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A method for the separation and purification of gallate decarboxylase

An acid decarboxylase, separation and purification technology, applied in the biological field, can solve problems such as instability

Active Publication Date: 2019-03-19
INST OF CHEM IND OF FOREST PROD CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this enzyme is unstable due to its sensitivity to oxygen, and so far, it has not been purified

Method used

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  • A method for the separation and purification of gallate decarboxylase
  • A method for the separation and purification of gallate decarboxylase
  • A method for the separation and purification of gallate decarboxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The crude extraction of embodiment 1 gallate decarboxylase (GAD)

[0041] Gallate decarboxylase (GAD) is an intracellular enzyme, that is, it is produced in the cell by biological cells. In order to obtain the intracellular enzyme, the simplest and most effective way to obtain the crude enzyme solution is to crush the cells and then centrifuge. From the research on the degradation process, in order to ensure the reproducibility of the experiment and the sustainability of the follow-up work, the bacterial cells under the optimal process above were treated with phosphoric acid containing 1mM dithiothreitol and 50mM sodium thiosulfate. Wash twice with salt buffer solution (pH 6.0), ultrasonically pulverize for 45 min at 4°C (5 s, interval 5 s), centrifuge for 20 min at 4°C (10000r min -1 ), that is, extract 100 mL of crude enzyme liquid from 12 bottles of fermentation liquid, and store it at 4°C for future use.

Embodiment 2

[0042] The salting-out of embodiment 2 gallate decarboxylase (GAD)

[0043] Effect of salt concentration on salting-out: take eight 18×180mm test tubes, add ammonium sulfate 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0g respectively, add 10mL distilled water to dissolve, stir evenly with a glass rod, make it fully Dissolve, then add 2mL of GAD crude enzyme solution respectively, seal and leave it in the refrigerator overnight to estimate the amount of flocs. It can be seen from Table 1 that when the concentration of ammonium sulfate is between 20% and 60%, the amount of flocs shows a continuous increase trend, and when the concentration of ammonium sulfate is between 60% and 80%, the amount of precipitation basically tends to be stable. For 60% ammonium sulfate, the concentrations of 60% and 70% ammonium sulfate can be selected for the two precipitations, so that the protein can be precipitated as much as possible.

[0044] Effect of precipitation time on salting-out: take five 18×...

Embodiment 3

[0046] Example 3 Dialysis of gallate decarboxylase (GAD): From the results in the second step, the approximate range of the protein in the crude enzyme solution is 50-70kD, so the molecular weight of the permeable protein is selected to be 3500-5000kD and 8000-14000kD respectively dialysis membrane for separation. Depend on Figure 4 It can be seen that the molecular weight of the protein extracted by ammonium sulfate is mainly distributed in the range of 50-100kD, and the protein content of 70-90kD is the highest. Therefore, the use of 80kD dialysis membrane filtration can retain large molecules and remove impurities of small molecules. Utilize the dialysis membrane filtration of 80kD size, investigate the influence of time with 500mL feed liquid as benchmark. Depend on Figure 5 It can be seen that the protein content in the permeate decreased with the increase of time. The decrease was not obvious in the first 6 hours, and it decreased sharply in the 8-16 hours, and then...

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Abstract

The invention discloses a method for separating and purifying gallic acid decarboxylase. Gallic acid is adopted as a substrate, gallic acid histidine decarboxylase is generated through substrate induction, crude extraction, salting out, dialysis and DEAE cellulose column and dextrangel column purifying and separating are carried out, and a gallic acid decarboxylase sample is obtained; the molecular weight of the gallic acid decarboxylase sample is preliminarily measured through polyacrylamide gel (PAGE) electro-phoresis, and the molecules of gallic acid decarboxylase are subjected to MALDI-TOF / TOF mass spectrum identification. Gallic acid decarboxylase is prepared through the method, gallic acid can be degraded, pyrogallic acid can be produced, and compared with a method for preparing gallic acid decarboxylase with gallic acid serving as a raw material, the method has the advantages that few by-products are generated, pollution is small, the requirement for equipment is low, the production process is easy to implement, the purity of prepared gallic acid decarboxylase is high, and the probability of industrial production can be met.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the preparation of gallic acid decarboxylase and a preparation method for the study of its enzymatic properties. Background technique [0002] Pyrogallic acid, also known as pyrogallol, pyrogallol, pyrobeiric acid, system name "1,2,3-trihydroxybenzene", white shiny crystalline powder, odorless, soluble in water, alcohol and ether , Slightly soluble in benzene, chloroform and carbon disulfide; easy to become light gray in the air, its aqueous solution is easy to darken in the air, faster in alkaline solution, slowly heating and sublimation begins. Pyrogallic acid has extremely strong reducibility, and can undergo substitution reaction on the benzene ring; it can form complex precipitation or color reaction with antimony, molybdenum, bismuth, titanium, cerium, iron, gold, tantalum, etc.; its phenolic Hydroxyl is easy to undergo methylation and can form electrophilic hydrogen bonds. Wh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12Q1/527
CPCC12N9/88C12Q1/527C12Y401/01059
Inventor 王成章李文君周昊陈虹霞陶冉
Owner INST OF CHEM IND OF FOREST PROD CHINESE ACAD OF FORESTRY
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