JP3100559B2 - Process for producing lacto-N-neotetraose - Google Patents
Process for producing lacto-N-neotetraoseInfo
- Publication number
- JP3100559B2 JP3100559B2 JP09058295A JP5829597A JP3100559B2 JP 3100559 B2 JP3100559 B2 JP 3100559B2 JP 09058295 A JP09058295 A JP 09058295A JP 5829597 A JP5829597 A JP 5829597A JP 3100559 B2 JP3100559 B2 JP 3100559B2
- Authority
- JP
- Japan
- Prior art keywords
- lnnt
- neotetraose
- reaction
- lacto
- producing
- 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.)
- Expired - Fee Related
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、グリコシダーゼに
よる糖転移反応によりラクト−Ｎ−トリオースＩＩ（Ｌ
ＮＴ−２）とガラクトース供与体からラクト−Ｎ−ネオ
テトラオース（ＬＮｎＴ）を合成することを特徴とする
ＬＮｎＴの製造法に関するものである。TECHNICAL FIELD The present invention relates to a lacto-N-triose II (L
The present invention relates to a method for producing LNnT, comprising synthesizing lacto-N-neotetraose (LNnT) from NT-2) and a galactose donor.
【０００２】[0002]
【従来の技術】ＬＮｎＴは、複合糖質のオリゴ糖基本単
位として知られている。従来、ＬＮｎＴは主として多段
階反応による化学合成法で造られており、合成手順が複
雑であるとともに、その収率も満足いくものではなかっ
た。その結果、合成されたＬＮｎＴは極めて高価とな
り、オリゴ糖研究の障害とさえなっていた。2. Description of the Related Art LNnT is known as a basic unit of oligosaccharide of glycoconjugate. Heretofore, LNnT has been produced mainly by a chemical synthesis method based on a multi-step reaction, the synthesis procedure has been complicated, and the yield thereof has not been satisfactory. As a result, the synthesized LNnT became extremely expensive and even hindered oligosaccharide research.
【０００３】[0003]
【発明が解決しようとする課題】このように、オリゴ糖
基本単位であるＬＮｎＴを安価に、しかも大量に製造す
る方法を見いだすことができれば、オリゴ糖の研究をよ
り一層促進することができる。As described above, if a method for producing LNnT, which is an oligosaccharide basic unit, at low cost and in large quantities can be found, research on oligosaccharides can be further promoted .
【０００４】[0004]
【課題を解決するための手段】本発明者らは、ＬＮｎＴ
を安価に、しかも大量に製造することの可能な方法を見
いだすべく、研究を重ねた結果、β−Ｄ−ガラクトシダ
ーゼなどのグリコシダーゼを用いる糖転移反応を応用す
ることでＬＮｎＴを安価に、しかも大量に製造すること
が可能であることを見いだし、本発明を完成させた。し
たがって、本発明は、グリコシダーゼによる糖転移反応
によりＬＮＴ−２とガラクトース供与体からＬＮｎＴを
合成することを特徴とするＬＮｎＴの製造法に関するも
のである。Means for Solving the Problems The present inventors have proposed LNnT
As a result of repeated studies to find a method capable of producing LNnT inexpensively and in large quantities, LNnT can be produced inexpensively and in large quantities by applying a glycosyltransfer reaction using a glycosidase such as β-D-galactosidase. It has been found that it can be manufactured, and the present invention has been completed. Therefore, the present invention relates to a method for producing LNnT, which comprises synthesizing LNnT from LNT-2 and a galactose donor by a glycosidase-mediated transglycosylation reaction.
【０００５】[0005]
【発明の実施の形態】本明細書において、「ラクト−Ｎ
−ネオテトラオース」または「ＬＮｎＴ」は、Ｇａｌβ
１−４βＧｌｃＮＡｃβ１−３Ｇａｌβ１−４Ｇｌｃを
意味する。また、「ラクト−Ｎ−トリオースＩＩ」また
は「ＬＮＴ−２」は、ＧｌｃＮＡｃβ１−３Ｇａｌβ１
−４Ｇｌｃを意味する。BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, "lacto-N
“Neotetraose” or “LNnT” is Galβ
1-4βGlcNAcβ1-3Galβ1-4Glc is meant. “Lact-N-triose II” or “LNT-2” is GlcNAcβ1-3Galβ1.
-4Glc.
【０００６】本発明の方法に使用するグリコシダーゼと
しては、好ましくはβ−Ｄ−ガラクトシダーゼを例示す
ることができる。このようなβ−Ｄ−ガラクトシダーゼ
は公知の酵素であり、ガラクトシル残基を位置選択的に
ＬＮＴ−２に導入でき、もってＬＮｎＴを合成できる可
能性のあるものであれば、特にその起源には制限されな
い。このようなβ−Ｄ−ガラクトシダーゼとしては、バ
チラス・サーキュランス由来のもの、連鎖球菌由来のも
の、大腸菌由来のものなどが既に報告され、一部は市販
されている（大和化成、生化学工業、シグマ、ニューイ
ングランドバイオラボなど各社から入手可能）。[0006] The glycosidase used in the method of the present invention is preferably β-D-galactosidase. Such β-D-galactosidase is a known enzyme, and its source is particularly limited as long as it can introduce a galactosyl residue regioselectively into LNT-2 and thus can synthesize LNnT. Not done. As such β-D-galactosidase, those derived from Bacillus circulans, those derived from streptococci, those derived from Escherichia coli and the like have already been reported, and some of them are commercially available (Daiwa Kasei, Seikagaku Kogyo, (Available from companies such as Sigma and New England Biolab).
【０００７】本発明の方法で原料として使用するＬＮＴ
−２は公知の化合物であり、化学的あるいは酵素的な合
成法で容易に調製可能である（J. Carbohydr. Chem.,7,
359-376(1988)、Carbohydr. Res.,200,269-285(1990)、
E.J.Biochem.,96,33-337(1979)）。また、ガラクトース
供与体としては、還元末端がガラクトースである糖質、
例えば乳糖や、合成基質であるｐＮｐ−β−Ｇａｌなど
を用いることができる。LNT used as a raw material in the method of the present invention
-2 is a known compound, which can be easily prepared by a chemical or enzymatic synthesis method (J. Carbohydr. Chem., 7,
359-376 (1988), Carbohydr. Res., 200, 269-285 (1990),
EJBiochem., 96, 33-337 (1979)). Further, as a galactose donor, a saccharide whose reducing end is galactose,
For example, lactose or a synthetic substrate such as pNp-β-Gal can be used.
【０００８】糖転移反応は、酢酸緩衝液などの緩衝液中
（ｐＨ４〜９）、ＬＮＴ−２ １モルに対しガラクトー
ス供与体を１〜１０モル、好ましくは１〜６モル程度使
用し、β−Ｄ−ガラクトシダーゼ０．０００１〜１００
単位（Ｕ）、好ましくは０．００１〜１０単位使用し、
反応温度１０〜９０℃で１〜１００時間程度反応させる
ことにより実施することができる。なお、反応の最適条
件は使用したβ−Ｄ−ガラクトシダーゼにより変動しう
るものであり、小規模試験により決定すればよい。ただ
し、副反応物の生成を抑制し、反応効率を高めるために
は、ＬＮＴ−２とガラクトース供与体を等モルか、ＬＮ
Ｔ−２ １モルに対しガラクトース供与体を１〜２モル
程度使用するのが好ましい。The glycosyltransfer reaction is carried out in a buffer such as an acetate buffer (pH 4 to 9) using a galactose donor in an amount of 1 to 10 mol, preferably 1 to 6 mol, per 1 mol of LNT-2. D-galactosidase 0.0001-100
Use units (U), preferably 0.001-10 units,
The reaction can be carried out at a reaction temperature of 10 to 90 ° C. for about 1 to 100 hours. The optimum conditions for the reaction may vary depending on the β-D-galactosidase used, and may be determined by a small-scale test. However, in order to suppress the generation of by-products and increase the reaction efficiency, LNT-2 and the galactose donor are equimolar or
It is preferable to use about 1 to 2 mol of the galactose donor per 1 mol of T-2.
【０００９】このようにして調製したＬＮｎＴは、オリ
ゴ糖の通常の精製法（各種カラムクロマトグラフィーな
ど）にて単離精製することができる。[0009] The LNnT thus prepared can be isolated and purified by an ordinary method for purifying oligosaccharides (various column chromatography, etc.).
【００１０】[0010]
【発明の効果】本発明の方法は、安価な基質と安価な酵
素からＬＮｎＴを合成しようとするものであり、使用す
る酵素も安定であることから、ＬＮｎＴの実用的な製造
法になりうるものである。The method of the present invention is intended to synthesize LNnT from an inexpensive substrate and an inexpensive enzyme, and the enzyme used is stable, so that it can be a practical method for producing LNnT. It is.
【００１１】[0011]
【実施例】以下、実施例を示し、本発明を具体的に説明
するが、本発明がこれに限定されないことは明らかであ
る。 実施例１：ＬＮｎＴの合成 ２０ｍＭ酢酸緩衝液（ｐＨ６．０）中に、ＬＮＴ−２
５ｍｇとラクトース１５．４ｍｇを添加し、さらにβ−
Ｎ−アセチルヘキソサミニダーゼの阻害剤であるナグス
タチン（Nagstatin：明治製菓社）を０．０５ｍＭおよ
び２５ｍＵのβ−Ｄ−ガラクトシダーゼ（バチラス・サ
ーキュランス由来：ニューイングランドバイオラボ社）
を加え、４０℃で２８時間反応させた。反応後、反応液
を沸騰水中で５分間加熱することにより反応を停止させ
た。EXAMPLES The present invention will be described below in detail with reference to examples, but it is apparent that the present invention is not limited to these examples. Example 1 Synthesis of LNnT LNT-2 in 20 mM acetate buffer (pH 6.0)
5 mg and lactose 15.4 mg were added, and β-
Nagstatin (Nagstatin: Meiji Seika), an inhibitor of N-acetylhexosaminidase, was added to 0.05 mM and 25 mU of β-D-galactosidase (from Bacillus circulans: New England Biolab).
Was added and reacted at 40 ° C. for 28 hours. After the reaction, the reaction was stopped by heating the reaction solution in boiling water for 5 minutes.
【００１２】このようにして得られた反応液を分取用Ｏ
ＤＳカラム（Ｗａｋｏｓｉｌ ＩＩ５Ｃ１８ＨＧ：和光
純薬社製、溶出溶媒：０．０２％ＴＦＡ／ＣＨ3Ｃ
Ｎ））にかけ、ＬＮｎＴを取得した。なお、得られたＬ
ＮｎＴの構造確認は、常法（Anal. Biochem.,219,378(1
994)）によりＡＢＥＥで標識後、ＨＰＬＣ分析において
市販のＬＮｎＴ（フナコシ社）と同一位置に溶出するこ
とを確認するとともに、ＦＡＢ−ＭＳで行った。ＦＡＢ
−ＭＳの結果を図１に示す。ＨＰＬＣ分析とＦＡＢ−Ｍ
Ｓの結果から、上記酵素反応で得られたものがＬＮｎＴ
であることを確認した。[0012] The reaction solution thus obtained is separated by O
DS column (Wakosil II5C18HG: manufactured by Wako Pure Chemical Industries, Ltd., elution solvent: 0.02% TFA / CH 3 C)
N)) to obtain LNnT. Note that the obtained L
The structure of NnT can be confirmed by a conventional method (Anal. Biochem., 219, 378 (1.
After labeling with ABEE according to 994)), HPLC analysis confirmed elution at the same position as commercially available LNnT (Funakoshi), and FAB-MS was performed. FAB
FIG. 1 shows the results of the MS. HPLC analysis and FAB-M
From the results of S, the result obtained by the above enzyme reaction was LNnT
Was confirmed.
【００１３】[0013]
【図１】図１は、ＦＡＢ−ＭＳの結果を示したものであ
る。FIG. 1 shows the results of FAB-MS.
───────────────────────────────────────────────────── フロントページの続き (58)調査した分野(Int.Cl.7，ＤＢ名) C12P 19/00 - 19/64 C12N 9/00 - 9/99 ＢＩＯＳＩＳ（ＤＩＡＬＯＧ) ＭＥＤＬＩＮＥ（ＳＴＮ) ＷＰＩ（ＤＩＡＬＯＧ)────────────────────────────────────────────────── ─── Continued on the front page (58) Fields surveyed (Int. Cl. 7 , DB name) C12P 19/00-19/64 C12N 9/00-9/99 BIOSIS (DIALOG) MEDLINE (STN) WPI (DIALOG) )
Claims (3)
ラクト−Ｎ−トリオースＩＩとガラクトース供与体から
ラクト−Ｎ−ネオテトラオースを合成することを特徴と
するラクト−Ｎ−ネオテトラオースの製造法。1. A method for producing lacto-N-neotetraose, comprising synthesizing lacto-N-neotetraose from lacto-N-triose II and a galactose donor by a glycosidase-mediated transglycosylation reaction.
ーゼである、請求項１記載の方法。2. The method according to claim 1, wherein the glycosidase is β-D-galactosidase.
る、請求項１記載の方法。3. The method according to claim 1, wherein the galactose donor is lactose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP09058295A JP3100559B2 (en) | 1997-02-26 | 1997-02-26 | Process for producing lacto-N-neotetraose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP09058295A JP3100559B2 (en) | 1997-02-26 | 1997-02-26 | Process for producing lacto-N-neotetraose |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH10234394A JPH10234394A (en) | 1998-09-08 |
| JP3100559B2 true JP3100559B2 (en) | 2000-10-16 |
Family
ID=13080239
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP09058295A Expired - Fee Related JP3100559B2 (en) | 1997-02-26 | 1997-02-26 | Process for producing lacto-N-neotetraose |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3100559B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8993740B2 (en) | 2010-02-19 | 2015-03-31 | Glycom A/S | Method for preparation of the tetrasaccharide lacto-N-neotetraose (LNnt) containing N-acetyllactosamine |
-
1997
- 1997-02-26 JP JP09058295A patent/JP3100559B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| Ｇｌｙｃｏｃｏｎｊｕｇａｔｅ Ｊｏｕｒｎａｌ，Ｖｏｌ．１６，Ｎｏ．３（１９９９），ｐ．１８９−１９５ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH10234394A (en) | 1998-09-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Efficient chemoenzymatic synthesis of an N-glycan isomer library | |
| Augé et al. | The use of an immobilised cyclic multi-enzyme system to synthesise branched penta-and hexa-saccharides associated with blood-group I epitopes | |
| EP0096547B1 (en) | Process for preparing uridine diphosphate-n-acetylgalactosamine | |
| JPH04507345A (en) | Method for producing oligosaccharide compounds using glycosidase from MOLLUSC | |
| Wong et al. | Enzymic synthesis of L-fucose and analogs | |
| JP3100559B2 (en) | Process for producing lacto-N-neotetraose | |
| JP2001046096A (en) | PRODUCTION OF GLYCOSIDE WITH alpha-GLUCOSIDASE AND NEW alpha- GLUCOSIDASE AND ITS PRODUCTION | |
| Murata et al. | Enzymic synthesis of 3′-O-and 6′-ON-acetylglucosaminyl-N-acetyllactosaminide glycosides catalyzed by β-N-acetyl-D-hexosaminidase from Nocardia orientalis | |
| JP2002017392A (en) | Method for producing aldohexose by using d-xylose isomerase | |
| WO1998011247A1 (en) | Processes for producing sugar nucleotides and complex carbohydrates | |
| Fujimoto et al. | Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains | |
| Wang et al. | Chemoenzymatic syntheses of tumor‐associated carbohydrate antigen Globo‐H and stage‐specific embryonic antigen 4 | |
| Nilsson et al. | Production of glucosamine containing disaccharides of the Lewis-A and-x types employing glycosidases | |
| Bojarová et al. | N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide | |
| JPH0430276B2 (en) | ||
| JPH0440997B2 (en) | ||
| JP3002113B2 (en) | Method for producing carbohydrate or complex carbohydrate | |
| JP3045509B2 (en) | Method for producing mannose-containing oligosaccharides | |
| JP3105306B2 (en) | Method for producing carbohydrate or complex carbohydrate | |
| JP3062592B2 (en) | Process for producing 2-acetylamino-4-O- (2-amino-2-deoxy-β-D-glucopyranosyl) -2-deoxy-D-glucose and salts thereof | |
| JP2949509B2 (en) | Sialyl lactose | |
| JPH05227982A (en) | Disaccharide and trisaccharide, preparation of these compounds and composition for hair growing containing these compounds | |
| JPH07508413A (en) | Purified sucrose-synthase, its production method and its use | |
| JP3062591B2 (en) | p-Nitrophenyl 2-acetylamino-4-O- (2-amino-2-deoxy-β-D-glucopyranosyl) -2-deoxy-β-D-glucopyranoside and salts thereof and a method for producing the same | |
| JP3030646B2 (en) | Method for producing oligosaccharides containing α1- → 3 linked L-fucose |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |