JPH0884587A - Glycolipid ceramide deacylase - Google Patents

Abstract

PURPOSE: To obtain a glycolipid ceramide deacylase capable of well acting on an enzyme having a wider substrate specificity than that of a conventionally known glycolipid ceramide deacylase, especially even on a neutral glycolipid. CONSTITUTION: This glycolipid ceramide deacylase has properties of actions on an intramolecular ceramide in a sphingoglycolipid to hydrolyze the ceramide into a sphingosine base and a fatty acid and produce a lysoglycolipid and the fatty acid, the substrate specificity for acting on a neutral glycolipid and an acidic glycolipid, 5-7 optimum pH, about 40 deg.C optimum temperature, the pH stability of being stable at a pH within the range of 4-9 by treatment at 5 deg.C for 16hr and the thermostability of being stable at 60 deg.C for 30min. A sphingolipid is treated with the resultant enzyme to afford a lysosphingolipid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel glycolipid ceramide deacylase having a wide range of substrate specificity. Furthermore, it relates to a method for enzymatically producing lysosphingolipid using the glycolipid ceramide deacylase.

[0002]

2. Description of the Related Art Conventionally, a microorganism belonging to the genus Nocardia is an enzyme that acts on an intramolecular ceramide in a glycolipid to hydrolyze the ceramide part into a sphingosine base and a fatty acid to produce a lysoglycolipid and a fatty acid. An enzyme to be produced (Japanese Patent Laid-Open No. 64-60379) is known. This enzyme is named glycolipid ceramide deacylase, but it is described in [Journal of Biochemistry].
Biochemistry), 103, 1-4 pages (198).
8)], GD1a, GM1, GM2, GM3 and so on, but it acts on gangliosides, which are so-called acidic glycolipids.
It has no effect on al Cer and Glc Cer, and La
c Cer, Gb3 Cer, or asialo (asia
lo) It has almost no effect on neutral glycolipids such as GM1. In addition, an enzyme that hydrolyzes the bond between the sphingosine base of ceramide and a fatty acid is ceramidase (EC).
3.5.1.13), though the [Journal of Biological Chemistry (Journal of Biolo
gical Chemistry), Volume 241, 3731-3737
Page (1966), Biochemistry
, Vol. 8, pp. 1692-1698 (1969), Biochimica Biophysica Actor (Biochimica
et Biophysica Acta), Volume 176, Volumes 339-347.
Page (1969), Science, Volume 178,
1100-1102 (1972)], this enzyme cannot hydrolyze the bond between the sphingosine base of the ceramide portion of the glycolipid and the fatty acid. That is, a conventionally known enzyme could not hydrolyze the bond between the sphingosine base of the ceramide portion of the neutral glycolipid and the fatty acid. In general, naturally occurring glycolipids (glycosphingolipids, etc.) show remarkable molecular diversity due to differences in fatty acids even in molecules having the same sugar chain. For example, forssman glycolipid (Gb5Cer) obtained from equine kidney has at least 10 kinds of molecular species depending on the difference in fatty acid moieties. Recently, it has been revealed that the mode of existence of glycolipids in lipid bilayer membranes, the expression of antigenicity, etc. are greatly influenced by fatty acid molecular species, and the structure of fatty acids is important in considering the physiological functions of glycolipids. It has started to be done. It has also been found that the substrate specificity of enzymes involved in the decomposition and synthesis of sphingolipids (glycolipids and sphingomyelins) depends on the fatty acid molecular species. In order to solve such a problem, development of a technique for easily converting a natural sphingolipid having a heterologous fatty acid molecular species into a glycolipid having a single fatty acid has been desired. Moreover, if a fluorescent substance can be prepared by introducing a fluorescent substance instead of the fatty acid of sphingolipid, it will not only be an important reagent that contributes to elucidation of intracellular metabolism and transport pathway of sphingolipid, but also sphingolipid synthesis. It is expected to be a highly sensitive substrate for enzymes and degrading enzymes. Known lysosphingolipid production methods include a hydrazine decomposition method and an alkaline hydrolysis method in an alcohol solvent. However, in these methods, in the case of a glycosphingolipid containing an amino sugar, the amide bond in the sugar chain portion is decomposed to produce de-N-acetyllysoglycolipid. In the case of a glycolipid containing sialic acid (ganglioside), the sialic acid moiety is deacylated. Therefore, after deacylation, it is necessary to attach a protecting group to the lipid portion, perform reacylation, and then remove the protecting group. These series of chemical operations require a lot of labor and technical skill. Moreover, it is very difficult to prepare a lyso form from a polysialoganglioside such as GQ1b having a plurality of sialic acids by the current chemical method. Further, in the case of sphingomyelin, there is a possibility that the choline phosphate group may be removed, which causes a problem in yield. Also,
A method for biologically obtaining lysosphingolipid is known (Japanese Patent Laid-Open No. 6-78782). This method produces many by-products in addition to the desired lysosphingolipid. Therefore, it is necessary to remove these by-products, and industrially there are problems in the steps of operation and yield.

[0003]

An object of the present invention is to provide an enzyme having a wider substrate specificity than the conventionally known glycolipid ceramide deacylase, particularly a glycolipid ceramide deacylase which also acts well on neutral glycolipids. To provide. Further, it is to provide a method for enzymatically and industrially producing a lysosphingolipid useful in the fields such as glycolipid engineering using the glycolipid ceramide deacylase.

[0004]

Means for Solving the Problems The present invention will be summarized. The first invention of the present invention relates to a glycolipid ceramide deacylase characterized by having the following physicochemical properties. (1) Action: It acts on the intramolecular ceramide in the glycosphingolipid to hydrolyze it into a sphingosine base and a fatty acid to produce a lysoglycolipid and a fatty acid. (2) Substrate specificity: It acts on neutral glycolipids and acidic glycolipids. (3) Optimum pH: The optimum pH is 5-7. (4) Optimum temperature: The optimum temperature is about 40 ° C. (5) pH stability: pH 4 to 4 after treatment at 5 ° C. for 16 hours
It is stable in the range of 9. (6) Thermal stability: Stable for 30 minutes at 60 ° C. A second invention of the present invention relates to a method for producing lysosphingolipid, which is characterized by treating the sphingolipid with the glycolipid ceramide deacylase of the first invention.

The present inventors collected various samples (eg, soil, seawater, fresh water, etc.) and used them for screening in order to obtain the glycolipid-related enzyme. In the process of this screening, surprisingly, a novel glycolipid ceramide dehydrogenase, which has never been known, which hydrolyzes neutral glycolipids and acidic glycolipids into sphingosine bases and fatty acids to produce lysoglycolipids and fatty acids The acylase activity was found. Furthermore, as a result of intensive investigations by the present inventors, a microorganism producing the enzyme of the present invention was identified, the enzyme was further purified, and the physicochemical properties of the enzyme were clarified. The present invention has been completed by treating sphingolipids with sucrose and succeeding in efficiently producing lysosphingolipids.

The present invention will be described in detail below. The method for producing the glycolipid ceramide deacylase of the present invention is not particularly limited and may be a microorganism or cell having the ability to produce the glycolipid ceramide deacylase of the present invention. For example, Pseudomonas SP TK-4 (Pseudomona
s sp. TK-4). The present strain is a strain newly obtained by the present inventors from soil, and its mycological properties are as follows. (1) Growth temperature range: ~ 41 ° C (2) Gram stain: Negative (3) Morphology: Bacillus (4) Motility: Positive (5) Growth under aerobic conditions: Positive (6) Anaerobic conditions Growth in Negative: Negative (7) Catalase: Positive (8) Oxidase: Positive (9) OF test: F (10) O / 129 Sensitivity test: Insensitive (11) Purification of dye: +/- (12) Growth in ammonium ion and glucose synthesis medium: positive (13) Utilization of amino acids as carbon source: Arg, As
n, His, Glu, Ser, Ala (14) 7.5% NaCl-containing broth medium (nutrient brot)
h) Growth in: Negative (15) Butanediol dehydrogenase activity: Positive (16) Gas production from glycerol: Positive (17) Gas production from glucose: Positive (18) Hydrogen sulfide from 2.5% peptone water Occurrence of: positive (19) assimilation of sugar: galactose, sucrose, arabinose (20) GC content: about 69.4% (21) having monopolar hairs. From the above results, this strain is identified as a bacterium belonging to the genus Pseudomonas.

This strain is designated as Pseudomonas sp. TK-4, is designated as G-182, and has been deposited as FERM BP-5096 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.

The enzyme of the present invention can be obtained, for example, by culturing the above-mentioned strain in a nutrient medium and separating the enzyme from the culture after culturing. The nutrient source to be added to the medium may be one that can be used by the strain to produce the enzyme of the present invention. As the carbon source, for example, glycerol, glucose, sucrose, molasses, etc. can be utilized, and as the nitrogen source, for example, yeast. Extract, peptone, corn steep liquor, meat extract, defatted soybean, ammonium sulfate, ammonium nitrate and the like are suitable. In addition, inorganic salts such as sodium salt, potassium salt, phosphate salt, magnesium salt, zinc salt and metal salts may be added. Further, glycolipids such as asialo GM1 were added to the medium in an amount of 0.
By adding 01 to 0.5%, the productivity of the enzyme of the present invention can be increased. In culturing the enzyme-producing bacterium of the present invention, the production amount of the enzyme greatly varies depending on the culturing conditions, but generally the culturing temperature is 20 to 35 ° C., and the pH of the medium is 5 to 5.
8 is good, and the enzyme of the present invention is produced by aerated stirring culture for 1 to 7 days. It is natural that the culture conditions are set so that the production amount of the enzyme of the present invention is maximized depending on the strain used, the composition of the medium and the like. Since the enzyme of the present invention produced by the above-mentioned strains exists mainly outside the cells, solid-liquid separation of the culture can be carried out, and a purified enzyme preparation can be obtained from the obtained supernatant by a commonly used purification means. it can. For example, it can be purified by salting out, organic solvent precipitation, ion exchange column chromatography, hydroxyapatite column chromatography, gel filtration column chromatography, freeze-drying and the like. The enzyme purity is, for example,
It can be assayed by polyacrylamide gel disc electrophoresis or the like.

The enzymatic and physicochemical properties of the glycolipid ceramide deacylase obtained by the present invention are as follows. (1) Measurement of enzyme activity The enzyme activity of the glycolipid ceramide deacylase is measured as follows. 10 μl of the enzyme solution was added to 10 μl of the substrate solution [50 mM acetate buffer containing 2 mM of Asialo GM1 and 0.6% (w / v) Triton X-100, pH 6.0] and reacted at 37 ° C. for 30 minutes. 100 ° C
The mixture is heated for 3 minutes to stop the enzymatic reaction, and the reaction solution is concentrated to dryness with a centrifugal concentrator. This is dissolved in 10 μl of 50% methanol and TLC plate (silica gel 60, manufactured by Merck).
Put on, chloroform / methanol / 0.02% CaC
After development with a ₁₂ aqueous solution (5: 4: 1), the glycolipid is colored by the orcinol-sulfuric acid method. In this TLC developing solvent, glycolipids deprived of fatty acids show a slightly slower Rf value than the native glycolipid. This spot is quantified using a TLC chromatoscanner (Shimadzu CS-9000, manufactured by Shimadzu Corporation) at a wavelength of 540 nm.

(2) Action It acts on the intramolecular ceramide in the glycosphingolipid to hydrolyze it into a sphingosine base and a fatty acid to produce a lysoglycolipid and a fatty acid.

(3) Substrate specificity The substrate specificity of the enzyme of the present invention was examined according to the method for measuring enzyme activity in (1). As shown in Table 1 below, not only ganglio acidic glycolipids but also ganglio type were identified. , Globo system,
It also acts on lacto-based glycosphingolipids and cerebrosides in which monosaccharides and ceramide are bound to produce lysoglycolipids and fatty acids. In Table 1, GM1 and asialo GM1 were prepared from bovine brain [Methods in Enzymology (Method
s in Enzymology), 83, 139-191 (1
982)], and other substrates are manufactured by Yatron.

[0012]

[Table 1] Table 1 ──────────────────────────── Substrate decomposition rate (%) ────────── ─────────────────── GM1 56.8 GM2 69.0 GM3 50.0 GD1b 43.4 GD3 51.2 Gb4 26.8 asialo GM1 72.5 LacCer 11 .2 GalCer 14.4 GlcCer 7.3 ─────────────────────────────

(4) Optimum pH The optimum pH of the enzyme of the present invention has a high activity in the vicinity of 5 to 7 as shown in FIG. The buffer used for activity measurement is
50 mM acetate buffer, p at pH 3.0 to 6.5
50 mM phosphate buffer, p for H6.5-9.0
50 mM glycine buffer is used in H10.
FIG. 1 is a diagram showing the optimum pH of the enzyme of the present invention, in which the vertical axis represents relative activity (%) and the horizontal axis represents reaction pH. In the figure, black circles represent acetate buffer, white triangles represent phosphate buffer, and white circles represent glycine buffer.

(5) Optimum temperature As shown in FIG. 2, the optimum temperature of the enzyme of the present invention shows maximum activity around 40 ° C. That is, FIG. 2 is a graph showing the optimum temperature of the enzyme of the present invention, in which the vertical axis represents relative activity (%) and the horizontal axis represents reaction temperature (° C.).

(6) pH Stability The enzyme of the present invention was kept at 5 ° C. for 16 hours at each pH, then the pH was returned to 6.0 and the enzyme activity was measured to examine the pH stability. PH 3.5-6.0 as a buffer
50 mM acetate buffer, 50 mM phosphate buffer at pH 6.0 to 8.0, 50 mM Tris-HCl buffer at pH 8.0 to 9.0, pH 9.0 to 9.0.
In 5 use 50 mM glycine buffer. FIG.
As shown in, the enzyme of the present invention is stable in the range of pH 4-9. That is, FIG. 3 is a diagram showing the pH stability of the enzyme of the present invention, in which the vertical axis represents residual activity (%) and the horizontal axis represents pH. In the figure, white circles represent acetate buffer, black circles represent phosphate buffer, white triangles represent Tris-hydrochloric acid buffer, and white squares represent glycine buffer.

(7) Thermostability When the thermostability of the enzyme of the present invention was examined, as shown in FIG. 4, 90% activity was retained even after treatment at 60 ° C. for 30 minutes. That is, FIG. 4 is a diagram showing the thermostability of the enzyme of the present invention, in which the vertical axis represents residual activity (%) and the horizontal axis represents temperature (° C.).

(8) Molecular weight The molecular weight of the enzyme of the present invention is about 52.0 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
00 is shown.

(9) Confirmation of Structure of Enzyme Reaction Product To confirm the decomposition product of the enzyme of the present invention, after digesting asialo GM1 with the enzyme, the decomposition product is purified by reverse phase HPLC and the first atom bombardment- It is subjected to mass spectrum (FAB-MS) analysis. First, 3 mg /
5 ml of asialo GM1 (C 18: 0, d 18: 1 molecular weight 1254) and 0.6% Triton X-100 5
0 mM acetate buffer pH 6.0, 1 ml of enzyme solution 200 μ
1 and 10 μl of toluene are added and the reaction is carried out at 37 ° C. for 3 days. After completion of the reaction, 5 volumes of chloroform / methanol (2: 1) were added for partitioning, the upper layer was taken and evaporated to dryness, and then dissolved in 500 μl of chloroform / methanol / water (3:48:47). And use it as a sample for reverse phase column chromatography. The column used is ODS-
80T (4.6 × 75mm, manufactured by Tosoh Corporation), flow velocity is 1m
1 / min, the fraction size is 1.5 ml. After adding the sample to the column, add methanol / water (6: 4) to 10 times.
ml, then gradient elution to 100% methanol in 60 minutes, and finally 5 ml of chloroform / methanol / water (60: 30: 4.5). The lysoasialo GM1 fractions were collected and used as a purified sample.
Subject to FAB-MS analysis (matrix is triethanolamine). The result is shown in FIG. That is, FIG. 5 is a diagram showing the results of FAB-MS analysis of the product obtained after digesting asialo GM1 with the enzyme of the present invention, in which the vertical axis represents relative intensity and the horizontal axis represents mass number / charge number (M / Z) is shown.
In the figure, an enlarged view of 600 to 1200 (M / Z) is shown in the central part, and asialo GM1 used as a substrate (upper part) in the upper part.
And the structure of the product lysoasialo GM1 (lower),
The molecular weight and the position of the signal [(M−H) ⁻ ] are shown. Sph represents sphingosine base and Cer represents ceramide.

As shown in FIG. 5, a signal 987 corresponding to the molecular weight 988 of lysoasialo GM1 is obtained as the strongest signal. In addition, a signal of 825 indicating that Gal was removed from the non-reducing end of the sugar chain portion of lysoasialo GM1, and from there, N-acetylgalactosamine (G
A signal at 622 is observed, indicating that alNAc) has dissociated. From these data, the reaction product of the enzyme of the present invention is lysoglycolipid, and asialo GM1 is present in the sugar chain portion thereof.
It is shown that it has a sugar chain portion of.

In addition to the results obtained by the above FAB-MS analysis, (I) the product obtained by digestion with the enzyme of the present invention is positive for ninhydrin reaction, (II) the product is endoglycoceramidase (EC 3.2.1.12.
3: an enzyme that hydrolyzes a glycoside bond between a sugar chain and ceramide or a sugar chain and sphingosine) produces a sugar chain portion of asialo GM1, (III) this sugar chain portion is ninhydrin negative (Ie G
It has been confirmed that the acetyl group of alNAc is not removed).

From the above results, the enzyme of the present invention acts on the intramolecular ceramide in the glycolipid to hydrolyze the ceramide portion into a sphingosine base and a fatty acid to catalyze the reaction of producing a lysoglycolipid and a fatty acid. It became clear.

In order to produce lysosphingolipid using the enzyme of the present invention, any sphingolipid acting on the enzyme can be used as a substrate. For example, GQ1, GT1, GD are used as acidic glycolipids.
1, various gangliosides and sulfatides such as GD3, GM1 and GM3, globoside as a neutral glycolipid, asialo GM1 and cerebroside, and sphingomyelin as a sphingolipid. Various lysosphingolipids can be obtained by suspending these substrates in a buffer and allowing this enzyme to act. For example, the substrate concentration in the reaction solution is 1 to 20 mg / ml, and the reaction temperature is 37 to 4
The reaction is carried out at 0 ° C at a reaction pH of 5.0 to 6.0, usually using an acetate buffer so that the final concentration of the buffer is 25 mM. Further, Triton X-100 as a surfactant is added to the reaction solution so that the final concentration is 0.8%.
After completion of the reaction, the reaction product and unreacted sphingolipid are separated by ODS reverse phase column chromatography. As the eluent, chloroform / methanol / water (5/4/1,
v / v) can be used. HPLC monitoring can be performed by analyzing the eluate by HPTLC. The developing solvent for HPTLC is chloroform / methanol / 10% acetic acid (5/4/1, v / v), color development is performed by the orcinol-sulfuric acid method for glycolipids and lysoglycolipids, and coomassie blue method for sphingomyelin and lysosphingomyelin. You can When it is desired to detect only lysosphingolipid, the ninhydrin method can be used.
Thus, the enzyme of the present invention can be used to produce lysosphingolipid.

Various derivatives can be obtained by reacylating the obtained lysosphingolipid. For example, the introduction of fatty acids into lysosphingolipids
In the presence of dicyclohexylcarbodiimide, fatty acid and N
A reacylated derivative can be obtained by a method of synthesizing an ester with -hydroxysuccinimide and reacting with lysosphingolipid, a method of synthesizing a fatty acid chloride and reacting with lysosphingolipid, and the like.

Further, by labeling the amino group of the sphingosine portion of the lysosphingolipid obtained using the enzyme of the present invention, a fluorescently labeled sphingolipid derivative (fluorescently labeled neosphingolipid) can be synthesized. For example, dansyl chloride, 4-fluoro-7-nitrobenzofurazan,
NBD-F), labeling with 10-pyrenedecanoic acid or the like is possible.

As described in detail above, the present invention provides a glycolipid ceramide deacylase, and further a method for producing a lysosphingolipid using the glycolipid ceramide deacylase. The enzyme is an enzyme having a very broad substrate specificity, and studies on the elucidation of the function of glycolipids,
It is also useful in fields such as glycolipid engineering. Furthermore,
Lysosphingolipids produced using the enzyme can produce substrates and reagents useful for cell engineering such as elucidation of intracellular metabolism and transport pathways of sphingolipids and elucidation of intracellular functions of sphingolipids. Become.

[0026]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but these examples are merely examples of the present invention, and the present invention is not limited thereto.

Example 1 0.5% peptone, 0.1% yeast extract, 0.2% Na
In a liquid medium containing Cl and 0.1% asialo GM1,
Pseudomonas SP TK-4 (FERM BP
-5096) was inoculated and cultured at 30 ° C. for 48 hours. After completion of the culture, the culture solution was centrifuged at 6000 rpm for 60 minutes to remove the cells, and a culture supernatant was obtained. All subsequent operations were performed at 5 ° C. Ammonium sulfate was added to the culture supernatant until it reached 80% saturation, left overnight, and then the precipitate was collected by centrifugation at 6000 rpm for 60 minutes. 20 mM acetate buffer pH containing a small amount of 0.2% Lubrol
It was dissolved in 6.0 and dialyzed against the same buffer overnight. The enzyme solution after dialysis is Toyopearl HW-55F
It was subjected to gel filtration chromatography (manufactured by Tosoh Corporation). Column size is 40 x 300 mm, elution is 0.2
20 mM with 20% Lubrol and 0.2M NaCl
The active fraction was collected using an acetate buffer pH 6.0 at a flow rate of 1 ml / min and a fraction size of 5 ml. Next, this fraction was subjected to column chromatography of DEAM (manufactured by Bekamond Co.) equilibrated with a 20 mM acetate buffer containing 6.0% of Lubrol at pH 6.0. The column size was 2.3 × 150 mm, elution was carried out using the same buffer solution at a flow rate of 2 ml / min, and the non-adsorbed fractions were collected. Next, this fraction was added to CM-5PW (manufactured by Tosoh Corporation) equilibrated with a 20 mM acetate buffer containing 6.0% of Lubrol at pH 6.0. Column size is 5 x 50 mm, elution is 0 at a flow rate of 0.5 ml / min and fraction size of 1.5 ml.
The gradient was adjusted to ˜1 M NaCl, and the active fractions were collected and subjected to gel filtration chromatography using a TSK-G3000SW (manufactured by Tosoh Corporation) column. Elution is 0.
20% containing 2% Lubrol and 0.2M NaCl
Using mM acetate buffer pH 6.0, flow rate 1.5 ml / m
In, the fraction size was 1.5 ml, and the active fractions were collected. Next, this active fraction was subjected to column chromatography of hydroxyapatite equilibrated with a 2 mM phosphate buffer solution pH 7.0 containing 0.2% rubrol. The column size was 1.4 × 70 mm, and elution was performed by gradient elution from a starting buffer solution to a final concentration of 400 mM phosphorus buffer solution pH 7.0. Next, the obtained active fractions were collected and used as a purified enzyme. The purified enzyme had an activity of hydrolyzing asialo GM1 and producing lyso asialo GM1 and a fatty acid.

Example 2 Production of Lysosphingolipid (1) Production of Lysoasialo GM1 Asialo GM1 was used as a sphingolipid. Asialo GM1 is a method of enzymology, No. 83
Vol. 139-191 (1982). 2.5 mg / ml asialo GM1
And containing this enzyme and 0.8% Triton X-100 25
The reaction was carried out by keeping the mM acetate buffer pH 6.0 at 37 ° C. for 3 days. After completion of the reaction, chloroform / methanol (2: 1) in an amount 5 times the reaction solution was added for partitioning, the upper layer was taken and evaporated to dryness, and then 500 μl of chloroform / methanol / water (3:48:47) was added. It melt | dissolved and it used for ODS reverse phase column chromatography. The reaction product and unreacted sphingolipid (asialo GM1) were separated by this chromatography. The column used is ODS-8
0T (4.6 × 75mm, manufactured by Tosoh Corporation), flow velocity is 1m
1 / min, the fraction size is 1.5 ml. The eluent is chloroform / methanol / water (5: 4: 1, v
/ V) was used. The HPLC eluate was monitored by analyzing the eluate by HPTLC. HPTL
The developing solvent for C was chloroform / methanol / 10% acetic acid (5: 4: 1, v / v), the color was developed by the orcinol sulfate method, and the ninhydrin method was used to detect only lysosphingolipid. Next, the lysoasialo GM1 fractions were collected and used as a purified sample, and subjected to FAB-MS analysis (the matrix is triethanolamine). The results are shown in Fig. 5. The vertical axis represents relative intensity, and the horizontal axis represents mass number / charge number (M /
Z) is shown. A signal 987 corresponding to the molecular weight 988 of lysoasialo GM1 was observed as the strongest signal. In addition, a signal of 825 indicating that Gal was deviated from the non-reducing end of the sugar chain portion of lysoasialo GM1,
Furthermore, from there, N-acetylgalactosamine (GalNA
A signal of 622 was observed, indicating that c) was missing. Furthermore, in the purified preparation, the reaction product was ninhydrin reaction positive, and when the product was treated with endoglycoceramidase, a sugar chain portion of asialo GM1 was produced. This sugar chain portion was ninhydrin negative. From these results, it was confirmed that the structure of the purified preparation of the reaction product was indeed lysoasialo GM1.

(2) Production of lysosphingomyelin Sphingomyelin [manufactured by Matreya] was used as a sphingolipid in the same manner as in Example 2- (1). However, a silica gel 60 column was used instead of the ODS-80T column. In addition, the color of HPTLC is
The Coomassie blue method and the ninhydrin method were used to detect only lysosphingolipids. The result of the FAB-MS analysis thus obtained is shown in FIG. The vertical axis represents relative intensity, and the horizontal axis represents mass number / charge number (M / Z). This Figure 6
FIG. 4 is an enlarged view of 400 to 550 (M / Z). As shown in FIG. 6, a signal 467 [(M + H) ⁺ ] corresponding to the molecular weight 466 of lysosphingomyelin was obtained as the strongest signal.

Example 3 Synthesis of derivative using lysosphingolipid (1) Introduction of single fatty acid into lysosphingolipid Introduction of fatty acid into lysosphingolipid (reacylation)
Was performed by synthesizing a fatty acid chloride and reacting it with lysosphingolipid. As a lysosphingolipid, G
Lyso GM1 prepared by the same method as in Example 2- (1) using M1 was used, and the fatty acids were C2: 0 and C14:
0, C16: 0, C18: 0, C22: 0, and C2
A molecular species of 4: 0 was used. 2 of lyso GM1 used in the reaction
~ 3 molar equivalents of fatty acid were placed in a small eggplant-shaped flask and 5-10 ml of thionyl chloride was added thereto. The mixture was heated to about 80 ° C. on a water bath while being refluxed with a cooling tube. At this time, a glass tube filled with calcium chloride was attached to the tip of the reflux tower in order to block water from the outside air. After completion of the reaction, thionyl chloride was removed by a nitrogen stream in the fume hood, and the mixture was left in a vacuum desiccator containing potassium hydroxide for 1 to 2 hours. After confirming that there was no odor of thionyl chloride, diethyl ether was added to the eggplant-shaped flask containing the reaction product and dissolved. To this, 1 μmol of Lyso GM1 previously dissolved in 1 ml of 0.3 M sodium hydrogen carbonate solution was added, and the mixture was stirred for 2 to 3 hours. The progress of the reaction was confirmed by TLC. After completion of the reaction, the reaction solution is dialyzed against ultrapure water to reacylate G
M1 was obtained. By this method, the target fatty acid could be introduced into Lyso GM1 with a yield of about 90% regardless of the length of the fatty acid carbon chain.

(2) Synthesis of Fluorescently Labeled Neosphingolipid A fluorescently labeled sphingolipid derivative (fluorescently labeled neosphingolipid) was synthesized by labeling the amino group of the sphingosine portion of lysosphingolipid. As lysosphingolipid, lyso GM was used as in Example 3- (1).
1 was used. In the case of labeling with dansyl chloride, lyso GM1 (1 μmol) was dissolved in 1 ml of 0.2 M sodium hydrogen carbonate solution, and an equal amount of 0.25% dansyl chloride acetone solution was added thereto. After leaving still at 37 ° C. for 1 hour in the dark, dialysis was performed against ultrapure water. On the other hand, NBD
In the case of labeling with -F, 500 μl of 0.5 nmol of lysoGM1 was added and the mixture was kept warm at 60 ° C. for 1 minute on a water bath. Immediately after completion of the reaction, the mixture was ice-cooled and dialyzed against ultrapure water at 4 ° C. for about 2 hours. In any labeling reaction, it is necessary to always shield the light. Confirmation of the synthesized fluorescence-labeled neosphingolipid and measurement of the synthetic yield were performed by TLC analysis. As a developing solvent, chloroform / methanol / 10% acetic acid (5: 4: 1, v / v) was used. The yield is dansylated G
Almost 100% in the case of M1 (dansyl-II ³ NeuAcα-Gg4-sphingosine), NBD-GM1 (NBD
-II ³ NeuAcα-Gg4-sphingosine) was 70 to 80%.

[0032]

INDUSTRIAL APPLICABILITY According to the present invention, a glycolipid ceramide deacylase having a very broad substrate specificity is provided. Furthermore, a method for producing lysosphingolipid from various sphingolipids by using the enzyme was provided. Furthermore, the lysosphingolipid thus obtained utilizes its free amino group to reintroduce a labeled fatty acid, or is directly fluorescently labeled, and further a protein having no sugar chain such as albumin. It can be converted into a sphingolipid derivative by binding with a conventional method, a substrate for measuring the activity of a sugar-related enzyme, and a ligand for affinity chromatography for purification, or an antigen for an anti-sphingolipid antibody, or It can be used to study the function of sphingolipids. Further, it can be used as a substrate and a reagent useful for elucidating intracellular metabolism and transport pathway of sphingolipid, elucidating intracellular function of sphingolipid, and the like.

[Brief description of drawings]

FIG. 1 is a graph showing the optimum pH of the glycolipid ceramide deacylase obtained by the present invention.

FIG. 2 is a graph showing the optimum temperature of the glycolipid ceramide deacylase obtained by the present invention.

FIG. 3 is a graph showing pH stability of the glycolipid ceramide deacylase obtained by the present invention.

FIG. 4 is a diagram showing the thermostability of the glycolipid ceramide deacylase obtained by the present invention.

FIG. 5 is a diagram showing a spectrum when lysoasialo GM1 obtained after digesting asialo GM1 with the glycolipid ceramide deacylase obtained by the present invention was subjected to FAB-MS analysis.

FIG. 6 is a diagram showing a spectrum when lysosphingomyelin obtained after digesting sphingomyelin with the glycolipid ceramide deacylase obtained by the present invention is subjected to FAB-MS analysis.

Claims (2)

[Claims] 1. A glycolipid ceramide deacylase having the following physicochemical properties. (1) Action: It acts on the intramolecular ceramide in the glycosphingolipid to hydrolyze it into a sphingosine base and a fatty acid to produce a lysoglycolipid and a fatty acid. (2) Substrate specificity: It acts on neutral glycolipids and acidic glycolipids. (3) Optimum pH: The optimum pH is 5-7. (4) Optimum temperature: The optimum temperature is about 40 ° C. (5) pH stability: pH 4 to 4 after treatment at 5 ° C. for 16 hours
It is stable in the range of 9. (6) Thermal stability: Stable for 30 minutes at 60 ° C. 2. A method for producing lysosphingolipid, which comprises treating a sphingolipid with the glycolipid ceramide deacylase according to claim 1.