Coding gene of phospholipase D and expression and application thereof

A phospholipase and encoding technology, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of long fermentation cycle and complicated culture conditions, and achieve short fermentation cycle, good stability of enzyme activity, and good acylation ability Effect

Inactive Publication Date: 2019-07-12
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the long fermentation period and complex culture conditions of wild bacteria, more and more researchers use heterologous expression methods to produce phospholipase D

Method used

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  • Coding gene of phospholipase D and expression and application thereof
  • Coding gene of phospholipase D and expression and application thereof
  • Coding gene of phospholipase D and expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The present invention provides a phospholipase D coding sequence (sspld), comprising 522 amino acids. The amino acid sequence is shown in SEQ ID NO. 1, and the gene encoding the phospholipase D has the nucleotides shown in SEQ ID NO. 2. The sequence is 1,566 nucleotides in length.

[0040] The present invention also provides a recombinant plasmid expressing phospholipase D, which includes the nucleotide sequence shown in SEQ ID NO. 2 and has a total length of 6916 nucleotides.

Embodiment 2

[0042] This example provides the construction of the recombinant plasmid pET-28a(+)-sspld and its expression method in E. coli BL21. The specific steps are as follows:

[0043] (1) Amplification of phospholipase D coding sequence

[0044] Using the phospholipase D coding sequence sspld from Streptomyces preserved in the laboratory as a template, design primers (P1, P2) to amplify the phospholipase D coding sequence:

[0045] Primer P1: 5’-CG AAGCTT ATGGCACGTCATCC-3’(Hind III)

[0046] Primer P2: 5’-CC GGATCC TTAGCCAGCCAGAT-3’(BamH I)

[0047] PCR amplification reaction is carried out in a 50μL system, and 25μL is added to the reaction system (Premix), 20μL ddH 2 O, 2μL of template DNA, 1.5μL of upstream and downstream primers. The reaction conditions were 30 cycles of pre-denaturation at 94°C for 3 minutes: denaturation at 94°C for 30 seconds, annealing at 52.4°C for 30 seconds, extension at 72°C for 1.5 minutes, and a final extension at 72°C for 10 minutes. The PCR products are i...

Embodiment 3

[0057] This embodiment provides a culture method for increasing the activity of phospholipase D through induction culture.

[0058] (1) The influence of induction temperature on enzyme production

[0059] When other fermentation induction conditions are the same, the recombinant bacteria are placed at 15, 20, 25, 30, 35 ℃ and other different temperature conditions for 8 hours after induction, according to the standard activity determination method to determine the phospholipase D under different induction temperature conditions Enzyme activity. by Figure 7 a It can be seen that the fermentation enzyme activity is highest when the induction temperature is 20℃.

[0060] (2) The effect of induction timing on enzyme production

[0061] The recombinant bacteria were cultured to OD600nm of 0.6, 0.8, 1.0, 1.2, 1.4 for IPTG induction, the induction time remained the same, and the enzyme activity of phospholipase D under different induction time conditions was measured according to the stand...

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Abstract

The invention provides a coding gene of phospholipase D and expression and application thereof, and provides a method for expressing recombinant phospholipase D by E.coli BL21 and producing phosphatidylserine by using the recombinant phospholipase D. The method includes construction of a recombinant plasmid pET-28a(+)-sspld, transformation of the recombinant plasmid into E.coli BL21 and catalyticproduction of phosphatidylserine by recombinant bacteria. The recombinant strain can successfully express the phospholipase D and the enzyme activity reaches 17.07 U / mL. By optimization of the induction conditions, the highest enzyme activity of the recombinant bacteria can reach 38.58 U / mL. The enzyme has good transphosphatidyl ability through HPLC detection and can synthesize the product phosphatidylserine by using lecithin and L-serine as substrates, the conversion rate can reach 28% and the yield can reach 1.34 g / L. The recombinant phospholipase D has short fermentation period and good catalytic performance, and lays a foundation for a practical application.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a phospholipase D encoding gene and its recombinant expression and application in the synthesis of phosphatidylserine. Background technique [0002] Phosphatidylserine (PS) was first extracted by Jordi Floch in 1942. The source of the extraction was bovine brain, and the molecular structure was subsequently analyzed [FOLCH J. Journal of Biological Chemistry, 1942, 146: 35-44 ]. It is a kind of phospholipid in phospholipids that can regulate the functional state of membrane proteins [Feng Leigang et al. Food Research and Development, 2006,27(9):191-193], and it occupies 10%-20% of the phospholipid bilayer of the cell membrane . In recent years, phosphatidylserine has been widely used in the medical and health care industries. It is mainly used to treat brain aging and repair brain damage, and has remarkable curative effects. It has attracted much attention as a "brain-specific...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/70C12P13/06
CPCC12N9/16C12Y301/04004C12P13/06
Inventor 龚劲松史劲松许正宏侯海娟陈杰鹏段丽丽
Owner JIANGNAN UNIV
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