JPH06153944A - Glucide hydrolase - Google Patents
Glucide hydrolaseInfo
- Publication number
- JPH06153944A JPH06153944A JP32897592A JP32897592A JPH06153944A JP H06153944 A JPH06153944 A JP H06153944A JP 32897592 A JP32897592 A JP 32897592A JP 32897592 A JP32897592 A JP 32897592A JP H06153944 A JPH06153944 A JP H06153944A
- Authority
- JP
- Japan
- Prior art keywords
- lacto
- bioside
- biosidase
- enzyme
- sugar chain
- 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.)
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- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は糖質の構造解析に有用な
新規エキソ型糖質加水分解酵素に関する。TECHNICAL FIELD The present invention relates to a novel exo-type sugar hydrolase useful for structural analysis of sugars.
【０００２】[0002]
【従来の技術】近年、高等動物由来の糖タンパク質、糖
脂質等の複合糖質中の糖鎖部分の構造と機能が研究され
ているが、このためには、特異性の高い糖加水分解酵素
が重要な役割を果す。従来、糖鎖の非還元末端から単糖
を遊離する酵素は種々の起源から単離され、糖鎖の構造
と機能の研究に利用されており、糖鎖の非還元末端から
二糖を遊離する酵素としては、デンプンからマルトース
を遊離するβ−アミラーゼやセルロースからセロビオー
スを遊離するセロビアーゼ等が知られている。糖鎖の非
還元末端には、しばしば、タイプ１の Galβ１-3GlcNAc
β１- 構造とタイプ２の Galβ１-4GlcNAcβ１- 構造の
２種類が見出される。従来この糖鎖構造の解析には、メ
チル化分析や核磁気共鳴スペクトル法などの分析法が用
いられてきたが、試料を大量に用いなければならないと
いう欠点を有していた。また微量の糖鎖試料で構造の推
定を行う方法として、タイプ１及び２の糖鎖を切断する
ストレプトコッカス（Streptococcus)6646K 又はナタマ
メのβ−ガラクトシダーゼと、タイプ２糖鎖のみを切断
するディプロコッカス ニューモニエ（Diprococcus pn
eumoniae) のβ−ガラクトシダーゼを併用する方法が知
られているが、ストレプトコッカス6646K 及びナタマメ
のβ−ガラクトシダーゼはいずれもタイプ２糖鎖の方を
速やかに加水分解するので、タイプ１の糖鎖を完全に加
水分解するには大量の酵素を用いて長時間反応させなけ
ればならないなど反応条件の設定がむずかしく、実際に
はタイプ１とタイプ２の糖鎖構造の識別は困難であり、
該ヘテロオリゴ糖に特異的に作用する酵素の提供が求め
られていた。本発明者らはタイプ１糖鎖構造に特異的に
作用して、その糖鎖中のラクト−Ｎ−ビオシド結合を加
水分解しラクト−Ｎ−ビオースを遊離するエキソ型糖質
加水分解酵素を見出し、該酵素をラクト−Ｎ−ビオシダ
ーゼと命名した〔プロシーディングズ オブ ザ ナシ
ョナル アカデミー オブ サイエンシーズ オブザ
ＵＳＡ（Proceedings of the National Academy of Sci
ences of the ＵＳＡ）、第８９巻、第８５１２頁（１
９９２）〕。2. Description of the Related Art In recent years, the structure and function of sugar chains in glycoconjugates such as glycoproteins and glycolipids derived from higher animals have been studied. Plays an important role. Conventionally, enzymes that release monosaccharides from the non-reducing end of sugar chains have been isolated from various sources and used for studying the structure and function of sugar chains, and release disaccharides from the non-reducing end of sugar chains. Known enzymes include β-amylase, which releases maltose from starch, and cellobiase, which releases cellobiose from cellulose. At the non-reducing end of the sugar chain, type 1 Galβ1-3GlcNAc is often used.
Two types are found, a β1-structure and a type 2 Galβ1-4GlcNAcβ1-structure. Conventionally, analysis methods such as methylation analysis and nuclear magnetic resonance spectroscopy have been used for the analysis of the sugar chain structure, but it has a drawback that a large amount of sample must be used. In addition, as a method for estimating the structure with a small amount of sugar chain sample, Streptococcus 6646K that cleaves type 1 and 2 sugar chains or β-galactosidase of ground bean and diprococcus pneumoniae that cleaves only type 2 sugar chains ( Diprococcus pn
eumoniae) β-galactosidase is known to be used in combination, but both Streptococcus 6646K and ground bean β-galactosidase hydrolyze type 2 sugar chains more rapidly, so type 1 sugar chains are completely hydrolyzed. It is difficult to set the reaction conditions such that a large amount of enzyme must be used for a long time to hydrolyze, and it is actually difficult to distinguish between the type 1 and type 2 sugar chain structures.
There has been a demand for providing an enzyme that specifically acts on the heterooligosaccharide. The present inventors have found an exo-type carbohydrate hydrolase that specifically acts on the type 1 sugar chain structure to hydrolyze the lacto-N-bioside bond in the sugar chain and release lacto-N-biose. The enzyme was named lacto-N-biosidase [Proceedings of the National Academy of Sciences of the
USA (Proceedings of the National Academy of Sci)
ences of the USA), 89, 8512 (1
992)].
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、該酵素
は菌体内に産生され、かつ、その生産量も少なく、ま
た、同じ培養条件下でラクト−Ｎ−ビオシダーゼよりも
大量にα−フコシダーゼが菌体内に生産され、該α−フ
コシダーゼとの分離も困難であることなどの問題を有し
ていた。本発明の目的は、高度に精製し易く、工業的製
造に適した糖鎖構造解析用試薬として有用な新規ラクト
−Ｎ−ビオシダーゼを提供することにある。However, the enzyme is produced in the bacterial cells and the production amount thereof is small, and α-fucosidase is produced in the bacterial cells in a larger amount than lacto-N-biosidase under the same culture conditions. And was difficult to separate from the α-fucosidase. An object of the present invention is to provide a novel lacto-N-biosidase that is highly purified and is useful as a reagent for sugar chain structure analysis suitable for industrial production.
【０００４】[0004]
【課題を解決するための手段】本発明を概説すれば、本
発明は下記の理化学的性質を有することを特徴とするエ
キソ型糖質加水分解酵素に関する。 （１）作用 下記式（化１）で表される糖鎖に作用してラクト−Ｎ−
ビオシド結合のみを加水分解する。Means for Solving the Problems The present invention will be summarized. The present invention relates to an exo-type sugar hydrolase characterized by having the following physicochemical properties. (1) Action Acting on a sugar chain represented by the following formula (Formula 1), lacto-N-
Hydrolyze only bioside bonds.
【０００５】[0005]
【化１】Ｇａｌβ１−３ＧｌｃＮＡｃβ１−ＲEmbedded image Galβ1-3GlcNAcβ1-R
【０００６】（Ｒは糖残基を表す） （２）基質特異性 ラクト−Ｎ−ビオシド結合に作用して、 Galβ１-3GlcN
Acを遊離するが、Ｎ−アセチルラクトサミニド結合（ G
alβ１-4GlcNAcβ1-R ）には作用しない。また、ｐ−ニ
トロフェニル−β−ラクト−Ｎ−ビオシドに作用して G
alβ１-3GlcNAcを遊離する。 （３）至適ｐＨ：５．５付近 （４）至適温度：６０℃付近 （５）分子量：約６×１０4 （ＳＤＳ−ポリアクリルア
ミドゲル電気泳動法による）(R represents a sugar residue) (2) Substrate specificity Galβ1-3GlcN acts on the lacto-N-bioside bond.
Releases Ac, but binds N-acetyllactosaminide (G
It does not act on alβ1-4GlcNAcβ1-R). It also acts on p-nitrophenyl-β-lacto-N-bioside to produce G
Release alβ1-3GlcNAc. (3) Optimum pH: around 5.5 (4) Optimum temperature: around 60 ° C (5) Molecular weight: about 6 × 10 4 (by SDS-polyacrylamide gel electrophoresis)
【０００７】なお、式（化１）で表される糖鎖とは、そ
の非還元末端にラクト−Ｎ−ビオース構造（ Galβ１-3
GlcNAcβ1 ）を有する糖鎖であり、Ｒで示される糖残基
とは、例えば単糖類、オリゴ糖類、多糖類、あるいは糖
タンパク質や糖脂質のオリゴ糖類等の糖類の残基であ
る。The sugar chain represented by the formula (Formula 1) has a lacto-N-biose structure (Galβ1-3
The sugar residue represented by R is a sugar chain having GlcNAcβ 1) and is, for example, a residue of a saccharide such as a monosaccharide, an oligosaccharide, a polysaccharide, or an oligosaccharide such as a glycoprotein or a glycolipid.
【０００８】本発明者らは、上記現状にかんがみ、工業
的製造に適したラクト−Ｎ−ビオシシド結合分解酵素を
探索中の所、ある種の放線菌がラクト−Ｎ−ビオシド結
合に特異的な新規エキソ型糖質加水分解酵素を主に菌体
外に産生することを見出し、本発明に到達した。In view of the above situation, the present inventors are searching for a lacto-N-biosideside decomposing enzyme suitable for industrial production, and as a result, some actinomycetes are specific for the lacto-N-bioside side bond. The inventors have found that the novel exo-type glycosyl hydrolase is produced mainly outside the cells, and arrived at the present invention.
【０００９】以下、本発明について詳細に説明する。本
発明に使用される菌株は、ラクト−Ｎ−ビオシダーゼ生
産能を有する菌株であればいかなる菌株でもよく、また
これらの菌株の変異株でもよい。本発明のラクト−Ｎ−
ビオシダーゼ生産能を有する菌株の具体例としては、例
えば、ストレプトミセス SP142が挙げられる。本菌株は
Streptomyces sp 142 と表示され、工業技術院微生物工
業技術研究所に、微工研菌寄第１０８０６号（ＦＥＲＭ
Ｐ−１０８０６）として寄託されている。The present invention will be described in detail below. The strain used in the present invention may be any strain as long as it has the ability to produce lacto-N-biosidase, and may be a mutant strain of these strains. Lacto-N- of the present invention
Specific examples of the strain having biosidase-producing ability include Streptomyces SP142. This strain is
It is displayed as Streptomyces sp 142, and is sent to the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology.
It has been deposited as P-10806).
【００１０】本発明のラクト−Ｎ−ビオシダーゼは、例
えば上述した菌を栄養培地中で培養し、該培養物から酵
素を分離することによって得られる。培養に当っては、
通常の微生物の培養方法が用いられる。培地に加える栄
養源は、本菌株が利用し、ラクト−Ｎ−ビオシダーゼを
生産するものであればよく、炭素源としては、例えばグ
リセロール、グルコース、ガラクトース、マルトース、
ラクトース、フコース、ムチンなどが利用でき、窒素源
としては、酵母エキス、ペプトン、コーンスティープリ
カー、肉エキス、脱脂大豆、硫安、塩化アンモニウムな
どが適当である。また、胃ムチン、卵白ムチンなどのム
チン型糖タンパク質の添加は、本発明の酵素の誘導に有
効である。その他にリン酸塩、カリウム塩、マグネシウ
ム塩、亜鉛塩などの無機質及び金属塩類を加えてもよ
い。本発明のラクト−Ｎ−ビオシダーゼ生産菌を培養す
るに当り、生産量は培養条件により大きく変動するが、
一般に培養温度は２０〜３５℃、培地のｐＨ５〜８が良
く、１日〜７日の通気かくはん培養で、本発明によるラ
クト−Ｎ−ビオシダーゼが生産される。培養条件は使用
する菌株、培地組成などに応じ、本発明のラクト−Ｎ−
ビオシダーゼの生産量が最大になるように設定するのは
当然である。The lacto-N-biosidase of the present invention can be obtained, for example, by culturing the above-mentioned bacterium in a nutrient medium and separating the enzyme from the culture. When culturing,
The usual method for culturing microorganisms is used. The nutrient source added to the medium may be one that is used by this strain and produces lacto-N-biosidase, and examples of the carbon source include glycerol, glucose, galactose, maltose, and the like.
Lactose, fucose, mucin, etc. can be used, and suitable nitrogen sources include yeast extract, peptone, corn steep liquor, meat extract, defatted soybean, ammonium sulfate, ammonium chloride and the like. Further, addition of mucin-type glycoprotein such as stomach mucin and egg white mucin is effective for inducing the enzyme of the present invention. In addition, inorganic salts such as phosphates, potassium salts, magnesium salts, zinc salts and metal salts may be added. In culturing the lacto-N-biosidase-producing bacterium of the present invention, the production amount varies greatly depending on the culture conditions.
Generally, the culturing temperature is 20 to 35 ° C., and the pH of the medium is preferably 5 to 8, and the lacto-N-biosidase according to the present invention is produced by aerated stirring culture for 1 to 7 days. The culturing conditions depend on the strain to be used, the composition of the medium, etc. and the lacto-N-
It is natural to set the maximum biosidase production.
【００１１】上述の放線菌によって生産された本発明の
ラクト−Ｎ−ビオシダーゼは、例えば豚胃ムチンを培地
に加えて培養すると主に菌体外に分泌される上に、他の
エキソグリコシダーゼ、特にα−フコシダーゼがほとん
ど生産されないので菌体内に生産される前出プロシーデ
ィングズ オブ ザ ナショナル アカデミー オブサ
ンエンシーズ オブ ザ ＵＳＡに記載の酵素に比べて
容易に精製を行うことができる。すなわち培養液を固液
分離し、得られた上清から通常用いられる精製手段によ
り精製酵素標品を得ることができる。例えば、塩析、有
機溶媒沈殿、イオン交換カラムクロマトグラフィー、疎
水結合カラムクロマトグラフィー、ハイドロキシアパタ
イトカラムクロマトグラフィー、ゲルろ過クロマトグラ
フィー、凍結乾燥などにより、精製を行い、ポリアクリ
ルアミドゲルディスク電気泳動的に単一な精製酵素標品
を得ることができる。The lacto-N-biosidase of the present invention produced by the above-mentioned actinomycetes is mainly secreted outside the cells when cultured with, for example, pig stomach mucin, and other exoglycosidases, particularly Since almost no α-fucosidase is produced, purification can be easily carried out as compared with the enzyme described in the above-mentioned Proceedings of the National Academy of Sun Sciences of the USA, which is produced in bacterial cells. That is, the culture solution is subjected to solid-liquid separation, and a purified enzyme preparation can be obtained from the obtained supernatant by a commonly used purification means. For example, purification is performed by salting out, organic solvent precipitation, ion exchange column chromatography, hydrophobic binding column chromatography, hydroxyapatite column chromatography, gel filtration chromatography, freeze-drying, etc. One purified enzyme preparation can be obtained.
【００１２】本発明により得られるラクト−Ｎ−ビオシ
ダーゼの酵素化学的及び理化学的性質は次のとおりであ
る。 （１）作用 ラクト−Ｎ−ビオシド結合に作用して、ラクト−Ｎ−ビ
オースを遊離する。 （２）基質特異性 本酵素は、人乳由来のラクト−Ｎ−テトラオース（ Gal
β１-3GlcNAcβ1-3Galβ1-4Glc) に作用してその非還元
末端からラクト−Ｎ−ビオースを遊離させるが、人乳由
来のラクト−Ｎ−ネオテトラオース（ Galβ１-4GlcNAc
β1-3Galβ1-4Glc）には作用しない。また牛胎児血清タ
ンパク質であるフェチュイン由来のラクト−Ｎ−ビオシ
ド結合を有する下記式（化２）：The enzymatic and physicochemical properties of lacto-N-biosidase obtained by the present invention are as follows. (1) Action It acts on the lacto-N-bioside bond to release lacto-N-biose. (2) Substrate specificity This enzyme is a human milk-derived lacto-N-tetraose (Gal
β1-3GlcNAcβ1-3Galβ1-4Glc) to release lacto-N-biose from its non-reducing end, but human milk-derived lacto-N-neotetraose (Galβ1-4GlcNAc
β1-3Gal β1-4Glc) does not act. Further, the following formula (Formula 2) having a lacto-N-bioside bond derived from fetuin which is a fetal bovine serum protein:
【００１３】[0013]
【化２】 [Chemical 2]
【００１４】（以下、式中Ｇは Gal、ＧＮはGlcNAc、Ｍ
は Manを示す）で表される複合型糖鎖に作用して、ラク
ト−Ｎ−ビオースを遊離するが、ラクト−Ｎ−ビオシド
結合を有しない複合型糖鎖には作用しない。すなわち、
本酵素はラクト−Ｎ−ビオシド結合に特異的で、糖鎖の
非還元末端からラクト−Ｎ−ビオースを遊離する。(Hereinafter, G is Gal, GN is GlcNAc, M
Represents Man, and liberates lacto-N-biose, but does not act on a complex-type sugar chain having no lacto-N-bioside bond. That is,
This enzyme is specific for lacto-N-bioside bond and releases lacto-N-biose from the non-reducing end of sugar chain.
【００１５】（３）至適ｐＨ及びｐＨ安定性 本酵素の至適ｐＨは図１の曲線で表されるごとくｐＨ
５．５付近に高い活性を有している。本酵素を４℃にお
いて、それぞれのｐＨで１６時間処理したときのｐＨ安
定性を図２に示した。図２より明らかなように本酵素は
ｐＨ４．０−１０．０の間で安定である。なお、図１は
本発明により得られるラクト−Ｎ−ビオシダーゼのｐＨ
（横軸）と相対活性（％、縦軸）の関係を表すグラフ、
図２はｐＨ（横軸）と残存活性（％、縦軸）との関係を
示すグラフであり、図２中、白丸印は酢酸−塩酸、黒丸
印は酢酸ナトリウム、白三角印はクエン酸ナトリウム、
黒三角印はリン酸ナトリウム、白四角印はグリシンナト
リウム、黒四角印は炭酸ナトリウムの各緩衝液を用いて
測定した。 （４）至適温度及び熱安定性 本酵素の作用最適温度は図３の曲線で表されるごとく６
０℃付近である。なお、図３は本発明のラクト−Ｎ−ビ
オシダーゼの相対活性（％、縦軸）と反応温度（℃、横
軸）との関係を表すグラフである。また、本酵素は、図
４に示すように、４５℃で少なくとも０．５時間安定で
あり、４℃で少なくとも６カ月間安定である。なお図４
は本発明のラクト−Ｎ−ビオシダーゼをそれぞれの温度
で０．５時間処理した後の残存活性（％、縦軸）と処理
温度（℃、横軸）との関係を表すグラフである。 （５）分子量 分子量は約６×１０4 である（ＳＤＳ−ポリアクリルア
ミドゲル電気泳動法による）(3) Optimum pH and pH stability The optimum pH of this enzyme is pH as shown by the curve in FIG.
It has a high activity around 5.5. The pH stability of this enzyme when treated at 4 ° C for 16 hours at each pH is shown in Fig. 2. As is clear from FIG. 2, this enzyme is stable between pH 4.0 and 10.0. In addition, FIG. 1 shows the pH of lacto-N-biosidase obtained by the present invention.
A graph showing the relationship between (horizontal axis) and relative activity (%, vertical axis),
FIG. 2 is a graph showing the relationship between pH (horizontal axis) and residual activity (%, vertical axis). In FIG. 2, white circles represent acetic acid-hydrochloric acid, black circles represent sodium acetate, white triangles represent sodium citrate. ,
The black triangle mark was measured using sodium phosphate, the white square mark was measured using sodium glycine, and the black square mark was measured using sodium carbonate buffer. (4) Optimum temperature and thermostability The optimum temperature for action of this enzyme is 6 as shown by the curve in FIG.
It is around 0 ° C. Note that FIG. 3 is a graph showing the relationship between the relative activity (%, vertical axis) of lacto-N-biosidase of the present invention and the reaction temperature (° C., horizontal axis). The enzyme is stable at 45 ° C for at least 0.5 hours and stable at 4 ° C for at least 6 months, as shown in Fig. 4. Figure 4
FIG. 3 is a graph showing the relationship between the residual activity (%, vertical axis) and the treatment temperature (° C., horizontal axis) after treating the lacto-N-biosidase of the present invention at each temperature for 0.5 hours. (5) Molecular weight The molecular weight is about 6 × 10 4 (by SDS-polyacrylamide gel electrophoresis).
【００１６】（６）酵素活性の測定 ラクト−Ｎ−ビオシダーゼ活性の測定は次のようにして
求めた。基質として下記式（化３）：(6) Measurement of enzyme activity The lacto-N-biosidase activity was measured as follows. The following formula (Formula 3) as a substrate:
【００１７】[0017]
【化３】Ｇａｌβ１−３ＧｌｃＮＡｃβ１−ｐＮＰEmbedded image Galβ1-3GlcNAcβ1-pNP
【００１８】〔以下、式中ｐＮＰはｐ−ニトロフェニル
基を意味する〕で表される構造の糖鎖を用いた。この基
質１mMを含む３５mMリン酸カリウム緩衝液、ｐＨ６．０
に酵素液を加え、最終液量０．１mlで３７℃で２０分間
反応させた後、１Ｍの炭酸ナトリウム溶液を加えて反応
を停止させ、４０５nmの吸光度を測定した。この条件下
で、１分間に１μmol のｐ−ニトロフェノールを生じる
酵素量を１単位とした。A sugar chain having a structure represented by [hereinafter, pNP means a p-nitrophenyl group] was used. 35 mM potassium phosphate buffer containing 1 mM of this substrate, pH 6.0
The enzyme solution was added to the reaction mixture, and the reaction was carried out at 37 ° C. for 20 minutes with a final solution volume of 0.1 ml, the reaction was stopped by adding a 1 M sodium carbonate solution, and the absorbance at 405 nm was measured. Under this condition, the amount of enzyme that produces 1 μmol of p-nitrophenol per minute was defined as 1 unit.
【００１９】本発明のラクト−Ｎ−ビオシダーゼを利用
して以下の事項を解明することができる。 （１）複合糖質中のラクト−Ｎ−ビオシル残基の役割を
知ることができる。 （２）本酵素を用いれば、複合糖質中のラクト−Ｎ−ビ
オシル基の有無を直接推定することができ、タイプ１糖
鎖とタイプ２糖鎖の識別が直接できる。 （３）糖鎖還元末端をあらかじめ還元ピリジルアミノ化
法〔ジャーナル オブバイオケミストリー（Journal of
Biochemistry)、第９５巻、第１９７〜２０３頁（１９
８４）〕にて蛍光標識した糖鎖を用いて、上記の酵素消
化法と２次元糖鎖マップ法〔アナリティカル バイオケ
ミストリー（Analytical Biochemistry)、第１７１巻、
第７３頁（１９８８）〕を組合せることによって、ラク
ト−Ｎ−ビオシル基も含めた糖鎖構造全体を、従来の数
百倍の感度で推定することができる。 （４）還元末端を〔 3Ｈ〕標識した糖鎖、あるいは、未
標識糖鎖を用いて、本酵素で酵素化を行い、酵素消化物
をゲルろ過クロマトグラフィーやイオン交換クロマトグ
ラフィーなどで分析することによって、糖鎖構造を推定
することができる。The following items can be elucidated by utilizing the lacto-N-biosidase of the present invention. (1) The role of lacto-N-biosyl residues in glycoconjugates can be known. (2) By using the present enzyme, the presence or absence of the lacto-N-biosyl group in the complex carbohydrate can be directly estimated, and the type 1 sugar chain and the type 2 sugar chain can be directly distinguished. (3) The reducing end of the sugar chain is preliminarily subjected to the reductive pyridyl amination method [Journal of Biochemistry (Journal of Biochemistry
Biochemistry), 95, 197-203 (19
84)] using a fluorescently labeled sugar chain, and the above-mentioned enzymatic digestion method and two-dimensional sugar chain mapping method [Analytical Biochemistry, Vol. 171,
Page 73 (1988)], the entire sugar chain structure including the lacto-N-biosyl group can be estimated with a sensitivity several hundred times that of the conventional method. (4) [ 3 H] -labeled sugar chain at the reducing end or unlabeled sugar chain is used to perform enzymatic reaction with this enzyme, and the enzyme digest is analyzed by gel filtration chromatography, ion exchange chromatography, etc. Thus, the sugar chain structure can be estimated.
【００２０】[0020]
【実施例】次に、実施例を挙げて本発明を説明するが、
本発明は以下の実施例の範囲のみに限定されるものでは
ない。EXAMPLES Next, the present invention will be described with reference to examples.
The present invention is not limited to the scope of the following examples.
【００２１】実施例１ （１）菌の培養と培養上清及び無細胞抽出液の調製 Streptomyces sp 142 〔微工研菌寄第１０８０６号（Ｆ
ＥＲＭ Ｐ−１０８０６）〕をペプトン０．３％、イー
ストエキス０．０１％、リン酸一カリウム０．１％、硫
酸マグネシウム７水和物０．０５％及び豚胃ムチン１％
を含む５００mlの液体培地（ｐＨ７．０）を用いて２５
〜２７℃で２日間培養した後、培養液を遠心分離して培
養上清及び菌体を得た。菌体を１mMのエチレンジアミン
四酢酸を含む１０mMリン酸ナトリウム緩衝液ｐＨ７．０
で洗浄後、同緩衝液に懸濁して超音波処理し、遠心分離
によって菌体残渣を除いて、無細胞抽出液を得た。上清
中の、すなわち菌体外に生産されたＮ−ラクト−ビオシ
ダーゼの活性は２．４mU／ml培養液、α−フコシダーゼ
の活性は０．０１mU／ml培養液であった。また、無細胞
抽出液の、すなわち菌体内に生産されたＮ−ラクト−ビ
オシダーゼの活性は０．５mU／ml培養液、α−フコシダ
ーゼの活性は０．００２mU／ml培養液であった。なお、
α−フコシダーゼの活性はジャーナル オブ バイオロ
ジカル ケミストリー（Journal of Biological Chemis
try)、第２６７巻、第１５２２頁（１９９２）に記載の
方法で行った。Example 1 (1) Culture of Bacteria and Preparation of Culture Supernatant and Cell-Free Extract Streptomyces sp 142 [Ministry of Industrial Science, Microbiology No. 10806 (F
ERM P-10806)] peptone 0.3%, yeast extract 0.01%, monopotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.05% and pig stomach mucin 1%.
25 with 500 ml of liquid medium (pH 7.0) containing
After culturing at ˜27 ° C. for 2 days, the culture solution was centrifuged to obtain a culture supernatant and bacterial cells. 10 mM sodium phosphate buffer pH 7.0 containing 1 mM ethylenediaminetetraacetic acid
After washing with, the cells were suspended in the same buffer and sonicated, and the cell residue was removed by centrifugation to obtain a cell-free extract. The activity of N-lacto-biosidase produced in the supernatant, that is, extracellularly, was 2.4 mU / ml culture medium, and the activity of α-fucosidase was 0.01 mU / ml culture medium. The activity of N-lacto-biosidase produced in the cells of the cell-free extract was 0.5 mU / ml, and the activity of α-fucosidase was 0.002 mU / ml. In addition,
The activity of α-fucosidase is reported in the Journal of Biological Chemis
try), vol. 267, page 1522 (1992).
【００２２】（２）酵素の調製 上記に得た培養上清５００mlを、０．５mMフェニルメタ
ンスルホニルフルオリド及び０．０５％ブリッジ（Bri
j) ５８を含む５０mMリン酸カリウム緩衝液ｐＨ７．５
に対して透析後、同緩衝液で平衡化したＱ−セファロー
スのカラム（２．５×１８cm）に供した。カラムをその
３倍容量の同一の緩衝液で洗浄し、溶出してきた素通り
画分を集めた。活性画分は０．１mMフェニルメタンスル
ホニルフルオリド及び０．０５％ブリッジ５８を含む５
０mM酢酸ナトリウム緩衝液ｐＨ５．５に対して透析後、
同緩衝液で平衡化したＳ−セファロースのカラム（１．
５×８．５cm）に供した。カラムをその３倍容量の同一
の緩衝液で洗浄した後、０−０．４ＭまでのＮａＣｌの
濃度勾配をかけて溶出した。活性画分を０．０５％ブリ
ッジ５８を含む５０mM酢酸ナトリウム緩衝液ｐＨ５．５
に対して透析後、同緩衝液で平衡化したＭｏｎｏ−Ｓカ
ラム（０．５×５．０cm）に供した。溶出は０−０．５
ＭまでのＮａＣｌの濃度勾配で行った。０．０５％ブリ
ッジ５８を含む５０mMリン酸カリウム緩衝液ｐＨ６．８
であらかじめ平衡化したＰ６ＤＧのカラムにかけて緩衝
液を交換した後、同緩衝液で平衡化したハイドロキシア
パタイトのカラム（０．６×４．０cm）に供した。溶出
はリン酸カリウム緩衝液の濃度を５０mM−５００mMまで
直線的に上げて行った。活性画分を限外ろ過（分画分子
量１万）にて濃縮後０．０５％ブリッジ５８及び０．２
Ｍ ＮａＣｌを含む５０mM酢酸ナトリウム緩衝液ｐＨ
５．５で平衡化したトヨパールＨＷ−５５Ｓのカラム
（１．５×９０cm）にかけた。同緩衝液で溶出し、活性
画分を集めて本発明のラクト−Ｎ−ビオシダーゼを得
た。この様にして得られたラクト−Ｎ−ビオシダーゼの
比活性は８０００ミリ単位／mgであり、菌体内に生産さ
れる酵素（前出プロシーディングズ オブ ザ ナショ
ナル アカデミー オブ サイエンシ−ズオブ ザ Ｕ
ＳＡ）の比活性、６８４ミリ単位／mgに比べてはるかに
精製された酵素標品であり、糖鎖構造解析用試薬として
十分使用可能であった。なお、上記酵素の比活性は前記
の測定法によって測定した。(2) Preparation of enzyme 500 ml of the culture supernatant obtained above was added to 0.5 mM phenylmethanesulfonyl fluoride and 0.05% bridge (Bri).
j) 50 mM potassium phosphate buffer solution containing 58, pH 7.5
After dialysis, the mixture was applied to a Q-Sepharose column (2.5 × 18 cm) equilibrated with the same buffer. The column was washed with 3 times its volume of the same buffer, and the eluted flow-through fraction was collected. Active fraction contains 0.1 mM phenylmethanesulfonyl fluoride and 0.05% bridge 58 5
After dialysis against 0 mM sodium acetate buffer pH 5.5,
A column of S-Sepharose equilibrated with the same buffer (1.
5 × 8.5 cm). The column was washed with 3 times its volume of the same buffer and then eluted by applying a gradient of NaCl from 0 to 0.4M. The active fraction contains 50% sodium acetate buffer containing 0.05% bridge 58, pH 5.5.
After dialysis, it was applied to a Mono-S column (0.5 × 5.0 cm) equilibrated with the same buffer. Elution is 0-0.5
A concentration gradient of NaCl up to M was performed. 50 mM potassium phosphate buffer pH 6.8 containing 0.05% bridge 58
The column was preliminarily equilibrated with P6DG to exchange the buffer solution, and then the column was applied to a hydroxyapatite column (0.6 × 4.0 cm) equilibrated with the same buffer solution. Elution was performed by linearly increasing the concentration of the potassium phosphate buffer solution to 50 mM-500 mM. The active fraction was concentrated by ultrafiltration (molecular weight cutoff of 10,000) and then concentrated to 0.05% bridge 58 and 0.2.
50 mM sodium acetate buffer pH containing M NaCl
It was applied to a Toyopearl HW-55S column (1.5 x 90 cm) equilibrated at 5.5. Elution with the same buffer and collection of active fractions gave the lacto-N-biosidase of the present invention. The specific activity of the lacto-N-biosidase thus obtained was 8,000 milliunits / mg, and the enzyme produced in the bacterial cells (Proceedings of the National Academy of Sciences of the U.
It was a much purified enzyme preparation compared to the specific activity of SA), 684 milliunits / mg, and was sufficiently usable as a reagent for sugar chain structure analysis. The specific activity of the above enzyme was measured by the above measuring method.
【００２３】実施例２ ピリジルアミン化糖鎖に対する
作用 基質として下記式（化４）：Example 2 Action on pyridylamine-modified sugar chain As a substrate, the following formula (Formula 4):
【００２４】[0024]
【化４】 [Chemical 4]
【００２５】で表される構造のピリジルアミノ化糖鎖
（宝酒造社製）を用いて実施例１で得た酵素を作用させ
た。３．３μＭの基質を含む８０mMリン酸緩衝液ｐＨ
６．０に本酵素５０μ単位を加えて３７℃、１６時間反
応を行った。反応終了後反応液をＨＰＬＣで分析し、下
記式（化５）：The enzyme obtained in Example 1 was allowed to act on a pyridyl aminated sugar chain having a structure represented by (manufactured by Takara Shuzo). 80 mM phosphate buffer pH with 3.3 μM substrate
50 µ units of the present enzyme was added to 6.0 and reacted at 37 ° C for 16 hours. After completion of the reaction, the reaction solution is analyzed by HPLC, and the following formula (Formula 5):
【００２６】[0026]
【化５】 [Chemical 5]
【００２７】で表される構造のピリジルアミノ化糖鎖が
生成している事を確認した。更に反応液を濃縮乾固し、
糖質ピリジルアミノ化装置・パルステーション（PALSTA
TION：宝酒造社製）にて還元ピリジルアミノ化した後、
ＨＰＬＣ分析を行い、式（化５）並びに下記式（化
６）：It was confirmed that a pyridyl aminated sugar chain having a structure represented by Further, the reaction solution is concentrated to dryness,
Glycopyridyl amination equipment / Pulsation (PALSTA
(TION: manufactured by Takara Shuzo)
HPLC analysis was performed and the formula (formula 5) and the following formula (formula 6):
【００２８】[0028]
【化６】Ｇ（β１−３）ＧＮ−ＰＡEmbedded image G (β1-3) GN-PA
【００２９】で表される構造のピリジルアミノ化糖鎖の
生成を確認した。It was confirmed that a pyridyl aminated sugar chain having a structure represented by
【００３０】[0030]
【発明の効果】本発明により、複合糖鎖の構造と機能の
解明に有用で、工業的製造にも適した新規ラクト−Ｎ−
ビオシダーゼが提供された。INDUSTRIAL APPLICABILITY According to the present invention, a novel lacto-N- which is useful for elucidating the structure and function of a complex sugar chain and is suitable for industrial production
Biosidase was provided.
【図１】本発明により得られるラクト−Ｎ−ビオシダー
ゼのｐＨと活性の関係を表すグラフである。FIG. 1 is a graph showing the relationship between pH and activity of lacto-N-biosidase obtained according to the present invention.
【図２】ラクト−Ｎ−ビオシダーゼを４℃において、そ
れぞれのｐＨで１６時間処理した後のｐＨと活性の関係
を示すグラフである。FIG. 2 is a graph showing the relationship between pH and activity after treating lacto-N-biosidase at 4 ° C. with each pH for 16 hours.
【図３】本発明のラクト−Ｎ−ビオシダーゼの相対活性
（％、縦軸）と反応温度（℃、横軸）との関係を表すグ
ラフである。FIG. 3 is a graph showing the relationship between the relative activity (%, vertical axis) of lacto-N-biosidase of the present invention and the reaction temperature (° C., horizontal axis).
【図４】本発明のラクト−Ｎ−ビオシダーゼをそれぞれ
の温度で０．５時間処理した後の残存活性（％、縦軸）
と処理温度（℃、横軸）との関係を表すグラフである。FIG. 4 Residual activity (%, vertical axis) after treating the lacto-N-biosidase of the present invention for 0.5 hours at each temperature.
It is a graph showing the relationship between the processing temperature (° C, horizontal axis).
Claims (1)
とするエキソ型糖質加水分解酵素。 （１）作用 下記式（化１）で表される糖鎖に作用してラクト−Ｎ−
ビオシド結合のみを加水分解する。 【化１】Ｇａｌβ１−３ＧｌｃＮＡｃβ１−Ｒ （Ｒは糖残基を表す） （２）基質特異性 ラクト−Ｎ−ビオシド結合に作用して、 Galβ１-3GlcN
Acを遊離するが、Ｎ−アセチルラクトサミニド結合（ G
alβ１-4GlcNAcβ1-R ）には作用しない。また、ｐ−ニ
トロフェニル−β−ラクト−Ｎ−ビオシドに作用して G
alβ１-3GlcNAcを遊離する。 （３）至適ｐＨ：５．５付近 （４）至適温度：６０℃付近 （５）分子量：約６×１０4 （ＳＤＳ−ポリアクリルア
ミドゲル電気泳動法による）1. An exo-type carbohydrate hydrolase having the following physicochemical properties. (1) Action Acting on a sugar chain represented by the following formula (Formula 1), lacto-N-
Hydrolyze only bioside bonds. Embedded image Galβ1-3GlcNAcβ1-R (R represents a sugar residue) (2) Substrate specificity Galβ1-3GlcN acts on the lacto-N-bioside bond.
Releases Ac, but binds N-acetyllactosaminide (G
It does not act on alβ1-4GlcNAcβ1-R). It also acts on p-nitrophenyl-β-lacto-N-bioside to produce G
Release alβ1-3GlcNAc. (3) Optimum pH: around 5.5 (4) Optimum temperature: around 60 ° C (5) Molecular weight: about 6 × 10 4 (by SDS-polyacrylamide gel electrophoresis)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32897592A JP2829810B2 (en) | 1992-11-16 | 1992-11-16 | Carbohydrate hydrolases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32897592A JP2829810B2 (en) | 1992-11-16 | 1992-11-16 | Carbohydrate hydrolases |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH06153944A true JPH06153944A (en) | 1994-06-03 |
| JP2829810B2 JP2829810B2 (en) | 1998-12-02 |
Family
ID=18216211
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP32897592A Expired - Fee Related JP2829810B2 (en) | 1992-11-16 | 1992-11-16 | Carbohydrate hydrolases |
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| Country | Link |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0739983A3 (en) * | 1995-04-27 | 1998-05-06 | Takara Shuzo Co. Ltd. | Gene encoding lacto-n-biosidase |
-
1992
- 1992-11-16 JP JP32897592A patent/JP2829810B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0739983A3 (en) * | 1995-04-27 | 1998-05-06 | Takara Shuzo Co. Ltd. | Gene encoding lacto-n-biosidase |
| US5908772A (en) * | 1995-04-27 | 1999-06-01 | Takara Shuzo Co., Ltd. | Gene encoding lacto-N-biosidase |
| CN1100878C (en) * | 1995-04-27 | 2003-02-05 | 宝酒造株式会社 | Gene encoding lacto-N-biosidase |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2829810B2 (en) | 1998-12-02 |
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