Process for the preparation of L-phosphinothricin by a coupled enzymatic reaction

ABSTRACT

A coupled enzyme reaction of a transaminase with GOT activity and of a transaminase with L-phosphinothricin transaminase activity produces phosphinothricin in virtually quantitative yield and virtually without any contamination whatever by a natural amino acid when the amino donor glutamate is employed in catalytic amounts and the amino donor aspartate is employed in approximately equimolar amounts relative to 4-(hydroxymethylphosphinyl)-2-oxobutyric acid.

INFORMATION

DETAILED DESCRIPTION OF THE INVENTION

This application is a continuation of application Ser. No. 08/273,615, filed Jul. 12, 1994, now abandoned, which is a continuation of application Ser. No. 08/130,148, filed Sep. 17, 1993, abandoned, which is a continuation of application Ser. No. 07/765,308, filed Sep. 25, 1991, abandoned.

EXAMPLE 1

Synthesis of L-phosphinothricin By Biotransformation with K-12

K-12 was cultivated in LB medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl) at 37° C. for 15 h. The bacterial cells were harvested by centrifugation at 5000 g for 10 min, washed twice in phosphate buffer (10 mM Na phosphate, pH=7.0, 10 mM NaCl) and then resuspended at a concentration of 100 mg/ml in the following solutions:

Solution 1

HMPB: glutamic acid: aspartic acid=1:1:1

100 mM HMPB

100 mM glutamic acid

100 mM aspartic acid

50 mM tris/HCl, pH=8.0.

Solution 2

HMPB: glutamic acid=1:4

100 mM HMPB

400 mM glutamic acid

50 mM tris/HCl, pH=8.0.

The cell suspensions were incubated with the reaction mixtures at 37° C. The L-phosphinothricin content in the reaction mixtures was determined with an amino-acid analyzer at various times during the course of the synthesis. Table 1 shows the percent conversion into L-phosphinothricin during the course of the reaction for both biotransformation mixtures. Whereas the L-PPT yield did not exceed 85% with a four-fold excess of the amino donor glutamic acid (Solution 2), it was possible to achieve conversion rates of almost 100% with equimolar use of the amino donors glutamic acid and aspartic acid (Solution 1).

EXAMPLE 2

Purification of Glutamate/Oxalacetate Transaminase from Pigs

EXAMPLE 3

Synthesis of L-phosphinothricin with Purified L-phosphinothricin-specific Transaminase from and GOT from Pigs at Various Glutamic Acid Concentrations

The L-phosphinothricin-specific transaminase (PST) from K-12 (see EP-A 0 344 683) and the GOT from pig hearts (see Example 2) were dissolved at a concentration of 1 mg/ml in phosphate buffer (20 mM Na phosphate, pH=7.0, 0.1 mM pyridoxal phosphate, 1 mM 2-ketoglutarate, 5 mM β-mercaptoethanol). Then the volumes in each case corresponding to an activity of 1 unit (=1 μmol of L-phosphinothricin/min in the case of PST and 1 μmol of glutamate/min in the case of GOT) of the two enzymes were mixed and incubated with the following reaction solutions at 37° C. for 1 h:

All reaction mixtures contained

100 mM HMPB

100 mM aspartic acid

50 mM tris/HC1, pH=8.0

and additionally

glutamic acid

in Experiment 1:100 mM

in Experiment 2:50 mM

in Experiment 3:20 mM

in Experiment 4:10 mM

in Experiment 5:5 mM

in Experiment 6: -

The L-phosphinothricin content in the various mixtures after the reaction time was determined with an amino-acid analyzer (Biotronic LC 5001). The L-phosphinothricin synthesis rates calculated therefrom as percent conversion are compiled in Table 2. The experiments show that it is possible, with a negligible adverse effect on the rate of L-phosphinothricin synthesis, to reduce the glutamic acid concentration to 0.2 times that of the HMPB or aspartate employed.

EXAMPLE 4

Synthesis of L-phosphinothricin with Purified PST () and GOT (Pig) at Various Enzyme Ratios

The two purified transaminases were incubated as described in Example 3 with a reaction solution composed of 100 mM HMPB, 20 mM glutamic acid, 100 mM aspartic acid and 50 mM tris/HCl, pH=8.0 at 37° C. Various ratios of the activities of the two enzymes were employed for this:

1. 1 unit of PST/10 units of GOT

2. 1 unit of PST/1 unit of GOT

3. 1 unit of PST/0 unit of GOT

The L-phosphinothricin content in the mixtures was determined at various times during the course of the synthesis reaction, measuring test samples in the amino-acid analyzer. The L-phosphinothricin syntheses calculated therefrom are shown in Table 3. The highest conversion was achieved with the enzyme ratio PST:GOT=1:1 (Mixture 2). The maximum PPT conversion rate achievable without addition of GOT (Mixture 3) was only about 20% because, in this case, only the glutamic acid but not the aspartic acid can be used for the synthesis of L-phosphinothricin.

EXAMPLE 5

Effect of Manganese Chloride on L-phosphinothricin Synthesis

The purified transaminases were mixed in the ratio 1:1 (1 unit of each) as described in Example 3 and incubated with reaction solution (100 mM HMPB, 20 mM glutamic acid, 100 mM aspartic acid, 50 mM tris/HCl, pH=8.0) in the presence of 1 mM MnCl2 without MnCl2 at 37° C. The course of L-phosphinothricin synthesis was measured as described in Example 4 and is summarized in Table 4. It was possible distinctly to increase the reaction rate by adding manganese chloride. The L-phosphinothricin conversion after a reaction time of 24 h was about 10% higher than that in the comparison mixture without manganese chloride.

EXAMPLE 6

Immobilization of the “PST” Transaminase on Silica Gel

50 ml of activated silica gel (silanization and glutaraldehyde activation as described in the literature: K. Mosbach, Methods in Enzymology, Vol. XLIV; Academic Press, New York, 1976, pages 139 and 140) are added to 50 ml of PST solution (purified PST from 10 mg of protein/ml of solution in 0.25 M potassium phosphate buffer, pH 8) and reaction is allowed to occur stirring gently for 3 h. The still moist catalyst is then filtered off and washed with 100 ml of 0.25 M phosphate buffer, pH 8. The biocatalyst prepared in this way can be employed for the reactions described below.

EXAMPLE 7

Immobilization of “GOT” Transaminase on Silica Gel

10 ml of a GOT solution (purified GOT from pigs, 8.4 mg of protein/10 ml of solution) are immobilized on 10 ml of silica gel as described in Example 6. The biocatalyst can be employed in this form for the coupled transamination.

EXAMPLE 8

Coupled Transamination with Immobilized PST and Immobilized GOT

180 mg (1 mmol) of HMPB, 133 mg (1 mmol) of L-aspartic acid, 29.4 mg (0.2 mmol) of L-glutamic acid, 10 mg of pyridoxal phosphate and 0.6 g of TRIS are made up with distilled water to 5 g of solution and adjusted to pH 8. To this are added 0.5 ml of immobilized PST (Example 6) and 0.5 ml of immobilized GOT (Example 7) and reaction is allowed to occur at pH 8 stirring cautiously at 26° C. for 48 h. The reaction mixture is investigated by HPLC (amino-acid analyzer) for the amino-acid content.

Yield (HPLC):

0.122 g of L-PPT (68% based on HMPB)

0.012 g of L-aspartic acid

0.028 g of L-glutamic acid

0.025 g of L-alanine

EXAMPLE 9

Coupled Transamination with Immobilized PST and Immobilized GOT

1 g (5.6 mmol) of HMPB, 715 mg (5.6 mmol) of L-aspartic acid, 163 mg (1.1 mmol) of L-glutamic acid and 10 mg of pyridoxal phosphate are made up with distilled water to 20 g of solution and adjusted to pH 8 with KHCO3. After addition of 1 ml of immobilized PST (Example 6) and 1 ml of GOT (Example 7), reaction is allowed to take place stirring gently at 36° C. for 72 h. The product solution is investivated by HPLC (AAA) for the amino-acid content.

Yield (HPLC):

0.58 g of L-PPT (58% based on HMPB)

0.30 g of L-aspartic acid

0.16 g of L-glutamic acid

0.02 g of L-alanine

EXAMPLE 10

Coupled Transamination with Immobilized PST and Free GOT

180 mg (1 mmol) of HMPB, 147 mg (1 mmol) of L-glutamic acid, 133 mg (1 mmol) of L-aspartic acid, 10 mg of pyridoxal phosphate and 0.7 g of TRIS are made up with distilled water to 50 g of solution, pH 8, and 0.5 ml of immobilized PST from the solution from Example 6 and 42 μl of free GOT from the GOT solution prepared in Example 7 are added. The reaction is carried out stirring cautiously at 36° C. The amino-acid content in the solution after 48 h is investigated by HPLC (AAA).

Yield (HPLC):

0.145 g of L-PPT (80% based on HMPB)

0.024 g of L-aspartic acid

0.147 g of L-glutamic acid

0.003 g of L-alanine

EXAMPLE 11

Coupled Transamination with Immobilized PST and Immobilized GOT

The reaction is carried out in analogy to Example 10. 0.5 ml of immobilized GOT (Example 6) is employed in place of free GOT. The amino-acid composition in the product solution corresponds to that from Example 10.

The L-PPT content was determined by thin-layer chromatography. It is about 80%.

CLAIMS

1. A process for the preparation of L-2-amino-4-(hydroxymethylphosphinyl)butyric acid of the formula (I): from 4-(hydroxymethylphosphinyl)-2-oxybutyric acid (HMPB) of the formula (II): in a coupled enzyme reaction using isolated enzymes comprising the following steps: (a) reacting aspartate and α-ketoglutarate with an amount of transaminase 1 effective to give oxaloacetate and glutamate, (b) reacting the glutamate produced with HMPB of the formula (II) with an amount of a suitable transaminase 2 to produce α-ketoglutarate and L-2-amino-4-(hydroxymethylphosphinyl)butyric acid and (c) recovering said L-2-amino-4-(hydroxymethyl phosphinyl)butyric acid, wherein the molar ratio of aspartate to HMPB is 0.8-1.2 to 1 and glutamate or α-ketoglutarate is added in catalytic amounts.

2. The process as claimed in claim 1, wherein glutamate or α-ketoglutarate is added in a molar ratio of 0.01-0.2 to 1 relative to HMPB.

3. The process as claimed in claim 1, wherein the transaminase 1 is a glutamate/oxalacetate transaminase.

4. The process as claimed in claim 3, wherein the glutamate/oxalacetate transaminase is obtained from Escherichia coli or Bacillus.

5. The process as claimed in claim 1, wherein the L-phosphinothricin-specific transaminase is obtained from Escherichia coli.

6. The process of claim 1, wherein oxaloacetate is decarboxylated to pyruvate in the presence of multiply charged cations.

7. The process of claim 6 wherein said multiply charged cations are selected from the group consisting of Al3+, Mg2+Mn2+, Zn2+, Fe2+, and Fe3+.

8. The process of claim 1, wherein one or more of the transaminases are present in immobilized form.

9. The process as claimed in claim 8, wherein the immobilized transaminase or transaminases are present in a columnar reactor.

10. The process as claimed in claim 1 wherein the molar ratio of aspartate to HMPB is equimolar.

11. The process as claimed in claim 1, wherein glutamate or α-ketoglutarate is added in a molar ratio of 0.05-0.2 to 1 relative to HMPB.

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