Title of Invention	"A PROCESS FOR THE PREPARATION OF MIXTURE OF 19 HYDROXYEICOSATETRAENOIC ACID AND 20 HYDROXYEICOSATETRAENOIC (19 HETE AND 20 HETE)"
Abstract	The invention relates to a process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid(19 HETE and 20 HETE). The process steps are: growing Candida apicola or Candida bombicola in a conventional growth medium consisting of carbon and nitrigen sources supplemented with arachidonic acid or poly unsaturated fatty acids for a pertiod of atleast 24 hrs, separating the biomass from the medium(broth) by conventional methods, extracting the separated broth with polar organic solvents, drying,hydrolysing by conventional acid hydrolysis method to obtain the products.

Title of Invention

"A PROCESS FOR THE PREPARATION OF MIXTURE OF 19 HYDROXYEICOSATETRAENOIC ACID AND 20 HYDROXYEICOSATETRAENOIC (19 HETE AND 20 HETE)"

Abstract

The invention relates to a process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid(19 HETE and 20 HETE). The process steps are: growing Candida apicola or Candida bombicola in a conventional growth medium consisting of carbon and nitrigen sources supplemented with arachidonic acid or poly unsaturated fatty acids for a pertiod of atleast 24 hrs, separating the biomass from the medium(broth) by conventional methods, extracting the separated broth with polar organic solvents, drying,hydrolysing by conventional acid hydrolysis method to obtain the products.

Full Text	The present invention relates a process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE). Arachidonic acid metabolites from all three enzymatic pathways are known to be vasoactive. A variety of cytochrome P450 metabolites affect vascular tone, including the co- hydroxylate products 19 HETE and 20 HETE. 20 HETE derived from arachidonic acid is released from activated neutrophils and contribute to vascular tone, in number of organ systems. Zou et al in 1994 have reported that inhibitors of renal vascular 20 HETE production impairs autoregulation of blood flow. Ma in 1993 have shown that 20 HETE is an endogenous vasoconstrictor of canine renal arcuate arteries, where as Escalante in 1993 has shown 20 HETE as an endothelium dependent vasoconstrictor in rabbit arteries. Pratt et al in 1998 have reported 20 HETE is a potent vasodilator of bovine coronary arteries. It also contributes to vascular tone in a number of organ systems , such as aorta,mesentric, cortical and renal arteries. They also report that bovine arteries when incubated with 20 HETE produce prostacyclin in response to increasing concentration of 20 HETE. Furthermore, 20 HETE was shown to activate MAPK ( mitogen activated protein kinase) which amplifies CPLA2 ( cytosolic phosopholipase A 2)activity and releases additional arachidonic acid by positive feed back mechanism which might play role in signaling processes involved in inflammation ,in cell growth,proliferation and differentiation. Schwartzmann in 1988 has shown 19 (s) HETE may contribute to the regulation of renal function by regulating Na* - K + ATPase which is essential for transtubular transport processes. There are no reports till date for microbial transforamation of 20 HETE and 19 HETE from extraneously added arachidonic acid. The reports of 20 HETE production are either by incubating arachidonic acid with mammalian cells or by totally synthetic forms. Most of the reported transformations involve oxidation of activated carbon (allylic) but oxidation of unactivated carbon is very difficult even by chemical methods. This is due to lack of reactivity at this terminal carbon. One of the chemical method for production of 19 HETE desciribed by Schwarrtzmann et al, 1988 is as follws : To a vigorously stirring - 40 ° C solution of methyl 14 -15 DHET (( 130mg, 0,369 mmole) in dichloroethane ( 4 ml) were added powedered , anhydrous sodium bicarbonate ( 40 mg, 0.387 mmole) and lead tetracaetate ( 171.6 mg 0.387 mmole) . After 20 minutes the reaction mixture was passed rapidly through a silica gel bed and the filter cake was washed with dry ether ( 10 ml) . The combined organic filterates were concentrated under reduced pressure on a rotary evaporator. The resultatnt oily aldehyde was used directly in the next reaction after drying azeotropically with benzene, n- Butyllithium ( 0.42 ml) was added dropwise with stirring to a -78 ° C solution of 5 ( R) -( t- butyldiphenyl -sililoxy ) hexyltriphenylphosphonium bromide in unhydrous THF ( 4 ml) under argon. After 45 minutes , a THF ( 2 ml) solution of above aldehyde was added slowly follwed after 2 minutes by dry hexamethylphosporamide ( 1.5 ml). The reaction mixture was warmed over 0 ° C , poured into 25 % aquetous ammonium acetate and extracted with ethyl acetate ( 4 xlO ml). The combined organic extracts were washed with water , brine and dried over sodium sulphate. Concentration and purification of the residue was done on silica gel column. Yield was approximatel 37 %. Biological methods require mammalian cells which are tedious and expensive and the productivity is very low. One of the method described by Escalante in 1989 is as follws : 20 HETE is prepared by incubating rat renal cortical microsome ( 3 mg) with arachidonic acid in presence of NADPH and indomethacin. Separated and purified by reverse and normal phase liquid chromatography as described Schwartzmann in 1988. For this rat aortic rings , male sprgue Dawley rats ( 300 -350 g) were killed by cervical dislocation and thoracic aorta was carefully removed and placed into cold kreb's bicarbonate buffer freed of periadventitial fat and cut into 3-4 mm wide rings. To ensure the integrity of the vascular endothelium, care was taken during the dissection to avoid streching or contact of instrument with the luminal surface of the ring. The aortic rings were mounted in 5 ml of water jacketed organ bath maintained at 37 °C and equilibriated for 1.5-2 hrs, in kreb's bicarbonate buffer gassed with 95 % O2 and 5 % CO2. The composition of the kreb's bicarbonate buffer was ( g/1) NaCl : 6.95; KC1 : 0.354; CaCI2; 0.280; KH2PO4 0.162; MgSO4, 7 H2O , 0.294; Na HCO3, 2.1 and dextrose 2.0. A minimum of four rings were used simultenously from each aorta . Basal tone was set at 2 g and adjusted accordingly over the equilibrium period. Tension was measured using glass model RPS 7C8A. This procedure provided optimal conditions for reproducible isomatic force development. There are always side products formed hence purification of the desired end product increases cost of the metabolite ( Sigma price of 10 jigs of 20 HETE is 78 $)• The main object of the present invention is to provide a new process for preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid ( 19 HETE and 20 HETE) which obviates the drawbacks as detailed above. Another object of the present invention is to provide one step transformation of arachidonic acid to a mixture of 19 HETE and 20 HETE.(This is in comparision with 10 steps chemical method for 19 (s) HETE, Falck et al 1988). Still another object of the present invention is to provide a process for the hydroxylation which is stereo specific, (in case of 19 HETE) Yet another object of the present invention is to provide a process wherein the conversion efficiency of Arachidonic acid to 19 HETE and 20 HETE is 100 times higher than that of mammalian cells as there are only two metabolites are formed and they are not metabolized further, In chemical as well as biological methods the yield is very low and in picomoles whereas microbial transformation has shown in milligrams levels. Yet another object is to provide a process for preparation of mixture of 19 hydroxyeicostetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE) in which yeast cells require less restricted conditions and cheaper carbon sources such as molasses, corn steep liquor etc., than mammalian cells or isolated P 450 from mammalian cells. Yeast cells are grown in conventional medium, containing glucose as carbon source with moderate temperature at 30°C and other conditions, whereas in chemical methods for preparation of the same requires temp. Ranging from -78 to 37°C. In biological methods it requires absolutely restricted conditions as described in prior arts. Accordingly the present invention provides a process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE) characterised in that Candia apicola (ATCC 96134) or Candida bombicola (ATCC 22214) are grown in conventional growth medium such as herein described with solution of arachidonic acid or Poly Unsaturated Fatty Acids (PUFA) prepared in alkanol for a period of 24 hrs in a known manner, separating the biomass from the medium (broth), by conventional methods, extracting the separated broth with polar organic solvents such as herein described by conventional methods, drying, acid hydrolyzing to obtain 19 HETE and 20 HETE. In one of the embodiments of the present invention, arachdonic acid or an oil containing polyunsaturated fatty acids is added to the medium at the time of the inoculation of the yeast sp. In another embodiment the polyunsaturated fatty acid (PUFA) may be such as linoleic, linolenic, arachidonic acid, eicosapentaenoic and docodahexaenoic acid. In still another embodiment the concentration of the arachidonic acid or PUFA may be ranging between 100 mg to 1000 mg/lit of the medium. In another embodiment the solvent used for supplementing of arachidonic acid or PUFA may be alkanols preferably ethanol. In another embodiment the solvent used for extraction of the mixture of products may be polar solvents exemplified by but not limited to ethyl acetate, chloroform, n-hexane preferably ethyl acetate. In a feature of the present invention the mixture of the 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acids may be separated to free hydroxy fatty acids by acid hydrolysis effected by using mineral acid such as molar HCL, H2 SO4, and nitric acid. In yet another feature of the present invention, the conventional media composition of the fermentor medium may be as given in table-1 The medium of growth as well fermentation consisted of (g 1" ) (Table Removed) In yet another feature of the present invention the yeast used may be optionally immobilized by conventional methods like using calcium alginate. The use of immobilized yeast enables continuous production. The process of the present invention is described hereinbelow with reference to examples which are illustrative in nature and should not be construed to limit the scope of the present invention in any manner. Example 1 The 19 HETE and 20 HETE were obtained from hydrolysis of the sophorolipid isolated from the fermentation of Candida apicola ATCC 96134 or Candida bombicola ATCC 22214. 500 mg of arachidonic acid in 5 ml of ethanol is added during inoculation. For the fermentation experiment cells were precultivated on medium as described above in a 250 ml flask containing 50 ml medium at 30 °C and 150 rpm.Late logarithamic cells ( 24 h) were used as an inoculum. These starter cultures ( 4-6 X 10 6 cells /ml) were used to inoculate conical flask ( 11) each containing 400 ml fermentation medium. Crude sophorolipid was extracted after 96 hrs. Sophorolipid was purified as described previously ( Hommel et al 1987 ). Crystalline crude sophorolipid was washed three times with n hexane ( 20 ml X 3) to remove residual substrate and dissolved in minimum volume of (1-3 ml) chilled ethyl acetate. The solvent was removed under reduced pressure and the resulting yellowish glycolipid was used as a substrate for acid hydrolysis. Acid hydrolysis was done essentially as described by Hommel, (1987) which is as follows, 20 mg of sophorolipid was hydrolysed with 1 ml of 1 M HC1 in a boiling water bath for 2 hrs. The liberated fatty acids were extracted with chloroform. Example 2 This example illustrate the purification of the products by fractionation on 500 mg Aminopropyl Sep - Pak Cartridges (Waters) by modification of the method described by Kaluzny et al (1985). Samples (0.5 ml chloroform) were applied to the cartridges which have been equilibrated with 5 ml hexane. Neutral lipids were eluted from the column with 25 ml chloroform / 2-propanol ( 2:1, v/v). Monohydroxylated fatty acids were eluted from the column with 25 ml of 2 % acetic acid in diethyl ether. Polar fatty acids were eluted with 25 ml of methanol. Ether fractions were rotary evaporated and the residues were taken in a minimum volum (0.5 ml) of chloroform and were separated on TLC (Keiselgel G60). Example 3 This example illustrates the confirmation of formation of end products. Methylated and silylated samples (1 µl) were analyzed by gas liquid chromatography equipped with a flame ionization detector( FID). Separation of fatty acid derivatives was achieved using BP -1 fused silica capillary column ( 25m x 0.22 mm with 0.25 mm coating) with N2 as the carrier gas. The temperature was initially 220 °C and an initial time of 5 min. which reached a final temperature at 280°C at ramp rate 2°C/min. Example 4 Methylated and silylated samples were identified on GC.MS. Gas liquid chromatography in combination with mass spectrometry was carried out with Finnigan Series automated quadruple mass spectrometry equipped with a BP -5 fused silica column ( 30 m x 0.25 mm with 0.25 mm coating). Parameters for gas liquid chromatography were : He carrier gas at 0.14 atm. head pressure; 10.1 split; injector temperature, 240°C; initial temperature, 220°C initial time , 5 min.; ramp rate 5°C min -1; final temperature, 280°C; final time 10 min; injection volume; 1µl. Mass spectroscopy parameters were : source temperature 240°C; manifold temperature 100°C, ionization current, 3.0 nm,scan range 70 to 650 atomic mass units in 1 scan. Results were confirmed with authentic standards. The main advantage of the present invention are 1. There is no reported method by which 19 HETE and 20 HETE can be prepared by microbial transformation. 2. In present method we report for biotransformation of Arachidonic acid into 19 HETE and 20 HETE. 3. In present method we also report that the given culture can hydroxylate other poly unsaturated fatty acids into respective ωand ω-l hydroxylated products. 4. In the present invention we also report for the simpler method for production of 19 HETE and 20 HETE. 5. Another advantage of the invention is there are no other side products from arachidonic acid other than 19 HETE and 20 HETE. 6. Advantage of the invention is, since there are only two end products from arachidonic acid, purification procedure is simpler and easier and economic. ( Dose not involve many steps). 7. Another advantage of the invention is , as biotransforming agent is yeast, it is easier to handle the system than mammalian cells. ( operations in mild conditions) 8. Another advantage is yeast can be grown on cheaper carbon source such as molasses keeping the end product value minimum. 9. This is very important as till today this is produced either by puerly chemical method ( multisteps and hazardous) or incubating arachidonic acid with mammalian cells. This invention is ecofriendly ( Green Technology) as most of the enzymatic reactions are carried out in aqeous medium and all other moderate conditions. 10. Another advantage of the present invention is use of immobilized system for continuous conversion of arachidonic acid ( or any other PUFA) into bioactive compounds such as 19 HETE and 20 HETE. 11. Another advantage of the present invention is it offers unusual activation at normally unreactive carbon centres where no conventional chemistry is applicable. We Claim: 1. A process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE) characterised in that Candia apicola (ATCC 96134) or Candida bombicola (ATCC 22214) are grown in conventional growth medium such as herein described with solution of arachidonic acid or Poly Unsaturated Fatty Acids (PUFA) prepared in alkanol for a period of 24 hrs in a known manner, separating the biomass from the medium (broth), by conventional methods, extracting the separated broth with polar organic solvents such as herein described by conventional methods, drying, acid hydrolyzing to obtain 19 HETE and 20 HETE. 2. A process as claimed in claim 1 wherein the poly unsaturated fatty acid (PUFA) are selected from linoleic, linolenic, arachidonic acid, eicosapentaenoic and docodahexaenoic acid. 3. A process as claimed in claims 1 & 2 wherein the concentration of the arachidonic acid or PUFA is in the range between 100 mg to 1000 mg/lit of the medium. 4. A process as claimed in claims 1 to 3 wherein the alkanol is ethanol. 5. A process as claimed in claims 1 to 4 wherein the polar organic solvent used for extraction of the mixture of products are selected from ethyl acetate, chloroform, n-hexane, preferably ethyl acetate. 6. A process as claimed in claims 1 to 5 wherein the hydrolysis is effected by using mineral acid selected from molar HCL, H2 SO4, and nitric acid. 7. A process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE) substantially as herein described with reference to the examples.

Full Text

The present invention relates a process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE).
Arachidonic acid metabolites from all three enzymatic pathways are known to be vasoactive. A variety of cytochrome P450 metabolites affect vascular tone, including the co- hydroxylate products 19 HETE and 20 HETE. 20 HETE derived from arachidonic acid is released from activated neutrophils and contribute to vascular tone, in number of organ systems. Zou et al in 1994 have reported that inhibitors of renal vascular 20 HETE production impairs autoregulation of blood flow. Ma in 1993 have shown that 20 HETE is an endogenous vasoconstrictor of canine renal arcuate arteries, where as Escalante in 1993 has shown 20 HETE as an endothelium dependent vasoconstrictor in rabbit arteries. Pratt et al in 1998 have reported 20 HETE is a potent vasodilator of bovine coronary arteries. It also contributes to vascular tone in a number of organ systems , such as aorta,mesentric, cortical and renal arteries. They also report that bovine arteries when incubated with 20 HETE produce prostacyclin in response to increasing concentration of 20 HETE. Furthermore, 20 HETE was shown to activate MAPK ( mitogen activated protein kinase) which amplifies CPLA2 ( cytosolic phosopholipase A 2)activity and releases additional arachidonic acid by positive feed back mechanism which might play role in signaling processes involved in

inflammation ,in cell growth,proliferation and differentiation. Schwartzmann in 1988 has shown 19 (s) HETE may contribute to the regulation of renal function by regulating Na* - K + ATPase which is essential for transtubular transport processes.
There are no reports till date for microbial transforamation of 20 HETE and 19 HETE from extraneously added arachidonic acid. The reports of 20 HETE production are either by incubating arachidonic acid with mammalian cells or by totally synthetic forms. Most of the reported transformations involve oxidation of activated carbon (allylic) but oxidation of unactivated carbon is very difficult even by chemical methods. This is due to lack of reactivity at this terminal carbon. One of the chemical method for production of 19 HETE desciribed by Schwarrtzmann et al, 1988 is as follws : To a vigorously stirring - 40 ° C solution of methyl 14 -15 DHET (( 130mg, 0,369 mmole) in dichloroethane ( 4 ml) were added powedered , anhydrous sodium bicarbonate ( 40 mg, 0.387 mmole) and lead tetracaetate ( 171.6 mg 0.387 mmole) . After 20 minutes the reaction mixture was passed rapidly through a silica gel bed and the filter cake was washed with dry ether ( 10 ml) . The combined organic filterates were concentrated under reduced pressure on a rotary evaporator. The resultatnt oily aldehyde was used directly in the next reaction after drying azeotropically with benzene, n- Butyllithium ( 0.42 ml) was added dropwise with stirring to a -78 ° C solution of 5 ( R) -( t- butyldiphenyl -sililoxy ) hexyltriphenylphosphonium bromide in unhydrous THF ( 4 ml) under

argon. After 45 minutes , a THF ( 2 ml) solution of above aldehyde was added slowly follwed after 2 minutes by dry hexamethylphosporamide ( 1.5 ml). The reaction mixture was warmed over 0 ° C , poured into 25 % aquetous ammonium acetate and extracted with ethyl acetate ( 4 xlO ml). The combined organic extracts were washed with water , brine and dried over sodium sulphate. Concentration and purification of the residue was done on silica gel column. Yield was approximatel 37 %.
Biological methods require mammalian cells which are tedious and expensive and the productivity is very low. One of the method described by Escalante in 1989 is as follws : 20 HETE is prepared by incubating rat renal cortical microsome ( 3 mg) with arachidonic acid in presence of NADPH and indomethacin. Separated and purified by reverse and normal phase liquid chromatography as described Schwartzmann in 1988. For this rat aortic rings , male sprgue Dawley rats ( 300 -350 g) were killed by cervical dislocation and thoracic aorta was carefully removed and placed into cold kreb's bicarbonate buffer freed of periadventitial fat and cut into 3-4 mm wide rings. To ensure the integrity of the vascular endothelium, care was taken during the dissection to avoid streching or contact of instrument with the luminal surface of the ring. The aortic rings were mounted in 5 ml of water jacketed organ bath maintained at 37 °C and equilibriated for 1.5-2 hrs, in kreb's bicarbonate buffer gassed with 95 % O2 and 5 % CO2. The composition of the kreb's bicarbonate buffer was ( g/1) NaCl : 6.95; KC1 : 0.354;

CaCI2; 0.280; KH2PO4 0.162; MgSO4, 7 H2O , 0.294; Na HCO3, 2.1 and dextrose 2.0. A minimum of four rings were used simultenously from each aorta . Basal tone was set at 2 g and adjusted accordingly over the equilibrium period. Tension was measured using glass model RPS 7C8A. This procedure provided optimal conditions for reproducible isomatic force development.
There are always side products formed hence purification of the desired end product increases cost of the metabolite ( Sigma price of 10 jigs of 20 HETE is 78
$)•
The main object of the present invention is to provide a new process for
preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid ( 19 HETE and 20 HETE) which obviates the drawbacks as detailed above.
Another object of the present invention is to provide one step transformation of arachidonic acid to a mixture of 19 HETE and 20 HETE.(This is in comparision with 10 steps chemical method for 19 (s) HETE, Falck et al 1988).
Still another object of the present invention is to provide a process for the hydroxylation which is stereo specific, (in case of 19 HETE) Yet another object of the present invention is to provide a process wherein the conversion efficiency of Arachidonic acid to 19 HETE and 20 HETE is 100 times

higher than that of mammalian cells as there are only two metabolites are formed and they are not metabolized further, In chemical as well as biological methods the yield is very low and in picomoles whereas microbial transformation has shown in milligrams levels.
Yet another object is to provide a process for preparation of mixture of 19 hydroxyeicostetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE) in which yeast cells require less restricted conditions and cheaper carbon sources such as molasses, corn steep liquor etc., than mammalian cells or isolated P 450 from mammalian cells.
Yeast cells are grown in conventional medium, containing glucose as carbon source with moderate temperature at 30°C and other conditions, whereas in chemical methods for preparation of the same requires temp. Ranging from -78 to 37°C. In biological methods it requires absolutely restricted conditions as described in prior arts.
Accordingly the present invention provides a process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE) characterised in that Candia apicola (ATCC 96134) or Candida bombicola (ATCC 22214) are grown in conventional growth medium such as herein described with solution of arachidonic acid or Poly Unsaturated Fatty Acids (PUFA)

prepared in alkanol for a period of 24 hrs in a known manner, separating the biomass from the medium (broth), by conventional methods, extracting the separated broth with polar organic solvents such as herein described by conventional methods, drying, acid hydrolyzing to obtain 19 HETE and 20 HETE.
In one of the embodiments of the present invention, arachdonic acid or an oil containing polyunsaturated fatty acids is added to the medium at the time of the inoculation of the yeast sp.
In another embodiment the polyunsaturated fatty acid (PUFA) may be such as linoleic, linolenic, arachidonic acid, eicosapentaenoic and docodahexaenoic acid.
In still another embodiment the concentration of the arachidonic acid or PUFA may be ranging between 100 mg to 1000 mg/lit of the medium.
In another embodiment the solvent used for supplementing of arachidonic acid or PUFA may be alkanols preferably ethanol.
In another embodiment the solvent used for extraction of the mixture of products

may be polar solvents exemplified by but not limited to ethyl acetate, chloroform, n-hexane preferably ethyl acetate.
In a feature of the present invention the mixture of the 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acids may be separated to free hydroxy fatty acids by acid hydrolysis effected by using mineral acid such as molar HCL, H2 SO4, and nitric acid.
In yet another feature of the present invention, the conventional media composition of the fermentor medium may be as given in table-1

The medium of growth as well fermentation consisted of (g 1" )

(Table Removed)
In yet another feature of the present invention the yeast used may be optionally immobilized by conventional methods like using calcium alginate. The use of

immobilized yeast enables continuous production.
The process of the present invention is described hereinbelow with reference to examples which are illustrative in nature and should not be construed to limit the scope of the present invention in any manner.
Example 1
The 19 HETE and 20 HETE were obtained from hydrolysis of the sophorolipid isolated from the fermentation of Candida apicola ATCC 96134 or Candida bombicola ATCC 22214.
500 mg of arachidonic acid in 5 ml of ethanol is added during inoculation. For the fermentation experiment cells were precultivated on medium as described above in a 250 ml flask containing 50 ml medium at 30 °C and 150 rpm.Late logarithamic cells ( 24 h) were used as an inoculum. These starter cultures ( 4-6 X 10 6 cells /ml) were used to inoculate conical flask ( 11) each containing 400 ml fermentation medium. Crude sophorolipid was extracted after 96 hrs. Sophorolipid was purified as described previously ( Hommel et al 1987 ). Crystalline crude sophorolipid was washed three times with n hexane ( 20 ml X 3) to remove residual substrate and dissolved in minimum volume of (1-3 ml) chilled ethyl acetate. The solvent was removed under reduced pressure and the resulting

yellowish glycolipid was used as a substrate for acid hydrolysis. Acid hydrolysis was done essentially as described by Hommel, (1987) which is as follows, 20 mg of sophorolipid was hydrolysed with 1 ml of 1 M HC1 in a boiling water bath for 2 hrs. The liberated fatty acids were extracted with chloroform.
Example 2
This example illustrate the purification of the products by fractionation on 500 mg Aminopropyl Sep - Pak Cartridges (Waters) by modification of the method described by Kaluzny et al (1985). Samples (0.5 ml chloroform) were applied to the cartridges which have been equilibrated with 5 ml hexane. Neutral lipids were eluted from the column with 25 ml chloroform / 2-propanol ( 2:1, v/v). Monohydroxylated fatty acids were eluted from the column with 25 ml of 2 % acetic acid in diethyl ether. Polar fatty acids were eluted with 25 ml of methanol. Ether fractions were rotary evaporated and the residues were taken in a minimum volum (0.5 ml) of chloroform and were separated on TLC (Keiselgel G60).
Example 3
This example illustrates the confirmation of formation of end products. Methylated and silylated samples (1 µl) were analyzed by gas liquid chromatography equipped with a flame ionization detector( FID). Separation of fatty acid derivatives was achieved using BP -1 fused silica capillary column ( 25m x 0.22

mm with 0.25 mm coating) with N2 as the carrier gas. The temperature was initially 220 °C and an initial time of 5 min. which reached a final temperature at 280°C at ramp rate 2°C/min.
Example 4
Methylated and silylated samples were identified on GC.MS. Gas liquid chromatography in combination with mass spectrometry was carried out with Finnigan Series automated quadruple mass spectrometry equipped with a BP -5 fused silica column ( 30 m x 0.25 mm with 0.25 mm coating). Parameters for gas liquid chromatography were : He carrier gas at 0.14 atm. head pressure; 10.1 split; injector temperature, 240°C; initial temperature, 220°C initial time , 5 min.; ramp rate 5°C min -1; final temperature, 280°C; final time 10 min; injection volume; 1µl. Mass spectroscopy parameters were : source temperature 240°C; manifold temperature 100°C, ionization current, 3.0 nm,scan range 70 to 650 atomic mass units in 1 scan. Results were confirmed with authentic standards.
The main advantage of the present invention are
1. There is no reported method by which 19 HETE and 20 HETE can be
prepared by microbial transformation.
2. In present method we report for biotransformation of Arachidonic acid into
19 HETE and 20 HETE.

3. In present method we also report that the given culture can hydroxylate
other poly unsaturated fatty acids into respective ωand ω-l hydroxylated
products.
4. In the present invention we also report for the simpler method for
production of 19 HETE and 20 HETE.
5. Another advantage of the invention is there are no other side products from
arachidonic acid other than 19 HETE and 20 HETE.
6. Advantage of the invention is, since there are only two end products from
arachidonic acid, purification procedure is simpler and easier and
economic. ( Dose not involve many steps).
7. Another advantage of the invention is , as biotransforming agent is yeast, it
is easier to handle the system than mammalian cells. ( operations in mild
conditions)
8. Another advantage is yeast can be grown on cheaper carbon source such as
molasses keeping the end product value minimum.
9. This is very important as till today this is produced either by puerly
chemical method ( multisteps and hazardous) or incubating arachidonic acid
with mammalian cells. This invention is ecofriendly ( Green Technology)

as most of the enzymatic reactions are carried out in aqeous medium and all other moderate conditions.
10. Another advantage of the present invention is use of immobilized system
for continuous conversion of arachidonic acid ( or any other PUFA) into
bioactive compounds such as 19 HETE and 20 HETE.
11. Another advantage of the present invention is it offers unusual activation at
normally unreactive carbon centres where no conventional chemistry is
applicable.

We Claim:
1. A process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and
20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE) characterised in that
Candia apicola (ATCC 96134) or Candida bombicola (ATCC 22214) are grown
in conventional growth medium such as herein described with solution of
arachidonic acid or Poly Unsaturated Fatty Acids (PUFA) prepared in alkanol for
a period of 24 hrs in a known manner, separating the biomass from the medium
(broth), by conventional methods, extracting the separated broth with polar
organic solvents such as herein described by conventional methods, drying, acid
hydrolyzing to obtain 19 HETE and 20 HETE.
2. A process as claimed in claim 1 wherein the poly unsaturated fatty acid (PUFA)
are selected from linoleic, linolenic, arachidonic acid, eicosapentaenoic and
docodahexaenoic acid.
3. A process as claimed in claims 1 & 2 wherein the concentration of the arachidonic acid or PUFA is in the range between 100 mg to 1000 mg/lit of the medium.
4. A process as claimed in claims 1 to 3 wherein the alkanol is ethanol.
5. A process as claimed in claims 1 to 4 wherein the polar organic solvent used for extraction of the mixture of products are selected from ethyl acetate, chloroform, n-hexane, preferably ethyl acetate.
6. A process as claimed in claims 1 to 5 wherein the hydrolysis is effected by using mineral acid selected from molar HCL, H2 SO4, and nitric acid.

7. A process for the preparation of mixture of 19 hydroxyeicosatetraenoic acid and 20 hydroxyeicosatetraenoic acid (19 HETE and 20 HETE) substantially as herein described with reference to the examples.

Documents:

1089-del-1999-abstract.pdf

1089-del-1999-claims.pdf

1089-del-1999-correspondence-others.pdf

1089-del-1999-correspondence-po.pdf

1089-del-1999-description (complete).pdf

1089-del-1999-form-1.pdf

1089-del-1999-form-19.pdf

1089-del-1999-form-2.pdf

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Patent Number

215843

Indian Patent Application Number

1089/DEL/1999

PG Journal Number

12/2008

Publication Date

21-Mar-2008

Grant Date

04-Mar-2008

Date of Filing

10-Aug-1999

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	ASMITA ASHUTOSH PRABHUNE	NATIONAL CHEMICAL LABORATORY, PUNE-411008, INDIA.
2	COLIN RATLEDGE	UNIVERSITY OF HULL, HULL, (UK) A BRITISH NATIONAL.

PCT International Classification Number

C07C 51/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA