Title of Invention	"A BIOCONVERSION PROCESS FOR THE PREPARATION OF ALPHA-KETO ACIDS AND L-AMINO ACIDS FORM RACEMIC MIXTURE OF AMINO ACIDS."
Abstract	This invention relates to a Bioconversion process for preparation of Alpha-Ketoacids and L-amino acids from racemic mixture of amino acids which comprises : a. Reacting mixture of D/DL amino acids as herein described with D-amino acid oxidase which is an intracellular enzyme locted in cell and Catalase enzyme as herein described at temperature in the range of 5-40°C for a period of 1 to 24 hours in a rotary shaker at revolution per minute ranging from 150 to 250 in alkali metal pyrophosphate buffer as herein described at a pH range of 6 to 9.5. b. During reaction, D-amino acid completely gets deaminated to alpha-keto acids and the reaction mixture contains both product. c. Separating alpha-ketoacids and L-amino acids from the said reaction mixture by ion- exchange chromatography.

Title of Invention

"A BIOCONVERSION PROCESS FOR THE PREPARATION OF ALPHA-KETO ACIDS AND L-AMINO ACIDS FORM RACEMIC MIXTURE OF AMINO ACIDS."

Abstract

This invention relates to a Bioconversion process for preparation of Alpha-Ketoacids and L-amino acids from racemic mixture of amino acids which comprises : a. Reacting mixture of D/DL amino acids as herein described with D-amino acid oxidase which is an intracellular enzyme locted in cell and Catalase enzyme as herein described at temperature in the range of 5-40°C for a period of 1 to 24 hours in a rotary shaker at revolution per minute ranging from 150 to 250 in alkali metal pyrophosphate buffer as herein described at a pH range of 6 to 9.5. b. During reaction, D-amino acid completely gets deaminated to alpha-keto acids and the reaction mixture contains both product. c. Separating alpha-ketoacids and L-amino acids from the said reaction mixture by ion- exchange chromatography.

Full Text	The present invention relates to a bioconversion process for preparation of alpha-Keto-acids and L-amino acids from racemic mixture of amino acids. The present invention finds applications in food and pharmaceutical industry. Particularly the invention finds applications in the production of alpha-ketoacids, 7-aminocephalosporanic acid, in the resolution of the racemic mixture of amino acids, and in the analytical determination of D-amino acids. Alpha-ketoacids, especially the ketoacid analogues of naturally occurring amino acids are of major importance in intermediary metabolism. Alpha ketoacids of essential amino acids are gaining importance as therapeutic agents and also as neutraceuticals, Patients suffering from acute uremia have a positive nitrogen balance and thus a poisonous excess of blood nitrogen that must be reduced as much as possible. This is done in an initial state of the disease, before dialysis is required, by administration of very restricted diet (low protein and high carbohydrate content). This diet is supplemented with the essential amino acids. If the carbon skeleton of essential amino acids could be given in "non nitrogen" form i.e. as the alpha-keto or alpha-hydroxy analogs of the amino acids, a more efficient treatment of this disease could be achieved, sine these analogs would be transaminated in the body to remove some of the excessive nitrogen, Alpha ketoacids are of continuing interest as intermediates in chemical syntheses , in the development of enzyme inhibitors and drugs, as model substrates of enzymes and in other ways . L~Amino acids are very important from nutritional point of view. In the synthetic route one always ends-up with the mixture of D- and L-isomers. Separation of these D- and L-isomers are very important. Enzymatic methods for the preparation of the optical antipodes of oi~ ammo acids from corresponding racemates fall into two general catagories a) those based upon a preliminary synthesis of some derivative of the racemates i.e N-acyl, ester or amide which is subsequently asymmetrically hydrolysed by the appropriate enzyme and b) those based upon a direct asymmetric oxidation or decarboxylation of one or the other antipode of the racemate. The potential drawback of the procedure (a) for the separation of racemic mixture on a large scale is, the preparation of a satisfactory derivative and then isolating the products of the enzymatic reaction, which poses difficulties in the way of economical recovery. It was therefore considered of value to turn to prodedure (b). For this purpose enzyme D-amino acid oxidase has been used . The reaction catalysed by the D-arrnno acid oxidase has got an added advantage that in a single step both the production of α-ketoaci ds and optically active L~amino acids can be achieved. D-amino acid oxidase catalyses the oxidative deamination of D- amino acids to the corresponding α-ketoacids with the liberation of ammonia and hydrogen peroxide. The hydrogen peroxide formed will be futher degraded by the enzyme catalase to water and molecular oxygen. Thus the enzyme catalase prevents the nonenzymatic decarboxylation of α-keto acids thereby the α-ketoaci ds will be the stable end product. The purified D-amino acid oxidase from mammalian sources (kidney and liver) and from microbial sources (Rhodotorula gracilis, Trigonopsis variabilis) could be used for the production of alpha-keto acids and optically active L-amino acids from the respective DL-amino acid mixture. Quite an amount of research work has already been done to convert the amino acid to the corresponding keto acid, using the purified and immobilized D-amino acid oxidase coupled to hydrogen peroxide scavenging system. Removal of the H2O2. is achieved with MnO2 or by inclusion of enzyme catalase. Addition of enzyme catalase is preferred over MnO2 to remove H2O2 since it is biological, nature friendly. Reference may be made to Cooper,A.J.L. , Ginos,Z. and Meister,A., Chem.Rew. 1983, 83, 321 wherein a survey of all the chemical synthetic methods for the preparation of alpha-ketoacids has been attempted. The draw backs of this methods are that ketoacids produced by the chemical methods are not preferable due to an enhanced emphasis on biotechnology based products and process developments. Reference may be made to Tosa T, Sano R and Chibata I., Agr,Biol.Chem. 1974, 18 (8), 1529 wherein D-amino acid oxidase from the hog kidney, immobilized onto a cyanogen bromide activated polysaccharide was suggested for the production of α-ketoacids. The drawbacks are mammalian D-amino acid oxidase can not be used for the biotechnological applications because of the limited raw material, unstability of the isloated enzyme due to loosely bound FAD. Reference may be made to Brodelius P, Nilsson K and Mosbach K ., Appl.Biochem. and Biotechnol. 1981, 6, 293 wherein the immobilized cells of Trigonopsis varaibi 1 is coimmobi1ized with manganese dioxide were employed for the production of alpha-ketoacids. The drawbacks are the use of manganese dioxide for the removal of hydrogen peroxide is not faesible for the commercial appliact ions. Reference may be made to Buto S Pollegioni L, D'Angivro L and Pilone M.S., Biotechnology and Bi oengi neer i ng. , 1994, 44, 1288 where in the purified D-amino acid oxidase from Rhodotorula gracilis was coimmobi1ized with catalase on Affinity-Gel 10 matrix and was used for the production of alpha-ketoacids. The drawbacks are isolation and purification of the enzymes is a cost effective process and isolated enzymes are very much unstable. Reference may be made to Parikh,J.R., Greenstein,J.P., Wintz,M., and Birnbaum,M ., J . Amer . Chem. Soc . , 1958, 20, 953 wherein hog renal D-amino acid oxidase was used for the resoultion of the racemic mixture of the amino acids. The drawbcks are mammalin enzyme Cannot be used for the industrial applications in a large scale. The main object of the present invention is to provide a bioconversion process for the preparation of alpha keto-acids from D-amino acids and simultaneous preparation of alpha-ketoacids and L-amino acids from racemic mixture from all the 20 naturally occurring d- amino acids and their synthetic analogues which obviates the drawbacks as described above. Accordingly, the present invention provides a bioconversion process for the preparation of alpha-Ketoacids and L-amino acids from racemic mixture of amino acids which comprises: a. Reacting mixture of D/DL amino acids as herein described with D- amino acid oxidase which is an intracellular enzyme located in cell and Catalase enzyme as herein described at temperature in the range of 5-40°C for a period of 1 to 24 hours in a rotary shaker at revolution per minute ranging from 150 to 250 in alkali metal pyrophosphate buffer as herein described at a pH range of 6 to 9.5 b. During reaction, D-amino acid completely gets deaminated to alpha-keto acids and the reaction mixture contains both product. e . Separating alpha ketoacids and L-amino acids from- the- said reaction mixture by ion-exchange chromatography In an embodiment of the_ present invention the concentration of the enzyme used for the bioconversion may range from 0.5% to 4% (W/v wet wt. of the yeast). In another embodiment of the present invention the biocatalyst used for the preparation of the ketoacids and L-amino acids were reused upto 20 cycles. The production of alpha-ketoacids and optically active L-amino acids by immobilized, purified D-amino acid oxidase is expensive because of the high cost of isolation and purification of the enzyme D-amino acid oxidase and the relative instability of the isolated enzyme. On the otherhand the whole cells containing the enzymes D-amino acid oxidase and catalase can be used for the conversion. This method has an advantage that the enzyme present in the whole cells tends to be more stable in its natural environment, than when isolated as described in the earlier processes. The potential drawback of this process is its lower enzyme activity due to diffusional and permeability barriers of the cells for D-amino acids. This consequently affects adversly the efficiency of catalysis as compared to that by the use of enzyme isolated from the whole cells. In our copending application no.Del/337/97 we have described and claimed a process for the preparation of stable intracellular D-amino acid oxidase and catalase for the preparation of -ketoacids and L-ammo acids. The following examples are given by way of illustration of the present invention and therefore should not be considered construded to limit the scope of the present invention. Example - 1 Four gram (wet wt) of glutaraldehyde stabilized CTAB permeabi1ized cells were incubated with 100 ml of 25mM D-pheny1alanine in 50mM sodium pyrophosphate buffer , pH 8.5 solution at 37°C in a metabolic shaker at 250 rpm. Aliquots of the reaction mixture were removed at various intervals and the production of phenylpyruvate was followed by the estimation of the phenyl pyruvate by dinitrophenylhydrazine (DNPH) method. The alioqot of the product formed was incubated with 0.25ml of C.1% DNPH solution in 2N HC1 for 10 min. at 25°C. After 10 min. 1 ml of 10% sodium hydroxide solution was added and the absorbance of the wine red colour formed was read at 450nm and the concentration of phenyl pyruvate was calculated. The results ( Table 1) indicate that D-phenylalanine was completly removed from the reaction mixture within 3 hours of incubation. There was no decarboxylated product of the ketoacid was formed during the bioconversion. Table 1: Bioconversion of D~phenylalanine to phenyl pyruvate using glutaraldehyde stabilized Cetyltrimethylammoniurn bromide permeabi1ized cells containing D-amino acid oxidase and catalase enzyme. (Table Removed) Example - 2 The glutaraldehyde stabilized cetyltrimethylammoniurn bromide (CTAB) permeabi1ized cells readily stoichiometrical1y converted D-amino acids to the corresponding α-keto acids from the racemic mixture of amino acids (DL-amino acids). The enzyme preparation stereospecifical1y converted only D-amino acids to the corresponding^-keto acids leaving behind the L- counter part. The glutaraldehyde treated CTAB permeabi1ized cells (4 G wet wt.) incubated with 100 ml of 50mM sodium pyrophosphate buffer pH 8.5, containing 50 mM DL-phenylalanine for 3 h. at 37° C. D-pheny1alanine was completely and stoichiometrical1y converted to phenyl pyruvate within three hours of the reaction. No decarboxylated product of phenyl pyruvate was formed during the bioconversion. After the reaction the cells were separated by centrifugation. The conversion of the D-pheny1alanine to phenyl pyruvate was monitored as described in example 1. Resolution of the racemic mixture was monitored by the optical rotation studies. The glutaraldehyde treated CTAB permeabi1ized cells (4 G wet wt.) were incubated with 100ml of 50rnM sodium pyrophosphate buffer pH 8,5, containing 25 mM D-methionine for 3 h. at 37° C. After the reaction the cells were separated by centrifugation and the conversion was monitored as described in example 1. The results in Table 2 indicate that D-isomer of the racemic mixture was removed completely and stlochiometriacl1y leaving behind the L-counterpart. Formation of the ketoacid was determined as described in example 1. Table -2 : Polarimetric studies of the amino acid solution before and after the D-amino acid oxidase reaction. (Table Removed) 2. Product of the D-amino acid oxidase reaction with D-methionine. (Table Removed) Example - 3. From the reaction mixture obtained (100ml) in Example. 2, products L-amino acids and o chromatography using Dowex-1 x-8-100 and Amberllite resins. The res-in was equi1ibriated with 50mM tris-HCl, pH 8.5. The keto acids were bound to the column and amino acids eluted out. Bound keto acids were eluted using 40% acetonitrile in 1 M HC1. Example - 4 In the example.1 and 2, since enzymes were stabilized within the cells by glutaraldehyde treatment, after the initial reaction (first cycle) the cells were collected either by centrifugation or filtration and reused as a source of enzymes for subsequent cycles. These microbial cells containing stabilized intracellular enzymes such as DAAO and catalase were repeatedly used upto 20 cycles without any loss in enzyme activity. Intracellular enzymes in general seem to be stabilized by the proximity of cellular structures or inclusion in cell membranes even when such structures or membranes are no longer alive or functional. Immobilization of DAAO and catalase within the cells by glutaraldehyde, which brings about the crosslinking of the enzymes with other intracellular components or to the cell debris maintains enzymes natural microenvironment rendering them more stable. Permeabi1ization of such glutaraldehyde treated cells by CTAB improves the mass transfer of substrates and products through the cell debris. The advantages of the present invention are: 1. The enzyme prepaprations described and claimed in our copending application no.Del/337/97 can be used directly for their application mainly to separate the L-amino acids by degrading D-amino acids from the racemic mixture and for the preparation of α-keto acids of both essential and non-essential amino acids from the corresponding racemic mixture of amino acids. 2. Since no step of isolation and purification of the enzymes D-ammo acid oxidase and catalase are involved in the process of the present invention and the cells are repeatedly used as a source of D-amino acid oxidase and catalase, the cost is considerably reduced. 3. The biocatalyst preapared as described and claimed in our copending application no.Del/337/97, could be separated from the reaction mixture from each cycle by centrifugation or filtration and reused as a source of enzymes for the subsequent cycles. 4. In the earlier methods exogeneous H2O2 degrading system viz., catalase and or Mn02 are included to obtain α-keto acids. However, in this system catalase is present in about 40 times more than that of D- amino acid oxidase. Therefore, additional H2O2 degrading system is not requi red. We Claim :- 1. A Bioconversiontprocess for preparation of Alpha-Ketoacids and L-amino acids from racemic mixture of amino acids which comprises: a. Reacting mixture of D/DL amino acids as herein described with D- amino acid oxidase which is an intracellular enzyme located in cell and Catalase enzyme as herein described at temperature an the range of 5-40°C for a period of 1 to 24 hours in a rotary shaker at revolution per minute ranging from 160 to 250 in alkali smetal pyrophosphate buffer as herein described at a pH range of 6 to 9.5 b. During reaction, D-amino acid completely gets deaminated to alpha-keto acids and the reaction mixture contains both product. c. Separating alpha-ketoacids and L-amino acids from the said reaction mixture by ion-exchange chromatography 2. A process as claimed in claim 1 wherein the enzyme deaminate all the 20 naturally occurring D-amino acid and their synthetic analogues. 2. A process as claimed in claims 1 and 2 wherein, the pH is maintained by alkali metal pyrophosphate buffer with concentration ranging from 0.005M to 0.1M and alkali-metal selected from sodium or potassium. 3. A process as claimed in claim (1) & (2), where-in the concentration of the enzymes (s) ranges from 0.5% to 4.0% (W/V wet wt of the yeast) 4. A Bioconversion process for the preparation of L- keto-acids and L- amino acids from racemic mixture of amino acids substantially as herein described with reference to the examples.

Full Text

The present invention relates to a bioconversion process for preparation of alpha-Keto-acids and L-amino acids from racemic mixture of amino acids.
The present invention finds applications in food and pharmaceutical industry. Particularly the invention finds applications in the production of alpha-ketoacids, 7-aminocephalosporanic acid, in the resolution of the racemic mixture of amino acids, and in the analytical determination of D-amino acids.
Alpha-ketoacids, especially the ketoacid analogues of naturally occurring amino acids are of major importance in intermediary metabolism. Alpha ketoacids of essential amino acids are gaining importance as therapeutic agents and also as neutraceuticals, Patients suffering from acute uremia have a positive nitrogen balance and thus a poisonous excess of blood nitrogen that must be reduced as much as possible. This is done in an initial state of the disease, before dialysis is required, by administration of very restricted diet (low protein and high carbohydrate content). This diet is supplemented with the essential amino acids. If the carbon skeleton of essential amino acids could be given in "non nitrogen" form i.e. as the alpha-keto or alpha-hydroxy analogs of the amino acids, a more efficient treatment of this disease could be achieved, sine these analogs would be transaminated in the body to remove some of the excessive nitrogen, Alpha ketoacids are of continuing interest as
intermediates in chemical syntheses , in the development of enzyme inhibitors and drugs, as model substrates of enzymes and in other ways .
L~Amino acids are very important from nutritional point of view. In the synthetic route one always ends-up with the mixture of D- and L-isomers. Separation of these D- and L-isomers are very important. Enzymatic methods for the preparation of the optical antipodes of oi~ ammo acids from corresponding racemates fall into two general catagories a) those based upon a preliminary synthesis of some derivative of the racemates i.e N-acyl, ester or amide which is subsequently asymmetrically hydrolysed by the appropriate enzyme and b) those based upon a direct asymmetric oxidation or decarboxylation of one or the other antipode of the racemate. The potential drawback of the procedure (a) for the separation of racemic mixture on a large scale is, the preparation of a satisfactory derivative and then isolating the products of the enzymatic reaction, which poses difficulties in the way of economical recovery. It was therefore considered of value to turn to prodedure (b). For this purpose enzyme D-amino acid oxidase has been used . The reaction catalysed by the D-arrnno acid oxidase has got an added advantage that in a single step both the production of α-ketoaci ds and optically active L~amino acids can be achieved.
D-amino acid oxidase catalyses the oxidative deamination of D-
amino acids to the corresponding α-ketoacids with the liberation of ammonia and hydrogen peroxide. The hydrogen peroxide formed will be futher degraded by the enzyme catalase to water and molecular oxygen. Thus the enzyme catalase prevents the nonenzymatic decarboxylation of α-keto acids thereby the α-ketoaci ds will be the stable end product.
The purified D-amino acid oxidase from mammalian sources (kidney and liver) and from microbial sources (Rhodotorula gracilis, Trigonopsis variabilis) could be used for the production of alpha-keto acids and optically active L-amino acids from the respective DL-amino acid mixture. Quite an amount of research work has already been done to convert the amino acid to the corresponding keto acid, using the purified and immobilized D-amino acid oxidase coupled to hydrogen peroxide scavenging system. Removal of the H2O2. is achieved with MnO2 or by inclusion of enzyme catalase. Addition of enzyme catalase is preferred over MnO2 to remove H2O2 since it is biological, nature friendly.
Reference may be made to Cooper,A.J.L. , Ginos,Z. and Meister,A., Chem.Rew. 1983, 83, 321 wherein a survey of all the chemical synthetic methods for the preparation of alpha-ketoacids has been attempted. The draw backs of this methods are that ketoacids produced by the chemical methods are not preferable due to an enhanced emphasis on biotechnology based products and process developments.
Reference may be made to Tosa T, Sano R and Chibata I.,
Agr,Biol.Chem. 1974, 18 (8), 1529 wherein D-amino acid oxidase from the hog kidney, immobilized onto a cyanogen bromide activated polysaccharide was suggested for the production of α-ketoacids. The drawbacks are mammalian D-amino acid oxidase can not be used for the biotechnological applications because of the limited raw material, unstability of the isloated enzyme due to loosely bound FAD.
Reference may be made to Brodelius P, Nilsson K and Mosbach K ., Appl.Biochem. and Biotechnol. 1981, 6, 293 wherein the immobilized cells of Trigonopsis varaibi 1 is coimmobi1ized with manganese dioxide were employed for the production of alpha-ketoacids. The drawbacks are the use of manganese dioxide for the removal of hydrogen peroxide is not faesible for the commercial appliact ions.
Reference may be made to Buto S Pollegioni L, D'Angivro L and Pilone M.S., Biotechnology and Bi oengi neer i ng. , 1994, 44, 1288 where in the purified D-amino acid oxidase from Rhodotorula gracilis was coimmobi1ized with catalase on Affinity-Gel 10 matrix and was used for the production of alpha-ketoacids. The drawbacks are isolation and purification of the enzymes is a cost effective process and isolated enzymes are very much unstable.
Reference may be made to Parikh,J.R., Greenstein,J.P., Wintz,M., and Birnbaum,M ., J . Amer . Chem. Soc . , 1958, 20, 953 wherein hog renal D-amino acid oxidase was used for the resoultion of the racemic mixture of the amino acids. The drawbcks are mammalin enzyme
Cannot be used for the industrial applications in a large scale.
The main object of the present invention is to provide a bioconversion process for the preparation of alpha keto-acids from D-amino acids and simultaneous preparation of alpha-ketoacids and L-amino acids from racemic mixture from all the 20 naturally occurring d- amino acids and their synthetic analogues which obviates the drawbacks as described above.
Accordingly, the present invention provides a bioconversion process for the preparation of alpha-Ketoacids and L-amino acids from racemic mixture of amino acids which comprises:
a. Reacting mixture of D/DL amino acids as herein described with D-
amino acid oxidase which is an intracellular enzyme located in cell and
Catalase enzyme as herein described at temperature in the range of 5-40°C
for a period of 1 to 24 hours in a rotary shaker at revolution per minute
ranging from 150 to 250 in alkali metal pyrophosphate buffer as herein
described at a pH range of 6 to 9.5
b. During reaction, D-amino acid completely gets deaminated to alpha-keto
acids and the reaction mixture contains both product.
e . Separating alpha ketoacids and L-amino acids from- the- said reaction mixture by ion-exchange chromatography In an embodiment of the_ present invention the concentration of the enzyme used for the bioconversion may range from 0.5% to 4% (W/v wet wt. of the yeast).
In another embodiment of the present invention the biocatalyst used for the preparation of the ketoacids and L-amino acids were reused upto 20 cycles.
The production of alpha-ketoacids and optically active L-amino acids by immobilized, purified D-amino acid oxidase is expensive because of the high cost of isolation and purification of the enzyme D-amino acid
oxidase and the relative instability of the isolated enzyme. On the otherhand the whole cells containing the enzymes D-amino acid oxidase and catalase can be used for the conversion. This method has an advantage that the enzyme present in the whole cells tends to be more stable in its natural environment, than when isolated as described in the earlier processes. The potential drawback of this process is its lower enzyme activity due to diffusional and permeability barriers of the cells for D-amino acids. This consequently affects adversly the efficiency of catalysis as compared to that by the use of enzyme isolated from the whole cells.
In our copending application no.Del/337/97 we have described and claimed a process for the preparation of stable intracellular D-amino acid oxidase and catalase for the preparation of -ketoacids and L-ammo acids.
The following examples are given by way of illustration of the present invention and therefore should not be considered construded to limit the scope of the present invention.
Example - 1
Four gram (wet wt) of glutaraldehyde stabilized CTAB permeabi1ized cells were incubated with 100 ml of 25mM D-pheny1alanine in 50mM sodium pyrophosphate buffer , pH 8.5 solution at 37°C in a metabolic shaker at 250 rpm. Aliquots of the reaction mixture were removed at various intervals and the production of phenylpyruvate was followed by
the estimation of the phenyl pyruvate by dinitrophenylhydrazine (DNPH)
method. The alioqot of the product formed was incubated with 0.25ml
of C.1% DNPH solution in 2N HC1 for 10 min. at 25°C. After 10 min. 1
ml of 10% sodium hydroxide solution was added and the absorbance of
the wine red colour formed was read at 450nm and the concentration of
phenyl pyruvate was calculated.
The results ( Table 1) indicate that D-phenylalanine was completly
removed from the reaction mixture within 3 hours of incubation. There
was no decarboxylated product of the ketoacid was formed during the
bioconversion.
Table 1: Bioconversion of D~phenylalanine to phenyl pyruvate using glutaraldehyde stabilized Cetyltrimethylammoniurn bromide permeabi1ized
cells containing D-amino acid oxidase and catalase enzyme.
(Table Removed)
Example - 2
The glutaraldehyde stabilized cetyltrimethylammoniurn bromide (CTAB) permeabi1ized cells readily stoichiometrical1y converted D-amino acids to the corresponding α-keto acids from the racemic mixture of amino acids (DL-amino acids). The enzyme preparation stereospecifical1y converted only D-amino acids to the corresponding^-keto acids leaving behind the L- counter part. The glutaraldehyde treated CTAB permeabi1ized cells (4 G wet wt.) incubated with 100 ml of 50mM sodium pyrophosphate buffer pH 8.5, containing 50 mM DL-phenylalanine for 3 h. at 37° C. D-pheny1alanine was completely and stoichiometrical1y converted to phenyl pyruvate within three hours of the reaction. No decarboxylated product of phenyl pyruvate was formed during the bioconversion. After the reaction the cells were separated by centrifugation. The conversion of the D-pheny1alanine to phenyl pyruvate was monitored as described in example 1. Resolution of the racemic mixture was monitored by the optical rotation studies.
The glutaraldehyde treated CTAB permeabi1ized cells (4 G wet wt.) were incubated with 100ml of 50rnM sodium pyrophosphate buffer pH 8,5, containing 25 mM D-methionine for 3 h. at 37° C. After the reaction the cells were separated by centrifugation and the conversion was monitored as described in example 1.
The results in Table 2 indicate that D-isomer of the racemic mixture was removed completely and stlochiometriacl1y leaving behind
the L-counterpart. Formation of the ketoacid was determined as
described in example 1.
Table -2 : Polarimetric studies of the amino acid solution before and after the D-amino acid oxidase reaction.
(Table Removed)
2. Product of the D-amino acid oxidase reaction with D-methionine.
(Table Removed)
Example - 3.
From the reaction mixture obtained (100ml) in Example. 2, products
L-amino acids and o chromatography using Dowex-1 x-8-100 and Amberllite resins. The res-in
was equi1ibriated with 50mM tris-HCl, pH 8.5. The keto acids were
bound to the column and amino acids eluted out. Bound keto acids were
eluted using 40% acetonitrile in 1 M HC1.
Example - 4
In the example.1 and 2, since enzymes were stabilized within the
cells by glutaraldehyde treatment, after the initial reaction (first
cycle) the cells were collected either by centrifugation or filtration
and reused as a source of enzymes for subsequent cycles. These
microbial cells containing stabilized intracellular enzymes such as
DAAO and catalase were repeatedly used upto 20 cycles without any loss
in enzyme activity.
Intracellular enzymes in general seem to be stabilized by the
proximity of cellular structures or inclusion in cell membranes even
when such structures or membranes are no longer alive or functional.
Immobilization of DAAO and catalase within the cells by
glutaraldehyde, which brings about the crosslinking of the enzymes
with other intracellular components or to the cell debris maintains
enzymes natural microenvironment rendering them more stable.
Permeabi1ization of such glutaraldehyde treated cells by CTAB
improves the mass transfer of substrates and products through the cell
debris.
The advantages of the present invention are:
1. The enzyme prepaprations described and claimed in our copending
application no.Del/337/97 can be used directly for their application
mainly to separate the L-amino acids by degrading D-amino acids from
the racemic mixture and for the preparation of α-keto acids of both
essential and non-essential amino acids from the corresponding racemic
mixture of amino acids.
2. Since no step of isolation and purification of the enzymes D-ammo
acid oxidase and catalase are involved in the process of the present
invention and the cells are repeatedly used as a source of D-amino
acid oxidase and catalase, the cost is considerably reduced.
3. The biocatalyst preapared as described and claimed in our copending
application no.Del/337/97, could be separated from the reaction
mixture from each cycle by centrifugation or filtration and reused as
a source of enzymes for the subsequent cycles.
4. In the earlier methods exogeneous H2O2 degrading system viz.,
catalase and or Mn02 are included to obtain α-keto acids. However, in
this system catalase is present in about 40 times more than that of D-
amino acid oxidase. Therefore, additional H2O2 degrading system is not
requi red.

We Claim :-
1. A Bioconversiontprocess for preparation of Alpha-Ketoacids and L-amino acids
from racemic mixture of amino acids which comprises:
a. Reacting mixture of D/DL amino acids as herein described with D-
amino acid oxidase which is an intracellular enzyme located in cell and
Catalase enzyme as herein described at temperature an the range of 5-40°C
for a period of 1 to 24 hours in a rotary shaker at revolution per minute
ranging from 160 to 250 in alkali smetal pyrophosphate buffer as herein
described at a pH range of 6 to 9.5
b. During reaction, D-amino acid completely gets deaminated to alpha-keto
acids and the reaction mixture contains both product.
c. Separating alpha-ketoacids and L-amino acids from the said reaction
mixture by ion-exchange chromatography
2. A process as claimed in claim 1 wherein the enzyme deaminate all the 20
naturally occurring D-amino acid and their synthetic analogues.
2. A process as claimed in claims 1 and 2 wherein, the pH is maintained by alkali metal pyrophosphate buffer with concentration ranging from 0.005M to 0.1M and alkali-metal selected from sodium or potassium.
3. A process as claimed in claim (1) & (2), where-in the concentration of the enzymes (s) ranges from 0.5% to 4.0% (W/V wet wt of the yeast)
4. A Bioconversion process for the preparation of L- keto-acids and L- amino acids from racemic mixture of amino acids substantially as herein described with reference to the examples.

Documents:

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1314-del-1999-complete specification (granted).pdf

1314-del-1999-correspondence-others.pdf

1314-del-1999-correspondence-po.pdf

1314-del-1999-description (complete).pdf

1314-del-1999-form-1.pdf

1314-del-1999-form-2.pdf

1314-del-1999-form-4.pdf

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Patent Number

191267

Indian Patent Application Number

1314/DEL/1999

PG Journal Number

42/2003

Publication Date

18-Oct-2003

Grant Date

26-Apr-2004

Date of Filing

30-Sep-1999

Name of Patentee

COUNCIL OF SCEINTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DLEHI 110001, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	SANTHOOR GURURAJA BHAT	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE- 570013,KARNATAKA
2	NAGAJYOTHI	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE- 570013,KARNATAKA
3	RAJENDRA UPADHYA	CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE- 570013,KARNATAKA

PCT International Classification Number

C07C 229/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA