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Method for preparing phosphatidylserine under catalysis of immobilized phospholipase D

A technology of phosphatidylserine and immobilized phospholipase, which is applied in the field of immobilized phospholipase D to catalyze the preparation of phosphatidylserine, can solve the problems of weak physical adsorption force, easy inactivation of enzyme molecules, and harsh cross-linking conditions, etc. Effects of temperature and pH stability and resistance to organic solvents, method simplicity, and ease of operation

Inactive Publication Date: 2014-08-06
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The adsorption method refers to the method of immobilizing the enzyme through the interaction of secondary bonds between the surface of the carrier and the surface of the enzyme. The operation is simple, the conditions are mild, and the enzyme activity is well maintained. Easy to lose; the cross-linking method is a method of cross-linking and bridging immobilized enzymes between enzyme molecules using bifunctional or multifunctional group reagents. Although the immobilized enzymes are very strong, due to harsh cross-linking conditions, enzyme molecules are easily inactivated

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  • Method for preparing phosphatidylserine under catalysis of immobilized phospholipase D

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Preparation of free phospholipase D.

[0035] (1) Seed culture: Take a Streptomyces racemochromogenes ATCC23954 glycerol bacterium stored at -80°C, put it into a 250mL Erlenmeyer shaker flask (containing 100mL seed medium) at the inoculation amount of 1v / v% under sterile conditions, and put it into the shaker After bed, culture at 30°C, 200rpm for 48h.

[0036] (2) Fermentation culture: the above-mentioned seed culture solution is inserted in the fermenter by 2v / v% inoculum, the initial fermentation medium is 5L fermentation medium, the pH of the fermentation liquid is controlled to be 7.0 with 25wt% ammonia water, and oxygen aeration is regulated The volume was 1VVM, the temperature was controlled at 30°C, and cultured at 500rpm for 24h.

[0037] (3) Treatment of crude enzyme liquid: vacuum filter the fermentation liquid filter paper to remove agglomerated bacteria in the fermentation liquid, collect the supernatant, which is the fermented crude enzyme liqu...

Embodiment 2

[0040] Example 2: Preparation of immobilized phospholipase D.

[0041](1) Take 1g of chitin as a carrier, add 5ml of glutaraldehyde solution of cross-linking reagent at a concentration of 3v / v% (the solvent in the glutaraldehyde solution is 50mM sodium acetate buffer solution with pH 5.5), soak at room temperature And stir for 4 hours, let it stand overnight, centrifuge to remove the supernatant; wash the carrier repeatedly with distilled water to remove residual glutaraldehyde, and filter with suction to obtain the cross-linked chitin carrier, which is stored at 4°C;

[0042] (2) Take out 1g of cross-linked chitin carrier, add 5ml of free phospholipase D enzyme solution (total enzyme activity is 26.5U), then add 5mL of 50mM, pH5.5 sodium acetate buffer solution, put it in a shaker, Under the conditions of a constant temperature of 30°C and a shaker speed of 200rpm, after adsorption and cross-linking for 6 hours, take out and filter, wash with sodium acetate buffer (50mM, pH5....

Embodiment 3

[0044] Example 3: Application of immobilized phospholipase D to catalyze the preparation of phosphatidylserine from soybean lecithin in a biphasic system.

[0045] Take one 50mL centrifuge tube, add 2.0mL butyl acetate (containing 40μmol soybean lecithin PC), 2.0mL pH5.550mM sodium acetate buffer (containing 10mM CaCl 2 , 1M L-serine and 0.96 U of the immobilized phospholipase D) prepared in Example 2 were placed in a constant temperature shaker at 30° C. and 200 rpm for reaction. The reaction time was 5 hours, and the concentration of substrate and product was detected by HPLC. The transesterification rate was 92%.

[0046] Filter out the immobilized phospholipase D in the reaction system, wash it, and add new trace buffer, L-serine, and butyl acetate containing 20mM PC to catalyze it. It is found that it can be recycled five times in the biphase and still maintain The transesterification rate is about 83% (mol / mol).

[0047] Product detection method:

[0048] High-perform...

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Abstract

The invention discloses a method for preparing phosphatidylserine under catalysis of immobilized phospholipase D. The method comprises the following steps: (1) with chitin as a vector and glutaraldehyde as a cross-linking reagent, immobilizing free phospholipase D to obtain immobilized enzyme of phospholipase D; (2) with the immobilized enzyme of the phospholipase D prepared in the step (1) as a catalyst, n-butyl acetate as an organic phase solvent, and 50mM of sodium acetate buffer solution of which the pH is 5.5 as a water phase solvent, reacting L-serine to react with soyabean lecithin at 20-60 DEG C according to the molar ratio of (10-100):1, thus obtaining phosphatidylserine; and at the end of reaction, filtering and recovering the phospholipase D for repeated use. By adopting the method disclosed by the invention, the ester transfer rate in the dual-phase catalytic system can be up to 78%, the immobilized enzyme can be repeatedly used, and the method is simple, easy to operate, and low in preparation cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing phosphatidylserine catalyzed by immobilized phospholipase D. Background technique [0002] Phosphatidylserine (PS) is an important phospholipid substance with outstanding brain health functions. Although naturally occurring PS is widely distributed, its content is very low. At present, the preparation of PS is mainly extraction method and enzymatic conversion method. The extraction method mainly extracts PS from animal brain tissue. In recent years, due to mad cow disease, the PS prepared by extraction method is facing the problem of food safety. In contrast, the enzymatic conversion method uses phospholipase D to catalyze the transesterification of lecithin to prepare PS. The reaction conditions are mild, the process is simple, and it is environmentally friendly. It is an ideal method for preparing PS. [0003] The enzymatic preparation of PS takes lecithin as...

Claims

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Application Information

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IPC IPC(8): C12P13/06C12N11/10
CPCY02P20/50
Inventor 严明高璐魏淼许琳郝宁李艳
Owner NANJING UNIV OF TECH
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