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Yeast-displayed polygalacturonase combined with ultrasonic treatment to extract laver polysaccharides

A technology of polygalactose and laver polysaccharides, which is applied in the field of bioengineering, can solve the problems of affecting the efficiency of laver cell crushing, less research on extracting laver polysaccharides, and inactivation of active substances, so as to achieve high product purity, short operation time, and extraction yield. high effect

Inactive Publication Date: 2011-12-21
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the chemical extraction method, the enzymatic hydrolysis method has the characteristics of simple operation, mild conditions, easy control, safety and reliability, and is currently widely used in the extraction and preparation of active substances, but the pure enzymatic extraction reaction speed is slow and the reaction time is long
Ultrasonic treatment of broken cells is also a commonly used method in the extraction of natural products and the preparation of active substances, but high-power, long-time ultrasonic treatment is likely to cause the inactivation of active substances; Polygalacturonic acid sulfate remains basically intact, only a small amount of voids are produced, which affects the efficiency of laver cell disruption, and polygalacturonic acid sulfate with too large molecular weight is insoluble in water, and it is difficult to separate from laver cell fragments
There are still few studies on the extraction of laver polysaccharides from laver by enzymatic hydrolysis and ultrasonic crushing

Method used

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  • Yeast-displayed polygalacturonase combined with ultrasonic treatment to extract laver polysaccharides
  • Yeast-displayed polygalacturonase combined with ultrasonic treatment to extract laver polysaccharides

Examples

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preparation example Construction

[0023] Preparation of Yeast Display Polygalacturonase

[0024] Synthesize the polygalacturonase gene (Genbank No.: HQ446162.1) and the cell wall α-lectin gene of Pichia pastoris GS115 (Genbank No.: M28164) by artificial synthesis. At the same time, polygalacturonase gene C The connecting peptide sequence GSSGGSGGSGGSGGSGS(linker) is added to the end, and the nucleotide sequence PGA-linker-α-agglutinin is obtained after connection, and EcoR I and Not I enzyme cutting sites are added at both ends of the sequence, wherein PGA is polygalacturonic acid Enzyme gene, α-agglutinin is cell wall α lectin gene.

[0025] Using the above artificially synthesized sequence as a template, PCR amplification was performed using the following primer pair,

[0026] Upstream primer: 5'-ATGTTGTTATCGACACACAGTATTGTTCTG-3';

[0027] Downstream primer: 5'-TTTTCCTTTTGCGGCCGCTAATGAAACG-3'

[0028] The PCR reaction system is: 1 μl of template DNA, 0.5 μl of high-fidelity DNA polymerase, 0.4 μl of dNTP ...

Embodiment 1

[0035] Example 1 Take 20 grams of laver dry powder with a particle size of 60 mesh and add it to 1000 grams of water, mix well, and prepare a suspension; then add 1 gram of yeast display polygalacturonase prepared according to the above method, and stir at 35°C React for 30 minutes with a stirring rate of 100 rpm; carry out ultrasonic crushing with an ultrasonic power of 730W, an ultrasonic treatment time of 65 minutes, and an ultrasonic treatment temperature of 30°C; centrifuge at 5000g for 10 minutes to remove the precipitate, and obtain the porphyra containing laver content. Supernatant: Add ammonium sulfate in the supernatant, make the concentration of ammonium sulfate in the supernatant reach 20% (W / V), leave standstill 12 hours in 4 ℃; Through 5000g centrifugation 10 minutes, remove precipitation, obtain containing A solution of laver polysaccharides.

[0036] The above method for determining the yield of laver polysaccharides was used to detect the extraction yield of l...

Embodiment 2

[0037] Example 2 Take 25 grams of laver dry powder with a particle size of 100 mesh and add it to 1000 grams of water, mix well, and prepare a suspension; then add 2 grams of yeast-displayed polygalacturonase prepared according to the above method, and stir at 37°C React for 45 minutes, the stirring rate is 200 rpm; carry out ultrasonic crushing, the ultrasonic power is 750W, the ultrasonic treatment time is 85 minutes, and the ultrasonic treatment temperature is 35°C; after centrifugation at 5000g for 10 minutes, the precipitate is removed to obtain the laver content. Supernatant: Add ammonium sulfate in the supernatant, make the concentration of ammonium sulfate in the supernatant reach 25% (W / V), leave standstill 12 hours in 4 ℃; Through 5000g centrifugal 10 minutes, remove precipitation, obtain containing A solution of laver polysaccharides.

[0038] The above method for determining the yield of laver polysaccharides was used to detect the extraction yield of laver polysac...

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Abstract

The invention discloses a method for extracting porphyra polysaccharide through ultrasonic treatment with cooperation of yeast display type polygalacturonase. The method comprises the following steps of: adding dry porphyra powder and yeast display type polygalacturonase to water, mixing uniformly, stirring to react at 35-37 DEG C for 30-45min, crushing at 28-35 DEG C for 1-2 hours by utilizing ultrasonic with power of 730-750W to obtain a cell disruption liquid, separating and purifying to obtain the porphyra polysaccharide. According to the method disclosed by the invention, and the polygalacturonase is displayed outside pichia pastoris cells and is cooperated with the ultrasonic to crush porphyra cells, thus the process is simple and convenient, the condition is gentle, the process is easy to be controlled and is safe and reliable, and higher-yield and higher-purity porphyra polysaccharide can be obtained.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for extracting laver polysaccharides through yeast-displayed polygalacturonase and ultrasonic treatment. Background technique [0002] Laver is rich in active polysaccharides composed of polygalacturonic acid sulfate, which exists in the cell wall of laver. Porphyra polysaccharides are rich in nutrients, not only have the physiological functions of general polysaccharides and dietary fiber, but also have various biological activities such as anticoagulant, blood lipid lowering, antimutation, antiaging and antiradiation. Since laver also contains a large amount of laver protein, which combines with laver polysaccharides and affects the separation of laver polysaccharides, for this reason, it is of great significance to study the extraction, separation and purification methods of laver polysaccharides in laver for subsequent research on its biological activity. [...

Claims

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Application Information

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IPC IPC(8): C08B37/00C12S3/02C12N15/55C12N15/81C12N9/16
Inventor 阮晖张延郭子堃徐娟周陈伟杜姗姗杨璐何国庆
Owner ZHEJIANG UNIV
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