WO1994006925A1 - Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase - Google Patents
Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase Download PDFInfo
- Publication number
- WO1994006925A1 WO1994006925A1 PCT/US1992/008002 US9208002W WO9406925A1 WO 1994006925 A1 WO1994006925 A1 WO 1994006925A1 US 9208002 W US9208002 W US 9208002W WO 9406925 A1 WO9406925 A1 WO 9406925A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- immobilized
- catalase
- glycolate oxidase
- acid
- glycolic acid
- Prior art date
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- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 title claims abstract description 105
- 108010062584 glycollate oxidase Proteins 0.000 title claims abstract description 74
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 title claims abstract description 71
- 102000016938 Catalase Human genes 0.000 title claims abstract description 55
- 108010053835 Catalase Proteins 0.000 title claims abstract description 55
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 10
- 102100038837 2-Hydroxyacid oxidase 1 Human genes 0.000 title claims abstract 9
- 230000001590 oxidative effect Effects 0.000 title description 2
- 238000006243 chemical reaction Methods 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 51
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000001301 oxygen Substances 0.000 claims abstract description 42
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 42
- 230000008569 process Effects 0.000 claims abstract description 26
- 239000000872 buffer Substances 0.000 claims abstract description 23
- 150000001412 amines Chemical class 0.000 claims abstract description 22
- 239000011324 bead Substances 0.000 claims abstract description 21
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims abstract description 4
- 239000003054 catalyst Substances 0.000 claims description 30
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 claims description 21
- 229940013640 flavin mononucleotide Drugs 0.000 claims description 21
- FVTCRASFADXXNN-UHFFFAOYSA-N flavin mononucleotide Natural products OP(=O)(O)OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-UHFFFAOYSA-N 0.000 claims description 21
- 239000011768 flavin mononucleotide Substances 0.000 claims description 21
- 235000019231 riboflavin-5'-phosphate Nutrition 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 18
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 claims description 6
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- 108010008488 Glycylglycine Proteins 0.000 claims description 3
- 229940043257 glycylglycine Drugs 0.000 claims description 3
- 125000003916 ethylene diamine group Chemical group 0.000 claims 2
- 102100038838 2-Hydroxyacid oxidase 2 Human genes 0.000 description 64
- 102000004190 Enzymes Human genes 0.000 description 56
- 108090000790 Enzymes Proteins 0.000 description 56
- 229960004275 glycolic acid Drugs 0.000 description 45
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 30
- 230000000694 effects Effects 0.000 description 28
- 239000011541 reaction mixture Substances 0.000 description 25
- 230000003647 oxidation Effects 0.000 description 24
- 238000007254 oxidation reaction Methods 0.000 description 24
- 239000000243 solution Substances 0.000 description 24
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 22
- 241000219315 Spinacia Species 0.000 description 17
- 235000009337 Spinacia oleracea Nutrition 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 14
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 11
- 241000228245 Aspergillus niger Species 0.000 description 11
- 235000019253 formic acid Nutrition 0.000 description 11
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 10
- 108010093096 Immobilized Enzymes Proteins 0.000 description 10
- -1 ethylenediamine Chemical class 0.000 description 10
- 235000006408 oxalic acid Nutrition 0.000 description 10
- 239000007983 Tris buffer Substances 0.000 description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 9
- 235000011130 ammonium sulphate Nutrition 0.000 description 9
- 238000001179 sorption measurement Methods 0.000 description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical class S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 8
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- 239000008188 pellet Substances 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 7
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- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 7
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- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- AEMRFAOFKBGASW-UHFFFAOYSA-M Glycolate Chemical compound OCC([O-])=O AEMRFAOFKBGASW-UHFFFAOYSA-M 0.000 description 3
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- 108030001056 (S)-2-hydroxy-acid oxidases Proteins 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- VILCJCGEZXAXTO-UHFFFAOYSA-N 2,2,2-tetramine Chemical compound NCCNCCNCCN VILCJCGEZXAXTO-UHFFFAOYSA-N 0.000 description 2
- 241000351920 Aspergillus nidulans Species 0.000 description 2
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
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- 235000002637 Nicotiana tabacum Nutrition 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 229920002873 Polyethylenimine Polymers 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- CBOQJANXLMLOSS-UHFFFAOYSA-N ethyl vanillin Chemical compound CCOC1=CC(C=O)=CC=C1O CBOQJANXLMLOSS-UHFFFAOYSA-N 0.000 description 2
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- 210000004185 liver Anatomy 0.000 description 2
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
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- CCBICDLNWJRFPO-UHFFFAOYSA-N 2,6-dichloroindophenol Chemical compound C1=CC(O)=CC=C1N=C1C=C(Cl)C(=O)C(Cl)=C1 CCBICDLNWJRFPO-UHFFFAOYSA-N 0.000 description 1
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- SHKUUQIDMUMQQK-UHFFFAOYSA-N 2-[4-(oxiran-2-ylmethoxy)butoxymethyl]oxirane Chemical compound C1OC1COCCCCOCC1CO1 SHKUUQIDMUMQQK-UHFFFAOYSA-N 0.000 description 1
- OORRCVPWRPVJEK-UHFFFAOYSA-N 2-oxidanylethanoic acid Chemical compound OCC(O)=O.OCC(O)=O OORRCVPWRPVJEK-UHFFFAOYSA-N 0.000 description 1
- 235000000832 Ayote Nutrition 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
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- 230000002378 acidificating effect Effects 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
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- ZLFRJHOBQVVTOJ-UHFFFAOYSA-N dimethyl hexanediimidate Chemical compound COC(=N)CCCCC(=N)OC ZLFRJHOBQVVTOJ-UHFFFAOYSA-N 0.000 description 1
- FRTGEIHSCHXMTI-UHFFFAOYSA-N dimethyl octanediimidate Chemical compound COC(=N)CCCCCCC(=N)OC FRTGEIHSCHXMTI-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
Definitions
- 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.
- Glycolate oxidase an enzyme commonly found in leafy green plants and mammalian cells, catalyzes the oxidation of glycolic acid to glyoxylic acid, with the concomitant production of hydrogen peroxide.
- 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.
- 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.
- This invention describes the preparation and use of an immobilized enzyme catalyst for the manufacture of glyoxylic acid from glycolic acid (hydroxyacetic acid).
- glycolic acid hydroxyacetic acid
- high selectivities (c 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.
- U.S.S.N. 07/422,011 filed Oct.
- 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, and 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 catalase (e.g., from Aspergillus niger. Aspergillus nidulans.
- 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; 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 prolonged storage in aqueous buffer; and 3) most importantly, the immobilized catalyst is stable to reaction conditions where oxygen is sparged into the reaction mixture to increase the rate of oxygen dissolution and reaction rate, where under similar reaction conditions the soluble glycolate oxidase is rapidly denatured.
- 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 attachment, physical adsorption, electrostatic binding, or affinity binding, (2) crosslinking with bifunctional or multifunctional reagents, (3) entrapment in gel matrices, polymers, emulsions, or some form of membrane, and (4) a combination of any of these methods.
- the stability of immobilized glycolate oxidase in aqueous buffers is much greater than the soluble enzyme (approaching the stability of ammonium sulfate-precipitated enzyme).
- 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 mixture; in this manner for immobilized glycolate oxidase the number of turnovers (i.e., the number of substrate molecules that are converted to product molecules per catalyst molecule before inactivation of the enzyme) as high as 10
- the immobilized glycolate oxidase used in the reaction should be present in an effective concentration, usually a concentration of about 0.001 to about 10.0 IU/mL, preferably about 0.1 to about 4 IU/mL.
- An IU IU/mL
- 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 maintained by a buffer, since enzyme activity varies with pH.
- the pH of the reaction 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
- an amine buffer capable of complexing the glyoxylic acid (by forming an imine which is more stable to chemical or enzymatic oxidation) is employed along with catalase to maximize product selectivity.
- Ethylenediamine, or less preferably, tris(hydroxymethyl)methylamine (hereinafter TRIS), piperazine, or glycylglycine improved the yield of glyoxylic acid.
- TRIS tris(hydroxymethyl)methylamine
- piperazine or glycylglycine
- 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.
- the exact value may be adjusted to obtain the desired pH.
- 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.
- acid for example hydrochloric or sulfuric acids.
- less basic amines such as TRIS it may be necessary to add a base to maintain the desired pH.
- the concentration of immobilized catalase should be 50 to 100,000 IU/mL, 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 catalase and glycolate oxidase concentrations be adjusted within the above ranges so that the ratio (measured in IU for each) of catalase: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 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 (measured at atmospheric pressure) per volume of reaction mixture per minute (vol/vol-min), and preferably between 0.2 and 2 vol/vol-min.
- reaction temperature is an important variable, in that it affects reaction rate and the stability of the enzymes.
- a reaction temperature of 0°C to 40°C may be used, but the preferred reaction temperature range is from 5°C to 15°C. Operating in the preferred temperature range maximizes recovered enzyme activity at the end of the reaction.
- amine buffer is most conveniently removed by use of an ion exchange resin.
- Suitable acidic cationic exchange resins include "AMBERLITE” CG120 or “AMBERLITE” IR120 (Rohm & Haas Co.), and “DOWEX” 50 (Dow Chemical Co.). The amine may then be recovered and subsequently recycled by treatment of the resin with strong base.
- glyoxylic acid is useful in the preparation of vanillin and ethylvanillin, as well as being used in ion exchange resins and as an acid catalyst in the pharmaceutical industry (Ullmanns). It is usually sold as a 50% (weight percent) aqueous solution. It is also to be understood that reference to glyoxylic acid in this application can also mean the glyoxylate anion, especially when the glyoxylic acid is present in a solution whose pH is greater than about 2.3.
- Glycolate oxidase from spinach was purified using selective ammonium sulfate fractionation followed by batch adsorption of the extract 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 4°C unless otherwise stated. At 25°C, 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 pulp was isolated by squeezing through 4 layers of cheesecloth; alternatively, a juice extractor (Nitantonio) may be used.
- 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
- the protein was dialysed for 16 hrs. vs. 4 L of 20 mM bicine (pH 8.0) containing 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 readings 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, P.. Phytochemistry. Vol. 14, 359-362 (1975)) added and the resulting mixture incubated for 10 minutes. Protein binding to the resin was monitored by following the decrease in protein concentration of the solution using the
- a 200 mL portion of the yeast suspension was transferred to a 400 mL capacity
- 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.
- LLB 280 nm filter
- LLB chart recorder
- This purification method has also been used to purify catalase from Aspergillus nidulans and Aspergillus niger.
- Glycolate oxidase immobilized on oxirane acrylic beads was 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
- 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 40°C, using as solvent an aqueous solution of H2SO4(0.01 N) and l-hydroxyethane-l,l-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 UN detector and 410 differential refractometer. UN analysis was performed at 210 nm. The retention times for oxalic acid, glyoxylic acid, glycolic acid, formic acid, and propionic acid (internal standard) were 4.29, 6.09, 7.77, 8.79, and 11.41 minutes, respectively.
- PION Poly(ethyleneimine)
- 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
- Eupergit C-30 ⁇ were all obtained from commercial sources.
- PAN-500 poly(acrylamide-co-N-acryloxysuccinimide) gel crosslinked with triethylenetetramine
- oxirane acrylic beads Eupergit C
- a solution containing 50 mM bicine buffer (pH 8.0) and 0.02 mM flavin mononucleotide 50 mM bicine buffer (pH 8.0) and 0.02 mM flavin mononucleotide
- 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 settled beads. This washing procedure was repeated a second time.
- a 10-mL mixture containing 100 mg (715,000 IU) of ammonium sulfate-precipitated Aspergillus niger catalase (Sigma C-3515) was centrifuged at 15,000 rpm for 10 minutes (Sorvall SS-34 rotor). The supernatant was discarded and the pellet dissolved in the buffer containing the glycolate oxidase. This enzyme solution was then added to the flask containing 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 bottle roller at 4-5 rpm for 16 hours at 15°C.
- 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 5°C in this same buffer.
- the co-immobilized enzyme catalyst had 7.2 IU of glycolate oxidase activity /gram Eupergit C and 5680 IU of catalase activity/gram Eupergit C.
- Example 3 Relative Stability of Soluble and Immobilized Glycolate Oxidase The stability of unimmobilized (soluble) glycolate oxidase versus glycolate oxidase immobilized on oxirane acrylic beads (Eupergit C) was measured by storing either form of the enzyme at 4°C in a buffered (pH 8.0) solution containing 2.0 mM flavin mononucleotide, then monitoring the enzyme activity with time. Additionally, the stability of the enzyme precipitated in 3.2 M ammonium sulfate, 2.0 mM flavin mononucleotide, and stored under similar conditions was also monitored. Enzyme
- Example 4 Sparged Co-Immobilized Enzyme Reaction Into a 2.5-cm ID x 20 cm glass column equipped with a 20-mm polyethylene bed support was placed 10 mL of a solution containing glycolic acid (0.25 M), ethylenediamine (0.33 M), propionic acid (0.075 M, HPLC internal standard), and flavin monunucleotide (0.2 mM). The column and its contents were cooled to 15°C, 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.
- Example 4 The reaction described in Example 4 was repeated, except that the same amounts of soluble, unimmobihzed glycolate oxidase and catalase were added to the reaction mixture. After 4 hours, the yields of glyoxylic 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 catalase were ⁇ 2% and 82% of their initial values, respectively, and no further reaction was observed at longer reaction times.
- Example 4 The reaction in Example 4 was repeated using 10 mL of a solution containing glycolic acid (0.75 M), ethylenediamine (0.86 M), propionic acid
- the yields of glyoxylic acid, oxalic acid, and formic acid were 93%, 0%, and 0.3%, respectively, with a 94% conversion of glycolic acid.
- the final activities of glycolate oxidase and catalase were 48% and 69% of their initial values.
- 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 standard), and flavin monunucleotide (0.01 mM), and the solution cooled to 15°C.
- 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 resulting mixture was stirred at 500 rpm and 15°C.
- 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 remaining liquid adsorbed on the catalyst was removed by briefly passing a stream of nitrogen through the column, then the catalyst was resuspended in 100 mL of a fresh 15°C solution containing glycolic acid (0.75 M), ethylenediamine (0.86 M), propionic acid (0.075 M HPLC internal standard), and flavin 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 acid are listed in the table below.
- 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 standard), and flavin mononucleotide (0.01 mM), and the solution cooled to 15°C.
- 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 15°C under 35, 70, 105, or 140 psig (242, 483, 724 or 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 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.
- Example 9 The reactions in Example 9 were repeated in a stirred autoclave reactor, except that no oxygen was bubbled through the reaction mixtures.
- Example 10 Enzymatic oxidation of Glycolic Acid Using Permeabilized Bakers Yeast with Immobilized 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 contained 100,000 IU of catalase activity, were used as catalyst.
- the reaction mixture was stirred at 400 rpm and 15°C 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 catalase activity, and yield of glyoxylic acid are listed in the table below.
- Example 7 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 35,000 IU of Aspergillus niger catalase co-immobilized, respectively, on Eupergit C.
- the reaction mixture was stirred at 400 rpm and 15°C under 70 psig of oxygen, while sparging oxygen through the mixture at 20 mL/min.
- the rates of glycolic acid oxidation obtained using different concentrations of glycolate oxidase is listed in the table below.
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- Organic Chemistry (AREA)
- 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)
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Abstract
Description
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Priority Applications (12)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU26543/92A AU2654392A (en) | 1992-09-18 | 1992-09-18 | Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase |
JP50803294A JP3145711B2 (en) | 1992-09-18 | 1992-09-18 | Production of glyoxylic acid by oxidation of glycolic acid in the presence of immobilized glycolate oxidase and catalase |
CZ95700A CZ70095A3 (en) | 1992-09-18 | 1992-09-18 | Preparation of glyoxylic acid by oxidizing glycollic acid in the presence of immobilized glycolate oxidase and catalase |
EP92920655A EP0662142A1 (en) | 1992-09-18 | 1992-09-18 | Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase |
BR9207165A BR9207165A (en) | 1992-09-18 | 1992-09-18 | Process for the production of glyoxylic acid |
CA002144637A CA2144637A1 (en) | 1992-09-18 | 1992-09-18 | Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase |
HU9500950A HU213760B (en) | 1992-09-18 | 1992-09-18 | Process for the production of glyoxylic acid by enzyme catalysed oxidation of glycolic acid |
PCT/US1992/008002 WO1994006925A1 (en) | 1992-09-18 | 1992-09-18 | Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase |
NZ244529A NZ244529A (en) | 1992-09-18 | 1992-09-28 | Production of glyoxylic acid comprising contacting glycolic acid, glycolate oxidase and catalase in an aqueous solution |
ZA927518A ZA927518B (en) | 1992-09-18 | 1992-09-30 | Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase |
PT100935A PT100935A (en) | 1992-09-18 | 1992-10-07 | PROCESS FOR THE PREPARATION OF GLIOXYLIC ACID BY OXIDACAO OF GLYCOLIC ACID IN THE PRESENCE OF GLYCOLATE OXIDASE AND CATALANASE IMMOBILIZED |
CN92112653A CN1086263A (en) | 1992-09-18 | 1992-10-26 | The production method of Glyoxylic acid hydrate |
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002144637A CA2144637A1 (en) | 1992-09-18 | 1992-09-18 | Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase |
HU9500950A HU213760B (en) | 1992-09-18 | 1992-09-18 | Process for the production of glyoxylic acid by enzyme catalysed oxidation of glycolic acid |
PCT/US1992/008002 WO1994006925A1 (en) | 1992-09-18 | 1992-09-18 | Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase |
CS7009592 | 1992-09-18 | ||
NZ244529A NZ244529A (en) | 1992-09-18 | 1992-09-28 | Production of glyoxylic acid comprising contacting glycolic acid, glycolate oxidase and catalase in an aqueous solution |
ZA927518A ZA927518B (en) | 1992-09-18 | 1992-09-30 | Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase |
NZ244592A NZ244592A (en) | 1991-10-04 | 1992-10-02 | Improving the selectivity of delignification of chemical pulp using an |
PT100935A PT100935A (en) | 1992-09-18 | 1992-10-07 | PROCESS FOR THE PREPARATION OF GLIOXYLIC ACID BY OXIDACAO OF GLYCOLIC ACID IN THE PRESENCE OF GLYCOLATE OXIDASE AND CATALANASE IMMOBILIZED |
CN92112653A CN1086263A (en) | 1992-09-18 | 1992-10-26 | The production method of Glyoxylic acid hydrate |
Publications (1)
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WO1994006925A1 true WO1994006925A1 (en) | 1994-03-31 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US1992/008002 WO1994006925A1 (en) | 1992-09-18 | 1992-09-18 | Production of glyoxylic acid by oxidizing glycolic acid in the presence of immobilized glycolate oxidase and catalase |
Country Status (10)
Country | Link |
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EP (1) | EP0662142A1 (en) |
JP (1) | JP3145711B2 (en) |
CN (1) | CN1086263A (en) |
AU (1) | AU2654392A (en) |
CZ (1) | CZ70095A3 (en) |
HU (1) | HU213760B (en) |
NZ (1) | NZ244529A (en) |
PT (1) | PT100935A (en) |
WO (1) | WO1994006925A1 (en) |
ZA (1) | ZA927518B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6323011B1 (en) | 1996-03-23 | 2001-11-27 | Institute Of Food Research | Production of vanillin |
US11806335B2 (en) | 2019-11-01 | 2023-11-07 | Lilac Therapeutics, Inc. | Heterocyclic carboxylate compounds as glycolate oxidase inhibitors |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109988784B (en) * | 2019-04-16 | 2021-02-02 | 台州学院 | A kind of method for immobilizing glycolate oxidase catalyzing synthesis of pyruvate |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991005868A1 (en) * | 1989-10-16 | 1991-05-02 | E.I. Du Pont De Nemours And Company | Production of glyoxylic acid by enzymatic oxidation of glycolic acid |
-
1992
- 1992-09-18 EP EP92920655A patent/EP0662142A1/en not_active Withdrawn
- 1992-09-18 JP JP50803294A patent/JP3145711B2/en not_active Expired - Fee Related
- 1992-09-18 CZ CZ95700A patent/CZ70095A3/en unknown
- 1992-09-18 WO PCT/US1992/008002 patent/WO1994006925A1/en not_active Application Discontinuation
- 1992-09-18 AU AU26543/92A patent/AU2654392A/en not_active Abandoned
- 1992-09-18 HU HU9500950A patent/HU213760B/en not_active IP Right Cessation
- 1992-09-28 NZ NZ244529A patent/NZ244529A/en not_active IP Right Cessation
- 1992-09-30 ZA ZA927518A patent/ZA927518B/en unknown
- 1992-10-07 PT PT100935A patent/PT100935A/en not_active Application Discontinuation
- 1992-10-26 CN CN92112653A patent/CN1086263A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991005868A1 (en) * | 1989-10-16 | 1991-05-02 | E.I. Du Pont De Nemours And Company | Production of glyoxylic acid by enzymatic oxidation of glycolic acid |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6323011B1 (en) | 1996-03-23 | 2001-11-27 | Institute Of Food Research | Production of vanillin |
US6664088B2 (en) | 1996-03-23 | 2003-12-16 | Plant Bioscience Limited | Production of vanillin |
US11806335B2 (en) | 2019-11-01 | 2023-11-07 | Lilac Therapeutics, Inc. | Heterocyclic carboxylate compounds as glycolate oxidase inhibitors |
US12168002B2 (en) | 2019-11-01 | 2024-12-17 | Lilac Therapeutics, Inc. | Heterocyclic carboxylate compounds as glycolate oxidase inhibitors |
Also Published As
Publication number | Publication date |
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CN1086263A (en) | 1994-05-04 |
EP0662142A1 (en) | 1995-07-12 |
HU213760B (en) | 1997-09-29 |
NZ244529A (en) | 1995-06-27 |
ZA927518B (en) | 1994-03-30 |
PT100935A (en) | 1994-06-30 |
HU9500950D0 (en) | 1995-05-29 |
JP3145711B2 (en) | 2001-03-12 |
JPH08501218A (en) | 1996-02-13 |
CZ70095A3 (en) | 1995-11-15 |
AU2654392A (en) | 1994-04-12 |
HUT72915A (en) | 1996-06-28 |
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