JP2009022220A - Method for producing ceramides - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing ceramides, in a selective, efficient and safe manner. <P>SOLUTION: The method for producing the ceramides includes a step for forming the ceramide, by allowing sphingomyelinase to act in an aqueous solution containing sphingomyelin, in the presence of a polar organic solvent miscible with water, and a step for collecting the formed ceramide. In one embodiment, the aqueous solution contains phosphatidylcholine besides them. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing ceramide useful for cosmetics, pharmaceuticals, lipid engineering, cell engineering and the like.

  Ceramide is a kind of sphingolipid in which sphingosine and a fatty acid are amide-bonded, and is abundant in skin and hair. In recent years, it has been clarified that ceramide functions as a moisturizing component of the skin, and is expected to be applied to the treatment of diseases such as atopic dermatitis.

  Methods for producing ceramide include organic synthesis, extraction from natural products, and a method of treating sphingomyelin with an enzyme. The production of ceramide by organic synthesis is a reaction that requires multistage and stereoselectivity, and requires a lot of technical work. Extraction from natural products is not high in yield, and tissues such as bovine brain cannot be used due to the problem of bovine spongiform encephalopathy (BSE).

  Enzymes such as phospholipase C and sphingomyelinase are used in the method for producing ceramide using an enzyme. Sphingomyelinase hydrolyzes sphingomyelin as a substrate to produce ceramide and phosphorylcholine. On the other hand, phospholipase C normally recognizes phosphatidylcholine more strongly as a substrate than sphingomyelin and hydrolyzes phosphatidylcholine to produce diacylglycerol and phosphorylcholine. However, the substrate selectivity of phospholipase C is not high, and the production of ceramide by phospholipase C applies this low substrate selectivity.

  On the other hand, sphingomyelin is similar in structure to phosphatidylcholine and is difficult to separate. Therefore, high-purity sphingomyelin derived from natural products is expensive, and its use at an industrial level is not realistic. From the above, in the production of ceramide at an industrial level by the enzymatic method, low-purity sphingomyelin containing a large amount of other phospholipids, particularly phosphatidylcholine, must be used. In this case, when phospholipase C is used, phosphatidylcholine mixed in low-purity sphingomyelin is preferentially used as a substrate to produce diacylglycerol, and ceramide production is reduced. For this reason, it is desirable to use sphingomyelinase in the enzymatic method.

  By the way, since sphingomyelin has high fat solubility, in order to dissolve or suspend sphingomyelin in the enzyme reaction solution, a surfactant is used, or a non-polar organic solvent such as diethyl ether is used. It is known to carry out enzymatic reactions in the system. In the use of a surfactant, it is desired to remove the surfactant after the enzyme reaction, but it is generally difficult to remove the surfactant. In addition, nonpolar organic solvents such as diethyl ether generally have a low flash point and are at risk of explosion. Therefore, in industrial ceramide production using enzymes, a selective, efficient and safe production method is desired without using additives such as surfactants or nonpolar organic solvents.

  Patent Document 1 discloses a method for measuring the activity of sphingomyelinase involved in the production of ceramide. Sphingomyelinase is allowed to act in a reaction solution containing radiolabeled sphingomyelin, sodium deoxycholate, and tris-hydrochloric acid suspended in ethanol, and the enzyme activity is measured with a scintillation counter. Here, ethanol is used only for the purpose of suspending the substrate, sphingomyelin.

  There are various reports on the production of ceramide using phospholipase C.

  Patent Document 2 describes a method for producing ceramide from sphingomyelin using erythrocytes as a raw material, using a phospholipase derived from Bacillus cereus. Enzymatic reaction is carried out in a liquid (two-layer system) containing the above phospholipase, sphingomyelin, Tris buffer and diethyl ether, and crude ceramide is obtained from the ether layer after completion of the reaction.

  In Patent Document 3, sphingomyelin or ceramide 2-aminoethylphosphonic acid is extracted from seafood, and ceramide is isolated by cleaving phosphonic acid using phospholipase C derived from Clostridium perfringens. A method is described. Reaction with ceramide 2-aminoethylphosphonic acid, phospholipase C, Tris buffer, and diethyl ether or a solution containing ethanol (two-layer system). After the reaction, ceramide is removed from the ether layer or organic layer (ethanol-ether layer). It has gained.

  Non-Patent Document 1 shows the hydrolysis activity of Clostridium perfringens-derived phospholipase C against various substrates. This document shows that the phospholipase derived from Clostridium perfringens shows the optimal activity against phosphatidylcholine when added with the surfactant sodium deoxycholate and is active against either phosphatidylcholine or sphingomyelin in the presence of ethanol. Have been observed. This document shows that phospholipase C derived from Clostridium perfringens partially decomposes sphingomyelin in a reaction solution containing 25% ethanol.

  A contribution to the activation of organic solvents in a non-polar solvent micro-water two-layer system using phospholipase C has been reported. Non-Patent Document 2 describes the synthesis of ceramide by Clostridium perfringens-derived phospholipase C using a fine water two-layer system. In sphingomyelin hydrolysis with phospholipase C, it has been reported that the reaction efficiency of the fine water two-layer system (water: organic solvent) is higher than that of the single layer (water-saturated toluene). This document reports that ethanol addition improves the hydrolysis rate in a fine water two-layer system containing a nonpolar solvent, but the amount added is 2% or less of the total reaction solvent.

  There is a study of phosphatidylinositol-specific phospholipase C (PI-PLC) for improving the activity of phospholipases using a polar organic solvent (Non-patent Document 3). In this study, isopropanol, dimethyl sulfoxide (DMSO), or dimethylformamide has been shown to activate PI-PLC. However, PI-PLC and sphingomyelinase clearly differ in protein homology and substrate recognition. Furthermore, in PI-PLC, a two-step mechanism is proposed in which the inositol phosphate moiety of the substrate phosphatidylinositol undergoes cyclic inositol phosphate as a reaction intermediate. On the other hand, the sphingomyelinase reaction is a one-step mechanism that does not go through an intermediate such as cyclic inositol phosphate. Therefore, the contribution of the inositol part of the substrate is essential in the enzyme reaction of PI-PLC, which is very different from the enzyme reaction of sphingomyelinase. From the above, activation of sphingomyelinase by polar organic solvents is significantly different from activation of PI-PLC.

  On the other hand, in Pseudomonas aureofaciens-derived phospholipase C, addition of 2% or more of ethanol has been reported to cause enzyme inhibition (Non-patent Document 4). Furthermore, in the research of lipase derived from Rhizomucor miehei, alcohols containing ethanol and tetrahydrofuran have been reported to inhibit enzyme activity (Non-patent Document 5).

As described above, there is no report on practical production of ceramide using sphingomyelinase as compared with the study on production of ceramide using phospholipase C. In addition, the addition of an organic solvent to the enzyme reaction solution does not always lead to an improvement in activation, and may be hindered. Furthermore, it is generally known that the addition of a polar organic solvent such as ethanol to the enzyme solution causes the enzyme to precipitate and decreases the enzyme activity in the reaction solution. Therefore, it is completely unexpected that the activity and selectivity of the enzyme are improved by adding only a polar organic solvent to the sphingomyelinase reaction solution found this time.
JP 2002-253225 A JP-A-8-294395 Japanese Patent Laid-Open No. 7-265089 EL Krug and C. Kent, Archives of Biochemistry and Biophysics, 231 (2), 400-410, 1984 L. Zhang et al., J. Biotechnology, 123, 93-105, 2006 Y. Wu et al., Biochemistry, 36, 8514-8521, 1997 S. Sonoki et al., J. Biochem., 80, 361-366, 1976 W. Tsuzuki et al., Biosci. Biotechnol. Biochem., 67 (8), 1660-1666, 2003

  As described above, the conventional enzymatic ceramide synthesis requires a lot of labor and technical skill for purification due to the use of additives such as surfactants or the production of by-products, and is never efficient. I could not say. In addition, since nonpolar organic solvents such as diethyl ether are used, there was a risk of explosion.

  Therefore, an object of the present invention is to provide a method for producing ceramide selectively, efficiently and safely.

  The present inventors improve the activity of the enzyme by acting sphingomyelinase in a single-layer reaction solution containing a polar organic solvent miscible with water, and do not act on the mixed phosphatidylcholine. We found that ceramide can be produced from sphingomyelin.

The present invention provides a method for producing ceramide. This method
A step of producing ceramide by reacting sphingomyelinase in an aqueous solution containing sphingomyelin in the presence of a polar organic solvent miscible with water; and a step of recovering the produced ceramide.

  In one embodiment, the aqueous solution further comprises phosphatidylcholine.

  According to the method of the present invention, the activity of sphingomyelinase that converts sphingomyelin into ceramide can be increased. Furthermore, ceramide can be selectively produced from sphingomyelin without using the mixed phosphatidylcholine as a substrate.

  According to the method of the present invention, ceramide is produced by allowing sphingomyelinase to act in an aqueous solution containing sphingomyelin in the presence of a polar organic solvent miscible with water.

  The origin of sphingomyelinase is not particularly limited. An enzyme derived from a microorganism is preferably used, and a sphingomyelinase derived from any microorganism that produces the enzyme can also be used. For example, a sphingomyelinase belonging to the genus Streptomyces, a sphingomyelinase derived from Staphylococcus, a sphingomyelinase derived from Bacillus, and the like can be mentioned. Examples of the sphingomyelinase derived from the genus Streptomyces include, for example, Streptomyces hachijoensis (which is also referred to as Streptomyces cinnamoneus) described later, and Streptomyces glinamus. Sphingomyelinase from (Streptomyces griseus) is included. Sphingomyelinase derived from the genus Staphylococcus includes sphingomyelinase derived from Staphylococcus aureus. The sphingomyelinase derived from Bacillus includes sphingomyelinase derived from Bacillus cereus.

  The sphingomyelinase may be an enzyme isolated from a microorganism that produces the enzyme, or a recombinant enzyme obtained by introducing a gene encoding sphingomyelinase into an arbitrary host and expressing it. Also good. The sphingomyelinase is preferably purified, but a crude enzyme extract such as an enzyme culture solution may be used within an acceptable range. Commercially available sphingomyelinase reagents can also be used.

  The origin of sphingomyelin used in the method of the present invention is not limited. It is preferably derived from a raw material containing more sphingomyelin. Examples include sphingomyelin preparations produced from cow brain, milk, chicken eggs, squid, fish, or animal red blood cells, or synthetically produced sphingomyelin preparations.

  The sphingomyelin preparation may be prepared from the raw materials as described above or synthesized by a method commonly used by those skilled in the art, or a commercially available product may be used. The purity of sphingomyelin used in the method of the present invention is also not limited. The sphingomyelin preparation may be mixed with other phospholipids, especially phosphatidylcholine. When phosphatidylcholine is mixed, the content ratio of sphingomyelin and phosphatidylcholine can vary. The amount of phosphatidylcholine may greatly exceed the amount of sphingomyelin. The sphingomyelin content may be 50% by mass or less or 10% by mass or less.

  Sphingomyelin contained in commercial phosphatidylcholine preparations can also be used in the method of the present invention.

  The enzymatic reaction with sphingomyelinase can be performed in a monolayer system in an aqueous solution. Solvents (eg, buffers) and added salts and their amounts for the preparation of aqueous solutions and their amounts can be readily determined by those skilled in the art in view of the reaction conditions, depending on the sphingomyelinase used.

  Examples of the polar organic solvent miscible with water include alcohols such as methanol, ethanol and isopropanol, ketones such as acetone, ethers such as tetrahydrofuran (THF) and dioxane, acetonitrile, and dimethyl sulfoxide (DMSO). Preferred are alcohols such as ethanol, methanol and isopropanol, ketones such as acetone, and ethers such as tetrahydrofuran and dioxane. Since ethanol is a food and food additive, it is safe and it is desirable to use ethanol in this method.

  The polar organic solvent is admixed with an aqueous solution containing the starting material for the production of ceramide (eg, the sphingomyelin preparation or phosphatidylcholine preparation described above). This mixing may be before or after the starting material or sphingomyelinase is added.

  The concentration of the polar organic solvent contained in the aqueous solution for the enzyme reaction by sphingomyelinase depends on the type of the polar organic solvent and sphingomyelinase used, the concentration of sphingomyelin as a substrate, and preferably 20% or more. More preferably, the concentration exceeds 25%, more preferably 30% or more, still more preferably 40% or more, preferably at most 80%, more preferably at most 70%. More preferably, it is at most 60%. In addition,% of the concentration of the polar organic solvent is represented by v / v.

  Although the reaction conditions of the present invention are not particularly limited, ceramide can be produced more efficiently by carrying out the reaction under the optimum conditions (pH, temperature, etc.) of the sphingomyelinase used. Usually, the pH is 5 to 13 and the temperature is 20 ° C to 60 ° C.

  The reaction time is not particularly limited, and the reaction time can be selected according to the type and amount of the substrate used and the amount of enzyme used.

  Confirmation of ceramide production from the enzyme reaction by sphingoemylinase can be performed by analysis such as thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).

  The produced ceramide can be recovered from the reaction solution without removing the polar organic solvent from the reaction solution. For example, the ceramide can be separated and fractionated by subjecting the reaction solution to column chromatography. When removing the polar organic solvent from the reaction solution, vacuum concentration or distillation may be used.

  According to the method of the present invention, even when sphingomyelin and other phospholipids such as phosphatidylcholine are mixed, sphingomyelinase selectively acts on sphingomyelin to produce ceramide, whereas phosphatidylcholine Other phospholipids such as can remain. Sphingomyelin and phosphatidylcholine are difficult to separate because they exhibit similar behavior, and their respective conversion products, ceramide and diacylglycerol, are also difficult to separate because they exhibit similar behavior. Therefore, by converting only sphingomyelin, the produced ceramide can be easily separated from other phospholipids such as phosphatidylcholine.

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

(Example 1: Preparation of enzyme solution)
In this example, sphingomyelinases derived from Streptomyces griseus, Streptomyces hachijoensis, Bacillus cereus, and Staphylococcus aureus were used (in order, SGSMase, SHSMase, BCSMase, and SASMase, respectively). Also called). Enzyme solutions of Sphingomyelinase (SGSMase and SHSMase) derived from Streptomyces griseus and Streptomyces honeyjoensis were prepared as follows. Sphingomyelinases (BCSMase and SASMase) derived from Bacillus cereus and Staphylococcus aureus were both purchased from SIGMA.

<Preparation of Streptomyces griseus-derived sphingomyelinase (SGSMase) enzyme solution>
The basic operation was performed based on Practical Streptomyces Genetics (The John Innes Foundation). A region having high homology was selected based on the amino acid sequence information of the existing sphingomyelinase, and primer sets (SEQ ID NO: 1 and SEQ ID NO: 2) for degenerate PCR were prepared. Using this degenerate PCR primer set, PCR was performed using Streptomyces griseus NBRC13350 strain chromosomal DNA as a template. The composition of the PCR reaction solution is as follows. Template chromosomal DNA 890 ng, 10 × PCR Buffer for KOD-plus- 25 μl, primer 300 nM, dNTP mixture 0.2 mM, MgSO ₄ 1 mM, DMSO 5% and KOD-plus-DNA Polymerase 1.0 unit, total volume of distilled water 250 μl It added so that it might become. PCR reaction conditions are as follows. Step 1; 94 ° C., 2 minutes; Step 2; 94 ° C., 15 seconds; Step 3; 68 ° C., 1 minute; Step 2 to Step 3 are repeated 30 cycles; Step 4; 68 ° C., 2 minutes. By this PCR, a specific amplification product of 0.5 kb was obtained. The amplification product was sequenced, and primer sets 1 (SEQ ID NO: 3 and SEQ ID NO: 4) and set 2 (SEQ ID NO: 5 and SEQ ID NO: 6) for inverse PCR were prepared based on the sequence information. Streptomyces griseus NBRC13350 strain chromosomal DNA was cleaved with SmaI and AluI, and the resulting fragments were self-ligated. PCR was performed using the SmaI-cleaved self-ligation product as a template and the inverse PCR primer set 1 to obtain an amplified fragment of 1.5 kb (the PCR reaction solution composition and conditions are the same as above). Similarly, PCR was performed using the AluI-cleaved self-ligation product as a template and the inverse PCR primer set 2 to obtain an amplified fragment of 1 kb (the PCR reaction solution composition and conditions are the same as above). The base sequences of the two kinds of amplified fragments thus obtained were determined to obtain the full-length base sequence of Streptomyces griseus NBRC13350-derived sphingomyelinase.

  Based on the obtained sequence information, a PCR primer set for expression (SEQ ID NO: 7 and SEQ ID NO: 8) was prepared, and PCR was carried out using Streptomyces griseus NBRC13350 strain chromosomal DNA as a template. The composition of the PCR reaction solution is the same as described above. PCR reaction conditions are as follows. Step 1; 98 ° C, 2 minutes; Step 2; 98 ° C, 15 seconds; Step 3; 56 ° C, 30 seconds; Step 4; 68 ° C, 1.5 minutes; Step 2 to Step 4 are repeated 30 cycles; Step 5; ℃, 2 minutes. This PCR yielded a specific amplification product of 1.4 kb. The resulting sphingomyelinase gene fragment for expression was treated with BglII and inserted into the BglII site of actinomycete plasmid pIJ702 (ATCC35287, obtained from American Type Culture Collection) to obtain recombinant plasmid pIJ702-SGSMase. Using the obtained recombinant plasmid pIJ702-SGSMase, according to the method described in “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Innes Foundation, 2000)”, Streptomyces lividans 1326 strain (Streptomyces lividans 1326 strain) Was transformed.

  The obtained transformant was cultured in a Trypticase Soy Broth (TSB) medium containing 1.5% w / v glucose, and the culture solution was filtered to obtain a culture enzyme solution. Ammonium sulfate was added to the culture enzyme solution to a final concentration of 60% w / v, and then an ammonium sulfate precipitate was obtained by centrifugation. This ammonium sulfate precipitate was dissolved in 20 mmol / L Tris buffer (pH 7.5) to obtain an SGSMase enzyme solution.

<Preparation of Sphingomyelinase (SHSMase) Enzyme Solution from Streptomyces Hachijoensis>
For PCR, a sense primer (SEQ ID NO: 9) having a BglII site added to the upstream region of the structural gene based on the gene sequence information of Sphingomyelinase derived from Streptomyces honeyjoensis, An antisense primer (SEQ ID NO: 10) in which a BglII site was added to the basin sequence was designed. Subsequently, PCR was performed using these primers and the chromosomal DNA of Streptomyces hachijoensis NBRC12782 as a template. The composition of the PCR reaction solution is as follows. Template chromosomal DNA 168ng, 10X PCR Buffer for KOD-plus-5μl, primer 300nM, dNTP mixture 0.2mM, MgSO ₄ 1mM, DMSO 5%, and KOD-plus-DNA Polymerase 1.0 unit, total volume of distilled water 50μl It added so that it might become. PCR reaction conditions are as follows. Step 1; 94 ° C., 2 minutes; Step 2; 94 ° C., 15 seconds; Step 3; 68 ° C., 1 minute; Step 2 to Step 3 are repeated 30 cycles; Step 4; By this PCR, a specific amplification product of about 1200 bp was obtained.

  This amplified fragment was digested with BglII and inserted into the BglII site of actinomycete plasmid pIJ702 (ATCC35287, obtained from American Type Culture Collection) to obtain recombinant plasmid pIJ702-SHSMase. Using this recombinant plasmid pIJ702-SHSMase, Streptomyces lividans strain 1326 was transformed according to the method described in “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Innes Foundation, 2000)”.

  From the obtained transformant, an SHSMase enzyme solution was prepared in the same manner as the above-mentioned preparation of the Streptomyces griseus-derived sphingomyelinase enzyme solution.

(Example 2: Activation of sphingomyelinase enzyme in the presence of ethanol)
For reactions with Sphingomyelinase (SGSMase and SHSMase) derived from Streptomyces griseus and Streptomyces honeyjoensis, glycine buffer (pH 10) and MgCl ₂ are final concentrations of 20 mmol / L and 1 mmol / L, respectively. An aqueous solution containing was prepared. In the case of the reaction using Bacillus cereus-derived and Staphylococcus aureus-derived sphingomyelinases (BCSMase and SASMase), Tris buffer (pH 7.5) was used instead of glycine buffer (pH 10). This aqueous solution contained milk-derived sphingomyelin (Avanti Polar Lipids) to a final concentration of 10 mg / mL. This aqueous solution was prepared to further contain ethanol at a final concentration of 10%, 20%, 30%, 40%, 50% or 60% (% is v / v).

  0.05 unit of sphingomyelinase was added to 200 μL of the aqueous solution containing sphingomyelin prepared as described above, and reacted at 37 ° C. and 200 rpm for 10 minutes. The reaction was stopped by adding 5 μL of 200 mmol / L EDTA.

Add ₂ μL of the above sphingomyelinase reaction solution to 18 μL of phosphatase reaction solution consisting of 50 mmol / L glycine buffer (pH 9.6), 2 mmol / L MgCl ₂ and 1 unit / mL bovine intestinal alkaline phosphatase, and add 100 rpm at 37 ° C. For 30 minutes. After the reaction, 180 μL of BIOMOL GREEN reagent (BIOMOL international) was added, and color was developed while gently stirring at room temperature for 20 minutes. After color development, free phosphoric acid was measured at an absorbance of 620 nm. The enzyme activity in the reaction solution containing ethanol was calculated so as to be relatively represented by 100 as the color developed by the reaction solution not containing ethanol (no addition). The results are shown in FIG.

  FIG. 1 relatively shows the activities of various sphingomyelinases in reaction solutions containing ethanol at various concentrations, where 100 is ethanol-free (no addition). In the figure, the rhombus is derived from Streptomyces griseus (SGSMase), the square is derived from Streptomyces hachijoensis (SHSMase), the triangle is derived from Bacillus cereus (BCSMase), and the circle is derived from Staphylococcus aureus (SASMase) ) Sphingomyelinase activity. In any sphingomyelinase, the enzyme activity increased as the ethanol content increased. In particular, it was remarkable when the concentration was 30% v / v or more, and an activity of 150 to 200% when no additive was added was observed.

(Example 3: Activation of sphingomyelinase enzyme in the presence of organic solvent)
The sphingomyelinase activity in the reaction solution was examined in the same manner as in Example 2 except that an aqueous solution was prepared so as to contain the organic solvent shown below instead of ethanol. The organic solvents used and their final concentrations are as follows (% is v / v): methanol 40%; isopropanol 40%; tetrahydrofuran (THF) 40%; acetone 40%; or dimethyl sulfoxide (DMSO) 40 %. For comparison, a reaction using a surfactant Triton X-100 instead of an organic solvent was also carried out (the concentration of Triton X-100 in the reaction solution was 0.1% w / v).

  Table 1 shows the enzyme activities of various sphingomyelinases (SGSMase, SHSMase, BCSMase, and SASMase) in the reaction solution containing the organic solvent. In the reaction solution containing 40% organic solvent, there is a slight difference depending on the enzyme used, but it shows almost the same activity as when no organic solvent was added (no addition), exceeding 150% (nearly 400%) Enzyme activity was also seen. In the presence of the surfactant Triton X-100, the activity of the enzyme was increased.

(Example 4: Conversion rate from sphingomyelin to ceramide in the presence of an organic solvent)
An aqueous solution having the same buffer and salt composition as in Example 2 above contained egg yolk-derived phosphatidylcholine (Nacalai Tesque, Inc .; containing 1.5% by mass of sphingomyelin) to a final concentration of 10 mg / mL. . Thus, it is estimated that this aqueous solution contains 0.15 mg / mL sphingomyelin. This aqueous solution was prepared to further contain an organic solvent or surfactant at a final concentration as shown in the table below.

200 μL of the aqueous solution thus prepared was dispensed, and 0.2 units of various sphingomyelinases were added and reacted at 37 ° C. and 200 rpm. After 30 minutes of reaction, 20 μL of the reaction solution was taken and added to 1 mL of a reaction stop solution composed of chloroform: methanol = 2: 1 (v / v). After centrifugation at 14,000 rpm for 5 minutes, 10 μL of the supernatant was subjected to HPLC, and the integrated value of sphingomyelin was calculated. HPLC analysis: column; Unisil Q NH ₂ 5 μM 4.6 mm × 250 mm, solvent; acetonitrile: methanol: 10 mmol / L ammonium phosphate = 1856: 874: 270, temperature; 37 ° C., flow rate; 1.3 mL / min, detection; UV 205 nm Measured with The ceramide conversion rate was calculated by {(100−integrated value of sphingomyelin after reaction) / initial integrated value of sphingomyelin} × 100.

  These results are shown in Tables 2 to 5 below for each enzyme.

  Table 2 shows SGSMase, Table 3 shows SHSMase, Table 4 shows BCSMase, and Table 5 shows the rate of ceramide production from sphingomyelin when SASMase is used. As shown in these tables, even if there is a lot of phosphatidylcholine, which is not a selective substrate for sphingomyelinase, and there is very little substrate sphingomyelin, the presence of organic solvent improves the rate of ceramide formation from sphingomyelin did. On the other hand, in the presence of a surfactant, the increase in the conversion rate of ceramide was not observed so much.

(Example 5: Sphingomyelinase selectivity improvement in the presence of ethanol)
An egg yolk-derived phosphatidylcholine (Nacalai Tesque; containing 1.5% by mass of sphingomyelin) was added to an aqueous solution containing 20 mmol / L Tris buffer pH 8.5 and 1 mmol / L MgCl ₂ at a final concentration of 10 mg / mL. (Thus, about 0.15 mg / mL of sphingomyelin will be contained). This aqueous solution was prepared to further contain 40% (v / v) ethanol or 0.1% (w / v) or 1% (w / v) Triton X-100.

  200 μL of the aqueous solution thus prepared was dispensed, 0.2 unit of Streptomyces griseus-derived sphingomyelinase (SGSMase) was added, and the mixture was reacted at 37 ° C. and 100 rpm for 60 minutes. The reaction was stopped by adding 10 μL of 250 mmol / L EDTA. 200 μL of chloroform: methanol = 2: 1 (v / v) was added, stirred and centrifuged, and 10 μL of the organic solvent layer was subjected to HPLC. Ceramide production rate from sphingomyelin and phosphatidylcholine residual rate were determined. The integrated values of phosphatidylcholine and sphingomyelin were calculated by HPLC analysis under the conditions shown in Example 4 above. The ceramide production rate was calculated as {(100-integrated value of sphingomyelin after reaction) / initial value of sphingomyelin integrated value} × 100, and the residual rate of phosphatidylcholine was calculated as (integrated value of phosphatidylcholine after reaction / integrated value of initial phosphatidylcholine) × 100. .

  Table 6 shows the ceramide production rate and the phosphatidylcholine residual rate in the reaction solution after the reaction for 60 minutes. In the reaction solution containing neither ethanol nor surfactant (no addition), the phosphatidylcholine residual rate was high, but the ceramide production rate was low. As for the surfactant, in the reaction solution containing Triton X-100 at a concentration of 1% w / v, the ceramide production rate was increased, but the decomposition of phosphatidylcholine was also observed. On the other hand, in the reaction solution containing ethanol, the ceramide production rate was high and phosphatidylcholine was hardly decomposed. Thus, it was found that sphingomyelinase uses only sphingomyelin as a substrate without using phosphatidylcholine as a substrate in the presence of ethanol.

(Example 6: Measurement of ceramide produced by sphingomyelinase in the presence of ethanol)
Egg yolk-derived phosphatidylcholine 300 mg (Nacalai Tesque; containing about 4.5 mg sphingomyelin) is dissolved in 800 μL of ethanol, and further dissolved in 1 mL of a reaction buffer consisting of 40 mmol / L glycine buffer (pH 10) and 2 mmol / L MgCl ₂ did. Streptomyces griseus-derived sphingomyelinase (SGSMase) was added in 0.6 units. 20 μL of the enzyme reaction solution was withdrawn at regular intervals and added to 300 μL of a reaction stop solution consisting of 200 μL of chloroform: methanol = 2: 1 (v / v) and 100 μL of 5 mmol / L EDTA. After stirring, the organic solvent layer was separated from the aqueous layer by centrifugation, and 10 μL of the organic solvent layer was subjected to HPLC analysis. HPLC analysis of sphingomyelin was performed under the conditions shown in Example 4 above. HPLC analysis of ceramide is as follows; column; Lichrospher 100 Diol 5 μM 4 mm × 150 mm, solvent A; hexane: isopropanol: acetic acid: triethylamine = 780: 30: 12.5: 0.665, solvent B; isopropanol: distilled water: acetic acid: triethylamine = 695: 117 : 12.5: 0.665, gradient (A%); 100% (0 to 2 minutes) 60% (10 minutes), flow rate: 1 mL / min, temperature: 55 ° C, detection: evaporative light scattering detection (ELSD).

  FIG. 2 shows changes over time in the amounts of sphingomyelin and ceramide in a sphingomyelinase reaction solution containing ethanol. In FIG. 2, squares indicate sphingomyelin (SM), and triangles indicate the amount of ceramide in the enzyme reaction solution as an integral value. As shown in FIG. 2, in the enzyme reaction solution containing ethanol, sphingomyelin decreased as the reaction progressed, whereas ceramide increased. From this, it was found that sphingomyelinase reacts with sphingomyelin to produce ceramide in the presence of ethanol.

  According to the method of the present invention, sphingomyelinase can improve the activity of converting sphingomyelin into ceramide, and can increase the production efficiency of ceramide. Furthermore, according to the method of the present invention, since sphingomyelinase does not use phosphatidylcholine that can be mixed with sphingomyelin as a substrate and can selectively produce ceramide using sphingomyelin as a substrate, low-purity sphingomyelin is used. However, ceramide can be produced efficiently. Therefore, labor for recovery and purification of ceramide can be suppressed even in industrial production, and inexpensive raw materials can be used.

It is a graph which shows relatively the activity of various sphingomyelinase in the reaction liquid containing ethanol at various concentrations, with no addition (ethanol-free) being 100. It is a graph which shows a time-dependent change of the quantity of sphingomyelin and ceramide in the sphingomyelinase reaction liquid containing ethanol.

Claims (2)

A method for producing ceramide, comprising:
A method comprising: producing a ceramide by reacting a sphingomyelinase in an aqueous solution containing sphingomyelin in the presence of a polar organic solvent miscible with water; and recovering the produced ceramide.   The method of claim 1, wherein the aqueous solution further comprises phosphatidylcholine.

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