JP2004000131A - Chitosanase and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chitosanase capable of efficiently producing low-molecular chitosan soluble in water even under neutral condition and having antimicrobial activity, to provide a DNA encoding said enzyme, to provide a method for producing said enzyme, to provide the low-molecular chitosan produced by said enzyme, and to provide pharmaceutical compositions or the like containing said low-molecular chitosan. <P>SOLUTION: The chitosanase is obtained by isolation and refining from at least one cultured product of Aspergillus oryzae, Aspergillus japonicus and Aspergillus sojae. <P>COPYRIGHT: (C)2004,JPO

Description

表１から、この酵素は、ｐＨ５．５においてはグリコールキチンよりもアセチル化度が１０〜３０％の部分アセチル化キトサン（キトサン９Ｂ、８Ｂ、７Ｂ）に対する加水分解活性が高く、アセチル化度が１％以下のキトサン（キトサン１０Ｂ）に対する加水分解活性がほとんどないことがわかる。カルボキシメチルセルロースナトリウムに対する加水分解活性はない。
１２）下記に示す部分アミノ酸配列を有する。配列は、Ｎ末端側から記す。
Ｇｌｕ−Ｍｅｔ−Ｔｈｒ−Ｐｒｏ−Ｔｙｒ−Ｌｅｕ−Ａｓｐ−Ｐｈｅ−Ｔｙｒ−Ａｒｇ−Ｌｅｕ−Ｍｅｔ−Ａｌａ−Ｔｙｒ−Ｇｌｎ　（配列表の配列番号１）
Ｉｌｅ−Ｖａｌ−Ｖａｌ−Ｇｌｙ−Ｍｅｔ−Ｐｒｏ−Ｉｌｅ−Ｔｙｒ−Ｇｌｙ−Ａｒｇ−Ｌｙｓ−Ｇｌｕ　（配列表の配列番号２）
Ａｌａ−Ｌｅｕ−Ｐｒｏ−Ｌｙｓ−Ｐｒｏ−Ｇｌｙ−Ａｌａ−Ｔｈｒ−Ｇｌｕ−Ｔｙｒ−Ｖａｌ−Ａｓｐ−Ｐｒｏ−Ｓｅｒ−Ｉｌｅ　　［配列表の配列番号３）
アスペルギルス・ジャポニクスＳＡＮＫ　１９２８８株により生産されるキチナーゼは、粗酵素液において、以下の性質を有する。
１）ＳＤＳ−ＰＡＧＥ電気泳動法にて分子量約４０，０００を示す。
２）等電点転記泳動法にて等電点ｐＩ約３．５を示す。
３）キトサン７Ｂを、ｐＨ３．５乃至ｐＨ１０．５にて加水分解する。
４）３）記載の加水分解活性の最適ｐＨはｐＨ４．０である。
５）キトサン７Ｂに対する酵素のｐＨ４．０、温度３７℃における１０分間の加水分解活性を１００％としたときの、他の基質に対する酵素のｐＨ４．０、温度３７℃における１０分間の加水分解活性が相対値として表２の値を示す。
【００７８】
【表２】
――――――――――――――――――――
基質　　　　　　　　　相対活性（％）
――――――――――――――――――――
キトサン１０Ｂ　　　　　８．２
キトサン　９Ｂ　　　　６７．６
キトサン　８Ｂ　　　　７４．４
キトサン　７Ｂ　　　１００
グリコールキトサン　　　８．２
グリコールキチン　　　２３．３
ＣＭ−セルロース　　　１１．４
リケナン　　　　　　　２５．３
――――――――――――――――――――
表２から、この酵素は、グリコールキチンよりもアセチル化度が１０〜３０％の部分アセチル化キトサン（キトサン９Ｂ、８Ｂ、７Ｂ）に対する加水分解活性が高く、アセチル化度が１％以下のキトサン（キトサン１０Ｂ）に対する加水分解活性は弱いことがわかる。
また、アスペルギルス・ジャポニクスＳＡＮＫ　１９２８８株により生産され、精製されたキチナーゼは、以下の性質を有する。
１）ＳＤＳ−ＰＡＧＥ電気泳動法にて分子量約４０，０００を示す。
２）等電点転記泳動法にて等電点ｐＩ約３．５を示す。
３）キトサン７Ｂを、ｐＨ３．５乃至ｐＨ１０．５にて加水分解する。
４）３）記載の加水分解活性の最適ｐＨはｐＨ５．０である。
５）３）記載の加水分解活性の最適温度はｐＨ５．０では６５℃である。
６）５０℃以下の温度で安定である。
７）ｐＨ４乃至ｐＨ９．５のｐＨ条件下で安定である。
８）キトサン７Ｂに対する酵素のｐＨ５．０、温度３７℃における１０分間の加水分解活性を１００％としたときの、他の基質に対する酵素のｐＨ５．０、温度３７℃における１０分間の加水分解活性が相対値として表３の値示す。表中、キトサン１０Ｂは１％アセチル化キチン、キトサン９Ｂは１０％アセチル化キチン、キトサン８Ｂは２０％アセチル化キチン、キトサン７Ｂは３０％アセチル化キチンである（いずれも加ト吉（株）製）。ＣＭ−セルロースは、カルボキシメチルセルロースナトリウムである（和光純薬社製）。グリコールキチンは、前述の方法により調製する。
【００７９】
【表３】
――――――――――――――――――
基質　　　　　　　　相対活性（％）
――――――――――――――――――
キトサン１０Ｂ　　　　　０
キトサン９Ｂ　　　　　５８．６
キトサン８Ｂ　　　　　７２．４
キトサン７Ｂ　　　　１００
グルコールキトサン　　　０
グリコールキチン　　　２８．３
ＣＭ−セルロース　　　　０
リケナン　　　　　　　　４．２
――――――――――――――――――
表３に示すとおり、アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼはキトサン７Ｂに対して最も加水分解活性が高く、アセチル化度が小さくなるにつれて加水分解活性は低下する。また、該精製キチナーゼはキチン加水分解活性を有し、セルラーゼ加水分解活性は無い。
９）下記に示す部分アミノ酸配列を有する。配列は、Ｎ末端側から記す。
Ｈｉｓ−Ｔｙｒ−Ｐｒｏ−Ｔｈｒ−Ａｓｐ−Ｓｅｒ−Ｔｒｐ−Ａｓｎ−Ａｓｐ−Ｖａｌ−Ｇｌｙ−Ｔｈｒ−Ａｓｎ−Ｖａｌ−Ｔｙｒ（配列表の配列番号１１）
Ａｌａ−Ｐｈｅ−Ｔｈｒ−Ａｓｎ−Ｔｈｒ−Ａｓｐ−Ｇｌｙ−Ｐｒｏ−Ｇｌｙ−Ｔｈｒ−Ａｌａ−Ｐｈｅ−Ｓｅｒ−Ｇｌｙ−Ｖａｌ（配列表の配列番号１２）
Ｌｅｕ−Ｓｅｒ−Ｇｌｎ−Ｍｅｔ−Ｔｈｒ−Ｐｒｏ−Ｔｙｒ−Ｌｅｕ−Ａｓｐ−Ｐｈｅ−Ｔｙｒ−Ａｓｎ−Ｌｅｕ−Ｍｅｔ−Ａｌａ−Ｔｙｒ−Ａｓｐ−Ｔｙｒ−Ａｌａ−Ｇｌｙ（配列表の配列番号１３）
アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株により生産されるキチナーゼは、粗酵素液において、以下の性質を有する。
１）ＳＤＳ−ＰＡＧＥ電気泳動法にて分子量約４０，０００を示す。
２）等電点電気泳動法にてｐＩ約４．５を示す。
３）キトサン７Ｂを、ｐＨ３．５乃至ｐＨ９．５にて加水分解する。
４）３）記載の加水分解活性の最適ｐＨはｐＨ４．５である。
５）キトサン７Ｂに対する酵素のｐＨ４．５、温度３７℃における１０分間の加水分解活性を１００％としたときの、他の基質に対する酵素のｐＨ４．５、温度３７℃における１０分間の加水分解活性が相対値として表４の値を示す。
【００８０】
【表４】
――――――――――――――――――――
基質　　　　　　　　相対活性（％）
――――――――――――――――――――
キトサン１０Ｂ　　　　　　０
キトサン９Ｂ　　　　　　６７．７
キトサン８Ｂ　　　　　　７１．６
キトサン７Ｂ　　　　　１００
グリコールキトサン　　　　２．９
グリコールキチン　　　　８１．９
ＣＭ―セルロース　　　　　０
リケナン　　　　　　　　１１．９
――――――――――――――――――――
表４から、この酵素は、アセチル化度が１０〜３０％の部分アセチル化キトサン（キトサン９Ｂ、８Ｂ、７Ｂ）に対する加水分解活性が高く、アセチル化度が１％以下のキトサン（キトサン１０Ｂ）に対する加水分解活性がほとんどないことがわかる。カルボキシメチルセルロースナトリウムに対する加水分解活性はない。
また、アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株により生産され、精製されたキチナーゼは、以下の性質を有する。
１）ＳＤＳ−ＰＡＧＥ電気泳動法にて分子量約４０，０００を示す。
２）等電点転記泳動法にて等電点ｐＩ約４．５を示す。
３）キトサン７Ｂを、ｐＨ３．５乃至ｐＨ９．５にて加水分解する。
４）３）記載の加水分解活性の最適ｐＨはｐＨ５．５およびｐＨ９．０である。
５）３）記載の加水分解活性の最適温度はｐＨ５．５では６０℃、ｐＨ９．０では３５℃である。
６）ｐＨ５．５およびｐＨ９．０において、４０℃以下の温度で安定である。
７）ｐＨ４．５乃至ｐＨ１０のｐＨ条件下で安定である。
８）キトサン８Ｂに対する酵素のｐＨ５．０、温度３７℃における１０分間の加水分解活性を１００％としたときの、他の基質に対する酵素のｐＨ５．０、温度３７℃における１０分間の加水分解活性が相対値として表５の値を示す。表中、キトサン１０Ｂは１％アセチル化キチン、キトサン９Ｂは１０％アセチル化キチン、キトサン８Ｂは２０％アセチル化キチン、キトサン７Ｂは３０％アセチル化キチンである（いずれも加ト吉（株）製）。ＣＭ−セルロースは、カルボキシメチルセルロースナトリウムである（和光純薬社製）。グリコールキチンは、前述の方法により調製する。
【００８１】
【表５】
――――――――――――――――――
基質　　　　　　　　相対活性（％）
――――――――――――――――――
キトサン１０Ｂ　　　　１４．３
キトサン９Ｂ　　　　　７３．６
キトサン８Ｂ　　　　１００
キトサン７Ｂ　　　　　９７．４
グルコールキトサン　　　０
グリコールキチン　　　２３．８
ＣＭ−セルロース　　　　０
リケナン　　　　　　　０
――――――――――――――――――
表５に示すとおり、アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼはキトサン７Ｂおよび８Ｂに対して最も加水分解活性が高く、アセチル化度が小さくなるにつれて加水分解活性は低下する。また、該精製キチナーゼはキチン加水分解活性を有し、セルラーゼ加水分解活性はない。
以上のことから、本発明のキチナーゼの有する性質としては以下のようなものが挙げられるが、これに限定されるものではない。
１）ＳＤＳ−ＰＡＧＥ電気泳動法にて分子量約４０，０００を示す。
２）等電点電気泳動法にて等電点ｐＩ３．０乃至５．０（好適にはｐＩ３．５乃至４．５）を示す。
３）キトサン７Ｂを、ｐＨ３．０乃至ｐＨ１１．０（好適にはｐＨ３．５乃至１０．５）にて加水分解する。
４）グリコールキチンを、ｐＨ３．０乃至ｐＨ１１．５（好適にはｐＨ４乃至ｐＨ１０．５）にて加水分解する；
５）０℃乃至８０℃で３）記載の加水分解活性を発揮する；
６）５０℃以下（好適には４５℃以下）の温度で安定である；
７）ｐＨ４．０乃至ｐＨ１０（好適にはｐＨ５乃至ｐＨ９．５）のｐＨ条件下で安定である。
【００８２】
また、本発明のキチナーゼを製造する方法も本発明に含まれる。
【００８３】
アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　　ＪＣＭ　２０６７株、アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株、アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株を初めとするキチナーゼ産生微生物を培地で培養することにより、キチナーゼを生産することができる。例えば、０．１〜５．０％マルトエクストラクト（Ｄｉｆｃｏ（株）製）、０．１〜１．０％イーストエキストラクト（Ｄｉｆｃｏ（株）製）、０．０５〜１．０％キチンあるいは０．０５〜１．０％キトサンの培地で、１６〜４５℃で１〜１５日間、１００〜２５０ｒｍｐで振とう培養する。
【００８４】
アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　ＪＣＭ　２０６７株、アスペルギルス・ジャポニクス　ＳＡＮＫ１９２８８株、アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の酵素は、次の各点が共通である。
１）弱酸性で最も高い部分アセチル化キトサン分解活性を有する。
２）３０％アセチル化キトサンに対する、ｐＨ４．０〜６．０、温度３７℃における１０〜３０分間の加水分解活性を１００％とすると、１％アセチル化キトサンに対する該活性は１５％以下である。
【００８５】
本発明によって得られるキトサン低分子キトサン及び各種塩溶液は、そのまま用いることもできるが、硫安沈殿・有機溶媒沈殿・遠心分離・凍結乾燥・限外濾過等により、濃縮することもできる。また、透析・限外濾過・ゲル濾過・カラムクロマトグラフィー等により精製と脱塩及び分子量分画をすることもできる。
【００８６】
このようにして作製した低分子キトサン塩酸塩は、水に対する溶解性に優れ、最大約１０質量％濃度の溶液とすることができる。また、低分子キトサン塩酸塩の１質量％水溶液は、ｐＨ４．９〜５．６を示し、アルカリを添加した場合に沈殿物（コロイド）が析出するｐＨはｐＨ６．６〜７．４である。
【００８７】
ここで作製した低分子キトサン塩酸塩が有する生理活性を、抗菌活性を指標として調べる。抗菌活性を有するための低分子キトサン９Ｂ、８Ｂ、７Ｂ各塩酸塩の最小阻止濃度（Ｍｉｎｉｍｕｍ　　Ｉｎｈｉｂｉｔｏｒｙ　　Ｃｏｎｃｅｎｔｒａｔｉｏｎ；　　ＭＩＣ）を測定する。高分子キトサン塩酸塩の抗菌活性と比較すると、グラム陽性菌・陰性菌にかかわらず、低分子キトサン塩酸塩は高分子キトサン塩酸塩と同等以上の活性を有している。したがって、低分子キトサンは高分子キトサンと同等以上の優れた生理活性を有している。このことから高分子キトサンが有するとされている抗菌活性は、本発明の低分子キトサンにおいても保持されているものと考えられる。また、皮膚欠損傷動物の治癒試験においても、治癒日数の短縮効果がみられ、動物における生理活性も有している．
このように優れた生理活性を有していることから、本発明で得られる低分子キトサンまたはその塩は、例えば、創傷・褥創用外用剤、床ずれ治療薬、アトピー性皮膚炎治療薬、ニキビ治療薬等の皮膚外用剤、入浴剤、化粧料、洗顔剤、基礎化粧品、虫歯予防薬等の口腔衛生剤、液状洗口剤、歯科向けの素材、腸内細菌改善薬等の整腸剤、院内感染予防薬等の医療分野、ダイエットフード、食品保存剤、防腐剤等の食品添加物分野、動物・魚類用飼料等の分野、植物活性増強剤などの農業分野、水等処理剤、金属吸着剤、原子力廃棄物吸着剤薬剤除法剤等に本発明の低分子キトサンを使用することが考えられるが、用途は特にこれらの例に限定されるものではない。また本発明の低分子キトサンまたはその塩の有効量を動物に投与することを含む、外傷、床ずれ，アトピー性皮膚炎等の疾患の治療方法も本発明に含まれる。また、これらの疾患を治療するための本発明の低分子キトサンまたはその塩の使用も本発明に含まれるものとする。
本発明によって得られる低分子キトサン又は低分子キトサン塩の用法は目的に応じて変わるが、上述のような用途に使用するには、さまざまな形態をとり得る。以下に形態を列挙するが、特に限定されるわけではない。低分子キトサンは、固体のまま用いることができる。また、１０質量％濃度の水溶液あるいは有機溶媒溶液、さらに適宜これを希釈して任意の濃度で用いることができる。希釈剤は水あるいは有機溶媒（アルコール類など）等を使用することができる。最終的にｐＨが６．５より酸性であれば溶液として、アルカリ性であればコロイド状化合物として使用できるが、塩の種類によっては弱アルカリ性でも溶液として使用できる。さらに、無機物あるいは有機物を共存させることにより、任意のｐＨで溶液として用いることができる。また、低分子キトサン溶液あるいは希釈溶液を１０質量％以下の任意の濃度においてクリーム基材と混合することにより、クリーム剤として使用することができる。低分子キトサン溶液あるいは希釈溶液を、任意の厚さを有するフィルムとして使用することができる。プラスチック様の硬さを有するフィルムは、低分子キトサン溶液を、さまざまな厚さを有する枠の中で乾燥させることにより得られる。また、柔軟性を有する軟質シートは、低分子キトサン溶液に例えばグリセリン等の溶剤を任意の割合で混合して、さまざまな厚さを有する枠の中で乾燥させることにより得られる。柔軟性の度合いは、例えばグリセリン等の溶剤を加える割合で調節される。さらに、キトサンーメチルセルロ−ス複合フィルムは、低分子キトサン溶液にメチルセルロースを混合し、さまざまな厚さを有する枠の中で乾燥させることにより得られる。
【００８８】
用量は、疾患の種類、症状、患者の性別、年令、剤型、投与法に応じて変わるが、安全性が高いので、任意の量を用いることができる。生体に投与する場合には２ｇ／ｋｇ体重以下が好ましく、それ以外の場合には、適宜調節される。
また、本発明のキチナーゼは、扱いにくい試料をより扱い易くする為にも有効に用いる事ができる。従来、甲殻類などを含む試料を、粉砕する，すり潰す、ミキサーにかけるなどの加工をした場合、粘度が非常に高くなり、扱いにくかった。これは、これら甲殻類に大量に含まれる高分子多糖（主成分は高分子部分アセチル化キトサン）が原因であった。たとえば、このような食品加工の過程に、本発明のキチナーゼを加えて反応させ、その液性成分中の高分子部分アセチル化キトサンを分解し、その液性成分中に高分子部分アセチル化キトサンを実質的に含まない、扱い易い試料を製造することが可能であり、キチナーゼを用いた該扱い易い試料の製造方法も本発明に含まれる。このような製造方法に用いる原材料としては、高分子部分アセチル化キトサンを含む試料であれば特に限定されないが、好ましくは、甲殻類を含む試料であり，より好ましくはエビおよびカニのいずれか一つまたは両方を含む試料である。原材料とキチナーゼの反応条件としては上記のキトサン分解活性を発揮する条件を適用することができる。反応時間は１０分以上であって、試料の粘度が低い状態になる時間であれば特に限定されないが、好ましくは１０分間から１０日間程度であり、より好ましくは１日間から５日間程度である。また、このような方法によって製造された試料も本発明に含まれる。
【００８９】
「その液性成分中に高分子部分アセチル化キトサンを実質的に含まない」とは、試料に高分子部分アセチル化キトサンが含有される事によりもたらされる、高い粘性が減少または消失した状態を指し、厳密に濃度として０％であることを指さないが、好ましくは高分子部分アセチル化キトサンの含有量が２０％以下であり、より好ましくは１０％以下であり、さらに好ましくは５％以下であり、最適には０％である。
【００９０】
【実施例】
以下に、実施例及び試験例を挙げるが、本発明の範囲はこれらに限定されるものではない。
【００９１】
実施例１．アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　　ＪＣＭ
２０６７株からのキチナーゼの精製
１）粗酵素液の調製
滅菌した表６の組成の培地１００ｍｌが入っている５００ｍｌ容の三角フラスコ（種フラスコ）にアスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　ＪＣＭ　２０６７株の菌体を接種し、２６℃にて８日間、１００ｒｐｍの振とう培養を行った。
【００９２】
【表６】
培地
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マルトエクストラクト　　　１０ｇ
イースト・エクストラクト　５ｇ
粉末キチン　　　　　　　　２ｇ
あるいは　コロイダルキトサン（キミツ化学（株）製）　　　２ｇ
純水で１，０００ｍｌとした。
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培養終了後、４℃、１０，０００×Ｇにて１０分間の遠心分離を行った。得られた上清を粗酵素液とした。
【００９３】
２）酵素活性測定法
キチナーゼの加水分解活性は以下のようにして測定した。
【００９４】
▲１▼部分アセチル化キトサンの加水分解反応
キトサン７Ｂ（３０％アセチル化キトサン、加ト吉（株）製）１２５ｍｇに水５０ｍｌおよび酢酸３０μｌを加え、粉末キトサンを完全に溶解させた。キトサン８Ｂ（２０％アセチル化キトサン、加ト吉（株）製）１２５ｍｇに水５０ｍｌおよび酢酸５０μｌを加え、粉末キトサンを完全に溶解させた。キトサン９Ｂ（１０％アセチル化キトサン、加ト吉（株）製）１２５ｍｇに水５０ｍｌおよび酢酸５０μｌを加え、粉末キトサンを完全に溶解させた。キトサン１０Ｂ（ほぼ完全な脱アセチル化キトサン、加ト吉（株）製）１２５ｍｇに水５０ｍｌおよび酢酸９０μｌを加え、粉末キトサンを完全に溶解させた。これらのキトサン水溶液１６０μｌおよび４００ｍＭ酢酸緩衝液（ｐＨ５．０）１００μｌの混合液に酵素液１４０μｌを加え撹拌して均一にし、３７℃で保温して２０分間酵素反応を行った。
【００９５】
▲２▼ランドル・モーガン法による遊離還元糖の定量
アセチルアセトン１０μｌを０．５Ｍ炭酸ナトリウム水溶液５００μｌに溶かした溶液４００μｌを酵素反応液に加えて反応を停止させた後、１００℃で２０分間保温した。氷冷後、Ｎ，Ｎ−ジメチルアミノベンズアルデビド０．８ｇをエタノール３０ｍｌ及び濃塩酸３０ｍｌに溶解した混合溶液４００μｌとエタノール１２００μｌを酵素反応溶液に加え、６５℃で１０分間発色反応をさせた。氷冷後、沈殿物を遠心分離し上清の５３０　ｎｍにおける吸光度を測定した。酵素反応１分間当たり１μｍｏｌのグルコサミンを生成する酵素活性を１単位とした。
【００９６】
３）粗精製酵素液の調製
１）で得られた粗酵素液５００ｍｌに、氷冷下攪拌しながら硫酸アンモニウム２８０ｇを徐々に加えた。混合溶液を一晩４℃で静置したのち、１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）３，０００ｍｌに対して１２時間ずつ５回透析した。これを、予め１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）で平衡化したＤＥＡＥトヨパール（東ソー（株）製）カラム（直径２．２ｃｍ×長さ２０ｃｍ）に添加し、吸着させた。１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）で該カラム十分洗浄した後、１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）６００ｍｌ中に０乃至０．４Ｍの塩化ナトリウムの直線的濃度勾配を作製して該カラムに吸着した成分を溶出させた。キトサン分解活性は塩化ナトリウム濃度が０．２０Ｍ乃至０．３０Ｍの画分（１３５ｍｌ）に溶出された。これを粗精製酵素画分とした。
【００９７】
４）精製酵素溶液の調製
３）で得られた活性画分１３５　ｍｌを２０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）３，０００ｍｌに対して１２時間ずつ３回透析した後、予め２０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）で平衡化したＭｏｎｏＱ（ファルマシア社製）カラム（直径７ｍｍ×長さ５ｃｍ）に添加し、吸着させた。該カラムを２０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）で十分洗浄した後、１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）２５０ｍｌ中に０乃至０．１５　Ｍの塩化ナトリウムの直線的濃度勾配を作製して該カラムに吸着した成分を溶出させた。キトサン分解活性は塩化ナトリウム濃度が０．０５４Ｍ乃至０．０５７Ｍの画分（１０ｍｌ）に溶出された。この
画分を精製酵素溶液とした。
【００９８】
５）精製酵素の分子量測定
１２．５％ポリアクリルアミドゲルを用いたＳＤＳ−ＰＡＧＥ電気泳動法（Ｌａｅｍｍｌｉ，　Ｕ．Ｋ．，　　Ｎａｔｕｒｅ，　２２７，　６８０（１９７０）参照）により、精製酵素の分子量を求めた。標準タンパク質として次のものを用いた：ａ．ホスホリラーゼ（ｐｈｏｓｐｈｏｒｙｌａｓｅ）、分子量９４，０００：ｂ．アルブミン（ａｌｂｕｍｉｎ）、分子量６７，０００：ｃ．オバルブミン（ｏｖａｌｂｕｍｉｎ）、分子量４３０００：ｄ．カルボニック・アンヒドラーゼ（ｃａｒｂｏｎｉｃ　ａｎｈｙｄｒａｓｅ）、分子量３０，０００：ｅ．トリプシン・インヒビター（ｔｒｙｐｓｉｎ　　ｉｎｈｉｂｉｔｏｒ）、分子量２０，１００：ｆ．α−ラクタルブミン（α−ｌａｃｔａｌｂｕｍｉｎ）、分子量１４，４００。
これら標準タンパクの移動度から求められた検量線を図１に記載した。図１に示す通り、精製酵素は分子量約４０，０００の単一バンドを示した。
【００９９】
６）精製酵素の等電点
精製酵素を等電点電気泳動（日本生化学会編「生化学実験講座１　タンパク質の化学Ｉ　分離精製、東京化学同人刊（１９７６年）」２６２乃至３１２頁参照）に供した。精製酵素の等電点は約ｐＩ４．５であった。
【０１００】
７）部分アミノ酸配列の決定
精製した酵素溶液を約１ｍｇ／ｍｌ程度まで限外濾過膜（ＡＤＶＡＮＴＥＣ社製ＵＬＴＲＡ　　ＦＩＬＴＥＲ　　ＵＮＩＴ　　ＵＳＹ−１、分子量分画１万）を用いて濃縮した。濃縮酵素液４００μｌにＤｅｎａｔｕｒｅ　ｂｕｆｆｅｒ（６Ｍ塩酸グアニジン、１０ｍＭ　　ＥＤＴＡ、０．１Ｍ炭酸水素アンモニウムｐＨ　７．８）４００μｌおよび５０ｍＭ　　Ｄｉｔｈｉｏｔｈｒｅｉｔｏｌ　８μｌを加え、９５℃で１０分間反応させた。室温まで冷却後、反応液にＤｅｎａｔｕｒｅ　ｂｕｆｆｅｒ溶液に溶解した５０ｍＭ　　Ｉｏｄｏａｃｅｔｏａｍｉｄｅ４０μｌを加え、暗所室温で１時間反応させた。この溶液をＳｌｉｄｅ−Ａ−Ｌｙｚｅｒ　　Ｍｉｎｉ　　Ｄｉａｌｙｓｉｓ　　Ｕｎｉｔｓ　Ｐｌｕｓ　Ｆｌｏａｔ（ＰＩＥＲＣＥ社製、分子量分画１万）で水１Ｌに対して透析した。得られた溶液にトリプシン（Ｍｏｄｉｆｉｅｄ、Ｐｒｏｍｅｇａ社製）８μｌを加え、３７℃で７時間酵素反応させた。反応液をＳＭＡＲＴ　　ｓｙｓｔｅｍ（Ｐｈａｒｍａｃｉａ　　Ｂｉｏｔｅｃｈ社製）にかけて分解アミノ酸のピークを分離した。分離条件を以下に示す。
カラム：Ｎｏｍｕｒａ　Ｃｈｅｍｉｃａｌ　Ｄｅｖｅｌｏｓｉｌ　　３００　ＣＤＳ−ＨＧ−５　（１ｍｍ×１５０ｍｍ）
Ａ　　ｂｕｆｆｅｒ：０．１％ＴＦＡ／Ｗａｔｅｒ
Ｂ　　ｂｕｆｆｅｒ：０．１％ＴＦＡ／Ａｃｅｔｏｎｉｔｒｉｌ
グラジエント：５→６０％Ｂ　１％／分．
Ｆｌｏｗ：５０μｌ／ｍｌ
分離した分解アミノ酸のうち、Ｂ　　ｂｕｆｆｅｒ濃度１５％程度の２つのピークおよび３５％程度のピークを分取した。この３つ（Ｎｏ．１〜Ｎｏ．３）についてアミノ酸配列解析装置（Ｐｒｏｃｉｓｅ　　ｃＬＣ、Ａｐｐｌｉｅｄ　　Ｂｉｏｓｙｓｔｅｍ社製）でアミノ酸配列を解析した。その結果得られた部分アミノ酸配列をアミノ末端側から記す。
Ｎｏ．１：Ｇｌｕ　　Ｍｅｔ　Ｔｈｒ　　Ｐｒｏ　Ｔｙｒ　　Ｌｅｕ　Ａｓｐ　　Ｐｈｅ　Ｔｙｒ　　Ａｒｇ　Ｌｅｕ　　Ｍｅｔ　Ａｌａ　　Ｔｙｒ　Ｇｌｎ（配列表の配列番号１）；
Ｎｏ．２：Ｉｌｅ　　Ｖａｌ　Ｖａｌ　　Ｇｌｙ　Ｍｅｔ　　Ｐｒｏ　Ｉｌｅ　　Ｔｙｒ　Ｇｌｙ　　Ａｒｇ　Ｌｙｓ　　Ｇｌｕ（配列表の配列番号２）；
Ｎｏ．３：Ａｌａ　　Ｌｅｕ　Ｐｒｏ　　Ｌｙｓ　Ｐｒｏ　　Ｇｌｙ　Ａｌａ　　Ｔｈｒ　Ｇｌｕ　　Ｔｙｒ　Ｖａｌ　　Ａｓｐ　Ｐｒｏ　　Ｓｅｒ　Ｉｌｅ（配列表の配列番号３）。
【０１０１】
実施例２．アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株からのキチナーゼの精製
１）粗酵素液の調製
滅菌した表７の組成の培地１００ｍｌが入っている５００ｍｌ容の三角フラスコ（種フラスコ）にアスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株の菌体を接種し、２６℃にて８日間、１００ｒｐｍの振とう培養を行った。培養終了後、４℃条件下、１０，０００×Ｇにて１０分間の遠心分離を行った。得られた上清を粗酵素液とした。
【０１０２】
【表７】
培地
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マルトエクストラクト　　　１０ｇ
イースト・エクストラクト　５ｇ
粉末キチン　　　　　　　　５ｇ
純水で１，０００ｍｌとした。
――――――――――――――――――――
２）精製酵素溶液の調製
１）で得られた粗酵素液２００ｍｌに、氷冷下攪拌しながら硫酸アンモニウム１１２ｇを徐々に加えた。混合溶液を一晩４℃で静置したのち、２０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）３，０００ｍｌに対して１２時間ずつ５回透析し、これを粗精製酵素画分とした。予め２０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）で平衡化したＭｏｎｏＱ（ファルマシア社製）カラム（直径７ｍｍ×長さ５ｃｍ）に粗精製酵素画分を添加し、吸着させた。該カラムを２０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）で十分洗浄した後、１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）２５０ｍｌ中に０乃至０．１５　Ｍの塩化ナトリウムの直線的濃度勾配を作製して該カラムに吸着した成分を溶出させた。キトサン分解活性は塩化ナトリウム濃度が０．０６３Ｍ乃至０．０６６Ｍの画分（１０ｍｌ）に溶出された。この画分を精製酵素溶液とした。
【０１０３】
３）精製酵素の分子量
実施例１　５）と同じ方法で分子量を測定した。本酵素は分子量約４０，０００の単一バンドを示した。
【０１０４】
４）精製酵素の等電点
実施例１　６）と同じ方法で等電点を測定した。本酵素の等電点は約ｐＩ３．５であった。
【０１０５】
実施例３．アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株からのキチナーゼの精製
１）粗酵素液の調製
滅菌した表８の組成の培地１００ｍｌが入っている５００ｍｌ容の三角フラスコ（種フラスコ）にアスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株の菌体を接種し、２６℃にて８日間、１００ｒｐｍの振とう培養を行った。培養終了後、４℃条件下、１０，０００×Ｇにて１０分間の遠心分離を行った。得られた上清を粗酵素液とした。
【０１０６】
【表８】
培地
――――――――――――――――――――
マルトエクストラクト　　　１０ｇ
イースト・エクストラクト　５ｇ
粉末キチン　　　　　　　　５ｇ
純水で１，０００ｍｌとした。
――――――――――――――――――――
２）精製酵素溶液の調製
１）で得られた粗酵素液２００ｍｌに、氷冷下攪拌しながら硫酸アンモニウム１１２ｇを徐々に加えた。混合溶液を一晩４℃で静置したのち、２０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）３，０００ｍｌに対して１２時間ずつ５回透析し、これを粗精製酵素画分とした。予め２０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）で平衡化したＭｏｎｏＱ（ファルマシア社製）カラム（直径７ｍｍ×長さ５ｃｍ）に粗精製酵素画分を添加し、吸着させた。該カラムを２０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）で十分洗浄した後、１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）２５０ｍｌ中に０乃至０．１５　Ｍの塩化ナトリウムの直線的濃度勾配を作製して該カラムに吸着した成分を溶出させた。キトサン分解活性は塩化ナトリウム濃度が０．０６２Ｍ乃至０．０６４Ｍの画分（１０ｍｌ）に溶出された。この画分を精製酵素溶液とした。
３）精製酵素の分子量
実施例１　５）と同じ方法で分子量を測定した。本酵素は分子量約４０，０００の単一バンドを示した。
【０１０７】
４）精製酵素酵素の等電点
実施例１　６）と同じ方法で等電点を測定した。本酵素の等電点は約ｐＩ４．５であった。
実施例４．　アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　　ＪＣＭ　２０６７株由来キチナーゼを用いた低分子キトサン塩酸塩の調製
キトサン９Ｂあるいはキトサン８Ｂあるいはキトサン７Ｂ、２．０ｇを水８００ｍｌに懸濁し、濃塩酸を加えてｐＨ２．５として粉末を完全に溶解した。飽和炭酸水素ナトリウム水でｐＨを５．０に合わせた後、水を加えて全量を１，０００　ｍｌとし、２００　ｍｌずつを５００　ｍｌ三角フラスコに移し、実施例１１）で調製した酵素液１０ｍｌを加え、４０℃で３日間８０ｒｐｍで振とうして酵素反応を行わせた。反応終了後、全ての反応液を回収し、氷冷下１％苛性ソーダ水でｐＨ１２以上としたのち、１０，０００ｒｐｍ、１０分間の遠心分離で沈殿物を得た。回収した沈殿物を２００ｍｌの水に懸濁し、氷冷下濃塩酸を加えてｐＨ２．５として溶解し、分子量分画１０，０００の透析膜を用いて、水に対して透析を行なった。透析終了後、溶液を濾過し、濾液を凍結乾燥した。低分子キトサン９Ｂ塩酸塩は、２．０　ｇの白色綿状固体として得られた。低分子キトサン８Ｂ塩酸塩が１．６　ｇの白色綿状固体として得られた。低分子キトサン７Ｂ塩酸塩が０．６　ｇの白色綿状固体として得られた。
【０１０８】
実施例５．　アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来キチナーゼを用いた低分子キトサン塩酸塩の調製
実施例４と同様の方法で低分子キトサン塩酸塩を調製した。酵素液は実施例２　１）で得られたものを用い、酵素反応はｐＨ４．０で行った。低分子キトサン９Ｂ塩酸塩は、１．３ｇの白色綿状固体として得られた。低分子キトサン８Ｂ塩酸塩が１．３ｇの白色綿状固体として得られた。低分子キトサン７Ｂ塩酸塩が０．９ｇの白色綿状固体として得られた。
【０１０９】
実施例６．　アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来キチナーゼを用いた低分子キトサン塩酸塩の調製
実施例４と同様の方法で低分子キトサン塩酸塩を調整した。但し、酵素液は実施例３で調整したものを用い、酵素反応はｐＨ４．５で行った。低分子キトサン９Ｂ塩酸塩は、２．１ｇの白色綿状固体として得られた。低分子キトサン８Ｂ塩酸塩が１．７ｇの白色綿状固体として得られた。低分子キトサン７Ｂ塩酸塩が１．０ｇの白色綿状固体として得られた。
【０１１０】
実施例７．　低分子キトサン塩酸塩のアセチル化度
実施例４〜６にて調製した低分子キトサン塩酸塩１００ｍｇを蒸留水１０ｍｌに溶解し、キトサナーゼ１０Ｕ（和光純薬（株）製）を加えて室温で３時間攪拌した。反応溶液を凍結乾燥し、乾燥固体をＤ_２Ｏに溶かして^１Ｈ−ＮＭＲを測定した。
アセチル基の３Ｈとアセチル基以外の、水酸基を除く７Ｈとの積分比から、アセチル化度を算出した。結果は表９に示す。
【０１１１】
【表９】

実施例８．　ゲル濾過法での分子量分画の決定
分子量マーカーとして、デキストランＴ１０（分子量１０，０００）、Ｔ４０（分子量４０，０００）、Ｔ７０（分子量７０，０００）、Ｔ１１０（分子量１１０，０００）、Ｔ２５０（分子量２５０，０００）（ファルマシア（株）製）の各１％溶液２００μｌを、あらかじめｐＨ４．５の２００ｍＭ酢酸緩衝液で膨潤させたゲル濾過材（Ｔｏｙｏｐｅａｒｌ　　ＨＷ５５　　Ｆｉｎｅ）をつめたカラム（８６×２ｃｍ）に展開し、１．７５ｍｌずつに分画した。水１容に濃硫酸８容を加えた硫酸液１．８ｍｌ、カルバゾール０．２５ｇをエタノール５０ｍｌに溶かした溶液６０μｌを各々の分画液２００μｌに加え、１００℃で１０分間保温した。氷冷後４２０ｎｍにおける吸光度を測定した。
【０１１２】
実施例４、５、６で作製した低分子キトサン塩酸塩の各５％溶液２００μｌを、同様にして１．７５ｍｌずつに分画した。各分画２８０μｌにｐＨ６．５の４００ｍＭ　ＭＯＰＳ−炭酸ナトリウム緩衝液１００μｌおよびキトサナーゼ１０Ｕ（和光純薬（株）製）を加えて、３７℃で１０分間酵素反応を行なった。遊離還元糖の定量は実施例１にしたがった。分子量マーカーと各低分子キトサンについて、最大吸光度を示す分画を１００％とし同一グラフ上にプロットした（図２〜４）。図から推定した低分子キトサンの分子量を表１０にまとめた。
【０１１３】
【表１０】

実施例９．アスペルギルス・オリザエ（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）由来のキチナーゼをコードするゲノムＤＮＡの分離
９−１）アスペルギルス・オリザエ（Ａｓｐｅｒｇｉｌｌｕｓ　ｏｒｙｚａｅ）のゲノムＤＮＡの分離アスペルギルス・オリザエ（Ａｓｐｅｒｇｉｌｌｕｓ　　ｏｒｙｚａｅ）ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　　ＪＣＭ　２０６７株を培地（１％　Ｍａｌｔ　Ｅｘｔｒａｃｔ，　０．５％　Ｙｅａｓｔ　　Ｅｘｔｒａｃｔ）１００ｍｌにて３０℃で４日間振盪培養した後、この培養液をろ過装置を用い、菌体を集菌した。続いて、この菌体を−８０℃に冷却した乳鉢に入れ、液体窒素を注いだ。液体窒素を加えながら乳棒を用いて菌体が粉末状になるまで十分砕いた。粉末状の菌体約１．５ｇを２０ｍｌの溶菌バッファー（６２．５ｍＭ　ＥＤＴＡ、５０ｍＭ　Ｔｒｉｓ−ＨＣｌ、５０ｍＭ　ＳＤＳ：ｐＨ８．０）に加え、穏やかに攪拌した。次に、これに１０ｍｌの９．５Ｍ　酢酸アンモニウムを添加し、さらに１５ｍｌのＴＥ飽和フェノールを添加し穏やかに攪拌した。この溶液を高速冷却遠心機（日立製ローターＮｏ．３）を用い、４℃，　　５０００ｒｐｍで５分間遠心し、上清を約２５ｍｌ回収した。回収した上清に１５ｍｌのクロロホルムを添加し、穏やかに攪拌した後、４℃，　　５０００ｒｐｍで１０分間遠心した。上清を新しいチューブに回収し、上清と等量の２−プロパノールを添加し攪拌した後、４℃，　　１００００ｒｐｍで１０分間遠心した。沈殿を４００μｌのＴＥに溶解した後、１ｍｌのマイクロチューブに移し、１０μｌの１０ｍｇ／ｍｌ　リボヌクレアーゼを添加し３７℃で２０分処理した。次いで、４０μｌの３Ｍ　酢酸ナトリウム、４００μｌ　クロロホルムを添加し攪拌し、室温で１００００ｒｐｍ，　　５分間遠心分離した。分離した上清をマイクロチューブに回収し、１ｍｌの１００％エタノール（−２０℃で冷却）を添加して穏やかに攪拌した。ガラス棒で糸状になったＤＮＡを巻き取り、エタノールをきった後に２００μｌのＴＥに溶解させた。
９−２）アスペルギルス・オリザエ・ゲノムＤＮＡライブラリーの作製
９−１）で得られたアスペルギルス・オリザエ・ゲノムＤＮＡ１０μｇを、制限酵素ＥｃｏＲＩ（宝酒造社製）１０μｌを用い、５０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、１０ｍＭ　ＭｇＣｌ_２、１ｍＭＤＴＴ及び１００ｍＭ　　ＮａＣｌからなる溶液中で、３７℃で約２時間切断処理した後、ＤＮＡを回収した。アスペルギルス・オリザエ・ゲノムＤＮＡのＥｃｏＲＩ切断産物と、同じくＥｃｏＲＩで切断されたベクター、ｐＵＣ１１８　ＥｃｏＲＩ／ＢＡＰ（宝酒造社製）をＲａｐｉｄ　　ＤＮＡ　Ｌｉｇａｔｉｏｎ　Ｋｉｔ（ロシュ・ダイアグノスティック社製）を用いてライゲーションを行い、アスペルギルス・オリザエのゲノムＤＮＡライブラリーとした。
９−３）ＤＩＧ（ジゴキシゲニン）標識ＤＮＡプローブの作製
実施例１で同定した内部アミノ酸配列（配列表の配列番号２）を元に作製したミックスプライマーＦ２１、およびＦ２１を用いてアスペルギルス・オリザエのゲノムライブラリーから同定したオリゴプライマーＰＲ５０を合成した。
【０１１４】
Ｆ２１：５’−ａｔｔｇｇｃａｔａｃｃａａｃａａｃｔａｔｃｔｔａｇａｇｃ−３’　（配列表の配列番号７）
ＰＲ５０：５’−ｔａａａｔｔｇａｃｃｃａｔａｔｃｃｔｃｔａｔｇｃａｔｔ−３’　（配列表の配列番号８）
この２種類のプライマーを用いて、アスペルギルス・オリザエのゲノムＤＮＡをテンプレートとしたＰＣＲにより、約８００ｂｐのＤＮＡを増やした。このＤＮＡは電気泳動により分離し、アガロースゲルから切り出した後、ＱＩＡｑｕｉｃｋ　Ｇｅｌ　　Ｅｘｔｒａｃｔｉｏｎ　　Ｋｉｔ　（キアゲン社製）を用いて精製した。こうして精製したＤＮＡをＤＩＧ　　ＤＮＡ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ　（ロシュ・ダイアグノスティック社製）を用いて標識した。標識の方法は以下の様に行った。まず、精製したＤＮＡを１００℃で１０分間加熱し、１本鎖にした後、氷上で急冷した。次いで１０μｌ　ヘキサヌクレオチド・ミックス（０．５Ｍ　　Ｔｒｉｓ、０．１Ｍ　ＭｇＣｌ_２、１ｍＭ　　Ｄｉｔｈｉｏｅｒｉｔｈｒｉｔｏｌ、２ｍｇ／ｍｌ　ＢＳＡ；ｐＨ７．２）、１０μｌ　ｄＮＴＰラベリング・ミックス（１ｍＭ　ｄＡＴＰ，　　１ｍＭ　ｄＣＴＰ，　　１ｍＭ　ｄＧＴＰ，　　０．６５ｍＭ　　ｄＴＴＰ，　０．３５ｍＭ　ＤＩＧ−１１−ｄＵＴＰ；　ｐＨ７．５）、５μｌ　　ＤＮＡポリメラーゼ（Ｋｌｅｎｏｗ　　ｆｒａｇｍｅｎｔ）を加え、滅菌水で全量が１００μｌになるように調整した。この反応液を３７℃で一晩反応させた後、１０μｌ　　０．２Ｍ　　ＥＤＴＡ（ｐＨ８．０）を添加して反応を停止させた。次いで、１０μｌ　　４Ｎ　　ＬｉＣｌと５００μｌ　　１００％エタノール（−２０℃で冷却させたもの）を加え、−７０℃で３０分間静置し、標識されたＤＮＡを沈殿させた。冷却遠心機で４℃、１５０００ｒｐｍ、１０分間遠心した。エタノールを捨て、１００μｌ　　７０％エタノール（−２０℃で冷却したもの）を添加してペレットを洗浄した後、１５０００ｒｐｍ、１０分間遠心し、ピペットで完全にエタノールを除去した。ペレットを乾燥した後、５０μｌ　　ＴＥに溶解し、２５ｍｌ　ＤＩＧ　　Ｅａｓｙ　　Ｈｙｂ（ロシュ・ダイアグノスティック社製）に加え、キチナーゼ遺伝子プローブとした。
９−４）キチナーゼ遺伝子を含む部分ＤＮＡライブラリーの作製
９−１）で得られたアスペルギルス・オリザエ・ゲノムＤＮＡ　　２０μｇを制限酵素ＥｃｏＲＩで切断処理を行った後、精製しＤＮＡを回収した。精製したＤＮＡをアガロースゲルで電気泳動により分離し、ナイロンメンブレンにブロッティングを行った。次いで、９−３）で得られたキチナーゼ遺伝子プローブを用い、サザンハイブリダイゼーションを行った結果、キチナーゼ遺伝子を含む一本の単一のバンドが得られた。そこで、次に９−１）で得られたアスペルギルス・オリザエ・ゲノムＤＮＡ　　８０μｇを制限酵素ＥｃｏＲＩで切断処理を行った後、精製しＤＮＡを回収した。このＤＮＡをアガロースゲルで電気泳動し、上記のサザンハイブリダイゼーションによって見られたバンドの位置と同じ位置をゲルから切り出し、ＤＮＡを抽出した。抽出したＤＮＡをｐＵＣ１１８　ＥｃｏＲＩ／ＢＡＰベクターにライゲーションを行い、キチナーゼ遺伝子を含むＤＮＡライブラリーとした。
９−５）キチナーゼ遺伝子含有ＤＮＡのスクリーニング
９−４）で得たＤＮＡライブラリーにより、大腸菌ＪＭ１０９株（宝酒造社製）を形質転換させた。寒天培地上に形質転換体コロニーを形成させ、その上に、ナイロントランスファーメンブレン（アマシャム社製）を重ねて、メンブレン上にコロニーの一部を移行させた。このメンブレンをアルカリ変性溶液（０．５Ｍ　ＮａＯＨ、３．０Ｍ　ＮａＣｌ）を浸したろ紙上に２０分間静置し、その後中和溶液（１Ｍ　Ｔｒｉｓ−ＨＣｌ　（ｐＨ７．５））を浸したろ紙上に１０分間置いた。さらに、２×ＳＳＣ溶液で振盪しながら洗浄した。次に、このメンブレンをＤＩＧ　　Ｅａｓｙ　　Ｈｙｂでプレハイブリダイゼーションを１時間行った。その後、メンブレンを９−３）で得られたキチナーゼ遺伝子プローブと３７℃で一晩ハイブリダイゼーションを行った。ハイブリダイゼーション後、常温の２×ＳＳＣバッファーで三回洗浄し、さらに５３℃に温めた２×ＳＳＣバッファーで二回洗浄した。その後、ＤＩＧ　　Ｗａｓｈ　　ａｎｄ　Ｂｌｏｃｋ　　Ｂｕｆｆｅｒ　　Ｓｅｔ（ロシュ・ダイアグノスティック社製）を用いて検出を行った。まず、Ｗａｓｈ　ｂｕｆｆｅｒ　（０．１Ｍ　マレイン酸、０．１５Ｍ　　ＮａＣｌ、０．３％　Ｔｗｅｅｎ　　２０：ｐＨ７．５）でメンブレンを順化した。次いで、このメンブレンをブロッキング溶液（０．１Ｍ　マレイン酸、０．１５Ｍ　　ＮａＣｌ：ｐＨ７．５に１０％ブロッキング試薬を含む溶液）に浸して６０分間ブロッキングを行い、非特異的な結合をブロッキングした。次に、メンブレンをブロッキング溶液に抗ジゴキシゲニン抗体を１００００倍希釈で加えた溶液に浸し、３７℃で３０分間反応させた後、Ｗａｓｈ　Ｂｕｆｆｅｒで二回洗浄した。その後、Ｄｅｔｅｃｔｉｏｎ　　ｂｕｆｆｅｒ　　（０．１Ｍ　Ｔｒｉｓ−ＨＣｌ、０．１Ｍ　ＮａＣｌ：ｐＨ９．５）でメンブレンを順化した。Ｄｅｔｅｃｔｉｏｎ　　ｂｕｆｆｅｒにＣＳＰＤ　（Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｃｅ　ｓｕｂｓｔｒａｔｅ）を１００倍希釈で加え、純化したメンブレンに滴下した後、３７℃で１５分間発色を安定させた。その後Ｌｕｍｉ−Ｆｉｌｍ　　Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ　　Ｄｅｔｅｃｉｏｎ　　Ｆｉｌｍ（コニカ社製）に感光させ、現像した。その結果、ポジティブシグナルを示すクローンを確認できた。
９−６）組換え体プラスミドＤＮＡの抽出と配列解析
９−５）で得たポジティブシグナルを示すクローンをＬ−ｂｒｏｔｈ（和光純薬工業社製）で一晩培養し、組換え体プラスミドのＤＮＡを抽出した。ＱＩＡｐｒｅｐ　　Ｓｐｉｎ　　Ｍｉｎｉｐｒｅｐ　　Ｋｉｔ　（キアゲン社製）を用い、プラスミドＤＮＡを抽出した。次いで、抽出したＤＮＡをこのプラスミド特有のユニバーサルプライマー、Ｍ１３Ｍ４，　Ｍ１３ＲＶを用いて配列解析を行い、その結果と９−３）で得られたキチナーゼ遺伝子プローブのヌクレオチド配列とをアラインメントし、キチナーゼの配列を含むことを確認した。キチナーゼの配列を含むことが確認できたプラスミドＤＮＡをＧＰＳ−１　　Ｇｅｎｏｍｅ　　Ｐｒｉｍｉｎｇ　Ｓｙｓｔｅｍ（ニューイングランド　バイオ・ラボ社製）を用いて、全体の配列を決定した。手順は、まず上記で抽出したプラスミドＤＮＡ　　４μｌに２μｌ　　１０×ＧＰＳ　　Ｂｕｆｆｅｒ　　（２５０ｍＭ　　Ｔｒｉｓ−ＨＣｌ；ｐＨ８．０、２０ｍＭ　ＤＴＴ、２０ｍＭ　ＡＴＰ、５００μｇ／ｍｌ　ＢＳＡ）、１μｌ　　ｐＧＰＳ１（Ｔｒａｎｓｐｒｉｍｅｒ−１　　Ｄｏｎｏｒ　ｐｌａｓｍｉｄ）、１１μｌ　滅菌水を加え混合した後、１μｌ　　ＴｎｓＡＢＣ　　Ｔｒａｎｓｐｏｓａｓｅを添加し３７℃で１０分間結合させた。その後、１μｌ　　Ｓｔａｒｔ　Ｓｏｌｕｔｉｏｎ　（３００ｍＭ　ｍａｇｎｅｓｉｕｍ　　ａｃｅｔａｔｅ）を添加し、３７℃で１時間反応させた後、７５℃で１０分間処理することで、反応を停止した。次いで、この反応産物１０μｌを大腸菌ＪＭ１０９株に形質転換し、アンピシリンとカナマイシンを含むＬＢ寒天培地に塗布し、３７℃で一晩培養した。生育してきたコロニーをＬＢ液体培地で一晩培養し、プラスミドＤＮＡを抽出した。この抽出したプラスミドＤＮＡをｐＧＰＳ１のプライマーであるＮプライマーおよびＳプライマーで配列をよみ、キチナーゼをコードするゲノム配列の全配列を決定した。
実施例１０．アスペルギルス・オリザエ由来のキチナーゼをコードするｃＤＮＡの同定
１０−１）アスペルギルス・オリザエの全ＲＮＡの精製
アスペルギルス・オリザエ（Ａｓｐｅｒｇｉｌｌｕｓ　　ｏｒｙｚａｅ）ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　　ＪＣＭ　２０６７株をＰｏｔａｔｏ　Ｄｅｘｔｒｏｓｅ培地２０ｍｌで３０℃、３日間前培養した。その後、液体培地（１％　Ｍａｌｔ　Ｅｘｔｒａｃｔ、０．５％　Ｙｅａｓｔ　　Ｅｘｔｒａｃｔ）に１％植菌し、３０℃で３日間培養した。培養した菌体を吸引集菌し、−８０℃で冷やした乳鉢（オートクレーブ滅菌済）に移した。液体窒素を加えながら、乳棒で菌体を破砕し、粉末状にした。完全に粉末状になった菌体をＲｎｅａｓｙ　Ｐｌａｎｔ　　Ｍｉｎｉ　　Ｋｉｔ　（キアゲン社製）を用いて次のように全ＲＮＡの精製を行った。まず粉末状の菌体約１ｇを４．５ｍｌ　　ＲＬＴ　ｂｕｆｆｅｒ　（β−　　Ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ）に加え、激しく振とうし菌体を溶解させ、氷上に置いた。０．５ｍｌずつＱＩＡｓｈｒｅｄｄｅｒスピンカラムに分注し、室温で１５０００ｒｐｍ、２分間遠心した。通過液に０．５倍量のエタノールを添加し、ピペッティングで攪拌した。その後、６７５μｌずつＲｎｅａｓｙ　Ｍｉｎｉスピンカラムに分注し、室温で１６０００ｒｐｍ、１５秒間遠心した。７００μｌのＲＷ１　　Ｗａｓｈ　　ｂｕｆｆｅｒを加え、同じように室温で１６０００ｒｐｍ、１５秒間遠心した。次に５００μｌ　　ＲＰＥ　Ｗａｓｈ　ｂｕｆｆｅｒを添加し、室温で１６０００ｒｐｍ、２分間遠心してＲＮＡを洗浄した。スピンカラムをマイクロチューブにセットし、５０μｌのＲｎａｓｅ　　ｆｒｅｅ　　ｗａｔｅｒでＲＮＡを溶出させ室温で１００００ｒｐｍ、１分間遠心して回収した。
１０−２）アスペルギルス・オリザエｃＤＮＡライブラリーの作製
アスペルギルス・オリザエのｃＤＮＡライブラリーは、ＳＭＡＲＴ　　ｃＤＮＡ　　Ｌｉｂｒａｒｙ　Ｃｏｎｓｔｒｕｃｔｉｏｎ　Ｋｉｔ　　（クロンテック社製）を用いて作製した。まず、滅菌した０．５ｍｌチューブに１０−１）で精製した全ＲＮＡ　　１μｇと１μｌ　　ＳＭＡＲＴ　ＩＶ　Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ、１μｌ　　ＣＤＳ　ＩＩＩ／３’　ＰＣＲ　　Ｐｒｉｍｅｒを混合し、７２℃で２分間インキュベートした。その後、氷上に２分間置き、冷却した。この冷却した試料に２μｌ　　５×　Ｆｉｒｓｔ−Ｓｔｒａｎｄ　Ｂｕｆｆｅｒ、１μｌ　　２０ｍＭ　　ＤＴＴ、１μｌ　　１０ｍＭ　　ｄＮＴＰ　　Ｍｉｘおよび１μｌ　　Ｐｏｗｅｒ　Ｓｃｒｉｐｔ　Ｒｅｖｅｒｓｅ　　Ｔｒａｎｓｃｒｉｐｔａｓｅを添加した。内容物を混合し、４２℃で１時間反応させた後、氷上に置き、ｆｉｒｓｔ−ｓｔｒａｎｄ　ＤＮＡ合成を停止させた。次いで、ＬＤ　ＰＣＲによるｃＤＮＡの増幅を行った。ＰＣＲチューブに前記のＦｉｒｓｔ−Ｓｔｒａｎｄ　ＤＮＡを２μｌ添加し、さらに次の試薬を添加した：８０μｌ　脱イオン水、１０μｌ　　１０×Ａｄｖａｎｔａｇｅ２　ＰＣＲ　　Ｂｕｆｆｅｒ、２μｌ　　５０×ｄＮＴＰ　Ｍｉｘ、２μｌ　　５’ＰＣＲ　Ｐｒｉｍｅｒ、２μｌ　　ＣＤＳ　ＩＩＩ／３’　ＰＣＲ　　Ｐｒｉｍｅｒ、２μｌ　　５０×　Ａｄｖａｎｔａｇｅ２　Ｐｏｌｙｍｅｒａｓｅ　Ｍｉｘ。このＰＣＲチューブを９５℃に予熱したサーマルサイクラーにセットし、９５℃で２０秒反応させた後、９５℃で５秒、６８℃で６分のサイクルを２０サイクル行った。この反応液（ｄｓ　ｃＤＮＡを含む）５０μｌを滅菌したチューブに取り、２μｌ　プロテアーゼＫを添加して４５℃で２０分間インキュベートし、プロテアーゼ処理を行った後、５０μｌの脱イオン水を添加した。１００μｌ　フェノール：クロロホルム：イソアミルアルコール混合液（２５：２４：１）を添加し、１分間混合して１４０００ｒｐｍで５分間遠心分離した。上清を新しいチューブに移し、１００μｌ　クロロホルム：イソアミルアルコール（２４：１）を添加した。１分間混合した後、再度１４０００ｒｐｍで５分間遠心分離した。上清を新しいチューブに移し、１０μｌ　　３Ｍ　酢酸ナトリウム、１．３μｌ　グリコーゲン及び２６０μｌ　　９５％エタノール（室温）を加え、直ちに室温で１４０００ｒｐｍで２０分間遠心分離した。ピペットで上清を除去し、８０％エタノール１００μｌでペレットを洗浄した後、ペレットを乾燥させた。ペレットを７９μｌ　脱イオン水に溶解した。続いて、制限酵素Ｓｆｉ　　Ｉによる処理を行った。７９μｌ　　ｃＤＮＡ、１０μｌ　　１０×　Ｓｆｉ　Ｂｕｆｆｅｒ、１０μｌ　　Ｓｆｉ　Ｅｎｚｙｍｅおよび１μｌ　　１００×　ＢＳＡを混合し、試験管を５０℃で２時間インキュベートした。その後、２μｌ　　１％　　ｘｙｌｅｎｅ　　ｃｙａｎｏｌ染色液を添加した。ＣＨＲＯＭＡ　ＳＰＩＮ−４００カラムを使い、ｃＤＮＡを分子ふるいにかけた。ＣＨＲＯＭＡ　ＳＰＩＮカラムは数回転倒混和してゲルマトリックスを懸濁させ、さらにピペットマンで懸濁した。カラムの蓋を取り、カラムが自然にドリップするように設置した。次にカラムバッファーを静かに添加し、重力で自然にドリップさせた。このバッファーのドリップが終了した後、マトリックスの上面中央にＳｆｉ　　Ｉで分解したｃＤＮＡサンプルをアプライし、マトリックスの表面に十分吸収させた。表面から液体がなくなり、ドリップが終了したら、６００μｌ　カラムバッファーを添加し、直ちに試験管１６本に１滴ずつフラクションを収集した。このフラクションの各３μｌをアガロースゲル電気泳動にかけ、ピークフラクションを判定した。ｃＤＮＡを含むフラクションを収集し、１本の新しい試験管に集めた。この試験管に１／１０倍量の酢酸ナトリウム（３Ｍ；ｐＨ４．８）、１．３μｌ　グリコーゲン、２．５倍量の９５％エタノール（−２０℃）を加え、軽く振って混合し、−２０℃で一晩インキュベートした。室温で１４０００ｒｐｍ、２０分間遠心分離し、上清を除去した。ペレットを完全に乾燥させた後、７μｌ　脱イオン水にペレットを再懸濁した。上記のｃＤＮＡ　０．５μｌを１μｌ　λＴｒｉｐｌＥｘ２ベクター、０．５μｌ　　１０×　Ｌｉｇａｔｉｏｎ　Ｂｕｆｆｅｒ、０．５μｌ　　ＡＴＰ、０．５μｌ　　Ｔ４　ＤＮＡ　Ｌｉｇａｓｅおよび２．０μｌ　脱イオン水と混合し、１６℃で一晩インキュベートした。次に、このライゲーションサンプルに対してλファージパッケージング反応を行った。パッケージングにはＧｉｇａｐａｃｋ　ＩＩＩ　　Ｇｏｌｄ　　Ｐａｃｋａｇｉｎｇ　Ｅｘｔｒａｃｔ（ＳＴＲＡＴＡＧＥＮＥ社製）を用いた。Ｐａｃｋａｇｉｎｇ　　Ｅｘｔｒａｃｔが入ったマイクロチューブに上記でライゲーションしたＤＮＡベクターを４μｌμｌ加え、静かにピペッティングで混合した後、室温で２時間インキュベートした。２時間後、５００μｌ　　ＳＭ　　ｂｕｆｆｅｒを加え、さらに２０μｌのクロロホルムを混合した後、軽く遠心分離することによりパッケージング液を調製した。次に、パッケージングしたファージを感染させるホスト細胞を培養した。Ｅ．　ｃｏｌｉ　ＸＬ１−Ｂｌｕｅの凍結ストックをＬＢ／ｔｅｔｒａｃｙｃｌｉｎｅアガープレートに植菌し、３７℃で一晩培養した。生育したシングルコロニーをＭｇＳＯ_４とｔｅｔｒａｃｙｃｌｉｎｅを含むプレートに移し、３７℃で一晩培養した。生育したシングルコロニーを１５ｍｌのＬＢ／ＭｇＳＯ_４／マルトースブロスに植菌し、培養液のＯＤ_６００が２．０になるまで３７℃で培養した。培養液を５０００ｒｐｍで５分間を遠心分離し、ペレットを７．５ｍｌの１０ｍＭ　ＭｇＳＯ_４に再懸濁した。パッケージング液を１×λ希釈バッファーで希釈した後、該パッケージング液　１μｌを、２００μｌ　　Ｅ．　　ｃｏｌｉ　　ＸＬ１−Ｂｌｕｅ懸濁液に添加し、３７℃で１５分間吸着させた。該反応液に、融解した２ｍｌのＬＢ／　　ＭｇＳＯ_４トップアガーを混合し、温めておいたＬＢ／　　ＭｇＳＯ_４プレートに均等に広げた。室温で１０分間冷やし、トップアガーを固めた後、３７℃で一晩培養した。得られたプラークをＳＭバッファーを加えて一晩振とうして溶出させ、アスペルギルス・オリザエｃＤＮＡライブラリーを含むファージを得た。
１０−３）キチナーゼ遺伝子含有ｃＤＮＡのスクリーニング
１０−２）で得たｃＤＮＡライブラリーを含むファージを大腸菌ＸＬ１−Ｂｌｕｅ株（クロンテック社製）に感染させた。希釈したファージ１μｌを培養した２００μｌ　　ＸＬ１−Ｂｌｕｅに添加し、３７℃で１０分から１５分間吸着させた。２ｍｌの融解したＬＢ／　　ＭｇＳＯ_４トップアガーを添加し、混合し、温めておいたＬＢ／　　ＭｇＳＯ_４プレートに注ぎ、プレートをまわしてトップアガーを均等に広げた。室温で１０分間冷やし、トップアガーを固めた後、３７℃で一晩インキュベートし、寒天培地上にプラークを形成させた。その上に、ナイロントランスファーメンブレン（アマシャム社製）を１分間ほど重ねて、ファージの一部をナイロンメンブレン上に移行させた。このメンブレンをろ紙上で２０分間乾燥させた後、アルカリ変性溶液（０．２Ｎ　ＮａＯＨ、１．５Ｍ　ＮａＣｌ）を浸したろ紙上に５分間静置し、その後中和溶液（０．４Ｍ　Ｔｒｉｓ−ＨＣｌ　（ｐＨ７．５）、２×ＳＳＣ）を浸したろ紙上に５分間置いた。さらに、中和溶液で１分間振盪しながら洗浄した後、２×ＳＳＣでさらに５分間洗浄した。フィルターをろ紙上で１時間程乾燥させ、８０℃のオーブンで２時間ベーキングを行った。その後、メンブレンをＸ−３で得られたＤＮＡプローブと３７℃で一晩ハイブリダイゼーションを行った。ハイブリダイゼーション後、常温の２×ＳＳＣバッファーで三回洗浄し、さらに５３℃に温めた２×ＳＳＣバッファーで二回洗浄した。その後、ＤＩＧ　　Ｗａｓｈ　　ａｎｄ　Ｂｌｏｃｋ　　Ｂｕｆｆｅｒ　Ｓｅｔ（ロシュ・ダイアグノスティック社製）を用いて検出を行った。まず、Ｗａｓｈ　ｂｕｆｆｅｒ　（０．１Ｍ　マレイン酸、０．１５Ｍ　　ＮａＣｌ、０．３％　Ｔｗｅｅｎ　　２０；ｐＨ７．５）でメンブレンを順化した。次いで、このメンブレンをブロッキング溶液（０．１Ｍ　マレイン酸、０．１５Ｍ　　ＮａＣｌ；ｐＨ７．５に１０％ブロッキング試薬を含む溶液）に浸して６０分間ブロッキングを行い、非特異的な結合をブロッキングした。次に、メンブレンをブロッキング溶液に抗ジゴキシゲニン抗体を１００００倍希釈で加えた溶液に浸し、３７℃で３０分間反応させた後、Ｗａｓｈ　Ｂｕｆｆｅｒで二回洗浄した。その後、Ｄｅｔｅｃｔｉｏｎ　　ｂｕｆｆｅｒ（０．１Ｍ　Ｔｒｉｓ−ＨＣｌ、０．１Ｍ　ＮａＣｌ；ｐＨ９．５）でメンブレンを順化した。Ｄｅｔｅｃｔｉｏｎ　　ｂｕｆｆｅｒにＣＳＰＤ　（Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｃｅ　ｓｕｂｓｔｒａｔｅ）を１００倍希釈で加え、純化したメンブレンに滴下した後、３７℃で１５分間発色を安定させた。その後Ｌｕｍｉ−Ｆｉｌｍ　　Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ　　Ｄｅｔｅｃｉｏｎ　　Ｆｉｌｍ（コニカ社製）に感光させ、現像し、検出した結果、ポジティブシグナルを示すクローンを検出できた。このポジティブを示すファージプラークを寒天培地ごとくり抜き、ＳＭバッファーに溶解させた後、大腸菌ＢＭ２５．８株に形質転換させ、形質転換大腸菌株　Ｅｓｃｈｅｒｉｃｈｉａ　　ｃｏｌｉ　　ＢＭ２５．８／ｐＴｒｉｐｌＥｘ−Ｃｈｉｔｉｎａｓｅ−ｃＤＮＡ　ＳＡＮＫ　７２１０２株を得た。この菌株は平成１４年１１月８日付けで、独立行政法人産業技術総合研究所特許生物寄託センターに国際寄託され、受託番号：ＦＥＲＭ　ＢＰ−８２３５を付与された。感染したファージＤＮＡであるλＴｒｉｐｌＥｘ−Ｃｈｉｔｉｎａｓｅ−ｃＤＮＡは、大腸菌ＢＭ２５．８株の細胞内で環状化し、組換えプラスミドｐＴｒｉｐｌＥｘ−Ｃｈｉｔｉｎａｓｅ−ｃＤＮＡとして存在する。得られたＥｓｃｈｅｒｉｃｈｉａ　　ｃｏｌｉ　　ＢＭ２５．８／ｐＴｒｉｐｌＥｘ−Ｃｈｉｔｉｎａｓｅ−ｃＤＮＡ　ＳＡＮＫ　７２１０２株のコロニーをＬＢ／Ａｍｐｉｃｉｌｉｎブロスで一晩培養し、組換え体プラスミドｐＴｒｉｐｌＥｘ−Ｃｈｉｔｉｎａｓｅ−ｃＤＮＡのＤＮＡを抽出した。ＱＩＡｐｒｅｐ　　Ｓｐｉｎ　　Ｍｉｎｉｐｒｅｐ　　Ｋｉｔ　（キアゲン社製）を用い、プラスミドＤＮＡを抽出した。次いで、該プラスミドＤＮＡ　　４μｌに２μｌ　　１０×ＧＰＳ　　Ｂｕｆｆｅｒ　　（２５０ｍＭ　　Ｔｒｉｓ−ＨＣｌ；ｐＨ８．０、２０ｍＭ　ＤＴＴ、２０ｍＭ　ＡＴＰ、５００μｇ／ｍｌ　　ＢＳＡ）、１μｌ　　ｐＧＰＳ１　（Ｔｒａｎｓｐｒｉｍｅｒ−１　Ｄｏｎｏｒ　　ｐｌａｓｍｉｄ）、１１μｌ　滅菌水を加え混合した後、１μｌ　　ＴｎｓＡＢＣ　　Ｔｒａｎｓｐｏｓａｓｅを添加し３７℃で１０分間結合させた。その後、１μｌ　　Ｓｔａｒｔ　Ｓｏｌｕｔｉｏｎ　（３００ｍＭ　ｍａｇｎｅｓｉｕｍ　　ａｃｅｔａｔｅ）を添加し、３７℃で１時間反応させた後、７５℃で１０分間処理することで、反応を停止した。次いで、この反応産物１０μｌを大腸菌ＪＭ１０９株に形質転換し、アンピシリンとカナマイシンを含むＬＢ寒天培地に塗布し、３７℃で一晩培養した。生育してきたコロニーをＬＢ液体培地で一晩培養し、プラスミドＤＮＡを抽出した。この抽出したプラスミドＤＮＡをｐＧＰＳ１のプライマーであるＮプライマーおよびＳプライマーで配列をよみ、アスペルギルス・オリザエのキチナーゼをコードするＤＮＡのヌクレオチド配列を決定した（配列表の配列番号４）。また、配列表の配列番号４に記載のヌクレオチド配列から、オープンリーディングフレーム（ＯＲＦ）は、ヌクレオチド番号２４２から２４４番目の“ＡＴＧ”を開始コドンとし、ヌクレオチド番号１４３６から１４３８番目の“ＴＡＧ”を終止コドンとする領域であり、コードするアミノ酸配列は配列表の配列番号５に示される配列であることが推察された。
実施例１１．アスペルギルス・オリザエ由来キチナーゼのＮ末端アミノ酸配列の決定
実施例１で精製した酵素溶液を約１ｍｇ／ｍｌ程度まで限外濾過膜（ＡＤＶＡＮＴＥＣ社製ＵＬＴＲＡ　　ＦＩＬＴＥＲ　　ＵＮＩＴ　　ＵＳＹ−１、分子量分画１万）を用いて濃縮した。濃縮酵素液４００μｌにＤｅｎａｔｕｒｅ　ｂｕｆｆｅｒ（６Ｍ塩酸グアニジン、１０ｍＭ　　ＥＤＴＡ、０．１Ｍ炭酸水素アンモニウムｐＨ　７．８）４００μｌおよび５０ｍＭ　　Ｄｉｔｈｉｏｔｈｒｅｉｔｏｌ８μｌを加え、９５℃で１０分間反応させた。室温まで冷却後、反応液にＤｅｎａｔｕｒｅ　ｂｕｆｆｅｒ溶液に溶解した５０ｍＭ　　Ｉｏｄｏａｃｅｔｏａｍｉｄｅ４０μｌを加え、暗所室温で１時間反応させた。この溶液をＳｌｉｄｅ−Ａ−Ｌｙｚｅｒ　　Ｍｉｎｉ　　Ｄｉａｌｙｓｉｓ　　Ｕｎｉｔｓ　Ｐｌｕｓ　Ｆｌｏａｔ（ＰＩＥＲＣＥ社製、分子量分画１万）で水１Ｌに対して透析した。得られた溶液にトリプシン（Ｍｏｄｉｆｉｅｄ、Ｐｒｏｍｅｇａ社製）８μｌを加え、３７℃で７時間酵素反応させた。得られた酵素反応液をＬＣ／ＭＳ　　（Ｐｅｎｃｏｎ／Ｑ−Ｔｏｆ２）で測定した。Ｉｎｃｌｕｄｅ　　ｍａｓｓとしてｍ／ｚ　　５３３を設定し、ＭＳ／ＭＳスペクトルを測定した。フラグメントイオンの帰属からＮ末端アミノ酸はＳｅｒであること、Ｎ末端にＳｅｒ−Ｓｅｒ−Ｇｌｙ−Ｌｅｕ−Ｌｙｓというアミノ酸配列を含むこと、および、Ｎ末端にアセチル基の存在が示唆された（配列表の配列番号１４のアミノ酸番号１から５に記載のアミノ酸配列）。この結果を配列表の配列番号５のアミノ酸配列と照らし合わせると、アスペルギルス・オリザエが産生するキチナーゼは、そのＮ末端、“Ｍｅｔ−Ｓｅｒ−Ｓｅｒ”、が翻訳後修飾をうけて、“Ａｃｅｔｙｌ−Ｓｅｒ−Ｓｅｒ”の形（配列表の配列番号１４）で産生されていることが示唆された。
実施例１２．アスペルギルス・オリザエのキチナーゼ遺伝子の発現
キチナーゼＯＲＦの５’末端と３’末端にそれぞれＢａｍＨＩサイトを接続させたプライマー、ＢａｍＨＩ−ｃＤＮＡおよびｃＤＮＡ−ＢａｍＨＩを設計し，合成したた。
【０１１５】
ＢａｍＨＩ−ｃＤＮＡ：５’−ｇａｇｇａｔｃｃａｔｇｔｃｔｔｃｃｇｇａｃｔａａａｇｔｃｇｇ−３’　（配列表の配列番号９）
ｃＤＮＡ−ＢａｍＨＩ：５’−ｃｔｇｇａｔｃｃａｇｃｇａａａｃｃｇｇｃｔｃｇｃａｇｇｔｔｇ−３’　（配列表の配列番号１０）
実施例１０−３）で得られた組換えプラスミド、ｐＴｒｉｐｌＥｘ−Ｃｈｉｔｉｎａｓｅ−ｃＤＮＡを鋳型とし、プライマーとして上記のＢａｍＨＩ−ｃＤＮＡおよびｃＤＮＡ−ＢａｍＨＩを用いてＰＣＲを行い、制限酵素サイトを導入したキチナーゼＤＮＡを増幅させた。さらにｐＣＲ　　２．１−ＴＯＰＯベクター（インヴィトロジェン社製）に挿入し、大腸菌ＪＭ１０９株に形質転換させ、増幅させた。ＪＭ１０９株を生育させたコロニーをＬｂ／Ａｍｐｉｃｉｌｉｎブロスで一晩培養し、プラスミドＤＮＡを抽出した。このプラスミドＤＮＡを制限酵素ＢａｍＨＩで処理し、キチナーゼＯＲＦを切断した。また、この断片を連結させるベクターとして、ｐＱＥ−６０（キアゲン社製）をＢａｍＨＩで切断し、さらにアルカリフォスファターゼで５’末端にあるリン酸基を除去する処理を行った。その後、ＴＯＰＯ　ＴＡ　Ｃｌｏｎｉｎｇ　　Ｋｉｔ（インヴィトロジェン社製）を用いて上記のキチナーゼＯＲＦとライゲーションを行った。該反応液を大腸菌ＪＭ１０９株に形質転換させ、Ｌｂ／Ａｍｐｉｃｉｌｉｎアガープレートで一晩培養した。生育してきたコロニーをＬｂ／Ａｍｐｉｃｉｌｉｎブロス３ｍｌに植菌し、振とうしながら一晩培養した。該培養液からプラスミドＤＮＡをＱＩＡｐｒｅｐ　　Ｓｐｉｎ　　Ｍｉｎｉｐｒｅｐ　　Ｋｉｔ（キアゲン社製）で抽出し、大腸菌Ｍ１５［ｐＲＥＰ４］株に形質転換した。Ｌｂ／Ａｍｐｉｃｉｌｉｎ、　Ｋａｎａｍａｉｃｉｎアガープレートで一晩培養し、生育してきたコロニーをＬｂ／Ａｍｐｉｃｉｌｉｎ、　Ｋａｎａｍａｉｃｉｎブロス２０ｍｌに植菌し、振とうしながら３７℃で一晩培養した。この培養液から２．５ｍｌをＬｂ／Ａｍｐｉｃｉｌｉｎ、　Ｋａｎａｍｉｃｉｎブロス１００ｍｌに植菌し、３７℃で培養した。培養開始後１時間後から３０分おきにＯＤ_６００を測定し、０．６−０．７の間になるまで培養後、１ｍＭ　　ＩＰＴＧを加えた。ＩＰＴＧ添加後、１８℃で培養を続け、４及び６時間後に集菌した。菌体に１０ｍｌ　ＰＢＳ（１３７ｍＭ　　ＮａＣｌ、８．１ｍＭ　　Ｎａ_２ＨＰＯ_４・１２Ｈ_２Ｏ、２．６８ｍＭ　ＫＣｌ、１．４７ｍＭ　ＫＨ_２ＰＯ_４）を添加し、ヴォルテックスをかけよく懸濁した。この懸濁液をソニケーションにより、菌体を破砕した後、４℃で８０００ｒｐｍ、５分間遠心分離した。上清１００μｌを１．５ｍｌチューブに取り、５０μｌ変性剤（６％　ＳＤＳ、２４％　　ｇｌｙｃｅｒｏｌ、１２％　β−メルカプトエタノール、０．３Ｍ　Ｔｒｉｓ−ＨＣｌ　（ｐＨ６．８）、０．１５％　　ＢＰＢ）を添加し１００℃で５分間処理した。この反応液を各サンプル１０μｌずつｅ−ＰＡＧＥＬ（ＡＴＴＯ社製）で電気泳動した後、Ｓｉｍｐｌｙ　Ｂｌｕｅ　ＳａｆｅＳｔａｉｎ（インヴィトロジェン社製）で染色、検出した。その結果、約４４ｋＤａ付近に濃いバンドが見られた。
【０１１６】
６時間培養し集菌した菌体の上清中にキチナーゼが存在することは、試験例１−６）に記載のシェールズ法により確認した。
【０１１７】
４時間培養し集菌した菌体の上清は、Ｈｉｓタグ精製を行った。カラムチューブにＮｉ−ＮＴＡアガロースを詰め、０．１Ｍ　Ｔｒｉｓ−ＨＣｌ　（ｐＨ８．０）約１０ｍｌで洗浄した後、上清をカラムに吸着させた。次いで、８ｍｌの１０ｍＭイミダゾールで非特異的な結合物を洗い流した後、８ｍｌの２００ｍＭイミダゾールでカラムに吸着している蛋白質を溶出させ、。この溶出液をｅ−ＰＡＧＥＬで電気泳動した結果、単一のバンドとして現れた。また、この溶出液と実施例１で得られたアスペルギルス・オリザエの培養物から精製したキチナーゼを１２．５％アクリルアミドゲルによってＳＤＳ電気泳動を行なった（図３０に示す）。溶出液に含まれる蛋白質の分子量は、アスペルギルス・オリザエ由来の精製キチナーゼよりも少し大きくなっているが、これはＨｉｓタグによるものと考えられ、溶出液に含まれるリコンビナント蛋白質がＨｉｓ融合リコンビナントキチナーゼであることが推察された。この溶出液のキトサン分解活性を試験例１−６）に記載のシェールズ法により測定したところ、２．０Ｕ／ｍｌであり、この溶出液に含まれるリコンビナント蛋白質がキチナーゼであることが確認された。なお、１分間に１μｍｏｌのアセチルグルコサミンを生成する酵素量を１Ｕと定義した。
【０１１８】
本発明においてアミノ酸配列が同定されたアスペルギルス・オリザエ由来のキチナーゼは、翻訳後修飾を受ける前の３９９アミノ酸からなるもの（配列表の配列番号５）および翻訳後修飾を受けた３９８アミノ酸からなるもの（配列表の配列番号１４）である。キチナーゼ遺伝子として報告（キチン・キトサン研究　第８巻　第２号　２３８−２３９頁　２００２年）されているアスペルギルス・オリザエ由来の遺伝子は４３１アミノ酸からなる蛋白質をコードするものであり、コードする蛋白質の活性も不明であるので、本発明のアスペルギルス・オリザエ由来のキチナーゼとは異なる分子であると推測される。
試験例１．アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　ＪＣＭ　２０６７株由来のキチナーゼの粗酵素液の諸性質
実施例１．１）で得られた粗酵素液について、活性測定を行なった。
１）加水分解活性
実施例１　２）の方法にしたがって、還元糖の定量を行った。キトサン７Ｂを基質として測定した場合、上清中１ｍｌあたり４．２×１０^−３Ｕの酵素活性を有していた。キトサン７Ｂを基質としたときの酵素活性を１００％としたときの、キトサン１０Ｂ分解活性の相対値を表１１に示す。キトサン７Ｂ分解活性を有し、キトサン１０Ｂ分解活性がほとんどないことが判明した。
【０１１９】
【表１１】

２）ｐＨ活性
キトサン７Ｂ溶液１６０μｌおよび４００ｍＭ緩衝液１００μｌの混合液に、酵素液１４０μｌを加え撹拌して均一にし、３７℃で２０分間酵素反応を行った。酵素反応後の遊離還元糖の定量は実施例１　２）記載の方法にしたがった。緩衝液は次のものを用いた：ｐＨ３．６乃至ｐＨ５．９の場合、酢酸−酢酸ナトリウム緩衝液：ｐＨ　５．９乃至ｐＨ８．２の場合、３−（Ｎ−Ｍｏｒｐｈｏｌｉｎｅ）　ｐｒｏｐａｎｅｓｕｌｆｏｎｉｃ　　ａｃｉｄ（以下「ＭＯＰＳ」と記す）−炭酸ナトリウム緩衝液：ｐＨ９．０乃至ｐＨ１０．２の場合、炭酸水素ナトリウム−炭酸ナトリウム緩衝液。最も活性が高かったｐＨ条件での加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図５に記載した。至適ｐＨはｐＨ５．０付近であった。
【０１２０】
３）培養上清中の酵素の温度活性
ｐＨ５．０の条件下において、種々の温度で加水分解活性を測定した。５分間予熱した実施例１で作製したキトサン７Ｂ溶液１６０μｌおよび４００ｍＭ酢酸緩衝液（ｐＨ５．０）１００μｌの混合液に、酵素液１４０μｌを加え撹拌して均一にし、１０分間酵素反応を行った。酵素反応後の遊離還元糖の定量は実施例１にしたがった。最も高い活性を示した温度条件で測定された加水分解活性を１００％とし、各温度における加水分解活性を相対値として図６にまとめた。
【０１２１】
４）培養上清中の酵素の温度安定性
培養上清中の酵素を種々の温度で３０分間処理した後、その加水分解活性を測定した。あらかじめ処理温度に保持した４００ｍＭ酢酸緩衝液（ｐＨ５．０）２００μｌに、酵素液２８０μｌを加え撹拌して均一にし、３０分間保温した。実施例１で作製したキトサン７Ｂ溶液１６０μｌに加温処理した酵素液２４０μｌを加え、３７℃で２０分間酵素反応を行った。遊離還元糖の定量は実施例１にしたがった。無処理（０℃保温）群の酵素および緩衝液の混合液が有する加水分解活性を１００％とし、各温度における加水分解活性を相対値として図７にまとめた。
【０１２２】
５）培養上清中の酵素のｐＨ安定性
培養上清４００μｌに、以下に述べる各ｐＨの４００ｍＭ緩衝液７０μｌ及び水９０μｌを添加し、３７℃にて１時間保温した。緩衝液は次のものを用いた：ｐＨ３．９乃至ｐＨ７．３の場合、酢酸−酢酸ナトリウム緩衝液：ｐＨ６．５乃至ｐＨ８．６の場合、ＭＯＰＳ−炭酸ナトリウム緩衝液：ｐＨ９．３乃至ｐＨ１０．４の場合、炭酸水素ナトリウム−炭酸ナトリウム緩衝液：ｐＨ１０．０乃至ｐＨ１１．４の場合、リン酸水素２ナトリウム−水酸化ナトリウム緩衝液。４００ｍＭ酢酸緩衝液（ｐＨ５．０）１００μｌおよび実施例１　２）記載の方法で作製したキトサン７Ｂ溶液１６０μｌの混合液に、加温した酵素混合液１４０μｌを加え撹拌して均一にし、３７℃で２０分間酵素反応を行った。遊離還元糖の定量は実施例１にしたがった。無処理（０℃保温）群の酵素および緩衝液の混合液が有する加水分解活性を１００％とし、各ｐＨにおける加水分解活性を相対値として図８にまとめた。
【０１２３】
６）培養上清中の酵素の基質選択性
次に、ｐＨ５．０における基質選択性を測定した。実施例２で調製した培養上清１０ｍｌを１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）１，０００　ｍｌに対して１２時間ごとに３回透析したものを酵素液として用いた。０．２５％　ｗｔ／ｖの基質溶液１６０μｌおよび４００ｍＭ酢酸緩衝液（ｐＨ５．０）および水１６０μｌの混合液に酵素液５０μｌを加え撹拌して均一にし反応を開始し、３７℃で保温して１０分間酵素反応を行った。酵素反応後の遊離還元糖の定量はシェールズ法（Ｉｍｏｔｏ，　Ｔ．　ａｎｄ　　Ｙａｇａｓｈｉｔａ，　　Ｋ．，　Ａｇｒｉｃ．　Ｂｉｏｌ．　　Ｃｈｅｍ．，　　３５，　１１５４（１９７１）参照）により行った。最大活性を示す基質を用いたときの加水分解活性を１００％としたときの、相対活性を表１２に示す。
【０１２４】
【表１２】
――――――――――――――――――――
基質　　　　　　　　　相対活性（％）
――――――――――――――――――――
キトサン１０Ｂ　　　　１２．７
キトサン９Ｂ　　　　　７８．９
キトサン８Ｂ　　　　　８２．３
キトサン７Ｂ　　　　　１００
グルコールキトサン　　１７．７
グルコールキチン　　　５２．５
ＣＭ−セルロース　　　　２．２
リケナン　　　　　　　３１．６
――――――――――――――――――――
試験例２．アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　　ＪＣＭ２０６７株由来の粗精製キチナーゼの諸性質
実施例１．２）で得られたアスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　　ＪＣＭ　２０６７株由来の粗精製キチナーゼの諸性質を調べた。
１）粗精製画分中の酵素が有する加水分解活性のｐＨ活性
実施例１で作製したキトサン７Ｂ溶液１６０μｌおよび４００ｍＭ緩衝液１００μｌの混合液に、酵素液１４０μｌを加え撹拌して均一にし反応を開始し、３７℃で保温して２０分間酵素反応を行った。酵素反応後の遊離還元糖の定量は実施例１にしたがった。緩衝液は次のものを用いた：ｐＨ３．３乃至ｐＨ５．９の場合、酢酸−酢酸ナトリウム緩衝液：ｐＨ５．９乃至ｐＨ８．５の場合、ＭＯＰＳ−炭酸ナトリウム緩衝液：ｐＨ９．２乃至ｐＨ１０．３の場合、炭酸水素ナトリウム−炭酸ナトリウム緩衝液。最も活性が高かったｐＨ条件での加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図９に記載した。至適ｐＨはｐＨ５．０付近であった。
【０１２５】
２）粗精製画分中の酵素が有する加水分解活性の温度活性
ｐＨ５．０の条件下において、種々の温度で加水分解活性を測定した。あらかじめ処理温度に保持した実施例１で作製したキトサン７Ｂ溶液１６０μｌおよび４００ｍＭ酢酸緩衝液（ｐＨ　５．０）１００μｌの混合液に、酵素液１４０μｌを加え撹拌して均一にし、１０分間酵素反応を行った。酵素反応後の遊離還元糖の定量は実施例１にしたがった。最も高い活性を示した温度条件で測定された加水分解活性を１００％とし、各温度における加水分解活性を相対値として図１０にまとめた。
【０１２６】
３）粗精製画分中の酵素の温度安定性
粗精製画分中の酵素を種々の温度で３０分間処理した後、その加水分解活性を測定した。処理温度に保持した、４００ｍＭ酢酸緩衝液（ｐＨ５．０）２００μｌおよび酵素液２８０μｌを加え撹拌して均一にし、３０分間保温した。実施例１で作製したキトサン７Ｂ溶液１６０μｌに加温処理した酵素液２４０μｌを加え、３７℃で２０分間酵素反応を行った。遊離還元糖の定量は実施例１にしたがった。無処理（０℃保温）群の酵素および緩衝液の混合液が有する加水分解活性を１００％とし、各温度における加水分解活性を相対値として図１１にまとめた。
【０１２７】
４）粗精製画分中の酵素のｐＨ安定性
粗精製画分中の酵素４００μｌに、以下に述べる各ｐＨの４００ｍＭ緩衝液１４０μｌおよび水２０μｌを添加し、３７℃にて１時間保温した。緩衝液は次のものを用いた：ｐＨ３．１乃至ｐＨ６．１の場合、酢酸−酢酸ナトリウム緩衝液：ｐＨ６．１乃至ｐＨ８．４の場合、ＭＯＰＳ−炭酸ナトリウム緩衝液：ｐＨ９．０乃至ｐＨ１０．３の場合、炭酸水素ナトリウム−炭酸ナトリウム緩衝液：ｐＨ１０．４乃至ｐＨ１１．４の場合、リン酸水素２ナトリウム−水酸化ナトリウム緩衝液。４００ｍＭ酢酸緩衝液（ｐＨ　５．０）　１００μｌおよび実施例１で作製したキトサン７Ｂ溶液１６０μｌ混合液に、加温した酵素混合液１４０μｌを加え撹拌して均一にし、３７℃で２０分間酵素反応を行った。遊離還元糖の定量は実施例１にしたがった。無処理（０℃保温）群の酵素および緩衝液の混合液が有する加水分解活性を１００％とし、各温度における加水分解活性を相対値として図１２にまとめた。
試験例３．アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　　ＪＣＭ２０６７株由来の精製キチナーゼの諸性質
実施例１．３）で得られたアスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　　Ｏｈａｒａ　　ＪＣＭ　２０６７株由来の精製キチナーゼの諸性質を調べた。
１）精製酵素が有する加水分解活性のｐＨ活性
実施例１　２）記載の方法によって作製したキトサン７Ｂ溶液１６０μｌおよび４００ｍＭ緩衝液１００μｌおよび水９０μｌの混合液に、酵素液５０μｌを加え撹拌して均一にし反応を開始し、３７℃で保温して４０分間酵素反応を行った。酵素反応後の遊離還元糖の定量は実施例１　２）記載の方法にしたがった。緩衝液は次のものを用いた：ｐＨ３．３乃至ｐＨ５．９の場合、酢酸−酢酸ナトリウム緩衝液：ｐＨ５．９乃至ｐＨ８．４の場合、ＭＯＰＳ−炭酸ナトリウム緩衝液：ｐＨ９．３乃至ｐＨ１０．４の場合、炭酸水素ナトリウム−炭酸ナトリウム緩衝液。最も活性が高かったｐＨ条件での加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図１３に記載した。至適ｐＨはｐＨ５．５付近であった。
また、０．２５％　ｗｔ／ｖのグリコールキチン溶液２４０μｌおよび４００ｍＭ緩衝液１５０μｌ及び水１３５μｌの混合液に、酵素液７５μｌを加え撹拌して均一にし反応を開始し、３７℃で保温して３０分間酵素反応を行った。酵素反応後の遊離還元糖の定量はシェールズ法（Ｉｍｏｔｏ，　Ｔ．　ａｎｄ　　Ｙａｇａｓｈｉｔａ，　　Ｋ．，　Ａｇｒｉｃ．　Ｂｉｏｌ．　　Ｃｈｅｍ．，　　３５，　１１５４（１９７１）参照）により行った。緩衝液は次のものを用いた：ｐＨ３．３乃至ｐＨ６．０の場合、酢酸−酢酸ナトリウム緩衝液：ｐＨ６．３乃至ｐＨ７．８の場合、リン酸水素１ナトリウム−リン酸水素２ナトリウム緩衝液：ｐＨ７．３乃至ｐＨ８．７の場合、トリス・塩酸緩衝液：ｐＨ９．３乃至ｐＨ１０．４の場合、炭酸水素ナトリウム−炭酸ナトリウム緩衝液：ｐＨ１０．６乃至ｐＨ１１．５の場合、リン酸水素２ナトリウム−水酸化ナトリウム緩衝液。最も活性が高かったｐＨ条件での加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図１４に記載した。至適ｐＨはｐＨ１０付近であった。
【０１２８】
２）精製酵素が有する加水分解活性の温度活性
ｐＨ５．５の条件下において、種々の温度で精製酵素の加水分解活性を測定した。あらかじめ処理温度に保持した１）と同様に調製したキトサン７Ｂ溶液１６０μｌおよび４００　ｍＭ酢酸緩衝液（ｐＨ５．５）１００μｌおよび水１００μｌの混合液に、酵素液４０μｌを加え撹拌して均一にし、１０分間酵素反応を行った。酵素反応後の遊離還元糖の定量は実施例１にしたがった。最も高い活性を示した温度条件で測定された精製酵素の加水分解活性を１００％とし、各温度における精製酵素の加水分解活性を相対値として図１５にまとめた。図１５に示す通り、精製酵素の加水分解活性の最適温度は、ｐＨ　５．５において６０℃であった。
【０１２９】
３）精製酵素の温度安定性
精製酵素を種々の温度で３０分間処理した後、その加水分解活性を測定した。あらかじめ処理温度に保持した４００ｍＭ酢酸緩衝液（ｐＨ　５．５）　２００μｌおよび水１８０μｌの混合液に、酵素液１００μｌを加え撹拌して均一にし、３０分間保温した。１）と同様に調製したキトサン７Ｂ溶液１６０μｌに加温処理した酵素液２４０μｌを加え、３７℃で４０分間酵素反応を行った。遊離還元糖の定量は実施例１にしたがった。無処理（０℃保温）群の精製酵素および緩衝液の混合液が有する加水分解活性を１００％とし、各温度における精製酵素の加水分解活性を相対値として図１６にまとめた。図１６に示す通り、精製酵素は４５℃以下で安定であった。
【０１３０】
４）精製酵素のｐＨ安定性
精製酵素１５０μｌに、以下に述べる各ｐＨの４００ｍＭ緩衝液５０μｌを添加し、３７℃にて１時間保温した。緩衝液は次のものを用いた：ｐＨ３．２乃至ｐＨ６．２の場合、酢酸−酢酸ナトリウム緩衝液：ｐＨ６．１乃至ｐＨ８．２の場合、ＭＯＰＳ−炭酸ナトリウム緩衝液：ｐＨ８．８乃至ｐＨ１０．１の場合、炭酸水素ナトリウム−炭酸ナトリウム緩衝液：ｐＨ９．６乃至ｐＨ１１．３の場合、リン酸水素２ナトリウム−水酸化ナトリウム緩衝液。４００ｍＭ酢酸緩衝液（ｐＨ５．５）１００μｌおよび実施例１で作製したキトサン７Ｂ溶液１６０μｌ及び水７０μｌの混合液に、加温した酵素混合液７０μｌを加え撹拌して均一にし、３７℃で４０分間酵素反応を行い、遊離還元糖の定量をした。無処理（０℃保温）群の精製酵素および緩衝液の混合液が有する加水分解活性を１００％とし、各温度における精製酵素の加水分解活性を相対値として図１７にまとめた。図１７に示す通り、精製酵素はｐＨ５乃至ｐＨ９．５で安定であった。
【０１３１】
５）精製酵素の基質特異性
精製酵素の種々の基質に対する加水分解活性を実施例５の方法にしたがって測定した。但し酵素反応は、ｐＨ５．５及びｐＨ１０．０で行なった。キトサン７Ｂに対する精製酵素の加水分解活性を１００％としたときの、他の各基質に対する精製酵素の加水分解活性を相対値として表１３に記載した。
【０１３２】
【表１３】

表１１に示す通り、精製酵素はキトサン７Ｂに対して最も加水分解活性が高く、アセチル化度が小さくなるにつれて加水分解活性は低下した。また、精製酵素はキチン加水分解活性を有し、セルラーゼ加水分解活性は見られなかった。
試験例４．アスペルギルス・ジャポニクス　ＳＡＮＫ１９２８８株由来のキチナーゼ粗酵素液の諸性質
１）実施例１　２）記載の方法にしたがって、還元糖の定量を行った。キトサン７Ｂを基質として測定した場合、上清中１ｍｌあたり３．４×１０^−３Ｕの酵素活性を有していた。キトサン７Ｂを基質としたときの酵素活性を１００％としたときの、キトサン１０Ｂ分解活性の相対値を表１４に示す。
【０１３３】
【表１４】

キトサン７Ｂ分解活性を有し、キトサン１０Ｂ分解活性がほとんどないことから、上清中にはキチナーゼが生産されていることが判明した。
【０１３４】
２）粗酵素液のｐＨ活性
実施例１　２）記載の方法にしたがって調製したキトサン７Ｂ溶液１６０μｌおよび４００ｍＭ緩衝液１００μｌの混合液に粗酵素液１４０μｌを加え撹拌して均一にし、３７℃で保温して２０分間酵素反応を行い、酵素反応後の遊離還元糖の定量を行なった。緩衝液は次のものを用いた：ｐＨ３．７乃至ｐＨ６．０の場合、酢酸−酢酸ナトリウム緩衝液：ｐＨ６．０乃至ｐＨ８．５の場合、ＭＯＰＳ−炭酸ナトリウム緩衝液：ｐＨ９．３乃至ｐＨ１０．４の場合、炭酸水素ナトリウム−炭酸ナトリウム緩衝液。最も活性が高かったｐＨ条件での加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図１８に記載した。至適ｐＨはｐＨ４．０付近であった。
【０１３５】
３）粗酵素の基質選択性
ｐＨ４．０における基質選択性を測定した。実施例２で得られた調製した培養上清１０ｍｌを１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）１，０００　ｍｌに対して１２時間ごとに３回透析したものを酵素液として用いた。０．２５％　ｗｔ／ｖの基質溶液１６０μｌおよび４００ｍＭ酢酸緩衝液（ｐＨ４．０）および水１６０μｌの混合液に酵素液５０μｌを加え撹拌して均一にし反応を開始し、３７℃で保温して１０分間酵素反応を行った。酵素反応後の遊離還元糖の定量はシェールズ法（Ｉｍｏｔｏ，　Ｔ．　ａｎｄ　　Ｙａｇａｓｈｉｔａ，　　Ｋ．，　Ａｇｒｉｃ．　Ｂｉｏｌ．　　Ｃｈｅｍ．，　　３５，　１１５４（１９７１）参照）により行った。最大活性を示す基質を用いたときの加水分解活性を１００％としたときの、相対活性を表１５に示す。
【０１３６】
【表１５】
―――――――――――――――――――――
基質　　　　　　　　　　　相対活性（％）
―――――――――――――――――――――
キトサン１０Ｂ　　　　　　　８．２
キトサン９Ｂ　　　　　　　６７．６
キトサン８Ｂ　　　　　　　７４．４
キトサン７Ｂ　　　　　　１００
グリコールキトサン　　　　　８．２
グリコールキチン　　　　　２３．３
ＣＭ−セルロース　　　　　１１．４
リケナン　　　　　　　　　２５．３
―――――――――――――――――――――
試験例５．アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来のキチナーゼ粗酵素液の諸性質
１）加水分解活性
実施例１　２）に記載の方法にしたがって還元糖の定量を行った。キトサン７Ｂを基質として測定した場合、上清中１ｍｌあたり２．６×１０^−３Ｕの酵素活性を有していた。キトサン７Ｂを基質としたときの酵素活性を１００％としたときの、キトサン１０Ｂ分解活性の相対値を表１６に示す。
【０１３７】
【表１６】

キトサン７Ｂ分解活性を有し、キトサン１０Ｂ分解活性がほとんどないことから、上清中にはキチナーゼが生産されていることが判明した。
【０１３８】
２）培養上清中の酵素のｐＨ活性
測定法は実施例６にしたがった。緩衝液は次のものを用いた：ｐＨ３．８乃至ｐＨ６．３の場合、酢酸−酢酸ナトリウム緩衝液：ｐＨ６．２乃至ｐＨ８．６の場合、ＭＯＰＳ−炭酸ナトリウム緩衝液：ｐＨ９．３乃至ｐＨ１０．４の場合、炭酸水素ナトリウム−炭酸ナトリウム緩衝液。最も活性が高かったｐＨ条件での加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図１９に記載した。至適ｐＨはｐＨ４．５付近であった。
【０１３９】
３）培養上清中の酵素の基質選択性
ｐＨ４．５における基質選択性を測定した。実施例４で調製した培養上清１０ｍｌを１０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）１，０００ｍｌに対して１２時間ごとに３回透析したものを酵素液として用いた。０．２５％ｗｔ／ｖの基質溶液１６０μｌおよび４００ｍＭ酢酸緩衝液（ｐＨ　４．５）および水１６０μｌの混合液に酵素液５０μｌを加え撹拌して均一にし反応を開始し、３７℃で保温して１０分間酵素反応を行った。酵素反応後の遊離還元糖の定量はシェールズ法（Ｉｍｏｔｏ，　Ｔ．　ａｎｄ　　Ｙａｇａｓｈｉｔａ，　　Ｋ．，　Ａｇｒｉｃ．　Ｂｉｏｌ．　　Ｃｈｅｍ．，　　３５，　１１５４（１９７１）参照）により行った。最大活性を示す基質を用いたときの加水分解活性を１００％としたときの、相対活性を表１７に示す。
【０１４０】
【表１７】
――――――――――――――――――
基質　　　　　　　　相対活性（％）
――――――――――――――――――
キトサン１０Ｂ　　　　　０
キトサン９Ｂ　　　　　６７．７
キトサン８Ｂ　　　　　７１．６
キトサン７Ｂ　　　　１００
グルコールキトサン　　　２．９
グリコールキチン　　　８１．９
ＣＭ−セルロース　　　　０
リケナン　　　　　　　１１．９
――――――――――――――――――
試験例６．　各低分子キトサン塩酸塩の水に対する溶解性
実施例４〜６にて調製した各低分子キトサン塩酸塩１００ｍｇを水１０ｍｌに溶かし、ｐＨを測定した。この溶液に１Ｎ苛性ソーダ水を少しずつ加え、溶液が白濁してコロイドが析出するｐＨを測定した。結果を表１８に示す。
【０１４１】
【表１８】

試験例７．　高分子キトサン塩酸塩の調製
キトサン９Ｂ、１ｇを２００ｍｌの水に懸濁し、濃塩酸でｐＨ２．５に合わせて、完全に溶解した。これを水に対して透析後、凍結乾燥を行い、５３７ｍｇの白色綿状固体として、高分子キトサン９Ｂ塩酸塩が得られた。高分子キトサン１０Ｂ及び８Ｂおよび７Ｂ塩酸塩も同じ方法で調製した。各高分子キトサン塩酸塩１００ｍｇを蒸留水５０ｍｌに溶解し、キトサナーゼ２０Ｕ（和光純薬（株）製）を加えて室温で３時間攪拌した。反応溶液を凍結乾燥し、乾燥固体をＤ_２Ｏに溶かして^１Ｈ−ＮＭＲを測定した。
アセチル基の３Ｈとアセチル基以外の、水酸基を除く７Ｈとの積分比から、アセチル化度を算出した。結果を表１９に示す。
【０１４２】
【表１９】

試験例８．低分子キトサンの抗菌活性
被検菌としてスタフィロコッカス・アウレウス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ　ａｕｒｅｕｓ）　２０９Ｐ　ＪＣ−１株及びシュードモナス・アエルギノーサ（Ｐｓｅｕｄｏｍｏｎａｓ　　ａｅｒｕｇｉｎｏｓａ）ＰＡＯ１株を被検菌とし、試験例７で作製した高分子キトサン塩酸塩並びに実施例４、実施例５および実施例６にて各種アスペルギルス族由来のキチナーゼを用いて調製した低分子キトサン塩酸塩のＭＩＣを測定した。結果は表１８に示した。
【０１４３】
使用培地
Ｍｕｅｌｌｅｒ　　Ｈｉｎｔｏｎ　　Ｂｒｏｔｈ（Ｄｉｆｃｏ（株）製）を用い、いずれもｐＨ６．５に調整した。
【０１４４】
試料菌液の調製
試験に使用する菌を上記培地で一晩培養し、菌数が２×１０^６になるように同培地で希釈した。
検体の調製
各低分子キトサン塩酸塩２０ｍｇを蒸留滅菌水に溶かし、１０ｍｌとした。
【０１４５】
試験方法
あらかじめ蒸留滅菌水を希釈液とし、検体溶液の倍数希釈系列を作製しておき、これに上記培地５０μｌ及び試験菌液５０μｌを加え、３７℃で１８時間培養し、ＭＩＣを判定した。結果は表２０に示した。
【０１４６】
【表２０】

試験例９．アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼの諸性質
実施例２．２）で得られたアスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼの諸性質を調べた。
【０１４７】
１）アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼが有する加水分解活性のｐＨ活性
アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼが有する加水分解活性のｐＨ活性を、試験例３　１）に記載の方法により測定した。最も活性が高かったｐＨ条件での加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図２０に記載した。至適ｐＨはｐＨ５．０付近であった。
【０１４８】
２）アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼが有する加水分解活性の温度活性
アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼが有する加水分解活性の温度活性を、試験例３　２）に記載の方法により測定した。最も活性が高かった温度条件での加水分解活性を１００％とし、各温度における酵素の加水分解活性を相対値として図２１にまとめた。至適温度はｐＨ５．０において６５℃であった。
【０１４９】
３）アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼの温度安定性
アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼの温度安定性を、試験例３　３）に記載の方法により測定した。無処置（０℃保温）群の該精製キチナーゼおよび緩衝液の混合液が有する加水分解活性を１００％とし、各温度における酵素の加水分解活性を相対値として図２２にまとめた。該精製キチナーゼは５０℃以下で安定であった。
【０１５０】
４）アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼのｐＨ安定性
アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼのｐＨ安定性を、試験例３　４）に記載の方法により測定した。無処置（０℃保温）群の該精製キチナーゼおよび緩衝液の混合液が有する加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図２３にまとめた。該精製キチナーゼはｐＨ４乃至ｐＨ９．５で安定であった。
【０１５１】
５）アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼの基質特異性
アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼの基質特異性を、試験例３　５）に記載の方法により測定した。キトサン７Ｂに対する、ｐＨ５．０、３７℃、１０分間の条件における該精製キチナーゼの加水分解活性を１００％としたときの、他の各基質に対する該精製キチナーゼの加水分解活性を相対値として表２１に示した。
【０１５２】
【表２１】
――――――――――――――――――
基質　　　　　　　　相対活性（％）
――――――――――――――――――
キトサン１０Ｂ　　　　　０
キトサン９Ｂ　　　　　５８．６
キトサン８Ｂ　　　　　７２．４
キトサン７Ｂ　　　　１００
グルコールキトサン　　　０
グリコールキチン　　　２８．３
ＣＭ−セルロース　　　　０
リケナン　　　　　　　　４．２
――――――――――――――――――
表２１に示すとおり、アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼはキトサン７Ｂに対して最も加水分解活性が高く、アセチル化度が小さくなるにつれて加水分解活性は低下した。また、該精製キチナーゼはキチン加水分解活性を有し、セルラーゼ加水分解活性は見られなかった。
【０１５３】
６）アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼの部分アミノ酸配列の決定
アスペルギルス・ジャポニクス　ＳＡＮＫ　１９２８８株由来の精製キチナーゼの部分アミノ酸配列の決定を、実施例１．７）記載の方法でおこなった。分離した分解アミノ酸のうち、Ｂ　　ｂｕｆｆｅｒ濃度２８％程度、３０％程度、３５％程度の３つのピークを分取した。この３つについてアミノ酸配列解析装置（Ｐｒｏｃｉｓｅ　　ｃＬＣ、Ａｐｐｌｉｅｄ　　Ｂｉｏｓｙｓｔｅｍ社製）で配列を解析した。結果をＮ末端側から記す。
Ｈｉｓ−Ｔｙｒ−Ｐｒｏ−Ｔｈｒ−Ａｓｐ−Ｓｅｒ−Ｔｒｐ−Ａｓｎ−Ａｓｐ−Ｖａｌ−Ｇｌｙ−Ｔｈｒ−Ａｓｎ−Ｖａｌ−Ｔｙｒ（配列表の配列番号１１）
Ａｌａ−Ｐｈｅ−Ｔｈｒ−Ａｓｎ−Ｔｈｒ−Ａｓｐ−Ｇｌｙ−Ｐｒｏ−Ｇｌｙ−Ｔｈｒ−Ａｌａ−Ｐｈｅ−Ｓｅｒ−Ｇｌｙ−Ｖａｌ（配列表の配列番号１２）
Ｌｅｕ−Ｓｅｒ−Ｇｌｎ−Ｍｅｔ−Ｔｈｒ−Ｐｒｏ−Ｔｙｒ−Ｌｅｕ−Ａｓｐ−Ｐｈｅ−Ｔｙｒ−Ａｓｎ−Ｌｅｕ−Ｍｅｔ−Ａｌａ−Ｔｙｒ−Ａｓｐ−Ｔｙｒ−Ａｌａ−Ｇｌｙ（配列表の配列番号１３）
試験例１０．アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼの諸性質
実施例３．２）で得られたアスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼの諸性質を調べた。
【０１５４】
１）アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼが有する加水分解活性のｐＨ活性
アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼが有する加水分解活性のｐＨ活性を、試験例３．１）に記載の方法により測定した。最も活性が高かったｐＨ条件での加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図２４に記載した。至適ｐＨはｐＨ５．５およびｐＨ９．０付近であった。
【０１５５】
２）アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼが有する加水分解活性の温度活性
アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼはアスペルギルス・オリザエおよびアスペルギルス・ジャポニクス由来のキチナーゼと異なり、至適ｐＨが２点存在する（試験例１０　１）参照）ため、アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼの温度活性はこの２点のｐＨにおいて、試験例３　２）に記載の方法により測定した。最も活性が高かった温度条件での加水分解活性を１００％とし、各温度における酵素の加水分解活性を相対値として図２５（ｐＨ５．５における温度活性）および図２６（ｐＨ９．０における温度活性）にまとめた。至適温度はｐＨ５．５において６０℃、ｐＨ９．０において３５℃であった。
【０１５６】
３）アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼの温度安定性
アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼはアスペルギルス・オリザエおよびアスペルギルス・ジャポニクス由来のキチナーゼと異なり、至適ｐＨが２点存在する（試験例１０　１）参照）ため、アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼの温度安定性はこの２点のｐＨにおいて、試験例３．３）に記載の方法により測定した。無処置（０℃保温）群の該精製キチナーゼおよび緩衝液の混合液が有する加水分解活性を１００％とし、各温度における酵素の加水分解活性を相対値として図２７（ｐＨ５．５における温度安定性）および図２８（ｐＨ９．０における温度安定性）にまとめた。該精製キチナーゼはいずれのｐＨにおいても４０℃以下で安定であった。
【０１５７】
４）アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼのｐＨ安定性
アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼのｐＨ安定性を試験例３．４）に記載の方法により測定した。無処置（０℃保温）群の該精製キチナーゼおよび緩衝液の混合液が有する加水分解活性を１００％とし、各ｐＨにおける酵素の加水分解活性を相対値として図２９にまとめた。該精製キチナーゼはｐＨ４．５乃至ｐＨ１０で安定であった。
【０１５８】
５）アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼの基質特異性
アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼの基質特異性を試験例３．５）に記載の方法により測定した。キトサン８Ｂに対する、ｐＨ５．０、３７℃、１０分間の条件における該精製キチナーゼの加水分解活性を１００％としたときの、他の各基質に対する該精製キチナーゼの加水分解活性を相対値として表２２に示した。
【０１５９】
【表２２】
――――――――――――――――――
基質　　　　　　　　相対活性（％）
――――――――――――――――――
キトサン１０Ｂ　　　　１４．３
キトサン９Ｂ　　　　　７３．６
キトサン８Ｂ　　　　１００
キトサン７Ｂ　　　　　９７．４
グルコールキトサン　　　０
グリコールキチン　　　２３．８
ＣＭ−セルロース　　　　０
リケナン　　　　　　　０
――――――――――――――――――
表２２に示すとおり、アスペルギルス・ソジャエ　ＳＡＮＫ　２２３８８株由来の精製キチナーゼはキトサン７Ｂおよび８Ｂに対して最も加水分解活性が高く、アセチル化度が小さくなるにつれて加水分解活性は低下した。また、該精製キチナーゼはキチン加水分解活性を有し、セルラーゼ加水分解活性は見られなかった。
製剤例１．５％キトサン軟膏製剤の作製
プロピレングリコール３．０ｇ、パラオキシ安息香酸メチル０．１ｇの精製水８０ｍｌ溶液を７０℃に加温し、実施例４にて調製した低分子キトサン９Ｂ塩酸塩５．０ｇを加えて溶解し、低分子キトサン９Ｂ溶液とした。ステアリン酸３．０ｇ、セタール１．０ｇ、モノステアリン酸グリセリン６．０ｇ、流動パラフィン５．０ｇ、パラオキシ安息香酸プロピル０．１ｇ、ステアリン酸ポリオキシル４０　２．５ｇを７０℃にて加温溶解後、同温度で上記の低分子キトサン９Ｂ溶液を加え、精製水をくわえて全量を１００ｇとし、混合乳化した。乳化後攪拌しながら室温まで冷却し、白色の５％低分子キトサン９Ｂ塩酸塩クリーム（５％キトサン軟膏と呼ぶ）を１００ｇ得た。
試験例１０．５％キトサン軟膏の外傷治癒試験
ラット皮膚欠損傷モデルでの５％キトサン軟膏の有効性を予知する目的で外傷治癒試験を行なった。
１）試験材料
被験物質としては、製剤例１で調製した５％キトサン軟膏を用いた。対照薬としては、ユーパスタ軟膏（１００ｇ中に精製白糖７０ｇ、ポピドンヨード３ｇを含有、添加物としてポリエチレングリコール、濃グリセリン、ポリオキシエチレン［１６０］ポリオキシプロピレン［３０］グリコール、プルラン、ヨウ化カリウムを含有、製造番号　ＢＡ１Ｓ、興和株式会社製）を使用した。試験に用いる動物としては、６週齢のＳｐｒａｇｕｅ−Ｄａｗｌｅｙ（ＳＤ）系雄性ラット（日本エスエルシー株式会社）を購入して使用した。動物は平均温度２３℃、平均湿度５５％の環境制御飼育装置（日本クレア株式会社）で固形飼料（マウス・ラット飼育用ＦＲ−２、株式会社船橋農場）および水道水を与え、照明時間７時−１９時の条件下で６日間馴化飼育管理を行い実験に用いた。
２）実験方法
７週齢のＳＤ系雄性ラット（体重１５７．６ｇ乃至２３５．９ｇ）を１群５匹で６群用いた。ラットの背部被毛を電気バリカンで除毛した後、ＥＢＡエバクリーム（東京田辺製薬株式会社製）を塗布し、１５分後に洗浄して脱毛した。背部脱毛部位を７０％エタノールで消毒後、ペントバルビタール・ナトリウム（４０ｍｇ／ｋｇ，ｉ．ｐ．）麻酔下に円形ポンチ（内径１５ｍｍ）を用いて背部正中線で対称の打ち抜き創を２個所作製した。欠損傷作製２４時間後に無処置群、基剤群、被験物質群および対照薬群に各群５匹で群分けし、その後被験物質および対照薬を欠損傷部２箇所に、約０．１ｇ／部位、一日２回、１０日間塗布した。ラットは実験期間中、個別ケージで飼育した。効果の判定はノギスを用いて欠損傷部の長径と短径を測定した。
【０１６０】
治療過程における面積変化は、（測定日の長径×短径／欠損傷作製後の長径×短径）×１００の式で面積比（％）を求め、面積比から治療面積（５−１５日目）を、完治率は面積比が５％以下を完治例とし治療日数を算出し、表２３に示した。実験結果は平均値±標準偏差で表した。無処理群の１３．７±１．３日に対して、５％キトサン軟膏群は１２．６±１．９日と治療日数を短縮した。対照薬群では１３．６±０．４日と治療日数の改善はみられなかった。
【０１６１】
【表２３】ラット皮膚欠損傷モデルの治療日数
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿
被験物質　　　　　　　　　　　　治療日数
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿
無処置　　　　　　　　　　　　　１３．７±１．３
５％キトサン　　　　　　　　　　１２．６±１．９
対照薬　　　　　　　　　　　　　１３．６±０．４
＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿＿
【０１６２】
【発明の効果】
以上述べたように、本発明のキチナーゼを用いる事により、分子量１万以上で、中性条件でも可溶な、本発明の低分子キトサン製造することができる。更に本発明の低分子キトサンは優れた抗菌性および外傷治癒効果を示し、本発明の低分子キトサンを有効成分として含有する医薬組成物は、外傷、床ずれ、アトピー性皮膚炎などの治療剤として有用である。また、本発明のキチナーゼを用いて、高分子アセチル化キトサンを含有することにより高い粘度を示す試料を原料として、粘度の低い扱い易い試料を製造することができる。このような試料の製造方法は食品加工の過程で有用である。
【０１６３】
【配列表フリーワード】
配列番号６：キチナーゼ遺伝子プローブ
配列番号７：ＰＣＲプライマー　Ｆ２１
配列番号８：ＰＣＲプライマー　ＰＲ５０
配列番号９：ＰＣＲプライマー　ＢａｍＨＩ−ｃＤＮＡ
配列番号１０：ＰＣＲプライマー　ｃＤＮＡ−ＢａｍＨＩ
【０１６４】
【配列表】

【図面の簡単な説明】
【図１】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の精製キチナーゼの分子量
【図２】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来のキチナーゼを用いて作製した低分子キトサンの分子量分布
【図３】アスペルギルス・ジャポニクス　ＳＡＮＫ１９２８８株由来のキチナーゼを用いて作製した低分子キトサンの分子量分布
【図４】アスペルギルス・ソジャエ　ＳＡＮＫ２２３８８株の培養上清中のキチナーゼを用いて作製した低分子キトサンの分子量分布
【図５】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７株培養上清中のキチナーゼの活性とｐＨとの関係
【図６】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７株培養上清中のキチナーゼの活性と温度との関係
【図７】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７株培養上清中のキチナーゼの温度安定性
【図８】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７株培養上清中のキチナーゼのｐＨ安定性
【図９】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の粗精製キチナーゼの活性とｐＨとの関係
【図１０】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の粗精製キチナーゼの活性と温度との関係
【図１１】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の粗精製キチナーゼのｐＨ安定性
【図１２】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の粗精製キチナーゼの温度安定性
【図１３】キトサン７Ｂを反応基質としたときのアスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の精製キチナーゼの活性とｐＨとの関係
【図１４】グリコールキチンを反応基質としたときのアスペルギルス・オリザエｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　Ｏｈａｒａ　ＪＣＭ２０６７由来の精製キチナーゼの活性とｐＨとの関係
【図１５】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の精製キチナーゼの活性と温度との関係
【図１６】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の精製キチナーゼの温度安定性
【図１７】アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の精製キチナーゼのｐＨ安定性
【図１８】アスペルギルス・ジャポニクス　ＳＡＮＫ１９２８８株培養上清中のキチナーゼの活性とｐＨとの関係
【図１９】アスペルギルス・ソジャエ　ＳＡＮＫ２２３８８株の培養上清中のキチナーゼの活性とｐＨとの関係
【図２０】グリコールキチンを反応基質としたときのアスペルギルス・ジャポニクス　ＳＡＮＫ１９２８８株由来の精製キチナーゼの活性とｐＨとの関係
【図２１】アスペルギルス・ジャポニクス　ＳＡＮＫ１９２８８株由来の精製キチナーゼの活性と温度との関係
【図２２】アスペルギルス・ジャポニクス　ＳＡＮＫ１９２８８株由来の精製キチナーゼの温度安定性
【図２３】アスペルギルス・ジャポニクス　ＳＡＮＫ１９２８８株由来の精製キチナーゼのｐＨ安定性
【図２４】グリコールキチンを反応基質としたときのアスペルギルス・ソジャエ
ＳＡＮＫ２２３８８株由来の精製キチナーゼの活性とｐＨとの関係
【図２５】ｐＨ５．５の条件における、アスペルギルス・ソジャエ　ＳＡＮＫ２２３８８株由来の精製キチナーゼの活性と温度との関係
【図２６】ｐＨ９．０の条件における、アスペルギルス・ソジャエ　ＳＡＮＫ２２３８８株由来の精製キチナーゼの活性と温度との関係
【図２７】ｐＨ５．５の条件における、アスペルギルス・ソジャエ　ＳＡＮＫ２２３８８株由来の精製キチナーゼの温度安定性
【図２８】ｐＨ９．０の条件における、アスペルギルス・ソジャエ　ＳＡＮＫ２２３８８株由来の精製キチナーゼの温度安定性
【図２９】アスペルギルス・ソジャエ　ＳＡＮＫ２２３８８株由来の精製キチナーゼのｐＨ安定性
【図３０】キチナーゼのＳＤＳ電気泳動図（１２％アクリルアミドゲル、ＣＢＢ染色、レーンＭ：マーカー、レーン１：Ｈｉｓ融合リコンビナントキチナーゼ、レーン２：アスペルギルス・オリザエ　ｖａｒ．　ｓｐｏｒｏｆｌａｖｕｓ　ＯｈａｒａＪＣＭ２０６７由来の精製キチナーゼ）[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a chitosan that is separated and purified from at least one culture of Aspergillus oryzae, Aspergillus japonics, Aspergillus sojae, and Aspergillus sojae. A DNA encoding the chitosan-degrading enzyme; a recombinant plasmid into which the DNA has been inserted; a host cell transformed with the recombinant plasmid; a method for producing the chitosan-degrading enzyme; A method for producing chitosan and a salt thereof, a low-molecular-weight chitosan and a salt thereof produced by the method, a pharmaceutical composition and a food containing the low-molecular-weight chitosan and a salt thereof, and a method for producing a sample using the chitosan-degrading enzyme; The present invention relates to a sample manufactured by the method.
[0002]
[Prior art]
Chitin (β-1,4-poly-N-acetylglucosamine) is a polysaccharide that exists in a very large amount in nature, and is mainly extracted and produced from crustaceans such as crabs and shrimps and squid molluscs on an industrial scale. . Chitosan (β-1,4-polyglucosamine) is a high molecular weight polysaccharide having an amino group, which is obtained by treating chitin with concentrated alkaline water, and is known to have various physiological activities (non-polysaccharide). See Patent Document 1.). Above all, it is known that chitosan has a strong antibacterial action as a substance derived from a natural product, and that its safety is much higher than that of a synthetic antibacterial agent (see Non-Patent Document 2). In particular, Staphylococcus aureus including Staphylococcus aureus (hereinafter referred to as “MRSA”) including methicillin cephem-resistant Staphylococcus aureus (Non-Patent Documents 3 to 8) and Escherichia coli ( It is known to be effective against bacteria such as Escherichia coli (see Non-Patent Document 9) and Pseudomonas aeruginosa (see Non-Patent Documents 10 and 11).
[0003]
Chitosan, which is produced by subjecting chitin extracted from crustaceans such as crabs and shrimp and squid molluscs to concentrated alkali treatment, is a high-molecular-weight polysaccharide with a molecular weight of 1,000,000 or more. Insoluble in water under alkaline conditions. And, when it is not soluble in water, the physiological activity of chitosan cannot be sufficiently exhibited. Further, when processing food materials including crustaceans, there is also a problem that the viscosity is extremely high due to these high molecular weight polysaccharides, and the subsequent processing is troublesome.
[0004]
Therefore, in order to increase the solubility in water, attempts have been made to derivatize high molecular weight chitosan (Non-Patent Document 12) or to reduce the molecular weight without impairing the biological activity of high molecular weight chitosan.
[0005]
When derivatization is performed, the structure may be different from that of a natural product, so that it may have properties different from that of natural chitosan, and safety test data or the like may be required for use. Furthermore, there is a problem that the removal and treatment of the organic solvent used in the derivatization reaction raises the production cost.
[0006]
Therefore, it is considered that in order to enhance the solubility in water, it is not a method of derivatization but a lower molecular weight is desirable. It is known that the antibacterial activity of chitosan is exerted not only by high molecular weight chitosan having a molecular weight of 1,000,000 or more but also by low molecular weight chitosan having a molecular weight of 10,000 or more (Non-Patent Document 13).
[0007]
On the other hand, it has been reported that chitosan having a molecular weight of 10,000 or less has side effects such as skin irritation (Patent Document 1), and a method for producing low molecular weight chitosan having a molecular weight of 10,000 or more has been studied.
[0008]
As a method for producing low molecular weight chitosan, hydrolysis of high molecular weight chitosan by a chemical or enzymatic method has been studied. Examples of the method for chemically producing low molecular weight chitosan include a method of treating with an oxidizing agent such as peracetic acid (Patent Document 2 and Patent Document 3), a method of ultrasonic treatment (Non-patent Document 14), and the like. However, it is known that a large amount of chitosan having a molecular weight of 500 to 30,000 is produced by a method of treating with an oxidizing agent, and the ratio of chitosan having a molecular weight of 10,000 or more is unknown. In addition, it is considered that it is difficult to increase the size of the apparatus by the ultrasonic treatment method, which is not suitable for mass production. Examples of the enzymatic production method of low molecular weight chitosan include a method using chitosanase (Patent Documents 4 and 5), a method using chitinase (Non-Patent Document 15), and the like.
[0009]
Examples of the bacterial chitinase include Bacillus genus, Corynebacterium genus (see Non-Patent Document 16), Cytophaga genus, and Achromobacter hyteropticum: Patent Document. 17), chitinase produced by the genus Flavobacterium and Micrococcus colpogenes (see Non-Patent Document 17), and chitinase A (GenBank Noes Acc) produced by Alteromonas sp. .D13762) are known. As a chitinase produced by actinomycetes, a chitinase produced by the genera Streptomyces, Nocardiopsis and Actinomyces is known (Non-Patent Document 18). As a chitinase produced by a filamentous fungus, a chitinase produced by the genus Aspergillus, the genus Rhizopus, the genus Talaromyces, the genus Trichoderma and the genus Penicillium is known. Aspergillus nidulans (Aspergillus nidulans), Aspergillus niger (Aspergillus niger), Aspergillus niger, and Aspergillus candidos (Aspergillus candius and non-patent literature, see Aspergillus candidus and Aspergillus candius). It has been known. Examples of the enzyme produced by yeast include a chitinase produced by Saccharomyces cerevisiae, and preferably, a chitinase produced by Saccharomyces cerevisiae (GenBank Accession No. M74070) and ORF D9487. Accession No. U28373).
[0010]
Thus, chitinases derived from many organisms are known, but there is no report that typical fermented foods, Aspergillus japonicus and Aspergillus sojae, produce chitinase. Similarly, regarding Aspergillus oryzae, which is also used for producing miso and soy sauce sake, etc., there has been a report that a gene considered to encode chitinase was presented at a conference (see Non-Patent Document 20). However, the biological activity of the protein encoded by the gene has not been reported, and its nucleotide sequence and amino acid sequence have not been confirmed in public databases or the like until recently.
[0011]
On the other hand, the method for producing low-molecular-weight chitosan using chitosanase also has a problem that the reaction control for obtaining low-molecular-weight chitosan having the same molecular weight is always complicated.
[0012]
[Patent Document 1]
JP 2000-169327 A
[Patent Document 2]
Japanese Patent No. 3076212
[Patent Document 3]
JP-A-5-65368
[Patent Document 4]
Japanese Patent No. 2763112
[Patent Document 5]
JP-A-11-322809
[Non-patent document 1]
"Chitin Chitosan Handbook", 1995, Gihodo, p. 302-376
[Non-patent document 2]
"Utilization of chitin and chitosan", 1998, Special Business Volume 46, No. 28, p. 106-111
[Non-Patent Document 3]
"Processing Technology", 1998, 33 volumes, p. 512-515
[Non-patent document 4]
"Academic report of Faculty of Agriculture, Meijo University", 1998, vol. 34, p. 25-34
[Non-Patent Document 5]
"Fiber Science", 1993, 35, p. 37-39
[Non-Patent Document 6]
"Osaka Prefectural Institute of Public Health Research Report Pharmaceutical Affairs Guide No. 29", 1995, p. 7-12
[Non-Patent Document 7]
"Processing Technology", 1997, Volume 32, p. 339-343
[Non-Patent Document 8]
"Processing Technology", 1998, 33 volumes, p. 530-532
[Non-Patent Document 9]
(Tokura, S et al.) "Macromolecular Symposia", 1997, Vol. 120, p. 1-9
[Non-Patent Document 10]
See, Loke, WK et al., "Journal of Biomedical Materials Research," 2000, vol. 53, p. 8-17
[Non-Patent Document 11]
Kim, HJ et al., "Journal of Biomaterials Science, Polymer Edition," 1999, 10, pp. 543-56.
[Non-Patent Document 12]
"Utilization of Chitin and Chitosan," Business Special Special Volume 46, No. 28, 1998, p. 78-81
[Non-patent document 13]
"Abstracts of the 74th Annual Meeting of the Japanese Society of Agricultural Chemistry" Lecture No. 2E183α, 2000
[Non-patent document 14]
"Studies on Chitin and Chitosan", 1999, vol. 5, p. 75-79
[Non-Patent Document 15]
“Abstracts of the 74th Annual Meeting of the Japanese Society of Agricultural Chemistry” Performance No. 2F313α, 2000
[Non-Patent Document 16]
Veldkamp, H., Nature, 1952, 169, p. 500
[Non-Patent Document 17]
Campbell, Jr., LL et al., "Journal of General Microbiology", 1951, vol. 5, p. 894
[Non-Patent Document 18]
Shota Nakagami et al., "Report of the Institute of Fermentation, Institute of Technology," 1966, vol. 30, p. 19
[Non-Patent Document 19]
Sherif, A.A., et al., "(Applied Microbiology and Biotechnology), 1991, 35, p. 228.
[Non-Patent Document 20]
"Studies on Chitin and Chitosan", 2002, Vol. 8, No. 2, p. 238-239
[0013]
[Problems to be solved by the invention]
The present inventors have conducted intensive studies on a method for efficiently producing low-molecular-weight chitosan that is soluble in water even under neutral conditions and has an antibacterial action. As a result, Aspergillus oryzae var. sporoflavus Ohara JCM 2067) or chitinase derived from Aspergillus japonicus SANK 19288 strain or Aspergillus sojae derived from Aspergillus sojae, and cloned from Aspergillus sojae derived from Aspergillus sojae, and cloned from SNK2 cDNA of Aspergillus sojae. By enzymatic reaction using the chitinase, a low molecular weight non-derivative chitosan having a molecular weight of 10,000 or more was successfully produced from a high molecular weight chitosan derived from crab and shrimp, thereby completing the present invention.
[0014]
According to the present invention, non-derivative low molecular weight chitosan can be supplied in a state having a molecular weight distribution of 10,000 or more.
[0015]
[Means for Solving the Problems]
The present invention
(1) A chitosan enzyme separated and purified from at least one culture of Aspergillus oryzae, Aspergillus japonics, Aspergillus sojae, and Aspergillus sojae.
(2) Aspergillus oryzae (Aspergillus oryzae) is Aspergillus oryzae (Aspergillus oryzae var. Sporoflavus Ohara JCM 2067) is a strain, Aspergillus japonicus (Aspergillus japonicus) is Aspergillus japonicus (Aspergillus japonicus) SANK 19288 strain, Aspergillus (1) The chitosan-degrading enzyme according to (1), wherein Aspergillus sojae is Aspergillus sojae SANK 22388 strain.
(3) The chitosan-degrading enzyme according to (1) or (2), which has a partial amino acid sequence of SEQ ID NOS: 1 to 3 in the sequence listing.
(4) The chitosan-degrading enzyme according to any one of (1) to (3), which has the following properties:
1) exhibiting a molecular weight of about 40,000 by SDS-PAGE electrophoresis;
2) show an isoelectric point pI of 3.5 to 4.5 by isoelectric focusing;
3) Hydrolyze 30% acetylated chitosan (viscosity 100-300 cps) at pH 3.5 to pH 10.5;
4) hydrolyzing glycol chitin at pH 3.0 to pH 10.5;
5) exhibit the hydrolysis activity described in 3) at 0 ° C to 80 ° C;
6) stable at temperatures below 45 ° C .;
7) stable under a pH condition of pH 5 to pH 9.5;
(5) Chitosan-degrading enzyme which is a protein according to any one of the following a) to d):
a) a protein consisting of the amino acid sequence of SEQ ID NO: 5 in the sequence listing;
b) a protein consisting of an amino acid sequence encoded by a nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing;
c) a protein comprising the amino acid sequence of a) or b), wherein one or several amino acids are substituted, deleted, inserted or added, and which has chitosan-degrading activity;
d) a protein comprising the amino acid sequence of a) or b),
(6) Chitosan-degrading enzyme which is a protein according to any one of the following a) to c):
a) a protein consisting of the amino acid sequence of SEQ ID NO: 14 in the sequence listing, wherein the α-amino group of the N-terminal serine residue is acetylated;
b) In the amino acid sequence of SEQ ID NO: 14 in the sequence listing, one or several amino acids are substituted, deleted, inserted or added, and the α-amino group of the N-terminal serine residue Is acetylated, and has a chitosan-degrading activity;
c) a protein comprising the amino acid sequence of SEQ ID NO: 14 in the sequence listing, wherein the α-amino group of the N-terminal serine residue is acetylated, and which has chitosan-degrading activity;
(7) DNA according to any one of the following a) to e):
a) DNA comprising a nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing;
b) DNA which hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above under stringent conditions and encodes a protein having chitosan-degrading activity ;
c) a DNA comprising a nucleotide sequence having 95% or more nucleotide sequence homology with the DNA described in a) above, and encoding a protein having chitosan-degrading activity;
d) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 5 in the sequence listing;
e) a DNA comprising a nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing,
(8) DNA according to any one of the following a) to c):
a) Recombinant plasmid retained by transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235), DNA inserted into pTriplEx-Chitinase-cDNA;
b) b) a protein which hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) under stringent conditions and encodes a protein having a chitosan-degrading activity. DNA;
c) a DNA comprising the DNA described in a) above and encoding a protein having chitosan-degrading activity;
(9) A chitosan-degrading enzyme which is a protein encoded by the DNA according to (7) or (8).
(10) Transformed E. coli Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA A recombinant plasmid carried by SANK 72102 (FERM BP-8235), a protein encoded by DNA inserted in pTriplEx-Chitinase-cDNA. A chitosan-degrading enzyme,
(11) The chitosan-degrading enzyme according to (5), (6), (9) or (10), which has the activity shown in the following a) to b):
a) an activity of hydrolyzing 30% acetylated chitosan (viscosity of 100 to 300 cps) under conditions of pH 3.5 to pH 10.5 and 0 ° C. to 80 ° C .;
b) an activity of hydrolyzing glycol chitin at pH 4.0 to pH 10.5, (12) a recombinant plasmid containing the DNA of (7) or (8),
(13) the recombinant plasmid of (12), which is an expression vector;
(14) a host cell transformed with the recombinant plasmid according to (12) or (13),
(15) The host cell according to (14), which is a prokaryotic cell or a eukaryotic cell.
(16) The host cell according to (14), which is a transformed Escherichia coli Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235).
(17) A method for producing a chitosan-degrading enzyme, comprising the following 1) and 2):
1) a step of culturing the cell according to any one of the following a) to e) under conditions for producing chitosan-degrading enzyme;
a) Aspergillus oryzae;
b) Aspergillus japonicus;
c) Aspergillus sojae;
d) a host cell according to any one of (14) to (16);
e) a cell that produces the chitosan-degrading enzyme according to any one of (5), (6), (9) to (11);
2) A step of separating and purifying chitosan-degrading enzyme from the culture product of 1).
(18) Aspergillus oryzae (Aspergillus oryzae) is Aspergillus oryzae (Aspergillus oryzae var. Sporoflavus Ohara JCM 2067) is a strain, Aspergillus japonicus (Aspergillus japonicus) is Aspergillus japonicus (Aspergillus japonicus) SANK 19288 strain, Aspergillus (17) The method according to (17), wherein the Aspergillus sojae is Aspergillus sojae SANK 22388 strain.
(19) a chitosan-degrading enzyme produced by the method according to (17) or (18),
(20) A method for producing low molecular weight chitosan, comprising the following steps 1) and 2):
1) The chitosan-degrading enzyme according to any one of (1) to (6), (9) to (11) and (19) is added to an acetylated chitosan aqueous solution having an acetylation degree of 10% to 30%. ;
2) performing an enzyme reaction for at least 10 minutes at a pH of 3 to 12, 10 ° C. to 60 ° C.
(21) Low molecular weight chitosan or a salt thereof produced by the method according to (20),
(22) The low molecular weight chitosan or a salt thereof according to (21), wherein the molecular weight is 40,000 to 250,000 and the degree of acetylation is 10%.
(23) The low molecular weight chitosan or a salt thereof according to (21), wherein the molecular weight is 10,000 to 110,000 and the degree of acetylation is 20%.
(24) The low molecular weight chitosan or a salt thereof according to (21), wherein the molecular weight is 10,000 to 70,000 and the degree of acetylation is 30%.
(25) A pharmaceutical composition comprising the low-molecular-weight chitosan or a salt thereof according to any one of (21) to (24) as an active ingredient,
(26) The pharmaceutical composition according to (25), which is a therapeutic agent for trauma, bedsore or atopic dermatitis.
(27) A cosmetic comprising the low-molecular-weight chitosan or a salt thereof according to any one of (21) to (24),
(28) A food having the low molecular weight chitosan or a salt thereof according to any one of (21) to (24),
(29) A treatment agent such as water having the low molecular weight chitosan or a salt thereof according to any one of (21) to (24),
(30) A method for producing a sample comprising the following steps 1) and 2), wherein the liquid component contains substantially no high molecular weight acetylated chitosan having a molecular weight of 1,000,000 or more:
1) The chitosan-degrading enzyme described in any one of (1) to (6), (9) to (11) and (19) is added to a sample containing a high molecular weight acetylated chitosan having a molecular weight of 1,000,000 or more. ;
2) performing an enzyme reaction at pH 3 to 12 and 10 ° C. to 60 ° C. for 10 minutes or more;
(31) The method according to (30), wherein the sample containing a polymer partially acetylated chitosan having a molecular weight of 1,000,000 or more is a sample containing crustaceans.
(32) The method according to (31), wherein the crustacean is one or both of shrimp and crab,
(33) A sample produced by the method according to any one of (30) to (32), wherein the liquid component contains substantially no high molecular weight acetylated chitosan having a molecular weight of 1,000,000 or more,
About.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[0017]
The present invention provides, as a filamentous fungus useful for producing a low molecular weight chitosan, a chitinase derived from Aspergillus oryzae or a chitinase derived from Aspergillus japonics, or a chitinase derived from Aspergillus japonicus, Aspergillus sojae sjajes, and Aspergillus sojae aspase. The present invention relates to a low molecular weight chitosan produced by the chitinase, a pharmaceutical composition containing the low molecular weight chitosan, and the like.
[0018]
In the present invention, chitinase refers to an enzyme having an activity of hydrolyzing chitin or partially acetylated chitosan. Hydrolysis of chitin produces N-acetylglucosamine and its oligosaccharide. This “activity of hydrolyzing chitin or partially acetylated chitosan” is defined as “chitosan-degrading activity”. Chitin refers to partially acetylated chitosan having a high acetylation ratio among the partially acetylated chitosans, and in the present invention, unless otherwise specified, these are also referred to as partially acetylated chitosan. In the present invention, both chitosan-degrading enzyme and chitinase refer to an enzyme having the above-described chitosan-degrading activity, and are used as synonyms. The chitinase of the present invention includes a protein having a chitosan-degrading activity in a culture of a microorganism that produces chitinase. Examples of such chitinases include a chitinase from Aspergillus oryzae, a chitinase from Aspergillus japonicus, and a chitinase from Aspergillus sojae, and Aspergillus sojae from Aspergillus sojae. More preferred are Aspergillus oryzae var. a chitinase from Aspergillus japonicus SANK 19288 strain or a chitinase from Aspergillus sojae (Aspergillus sojae) from Chiba sp. oryzae) var. sporoflavus Ohara JCM 2067).
Further, another example of the chitinase of the present invention includes a protein containing all the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing and having chitosan-degrading activity. The protein may include all of them as an internal amino acid sequence, regardless of the order of each amino acid sequence. Preferred among such chitinases include all the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing, and the N-terminal sequence is “Ser-Ser-Gly-Leu- Lys (amino acids 1 to 5 of SEQ ID NO: 14 in the sequence listing) "and a protein having chitosan-degrading activity. More preferably, it contains all of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing, and the N-terminal sequence consists of Ser-Ser-Gly-Leu-Lys-, and , An N-terminal acetylated protein having chitosan-degrading activity.
[0019]
Another example of the chitinase of the present invention is a protein that contains all of the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 in the sequence listing and has chitosan-degrading activity.
[0020]
Further, in the protein consisting of the amino acid sequence of SEQ ID NO: 5 in the sequence listing, a protein in which one or several amino acid residues are substituted, deleted, inserted and / or added at one or several sites is also included. , As long as it has chitosan-degrading activity. Several means not more than ten, preferably not more than five. As an example in which a protein having a substituted amino acid sequence has an activity equivalent to that of a natural protein, for example, a nucleotide sequence corresponding to cysteine of the interleukin 2 (IL-2) gene is converted into a nucleotide sequence corresponding to serine. It is known that the obtained protein retains IL-2 activity (Wang, A. et al. (1984) Science 224, 1431-1433).
[0021]
Another example of the chitinase of the present invention is a protein comprising the amino acid sequence of SEQ ID NO: 5 in the sequence listing. Also, a protein comprising the amino acid sequence of SEQ ID NO: 5 in the sequence listing is included in the present invention as long as it has chitosan-degrading activity.
[0022]
Another example of the chitinase of the present invention is a protein comprising the amino acid sequence of SEQ ID NO: 14 in the sequence listing, wherein the N-terminal α-amino group is acetylated. Further, a protein comprising such a protein is also included in the present invention as long as it has chitosan-degrading activity.
[0023]
Suitable chitinases of the present invention include those described in Aspergillus oryzae var. A chitinase derived from Aspergillus japonicus SANK19288 strain, a chitinase derived from Sporoflavus Ohara JCM 2067), a chitinase derived from Aspergillus sojae, a protein sequence described in Aspergillus sojae, and a sequence described in SEQ ID NO: 5 from the amino acid sequence of Aspergillus sojae. And a protein comprising the amino acid sequence of SEQ ID NO: 14 in the sequence listing, in which the N-terminal α-amino group is acetylated. More preferable is Aspergillus oryzae var. sporoflavus Ohara JCM 2067) and a protein comprising the amino acid sequence of SEQ ID NO: 14 in the sequence listing and having an acetylated N-terminal α-amino group.
In the present invention, the “DNA of the present invention” refers to a DNA encoding the chitinase of the present invention. The DNA may be in any form known so far, such as cDNA, genomic DNA, artificially modified DNA, and chemically synthesized DNA.
[0024]
Examples of the DNA of the present invention include a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing, which hybridizes under stringent conditions and decomposes chitosan. DNA encoding a protein having activity.
[0025]
Another example of the DNA of the present invention includes a nucleotide sequence homology of 70% or more, preferably 80% or more, more preferably 95% or more with the nucleotide sequence of nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing. DNA having the following formula: Such DNAs include mutant DNAs found in nature, artificially modified mutant DNAs, homologous DNAs derived from heterologous organisms, and the like.
[0026]
Still another example of the nucleotide of the present invention is a DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 5 in the sequence listing. The codon corresponding to the desired amino acid may be arbitrarily selected, and can be determined according to a conventional method, taking into account, for example, the codon usage of the host to be used. (Grantham, R. et al. (1981) Nucleic Acids Res. 9, 143-174). Furthermore, partial modification of the codons of these nucleotide sequences can be performed by a site-specific mutagenesis method (site specific mutagenesis / Mark, DF, et al.) Using a primer consisting of a synthetic oligonucleotide encoding the desired modification in a conventional manner. et al. (1984) Proc. Natl. Acad.
Sci. USA 81, 5662-5666).
[0027]
Another example of the DNA of the present invention is a DNA having a nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing. In addition, a DNA comprising a DNA comprising a nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing is also included in the present invention as long as it comprises a region encoding a protein having chitosan degrading activity. It is.
[0028]
Transformed E. coli Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 carrying the recombinant plasmid into which the DNA of the present invention has been inserted is a patent issued by the National Institute of Advanced Industrial Science and Technology on November 8, 2002. It has been internationally deposited at the Organism Depositary Center (1-1-1, Higashi 1-1-1, Tsukuba, Ibaraki, Japan) and is assigned the accession number FERM BP-8235. Therefore, the DNA of the present invention can be obtained from the strain. In addition, DNAs that hybridize under stringent conditions with DNAs inserted into the recombinant plasmids carried by the deposited strains are also included in the present invention as long as the encoded proteins have chitosan-degrading activity. In addition, a DNA comprising the above nucleotide sequence is also included in the present invention. Furthermore, proteins encoded by such DNAs are also included in the chitinase of the present invention.
[0029]
Preferable examples of the DNA of the present invention include a DNA having the nucleotide sequence shown in nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the Sequence Listing, and a transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK72102 (FERM). BP-8235), which is a DNA inserted into a recombinant plasmid, pTriplEx-Chitinase-cDNA, and most preferably transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP- 8235), the DNA inserted into the recombinant plasmid pTriplEx-Chitinase-cDNA A.
[0030]
The chitinase of the present invention includes a protein comprising an amino acid sequence encoded by the DNA of the present invention. Further, in order to prepare a modified form of the chitinase of the present invention in which any one or two or more amino acids are deleted, a method in which DNA is cut from the end using exonuclease Bal31 or the like (Toshimi Kishimoto et al. The method can be performed according to the method for cascade chemistry experiment, Gene Research Method II, "335-354", the cassette mutation method (Toshimitsu Kishimoto, "New Life Chemistry Experiment, Nucleic Acid III, Recombinant DNA Technology", 242-251). As described above, even a protein obtained by a genetic engineering technique based on the DNA of the present invention is included in the present invention as long as it has chitosan-degrading activity. Such a chitinase does not necessarily have to have all of the amino acid sequence described in SEQ ID NO: 5 in the sequence listing. For example, even if the protein has a partial sequence, as long as the protein exhibits chitosan-degrading activity, Included in the chitinase of the present invention. Further, the DNA encoding such a chitinase is also included in the present invention.
[0031]
The chitinase used in the present invention may be purified from a chitinase-producing bacterium, roughly purified, a lysate of the bacterium, or a culture supernatant of the bacterium as it is. When culturing a chitinase-producing bacterium, it is preferable to add powdered chitin, powdered chitosan, colloidal chitin, colloidal chitosan, etc. to the medium in addition to a carbon source and a nitrogen source, and to culture. After the culture of the chitinase-producing bacteria, centrifugation is performed, and the culture supernatant from which the cells have been removed can be used as it is as a crude enzyme solution. Further, a crude enzyme solution may be roughly purified by ion exchange chromatography or the like, or a purified product may be used.
[0032]
A chitinase-producing bacterium refers to a microorganism that has chitinase-producing ability essentially innately, and includes a microorganism that accumulates chitinase in a cell and a microorganism that secretes chitinase outside the cell. When a culture supernatant of a chitinase-producing bacterium or a chitinase purified from the culture supernatant is used, a bacterium that secretes chitinase outside the cells can be used.
[0033]
Examples of the chitinase-producing bacterium used in the present invention include Aspergillus oryzae, Aspergillus japonics, and Aspergillus sojae, preferably Aspergillus sojae. sporoflavus Ohara JCM 2067 strain, Aspergillus japonics SANK 19288 or Aspergillus sojae SANK 22388 strain. The cultivation of the chitinase-producing bacterium can be carried out using a usual medium using a culture apparatus and medium. For the culture, a method such as liquid culture or solid culture can be appropriately selected. In the case of liquid culture, flask culture or culture using a fermenter can be performed, and after the start of culture, use a batch culture method without additional medium or a fed-batch culture method in which a medium is added as needed during culture. Can be. A carbon source and a nitrogen source can be added to the medium, and vitamins and trace metals can be added as needed. Examples of the carbon source include monosaccharides such as glucose, mannose, galactose and fructose; maltose, cellobiose, disaccharides such as isomaltose, lactose and sucrose; polysaccharides such as starch; and malt extract. It is not limited to these as long as the fungus grows. Examples of the nitrogen source include inorganic nitrogen such as ammonia, ammonium sulfate, and ammonium nitrate; and organic nitrogen such as yeast extract, malt extract, corn steep liquor, and peptone, but are not limited thereto as long as the chitinase-producing bacteria grow. In addition, powdered chitin or colloidal chitin can be added to the medium to increase the amount of chitinase produced by the chitinase-producing bacterium. The amount of the composition in these media can be appropriately selected. The culture temperature, pH, and aeration and stirring amount can be appropriately selected so as to be suitable for chitinase production.
[0034]
As a chitinase used in the present invention, Aspergillus oryzae or Aspergillus japonicus or Aspergillus sojae (Aspergillus sojae) and chitinase derived from Aspergillus sojae is preferably used, and a chitinase derived from Aspergillus sojae is preferably used. . A chitinase derived from sporoflabus Ohara JCM 2067 strain or Aspergillus japonics SANK 19288 or Aspergillus sojae SANK 22388 strain can be used. Chitinase may be produced by these chitinase-producing bacteria themselves, or may be produced by a mutant or modified form thereof.Furthermore, a gene encoding chitinase of these chitinase-producing bacteria may be introduced into a host. It may be a recombinant protein produced from the obtained transformant.
Obtaining chitinase-producing bacteria
Aspergillus oryzae var. sporoflabus Ohara JCM 2067 strain is a microorganism collection preservation facility (JCM: Japan Collection of Microorganisms; RIKEN Institute of Bioscience and Biotechnology, Japan; address: 2-1-1, Hirosawa, Wako, Saitama, Japan, homepage address <http: // www. jcm.liken.go.jp / >>).
[0035]
The bacteriological properties of Aspergillus japonics SANK 19288 strain and Aspergillus sojae SANK 22388 strain are shown below.
[0036]
Aspergillus japonics SANK 19288 strain and Aspergillus sojae SANK 22388 strain were clicked and described in Pitt's literature (Klich, MA and Pitt, J.I. (1988) Laboratories gasoline gasoline gasoline gasoline gasoline gasoline sampling system. According to Division of Food Processing, North Ryde NSW, Australia, three types of media (CYA medium, MEA medium, CY20S medium) were inoculated to observe mycological properties.
[0037]
The display of the color tone is described in "Metune Handbook of Color" (Kornerup, A. and Wanscher, JH (1978), Method Handbook of Color (3rd edition), Ernie Methuen, London).
[0038]
The compositions of the three types of media (CYA medium, MEA medium, CY20S medium) are as follows. CYA medium [Czapek Yeast Extract Agar medium] (K2HPO4 1.0 g, * Zapek concentrated solution 10 ml, yeast extract 5 g, sucrose 30 g, agar 15 g, distilled water 1000 ml)
* Zapec concentrate (30 g of NaNO3, 5 g of KCl, 5 g of MgSO4.7H2O, 0.1 g of FeSO4.7H2O 0.1 g, 0.1 g of ZnSO4.7H2O, 0.05 g of CuSO4.5H2O 0.05 g distilled water 100 ml)
MEA medium [Malt Extract Agar medium] (malt extract 20 g, peptone 1 g, glucose 20 g, agar 20 g, distilled water 1000 ml)
CY20S medium [Czapek Yeast ExtractAgar with 20% Sucrose] medium] (K2HPO4 1.0 g, * Zapec concentrate 10 ml, yeast extract 5 g, sucrose 200 g, agar) Distilled water
1000 ml)
1) Mycological properties
a) Mycological properties of Aspergillus japonicus SANK 19288 strain
The colony in the CYA medium has a diameter of 45 to 60 mm after culturing at 25 ° C. for one week. The hypha is white. Conidia formation is strong at the center, and the color of the formation part is from olive brown (4F4) to black. The color of the back surface is grayish yellow (4B4) to yellowish white (4A2). Sclerotia are not observed. No exudate or soluble dye is observed. Colonies in the CY20S medium are 57 to 68 mm in diameter at 25 ° C. for one week of culture. Colonies resemble colonies in CYA medium. The color of the back surface is pastel yellow (3A4). Colonies in MEA medium have a diameter of 51 to 57 mm at 25 ° C. for one week of culture. Conidia formation is strong, and the color of the formation part is black. The color of the back is colorless. The colony of the CYA medium cultured at 37 ° C. for one week has a diameter of 13 to 20 mm. Does not grow at 5 ° C.
[0039]
Conidial heads are radial, conidiophores are smooth, colorless or brown on top, 4-11 μm wide. The apex is spherical to elliptical and 19 to 50 μm wide. Aspergilla is single row. The phialide covers 3/4 of the apex and is 7 to 10 x 3.5 to 4.5 μm. Conidia are oval to spherical, covered with rather sparse spines, and are 3.5 to 4.5 x 3.5 to 4 μm in diameter.
[0040]
Based on the above mycological properties, a search was made for a bacterium corresponding to the present bacterium. Aspergillus japonicus var accuritus (Aspergillus japonicus var. Aculeatus (Iizuka) Al-Musallam) described in the literature of Crick and Pitt. Almost matched. Accordingly, this strain was identified as Aspergillus japonics var. Aculeatus (Iizuka) Al-Musallam.
[0041]
The Aspergillus japonicus SANK 19288 strain was designated as Aspergillus japonics var Acuricus SANK 19288 on September 13, 2001 by the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (Tsukuba, Ibaraki, Japan). 1-1 Deposited at Chuo No. 6) and deposited under accession number FERM BP-7736.
[0042]
b) Mycological properties of Aspergillus sojae SANK 22388 strain
Colonies in the CYA medium are 46 to 52 mm in diameter at 25 ° C. for one week of culture. Colonies are fluffy. The mycelium is white and dense. Conidia formation is vigorous, and the color of the formation part is olive (2F-E7). The color of the back surface is yellowish white (2A2). Colonies in CY20S medium are 42 to 45 mm in diameter at 25 ° C. for one week of culture. Colonies resemble colonies in CYA medium. Conidia formation is vigorous, and the color of the forming part is olive yellow (3D6) to olive (3E7). Colonies on MEA medium are 43 to 47 mm in diameter at 25 ° C. for one week of culture. Colonies are slightly fluffy and slightly sparse. The mycelium is white and unobtrusive. Conidia formation is vigorous, and the color of the formation part is olive (1E8). The color of the back is colorless. The colony of the CYA medium cultured at 37 ° C. for one week has a diameter of 52 to 56 mm. Does not grow at 5 ° C.
[0043]
The conidium head is radial, the conidiophore is rough, colorless and 7 to 12 μm wide. The crowns are pear-shaped or spherical, 24 to 41 μm wide. Aspergilla is single row, sometimes double row. The metre is 8 to 12 × 4 to 8 μm. The phialide is 10-11 × 4.5-6 μm. Conidia are spherical, rough, 5 to 6.5 μm in diameter.
[0044]
Based on the above mycological properties, a search was made for a bacterium corresponding to the present bacterium. As a result, the bacterium almost coincided with the property of Aspergillus sojae Sakaguchi & Yamada described in the literature of Crick and Pitt. Therefore, this strain was identified as Aspergillus sojae Sakaguchi & Yamada.
[0045]
The Aspergillus sojae SANK 22388 strain was designated as Aspergillus sojae SANK 22388 on September 13, 2001 by the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary (1-1-1, Higashi, Tsukuba, Ibaraki, Japan). Central deposit No. 6) and deposited under accession number FERM BP-7778.
[0046]
As is well known, filamentous fungi are susceptible to mutation in the natural world or by artificial manipulation (eg, ultraviolet irradiation, irradiation, chemical treatment, etc.), and Aspergillus oryzae var. Sporoflabus Ohara JCM of the present invention. 2067), Aspergillus japonix SANK 19288 and Aspergillus sojae SANK 22388. Aspergillus oryzae var. Sporoflabus Ohara JCM 2067 strain, Aspergillus japonicus SANK 19288 strain and Aspergillus sojae SANK 22388 strain, all of which are referred to in the present invention. These mutants also include those obtained by genetic methods, for example, recombination, transduction, transformation and the like. That is, all strains of Aspergillus oryzae var. Sporoflabus Ohara JCM 2067, chitinase-producing strains, mutants thereof, and strains that are not clearly distinguished therefrom are all included in Aspergillus oryzaris ovars. Included. Aspergillus japonicus SANK 19288 strain, chitinase-producing Aspergillus japonicus SANK 19288 strain, mutants thereof and strains not clearly distinguished therefrom are all included in Aspergillus japonicus SANK 19288 strain. Aspergillus sojae SANK 22388, a chitinase-producing Aspergillus sojae SANK 22388 strain, mutants thereof and strains not clearly distinguished therefrom are all included in Aspergillus sojae SANK 22388 strain.
The chitinase of the present invention can also be obtained by transforming a host cell with a recombinant plasmid in which the DNA of the present invention is inserted into a vector, and obtaining a culture product of the transformed cell. Thus, the recombinant plasmid in which the DNA of the present invention is inserted into an appropriate vector is also included in the present invention. As a vector used for such a purpose, various generally known vectors can be used. Suitable examples include, but are not limited to, vectors for prokaryotic cells, vectors for eukaryotic cells, vectors for mammalian cells, and the like. Such recombinant plasmids can transform other prokaryotic or eukaryotic host cells. Furthermore, by using a vector having an appropriate promoter sequence and / or a sequence related to expression of a trait, or by introducing such a sequence, an expression vector can be used to express the gene in each host. It is. Such an expression vector is a preferred embodiment of the recombinant plasmid of the present invention.
[0047]
Host cells can be obtained by introducing the recombinant plasmid of the present invention into various cells. Such cells may be prokaryotic or eukaryotic as long as they are capable of introducing the plasmid.
[0048]
Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cells are transformed with a plasmid vector that contains replicons or origins of replication from species compatible with the host and regulatory sequences. Further, the vector is preferably a vector having a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.
[0049]
For example, K12 strain or the like is often used as Escherichia coli, and pBR322 or a pUC-type plasmid is generally used as a vector, but is not limited thereto, and any known various strains and vectors can be used.
[0050]
Examples of the promoter include, in Escherichia coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter, and the like. Any promoter can be used to produce the chitinase of the present invention.
[0051]
As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) is used, but it is not limited thereto. is not.
[0052]
By connecting a DNA sequence encoding the signal peptide sequence of α-amylase of Bacillus subtilis as a promoter, extracellular secretory expression becomes possible.
[0053]
Eukaryotic host cells include cells such as vertebrates, insects, and yeast. Examples of vertebrate cells include mammalian-derived cells, such as monkey COS cells (Gluzman, Y. (1981). ) Dihydrofolate reductase deficient strains of Cell 23, 175-182, ATCC CRL-1650) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LA (1980)). Natl. Acad. Sci. USA 77, 4126-4220), etc., but is not limited thereto.
[0054]
As a vertebrate cell expression promoter, a promoter having an upstream site of a gene to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used. May be provided. Examples of the expression vector include, but are not limited to, pSV2dhfr having an early promoter of SV40 (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864).
[0055]
As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and further has a transcription promoter, a transcription termination signal, and an RNA splice site. Can be used. The expression vector is prepared by the diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res, 11, 1295-1308), and calcium phosphate-DNA coprecipitation method (Graham, FL. and van der Eb, AJ (1973) Virology 52, 456-457) and electric pulse perforation (Neumann, E. et al. (1982) EMBO J. 1, 841-845) and the like. Thus, the desired transformed cells can be obtained. When a CHO cell is used as a host cell, a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker together with an expression vector, for example, pRSVneo (Sambrook, J. et al. (1989): "Molecular Cloning") A Laboratory Manual "Cold Spring Harbor Laboratory, NY) and pSV2-neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-3 Trans. Then, by selecting a G418-resistant colony, a transformed cell stably producing the chitinase of the present invention can be obtained.
[0056]
When insect cells are used as host cells, ovarian cell-derived cell lines (Sf-9 or Sf-21) of Spodoptera frugiperda of Lepidoptera and Noctuidae, and High Five cells (Wickham, T. J. et al.) Derived from egg cells of Trichoplusia ni. al, (1992) Biotechnol. Prog. I: 391-396) and the like, and pVL1392 / 1393 using a polyhedrin protein promoter of autographer nuclear polyhedrosis virus (AcNPV) as a baculovirus transfer vector. (Kidd, IM and VC Emery (1993) The use of baculoviruses as expression vec). ors. Applied Biochemistry and Biotechnology 42, 137-159). In addition, vectors using baculovirus P10 or a promoter of the same basic protein can also be used. Furthermore, the recombinant protein can be expressed as a secreted protein by linking the secretory signal sequence of the AcNPV envelope surface protein GP67 to the N-terminal side of the target protein (Zhe-mei Wang, et al. (1998)). Biol. Chem., 379, 167-174).
[0057]
As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well known. Among them, yeast of the genus Saccharomyces, for example, baker's yeast Saccharomyces cerevisiae and petroleum yeast Pichia pastoris are preferable. Examples of expression vectors for eukaryotic microorganisms such as yeast include, for example, a promoter for an alcohol dehydrogenase gene (Bennetzen, JL and Hall, BD (1982) J. Biol. Chem. 257, 3018-3025). ) Or a promoter of an acid phosphatase gene (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5). When expressed as a secretory protein, it can be expressed as a recombinant having a secretory signal sequence and a cleavage site of an endogenous protease or a known protease possessed by the host cell at the N-terminal side. For example, in a system in which human mast cell tryptase of a trypsin-type serine protease is expressed in petroleum yeast, the activity is achieved by connecting the secretory signal sequence of α-factor of yeast and the cleavage site of KEX2 protease possessed by petroleum yeast to the N-terminal side and expressing it. It is known that type tryptase is secreted into the medium (Andrew, L. Niles, et al. (1998) Biotechnol. Appl. Biochem. 28, 125-131).
[0058]
The transformant obtained as described above can be cultured according to a conventional method, and the chitinase of the present invention is produced intracellularly or extracellularly by the culture. As the medium used for the culture, various types commonly used depending on the host cell used can be appropriately selected. For example, in the case of the above-mentioned COS cells, RPMI1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) A medium obtained by adding a serum component such as fetal bovine serum to a medium such as the above can be used as necessary. Culture conditions may be such that the CO2 concentration is in the range of 0 to 50%, preferably 1 to 10%, and more preferably 5%. The culture temperature may be 0 to 99 ° C., preferably 20 to 50 ° C., and more preferably 35 to 40 ° C.
[0059]
The chitinase of the present invention, which is produced as a recombinant protein inside or outside the cells of a transformant by the above culture, can be used in a physicochemical property, a chemical property, a biochemical property (enzyme activity) of the protein from a culture product. And the like ("Biochemical Data Book II", paragraphs 1175 to 1259, 1st edition, 1st edition, issued by Tokyo Chemical Co., Ltd. on June 23, 1980; Biochemistry, vol. 25, No. 25, p8274-8277 (1986); Eur. J. Biochem., 163, p313-321 (1987), etc.). Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, freeze-thaw method, ultrasonic crushing, ultrafiltration, gel filtration, Examples thereof include various types of liquid chromatography such as adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis, and combinations thereof. As described above, the desired recombinant protein can be produced on an industrial scale in high yield. In addition, by connecting histidine consisting of 6 residues to the recombinant protein to be expressed, it can be efficiently purified with a nickel affinity column. By combining the above methods, the chitinase of the present invention can be easily produced in high yield and high purity in large quantities.
[0060]
Chitinase produced by the above method can also be mentioned as a preferred example of the present invention.
The low molecular weight chitosan or a salt thereof used in the present invention dissolves in an amount of 10 g or more in 100 g of water at 0 to 100 ° C. Here, the water may be distilled water, tap water, or well water. Furthermore, 10 g or more of low molecular weight chitosan or a salt thereof is dissolved in 100 g of water under a condition of pH 1.0 to 7.0. In addition, low molecular weight chitosan has antibacterial activity.
[0061]
Partially acetylated chitosan refers to chitosan in which some of the amino groups of chitosan are acetylated, and partially acetylated chitosan having a low degree of acetylation can be produced by deacetylating chitin with an alkali. . The degree of acetylation of the partially acetylated chitosan can be controlled by the alkali concentration, reaction time and the like. Further, those having a molecular weight of 1,000,000 or more are particularly referred to as polymer partially acetylated chitosan. Examples of the partially acetylated chitosan having a molecular weight of 1,000,000 or more include 30%, 20%, and 10% acetylated chitosan derived from shrimp and crab (trade names: Chitosan 7B, 8B, 9B (all manufactured by Katoyoshi Co., Ltd.)) The natural chitin contained in shrimp and crab is a polymer partially acetylated chitosan having a higher degree of acetylation, and these can also be used as partially acetylated chitosan.
[0062]
The molecular weight of the polymer partially acetylated chitosan can be measured by high performance liquid chromatography using gel permeation chromatography (GPC) (pullulan is used as a standard substance) (Takiguchi et al., Chitin-Chitosan Study, p. 75-). 79, 1999).
[0063]
The acetylation degree of the polymer partially acetylated chitosan can be measured by performing PVSK colloid titration with a 1/400 N polyvinyl potassium sulfate solution using a toluidine blue solution as an indicator (Toei K. et al. Anal. Chem. Acta 83, 59, 1976). The degree of acetylation measured by this method refers to the percentage of acetylated amino groups (for example, 1% acetylation means that one out of 100 amino groups is acetylated. ).
[0064]
The degree of acetylation of chitosan is ¹ It can also be calculated from the integral ratio of 3H of the acetyl group obtained by H-NMR and 7H excluding the hydroxyl group other than the acetyl group.
[0065]
The hydrolysis activity of chitinase can be measured according to the Randle-Morgan method (Randle CJM et al. Biochem. J. GL 586-589, 1955). It can also be measured according to the modified Schales method (Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)).
[0066]
The present invention relates to a method for producing low molecular weight chitosan.
[0067]
The chitinase used in the method is a hydrolyzate using 30% acetylated chitosan (viscosity: 100 to 300 cps) as a substrate under the reaction conditions of pH 4.0 to 5.5 and a temperature of 37 ° C for 10 to 30 minutes. When the degrading activity is 100%, the hydrolysis activity is lower than 100% when glycol chitin is used as the substrate, and the chitosan (viscosity of 50 to 200 cps) having a degree of acetylation of 1% or less is used as the substrate. The hydrolysis activity of chitinase is 15% or less.
[0068]
The amount and concentration of the enzyme used in the method are not particularly limited.
[0069]
In this method, the aqueous solution of the partially acetylated chitosan polymer as a substrate for chitinase is preferably 0.1 to 5% by mass, preferably 0.25 to 2% by mass, more preferably 0.5 to 1% by mass. It is. The aqueous solution can be used as a solution dissolved in water under acidic conditions (for example, pH 1 to 6). Further, the solution may be further weakly acidic (eg, pH 4.0 to 6.0) by adding an alkali (eg, sodium carbonate, sodium hydrogen carbonate, caustic soda, etc.) to the solution, and used in the method.
[0070]
Acids used to acidify the polymer partially acetylated chitosan solution and dissolve it in water include hydrochloric acid, acetic acid, trifluoroacetic acid, paratoluenesulfonic acid, acrylic acid, lactic acid, amino acid, citric acid, salicylic acid, and glucuronic acid. , Glycolic acid and the like, but are not limited thereto as long as an acidic solution can be prepared.
[0071]
The pH range of the reaction solution when producing low molecular weight chitosan using the chitinase of the present invention may be in the range of pH 3 to 12, preferably in the range of pH 3 to 7, and more preferably in the range of pH 3 to 7. The pH is in the range of 4-6. The temperature of the reaction solution may be 0 ° C to 70 ° C, preferably 10 ° C to 60 ° C, and more preferably 40 ° C to 50 ° C. The reaction time may be 10 minutes to 7 days, preferably 3 hours to 5 days, more preferably 1 to 3 days.
[0072]
Various low-molecular-weight chitosan salts having a molecular weight of 10,000 or more and less than 1,000,000 are produced by converting the polymer partially acetylated chitosan into an aqueous solution using various acids and then performing a hydrolysis reaction with the chitinase of the present invention. Depending on the acid used, the type of salt may be hydrochloride, acetate, trifluoroacetate, paratoluenesulfonate, acrylate, lactate, amino acid salt, citrate, salicylate, glucuronate, glycol Examples include, but are not limited to, acid salts.
According to the present invention, low-molecular-weight chitosan or a salt thereof that is soluble in 10 g or more in 100 g of water at 0 to 100 ° C is provided.
[0073]
Further, the present invention provides a pharmaceutical composition comprising low-molecular-weight chitosan that is soluble in 10 g or more in 100 g of water at 0 to 100 ° C.
[0074]
The hydrolysis activity of chitinase can be measured according to the Randle-Morgan method (Randle CJM et al. Biochem. J. GL 586-589, 1955). It can also be measured according to the modified Schales method (Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)).
[0075]
Glycol chitin is available from Senzyu and S.M. Okimatsu, Nippon Nogeikagakuku Kaishi, 23, 432 (1950).
Specific properties of the chitinase obtained from the chitinase-producing bacteria will be described below, but the properties of the chitinase of the present invention are not limited thereto.
[0076]
Aspergillus oryzae var. The chitinase produced and purified by Sporoflavus Ohara JCM 2067) strain has the following properties.
1) It shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) Isoelectric point pI 4.5 is shown by isoelectric focusing.
3) Chitosan 7B (manufactured by Katoyoshi) is hydrolyzed at pH 3.5 to pH 10.5.
4) Glycol chitin is hydrolyzed at pH 3.0 to pH 10.5.
5) Exhibits the hydrolysis activity described in 3) below 80 ° C.
6) The optimum pH for the hydrolysis activity described in 3) is pH 5.5.
7) The optimum pH for the hydrolysis activity described in 4) is pH 10.0.
8) The optimum temperature of the hydrolysis activity described in 3) is 60 ° C. at pH 5.5.
9) Stable at temperatures below 45 ° C.
10) Stable under pH conditions of pH 5 to pH 9.5.
11) pH 5.5 or pH 10.0 of the enzyme with respect to another substrate at a temperature of 37 ° C., assuming that the hydrolysis activity of the enzyme for chitosan 7B at pH 5.5 or pH 10.0 at a temperature of 37 ° C. for 10 minutes is 100%. Are shown in Table 1 as relative values. In the table, chitosan 10B is 1% acetylated chitin, chitosan 9B is 10% acetylated chitin, chitosan 8B is 20% acetylated chitin, and chitosan 7B is 30% acetylated chitin (all manufactured by Katoyoshi Co., Ltd.). CM-cellulose is carboxymethylcellulose sodium (manufactured by Wako Pure Chemical Industries, Ltd.). Glycol chitin is prepared by the method described above.
[0077]
[Table 1]

From Table 1, it can be seen that at pH 5.5, this enzyme has a higher degree of hydrolysis activity on partially acetylated chitosan (chitosan 9B, 8B, 7B) having a degree of acetylation of 10 to 30% than glycol chitin, and has a degree of acetylation of 1%. % Of chitosan (chitosan 10B) has almost no hydrolysis activity. No hydrolytic activity on sodium carboxymethylcellulose.
12) It has a partial amino acid sequence shown below. The sequence is shown from the N-terminal side.
Glu-Met-Thr-Pro-Tyr-Leu-Asp-Phe-Tyr-Arg-Leu-Met-Ala-Tyr-Gln (SEQ ID NO: 1 in Sequence Listing)
Ile-Val-Val-Gly-Met-Pro-Ile-Tyr-Gly-Arg-Lys-Glu (SEQ ID NO: 2 in Sequence Listing)
Ala-Leu-Pro-Lys-Pro-Gly-Ala-Thr-Glu-Tyr-Val-Asp-Pro-Ser-Ile [SEQ ID NO: 3 in Sequence Listing]
Chitinase produced by Aspergillus japonicus SANK 19288 has the following properties in a crude enzyme solution.
1) It shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) An isoelectric point pI of about 3.5 is shown by isoelectric point transfer electrophoresis.
3) Chitosan 7B is hydrolyzed at pH 3.5 to pH 10.5.
4) The optimum pH for the hydrolysis activity described in 3) is pH 4.0.
5) Assuming that the hydrolysis activity of the enzyme for chitosan 7B at pH 4.0 and a temperature of 37 ° C. for 10 minutes is 100%, the hydrolysis activity of the enzyme for another substrate at a pH of 4.0 and a temperature of 37 ° C. for 10 minutes is The values in Table 2 are shown as relative values.
[0078]
[Table 2]
――――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――――
Chitosan 10B 8.2
Chitosan 9B 67.6
Chitosan 8B 74.4
Chitosan 7B 100
Glycol chitosan 8.2
Glycol chitin 23.3
CM-cellulose 11.4
Lichenan 25.3
――――――――――――――――――――
From Table 2, this enzyme has a higher hydrolysis activity on partially acetylated chitosan (chitosan 9B, 8B, 7B) having a degree of acetylation of 10 to 30% than glycol chitin, and chitosan having a degree of acetylation of 1% or less ( It can be seen that the hydrolysis activity for chitosan 10B) is weak.
Chitinase produced and purified by Aspergillus japonicus SANK 19288 strain has the following properties.
1) It shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) An isoelectric point pI of about 3.5 is shown by isoelectric point transfer electrophoresis.
3) Chitosan 7B is hydrolyzed at pH 3.5 to pH 10.5.
4) The optimum pH for the hydrolysis activity described in 3) is pH 5.0.
5) The optimum temperature of the hydrolysis activity described in 3) is 65 ° C. at pH 5.0.
6) It is stable at a temperature of 50 ° C. or less.
7) Stable under pH conditions of pH 4 to pH 9.5.
8) Assuming that the hydrolysis activity of the enzyme for chitosan 7B at pH 5.0 and a temperature of 37 ° C. for 10 minutes is 100%, the hydrolysis activity of the enzyme for another substrate at a pH of 5.0 and a temperature of 37 ° C. for 10 minutes is The values in Table 3 are shown as relative values. In the table, chitosan 10B is 1% acetylated chitin, chitosan 9B is 10% acetylated chitin, chitosan 8B is 20% acetylated chitin, and chitosan 7B is 30% acetylated chitin (all manufactured by Katoyoshi Co., Ltd.). CM-cellulose is carboxymethylcellulose sodium (manufactured by Wako Pure Chemical Industries, Ltd.). Glycol chitin is prepared by the method described above.
[0079]
[Table 3]
――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――
Chitosan 10B 0
Chitosan 9B 58.6
Chitosan 8B 72.4
Chitosan 7B 100
Glucol chitosan 0
Glycol chitin 28.3
CM-cellulose 0
Lichenan 4.2
――――――――――――――――――
As shown in Table 3, purified chitinase derived from Aspergillus japonix SANK 19288 strain has the highest hydrolysis activity on chitosan 7B, and the hydrolysis activity decreases as the degree of acetylation decreases. The purified chitinase has chitin hydrolyzing activity and no cellulase hydrolyzing activity.
9) It has the partial amino acid sequence shown below. The sequence is shown from the N-terminal side.
His-Tyr-Pro-Thr-Asp-Ser-Trp-Asn-Asp-Val-Gly-Thr-Asn-Val-Tyr (SEQ ID NO: 11 in Sequence Listing)
Ala-Phe-Thr-Asn-Thr-Asp-Gly-Pro-Gly-Thr-Ala-Phe-Ser-Gly-Val (SEQ ID NO: 12 in Sequence Listing)
Leu-Ser-Gln-Met-Thr-Pro-Tyr-Leu-Asp-Phe-Tyr-Asn-Leu-Met-Ala-Tyr-Asp-Tyr-Ala-Gly (SEQ ID NO: 13 in Sequence Listing)
Chitinase produced by Aspergillus sojae SANK 22388 strain has the following properties in a crude enzyme solution.
1) It shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) shows a pI of about 4.5 by isoelectric focusing.
3) Chitosan 7B is hydrolyzed at pH 3.5 to pH 9.5.
4) The optimum pH for the hydrolysis activity described in 3) is pH 4.5.
5) Assuming that the hydrolysis activity of the enzyme for chitosan 7B at pH 4.5 and a temperature of 37 ° C. for 10 minutes is 100%, the hydrolysis activity of the enzyme for other substrates at a pH of 4.5 and a temperature of 37 ° C. for 10 minutes is The values in Table 4 are shown as relative values.
[0080]
[Table 4]
――――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――――
Chitosan 10B 0
Chitosan 9B 67.7
Chitosan 8B 71.6
Chitosan 7B 100
Glycol chitosan 2.9
Glycol chitin 81.9
CM-cellulose 0
Lichenan 11.9
――――――――――――――――――――
From Table 4, it can be seen that this enzyme has a high hydrolysis activity on partially acetylated chitosan (chitosan 9B, 8B, 7B) having a degree of acetylation of 10 to 30%, and has a high activity on chitosan (chitosan 10B) having a degree of acetylation of 1% or less. It can be seen that there is almost no hydrolysis activity. No hydrolytic activity on sodium carboxymethylcellulose.
Chitinase produced and purified by Aspergillus sojae SANK 22388 strain has the following properties.
1) It shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) An isoelectric point pI of about 4.5 is shown by the isoelectric point transfer electrophoresis method.
3) Chitosan 7B is hydrolyzed at pH 3.5 to pH 9.5.
4) The optimum pH for the hydrolysis activity described in 3) is pH 5.5 and pH 9.0.
5) The optimum temperature of the hydrolysis activity described in 3) is 60 ° C. at pH 5.5 and 35 ° C. at pH 9.0.
6) It is stable at a temperature of 40 ° C. or less at pH 5.5 and pH 9.0.
7) It is stable under pH conditions of pH 4.5 to pH 10.
8) Assuming that the hydrolysis activity of the enzyme for chitosan 8B at pH 5.0 and a temperature of 37 ° C. for 10 minutes is 100%, the hydrolysis activity of the enzyme for other substrates at pH 5.0 and a temperature of 37 ° C. for 10 minutes is The values in Table 5 are shown as relative values. In the table, chitosan 10B is 1% acetylated chitin, chitosan 9B is 10% acetylated chitin, chitosan 8B is 20% acetylated chitin, and chitosan 7B is 30% acetylated chitin (all manufactured by Katoyoshi Co., Ltd.). CM-cellulose is carboxymethylcellulose sodium (manufactured by Wako Pure Chemical Industries, Ltd.). Glycol chitin is prepared by the method described above.
[0081]
[Table 5]
――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――
Chitosan 10B 14.3
Chitosan 9B 73.6
Chitosan 8B 100
Chitosan 7B 97.4
Glucol chitosan 0
Glycol chitin 23.8
CM-cellulose 0
Lichenan 0
――――――――――――――――――
As shown in Table 5, purified chitinase derived from Aspergillus sojae SANK 22388 strain has the highest hydrolysis activity on chitosan 7B and 8B, and the hydrolysis activity decreases as the degree of acetylation decreases. The purified chitinase has chitin hydrolyzing activity and no cellulase hydrolyzing activity.
From the above, the properties of the chitinase of the present invention include the following, but are not limited thereto.
1) It shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) shows an isoelectric point of 3.0 to 5.0 (preferably 3.5 to 4.5) by isoelectric focusing.
3) Hydrolyze chitosan 7B at pH 3.0 to pH 11.0 (preferably pH 3.5 to 10.5).
4) hydrolyzing the glycol chitin at pH 3.0 to pH 11.5 (preferably pH 4 to pH 10.5);
5) exhibit the hydrolysis activity described in 3) at 0 ° C to 80 ° C;
6) stable at temperatures below 50 ° C. (preferably below 45 ° C.);
7) It is stable under pH conditions of pH 4.0 to pH 10 (preferably pH 5 to pH 9.5).
[0082]
Further, a method for producing the chitinase of the present invention is also included in the present invention.
[0083]
Aspergillus oryzae var. Chitinase can be produced by culturing chitinase-producing microorganisms such as sporoflabus Ohara JCM 2067 strain, Aspergillus japonicus SANK 19288 strain, Aspergillus sojae SANK 22388 strain in a medium. For example, 0.1-5.0% malt extract (manufactured by Difco), 0.1-1.0% yeast extract (manufactured by Difco), 0.05-1.0% chitin or Incubate with shaking in a medium of 0.05-1.0% chitosan at 16-45 ° C. for 1-15 days at 100-250 rpm.
[0084]
Aspergillus oryzae var. Enzymes derived from sporoflabus Ohara JCM 2067 strain, Aspergillus japonicus SANK19288 strain, and Aspergillus sojae SANK 22388 strain have the following common features.
1) It has the highest partially acetylated chitosan-decomposing activity in weak acidity.
2) Assuming that the hydrolysis activity for 30% acetylated chitosan at pH 4.0 to 6.0 and a temperature of 37 ° C. for 10 to 30 minutes is 100%, the activity for 1% acetylated chitosan is 15% or less.
[0085]
The chitosan low molecular weight chitosan and various salt solutions obtained by the present invention can be used as they are, but can also be concentrated by ammonium sulfate precipitation, organic solvent precipitation, centrifugation, lyophilization, ultrafiltration, or the like. Further, purification, desalting, and molecular weight fractionation can also be performed by dialysis, ultrafiltration, gel filtration, column chromatography, or the like.
[0086]
The low-molecular-weight chitosan hydrochloride thus prepared is excellent in solubility in water, and can be a solution having a maximum concentration of about 10% by mass. A 1% by mass aqueous solution of low molecular weight chitosan hydrochloride has a pH of 4.9 to 5.6, and the pH at which a precipitate (colloid) precipitates when an alkali is added is pH 6.6 to 7.4.
[0087]
The physiological activity of the low molecular weight chitosan hydrochloride prepared here is examined using the antibacterial activity as an index. The minimum inhibitory concentration (MIC) of each of the low molecular weight chitosan 9B, 8B, 7B hydrochloride to have antibacterial activity is measured. Compared to the antibacterial activity of high-molecular-weight chitosan hydrochloride, low-molecular-weight chitosan hydrochloride has an activity equal to or higher than high-molecular-weight chitosan hydrochloride regardless of gram-positive bacteria or negative bacteria. Therefore, low molecular weight chitosan has excellent physiological activity equal to or higher than high molecular weight chitosan. From this, it is considered that the antibacterial activity considered to be possessed by the high molecular weight chitosan is retained in the low molecular weight chitosan of the present invention. Also, in the healing test of the animal with skin deficiency, the effect of shortening the number of healing days is observed, and the animal has physiological activity.
Because of having such excellent physiological activity, the low-molecular-weight chitosan or a salt thereof obtained according to the present invention can be used, for example, for external preparations for wounds / pulmonary wounds, drugs for bedsores, drugs for atopic dermatitis, acne Skin external preparations such as therapeutic agents, bath preparations, cosmetics, facial cleansers, basic cosmetics, oral hygiene agents such as anti-caries agents, liquid mouthwashes, materials for dentistry, intestinal preparations such as intestinal bacteria improving agents, hospital infections Medical fields such as prophylactics, diet foods, food preservatives, food additives such as preservatives, animal and fish feeds, etc., plant activity enhancers and other agricultural fields, water treatment agents, metal adsorbents, It is conceivable to use the low-molecular-weight chitosan of the present invention as a chemical remover for a nuclear waste adsorbent or the like, but the application is not particularly limited to these examples. The present invention also includes a method for treating diseases such as trauma, bedsores, and atopic dermatitis, which comprises administering an effective amount of the low molecular weight chitosan of the present invention or a salt thereof to an animal. The use of the low-molecular-weight chitosan of the present invention or a salt thereof for treating these diseases is also included in the present invention.
The usage of the low molecular weight chitosan or the low molecular weight chitosan salt obtained according to the present invention varies depending on the purpose, but may take various forms for use in the above-mentioned applications. The forms are listed below, but are not particularly limited. Low molecular weight chitosan can be used as a solid. Further, an aqueous solution or an organic solvent solution having a concentration of 10% by mass, which is appropriately diluted, can be used at an arbitrary concentration. As the diluent, water or an organic solvent (such as alcohols) can be used. Finally, if the pH is more than 6.5, it can be used as a solution if it is alkaline, and if it is alkaline, it can be used as a colloidal compound. Furthermore, by allowing an inorganic substance or an organic substance to coexist, it can be used as a solution at an arbitrary pH. Also, a low molecular weight chitosan solution or a diluted solution can be used as a cream by mixing it with a cream base at an arbitrary concentration of 10% by mass or less. A low molecular weight chitosan solution or a dilute solution can be used as a film having an arbitrary thickness. Films with plastic-like hardness are obtained by drying a low molecular weight chitosan solution in frames of various thicknesses. A flexible sheet having flexibility can be obtained by mixing a low molecular weight chitosan solution with a solvent such as glycerin in an arbitrary ratio and drying the mixture in frames having various thicknesses. The degree of flexibility is adjusted by adding a solvent such as glycerin. Furthermore, a chitosan-methylcellulose composite film is obtained by mixing methylcellulose with a low molecular weight chitosan solution and drying in a frame having various thicknesses.
[0088]
The dose varies depending on the type and condition of the disease, the sex of the patient, age, dosage form, and administration method, but any amount can be used because it is highly safe. When administered to a living body, the weight is preferably 2 g / kg or less, and in other cases, it is appropriately adjusted.
In addition, the chitinase of the present invention can be effectively used to make difficult-to-handle samples easier to handle. Conventionally, when a sample containing crustaceans and the like is subjected to processing such as crushing, grinding, and placing in a mixer, the viscosity becomes extremely high and it is difficult to handle. This was attributable to the high molecular weight polysaccharides (the main component was high molecular weight partially acetylated chitosan) contained in these crustaceans. For example, in the course of such food processing, the chitinase of the present invention is added and reacted to decompose the high-molecular-weight acetylated chitosan in the liquid component, and convert the high-molecular-weight acetylated chitosan into the liquid component. It is possible to produce an easy-to-handle sample that is substantially free, and the present invention also includes a method for producing the easy-to-handle sample using chitinase. The raw material used in such a production method is not particularly limited as long as it is a sample containing a polymer partially acetylated chitosan, but is preferably a sample containing crustaceans, and more preferably one of shrimp and crab. Or a sample containing both. As the reaction conditions of the raw material and the chitinase, the conditions that exhibit the above-described chitosan decomposition activity can be applied. The reaction time is not less than 10 minutes and is not particularly limited as long as the viscosity of the sample becomes low, but is preferably about 10 minutes to 10 days, more preferably about 1 to 5 days. The present invention also includes a sample manufactured by such a method.
[0089]
“The liquid component is substantially free of high-molecular-weight acetylated chitosan” refers to a state in which a sample has high-molecular-weight acetylated chitosan and the high viscosity is reduced or eliminated. Although it does not mean that the concentration is strictly 0%, the content of the polymer partially acetylated chitosan is preferably 20% or less, more preferably 10% or less, and still more preferably 5% or less. Yes, optimally at 0%.
[0090]
【Example】
Examples and test examples will be described below, but the scope of the present invention is not limited to these.
[0091]
Embodiment 1 FIG. Aspergillus oryzae var. sporoflabus Ohara JCM
Purification of chitinase from 2067 strain
1) Preparation of crude enzyme solution
Aspergillus oryzae var. Is placed in a 500 ml Erlenmeyer flask (seed flask) containing 100 ml of the sterilized medium having the composition shown in Table 6. Cells of sporoflabus Ohara JCM 2067 strain were inoculated, and shaking culture was performed at 26 ° C. for 8 days at 100 rpm.
[0092]
[Table 6]
Culture medium
――――――――――――――――――――――――――――――
Malt Extract 10g
Yeast Extract 5g
2g powdered chitin
Or colloidal chitosan (made by Kimitsu Chemical Co., Ltd.) 2 g
The volume was adjusted to 1,000 ml with pure water.
――――――――――――――――――――――――――――――
After completion of the culture, centrifugation was performed at 10,000 ° G for 10 minutes at 4 ° C. The obtained supernatant was used as a crude enzyme solution.
[0093]
2) Enzyme activity measurement method
The hydrolysis activity of chitinase was measured as follows.
[0094]
(1) Hydrolysis of partially acetylated chitosan
50 ml of water and 30 μl of acetic acid were added to 125 mg of chitosan 7B (30% acetylated chitosan, manufactured by Katoyoshi) to completely dissolve the powdered chitosan. 50 ml of water and 50 μl of acetic acid were added to 125 mg of chitosan 8B (20% acetylated chitosan, manufactured by Katoyoshi Co., Ltd.) to completely dissolve the powdered chitosan. 50 ml of water and 50 μl of acetic acid were added to 125 mg of chitosan 9B (10% acetylated chitosan, manufactured by Katoyoshi Co., Ltd.) to completely dissolve the powdered chitosan. To 125 mg of chitosan 10B (almost completely deacetylated chitosan, manufactured by Katoyoshi Co., Ltd.), 50 ml of water and 90 μl of acetic acid were added to completely dissolve the powdered chitosan. 140 μl of the enzyme solution was added to a mixture of 160 μl of the chitosan aqueous solution and 100 μl of 400 mM acetate buffer (pH 5.0), and the mixture was stirred to make the mixture uniform, and the enzyme reaction was carried out at 37 ° C. for 20 minutes.
[0095]
(2) Determination of free reducing sugars by Randle-Morgan method
The reaction was stopped by adding 400 μl of a solution prepared by dissolving 10 μl of acetylacetone in 500 μl of a 0.5 M aqueous sodium carbonate solution to the enzyme reaction solution, followed by keeping the temperature at 100 ° C. for 20 minutes. After cooling on ice, 400 μl of a mixed solution of 0.8 g of N, N-dimethylaminobenzaldevidide in 30 ml of ethanol and 30 ml of concentrated hydrochloric acid and 1200 μl of ethanol were added to the enzyme reaction solution, and a color reaction was performed at 65 ° C. for 10 minutes. After cooling on ice, the precipitate was centrifuged, and the absorbance of the supernatant at 530 nm was measured. The enzymatic activity for producing 1 μmol of glucosamine per minute of the enzymatic reaction was defined as one unit.
[0096]
3) Preparation of crude enzyme solution
To 500 ml of the crude enzyme solution obtained in 1), 280 g of ammonium sulfate was gradually added while stirring under ice cooling. After allowing the mixed solution to stand overnight at 4 ° C., it was dialyzed 5 times for 12 hours each against 3,000 ml of 10 mM Tris / hydrochloric acid buffer (pH 7.5). This was added to a DEAE Toyopearl (manufactured by Tosoh Corporation) column (2.2 cm in diameter × 20 cm in length), which had been equilibrated with 10 mM Tris / hydrochloric acid buffer (pH 7.5) in advance, and adsorbed. After sufficiently washing the column with 10 mM Tris / HCl buffer (pH 7.5), a linear concentration gradient of 0 to 0.4 M sodium chloride was prepared in 600 ml of 10 mM Tris / HCl buffer (pH 7.5). The components adsorbed on the column were eluted. Chitosan-degrading activity was eluted in a fraction (135 ml) having a sodium chloride concentration of 0.20 M to 0.30 M. This was used as a crudely purified enzyme fraction.
[0097]
4) Preparation of purified enzyme solution
135 ml of the active fraction obtained in 3) was dialyzed three times for 12 hours each against 3,000 ml of 20 mM Tris / HCl buffer (pH 7.5), and then 20 mM Tris / HCl buffer (pH 7.5). It was added to a MonoQ (Pharmacia) column (diameter: 7 mm x length: 5 cm) equilibrated in and adsorbed. After sufficiently washing the column with 20 mM Tris / hydrochloric acid buffer (pH 7.5), a linear concentration gradient of 0 to 0.15 M sodium chloride in 250 ml of 10 mM Tris / hydrochloric acid buffer (pH 7.5) is prepared. The components adsorbed on the column were eluted. The chitosan-degrading activity was eluted in a fraction (10 ml) having a sodium chloride concentration of 0.054 M to 0.057 M. this
The fraction was used as a purified enzyme solution.
[0098]
5) Measurement of molecular weight of purified enzyme
The molecular weight of the purified enzyme was determined by SDS-PAGE electrophoresis using 12.5% polyacrylamide gel (see Laemmli, UK, Nature, 227, 680 (1970)). The following were used as standard proteins: a. Phosphorylase, molecular weight 94,000: b. Albumin, molecular weight 67,000: c. Ovalbumin, molecular weight 43000: d. Carbonic anhydrase, molecular weight 30,000: e. Trypsin inhibitor, molecular weight 20,100: f. α-lactalbumin, molecular weight 14,400.
The calibration curve obtained from the mobility of these standard proteins is shown in FIG. As shown in FIG. 1, the purified enzyme showed a single band having a molecular weight of about 40,000.
[0099]
6) Isoelectric point of purified enzyme
The purified enzyme was subjected to isoelectric focusing (see “Biochemical Experiment Course 1 Protein Chemistry I Separation and Purification, edited by The Society of Biochemistry, Japan, Tokyo Kagaku Dojin (1976)”, pp. 262 to 312). The isoelectric point of the purified enzyme was about pI4.5.
[0100]
7) Determination of partial amino acid sequence
The purified enzyme solution was concentrated to about 1 mg / ml using an ultrafiltration membrane (ULTRA FILTER UNIT USY-1, manufactured by ADVANTEC, molecular weight fraction 10,000). 400 μl of Denature buffer (6 M guanidine hydrochloride, 10 mM EDTA, 0.1 M ammonium bicarbonate pH 7.8) and 8 μl of 50 mM Dithiothreitol were added to 400 μl of the concentrated enzyme solution, and the mixture was reacted at 95 ° C. for 10 minutes. After cooling to room temperature, 40 μl of 50 mM iodoacetoamide dissolved in Denatur buffer solution was added to the reaction solution, and the mixture was reacted at room temperature in the dark for 1 hour. This solution was dialyzed against 1 L of water with Slide-A-Lyzer Mini Dialysis Units Plus Float (PIERCE, molecular weight fraction 10,000). To the obtained solution, 8 μl of trypsin (Modified, manufactured by Promega) was added, and an enzyme reaction was performed at 37 ° C. for 7 hours. The reaction solution was applied to a SMART system (manufactured by Pharmacia Biotech) to separate the peak of the decomposed amino acid. The separation conditions are shown below.
Column: Nomura Chemical Develosil 300 CDS-HG-5 (1 mm x 150 mm)
A buffer: 0.1% TFA / Water
B buffer: 0.1% TFA / Acetonitril
Gradient: 5 → 60% B 1% / min.
Flow: 50 μl / ml
Among the separated amino acids, two peaks with a B buffer concentration of about 15% and a peak with a B buffer concentration of about 35% were collected. The amino acid sequences of these three samples (No. 1 to No. 3) were analyzed using an amino acid sequence analyzer (Procise cLC, manufactured by Applied Biosystem). The partial amino acid sequence obtained as a result is described from the amino terminal side.
No. 1: Glu Met Thr Pro Tyr Leu Asp Phe Tyr Arg Leu Met Ala Tyr Gln (SEQ ID NO: 1 in Sequence Listing);
No. 2: Ile Val Val Gly Met Pro Ile Tyr Gly Arg Lys Glu (SEQ ID NO: 2 in Sequence Listing);
No. 3: Ala Leu Pro Lys Pro Gly Ala Thr Glu Tyr Val Asp Pro Ser Ile (SEQ ID NO: 3 in Sequence Listing).
[0101]
Embodiment 2. FIG. Purification of chitinase from Aspergillus japonicus SANK 19288 strain
1) Preparation of crude enzyme solution
Cells of Aspergillus japonicus SANK 19288 strain were inoculated into a 500 ml Erlenmeyer flask (seed flask) containing 100 ml of the sterilized medium having the composition shown in Table 7, and cultured at 26 ° C. for 8 days with shaking at 100 rpm. Was. After completion of the culture, centrifugation was performed at 10,000 × G for 10 minutes at 4 ° C. The obtained supernatant was used as a crude enzyme solution.
[0102]
[Table 7]
Culture medium
――――――――――――――――――――
Malt Extract 10g
Yeast Extract 5g
5g powdered chitin
The volume was adjusted to 1,000 ml with pure water.
――――――――――――――――――――
2) Preparation of purified enzyme solution
To 200 ml of the crude enzyme solution obtained in 1), 112 g of ammonium sulfate was gradually added while stirring under ice cooling. After the mixed solution was allowed to stand at 4 ° C. overnight, it was dialyzed 5 times for 12 hours each against 3,000 ml of 20 mM Tris / hydrochloric acid buffer (pH 7.5) to obtain a crudely purified enzyme fraction. The crude enzyme fraction was added to a MonoQ (Pharmacia) column (diameter 7 mm × length 5 cm), which had been equilibrated with 20 mM Tris / hydrochloric acid buffer (pH 7.5), and adsorbed. After sufficiently washing the column with 20 mM Tris / hydrochloric acid buffer (pH 7.5), a linear concentration gradient of 0 to 0.15 M sodium chloride in 250 ml of 10 mM Tris / hydrochloric acid buffer (pH 7.5) is prepared. The components adsorbed on the column were eluted. The chitosan-degrading activity was eluted in a fraction (10 ml) having a sodium chloride concentration of 0.063 M to 0.066 M. This fraction was used as a purified enzyme solution.
[0103]
3) Molecular weight of purified enzyme
The molecular weight was measured in the same manner as in Example 15). This enzyme showed a single band with a molecular weight of about 40,000.
[0104]
4) Isoelectric point of purified enzyme
The isoelectric point was measured in the same manner as in Example 16). The isoelectric point of this enzyme was about pI 3.5.
[0105]
Embodiment 3 FIG. Purification of chitinase from Aspergillus sojae SANK 22388 strain
1) Preparation of crude enzyme solution
The cells of Aspergillus sojae SANK 22388 strain were inoculated into a 500 ml Erlenmeyer flask (seed flask) containing 100 ml of the sterilized medium having the composition shown in Table 8, and cultured at 26 ° C. for 8 days with shaking at 100 rpm. Was. After completion of the culture, centrifugation was performed at 10,000 × G for 10 minutes at 4 ° C. The obtained supernatant was used as a crude enzyme solution.
[0106]
[Table 8]
Culture medium
――――――――――――――――――――
Malt Extract 10g
Yeast Extract 5g
5g powdered chitin
The volume was adjusted to 1,000 ml with pure water.
――――――――――――――――――――
2) Preparation of purified enzyme solution
To 200 ml of the crude enzyme solution obtained in 1), 112 g of ammonium sulfate was gradually added while stirring under ice cooling. After the mixed solution was allowed to stand at 4 ° C. overnight, it was dialyzed 5 times for 12 hours each against 3,000 ml of 20 mM Tris / hydrochloric acid buffer (pH 7.5) to obtain a crudely purified enzyme fraction. The crude enzyme fraction was added to a MonoQ (Pharmacia) column (diameter 7 mm × length 5 cm), which had been equilibrated with 20 mM Tris / hydrochloric acid buffer (pH 7.5), and adsorbed. After sufficiently washing the column with 20 mM Tris / hydrochloric acid buffer (pH 7.5), a linear concentration gradient of 0 to 0.15 M sodium chloride in 250 ml of 10 mM Tris / hydrochloric acid buffer (pH 7.5) is prepared. The components adsorbed on the column were eluted. The chitosan degrading activity was eluted in a fraction (10 ml) having a sodium chloride concentration of 0.062M to 0.064M. This fraction was used as a purified enzyme solution.
3) Molecular weight of purified enzyme
The molecular weight was measured in the same manner as in Example 15). This enzyme showed a single band with a molecular weight of about 40,000.
[0107]
4) Isoelectric point of purified enzyme
The isoelectric point was measured in the same manner as in Example 16). The isoelectric point of this enzyme was about pI4.5.
Embodiment 4. FIG. Aspergillus oryzae var. Preparation of low molecular weight chitosan hydrochloride using chitinase derived from sporoflabus Ohara JCM 2067 strain
2.0 g of chitosan 9B or 8B or 7B was suspended in 800 ml of water, and concentrated hydrochloric acid was added to adjust the pH to 2.5, and the powder was completely dissolved. After adjusting the pH to 5.0 with saturated aqueous sodium hydrogen carbonate, water was added to make the total volume 1,000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, and 10 ml of the enzyme solution prepared in Example 11) was added. In addition, the mixture was shaken at 40 rpm at 80 rpm for 3 days to carry out an enzyme reaction. After the completion of the reaction, all the reaction solutions were collected, adjusted to pH 12 or more with 1% sodium hydroxide solution under ice cooling, and centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The collected precipitate was suspended in 200 ml of water, dissolved in ice-cooled concentrated hydrochloric acid to adjust the pH to 2.5, and dialyzed against water using a dialysis membrane having a molecular weight cut-off of 10,000. After completion of the dialysis, the solution was filtered, and the filtrate was freeze-dried. Low molecular weight chitosan 9B hydrochloride was obtained as 2.0 g of a white flocculent solid. Low molecular weight chitosan 8B hydrochloride was obtained as 1.6 g of a white flocculent solid. Low molecular weight chitosan 7B hydrochloride was obtained as 0.6 g of a white flocculent solid.
[0108]
Embodiment 5 FIG. Preparation of low molecular weight chitosan hydrochloride using chitinase derived from Aspergillus japonicus SANK 19288 strain
Low-molecular-weight chitosan hydrochloride was prepared in the same manner as in Example 4. The enzyme solution used in Example 21) was used, and the enzyme reaction was carried out at pH 4.0. Low molecular weight chitosan 9B hydrochloride was obtained as 1.3 g of a white flocculent solid. Low molecular weight chitosan 8B hydrochloride was obtained as 1.3 g of a white flocculent solid. Low molecular weight chitosan 7B hydrochloride was obtained as 0.9 g of a white flocculent solid.
[0109]
Embodiment 6 FIG. Preparation of low molecular weight chitosan hydrochloride using chitinase derived from Aspergillus sojae SANK 22388 strain
A low molecular weight chitosan hydrochloride was prepared in the same manner as in Example 4. However, the enzyme solution prepared in Example 3 was used, and the enzyme reaction was performed at pH 4.5. Low molecular weight chitosan 9B hydrochloride was obtained as 2.1 g of a white flocculent solid. Low molecular weight chitosan 8B hydrochloride was obtained as 1.7 g of a white flocculent solid. Low molecular weight chitosan 7B hydrochloride was obtained as 1.0 g of a white flocculent solid.
[0110]
Embodiment 7 FIG. Acetylation degree of low molecular weight chitosan hydrochloride
100 mg of the low molecular weight chitosan hydrochloride prepared in Examples 4 to 6 was dissolved in 10 ml of distilled water, 10 U of chitosanase (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution is freeze-dried, ₂ Dissolve in O ¹ 1 H-NMR was measured.
The degree of acetylation was calculated from the integral ratio of 3H of the acetyl group to 7H other than the acetyl group, excluding the hydroxyl group. The results are shown in Table 9.
[0111]
[Table 9]

Embodiment 8 FIG. Determination of molecular weight fraction by gel filtration
As a molecular weight marker, dextran T10 (molecular weight 10,000), T40 (molecular weight 40,000), T70 (molecular weight 70,000), T110 (molecular weight 110,000), T250 (molecular weight 250,000) (manufactured by Pharmacia Co., Ltd.) ) Was developed on a column (86 × 2 cm) packed with a gel filtration material (Toyopearl HW55 Fine) which had been swollen in advance with a 200 mM acetate buffer at pH 4.5, and fractionated into 1.75 ml portions. did. 1.8 ml of a sulfuric acid solution obtained by adding 8 volumes of concentrated sulfuric acid to 1 volume of water, and 60 μl of a solution obtained by dissolving 0.25 g of carbazole in 50 ml of ethanol were added to 200 μl of each fraction, and the mixture was kept at 100 ° C. for 10 minutes. After cooling on ice, the absorbance at 420 nm was measured.
[0112]
200 μl of each 5% solution of the low-molecular-weight chitosan hydrochloride prepared in Examples 4, 5, and 6 was fractionated in 1.75 ml in the same manner. 100 μl of 400 mM MOPS-sodium carbonate buffer at pH 6.5 and 10 U of chitosanase (manufactured by Wako Pure Chemical Industries, Ltd.) were added to 280 μl of each fraction, and an enzyme reaction was performed at 37 ° C. for 10 minutes. The quantification of free reducing sugars was as in Example 1. For the molecular weight marker and each low molecular weight chitosan, the fraction showing the maximum absorbance was set to 100% and plotted on the same graph (FIGS. 2 to 4). Table 10 summarizes the molecular weight of the low molecular weight chitosan estimated from the figure.
[0113]
[Table 10]

Embodiment 9 FIG. Isolation of genomic DNA encoding chitinase from Aspergillus oryzae
9-1) Isolation of genomic DNA of Aspergillus oryzae Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 strain was shake-cultured in 100 ml of a medium (1% Malt Extract, 0.5% Yeast Extract) at 30 ° C. for 4 days, and the culture was collected using a filtration device to collect cells. Subsequently, the cells were placed in a mortar cooled to -80 ° C, and liquid nitrogen was poured. The cells were crushed sufficiently using a pestle while adding liquid nitrogen until the cells became powdery. About 1.5 g of the powdered bacterial cells was added to 20 ml of a lysis buffer (62.5 mM EDTA, 50 mM Tris-HCl, 50 mM SDS: pH 8.0) and gently stirred. Next, 10 ml of 9.5 M ammonium acetate was added thereto, and further 15 ml of TE-saturated phenol was added, followed by gentle stirring. This solution was centrifuged at 5,000 rpm for 5 minutes at 4 ° C. using a high-speed cooling centrifuge (Rotor No. 3 manufactured by Hitachi), and about 25 ml of the supernatant was recovered. 15 ml of chloroform was added to the collected supernatant, and the mixture was gently stirred and then centrifuged at 4 ° C. and 5000 rpm for 10 minutes. The supernatant was collected in a new tube, an equal amount of 2-propanol to the supernatant was added, the mixture was stirred, and then centrifuged at 10,000 rpm at 4 ° C for 10 minutes. After dissolving the precipitate in 400 µl of TE, the precipitate was transferred to a 1 ml microtube, and 10 µl of 10 mg / ml ribonuclease was added, followed by treatment at 37 ° C for 20 minutes. Next, 40 μl of 3M sodium acetate and 400 μl of chloroform were added, stirred, and centrifuged at 10,000 rpm for 5 minutes at room temperature. The separated supernatant was collected in a microtube, and 1 ml of 100% ethanol (cooled at −20 ° C.) was added thereto, followed by gentle stirring. The thread-shaped DNA was wound up with a glass rod, and after ethanol was removed, the DNA was dissolved in 200 μl of TE.
9-2) Preparation of Aspergillus oryzae genomic DNA library
10 μg of Aspergillus oryzae genomic DNA obtained in 9-1) was mixed with 10 μl of restriction enzyme EcoRI (manufactured by Takara Shuzo Co., Ltd.) to obtain 50 mM Tris-HCl (pH 7.5) and 10 mM MgCl 2. ₂ After a cleavage treatment at 37 ° C. for about 2 hours in a solution consisting of 1 mM DTT and 100 mM NaCl, DNA was recovered. The EcoRI digested product of Aspergillus oryzae genomic DNA and the vector also digested with EcoRI, pUC118 EcoRI / BAP (manufactured by Takara Shuzo) were ligated using Rapid DNA Ligation Kit (manufactured by Roche Diagnostics). A genomic DNA library of Aspergillus oryzae was used.
9-3) Preparation of DIG (digoxigenin) -labeled DNA probe
The oligo primer PR50 identified from the genomic library of Aspergillus oryzae was synthesized using the mixed primers F21 and F21 prepared based on the internal amino acid sequence (SEQ ID NO: 2 in the sequence listing) identified in Example 1.
[0114]
F21: 5'-attggcataccaacactactttataggc-3 '(SEQ ID NO: 7 in Sequence Listing)
PR50: 5'-taaaattgacccatatcctcttatgcatt-3 '(SEQ ID NO: 8 in Sequence Listing)
Using these two primers, PCR using Aspergillus oryzae genomic DNA as a template to increase the DNA of about 800 bp. This DNA was separated by electrophoresis, cut out from an agarose gel, and then purified using QIAquick Gel Extraction Kit (Qiagen). The DNA thus purified was labeled with DIG DNA Labeling Kit (Roche Diagnostics). Labeling was performed as follows. First, the purified DNA was heated at 100 ° C. for 10 minutes to form a single strand, and then rapidly cooled on ice. Then 10 μl hexanucleotide mix (0.5 M Tris, 0.1 M MgCl 2) ₂ 1 mM Dithioerithritol, 2 mg / ml BSA; pH 7.2, 10 μl dNTP labeling mix (1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM DIG-11-dUTP; pH 7.5), 5 μl DNA A polymerase (Klenow fragment) was added, and the total volume was adjusted to 100 μl with sterile water. After the reaction solution was reacted at 37 ° C. overnight, 10 μl of 0.2 M EDTA (pH 8.0) was added to stop the reaction. Then, 10 μl of 4N LiCl and 500 μl of 100% ethanol (cooled at −20 ° C.) were added, and the mixture was allowed to stand at −70 ° C. for 30 minutes to precipitate labeled DNA. The mixture was centrifuged at 4 ° C. and 15000 rpm for 10 minutes in a cooling centrifuge. The ethanol was discarded, and 100 μl of 70% ethanol (cooled at −20 ° C.) was added to wash the pellet, followed by centrifugation at 15,000 rpm for 10 minutes, and complete removal of the ethanol with a pipette. After the pellet was dried, the pellet was dissolved in 50 μl of TE and added to 25 ml of DIG Easy Hyb (manufactured by Roche Diagnostics) to obtain a chitinase gene probe.
9-4) Preparation of partial DNA library containing chitinase gene
20 μg of Aspergillus oryzae genomic DNA obtained in 9-1) was cleaved with a restriction enzyme EcoRI, and then purified to recover DNA. The purified DNA was separated by electrophoresis on an agarose gel, and blotted on a nylon membrane. Next, as a result of Southern hybridization using the chitinase gene probe obtained in 9-3), one single band containing the chitinase gene was obtained. Then, 80 μg of Aspergillus oryzae genomic DNA obtained in 9-1) was cleaved with a restriction enzyme EcoRI, and then purified to recover DNA. This DNA was electrophoresed on an agarose gel, and the same position as that of the band observed by the above-mentioned Southern hybridization was cut out from the gel, and the DNA was extracted. The extracted DNA was ligated to a pUC118 EcoRI / BAP vector to obtain a DNA library containing a chitinase gene.
9-5) Screening of DNA containing chitinase gene
Escherichia coli JM109 strain (Takara Shuzo) was transformed with the DNA library obtained in 9-4). A transformant colony was formed on an agar medium, and a nylon transfer membrane (manufactured by Amersham) was overlaid thereon, and a part of the colony was transferred onto the membrane. This membrane is allowed to stand on a filter paper soaked with an alkali denaturing solution (0.5 M NaOH, 3.0 M NaCl) for 20 minutes, and then on a filter paper soaked with a neutralization solution (1 M Tris-HCl (pH 7.5)). Placed for 10 minutes. Further, the plate was washed with a 2 × SSC solution while shaking. Next, the membrane was pre-hybridized with DIG Easy Hyb for 1 hour. Thereafter, the membrane was hybridized with the chitinase gene probe obtained in 9-3) overnight at 37 ° C. After hybridization, the plate was washed three times with 2 × SSC buffer at normal temperature, and further twice with 2 × SSC buffer warmed to 53 ° C. Thereafter, detection was performed using DIG Wash and Block Buffer Set (manufactured by Roche Diagnostics). First, the membrane was acclimated with Wash buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween 20: pH 7.5). Next, this membrane was immersed in a blocking solution (0.1 M maleic acid, 0.15 M NaCl: a solution containing 10% blocking reagent in pH 7.5) to perform blocking for 60 minutes to block non-specific binding. Next, the membrane was immersed in a solution obtained by adding an anti-digoxigenin antibody to the blocking solution at a 10000-fold dilution, reacted at 37 ° C. for 30 minutes, and then washed twice with a Wash Buffer. Thereafter, the membrane was acclimated with a detection buffer (0.1 M Tris-HCl, 0.1 M NaCl: pH 9.5). CSPD (Chemilluminance substrate) was added to the detection buffer at 100-fold dilution, and the solution was added dropwise to the purified membrane, and the color development was stabilized at 37 ° C. for 15 minutes. Thereafter, the resultant was exposed to a Lumi-Film Chemiluminescent Defection Film (manufactured by Konica) and developed. As a result, a clone showing a positive signal was confirmed.
9-6) Extraction and sequence analysis of recombinant plasmid DNA
The clone showing the positive signal obtained in 9-5) was cultured overnight in L-broth (manufactured by Wako Pure Chemical Industries, Ltd.), and the DNA of the recombinant plasmid was extracted. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (manufactured by Qiagen). Subsequently, the extracted DNA was subjected to sequence analysis using the universal primers M13M4 and M13RV unique to this plasmid, and the results were aligned with the nucleotide sequence of the chitinase gene probe obtained in 9-3), and the sequence of the chitinase was determined. I confirmed that it included. The entire sequence of the plasmid DNA confirmed to contain the chitinase sequence was determined using GPS-1 Genome Priming System (New England Biolabs). The procedure was as follows. First, 4 μl of the plasmid DNA extracted above was added to 2 μl of 10 × GPS Buffer (250 mM Tris-HCl; pH 8.0, 20 mM DTT, 20 mM ATP, 500 μg / ml BSA), 1 μl pGPS1 (Transprimer-1 Donor plasmamid), 11 μl After adding sterile water and mixing, 1 μl TnsABC Transposase was added and allowed to bind at 37 ° C. for 10 minutes. Thereafter, 1 μl Start Solution (300 mM magnesium acetate) was added, the reaction was performed at 37 ° C. for 1 hour, and the reaction was stopped at 75 ° C. for 10 minutes. Next, 10 μl of this reaction product was transformed into Escherichia coli JM109 strain, applied to an LB agar medium containing ampicillin and kanamycin, and cultured at 37 ° C. overnight. The growing colonies were cultured overnight in an LB liquid medium, and plasmid DNA was extracted. The sequence of the extracted plasmid DNA was read using N and S primers, which are pGPS1 primers, and the entire genomic sequence encoding chitinase was determined.
Embodiment 10 FIG. Identification of cDNA encoding chitinase from Aspergillus oryzae
10-1) Purification of total RNA of Aspergillus oryzae
Aspergillus oryzae var. sporoflabus Ohara JCM 2067 strain was pre-cultured in Potato Dextrose medium (20 ml) at 30 ° C for 3 days. Thereafter, 1% of the cells was inoculated into a liquid medium (1% Malt Extract, 0.5% Yeast Extract) and cultured at 30 ° C. for 3 days. The cultured cells were collected by suction and transferred to a mortar (autoclaved) cooled at -80 ° C. While adding liquid nitrogen, the cells were crushed with a pestle to make a powder. The completely powdered microbial cells were purified using Rneasy Plant Mini Kit (Qiagen) as follows. First, about 1 g of powdered bacterial cells was added to 4.5 ml RLT buffer (β-Mercaptoethanol), and the cells were shaken vigorously to dissolve the cells and placed on ice. Each 0.5 ml was dispensed into a QIAshredder spin column, and centrifuged at room temperature at 15,000 rpm for 2 minutes. A 0.5-fold amount of ethanol was added to the passing solution, and the mixture was stirred by pipetting. Thereafter, 675 μl was dispensed into a Rneasy Mini spin column, and centrifuged at room temperature at 16,000 rpm for 15 seconds. 700 μl of RW1 Wash buffer was added and centrifuged at 16,000 rpm for 15 seconds at room temperature in the same manner. Next, 500 μl RPE Wash buffer was added, and the RNA was washed by centrifugation at room temperature at 16,000 rpm for 2 minutes. The spin column was set in a microtube, and RNA was eluted with 50 μl of Rnase free water, and collected by centrifugation at 10,000 rpm for 1 minute at room temperature.
10-2) Preparation of Aspergillus oryzae cDNA library
The Aspergillus oryzae cDNA library was prepared using SMART cDNA Library Construction Kit (Clontech). First, 1 μg of total RNA purified in 10-1) and 1 μl SMART IV Oligonucleotide, 1 μl CDS III / 3 ′ PCR Primer were mixed in a sterilized 0.5 ml tube, and incubated at 72 ° C. for 2 minutes. Then, it was placed on ice for 2 minutes and cooled. To this cooled sample, 2 μl 5 × First-Strand Buffer, 1 μl 20 mM DTT, 1 μl 10 mM dNTP Mix and 1 μl Power Script Reverse Transcriptase were added. The contents were mixed and reacted at 42 ° C. for 1 hour, and then placed on ice to stop first-strand DNA synthesis. Next, cDNA was amplified by LD PCR. 2 μl of the First-Strand DNA was added to the PCR tube, and the following reagents were further added: 80 μl deionized water, 10 μl 10 × Advantage2 PCR Buffer, 2 μl 50 × dNTP Mix, 2 μl 5 ′ PCR Primer, 2 μl CDS III / 3 ′ PCR Primer, 2 μl 50 × Advantage 2 Polymerase Mix. The PCR tube was set in a thermal cycler preheated to 95 ° C., reacted at 95 ° C. for 20 seconds, and then subjected to 20 cycles of 5 seconds at 95 ° C. and 6 minutes at 68 ° C. 50 μl of this reaction solution (including ds cDNA) was placed in a sterilized tube, 2 μl of protease K was added, the mixture was incubated at 45 ° C. for 20 minutes, protease treatment was performed, and 50 μl of deionized water was added. 100 μl of a phenol: chloroform: isoamyl alcohol mixture (25: 24: 1) was added, mixed for 1 minute, and centrifuged at 14000 rpm for 5 minutes. The supernatant was transferred to a new tube and 100 μl chloroform: isoamyl alcohol (24: 1) was added. After mixing for 1 minute, the mixture was centrifuged again at 14000 rpm for 5 minutes. The supernatant was transferred to a new tube, 10 μl of 3 M sodium acetate, 1.3 μl of glycogen and 260 μl of 95% ethanol (room temperature) were added, and the mixture was immediately centrifuged at room temperature at 14000 rpm for 20 minutes. The supernatant was removed with a pipette, and the pellet was washed with 100 μl of 80% ethanol, and then the pellet was dried. The pellet was dissolved in 79 μl deionized water. Subsequently, treatment with the restriction enzyme Sfi I was performed. 79 μl cDNA, 10 μl 10 × Sfi Buffer, 10 μl Sfi Enzyme and 1 μl 100 × BSA were mixed and the tubes were incubated at 50 ° C. for 2 hours. Then, 2 μl of 1% xylene cyanol staining solution was added. The cDNA was molecular sieved using a CHROMA SPIN-400 column. The CHROMA SPIN column was mixed several times by inversion to suspend the gel matrix, and further suspended with a Pipetman. The lid of the column was removed and the column was set up to drip naturally. The column buffer was then gently added and allowed to drip naturally by gravity. After the drip of this buffer was completed, a cDNA sample degraded with Sfi I was applied to the center of the upper surface of the matrix, and was sufficiently absorbed on the surface of the matrix. When the liquid was removed from the surface and the drip was completed, 600 μl column buffer was added and fractions were immediately collected drop by drop into 16 test tubes. Each 3 μl of this fraction was subjected to agarose gel electrophoresis, and the peak fraction was determined. Fractions containing cDNA were collected and collected in one new tube. To this test tube, add 1/10 volume of sodium acetate (3M; pH 4.8), 1.3 μl glycogen, and 2.5 volume of 95% ethanol (−20 ° C.), shake gently, and mix. Incubated overnight at ° C. The mixture was centrifuged at room temperature at 14000 rpm for 20 minutes, and the supernatant was removed. After the pellet was completely dried, the pellet was resuspended in 7 μl deionized water. 0.5 μl of the above cDNA was mixed with 1 μl λ TriplEx2 vector, 0.5 μl 10 × Ligation Buffer, 0.5 μl ATP, 0.5 μl T4 DNA Ligase and 2.0 μl deionized water and incubated at 16 ° C. overnight. Next, a λ phage packaging reaction was performed on this ligation sample. Gigapack III Gold Packaging Extract (manufactured by STRATAGENE) was used for packaging. The DNA vector ligated as described above was added to a microtube containing Packing Extract in an amount of 4 μl / l, mixed gently by pipetting, and then incubated at room temperature for 2 hours. Two hours later, 500 µl of SM buffer was added, and 20 µl of chloroform was further mixed, followed by light centrifugation to prepare a packaging solution. Next, host cells to be infected with the packaged phage were cultured. E. FIG. The frozen stock of E. coli XL1-Blue was inoculated on LB / tetracycline agar plate and cultured at 37 ° C. overnight. Single grown colonies ₄ And tetracycline, and cultured overnight at 37 ° C. A single colony that grew was cultivated in 15 ml of LB / MgSO. ₄ / Inoculate maltose broth and OD ₆₀₀ Was cultured at 37 ° C. until the pH of the mixture became 2.0. The culture was centrifuged at 5000 rpm for 5 minutes and the pellet was reconstituted with 7.5 ml of 10 mM MgSO4. ₄ Was resuspended. After diluting the packaging solution with 1 × λ dilution buffer, 1 μl of the packaging solution was added to 200 μl of E. coli. coli XL1-Blue suspension and allowed to adsorb at 37 ° C. for 15 minutes. Add 2 ml of molten LB / MgSO 4 to the reaction solution. ₄ Mix top agar and warm LB / MgSO ₄ Spread evenly on plate. After cooling at room temperature for 10 minutes to solidify the top agar, the cells were cultured overnight at 37 ° C. The obtained plaque was added with SM buffer and shaken overnight to elute it, thereby obtaining a phage containing an Aspergillus oryzae cDNA library.
10-3) Screening of cDNA containing chitinase gene
The phage containing the cDNA library obtained in 10-2) was infected into Escherichia coli XL1-Blue strain (Clontech). 1 μl of the diluted phage was added to the cultured 200 μl XL1-Blue, and adsorbed at 37 ° C. for 10 to 15 minutes. 2 ml of melted LB / MgSO ₄ Add top agar, mix and warm LB / MgSO 4 ₄ Pour into a plate, rotate the plate and spread the top agar evenly. After cooling at room temperature for 10 minutes to solidify the top agar, the mixture was incubated at 37 ° C. overnight to form plaque on an agar medium. A nylon transfer membrane (manufactured by Amersham) was overlaid thereon for about 1 minute to transfer a part of the phage onto the nylon membrane. After drying the membrane on a filter paper for 20 minutes, the membrane is allowed to stand on a filter paper soaked with an alkali denaturing solution (0.2 N NaOH, 1.5 M NaCl) for 5 minutes, and then neutralized solution (0.4 M Tris-HCl) (PH 7.5, 2 × SSC) was placed on the filter paper soaked for 5 minutes. After washing with a neutralizing solution for 1 minute while shaking, it was further washed with 2 × SSC for 5 minutes. The filter was dried on a filter paper for about 1 hour and baked in an oven at 80 ° C. for 2 hours. Thereafter, the membrane was hybridized with the DNA probe obtained in X-3 at 37 ° C. overnight. After hybridization, the plate was washed three times with 2 × SSC buffer at normal temperature, and further twice with 2 × SSC buffer warmed to 53 ° C. Thereafter, detection was performed using DIG Wash and Block Buffer Set (manufactured by Roche Diagnostics). First, the membrane was acclimated with Wash buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% Tween 20; pH 7.5). Next, the membrane was immersed in a blocking solution (0.1 M maleic acid, 0.15 M NaCl; a solution containing 10% blocking reagent at pH 7.5) to perform blocking for 60 minutes to block nonspecific binding. Next, the membrane was immersed in a solution obtained by adding an anti-digoxigenin antibody to the blocking solution at a 10000-fold dilution, reacted at 37 ° C. for 30 minutes, and then washed twice with a Wash Buffer. Thereafter, the membrane was acclimated with a detection buffer (0.1 M Tris-HCl, 0.1 M NaCl; pH 9.5). CSPD (Chemilluminance substrate) was added to the detection buffer at 100-fold dilution, and the solution was added dropwise to the purified membrane, and the color development was stabilized at 37 ° C. for 15 minutes. After that, it was exposed to a Lumi-Film Chemiluminescent Decimation Film (manufactured by Konica), developed, and detected. As a result, a clone showing a positive signal could be detected. A phage plaque showing this positive was cut out along with the agar medium, dissolved in SM buffer, transformed into Escherichia coli BM25.8 strain, and transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 strain was obtained. Was. This strain was internationally deposited on November 8, 2002 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, under the accession number: FERM BP-8235. The infected phage DNA, λTriplEx-Chitinase-cDNA, cyclizes in the cells of Escherichia coli BM25.8 strain and exists as a recombinant plasmid pTriplEx-Chitinase-cDNA. A colony of the obtained Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 strain was cultured overnight in LB / Ampilicin broth, and DNA of the recombinant plasmid pTriplEx-Chitinase-cDNA was extracted. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (manufactured by Qiagen). Next, 2 μl of 10 × GPS Buffer (250 mM Tris-HCl; pH 8.0, 20 mM DTT, 20 mM ATP, 500 μg / ml BSA), 1 μl of pGPS1 (Transprimer-1 Donor plasmid), and 11 μl of sterilized water were added to 4 μl of the plasmid DNA. After that, 1 μl TnsABC Transposase was added, and binding was performed at 37 ° C. for 10 minutes. Thereafter, 1 μl Start Solution (300 mM magnesium acetate) was added, the reaction was performed at 37 ° C. for 1 hour, and the reaction was stopped at 75 ° C. for 10 minutes. Next, 10 μl of this reaction product was transformed into Escherichia coli JM109 strain, applied to an LB agar medium containing ampicillin and kanamycin, and cultured at 37 ° C. overnight. The growing colonies were cultured overnight in an LB liquid medium, and plasmid DNA was extracted. The sequence of the extracted plasmid DNA was read with N and S primers as primers of pGPS1, and the nucleotide sequence of the DNA encoding chitinase of Aspergillus oryzae was determined (SEQ ID NO: 4 in the sequence listing). Also, from the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, the open reading frame (ORF) uses the “ATG” at nucleotides 242 to 244 as a start codon and terminates “TAG” at nucleotides 1436 to 1438. It was a codon region, and it was inferred that the encoded amino acid sequence was the sequence shown in SEQ ID NO: 5 in the sequence listing.
Embodiment 11 FIG. Determination of N-terminal amino acid sequence of chitinase from Aspergillus oryzae
The enzyme solution purified in Example 1 was concentrated to about 1 mg / ml using an ultrafiltration membrane (ULTRA FILTER UNIT USY-1, manufactured by ADVANTEC, molecular weight fraction 10,000). 400 μl of Denatur buffer (6 M guanidine hydrochloride, 10 mM EDTA, 0.1 M ammonium bicarbonate, pH 7.8) and 8 μl of 50 mM Dithiothreitol were added to 400 μl of the concentrated enzyme solution, and the mixture was reacted at 95 ° C. for 10 minutes. After cooling to room temperature, 40 μl of 50 mM iodoacetoamide dissolved in Denatur buffer solution was added to the reaction solution, and the mixture was reacted at room temperature in the dark for 1 hour. This solution was dialyzed against 1 L of water with Slide-A-Lyzer Mini Dialysis Units Plus Float (PIERCE, molecular weight fraction 10,000). To the obtained solution, 8 μl of trypsin (Modified, manufactured by Promega) was added, and an enzyme reaction was performed at 37 ° C. for 7 hours. The obtained enzyme reaction solution was measured by LC / MS (Pencon / Q-Tof2). MS / MS spectrum was measured by setting m / z 533 as Include mass. Assignment of the fragment ion suggested that the N-terminal amino acid was Ser, that the N-terminus contained the amino acid sequence of Ser-Ser-Gly-Leu-Lys, and that the N-terminus had an acetyl group (see Sequence Listing). Amino acid sequence of amino acid numbers 1 to 5 of SEQ ID NO: 14). When this result is compared with the amino acid sequence of SEQ ID NO: 5 in the sequence listing, the chitinase produced by Aspergillus oryzae has its N-terminus, "Met-Ser-Ser", undergoes post-translational modification and becomes "Acetyl-Ser". -Ser "(SEQ ID NO: 14 in the sequence listing).
Embodiment 12 FIG. Expression of chitinase gene of Aspergillus oryzae
Primers having BamHI sites connected to the 5 'end and 3' end of the chitinase ORF, BamHI-cDNA and cDNA-BamHI were designed and synthesized.
[0115]
BamHI-cDNA: 5'-gaggatccatgtctttccgggtaaaagtcgg-3 '(SEQ ID NO: 9 in Sequence Listing)
cDNA-BamHI: 5′-ctggatccagcgaaaaccggctcgcaggttg-3 ′ (SEQ ID NO: 10 in Sequence Listing)
PCR was performed using the recombinant plasmid obtained in Example 10-3), pTriplEx-Chitinase-cDNA as a template, and the above-described BamHI-cDNA and cDNA-BamHI as primers, to obtain a chitinase DNA into which a restriction enzyme site was introduced. Amplified. Furthermore, it was inserted into pCR 2.1-TOPO vector (manufactured by Invitrogen), transformed into E. coli JM109 strain, and amplified. The colony in which the JM109 strain was grown was cultured overnight in Lb / Ampilicin broth to extract plasmid DNA. This plasmid DNA was treated with the restriction enzyme BamHI to cut the chitinase ORF. In addition, as a vector to which this fragment was ligated, pQE-60 (manufactured by Qiagen) was digested with BamHI, and further treated with alkaline phosphatase to remove the phosphate group at the 5 'end. Thereafter, ligation was performed with the above-mentioned chitinase ORF using TOPO TA Cloning Kit (manufactured by Invitrogen). The reaction solution was transformed into Escherichia coli JM109 strain and cultured overnight on an Lb / Ampilicin agar plate. The growing colony was inoculated into 3 ml of Lb / Ampilicin broth and cultured overnight with shaking. Plasmid DNA was extracted from the culture with QIAprep Spin Miniprep Kit (manufactured by Qiagen) and transformed into Escherichia coli M15 [pREP4] strain. The cells were cultured overnight on an Lb / Ampilicin, Kanamaicin agar plate, and the growing colonies were inoculated into 20 ml of Lb / Ampilicin, Kanamaicin broth and cultured at 37 ° C. overnight with shaking. 2.5 ml of this culture solution was inoculated into 100 ml of Lb / Ampicilin, Kanamicin broth, and cultured at 37 ° C. OD every 30 minutes from 1 hour after the start of culture ₆₀₀ Was measured, and after culturing until it was between 0.6 and 0.7, 1 mM IPTG was added. After the addition of IPTG, the culture was continued at 18 ° C., and the cells were collected after 4 and 6 hours. The cells were added to 10 ml of PBS (137 mM NaCl, 8.1 mM Na ₂ HPO ₄ ・ 12H ₂ O, 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ ) Was added and vortexed to suspend well. The suspension was disrupted by sonication to disrupt cells, and then centrifuged at 4 ° C. at 8,000 rpm for 5 minutes. Take 100 μl of the supernatant into a 1.5 ml tube, and add 50 μl denaturant (6% SDS, 24% glycerol, 12% β-mercaptoethanol, 0.3 M Tris-HCl (pH 6.8), 0.15% BPB) Then, it was treated at 100 ° C. for 5 minutes. The reaction solution was subjected to electrophoresis using 10 μl of each sample on e-PAGE (manufactured by ATTO), and then stained and detected with Simply Blue SafeStain (manufactured by Invitrogen). As a result, a dark band was observed at about 44 kDa.
[0116]
The presence of chitinase in the supernatant of the cells collected after culturing for 6 hours was confirmed by the Shales method described in Test Example 1-6).
[0117]
The supernatant of the cells cultured and collected for 4 hours was subjected to His tag purification. The column tube was filled with Ni-NTA agarose, washed with about 10 ml of 0.1 M Tris-HCl (pH 8.0), and the supernatant was adsorbed to the column. Then, after washing away non-specific binding substances with 8 ml of 10 mM imidazole, the protein adsorbed on the column was eluted with 8 ml of 200 mM imidazole. As a result of electrophoresis of the eluate by e-PAGE, a single band appeared. Chitinase purified from the eluate and the culture of Aspergillus oryzae obtained in Example 1 was subjected to SDS electrophoresis on a 12.5% acrylamide gel (shown in FIG. 30). Although the molecular weight of the protein contained in the eluate is slightly larger than that of the purified chitinase derived from Aspergillus oryzae, this is considered to be due to the His tag, and the recombinant protein contained in the eluate is a His fusion recombinant chitinase. That was speculated. When the chitosan-decomposing activity of this eluate was measured by the Schells method described in Test Example 1-6), it was 2.0 U / ml, and it was confirmed that the recombinant protein contained in this eluate was chitinase. The amount of the enzyme that produces 1 μmol of acetylglucosamine per minute was defined as 1 U.
[0118]
The chitinase derived from Aspergillus oryzae whose amino acid sequence has been identified in the present invention comprises 399 amino acids before undergoing post-translational modification (SEQ ID NO: 5 in the sequence listing) and 398 amino acids subjected to post-translational modification ( SEQ ID NO: 14) in the sequence listing. The gene derived from Aspergillus oryzae reported as a chitinase gene (Chitin and Chitosan Research Vol. 8, No. 2, pp. 238-239, 2002) encodes a protein consisting of 431 amino acids, and the activity of the encoded protein is also high. Since it is unknown, it is assumed that the molecule is different from the chitinase derived from Aspergillus oryzae of the present invention.
Test Example 1 Aspergillus oryzae var. Various properties of crude enzyme solution of chitinase derived from sporoflabus Ohara JCM 2067 strain
The activity of the crude enzyme solution obtained in Example 1.1) was measured.
1) Hydrolytic activity
According to the method of Example 12), the amount of reducing sugar was quantified. When measured using chitosan 7B as a substrate, 4.2 × 10 ^-3 U enzyme activity. Table 11 shows the relative values of the chitosan 10B degrading activity when the enzyme activity when using chitosan 7B as a substrate is 100%. It was found that it had chitosan 7B degrading activity and had little chitosan 10B degrading activity.
[0119]
[Table 11]

2) pH activity
140 μl of the enzyme solution was added to a mixture of 160 μl of the chitosan 7B solution and 100 μl of a 400 mM buffer, and the mixture was stirred to make the mixture uniform, and the enzyme reaction was performed at 37 ° C. for 20 minutes. Quantification of free reducing sugar after the enzymatic reaction followed the method described in Example 12). The following buffer solution was used: pH 3.6 to pH 5.9, acetic acid-sodium acetate buffer: pH 5.9 to pH 8.2, 3- (N-morpholine) propanesulfonic acid (hereinafter referred to as “buffer”). MOPS ")-Sodium carbonate buffer: in the case of pH 9.0 to pH 10.2, sodium hydrogen carbonate-sodium carbonate buffer. The hydrolysis activity under the highest pH condition was defined as 100%, and the hydrolysis activity of the enzyme at each pH is shown in FIG. 5 as a relative value. The optimum pH was around pH 5.0.
[0120]
3) Temperature activity of enzyme in culture supernatant
The hydrolysis activity was measured at various temperatures under the condition of pH 5.0. 140 μl of the enzyme solution was added to a mixture of 160 μl of the chitosan 7B solution prepared in Example 1 preheated for 5 minutes and 100 μl of 400 mM acetate buffer (pH 5.0), and the mixture was stirred to make the mixture uniform, followed by an enzyme reaction for 10 minutes. The amount of the free reducing sugar after the enzymatic reaction was determined according to Example 1. The hydrolysis activity measured under the temperature condition showing the highest activity was taken as 100%, and the hydrolysis activity at each temperature was summarized as a relative value in FIG.
[0121]
4) Temperature stability of enzyme in culture supernatant
After treating the enzyme in the culture supernatant at various temperatures for 30 minutes, its hydrolysis activity was measured. 280 μl of the enzyme solution was added to 200 μl of a 400 mM acetate buffer (pH 5.0) previously maintained at the processing temperature, and the mixture was stirred to make the mixture uniform, and kept warm for 30 minutes. 240 μl of the heated enzyme solution was added to 160 μl of the chitosan 7B solution prepared in Example 1, and an enzyme reaction was carried out at 37 ° C. for 20 minutes. The quantification of free reducing sugars was as in Example 1. The hydrolysis activity of the mixture of the enzyme and the buffer solution in the untreated group (incubated at 0 ° C.) is defined as 100%, and the hydrolysis activity at each temperature is shown in FIG. 7 as a relative value.
[0122]
5) pH stability of enzyme in culture supernatant
To 400 µl of the culture supernatant, 70 µl of a 400 mM buffer solution of each pH described below and 90 µl of water were added, and the mixture was kept at 37 ° C for 1 hour. The buffers used were: pH 3.9 to pH 7.3, acetic acid-sodium acetate buffer: pH 6.5 to pH 8.6, MOPS-sodium carbonate buffer: pH 9.3 to pH 10. In the case of pH 4, sodium hydrogen carbonate-sodium carbonate buffer: in the case of pH 10.0 to pH 11.4, disodium hydrogen phosphate-sodium hydroxide buffer. To a mixture of 100 μl of a 400 mM acetate buffer (pH 5.0) and 160 μl of a chitosan 7B solution prepared by the method described in Example 12), 140 μl of a heated enzyme mixture was added, and the mixture was stirred to homogenize. The enzyme reaction was performed for minutes. The quantification of free reducing sugars was as in Example 1. The hydrolysis activity of the mixture of the enzyme and the buffer in the untreated (incubated at 0 ° C.) group was defined as 100%, and the hydrolysis activity at each pH was summarized as a relative value in FIG.
[0123]
6) Substrate selectivity of enzyme in culture supernatant
Next, the substrate selectivity at pH 5.0 was measured. 10 ml of the culture supernatant prepared in Example 2 was dialyzed three times every 12 hours against 1,000 ml of 10 mM Tris / hydrochloric acid buffer (pH 7.5) and used as an enzyme solution. To a mixture of 160 μl of a 0.25% wt / v substrate solution and 160 μl of 400 mM acetate buffer (pH 5.0) and 160 μl of water, add 50 μl of the enzyme solution, stir to homogenize, start the reaction, and incubate at 37 ° C. The enzyme reaction was performed for minutes. Quantification of the free reducing sugar after the enzymatic reaction was performed by the Shales method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). Table 12 shows the relative activities when the hydrolysis activity when the substrate showing the maximum activity was used was taken as 100%.
[0124]
[Table 12]
――――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――――
Chitosan 10B 12.7
Chitosan 9B 78.9
Chitosan 8B 82.3
Chitosan 7B 100
Gluchor chitosan 17.7
Glucol chitin 52.5
CM-cellulose 2.2
Lichenan 31.6
――――――――――――――――――――
Test Example 2. Aspergillus oryzae var. Properties of partially purified chitinase from sporoflabus Ohara JCM2067 strain
Aspergillus oryzae var. Obtained in Example 1.2). Various properties of the crude purified chitinase derived from sporoflabus Ohara JCM 2067 strain were examined.
1) pH activity of hydrolysis activity of enzyme in crudely purified fraction
140 μl of the enzyme solution was added to a mixed solution of 160 μl of the chitosan 7B solution prepared in Example 1 and 100 μl of a 400 mM buffer, and the mixture was stirred to make the reaction uniform, and the reaction was started. The amount of the free reducing sugar after the enzymatic reaction was determined according to Example 1. The buffer used was: pH 3.3 to pH 5.9, acetic acid-sodium acetate buffer: pH 5.9 to pH 8.5, MOPS-sodium carbonate buffer: pH 9.2 to pH 10. In case 3, sodium bicarbonate-sodium carbonate buffer. The hydrolysis activity under the pH condition where the activity was highest was defined as 100%, and the hydrolysis activity of the enzyme at each pH is shown in FIG. 9 as a relative value. The optimum pH was around pH 5.0.
[0125]
2) Temperature activity of hydrolysis activity of enzyme in crude fraction
The hydrolysis activity was measured at various temperatures under the condition of pH 5.0. 140 μl of the enzyme solution is added to a mixture of 160 μl of the chitosan 7B solution prepared in Example 1 and 100 μl of 400 mM acetate buffer (pH 5.0) previously maintained at the processing temperature, and the mixture is stirred to homogenize, and the enzyme reaction is performed for 10 minutes. Was. The amount of the free reducing sugar after the enzymatic reaction was determined according to Example 1. The hydrolysis activity measured under the temperature condition showing the highest activity was taken as 100%, and the hydrolysis activity at each temperature was summarized as a relative value in FIG.
[0126]
3) Temperature stability of enzyme in crude fraction
After treating the enzyme in the partially purified fraction at various temperatures for 30 minutes, its hydrolysis activity was measured. 200 μl of a 400 mM acetate buffer (pH 5.0) and 280 μl of an enzyme solution maintained at the treatment temperature were added, and the mixture was stirred to make uniform, and kept warm for 30 minutes. 240 μl of the heated enzyme solution was added to 160 μl of the chitosan 7B solution prepared in Example 1, and an enzyme reaction was carried out at 37 ° C. for 20 minutes. The quantification of free reducing sugars was as in Example 1. The hydrolysis activity of the mixture of the enzyme and the buffer in the untreated (incubated at 0 ° C.) group was defined as 100%, and the hydrolysis activity at each temperature is shown in FIG. 11 as a relative value.
[0127]
4) pH stability of enzyme in crude fraction
To 400 μl of the enzyme in the crude purified fraction, 140 μl of a 400 mM buffer solution of each pH described below and 20 μl of water were added, and the mixture was kept at 37 ° C. for 1 hour. The buffers used were: pH 3.1 to pH 6.1, acetic acid-sodium acetate buffer: pH 6.1 to pH 8.4, MOPS-sodium carbonate buffer: pH 9.0 to pH 10. In the case of 3, sodium hydrogen carbonate-sodium carbonate buffer: in the case of pH 10.4 to pH 11.4, disodium hydrogen phosphate-sodium hydroxide buffer. 140 μl of the heated enzyme mixture was added to 100 μl of 400 mM acetate buffer (pH 5.0) and 160 μl of the chitosan 7B solution prepared in Example 1, and the mixture was stirred and homogenized, and the enzyme reaction was carried out at 37 ° C. for 20 minutes. Was. The quantification of free reducing sugars was as in Example 1. The hydrolysis activity of the mixture of the enzyme and the buffer in the untreated (incubated at 0 ° C.) group was defined as 100%, and the hydrolysis activity at each temperature is shown as a relative value in FIG.
Test Example 3 Aspergillus oryzae var. Properties of purified chitinase from sporoflabus Ohara JCM2067 strain
Aspergillus oryzae var. Obtained in Example 1.3). Various properties of the purified chitinase derived from sporoflabus Ohara JCM 2067 strain were examined.
1) pH activity of hydrolysis activity of purified enzyme
Example 1 50 μl of an enzyme solution was added to a mixture of 160 μl of a chitosan 7B solution prepared by the method described in 2), 100 μl of a 400 mM buffer solution and 90 μl of water, and the mixture was stirred to homogenize the reaction. The enzyme reaction was performed for minutes. Quantification of free reducing sugar after the enzymatic reaction followed the method described in Example 12). The buffer used was: pH 3.3 to pH 5.9, acetic acid-sodium acetate buffer: pH 5.9 to pH 8.4, MOPS-sodium carbonate buffer: pH 9.3 to pH 10. In case 4, sodium bicarbonate-sodium carbonate buffer. The hydrolysis activity under the pH condition where the activity was highest was defined as 100%, and the hydrolysis activity of the enzyme at each pH was shown as a relative value in FIG. The optimum pH was around pH 5.5.
75 μl of the enzyme solution was added to a mixture of 240 μl of a 0.25% wt / v glycol chitin solution, 150 μl of a 400 mM buffer solution and 135 μl of water, and the mixture was stirred to homogenize the reaction. The reaction was started at 37 ° C. for 30 minutes. An enzymatic reaction was performed. Quantification of the free reducing sugar after the enzymatic reaction was performed by the Shales method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). The buffers used were: pH 3.3 to pH 6.0, acetic acid-sodium acetate buffer: pH 6.3 to pH 7.8, monosodium hydrogen phosphate-disodium hydrogen phosphate buffer : PH 7.3 to pH 8.7, Tris / hydrochloric acid buffer: pH 9.3 to pH 10.4, sodium bicarbonate-sodium carbonate buffer: pH 10.6 to pH 11.5, hydrogen phosphate 2 Sodium-sodium hydroxide buffer. The hydrolysis activity under the pH condition where the activity was the highest was defined as 100%, and the hydrolysis activity of the enzyme at each pH was shown as a relative value in FIG. The optimum pH was around pH 10.
[0128]
2) Temperature activity of hydrolysis activity of purified enzyme
The hydrolysis activity of the purified enzyme was measured at various temperatures under the condition of pH 5.5. 40 μl of the enzyme solution was added to a mixture of 160 μl of the chitosan 7B solution, 100 μl of 400 mM acetate buffer (pH 5.5), and 100 μl of water, which had been prepared in the same manner as in 1) and stirred to homogenize for 10 minutes. An enzymatic reaction was performed. The amount of the free reducing sugar after the enzymatic reaction was determined according to Example 1. The hydrolytic activity of the purified enzyme measured under the temperature condition showing the highest activity was taken as 100%, and the hydrolytic activity of the purified enzyme at each temperature was summarized as a relative value in FIG. As shown in FIG. 15, the optimum temperature for the hydrolysis activity of the purified enzyme was 60 ° C. at pH 5.5.
[0129]
3) Temperature stability of purified enzyme
After treating the purified enzyme at various temperatures for 30 minutes, its hydrolysis activity was measured. 100 μl of the enzyme solution was added to a mixed solution of 200 μl of 400 mM acetate buffer (pH 5.5) and 180 μl of water which had been kept at the processing temperature in advance, and the mixture was stirred to make the mixture uniform, and kept warm for 30 minutes. 240 μl of the heated enzyme solution was added to 160 μl of the chitosan 7B solution prepared in the same manner as in 1), and the enzyme reaction was carried out at 37 ° C. for 40 minutes. The quantification of free reducing sugars was as in Example 1. The hydrolysis activity of the mixture of the purified enzyme and the buffer solution in the untreated (0 ° C kept) group was defined as 100%, and the hydrolysis activity of the purified enzyme at each temperature is shown in FIG. 16 as a relative value. As shown in FIG. 16, the purified enzyme was stable at 45 ° C. or lower.
[0130]
4) pH stability of purified enzyme
To 150 μl of the purified enzyme, 50 μl of a 400 mM buffer at each pH described below was added, and the mixture was kept at 37 ° C. for 1 hour. The buffers used were: pH 3.2 to pH 6.2, acetic acid-sodium acetate buffer: pH 6.1 to pH 8.2, MOPS-sodium carbonate buffer: pH 8.8 to pH 10. In the case of 1, sodium bicarbonate-sodium carbonate buffer: in the case of pH 9.6 to pH 11.3, disodium hydrogen phosphate-sodium hydroxide buffer. 70 μl of a heated enzyme mixture was added to a mixture of 100 μl of a 400 mM acetate buffer (pH 5.5), 160 μl of a chitosan 7B solution prepared in Example 1, and 70 μl of water, and the mixture was stirred and homogenized. The reaction was performed, and the amount of free reducing sugar was determined. The hydrolysis activity of the mixture of the purified enzyme and the buffer solution in the untreated group (incubated at 0 ° C.) was defined as 100%, and the hydrolysis activity of the purified enzyme at each temperature is shown in FIG. 17 as a relative value. As shown in FIG. 17, the purified enzyme was stable at pH 5 to pH 9.5.
[0131]
5) Substrate specificity of purified enzyme
The hydrolysis activity of the purified enzyme on various substrates was measured according to the method of Example 5. However, the enzyme reaction was performed at pH 5.5 and pH 10.0. Table 13 shows the relative values of the hydrolysis activity of the purified enzyme for each of the other substrates, assuming that the hydrolysis activity of the purified enzyme for chitosan 7B was 100%.
[0132]
[Table 13]

As shown in Table 11, the purified enzyme had the highest hydrolysis activity for chitosan 7B, and the hydrolysis activity decreased as the degree of acetylation decreased. In addition, the purified enzyme had chitin hydrolysis activity, and no cellulase hydrolysis activity was observed.
Test example 4. Properties of Chitinase Crude Enzyme Solution from Aspergillus japonicus SANK19288
1) Example 1 The reducing sugar was quantified according to the method described in 2). When measured using chitosan 7B as a substrate, 3.4 × 10 ^-3 U enzyme activity. Table 14 shows the relative value of the chitosan 10B decomposition activity when the enzyme activity when chitosan 7B was used as a substrate was 100%.
[0133]
[Table 14]

Since it had chitosan 7B degrading activity and little chitosan 10B degrading activity, it was found that chitinase was produced in the supernatant.
[0134]
2) pH activity of crude enzyme solution
Example 1 140 μl of a crude enzyme solution was added to a mixture of 160 μl of a chitosan 7B solution prepared according to the method described in 2) and 100 μl of a 400 mM buffer solution, and the mixture was stirred to homogenize the mixture. The amount of free reducing sugar after the enzyme reaction was quantified. The buffers used were: pH 3.7 to pH 6.0, acetic acid-sodium acetate buffer: pH 6.0 to pH 8.5, MOPS-sodium carbonate buffer: pH 9.3 to pH 10. In case 4, sodium bicarbonate-sodium carbonate buffer. The hydrolysis activity under the pH condition where the activity was the highest was defined as 100%, and the hydrolysis activity of the enzyme at each pH was shown as a relative value in FIG. The optimum pH was around pH 4.0.
[0135]
3) Substrate selectivity of crude enzyme
The substrate selectivity at pH 4.0 was measured. 10 ml of the culture supernatant prepared in Example 2 was dialyzed 3 times every 12 hours against 1,000 ml of 10 mM Tris / hydrochloric acid buffer (pH 7.5), and used as the enzyme solution. 50 μl of the enzyme solution was added to a mixture of 160 μl of a 0.25% wt / v substrate solution and 160 μl of 400 mM acetate buffer (pH 4.0) and water, and the mixture was stirred to homogenize the reaction. The reaction was started at 37 ° C. The enzyme reaction was performed for minutes. Quantification of the free reducing sugar after the enzymatic reaction was performed by the Shales method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). Table 15 shows the relative activities when the hydrolysis activity when the substrate showing the maximum activity was used was taken as 100%.
[0136]
[Table 15]
―――――――――――――――――――――
Substrate Relative activity (%)
―――――――――――――――――――――
Chitosan 10B 8.2
Chitosan 9B 67.6
Chitosan 8B 74.4
Chitosan 7B 100
Glycol chitosan 8.2
Glycol chitin 23.3
CM-cellulose 11.4
Lichenan 25.3
―――――――――――――――――――――
Test Example 5. Properties of crude enzyme solution of chitinase from Aspergillus sojae SANK 22388 strain
1) Hydrolytic activity
The amount of reducing sugar was determined according to the method described in Example 12). When measured using chitosan 7B as a substrate, 2.6 × 10 ^-3 U enzyme activity. Table 16 shows the relative values of the chitosan 10B degrading activity when the enzyme activity when using chitosan 7B as a substrate is 100%.
[0137]
[Table 16]

Since it had chitosan 7B degrading activity and little chitosan 10B degrading activity, it was found that chitinase was produced in the supernatant.
[0138]
2) pH activity of enzyme in culture supernatant
The measuring method followed Example 6. The buffers used were: pH 3.8 to pH 6.3, acetic acid-sodium acetate buffer: pH 6.2 to pH 8.6, MOPS-sodium carbonate buffer: pH 9.3 to pH 10. In case 4, sodium bicarbonate-sodium carbonate buffer. The hydrolysis activity under the pH condition where the activity was the highest was defined as 100%, and the hydrolysis activity of the enzyme at each pH was shown as a relative value in FIG. The optimum pH was around pH 4.5.
[0139]
3) Substrate selectivity of enzyme in culture supernatant
The substrate selectivity at pH 4.5 was measured. 10 ml of the culture supernatant prepared in Example 4 was dialyzed three times every 12 hours against 1,000 ml of 10 mM Tris / hydrochloric acid buffer (pH 7.5) and used as an enzyme solution. To a mixture of 160 μl of a 0.25% wt / v substrate solution and 160 μl of 400 mM acetate buffer (pH 4.5) and 160 μl of water, add 50 μl of the enzyme solution, stir to homogenize, start the reaction, and maintain the temperature at 37 ° C. The enzyme reaction was performed for 10 minutes. Quantification of the free reducing sugar after the enzymatic reaction was performed by the Schles method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). Table 17 shows the relative activities when the hydrolysis activity when the substrate showing the maximum activity was used was taken as 100%.
[0140]
[Table 17]
――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――
Chitosan 10B 0
Chitosan 9B 67.7
Chitosan 8B 71.6
Chitosan 7B 100
Gluchor chitosan 2.9
Glycol chitin 81.9
CM-cellulose 0
Lichenan 11.9
――――――――――――――――――
Test Example 6. Solubility of each low molecular weight chitosan hydrochloride in water
100 mg of each low molecular weight chitosan hydrochloride prepared in Examples 4 to 6 was dissolved in 10 ml of water, and the pH was measured. 1N caustic soda water was added little by little to this solution, and the pH at which the solution became cloudy and a colloid precipitated was measured. The results are shown in Table 18.
[0141]
[Table 18]

Test Example 7 Preparation of polymeric chitosan hydrochloride
1 g of chitosan 9B was suspended in 200 ml of water, adjusted to pH 2.5 with concentrated hydrochloric acid, and completely dissolved. This was dialyzed against water and freeze-dried to obtain 537 mg of a high-molecular-weight chitosan 9B hydrochloride as a white flocculent solid. Polymeric chitosan 10B and 8B and 7B hydrochloride were prepared in the same manner. 100 mg of each polymer chitosan hydrochloride was dissolved in 50 ml of distilled water, 20 U of chitosanase (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution is freeze-dried, ₂ Dissolve in O ¹ 1 H-NMR was measured.
The degree of acetylation was calculated from the integral ratio of 3H of the acetyl group to 7H other than the acetyl group, excluding the hydroxyl group. The results are shown in Table 19.
[0142]
[Table 19]

Test Example 8. Antibacterial activity of low molecular weight chitosan
As the test bacteria, Staphylococcus aureus (Staphylococcus aureus) 209P JC-1 strain and Pseudomonas aeruginosa PAO1 strain were used as test bacteria, and the polymer chitosan hydrochloride prepared in Test Example 7 and Example 4, The MICs of low molecular weight chitosan hydrochloride prepared in Examples 5 and 6 using various Aspergillus-derived chitinases were measured. The results are shown in Table 18.
[0143]
Medium used
All were adjusted to pH 6.5 using Mueller Hinton Broth (manufactured by Difco).
[0144]
Preparation of sample bacterial solution
The bacteria used for the test were cultured overnight in the above medium, and the number of bacteria was 2 × 10 ⁶ And diluted with the same medium.
Sample preparation
20 mg of each low molecular weight chitosan hydrochloride was dissolved in distilled sterilized water to make 10 ml.
[0145]
Test method
Diluted sterilized water was used as a diluent in advance to prepare a multiple dilution series of the sample solution, and 50 μl of the above medium and 50 μl of the test bacterial solution were added thereto, and cultured at 37 ° C. for 18 hours to determine the MIC. The results are shown in Table 20.
[0146]
[Table 20]

Test Example 9. Properties of purified chitinase from Aspergillus japonicus SANK 19288 strain
Various properties of the purified chitinase derived from Aspergillus japonicus SANK 19288 obtained in Example 2.2) were examined.
[0147]
1) pH activity of hydrolysis activity of purified chitinase derived from Aspergillus japonicus SANK 19288 strain
The pH activity of the hydrolysis activity of purified chitinase derived from Aspergillus japonicus SANK 19288 strain was measured by the method described in Test Example 31). The hydrolysis activity under the pH condition where the activity was highest was defined as 100%, and the hydrolysis activity of the enzyme at each pH is shown in FIG. 20 as a relative value. The optimum pH was around pH 5.0.
[0148]
2) Temperature activity of hydrolysis activity of purified chitinase derived from Aspergillus japonicus SANK 19288 strain
The temperature activity of the hydrolysis activity of the purified chitinase derived from Aspergillus japonicus SANK 19288 strain was measured by the method described in Test Example 32). The hydrolysis activity under the temperature condition where the activity was the highest was taken as 100%, and the hydrolysis activity of the enzyme at each temperature was summarized as a relative value in FIG. The optimum temperature was 65 ° C. at pH 5.0.
[0149]
3) Temperature stability of purified chitinase from Aspergillus japonicus SANK 19288 strain
The temperature stability of purified chitinase derived from Aspergillus japonicus SANK 19288 strain was measured by the method described in Test Example 33). The hydrolytic activity of the mixture of the purified chitinase and the buffer in the untreated (incubated at 0 ° C.) group was defined as 100%, and the hydrolytic activity of the enzyme at each temperature is shown in FIG. 22 as a relative value. The purified chitinase was stable below 50 ° C.
[0150]
4) pH stability of purified chitinase derived from Aspergillus japonicus SANK 19288 strain
The pH stability of purified chitinase derived from Aspergillus japonicus SANK 19288 strain was measured by the method described in Test Example 34). The hydrolysis activity of the mixture of the purified chitinase and the buffer in the untreated (incubated at 0 ° C.) group was defined as 100%, and the hydrolysis activity of the enzyme at each pH is shown in FIG. 23 as a relative value. The purified chitinase was stable between pH 4 and pH 9.5.
[0151]
5) Substrate specificity of purified chitinase from Aspergillus japonicus SANK 19288 strain
The substrate specificity of purified chitinase derived from Aspergillus japonicus SANK 19288 strain was measured by the method described in Test Example 35). Table 21 shows, as relative values, the hydrolysis activity of the purified chitinase with respect to other substrates, assuming that the hydrolysis activity of the purified chitinase with respect to chitosan 7B at pH 5.0, 37 ° C. and 10 minutes was 100%. Indicated.
[0152]
[Table 21]
――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――
Chitosan 10B 0
Chitosan 9B 58.6
Chitosan 8B 72.4
Chitosan 7B 100
Glucol chitosan 0
Glycol chitin 28.3
CM-cellulose 0
Lichenan 4.2
――――――――――――――――――
As shown in Table 21, the purified chitinase derived from Aspergillus japonicus SANK 19288 had the highest hydrolysis activity on chitosan 7B, and the hydrolysis activity decreased as the degree of acetylation decreased. Further, the purified chitinase had chitin hydrolyzing activity, and no cellulase hydrolyzing activity was observed.
[0153]
6) Determination of partial amino acid sequence of purified chitinase derived from Aspergillus japonicus SANK 19288 strain
The partial amino acid sequence of purified chitinase derived from Aspergillus japonicus SANK 19288 strain was determined by the method described in Example 1.7). Among the separated decomposed amino acids, three peaks having a B buffer concentration of about 28%, about 30%, and about 35% were collected. The sequences of the three were analyzed using an amino acid sequence analyzer (Procise cLC, manufactured by Applied Biosystem). The results are shown from the N-terminal side.
His-Tyr-Pro-Thr-Asp-Ser-Trp-Asn-Asp-Val-Gly-Thr-Asn-Val-Tyr (SEQ ID NO: 11 in Sequence Listing)
Ala-Phe-Thr-Asn-Thr-Asp-Gly-Pro-Gly-Thr-Ala-Phe-Ser-Gly-Val (SEQ ID NO: 12 in Sequence Listing)
Leu-Ser-Gln-Met-Thr-Pro-Tyr-Leu-Asp-Phe-Tyr-Asn-Leu-Met-Ala-Tyr-Asp-Tyr-Ala-Gly (SEQ ID NO: 13 in Sequence Listing)
Test Example 10. Properties of purified chitinase from Aspergillus sojae SANK 22388 strain
Various properties of the purified chitinase derived from Aspergillus sojae SANK 22388 obtained in Example 3.2) were examined.
[0154]
1) pH activity of hydrolysis activity of purified chitinase derived from Aspergillus sojae SANK 22388 strain
The pH activity of the hydrolysis activity of the purified chitinase derived from Aspergillus sojae SANK 22388 strain was measured by the method described in Test Example 3.1). The hydrolysis activity under the pH condition where the activity was highest was defined as 100%, and the hydrolysis activity of the enzyme at each pH is shown in FIG. 24 as a relative value. The optimum pH was around pH 5.5 and pH 9.0.
[0155]
2) Temperature activity of hydrolysis activity of purified chitinase derived from Aspergillus sojae SANK 22388 strain
The purified chitinase derived from Aspergillus sojae SANK 22388 strain is different from the chitinase derived from Aspergillus oryzae and Aspergillus japonix, and has two optimum pHs (see Test Example 101). The temperature activity of the purified chitinase was measured at these two pH values by the method described in Test Example 32). FIG. 25 (temperature activity at pH 5.5) and FIG. 26 (temperature activity at pH 9.0) with the hydrolysis activity under the temperature condition of the highest activity as 100% and the hydrolysis activity of the enzyme at each temperature as a relative value. Summarized in The optimum temperature was 60 ° C at pH 5.5 and 35 ° C at pH 9.0.
[0156]
3) Temperature stability of purified chitinase from Aspergillus sojae SANK 22388 strain
The purified chitinase derived from Aspergillus sojae SANK 22388 strain is different from the chitinase derived from Aspergillus oryzae and Aspergillus japonix, and has two optimum pHs (see Test Example 101). The temperature stability of the purified chitinase was measured at these two pH values by the method described in Test Example 3.3). The hydrolysis activity of the mixture of the purified chitinase and the buffer in the untreated (incubated at 0 ° C.) group was defined as 100%, and the hydrolysis activity of the enzyme at each temperature was defined as a relative value. ) And FIG. 28 (temperature stability at pH 9.0). The purified chitinase was stable at 40 ° C. or less at any pH.
[0157]
4) pH stability of purified chitinase from Aspergillus sojae SANK 22388 strain
The pH stability of purified chitinase derived from Aspergillus sojae SANK 22388 strain was measured by the method described in Test Example 3.4). The hydrolysis activity of the mixture of the purified chitinase and the buffer in the untreated (incubated at 0 ° C.) group was defined as 100%, and the hydrolysis activity of the enzyme at each pH was summarized as a relative value in FIG. The purified chitinase was stable between pH 4.5 and pH 10.
[0158]
5) Substrate specificity of purified chitinase from Aspergillus sojae SANK 22388 strain
The substrate specificity of the purified chitinase derived from Aspergillus sojae SANK 22388 strain was measured by the method described in Test Example 3.5). Assuming that the hydrolysis activity of the purified chitinase with respect to chitosan 8B under the conditions of pH 5.0, 37 ° C. and 10 minutes is 100%, the hydrolysis activity of the purified chitinase with respect to other substrates is shown as a relative value in Table 22. Indicated.
[0159]
[Table 22]
――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――
Chitosan 10B 14.3
Chitosan 9B 73.6
Chitosan 8B 100
Chitosan 7B 97.4
Glucol chitosan 0
Glycol chitin 23.8
CM-cellulose 0
Lichenan 0
――――――――――――――――――
As shown in Table 22, purified chitinase derived from Aspergillus sojae SANK 22388 strain had the highest hydrolysis activity on chitosan 7B and 8B, and the hydrolysis activity decreased as the degree of acetylation decreased. Further, the purified chitinase had chitin hydrolyzing activity, and no cellulase hydrolyzing activity was observed.
Formulation Example Preparation of 1.5% Chitosan Ointment Formulation
A solution of 3.0 g of propylene glycol and 0.1 g of methyl parahydroxybenzoate in 80 ml of purified water was heated to 70 ° C., and 5.0 g of the low-molecular-weight chitosan 9B hydrochloride prepared in Example 4 was added to dissolve the solution. The solution was a chitosan 9B solution. 3.0 g of stearic acid, 1.0 g of cetal, 6.0 g of glyceryl monostearate, 5.0 g of liquid paraffin, 0.1 g of propyl paraoxybenzoate, and 2.5 g of polyoxyl 40 stearate were heated and dissolved at 70 ° C. At the same temperature, the low-molecular-weight chitosan 9B solution was added, and purified water was added to make the total amount 100 g, followed by mixing and emulsification. After the emulsification, the mixture was cooled to room temperature with stirring to obtain 100 g of a white 5% low molecular weight chitosan 9B hydrochloride cream (referred to as 5% chitosan ointment).
Test Example 1 Trauma healing test of 0.5% chitosan ointment
A wound healing test was performed to predict the efficacy of 5% chitosan ointment in a rat skin defect injury model.
1) Test material
As the test substance, 5% chitosan ointment prepared in Preparation Example 1 was used. As a control, Eupasta ointment (containing 70 g of purified sucrose and 3 g of povidone iodine in 100 g, containing polyethylene glycol, concentrated glycerin, polyoxyethylene [160] polyoxypropylene [30] glycol, pullulan, and potassium iodide as additives) , Serial number BA1S, manufactured by Kowa Co., Ltd.). As the animal used for the test, a 6-week-old Sprague-Dawley (SD) male rat (Japan SLC, Inc.) was purchased and used. The animals were fed solid feed (FR-2 for rearing mice and rats, Funabashi Farm Co., Ltd.) and tap water using an environmental control rearing device (CLEA Japan) with an average temperature of 23 ° C. and an average humidity of 55%, and lighting time was 7:00. The animals were acclimated and bred for 6 days under the condition of -19 o'clock, and used for the experiment.
2) Experimental method
Six groups of 7-week-old male SD rats (body weight: 157.6 g to 235.9 g) were used, with 5 rats per group. After removing the hair from the back of the rat with an electric hair clipper, EBA Eva Cream (manufactured by Tokyo Tanabe Seiyaku Co., Ltd.) was applied, and after 15 minutes, the hair was washed and depilated. After disinfecting the back hair loss site with 70% ethanol, two symmetrical punch wounds were made at the midline of the back using a circular punch (inner diameter 15 mm) under pentobarbital sodium (40 mg / kg, ip) anesthesia. . Twenty-four hours after the preparation of the incomplete injury, the animals were divided into 5 groups each in a non-treatment group, a base group, a test substance group, and a control drug group. The site was applied twice a day for 10 days. Rats were housed in individual cages throughout the experiment. The effect was determined by measuring the major axis and minor axis of the damaged part using a caliper.
[0160]
The area change in the treatment process is obtained by calculating the area ratio (%) by the formula of (major axis × minor axis / major axis × minor axis after missing damage preparation) × 100 on the measurement day, and calculating the area to be treated (day 5-15) from the area ratio. ) And the number of treatment days was calculated with the area ratio of 5% or less as a completely cured case. The experimental results were expressed as mean ± standard deviation. The 5% chitosan ointment group reduced the number of treatment days to 12.6 ± 1.9 days, compared to 13.7 ± 1.3 days in the untreated group. No improvement in the number of treatment days was observed in the control group, at 13.6 ± 0.4 days.
[0161]
Table 23: Days of treatment in rat skin deficiency injury model
________________________
Test substance Treatment days
________________________
No treatment 13.7 ± 1.3
5% chitosan 12.6 ± 1.9
Control drug 13.6 ± 0.4
________________________
[0162]
【The invention's effect】
As described above, by using the chitinase of the present invention, the low-molecular-weight chitosan of the present invention, which has a molecular weight of 10,000 or more and is soluble even under neutral conditions, can be produced. Furthermore, the low molecular weight chitosan of the present invention exhibits excellent antibacterial properties and wound healing effects, and the pharmaceutical composition containing the low molecular weight chitosan of the present invention as an active ingredient is useful as a therapeutic agent for trauma, bedsores, atopic dermatitis, etc. It is. Further, by using the chitinase of the present invention, a sample having high viscosity can be produced from a sample having high viscosity by containing high molecular acetylated chitosan as a raw material. Such a method for producing a sample is useful in the process of food processing.
[0163]
[Sequence List Free Word]
SEQ ID NO: 6: Chitinase gene probe
SEQ ID NO: 7: PCR primer F21
SEQ ID NO: 8: PCR primer PR50
SEQ ID NO: 9: PCR primer BamHI-cDNA
SEQ ID NO: 10: PCR primer cDNA-BamHI
[0164]
[Sequence list]

[Brief description of the drawings]
FIG. 1. Aspergillus oryzae var. Molecular weight of purified chitinase from sporoflabus Ohara JCM2067
FIG. 2. Aspergillus oryzae var. Molecular weight distribution of low molecular weight chitosan prepared using chitinase derived from sporoflabus Ohara JCM2067
FIG. 3: Molecular weight distribution of low molecular weight chitosan prepared using chitinase derived from Aspergillus japonicus SANK19288 strain
FIG. 4: Molecular weight distribution of low molecular weight chitosan prepared using chitinase in culture supernatant of Aspergillus sojae SANK22388 strain
FIG. 5. Aspergillus oryzae var. Relationship between chitinase activity and pH in culture supernatant of sporoflabus Ohara JCM2067 strain
FIG. 6. Aspergillus oryzae var. Relationship between chitinase activity and temperature in culture supernatant of sporoflabus Ohara JCM2067 strain
FIG. 7: Aspergillus oryzae var. Temperature stability of chitinase in sporoflabus Ohara JCM2067 culture supernatant
FIG. 8. Aspergillus oryzae var. pH stability of chitinase in sporoflabus Ohara JCM2067 culture supernatant
FIG. 9. Aspergillus oryzae var. Relationship between activity and pH of partially purified chitinase from sporoflabus Ohara JCM2067
FIG. 10. Aspergillus oryzae var. Relationship between activity and temperature of partially purified chitinase from sporoflabus Ohara JCM2067
FIG. 11. Aspergillus oryzae var. pH stability of crude purified chitinase from sporoflabus Ohara JCM2067
FIG. 12. Aspergillus oryzae var. Temperature stability of partially purified chitinase from sporoflabus Ohara JCM2067
FIG. 13: Aspergillus oryzae var. Using chitosan 7B as a reaction substrate. Relationship between activity and pH of purified chitinase from sporoflabus Ohara JCM2067
FIG. 14: Aspergillus oryzae var. Using glycol chitin as a reaction substrate. Relationship between activity and pH of purified chitinase from sporoflabus Ohara JCM2067
FIG. 15. Aspergillus oryzae var. Relationship between temperature and activity of purified chitinase from sporoflabus Ohara JCM2067
FIG. 16: Aspergillus oryzae var. Temperature stability of purified chitinase from sporoflabus Ohara JCM2067
FIG. 17. Aspergillus oryzae var. pH stability of purified chitinase from sporoflabus Ohara JCM2067
FIG. 18: Relationship between chitinase activity and pH in culture supernatant of Aspergillus japonicus SANK19288 strain
FIG. 19: Relationship between chitinase activity and pH in culture supernatant of Aspergillus sojae SANK22388 strain
FIG. 20: Relationship between activity of purified chitinase derived from Aspergillus japonicus SANK19288 strain and glycol pH using glycol chitin as a reaction substrate
FIG. 21: Relationship between activity of purified chitinase derived from Aspergillus japonicus SANK19288 strain and temperature.
FIG. 22. Temperature stability of purified chitinase from Aspergillus japonicus SANK19288 strain
FIG. 23. pH stability of purified chitinase from Aspergillus japonicus SANK19288 strain
FIG. 24: Aspergillus sojae using glycol chitin as a reaction substrate
Relationship between activity and pH of purified chitinase derived from SANK22388 strain
FIG. 25: Relationship between the activity of purified chitinase derived from Aspergillus sojae SANK22388 strain and the temperature under the condition of pH 5.5.
FIG. 26 shows the relationship between the activity of purified chitinase derived from Aspergillus sojae SANK22388 strain and the temperature under the condition of pH 9.0.
FIG. 27 shows the temperature stability of purified chitinase derived from Aspergillus sojae SANK22388 strain at pH 5.5.
FIG. 28 shows the temperature stability of purified chitinase derived from Aspergillus sojae SANK22388 strain at pH 9.0.
FIG. 29: pH stability of purified chitinase from Aspergillus sojae SANK22388 strain
FIG. 30 SDS electrophoretogram of chitinase (12% acrylamide gel, CBB staining, lane M: marker, lane 1: His fusion recombinant chitinase, lane 2: purified chitinase from Aspergillus oryzae var. Sporoflavas Ohara JCM2067)

Claims (33)

A chitosan-degrading enzyme separated and purified from a culture of at least one of Aspergillus oryzae, Aspergillus japonicus, Aspergillus sojae and Aspergillus sojae. Aspergillus oryzae (Aspergillus oryzae) is Aspergillus oryzae (Aspergillus oryzae var. Sporoflavus Ohara JCM 2067) is a strain, Aspergillus japonicus (Aspergillus japonicus) is Aspergillus japonicus (Aspergillus japonicus) SANK19288 strain, Aspergillus Sojae (Aspergillus The chitosan-degrading enzyme according to claim 1, wherein the sojae is Aspergillus sojae SANK # 22388 strain. The chitosan-degrading enzyme according to claim 1 or 2, having a partial amino acid sequence of SEQ ID NOS: 1 to 3 in the sequence listing. The chitosan degrading enzyme according to any one of claims 1 to 3, which has the following properties:
1) exhibiting a molecular weight of about 40,000 by SDS-PAGE electrophoresis;
2) show an isoelectric point pI of 3.5 to 4.5 by isoelectric focusing;
3) Hydrolyze 30% acetylated chitosan (viscosity 100-300 cps) at pH 3.5 to pH 10.5;
4) hydrolyzing glycol chitin at pH 3.0 to pH 10.5;
5) exhibit the hydrolysis activity described in 3) at 0 ° C to 80 ° C;
6) stable at temperatures below 45 ° C .;
7) Stable under pH conditions of pH 5 to pH 9.5. Chitosan-degrading enzyme which is a protein according to any one of the following a) to d):
a) a protein consisting of the amino acid sequence of SEQ ID NO: 5 in the sequence listing;
b) a protein consisting of an amino acid sequence encoded by a nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing;
c) a protein comprising the amino acid sequence of a) or b), wherein one or several amino acids are substituted, deleted, inserted or added, and which has chitosan-degrading activity;
d) A protein comprising the amino acid sequence of a) or b). Chitosan-degrading enzyme which is a protein according to any one of the following a) to c):
a) a protein consisting of the amino acid sequence of SEQ ID NO: 14 in the sequence listing, wherein the α-amino group of the N-terminal serine residue is acetylated;
b) In the amino acid sequence of SEQ ID NO: 14 in the sequence listing, one or several amino acids are substituted, deleted, inserted or added, and the α-amino group of the N-terminal serine residue Is acetylated, and has a chitosan-degrading activity;
c) A protein comprising the amino acid sequence of SEQ ID NO: 14 in the sequence listing, wherein the α-amino group of the N-terminal serine residue is acetylated, and which has chitosan-degrading activity. DNA according to any one of the following:
a) DNA comprising a nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing;
b) DNA which hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above under stringent conditions and encodes a protein having chitosan-degrading activity ;
c) a DNA comprising a nucleotide sequence having 95% or more nucleotide sequence homology with the DNA described in a) above, and encoding a protein having chitosan-degrading activity;
d) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 5 in the sequence listing;
e) DNA comprising a nucleotide sequence represented by nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing. DNA according to any one of the following a) to c):
a) Recombinant plasmid retained by transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235), DNA inserted into pTriplEx-Chitinase-cDNA;
b) b) a protein which hybridizes with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) under stringent conditions and encodes a protein having a chitosan-degrading activity. DNA;
c) A DNA comprising the DNA described in a) above and encoding a protein having chitosan degrading activity. A chitosan-degrading enzyme which is a protein encoded by the DNA according to claim 7 or 8. Recombinant plasmid carried by transformed Escherichia coli Escherichia coli BM25.8/pTriplEx-Chitinase-cDNA@SANK 72102 (FERM @ BP-8235), chitosan digestion which is a protein encoded by DNA inserted in pTriplEx-Chitinase-cDNA enzyme. The chitosan-degrading enzyme according to claim 5, 6, 9, or 10 having an activity shown in the following a) to b):
a) an activity of hydrolyzing 30% acetylated chitosan (viscosity of 100 to 300 cps) under conditions of pH 3.5 to pH 10.5 and 0 ° C. to 80 ° C .;
b) Activity of hydrolyzing glycol chitin at pH 4.0 to pH 10.5. A recombinant plasmid comprising the DNA according to claim 7. The recombinant plasmid according to claim 12, which is an expression vector. A host cell transformed with the recombinant plasmid according to claim 12 or 13. The host cell according to claim 14, which is a prokaryotic cell or a eukaryotic cell. The host cell according to claim 14, which is a transformed Escherichia coli Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235). A method for producing a chitosan-degrading enzyme, comprising the following 1) and 2):
1) a step of culturing the cell according to any one of the following a) to e) under conditions for producing chitosan-degrading enzyme;
a) Aspergillus oryzae;
b) Aspergillus japonicus;
c) Aspergillus sojae;
d) a host cell according to any one of claims 14 to 16;
e) a cell that produces the chitosan-degrading enzyme according to any one of claims 5, 6, 9, and 11;
2) A step of separating and purifying chitosan-degrading enzyme from the culture product of 1). Aspergillus oryzae (Aspergillus oryzae) is Aspergillus oryzae (Aspergillus oryzae var. Sporoflavus Ohara JCM 2067) is a strain, Aspergillus japonicus (Aspergillus japonicus) is Aspergillus japonicus (Aspergillus japonicus) SANK 19288 strain, Aspergillus Sojae ( 18. The method according to claim 17, wherein Aspergillus sojae is Aspergillus sojae SANK strain 22388. A chitosan-degrading enzyme produced by the method according to claim 17. A method for producing low molecular weight chitosan, comprising the following steps 1) and 2):
1) The chitosan-degrading enzyme according to any one of claims 1 to 6, 9 to 11, and 19 is added to an aqueous solution of acetylated chitosan having a degree of acetylation of 10% to 30%. ;
2) Enzymatic reaction is performed for 10 minutes or more at pH 3 to 12, 10 ° C to 60 ° C. A low molecular weight chitosan or a salt thereof produced by the method according to claim 20. 22. The low molecular weight chitosan or a salt thereof according to claim 21, which has a molecular weight of 40,000 to 250,000 and a degree of acetylation of 10%. 22. The low molecular weight chitosan or a salt thereof according to claim 21, which has a molecular weight of 10,000 to 110,000 and a degree of acetylation of 20%. 22. The low molecular weight chitosan or a salt thereof according to claim 21, which has a molecular weight of 10,000 to 70,000 and a degree of acetylation of 30%. A pharmaceutical composition comprising the low-molecular-weight chitosan according to any one of claims 21 to 24 or a salt thereof as an active ingredient. 26. The pharmaceutical composition according to claim 25, which is a therapeutic agent for trauma, bedsore or atopic dermatitis. A cosmetic comprising the low-molecular-weight chitosan according to any one of claims 21 to 24 or a salt thereof. A food comprising the low molecular weight chitosan or a salt thereof according to any one of claims 21 to 24. 25. A treating agent such as water comprising the low molecular weight chitosan or a salt thereof according to any one of claims 21 to 24. A method for producing a sample comprising the following steps 1) and 2), wherein the liquid component does not substantially contain a polymer partially acetylated chitosan having a molecular weight of 1,000,000 or more:
1) A sample containing a polymer partially acetylated chitosan having a molecular weight of 1,000,000 or more is set forth in claim 1, claim 2, claim 3, claim 4, claim 5, claim 6, claim 9, claim 9, or claim 10. Addition of the chitosan-degrading enzyme according to any one of claim 11 and claim 19;
2) Enzymatic reaction is carried out for 10 minutes or more under conditions of pH 3 to 12, 10 ° C to 60 ° C. 31. The method according to claim 30, wherein the sample containing a high molecular weight partially acetylated chitosan having a molecular weight of 1,000,000 or more is a sample containing crustaceans. The method according to claim 31, wherein the crustacean is one or both of shrimp and crab. A sample produced by the method according to any one of claims 30 to 32, wherein the liquid component does not substantially contain a high molecular weight acetylated chitosan having a molecular weight of 1,000,000 or more.

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