JP7533918B2 - Method for quantifying phospholipase D and ethanolamine-type plasmalogen - Google Patents

Description

The present invention relates to a method for quantifying phospholipase D and ethanolamine-type plasmalogen, an expression vector, a transformant into which the expression vector has been introduced, 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. Such plasmalogens are present in all mammalian tissues and are said to account for approximately 18% of the phospholipids in the human body, and are found in particularly large amounts in brain nerve cells, cardiac muscle, lymphocytes, macrophages, etc.

In recent years, it has been disclosed that plasmalogens are significantly decreased in the brains of patients with Alzheimer's dementia and mild cognitive impairment (MCI) (see Non-Patent Documents 1 and 2), and that plasmalogen concentrations are decreased in the blood of patients with Alzheimer's dementia (see Non-Patent Documents 3 and 4), and the relationship between Alzheimer's dementia and plasmalogens has attracted attention.

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 taken.

In addition, a method for detecting and quantifying ethanolamine-type plasmalogens has also been proposed that involves hydrolyzing ethanolamine-type plasmalogens in a sample with phospholipase A1 to generate ethanolamine-type lysoplasmalogens, which are then treated with phospholipase D (see Patent Documents 2-4 and Non-Patent Document 5).

International Publication No. 2012/090625 JP 2016-111929 A JP 2016-45112 A International Publication No. 2014/013538

Guan Z, et al., J Neuropathol Exp Neurol; 58(7):740-747 (1999) Fujino et al., J Alzheimers Dis Parkinsonism; 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) Sakasegawa SI et al., Biotechnol Lett;38(1):109-16(2016)

Conventional methods for measuring plasmalogen require large and expensive equipment, such as high-performance liquid chromatography. Ethanolamine-type plasmalogen is used as an indicator of Alzheimer's dementia. To measure this ethanolamine-type plasmalogen, it is first hydrolyzed with phospholipase A1, but there are problems such as the two-step reaction being complicated and cumbersome, as well as high costs, and the difficulty of producing phospholipase A1 using recombinant Escherichia coli. In addition, the phospholipases known so far have low substrate specificity, particularly for ethanolamine-type plasmalogen, and have the problem of hydrolyzing phospholipids other than plasmalogen, which is a biomarker for Alzheimer's dementia. Therefore, the object of the present invention is to provide phospholipase D with high substrate specificity for ethanolamine-type plasmalogen, and to provide a method for quantifying ethanolamine-type plasmalogen using such phospholipase D.

The present inventors conducted extensive research to solve the above problems and found that phospholipase D derived from Streptomyces polychromogenes or the AK461 strain of the Streptomyces genus, which is closely related to Streptomyces racemochromogenes, has high substrate specificity for ethanolamine-type plasmalogens. Furthermore, they found that ethanolamine-type plasmalogens can be detected or quantified, and can be selectively quantified, by using such phospholipase D, and thus completed 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 to the amino acid sequence set forth in SEQ ID NO:1 and having a hydrolysis action on a phosphate ester bond in an ethanolamine-type plasmalogen molecule;
(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 which has a hydrolysis effect on the phosphate ester bond in an ethanolamine-type plasmalogen molecule;
[2] Phospholipase D having a hydrolytic effect on a phosphate ester bond in an ethanolamine-type plasmalogen molecule, and having the following properties (2-1) to (2-5).
(2-1) Has a hydrolytic effect on the phosphate ester bond in the ethanolamine-type plasmalogen molecule;
(2-2) Molecular weight is 50,000 to 60,000;
(2-3) Optimum temperature: 40 to 60° C. under conditions of pH 7.0 and 30 minutes of reaction;
(2-4) Optimum pH: pH 5.5 to 7.5 under reaction conditions of 37°C and 30 minutes;
(2-5) 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 polychromogenes or Streptomyces racemochromogenes.
[4] Phospholipase D according to [2] or [3] above, characterized in that, when the hydrolysis activity towards phosphate ester bonds when ethanolamine-type plasmalogen is used as a substrate is taken as 100, the relative activity when ethanolamine-type lysoplasmalogen is used as a substrate is 5 or less, the relative activity when phosphatidylethanolamine is used as a substrate is 5 or less, and/or the relative activity when lysophosphatidylethanolamine is used as a substrate is 5 or less.
[5] A method for quantifying ethanolamine-type plasmalogen in a sample, comprising a step of reacting the sample with phospholipase D according to any one of [1] to [4] above.
[6] An expression vector comprising a polynucleotide encoding the phospholipase D according to [1] above.
[7] A transformant incorporating 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 ethanolamine-type plasmalogens. Therefore, the phospholipase D of the present invention makes it possible to easily and quickly detect or quantify ethanolamine-type plasmalogens.

1 is a molecular phylogenetic tree based on approximately 1,500 base pairs of the 16S ribosomal DNA gene of the AK461 strain, prepared in Example 2. 1 shows the results of SDS-PAGE analysis of the purified phospholipase D of the present invention (hereinafter also referred to as "PlsEtn-PLD") (DEAE fraction) in Example 3. FIG. 1 shows the results of measuring phospholipase D activity using purified PlsEtn-PLD (DEAE fraction) in Example 5. The activity is shown as a relative activity when the phospholipase D activity when PlsEtn 22:6 is used as a substrate is taken as 100. This shows the results of investigating the optimum temperature of purified PlsEtn-PLD in Example 5. This shows the results of investigating the optimum pH of purified PlsEtn-PLD in Example 5. FIG. 13 shows the detection results of ethanolamine-type plasmalogen separated from scallops in Example 8. FIG. 13 shows the detection results of soybean-derived phosphatidylethanolamine (PE) in a comparative example. FIG. 13 shows the detection results of ethanolamine-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 to the amino acid sequence set forth in SEQ ID NO:1 and having a hydrolysis action on a phosphate ester bond in an ethanolamine-type plasmalogen molecule;
(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 which has a hydrolysis effect on the phosphate ester bond in an ethanolamine-type plasmalogen molecule;
The present invention is a phospholipase D comprising a polypeptide according to any one of (1-1) to (1-3) above, hereinafter also referred to as "the present phospholipase D-(1)." As described below, the 26 amino acids on the N-terminal side of SEQ ID NO: 1, i.e., the 1st to 26th amino acids in SEQ ID NO: 1, are considered to be a Sec secretion signal. Therefore, the phospholipase D may be a sequence excluding amino acids considered to be a Sec secretion signal, such as the 1st to 26th amino acids in SEQ ID NO: 1, for example, a polypeptide having an amino acid sequence from the 27th to 528th amino acids in SEQ ID NO: 1, or a polypeptide having another secretion signal sequence bound to the N-terminal side of a polypeptide having an amino acid sequence from the 27th to 528th amino acids in SEQ ID NO: 1.

In addition, the phospholipase D-(2) of the present invention is
(2-1) Has the effect of hydrolyzing phosphate esters in ethanolamine-type plasmalogens;
(2-2) Molecular weight is 50,000 to 60,000;
(2-3) Optimum temperature: 40 to 60° C. under conditions of pH 7.0 and 30 minutes of reaction;
(2-4) Optimum pH is 5.5 to 7.5 under conditions of 37°C and 30 minutes of reaction;
(2-5) derived from a microorganism belonging to the genus Streptomyces;
The present phospholipase D has the properties of (2-1) to (2-5) and has a hydrolysis effect on the phosphate ester bond in the ethanolamine-type plasmalogen molecule, 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."

The ethanolamine-type plasmalogen (hereinafter also referred to as "PlsEtn") in this specification is one of the subclasses of glycerophospholipids, and is an alkenyl ether-type glycerophospholipid having a hydrocarbon chain bonded to an alkenyl (vinyl) ether at the C1 (sn-1) position of the glycerophospholipid and a fatty acid ester bond at the C2 (sn-2) position, in which the base is ethanolamine, and the structure is shown in the following formula (I).

In the formula, R ¹ and R ² are each independently an aliphatic hydrocarbon group. R ¹ is usually an aliphatic hydrocarbon group having 1 to 20 carbon atoms, such as a dodecyl group, a tetradecyl group, a hexadecyl group, an octadecyl group, or an icosanyl group. R ² is usually an aliphatic hydrocarbon group derived from a fatty acid residue, such as an octadecadienoyl group, an octadecatrienoyl group, an icosaretraenoyl group 20:4, an icosapentaenoyl group, a docosatetraenoyl group, a docosapentaenoyl group, or a docosahexaenoyl group 22:6.

The present phospholipase D is an enzyme that has the ability to hydrolyze the phosphate ester bond in the ethanolamine-type plasmalogen molecule, specifically, to hydrolyze the sn-3 phosphate ester in the ethanolamine-type plasmalogen molecule to release plasmonylphosphatidic acid and ethanolamine, as shown in the following formula (II).

Whether or not an ethanolamine-type plasmalogen has the ability to hydrolyze to phosphate esters can be determined, for example, by subjecting the ethanolamine-type plasmalogen to the action of phospholipase D and quantifying the amount of ethanolamine released by hydrolysis. Ethanolamine can be quantified, for example, by subjecting ethanolamine to the action of ethanolamine oxidase (EAO: JP 2014-233219 A) and quantifying the amount of hydrogen peroxide generated.

The ethanolamine oxidase may be any enzyme that catalyzes the oxidation of ethanolamine to hydrogen peroxide, ammonia, and glycolaldehyde, and examples of the ethanolamine oxidase that can be used include monoethanolamine oxidase (EC 1.4.3.8), monoamine oxidase (EC 1.4.3.4), and primary amine oxidase (EC 1.4.3.21).

Methods for quantifying the hydrogen peroxide include spectrophotometry and a method using a hydrogen peroxide electrode. Specifically, for example, a method for measuring the absorbance (550 nm) based on a quinone dye, which is a blue-purple pigment produced by quantitatively oxidatively condensing 4-aminoantipyrine (4-AA) and N,N-bis(4-sulfobutyl)-3-methylaniline, disodium (TODB) using the action of peroxidase (POD). Since the amount of quinone dye and the amount of liberated ethanolamine have a 1:1 relationship, the amount of ethanolamine is measured by measuring the amount of quinone dye. In this specification, the amount of enzyme that liberates 1 μmol of ethanolamine per minute is defined as 1 U.

In the above-mentioned polypeptide of (1-2) of the present phospholipase D-(1), "a polypeptide having at least 85% identity with the amino acid sequence set forth in SEQ ID NO: 1 and having a hydrolysis effect on the phosphate ester bond in the ethanolamine-type plasmalogen molecule" can be a polypeptide having at least 85%, 90% or more, 93% or more, 95% or more, or 98% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having a hydrolysis effect on the phosphate ester bond in the ethanolamine-type plasmalogen molecule.

The "polypeptide in which one or several amino acids have been added, substituted, deleted and/or inserted" in the above-mentioned polypeptide of phospholipase D-(1) (1-3) of the present invention may include a polypeptide in which any number of amino acids have been added, substituted, deleted and/or inserted, for example, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acid. The above-mentioned "polypeptide to which one or several amino acids have been added, substituted, deleted and/or inserted" is preferably a polypeptide to which one or several amino acids have been added, substituted, deleted and/or inserted other than H at positions 192 and 462, K at positions 194 and 464, and D at positions 199 and 469 in SEQ ID NO: 1, preferably other than HSKLVVVD at positions 192 to 199, and HxKxxxxD motif (H is histidine, K is lysine, D is aspartic acid, S is serine, L is leucine, V is valine, and x is any amino acid: HSK motif) at positions 462 to 469. The above-mentioned HSK motif is an amino acid sequence that contributes to a catalytic reaction in phospholipase D.

Preferable examples of the above-mentioned polypeptide (1-2) or (1-3) of the present phospholipase D-(1) include polypeptides having a relative activity of 5 or less, preferably 3 or less, when ethanolamine-type lysoplasmalogen (LyPlsEtn) is used as a substrate, a relative activity of 5 or less, preferably 3 or less, when phosphatidylethanolamine (PE) is used as a substrate, and/or a relative activity of 5 or less, preferably 3 or less, when lysophosphatidylethanolamine is used as a substrate, when the hydrolysis activity to phosphate ester when ethanolamine-type plasmalogen is used as a substrate is taken as 100.

The molecular weight of the present phospholipase D-(2) may vary slightly depending on electrophoresis conditions, etc., but can be 50,000 to 60,000 (Da), preferably 52,000 to 54,000 (Da) in SDS-PAGE. In addition, the isoelectric point (calculated by Genetyx) can be 6 to 6.5. This isoelectric point may differ from the actually measured value.

The optimum temperature for the present phospholipase D-(2) is 40 to 60°C, preferably 45°C to 55°C, under conditions of pH 7.0 and 30 minutes of reaction.

The phospholipase D-(2) has an optimum pH of 5.5 to 7.5, preferably 6.0 to 7.0, when reacted at 37°C for 30 minutes.

The phospholipase D-(2) of the present invention is derived from a microorganism belonging to the genus Streptomyces. Examples of the microorganism belonging to the genus Streptomyces include Streptomyces polychromogenes, Streptomyces racemochromogenes, or microorganisms closely related thereto, as well as Streptomyces katrae, Streptomyces flavotricini, Streptomyces toxytricini, Streptomyces yangpuensis, Streptomyces amritsarensis, Streptomyces cinnamonensis, Streptomyces virginiae, Streptomyces virginiae), Streptomyces cirratus, Streptomyces nojiriensis, Streptomyces colombiensis, Streptomyces nashvillensis, Streptomyces cellostaticus, and Streptomyces vietnamensis. Whether or not the microorganisms are 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 phospholipase D having a relative activity value of 5 or less, preferably 3 or less, when ethanolamine-type lysoplasmalogen (LyPlsEtn) is used as a substrate, a relative activity value of 5 or less, preferably 3 or less, when phosphatidylethanolamine (PE) is used as a substrate, and/or a relative activity value of 5 or less, preferably 3 or less, when lysophophatidylethanolamine is used as a substrate, when the hydrolysis activity to phosphate ester when ethanolamine-type plasmalogen is used as a substrate is taken as 100.

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

[Method for quantifying ethanolamine-type plasmalogen]
The method for quantifying ethanolamine-type plasmalogen in a sample of the present invention may be a method for quantifying ethanolamine-type plasmalogen in a sample (hereinafter also referred to as the "method for quantifying PlsEtn") that includes a step of reacting the sample with the above-mentioned phospholipase D, and makes it possible to accurately quantify ethanolamine-type plasmalogen based on the substrate specificity of the present phospholipase D. Specifically, a method that performs the following steps (A) to (D) can be mentioned.
1) a step (A) of hydrolyzing a sample using the present phospholipase D;
2) a step (B) of treating the treated solution obtained in the step (A) with ethanolamine oxidase;
3) a step (C) of treating the treatment solution obtained in the step (B) with peroxidase and a color-developing agent or a fluorescent reagent;
4) a step (D) of subjecting the treated liquid obtained in the step (C) to measurement using a spectrophotometer or a fluorometer;

Examples of the above 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 by a normal blood collection method (e.g., syringe blood collection or vacuum blood collection) by a known method such as centrifuging (e.g., 1000 x g for 5 minutes) and recovering the supernatant. When collecting blood, anticoagulants and glycolysis inhibitors such as EDTA, sodium fluoride, sodium citrate, sodium heparin, and monoiodoacetic acid can also be used.

The above 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.

Examples of the color former include a combination of phenol or its derivatives or an aniline derivative with 4-aminoantipyrine, a leuco dye, a diphenylamine derivative, a triaryl imidazole derivative, etc., and a combination of 4-aminoantipyrine (4-AA) with a Trinder reagent such as N-ethyl-N-(2-hydroxy-3-sulfopropyl)-toluidine (TOOS) or N,N-bis(4-sulfobutyl)-3-methylaniline (TODB) is preferred.

An example of the fluorescent reagent is Amplex Red.

The principle of quantification of ethanolamine-type plasmalogen when a color developer is used is as follows. First, ethanolamine is produced in the above-mentioned step (A). When the treatment liquid obtained in step (A) is treated with ethanolamine oxidase, hydrogen peroxide (H ₂ O ₂ ) is produced. When further treated with peroxidase, the produced hydrogen peroxide and fluorescent reagent are converted to a quinone dye by the peroxidase, making it possible to quantify ethanolamine using a spectrophotometer. In this way, it is possible to quantify ethanolamine-type plasmalogen.

The above steps (A) to (C) can be carried out in an appropriate buffer solution, for example, 0.2 M Tris-HCl buffer (pH 7.0, 37°C) for step (A) and 0.2 M Bis-Tris-HCl buffer (pH 7.5, 45°C) for step (B). Step (A) may be carried out in the presence of a surfactant to increase the solubility of the ethanolamine-type plasmalogen, and step (C) may be carried out in the presence of a surfactant to suppress turbidity of the color-developing solution. Such surfactants are preferably nonionic surfactants, more preferably polyoxyethylene alkylphenyl ether, and even more preferably Triton X-100. The amount of surfactant used may be, for example, 0.001 to 10% by mass.

In addition, hydrogen peroxide (H ₂ O ₂ ) produced by treating the treatment solution obtained in step (A) with ethanolamine oxidase may be quantified using known methods such as the electrode method, the absorption method, the luminescence method, and the oxidation-reduction method using KMnO ₄ , in addition to the above.

The method for quantifying ethanolamine plasmalogen involves creating a calibration curve by steps (A) to (D) above using ethanolamine plasmalogen of known concentration as a sample, and then using the sample to be measured to compare the measured values obtained by steps (A) to (D) above to calculate the amount of ethanolamine plasmalogen.

[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 an example of the polynucleotide encoding the present phospholipase D is a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1, such as a polynucleotide consisting of the base sequence of SEQ ID NO: 2. In addition, since the 1st to 78th bases (corresponding to the 1st to 26th amino acids in SEQ ID NO: 1) of the base sequence of SEQ ID NO: 2 are considered to be a base sequence encoding a secretion signal, when transforming with the vector of the present invention and secreting the present phospholipase D outside the cell, the bases predicted to be the secretion signal, such as the 1st to 78th bases, may be deleted in whole or in part, or the bases predicted to be the secretion signal, such as the 1st to 78th bases, may be replaced in whole or in part with a base sequence encoding another secretion signal.

The type of expression vector may be of any form, such as circular or linear. In addition, it can be appropriately selected depending on the host cell to be used, and examples of such vectors 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, pUC702 vectors, pIJ680 vectors, pIJ702 vectors, pTONa5 vectors, pTONashort vectors, pTip vectors, pNit vectors, and the like. Furthermore, when a gram-positive bacterium is used as a 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 a host, examples of the vector include the YRp7 vector, pYC1 vector, and YEp13 vector.

In addition, such an expression vector may contain a control sequence such as a promoter or terminator, or a selection marker sequence such as a drug resistance gene or a reporter gene. Furthermore, it may contain a secretion signal sequence to facilitate secretion outside the host cell. Examples of secretion signals include an OmpA signal (functions as a periplasmic transport signal), a pelB signal (functions as a periplasmic transport signal), and affinity and solubility tags include an MBP tag (also functions as a periplasmic transport signal), a GST tag, a TF tag, a Halo tag, a His tag, a SKIK tag (Kato et al., J. Biosci. Bioeng. 10.1016/j.jbiosc.2016.12.004 (2017)), a Profinity eXact fusion tag, etc.

The polynucleotide may be a naturally occurring polynucleotide such as that of a microorganism belonging to the genus Streptomyces, or an artificially synthesized polynucleotide, and may be appropriately selected depending on the type of microorganism into which the expression vector is to be introduced. Sequence information may be appropriately obtained by searching known literature or databases such as NCBI (www.ncbi.nlm.nih.gov/guide/). The polynucleotide may be prepared by known techniques such as chemical synthesis or PCR amplification based on the base sequence information of the polynucleotide. The codons selected to code for 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 obtained by introducing the above-mentioned expression vector into 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 of the host 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, and Pseudomonas putida. Examples of suitable fungi include gram-negative bacteria such as Saccharomyces cerevisiae and Pichia pastoris, yeasts such as Aspergillus oryzae and Aspergillus nigar, and filamentous fungi of the genus Aspergillus.

Methods for introducing the present expression vector into a host can be known, including chemical methods such as the competent cell method, lipofection method, calcium phosphate coprecipitation method, and liposome method; biological methods such as methods using viral vectors, methods using specific receptors, and cell fusion method; and physical methods such as electroporation method, microinjection method, gene gun method, and ultrasonic gene transfer method.

[Method of producing phospholipase D]
The method for producing phospholipase D of the present invention may be any method for producing phospholipase D that includes a step of culturing the present transformant to produce phospholipase D, and the present phospholipase D can be produced efficiently by such a method. The culture method can be a method generally used for culturing host cells. For example, when the host is Escherichia coli or actinomycetes, the host can be cultured under aerobic or anaerobic conditions such as shaking culture or aeration and stirring culture 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. The present phospholipase D can be recovered from the culture solution or from the disrupted present transformant. Examples of such a recovery method include known protein recovery methods, such as centrifugation followed by gel filtration, ion exchange, affinity chromatography, and the like.

The present invention will be described 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 ethanolamine-type plasmalogens)
Based on past experience, the present inventors focused on actinomycetes as a microorganism that produces phospholipase D (PlsEtn-PLD) with high substrate specificity for ethanolamine-type plasmalogens and conducted a search using the following steps.
1) An actinomycete strain stocked in glycerol (stored at -80°C) was spread onto an agar medium using a platinum loop and cultured at 28°C.
2) The grown colonies were taken 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) MgSO ₄ ·7H ₂ O: 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). Then, the mixture was shake-cultured at 28°C and 160 strokes per minute for 72 hours. 1 mL of the culture solution was inoculated into 100 mL of MPD medium and shake-cultured at 28°C and 160 rpm for 72 hours.

Next, the hydrolysis action of phospholipase D (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 an enzyme sample.
2) The following enzyme reaction solution was incubated at 37° C. for 30 minutes. PlsEtn (Avanti Polar Lipids: AVT) and scallop-derived PlsEtn (Rheology Functional Food Research Institute) were used as substrates.
3) The following color reaction solution was added and incubated at 45°C for 10 minutes.
4) After the reaction, the mixture was centrifuged (21,600×g, 5 minutes, 4° C.) and 200 μL of the centrifuged supernatant was transferred to a 96-well microwell plate (96MP), and the absorbance at 550 nm (A550) was measured using a microplate reader (Thermo Fisher).

(Enzyme reaction solution)
0.2M Tris-HCl (pH 7.0, 37°C) 25μL
0.01M CaCl2 _5μL
1% substrate/0.1% TritonX-100 10μL
Enzyme sample 10 μL

(Color reaction solution)
0.2M BisTris-HCl (pH 7.5, 45°C) 100μL
50U/mL peroxidase (POD) 20μL
0.2% (w/v) TODB 20μL
0.3% (w/v) 4-AA 20μL
2% TritonX-100 10μL
0.06U/mL ethanolamine oxidase 20μL
Ultrapure water (UPW) 10μL

Among the strains for which phospholipase D activity against PlsEtn was confirmed by the above method, a substrate specificity test was conducted on strains with high activity. In the substrate specificity test, phospholipase D activity against seven types of lipids was measured: scallop-derived PlsEtn (Rheology Functional Food Research Institute), PlsEtn22:6 (AVT), PlsEtn20:4 (AVT), PlsEtn18:1 (AVT), PE (1-Hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine) (AVT), LPE (L-α-Lysophosphatidylethanolamine) (SRL), and LyPlsEtn (1-O-1'-(Z)-Octadecenyl-2-hydroxy-sn-glycero-3-phosphoethanolamine) (AVT). Phospholipase D activity was measured in the same manner as above. Among them, the AK461 strain was selected as a PlsEtn-PLD producing bacterium because it showed the highest phospholipase D activity against PlsEtn.

(Taxonomic identification of the AK461 strain)
The AK461 strain selected in Example 1 was taxonomically identified. First, the AK461 strain was cultured in ISP2 medium (Nihon Seiyaku Co., Ltd.) at 30°C for 72 hours. After the culture, DNA was extracted using achromopeptidase (Wako Pure Chemical Industries, Ltd.). The extracted DNA was used as a template for PCR, and PCR was performed using primers described in the literature (Yasuyoshi Nakagawa and Hiroko Kawasaki, Japanese Society of Actinomycetes, Tokyo: Japan Society Management Center, 88-117 (2001)), to amplify 16S rDNA by about 1500 bp.

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

As a result of a BLAST homology search, the 16S rDNA partial sequence of the AK461 strain showed a homology rate of 99.8% to the type strain (NBRC13072) of Streptomyces polychromogenes or the type strain (NBRC12906) of Streptomyces racemochromogenes.

Furthermore, simple morphological observation revealed wrinkled colonies with a pale yellow color on both the front and back. Microscopic observation also revealed the formation of aerial hyphae and chain spores.

From the phylogenetic tree in Figure 1 and the results of simple morphological observation, it was confirmed that the AK461 strain belongs to Streptomyces, and that the most closely related microorganism is Streptomyces polychromogenes or Streptomyces racemochromogenes.

(Purification of PlsEtn-PLD)
PlsEtn-PLD produced by the AK461 strain was purified by the following method.
(1) A colony of the AK461 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 28°C and 160 rpm for 2 to 3 days (preculture). 1 mL of the preculture was inoculated into 100 mL of MPD flask medium. Shaking culture was performed at 28°C and 160 rpm for 3 to 4 days (main culture). Unless otherwise specified, all of the following purification steps of PlsEtn-PLD were performed on ice or at 4°C.
(2) The culture medium of the AK461 strain obtained above was centrifuged (18,800×g, 30 minutes) to recover a culture supernatant (Culture sup.) from the culture medium.
(3) 1M 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 60% (w/v) saturation on ice, and the resulting precipitate was collected by centrifugation (18,800×g, 30 min.) The precipitate was suspended in 20 mM Tris-HCl buffer (pH 8.0) to obtain a crude enzyme solution (60% AS).
(4) Powdered ammonium sulfate was added to the crude enzyme solution obtained in (3) to a final concentration of 1 M on ice, and the mixture was completely dissolved while stirring with a stirrer, followed by column chromatography under the following conditions.

(5) The crude enzyme solution obtained in (4) was applied to a Toyopearl (registered trademark)-Phenyl-650M column (Tosoh Corporation) that had been previously equilibrated with 1M ammonium sulfate/20 mM Tris-HCl buffer (pH 8.0). After washing the column with the same buffer, the buffer was changed to 20 mM MES-NaOH (pH 5.5), and elution was performed with a linear gradient of ammonium sulfate (1M to 0M) to obtain an active fraction (Phenyl).

(6) The active fraction (phenyl) obtained in (5) was applied to a Toyopearl-MX-Trp-650M column (Tosoh Corporation) previously equilibrated with 20 mM MES-NaOH (pH 5.5). After washing the column with the same buffer, the buffer was changed to 20 mM Tris-HCl (pH 8.0), and elution was performed with a linear gradient of sodium chloride (0 M to 0.5 M) to obtain an active fraction (Mx-Trp).
(7) The active fraction (MX-Trp) obtained in (6) was applied to a Toyopearl DEAE-650M column (Tosoh Corporation) previously equilibrated with 20 mM Tris-HCl buffer (pH 9.0). After washing the column with the same buffer, it was eluted with a linear gradient of sodium chloride (0 M to 2 M) to obtain the active fraction (DEAE).

The yield, yield, phospholipase D activity, etc. in each purification step are shown in Table 1. Phospholipase D activity was measured in the same manner as in Example 1. The above steps yielded purified PlsEtn-PLD with a 925-fold improvement in purification fold and specific activity. The results of analyzing the purified PlsEtn-PLD (DEAE fraction) by SDS-PAGE (12% (w/v) polyacrylamide gel) are shown in Figure 2. The left lane (M: Marker) is a molecular weight marker, and the right lane shows the band of the activated fraction (DEAE). As a result, four bands of approximately 66 kDa, approximately 53 kDa, approximately 45 kDa, and approximately 32 kDa were observed.

(Analysis of the internal amino acid sequence of purified PlsEtn-PLD)
The gel of the four bands indicated by the arrows in the SDS-PAGE shown in Figure 2 was excised and in-gel digested with trypsin. The obtained peptide samples were then subjected to analysis of the internal amino acid sequences by liquid chromatography mass spectrometry (LC-MS/MS). As a result, the internal amino acid sequence analysis by LC-MS/MS of the 53 kDa band showed that the seven peptide sequences shown in Table 2 were homologous to parts of the amino acid sequences of proteins in the database. On the other hand, no proteins were found to be homologous to the 66 kDa, 45 kDa, and 32 kDa bands in the LC-MS/MS analysis.

The seven peptide sequences hit by the internal amino acid sequence analysis of the 53 kDa band by LC-MS/MS were analyzed by the proteomics support system "ProteinLynx Global Server (PLGS)". As a result, as shown in Table 3, it was revealed that phospholipase D of Streptomyces racemochromogenes (Uniprot entry No. E0D7I2: SEQ ID NO: 3) had the highest PLGS score of 688. Therefore, it was considered that the protein of the 53 kDa band was highly likely to be PlsEtn-PLD.

(Properties of purified PlsEtn-PLD)
The substrate specificity, optimum temperature, and optimum pH of the purified PlsEtn-PLD were examined.
Substrate specificity of PlsEtn-PLD The phospholipase D activity of the PlsEtn-PLD purified above was measured in the same manner as in Example 1 to examine the substrate specificity. PlsEtn22:6, PlsEtn20:4, PlsEtn18:1, phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), and ethanolamine-type lysoplasmalogen (LyPlsEtn) were used as substrates. PlsEtn22:6, PlsEtn20:4, and PlsEtn18:1 are shown in formulas (III) to (V).

(1) PlsEtn22:6
1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine
CAS Number:206059-98-5

(2) PlsEtn20:4
1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphoethanolamine
CAS Number:144371-69-7

(3) PlsEtn18:1
1-(1Z-octadecenyl)-2-oleoyl-sn-glycero-3-phosphoethanolamine
CAS Number:144371-68-6

Figure 3 shows the relative activity when PlsEtn 22:6 was used as a substrate, with the phospholipase D activity taken as 100. As is clear from Figure 3, the relative activity when PlsEtn 20:4 was used as a substrate was about 15, and the relative activity when PlsEtn 18:1 was used as a substrate was about 8. On the other hand, when phospholipase D activity was measured using phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), and ethanolamine-type lysoplasmalogen (LyPlsEtn) as substrates in the same manner as in Example 1, the relative activity when the phospholipase D activity was taken as 100 was almost 0 in all cases. Thus, it was revealed that the purified PlsEtn-PLD has extremely high substrate specificity for ethanolamine-type plasmalogens.

◆ Optimum temperature of PlsEtn-PLD The purified PlsEtn-PLD was added to a solution of 0.2% PlsEtn 22:6 as a substrate and 50 mM Tris-HCl (pH 7.0, each temperature) to make 10 or 20% (v/v) to prepare a reaction solution with a total volume of 50 μL. This reaction solution was reacted at 37°C for 30 minutes. Thereafter, the hydrolysis activity was measured. The results are shown in FIG. 4. In FIG. 4, the horizontal axis is temperature, and the vertical axis is ΔA550 (the difference between the absorbance at 550 nm of the sample and the absorbance of the blank solution, the same below). As is clear from FIG. 4, it was revealed that the purified PlsEtn-PLD has a hydrolysis activity at 30°C to 70°C, and the hydrolysis activity is particularly high at 40°C to 60°C.

Optimal pH of PlsEtn-PLD
The purified PlsEtn-PLD was added to a solution of 0.2% PlsEtn 22:6 as a substrate and a buffer solution (pH 4.5, 5, 6, 6.5, 7, 7.5, 8, or 9, temperature 37 ° C.) selected from acetic acid-Na acetate (Na acetate in the graph: pH 4.5-6), BisTris-HCl (Bis Tris HCl in the graph: pH 6-7), or Tris-HCl (Tris HCl in the graph: pH 7-9) at 10 or 20% (v/v), and a reaction solution with a total volume of 50 μL was prepared. This reaction solution was subjected to an enzyme reaction for 30 minutes under each pH condition. The subsequent color reaction was performed in the same manner as in Example 1, and the enzyme activity was measured. The results are shown in FIG. 5. In FIG. 5, the horizontal axis is pH and the vertical axis is ΔA550. Based on FIG. 5, the purified PlsEtn-PLD had hydrolytic activity even when treated at pH 5-8.5, particularly pH 6-7.

(Cloning of PlsEtn-PLD)
Cloning of PlsEtn-PLD derived from the AK461 strain was carried out by the following method.

(Preparation of Genomic DNA)
The AK461 strain was cultured at 28°C for 2 days using MPD medium in the same manner as in Example 1, and the bacteria were collected. Next, 5mL of SET buffer and 10mg of lysozyme (final concentration 2mg/mL) were added to the bacteria, mixed by inversion, and incubated at 37°C for 2 hours. Next, 670μL of 10% SDS solution (0.2N NaOH) (final concentration 1%) and 1mL of 3mg/mL proteinase K (final concentration 0.5mg/mL) were added to the bacteria, and the mixture was incubated at 55°C for 2 hours while slowly rotating in a hybridizer. 4mL of 5M NaCl and 10mL of chloroform were added to the solution and stirred, and after centrifugation, the supernatant was collected in a separate tube, 0.6 times the volume of the supernatant of 2-propanol was added, and the mixture was centrifuged (4,000×g, 5 minutes, 4°C). The precipitated genomic DNA was collected and dissolved in 10 mM Tris-HCl buffer (pH 7.5). RNase A was added to the mixture to a final concentration of 0.1 mg/mL, and the mixture was treated at 37°C for 20 minutes. 500 μL of 13% PEG solution containing 0.8 M NaCl was then added, stirred, and centrifuged. The supernatant was removed, and the precipitate was dissolved in 500 μL of 10 mM Tris-EDTA buffer (pH 7.5), after which 500 μL of a phenol/chloroform (1:1, v/v) mixture was added, stirred, and centrifuged to separate the aqueous phase. 0.1 times the volume of 3 M sodium acetate (pH 5.2) and 2 times the volume of ethanol were added and mixed to the aqueous phase, and the genomic DNA of the AK461 strain was collected. The recovered DNA was immersed in 70% (v/v) ethanol and then dissolved in a solution of 10 mM Tris-HCl buffer (pH 7.5) to prepare a genomic DNA (gDNA) solution.

(Cloning of partial base sequence of PlsEtn-PLD derived from AK461 strain)
1. PCR amplification PCR was performed using the genomic DNA of the AK461 strain prepared above as a template. The PCR amplified product was then subcloned and sequenced to analyze the partial base sequence of the PlsEtn-PLD gene.

First, among the codons corresponding to the internal amino acid sequence revealed in Example 4, codons with a codon usage frequency of 10% or more in the genus Streptomyces were selected, and a forward primer (PlsEtn-FW1) described in SEQ ID NO: 4 and a reverse primer (PlsEtn-RV1) described in SEQ ID NO: 5 were designed.

A PCR reaction was performed using the above PCR primers. The composition of the PCR reaction solution is as follows:

2xGflex PCR buffer 12.5μL
10μM PlsEtn-FW1 0.75μL
10μM PlsEtn-RV1 0.75μL
33.27ng/μL AK461 gDNA 2μL
1.25U/μL Gflex DNA polymerase 0.5μL
Ultrapure water (UPW) 8.5μL

The PCR reaction conditions were as follows:
Step 1: 94°C, 2 minutes;
Step 2: 98°C, 10 seconds;
Step 3: 65°C, 30 seconds;
Step 4: 68°C, 2 minutes;
Repeat steps 2 to 4 for 30 cycles;
Step 5: 68°C, 2 minutes;

2. Subcloning and sequencing of PCR amplified product In order to perform DNA sequencing of the 1400 bp PCR amplified product obtained above, subcloning was performed into a pMD20-T vector using Mighty TA-cloning Reagent SET for PrimeSTAR (registered trademark) by the following method. Next, the product was transformed into Escherichia coli (HST08) and cultured, white colonies were selected by blue-white colony selection, and colonies in which a band of the target size was confirmed by colony PCR were cultured, and the plasmid was purified using a High Pure Plasmid Isolation kit (Roche Diagnostics). The purified plasmid was sequenced using the forward primer (M13R-pUC) described in SEQ ID NO: 6 and the reverse primer (M13M4) described in SEQ ID NO: 7.

(Analysis of unknown regions of PlsEtn-PLD)
To analyze the unknown region of PlsEtn-PLD, inverse PCR was performed using restriction enzyme-digested genomic DNA of the AK461 strain as a template, followed by cloning and sequencing.

First, referring to the partial base sequence obtained in "2. Subcloning and sequencing of PCR amplified products" above, a forward primer (PlsEtn-FW2) described in SEQ ID NO: 8 and a reverse primer (PlsEtn-RV2) described in SEQ ID NO: 9 were designed as primers for Inverse PCR. Next, the genomic DNA of the AK461 strain was digested with six types of restriction enzymes (BamHI, EcoRI, KpnI, NdeI, PstI, SmaI). Digestion was then confirmed by agarose electrophoresis, and the restriction enzymes and the genomic DNA of the AK461 strain were removed by phenol-chloroform extraction and ethanol precipitation. Then, self-ligation, phenol-chloroform extraction, and ethanol precipitation were performed to prepare a template DNA for Inverse PCR.

Inverse PCR was performed using the template DNA for Inverse PCR obtained above and the primers for Inverse PCR. The composition of the PCR reaction solution is as follows:

2xGflex PCR buffer 12.5μL
10μM PlsEtn-FW2 0.75μL
10μM PlsEtn-RV2 0.75μL
Ligation reaction solution 2 μL
1.25U/μL Glex DNA polymerase 0.5μL
Ultrapure water (UPW) 8.5μL

The PCR reaction conditions were as follows:
Step 1: 94°C, 2 minutes;
Step 2: 98°C, 10 seconds;
Step 3: 65°C, 30 seconds;
Step 4: 68°C, 2 minutes;
Repeat steps 2 to 4 for 30 cycles;
Step 5: 68°C, 2 minutes;

The PCR amplified product was then cloned and sequenced using the same method as described above in "Cloning of partial base sequence of PlsEtn-PLD derived from AK461 strain."

The sequence analysis above showed that the amino acid sequence of PlsEtn-PLD is a sequence consisting of 528 amino acids as described in SEQ ID NO:1, and the Signal P program predicted that the N-terminal 26 amino acids are a Sec secretion signal. The base sequence encoding the amino acid sequence of PlsEtn-PLD was confirmed to be the base sequence as described in SEQ ID NO:2. The estimated molecular weight by the GENETYX-MAC program was 52,963 Da, and the estimated isoelectric point was 6.16. This isoelectric point is an estimated value and may differ from the actual measured value. In addition, it had two HKD motifs (positions 192 to 199 and 462 to 469 in SEQ ID NO:1).

(Expression and preparation of PlsEtn-PLD in E. coli)
PCR was performed using the forward primer (PlsEtn-FW3) described in SEQ ID NO: 10 and the reverse primer (PlsEtn-RV3) described in SEQ ID NO: 11, and the sequence (the base sequence shown in SEQ ID NO: 12) excluding the sequence thought to be the Sec signal sequence of the PlsEtn-PLD gene consisting of the base sequence described in SEQ ID NO: 2 was amplified so as to add an NdeI site to the 5' end and a HindIII site to the 3' end. The PCR by-product was digested with NdeI and HindIII and inserted into the NdeI-HindIII site of the expression vector pET24a(+) to obtain a recombinant plasmid. Next, the obtained recombinant plasmid was transformed into E. coli BL21(DE3) to obtain a recombinant E. coli. The obtained recombinant E. coli was cultured in 100 mL of Overnight Express TB medium (Novagen) containing 30 μg/mL kanamycin at 30° C. or 37° C. for 24 hours. The obtained culture solution was centrifuged to recover the cells. The cells were suspended in 20 mM Tris-HCl buffer (pH 7.0) and then disrupted by ultrasonication, and the centrifugal supernatant was used as the live enzyme (approximately 0.04 U/mL).

Next, PlsEtn, LyPlsEtn, POPE, and LPE were used as substrates, and phospholipase D activity was measured by adding 10 μL of the above-mentioned live enzyme or heat-inactivated Ctrl. At the same time, a control without enzyme (no enzyme (Ctrl)) was also performed. Phospholipase D activity was measured in the same manner as in Example 1. As shown in Table 4, it was confirmed that PlsEtn-PLD has high specificity for PlsEtn.
POPE: 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids)
LPE: Lysophosphatidylethanolamine (L-α-Lysophosphatidylethanolamine, Egg) (DOOSAN Serdary Research Laboratories)

(Creating a calibration curve for scallop ethanolamine-type plasmalogen)
Ethanolamine-type plasmalogen (scallop-derived PlsEtn) separated from scallops was detected using the PlsEtn-PLD prepared in Example 7. The scallop-derived PlsEtn was quantified by quantifying the ethanolamine obtained by hydrolysis of PlsEtn as described in Example 1.

1. Preparation of Scallop-Derived PlsEtn 1 mg of scallop-derived PlsEtn (Rheology Functional Food Research Institute) separated from edible parts of scallops was added to 1 mL of 50 mM Tris-HCl buffer and emulsified with ultrasound.
2. Preparation of PlsEtn-PLD Solution 15 mL of PlsEtn-PLD prepared in Example 7 was lyophilized and then dissolved in 1.5 mL of 50 mM Tris-HCl buffer (10-fold concentrated).
3. Measurement of Phospholipase D Activity Phospholipase D activity was measured by measuring fluorescence intensity according to the following method.
1) PlsEtn-PLD (0.042 U/mL) was concentrated 10-fold to prepare a 0.42 U/mL enzyme sample.
2) Scallop-derived PlsEtn (Rheology Functional Food Research Institute), soybean-derived phosphatidylethanolamine (Sigma-Aldrich), and plasma were dispensed into a 96-well plate.
3) The following fluorescent enzyme reaction solution and enzyme sample were added and shaken for 1 minute.
[Fluorescence method enzyme reaction solution]
50mM Tris-HCl (pH 7.4)
50 mM NaCl
0.75mM _CaCl2
0.2% TritonX-100
2.5U/mL peroxidase (POD)
1U/mL Tyramine oxidase 50μM Amplex Red
Total volume of the above: 100 μL
0.42U/mL enzyme sample 10μL
4) The mixture was incubated at 37°C for 10 minutes.
5) After the reaction, the fluorescence intensity (EX 535 nm, EM 595 nm) was measured using a plate reader (BECKMAN COULTER).

As the substrate, 10, 15, 20, 25, or 50 μg of scallop-derived PlsEtn was used. The results are shown in Figure 6.

As shown in Figure 6, it was confirmed that scallop-derived plsEtn can be detected using the PlsEtn-PLD prepared in Example 7, and that the fluorescence intensity increases in a concentration-dependent manner with scallop-derived PlsEtn.

[Comparative Example]
It is known that soybeans do not contain plasmalogens, so measurements were performed using soybean-derived phosphatidylethanolamine (PE) as a comparative example.

1. Preparation of soybean-derived phosphatidylethanolamine (PE) 1 mg of soybean-derived phosphatidylethanolamine (PE) was added to 1 mL of 50 mM Tris-HCl buffer (pH 7.4) and emulsified with ultrasonic waves.
2. Analysis by Fluorescence Spectrophotometer The reaction was carried out in the same manner as in Example 8 above, except that 10, 15, 20, 25 or 50 μg of soybean-derived phosphatidylethanolamine (PE) was used instead of scallop-derived PlsEtn. The results are shown in FIG.

As shown in Figure 7, even when the amount of soybean-derived PE increased, the fluorescence intensity hardly increased at all and remained almost constant. Therefore, it was revealed that PlsEtn-PLD does not show activity against phosphatidylethanolamine (PE).

As described above, it was confirmed that PlsEtn can be specifically quantified by using the PlsEtn-PLD prepared in Example 7.

(Quantitative determination of ethanolamine-type plasmalogen in plasma)
1. Preparation of plasma Since serum and plasma are not cell membranes, they contain very little plasmalogen compared to red blood cells and white blood cells. Therefore, it has been difficult to measure the amount of plasmalogen in serum or plasma using conventional techniques. On the other hand, in recent years, plasmalogen has been considered to be a biomarker for Alzheimer's dementia, and quantifying plasmalogen in serum or plasma is important for determining the risk of developing diseases such as Alzheimer's dementia. Therefore, we investigated whether it is possible to quantify ethanolamine-type plasmalogen in plasma using PlsEtn-PLD prepared in Example 7.

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.
2. Preparation of Plasma Samples The plasma collected above was stored frozen, and before measurement, it was thawed and adjusted with 50 mM Tris-HCl buffer.
3. Analysis by Fluorescence Spectrophotometer The reaction was carried out in the same manner as in Example 8, except that 20, 30, 40 or 50 μL of the above plasma sample was used instead of scallop-derived PlsEtn. The results are shown in FIG.

As shown in Figure 8, it was confirmed that the fluorescence intensity increased in proportion to the amount of plasma sample. Combined with the results of Example 8, it was confirmed that ethanolamine-type plasmalogen in plasma can be quantified using PlsEtn-PLD prepared in Example 7.

By using the phospholipase D of the present invention, it is possible to inexpensively detect or quantify ethanolamine-type plasmalogens contained in plasma and the like, and this has great industrial utility.

Claims (6)

A phospholipase D having an ethanolamine-type plasmalogen as a substrate, 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 95 % identity to the amino acid sequence set forth at positions 27 to 528 in SEQ ID NO: 1, having a hydrolytic activity on phosphate ester bonds in an ethanolamine-type plasmalogen molecule, and having a relative activity value of 5 or less when ethanolamine-type lysoplasmalogen is used as a substrate, a relative activity value of 5 or less when phosphatidylethanolamine is used as a substrate, and/or a relative activity value of 5 or less when lysophosphatidylethanolamine is used as a substrate, when the hydrolytic activity on phosphate ester bonds when ethanolamine-type plasmalogen is used as a substrate is taken as 100;
(1-3) A polypeptide in which one or several amino acids have been added, substituted, deleted and/or inserted in the amino acid sequence described in positions 27 to 528 in SEQ ID NO: 1, which has a hydrolytic activity on a phosphate ester bond in an ethanolamine-type plasmalogen molecule, and has a relative activity value of 5 or less when ethanolamine-type lysoplasmalogen is used as a substrate, a relative activity value of 5 or less when phosphatidylethanolamine is used as a substrate, and/or a relative activity value of 5 or less when lysophosphatidylethanolamine is used as a substrate, when the hydrolytic activity on a phosphate ester bond when ethanolamine-type plasmalogen is used as a substrate is taken as 100; A phospholipase D having a hydrolysis action on a phosphate ester bond in an ethanolamine-type plasmalogen molecule, the phospholipase D being derived from a microorganism belonging to Streptomyces that shows 99.8% or more homology with a 16S rDNA partial sequence of Streptomyces polychromogenes, Streptomyces racemochromogenes, or the type strain (NBRC13072) of Streptomyces polychromogenes or the type strain (NBRC12906) of Streptomyces racemochromogenes;
Phospholipase D having the following properties (2-1) to (2-4):
(2-1) has a hydrolytic activity on phosphate ester bonds in ethanolamine-type plasmalogen molecules, and when the hydrolytic activity on phosphate ester bonds when ethanolamine-type plasmalogen is used as a substrate is taken as 100, the relative activity value when ethanolamine-type lysoplasmalogen is used as a substrate is 5 or less, the relative activity value when phosphatidylethanolamine is used as a substrate is 5 or less, and/or the relative activity value when lysophosphatidylethanolamine is used as a substrate is 5 or less;
(2-2) Molecular weight is 50,000 to 60,000;
(2-3) Optimum temperature: 40 to 60° C. under conditions of pH 7.0 and 30 minutes of reaction;
(2-4) Optimum pH: pH 5.5 to 7.5 under reaction conditions of 37°C and 30 minutes; A method for quantifying ethanolamine-type plasmalogen in a sample, comprising a step of reacting the sample with the phospholipase D according to claim 1 or 2 . An expression vector containing a polynucleotide encoding the phospholipase D of claim 1. A transformant comprising the expression vector according to claim 4 . A method for producing phospholipase D, comprising the step of culturing the transformant according to claim 5 to produce phospholipase D.