WO2012105565A1

WO2012105565A1 - Enzyme and method of production therefor

Info

Publication number: WO2012105565A1
Application number: PCT/JP2012/052159
Authority: WO
Inventors: 大助杉森; 功大加納
Original assignee: 国立大学法人福島大学
Priority date: 2011-02-01
Filing date: 2012-01-31
Publication date: 2012-08-09
Also published as: WO2012104987A1

Abstract

Provided are a novel PLA1 and a method of production therefor. The enzyme hydrolyzes, with priority, the acyl group at the sn-1 position of a phospholipid to generate one or more of the following: a lysophospholipid, glycerol-3-phosphate, and a glycerol-3-phosphoric ester compound. The enzyme contains any one of the polypeptides described in (a) to (c) below. (a) a polypeptide having the amino acid sequence represented by SEQ ID NO:2. (b) a polypeptide having an amino acid sequence obtained by substituting, inserting, deleting and/or adding one or more amino acids in the amino acid sequence represented by SEQ ID NO:2, (c) a polypeptide having at least 75% homology with the amino acid sequence represented by SEQ ID NO:2. Thus, it is possible to provide a novel phospholipase A1 having high enzymatic activity. The PLA1 has substrate specificity with respect to phospholipids.

Description

Enzyme and production method thereof

The present invention relates to a novel phospholipase A1 and a method for producing the same.

Phospholipase A1 is an enzyme that acts on phospholipid (glycerol phospholipid) to produce lysophospholipid (lyso form). C. Known as 3.1.1.32. Phospholipase is a general term for enzymes that hydrolyze phospholipids. Phospholipids have a fatty acid ester-bonded to one α (sn-1) and β (sn-2) hydroxyl groups of glycerol, and the other α-position hydroxyl group via a phosphate group is choline. , Ethanolamine, inositol, and the like.

The enzyme that hydrolyzes the fatty acid ester bond at the α-position of the glycerol group in glycerol phospholipid is called phospholipase A1 (PLA1), and the enzyme that hydrolyzes the fatty acid ester group at the β-position of the glycerol group is called phospholipase A2 (PLA2). An enzyme having both phospholipase A1 activity and phospholipase A2 activity is referred to as phospholipase B (PLB).

In addition, a phospholipid from which only one of α-position or β-position fatty acyl group in phospholipid is removed is called lysophospholipid, and acts on lysophospholipid to hydrolyze the remaining fatty acid ester bond. Is also included in the PLB because the decomposition product is the same as in the case of the PLB. The action on lysophospholipid is called lysophospholipase activity.

On the other hand, an enzyme that hydrolyzes an ester bond between a glycerol group and a phosphate group of a phospholipid is called phospholipase C (PLC). An enzyme that hydrolyzes the bond between a phosphate group and choline, ethanolamine, or the like is referred to as phospholipase D (PLD).

It has been reported that phosphatidylinositol is contained in soybean lecithin and is deeply involved in cell information transmission. In recent years, it has been reported that the intake of phosphatidylinositol decreases the blood triacylglycerol (TG) concentration and increases the HDL-C (high density lipoprotein cholesterol, generally referred to as good cholesterol) concentration (Non-patent Document). 1, 2). Therefore, its physiological action is attracting attention along with its metabolic derivatives, lysophosphatidylinositol and glyceryl phosphorylinositol.

In addition, lysophosphatidylethanolamine has been reported to have a neuronal differentiation inducing action and an action to protect neuronal cells from apoptosis (apoptosis inhibitory action) as a neuronal nutrient (Non-patent Documents 3 and 4). . In addition to this, it has been reported that lysophosphatidylethanolamine was found to have a translocational activity suppression effect, a nerve activation effect, a cytoprotective effect, and a central nerve calming effect (Non-patent Document 4). From these reports, lysophosphatidylethanolamine is expected to be used for the prevention and treatment of Alzheimer's disease that occurs after ischemic brain injury and neurological disorders such as neuronal cell death.

Furthermore, it has been reported that lysophosphatidylinositol has an antifungal action (Patent Document 1). In addition, lysophosphatidic acid is known to have a vasoconstrictive action (Non-patent Document 5), a strong cell proliferation action and a DNA synthesis promoting action (Non-Patent Document 6). 1-lysophosphatidic acid has been reported to be effective in hair formation (hair growth, hair growth) (Non-patent Document 7). In addition, lysophosphatidylserine has been reported to be involved in mast cell activation (allergy and atopic dermatitis) (Non-patent Document 5). In addition, lysophosphatidylglycerol has been reported to have an excellent noodle modification action as a noodle modifier (Patent Document 2). In addition to this, lysophosphatidylglycerol has a strong surface activity and is known to have a strong foaming action. For example, strengthening O / W emulsification with strong emulsifying power, improving thermal stability of O / W emulsions, forming emulsions with excellent storage stability, excellent binding properties with proteins and starches, antibacterial effects, excellent Moisturizing properties and antioxidative effects are known.

Soy lysolecithin has (1) strong O / W emulsifying properties, (2) high emulsion stability in the presence of acids and in the presence of salts, and (3) excellent ability to bind to proteins and starch. It has been described that the demand has been increasing in recent years because it has such features as (4) excellent mold release action and the like (Patent Document 3).

Phosphatidylethanolamine is a component of phospholipids contained in soybean lecithin and egg yolk lecithin, and is known to have an emulsion stabilizing effect. In addition, a lot of unsaturated fatty acids are bound to phosphatidylethanolamine, particularly in the β (sn-2) position of egg yolk lecithin, and when this unsaturated fatty acid is liberated, it becomes a source of bitterness. Therefore, in food processing, it can be said that it is desirable that hydrolysis of phosphatidylethanolamine does not occur. However, no enzyme suitable for this purpose has been found so far. In addition, no enzyme capable of controlling substrate specificity by pH has been found so far.

PLA1 is widely distributed in the biological world, and in particular, those derived from microorganisms and snake venoms have been studied in detail, and there are sales as biochemical reagents and sales as industrial enzymes. As industrial PLA1, there is PLA1 (manufactured by Mitsubishi Chemical Foods) derived from Aspergillus oryzae. Aspergillus aspergillus oryzae-derived PLA1 has a problem of being restricted in use because its working pH is in the acidic range. Further, since metal ions such as Ca ²⁺ ions are required for the catalytic action, there is a problem depending on applications. Furthermore, the kind of phospholipid which can act is not specified (patent document 5). In order to solve this problem, a filamentous fungus Celiridium unicorn Sutton-derived PLA1 showing activity in the neutral range has been developed and disclosed (Patent Document 6). However, the point that addition of Ca salt is still necessary and the problem of hydrolysis of phosphatidylethanolamine have not yet been solved. From the above circumstances, development of more useful enzymes that solve these problems is desired.

In addition, glycerol-3-phosphocholine is expected to prevent dementia (Patent Document 4). However, no enzyme or technology for efficiently producing a glycerol-3-phosphate compound such as glycerol-3-phosphocholine has been developed.

JP-A-6-256366 JP 2008-11745 A JP-A-10-287686 International Publication No. 2007/010892 JP-A-6-62850 Japanese Patent Laid-Open No. 2004-261022

The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel PLA 1 and a manufacturing method thereof.

As a result of searching for microorganisms that produce such enzymes for the purpose of obtaining novel PLA1 (enzymes), the present inventors have found PLA1 having novel properties from microorganisms belonging to the genus Streptomyces. I found it.

[enzyme]
The enzyme which concerns on this invention contains the polypeptide in any one of the following (a) to (c).
(A) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2.
(C) a polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2.

Furthermore, the enzyme according to the present invention has the following physicochemical properties (1) and (2).

(1) Action One kind selected from lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate compound by preferentially hydrolyzing the sn-1 position acyl group of phospholipid The above is generated.

(2) Substrate specificity When egg yolk lecithin (L) (manufactured by Nacalai Tesque) is used as a substrate, and the hydrolysis activity under the condition of 5 minutes at 50 ° C at pH 7.2 is 100%,
50% or more (upper limit is 200%) with respect to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (1-PElmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine: POPE),
1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate (1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphate: POPA) of 500% or more (upper limit is 600%),
150% or more (upper limit is 350%) with respect to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (1-PClmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: POPC),
300% or more based on L-α-phosphatidylinositol (PI) (upper limit is 450%),
300% or more (upper limit is 700%) with respect to L-α-phosphatidyl-L-serine (L-α-Phosphatidyl-L-serine: PS),
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac- (1-glycerol) (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac- (1-glycerol): POPG ) 100% or more (upper limit is 300%)
100% or more (upper limit is 150%) with respect to soybean-derived L-α-Phosphatidylcholine (SB-PC),
30% or more of soybean lecithin (SBL) (upper limit is 150%)
250% or more (upper limit is 350%) with respect to L-α-Lysophosphatidylcholine (LPC),
1- (1Z-Octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PlsPE (C18, 20: 4) 1- (1Z-Octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine) ) Over 50% (upper limit is 90%)
1-hexadecyl-2- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (1-hexadecyl-2- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine: PlsPE (C16, 18: 1)) ) 200% or more (upper limit is 300%)
Against 1- (1Z-Octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphocholine (PlSPC (C18, 20: 4)) 0%,
10% or less for soybean oil and olive oil (lower limit is 0%),
4-nitrophenyl butyrate (pNPB), 4-nitrophenyl octanoate (pNPO), 4-nitrophenyl decanoate (pNPD), 4-nitro 4-Nitorophenyl laurate (pNPL), 4-nitrophenyl myristate (pNPM), 4-nitrophenyl palmitate (pNPP) and 4-nitrophenyl stearate (4 -Nitorophenyl stearate (pNPS) has a relative activity of 15% or less (lower limit is 0%).

A preferable form of the above enzyme is that when egg yolk lecithin (L) (manufactured by Nacalai Tesque) is used as a substrate and the hydrolysis activity is 100% at 37 ° C. for 5 minutes at pH 5.6. A hydrolysis activity of 50% or more is exhibited within the range of 1 to pH 10.0.

A preferred form of the above enzyme is when egg yolk lecithin (L) (manufactured by Nacalai Tesque) is used as a substrate, and the hydrolysis activity under the condition of pH 9.0 and 50 ° C. for 5 minutes is defined as 100%.
50% or less (lower limit is 30%) with respect to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
350% or more with respect to 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA) (upper limit is 400%),
400 for 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol): PG) % Or more (upper limit is 450%),
450% or more with respect to L-α-phosphatidylserine (PS) (upper limit is 480%),
100% or more (upper limit is 140%) with respect to 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
350% or more (upper limit is 380%) with respect to L-α-phosphatidylinositol (PI),
More than 300% (upper limit is 360%) of soybean phosphatidylcholine (SB-PC) (manufactured by Sigma-Aldrich)
It has a relative activity of 75.0% or more (upper limit is 90%) with respect to soybean lecithin (SBL) (manufactured by Wako Pure Chemical Industries, Ltd.).

A preferred form of the above enzyme is when egg yolk lecithin (L) (manufactured by Nacalai Tesque) is used as a substrate and the hydrolysis activity under the condition of 5 minutes at 50 ° C. at pH 5.6 is 100%.
100% or more (upper limit is 140%) based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
100% or less (lower limit is 60%) with respect to 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA),
150 against 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol): PG) % Or more (upper limit is 190%)
150% or more with respect to L-α-phosphatidylserine (PS) (upper limit is 190%),
100% or more based on 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (upper limit is 120%),
250% or more (upper limit is 290%) with respect to L-α-phosphatidylinositol (L-α-Phosphatidylinositol: PI),
More than 250% (upper limit is 290%) with respect to soybean phosphatidylcholine (L-α-phosphatidylcholine: SB-PC) (manufactured by Sigma-Aldrich)
It has a relative activity of 250% or more (upper limit is 280%) with respect to soybean lecithin (SBL) (manufactured by Wako Pure Chemical Industries, Ltd.)
The relative activity with respect to triglyceride (Olive Oil, manufactured by Wako Pure Chemical Industries, Ltd.) is almost 0%.

In a preferred form of the above enzyme, the isoelectric point calculated from the mature amino acid sequence excluding the secretory signal sequence is in the range of 5.7 to 5.8.

A preferred form of the above enzyme has a molecular weight in the range of 25,000 to 30,000 as measured by SDS-PAGE, and a molecular weight in the range of 25,000 to 30,000 as analyzed from the amino acid composition. It is.

The preferred form of the enzyme is derived from a microorganism belonging to the genus Streptomyces.

[Polynucleotide]
The polynucleotide according to the present invention is a polynucleotide encoding the above enzyme.

The preferable form of said polynucleotide contains the polynucleotide in any one of the following (a) to (c).
(A) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.
(B) A polynucleotide that hybridizes with a base sequence complementary to the base sequence described in SEQ ID NO: 1 under stringent conditions.
(C) a polynucleotide having at least 75% sequence identity to the base sequence set forth in SEQ ID NO: 1.

The preferred form of the above polynucleotide is derived from a microorganism belonging to the genus Streptomyces.

[vector]
The vector according to the present invention is a vector containing the above-described polynucleotide.

[Transformant]
The transformant according to the present invention is one or more selected from phospholipids, lysophospholipids, glycerol-3-phosphate, and glycerol-3-phosphate compounds into which the above-described polynucleotide or the above-described vector is introduced. It is a transformant possessing the ability to produce an enzyme that produces.

A preferred form of the transformant is a microorganism in which the host of the transformant belongs to the genus Streptomyces.

[Enzyme production method]
The method for producing an enzyme according to the present invention acts on a phospholipid, and hydrolyzes the phospholipid to select one or more selected from lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate ester compound. A step of obtaining a transformant producing the enzyme by introducing the polynucleotide or the vector described above into a host; and culturing the transformant A step of producing the enzyme.

In a preferred embodiment of the enzyme production method, the host is a microorganism belonging to the genus Streptomyces.

According to the present invention, it is possible to provide a novel phospholipase A1 having a high enzyme activity. Furthermore, it is possible to provide a method for efficiently producing this enzyme by a microorganism. Thereby, a novel phospholipid processing agent having high PLA1 activity and having substrate specificity for phospholipid can be obtained without the addition of a metal salt. In addition, glycerol-3-phosphate compounds such as lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphocholine can be efficiently produced. In addition, highly purified phosphatidylethanolamine, glycerol-3-phosphate, or glycerol-3-phosphocholine can be efficiently produced.

2 is an electrophoresis photograph showing the results of SDS-PAGE analysis of a purified fraction obtained from a culture solution of Streptomyces albidoflavus. It is a graph which shows the relative activity of phospholipase A1 about the purified enzyme derived from Streptomyces albidoflavus in various pH on the basis of the enzyme activity when pH is 5.6 (100%) . It is a graph which shows the relative activity of phospholipase A1 about the purified enzyme derived from Streptomyces albidoflavus in various temperature on the basis of the enzyme activity in case reaction temperature is 50 degreeC (100%). . It is a graph which shows the residual activity about the refinement | purification enzyme derived from Streptomyces albidoflavus after the process by various temperature on condition of pH 5.6 and 30 minutes. It is a graph which shows the residual activity about the refinement | purification enzymes derived from Streptomyces albidoflavus after the process by various pH on conditions of 4 degreeC for 3 hours. It is a figure which shows a part of deduced amino acid sequence about the base sequence and structural gene of the area | region containing the phospholipase A1 gene derived from Streptomyces albidoflavus (Streptomyces albidoflavus). It is a figure which shows a part of deduced amino acid sequence (the continuation of FIG. 6A) about the base sequence and structural gene of the area | region containing the phospholipase A1 gene derived from Streptomyces albidoflavus (Streptomycesalbidoflavus). Based on the enzyme activity of PS in Table 2 (conditions at pH 9.0, 50 ° C., 5 minutes) (100%), and based on the enzyme activity of PI in Table 3 (conditions at pH 5.6, 50 ° C., 5 minutes) 100%) is a graph depicting the relative activities of other substrates converted again. It is the graph which re-converted the relative activity of the other substrate on the basis of the enzyme activity of POPA in Table 5 and Table 6 (conditions of pH 7.2, 50 ° C., 5 minutes) as a standard (100%). 3 is a graph showing the change over time in the production concentration of lysophosphatidylcholine (a sum of both 1-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphocholine). 2 is a graph showing changes over time in the production concentrations of 1-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphocholine. 6 is a graph showing the change over time in the molar ratio of 1-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphocholine. It is a ribbon model which shows the three-dimensional structure of phospholipase A1 derived from Streptomyces albidoflavus. It is an enlarged view of the part (active center) enclosed with the broken line of FIG. 12A.

[enzyme]
In the present specification, the enzyme is not limited to a purified enzyme, but includes a crude product, an immobilized product, and the like. The enzyme is purified by a method well known to those skilled in the art, such as ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, etc., using a microorganism culture solution. Thereby, enzymes with various degrees of purification (including enzymes purified to approximately one) can be obtained.

In this specification, the microorganism refers to any of wild strains, mutant strains (for example, those induced by ultraviolet irradiation), recombinants induced by genetic engineering techniques such as cell fusion or genetic recombination methods, and the like. Or a stock of Genetically engineered microorganisms such as recombinants are known to those skilled in the art, for example, as described in Molecular Cloning A Laboratory Manual, 2nd edition (Sambrook, J. et al., Cold Spring Harbor Press, 1989). Can be easily created using various techniques. The culture solution of microorganisms means both a culture solution containing microbial cells and a culture solution from which microbial cells have been removed by centrifugation or the like.

[Phospholipase A1]
The enzyme that hydrolyzes the fatty acid ester bond at the α-position (sn-1 position) of the glycerol group in the phospholipid (glycerol phospholipid) is called phospholipase A1 (PLA1), and the β-position (sn-2 position) of the glycerol group The enzyme that hydrolyzes the fatty acid ester group is referred to as A2 (PLA2). An enzyme having both phospholipase A1 activity and phospholipase A2 activity is referred to as phospholipase B (PLB). That is, PLB has enzyme activity at both the α-position and the β-position.

Accordingly, PLA1 generates at least one, preferably all three of glycerol-3-phosphate compounds such as 2-lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphocholine from phospholipids. Is an enzyme.

The PLA1 activity can be confirmed, for example, as follows, but the confirmation method is not limited to this. Specifically, for example, PLA1 activity can be confirmed by measuring the amount of free fatty acid produced as a result of the enzyme reaction.

Specifically, first, 1 g of egg yolk lecithin (L) (manufactured by Nacalai Tesque) was dissolved in 10 mL of 0.02% (w / v) Triton X-100 (manufactured by Nacalai Tesque). % (W / v) egg yolk lecithin (L) is prepared. 0.025 mL of this 10% (w / v) egg yolk lecithin (L), 0.060 mL of 0.2 M Tris-HCl buffer (pH 8.0), and 0.5 M disodium EDTA (Wako Pure) Add 0.005 mL of Yakuhin Kogyo). And after preheating at 37 degreeC for 5 minute (s), 0.010 mL of samples which confirm enzyme activity are added, and it is made to react at 37 degreeC for 5 minutes. After the enzyme reaction, the reaction is stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution can be determined using, for example, “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.) which is a free fatty acid measurement kit, as described in the instructions attached to the kit. Measure as follows. The amount of enzyme that produces 1 μmol of free fatty acid per minute is defined as 1 unit.

The enzyme according to the present invention is selected from lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate ester compound by preferentially hydrolyzing the sn-1 position acyl group of phospholipid. An enzyme that produces more than a species, comprising a polypeptide according to any one of (a) to (c) below.
(A) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2.
(C) a polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2.

(2) Substrate specificity When egg yolk lecithin (L) is used as a substrate and the hydrolysis activity under the condition of pH 7.2 and 50 ° C. for 5 minutes is defined as 100%, under the same pH, temperature and time conditions, Each of the following substrates has the following relative activity.

50% or more (upper limit is 200%) with respect to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) Has relative activity.

More than 500% (upper limit is 600%) with respect to 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate (1-PALMitoyl-2-oleoyl-sn-glycerol-3-phosphate: POPA) Has relative activity.

Furthermore, it is 150% or more (upper limit is 350%) with respect to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Has relative activity.

Further, it has a relative activity of 300% or more (upper limit is 450%) with respect to L-α-phosphatidylinositol (PI).

Further, it has a relative activity of 300% or more (upper limit is 700%) with respect to L-α-phosphatidyl-L-serine (PS).

In addition, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac- (1-glycerol) (1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac- (1-glycerol) : POPG) has a relative activity of 100% or more (upper limit is 300%).

Also, it has a relative activity of 100% or more (upper limit is 150%) with respect to soybean-derived L-α-phosphatidylcholine (SB-PC).

Also, it has a relative activity of 30% or more (upper limit is 150%) with respect to soybean lecithin (SBL).

Also, it has a relative activity of 250% or more (upper limit is 350%) with respect to L-α-Lysophosphatidylcholine (LPC).

Further, 1- (1Z-octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine (1- (1Z-Octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine: PlsPE (C18, 20: 4)) has a relative activity of 50% or more (upper limit is 90%).

Also, 1-hexadecyl-2- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine: PlsPE (C16, 18: 1)) with a relative activity of 200% or more (upper limit is 300%).

In addition, 1- (1Z-octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphocholine (1- (1Z-Octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphocholine: PlsPC (C18, 20: 4) 0% relative activity.

Moreover, it has a relative activity of 10% or less (lower limit is 0%) with respect to soybean oil and olive oil.

In addition, 4-nitrophenylbutyrate (pNPB), 4-nitrophenyloctanoate (pNPO), 4-nitrophenyldecanoate (pNPD), 4-nitrophenyl Laurate (4-Nitorophenyl laurate: pNPL), 4-nitrophenyl myristate (pNPM), 4-nitrophenyl palmitate (pNPP) and 4-nitrophenyl stearate (4-N Nitorophenyl stearate (pNPS) has a relative activity of 15% or less (lower limit is 0%).

For example, some secretory hydrolases derived from the Streptomyces albus strain J1074 show 99% or more sequence identity with the amino acid sequence of the enzyme (PLA1) according to the present invention. However, the above-described secreted hydrase is merely presumed to be a lipase, and the physicochemical properties of the enzyme according to the present invention have not been clarified, and even a person skilled in the art is in accordance with the present invention. It is impossible to estimate the enzyme. This is clear from the fact that the enzyme according to the present invention does not exhibit lipase activity and hardly exhibits esterase activity. Even if such homology is high, other factors such as the three-dimensional structure have a great influence on the physicochemical properties of the enzyme. Therefore, an enzyme showing 99% or more sequence identity with the amino acid sequence of the enzyme according to the present invention. Even so, it is considered that the substrate has no substrate specificity like the enzyme according to the present invention. The three-dimensional structure of the enzyme (PLA1) according to the present invention will be described later as an example.

For example, the above-mentioned enzymes include, as buffer solutions, acetic acid-sodium acetate buffer (pH 4.1 to 5.6), Bistris-HCl (BisTris-HCl) buffer (pH 5.6 to 7.2), Tris-hydrochloric acid. (Tris-HCl) buffer solution (pH 7.2 to 8.8) and glycine-sodium hydroxide buffer solution (pH 8.8 to 10.5) are used to react the above egg yolk lecithin (L) with the enzyme. When placed below, it may exhibit PLA1 activity within its pH range (pH 4.1-10.5). The optimum pH is around pH 5.6, but shows substantially 100% activity at pH 5-8.

For example, when the hydrolysis activity at pH 5.6 is 100% under the condition where egg yolk lecithin (L) and the above enzyme are reacted at 37 ° C. for 5 minutes as described above, the enzyme has a pH of 4. It is preferable to exhibit an activity of 50% or more within the range of 1 to pH 10.

The above enzyme can act at about 20 to 65 ° C., for example, under the reaction conditions with the above egg yolk lecithin (L). The optimum temperature can be within this range. Preferably it is in the range of about 30-55 ° C, more preferably in the range of 40-55 ° C, and even more preferably about 50 ° C.

For example, when the enzyme is treated with 120 mM acetic acid-sodium acetate buffer (pH 5.6) for 30 minutes, it can be stable with almost no decrease in activity from 4 ° C. to 40 ° C. and 45 ° C. However, an activity of about 80% (for example, 75%) or more remains.

For example, when the above phospholipid and the above enzyme solution are placed under reaction conditions using an acetic acid-sodium acetate buffer (pH 5.6) as a buffer, the above enzyme has 100 mM EDTA. Are not inhibited, and preferably exhibit almost the same activity as when EDTA is not added. In the presence of 10 mM Ca ²⁺ and Zn ²⁺ , it is preferable to exhibit about 80% activity (for example, about 80 to 95% activity). On the other hand, 10 mM Fe ³⁺ and Fe ²⁺ can inhibit the activity.

Furthermore, the above enzyme preferably has the following substrate specificity.

That is, when egg yolk lecithin (L) is used as a substrate and the hydrolysis activity under the conditions of pH 9.0 and 50 ° C. for 5 minutes is 100%, the following substrate is used under the same pH, temperature and time conditions. The following relative activities are preferable.

It has a relative activity of 50% or less (lower limit is 30%) with respect to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). preferable.

In addition, it has a relative activity of 350% or more (upper limit is 400%) with respect to 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA). It is preferable.

Also, for 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol): PG) Preferably having a relative activity of 400% or more (upper limit is 450%).

Further, it preferably has a relative activity of 450% or more (upper limit is 480%) with respect to L-α-phosphatidylserine (PS).

Furthermore, it has a relative activity of 100% or more (upper limit is 140%) with respect to 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). It is preferable to have.

Further, it preferably has a relative activity of 350% or more (upper limit is 380%) with respect to L-α-phosphatidylinositol (PI).

In addition, it preferably has a relative activity of 300% or more (upper limit is 360%) with respect to soybean phosphatidylcholine (SB-PC) (manufactured by Sigma-Aldrich).

Moreover, it is preferable to have a relative activity of 75.0% or more (upper limit is 90%) with respect to soybean lecithin (SBL) (manufactured by Wako Pure Chemical Industries, Ltd.).

That is, when egg yolk lecithin (L) is used as a substrate and the hydrolysis activity under the condition of pH 5.6 and 50 ° C. for 5 minutes is 100%, the following substrate is used under the same pH, temperature and time conditions. The following relative activities are preferable.

It has a relative activity of 100% or more (upper limit is 140%) against 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). preferable.

Also, it has a relative activity of 100% or less (lower limit is 60%) with respect to 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA). It is preferable.

Also, for 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol): PG) The relative activity is preferably 150% or more (upper limit is 190%).

Further, it is preferable to have a relative activity of 150% or more (upper limit is 190%) with respect to L-α-phosphatidylserine (PS).

Furthermore, it has a relative activity of 100% or more (upper limit is 120%) with respect to 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). It is preferable to have.

Further, it preferably has a relative activity of 250% or more (upper limit is 290%) with respect to L-α-phosphatidylinositol (PI).

In addition, it preferably has a relative activity of 250% or more (upper limit is 290%) with respect to soybean phosphatidylcholine (SB-PC) (manufactured by Sigma-Aldrich).

Moreover, it is preferable to have a relative activity of 250% or more (upper limit is 280%) with respect to soybean lecithin (SBL) (manufactured by Wako Pure Chemical Industries, Ltd.).

Further, the relative activity with respect to triglyceride (olive oil) is preferably about 0% (for example, 5% or less, 3% or less, 1% or less, or the detection limit or less).

The above enzyme may vary slightly depending on electrophoresis conditions, etc., but the molecular weight in SDS-PAGE is within the range of 25,000 to 30,000 (for example, about 28,000 or about 27,000). Is preferred. The enzyme preferably has a molecular weight calculated from the amino acid composition in the range of 25,000 to 30,000. For example, a natural enzyme derived from Streptomyces albidoflavus strain NA297 has a molecular weight of about 28,000, specifically 28,000 in SDS-PAGE. The natural enzyme derived from this Streptomyces albidoflavus NA297 strain has a molecular weight of 27,199 calculated from its amino acid composition.

The above enzyme preferably exhibits an isoelectric point in the range of 5.7 to 5.8 (eg, 5.72). The isoelectric point of the enzyme can be calculated by GENETYX from the mature amino acid sequence excluding the secretory signal sequence.

One embodiment of the above enzyme, that is, phospholipase A1, consists of the amino acid sequence shown in SEQ ID NO: 2. The phospholipase A1 preferably has an amino acid sequence from the 34th position to the 269th position of SEQ ID NO: 2 (also referred to herein as "the amino acid sequence described in SEQ ID NO: 2").

As long as the enzyme has PLA1 activity, one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 are substituted, deleted, inserted, And / or an enzyme having an added amino acid sequence. Those skilled in the art, for example, site-directed mutagenesis (Nucleic AcidＡRes., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual). , 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1989; PCR: A Practical Approach, IRL Press, 1991, pp. 200), etc., as appropriate, and / or By introducing additional mutations, the structure of the protein can be altered. In the present specification, the number of amino acid residues that can be substituted, deleted, inserted and / or added is usually 50 or less, such as 30 or less, 20 or less, preferably 16 or less, more preferably 5 or less, and even more preferably. Is 0-3. In addition, amino acid mutations include not only artificially mutated enzymes but also naturally mutated enzymes as long as they have PLA1 activity, and are included in the enzyme (PLA1).

A protein having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having homology to the amino acid sequence shown in SEQ ID NO: 2 is also included in the enzyme (PLA1) as long as it has PLA1 activity. . PLA1 is preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 75% of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence set forth in SEQ ID NO: 2. It may be a protein having an amino acid sequence with 90%, even more preferably at least 95%, even more preferably at least 99% homology.

Protein homology (homology) search, for example, databases such as SWISS-PROT, PIR, DAD and other protein amino acid sequences, DNA databases such as DDBJ, EMBL, Gene-Bank, etc., programs such as BLAST and FASTA For example, it can be performed through the Internet. Confirmation of the activity of the protein can be performed using the procedure described above.

The supply source of PLA1 is not particularly limited, but PLA1 can be obtained from living cells such as microorganisms. Examples of such microorganisms include microorganisms belonging to the genus Streptomyces. Preferably, Streptomyces albidoflavus (Streptomyces albidoflavus) and Streptomyces albus (Streptomycesalbus), most preferably Streptomyces albidoflavus NA297 (accession number: NITE BP-1014 strain), hereinafter referred to as “NA297 strain” It is said.)

For example, the Streptomyces albidoflavus NA297 (Accession Number: NITE BP-1014) is liquid-cultured in an appropriate nutrient medium to secrete the above enzyme outside the cells. Those treated with lyophilization, salting out, organic solvent, etc. can be produced as PLA1 enzyme preparations.

The microorganism that can be used in the production of the PLA1 enzyme preparation is not limited to Streptomyces albidoflavus NA297, and may be a microorganism that belongs to the genus Streptomyces and can produce PLA1. In addition, natural or artificial mutants of these species or gene fragments necessary for the expression of PLA1 activity can be artificially extracted and used for the production of PLA1 even in other species incorporating them. . Moreover, even if it does not belong to Streptomyces genus, if it is a microorganism which can produce said PLA1, it can also be used.

The production of PLA1 enzyme preparation using Streptomyces albidoflavus NA297 will be described as an example.

Since this bacterium is liquid-cultured in a nutrient medium, the above enzyme is secreted to the outside of the microbial cell. Therefore, the culture supernatant is treated with lyophilization, salting out, an organic solvent, or the treated product is immobilized. Or the like to produce an enzyme preparation. More specifically, first, the bacterium is cultured in a suitable medium, for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to secrete the enzyme. Here, as the carbon source, starch, starch hydrolysate, sugars such as glucose and sucrose, alcohols such as glycerol, organic acids (for example, acetic acid and citric acid) or salts thereof (for example, sodium salt), and the like. Can be mentioned. Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, and soybean powder, and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, and urea. Examples of inorganic salts include sodium chloride, monopotassium phosphate, magnesium sulfate, manganese chloride, calcium chloride, and ferrous sulfate. The concentration of the carbon source is, for example, in the range of 1 to 20% (w / v), preferably 1 to 10% (w / v). The concentration of the nitrogen source is, for example, in the range of 1 to 20% (w / v), preferably 1 to 10% (w / v). The culture temperature is preferably a temperature at which the above enzyme is stable and the cultured microorganism can sufficiently grow, and is preferably 20 to 37 ° C., for example. The culture time is preferably a time during which the enzyme is sufficiently produced, for example, about 1 to 7 days. The culture can be preferably performed under aerobic conditions, for example, with aeration stirring or shaking.

PLA1 is fractionated by protein solubility (precipitation with organic solvents, salting out with ammonium sulfate, etc.); cation exchange, anion exchange, gel filtration, hydrophobic chromatography; affinity chromatography using chelates, dyes, antibodies, etc. It can be purified by appropriately combining known methods such as graphy. For example, after recovering the culture supernatant of the microorganism, it can be purified by ammonium sulfate precipitation, further anion exchange chromatography, hydrophobic chromatography, and / or cation exchange chromatography. Thereby, it can be purified to almost a single band in polyacrylamide gel electrophoresis (SDS-PAGE). That is, the enzyme (PLA1) can be estimated as a monomer by HPLC analysis and gel filtration chromatography analysis.

[Polynucleotide encoding PLA1]
The polynucleotide according to the present invention encodes the above PLA1. This polynucleotide preferably includes the polynucleotide described in any of (a) to (c) below.
(A) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.
(B) A polynucleotide that hybridizes with a base sequence complementary to the base sequence described in SEQ ID NO: 1 under stringent conditions.
(C) a polynucleotide having at least 80% sequence identity to the base sequence set forth in SEQ ID NO: 1.

The above polynucleotide may be an artificial molecule including an artificial nucleotide derivative in addition to a natural polynucleotide such as DNA or RNA. The polynucleotide may be a DNA-RNA chimeric molecule. The above-mentioned polynucleotide encoding PLA1 has, for example, the base sequence from position 1 to position 816 of SEQ ID NO: 1 (also referred to herein as “base sequence described in SEQ ID NO: 1”). The base sequence described in SEQ ID NO: 1 encodes a protein including the amino acid sequence described in SEQ ID NO: 2, and the protein including this amino acid sequence constitutes a preferred form of PLA1.

The above-mentioned polynucleotide encoding PLA1 includes an amino acid in which one or more amino acids are substituted, deleted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 2 as described above, and PLA1 activity Also included are polynucleotides that encode proteins having: A person skilled in the art can introduce substitution, deletion, insertion, and / or addition mutation as appropriate into the polynucleotide having the base sequence described in SEQ ID NO: 1 using a site-directed mutagenesis method (described above). Thus, it is possible to obtain a homologue of a polynucleotide.

The above-mentioned polynucleotide encoding PLA1 is also a protein capable of hybridizing under stringent conditions with a polynucleotide having a base sequence complementary to the polynucleotide having the base sequence shown in SEQ ID NO: 1 and having PLA1 activity Also included are polynucleotides encoding.

The target gene in the polynucleotide is based on the nucleotide sequence information described in the present specification, preferably the above microorganism, preferably a microorganism belonging to the genus Streptomyces, more preferably Streptomyces albidoflavus (Streptomyces). albidoflavus) NA297. PCR or hybridization screening can be used for obtaining the gene.

Polynucleotides can also be obtained by chemically synthesizing the full length of a gene by DNA synthesis. Moreover, based on said base sequence information, the polynucleotide which codes said PLA1 derived from organisms other than the above can also be acquired. For example, by designing a probe using the above base sequence or a part of the base sequence and performing hybridization under conditions stringent to DNA prepared from other organisms, PLA1 derived from various organisms can be obtained. The encoding polynucleotide can be isolated.

Furthermore, based on the above base sequence information, PCR primers are designed from regions with high homology using sequence information registered in DNA databases such as DNA Databank of Japan (DDBJ), EMBL, and Gene-Bank. You can also By using such a primer and performing PCR using chromosomal DNA or cDNA as a template, the polynucleotide encoding PLA1 can also be isolated from various organisms. Similarly, the polynucleotide encoding PLA1 can also be isolated from various organisms by performing PCR using DNA or RNA extracted from the environment as a template.

The polynucleotide capable of hybridizing under stringent conditions is a sequence of at least 20, preferably at least 30, for example, 40, 60, or 100 consecutive sequences in the nucleotide sequence set forth in SEQ ID NO: 1. The probe is designed by selecting one or more, and includes the conditions described in the manual (for example, cleaning conditions: 42 ° C., 0.5 × SSC) using, for example, ECL directic acid labeling and detection system (manufactured by GE Healthcare) In primary wash buffer), it refers to a hybridizing polynucleotide.

More specifically, the “stringent conditions” are usually conditions of 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS. More preferably, the conditions are 65 ° C., 0.1 × SSC, and 0.1% SDS, but are not particularly limited to these conditions.

There are a plurality of factors such as temperature and salt concentration as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.

Furthermore, the above-mentioned polynucleotide encoding PLA1 is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably the amino acid sequence set forth in SEQ ID NO: 2. Comprises a polynucleotide encoding a protein having an amino acid sequence with at least 95%, even more preferably at least 99% homology, and having PLA1 activity. The protein homology search is as described above.

The polynucleotide encoding PLA1 is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably the nucleotide sequence set forth in SEQ ID NO: 1. Also included are polynucleotides that have a base sequence with at least 95%, even more preferably at least 99% sequence identity, and encode a protein having PLA1 activity. The determination and search of the sequence identity of the base sequence is also as described above.

The polynucleotide encoding PLA1 can be expressed in the same or different host using genetic recombination technology.

[Vector and transformant]
The vector according to the present invention contains the above-described polynucleotide. In addition, any of glycerol-3-phosphate ester compounds such as lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphocholine acts on phospholipids by introducing a polynucleotide or a vector into a host. A transformant possessing the ability to produce an enzyme that produces one or more can be made.

A procedure for producing a transformant and construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al. , Molecular Cloning: A Laboratory Manual 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). In particular, for actinomycetes, it can be performed with reference to “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000)”.

In order to express a polynucleotide encoding PLA1 in a microorganism, this DNA is first introduced into a plasmid vector or a phage vector that is stably present in the microorganism, and the genetic information is transcribed and translated. Therefore, it is preferable to incorporate a promoter corresponding to a unit for controlling transcription / translation 5 'upstream of the DNA strand. Further, it is preferable to incorporate a terminator, which is a unit for controlling transcription / translation, downstream of the 3 ′ side of the DNA strand. More preferably, both the promoter and terminator are incorporated at each site. As the promoter and terminator, promoters and terminators known to function in microorganisms used as hosts are used. Regarding the vectors, promoters, and terminators that can be used in these various microorganisms, “Basic Course of Microbiology 8, Genetic Engineering, Kyoritsu Shuppan”, especially regarding actinomycetes, “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000) It is possible to use this method. Moreover, it can be efficiently secreted and produced extracellularly by using a signal sequence as necessary. The signal sequence used at this time may be the above PLA1 or the other.

The host to be transformed is not particularly limited as long as it is a living organism that can be transformed with a vector containing a polynucleotide encoding PLA1 and express PLA1 activity. For example, Escherichia coli, Bacillus subtilis, bacteria, actinomycetes, yeast, mold and the like can be mentioned. More specifically, for example, the genus Escherichia, the genus Bacillus, the genus Pseudomonas, the genus Serratia, the genus Brevibacterium, the genus Corynebacterium, Bacteria for which host vector systems such as Streptococcus and Lactobacillus are developed; Actinomycetes for which host vector systems such as Rhodococcus and Streptomyces are developed; Saccharomyces (Saccharomyces) Saccharomyces), Kluyveromyces, Shizosa S. genus Schizosaccharomyces, genus Zygosaccharomyces, genus Yarrowia, genus Trichosporon, genus Rhodosporidium, genus Rhodospodium, genus C And yeasts that have been developed for host vector systems such as the genus Neurospora, the genus Aspergillus, the genus Cephalosporum, the genus Trichoderma, and the like. Escherichia coli is preferred for ease of gene recombination, and actinomycetes are preferred for ease of gene expression.

In addition to microorganisms, various host / vector systems have been developed in plants and animals. For example, insects such as moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes and other plants. Systems for expressing large amounts of heterologous proteins have been developed and may be used.

The obtained transformant can be used for production of an enzyme preparation (PLA1) as described above. Specifically, the transformant is subjected to liquid culture in an appropriate nutrient medium, the expressed PLA1 is secreted outside the cell, and the culture supernatant is treated with lyophilization, salting out, an organic solvent, etc. Can be manufactured.

Depending on the host cell, the culture conditions may vary, but the culture can be performed under conditions commonly used by those skilled in the art. For example, when an actinomycete such as Streptomyces is used as a host, a tryptic soy medium containing thiostrepton (for example, manufactured by Becton Dickinson) can be used. The enzyme produced by the transformant can be further purified as described above.

[Advantages of PLA1]
The advantages of the preferred form of PLA 1 that can be produced as described above will be described below.

(1) Conventional PLA1 has a low catalytic activity in the absence of metal ions such as calcium ions. Therefore, in food processing, it is necessary to add a metal salt to a food material and perform enzyme treatment. In contrast, PLA1 of the present invention does not require the addition of a metal salt for the enzyme reaction. Therefore, the food material can be processed as it is, and the range of use can be expanded, and it can be expected to prevent the deterioration of food quality due to the metal salt and improve the safety. Furthermore, when the enzyme treatment is performed using a reactor, it is not necessary to add a metal salt, so that scales do not adhere to the reactor, maintenance is easy, and running costs are kept low.

(2) Whereas the conventional filamentous fungus Celidium unicorn Sutton-derived PLA1 (Patent Document 6) has low activity against PE (phosphatidylethanolamine), the PLA1 of the present invention includes various types of PE. It can act on phospholipids, especially on PI (phosphatidylinositol), PG (phosphatidylglycerol), and PS (phosphatidylserine). It works well for PI, PG, PS, DOPE, L at pH 5.6, and works well for PI, PG, PS, DMPA at pH 9.0. By controlling the pH, the acting substrate can be controlled. That is, it is possible to suppress the enzyme action to high purity phosphatidylcholine (for example, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine: DPPC) under reaction conditions of pH 9.0 and 50 ° C. Further, at 37 ° C., phosphatidylcholine having a high purity (for example, 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine: DPPC), phosphatidylethanolamine (for example, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine: DOPE), It is possible to suppress the enzyme action to phosphatidic acid (for example, 1,2-Dimyristoyl-sn-glycerol-3-phosphate: DMPA) low. More than 250% (upper limit is 290%) for soybean phosphatidylcholine (SB-PC) (Sigma Aldrich), more than 250% (upper limit is 280%) for soybean lecithin (SBL) (Wako Pure Chemical Industries, Ltd.) It is easy to use for lecithin with low purity as in (1) because it can act well. Furthermore, since it does not have lipase activity and esterase activity, there is an advantage that side reactions that hydrolyze glycerides and esters that coexist with phospholipids do not occur. For example, when PLA1 of the present invention is used in an oil refining process, it does not act on glycerides or esters but acts only on phospholipids, so that it can be expected that the yield of fats and oils will be improved.

(3) PLA1 of the present invention exhibits substantially 100% activity in the pH range of 5-8. Furthermore, since it exhibits an activity of 50% or more with respect to the maximum activity in the range of pH 4.1 to pH 10, the usable pH range is extremely wide compared to the conventional PLA1, and it can be used under weakly acidic to alkaline conditions. Therefore, reaction control is easy and a wide range of applications can be expected.

(4) PLA1 of the present invention exhibits an activity of 50% or more in a range exceeding the range of 40 to 55 ° C. Furthermore, the conventional enzyme (PLA1 manufactured by Mitsubishi Chemical Foods) has an activity of 50% or less at 30 ° C. or lower, whereas PLA1 of the present invention exhibits an activity of 50% or higher at 20 ° C. Low temperature processing is possible. Therefore, the usable temperature range is wide as compared with the conventional PLA1.

(5) Since it is not inhibited by Mn ²⁺ , Co ²⁺ , Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , DTT (dithiothreitol), and EDTA, the reaction is easily controlled.

(6) The conventional PLA1 (PLA1 manufactured by Mitsubishi Chemical Foods) has a stable temperature of 55 ° C., whereas the stable temperature of the PLA1 of the present invention is 40 to 45 ° C. Therefore, it is possible to deactivate the enzyme at a lower temperature.

(7) The same enzyme activity was observed in the NBRC standard strain (Streptomyces albidoflavus NBRC13010). Therefore, billing at the species level is possible.

[Use of PLA1]
Below, the utilization method about the preferable form of PLA1 is demonstrated.

(1) PLA1 can decompose the cell membrane. PLA1 then lyses the cells. Therefore, it can be used for cell degrading agents and cell lysing agents including detergents such as egg yolk stains and blood stains.

(2) Since lysophospholipids are extremely excellent in emulsifying properties, they become emulsifiers and surfactants that can be used in various fields. In addition, lysophospholipids have been discovered as various physiological functions in recent years, and thus can be used as pharmaceuticals and supplements. Therefore, various highly functional lysophospholipids can be produced using PLA1 of the present invention.

(3) In the purification of fats and oils, phospholipids that become impurities are efficiently decomposed using PLA1 of the present invention, and physical separation from the fats and oils becomes possible.

(4) By adding PLA1 of the present invention to flour such as bread dough or noodle dough, the phospholipid originally contained in the flour is converted to lysolecithin. By forming lysolecithin in the dough, it binds to proteins and starch to help form a gluten network, improve the physical properties of bread and noodles, and prevent aging.

(5) Cyclic phosphatidic acid having excellent properties and effects as a cosmetic material can be produced from lysophospholipid. Therefore, cyclic phosphatidic acid can be efficiently produced by using PLA1 of the present invention.

(6) Glycerol-3-phosphocholine is expected to prevent dementia. Other glycerol-3-phosphate compounds can be expected to have high functions. Therefore, the PLA1 of the present invention can produce a highly functional glycerol-3-phosphate ester.

(7) Glycerol-3-phosphate is a glycolytic metabolic intermediate. Therefore, PLA1 of the present invention can produce glycerol-3-phosphate having the possibility of enhancing metabolism.

Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited to these examples.

[Method for measuring enzyme activity of PLA1]
In this example, the enzyme activity was basically measured as follows. This method is hereinafter referred to as “PLA1 activity standard measurement method”. This method illustrates the case where egg yolk lecithin (L) is used as a substrate.

1% egg yolk lecithin (L) (manufactured by Nacalai Tesque) was dissolved in 10 mL of 0.02% (w / v) Triton X-100 (manufactured by Nacalai Tesque), and 10% (w / v) egg yolk lecithin ( L) was prepared. 0.025 mL of this 10% (w / v) egg yolk lecithin (L), 0.060 mL of 0.2 M Tris-HCl buffer (pH 8.0), and 0.5 M disodium EDTA (Wako Pure) Yakuhin Kogyo Co., Ltd.) was added. And after preheating at 37 degreeC for 5 minute (s), 0.010 mL of samples containing an enzyme were added and it was made to react at 37 degreeC for 5 minutes. After the enzyme reaction, the enzyme reaction was stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution was determined using “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.), a free fatty acid measurement kit, as described in the instructions attached to the kit. Measured. The amount of enzyme that produces 1 μmol of free fatty acid per minute was defined as 1 unit.

[Example 1: Purification of Streptomyces albidoflavus-derived enzyme]
(A) Cultivation 500 mL of tryptic soy medium (Becton Dinkinson) is prepared, and 50 ml each is dispensed into a 500 mL baffle Erlenmeyer flask, and further 1% soybean lecithin and 0.1% Tween (Tween) After adding 80, steam sterilization was performed at 121 ° C. for 15 minutes. Inoculate a φ18 test tube (18 x 180 mm) containing 5 mL of tryptic soy medium (manufactured by Becton Dinkinson), taking an appropriate amount of a colony of Streptomyces albidoflavus NA297 previously grown on a plate medium. The mixture was then cultured with shaking at 28 ° C. until good growth was obtained. 0.5 ml of this culture solution was inoculated into 50 mL of the previously sterilized medium and cultured with shaking at 28 ° C. for 55 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 (15,000 rpm, 30 minutes, 4 minutes). C). This precipitate was solubilized with 30 ml of 20 mM Tris-HCl (Tris-HCl) buffer (pH 9.0) and dialyzed with 20 mM Tris-HCl (Tris-HCl) buffer (pH 9.0) to obtain a crude enzyme solution. It was.

(C) Toyopearl Phenyl-650M column chromatography To the crude enzyme solution obtained in (b) above, 1.5 M ammonium sulfate was added at a final concentration, and 20 mM Tris-HCl (Tris-HCl) buffer containing 1.5 M ammonium sulfate. The sample was applied to a Toyopearl Phenyl-650M column (inner diameter 26 mm, height 38 mm, manufactured by Tosoh Corporation) previously equilibrated with a liquid (pH 8.0). After washing the column with the same buffer, the active fraction was eluted with a linear gradient of ammonium sulfate (from 1.5 M to 0 M).

(D) HiTrap SP HP column chromatography The active fractions obtained in (c) above were collected and concentrated and desalted using Viva spin (manufactured by Sartorius). To this was added 20 mM MES-sodium hydroxide buffer (pH 6.0). This was applied to a HiTrap SP (5 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 20 mM MES-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 1M).

(E) HiTrap Q HP column chromatography The active fractions obtained in (d) above were collected and concentrated and desalted using Viva spin. To this was added 20 mM Tris-HCl buffer (pH 9.0). After applying to HiTrap Q HP (5 ml) column (manufactured by GE Healthcare Biosciences) pre-equilibrated with 20 mM Tris-HCl buffer (pH 9.0), the column was washed with the same buffer. The active fraction was eluted with a linear gradient of sodium chloride (0M to 1M).

(F) SDS-PAGE
The active fractions eluted in (e) above were collected and analyzed by SDS-PAGE (15% (w / v) polyacrylamide gel).

FIG. 1 is an electrophoresis photograph showing the results of analysis by SDS-PAGE of the eluted fraction. Lane 1 (left side of the figure) is a molecular weight marker, and lane 2 (right side of the figure) shows a band of the eluted fraction. As shown in FIG. 1, a single band was observed in the elution fraction.

As described above, an electrophoretically purified enzyme was obtained from Streptomyces albidoflavus NA297 strain.

This enzyme was estimated to be a monomer by HPLC analysis and gel filtration chromatography analysis.

[Example 2: Measurement of properties of enzyme derived from Streptomyces albidoflavus]
(PLA1 activity)
The purified enzyme obtained in Example 1 and 1-palmitoyl-2-oleoylphosphatidinecholine as a substrate were subjected to an enzymatic reaction under the reaction conditions of “PLA1 activity standard measurement method”.

Thereafter, the reaction solution was extracted with chloroform / methanol (2/1, v / v). The extract was analyzed by gas chromatography and the fatty acid produced by hydrolysis was quantitatively analyzed to confirm that it had PLA1 activity.

(Enzymological properties)
The enzymatic properties of the purified enzyme obtained in Example 1 were examined.

(1) Working pH
As the buffer solution, acetic acid-sodium acetate buffer (pH 4.1-5.6), Bistris-HCl (BisTris-HCl) buffer (pH 5.6-7.2), Tris-HCl (Tris-HCl) buffer ( According to the above “PLA1 activity standard measurement method” except that the pH was changed using pH 7.2 to 8.8) and glycine-sodium hydroxide buffer (pH 8.8 to 10.5). LLA was used as a substrate and PLA1 activity was measured. As a result, the enzyme obtained in Example 1 was found to have an optimum pH of the reaction of 5.0 to 8.0.

FIG. 2 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 5.6 (100%). As can be seen from the graph of FIG. 2, this enzyme showed an activity with a relative activity of 50% or more with respect to the maximum activity (100%) in a wide range from pH 4.1 to pH 10.0.

(2) Action temperature According to the above “PLA1 activity standard measurement method” except that the reaction temperature during the enzyme reaction was changed, the yolk lecithin (L) was used as a substrate at pH 5.6 for 5 minutes, and PLA1 activity was measured. It was measured.

FIG. 3 is a graph showing enzyme activities at various reaction temperatures as relative activities based on the activity (100%) when the reaction temperature is 50 ° C. As shown in the graph of FIG. 3, this enzyme exhibited activity at 20 to 60 ° C., and the optimum temperature for the reaction was around 50 ° C. (eg, 45 to 55 ° C.).

(3) Temperature stability After treating the purified enzyme obtained in Example 1 in 120 mM acetic acid-sodium acetate buffer (pH 5.6) at various temperatures for 30 minutes, the residual activity was measured as described in “PLA1 activity standard measurement”. According to the “Method”, egg yolk lecithin (L) was used as a substrate and measured at 50 ° C. for 5 minutes at pH 5.6. Based on the enzyme activity before the temperature treatment as a reference (100%), the remaining activity was shown as the remaining ratio of the enzyme activity after each temperature treatment.

FIG. 4 is a graph showing the residual activity of the enzyme after treatment at various temperatures. As shown in the graph of FIG. 4, after the treatment at a temperature of 4 ° C. to 40 ° C., 90% or more of the enzyme remained before the treatment. After the treatment at 45 ° C., about 80% (about 75 to 80%) of the activity before the treatment remained.

(4) pH stability The residual activity after treating the purified enzyme obtained in Example 1 in various buffer solutions at 120 ° C. at 4 ° C. for 3 hours in each buffer solution (see “Action pH” column) According to the “PLA1 activity standard measurement method”, egg yolk lecithin (L) was used as a substrate, and measurement was carried out at 50 ° C. for 5 minutes at pH 5.6. Based on the enzyme activity before treatment as a reference (100%), the remaining activity was shown as the remaining ratio of enzyme activity after each treatment.

FIG. 5 is a graph showing the residual activity of the enzyme after treatment at various pHs. As shown in the graph of FIG. 5, this enzyme remained active from pH 3.99 to 10.5.

(5) Effects of various metal salts and chemical substances such as EDTA In the enzyme reaction, various metal ions or EDTA are added, and egg yolk lecithin (L) is used as a substrate in accordance with the above “PLA1 activity standard measurement method”. PLA1 activity was measured at 50 ° C for 5 minutes at pH 5.6.

Table 1 shows the results of relative activities with various additives added, assuming that the condition in which no chemical substance is added is 100%. The enzyme obtained in Example 1 is not inhibited by 50 mM EDTA and exhibits the same activity as when not added, and is hardly affected by 100 mM EDTA. Moreover, it was not inhibited by 2 mM dithiothreitol. Further, ^{10 mM} Ca 2+, in the ^{Zn 2+} presence, showed about 80% active. On the other hand, it was confirmed that the activity could be inhibited by 10 mM Fe ³⁺ , Fe ²⁺ , 2 mM 2-mercaptoethanol, iodoacetamide, PMSF (phenylmethylsulfonyl fluoride), and SDS.

(6) Substrate specificity PLA1 activity was measured according to the above "PLA1 activity standard measurement method" except that various phospholipids were used as a substrate instead of egg yolk lecithin (L).

Table 2 shows the relative values when the yolk lecithin (L) is used as a substrate and the hydrolysis activity at 100 ° C. for 5 minutes at 50 ° C. with a pH of 9.0 and a final concentration of 1.0% Triton X-100. It is the result of activity.

Table 3 shows the results when the yolk lecithin (L) was used as a substrate, the hydrolysis activity at 100 ° C. for 5 minutes at 50 ° C. with a pH of 5.6 and a final concentration of 1.0% Triton X-100. It is the result of relative activity.

According to the above “PLA1 activity standard measurement method” except that various phospholipids were used as a substrate instead of egg yolk lecithin (L), PLA1 was prepared in a reaction solution containing a final concentration of 0.005% Triton X-100. Activity was measured.

Table 4 shows the relative results when the yolk lecithin (L) is used as a substrate and the hydrolysis activity at pH 8.0, final concentration 0.23% Triton X-100 at 37 ° C. for 5 minutes is defined as 100%. It is the result of activity.

Tables 5 and 6 show the case where egg yolk lecithin (L) is used as a substrate and the hydrolysis activity at pH 7.2, final concentration 1% Triton X-100 at 50 ° C. for 5 minutes is defined as 100%. It is a result of relative activity with respect to various substrates. The composition of the reaction solution when the substrate is shown in Table 5 and the soybean oil and olive oil shown in Table 6 is shown in Table 7, and the substrates are pNPB, pNPO, pNPD, pNPL, pNPM, pNPP, pNPS (hereinafter “ The composition of the reaction solution in the case of “pNPB” is shown in Table 8.

The enzyme solutions in Table 7 are obtained by diluting 1279 U / ml (106.6 U / mg) enzyme solution 2000 times with 20 mM Tris-HCl buffer (pH 9.0). The enzyme activity before dilution was measured at pH 7.2 and 50 ° C. using egg yolk lecithin (L) as a substrate according to the above “PLA1 activity standard measurement method” (see Table 7).

To the reaction solution using soybean oil (containing a final concentration of 10 mM CaCl ₂ ) and olive oil (containing a final concentration of 10 mM CaCl ₂ ) as a substrate, 0.5 M EDTA was not added, but instead 0.1 M CaCl ₂ was added. The final concentration was adjusted to 10 mM CaCl ₂ by adding 10 μl.

The enzyme solutions in Table 8 are obtained by diluting the above 1279 U / ml (106.6 U / mg) enzyme solution 4000 times with 20 mM Tris-HCl buffer solution (pH 9.0).

When pNPB or the like is used as a substrate, 150 μl of ethanol is used as a stop solution for enzyme reaction at pH 7.2, 50 ° C. for 5 minutes, and the absorbance (A ₄₀₀ ) based on p-Nitrophenol at a wavelength of 400 nm is measured. The relative activity was calculated by Note that 14700 M ⁻¹ cm ⁻¹ was used as the molar extinction coefficient.

As described above, the enzyme obtained in Example 1 exhibited substrate specificity as shown in Tables 2 to 6, FIG. 7 and FIG.

(7) Molecular Weight According to the SDS-PAGE method (15% (w / v) polyacrylamide gel), the molecular weight of the purified enzyme obtained in Example 1 was measured. As a result, the molecular weight of the purified enzyme was about 28,000 ( FIG. 1). The unit is Da (Dalton).

(8) Isoelectric point As a result of predicting the isoelectric point of the enzyme based on the mature amino acid sequence excluding the secretory signal sequence of the enzyme using Genetyx, the isoelectric point of the enzyme was 5.72.

(9) Production of lysophospholipid (lyso form) Lysophospholipid was produced by allowing the purified enzyme obtained in Example 1 to act on L-α-phosphatidylcholine (PC) (manufactured by Nacalai Tesque, purity 98%). Table 9 shows the composition of the reaction solution.

The enzyme solutions in Table 9 are obtained by diluting 1279 U / ml (106.6 U / mg) enzyme solution 1500 times with 20 mM Tris-HCl buffer solution (pH 9.0). The enzyme activity of the enzyme solution before dilution was measured at pH 7.2 and 50 ° C. using egg yolk lecithin (L) as a substrate according to the above “PLA1 activity standard measurement method” (see Table 7). That is, the enzyme activity of the enzyme solution before dilution was measured by diluting 2000 times with egg yolk lecithin (L).

Specifically, the above reaction was performed at pH 7.2 and 37 ° C. Then, 50 μl of the reaction solution is taken out at regular time intervals (0 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes), and a solution of CHCl ₃ : CH ₃ OH (volume ratio 2: 1) is added to the reaction solution. Then, the reaction was stopped, the organic phase was extracted, the solvent was removed with a vacuum concentrator, and the lyso form was dissolved again with the solvent, and then analyzed by HPLC. The results are shown in FIGS. As is clear from FIGS. 9 to 11, a lysophospholipid with a yield of 16.5% was obtained in the reaction for 20 minutes. This yield was calculated from the mass (w / v%) of lysophospholipid (lyso form) generated from the mass 0.5 w / v% of the substrate (phospholipid) charged in the reaction solution shown in Table 9. . The breakdown is about 2-acyl-sn-glycero-3-phosphocholine (2-Acyl-sn-glycero-3-phosphocholine), 1-acyl-sn-glycero-3-phosphocholine (1-Acyl -sn-glycero-3-phosphocholine) was about 20%.

Table 10 shows the manufacturers and product numbers of the substrates described in this specification.

(10) Three-dimensional structure When the purified enzyme obtained in Example 1 was analyzed by X-ray crystal structure, the three-dimensional structure (ribbon model) of phospholipase A1 derived from Streptomyces albidoflavus is shown in FIG. 12A. It turned out to be something like this. The resolution of the X-ray crystal structure analysis was 2.2 mm. From this three-dimensional structure, it was found that the above phospholipase A1 has seven α-helices, six β-sheets, and three SS bonds (disulfide bonds). As shown in FIG. 12B, the above-mentioned phospholipase A1 is mainly Ser11, Ser216, His218 (the mature enzyme is from position 34 to position 269 of SEQ ID NO: 2, and therefore the amino acid residue at position 34 of SEQ ID NO: 2). When the group is counted as 1, Ser11, Ser216, and His218 are obtained. In SEQ ID NO: 2, Ser at position 44, Ser at position 249, and His at position 251 are located in the active center, and phospholipid hydrolysis reaction occurs. It is thought that it catalyzes. This was supported by the results that the mutant enzymes in which Ser11, Ser216, and His218 were substituted with each alanine lost the activity of the enzyme produced as an enzyme.

[Example 3: Analysis of N-terminal amino acid sequence of enzyme derived from Streptomyces albidoflavus]
Using the purified enzyme obtained in Example 1, the N-terminal amino acid sequence was analyzed with a protein sequencer. The internal amino acid sequence was analyzed by nanoLC-MS / MS. From the analysis, it was confirmed that the N-terminal amino acid sequence of this purified enzyme was as shown in SEQ ID NO: 3. The internal amino acid sequences were confirmed to be those shown in SEQ ID NOs: 4 and 5. SEQ ID NO: 3 is a sequence shown from position 34 of SEQ ID NO: 2 (positions 1 to 33 are secretory signal sequences that have been removed by the mature (purified) enzyme). SEQ ID NO: 4 is the sequence shown from position 145 of SEQ ID NO: 2. SEQ ID NO: 5 is the sequence shown from position 165 of SEQ ID NO: 2.

[Example 4: Isolation of chromosomal DNA of Streptomyces albidoflavus NA297]
Streptomyces albidoflavus NA297 was added to YEME medium (0.3% yeast extract, 0.5% peptone, 0.3% malt extract, 1% glucose, 34% sucrose, 5 mM MgCl ₂ , 0.5 % Glycine) was 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.

3 ml of isopropanol was added to and mixed with this aqueous phase to collect the DNA fraction, which was dissolved in 500 μl of a solution consisting of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA. RNase A was added to 20 μg / ml and treated at 37 ° C. for 1 hour. Then, 500 μl of 13% PEG solution containing 0.8 M NaCl was added and stirred, and 500 μl of aqueous phase was collected by centrifugation. . 500 μl of a phenol / chloroform mixed solution was added thereto and stirred, and 500 μl of an 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.

The DNA was immersed in 70% (v / v) ethanol for 10 minutes and then dissolved in 200 μl of a solution composed of 10 mM Tris-HCl (Tris-HCl) buffer (pH 8.0) and 1 mM EDTA.

[Example 5: Cloning of the core region of PLA1 gene derived from Streptomyces albidoflavus NA297]
Based on the N-terminal and internal amino acid sequences of PLA1 and the codons used in the genus Streptomyces, degenerate oligonucleotide primer for PCR S1 sense primer “primer S1” (SEQ ID NO: 6), and three types of A1 antisense primers “Primer A1-1” (SEQ ID NO: 7) and “primer A1-2” (SEQ ID NO: 8) were designed. Here, s in the sequence represents c or g, w represents a or t, and k represents g or t.

The PCR reaction solution composition is as follows.

Distilled water was added to 50 μl of template chromosomal DNA 50 ng, 2 × MightyAmp Buffer 25 μL obtained in Example 4, 300 nM of each primer, and MightyAmp DNA Polymerase 1.25 unit.

PCR reaction conditions are as follows.

Step 1: 98 ° C., 2 minutes;
Step 2: 98 ° C., 10 seconds;
Step 3: 65 ° C., 15 seconds;
Step 4: 68 ° C., 1 minute;
Repeat steps 2 to 4 for 30 cycles;
Step 4: 68 ° C., 1 minute.

A specific amplification product of about 300 bp was obtained by PCR using SEQ ID NOs: 6-8.

The PCR reaction solution was subjected to agarose gel electrophoresis, the target 300 bp band portion was cut out, and bound to pMD20-T Vector using pMD20-T Vector (manufactured by TAKARA) to transform Escherichia coli. The transformed strain is cultured in LB medium (tryptone 1%, yeast extract 0.5%, sodium chloride 0.4%, pH 7.2) containing ampicillin 50 μg / ml, and using Miniprep DNA Purification Kit (TaKaRa). Plasmid for DNA sequencing was extracted and purified.

Subsequently, the base sequence of the inserted fragment was determined by an automatic sequencer using M13 primerM4 derived from a vector (pMD20-T Vector). This base sequence is shown in SEQ ID NO: 9.

[Example 6: Cloning around the core region of PLA1 gene derived from Streptomyces albidoflavus NA297]
In order to clarify the sequence of the peripheral region of the gene sequence determined in Example 5, a DNA fragment including the upstream side and the downstream side was amplified by inverse PCR.

The chromosomal DNA obtained in Example 4 was completely digested with SacII, and Ligation high Ver. 2 (manufactured by Toyobo Co., Ltd.). Using this as a template, Inverse PCR using sense primer SE1 “primer SE1” (SEQ ID NO: 10) and antisense primer AN1 “primer AN1” (SEQ ID NO: 11) prepared based on the partial gene sequence of phospholipase A1 Went.

The PCR reaction solution composition is as follows.

Distilled water was added to a template DNA of 200 ng, 2 × MightyAmp Buffer 25 μl, each primer 300 nM, and MightyAmp DNA Polymerase 1.25 unit to a total volume of 50 μl.

PCR reaction conditions are as follows.

Step 1: 98 ° C., 2 minutes;
Step 2: 98 ° C., 10 seconds;
Step 3: 68 ° C., 4 minutes;
Repeat steps 2 to 3 for 30 cycles;
Step 4: 68 ° C., 7 minutes.

By this PCR, a specific amplification product of about 3,000 bp was obtained, which was cloned with the pMD20-T vector (manufactured by TaKaRa) and the base sequence was determined.

Based on the base sequence determined above together with the base sequence determined in Example 5, the base sequence on the 5 'region side of the region containing the PLA1 gene derived from Streptomyces albidoflavus was determined. Further, the amino acid sequence of the structural gene portion was deduced from the base sequence (SEQ ID NO: 12).

[Example 7: Cloning around the core region of PLA1 gene derived from Streptomyces albidoflavus NA297]
Since the digestion site of SacII used for inverse PCR was contained in the PLA1 gene, the 3 ′ region side (C-terminal side) was determined only halfway. Therefore, in order to clarify the full-length sequence of the PLA1 gene sequence determined in Examples 5 and 6, the amino acid sequence which showed 100% homology with BLAST (Lipase_GDSL (DDBJ accession estimated by genome analysis of Streptomyces albus J1074) The base sequence was identified from the number D6BAL1)), the primer A2 (SEQ ID NO: 13) on the 3 ′ region side (C-terminal side) was designed, and the DNA fragment was amplified by PCR with the S1 primer (SEQ ID NO: 6).

The PCR reaction solution composition is as follows.

PCR reaction conditions are as follows.

Step 1: 98 ° C., 2 minutes;
Step 2: 98 ° C., 10 seconds;
Step 3: 68 ° C, 1 minute;
Repeat steps 2 to 3 for 30 cycles;
Step 4: 68 ° C., 5 minutes.

This PCR yielded a specific amplification product of about 700 bp, which was cloned with the pMD20-T vector (TaKaRa) and the base sequence was determined.

Based on the base sequence determined above in combination with the base sequences determined in Examples 5 and 6, the base sequence of the region containing the PLA1 gene derived from Streptomyces albidoflavus NA297 was determined (FIG. 6A and FIG. 6). 6B (hereinafter collectively referred to as FIG. 6)). Further, the amino acid sequence of the structural gene portion was deduced from the base sequence (FIG. 6, SEQ ID NOs: 14 and 15).

FIG. 6 shows the base sequence of the region containing the determined PLA1 gene, and the deduced amino acid sequence of the structural gene deduced from this base sequence. Is shown. The arrangement in FIG. 6 is DDBJ ACCESSION No. It is registered privately as AB605634.

From the results of sequence analysis shown in FIG. 6, it was revealed that the structural gene encoding PLA1 is composed of 807 bp nucleotides and encodes 269 amino acids. 236 residues are positions 34 to 269 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6). Positions 1-33 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6) are a secretory signal sequence.

The N-terminal and internal amino acid sequences of the purified enzyme derived from Streptomyces albidoflavus NA297 determined in Example 3 were present in the above deduced amino acid sequence and were completely consistent (see FIG. 6). Underlined).

That is, the amino acid sequence of SEQ ID NO: 3 is a sequence shown from position 34 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6). The amino acid sequence of SEQ ID NO: 4 is the sequence shown from position 145 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6). The amino acid sequence of SEQ ID NO: 5 is the sequence shown from position 165 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6).

By using the sequence similarity search program BLAST, the above deduced amino acid sequences were compared with sequences in four types of protein sequence databases (PTR, PRF, UNI-PROT and SWISS-PROT). As a result, the mature enzyme excluding the secretory signal sequence at positions 1 to 33 of the amino acid sequence of SEQ ID NO: 14 (FIG. 6) among the above estimated amino acid sequences is Lipase_GDSL (DDBJ accession) estimated by genome analysis of Streptomyces albus J1074. No. D6BAL1) was 100% consistent. However, the second and third residues at the N-terminal of the signal sequence were different.

Also, the purified enzyme derived from Streptomyces albidoflavus (ie, PLA1) was calculated based on the amino acid composition of this deduced amino acid sequence, and was estimated to have a molecular weight of 27,199.

[Example 8: Preparation of recombinant plasmid containing PLA1 gene derived from Streptomyces albidoflavus]
In order to express PLA1 derived from Streptomyces albidoflavus in actinomycetes, a recombinant plasmid used for transformation was prepared.

The restriction enzyme sites that can be used for recombination were NheI and BglII, but the BglII recognition sequence was replaced by the following method because the BglII site was included in the structural gene of PLA1 derived from Streptomyces albidoflavus. . First, a sense primer C1 “primer C1” (SEQ ID NO: 16) in which an NheI site is added to the upstream region of the structural gene of PLA1 derived from Streptomyces albidoflavus, and a site containing the BglII site in this structural gene Antisense primer C2 “primer C2” (SEQ ID NO: 17) was designed. The antisense primer C2 “primer C2” was designed so that the BglII recognition sequence was eliminated and the designated amino acid was not changed. That is, the 696th g in SEQ ID NO: 1 was substituted with a (the designated amino acid remained glutamic acid). Subsequently, PCR was performed using these primers with the chromosomal DNA obtained in Example 4 as a template.

The PCR reaction solution composition is as follows.

Template chromosomal DNA 200 ng, 10 × PCR Buffer 2.5 μl, primers 1200 nM each, dNTP mixture 0.3 mM, MgCl ₂ 1.2 mM, DMSO 4%, and KOD DNA Polymerase 1.25 units, distilled water 25 μl in total It added so that it might become.

PCR reaction conditions are as follows.

Step 1: 98 ° C., 2 minutes;
Step 2: 98 ° C., 15 seconds;
Step 3: 72 ° C., 2 seconds;
Step 4: 74 ° C., 25 seconds;
Repeat steps 2 to 4 for 30 cycles;
Step 5: 74 ° C., 10 seconds.

A specific amplification product of about 600 bp was obtained by this PCR.

Similarly, sense primer C3 “primer C3” (SEQ ID NO: 18) designed to replace the BglII recognition sequence in this structural gene, and an antisense primer in which a BglII site is added to the downstream sequence of the PLA1 structural gene C4 “primer C4” (SEQ ID NO: 19) was designed. Subsequently, PCR was performed using these primers with the chromosomal DNA obtained in Example 4 as a template.

The PCR reaction solution composition is as follows.

PCR reaction conditions are as follows.

A specific amplification product of about 100 bp was obtained by this PCR.

Next, a sense primer C1 “primer C1” (SEQ ID NO: 16) with an NheI site added to the upstream region sequence of the PLA1 structural gene, and an antisense primer C4 “with a BglII site added to the downstream region sequence of the PLA1 structural gene. Using “primer C4” (SEQ ID NO: 19), PCR was performed using the amplification products of about 600 bp and about 100 bp obtained by the above PCR as templates.

The PCR reaction solution composition is as follows.

Template 600 bp DNA 300 ng, Template 100 bp DNA 340 ng, 10 × PCR Buffer 2.5 μl, Primer 1200 nM, dNTP mixture 0.3 mM, MgCl ₂ 1.2 mM, DMSO 4%, and KOD DNA Polymerase 1.25 units, distilled water The total amount was 25 μl.

PCR reaction conditions are as follows.

Step 1: 98 ° C., 2 minutes;
Step 2: 98 ° C., 15 seconds;
Step 3: 72 ° C., 2 seconds;
Step 4: 74 ° C., 25 seconds;
Repeat steps 2 to 4 for 35 cycles;
Step 5: 74 ° C., 10 seconds.

A specific amplification product of about 700 bp was obtained by this PCR.

The amplified fragment was digested with NheI and BglII and inserted into the NheI-BglII site of the actinomycete plasmid as an expression vector to obtain a recombinant plasmid. Here, the signal sequence and the terminator are those of the phospholipase enzyme gene, but those derived from other genes may be used in appropriate combination.

[Example 9: Production of recombinant actinomycetes expressing PLA1 gene derived from Streptomyces albidoflavus NA297]
Using the recombinant plasmid obtained in Example 8, protoplastized Streptomyces lividans (Streptomyces lividans) according to the method described in “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000)”. 1326 was transformed to obtain recombinant actinomycetes.

[Example 10: Measurement of enzyme activity of recombinant actinomycetes expressing PLA1 gene derived from Streptomyces albidoflavus NA297]
The recombinant actinomycetes obtained in Example 9 were cultured in four 100 mL tryptic soy media (Becton 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 recovered precipitate is dissolved in 20 mM Tris-HCl (Tris-HCl) buffer (pH 8.0), dialyzed using 20 mM Tris-HCl (Tris-HCl) buffer (pH 8.0) as an external solution, and then enzyme solution. Got.

The enzyme activity of this enzyme solution was measured at pH 5.6 and 50 ° C. using egg yolk lecithin (L) as a substrate according to the above “PLA1 activity standard measurement method”. As a result, a culture supernatant showing strong activity exceeding 100 U / ml (lower limit is 60 U / ml) was obtained. Moreover, the enzyme activity (80 mL) after ammonium sulfate fractionation showed a stronger activity (244 U / ml or more, the lower limit was 200 U / ml). In addition, PLA1 activity was not detected in actinomycetes transformed using the plasmid as a vector as it was.

According to the present invention, a novel PLA 1 and a manufacturing method thereof are provided. Glycerol-3-phosphate compounds such as lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphocholine produced by causing PLA1 to act on phospholipids are useful as base materials for foods and cosmetics. is there. The enzyme according to the present invention works particularly well on PI, PG, PS, DOPE, L at pH 5.6 and on PI, PG, PS, DMPA at pH 9.0. In addition, lysophosphatidylinositol (LPI) is an antifungal agent, lysophosphoglycerol (LPG) is a foaming stabilizer, starch aging inhibitor, noodle modifier, lysophosphatidylethanolamine (LPE): neurotrophic action ( Stress, depression, dementia, etc.), lysophosphatidic acid (LPA): promotion of fertilized egg implantation (implantation improving agent), hair formation (hair growth), strong cell proliferation action, lysophosphatidylserine (LPS): mast cell It is effective in producing lysolecithin that exhibits many physiological activities such as promoting the activation of (involved in allergy and atopy). Further, by mixing the enzyme according to the present invention with a food material and hydrolyzing phospholipids present in the material, it becomes possible to enhance the functionality or improve the physical properties of the material. Furthermore, glycerol-3-phosphocholine (GPC), which can be produced using the enzyme according to the present invention, is expected to prevent dementia and the like. Glycerol-3-phosphoinositol (GPI) can be expected to be useful because it has an effect of increasing good cholesterol.

Name of Contracting Organization: National Institute for Product Evaluation Technology Patent Microbiology Depositary Center (NPMD)
Contracted organization address: Japan 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan
Date of entrustment: January 26, 2011 Accession number: NITE BP-1014

SEQ ID NO: 1: PLA1 gene SEQ ID NO: 2: Full length of PLA1 (polypeptide) SEQ ID NO: 3: N-terminal sequence of PLA1 SEQ ID NO: 4: Internal sequence of PLA1 SEQ ID NO: 5: Internal sequence of PLA1 SEQ ID NO: 6: Primer S1
SEQ ID NO: 7: Primer A1-1
SEQ ID NO: 8: Primer A1-2
SEQ ID NO: 9: internal sequence of PLA1 gene SEQ ID NO: 10: primer SE1
Sequence number 11: Primer AN1
SEQ ID NO: 12: part of PLA1 (polypeptide) SEQ ID NO: 13: primer A2
SEQ ID NO: 14: PLA1 gene SEQ ID NO: 15: Full length of PLA1 (polypeptide) SEQ ID NO: 16: Primer C1
Sequence number 17: Primer C2
SEQ ID NO: 18: Primer C3
SEQ ID NO: 19: Primer C4

Claims

An enzyme comprising the polypeptide according to any one of the following (a) to (c) and having the following physicochemical properties (1) and (2):
(A) A polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.
(B) A polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2.
(C) a polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2.
(1) Action By preferentially hydrolyzing the sn-1 position acyl group of phospholipid, one or more selected from lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate compound Generate.
(2) Substrate specificity When egg yolk lecithin (L) is used as a substrate and the hydrolysis activity under the condition of pH 7.2 and 50 ° C. for 5 minutes is defined as 100%,
50% or more based on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE),
500% or more based on 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate (POPA),
150% or more based on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
300% or more based on L-α-phosphatidylinositol (PI),
300% or more based on L-α-phosphatidyl-L-serine (PS),
100% or more based on 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac- (1-glycerol) (POPG),
100% or more based on soybean-derived L-α-phosphatidylcholine (SB-PC),
30% or more of soybean lecithin (SBL)
250% or more based on L-α-lysophosphatidylcholine (LPC),
50% or more based on 1- (1Z-octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PlsPE (C18, 20: 4)),
200% or more based on 1-hexadecyl-2- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (PlsPE (C16, 18: 1)),
0% to 1- (1Z-octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphocholine (PlsPC (C18, 20: 4)),
10% or less of soybean oil and olive oil,
4-nitrophenyl butyrate (pNPB), 4-nitrophenyl octanoate (pNPO), 4-nitrophenyl decanoate (pNPD), 4-nitrophenyl laurate (pNPL), 4-nitrophenyl myristate (pNPM) ), 4-nitrophenyl palmitate (pNPP) and 4-nitrophenyl stearate (pNPS).
When egg yolk lecithin (L) is used as a substrate and the hydrolysis activity under the conditions of pH 5.6 and 37 ° C. for 5 minutes is defined as 100%, 50% or more of water is added within the range of pH 4.1 to pH 10.0. The enzyme according to claim 1, which exhibits a degrading activity.
Furthermore, the enzyme of Claim 1 or 2 which has the following substrate specificities.
When egg yolk lecithin (L) is used as a substrate and the hydrolysis activity under the condition of pH 9.0 and 50 ° C. for 5 minutes is defined as 100%,
50% or less based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
350% or more based on 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA),
400% or more based on 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (PG),
450% or more based on L-α-phosphatidylserine (PS),
100% or more based on 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
350% or more based on L-α-phosphatidylinositol (PI),
More than 300% of soybean phosphatidylcholine (SB-PC)
It has a relative activity of 75.0% or more with respect to soybean lecithin (SBL).
Furthermore, the enzyme as described in any one of Claims 1 thru | or 3 which has the following substrate specificity.
When egg yolk lecithin (L) is used as a substrate and the hydrolysis activity under the condition of 5 minutes at 50 ° C. at pH 5.6 is 100%,
100% or more based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
100% or less based on 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA),
150% or more based on 1,2-diacyl-sn-glycero-3-phospho- (1-rac-glycerol) (PG),
150% or more based on L-α-phosphatidylserine (PS),
100% or more based on 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
250% or more based on L-α-phosphatidylinositol (PI),
More than 250% of soybean phosphatidylcholine (SB-PC)
Has a relative activity of 250% or more with soybean lecithin (SBL),
Relative activity to triglycerides is 0%.
The enzyme according to any one of claims 1 to 4, wherein the isoelectric point calculated from the amino acid sequence is in the range of 5.7 to 5.8.
6. The molecular weight as measured by SDS-PAGE is in the range of 25,000 to 30,000, and the molecular weight as analyzed from the amino acid composition is in the range of 25,000 to 30,000. The enzyme as described in any one of these.
The enzyme according to any one of claims 1 to 6, which is derived from a microorganism belonging to the genus Streptomyces.
A polynucleotide encoding the enzyme according to any one of claims 1 to 7.
The polynucleotide according to claim 8, comprising the polynucleotide according to any one of the following (a) to (c).
(A) A polynucleotide comprising the base sequence set forth in SEQ ID NO: 1.
(B) A polynucleotide that hybridizes with a base sequence complementary to the base sequence described in SEQ ID NO: 1 under stringent conditions.
(C) a polynucleotide having at least 80% sequence identity to the base sequence set forth in SEQ ID NO: 1.
The polynucleotide according to claim 8 or 9, which is derived from a microorganism belonging to the genus Streptomyces.
A vector comprising the polynucleotide according to any one of claims 8 to 10.
A polynucleotide according to any one of claims 8 to 10 or a vector according to claim 11 is introduced, and from phospholipid to lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate ester compound A transformant having the ability to produce an enzyme that produces one or more selected from the group consisting of
The transformant according to claim 12, wherein the host of the transformant is a microorganism belonging to the genus Streptomyces.
This is a method for producing an enzyme that acts on phospholipid and hydrolyzes the phospholipid to produce one or more selected from lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate ester compound. And
A step of introducing the polynucleotide according to any one of claims 8 to 10 or the vector according to claim 11 into a host to obtain a transformant that produces the enzyme; and culturing the transformant And producing the enzyme as described above;
Manufacturing method.
The production method according to claim 14, wherein the host is a microorganism belonging to the genus Streptomyces.