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Method for synthesizing phosphatidylserine by using immobilized biocatalyst

A phosphatidylserine and biocatalyst technology, applied in the field of biochemical science and technology, can solve the problems of deactivation, decreased reaction rate, easy aggregation and agglomeration, and achieves the effect of ensuring the quality of synthetic products, mild reaction conditions and strong operability

Pending Publication Date: 2020-10-16
上海云洛生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since phospholipase D is a water-soluble enzyme, it is easy to aggregate and agglomerate when in contact with organic solvents, or even inactivate, so the reaction rate will decrease.

Method used

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  • Method for synthesizing phosphatidylserine by using immobilized biocatalyst
  • Method for synthesizing phosphatidylserine by using immobilized biocatalyst
  • Method for synthesizing phosphatidylserine by using immobilized biocatalyst

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Preparation of immobilized phospholipase D

[0030] (1) Cut the ZnO nanowire / mesoporous silica composite carrier into small particles (less than 8mm 3 ), the carrier was immersed in an aqueous solution containing 10 mg / ml anionic cross-linker (polyethylene glycol 600), and after 30 min, the sample was taken out of the solution and washed three times with distilled water to remove free cross-linker in the water.

[0031] (2) Add phospholipase D powder to 20ml of 100mM, pH 6.0-8.0 sodium acetate buffer, keep the mixture stirring for 15 minutes, then collect the supernatant by centrifugation, and prepare PLD solutions with different concentrations (1-5mg / ml).

[0032] (3) Soak the ZnO nanowire / mesoporous silica composite carrier adsorbed with polyethylene glycol 600 in 10ml of free PLD solution with pH 6.0-8.0 for 20-30 hours, keep the temperature at 15°C, after that, The samples were washed with deionized water and sodium acetate buffer solution, and stor...

Embodiment 2

[0033] Example 2: Determination of Immobilized Enzyme Loading Capacity

[0034] In order to measure the loading capacity of PLD in the immobilized phospholipase described in Example 1, the protein content in the enzyme liquid detects with Bradford method, uses bovine serum albumin as standard protein, draws the standard curve of protein concentration and absorbance; Initial free PLD protein concentration C 1 (mol / ml), PLD protein concentration C in the system after the immobilization reaction2 (mol / ml), PLD protein concentration in the buffer solution used to wash the sample C 3 (mol / ml); the initial volume of the reaction system V 1 (ml), the buffer volume V used to wash the samples 2 (ml), the mass of the composite carrier used is M (g), then the enzyme load per unit carrier is:

[0035] Loading capacity (mg / g) =

[0036] Experimental results such as figure 1 As shown, in the immobilization reaction process, when the free PLD concentration is 1-4mg / ml, the enzyme load i...

Embodiment 3

[0037] Example 3: Enzyme activity assay of free PLD and immobilized PLD

[0038] For the convenience of experiments, the hydrolysis activity of the PLD of the present invention is used to determine its activity, according to the method similar to that of D'Arrigo et al. Anal. Chim. Acta 304 (1995) 249–254.) to measure the release of nitrophenol from p-phosphatidyl-p-nitrophenol (PpNP) by PLD at 405 nm. The enzyme activity reaction system consists of 50 μl substrate solution (10mM PpNP in 10mM pH7.0 Tris / HCl, 5% Triton X-100, 5mM SDS), 400μl 0.1M pH5.5 sodium acetate buffer (containing 20mM CaCl2), and 50 μl of free PLD enzyme. After incubation at 30°C for 10 minutes, 100 μl of 1M pH 7.0 Tris / HCl (containing 0.1M ethylenediaminetetraacetic acid) buffer was added to terminate the reaction. The activity measurement of the immobilized PLD enzyme was carried out under the same conditions as the free PLD enzyme. The reaction was started with 25 mg (wet weight) of immobilized PLD, ...

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Abstract

The invention discloses a method for synthesizing phosphatidylserine by using an immobilized biocatalyst. The method comprises the following steps of (1) using a ZnO nanowire / mesoporous silica compound as a carrier, and immobilizing free phospholipase D to obtain immobilized phospholipase D; and (2) using the immobilized phospholipase D obtained in the step (1) as a nano biocatalyst; firstly mixing and stirring L-serine and CaCl2 in a 25 DEG C water solution until the solution is clear; then, adding substrate phosphatidylcholine dissolved in butyl acetate; putting the mixture in a constant-temperature shaking table at the temperature of 35 to 55 DEG C, and performing reaction at 400 rpm to prepare phosphatidylserine; and after the reaction is finished, performing filtration to recover theimmobilized phospholipase D to be repeatedly used. According to the method provided by the invention, the enzyme loading capacity of the immobilized phospholipase D can reach 150 mg / g carrier; and thetransesterification rate reaches 96.4 percent. The temperature and pH stability of the immobilized phospholipase D are improved; the repeatability is good; and the industrial production cost is effectively reduced.

Description

technical field [0001] The invention relates to the technical field of biochemical technology, in particular to a method for preparing and synthesizing phosphatidylserine by using highly active and stable immobilized phospholipase D as a biocatalyst. Background technique [0002] In recent years, with the in-depth research on phospholipids, people have become more and more aware of the medicinal and nutritional value of phospholipids, and have widely used them in the fields of food, medicine and health products. Taking phosphatidylserine (PS) as an example, it is an important phospholipid substance, usually located in the inner layer of the cell membrane, is an important component of the cell membrane, and can participate in a series of membrane functional reactions. Especially in the nervous system of the human body, phosphatidylserine (PS) is one of the important components of the brain cell membrane, because of its obvious effects on improving memory, relieving stress, re...

Claims

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Application Information

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IPC IPC(8): C12P13/04C12N11/14C12N11/04
CPCC12P13/04C12N11/14C12N11/04C12N9/16C12Y301/04004
Inventor 张永志
Owner 上海云洛生物技术有限公司
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