JPH10501984A - Glyoxylic acid / aminomethylphosphonic acid mixture produced using a transformed microorganism - Google Patents

Abstract

  (57) [Summary] Modifications include microbial cell catalysts expressing glycolate oxidase (eg, Pichia pastoris, Hansenula polymorpha, Aspergillus nidulans or Escherichia coli) and glycolic acid in aqueous solution in the presence of aminomethylphosphonic acid, including the use of endogenous catalase. And improved enzymatic methods for reacting oxygen; soluble catalase may also be included. The resulting mixture is a useful intermediate in the production of N- (phosphonomethyl) glycine.

Description

DETAILED DESCRIPTION OF THE INVENTION Glyoxylic acid / aminomethylphosphonic acid mixture produced using a transformed microorganism Compound                                Background of the Invention 1. Field of the Invention:   The present invention relates to a mixture of glyoxylic acid and aminomethylphosphonic acid (AMPA). Regarding the production method, the method involves glycolic acid and oxygen in an aqueous solution, such as AMPA, The enzyme glycolate oxidase from spinach ((S) -2-hydrogen Genetically engineered to express xy-acid oxidase, EC 1.1.1.35) In the presence of a transformed microorganism and a catalyst containing catalase (EC 1.11.1.6) In the reaction. Glyoxylic acid / aminomethyl produced by this method The phosphonic acid mixture provides a wide range of postemergence herbicides, N- (phosphonomethyl) glycol. It is a useful intermediate in the production of syn. 2. Description of related technology   Glycolate, an enzyme normally found in leafy green plants and mammalian cells. Toxidase catalyzes the oxidation of glycolic acid to glyoxylic acid and produces hydrogen peroxide. Involves the production of:       HOCH_TwoCO_TwoH + O_Two→ OCHCO_TwoH + H_TwoO_Two N. E. FIG. Tolbert et al. ,J. Biol. Chem. , Vol. 181, 905-914 (1949) describes an enzyme first extracted from tobacco leaves. Reported that it is glycolic acid, glyoxylic acid Formic acid and CO via intermittent formation_TwoCatalyzed oxidation to Such as ethylenediamine Addition of certain compounds limited further oxidation of intermediate glyoxylic acid . Oxidation is at a pH of about 8, typically about 3-40 mM (mmol) glycolic acid concentration. Made with degrees. Optimal pH for glycolate oxidation reported to be 8.9 Was done. Oxalic acid (100 mM) inhibits the catalysis of glycolate osidase It was reported. Similarly, K. E. FIG. Richardson and N.M. E. FIG. Tolbert (J. Biol. Chem. , Vol. 236,1280-12 84 (1961)) is tris (hydroxymethyl) aminomethane (TRIS) -Containing buffer in glycolate oxidase-catalyzed oxidation of glycolic acid It was shown to inhibit the production of oxalic acid. C. O. Clagett, N .; E. FIG. T olbert and R.S. H. Burris (J. Biol. Chem. , V ol. 178, 977-987 (1949)) describes the production of glycolic acid using oxygen. The optimal pH for glycolate oxidase catalyzed oxidation is about 7.8-8.6 Reported that the optimum temperature was 35-40 ° C.   I. Zelitch and S.M. Ochoa (J. Biol. Chem. ( Vol. 201, 707-718 (1953)) and J. Am. C. Robinsos net et al. , (J. Biol. Chem. , Vol. 237, 2001- 2009 (1962)) is a spinach glycolate oxidase of glycolic acid. Acid and CO in case-catalyzed oxidation_TwoIs H_TwoO_TwoAnd non-enzyme of glyoxylic acid It was reported to arise from elementary reactions. They are H_TwoO_TwoIs an enzyme that catalyzes the decomposition of Catalase is added to formic acid and CO_TwoGlyoxyl by suppressing the formation of Acid yield Was observed to improve significantly. Addition of FMN (flavin mononucleotide) Has also been found to greatly enhance the stability of glycolate oxidase .   N. A. Frigerio and H.S. A. Harbury (J. Biol . Chem . , Vol. 231, 135-157 (1958)) is spinach Preparation and properties of glycolate oxidase isolated from cormorants were reported. The purified enzyme was found to be very unstable in solution; this instability was Relatively weak binding of rabin mononucleotide (FMN) to the enzyme active site; Enzymatically active tetramer and / or octamer, irreversibly aggregate and precipitate Attributable to dissociation into inert monomers and dimers. FMN (Flavi) into enzyme solution Mononucleotide) greatly improves its stability and increases protein concentration. Alternatively, high ion concentrations retained the enzyme as an octamer or tetramer.   Multiple studies on glycolate oxidase-catalyzed oxidation of glycolic acid There are a number of other references. Enzyme isolation (and assay) is described in the following references: Listed: I. Zelitch,Methods of Enzymolo gy , Vol. 1, Academic Press, New York, 195 5, p. 528-531 (from spinach and tobacco leaves); Nishi mura et al. ,Arch. Biochem. Biophys. , Vo l. 222, 397-402 (1983) (from pumpkin cotyledons); Aske r and d. Davies,Biochim. Biophys. Acta, Vol. 761, 103-108 (1983) (from rat liver), and M . J. Emes and K.S. H. Erismann,Int. J. Biochem . , Vol. 16, 1373-1378 (1984) (REM From Naminor L). The structure of the enzyme was also reported Was: E. Cederlund et al. ,Eur. J. Biochem. , Vol. 173, 523-530 (1988) and Y. Lindquist a nd C.I. Branden,J. Biol. Chem. , Vol. 264,36 24-3628 (1989).                                Summary of the Invention   The present invention uses oxygen in an aqueous solution, and uses AMPA and two catalysts: Enzyme glycolate oxidase from spinach ((S) -2-hydroxy acid Genetically engineered transformation expressing oxidase, EC 1.1.1.3.15) Glycol in the presence of microorganisms and catalase (EC 1.11.1.6) Glyoxylic acid (or their salts) and aminomethyl It relates to the preparation of a mixture of sulphonic acids (AMPA) (or their salts). like that A mixture of glyoxylic acid and AMPA is a post-emergence herbicide, N- (phosphonomethyl). ) Useful for the production of glycine.                         Brief description of biological deposit Applicants have made the following biological deposits under the terms of the Budapest Treaty:   "NRRL" as used herein is 11815 N.V. University   Street, Peoria, IL 61604 U.S. S. Nor where A the region Regional Research Laboratory, A gricultural Research Service Culture   Collection international deposition Say "NRRL No." is the accession number of the culture deposited with NRRL. No.                          Description of the preferred embodiment   A related patent, US Pat. No. 5,135,860 (August 4, 1992), “ Process for Preparing Glyoxylic Acid / Aminoethylphosphonic Acid Methods ” (PCT / US92 / 09419) is oxygen, aminoethylphosphonic acid (AMP) A) and in the presence of the soluble enzymes glycolate oxidase and catalase Describes a method for the enzymatic conversion of glycolic acid to glyoxylic acid in You. Addition of AMPA to enzymatic oxidation of glycolic acid in the pH range of 7-9 Is not expected to result in a high yield of the resulting glyoxylic acid, which is Production of oxidation-resistant N-substituted hemiaminal and / or imine complexes with xylates This was because it was thought that an unprotonated amine was necessary for the formation. AMPA Professional The pKa of the toned amine is reported to be 10.8 (Lange's Hand Book of Chemistry, J.C. A. Dean, ed. , McGra w-Hill, New York, 1979, 12th edition), Therefore, AMPA exists mainly as a protonated ammonium ion in the reaction mixture. , Glyoxylate and It was not expected to form such a protected complex. The present invention uses this method. An improvement to the above method is provided in the form of whole cells of the microorganism as a catalyst.   Previously reported production of glyoxylic acid / aminomethylphosphonic acid mixtures The use of soluble enzymes as catalysts to raise several problems: Recovery of the catalyst is not easy, and the enzyme activity is limited to that of immobilized enzyme or whole-cell catalytic system Not as stable, soluble glycolate oxidase converts acid into the reaction mixture. For the spraying of elemental (required to increase the rate of oxygen dissolution and thus the reaction rate) Not stable. A second related patent application, U.S. Pat. S. S. N. 08 / 085,488, “Glycolate Oxidase Prod action "(PCT / US94 / ) Is well known in the art. Spinach with endogenous catalase, using genetic engineering techniques commonly known to practitioners Aspergillus nidulans expressing glycolate oxidase from Saw The construction of several transformants has been described. In the present invention, glyoxylic acid / a These beyond the use of soluble enzymes for the production of minomethylphosphonic acid mixtures Some advantages in the case of using a whole-cell catalyst are:   (1) All-catalyst The catalyst is easily removed from the reaction mixture at the end of the reaction for reuse. Although recovered, soluble enzymes are only recovered with great difficulty and loss of activity. I;   (2) they relate to the catalyst turnover obtained for soluble enzymes, and More stable than soluble enzyme both in terms of recovered enzyme activity at the end of the reaction ; And   (3) Most importantly, they indicate that oxygen or oxygen-containing gas has a rate of oxygen dissolution. And reaction conditions sparged into the reaction mixture to increase the reaction rate Stable to soluble glycolate oxidase under similar reaction conditions Denatures quickly.   Transformed Aspergillus nidulans first produces glycolate oxidase. The encoding spinach gene is cloned, and this gene is then Into an Aspergillus nidulans strain that produces endogenous catalase And manufactured by. The resulting transformants should be grown in shake flasks or fermenters Cultured in a concentrated medium or SYG concentrated medium, further comprising glycolate oxidase and / or Are oleic acid (OL), hydroxyacetic acid (HA) to increase the expression of catalase. ) Or corn immersion liquid is added to the medium. Then various transformations The recombinants were converted to whole cells (untreated) for catalase and glycolate oxidase activity. ) And assay as a catalyst for the oxidation of glycolic acid to glyoxylic acid. Screening is performed by performing a reaction using cells.   When used as a catalyst for the oxidation of glycolic acid to glyoxylic acid, Aspergillus nidulans cells are either not pre-treated or Permeabilized to increase the accessibility of the reaction mixture to the enzymes; Some may be from exposure to the reaction mixture or any of its components, or may be necessary. Perform by freezing and thawing used to store whole cell catalyst until needed. Can be.   Transformed Aspergillus as a whole-cell catalyst for glyoxylic acid production The use of nislans has been shown previously (PCT / US93 / 00777, "Oxid ation of Glycolic Acid o Glyoxylic Aciding a Microbial C cell Transformant as Catalyst ”), where Amine buffers that can form chemical adducts with lioxylic acid (eg, (Diamine or TRIS) yields glyoxylic acid yields as high as 98%. In that case, the concentration of endogenous catalase in the cells is due to the byproduct hydrogen peroxide. Was sufficient to limit the oxidation of glyoxylate to formic acid. To hydrogen peroxide In which AMPA is not very effective in protecting glyoxylate from oxidation by In the case of the invention, endogenous A. The concentration of Nizlans catalase is Not enough to give the desired high yield of sylate. Glycolate oxy Additional to the AMPA-containing reaction mixture using these whole cells as a source of Soluble catalase (eg, A. niger or S. cerevisiae) (From S. cerevisiae) using soluble or immobilized enzymes Glyoxylate yields similar to those achieved when That was found.   Transformation as a catalyst for oxidation of a Grecolate / AMPA mixture Nizura When using a glance, glyoxylase can be used in cells containing glycolate oxidase. If both phosphate and hydrogen peroxide are produced and no soluble catalase is added, Glyoxylate is rapidly oxidized to formate (eg: in Example 2 87% formate, 1.7% glyoxylate). Glyoxylate high It is anticipated that yields can be obtained simply by adding soluble catalase to the reaction. It is not (1) glyoxylate and hydrogen peroxide produced throughout the course of the reaction. Is greater in cells than in the surrounding aqueous solution, (2) A. D Z The concentration of endogenous catalase in the lance cells is typically added to the normal reaction mixture. (2) 15% of the concentration of soluble catalase obtained, and In order to be decomposed into water and oxygen by the enzyme, hydrogen peroxide needs to be This is because it must diffuse into the liquid. But just react with soluble catalase The mixture contains only soluble catalase and soluble glycolate oxidase as catalysts. When used at the same concentration as previously used (rice) National Patent No. 5,135,860, PCT / US92 / 09419), 94% High glyoxylate yields were obtained.   The second microbial cell catalyst used in the present invention is Hansenula polymorpha. (Methylotrophic yeast), which is spinach with endogenous catalase. Expressing the glycolate oxidase enzyme from E. coli. Enough glycolate oxida Some H. cerevisiae strains with protease activity The polymorpha transformant is glycolate The DNA for the oxidase is placed in an expression vector, formate dehydrogenase ( FMD) by insertion under the control of a promoter. This vector Using H. Polymorpha is transformed to produce large amounts of glycolate oxidase. A strain to be produced is selected. Polymorpha G01 (NRRL Y-21065 ).   H. Pormorpha cell catalysts are typically first transformed with H. Contact of polymorpha Seed material can be grown in 500 ml YPD (Difco), pH 4.4. And manufactured by. The culture was then added to 10 L of Yeast Nitroge. n Base (YBN, Difco) w / o amino acid (14 g), ammonium sulfate (50 g) and methanol (1 00) at pH 5.0. Fermenter at 37 ° C, 4 Agitation rate of 00 rpm, constant pH of 5.0, 40% Operated for 42.5 hours in dissolved oxygen (control) and 14 psig air. fermentation At the end of the reaction, 1.0 kg of glycerol is added, the cells are harvested by centrifugation and It was frozen in body nitrogen and stored at -80 ° C.   The third microbial cell catalyst used in the present invention, together with endogenous catalase, Pichia pastori expressing glycolate oxidase enzyme from spinach (Methylotrophic yeast). Spinach glycolate The DNA fragment containing the oxidase gene was Pastoris expression vector (PHIL-D4) in methanol-inducible alcohol oxidase I promoter By inserting the plasmid under the control of the P. having sufficient glycolate oxidase activity Some traits of Pastoris A transformant was produced. Chromosomal alcohol oxidase of linearized plasmid pMP1 Incorporation into the I locus and alcohol by the glycolate oxidase gene To allow for replacement of the oxidase gene, P. Pastoris strain GTS11 5 (NRRL Y-15851) was transformed with plasmid pMP1 and selected. Was done. The pool of such transformants is then integrated into the expression cassette. Selected for the maximum number of P. Pastoris strain GS115-MSP10 High copy number transformants were isolated and isolated from NRRL, Peoria, Illin. ois deposit.   P. Pastoris cells are typically 100 mL containing 1% glycerose. Produced by growing the inoculum in YNB. 30 ° C After growing for 48 hours in yeast nigrogen base (YBN) w / o amino acids (134 g), glycerose (100 g) and biotin (20 m The cells were transferred into a fermenter containing 10 L of medium containing g). Fermentation is pH 5.0 (NH_FourOH), 30 ° C, 200 rpm agitation speed, 5 slpm air Solution, 5 psig air and dissolved oxygen held above 50% saturation I drove. Once glycerol is depleted, glycerol is reconstituted with methanol (50 g). The cells are induced by growing in the same medium except for replacing Oxidase was expressed. Glycolate oxidase activity during induction Followed by elementary assay. 24 hours after induction, use glycerol (1 kg) Cells were harvested after treatment. Following harvest, cells were frozen in liquid nitrogen and Stored at ° C.   A. Unlike Nislance, H. Polymorpha and P. Transformation of pastoris Before the cells are used as a catalyst for the oxidation of glycolic acid to glyoxylic acid Required permeabilization. Permeable A variety of known methods for producing cells with sufficient glycolate oxidase activity Useful for production (Felix, H .;Anal. Biochemist ry , Vol. 120, 211-234, (1982)); A 10% by weight suspension of wet cells was combined with 0.1% (v / v) "TRITON" X Suspended in -100/20 mM phosphate buffer (pH 7.0) for 15 minutes, Frozen in liquid nitrogen, thawed, 20 mM phosphate / 0.1 mM FMN buffer ( (pH 7.0).   H. as a catalyst for the oxidation of glycolic acid / AMPA mixtures Polimo Rufa and P.M. If any of the Pastoris transformed cells are used, A. Nigel catalase addition is required for high yield glyoxylic acid production Was found. H. Polymorpha or P. Accessibility of Pastoris permeabilized cells Effective catalase activity is described in About 10 times higher than that of Nizlance, Endothelial Catalase of Methylotrophic Yeast in a Reaction Mixture Containing AMPA In the decomposition of hydrogen peroxide, a by-product of Nizuransu or A. From Nigel Was less effective than catalase. Soluble A. as a supplemental catalase source D Gel catalase or Saccharomyces cerevisiae (Saccharo) Myces cerevisiae) permeabilized whole cells were added with catalase. Significant increase in glyoxylic acid production compared to reaction experiments when not added Addition occurred.   The fourth microbial cell catalyst used in the present invention is a spinach with endogenous catalase. Escherichia coli expressing the glycolate oxidase enzyme from Panax scherichia boli (bacteria). That's it Transformation Coli is described in Maceraux et al. ,Biochem. Biophys. Acta , Vol. 1132, 11-16 (1992) Manufactured as follows. Transformation expressing glycolate oxidase activity Ko Li did not permeabilize before use as a catalyst in the present invention.   Many of the drawbacks of using soluble enzymes as catalysts in this application are Transformed microbes removed by using whole cells (impermeable or permeabilized) . For recovery and reuse of whole-cell catalyst, centrifuge or filter catalyst from reaction mixture And recycle it to a new reaction mixture Was easily performed by this method;⁶Expensive glycolate oxidizer The number of revolutions for ze was obtained. Without rapidly denaturing glycolate oxidase Bubble oxygen through the reaction mixture (as observed when using soluble enzymes) Deposition is at least 10 times less than the reaction without foaming the reaction mixture. Increase the response speed, and an increase in this speed significantly increases the manufacturing costs for this method. To lower.   Glycolate oxidase activity used in the reaction Is an effective concentration, usually from 0.01 to about 100 IU / ml, preferably Must be present at a concentration of about 0.1 to about 10 IU / ml. IU (international Is defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute. Is defined. The assay for this enzyme is described in Zelitch and S.M. Ocho a,J. Biol. Chem. , Vol. 201, 707-718 (1953) Found in The process involves recovering or recycling glycolate oxy. It is also used in assays for the activity of dase. Catalase concentration is 1mL reaction mixture 50 to 100,000 IU per product, preferably 500 to 14,000 IU / m Must be L. Both glycolate oxidase and catalase enzymes Preferably, they are present in the same microbial cell (in the case of the appended examples, A. nizuran H. Polymorpha, P.M. Pastoris or E. coli. Cori). Endogenous in cells Catalase concentration is insufficient to effectively decompose the produced hydrogen peroxide In some cases (as in the appended examples), additional sources of catalase (eg, soluble as Pergillus niger catalase or whole cells of Saccharomyces cerevisiae) Can be added to supplement the endogenous catalase present. More catalase And glycolate oxidase concentration, catalase versus glycolate oxidase Of at least about 250: 1 (measured in IU for each) Must be adjusted within the above range. Flavin mononucleotide (FM N) is a component that is optionally added and is 0.0 to 2.0 mM, preferably 0.1 to 2.0 mM. Used at a concentration of 01-0.2 mM.   In view of the above-mentioned effective concentration range, the improved method of the present invention falls within these effective ranges. All microorganisms that express glycolate oxidase and / or catalase Includes and is intended to include the use of cellular catalysts, ie, whole cell catalysts. It should be recognized that Thus, for the purposes of the present invention, the term The term "cell catalyst" actually gives, for example, the effective concentration ranges indicated above. The genetically engineered transformed microorganism of the preferred embodiment, as well as strong expression capacity Selected strains of natural or mutant microorganisms and / or mixtures thereof Including but not limited to objects.   Glycolic acid (2-hydroxyacetic acid) is commercially available. Place of this reaction The initial concentration is in the range of 0.10M to 2.0M, preferably 0.25M to 1M. . 0M. It is as such or its compatible salt, i.e. water-soluble The cation is the desired conversion of glycolic acid to glyoxylic acid, or N- (phosphonomethyl) glycine of oxylic acid product and aminomethylphosphonic acid Can be used as a salt that does not conflict with the reaction for forming Suitable and compatible The salt-forming cationic group of is easily determined by experimentation. Typical such Salts are alkali metals, alkaline earth metals, ammonium, substituted ammonium, And sulfonium and substituted phosphonium salts.   The conversion of glycolic acid to glyoxylic acid is simple and, preferably, in aqueous media. Done in Aminomethylphosphonic acid (AMPA) or a suitable salt thereof is 0.0 1 / 1.0 to 3.0 / 1.0, preferably 0.25 / 1.0 to 1.05 / 1.0 To give a molar ratio of AMPA / glycolic acid (starting amount) in the range of You. After combining AMPA and glycolic acid in aqueous solution, the pH of the resulting mixture Is adjusted to a value of 6 to 10, preferably 7.0 to 8.5. Within this pH range, Compatible non-violating salts including alkali metal hydroxides, carbonates, bicarbonates and phosphates The exact pH can be adjusted by addition of groups to obtain the desired pH. Reaction proceeds And the pH of the reaction mixture drops slightly, thus increasing the pH range of maximum enzyme activity. The reaction starts at about 9.0 to 8.5, around the maximum of the pH, and allows the pH to drop during the reaction. Is often useful. Since enzyme activity changes with pH, PH can be maintained by adding non-violent inorganic or organic buffer separately. it can.   Glycolic and glyoxylic acids are highly dissociated in water and have a p-value of 7-10. In H, most if not substantially complete glycolate and It is understood that it exists as a xylate ion. Glyoxylic acid (and its conjugates) Base, glyoxylate anion) is a hydrate, for example (HO)_TwoCHCOOH and And / or hemiacetal HOOCCH (OH) OCH (OH) COOH Wherein the composition and the corresponding anion are N- (phosphonome Glyoxylated for this purpose to be a suitable reactant for the production of til) glycine Equivalent to silicic acid and its anions also recognized by those skilled in the art Will be done.   Oxygen (O 2 O) which is an oxidizing agent for the addition of glycolic acid to glyoxylic acid_Two) Is It reacts as a gas and disturbs the liquid at the gas-liquid interface. Oxygen is sparged through a membrane that is permeable or through the reaction mixture (bubble Standing). Under most conditions, the reaction rate is oxygen Seems to be at least partially controlled by the rate at which . Thus, oxygen can be added to the reaction as air, but in relatively pure form. It is preferred to use oxygen, and even to use high pressure. The upper limit of oxygen pressure is unknown However, an oxygen pressure up to 50 atm can be used, with an upper limit of 15 atm being preferred. High Disturbance is important to maintain a high oxygen dissolution (and therefore reaction) rate. Such as stirring Any simple form of perturbation can be used.   Reaction temperature is an important variable in that it affects reaction rate and enzyme stability. Reaction temperatures of about 0 ° C to 40 ° C can be used, but preferred reaction temperatures are 5 ° C to It is in the range of 15 ° C. Operation in the preferred temperature range is recovered at the end of the reaction Maximize enzyme activity. The temperature must not be so low that the aqueous solution begins to freeze. The temperature is Use a jacketed reaction vessel and pass liquid of the appropriate temperature through the jacket. Can be controlled by ordinary methods such as There is no. Reaction vessels can be constructed from any material that is inert to the reaction components You.   Reaction solution and O_TwoFollowing the termination of contact, the microbial cell catalyst was decanted and filtered. It can be removed by filtration or centrifugation and reused. Optionally, remove the solution Extracts flavin mononucleotide (FMN) by contacting with activated carbon be able to. Glyoxylic acid and aminomethylphosphonic acid (with the corresponding imine Which appears to be in equilibrium) The solution can be prepared using a method known in the art for the production of N- (phosphonomethyl) glycine. Process according to any one of the above methods.   Catalytic hydrogenation is from mixtures containing glyoxylic acid and aminomethylphosphonic acid It is a preferred method for producing N- (phosphonomethyl) glycine. This eye Suitable hydrogenation catalysts are various platinum group metals such as iridium, osmium, Didium, ruthenium, platinum and palladium; also various other transition metals such as copper Including (but not limited to) Baltic, Copper, Nickel and Zinc. Touch The medium can be unsupported, for example, Raney nickel or platinum oxide; Or it can be, for example, platinum on carbon, palladium on alumina or kieselguhr It can be supported like supported nickel. Palladium on carbon, key Zelgur-supported nickel and Raney nickel are preferred. Hydrogenation is 4-11, good Preferably, it can be performed at a pH of 5-10. Wide range of hydrogenation temperature and pressure Can be changed. The temperature is generally between 0 ° C and 150 ° C, preferably between 20 ° C and 90 ° C. And H_TwoThe pressure is generally from about atmospheric pressure to about 100 atmospheres, preferably 1-1. It is within the range of 0 atm. No matter which reduction method is used, it can be used as a herbicide after germination. N- (phosphonomethyl) glycine for any of the times known in the art. It can also be recovered from the reducing solution by a recovery method.   In the following examples, which serve to further illustrate the invention, glyoxylate , Formate and oxalate yields, and glycolate recovery yields The percentage is based on the total amount of glycolic acid present at the beginning of the reaction. Anti The reaction mixture was analyzed using high pressure liquid chromatography: organic acid analysis was performed using Bi. o-Rad HPX-87H column AMPA and N- (phosphonomethyl) glycine were obtained using Bio-Rad.   Analyze using Aminex Glyphosate Analysis column did.   The transformed microbial cells consisted of a magnetic stir bar, and 2,6-dichlorophenol- 0.12 mM for indophenol (DCIP) and TRIS buffer (pH 8.3) of 3-mL quartz containing 2.0 mL solution which is 80 mM with respect to Glycorelation can be achieved by accurately weighing about 5-10 mg of wet cells into a cuvette. Assayed for oxidase activity. Cover the cuvette with a rubber septum The solution was deoxygenated by bubbling nitrogen for 5 minutes. Then 40 μl of 1.0 M glycolic acid / 1.0 M TRIS (pH 8.3) was cuvette with a syringe. At 605 nm (ε = 22,000) while stirring the mixture. The change in absorption associated with was measured.   Catalase activity was determined using a 3-mL solution containing a magnetic stir bar and 2.0 mL of distilled water. Accurately weigh about 2-5 mg of wet cells into a quartz cuvette, then add 50 m 1.0 mL of 59 mM hydrogen peroxide in M phosphate buffer (pH 7.0) , 240 nm (ε = 39.4) to measure the change in absorption with time Assayed. A. cultivated in various media Nizrans wet cells (non-permeant The glycolate oxidase and catalase activities of 0.5-4.0 DCIP IU per gram of wet cells for dase, 500-7000 IU per gram of wet cells for endogenous catalase Was in the range. H. cultivated in various media. Polymorpha or P. Pastries Glycolate oxidase and catalyzer of humid cells (permeabilized) Lase activity was 2 per gram of wet cells for glycolate oxidase. 1 gram of wet fines for 0-60 DCIP IG, as well as endogenous catalase The range was 30,000-80,000 IU per cell.                                 Example 1   300-mL EZE-Seal stirred autoclave reactor (Autocla) ve Engineers), glycolic acid (0.500M), aminomethyl Phosphonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and And flavin mononucleotide (0.01 mM) at pH 8.3 (50% NaOH). 100 ml of the solution contained in the control) was charged and the solution was cooled to 5 ° C. Next 26 g of frozen (−80 ° C.) Aspergillus nidulans FTI7 SYSL / OL (124 IU glycolate oxidase and 57,800 IU Catalase) and 1.0 × 10⁶Units of Aspergillus niger soluble catala (Sigma) was added, the cells were thawed at 5 ° C., and the pH of the resulting mixture was adjusted to Re-adjusted to 8.3 with 50% NaOH. The mixture is brought to 400 rpm and 5 ° C. And bubbled oxygen through the mixture at 50 mL / min under 120 psig oxygen. While stirring. Take 0.40 mL aliquots of the reaction mixture at regular intervals. Tori, Millipore "ULTRAFREE" -MC 10,000NMWL   An aliquot was filtered using a Filter Unit and the filtrate was analyzed by HPLC. The reaction was monitored by analysis. 10 hours later, glyoxylic acid, oxalic acid and And formic acid yields were 91%, 0% and 7.9%, respectively, with 2.5% Luic acid was recovered. Glycolate oxidase and Aspergillus nigercata The final activities of the lase were 11% and 87% of their initial value.                             Example 2 (comparison)   Omit the addition of Aspergillus niger soluble catalase (Sigma) Other than that, the reaction described in Example 1 was repeated. After 22 hours, glyoxylic acid, shu The yields of acid and formic acid were 1.7%, 0% and 87.4%, respectively, The acid was completely converted. At the end of the reaction Aspergillus nidulans FT17S Glycolate oxidase or catalase activity detectable in YCSL / OL cells There was no sex.                                 Example 3   300-mL EZE-Seal stirred autoclave reactor (Autocla) ve Engineers), glycolic acid (0.500 M), aminomethyl Phosphonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and And flavin mononucleotide (0.01 mM) at pH 8.3 (50% NaOH). 100 ml of the solution contained in the control) was charged and the solution was cooled to 5 ° C. Next 26 g of frozen (−80 ° C.) Aspergillus nidulans FT17 SYSL / OL (124 IU glycolate oxidase and 57,800 IU Catalase) and 5.6 × 10^FiveUnits of Aspergillus niger soluble catala (Sigma) was added, the cells were thawed at 5 ° C., and the pH of the resulting mixture was adjusted to Re-adjusted to 8.3 with 50% NaOH. The mixture is brought to 400 rpm and 5 ° C. And bubbled oxygen through the mixture at 50 mL / min under 120 psig oxygen. While stirring. After 10 hours, the yield of glyoxylic acid, oxalic acid and formic acid was 83%, 0% and 9.4% respectively, 4.4% glycolic acid recovered . Glycolate oxidase And Aspergillus niger catalase have a final activity of 17% of their initial value. And 88%.                                 Example 4   300-mL EZE-Seal stirred autoclave reactor (Autocla) ve Engineers), glycolic acid (0.500M), aminomethyl Phosphonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and And flavin mononucleotide (0.01 mM) at pH 8.3 (50% NaOH). 100 ml of the solution contained in the control) was charged and the solution was cooled to 5 ° C. Next 15 g of frozen (−80 ° C.) Aspergillus nidulans FT17 SYSL / OL (72 IU glycolate oxidase and 33,300 IU Talase) and 1.4 × 10⁶Units of Aspergillus niger soluble catalase (Sigma) was added, the cells were thawed at 5 ° C., and the pH of the resulting mixture was adjusted to 5 Readjusted to 8.3 with 0% NaOH. This mixture is placed at 400 rpm and 5 ° C. To bubble oxygen through the mixture at 50 mL / min under 120 psig oxygen. While stirring. After 17 hours, the yield of glyoxylic acid, oxalic acid and formic acid was lower 76%, 0% and 4.1% respectively, 16.8% glycolic acid recovered . Final activity of glycolate oxidase and Aspergillus niger catalase The sex was 7% and 49% of their initial value.                                 Example 5   300-mL EZE-Seal stirred autoclave reactor (Autocla) Ve Engineers), glycolic acid (0.500 M), aminomethylphos Sulfonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and flavin mononucleotide (0.01 mM) at pH 8. 3 (adjusted with 50% NaOH), load 100 mL of solution containing Was cooled to 5 ° C. Frozen (-80 ° C) Aspergillus nidulans FT17S YCSL / OL (26 g) 124 IU glycolate oxidase and 57,80 0IU catalase) is thawed at 5 ° C., then 2 × 100 mL of KH at 5 ° C._Two PO_Four(50 mM, pH 7.0) / FMN (0.01 mM) buffer, wash 1.4 x 10 purified cells⁶Units of Aspergillus niger soluble catalase (Sigma) with the reactor. The pH of the resulting mixture is adjusted to 50% NaOH Readjust to 8.3 at 400 rpm and 5 ° C. under 120 psig oxygen. And stirred while bubbling oxygen through the mixture at 50 mL / min. 11.5 o'clock After a while, the yields of glyoxylic acid, oxalic acid and formic acid are 94%, 3.5% and 94%, respectively. And 2.7%, and 1.3% of glycolic acid was recovered. Glycolate oxy The final activities of Dase and Aspergillus niger catalase are based on their initial values. 7% and 77%.                                 Example 6   300-mL EZE-Seal stirred autoclave reactor (Autocla) Ve Engineers), glycolic acid (0.500 M), aminomethylphos Sulfonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and Flavin mononucleotide (0.01 mM) was adjusted to pH 8.3 (50% NaOH). 100 mL of the solution contained in section) was charged and the solution was cooled to 5 ° C. new Well harvested Aspergillus nidulans FT17SYCSL / OL (37g , 44 IU glycolate oxidase and 57,800 IU catalase) at 5 ° C. At 4x 100 mL KH_TwoPO_Four(50 mM, pH 7.0) / FMN (0.01 mM) buffer Wash with the buffer and wash the washed cells at 1.4 x 10⁶Units of Aspergillus niger Added to the reactor with soluble catalase (Sigma). PH of the resulting mixture Is readjusted to 8.3 with 50% NaOH and then at 400 rpm and 5 ° C. Bubble oxygen through the mixture at 50 mL / min under 120 psig oxygen. And stirred. After 15 hours, the yields of glyoxylic acid, oxalic acid and formic acid were respectively 84%, 0% and 9.4%, 6.4% glycolic acid was recovered.   At the completion of the reaction, the reaction mixture is centrifuged at 5 ° C. and the supernatant is decanted. Was done. 100 mL of the obtained pellet of Aspergillus nidulans cells And resuspended in a fresh reaction mixture at 5 ° C under the same conditions as described above. Was repeated. After 25 hours, the yields of glyoxylic acid, oxalic acid and formic acid are respectively This was 59%, 1.1% and 1.6%, and 43% of the glycolic acid was recovered. At this point, the assay for glycolate oxidase in the cells has no residual activity. Was shown.                                 Example 7   300-mL EZE-Seal stirred autoclave reactor (Autocla) Ve Engineers), glycolic acid (0.500 M), aminomethylphos Contains sulphonic acid (0.375M) and flavin mononucleotide (0.01mM) And 100 mL of the solution (pH 8.3, 50 % NaOH) and the solution was cooled to 5 ° C. Frozen (-80 ℃) Lugils nizuransu FT17SYCSL / OL (26g) 124IU glyco Rate oxidase and 57,800 IU catalase) at 3 ° C. Then 5 ° C 4x100mL KH at_TwoPO_Four(50 mM, pH 7.0) / FMN (0. 01 mM) buffer and wash the washed cells at 1.4 × 10 4⁶Unit Asperghi Added to the reactor with Russ Nigel soluble catalase (Sigma). can get Mix the mixture at 400 rpm and 5 ° C. under 120 psig of oxygen in 50 mL The mixture was stirred while bubbling oxygen through the mixture at / min. 11 hours later, glyoxy The yields of luic, oxalic and formic acids were 89%, 4.5% and 2.0%, respectively. Glycolic acid was completely converted.                                 Example 8   Magnetic in 3 oz Fischer-Porter glass aerosol reactor A stirring rod, glycolic acid (0.500 M), aminomethylphosphonic acid (0 . 375M), isobutyric acid (0.100M, HPLC internal standard) and flavin mono Nucleotides (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) The contained 10 mL aqueous solution was charged and the solution was cooled to 5 ° C. The container is then filled with 0. Permeability by processing with 1% "TRITON" X-100 / l freeze-thaw 0.47 g of the transformed Hansenula polymorpha GO1 (10 IU glyco Rate oxidase and 22,100 IU catalase), and 1.4 x 10⁶single Aspergillus niger soluble catalase (Sigma) was added. Obtained The pH of the resulting mixture was readjusted to 8.3 with 50% NaOH and the reaction vessel was then Sealed and cooled the reaction mixture to 5 ° C. Pressurized to 70 psig 5 times with stirring The container is flushed with oxygen by evacuating to atmospheric pressure and then the container is pumped to 70 ps. Pressurized to ig oxygen and the mixture was stirred at 5 ° C. To monitor the progress of the reaction For analysis by HPLC, At regular intervals through the sampling port, by syringe (without loss of pressure in the container) A) An aliquot (0.10 mL) was removed. 16 hours later, glyoxylate, The HPLC yields of formate and oxalate were 90.1%, 1.3% and 1.3%, respectively. And 5.9%, leaving 3.0% glucose. Residual glycolate oxida Ase activity and total catalase activity were 86% and 136% of their initial values, respectively. %Met.                             Example 9 (comparison)   1.4x10⁶Units of Aspergillus niger soluble catalase (Sigma The reaction in Example 8 was repeated except that the addition of ()) was omitted. 16 hours later, The HPLC yields of reoxylate, formate and oxalate were respectively 57. 6%, 32.5% and 2.6%, leaving 8.9% glucose. Glico Residual permeabilizing cell activities of rate oxidase and catalase are at their initial values. They were 60% and 378%, respectively.                            Example 10 (comparison)   1.4x10⁶Units of Aspergillus niger soluble catalase (Sigma ) Was added at 1.4 × 10⁶1.67 g permeability with unit catalase activity (0.1% “TRITON” X-100 / l freeze-thaw) Saccharomyces The reaction in Example 8 was repeated except that the cells were replaced with Levisiae cells. 16 hours Thereafter, the HPLC yields of glyoxylate, formate and oxalate were respectively 67.2%, 19.7% and 6.0%, and no glycolate remained.                                Example 11   3 oz Fischer-Porter glass aerosol reactor Inside, a magnetic stir bar, glycolic acid (0.500M), aminomethylphospho Acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and Bin mononucleotide (0.01 mM) pH 8.3 (adjusted with 50% NaOH) Was added and the solution was cooled to 5 ° C. Then container And 0.1% “TRITON” X-100 / l freeze-thaw. 0.75 g of transformed Pichia pastoris GS115-MSP10 (13.2 IU glycolate oxidase and 21,200 IU catalase) added. The pH of the resulting mixture was readjusted to 8.3 with 50% NaOH, The reaction vessel was then sealed and the reaction mixture was cooled to 5 ° C. 5 times with stirring, 70 Flushing the container with oxygen by pressurizing to psig and evacuating to atmospheric pressure, then The vessel was pressurized to 70 psig oxygen and the mixture was stirred at 5 ° C. The progress of the reaction Through the sampling port at regular intervals for analysis by HPLC for monitoring Aliquot (0.10 mL) by syringe (without loss of pressure in the container) I took it out. After 16 hours, the glyoxylate, formate and oxalate H PLC yields were 30.5%, 59.2% and 10.7%, respectively, 0.8% Glucose remained.                                Example 12   300-mL EZ equipped with Dispersimax Impeller E-Seal stirred autoclave reactor (Autoclave Engineer) rs), glycolic acid (0.500M), aminomethylphosphonic acid (0.37M) 5M), isobutyric acid (0.100M, HPLC internal standard) and flavin mononucleotide Otide (0.01 mM) was added at pH8. 3 (adjusted with 50% NaOH), load 100 mL of solution and bring the solution to 5 ° C. And cooled. Then process with 0.1% "TRITON" X-100 / l freeze-thaw 10 g of transformed Pichia pastoris NRRL permeabilized by Y-21001 (391 IU glycolate oxidase and 457,000 IU Catalase), and 1.4 x 10⁶Unit of Aspergillus niger soluble mosquito Talase (Sigma) was added to the vessel. The pH of the resulting mixture is adjusted to 50% NaO Adjusted to 8.3 with H. At 1000 rpm and 5 ° C. Stir under 120 psig of oxygen, which mixes through the action of the turbine impeller Oxygen was bubbled through the object. 0.40 mL aliquots of the reaction mixture at regular intervals The Millipore “ULTRAFREE” -MC 10,000. The aliquot was filtered using NMWL Filter Unit and the filtrate was HPL The reaction was monitored by analysis with C. One hour later, glyoxylic acid, The yields of uric acid and formic acid were 89.8%, 6.0% and 2.9%, respectively. 7% glycolic acid was recovered. Glycolate oxidase and permeabilized cells And catalase final activities were 117% and 78% of their initial values.   Microbial cell catalyst was recovered from the above reaction mixture by centrifugation. Further processing Without removing the cell pellet, add 100 mL of fresh reaction mixture and an additional 1.4. x10⁶Mix with Aspergillus niger soluble catalase and repeat the reaction. I returned. After 1.0 hour, the yield of glyoxylic acid, oxalic acid and formic acid was 8 7.8%, 5.0% and 5.3%, with 2.9% glycolic acid recovered . Permeabilization-The ultimate activity of cellular glycolate oxidase and catalase is 172 of their initial values % And 61%.                                Example 13   Mixing glyoxylic acid and AMPA produced via the reaction described in Example 2 The product is centrifuged to remove Aspergillus nidulans whole cell catalyst and then Am icon "CENTRIPREP" 10 concentrator (10,000 molecular weight cut-off ) To remove soluble Aspergillus niger catalase. Get The resulting solution was stirred with activated carbon (0.50 g) to remove FMN, filtered, and filtered. 0mL, 316SS Cup Autoclave Engineers EZE -Seal reactor was charged with 0.50 g of 10% Pd on activated carbon. Reactor Flushed with nitrogen and then charged with hydrogen at 300 psig, Stirred at pm and 27 ° C. After 22 hours, N- (phosphonomethyl) glycyl Yield (based on AMPA) was 80%.                                Example 14   Mixing of glyoxylic acid and AMPA produced via the reaction described in Example 4 The product is centrifuged to remove Aspergillus nidulans whole cell catalyst and then Am icon "CENTRIPREP" 10 concentrator (10,000 molecular weight cut-off ) To remove soluble Aspergillus niger catalase. Get The resulting solution was stirred with activated carbon (0.50 g) to remove FMN, filtered, and filtered. 0mL, 316SS Cup Autoclave Engineers EZE -Seal reactor was charged with 0.50 g of 10% Pd on activated carbon. Reactor Flushed with nitrogen and then charged with hydrogen at 300 psig, Stirred at pm and 27 ° C. After 22 hours, N- (phosphonomethyl) glyc The yield of syn (based on AMPA) was 89%.                                Example 15   Mixing glyoxylic acid and AMPA produced via the reaction described in Example 6 The product is centrifuged to remove Aspergillus nidulans whole cell catalyst and then Am icon "CENTRIPREP" 10 concentrator (10,000 molecular weight cut-off ) To remove soluble Aspergillus niger catalase. Get The resulting solution was stirred with activated carbon (0.50 g) to remove FMN, filtered, and filtered. 0.50 g of activated carbon was placed on a 0 mL glass liner. And 10% Pd. Seal the glass liner in an autoclave, Flush with nitrogen, then charge with hydrogen at 1000 psig and bring to 27 ° C. And mixed by rocking. After 11 hours, N- (phosphonomethyl) glyci Yield (based on AMPA) was 76%.                                Example 16   300ML EZE equipped with Dispersimax Impeller -Seal stirred autoclave reactor (Autoclave Engineer) s), glycolic acid (0.500M), aminomethylphosphonic acid (0.375M) M), isobutyric acid (0.100 M), HPLC internal standard) and flavin mononucleotide 10 containing Otide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) 0 mL of the solution was charged and the solution was cooled to 5 ° C. Then 30 g of the transformed E. Ko Li (25.2 IU glycolate oxidase and 39,900 IU catalase) Is added to the vessel and the mixture is subjected to 120 psig acid at 1000 rpm and 5 ° C. Agitating the mixture through the mixture through the action of the turbine impeller. I whipped the element. Take 0.40 mL aliquots of the reaction mixture at regular intervals , Millipore “ULTRAFREE” -MC 10,000NMWL F Filter an aliquot using ilter Unit and analyze the filtrate by HPLC To monitor the reaction. After 19 hours, glyoxylic acid, oxalic acid and ants The acid yields were 8.6%, 8.3% and 1.1%, respectively, with 57.3% Cholic acid remained. Recovery activity of microbial glycolate oxidase and catalase Were 10% and 68% of their initial values, respectively.                                Example 17   Higher glycolate oxidase activity (72 IU glycolate oxidase) And 29.600 IU catalase). The reaction described in Example 16 was repeated using E. coli. 23 hours later, glyoxyl The yields of acid, oxalic acid and formic acid were 18.3%, 1.9% and 2.9%, respectively. And 42.6% glycolic acid remained. Microbial glycolate oxidase; and Catalase recovery activities were 18% and 71% of their initial values, respectively.   Thus, the present invention has been described and illustrated with some particularity. The scope of the claim should not be limited in that way, and the representation of each element of the claims and A range commensurate with their equivalent should be given.

[Procedure for Amendment] Article 184-8 of the Patent Act [Submission Date] May 22, 1996 [Content of Amendment] The second related patent application filed on July 1, 1993 S. S. N. 08 / 085,488, "Glycolate Oxidase Production" (PCT / US94 / 07081, WO 95/01444) uses endogenous catalase and glycosynthesis from spinach using genetic engineering techniques commonly known to those skilled in the art. The construction of several transformants of Aspergillus nidulans expressing rate oxidase has been described. Some advantages of the use of these all-cell catalysts over the use of soluble enzymes for the production of glyoxylic acid / aminomethylphosphonic acid mixtures in the present invention include: (1) the termination of the reaction Sometimes the reaction mixture is easily recovered for reuse, but the soluble enzymes are recovered only with great difficulty and loss of activity; (2) they are the catalyst turnover obtained for the soluble enzymes And more stable than the soluble enzyme both in terms of recovered enzyme activity at the end of the reaction; and (3) most importantly, that oxygen or oxygen-containing gases increase the rate of oxygen dissolution and reaction rate Is stable to the reaction conditions applied to the reaction mixture, and under similar reaction conditions the soluble glycolate oxidase denatures rapidly. Transformed Aspergillus nidulans was produced by first cloning the spinach gene encoding glycolate oxidase and then introducing this gene into a strain of Aspergillus nidulans that already produces endogenous catalase. The resulting transformants are cultured in minimal media or SYG-enriched media in shake flasks or fermenters, and oleic acid (OL), hydroxyacetic acid (HA) to increase the expression of glycolate oxidase and / or catalase. Alternatively, various reagents, such as corn immersion liquid, are added to the medium. The various transformants are then screened by assaying whole cells (untreated) for catalase and glycolate oxidase activity and performing a reaction using the cells as a catalyst for the oxidation of glycolic acid to glyoxylic acid. . When used as a catalyst for the oxidation of glycolic acid to glyoxylic acid, Aspergillus nidulans cells are not pretreated or permeabilized to increase the accessibility of the reaction mixture to the enzyme inside the cells; Some of the permeabilization may be effected, possibly from exposure to the reaction mixture or any of its components, or by freezing and thawing used to preserve the whole cell catalyst until needed. H. cultivated in various media. Polymorpha or P. Glycolate oxidase and catalase activity of Pastoris wet cells (permeabilized) was 20-60 DCIP IG per gram of wet cells for glycolate oxidase, and 30,000-80,00 per gram of wet cells for endogenous catalase. It was in the range of 0 IU. Example 1 In a 300-mL EZE-Seal stirred autoclave reactor (Autoclave Engineers), glycolic acid (0.500M), aminomethylphosphonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and A 100 mL solution containing flavin mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) was charged and the solution was cooled to 5 ° C. The reactor was then charged with 26 g of frozen (-80 ° C) Aspergillus nidulans FT17 SYCSL / OL (124 IU glycolate oxidase and 57,800 IU catalase) and 1.0 × 10 ⁶ units of Aspergillus niger soluble catalase (Sigma) and 5 ° C. The cells were thawed in and the pH of the resulting mixture was readjusted to 8.3 with 50% NaOH. The mixture was stirred at 400 rpm and 5 ° C. under 120 psig of oxygen while bubbling oxygen through the mixture at 50 mL / min. At regular intervals, 0.40 mL aliquots of the reaction mixture were taken, the aliquots were filtered using a Millipore "ULTRAFREE" -MC 10,000 NMWL Filter Unit, and the reaction was monitored by analyzing the filtrate by HPLC. After 10 hours, the yields of glyoxylic acid, oxalic acid, and formic acid were 91%, 0%, and 7.9%, respectively, and 2.5% glycolic acid was recovered. The final activities of glycolate oxidase and Aspergillus niger catalase were 11% and 87% of their initial values. Example 2 (Comparative) The reaction described in Example 1 was repeated except that the addition of Aspergillus niger soluble catalase (Sigma) was omitted. After 22 hours, the yields of glyoxylic acid, oxalic acid and formic acid were 1.7%, 0% and 87.4%, respectively, and the glycolic acid was completely converted. At the end of the reaction there was no detectable glycolate oxidase or catalase activity in Aspergillus nidulans FT17S YCSL / OL cells. Example 3 In a 300-mL EZE-Seal stirred autoclave reactor (Autoclave Engineers), glycolic acid (0.500M), aminomethylphosphonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and A 100 mL solution containing flavin mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) was charged and the solution was cooled to 5 ° C. The reactor was then charged with 26 g of frozen (−80 ° C.) Aspergillus nidulans FT17 SYCSL / OL (124 IU glycolate oxidase and 57,800 IU catalase) and 5.6 × 10 ⁵ units of Aspergillus niger soluble catalase (Sigma). The cells were thawed in and the pH of the resulting mixture was readjusted to 8.3 with 50% NaOH. The mixture was stirred at 400 rpm and 5 ° C. under 120 psig of oxygen while bubbling oxygen through the mixture at 50 mL / min. After 10 hours, the yields of glyoxylic acid, oxalic acid, and formic acid were 83%, 0%, and 9.4%, respectively, and 4.4% glycolic acid was recovered. The final activities of glycolate oxidase and Aspergillus niger catalase were 17% and 88% of their initial values. Example 4 In a 300-mL EZE-Seal stirred autoclave reactor (Autoclave Engineers), glycolic acid (0.500M), aminomethylphosphonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and A 100 mL solution containing flavin mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) was charged and the solution was cooled to 5 ° C. 15 g of frozen (-80 ° C) Aspergillus nidulans FT17 SYCSL / OL (72 IU glycolate oxidase and 33,300 IU catalase) and 1.4 × 10 ⁶ units of Aspergillus niger soluble catalase (Sigma) were then added to the reactor and added at 5 ° C. The cells were thawed in and the pH of the resulting mixture was readjusted to 8.3 with 50% NaOH. The mixture was stirred at 400 rpm and 5 ° C. under 120 psig of oxygen while bubbling oxygen through the mixture at 50 mL / min. After 17 hours, the yields of glyoxylic acid, oxalic acid and formic acid were 76%, 0% and 4.1%, respectively, and 16.8% glycolic acid was recovered. The final activities of glycolate oxidase and Aspergillus niger catalase were 7% and 49% of their initial values. Example 5 In a 300-mL EZE-Seal stirred autoclave reactor (Autoclave Engineers), glycolic acid (0.500M), aminomethylphosphonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and A 100 mL solution containing flavin mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) was charged and the solution was cooled to 5 ° C. Frozen (−80 ° C.) Aspergillus nidulans FT17SYCSL / OL (26 g, 124 IU glycolate oxidase and 57,800 IU catalase) is thawed at 5 ° C., then ₂ × 100 mL KH ₂ PO ₄ (50 mM, pH 7.0) / 5 ° C. / Washed with FMN (0.01 mM) buffer and the washed cells were added to the reactor along with 1.4 × 10 ⁶ units of Aspergillus niger soluble catalase (Sigma). The pH of the resulting mixture was readjusted to 8.3 with 50% NaOH and stirred at 400 rpm and 5 ° C. under 120 psig of oxygen at 50 mL / min while bubbling oxygen through the mixture. After 11.5 hours, the yields of glyoxylic acid, oxalic acid, and formic acid were 94%, 3.5%, and 2.7%, respectively, and 1.3% glycolic acid was recovered. The final activities of glycolate oxidase and Aspergillus niger catalase were 7% and 77% of their initial values. Example 6 In a 300-mL EZE-Seal stirred autoclave reactor (Autoclave Engineers), glycolic acid (0.500M), aminomethylphosphonic acid (0.375M), isobutyric acid (0.100M, HPLC internal standard) and A 100 mL solution containing flavin mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) was charged and the solution was cooled to 5 ° C. Freshly harvested Aspergillus nidulans FT17SYCSL / OL (37 g, 44 IU glycolate oxidase and 57,800 IU catalase) was added at 4 ° C. in 4 × 100 mL of KH ₂ PO ₄ (50 mM, pH 7.0) / FMN (0.01 mM) buffer. And washed cells were added to the reactor along with 1.4 × 10 ⁶ units of Aspergillus niger soluble catalase (Sigma). The pH of the resulting mixture was readjusted to 8.3 with 50% NaOH and then stirred at 400 rpm and 5 ° C. under 120 psig of oxygen at 50 mL / min while bubbling oxygen through the mixture. After 15 hours, the yields of glyoxylic acid, oxalic acid and formic acid were 84%, 0% and 9.4%, respectively, and 6.4% glycolic acid was recovered. Upon completion of the reaction, the reaction mixture was centrifuged at 5 ° C. and the supernatant was decanted. The resulting pellet of Aspergillus nidulans cells was resuspended in 100 mL of fresh reaction mixture at 5 ° C. and the reaction was repeated under the same conditions as described above. After 25 hours, the yields of glyoxylic acid, oxalic acid, and formic acid were 59%, 1.1%, and 1.6%, respectively, and 43% of the glycolic acid was recovered. Assay of glycolate oxidase in the cells at this point showed no residual activity. Example 7 A 300-mL EZE-Seal stirred autoclave reactor (Autoclave Engineers) contains glycolic acid (0.500 M), aminomethylphosphonic acid (0.375 M), flavin mononucleotide (0.01 mM), 100 mL of solution (pH 8.3, adjusted with 50% NaOH) to which no HPLC internal standard was added was charged and the solution was cooled to 5 ° C. Frozen (−80 ° C.) Aspergillus nidulans FT17SYCSL / OL (26 g, 124 IU glycolate oxidase and 57,800 IU catalase) is thawed at 5 ° C., then 4 × 100 mL KH ₂ PO ₄ (50 mM, pH 7.0) / 5 ° C. / Washed with FMN (0.01 mM) buffer and washed cells were added to the reactor along with 1.4 × 10 ⁶ units of Aspergillus niger soluble catalase (Sigma). The resulting mixture was stirred at 400 rpm and 5 ° C. under 120 psig of oxygen while bubbling oxygen through the mixture at 50 mL / min. After 11 hours, the yields of glyoxylic acid, oxalic acid and formic acid were 89%, 4.5% and 2.0%, respectively, and the glycolic acid was completely converted. Example 8 A magnetic stir bar in a 3 oz Fischer-Porter glass aerosol reaction vessel and glycolic acid (0.500 M), aminomethylphosphonic acid (0.375 M), isobutyric acid (0.100 M, HPLC internal standard) and flavin 10 mL of an aqueous solution containing the mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) was charged and the solution was cooled to 5 ° C. The container is then filled with 0. 0.47 g of transformed Hansenula polymorpha GO1 (10 IU glycolate oxidase and 22,100 IU catalase) permeabilized by treatment with 1% “TRITON” X-100 / l freeze-thaw, and 1.4 × 10 ⁶ Units of Aspergillus niger soluble catalase (Sigma) were added. The pH of the resulting mixture was readjusted to 8.3 with 50% NaOH, then the reaction vessel was sealed and the reaction mixture was cooled to 5 ° C. The vessel was flushed of oxygen by pressurizing to 70 psig and evacuating to atmospheric pressure five times with stirring, then pressurizing the vessel to 70 psig of oxygen and stirring the mixture at 5 ° C. Aliquots (0.10 mL) were removed by syringe (without loss of pressure in the vessel) via the sampling port at regular intervals for analysis by HPLC to monitor the progress of the reaction. After 16 hours, the HPLC yields of glyoxylate, formate and oxalate were 90.1%, 1.3% and 5. 9%, leaving 3.0% glucose. Residual glycolate oxidase activity and total catalase activity were 86% and 136% of their initial values, respectively. Example 9 (Comparative) The reaction of Example 8 was repeated except that the addition of 1.4 × 10 ⁶ units of Aspergillus niger soluble catalase (Sigma) was omitted. After 16 hours, the HPLC yield of glyoxylate, formate and oxalate was 57.75 respectively. 6%, 32.5% and 2.6%, leaving 8.9% glucose. The residual permeabilized cell activity of glycolate oxidase and catalase was 60% and 378% of their initial values, respectively. Example 10 (comparative) The addition of 1.4 × 10 ⁶ units of Aspergillus niger soluble catalase (Sigma) was followed by permeabilization of 1.67 g with 1.4 × 10 ⁶ units of catalase activity (0.1% “TRITON” X The reaction in Example 8 was repeated, except that the cells were replaced with Saccharomyces cerevisiae cells. After 16 hours, the HPLC yields of glyoxylate, formate and oxalate were 67.2%, 19.7% and 6.0%, respectively, with no glycolate remaining. Example 11 A magnetic stir bar and glycolic acid (0.500 M), aminomethylphosphonic acid (0.375 M), isobutyric acid (0.100 M, HPLC internal standard) and flavin in a 3 oz Fischer-Porter glass aerosol reaction vessel. 10 mL of an aqueous solution containing the mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) was charged and the solution was cooled to 5 ° C. The container is then filled with 0. 0.75 g of transformed Pichia pastoris GS115-MSP10 (13.2 IU glycolate oxidase and 21,200 IU catalase) permeabilized by treatment with 1% "TRITON" X-100 / l freeze-thaw were added. . The pH of the resulting mixture was readjusted to 8.3 with 50% NaOH, then the reaction vessel was sealed and the reaction mixture was cooled to 5 ° C. The vessel was flushed with oxygen by pressurizing to 70 psig and evacuating to atmospheric pressure five times with stirring, then pressurizing the vessel to 70 psig oxygen and stirring the mixture at 5 ° C. Aliquots (0.10 mL) were removed by syringe (without loss of pressure in the vessel) via the sampling port at regular intervals for analysis by HPLC to monitor the progress of the reaction. After 16 hours, the HPLC yields of glyoxylate, formate and oxalate were 30.5%, 59.2% and 10.7%, respectively, leaving 0.8% glucose. Example 12 Glycolic acid (0.500 M), aminomethylphosphonic acid (0.375 M), isobutyric acid (0 M) in a 300-mL EZ E-Seal stirred autoclave reactor (Autoclave Engineers) equipped with a Dispersimax Impeller. A 100 mL solution containing .100M, HPLC internal standard) and flavin mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) was charged and the solution was cooled to 5 ° C. 10 g of transformed Pichia pastoris NRRL Y-21 001 (391 IU glycolate oxidase and 457,000 IU catalase) permeabilized by treatment with 0.1% "TRITON" X-100 / 1 freeze-thaw, And 1.4 × 10 ⁶ units of Aspergillus niger soluble catalase (Sigma) were added to the vessel. The pH of the resulting mixture was readjusted to 8.3 with 50% NaOH. The mixture was stirred at 1000 rpm and 5 ° C. under 120 psig of oxygen, which bubbled oxygen through the mixture through the action of the turbine impeller. At regular intervals, 0.40 mL aliquots of the reaction mixture were taken, the aliquots were filtered using a Millipore "ULTRAFREE" -MC 10,000 NMWL Filter Unit, and the reaction was monitored by analyzing the filtrate by HPLC. After 1 hour, the yields of glyoxylic acid, oxalic acid, and formic acid were 89.8%, 6.0%, and 2.9%, respectively, and 1.7% glycolic acid was recovered. The final activity of glycolate oxidase and catalase in the permeabilized cells was 117% and 78% of their initial value. Microbial cell catalyst was recovered from the above reaction mixture by centrifugation. Without further treatment, the cell pellet was mixed with 100 mL of the fresh reaction mixture and an additional 1.4 × 10 ⁶ units of Aspergillus niger soluble catalase, and the reaction was repeated. After 1.0 hour, the yields of glyoxylic acid, oxalic acid, and formic acid were 87.8%, 5.0%, and 5.3%, respectively, and 2.9% glycolic acid was recovered. Permeabilization-The final activities of cellular glycolate oxidase and catalase were 172% and 61% of their initial values. Example 13 A mixture of glyoxylic acid and AMPA produced via the reaction described in Example 2 was centrifuged to remove Aspergillus nidulans whole cell catalyst, and then an Amicon "CENTRIPREP" 10 concentrator (10,000) (Molecular weight cut off) to remove soluble Aspergillus niger catalase. The resulting solution was stirred with activated carbon (0.50 g) to remove FMN, filtered and charged to a 300 mL, 316 SS cup Autoclave Engineers EZE-Seal reactor with 0.50 g of 10% Pd on activated carbon. The reactor was flushed with nitrogen and then charged with hydrogen at 300 psig and stirred at 1000 rpm and 27 ° C. After 22 hours, the yield of N- (phosphonomethyl) glycine (based on AMPA) was 80%. Example 14 A mixture of glyoxylic acid and AMPA produced via the reaction described in Example 4 was centrifuged to remove Aspergillus nidulans whole cell catalyst, and then an Amicon "CENTRIPREP" 10 concentrator (10,000) (Molecular weight cut off) to remove soluble Aspergillus niger catalase. The resulting solution was stirred with activated carbon (0.50 g) to remove FMN, filtered and charged to a 300 mL, 316 SS cup Autoclave Engineers EZE-Seal reactor with 0.50 g of 10% Pd on activated carbon. The reactor was flushed with nitrogen and then charged with hydrogen at 300 psig and stirred at 1000 rpm and 27 ° C. After 22 hours, the yield of N- (phosphonomethyl) glycine (based on AMPA) was 89%. Example 15 A mixture of glyoxylic acid and AMPA produced via the reaction described in Example 6 was centrifuged to remove Aspergillus nidulans whole cell catalyst, and then an Amicon "CENTRIPREP" 10 concentrator (10,000) (Molecular weight cut off) to remove soluble Aspergillus niger catalase. The resulting solution was stirred with activated carbon (0.50 g) to remove FMN, filtered and placed in a 300 mL glass liner with 0.50 g of 10% Pd on activated carbon. The glass liner was sealed in an autoclave, flushed with nitrogen, then charged with hydrogen at 1000 psig and mixed by rocking at 27 ° C. After 11 hours, the yield of N- (phosphonomethyl) glycine (based on AMPA) was 76%. Example 16 Glycolic acid (0.500 M), aminomethylphosphonic acid (0.375 M), isobutyric acid (0.100 M) in a 300 ML EZE-Seal stirred autoclave reactor (Autoclave Engineers) equipped with a Dispersimax Impeller. , HPLC internal standard) and 100 mL of a solution containing flavin mononucleotide (0.01 mM) at pH 8.3 (adjusted with 50% NaOH) and the solution was cooled to 5 ° C. Then 30 g of the transformed E. E. coli (25.2 IU glycolate oxidase and 39,900 IU catalase) was added to the vessel and the mixture was stirred at 1000 rpm and 5 ° C. under 120 psig of oxygen, which bubbled oxygen through the mixture via the action of the turbine impeller. . At regular intervals, 0.40 mL aliquots of the reaction mixture were removed, the aliquots were filtered using a Millipore "ULTRAFREE" -MC 10,000 NMWL Filter Unit and the reaction was monitored by analyzing the filtrate by HPLC. After 19 hours, the yields of glyoxylic acid, oxalic acid, and formic acid were 8.6%, 8.3%, and 1.1%, respectively, leaving 57.3% glycolic acid. The recovery activities of microbial glycolate oxidase and catalase were 10% and 68% of their initial values, respectively. Transformation of a second growth of 30 g with higher glycolate oxidase activity than Example 17 (72 IU glycolate oxidase and 29.600 IU catalase) The reaction described in Example 16 was repeated using E. coli. After 23 hours, the yields of glyoxylic acid, oxalic acid and formic acid were 18.3%, 1.9% and 2.9%, respectively, leaving 42.6% glycolic acid. The microbial glycolate oxidase and catalase recovery activities were 18% and 71% of their initial values, respectively. Claims 1. Comprising oxidizing glycolic acid with oxygen in an aqueous solution containing aminomethylphosphonic acid and the enzymes glycolate oxidase and catalase, wherein the improvement comprises: (a) sparging the oxygen in the aqueous solution; A catalyst is added in the form of a transformed microbial cell that expresses glycolate oxidase, and the microbial cell catalyst is a transformant of Aspergillus nidulans, a transformant of Escherichia coli, a NRRL-Y- Selected from the group consisting of Pichia pastoris transformant GS115-MSP10 designated 21001 and NRRL-Y-21065 transformant G01 of Hansenula polymorpha designated as NRRL-Y-21065. Containing glyoxylic acid and aminomethylphos Process for the preparation of a mixture of phonic acids. 2. The method of claim 1 wherein said microbial cell catalyst also expresses endogenous catalase. 3. The method of claim 1, further comprising adding a soluble catalase to the mixture. 4. The method of claim 1 wherein said transformed microbial cell is Aspergillus nidulans T17 designated NRRL-21000.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI C12R 1:78) (C12P 7/40 C12R 1:19) (C12P 7/40 C12R 1:84) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AM, AU, BB, BG, BR, BY, CA, CN, CZ, FI, GE, HU, JP, KE, KG, KP, KR, KZ, LK, LV, MD, MG, MN, MW, NO, NZ, PL, RO, RU, SD, SI, SK, TJ, TT, UA, US, UZ, VN

Claims (1)

[Claims]   1. Aminomethylphosphonic acid and enzymes glycolate oxidase and Oxidizing glycolic acid with oxygen in an aqueous solution in the presence of an enzyme Here, improvements include the use of microbial cell catalysts that express glycolate oxidase. A method for producing a mixture of glyoxylic acid and aminomethylphosphonic acid.   2. The method of claim 1 wherein said microbial cell catalyst also expresses endogenous catalase. .   3. 3. The method of claim 2, wherein soluble catalase is also used.   4. The microbial cell catalyst is Aspergillus, Hansenul (Hansenul) 2. The method of claim 1, wherein the method is selected from the group consisting of: a) and Pichia.   5. The microbial cell catalyst is Pichia Pastoris. The method of range 1.   6. The microbial cell catalyst is Hansenula polymorpha The method of claim 1.   7. The microbial cell catalyst is Aspergillus nidulans The method of claim 1 wherein:   8. The microbial cell catalyst is Escherichia coli. The method of range 1.