JP3100559B2 - Process for producing lacto-N-neotetraose - Google Patents

Process for producing lacto-N-neotetraose

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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
Application number
JP09058295A
Other languages
Japanese (ja)
Other versions
JPH10234394A (en
Inventor
健臣 村田
泰市 碓氷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yamasa Corp
Original Assignee
Yamasa Corp
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Yamasa Corp filed Critical Yamasa Corp
Priority to JP09058295A priority Critical patent/JP3100559B2/en
Publication of JPH10234394A publication Critical patent/JPH10234394A/en
Application granted granted Critical
Publication of JP3100559B2 publication Critical patent/JP3100559B2/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

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Description

【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION

【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
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/CH3
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]

【図面の簡単な説明】[Brief description of the drawings]

【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)

(57)【特許請求の範囲】(57) [Claims] 【請求項1】 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.
【請求項2】 グリコシダーゼがβ−D−ガラクトシダ
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.
【請求項3】 ガラクトース供与体がラクトースであ
る、請求項1記載の方法。
3. The method according to claim 1, wherein the galactose donor is lactose.
JP09058295A 1997-02-26 1997-02-26 Process for producing lacto-N-neotetraose Expired - Fee Related JP3100559B2 (en)

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
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Country Status (1)

Country Link
JP (1) JP3100559B2 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Glycoconjugate Journal,Vol.16,No.3(1999),p.189−195

Also Published As

Publication number Publication date
JPH10234394A (en) 1998-09-08

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