Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant phospholipase D mutant and application thereof in synthesis of phosphatidylserine

A technology of phosphatidylserine and mutants, applied in the field of bioengineering, can solve the problems of reducing PS yield, increasing cost, and failure to successfully transform lecithin, etc., and achieve the goal of accelerating the industrialization process, improving production capacity, and high transphosphatidyl activity Effect

Active Publication Date: 2021-11-12
JIANGNAN UNIV
View PDF8 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reaction system of enzyme-catalyzed generation of PS will inevitably have the participation of water, which leads to the inability of some lecithin to be successfully converted into PS, thereby reducing the yield of PS to a large extent and increasing the cost of preparation.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant phospholipase D mutant and application thereof in synthesis of phosphatidylserine
  • Recombinant phospholipase D mutant and application thereof in synthesis of phosphatidylserine
  • Recombinant phospholipase D mutant and application thereof in synthesis of phosphatidylserine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: Expression and purification of SrPLD

[0068] Construction of genetically engineered bacteria and protein expression:

[0069] Taking the nucleotide sequence (shown in SEQ ID NO.2) of the target protein coding gene in Streptomyces racemochromogenes as template, using F1 and R1 as primers (the underlines are respectively BamH I and EcoR I restriction enzyme cut sites Point) PCR amplification, the amplification conditions are: 95°C for 5min, 29 cycles (98°C for 10s, 55°C for 15s, 72°C for 2min), 72°C for 5min.

[0070] F1: aaatgggtcgc ggatcc ttggcacgcacc;

[0071] R1: gacggagctc gaattc tcaggcctggcaga.

[0072] Obtain the cDNA sequence of the coding region of the PLD gene, recover the PCR product and connect it with the pET-28a(+) plasmid vector that has undergone the same double digestion through homologous recombination to obtain the recombinant expression plasmid pET-28a(+)-PLD, and convert the recombinant plasmid pET-28a(+) to -28a(+)-PLD was trans...

Embodiment 2

[0075] Example 2: Recombinant bacteria E.coli BL21 / pET-28a(+)-Sr MBP Construction of PLD and Phospholipase D Sr MBP Expression of PLD

[0076] (1) Recombinant bacteria E.coli BL21 / pET-28a(+)-Sr MBP Construction of PLDs

[0077] In order to solve the problem of heterologous expression of inclusion bodies in SrPLD, our research group found that SrPLD contains a signal peptide sequence (the nucleic acid sequence of the signal peptide is shown in SEQ ID NO.6), so a recombinant plasmid that removes the signal peptide should be constructed. Using the recombinant plasmid pET-28a(+)-PLD prepared in Example 1 as a template, using F2 and R2 as primers (the underlines are respectively BamH I and EcoR I restriction enzyme sites) PCR amplification, the amplification conditions are: : 95°C for 5min, 29 cycles (98°C for 10s, 55°C for 15s, 72°C for 2min), 72°C for 5min.

[0078] F2: atgggtcgc ggatcc gcttcgccga;

[0079] R2: gacggagctc gaattc tcacgcttggcac.

[0080] After the PCR pro...

Embodiment 3

[0090] Example 3: Preparation of Phosphatidylserine by Whole Cells

[0091] Add 0.08g of whole cells E.coli BL21 / pET-28a(+)-Sr expressing PLD protein after induction culture to 10mL small brown bottle MBP PLD or E.coli BL21 / pET-28a(+)-PLD, 0.24g L-serine, 0.3g phosphatidylcholine (PC), 1mL acetic acid-sodium acetate buffer pH6.0, 3mL ethyl acetate, in React at 40° C. and 400 rpm in a constant temperature shaker for 12 hours, and the reaction pH is 6.0. The converted reaction solution sample was centrifuged at 12000rpm for 5-10min, the upper organic phase was absorbed, and the organic phase was volatilized and then dissolved with the mobile phase (n-hexane / isopropanol / acetic acid=8 / 8 / 1), passed through 0.22μm After the organic membrane, high-phase liquid chromatography (HPLC) analysis was performed.

[0092] The obtained E.coli BL21 / pET-28a(+)-Sr MBP PLD and E.coli BL21 / pET-28a(+)-PLD were used to prepare phosphatidylserine in whole cells, and the results were as follows: us...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a recombinant phospholipase D mutant and application thereof in synthesis of phosphatidylserine, and belongs to the technical field of bioengineering. Recombinant phospholipase is constructed firstly, wherein the recombinant phospholipase is obtained by removing signal peptide from the C end of PLD and adding a label MBP through connecting peptide on the basis of phospholipase from streptomyces spike chromogenes, and the recombinant phospholipase mutant is obtained by mutating one or more of amino acid at the 379 site, the 169 site and the 185 site of the recombinant phospholipase. The recombinant phospholipase mutant shows high trans-phosphatidylactivity, the conversion rate of phosphatidylcholine can reach 85.8%, the yield of phosphatidylserine can reach 72.12%, and the yield of phosphatidylserine can reach 57.69g / L. By means of the method, the production capacity of a unit catalyst and the reaction efficiency of the catalyst are improved, and the reaction cost is reduced.

Description

technical field [0001] The invention relates to a recombinant phospholipase D mutant and its application in the synthesis of phosphatidylserine, belonging to the technical field of bioengineering. Background technique [0002] Phosphatidylserine (Phosphatidylserine, PS) is the only phospholipid that can regulate the function of key protein in cell membrane. Dementia, improving the brain function of the elderly; at the same time, repairing brain damage and treating children with ADHD have very obvious effects. The content of natural phosphatidylserine in nature is very rare, and the extraction process is complicated, which is not enough to meet people's needs. Therefore, it is of great significance to prepare high-purity and high-quality phosphatidylserine products. [0003] There are many preparation methods for phosphatidylserine, among which extraction and enzymatic conversion are the main methods. The biological enzymatic method mainly refers to using phosphatidylcholin...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21C12P7/64C12P13/06C12R1/19
CPCC12N9/16C12N15/70C12P7/6481C12P13/06C12Y301/04004C12N2800/101
Inventor 吴静齐娜刘立明宋伟胡贵鹏周怡雯陈修来刘佳高聪
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com