JP4439153B2 - Thermostable mannose isomerase and method for producing mannose using the enzyme - Google Patents

Description

本発明の酵素：アグロバクテリウム・ラディオバクターＭ３６由来
酵素Ａ：シュードモナス・エスピーＡＭ−９５８２（特許第３００３００９号）
由来の酵素
酵素Ｂ：シュードモナス・エスピーＮ−２５（特開平６−２９２５７８）由来の
酵素
酵素Ｃ：サーモモノスポラ・エスピーＭＢＬ１０００３（特開平１１−７５８３
６）由来の酵素
表１の結果から、本発明の酵素は、▲１▼その由来の微生物において酵素Ａ〜Ｃとは異なること、▲２▼基質特異性や最適ｐＨ、最適温度などにおいて酵素Ｂや酵素Ｃと異なること、▲３▼安定温度やカルシウムイオンによる熱安定化作用等において酵素Ａと異なること、▲４▼マンノースに対するＫｍ値において酵素Ａと異なることが、それぞれ分かる。
従って、本発明の酵素は、公知の酵素と比較すると、マンノースをフラクトースに異性化するという共通の作用を示すものの、その理化学的性質が異なっているから、酵素タンパク質としては異なる物質であるといえる。
以上により、本発明の酵素は、従来知られている酵素とは全く異なる新規なマンノースイソメラーゼである。
［発明の実施の形態］
以下、本発明の実施の形態を更に説明する。
（イ） 菌株
本発明に使用するアグロバクテリウム・ラディオバクターＭ３６株は、本発明者らによって土壌中より発見された菌種であり、工業技術院生命工学工業技術研究所（日本国茨城県つくば市東１丁目１番３号）に、ＦＥＲＭ Ｐ−１７３７７として寄託されている（平成１１年４月２３日）。
アグロバクテリウム・ラディオバクターＭ３６株は、以下の菌学的諸性質を有する。
（１）形態
細胞の形及び大きさ：０．８×１．２〜１．５μｍのかん菌
運動性：＋
グラム染色性：−
胞子形成：−
コロニー形態：円形、低凸状、金縁なめらか、光沢、半透明
（２）生理学的性質
硝酸塩還元：＋
インドール産生：−
ブドウ糖酸性化：−
３−ケト乳糖産生：＋
アルギニンジヒドロラーゼ：−
ウレアーゼ：−
エスタリン加水分解：＋
ゼラチン加水分解：−
β−ガラクトシダーゼ：＋
オキシダーゼ：＋
カタラーゼ：＋
基質資化能：
ブドウ糖：＋
Ｌ−アラビノース：＋
Ｄ−マンノース：＋
Ｄ−マンニトール：＋
Ｎ−アセチル−Ｄ−グルコサミン：＋
マルトース：＋
グルコン酸カリウム：＋
ｎ−カプリン酸：−
アジピン酸：−
ｄｌ−リンゴ酸：＋
クエン酸ナトリウム：−
酢酸フェニル：−
Ｏ／Ｆ試験：−
（ロ） マンノースイソメラーゼの産出
マンノースイソメラーゼを産出する微生物としては、上記の菌株が有利に用いられる。また、その他のアグロバクテリウム属に属する微生物も利用でき、例えば、Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒａｄｉｏｂａｃｔｅｒ（ＩＡＭ１２０４８）、Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（ＩＡＭ１３１２９）、等が挙げられる。また、これらの変異株や組換え微生物が産生する酵素も利用できる。分泌の場所は特に制限されず、菌体内、菌体外の何れでもよい。
この酵素は、以下のような、通常の培養手段により産出される。
フラクトース、グルコース、マンノース、ガラクトース、異性化糖、蔗糖、マルトース、キシロース、ソルビトール、マンニトール、澱粉など種々な糖、糖アルコール及び澱粉やデキストリンなどを炭素源とし、それに微生物の増殖に必要な窒素化合物（酵母エキス、魚肉エキス、ペプトン、アミノ酸、などの有機窒素源、あるいは硫酸アンモニウム、塩化アンモニウム、尿素、硝酸アンモニウムなどの無機窒素源）、ミネラルを加えた培地で、２０〜４０℃、好ましくは３０℃前後で好気条件下において、１〜５日、好ましくは１〜３日培養する。
分泌の場所が菌体内の場合、培養後、濾過又は遠心分離により菌体を回収し、そのまま使用するか、又は超音波若しくは自己消化法等により該酵素を抽出し使用する。又、分泌の場所が菌体外の場合、培養後、濾過又は遠心分離により菌体を除去し、濾液又は上清をそのまま使用する。抽出された酵素は、必要により、硫酸アンモニウム、アセトン、メタノール、エタノール等で沈澱し、精製濃縮し、又は乾燥保存する。
（ハ） マンノースの製造
ａ） フラクトースを原料とする場合
フラクトースに、上記（ロ）で示した菌体や菌体処理物などを作用させると、マンノースを製造することができる。
菌体処理物としては、▲１▼菌体を通常の方法で固定化したもの、▲２▼菌体を機械的、酵素的、若しくは、界面活性剤や有機溶剤などで処理したもの、又は、それらを固定化したもの、▲３▼菌体を破砕して破砕残さを除去したもの、又は、それを固定化したもの、▲４▼酵素精製物、又は、それを固定化したもの、等が挙げられる。
工業的には、菌体を固定化処理するか、又は菌体から抽出した酵素を適当な担体に固定し、固定化酵素として使用するのが有効である。
菌体処理物の固定化は、ポリアクリルアミド、アルギン酸カルシウム、カラギーナン、イオン交換担体、キトサンビーズなどを用いた通常の固定化方法が使用できる。
本酵素を用い、フラクトースからマンノースを製造する反応は、フラクトースまたは１〜７５％のフラクトース含有液を用いて、ｐＨ５．５〜１０、温度６５℃以下で行うのがよい。閉鎖した反応系で本酵素反応を行なった場合、反応平衡時ではフラクトース：マンノース＝約７５：約２５となる。
ｂ） グルコースを原料とする場合
グルコースを原料とする場合には、グルコースからフラクトースを生成する反応と該フラクトースからマンノースを生成する反応との二種類の反応を生起させることに行われる。
従って、本方法の場合、本酵素のマンノースイソメラーゼの他に、グルコースイソメラーゼを併用するので、両酵素の作用条件が一致することが必要である。
前述したように、本酵素のマンノースイソメラーゼの作用条件は、グルコースイソメラーゼの作用条件（ｐＨ６．５〜７、温度６０℃）と一致するので問題はない。閉鎖した反応系で酵素反応を行なった場合、反応平衡時ではグルコース：フラクトース：マンノース＝約５０：約４０：約１０となる。
上記の二種類の反応に順じて、固定相式クロマト分離装置と疑似移動相式クロマト分離装置とを組み合わせて、連続反応製造とすることも当然可能である。
［実施例］
以下、実施例によって本発明を詳細に説明するが、本発明はこれらに限定されない。特別に標記がない場合、百分率はＷ／Ｖ％である。
実施例１ 菌体の培養
ポリペプトン １０％、酵母エキス５％、ＮａＣｌ ５％からなる培地（ｐＨ７．０）１００ｍｌを５００ｍｌ容量バッフル付き三角フラスコに入れ、常法により滅菌後、アグロバクテリウム・ラディオバクターＭ３６（ＦＥＲＭ Ｐ−１７３７７）を接種し、３０℃で２４時間振どう培養した。培養液を遠心分離機にて１２０００ｒｐｍの回転数で遠心分離し、菌体を回収した。得られた菌体は１００ｍＭのリン酸緩衝液（ｐＨ７．０）２５ｍｌで２回洗浄した。１０ｍｌのリン酸緩衝液で菌体を懸濁した後、超音波破砕を行ない、遠心分離し、沈澱と粗酵素液に分けた。粗酵素溶液のマンノースイソメラーゼ活性は０．６３単位／ｍｌであった。
実施例２ マンノース変換率の測定
粗酵素液５００μｌに２０％フラクトース溶液５００μｌを加え、５０℃で２４時間反応を行なった。反応液の糖組成はＨＰＬＣによって確認した。ＨＰＬＣの条件を下記に示した。反応液中のマンノースを定量したところ、フラクトースからの変換率は２５．０％であった。
ＨＰＬＣ条件
ポンプ機種：日本分光（株）製 ＰＵ−１５８０
カラム：資生堂（株）製 ＣＡＰＣＥＬＬ ＰＡＫ ＮＨ２ ＵＧ８０
検出器：昭和電工（株）製 ＲＩ−７１型 示差屈折計
溶離液：アセトニトリル：水＝８０：２０
流速：１．０ｍｌ／ｍｉｎ
実施例３ アグロバクテリウム（Ａｇｒｏｂａｃｔｅｒｉｕｍ）属の微生物由来のマンノースイソメラーゼの酵素活性
アグロバクテリウムに属する微生物として、Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒａｄｉｏｂａｃｔｅｒ（Ｍ３６）、Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒａｄｉｏｂａｃｔｅｒ（ＩＡＭ１２０４８）、Ａｇｒｏｂａｃｔｃｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（ＡＭ１３１２９）を用いた。これらの微生物から実施例１記載の方法に従って粗酵素液を調製し、マンノースイソメラーゼ活性について測定した。
尚、酵素活性の測定は以下の方法で行った。
１００ｍＭリン酸緩衝液（ｐＨ７．０）に溶解した２００ｍＭのマンノース溶液２００μｌに適宜希釈した酵素溶液２００μｌを加え、５０℃で酵素反応を行なった。次いで、酵素反応を１０分間の煮沸によって停止させ、ＨＰＬＣによって糖組成の分析を行なった。この条件下で、１分間に１μ ｍｏｌのフラクトースを生成する酵素量を１単位とした。結果は表２に示した。

上記の結果から、アグロバクテリウム属に属するいずれの微生物にもマンノースイソメラーゼ活性が認められ、特に、Ａｇｒｏｂａｃｔｅｒｉｕｍ ｒａｄｉｏｂａｃｔｅｒ（Ｍ３６）の酵素活性が高いことが分かる。
実施例４ マンノースイソメラーゼの酵素特性
アグロバクテリウム・ラディオバクターＭ３６由来のマンノースイソメラーゼについて、実施例１に準じて粗酵素液を調製した後、硫安分画、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によって酵素精製を行い以下の試験に供した。
（１）基質特異性
基質には、Ｄ−マンノース、Ｄ−フラクトース、Ｄ−リキソース、Ｄ−キシルロース、Ｄ−グルコース、Ｄ−ガラクトース、Ｌ−アラビノース、Ｄ−キシロース、Ｄ−リボース、Ｌ−ラムノース、Ｌ−フコース、Ｄ−フコースの各高純度品（試薬）を用いた。１００ｍＭリン酸緩衝液（ｐＨ７．０）に溶解させた２００ｍＭの各基質２００μｌに適宜希釈した酵素溶液２００μｌを加え、５５℃で３時間反応させ、ＨＰＬＣで解析した。その結果、本マンノースイソメラーゼはＤ−マンノース、Ｄ−フラクトース、Ｄ−リキソース及びＤ−キシルロースに作用し、Ｄ−マンノースとＤ−フラクトース及びＤ−リキソースとＤ−キシルロースをそれぞれ相互変換した。また、Ｄ−グルコース、Ｄ−ガラクトース、Ｌ−アラビノース、Ｄ−キシロース、Ｄ−リボース、Ｌ−ラムノース、Ｌ−フコース、Ｄ−フコースには実質的に作用しないことが判明した。
（２）最適温度：
１００ｍＭリン酸緩衝液（ｐＨ７．０）に溶解させた２００ｍＭマンノース溶液２００μｌに適宜希釈した酵素溶液２００μｌを加え、４０、５０、５５、６０、６５℃で３０分間反応させた。酵素失活させた後、生成したフラクトース量をＨＰＬＣによって定量した。結果を図１に示す。
図１から明らかなように、本酵素の最適温度は５５℃〜６０℃であった。
（３）最適ｐＨ：
１００ｍＭの各種ｐＨの緩衝液に溶解させた２００ｍＭマンノース溶液２００μｌに適宜希釈した酵素溶液２００μｌを加え、５０℃で３０分間反応させた。酵素失活させた後、生成したフラクトース量をＨＰＬＣによって定量した。結果を図２に示す。
図２から明らかなように、本酵素の最適ｐＨは７〜９であった。
（４）安定温度
適宜希釈した酵素溶液２００μｌを４０、５０、５５、６０、６５℃で１０分間インキュベートした後、１００ｍＭリン酸緩衝液（ｐＨ７．０）に溶解させた２００ｍＭマンノース溶液２００μｌを加えて反応させた。酵素失活させた後、生成したフラクトース量をＨＰＬＣによって定量した。結果を図３に示す。
図３から明らかなように、本酵素は約６０℃まで安定であった。
（５）安定ｐＨ
各種ｐＨの緩衝液で適宜希釈した酵素溶液２００μｌを２５℃で３時間インキュベートした後、１００ｍＭリン酸緩衝液（ｐＨ７．０）に溶解させた２００ｍＭマンノース溶液２００μｌを加えて反応させた。酵素失活させた後、生成したフラクトース量をＨＰＬＣによって定量した。結果を図４に示す。
図４から明らかなように、本酵素はｐＨ約６〜１０まで安走であった。
（６）分子量と等重点
ゲル濾過ＨＰＬＣによる分子量は約１４０，０００、等電点電気泳動による等重点は約５．２であった。
（７）カルシウムイオンによる熱安定化作用
塩化カルシウムを終濃度５ｍＭになるように含む２５ｍＭのＰＩＦＥＳ（ピペラジン−Ｎ，Ｎ′−ビス（２−エタンスルホン酸））緩衝液（ｐＨ７．０）で適宜希釈した酵素溶液を５５℃、６０℃、６２．５℃で１０分間インキュベートした。次いで２５ｍＭのＰＩＦＥＳ緩衝液に溶解した２００ｍＭのマンノース溶液２００μｌに本酵素溶液２００μｌを加え、５５℃で３０分間反応させ、反応停止後生成したフラクトースをＨＰＬＣで定量し、各温度のカルシウムイオンの安定化作用を以下の安定化率により評価した。
安定化率（％）＝（カルシウム添加の反応系におけるフラクトース変換率／カルシウム未添加の反応系におけるフラクトース変換率）×１００
結果を表３に示した。

表３の結果から、各温度条件においてフラクトース変換率は実質的に変化なく、本酵素は、カルシウムイオンにより殆ど安定化されないことが判明した。
（８）Ｋｍ（ミカエリス定数）値の測定
酵素反応速度論に基づき、Ｈａｎｅｓ−Ｗｏｏｌｆプロットにより本酵素のマンノースに対するＫｍ値を求めたところ、約０．０１Ｍであった。
実施例５ マンノース生成反応時の特性
実施例１で調製したマンノースイソメラーゼを用いて酵素の諸性質について確認した。
（１）反応温度の影響
フラクトース各２００ｍｇに、１００ｍＭリン酸緩衝液（ｐＨ７．０）、マンノースイソメラーゼ０．２単位を加え、全量を１ｍｌとし、４５、５０、５５、６０℃で３日間反応させた。経時的にサンプリングを行ない、生成したマンノース量をＨＰＬＣによって定量した。結果を図５に示す。
図５から明らかなように、反応初期では６０℃反応が最も良かったが、反応終期では５５℃の反応が最も良く、７２時間目で変換率２５．６％に達した。
（２）反応ｐＨの影響
フラクトース各２００ｍｇに、１００ｍＭの各種酸緩衝液、マンノースイソメラーゼ０．２単位を加え、全量を１ｍｌとし、５０℃で２４時間反応させた。反応開始２４時間目にサンプリングを行ない、生成したマンノース量をＨＰＬＣによって定量した。結果を図６に示す。
図６から明らかなように、ｐＨ６．５〜９．５という、幅広いｐＨにおいて実質的なマンノース生産が可能であることが明らかにされた。
（３）高濃度基質での反応
フラクトース ５００ｍｇに１００ｍＭリン酸緩衝液（ｐＨ７．０）、マンノースイソメラーゼ４、２、１、０．５単位を加え、水で全量を１ｍｌとし、５５℃で２日間反応させた。経時的にサンプリングを行ない、生成したマンノース量をＨＰＬＣによって定量した。結果を図７に示す。
図７から明らかなように、本酵素は、高濃度のフラクトース溶液（５０％）を効率良くマンノースに変換することがわかった（変換率約２５％）。
実施例６ フラクトースからマンノースの製造
実施例１の方法で調製したマンノースイソメラーゼを硫酸アンモニウムによる分画処理を行なった。脱塩して部分精製した酵素溶液５ｍｌを１００ｍＭリン酸緩衝液（ｐＨ７．０）で２倍希釈し、キトサンビーズ（富士紡績（株）製、「キトパールＢＣＷ−３５０５」８ｍｌを加え、３０℃で２時間振とうさせて固定化を行なった。酵素を固定化したキトサンビーズをカラムに充填し、１００ｍＭリン酸緩衝液（ｐＨ７．０）５０ｍｌを通液した。通液した液には酵素の溶出は確認できず、酵素は完全にキトサンビーズに吸着されたことがわかった。５５℃で保温したカラムに２０％フラクトース溶液を流速０．２５ｍｌ／分で３日間循環させた。反応３日目に反応液を回収し、生成したマンノース量をＨＰＬＣによって定量した。その結果、フラクトースからの変換率は２４．５％であった。
実施例７ グルコースからマンノースの製造
実施例１で調製したマンノースイソメラーゼと市販のストレブチマイセル・ムリヌス由来のグルコースイソメラーゼ（スウィートザイムＴ（ノボノルディスク バイオインダストリー（株）製））を用い、グルコースからマンノースの生産試験を行なった。
グルコース４００ｍｇに、１００ｍＭリン酸緩衝液（ｐＨ７．０）、マンノースイソメラーゼ０．４単位及びグルコースイソメラーゼ０．４単位を加え、全量を１ｍｌとし、５０℃で３日間反応させた。反応３日目に酵素を失活させ、生成したマンノース量をＨＰＬＣによって定量した。その結果、グルコース：フラクトース：マンノース比は４９．１：４０．７：１０．２であった。
［産業上の利用可能性］
（１）本発明のマンノースイソメラーゼは、その諸性質からこれまでにない新規なものであり、遺伝子操作の点から有用である。
（２）本発明のマンノースイソメラーゼは、広い作用ｐＨ域を有し、高濃度のフラクトース溶液において、効率良くマンノースへ変換することが出来る。
（３）本マンノースイソメラーゼの作用条件は、グルコースイソメラーゼの作用条件と一致しているので、グルコースイソメラーゼを併用することにより、フラクトースからだけではなくて、グルコースからマンノースの製造も可能である点で有利である。
（４）従って、安価なフラクトースやグルコースから、マンノースを低コストで大量に製造することが出来るので、工業的に有利である。
（５）なお、アグロバクテリウム属の微生物が、マンノースイソメラーゼを生産することについては未だ知られておらず、本発明のものが最初であり、その技術的価値は極めて高い。
【図面の簡単な説明】
［図１］ 本発明のマンノースインメラーゼの最適温度を示す。
［図２］ 本発明のマンノースイソメラーゼの最適ｐＨを示す。
［図３］ 本発明のマンノースイソメラーゼの安定温度を示す。
［図４］ 本発明のマンノースイソメラーゼの安定ｐＨを示す。
［図５］ 本発明のマンノースイソメラーゼを用いた、フラクトースからマンノースを得る反応での反応温度の影響を示す。
［図６］ 本発明のマンノースイソメラーゼを用いた、フラクトースからマンノースを得る反応でのｐＨの影響を示す。
［図７］ 本発明のマンノースイソメラーゼを用いた、フラクトースからマンノースを得る反応での酵素濃度の影響を示す。[Technical field]
The present invention relates to a method for producing mannose using fructose or glucose as a raw material and using a novel mannose isomerase, and a method for producing the mannose isomerase.
[Background technology]
Mannose is a hexose that is abundant after glucose and glucosamine. Mannose exists as a component of cell membranes and cell walls of yeast, bacteria, plants and animals, and is contained in large amounts in N-linked sugar chains of glycoproteins and rarely exists in free form.
Mannose is said to be inferior to glucose and galactose as a biological nutrient source, but is useful as a raw material for pharmaceutical synthesis, mannitol, or as an animal cell culture raw material.
In addition, research on the suppression of Salmonella colonization by the addition of mannose feed has been carried out by a group of Texas laboratories of the US Department of Agriculture, which has been reported to significantly suppress the growth of intestinal Salmonella and is expected to be used as poultry feed. [Poultry Science. 68 . p. 1357, 1989].
Mannose can be produced by acid degradation or enzymatic degradation of mannose-containing polysaccharides (such as glucomannan and galactomannan), or by allowing glucose to act on glucose at high temperatures using molybdate as a catalyst. However, its use has been limited due to high manufacturing costs.
Therefore, as a method for producing mannose at low cost, inexpensive fructose or glucose is used as a raw material, and mannose isomerase (an enzyme that interconverts fructose and mannose) is allowed to act on fructose, or glucose isomerase (glucose and glucose) are used. For example, a method of allowing mannose isomerase to act on an enzyme that interconverts fructose).
The above mannose isomerase was first discovered in 1956 by Palleroni and Dodoroff et al. In Psudomonas saccharophila [J. Biol. Chem. , 218 . p. 535, 1956], then Streptomyces aerocollignes [Agric. Biol. Chem. 31 , P. 435, 1967], Mycobacterium smegmatis [J. Bacteriol. , 101 , P. 777, 1970] has been reported.
However, mannose isomerase derived from these microorganisms has a disadvantage that, in addition to low heat resistance (optimum temperature: 35 to 40 ° C.), the conversion efficiency from fructose to mannose is low and it is not suitable for industrial use.
On the other hand, as mannose isomerase having high heat resistance and excellent conversion rate from fructose to machinose, one derived from Pseudomonas (JP-A-6-292578, Japanese Patent No. 3003009) and thermomono Known from Spora (Thermomonospora) (Japanese Patent Laid-Open No. 11-75836) and the like are known. These enzymes have heat resistance suitable for industrial use, and the conversion rate from fructose to mannose is about 25%.
However, there is nothing other than the above that has high heat resistance and excellent conversion rate, and it is still inadequate and its development is awaited.
[Disclosure of the Invention]
Currently, mannose is a very expensive carbohydrate because it is produced by the decomposition and chemical synthesis of mannan. Therefore, mannose for foods, food additives, or feed additives could not be produced industrially. As a method for producing mannose at low cost, a method using mannose isomerase can be considered. However, handling such as low conversion of fructose to mannose and low heat and pH stability often do not meet the standards at the manufacturing site.
In order to conform to various standards at the manufacturing site, and in heat-resistant studies of enzymes using protein engineering, thermostable mannose isomerase from various sources is required.
In view of the above situation, the present inventors searched for microorganisms that produce mannose from fructose from the soil, and found a new microorganism that produces mannose isomerase that was not known at all. Thus, it was identified as Agrobacterium radiobacter from its bacteriological properties, and the present invention was completed.
That is, the present invention is as follows.
1. Mannose isomerase having the following physicochemical properties
(1) Action
Interconverts mannose and fructose, and lyxose and xylulose.
(2) Substrate specificity
Acts on D-mannose, D-fructose, D-lyxose and D-xylulose, D-glucose, D-galactose, L-arabinose, D-xylose, D-ribose, L-rhamnose, L-fucose, D-fucose Does not substantially work.
(3) Optimal temperature: 55-60 ° C
(4) Optimum pH: 7-9
(5) Stable temperature: up to about 60 ° C
(6) Stable pH: about 6-10
(7) Molecular weight: about 140,000 (gel filtration HPLC)
(8) Isoelectric point: about 5.2 (isoelectric focusing)
(9) Thermal stabilization by Ca ions: substantially not recognized
(10) Km value for mannose: about 0.01M
2. Mannose isomerase from the genus Agrobacterium.
3. The mannose isomerase having the physicochemical properties described in 1 above, which is produced by Agrobacterium radiobacter M36.
4). A method for producing a novel mannose isomerase, which comprises culturing a microorganism that produces the mannose isomerase according to 1 above, producing the mannose isomerase, and collecting the mannose isomerase.
5). A method for producing mannose, which comprises producing mannose by reacting the mannose isomerase described in 1 above with a fructose-containing liquid, and collecting the mannose.
6). A method for producing mannose, which comprises producing mannose by allowing the mannose isomerase and glucose isomerase described in 1 above to act on a glucose-containing solution and collecting the mannose.
The strain found by the present inventors was identified as Agrobacterium radiobacter due to its bacteriological properties, and the present inventors named it Agrobacterium radiobacter M36 (Agrobacterium radiobacter M36).
Agrobacterium radiobacter is not classified as biosafety level 2 or higher in the National Institute of Preventive Health, “Safety Control Regulations for Pathogens,” etc. Therefore, it does not require containment of P2 or higher, is frequently used in gene recombination techniques, and is a highly safe bacterium.
The enzyme derived from Agrobacterium radiobacter M36 used in the present invention exhibits heat resistance and conversion efficiency that can be used for industrial production of mannose. In addition, a technique for dramatically improving enzyme productivity of a specific microorganism by genetic manipulation is known. The present inventors have established information on the gene sequence relating to this enzyme (Japanese Patent Application No. 11-286034) and a technique for improving enzyme productivity (Japanese Patent Application No. 11-060904), and mannose by using an inexpensive enzyme. The prospects for low-cost manufacturing are on the rise.
The action conditions of the present mannose isomerase in the above reaction (pH 6.5 to 9.5, reaction temperature 60 ° C .: see Example 5) are the same as those for glucose isomerase (pH 6.5 to 7, temperature 6.0 ° C.). Therefore, it is advantageous in that mannose can be produced from glucose by using glucose isomerase together.
It is not yet known that microorganisms belonging to the genus Agrobacterium produce mannose isomerase, and the present invention is the first one.
Table 1 shows the results of comparing the physicochemical properties of the enzyme of the present invention with known enzymes.

Enzyme of the present invention: derived from Agrobacterium radiobacter M36
Enzyme A: Pseudomonas sp. AM-9582 (Patent No. 3003009)
Derived enzyme
Enzyme B: derived from Pseudomonas sp. N-25 (JP-A-6-292578)
enzyme
Enzyme C: Thermomonospora SP MBL10003 (Japanese Patent Laid-Open No. 11-7583)
6) Derived enzyme
From the results of Table 1, the enzyme of the present invention is (1) different from enzymes A to C in microorganisms derived from the enzyme, and (2) is different from enzymes B and C in substrate specificity, optimum pH, optimum temperature, etc. It can be seen that (3) it is different from enzyme A in the stabilization temperature, heat stabilization action by calcium ions, etc., and (4) Km value for mannose is different from enzyme A.
Therefore, the enzyme of the present invention shows a common action of isomerizing mannose into fructose as compared with known enzymes, but has different physicochemical properties, so it can be said that it is a different substance as an enzyme protein. .
As described above, the enzyme of the present invention is a novel mannose isomerase which is completely different from conventionally known enzymes.
[Embodiment of the Invention]
Hereinafter, embodiments of the present invention will be further described.
(I) Strains
The Agrobacterium radiobacter M36 strain used in the present invention is a bacterial species discovered in the soil by the present inventors, and is a biotechnological laboratory of the National Institute of Advanced Industrial Science and Technology (1 East East, Tsukuba City, Ibaraki, Japan). No. 3) is deposited as FERM P-17377 (April 23, 1999).
Agrobacterium radiobacter M36 strain has the following mycological properties.
(1) Form
Cell shape and size: 0.8 × 1.2 to 1.5 μm bacilli
Mobility: +
Gram staining:-
Sporulation:-
Colony form: circular, low convex, smooth gold edge, gloss, translucent
(2) Physiological properties
Nitrate reduction: +
Indole production:-
Glucose acidification:-
3-keto lactose production: +
Arginine dihydrolase:-
Urease:-
Estarin hydrolysis: +
Gelatin hydrolysis:-
β-galactosidase: +
Oxidase: +
Catalase: +
Substrate utilization ability:
Glucose: +
L-arabinose: +
D-Mannose: +
D-mannitol: +
N-acetyl-D-glucosamine: +
Maltose: +
Potassium gluconate: +
n-Capric acid:-
Adipic acid:-
dl-malic acid: +
Sodium citrate:-
Phenyl acetate:-
O / F test:-
(B) Mannose isomerase production
As the microorganism producing mannose isomerase, the above strains are advantageously used. Other microorganisms belonging to the genus Agrobacterium can also be used, and examples thereof include Agrobacterium radiobacter (IAM12048), Agrobacterium tumefaciens (IAM13129), and the like. In addition, enzymes produced by these mutant strains and recombinant microorganisms can also be used. The location of secretion is not particularly limited, and may be inside or outside the fungus body.
This enzyme is produced by the usual culture means as follows.
Fractose, glucose, mannose, galactose, isomerized sugar, sucrose, maltose, xylose, sorbitol, mannitol, starch and other sugars, sugar alcohols and starch and dextrin are used as carbon sources and nitrogen compounds necessary for the growth of microorganisms ( Organic nitrogen sources such as yeast extract, fish extract, peptone, amino acids, etc., or inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, urea, ammonium nitrate, etc.), mineral added medium, 20-40 ° C., preferably around 30 ° C. Under aerobic conditions, the cells are cultured for 1 to 5 days, preferably 1 to 3 days.
When the location of secretion is in the microbial cells, the microbial cells are collected by filtration or centrifugation after culturing and used as they are, or the enzyme is extracted and used by ultrasonic or self-digestion. If the secretion site is outside the cells, the cells are removed by filtration or centrifugation after cultivation, and the filtrate or supernatant is used as it is. If necessary, the extracted enzyme is precipitated with ammonium sulfate, acetone, methanol, ethanol, etc., purified and concentrated, or stored dry.
(C) Mannose production
a) When using fructose as a raw material
Mannose can be produced by allowing the fungus body or the treated cell body shown in (b) above to act on fructose.
As the processed bacterial cells, (1) those obtained by immobilizing the bacterial cells in a normal manner, (2) those obtained by treating the bacterial cells mechanically, enzymatically, or with a surfactant or an organic solvent, or Those that have been immobilized, (3) those obtained by crushing the cells and removing the crushing residue, those obtained by immobilizing them, (4) purified enzyme products, those obtained by immobilizing them, etc. Can be mentioned.
Industrially, it is effective to immobilize bacterial cells or to immobilize an enzyme extracted from the bacterial cells on an appropriate carrier and use it as an immobilized enzyme.
For immobilization of the treated bacterial cell, a normal immobilization method using polyacrylamide, calcium alginate, carrageenan, ion exchange carrier, chitosan beads, or the like can be used.
The reaction for producing mannose from fructose using this enzyme is preferably carried out using fructose or a 1-75% fructose-containing liquid at a pH of 5.5 to 10 and a temperature of 65 ° C. or lower. When this enzyme reaction is carried out in a closed reaction system, fructose: mannose = about 75: about 25 at the time of reaction equilibrium.
b) When using glucose as a raw material
When glucose is used as a raw material, it is carried out by causing two types of reactions: a reaction for producing fructose from glucose and a reaction for producing mannose from the fructose.
Therefore, in the case of this method, since glucose isomerase is used in addition to the mannose isomerase of this enzyme, it is necessary that the operating conditions of both enzymes match.
As described above, there is no problem because the mannose isomerase operating conditions of this enzyme match the glucose isomerase operating conditions (pH 6.5-7, temperature 60 ° C.). When an enzyme reaction is performed in a closed reaction system, glucose: fructose: mannose = about 50: about 40: about 10 at the time of reaction equilibrium.
Of course, it is possible to produce a continuous reaction by combining a stationary phase chromatographic separator and a pseudo mobile phase chromatographic separator in accordance with the above two types of reactions.
[Example]
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these. Unless otherwise indicated, the percentage is W / V%.
Example 1 Cell culture
100 ml of a medium (pH 7.0) composed of 10% polypeptone, 5% yeast extract, and 5% NaCl is placed in a 500 ml Erlenmeyer flask with a baffle, sterilized by a conventional method, and then Agrobacterium radiobacter M36 (FERM P-17377) And cultured with shaking at 30 ° C. for 24 hours. The culture solution was centrifuged with a centrifuge at 12000 rpm, and the cells were collected. The obtained bacterial cells were washed twice with 25 ml of 100 mM phosphate buffer (pH 7.0). After suspending the cells with 10 ml of phosphate buffer, the cells were sonicated, centrifuged, and separated into a precipitate and a crude enzyme solution. The mannose isomerase activity of the crude enzyme solution was 0.63 units / ml.
Example 2 Mannose conversion rate measurement
To 500 μl of the crude enzyme solution, 500 μl of a 20% fructose solution was added and reacted at 50 ° C. for 24 hours. The sugar composition of the reaction solution was confirmed by HPLC. The HPLC conditions are shown below. When the mannose in the reaction solution was quantified, the conversion from fructose was 25.0%.
HPLC conditions
Pump model: PU-1580 manufactured by JASCO Corporation
Column: manufactured by Shiseido Co., Ltd. CAPCELL PAK NH2 UG80
Detector: RI-71 type differential refractometer manufactured by Showa Denko KK
Eluent: acetonitrile: water = 80: 20
Flow rate: 1.0 ml / min
Example 3 Enzymatic activity of mannose isomerase from microorganisms of the genus Agrobacterium
As microorganisms belonging to Agrobacterium, Agrobacterium radiobacter (M36), Agrobacterium radiobacter (IAM12048), and Agrobacterium tumefaciens (AM13129) were used. A crude enzyme solution was prepared from these microorganisms according to the method described in Example 1, and the mannose isomerase activity was measured.
The enzyme activity was measured by the following method.
200 μl of an appropriately diluted enzyme solution was added to 200 μl of a 200 mM mannose solution dissolved in 100 mM phosphate buffer (pH 7.0), and an enzyme reaction was performed at 50 ° C. The enzyme reaction was then stopped by boiling for 10 minutes and the sugar composition was analyzed by HPLC. Under this condition, the amount of enzyme that produces 1 μmol of fructose per minute was defined as 1 unit. The results are shown in Table 2.

From the above results, it is understood that any microorganism belonging to the genus Agrobacterium has mannose isomerase activity, and in particular, the enzyme activity of Agrobacterium radiobacter (M36) is high.
Example 4 Enzymatic properties of mannose isomerase
For mannose isomerase derived from Agrobacterium radiobacter M36, after preparing a crude enzyme solution according to Example 1, enzyme purification by ammonium sulfate fractionation, ion exchange chromatography and gel filtration chromatography was performed for the following tests. did.
(1) Substrate specificity
Substrates include D-mannose, D-fructose, D-lyxose, D-xylulose, D-glucose, D-galactose, L-arabinose, D-xylose, D-ribose, L-rhamnose, L-fucose, D- Each high-purity product (reagent) of fucose was used. 200 μl of appropriately diluted enzyme solution was added to 200 μl of each 200 mM substrate dissolved in 100 mM phosphate buffer (pH 7.0), reacted at 55 ° C. for 3 hours, and analyzed by HPLC. As a result, the mannose isomerase acted on D-mannose, D-fructose, D-lyxose and D-xylulose, and D-mannose and D-fructose and D-lyxose and D-xylulose were interconverted, respectively. It was also found that D-glucose, D-galactose, L-arabinose, D-xylose, D-ribose, L-rhamnose, L-fucose and D-fucose do not substantially act.
(2) Optimal temperature:
200 μl of an appropriately diluted enzyme solution was added to 200 μl of 200 mM mannose solution dissolved in 100 mM phosphate buffer (pH 7.0), and reacted at 40, 50, 55, 60, and 65 ° C. for 30 minutes. After enzyme inactivation, the amount of fructose produced was quantified by HPLC. The results are shown in FIG.
As is clear from FIG. 1, the optimum temperature of this enzyme was 55 ° C. to 60 ° C.
(3) Optimal pH:
200 μl of appropriately diluted enzyme solution was added to 200 μl of 200 mM mannose solution dissolved in 100 mM various pH buffers, and reacted at 50 ° C. for 30 minutes. After enzyme inactivation, the amount of fructose produced was quantified by HPLC. The results are shown in FIG.
As is clear from FIG. 2, the optimum pH of this enzyme was 7-9.
(4) Stable temperature
After 200 μl of appropriately diluted enzyme solution was incubated at 40, 50, 55, 60, and 65 ° C. for 10 minutes, 200 μl of 200 mM mannose solution dissolved in 100 mM phosphate buffer (pH 7.0) was added and reacted. After enzyme inactivation, the amount of fructose produced was quantified by HPLC. The results are shown in FIG.
As is apparent from FIG. 3, the enzyme was stable up to about 60 ° C.
(5) Stable pH
After incubating 200 μl of an enzyme solution appropriately diluted with various pH buffer solutions at 25 ° C. for 3 hours, 200 μl of 200 mM mannose solution dissolved in 100 mM phosphate buffer (pH 7.0) was added and reacted. After enzyme inactivation, the amount of fructose produced was quantified by HPLC. The results are shown in FIG.
As is clear from FIG. 4, the enzyme was at a low pH of about 6-10.
(6) Molecular weight and equal priority
The molecular weight by gel filtration HPLC was about 140,000, and the isoweight by isoelectric focusing was about 5.2.
(7) Thermal stabilization by calcium ions
An enzyme solution appropriately diluted with 25 mM PIFES (piperazine-N, N′-bis (2-ethanesulfonic acid)) buffer (pH 7.0) containing calcium chloride to a final concentration of 5 mM was obtained at 55 ° C. and 60 ° C. And incubated at 62.5 ° C. for 10 minutes. Next, 200 μl of this enzyme solution is added to 200 μl of 200 mM mannose solution dissolved in 25 mM PIFES buffer, reacted at 55 ° C. for 30 minutes, and the fructose produced after the reaction is stopped is quantified by HPLC to stabilize calcium ions at each temperature. The action was evaluated by the following stabilization rate.
Stabilization rate (%) = (fructose conversion rate in a reaction system with addition of calcium / fructose conversion rate in a reaction system without addition of calcium) × 100
The results are shown in Table 3.

From the results in Table 3, it was found that the fructose conversion rate was not substantially changed under each temperature condition, and this enzyme was hardly stabilized by calcium ions.
(8) Measurement of Km (Michaelis constant) value
Based on the enzyme kinetics, the Km value of the enzyme with respect to mannose was determined by Hanes-Woolf plot and found to be about 0.01M.
Example 5 Characteristics during mannose formation reaction
Various properties of the enzyme were confirmed using the mannose isomerase prepared in Example 1.
(1) Effect of reaction temperature
To 200 mg of fructose, 100 mM phosphate buffer (pH 7.0) and 0.2 unit of mannose isomerase were added to make a total volume of 1 ml, and reacted at 45, 50, 55, and 60 ° C. for 3 days. Sampling was performed over time, and the amount of mannose produced was quantified by HPLC. The results are shown in FIG.
As apparent from FIG. 5, the reaction at 60 ° C. was the best at the beginning of the reaction, but the reaction at 55 ° C. was the best at the end of the reaction, reaching a conversion rate of 25.6% at 72 hours.
(2) Effect of reaction pH
To 200 mg of fructose, 100 mM of various acid buffers and 0.2 units of mannose isomerase were added to make a total volume of 1 ml and reacted at 50 ° C. for 24 hours. Sampling was performed 24 hours after the start of the reaction, and the amount of mannose produced was quantified by HPLC. The results are shown in FIG.
As is clear from FIG. 6, it was revealed that substantial mannose production is possible in a wide pH range of pH 6.5 to 9.5.
(3) Reaction with high concentration substrate
To 500 mg of fructose, 100 mM phosphate buffer (pH 7.0) and mannose isomerase 4, 2, 1, 0.5 units were added, the total volume was made up to 1 ml with water, and the mixture was reacted at 55 ° C. for 2 days. Sampling was performed over time, and the amount of mannose produced was quantified by HPLC. The results are shown in FIG.
As is clear from FIG. 7, this enzyme was found to efficiently convert a high-concentration fructose solution (50%) to mannose (conversion rate of about 25%).
Example 6 Mannose production from fructose
The mannose isomerase prepared by the method of Example 1 was fractionated with ammonium sulfate. 5 ml of the desalted and partially purified enzyme solution was diluted 2-fold with 100 mM phosphate buffer (pH 7.0), and 8 ml of chitosan beads (Fujibo Co., Ltd., “Chitopearl BCW-3505”) was added at 30 ° C. The column was filled with chitosan beads with immobilized enzyme, and 50 ml of 100 mM phosphate buffer (pH 7.0) was passed through the column. The enzyme was completely adsorbed to the chitosan beads, and a 20% fructose solution was circulated for 3 days at a flow rate of 0.25 ml / min through a column kept at 55 ° C. On the third day of the reaction The reaction solution was recovered, and the amount of mannose produced was quantified by HPLC, and as a result, the conversion rate from fructose was 24.5%.
Example 7 Mannose production from glucose
Mannose isomerase prepared in Example 1 and a commercially available glucose isomerase derived from Streptomyces murinus (Sweetzyme T (manufactured by Novo Nordisk Bioindustry Co., Ltd.)) were used to produce mannose from glucose.
To 400 mg of glucose, 100 mM phosphate buffer (pH 7.0), 0.4 unit of mannose isomerase and 0.4 unit of glucose isomerase were added to make a total volume of 1 ml and reacted at 50 ° C. for 3 days. On the third day of the reaction, the enzyme was inactivated and the amount of mannose produced was quantified by HPLC. As a result, the glucose: fructose: mannose ratio was 49.1: 40.7: 10.2.
[Industrial applicability]
(1) The mannose isomerase of the present invention is an unprecedented new one due to its properties and is useful from the viewpoint of gene manipulation.
(2) The mannose isomerase of the present invention has a wide working pH range and can be efficiently converted to mannose in a high-concentration fructose solution.
(3) Since the operating conditions of the present mannose isomerase coincide with the operating conditions of glucose isomerase, it is advantageous in that mannose can be produced not only from fructose but also from glucose by using glucose isomerase in combination. It is.
(4) Therefore, mannose can be produced in large quantities at low cost from inexpensive fructose and glucose, which is industrially advantageous.
(5) It is not yet known that microorganisms belonging to the genus Agrobacterium produce mannose isomerase, and the present invention is the first one, and its technical value is extremely high.
[Brief description of the drawings]
FIG. 1 shows the optimum temperature of the mannose imerase of the present invention.
FIG. 2 shows the optimum pH of the mannose isomerase of the present invention.
FIG. 3 shows the stable temperature of the mannose isomerase of the present invention.
FIG. 4 shows the stable pH of the mannose isomerase of the present invention.
FIG. 5 shows the influence of reaction temperature in a reaction for obtaining mannose from fructose using the mannose isomerase of the present invention.
FIG. 6 shows the influence of pH in a reaction for obtaining mannose from fructose using the mannose isomerase of the present invention.
FIG. 7 shows the influence of enzyme concentration in a reaction for obtaining mannose from fructose using the mannose isomerase of the present invention.

Claims (4)

A mannose isomerase produced by Agrobacterium radiobacter M36 (Accession No. FERM BP-7206) having the following physicochemical properties:
(1) Action It interconverts mannose and fructose, and lyxose and xylulose.
(2) Substrate specificity
Acts on D-mannose, D-fructose, D-lyxose and D-xylulose, D-glucose, D-galactose, L-arabinose, D-xylose, D-ribose, L-rhamnose, L-fucose, D-fucose Does not work.
(3) Optimal temperature: 55-60 ° C
(4) Optimum pH: 7-9
(5) Stable temperature: up to 60 ° C (6) Stable pH: 6-10
(7) Molecular weight: 140,000 (gel filtration HPLC)
(8) Isoelectric point: 5.2 (isoelectric focusing)
(9) Thermal stabilization by Ca ions: not observed (10) Km value for mannose: 0.01M   A method for producing a novel mannose isomerase, comprising culturing a microorganism producing mannose isomerase according to claim 1, producing the mannose isomerase, and collecting the mannose isomerase.   A mannose isomerase according to claim 1 is allowed to act on a fructose-containing liquid to produce mannose, which is collected.   A method for producing mannose, comprising producing mannose by allowing the mannose isomerase and glucose isomerase according to claim 1 to act on a glucose-containing solution, and collecting the mannose.

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