Recombination strain for expressing phospholipase D and application

A recombinant strain and phospholipase technology, applied in the biological field, can solve the problems of gene cloning and expression research, and achieve the effects of good enzyme activity stability, good ability to transfer phosphatidyl groups, and improved enzyme activity

Active Publication Date: 2018-12-07
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research on phospholipase D at home and abroad is mostly in the stage of isolation and screening of strains, isolation and purification of phospholipase D, and research on physical and chemical properties. In terms of molecular biology, there are relatively few studies on the cloning and expression of related genes.

Method used

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  • Recombination strain for expressing phospholipase D and application
  • Recombination strain for expressing phospholipase D and application
  • Recombination strain for expressing phospholipase D and application

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0047] The present invention provides a phospholipase D, comprising 541 amino acids, the amino acid sequence shown in SEQ ID NO.1, the gene encoding the phospholipase D has the nucleotide sequence shown in SEQ ID NO.2, the full length is 1,623 nucleotides.

[0048] The present invention also provides a recombinant plasmid expressing phospholipase D, including the nucleotide sequence shown in SEQ ID NO.2, a signal peptide gene for extracellular expression of phospholipase D, and ribosomes for expressing phospholipase D A binding site, a promoter for expressing phospholipase D, wherein the nucleotide sequence of the signal peptide gene is shown in SEQ ID NO.3, with a full length of 96 nucleotides encoding 32 amino acids; ribosome binding site The nucleotide sequence of the spot (RBS) is shown in SEQ ID NO.4, with a total of 15 nucleotides; the nucleotide sequence of the promoter is shown in SEQ ID NO.5, with a total length of 372 nucleotides.

Embodiment 2

[0050] This embodiment provides the construction of the recombinant plasmid pMA5-pld and its expression method in Bacillus subtilis, the specific steps are as follows:

[0051] (1) Amplification of phospholipase D coding sequence

[0052] Using the recombinant plasmid pET-28a(+)-spld containing the phospholipase D coding gene as a template, design primers (P1, P2) to amplify the phospholipase D coding sequence:

[0053] Primer P1: 5'-CG GGATCC ATGGCACGTCATCCGC-3' (BamHI)

[0054] Primer P2: 5'-CG ACGCGT TTAATCCTGACAAATA-3'(MluI)

[0055] The PCR amplification reaction was carried out in a 50 μL system, and 25 μL was added to the reaction system (Premix), 20 μL ddH 2 O, 2 μL template DNA, 1.5 μL each for upstream and downstream primers. The reaction conditions were 30 cycles of denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 s, annealing at 58.4°C for 30 s, extension at 72°C for 1.5 min, and a total of 30 cycles; final extension at 72°C for 10 min. The...

Embodiment 3

[0061] This embodiment provides the construction of the recombinant plasmid pMA5-npld and its expression method in Bacillus subtilis, the specific steps are as follows:

[0062] (1) Amplification of NprB signal peptide sequence

[0063] Using the B. subtilis168 genome as a template, design primers (P3, P4) to amplify the NprB signal peptide sequence:

[0064] Primer P3: 5'-CG GGATCC CGCAACTTGACCAAGAC-3' (BamH I)

[0065] Primer P4: 5'-TTGCGCGGATGACGTGCCATAGCAGCTGAGGCATGTGTTA-3'

[0066] The PCR amplification reaction was carried out in a 50 μL system, and 25 μL was added to the reaction system (Premix), 20 μL ddH 2 O, 2 μL template DNA, 1.5 μL each for upstream and downstream primers. The reaction conditions were 30 cycles of denaturation at 94°C for 3 minutes: denaturation at 94°C for 30 s, annealing at 54.6°C for 30 s, extension at 72°C for 0.5 min, and a total of 30 cycles; final extension at 72°C for 10 min. The PCR products were identified by nucleic acid electrop...

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Abstract

The invention relates to phospholipase D having an amino acid sequence shown as SEQ ID NO.1. The invention further relates to a gene sequence coding the phospholipase D. The nucleotide sequence of thegene is shown as SEQ ID NO.2. The invention provides a method for improving the expression level of the phospholipase D by virtue of a system modification expression component. The method comprises the following steps: screening and replacing signal peptides, ribosome binding sites and promoters, and transforming the constructed recombinant plasmid into host bacteria, wherein the recombination strain is capable of successfully expressing the phospholipase D. The phospholipase D disclosed by the invention has excellent phosphatidyl transformation ability, and the product phosphatidylserine canbe synthesized by taking lecithin and L-serine as the substrate. The recombinant bacteria have excellent enzyme activity stability, the fermentation cycle is short, and a foundation is laid for large-scale industrial production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant bacterial strain expressing phospholipase D and its application. Background technique [0002] Phosphatidylserine, a phospholipid that regulates the functional state of membrane proteins, is found in both animal and plant tissues and has been used as a food additive to prevent Alzheimer's disease. Phosphatidylserine has received a lot of attention in recent years because of its positive effects in improving memory, preventing muscle pain, and treating depression. Normally, people can obtain phosphatidylserine from animal and plant tissues through extraction and separation, and can also use a series of chemical reactions to synthesize phosphatidylserine. However, due to the low content of phosphatidylserine in animal cells and plant tissues, and the separation and extraction process will cause the loss of phosphatidylserine, resulting in a low yield of phosphatidylserin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/75C12N15/81C12N15/77C12N1/21C12N1/19C12P13/06C12R1/125C12R1/84C12R1/15
CPCC12N9/16C12N15/75C12N15/77C12N15/815C12P13/06C12Y301/04004C12P7/6481
Inventor 史劲松许正宏龚劲松侯海娟张晓梅李恒
Owner JIANGNAN UNIV
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