Gene mining and application method of phospholipase D

Abstract

The invention provides a phospholipase D acquired based on a gene mining method, as well as a gene coding sequence and application method thereof. A phospholipase D gene has a full length of 1,614bp, encodes 538 amino acids, uses plasmid pET28a (+) as an expression vector, and uses E. coli BL21 (DE3) as an expression host, to realize efficient expression of phospholipase D. The phospholipase D gene is simple and convenient in gene mining method; a recombinant strain constructed by the mined target gene can be fermented and induced to excrete the phospholipase D. For the phospholipase D, an optimum reaction temperature is 60 DEG C; an optimum reaction pH is 7.5; and Ca<2+> has good activating and promoting effects on enzyme activity of the phospholipase D; the phospholipase D can modify phospholipids, and can catalyze substrates phosphatidylcholine and L-serine to synthesize phosphatidylserine, thereby having good application prospects in food, medicine and healthcare industries.

Description

technical field

[0001] The invention provides a phospholipase D obtained based on a database gene mining method and an application method thereof, belonging to the field of industrial biotechnology. Background technique

[0002] Phospholipase D (PLD) is a general term for a class of enzymes that catalyze the hydrolysis of phosphodiester bonds and base exchange reactions. The main substrate is phosphatidylcholine (PC). PLD can hydrolyze PC to generate phosphatidic acid (PA) and bile Alkali, and many rare phospholipids and phospholipid derivatives with good functions can be prepared by using its phosphatidyl transfection activity, which are widely used in the fields of food, medicine and health products.

[0003] Phospholipase D has a wide range of sources. It was first reported that it was derived from cabbage leaves, and then it was found in other animals, plants, and microorganisms. It was also reported that PLD was obtained by cloning yeast and bacteria. In plants, phosph...

Examples

Embodiment 1

[0044] This example illustrates the method for screening phospholipase D based on database gene mining technology.

[0045] According to the literature on the exogenous expression of phospholipase D, the amino acid sequence of phospholipase D with high phosphatidyltransfer activity verified by experiments was selected as a template, and BLAST was performed in the NCBI database, and the screening was carried out according to the set screening criteria. A series of phospholipase D amino acid sequences are obtained, and then the amino acid sequence of the target phospholipase D is obtained by constructing a phylogenetic tree analysis and screening, thereby obtaining its coding gene sequence.

Embodiment 2

[0047] This example illustrates the cloning method of the gene encoding phospholipase D.

[0048] With the synthetic nucleotide sequence design primer (P1, P2), the coding gene of phospholipase D is amplified by PCR:

[0049] Primer P1: 5'-CG GGA TCC ATG CTG CGT CGC CTG CAT

[0050] Primer P2: 5'-CCC AAG CTT TTA CAG ACT ACA CAC ACC

[0051] The PCR amplification reaction was carried out in a 50 μL system, and 25 μL Ex Taq (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 57°C for 30 s, extension at 72°C for 1 min, and a total of 30 cycles; final extension at 72°C for 10 min. The PCR products were identified by nucleic acid electrophoresis, recovered and purified by tapping the gel, and the recovered products were cloned into the pMD19-T vector to construct a recombinant cloning plasmid, which was transforme...

Embodiment 3

[0053] This example takes pET-28a(+) as an example to illustrate the construction process of the recombinant expression plasmid.

[0054] pET-28a(+) has a T7 promoter and 6×His-tag tag, and the pET-28a(+) plasmid and the verified recombinant cloned plasmid containing the target gene are double digested with BamH I and Hind III at the same time, After digestion at 37°C for 3 hours, carry out nucleic acid electrophoresis verification and tap gel to recover the target gene and target plasmid. The recovered product was ligated with T4 DNA ligase at 16°C overnight, and the ligated product was transformed into E.coli JM109 competent cells and spread on the Cultivate in LB solid culture of mycin resistance (50mg / mL) for 8-10h, pick multiple positive transformants and culture them in LB liquid medium containing kanamycin (50mg / mL), at 37°C, 220rpm The plasmid was extracted and named pET-28a(+)-pld after culturing under the condition for 10-12 hours.

technical field

[0001] The invention provides a phospholipase D obtained based on a database gene mining method and an application method thereof, belonging to the field of industrial biotechnology. Background technique

[0002] Phospholipase D (PLD) is a general term for a class of enzymes that catalyze the hydrolysis of phosphodiester bonds and base exchange reactions. The main substrate is phosphatidylcholine (PC). PLD can hydrolyze PC to generate phosphatidic acid (PA) and bile Alkali, and many rare phospholipids and phospholipid derivatives with good functions can be prepared by using its phosphatidyl transfection activity, which are widely used in the fields of food, medicine and health products.

[0003] Phospholipase D has a wide range of sources. It was first reported that it was derived from cabbage leaves, and then it was found in other animals, plants, and microorganisms. It was also reported that PLD was obtained by cloning yeast and bacteria. In plants, phosph...

Examples

Embodiment 1

[0044] This example illustrates the method for screening phospholipase D based on database gene mining technology.

[0045] According to the literature on the exogenous expression of phospholipase D, the amino acid sequence of phospholipase D with high phosphatidyltransfer activity verified by experiments was selected as a template, and BLAST was performed in the NCBI database, and the screening was carried out according to the set screening criteria. A series of phospholipase D amino acid sequences are obtained, and then the amino acid sequence of the target phospholipase D is obtained by constructing a phylogenetic tree analysis and screening, thereby obtaining its coding gene sequence.

Embodiment 2

[0047] This example illustrates the cloning method of the gene encoding phospholipase D.

[0048] With the synthetic nucleotide sequence design primer (P1, P2), the coding gene of phospholipase D is amplified by PCR:

[0049] Primer P1: 5'-CG GGA TCC ATG CTG CGT CGC CTG CAT

[0050] Primer P2: 5'-CCC AAG CTT TTA CAG ACT ACA CAC ACC

[0051] The PCR amplification reaction was carried out in a 50 μL system, and 25 μL Ex Taq (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 57°C for 30 s, extension at 72°C for 1 min, and a total of 30 cycles; final extension at 72°C for 10 min. The PCR products were identified by nucleic acid electrophoresis, recovered and purified by tapping the gel, and the recovered products were cloned into the pMD19-T vector to construct a recombinant cloning plasmid, which was transforme...

Embodiment 3

[0053] This example takes pET-28a(+) as an example to illustrate the construction process of the recombinant expression plasmid.

[0054] pET-28a(+) has a T7 promoter and 6×His-tag tag, and the pET-28a(+) plasmid and the verified recombinant cloned plasmid containing the target gene are double digested with BamH I and Hind III at the same time, After digestion at 37°C for 3 hours, carry out nucleic acid electrophoresis verification and tap gel to recover the target gene and target plasmid. The recovered product was ligated with T4 DNA ligase at 16°C overnight, and the ligated product was transformed into E.coli JM109 competent cells and spread on the Cultivate in LB solid culture of mycin resistance (50mg / mL) for 8-10h, pick multiple positive transformants and culture them in LB liquid medium containing kanamycin (50mg / mL), at 37°C, 220rpm The plasmid was extracted and named pET-28a(+)-pld after culturing under the condition for 10-12 hours.

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