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A method for immobilizing phospholipase d to improve the activity of phosphatidylglycerol synthesis

A phosphatidylglycerol, synthesis reaction technology, applied in biochemical equipment and methods, immobilized on/in organic carriers, enzymes, etc., can solve problems such as high cost, increase PG production cost, etc., achieve enzyme activity improvement, low Good cost and stability

Active Publication Date: 2020-04-21
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Considering the expensive price of phospholipase D, the existing immobilization technology will undoubtedly increase the production cost of PG

Method used

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  • A method for immobilizing phospholipase d to improve the activity of phosphatidylglycerol synthesis
  • A method for immobilizing phospholipase d to improve the activity of phosphatidylglycerol synthesis
  • A method for immobilizing phospholipase d to improve the activity of phosphatidylglycerol synthesis

Examples

Experimental program
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Effect test

Embodiment 1

[0049] This embodiment provides a method for immobilizing phospholipase D to improve the activity of phosphatidylglycerol synthesis. The method is carried out according to the following steps:

[0050] Step 1, blotting of phospholipase D:

[0051] Add 0.05 g of glycerol to 1 mL of acetate buffer (0.2 M, pH 5.5) containing phospholipase D at a concentration of 0.05 mg / mL, and blot for 60 min under gentle stirring in an ice bath to obtain enzyme-substrate complexes. substance (imprinted enzyme) solution.

[0052] Step 2, precipitation of phospholipase D-substrate complex:

[0053] Add 8 mL of supercooled (below 0°C) anhydrous precipitant dropwise to the enzyme-substrate complex solution obtained in step 1 (methanol, ethanol, acetone, propanol and isopropanol were used as precipitants respectively), dropwise After completion, the precipitation was continued for 10 min under gentle stirring in an ice bath to obtain a mixed solution containing phospholipase D-substrate complex ag...

Embodiment 2

[0065] This embodiment provides a method for immobilizing phospholipase D to improve the activity of phosphatidylglycerol synthesis. The method is carried out according to the following steps:

[0066] Step 1, blotting of phospholipase D:

[0067] Same as Step 1 of Example 1.

[0068] Step 2, precipitation of phospholipase D-substrate complex:

[0069] Add 8 mL of supercooled (below 0°C) anhydrous precipitant absolute ethanol dropwise to the enzyme-substrate complex solution obtained in step 1. After the dropwise addition, continue to precipitate under gentle stirring in an ice bath for 10 min to obtain Phospholipase D-substrate complex aggregate mixed solution.

[0070] Step 3, cross-linking of phospholipase D-substrate complex aggregates:

[0071] Under ice-bath conditions, add water-soluble cross-linking agent mass concentration to the mixed solution obtained in step 2 and be 25% glutaraldehyde, adjust the glutaraldehyde mass concentration in the reaction solution to be ...

Embodiment 3

[0081] This embodiment provides a method for immobilizing phospholipase D to improve the activity of phosphatidylglycerol synthesis. The method is carried out according to the following steps:

[0082] Step 1, blotting of phospholipase D:

[0083] Add 0.1 mL of ether solution containing phosphatidylcholine at a concentration of 1 mg / mL to 1 mL of acetate buffer solution containing phospholipase D at a concentration of 0.05 mg / mL (concentration is 0.2 M, pH value is 5.5), under gentle stirring in an ice bath, After blotting for 60 min, a solution containing the enzyme-substrate complex (imprinted enzyme) was obtained.

[0084] Step 2, precipitation of phospholipase D-substrate complex:

[0085] Add 8 mL of supercooled (below 0°C) anhydrous precipitant absolute ethanol dropwise to the enzyme-substrate complex solution obtained in step 1. After the dropwise addition, continue to precipitate under gentle stirring in an ice bath for 10 min to obtain Phospholipase D-substrate comp...

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Abstract

The invention provides a method for immobilizing phospholipase D to improve the reaction activity of phosphatidylglycerol synthesis. The method is carried out according to the following steps: step 1, adding the imprinted substrate to the phospholipase D solution for coordination to obtain the enzyme-substrate Complex solution (imprinted enzyme); step 2, adding a precipitating agent to precipitate the enzyme-substrate complex to obtain a mixed solution containing enzyme-substrate complex aggregates; step 3, adding a water-soluble cross-linking agent to the mixed solution after precipitation Perform a cross-linking reaction to obtain cross-linked enzyme-substrate complex aggregates; step 4, wash the cross-linked enzyme-substrate complex aggregates, and obtain cross-linked imprinted phospholipase D aggregates after removing the substrate. In the method of the present invention, after the imprinting action between the phospholipase D molecule and the substrate molecule is completed, the precipitation method is used to "fossilize" the "superactivated" structure of the enzyme molecule, and then add an appropriate water-soluble cross-linking agent to carry out cross-linking of the enzyme molecule , achieving intermolecular cross-linking and intramolecular cross-linking of "superactivated" structural enzyme molecules to form cross-linked imprinted phospholipase D aggregates.

Description

technical field [0001] The invention belongs to the field of synthesis of phospholipids, relates to phospholipase D, in particular to a method for immobilizing phospholipase D for improving the reaction activity of phosphatidylglycerol synthesis. Background technique [0002] In recent years, with the in-depth research on phospholipids, the medical and nutritional value of phospholipids has been more clearly understood, and it has been widely used in the fields of food, medicine and health products. Taking phosphatidylglycerol (PG) as an example, it can reduce the surface tension of the lung and stabilize the function of the alveoli, and has a wide range of applications in the diagnosis and treatment of lung diseases. At the same time, due to its unique structure and physical and chemical properties, PG is also an important intermediate product for the artificial synthesis of carotene lipids. [0003] However, the natural PG obtained from nature has complex structural compo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/02C12N9/16
CPCC12N9/16C12N11/02C12Y301/04004
Inventor 张小里王姣李冰麟段丹丹赵彬侠董文博
Owner NORTHWEST UNIV
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