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Novel method for extracting and purifying beer malt root phospholipase D

A technology of phospholipase and a new method, applied in biochemical equipment and methods, hydrolytic enzymes, pharmaceutical formulations, etc., can solve unseen problems and achieve the effect of high enzyme activity

Inactive Publication Date: 2009-05-20
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, although it has been reported (Zhang Yuhong, Wang Xuedong, Wang Li. Research on the Extraction Process of Compound Phosphatase. Shandong Pharmaceutical Industry, 2000, 19(13): 2), organic substances such as ethanol of phospholipase D in malt root can be used. Solvent crude extracts have been used to develop related drugs, but there is no report on the use of phospholipase D purified by affinity chromatography to develop related drugs

Method used

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  • Novel method for extracting and purifying beer malt root phospholipase D
  • Novel method for extracting and purifying beer malt root phospholipase D
  • Novel method for extracting and purifying beer malt root phospholipase D

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] : The crushing treatment of beer malt root

[0046] An appropriate amount of malt root is crushed through an 80-mesh sieve, then a small amount of quartz sand is added to the mortar for mechanical grinding for 15 minutes, and then compound enzymes (1.0% cellulase and 0.6% pectinase) are added at pH 5.5, 40 Under the condition of ℃, enzymatic hydrolysis for 4 hours to break the cell wall to obtain the sample to be separated and purified.

Embodiment 2

[0047] : Extraction and separation of phospholipase D

[0048] The sample after the wall breaking treatment was added with a material-to-water ratio of 1:11, extracted in water for 24 hours at 20°C and pH 5.5, centrifuged, the supernatant was taken, and 1:4 alcohol was added for precipitation. Leave it for 3 hours, centrifuge to remove the supernatant, and harvest the extract-like precipitate.

Embodiment 3

[0049] : Purification of Phospholipase D

[0050] The invention adopts epichlorohydrin to activate the carrier and the coupling ligand. Take Sepharose 4B with a precipitation volume of 10mL in a G2 funnel, filter it to semi-dry, first rinse with about 100mL 0.5mol / L NaCl solution, and then wash with 100mL distilled water to remove the protective agent and preservative therein. Drain about 6g semi-dry product, put it in a 50mL Erlenmeyer flask, add 6.5mL 2mol / L NaOH, 2mL epichlorohydrin and 15mL 56% dimethyl sulfoxide, mix well, shake gently at 40°C for 2 hours, then transfer the gel to the G2 funnel, rinse with distilled water to remove excess reagent, and finally use about 100mL 0.2mol / L Na 2 CO 3 Wash with pH 9.5 buffer. Conjugation experiments were then performed as soon as possible. Transfer the activated Sepharose 4B to the Erlenmeyer flask. With 10mL 0.2mol / L Na 2 CO 3 , 2 g of phosphatidylcholine was dissolved in a pH9.5 buffer, and then transferred to a Erlenmey...

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Abstract

The invention relates to a new method that is used for extracting, separating and purifying phospholipase D from beer malt roots, takes the raw material of beer malt root dregs, and implements wall breaking processing of cell structure through grinding, water extraction, and sedimentation of a supernatant fluid with a polar organic solvent; the purification adopts the affinity chromatography, uses Sepharose 4B for purifying components that contain target compounds, and adopts an affinity eluting solvent with the concentration of formic acid 0.1 mol per liter, the concentration of KCl 0.5 mol per liter and the pH value of 2.5; and distillation cut of protein peak is collected, and a fluid collection that is obtained by the affinity chromatography is dialyzed, refrigerated and dried in vacuum, thus obtaining dry powder. By measuring enzyme activity with high-efficiency liquid chromatogram, the phospholipase D achieves the purity of 96.7 percent. The phospholipase D can be used as raw materials for preparing high-purity phospholipase D medicaments, and has well development prospect.

Description

Technical field: [0001] The present invention relates to a new preparation method of phospholipase D (Phospholipase D) in beer malt root, in particular, relates to a new extraction and purification method of phospholipase D in beer malt root; in the application. Background technique: [0002] Barley is soaked, germinated, dried and rooted to obtain dry malt, and the by-product barley root accounts for about 3%-5% of the total feeding amount. There are hundreds of breweries and professional malt manufacturers in my country, and the annual output of malt roots is very large, reaching more than 100,000 tons. At present, the price of malt root is low, and it contains more valuable components, but there are few studies on malt root at home and abroad, and it has a slightly bitter taste, so there is almost no deep processing. For a long time, the malt root in my country has been directly used as feed, and the rest has been treated as garbage. Because it contains hordeine, there...

Claims

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Application Information

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IPC IPC(8): C12N9/16A61K38/46C12Q1/44
Inventor 贠建民韩丽毕阳
Owner GANSU AGRI UNIV
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