JP2010200690A - New sphingolipid and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new sphingolipid and a method for producing the lipid. <P>SOLUTION: There are provided the new sphingolipid expressed by general formula (1) and obtained by being extracted from a cultured cell of a marine eukaryotic microorganism, specifically Labyrinthula, more specifically Thraustochytrium aureum, and purifying the extract, and the method for producing the lipid. In the formula, m is 15-n when n is 1 to 14, or m is 16-n when n is 1 to 15. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel sphingolipid and a method for producing the same. Specifically, the sphingolipid of the present invention is a ceramide to which phosphorylethanolamine is bound, and relates to a method for producing the sphingolipid from Labyrinthula, which is a marine eukaryotic microorganism.

  Sphingolipid refers to a lipid having a sphingosine skeleton, and is known to be widely distributed in mammalian plasma membranes. Many sphingolipids are present in the outer envelope of the plasma membrane and form a plasma membrane microregion (raft), which is thought to be involved in signal transduction between cells. Sphingomyelin (SM) conjugated with ceramide and choline phosphate is widely distributed in mammals and is metabolized not only to raft formation but also to ceramide and sphingosine 1-phosphate (S1P). (Non-Patent Document 1). In addition, ethanolamine phosphoceramide (EPC) bound with phosphorylethanolamine instead of choline phosphate is a freshwater snail (Non-patent Document 2), insects including Drosophila (Non-patent Document 3), and some It is reported that it is widely distributed in bacteria (Non-patent Document 4), eukaryotic microorganisms trypanosome (Non-patent Document 5), Paramecium (Non-patent Document 6), and the like. This EPC has a role as a precursor of SM (Non-patent Document 7) and a raft as an SM isotype, suggesting the possibility of signal transduction (Non-patent Document 3). In addition, as a result of EPC deficiency experiments conducted in Drosophila, individuals with a 70% reduction in EPC were confirmed to have reduced life span, membrane tissue changes, membrane lipids, and membrane protein oxidation associated with premature aging (non-patented) Reference 8). This result suggests that EPC may play an important role in the suppression of aging, and the use of EPC as a functional lipid is expected.

On the other hand, Labyrinthulas have been reported to produce and accumulate abundant DHA (Patent Document 1, Non-Patent Document 9), and may have unique lipid metabolism pathways and membrane functions. Examples of the technique using Labyrinthula as a source of DHA include, for example, a technique utilizing S3-2 strain (accession number FERM BP-5034) which is a microorganism belonging to the genus Labyrinthula (see Patent Documents 2 and 3), Schizochytrium genus SR21 strain (FERM BP-5034) and its utilization technology (refer to Patent Documents 4 to 6) and the like.
However, in Labyrinthula, sphingolipids that play an important role in membrane function as described above have hardly been analyzed. In addition, since there is a report that the metabolism of glycerophospholipid and SM interacts closely in human neurons (Non-patent Document 1), the dynamics of sphingolipid affects the metabolism and function of DHA. The possibility of giving Therefore, elucidating the molecular species and function of labyrinthula sphingolipids is indispensable for investigating the function of membrane lipids in labyrinthula.

JP 2007-143479 A JP 2001-275656 A Japanese Patent Laid-Open No. 2003-000292 JP-A-9-000284 Japanese Patent Laid-Open No. 10-072590 JP-A-10-310556 JP 2005-287380 A

Akhlaq A. Farooqui, Lloyd A. Horrochs, Tahira Farooqui. J. Neurosci. Res 85, 1834-1850 (2007) Rikukai Sugita: Oreoscience 653, 13-21 (2003) Anton Rietveld, et.al. J. Bio. Chem. 274, 12049-12054 (1999) Takashi Naka, et al. Biochimi. Biophys. Acta1635, 83-92 (2003) Shaheen S. Sutterwala, et. Al. Molecular Microbiology 70, 281-296 (2008) Enda S. Kaneshiro, et. Al. Jounalof Lipid Research 38 2399-2410 (1997) Monique Malgat, et.al.jounal of Lipid Research 27 251-260 (1986) Raghavendra Pralhada Rao, et.al.PNAS 104 11364-11369 (2007) Barbara B. Ellenbogen, S. Aaronson, Comp. Biochem. Physiol. 29, 805 (1969)

  An object of the present invention is to provide a novel sphingolipid and a method for producing the same. Specifically, it is an object to provide a method for producing the sphingolipid from the sphingolipid represented by Chemical Formula 1 and Labyrinthulas.

  As a result of intensive studies on the sphingolipids of Labyrinthula in view of the above-mentioned situation, the present inventors have found a novel sphingolipid and have completed the present invention.

Accordingly, the gist of the present invention is the sphingolipid described in the following (1)).
(1) A sphingolipid represented by the following general formula (1).
(Here, when n = 1-14, m = 15-n or when n = 1-15, m = 16-n)

Moreover, this invention makes a summary the manufacturing method of the sphingolipid of the following (2)-(4) description.
(2) In the production of the sphingolipid according to (1), a marine eukaryotic microorganism is cultured and purified.
(3) The method for producing a novel sphingolipid according to (2) above, wherein the marine eukaryotic microorganism is Labyrinthula.
(4) labyrinthulean is Thraustochytrium Aureumu a (Thraustochytrium aureu m, hereinafter sometimes abbreviated as "T. Aureum".), Wherein (3) new sphingolipids method according.
(4) The method for producing a novel sphingolipid according to (3) or (4), wherein the labyrinthula is Thraustochytrium aureum ATCC34304 ( Thraustochytrium aureum ATCC34304).

  According to the present invention, novel sphingolipids are provided, and substances useful for pharmaceuticals, cosmetics, foods and the like are provided.

The result of the weak alkali degradation test of the lipid extracted from T. aureum in Example 1 is represented (in the figure, PC: phosphatidylcholine, GalCer: galactosylceramide (sphingoglycolipid), FA: fatty acid). The results of SCDase treatment of the phospholipid fraction of T. aureum in Example 2 are shown (in the figure, SM: Sphingomyelin, C12 NBD-FA: (I) TLC colored with ninhydrin reagent, (II) transillumination TLC). The result of SCDase treatment of the glycolipid fraction of T. aureum in Example 2 is shown ((1): glycolipid fraction SCDace treatment, (2): boiling treatment of glycolipid fraction, (3): Sphingomyelin SCDace treatment, (4): Sphingomyelin boiling treatment, SM: Sphingomyelin, (I): Hydrolyzed TLC, (II): TLC after condensation reaction). The separation results of alkali-resistant phospholipids in Example 3 are shown ((I) color development with 50% sulfuric acid, (II) color development with dittmer reagent). The separation result by normal phase HPLC of the alkali-resistant phospholipid in Example 3 is shown (A: peak A in the figure). The result of weak alkali decomposition test of peak A in Example 4 is shown (in the figure, A: peak A, PC: phosphatidylcholine, SM: sphingomyelin, FA: fatty acid, +: alkaline decomposition,-: alkali Undecomposed; developed with 50% sulfuric acid). The results of SCDase treatment of peak A in Example 4 are shown (in the figure, SM: sphingomyelin, 1: peak A SCDace treatment, 2: peak A boiling enzyme treatment, 3: sphingomyelin SCDace treatment, 4: sphingomyelin boiling Enzyme treatment, (I): hydrolysis reaction, (II): condensation reaction). The confirmation result of the functional group by TLC of peak A in Example 5 is shown (in the figure, A: peak A, PC: phosphatidylcholine, PE: phosphatidylethanolamine, SM: sphingomyelin, (I): color development by Dittmer reagent, (II): Color development by ninhydrin reagent). The separation result of this invention lipid by reverse phase HPLC in Example 6 is shown. The FAB-MS analysis result of the peak 1 in Example 6 is shown. The FAB-MS analysis result of the peak 2 in Example 6 is shown.

The present invention relates to a novel sphingolipid represented by the following general formula (1).
(Here, when n = 1-14, m = 15-n or when n = 1-15, m = 16-n)

The novel sphingolipid of the present invention can be obtained by culturing, extracting and purifying marine eukaryotic microorganisms. Specifically, marine eukaryotic microorganisms are Labyrinthulas. Labyrinthula, Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Examples include the genus Thraustochytrium, Ulkenia, Aurantiochytrium, preferably microorganisms belonging to the genus Thraustochytrium, particularly preferably Thraustochytrium aurem It is done. These may be strains handed over from a storage organization, or subcultures thereof. Thraustochytrium Au les um ATCC28211, Thraustochytrium Aureumu ATCC34304, Schizochytrium (Schizochytorium) sp. M-8 (FERM P-19755, refer to Patent Document 7) strain are exemplified.
The novel sphingolipid of the present invention can be obtained by culturing the marine eukaryotic microorganism, performing extraction and purification operations.

  In order to obtain the novel sphingolipids of the present invention, marine eukaryotic microorganisms are cultured. Culturing is carried out by a conventional method using a commonly used solid medium or liquid medium. Examples of the medium used at this time include glucose, fructose, saccharose, starch, glycerin and the like as the carbon source, and yeast extract, corn steep liquor, polypeptone, sodium glutamate, urea, ammonium acetate, ammonium sulfate, ammonium nitrate, and chloride as the nitrogen source. It is a medium in which ammonium, sodium nitrate, etc. and other necessary components such as potassium phosphate as an inorganic salt are appropriately combined, and is not particularly limited as long as it is usually used for culturing Labyrinthula, but particularly preferably Yeast extract / glucose medium (GY medium) is used. After preparation, the medium is adjusted to a pH of 3.0 to 8.0 and then sterilized by an autoclave or the like. The culture may be performed at 10 to 40 ° C., preferably 15 to 35 ° C., for 1 to 14 days by aeration and agitation culture, shaking culture, or stationary culture.

Next, the cultured microorganisms are dried by an appropriate drying method, and lipids are extracted therefrom. At this time, freeze drying is particularly preferably used as a drying method. Lipids are extracted from the lyophilized cells using an appropriate method.
The lipid extraction method is not particularly limited as long as it is a commonly used method, but a solvent extraction, pH adjustment or separation or removal of protein components by enzyme treatment, supercritical extraction, or a combination of these is used. Preferably, extraction with an organic solvent, enzymatic treatment followed by extraction with an organic solvent, or supercritical carbon dioxide extraction is used.
As a solvent used in the solvent extraction, an appropriate organic solvent, for example, alcohols such as methanol, ethanol, propanol, isopropanol, butanol, propylene glycol, butylene glycol, methyl acetate, ethyl acetate, acetone, chloroform, toluene, pentane, hexane, Cyclohexane or the like can be used alone or in combination of two or more. Preferably, the lipid is extracted using a chloroform / methanol mixture. At this time, the solvent mixing ratio or the bacterial cell amount: solvent ratio may be set arbitrarily, but extraction is particularly preferably performed using a Folch partition, that is, a solvent of chloroform / methanol = 2: 1.

  The lipid extracted in this manner is subjected to a weak alkali decomposition treatment, followed by Folch partitioning, and the lower layer is recovered. At this time, weak alkaline decomposition may be carried out by adding 0.1 to 10 ml of 0.01 to 0.5 N potassium hydroxide or sodium hydroxide per 5 mg of lipid and incubating at 30 to 50 ° C. for 1 to 24 hours. The lower layer of Folch is then separated by normal phase chromatography. At this time, a silica column is used for the column, and the solvent is passed in the order of chloroform, acetone, and methanol. By this operation, the target phospholipid is eluted in the methanol fraction. Next, by subjecting the obtained phospholipid fraction to normal phase high performance liquid chromatography (HPLC), the target substance can be obtained from a peak at a retention time of about 23 minutes. Furthermore, each molecular species can be separated by using reverse phase HPLC. At this time, the novel sphingolipid of the present invention can be obtained particularly preferably by using an ODS column and collecting peaks at retention times of around 22 minutes and 27 minutes.

  The novel sphingolipid of the present invention thus obtained is useful as a raw material for pharmaceuticals, cosmetics, foods and the like.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

[Weak alkaline degradation test of neutral lipid, glycolipid and phospholipid fractions of T. aureum]
First, the cells were cultured and collected. That is, Thraustochytrium aureum ATCC 34304 cells were cultured with shaking in 200 ml of GY medium (prepared with the composition shown in Table 1) at 25 ° C. for 4 days, and then centrifuged at 8,000 rpm for 5 minutes to remove the supernatant. Add an appropriate amount of 0.9% NaCl and suspend well, then centrifuge at 8,000 rpm for 5 minutes to remove the supernatant, add an appropriate amount of ultrapure water, suspend well, and then centrifuge at 8,000 rpm for 5 minutes. After removing, the cells were collected and lyophilized.

  Next, total lipids were extracted from the collected cells by Folch partitioning. That is, 20 ml of chloroform / methanol = 2/1 was added to the cells, and the cells were crushed with ultrasonic waves, followed by centrifugation at 1,800 rpm for 5 minutes, and the supernatant was collected. After repeating this three times, 15 ml of 0.9% NaCl was added, mixed well and allowed to stand overnight at 4 ° C., and the lower layer was collected and concentrated on a rotary evaporator.

  Next, the obtained lipid was separated into each lipid fraction. That is, Sep-Pak-Silica (manufactured by Waters) was equilibrated with 30 ml of chloroform, and the concentrated and dried total lipid was dissolved in 2 ml of chloroform and loaded onto the column. At this time, 30 ml of chloroform, 50 ml of acetone, and 20 ml of methanol were eluted in this order, and 10 ml fractions were collected. Lipids contained in the collected fractions were confirmed by thin layer chromatography. At this time, n-hexane / ether / acetic acid = 80/20/1 (v / v / v) was used as a developing solvent for neutral lipids, and chloroform / methanol / water = 65/25 / for glycolipids and phospholipids. 4 (v / v / v) was used. Further, 50% sulfuric acid, orcinol sulfuric acid, and Dittmer reagent (manufactured by Sigma) were used as the coloring reagent. Furthermore, galactosylceramide (GalCer) as a positive control, phosphatidylcholine (PC) as a negative control, and fatty acid (FA, specifically, linoleic acid (manufactured by sigma)) as a control were simultaneously loaded.

  Next, each of the collected lipid fractions was concentrated with an evaporator and subjected to a weak alkaline decomposition test to confirm the presence of sphingolipids. That is, about 0.5 mg was taken from each of the collected lipid fractions, concentrated with N2 gas, 100 μl of 0.1 N KOH was added, and the mixture was dissolved by ultrasonication, followed by incubation at 37 ° C. overnight. To this was added 1 μl of 10 N acetic acid for neutralization, 240 μl of chloroform and 240 μl of 0.9% NaCl were added, and the mixture was mixed well with a vortex mixer and centrifuged at 15,000 rpm for 3 minutes to recover the lower layer. This was confirmed by thin layer chromatography (TLC). At this time, chloroform / methanol / water = 65/25/4 was used as a developing solvent, and 50% sulfuric acid was used as a coloring reagent. The results are shown in FIG.

  As a result of separation using a sep pak silica column, it was found that the chloroform fraction contained neutral lipids, the acetone fraction contained glycolipids, and the methanol fraction contained phospholipids. Therefore, each fraction was made into a neutral lipid fraction, a glycolipid fraction, and a phospholipid fraction, and each of the fractions was subjected to a weak alkaline decomposition treatment. From the glycolipid fraction and the phospholipid fraction, The presence of a band that was not decomposed by alkali was confirmed (FIG. 1). From this result, it was suggested that sphingolipid may exist in the glycolipid fraction and the phospholipid fraction.

[SCDase treatment of glycolipid and phospholipid fraction of T. aureum]
In order to confirm whether the alkali-resistant lipid present in the glycolipid and phospholipid fractions suggested in Example 1 is a sphingolipid, a hydrolysis reaction using SCDase and a fatty acid condensation reaction were performed.
That is, about 50 μg of the phospholipid fraction obtained in Example 1 was taken, 5 μl of 2% triton X-100 (manufactured by Sigma) was added, and the mixture was concentrated by a centrifugal evaporator. Next, 5 μl of 4 × reaction buffer 1 (100 mM sodium acetate buffer (pH 5.5), 20 mM CaCl ₂ ) and 10 μl of ultrapure water were added for sonication. 5 μl of sphingolipid ceramide deacylase (SCDase) (from Shewanella alga G8, 7 mU, Furusato, M. et. Al. (2002) J. Biol. Chem. 277, 17300-17307) And mixed well with a vortex mixer. This was incubated overnight at 37 ° C., concentrated and dried with a centrifugal evaporator, dissolved in 5 μl of chloroform / methanol = 2/1, and the reaction product was confirmed by TLC. At this time, chloroform / methanol / 0.02% CaCl ₂ = 5/4/1 (v / v / v) was used as a developing solvent, and a ninhydrin reagent was used as a coloring reagent. The results are shown in FIG.

Next, a condensation reaction of C12 NBD-fatty acid with SCDase was performed. That is, about 50 μg of the glycolipid fraction obtained in Example 1 was taken, and 5 μl of 0.4% triton X-100, 1 nmol 12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl ) Amino) dodecanoic acid (C12 NBD-FA Molecular Probes) was added and concentrated with a centrifugal evaporator. 5 μl of 4 × reaction buffer 2 (100 mM Tris-HCl buffer (pH 7.5), 20 mM MgCl ₂ ) and 10 μl of ultrapure water were added to the concentrate for sonication. 5 μl of SCDase (7 mU) was added thereto and mixed well with a vortex mixer. This was incubated overnight at 37 ° C., concentrated and dried with a centrifugal evaporator, dissolved in 5 μl of chloroform / methanol = 2/1, loaded on TLC, and the reaction product was confirmed with a transilluminator. At this time, chloroform / methanol / 0.02% CaCl ₂ = 5/4/1 (v / v / v) was used as a developing solvent. The results are shown in FIG.

  As a result, it was confirmed that both hydrolysis and condensation reactions occurred in the phospholipid fraction (FIG. 2). On the other hand, neither hydrolysis nor condensation reaction could be confirmed for the glycolipid fraction (FIG. 3). From these results, it was considered very likely that the alkali-resistant lipid contained in the phospholipid fraction is a sphingolipid.

[Purification of alkali-resistant phospholipid fraction]
From 7.4 L of GY medium, 3.797 g of dried cells were recovered, and 361.6 mg of total lipid was obtained. The total lipid was subjected to weak alkali treatment and Folch partitioning was performed. The Folch lower layer was separated into neutral lipid, glycolipid, and phospholipid by sep pak silica. The results are shown in FIGS.

  As a result, as shown in FIG. 4, two bands considered to be alkali-resistant phospholipids were obtained from the methanol fraction.

[Separation of sphingolipids by normal phase HPLC]
The phospholipid recovered in Example 3 was dissolved in n-hexane / isopropanol = 3/1 (20 μg / μl), and 20 μl of this was loaded on high performance liquid chromatography to separate the sphingolipid. At this time, an Inertsil HPLC column (NH2 6 μm, 250 × 4.6 mm; manufactured by GL Sciences) was used as the column, and acetonitrile / methanol / 0.2% triethyleneamine (pH 4.0) = 67/22/11 (v / v / v) was used, the flow rate was 1 ml / min, and the detection wavelength was 210 nm. The most prominent peak of this chromatogram (peak A; FIG. 5) was collected. About the fraction of the collect | recovered peak, the weak fraction by a weak alkali decomposition | disassembly test and SCDace process was confirmed like Example 1 and 2. The results are shown in FIG. 6 (weak alkali decomposition test) and FIG. 7 (SCDace treatment).

  As a result, the peak A did not disappear even after the weak alkali treatment (FIG. 6). Therefore, it was confirmed that the peak A is a lipid resistant to alkali. In addition, it was confirmed that both the hydrolysis reaction and the condensation reaction occurred in the peak A when the hydrolysis reaction using SCDase and the condensation reaction of the fluorescent fatty acid were performed respectively (FIG. 7).

[Confirmation of functional groups by TLC of recovered lipid fraction]
The peak A obtained in Example 4 was developed by TLC, and the presence of a functional group was confirmed by coloration with each coloring reagent. That is, about 50 μg of the lipid fraction collected in Example 3 was taken and loaded onto TLC. At this time, chloroform / methanol / water = 65/25/4 (v / v / v) was used as a developing solvent, and Dittmer reagent and ninhydrin reagent were used as coloring reagents. The results are shown in FIG.

  As a result, the peak A was confirmed to have a blue color by the Dittmer reagent and a red color by the ninhydrin reagent. This revealed that peak A has a phosphate group and a free amino group in the molecule.

[Separation and structural analysis of recovered lipid fractions into various molecular species]
The lipid fraction collected in Example 5 was separated into each molecular species by reverse phase HPLC. That is, the sample was dissolved in methanol (20 μg / μl), and 20 μl of this sample was loaded onto the HPLC. At this time, YMC-Pack ODS-AQ column (150x6.0 mm, S-120mm; manufactured by YMC) was used for the column, and methanol / 1 mM potassium phosphate buffer (pH 7.4) = 9/1 was used for the moving bed. (v / v) was used, and the detection wavelength was 210 nm at a flow rate of 1 ml / min. Each peak was collected and the recovered material was confirmed by TLC. The results are shown in FIG.

  As a result, a remarkable peak was observed around retention time 22 minutes and 27 minutes (FIG. 9). These two peaks were collected as peak 1 and peak 2, and a part thereof was loaded onto TLC to confirm the functional group and Rf value. As a result, it was confirmed that both peak 1 and peak 2 were ninhydrin positive and the Rf value was consistent with that of peak A.

  Further, structural analysis was performed on peak 1 and peak 2 by NMR and FAB-MS. NMR was measured for 1H-NMR (600 MHz, CD3OD) and 13C-NMR (125 MHz, CD3OD). FAB-MS was performed under the conditions of Ion mode: FAB-, Matrix: triethanolamine. The results of FAB-MS are shown in FIG. 10 (Peak 1) and FIG. 11 (Peak 2), respectively.

  As a result of NMR analysis, it was confirmed that both peak 1 and peak 2 were ethanolamine phosphoceramide (EPC) in which phosphorylethanolamine group was bonded to ceramide. In addition, it was confirmed that the sphingosine portion constituting ceramide has an unsaturated bond at the 4, 8, and 10 positions and a methyl group at the 9 position. In addition, fatty acids were found to be a-hydroxy acids with a hydroxyl group added at the 2-position and an unsaturated bond at the 3-position (Table 2).

  As a result of mass spectrometry, it was revealed that peak 1 has a molecular weight of 684 and peak 2 has a molecular weight of 698 (FIGS. 10 and 11). From this, it is considered that the number of carbons in the ceramide part is 34 at peak 1 and 35 at peak 2. These two lipids are different in one of sphingosine or fatty acid carbon chain. .

  From the above results, it was determined that the chemical structure is represented by the general formula (1). This was a new compound.

  The present invention provides a novel sphingolipid and a method for producing the same. The novel sphingolipid of the present invention is expected to be widely used in research fields such as pharmaceuticals, cosmetics, and foods, and in production sites.

Claims (5)

A sphingolipid represented by the following general formula (1).
(Here, when n = 1-14, m = 15-n or when n = 1-15, m = 16-n) The method for producing sphingolipid according to claim 1, wherein marine eukaryotic microorganisms are cultured and purified. The method for producing sphingolipid according to claim 2, wherein the marine eukaryotic microorganism is Labyrinthula. The method for producing a novel sphingolipid according to claim 3, wherein the Labyrinthula is Thraustochytrium aureum . The method for producing a novel sphingolipid according to claim 3 or 4, wherein the Labyrinthula is Thraustochytrium aureum ATCC 34304.