JP4204857B2 - Chitosan degrading enzyme and its use - Google Patents

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 ＪＣＭ２０６７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
――――――――――――――――――――――――――――――
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。
――――――――――――――――――――――――――――――
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
――――――――――――――――――――
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Ｄ₂Ｏ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 MgCl₂、1mM DTT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 MgCl₂、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MgSO₄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/MgSO₄/マルトースブロスに植菌し、培養液のOD₆₀₀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 MgSO₄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/ MgSO₄トップアガーを混合し、温めておいたLB/ MgSO₄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/ MgSO₄トップアガーを添加し、混合し、温めておいたLB/ MgSO₄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 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。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OD₆₀₀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 Na₂HPO₄・12H₂O、2.68mM KCl、1.47mM KH₂PO₄)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 ＪＣＭ
２０６７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Ｄ₂Ｏ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２×１０⁶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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７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 Ohara ＪＣＭ２０６７由来の精製キチナーゼ）[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a chitosan-degrading enzyme that is separated and purified from at least one culture of Aspergillus oryzae, Aspergillus japonicus, Aspergillus sojae. Further, DNA encoding the chitosan-degrading enzyme, a recombinant plasmid inserted with the DNA, a host cell transformed with the recombinant plasmid, a method for producing the chitosan-degrading enzyme, and a low molecule using the chitosan-degrading enzyme Method for producing chitosan and a salt thereof, low-molecular chitosan produced by the method and a salt thereof, a pharmaceutical composition containing the low-molecular chitosan and a salt thereof, a food, 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 naturally abundant polysaccharide that is extracted and produced on an industrial scale mainly from crustaceans such as crabs and shrimps and squid soft shells. . Chitosan (β-1,4-polyglucosamine) is a polymeric polysaccharide having an amino group obtained by treating chitin with concentrated alkaline water, and is known to have various physiological activities (non- (See Patent Document 1). Among these, chitosan is known to have a strong antibacterial action as a natural product-derived substance, and is extremely safer than synthetic antibacterial agents (see Non-Patent Document 2). In particular, Staphylococcus aureus (see Non-Patent Document 3 to Non-Patent Document 8), Escherichia coli (including Non-patent Document 3 to Non-Patent Document 8), including methichilin resistant s. It is known to be effective against bacteria such as Escherichia coli (see Non-Patent Document 9) and Pseudomonas aeruginosa (see Non-Patent Document 10 and Non-Patent Document 11).
[0003]
Chitosan, which is produced by treating the chitin extracted from crustaceans such as crab and shrimp and squid soft shell with concentrated alkali, is a high molecular weight polysaccharide with a molecular weight of 1 million or more, and is soluble in water under acidic conditions. Does not dissolve in water under alkaline conditions. And in the state which does not melt | dissolve in water, the physiological activity of chitosan cannot fully be exhibited. In addition, when processing food materials containing crustaceans, there has been a problem that these polymer polysaccharides are in a very high viscosity state, and the subsequent processing is troublesome.
[0004]
In order to increase the solubility in water, attempts have been made to derivatize high molecular chitosan (Non-patent Document 12) or to lower the molecular weight without impairing the physiological activity of high molecular chitosan.
[0005]
If derivatization is performed, the structure may be different from that of a natural product, so that it may have a property different from that of natural chitosan, or safety test data 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, in order to increase the solubility in water, it is considered that low molecular weight is desirable rather than derivatization. It is known that the antibacterial activity of chitosan is exhibited not only by high molecular chitosan having a molecular weight of 1 million or more but also by low molecular chitosan having a molecular weight of 10,000 or more (Non-patent Document 13).
[0007]
On the other hand, chitosan having a molecular weight of 10,000 or less has been reported to have side effects such as skin irritation (Patent Document 1), and a method for producing a low-molecular chitosan having a molecular weight of 10,000 or more has been studied.
[0008]
As a method for producing low-molecular chitosan, hydrolysis of high-molecular chitosan by a chemical or enzymatic method has been studied. Examples of the chemical production method of low molecular 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 chitosan having a molecular weight of 500 to 30,000 is produced in a large amount by the method of treating with an oxidizing agent, and the ratio of chitosan having a molecular weight of 10,000 or more is not clear. In addition, it is difficult to increase the size of the apparatus by the ultrasonic processing method, and it is considered unsuitable for mass production. Examples of the enzymatic production method of low-molecular chitosan include a method using chitosanase (Patent Documents 4 and 5), a method using chitinase (Non-Patent Document 15), and the like.
[0009]
In addition, as the chitinase derived from bacteria, the genus Bacillus, the genus Corynebacterium (see Non-Patent Document 16), the genus Cytophaga, the Acromobacter hyteropticum (patent document) 17), chitinases produced by the genus Flavobacterium and Micrococcus colpogenes (see non-patent document 17) and chitinases A produced by Alteromonas sp. (GenBank Accession No. .D13762) is known. As chitinases produced by actinomycetes, chitinases produced by the genera Streptomyces, Nocardiopsis, and Actinomyces are known (Non-patent Document 18). As chitinases produced by filamentous fungi, chitinases produced by the genera Aspergillus, Rhizopus, Talaromyces, Trichoderma, and Penicillium are known. Examples of enzymes produced by Aspergillus include Aspergillus nidulans, Aspergillus niger, Aspergillus candidus (see Non-Patent Document 19), and Aspergillus fumigatus produced by Aspergillus fumigatus. It has been known. Examples of enzymes produced by yeast include chitinases produced by Saccharomyces cerevisiae, and chitinases produced by Saccharomyces cerevisiae (GenBank Accession No. M74070) and ORF D9481.7 (GenBank Accession No. U28373).
[0010]
As described above, 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 in the production of miso and soy sauce sake, there is a report that there was an academic conference on a gene thought to encode chitinase (see Non-Patent Document 20). However, the biological activity of the protein encoded by the gene has not been reported, and the nucleotide sequence and amino acid sequence have not been confirmed in public databases until now.
[0011]
On the other hand, the method for producing low-molecular chitosan using chitosanase also has a problem that the reaction control for obtaining low-molecular chitosan having the same molecular weight is 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]
Japanese Patent Laid-Open No. 11-322809
[Non-Patent Document 1]
“Chitin / Chitosan Handbook”, 1995, Gihodo, p.302-376
[Non-Patent Document 2]
“Utilization of chitin and chitosan”, Special Issue of Business World, Volume 46, 28, 1998, p.106-111
[Non-Patent Document 3]
“Processing Technology” 1998, 33, pp. 512-515
[Non-Patent Document 4]
"Meijo University Academic Report, 1998", 34, p.25-34
[Non-Patent Document 5]
Textile Science, 1993, 35, p.37-39
[Non-Patent Document 6]
"Osaka Prefectural Institute of Public Health Research Report Pharmaceutical Affairs No.29", 1995, p.7-12
[Non-Patent Document 7]
“Processing Technology” 1997, 32, p.339-343
[Non-Patent Document 8]
"Processing Technology" 1998, 33, p.530-532
[Non-patent document 9]
Tokura, S et al. “Macromolecular Symposia” 1997, 120, p.1-9
[Non-Patent Document 10]
Loke, WK et al. “Journal of Biomedicinal Materials Research” 2000, 53, p.8-17
[Non-Patent Document 11]
Kim, HJ et al., “Journal of Biomaterials Science, Polymer Edition” 1999, 10, p.543-56
[Non-Patent Document 12]
“Utilization of chitin and chitosan” Special Issue 46, No. 28, 1998, p.78-81
[Non-Patent Document 13]
"Abstracts of the 74th Annual Meeting of the Japanese Society for Agricultural Chemistry" Lecture number 2E183α, 2000
[Non-Patent Document 14]
"Research on chitin and chitosan" 1999, Volume 5, p.75-79
[Non-Patent Document 15]
“Abstracts of the 74th Annual Meeting of the Japanese Society for Agricultural Chemistry” Lecture number 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, Volume 5, p.894
[Non-Patent Document 18]
Shota Nakagami et al., “Technology Institute Fermentation Laboratory Report”, 1966, Volume 30, p.19
[Non-Patent Document 19]
Sherif, AA, et al., “(Applied Microbiology and Biotechnology)” 1991, 35, p.228
[Non-Patent Document 20]
“Research 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 extensive studies on a method for efficiently producing a low-molecular chitosan that is soluble in water under neutral conditions and has an antibacterial action. As a result, Aspergillus oryzae var. Sporoflavus Ohara A chitinase derived from the JCM 2067) strain or a chitinase derived from the Aspergillus japonicus SANK 19288 strain or a chitinase derived from the Aspergillus sojae SANK 22388 strain, and a cDNA encoding the chitinase is cloned. Through the enzymatic reaction using chitinase, we succeeded in producing a low molecular weight non-derivative chitosan having a molecular weight of 10,000 or more from crab / shrimp-derived high molecular chitosan, and completed the present invention.
[0014]
According to the present invention, non-derivative low-molecular 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 degrading enzyme that is separated and purified from at least one culture of Aspergillus oryzae, Aspergillus japonicus, Aspergillus sojae,
(2) Aspergillus oryzae is Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 strain, Aspergillus japonicus is Aspergillus japonicus strain 19 Aspergillus japonicus 19 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 exhibits the following properties:
1) Shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis;
2) Isoelectric point pI3.5 to 4.5 is shown by isoelectric focusing method;
3) Hydrolyze 30% acetylated chitosan (viscosity 100-300 cps) at pH 3.5 to pH 10.5;
4) Hydrolyze glycol chitin at pH 3.0 to pH 10.5;
5) exhibits the hydrolytic 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,
(5) Chitosan-degrading enzyme, which is a protein according to any one of the following a) to d):
a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing;
b) a protein comprising an amino acid sequence encoded by the nucleotide sequence shown in nucleotide numbers 242-1438 of SEQ ID NO: 4 in the sequence listing;
c) a protein comprising the amino acid sequence described in a) or b), wherein one or several amino acids are substituted, deleted, inserted or added, and has chitosan degrading activity;
d) a protein comprising the amino acid sequence described in a) or b),
(6) Chitosan degrading enzyme, which is a protein according to any one of the following a) to c):
a) a protein comprising the amino acid sequence set forth in 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 set forth in SEQ ID NO: 14 in the Sequence Listing, an amino acid sequence consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added, and the N-terminal serine residue α-amino group Wherein the protein is acetylated and has chitosan degrading activity;
c) a protein comprising the amino acid sequence set forth in SEQ ID NO: 14 of the Sequence Listing, wherein the α-amino group of the N-terminal serine residue is acetylated, and has chitosan degrading activity;
(7) DNA according to any one of the following a) to e):
a) DNA consisting of the nucleotide sequence shown in nucleotide numbers 242-1438 of SEQ ID NO: 4 in the sequence listing;
b) DNA that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above 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 set forth in SEQ ID NO: 5 in the Sequence Listing;
e) DNA comprising the nucleotide sequence shown in nucleotide numbers 242-1438 of SEQ ID NO: 4 in the sequence listing;
(8) DNA according to any one of the following a) to c):
a) a recombinant plasmid carried by transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235), DNA inserted into pTriplEx-Chitinase-cDNA;
b) b) hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above and encodes a protein having chitosan degrading activity, DNA to do;
c) 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) A transformed plasmid carried by transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235), a protein encoded by DNA inserted into 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) Activity of hydrolyzing 30% acetylated chitosan (viscosity 100 to 300 cps) under the conditions of pH 3.5 to pH 10.5 and 0 ° C. to 80 ° C .;
b) activity to hydrolyze glycol chitin at pH 4.0 to pH 10.5;
(12) A recombinant plasmid containing the DNA according to (7) or (8),
(13) The recombinant plasmid according to (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 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) to 2):
1) A step of culturing the cell according to any one of the following a) to e) under conditions for producing a chitosan degrading enzyme;
a) Aspergillus oryzae;
b) Aspergillus japonicus;
c) Aspergillus sojae;
d) The host cell according to any one of (14) to (16);
e) a cell producing 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 is Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 strain, Aspergillus japonicus is Aspergillus japonicus 19 -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 a low-molecular 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) Perform enzyme reaction for 10 minutes or more under the conditions of pH 3 to 12, 10 ° C to 60 ° C.
(21) Low-molecular chitosan produced by the method according to (20) or a salt thereof,
(22) The low molecular weight chitosan or salt thereof according to (21), wherein the molecular weight is 40,000 to 250,000 and the degree of acetylation is 10%,
(23) Low molecular 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 chitosan or a salt thereof according to (21), having a molecular weight of 10,000 to 70,000 and a degree of acetylation of 30%,
(25) A pharmaceutical composition comprising the low-molecular chitosan or 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, bed sores or atopic dermatitis
(27) A cosmetic comprising the low-molecular chitosan or a salt thereof according to any one of (21) to (24),
(28) A food having the low-molecular chitosan or salt thereof according to any one of (21) to (24),
(29) A treatment agent such as water having the low-molecular chitosan or a salt thereof according to any one of (21) to (24),
(30) A method for producing a sample which contains the following steps 1) and 2) and which does not substantially contain a polymer partially acetylated chitosan having a molecular weight of 1 million or more in the liquid component:
1) The chitosan degrading enzyme according to any one of (1) to (6), (9) to (11) and (19) is added to a sample containing a high molecular weight partially acetylated chitosan having a molecular weight of 1 million or more. ;
2) Perform an enzyme reaction for 10 minutes or more under conditions of pH 3 to 12, 10 ° C to 60 ° C.
(31) The method according to (30), wherein the sample containing a polymer partially acetylated chitosan having a molecular weight of 1 million 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 which is produced by the method of any one of (30) to (32) and which does not substantially contain a polymer partially acetylated chitosan having a molecular weight of 1 million or more in its liquid component,
About.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
[0017]
As a filamentous fungus useful for producing low-molecular chitosan, chitinase derived from Aspergillus oryzae, chitinase derived from Aspergillus japonicus or chitinase derived from Aspergillus sojae, The present invention relates to a low molecular chitosan produced by the chitinase, a pharmaceutical composition containing the low molecular chitosan, and the like.
[0018]
In the present invention, chitinase refers to an enzyme having an activity of hydrolyzing chitin or partially acetylated chitosan. When chitin is hydrolyzed, N-acetylglucosamine and its oligosaccharide are produced. This “activity for hydrolyzing chitin or partially acetylated chitosan” is defined as “chitosan decomposing activity”. Chitin refers to a partially acetylated chitosan having a high acetylation rate. 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 enzymes having the 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 producing chitinase. Examples of such chitinases include chitinases derived from Aspergillus oryzae, chitinases derived from Aspergillus japonicus or chitinases derived from Aspergillus sojae. More preferred are chitinase from Aspergillus oryzae var. Sporoflavus Ohara JCM 2067), chitinase from Aspergillus japonicus SANK 19288 strain or Aspergillus sojae 23 strain Aspergillus sojae It is a chitinase, and more preferably a chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara JCM 2067).
Further, another example of the chitinase of the present invention includes a protein having 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 be any protein as long as it contains all of these 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-”. A protein consisting of Lys (amino acid numbers 1 to 5 of SEQ ID NO: 14 in the sequence listing) "and having chitosan degrading activity. More preferably, it includes 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 consists of Ser-Ser-Gly-Leu-Lys-, and And N-terminal acetylated protein having chitosan degrading activity.
[0019]
Further, another example of the chitinase of the present invention includes a protein having all the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 in the sequence listing and having chitosan degrading activity.
[0020]
Further, in the protein comprising the amino acid sequence set forth in 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. As long as it has chitosan degrading activity, it is included in the present invention. The term “several” means a number not exceeding 10, and preferably not exceeding 5. 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 an interleukin 2 (IL-2) gene is converted into a nucleotide sequence corresponding to serine. The resulting protein is known to retain 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 consisting of the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing. Moreover, even if it is a protein which consists of an amino acid sequence of sequence number 5 of a sequence table, as long as it has chitosan degradation activity, it is included in this invention.
[0022]
Another example of the chitinase of the present invention is a protein comprising the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing and acetylated at the N-terminal α-amino group. A protein comprising such a protein is also included in the present invention as long as it has chitosan degrading activity.
[0023]
Suitable as the chitinase of the present invention is chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara JCM 2067, chitinase derived from Aspergillus japonicus SANK19288, Aspergillus sojae 23A88 Strain-derived chitinase, a protein comprising the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing, and a protein comprising the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing, wherein the N-terminal α-amino group is acetylated. More preferred is a chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara JCM 2067) and the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing, and having an N-terminal α-amino group Acetylated That is the protein.
In the present invention, the “DNA of the present invention” refers to DNA encoding the chitinase of the present invention. The DNA may take any form as long as it is currently known, such as cDNA, genomic DNA, artificially modified DNA, and chemically synthesized DNA.
[0024]
As an example of the DNA of the present invention, it hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence shown in nucleotide numbers 242 to 1438 of SEQ ID NO: 4 in the sequence listing, and has chitosan degradation. Examples thereof include DNA encoding a protein having activity.
[0025]
Another example of the DNA of the present invention is a nucleotide sequence homology of 70% or more, preferably 80% or more, more preferably 95% or more with the nucleotide sequence shown in nucleotide numbers 242-1438 of SEQ ID NO: 4 in the Sequence Listing. DNA having Such DNA includes mutant DNA found in nature, artificially modified mutant DNA, homologous DNA derived from heterologous organisms, and the like.
[0026]
Still another example of the nucleotide of the present invention is DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing. The codon corresponding to the desired amino acid may be selected arbitrarily and can be determined according to a conventional method in consideration of the codon usage frequency of the host to be used, for example. (Grantham, R. et al. (1981) Nucleic Acids Res. 9, 143-174). Furthermore, partial modification of the codons of these nucleotide sequences is carried out by site-directed mutagenesis / Mark, DF et al., Using a primer comprising a synthetic oligonucleotide encoding a desired modification according to a conventional method. (1984) Proc. Natl. Acad. Sci. USA 81, 5662-5666).
[0027]
Another example of the DNA of the present invention includes DNA consisting of the nucleotide sequence shown in nucleotide numbers 242-1438 of SEQ ID NO: 4 in the sequence listing. In addition, a DNA comprising a DNA comprising the nucleotide sequence shown in 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 contains a region encoding a protein having chitosan degrading activity. It is.
[0028]
The transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102, which holds the recombinant plasmid inserted with the DNA of the present invention, is a patent of the National Institute of Advanced Industrial Science and Technology as of November 8, 2002. It is deposited internationally at the Biological Depositary Center (Higashi 1-1-1, Chuo 6th, Tsukuba City, Ibaraki, Japan) and is given the deposit number FERM BP-8235. Therefore, the DNA of the present invention can also be obtained from the strain. In addition, DNA that hybridizes under stringent conditions with DNA inserted into the recombinant plasmid held by the deposited strain is also included in the present invention as long as the encoded protein has chitosan degrading activity. Further, DNA comprising the above nucleotide sequence is also included in the present invention. Furthermore, the protein encoded by such DNA is also included in the chitinase of the present invention.
[0029]
Suitable as the DNA of the present invention is DNA consisting of the nucleotide sequence shown in nucleotide numbers 242-1438 of SEQ ID NO: 4 in the sequence listing, and transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK72102 (FERM BP-8235) is a DNA inserted into a recombinant plasmid, pTriplEx-Chitinase-cDNA, optimally transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP- 8235) is a DNA inserted into a recombinant plasmid, pTriplEx-Chitinase-cDNA.
[0030]
The chitinase of the present invention can include a protein having an amino acid sequence encoded by the DNA of the present invention. In addition, in order to produce a modified form in which any one or more amino acids are deleted in the chitinase of the present invention, a method of cutting DNA from the end using exonuclease Bal31 or the like (Toshimitsu Kishimoto et al. “ Follow-up Biochemistry Experiment Course 1, Genetic Research Method II ”335-354), cassette mutation method (Toshimitsu Kishimoto,“ Neochemistry Experiment Course 2, Nucleic Acid III Recombinant DNA Technology ”242-251), etc. Thus, even a protein obtained by genetic engineering techniques 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 is not necessarily required to have all of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing. For example, even a protein consisting of a partial sequence thereof is not limited as long as the protein exhibits chitosan degrading activity. It is included in the chitinase of the present invention. Further, DNA encoding such a chitinase is also included in the present invention.
[0031]
The chitinase used in the present invention may be a product purified from a chitinase-producing bacterium, a crudely purified product, a cell disruption solution, or a cell culture supernatant as it is. When culturing chitinase-producing bacteria, it is preferable to add powder chitin, powder chitosan, colloidal chitin, colloidal chitosan and the like in addition to the carbon source and nitrogen source in the medium. Centrifugation is performed after completion of the culture of the chitinase-producing bacteria, and the culture supernatant from which the cells have been removed can be used as a crude enzyme solution as it is. Moreover, the crude enzyme solution can be roughly purified by ion exchange chromatography or the like, or purified.
[0032]
A chitinase-producing bacterium refers to a microorganism having an inherently innate ability to produce chitinase, and includes a microorganism that accumulates chitinase in the microbial cell, a microorganism that secretes the microbial cell outside the microbial cell, and the like. When using a chitinase-producing bacterium culture supernatant or a chitinase purified from the culture supernatant, a bacterium that secretes chitinase outside the cell body can be used.
[0033]
Examples of the chitinase producing bacterium used in the present invention include Aspergillus oryzae, Aspergillus japonicus or Aspergillus sojae, preferably Aspergillus oryzae var. Examples include sporoflavus Ohara JCM 2067 strain, Aspergillus japonicus SANK 19288 or Aspergillus sojae SANK 22388 strain. A chitinase-producing bacterium can be cultured using a normal medium using a culture apparatus and a medium. The culture can be appropriately selected from methods such as liquid culture and solid culture. In the case of liquid culture, flask culture or culture using a fermenter can be performed, and after the start of culture, a batch culture method in which no medium is added or a fed-batch culture method in which a medium is appropriately added during the culture should be used. Can do. A carbon source and a nitrogen source can be added to the medium, and vitamins, trace metals, and the like can be added as necessary. Examples of the carbon source include monosaccharides such as glucose, mannose, galactose and fructose, disaccharides such as maltose, cellobiose, isomaltose, lactose and sucrose, polysaccharides such as starch, and malt extract. It is not limited to these as long as the fungus grows. As the nitrogen source, inorganic nitrogen such as ammonia, ammonium sulfate, and ammonium nitrate, and organic nitrogen such as yeast extract, malto extract, corn steep liquor, and peptone are used, but are not limited to these as long as the chitinase-producing bacteria grow. In addition, in order to increase the amount of chitinase produced by the chitinase-producing bacterium, powdered chitin or colloidal chitin can be added to the medium. The amount of the composition in these media can be appropriately selected. The culture temperature, pH, and aeration stirring amount can be appropriately selected so as to be suitable for chitinase production.
[0034]
As the chitinase used in the present invention, chitinase derived from Aspergillus oryzae, Aspergillus japonicus or Aspergillus sojae can be used, and preferably Aspergillus oryzae sporoflavus Ohara Chitinase derived from JCM 2067 strain, Aspergillus japonicus SANK 19288 or Aspergillus sojae SANK 22388 strain can be used. The chitinase may be produced by the chitinase-producing bacterium itself, or may be produced by a mutant or modified product thereof. Further, a gene encoding the chitinase of the chitinase-producing bacterium is introduced into the host. The recombinant protein produced from the obtained transformant may be sufficient.
Obtaining chitinase-producing bacteria
Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 strain is the National Institute for Biological Sciences, Microorganism Preservation Facility (JCM: Japan Collection of Microorganisms; address 2-1 Hirosawa, Wako, Saitama, Japan 351-0198) It can be purchased from the website address <http://www.jcm.riken.go.jp/>).
[0035]
The mycological properties of Aspergillus japonicus SANK 19288 and Aspergillus sojae SANK 22388 are shown below.
[0036]
Aspergillus Japonix SANK 19288 and Aspergillus sojae SANK 22388 strain Click and Pit literature (Klich, MA and Pitt, JI (1988) A laboratory guide to the common Aspergillus species and their teleomorphs. CSIRO, Division of Food Processing, North Ryde NSW, Austraria.) Was inoculated into three types of medium (CYA medium, MEA medium, CY20S medium) and the mycological properties were observed.
[0037]
The display of the color tone was according to “Metune Handbook of Color” (Kornerup, A. and Wanscher, JH (1978) Methuen handbook of color (3rd. Edition). Erye Methuen, London.).
[0038]
The composition of the three types of medium (CYA medium, MEA medium, CY20S medium) is as follows.
CYA Medium [Czapek Yeast Extract Agar Medium] (K2HPO4 1.0 g, * Zapec Concentrate 10 ml, Yeast Extract 5 g, Sucrose 30 g, Agar 15 g, Distilled Water 1000 ml)
* Zapec concentrate (NaNO3 30 g, KCl 5 g, MgSO4 ・ 7H2O 5 g, FeSO4 ・ 7H2O 0.1 g, ZnSO4 ・ 7H2O 0.1 g, 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 Extract Agar with 20% Sucrose Medium] (K2HPO4 1.0 g, * Zapec Concentrate 10 ml, Yeast Extract 5 g, Sucrose 200 g, Agar 15 g, Distillation (1000 ml of water)
1) Mycological properties
a) Mycological properties of Aspergillus japonics SANK 19288 strain
Colonies in CYA medium are 45-60 mm in diameter at 25 ° C. for 1 week. The mycelium is white. Conidia formation is vigorous in the center, and the color of the formation is olive brown (4F4) or black. The back color is grayish yellow (4B4) or yellowish white (4A2). No sclerotia is observed. No leachate or soluble pigment is observed. Colonies in CY20S medium have a diameter of 57 to 68 mm when cultured for 1 week at 25 ° C. The colony resembles that of CYA medium. The back color is Pastoral Yellow (3A4). Colonies in MEA medium are 51 to 57 mm in diameter at 25 ° C. for 1 week. Conidia formation is vigorous, and the color of the formation part is black. The color on the back is colorless. Colonies of CYA medium cultured at 37 ° C for 1 week are 13 to 20 mm in diameter. Does not grow at 5 ° C.
[0039]
Conidia head is radial, conidia pattern is smooth, colorless or brown at the top, width 4-11 μm. The crest is spherical or elliptical and has a width of 19 to 50 μm. Aspergillus is a single row. The phialide covers 3/4 of the summit and is 7-10 × 3.5-4.5 μm. The conidia are oval or spherical, covered with somewhat sparse spines, and have a diameter of 3.5 to 4.5 x 3.5 to 4 μm.
[0040]
Based on the above bacteriological properties, a search was made for a bacterium corresponding to this bacterium. The properties of Aspergillus japonicus var. Aculeatus (Iizuka) Al-Musallam described in the click and pit literature. Almost matched. Therefore, this strain was identified as Aspergillus japonicus var. Aculeatus (Iizuka) Al-Musallam.
[0041]
In addition, Aspergillus Japonix SANK 19288 was changed to Aspergillus Japonix Bar Accuritus SANK 19288 on September 13, 2001 at the National Institute of Advanced Industrial Science and Technology (AIST) 1-1 Centrally deposited at 6) and assigned the deposit number FERM BP-7736.
[0042]
b) Mycological properties of Aspergillus sojae SANK 22388 strain
Colonies in CYA medium are 46 to 52 mm in diameter at 25 ° C. for 1 week. The colony is fluffy. The mycelium is white and dense. Conidia formation is vigorous, and the color of the formation part is olive (2F-E7). The reverse side is yellowish white (2A2). Colonies in CY20S medium have a diameter of 42 to 45 mm when cultured for 1 week at 25 ° C. The colony resembles that of CYA medium. Conidia formation is vigorous, and the color of the formation part is olive yellow (3D6) to olive (3E7). Colonies in MEA medium are 43-47 mm in diameter at 25 ° C. for 1 week. The colony is slightly fluffy and slightly sparse. The mycelium is white and inconspicuous. Conidia formation is vigorous, and the color of the formation part is olive (1E8). The color on the back is colorless. The colonies of CYA medium cultured at 37 ° C. for 1 week are 52 to 56 mm in diameter. Does not grow at 5 ° C.
[0043]
The conidia head is radial, the conidia pattern is rough, colorless, and the width is 7 to 12 μm. The summit has a western shape or a spherical shape, and a width of 24 to 41 μm. Aspergillus is single-row, sometimes double-row. The metre is 8-12 × 4-8 μm. The phialide is 10 to 11 × 4.5 to 6 μm. The conidia are spherical, rough, and have a diameter of 5 to 6.5 μm.
[0044]
Based on the above bacteriological properties, when the bacteria corresponding to this bacterium were searched, the properties of Aspergillus sojae Sakaguchi & Yamada described in the click and pit literature were almost identical. Therefore, this strain was identified as Aspergillus sojae Sakaguchi & Yamada.
[0045]
In addition, Aspergillus sojae SANK 22388 strain was changed to Aspergillus sojae SANK 22388 strain on September 13, 2001, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1-1-1 Tsukuba City East, Ibaraki Prefecture, Japan). Deposited internationally in the center No. 6) and given the accession number FERM BP-7738.
[0046]
As is well known, filamentous fungi are susceptible to mutation in nature or by artificial manipulation (for example, ultraviolet irradiation, radiation irradiation, chemical treatment, etc.), and Aspergillus oryzae var. Sporoflavus Ohara JCM of the present invention. 2067) strain, Aspergillus japonics SANK 19288 strain and Aspergillus sojae SANK 22388 strain are the same. Aspergillus oryzae (Aspergillus oryzae var. Sporoflavus Ohara JCM 2067) strain, Aspergillus japonics SANK 19288 strain and Aspergillus sojae SANK 22388 strain include all the mutant strains. These mutant strains also include those obtained by genetic methods such as recombination, transduction, transformation and the like. That is, the Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 strain that produces chitinase, the mutants thereof, and the strains that are not clearly distinguished from them all belong to the Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 strain. Is included. Aspergillus japonics SANK 19288 strains producing chitinase, mutants thereof and strains not clearly distinguished from them are all included in the Aspergillus japonics SANK 19288 strain. The Aspergillus sojae SANK 22388 strain, the mutants thereof, and the strains not clearly distinguished from them are all encompassed by the Aspergillus sojae SANK 22388 strain.
The chitinase of the present invention can also be obtained from a culture product of a transformed cell obtained by transforming a host cell with a recombinant plasmid in which the DNA of the present invention is inserted into a vector. Thus, a 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, prokaryotic vectors, eukaryotic vectors, mammalian cell vectors, and the like. Such recombinant plasmids can transform other prokaryotic or eukaryotic host cells. Furthermore, it is possible to express a gene in each host by using a vector having an appropriate promoter sequence and / or a sequence involved in expression, or by introducing such a sequence into an expression vector. It is. Such an expression vector is a preferred embodiment of the recombinant plasmid of the present invention.
[0047]
A host cell can be obtained by introducing the recombinant plasmid of the present invention into various cells. Such a cell may be a prokaryotic cell or a eukaryotic cell as long as it can introduce a plasmid.
[0048]
Examples of the prokaryotic host include Escherichia coli and Bacillus subtilis. To transform a gene of interest into these host cells, the host cell is transformed with a plasmid vector containing a replicon or origin of replication from a species compatible with the host and regulatory sequences. The vector preferably has a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.
[0049]
For example, K12 strain and the like are often used as E. coli, and pBR322 and pUC-type plasmids are generally used as vectors. However, the present invention is not limited thereto, and any known various strains and vectors can be used.
[0050]
Examples of the promoter in Escherichia coli include tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter, etc. Any promoter can be used to produce the chitinase of the present invention.
[0051]
As Bacillus subtilis, for example, 207-25 strain is preferable, and pTUB228 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) is used as a vector, but is not limited thereto. is not.
[0052]
As a promoter, secretory expression outside the cell can be achieved by ligating a DNA sequence encoding a signal peptide sequence of Bacillus subtilis α-amylase.
[0053]
Eukaryotic host cells include cells such as vertebrates, insects, and yeasts. Examples of vertebrate cells include mammalian cells such as COS cells (Gluzman, Y. (1981 Cell 23, 175-182, ATCC CRL-1650) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, LA (1980) Proc. Natl Acad. Sci. USA 77, 4126-4220) is often used, but is not limited thereto.
[0054]
As an expression promoter for vertebrate cells, a promoter that is usually located upstream of the gene to be expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, and the like can be used. You may have. Examples of the expression vector include, but are not limited to, pSV2dhfr (Subramani, S. et al. (1981) Mol. Cell. Biol. 1, 854-864) having the SV40 early promoter.
[0055]
For example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, and can self-propagate in the COS cell. Furthermore, a transcription promoter, a transcription termination signal, and an RNA splice site Can be used. The expression vector includes diethylaminoethyl (DEAE) -dextran method (Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res, 11, 1295-1308), 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), etc. Thus, a desired transformed cell can be obtained. When a CHO cell is used as a host cell, an expression vector and a vector capable of expressing a neo gene that functions as an antibiotic G418 resistance marker, such as 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, 327-341) By selecting resistant colonies, transformed cells that stably produce the chitinase of the present invention can be obtained.
[0056]
When insect cells are used as host cells, cell lines derived from ovarian cells of Spodoptera frugiperda (Sf-9 or Sf-21) of Lepidoptera gourdaceae or high five cells derived from egg cells of Trichoplusia ni (Wickham, TJ et al, ( 1992) Biotechnol. Prog. I: 391-396) is often used as a host cell, and pVL1392 / 1393 using a polyhedrin protein promoter of autographer nuclear polyhedrosis virus (AcNPV) is often used as a baculovirus transfer vector. (Kidd, IM and VC Emery (1993) The use of baculoviruses as expression vectors. Applied Biochemistry and Biotechnology 42, 137-159). In addition, vectors using baculovirus P10 and promoters of the same basic protein can also be used. Furthermore, it is also possible to express a recombinant protein as a secreted protein by connecting the secretory signal sequence of the envelope surface protein GP67 of AcNPV 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, and among them, Saccharomyces yeasts such as baker's yeast Saccharomyces cerevisiae and petroleum yeast Pichia pastoris are preferable. Examples of expression vectors for eukaryotic microorganisms such as yeast include alcohol dehydrogenase gene promoters (Bennetzen, JL and Hall, BD (1982) J. Biol. Chem. 257, 3018-3025) and acid phosphatase gene. A promoter (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1-5) and the like can be preferably used. When expressed as a secreted protein, it can also be expressed as a recombinant having a secretory signal sequence and an endogenous protease possessed by the host cell or a known protease cleavage site on 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, it is activated by connecting the yeast α-factor secretion signal sequence to the N-terminal side of the yeast yeast factor KEX2 protease cleavage site. Type tryptase is known to be 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 commonly used media can be appropriately selected depending on the employed host cells. For example, in the case of the COS cells, RPMI1640 medium and Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”). A medium supplemented with a serum component such as fetal calf serum can be used if necessary. As culture conditions, the CO2 concentration may be 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., more preferably 35 to 40 ° C.
[0059]
The chitinase of the present invention produced as a recombinant protein inside or outside of the transformant by the above-described culture is the physicochemical property, chemical property, biochemical property (enzyme activity) of the protein from the culture product. Etc.), etc. ("Biochemistry Data Book II", 1175-1259, 1st edition, 1st print, published on June 23, 1980 by Tokyo Chemical Co., Ltd .; Biochemistry, vol. 25, No. 25, p8274-8277 (1986); see Eur. J. Biochem., 163, p313-321 (1987), etc.). Specific examples of the method include normal reconstitution treatment, treatment with a protein precipitating agent (salting out method), centrifugation, osmotic shock method, freeze-thaw method, ultrasonic crushing, ultrafiltration, gel filtration, Examples include adsorption chromatography, ion exchange chromatography, affinity chromatography, various liquid chromatography such as high performance liquid chromatography (HPLC), dialysis methods, combinations thereof, and the like. As described above, a desired recombinant protein can be produced on an industrial scale in a high yield. Further, by connecting histidine consisting of 6 residues to a 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 large quantities with high yield and high purity.
[0060]
A chitinase produced by the above method can also be mentioned as a preferred example of the present invention.
10 g or more of low molecular chitosan or a salt thereof used in the present invention is dissolved in 100 g of water at 0 to 100 ° C. The water here is preferably distilled water, tap water, or well water. Furthermore, 10 g or more of low-molecular chitosan or a salt thereof is dissolved in 100 g of water under the condition of pH 1.0 to 7.0. Low molecular chitosan has antibacterial activity.
[0061]
Partially acetylated chitosan means that part of the amino group of chitosan is acetylated. Partially acetylated chitosan having a low degree of acetylation can be produced by deacetylating chitin with an alkali. . The degree of acetylation of partially acetylated chitosan can be controlled by alkali concentration, reaction time, and the like. Further, those having a molecular weight of 1 million or more are particularly referred to as polymer partially acetylated chitosan. Examples of polymer partially acetylated chitosan having a molecular weight of 1 million or more include, for example, 30%, 20%, 10% acetylated chitosan derived from shrimp crabs (trade names: Chitosan 7B, 8B, 9B (all manufactured by Katoyoshi Co., Ltd.)) However, 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 partially polymerized acetylated chitosan can be measured by high performance liquid chromatography using gel permeation chromatography (GPC) (with pullulan as a standard substance) (Takiguchi et al., Chitin Chitosan Research p.75-). 79, 1999).
[0063]
The degree of acetylation of the polymer partially acetylated chitosan can be measured by performing a PVSK colloid titration with a 1/400 N potassium 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 is the percentage of amino groups that are acetylated (for example, 1% acetylation means that one 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 hydrolytic activity of chitinase can be measured according to the Randle-Morgan method (Randle CJM et al. Biochem. J. GL 586-589, 1955). Moreover, it can also measure 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 a low-molecular chitosan.
[0067]
The chitinase used in the method is hydrolyzed when 30% acetylated chitosan (viscosity 100 to 300 cps) is used as a substrate under reaction conditions of pH 4.0 to 5.5 and temperature of 37 ° C. for 10 to 30 minutes. When the degradation activity is 100%, the hydrolysis activity when glycol chitin is used as a substrate is less than 100%, and chitosan (viscosity 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]
There are no particular restrictions on the amount and concentration of enzyme used in the method.
[0069]
In the method, the aqueous solution of the polymer partially acetylated chitosan serving as a substrate for chitinase is 0.1 to 5% by mass, preferably 0.25 to 2% by mass, and more preferably 0.5 to 1% by mass. It is. The aqueous solution can also be used as a solution dissolved in water under acidic conditions (for example, pH 1 to 6). Moreover, it can also use for this method as a solution which added alkali (for example, sodium carbonate, sodium hydrogencarbonate, caustic soda, etc.) to this solution, and was made weakly acidic (for example, pH 4.0-6.0).
[0070]
Acids used to make the polymer partially acetylated chitosan solution acidic and dissolved in water include hydrochloric acid, acetic acid, trifluoroacetic acid, p-toluenesulfonic acid, acrylic acid, lactic acid, amino acid, citric acid, salicylic acid, glucuronic acid , Glycolic acid and the like can be mentioned, but are not limited thereto as long as an acidic solution can be produced.
[0071]
The pH range of the reaction solution when producing a low-molecular chitosan using the chitinase of the present invention can include a pH range of 3 to 12, preferably a pH range of 3 to 7, and more preferably It is in the range of pH 4-6. The temperature of the reaction solution may be 0 ° C to 70 ° C, preferably 10 ° C to 60 ° C, and more preferably 40-50 ° C. Although reaction time can mention 10 minutes-7 days, Preferably it is 3 hours-5 days, More preferably, it is 1-3 days.
[0072]
Various low-molecular chitosan salts having a molecular weight of 10,000 or more and less than 1,000,000 are produced by making a 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 salt type is hydrochloride, acetate, trifluoroacetate, p-toluenesulfonate, acrylate, lactate, amino acid salt, citrate, salicylate, glucuronate, glycol Examples of the acid salt include, but are not limited to.
The present invention provides a low-molecular chitosan or a salt thereof that dissolves 10 g or more in 100 g of water at 0 to 100 ° C.
[0073]
Furthermore, this invention provides the pharmaceutical composition containing the low molecular chitosan which melt | dissolves 10g or more with respect to 100g of water of 0-100 degreeC.
[0074]
The hydrolytic activity of chitinase can be measured according to the Randle-Morgan method (Randle CJM et al. Biochem. J. GL 586-589, 1955). Moreover, it can also measure according to the modified Schales method (Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)).
[0075]
Glycolchitin can be produced by the method described in R. Senzyu and S. Okimatsu, Nippon Nogeikagaku Kaishi, 23, 432 (1950).
Specific properties of the chitinase obtained from the chitinase-producing bacterium are shown below, but the properties of the chitinase of the present invention are not limited to these.
[0076]
The chitinase produced and purified by Aspergillus oryzae (var. Sporoflavus Ohara JCM 2067) strain has the following properties.
1) Shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) The isoelectric point pI4.5 is shown by isoelectric focusing.
3) Chitosan 7B (manufactured by Katoyoshi Co., Ltd.) is hydrolyzed at pH 3.5 to pH 10.5.
4) Glycol chitin is hydrolyzed at pH 3.0 to pH 10.5.
5) The hydrolysis activity described in 3) is exhibited at 80 ° C. or lower.
6) The optimum pH of the hydrolysis activity described in 3) is pH 5.5.
7) The optimum pH of the hydrolysis activity described in 4) is pH 10.0.
8) The optimum temperature for the hydrolytic activity described in 3) is 60 ° C. at a pH of 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 for other substrates at a temperature of 37 ° C. when the hydrolysis activity for 10 minutes at chitosan 7B at pH 5.5 or pH 10.0 at a temperature of 37 ° C. is taken as 100%. The hydrolytic activity for 10 minutes in Table 1 shows the values 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 sodium carboxymethyl cellulose (manufactured by Wako Pure Chemical Industries, Ltd.). Glycolchitin is prepared by the method described above.
[0077]
[Table 1]

From Table 1, this enzyme has a high hydrolytic activity for partially acetylated chitosan (chitosan 9B, 8B, 7B) having a degree of acetylation of 10 to 30% than glycol chitin at pH 5.5, and the degree of acetylation is 1 % Of chitosan (chitosan 10B) is hardly hydrolyzed. There is no hydrolysis activity for sodium carboxymethylcellulose.
12) It has the partial amino acid sequence shown below. The sequence is described 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 the sequence listing)
Ile-Val-Val-Gly-Met-Pro-Ile-Tyr-Gly-Arg-Lys-Glu (SEQ ID NO: 2 in the sequence listing)
Ala-Leu-Pro-Lys-Pro-Gly-Ala-Thr-Glu-Tyr-Val-Asp-Pro-Ser-Ile [SEQ ID NO: 3 in the Sequence Listing]
Chitinase produced by Aspergillus japonics SANK 19288 has the following properties in the crude enzyme solution.
1) Shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) Isoelectric point pI of about 3.5 is shown by isoelectric point transfer electrophoresis.
3) Hydrolyze chitosan 7B at pH 3.5 to pH 10.5.
4) The optimum pH of the hydrolysis activity described in 3) is pH 4.0.
5) When the enzyme activity for chitosan 7B at pH 4.0 at 37 ° C. for 10 minutes is defined as 100%, the enzyme activity for other substrates at pH 4.0 at 10 ° C. for 10 minutes at 37 ° C. 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
Glycolchitin 23.3
CM-cellulose 11.4
Rikennan 25.3
――――――――――――――――――――
From Table 2, this enzyme has higher hydrolytic activity for 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 hydrolytic activity for chitosan 10B) is weak.
Moreover, the chitinase produced and purified by Aspergillus japonics SANK 19288 has the following properties.
1) Shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) Isoelectric point pI of about 3.5 is shown by isoelectric point transfer electrophoresis.
3) Hydrolyze chitosan 7B at pH 3.5 to pH 10.5.
4) The optimum pH of the hydrolysis activity described in 3) is pH 5.0.
5) The optimum temperature for the hydrolytic activity described in 3) is 65 ° C. at pH 5.0.
6) Stable at a temperature of 50 ° C. or lower.
7) Stable under pH conditions of pH 4 to pH 9.5.
8) When the enzyme activity for chitosan 7B is 5.0% and the hydrolysis activity for 10 minutes at a temperature of 37 ° C. is 100%, the enzyme activity for other substrates is pH 5.0 and the hydrolysis activity for 10 minutes at a temperature of 37 ° C. 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 sodium carboxymethyl cellulose (manufactured by Wako Pure Chemical Industries, Ltd.). Glycolchitin 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
Glycolchitin 28.3
CM-cellulose 0
Rikennan 4.2
――――――――――――――――――
As shown in Table 3, purified chitinase derived from Aspergillus japonics SANK 19288 strain has the highest hydrolysis activity against 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 described 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 the Sequence Listing)
Ala-Phe-Thr-Asn-Thr-Asp-Gly-Pro-Gly-Thr-Ala-Phe-Ser-Gly-Val (SEQ ID NO: 12 in the 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 the Sequence Listing)
The chitinase produced by Aspergillus sojae SANK 22388 has the following properties in the crude enzyme solution.
1) Shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) pI of about 4.5 is shown by isoelectric focusing.
3) Hydrolyze chitosan 7B at pH 3.5 to pH 9.5.
4) The optimum pH of the hydrolysis activity described in 3) is pH 4.5.
5) When the hydrolysis activity of chitosan 7B for enzyme at pH 4.5 at a temperature of 37 ° C. for 10 minutes is defined as 100%, the enzyme has a hydrolysis activity for other substrates of pH 4.5 at a temperature of 37 ° C. for 10 minutes. 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
Glycolchitin 81.9
CM-cellulose 0
Rikennan 11.9
――――――――――――――――――――
From Table 4, this enzyme has high hydrolytic activity for partially acetylated chitosan (chitosan 9B, 8B, 7B) having a degree of acetylation of 10 to 30%, and chitosan (chitosan 10B) having a degree of acetylation of 1% or less. It can be seen that there is almost no hydrolysis activity. There is no hydrolysis activity for sodium carboxymethylcellulose.
The chitinase produced and purified by Aspergillus sojae SANK 22388 has the following properties.
1) Shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) Isoelectric point pI of about 4.5 is shown by isoelectric point transfer electrophoresis.
3) Hydrolyze chitosan 7B at pH 3.5 to pH 9.5.
4) The optimum pH of the hydrolytic activity described in 3) is pH 5.5 and pH 9.0.
5) The optimum temperature for the hydrolytic 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 lower at pH 5.5 and pH 9.0.
7) Stable under pH conditions of pH 4.5 to pH 10.
8) When the hydrolysis activity of chitosan 8B for enzyme at pH 5.0 at a temperature of 37 ° C. for 10 minutes is defined as 100%, the enzyme has a hydrolysis activity for other substrates of pH 5.0 at a temperature of 37 ° C. for 10 minutes. 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 sodium carboxymethyl cellulose (manufactured by Wako Pure Chemical Industries, Ltd.). Glycolchitin 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
Glycolchitin 23.8
CM-cellulose 0
Rikenan 0
――――――――――――――――――
As shown in Table 5, purified chitinase derived from Aspergillus sojae SANK 22388 strain has the highest hydrolysis activity against 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 following properties can be mentioned as the properties of the chitinase of the present invention, but the present invention is not limited thereto.
1) Shows a molecular weight of about 40,000 by SDS-PAGE electrophoresis.
2) Isoelectric point pI3.0 to 5.0 (preferably pI3.5 to 4.5) is shown by isoelectric focusing.
3) Chitosan 7B is hydrolyzed at pH 3.0 to pH 11.0 (preferably pH 3.5 to 10.5).
4) Hydrolyze glycol chitin at pH 3.0 to pH 11.5 (preferably pH 4 to pH 10.5);
5) exhibits the hydrolytic activity described in 3) at 0 ° C. to 80 ° C .;
6) stable at a temperature of 50 ° C. or lower (preferably 45 ° C. or lower);
7) Stable under pH conditions of pH 4.0 to pH 10 (preferably pH 5 to pH 9.5).
[0082]
Moreover, the method for producing the chitinase of the present invention is also included in the present invention.
[0083]
A chitinase can be produced by culturing chitinase-producing microorganisms, such as Aspergillus oryzae var. For example, 0.1 to 5.0% malto extract (manufactured by Difco), 0.1 to 1.0% yeast extract (manufactured by Difco), 0.05 to 1.0% chitin or Culture with shaking at 100 to 250 rpm in a medium of 0.05 to 1.0% chitosan at 16 to 45 ° C. for 1 to 15 days.
[0084]
Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 strain, Aspergillus Japonix SANK19288 strain and Aspergillus sojae SANK 22388 strain have the following common points.
1) It is weakly acidic and has the highest partially acetylated chitosan degrading activity.
2) If the hydrolysis activity for 30% acetylated chitosan at pH 4.0-6.0 and temperature of 37 ° C. for 10-30 minutes is 100%, the activity for 1% acetylated chitosan is 15% or less.
[0085]
The chitosan low-molecular 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 be performed by dialysis, ultrafiltration, gel filtration, column chromatography, or the like.
[0086]
The low-molecular chitosan hydrochloride thus prepared has excellent solubility in water and can be made into a solution having a maximum concentration of about 10% by mass. Moreover, the 1 mass% aqueous solution of low molecular weight chitosan hydrochloride shows pH 4.9-5.6, and the pH at which a deposit (colloid) precipitates when alkali is added is pH 6.6-7.4.
[0087]
The physiological activity of the low molecular chitosan hydrochloride prepared here is examined using antibacterial activity as an index. The minimum inhibitory concentration (Minimum Inhibitory Concentration; MIC) of each low molecular weight chitosan 9B, 8B, 7B hydrochloride for antibacterial activity is measured. Compared with the antibacterial activity of high molecular chitosan hydrochloride, low molecular chitosan hydrochloride has an activity equal to or higher than that of high molecular chitosan hydrochloride, regardless of whether it is a gram-positive or negative bacterium. Therefore, low molecular chitosan has excellent physiological activity equivalent to or higher than that of high molecular chitosan. From this, it is considered that the antibacterial activity which is assumed to be possessed by the high molecular chitosan is also retained in the low molecular chitosan of the present invention. In addition, in the healing test of animals lacking skin damage, the effect of shortening the healing days was observed, and it has physiological activity in animals.
Since it has such an excellent physiological activity, the low-molecular chitosan obtained by the present invention or a salt thereof is, for example, an external preparation for wounds / wound wounds, a bed socking agent, an atopic dermatitis therapeutic agent, an acne. Skin preparations such as therapeutic agents, bath preparations, cosmetics, facial cleansers, basic cosmetics, oral hygiene agents such as anti-cavities, liquid mouthwashes, dental materials, intestinal preparations such as enteric bacteria improving agents, hospital infections Medical fields such as preventive drugs, food additives such as diet foods, food preservatives, preservatives, fields such as animal and fish feed, agricultural fields such as plant activity enhancers, water treatment agents, metal adsorbents, Although it is possible to use the low molecular weight chitosan of this invention for a nuclear waste adsorbent chemical | medical agent method etc., a use is not specifically limited to these examples. Further, the present invention also includes a method for treating diseases such as trauma, bed sores and atopic dermatitis, which comprises administering an effective amount of the low molecular weight chitosan or a salt thereof of the present invention to an animal. In addition, the use of the low-molecular chitosan or a salt thereof of the present invention for treating these diseases is also included in the present invention.
The usage of the low-molecular chitosan or low-molecular chitosan salt obtained by the present invention varies depending on the purpose, but can be in various forms for use in the above-mentioned applications. The forms are listed below, but are not particularly limited. The low molecular chitosan can be used as a solid. In addition, a 10% by weight aqueous solution or organic solvent solution can be used by diluting it appropriately and using any concentration. As the diluent, water or an organic solvent (alcohol or the like) can be used. If the pH is finally more acidic than 6.5, it can be used as a solution, and if it is alkaline, it can be used as a colloidal compound. Furthermore, it can be used as a solution at an arbitrary pH by coexisting inorganic or organic substances. Moreover, it can be used as a cream agent by mixing a low molecular chitosan solution or a diluted solution with a cream base at an arbitrary concentration of 10% by mass or less. A low molecular chitosan solution or a diluted solution can be used as a film having an arbitrary thickness. A film having a plastic-like hardness can be obtained by drying a low-molecular chitosan solution in a frame having various thicknesses. Moreover, the soft sheet | seat which has a softness | flexibility is obtained by mixing solvents, such as glycerol, for example in a low molecular chitosan solution in arbitrary ratios, and making it dry in the frame which has various thickness. The degree of flexibility is adjusted by the ratio of adding a solvent such as glycerin. Furthermore, a chitosan-methyl cellulose composite film can be obtained by mixing methyl cellulose into a low molecular chitosan solution and drying it in frames having various thicknesses.
[0088]
The dose varies depending on the type of disease, symptoms, patient's sex, age, dosage form, and administration method, but since the safety is high, any amount can be used. When administered to a living body, 2 g / kg body weight or less is preferable, and in other cases, it is adjusted appropriately.
In addition, the chitinase of the present invention can be used effectively to make it difficult to handle difficult samples. Conventionally, when samples such as crustaceans were processed by crushing, crushing, and applying to a mixer, the viscosity became very high and difficult to handle. This was caused by a high molecular weight polysaccharide (the main component is a partially polymerized acetylated chitosan) contained in these crustaceans. For example, in the process of food processing, the chitinase of the present invention is added and reacted to decompose the polymer partially acetylated chitosan in the liquid component, and the polymer partially acetylated chitosan in the liquid component. It is possible to produce a sample that is substantially free and easy to handle, and a method for producing the easy sample using chitinase is also included in the present invention. 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, more preferably any one of shrimp and crab. Or a sample containing both. As the reaction conditions for the raw material and chitinase, the above-mentioned conditions for exhibiting chitosan degrading activity can be applied. The reaction time is 10 minutes or longer and is not particularly limited as long as the viscosity of the sample is low, but is preferably about 10 minutes to 10 days, more preferably about 1 day to 5 days. A sample produced by such a method is also included in the present invention.
[0089]
“Substantially free of polymer partially acetylated chitosan in its liquid component” refers to a state in which high viscosity is reduced or eliminated due to the sample containing polymer partially acetylated chitosan. Strictly, it does not indicate that the concentration is 0%, but the content of the polymer partially acetylated chitosan is preferably 20% or less, more preferably 10% or less, and even more preferably 5% or less. Yes, optimally 0%.
[0090]
【Example】
Examples and test examples are given below, but the scope of the present invention is not limited to these examples.
[0091]
Example 1. Purification of chitinase from Aspergillus oryzae var. Sporoflavus Ohara JCM2067
1) Preparation of crude enzyme solution
Inoculate cells of Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 strain into a 500 ml Erlenmeyer flask (seed flask) containing 100 ml of a sterilized medium having the composition shown in Table 6 and shake at 100 rpm for 8 days at 26 ° C. Culture was performed.
[0092]
[Table 6]
Culture medium
――――――――――――――――――――――――――――――
Marut Extract 10g
East Extract 5g
Powdered chitin 2g
Or colloidal chitosan (Kimitsu Chemical Co., Ltd.) 2g
Made up to 1,000 ml with pure water.
――――――――――――――――――――――――――――――
After completion of the culture, centrifugation was performed at 4 ° C. and 10,000 × G for 10 minutes. 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 reaction 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 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 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. 50 ml of water and 90 μl of acetic acid were added to 125 mg of chitosan 10B (almost completely deacetylated chitosan, manufactured by Katoyoshi Co., Ltd.) to completely dissolve the powdered chitosan. To the mixed solution of 160 μl of these chitosan aqueous solution and 100 μl of 400 mM acetate buffer (pH 5.0), 140 μl of enzyme solution was added and stirred to homogenize, and incubated at 37 ° C. for 20 minutes.
[0095]
(2) Determination of free reducing sugars by Randle Morgan method
400 μl of a solution obtained by dissolving 10 μl of acetylacetone in 500 μl of 0.5 M sodium carbonate aqueous solution was added to the enzyme reaction solution to stop the reaction, and then kept at 100 ° C. for 20 minutes. After cooling with ice, 400 μl of a mixed solution in which 0.8 g of N, N-dimethylaminobenzaldevid was dissolved 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 carried out at 65 ° C. for 10 minutes. After cooling with ice, the precipitate was centrifuged, and the absorbance of the supernatant at 530 nm was measured. Enzyme activity to produce 1 μmol of glucosamine per minute of enzyme reaction was defined as 1 unit.
[0096]
3) Preparation of crude purified enzyme solution
To 500 ml of the crude enzyme solution obtained in 1), 280 g of ammonium sulfate was gradually added with stirring under ice cooling. The mixed solution was allowed to stand at 4 ° C. overnight, and then dialyzed 5 times for 12 hours against 3,000 ml of 10 mM Tris / HCl buffer (pH 7.5). This was added to a DEAE Toyopearl (Tosoh Corp.) column (diameter 2.2 cm × length 20 cm) previously equilibrated with 10 mM Tris / HCl buffer (pH 7.5) and adsorbed. After thoroughly washing the column with 10 mM Tris / HCl buffer (pH 7.5), a linear concentration gradient of 0 to 0.4 M sodium chloride in 600 ml of 10 mM Tris / HCl buffer (pH 7.5) was prepared. The components adsorbed on the column were eluted. Chitosan degrading activity was eluted in fractions (135 ml) having a sodium chloride concentration of 0.20M to 0.30M. 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 3 times for 12 hours against 3,000 ml of 20 mM Tris / HCl buffer (pH 7.5), and then 20 mM Tris / HCl buffer (pH 7.5) in advance. And adsorbed on a MonoQ (Pharmacia) column (diameter 7 mm × length 5 cm) equilibrated in (1). After thoroughly washing the column with 20 mM Tris / HCl buffer (pH 7.5), a linear concentration gradient of 0 to 0.15 M sodium chloride in 250 ml of 10 mM Tris / HCl buffer (pH 7.5) was prepared. Then, the components adsorbed on the column were eluted. The chitosan degrading activity was eluted in the fraction (10 ml) having a sodium chloride concentration of 0.054M to 0.057M. This fraction was used as a purified enzyme solution.
[0098]
5) Molecular weight measurement 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 with a molecular weight of about 40,000.
[0099]
6) Isoelectric point of purified enzyme
The purified enzyme was subjected to isoelectric focusing (see Biochemical Experimental Course 1, Protein Chemistry I Separation and Purification, Tokyo Chemical Doujin (1976), pages 262 to 312) edited by the Japanese Biochemical Society). 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 reacted at 95 ° C. for 10 minutes. After cooling to room temperature, 40 μl of 50 mM Iodoacetoamide dissolved in Denature buffer solution was added to the reaction solution, and reacted at room temperature in the dark for 1 hour. This solution was dialyzed against 1 L of water using Slide-A-Lyzer Mini Dialysis Units Plus Float (manufactured by PIERCE, molecular weight fraction 10,000). 8 μl of trypsin (Modified, Promega) was added to the resulting solution, and the enzyme reaction was carried out at 37 ° C. for 7 hours. The reaction solution was applied to SMART system (Pharmacia Biotech) to separate peaks of degraded amino acids. The separation conditions are shown below.
Column: Nomura Chemical Develosil 300 CDS-HG-5 (1mm x 150mm)
A buffer: 0.1% TFA / Water
B buffer: 0.1% TFA / Acetonitril
Gradient: 5 → 60% B 1% / min.
Flow: 50μl / ml
Among the separated degraded amino acids, two peaks having a B buffer concentration of about 15% and about 35% were collected. The amino acid sequences of these three (No. 1 to No. 3) were analyzed with 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 the Sequence Listing);
No. 2: Ile Val Val Gly Met Pro Ile Tyr Gly Arg Lys Glu (SEQ ID NO: 2 in the Sequence Listing);
No. 3: Ala Leu Pro Lys Pro Gly Ala Thr Glu Tyr Val Asp Pro Ser Ile (SEQ ID NO: 3 in the Sequence Listing)
[0101]
Example 2 Purification of chitinase from Aspergillus japonics SANK 19288 strain
1) Preparation of crude enzyme solution
A 500 ml Erlenmeyer flask (seed flask) containing 100 ml of the sterilized medium with the composition shown in Table 7 is inoculated with Aspergillus japonicus SANK 19288 strain and cultured at 26 ° C for 8 days with shaking at 100 rpm. It was. After completion of the culture, centrifugation was performed at 10,000 × G for 10 minutes under 4 ° C. conditions. The obtained supernatant was used as a crude enzyme solution.
[0102]
[Table 7]
Culture medium
――――――――――――――――――――
Marut Extract 10g
East Extract 5g
Powdered chitin 5g
Made up to 1,000 ml with pure water.
――――――――――――――――――――
2) Preparation of purified enzyme solution
112 g of ammonium sulfate was gradually added to 200 ml of the crude enzyme solution obtained in 1) while stirring under ice cooling. The mixed solution was allowed to stand overnight at 4 ° C., and then dialyzed 5 times for 12 hours against 3,000 ml of 20 mM Tris / HCl buffer (pH 7.5) to obtain a crude purified enzyme fraction. The crudely purified enzyme fraction was added and adsorbed onto a MonoQ (Pharmacia) column (diameter 7 mm × length 5 cm) equilibrated in advance with 20 mM Tris / HCl buffer (pH 7.5). After thoroughly washing the column with 20 mM Tris / HCl buffer (pH 7.5), a linear concentration gradient of 0 to 0.15 M sodium chloride in 250 ml of 10 mM Tris / HCl buffer (pH 7.5) was prepared. Then, the components adsorbed on the column were eluted. Chitosan degrading activity was eluted in fractions (10 ml) having a sodium chloride concentration of 0.063M to 0.066M. This fraction was used as a purified enzyme solution.
[0103]
3) Molecular weight of purified enzyme
Example 1 The molecular weight was measured by the same method as 5). The enzyme showed a single band with a molecular weight of about 40,000.
[0104]
4) Isoelectric point of purified enzyme
Example 1 The isoelectric point was measured by the same method as 6). The isoelectric point of this enzyme was about pI3.5.
[0105]
Example 3 FIG. Purification of chitinase from Aspergillus sojae SANK 22388 strain
1) Preparation of crude enzyme solution
A 500 ml Erlenmeyer flask (seed flask) containing 100 ml of the sterilized medium with the composition shown in Table 8 was inoculated with Aspergillus sojae SANK 22388 strain and cultured at 26 ° C for 8 days with shaking at 100 rpm. It was. After completion of the culture, centrifugation was performed at 10,000 × G for 10 minutes under 4 ° C. conditions. The obtained supernatant was used as a crude enzyme solution.
[0106]
[Table 8]
Culture medium
――――――――――――――――――――
Marut Extract 10g
East Extract 5g
Powdered chitin 5g
Made up to 1,000 ml with pure water.
――――――――――――――――――――
2) Preparation of purified enzyme solution
112 g of ammonium sulfate was gradually added to 200 ml of the crude enzyme solution obtained in 1) while stirring under ice cooling. The mixed solution was allowed to stand overnight at 4 ° C., and then dialyzed 5 times for 12 hours against 3,000 ml of 20 mM Tris / HCl buffer (pH 7.5) to obtain a crude purified enzyme fraction. The crudely purified enzyme fraction was added and adsorbed onto a MonoQ (Pharmacia) column (diameter 7 mm × length 5 cm) equilibrated in advance with 20 mM Tris / HCl buffer (pH 7.5). After thoroughly washing the column with 20 mM Tris / HCl buffer (pH 7.5), a linear concentration gradient of 0 to 0.15 M sodium chloride in 250 ml of 10 mM Tris / HCl buffer (pH 7.5) was prepared. Then, the components adsorbed on the column were eluted. The chitosan degrading activity was eluted in the 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
Example 1 The molecular weight was measured by the same method as 5). The enzyme showed a single band with a molecular weight of about 40,000.
[0107]
4) Isoelectric point of purified enzyme
Example 1 The isoelectric point was measured by the same method as 6). The isoelectric point of this enzyme was about pI4.5.
Example 4 Preparation of low molecular weight chitosan hydrochloride using chitinase from Aspergillus oryzae var. Sporoflavus Ohara JCM 2067
Chitosan 9B or chitosan 8B or chitosan 7B, 2.0 g was suspended in 800 ml of water, 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 a total volume of 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 enzyme reaction was performed by shaking at 80 rpm for 3 days at 40 ° C. After completion of the reaction, all the reaction liquids were collected, adjusted to pH 12 or higher with 1% sodium hydroxide water under ice cooling, and then a precipitate was obtained by centrifugation at 10,000 rpm for 10 minutes. The collected precipitate was suspended in 200 ml of water, concentrated hydrochloric acid was added under ice-cooling to obtain a pH of 2.5, and dialyzed against water using a dialysis membrane having a molecular weight fraction of 10,000. After dialysis, the solution was filtered and the filtrate was lyophilized. Low molecular 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 from Aspergillus japonics SANK 19288 strain
Low molecular chitosan hydrochloride was prepared in the same manner as in Example 4. The enzyme solution obtained in Example 21 1) was used, and the enzyme reaction was performed at pH 4.0. Low molecular 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]
Example 6 Preparation of low molecular weight chitosan hydrochloride using chitinase from Aspergillus sojae SANK 22388 strain
Low molecular 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 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 fluffy solid. Low molecular weight chitosan 7B hydrochloride was obtained as 1.0 g of a white flocculent solid.
[0110]
Example 7 Degree of acetylation of low molecular chitosan hydrochloride
100 mg of low-molecular chitosan hydrochloride prepared in Examples 4 to 6 was dissolved in 10 ml of distilled water, and 10 U of chitosanase (manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred at room temperature for 3 hours. The reaction solution is lyophilized and the dried solid is D ₂ Dissolved in O ¹ 1 H-NMR was measured.
The degree of acetylation was calculated from the integral ratio of 3H of the acetyl group and 7H other than the acetyl group and excluding the hydroxyl group. The results are shown in Table 9.
[0111]
[Table 9]

Example 8 FIG. Determination of molecular weight fraction in gel filtration method
As molecular weight markers, 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) (Pharmacia Co., Ltd.) 200 μl of each 1% solution of 1) was developed on a column (86 × 2 cm) packed with a gel filter (Toyopearl HW55 Fine) previously swollen with 200 mM acetate buffer at pH 4.5 and fractionated into 1.75 ml fractions. did. 60 ml of a solution obtained by dissolving 1.8 ml of concentrated sulfuric acid in 1 volume of water and 0.25 g of carbazole in 50 ml of ethanol was added to 200 μl of each fraction and kept at 100 ° C. for 10 minutes. Absorbance at 420 nm was measured after ice cooling.
[0112]
200 μl of each 5% solution of low-molecular chitosan hydrochloride prepared in Examples 4, 5, and 6 was fractionated into 1.75 ml in the same manner. To 280 μl of each fraction, 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, and an enzyme reaction was carried out at 37 ° C. for 10 minutes. The amount of free reducing sugar was determined according to Example 1. The molecular weight marker and each low molecular chitosan were plotted on the same graph with the fraction showing the maximum absorbance as 100% (FIGS. 2 to 4). The molecular weights of the low-molecular chitosan estimated from the figure are summarized in Table 10.
[0113]
[Table 10]

Example 9 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 in 100 ml of medium (1% Malt Extract, 0.5% Yeast Extract) at 30 ° C. After shaking culture for a day, the cells were collected from the culture using a filtration device. Subsequently, the cells were placed in a mortar cooled to -80 ° C., and liquid nitrogen was poured. While adding liquid nitrogen, the cells were sufficiently crushed using a pestle until the cells became powdery. About 1.5 g of powdered cells were added to 20 ml of 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 to this, and another 15 ml of TE saturated phenol was added and gently stirred. This solution was centrifuged at 4 ° C. and 5000 rpm for 5 minutes using a high-speed cooling centrifuge (Hitachi rotor No. 3), and about 25 ml of the supernatant was recovered. 15 ml of chloroform was added to the collected supernatant and gently stirred, and then centrifuged at 4 ° C. and 5000 rpm for 10 minutes. The supernatant was collected in a new tube, and 2-propanol equivalent to the supernatant was added and stirred, followed by centrifugation at 4 ° C. and 10,000 rpm for 10 minutes. The precipitate was dissolved in 400 μl of TE, then transferred to a 1 ml microtube, 10 μl of 10 mg / ml ribonuclease was added, and treated at 37 ° C. for 20 minutes. Next, 40 μl of 3M sodium acetate and 400 μl chloroform were added and stirred, followed by centrifugation 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 and gently stirred. The DNA in the form of a thread was wound up with a glass rod, and after ethanol was removed, it was dissolved in 200 μl of TE.
9-2) Preparation of Aspergillus oryzae genomic DNA library
Using 10 μl of the restriction enzyme EcoRI (Takara Shuzo), 10 μg of Aspergillus oryzae genomic DNA obtained in 9-1), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ After digestion in a solution consisting of 1 mM DTT and 100 mM NaCl at 37 ° C. for about 2 hours, DNA was recovered. The Aspergillus oryzae genomic DNA EcoRI cleavage product and pUC118 EcoRI / BAP (Takara Shuzo), also digested with EcoRI, were ligated using Rapid DNA Ligation Kit (Roche Diagnostics), The 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 primer F21 prepared based on the internal amino acid sequence identified in Example 1 (SEQ ID NO: 2 in the sequence listing) and F21.
[0114]
F21: 5'-attggcataccaacaactatcttagagc-3 '(SEQ ID NO: 7 in the sequence listing)
PR50: 5'-taaattgacccatatcctctatgcatt-3 '(SEQ ID NO: 8 in the sequence listing)
Using these two types of primers, about 800 bp of DNA was increased by PCR using Aspergillus oryzae genomic DNA as a template. This DNA was separated by electrophoresis, cut out from an agarose gel, and then purified using a QIAquick Gel Extraction Kit (manufactured by Qiagen). The DNA thus purified was labeled using a DIG DNA Labeling Kit (Roche Diagnostics). The labeling method was 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 , 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 polymerase ( Klenow fragment) was added, and the whole amount was adjusted to 100 μl with sterilized water. This reaction solution was reacted at 37 ° C. overnight, and 10 μl 0.2M EDTA (pH 8.0) was added to stop the reaction. Subsequently, 10 μl 4N LiCl and 500 μl 100% ethanol (cooled at −20 ° C.) were added, and allowed to stand at −70 ° C. for 30 minutes to precipitate the labeled DNA. Centrifugation was performed at 4 ° C. and 15000 rpm for 10 minutes in a cooling centrifuge. The ethanol was discarded, and 100 μl 70% ethanol (cooled at −20 ° C.) was added to wash the pellet, followed by centrifugation at 15000 rpm for 10 minutes, and the ethanol was completely removed with a pipette. The pellet was dried, dissolved in 50 μl TE, added to 25 ml DIG Easy Hyb (Roche Diagnostics), and used as a chitinase gene probe.
9-4) Preparation of partial DNA library containing chitinase gene
After 20 μg of Aspergillus oryzae genomic DNA obtained in 9-1) was cleaved with the restriction enzyme EcoRI, it was purified and DNA was recovered. 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 the restriction enzyme EcoRI and purified to recover the DNA. This DNA was electrophoresed on an agarose gel, and the same position as the band position observed by the Southern hybridization was cut out from the gel to extract the DNA. The extracted DNA was ligated to the pUC118 EcoRI / BAP vector to obtain a DNA library containing a chitinase gene.
9-5) Screening for DNA containing chitinase gene
E. 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 to transfer part of the colony onto the membrane. This membrane is allowed to stand for 20 minutes on a filter paper soaked with an alkali-denaturing solution (0.5M NaOH, 3.0M NaCl), and then for 10 minutes on a filter paper soaked with a neutralization solution (1M Tris-HCl (pH7.5)). placed. Further, it was washed while shaking with 2 × SSC solution. Next, this membrane was prehybridized with DIG Easy Hyb for 1 hour. Thereafter, the membrane was hybridized overnight with the chitinase gene probe obtained in 9-3) at 37 ° C. After hybridization, the plate was washed three times with normal temperature 2 × SSC buffer and further washed twice with 2 × SSC buffer warmed to 53 ° C. Thereafter, detection was performed using DIG Wash and Block Buffer Set (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: solution containing 10% blocking reagent in pH 7.5) for blocking for 60 minutes to block nonspecific binding. Next, the membrane was immersed in a solution obtained by adding 10000-fold diluted anti-digoxigenin antibody to a blocking solution, reacted at 37 ° C. for 30 minutes, and then washed twice with Wash Buffer. Thereafter, the membrane was acclimated with Detection buffer (0.1 M Tris-HCl, 0.1 M NaCl: pH 9.5). CSPD (Chemiluminescence substrate) was added to the detection buffer at a 100-fold dilution, dropped onto 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 Detecion 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 (Qiagen). Next, the extracted DNA was subjected to sequence analysis using universal primers peculiar to this plasmid, M13M4, M13RV, and the result was aligned with the nucleotide sequence of the chitinase gene probe obtained in 9-3) to obtain the chitinase sequence. Confirmed to include. The entire sequence of the plasmid DNA that was confirmed to contain the chitinase sequence was determined using the GPS-1 Genome Priming System (manufactured by New England Biolabs). The procedure is as follows: 4 μl of the plasmid DNA extracted above is 2 μl 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 plasmid), 11 μl After adding sterilized 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 and reacted at 37 ° C. for 1 hour, followed by treatment at 75 ° C. for 10 minutes to stop the reaction. Next, 10 μl of this reaction product was transformed into E. coli strain JM109, applied to an LB agar medium containing ampicillin and kanamycin, and cultured at 37 ° C. overnight. The grown colonies were cultured overnight in LB liquid medium, and plasmid DNA was extracted. The extracted plasmid DNA was sequenced with N and S primers, which are pGPS1 primers, and the entire genomic sequence encoding chitinase was determined.
Example 10 Identification of cDNA encoding chitinase from Aspergillus oryzae
10-1) Purification of total RNA of Aspergillus oryzae
Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 strain was pre-cultured in 20 ml of Potato Dextrose medium at 30 ° C. for 3 days. Thereafter, 1% inoculation was carried out in 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 form a powder. Total RNA was purified from the completely powdered cells using Rneasy Plant Mini Kit (Qiagen) as follows. First, about 1 g of powdered cells were added to 4.5 ml RLT buffer (β-Mercaptoethanol), and vigorously shaken cells were dissolved and placed on ice. 0.5 ml each was dispensed onto a QIAshredder spin column and centrifuged at 15000 rpm for 2 minutes at room temperature. 0.5 times the amount of ethanol was added to the passing solution, and the mixture was stirred by pipetting. Thereafter, 675 μl each was dispensed onto an Rneasy Mini spin column and centrifuged at 16000 rpm for 15 seconds at room temperature. 700 μl of RW1 Wash buffer was added, and similarly centrifuged at 16000 rpm for 15 seconds at room temperature. Next, 500 μl RPE Wash buffer was added, and the RNA was washed by centrifugation at 16000 rpm for 2 minutes at room temperature. The spin column was set in a microtube, RNA was eluted with 50 μl of Rnase free water, and collected by centrifugation at 10000 rpm for 1 minute at room temperature.
10-2) Preparation of Aspergillus oryzae cDNA library
Aspergillus oryzae cDNA library was prepared using SMART cDNA Library Construction Kit (Clontech). First, 1 μg of the total RNA purified in 10-1), 1 μl SMART IV Oligonucleotide, and 1 μl CDS III / 3 ′ PCR Primer were mixed in a sterile 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 was added 2 μl 5 × First-Strand Buffer, 1 μl 20 mM DTT, 1 μl 10 mM dNTP Mix and 1 μl Power Script Reverse Transcriptase. The contents were mixed and reacted at 42 ° C. for 1 hour, and then placed on ice to stop first-strand DNA synthesis. Subsequently, cDNA was amplified by LD PCR. 2 μl of the First-Strand DNA was added to the PCR tube, and the following reagents were 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, 2μl 50x Advantage2 Polymerase Mix. This 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 95 ° C. for 5 seconds and 68 ° C. for 6 minutes. 50 μl of this reaction solution (containing ds cDNA) was placed in a sterilized tube, 2 μl protease K was added and incubated at 45 ° C. for 20 minutes. After protease treatment, 50 μl deionized water was added. 100 μl 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, it was centrifuged again at 14000 rpm for 5 minutes. The supernatant was transferred to a new tube, 10 μl 3M sodium acetate, 1.3 μl glycogen and 260 μl 95% ethanol (room temperature) were added and immediately centrifuged at 14000 rpm for 20 minutes at room temperature. After removing the supernatant with a pipette and washing the pellet with 100 μl of 80% ethanol, the pellet was dried. The pellet was dissolved in 79 μl deionized water. Subsequently, treatment with 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. Thereafter, 2 μl of 1% xylene cyanol staining solution was added. CDNA was screened using a CHROMA SPIN-400 column. The CHROMA SPIN column was mixed by inversion several times to suspend the gel matrix, and further suspended with Pipetman. The lid of the column was removed and the column was placed so that it drip naturally. Column buffer was then added gently and allowed to drip naturally by gravity. After the drip of the buffer was completed, a cDNA sample decomposed with Sfi I was applied to the center of the upper surface of the matrix and sufficiently absorbed on the surface of the matrix. When there was no liquid on the surface and the drip was finished, 600 μl column buffer was added and immediately fractions collected in 16 test tubes. Each 3 μl of this fraction was subjected to agarose gel electrophoresis to determine the peak fraction. Fractions containing cDNA were collected and collected in a new tube. Add 1/10 volume of sodium acetate (3M; pH 4.8), 1.3 μl glycogen, 2.5 volumes of 95% ethanol (-20 ° C) to this test tube, mix gently and shake at -20 ° C. Incubated overnight. Centrifugation was carried out at 14000 rpm for 20 minutes at room temperature, 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 λTriplEx2 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. For packaging, Gigapack III Gold Packaging Extract (manufactured by STRATAGENE) was used. 4 μl μl of the ligated DNA vector was added to the microtube containing Packaging Extract, gently mixed by pipetting, and incubated at room temperature for 2 hours. Two hours later, 500 μl SM buffer was added, and further 20 μl of chloroform was mixed, followed by light centrifugation to prepare a packaging solution. Next, host cells to infect the packaged phage were cultured. A frozen stock of E. coli XL1-Blue was inoculated into LB / tetracycline agar plates and cultured overnight at 37 ° C. Growing single colony MgSO _Four And transferred to a plate containing tetracycline and cultured overnight at 37 ° C. Growing single colonies in 15 ml LB / MgSO _Four / Inoculate maltose broth and culture OD ₆₀₀ The cells were cultured at 37 ° C. until 2.0 was reached. Centrifuge the culture at 5000 rpm for 5 min and transfer the pellet to 7.5 ml of 10 mM MgSO. _Four Resuspended. After diluting the packaging solution with 1 × λ dilution buffer, 1 μl of the packaging solution was added to 200 μl E. coli XL1-Blue suspension and adsorbed at 37 ° C. for 15 minutes. The reaction was charged with 2 ml of melted LB / MgSO. _Four LB / MgSO mixed with top agar and warmed _Four Spread evenly on the plate. After cooling at room temperature for 10 minutes to harden the top agar, it was cultured overnight at 37 ° C. The obtained plaque was eluted by shaking overnight with the addition of SM buffer to obtain a phage containing the Aspergillus oryzae cDNA library.
10-3) Screening of chitinase gene-containing cDNA
The phage containing the cDNA library obtained in 10-2) was infected with E. coli XL1-Blue strain (Clontech). 1 μl of diluted phage was added to 200 μl XL1-Blue cultured and adsorbed at 37 ° C. for 10 to 15 minutes. 2 ml of melted LB / MgSO _Four Add top agar, mix and warm LB / MgSO _Four Pour onto the plate and rotate the plate to spread the top agar evenly. After cooling at room temperature for 10 minutes to solidify the top agar, it was incubated overnight at 37 ° C. to form plaques on the agar medium. On top of that, a nylon transfer membrane (manufactured by Amersham) was overlaid for about 1 minute, and a part of the phage was transferred onto the nylon membrane. The membrane was dried on filter paper for 20 minutes, then left on filter paper soaked with an alkali-denaturing solution (0.2N NaOH, 1.5M NaCl) for 5 minutes, and then neutralized solution (0.4M Tris-HCl (pH 7. 5), 2 × SSC) was placed on a filter paper soaked for 5 minutes. Further, after washing with a neutralizing solution for 1 minute while shaking, the plate was further washed with 2 × SSC for 5 minutes. The filter was dried on the filter paper for about 1 hour and baked in an oven at 80 ° C. for 2 hours. Thereafter, the membrane was hybridized overnight with the DNA probe obtained in X-3 at 37 ° C. After hybridization, the plate was washed 3 times with normal temperature 2 × SSC buffer and further washed twice with 2 × SSC buffer warmed to 53 ° C. Thereafter, detection was performed using DIG Wash and Block Buffer Set (Roche Diagnostics). First, the membrane was conditioned with a 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 at pH 7.5) for blocking for 60 minutes to block non-specific binding. Next, the membrane was immersed in a solution obtained by adding 10000-fold diluted anti-digoxigenin antibody to a blocking solution, reacted at 37 ° C. for 30 minutes, and then washed twice with Wash Buffer. Thereafter, the membrane was acclimated with Detection buffer (0.1 M Tris-HCl, 0.1 M NaCl; pH 9.5). CSPD (Chemiluminescence substrate) was added to the detection buffer at a 100-fold dilution, dropped onto 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 Detecion Film (manufactured by Konica), developed, and detected. As a result, a clone showing a positive signal could be detected. This positive phage plaque is cut out together with the agar medium, dissolved in SM buffer, and then transformed into Escherichia coli BM25.8 strain to obtain transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 strain. It was. This strain was deposited internationally at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, on November 8, 2002, and was given the deposit number: FERM BP-8235. Infected phage DNA, λTriplEx-Chitinase-cDNA is circularized in cells of E. coli BM25.8 strain and exists as a recombinant plasmid pTriplEx-Chitinase-cDNA. The obtained Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 colony was cultured overnight in LB / Ampicilin broth, and the DNA of the recombinant plasmid pTriplEx-Chitinase-cDNA was extracted. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen). Next, add 2 μl 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 plasmid), 11 μl sterile water to 4 μl of the plasmid DNA and mix. Then, 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 and reacted at 37 ° C. for 1 hour, followed by treatment at 75 ° C. for 10 minutes to stop the reaction. Next, 10 μl of this reaction product was transformed into E. coli strain JM109, applied to an LB agar medium containing ampicillin and kanamycin, and cultured at 37 ° C. overnight. The grown colonies were cultured overnight in LB liquid medium, and plasmid DNA was extracted. The extracted plasmid DNA was sequenced with N and S primers which are primers for pGPS1, and the nucleotide sequence of DNA encoding Aspergillus oryzae chitinase was determined (SEQ ID NO: 4 in the Sequence Listing). Further, from the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing, the open reading frame (ORF) starts from the nucleotide number 242 to the 244th “ATG” and starts from the nucleotide number 1436 to the 1438th “TAG”. It was a region to be a codon, and the encoded amino acid sequence was assumed to be the sequence shown in SEQ ID NO: 5 in the sequence listing.
Example 11 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 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 reacted at 95 ° C. for 10 minutes. After cooling to room temperature, 40 μl of 50 mM Iodoacetoamide dissolved in Denature buffer solution was added to the reaction solution, and reacted at room temperature in the dark for 1 hour. This solution was dialyzed against 1 L of water using Slide-A-Lyzer Mini Dialysis Units Plus Float (manufactured by PIERCE, molecular weight fraction 10,000). 8 μl of trypsin (Modified, Promega) was added to the resulting solution, and the enzyme reaction was carried out 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. Fragment ion assignments suggested that the N-terminal amino acid is Ser, that the N-terminus contains the amino acid sequence Ser-Ser-Gly-Leu-Lys, and that an acetyl group exists at the N-terminus (see Sequence Listing). The 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 is subjected to post-translational modification at its N-terminus, “Met-Ser-Ser”, and “Acetyl-Ser” -Ser ”(SEQ ID NO: 14 in the sequence listing).
Example 12 Expression of Aspergillus oryzae chitinase gene
Primers, BamHI-cDNA and cDNA-BamHI were designed and synthesized by connecting BamHI sites to the 5 'and 3' ends of chitinase ORF, respectively.
[0115]
BamHI-cDNA: 5'-gaggatccatgtcttccggactaaagtcgg-3 '(SEQ ID NO: 9 in the sequence listing)
cDNA-BamHI: 5'-ctggatccagcgaaaccggctcgcaggttg-3 '(SEQ ID NO: 10 in the sequence listing)
PCR was performed using the recombinant plasmid obtained in Example 10-3), pTriplEx-Chitinase-cDNA as a template, and the above BamHI-cDNA and cDNA-BamHI as primers, and chitinase DNA introduced with restriction enzyme sites was obtained. Amplified. Further, it was inserted into a pCR 2.1-TOPO vector (manufactured by Invitrogen), transformed into Escherichia coli JM109 strain, and amplified. The colony on which the JM109 strain was grown was cultured overnight in Lb / Ampicilin broth, and plasmid DNA was extracted. This plasmid DNA was treated with the restriction enzyme BamHI to cleave the chitinase ORF. Further, as a vector for ligating this fragment, pQE-60 (Qiagen) was cleaved with BamHI and further treated with alkaline phosphatase to remove the phosphate group at the 5 ′ end. Thereafter, the above chitinase ORF was ligated using TOPO TA Cloning Kit (manufactured by Invitrogen). The reaction solution was transformed into E. coli strain JM109 and cultured overnight on an Lb / Ampicilin agar plate. The grown colonies were inoculated into 3 ml of Lb / Ampicilin broth and cultured overnight with shaking. Plasmid DNA was extracted from the culture with QIAprep Spin Miniprep Kit (Qiagen) and transformed into Escherichia coli M15 [pREP4]. The cells were cultured overnight on Lb / Ampicilin and Kanamaicin agar plates, and the grown colonies were inoculated into 20 ml of Lb / Ampicilin and Kanamaicin broth, and cultured overnight at 37 ° C. with shaking. 2.5 ml of this culture solution was inoculated into 100 ml of Lb / Ampicilin and Kanamicin broth and cultured at 37 ° C. OD every 30 minutes from 1 hour after the start of culture ₆₀₀ Was measured, and 1 mM IPTG was added after culturing until 0.6-0.7. After the addition of IPTG, the culture was continued at 18 ° C., and the cells were collected after 4 and 6 hours. 10 ml PBS (137 mM NaCl, 8.1 mM Na ₂ HPO _Four ・ 12H ₂ O, 2.68 mM KCl, 1.47 mM KH ₂ PO _Four ) Was added, and vortexed and well suspended. The suspension was disrupted by sonication, and then centrifuged at 4 ° C. and 8000 rpm for 5 minutes. Take 100 μl of the supernatant in a 1.5 ml tube, add 50 μl denaturing agent (6% SDS, 24% glycerol, 12% β-mercaptoethanol, 0.3M Tris-HCl (pH 6.8), 0.15% BPB) at 100 ° C. Treated for 5 minutes. The reaction solution was electrophoresed with 10 μl of each sample with e-PAGEL (manufactured by ATTO), and then stained and detected with Simply Blue SafeStain (manufactured by Invitrogen). As a result, a dark band was observed around 44 kDa.
[0116]
The presence of chitinase in the supernatant of the cells cultured and collected for 6 hours was confirmed by the Schaels method described in Test Example 1-6).
[0117]
The cell supernatant collected after 4 hours of culture was subjected to His tag purification. The column tube was filled with Ni-NTA agarose and washed with about 10 ml of 0.1 M Tris-HCl (pH 8.0), and then the supernatant was adsorbed onto the column. Next, after washing away non-specific binders 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 this eluate by e-PAGEL, it appeared as a single band. The eluate and chitinase purified from the culture of Aspergillus oryzae obtained in Example 1 were subjected to SDS electrophoresis using a 12.5% acrylamide gel (shown in FIG. 30). The molecular weight of the protein contained in the eluate is slightly larger than that of the purified chitinase derived from Aspergillus oryzae, which is thought to be due to the His tag, and the recombinant protein contained in the eluate is the His fusion recombinant chitinase. It was inferred. The chitosan degrading activity of this eluate was measured by the Schaels method described in Test Example 1-6). As a result, it was 2.0 U / ml, and it was confirmed that the recombinant protein contained in this eluate was chitinase. The amount of 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, consists of 399 amino acids before undergoing post-translational modification (SEQ ID NO: 5 in the sequence listing) and 398 amino acids that have undergone post-translational modification ( It is SEQ ID NO: 14) in the sequence listing. The gene derived from Aspergillus oryzae reported as a chitinase gene (chitin / 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 Since it is unknown, it is presumed to be a different molecule from the chitinase derived from Aspergillus oryzae of the present invention.
Test Example 1 Properties of crude enzyme solution of chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara JCM 2067
The activity of the crude enzyme solution obtained in Example 1.1) was measured.
1) Hydrolysis activity
Example 1 Reducing sugar was quantified according to the method of 2). When measured using chitosan 7B as a substrate, 4.2 × 10 4 per ml in the supernatant ^-3 It had U enzyme activity. Table 11 shows the relative values of chitosan 10B degrading activity when the enzyme activity is 100% when chitosan 7B is used as a substrate. It was found to have chitosan 7B degrading activity and almost no chitosan 10B degrading activity.
[0119]
[Table 11]

2) pH activity
To a mixed solution of 160 μl of chitosan 7B solution and 100 μl of 400 mM buffer, 140 μl of enzyme solution was added and stirred to homogenize, and the enzyme reaction was carried out at 37 ° C. for 20 minutes. Quantification of the free reducing sugar after the enzyme reaction was in accordance with the method described in Example 12 2). The following buffer solutions were used: pH 3.6 to pH 5.9, acetic acid-sodium acetate buffer solution: pH 5.9 to pH 8.2, 3- (N-Morpholine) propanesulfonic acid (hereinafter “ "MOPS")-Sodium carbonate buffer: sodium bicarbonate-sodium carbonate buffer in the case of pH 9.0 to 10.2. The hydrolysis activity under the pH condition with the highest activity was taken as 100%, and the hydrolysis activity of the enzyme at each pH is shown as a relative value in FIG. The optimum pH was around pH 5.0.
[0120]
3) Temperature activity of enzyme in culture supernatant
Hydrolysis activity was measured at various temperatures under the condition of pH 5.0. To a mixed solution of 160 μl of the chitosan 7B solution prepared in Example 1 and 100 μl of 400 mM acetate buffer (pH 5.0) that had been preheated for 5 minutes, 140 μl of the enzyme solution was added and stirred to make it uniform for 10 minutes. The amount of free reducing sugar after the enzyme reaction was in accordance with 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 in FIG. 6 as relative values.
[0121]
4) Temperature stability of enzyme in culture supernatant
After the enzyme in the culture supernatant was treated at various temperatures for 30 minutes, its hydrolysis activity was measured. 280 μl of the enzyme solution was added to 200 μl of 400 mM acetate buffer (pH 5.0) previously maintained at the treatment temperature, and the mixture was stirred to make it 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 the enzyme reaction was performed at 37 ° C. for 20 minutes. The amount of free reducing sugar was determined according to Example 1. The hydrolysis activity possessed by the mixture of the enzyme and buffer solution of the untreated (0 ° C. incubation) group was defined as 100%, and the hydrolysis activity at each temperature is shown in FIG. 7 as relative values.
[0122]
5) pH stability of enzyme in culture supernatant
To 400 μl of the culture supernatant, 70 μl of a 400 mM buffer solution having each pH described below and 90 μl of water were added and incubated at 37 ° C. for 1 hour. The following buffers were used: 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 4, sodium bicarbonate-sodium carbonate buffer solution: in the case of pH 10.0 to pH 11.4, disodium hydrogen phosphate-sodium hydroxide buffer solution. To a mixed solution of 100 μl of 400 mM acetate buffer (pH 5.0) and 160 μl of the chitosan 7B solution prepared by the method described in Example 1 2), 140 μl of the warmed enzyme mixture was added and stirred to homogenize. The enzyme reaction was performed for a minute. The amount of free reducing sugar was determined according to Example 1. The hydrolysis activity of the mixed solution of the enzyme and buffer solution of the untreated (0 ° C. incubation) 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 / HCl buffer (pH 7.5), and used as an enzyme solution. 50 μl of enzyme solution was added to a mixture of 160 μl of 0.25% wt / v substrate solution and 400 mM acetate buffer (pH 5.0) and 160 μl of water, and the mixture was stirred to homogenize the reaction. The enzyme reaction was performed for a minute. The free reducing sugar after the enzymatic reaction was quantified by the Schers method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). Table 12 shows the relative activities when the hydrolysis activity when using the substrate exhibiting the maximum activity is 100%.
[0124]
[Table 12]
――――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――――
Chitosan 10B 12.7
Chitosan 9B 78.9
Chitosan 8B 82.3
Chitosan 7B 100
Glucol chitosan 17.7
Glucol chitin 52.5
CM-cellulose 2.2
Rikennan 31.6
――――――――――――――――――――
Test Example 2 Aspergillus oryzae var. Sporoflavus Ohara JCM
Properties of roughly purified chitinase from 2067 strain
Various properties of the crude purified chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 obtained in Example 1.2) were examined.
1) The pH activity of the hydrolysis activity of the enzyme in the crudely purified fraction
To the mixed solution of 160 μl of the chitosan 7B solution prepared in Example 1 and 100 μl of 400 mM buffer, 140 μl of the enzyme solution was added and stirred to make the reaction uniform, and the reaction was continued at 37 ° C. for 20 minutes. The amount of free reducing sugar after the enzyme reaction was determined according to Example 1. The following buffers were used: 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 of 3, sodium bicarbonate-sodium carbonate buffer. The hydrolysis activity under the pH condition with the highest activity was taken 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 5.0.
[0125]
2) Temperature activity of the hydrolysis activity of the enzyme in the crudely purified fraction
Hydrolysis activity was measured at various temperatures under the condition of pH 5.0. 140 μl of the enzyme solution was added to the mixed solution of 160 μl of the chitosan 7B solution prepared in Example 1 and 100 μl of 400 mM acetate buffer (pH 5.0), which had been kept at the treatment temperature in advance, and the mixture was stirred and homogenized for 10 minutes. It was. The amount of free reducing sugar after the enzyme 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 the enzyme in the crudely purified fraction
The enzyme in the crudely purified fraction was treated at various temperatures for 30 minutes, and then its hydrolysis activity was measured. 200 μl of 400 mM acetate buffer (pH 5.0) and 280 μl of enzyme solution kept at the treatment temperature were added, and the mixture was stirred and homogenized, 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 the enzyme reaction was performed at 37 ° C. for 20 minutes. The amount of free reducing sugar was determined according to Example 1. The hydrolysis activity of the mixed solution of the enzyme and buffer solution of the untreated (0 ° C. incubation) group was defined as 100%, and the hydrolysis activity at each temperature is shown in FIG. 11 as relative values.
[0127]
4) pH stability of the enzyme in the crudely purified fraction
To 400 μl of the enzyme in the crudely purified fraction, 140 μl of 400 mM buffer solution having each pH described below and 20 μl of water were added, and the mixture was incubated at 37 ° C. for 1 hour. The following buffers were used: 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. To the mixed solution of 100 mM of 400 mM acetate buffer (pH 5.0) and 160 μl of the chitosan 7B solution prepared in Example 1, 140 μl of the warmed enzyme mixture was added and stirred uniformly, and the enzyme reaction was performed at 37 ° C. for 20 minutes. The amount of free reducing sugar was determined according to Example 1. The hydrolysis activity of the mixed solution of the enzyme and buffer solution of the untreated (0 ° C. incubation) group was taken as 100%, and the hydrolysis activity at each temperature was summarized in FIG. 12 as relative values.
Test Example 3 Properties of purified chitinase from Aspergillus oryzae var. Sporoflavus Ohara JCM2067
Various properties of the purified chitinase derived from Aspergillus oryzae var. Sporoflavus Ohara JCM 2067 obtained in Example 1.3) were examined.
1) pH activity of hydrolysis activity of purified enzyme
Example 1 To a mixed solution of 160 μl of chitosan 7B solution prepared by the method described in 2) and 100 μl of 400 mM buffer and 90 μl of water, 50 μl of enzyme solution was added and stirred to homogenize, and the reaction was started. The enzyme reaction was performed for a minute. Quantification of the free reducing sugar after the enzyme reaction was in accordance with the method described in Example 12 2). The following buffers were used: 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 the case of 4, sodium bicarbonate-sodium carbonate buffer. The hydrolysis activity under the pH condition with the highest activity was taken 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.
In addition, 75 μl of enzyme solution was added to a mixed solution of 240 μl of 0.25% wt / v glycol chitin solution and 150 μl of 400 mM buffer and 135 μl of water, and the mixture was stirred to homogenize the reaction, and kept at 37 ° C. for 30 minutes. Enzymatic reaction was performed. The free reducing sugar after the enzymatic reaction was quantified by the Schers method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). The following buffers were used: 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 with the highest activity was taken as 100%, and the hydrolysis activity of the enzyme at each pH is shown as a relative value in FIG. The optimum pH was around pH 10.
[0128]
2) Temperature activity of hydrolysis activity of purified enzyme
Under conditions of pH 5.5, the hydrolysis activity of the purified enzyme was measured at various temperatures. Add 40 μl of enzyme solution to a mixture of 160 μl of chitosan 7B solution and 100 μl of 400 mM acetate buffer (pH 5.5) and 100 μl of water prepared in the same manner as in 1) previously maintained at the treatment temperature, and stir to homogenize for 10 minutes. Enzymatic reaction was performed. The amount of free reducing sugar after the enzyme reaction was determined according to Example 1. The hydrolysis activity of the purified enzyme measured under the temperature condition showing the highest activity was taken as 100%, and the hydrolysis 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
The purified enzyme was treated at various temperatures for 30 minutes and then its hydrolysis activity was measured. To a mixed solution of 200 μl of 400 mM acetate buffer (pH 5.5) and 180 μl of water previously maintained at the treatment temperature, 100 μl of the enzyme solution was added and stirred to homogenize, 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 amount of free reducing sugar was determined according to Example 1. The hydrolysis activity of the mixed solution of the purified enzyme and the buffer solution of the untreated (0 ° C. incubation) group was defined as 100%, and the hydrolysis activity of the purified enzyme at each temperature is shown as a relative value in FIG. 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 solution having each pH described below was added and incubated at 37 ° C. for 1 hour. The following buffers were used: 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 hydrogen carbonate-sodium carbonate buffer: in the case of pH 9.6 to pH 11.3, disodium hydrogen phosphate-sodium hydroxide buffer. To a mixed solution of 100 μl of 400 mM acetate buffer (pH 5.5) and 160 μl of the chitosan 7B solution prepared in Example 1 and 70 μl of water, 70 μl of the warmed enzyme mixture was added and stirred to homogenize, and the enzyme was incubated at 37 ° C. for 40 minutes. Reaction was performed and free reducing sugar was quantified. The hydrolysis activity of the mixed solution of the purified enzyme and the buffer solution of the untreated (0 ° C. incubation) group was defined as 100%, and the hydrolysis activity of the purified enzyme at each temperature was summarized as a relative value in FIG. As shown in FIG. 17, the purified enzyme was stable at pH 5 to pH 9.5.
[0131]
5) Substrate specificity of the purified enzyme
The hydrolysis activity of the purified enzyme with respect to 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 hydrolytic activity of the purified enzyme with respect to other substrates as relative values when the hydrolytic activity of the purified enzyme with respect to chitosan 7B is 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. Further, the purified enzyme had chitin hydrolyzing activity and no cellulase hydrolyzing activity was observed.
Test Example 4 Properties of Chitinase Crude Enzyme Solution from Aspergillus japonics SANK19288
1) Reducing sugar was quantified according to the method described in Example 1 2). When measured using chitosan 7B as a substrate, 3.4 × 10 5 per ml in the supernatant ^-3 It had U enzyme activity. Table 14 shows the relative values of chitosan 10B degrading activity when the enzyme activity when chitosan 7B is used as a substrate is 100%.
[0133]
[Table 14]

Since it has chitosan 7B degradation activity and almost no chitosan 10B degradation activity, it was found that chitinase was produced in the supernatant.
[0134]
2) pH activity of the crude enzyme solution
Example 1 2) 140 μl of crude enzyme solution was added to a mixture of 160 μl of chitosan 7B solution and 100 μl of 400 mM buffer prepared according to the method described in the above, and the mixture was stirred and homogenized. Quantification of the free reducing sugar after the enzyme reaction was performed. The following buffers were used: 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 the case of 4, sodium bicarbonate-sodium carbonate buffer. The hydrolysis activity under the pH condition with the highest activity was taken as 100%, and the hydrolysis activity of the enzyme at each pH is shown as a relative value in FIG. The optimum pH was around pH 4.0.
[0135]
3) Substrate selectivity of crude enzyme
Substrate selectivity at pH 4.0 was measured. 10 ml of the prepared culture supernatant obtained in Example 2 was dialyzed 3 times every 12 hours against 1,000 ml of 10 mM Tris / HCl buffer (pH 7.5), and used as an enzyme solution. 50 μl of enzyme solution was added to a mixture of 160 μl of a substrate solution of 0.25% wt / v and 400 mM acetate buffer (pH 4.0) and 160 μl of water, and the mixture was stirred to homogenize the reaction. The enzyme reaction was performed for a minute. The free reducing sugar after the enzymatic reaction was quantified by the Schers method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). Table 15 shows the relative activities when the hydrolysis activity when using the substrate exhibiting the maximum activity is 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
Glycolchitin 23.3
CM-cellulose 11.4
Rikennan 25.3
―――――――――――――――――――――
Test Example 5. Properties of chitinase crude enzyme solution derived from Aspergillus sojae SANK 22388 strain
1) Hydrolysis activity
Example 1 Reducing sugars were quantified according to the method described in 2). When measured using chitosan 7B as a substrate, 2.6 × 10 6 per ml in the supernatant ^-3 It had U enzyme activity. Table 16 shows the relative values of chitosan 10B degrading activity when the enzyme activity when chitosan 7B is used as a substrate is 100%.
[0137]
[Table 16]

Since it has chitosan 7B degradation activity and almost no chitosan 10B degradation activity, it was found that chitinase was produced in the supernatant.
[0138]
2) pH activity of enzyme in culture supernatant
The measurement method was in accordance with Example 6. The following buffers were used: 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 the case of 4, sodium bicarbonate-sodium carbonate buffer. The hydrolysis activity under the pH condition with the highest activity was taken as 100%, and the hydrolysis activity of the enzyme at each pH is shown as a relative value in FIG. The optimum pH was around pH 4.5.
[0139]
3) Substrate selectivity of enzyme in culture supernatant
Substrate selectivity at pH 4.5 was measured. 10 ml of the culture supernatant prepared in Example 4 was dialyzed 3 times every 12 hours against 1,000 ml of 10 mM Tris / HCl buffer (pH 7.5) and used as the enzyme solution. 50 μl of enzyme solution was added to a mixture of 160 μl of 0.25% wt / v substrate solution and 400 mM acetate buffer (pH 4.5) and 160 μl of water, and the mixture was stirred to homogenize the reaction and kept at 37 ° C. for 10 minutes. Enzymatic reaction was performed. The free reducing sugar after the enzymatic reaction was quantified by the Schers method (see Imoto, T. and Yagashita, K., Agric. Biol. Chem., 35, 1154 (1971)). Table 17 shows the relative activities when the hydrolysis activity when using the substrate exhibiting the maximum activity is 100%.
[0140]
[Table 17]
――――――――――――――――――
Substrate Relative activity (%)
――――――――――――――――――
Chitosan 10B 0
Chitosan 9B 67.7
Chitosan 8B 71.6
Chitosan 7B 100
Glucol Chitosan 2.9
Glycolchitin 81.9
CM-cellulose 0
Rikennan 11.9
――――――――――――――――――
Test Example 6. Solubility of each low molecular weight chitosan hydrochloride in water
100 mg of each low molecular chitosan hydrochloride prepared in Examples 4 to 6 was dissolved in 10 ml of water, and the pH was measured. 1N sodium hydroxide water was added little by little to this solution, and the pH at which the solution became cloudy and colloids were measured 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 polymeric chitosan 9B hydrochloride as 537 mg of a white flocculent solid. Polymeric chitosan 10B and 8B and 7B hydrochloride were also prepared in the same way. 100 mg of each polymer chitosan hydrochloride was dissolved in 50 ml of distilled water, chitosanase 20U (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution is lyophilized and the dried solid is D ₂ Dissolved in O ¹ 1 H-NMR was measured.
The degree of acetylation was calculated from the integral ratio of 3H of the acetyl group and 7H other than the acetyl group and excluding the hydroxyl group. The results are shown in Table 19.
[0142]
[Table 19]

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

Test Example 9 Properties of purified chitinase from Aspergillus japonics SANK 19288
Various properties of the purified chitinase derived from Aspergillus japonics SANK 19288 obtained in Example 2.2) were examined.
[0147]
1) Hydrolytic activity pH activity of purified chitinase derived from Aspergillus japonics SANK 19288 strain
The hydrolytic activity pH activity of the purified chitinase derived from Aspergillus japonics SANK 19288 was measured by the method described in Test Example 31). The hydrolysis activity under the pH condition with the highest activity was taken 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 5.0.
[0148]
2) Temperature activity of hydrolysis activity of purified chitinase derived from Aspergillus japonics SANK 19288 strain
The temperature activity of the hydrolysis activity of the purified chitinase derived from Aspergillus japonics SANK 19288 was measured by the method described in Test Example 32). The hydrolysis activity under the temperature condition with the highest activity 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 japonics SANK 19288 strain
The temperature stability of purified chitinase derived from Aspergillus japonics SANK 19288 was measured by the method described in Test Example 33). The hydrolysis activity possessed by the mixture of the purified chitinase and the buffer solution of the untreated (0 ° C. incubation) group was defined as 100%, and the hydrolysis activity of the enzyme at each temperature is shown in FIG. 22 as relative values. The purified chitinase was stable at 50 ° C. or lower.
[0150]
4) pH stability of purified chitinase from Aspergillus japonics SANK 19288 strain
The pH stability of purified chitinase derived from Aspergillus japonics SANK 19288 was measured by the method described in Test Example 34). The hydrolysis activity possessed by the mixture of the purified chitinase and the buffer solution of the untreated (0 ° C. incubation) group was defined as 100%, and the hydrolysis activity of the enzyme at each pH was summarized in FIG. 23 as relative values. The purified chitinase was stable at pH 4 to pH 9.5.
[0151]
5) Substrate specificity of purified chitinase from Aspergillus japonics SANK 19288 strain
The substrate specificity of purified chitinase derived from Aspergillus japonics SANK 19288 was measured by the method described in Test Example 35). Table 21 shows the hydrolysis activity of the purified chitinase relative to each of the other substrates as relative values when the hydrolysis activity of the purified chitinase on chitosan 7B under conditions of pH 5.0, 37 ° C. and 10 minutes is defined as 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
Glycolchitin 28.3
CM-cellulose 0
Rikennan 4.2
――――――――――――――――――
As shown in Table 21, purified chitinase derived from Aspergillus japonics SANK 19288 strain had the highest hydrolysis activity against chitosan 7B, and the hydrolysis activity decreased as the degree of acetylation decreased. 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 from Aspergillus japonics SANK 19288 strain
The partial amino acid sequence of purified chitinase derived from Aspergillus japonics SANK 19288 was determined by the method described in Example 1.7). Among the separated degraded amino acids, three peaks having a B buffer concentration of about 28%, about 30%, and about 35% were collected. The sequences of these three were analyzed with 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 the Sequence Listing)
Ala-Phe-Thr-Asn-Thr-Asp-Gly-Pro-Gly-Thr-Ala-Phe-Ser-Gly-Val (SEQ ID NO: 12 in the 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 the Sequence Listing)
Test Example 10 Properties of purified chitinase from Aspergillus sojae SANK 22388
Various properties of the purified chitinase derived from Aspergillus sojae SANK 22388 obtained in Example 3.2) were examined.
[0154]
1) Hydrolysis activity pH activity of purified chitinase 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 with the highest activity was taken as 100%, and the hydrolysis activity of the enzyme at each pH is shown as a relative value in FIG. 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 differs from chitinases derived from Aspergillus oryzae and Aspergillus japonics, and has two optimum pHs (see Test Example 101)), and therefore derived from Aspergillus sojae SANK 22388 strain The temperature activity of the purified chitinase was measured by the method described in Test Example 32) at these two pH points. FIG. 25 (temperature activity at pH 5.5) and FIG. 26 (temperature activity at pH 9.0) with the hydrolytic activity under the temperature condition with 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 differs from chitinases derived from Aspergillus oryzae and Aspergillus japonics, and has two optimum pHs (see Test Example 101)), and therefore derived from Aspergillus sojae SANK 22388 strain The temperature stability of the purified chitinase was measured at the pH at these two points by the method described in Test Example 3.3). The hydrolytic activity of the mixture of the purified chitinase and buffer in the untreated (0 ° C. incubation) group is defined as 100%, and the hydrolytic activity of the enzyme at each temperature is shown as a relative value. FIG. 27 (temperature stability at pH 5.5) ) And FIG. 28 (temperature stability at pH 9.0). The purified chitinase was stable at 40 ° C. or lower 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 possessed by the mixture of the purified chitinase and the buffer solution in the untreated (0 ° C. incubation) group was defined as 100%, and the hydrolysis activity of the enzyme at each pH was summarized in FIG. 29 as relative values. The purified chitinase was stable at pH 4.5 to pH 10.
[0158]
5) Substrate specificity of purified chitinase from Aspergillus sojae SANK 22388 strain
The substrate specificity of purified chitinase derived from Aspergillus sojae SANK 22388 was measured by the method described in Test Example 3.5). Table 22 shows the hydrolytic activity of the purified chitinase relative to other substrates as relative values when the hydrolytic activity of the purified chitinase on chitosan 8B under conditions of pH 5.0, 37 ° C. and 10 minutes is defined as 100%. 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
Glycolchitin 23.8
CM-cellulose 0
Rikenan 0
――――――――――――――――――
As shown in Table 22, purified chitinase derived from Aspergillus sojae SANK 22388 strain had the highest hydrolysis activity against chitosan 7B and 8B, and the hydrolysis activity decreased as the degree of acetylation decreased. The purified chitinase had chitin hydrolyzing activity and no cellulase hydrolyzing activity was observed.
Formulation Example 1. Preparation of 5% chitosan ointment formulation
A solution of 3.0 g of propylene glycol and 0.1 g of methyl paraoxybenzoate in 80 ml of purified water is heated to 70 ° C. and dissolved by adding 5.0 g of low molecular chitosan 9B hydrochloride prepared in Example 4 to obtain a low molecular chitosan 9B solution. It was. Stirrate 3.0g, Settal 1.0g, Glycerol monostearate 6.0g, Liquid paraffin 5.0g, Propyl paraoxybenzoate 0.1g, Polyoxyl stearate 40 2.5g was heated and dissolved at 70 ° C. The molecular chitosan 9B solution was added, and purified water was added to make the total amount 100 g, and mixed and emulsified. After emulsification, the mixture was cooled to room temperature with stirring to obtain 100 g of white 5% low molecular weight chitosan 9B hydrochloride cream (referred to as 5% chitosan ointment).
Test Example 10.5% chitosan ointment wound healing test
A trauma healing test was conducted to predict the effectiveness of 5% chitosan ointment in a rat skin defect model.
1) Test material
As a test substance, 5% chitosan ointment prepared in Preparation Example 1 was used. As a control drug, Eupasta ointment (containing 100 g of purified white sugar and 3 g of popidone iodine in 100 g, polyethylene glycol, concentrated glycerin, polyoxyethylene [160] polyoxypropylene [30] glycol, pullulan, potassium iodide as additives , Production number BA1S, manufactured by Kowa Co., Ltd.). As an animal used for the test, 6-week-old Sprague-Dawley (SD) male rats (Japan SLC Co., Ltd.) were purchased and used. Animals are fed solid feed (FR-2 for breeding mice and rats, Funabashi Farm Co., Ltd.) and tap water with an environmentally controlled breeding device (CLEA Japan) with an average temperature of 23 ° C and an average humidity of 55%. The animals were acclimatized for 6 days under the condition of -19: 00 and used for the experiment.
2) Experimental method
Seven groups of 7-week-old SD male rats (body weight: 157.6 g to 235.9 g) were used in groups of 5 per group. After removing the rat's back hair with an electric clipper, EBA Eva Cream (manufactured by Tokyo Tanabe Seiyaku Co., Ltd.) was applied, and after 15 minutes, the hair was washed and depilated. After sterilizing the back hair loss site with 70% ethanol, two symmetrical punched wounds were made on the back midline using a circular punch (inner diameter 15 mm) under pentobarbital sodium (40 mg / kg, ip) anesthesia. 24 hours after the absence of damage, group 5 groups each into the no-treatment group, base group, test substance group, and control drug group, and then add the test substance and control drug to the 2 lesions at about 0.1 g / site. And 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 change in area during the treatment process was calculated by the following formula: (Actual diameter of the measurement day x Minor axis / Long diameter after preparation of missing damage x Minor axis) x 100. ), The complete cure rate was calculated with the area ratio of 5% or less as the complete cure case, and the treatment days were calculated and shown in Table 23. Experimental results were expressed as mean ± standard deviation. The treatment time was shortened to 12.6 ± 1.9 days in the 5% chitosan ointment group, compared to 13.7 ± 1.3 days in the untreated group. In the control group, there was no improvement in the number of treatment days at 13.6 ± 0.4 days.
[0161]
[Table 23]
Treatment days for rat skin defect model
_____________________
Test substance Treatment days
_____________________
No treatment 13.7 ± 1.3
5% chitosan 12.6 ± 1.9
Control 13.6 ± 0.4
_____________________
[0162]
【The invention's effect】
As described above, by using the chitinase of the present invention, the low molecular chitosan of the present invention having a molecular weight of 10,000 or more and soluble under neutral conditions can be produced. Furthermore, the low molecular chitosan of the present invention exhibits excellent antibacterial properties and wound healing effects, and the pharmaceutical composition containing the low molecular chitosan of the present invention as an active ingredient is useful as a therapeutic agent for trauma, bed sores, atopic dermatitis and the like. It is. Moreover, by using the chitinase of the present invention, a sample having a high viscosity by containing a polymer acetylated chitosan can be used as a raw material to produce a sample with a low viscosity and easy to handle. Such a method for producing a sample is useful in the process of food processing.
[0163]
[Sequence Listing Free Word]
SEQ ID NO: 6: Chitinase gene probe
SEQ ID NO: 7: PCR primer F21
Sequence number 8: PCR primer PR50
SEQ ID NO: 9: PCR primer BamHI-cDNA
SEQ ID NO: 10: PCR primer cDNA-BamHI
[0164]
[Sequence Listing]

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

Claims (13)

Chitinase, which is a protein according to any one of the following a) to d):
a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing;
b) a protein comprising an amino acid sequence encoded by the nucleotide sequence shown in nucleotide numbers 242-1438 of SEQ ID NO: 4 in the sequence listing;
c) a protein comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence described in a) or b) and having chitinase activity ;
d) A protein comprising the amino acid sequence described in a) or b). Chitinase, which is a protein according to any one of the following a) to c):
a) a protein comprising the amino acid sequence set forth in 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 set forth in SEQ ID NO: 14 in the Sequence Listing, an amino acid sequence consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added, and the N-terminal serine residue α-amino group A protein characterized in that is acetylated and has chitinase activity ;
c) A protein comprising the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing, wherein the α-amino group of the N-terminal serine residue is acetylated, and has a chitinase activity . DNA according to any one of the following:
a) DNA consisting of the nucleotide sequence shown in nucleotide numbers 242-1438 of SEQ ID NO: 4 in the sequence listing;
b) DNA characterized by hybridizing under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above and encoding a protein having chitinase 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 chitinase activity ;
d) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing;
e) DNA comprising the nucleotide sequence shown in nucleotide numbers 242-1438 of SEQ ID NO: 4 in the sequence listing. DNA according to any one of the following a) to c):
a) a recombinant plasmid carried by transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235), DNA inserted into pTriplEx-Chitinase-cDNA;
b) b) hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence of the DNA described in a) above, and encodes a protein having chitinase activity DNA;
c) A DNA comprising the DNA described in a) above and encoding a protein having chitinase activity . A chitinase, which is a protein encoded by the DNA according to claim 3 or 4 . A recombinant plasmid held by transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235), chitinase which is a protein encoded by DNA inserted into pTriplEx-Chitinase-cDNA. A recombinant plasmid comprising the DNA according to claim 3 or 4 . The recombinant plasmid according to claim 7 , which is an expression vector. A host cell transformed with the recombinant plasmid according to claim 7 or 8 . Host cell according to claim 9 , characterized in that it is a prokaryotic cell or a eukaryotic cell. The host cell according to claim 9, which is transformed Escherichia coli BM25.8 / pTriplEx-Chitinase-cDNA SANK 72102 (FERM BP-8235). A method for producing chitinase comprising the following 1) to 2):
1) A step of culturing the cells described in a) or b ) below under conditions that produce chitinase ;
a) a host cell according to any one of claims 9 to 11 ;
b) a cell producing the chitinase according to any one of claims 1, 2, 5 and 6 ;
2) A step of separating and purifying chitinase from the culture product of 1). A chitinase produced by the method according to claim 12 .

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