Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
PRODUCTION OF GLYOXYLIC ACID BY OXIDIZING GLYCOLIC
ACID IN THE PRESENCE OF IMMOBTT T7;Fn GLYCOLATE
- OXIDASE AND CATALASE
BACKGROUND OF THE INVENTION
1. Field of the Invention:
This invention relates to an improved process for the production of
glyoxylic acid by the enzyme catalyzed oxidation of glycolic acid. More
specifically, the present invention relates to the use of glycolate oxidase and
catalase immobilized on an insoluble support as catalyst.
2. Description of the Related Art:
Glycolate oxidase, an enzyme commonly found in leafy green
plants and m~mm~ n cells, catalyzes the oxidation of glycolic acid to glyoxylic
lS acid, with the concomitant production of hydrogen peroxide. N. E. Tolbert et al.,
J. Biol. Chem., Vol. 181, 905-914 (1949) first reported an enzyme, extracted from
tobacco leaves, which catalyzed the oxidation of glycolic acid to formic acid and
C2 via the intermediate formation of glyoxylic acid. The addition of certain
compounds, such as ethylenediamine, limited the further oxidation of the
intermediate glyoxylic acid. The oxidations were carried out at a pH of about 8,typically using glycolic acid concentrations of about 3-40 mM (millimolar). The
optimum pH for the glycolate oxidation was reported to be 8.9. Oxalic acid (100
mM) was reported to inhibit the catalytic action of the glycolate oxidase.
Similarly, K. E. Richardson and N. E. Tolbert, J. Biol. Chem., Vol. 236,
1280-1284 (1961) showed that buffers containing
tris(hydroxymethyl)aminomethane inhibited the formation of oxalic acid in the
glycolate oxidase catalyzed oxidation of glycolic acid. C. O. Clagett, N. E.
Tolbert and R. H. Burris, J. Biol. Chem., Vol. 178, 977-987 (1949) reported thatthe optimum pH for the glycolate oxidase catalyzed oxidation of glycolic acid
with oxygen was about 7.8 - 8.6, and the optimum temperature was 35-40C.
I. Zelitch and S. Ochoa, J. Biol. Chem., Vol. 201, 707-718 (1953),
and J. C. Robinson et al., J. Biol. Chem., Vol. 237, 2001-2009 (1962), reported
that the formation of formic acid and CO2 in the spinach glycolate
oxidase-catalyzed oxidation of glycolic acid resulted from the nonenzymatic
3~ reaction of H22 with glyoxylic acid. They observed that addition of c~t~l~ce, an
enzyme that catalyzes the decomposition of H22 greatly improved the yields of
glyoxylic acid by suppressing the formation of formic acid and CO2. The
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addition of FMN (flavin mononucleotide) was also found to greatly increase the
stability of the glycolate oxidase.
N. A. Frigerio and H. A. Harbury, J. Biol. Chem., Vol. 231, 135-157
(1958) have reported on the preparation and properties of glycolic acid oxicl~ces isolated from spinach. The purified enzyme was found to be very unstable in
solution; this instability was ascribed to the relatively weak binding of flavinmononucleotide (FMN) to the enzyme active site, and to the dissociation of
enzymatically active tetramers and/or octamers of the enzyme to
enzymatically-inactive monomers and dimers, which irreversibly aggregate and
precipitate. The addition of FMN (flavin mononucleotide) to solutions of the
enzyme greatly increased its stability, and high
protein concentrations or high ionic strength maintained the enzyme as octamers
or tetramers.
There are numerous other references to the oxidation of glycolic
acid catalyzed by glycolic acid oxidase, for example:
Isolation of the enzyme (usually includes an assay method):
I. Zelitch in Methods of Enzymologv. Vol. 1, Academic Press, New York,
1955, p. 528-532, from spinach and tobacco leaves.
M. Nichimllra et al., Arch. Biochem. Biophys., Vol. 222, 397-402 (1983),
from pumpkin cotyledons.
H. Asker and D. Davies, Biochim. Biophys. Acta, Vol. 761, 103-108
(1983), from rat liver.
M. J. Emes and K. H. Eri~m~nn, Int. J. Biochem., Vol. 16, 1373-1378
(1984), from Lemna Minor L.
Structure of the enzyme:
E. Cederlund et al., Eur. J. Biochem., Vol. 173, 523-530 (1988).
Y. Lindquist and C. Branden, J. Biol. Chem. Vol. 264, 3624-3628, (1989).
SUMMARY OF THE lNVENTION
This invention relates to a process for the production of glyoxylic
acid (OCHCOOH) where glycolic acid (HOCH2COOH) (200 to about 2500
mM) and oxygen are reacted in an aqueous solution (pH 7 to 10), in the presence
of a catalyst consisting of glycolate oxidase ((S)-2-hydroxy-acid oxidase, EC
1.1.3.15) and catalase (EC 1.11.1.6) immobilized on an insoluble support. Under
optimum conditions, very high yields of glyoxylic acid are obtained at high
conversion of glycolic acid, and the immobilized enzyme catalyst can be
recovered and reused.
.
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DETAILED DESCRIPTION OF THE INVENTION
This invention describes the preparation and use of an
immobilized enzyme catalyst for the manllf~cture of glyoxylic acid from glycolicacid (hydroxyacetic acid). Although the enzyme-catalyzed reaction of glycolic
5 acid with oxygen has been known for many years, high selectivities (¢ 99%) to
glyoxylic acid have not been previously obtained, nor has the oxidation of glycolic
acid been performed at concentrations of 0.20M to 2.5M. A previous, commonly
assigned, application, U.S.S.N. 07/422,011, filed Oct. 16, 1989, '~roduction of
Glyoxylic Acid from Glycolic Acid", described a process for the enzymatic
n conversion of glycolic acid to glyoxylic acid in the presence of oxygen, an amine
buffer, and the soluble enzymes glycolate oxidase and catalase. This process
demonstrated the unexpected synergistic effect of using both c~t~ e (to destroy
by-product hydrogen peroxide) and an amine buffer capable of forming a
chemical adduct with the glyoxylic acid produced (limiting its further oxidation)
15 and is herein incorporated by reference for such purpose. Neither the separate
addition of catalase or an amine buffer were found to produce the h~igh selectivity
observed when both were present, and the almost quantitative
yields of glyoxylic acid obtained were more than expected from a simple additiveeffect of using c~t~l~ce or amine buffer alone. The instant invention is viewed as
20 an improvement to the above process in that an immobilized enzyme catalyst is provided for this process.
A previously-reported use of soluble enzymes as catalysts poses
several problems: catalyst recovery for reuse is not easily performed, catalyst
stability is not as good as can be obtained with immobilized enzyme systems, and25 soluble enzymes are not stable to the sparging of the reaction mixture with
oxygen (required to increase the rate of oxygen dissolution and, thus, reaction
rate). A catalyst preparation has now been developed which involves the
simultaneous immobilization of the two enzymes; i.e., glycolate oxidase (e.g.,
from spinach or beet leaves, isolated or obtained from commercial sources) and
30 catalase (e.g., from Aspergillus niger, Aspergil]us nidulans, Saccharomyces
cerevisae (Baker's yeast), or bovine liver, isolated or obtained from commercialsources), on a solid support (e.g. commercially available oxirane acrylic beads).
Several advantages are offered by the use of this immobilized enzyme catalyst inthe previously described process:
35 1) the immobilized catalyst is easily recovered from the reaction mixture at the
conclusion of the reaction for reuse, whereas the soluble enzyme is only
recovered with great difficulty and loss of activity;
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2) the immobilized catalyst is more stable than the soluble enzyme, both for
the number of catalyst turnovers obtained versus the soluble enzyme, as well as
for recovered enzyme activity at the conclusion of a reaction or after prolongedstorage in aqueous buffer; and h'
s 3) most importantly, the immobilized`,;catalyst is stable to reaction conditions
where oxygen is sparged into the reaction mixture to increase the rate of oxygendissolution and reaction rate, where under similar reaction conditions the soluble
glycolate oxidase is rapidly denatured.
No one method of immobilization can be chosen for a particular
lo enzyme with the expectation that the immobilization will be successful.
Furthermore, the expectation for successful co-immobilization of more than one
enzyme is even less predictable. It is generally agreed by those skilled in the art
that a successful immobilization of any one enzyme must be discovered by
screening a variety of methods, and an optimal result obtained by trial and error.
In the case of glycolate oxidase, there have been no reports of attempts at
immobilization. The immobilization of enzymes can be performed~using a
variety of techniques, including: (1) binding of the enzyme to a carrier or
support, via covalent ~tt~chment, physical adsorption, electrostatic binding, oraffinity binding, (2) cro~linking with bifunctional or multifunctional reagents, (3)
en~ ent in gel matrices, polymers, emulsions, or some form of membrane,
and (4) a combination of any of these methods. Detailed descriptions of many of
these methods of enzyme immobilization, and the various factors affecting the
choice of a method of immobilization, are collected in the following volumes of
Methods in Enzymology, K. Mosbach (ed.), Academic Press, New York: Vol. 44
2s (1976), Vol. 135 (1987), Vol. 136 (1987), Vol. 137 (1988), and the references
therein.
A variety of methods of immobilization of glycolate oxidase were
e~mined, and the optimal
results for a number of these procedures are listed in the Examples. Covalent
~tt~chment of the protein to oxirane acrylic beads (Eupergit C), cyanogen
bromide-activated agarose, and poly(acrylamide-co-N-acryloxysuccinimide) gel
crosslinked with triethylenetetramine (PAN-500) produced active immobilized
enzyme, as did physical adsorption to the resin XAD-8 and phenyl agarose. Ionic
binding to various supports was unsuccessful, as were attempts at crosslinking the
3s protein with glutaraldehyde, dimethyl adipimidate, dimethylsuberimidate, or
1,4-butanediol diglycidyl ether. Of the different forms of active immobilized
glycolate oxidase, only the oxirane acrylic bead-enzyme was useful as a catalystfor the oxidation of glycolic acid. The physically-adsorbed enzyme rapidly
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desorbed from the support in the reaction mixture cont~ining 0.75 M glycolic
acid and 0.79 M ethylenediamine at pH 9.0, while the cyanogen
brornide-activated agarose reacted with the ethylene diamine, again releasing the
covalently-bound enzyme into the reaction mixture. The specific activity of the
enzyrne attached to PAN-500 gel was too low to be useful as a practical catalystin the reaction.
The immobilization of glycolate oxidase on oxirane acrylic beads
Eupergit C and Eupergit C-250L (Rohm Pharma) resulted in a catalyst which
was stable to the reaction conditions, and had a sufficiently high specific activity
(units of enzyme activity/gram of catalyst) to be useful in this application.
C~t~l~ce was also immobilized on oxirane acrylic beads, and the two separate
catalysts used together, or both enzymes were co-immobilized on the same
support, and this single catalyst added to the reaction mixture (the latter method
being preferred).
S Many of the deficiencies of the soluble enzymes were elimin~ted
by employing the immobilized enzyme catalyst. The stability of im~obilized
glycolate nxi~ e in aqueous buffers is much greater than the soluble enzyme
(appro~ ing the stability of ammonium sulfate-precipitated enzy~ne). Recovery
and reuse of the co-immobilized catalyst was easily performed by simply filtering
the catalyst away from the reaction mixture and recycling it to fresh reaction
re; in this manner for immobilized glycolate oxidase the number of
turnovers (i.e., the number of substrate molecules that are converted to productmolecules per catalyst molecule before inactivation of the enzyme) as high as 107
(mol/mol) have been obtained. Finally, the ability to bubble oxygen through the
2s reaction mixture without denaturing the enzyme catalyst resulted in increases in
the reaction rate of at least ten-fold, and this increase in rate significantly
reduces the cost of m~nllf~cture for this process.
The immobilized glycolate oxidase used in the reaction should be
present in an effective concentration, usually a concentration of about 0.001 toabout 10.0 IU/mL, preferably about 0.1 to about 4 IU/mL. An IU
(International Unit) is defined as the amount of enzyme that will catalyze the
transformation of one micromole of substrate per minute. A procedure for the
assay of this enzyme is found in I. Zelitch and S. Ochoa, J. Biol. Chem., Vol. 201,
707-718 (1953). This method is also used to assay the activity of recovered or
recycled glycolate oxidase.
The pH of the reaction solution should be between 7 and 10,
preferably between 8.0 and 9.5. The pH can be m~int~ined by a buffer, since
enzyme activity varies with pH. The pH of the reaction decreases slightly as the
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reaction proceeds, so it is often useful to start the reaction near the high end of
the m~imllm enzyme activity pH range, about
9.0-9.5, and allow it to drop during the reaction. As has been previously
described in U.S.S.N. 07/422,011, filed October 16, 1989, an amine buffer
5 capable of complexing the glyoxylic acid (by forming an imine which is more
stable to chemical or enzymatic oxidation) is employed along with c~t~l~ce to
m~imi7e product selectivity. Ethylene~ mine, or less preferably,
tris(hydroxymethyl)methylamine (hereinafter TRIS), piperazine, or glycylglycine
improved the yield of glyoxylic acid. These amines are used in a molar ratio of
amine/glycolic acid (starting amount) of 1.0 to 3.0, preferably 1.05 to 1.33.
Within this range, the exact value may be adjusted to obtain the desired pH.
With very basic amines used at high amine to glycolic acid ratios, it may be
necessary to adjust the pH, as by adding acid, for example hydrochloric or
sulfuric acids. With less basic amines such as TRIS, it may be necessary to add a
base to m~int~in the desired pH.
The concentration of immobilized catalase should be~S0 to 100,000
IU/rnL, preferably 350 to 14,000 IU/mL. It is preferred that the enzymes be
co-immobilized to limit the amount of catalyst added to the reaction, and that
the c~t~l~ce and glycolate oxidase concentrations be adjusted within the above
ranges so that the ratio (measured in IU for each) of c~t~l~ce:glycolate oxidase is
at least about 250:1. Flavinmononucleotide (FMN) is an optional added
ingredient, used at a concentration of 0.0 to 2.0 mM, preferably 0.01 to 0.2 mM.The reaction rate is at least partially controlled by the rate at
which oxygen can be dissolved into the aqueous medium. Oxygen can be added
2s to the reaction as the oxygen in air, but it is preferred to use a relatively pure
form of oxygen, and
to 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. Sparging (bubbling) oxygen through the reaction
mixture is necessary to maintain a high oxygen dissolution (and hence reaction)
rate. Oxygen is sparged through the reaction mixture at a rate of 0.05 to 5
volumes of oxygen (measure ~ at atmospheric pressure) per volume of reaction
mixture per minute (vol/vol-min), and preferably between 0.2 and 2 vol/vol-min.
Additionally, a convenient form of agitation is useful, such as stirring.
The reaction temperature is an important variable, in that it affects
reaction rate and the stability of the enzymes. A reaction temperature of 0C to40C may be used, but the preferred reaction temperature range is from 5C to
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15C. Operating in the preferred temperature range maximizes recovered
enzyme activity at the end of the reaction.
Upon completion of the reaction and removal of the enzyme
catalyst by filtration or centrifugation, the amine buffer is most conveniently
s removed by use of an ion exchange resin. Suitable acidic cationic exchange
resins include "AMBERLITE" CG120 or "AMBERLITE" IR120 (Rohm & Haas
Co.), and '~OWEX" 50 (Dow Chemical Co.). The amine may then be recovered
and subsequently recycled by treatment of the resin with strong base.
The product, glyoxylic acid, is useful in the preparation of vanillin
l0 and ethylvanillin, as well as being used in ion exchange resins and as an acid
catalyst in the pharmaceutical industry (Ullm~nn~). It is usually sold as a 50%
(weight percent) aqueous solution. It is also to be understood that reference toglyoxylic acid in this application can also mean
the glyoxylate anion, especially when the glyoxylic acid is present in a solution
5 whose pH is greater than about 2.3.
Purification of Glycolate Oxidase from Spinach Leaves
Glycolate oxidase from spinach was purified using selective
ammonium sulfate fractionation followed by batch adsorption of the extract
20 using DEAE cellulose. The latter procedure resulted in the adsorption of
virtually all plant proteins except glycolate oxidase. All steps in the purification
were performed at 4C unless otherwise stated. At 25C, two bushels (16 kg) of
fresh spinach were chopped into fine particles using a Fitz Mill grinder fitted
with a 0.5 inch mesh screen. The liquid fraction (ca. 6 L) of the resulting pulp2s was isolated by squeezing through 4 layers of cheesecloth; alternatively, a juice
extractor (Vitantonio) may be used. To the liquid fraction was added 5.6 g of
dithiothreitol (5 mM final concentation), then the pH was adjusted to 5.2 by
adding 5-20 mL of 20~ acetic acid. After a 10 minute incubation, the reslllting
mixture was centrifuged at 13,000 g for 25 min. at 4C using a GS-3 rotor
30 (Sorvall). The pellet was discarded, and the pH of the supernatant adjusted to
7.5 - 8.0 using 15-20 mL of 6 N potassium hydroxide (Zelitch, I., Ochoa, S., J.Biol.
Chem., Vol. 201, 707 (1953); Frigerio, N.A., Harbury, H.A., J.Biol. Chem., Vol.
231, 135 (1958)). The supernatant (approx. 5.5 L) was then concentrated 5-fold
using a Pelicon (Millipore) ultrafiltration apparatus fitted with a 100,000 MW
3s membrane cassette; the final volume of concentrate was approx. 1.1 L. To the
concentrate was then added solid ammonium sulfate (154 g) slowly over 10 min.
After all the ammonium sulfate dissolved, the resulting precipitate was removed
by centrifuging for 25 min. at 13,000 g. The pellet was discarded, and 77 g of
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ammonium sulfate was added to the supernatant (approx. 1.1 L), which was then
centrifuged as before. The resulting protein pellet was collected and the
supernatant discarded (Zelitch (1953); Frigerio (1958)).
The protein pellet was dissolved in approx. 200 mL of 20 mM
bicine buffer (pH 8.0). Using Spectropor 2 dialysis tubing (12,000-14,000
MWCO), the protein was dialysed for 16 hrs. vs. 4 L of 20 mM bicine (pH 8.0)
cont~ining 2 mM FMN. The conductivity of the protein solution was measured
relative to the conductivity of fresh bicine buffer using a conductivity meter, and
if the re~-lings were not equivalent, the protein solution was dialysed an
additional 4 hrs, then tested as before. The dialysed protein solution (approx.
250 ml) was stirred in a beaker using either a magnetic stir bar or overhead
stirrer, then 25 g of pre-swollen DEAE cellulose (Sigma) (Kerr, M. W., Groves,
D., Phytochemistry, Vol. 14, 359-362 (1975)) added and the resulting mixture
incubated for 10 minutes. Protein binding to the resin was monitored by
lS following the decrease in protein concentration of the solution using the
Bradford assay (Bio-Rad). When the protein concentration of the sypernatant
was reduced to trace levels (0.2 mg/mL), the unbound protein was recovered
from the mixture by vacuum filtration through a 11-cm Wh~tm~n #1 filter disk in
a 13 cm diameter Buchner funnel (Cole-Parmer). To m~ximi7e enzyme
recovery, the resin cake was washed with 100 mL of 20 mM bicine (pH 8.0).
Flavin mononucleotide (FMN, Sigma) was added to the protein solution to a 2
mM final concentration, then 240 g of solid ammonium sulfate
was added to the enzyme solution (approx. 400 mL) gradually over 15 minutes
with stirring while m~int~ining the pH at 8.0 by the dropwise addition of 5 N
pot~ m hydroxide. The resulting precipitated glycolate oxidase was stored in
the dark at 4C until needed.
Purification of Catalase from Fresh Bakers Yeast
All steps of the purification were performed at 4C. Fresh Baker's
yeast (1 lb., Universal Foods-Red Star), was suspended in 450 mL of 20 mM Tris
buffer (pH 7.5) cont~ining 1 mM phenylmethysulfonyl-fluoride (PMSF, Sigma).
A 200 mL portion of the yeast suspension was transferred to a 400 mL capacity
Bead Beater blender containing 200 mL of glass beads (0.5 mm
diameter-Biospec Products), and after 5 min. of continuous mixing, the lysate was
transferred to a receiving vessel (on ice). The rem~ining yeast suspension was
processed in the same manner.
Cell debris was removed by centrifuging the extract at 4C for 45
min.-1 hr. at 13,000 g in a GS-3 rotor (Sorvall). The supernatant (400 mL) was
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collected and 90.4 g of solid ammonium sulfate dissolved into the solution to
achieve 40~o saturation, then after incubation for 10 min. on ice the suspensionwas centrifuged at 4C for 25 min. at 13,000 g in a GSA rotor (Sorvall). The
pellet was discarded, then 48 g of solid ammonium sulfate was added to the
5 supernatant (400 mL) to achieve a final 60~o saturation (Seah, T.C.M., Kaplan,J.G. J. Biol. Chem. Vol. 218, No. 8, 2880 (1973)). This mixture was incubated
and cen~lifuged as before, and the resulting protein pellet was dissolved in a
minim~l volume of 20 mM TRIS (pH 7.5). Using Spectropor 2 dialysis tubing,
the protein solution was dialysed against 20 mM TRIS (pH 7.5); the dialysis
o buffer was discarded and replenished after 16 hrs. The dialysis was continued for
an additional 4 hrs., then the dialysed protein (100 mL) was recovered.
A 50-mL portion of the dialysed protein was loaded onto a radial
flow chromatography column (Sepragen) packed with 100 mL of Q Sepharose
fast flow ion exchange resin (Pharmacia), and the unbound protein eluted with
20 mM TRIS (pH 7.5) at 10 mL/min. Protein elution was monitored using a
flow cell fitted with a 280 nm filter (LKB) linked to a chart recorder~(LKB);
10-15 mL column fractions were collected using an LKB fraction collector.
When all unbound protein had eluted from the column, a 400 mL linear gradient
of NaCl from 0-500 mM dissolved in 20 mM TRIS (pH 7.5) was started at 10
20 mL/min., and fractions assayed for catalase activity by monitoring for the
disappearance of peroxide at 240 nm. Fractions with catalase activity were
pooled, and ammonium sulfate added to a final concentration of 80% saturation.
The resulting precipitated catalase was stored at 4C.
This purification method has also been used to purify catalase from
2s Aspergillus nidulans and Aspergillus niger.
Enzy~ne Assays for Glycolate Oxidase and Catalase Immobilized on Oxirane
Acrylic Beads.
Glycolate oxidase immobilized on oxirane acrylic beads was
30 assayed by accurately weighing ca. 5-10 mg of the treated beads into a 3-mL
quartz cuvette containing a magnetic stirring bar, then 2.0 mL of a solution which
was 0.12 mM in
2,6-dichlorophenol-indophenol and 80 mM in TRIS buffer (pH 8.3) was added.
The cuvette was capped with a rubber septum and the solution deoxygenated by
3~ bubbling with nitrogen for 5 min. To the cuvette was then added by syringe 40mL of 1.0 M glycolic acid/1.0 M TRIS (pH 8.3), and the mixture stirred while
measuring the change in adsorption with time at 605 nm (e = 22,000).
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Catalase activity was assayed by accurately weighing ca. 2-5 mg of
the treated beads into a 3 mL quartz cuvette containing a magnetic stirring bar,then adding 2.0 mL of a distilled water, and 1.0 mL of 59 mM hydrogen peroxide
in 50 mM phosphate buffer (pH 7.0) and measuring the change in absorption
with time at 240 nm (e = 39.4). Activities of immobilized glycolate oxidase and
catalase were typically 6 IU/gram beads and 6000 IU/gram beads, respectively.
HPLC Analysis for Glycolic. Glyoxylic. Oxalic. and Formic Acid
Samples for analysis were prepared by mixing 0.100 mL of the
reaction mixture with 0.300 mL of 0.1 N H2SO4, then filtering the resulting
solution through a Millipore Ultrafree MC filter unit (10,000 mw cutoff).
Analyses for glycolic acid, glyoxylic acid, oxalic acid and formic acid were
performed by high performance liquid chromatography (HPLC) on a Bio-Rad
Aminex HPX-87H column (300 x 7.8 mm) at 40C, using as solvent an aqueous
solution of H2SO4(0.01 N) and 1-hydroxyethane-1,1-diphosphonic acid (0.1 mM)
at 1.0 mL/minute. The instrument was a Waters 840 HPLC system with Model
510 pumps, a 712 WISP autosampler, and, in sequence, a 490E UV detector and
410 differential refractometer. UV analysis was performed at 210 nm. The
retention times for oxalic acid, glyoxylic acid, glycolic acid, formic acid, andpropionic acid (internal standard) were 4.29, 6.09, 7.77, 8.79, and 11.41 mimltec~
respectively.
Example 1
Immobilization of Glycolate Oxidase
on Various Supports
Poly(ethyleneimine) (PEI), poly(ethyl-eneimine) on silica gel,
benzylated poly(ethyl-eneimine) on silica gel, Bio-Rex 70, CH Sepharose 4B,
XAD-4, XAD-8, Phenyl Agarose, Eupergit C, Eupergit C-250L and Eupergit
C-30N were all obtained from commercial sources. PAN-500
(poly(acrylamide-co-N-acryloxysuccinimide)) gel crosslinked with
triethylenetetramine) was prepared and used to immobilize glycolate oxidase
according to the procedures described by Pollack, A., et al. J. Am. Chem. Soc.,
1980, 102, 6324-6336. For those supports which bind protein by physical
adsorption, immobilizations were performed by washing the support with an
aqueous buffer at pH 5-10 as appropriate, then exposing the support to a
buffered solution of the enzymes for a predetermined time at either 5C or 25C,then washing the support with fresh buffer 3-4 times to remove any unadsorbed
enzyme and assaying the support for glycolate oxidase activity. For supports used
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in conjunction with glutaraldehyde, the procedure outlined above was repeated
except that prior to addition of the enzyme, the supports were treated with 5%
aqueous glutaraldehyde. A detailed procedure for the immobilization of
glycolate oxidase on Eupergit is given in Example 2. The yields of immobilized
5 glycolate oxidase listed in the table below were obtained by optimi7.ing the
immobilization conditions
for each support, and are based on the total amount of enzyme activity added
during each procedure.
10 SUPPORT ~o YIELD ACTIVITY ATI`ACHMENT
PEI 0 0 ionicadsorption
PEI/glutaraldehyde 0 0 covalent
PEI-silica/glutar. 0 0 covalent
Bio-Rex 70 0 0 ionic adsorption
lS CH Sepharose 4B 0 0 ionic adsorption
CNBr-Agarose 20 5.0 IU/mL covalent
PAN-500 gel 19 0.14IU/mL covalent
XAD~ O O physicaladsorpt.
XAD-8 4 0.76 IU/gr physicaladsorpt.
PEI-silica/benzyl. 0 0 physicaladsorpt.
Phenyl Agarose 3 0.62 IU/gr physical adsorpt.
EupergitC 17 7.8 IU/gr covalent
EupergitC-250L 8 5.0IU/gr covalent
Example 2
Co-immobilization of Glycolate Oxidase
and Catalase on Eupergit C
Into a 125-mL erlenmeyer flask was weighed 10.0 g of oxirane
acrylic beads (Eupergit C). To the flask was then added ca. 75 mL of a solution
cont~ining 50 mM bicine buffer (pH 8.0) and 0.02 mM flavin mononucleotide,
and the oxirane acrylic beads were then suspended in the buffer by swirling the
contents of the flask. After the beads had settled to the bottom of the flask, the
fines which floated to the top of the mixture were removed by pipet, along with
as much of the supernatant which could be removed without disturbing the
3~ settled beads. This washing procedure was repeated a second time.
A 100-mL, ammonium sulfate-precipitated glycolate oxidase
mixture, cont~ining 327 IU of glycolate oxidase activity (isolated from fresh
spinach leaves), was centrifuged at 12,000 rpm for 20 min (Sorvall GSA rotor at
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4C). The supernatant was discarded and the pellet dissolved in 50 mL of 50 mM
bicine (pH 8.0), 0.02 mM flavi~i mononucleotide buffer. A 10-mL mixture
cont~ining 100 mg (715,000 IU) of ammonium sulfate-precipitated Aspergillus
niger catalase (Sigma C-3515) was centrifuged at 15,000 rpm for 10 mimltes
(Sorvall SS-34 rotor). The supernatant was discarded and the pellet dissolved inthe buffer cont~ining the glycolate oxidase. This enzyme solution was then
added to the flask cont~ining the washed oxirane acrylic beads, and the final
volume adjusted to 125 mL with additional buffer. The resulting mixture was
transferred to a 250-mL polypropylene bottle, which was capped and placed on a
lo bottle roller at 4-5 rpm for 16 hours at 15C. The mixture was then poured into a
chromatography column equipped with a fritted bed support, allowed to drain,
and the immobilized enzymes were washed three times with 30 mL of the
bicine/FMN buffer and stored at 5C in this same buffer. The co-immobilized
enzyme catalyst had 7.2 IU of glycolate oxidase activity/gram Eupergit C and
lS 5680 IU of catalase activity/gram Eupergit C.
Example 3
Relative Stability of Soluble
and Immobilized Glycolate Oxidase
The stability of ~lnimmobilized (soluble) glycolate oxid~e versus
glycolate oxidase immobilized on oxirane acrylic beads (Eupergit C) was
measured by storing either form of the enzyme at 4C in a buffered (pH 8.0)
solution cont~ining 2.0 mM flavin mononucleotide, then monitoring the enzyrne
activity with time. Additionally, the stability of the enzyme precipitated in 3.2 M
~mmonillm sulfate, 2.0 mM flavin mononucleotide, and stored under similar
conditions was also monitored.
Enzyme
Recovery 1 dav 2days 3 months 6 months
soluble 50~ 0 0 0
immobilized 92~o 95~o 50~o 50~o
precipitated 100~o 100~o 95~ 85~o
Example 4
Sparged Co-Immobilized Enzyme Reaction
Into a 2.5-cm ID x 20 cm glass column equipped with a 20-mrn
polyethylene bed support was placed 10 mL of a solution cont~ining glycolic acid(0.25 M), ethylenediamine (0.33 M), propionic acid (0.075 M, HPLC internal
st~n~l~rd), and flavin monunucleotide (0.2 mM). The column and its contents
.
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were cooled to 15C, then 2.5 IU of spinach glycolate oxidase and 27,000 IU of
Aspergillus niger catalase (co-immobilized on Eupergit C) were added to the
solution. Oxygen was then passed through the porous bed support and bubbled
through the reaction mixture at a rate of 10 mL/min. The reaction was
monitored by taking a 100 mL aliquot of the reaction mixture at regular intervals,
mixing the aliquot with 300 mL of 0.1 N sulfuric acid to quench the reaction,
filtering the aliquot and analyzing by HPLC. After 5.5 hours, the yields of
glyoxylic acid, oxalic acid, and formic acid were 98%, 2%, and 0%, respectively,with complete conversion of glycolic acid. The final activities of glycolate
oxidase and catalase were 95% and 65% of their initial values.
Comparative Example 1
Sparged Soluble Enzyme Reaction
The reaction described in Example 4 was repeated, except that the
same amounts of soluble, unimmobilized glycolate oxidase and catalase were
added to the reaction mixture. After 4 hours, the yields of glyoxyli~ acid, oxalic
acid, and formic acid were 43%, 0%, and 0%, respectively, with a 46%
conversion of glycolic acid. The final activities of glycolate oxidase and c~t~l~.ce
were ~2% and 82% of their initial values, respectively, and no further reaction
was observed at longer reaction times.
Example 5 )~
Sparged, Separately Immobilized Enzymes Reaction
The reaction in Example 4 was repeated using 10 mL of a solution
cont~ining glycolic acid (0.75 M), ethylenediamine (0.86 M), propionic acid
(0.075 M, HPLC internal standard), flavin mononucleotide (0.2 mM), 2.5 IU of
spinach glycolate oxidase immobilized on Eupergit C, and 14,000 IU of
Aspergillus niger catalase immobilized on Eupergit C (the two enzymes were not
co-immobilized on the same support). After 20 hours, the yields of glyoxylic acid,
oxalic acid, and formic acid were 99%, 0.2%, and 0.5%, respectively, with a 100%conversion of glycolic acid. The final activities of glycolate oxidase and c~t~l~ce
were 72% and 66% of their initial values, respectively.
Example 6
Fixed-bed Co-Immobilized Enzyme Reactor
Into a Kontes Airlift Bioreactor was placed 400 mL of a solution of
0.75 M glycolic acid, 0.86 M ethylenediamine, 0.075 M propionic acid (HPLC
internal standard), and 0.01 mM flavin mononucleotide tpH 9.0). Wet oxygen
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was bubbled through the solution in the bioreactor, and a peristaltic pump was
used to recirculate the oxygenated solution (at 40 mL/min) from the bioreactor
through a jacketed 1-cm ID x 30-cm chromatography column cont~ining spinach
glycolate oxidase (13.9 IU) and Asper~illus ni~er catalase (56,000 IU)
co-immobilized on Eupergit C oxirane acrylic beads (21-mL fixed bed volume).
The contents of the bioreactor and jacketed chromatography column were
m~intz3ined at 15C by recirculating 50:50 ethylene glycol/water through the
jacket of the reactor and column using a refrigerated bath/circulator set at 10C.
After 377 hours, the yields of glyoxylic acid, oxalic acid, and formic acid were93%, 0~o, and 0.3%, respectively, with a 94% conversion of glycolic acid. The
ffnal activities of glycolate oxidase and catalase were 48% and 69% of their
initial values.
Example 7
Oxidation of Glycolic Acid using
Co-Immobilized Glycolate Oxidase/C~tzll~ce
in a Stirred Autoclave Reactor
A 300-mL EZE-Seal stirred autoclave (Autoclave Engineers) was
charged with 100 mL of a solution cont~ining glycolic acid (0.75 M),
ethylenediamine (0.86 M, pH 9.0), propionic acid (0.075 M, HPLC internal
standard), and flavin monunucleotide (0.01 mM), and the solution cooled to
15C. To the autoclave was then added 89 IU of spinach glycolate oxidase and
72,600 IU of Aspergillus niger catalase co-immobilized on ca. 28 g of Eupergit C.
The resllltin~ mixture was stirred at 500 rpm and 15C. under 70 psig (483 kPa)
of oxygen, while bubbling oxygen through the mixture at 100 mL/min. The
reaction was monitored by taking a 100 uL aliquot of the reaction mixture at
regular intervals, mixing the aliquot with 300 mL of 0.1 N sulfuric acid to quench
the reaction, filtering the aliquot and analyzing by HPLC. After 3 hours, the
yields of glyoxylic acid, oxalic acid, and formic acid were 100~o, 0~o, and 0%,
respectively, with complete conversion of glycolic acid. The final activities ofglycolate oxidase and catalase were 100~o and 100% of their initial values.
Example 8
Recovery and Reuse of Co-Immobilized Glycolate Oxidase/Catalase in a Stirred
Autoclave Reactor
The immobilized enzyme catalyst was recovered from the reaction
described in Example 7 by filtering the reaction mixture through a 2.5-cm ID x 20
cm glass column equipped with a 20-mm polyethylene bed support. The
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rem~ining liquid adsorbed on the catalyst was removed by briefly passing a
stream of nitrogen through the column, then the catalyst was resuspended in 100
rnL of a fresh 15C solution containing glycolic acid (0.75 M), ethylene(li~rnine
(0.86 M), propionic acid (0.075 M HPLC internal standard), and flavin
s mononucleotide (0.01 mM). The 300-mL autoclave reactor was again charged
with this
reaction mixture, and the reaction repeated. This catalyst recovery procedure
was performed for 10 consecutive batch reactions, and the reaction time, the
recovery of glycolate oxidase (G.O.) and catalase activity, and yield of glyoxylic
l0 acid are listed in the table below.
Glyoxylic
Run iY Time (hours) G.O.(~o) catalase (5'o) acid(~o)
3 100 100 100
2 2 100 100 96
3 2 98 70 97
4 2 64 100 100
2 80 92 100
6 2 77 77 98
7 2.5 78 96 100
8 2.5 75 100 100
9 3 77 89 100
3 85 100 93
Example 9
2s Reaction Rates for Sparged Enzymatic Oxidations
of Glycolic Acid in a Stirred Autoclave Reactor
A 300-mL EZE-Seal stirred autoclave (Autoclave Engineers) was
charged with 100 mL of a solution containing glycolic acid (0.75 M),
ethylenediamine (0.86 M, pH 9.0), propionic acid (0.075 M, HPLC internal
30 standard), and flavin mononucleotide (0.01 mM), and the solution cooled to
15C. To the autoclave was then added 41 IU of spinach glycolate oxidase and
42,800 IU of Aspergillus niger catalase co-immobilized on ca. 15 g of Eupergit C.
The resulting mixture was stirred at 400 rpm and 15C under 35, 70, 105, or 140
psig (242, 483, 724 or
3s 965 kPa) of oxygen, while bubbling oxygen through the mixture at 50 mL/min.
The reaction was monitored by taking a 100 mL aliquot of the reaction mixture
at regular intervals, mixing the aliquot with 300 mL of 0.1 N sulfuric acid to
quench the reaction, filtering the aliquot and analyzing by HPLC. The rates for
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reactions run under 35, 70, 105, or 140 psig (242, 483, 724 or 965 kPa) of oxygen
were 0.48, 0.54, 0.53, and 0.57 mmol/min of glycolic acid, respectively.
Comparative Example 2
Reaction Rates for Non-Sparged Enzymatic Oxidations
of Glycolic Acid in a Stirred AutocLave Reactor
The reactions in Example ~ were repeated in a stirred autoclave
reactor, except that no oxygen was bubbled through the reaction n~ixtures. The
rates for reactions run under 35, 70, or 105 psig (242, 483, or 724 kPa) of oxygen
were 0.032, 0.053, and 0.071 rnmol/min of glycolic acid, respectively.
Example 10
Enzymatic oxidation of Glycolic Acid Using Permeabilized Bakers Yeast with
Imrnobilized
Glycolate Oxidase
The procedures described in Examples 7 and 8 were repeated,
except that 50 IU of spinach glycolate oxidase immobilized on ca. 15 g of
Eupergit C, and 4.0 g of fresh Saccharomyces cerevisiae (Bakers yeast, Red Star
brand, Universal Foods) which had been permeabilized with isopropanol and
cont~ined 100,000 IU of catalase activity, were used as catalyst. The reaction
ll~LLLule was stirred at 400 rpm and 15C under 70 psig (483 kPa) of oxygen, while
bubbling oxygen through the mixture at 20 mL/min. Six consecutive batch
reactions were run, and the reaction time, the recovery of glycolate oxidase
(G.O.) and c~t~l~ce activity, and yield of glyoxylic acid are listed in the table
below.
2s
Glyoxylic
Run # Time(hours) G.O.(~o) Catalase (%) Acid(~o)
2.5 81 62 100
2 3 97 30 100
3 3 73 54 100
4 3 51 53 96
4 49 57 98
6 6 24 45 97
Example l 1
Dependence of the Rate of Glycolic Acid
Oxidation on Oxygen Sparge Rate
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The procedure described in Example 7 was repeated using 52 IU
of spinach glycolate oxidase and 95,000 IU of Aspergillus niger c~t~l~ce
co-immobilized on ca. 18 g of Eupergit C. The reaction mixture was stirred at
500 rpm and 15C under 70 psig (483 kPa) of oxygen, while sparging oxygen
5 through the mixture at 5 - 50 mL/min. The rates of glyolic acid oxidation at
different oxygen sparge rates are listed in the table below.
mL O~/min mmol O21min mmol glycolic acid/rnin
0.40 0.22
l0 10 0.57 0.45
0.70 0.67
0.79 0.89
0.78 1.11
0.99 1.34
15 50 1.10 2.23
Example 12
Dependence of the Rate of Glycolic Acid
Oxidation on Autoclave Stirring Rate
The procedure described in Example 7 was repeated using 50 IU
of spinach glycolate oxidase and 47,000 IU of ~spergillus niger c~t~ e
co-immobilized on ca. 15 g of Eupergit C. The reaction mixture was stirred at
100-500 rpm and 15C under 70 psig(483 kPa) of oxygen, while sparging oxygen
through the mixture at 20 mL/min. The rates of glycolic acid oxidation at
different stirring rates is listed in the table below.
rpm mmol glycolic acid/min
100 0.08
200 0.15
300 0.22
400 0.43
500 0.45
Example 13
3~ Dependence of the Rate of Glycolic Acid
Oxidation on Glycolate Oxidase Concentration
The procedure described in Example 7 was repeated using 80, 60,
40, or 20 IU of spinach glycolate oxidase and 1,400,000, 1,000,000, 70,000, or
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--18--
35,000 IU of Aspergillus ni~er catalase co-immobilized, respectively, on Eupergit
C. The reaction mixture was s~ir~ed at 400 rpm and 15C under 70 psig of
oxygen, while sparging oxyge~i through the mixture at 20 mL/min. The rates of
glycolic acid oxidation obtained using different concentrations of glycolate
s oxidase is listed in the table below.
Glycolate Oxidase
(IU/mL) mmol glycolic acid/min
0.2 0.20
0.4 0.30
0.6 0.36
0.8 0.57
Having thus described and exemplified the invention with a certain
lS degree of particularity, it should be appreciated that the following claims are not
to be so limited but are to be afforded a scope commensurate with the wording
of each element of the claim and equivalents thereof.
