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Recombinant phospholipase D and application thereof for synthesizing phosphatidylserine or other phospholipids

A phospholipase and phospholipid technology, applied in the field of functional enzyme screening, can solve the problems of overnight incubation, unfavorable PLD production or application, low yield, etc., and achieve the effect of improving transesterification activity

Active Publication Date: 2019-12-13
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although PLDs exhibit good catalytic potential, as a cytotoxic protein, the low yield limits their industrial application prospects
The formation of inclusion bodies is another factor that limits the industrial application of PLD. If the inclusion body protein wants to form a correctly folded soluble protein, it generally needs to add chemical reagents such as urea and guanidine chloride for refolding, usually overnight incubation and shaking / Stirring, some even need microwave heating as an auxiliary means, which is extremely unfavorable to the production or application of PLD

Method used

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  • Recombinant phospholipase D and application thereof for synthesizing phosphatidylserine or other phospholipids
  • Recombinant phospholipase D and application thereof for synthesizing phosphatidylserine or other phospholipids
  • Recombinant phospholipase D and application thereof for synthesizing phosphatidylserine or other phospholipids

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Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1: Screening and preparation of recombinant PLD

[0028] 1. PCR amplification of phospholipase D fragment

[0029] Firstly, the two PLD proteins obtained by the applicant were amplified by PCR method, namely S.griseofuscusstrain PLDsg / G213S and S.roseus, PLDsr, with a size of about 1500-1600bp. The amino acid sequence of PLDsg / G213S is SEQ ID NO :3, the sequence of the coding gene is SEQ ID NO:4; the amino acid sequence of S.roseus, PLDsr is SEQ ID NO:5, and the sequence of the coding gene is SEQ ID NO:6. Then the PCR product was recovered, and the concentrations of the two fragments were measured with a micro-nucleic acid analyzer. Get the same amount of PCR product solutions that have been amplified again and mix them with enzyme DNsae I to cut into fragments, and the enzyme-cut products are detected by agarose gel electrophoresis ( figure 1 A). According to the materials and methods, 0.3 μL DNase I (diluted to 1U / μL, diluted immediately after use) at 15...

Embodiment 2

[0039] Embodiment 2: Recombinant PLDs enzyme synthesis PS and DHA-PS

[0040] In order to verify the ability of the recombinant PLDs enzyme of the present invention and the parent enzyme to synthesize PS, a two-phase reaction system was used to synthesize PS with phosphatidylcholine PC and L-serine as substrates. The parent enzymes PLDsr and PLDsg / G213S were used as controls, and Reom-12, Recom-34 and Recom-35 were used as experimental groups, and the same weight of enzyme powder, 1M L-serine and 50mM CaCl 2 Dissolve in 1mL HAc-NaAc buffer (20mM, pH 6.0), and the organic phase contains PC (dissolved in anhydrous ether, 20mg / mL). The enzymatic reaction time was optimized, and after 6 hours of reaction, the organic phase was collected by centrifugation, and the reaction results were detected by thin-layer chromatography and HPLC.

[0041] The result is as Figure 4 As shown in a, the first lane from left to right is the substrate phosphatidylcholine PC, the second and third la...

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Abstract

The invention provides a recombinant phospholipase D, which is a novel PLD (phospholipase D) screened through an escherichia coli surface display technology, a high throughput screening technology anda DNA gene recombination technology. The amino acid sequence of the recombinant phospholipase D provided by the invention is SEQ ID NO:1. The transesterification vitality of the recombinant phospholipase PLDr34 is improved by 3.24 times, a conversion rate is 80.3%, selectivity is 86.8%, and the recombinant phospholipase D is an ideal catalyst synthesized by PS (phosphatidylserine), DHA-PS and other rare phospholipids.

Description

technical field [0001] The invention belongs to the technical field of functional enzyme screening, in particular to a recombinant phospholipase D and its application in synthesizing phosphatidylserine, DHA-phosphatidylserine or other phospholipids. Background technique [0002] Phospholipids widely exist in the tissue structure of organisms and are an important part of cell membranes. They are composed of a hydrophilic phosphate group head and a hydrophobic fatty acid chain tail. Currently, four major groups have been identified: ethanolamine, inositol, serine, and choline. These groups form the most important phospholipids, namely phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylcholine (PC). PC is the most important natural phospholipid, but it cannot meet human nutrition or other needs. Synthesizing phospholipids with specific configurations from natural phospholipids has important industrial application value. [0003] ...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21C12P13/06C12P7/64C12R1/19
CPCC12N9/16C12Y301/04004C12P13/06C12P7/6481
Inventor 毛相朝张海洋孙建安薛长湖
Owner OCEAN UNIV OF CHINA
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