WO2014013538A1

WO2014013538A1 - Method for hydrolyzing plasmalogen, method for measuring plasmalogen, phospholipase-containing composition, and method for producing phospholipase

Info

Publication number: WO2014013538A1
Application number: PCT/JP2012/068073
Authority: WO
Inventors: 信一酒瀬川; 松本　英之; 大助杉森
Original assignee: 国立大学法人福島大学; 旭化成ファーマ株式会社
Priority date: 2012-07-17
Filing date: 2012-07-17
Publication date: 2014-01-23
Also published as: JP5926801B2; JPWO2014013538A1

Abstract

Provided is a novel method for hydrolyzing plasmalogen. Plasmalogen is hydrolyzed into lysoplasmalogen using a phospholipase having at least the properties (a) to (c) mentioned below: (a) the specific activity of the phospholipase for ethanolamine-type plasmalogen (C18, 20:4) is 0.66 ± 0.2 U/mg; (b) the molecular weight of the phospholipase ranges from 25 to 30 kDa as measured by an SDS-PAGE method; and (c) the phospholipase is derived from an actinomycete belonging to the genus Streptomyces.

Description

Plasmalogen hydrolysis method, plasmalogen measurement method, phospholipase-containing composition, phospholipase production method

The present invention relates to a method for hydrolyzing plasmalogen to lysoplasmalogen using a phospholipase having a novel action, a method for measuring plasmalogen using the above-mentioned phospholipase, and a method for hydrolysis containing the above-mentioned phospholipase or It is related with the composition for a measurement, and the method of manufacturing said phospholipase. The technical fields in which the present invention can be used include, for example, diagnostic techniques for diseases associated with plasmalogen and / or lysoplasmalogen, and production techniques for foods and foods containing plasmalogen and / or lysoplasmalogen.

The known phospholipase A2 (EC 3.1.1.4) is an enzyme having a molecular weight of 14 to 18.5 kDa that exhibits the action of hydrolyzing the sn-2 position of phospholipid to form lysophospholipid in the presence of calcium ions. Yes (Non-Patent Document 1).

Since plasmalogen is one of phospholipids, it has been considered that an enzyme having an action of hydrolyzing the sn-2 position of plasmalogen to form lysoplasmalogen is phospholipase A2 (Non-patent Document 2). .

There is phospholipase A1 (EC 3.1.1.32) as another known enzyme having an action of hydrolyzing the sn-2 position of phospholipid to form lysophospholipid. Phospholipase A1 mainly has the action of hydrolyzing the sn-1 position of phospholipids to give lysophospholipids, but also has the action of hydrolyzing the sn-2 position to make lysophospholipids (Non-patent Document 1). As phospholipase A1 derived from microorganisms, phospholipase A1 (non-patent document 3), which requires calcium ions for its action, and phospholipase A1 (non-patent document 4), whose action is activated by calcium ions, are known. It has been reported that the molecular weight of microorganism-derived phospholipase A1 by the SDS-PAGE method is 35 kDa (Non-patent Document 4).

The problem to be solved by the present invention is to provide a method for hydrolyzing plasmalogen to lysoplasmalogen using phospholipase, a method for measuring plasmalogen using phospholipase, a composition containing phospholipase, and a method for producing phospholipase. It is.

The present inventors tried to hydrolyze plasmalogen into lysoplasmalogen by the action of phospholipase A2 according to the information in Non-Patent Document 2. However, the present inventors have found that phospholipase A2 does not have an action of efficiently hydrolyzing plasmalogen to lysoplasmalogen even in the presence of calcium ions, which is different from conventional common knowledge.

Next, the present inventors have reported at the 2011 Annual Meeting of the Japanese Society of Agricultural Chemistry (lecture number: 2C10a05) that the action of phospholipase A1 and phospholipase A2 is combined, and the molecular weight by the SDS-PAGE method is about 40 kDa. sp. An attempt was made to hydrolyze plasmalogen to lysoplasmalogen by the action of NA684-derived phospholipase (sometimes referred to herein as PLB).

However, it has been found that PLB may not have an action of efficiently hydrolyzing plasmalogen into a fatty acid and lysoplasmalogen even in the presence of calcium ions.

Therefore, the present inventors tried to hydrolyze plasmalogen to lysoplasmalogen by the action of phospholipase A1, which has both actions of phospholipase A1 and phospholipase A2, but the action of phospholipase A1 is stronger than that of phospholipase A2. . As a result, the present inventors have found that this phospholipase has an action of efficiently hydrolyzing plasmalogen into lysoplasmalogen, which was not foreseeable from conventional common sense. Furthermore, this phospholipase
(I) In the absence of calcium ions, the relative activity to ethanolamine-type plasmalogen (C18, 18: 1) is 19 ± 5% relative to dipalmitoylphosphocholine,
(Ii) in the absence of calcium ions, the relative activity against ethanolamine-type plasmalogen (C18, 20: 4) is 29 ± 13% relative to 1-palmitoyl-2-oleoyl-phosphocholine;
(Iii) the molecular weight by SDS-PAGE method is in the range of about 25-30 kDa,
In this respect, it was found that the known phospholipase A1 and phospholipase A2 are clearly different.

Thus, the present inventors have found a novel method for hydrolyzing plasmalogen to lysoplasmalogen using the above phospholipase, and a novel method for measuring plasmalogen using the above phospholipase, and The present invention was completed by creating a composition containing the above phospholipase and a method for producing the above phospholipase.

According to the present invention, plasmalogen can be efficiently hydrolyzed to lysoplasmalogen.

An action of hydrolyzing plasmalogen (Pls) of a novel phospholipase (PL (A)) to lysoplasmalogen (lyPls) and a method for measuring ethanolamine-type plasmalogen (PlsEtn) using a known lysoplasmalogogenase It is a simplified diagram. It is a simplified diagram showing an action of hydrolyzing Pls of PL (A) to lyPls and a method of measuring PlsEtn using Lysophospholipase D. It is a simplified diagram showing the activity measurement method of each phospholipase (PL (A), PLB, PLA2 II L, PLA2 Nagase, and LIPOMOD 699L). 2 is a graph showing a comparison of the reaction rate of PL (A) with respect to sn-1 and sn-2 positions of phosphatidylcholine. 2 is an electrophoresis photograph showing the results of SDS-PAGE of each phospholipase. It is a photograph which shows the comparison with respect to PlsEtn extracted from the pig brain of each phospholipase by thin layer chromatography (TLC). However, it is in the presence of calcium ions. It is a photograph which shows the comparison of the effect | action with respect to PlsEtn (Formula 1) of each phospholipase by TLC. However, it is in the presence of calcium ions. It is a photograph which shows the comparison with respect to PlsEtn extracted from the pig brain of each phospholipase by TLC. However, it is in the absence of calcium ions. It is a photograph which shows the comparison of the effect | action with respect to PlsEtn (Formula 1) of each phospholipase by TLC. However, it is in the absence of calcium ions. It is a photograph which shows the result of having compared the relationship between the amount of PL (A) enzyme and the effect on PlsEtn (chemical formula 1) by TLC. It is a photograph which shows the result of having compared the relationship of the effect | action to PlsEtn of pH and PL (A) by TLC. It is a calibration curve of PlsEtn (Chemical Formula 4) using the action of hydrolyzing Pls of PL (A) to lyPls and known lysoplasmalogogenase. It is a photograph showing that Streptomyces avermitilis JCM 5070-derived PL (A) by TLC acts on PlsEtn (Chemical Formula 4). However, it is in the absence of calcium ions.

The present invention will be specifically described below.

The present invention relates to a method of hydrolyzing plasmalogen to lysoplasmalogen using phospholipase, a method of measuring the content of plasmalogen at an unknown concentration in a sample using phospholipase, a composition containing phospholipase, and a phospholipase It is a method to do.

The plasmalogen (sometimes abbreviated as plasmalogen, Pls) of the present invention includes known Pls. Preferred Pls of the present invention is an alkenyl acyl type glycerophospholipid (alkenyl acyl type ether phospholipid) in which fatty acid is vinyl ether-bonded at the sn-1 (C1) position among glycerophospholipids, and is represented by the following (Chemical Formula 1). The

(Wherein R ₁ and R ₂ are each independently a lower or higher alkyl group or an alkenyl group, and X is hydrogen, choline, ethanolamine, serine, inositol, ethanol, or a derivative thereof)
In the present invention, Pls in which X is choline or ethanolamine in the above formula is particularly preferable. In the present specification, in the above formula, the case where X is choline is referred to as choline-type plasmalogen (sometimes abbreviated as PlsCho), and the case where X is ethanolamine is sometimes referred to as ethanolamine-type plasmalogen (PlsEtn). ). In the present specification, the simple description of Pls includes at least PlsCho and PlsEtn, and PlsEtn is preferred in the present invention.

The lysoplasmagen of the present invention (sometimes abbreviated as lysoplasmagen, lyPls) includes known lyPls. A preferred lyPls in the present invention is an alkenyl acyl type glycerophospholipid (alkenyl acyl type ether phospholipid) in which a fatty acid is vinyl ether-bonded at the sn-1 position among glycerophospholipids, and is represented by the following (Chemical Formula 2).

(Wherein R ₁ is a lower or higher alkyl group or an alkenyl group, and X is hydrogen, choline, ethanolamine, serine, inositol, ethanol, or a derivative thereof)
In the present invention, lyPls in which X is choline or ethanolamine in the above formula is particularly preferable. In the present specification, in the above formula, the case where X is choline is referred to as choline-type lysoplasmalogen (sometimes abbreviated as lyPlsCho), and the case where X is ethanolamine is referred to as ethanolamine-type lysoplasmalogen (lyPlsEtn). There is). In the present specification, the simple description of lyPls includes at least lyPlsCho and lyPlsEtn, and lyPlsEtn is preferred in the present invention.

In the present invention, the phospholipase used for the method of hydrolyzing Pls to lyPls and / or the method of measuring Pls preferably has any of the following characteristics and properties. In other words, phospholipase has an action (property) to hydrolyze phospholipids into fatty acids and other lipophilic substances, specifically, an action to hydrolyze an acyl group at the sn-1 position of phospholipid (phospholipase A1). ), The action of hydrolyzing the acyl group at the sn-2 position of the phospholipid (phospholipase A2 (phospholipase A2)), the action of hydrolyzing the acyl group at the sn-1 position and the sn-2 position of the phospholipid, It preferably has one or more of the actions of hydrolyzing the acyl group at the sn-1 position or sn-2 position (phospholipase B). The presence or absence of the property is not particularly limited. Further, it is more preferable that any one or more of the characteristics and properties described in the present specification are provided. More preferably, it has characteristics (a) to (c) described below, particularly preferably (a) to (e), and more preferably (a) to (m). The phospholipase is sometimes referred to herein as PL (A).

In the present invention, the experimental method is, for example, “Protein / Enzyme Experimental Method, Revised 2nd Edition, Takeichi Horio, 1994 Nankodo”, “Does not fail in bio-experiments! Tips for detection and quantification, edited by Tatsuya Moriyama , Yodosha, 2005 "," Bio-Experiment Illustrated <5> Protein is not annoying, Takato Nishiyama, Shujunsha, 2003 1st Edition 5th Edition "and commercial kits Although it can be carried out by following the procedure manual, the measured value can change depending on the measurement conditions and the accuracy of the equipment used.

(A) Specific activity with respect to PlsEtn (C18, 20: 4) is 0.66 ± 0.2 U / mg.

Here, “PlsEtn (C18, 20: 4)” means PlsEtn in which the fatty acid at the sn-1 position has 18 carbon atoms, the fatty acid at the sn-2 position has 20 carbon atoms, and the number of double bonds is 4. Means. An example of such PlsEtn is 1- (1Z-octadedecyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine (Chemical Formula 3). This PlsEtn is, for example, Avanti Polar Lipids, Inc. Can be purchased as part number 852469.

Here, “specific activity (U / mg)” is calculated by the following (Equation 1) by measuring activity (U / mL) and protein concentration (mg / mL).

Activity (U / mL) can be measured by the following method.

<First reaction reagent mixture>
80 mM Tris-HCl buffer pH 8.0
50 mM calcium chloride 4 mM ATP
4 mM CoA
1.06U / mL Acyl-CoA Synthetase
2 mM PlsEtn (C18, 20: 4)
<Second reaction reagent mixture>
40 mM PIPES-NaOH buffer pH 7.5
0.06% 4-AA
0.04% Phenol 4.5U / mL peroxidase
30U / mL Acyl-CoA Oxidase
0.2% Triton X-100
20 mM ATP
0.1 mM FAD
Acyl-CoA Synthetase (EC 6.2.1.3) and Acyl-CoA Oxidase (EC 1.3.3.6) can be obtained from Asahi Kasei Pharma Corporation (part numbers are T-16 and T17, respectively) .

<Reaction stop solution>
0.1M EDTA solution pH 8.0 containing 0.5% SDS (sodium dodecyl sulfate)
<PL (A) Dissolution Solution>
10 mM Tris-HCl buffer pH 8.0 containing 0.05% BSA
<Measurement operation method>
(1) 0.50 mL of the first reaction reagent mixture is accurately dispensed into a small test tube and pre-warmed at 37 ° C.

(2) After 5 minutes, 50 μL of PL (A) diluted with an enzyme lysis dilution buffer to an appropriate concentration is accurately added and mixed, and the first reaction is started at 37 ° C. In the blind test, 50 μL of enzyme lysis dilution buffer is added instead of the enzyme sample solution.

(3) After 10 minutes, 0.50 mL of 20 mM N-ethylmaleimide (NEM) solution was added and mixed. After 15 seconds, 0.50 mL of the second reaction reagent mixture was added and mixed, and the second reaction was performed at 37 ° C. To start.

(4) After 5 minutes, add 1.50 mL of the reaction stop solution and mix to stop the reaction.

(5) The absorbance at 500 nm is measured. The obtained absorbance is As for the sample solution and Ab for the blind solution. The absorbance range is ΔA = (As−Ab) ≦ 0.25 Abs.

<Calculation>
The activity is calculated according to the following (Equation 2). One unit (U) is defined as the amount of enzyme that PL (A) hydrolyzes PlsEtn (C18, 20: 4) and produces 1 μmol of fatty acid per minute under the above conditions.

The protein concentration (mg / mL) may be measured by, for example, an ultraviolet absorption method, a Bradford method, a Lowry method, a BCA method or the like, but the ultraviolet absorption method is preferable because it is easy.

In the present invention, the specific activity of PL (A) with respect to PlsEtn (C18, 20: 4) only needs to be present, so there is no lower limit, but if it is provided, it is 0.1 U / mg, even 0.2 U / mg. It may be 0.3 U / mg or 0.4 U / mg, but 0.46 U / mg is most preferable. In the present invention, the specific activity of PL (A) with respect to PlsEtn (C18, 20: 4) is preferably as high as possible, so there is no upper limit, but if it is provided, it is 200 U / mg, may be 100 U / mg, and may be 50 U / mg. It may be 10 U / mg, 5 U / mg, 2 U / mg, 1 U / mg, and most preferably 0.86 U / mg.

(B) The molecular weight determined by SDS-PAGE is in the range of about 25-30 kDa.

The molecular weight measurement by SDS-PAGE (Poly-Acrylamide Gel Electrophoresis) method in the present invention refers to a protein (polypeptide) that has been denatured (forms an SDS-protein complex) by sodium dodecyl sulfate (SDS). The molecular weight measurement method utilizes the fact that each polypeptide can be separated according to its mobility by applying a voltage in a gel polymerized with acrylamide.

In addition, in the molecular weight measurement by SDS-PAGE method, when adding the type, number, pH, concentration, and voltage of the electrophoresis buffer such as the type and number of molecular weight markers to be used, polyacrylamide content of polyacrylamide gel, size, manufacturing method, etc. The measured value may contain an error depending on the method of staining, decoloring, etc. of the polypeptide, such as temperature, current, and time.

In the present invention, the molecular weight of the phospholipase by SDS-PAGE is not limited as long as it is in the range of about 25 to 30 kDa, but the lower limit is about 25 kDa, preferably 26 kDa, and more preferably 27 kDa. The upper limit is about 30 kDa, preferably 29 kDa, and more preferably 28 kDa.

(C) It is derived from actinomycetes belonging to the genus Streptomyces.

(F) Derived from Streptomyces albidoflavus.

(G) Derived from Streptomyces avermitilis.

In the present invention, the phospholipase may be derived from a microorganism belonging to the genus Streptomyces (actinomycetes), but is preferably a phospholipase derived from Streptomyces albidoflavus or Streptomyces avermitilis; Most preferably, it is derived from avermitilis JCM 5070 (= DSM 46492).

For example, whether or not a strain isolated from soil, lakes, seas, the surface of a living organism or inside a body cavity is a microorganism belonging to the genus Streptomyces, for example, “Bergey's Manual 2nd edition (2001)”, “Classification of microorganisms”・ Methods of identification experiment-Focusing on molecular genetics and molecular biology (Springer Lab Manual) Springer Fairlark Tokyo, September 2001 ", etc., commercially available bacteria identification test products (for example, BIOMERIEUX) ), A method entrusted to “Techno Suruga Lab Co., Ltd. (Shizuoka City, Shizuoka City)” and the like.

Furthermore, whether or not those strains are Streptomyces albidoflavus or Streptomyces avermitilis, "Stackebrandt E., Ebers J .: Taxonomic parameters revised: tert. It may be determined by a method or the like. That is, if DNA-DNA hybridization has 70% or more homology, or 16S rRNA is 98.5% or more identical, it can be judged as a cognate homolog. Preferably, if homology of 70% or more is found in DNA-DNA hybridization, it is judged as a cognate homolog.

(D) In the absence of calcium ions, the relative activity with respect to PlsEtn (C18, 18: 1) is 19 ± 5% with respect to dipalmitoylphosphocholine.

Here, “PlsEtn (C18, 18: 1)” means PlsEtn in which the fatty acid at the sn-1 position has 18 carbon atoms, the fatty acid at the sn-2 position has 18 carbon atoms, and the number of double bonds is 1. means. An example of such PlsEtn is 1- (1Z-octadecenyl) -2-oleoyl-sn-glycero-3-phosphoethanolamine (Chemical Formula 4). This PlsEtn is, for example, Avanti Polar Lipids, Inc. Can be purchased as part number 852758.

Here, “dipalmitoylphosphocholine (DPPC)” is phosphatidylcholine in which the fatty acid at the sn-1 position has 16 carbon atoms and the fatty acid at the sn-2 position has 16 carbon atoms. An example of such phosphatidylcholine is 1,2-dipalmitoyyl-sn-glycero-3-phosphocholine (Chemical Formula 5). This PlsEtn is, for example, Avanti Polar Lipids, Inc. Can be purchased as part number 850355.

Here, the relative activity means the relative value (%) of the reaction rate (hydrolysis rate (acting rate)) of PL (A) to PlsEtn (C18, 18: 1) and DPPC. 3).

The relative activity (%) of PlsEtn (C18, 18: 1) in the present invention with respect to DPPC is 19 ± 5%, but it may be 9% or more, 11% or more, 12% or more, or 13% or more. It may be 14% or more, preferably 15% or more, and more preferably 16% or more. Needless to say, the higher the relative activity, the better, so the upper limit is not particularly set. However, if the upper limit is set, it is 100% or less, 80% or less, 60% or less, or 40% or less, It may be 30% or less, may be 24% or less, may be 21% or less, and may be 19% or less.

(E) In the absence of calcium ions, the relative activity against PlsEtn (C18, 20: 4) is 29 ± 13% relative to 1-palmitoyl-2-oleoyl-phosphocholine.

Here, “1-palmitoyl-2-oleoyl-phosphocholine (POPC)” means that the number of carbon atoms of the fatty acid at the sn-1 position is 16, the number of carbon atoms of the fatty acid at the sn-2 position is 18, and the number of double bonds Is phosphatidylcholine in which 1. An example of such phosphatidylcholine is 1-palmitoyyl-2-oleoyl-sn-glycero-3-phosphocholine (Chemical Formula 6). This POPC is available, for example, from Avanti Polar Lipids, Inc. Can be purchased as part number 850457.

Here, the relative activity means the relative value (%) of the reaction rate (hydrolysis rate (rate of action)) of PL (A) to PlsEtn (C18, 20: 4) and POPC. 4).

In the present invention, PlsEtn (C18, 20: 4) has a relative activity (%) to POPC of 29 ± 13%, preferably 16% or more, and more preferably 20% or more. It goes without saying that the higher the relative activity, the better. Therefore, the upper limit is not particularly set. However, if the upper limit is set, it is 42% or less, preferably 35% or less, and may be 30% or less.

The origin of the calcium ion in the above (d) and (e) is not limited by the counter ion (counter ion), and examples thereof include chloride, bromide, sulfate, acetate, nitrate and the like.

In the present invention, “non-existence” means that calcium ions are not intentionally present. In addition, it means that water, pH buffer, Pls, phospholipase and container in the present invention in the composition for hydrolyzing Pls to lyPls do not intentionally contain calcium ions. That is, the case where calcium ions are present in the composition for hydrolyzing Pls to lyPls without our fault is also included in the “absence” in the present invention. The concentration of calcium ions in the composition for hydrolyzing Pls to lyPls may be 10 μM or less, preferably 1 μM or less, more preferably 0.1 μM or less, and most preferably 0 μM.

(H) having the amino acid sequence set forth in SEQ ID NO: 1.

(I) It has an amino acid sequence in which one or more amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, and has an action of hydrolyzing Pls to lyPls.

(J) having an amino acid sequence in which one or more amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, and further the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10 And has an action of hydrolyzing Pls to lyPls.

(K) having the amino acid sequence set forth in SEQ ID NO: 2.

(L) It has an amino acid sequence in which one or more amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 2, and has an action of hydrolyzing Pls to lyPls.

(M) having an amino acid sequence in which one or more amino acids are mutated, deleted, or added in the amino acid sequence shown in SEQ ID NO: 2, and further the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10 And has an action of hydrolyzing Pls to lyPls.

That is, PL (A) of the present invention consists of the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, for example, and in addition, an amino acid sequence substantially equivalent to the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 Or consisting of an amino acid sequence in which some amino acids not involved in catalysis are mutated or various amino acid residues are added, and this amino acid sequence is the amino acid sequence shown in SEQ ID NO: 9 and SEQ ID NO: 10 A phospholipase containing an amino acid sequence and having an action of hydrolyzing Pls to lyPls can be used. The preferred homology of such amino acid sequences is 50% or more, preferably 60%, more preferably 70% or more, particularly preferably 80% or more, and most preferably 90% or more.

It is also preferable to add a functional enzyme such as thioredoxin enzyme or other amino acid sequence to the N-terminal side and / or C-terminal side of PL (A) of the present invention, or to make a fusion enzyme, and the part to be added Examples include a case where a portion called a tag that can be purified or confirmed by fusing is fused, and in some cases, even if the tag portion is deleted, all or a part of the portion remains. For example, about 20 signal peptides for transporting the PL (A) of the present invention to the outside of a cell or the periplasm, or addition of 4 to 10 His for efficient purification may be used. They may be added in series. In addition, several protease recognition amino acid sequences can be arranged and added between these amino acid sequences. Similar to the addition example described above, deletion or substitution can be performed, for example, when there is a domain consisting of several amino acids unrelated to the essential function of PL (A) of the present invention, or When there is a gap consisting of a plurality of amino acids in the amino acid sequence shown in SEQ ID NO: 1, those deletions can be combined. It is also possible to combine deletion, substitution or addition as appropriate. Examples of the additional amino acid residue include a signal peptide, a TEE sequence, an S tag, or a His tag. When the amino acid described in SEQ ID NO: 1 or SEQ ID NO: 2 is deleted, for example, an example in which Met on the N-terminal side or Lys on the C-terminal side is deleted in order. In the amino acid sequence shown in SEQ ID NO: 1, phospholipase modified after translation, such as deletion of Met at the N-terminus or modification of the N-terminus with an acyl group or an alkyl group is also PL (A) of the present invention. . Also, the PL (A) of the present invention is chemically modified with succinic anhydride, PEG or the like by a known method, and changes are made so that the properties such as optimum pH and stability of the PL (A) of the present invention can be easily used. It is also possible to make it. A preferred amino acid sequence of PL (A) of the present invention is the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

In the case of SEQ ID NO: 1, the molecular weight of PL (A) of the present invention is 27199, and in the case of SEQ ID NO: 2, the molecular weight of PL (A) of the present invention is estimated to be 27565. The molecular weight changes due to the addition of the tag portion described above or the deletion of some amino acids.

If the base sequence of PL (A) of the present invention is required, one or a plurality of amino acid sequences described in SEQ ID NO: 1 or SEQ ID NO: 2 and amino acid sequences described in SEQ ID NO: 1 or SEQ ID NO: 2 are used. A base sequence encoding PL (A) may be used. A preferred base sequence of the phospholipase used in the method of hydrolyzing Pls of the present invention to lyPls and / or the method of measuring Pls is the base sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4, and SEQ ID NO: 1 or SEQ ID NO: 2, respectively. Is encoded.

As the base sequence encoding the amino acid sequence shown in SEQ ID NO: 9 and SEQ ID NO: 10, any base sequence encoding the amino acid sequence shown in SEQ ID NO: 9 and SEQ ID NO: 10 may be selected from the genetic code table. The base sequence encoding the amino acid sequence shown in SEQ ID NO: 9 is the base sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, and the base sequence encoding the amino acid sequence shown in SEQ ID NO: 10 is SEQ ID NO: 13 or This is the base sequence set forth in SEQ ID NO: 14.

The Pls of the present invention is preferably Pls in the sample. The origin of Pls of the present invention is not particularly limited, but Pls in the sample is preferable. Samples may include plasma, serum, urine, research samples, and biological samples including whole blood, plasma, serum, blood cells, spinal fluid, lymph fluid, urine, etc. that are expected to contain Pls Samples and extracts thereof can be mentioned, and these samples are preferably samples that are expected to contain Pls. Examples of other samples include extracts from organisms such as sea squirts, krill, and shellfish, seawater, natural water, fruit juice, beverages, and waste liquids. Pls in such a sample is expected to contain Pls such as (Chemical Formula 3) and (Chemical Formula 4), for example, and other Pls are also considered to be mixed.

The determination of the possibility of Pls being included in the sample includes the case where the presence or absence of the possibility of Pls being included in the sample can be determined before the measurement method of the present invention is performed (for example, normal Human blood contains Pls (Plasmalogens in human serum positively correlated with high-density lipoprotein and decree with aging. Maeba et al., P. 14th-Mal. The presence or absence of the possibility may be determined by a conventional technique such as JP-A-2007-33410 and LC-MS (JP-A-2011-136926).

The amount of PL (A) used in the method of hydrolyzing Pls of the present invention to lyPls and / or the method of measuring Pls is not particularly limited as long as Pls can be hydrolyzed to lyPls and / or Pls can be measured. It is determined so that favorable results can be obtained according to the amount of Pls contained in the sample, the degree of hydrolysis of the target Pls, the equipment used, the purity of PL (A), and / or economic circumstances. obtain. Furthermore, when using a pH buffer, a metal ion, and a surfactant, the same applies to the type and amount thereof.

The amount of PL (A) used in the method of hydrolyzing Pls of the present invention to lyPls and / or the method of measuring Pls is, for example, that the amount of Pls contained in the sample is 2 μmol or less, and all of them is 37 ° C. In the case of the conditions for hydrolysis in 10 minutes, the lower limit is 0.05 mU or more, preferably 0.1 mU or more, more preferably 0.5 mU or more, and it is clear that the larger the amount of phospholipase, the better the hydrolysis efficiency. An upper limit is not particularly provided, but if an upper limit is provided for economic reasons, for example, it is 10 U or less, preferably 5 U or less, more preferably 1 U or less.

The method of hydrolyzing Pls of the present invention to lyPls and / or the method of measuring Pls may be performed in a liquid, a gas phase, a solid phase, or the like or on each critical surface, but may be performed in a liquid. preferable. The liquid may be an aqueous solution, an organic solvent, etc., and the measurement method of the present invention is preferably carried out in an aqueous solution. However, an aqueous medium containing an appropriate organic solvent may be used, and in such a case, an appropriate pH buffering agent may be used. Is preferably used. When a pH buffer is used, the type of the pH buffer is not particularly limited as long as the target pH can be maintained, and Pls can be hydrolyzed to lyPls and / or Pls can be measured. Good pH buffer, Tris / HCl buffer Liquid, potassium phosphate buffer, acetic acid / NaOH buffer, citric acid / NaOH buffer. The pH for carrying out the present invention is not particularly limited as long as Pls can be hydrolyzed to lyPls and / or Pls can be measured, but the lower limit is pH 4 or more, preferably pH 5 or more, more preferably pH 6 or more, and the upper limit. The pH is 11 or less, preferably 10.5 or less, more preferably 10 or less. The concentration of the pH buffer is not particularly limited as long as the target pH can be maintained and Pls can be hydrolyzed to lyPls and / or Pls can be measured, but the lower limit is 3 mM or more, preferably 5 mM or more, more preferably 10 mM or more. The upper limit is 500 mM or less, preferably 200 mM or less, more preferably 100 mM or less.

As another preferred embodiment of the method of hydrolyzing Pls of the present invention to lyPls and / or the method of measuring Pls, for example, an enzyme (PL (A), etc.) is immobilized using a sol-gel method or a sensor is prepared. Can be mentioned. In order to obtain a sol-gel, for example, a polysaccharide such as agar may be used. When the sol-gel and the emulsion are distinguished, the emulsion may be implemented as an emulsion. In order to obtain an emulsion, for example, an organic solvent or the like may be used, and if an amphiphilic substance is used, micelles can be used. In any case, when a pH buffer is used, it is the same as described above.

In the method of hydrolyzing Pls of the present invention to lyPls and / or the method of measuring Pls, the reaction time is not particularly limited as long as Pls in a sample can be hydrolyzed to lyPls and / or Pls can be measured. The lower limit is 15 seconds or longer, preferably 1 minute or longer, more preferably 3 minutes or longer. Although there is no particular upper limit, it is preferably 30 minutes or less, more preferably 15 minutes or less, and particularly preferably 10 minutes or less.

In the method of hydrolyzing Pls of the present invention to lyPls and / or the method of measuring Pls, the temperature is not particularly limited as long as it is a temperature at which Pls in a sample can be hydrolyzed to lyPls and / or a temperature at which Pls can be measured. However, it is preferably within the range of the working temperature of the phospholipase used, the lower limit is 10 ° C. or higher, preferably 20 ° C. or higher, more preferably 25 ° C. or higher, and the upper limit is 70 ° C. or lower, preferably 60 ° C. or lower, more preferably 50 ° C. It is as follows.

The method for measuring Pls of the present invention includes a method of hydrolyzing Pls to lyPls using PL (A) having the characteristics and properties described herein. That is, the method for measuring Pls of the present invention is as follows.
<Step 1> A step of hydrolyzing Pls to lyPls using phospholipase (PL (A)) having the properties and properties described in the present specification,
including.

The method for measuring Pls of the present invention may further include a known step for measuring lyPls different from <Step 1>. An example of such a process is
<Step 2-1> lysoplasmalogenase (EC 3.3.2.2; EC 3.3.2.5, alkenyl hydrolase, (Biochimica et Biophysica Acta, 1437, 1999, pages 142 to 156, The journal of pharmacology) chemistry, 286, 24916-24930, 2011), which may be abbreviated as “lyPls as”),
<Step 2-2> A method using thin layer chromatography (TLC), etc.
<Step 2-3> Method using Lysophospholipase D (Autotaxin, The journal of biological chemistry, Vol. 277, 2002, pages 39436-39442),
<Step 2-4> Method using HPLC (Japanese Patent Application Laid-Open No. 2007-33410),
<Step 2-5> Method using LC-MS (Japanese Patent Laid-Open No. 2011-136926),
Etc.

<Step 2-1> is preferable from the viewpoint that Pls can be accurately measured without erasing even if a substance other than Pls is mixed in the sample. <Step 2-2> is preferable from the viewpoint that materials necessary for measurement are inexpensive and easily available and can be easily implemented by those skilled in the art. <Step 2-3> is preferable from the viewpoint that one kind of enzyme is used as compared with <Step 2-1>. <Step 2-4> is preferable from the viewpoint of accurate quantification. <Step 2-5> is preferable from the viewpoint that the molecular species of Pls (R _{1 of} “Chemical Formula 1”) can also be specified.

Among the methods for measuring Pls of the present invention including <Step 1> and <Step 2-1>, a method for measuring PlsEtn is shown in a simplified manner in FIG. The lyPls as used here is described in “Biochimica et Biophysica Acta, 1437, 1999, pages 142 to 156” or “The journal of biologics, 286, 24916 to 24930, 2011”. Use rat-derived lyPls as. Glycerophylphospholine phosphatase (GPCP, EC 3.1.4.2) can be purchased from Asahi Kasei Pharma Corporation (product number T-33). Ethanolamine oxidase may be produced by the method of Bioscience, Biotechnology, and Biochemistry 2008, Vol. 72, pages 2732-2738, and quantified by the method described in Japanese Patent No. 4244168. In addition, when measuring PlsCho, instead of the above-mentioned Ethanolamine oxidase, Choline Oxidase is purchased from Asahi Kasei Pharma Corporation (product number T-05) and quantified by the method described in JP-A-58-28283. Good.

H ₂ O ₂ is Peroxidase (POD, EC 1.11.1. 7), a chromogen of a Trinder reagent such as a phenol derivative, an aniline derivative or a toluidine derivative described later, 4-aminoantipyrine or 3-methyl-2-benzo. It can be quantified by a known method such as using a coupler such as thiazolinone hydrazone. H ₂ O ₂ can also be analyzed by a fluorescence method or an electrode method. In the fluorescence method, compounds that emit fluorescence upon oxidation, such as homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, diacetylfluorescin derivatives, and the like, and in the chemiluminescence method, luminol, lucigenin, isoluminol, Pyrogallol can be used.

When measuring H ₂ O ₂ with an electrode, the electrode is not particularly limited as long as it is a material that can exchange electrons with H ₂ O _2. For example, platinum, gold, silver and the like can be used to measure electrodes. As a method, a known method such as amperometry, potentiometry, coulometry, etc. can be used. Further, an electron carrier is interposed in the reaction between the oxidase or substrate and the electrode, and the resulting oxidation, reduction current or electric current thereof is obtained. The amount may be measured. Any substance having an electron transfer function can be used as the electron carrier, and examples thereof include substances such as ferrocene derivatives and quinone derivatives. The oxide obtained by interposing an electron carrier between the H ₂ O ₂ and the electrode generated by oxidase reaction, may measure the reduction current or the electrical quantity. In the present invention, buffers, enzyme stabilizers, preservatives, and the like are appropriately used as analytical reagents as necessary.

The amount of lyPlsase enzyme used in <Step 2-1> is 4.2 U / mL as described in Biochimica et Biophysica Acta, Volume 1437, 1999, pages 142-156.

The lyPls ase used in <Step 2-1> is “Tmem86b (inventor 註: gene of lyPls ase), foundlybesided in The journal of biologic chemistry, 286, 24916-24930, 2011”. "Including humans, rice, rats, cows, dogs, and zebrafish.", human, mouse, rat, cow, dog, and sebrafish are used.

The TLC of <Step 2-2> may be performed by appropriately combining known methods described in, for example, “No failure in bio-experiments! Tips for detection and quantification, Tatsuya Moriyama, Yodosha, 2005”. Examples of the detection method include a UV lamp and a coloration method. Examples of the colorant include anis-sulfuric acid, phosphomolybdic acid, iodine, ninhydrin solution, chameleon solution, 2,4-dinitrophenylhydrazine solution, bromocresol green solution, and Dragendorfff. A known reagent such as a reagent can be determined so as to obtain a preferable result according to the purpose, sample, apparatus used, and the like.

Of the methods for measuring Pls of the present invention including <Step 1> and <Step 2-3>, a method for measuring PlsEtn is shown in a simplified manner in FIG. As the Lysophospholipase D used here, for example, a human-derived enzyme described in “The journal of biochemical chemistry, 277, 39436-39442, 2002” can be used. The case of measuring Ethanolamine oxidase and PlsCho is the same as in the case of <Step 2-1>. The amount of Lysophospholipase D enzyme used in <Step 2-3> is the same as in <Step 2-1>.

<Step 1> and <Step 2-1>, <Step 2-2> or <Step 2-3> can be carried out in different reaction vessels (phases), but in the same reaction vessel (phase). Is preferred. <Step 1> and <Step 2-1>, <Step 2-2> or <Step 2-3> can be performed discontinuously, but are preferably performed continuously. <Step 1> and <Step 2-1>, <Step 2-2> or <Step 2-3> are followed by <Step 1>, <Step 1>, <Step 2-1>, and <Step 2-2 or <Step 2-3> may be performed, and <Step 1> and <Step 2-1>, <Step 2-2> or <Step 2-3> may be performed simultaneously. <Step 1> and <Step 2-4> or <Step 2-5> are usually performed in the order of <Step 1>, <Step 2-4> or <Step 2-5>. These reaction vessels (phases) and the order of execution can be determined so as to obtain preferable results according to the purpose, sample, apparatus used, and the like.

The aspects of pH, reaction time, reaction temperature, etc. in <Step 2-1> and <Step 2-3> are the same as in <Step 1>, but <Step 1> and <Step 2-1> or <Step 2> The aspect of -2> such as pH, reaction time, reaction temperature and the like is not necessarily uniform with <Step 1>, and may be uneven with <Step 1> within the range described in this specification.

PL (A) for hydrolyzing Pls having the characteristics and properties of the present invention to lyPls and / or PL (A) for measuring Pls is a composition for hydrolyzing Pls to lyPls and / or It can be a composition for measuring Pls.

The composition of the present invention may be a composition (phospholipase-containing composition) containing the following component (I), but (I) having the characteristics and properties of the present invention is preferable. The composition of the present invention may be a composition containing the components (I), (V) and (IV), but a composition containing the components (I) to (IV) is more preferred. The composition may be any of a composition for hydrolyzing Pls to lyPls, a composition for measuring Pls, and a composition for hydrolyzing Pls to lyPls and measuring Pls.

(I) PL (A)
(II) Known lyPls as
(III) GPCP
(IV) Ethanolamine oxidase
(V) Lysophospholipase D
If necessary, these compositions may be used as peroxidase, catalase (EC 1.11.1.6), chromogens of Trinder reagents such as phenol derivatives, aniline derivatives, toluidine derivatives, 4-aminoantipyrine (4-AA). Alternatively, a coupler such as 3-methyl-2-benzothiazolinone hydrazone may be contained.

As the chromogen of the Trinder type reagent, phenol derivatives, aniline derivatives, toluidine derivatives and the like can be used. Specific examples include N, N dimethylaniline, N, N diethylaniline, 2,4 dichlorophenol, N-ethyl- N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-3,5 dimethylaniline (MAPS), N-ethyl-N- (2- Hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS) (manufactured by Doujin Chemical Laboratory) Etc. Hydrogen peroxide can be colored using a leuco reagent in the presence of peroxidase. Specific examples of this reagent include o-dianisidine, o-tolidine, 3,3 diaminobenzidine, 3,3,5,5-tetramethylbenzidine (manufactured by Dojindo Laboratories), N- (carboxymethylaminocarbonyl). ) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67) (manufactured by Wako Pure Chemical Industries, Ltd.) It is done.

The composition for hydrolyzing Pls of the present invention to lyPls and / or the composition for measuring Pls may not contain calcium ions. When the composition is an aqueous solution, carbon dioxide in the air may dissolve in the composition over time. At this time, if calcium ions are contained in the composition, the calcium ions may be precipitated as calcium carbonate and the composition may become cloudy. However, the composition and Pls for hydrolyzing Pls of the present invention to lyPls are measured. The composition for this purpose does not need to contain calcium ions, and therefore has an advantage that such a change with time does not easily occur.

Further, in the composition of the present invention, among the above components, (I) and (II), (III) and (IV) are preferably combined with (V) immediately before measurement to become the composition of the present invention. . That is, in order to obtain the composition of the present invention having all the components necessary for measurement, at least (I), (II), (III) and (IV) and (V) are separated before the measurement. It is preferable. A preferable form is divided into three reagents, the first reagent contains (I) and (II), the second reagent contains (III) and (IV), and the third reagent contains (V). When these are united immediately before the measurement to obtain a composition, the uniting order is arbitrary, and may be united one by one or may be united at the same time. The most preferred form is divided into two reagents, the first reagent contains (I), (II), (III) and (IV), and the second reagent contains (V). The first reagent and the second reagent are kept separate until just before the measurement. These are united immediately before the measurement to obtain a composition.

An effective amount of the components (I) to (V) contained in the composition of the present invention, that is, the composition can be a composition for hydrolyzing Pls to lyPls and / or a composition for measuring Pls. Therefore, the effective addition amount and pH buffering agent conditions are the same as those in the measurement method of the present invention.

The composition of the present invention preferably contains a pH buffer as appropriate. It is also preferable to include a calibration reagent comprising at least a known amount of Pls.

In the present invention, the calibration reagent is preferably a reagent containing at least a known amount of Pls, but is preferably a reagent containing a pH buffer, a preservative such as sodium azide and antibiotics, and a stabilizer such as sugar. When a pH buffering agent is included, conditions such as the type and concentration are the same as in the measurement method of the present invention. When sodium azide or antibiotics are included, the type and concentration are not limited as long as they have a preservative effect. For example, in the case of sodium azide, the lower limit is 0.005% or more, preferably 0.01% or more, more preferably It is 0.03% or more, and the upper limit is 1% or less, preferably 0.5% or less, and more preferably 0.1% or less. For example, in the case of antibiotics, the lower limit is 5 μg / mL or more, preferably 10 μg / mL or more, more preferably 30 μg / mL or more, and the upper limit is 100 μg / mL or less, preferably 75 μg / mL or less, more preferably 60 μg / mL or less. is there. When a stabilizer is included, conditions such as the type and concentration are the same as those of the above-described phospholipase stabilizer. As the calibration method, a single inspection quantity, a multi-inspection quantity (a broken line or a spline), a linear regression of the multi-inspection quantity, and the like can be selected.

The known amount is not particularly limited, and may be selected in order to accurately measure Pls in the sample. The same applies to the known amount of calibration reagent when a plurality of calibration reagents are used. For example, in the case of Pls, the lower limit is 0.00 μM or more, preferably 10 μM or more, more preferably 50 μM or more, and the upper limit is 500 μM or less, preferably 200 μM or less, more preferably 150 μM or less.

The composition and calibration reagent for Pls measurement of the present invention are liquid products, frozen products of liquid products, freeze-dried products of liquid products, or dried products of liquid products (heat-dried and / or air-dried and / or dried under reduced pressure, etc.) According to). Liquid frozen products are preferred, liquid freeze-dried products are more preferred, and liquid products are most preferred. In another embodiment, a frozen liquid product may be preferable. As yet another aspect, lyophilization of a liquid product may be preferred. The composition for hydrolyzing Pls of the present invention to lyPls and / or the composition for measuring Pls may be a composition of one reagent, but usually it is separated into two or more reagents as described above. Is preferred. For the purpose of improving reagent quality, salts such as NaCl and KCl, surfactants such as TX-100 and Tween 20, and / or preservatives such as sodium azide and antibiotics may be mixed. For example, when using POC (point of care) in capillaries or as an enzyme sensor, the concentration of each component is preferably higher than usual. For example, it may be fixed or soaked in paper or a film. Or a gel-sol composition. When mixing a salt, the type and concentration are not limited, but it is usually in the range of 5 to 200 mM, and when mixing a surfactant, the type and concentration are not limited, but usually 0.001% to 2%. Yes, when the preservative is mixed, it is the same as the case of the calibration reagent.

The method for producing a phospholipase of the present invention is a method for producing any one of the following <1> to <3>, wherein a step of forming a phospholipase based on a nucleotide sequence encoding phospholipase and a phospholipase are obtained. A process for producing a phospholipase comprising a step.

<1> Consists of the amino acid sequence set forth in SEQ ID NO: 2 and has an action of hydrolyzing Pls to lyPls.

<2> The amino acid sequence described in SEQ ID NO: 2 consists of an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence is described in SEQ ID NO: 9 and SEQ ID NO: 10 It has the effect | action which hydrolyzes Pls to lyPls including the amino acid sequence of.

<3> It has the following characteristics (a) to (d).

(A) In the absence of calcium ions, the relative activity against PlsEtn (C18, 18: 1) is 19 ± 5% relative to dipalmitoylphosphocholine.

(B) In the absence of calcium ions, the relative activity to PlsEtn (C18, 20: 4) is 29 ± 13% relative to 1-palmitoyl-2-oleoyl-phosphocholine.

(C) The molecular weight determined by SDS-PAGE is in the range of about 25-30 kDa.

(D) It is derived from actinomycetes belonging to the genus Streptomyces.

Here, the action of hydrolyzing Pls to lyPls and the characteristics (a) to (d) are the same as described above. That is, the method for producing phospholipase is the method for producing PL (A) of the present invention. The amino acid sequences described in SEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 10 are the same as described above. The nucleotide sequences encoding the amino acid sequences described in SEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 10, and preferred nucleotide sequences SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 The base sequence is the same as described above.

In producing such PL (A) of the present invention, a step of forming PL (A) based on the base sequence encoding PL (A) can be used. As this step, a cell-free protein synthesis system containing a base sequence encoding PL (A), preferably a step using cells containing a base sequence encoding PL (A), or natural PL (A) is formed. Examples include a step of using a microorganism having a base sequence encoding PL (A) such as a microorganism, a step of using a transformant into which a base sequence encoding PL (A) is introduced, and the like.

As a typical method for producing PL (A) of the present invention, there is an example in which PL (A) is formed using a natural microorganism that forms PL (A). Examples of natural microorganisms that can be prepared include actinomycetes belonging to the genus Streptomyces, more preferably Streptomyces avermitilis, most preferably Streptomyces avermitilis JCM 5070 (= DSM 46492). The method for obtaining, identifying and judging these microorganisms is as described above.

The natural microorganism that forms the PL (A) of the present invention can be a mutant strain further treated with a drug such as NTG, ultraviolet rays, and / or radiation. The mutant strain can improve the productivity of the PL (A) of the present invention and can form a mutant of the PL (A) of the present invention, and is excellent in stability, productivity, reactivity and the like. It is also possible to form mutants having different properties. In the case of employing a process using cells containing a base sequence encoding PL (A) of the present invention, a transformant is prepared by inserting the above base sequence into a vector and introducing it into a host microorganism. The process of forming a protein using a converter is illustrated. The transformant introduced with the base sequence encoding PL (A) of the present invention includes cells or microorganisms into which a recombinant phage or recombinant plasmid, which is a vector having the base sequence inserted, is introduced into a host. The base sequence encoding PL (A) of the present invention can be used by synthesizing a part or all of the base sequence. Preferably, the base sequence encoding PL (A) of the present invention is obtained from a gene donor. To use. Examples of gene donors include actinomycetes belonging to the genus Streptomyces, more preferably Streptomyces avermitilis, most preferably Streptomyces avermitilis JCM 5070 (= DSM 46492).

As a vector for inserting a base sequence encoding PL (A) of the present invention, a phage or plasmid constructed for gene recombination among phages or plasmids that can autonomously grow in the host microorganism is suitable. For example, when a microorganism belonging to E. coli is used as a host, λgt · λC, λgt · λB, and the like can be used. As plasmid vectors, for example, when Escherichia coli is used as a host, Novagen's pET vector, or pBR322, pBR325, pACYC184, pUC system, and Bacillus subtilis as hosts, pWH1520, pUB110, pKH300PLK, When actinomycetes are used as hosts, pIJ680 and pIJ702 can be used. When yeasts, particularly Saccharomyces cerevisiae, are used as hosts, YRp7, pYC1, YEp13 and the like can be used. In the present invention, plasmid vectors using Escherichia coli and actinomycetes as hosts are preferred. The promoter is not particularly limited as long as it can be expressed in the host.

A vector fragment is prepared by cleaving such a vector with a restriction enzyme that produces the same end as the end of the base sequence generated by the restriction enzyme used to cleave the base sequence encoding PL (A) of the present invention. The base sequence fragment encoding the PL (A) of the present invention is ligated with a fragment of the base sequence encoding the PL (A) of the present invention by DNA ligase according to a conventional method, and the base sequence encoding the PL (A) of the present invention is inserted into the target vector. Recombinant phage or recombinant plasmid. The host into which the recombinant plasmid is introduced may be any cell or microorganism that can stably and autonomously propagate the recombinant plasmid, and Escherichia coli B strain, K strain, C strain and lysogens thereof can be used. In addition, when the host microorganism is a microorganism belonging to the genus Bacillus, Bacillus subtilis, Bacillus megaterium, etc., a microorganism belonging to actinomycetes, Streptomyces lividans TK24, etc., a microorganism belonging to Saccharomyces cerevisiae, Saccharomyces cerevisiae INVSC1 etc. can be used. In the present invention, it is preferable to use Escherichia coli or actinomycetes as a host microorganism.

The PL (A) of the present invention may be formed by culturing a transformant introduced with a base sequence encoding PL (A) or a microorganism having a base sequence encoding PL (A).

In order to obtain the PL (A) of the present invention from such a microorganism, for example, “Catalogue of Strains, fifth edition, 1993, Deutsche SammLung von Mikroorganisundund Zellkulturen GMBER” The microorganisms were cultured by the method described in the American Type Culture Collection, etc., and for example, “Protein / Enzyme Basic Experiment (Revised 2nd Edition, Takeichi Horio, 1994) Can be obtained without purification or purification by the method described in `` Nankodo ''). Properly it can be purified. That is, the PL (A) of the present invention can be produced by a method including the step of obtaining the PL (A) of the present invention formed as described above. It is also preferable to use PL (A) in which impurities such as cultured impurities and cell debris are lightly removed remain. Furthermore, it is preferable that the crude protein of the protein of the present invention does not substantially contain impurities depending on the purpose and application, but usually, for example, 50% or more, 70% or more, 95% or more of various kinds It is exemplified to make it pure. The purity may be confirmed by a known method such as SDS-PAGE or HPLC.

PL (A) of the present invention is obtained by culturing natural microorganisms that form PL (A), microorganisms of transformants into which a base sequence encoding PL (A) has been introduced, and the like. Can be manufactured. First, microorganisms and the like are cultured in a nutrient medium to form PL (A) in the microbial cells or in the culture solution, and when formed in the microbial cells, the obtained culture is filtered or centrifuged after completion of the culture. Bacteria are collected by the means described above. Next, this bacterial cell is destroyed by a mechanical method or an enzymatic method such as lysozyme, and EDTA and / or an appropriate surfactant is added as necessary to concentrate or concentrate PL (A). Without application, the PL (A) of the present invention is precipitated and recovered by applying a fractional precipitation method using an organic solvent such as acetone, methanol, ethanol or the like, a salting out method using ammonium sulfate, sodium chloride, or the like. The precipitate is subjected to dialysis and isoelectric precipitation, if necessary, and then subjected to gel filtration, adsorption chromatography such as affinity chromatography, ion exchange chromatography or hydrophobic chromatography to obtain the PL ( A) can be obtained. Moreover, it can carry out combining these methods suitably. Further, when the PL (A) of the present invention is formed in a culture solution, the cells are removed by means of filtration or centrifugation, and the culture solution is formed in the cells. The same processing may be performed.

The PL (A) of the present invention obtained by these methods can be used as a stabilizer, such as ultrafiltration concentration, lyophilization, etc., with or without various salts, saccharides, proteins, lipids, surfactants and the like. According to the method, the liquid or solid PL (A) of the present invention can be obtained, and may be freeze-dried as appropriate. In this case, 0.5 to 0.5% of saccharose, mannitol, sodium chloride, albumin or the like is used as a stabilizer. About 10% may be added.

The base sequence described in SEQ ID NO: 4 is Nat. Biotechnol. 21, 526-531, 2003, but the nature of the protein encoded by the base sequence has not been elucidated so far and was simply expected as a hydrolase. That is, the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 encoded by the base sequence shown in SEQ ID NO: 4 has an action of hydrolyzing Pls to lyPls (the property of the present invention as PL (A)) It has never been known before.

Hereinafter, the present invention will be described based on examples and the like, but the scope of the present invention is not construed as being limited to the following examples. The technique described below is, for example, the method of Maniatis et al. (Maniatis, T., et al. Molecular Cloning. Cold Spring Harbor Laboratory (1982, 1989), (non-) patent literature described in this specification, It can be carried out by following the procedures attached to various commercially available enzymes or kits.

The phospholipase A2 used in this example is as follows.

PLA2 II L (Lot 1001A, Asahi Kasei Pharma Corporation, derived from Streptomyces avermitilis, product number T-194). PLA2 Nagase (manufacturing number 7907851, derived from Nagase ChemteX Corporation, Streptomyces avermitilis). LIPOMOD 699L (Batch No. 90825437, Biocatalyst, derived from porcine pancreas, product number L699L).

Unless otherwise specified, the reagents used in this example are those manufactured by Wako Pure Chemical Industries, Ltd., Sigma Aldrich, Takara Bio, etc., and those that are readily available in the market are used.

Measured values shown below may vary depending on measurement conditions, accuracy of equipment used, reagent manufacturer and purity.

[Example 1: Activity measurement method]
A method for measuring the activity of each phospholipase (PL (A), PLB, PLA2 II L, PLA2 Nagase, and LIPOMOD 699L) is simply shown in FIG. Acyl-CoA Synthetase (EC 6.2.1.3) and Acyl-CoA Oxidase (EC 1.3.3.6) were obtained from Asahi Kasei Pharma Corporation (product numbers T-16 and T17, respectively).

<First reaction reagent mixture>
80 mM Tris-HCl buffer pH 8.0
50 mM (0 mM (*)) Calcium chloride 4 mM ATP
4 mM CoA
1.06U / mL Acyl-CoA Synthetase
2 mM dipalmitoylphosphocholine (DPPC)
(*) In other examples, it may be 0 mM.

<Second reaction reagent mixture>
40 mM PIPES-NaOH buffer pH 7.5
0.06% 4-AA
0.04% Phenol 4.5U / mL peroxidase
30U / mL Acyl-CoA Oxidase
0.2% Triton X-100
20 mM ATP
0.1 mM FAD
<Reaction stop solution>
0.1M EDTA solution pH 8.0 containing 0.5% SDS (sodium dodecyl sulfate)
<Enzyme dissolution dilution>
10 mM Tris-HCl buffer pH 8.0 containing 0.05% BSA
<Measurement operation method>
(1) 0.50 mL of the first reaction reagent mixture is accurately dispensed into a small test tube and pre-warmed at 37 ° C.

(2) After 5 minutes, 50 μL of an enzyme sample solution (PL (A), PLB, PLA2 II L, PLA2 Nagase or LIPOMOD 699 L) diluted with an enzyme dissolution dilution buffer to an appropriate concentration was accurately added and mixed, Start the first reaction at 37 ° C. In the blind test, 50 μL of enzyme lysis dilution buffer is added instead of the enzyme sample solution.

(3) After 10 minutes, 0.50 mL of 20 mM NEM solution is added and mixed. After 15 seconds, 0.50 mL of the second reaction reagent mixed solution is added and mixed, and the second reaction is started at 37 ° C.

<Calculation>
Activity is calculated according to (Equation 5) below. One unit (U) was defined as a reaction rate at which each phospholipase hydrolyzes a substrate (DPPC in Example 1) to produce 1 μmol of fatty acid per minute under the conditions of this example.

<Result>
As shown in FIG. 1, the activity measurement method of this example is a method for measuring fatty acids produced by the action of each phospholipase. Therefore, the activity of PL (A) could be measured by the activity measurement method of this example, indicating that PL (A) acts on DPPC to produce at least fatty acids, like other PLA2. ing.

The activity of each phospholipase measured in Example 1 is shown in Table 1.

In this example, each phospholipase shown in Table 1 was appropriately diluted and used. That is, the display activity of each phospholipase was not employed in this example.

[Reference Example 1: Preparation of Streptomyces albidoflavus-derived PL (A)]
[Reference Example 1-1: Isolation of Chromosomal DNA of Streptomyces albidoflavus NA297]
Streptomyces albidoflavus NA297 with 50 mL of YEME medium (0.3% yeast extract, 0.5% peptone, 0.3% malt extract, 1% glucose, 34% sucrose, 5 mM MgCl ₂ , 0.5% glycine) The cells were cultured at 28 ° C. for 4 days and collected. Next, the cells were suspended in 5 mL of a solution consisting of 75 mM NaCl, 25 mM EDTA, 20 mM Tris-HCl buffer (pH 7.5) and 1 mg / ml lysozyme, and treated overnight at 37 ° C. To this, 750 μL of 10% (w / v) SDS and 5 mg of proteinase K were added and treated at 55 ° C. for 2 hours. To this solution, 7.5 mL of chloroform was added and stirred, and 5 mL of the aqueous phase was collected by centrifugation. To this aqueous phase, 3 mL of isopropanol was added and mixed to collect the DNA fraction, which was dissolved in 500 μL of a solution composed of 10 mM squirrel-hydrochloric acid buffer (pH 8.0) and 1 mM EDTA. RNase A was added to this so that it might become 20 micrograms / mL, and after processing at 37 degreeC for 1 hour, 500 microliters of 13% PEG solutions containing 0.8M NaCl were added and stirred, and 500 microliters of water phases were fractionated by centrifugation. . To this was added 500 μL of a phenol / chloroform mixed solution and stirred, and 500 μL of the aqueous phase was collected by centrifugation. To this aqueous phase, 50 μL of 3M sodium acetate (pH 5.2) and 1 mL of ethanol were added and mixed to recover DNA. This DNA was immersed in 70% (v / v) ethanol for 10 minutes and then dissolved in 200 μL of a solution consisting of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA to obtain template chromosome DNA.

[Reference Example 1-2: Preparation of recombinant plasmid containing Streptomyces albidoflavus NA297PL (A) gene]
A sense primer “primer S” (SEQ ID NO: 5) was synthesized as an oligo for PCR, and “primer AS” (SEQ ID NO: 6) was synthesized as an antisense primer. Template chromosomal DNA 50 ng obtained in Reference Example 1-1, 10 × PCR Buffer 2.5 μL, each primer 1200 nM, dNTPs 0.3 mM each, MgCl ₂ , 1.2 mM, DMSO 4%, KOD DNA Polymerase 1.25 units, Distilled water was added to a total volume of 25 μL. PCR reaction conditions are as follows.

Step 1: 98 ° C., 2 minutes;
Step 2: 98 ° C., 15 seconds;
Step 3: 72 ° C., 15 seconds;
Step 4: 74 ° C., 60 seconds;
Repeat steps 2 to 4 for 30 cycles;
Step 5: 74 ° C., 4 minutes.

A specific amplification product of about 900 bp was obtained by this PCR. The ⁶⁸⁹ AGATCT ⁶⁹⁴ sequence in the amplified product is the Kunkel method (Kunkel, T.A. (1985) Rapid and effective site-specific mutations with the phenotype of the United States of America. Page) ⁶⁸⁹ AAATCT ⁶⁹⁴ .

The amplified fragment was digested with NheI and BglII, and inserted into the NheI-BglII site of the actinomycete plasmid (pMD20-T vector (manufactured by TaKaRa)) as an expression vector to obtain a recombinant plasmid.

[Reference Example 1-3: Production of recombinant actinomycetes expressing PL (A) gene derived from Streptomyces albidoflavus NA297]
Using the recombinant plasmid obtained in Reference Example 1-2, the protoplast actinomycete Streptomyces lividans) 1326 was transformed according to the method described in “PRACTICAL STREPTOMY CES GENETICS (Kieser et al., John Inns Foundation, 2000)”. The recombinant actinomycetes were obtained after conversion.

[Reference Example 1-4: Culture of recombinant actinomycetes expressing PL (A) gene derived from Streptomyces albidoflavus NA297]
The recombinant actinomycetes obtained in Reference Example 1-3 were cultured in 100 mL × 4 tryptic soy media (Pecton Dickinson) containing 12 μg / mL thiostrepton. The supernatant was recovered from the obtained culture solution (340 mL) by centrifugation (15000 rpm, 5 minutes, 4 ° C.), and the precipitate was recovered by ammonium sulfate fractionation. The collected precipitate was dissolved in 20 mM squirrel-hydrochloric acid buffer (pH 8.0) and dialyzed with 20 mM Tris-hydrochloric acid buffer (pH 8.0) as an external solution to obtain an enzyme solution. The concentration of the obtained PL (A) was about 2.5 U / mL, and it was appropriately concentrated with 10-kDa centrifugal filter device (Millipore) or the like, and the PL (A) of the present invention was used in Examples 1 to 11. Used as.

[Reference Example 2: Method for preparing PLB]
Lecture number of the 2011 Annual Meeting of the Japanese Society for Agricultural Chemistry: 2C10a05 (March 23, 2011 9: 44-), Streptomyces sp. A large amount of NA684-derived novel phospholipase B was expressed and analyzed by a method published by Yusaku Matsumoto and Daisuke Sugimori (Fukushima Univ., Riko) and used in the examples.

(A) Culture NB medium “1% peptone (Becton Dinkinson), 1% meat extract (Kyokuto Pharmaceutical Co., Ltd.), 0.5% sodium chloride (Wako Pure Chemical Industries, Ltd.) , PH 7.2 ", 300 mL, dispense 100 ml into a 500 mL Erlenmeyer flask, add 1% soybean lecithin and 0.1% Tween 80, and steam sterilize at 121 ° C for 15 minutes. went. A suitable amount of a colony of Streptomyces sp. NA684 previously grown on a plate medium was taken and put in a φ18 test tube (18 × 180 mm) containing 5 mL of tryptic soy medium (Becton Dinkinson). Inoculated and cultured with shaking at 28 ° C. until good growth was obtained. 1 ml of this culture solution was inoculated into 100 mL of the previously sterilized medium and cultured with shaking at 28 ° C. for 108 hours. The supernatant was recovered from this culture using a centrifuge.

(B) Ammonium sulfate fraction To the culture supernatant collected in (a) above, ammonium sulfate was added so as to be 80% (w / v) saturated, and the resulting precipitate was centrifuged (10,000 rpm, 30 minutes, 4 minutes). C). This precipitate was solubilized and dialyzed against 20 mM Tris-HCl buffer (pH 9.0) to obtain a crude enzyme solution.

(C) DEAE-Toyopearl column chromatography The “DEAE-Toyopearl 650M column” (inner diameter: 26 mm, height) obtained by previously equilibrating the crude enzyme solution obtained in (b) above with 20 mM Tris-HCl buffer (pH 9.0). 55 mm, manufactured by Tosoh Corporation). After washing the column with the same buffer, the active fraction was eluted with a linear gradient of sodium chloride (from 0 M to 1 M).

(D) HiTrap Q column chromatography The active fractions obtained in (c) above were collected and concentrated and desalted using Viva spin (manufactured by Sartorius). To this, 20 mM Tris-HCl buffer (pH 9.0) was added. This was applied to a “HiTrap Q” (5 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 20 mM Tris-HCl buffer (pH 9.0), and the column was washed with the same buffer. The active fraction was eluted with a linear gradient of sodium chloride (0M to 1M).

(E) RESOURCE PHE column chromatography The active fractions obtained in (d) above were collected and concentrated and desalted using Viva spin. To this, 20 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate was added. After applying to a “RESOURCE PHE” (1 ml) column (manufactured by GE Healthcare Biosciences) previously equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate, the column was washed with the same buffer. The active fraction was eluted with a linear gradient of ammonium sulfate (1M to 0M).

(F) Mono S column chromatography The active fractions obtained in (e) above were collected and concentrated and desalted using Viva spin. To this, 20 mM female-sodium hydroxide buffer (pH 6.0) was added. This was applied to a “Mono S” (1 ml) column (manufactured by GE Healthcare Biosciences) previously equilibrated with 20 mM female-sodium hydroxide buffer (pH 6.0), and the column was washed with the same buffer. The active fraction was eluted with a linear gradient of sodium (0M to 0.5M).

(G) SDS-PAGE
The active fractions eluted in (e) above were collected and analyzed by SDS-PAGE (12% (w / v) polyacrylamide gel).

As described above, electrophoretically purified enzyme was obtained from Streptomycessp. NA684 strain.

[Example 2: Substrate specificity 1 of PL (A)]
In this example, the reaction rate (hydrolysis rate) of PL (A) with respect to the sn-1 and sn-2 positions of phosphatidylcholine was compared.

<Reaction solution>
100 mM Tris-HCl buffer (pH 7.2)
0.50% Phosphatidylcholine (Nacalai Tesque, product number 20342-52)
25 mM EDTA
1% Triton X-100
0.9mU / mL PL (A)
The reaction was carried out at 37 ° C. At 0, 5, 10, 20, and 30 minutes after the start of the reaction, 50 μL of the <reaction solution> was taken out, and 50 μL of a solution of CHCl ₃ : CH ₃ OH (volume ratio 2: 1) was added to this <reaction solution> and mixed. The reaction was stopped. The amount of lysophospholipid extracted into the organic phase was measured by HPLC.

<HPLC conditions>
Systech, Alltima Silica 3 μm, 100 mm × 4.6 mm
Eluent A: CHCl ₃ / MeOH / H ₂ O (80/18/2)
Eluent B: CHCl ₃ / MeOH / H ₂ O (60/34/6)
Gradient: 0 min, 100% A to 15 min linear gradient of 100% B Detector: ELSD
Flow rate: 1 ml / min
Column temperature: 25 ° C
<Result>
In the hydrolysis reaction at 37 ° C. for 20 minutes, lysophospholipids could be obtained with a yield of about 16.5%. The breakdown is about 80% of 2-acyl-sn-glycero-3-phosphocholine (1-Acyl-sn-glycero-3-phosphocholine), 1-acyl-sn-glycose-3-phosphocholine (1-AcyL- sn-glycero-3-phosphocholine) was about 20%, and the ratio was constant for at least 5 to 30 minutes after the start of the reaction (FIG. 4).

Therefore, it was shown that the reaction rate ratio of PL (A) to the sn-1 and sn-2 positions of phosphatidylcholine was about 4: 1.

[Example 3: Substrate specificity 2 and specific activity of PL (A)]
In this example, PlsEtn (C18, 18: 1) was compared with the reaction rate (hydrolysis rate) of PL (A) against DPPC, and the specific activity was measured.

<First reaction reagent mixture 2>
80 mM Tris-HCl buffer pH 8.0
5 or 0 mM calcium chloride 4 mM ATP
4 mM CoA
1.06U / mL Acyl-CoA Synthetase
2 mM PlsEtn (C18, 18: 1)
First, the activity of PL (A) was measured by the method of Example 1. Next, a <first reaction reagent mixed solution> of 0 mM calcium chloride (no calcium ion) was prepared, and the activity of PL (A) was measured by the method of Example 1. Next, the activity of PL (A) was measured in the same manner as in Example 1 except that <first reaction reagent mixture 2> was used instead of <first reaction reagent mixture>. Finally, the activity of PL (A) was measured in the same manner as in Example 1 except that <First Reaction Reagent Mixture 2> containing 0 mM calcium chloride (no calcium ion) was used. The relative activity was calculated by (Equation 3). n = 5.

As shown in Table 2, the relative activity of PL (A) with respect to PlsEtn (C18, 18: 1) was 19 ± 5% (average ± SD) with respect to DPPC.

[Example 4: Substrate specificity 3 of PL (A)]
In this example, the reaction rate (hydrolysis rate) of PL (A) against PlsEtn (C18, 20: 4) and POPC was compared.

<Reaction liquid 1>
120 mM Tris-HCl buffer (pH 7.2)
2.5% PlsEtn (C18, 20: 4)
1 mM EDTA
1% Triton X-100
<Reaction liquid 2>
120 mM Tris-HCl buffer (pH 7.2)
2.5% POPC
1 mM EDTA
1% Triton X-100
5 μL of 5 mU / mL PL (A) was added to 100 μL of <Reaction Solution 1> and <Reaction Solution 2> at 50 ° C. to initiate the reaction. Five minutes after the start of the reaction, <Reaction Solution 1> and <Reaction Solution 2> were boiled to stop the reaction. The amount of free fatty acid contained in <Reaction Solution 1> and <Reaction Solution 2> was measured with “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.) which is a free fatty acid measurement kit. The relative activity was calculated by (Equation 4). n = 5.

<Result>
It was revealed that PL (A) acts on PlsEtn (C18, 20: 4) to produce fatty acids. Further, the relative activity of PL (A) of the present invention with respect to PlsEtn (C18, 20: 4) was 29 ± 13% (mean ± SD) with respect to POPC.

[Example 5: Molecular weight measurement of each phospholipase by SDS-PAGE method]
<SDS-PAGE sample buffer>
125 mM Tris-HCl buffer (pH 6.8)
4 (W / V)% SDS
10 (W / V)% Sucrose 0.01 (W / V)% Bromophenol Blue 10 (W / V)% 2-Mercaptoethanol <Migration buffer>
3g / L Tris
14.4 g / L Glycine
1g / L SDS
Each phospholipase (PL (A), PLB, PLA2 II L, PLA2 Nagase, and LIPOMOD 699L) solution was diluted with distilled water so that the absorbance at 280 nm was about 1.0 to obtain each phospholipase aqueous solution. Next, 20 μL of each phospholipase aqueous solution and 20 μL of <SDS-PAGE sample buffer> were mixed and denatured by heating at 99 ° C. for 20 minutes. Each denatured phospholipase was cooled to room temperature, and 10 μL was measured for molecular weight by SDS-PAGE. As a molecular weight marker, SDS-PAGE standard Broad range (Bio RAD, product number 161-0317) was used. As the polyacrylamide gel, e-PAGEEL (Ato Corporation, product number E-T15S) was used. The electrophoresis buffer is as described above. Electrophoresis was performed at room temperature and constant current (20 mA) for about 60 minutes. Ez Stain Aqua (Ato Co., product number AE-1340) was used for polypeptide staining. Pure water was used for decolorization.

Lanes

1 and 7 in FIG. 5 are markers. Lanes 2 to 6 are phospholipases, and the molecular weight by SDS-PAGE method is
Lane 2 PL (A) Range of about 25-30 kDa Lane 3 PLB Range of about 43-45 kDa Lane 4 PLA2 II L Range of about 12-14 kDa Lane 5 PLA2 Nagase Range of about 12-14 kDa Lane 6 LIPOMOD 699L About 12-14 kDa It became the range.

The molecular weight of PLA2 II L, PLA2 Nagase and PLIPMOD 699L by SDS-PAGE was the same as that of the known phospholipase A2. On the other hand, the molecular weight of PL (A) by SDS-PAGE was in the range of about 25-30 kDa, which was clearly different from phospholipase A1 and phospholipase A2.

[Example 6-1: Action of each phospholipase on PlsEtn (in the presence of calcium ion)]
In this example, the effects of each phospholipase (PL (A), PLB, PLA2 II L, PLA2 Nagase, LIPOMOD 699L) on PlsEtn extracted from pig brain were compared in the presence of calcium ions.

Note that PlsEtn (Brain, Porcine, Avanti Polar Lipids, Inc., part number 840022) extracted from pig brain is a natural product and a plurality of PlsEtns including (Chemical Formula 3) and (Chemical Formula 4) are mixed. Conceivable. That is, it is considered that PlsEtn, a molecular species that is easily acted by each phospholipase, and PlsEtn, a molecular species that is difficult to act.

<Reaction solution>
10 mM Tris-HCl buffer pH 7.5
PlsEtn extracted from 2 mM pig brain (or lyPls)
2 mM CaCl ₂
10 U / mL Each phospholipase <reaction solution> was reacted at 37 ° C., and 10 minutes after the start of the reaction, an equal amount of a solution of CHCl ₃ : CH ₃ OH (volume ratio 2: 1) was added to the <reaction solution> and mixed to react. Stopped. The phospholipid extracted into the organic phase was used as a TLC sample. In the blind test, each phospholipase was not added to the <reaction solution>.

<Method of TLC>
3 μL was spotted on thin layer chromatography (TLC, TLC silica gel 60, MERCK) and developed with a developing solvent (chloroform: methanol: distilled water = 65: 25: 4) for about 2 to 3 hours. A suitable amount of ninhydrin spray (Wako Pure Chemical Industries, Ltd., product number 145-08601) was sprayed on the developed TLC and heated in a dry heat oven at 100 ° C. for about 15 to 30 seconds to detect lyPlsEtn and PlsEtn. The TLC conditions in other examples are the same as those in this example.

Here, it is 1-O-1 ′-(Z) -octadecenyl-2-hydroxy-sn-glycero-3-phosphoethanolamine ((chemical formula 7) Avanti Polar Lipids, Inc. product number 852471) used as a preparation of lyPlsEtn. .

<Result>
Lanes 1 to 7 in FIG. 6 are as follows.

Lane 1 is a phospholipid extracted into the organic phase from <reaction solution> (blind) in which each phospholipase was not allowed to act on PlsEtn extracted from pig brain.

Lane 2 is a phospholipid extracted from the <reaction solution> (blind) in which the phospholipase was not allowed to act on lyPlsEtn (Chemical Formula 7) to the organic phase.

Lane 3 is phospholipid extracted into the organic phase from <reaction solution> in which PL (A) was allowed to act on PlsEtn extracted from pig brain.

Lane 4 is a phospholipid extracted into the organic phase from the <reaction solution> in which PLB was allowed to act on PlsEtn extracted from pig brain.

Lane 5 is a phospholipid extracted in the organic phase from the <reaction solution> in which PLA2 II L is allowed to act on PlsEtn extracted from pig brain.

Lane 6 is a phospholipid extracted into the organic phase from <reaction solution> in which PLA2 Nagase was allowed to act on PlsEtn extracted from pig brain.

Lane 7 is a phospholipid extracted from the <reaction solution> in which LIPOMOD 699L was allowed to act on PlsEtn extracted from pig brain to the organic phase.

The results of quantifying TLC spots with a densitometer are shown in Table 3.

It was Lane 3 (PL (A)) that showed that PlsEtn extracted from pig brain disappeared and the mobility changed to the same position as lyPlsEtn (Lane 2). Lane 6 (PLA2 Nagase) and Lane 7 (LIPOMOD 699L) showed the same effect, but the degree was weaker than that of PL (A).

[Example 6-2: Action of each phospholipase on PlsEtn (in the presence of calcium ion)]
In this example, the effects of each phospholipase (PL (A), PLB, PLA2 II L, PLA2 Nagase, LIPOMOD 699L) on PlsEtn (Chemical Formula 1) were compared in the presence of calcium ions.

<Reaction solution>
10 mM Tris-HCl buffer pH 7.5
2 mM PlsEtn (Chemical Formula 1) (or lyPls (Chemical Formula 7))
2 mM CaCl ₂
10 U / mL Each phospholipase <reaction solution> was reacted at 37 ° C., and 10 minutes after the start of the reaction, a solution of CHCl ₃ : CH ₃ OH (volume ratio 2: 1) was added in the same amount as <reaction solution> and mixed to react. Stopped. The phospholipid extracted into the organic phase was used as a TLC sample.

<Result>
Lanes 1 to 7 in FIG. 7 are as follows.

Lane 1 is a phospholipid extracted from an organic phase from <reaction solution> (blind) in which each phospholipase was not allowed to act on PlsEtn (Chemical Formula 4).

Lane 3 is a phospholipid extracted into an organic phase from a <reaction solution> in which PL (A) is allowed to act on PlsEtn (Chemical Formula 4).

Lane 4 is a phospholipid extracted from the <reaction solution> in which PLB was allowed to act on PlsEtn (chemical formula 4) to the organic phase.

Lane 5 is a phospholipid extracted into an organic phase from <reaction solution> in which PLA2 II L is allowed to act on PlsEtn (Chemical Formula 4).

Lane 6 is a phospholipid extracted into an organic phase from <reaction solution> in which PLA2 Nagase was allowed to act on PlsEtn (Chemical Formula 4).

Lane 7 is a phospholipid extracted from the <reaction solution> in which LIPOMOD 699L was allowed to act on PlsEtn (Chemical Formula 4) to the organic phase.

The results of quantifying TLC spots with a densitometer are shown in Table 4.

It was Lane 3 (PL (A)) that PlsEtn (Chemical Formula 4) disappeared and the mobility changed to the same position as lyPlsEtn (Chemical Formula 7). Lane 6 (PLA2 Nagase) and Lane 7 (LIPOMOD 699L) showed the same effect, but the degree was weaker than that of PL (A).

[Example 7-1: Action of each phospholipase on Pls (in the absence of calcium ion)]
In this example, the effect of each phospholipase (PL (A), PLB, PLA2 II L, PLA2 Nagase, LIPOMOD 699L) on PlsEtn extracted from pig brain was compared in the absence of calcium ions.

<Reaction solution>
10 mM Tris-HCl buffer pH 7.5
EtnPls extracted from 2 mM pig brain (or lyPls)
10 U / mL Each phospholipase <reaction solution> was reacted at 37 ° C., and 10 minutes after the start of the reaction, a solution of CHCl ₃ : CH ₃ OH (volume ratio 2: 1) was added in the same amount as <reaction solution> and mixed to react. Stopped. The phospholipid extracted into the organic phase was used as a TLC sample. In the blind test, each phospholipase was not added to the <reaction solution>.

<Result>
Lanes 1 to 7 in FIG. 8 are as follows.

The results of quantifying TLC spots with a densitometer are shown in Table 5.

PL (A) strongly showed the effect that PlsEtn extracted from pig brain disappeared even in the absence of calcium, and the mobility changed to the same position as lyPlsEtn (Chemical Formula 7) (lane 3).

[Example 7-2: Action of each phospholipase on PlsEtn (in the absence of calcium ion)]
In this example, the effects of each phospholipase (PL (A), PLB, PLA2 II L, PLA2 Nagase, LIPOMOD 699L) on PlsEtn (Chemical Formula 4) were compared in the presence of calcium ions.

<Reaction solution>
10 mM Tris-HCl buffer pH 7.5
2 mM PlsEtn (Chemical formula 4) (or lyPls (Chemical formula 7))
10 U / mL Each phospholipase <reaction solution> was reacted at 37 ° C., and 10 minutes after the start of the reaction, a solution of CHCl ₃ : CH ₃ OH (volume ratio 2: 1) was added in the same amount as <reaction solution> and mixed to react. Stopped. The phospholipid extracted into the organic phase was used as a TLC sample. In the blind test, each phospholipase was not added to the <reaction solution>.

<Result>
Lanes 1 to 7 in FIG. 9 are as follows.

The results of quantifying TLC spots with a densitometer are shown in Table 6.

In the absence of calcium, PlsEtn (Chemical Formula 4) disappeared, and it was Lane 3 (PL (A)) that strongly showed the action of changing mobility at the same position as lyPlsEtn (Chemical Formula 7). Lane 7 (LIPOMOD 699L) also showed the same effect, but the degree was weaker than that of PL (A).

[Example 8: Product when PL (A) is allowed to act on Pls]
In Example 6-2 and Example 7-2, the product when PL (A) was allowed to act on PlsEtn (Chemical Formula 4) (ie, the substance indicated by the arrows in FIGS. 7 and 9) was confirmed by LC / MS. .

<LC conditions>
Equipment Waters, UPLC
Column Waters, ACQUITY UPLC HSS C18 1.8 μm
(2.1 mm ID x 50 mm)
Column temperature 40 ° C
Detection 220nm
Mobile phase A = water (0.1% HCOOH) B = isopropyl alcohol gradient 0 min A 80% B 20%
10 minutes A 0% B 100%
13 minutes A 0% B 100%
13.1 minutes A 80% B 20%
15 minutes A 80% B 20%
Injection volume 1μL
<Conditions for MS>
Equipment Waters, Synapt G2
Ionization ESI +
Scan range m / z 100-1500
<Result>
In MS, one kind of ion considered to be PlsEtn (Chemical Formula 4) (m / z 730.55) and one type of ion considered to be lyPlsEtn (Chemical Formula 7) (m / z 466.31) are detected, and the standard PlsEtn is detected. Consistent with (Chemical Formula 4) and lyPlsEtn (Chemical Formula 7). Therefore, it was confirmed that the product of PL (A) acting on PlsEtn (Chemical Formula 4) was lyPlsEtn (Chemical Formula 7).

[Example 9: Relationship between the amount of PL (A) enzyme and the effect on Pls]
<Reaction solution>
10 mM Tris-HCl buffer pH 7.5
2 mM PlsEtn (Chemical formula 4) (or lyPls (Chemical formula 7))
0 ~ 0.5U / mL PL (A)
1 mL each of <reaction solution> containing 0, 0.005, 0.0067, 0.01, 0.02, 0.05, and 0.5 U / mL PL (A) was prepared. After reacting these <reaction liquid> at 37 ° C. for 10 minutes, 1 mL of a solution of CHCl ₃ : CH ₃ OH (volume ratio 2: 1) was added to and mixed with the <reaction liquid> to stop the reaction. The phospholipid extracted into the organic phase was used as a TLC sample.

<Result>
Lanes 1 to 8 in FIG. 10 are as follows.

Lane 1 is a phospholipid extracted into an organic phase from <reaction solution> in which PL (A) is allowed to act on PlsEtn at 0.5 U / mL.

Lane 2 is a phospholipid extracted into an organic phase from <reaction solution> in which PL (A) was allowed to act at 0.05 U / mL on PlsEtn.

Lane 3 is phospholipid extracted into the organic phase from <reaction solution> in which PL (A) was allowed to act on PlsEtn at 0.02 U / mL.

Lane 4 is a phospholipid extracted into the organic phase from <reaction solution> in which PL (A) was allowed to act on PlsEtn at 0.01 U / mL.

Lane 5 is a phospholipid extracted into the organic phase from <reaction solution> in which PL (A) was allowed to act on PLsEtn at 0.0067 U / mL.

Lane 6 is phospholipid extracted into the organic phase from <reaction solution> in which PL (A) was allowed to act on PlsEtn at 0.005 U / mL.

Lane 7 is phospholipid extracted into the organic phase from <reaction solution> in which PL (A) was not allowed to act on lyPlsEtn (0 U / mL).

Lane 8 is a phospholipid extracted from the <reaction solution> in which PL (A) was not allowed to act on Pls (0 U / mL) to the organic phase.

From this example, it was found that at least 0.5 U / mL PL (A) can hydrolyze 2 mM PlsEtn to lyPls at 37 ° C. for 10 minutes. That is, it was found that at least 0.5 mU of PL (A) can hydrolyze 2 μmol of PlsEtn to lyPls at 37 ° C. for 10 minutes.

[Example 10-1: Relationship between pH and effect of PL (A) on PlsEtn]
<Reaction solution>
50 mM each buffer 2 mM PlsEtn (Chemical formula 4) (or lyPls (Chemical formula 7))
0 or 0.5 U / mL PL (A)
<Reaction solution> containing citrate-NaOH buffer (

pH

4, 5, 6), potassium phosphate buffer pH 7, Tris-hydrochloric acid buffer (pH 8, 9), glycine-NaOH buffer pH 10 as each buffer solution Prepared. After reacting these <reaction liquid> at room temperature for 10 minutes, 1 mL of a solution of CHCl ₃ : CH ₃ OH (volume ratio 2: 1) was added and mixed with the <reaction liquid> to stop the reaction. The phospholipid extracted into the organic phase was used as a TLC sample.

<Result>
Lanes 1 to 18 in FIG. 11 are as follows.

In lanes 1 to 3, PL (A) was not allowed to act on PlsEtn in the <reaction solution> of the citrate-

NaOH buffer pH

4, 5, 6 in order (0 U / mL) from the <reaction solution> to the organic phase. Extracted phospholipid.

Lane 4 is phospholipid extracted into the organic phase from <reaction solution> in which PL (A) was not allowed to act on PlsEtn in <reaction solution> of potassium phosphate buffer pH 7.

In lanes 5 to 7, PL (A) was not allowed to act on PlsEtn in <reaction solution> in Tris-HCl buffer pH 7.5, 8, 9 in order (0 U / mL) from <reaction solution> to the organic phase It is a phospholipid extracted.

Lane 8 is a phospholipid extracted from the <reaction solution> in which PL (A) was not allowed to act on PlsEtn in the <reaction solution> in glycine-NaOH buffer pH 10 (0 U / mL).

Lanes 9 to 11 are phospholipids extracted in the organic phase from <reaction solution> in which PL (A) was allowed to act on PlsEtn in <reaction solution> of citrate-

NaOH buffer pH

4, 5, 6 in order. .

Lane 12 is a phospholipid extracted into an organic phase from a <reaction solution> obtained by allowing PL (A) to act on PlsEtn in a <reaction solution> in a potassium phosphate buffer pH 7.

Lanes 13 to 15 are phospholipids extracted in the organic phase from <reaction solution> in which PL (A) was allowed to act on PlsEtn in <reaction solution> of Tris-HCl buffer pH 7.5, 8, 9 in order. is there.

Lane 16 is phospholipid extracted into the organic phase from <reaction solution> in which PL (A) was allowed to act on PlsEtn in <reaction solution> of glycine-NaOH buffer pH 10.

Lane 17 is a phospholipid extracted from the <reaction solution> in which the PL (A) was not allowed to act on lyPlsEtn in the <reaction solution> in Tris-HCl buffer pH 7.5 (0 U / mL).

Lane 18 is a phospholipid extracted from the <reaction solution> in which PL (A) was not allowed to act on PlsEtn in <reaction solution> in Tris-HCl buffer pH 7.5 (0 U / mL) to the organic phase.

FIG. 11 shows that PL (A) can hydrolyze PlsEtn to lyPls in the pH range of 4 to 10 under the conditions of this example. And it turned out that the effect | action is so strong that pH is high. As is clear from lanes 1 to 8, PlsEtn did not spontaneously change to lyPls due to pH change.

[Example 11: Pls measurement in sample]
Pls in the sample was measured by the method shown in FIG.

<Reaction liquid 1>
50 mM BES-NaOH pH 7.5
10U / mL Peroxidase
50U / mL Ethanolamine oxidase
5U / mL GPCP
1U / mL PL (A)
<Reaction liquid 2>
50 mM BES-NaOH pH 7.5
4.2 U / mL lyPls as
12 μM DA67
Here, Peroxidase was purchased from SIGMA as product number P8375. Ethanolamine oxidase was produced by the method of Bioscience, Biotechnology, and Biochemistry 2008, 72, 2732-2738. GPPC (Glycerophylcholine phosphatase) was obtained as part number T-33 from Asahi Kasei Pharma Corporation. LyPlsase was produced as a recombinant Escherichia coli from rats by the method described in The journal of biochemical chemistry, 2011, 286, 24916-24930. DA67 was purchased from Wako Pure Chemical Industries, Ltd. as part number 046-22341.

<Sample>
PlsEtn (chemical formula 4) was added to a 10% aqueous solution of dodecyl maltoside (Dojindo Laboratories, product number 347-06163) so as to be 0 to 200 μM.

<Measurement>
Measurement was performed using a Hitachi 7080 automatic analyzer. The amount of the sample was 12 μL, the amount of <Reaction Solution 1> was 180 μL, the amount of <Reaction Solution 2> was 45 μL, the reaction temperature was 37 ° C., and the absorbance difference at 660 nm (subwavelength 750 nm) was measured as a one-point end assay.

<Result>
FIG. 12 shows a calibration curve of a sample prepared by adding PlsEtn (Chemical Formula 4) to a 10% dodecyl maltoside aqueous solution at 0 to 200 μM. The calibration curve was represented by the correlation formula Y = 2.724X-52.962, and R ² = 0.99991. It was shown that PlsEtn in the sample could be measured in this example. The section became −53 because <Reaction Solution 1> and <Reaction Solution 2> were colored and suspended.

[Example 12: Preparation method of Streptomyces avermitilis JCM 5070-derived PL (A)]
[Example 12-1: Separation of chromosomal DNA of Streptomyces avermitilis JCM 5070]
Streptomyces avermitilis JCM 5070 was used in 5 mL of LB medium and cultured at 28 ° C. for 2 days for collection. Next, this bacterial cell was suspended in 250 μL of PIA buffer of QIAprep Miniprep (QIAGEN), 250 μL of P2 buffer was added, and the mixture was stirred 5 times to obtain template chromosomal DNA.

[Example 12-2: Preparation of recombinant plasmid containing PL (A) gene derived from Streptomyces avermitilis JCM 5070]
A sense primer “primer S” (SEQ ID NO: 7) was synthesized as an oligo for PCR, and “primer AS” (SEQ ID NO: 8) was synthesized as an antisense primer. Template chromosomal DNA 50 ng obtained in Example 12-1, 10 × PCR Buffer 2.5 μL, each primer 1200 nM, dNTPs 0.3 mM each, MgCl ₂ , 1.2 mM, DMSO 4%, KOD DNA Polymerase 1.25 units, Distilled water was added to a total volume of 25 μL. PCR reaction conditions are as follows.

A specific amplification product of about 810 bp was obtained by this PCR. This amplified fragment was digested with NdeI and EcoRI and inserted into the NdeI-EcoRI sites of expression vectors pET21a (+) and pET24a (+) to obtain a recombinant plasmid.

[Example 13-3: Preparation of recombinant Escherichia coli expressing a Streptomyces avermitilis JCM 5070-derived PL (A) gene]
The recombinant plasmid obtained in Example 12-2 was transformed into E. coli BL21 (DE3) to obtain recombinant E. coli.

[Example 13-4: Culture of recombinant actinomycetes expressing PL (A) gene derived from Streptomyces avermitilis JCM 5070]
Recombinant E. coli obtained in Example 13-3 was mixed with 50 μg / mL ampicillin (in the case of E. coli transformed with pET21a (+)) or 30 μg / mL kanamycin (in the case of E. coli transformed with pET24a (+)). The culture was carried out at 34 ° C. for 24 hours in 100 mL of Overnight Express TB medium (manufactured by Novagen). The obtained culture solution was centrifuged to recover the cells. The cells were suspended in 20 mM Tris-HCl buffer (pH 7.5), sonicated, and the centrifuged supernatant was used in Example 14 as it was (about 0.1 U / mL). Centrifugal supernatant obtained by culturing untransformed E. coli (no antibiotics added) and sonication was used as a negative control.

[Example 14: Action of Streptomyces avermitilis JCM 5070-derived PL (A) on PlsEtn (in the absence of calcium ions)]
In this example, it was confirmed by TLC that Streptomyces avermitilis JCM 5070-derived PL (A) acts on PlsEtn (chemical formula 4) in the absence of calcium ions.

<Reaction solution>
10 mM Tris-HCl buffer pH 7.5
2 mM PlsEtn (Chemical formula 4) (or lyPls (Chemical formula 7))
Culture solubilized centrifuge supernatant <Reaction solution> is reacted at 25 ° C. or 30 ° C., and 10 minutes after the start of the reaction, a solution of CHCl ₃ : CH ₃ OH (volume ratio 2: 1) is added in the same amount as <Reaction solution>. The reaction was stopped by mixing. The phospholipid extracted into the organic phase was used as a TLC sample.

<Result>
Lanes 1 to 8 in FIG. 13 are as follows.

Lane 1 is a phospholipid extracted into an organic phase from <reaction solution> in which PL (A) was not allowed to act on PlsEtn (Chemical Formula 4).

Lane 2 is a phospholipid extracted into the organic phase from <reaction solution> in which PL (A) was not allowed to act on lyPlsEtn (Chemical Formula 7).

Lane 3 is a phospholipid extracted into the organic phase from <reaction solution> in which PL (A) derived from E. coli transformed with pET21a (+) was allowed to act on PlsEtn (Chemical Formula 4) at 25 ° C.

Lane 4 is a phospholipid extracted into the organic phase from <reaction solution> in which PL (A) derived from E. coli transformed with pET24a (+) was allowed to act on PlsEtn (Chemical Formula 4) at 25 ° C.

Lane 5 is a phospholipid extracted from the <reaction solution> in which a negative control was allowed to act on PlsEtn (Chemical Formula 4) to the organic phase at 25 ° C.

Lane 6 is a phospholipid extracted into an organic phase from a <reaction solution> in which PL (A) derived from Escherichia coli transformed with pET21a (+) was allowed to act on PlsEtn (Chemical Formula 4) at 30 ° C.

Lane 7 is a phospholipid extracted into the organic phase from <reaction solution> in which PL (A) derived from E. coli transformed with pET24a (+) was allowed to act on PlsEtn (Chemical Formula 4) at 30 ° C.

Lane 8 is a phospholipid extracted at 30 ° C. into the organic phase from the <reaction solution> in which negative control was allowed to act on PlsEtn (Chemical Formula 4).

The results of quantifying TLC spots with a densitometer are shown in Table 7.

It was confirmed that Streptomyces avermitilis JCM 5070-derived PL (A) acts on PlsEtn (Chemical Formula 4) in the absence of calcium ions.

The present invention can provide a method for hydrolyzing plasmalogen to lysoplasmalogen, a method for measuring plasmalogen, a composition for hydrolysis and / or measurement, and a method for producing phospholipase.

[Streptomyces albidoflavus NA297 (Accession number: NITE BP-1014)]
Accession Number: NITE BP-1014
Name of depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary (NPMD)
Name of depositary institution: Japan 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan
Date of deposit: January 26, 2011.
[Streptomyces sp. NA684 (Accession number: NITE BP-1015)]
Accession Number: NITE BP-1015
Name of depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary (NPMD)
Name of depositary institution: Japan 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan
Date of deposit: January 26, 2011.

SEQ ID NO: 1: Full length of amino acid sequence of PL (A) SEQ ID NO: 2: Full length of amino acid sequence of other PL (A) SEQ ID NO: 3: Base sequence of PL (A) gene SEQ ID NO: 4: Other PL (A) Base sequence of gene SEQ ID NO: 5: Primer S
Sequence number 6: Primer AS
SEQ ID NO: 7: other primer S
SEQ ID NO: 8: other primer AS
SEQ ID NO: 9: Partial amino acid sequence of PL (A) SEQ ID NO: 10: Partial amino acid sequence of other PL (A) SEQ ID NO: 11: Base sequence encoding the amino acid sequence of SEQ ID NO: 9 : Other base sequence encoding the amino acid sequence set forth in SEQ ID NO: 9 SEQ ID NO: 13: Base sequence encoding the amino acid sequence set forth in SEQ ID NO: 10 SEQ ID NO: 14: Other base sequence encoding the amino acid sequence set forth in SEQ ID NO: 10 Base sequence

Claims

A method for hydrolyzing a plasmalogen, comprising hydrolyzing a plasmalogen to a lysoplasmalogen using a phospholipase having at least the following characteristics (a) to (c):
(A) Specific activity with respect to ethanolamine type plasmalogen (C18, 20: 4) is 0.66 ± 0.2 U / mg.
(B) The molecular weight by SDS-PAGE is in the range of 25-30 kDa.
(C) It is derived from actinomycetes belonging to the genus Streptomyces.
The method for hydrolyzing plasmalogen according to claim 1, wherein the phospholipase further has the following properties (d) and (e).
(D) In the absence of calcium ions, the relative activity against ethanolamine-type plasmalogen (C18, 18: 1) is 19 ± 5% relative to dipalmitoylphosphocholine.
(E) In the absence of calcium ions, the relative activity to ethanolamine-type plasmalogen (C18, 20: 4) is 29 ± 13% relative to 1-palmitoyl-2-oleoyl-phosphocholine.
The method for hydrolyzing a plasmalogen according to claim 1 or 2, wherein the phospholipase further has the following property (f).
(F) It is derived from Streptomyces albidoflavus.
The method for hydrolyzing a plasmalogen according to claim 1 or 2, wherein the phospholipase further has the following property (g).
(G) It is derived from Streptomyces avermitilis.
The method for hydrolyzing a plasmalogen according to any one of claims 1, 2, and 3, wherein the phospholipase further has any of the following properties (h) to (j).
(H) having the amino acid sequence set forth in SEQ ID NO: 1.
(I) It has an amino acid sequence in which one or a plurality of amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, and has an action of hydrolyzing plasmalogen to lysoplasmalogen.
(J) having an amino acid sequence in which one or more amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, and further the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10 And has an action of hydrolyzing plasmalogen to lysoplasmalogen.
The method for hydrolyzing a plasmalogen according to any one of claims 1, 2, and 4, wherein the phospholipase further has any of the following properties (k) to (m).
(K) having the amino acid sequence set forth in SEQ ID NO: 2.
(L) It has an amino acid sequence in which one or more amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 2 and has an action of hydrolyzing plasmalogen into lysoplasmalogen.
(M) having an amino acid sequence in which one or more amino acids are mutated, deleted, or added in the amino acid sequence shown in SEQ ID NO: 2, and further the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10 And has an action of hydrolyzing plasmalogen to lysoplasmalogen.
The method for hydrolyzing a plasmalogen according to any one of claims 1 to 6, wherein the plasmalogen is an ethanolamine type plasmalogen, and the lysoplasmalogen is an ethanolamine type lysoplasmalogen. .
The method for hydrolyzing plasmalogen according to any one of claims 1 to 7, wherein the plasmalogen is a plasmalogen in a sample.
A method for measuring plasmalogen, comprising measuring plasmalogen using a phospholipase having at least the following characteristics (a) to (c):
(A) Specific activity with respect to ethanolamine type plasmalogen (C18, 20: 4) is 0.66 ± 0.2 U / mg.
(B) The molecular weight by SDS-PAGE is in the range of 25-30 kDa.
(C) It is derived from actinomycetes belonging to the genus Streptomyces.
The method for measuring plasmalogen according to claim 9, wherein the phospholipase further has the following properties (d) and (e).
(D) In the absence of calcium ions, the relative activity against ethanolamine-type plasmalogen (C18, 18: 1) is 19 ± 5% relative to dipalmitoylphosphocholine.
(E) In the absence of calcium ions, the relative activity to ethanolamine-type plasmalogen (C18, 20: 4) is 29 ± 13% relative to 1-palmitoyl-2-oleoyl-phosphocholine.
The method for measuring plasmalogen according to claim 9 or 10, wherein the phospholipase further has the following property (f).
(F) It is derived from Streptomyces albidoflavus.
The method for measuring plasmalogen according to claim 9 or 10, wherein the phospholipase further has the following property (g).
(G) It is derived from Streptomyces avermitilis.
The method for measuring plasmalogen according to any one of claims 9, 10, and 11, wherein the phospholipase further has any of the following properties (h) to (j).
(H) having the amino acid sequence set forth in SEQ ID NO: 1.
(I) It has an amino acid sequence in which one or a plurality of amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, and has an action of hydrolyzing plasmalogen to lysoplasmalogen.
(J) having an amino acid sequence in which one or more amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, and further the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10 And has an action of hydrolyzing plasmalogen to lysoplasmalogen.
The method for measuring plasmalogen according to any one of claims 9, 10, and 12, wherein the phospholipase further has any of the following properties (k) to (m).
(K) having the amino acid sequence set forth in SEQ ID NO: 2.
(L) It has an amino acid sequence in which one or more amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 2 and has an action of hydrolyzing plasmalogen into lysoplasmalogen.
(M) having an amino acid sequence in which one or more amino acids are mutated, deleted, or added in the amino acid sequence shown in SEQ ID NO: 2, and further the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10 And has an action of hydrolyzing plasmalogen to lysoplasmalogen.
The method for measuring plasmalogen according to any one of claims 9 to 14, wherein the plasmalogen is an ethanolamine type plasmalogen, and the lysoplasmalogen is an ethanolamine type lysoplasmalogen.
The plasmalogen measuring method according to any one of claims 9 to 15, wherein the plasmalogen is a plasmalogen in a sample.
A phospholipase-containing composition comprising a phospholipase having at least the following properties (a) to (c):
(A) Specific activity with respect to ethanolamine type plasmalogen (C18, 20: 4) is 0.66 ± 0.2 U / mg.
(B) The molecular weight by SDS-PAGE is in the range of 25-30 kDa.
(C) It is derived from actinomycetes belonging to the genus Streptomyces.
The phospholipase-containing composition according to claim 17, wherein the phospholipase further has the following properties (d) and (e):
(D) In the absence of calcium ions, the relative activity against ethanolamine-type plasmalogen (C18, 18: 1) is 19 ± 5% relative to dipalmitoylphosphocholine.
(E) In the absence of calcium ions, the relative activity to ethanolamine-type plasmalogen (C18, 20: 4) is 29 ± 13% relative to 1-palmitoyl-2-oleoyl-phosphocholine.
The phospholipase-containing composition according to claim 17 or 18, wherein the phospholipase further has the following property (f).
(F) It is derived from Streptomyces albidoflavus.
The phospholipase-containing composition according to claim 17 or 18, wherein the phospholipase further has the following property (g):
(G) It is derived from Streptomyces avermitilis.
The phospholipase-containing composition according to any one of claims 17, 18, and 19, wherein the phospholipase further has any of the following properties (h) to (j):
(H) having the amino acid sequence set forth in SEQ ID NO: 1.
(I) It has an amino acid sequence in which one or a plurality of amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, and has an action of hydrolyzing plasmalogen to lysoplasmalogen.
(J) having an amino acid sequence in which one or more amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 1, and further the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10 And has an action of hydrolyzing plasmalogen to lysoplasmalogen.
The phospholipase-containing composition according to any one of claims 17, 18, and 20, wherein the phospholipase further has any of the following properties (k) to (m):
(K) having the amino acid sequence set forth in SEQ ID NO: 2.
(L) It has an amino acid sequence in which one or more amino acids are mutated, deleted or added in the amino acid sequence shown in SEQ ID NO: 2 and has an action of hydrolyzing plasmalogen into lysoplasmalogen.
(M) having an amino acid sequence in which one or more amino acids are mutated, deleted, or added in the amino acid sequence shown in SEQ ID NO: 2, and further the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 10 And has an action of hydrolyzing plasmalogen to lysoplasmalogen.
23. The phospholipase-containing composition according to any one of claims 17 to 22, wherein the composition is a composition for hydrolyzing plasmalogen to lysoplasmalogen.
The phospholipase-containing composition according to any one of claims 17 to 23, which is a composition for measuring plasmalogen.
25. The phospholipase-containing composition according to any one of claims 17 to 24, wherein the plasmalogen is an ethanolamine type plasmalogen, and the lysoplasmalogen is an ethanolamine type lysoplasmalogen.
The phospholipase-containing composition according to any one of claims 17 to 25, wherein the plasmalogen is a plasmalogen in a sample.
A method for producing any one of the following phospholipases from <1> to <3>, comprising a step of forming the phospholipase based on a base sequence encoding the phospholipase, and a step of obtaining the phospholipase. A method for producing a characteristic phospholipase.
<1> It has the amino acid sequence set forth in SEQ ID NO: 2 and has an action of hydrolyzing plasmalogen to lysoplasmalogen.
<2> The amino acid sequence of SEQ ID NO: 2 has an amino acid sequence in which one or more amino acids are deleted, substituted or added, and the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10 And has an action of hydrolyzing plasmalogen to lysoplasmalogen.
<3> It has the following characteristics (a) to (d).
(A) In the absence of calcium ions, the relative activity to ethanolamine-type plasmalogen (C18, 18: 1) is 19 ± 5% relative to dipalmitoylphosphocholine.
(B) In the absence of calcium ions, the relative activity against ethanolamine-type plasmalogen (C18, 20: 4) is 29 ± 13% relative to 1-palmitoyl-2-oleoyl-phosphocholine.
(C) The molecular weight determined by SDS-PAGE is in the range of 25-30 kDa.
(D) It is derived from actinomycetes belonging to the genus Streptomyces.
The method for producing a phospholipase according to claim 27, wherein in the step of forming the phospholipase, a microorganism that produces a phospholipase belonging to the genus Streptomyces and having an action of hydrolyzing a plasmalogen to a lysoplasmalogen is used. .
The method for producing a phospholipase according to claim 27 or 28, further comprising a step of purifying the obtained phospholipase.
The method for producing phospholipase according to any one of claims 27 to 29, wherein the base sequence encoding the phospholipase is the base sequence shown in SEQ ID NO: 4.
The method for producing phospholipase according to any one of claims 27 to 30, wherein the phospholipase is derived from Streptomyces avermitilis.