; CaCO3 -3.0; K2HPO4 -1.0; (NH4)2 SO4; -2.0 MgSO4-1.0; NaCl-1.0 PT / 2025 / 10079 8 PM-8 Starch-10.0; Casein-10.0; Peptone-1.0; Yeast extract-1.0 9 PM-9 KNO3-1.0; K2HPO4 -0.5; MgSO4-0.5; NaCl-0.5g; FeSO4-0.010; Starch-20.0 10 PM-10 Oatmeal-10.0; MgSO4-1.0; KCl-1.0; KH2PO4-0.5; K2HP04-0.5; CaCl2-2.0; Yeast extract-4.0; Malt extract-4.0 11 PM-11 Starch-24.0; Dextrin-46.0; Yeast extract-5.0; Peptone-4.0; Soyabean meal-5.0; K2HPO4-1.0; CaCO3 -1.0; MgSO4-1.0 12 PM-12 yeast extract-1.0; beef extract-1.0; casamino acids-2.0; glucose- 10.0 Table 5: Components used for modified production medium Ingredients Composition (g / L) Soluble starch 24 Dextrin 46 Peptone 02 Soyabean meal 05 Yeast extract 7.5 K2HPO41.5 CaCO31.5 MgSO401 pH 7.0 Example 5: Effect of pH on Actinomycin D To study the effect of pH on the yield of Actinomycin D, the selected modified media was prepared in 500 mL Erlenmeyer flask with volume 100 mL; the pH of the selected media was adjusted to 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 using weak acid and base autoclaved, inoculated with seed culture and placed on rotary shaker at 28±2oC. The flasks were terminated after 2- 15 days and extracted using ethyl acetate solvent in rotary evaporator; quantified by HPLC. After HPLC quantification higher yield was observed at pH 7.0. Example 6: Effect of inoculum size on production of Actinomycin D To study the effect of inoculum percentage on the yield of Actinomycin D, different inoculum size i.e.5%, 10%, 15%, 20% were used. The modified medium was prepared in 500 mL Erlenmeyer flasks with volume 100 mL, autoclaved and inoculated with different PT / 2025 / 10079 inoculum as mentioned above, kept on rotary shaker at 28±2oC. The flasks were terminated after 2-15 days and broth was extracted using ethyl acetate solvent extraction system, dried on rotary evaporator using boiling point of solvent used. The samples were prepared in HPLC grade methanol and then quantified using HPLC. After HPLC quantification higher yield was observed with inoculum size of 10%. Example 7: Effect of air to volume ratio on the production Actinomycin D Aeration plays an important role on the overall yield of the secondary metabolites in case of aerobic fermentation. To study the effect of aeration, different air to volume ratio were used. The modified media was prepared in 1000mL Erlenmeyer flasks with different volume i.e.50 mL, 100mL, 200mL, 300mL, 400mL and inoculated with seed culture placed on rotary shaker with temperature maintained at 28±2oC. The flasks were terminated after 2-15 days and extracted using ethyl acetate solvent extraction method. The samples were prepared in HPLC grade methanol and then quantified. Example 8: Effect of temperature on Actinomycin D Temperature can be an important factor to play to increase the yield of Actinomycin D. Different temperatures were used i.e.10oC, 20oC, 30oC, 40oC to study its effect. Modified media was prepared in 500mL Erlenmeyer flasks with media volume 100mL, autoclaved and inoculated with seed culture and on rotary shaker maintained at different temperature i.e.10oC, 20oC, 30oC, 40oC. The flasks were terminated after 2-15 days and extracted using ethyl acetate solvent. The samples were quantified by HPLC. After HPLC quantification higher yield was observed at temperature 28oC. Example 9: Effect of carbon sources on Production of actinomycin D Different carbon sources were used to enhance the yield of actinomycin D like Glucose, Fructose, Sucrose, Mannitol, Glycerol, Pectin, Lactose, Maltose, Mannose, Gluconic acid, Chitosan, Dextrin, Starch, Xylose, Molasses, Corn steep liquor, Inositol, Chitin, Sorbitol. Out of these carbon sources, starch and dextrin in combination were found the main carbon sources to increase the yield of actinomycin D at maximum level. Example 10: Effect of nitrogen sources on Production of actinomycin D In addition to carbon sources, different nitrogen sources were also used to observe their effect on the yields of actinomycin D. Out of different nitrogen sources like soyabean meal, yeast PT / 2025 / 10079 extract, peptone, sodium / potassium nitrate, casein, casein hydrolysate and ammonium nitrate, urea, arginine, asparagine, ammonium phosphate, potassium nitrate, ammonium sulphate, beef extract; yeast extract, soyabean meal, peptone were found to be the suitable nitrogen sources for the maximum yield of actinomycin D and its analogues. Example 10: Production of actinomycin D The time course of Actinomycin D and its analogues production was evaluated by transferring the freshly grown cultures in seed inoculum and further to the production medium. The growth and production were evaluated by sampling the fermentation broth after regular intervals. Antibiotic complex formation starts after 24 hours until 12 days of incubation. Actinomycin D and its analogues were produced in 5L to 500L stirred tank bioreactor using modified production medium PM-11 (Table 5) with combination of other ingredients providing additional carbon / nitrogen / minerals and elicitations. The inoculum was prepared using freshly grown culture of vegetative stage in conical flask using PM-2 medium (Table 3). The 5-10% of 2-4 days old inoculum was transferred to the fermenter. For the efficient growth the volume of air used was 0.05 to 1.5 (vvm) with agitation speed of 50 to 400 RPM at 28oC±2oC for 2-15 days. Fermentation was carried out by transferring the pre- inoculum prepared in a narrow mouth 1L Erlenmeyer flask containing 200 mL medium and inoculating at about 28oC±2oC for about 2-12 days on a rotating shaker at 200 rpm to the production medium (Constituents in g / L: Soluble starch 24.0; dextrin 46; peptone 2.0; soyabean meal 5.0; yeast extract 7.5; K2HPO41.5; CaCO31.5; MgSO41.0; pH 7.0±0.2). The culture was allowed to grow at 28oC±2oC under shaking conditions and production of Actinomycin D was monitored for 12 days during fermentation. Optimum yield of 3.0g / L has been obtained with the productivity rate of 15mg / L / h. Example 11: Strain development The protoplasts were prepared from the culture of Streptomyces marokkonensis using enzymatic digestion to remove the cell wall, ensuring the formation of stable, viable protoplasts. The resulting protoplasts were then subjected to UV-induced mutagenesis under a 15 watt UV lamp for 10 minutes with a distance of 50 cm, ensuring uniform exposure to induce genetic variations. Following UV treatment, the protoplasts were carefully recovered in an osmotic stabilizing medium to enhance their survival and were subsequently plated on a suitable regeneration medium to assess viability. PT / 2025 / 10079 Example 12: Production and purification of actinomycin D using modified strain The UV mutant culture was used as inoculum for seed production as given in Table 3 and then after 48-72 hours, the seed was transferred to a modified production medium (composition as given in the previous example). With the UV mutant culture, the yield of actinomycin D has been enhanced to ~4.0 g / L within 10 days Example 13: Downstream processing of fermented broth (a) Extraction, isolation, and characterization of metabolites The fermented broth (25 L) recovered after fermentation was extracted with equal volume of ethyl acetate (EtOAc) three times at room temperature. Ethyl acetate was evaporated using rotatory evaporator giving orange red colored-oily residue which on partitioning between methanol (250 mL) and hexane (250 mL) separated out the oil portion in hexane layer and a red colour solid substance in the methanol layer. The methanol fraction (15.9 g) was subjected to column chromatography on Al2O3 and eluted by a stepwise hexane ethyl Thus, obtained fractions were pooled to three fractions (Fr.1 Fr.3) based on the similar TLC profiles. Fr.2 (5.56 g) obtained as yellow color solid showed one major peak along with two minor signals on the HPLC chromatogram (Figure 1). On HPLC, 0.1 g of the Fraction 2 was separated on RP-C18 column (SunFire Prep C18 OBD 10 µm 19X250 mm) 2O isocratic solvent system (Figure 2) to give Compound Actinomycin D (tR 47.1 min, 46 mg) as major constituent along with two minorcompounds Actinomyci tR 27.28 min, 7.0 mg), Actinomycin X2 (tR 42.54 min, 9.0mg) as pure compounds. (b) Purification and characterization of Actinomycin D from fermentation broth The fermented broth was homogenized followed by separation of cell biomass by filtration and filtrate was used for direct binding with macroporous adsorbent resin (Diaion HP20). Diaion HP20 column (3 cm x 60 cm, 150 mL) was prepared by soaking Diaion HP20 resin (Sigma Aldrich, USA) in Milli-Q water. Finally, the column was eluted with methanol at a flow rate of 2 mL / min, collecting 30 mL fractions. Each fraction was checked for its compositions by TLC using 100% EtOAc for TLC development. Anisaldehyde stain was used for detection of the fractions and all the fractions with actinomycin D were combined and concentrated to dryness to give orange red color powder (actinomycin D mg). The purity and recovery of pure compound was compared with the conventional solvent extraction method to validate the results (Table 6). The structure of the Actinomycin D was confirmed PT / 2025 / 10079 Based on1H,13C NMR and MS data and further their comparison with literature values. Experiments were performed on a Waters 1525 HPLC system (Waters, Milford, MA, USA) equipped with a binary gradient pump, auto sampler, column oven, photodiode array detector, and data acquirement and processing was operated by the Waters Empower 3 software 5 (Waters, Milford, MA, USA). All chemicals and reagents used in the study were of high purity. Ultra deionized water utilized in the study was obtained by a Milli-Q water were from CDH, HPLC grade Acetic acid, purchased from Merck, India, and was used as the mobile phase, whereas LC MS grade methanol was used for sample preparation. 1H and 13C10 spectroscopic data in Pyridine-D5 were recorded on Bruker-Advance DPX FT-NMR 400 MHz instruments (100 MHz for13C NMR). Example 12: Comparison of single step purification with conventional solvent extraction method 15 Present invention involves direct purification of Actinomycin D from the fermented broth without using solvent extraction in the process as compared to the conventional process used for the purification in the prior art. Present process yields (4.0 g / L) are better as compared to the conventional method used for purification of actinomycin D i.e.2.1g / L. Present process also reduces the time and solvent cost used in the conventional method. In terms of recovery 20 rate is also high i.e. >90% as compared to percent recovery in conventional method. Purity of the product in study is equivalent to the conventional method i.e >95%. Table 6: Comparison of different parameters of direct purification method with solvent extraction method Parameter Solvent extraction Direct purification method method (present invention) Yields 2.1 g / L 4.0 g / L Recovery (purified ACT D) ~50% >90% Purity >95% >95% 25 After fermentation, initially, the portion of the whole broth was evaluated for antimicrobial activity against panel of pathogens such as Staphylococcus aureus etc. using disk diffusion method. The broth showed prominent zone of inhibition against Staphylococcus aureus. PT / 2025 / 10079 Then, 1 mL of the whole culture broth were diluting with equal volumes of HPLC methanol, standing for 10 h, finally centrifugation for 10 min. Towards the goal of the dereplication of active secondary metabolites in the culture broth, the above supernatant was investigated by HPLC-PDA followed by LC-MS-DNP analysis. This study was 5 intended to elucidation of the main secondary metabolites present in broths before purification required. This initial analysis revealed a major constituents UV absorption maxima, molecular ions and fragmentations, a search of the Dictionary of Natural Products 10 database (American Chemical Society) showed that a number of common features associated with chromo peptide that to be further confirmed.

Claims

PT / 2025 / 10079 WE CLAIM:

1. A process for the preparation of Actinomycin of Formula IFormula I 5 from Streptomyces marokkonensis MTCC-25511 strain comprising the steps of: (i) isolating the Streptomyces marokkonensis MTCC-25511 strain from soil sample of cold deserts of NW Himalayas 34.57450N, 76.23200E (Barchey) India; (ii) preparing the seed inoculum from the isolated strain Streptomyces marokkonensis MTCC-25511 strain as obtained in step (i) and transferring 5-10% of three days 10 old seed culture to the production medium containing one or more carbon source (2 to 5% w / v), nitrogen source (0.1 to 1%) and trace salts at pH in the range of 5.0 to 10.0 followed by incubating at a temperature in the range of 10°C-40oC for a period in the range of 2-15 days on a rotating shaker at speed in the range of 50-400 rpm to obtain fermented broth; 15 (iii) fermentation was initiated in 5-500L bioreactor by inoculation of 10% inoculum as obtained in step (ii) in production medium having two carbon sources i.e. starch (24g / L) and dextrin (46g / L) with nitrogen source i.e. soyabean meal (5g / L), yeast extract (7.5g / L), peptone2g / L) along with trace elements by providing 10 LPM air at 100 rpm agitation rate. 20 2. The process as claimed in claim 1, wherein major compounds of Formula 1 were purified from the crude solvent extract as well as directly from the fermented broth by open glass gravity column in a single step;PT / 2025 / 10079 (i) liquid-liquid extraction of fermented broth of 2-15 days as obtained in Claim 1steps (i to iii) using selected solvents from the group comprising ethyl acetate,to obtain actinomycins of Formula I;5 (ii) purifying the extract obtained from step (i) by column chromatography on Silicagel or Al2O3 and elution by a stepwise hexane ethyl acetate gradient followedby HPLC purification on C18 column; (iii) concentrating the fermented broth under reduced pressure and purification bydirect single step involving binding of the metabolites of fermented broth with 10 macroporous resin and elution of purified compound Actinomycin D using methanol / water / acetone.

3. The process as claimed in Claim 1-2, wherein formula I is selected from the groupcomprising of compounds Actinomycin D, Actinomycin X2 and Actinomycin 0ß 15groupcomprising monosaccharides, disaccharides and polysaccharides like Glucose, fructose, sucrose, mannitol, glycerol, gluconic acid, pectin, lactose, maltose, mannose, chitosan, 20 dextrin, molasses, starch, xylose, molasses, corn steep liquor, inositol, chitin, sorbitol either alone or combination thereof.

5. The process as claimed in claims 1-4, wherein nitrogen sources are selected from thegroup comprising beef extract, meat solubles, corn meal, casein, soyabean meal, yeast 25 extract, n / z amine a, n / z amine b, casein hydrolysate, enzyme-hydrolyzed casein, peptone, sodium nitrate, valine, ammonium nitrate, urea, arginine, asparagine,PT / 2025 / 10079 ammonium phosphate, potassium nitrate, ammonium sulphate, nitrate salts either alone or combination thereof.

6. The process as claimed in claims 1-5, wherein trace salts are selected from the group 5 comprising calcium carbonate, magnesium sulphate, di-potassium hydrogen phosphate, ferrous sulphate, potassium chloride and sodium chloride, ammonium sulphate / nitrate.

7. The process as claimed in Claim 1-6, wherein the major compound Actinomycin D has been purified by both conventional purification methods such as solvent and direct single 10 step purification by using macroporous adsorbent resin ; (i) Purification by conventional method involved column chromatography on Silica gel or Al2O3and elution by a stepwise hexane ethyl ac followed by purification by HPLC purification on methanol / water / acetonitrile 15 column to give Compound Actinomycin D (46 mg) as major constituent along with ctinomycin X2 as pure compounds; (ii) Purification by direct single step involved binding of the metabolites of fermented broth with macroporous adsorbent resin and elution of purified compound Actinomycin D using methanol / water / acetone. 20 8. The process as claimed in Claims 1-7, wherein the major compound Actinomycin D has been purified by conventional solvent and single step purification in time period 2-15 days with yield of 4.0 g / L with a modified stain Streptomyces marokkonensis MTCC- 25511 in 10 days. 25 9. The method as claimed in Claims 1-8, wherein the Actinomycin D productivity is (~16.7mg / L / h) , recovery is 90% & purity is >95%.

Images