Method for extracting and purifying lignin peroxidase by using reverse micelles

A peroxidase and reverse micelle technology, which is applied in the field of extraction and purification of enzymes, can solve the problems of reducing the effectiveness of enzyme catalysis, hindering the progress of reverse micelle extraction and purification enzymes, and volatile inactivation of enzyme proteins, and achieves convenient operation. , The effect of short extraction time and high degree of purification

Inactive Publication Date: 2012-07-04
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the reverse micellar extraction technology is limited to the application of chemical surfactants. Commonly used chemically synthesized surfactants are prone to strong electrostatic interactions with the surface of enzyme proteins during enzyme-catalyzed reactions, which inactivates the variability of enzyme proteins, thereby reducing Efficiency of enzyme catalysis hinders progress in purification of enzymes by reverse micellar extraction

Method used

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  • Method for extracting and purifying lignin peroxidase by using reverse micelles
  • Method for extracting and purifying lignin peroxidase by using reverse micelles
  • Method for extracting and purifying lignin peroxidase by using reverse micelles

Examples

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Embodiment 1

[0026] A method for reverse micellar extraction and purification of lignin peroxidase of the present invention, specifically comprising the following steps:

[0027] (1) Preparation of reverse micelle solution: the reverse micelle solution of this example includes biosurfactant, isooctane and n-hexanol, wherein the surfactant is rhamnolipid; After cultivation, it was dissolved in a mixture of isooctane and n-hexanol with a volume ratio of 1:1, and prepared into a 40mL reverse micellar solution with a concentration of 2.75mmol / L (rhamnolipid-n-hexanol-isooctane solution ).

[0028] (2) Pre-extraction process: put 100mL of conventional liquid fermentation medium after autoclaving in a 500mL Erlenmeyer flask, and inoculate 4×10 8 Put the spores of Phanerochaete chrysosporium BKM-F-1767 (not limited to this strain, other Phanerochaete chrysosporium strains are acceptable) into the liquid fermentation medium, and place on a shaking table at a temperature of 37°C and a speed of 150...

Embodiment 2

[0035] A method for reverse micellar extraction and purification of lignin peroxidase of the present invention, specifically comprising the following steps:

[0036] (1) Preparation of reverse micelle solution: the reverse micelle solution of this example includes biosurfactant, isooctane and n-hexanol, wherein the surfactant is rhamnolipid; After cultivation, it was dissolved in a mixture of isooctane and n-hexanol with a volume ratio of 1:1.2, and prepared into a 40mL reverse micellar solution with a concentration of 3.0mmol / L (rhamnolipid-n-hexanol-isooctane solution ).

[0037] (2) Pre-extraction process: put 100mL of conventional liquid fermentation medium after autoclaving in a 500mL Erlenmeyer flask, and inoculate 5×10 8 Put the spores of Phanerochaete chrysosporium BKM-F-1767 (not limited to this strain, other Phanerochaete chrysosporium strains are acceptable) into the liquid fermentation medium, and place on a shaker at 38°C and 180r / min. Cultivate for 8 days, use ...

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Abstract

The invention discloses a method for extracting and purifying a lignin peroxidase by using reverse micelles. The method comprises the following steps of: preparing a reverse micelle solution containing a biosurfactant, isooctane and n-hexyl alcohol, wherein the biosurfactant is rhamnolipid; mixing a prepared crude solution of the lignin peroxidase with the reverse micelle solution, stirring, centrifuging, then, standing to carry out phase splitting, and distilling to obtain an upper organic phase; and mixing the upper organic phase with a buffer solution, stirring, centrifuging, then, standing to carry out phase splitting, distilling to obtain a lower water phase, i.e. a purified lignin peroxidase solution. The method disclosed by the invention has the advantages of environment-friendliness and safety, simplicity and convenience in operation, and low cost.

Description

technical field [0001] The invention relates to a method for extracting and purifying enzymes, in particular to a method for reverse micelles extracting and purifying lignin peroxidase. Background technique [0002] Lignin is a complex spherical aromatic polymer composed of phenylpropane structural units. Its difficulty in degradation slows down the speed of carbon cycle on the earth. Lignin peroxidase ( Lignin Peroxidase , LiP), manganese peroxidase ( Manganese Peroxidase , MnP) and laccase ( Laccase ) constitute the lignin degradation system, and studies have shown that LiP plays a key role in lignin degradation. This enzyme has a wide range of substrates and strong oxidizing ability. The use of this enzyme to degrade various pollutants has become a research hotspot in environmental pollution control in the world today. At present, the purification methods of LiP mostly use salting out precipitation, gel filtration chromatography, and ion exchange chromatography. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08
Inventor 袁兴中彭馨黄华军郭灵芝曾光明
Owner HUNAN UNIV
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