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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  • Abstract
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Problems solved by technology

However, since phospholipase D is a water-soluble enzyme, it is easy to aggregate and agglomerat

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

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Example Embodiment

[0029] Example 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 is immersed in an aqueous solution containing 10mg / ml anionic crosslinker (polyethylene glycol 600). After 30 minutes, the sample is taken out of the solution and washed three times with distilled water to remove the free crosslinker in the water.

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

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

Example Embodiment

[0033] Example 2: Determination of immobilized enzyme loading

[0034] In order to determine the loading amount of PLD in the immobilized phospholipase described in Example 1, the protein content in the enzyme solution was detected by Bradford method, and bovine serum albumin was used as the standard protein to draw a standard curve of protein concentration and absorbance; the measurement was used for reaction Initial free PLD protein concentration C 1 (Mol / ml), the PLD protein concentration C in the system after the immobilization reaction is over 2 (Mol / ml), the PLD protein concentration C in the buffer solution used to wash the sample 3 (Mol / ml); initial volume of reaction system V 1 (Ml), the volume of buffer used to wash the sample V 2 (Ml), the mass of the composite carrier used is M (g), then the enzyme load per unit carrier is:

[0035] Load (mg / g)=

[0036] The experimental results are as figure 1 As shown, in the process of immobilization reaction, when the free PLD concent...

Example Embodiment

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

[0038] In order to facilitate the experiment, the hydrolytic activity of the PLD of the present invention is used to determine its activity, according to a similar method to D'Arrigo et al. (P. D'Arrigo, V. Piergianni, D. Scarcelli, S. Servi, A spectrophotometricassay for phospholipase D, The procedure described in Anal. Chim. Acta 304 (1995) 249-254.) measures the nitrophenol released by PLD from p-phosphatidyl-p-nitrophenol (PpNP) at 405 nm. The enzyme activity reaction system consists of 50μl substrate solution (10mM PpNP, 5% Triton X-100, 5mM SDS in 10mM pH7.0 Tris / HCl), 400μl 0.1M pH5.5 sodium acetate buffer (containing 20mM CaCl2), And 50μl free PLD enzyme. After incubating at 30°C for 10 minutes, add 100μl of 1M pH7.0 Tris / HCl (containing 0.1M ethylenediaminetetraacetic acid) buffer to stop the reaction. The activity measurement of the immobilized PLD enzyme was carried out under the same c...

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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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