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Glycolic acid oxidase preparation, preparation method and applications

A glycolate oxidase, glycolic acid technology, applied in oxidoreductase, chemical industry, sustainable manufacturing/processing and other directions, can solve the problem that glycolate oxidase has not been reported in literature and so on

Inactive Publication Date: 2008-11-19
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the above limitations, and through extensive screening, it is found that there is a high-activity glycolate oxidase in the plant Alfalfa sativa (commonly known as "grass head"), and the glycolate oxidase of this plant has not been reported in the literature

Method used

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  • Glycolic acid oxidase preparation, preparation method and applications
  • Glycolic acid oxidase preparation, preparation method and applications
  • Glycolic acid oxidase preparation, preparation method and applications

Examples

Experimental program
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Embodiment 1

[0031] The preparation of embodiment 1 Medicago clover glycolate oxidase

[0032] 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.

Embodiment 2

[0033] The preparation of embodiment 2 magnetic nanomaterials

[0034] Weigh 3.6g of 1,6-hexanediamine, 4.0g of anhydrous sodium acetate, FeCl 3 ·6H 2 O 1.0g, put in a conical flask, add 30ml of ethylene glycol, make it miscible under magnetic stirring, form a transparent liquid, transfer it to a high-pressure reaction kettle, and take it out after standing at 200°C for 6 hours, and the bottom of the kettle can be seen There are black precipitates, which are the required magnetic nanoparticles. Wash the obtained magnetic powder particles with hot water and ethanol (3 times), wash away the residual solvent and 1,6-hexamethylenediamine, dry at 50°C, and set aside.

Embodiment 3

[0035] Example 3 Magnetic Nanomaterials Immobilized Glycolate Oxidase

[0036] Weigh 500mg of magnetic powder, place it in a 100ml round bottom glass flask, add 50ml of Tris-HCl buffer (pH9.0, 100mM, containing 0.1mM FMN), suspend the magnetic powder by ultrasonication, 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.

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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 [0001] 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 [0002] Glyoxylic acid (GA), also known as diglycolic acid and formylformic acid, is the simplest form of aldehyde acid. It contains two functional groups, aldehyde group and carboxyl group, 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 subs...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N11/14C12P7/40
CPCY02P20/50
Inventor 潘江朱虹胡彬许建和
Owner EAST CHINA UNIV OF SCI & TECH
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