Indian Patents. 208723:"PROCESS FOR PRODUCING 6-HYDROXYNICOTING ACID USING A MICROBIAL STRAIN YERSINIA PSEUDOTUBERCULOSIS MTCC 5129"

Title of Invention	"PROCESS FOR PRODUCING 6-HYDROXYNICOTING ACID USING A MICROBIAL STRAIN YERSINIA PSEUDOTUBERCULOSIS MTCC 5129"
Abstract	Disclosed herein is a process for producing 6-hydroxynicotinic acid via biotransformation, the process comprises, hydroxylation of nicotinic acid in aqueous medium using a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme activity under mild reaction conditions.

Full Text	Field of the Invention This Invention in general relates to the process for preparation of 6-substituted nicotinic acid via biotransformation, more particularly the invention relates to the process for preparation of 6-hydroxynicotinic acid comprising, microbiological hydroxylation of nicotinic acid employing novel Yersinia pseudotuberculosis MTCC 5129 cells. Background of the Invention 6-Hydroxynicotinic acid is an important derivative of nicotinic acid. It is generally used for the preparation of several new neonicotinoid insecticides, which has become an important pest control agent on many crops, and is also used for the synthesis of new generation insecticides, "halonicotinylimidazolidine" e.g. Imadacloprid and pharmaceutical ingredients e.g. pyridyl-pyridazinone compounds effective for the treatment of congestive heat failure and the antilipolytic drug, N-6-chloronicotinyl-d, 1-homocysteine thiolactone. Several chemical and biological processes are disclosed in prior patent disclosures for the production of 6-hydroxynicotinic acid by using different chemical agent and biological agent. United States Patent No. 4,609,734 to Quarroz, et al., discloses the chemical synthesis for producing 6-hydroxynicotinic acid, the process disclosed adding acetic acid anhydride or propionic acid anhydride and Et3N or tributylamine and CCl4 or petrolether in a flask, and isocinchomeronic acid-N-oxide was added by portions to this solution. The temperature was raised to 50°C. After completion of the reaction the solvents were distilled off. About 100 ml of 20% KOH was added drop by drop to the viscous residue and the acetate was saponified over a 15 minute period at 80°C. After removal of the Et3N by extraction, the aqueous solution was acidified with concentrated HC1. The precipitate was sucked off, washed with water and dried under a vacuum. 11.0 g of 6-hydroxy nicotinic acid was obtained, which was pure according to NMR. Above discussed processes have the disadvantages of low product yield due to formation of undesirable by-products and also chemical synthesis of 6-hydroxynicotinic acid is difficult and expensive due to the separation of by-product. There are various biological processes disclosed in the prior patents, which have in one way or the other overcome the disadvantages associated with the chemical synthesis. By using microorganism or enzymes, biotransformation of nicotinic acid to 6-hydroxynicotinic acid make comparatively easier and cost efficient processes than chemical synthesis. United States Patent No. 5,082,777 to Lehky, et al., discloses a process for the production of 6-hydroxynicotinic acid from nicotinic acid. The hydroxylation is carried out enzymatically in the presence of a microorganism of the species Pseudomonas, Bacillus or Achromobacter, the process is carried out at 20° to 40°C and a pH of 5.5 to 9.0 under aerobic conditions, the concentration of the nicotinic acid being 0.5 to 10 percent by weight, based on the weight of the aqueous solution, which is effective to substantially prevent the enzymatic conversion of the 6-hydroxynicotinic acid. United States Patent No. 4,738,924 to Kulla, et al., discloses a process for the production of 6-hydroxynicotinic acid by enzymatic hydroxylation of nicotinic acid in the presence of equivalent quantities of magnesium or barium ions with the help of nicotinic acid-hydroxylating microorganisms, such as Pseudomonas, Bacillus or Achromobacter, for example, Achromobacter xylosoxydans at 20° to 40°C and a pH of 5.5 to 9.0 under aerobic conditions United States Patent No. 5,151,351 to Hoeks, et al., discloses a process for the production of 6-hydroxynicotinic acid by microbiological hydroxylation of nicotinic acid under aerobic conditions, by maintaining a specific concentration range during the addition of mcotinic acid, the biomass formation can take place in the same process step as the product formation, without product losses occurring by further decomposition. The microorganisms used in the process are Achromobacter xylosoxydans of strain DSM 2783, Pseudomonas putida of strain NCIB 10521 and Pseudomonas putida of strain NCIB 8176. United States Patent No. 5,264,362 to Kiener, et al., discloses microbiological process for the production of 6-hydroxynicotinic acid. The process comprises biotransformation of 3-cyanopyridine with the microorganisms to 6-hydroxynicotinic acid. Preferably the effective enzymes of the microorganisms are induced with 3-cyanopyridine. Preferably the reaction takes place under substrate addition once or continuously so that the substrate concentration does not exceed 20 percent by weight. Preferably the reaction is performed at a pH of 4 to 10 and a temperature of 10° to 50°C. Known literature disclosing various processes for producing 6-hydroxynicotinic acid using microorganism include the ones reported in J Biol Chem (1943) 147, 785-791 and J Biol Chem (1957) 228, 923- 945, wherein disclosed is a process comprising biotransformation of nicotinic acid to 6-hydroxynicotinic acid using live microorganisms of the genera. Pseudomonas, Bacillus. All the bioconversion processes of production of 6-hydroxynicotinic acid from nicotinic acid use a bio-mass suspension of the corresponding microorganisms, which are obtained in a separate process step by multiplication of starter culture. The actual hydroxylation takes place either in a batch process with single addition of nicotinic acid as the sodium salt or in a continuous process in which nicotinic acid is added as the magnesium or barium salts and the 6-hydroxynicotinic acid is isolated as slightly soluble magnesium or barium salt. Since the multiplication of the microorganisms is inhibited by the nicotinic acid concentrations used in such processes, it is necessary in both processes to obtain the total amount of the effective biomass in an upstream process step. The continuous process according to United States Patent No. 4,738,924 yields magnesium or barium salts from which the 6-hydroxynicotinic acid is released by acid addition. In this case, the magnesium or barium salt of the added acid results as waste, which has to be disposed off, but especially soluble barium salts are highly toxic for higher microorganisms. The nicotinic acid hydroxylase can also be isolated from cell extracts and the enzyme preparations can be used for the hydroxylation of nicotinic acid. This has been done and small quantities of 6-hydroxynicotinic acid were actually obtained (Behrmen, EJ. and Stainier, R.Y., J Biol Chem (1957) 228, 923- 945). Apart from the high costs of enzyme isolation and/or the instability of the nicotinic acid hydroxylase it was still necessary to take care of the requirements of the cofactor and electron transport systems. In case of many fermentation and enzyme reactions, the product concentration in the reaction solution are very low and in the case of product isolation, it is necessary therefore to process large volumes of materials. Such process leads to high cost of processing, investment and effluent treatment. The enzymatic hydroxylation can be carried out with 0.1 percent by weight up to a saturated nicotinic acid solution. The stability of the enzyme located in the cells, however, has been considerably lowered in the case of higher substrate concentrations. The problems associated with the above prior art can be overcome by using a novel microorganism and through developed techniques. The present invention provides a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid using a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme. The process disclosed herein the present invention performs in an economic manner with high yield and product purity, which overcome the above stated disadvantages. Summary of the Invention In accordance with the principle embodiment of the present invention, there is provided a process for producing 6-hydroxynicotinic acid via biotransformation of nicotinic acid using a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells under mild reaction conditions e.g. atmospheric pressure, low temperature, near neutral pH to hydroxylate nicotinic acid to 6-hydroxynicotinic acid without any by-product formation. A process for producing 6-hydroxynicotinic acid comprising biotransformation of nicotinic acid to 6-hydroxynicotinic acid by hydroxylation of nicotinic acid in aqueous medium using a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells at atmospheric pressure and a temperature of about 25-35°C. In accordance with one preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein the process comprises hydroxylation of nicotinic acid in aqueous medium at a temperature of 25-35°C and pH 6.0-7.5 with Yersinia pseudotuberculosis MTCC 5129 cells, an isolated bacterial strain which is capable of specific hydroxylation of nicotinic acid to 6-hydroxnicotinic acid. In accordance with one another preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein the process comprises hydroxylation of nicotinic acid to produce 100 g 6-hydroxynicotinic acid per litre of reaction mixture by using Yersinia pseudotuberculosis MTCC 5129 cells under mild reaction conditions e.g. atmospheric pressure, pH in the range of 6.0 to 7.5, temperature 25-35°C. In accordance with one another preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein the cell mass of Yersinia pseudotuberculosis MTCC 5129 was reused for the production of 30-50 g of 6-Hydroxynicotinic acid per litre of reaction mixture without any by-product formation. In accordance with yet another preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme obtained by growing the cells in a conventional nutrient medium in presence of nicotinic acid as an inducer, wherein the conventional nutrient medium consists of glucose (0.5 to 2 g %), inorganic nutrients KH2PO4 (0.05 to 0.5 g %), Na2HPO4 (0.05 to 0.5 g %) MgSO4.7H2O (0.02 to 0.1 g %), FeSO4.7H2O and Na2MoO4.2H2O (0.001 to 0.005 g %), peptone, yeast extract in combination or alone (Complex nitrogen source) (0.5 to 2 g %) and nicotinic acid (0.1 to 1 g %). The microorganism was grown under aerobic conditions for 24 to 44 hrs in shaking flask or in a fermenter. In accordance with yet another preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme obtained by growing the cells in a conventional nutrient medium in presence of nicotinic acid as an inducer, wherein the inducer capable of inducing nicotinic acid hydroxylase is nicotinic acid (0.1 to 1 g%). In still another preferred embodiment of the present invention, there are provided conditions under which Yersinia pseudotuberculosis MTCC 5129 could be cultivated to produce cells having nicotinic acid hydroxylase enzyme may be varied over relatively wide range, the carbon sources (0.5 to 2.0 g %) used for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are selected from a member group consisting of glucose, sucrose, and sodium citrate, more preferred carbon source is glucose and nitrogen sources (0.5 to 2.0 g %) used for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are peptone and yeast extract. In still another preferred embodiment of the present invention, there is provided a process for the bio-transformation of nicotinic acid to 6-hydroxynictinic acid aerobically in an aqueous medium containing Yersinia pseudotuberculosis MTCC 5129 cells at a pH and temperature which are effective to achieve such enzymatic hydroxylation, wherein the typical pH range at which Yersinia pseudotuberculosis MTCC 5129 may be grown is 5 to 9 and the typical temperature range is 15-40°C, more preferred pH and temperature range is pH 7 to 8 and at 28-35°C. In still another preferred embodiment of the present invention, there is provided a process for biological conversion of nicotinic acid to 6-hydrxynicotinic acid by using immobilized cells of Yersinia pseudotuberculosis MTCC 5129, wherein immobilization can be conducted by suspending the cells in a suitable aqueous medium (e.g. water, physiological saline, buffer solution etc.) containing an acrylamide series monomer (e.g. acrylamide) and a cross linking agent (e.g. N,N'methylene bisacrylamide) adding a suitable polymerization initiator (e.g. ammonium persulphate) and a polymerization accelerator (e.g. N,N,N',N'-terra methyl ethylene diamine) to the suspension and conducting polymerization at about 0-30°C, preferably at 0-15°C at a pH of about 5-8, preferably at 6-7. In still another preferred embodiment of the present invention, there is provided a process for biological conversion of nicotinic acid to 6-hydroxynicotinic acid wherein about 98% conversion of nicotinic acid to 6-hydroxynicotinic acid can be achieved at 10% nicotinic acid concentration in the first use of these polyacrylamide immobilized cells under mild reaction conditions e.g. atmospheric pressure, pH in the range of 6.0 to 7.5, temperature 25-35°C. In still another preferred embodiment of the present invention, there is provided a bioconversion of the nicotinic acid to 6-hydroxynicotinic acid by using polyacrylamide immobilized cells of Yersinia pseudotuberculosis MTCC 5129, wherein these cells can be repeatedly used up to four successive batches at 5% nicotinic acid concentration under mild reaction conditions e.g. atmospheric pressure, pH in the range of 6.0 to 7.5, temperature 25- 35°C without considerable loss in enzyme activity. Detailed Description of the Invention The present invention aims to screening a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme for use of the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, which is isolated from the soil. Hydroxylation of nicotinic acid by means of biological conversion to get 6-hydroxynicotinic acid using the novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells, which makes the process more easier, time efficient and cost efficient and also prevent the formation of byproduct. The novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 disclosed herein the present invention is used as free cells or immobilized form on various supports such as polyacrylamide, calcium alginate, agar etc. Used novel microbial strain for hydroxylation of nicotinic acid to 6-hydroxynicotinic acid in aqueous medium is isolated and washed with tap water to avoid the cost of saline or buffer at commercial scale without affecting the enzyme activity. The microorganism used herein the present invention is isolated from a soil sample near pyridine plant site at Jubilant Organosys Limited, Gajraula, Uttar Pradesh, India and identified as Yersinia pseudotuberculosis by the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh-160036, India. The isolated microbial culture is deposited at Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh-160036, India and has been given the accession no. as Yersinia pseudotuberculosis MTCC 5129. The morphological and biochemical characteristics of bacterial strain are given in Table 1 Table 1 Morphological and biochemical characteristics of Yersinia pseudotuberculosis MTCC 5129 (Table Removed) Preferred embodiments are further illustrated in the following examples. These examples illustrate particular embodiments of the invention and are not intended to limit the scope of the invention in any way. Example 1 Yersinia pseudotuberculosis MTCC 5129 was cultivated at 30°C for 24 hrs with shaking at 200 RPM in an Erlenmeyer flask containing 50 ml of nutrient medium consisting of 1.0 g of glucose, 0.5 g of yeast extract, 0.5 g of peptone, 0.3 g of KH2PO4, 0.5 g of Na2HPO4, 0.02 g of MgSO4.7H2O, 0.001 g of FeSO4.7H2O, 0.001 g of Na2MoO4.2H2O and 0.4 g of nicotinic acid per 100 ml of tap water. pH was adjusted to 7.2. Cells were harvested by centrifugation followed by washing with tap water at 10,000 rpm at 20°C for 7 min. The washed cells then suspended in tap water. A small portion of the cell suspension was used for measuring the dry weight of the bacterial cells and the cell-yield was 3.0 g per liter of medium. The washed cells obtained as above were used for conversion of nicotinic acid to 6-hydroxynicotinic acid. In the reaction, washed cells of Yersinia pseudotuberculosis MTCC 5129 (60 mg dry weight equivalent) were suspended in 20 ml of 10 % nicotinic acid solution (pH adjusted to 6.2 with NaOH) in a 250 ml Erlenmeyer flask and the reaction was allowed to proceed at 30°C and 200 rpm in a orbital shaker incubator for 24 hours. After completion of the reaction, cell mass was removed through centrifugation. The supernatant obtained was then acidified to pH 1.5 with H2SO4/HC1, which resulted to white precipitate. The precipitated product was recovered by filtration and then dried at 80°C for 12 hours. The dried weight of product was 2.22 g, which contained 99.4% of 6-hydroxynicotinic acid according to HPLC analysis and the corresponding yield was 98% calculated on basis of nicotinic acid used. Example 2 1.35 litres of nutrient solution with the composition and pH described in example 1 was filled into a 3.0 litres fermenter and sterilized at 121°C for 30 min. The nutrient media was then inoculated with 150 ml of Yersinia pseudotuberculosis MTCC 5129 cell inoculum that had already been prepared separately using the same media composition and pH in an orbital shaker incubator at 30°C and 200 rpm for 18 hours. The fermenter was run at 30°C and at 200 rpm with an air pressure of 1.5 liter per min. After 24 hrs, fermentation was stopped and the cell mass was harvested followed by washing with tap water by centrifugation at 10000 rpm and 20°C for 7 min. A small portion of the cell suspension was used for measuring the dry weight of the bacterial cells and the cell-yield was 3.0 g per liter of medium. The washed cells was then used to hydroxylate 150 g of nicotinic acid dissolved in 1.5 lit of tap water (pH adjusted to 6.2 using NaOH) in the same fermenter used for culture grown at 30°C and 400 rpm with a air pressure 1.5 liter per min. After 48 hrs, the reaction was stopped and the cell mass was separated by centrifugation. The supernatant was then subjected to acidification using H2SO4/HCL to pH 1.5 that resulted to white precipitate formation. The precipitate was recovered by filtration under suction and then allowed to dry at 80°C for 12 hrs. The dry product was 167 g and according to HPLC analysis it contained 98% of 6-hydroxynicotinic acid. This corresponded to a yield of 98.5% that calculated on the basis of nicotinic acid used. Example 3 The separated cells after completion of the reaction as stated in example 2 was reused for conversion of nicotinic acid to 6-hydroxynicotinic acid at 5% substrate concentration in 1.5 liter of tap water (pH was adjusted to 6.2 by addition of NaOH). The reaction was allowed to proceed at 400 rpm, 30°C and 1.5 liter per min. air pressure for 48 hours. After the stoppage of the reaction, the cell mass was separated by centrifugation and the supernatant was acidified to pH 1.5 by addition of HaSOvHCl which resulted to white precipitate that is recovered by filtration under suction. The product thus obtained was further subjected to dry at 80°C for 12 hours. The dried product was 83.5 gram, which corresponded to 98.5% yield on the basis of substrate consumed. According to HPLC analysis the product purity was 99.60%. Example 4 Eight litres of nutrient solution with the composition and pH described in example 1 and example 2 filled into a 20 liter fermenter after sterilization at 120°C for 20 minutes. The nutrient medium was then inoculated with 2.0 litres Yersinia pseudotuberculosis MTCC 5129 inoculum that already prepared separately using the same medium composition in orbital shaker incubator at 30°C and 200 rpm for 18 hours. The fermenter was run at 30°C and at 200 rpm. After 24 hours fermentation was stopped and the cell mass was harvested followed by washing with tap water by centrifugation at 10000 rpm and at 20°C for 7 minutes. A small portion of the cultured cells was used for measuring the cell dried weight and the obtained cell yield was 2.8 g per liter of the medium. The washed cells was then used to hydroxylate 800 gram of nicotinic acid dissolved in 10 liter of tap water (pH was adjusted to 6.2 using NaOH) in a stirred tank reactor with 200 rpm at 30°C and 0.5 kg air pressure per minute. After 44 hours, the reaction was stopped and cell mass was separated by centrifugation. The supernatant was then subjected to acidification using H2SO4/HC1 to the pH 1.5 that resulted to white precipitate formation. The precipitate was recovered by filtration under suction and then allowed to dry at 80°C for 12 hours. The dried product was 880 gram and according to HPLC analysis it contained 98.9% of 6-hydroxynicotinic acid. This corresponded to a yield of 97.34% that calculated on the basis of nicotinic acid used. Example 5 The separated cells after completion of the reaction as stated in example 4 was reused for conversion of nicotinic acid to 6-hydroxynicotinic acid at 3% substrate concentration in 10 liter of tap water (pH was adjusted to 6.2 by addition of NaOH). The reaction was allowed to proceed at 200 rpm, 30°C and 0.5 kg air pressure per minute for 30 hours. After the stoppage of the reaction, the cell mass was separated by centrifugation and the supernatant was acidified to pH 1.5 by addition of H2SO4/HC1 which resulted to white precipitate that is recovered by filtration under suction. The product thus obtained was further subjected to dry at 80°C for 12 hours. The dried product was 317 gram, which corresponded to 93.5% yield on the basis of substrate consumed. According to HPLC analysis the product purity was 99.20%. Example 6 The washed cells of Yersinia pseudotuberculosis MTCC 5129 (1.2 g dry weight equivalent) prepared as given in example 1 were suspended in 10 ml of tap water and added to 20 ml of a solution of monomer (3.9 g acrylamide) and cross linker (0.39 g N,N' methylene bisacrylamide) in tap water to prepare uniform suspension. The polymerization was initiated by adding 0.1 ml of N,N,N',N'-tetra methyl ethylene diamine (99%) and 0.3 ml of ammonium persulphate solution (10% w/v in water). The polymerization was performed in a ice water bath (10°C) for 1 h. The gel block was cut into cubes (2 X 2.5 X 2.5 mm3) and these cubes were then thoroughly washed with water in order to remove unpolymerized monomers and residues. By using the immobilized cells as obtained above, the conversion of nicotinic acid to 6-hydroxynicotinic acid was conducted in a shaking flask. Reaction was carried out taking 20 g gel beads in 20 ml of 10% nicotinic acid solution (pH 6.2) in a 250 ml Erlenmeyer flask at 30°C and 200 rpm in a orbital incubator shaker for 30 h. After completion of reaction the reaction solution was separated out simply by decantation and left polyacrylamide gel beads were again subjected to reaction with 20 ml of 5% nicotinic acid solution in water keeping other conditions same as described in first use. This process was repeated for another three batches with 20 ml of 5% nicotinic acid solution in water under similar conditions as in first reuse. The clear reaction solution resulted from each batch after decantation was acidified to pH 1.5 with H2SO4/HC1 separately with resulted white precipitate of 6-hydroxynicotinic acid. According to HPLC analysis the product purity was 99.6%. The results are given in the Table 2 Table 2 (Table Removed) It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be falling within the scope of the invention, which is limited only by the following claims. We Claim: 1. A process for producing 6-hydroxynicotinic acid comprising: biotransformation of nicotinic acid to 6-hydroxynicotinic acid by hydroxylation of nicotinic acid in aqueous medium using a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells at atmospheric pressure and a temperature of 25 to 35°C and at pH 6 to 7.5. 2. The process according to claim 1, wherein the aqueous medium is tap water. 3. The process according to any of claims 1 or 2, wherein the Yersinia pseudotuberculosis MTCC 5129 cells are recoverable and reusable in the process. 4 The process according to any of claims 1 through to 3, wherein said bacterial strain Yersinia pseudotuberculosis MTCC 5129 cells is isolated from soil, which is capable of specific hydroxylation of nicotinic acid to 6-hydroxnicotinic acid. 5. The process according to claim 4, wherein said bacterial strain Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme is obtained by growing the cells in a nutrient medium, the nutrient medium comprising a carbon source, a nitrogen source, inorganic salts, inducer and recovering the cells by centrifugation followed by washing with tap water. 6. The process according to claim 5, wherein the carbon sources used for growing the strain Yersinia pseudotuberculosis MTCC 5129 are selected from a group comprising glucose, sucrose, and sodium citrate. 7. The process according to claim 6, wherein the carbon sources used for growing the strain Yersinia pseudotuberculosis MTCC 5129 are in the range of 0.5 to 2.0 g %. 8. The process according to claim 5, wherein the nitrogen sources used for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are peptone and yeast extract. 9. The process according to claim 8, wherein the nitrogen sources used for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are in the range of 0.5 to 2.0 g%. 10. The process according to claim 5, wherein the inorganic nutrients used for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are selected from a group comprising phosphate, sulphate, potassium, sodium, magnesium, molybdenum, iron and mixture thereof. 11. The process according to claim 10, wherein the inorganic nutrients used for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are in the range of 0.001 to 0.5 g %. 12. The process according to claim 5, wherein the inducer is nicotinic acid. 13. The process according to claim 12, wherein the range of the nicotinic acid is 0.1 to 1 g%. 14. The process according to claim 5, wherein the said microbial strain Yersinia pseudotuberculosis MTCC 5129 cells are grown at a pH of 5 to 9. 15. The process according to claim 14, wherein the said microbial strain Yersinia pseudotuberculosis MTCC 5129 cells are grown at a pH of 7 to 8. 16. The process according to claim 5, wherein the said microbial strain Yersinia pseudotuberculosis MTCC 5129 cells are grown at a temperature of l5-40°C. 17. The process according to claim 16, wherein the said microbial strain Yersinia pseudotuberculosis MTCC 5129 cells are grown at a temperature of 28-35°C. 18. The process according to any of claims 1 through to 17, wherein said bacterial strain Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme, obtained by growing the cells in a nutrient medium comprising glucose (0.5 to 2 g %), inorganic nutrients KH2PO4 (0.05 to 0.5 g %), Na2HPO4 (0.05 to 0.5 g %) MgSO4.7H2O (0.02 to 0.1 g %), FeSO4.7H2O and Na2MoO4.2H2O (0.001 to 0.005 g %), peptone, yeast extract in combination or alone (Complex nitrogen source) (0.5 to 2 g %) and nicotinic acid (0.1 to 1 g %), was grown under aerobic conditions for 24 to 44 hrs in shaking flask or in a fermenter at a temperature of 15-40 and at the pH of 5 to 9. 19. The process according to any of claims 1 through to 18, wherein the strain Yersinia pseudotuberculosis MTCC 5129 is used as free cells or in immobilized form. 20. A process for producing 6-hydroxynicotinic acid according to any of the preceding claims and supported by the description and the examples.

Full Text

Field of the Invention
This Invention in general relates to the process for preparation of 6-substituted nicotinic acid via biotransformation, more particularly the invention relates to the process for preparation of 6-hydroxynicotinic acid comprising, microbiological hydroxylation of nicotinic acid employing novel Yersinia pseudotuberculosis MTCC 5129 cells.
Background of the Invention
6-Hydroxynicotinic acid is an important derivative of nicotinic acid. It is generally used for the preparation of several new neonicotinoid insecticides, which has become an important pest control agent on many crops, and is also used for the synthesis of new generation insecticides, "halonicotinylimidazolidine" e.g. Imadacloprid and pharmaceutical ingredients e.g. pyridyl-pyridazinone compounds effective for the treatment of congestive heat failure and the antilipolytic drug, N-6-chloronicotinyl-d, 1-homocysteine thiolactone.
Several chemical and biological processes are disclosed in prior patent disclosures for the production of 6-hydroxynicotinic acid by using different chemical agent and biological agent.
United States Patent No. 4,609,734 to Quarroz, et al., discloses the chemical synthesis for producing 6-hydroxynicotinic acid, the process disclosed adding acetic acid anhydride or propionic acid anhydride and Et3N or tributylamine and CCl4 or petrolether in a flask, and isocinchomeronic acid-N-oxide was added by portions to this solution. The temperature was raised to 50°C. After completion of the reaction the solvents were distilled off. About 100 ml of 20% KOH was added drop by drop to the viscous residue and the acetate was saponified over a 15 minute period at 80°C. After removal of the Et3N by extraction, the aqueous solution was acidified with concentrated HC1. The precipitate was sucked off, washed with water and dried under a vacuum. 11.0 g of 6-hydroxy nicotinic acid was obtained, which was pure according to NMR.
Above discussed processes have the disadvantages of low product yield due to formation of undesirable by-products and also chemical synthesis of 6-hydroxynicotinic acid is difficult and expensive due to the separation of by-product.
There are various biological processes disclosed in the prior patents, which have in one way or the other overcome the disadvantages associated with the chemical synthesis. By using microorganism or enzymes, biotransformation of nicotinic acid to 6-hydroxynicotinic acid make comparatively easier and cost efficient processes than chemical synthesis.
United States Patent No. 5,082,777 to Lehky, et al., discloses a process for the production of 6-hydroxynicotinic acid from nicotinic acid. The hydroxylation is carried out enzymatically in the presence of a microorganism of the species Pseudomonas, Bacillus or Achromobacter, the process is carried out at 20° to 40°C and a pH of 5.5 to 9.0 under aerobic conditions, the concentration of the nicotinic acid being 0.5 to 10 percent by weight, based on the weight of the aqueous solution, which is effective to substantially prevent the enzymatic conversion of the 6-hydroxynicotinic acid.
United States Patent No. 4,738,924 to Kulla, et al., discloses a process for the production of 6-hydroxynicotinic acid by enzymatic hydroxylation of nicotinic acid in the presence of equivalent quantities of magnesium or barium ions with the help of nicotinic acid-hydroxylating microorganisms, such as Pseudomonas, Bacillus or Achromobacter, for example, Achromobacter xylosoxydans at 20° to 40°C and a pH of 5.5 to 9.0 under aerobic conditions
United States Patent No. 5,151,351 to Hoeks, et al., discloses a process for the production of 6-hydroxynicotinic acid by microbiological hydroxylation of nicotinic acid under aerobic conditions, by maintaining a specific concentration range during the addition of mcotinic acid, the biomass formation can take place in the same process step as the product formation, without product losses occurring by further decomposition. The microorganisms used in the process are Achromobacter
xylosoxydans of strain DSM 2783, Pseudomonas putida of strain NCIB 10521 and Pseudomonas putida of strain NCIB 8176.
United States Patent No. 5,264,362 to Kiener, et al., discloses microbiological process for the production of 6-hydroxynicotinic acid. The process comprises biotransformation of 3-cyanopyridine with the microorganisms to 6-hydroxynicotinic acid. Preferably the effective enzymes of the microorganisms are induced with 3-cyanopyridine. Preferably the reaction takes place under substrate addition once or continuously so that the substrate concentration does not exceed 20 percent by weight. Preferably the reaction is performed at a pH of 4 to 10 and a temperature of 10° to 50°C.
Known literature disclosing various processes for producing 6-hydroxynicotinic acid using microorganism include the ones reported in J Biol Chem (1943) 147, 785-791 and J Biol Chem (1957) 228, 923- 945, wherein disclosed is a process comprising biotransformation of nicotinic acid to 6-hydroxynicotinic acid using live microorganisms of the genera. Pseudomonas, Bacillus.
All the bioconversion processes of production of 6-hydroxynicotinic acid from nicotinic acid use a bio-mass suspension of the corresponding microorganisms, which are obtained in a separate process step by multiplication of starter culture. The actual hydroxylation takes place either in a batch process with single addition of nicotinic acid as the sodium salt or in a continuous process in which nicotinic acid is added as the magnesium or barium salts and the 6-hydroxynicotinic acid is isolated as slightly soluble magnesium or barium salt. Since the multiplication of the microorganisms is inhibited by the nicotinic acid concentrations used in such processes, it is necessary in both processes to obtain the total amount of the effective biomass in an upstream process step. The continuous process according to United States Patent No. 4,738,924 yields magnesium or barium salts from which the 6-hydroxynicotinic acid is released by acid addition. In this case, the magnesium or barium salt of the added acid results as waste, which has to be disposed off, but especially soluble barium salts are highly toxic for higher microorganisms.
The nicotinic acid hydroxylase can also be isolated from cell extracts and the enzyme preparations can be used for the hydroxylation of nicotinic acid. This has been done and small quantities of 6-hydroxynicotinic acid were actually obtained (Behrmen, EJ. and Stainier, R.Y., J Biol Chem (1957) 228, 923- 945). Apart from the high costs of enzyme isolation and/or the instability of the nicotinic acid hydroxylase it was still necessary to take care of the requirements of the cofactor and electron transport systems.
In case of many fermentation and enzyme reactions, the product concentration in the reaction solution are very low and in the case of product isolation, it is necessary therefore to process large volumes of materials. Such process leads to high cost of processing, investment and effluent treatment.
The enzymatic hydroxylation can be carried out with 0.1 percent by weight up to a saturated nicotinic acid solution. The stability of the enzyme located in the cells, however, has been considerably lowered in the case of higher substrate concentrations.
The problems associated with the above prior art can be overcome by using a novel microorganism and through developed techniques.
The present invention provides a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid using a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme. The process disclosed herein the present invention performs in an economic manner with high yield and product purity, which overcome the above stated disadvantages.
Summary of the Invention
In accordance with the principle embodiment of the present invention, there is provided a process for producing 6-hydroxynicotinic acid via biotransformation of nicotinic acid using a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells under
mild reaction conditions e.g. atmospheric pressure, low temperature, near neutral pH to hydroxylate nicotinic acid to 6-hydroxynicotinic acid without any by-product formation.
A process for producing 6-hydroxynicotinic acid comprising biotransformation of nicotinic acid to 6-hydroxynicotinic acid by hydroxylation of nicotinic acid in aqueous medium using a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells at atmospheric pressure and a temperature of about 25-35°C.
In accordance with one preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein the process comprises hydroxylation of nicotinic acid in aqueous medium at a temperature of 25-35°C and pH 6.0-7.5 with Yersinia pseudotuberculosis MTCC 5129 cells, an isolated bacterial strain which is capable of specific hydroxylation of nicotinic acid to 6-hydroxnicotinic acid.
In accordance with one another preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein the process comprises hydroxylation of nicotinic acid to produce 100 g 6-hydroxynicotinic acid per litre of reaction mixture by using Yersinia pseudotuberculosis MTCC 5129 cells under mild reaction conditions e.g. atmospheric pressure, pH in the range of 6.0 to 7.5, temperature 25-35°C.
In accordance with one another preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein the cell mass of Yersinia pseudotuberculosis MTCC 5129 was reused for the production of 30-50 g of 6-Hydroxynicotinic acid per litre of reaction mixture without any by-product formation.
In accordance with yet another preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid
hydroxylase enzyme obtained by growing the cells in a conventional nutrient medium in presence of nicotinic acid as an inducer, wherein the conventional nutrient medium consists of glucose (0.5 to 2 g %), inorganic nutrients KH2PO4 (0.05 to 0.5 g %), Na2HPO4 (0.05 to 0.5 g %) MgSO4.7H2O (0.02 to 0.1 g %), FeSO4.7H2O and Na2MoO4.2H2O (0.001 to 0.005 g %), peptone, yeast extract in combination or alone (Complex nitrogen source) (0.5 to 2 g %) and nicotinic acid (0.1 to 1 g %). The microorganism was grown under aerobic conditions for 24 to 44 hrs in shaking flask or in a fermenter.
In accordance with yet another preferred embodiment of the present invention, there is provided a process for the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, wherein Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme obtained by growing the cells in a conventional nutrient medium in presence of nicotinic acid as an inducer, wherein the inducer capable of inducing nicotinic acid hydroxylase is nicotinic acid (0.1 to 1 g%).
In still another preferred embodiment of the present invention, there are provided conditions under which Yersinia pseudotuberculosis MTCC 5129 could be cultivated to produce cells having nicotinic acid hydroxylase enzyme may be varied over relatively wide range, the carbon sources (0.5 to 2.0 g %) used for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are selected from a member group consisting of glucose, sucrose, and sodium citrate, more preferred carbon source is glucose and nitrogen sources (0.5 to 2.0 g %) used for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are peptone and yeast extract.
In still another preferred embodiment of the present invention, there is provided a process for the bio-transformation of nicotinic acid to 6-hydroxynictinic acid aerobically in an aqueous medium containing Yersinia pseudotuberculosis MTCC 5129 cells at a pH and temperature which are effective to achieve such enzymatic hydroxylation, wherein the typical pH range at which Yersinia pseudotuberculosis MTCC 5129 may be grown is 5 to 9 and the typical temperature range is 15-40°C, more preferred pH and temperature range is pH 7 to 8 and at 28-35°C.
In still another preferred embodiment of the present invention, there is provided a process for biological conversion of nicotinic acid to 6-hydrxynicotinic acid by using immobilized cells of Yersinia pseudotuberculosis MTCC 5129, wherein immobilization can be conducted by suspending the cells in a suitable aqueous medium (e.g. water, physiological saline, buffer solution etc.) containing an acrylamide series monomer (e.g. acrylamide) and a cross linking agent (e.g. N,N'methylene bisacrylamide) adding a suitable polymerization initiator (e.g. ammonium persulphate) and a polymerization accelerator (e.g. N,N,N',N'-terra methyl ethylene diamine) to the suspension and conducting polymerization at about 0-30°C, preferably at 0-15°C at a pH of about 5-8, preferably at 6-7.
In still another preferred embodiment of the present invention, there is provided a process for biological conversion of nicotinic acid to 6-hydroxynicotinic acid wherein about 98% conversion of nicotinic acid to 6-hydroxynicotinic acid can be achieved at 10% nicotinic acid concentration in the first use of these polyacrylamide immobilized cells under mild reaction conditions e.g. atmospheric pressure, pH in the range of 6.0 to 7.5, temperature 25-35°C.
In still another preferred embodiment of the present invention, there is provided a bioconversion of the nicotinic acid to 6-hydroxynicotinic acid by using polyacrylamide immobilized cells of Yersinia pseudotuberculosis MTCC 5129, wherein these cells can be repeatedly used up to four successive batches at 5% nicotinic acid concentration under mild reaction conditions e.g. atmospheric pressure, pH in the range of 6.0 to 7.5, temperature 25- 35°C without considerable loss in enzyme activity.
Detailed Description of the Invention
The present invention aims to screening a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme for use of the biotransformation of nicotinic acid to 6-hydroxynicotinic acid, which is isolated from the soil.
Hydroxylation of nicotinic acid by means of biological conversion to get 6-hydroxynicotinic acid using the novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells, which makes the process more easier, time efficient and cost efficient and also prevent the formation of byproduct.
The novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 disclosed herein the present invention is used as free cells or immobilized form on various supports such as polyacrylamide, calcium alginate, agar etc.
Used novel microbial strain for hydroxylation of nicotinic acid to 6-hydroxynicotinic acid in aqueous medium is isolated and washed with tap water to avoid the cost of saline or buffer at commercial scale without affecting the enzyme activity.
The microorganism used herein the present invention is isolated from a soil sample near pyridine plant site at Jubilant Organosys Limited, Gajraula, Uttar Pradesh, India and identified as Yersinia pseudotuberculosis by the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh-160036, India. The isolated microbial culture is deposited at Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh-160036, India and has been given the accession no. as Yersinia pseudotuberculosis MTCC 5129. The morphological and biochemical characteristics of bacterial strain are given in Table 1
Table 1
Morphological and biochemical characteristics of Yersinia pseudotuberculosis MTCC 5129
(Table Removed)
Preferred embodiments are further illustrated in the following examples. These examples illustrate particular embodiments of the invention and are not intended to limit the scope of the invention in any way.
Example 1
Yersinia pseudotuberculosis MTCC 5129 was cultivated at 30°C for 24 hrs with shaking at 200 RPM in an Erlenmeyer flask containing 50 ml of nutrient medium consisting of 1.0 g of glucose, 0.5 g of yeast extract, 0.5 g of peptone, 0.3 g of KH2PO4, 0.5 g of Na2HPO4, 0.02 g of MgSO4.7H2O, 0.001 g of FeSO4.7H2O, 0.001 g of Na2MoO4.2H2O and 0.4 g of nicotinic acid per 100 ml of tap water. pH was adjusted to 7.2. Cells were harvested by centrifugation followed by washing with tap water at 10,000 rpm at 20°C for 7 min. The washed cells then suspended in tap water. A small portion of the cell suspension was used for measuring the dry weight of the bacterial cells and the cell-yield was 3.0 g per liter of medium. The washed cells obtained as above were used for conversion of nicotinic acid to 6-hydroxynicotinic acid. In the reaction, washed cells of Yersinia pseudotuberculosis MTCC 5129 (60 mg dry weight equivalent) were suspended in 20 ml of 10 % nicotinic acid solution (pH adjusted to 6.2
with NaOH) in a 250 ml Erlenmeyer flask and the reaction was allowed to proceed at 30°C and 200 rpm in a orbital shaker incubator for 24 hours. After completion of the reaction, cell mass was removed through centrifugation. The supernatant obtained was then acidified to pH 1.5 with H2SO4/HC1, which resulted to white precipitate. The precipitated product was recovered by filtration and then dried at 80°C for 12 hours. The dried weight of product was 2.22 g, which contained 99.4% of 6-hydroxynicotinic acid according to HPLC analysis and the corresponding yield was 98% calculated on basis of nicotinic acid used.
Example 2
1.35 litres of nutrient solution with the composition and pH described in example 1 was filled into a 3.0 litres fermenter and sterilized at 121°C for 30 min. The nutrient media was then inoculated with 150 ml of Yersinia pseudotuberculosis MTCC 5129 cell inoculum that had already been prepared separately using the same media composition and pH in an orbital shaker incubator at 30°C and 200 rpm for 18 hours. The fermenter was run at 30°C and at 200 rpm with an air pressure of 1.5 liter per min. After 24 hrs, fermentation was stopped and the cell mass was harvested followed by washing with tap water by centrifugation at 10000 rpm and 20°C for 7 min. A small portion of the cell suspension was used for measuring the dry weight of the bacterial cells and the cell-yield was 3.0 g per liter of medium. The washed cells was then used to hydroxylate 150 g of nicotinic acid dissolved in 1.5 lit of tap water (pH adjusted to 6.2 using NaOH) in the same fermenter used for culture grown at 30°C and 400 rpm with a air pressure 1.5 liter per min. After 48 hrs, the reaction was stopped and the cell mass was separated by centrifugation. The supernatant was then subjected to acidification using H2SO4/HCL to pH 1.5 that resulted to white precipitate formation. The precipitate was recovered by filtration under suction and then allowed to dry at 80°C for 12 hrs. The dry product was 167 g and according to HPLC analysis it contained 98% of 6-hydroxynicotinic acid. This corresponded to a yield of 98.5% that calculated on the basis of nicotinic acid used.
Example 3
The separated cells after completion of the reaction as stated in example 2 was reused for conversion of nicotinic acid to 6-hydroxynicotinic acid at 5% substrate concentration in 1.5 liter of tap water (pH was adjusted to 6.2 by addition of NaOH). The reaction was allowed to proceed at 400 rpm, 30°C and 1.5 liter per min. air pressure for 48 hours. After the stoppage of the reaction, the cell mass was separated by centrifugation and the supernatant was acidified to pH 1.5 by addition of HaSOvHCl which resulted to white precipitate that is recovered by filtration under suction. The product thus obtained was further subjected to dry at 80°C for 12 hours. The dried product was 83.5 gram, which corresponded to 98.5% yield on the basis of substrate consumed. According to HPLC analysis the product purity was 99.60%.
Example 4
Eight litres of nutrient solution with the composition and pH described in example 1 and example 2 filled into a 20 liter fermenter after sterilization at 120°C for 20 minutes. The nutrient medium was then inoculated with 2.0 litres Yersinia pseudotuberculosis MTCC 5129 inoculum that already prepared separately using the same medium composition in orbital shaker incubator at 30°C and 200 rpm for 18 hours. The fermenter was run at 30°C and at 200 rpm. After 24 hours fermentation was stopped and the cell mass was harvested followed by washing with tap water by centrifugation at 10000 rpm and at 20°C for 7 minutes. A small portion of the cultured cells was used for measuring the cell dried weight and the obtained cell yield was 2.8 g per liter of the medium. The washed cells was then used to hydroxylate 800 gram of nicotinic acid dissolved in 10 liter of tap water (pH was adjusted to 6.2 using NaOH) in a stirred tank reactor with 200 rpm at 30°C and 0.5 kg air pressure per minute. After 44 hours, the reaction was stopped and cell mass was separated by centrifugation. The supernatant was then subjected to acidification using H2SO4/HC1 to the pH 1.5 that resulted to white precipitate formation. The precipitate was recovered by filtration under suction and then allowed to dry at 80°C for 12 hours. The dried product was 880 gram and according to HPLC analysis it contained 98.9% of 6-hydroxynicotinic acid. This corresponded to a yield of 97.34% that calculated on the basis of nicotinic acid used.
Example 5
The separated cells after completion of the reaction as stated in example 4 was reused for conversion of nicotinic acid to 6-hydroxynicotinic acid at 3% substrate concentration in 10 liter of tap water (pH was adjusted to 6.2 by addition of NaOH). The reaction was allowed to proceed at 200 rpm, 30°C and 0.5 kg air pressure per minute for 30 hours. After the stoppage of the reaction, the cell mass was separated by centrifugation and the supernatant was acidified to pH 1.5 by addition of H2SO4/HC1 which resulted to white precipitate that is recovered by filtration under suction. The product thus obtained was further subjected to dry at 80°C for 12 hours. The dried product was 317 gram, which corresponded to 93.5% yield on the basis of substrate consumed. According to HPLC analysis the product purity was 99.20%.
Example 6
The washed cells of Yersinia pseudotuberculosis MTCC 5129 (1.2 g dry weight equivalent) prepared as given in example 1 were suspended in 10 ml of tap water and added to 20 ml of a solution of monomer (3.9 g acrylamide) and cross linker (0.39 g N,N' methylene bisacrylamide) in tap water to prepare uniform suspension. The polymerization was initiated by adding 0.1 ml of N,N,N',N'-tetra methyl ethylene diamine (99%) and 0.3 ml of ammonium persulphate solution (10% w/v in water). The polymerization was performed in a ice water bath (10°C) for 1 h. The gel block was cut into cubes (2 X 2.5 X 2.5 mm3) and these cubes were then thoroughly washed with water in order to remove unpolymerized monomers and residues.
By using the immobilized cells as obtained above, the conversion of nicotinic acid to 6-hydroxynicotinic acid was conducted in a shaking flask. Reaction was carried out taking 20 g gel beads in 20 ml of 10% nicotinic acid solution (pH 6.2) in a 250 ml Erlenmeyer flask at 30°C and 200 rpm in a orbital incubator shaker for 30 h. After completion of reaction the reaction solution was separated out simply by decantation and left polyacrylamide gel beads were again subjected to reaction with 20 ml of 5% nicotinic acid solution in water keeping other conditions same as described in first use. This process was repeated for another three batches with 20 ml of 5% nicotinic acid
solution in water under similar conditions as in first reuse. The clear reaction solution resulted from each batch after decantation was acidified to pH 1.5 with H2SO4/HC1 separately with resulted white precipitate of 6-hydroxynicotinic acid. According to HPLC analysis the product purity was 99.6%. The results are given in the Table 2
Table 2

(Table Removed)
It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be falling within the scope of the invention, which is limited only by the following claims.

We Claim:
1. A process for producing 6-hydroxynicotinic acid comprising:
biotransformation of nicotinic acid to 6-hydroxynicotinic acid by
hydroxylation of nicotinic acid in aqueous medium using a novel microbial strain, Yersinia pseudotuberculosis MTCC 5129 cells at atmospheric pressure and a temperature of 25 to 35°C and at pH 6 to 7.5.

2. The process according to claim 1, wherein the aqueous medium is tap
water.
3. The process according to any of claims 1 or 2, wherein the Yersinia
pseudotuberculosis MTCC 5129 cells are recoverable and reusable in the process.
4 The process according to any of claims 1 through to 3, wherein said bacterial strain Yersinia pseudotuberculosis MTCC 5129 cells is isolated from soil, which is capable of specific hydroxylation of nicotinic acid to 6-hydroxnicotinic acid.
5. The process according to claim 4, wherein said bacterial strain Yersinia
pseudotuberculosis MTCC 5129 cells having nicotinic acid hydroxylase enzyme is
obtained by growing the cells in a nutrient medium, the nutrient medium comprising a
carbon source, a nitrogen source, inorganic salts, inducer and recovering the cells by
centrifugation followed by washing with tap water.
6. The process according to claim 5, wherein the carbon sources used for
growing the strain Yersinia pseudotuberculosis MTCC 5129 are selected from a group
comprising glucose, sucrose, and sodium citrate.
7. The process according to claim 6, wherein the carbon sources used for
growing the strain Yersinia pseudotuberculosis MTCC 5129 are in the range of 0.5 to
2.0 g %.
8. The process according to claim 5, wherein the nitrogen sources used for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are peptone and yeast extract.
9. The process according to claim 8, wherein the nitrogen sources used for
cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are in the range of 0.5 to
2.0 g%.
10. The process according to claim 5, wherein the inorganic nutrients used
for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are selected from a
group comprising phosphate, sulphate, potassium, sodium, magnesium, molybdenum,
iron and mixture thereof.
11. The process according to claim 10, wherein the inorganic nutrients used
for cultivating the strain Yersinia pseudotuberculosis MTCC 5129 are in the range of
0.001 to 0.5 g %.
12. The process according to claim 5, wherein the inducer is nicotinic acid.
13. The process according to claim 12, wherein the range of the nicotinic
acid is 0.1 to 1 g%.
14. The process according to claim 5, wherein the said microbial strain
Yersinia pseudotuberculosis MTCC 5129 cells are grown at a pH of 5 to 9.
15. The process according to claim 14, wherein the said microbial strain
Yersinia pseudotuberculosis MTCC 5129 cells are grown at a pH of 7 to 8.
16. The process according to claim 5, wherein the said microbial strain
Yersinia pseudotuberculosis MTCC 5129 cells are grown at a temperature of l5-40°C.
17. The process according to claim 16, wherein the said microbial strain Yersinia pseudotuberculosis MTCC 5129 cells are grown at a temperature of 28-35°C.
18. The process according to any of claims 1 through to 17, wherein said
bacterial strain Yersinia pseudotuberculosis MTCC 5129 cells having nicotinic acid
hydroxylase enzyme, obtained by growing the cells in a nutrient medium comprising
glucose (0.5 to 2 g %), inorganic nutrients KH2PO4 (0.05 to 0.5 g %), Na2HPO4 (0.05 to
0.5 g %) MgSO4.7H2O (0.02 to 0.1 g %), FeSO4.7H2O and Na2MoO4.2H2O (0.001 to
0.005 g %), peptone, yeast extract in combination or alone (Complex nitrogen source)
(0.5 to 2 g %) and nicotinic acid (0.1 to 1 g %), was grown under aerobic conditions for

24 to 44 hrs in shaking flask or in a fermenter at a temperature of 15-40 and at the pH
of 5 to 9.
19. The process according to any of claims 1 through to 18, wherein the
strain Yersinia pseudotuberculosis MTCC 5129 is used as free cells or in immobilized
form.
20. A process for producing 6-hydroxynicotinic acid according to any of the
preceding claims and supported by the description and the examples.

Documents:

826-del-2004-abstract.pdf

826-del-2004-claims.pdf

826-del-2004-correspondence-others.pdf

826-del-2004-correspondence-po.pdf

826-del-2004-description (complete).pdf

826-del-2004-form-1.pdf

826-del-2004-form-19.pdf

826-del-2004-form-2.pdf

826-del-2004-form-26.pdf

826-del-2004-form-3.pdf

826-del-2004-form-5.pdf

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Patent Number

208723

Indian Patent Application Number

826/DEL/2004

PG Journal Number

40/2007

Publication Date

05-Oct-2007

Grant Date

07-Aug-2007

Date of Filing

05-May-2004

Name of Patentee

M/S JUBILANT ORGANOSYS LIMITED

Applicant Address

PLOT 1A SECTOR 16 A, NOIDA-201 301, U.P., INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	NATH, TANKESWAR	JUBILANT ORGANOSYS LTD., BHARTIAGRAM, GAJRAULA, DISTRICT MORADABAD-244 223, U.P, INDIA.
2	GOEL, PRABHAT	JUBILANT ORGANOSYS LTD., BHARTIAGRAM, GAJRAULA, DISTRICT MORADABAD-244 223, U.P,INDIA.
3	MISHRA, SHIV KUMAR	JUBILANT ORGANOSYS LTD., BHARTIAGRAM, GAJRAULA, DISTRICT MORADABAD-244 223, U.P, INDIA.
4	AGARWAL, ASHUTOSH	JUBILANT ORGANOSYS LTD., BHARTIAGRAM, GAJRAULA, DISTRICT MORADABAD-244 223, U.P, INDIA.

PCT International Classification Number

C12N 1/20

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA