The invention provides a process for the production of N-(phosphonomethyl)glycine, also known as glyphosate. The process comprises hydrogenating a mixture containing glyoxylic acid and aminomethylphosphonic acid, the mixture having been enzymatically prepared in situ by the reaction of glycolic acid and oxygen in an aqueous solution containing aminomethylphosphonic acid and the enzymes glycolate oxidase and catalase.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to the preparation of N-(posphonomethyl)glycine by 
the hydrogenation of mixtures produced by the reaction of glycolic acid 
and oxygen in an aqueous solution containing aminomethylphosphonic acid 
(AMPA) and the enzymes glycolate oxidase ((S)-2-hydroxy-acid oxidase, EC 
1.1.3.15) and catalase (EC 1.11.1.6). N-(Phosphonomethyl)glycine is a 
broad-spectrum, post-emergent herbicide useful in controlling the growth 
of a wide variety of plants. 
2. Description of the Related Art 
Numerous methods are known for preparing N-(phosphonomethyl)glycine from 
aminomethylphosphonic acid and glyoxylic acid. One such method, described 
in Rogers et al., European Patent Application 186,648, involves 
condensation of glyoxylic acid or a salt thereof with 
aminomethylphosphonic acid or a salt thereof to form an intermediate 
product, generally regarded as an aldimine (Schiff base), which without 
isolation is reduced, as by catalytic hydrogenation, to 
N-(phosphonomethyl)glycine. A second method, described in Gaertner, U.S. 
Pat. No. 4,094,928, isolates these same intermediate 
carbonylaldiminomethanephosphonates by the reaction of glyoxylic acid 
esters with aminomethylphosphonate esters in a non-aqueous solvent; after 
azeotropic distillation of water and removal of the solvent, the 
carbonylaldiminomethanephosphonate ester is reduced and the ester groups 
hydrolyzed to produce N-(phosphonomethyl)glycine. 
The above routes to N-(phosphonomethyl)glycine suffer in that glyoxylic 
acid is a rather costly starting material, and other less expensive routes 
to the desired material are practiced. Existing methods for the 
preparation of glyoxylic acid, such as hydrolysis of a dihaloacetic acid, 
electrolytic reduction of oxalic acid, oxidation of glyoxal, catalytic 
oxidation of ethylene or acetaldehyde, and ozonolysis of maleic acid, its 
esters or anhydride, present one or more difficulties in practice, e.g. 
costly separation/purification steps, low yields, or large waste streams. 
The method described in Gaertner is also disadvantageous in that it 
requires several additional steps (with corresponding losses in yield), 
and the unnecessary isolation of an intermediate. 
Another method for the synthesis of N-(phosphonomethyl)glycine, disclosed 
in Kleiner, U.S. Pat. No. 4,670,191, comprises the reaction of 
aminomethylphosphonic acid or a salt thereof with about two molar 
equivalents of glyoxylic acid in aqueous medium. The excess glyoxylic acid 
evidently functions as a reducing agent, converting an intermediate 
glyoxylic acid-aminomethylphosphonic acid reaction product to the desired 
N-(phosphonomethyl)glycine, and is itself oxidized to one or more 
by-products, including CO.sub.2. Similarly, Fields et al., in U.S. Pat. 
No. 4,851,159 prepare N-(phosphonomethyl)glycine by heating an 
N-acylaminomethylphosphonic acid with glyoxylic acid or a derivative 
thereof. The mole ratio of the glyoxylic to the N-acylamino component is 
preferably 2 to 1; otherwise at smaller ratios the yield suffers. 
The Kleiner and Fields et al. processes entail the disadvantages of not 
only employing relatively expensive glyoxylic acid but of employing it as 
a sacrificial reductant (ca. one mole of glyoxylate employed as reductant 
for every mole of N-(phosphonomethyl)glycine produced) as well as the 
condensing agent for the amino-(or N-acylamino) methylphosphonic acid. 
SUMMARY OF THE INVENTION 
The process for preparing N-(phosphonomethyl)glycine according to the 
present invention involves hydrogenating a mixture, wherein the mixture is 
enzymatically produced by reacting glycolic acid and oxygen in an aqueous 
solution containing aminomethylphosphonic acid (AMPA) and the enzymes 
glycolate oxidase and catalase. It should be appreciated for purposes of 
this invention that mixtures, so produced, inherently result in a 
distribution of oxidation by-products in addition to the desired glyoxylic 
acid component (including by way of example but not limited thereto, 
oxalate, formate, and carbon dioxide). Also present in such mixtures will 
be unreacted glycolate as well as various additives such as flavin 
mononucleotide (hereinafter referred to as FMN) or the like, all of which 
may or may not influence the desired hydrogenation reaction (again by way 
of example, but not limited thereto, it has been found that both formate 
and FMN lower the recovered carbon balance when present during the 
hydrogenation of glyoxylic acid in the presence AMPA). 
Thus the present invention provides an improved process for preparing 
N-(phosphonomethyl)glycine comprising the step of reducing a mixture of 
glyoxylic acid and aminomethylphosphonic acid by hydrogenation; said 
mixture being enzymatically generated in situ in an aqueous solution by 
incorporating into the aqueous solution glycolic acid, a first catalyst 
adapted to catalyze the oxidation of glycolic acid with oxygen to 
glyoxylic acid and hydrogen peroxide, and a second catalyst adapted to 
catalyze the decomposition of hydrogen peroxide, adjusting the pH of the 
solution to between 7 and about 10, contacting the solution with a source 
of oxygen at an effective temperature and sufficient time to convert at 
least a portion of the glycolic component to the glyoxylic component in 
the presence of aminomethylphosphonic acid, and ceasing contacting the 
solution with oxygen prior to the reducing step. 
Preferably, the catalysts are enzymatic; more preferably the first enzyme 
is glycolate oxidase ((S)-2-hydroxy-acid oxidase, EC 1.1.3.15) and the 
second enzyme is catalase (EC 1.11.1.6). After the contacting of the 
solution with O.sub.2 in the presence of the catalysts/enzymes is ceased, 
the catalysts/enzymes are removed, as by filtration or centrifugation, 
before the solution is subjected to reducing conditions for the production 
of N-(phosphonomethyl)glycine. 
Thus, by obviating the need to prepare glyoxylic acid in a separate step, 
the present invention provides for a more efficient and economic process 
for the production of N-(phosphonomethyl)glycine. 
It is an object of this invention to provide an improved process for the 
production of N-(phosphonomethyl)glycine by reduction of mixtures of 
glyoxylic acid and aminomethylphosphonic acid which avoids the need to 
separately prepare glyoxylic acid. 
Another object is to provide such a process wherein glyoxylic acid is 
enzymatically generated in situ in the presence of aminomethylphosphonic 
acid from a readily available precursor thereof, namely glycolic acid, 
thereby affording a more efficient and economic process for the production 
of N-(phosphonomethyl)glycine. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The improved process for the production of N-(phosphonomethyl)glycine 
according to the present invention involves the reduction of a mixture 
containing glyoxylic acid (or a suitable derivative thereof) with 
aminomethylphosphonic acid (AMPA) (or a suitable derivative thereof). 
Prefferably, the mixture is prepared by catalytically oxidizing a glycolic 
acid component or a suitable salt thereof by contacting the glycolic acid 
component with a source of molecular oxygen in the presence of AMPA and a 
catalyst effective to catalyze the reaction of glycolic acid with O.sub.2 
to form glyoxylic acid. One such catalyst is a naturally-occurring enzyme 
glycolate oxidase (EC 1.1.3.15), also known as glycolic acid oxidase, 
which is capable of catalyzing acid the reaction to produce glyoxylic acid 
in high yields at high glycolic acid conversions in aqueous media under 
mild conditions of pH and temperature, i.e., 
EQU HOCH.sub.2 CO.sub.2 H+O.sub.2 .fwdarw.OCHCO.sub.2 H+H.sub.2 O.sub.2 
Optimal results in the use of glycolate oxidase as a catalyst for the 
oxidative conversion of glycolic acid to glyoxylic acid are obtained by 
incorporating into the reaction solution a catalyst for the decomposition 
of hydrogen peroxide. One such peroxide-destroying catalyst which is 
effective in combination with glycolate oxidase is the enzyme catalase 
(E.C. 1.11.1.6). Catalase catalyzes the decomposition of hydrogen peroxide 
to water and oxygen, and it is believed to improve yields of glyoxylic 
acid by accelerating the decomposition of the hydrogen peroxide produced 
as a byproduct with glyoxylic acid in the glycolate oxidase-catalyzed 
reaction of glycolic acid with O.sub.2. The concentration of catalase 
should be 50 to 50,000 IU/mL, preferably 500 to 15,000 IU/mL. It is 
preferred that the catalase and glycolate oxidase concentrations be 
adjusted within the above ranges so that the ratio (measured in IU for 
each enzyme) of catalase to glycolate oxidase is at least about 250:1. 
Another optional but often beneficial ingredient in the reaction solution 
is flavin mononucleotide (FMN), which is generally used at a concentration 
of 0.0 to about 2.0 mM, preferably about 0.01 to about 0.2 mM. It is 
believed the FMN increases the productivity of the glycolate oxidase, by 
which is meant the amount of glycolic acid converted to glyoxylic acid per 
unit of enzyme. It is to be understood that the concentration of added FMN 
is in addition to any FMN present with the enzyme, because FMN is often 
also added to the enzyme during the preparation of the enzyme. The 
structure of FMN and a method for its analysis is found in K. Yagai, 
Methods of Biochemical Analysis, Vol. X, Interscience Publishers, New 
York, 1962, p. 319-355, which is hereby included by reference. 
Glycolic acid (2-hydroxyacetic acid) is employed in the present reaction at 
an initial concentration in the range of 0.10M to 2.0M, preferably between 
0.25M and 1.0M. It can be used as such or as a compatible salt thereof, 
that is, a salt that is water-soluble and whose cation does not interfere 
with the desired conversion of glycolic acid to glyoxylic acid, or the 
subsequent reaction of the glyoxylic acid product with the 
aminomethylphosphonic acid to form N-(phosphonomethyl)glycine. Suitable 
and compatible salt-forming cationic groups are readily determined by 
trial. Representative of such salts are the alkali metal, alkaline earth 
metal, ammonium, substituted ammonium, phosphonium, and substituted 
phosphonium salts. 
The conversion of glycolic acid to glyoxylic acid is conveniently and 
preferably conducted in aqueous media. Aminomethylphosphonic acid (AMPA), 
or a suitable salt thereof, is added to produce a molar ratio of 
AMPA/glycolic acid (starting amount) in the range of from 0.01/1.0 to 
3.0/1.0, preferably from 0.25/1.0 to 1.05/1.0. After combining AMPA and 
glycolic acid in an aqueous solution, the pH of the resulting mixture is 
adjusted to a value between 6 and 10, preferably between 7.0 and 8.5. 
Within this pH range, the exact value may be adjusted to obtain the 
desired pH by adding any compatible, non-interfering base, including 
alkali metal hydroxides, carbonates, bicarbonates and phosphates. The pH 
of the reaction mixture decreases slightly as the reaction proceeds, so it 
is often useful to start the reaction near the high end of the maximum 
enzyme activity pH range, about 9.0-8.5, and allow it to drop during the 
reaction. The pH can optionally be maintained by the separate addition of 
a non-interfering inorganic or organic buffer, since enzyme activity 
varies with pH. 
It is understood that glycolic and glyoxylic acids are highly dissociated 
in water, and at pH of between 7 and 10 are largely if not substantially 
entirely present as glycolate and glyoxylate ions. It will be also be 
appreciated by those skilled in the art that glyoxylic acid (and its 
conjugate base, the glyoxylate anion) may also be present as the hydrate, 
e.g. (HO).sub.2 CHCOOH and/or as the hemiacetal, HOOCCH(OH)OCH(OH)COOH, 
which compositions and their anionic counterparts are equivalent to 
glyoxylic acid and its anion for the present purpose of being suitable 
reactants for N-(phosphonomethyl)glycine formation. 
Oxygen (O.sub.2), the oxidant for the conversion of the glycolic acid to 
glyoxylic acid, may be added as a gas to the reaction by agitation of the 
liquid at the gas-liquid interface or through a membrane permeable to 
oxygen. It is believed that under most conditions, the reaction rate is at 
least partially controlled by the rate at which oxygen can be dissolved 
into the aqueous medium. Thus, although oxygen can be added to the 
reaction as air, it is preferred to use a relatively pure form of oxygen, 
and even use elevated pressures. Although no upper limit of oxygen 
pressure is known, oxygen pressures up to 50 atmospheres may be used, and 
an upper limit of 15 atmospheres is preferred. Agitation is important to 
maintaining a high oxygen dissolution (hence reaction) rate. Any 
convenient form of agitation is useful, such as stirring. On the other 
hand, as is well known to those skilled in the enzyme art, high shear 
agitation or agitation that produces foam may decrease the activity of the 
enzyme(s), and should be avoided. 
The reaction temperature is an important variable, in that it affects 
reaction rate and the stability of the enzymes. A reaction temperature of 
0.degree. C. to 40.degree. C. may be used, but the preferred reaction 
temperature range is from 5.degree. C. to 15.degree. C. Operating in the 
preferred temperature range maximizes recovered enzyme activity at the end 
of the reaction. The temperature should not be so low that the aqueous 
solution starts to freeze. Temperature can be controlled by ordinary 
methods, such as, but not limited to, by using a jacketed reaction vessel 
and passing liquid of the appropriate temperature through the jacket. The 
reaction vessel may be constructed of any material that is inert to the 
reaction ingredients. 
Upon completion of the reaction, the enzymes may be removed by filtration 
or centrifugation and reused. Alternatively, they can be denatured by 
heating, e.g., to 70.degree. C. for 5 minutes, and/or they can be allowed 
to remain in the reaction mixture if their presence in the subsequent 
steps of converting the glyoxylic-aminomethylphosphonic acid mixture to 
N-(phosphonomethyl)glycine and of recovering N-(phosphonomethyl)glycine 
from the reaction mixture is not objectionable. Flavin mononucleotide 
(FMN) may optionally be removed by contacting the solution with 
decolorizing carbon. 
Following the cessation of contacting the reaction solution with O.sub.2 
and preferably following the removal of the enzyme glycolate oxidase and 
the enzyme catalase when present, the solution containing glyoxylic acid 
and aminomethylphosphonic acid (which are believed to be in equilibrium 
with the corresponding imine), is treated in accordance with any of the 
processes known to the art for producing N-(phosphonomethyl)glycine. 
Catalytic hydrogenation is a preferred method for preparing 
N-(phosphonomethyl)glycine from a mixture of glyoxylic acid and 
aminomethylphosphonic acid. Catalysts suitable for this purpose include 
(but are not limited to) the various platinum metals, such as iridium, 
osmium, rhodium, ruthenium, platinum, and palladium; also various other 
transition metals such as cobalt, copper, nickel and zinc. The catalyst 
may be unsupported, for example as Raney nickel or platinum oxide; or it 
may be supported, for example as platinum on carbon, palladium on alumina, 
or nickel on kieselguhr. Palladium on carbon, nickel on kieselguhr and 
Raney nickel are preferred. 
The hydrogenation can be performed at a pH of from 4 to 11, preferably from 
5 to 10. Within this pH range, the exact value may be adjusted to obtain 
the desired pH by adding any compatible, non-interfering base or acid. 
Suitable bases include, but are not limited to, alkali metal hydroxides, 
carbonates, bicarbonates and phosphates, while suitable acids include, but 
are not limited to, hydrochloric, sulfuric, or phosphoric acid. 
The hydrogenation temperature and pressure can vary widely. The temperature 
may generally be in the range of 0.degree. C. to 150.degree. C., 
preferably from 20.degree. C. to 90.degree. C., while the H.sub.2 pressure 
is generally in the range of from about atmospheric to about 100 
atmospheres, preferably from 1 to 10 atmospheres. The hydrogenation 
catalyst is employed at a minimum concentration sufficient to obtain the 
desired reaction rate and total conversion of starting materials under the 
chosen reaction conditions; this concentration is easily determined by 
trial. The catalyst may be used in amounts of from 0.001 to 20 or more 
parts by weight of catalyst per 100 parts of combined weight of the 
glyoxylic acid and AMPA employed in the reaction. 
N-(Phosphonomethyl)glycine, useful as a post-emergent herbicide, may be 
recovered from the reduced solution, whatever the reducing method 
employed, by any of the recovery methods known to the art, including those 
disclosed in the U.S. Pat. Nos. 4,851,159 and 4,670,191 and in European 
Patent Applications 186 648 and 413 672.

In the following Examples, which serve to further illustrate the invention, 
the yields of glyoxylate, formate and oxalate, and the recovered yield of 
glycolate, are percentages based on the total amount of glycolic acid 
present at the beginning of the reaction. Analyses of reaction mixtures 
were performed using high pressure liquid chromatography. Organic acid 
analyses were performed using a Bio-Rad HPX-87H column, and AMPA and 
N-(phosphonomethyl)glycine were analyzed using a Bio-Rad Aminex glyphosate 
analysis column. Reported yields of N-(phosphonomethyl)glycine are based 
on either glyoxylate or AMPA, depending on which was the limiting reagent 
in the reaction. 
EXAMPLE 1 
Into a 3 oz. Fischer-Porter glass aerosol reaction vessel was placed a 
magnetic stirring bar and 10 mL of an aqueous solution containing glycolic 
acid (0.50M), aminomethylphosphonic acid (AMPA, 0.40 m), FMN (0.01 mM), 
butyric acid (HPLC internal standard, 0.10M), glycolate oxidase (from 
spinach, 1.0 IU/mL), and catalase (from Aspergillus niger, 14,000 IU/mL) 
at pH 8.5. The reaction vessel was sealed and the reaction mixture was 
cooled to 5.degree. C., then the vessel was flushed with oxygen by 
pressurizing to 70 psig and venting to atmospheric pressure five times 
with stirring. The vessel was then pressurized to 70 psig of oxygen and 
the mixture stirred at 5.degree. C. Aliquots (0.10 mL) were removed by 
syringe through a sampling port (without loss of pressure in the vessel) 
at regular intervals for analysis by HPLC to monitor the progress of the 
reaction. After 17.5 h, the HPLC yields of glyoxylate, formate, and 
oxalate were 91.0%, 2.9%, and 2.9%, respectively, and 4.1% glycolate 
remained. The final pH of the reaction mixture was 6.7. 
The resulting mixture of glyoxylic acid (0.46M) and AMPA (0.40M) was 
filtered using an Amicon Centriprep 10 concentrator (10,000 molecular 
weight cutoff) to remove the soluble enzymes, then the filtrate was placed 
in a 3-oz. Fischer-Porter bottle equipped with a magnetic stirrer bar. To 
the bottle was then added 0.100 g of 10% Pd/C and the bottle sealed, 
flushed with nitrogen gas, then pressurized to 50 psi with hydrogen and 
stirred at 25.degree. C. After 17 h, the concentration of 
N-(phosphonomethyl)glycine (determined by HPLC) was 0.29M (72% yield based 
on AMPA). 
EXAMPLE 2 
The enzymatic oxidation of glycolic acid in Example 1 was repeated, using 
10 mL of an aqueous solution containing glycolic acid (0.25M), 
aminomethylphosphonic acid (AMPA, 0.20M), FMN (0.01 mM), butyric acid 
(HPLC internal standard, 0.10M), glycolate oxidase (from spinach, 1.0 
IU/mL), and catalase (from Aspergillus niger, 14,000 IU/mL) at pH 8.5. 
After 6 h, the HPLC yields of glyoxylate, formate, and oxalate were 92.3%, 
4.36%, and 5.5%, respectively, and no glycolate remained. The final pH of 
the reaction mixture was 6.7. 
The resulting mixture of glyoxylic acid (0.23M) and AMPA (0.20M) was 
filtered using an Amicon Centriprep 10 concentrator (10,000 molecular 
weight cutoff) to remove the soluble enzymes, then the filtrate was placed 
in a 3-oz. Fischer-Porter bottle equipped with a magnetic stirrer bar. To 
the bottle was then added 0.100 g of 10% Pd/C and the bottle sealed, 
flushed with nitrogen gas, then pressurized to 50 psi with hydrogen and 
stirred at 25.degree. C. After 17 h, the concentration of 
N-(phosphonomethyl)glycine (determined by HPLC) was 0.13M (66% yield based 
on AMPA). 
EXAMPLE 3 
The enzymatic oxidation of glycolic acid in Example 1 was repeated, using 
10 mL of an aqueous solution containing glycolic acid (0.75 M), 
aminomethylphosphonic acid (AMPA, 0.60M), FMN (0.01 mM), butyric acid 
(HPLC internal standard, 0.10M), glycolate oxidase (from spinach, 2.0 
IU/mL), and catalase (from Aspergillus niger, 14,000 IU/mL) at pH 8.5. 
After 40 h, the HPLC yields of glyoxylate, formate, and oxalate were 
83.2%, 2.3%, and 7.5%, respectively, and no glycolate remained. The final 
pH of the reaction mixture was 6.8. 
The resulting mixture of glyoxylic acid (0.62M) and AMPA (0.60M) was 
filtered using an Amicon Centriprep 10 concentrator (10,000 molecular 
weight cutoff) to remove the soluble enzymes, then the filtrate was placed 
in a 3-oz. Fischer-Porter bottle equipped with a magnetic stirrer bar. To 
the bottle was then added 0.100 g of 10% Pd/C and the bottle sealed, 
flushed with nitrogen gas, then pressurized to 50 psi with hydrogen and 
stirred at 25.degree. C. After 24 h, the concentration of 
N-(phosphonomethyl)glycine (determined by HPLC) was 0.42M (70% yield based 
on AMPA). 
EXAMPLE 4 
The enzymatic oxidation of glycolic acid in Example 1 was repeated, using 
10 mL of an aqueous solution containing glycolic acid (1.0M), 
aminomethylphosphonic acid (AMPA, 0.80M), FMN (0.01 mM), butyric acid 
(HPLC internal standard, 0.10M), glycolate oxidase (from spinach, 2.0 
IU/mL), and catalase (from Aspergillus niger, 14,000 IU/mL) at pH 8.5. 
After 66 h, the HPLC yields of glyoxylate, formate, and oxalate were 
78.9%, 2.2%, and 12.1%, respectively, and 2.0% glycolate remained. The 
final pH of the reaction mixture was 6.9. 
The resulting mixture of glyoxylic acid (0.79M) and AMPA (0.80M) was 
filtered using an Amicon Centriprep 10 concentrator (10,000 molecular 
weight cutoff) to remove the soluble enzymes, then the filtrate was placed 
in a 3-oz. Fischer-Porter bottle equipped with a magnetic stirrer bar. To 
the bottle was then added 0.100 g of 10% Pd/C and the bottle sealed, 
flushed with nitrogen gas, then pressurized to 50 psi with hydrogen and 
stirred at 25.degree. C. After 23 h, the concentration of 
N-(phosphonomethyl)glycine (determined by HPLC) was 0.51M (65% yield based 
on glyoxylic acid). 
EXAMPLE 5 
The enzymatic oxidation of glycolic acid in Example 1 was repeated, using 
10 mL of an aqueous solution containing glycolic acid (025M), 
aminomethylphosphonic acid (AMPA, 0.263M), FMN (0.01 mM), butyric acid 
(HPLC internal standard, 0.25M), glycolate oxidase (from spinach, 1.0 
IU/mL), and catalase (from Aspergillus niger, 14,000 IU/mL) at pH 7.0 and 
15.degree. C. After 8 h, the HPLC yields of glyoxylate, formate, and 
oxalate were 82.8%, 0.9%, and 2.1%, respectively, and 13.9% glycolate 
remained. The final pH of the reaction mixture was 6.6. 
This mixture of glyoxylic acid (0.21M) and AMPA (0.263M) was filtered using 
an Amicon Centriprep 10 concentrator (10,000 molecular weight cutoff) to 
remove the soluble enzymes, then the filtrate and 50 mg of 10% Pd/C were 
placed in a stainless steel pressure vessel equipped with glass liner. The 
vessel was sealed, flushed with nitrogen gas, then pressurized to 1000 psi 
with hydrogen gas and shaken at 25.degree. C. The pressure in the vessel 
fell to a stable value in the first 0.5 h of reaction, and the vessel was 
then repressurized to 1000 psi. After 4 h, the pressure in the vessel was 
vented, and the vessel flushed with nitrogen. The concentration of 
N-(phosphonomethyl)glycine (determined by HPLC) was 0.16M (76% yield based 
on glyoxylic acid). 
EXAMPLE 6 
The enzymatic oxidation of glycolic acid in Example 5 was repeated at pH 8. 
After 8 h, the HPLC yields of glyoxylate, formate, and oxalate were 86.7%, 
1.8%, and 4.1%, respectively, and 13.2% glycolate remained. The final pH 
of the reaction mixture was 6.7. 
This mixture of glyoxylic acid (0.22M) and AMPA (0.263M) was hydrogenated 
at 1000 psi using the same procedure as described in Example 5. After 4 h, 
the concentration of N-(phosphonomethyl)glycine (determined by HPLC) was 
0.14M (64% yield based on glyoxylic acid). 
EXAMPLE 7 
The enzymatic oxidation of glycolic acid in Example 5 was repeated at pH 9. 
After 7 h, the HPLC yields of glyoxylate, formate, and oxalate were 70.0%, 
5.6%, and 11.1%, respectively, and no glycolate remained. The final pH of 
the reaction mixture was 6.8. 
This mixture of glyoxylic acid (0.18M) and AMPA (0.263M) was hydrogenated 
at 1000 psi using the same procedure as described in Example 5. After 4 h, 
the concentration of N-(phosphonomethyl)glycine (determined by HPLC) was 
0.094M (52% yield based on AMPA). 
EXAMPLE 8 
The enzymatic oxidation of glycolic acid in Example 5 was repeated at pH 
8.5, and using initial concentrations of glycolic acid and AMPA of 0.50M 
and 0.40M, respectively. After 16.5 h, the HPLC yields of glyoxylate, 
formate, and oxalate were 85.4%, 3.5%, and 6.3%, respectively, and 1.4% 
glycolate remained. The final pH of the reaction mixture was 7.0. 
This mixture of glyoxylic acid (0.43M) and AMPA (0.40M) was hydrogenated at 
1000 psi using the same procedure as described in Example 5. After 4 h, 
the concentration of N-(phosphonomethyl)glycine (determined by HPLC) was 
0.30M (75% yield based on AMPA). 
EXAMPLE 9 
The enzymatic oxidation of glycolic acid in Example 1 was repeated, using 
10 mL of an aqueous solution containing glycolic acid (0.50M), 
aminomethylphosphonic acid (AMPA, 0.375M), FMN (0.01 mM), butyric acid 
(HPLC internal standard, 0.10M), glycolate oxidase (from spinach, 1.0 
IU/mL), and catalase (from Aspergillus niger, 14,000 IU/mL) at pH 8.5. 
After 17 h, the HPLC yields of glyoxylate, formate, and oxalate were 
87.1%, 1.9%, and 2.1%, respectively, and 8.9% glycolate remained. The 
final pH of the reaction mixture was 6.7. 
The resulting mixture of glyoxylic acid (0.435M) and AMPA (0.375M) was 
filtered using an Amicon Centriprep 10 concentrator (10,000 molecular 
weight cutoff) to remove the soluble enzymes, then the filtrate was mixed 
with 50 mg of decolorizing carbon (to remove FMN) and again filtered. The 
resulting filtrate was placed in a 3-oz. Fischer-Porter bottle equipped 
with a magnetic stirrer bar. To the bottle was then added 0.100 g of 10% 
Pd/C and the bottle sealed, flushed with nitrogen gas, then pressurized to 
50 psi with hydrogen and stirred at 25.degree. C. After 17 h, the 
concentration of N-(phosphonomethy)glycine (determined by HPLC) was 0.372M 
(99% yield based on AMPA).