JP6823780B2 - Manufacturing method of ceramide - Google Patents

Description

The present invention relates to a method for producing ceramide from glycosphingolipid using endoglycoceramidelase. More specifically, the present invention relates to a method for efficiently producing ceramide from glycosphingolipid using endoglycoceramidese in the substantially absence of a surfactant.

Ceramide exists in the stratum corneum and has a moisturizing function of retaining water and a barrier function of protecting the skin from external stimuli. It is known that when the amount of ceramide in the stratum corneum decreases due to aging or the like, the water retention function and the barrier function of the stratum corneum decrease, the skin becomes dry easily, and skin aging is promoted. In addition, it has been reported that in skin diseases such as dry skin, rough skin, atopic dermatitis, senile xerosis, and psoriasis, a decrease in ceramide in the stratum corneum causes a decrease in moisturizing function and barrier function. ..

It is known that external supplementation of ceramide is effective for reducing the water retention function and barrier function of the stratum corneum. In recent years, cosmetics and supplements containing ceramide have been attracting attention, and the demand for ceramide raw materials is on the rise.

Conventionally, as a method for producing ceramide, endoglycoceramidese (hereinafter, sometimes abbreviated as "EGCase") that hydrolyzes the sugar-ceramide bond of glycosphingolipid is used to release ceramide from glycosphingolipid. The method is known. It has been reported that the EGCase used in this method is produced by Rhodococcus microorganisms, Corynebacterium microorganisms, leeches, earthworms, shijimi, jellyfish, hydra, etc., and various EGCase genes have been reported so far. Variants of EGCase that have been cloned and have improved activity and substrate specificity have also been developed (see, for example, Patent Documents 1 to 3).

However, EGCase is a protein having extremely strong hydrophobicity, and in the absence of a surfactant, the molecules aggregate with each other, and the hydration resolution exhibited is low (see Non-Patent Document 1). In general, in substance production using an enzymatic reaction, lengthening the reaction time is effective in improving the yield in a system with low reaction efficiency, but EGCase acts on glycosphingolipid in the absence of a surfactant. It has also been reported that the yield of ceramide does not improve so much even if the reaction time is lengthened. Therefore, conventionally, when liberating ceramide from glycosphingolipid using EGCase, it has been indispensable to add a surfactant such as Triton X-100 to the reaction system.

On the other hand, since surfactants may show cytotoxicity, it is desirable that ceramides provided as raw materials for cosmetics and foods do not contain surfactants and the like. However, since surfactants are highly compatible with lipids, it is difficult to separate the released ceramides and surfactants after the reaction when EGCase is reacted with sphingoglycolipids to release ceramides in the presence of surfactants. There is a drawback that a complicated purification step is required to separate the surfactant and the ceramide.

Shin Ito, Oil Chemistry, Vol.40 (1991) No.5, p.352-360

JP-A-2007-312677 Japanese Unexamined Patent Publication No. 9-70294 Japanese Patent Application Laid-Open No. 6-7161

An object of the present invention is to provide a method for efficiently producing ceramide from glycosphingolipid using EGCase without substantially adding a surfactant.

The present inventor has conducted diligent studies to solve the above problems, and found that when ceramide is released from glycosphingolipid using EGCase in the absence of a surfactant, EGCase is used in the presence of ceramide. We have found that ceramide can be efficiently produced by initiating the hydrolysis reaction of glycosphingolipid. The present invention has been completed by further studies based on such findings.

That is, the present invention provides the following methods for producing ceramide.
Item 1. A method of producing ceramides from glycosphingolipids using endoglycoceramidese in the virtually absence of surfactants.
A method for producing ceramide, which comprises initiating a hydrolysis reaction of glycosphingolipid by endoglycoceramidese in the presence of ceramide.
Item 2. Item 2. The production method according to Item 1, which comprises the following steps 1 to 3.
The first step, in which glycosphingolipids are allowed to act on endoglycoceramides to liberate ceramides in the absence of a detergent.
With respect to the second step of recovering the ceramide released in the first step and the remaining glycosphingolipid, and the mixture of ceramide and glycosphingolipid obtained in the second step, substantially no surfactant is used. A third step in which endoglycoceramidese is allowed to act in the presence to release ceramide from the glycosphingolipid.
Item 3. Item 2. The production method according to Item 2, wherein the second step is performed by a bri-dialer method.
Item 4. Item 8. The production method according to any one of Items 1 to 3, wherein the glycosphingolipid is cerebroside.
Item 5. Item 8. The production method according to any one of Items 1 to 4, wherein the glycosphingolipid is derived from konjac.
Item 6. Item 4. The production method according to Item 4 or 5, wherein the endoglycoceramidase is endoglycoceramidase I.

According to the production method of the present invention, ceramide can be efficiently released from glycosphingolipid by EGCase without substantially using a surfactant, so that the surfactant is substantially mixed. However, highly safe ceramide can be obtained in high yield.

In a preferred embodiment of the production method of the present invention, the first step of allowing EGCase to act on glycosphingolipid in the substantially absence of a surfactant, the ceramide released after the step and the remaining glycosphingolipid. By carrying out the second step of recovering the ceramide and the third step of allowing EGCase to act on the mixture of ceramide and glycosphingolipid obtained in the second step in the substantially absence of a surfactant. It is possible to dramatically improve the yield of ceramide.

In Reference Test Example 1, after EG Case I was allowed to act on plant-derived glucosylceramide (soybean-derived (S), konjac-derived (K), corn-derived (C), and tamogidake-derived (T)) in the presence of Triton X-100. The results of copper phosphoric acid color development by subjecting the recovered lipid fraction to TLC are shown. In Reference Test Example 2, the lipid fraction recovered after allowing EGCase I to act on konjac-derived glucosylceramide in the presence of 0.2% by weight of Triton X-100 is subjected to TLC to develop copper phosphoric acid color. In Test Example 1, the lipid fraction (lanes 2 to 5) recovered after allowing EGCase I to act once on konjac-derived glucosylceramide in the absence of a surfactant, and the lipid fraction were allowed to act again on EGCase I. After that, the collected lipid fractions (lanes 6 to 9) were subjected to TLC to show the results of copper phosphoric acid color development.

The production method of the present invention is a method for producing glycosphingolipid from glycosphingolipid using endoglycoceramidese in the absence of a surfactant, which is a method for producing glycosphingolipid by endoglycoceramidese in the presence of ceramide. It is characterized by initiating a hydrolysis reaction. Hereinafter, the production method of the present invention will be described in detail.

[Material used]
Glycosphingolipid In the production method of the present invention, glycosphingolipid is used as a substrate as a raw material for producing ceramide. Glycosphingolipid is a glycolipid in which a sugar is bound to a primary alcoholic hydroxy group of ceramide having a structure in which a fatty acid is amide-bonded to sphingoid.

The origin of the glycosphingolipid used in the present invention is not particularly limited and may be derived from a plant, an animal, or a microorganism, but a plant-derived one is preferable.

Specific examples of the plant from which the glycosphingolipid used in the present invention is derived include almonds, sea lettuce, green laver, red sardine, acacia, akane, red grapes, and red pine (pine tar, amber, and copal. Same), Agarix, Akinonogeshi, Akebi, Asagao, Azalea, Hydrangea, Ashitaba, Azuki, Asparagus, Acerola, Asenyaku, Anis, Avocado, Amacha, Amachazul, Amariris, Artea, Arnica, Aloe, Angelica, Anzu, Ansokko Izayoibara, Ichii, Ichijiku, Ginkgo, Iran Iran, Uikyo, Oolong tea, Ukon, Usvenia oyster, Utsubogusa, Udo, Ume, Urajirogashi, Wenshu mikan, Agetsu, Eshalot, Ezokogi, Enishida, , Sea lettuce, Okera, Osmanthus, Otogirisou, Odorikosou, Onidokoro, Olive, Oregano, Orange (including orange peel), Carnation, Cacao, Kaki, Kakidooshi, Kakkon, Kashiwa, Katakuri, Pumpkin, Chamomile, Kamukam, Chamomile, Karasuuri , Karin, Garsina, Cardamon, Kistrawberry, Kiwi, Kikyo, Cabbage (including kale), Caraway, Cucumber, Kinkan, Ginnan, Guava, Kuco, Kuzu, Kuchinashi, Kumin, Cranberry, Walnut, Grapefruit, Clove, Black pine, Black bean , Chlorella, Ketsumeishi, Gennoshoco, Kokemomo, Kosho, Cosmos, Gobo, Wheat (including wheat germ), Sesame, Komatsuna, Rice (including rice bran), Copal, Konnaku (Konnyaku potato) (including amber tobi powder), Comb , Salmonberry, Cypress, Pomegranate, Satsumaimo, Satoimo, Satoukibi, Satodaikon, Saffron, Zabon, Sanzashi, Sansho, Shiitake, Cyclamen, Shiso, Shimeji, Jagaimo, Shakuyaku, Jasmine, Juzudama, Shungiku, Shoga , Shirakashi, Jinchoge, Shinnamon, Watermelon, Suitopy, Sugina, Staranis, Starapple, Sudachi, Stevia, Sumomo, Sage (Salvia), Zeniaoi, Green laver, Senkyu, Senburi, Soba, Solamame, Daikon, Soybean (including okara) ), Daidai, Thyme, Takenoko, Onion, Taragon, Taloymo, Tanjin, Dandelion, Chicoli, Tsukimisou , Tsukushi, Tsubaki, Tsubokusa, Tsumekusa, Tsurukusa, Tsuruna, Tsubaki, Dill, Tenjikuaoi (Geranium), Touga, Togarashi, Touki, Tochu Kasou, Corn, Dokudami, Tokon, Tochu, Toneriko, Nagaimo, Nazuna , Chinese chive, leek, carrot, garlic, green onion, horseradish, horseradish, nobile, barbena, palm, pineapple, hibiscus, scallions, basil, parsley, hadakamugi, scallions, scallions, bananas, banaba, vanilla, paprika, hamamelis, beet , Higanbana, Hishi, Hijiki, Pistachio, Hyssop (Yanagihakka), Chive, Chive, Chive, Hiba, Himashi, Sunflower, Biwa, Phalaenopsis, Fenegrik, Fukinotou, Blackberry, Plum, Blueberry (including bilberry), Prune, Hechima Chinese chive, veradonna, bergamot, horseradish, spinach, scallions, bodaiju, button, hop, jojoba, maitake, maou, maca, macadamian nuts, matatabi, marigold, mango, mitsuba, mimosa, myoga, mira, murasaki, mace, melissa , Merilot, melon, men (including cottonseed oil cake), moyashi, yagurumasou, yamaimo, yamayuri, yamayomogi, eucalyptus, yukinoshita, yuzu, lily, yokuinin, yomena (aster), yomogi, lime, limegi, lilac, raspberry, lakkasei , Rakkyo, apple (including apple fiber), lindow, reishi, lettuce, lemon, mace, leek, rose hip, rosemary, laurier, wakegi, wasabi (including horseradish) and the like. Among these, konnyaku, sweet potato, potato, sweet potato, yama potato, naga potato and other potato-derived potatoes are preferable, and konnyaku is more preferable.

In the glycosphingolipid used in the present invention, the structure of the sphingoid moiety is not particularly limited, but specifically, 4-sphingosine (sphingosine), 4-hydroxysphingosine (phytosphingosine), 4-hydroxy -Trans-8-sphingosine, 4-hydroxy-cis-8-sphingosine, sphingosine, trans-8-sphingosine, cis-8-sphingosine, trans-4-sphingosine, trans-4, trans-8-sphingosine, Examples thereof include trans-4 and cis-8-sphingosine. Among these, preferably, sphingoids constituting konjac-derived glycosphingolipids, specifically, trans-4, cis-8-sphingadienin, and 4-hydroxy-cis-8-sphingenin can be mentioned. ..

In the glycosphingolipid used in the present invention, the number of carbon atoms of the fatty acid bonded to the sphingoid moiety is not particularly limited, but for example, 2 to 30, preferably 14 to 26, and more preferably 18 to 24 are used. Can be mentioned. Further, the fatty acid may be any of a saturated fatty acid, an unsaturated fatty acid containing a carbon-carbon double bond and / or a carbon-carbon triple bond, and an α-hydroxy fatty acid.

In the sphingo glycolipid used in the present invention, as the fatty acid bound to the sphingoid moiety, specifically, hexadecanoic acid (C16: 0), octadecanoic acid (C18: 0), icosanoic acid (C20: 0). , Heneicosanoic acid (C21: 0), docosanoic acid (C22: 0), tricosanoic acid (C23: 0), tetradocosanoic acid (C24: 0), pentacosanoic acid (C25: 0), hexadocosanoic acid (C26: 0), heptacosan Acids (C27: 0), octadocosanoic acid (28: 0), cis-9-octadecenoic acid (C18: 1) and the like can be mentioned. In the notation "CX: Y" shown in parentheses of the fatty acid, CX indicates the number of carbon atoms per molecule, Y indicates the number of unsaturated bonds per molecule, and for example, "C16: 0" It represents a fatty acid having 16 carbon atoms and 0 unsaturated bonds. Among these fatty acids, preferred are fatty acids constituting the glycosphingolipid derived from konjac, and specific examples thereof include octadecane acid, docosanoic acid, and icosanoic acid.

The structure of the sugar moiety bound to the glycosphingolipid used in the present invention is not particularly limited, and may be a monosaccharide or a sugar chain.

When the sugar moiety is a monosaccharide in the glycosphingolipid used in the present invention, the type of constituent sugar is not particularly limited, and specific examples thereof include glucose. Among these monosaccharides, glucose is preferable.

When the sugar portion of the sphingo glycolipid used in the present invention is a sugar chain, the type of the constituent sugar chain is not particularly limited, and for example, the ceramide-side terminal of the sugar chain is composed of a glucose residue. Any sugar chain can be used. Specifically, lactose, a gala system in which a sugar chain and a ceramide are bonded by a galactoside bond, a glybo system having a Galα1-4Gal unit, an isoglobo system having a Galα1-3Gal unit, and Galβ1 Examples thereof include a lacto system having a -3GlcNAc unit, a neolacto system having a Galβ1-4GlcNAc unit, and a ganglio system having a sialic acid residue or a GalNAcβ1-4Gal unit.

When the glycosphingolipid used in the present invention is classified according to the structure of the sugar moiety, specifically, cerebrosides such as Glcβ1-1Cer (glucosylceramide); Galβ1-4Glcβ1-1Cer (lactosylceramide); Galα-1 Globo-based glycosphingolipids such as -4Galβ1-4Glcβ1-1Cer, GalNAcβ1-3Galα-1-4Galβ1-4Glcβ1-1Cer, GalNAcα-1-3GalNAcβ1-3Galα-1-4Galβ1-4Glcβ1-1Cer; GalNAcβ1-3Galα-1-3Galβ1- Isoglobo glycosphingolipids such as 4Glcβ1-1Cer; lactoglycosphingolipids such as GlcNAcβ1-3Galβ1-4Glcβ1-1Cer, Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer Glycolipids; NeuAcα-2-3Galβ1-4Glcβ1-1Cer, GalNAcβ1-4 (NeuAcα-2-3) Galβ1-4Glcβ1-1Cer, Galβ1-3GalNAcβ1-4 (NeuAcα2-3) Galβ1-4Glcβ1-1Cer, NeuAcα2-3Galβ1-3GalNAc -4 (NeuAcα2-3) Galβ1-4Glcβ1-1Cer, GalNAcβ1-4 (NeuAcα-2-8NeuAcα-2-3) Galβ1-4Glcβ1-1Cer, NeuAcα-2-8 NeuAcα-2-3Galβ1-4Glcβ1-1Cer, Galβ1-3GalNAcβ1 Examples thereof include ganglio glycosphingolipids such as -4Galβ1-4Glcβ1-1Cer and GalNAcβ1-4Galβ1-4Glcβ1-1Cer. In the present specification, "Cer" is an abbreviation for ceramide, "Glc" is glucose, "Gal" is galactose, "GlcNAc" is N-acetylglucosamine, "GalNAc" is N-acetylgalactosamine, and "NeuAc" is an abbreviation for sialsan. Is.

Among these, glucosylceramide, lactosylceramide, and more preferably glucosylceramide are preferable from the viewpoint of producing ceramide more efficiently. Glucosylceramide is a cerebroside that is abundant in glycosphingolipids derived from konjac.

Glycosphingolipids can be obtained from the above-mentioned derived plants by a known extraction method. In addition, glycosphingolipids are commercially available, and commercially available products may be used.

In the production method of the present invention, glycosphingolipids having one type of structure or origin may be used alone, or two or more types of structures or origins may be used in combination.

The endoglycoceramidase production method of the present invention, in order to release the ceramide from glycosphingolipids uses EGCase. EGCase is an enzyme that hydrolyzes the sugar-ceramide bond of glycosphingolipids.

Three molecular species (EGCaseI, EGCaseII, and EGCaseIII) having different isoelectric points and molecular weights are known for EGCase, and it is known that the substrate specificity differs depending on the molecular species. In the production method of the present invention, the molecular species of EGCase to be used may be appropriately set according to the structure of glycosphingolipid as a substrate. For example, when cerebroside, particularly glycosphingolipid derived from konjak, is used as the glycosphingolipid, EGCase I is preferably used.

EGCase is known to be produced by Rhodococcus microorganisms, Corynebacterium microorganisms, leeches, earthworms, freshwater clams, jellyfish, hydra, etc., and its amino acid sequence and its gene sequence have also been clarified. In the production method of the present invention, EGCases derived from these organisms may be used, or mutants obtained by modifying EGCases derived from these organisms may be used.

In the production method of the present invention, the EGCase may be obtained by culturing or breeding an organism capable of producing EGCase, or may be obtained by a genetic engineering method. In addition, EGCase is commercially available, and a commercially available product may be used.

In the production method of the present invention, one type of EGCase may be used alone, or two or more types may be used in combination.

Ceramide In the production method of the present invention, ceramide is allowed to coexist at the time when the hydrolysis reaction of glycosphingolipid by EGCase is started. By allowing ceramide to be present in advance at the start of the reaction with EGCase in this way, it is possible to efficiently hydrolyze glycosphingolipids and release ceramide in high yield in the substantially absence of a surfactant. become.

In the production method of the present invention, the type of ceramide that is pre-existing in the system at the start of the reaction is not particularly limited, and the origin, sphingoid structure, bound fatty acid structure, etc. are the above-mentioned glycosphingolipids. It is the same as the one illustrated in the column of.

In the production method of the present invention, as the ceramide pre-existing in the system at the start of the reaction, one having one type of structure or origin may be used alone, or two or more types of structures or origins may be used in combination. You may.

Further, in the production method of the present invention, the origin of the ceramide pre-existing in the system at the start of the reaction may be different from that of glycosphingolipid used as a substrate, but is preferably the same as that of glycosphingolipid. .. That is, for example, when a glycosphingolipid derived from konjac is used as a substrate, it is preferable that the ceramide previously present in the system at the start of the reaction is derived from konjac. In this way, if the origin of the glycosphingolipid used as a substrate is the same as that of the ceramide pre-existing in the system at the start of the reaction, all the ceramides released in the system after the reaction are composed of the same species. Therefore, it becomes possible to homogenize the structure of the ceramide recovered after the reaction.

[Reaction conditions]
Hydrolysis reaction of glycosphingolipid by EGCase in the substantial absence of a surfactant In the production method of the present invention, glycosphingolipid and glycosphingolipid coexist in the absence of a surfactant. Initiate the hydrolysis reaction of glycosphingolipid by EGCase.

In the production method of the present invention, "substantially in the absence of a surfactant" means an interface required for improving the reaction efficiency in the hydrolysis reaction of sphingoli glycol by EGCase in the absence of ceramide. It means that the concentration of the activator is less than the concentration of the activator (that is, the concentration of the surfactant does not affect the reaction efficiency), and specifically, the surface activity in the reaction solution in the production method of the present invention. The permissible range of the concentration of the agent is usually 0.02% by weight or less, preferably 0% by weight.

In the production method of the present invention, glycosphingolipid, ceramide, and EGCase are added to the reaction solvent, and the hydrolysis reaction of glycosphingolipid by EGCase is started in the substantially absence of a surfactant, and the glycosphingolipid is used as a reaction solvent. Release ceramide.

The composition of the reaction solvent is not particularly limited as long as the hydrolysis reaction of sphingo glycolipid by EGCase is possible, and for example, water; acetate buffer, citrate buffer, phosphate buffer, boric acid. A buffer solution such as a buffer solution; a physiological saline solution and the like can be mentioned.

The concentration of glycosphingolipid in the reaction solvent (concentration at the start of the reaction) is not particularly limited, and examples thereof include 1 to 30% by weight, preferably 3 to 25% by weight, and more preferably 5 to 25% by weight. ..

The concentration of ceramide in the reaction solvent (concentration at the start of the reaction) is not particularly limited, and examples thereof include 1 to 30% by weight, preferably 3 to 25% by weight, and more preferably 5 to 25% by weight.

The ratio of glycosphingolipid and ceramide coexisting in the reaction solvent at the start of the reaction is not particularly limited, but from the viewpoint of more efficiently advancing the hydrolysis reaction of glycosphingolipid by EGCase, glycosphingolipid The ratio of ceramide to 1 mol of lipid is usually 0.5 to 2.0 mol, preferably 0.6 to 1.5 mol, and more preferably 0.8 to 1.2 mol.

The concentration of EGCase in the reaction solvent (concentration at the start of the reaction) may be appropriately set in consideration of the type of EGCase used, the reaction time, etc., and is, for example, 0.1 to 1.0 mg / mL, preferably 0.2 to 0.8. Examples thereof include mg / mL, more preferably 0.4 to 0.6 mg / mL.

In the production method of the present invention, the reaction time of the hydrolysis reaction of glycosphingolipid by EGCase may be appropriately set in consideration of the concentration of glycosphingolipid and EGCase added to the reaction solvent, the reaction temperature and the like. For example, 12 hours or more, preferably 12 to 20 hours, and more preferably 14 to 18 hours.

In the production method of the present invention, the reaction temperature of the hydrolysis reaction of glycosphingolipid by EGCase may be appropriately set within the operating temperature range of EGCase to be used, but is usually 20 to 40 ° C, preferably 25 to 39 ° C. More preferably, it is 35 to 38 ° C.

A preferred embodiment Further, as a preferred embodiment of the production method of the present invention, a method including the following steps 1 to 3 can be mentioned.
First step: In the absence of a surfactant, sphingolipids are allowed to act on endoglycoceramides to liberate ceramides.
Second step: The ceramide released in the first step and the remaining glycosphingolipid are recovered.
Third step: Endoglycoceramidese is allowed to act on the mixture of ceramide and glycosphingolipid obtained in the second step in the substantially absence of a surfactant to release ceramide from the glycosphingolipid. ..

The first and second steps correspond to the preparation steps of the raw materials (glycosphingolipid and ceramide) used in the production method of the present invention, and the third step corresponds to the above-mentioned "substantially absent of surfactant". Corresponds to "Glycosphingolipid hydrolysis reaction by EGCase".

In the first step, glycosphingolipid and EGCase are added to the reaction solvent, and the hydrolysis reaction of glycosphingolipid by EGCase is carried out in the substantially absence of a surfactant to release ceramide from glycosphingolipid. In the first step, since the hydrolysis reaction of glycosphingolipid by EGCase is carried out in the absence of ceramide, the hydrolysis efficiency of glycosphingolipid is low, and after the reaction, the hydrolysis reaction with ceramide released from the glycosphingolipid is carried out. The glycosphingolipid remaining without receiving the reaction is contained in the reaction solvent.

The hydrolysis rate of glycosphingolipid achieved in the first step is not particularly limited, but is 10 to 60 mol%, preferably 30 to 60 mol%, based on the total amount of glycosphingolipid added to the reaction solvent. Furthermore, it is desirable to set the conditions so as to release ceramide from preferably 40 to 60 mol% of glycosphingolipid. By liberating ceramide from glycosphingolipid at such a hydrolysis rate in the first step, it becomes possible to efficiently proceed with the hydrolysis reaction in the third step described later.

The composition of the reaction solvent used in the first step is not particularly limited as long as the hydrolysis reaction of sphingo glycolipid by EGCase is possible, and for example, water; acetate buffer, citrate buffer, etc. Buffer solutions such as phosphate buffer and borate buffer; physiological saline and the like can be mentioned.

In the first step, the concentration of glycosphingolipid added to the reaction solvent (concentration at the start of the reaction) is not particularly limited, but is, for example, 1 to 60% by weight, preferably 5 to 60% by weight, more preferably. Is 10 to 60% by weight.

The concentration of EGCase added to the reaction solvent (concentration at the start of the reaction) in the first step is appropriately determined in consideration of the type of EGCase used, the reaction time, the hydrolysis rate of glycosphingolipid to be achieved, and the like. It may be set, and examples thereof include 0.1 to 1.0 mg / mL, preferably 0.2 to 0.8 mg / mL, and more preferably 0.4 to 0.6 mg / mL.

In the first step, the reaction time of the glycosphingolipid hydrolysis reaction by EGCase takes into consideration the glycosphingolipid added to the reaction solvent, the reaction temperature, the hydrolysis rate of glycosphingolipid to be achieved, and the like. It may be set as appropriate, and examples thereof include 12 hours or more, preferably 12 to 20 hours, and more preferably 14 to 18 hours.

In the first step, the reaction temperature of the hydrolysis reaction of glycosphingolipid by EGCase may be appropriately set within the operating temperature range of EGCase to be used, but is usually 20 to 40 ° C, preferably 25 to 39 ° C, and further. The temperature is preferably 35 to 38 ° C.

In the second step, after the first step, the ceramide liberated in the reaction solvent and the remaining glycosphingolipid are recovered.

Recovery of ceramide and glycosphingolipid can be performed according to a known lipid recovery method. Specifically, as a method for recovering ceramide and glycosphingolipid, the Bligh-Dyer method (EG Bligh, WJ Dye. A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 3 , 1959, 911-917.), Folch method (Folch J., M.Lees, GH Sloanestanley, J. Biol. Chem., 226, 1957, 497-509.) And other solvent extraction methods. .. Among these solvent extraction methods, the bri-dialer method is preferable.

In the third step, EGCase is allowed to act on the mixture of ceramide and glycosphingolipid recovered in the second step in the substantially absence of a surfactant to release ceramide from the glycosphingolipid.

In the third step, the mixture of ceramide and glycosphingolipid recovered in the second step and EGCase are added to the reaction solvent, and the glycosphingolipid is hydrated by EGCase in the substantially absence of the surfactant. The degradation reaction is initiated to release ceramide from glycosphingolipid.

The third step is a step of carrying out the "hydrolysis reaction of glycosphingolipid by EGCase in the substantially absence of a surfactant", and is a reaction solvent to be used, glycosphingolipid, ceramide and ceramide in the reaction solvent. The concentration of EGCase, reaction temperature, reaction time, etc. are as described above.

Further, in the third step, by adding the mixture of ceramide and glycosphingolipid recovered in the second step to the reaction solvent, an efficient hydrolysis reaction of glycosphingolipid and glycosphingolipid as substrates Since the ceramide necessary for advancing is supplied, it is not necessary to separately add ceramide and / or glycosphingolipid to the reaction solvent in addition to the mixture recovered in the second step, but the above-mentioned Ceramide and / or glycosphingolipid may be added separately in addition to the mixture recovered in the second step.

[Recovery of ceramide after reaction]
Thus, after liberating ceramide from glycosphingolipid by EGCase in the substantially absence of a surfactant, ceramide is recovered from the reaction solvent by a known lipid extraction method such as the Bry-Dyer method or the Volk method. To obtain ceramide.

The ceramide thus obtained can be used as a raw material for cosmetics, foods and pharmaceuticals.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not construed as being limited to the following examples.

Experimental material
・Glucocerebroside ceramide (NS170302 Glucosylceramide, from Konjac, purity (99% (TLC) Nagara Science Co., Ltd.), soybean-derived glucosylceramide (NS170602 Glucosylceramide, from Soybean, purity (99% (TLC) Nagara Co., Ltd.) Science), Glucosylceramide derived from Tamogi mushroom (NS170502 Glucosylceramide, from Tamogitake, Purity (99% (TLC) Nagara Science Co., Ltd.), Glucocerebroside derived from corn (NS170202 Glucosylceramide, from Maize, Purity (99% (TLC) Co., Ltd.) Nagara Science) was purchased and used for the following experiments.

・ EGCaseI
As EGCaseI, the one obtained by the following genetic engineering method was used. First, the vector pTipEGCI (Journal of Lipid Reseach Vol.53 p2242) constructed by inserting the gene of Rhodococcus equi M-750 strain-derived EGCaseI into the multicloning sites NdeI and HindIII sites of pTipLCH (Patent No. 3944577 and Patent No. 3793812). -2251, 2012) was transformed into Rhodococcus erthropolis L-88 strain (Patent No. 3876310). The transformation was carried out by the electroporation method (electroporation method) according to the method described in Japanese Patent No. 3876310, etc., and the conditions were as follows. A mixture of 400 μL Rhodococcus erthropolis L-88 competent cells and pTip-EGCI was transferred to an electroporation cuvette (Bio-Rad: 0.2 cm gap cuvette), and an electric field was used using the company's gene transfer device Gene Pulser II. The intensity was 12.5 kV / cm, and the pulse controller was set to give an electric pulse with a capacitance of 25 μF and an external resistance of 400 Ω. A mixed solution of electropulse-treated cells and DNA was mixed with 1 ml of LB medium, cultured at 30 ° C. for 4 hours, collected, and applied to 17.5 μg / ml LB agar medium containing chloramphenicol. The product was cultured at ° C to obtain a transformant.

The obtained transformant was inoculated into 10 ml of LB medium containing 17.5 μg / ml of chloramphenicol and cultured at 28 ° C for 2 days, and then 5 ml of the cultured bacterial solution was scaled up to 200 ml and further 28 ° C. Incubated for 2 days and used as a seed. Add the above seeds to 1 L of LB medium containing 17.5 μg / ml chloramphenicol, add thiostreptone to a final concentration of 0.5 μg / ml, and incubate at 28 ° C for 1 day to express EGCase I. Induced.

Centrifuge the culture medium to collect the cells, suspend the cells in a buffer for cell disruption (50 mM sodium phosphate buffer, 10% glycerol, 0.3 mM NaCl (pH 8.0)), and lysozyme (final concentration 2 mg / ml). , Benzonate (750 units) was added, and the cells were allowed to stand on ice for 30 minutes and then ultrasonically disrupted to disrupt the cells. The supernatant obtained by centrifuging the crushed solution was used as a crude protein solution for protein purification.
The crude protein solution obtained above was adsorbed on His-Select HF Nickel Affinity Gel (manufactured by Sigma) equilibrated with the above cell disruption buffer, and then washed buffer (50 mM sodiumphosphate buffer, 10% glycerol, 0.3). The carrier was washed with mM NaCl (pH 6.0)). The protein was eluted by forming a concentration gradient of 0-400 mM imidazole in wash buffer. The EGCI eluted fraction is dialyzed against 20 mM sodiumacetate buffer (pH 5.5), then the insoluble material is removed by centrifugation, and the protein concentration is adjusted to 6.6 mg / ml by centrifugation (Amicon Ultra 30K, Millipore). It was concentrated to give a concentrated EGCase I solution. After confirming the enzyme activity of the concentrated EGCase I solution, 50 μL of the concentrated solution was dispensed into 0.2 mL microtubes and stored at -80 ° C until use. It has been confirmed that the obtained concentrated EGCase I solution does not show inactivation of enzyme activity even when stored at -80 ° C for 1 year.

Further, in the experiments shown below, the EGCase I solution used was an appropriately diluted EGCase I solution.

Identification and quantification of ceramide and glucosyl ceramide Identification and quantification of ceramide and glucosyl ceramide were carried out according to the following methods.
Samples are linearly spotted 5 mm wide on a thin layer 1.5 cm from the bottom of a normal phase silica gel TLC plate (5 cm) and plated in a developing layer saturated with a mixed solvent of chloroform, methanol and acetic acid. After developing to the top, it was sufficiently dried and sprayed with a copper phosphorus reagent consisting of an aqueous solution containing 10% by weight copper sulfate and 8% by weight phosphoric acid. In Reference Test Example 1 below, a solvent having a volume ratio of chloroform: methanol: acetic acid of 76: 3.6: 0.4 was used as the mixed solvent, and in Reference Test Example 2 and Test Example 1 below, chloroform: methanol: acetic acid. The one with a capacity ratio of 99: 2: 0.4 was used. The TLC plate sprayed with copper phosphorus reagent was then heated on a hot plate heated to 180 ° C. Lipid components such as ceramide and glucosylceramide separated on the TLC plate were identified by exhibiting a specific dark blue color.

In addition, in order to quantify the amount of ceramide and glucosylceramide, the digital image (TIF) captured by the image scanner from the colored plate is analyzed by image analysis software (JustTLC, SWEDAY, Sodara, Sanby, Sweden) and the same. The ceramide and glucosylceramide bands on the lane were quantified according to the degree of color development. The yield of ceramide was calculated from the sum of the quantified bands according to the following formula. In addition, the purified unknown amount of ceramide was also developed and colored after spotting a certain amount on TLC by the same method, and the coloration of the band was quantified. At that time, by spotting different known amounts of glucosylceramide in different 3 lanes, the coloration of unknown amount of ceramide is converted from the calibration curve of the coloration of glucosylceramide in 3 lanes to calculate the ceramide content. did.

Reference test example 1
Purified glucosylceramide derived from plants (soybean-derived (S) 100 μg, konjak-derived (K) 100 μg, corn-derived (C) 100 μg, tamogidake-derived (T) 50 μg) as a substrate, and EGCase I solution 50 μL (6.6 mg / mL) ), Triton X-100 (final concentration 0.02% by weight), and 440 μL of 0.1 M aqueous sodium acetate solution (pH 5.0) were added, and the mixture was incubated at 37 ° C. for 16 hours. Then, the lipid fraction was recovered by the Bligh-Dyer extraction method, and the obtained lipid fraction was concentrated to dryness to quantify the amounts of ceramide and glucosylceramide.

The results obtained are shown in Table 1 and FIG. When the reaction was carried out under the same conditions as above in the absence of TritonX-100, the yield of ceramide was 45-60% (results omitted), but in the presence of 0.02% by weight TritonX-100, ceramide. The yield was improved, and when konjac-derived glucosylceramide was used as a substrate, the yield was increased to 82.5%.

Reference test example 2
The following tests were conducted to clarify the effect of the solubilizer on the hydrolysis efficiency of glucosylceramide by EGCase I. Using 100 μg of purified konjac-derived glucosylceramide as a substrate, 50 μL (6.6 mg / mL) of EGCase I solution, a solubilizer, and 440 μL of 0.1 M aqueous sodium acetate solution (pH 5.0) were added, and the mixture was incubated at 37 ° C. for 16 hours. .. As the solubilizer, sodium deoxycholate (final concentration 0.1% by weight), dimethylsulfoxide (final concentration% by weight), and Triton X-100 (final concentration 0.02% by weight or 0.2% by weight) were used. .. For comparison, incubation was carried out under the same conditions as above without adding a solubilizer. Then, the lipid fraction was recovered by the Bligh-Dyer extraction method, and the obtained lipid fraction was concentrated to dryness to quantify the amounts of ceramide and glucosylceramide.

The results obtained are shown in Table 2, and the results when Triton X-100 (final concentration 0.02% by weight) was added as a solubilizer are shown in FIG. In the absence of the solubilizer, the yield of ceramide was 57.1%, but in the presence of Triton X-100, the yield of ceramide was improved, and in the presence of Triton X-100 with a final concentration of 0.02% by weight. Then, the yield of ceramide was improved to about 100%. In addition, in the presence of sodium deoxycholate or dimethylsulfoxide, no significant improvement in the yield of ceramide was observed.

Test Example 1
Prepare 10 threaded round-bottomed glass test tubes (16 x 100 m / m) containing 100 μL (135 nmol, 100 μg) of a chloroform solution of glucoside ceramide derived from konjak, and use a centrifugal evaporator (EYELA, CVE-100). The mixture was centrifuged at 40 ° C. for several minutes to dry. Then, 450 μL of 0.1 M sodium acetate buffer (pH 5.0) was added to each tube, stirred with a Volx mixer until the substrate solution was visually uniformly suspended, and then a small ultrasonic cleaner (Elma Ultrasonic Cleaner, 460). Processed with / H) for 5 seconds. Then, 50 μL (2.6 mg / mL, 4.0 mg / mL, 5.3 mg / mL, or 6.6 mg / mL) of EGCase I solution is added to each test tube, stirred with a vortex mixer, and shaken at 37 ° C. for 16 hours. The enzyme reaction was carried out (the above is the first step).

Then, 1.3 mL of water, 2 mL of chloroform, and 2 mL of methanol were added to each test tube, stirred with a vortex mixer, and centrifuged at 2,500 rpm for 5 minutes to separate into two layers (Bligh-). Dyer extraction method). The lower organic layer was separated, 2 mL of chloroform was added to the upper layer, the mixture was stirred with a vortex mixer, and then centrifuged at 2,500 rpm for 5 minutes. Further, stirring and centrifugation with 2 mL of chloroform were repeated twice, and all the lower layers from the 10 test tubes collected by separating the lower layers were combined and concentrated to dryness with a rotary evaporator. The concentrated dry matter was dissolved in 5 mL of chloroform, 0.5 mL each was dispensed into 10 test tubes, and the lipid fraction was centrifugally concentrated to dryness with a centrifugal evaporator (the above, the second step). ..

Then, 450 μL of 0.1 M sodium acetate buffer (pH 5.0) was added to each test tube, and 50 μL (2.6 mg / mL, 4.0 mg / mL, 5.3 mg / mL, or 6.6 mg) of EGCase I solution was operated in the same manner as above. / mL) was added, and the mixture was shaken at 37 ° C. for 16 hours for an enzymatic reaction (the above, the third step).

The amounts of ceramide and glucosylceramide were quantified for the lipid fraction after the second step and the lipid fraction recovered and concentrated by the Bligh-Dyer extraction method after the third step, and the yield of ceramide was calculated. ..

The results obtained are shown in Table 3 and FIG. In the lipid fraction obtained after the second step (that is, when EGCase I was allowed to act on the glucoside ceramide only once in the absence of a surfactant), the yield of ceramide was 55 even if the EGCase I used was increased. It stayed below about%. On the other hand, in the lipid fraction obtained after the third step, ceramide was obtained in a yield of about 100%.

From the above results, when ceramide is produced from glycosphingolipid using endoglycoceramidese in the absence of a surfactant, the hydrolysis reaction of glycosphingolipid by endoglycoceramidese in the presence of ceramide. It was clarified that the yield of ceramide can be remarkably improved by starting the above. In particular, in the substantially absence of the surfactant, the step of allowing the glycoceramide to act on the glycosphingolipid, and again in the substantially absence of the surfactant on the glycosphingolipid and ceramide obtained by the step. It was also clarified that the yield of ceramide was dramatically improved by carrying out the step of acting endoglycoceramidese.

Claims (6)

Under conditions concentrations below 0.02% by weight of a surfactant, a method for producing the ceramide from glycosphingolipids using endoglycoceramidase,
A method for producing ceramide, which comprises initiating a hydrolysis reaction of glycosphingolipid by endoglycoceramidese in the presence of ceramide. The production method according to claim 1, which comprises the following steps 1 to 3.
The first step under conditions concentrations below 0.02 wt% of a surfactant, which by the action of endoglycoceramidase in glycosphingolipid to liberate the ceramide,
The concentration of the surfactant is 0 with respect to the second step of recovering the ceramide released in the first step and the remaining glycosphingolipid, and the mixture of ceramide and glycosphingolipid obtained in the second step. .02 under wt% following conditions, by the action of endoglycoceramidase, third step of releasing ceramide from the glycosphingolipid. The manufacturing method according to claim 2, wherein the second step is performed by a bri-dear method. The production method according to any one of claims 1 to 3, wherein the glycosphingolipid is cerebroside. The production method according to any one of claims 1 to 4, wherein the glycosphingolipid is derived from konjac. The production method according to claim 4 or 5, wherein the endoglycoceramidase is endoglycoceramidase I.