Title of Invention

A PROCESS FOR THE HYDROXYLATION OF NICOTINIC ACID TO 6-HYDROXYNICOTINIC ACID

Abstract

A process for the hydroxylation of nicotinic acid to 6-hydroxynicontinic acid The invention relates to A process for the hydroxylation of nicotinic acid to 6-hydroxynicontinic acid. The present invention provides a new strain of Pseudomonas sp. Strain MTCC 5126 having high nicotinic acid hydroxylase activity and capable of converting nicotinic acid to 6-hydroxynicotinic acid, the improvement which comprises the use of whole cell nicotinic acid hydroxylase which is obtained by culturing the microorganism in a nutrient medium containing glucose, yeast extract, peptone, mineral salts, and nicotinic acid or nicotinamide inducer. The main advantages of the present invention are: (i) the resting cells of the new strain Pseudomonas sp. Strain MTCC 5126 can be used to produce high concentration of the 6-hydroxynicotinic acid (10 - 20 percent by weight, based on nicotinic acid used) with practically quantitative yield, as distinct from its prior art analogues wherein final concentration of the 6-hyhdroxynicotinic acid is 10 percent by weight or less, (2) the resting cells of the strain MTCC 5126 with high nicotinic acid hydroxylase activity can be prepared in unspecialized nutrient medium. Both the said advantages of the present invention will reduce the high cost involved in fermentation and product recovery.

Full Text

The present invention provides a new strain of Pseudomonas sp. strain RIJ12N having high nicotinic acid hydroxylase activity and capable of converting nicotinic acid to 6- hydroxynicotinic acid, the improvement which comprises the use of whole cell nicotinic acid hydroxylase which is obtained by culturing the microorganism in a nutrient medium containing glucose, ycast extract, peptone, mineral salts, and nicotinic acid or nicotinamide inducer. The main usage of the invention is to provide a process for the preparation of the Pseiidomonas sp. strain RLJ12N cells having high nicotinic acid hydroxylase aciivity which can be used under mild reaction conditions e.g. atmospheric pressure, temperature (10 to 40° C), pH 5.5-9.0 to hydoxylase nicotinic acid to 6-hydroxynicotinic acid. Several methods for the production of 6-hydroxynicotinic acid by organic syntheses are known(Lehky, US Patent No. 5,082,777(1992), Briancourt el al. I Chim. Ther. 8(2), pp226-232 (1973); Quarroz, Swiss Application No, 7731/80). However, the chemical syntheses of 6-hydroxynicotinic acid is difficult and cost intensive because of poor yield and the necessity of the separation of by-products that are produced by non-regioselective hydroxylations. Enzymatic conversion of nicotinic acid to 6-hydroxynicotinic acid under aerobic condition in aqueous solution using whole cells (either growing or resting cells) of the genera Psendomonas, Bacillus or Achramobacher are known. Hoek et al. [US Patent No. 5,151,351(1992)] describes a process for the production of 6-hydroxynicotinic acid from nicotinic acid (as soluble sodium or potassium salt) using growing cells of Achromobacler xylosoxydans DSM2783 under aerobic conditions at 30°C, whereby 74g/l of 6-hydroxynicotinic acid with practically quantitative conversion of nicotinic acid was produced. A patent by Lehky etal. [US Patent No. 5,082,777 (1992)] claims that nicotinic acid can be converted to 6-hydroxynicotinic acid with whole cell nicotinic acid hydroxylase of the genera Pscmhmonas, Bacillus or Achmmohaclcr at 30-35' C under aerobic conditions. The process reveals that Achromobacter xylosoxydins DSM 2783, Pseudomonasputiaa NCIB 10521 and P. pulida NC1P 8176 could produce 3.5 percent by weight (93.7% yield), 0.542 percent by weight (97.5% yield) and 10 percent by weight (95.4% yield) of 6-hydroxynicotinic acid, respectively. A patent by Kiener et al. [US Patent No 5,516,661(1996)] describe:; the use of resting cells of Arthrobacter crystallopoietes DSM 7202, Pseudomonas acklovorans DSM 7203, Alcaligenesfaecalix DSM 7204 and P. acidovorans DSM 7205 to produce 3.14 percent by weight (41% yield), 6.05 percent by weight (80% yield), 5.6 percent by weight (73 % yield) and 6.4 percent by weight (85% yield) of 6-hydroxynicotinic acid, respectively. A patent by Kulla et al. [US Patent No 4,738,924 (1998 ) describe:; the use of resting cells of Achromobacter xylosoxyJans DSM 2783 to convert nicotinic acid in the form of easily soluble magnesium or barium salt to 6-hydroxynicolinic acitl, which is isolated as sparingly soluble magnesium or barium salt. The said patent claims that the resting cells of Achromohacter xylosoxydans DSM 2783 could produce 8.0 percent by weight of 6- hydroxynicotinic acid ( 91.6% yield) from magnesium nicotinate and 5.7 percent by weight of 6-hydroxynicotinic acid (64.7% yield) from barium nicotinate at 30°C. In the above mentioned processes, although the conversion of nicotinic acid to 6- hydroxynicotinic acid is quantitative in some cases, the final concentration of 6- Hydroxynicotinic acid in the reaction mixture is 10 percent by weight or less. However, a high product concentration in the reaction mixture is always desired for favorable cost economics, because the isolation of the product from a diluted reaction mixture leads to high costs of processing, investment and sewage treatment. The main objective of the present invention is to provide a process for the hydroxylation of nicotinic acid to 6-hydroxynicotinic acid using whole cells of novel Pseudomonas sp. Strain MTCC 5126 having high nicotinic acid hydroxylase activity. Accordingly, the present invention provides a process for the hydroxylation of nicotinic acid to 6-hydroxynicontinic acid which comprises contacting nicotinic acid in the form of soluble or potassium salt in aqueous solution at pH 5.5-9.0 and temperature ranging between 10 to 40°C with whole cells of Pseudomonas sp. Strain MTCC 5126 having characteristics as herein described and having high nicotinic acid hydroxylase activity obtained by growing the cells under aerobic conditions characterized in that aqueous solution of soluble sodium or potassium salt is added at the beginning of the reaction or added continuously or by portions in a conventional nutrient medium containing glucose, an organic nitrogen source, phosphates, salts, and nicotinic acid or nicotinamide inducer, separating the cells from the reaction mixture by known method, and recovering the 6-hydroxynicotinic acid from the said supernatant by precipitation with concentrated hydrochloric acid or sulfuric acid. The conventional nutrient medium used for growing the Pseudomonas sp. Strain MTCC 5126 having high nicotinic acid hydroxylase activity consists of glucose, Na2HPO4, KH2PO4, MgSO4, an organic nitrogen source, ferrous sulfate, sodium molybdate, and nicotinic acid or nicotinamide (inducer). The microorganism was grown under aerobic conditions for 10-40h in shaking flasks. In an embodiment of the present invention, in cultivation of Pseudomonas sp. Strain MTCC 5126 as used herein, the sterilized nutrient medium containing a carbon source e.g. glucose, an organic nitrogen source e.g. yeast extract, peptone, casein hydrolysate, glutamic acid (alone or in combination) , an inducer eg. nicotinic acid or nicotinamidc, phosphate and mineral salts. The cultivation is aerobically carried out with stirring at a pH value of 6 to 9 and at a temperature of 20 to 40"C, preferably al 25 to 35 °C, for a period of 10 to 40h. The resulting cells separated by centrifugation of the culturing broth is used for hydroxylation of nicotinic acid to 6-hydroxynicotinic acid In another embodiment of the present invention, the cells with high nicotinic acid hydroxylase can be produced in the unsterilized nutrient medium In another embodiment of the present invention, the nicotinic acid as soluble sodium or potassium salt acted on by I'.semhnionus sp strain RLJ12N by reaction in aqueous medium generally containing 10 to 20 percent by weight of the nicotinic acid at a temperature of from 10 to 40°C, and preferably 20 to 30°C, at a pH from 5.5 to 9.0, and preferably 6.0 to 8.0, for a period of from 10 to 48h under aerobic conditions, results in formation of 6-hydroxynicotinic acid at an yield of 99%, (based on the nicotinic acid used) by appropriately controlling the reaction conditions In another embodiment of the present invention, the 6-hydroxynicolinic acid thus formed by the action of the resting cells of Pseudomonas sp strain RLJ12N with nicotinic acid (as soluble sodium or potassium salt), can be recovered from the reaction mixture after removal of the cells by centrifugation and then adjusting the pi f of the said filtrate to pH 1.2 with concentrated hydrochloric acid or sulfuric acid, whereby white crystals of 6-hydroxynicotinic acid is precipitated out. The crystals are then separated by filtration using suction, washed with ice cold water and dried at 50°C under vacuum. In yet another embodiment of the present invention the new bacterium used was isolated from a petroleum contaminated soil sample of the Borholla oil Held, Assam, by enrichment culture technique using n-hexadecane as the carbon source The taxonomic description (to the extend presently known) of he said new organism is as follows: I.Cell shape and arrangement: small rods, arranged i n d i v i d u a l l y 2. Pigment formation in nutrient agar: 3.Gram reaction: 4.Oxidase test: S.Catalase test: 6.1ndole production: 7. Methyl red test: 8.Nitrate reduction: Q.HjS production: 10.Glucose fermentation: 11.Gelatine hydrolysis: 12. Starch hydrolysis: 13.Growth in Pseudomonas isolation agar ( H1MEDIA M406): 14.Oxygen relationship: H.Growthat 4"C 42°C positive negative positive positive negative negative positive negative no gas, no acid formed positive negative good growth strictly aerobic nil good growth Such the new strain agrees in the tested characteristics wish the bacterial genus I'.wiitlomonax. The cited strain of Pst'iidomonas sp is deposited al RRl. Jorhat qulture collection center giving an accession number RI.J12N. The present invention is described in greater detail with reference to the following examples Example 1. (a) cultivation of the biomass A nutrient medium was prepared from Ig glucose, 0.25g NaiHPO^HiO, 0.2g KH2PO,, O.OSg MgSO.,.7H2O, Img sodium molybdate, I nig 1 eSO.,.7ll2O, ().5g peptone, 0.5g yeast extract, 0.4g nicotinic acid and 100ml water The pH of the medium was adjusted to pH 72 and sterilized for 20 min at 10 Ib pressure. The said medium (30ml) in 250 ml conical flask was inoculated w th a loopful of the Pseudomonas sp. strain RLJ12N from an agar slant and cultured for I8h at 30°C and 200 rev/min. At the end of cultivation, cells were harvested by oentrifugation (lOOOOg for ISmin), washed with 0.85% NaCl solution, and kept at 4°C until use (b) hydoxylqtion of nicotinic acid The said washed cells of the I'sciii/ontoiuvi sp. strain R I J I 2 N from 10ml culture broth (corresponding to 45mg of dry weight) were suspended in IOml of sodium nicotinate solution (pH 6.5) which was prepared by suspending Ig of nicolinic acid in 8ml water, adjusting the pH with 9M NaOH to pH 6 5 under stirring condition and then making up the volume to 10ml with water. The reaction was carried out in a shaker (200 rev/min) at 30°C for a period of 18h. At the end of the reaction, the cells were separated from the reaction mixture by centrifugation at lOOOOg for 15 min. 6- Hydroxynicotinic acid was precipitated out from the filtrate thus obtained by acidification with 4M HCI up to pM 1.5. The white crystals of 6-hydroxynicolinic acid was filtered out using suction, washed with 5ml of ice cold water and dried at 50°C under vacuum. The yield of 6-hydroxynicotinic acid was 1.12g, which according to HPLC analysis contains 99.1 percent 6-hydroxynicolinic acid. This corresponds to yield of 99 percent, calculated on the nicotinic acid used. Example 2 The resting cells of the I'seudomonas sp. strain RI.J12N we-e prepared using the same procedure as given in Example 1. The said washed cells of the I'si'miontoiias sp, strain RLJ12N from 10ml culture broth (corresponding to 45 mg of dry weight) were suspended in 10ml of potassium nicotinate solution (pH 6.5) waich was prepared by suspending Ig of nicotinic acid in 8ml water, adjusting the pH with 9M KOFI to pH 6.5 under stirring condition and then making up the volume to IOml with water. The reaction was carried out in a shaker (200 rev/min) at 30°C for a period of 18h. At the end of the reaction, the cells were separated from the reaction mixture by centrifugation at lOOOOg for 15 min. 6-Hydroxynicotinic acid was precipitated out from the filtrate thus obtained by acidification with 4M HCI up to pH 1.5. The white crystals of 6-hydroxynicotinic acid was filtered out using suction, washed with 5ml of ice cold water and ^ried at 50°C under vacuum. The yield of 6-hydroxynicotinic acid was 1.12g, which according to HPLC analysis contains 992 percent 6- hydroxynicotinic acid. This corresponds to yield of 99.1 percent, calculated on the nicotinic acid used. Example 3 The resting cells of the Psendomonas sp. strain RLJ12N were prepared using the same procedure as given in Example 1 The said washed cells of the Pswidomonas sp. strain RLJ12N from 10ml culture broth (corresponding to 45mg of dry weight) were suspended in 10ml of sodium nicotinate solution (pH 65) which was prepared by suspending 1.5g of nicotinic acid in 8ml water, adjusting the pi I with 9M NaOH to pH 6.5 under stirring condition and then making up the volume to 10ml with water. The reaction was carried out in a shaker (200 rev/min) at 30"C for a period of 32h. At the end of the reaction, the cells were separated from the reaction mixture by centrifugation at lOOOOg for 15 min. 6-Hydroxynicotinic acid was precipitated out from the filtrate thus obtained by acidification with 4M HC1 up to pH 1.5. The white crystals of 6- hydroxynicotinic acid was filtered out by using suction, washed with 5ml of ice cold water and dried at 50°C under vacuum. The yield of 6-hydroxynicolinic acid was 1.67g, which according to HPLC analysis contains 99.2 percent 6-hydroxynicotinic acid. This corresponds to yield of 98.5 percent, calculated on the nicotinic acid used. Example 4 The resting cells of the Pseudomonas sp. strain RLJ12N were prepared using the same procedure as given in Example 1. The said washed cells of the l*senJoniomts sp. strain RLJ12N from I Oml culture broth (corresponding to 45mg of dry weight) were suspended in 10ml of potassium nicotinate solution (pH 6.5) which was prepared by suspending 1.5g of nicotinic acid in 8ml water, adjusting the pH with 9M KOH to pll 6.5 under stirring condition and then making up the volume to 10ml with water. The reaction was carried out in a shaker (200rev/min) at 30°C for a period of 32h At the end of the reaction, the cells were separated from the reaction mixture by centrifugation at lOOOOg for 15 min. 6-Hydroxynicotinic acid was precipitated out from the filtrate by acidification with 4M HC1 up to pH 1.5. The white crystals of 6-hydroxynicotinic acid was filtered out by using suction, washed with 5ml of ice cold water and dried ,U 50°C under vacuum. The yield of 6-hydroxynicotinic acid was 1.67g, which according to IIPLC analysis contains 99.2 percent 6-hydroxynicotintc acid. This corresponds to yield of 98.5 percent, calculated on the nicotinic acid used. Example 5 The resting cells of the Pseuclomonas sp. strain RLJ12N were prepared using the same procedure as given in Example 1. The said washed cells of the l\\L'iuiomonas sp. strain RLJ12N from 10ml culture broth (corresponding to 45mg of dry weight) were suspended in 10ml of sodium nicotinate solution (pH 6.5) which was prepared by suspending 1,8g of nicotinic acid in 8ml water, adjusting the pH with 9M NaOll tc pi I 6.5 under stirring condition and then making up the volume to lOrnl with water The reaction was carried out in a shaker (200rev/min) at 30°C for a period of 36h. At the end of the reaction, the cells were separated from the reaction mixture by centrifugation at lOOOOg for 15 min. 6- Hydroxynicotinic acid was precipitated out from the filtrate by acidification with 4M I Id up to pH 1.5. The white crystals of 6-hydroxynicolinic acid was filtered out by using suction, washed with 5ml of ice cold water and dried at 50"C under vacuum. The yield of 6-hydroxynicotinic acid was 2g, which according to HPLC analysis contains 99.2 percent 6-hydroxynicotinic acid. This corresponds to yield of 98.3 percent, calculated on the nicotinic acid used. Example6 (a) cultivation of biomass The Pseudomona.? sp. strain RLJ12N was cultivated in the same medium and under the same conditions as given in Example!, except that (he freshly prepared medium used for preparation of the ceils was not sterilized. The rest of the procedure for harvesting the cells was same as that given in Example 1. (b) hydroxylation of nicotinic acid The said washed cells of the Pseuilomonas sp. strain RLJ12N from 10ml culture broth (corresponding to 48mg of dry weight) were suspended in 10ml of sodium nicotinate solution (pH 6.5) which was prepared by suspending Ig of nicothic acid in 8ml water, adjusting the pH with 9M NaOH to pH 6.5 under stirring condition and then making up the volume to 10ml with water. The reaction was carried out in a shaker (200 rev/min) at 30°C for a period of 18h. At the end of the reaction, the cells were separated from the reaction mixture by centrifugation at lOOOOg for 15 min. 6-Hydroxynicotinic acid was precipitated from the filtrate thus obtained by acidification with 4M HC1 up to pH 1.5. The white crystals of 6-hydroxynicotinic acid was filtered out by using suction, washed with 5ml of ice cold water and dried at 50°C under vacuum. The yield of 6- hydroxynicotinic acid was 1.1 Ig, which according to HPLC analysis contains 99.2 percent 6-hydroxynicotinic acid. This corresponds to yield of 98.2 percent, calculated on the nicotinic acid used. While the invention has been described in detail and with reference lo specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. The main advantages of the present invention are: (1) the resting cells of the new strain J'seiuhmonas sp. strain RIJ12N can be used to produce high concentration of the 6-hydroxynicotinic acid ( 10-20 percent by weight, based on nicotinic acid used) with practically quantitative yield, as distinct from its prior art analogues wherein final concentration of the 6-hydroxynicotinic acid is 10 percent by weight or less , (2) the resting cells of the strain R I J I 2N with high nicotinic acid hydroxylase activity can be prepared in unsterilized nutrient medium. Both the said advantages of the present invention will reduce the high cost involved in fermentation and product recovery . We Claim: 1. A process for the hydroxylation of nicotinic acid to 6-hydroxynicontinic acid which comprises; contacting nicotinic acid in the form of soluble or potassium salt in aqueous solution at pH 5.5-9.0 and temperature ranging between 10 to 40°C with whole cells of Pseudomonas sp. Strain MTCC 5126 having characteristics as herein described and having high nicotinic acid hydroxylase activity obtained by growing the cells under aerobic conditions characterized in that aqueous solution of soluble sodium or potassium salt is added at the beginning of the reaction or added continuously or by portions in a conventional nutrient medium containing glucose, an organic nitrogen source, phosphates, salts, and nicotinic acid or nicotinamide inducer, separating the cells from the reaction mixture by known method, and recovering the 6-hydroxynicotinic acid from the said supernatant by precipitation with concentrated hydrochloric acid or sulfuric acid. 2. A process as claimed in claim 1, wherein the organic nitrogen source used to prepare intracellular nicotinic acid hydroxylase in said Pseudomonas sp. Strain MTCC 5126 is selected from a member or combination of the members of the group consisting of peptone, beef extract, yeast extract, casein hydrolysate and glutamic acid. 3. A process as claimed in claim 1, wherein the said strain MTCC 5126 is cultivated at pH 6.0 to 9.0 to prepare cells containing intracellular nicotinic acid hydroxylase. 4. A process as claimed in claim 1, wherein the said strain MTCC 5126 is cultured at 20 to 40°C to prepare cells containing intracellular nicotinic acid hydroxylase. 5. A process as claimed in claim 1, wherein the known method used for recovering the cells of the said strain MTCC 5126 is centrifugation. 6. A process for the hydroxylation of nicotinic acid to 6-hydroxynicontinic acid substantially as herein described with reference to the examples.

Documents:

251-DEL-2003-Abstract-(23-12-2008).pdf

251-DEL-2003-Abstract-(24-11-2008).pdf

251-DEL-2003-Abstract-(29-12-2008).pdf

251-del-2003-abstract.pdf

251-DEL-2003-Claims-(23-12-2008).pdf

251-DEL-2003-Claims-(24-11-2008).pdf

251-DEL-2003-Claims-(29-12-2008).pdf

251-del-2003-claims.pdf

251-del-2003-complete specification (granted).pdf

251-DEL-2003-Correspondence-Others-(23-12-2008).pdf

251-DEL-2003-Correspondence-Others-(24-11-2008).pdf

251-DEL-2003-Correspondence-Others-(29-12-2008).pdf

251-del-2003-correspondence-others.pdf

251-del-2003-correspondence-po.pdf

251-DEL-2003-Description (Complete)-(23-12-2008).pdf

251-DEL-2003-Description (Complete)-(24-11-2008).pdf

251-DEL-2003-Description (Complete)-(29-12-2008).pdf

251-del-2003-description (complete)-29-12-2008.pdf

251-del-2003-description (complete).pdf

251-DEL-2003-Form-1-(24-11-2008).pdf

251-del-2003-form-1.pdf

251-del-2003-form-18.pdf

251-DEL-2003-Form-2-(24-11-2008).pdf

251-del-2003-form-2.pdf

251-DEL-2003-Form-3-(24-11-2008).pdf

251-del-2003-form-3.pdf