Method for quantifying phospholipase D and choline-type plasmalogen

Abstract

To obtain a novel phospholipase D against choline-type plasmalogen.SOLUTION: Prepared is a phospholipase D comprising any of the described polypeptides: (1-1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (1-2) a polypeptide having at least 85% identity with the amino acid sequence set forth in SEQ ID NO: 1, and having a hydrolytic activity to a phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-bonded to the sn-2 position of a glycerol skeleton; and (1-3) a polypeptide in which one or several amino acids are added, substituted, deleted and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and having a hydrolytic activity to a phosphate ester bond in a choline-type plasmalogen molecule.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying phospholipase D and choline-type plasmalogen, an expression vector, a transformant containing the expression vector, and a method for producing phospholipase D, which includes a step of culturing the transformant to produce phospholipase D. [Background technology]

[0002] Plasmalogen is a type of phospholipid that has functions such as antioxidant activity, ion transport, and cholesterol excretion. Choline-type plasmalogens are found in large amounts in mammals, mainly in cardiac muscle and muscle, and also in the brain.

[0003] In recent years, it has been disclosed that plasmalogens are significantly reduced in the brains of patients with Alzheimer's disease and mild cognitive impairment (MCI) (see Non-Patent Documents 1 and 2), and that plasmalogen concentrations are reduced in the blood of patients with Alzheimer's disease (see Non-Patent Documents 3 and 4), drawing attention to the relationship between Alzheimer's disease and plasmalogens. Furthermore, the relationship between choline-type plasmalogens and disease has also been attracting attention, as reported in a report on a relationship between the blood concentration of choline-type plasmalogens, in which oleic acid is ester-bonded at the sn-2 position, and coronary artery disease (see Non-Patent Document 5).

[0004] In this context, research into the measurement of plasmalogens has also progressed. For example, a test method for determining whether a test blood sample is derived from a mammal with dementia (see Patent Document 1) has been proposed, which includes the steps of (A) measuring the amount of plasmalogen contained in red blood cells contained in the test blood sample using high-performance liquid chromatography, and (B) (i) comparing the amount of plasmalogen contained in red blood cells contained in the test blood sample with (ii) the amount of plasmalogen contained in red blood cells derived from a healthy mammal of the same species as the mammal from which the test blood sample was collected.

[0005] Furthermore, although phospholipase D derived from Streptomyces sp. has high phospholipase D activity against lysophosphatidylcholine and phosphatidylcholine, it has been proposed as a phospholipase D that acts on choline-type plasmalogens to liberate choline based on the discovery of phospholipase D activity against choline-type plasmalogens (see Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2012 / 090625 Brochure [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-082991 [Non-patent literature]

[0007] [Non-Patent Document 1] Guan Z, et al., J Neuropathol Exp Neurol; 58(7):740-747 (1999) [Non-patent document 2] Fujino et al., J Alzheimers Dis Parkinsonism; 2018,8(1) DOI: 10.4172 / 2161-0460.1000419 [Non-patent document 3] Goodenowe DB et al., J Lipid Res Nov;48(11): 2485-2498 (2007) [Non-patent document 4] Oma et al., Dement Geriatr Cogn Disord Extra;2:298-303 (2012) [Non-Patent Document 5] Nishimukai et al., Clinica Chimica Acta; 437(1):147-154 (2014) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to obtain a novel phospholipase D enzyme that acts on a choline-type plasmalogen having oleic acid ester-linked at the sn-2 position (hereinafter also referred to as "PlsCho(sn-2 C18:1)"). [Means for solving the problem]

[0009] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that phospholipase D derived from the Streptomyces A746 strain, which is closely related to Streptomyces griseoflavus, has high substrate specificity for choline-type plasmalogens in which oleic acid is ester-linked at the sn-2 position. Furthermore, they have found that the use of such phospholipase D makes it possible to selectively detect or quantify choline-type plasmalogens in which oleic acid is ester-linked at the sn-2 position, thereby completing the present invention.

[0010] That is, the present invention is as follows. [1] A phospholipase D comprising a polypeptide according to any one of (1-1) to (1-3) below: (1-1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (1-2) A polypeptide having at least 85% identity with the amino acid sequence set forth in SEQ ID NO: 1 and having hydrolysis activity for the phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-bonded to the sn-2 position of the glycerol backbone; (1-3) A polypeptide in which one or several amino acids are added, substituted, deleted, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and which has hydrolytic activity against phosphate ester bonds in the choline-type plasmalogen molecule; [2] A phospholipase D having hydrolytic activity on a phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-linked at the sn-2 position, the phospholipase D having the following properties (2-1) to (2-4): (2-1) It has hydrolytic activity against the phosphate ester bond in the choline-type plasmalogen molecule, in which oleic acid is ester-linked at the sn-2 position; (2-2) The molecular weight measured by SDS-PAGE analysis is 38,000 to 50,000; (2-3) The optimum temperature is 40 to 60°C under the reaction conditions of pH 7.2 and 2.5 minutes; (2-4) Derived from microorganisms belonging to the genus Streptomyces; [3] The phospholipase D according to [2] above, which is derived from a microorganism closely related to Streptomyces griseoflavus, Streptomyces griseorubens, or Streptomyces griseoincarnatus. [4] The phospholipase D according to any one of [1] to [3] above, characterized in that, when the hydrolysis activity towards phosphate ester bonds is taken as 100 when a choline-type plasmalogen having oleic acid ester-bonded to the sn-2 position as a substrate is used, the relative activity when a choline-type plasmalogen having arachidonic acid ester-bonded to the sn-2 position as a substrate is used is 10 or less, and the relative activity when a choline-type plasmalogen having docosahexaenoic acid ester-bonded to the sn-2 position as a substrate is used is 10 or less. [5] A method for quantifying a choline-type plasmalogen in which oleic acid is ester-bonded to the sn-2 position in a sample, the method comprising the step of reacting the sample with phospholipase D described in any one of [1] to [4] above. [6] An expression vector containing a polynucleotide encoding the phospholipase D according to [1] above. [7] A transformant containing the expression vector described in [6] above. [8] A method for producing phospholipase D, comprising the step of culturing the transformant according to [7] above to produce phospholipase D. [Effects of the Invention]

[0011] The phospholipase D of the present invention can hydrolyze choline-type plasmalogens in which oleic acid is ester-linked at the sn-2 position. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a molecular phylogenetic tree based on approximately 1,500 base pairs of 16S ribosomal DNA of the A746 strain prepared in Example 2. [Figure 2] 1 shows the results of analyzing purified phospholipase D (DEAE fraction) by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in Example 3. [Figure 3] FIG. 1 shows the results of measuring phospholipase D activity using purified phospholipase D (DEAE fraction) in Example 4. The phospholipase D activity is shown as a relative activity, with the phospholipase D activity when PlsCho having oleic acid ester-linked at the sn-2 position as the substrate being set at 100. [Figure 4] 1 shows the results of investigating the optimum temperature of purified phospholipase D in Example 4. [Figure 5] 1 shows the results of investigating the optimum pH of purified phospholipase D in Example 4. [Figure 6] FIG. 1 shows the results of investigating the importance of disulfide bonds in purified phospholipase D in Example 4. [Figure 7] FIG. 1 shows the results of investigating the metal ion requirement of purified phospholipase D in Example 4. [Figure 8] FIG. 1 shows the results of SDS-PAGE analysis of the enzyme PlsCho-PLD solution obtained by expression in Escherichia coli in Example 6. [Figure 9]FIG. 10 shows the results of Western blot analysis using an anti-histidine tag antibody of the enzyme PlsCho-PLD solution obtained by expression in E. coli in Example 6. [Figure 10] FIG. 10 shows the results of detecting PlsCho-PLD activity using PlsCho (sn-2 C18:1) as a substrate in Example 7. [Figure 11] 1 shows the results of examining substrate specificity in Example 8. [Figure 12] FIG. 10 shows the results of detecting choline-type plasmalogen in plasma in Example 9. DETAILED DESCRIPTION OF THE INVENTION

[0013] [Phospholipase D] The phospholipase D-(1) of the present invention is (1-1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (1-2) A polypeptide having at least 85% identity with the amino acid sequence set forth in SEQ ID NO: 1 and having hydrolysis activity for the phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-bonded to the sn-2 position of the glycerol backbone; (1-3) A polypeptide in which one or several amino acids are added, substituted, deleted, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and which has hydrolytic activity against phosphate ester bonds in the choline-type plasmalogen molecule; The present invention relates to a phospholipase D comprising a polypeptide according to any one of (1-1) to (1-3), and is hereinafter also referred to as "the present phospholipase D-(1)."

[0014] The phospholipase D-(2) of the present invention is (2-1) It has hydrolytic activity against the phosphate ester bond in the choline-type plasmalogen molecule, in which oleic acid is ester-linked at the sn-2 position; (2-2) The molecular weight measured by SDS-PAGE analysis is 38,000 to 50,000; (2-3) The optimum temperature is 40 to 60°C under the reaction conditions of pH 7.2 and 2.5 minutes; (2-4) Derived from microorganisms belonging to the genus Streptomyces; The present invention relates to a phospholipase D having the properties (2-1) to (2-4) and having hydrolysis activity for the phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-linked at the sn-2 position, and is hereinafter also referred to as "the present phospholipase D-(2)." In addition, the present phospholipase D-(1) and the present phospholipase D-(2) are hereinafter collectively referred to as "the present phospholipase D" or "the present PLD."

[0015] The choline-type plasmalogen herein, in which oleic acid is ester-bonded at the sn-2 position, is one of the subclasses of glycerophospholipids, and is an alkenyl ether-type glycerophospholipid having an alkenyl (vinyl ether)-bonded hydrocarbon chain at the C1 (sn-1) position of the glycerophospholipid and an ester bond of oleic acid (18 carbon atoms with one double bond, 18:1) at the C2 (sn-2) position, in which the base is choline, and its structure is shown in the following formula (I).

[0016] [ka]

[0017] In the formula, R 1 is usually an aliphatic hydrocarbon group having 1 to 20 carbon atoms, and examples thereof include a dodecyl group, a tetradecyl group, a hexadecyl group, an octadecyl group, and an icosanyl group. 2 is an unsaturated aliphatic hydrocarbon group derived from an unsaturated fatty acid having 18 carbon atoms and one double bond, such as an octadecenoyl group.

[0018] The present phospholipase D is an enzyme that has the activity of hydrolyzing the phosphate ester bond within a choline-type plasmalogen molecule in which oleic acid is ester-bonded at the sn-2 position, specifically, the activity of hydrolyzing the sn-3 phosphate ester within a choline-type plasmalogen molecule in which oleic acid is ester-bonded at the sn-2 position, thereby releasing alkenyl ether lipid and choline, as shown in the following formula (II).

[0019] [ka]

[0020] Whether or not phospholipase D has the activity to hydrolyze the phosphate ester in the PlsCho(sn-2 C18:1) molecule can be determined by, for example, allowing phospholipase D to act on PlsCho(sn-2 C18:1) and quantifying the amount of choline released by hydrolysis. The quantification of choline will be described later.

[0021] The molecular weight of the present phospholipase D-(2) measured by SDS-PAGE analysis may vary slightly depending on electrophoresis conditions, etc., but is typically 38,000 to 50,000, preferably 42,000 to 45,000.The isoelectric point (calculated by Genetyx) may be 5.2 to 5.6, preferably 5.44.

[0022] The present phospholipase D-(2) has an optimum temperature of 40 to 60°C, preferably 45 to 55°C, under reaction conditions of pH 7.2 and 2.5 minutes.

[0023] The present phospholipase D-(2) has an optimum pH of 3.5 to 4.5, preferably 5.5 to 8.0, and more preferably 6.5 to 7.5 under reaction conditions of 37°C and 2.5 minutes.

[0024] The present phospholipase D-(2) is derived from a microorganism belonging to the genus Streptomyces. Examples of microorganisms belonging to the genus Streptomyces include microorganisms closely related to Streptomyces griseoflavus, Streptomyces griseorubens, or Streptomyces griseoincarnatus, as well as Streptomyces althioticus, Streptomyces almquistii, Streptomyces heliomycini, Streptomyces viridochromogenes, Streptomyces flaveolus, Streptomyces Examples of the microorganisms include Streptomyces ambofaciens, Streptomyces albogriseolus, Streptomyces viridodiastaticus, and Streptomyces coelicolor. Whether or not the microorganisms are closely related can be determined by, for example, estimating a phylogenetic tree using the neighbor-joining method or the like and performing molecular phylogenetic analysis.

[0025] Furthermore, the above-mentioned phospholipase D-(2) of the present invention is preferably a polypeptide having a relative activity of 10 or less, preferably 5 or less, and more preferably 3 or less, when a choline-type plasmalogen having arachidonic acid ester-linked to the sn-2 position as a substrate is used, where the hydrolysis activity toward phosphate ester bonds when reacted under reaction conditions of pH 7.2, 50°C, and 2.5 minutes using PlsCho (sn-2 C18:1) as a substrate is 100, and a relative activity of 10 or less, preferably 5 or less, and more preferably 3 or less, when a choline-type plasmalogen having docosahexaenoic acid ester-linked to the sn-2 position as a substrate is used, where the hydrolysis activity toward phosphate ester bonds when reacted under reaction conditions of pH 7.2, 50°C, and 2.5 minutes is 100.

[0026] When the present phospholipase D-(2) is prepared from a microorganism belonging to the genus Streptomyces, the microorganism is cultured, the culture supernatant is recovered, and the resulting phospholipase D-(2) can be purified by a known enzyme purification method, for example, ammonium sulfate precipitation, and an appropriate combination of anion exchange chromatography, hydrophobic chromatography, cation exchange chromatography, gel filtration chromatography, and / or affinity chromatography.

[0027] The "polypeptide having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 1 and having hydrolysis activity for the phosphate ester bond in the PlsCho (sn-2 C18:1) molecule" in the above-mentioned polypeptide of the present phospholipase D-(1) (1-2) includes a polypeptide having at least 85%, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence set forth in SEQ ID NO: 1 and having hydrolysis activity for the phosphate ester bond in the PlsCho (sn-2 C18:1) molecule. The "polypeptide having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 1 and having hydrolysis activity for the phosphate ester bond within the PlsCho(sn-2 C18:1) molecule" preferably maintains identity with the corresponding positions in the amino acid sequence set forth in SEQ ID NO: 1 at H positions 81 and 258, K positions 83 and 260, and D positions 88 and 265, preferably at positions 81 to 88, HRKVLLTD, and 258 to 265, respectively, of the HxKxxxxD motif (H is histidine, K is lysine, D is aspartic acid, R is arginine, V is valine, L is leucine, T is threonine, and x is any amino acid; HKD motif) of HTKIITVD. The HKD motif is an amino acid sequence that contributes to the catalytic reaction of phospholipase D.

[0028] The "polypeptide having one or more amino acids added, substituted, deleted, and / or inserted" in the above-mentioned polypeptide of the present phospholipase D-(1)(1-3) includes a polypeptide having any number of amino acids added, substituted, deleted, and / or inserted, such as 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1. The "polypeptide having one or more amino acids added, substituted, deleted, and / or inserted" is preferably a polypeptide having one or more amino acids added, substituted, deleted, and / or inserted other than H at positions 81 and 258, K at positions 83 and 260, and D at positions 88 and 265 in SEQ ID NO: 1, preferably other than the HxKxxxxD motifs of HRKVLLTD at positions 81 to 88 and HTKIITVD at positions 258 to 265.

[0029] Preferred examples of the (1-2) or (1-3) polypeptide of the present phospholipase D-(1) include polypeptides that, when PlsCho (sn-2 C18:1) is used as a substrate and the hydrolysis activity toward phosphate ester bonds is reacted under reaction conditions of pH 7.2, 50°C, and 2.5 minutes, taken as 100, have a relative activity of 10 or less, preferably 5 or less, and more preferably 3 or less, when a choline-type plasmalogen having arachidonic acid bound to the sn-2 position is used as a substrate, and have a relative activity of 10 or less, preferably 5 or less, and more preferably 3 or less, when a choline-type plasmalogen having docosahexaenoic acid bound to the sn-2 position is used as a substrate.

[0030] When the present phospholipase D is reacted with PlsCho (sn-2 C18:1), it is preferable to use a buffer such as Tris-HCl buffer, acetate buffer, citrate buffer, BisTris-HCl buffer, or HEPES buffer. The pH during the reaction can be 3.5 to 4.5 or 5.5 to 8.0, preferably 6.5 to 7.5, and the temperature during the reaction can be 40 to 60°C, preferably 45 to 55°C.

[0031] (Quantitative method for PlsCho (sn-2 C18:1)) The method for quantifying PlsCho (sn-2 C18:1) in a sample of the present invention may be any method for quantifying PlsCho (sn-2 C18:1) in a sample that includes the step of reacting the sample with the above-mentioned phospholipase D. The substrate specificity of this phospholipase D allows for accurate quantification of PlsCho (sn-2 C18:1). An example of a method for quantifying PlsCho (sn-2 C18:1) in a sample is the method disclosed in Japanese Patent Application Laid-Open No. 2014-82991, which utilizes a color reaction. Specifically, after reacting the sample (substrate) with phospholipase D, the enzyme reaction is terminated by heat inactivation. A solution containing choline oxidase, peroxidase, and a color reagent is then added to induce a color reaction, and the absorbance is measured. Next, a calibration curve is created using a sample of known concentration of PlsCho (sn-2 C18:1), and the amount of PlsCho (sn-2 C18:1) can be calculated by comparing the calibration curve with the measured value obtained using the sample to be measured.

[0032] Examples of the sample include body fluids such as plasma, serum, blood, saliva, cerebrospinal fluid, urine, lymph, and sweat collected from a subject. Serum and plasma can be obtained by treating blood obtained from a subject by a conventional blood collection method (e.g., syringe blood collection or vacuum blood collection) with a known method, such as centrifuging (e.g., 1000 × g, 5 minutes) and recovering the supernatant. When collecting blood, anticoagulants or glycolysis inhibitors such as EDTA, sodium fluoride, sodium citrate, sodium heparin, and monoiodoacetic acid can also be used.

[0033] The above-mentioned subjects include mammals such as humans, dogs, cats, monkeys, cows, horses, sheep, goats, pigs, mice, rats, hamsters, and rabbits, as well as birds, fish, amphibians, marine organisms such as scallops, mollusks such as sea cucumbers, and various microorganisms including lactic acid bacteria, molds, and yeasts.

[0034] [Expression vector] The expression vector of the present invention may be any expression vector (hereinafter also referred to as the present expression vector) containing a polynucleotide encoding the present phospholipase D, and examples of polynucleotides encoding the present phospholipase D include polynucleotides encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. Examples of polynucleotides encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 include the nucleotide sequence set forth in SEQ ID NO: 2 or polynucleotides having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more identity to the nucleotide sequence set forth in SEQ ID NO: 2.

[0035] Expression vectors may be of any form, such as circular or linear. Furthermore, they can be appropriately selected depending on the host cells used, and examples thereof include plasmid vectors, viral vectors, and phages. Commercially available expression vectors can also be used. Examples of plasmid vectors include, for example, pET vectors such as pET24a and pET22b, pMAL vectors such as pMAL-p5x, pGEX vectors, pCold vectors, pBR vectors such as pFN18, pPAL7, pBR322, and pBR325, pUC vectors such as pACYC184 vectors, pUC12, pUC13, pUC18, pUC19, and pUC118, and BluescriptKS+ vectors when using actinomycetes as the host. Examples of plasmid vectors include pUC702 vectors, pIJ680 vectors, pIJ702 vectors, pTONa5 vectors, pTONashort vectors, pTip vectors, and pNit vectors when using actinomycetes as the host. Further, when a Gram-positive bacterium is used as the host, examples of the vector include the pBIC vector, pWH1520 vector, pMM1522 vector, and pHIS1522 vector. When a yeast, particularly Saccharomyces cerevisiae, is used as the host, examples of the vector include the YRp7 vector, pYC1 vector, and YEp13 vector.

[0036] Such expression vectors may also contain polynucleotides encoding regulatory sequences such as promoters and terminators, tags such as affinity and solubility tags, and selection markers such as drug resistance genes and reporter genes. Furthermore, they may contain polynucleotides encoding secretion signals to facilitate secretion outside of host cells. Examples of secretion signals include the Tat secretion signal shown in SEQ ID NO: 3, the OmpA signal (which functions as a periplasmic localization signal), and the pelB signal (which functions as a periplasmic localization signal). Examples of affinity and solubility tags include the MBP tag (which also functions as a periplasmic localization signal), GST tag, TF tag, Halo tag, His tag, SKIK tag (Kato et al., J. Biosci. Bioeng. 10.1016 / j.jbiosc.2016.12.004 (2017)), and the Profinity eXact fusion tag. The polynucleotides encoding the above-mentioned affinity and solubility tags and other tags, and selection markers such as drug resistance genes and reporter genes may be located at either the 5'-end or 3'-end of the polynucleotide encoding the present phospholipase D within the above-mentioned expression vector.

[0037] The polynucleotide may be a naturally occurring polynucleotide, such as a microorganism belonging to the genus Streptomyces, or an artificially synthesized polynucleotide, and can be selected appropriately depending on the type of microorganism into which the expression vector is to be introduced. Sequence information can be obtained appropriately by searching publicly known literature or databases such as NCBI (www.ncbi.nlm.nih.gov / guide / ). The polynucleotide can be prepared by publicly known techniques, such as chemical synthesis or PCR amplification, based on the base sequence information of the polynucleotide. Note that the codons selected to encode amino acids may be optimized for expression depending on the type of host cell used.

[0038] [Transformants] The transformant of the present invention may be a transformant containing the above-described expression vector of the present invention as a host (hereinafter also referred to as "the transformant of the present invention"). The host can be appropriately selected depending on the expression vector used, and examples thereof include Escherichia coli such as BL21 and SHuffle, actinomycetes belonging to the genus Streptomyces such as Streptomyces lividans, actinomycetes such as Rhodococcus erythropolis L88, gram-positive bacteria such as Bacillus subtilis, Bacillus megaterium, Brevibacillus (Bacillus brevis), and Corynebacterium glutamicum, gram-negative bacteria such as Pseudomonas putida, and Saccharomyces cerevisiae. Examples of suitable fungi include yeasts such as Pichia pastoris, Aspergillus oryzae, Aspergillus niger, and other filamentous fungi of the genus Aspergillus.

[0039] The expression vector of the present invention can be introduced into a host cell to produce the transformant of the present invention by a known method, and examples of such known methods include chemical methods such as the competent cell method, lipofection, calcium phosphate co-precipitation, and liposome method; biological methods such as methods using viral vectors, methods using specific receptors, and cell fusion; and physical methods such as electroporation, microinjection, gene gun method, and ultrasonic gene transfer.

[0040] [Method of producing phospholipase D] The method for producing phospholipase D of the present invention may be any method comprising the step of culturing the present transformant to produce phospholipase D, and such a method can efficiently produce the present phospholipase D. The culture method can be a conventional method used for culturing host cells. For example, when the host is Escherichia coli or actinomycetes, the culture can be performed at a temperature of 10 to 45°C, preferably 20 to 42°C, and more preferably 25 to 37°C, at a pH of 5.5 to 8.5, preferably 6.2 to 7.5, for a culture time of 10 to 80 hours, preferably 10 to 48 hours, under aerobic or anaerobic conditions such as shaking culture or aeration and stirring culture. The present phospholipase D can be recovered from the culture medium or from disrupted present transformant. Examples of such recovery methods include known protein recovery methods, such as centrifugation followed by gel filtration, ion exchange, or affinity chromatography.

[0041] The present invention will be described in more detail below with reference to examples. The examples are not limiting. [Example]

[0042] (Search for microorganisms that produce phospholipase D with high substrate specificity for PlsCho (sn-2 C18:1)) Based on their previous experience, the present inventors focused on actinomycetes as a microorganism that produces phospholipase D (also referred to as "PlsCho-PLD" in this specification) with high substrate specificity for PlsCho (sn-2 C18:1) and conducted a search using the following steps. 1) Glycerol stocks (stored at -80°C) of actinomycete strains were smeared on ISP2 agar medium using a platinum loop and cultured at 28°C. 2) Grown colonies were picked with a platinum loop and inoculated into 5 mL of MPD medium (1% (w / v) glucose, 0.75% (w / v) malt extract, 0.75% (w / v) peptone, 0.3% (w / v) NaCl, 0.1% (w / v) MgSO4·7H2O: pH 7.0) or ISP2 medium (1% (w / v) malt extract, 0.4% (w / v) yeast extract, 0.4% (w / v) glucose: pH 7.2). The culture was then cultured at 28°C and 160 strokes per minute (spm) for 72 hours with shaking. 1 mL of the culture was inoculated into 100 mL of MPD medium and cultured at 28°C and 160 rpm for 72 hours with shaking.

[0043] Next, the hydrolysis activity of phospholipase D on the phosphate ester bond in the choline-type plasmalogen molecule (phospholipase D activity) was measured by the following method. 1) The culture medium was centrifuged (21,600 × g, 10 minutes, 4°C), and the resulting supernatant was used as the enzyme sample. The enzyme sample was also heat-inactivated at 100°C for 10 minutes to prepare a heat-inactivated control (ctrl). 2) The enzyme reaction solution shown in Table 1 below was incubated at 37°C for 5 minutes, and then 5 μL of the enzyme sample was added and reacted at 37°C for 30 minutes. In the table, PlsCho means PlsCho in which oleic acid is ester-linked at the sn-2 position (PlsCho(sn-2 C18:1)), and Tx100 means Triton X-100.

[0044] [Table 1]

[0045] 3) 200 μL of the color reaction solution shown in Table 2 below was added and incubated at 37° C. for 10 minutes. In Table 2, POD stands for peroxidase, COD stands for choline oxidase, 4-AA stands for 4-aminoantipyrine, and TODB stands for N,N-bis(4-sulfobutyl)-3-methylaniline.

[0046] [Table 2]

[0047] 4) Immediately after color development, 200 μL of the mixture was taken and the absorbance (A550) at 550 nm was measured using a microplate reader (Thermo Fisher). The choline concentration in the enzyme reaction mixture was determined from a calibration curve prepared using known concentrations of choline. The amount of enzyme liberating 1 μmol of choline per minute was defined as 1 U.

[0048] Among the strains for which phospholipase D activity toward PlsCho (sn-2 C18:1) with oleic acid ester-linked at the sn-2 position was confirmed, a substrate specificity test was performed on strains with high activity. In the substrate specificity test, phospholipase D activity toward three types of lipids was measured: PlsCho (sn-2 C18:1) with oleic acid ester-linked at the sn-2 position (PlsCho(sn-2 C18:1)) (Avanti Polar Lipids), PlsCho (sn-2 C20:4) with C20:4 at the sn-2 position (PlsCho(sn-2 C20:4)) (Avanti Polar Lipids), and PlsCho (sn-2 C22:6) with C22:6 at the sn-2 position (PlsCho(sn-2 C22:6)) (Avanti Polar Lipids). Phospholipase D activity was measured as described above. Among them, strain A746 was selected as a PlsCho-PLD producer because it showed the highest phospholipase D activity toward PlsCho (sn-2 C18:1), in which oleic acid was ester-linked at the sn-2 position. [Example]

[0049] (Taxonomic identification of strain A746) The A746 strain selected in Example 1 was taxonomically identified. First, the A746 strain was cultured in ISP2 medium (Nihon Pharmaceutical Co., Ltd.) at 30°C for 72 hours. After the culture, DNA was extracted using achromopeptidase (Wako Pure Chemical Industries, Ltd.). Using the extracted DNA as a template for PCR, PCR was performed using primers described in the literature (Nakagawa Yasuyoshi and Kawasaki Hiroko, Japanese Society of Actinomycetes, Tokyo: Japan Society Office, pp. 88-117 (2001)), and approximately 1500 bp of 16S rDNA was amplified.

[0050] The amplified 16S rDNA sequences were determined using ChromasPro 2.1 (Technelysium). The amplified 16S rDNA sequences were compared to the 16S rDNA sequences of known microorganisms obtained from the international nucleotide sequence databases DDBJ, ENA (EMBL), and GenBank using BLAST homology searches to compare the degree of DNA homology with known microorganisms. A phylogenetic tree was created using the Clustal W program described in a literature review (Saitou & Nei, 1987; Mol. Biol. Evol. 4406-425), known as the neighbor-joining method. The phylogenetic tree is shown in Figure 1. The line in the upper left corner represents the scale bar, the numbers at the junctions of the phylogenetic branches represent bootstrap values, and the T at the end of the strain name indicates the type strain of that species.

[0051] As a result of a BLAST homology search, the 16S rDNA partial sequence of the A746 strain showed 99.8% identity to the type strains NBRC 12780 of Streptomyces griseorubens and NBRC 13044 of Streptomyces griseoflavus, and 99.5% identity to the type strain LMG19316 of Streptomyces griseoincarnatus. Furthermore, it showed 99.2%, 99.3%, 99.3%, 99.1%, and 99.1% homology to the type strain NRRL_B-3981 of Streptomyces althioticus, the type strain M27245 of Streptomyces ambofaciens, the type strain AB184712 of Streptomyces flaveolus, the type strain AB184712 of Streptomyces heliomycini, and the type strain AB184317 of Streptomyces viridodiastaticus, respectively.

[0052] Furthermore, simple morphological observation revealed powdery colonies with a gray surface and a pale yellow back. Microscopic observation revealed the formation of aerial mycelia (1 μm wide) and chain spores.

[0053] From the phylogenetic tree in Figure 1 and the results of simple morphological observation, it was confirmed that the A746 strain belongs to the genus Streptomyces, and that its most closely related microorganism is Streptomyces griseorubens or Streptomyces griseoflavus. [Example]

[0054] (Purification of PlsCho-PLD) PlsCho-PLD produced by the A746 strain was purified by the following method. (1) A colony of the A746 strain grown on an MPD plate was picked with a platinum loop and inoculated into 5 mL of MPD test tube medium. Shaking culture was performed at 30°C and 160 rpm for 4 days (preculture). 1 mL of the preculture was inoculated into 100 mL of MPD flask medium. Shaking culture was performed at 30°C and 160 rpm for 4 days (main culture). Unless otherwise noted, all of the following PlsCho-PLD purification steps were performed on ice or at 4°C. (2) The culture medium of the A746 strain obtained above was centrifuged (18,800×g, 30 minutes) to recover the culture supernatant (Cult.sup.). (3) 1 M Tris-HCl (pH 8.0) was added to the collected culture supernatant to a final concentration of 20 mM, and then powdered ammonium sulfate was added to 70% (w / v) saturation on ice. The resulting precipitate was collected by centrifugation (18,800 × g, 30 min), and suspended in 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution (70% sat. AS). (4) The crude enzyme solution obtained in (3) was dialyzed.

[0055] (5) The dialyzed solution obtained in (4) was applied to a Toyopearl®-DEAE650M column (Tosoh Biosciences) pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0) for anion exchange chromatography. After washing the column with the same buffer, the buffer was changed to 20 mM Tris-HCl buffer (pH 8.0). Elution was performed with a linear gradient of NaCl (0 to 1 M) to obtain an active fraction (DEAE650M).

[0056] (6) To the active fraction (DEAE650M) obtained in (5), ammonium sulfate powder was added to a sample to a final concentration of 1.5 M. The sample was applied to a TOYOPEARL PPG-600M column (Tosoh Biosciences) pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 1.5 M ammonium sulfate. After washing the column with the same buffer, the active fraction was eluted with a linear gradient of ammonium sulfate (1.5 M to 0 M) to obtain the active fraction (PPG600M). (7) The active fraction (PPG600M) obtained in (6) was applied to a Toyopearl Butyl-650M column (Tosoh Biosciences) pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 1.5 M ammonium sulfate. After washing the column with the same buffer, the active fraction was eluted with a linear gradient of ammonium sulfate (1.5 M to 0 M) in 20 mM Tris-HCl (pH 8.0) containing 1% Triton X-100 to obtain the active fraction (Butyl650M). (8) The active fraction (Butyl 650M) obtained in (7) was filtered through Amicon-100k (Merck Millipore), and the filtrate was collected. (9) The filtrate obtained in (8) was buffer-exchanged and concentrated using an Amicon Ultra-4 filter with a 30 kDa cutoff (Amicon-30k: Merck Millipore) in 20 mM Tris-HCl (pH 8.0) + 0.01% (w / v) Triton X-100. The volume of the concentrated sample was kept below 250 μL. (10) The sample obtained in (9) was applied to a Superdex200 Increase 10 / 300GL column (GE Healthcare Biosciences) pre-equilibrated with 20 mM Tris-HCl (pH 8.0) + 0.01% (w / v) Triton X-100, and size exclusion chromatography was performed with the same buffer to elute the active fraction (Superdex). (11) The active fraction (Superdex) obtained in (10) was applied to a TSKgel BioAssist Phenyl (Tosoh Corporation) column pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 1.5 M ammonium sulfate. After washing the column with the same buffer, the active fraction was eluted with a linear gradient of ammonium sulfate (1.5 M to 0 M) to obtain the active fraction (TSKPhe).

[0057] Table 3 shows the yield, yield, phospholipase D activity, and other data for each purification step. Phospholipase D activity was measured using the same method as in Example 1. Through these steps, purified PlsCho-PLD was obtained with an 8014-fold improvement in purification fold and a 1343-fold improvement in specific activity (Sp.act.). The molecular weight of the purified PlsCho-PLD (TSKPhe fraction) was measured by SDS-PAGE (12% (w / v) polyacrylamide gel) analysis, and the results are shown in Figure 2. The left lane (M: Marker) is a molecular weight marker, and the right lane shows the band of the active fraction (TSKPhe). As a result, three bands of approximately 90 kDa, approximately 43 kDa, and approximately 30 kDa, as indicated by the arrows, were observed.

[0058] [Table 3] [Example]

[0059] (Properties of purified PlsCho-PLD) The substrate specificity, optimum temperature, and optimum pH of the purified PlsCho-PLD were investigated. ◆Substrate specificity of PlsCho-PLD The phospholipase D activity of the purified PlsCho-PLD was measured in the same manner as in Example 1, except that the reaction was carried out using the enzyme reaction solution shown in Table 4 below at pH 7.2, 50°C for 2.5 minutes, to examine the substrate specificity. [Table 4]

[0060] The substrates used were PlsCho (sn-2 C18:1) (Avanti Polar Lipids), which has oleic acid ester-linked at the sn-2 position; PlsCho (sn-2 C20:4) (Avanti Polar Lipids), which has arachidonic acid ester-linked at the sn-2 position; PlsCho (sn-2 C22:6) (Avanti Polar Lipids), which has docosahexaenoic acid ester-linked at the sn-2 position; and sphingomyelin (SM; Sigma-Aldrich). PlsCho (sn-2 C18:1), PlsCho (sn-2 C20:4), and PlsCho (sn-2 C22:6) are shown in formulas (III) to (V).

[0061] (1) PlsCho(sn-2 C18:1) 1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphocholine

[0062] [ka]

[0063] (2) PlsCho(sn-2 C20:4) 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine

[0064] [ka]

[0065] (3) PlsCho(sn-2 C22:6) 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine

[0066] [ka]

[0067] Figure 3 shows the relative activity of phospholipase D enzymes using PlsCho (sn-2 C18:1) as a substrate, with the activity set at 100. The horizontal axis of Figure 3 indicates PlsCho18:1 when PlsCho (sn-2 C18:1) was used as a substrate, PlsCho20:4 when PlsCho (sn-2 C20:4) was used as a substrate, and PlsCho22:6 when PlsCho (sn-2 C22:6) was used as a substrate. As is clear from Figure 3, the relative activity was below the detection limit when PlsCho (sn-2 C20:4) or PlsCho (sn-2 C22:6) was used as a substrate. Similarly, the relative activity of SM was also below the detection limit. Therefore, it was revealed that the PlsCho-PLD produced by the A746 strain purified in Example 3 is specific to PlsCho (sn-2 C18:1) among choline-type plasmalogens.

[0068] ◆Optimum temperature of PlsCho (sn-2 C18:1) The purified PlsCho-PLD was added to a substrate solution containing 0.1% PlsCho (sn-2 18:1), 0.01% Triton X-100, and 50 mM Tris-HCl (pH 7.2, at each temperature) at a concentration of 2 (v / v)% to prepare a reaction solution shown in Table 5 below, with a total volume of 50 μL. This reaction solution was allowed to react for approximately 2.5 minutes. The hydrolytic activity was then measured. The results are shown in Figure 4. In Figure 4, the horizontal axis represents temperature (20, 30, 37, 40, 50, 60, and 70°C), and the vertical axis represents relative activity, with the activity at 50°C set at 100. As is clear from Figure 4, purified PlsCho-PLD exhibited high hydrolytic activity between 25°C and 65°C, with particularly high hydrolytic activity at 30°C to 60°C, preferably between 40°C and 60°C, and particularly around 50°C.

[0069] [Table 5]

[0070] ◆Optimal pH of PlsCho-PLD The purified PlsCho-PLD was added to a 50 μL reaction solution containing 0.1% PlsCho (sn-2 C18:1) substrate in a 50 mM buffer solution (pH 4, 5, 6, 7, 7.2, 8, 9, or 10; temperature 37°C) selected from acetate buffer (pH 4-5), citrate buffer (pH 4-6), BisTris-HCl buffer (BisTris: pH 6-7), HEPES buffer (HEPES: pH 7-8), Tris-HCl buffer (Tris: pH 7.2-9), or glycine buffer (pH 9-10) at 2% (v / v). The reaction solution was subjected to an enzymatic reaction for approximately 2.5 minutes at each pH. The enzymatic activity was then measured as in Example 1. The results are shown in Figure 5. In Figure 5, the horizontal axis shows pH, and the vertical axis shows relative activity, with the activity in Tris-HCl buffer at pH 7.2 set at 100. Figure 5 reveals that purified PlsCho-PLD has high hydrolytic activity around pH 4 and at pH 6 to 8, particularly at pH 6.5 to 7.5.

[0071] The importance of disulfide bonds The effect of disulfide bonds on the hydrolytic activity of PlsCho-PLD was examined. Purified PlsCho-PLD was added to a 50 mM Tris-HCl (pH 7.2, 50°C) solution at 2 (v / v)% and 1 mM inhibitors (dithiothreitol (DTT) as a reducing agent, 2-mercaptoethanol (2ME) as a reducing agent, iodobutyric acid (IAA) which irreversibly modifies cysteine ​​thiol groups and inhibits disulfide bonds, phenylmethylsulfonyl fluoride (PMSF) as a serine protease inhibitor, ethylenediaminetetraacetic acid (EDTA) as a metal chelator, or glycoletherdiaminetetraacetic acid (EGTA) as a metal chelator) to a total volume of 49 μL. After incubation at 50°C for approximately 5 minutes, the substrate 0.1% PlsCho (sn-2 18:1) / 0.001% Triton X-100 was added and the enzymatic reaction was carried out at 50°C for approximately 2.5 minutes. The enzymatic reaction was then carried out as in Example 1, and the hydrolytic activity was measured. The results are shown in Figure 6. In Figure 6, the horizontal axis shows the inhibitor activity, and the vertical axis shows the relative activity, with the activity in the reaction with water set at 100. As is clear from Figure 6, the activity was inhibited by reducing agents and alkylating agents that cleave the disulfide bond formed between the thiol groups of cysteines, demonstrating the importance of disulfide bonds in expressing the activity of this enzyme, PlsCho-PLD.

[0072] ◆ Metal ion requirements Metal ions (Ca 2+ ,Mg 2+ ,Mn 2+ ,Zn 2+ ,Co 2+ ,Al 3+ ,Fe 2+ ,Fe 3+ ,Cu 2+ The purified PlsCho-PLD was subjected to an enzymatic reaction at 50°C for approximately 2.5 minutes using the reaction mixture shown in Table 6 below, followed by the same procedure as in Example 1. The effect of metal ions on the hydrolytic activity of PlsCho-PLD was examined. The results are shown in Figure 7. Relative activity was calculated by comparing with that of the enzyme incubated in the absence of metal ions (free). EDTA (final concentration 2 mM) was also added as a chelating agent to evaluate metal ion requirement.

[0073] [Table 6]

[0074] As is clear from FIG. 7, the purified PlsCho-PLD was an enzyme that did not require metal ions. [Example]

[0075] (Search for PlsCho-PLD genes) The PlsCho-PLD gene from the A746 strain was searched for by the following method.

[0076] Using Hiseq® 2500 (Illumina), we performed de novo analysis of the draft genome using 250PE (paired-end) to create a CDS list, and found one gene that was predicted to belong to the phospholipase D superfamily.

[0077] Based on the above analysis, the amino acid sequence of PlsCho-PLD is a 387-amino acid sequence as set forth in SEQ ID NO:4. The Signal P program predicted that it contains a 47-amino acid Tat secretion signal at the N-terminus as set forth in SEQ ID NO:3. Therefore, the mature (active) sequence of PlsCho-PLD is a 340-amino acid sequence as set forth in SEQ ID NO:1. The molecular weight estimated by the GENETYX-MAC program was 38,568 and the isoelectric point estimated was 5.44. These isoelectric points are estimates and may differ from the measured values. Furthermore, the PlsCho-PLD protein contained an HKD motif (HxKxxxxD: H is histidine, K is lysine, D is aspartic acid, and x is any amino acid) at two positions (positions 81-88 and 258-265 in the amino acid sequence set forth in SEQ ID NO:1). Genome analysis revealed no gene products with a consensus sequence for known phospholipase Ds other than the 387-amino acid sequence set forth in SEQ ID NO:4. [Example]

[0078] (Expression of PlsCho-PLD in E. coli) Genomic DNA was obtained from the A746 strain in the same manner as described in Japanese Patent Application No. 2020-36298. Using the obtained genomic DNA as a template, PCR was performed using the forward primer PlsCho-FW1 described in SEQ ID NO: 5 and the reverse primer PlsCho-RV described in SEQ ID NO: 6. In the cDNA of the mature sequence excluding the Tat signal of the gene presumed to be PlsCho-PLD, an NcoI site was added to the 5' end and a nucleotide sequence encoding a HindIII site was added to the 3' end. The obtained PCR by-product was digested with NcoI and HindIII and inserted into the NcoI-HindIII site of the expression vector pET22b to obtain a recombinant plasmid (pET22b / PlsCho-PLD).

[0079] Using this recombinant plasmid (pET22b / PlsCho-PLD) as a template, a first inverse PCR was performed using the forward primer 22bPlCPLDHis.Fw described in SEQ ID NO: 7 and the reverse primer 22bPlCPLDHis.Rv described in SEQ ID NO: 8 to generate a plasmid (pET22b / PlsCho-PLD-His) containing a polynucleotide encoding PlsCho-PLD-His with a histidine tag attached to the C-terminus.

[0080] Using the obtained plasmid (pET22b / PlsCho-PLD-His) as a template, inverse PCR was performed using the forward primer 22bPlsChoN_delFw. (SEQ ID NO: 9) and the reverse primer 22bPlsChoN_delRv. (SEQ ID NO: 10) to generate a recombinant plasmid (pET22b / PlsCho-PLD-His: no signal) in which only the region encoding the pelB signal sequence from pET22b was deleted. pET22b was used as a control plasmid. Next, the obtained recombinant plasmid or the control plasmid pET22b was transformed into E. coli BL21(DE3) to obtain recombinant E. coli. The obtained recombinant E. coli was cultured in 100 mL of ZYM5052 medium (Studier, FW Protein Expr. Purif. 41, 207-234) containing 30 μg / mL ampicillin at 30°C for 24 hours. The resulting culture was centrifuged to collect the bacterial cells. The cells were suspended in 20 mM TrisHCl buffer (pH 7.0) and then sonicated to obtain bacterial lysates from E. coli transformed with each plasmid. Each lysate was then centrifuged at 14,000 rpm for 15 minutes to obtain supernatants containing the enzyme PlsCho-PLD (approximately 0.04 U / mL) expressed in E. coli or used as a control.

[0081] The molecular weight of the enzyme PlsCho-PLD expressed in E. coli was measured by SDS-PAGE analysis, and the results are shown in Figure 8. In Figure 8, the left and right ends are markers, "+" indicates the enzyme PlsCho-PLD solution expressed in E. coli, and "-" indicates the case where the control supernatant was applied. As shown in Figure 8, a band of approximately 43 kDa was increased in the "+" lane for the enzyme PlsCho-PLD solution expressed in E. coli, indicating that the molecular weight of the expressed PlsCho-PLD (including the histidine tag) was approximately 43.3 kDa.

[0082] Furthermore, the results of Western blot analysis of each of the bacterial lysates and supernatants (enzyme PlsCho-PLD solution expressed in E. coli or control supernatant) are shown in Figure 9. In Figure 9, "+" indicates the application of bacterial lysates obtained by transforming E. coli with the recombinant plasmid or their centrifuged supernatant (enzyme PlsCho-PLD solution expressed in E. coli), and "-" indicates the application of bacterial lysates obtained by transforming E. coli with the control plasmid or their centrifuged supernatant (control supernatant). The primary antibody used was His-Tag(D3I1O)XPRabbit mAb (registered trademark: Cell Signaling Technology).

[0083] As shown in Figure 9, a band was detected at around 43 kDa, confirming that the molecular weight of PlsCho-PLD was approximately 43 kDa. [Example]

[0084] (Creating a calibration curve) A calibration curve for PlsCho-PLD was prepared using the solution of PlsCho-PLD enzyme expressed in E. coli prepared in Example 6. PlsCho-PLD was quantified by quantifying choline obtained by hydrolysis of PlsCho as described in Example 1.

[0085] (Measurement of phospholipase D activity) Phospholipase D activity was measured by measuring fluorescence intensity using the following method. 1) The enzyme PlsCho-PLD solution expressed in E. coli prepared in Example 6 was centrifuged, and the resulting supernatant was used as an enzyme sample. 2) PlsCho-PLD was dispensed into a 96-well plate. 3) The fluorometric enzyme reaction solution and enzyme sample shown in Table 7 below were added and shaken for about 1 minute.

[0086] [Table 7] 4) The mixture was kept at 50°C for about 60 minutes. 5) After the reaction, the fluorescence intensity (EX 535 nm, EM 595 nm) was measured using a plate reader (BECKMAN COULTER).

[0087] The substrate used was 5, 25, or 50 μg of PlsCho(sn-2 C18:1). The results are shown in FIG.

[0088] As shown in Figure 10, it was confirmed that the fluorescence intensity increased in a concentration-dependent manner depending on PlsCho (sn-2 C18:1) when the enzyme PlsCho-PLD solution expressed in E. coli prepared in Example 6 was used, and that PlsCho (sn-2 C18:1) could be detected. [Example]

[0089] (substrate specificity) In Example 7, PlsCho (sn-2 C18:1) was used as the substrate. Substrate specificity was also examined using PlsCho (sn-2 C20:4), PlsCho (sn-2 C22:6), and phosphatidylcholine (PtdCho). The PlsCho-PLD enzyme solution expressed in E. coli prepared in Example 6 was reacted with substrates (10 μg each) for 30 minutes in a manner similar to that described in Example 7, and the activity of PlsCho-PLD was measured. The results are shown in Figure 11. In the figure, C18:1, C20:4, and C22:6 represent the results obtained using PlsCho (sn-2 C18:1), PlsCho (sn-2 C20:4), and PlsCho (sn-2 C22:6), respectively. The vertical axis indicates the relative activity, with C18:1 set to 1.

[0090] As shown in FIG. 11, it was revealed that the enzyme PlsCho-PLD solution expressed in E. coli prepared in Example 6 had extremely high substrate specificity for PlsCho (sn-2 C18:1). [Example]

[0091] (Quantitative determination of choline-type plasmalogens in plasma) 1. Plasma Preparation Because serum and plasma are not cell membranes, they contain significantly less plasmalogen than red blood cells and white blood cells. Therefore, measuring the amount of plasmalogen in serum or plasma has been difficult using conventional techniques. Meanwhile, plasmalogens have recently been considered to be biomarkers for coronary artery disease, and quantifying choline-type plasmalogen in serum or plasma is important for determining the risk of developing diseases such as coronary artery disease. Therefore, we investigated whether choline-type plasmalogen in plasma can be quantified using the PlsCho-PLD solution of the enzyme expressed in Escherichia coli prepared in Example 6.

[0092] Plasma was prepared according to the method described in JP 2016-111929 A. Briefly, venous blood was collected using a heparin-containing blood collection tube (Terumo Corporation), centrifuged at 1000 × g for 5 minutes, and the supernatant (i.e., plasma) was collected.

[0093] (Plasma sample preparation) The plasma collected above was stored frozen, thawed, and adjusted with 50 mM Tris-HCl buffer (pH 7.4) before measurement.

[0094] (Analysis by fluorometer) The reaction was carried out in the same manner as in Example 7, except that 20, 30, or 40 μL of the above plasma sample was used instead of PlsCho (sn-2 C18:1). The results are shown in FIG.

[0095] (result) As shown in Figure 12, it was confirmed that the fluorescence intensity increased in proportion to the amount of plasma sample. Combined with the results of Examples 7 and 8, it was confirmed that the choline-type plasmalogen PlsCho (sn-2 C18:1) in plasma can be specifically quantified by using the PlsCho-PLD enzyme solution expressed in E. coli prepared in Example 6. [Industrial Applicability]

[0096] Use of the phospholipase D of the present invention makes it possible to specifically detect or quantify PlsCho (sn-2 C18:1) at low cost, and is therefore highly useful industrially.

Claims

1. Use of phospholipase D, comprising a polypeptide according to any one of (1-1) to (1-3) below, for quantifying a choline-type plasmalogen in which oleic acid is ester-linked to the sn-2 position. (1-1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (1-2) A polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 1 and having hydrolytic activity towards a phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-bonded to the sn-2 position of the glycerol backbone, wherein the hydrolytic activity towards a phosphate ester bond when a choline-type plasmalogen in which oleic acid is ester-bonded to the sn-2 position is used as a substrate is taken as 100, and the relative activity when a choline-type plasmalogen in which arachidonic acid is ester-bonded to the sn-2 position is used as a substrate is 10 or less, and the relative activity when a choline-type plasmalogen in which docosahexaenoic acid is ester-bonded to the sn-2 position is used as a substrate is 10 or less; (1-3) A polypeptide in which one or several amino acids are added, substituted, deleted and / or inserted in the amino acid sequence set forth in SEQ ID NO: 1, and has hydrolytic activity towards phosphate ester bonds in the choline-type plasmalogen molecule, and when the hydrolytic activity towards phosphate ester bonds when a choline-type plasmalogen in which oleic acid is ester-bonded at the sn-2 position is used as a substrate is taken as 100, the relative activity value when a choline-type plasmalogen in which arachidonic acid is ester-bonded at the sn-2 position is used as a substrate is 10 or less, and the relative activity value when a choline-type plasmalogen in which docosahexaenoic acid is ester-bonded at the sn-2 position is used as a substrate is 10 or less;

2. A method for quantifying a choline-type plasmalogen in which oleic acid is ester-bonded to the sn-2 position in a sample, the method comprising the step of reacting the sample with a phospholipase D containing a polypeptide according to any one of (1-1), (1-2'), and (1-3') below; (1-1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1; (1-2') A polypeptide having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 1 and having hydrolysis activity for the phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-bonded to the sn-2 position of the glycerol backbone; (1-3') A polypeptide in which one or several amino acids are added, substituted, deleted and / or inserted in the amino acid sequence described in SEQ ID NO: 1, and the choline-type plasmalogen molecule. A polypeptide having hydrolytic activity against a phosphate ester bond.

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