JP7773142B2 - Method for quantifying phospholipase D and choline-type plasmalogen - Google Patents

Description

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.

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 muscles, and are also found in the brain.

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 is also drawing attention, as evidenced by a report of a correlation between blood concentrations 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).

Amid this, research into measuring plasmalogens has also progressed. For example, a testing method has been proposed for determining whether a test blood sample is derived from a mammal with dementia (see Patent Document 1), which includes: (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.

In addition, although phospholipase D derived from Streptomyces sp. has high phospholipase D activity toward lysophosphatidylcholine and phosphatidylcholine, based on the discovery of phospholipase D activity toward choline-type plasmalogens, it has been proposed as a phospholipase D that acts on choline-type plasmalogens to liberate choline (see Patent Document 2).

International Publication No. 2012/090625 JP 2014-082991 A

Guan Z, et al., J Neuropathol Exp Neurol; 58(7):740-747 (1999) Fujino et al., J Alzheimers Dis Parkinsonism; 2018,8(1) DOI: 10.4172/2161-0460.1000419 Goodenowe DB et al., J Lipid Res Nov;48(11): 2485-2498 (2007) Oma et al., Dement Geriatr Cogn Disord Extra;2:298-303 (2012) Nishimukai et al., Clinica Chimica Acta; 437(1):147-154 (2014)

The objective of the present invention is to obtain a new phospholipase D that acts on a choline-type plasmalogen in which oleic acid is ester-linked at the sn-2 position (hereinafter also referred to as "PlsCho (sn-2 C18:1)").

As a result of extensive research aimed at solving the above-mentioned problems, the inventors discovered 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 discovered 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.

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 a phosphate ester bond 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-bonded to the sn-2 position, and having the following properties (2-1) to (2-4).
(2-1) has hydrolytic activity against the phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-bonded to the sn-2 position;
(2-2) The molecular weight measured by SDS-PAGE analysis is 38,000 to 50,000;
(2-3) Optimum temperature: 40 to 60°C under reaction conditions of pH 7.2 and 2.5 minutes;
(2-4) Derived from a microorganism 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 when a choline-type plasmalogen having oleic acid ester-bonded to the sn-2 position as a substrate is taken as 100, the relative activity when a choline-type plasmalogen having arachidonic acid ester-bonded to the sn-2 position as a substrate 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 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 the sample, comprising a step of reacting the sample with the phospholipase D according to 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.

The phospholipase D of the present invention makes it possible to hydrolyze choline-type plasmalogens in which oleic acid is ester-linked at the sn-2 position.

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. 1 shows the results of analyzing purified phospholipase D (DEAE fraction) by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in Example 3. FIG. 1 shows the results of measuring phospholipase D activity using purified phospholipase D (DEAE fraction) in Example 4. The activity is shown as a relative activity when the phospholipase D activity when PlsCho having oleic acid ester-linked at the sn-2 position was used as a substrate was taken as 100. 1 shows the results of investigating the optimum temperature of purified phospholipase D in Example 4. 1 shows the results of investigating the optimum pH of purified phospholipase D in Example 4. FIG. 1 shows the results of investigating the importance of disulfide bonds in purified phospholipase D in Example 4. FIG. 1 shows the results of investigating the metal ion requirement of purified phospholipase D in Example 4. FIG. 10 shows the results of SDS-PAGE analysis of the enzyme PlsCho-PLD solution obtained by expression in Escherichia coli in Example 6. 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 Escherichia coli in Example 6. FIG. 10 shows the results of detecting PlsCho-PLD activity using PlsCho (sn-2 C18:1) as a substrate in Example 7. This shows the results of investigating substrate specificity in Example 8. FIG. 10 shows the results of detecting choline-type plasmalogen in plasma in Example 9.

[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 a phosphate ester bond 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)."

The phospholipase D-(2) of the present invention is
(2-1) has hydrolytic activity against the phosphate ester bond in a choline-type plasmalogen molecule in which oleic acid is ester-bonded to the sn-2 position;
(2-2) The molecular weight measured by SDS-PAGE analysis is 38,000 to 50,000;
(2-3) Optimum temperature: 40 to 60°C under reaction conditions of pH 7.2 and 2.5 minutes;
(2-4) Derived from a microorganism 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)."
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."

The choline-type plasmalogen herein, in which oleic acid is ester-bonded at the sn-2 position, is a subclass 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 to oleic acid (18 carbon atoms with one double bond, 18:1) at the C2 (sn-2) position, as long as the base is choline. The structure is shown in formula (I) below.

In the formula, ^R1 is typically an aliphatic hydrocarbon group having 1 to 20 carbon atoms, such as a dodecyl group, a tetradecyl group, a hexadecyl group, an octadecyl group, an icosanyl group, etc. In the formula, ^R2 is an unsaturated aliphatic hydrocarbon group having 18 carbon atoms and one double bond derived from an unsaturated fatty acid, such as an octadecenoyl group.

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).

Whether phospholipase D has the activity to hydrolyze the phosphate ester within the PlsCho (sn-2 C18:1) molecule can be determined, for example, by allowing phospholipase D to act on PlsCho (sn-2 C18:1) and quantifying the choline released by hydrolysis. The quantification of choline is described below.

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 between 38,000 and 50,000, and preferably between 42,000 and 45,000. The isoelectric point (calculated using Genetyx) is typically between 5.2 and 5.6, and preferably 5.44.

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.

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.

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, and Streptomyces ambofaciens. Examples of related microorganisms include Streptomyces ambofaciens, Streptomyces albogriseolus, Streptomyces viridodiastaticus, and Streptomyces coelicolor. Whether or not a microorganism is closely related can be determined by, for example, estimating a phylogenetic tree using the neighbor-joining method and performing molecular phylogenetic analysis.

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 at the sn-2 position is used as a substrate, when the hydrolysis activity toward phosphate ester bonds is determined to be 100 when reacted under reaction conditions of pH 7.2, 50°C, and 2.5 minutes using PlsCho (sn-2 C18:1) as a substrate. The 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 at the sn-2 position is used as a substrate, can be 10 or less, preferably 5 or less, and more preferably 3 or less, when a choline-type plasmalogen having docosahexaenoic acid ester-linked at the sn-2 position is used as a substrate.

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

In the above-mentioned polypeptide of (1-2) of the present phospholipase D-(1), "a polypeptide having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 1 and having hydrolytic activity against the phosphate ester bond in the PlsCho (sn-2 C18:1) molecule" can include 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 hydrolytic activity against 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 phosphate ester bonds within a 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 HRKVLLTD positions 81 to 88, and HTKIITVD positions 258 to 265 (HKD 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). The HKD motif is an amino acid sequence that contributes to the catalytic reaction of phospholipase D.

The "polypeptide with one or more amino acids added, substituted, deleted, and/or inserted" in the above-mentioned polypeptide of the present phospholipase D-(1) (1-3) can be a polypeptide with 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 with one or more amino acids added, substituted, deleted, and/or inserted" is preferably a polypeptide with 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.

Preferable examples of the present phospholipase D-(1) polypeptide (1-2) or (1-3) 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, exhibit 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 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.

When reacting the present phospholipase D 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 reaction can be 3.5 to 4.5, or 5.5 to 8.0, preferably 6.5 to 7.5. The temperature during reaction can be 40 to 60°C, preferably 45 to 55°C.

(Method for quantification of 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 a step of reacting the sample with the above-mentioned phospholipase D, and the substrate specificity of this phospholipase D makes it possible to accurately quantify 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, and then a solution containing choline oxidase, peroxidase, and a color reagent is added to cause 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.

Examples of the above-mentioned samples 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 using a conventional blood collection method (e.g., syringe blood collection or vacuum blood collection) using a known method, such as centrifuging (e.g., 1000 x g for 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.

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.

[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 the polynucleotide encoding the present phospholipase D include a polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. Examples of the polynucleotide encoding the 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 a polynucleotide 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.

The expression vector may be of any form, such as circular or linear. It can be selected appropriately depending on the host cell to be used, and examples include plasmid vectors, viral vectors, and phages. Commercially available expression vectors can also be used. Examples of plasmid vectors include, for example, when Escherichia coli is used as a host, 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, pACYC184 vectors, pUC vectors such as pUC12, pUC13, pUC18, pUC19 and pUC118, and Bluescript KS+ vectors; and when actinomycetes is used as a host, examples of the plasmid vector include pUC702 vectors, pIJ680 vectors, pIJ702 vectors, pTONa5 vectors, pTONashort vectors, pTip vectors, and pNit vectors. Furthermore, when a Gram-positive bacterium is used as the host, examples of vectors include the pBIC vector, pWH1520 vector, pMM1522 vector, and pHIS1522 vector. When a yeast, particularly Saccharomyces cerevisiae, is used as the host, examples of vectors include the YRp7 vector, pYC1 vector, and YEp13 vector.

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 the host cells. Examples of secretion signals include the Tat secretion signal shown in SEQ ID NO: 3, as well as 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 Profinity eXact fusion tag. The polynucleotides encoding the affinity and solubility tags, drug resistance genes, reporter genes, and other selection markers may be located at either the 5' end or the 3' end of the polynucleotide encoding the phospholipase D within the expression vector.

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 produced using 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.

[Transformant]
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 to be 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 and Pichia cerevisiae, and filamentous fungi of the genus Aspergillus such as Aspergillus oryzae and Aspergillus nigar.

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

[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, 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.

The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

(Search for microorganisms that produce phospholipase D with high substrate specificity for PlsCho (sn-2 C18:1))
Based on their past experience, the present inventors focused on actinomycetes as microorganisms that produce 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 stock (stored at -80°C) of actinomycete strain was smeared on ISP2 agar medium using a platinum loop and cultured at 28°C.
2) Grown colonies were collected 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 with shaking at 28°C and 160 strokes per minute (spm) for 72 hours. 1 mL of the culture was inoculated into 100 mL of MPD medium and cultured with shaking at 28°C and 160 rpm for 72 hours.

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.

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 is peroxidase, COD is choline oxidase, 4-AA is 4-aminoantipyrine, and TODB is N,N-bis(4-sulfobutyl)-3-methylaniline.

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

Among the strains confirmed to have phospholipase D activity toward PlsCho (sn-2 C18:1) in which oleic acid is ester-linked at the sn-2 position, a substrate specificity test was conducted on strains with high activity. In the substrate specificity test, phospholipase D activity toward three types of lipids was measured: PlsCho in which oleic acid is ester-linked at the sn-2 position (PlsCho (sn-2 C18:1)) (Avanti Polar Lipids), PlsCho with C20:4 at the sn-2 position (PlsCho (sn-2 C20:4)) (Avanti Polar Lipids), and PlsCho 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 these, strain A746 was selected as a PlsCho-PLD producer, as 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.

(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.

The base sequence of the amplified 16S rDNA was determined using ChromasPro 2.1 (Technelysium). The base sequence of the amplified 16S rDNA was compared using a BLAST homology search with the 16S rDNA base sequences of known microorganisms obtained from the international base sequence databases DDBJ, ENA (EMBL), and GenBank to compare the degree of DNA homology with known microorganisms. A phylogenetic tree was created using the Clustal W program described in a literature reference (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 is the scale bar, the numbers at the phylogenetic branch branches are bootstrap values, and the T at the end of the strain name indicates the type strain of that species.

A BLAST homology search showed that the 16S rDNA partial sequence of strain A746 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.

Furthermore, simple morphological observation revealed powdery colonies with a gray surface and a pale yellow underside. Microscopic observation also revealed the formation of aerial mycelia (hyphal width 1 μm) and chain spores.

The phylogenetic tree in Figure 1 and the results of simple morphological observation confirmed that strain A746 belongs to the genus Streptomyces, and that its most closely related microorganisms are Streptomyces griseorubens or Streptomyces griseoflavus.

(Purification of PlsCho-PLD)
PlsCho-PLD produced by strain A746 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 specified, 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 minutes). This precipitate was 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.

(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 M to 1 M) to obtain the active fraction (DEAE650M).

(6) To the active fraction (DEAE650M) obtained in (5), ammonium sulfate powder was added to the 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, and 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 subjected to buffer exchange and concentration using an Amicon Ultra-4 (Amicon-30k: Merck Millipore) with a 30 kDa cut-off and 20 mM Tris-HCl (pH 8.0) + 0.01% (w/v) Triton X-100. The volume of the concentrated sample was 250 μL or less.
(10) The sample obtained in (9) was applied to a Superdex 200 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, thereby obtaining 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 an active fraction (TSKPhe).

The yield, yield, phospholipase D activity, etc. for each purification step are shown in Table 3. Phospholipase D activity was measured using the same method as in Example 1. Through the above 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.

(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 PlsCho-PLD purified above was used to measure phospholipase D activity and examine substrate specificity in the same manner as in Example 1, except that the enzyme reaction solution shown in Table 4 below was used and the reaction was carried out at pH 7.2, 50°C for 2.5 minutes.

The substrates used were PlsCho (sn-2 C18:1) (Avanti Polar Lipids) with oleic acid ester-linked at the sn-2 position, PlsCho (sn-2 C20:4) (Avanti Polar Lipids) with arachidonic acid ester-linked at the sn-2 position, PlsCho (sn-2 C22:6) (Avanti Polar Lipids) with 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).

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

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

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

Figure 3 shows the relative activity of phospholipase D when PlsCho (sn-2 C18:1) is used as a substrate, with the activity taken as 100. On the horizontal axis of Figure 3, PlsCho18:1 represents the activity when PlsCho (sn-2 C18:1) is used as a substrate, PlsCho20:4 represents the activity when PlsCho (sn-2 C20:4) is used as a substrate, and PlsCho22:6 represents the activity when PlsCho (sn-2 C22:6) is used as a substrate. As is clear from Figure 3, the relative activity when PlsCho (sn-2 C20:4) or PlsCho (sn-2 C22:6) is used as a substrate was below the detection limit. Similarly, the relative activity with 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.

◆ Optimum Temperature for PlsCho (sn-2 C18:1) Purified PlsCho-PLD was added to a substrate solution of 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, 70°C), and the vertical axis represents the relative activity, with the activity at 50°C set at 100. As is clear from FIG. 4, the purified PlsCho-PLD has high hydrolytic activity at 25°C to 65°C, and the hydrolytic activity is particularly high at 30°C to 60°C, preferably 40 to 60°C, and particularly around 50°C.

Optimal pH of PlsCho-PLD
The substrate was 0.1% PlsCho (sn-2 C18:1) and a buffer solution (pH 4, 5, 6, 7, 7.2, 8, 9, or 10: temperature 37 ° C.) selected from acetate buffer (Acetate: pH 4-5), citrate buffer (Citric: 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 (Glycine: pH 9-10). Purified PlsCho-PLD was added to a 50 mM solution to a concentration of 2% (v/v), and a reaction solution with a total volume of 50 μL was prepared. This reaction solution was subjected to an enzymatic reaction for about 2.5 minutes under each pH condition. The subsequent reaction was carried out in the same manner as in Example 1, and the enzyme activity was measured. The results are shown in Figure 5. In Figure 5, the horizontal axis represents pH, and the vertical axis represents relative activity when the activity in the reaction at pH 7.2 in Tris-HCl buffer is set at 100. Figure 5 reveals that the purified PlsCho-PLD has high hydrolytic activity around pH 4 and at pH 6 to 8, particularly at pH 6.5 to 7.5.

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 solution of Tris-HCl (pH 7.2, 50°C) to a concentration of 2 (v/v)% and 1 mM inhibitors (dithiothreitol: DTT, a reducing agent; 2-mercaptoethanol: 2ME, a reducing agent; iodobutyric acid: IAA, which irreversibly modifies cysteine thiol groups and inhibits disulfide bonds; phenylmethylsulfonyl fluoride: PMSF, a serine protease inhibitor; ethylenediaminetetraacetic acid: EDTA, a metal chelating agent; or glycoletherdiaminetetraacetic acid: EGTA, a metal chelating agent) to a total volume of 49 μL. After incubating 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 enzyme reaction was carried out at 50°C for approximately 2.5 minutes. The subsequent reaction was carried out in the same manner as in Example 1, and the hydrolytic activity was measured. The results are shown in Figure 6. In Figure 6, the horizontal axis represents the inhibitor and the vertical axis represents 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 the expression of the activity of this enzyme, PlsCho-PLD.

◆ Metal ion requirement The purified PlsCho-PLD described above and metal ions (Ca ²⁺ , Mg ²⁺ , Mn ²⁺ , Zn ²⁺ , Co ²⁺ , Al ³⁺ , Fe ²⁺ , Fe ³⁺ , Cu ²⁺ final concentration 2 mM) were subjected to an enzymatic reaction in the reaction solution shown in Table 6 below at 50°C for approximately 2.5 minutes. The subsequent reaction was carried out in the same manner as in Example 1, and 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 under conditions without metal ions (free). In addition, EDTA (final concentration 2 mM) was added as a chelating agent to evaluate the metal ion requirement.

As is clear from Figure 7, the purified PlsCho-PLD was an enzyme that did not require metal ions.

(Search for the PlsCho-PLD gene)
The PlsCho-PLD gene derived from the A746 strain was searched for by the following method.

A draft genome analysis was performed using a Hiseq® 2500 (Illumina) de novo analysis with 250PE (paired end) to create a CDS list. Among the CDS, one gene predicted to belong to the phospholipase D superfamily was identified.

The above analysis revealed that the amino acid sequence of PlsCho-PLD is a sequence consisting of 387 amino acids as set forth in SEQ ID NO:4, and that it contains a Tat secretion signal consisting of 47 amino acids as set forth in SEQ ID NO:3 at the N-terminus, as predicted by the Signal P program. Therefore, the mature (active) sequence of PlsCho-PLD is a sequence consisting of 340 amino acids as set forth in SEQ ID NO:1. The GENETYX-MAC program predicted a molecular weight of 38,568 and an isoelectric point of 5.44. This is an estimated value and may differ from the measured value. Furthermore, the HKD motif (HxKxxxxD: H is histidine, K is lysine, D is aspartic acid, and x is any amino acid) was present at two locations (positions 81-88 and 258-265 in the amino acid sequence set forth in SEQ ID NO:1). Furthermore, as a result of genome analysis, no gene products with a consensus sequence among known phospholipase Ds were found other than the sequence consisting of 387 amino acids set forth in SEQ ID NO: 4.

(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, and 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 base 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).

Using this recombinant plasmid (pET22b/PlsCho-PLD) as a template, a first round of 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.

Using the plasmid obtained above (pET22b/PlsCho-PLD-His) as a template, inverse PCR was performed using the forward primer 22bPlsChoN_delFw. described in SEQ ID NO: 9 and the reverse primer 22bPlsChoN_delRv. described in 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 had been 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 resulting recombinant E. coli was cultured for 24 hours at 30°C in 100 mL of ZYM5052 medium (Studier, F.W. Protein Expr. Purif. 41, 207-234) containing 30 μg/mL ampicillin. The resulting culture was centrifuged to collect the bacterial cells. The bacterial cells were suspended in 20 mM TrisHCl buffer (pH 7.0) and then sonicated to obtain bacterial lysates derived from E. coli transformed with each plasmid. Each bacterial lysate was then centrifuged at 14,000 rpm for 15 minutes to obtain the supernatant, which was used as either a solution of the enzyme PlsCho-PLD (approximately 0.04 U/mL) expressed in E. coli or a control supernatant.

Figure 8 shows the results of SDS-PAGE analysis of the molecular weight of the enzyme PlsCho-PLD expressed in E. coli. In Figure 8, the left and right edges 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, revealing that the molecular weight of the expressed PlsCho-PLD (including the histidine tag) is approximately 43.3 kDa.

Furthermore, Figure 9 shows 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). In Figure 9, "+" indicates the application of bacterial lysate obtained by transforming E. coli with the recombinant plasmid or its centrifuged supernatant (enzyme PlsCho-PLD solution expressed in E. coli), and "-" indicates the application of bacterial lysate obtained by transforming E. coli with the control plasmid or its centrifuged supernatant (control supernatant). The primary antibody used was His-Tag(D3I1O)XPRabbit mAb (registered trademark: Cell Signaling Technology).

As shown in Figure 9, a band was detected around 43 kDa. These results also confirmed that the molecular weight of PlsCho-PLD is approximately 43 kDa.

(Creating a calibration curve)
A calibration curve for PlsCho-PLD was prepared using the enzyme PlsCho-PLD solution 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.

(Measurement of Phospholipase D Activity)
Phospholipase D activity was measured by measuring the fluorescence intensity according to 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.

4) The mixture was kept at 50°C for approximately 60 minutes.
5) After the reaction, the fluorescence intensity (EX 535 nm, EM 595 nm) was measured using a plate reader (Beckman Coulter).

The substrate used was 5, 25, or 50 μg of PlsCho (sn-2 C18:1). The results are shown in Figure 10.

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

(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 the same manner as described in Example 7, and the activity of PlsCho-PLD was measured. The results are shown in FIG. 11. In the figure, C18:1, C20:4, and C22:6 represent the results when PlsCho (sn-2 C18:1), PlsCho (sn-2 C20:4), and PlsCho (sn-2 C22:6) were used as substrates, respectively. The vertical axis indicates the relative activity when the activity in the case of C18:1 is set to 1.

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

(Quantitation of choline-type plasmalogen in plasma)
1. Preparation of Plasma 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 it was possible to quantify choline-type plasmalogen in plasma using the PlsCho-PLD solution of the enzyme expressed in Escherichia coli prepared in Example 6.

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.

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

(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.

(result)
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 enzyme PlsCho-PLD solution expressed in E. coli prepared in Example 6 can be used to specifically quantify PlsCho (sn-2 C18:1), a choline-type plasmalogen in plasma.

The phospholipase D of the present invention enables inexpensive and specific detection or quantification of PlsCho (sn-2 C18:1), making it highly useful industrially.

Claims (2)

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 ; 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 .