JPH0499494A - Production of sphingoglycolipid - Google Patents
Production of sphingoglycolipidInfo
- Publication number
- JPH0499494A JPH0499494A JP2214116A JP21411690A JPH0499494A JP H0499494 A JPH0499494 A JP H0499494A JP 2214116 A JP2214116 A JP 2214116A JP 21411690 A JP21411690 A JP 21411690A JP H0499494 A JPH0499494 A JP H0499494A
- Authority
- JP
- Japan
- Prior art keywords
- ceramide
- glycosphingolipids
- oligosaccharide
- glycosphingolipid
- derived
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 6
- 229940106189 ceramide Drugs 0.000 claims abstract description 49
- 150000002339 glycosphingolipids Chemical class 0.000 claims abstract description 44
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 claims abstract description 41
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 claims abstract description 41
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 claims abstract description 41
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 claims abstract description 39
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 22
- 150000002482 oligosaccharides Chemical class 0.000 claims abstract description 22
- 241000283690 Bos taurus Species 0.000 claims abstract description 18
- 210000004556 brain Anatomy 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 14
- 235000000346 sugar Nutrition 0.000 claims abstract description 13
- OIZGSVFYNBZVIK-FHHHURIISA-N 3'-sialyllactose Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)O[C@@H]1[C@@H](O)[C@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@@H]1O OIZGSVFYNBZVIK-FHHHURIISA-N 0.000 claims abstract description 6
- 150000001783 ceramides Chemical class 0.000 claims description 8
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 5
- IESOVNOGVZBLMG-BUZVEHKISA-N alpha-Neup5Ac-(2->8)-alpha-Neup5Ac-(2->3)-beta-D-Galp-(1->4)-beta-D-Glcp Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)O[C@H](CO)[C@@H](O)[C@H]1[C@H](NC(C)=O)[C@@H](O)C[C@@](C(O)=O)(O[C@@H]2[C@H]([C@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)O[C@@H]3CO)O)O[C@H](CO)[C@@H]2O)O)O1 IESOVNOGVZBLMG-BUZVEHKISA-N 0.000 claims description 5
- WPIHMWBQRSAMDE-YCZTVTEBSA-N beta-D-galactosyl-(1->4)-beta-D-galactosyl-N-(pentacosanoyl)sphingosine Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCC(=O)N[C@@H](CO[C@@H]1O[C@H](CO)[C@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@H](O)[C@H]1O)[C@H](O)\C=C\CCCCCCCCCCCCC WPIHMWBQRSAMDE-YCZTVTEBSA-N 0.000 claims description 5
- 150000002270 gangliosides Chemical class 0.000 claims description 5
- 239000008101 lactose Substances 0.000 claims description 5
- PFJKOHUKELZMLE-VEUXDRLPSA-N ganglioside GM3 Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@@H]([C@H](O)/C=C/CCCCCCCCCCCCC)NC(=O)CCCCCCCCCCCCC\C=C/CCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O1 PFJKOHUKELZMLE-VEUXDRLPSA-N 0.000 claims description 4
- 229930186217 Glycolipid Natural products 0.000 abstract description 11
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 abstract description 6
- 238000006911 enzymatic reaction Methods 0.000 abstract description 6
- 241000316848 Rhodococcus <scale insect> Species 0.000 abstract description 5
- 150000002632 lipids Chemical class 0.000 abstract description 5
- 239000006227 byproduct Substances 0.000 abstract description 4
- 125000001549 ceramide group Chemical group 0.000 abstract description 2
- 229930182470 glycoside Natural products 0.000 abstract description 2
- 230000000813 microbial effect Effects 0.000 abstract 1
- 239000000203 mixture Substances 0.000 description 9
- 108010005965 endoglycoceramidase Proteins 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- AWDRATDZQPNJFN-VAYUFCLWSA-N taurodeoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 AWDRATDZQPNJFN-VAYUFCLWSA-N 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000004809 thin layer chromatography Methods 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 229930014626 natural product Natural products 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 108010085659 ceramide glycanase Proteins 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- -1 D+b and GT+b Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000545744 Hirudinea Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 150000001414 amino alcohols Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 229930183167 cerebroside Natural products 0.000 description 1
- 150000001784 cerebrosides Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 241001233061 earthworms Species 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 150000002298 globosides Chemical class 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002337 glycosamines Chemical class 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、オリゴ環とセラミドを出発原料とし、糖脂質
（グリコスフィンゴリピド）の糖鎖とセラミドとの間の
グリコシド結合を切断してオリゴ糖を遊離する酵素を用
いて酵素的にスフィンゴ糖脂質を製造する方法に関する
。Detailed Description of the Invention [Field of Industrial Application] The present invention uses an oligo ring and ceramide as starting materials, and cleaves the glycosidic bond between the sugar chain of a glycolipid (glycosphingolipid) and the ceramide to produce an oligomer. This invention relates to a method for enzymatically producing glycosphingolipids using an enzyme that releases sugar.
スフィンゴ糖脂質は一種の不飽和アミノアルコールであ
るスフィンゴシンまたはその誘導体をアグリコンとする
配糖体で、通常この部分は脂肪酸がアミド結合したセラ
ミドと呼ばれる脂質構造となっている。糖鎖はグルコー
スやガラクトースなどの中性糖だけを含むセレブロシド
、Ｎ−アセチルグリコサミンやＮ−アセチルガラクトサ
ミンなどのアミノ糖を含むグロボシド、シアル酸を含む
ガングリオシド、硫酸エステルを含むスルファチドなど
がある。スフィンゴ糖脂質は動物界の代表的な糖脂質で
、セラミドの構造や糖鎖の構造が異なる多くの分子種が
ある。このスフィンゴ糖脂質は細胞膜表面に存在して、
細胞の識別や情報の受容と応答、分化、増殖、悪性変化
、免疫、レセプター機能など重要な膜機能に関与してお
り、さらに、ウィルスや細菌に対する感染阻止活性が認
められるなど、生化学的に重要な意義を持つ物質である
。Glycosphingolipids are glycosides whose aglycones are sphingosine, a type of unsaturated amino alcohol, or its derivatives, and this moiety usually has a lipid structure called ceramide in which fatty acids are bonded with amide bonds. Sugar chains include cerebrosides containing only neutral sugars such as glucose and galactose, globosides containing amino sugars such as N-acetylglycosamine and N-acetylgalactosamine, gangliosides containing sialic acid, and sulfatides containing sulfate esters. Glycosphingolipids are representative glycolipids in the animal kingdom, and there are many molecular types with different ceramide structures and sugar chain structures. This glycosphingolipid exists on the surface of the cell membrane,
It is involved in important membrane functions such as cell identification, information reception and response, differentiation, proliferation, malignant changes, immunity, and receptor function.Furthermore, it is biochemically known to have infection inhibiting activity against viruses and bacteria. It is a substance with important significance.
従来、スフィンゴ糖脂質は天然物からの抽出や化学的な
合成によって得られている。特に、化学的な合成でラク
トシルセラミドやガングリオシドＧＭ３などが大量に製
造されている。しかし、その操作は煩雑で、かつ危険性
が高く、さらに、副産物が大量に生成するという問題も
ある。Conventionally, glycosphingolipids have been obtained by extraction from natural products or chemical synthesis. In particular, lactosylceramide and ganglioside GM3 are produced in large quantities through chemical synthesis. However, the operation is complicated and highly dangerous, and there is also the problem that a large amount of by-products are produced.
本発明者らは、任意のオリゴ環とセラミドを出発原料と
し、種々のスフィンゴ糖脂質を面側に合成する方法につ
いて鋭意検討を行った結果、糖脂ｆ（グリコスフィンゴ
リピド）の糖鎖とセラミドとの間のグリコシド結合を切
断してオリゴ糖を遊離する酵素を用いることによって目
的とするスフィンゴ糖脂質を合成し得ることを見出し、
本発明を成すに至った。The present inventors conducted intensive studies on methods for synthesizing various glycosphingolipids on the surface side using arbitrary oligo rings and ceramides as starting materials. discovered that it is possible to synthesize the desired glycosphingolipid by using an enzyme that releases oligosaccharides by cleaving the glycosidic bonds between
The present invention has been accomplished.
従って本発明は、糖脂質（グリコスフィンゴリピド）の
糖鎖とセラミドとの間のグリコシド結合を切断してオリ
ゴ糖を遊離する酵素を用い、任意のオリゴ環とセラミド
を出発原料として種々のスフィンゴ糖脂質を合成するス
フィンゴ糖脂質の新規な製造方法を提供することを課題
とする。Therefore, the present invention uses an enzyme that releases oligosaccharides by cleaving the glycosidic bonds between the sugar chains of glycosphingolipids and ceramides, and uses arbitrary oligo rings and ceramides as starting materials to produce various sphingosaccharides. An object of the present invention is to provide a novel method for producing glycosphingolipids by synthesizing lipids.
本発明では、スフィンゴ糖脂質を構成する任意のオリゴ
環とセラミドを基質とし、糖脂質（グリコスフィンゴリ
ピド）の糖鎖とセラミドとの間のグリコシド結合を切断
してオリゴ糖をＭＨする酵素（エンドグリコセラミダー
ゼ、あるいはセラミドグリカナーゼと称して市販されて
いる〕を作用させて種々のスフィンゴ糖脂質を酵素的に
合成する。In the present invention, an enzyme (endo) that uses any oligo ring constituting a glycosphingolipid and ceramide as a substrate and cleaves the glycosidic bond between the sugar chain of the glycosphingolipid and the ceramide to MH the oligosaccharide. Various glycosphingolipids are enzymatically synthesized by the action of glycoceramidase (commercially available as ceramide glycanase).
本発明におけるオリゴ環には、シアリルラクトース、ジ
シアリルラクトース、ラクトース、牛脳糖脂質混合物か
ら酵素的にセラミドを遊離させて得られるオリゴ糖混合
物等が用いられ、またセラミドは牛脳由来のセラミドを
例示することができる。これらの反応は、まず、酵素反
応溶液のｐＨを４．０〜８．０に調整して１０−Ｕ〜Ｉ
Ｕ／ｄの糖脂質（グリコスフィンゴリピド）の糖鎖とセ
ラミドとの間のグリコシド結合を切断してオリゴ環とセ
ラミドとを生成する酵素を添加し、温度２０〜５０°Ｃ
で２〜４８時間酵素反応を行う、基質濃度はオリゴ環５
〜５０％（Ｗ／Ｖ）、セラミド０．１〜５％体／ν）と
し、さらに、Ｔｒｉｔｏｎ Ｘ−１００やタウロデオキ
シコール酸などの界面活性剤を０．５〜３％（圓ハ）添
加することが好ましい
本発明で用いる酵素は、糖脂質（グリコスフィンゴリピ
ド）の糖鎖とセラミドとの間のグリコシド結合を切断し
てオリゴ環とセラミドとを生成する酵素であって、ロド
コッカス属（Ｒｈｏｄｏｃｏｃｃｕｓｓｐ、）に属する
微生物が菌体外に分泌することが知られており〔特開昭
６２−６９９８１号、特開昭６２−１２２５８７号、特
開平１−３０９６７７号、化学と工業、邦、９４６９４
９ （１９９０） Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｂ
ｉｏｌｏｇｉｃａｌ Ｃｈｅ＋ｍ１ｓｔｒｙ ２６１
（３０）、 １４２７８−１４２８２（１９８６）
、同２６４ （１６）９５１０−９５１９（１９８９）
）、「エンドグリコセラミダーゼ」として市販（三菱
化成工業■製）されている。For the oligo ring in the present invention, sialyllactose, disialyllactose, lactose, an oligosaccharide mixture obtained by enzymatically releasing ceramide from a bovine brain glycolipid mixture, etc. are used, and the ceramide is a ceramide derived from bovine brain. I can give an example. In these reactions, first, the pH of the enzyme reaction solution is adjusted to 4.0 to 8.0, and 10-U to I
An enzyme that cleaves the glycosidic bond between the sugar chain of U/d glycosphingolipid and ceramide to generate an oligo ring and ceramide is added, and the temperature is 20 to 50°C.
The enzyme reaction is carried out for 2 to 48 hours at a substrate concentration of oligo ring 5.
-50% (W/V), ceramide 0.1-5% body/ν), and further added 0.5-3% (W/V) of surfactants such as Triton X-100 and taurodeoxycholic acid. The enzyme preferably used in the present invention is an enzyme that cleaves the glycosidic bond between the sugar chain of a glycolipid (glycosphingolipid) and ceramide to produce an oligo ring and ceramide. It is known that microorganisms belonging to .
9 (1990) The Journal of B
iological Che+m1stry 261
(30), 14278-14282 (1986)
, 264 (16) 9510-9519 (1989)
), is commercially available as "endoglycoceramidase" (manufactured by Mitsubishi Chemical Industries, Ltd.).
また、ヒル及びミミズからも同様の酵素が見出されてお
り（Ｂｉｏｃｈｅｍｉｃａｌ ａｎｄ Ｂｉｏｐｈｙｓ
ｉｃａｌ Ｒｅ５ｅａｒｃｈｃｏ＋ｕ＋ｕｎｉｃａｔｉ
ｏｎｓｊＪｌ （１）、 ３５３−３５９（１９８６）
３「セラミド グリカナーゼＪの商品名（バイオファー
ム社）で試薬として市販されている。Similar enzymes have also been found in leeches and earthworms (Biochemical and Biophys
ical Re5earchco+u+unicati
onsjJl (1), 353-359 (1986)
3. It is commercially available as a reagent under the trade name of Ceramide Glycanase J (Biopharm).
基質として用いるオリゴ環は合成品あるいは天然物由来
のオリゴ環でよく、合成するスフィンゴ糖脂質の目的に
あったものを適宜選択することができる。また、基質と
しで用いるセラミドも合成品あるいは天然物由来のセラ
ミドでよく、合成するスフィンゴ糖脂質の目的にあった
ものを適宜選択することができる。The oligo ring used as a substrate may be a synthetic product or an oligo ring derived from a natural product, and can be appropriately selected according to the purpose of the glycosphingolipid to be synthesized. Furthermore, the ceramide used as the substrate may be a synthetic product or a ceramide derived from a natural product, and can be appropriately selected according to the purpose of the glycosphingolipid to be synthesized.
例えば、前記したようにオリゴ糖としてラクトースヲ用
い、セラミドとして牛脳由来のセラミドを用いることに
よってラクトシルセラミドが生成し・オリゴ糖としてシ
アリルラクトースを用い、セラミドとして牛脳由来のセ
ラミドを用いることによってガングリオシドＧ？１３が
生成し、オリゴ糖としてジシアリルラクトースを用い、
セラミドとして牛脳由来のセラミドを用いることによっ
てガングリオシドＧＤ３が化成する。オリゴ糖として牛
１１１ｉｉ［脂質混合物からセラミドを遊離させて得ら
れるオリゴ糖混合物を用い、セラミドとして牛脳由来の
セラミドを用いることによりＧＭ＋、 ＣＤｌ−、ＣＤ
ｌｂ。For example, as mentioned above, lactosylceramide is produced by using lactose as the oligosaccharide and ceramide derived from bovine brain as the ceramide, and ganglioside is produced by using sialyllactose as the oligosaccharide and ceramide derived from bovine brain as the ceramide. G? 13 is produced, using disialyllactose as the oligosaccharide,
Ganglioside GD3 is chemically synthesized by using ceramide derived from bovine brain as ceramide. By using bovine 111ii [an oligosaccharide mixture obtained by releasing ceramide from a lipid mixture as the oligosaccharide and using ceramide derived from bovine brain as the ceramide, GM+, CDl-, CD
lb.
ＧＴ、ｂ等もとの牛脳糖脂質と同しスフィンゴ糖脂質を
生成する。It produces the same glycosphingolipids as the original bovine brain glycolipids such as GT and b.
このようにして生成したスフィンゴ糖脂質は、薄層クロ
マトグラフィーによって確認できる。Glycosphingolipids produced in this way can be confirmed by thin layer chromatography.
本発明の方法によって得られる反応生成物は、副産物の
生成が少なくスフィンゴ糖脂質の含量が高いので、反応
に関与しなかった原料のオリゴ環、セラミド、糖脂質（
グリコスフィンゴリピド）の糖鎖とセラミド結合を切断
してオリゴ糖を遊離する酵素あるいは緩衝液の成分、界
面活性剤等を除き、これをこのままあるいは乾燥させて
スフィンゴ糖脂質として用いることができる。また、こ
の反応生成物からスフィンゴ糖脂質を単離してもよい。The reaction product obtained by the method of the present invention has a high content of glycosphingolipids with few by-products, so it is free from the oligo rings, ceramides, and glycolipids of the raw materials that did not participate in the reaction.
By removing enzymes, buffer components, surfactants, etc. that cleave the sugar chains and ceramide bonds of glycosphingolipids and liberating oligosaccharides, the glycosphingolipids can be used as they are or after being dried as glycosphingolipids. Additionally, glycosphingolipids may be isolated from this reaction product.
単離方法としては、有機化合物の精製単離に用いられる
クロマトグラフィー等が用いられる。As an isolation method, chromatography or the like used for purifying and isolating organic compounds is used.
以下に実施例を示して本発明の詳細な説明する。The present invention will be described in detail below with reference to Examples.
実施例（１）（ラクトシルセラミドの合成）ラクトース
３００■、セラミド（牛脳由来）１０■を２χ（Ｗ／Ｖ
）タウロデオキシコール酸を含むｐＨ５，０の０．１Ｍ
リン酸緩衝液７００μ２に濁した。この懸濁液にロドコ
ッカス由来の「エンドグリコセラミダーゼ」 （三菱化
成工業■製）５ｓ＋Ｕを添加し３０°Ｃにて２４時間反
応させた０反応後反応液から、ラクトース、タラロブキ
シコール酸、［エンドグリコセラミダーゼ」、及び緩衝
液をＯＤＳ （ｓｅｐ−ｐａｃｋ Ｃ１Ｂ）を用いて除
去しスフィンゴ糖脂質画分を得た。得られた両分につい
て薄層クロマトグラフィーを行ない牛乳由来のラクトシ
ルセラミドと移動度の等しいスフィンゴ糖脂質が生成し
ていることを確認した。Example (1) (Synthesis of lactosylceramide) 300 μ of lactose and 10 μ of ceramide (derived from bovine brain) were mixed into 2× (W/V
) 0.1M at pH 5.0 containing taurodeoxycholic acid
The mixture was suspended in 700μ2 of phosphate buffer. To this suspension, "endoglycoceramidase" derived from Rhodococcus (manufactured by Mitsubishi Chemical Industries, Ltd.) 5s+U was added and reacted at 30°C for 24 hours. From the reaction solution, lactose, talaloboxycholic acid, [ "Endoglycoceramidase" and the buffer were removed using ODS (sep-pack C1B) to obtain a glycosphingolipid fraction. Thin layer chromatography was performed on both of the obtained samples, and it was confirmed that glycosphingolipids having the same mobility as milk-derived lactosylceramide were produced.
実施例（２）（ガングリオシドＧＭ３の合成）シアリル
ラクトース３００［、セラミド（牛脳由来）１０■を２
χ（−／ν）タウロデオキシコール酸を含むｐＨ５，０
，０，１ＭＩＪ ン酸緩衝液７００ＩＩＪ！に熔解した
。Example (2) (Synthesis of ganglioside GM3) sialyllactose 300 [, ceramide (derived from bovine brain) 10]
pH 5.0 containing χ(-/ν) taurodeoxycholic acid
,0,1MIJ acid buffer 700IIJ! It melted into
この溶液にロドコッカス由来の「エンドグリコセラミダ
ーゼ」５■Ｕを添加し３０”Ｃにおいて２４時間反応さ
せた０反応後シアリルラクトース、タウロデオキシコー
ル酸、［エンドグリコセラミダーゼ」、及び緩衝液を０
ＤＳ（ｓｅｐ−ｐａｃｋ Ｃｌ８）を用いて除去し、ス
フィンゴ糖脂質画分を得た。得られた百分について薄層
クロマトグラフィーを行ない牛乳由来のガングリオシド
ＧＭ３と移動度の等しいスフィンゴ糖脂質が生成してい
ることを確認した。To this solution, 5 U of "endoglycoceramidase" derived from Rhodococcus was added and reacted at 30"C for 24 hours. After the reaction, sialyllactose, taurodeoxycholic acid, "endoglycoceramidase" and buffer solution were added to 0.
It was removed using DS (sep-pack Cl8) to obtain a glycosphingolipid fraction. The obtained percentage was subjected to thin layer chromatography and it was confirmed that glycosphingolipid having the same mobility as milk-derived ganglioside GM3 was produced.
実施例（３）（ガングリオシドＧＤ３の合成）ジシアリ
ルラクトース３００■、セラミド（牛脳由来）１０■を
２２ （Ｗ／Ｖ）タウロデオキシコール酸を含むｐＨ５
，０，０，ＩＭ リフ酸緩衝液７００ｕｌに溶解した。Example (3) (Synthesis of ganglioside GD3) 300μ of disialyllactose, 10μ of ceramide (derived from bovine brain), pH 5 containing 22 (W/V) taurodeoxycholic acid
,0,0,IM Dissolved in 700ul of phosphate buffer.
この溶液にロドコッカス由来の「エンドグリコセラミダ
ーゼ」５−Ｕを添加し３０’Ｃで２４時間酵素反応させ
た０反応後ジシアリルラクトース、タウロデオキシコー
ル酸、［エンドグリコセラミダーゼ」、及び緩衝液を０
ＤＳ（ｓｅｐ−ｐａｃｋ Ｃ１Ｂ）を用いて除去しスフ
ィンゴ糖脂質画分を得た。得られた画分について薄層ク
ロマトグラフィーを行ない牛乳由来のカンクリオシドＧ
Ｄ３と移動度の等しいスフィンゴ糖脂質が生成している
ことを確認した。5-U of "endoglycoceramidase" derived from Rhodococcus was added to this solution, and enzymatic reaction was carried out at 30'C for 24 hours. After the reaction, disialyllactose, taurodeoxycholic acid, "endoglycoceramidase" and the buffer solution were added to the solution.
It was removed using DS (sep-pack C1B) to obtain a glycosphingolipid fraction. The obtained fractions were subjected to thin layer chromatography to detect milk-derived cancrioside G.
It was confirmed that a glycosphingolipid with the same mobility as D3 was produced.
実施例（４）
牛脳糖脂質混合物よりセラミドを遊離させて得られるオ
リゴ糖混合物３００ｍｇ 、セラミド（牛脳由来）１０
■、２％（ｗ／ｖ）タウロデオキシコール［含量ｐＨ５
，０，０，１？ｌＩＪ：／酸１ａ衝液７００μｆにｉ解
した。この溶液にロドコッカス由来の「エンドグリコセ
ラミダーゼ」５−ｕを添加し３０”Ｃで２４時間反応さ
せた０反応後、未反応の牛脳糖脂質混合物よりセラミド
を遊離させて得られるオリゴ糖混合物、タウロデオキシ
コール酸、Ｅエンドグリコセラミダーゼ」、及び緩衝液
を００５（ｓｅｐ−ｐａｃｋ Ｃｌ８）を用いて除去し
スフィンゴ糖脂質画分を得た。得られた両分について薄
層クロマトグラフィーを行ないＧＭ＋、 ＧＤ＋−、Ｇ
Ｄ＋ｂ、 ＧＴ＋ｂ等もとの糖脂質と移動度の等しいス
フィンゴ糖脂質を生成していることを確認した。Example (4) 300 mg of oligosaccharide mixture obtained by liberating ceramide from a bovine brain glycolipid mixture, 10 ceramides (derived from bovine brain)
■, 2% (w/v) taurodeoxycol [content pH 5
,0,0,1? lIJ:/I was dissolved in 700 μf of acid 1a buffer solution. After adding 5-u of "endoglycoceramidase" derived from Rhodococcus to this solution and reacting at 30"C for 24 hours, an oligosaccharide mixture obtained by releasing ceramide from the unreacted bovine brain glycolipid mixture, Taurodeoxycholic acid, E-endoglycoceramidase, and buffer were removed using 005 (sep-pack Cl8) to obtain a glycosphingolipid fraction. Thin layer chromatography was performed on both obtained fractions to determine GM+, GD+-, and G.
It was confirmed that glycosphingolipids such as D+b and GT+b, which have the same mobility as the original glycolipids, were produced.
本発明の方法によるとオリゴ糖とセラミドとを糖脂質（
グリコスフィンゴリピド）の糖鎖とセラミドとの間のグ
リコシド結合を切断してオリゴ垢を遊離する酵素を使用
し、酵素反応によってスフィンゴ糖脂質を合成するので
、比較的簡単な操作によってスフィンゴ糖脂質を合成す
ることができる。しかも反応副産物の生成が少（、反応
液から未反応の原料を除くだけで純度の高いスフィンゴ
糖脂質を得ることができ、精製が容易である。According to the method of the present invention, oligosaccharides and ceramides are combined into glycolipids (
Glycosphingolipids are synthesized through an enzymatic reaction using an enzyme that cleaves the glycosidic bonds between the sugar chains of glycosphingolipids and ceramide to release oligosaccharides, so glycosphingolipids can be synthesized through relatively simple operations. Can be synthesized. In addition, only a small amount of reaction by-products are produced (highly pure glycosphingolipids can be obtained simply by removing unreacted raw materials from the reaction solution, and purification is easy).
Claims (4)
ンゴリピド）の糖鎖とセラミドとの間のグリコシド結合
を切断してオリゴ糖を遊離する酵素を用いて酵素的に反
応させてスフィンゴ糖脂質を得ることを特徴とするスフ
ィンゴ糖脂質の製造方法。(1) Oligosaccharides and ceramides are enzymatically reacted using an enzyme that releases oligosaccharides by cleaving the glycosidic bonds between the sugar chains of glycosphingolipids and ceramides to produce glycosphingolipids. A method for producing a glycosphingolipid, characterized in that it obtains a glycosphingolipid.
として牛脳由来のセラミドを用い、ガングリオシドＧＭ
３を得ることを特徴とする請求項（１）によるスフィン
ゴ糖脂質の製造方法。(2) Using sialyllactose as the oligosaccharide and ceramide derived from bovine brain as the ceramide, ganglioside GM
3. The method for producing glycosphingolipids according to claim 1, characterized in that 3 is obtained.
ドとして牛脳由来のセラミドを用い、ガングリオシドＧ
Ｄ３を得ることを特徴とする請求項（１）によるスフィ
ンゴ糖脂質の製造方法。(3) Using disialyllactose as the oligosaccharide and ceramide derived from bovine brain as the ceramide, ganglioside G
The method for producing glycosphingolipids according to claim 1, characterized in that D3 is obtained.
脳由来のセラミドを用い、ラクトシルセラミドを得るこ
とを特徴とする請求項（１）によるスフィンゴ糖脂質の
製造方法。(4) The method for producing glycosphingolipids according to claim (1), characterized in that lactosylceramide is obtained by using lactose as the oligosaccharide and ceramide derived from bovine brain as the ceramide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21411690A JP2832746B2 (en) | 1990-08-13 | 1990-08-13 | Method for producing glycosphingolipid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21411690A JP2832746B2 (en) | 1990-08-13 | 1990-08-13 | Method for producing glycosphingolipid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0499494A true JPH0499494A (en) | 1992-03-31 |
| JP2832746B2 JP2832746B2 (en) | 1998-12-09 |
Family
ID=16650500
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP21411690A Expired - Fee Related JP2832746B2 (en) | 1990-08-13 | 1990-08-13 | Method for producing glycosphingolipid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2832746B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0730033A3 (en) * | 1995-03-03 | 1998-03-11 | Snow Brand Milk Products Co., Ltd. | Method of producing glycosphingolipid |
| JP2008502329A (en) * | 2004-06-01 | 2008-01-31 | ネオス テクノロジーズ インコーポレイティッド | Mutant endoglycoceramidase with enhanced synthetic activity |
| WO2008123070A1 (en) * | 2007-03-27 | 2008-10-16 | Snow Brand Milk Products Co., Ltd. | Method for production of lactosylceramide |
-
1990
- 1990-08-13 JP JP21411690A patent/JP2832746B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0730033A3 (en) * | 1995-03-03 | 1998-03-11 | Snow Brand Milk Products Co., Ltd. | Method of producing glycosphingolipid |
| JP2008502329A (en) * | 2004-06-01 | 2008-01-31 | ネオス テクノロジーズ インコーポレイティッド | Mutant endoglycoceramidase with enhanced synthetic activity |
| WO2008123070A1 (en) * | 2007-03-27 | 2008-10-16 | Snow Brand Milk Products Co., Ltd. | Method for production of lactosylceramide |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2832746B2 (en) | 1998-12-09 |
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