JP3793269B2 - β-N-acetylgalactosaminidase - Google Patents

Description

【００１５】
【表２】

【００１６】
【表３】

【００１７】
表１〜表３の菌学的性質をもとに、Bergey' s Manual of Systematic Bacteriology 第２巻（Williams and Wilkins, Baltimore, 1986）を参照して、本菌はバシラス属に属する菌株であることがわかった。バシラス属の中ではバシラス・スファエリカス（Bacillus sphaericus)に最も類似しているが、胞子の形などが異なっているため、本菌をバシラス・スファエリカスと同定することはできなかった。そこで、発明者は本菌をBacillus sp. AT173-1株と命名した。
【００１８】
以下、本菌から本β−Ｎ−アセチルガラクトサミニダーゼを生産および採取するための条件について説明する。
【００１９】
（ｂ）培養条件
本菌の培養条件は特に限定されず、通常の液体培地または固体培地が用いられる。炭素源としては、各種の糖類が用いられ、アミノ糖、グルコース、糖蜜などが用いられる。窒素源としては、ペプトン、酵母エキス、大豆粉、コーン・スティープ・リカー、カゼイン、肉エキス、アミノ酸などが用いられるがこれらに限定されない。上記炭素源や窒素源の他、各種の無機塩類（例えば、マグネシウム塩、カリウム塩、ナトリウム塩）およびビタミン類などが必要に応じて添加され得る。本菌の生育好適ｐＨは６〜８であるがこれに限定されない。
【００２０】
本菌の培養温度は本菌が生育できる温度であれば特に制限はないが、培養温度２０℃〜４５℃が好ましく、特に３０℃が好ましい。液体培養の場合は、通常２４時間〜７２時間、好気的に撹拌または振盪しながら培養を行う。培養過程における菌体増殖経時変化は、例えば、培養懸濁液の濁度変化（通常は６６０ｎｍの吸光度の計測）をモニターして、知り得る。本β−Ｎ−アセチルガラクトサミニダーゼは、主として菌体外に分泌される。
【００２１】
（ｃ）β−Ｎ−アセチルガラクトサミニダーゼの採取方法
上記培養液から本発明の酵素を採取、精製するには、既知の精製法が単独もしくは併用して利用され得る。例えば、培養液を濾過または遠心分離にかけて菌体を除き、得られた上清を、硫安、芒硝などによる塩析、またはメタノール、エタノール、アセトンなどの有機溶媒による沈殿などに供することにより分画、濃縮し、本β−Ｎ−アセチルガラクトサミニダーゼを含有する粗酵素液が得られる。さらにこの粗酵素液は、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、および／またはその他の各種クロマトグラフィーを行うことにより精製される。好ましい精製法の概要を以下に示す。
【００２２】
（１）培養液を遠心分離して菌体を除去し、上清液を得る。
（２）次いで、得られた上清液を硫安（８０％飽和溶液）で塩析する。
（３）次いで、得られた塩析液をｐＨ８．０のトリス・塩酸バッファーで透析し、ＱＡＥ−トヨパールカラムクロマトグラフィーにかける。
（４）上記（３）で得られた活性画分に、最終２５％飽和濃度になるように硫安溶液を加え、フェニルセファロースカラムクロマトグラフィーにかける。
（５）上記（４）で得られた活性画分をｐＨ６．０の酢酸バッファーで透析し、Ｑ−セファロースカラムクロマトグラフィーにかける。
（６）上記（５）で得られた活性画分を限外濾過膜で濃縮し、セファクリルＳ−２００カラムクロマトグラフィーにかける。
（７）上記（６）で得られた活性画分に、最終２５％飽和濃度になるように硫安溶液を加え、リソースＰＨＥカラムクロマトグラフィーにかける。
【００２３】
（ｄ）本β−Ｎ−アセチルガラクトサミニダーゼの活性測定法
本明細書においては、本β−Ｎ−アセチルガラクトサミニダーゼ活性は、パラニトロフェニル−Ｎ−アセチル−β−Ｄ−ガラクトサミニドを基質として用い、１分間に１マイクロモルのパラニトロフェノールを遊離させる酵素量を１単位（Ｕ）として測定する。具体的には本明細書においては、ｐＨ６．０の５０ｍ Ｍ酢酸バッファーに溶解させた２．５ｍＭのパラニトロフェニル−Ｎ−アセチル−β−Ｄ−ガラクトサミニド溶液０．４ｍｌに、あらかじめジチオスレイトールを０．１％になるように加えて室温で１時間放置した試料溶液０．１ｍ１を加え、３７℃で１０分間反応させる。その後、２％炭酸ナトリウム溶液を２．５ｍｌ加えて反応を停止し、この反応液の波長４２０ｎｍにおける吸光度（パラニトロフェノールの遊離に伴って増加する）を測定する。
【００２４】
【発明の実施の形態】
本発明のβ−Ｎ−アセチルガラクトサミニダーゼは、Ｎ−アセチル−β−Ｄ−ガラクトサミニド結合を特異的に加水分解するエキソグリコシダーゼである。そのため、糖鎖の非還元末端がＮ−アセチル−β−Ｄ−ガラクトサミニドのとき、該グリコシド結合を加水分解して、非還元末端からＮ−アセチル−Ｄ−ガラクトサミンを遊離させるが、その糖鎖の非還元末端がＮ−アセチル−β−Ｄ−グルコサミニドのとき、本酵素は作用せず、遊離単糖も生じさせない。
【００２５】
以下の実施例にて、本発明をさらに詳細に説明するが、これはなんら本発明を限定するものではない。
【００２６】
【実施例】
実施例１
（培養上清の調製）
１００ｍｌの培地（０．０６２５％Ｎ−アセチル−Ｄ−ガラクトサミン、１％脱脂大豆製ペプトン、１％酵母エキス、０．１％Ｋ₂ＨＰＯ₄および０．０２％ＭｇＳＯ₄・７Ｈ₂Ｏ）を含有する５００ｍｌ容振盪フラスコ８０本に、本菌を植菌し、３０℃で２日間、往復振盪培養を行った。培養終了後、この培養液を回収し、１０，０００ｒｐｍで１０分間遠心分離して菌体を除き、培養上清を７３６０ｍｌ得た。
【００２７】
実施例２
（培養上清中のβ−Ｎ−アセチルガラクトサミニダーゼ活性およびβ−Ｎ−アセチルグルコサミニダーゼ活性）
上記実施例１で得られた上清中の本β−Ｎ−アセチルガラクトサミニダーゼの活性は上記（ｄ）に記載の方法に準じて測定した。β−Ｎ−アセチルグルコサミニダーゼ活性は、基質にパラニトロフェニル−Ｎ−アセチル−β−Ｄ−グルコサミニドを用い、その他の条件は上記（ｄ）に記載の方法に準じて測定した。結果を図１に示す。図中の横軸は反応時間を、縦軸はパラニトロフェノールの生成量に伴う４２０ｎｍの吸光度をそれぞれ表し、●はパラニトロフェニル−Ｎ−アセチル−β−Ｄ−ガラクトサミニドを、および○はパラニトロフェニル−Ｎ−アセチル−β−Ｄ−グルコサミニドをそれぞれ基質としたときの数値を示す。パラニトロフェニル−Ｎ−アセチル−β−Ｄ−ガラクトサミニドを基質としたときには、遊離したパラニトロフェノールの増加に伴う４２０ｎｍの吸光度の増加がみられたが、パラニトロフェニル−Ｎ−アセチル−β−Ｄ−グルコサミニドを基質としたときには、遊離したパラニトロフェノールの増加に伴う４２０ｎｍの吸光度の増加はまったくみられなかった。上清中の本酵素の活性は、０.０８８Ｕ/ｍｌであった。
【００２８】
実施例３
（本酵素の調製）
すべてのカラムクロマトグラフィー操作は、４℃で行った。上記実施例１で得られた培養上清に硫安を８０％飽和になるまで加え、生じた沈澱を遠心分離して回収した。この沈澱を、ｐＨ６．０、５０ｍＭの酢酸バッファーに溶解し、次いでｐＨ８．０、５０ｍＭのトリス・塩酸バッファーで透析した。この酵素液を、ｐＨ８．０、５０ｍＭトリス・塩酸バッファーで平衡化したＱＡＥ−トヨパール５５０Ｃカラム（東ソー株式会社製）に吸着させ、０．１〜１Ｍの食塩濃度勾配をかけて溶出した。得られた本酵素画分に最終濃度で２５％飽和になるように硫安溶液を加えて、２５％飽和硫安を含むｐＨ８．０、５０ｍＭトリス・塩酸バッファーで平衡化したフェニルセファロースＨＰカラム（ファルマシアバイオテク社製）に吸着させ、２５〜０％飽和の硫安濃度勾配をかけて溶出した。得られた本酵素画分をｐＨ６．０、５０ｍＭの酢酸バッファーで透析し、この透析液をｐＨ６．０、５０ｍＭの酢酸バッファーで平衡化したＱ−セファロースＦＦカラムに吸着させ、０〜０．５Ｍの食塩濃度勾配をかけて溶出した。得られた本酵素画分を限外濾過膜で濃縮し、その濃縮液を、０．１５Ｍの食塩を含むｐＨ８．０、５０ｍＭトリス・塩酸バッファーで平衡化したセファクリルＳ−２００カラム（ファルマシアバイオテク社製）にかけた。得られた本酵素画分に最終濃度で、２５％飽和になるように硫安溶液を加えて、２５％飽和硫安を含むｐＨ８．０、５０ｍＭトリス・塩酸バッファーで平衡化したリソースＰＨＥカラム（ファルマシアバイオテク社製）に吸着させ、２５〜０％飽和の硫安濃度勾配をかけて溶出した。
【００２９】
以上の操作により、実施例１で得られた培養上清７３６０ｍｌ（計６４７Ｕ）から、収率１．５％で８７００倍に精製された本β−Ｎ−アセチルガラクトサミニダーゼを含む溶液を２．５ｍｌ（９．５６Ｕ）得た。本酵素の比活性は、タンパク質１ｍｇあたり１５４Ｕであった。本精製酵素液は、後述するＳＤＳゲル電気泳動および等電点電気泳動で単一のバンドを示した。上記（ｃ）に記載の（１）〜（７）に対応した本実施例１および３の各精製工程における酵素の精製度合いを、表４に示す。表中の活性値は、いずれも上記（ｄ）に記載の測定方法に準じて測定した値である。
【００３０】
【表４】

【００３１】
以下の実施例では、上記実施例３で、得られた酵素液またはその濃縮液を用いた。
【００３２】
実施例４
（本酵素の基質特異性）
種々のピリシルアミノ化糖鎖を基質として、それらに対する本酵素の反応性を比較した。結果を表５に示す。
【００３３】
【表５】

【００３４】
表５中の基質（１）、（２）および（５）は宝酒造（株）製のものを、（４）はアジノキ（株）製のものを使用した。基質（３）は、ピリシルアミノ化ラクト−Ｎ−ネオテトラオース（Ｇａｌβ１−４ＧｌｃＮＡｃβ１−３Ｇａｌβ１−４Ｇｌｃ−ＰＡ：宝酒造（株）製）に、β−ガラクトシダーゼ（アスペルギルス・エスピー由来、東洋紡（株）製）を作用させて調製した。
【００３５】
基質（１）、（２）、（３）および（５）に対する反応は、以下に示す方法で行った。２０ｐｍｏｌの基質、２０μｇの牛血清アルブミン（シグマ社製）および１μｇのジチオスレイトールを含む、４μｌのｐＨ６．０、５０ｍＭの酢酸バッファーに６μｌの１４Ｕ／ｍｌ本酵素液を加え、３７℃で６時間反応させた。そしてこの反応液に４０μｌの１％トリフルオロ酢酸を加えて反応を停止した。基質（４）に対する反応は、基質の量を１０ｐｍｏｌとした以外は、上記反応条件にしたがった。得られた反応液は、高速液体クロマトグラフィーで分析し、分解率を計算した。
【００３６】
非還元末端にＮ−アセチル−β−ガラクトサミニド結合を有する基質（１）、（２）および（４）は、非還元末端に存在するすべてのＮ−アセチル−β−ガラクトサミニド結合が完全に加水分解されたのにたいして、非還元末端にＮ−アセチル−β−グルコサミニド結合を有する基質（３）および（５）はまったく加水分解されなかった。
【００３７】
実施例５
（本酵素の至適ｐＨおよび安定ｐＨ）
（１）至適ｐＨ
２５ｍＭブリトン−ロビンソン広域バッファーにより、ｐＨを４．０、５．０、５．５、６．０、６．５、７．０、８．０および９．０に調製した以外は、上記（ｄ）に記載の方法に準じて、各ｐＨにおける本β−Ｎ−アセチルガラクトサミニダーゼ活性を測定した。結果を図２に示す。図中の横軸は反応ｐＨを示し、縦軸は、得られた最大活性値を１００とした場合の相対活性（％）を示す。本酵素の至適ｐＨは６．０であった。
【００３８】
（２）安定ｐＨ範囲
５３ｍＭブリトン−ロビンソン広域バッファーにより、本酵素液のｐＨを３．０から１１．０まで０．５間隔で調整し、２５℃で１時間保持した後の本酵素の残存活性を上記（ｄ）に記載の方法に準じて測定した。結果を図３に示す。図中の横軸は保持ｐＨを示し、縦軸は最初の活性を１００とした場合の残存活性（％）を示す。ｐＨ５．５〜９．５の範囲で本酵素は安定であった。
【００３９】
実施例６
（本酵素の作用適温の範囲および熱安定性）
（１）作用適温の範囲
種々の反応温度条件（３０、３７、４０、４５、５０、５５℃）下での本酵素活性を上記（ｄ）に記載の方法に準じて測定した。結果を図４に示す。図中の横軸は反応温度を示し、縦軸は、得られた最大活性値を１００とした場合の相対活性（％）を示す。本酵素の至適温度は４５℃であった。
【００４０】
（２）熱安定性
種々の温度条件（２５、３０、３５、４０、４５、５０、５５℃）下、０．２％牛血清アルブミンを含むｐＨ６．０の５０ｍＭ酢酸バッファーに溶解した０．１４Ｕ／ｍｌの本酵素液を１５分間保持した後、本酵素の残存活性を上記（ｄ）に記載の方法に準じて測定した。結果を図５に示す。図中の横軸は保持温度を表し、縦軸は最初の活性を１００とした場合の残存活性（％）を表す。本酵素は４０℃まで安定であった。
【００４１】
実施例７
（本酵素の分子量）
一般的なＳＤＳゲル電気泳動法を用いて、本酵素の分子量を測定した。本酵素を含む試料を、分子量既知の種々のタンパク質を含む標準試料と同一電気泳動ゲル上で分析し、それらの移動度と分子量の関係に基づいて分子量を測定した。ゲルはファルマシアバイオテク社製ファーストゲル・グラジエント１０−１５を用いた。図６に各標準タンパク質の移動度（横軸）と分子量（縦軸；×１０⁴）の関係を示す。図中のＡ、Ｂ、Ｃ、Ｄ、ＥおよびＦは、それぞれウサギ筋肉ホスホリラーゼｂ（分子量９４，０００）、ウシ血清アルブミン（分子量６７，０００）、ニワトリ卵白アルブミン（分子量４３，０００）、ウシ赤血球カルボニック・アンヒドラーゼ（分子量３０，０００）、大豆トリプシン・インヒビター（分子量２０，１００）および牛乳α−ラクトアルブミン（分子量１４，４００）を示す。ＳＤＳゲル電気泳動法により測定した本酵素の分子量は、約４８，０００である。
【００４２】
実施例８
（本酵素の等電点）
常法の等電点電気泳動法により本酵素の等電点を測定した。本酵素を、分子量既知の種々のタンパク質を含む標準試料と同一電気泳動ゲル上で分析し、それらの移動度と等電点の関係に基づく検量線から、本酵素の等電点を測定した。本酵素の等電点は約４．３であった。
【００４３】
【発明の効果】
本発明によれば、糖鎖の非還元末端のＮ−アセチル−β−Ｄ−ガラクトサミニド結合を特異的に加水分解するβ−Ｎ−アセチルガラクトサミニダーゼ、およびその生産方法が提供される。
【００４４】
本β−Ｎ−アセチルガラクトサミニダーゼは、糖鎖の非還元末端にあるβ−グリコシド結合したＮ−アセチル−Ｄ−ガラクトサミンを遊離させるが、非還元末端にあるβ−グリコシド結合したＮ−アセチル−Ｄ−グルコサミンをまったく遊離させないので、構造解析が望まれる糖鎖の非還元末端にあるβ−グリコシド結合したＮ−アセチル−Ｄ−ガラクトサミンの存在の有無を容易に判定し得、糖鎖の構造決定に非常に有用である。本酵素は微生物の培養によって生産されるため、簡便な工程で大量かつ安価に提供され得る。
【図面の簡単な説明】
【図１】培養上清中の本β−Ｎ−アセチルガラクトサミニダーゼ活性およびβ−Ｎ−アセチルグルコサミニダーゼ活性を示す。
【図２】ｐＨ４．０〜９．０の範囲で本β−Ｎ−アセチルガラクトサミニダーゼ活性を測定した場合の相対活性を示す。
【図３】本β−Ｎ−アセチルガラクトサミニダーゼ活性をｐＨ３．０〜１１．０の各条件下、２５℃で１時間保持した後に測定した残存活性を示す。
【図４】３０〜５５℃の温度範囲で本β−Ｎ−アセチルガラクトサミニダーゼ活性を測定した場合の相対活性を示す。
【図５】本β−Ｎ−アセチルガラクトサミニダーゼを０．２％牛血清アルブミンを含む５０ｍＭ酢酸バッファー（ｐＨ６．０）中、２５〜５５の各温度条件下で、１５分間保持した後に測定した残存活性を示す。
【図６】ＳＤＳ電気泳動により測定した本β−Ｎ−アセチルガラクトサミニダーゼの分子量を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention releases β-glycoside-bonded N-acetyl-D-galactosamine at the non-reducing end of the sugar chain, but does not release β-glycoside-bonded N-acetyl-D-glucosamine at the non-reducing end. The present invention relates to a glycosidase, β-N-acetylgalactosaminidase, and a method for producing the same.
[0002]
[Prior art]
Many sugar chains containing an N-acetyl-β-D-galactosaminide bond are present in glycolipids and glycoproteins in vivo. Recent studies have revealed that these sugar chains play an important role in the living body, and accordingly, a simpler and more accurate sugar chain structure analysis technique has been demanded. One such glycan structure analysis technique is promising as a technique using exoglycosidase (Pauline Rudd (translated by Kenji Kobayashi), Chemistry and Biology, 32, 661, (1994)); Sano Tsubaki, Satoshi Miyamura, “BIO VIEW”, No. 13, p. 13, published by Takara Shuzo (1994)). These utilize the substrate specificity of exoglycosidase. For example, β-galactosidase is added to a sugar chain of unknown structure and hydrolyzed, and then monosaccharide is released from the sugar chain and / or the substrate sugar chain. By paying attention to the structural change and confirming these, the presence of β-galactoside bond at the non-reducing end of the sugar chain (oligosaccharide) is determined.
[0003]
By the way, it is known that most of the enzymes acting on the N-acetyl-β-D-galactosaminide bond so far known also act on the N-acetyl-β-D-glucosaminide bond (therefore, β-N -The enzyme acting only on the N-acetyl-β-D-galactosaminide bond is named by Hooghwinkel et al. (Named acetylhexosaminidase (EC 3.2.1.52)) ( GJM Hooghwinkel et al., Hoppe-Seyler's Z. Physiol. Chem., 353, 839, (1972); JA Cabezas, Biochem. J, 261, 1059-1060 (1989)). In this Hooghwinkel et al. Report, although experimental results suggesting that there exists an enzyme that acts only on the N-acetyl-β-D-galactosaminide bond, the enzyme is not actually separated. Therefore, there has been no known example in which β-N-acetylgalactosaminidase (EC 3.2.1.53) has been actually separated and purified.
[0004]
N-acetyl-β-D-glucosaminide linkages are present in glycolipids and glycoproteins as well as N-acetyl-β-D-galactosaminide linkages. Therefore, when a conventionally known β-N-acetylhexosaminidase is used in the sugar chain structure analysis method, the monosaccharide is released from the substrate sugar chain and / or the substrate sugar chain after the enzyme reaction. In addition to the step of confirming the structural change, a complicated analysis step for determining whether the non-reducing end of the substrate sugar chain is N-acetyl-D-galactosamine or N-acetyl-D-glucosamine must be further added. is the current situation.
[0005]
[Problems to be solved by the invention]
The present invention solves the above-mentioned conventional problems. The object of the present invention is to release β-glycoside-bonded N-acetyl-D-galactosamine at the non-reducing end of the sugar chain. It is to provide β-N-acetylgalactosaminidase, which is an exoglycosidase that does not release β-glycoside-linked N-acetyl-D-glucosamine in the above, and a method for producing the same.
[0006]
[Means for Solving the Problems]
The present inventors have, belong to Bacillus (Bacillus) genus are Gram-positive bacteria, from freshly isolated strains, the N- acetyl-beta-D-galactosaminidic bonds at the non-reducing end of a sugar chain by hydrolysis, An exo that liberates N-acetyl-D-galactosamine but does not act at all on the N-acetyl-β-D-glucosaminide linkage at the non-reducing end of the sugar chain and therefore does not liberate N-acetyl-β-D-glucosamine The present inventors have found β-N-acetylgalactosaminidase, a glycosidase, and completed the present invention.
[0007]
The β-N-acetylgalactosaminidase of the present invention (hereinafter referred to as the present β-N-acetylgalactosaminidase or the present enzyme) hydrolyzes the N-acetyl-β-D-galactosaminide bond at the non-reducing end of the sugar chain. Thus, N-acetyl-D-galactosamine is released, but N-acetyl-β-D-glucosaminide linkage at the non-reducing end of the sugar chain is not affected at all, and therefore N-acetyl-D-glucosamine is not released. It is an exoglycosidase characterized by this. This achieves the above object.
[0008]
Preferred β-N-acetylgalactosaminidase of the present invention further has the following characteristics.
(1) Optimum pH: 6.0.
(2) Stable pH range: pH is 5.5 to 9.5 when held at 25 ° C. for 1 hour.
(3) Range of temperature suitable for action: The optimum temperature is 45 ° C.
(4) Thermal stability: Stable to 40 ° C. when held at pH 6.0 for 15 minutes.
(5) Molecular weight by SDS gel electrophoresis: about 48,000.
(6) Isoelectric point by isoelectric focusing: 4.3.
[0009]
This β-N-acetylgalactosaminidase is produced by bacteria of the genus Bacillus. A particularly preferred Bacillus bacterium is Bacillus sp. AT173-1 strain (Life Research Bacteria No. 15390) deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (abbreviated as Life Research Institute) on January 12, 1996; FERM P-15390).
[0010]
The production method of the present β-N-acetylgalactosaminidase comprises the steps of culturing a Bacillus bacterium that produces the present β-N-acetylgalactosaminidase, and the present β-N-acetylgalactosaminidase from the culture obtained by the cultivation step. Collecting N-acetylgalactosaminidase. This achieves the above object.
[0011]
A preferred production method of the present β-N-acetylgalactosaminidase is a method using the above Bacillus sp. AT173-1 strain.
Hereinafter, the present invention will be described in detail.
[0012]
The present β-N-acetylgalactosaminidase is produced by a bacterium belonging to the genus Bacillus , in particular, Bacillus sp. AT173-1 strain (Life Research Bacteria No. 15390; FERM P-15390) (hereinafter referred to as the present bacterium). This bacterium is a bacterium isolated by the inventors from soil in Kosai-cho, Koka-gun, Shiga Prefecture. The bacteriological properties of this bacterium are shown below.
[0013]
(A) Morphology and physiological properties Tables 1 to 3 show the results of culturing this bacterium. The culture temperature is 30 ° C. unless otherwise specified. The form of this bacterium was examined by culturing in tryptic soy medium (manufactured by DIFCO).
[0014]
[Table 1]

[0015]
[Table 2]

[0016]
[Table 3]

[0017]
Based on the bacteriological properties of Tables 1 to 3, referring to Bergey's Manual of Systematic Bacteriology Volume 2 (Williams and Wilkins, Baltimore, 1986), the bacterium must belong to the genus Bacillus I understood. Among the genus Bacillus, it is most similar to Bacillus sphaericus ( Bacillus sphaericus ), but because of the different spore shape, it was not possible to identify this bacterium as Bacillus sphaericus. Therefore, the inventors named this bacterium Bacillus sp. AT173-1 strain.
[0018]
Hereinafter, conditions for producing and collecting the present β-N-acetylgalactosaminidase from the bacterium will be described.
[0019]
(B) Culture conditions The culture conditions of the present bacterium are not particularly limited, and a normal liquid medium or solid medium is used. As the carbon source, various saccharides are used, and amino sugar, glucose, molasses and the like are used. Examples of the nitrogen source include, but are not limited to, peptone, yeast extract, soybean flour, corn steep liquor, casein, meat extract, and amino acid. In addition to the above carbon source and nitrogen source, various inorganic salts (eg, magnesium salt, potassium salt, sodium salt), vitamins, and the like can be added as necessary. The preferred growth pH of the bacterium is 6-8, but is not limited thereto.
[0020]
The culture temperature of the bacterium is not particularly limited as long as the bacterium can grow, but the culture temperature is preferably 20 ° C to 45 ° C, particularly preferably 30 ° C. In the case of liquid culture, the culture is usually performed with aerobic stirring or shaking for 24 to 72 hours. Changes in cell growth over time in the culture process can be known, for example, by monitoring changes in turbidity of the culture suspension (usually measurement of absorbance at 660 nm). This β-N-acetylgalactosaminidase is mainly secreted outside the cells.
[0021]
(C) Method for collecting β-N-acetylgalactosaminidase In order to collect and purify the enzyme of the present invention from the culture medium, known purification methods can be used alone or in combination. For example, the culture medium is filtered or centrifuged to remove the cells, and the obtained supernatant is fractionated by subjecting it to salting out with ammonium sulfate, sodium sulfate, etc., or precipitation with an organic solvent such as methanol, ethanol, acetone, etc. By concentrating, a crude enzyme solution containing the present β-N-acetylgalactosaminidase is obtained. Further, the crude enzyme solution is purified by performing ion exchange chromatography, gel filtration chromatography, and / or other various chromatography. An outline of a preferred purification method is shown below.
[0022]
(1) The culture solution is centrifuged to remove the cells and obtain a supernatant.
(2) Next, the obtained supernatant is salted out with ammonium sulfate (80% saturated solution).
(3) Next, the obtained salting-out solution is dialyzed against Tris / hydrochloric acid buffer of pH 8.0 and subjected to QAE-Toyopearl column chromatography.
(4) An ammonium sulfate solution is added to the active fraction obtained in the above (3) so that the final concentration is 25%, and subjected to phenyl sepharose column chromatography.
(5) The active fraction obtained in (4) above is dialyzed against pH 6.0 acetate buffer and subjected to Q-Sepharose column chromatography.
(6) The active fraction obtained in (5) above is concentrated with an ultrafiltration membrane and subjected to Sephacryl S-200 column chromatography.
(7) An ammonium sulfate solution is added to the active fraction obtained in the above (6) so as to have a final saturated concentration of 25%, and subjected to resource PHE column chromatography.
[0023]
(D) Method for measuring activity of the present β-N-acetylgalactosaminidase In the present specification, the present β-N-acetylgalactosaminidase activity is determined using paranitrophenyl-N-acetyl-β-D-galactosaminide as a substrate. The amount of enzyme that liberates 1 micromole of paranitrophenol per minute is measured as 1 unit (U). Specifically, in this specification, dithiothreitol was previously added to 0.4 ml of a 2.5 mM paranitrophenyl-N-acetyl-β-D-galactosaminide solution dissolved in 50 mM acetate buffer at pH 6.0. Add 0.1 ml of the sample solution which was added to 0.1% and left at room temperature for 1 hour, and react at 37 ° C. for 10 minutes. Thereafter, 2.5 ml of a 2% sodium carbonate solution is added to stop the reaction, and the absorbance of the reaction solution at a wavelength of 420 nm (which increases with the release of paranitrophenol) is measured.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
The β-N-acetylgalactosaminidase of the present invention is an exoglycosidase that specifically hydrolyzes N-acetyl-β-D-galactosaminide linkages. Therefore, when the non-reducing end of the sugar chain is N-acetyl-β-D-galactosaminide, the glycosidic bond is hydrolyzed to release N-acetyl-D-galactosamine from the non-reducing end. When the non-reducing end is N-acetyl-β-D-glucosaminide, the enzyme does not act and does not generate free monosaccharides.
[0025]
The following examples further illustrate the present invention in detail but are not intended to limit the present invention in any way.
[0026]
【Example】
Example 1
(Preparation of culture supernatant)
Contains 100 ml of medium (0.0625% N-acetyl-D-galactosamine, 1% defatted soybean peptone, 1% yeast extract, 0.1% K ₂ HPO ₄ and 0.02% MgSO ₄ .7H ₂ O) The bacteria were inoculated into 80 500 ml shake flasks to be subjected to reciprocal shaking culture at 30 ° C. for 2 days. After completion of the culture, this culture solution was collected and centrifuged at 10,000 rpm for 10 minutes to remove the cells and obtain 7360 ml of the culture supernatant.
[0027]
Example 2
(Β-N-acetylgalactosaminidase activity and β-N-acetylglucosaminidase activity in the culture supernatant)
The activity of the present β-N-acetylgalactosaminidase in the supernatant obtained in Example 1 was measured according to the method described in (d) above. β-N-acetylglucosaminidase activity was measured according to the method described in (d) above using paranitrophenyl-N-acetyl-β-D-glucosaminide as a substrate. The results are shown in FIG. In the figure, the horizontal axis represents the reaction time, the vertical axis represents the absorbance at 420 nm with the amount of paranitrophenol produced, ● represents paranitrophenyl-N-acetyl-β-D-galactosaminide, and ○ represents paranitro. The numerical values when phenyl-N-acetyl-β-D-glucosaminide is used as a substrate are shown. When paranitrophenyl-N-acetyl-β-D-galactosaminide was used as a substrate, an increase in absorbance at 420 nm was observed with the increase in liberated paranitrophenol, but paranitrophenyl-N-acetyl-β-D. -When glucosaminide was used as a substrate, no increase in absorbance at 420 nm was observed with the increase in liberated paranitrophenol. The activity of this enzyme in the supernatant was 0.088 U / ml.
[0028]
Example 3
(Preparation of this enzyme)
All column chromatography operations were performed at 4 ° C. Ammonium sulfate was added to the culture supernatant obtained in Example 1 until 80% saturation, and the resulting precipitate was collected by centrifugation. The precipitate was dissolved in pH 6.0, 50 mM acetate buffer, and then dialyzed against pH 8.0, 50 mM Tris-HCl buffer. This enzyme solution was adsorbed on a QAE-Toyopearl 550C column (manufactured by Tosoh Corporation) equilibrated with 50 mM Tris / HCl buffer, and eluted with a 0.1-1 M salt concentration gradient. A phenyl sepharose HP column (Pharmacia Biotech) was added to the obtained enzyme fraction so that an ammonium sulfate solution was added to a final concentration of 25% and equilibrated with 50 mM Tris / HCl buffer containing 25% saturated ammonium sulfate. And eluted with a gradient of ammonium sulfate concentration of 25 to 0% saturation. The obtained enzyme fraction was dialyzed against a pH 6.0, 50 mM acetate buffer, and the dialysate was adsorbed onto a Q-Sepharose FF column equilibrated with a pH 6.0, 50 mM acetate buffer. The elution was carried out using a salt concentration gradient. The obtained enzyme fraction was concentrated with an ultrafiltration membrane, and the concentrated solution was equilibrated with pH 8.0, 50 mM Tris / HCl buffer containing 0.15 M sodium chloride (Pharmacia Biotech). Made). A resource PHE column (Pharmacia Biotech) was prepared by adding an ammonium sulfate solution to the obtained enzyme fraction to a final concentration of 25% saturation and equilibrating with a pH 8.0, 50 mM Tris / HCl buffer solution containing 25% saturated ammonium sulfate. And eluted with a gradient of ammonium sulfate concentration of 25 to 0% saturation.
[0029]
By the above operation, a solution containing the present β-N-acetylgalactosaminidase purified 8700 times in a yield of 1.5% from 7360 ml (total 647 U) of the culture supernatant obtained in Example 1 was obtained. 5 ml (9.56 U) was obtained. The specific activity of the enzyme was 154 U / mg protein. This purified enzyme solution showed a single band by SDS gel electrophoresis and isoelectric focusing described later. Table 4 shows the degree of enzyme purification in each purification step of Examples 1 and 3 corresponding to (1) to (7) described in (c) above. The activity values in the table are values measured according to the measurement method described in (d) above.
[0030]
[Table 4]

[0031]
In the following examples, the enzyme solution or concentrated solution obtained in Example 3 was used.
[0032]
Example 4
(Substrate specificity of this enzyme)
The reactivity of this enzyme with various pyrisylaminated sugar chains as substrates was compared. The results are shown in Table 5.
[0033]
[Table 5]

[0034]
Substrates (1), (2) and (5) in Table 5 were manufactured by Takara Shuzo Co., Ltd., and (4) was manufactured by Ajinoki Co., Ltd. Substrate (3) is pyricylaminated lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-PA: manufactured by Takara Shuzo Co., Ltd.), β-galactosidase (derived from Aspergillus sp., Manufactured by Toyobo Co., Ltd.) It was prepared by acting.
[0035]
Reactions with respect to the substrates (1), (2), (3) and (5) were carried out by the method shown below. 6 μl of 14 U / ml of this enzyme solution was added to 4 μl of pH 6.0, 50 mM acetate buffer containing 20 μmol of substrate, 20 μg of bovine serum albumin (Sigma) and 1 μg of dithiothreitol, and the mixture was incubated at 37 ° C. for 6 hours. Reacted. Then, 40 μl of 1% trifluoroacetic acid was added to the reaction solution to stop the reaction. The reaction with respect to the substrate (4) followed the above reaction conditions except that the amount of the substrate was 10 pmol. The obtained reaction solution was analyzed by high performance liquid chromatography, and the decomposition rate was calculated.
[0036]
Substrates (1), (2) and (4) having an N-acetyl-β-galactosaminide bond at the non-reducing end completely hydrolyze all N-acetyl-β-galactosaminide linkages present at the non-reducing end. In contrast, the substrates (3) and (5) having an N-acetyl-β-glucosaminide bond at the non-reducing end were not hydrolyzed at all.
[0037]
Example 5
(Optimal pH and stable pH of this enzyme)
(1) Optimum pH
(D) except that the pH was adjusted to 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0 and 9.0 with 25 mM Briton-Robinson broad-area buffer. The β-N-acetylgalactosaminidase activity at each pH was measured according to the method described in (1). The results are shown in FIG. In the figure, the horizontal axis indicates the reaction pH, and the vertical axis indicates the relative activity (%) when the obtained maximum activity value is 100. The optimum pH of this enzyme was 6.0.
[0038]
(2) Residual activity of the enzyme after adjusting the pH of the enzyme solution from 3.0 to 11.0 at 0.5 intervals with a stable pH range of 53 mM Briton-Robinson broad buffer and holding at 25 ° C. for 1 hour Was measured according to the method described in (d) above. The results are shown in FIG. The horizontal axis in the figure represents the retained pH, and the vertical axis represents the residual activity (%) when the initial activity is 100. The enzyme was stable in the range of pH 5.5 to 9.5.
[0039]
Example 6
(Range of temperature and heat stability of this enzyme)
(1) Range of temperature suitable for action The enzyme activity under various reaction temperature conditions (30, 37, 40, 45, 50, 55 ° C.) was measured according to the method described in (d) above. The results are shown in FIG. In the figure, the horizontal axis indicates the reaction temperature, and the vertical axis indicates the relative activity (%) when the obtained maximum activity value is 100. The optimum temperature for this enzyme was 45 ° C.
[0040]
(2) Thermal stability 0.14U dissolved in 50 mM acetate buffer at pH 6.0 containing 0.2% bovine serum albumin under various temperature conditions (25, 30, 35, 40, 45, 50, 55 ° C.) / Ml of the enzyme solution was maintained for 15 minutes, and the residual activity of the enzyme was measured according to the method described in (d) above. The results are shown in FIG. The horizontal axis in the figure represents the holding temperature, and the vertical axis represents the remaining activity (%) when the initial activity is 100. The enzyme was stable up to 40 ° C.
[0041]
Example 7
(Molecular weight of this enzyme)
The molecular weight of this enzyme was measured using general SDS gel electrophoresis. Samples containing this enzyme were analyzed on the same electrophoresis gel as standard samples containing various proteins with known molecular weights, and molecular weights were measured based on the relationship between their mobility and molecular weight. The gel used was First Gel Gradient 10-15 manufactured by Pharmacia Biotech. FIG. 6 shows the relationship between the mobility (horizontal axis) of each standard protein and the molecular weight (vertical axis; × 10 ⁴ ). A, B, C, D, E and F in the figure are rabbit muscle phosphorylase b (molecular weight 94,000), bovine serum albumin (molecular weight 67,000), chicken ovalbumin (molecular weight 43,000), and bovine erythrocytes, respectively. Carbonic anhydrase (molecular weight 30,000), soybean trypsin inhibitor (molecular weight 20,100) and milk α-lactalbumin (molecular weight 14,400) are shown. The molecular weight of the enzyme measured by SDS gel electrophoresis is about 48,000.
[0042]
Example 8
(Isoelectric point of this enzyme)
The isoelectric point of the enzyme was measured by a conventional isoelectric focusing method. The enzyme was analyzed on the same electrophoresis gel as a standard sample containing various proteins with known molecular weights, and the isoelectric point of the enzyme was measured from a calibration curve based on the relationship between their mobility and isoelectric point. The isoelectric point of this enzyme was about 4.3.
[0043]
【The invention's effect】
According to the present invention, β-N-acetylgalactosaminidase that specifically hydrolyzes the N-acetyl-β-D-galactosaminide bond at the non-reducing end of a sugar chain, and a method for producing the β-N-acetylgalactosaminidase are provided.
[0044]
This β-N-acetylgalactosaminidase liberates β-glycosidically linked N-acetyl-D-galactosamine at the non-reducing end of the sugar chain, but β-glycosidically linked N-acetyl- at the non-reducing end. Since D-glucosamine is not released at all, the presence or absence of β-glycoside-bonded N-acetyl-D-galactosamine at the non-reducing end of the sugar chain whose structural analysis is desired can be easily determined, and the structure of the sugar chain is determined. Very useful to. Since the present enzyme is produced by culturing microorganisms, it can be provided in a large amount and at a low cost by a simple process.
[Brief description of the drawings]
FIG. 1 shows the present β-N-acetylgalactosaminidase activity and β-N-acetylglucosaminidase activity in culture supernatants.
FIG. 2 shows the relative activity when the present β-N-acetylgalactosaminidase activity is measured in the range of pH 4.0 to 9.0.
FIG. 3 shows the residual activity measured after maintaining this β-N-acetylgalactosaminidase activity at 25 ° C. for 1 hour under the conditions of pH 3.0 to 11.0.
FIG. 4 shows the relative activity when the present β-N-acetylgalactosaminidase activity is measured in the temperature range of 30 to 55 ° C.
[Fig. 5] This β-N-acetylgalactosaminidase was measured after being kept for 15 minutes under a temperature condition of 25 to 55 in 50 mM acetate buffer (pH 6.0) containing 0.2% bovine serum albumin. Shows residual activity.
FIG. 6 shows the molecular weight of the present β-N-acetylgalactosaminidase measured by SDS electrophoresis.

Claims (4)

An exoglycosidase that releases β-glycoside-bonded N-acetyl-D-galactosamine at the non-reducing end of the sugar chain, but does not release β-glycoside-bonded N-acetyl-D-glucosamine at the non-reducing end. β-N-acetylgalactosaminidase having the following characteristics:
PH optimum: 6.0;
Stable pH range: pH 5.5 to 9.5 when held at 25 ° C. for 1 hour;
Range of optimal working temperature: the optimal temperature is 45 ° C;
Thermal stability: stable to 40 ° C. when held at pH 6.0 for 15 minutes;
Molecular weight in SDS gel electrophoresis: about 48,000; and
Isoelectric point in isoelectric focusing: 4.3;
Β-N-acetylgalactosaminidase having The β-N-acetylgalactosaminidase according to claim 1, which is produced by a bacterium belonging to the genus Bacillus. The following steps:
(A) culturing a Bacillus bacterium that produces the β-N-acetylgalactosaminidase; and (b) the β-N-acetylgalactosaminidase from the culture obtained by the culturing step (a). A method for producing β-N-acetylgalactosaminidase according to claim 1 , comprising the step of: The method according to claim 3 , wherein the Bacillus bacterium is Bacillus sp. AT173-1 strain (Nippon Kenkyu No. 15390; FERM P-15390).

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