CN1086263A

CN1086263A - The production method of Glyoxylic acid hydrate

Info

Publication number: CN1086263A
Application number: CN92112653A
Authority: CN
Inventors: D·L·安敦; R·迪科西莫; J·E·加瓦根
Original assignee: EI Du Pont de Nemours and Co
Current assignee: EIDP Inc
Priority date: 1992-09-18
Filing date: 1992-10-26
Publication date: 1994-05-04
Also published as: JP3145711B2; AU2654392A; HUT72915A; HU213760B; EP0662142A1; PT100935A; WO1994006925A1; ZA927518B; NZ244529A; HU9500950D0; JPH08501218A; CZ70095A3

Abstract

The invention describes a kind of production method of Glyoxylic acid hydrate: in the aqueous solution, under the aerobic situation, form the amine damping fluid of chemical adducts with an energy and Glyoxylic acid hydrate; Hydroxyl oxidize enzyme and catalase by co-immobilization on an insoluble carrier.Best insoluble fixation support is Bupergit C-250L and EupergitC (oxyethane vinylformic acid bead).

Description

The production method of Glyoxylic acid hydrate

The invention relates to a kind of method of the production Glyoxylic acid hydrate that has improved, this method is to utilize the enzymatic oxidation oxyacetic acid, specifically, relates to and adopts the hydroxyl oxidize enzyme that is fixed on the insoluble carrier and hydrogen peroxide alcohol as catalyzer.

The hydroxyl oxidize enzyme generally is present in the leafy green plants or mammalian cell, the catalyzed oxidation oxyacetic acid generates Glyoxylic acid hydrate, and produce hydrogen peroxide simultaneously, Tao Baite (N.E.Tolbert) is at " J.Biol.Chem. " Vol.181, at first reported a kind of enzyme 905-914(1949), it gets by extracting in the tobacco leaf, it can form formic acid and carbonic acid gas by the intermediate catalyzed oxidation oxyacetic acid that generates Glyoxylic acid hydrate, and add some specific compounds, as quadrol, just can limit the continuation oxidation of intermediate product Glyoxylic acid hydrate, oxidation approximately is to carry out under PH8, the oxyacetic acid concentration that the typical case uses is about 3-40mm, and best hydroxyl oxidize pH value is 8.9.And it is reported that the oxalic acid of 100mm can suppress the katalysis of hydroxyl oxidize enzyme.Similarly, Li Chasen (K.E.Richardson) and Tao Baite are at " J.Biol.Chem. " Vol 236, report 1280-1284(1961), tris buffer can suppress the formation of oxalic acid in hydroxyl oxidize enzymatic oxidation oxyacetic acid; Gram Lay Gothic (C.O.Clagett), Tao Baite and Bai Li, (K.H.Burris) at " J.Biol.Chem. " Vol178, report 977-887(1949), the hydroxyl oxidize enzyme is 7.8～8.6 with the optimum PH value range of oxygen catalyzed oxidation oxyacetic acid, optimum temperature range is 35 ℃～40 ℃;

Cha Like (I.Zelitch) He Aoke (S.Ochoa) is at " J.Biol.Chem. " Vol 707-718(1953) in and Luo Binxun (J.C.Robinson etc. are at " J.Biol.Chem. " Vol 237, report 2001-2009(1962), in spinach oxyacetic acid oxidation catalysis process, the generation of formic acid and carbonic acid gas is because non-enzyme reaction hydrogen peroxide and Glyoxylic acid hydrate reaction cause.They find, add a kind of catalase that can make hydrogen peroxide degradation, can improve the output of Glyoxylic acid hydrate by the generation that suppresses formic acid and carbonic acid gas greatly.Add the FMN(vitamin B2 phosphate) also can improve the stability of hydroxyl oxidize enzyme greatly.

Good fortune league (unit of length) (N.A.Frigerio) and Haber (H.A.Harbury) are at " J.Biol.Chem. " Vol 231, the preparation and the character of relevant glycollate oxidase have been reported 135-157(1958), and find that sublimed enzyme is very unstable in solution, and think this unstable be since FMN is connected with zymophore power too a little less than and the tetramer of enzymatic activity or octamer is degraded into the inactive single polymers of enzyme catalysis or the double focusing thing causes, and formed irreversible cohesion and precipitation.In enzyme liquid, add FMN and can improve its activity.And high density albumen or high ionic strength can keep enzyme to be tetramer or octamer form.In fact, the bibliographical information that the oxyacetic acid catalyzed oxidation generates Glyoxylic acid hydrate is a lot, for example:

The separation of enzyme (generally including a kind of analytical procedure)

I.Zelitch in Methods of Enzymology.Vol.1, Academic Press, New York, 1955, p.528-532, from the spinach tobacco leaf

M.Nishimura et al., Arch.Biochem.Biophys., Vol.222,397-402(1983), from pumpkin, pig ear

H.Asker and D.Davies, Biochim.Biophys.Acta, Vol.761,103-108(1983), from the mouse liver

M.J.Emes and K.H.Erismann, Int.J.Biochem., Vol.16,1373-1378(1984), from lemon

Enzymatic structure

E.Cederlund et al.，Eur.J.Biochem.，Vol.173，523-530（1988）.

Y.Lindquist and C.Branden，J.Diol.Chem.Vol.264，3624-3628，（1989）：

The present invention relates to the production method of a kind of Glyoxylic acid hydrate (OCHCOOH), this method is oxyacetic acid (HOCH ₂COOH) (the about 2500mM of 200-) is at hydroxyl oxidize enzyme ((S)-2-hydroxy acid oxidase, ECl.1.3.15) and catalase (EC1.11.1.6) be fixed under the situation on the insoluble carrier, (PH7-10) and oxygen reaction in the aqueous solution, under the top condition, can obtain the Glyoxylic acid hydrate of high yield, the oxyacetic acid transformation efficiency is very high simultaneously.And immobilized enzyme can be regenerated and utilization again.

The invention describes a kind of preparation of immobilized enzyme and utilize it to produce Glyoxylic acid hydrate from oxyacetic acid, though oxyacetic acid and oxidase catalyzed reaction are for many years known, but never obtained 99% highly selective, the oxidation operation concentration that does not also have oxyacetic acid is in the low scope of 0.20～2.5M.Previous co-applications " U.S.S.N, 07/422,011, Oct.16 submits to, 1989, produces Glyoxylic acid hydrate from oxyacetic acid " method that a kind of enzyme catalysis conversion of hydroxymethyl guanidine-acetic acid generates Glyoxylic acid hydrate has been described.It is under the aerobic situation, carries out in ammonia damping fluid and hydroxyl oxidize enzyme and the catalase liquid.This method has shown a kind of unexpected use catalase (destroying the by product hydrogen peroxide) and the synergistic effect of ammonia damping fluid, thereby more effectively formed the chemical adducts (having limited its further oxidation) with Glyoxylic acid hydrate, this article is incorporated this paper as a reference into.No matter be to add catalase separately or the ammonia damping fluid all can not produce the highly selective that both add simultaneously, productive rate is also any also high a lot of than independent adding simultaneously.The present invention has done improvement under aforesaid method, promptly used immobilized enzyme as catalyzer.

The lytic enzyme that utilizes of previous report had many problems as the method for catalyzer, for example, and the regeneration of catalyzer and do not reuse operation so easily; The stability of catalyzer is also not as immobilized enzyme, and lytic enzyme also less stable under drum oxygen (thereby the solubleness that improves oxygen improves speed of response) situation in reactant.Developed a kind of Preparation of catalysts method now, it comprises simultaneously hydroxyl oxidize enzyme (for example: separate from spinach or beet or from the commercial channel acquisition) (for example: commercial available oxyethane vinylformic acid bead) is fixed on same solid carrier with these two kinds of enzymes of catalase (for example: separate also and can obtain from the commercial channel) from aspergillus soap stock, structure nest aspergillus or cereuisiae fermentum or beef liver.As catalyzer following advantage is arranged with this immobilized enzyme:

1. after reaction finishes, immobilized enzyme regeneration and utilizing again from reaction mixture easily, and lytic enzyme regeneration is very difficult, and easy inactivation.

2. immobilized enzyme is than stable many of lytic enzyme.Compare with lytic enzyme, no matter be the catalyzer turnover ratio that obtains or after the reaction or store back gained activity for a long time in damping fluid, immobilized enzyme is all much higher.

3. more importantly, the stability of immobilized enzyme is not subjected to increasing the influence that dissolved oxygen and speed of response rouses the reaction conditions of oxygen, and equally under the situation, lytic enzyme can very fast inactivation.

To certain specific enzyme, there is not a kind of selectable process for fixation can expect that its immobilization is successful, and, above a kind of co-immobilization of enzyme, just more cannot predict its success.Therefore, this area the professional it has been generally acknowledged that, wants a kind of enzyme of successfully immobilization, must use diverse ways to attempt, and optimal results often wants repetition test to obtain.For the hydroxyl oxidize enzyme, there is not the report of relevant immobilization trial aspect.The immobilization of enzyme can be with different technology, comprise 1) by covalently bound, physical adsorption, static connects or affine connection is attached to enzyme on the carrier, 2) and difunctional or poly functional reagent is crosslinked, 3) be embedded in the film of agar, polymkeric substance, latex or other kind 4) combination of aforesaid method.The detailed description of relevant process for fixation and influence the reference that factor that process for fixation selects can be consulted following data and this paper:

Methods in Enzymology，K.Mosbach（ed.），Academic Press，New York：Vol.44（1976），Vol.135（1987），Vol.136（1987），Vol.137（1988），and the references

With different process for fixation fix the hydroxyl oxidize enzyme, the optimum of these methods all shows in an embodiment.Covalently bound to oxyethane vinylformic acid bead (Ewpergit C) proteolytic enzyme, the crosslinked acrylamide/N-propenyloxy group succinimide copolymer gel (PAN-500) of the agarose of cyanogen bromide-activated and triethylamine can both produce active immobilized enzyme.As XAO-8 resin and phenyl sepharose are arrived in physical adsorption.Also very successful with the method that ion is connected on the various carriers, as what attempted protein cross is arrived glutaraldehyde, dimethyl adipoyl diamines, dimethyl-octa two acid diamides or 1-4 butanediol diglycidyl ether etc.In these different immobilized active hydroxyl oxidize enzymes, have only the enzyme on the oxyethane vinylformic acid bead to use as the catalyzer of oxyacetic acid oxidation.Enzyme in the physical adsorption, in the reaction mixture of PH9.0, the concentration of oxyacetic acid be 0.75m and quadrol concentration when being 0.79M with regard to desorb, and the agar of cyanogen bromide-activated and reacting ethylenediamine discharge the covalent attachment enzyme once more, enter reaction mixture.The enzymic activity that is attached on the PAN-500 is too low, so that can not use as catalyzer in real reaction.

Immobilization is combined in oxyethane vinylformic acid bead Eupergit C and Eupergit C-250L(120hm Pharma) on the hydroxyl oxidize enzyme very stable under reaction conditions, and active (enzyme activity unit/gram catalyzer) is very high, in actual procedure of great use.Catalase also is fixed on the oxyethane vinylformic acid bead, and these two kinds are separated immobilized catalyzer and use together, perhaps two kinds of enzymes are fixed on the identical carrier together, is added to (a kind of method in back is better) in the reaction mixture again.

Adopt the method for immobilized enzyme catalysis agent to eliminate a lot of weak points of lytic enzyme, in water buffered soln, immobilization hydroxyl oxidize enzyme than lytic enzyme stable many (stable enzymes) near ammonium sulfate precipitation.Simple filtration is with regard to recyclable these immobilized catalysts from reaction solution.Adding fresh reactant liquid more just can re-use.In this way, the oxidasic turnover ratio of immobilization (being the mole number that every mol catalyst institute energy conversion of substrate becomes product before the enzyme deactivation) can be up to 10 ⁷(mol/mol).Because drum oxygen can not make the enzyme catalyst inactivation in reaction solution, thereby can make speed of response increase at least 10 trainings, thereby reduce the production cost of this processing method significantly by adding big drum oxygen amount.

This immobilized hydroxyl oxidize enzyme must use in an effective concentration range in reaction, is generally 0.001～10.0IU/ml, best 0.1～4IU/ml." IU " (international unit) is defined as the amount that per minute catalysis one micromole's substrate conversion becomes the required enzyme of product.The analytical procedure of this kind of enzyme can find in following document:

I.Zelitch and S.Ochoa，J.Biot.Chem.Vol.201，707-718（1953）

That this method also is used for analyzing recovery or round-robin hydroxyl oxidize enzymic activity.

The pH value of reaction solution should be in the 7-10 scope, and preferably at 8.0-9.5, because enzymic activity becomes with pH value, we can keep the solution pH value with damping fluid.When reaction was carried out, the pH value of reaction descended slightly, and PH is near the most significant end of enzymic activity PH scope when beginning so react usually, and approximately 9.0-9.5 allows it during reaction descend.As what before in " U.S.S.N.07/422; 011; filed October 16 1989 ", described, adopt a kind of energy and Glyoxylic acid hydrate to be combined into the ammonia damping fluid of mixture (forming a kind of) and catalase together, can improve product selectivity to greatest extent chemistry or the more stable imonium of oxydasis.Some trihydroxymethylaminomethane (hereinafter being designated as TRIS) of quadrol or weak effect, piperazine, or the N-glycylglycine can both improve the output of Glyoxylic acid hydrate.These amine are generally 1.0-3.0, best 1.05-1.33 to the employed ratio of every mole of oxyacetic acid (initial concentration).In this scope, can accurately regulate pH value in desired scope.When amine own is too high to the ratio of oxyacetic acid, be necessary to regulate pH value, can adopt to add sour example hydrochloric acid or vitriolic method, when with the lower amine of alkalescence,, be necessary to adopt the method that adds alkali to keep required pH value as TRIS.

The concentration of immobilized catalyst should be at 50-100000IU/ml, and the best is at 350-14000IU/ml.Preferably enzyme is fixed together jointly and joins catalytic amount in the reaction, and catalase and hydroxyl oxidize determining alcohol can be adjusted in the above-mentioned scope so that the ratio of catalase and hydroxyl oxidize enzyme (is unit with IU) was at least about 250: 1 with restriction.FMN is best adding composition, and concentration is 0.0-2.0mM, and the best is 0.01-0.2mM.

Speed of response is controlled by the dissolved oxygen amount part in the water medium at least.Can add oxygen by the method that adds air, but preferably use pure relatively oxygen, and pressurization.Though the upper limit that oxygen is pressed does not know that also can use 50 normal atmosphere, preferably 15 normal atmosphere are as the upper limit.For keeping high dissolved oxygen rate (thereby keeping reaction), drum oxygen is necessary in reactant.Drum oxygen speed is oxygen (normal atmosphere is measured down)/every volumetric reaction mixture of 0.05-5 volume.Per minute and be preferably in 0.2-2Vol/Vol.min.In addition, simple agitation is essential.

Temperature of reaction is a very important variable, and it influences the stability of speed of response and enzyme.Temperature of reaction can be at 0 ℃-40 ℃, but the best is 5 ℃-15 ℃.Operate in this optimum range, reaction finishes back enzymic activity rate of recovery maximum.

Can remove enzyme catalyst by filtration or centrifugation method after reaction finishes, the amine damping fluid can be removed by ion-exchange-resin process easily.The acidic cation-exchange resin that is fit to has " AMBERLITE " CG120 or AMBERLITE " IR120(Rohm ﹠ Haas Co.) and " DOWEX " 50(Dow Chemical Co.).And then reclaim and recycle amine with the highly basic process resin.

The product Glyoxylic acid hydrate is used in the preparation of Vanillin and vanillal, equally also be used in ion exchange resion and in pharmaceutical industries as an acidic catalyst.General it with the 50%(weight percent) the aqueous solution sell.Need to prove that the Glyoxylic acid hydrate in these are used also can refer to the Glyoxylic acid hydrate negatively charged ion, especially when the height of the pH value in the solution 2.3 the time.

Purification of hydroxy oxydase from spinach

Hydroxyl oxidize enzyme in the spinach can add that the DEAE Mierocrystalline cellulose criticizes adsorbing and extracting with classification sulphur ammonium precipitation.A kind of method in back is based on all outer vegetable-proteins of DEAE cellulose adsorption hydroxyl-removal oxydase.Except as otherwise noted, all purification process temperature are all carried out under 4 ℃.At 25 ℃, the fresh spinach of 2 bushels (16Kg) is milled to fine particle with " Fitz Mill " masher that 0.5 inch mesh sieve is housed.Last juice liquid portion (about 6L) can separate by 4 layers of cheese cloth milling process.Also can use juice extraction device (Vitantonio) instead separates.The dithiothreitol (DTT) (ultimate density of 5mM) that adds 5.6 grams is regulated pH value to 5.2 with the acetate of 5-20mL 20% then in this liquid.Hatch through 10 minutes, final mixture is at 4 ℃, GS-3 rotary head (Sorvall), under the 13000g centrifugal 25 minutes.Abandon throw out, supernatant liquor is regulated pH value to 7.5-8.0(Zelitch with the 6N potassium hydroxide of 15～20ml, I.Ochoa, and S., J.Biol.Chem., Vol, 201,707(1953); Frigerio, N.A., Harbury, H.A., J.Biol.Chem., Vol.231,135(1958)). then supernatant liquor (about 5.5L) with the 100000MW(molecular weight is housed) diaphragm-operated Pelicon(Millipore) ultra-fine filter concentrates 5 times; Final enriched material volume approximately is 1.1 liters.More than 10 minutes, slowly add the ammonium sulfate of 154 grams in concentrated solution again, behind the sulphur ammonium CL, precipitation was removed with 13000g in centrifugal 25 minutes.Abandon precipitation, in supernatant liquor (about 1.1L), add 77 gram ammonium sulfate, the same centrifugal, collect albumen precipitation, supernatant liquor is abandoned (Zelitch(1953); Frigerio(1958)).

Albumen precipitation is dissolved in 20mM N-two (hydroxyethyl) glycine buffer (PH8.0) of about 200ml, with Spectroporz dialysis tube (1200-14000 MWCO) 20mM N-two (hydroxyethyl) glycine that 4L contains 2mM FMN was permeated protein liquid 16 hours, leading relevant protein liquid electricity with the conductivity meter survey with fresh N-two (hydroxyethyl) glycine buffer electricity leads, if reading does not wait, then continued the infiltration protein liquid 4 hours, and ditto detected then.Protein liquid after the dialysis (about 250ml) is contained in the beaker with magnetic force or top mechanical stirring, adds 25 gram preswollen DEAZ resins (Sigma) (Kerr.M.W., Groves, D., phytochemistry, Vol.14,359-362(1975)) then.Mixture was hatched 10 minutes, and the available Bradford analysis of protein of the reduction of protein concentration method detects in the solution, thus the absorption of monitoring albumen on resin.When protein concentration in the supernatant liquor drops to tracking level (0.2mg/ml), do not have bonded albumen with vacuum filtration process by a Whatman who is contained on the Buchner funnel of 13cm diameter ^#1 filtering table reclaims from mixture.For reclaiming enzyme to greatest extent, the resin cake washes with N-two (hydroxyethyl) glycine (PH8.0) of 100ml 20mM.Adding ultimate density in protein solution is the FMN(Sigma of 2mM), and then, stir the ammonium sulfate (approximately 400ml) in enzyme liquid that slowly add 240 grams more than 15 minutes, simultaneously, potassium hydroxide that must Dropwise 5 N is 8.0 to keep pH value.Last sedimentary hydroxyl oxygen people enzyme is stored in 4 ℃ of dark places until use.

Purifying catalase from the soft tommy yeast

All purification process are all carried out under 4 ℃.Fresh bread yeast (11b, General Foods Corporation-Red Star) is suspended in contains the 1mM phenylmethyl sulfonylfluoride (PMSF is in the Tris damping fluid (PH7.5) of 450ml 20mM Sigma).Getting 200ml Yeast protein suspension, to transfer to a capacity be 400ml, be equipped with in the homogenizer of 200ml granulated glass sphere (0.5mm diameter biological product), mixed continuously through 5 minutes, lysate is transferred to (in ice) in the receiving vessel, remaining yeast suspension is handled the same.

Cell debris can pass through at 4 ℃, 13000g, GS-3 rotary head (Sorvoall) centrifugal 45 minutes-1 hour down.Collect supernatant liquor (400ml) and add 90.4 gram solid ammonium sulfates, hatched on ice 10 minutes to reach 40% degree of unsaturation, again at 4 ℃, 13000g, GSA rotary head (Sorvall) centrifugal 25 minutes down.Abandon throw out, the solid ammonium sulfates that add 48 grams in the supernatant liquor (400ml) are to reach final 60% saturation ratio (Seach, T.C.M., Daplan, J.G.J.Biol.Chem.Vol.218, No.8.2880(1973)).Mixture is with preincubation and centrifugal, and final albumen precipitation is dissolved in the 20mM TRIS(PH7.5 of a minimum volume) in the liquid.Use Spectropor 2 dialysis tubes, protein solution is by the TRIS(PH7.5 of 20mM) dialysis; Discard dialyzate after 16 hours, and the new TRIS liquid of replacement, continue dialysis 4 hours, reclaim dialysis albumen (100mL) then.

Get above-mentioned albumen dialyzate 50ml and join warp-wise and flow and 100ml Q Sepharose to be housed to wander about as a refugee fast in sub-exchange resin (Pharmacia) chromatographic column (Sepragen), be provided with the TRIS(PH7.5 of the albumen of absorption with 20mM) with 10ml/min.Speed flows out.Flow out the flow assay cell monitoring that 280nm strainer (LKB) be housed of albumen with the connection chart recording instrument; Every 10-15ml collects the post effluent liquid with the LKB Fraction Collector.When all do not have conjugated protein outflow post after, with 20mM TRIS liquid (PH7.5) linear gradient elution that contains 0～500mM NaCl of 400ml, flow velocity 10ml/min, every part enzyme activity assay are used in the method that superoxide disappears under the 240nm and detect.Collection has the part of catalase activity, adds ammonium sulfate matter ultimate density and reaches 80% saturation ratio, 4 ℃ of storages of final sedimentary catalase.

This method equally also is applicable to purifying catalase from structure nest aspergillus and aspergillus soap stock.Be fixed on hydroxyl oxidize enzyme and catalase activity analysis on the oxyethane vinylformic acid bead

Be fixed on the hydroxyl oxidize enzyme on the oxyethane vinylformic acid bead, available laxative remedy is analyzed active: the bead that the about 5-10mg of weighing handled joins in the quartz curette that is placed with magnetic stirring bar of 3ml, add 2.0mL then and contain 2 of 0.12mM, the 80mM Tris damping fluid (PH8.3) of 6-dichlorobenzene indophenols.Cover quartz curette with a rubber septum,, and then add the oxyacetic acid of 40mL 1.0M/1.0MTRis(PH8.3) in quartz curette, stir, simultaneously at 605nm(e=22000 with syringe with bubble oxygen denitrification 5 minutes) measure absorbancy down and change.

The hydrogen peroxide method of analyzing enzyme is as follows: the bead that accurate weighing 2-5mg handled joins in the quartz curette of magnetic stirring bar, add the distilled water of 2.0mL and the 50mM phosphoric acid buffer (PH20) that 1.0mL contains the 59mM hydrogen peroxide then, and at 240nm(e=39.4) absorbancy measured over time down.Usually, immobilized hydroxyl oxidize enzyme and catalatic activity are respectively 6IU/ gram bead and 6000IU/ gram bead.

High pressure liquid chromatography (HPLC) is analyzed oxyacetic acid, Glyoxylic acid hydrate.

Oxalic acid and formic acid

Analytic sample is prepared as follows: the 0.1N sulfuric acid of the reaction mixture of 0.100mL and 0.300mL is mixed, filter by Millipore Ultrafre MC strainer (isolating 10000Mw) then.(300 * 7.8mm) 40 ℃ are analyzed oxyacetic acid, Glyoxylic acid hydrate, oxalic acid and formic acid is 0.01N sulfuric acid and 0.1mM1-hydroxyl 2 alkane-1 as solvent with a Bio-Rad Aminex HPX-87H post by HPLC, the aqueous solution of 1-bisphosphate, flow velocity is that the 1.0ml/min. instrument is a water 840HPLC system, it is with 510 type pumps, 712WISP self-actuated sampler and 490E UV(ultraviolet correspondingly) detector and 410 differential refractometers.The retention time of oxalic acid, Glyoxylic acid hydrate, oxyacetic acid, formic acid and propionic acid (international standard) is respectively 4.29,6.09,7.07,8.79 and 11.41 minutes.

Embodiment one

The immobilization of hydroxyl oxidize enzyme

To different carriers

Polymine (PEI), the PEI on silica gel, the benzyl PEI on silica gel, Bio-Rex760, CHSepharose 4B, XAD-4, XAD-8, the benzene agarose, Eupergit C, Eupergit C-250L and Eupergit C-30N can both obtain from the commercial channel.Preparation PAN-500) the crosslinked acrylamide/N-propenyloxy group succinic diamide copolymer gel of triethylamine) and with it fixes the hydroxyl oxidize enzyme, method can be according to the step of describing in " Pollack, A etc. be at J.Am.Chem.Soc., 1980; 102,6324-6336 ".Utilize the protein-bonded carrier of physical adsorption for those, the immobilization operation can be used the water damping fluid washing of PH5-10 earlier, scheduled time in 5 ℃ or the 25 ℃ buffered soln that carrier are immersed in enzyme then, do not have bonded albumen with fresh buffer solution for cleaning carrier 3-4 time with removal again, analyze the hydroxyl oxidize enzymic activity of carrier.For the carrier that connects with glutaraldehyde method, repeat aforesaid method, just before adding enzyme, carrier is handled with 5% glutaraldehyde water solution.A kind of detailed with Eupergit do carrier fixedly the method for hydroxyl oxidize enzyme in example 2, provide, the hydroxyl oxidize enzyme is as shown in the table to the immobilization rate under the different carriers top condition, and they are based on the activity of whole enzyme in per step.

The active mode of connection of carrier immobilized rate

PEI O O ionic adsorption

PEI/ glutaraldehyde 00 is covalently bound

PEI-silica gel/glutaraldehyde 00 is covalently bound

Bio-Rex70 00 ionic adsorption

CH Sepharose 4B 00 ionic adsorption

20 5.0IU/mL are covalently bound for CNBr-agar

19 0.14IU/mL are covalently bound for PAN-500 glue

XAD-4 00 physical adsorptions

XAD-8 4 0.76IU/gr physical adsorptions

00 physical adsorptions of PEI-silica gel/benzene

The 3 0.62IU/gr physical adsorptions of phenyl agar

EupergitC 17 7.8IU/gr are covalently bound

Eupergit C-250L 8 5.0IU/gr are covalently bound

Embodiment two

While immobilization hydroxyl oxidize enzyme and catalase on Eupergit C

The oxyethane vinylformic acid bead (EupergitC) of 10.0g of weighing is put into the erlenmeyer flask of a 125ml, add the about 75mL of FMN solution that contains 50mm glycine buffer (PH8.0) and 0.02mM then, sway flask, oxyethane acryllic acid bead is suspended.After bead deposits to drag, float the fine particle of staying on the mixture top and remove, and under the situation of not disturbance bottom bead, remove supernatant liquor as much as possible simultaneously with transfer pipet.Repeated washing once.

Is the activity with the ammonium sulfate precipitation gained that hydroxyl oxidize enzyme (separating from fresh spinach) the mixed solution 100mL of 327IU is at 12000rpm(Sorvall, the GSA rotary head, 4 ℃) centrifugal 20 minutes, abandoning supernatant, the cake piece is with containing 50mM glycine buffer (PH8.0) and 0.02mMFMN damping fluid 50mL dissolving.With ammonium sulfate precipitation isolating 100mg catalase (Sigma(-3515 from the aspergillus soap stock, mixed solution 10mL 715000Iu) is at 15000rpm(Sorvall SS-34 rotor) centrifugal 10 minutes, abandoning supernatant, the cake piece dissolves with the oxidasic damping fluid of above-mentioned hydroxyl, then this kind of enzyme liquid is joined in the oxyethane vinylformic acid bead flask that has cleaned, regulate final volume 125mL with damping fluid.Again this mixed solution is transferred to～polypropylene vial with cover of 250mL in, then this bottle is placed on one bottle of vibrator 15 ℃ of 4～5rpm vibrations 16 hours down.Again mixture is poured in the chromatographic column that the porous support bed is housed, let things run their course.With this immobilized enzyme of glycine/FMN buffer solution for cleaning of 30mL three times, in 5 ℃ of same damping fluids, preserve then.The co-immobilization enzyme catalyst of Huo Deing like this, the activity of hydroxyl oxidize enzyme are that 7.2IU/ gram EupergitC and catalase activity are 5680IU/ gram Eupergit C.

Embodiment three

The hydroxyl oxidize enzyme stability of lytic enzyme and respective fixation enzyme

Do not have fixed (dissolved) hydroxyl oxidize enzyme can be stored in enzyme acquisition in the damping fluid (PH8.0) that contains 2.0mMFMN under 4 ℃ by measurement, detect enzymic activity over time the stability that is fixed on the hydroxyl oxidize enzyme on the oxyethane vinylformic acid bead (Eupergit C).In addition, also monitored the enzyme stability that in 3.2M ammonium sulfate and 2.0mM FMN liquid, precipitates and store under the same conditions under the same conditions.The result is as follows:

1 day 2 day 6 months March of the enzyme rate of recovery

Under the dissolving 50% 000

Immobilized 92% 95% 50% 50%

Sedimentary 100% 100% 95% 85%

Embodiment four

The enzyme reaction of drum oxygen co-immobilization

In the long glass column that 20mm polyethylene bed carrier is housed of a 2.5cm internal diameter * 20cm, put into hydroxyl acetate (0.25M), quadrol (0.33M), propionic acid (0.075M, HPLC internal standard substance) and FMN(0.2mM) liquid 10ml.Pillar and content are cooled to 15 ℃, add spinach hydroxyl oxidize enzyme (2.5IU) and the co-immobilization enzyme of aspergillus soap stock catalase (27000IU) on Euperigt C then.From porous support bed drum oxygen, each fixed interval is got the reaction mixture monitoring of 100ml five equilibrium with 10ml/min speed.Method is that the 0.1N sulfuric acid of like this separatory and 300mL is mixed with termination reaction, filters like this separatory and uses high pressure liquid chromatographic analysis.5.5 after hour, the productive rate of Glyoxylic acid hydrate, oxalic acid and formic acid is respectively 98%, 2% and 0%.Correspondingly oxyacetic acid all transforms.Final hydroxyl oxidize enzyme and catalatic activity are respectively initial 95% and 65%.

The comparative example one

The reaction of drum oxygen lytic enzyme

Repeat the reaction of embodiment four, that just uses same quantity instead does not have immobilized hydroxyl oxidize enzyme and a catalase.After 4 hours, the productive rate of Glyoxylic acid hydrate, oxalic acid and formic acid is respectively 43%, 0% and 0%, and correspondingly the transformation efficiency of oxyacetic acid is 46%, final hydroxyl oxidize enzyme and catalatic activity are respectively initial 2% and 82% correspondingly, when prolonging reaction with not observed further reaction.

Embodiment five

The independent immobilized enzyme reaction of drum oxygen

Repeat the reaction of embodiment four, with hydroxyl acetate (0.75M), quadrol (0.86M), propionic acid (0.075M, the MPLC internal standard substance), FMN 0.2mM liquid 10mL is fixed on the hydroxyl oxidize enzyme (2.5IU) on the EupergitC and is fixed on aspergillus soap stock catalase (1400IU) on another Eupergit C.After 20 hours, the productive rate of Glyoxylic acid hydrate, oxalic acid and formic acid is respectively 99%, 0.2% and 0.5%.Correspondingly, the oxyacetic acid transformation efficiency is 100%, and hydroxyl oxidize enzyme and catalatic final activity are initial 72% and 66%.

Embodiment six

Co-immobilization enzyme fixed-bed reactor

In a gas lift formula biochemical reactor, put into the following mixed solution of 400ml: oxyacetic acid 0.75M, quadrol 0.86M, propionic acid (mark in the HPL C) 0.075M, FMN 0.01M(PH9.0).Wet oxygen passes through the solution bubbling in biochemical reactor, with a peristaltic pump from the circulate solution (40ml/min) of oxygen enrichmentization of the chromatographic column of biochemical reactor by a jacket layer 1 cm x internal diameter 30cm, be equipped with in the chromatographic column contain spinach hydroxyl oxidize enzyme (13.9Iu) and aspergillus soap stock catalase (56000Iu) co-immobilization at Eupergit C bead (21ml fixed bed volume).Last biochemical reactor inclusion and jacket layer chromatographic column remain on 15 ℃ with-10 ℃ of refrigeration circulating device for shower by 50: 50 glycol/water of interlayer circulation, and after 377 hours, the productive rate of Glyoxylic acid hydrate, oxalic acid and formic acid is respectively 93%, 0% and 0.3%.Correspondingly the transformation efficiency of oxyacetic acid is 94%.Hydroxyl oxidize enzyme and catalatic final activity are initial 48% and 69%.

Embodiment seven

In an autoclave reactor that stirs, the solution that 100ml contains following material is housed in the stirred autoclave of a 300ml EZE sealing: oxyacetic acid 0.75M with co-immobilization hydroxyl oxidize enzyme/hydrogen peroxide oxydasis oxyacetic acid, quadrol 0.86M(PH9.0), propionic acid (HPLC internal standard substance) 0.075M, FMN0.01mM solution is cooled to 15 ℃, in reactor, add co-immobilization then the spinach hydroxyl oxidize enzyme of 89IU and the aspergillus soap stock catalase Eupergit C bead (about 28 grams) of 72600IU.15 ℃, 500rpm stirs.At 70Psig(48Kpa) oxygen depresses by mixture with 100ml/min speed drum oxygen.Reaction monitoring mixes with 300mL 0.1N sulfuric acid with the quenching reaction for regularly getting 100 μ l reactant five equilibriums, filters five equilibrium, analyzes with HPLC.After 3 hours, the productive rate of oxyacetic acid, oxalic acid and formic acid is respectively 100%, 0% and 0%, and correspondingly oxyacetic acid transforms fully.Hydroxyl oxidize enzyme and catalatic final activity are respectively initial 100% and 100%.

Embodiment eight

In an autoclave reactor that stirs, reclaim and utilize again the hydroxyl oxidize enzyme/catalase of co-immobilization.

It is as follows to reclaim the immobilized enzyme method in the reaction of describing from embodiment seven: by 2.5cm internal diameter * 20cm long 20mm polyethylene support bed glass column filter reaction mixture is housed, the residual liquid that is adsorbed on the catalyzer removes by a nitrogen gas stream in post momently, then compound is suspended in the fresh solution of 15 ℃ of 100ml, this solution hydroxyl acetate 0.75M, quadrol 0.86M, propionic acid 0.075M(HPLC internal standard substance, and FMN 0.01mM.They are reinstalled the autoclave reactor of 300ml, so reaction repeats again.This catalyst recovery method by operate continuously 10 batches, and reaction times, the productive rate of hydroxyl oxidize enzyme (G.O) and the catalatic rate of recovery and Glyoxylic acid hydrate is as shown in the table

Glyoxylic acid hydrate

The round # time (hour) G.O. (%) catalase (%) acid (%)

1 3 100 100 100

2 2 100 100 96

3 2 98 70 97

4 2 64 100 100

5 2 80 92 100

6 2 77 77 98

7 2.5 78 96 100

8 2.5 75 100 100

9 3 77 89 100

10 3 85 100 93

Embodiment nine

The speed of response of oxyacetic acid under the oxidation of drum oxygenase in stirring autoclave

The solution that 100ml contains following material is housed: oxyacetic acid 0.75M, quadrol 0.86M(PH9.0 in the autoclave that-300ml EZE sealing is stirred), propionic acid (HPLC internal standard substance) 0.075M and FMN0.01mM.Solution is cooled to 15 ℃.In still, add then co-immobilization on about 15 gram EupergitC 14Iu spinach hydroxyl oxidize enzyme and the aspergillus soap stock catalase of 42800Iu.15 ℃, 400rpm stirs, the oxygen of drum oxygen press be respectively 35,70,105 and 140PSig(242,483,724 and 965KPa), gas speed is 50ml/min.Monitoring method is with experiment four, and with respect to above-mentioned oxygen pressure, the speed of response of oxyacetic acid should be 0.48,0.54,0.53 and 0.57mmol/min mutually.

The comparative example 2

Do not rouse oxydasis speed of response under the oxygen

Repeat embodiment nine, just do not rouse oxygen in the reaction solution.Oxygen press be 35,70 and 105psig under, the speed of response of oxyacetic acid is respectively 0.032,0.053 and 0.071mmol/min.

Embodiment ten

Bread yeast and immobilization hydroxyl oxidize oxydasis oxyacetic acid with infiltration

Repeat example seven and eight, just the spinach hydroxyl oxidize enzyme of 50IU is fixed on the Eupergit C of about 15 grams, and the fresh beer yeast (bread yeast, Red Star trade mark, General Foods Corporation) of 4 grams are with the Virahol infiltration and contain the 100000Iu catalase activity.They are used as catalyzer.Mixture stirs under 15 ℃, 400rpm, and oxygen is pressed 70 PSig(483Kpa).Drum oxygen speed is 20ml/min.The yield results example table of large quantities of successive reaction time, hydroxyl oxidize enzyme (G.0) and the catalase rate of recovery and Glyoxylic acid hydrate is as follows:

Glyoxylic acid hydrate

The round # time (hour) G.O. (%) catalase (%) acid (%)

1 2.5 81 62 100

2 3 97 30 100

3 3 73 54 100

4 3 51 53 96

5 4 49 57 98

6 6 24 45 97

Embodiment 11

The rate of oxidation of oxyacetic acid is to the dependence of drum oxygen speed

Repeat embodiment seven, use is immobilized in the aspergillus soap stock catalase bead reaction mixture of the spinach hydroxyl oxidize enzyme of the 52Iu on about 18 gram Eupergit C and 95000IU at 70Psig(48KPa) oxygen press and 15 ℃ under, rotating speed with 500rpm stirs, and drum oxygen speed is 5-50ml/min.The rate of oxidation of oxyacetic acid is as shown in the table under difference drum oxygen speed.

MLO ₂/ min mmolO ₂/ min mmol oxyacetic acid/min

5 0.40 0.22

10 0.57 0.45

15 0.70 0.67

20 0.79 0.89

25 0.78 1.11

30 0.99 1.34

50 1.10 2.23

Embodiment 12

The rate of oxidation of oxyacetic acid is to the dependence of the stirring velocity of autoclave.

Repeat embodiment seven, with spinach hydroxyl oxidize enzyme and the 47000IU aspergillus soap stock catalase of 50IU co-immobilization on about 15g Eupergit C, the stirring velocity of reactant is 100-500rpm, and 15 ℃, 70psig(483KPa) oxygen is pressed.Drum oxygen speed is 20ml/min, and oxyacetic acid is as follows with the oxidation rate of rotation speed change:

Rpm mmol oxyacetic acid/min

100 0.08

200 0.15

300 0.22

400 0.43

500 0.45

Embodiment 13

The oxidation rate of oxyacetic acid is to the dependence of hydroxyl oxidize enzyme concn

Repeat embodiment seven, with co-immobilization hydroxyl oxidize enzyme and catalase, the former is respectively 80,60 at activity, and 40 and 20IU, correspondingly the latter is 1400000,1000000,70000 or 35000IU.15 ℃ of 400rpm stir, and oxygen is pressed 70psig.Drum oxygen speed 20ml/min.As shown in the table with the different oxidation rates of hydroxyl oxidize enzyme concn.

The hydroxyl oxidize enzyme

(IU/mL) mmol Glyoxylic acid hydrate/min

0.2 0.20

0.4 0.30

0.6 0.36

0.8 0.57

Above we describe and have enumerated the present invention quite particularly.Should, following claim is not a limited range, and just makes great efforts to propose the scope of the invention.

Claims

1, a kind of production method of oxyacetic acid, it comprises: in the PH7-10 aqueous solution, be immobilized in by hydroxyl oxidize enzyme, catalase in the presence of the catalyzer that constitutes on the insoluble carrier, oxyacetic acid is contacted with oxygen, wherein, the starting point concentration of oxyacetic acid is 200mm-2500mM, a kind of amine damping fluid that can form chemical adducts with Glyoxylic acid hydrate, and the initial molar ratio of amine and oxyacetic acid is 1.0-3.0.

2, the process of claim 1 wherein hydroxyl oxidize enzyme and catalase by co-immobilization on same insoluble carrier.

3, the process of claim 1 wherein that immobilized hydroxyl oxidize enzymic activity is 0.01-10.0IU/mL.

4, the method for claim 3, wherein immobilized catalase activity is 50-1000000IU/mL.

5, the method for claim 4 wherein is reflected under 0 ℃-40 ℃ and carries out.

6, the method for claim 5, wherein oxygen is pressed and is the 1-50 normal atmosphere.

7, the method for claim 6, the activity of wherein immobilized hydroxyl oxidize enzyme are 0.1-41U/mL.

8, the method for claim 7, wherein immobilized hydrogen peroxide activity is 350-14000IU/mL.

9, the method for claim 8 wherein is reflected under 5-15 ℃ and carries out.

10, the method for claim 9, wherein the amine damping fluid is selected from quadrol, trihydroxymethylaminomethane, piperazine, N-glycylglycine and their mixture.

11, the method for claim 10, wherein hydroxyl oxidize enzyme and catalase are immobilized on the oxyethane vinylformic acid bead by covalently bound.

12, the process of claim 1 wherein that immobilized catalase and immobilized hydroxyl oxidize activity ratio (is that unit measures with IU) were at least 250: 1.

13, the process of claim 1 wherein that the starting point concentration of FMN is 0-2.0mM.

14, the process of claim 1 wherein that the amine damping fluid is a quadrol.

15, the process of claim 1 wherein that the amine damping fluid is two (methylol) methylamine.

16, the process of claim 1 wherein that the amine damping fluid is a piperazine.

17, the process of claim 1 wherein that the amine damping fluid is the N-glycylglycine.