CN101307307A - A kind of glycolic acid oxidase preparation, preparation method and application - Google Patents

Abstract

The invention discloses a yellow clover glycolic acid oxidase preparation, a preparation method thereof and an application for preparing glyoxylic acid by oxidizing glycolic acid. The glycolic acid oxidase preparation is prepared by the steps of ammonium sulfate precipitation and freeze-drying from the juice of the plant Medicago sativa; the enzyme protein obtained by the ammonium sulfate precipitation can also be dissolved and dialyzed to remove salt, and uploaded on the magnetic nanoparticle material , the magnetic powder-immobilized glycolate oxidase preparation can be prepared. Glycolic acid oxidase is used as a biocatalyst to catalyze the limited oxidation of glycolic acid to glyoxylic acid under mild conditions. When the substrate concentration is 50-1000 mM, the yield of glyoxylic acid can reach up to 98.9%. If the glycolate oxidase immobilized by magnetic powder is used as a catalyst, the immobilized enzyme can be easily separated from the reaction system by the action of a magnetic field after the reaction is finished, and can be used repeatedly.

Description

A kind of glycolic acid oxidase preparation, preparation method and application

technical field

The invention belongs to the field of biochemical industry, and relates to a Medicago clover glycolate oxidase preparation, a preparation method and its application for catalyzing the oxidation of glycolic acid to produce glyoxylic acid.

Background technique

Glyoxylic acid (GA), also known as diglycolic acid and formylformic acid, is the simplest form of aldehyde acid. The molecule contains two functional groups, aldehyde and carboxyl, so it has the dual characteristics of aldehyde and carboxylic acid. , can react with aldehydes and acids at the same time, and sometimes cyclization reactions can occur, and it is an important intermediate in organic synthesis. Starting from glyoxylic acid, dozens of fine chemical products with a wide range of uses can be derived, which are widely used in spices, medicine, pesticides, food additives and other industries. With the expansion of the application scope of glyoxylic acid and the development of subsequent products, its market capacity continues to increase, and higher requirements are put forward for the quality of products. Accelerating the research and development of glyoxylic acid and its series products is of great significance to the development of my country's glyoxylic acid industry.

The synthesis methods of glyoxylic acid include chemical synthesis and biosynthesis. The most commonly used chemical synthesis methods in industry are electrolytic reduction of oxalic acid, nitric acid oxidation of glyoxal, and ozonation of maleic acid (anhydride). Generally speaking, the chemical synthesis method is complex in process, high in production cost, large in energy consumption, and has relatively serious environmental pollution. Using glycolic acid as a substrate and using glycolic acid oxidase as a biocatalyst to biosynthesize glycolic acid into glyoxylic acid is a research hotspot in the production of glyoxylic acid. Compared with chemical synthesis, it has low energy consumption, It has unique advantages such as less by-products, less environmental pollution, and relatively simple subsequent separation of products. With the continuous strengthening of environmental protection measures, it is an inevitable trend to synthesize glyoxylic acid through biological methods.

So far, in the study of biosynthesizing glyoxylic acid, the enzyme preparation used is mainly spinach glycolate oxidase recombined by genetic engineering. For example, document J.Org.Chem., 1993,58:2253-2259 report: U.S. DuPont Company uses free spinach glycolic acid oxidase as catalyst, in the presence of additives such as catalase and FMN, ethylenediamine, catalyzes Glyoxylic acid is oxidized to form glyoxylic acid. Due to the poor stability of the free spinach glycolate oxidase, the reaction needs to be carried out at low temperature (15° C.), and the free enzyme cannot be reused. After the reaction is completed, the free glycolate oxidase is denatured and precipitated by heating the reaction liquid, and the product glyoxylic acid can be extracted from the supernatant after centrifuging to remove the enzyme precipitate.

Later, DuPont directly used recombinant microbial cells containing enzymes as catalysts to catalyze the oxidation of glycolic acid to synthesize glyoxylic acid [J.Org.Chem., 1995, 60: 3957-3963]. After the reaction, the whole cell catalyst can be recycled reuse. Due to the serious cell wall permeability barrier, the whole cell catalyst must be permeabilized with chemical reagents before use. Considering the stability of the enzyme, the reaction still needs to be carried out at low temperature (5°C).

The low temperature reaction requires additional energy consumption. In order to improve the temperature stability of the enzyme, it is necessary to modify or immobilize the enzyme. The existing patent report (David L.Anton, et al.US Patent 005,439,813A) of the immobilization of spinach glycolate oxidase, the carrier that selects for use is epoxy resin, and the recovery rate of enzyme activity is low, only 17%, and commercialization Epoxy resins are expensive and not suitable for mass production applications.

Aiming at the above limitations, the present invention finds through extensive screening that there is a high-activity glycolate oxidase in the plant Alfalfa sativa (commonly known as "grass head"). The glycolate oxidase of this plant has not been reported in the literature. Compared with spinach glycolate oxidase, Medicago clover glycolate oxidase has excellent performance, sufficient raw materials, easy enzyme extraction, and is a good biocatalyst for biosynthesis of glyoxylate; we have further developed a simple, low-cost and high-efficiency Efficient enzyme immobilization method, using self-prepared nano-magnetic powder materials, can easily realize the immobilization of glycolate oxidase by simple physical adsorption method; after the enzyme-catalyzed reaction is completed, it can be easily immobilized by using a magnet The immobilized enzyme is recovered and reused, the process is very simple, and the economic cost is relatively low.

Contents of the invention

The object of the invention is to provide a kind of Medicago clover glycolate oxidase preparation;

The object of the present invention also provides a kind of preparation method of above-mentioned Medicago clover glycolate oxidase preparation;

Another object of the present invention is to provide a kind of above-mentioned Medicago clover glycolate oxidase preparation.

The glycolic acid oxidase preparation of the invention is a glycolic acid oxidase that can catalyze the oxidation of glycolic acid to generate glyoxylic acid, which is obtained by extracting from the broken liquid of alfalfa plants by ammonium sulfate precipitation. The isoelectric point pI of the glycolic acid oxidase is >9.0, the optimum pH and reaction temperature of the reaction are 9.0 and 15°C respectively, and the apparent Michaelis constants K _m and V _max are 0.138mmol/L and 0.173mmol· min ^-1 /g protein.

According to the feature that glycolate oxidase is a key enzyme in the photorespiration pathway of higher plants, the inventors compared the content and activity of glycolate oxidase in various _C3 plants with strong photorespiration.

Glycolic acid oxidase screening method of the present invention is briefly described as follows:

(1) Preliminary screening: Weigh fresh leaves of different types of _C3 plants with the same weight, add pre-cooled buffer solution, grind to obtain plant cell crushing liquid, and measure glycolate oxidase activity and protein content in the crushing liquid.

(2) Re-screening: Select several plants with relatively high activity of glycolate oxidase for detailed investigation. The crushed plant cell solution obtained by grinding is taken, and glycolic acid is added to carry out the conversion reaction, and the amount of products and by-products in the reaction process is monitored by ion chromatography. Finally, plants with high product content and low by-product content were selected as the new source of glycolate oxidase.

The activity of free glycolate oxidase in the plant cell disruption liquid was determined by the following method:

Take 2.5ml of K ₂ HPO ₄ -KH ₂ PO ₄ buffer solution (100mM, pH 8.0) preheated at 30°C, put it in a quartz cuvette with an optical path of 1.0cm, add 0.3ml of phenylhydrazine hydrochloride solution (50mM , pH8.0) and 0.1ml of enzyme solution, mixed well and used as a blank, set to zero at 324nm. Add 0.1ml of glycolic acid solution (100mM, pH 8.0), mix immediately to start the reaction, start timing when the reading changes, and record the change in absorbance value at intervals of 30s. Under the above reaction conditions, the amount of catalyst (enzyme) required to change the absorbance value by 0.1 unit within 1 minute is defined as one enzyme activity unit (1U).

The activity of immobilized glycolate oxidase was measured by the following method:

Weigh a certain amount of immobilized enzyme, place it in a test tube, add 2ml of Tris-HCl buffer containing 50mM glycolic acid (pH9.0, 100mM, containing 0.1mM flavin mononucleotide FMN ), 30°C, 160rpm after 30min reaction, sample 100μL, dilute the reaction supernatant to an appropriate multiple, take 0.4ml and place it in a test tube, add 0.2ml of 1% 3-methyl-2-benzothiazolone hydrazone (referred to as MBTH) solution, after shaking well, keep it at 30°C for 10min, then add 2.5ml of ferric chloride solution (0.2%, w/v), shake it up, keep it at 30°C for 30min, and place it on the ultraviolet-visible spectrophotometer at 610nm. Measure the absorbance. 0.4ml of glycine-HCl buffer solution (100mM, pH4.0) plus 0.2ml of 1% MBTH solution and 2.5ml of 0.2% ferric chloride solution (incubated for the same time) were used as blank control. Calculate the molar amount of glyoxylic acid produced by the reaction according to the absorbance value, and then convert it into the enzyme activity determined by the phenylhydrazine hydrochloride method according to the corresponding relationship between the two.

Through repeated screening and comparison, it was finally found that Medicago sativa was an ideal new enzyme source for glycolate oxidase.

The preparation method of the enzyme preparation of the present invention is to use the fresh leaves of Medicago clover as the enzyme source, perform ammonium sulfate fractional precipitation after cell crushing, buffer soaking, and extraction, and then freeze-dry after adding an appropriate amount of lactose as a protective agent in the enzyme protein precipitation The crude enzyme preparation of glycolate oxidase can be obtained. The weight ratio of the crushed supernatant of the fresh leaves of Medicago clover and ammonium sulfate is 1: (0.1～0.6); the weight ratio of protein and lactose is 1: (0.5～2); the difference between protein and crude enzyme powder preparation is that the latter is A mixture of egg whites and lactose.

The buffer mentioned here refers to phosphate buffer or Tris-HCl buffer, and the pH of the buffer is 6-9. Said Tris represents trishydroxymethylaminomethane.

On this basis, the ammonium sulfate-precipitated crude enzyme protein preparation is further dissolved and dialyzed to remove salt, then magnetic nanoparticles are added, and the enzyme is immobilized by physical adsorption to obtain a magnetic powder-immobilized glycolate oxidase preparation. The mass ratio of the enzyme protein to the carrier is 1: (10-100), the immobilization temperature is 15°C-30°C, and the immobilization time is 12-24 hours. The weight ratio of the protein to the dialysis buffer is (1-5):1000; the dialysis time is 5-12 hours; the dialysis membrane pore size is 12000-14000Da.

The magnetic nanoparticles mentioned here are aminated magnetic powder particles prepared by hydrothermal synthesis. The preparation method is: mix 1,6-hexanediamine, anhydrous sodium acetate and FeCl ₃ 6H ₂ O in a certain proportion , dissolved in ethylene glycol, wherein the content of 1,6-hexanediamine is 8-12% (w/v), the content of anhydrous sodium acetate is 9-15% (w/v), FeCl ₃ ·6H The content of ₂ O is 2 to 5 (w/v). Transfer the mixed solution to a high-pressure reactor, and place it in a high-temperature environment of 170-250°C for 4-10 hours before taking it out. The black precipitate formed in the reactor is the required magnetic nano-particles. Wash with ethanol repeatedly, dry, and set aside.

The technique of preparing glyoxylic acid by catalyzing the oxidation reaction of glycolic acid with glycolic acid oxidase preparation is as follows:

Mix glycolic acid solution, ethylenediamine solution and buffer solution with pH adjusted in advance, add catalase, flavin mononucleotide FMN and glycolic acid oxidase preparation prepared as above, and react at constant temperature. The concentration of substrate glycolic acid is 50mM～1000mM; the molar ratio of ethylenediamine and glycolic acid is (1～1.2): 1; the consumption of glycolic acid oxidase preparation is 10～100U/mol glycolic acid; The concentration is (1～10)×10 ⁴ U/ml; the concentration of FMN is 0.01～0.1mM; the pH of the reaction solution is 7.0～10.0; the reaction temperature is 10～35°C, and the reaction time is 0.2～72h.

Description of drawings

Fig. 1 Medicago clover glycolate oxidase crude enzyme powder catalyzes glycolic acid oxidation reaction process;

Fig. 2 The batch reaction of the production of glyoxylic acid by the oxidation of glycolic acid catalyzed by the magnetic powder immobilized enzyme preparation.

In Fig. 1, (●): glycolic acid; (○): glyoxylic acid; (▲): formic acid; (□): oxalic acid.

Detailed ways

The following examples will help to understand the present invention, but do not limit the content and scope of the present invention.

The preparation of embodiment 1 Medicago clover glycolate oxidase

Weigh 100g of fresh alfalfa, cool to 4°C, add 140ml of pre-cooled potassium phosphate buffer (100mM, pH8.0), add a small amount of quartz sand to aid research, and filter with four layers of gauze. Centrifuge at 8000rpm for 8min, the volume of the supernatant is about 200ml, put it in an ice bath, stir with low-speed magnetic force, slowly add 9.35g of finely ground ammonium sulfate powder, let it stand for 30min, centrifuge at 13000rpm for 8min, take the supernatant, about 170ml, then slowly add 9.52g Grind fine ammonium sulfate powder, let it stand for 30 minutes, centrifuge at 13000rpm for 8 minutes, take the precipitate, add 5g of lactose powder as a freeze-drying protection agent, freeze-dry to make a crude enzyme preparation, the activity is about 297U/g, and store at 4°C.

The preparation of embodiment 2 magnetic nanomaterials

Weigh 3.6g of 1,6-hexamethylenediamine, 4.0g of anhydrous sodium acetate, 1.0g of FeCl ₃ 6H ₂ O, put them in a conical flask, add 30ml of ethylene glycol, make it miscible under magnetic stirring, and form The transparent liquid was transferred to a high-pressure reaction kettle, and it was taken out after standing for 6 hours at 200°C. There was a black precipitate at the bottom of the kettle, which was the required magnetic nanoparticles, and the obtained magnetic powder was washed with hot water and ethanol (3 times). Granules, wash off residual solvent and 1,6-hexanediamine, dry at 50°C, and set aside.

Example 3 Magnetic Nanomaterials Immobilized Glycolate Oxidase

Weigh 500mg of magnetic powder, put it in a 100ml round bottom glass flask, add 50ml of Tris-HCl buffer solution (pH9.0, 100mM, containing 0.1mM FMN), ultrasonically suspend the magnetic powder, then add 1.0g of glycolic acid oxidase crude enzyme powder (about 297 U), stirred slowly at 15°C and 160 rpm for 24 hours, sucked the obtained immobilized enzyme to the bottom of the container with a magnet, and removed the supernatant with a pipette. The obtained immobilized enzyme was repeatedly washed with 100 ml Tris-HCl buffer solution (pH 9.0, 100 mM) containing 0.1 mMFN until no protein was detected in the washing solution, and stored at 4°C. The immobilized enzyme activity is about 214U/g.

Embodiment 4 oxidase preparation catalyzes the conversion of glycolic acid

Weigh 0.4g of crude enzyme powder and dissolve it in 4ml of potassium phosphate buffer (100mM, pH8.0), add 0.1g of catalase (about 26800U); then add 1ml of 525mM ethylenediamine solution (KPB diluted, hydrochloric acid adjusted pH to 8.0), and 5ml of 100mM glycolic acid solution (diluted with KPB, adjusted to pH 8.0 with NaOH), reacted at 15°C for 5h, the results are shown in Figure 1, the conversion rate of glycolic acid reached 88.7%, and the concentration of product glyoxylic acid Up to 43.3mmol/L.

Embodiment 5 Magnetic powder immobilized enzyme catalyzes the transformation of glycolic acid

Add Tris-HCl buffer solution (pH9.0, 500mM, containing 0.1mMFMN), glycolic acid, ethylenediamine (molar ratio to glycolic acid is 1.05:1), 50mg catalase (1.31 ×10 ⁶ IU) and immobilized glycolate oxidase, the total volume of the reaction solution is 10ml. 30°C, 200rpm constant temperature shaking reaction, using the MBTH method to regularly detect the product concentration, until the product concentration no longer increases, the results are shown in Table 1.

Table 1 The conversion reaction of glycolic acid catalyzed by magnetic powder immobilized enzyme

Embodiment 6 immobilized enzyme produces the batch reaction of glyoxylic acid

Add 50ml of Tris-HCl buffer solution (pH9.0, 100mM, containing 0.1mMFMN) containing glycolic acid 100mM, ethylenediamine 102mM, catalase 50mg (1.31×10 ⁶ IU) into a round bottom flask, 107U of fixed Glycolate oxidase was synthesized, incubated in a water bath at 30°C, stirred at a constant speed of 200rpm, and the concentration of the product was detected by MBTH method. After the substrate glycolic acid was completely converted, the immobilized enzyme was absorbed at the bottom of the reactor with a magnet, and the immobilized enzyme was washed with Tris-HCl buffer (pH 9.0, 100 mM, containing 0.1 mM FMN) after removing the reaction supernatant ( 2×100ml), and then add fresh buffer solution containing substrate, and start the reaction again. The results are shown in Figure 2. After four batches of reactions, after about 70 hours, the amount of glyoxylic acid obtained was 19.79 mmol, and the yield was 98.9%. The residual activity of the immobilized enzyme was 70% of the initial activity, and the half-life of the immobilized enzyme was about 117h under the reaction conditions.

Claims (9)

1. A glycolic acid oxidase preparation, characterized in that: described glycolic acid oxidase is the glycolic acid oxidation that can catalyze the oxidation of glycolic acid to generate glyoxylic acid obtained by extracting from the broken liquid of Medicago clover plants by adopting the ammonium sulfate precipitation method enzyme. 2. Glycolic acid oxidase preparation as claimed in claim 1, is characterized in that: the isoelectric point pI of described glycolic acid oxidase>9.0, and the optimum pH of reaction and reaction temperature are respectively 9.0 and 15 ℃, table The apparent Michaelis constants K _m and V _max were 0.138mmol/L and 0.173mmol·min ^-1 /g protein, respectively. 3. a preparation method of glycolic acid oxidase preparation as claimed in claim 1, is characterized in that described glycolic acid oxidase adopts following (1) or (2) step to obtain the crude enzyme powder of glycolic acid oxidase respectively Preparation or magnetic powder immobilized enzyme preparation: (1) Glycolic acid oxidase crude enzyme powder preparation: Crush and centrifuge the leaves of fresh black alfalfa, collect the supernatant, slowly add ammonium sulfate powder until it is completely dissolved, continue to stir slowly for 30 minutes, collect the precipitated protein, add an appropriate amount of lactose as a freeze-drying protective agent, and freeze-dry to prepare the crude enzyme Powder preparation; the weight ratio of the supernatant to ammonium sulfate is 1: (0.1-0.6); the weight ratio of protein to lactose is 1: (0.5-2); (2) Magnetic powder immobilized glycolate oxidase preparation: Crush and centrifuge the leaves of fresh alfalfa, collect the supernatant, slowly add ammonium sulfate powder, collect the precipitated protein, dissolve it in a buffer solution with a pH of 6-9, dialyze to remove ammonium sulfate, and then add magnetic nanoparticles as immobilization The carrier is made of magnetic powder immobilized enzyme preparation by adsorption and washing; the weight ratio of the supernatant to ammonium sulfate is 1: (0.1-0.6); the weight ratio of protein to buffer is (1-5): 1000; The dialysis time is 5-12 hours; the dialysis membrane pore size is 12000-14000Da. 4. the preparation method of glycolic acid oxidase preparation as claimed in claim 3 is characterized in that when step (2) carries out ammonium sulfate precipitation enzyme, the amount of added ammonium sulfate is the degree of saturation of 10%～70%; The buffer is phosphate buffer or Tris-HCl buffer. 5. The method according to claim 3, characterized in that the magnetic nanoparticle immobilized carrier described in step (2) is the aminated magnetic nanoparticle prepared by hydrothermal synthesis; the weight ratio of enzyme protein to magnetic carrier The ratio is 1:(10-100), the immobilization temperature is 15°C-30°C, and the immobilization time is 12-24 hours. 6. The method according to claim 5, characterized in that the preparation method of said magnetic nanoparticles comprises the steps of: The content of 1,6-hexanediamine is 8-12% (w/v), the content of anhydrous sodium acetate is 9-15% (w/v) and the content of FeCl ₃ ·6H ₂ O is 2-5 % (w/v) ethylene glycol solution at 170-250° C. for 4-10 hours of standing reaction to generate black precipitated magnetic nanoparticles, which are washed repeatedly with hot water and ethanol in sequence, dried, and set aside. 7. A glycolic acid oxidase preparation as claimed in claim 1 is used to catalyze the oxidation of glycolic acid to prepare glyoxylic acid. 8. The application as claimed in claim 7, characterized in that: the preparation is: the glycolic acid solution whose pH is 7.0 to 10.0 at a pH of 50mM to 1000mM is added as described in claim 3 (1) or (2). The glycolate oxidase preparation is reacted at 10-35° C. for 0.2-72 hours; wherein, the amount of the glycolate oxidase preparation based on the substrate amount is 10-100 U/mol glycolic acid. 9. application as claimed in claim 8, is characterized in that: when preparing, add appropriate ethylenediamine, flavin mononucleotide and catalase, the mol ratio of ethylenediamine and glycolic acid is: (1～1.2 ): 1; the concentration of flavin mononucleotide is 0.01～0.1mM; the amount of catalase added is (1～10)×104U/ml.

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