CA1291925C - Process for preparing l-amino acids by transamination - Google Patents
Process for preparing l-amino acids by transaminationInfo
- Publication number
- CA1291925C CA1291925C CA000535341A CA535341A CA1291925C CA 1291925 C CA1291925 C CA 1291925C CA 000535341 A CA000535341 A CA 000535341A CA 535341 A CA535341 A CA 535341A CA 1291925 C CA1291925 C CA 1291925C
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- amino group
- alpha
- acid
- group donor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Abstract of the disclosure The yield of L-amino acids by transamination of .alpha.-keto acids by means of microorganisms can be substantially increased by passing in a gas.
Description
HOECHST AKTIENGESELLSCHAFT HOE 86/F 083 Dr.KH/je Process for preparing L-amino acids by transamination Amino acids have a versatile application profile. They are used as a supplement in animal nutrition, as a food additive for man or as an ingredient of infusion solu-tions. A further use of L-phenylalanine is as a build-ing block for the synthesis of the sweetener aspartam, which is composed of methyl phenylalaninate and aspartic acid.
The preparation of L-amino acids by biotransformation with transaminases is known per se. European Patent App-lication 152,275 describes, in particular, a process for preparing phenylalanine by transamination by means of a genetically engineered microorganism which is dis-tinguished by overproduction of aminotransferase. The transamination reaction is carried out at a temperature of at least 40C, since the microorganism cells become more permeable at these higher temperatures.
According to European Patent Application 135,846 (US
Patents 4,518,692 and 4,525,454), L-amino acids are pre-pared by reacting ~-keto acids with L-aspartic acid in the presence of a transaminase which was isolated from E. coli. The products are the ~-amino acid correspond-ing to the keto acid and oxalacetate from the aspartic acid. 9y removing oxalacetate from the reaction medium after decarboxylation, the reaction equilibrium is shif-ted towards the side of the end product. Due to its in-stability, oxalacetate decarboxylates in aqueous solution.This reaction can be accelerated thermally, chemically or enzymatically.
The seLection and mutation of microorganisms from the series comprising E. coli, Paracoccus denitrificans, Torula, Rhodotorula and Streptomyces for the preparation of L-phenylalanine from phenylpyruvic acid in an improved ~k yield is illustrated in German Patent Application 3,423,936.
It has now been found, surprisingly, that a yield of the desired amino acid of up to 100% can be achieved after a short reaction time by simply passing gas into the re-action solution in which the transamination is carried out.
The invention thus relates to a process for the micro-bial preparation of L-amino acids by transamination of an ~-keto acid by means of an amino group donor, which comprises using asparagine, aspartic acid, glutamine and glutamic acid as the amino group donor and passing gas into the reaction solution.
The invention is explained in detail below and defined in the patent claims.
Numerous microorganisms are capable of converting ~-keto acids into a L-amino acids by biotransformation. These microorganisms can be employed according to the invention.
Preferably, however, Paracoccus denitrificans DSM 65 and Streptomyces isolated from soil samples are used. The best results are obtained with E. coli ATTC 11303. It is advantageous, but not absolutely necessary, to choose, by selection and mutation in a manner known per se, especi-ally according to the process of German Patent Appli-cation 3,423,936, in the presence of increasing quantities of the corresponding ~-keto acid in the culture media for the further work, microorganisms which effect the bio-transformation in h;gher yields due to their adaptation to the ~-keto acid.
~y selection, for example, transaminase activities of 100-200 ~mol/min x l of culture fluid can be taken as the basis. In this way, up to 60 g/l of ~-keto acid can be transaminated with approximately 100~ yield to give the corresponding amino acid by the process according to the invention.
The microorganisms are advantageously cultured in a nut-rient medium, which is optimized for their growth, under appropriate favorable temperature and aeration conditions up to a moist weight of about 4 to 10 g/l of nutrient solution. The most favorable conditions for the parti-cular microorganism are either known to those skilled in the art or can be determined in simpLe preLiminary experi-ments. The cells are then employed for the amination ofthe keto acids in the nutrient solution or after separa-tion from the nutrient solution. The transamination can be carried out with whole cells or with digested cells, the usuaL digestion methods being applied. For reasons of easing the work, intact cells are used preferably.
It is also possible to employ the microorganisms in a fixed form. For fixing, the known processes can be used, advantageously the methods according to German Offenle-gungsschriften 3,237,341 (US Patent 4,603,111) and 3,243,591 ~US Patent 4,542,069).
The microorganisms are suspended in a buffer which is physiological for the cells, the -keto acid and the amino group donor being added. Depending on the quan-tity of the microorganisms, the enzymatic activity addedto the batch can vary within wide ranges. Appropriately, it is between 10 and 20,000 ~mol/min x l. Preferably, the batch contains cell quantities having an enzyme ac-tivity of 1500-2000 ~mol/min x l.
The amino group donors used are amino acids, for example glycine, alanine, valine, leucine and especially aspara-gine, aspartic acid, glutamine and glutamic acid. These amino scids are employed in the form of their free acid or of suitable salts (in accordance with the medium used). The amino group donor is employed in equimolar amounts or in an excess relative to the ~-keto ac;d.
Ratios of 1:1 to 5:1, advantageously 1:1 to 2:1, have proved suitable.
lZ9~
The reaction components can be added simultaneously to the reaction batch, as a soLution in water or by adding the solid substances. However, a staggered or continuous addition in quantities of 1-4,5%, in particular 1.5-2Yo, each rela-tive to the weight of the reaction batch, over a periodof 1-90 hours, preferably 2-40 hours, is preferred.
Advantageously, the process is carried out at a pH be-tweer 5 and 9, especially 7 and 8.5. Moreover, it is ap-propriate to carry out the transamination in a tempera-ture range from 20 to 65C. At lower temperatures, the enzyme reaction increasingly slows down, whereas the enzyme is progressively deactivated at higher tempera-tures.
The most advantageous procedure depends on the particular microorganism and can easily be fixed by simple prelim-inary experiments.
It has proved to be particularly advantageous to make the microorganisms more permeable before or during the transamination reaction. This can be effected by adding suitable agents, such as toluene, cetyltrimethylammonium bromide, dimethyl sulfoxide etc., to the incubation medium.
Surprisingly, high conversion rates in a short time are achieved when gas is passed into the reaction batches described, wherein the biotransformation takes place.
The rate of passing in gas is in the range of 0.1-15 vol-umes per volume per minute (VVm), advantageously in the range of 0.2-3 VVm. In principle, all gases can be used which do not significantly reduce the enzyme activity of the microorganism. Suitable examples are compressed air, pure oxygen, nitrogen and also various rare gases, such as helium, neon, argon or krypton.
8ecause of their low price and accessibility, compressed air and pure nitrogen are preferred.
S
Using the process according to the invention, in prin-ciple all ~-keto acids can be aminated. Preferably, amino acids are used which are incorporated into natural proteins, in particular those listed in the table.
Precursor, End product ~-keto acid natural amino acid Pyruvate Alanine Dimethylpyruvate Valine 10 Isopropylpyruvate Leucine Ethylmethylpyruvate Isoleucine Hydroxypyruvate Serine Phenylpyruvate Phenylalanine 4-Hydroxyphenylpyruvate Tyrosine 15 Indolepyruvate Tryptophan The examples which follow serve to illustrate the inven-tion further, which has been described. Unless other-wise stated, percentage data relate to the weight.
Example 1 Escherichia coli ATTC 11303 was cultured by conventional methods and mutagenized with N-methyl-N-nitro-N-nitro-Z5 guanidine ~MNG). The cells treated with MNG were spreadupon an autocLaved agar of the following composition:
Fumaric acid 5 g/l Meat extract 20 g/l 30 Aspartic acid 20 g/l KHzP04 2 g/l MgS04 x 7 H20 CaCl2 x 2 HzO 0.1 g/l Agar 20 g/l The pH was adjusted to 7.2 with sodium hydroxide solution.
A ster;le-filtered solution of phenylpyruvate was poured into the still hot agar, such that an end concentration of 24 g/l of phenylpyruvate was reached. The plates were incubated for 4 cdays at 37C. Colonies of a diameter of > 1 mm were isolated. 20% of the growing strains had a transaminase activity higher than that of the ori-ginal strain.
The transaminase activity was determined by means of the Sigma test kit G 0390. Instead of ~-ketoglutarate, 12 mmol/l of sodium phenylpyruvate were used.
Example 2 The Escherichia coli ATCC 11303 mutant selectioned accord-ing to Example 1 was cultured in the nutrient solution of Example 1 without agar. It had a transaminase activity of 170 ~mol/min x l after 20 hours' growth at 37QC. The cells were centrifuged off, washed with 50 mmol/l of pH 7.4 phosphate buffer and suspended in 30 mmolar phos-phate buffer (pH 7.4), such that the suspension contained 1500 ~mol/min x l of transaminase activity. 24 g/l of Na phenylpyruvate and 20 g/l of aspartic acid were added to the cell suspension.
After an incubation period of 6 hours at 37C, the reac-tion mixture contained 21 g/l of L-phenylalanine. The solut;on was fi(tered and concentrated to 1:10 by evapora-tion of water. Phenylalanine was crystallized at pH 5.5 and 5C. The amino acid was determined qualitatively and quantitatively by HPLC on an RP 18 column.
Example 3 Cell material obtained according to Example 1 was sus-pended in 100 ml of a solution of 42 g/l of phenylpyru-vate, 34 g/l of aspartic acid and 10 mmol/l of pH 7.4phosphate buffer, such that the enzyme activity in the solution corresponded to 1500 ~mol/l x min. One half of the reaction batch was stirred at 37C, whereas 1 l of compressed air per liter of reaction buffer and per 1~91~;25 minute was additionally passed into the other half.
After 4 hours, the non-aerated reaction vessel contained 16 5 g/l of phenylalanine, whereas 26.9 g/l were mea-sured in the aerated vessel.
Example 4 Cells of Paracoccus denitrificans DSM 65, the quantity of which corresponds to an enzyme activity of 10û ~mol/l x min, were suspended in 100 ml of an aqueous solution of the following composition: 90 mmol/l of aspartic acid, 27 ~moL/l of N-cetyl-N,N,N-trimethylammonium bromide, 20 mmol/l of 4-hydroxyphenylpyruvate and 30 mmol/l of phosphate buffer (pH 7.4).
After 20 hours' incubation at 30C while passing 0.5 VVm of nitrogen in, 18 mmol/l of L-tyrosine were measured.
Example 5 Cell material as in Example 3 was incubated in 100 ml of an aqueous solution of 90 mmol/l of aspartic acid, 27 ~mol/l of N-cetyl-N,N,N-trimethylammonium bromide, 35 mmol/l of dimethylpyruvate and 30 mmol/l of phosphate buffer (pH
7.4). After 20 hours at 40C while passing in 1 VVm of compressed air, 30 mmol/l of valine were measured.
Example 6 Cell material as in Example 3 was incubated in 100 ml of an aqueous solution of 320 mmol/l of sodium phenylpyru-vate, 340 mmol/l of aspartic acid, 10 ~mol/l of toluene and 10 mmol/l of Tris/HCl buffer (pH 7.4). After 5 hours of pass;ng in 0.5 VVm of oxygen, a phenylalanine content of 150 mmol/l was measured at 37C, and a phenylalanine content of 270 mmol/l was measured at 50C.
Example 7 Cell material as in Example 3 was suspended in 100 ml of an aqueous solution of 30 g/l of sodium phenylpyruvate, 28 g/l of aspartic acid and 10 mmol/l of Tris/HCl buffer (pH 7.4). The suspension was incubated at 50C while passing in 0.5 VVm of compressed air. After 2 hours, a phenylalanine concentration of 25 g/l was measured. 30 9 of solid sodium phenylpyruvate and 26 9 of solid sodium aspartate were then metered in per litre of reaction volume. After a further 2 hours, a phenylalanine concen-tration of 45 g/l was measured.
The preparation of L-amino acids by biotransformation with transaminases is known per se. European Patent App-lication 152,275 describes, in particular, a process for preparing phenylalanine by transamination by means of a genetically engineered microorganism which is dis-tinguished by overproduction of aminotransferase. The transamination reaction is carried out at a temperature of at least 40C, since the microorganism cells become more permeable at these higher temperatures.
According to European Patent Application 135,846 (US
Patents 4,518,692 and 4,525,454), L-amino acids are pre-pared by reacting ~-keto acids with L-aspartic acid in the presence of a transaminase which was isolated from E. coli. The products are the ~-amino acid correspond-ing to the keto acid and oxalacetate from the aspartic acid. 9y removing oxalacetate from the reaction medium after decarboxylation, the reaction equilibrium is shif-ted towards the side of the end product. Due to its in-stability, oxalacetate decarboxylates in aqueous solution.This reaction can be accelerated thermally, chemically or enzymatically.
The seLection and mutation of microorganisms from the series comprising E. coli, Paracoccus denitrificans, Torula, Rhodotorula and Streptomyces for the preparation of L-phenylalanine from phenylpyruvic acid in an improved ~k yield is illustrated in German Patent Application 3,423,936.
It has now been found, surprisingly, that a yield of the desired amino acid of up to 100% can be achieved after a short reaction time by simply passing gas into the re-action solution in which the transamination is carried out.
The invention thus relates to a process for the micro-bial preparation of L-amino acids by transamination of an ~-keto acid by means of an amino group donor, which comprises using asparagine, aspartic acid, glutamine and glutamic acid as the amino group donor and passing gas into the reaction solution.
The invention is explained in detail below and defined in the patent claims.
Numerous microorganisms are capable of converting ~-keto acids into a L-amino acids by biotransformation. These microorganisms can be employed according to the invention.
Preferably, however, Paracoccus denitrificans DSM 65 and Streptomyces isolated from soil samples are used. The best results are obtained with E. coli ATTC 11303. It is advantageous, but not absolutely necessary, to choose, by selection and mutation in a manner known per se, especi-ally according to the process of German Patent Appli-cation 3,423,936, in the presence of increasing quantities of the corresponding ~-keto acid in the culture media for the further work, microorganisms which effect the bio-transformation in h;gher yields due to their adaptation to the ~-keto acid.
~y selection, for example, transaminase activities of 100-200 ~mol/min x l of culture fluid can be taken as the basis. In this way, up to 60 g/l of ~-keto acid can be transaminated with approximately 100~ yield to give the corresponding amino acid by the process according to the invention.
The microorganisms are advantageously cultured in a nut-rient medium, which is optimized for their growth, under appropriate favorable temperature and aeration conditions up to a moist weight of about 4 to 10 g/l of nutrient solution. The most favorable conditions for the parti-cular microorganism are either known to those skilled in the art or can be determined in simpLe preLiminary experi-ments. The cells are then employed for the amination ofthe keto acids in the nutrient solution or after separa-tion from the nutrient solution. The transamination can be carried out with whole cells or with digested cells, the usuaL digestion methods being applied. For reasons of easing the work, intact cells are used preferably.
It is also possible to employ the microorganisms in a fixed form. For fixing, the known processes can be used, advantageously the methods according to German Offenle-gungsschriften 3,237,341 (US Patent 4,603,111) and 3,243,591 ~US Patent 4,542,069).
The microorganisms are suspended in a buffer which is physiological for the cells, the -keto acid and the amino group donor being added. Depending on the quan-tity of the microorganisms, the enzymatic activity addedto the batch can vary within wide ranges. Appropriately, it is between 10 and 20,000 ~mol/min x l. Preferably, the batch contains cell quantities having an enzyme ac-tivity of 1500-2000 ~mol/min x l.
The amino group donors used are amino acids, for example glycine, alanine, valine, leucine and especially aspara-gine, aspartic acid, glutamine and glutamic acid. These amino scids are employed in the form of their free acid or of suitable salts (in accordance with the medium used). The amino group donor is employed in equimolar amounts or in an excess relative to the ~-keto ac;d.
Ratios of 1:1 to 5:1, advantageously 1:1 to 2:1, have proved suitable.
lZ9~
The reaction components can be added simultaneously to the reaction batch, as a soLution in water or by adding the solid substances. However, a staggered or continuous addition in quantities of 1-4,5%, in particular 1.5-2Yo, each rela-tive to the weight of the reaction batch, over a periodof 1-90 hours, preferably 2-40 hours, is preferred.
Advantageously, the process is carried out at a pH be-tweer 5 and 9, especially 7 and 8.5. Moreover, it is ap-propriate to carry out the transamination in a tempera-ture range from 20 to 65C. At lower temperatures, the enzyme reaction increasingly slows down, whereas the enzyme is progressively deactivated at higher tempera-tures.
The most advantageous procedure depends on the particular microorganism and can easily be fixed by simple prelim-inary experiments.
It has proved to be particularly advantageous to make the microorganisms more permeable before or during the transamination reaction. This can be effected by adding suitable agents, such as toluene, cetyltrimethylammonium bromide, dimethyl sulfoxide etc., to the incubation medium.
Surprisingly, high conversion rates in a short time are achieved when gas is passed into the reaction batches described, wherein the biotransformation takes place.
The rate of passing in gas is in the range of 0.1-15 vol-umes per volume per minute (VVm), advantageously in the range of 0.2-3 VVm. In principle, all gases can be used which do not significantly reduce the enzyme activity of the microorganism. Suitable examples are compressed air, pure oxygen, nitrogen and also various rare gases, such as helium, neon, argon or krypton.
8ecause of their low price and accessibility, compressed air and pure nitrogen are preferred.
S
Using the process according to the invention, in prin-ciple all ~-keto acids can be aminated. Preferably, amino acids are used which are incorporated into natural proteins, in particular those listed in the table.
Precursor, End product ~-keto acid natural amino acid Pyruvate Alanine Dimethylpyruvate Valine 10 Isopropylpyruvate Leucine Ethylmethylpyruvate Isoleucine Hydroxypyruvate Serine Phenylpyruvate Phenylalanine 4-Hydroxyphenylpyruvate Tyrosine 15 Indolepyruvate Tryptophan The examples which follow serve to illustrate the inven-tion further, which has been described. Unless other-wise stated, percentage data relate to the weight.
Example 1 Escherichia coli ATTC 11303 was cultured by conventional methods and mutagenized with N-methyl-N-nitro-N-nitro-Z5 guanidine ~MNG). The cells treated with MNG were spreadupon an autocLaved agar of the following composition:
Fumaric acid 5 g/l Meat extract 20 g/l 30 Aspartic acid 20 g/l KHzP04 2 g/l MgS04 x 7 H20 CaCl2 x 2 HzO 0.1 g/l Agar 20 g/l The pH was adjusted to 7.2 with sodium hydroxide solution.
A ster;le-filtered solution of phenylpyruvate was poured into the still hot agar, such that an end concentration of 24 g/l of phenylpyruvate was reached. The plates were incubated for 4 cdays at 37C. Colonies of a diameter of > 1 mm were isolated. 20% of the growing strains had a transaminase activity higher than that of the ori-ginal strain.
The transaminase activity was determined by means of the Sigma test kit G 0390. Instead of ~-ketoglutarate, 12 mmol/l of sodium phenylpyruvate were used.
Example 2 The Escherichia coli ATCC 11303 mutant selectioned accord-ing to Example 1 was cultured in the nutrient solution of Example 1 without agar. It had a transaminase activity of 170 ~mol/min x l after 20 hours' growth at 37QC. The cells were centrifuged off, washed with 50 mmol/l of pH 7.4 phosphate buffer and suspended in 30 mmolar phos-phate buffer (pH 7.4), such that the suspension contained 1500 ~mol/min x l of transaminase activity. 24 g/l of Na phenylpyruvate and 20 g/l of aspartic acid were added to the cell suspension.
After an incubation period of 6 hours at 37C, the reac-tion mixture contained 21 g/l of L-phenylalanine. The solut;on was fi(tered and concentrated to 1:10 by evapora-tion of water. Phenylalanine was crystallized at pH 5.5 and 5C. The amino acid was determined qualitatively and quantitatively by HPLC on an RP 18 column.
Example 3 Cell material obtained according to Example 1 was sus-pended in 100 ml of a solution of 42 g/l of phenylpyru-vate, 34 g/l of aspartic acid and 10 mmol/l of pH 7.4phosphate buffer, such that the enzyme activity in the solution corresponded to 1500 ~mol/l x min. One half of the reaction batch was stirred at 37C, whereas 1 l of compressed air per liter of reaction buffer and per 1~91~;25 minute was additionally passed into the other half.
After 4 hours, the non-aerated reaction vessel contained 16 5 g/l of phenylalanine, whereas 26.9 g/l were mea-sured in the aerated vessel.
Example 4 Cells of Paracoccus denitrificans DSM 65, the quantity of which corresponds to an enzyme activity of 10û ~mol/l x min, were suspended in 100 ml of an aqueous solution of the following composition: 90 mmol/l of aspartic acid, 27 ~moL/l of N-cetyl-N,N,N-trimethylammonium bromide, 20 mmol/l of 4-hydroxyphenylpyruvate and 30 mmol/l of phosphate buffer (pH 7.4).
After 20 hours' incubation at 30C while passing 0.5 VVm of nitrogen in, 18 mmol/l of L-tyrosine were measured.
Example 5 Cell material as in Example 3 was incubated in 100 ml of an aqueous solution of 90 mmol/l of aspartic acid, 27 ~mol/l of N-cetyl-N,N,N-trimethylammonium bromide, 35 mmol/l of dimethylpyruvate and 30 mmol/l of phosphate buffer (pH
7.4). After 20 hours at 40C while passing in 1 VVm of compressed air, 30 mmol/l of valine were measured.
Example 6 Cell material as in Example 3 was incubated in 100 ml of an aqueous solution of 320 mmol/l of sodium phenylpyru-vate, 340 mmol/l of aspartic acid, 10 ~mol/l of toluene and 10 mmol/l of Tris/HCl buffer (pH 7.4). After 5 hours of pass;ng in 0.5 VVm of oxygen, a phenylalanine content of 150 mmol/l was measured at 37C, and a phenylalanine content of 270 mmol/l was measured at 50C.
Example 7 Cell material as in Example 3 was suspended in 100 ml of an aqueous solution of 30 g/l of sodium phenylpyruvate, 28 g/l of aspartic acid and 10 mmol/l of Tris/HCl buffer (pH 7.4). The suspension was incubated at 50C while passing in 0.5 VVm of compressed air. After 2 hours, a phenylalanine concentration of 25 g/l was measured. 30 9 of solid sodium phenylpyruvate and 26 9 of solid sodium aspartate were then metered in per litre of reaction volume. After a further 2 hours, a phenylalanine concen-tration of 45 g/l was measured.
Claims (9)
1. A process for the microbial preparation of L-amino acids which comprises transaminating an .alpha.-keto acid by means of an amino group donor in aqueous solution, the amino group donor being selected from the group consisting of asparagine, aspartic acid, glutamine and glutamic acid, and passing gas into the reaction solution.
2. The process as claimed in claim 1, wherein 0.1-15 volumes per volume per minute (W m) of gas are passed into the reaction solution.
3. The process as claimed in claim 2, wherein 0.2-3 VVm of gas are passed into the reaction solution.
4. The process as claimed in claim 1 wherein the .alpha.-keto acid and the amino group donor are added to the reaction medium simultaneously and staggered over a period of 1-90 hours.
5. The process as claimed in claim 2 or 3, wherein the .alpha.-keto acid and the amino group donor are added to the reaction medium simultaneously and staggered over a period of 1-90 hours.
6. The process as claimed in claim 4, wherein the addition is carried out over a period of 2-40 hours.
7. The process as claimed in claim 1, wherein the amino group donor and the .alpha.-keto acid are employed in a ratio of 1:1 to 5:1 (mol:mol).
8. The process as claimed in claim 2, 3 or 4, wherein the amino group donor and the .alpha.-keto acid are employed in a ratio of 1:1 to 5:1 (mol:mol).
9. The process as claimed in claim 7, wherein the amino group donor and the .alpha.-keto acid are employed in a ratio of 1:1 to 2:1 (mol:mol).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3613952.1 | 1986-04-24 | ||
| DE19863613952 DE3613952A1 (en) | 1986-04-24 | 1986-04-24 | METHOD FOR PRODUCING L-AMINO ACIDS BY TRANSAMINATION |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1291925C true CA1291925C (en) | 1991-11-12 |
Family
ID=6299476
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000535341A Expired - Fee Related CA1291925C (en) | 1986-04-24 | 1987-04-23 | Process for preparing l-amino acids by transamination |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0242833B1 (en) |
| JP (1) | JPS62257392A (en) |
| AU (1) | AU7191387A (en) |
| CA (1) | CA1291925C (en) |
| DE (2) | DE3613952A1 (en) |
| DK (1) | DK206287A (en) |
| FI (1) | FI871761A (en) |
| IL (1) | IL82297A0 (en) |
| NO (1) | NO871687L (en) |
| PT (1) | PT84743B (en) |
| ZA (1) | ZA872880B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3721838A1 (en) * | 1987-07-02 | 1989-01-12 | Hoechst Ag | METHOD FOR PRODUCING L-PHENYLALANINE FROM BENZYLIDENE HYDANTOINE |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4600692A (en) * | 1983-02-10 | 1986-07-15 | Purification Engineering, Inc. | Immobilized cells for preparing phenylalanine |
| NL8401049A (en) * | 1983-08-05 | 1985-03-01 | Grace W R & Co | PROCESS FOR THE PREPARATION OF L-AMINO ACIDS FROM ALFA-KETO ACIDS. |
| HU193902B (en) * | 1983-09-01 | 1987-12-28 | Genetics Inst | Process for preparing l-amino acids by means of transamination |
| GB2152503B (en) * | 1984-01-05 | 1986-03-19 | Grace W R & Co | Process for producing l-phenylalanine |
| GB8403244D0 (en) * | 1984-02-07 | 1984-03-14 | Searle & Co | Aminoacids via bioconversion |
| DE3423936A1 (en) * | 1984-06-29 | 1986-01-02 | Hoechst Ag, 6230 Frankfurt | METHOD FOR PRODUCING L-PHENYLALANINE |
| BE902313A (en) * | 1985-04-29 | 1985-08-16 | Genex Corp | Phenylalanine ammonia lyase enzyme prepn. - by cultivating microorganism, e.g. Rhodotorula, aerobically and then under static conditions anaerobically |
-
1986
- 1986-04-24 DE DE19863613952 patent/DE3613952A1/en not_active Withdrawn
-
1987
- 1987-04-18 EP EP87105790A patent/EP0242833B1/en not_active Expired - Lifetime
- 1987-04-18 DE DE8787105790T patent/DE3776717D1/en not_active Expired - Fee Related
- 1987-04-22 FI FI871761A patent/FI871761A/en not_active Application Discontinuation
- 1987-04-22 IL IL82297A patent/IL82297A0/en unknown
- 1987-04-23 CA CA000535341A patent/CA1291925C/en not_active Expired - Fee Related
- 1987-04-23 JP JP62098759A patent/JPS62257392A/en active Pending
- 1987-04-23 PT PT84743A patent/PT84743B/en unknown
- 1987-04-23 AU AU71913/87A patent/AU7191387A/en not_active Abandoned
- 1987-04-23 NO NO871687A patent/NO871687L/en unknown
- 1987-04-23 ZA ZA872880A patent/ZA872880B/en unknown
- 1987-04-23 DK DK206287A patent/DK206287A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| PT84743A (en) | 1987-05-01 |
| JPS62257392A (en) | 1987-11-09 |
| NO871687D0 (en) | 1987-04-23 |
| DK206287D0 (en) | 1987-04-23 |
| DE3613952A1 (en) | 1987-10-29 |
| ZA872880B (en) | 1987-11-25 |
| EP0242833B1 (en) | 1992-02-19 |
| AU7191387A (en) | 1987-10-29 |
| NO871687L (en) | 1987-10-26 |
| PT84743B (en) | 1989-06-08 |
| IL82297A0 (en) | 1987-10-30 |
| DK206287A (en) | 1987-10-25 |
| FI871761A0 (en) | 1987-04-22 |
| FI871761A (en) | 1987-10-25 |
| EP0242833A1 (en) | 1987-10-28 |
| DE3776717D1 (en) | 1992-03-26 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |