CN101307307A - Glycolic acid oxidase preparation, preparation method and applications - Google Patents
Glycolic acid oxidase preparation, preparation method and applications Download PDFInfo
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- CN101307307A CN101307307A CNA2008100404989A CN200810040498A CN101307307A CN 101307307 A CN101307307 A CN 101307307A CN A2008100404989 A CNA2008100404989 A CN A2008100404989A CN 200810040498 A CN200810040498 A CN 200810040498A CN 101307307 A CN101307307 A CN 101307307A
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Abstract
The invention discloses a sickle alfalfa glycolic acid oxidase preparation, a preparation method thereof and the application of the preparation to preparing a glyoxylic acid through glycolic acid oxidation. The glycolic acid oxidase preparation is obtained through the steps of precipitating sickle alfalfa juice via ammonium sulfate, freezing, drying, etc. The glycolic acid oxidase preparation immobilized through magnetic powder can also be obtained through the steps of dissolving and dialyzing pheron obtained from ammonium sulfate precipitation for salt elimination, and uploading to magnetic nano granular material. The glycolic acid oxidase is used as a biocatalyst to catalyze the glycolic acid into a glyoxylic acid through limited oxidation. The yield of the glyoxylic acid reaches a highest value of 98.9 percent when the concentration of a substrate is 50-1000mM. If the glycolic acid oxidase immobilized through magnetic powder is used as a catalyst, the immobilized enzyme is every easy to be separated from a reaction system through the action of a magnetic field, thereby bringing about repeated use.
Description
Technical field
The invention belongs to biological chemical field, relate to bur clover glycolate oxidase preparation, preparation method and be used for the application that the catalysis ethanol acid oxidase is produced oxoethanoic acid.
Background technology
Oxoethanoic acid (Glyoxylic acid, GA), have another name called gyloxylic acid, formyl formic acid, be the simplest aldehydic acid, comprise two kinds of functional groups of aldehyde radical and carboxyl in the molecule, thereby have the dual nature of aldehyde and carboxylic acid concurrently, the reaction of aldehyde, acid can take place simultaneously, sometimes also cyclization can take place, be a kind of important organic synthesis intermediate.From oxoethanoic acid, can derive tens kinds of fine chemical products that extensive use is arranged, be widely used in the industries such as spices, medicine, agricultural chemicals, foodstuff additive.Along with the expansion of oxoethanoic acid range of application and the exploitation of successor, its market capacity continues to increase, to the higher requirement of quality proposition of product.Quicken development, exploitation oxoethanoic acid and series product thereof, the oxoethanoic acid industry that develops China is had great importance.
The synthetic method of oxoethanoic acid comprises chemical synthesis and biological synthesis process.Industrial the most frequently used chemical synthesis has acid by electrolytic reduction of oxalic method, oxalic dialdehyde nitric acid oxidation method, toxilic acid (acid anhydride) ozonization method.Generally speaking, the chemical synthesis complex process, the production cost height, energy consumption is big, and has than serious environmental and pollute.With the oxyacetic acid is substrate, with glycolate oxidase as biological catalyst, the biological synthesis process that carries out oxyacetic acid bio-transformation synthesizing glyoxalic acid is the research focus of produces tartronic now, compare with chemical synthesis, it is low to have energy consumption, distinct advantages such as by product is few, and environmental pollution is little, and the later separation of product is simple relatively.Along with the continuous reinforcement of environmental practice, be a kind of inexorable trend by Production of Glyoxylic Acid Using Microbial Methods.
Up to the present, in the research of Production of Glyoxylic Acid Using Microbial Methods, the zymin of use mainly is the spinach glycolate oxidase of genetically engineered reorganization.For example, document J.Org.Chem., 1993, the 58:2253-2259 report: du pont company uses free spinach glycolate oxidase as catalyzer, in the presence of additives such as catalase and FMN, quadrol, catalysis ethanol acid oxidase synthesizing glyoxalic acid.Because the less stable of free spinach glycolate oxidase, reaction need to carry out under low temperature (15 ℃), resolvase can't be reused.After reaction finishes, by to the reaction solution heat treated, make free glycolate oxidase sex change precipitation, the post precipitation of centrifugal removal enzyme could extract the product oxoethanoic acid from supernatant liquor.
Thereafter, E.I.Du Pont Company directly uses the recombinant microorganism cell that contains enzyme as catalyzer, catalysis ethanol acid oxidase synthesizing glyoxalic acid [J.Org.Chem., 1995,60:3957-3963], and after reaction finished, the intact cell catalysis agent can be reclaimed repeated use.Because have the more serious penetrating obstacle of cell walls, the intact cell catalysis agent must be carried out penetrating processing with chemical reagent before use, considers the stability of enzyme, reaction still needs to carry out under low temperature (5 ℃) condition.
Low-temp reaction needs the additive decrementation energy, in order to improve the temperature stability of enzyme, is necessary enzyme is transformed or carried out immobilization.The immobilization of spinach glycolate oxidase has patent report (David L.Anton, et al.US Patent 005,439,813A), the carrier of selecting for use is a Resins, epoxy, and enzymatic activity recovery is low, only be 17%, and commercial Resins, epoxy costs an arm and a leg, be unsuitable for large-scale production and application.
The present invention is directed to above limitation, by extensive screening, find to exist in the plant bur clover (being commonly called as " toothed burclover ") glycolate oxidase of high vigor, this plant GIycoIate Oxidase is not seen bibliographical information as yet.Compare with the spinach glycolate oxidase, bur clover glycolate oxidase excellent property, the raw material abundance, the extraction of enzyme is easy, is the good biological catalyzer of biosynthesizing oxoethanoic acid; We have also further developed a kind of easy, low-cost and high efficiency enzyme immobilization method, use the nano-magnetic powder material of preparation voluntarily, can realize the immobilization of glycolate oxidase at an easy rate by the simple physics adsorption method; After enzymic catalytic reaction is finished, to use magnet can carry out the recovery of immobilized enzyme at an easy rate, and reuse, technological process is very simple, and Financial cost is also relatively low.
Summary of the invention
The purpose of this invention is to provide a kind of bur clover glycolate oxidase preparation;
Purpose of the present invention also provides a kind of preparation method of above-mentioned bur clover glycolate oxidase preparation;
Another object of the present invention provides a kind of above-mentioned bur clover glycolate oxidase preparation.
But glycolate oxidase preparation of the present invention is to adopt ammonium sulfate precipitation method to extract the glycolate oxidase that the catalytic oxidation of alcohol acid that obtains generates oxoethanoic acid in the broken liquid of bur clover plant.The iso-electric point pI of described glycolate oxidase＞9.0, the optimal pH of reaction and temperature of reaction are respectively 9.0 and 15 ℃, apparent Michaelis-Menton constant K
mWith V
MaxBe respectively 0.138mmol/L and 0.173mmolmin
-1/ g albumen.
According to glycolate oxidase is this feature of key enzyme in the higher plant photorespiration approach, and the inventor has a C that breathes than high light to multiple
3Plant has carried out the comparison of glycolate oxidase content and vigor.
Glycolate oxidase screening method of the present invention is summarized as follows:
(1) primary dcreening operation: the different sorts C that takes by weighing identical weight
3The fresh blade of plant, the damping fluid of adding precooling grinds and obtains the broken liquid of vegetable cell, measures glycolate oxidase vigor and protein content in the broken liquid.
(2) multiple sieve: selected several glycolate oxidase vigor carry out detailed investigation than higher plant.Get and grind the broken liquid of the vegetable cell that obtains, add oxyacetic acid and carry out conversion reaction, the amount of product and by product in the use chromatography of ions monitoring reaction course.Select the low plant of product amount height, amount of by-products as new glycolate oxidase source at last.
Adopt following method to measure the vigor of free glycolate oxidase in the broken liquid of vegetable cell:
Get the K of 30 ℃ of preheatings of 2.5ml
2HPO
4-KH
2PO
4(100mM pH8.0), places the quartz colorimetric utensil of optical path 1.0cm to damping fluid, and (50mM pH8.0) and 0.1ml enzyme liquid, as blank, returns to zero at the 324nm place behind the mixing to add the phenylhydrazine hydrochloride solution of 0.3ml.The glycollic acid solution of adding 0.1ml (100mM, pH8.0), mixing begins reaction immediately, picks up counting when reading changes, and record 30s is the variable quantity of light absorption value at interval.Under above-mentioned reaction conditions, light absorption value changes 0.1 needed catalyzer of unit (enzyme) amount and is defined as an enzyme activity unit (1U) in 1 minute.
Adopt following method to measure the vigor of immobilization glycolate oxidase:
Take by weighing the immobilized enzyme of certain mass, place test tube, adding 2ml contains Tutofusin tris hydrochloric acid (Tris-HCl) damping fluid (pH9.0 of 50mM oxyacetic acid, 100mM, contain 0.1mM vitamin B2 phosphate FMN), 30 ℃, sampling 100 μ L behind the 160rpm reaction 30min will reacts after supernatant liquor dilutes suitable multiple, get 0.4ml and place test tube, 1%3-methyl-2-[4-morpholinodithio ketone hydrazone (the being called for short MBTH) solution that adds 0.2ml, shake up the back in 30 ℃ the insulation 10min, add then 2.5ml ferric chloride Solution (0.2%, w/v), shake up the back in 30 ℃ of insulation 30min, the 610nm place measures light absorption value on ultraviolet-visible spectrophotometer.(100mM pH4.0) adds the 1%MBTH solution of 0.2ml, 0.2% ferric chloride Solution of 2.5ml (being incubated the identical time) as blank with 0.4ml glycine-HCl damping fluid.Calculate the molar weight that reaction generates oxoethanoic acid according to light absorption value size, and then be scaled the enzyme activity that the phenylhydrazine hydrochloride method is measured according to the two corresponding relation.
By repeated screening, comparison, find that finally bur clover is the desirable new enzyme source of glycolate oxidase.
The preparation method of zymin of the present invention is to use the fresh blade of bur clover as the enzyme source; after soaking, extract, cytoclasis, damping fluid carry out ammonium sulfate precipitation; in zymoprotein precipitation, add an amount of lactose as protective material after, carry out the crude zyme preparation that lyophilize can obtain glycolate oxidase.The weight ratio of broken supernatant liquor of the fresh blade of bur clover and ammonium sulfate is 1: (0.1～0.6); The weight ratio of protein and lactose is 1: (0.5～2); The difference of protein and thick enzyme powder preparation is that the latter is the mixture of albumen and lactose.
Here said damping fluid is meant phosphate buffered saline buffer or Tris-HCl damping fluid, and pH of buffer is 6～9.Described Tris represents Tutofusin tris.
On this basis, further the thick zymoprotein preparation of ammonium sulfate precipitation is dissolved and the desalination of dialysing after, add magnetic nanoparticle, carry out the immobilization of enzyme by physical adsorption, can obtain magnetic immobilization glycolate oxidase preparation.The mass ratio of zymoprotein and carrier is 1: (10～100), immobilization temperature are 15 ℃～30 ℃, and the immobilization time is 12～24 hours.Described protein and dialysis buffer liquid weight ratio are (1～5): 1000; Dialysis time 5～12h; Dialysis membrane aperture 12000～14000Da.
Here said magnetic nanoparticle is the amination magnetic powder particle for preparing gained by hydrothermal synthesis method, and its preparation method is: with 1, and 6-hexanediamine, anhydrous sodium acetate and FeCl
36H
2O mixes by a certain percentage, is dissolved in the ethylene glycol, and wherein 1, the content of 6-hexanediamine is 8～12% (w/v), the content of anhydrous sodium acetate is 9～15% (w/v), FeCl
36H
2The content of O is 2～5 (w/v).Mixing solutions is transferred in the autoclave, and in 170～250 ℃ of hot environments, standing and reacting was taken out after 4～10 hours, and the black precipitate that generates in the reactor is required magnetic nanoparticle, uses hot water and ethanol repetitive scrubbing successively, and oven dry is standby.
Technology with glycolate oxidase preparation catalysis ethanol acid oxidase prepared in reaction oxoethanoic acid is as follows:
The glycollic acid solution, the ethylenediamine solution that regulate the pH value are in advance mixed the glycolate oxidase preparation that adds catalase, vitamin B2 phosphate FMN and prepare as mentioned above, isothermal reaction with damping fluid.The concentration of substrate oxyacetic acid is 50mM～1000mM; The mol ratio of quadrol and oxyacetic acid is (1～1.2): 1; The consumption of glycolate oxidase preparation is 10～100U/mol oxyacetic acid; Catalatic concentration is (1～10) * 10
4U/ml; The concentration of FMN is 0.01～0.1mM; Reaction solution pH is 7.0～10.0; Temperature of reaction is 10～35 ℃, and the reaction times is 0.2～72h.
Description of drawings
The thick enzyme powder of Fig. 1 bur clover glycolate oxidase catalysis ethanol acid oxidase reaction process;
Fig. 2 magnetic immobilized enzyme preparation catalysis ethanol acid oxidase is produced batch reaction of oxoethanoic acid.
Among Fig. 1, (●): oxyacetic acid; (zero): oxoethanoic acid; (▲): formic acid; (): oxalic acid.
Embodiment
Following embodiment will help to understand the present invention, but not limit content of the present invention and scope.
The preparation of embodiment 1 bur clover glycolate oxidase
Take by weighing the fresh bur clover of 100g, be cooled to 4 ℃, (100mM pH8.0), adds a small amount of quartz sand assistant research fellow, four layers of filtered through gauze to the potassium phosphate buffer of adding 140ml precooling.The centrifugal 8min of 8000rpm, the about 200ml of supernatant liquor volume, ice bath; the low speed magnetic agitation slowly adds the ammonium sulfate powder 9.35g of porphyrize, leaves standstill 30min; the centrifugal 8min of 13000rpm gets supernatant liquor, about 170ml; slowly add the ammonium sulfate powder of 9.52g porphyrize again, leave standstill 30min, the centrifugal 8min of 13000rpm; get precipitation, add the 5g lactose powder, make crude zyme preparation after the lyophilize as lyophilized vaccine; the about 297U/g of vigor, 4 ℃ of preservations.
The preparation of embodiment 2 magnetic Nano materials
Take by weighing 1,6-hexanediamine 3.6g, anhydrous sodium acetate 4.0g, FeCl
36H
2O 1.0g places Erlenmeyer flask, adds 30ml ethylene glycol, make it miscible under the magnetic agitation, form transparent liquid, be transferred in the autoclave, take out after 6 hours in 200 ℃ of standing and reacting, as seen black precipitate is arranged at the bottom of the still, be required magnetic nanoparticle, with the magnetic powder particle of hot water and ethanol cleaning (3 times) gained, the solvent that flush away is residual and 1, the 6-hexanediamine, 50 ℃ of oven dry, standby.
Embodiment 3 magnetic Nano material immobilization glycolate oxidases
Take by weighing the 500mg magnetic, place 100ml round bottom glass flask, add 50ml Tris-HCl damping fluid (pH9.0,100mM contains 0.1mM FMN), ultrasonic magnetic is suspended after, add the thick enzyme powder of 1.0g glycolate oxidase (about 297U), slowly stir 24h in 15 ℃, 160rpm, the immobilized enzyme that obtains is inhaled at container bottom, remove supernatant liquor with suction pipe with magnet.(pH9.0,100mM) immobilized enzyme that obtains of repetitive scrubbing detects less than till the albumen 4 ℃ of preservations to washings to contain the Tris-HCl buffered soln of 0.1mMFMN with 100ml.The about 214U/g of immobilized enzyme vigor.
The conversion of embodiment 4 oxidase preparation catalysis ethanol acid
Take by weighing the thick enzyme powder of 0.4g be dissolved in the 4ml potassium phosphate buffer (100mM, pH8.0) in, add 0.1g catalase (about 26800U); Ethylenediamine solution (the KPB dilution that adds 1ml 525mM again, hydrochloric acid is regulated pH to 8.0), and the glycollic acid solution of 5ml 100mM (KPB dilution, NaOH regulates pH to 8.0), 15 ℃ of reaction 5h, the result as shown in Figure 1, the transformation efficiency of oxyacetic acid reaches 88.7%, the concentration of product oxoethanoic acid reaches 43.3mmol/L.
The conversion of embodiment 5 magnetic immobilized enzyme catalysis oxyacetic acids
In the 50ml Erlenmeyer flask, add Tris-HCl damping fluid (pH9.0,500mM contain 0.1mMFMN), oxyacetic acid, quadrol (with the mol ratio of oxyacetic acid be 1.05: 1), 50mg catalase (1.31 * 10
6IU) and the immobilization glycolate oxidase, the reaction solution cumulative volume is 10ml.30 ℃, the reaction of 200rpm isothermal vibration uses the MBTH legal time to detect production concentration, till no longer increasing to production concentration, the results are shown in Table 1.
The conversion reaction of table 1 magnetic immobilized enzyme catalysis oxyacetic acid
Embodiment 6 immobilized enzyme are produced batch reaction of oxoethanoic acid
In round-bottomed flask, add 50ml and contain oxyacetic acid 100mM, quadrol 102mM, catalase 50mg (1.31 * 10
6IU) Tris-HCl damping fluid (pH9.0,100mM contain 0.1mMFMN), the immobilization glycolate oxidase of 107U, 30 ℃ of water bath heat preservations, 200rpm constant speed stirring reaction, the MBTH legal time is detected production concentration.After the substrate oxyacetic acid transforms fully, immobilized enzyme is inhaled at reactor bottom, removed after the reaction supernatant liquor with Tris-HCl damping fluid (pH9.0 with magnet, 100mM, contain 0.1mM FMN) and the washing immobilized enzyme (2 * 100ml), and then add the fresh buffer that contains substrate, begin reaction once more.The result as shown in Figure 2, through four batch reactions, after about 70 hours, obtaining product oxoethanoic acid amount is 19.79mmol, yield is 98.9%.The residual vigor of immobilized enzyme is 70% of an initial vigor, and the transformation period of immobilized enzyme is approximately 117h under this reaction conditions.
Claims (9)
1. glycolate oxidase preparation is characterized in that: but described glycolate oxidase is to adopt ammonium sulfate precipitation method to extract the glycolate oxidase that the catalytic oxidation of alcohol acid that obtains generates oxoethanoic acid in the broken liquid of bur clover plant.
2. glycolate oxidase preparation as claimed in claim 1 is characterized in that: the iso-electric point pI of described glycolate oxidase＞9.0, the optimal pH of reaction and temperature of reaction are respectively 9.0 and 15 ℃, apparent Michaelis-Menton constant K
mWith V
MaxBe respectively 0.138mmol/L and 0.173mmolmin
-1/ g albumen.
3. the preparation method of a glycolate oxidase preparation as claimed in claim 1 is characterized in that described glycolate oxidase adopts following (1) or (2) step to obtain the thick enzyme powder preparation or the magnetic immobilized enzyme preparation of glycolate oxidase respectively:
(1) the thick enzyme powder preparation of glycolate oxidase:
With fresh bur clover blade fragmentation, centrifugal, collect supernatant liquor, slowly add after the ammonium sulfate powder treats to dissolve fully, continue slowly to stir 30min, the protein of collecting precipitation adds an amount of lactose as lyophilized vaccine, makes thick enzyme powder preparation after the lyophilize; The weight ratio of described supernatant liquor and ammonium sulfate is 1: (0.1～0.6); The weight ratio of protein and lactose is 1: (0.5～2);
(2) magnetic immobilization glycolate oxidase preparation:
With fresh bur clover blade fragmentation, centrifugal, collect supernatant liquor, slowly add the ammonium sulfate powder, the protein of collecting precipitation, be dissolved in pH and be in 6～9 the damping fluid, ammonium sulfate is removed in dialysis, adds magnetic nanoparticle then as fixation support, makes magnetic immobilized enzyme preparation by absorption and washing; The weight ratio of described supernatant liquor and ammonium sulfate is 1: (0.1～0.6); Protein and damping fluid weight ratio are (1～5): 1000; Dialysis time 5～12h; Dialysis membrane aperture 12000～14000Da.
4. the preparation method of glycolate oxidase preparation as claimed in claim 3, when it is characterized in that step (2) is carried out the ammonium sulfate precipitation enzyme, add ammonium sulfate amount be 10%～70% saturation ratio; Described damping fluid is phosphate buffered saline buffer or Tris-HCl damping fluid.
5. method as claimed in claim 3 is characterized in that the magnetic nano particle immobilized carrier described in the step (2) is for preparing the amination magnetic nanoparticle of gained with hydrothermal synthesis method; The weight ratio of zymoprotein and magnetic carrier is 1: (10～100), immobilization temperature are 15 ℃～30 ℃, and the immobilization time is 12～24 hours.
6. method as claimed in claim 5 is characterized in that comprising the steps: the preparation method of described magnetic nanoparticle
With 1, the content of 6-hexanediamine is that the content of 8～12% (w/v), anhydrous sodium acetate is 9～15% (w/v) and FeCl
36H
2The content of O is the magnetic nanoparticle of the black precipitate that generates after 4～10 hours in 170～250 ℃ of standing and reacting of the ethylene glycol solution of 2～5% (w/v), uses hot water and ethanol repetitive scrubbing successively, and oven dry is standby.
7. a glycolate oxidase preparation as claimed in claim 1 is applied to catalytic oxidation of alcohol acid preparation oxoethanoic acid.
8. application as claimed in claim 7, it is characterized in that: described being prepared as: be 7.0～10.0 glycollic acid solution at the pH of 50mM～1000mM, add as claim 3 (1) or (2) described glycolate oxidase preparation, at 10～35 ℃ of reaction 0.2～72h; Wherein, the glycolate oxidase preparation usage quantity based on amount of substrate is 10～100U/mol oxyacetic acid.
9. application as claimed in claim 8 is characterized in that: add an amount of quadrol, vitamin B2 phosphate and catalase in the preparation, the mol ratio of quadrol and oxyacetic acid is: (1～1.2): 1; The concentration of vitamin B2 phosphate is 0.01～0.1mM; Catalatic addition is (1～10) * 104U/ml.
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| CN108949787A (en) * | 2018-07-05 | 2018-12-07 | 上海海洋大学 | A kind of goldfish Tgf2 transposase and its preparation and store method |
| CN109470637A (en) * | 2018-10-08 | 2019-03-15 | 浙江大学 | A method of measurement alcohol dehydrogenase activity |
| CN109988784A (en) * | 2019-04-16 | 2019-07-09 | 台州学院 | A kind of method of the oxidase catalyzed synthesis pyruvic acid of immobilization Glycolic acid |
| CN110452935A (en) * | 2019-08-28 | 2019-11-15 | 精晶药业股份有限公司 | A kind of method that enzyme process prepares glyoxalic acid |
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2008
- 2008-07-11 CN CNA2008100404989A patent/CN101307307A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949787A (en) * | 2018-07-05 | 2018-12-07 | 上海海洋大学 | A kind of goldfish Tgf2 transposase and its preparation and store method |
| CN109470637A (en) * | 2018-10-08 | 2019-03-15 | 浙江大学 | A method of measurement alcohol dehydrogenase activity |
| CN109470637B (en) * | 2018-10-08 | 2020-04-17 | 浙江大学 | Method for determining activity of ethanol dehydrogenase |
| CN109988784A (en) * | 2019-04-16 | 2019-07-09 | 台州学院 | A kind of method of the oxidase catalyzed synthesis pyruvic acid of immobilization Glycolic acid |
| CN110452935A (en) * | 2019-08-28 | 2019-11-15 | 精晶药业股份有限公司 | A kind of method that enzyme process prepares glyoxalic acid |
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Open date: 20081119 |