NO871687L - PROCEDURE FOR THE PREPARATION OF L-AMINO ACIDS BY TRANSAMINATION. - Google Patents

Description

The present invention relates to a method for the production of L-amino acids by transamination.

Amino acids have a diverse application profile. They find use as a supplement in animal nutrition, as a food additive for humans or as a component of infusion solutions. L-phenylalanine finds a further application as the synthetic building block for the sweetener Aspartame, which consists of phenylalanine methyl ester and aspartic acid.

The production of L-amino acids by biotransformation with transaminases is known per se. In the European patent application 152 275, in particular, a method for the production of phenylalanine by transamination by means of a genetically modified microorganism which is characterized by overproduction of aminotransferase is described. The transamination reaction is carried out at at least 40°C, as the microorganism cells are more easily penetrated at these higher temperatures.

According to the European patent application 135,846 (US 4,518,692; US 45 25 454), the preparation of the L-amino acids takes place in such a way that cx-keto acids are reacted with L-aspartic acid in the presence of a transaminase which is isolated from E. Coli. From the aspartic acid, the cx-amino acid corresponding to the keto acid, as well as oxalacetate, is formed. When oxaloacetate is removed from the reaction medium after decarboxylation, the reaction equilibrium shifts in the direction of the end product. Oxaloacetate is decarboxylated in aqueous solution due to its instability. This reaction can be accelerated thermally, chemically or enzymatically.

The selection and mutation of microorganisms from the group E. coli, Paracoccus denitrificans, Torula, Rhodotorula and Streptomyces for the production of L-phenylalanine from phenylpyruvic acid with an improved yield is described in the German patent application No. DE 3 423 936.

Surprisingly, it has now been found that by simple gas treatment of the reaction solution in which the transamination is carried out after a short reaction time, a yield of the desired amino acid of up to 100% can be achieved.

The invention therefore relates to a method for the microbial production of L-amino acids by transamination of a cx-keto acid using an amino group donor which is characterized in that asparagine, aspartic acid, glutamine and glutamic acid are used as amino group donors and that the reaction solution is treated with gas.

In what follows, the invention is described in more detail and also defined in the patent claims.

Numerous microorganisms are capable of converting α-keto acids into L-amino acids by biotransformation. These microorganisms can be used according to the invention. Preferably, however, work is done with Paracoccus denitrificans DSM 65, as well as with Streptomyceters isolated from soil samples. The best results are obtained with E. coli ATTC 11303. It is advantageous, but not absolutely necessary, that by selection and mutation in a manner known per se, in particular by the method according to the German patent application DE 3 423 936, for the further work microorganisms are selected against increasing amounts of the corresponding cx-keto acid in the culture media, which, due to their adaptation to the ot-keto acid, carry out the bio-transformation with better yields.

For example, selection can be based on transaminase activities of 100-200 jjmol/min'1 culture fluid. In this way, with the method according to the invention, up to 60 g/l cx-keto acid can be transaminated with approx. 100$ dividend to the corresponding amino acid.

The microorganisms are advantageously grown in a nutrient medium that is optimal for their growth under correspondingly favorable temperature and ventilation conditions up to a wet weight of approx. 4 to 10 g/l nutrient solution. The favorable conditions for any microorganism are either known to the person skilled in the art or can be determined in simple preliminary experiments. The cells are then used for amination of the keto acids in the nutrient solution or separated from the nutrient solution. Tranamination can be carried out with whole cells or with opened cells, whereby normal opening methods are used. In order to facilitate the work, it is preferable to work with intact cells. It is also possible to use the microorganisms in fixed form. For fixation, the usual methods come into consideration, advantageously the methods according to German specification documents 32 37 341 (US 4 603 111) and 32 43 591 (US 4 542 069).

The microorganisms are suspended in a physiological buffer for the cells with the addition of the cx-keto acid and the amino group donor. Depending on the quantity of the microorganisms, the enzymatic activity added to the mixture can vary within wide ranges. Appropriately, it lies between 10 and 20,000 pmol/min'1. Preferably, the mixture contains amounts of cells with an enzyme activity of 1500-2000 jjmol/min*l. Amino acids are used as amino group donors, such as e.g. glycine, alanine, valine, leucine, especially asparagine, aspartic acid, glutamine and glutamic acid. These amino acids are used in the form of their free acid or suitable salts (in accordance with the medium used). The amino group donor is used in equimolar amounts, respectively in excess, in relation to the cx-keto acid. Ratios from 1:1 to 5:1, advantageously 1:1 to 2:1, have been found suitable. The addition of the reaction components to the reaction mixture can take place as a solution in water or by adding the solids at the same time. However, a stepwise addition in amounts of 1-2$, especially 1.5-4.5$, calculated on the basis of the weight of the reaction mixture, over a period of 1-90 hours, preferably 2-40 hours, is preferred. It is beneficial to work at a pH value between 5 and 9, especially between 7 and 8.5. It is very appropriate to carry out the transamination in a temperature range of 20-65°C. At lower temperatures, the enzyme reaction proceeds more and more slowly, while at higher temperatures the enzyme is increasingly deactivated. The most favorable method depends on the microorganism in question and can be easily determined by simple experiments. ;It has proven particularly appropriate to permeabilize the microorganisms before or during the transamination reaction. This can be done by adding suitable agents, such as toluene, cetyltrimethylammonium bromide, dimethylsulfoxide etc. to the incubation medium. ;High turnover rates within a short time are surprisingly achieved when the aforementioned reaction mixture, in which the biotransformation takes place, is treated with gas. The gas treatment takes place in the range from 0.1-15 VVm, advantageously in the range 0.2-3 VVm. In principle, all gases can be used which do not significantly reduce the enzyme activity of the microorganism. Examples are compressed air, pure oxygen, nitrogen, but also various noble gases, such as helium, neon, argon or krypton. ;Due to the favorable price and availability, compressed air and pure nitrogen are preferred. With the method according to the invention, in principle all α-keto acids can be aminated. Preferably, amino acids are used which are incorporated into natural proteins, especially those listed in the following table. ; The following examples will illustrate the invention in more detail. Percentages indicate, unless otherwise stated, weight-$. Example 1 Escherichia coli ATTC 11303 was grown by conventional methods and mutagenized with N-methyl-N-nitro-N-nitro-guanidine (MNG). The MNG-treated cells were applied to an autoclaved agar with the following composition: ; The pH was adjusted to 7.2 with caustic soda. A sterile filtered solution of phenylpyruvate was poured into the still warm agar, so that a final concentration of 24 g/l phenylpyruvate was obtained. The plates were incubated at 37°C for 4 days. Colonies with a diameter >1 mm were isolated. 20% of the cultivated strains had an elevated transaminase activity compared to the starting strain. The transaminase activity determination was carried out with "Sigma Test kit G 0390". Instead of oc-ketoglutarate, 12 mmol/l phenylpyruvate sodium salt was used. ;Example 2;The mutants of Escherichia coli ATCC 11303 selected according to example 1 were grown in the nutrient solution from example 1 without agar. They had a transaminase activity of 170 µmol/min'1 after 20 hours of cultivation at 37°C. The cells were centrifuged, washed with 50 mmol/l phosphate buffer pH 7.4 and suspended in 30 mmol/l phosphate buffer (pH 7.4), so that the suspension contained a transaminase activity of 1500 jjmol/min*l. 24 g/l Na-phenylpyruvate and 20 g/l aspartic acid were added to the cell suspension.

After an incubation time of 6 hours at 37°C, the reaction mixture contained 21 g/l L-phenylalanine. The solution was filtered and by evaporation of the water concentrated to 1:10. Phenylalanine was crystallized at pH 5.5 and 5°C. The qualitative and quantitative determination of the amino acid took place by HPLC on an "RP 18" column.

Example 3

The cell material obtained in example 1 was suspended in 100 ml of a solution of 42 g/l phenylpyruvate, 34 g/l aspartic acid and 10 mmol/l phosphate buffer pH 7.4, so that the enzyme activity in the solution corresponded to 1500 pmol/1'min. One half of the reaction mixture was stirred at 37° C, while the other half was additionally treated with 1 1 compressed air per liter of reaction buffer and minute. After 4 hours, the non-aerated reaction vessel contained 16.5 g/l of phenylalanine, while the aerated vessel contained 26.9 g/l.

Example 4

Cells of Paracoccus denitrificans DSM 65, the amount of which corresponded to an enzyme activity of 100 pmol/1'min, were suspended in 100 ml of an aqueous solution of the following composition: 90 mmol/1 aspartic acid, 27 pmol/1 N-cetyl-N,N ,N-trimethyl-ammonium bromide, 20 mmol/1 4-hydroxyphenylpyruvate and 30 mmol/1 phosphate buffer (pH 7.4).

After 20 hours of incubation at 30°C and a gas treatment with 0.5 VVm of nitrogen, 18 mmol/1 L-tyrosine was measured.

Example 5

The cell material as in example 3 was incubated in 100 ml of an aqueous solution of 90 mmol/1 aspartic acid, 27 jjmol/1 N-cetyl-N,N,N-trimethylammonium bromide, 35 mmol/1 dimethylpyruvate and 30 mmol/1 phosphate buffer (pH 7.4). After 20 hours at 40° C and a gas treatment of 1 VVm with compressed air, 30 mmol/1 valine was measured.

Example 6

The cell material as in example 3 was incubated in 100 ml of an aqueous solution of 320 mmol/l phenylpyruvate sodium salt, 340 mmol/l aspartic acid, 10 jjmol/l toluene and 10 mmol/l tris/HCl buffer (pH 7.4). After 5 hours with a gas treatment of 0.5 VVm with oxygen, a phenylalanine content of 150 mmol/1 could be measured at 37°C and a phenylalanine content of 270 mmol/1 at 50°C.

Example 7

The cell material as in example 3 was suspended in 100 ml of an aqueous solution of 30 g/l phenylpyruvate sodium salt, 28 g/l aspartic acid and 10 mmol/l tris/HCl buffer (pH 7.4). The suspension was incubated at 50° C. under gas treatment with 0.5 VVm compressed air. After 2 hours, a phenylalanine concentration of 25 g/l could be measured. 30 g of solid phenylpyruvate sodium salt and 26 g of solid aspartic acid sodium salt were then added per liter reaction volume. After a further 2 hours, a phenylalanine concentration of 45 g/l could be measured.

Claims (7)

1. Process for microbial production of L-amino acids by transamination of a cx-keto acid using an amino group donor in aqueous solution, characterized in that asparagine, aspartic acid, glutamine and glutamic acid are used as amino group donors and that the reaction solution is gas treated. 2. Method according to claim 1, characterized in that the reaction solution is gas-treated with 0.1-15 VVm. 3. Method according to claim 2, characterized in that the reaction solution is gas-treated with 0.2-3 VVm. 4. Method according to one or more of claims 1 to 3, characterized in that the cx-keto acid and the amino group donor are added simultaneously to the reaction medium in portions over a period of 1-90 hours. 5. Method according to claim 4, characterized in that the addition takes place in 2-40 hours. 6. Method according to one or more of claims 1 to 5, characterized in that the amino group donor and the cx-keto acid are used in a ratio of 1:1 to 5:1 (mol:mol). 7. Method according to claim 6, characterized in that the amino group donor and the cx-keto acid are used in a ratio of 1:1 to 2:1 (mol:mol).