JP4122208B2 - Polymer cyclodextran, method for producing the same and microorganism used therefor - Google Patents

Description

【００２６】
上記４菌株のそれぞれをＬＢ Ｂｒｏｔｈ（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ，ＮＪ，ＵＳＡ）＋寒天培地に植菌し、３０℃で１日培養した。培養物より採取した菌体からゲノムＤＮＡを抽出した。ゲノムＤＮＡの抽出には、ＰｒｅｐＭａｎ^TMＭｅｔｈｏｄ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ，ＣＡ，ＵＳＡ）を使用した。
抽出したＤＮＡを鋳型としてＰＣＲにより１６Ｓ リボソーマルＲＮＡ遺伝子（１６Ｓ ｒＤＮＡ）のうち５’末端側約５００ｂｐの領域を増幅し、塩基配列をシーケンスして解析に使用した。ＰＣＲ産物の精製、サイクルシークエンスには、ＭｉｃｒｏＳｅｑ^TM ５００ １６Ｓ ｒＤＮＡ Ｂａｃｔｅｒｉａｌ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ，ＣＡ，ＵＳＡ）を使用した。
サーマルサイクラーには、ＧｅｎｅＡｍｐ ＰＣＲ Ｓｙｓｔｅｍ ９６００（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ，ＣＡ，ＵＳＡ）、ＤＮＡシーケンサーには、ＡＢＩ ＰＲＩＳＭ ３７７ ＤＮＡ Ｓｅｑｕｅｎｃｅｒ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ，ＣＡ，ＵＳＡ）を使用した。
また、ゲノムＤＮＡ抽出からサイクルシークエンスまでの操作に関しては、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社のプロトコール（Ｐ／Ｎ４３０８１３２Ｒｅｖ．Ａ）に従った。
【００２７】
得られた１６Ｓ ｒＤＮＡの塩基配列を使って相同性検索、系統樹の作成をして検体の近縁種及び帰属分類群の検討を行った。相同性検索及び系統樹の作成には、ＭｉｃｒｏＳｅｑ^TM Ｍｉｃｒｏｂｉａｌ ＩｄｅｎｔｉｆｉｃａｔｉｏｎＳｙｓｔｅｍ Ｓｏｆｔｗａｒｅ Ｖ．１．４．１及びＭｉｃｒｏＳｅｑ^TM Ｂａｃｔｅｒｉａｌ ５００ Ｌｉｂｒａｒｙ ｖ．００２３（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ，ＣＡ，ＵＳＡ）を使用した。ＭｉｃｒｏＳｅｑ^TM Ｂａｃｔｅｒｉａｌ ５００ Ｌｉｂｒａｒｙでの相同率が９７％以下であった場合は、ＢＬＡＳＴを用いてＤＮＡ塩基配列データベース（ＧｅｎＢａｎｋ）に対して相同性検索を行った。
配列表の配列番号１にバチルス・エスピー３３０Ｋ株の１６Ｓ ｒＤＮＡの塩基配列を、配列番号２に同３５０Ｋ株の１６Ｓ ｒＤＮＡの塩基配列を、配列番号３に同３６０Ｋ株の１６Ｓ ｒＤＮＡの塩基配列を、配列番号４に同８６０Ｋ株の１６Ｓ ｒＤＮＡの塩基配列を、それぞれ示す。
【００２８】
バチルス・エスピー３５０Ｋ株、同３６０Ｋ株、同８６０Ｋ株の１６Ｓ ｒＤＮＡ塩基配列は、互いに一致し、バチルス・エスピー３３０Ｋ株の１６Ｓ ｒＤＮＡ塩基配列は、同３５０Ｋ株、同３６０Ｋ株、同８６０Ｋ株の配列に９９．１％の相同性があった。
ＭｉｃｒｏＳｅｑを用いた解析の結果、バチルス・エスピー３３０Ｋ株の１６Ｓ ｒＤＮＡの部分塩基配列は、９２．９１％でＢａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓの１６Ｓ ｒＤＮＡに対し最も高い相同性を示した。また、バチルス・エスピー３５０Ｋ株、同３６０Ｋ株及び同８６０Ｋ株の１６Ｓ ｒＤＮＡの部分塩基配列は、９３．１０％でＢａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓの１６Ｓ ｒＤＮＡに対し最も高い相同性を示した。
【００２９】
ＢＬＡＳＴを用いたＧｅｎＢａｎｋに対する相同性検索の結果、バチルス・エスピー３３０Ｋ株の１６Ｓ ｒＤＮＡは９８．１％で、同３５０Ｋ株、同３６０Ｋ株及び同８６０Ｋ株の１６Ｓ ｒＤＮＡは９８．７％で、Ｂａｃｉｌｌｕｓ ｓｐ． ＭＮ−００３株に対し最も高い相同性を示した。
一方、分子系統樹上では、図２に示す通り、バチルス・エスピー３３０Ｋ株、同３５０Ｋ株、同３６０Ｋ株、同８６０Ｋ株共にＢａｃｉｌｌｕｓ ｏｌｅｒｏｎｉｕｓの１６Ｓ ｒＤＮＡとクラスターを形成したが、帰属種の推定に有効なほどの近縁種は示されなかった。
以上の結果から、現時点では、バチルス・エスピー３３０Ｋ株、同３５０Ｋ株、同３６０Ｋ株、同８６０Ｋ株の１６Ｓ ｒＤＮＡと一致する塩基配列は登録されていないと考えられる。これら４株は、いずれもＢａｃｉｌｌｕｓ ｓｐ．に帰属することが妥当であると結論された。
【００３０】
実施例３
この例では、バチルス・エスピー３３０Ｋ株、同３５０Ｋ株、同３６０Ｋ株及び同８６０Ｋ株の４菌株を用いて、高分子サイクロデキストランの生産を行った。
まず、前記の０．５％デキストランＴ５００（ファルマシア社製）を含む液体培地５ｍＬを試験管に入れて１２１℃で２０分オートクレーブ滅菌したものに、各菌株の１白金耳を接種し、振盪しながら３０℃で２４時間培養した。
次に、デキストランＴ５００（ファルマシア社製）０．５％、酵母エキス（バクト社製）０．５％、トリプトン（バクト社製）１．０％、ＮａＣｌ １．０％を含む液体培地１００ｍｌを５００ｍｌ容三角フラスコに入れ、１２１℃で２０分間オートクレーブ滅菌したものに、上記の種培養液１ｍｌを加え、３０℃で２４〜４８時間振盪培養した。
【００３１】
培養終了後、１０，０００ｒｐｍで２０分間遠心分離して集めた菌体を−８０℃で凍結し、これに界面活性剤Ｂ−ＰＥＲ^TM Ｒｅａｇｅｎｔ（ＰＩＥＲＣＥ社製）３ｍｌを加えて溶菌し、これを粗酵素液とした。
この粗酵素液に４％ デキストランＴ−４０（ファルマシア社製）、２０ｍＭＣａＣｌ₂ 、８０ｍＭ 酢酸ナトリウム緩衝液（ｐＨ５．５）を加え、全量が４０ｍｌになるように調整し、４０℃で２４〜４８時間ゆるやかに振盪しながら反応を行った。
【００３２】
次いで、１００℃で１０分加熱して反応を終了させた後、冷却し、８，０００ｒｐｍで２０分間遠心し、沈殿を取り除いた。
遠心上清にＨＢＤａｓｅを加え、２０ｍＭ 酢酸ナトリウム緩衝液（ｐＨ５．５）中にて３０℃で２４時間反応を行い、残存した直鎖のオリゴ糖又はデキストランをすべてグルコースまで分解した。次いで、１００℃で１０分間加熱後、冷却して８，０００ｒｐｍで２０分間遠心し、沈殿を取り除いた。
【００３３】
このようにして得た遠心上清を逆相カラムＳｅｐ−Ｐａｋ Ｃ１８カートリッジ（ウォーターズ社製）に通し、水で洗浄し、吸着しない直鎖のオリゴ糖又はデキストランを除いた。さらに、２０％エタノールでカラムに吸着している画分を溶出した。
サイクロデキストランを含む画分を高速液体クロマトグラフＰＵ−９８０（日本分光社製）とＡｍｉｄｅ−８０カラム（４．６ｍｍ×２５ｃｍ）（東ソー社製）を用いて５５％アセトニトリルで流速０．６ｍｌ／分で溶出し、溶出液を０．６ｍｌずつ分取分画した。
それぞれの画分を１０μＬ〜５０μＬずつとり、再び高速液体クロマトグラフＰＵ−９８０（日本分光社製）とＡｍｉｄｅ−８０カラム（４．６ｍｍ×２５ｃｍ）（東ソー社製）を用いて５５％アセトニトリルで流速１ｍｌ／分及び噴霧蒸発光散乱検出器ＳＥＤＥＸ５５（ＳＥＤＥＲＥ社製）で分析ソフトウェアＢＯＷＩＮ（日本分光社製）を用いて分析し、目的のオリゴ糖（図１に示したａ、ｂ、ｃ、ｄ、ｅ、ｆ）を含むフラクションがどれであるかを分析した。
図１に示したａ、ｂ、ｃ、ｄ、ｅ、ｆを含むフラクションにつき各々、遠心濃縮機によりアセトニトリルを除去した後、凍結乾燥した。
【００３４】
このようにして得られたオリゴ糖の構造を¹³Ｃ ＮＭＲで解析した。具体的には０．５〜１．４ｍｇ／０．６ｍｌの濃度の環状オリゴ糖溶液になるように、重水（Ｄ₂ Ｏ）（アルドリッチ社製）に溶かし、指標として０．０２５％ ３−（ｔｒｉｍｅｔｈｙｌｓｉｌｙｌ）−１−ｐｒｏｐａｎｅｓｕｌｐｈｏｎｉｃ ａｃｉｄ（ＤＳＳ）を加え、６００ＭＨｚのＮＭＲ装置（ＤＲＸ６００）（Ｂｒｕｋｅｒ社製）により２９８Ｋで、完全デカップリングの条件で¹³Ｃ一次元スペクトルを測定した。
対象として既に構造解析がなされ、８個のグルコースがα−１，６結合で環状につながった構造をしていると明らかになっているＢａｃｉｌｌｕｓ ｃｉｒｃｕｌａｎｓ Ｔ−３０４０株が生産するＣＩ−８も同じ条件で¹³Ｃ ＮＭＲで解析した。その結果、図３に示すように、本発明に係る４菌株が生産するオリゴ糖のうち、ｂ、ｃ、ｄ、ｅ、ｆは、ＣＩ−８と全く同様のα−１，６結合のグルコースを示すシグナルのみ現れた。また、ピークａについては、収量が少なく夾雑物が多いために、シグナルに乱れが生じたが、概ねα−１，６結合のグルコースを示すシグナルのみであると考えられた。
【００３５】
次に、上記で得られたオリゴ糖の質量分析を行った。ａ、ｂ、ｃ、ｄ、ｅ、ｆの凍結乾燥標品それぞれ０．１ｇを５μＬのＨ₂ Ｏで溶解し、マトリックスにグリセロールを用い、ＨＸ−１１０Ａ／１１０Ａ（ＪＥＯＬ社製）で二次イオン質量分析法（ＳＩＭＳ）で測定を行った。測定は、正イオンモード及び負イオンモードで実施した。正イオンモードで測定した結果を図４に示す。
サイクロデキストランは、同じグルコース分子より成る直鎖のイソマルトオリゴ糖よりもＨ₂ Ｏが１分子少なく、それぞれのサイクロデキストランの分子量は、ＣＩ−７が１１３４、ＣＩ−８が１２９６、ＣＩ−９が１４５８、ＣＩ−１０が１６２０、ＣＩ−１１が１７８２、ＣＩ−１２が１９４４と計算される。
質量分析によるオリゴ糖ａ、ｂ、ｃ、ｄ、ｅ、ｆの推定分子量はａがＣＩ−７、ｂがＣＩ−８、ｃがＣＩ−９、ｄがＣＩ−１０、ｅがＣＩ−１１、ｆがＣＩ−１２の分子量に一致した。
【００３６】
前記¹³Ｃ ＮＭＲ分析及び上記の質量分析の結果、バチルス・エスピー３３０Ｋ株、同３５０Ｋ株、同３６０Ｋ株及び同８６０Ｋ株が原料のデキストランより生産するオリゴ糖ａ、ｂ、ｃ、ｄ、ｅ、ｆはすべてグルコースがα−１，６結合で環状につながった環状イソマルトオリゴ糖、すなわちサイクロデキストランであり、それらの分子式は下記のように示されることが判明した。
ａ： Ｃ₄₂Ｈ₇₀Ｏ₃₅
ｂ： Ｃ₄₈Ｈ₈₀Ｏ₄₀
ｃ： Ｃ₅₄Ｈ₉₀Ｏ₄₅
ｄ： Ｃ₆₀Ｈ₁₀₀ Ｏ₅₀
ｅ： Ｃ₆₆Ｈ₁₁₀ Ｏ₅₅
ｆ： Ｃ₇₂Ｈ₁₂₀ Ｏ₆₀
【００３７】
上記４菌株は、グルコース１０〜１２分子が結合したＣＩ−１０、ＣＩ−１１及びＣＩ−１２のサイクロデキストランを主として生産するが、その他に少量であるが、ＣＩ−７、ＣＩ−８、ＣＩ−９も生産する。さらに、前記の如く、ピークｆ以降にＣＩ−１３〜ＣＩ−１６などの高分子環状オリゴ糖を生産する可能性もある。
以上の結果から、バチルス・エスピー３３０Ｋ株、同３５０Ｋ株、同３６０Ｋ株及び８６０Ｋ株は、高分子サイクロデキストランを中心に生産する新規のバチルス属菌株であることが明らかになった。
【００３８】
実施例４
次に、本発明に係る高分子サイクロデキストラン（ＣＩ）の機能について調べた。はじめに、ＣＩの抗齲蝕能を調べた。
実施例３で得た高分子サイクロデキストランＣＩ−１０及びＣＩ−１１について、ロイコノストック・メセンテロイデス菌由来のデキストランスクラーゼ阻害効果を測定した。すなわち、反応液２０μｌ中に、終濃度５％スクロースを含む２０ｍＭ 酢酸緩衝液（ｐＨ５．２）にＣＩを最終濃度０〜１ｍＭになるように添加し、デキストランスクラーゼを加え、３０℃で１０分酵素反応を行った後、スクロースの分解によって生じるフラクトースの量をネルソン・ソモギー法により還元糖量として測定した。ここで用いたロイコノストック・メセンテロイデス由来のデキストランスクラーゼは、Ｌｅｕｃｏｎｏｓｔｏｃ ｍｅｓｅｎｔｅｒｏｉｄｅｓ ＮＲＲＬ Ｂ−５１２Ｆ株をスクロースを加えた培地中で培養することにより得られた酵素を用いた。
ＣＩ−１０、ＣＩ−１１並びに対照としてのＣＩ−８によるデキストランスクラーゼ阻害試験の結果を図５に示す。図５から明らかなように、ＣＩ−１０及びＣＩ−１１もＣＩ−８と同様に、１ｍＭの濃度で約５０％の阻害が見られ、抗齲蝕性を有すると認められる。なお、ＣＩ−１２以上のものについても、その構造から他のＣＩと同様に、デキストランスクラーゼ活性阻害があるものと推定できる。
【００３９】
次に、ＣＩ−１０及びＣＩ−１１のストレプトコッカス・ミュータンス菌由来のグルコシルトランスフェラーゼ（ＧＴＦ）阻害効果について調べた。すなわち、ＣＩによるＧＴＦ活性の阻害は、反応液２０μｌ中に終濃度５％のスクロースを含む２０ｍＭ 酢酸緩衝液（ｐＨ５．５）又は２０ｍＭ リン酸ナトリウム緩衝液（ｐＨ７．０）にＣＩを最終濃度０〜２ｍＭになるように添加し、ＧＴＦを加え、３７℃で１８時間酵素反応を行った後、生じた不溶性グルカンを遠心で集めて、該グルカンの量をフェノール硫酸法で全糖量を定量することにより測定した。なお、ここで用いたストレプトコッカス・ミュータンス由来のＧＴＦは、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｍｕｔａｎｓを培養することによって得られたものである。
ＣＩ−１０、ＣＩ−１１並びに対照としてのＣＩ−８によるグルコシルトランスフェラーゼ阻害試験の結果を図６に示す。図６から明らかなように、ＣＩ−１０及びＣＩ−１１の場合も、ＣＩ−８と同様に、生成するグルカン量の減少が見られ、抗齲蝕性を有すると考えられる。なお、ＣＩ−１２以上のものについても、その構造から他のＣＩと同様にＧＴＦ活性阻害があるものと推定できる。
【００４０】
さらに、ＣＩ−１０、ＣＩ−１１及びＣＩ−１２のビクトリアブルーに対する包接試験を行った。すなわち、反応液５０μｌ中に終濃度０．０３２ｍＭのビクトリアブルーＢ（ワコー社製）、１００ｍＭ リン酸ナトリウム緩衝液（ｐＨ７．０）、１．２ｍＭのＣＩ、あるいはサイクロデキストリン（ＣＤ）又はグルコース（Ｇｌｃ）を加え、室温で保持して６２０ｎｍにおける吸光度を測定した。
その結果、図７に示したように、対照として用いたＣＩ−７、ＣＩ−８、ＣＩ−９、並びにＣＩ−１１、ＣＩ−１２及びα−ＣＤは、グルコース添加の反応液や無添加の反応液の場合と同様に、６２０ｎｍにおける吸光度の減少が見られ、顕著なビクトリアブルーＢの包接能は見出せなかった。しかし、ＣＩ−１０を加えた反応液は、２２時間経過した後も、反応開始時に比較して、約５０％の吸光度を示した。これはβ−ＣＤ及びγ−ＣＤよりも高い値であり、ＣＩ−１０は有効にビクトリアブルーを包接するものと考えられる。
【００４１】
ＣＩ−７、ＣＩ−８、ＣＩ−９などの低分子サイクロデキストランの包接能が低い理由は、同じグルコース分子数のサイクロデキストリンに比べて環の口径が大きく、浅い構造をしているため、物質を保持する力が弱いものと考察される。
一方、口径の大きいＣＩ−１０に高い包接能を有している理由として、分子が大きくなったために、よじれた構造をとっている可能性が推定できる。ＣＩ−１１、ＣＩ−１２も、８の字のようなよじれた構造を形成する可能性が推察できることから、包接能を有する物質が今後見出されることが期待できる。すなわち、本発明に係る４菌株は、前記の如く、ＣＩ−１３〜ＣＩ−１６という高分子のＣＩを生産する可能性があり、これらの物質にも包接能が期待できる。
【００４２】
ＣＩ−１０、ＣＩ−１１及びＣＩ−１２にビクトリアブルーを加えて吸収スペクトルの変化を測定した。すなわち、反応液５０μｌ中に終濃度０．０３２ｍＭのビクトリアブルーＢ（ワコー社製）、１００ｍＭ リン酸ナトリウム緩衝液（ｐＨ７．０）、１．２ｍＭのＣＩ、あるいはＣＤ又はグルコースを加え、室温で６時間保持した後に３００ｎｍから８００ｎｍにおける吸収スペクトルの変化を分光光度計 ＵＶ／ＶＩＳ Ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｅｒ Ｖ−５５０）（日本分光社製）を用いて測定した。
その結果を図８に示す。この図から、ビクトリアブルーに対する高い包接能を示したＣＩ−１０を加えた反応液は、吸収スペクトルの形に顕著な変化が見られ、ＣＩ−１０が有効にビクトリアブルーを包接することの裏付けになると考えられた。
【００４３】
【発明の効果】
本発明により新規な高分子サイクロデキストランが提供される。この物質は、バチルス属菌株であるバチルス・エスピー３３０Ｋ株、同３５０Ｋ株、同３６０Ｋ株及び同８６０Ｋ株を培養することにより得られる。
これらの菌株は、従来のサイクロデキストラン生産菌株と比べて、より高分子のサイクロデキストランを中心に生産するので、高分子サイクロデキストランの生産に適している。
【００４４】
しかも、本発明のバチルス属菌株が生産する高分子サイクロデキストランは、抗齲蝕剤として利用できる。また、当該高分子サイクロデキストランのうち、ＣＩ−１０は包接能を持ち、かつ高い水溶性であることから、食品、医薬品、化成品などに応用できる。ＣＩ−１０が包接する化合物は、γ−ＣＤにもよく包接されることから、これまでにγ−ＣＤに包接されることが知られている物質をＣＩ−１０が有効に包接する可能性がある。また、当該高分子サイクロデキストランは、サイクロデキストリンに比べて水溶性が非常に高いことから、不溶性物質の包接による可溶化の効果がサイクロデキストリンよりも高いことが期待できる。
【００４５】
【配列表】

【図面の簡単な説明】
【図１】 (a)バチルス・エスピー３３０Ｋ株、(b)同３５０Ｋ株、(c)同３６０Ｋ株及び(d)同８６０Ｋ株が生産する、ＨＢＤａｓｅで分解されないオリゴ糖を示すＨＰＬＣ分析の図である。
【図２】 (a)バチルス・エスピー３３０Ｋ株、(b)同３５０Ｋ株、(c)同３６０Ｋ株並びに(d)同８６０Ｋ株の分子系統樹を示す図である。
【図３】 バチルス・エスピー３３０Ｋ株、同３５０Ｋ株、同３６０Ｋ株並びに同８６０Ｋ株がが生産する、ＨＢＤａｓｅで分解されないオリゴ糖ａ、ｂ、ｃ、ｄ、ｅ、ｆ及び既知のサイクロデキストランＣＩ−８について¹³Ｃ ＮＭＲ分析した結果を示す図である。
【図４】 バチルス・エスピー３３０Ｋ株、同３５０Ｋ株、同３６０Ｋ株並びに同８６０Ｋ株が生産する、ＨＢＤａｓｅで分解されないオリゴ糖ａ、ｂ、ｃ、ｄ、ｅ、ｆを質量分析した結果を示す図((a)〜(f))である。
【図５】 高分子サイクロデキストランＣＩ−１０及びＣＩ−１１のデキストランスクラーゼ活性の阻害作用を既知のサイクロデキストランＣＩ−８と比較した図である。
【図６】 高分子サイクロデキストランＣＩ−１０及びＣＩ−１１のＧＴＦ阻害作用を既知のサイクロデキストランＣＩ−８と比較した図である。(a)はｐＨ５．５、(b)はｐＨ７．０での結果を示す。
【図７】 高分子サイクロデキストランＣＩ−１０、ＣＩ−１１、ＣＩ−１２のビクトリアブルーの包接能を既知のサイクロデキストランＣＩ−７、ＣＩ−８、ＣＩ−９及びα−ＣＤ、β−ＣＤ、γ−ＣＤ、グルコースと比較した図である。
【図８】 ビクトリアブルーに高分子サイクロデキストランＣＩ−１０、ＣＩ−１１又はＣＩ−１２を加えた溶液の吸収スペクトルを、既知のサイクロデキストランＣＩ−７、ＣＩ−８、ＣＩ−９及びα−ＣＤ、β−ＣＤ、γ−ＣＤ、グルコースを加えた場合と比較した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a polymer cyclodextran, a method for producing the same, and a microorganism used therefor. More specifically, the present invention relates to a polymer cyclodextran having a structure in which glucose molecules are connected by α-1,6 bonds, a method for producing the same, a novel microorganism used therefor, and a use thereof.
[0002]
[Prior art]
Conventionally, only two types of cyclodextran-producing bacteria have been reported: Bacillus circulans T-3040 (patent document 1) and Bacillus circulans U-155 (non-patent document 1). In addition, all of these are relatively low-molecular cyclodextran such as cyclic oligosaccharide CI-7 in which 7 molecules of glucose are linked by α-1,6 bonds, CI-8 of 8 molecules of glucose, and CI-9 of 9 molecules of glucose. It is known to produce mainly. Cyclic oligosaccharides CI-7, CI-8, and CI-9 have been reported to have anti-caries action and weak inclusion ability. Although the anti-caries ability can be expected to be effective, the inclusion ability is considerably weaker than cyclodextrin, which is the same cyclic oligosaccharide, and the effectiveness as an inclusion substance cannot be expected (Patent Document 2). In addition, a technique for producing a branched cyclodextran by imparting a side chain to these CI-7, CI-8, and CI-9 has also been attempted, but a product having significantly improved inclusion ability has not yet been obtained.
[0003]
[Patent Document 1]
JP-A-6-197783
[Non-Patent Document 1]
DDBJ / EMBL / Genbank DNA database accession number D88360
[Patent Document 2]
JP-A-8-59484
[0004]
[Problems to be solved by the invention]
Since cyclodextran has a structure of α-1,6 bonds, it is extremely water-soluble. Therefore, if this substance has an inclusion ability, it is expected to be widely used for foods, pharmaceuticals, chemical products and the like.
An object of the present invention is to develop a novel cyclodextran having a different number of glucose bonded to the cyclic oligosaccharide and having not only caries resistance but also inclusion ability.
Furthermore, an object of the present invention is to provide a method for searching for a microorganism that produces such a novel cyclodextran and a method for producing a novel cyclodextran using the microorganism.
[0005]
[Means for Solving the Problems]
The inventors of the present invention have succeeded in searching for a group of microorganisms capable of producing a high-molecular cyclodextran from a sample collected in a sugar factory. And these microorganisms belong to the genus Bacillus (Bacillus sp.), And it discovered that 10 or more glucose molecules from dextran produce the high molecular cyclodextran which forms a cyclic structure by alpha-1,6 coupling | bonding. Thus, the present invention has been completed.
[0006]
  The present invention according to claim 1 provides glucose 1012It is a polymer cyclodextran in which a molecule forms a cyclic structure by α-1,6 bonds.
  The present invention described in claim 2Bacillus sp. 330K strain (FERM P-19080), 350K strain (FERM P-19081), 360K strain (FERM P-19082) or 860K strain (FERM P-19083)Is cultivated in a medium containing dextran, and the polymer cyclodextran according to claim 1 is collected from the culture.
  The present invention described in claim 3Bacillus sp. 330K strain (FERM P-19080), 350K strain (FERM P-19081), 360K strain (FERM P-19082) or 860K strain (FERM P-19083)The polymer cyclodextran according to claim 1, which is obtained by lysing a microorganism obtained by culturing a microorganism in a medium containing dextran, reacting with dextran, and collecting the polymer cyclodextran according to claim 1 from the reaction product. In the manufacturing method of cyclodextranis there.
[0007]
  Claim4The described invention belongs to the genus Bacillus and has the ability to produce the high-molecular cyclodextran according to claim 1, the Bacillus sp. Strain 330K (FERM P-19080), the same 350K strain (FERM P-19081), the same 360K. Strain (FERM P-19082) or 860K strain (FERM P-19083).
  Claim5The present invention described is an anti-cariogenic agent characterized by containing the polymeric cyclodextran according to claim 1 as an active ingredient.
  Claim6The described invention is a water-soluble cyclic oligosaccharide composition having an inclusion ability containing, as an active ingredient, a polymeric cyclodextran in which 10 molecules of glucose form a cyclic structure by α-1,6 bonds.
  Claim7The present invention as described is a food or drink comprising the polymer cyclodextran according to claim 1 as an active ingredient.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
  Glucose 10 according to claim 112A polymer cyclodextran in which molecules form a cyclic structure by α-1,6 bonds can be produced by the methods according to claims 2 and 3.
  All the bacteria producing the high-molecular cyclodextran belong to the genus Bacillus. These microorganisms are strains isolated from a sugar factory, and include Bacillus 330K strain, 350K strain, 360K strain, and 860K strain.
  In the following, the present invention will be described in detail.
[0009]
The polymer cyclodextran of the present invention (hereinafter sometimes abbreviated as CI) is a polymer cyclodextran (hereinafter referred to as CI-10) in which 10 molecules of glucose form a cyclic structure by α-1,6 bonds. ), A polymer cyclodextran (hereinafter referred to as CI-11) in which 11 molecules of glucose form a cyclic structure by α-1,6 bonds, and 12 molecules of glucose form a cyclic structure by α-1,6 bonds. Polymer cyclodextran (hereinafter referred to as CI-12), a polymer cyclodextran (hereinafter referred to as CI-13) in which 13 molecules of glucose form a cyclic structure by α-1,6 bonds, glucose A polymer cyclodextran (hereinafter referred to as CI-14), in which 14 molecules form a cyclic structure by α-1,6 bonds, gluco 15 cyclohextran (hereinafter referred to as CI-15) in which 15 molecules form a cyclic structure by α-1,6 bonds and 16 molecules of glucose form a cyclic structure by α-1,6 bonds. A high-molecular cyclodextran (hereinafter referred to as CI-16).
[0010]
The Bacillus genus strain which is a CI producing bacterium can be obtained by, for example, the following method.
Samples are collected at a sugar factory and suspended in physiological saline to make appropriate dilutions. Next, the diluted solution is smeared on a plate medium (pH 7.0) containing 0.2% blue dextran, and a transparent halo is formed by decomposition of blue dextran from a large number of colonies grown on the plate. Get a colony.
The obtained strain is further subjected to a selection test to obtain a cyclic oligosaccharide that is not degraded by a multi-branched dextran hydrolase (hereinafter referred to as HBDase), and that is higher in molecular weight than CI-9, which is a known cyclic oligosaccharide. Select strains that can be produced in the center.
[0011]
The acquisition method is summarized as follows.
(1) Isolation of bacteria that form halos on a blue dextran plate medium from nature, (2) After culturing in a liquid culture solution containing dextran, dextran is again added to the lysate and incubated to produce oligosaccharides. Separation, (3) Separation of bacteria that adsorb oligosaccharides in the reaction solution to reverse phase C18 column and elute with 20% ethanol, (4) Cyclic oligosaccharide solution in which the 20% ethanol elution fraction is not decomposed by HBDase Separation of bacteria to be produced, (5) When the cyclic oligosaccharide is analyzed by HPLC using an Amide-80 column, a strain that mainly produces high molecular cyclodextran detected after the retention time at which CI-9 elutes To select.
[0012]
The detail of the acquisition method of the Bacillus genus strain which concerns on this invention is shown below.
A sample is collected at a sugar factory, suspended in a raw saline solution, and a diluted solution is appropriately prepared. The diluted solution is 2 g of blue dextran (manufactured by Pharmacia), 5 g of yeast extract (manufactured by Bacto), Stain 10g of tryptone (manufactured by Bacto), 10g of NaCl, 15g of agar (manufactured by Bacto), smear on a plate medium adjusted to pH 7.0, and from the colonies grown on the plate, halos caused by the degradation of blue dextran Forming colonies are primarily screened as dextranase producing strains.
Next, the selected strain contains 5 g of dextran T500 (manufactured by Pharmacia), 5 g of yeast extract (manufactured by Bacto), 10 g of tryptone (manufactured by Bacto), and 10 g of NaCl, and a pH adjusted to 7.0. After inoculating the medium and culturing with shaking at 30 ° C. overnight for 2 days, the cells are collected by centrifugation, lysed by adding a surfactant B-PER Reagent (manufactured by PIERCE), To do. Dextran T40 (Pharmacia) is added thereto, and the reaction is carried out in 20 mM sodium acetate buffer (pH 5.5) at 40 ° C. for 24 to 48 hours with gentle shaking.
[0013]
HBDase is added to this, and the reaction is carried out overnight at 30 ° C. in 20 mM sodium acetate buffer (pH 5.5) to decompose all remaining linear oligosaccharides or dextran to glucose. HBDase may be obtained by culturing Sphingobacterium sp. V-54 (FERM P-16086) described in JP-A-10-229876, or Escherichia coli JM109 described in JP-A-2001-054382. It may be obtained by culturing an E. coli transformant introduced with a multi-branched dextran hydrolase gene such as (pKK223-3-3) (FERM P-17510).
This reaction solution is passed through a reverse phase column such as a Sep-Pak C18 cartridge (manufactured by Waters) and washed with water to remove non-adsorbed linear oligosaccharide or dextran. Next, the fraction adsorbed on the column is eluted with 20% ethanol.
The order of the reverse phase column process and the HBDase process may be interchanged.
[0014]
High-performance liquid chromatograph PU-980 (manufactured by JASCO Corporation) and Amide-80 column (4.6 mm × 25 cm) obtained by subjecting the reaction solution to reverse phase column processing such as HBDase treatment and Sep-Pak C18 cartridge (manufactured by Waters). (Tosoh Corporation) was analyzed with 55% acetonitrile at a flow rate of 1 ml / min and a spray evaporation light scattering detector SEDEX55 (SIDERE) using analysis software BOWIN (manufactured by JASCO Corporation) and not decomposed by HBDase If the oligosaccharide peak is detected after the retention time at which CI-9 elutes, it can be presumed to be a macromolecular oligosaccharide-producing bacterium.
FIG. 1 shows a high-performance liquid chromatograph of HBDase non-degradable oligosaccharide produced by the Bacillus genus strain 330K, 350K, 360K or 860K isolated as a macromolecular oligosaccharide-producing bacterium by the above method ( The measurement result by HPLC) is shown. In any strain, oligosaccharide peaks d, e, and f can be observed at a retention time after CI-9. FIG. 1 shows data up to a measurement time of 20 minutes, but it is considered that there is a possibility that polymer cyclic oligosaccharides eluting after the peak f are generated.
[0015]
The strains thus obtained are Bacillus sp. 330K strain, 350K strain, 360K strain and 860K strain. These strains have the bacteriological properties described later, and further amplify a specific region of the 16S ribosomal RNA gene (16S rDNA) by PCR using genomic DNA extracted from the cells as a template, and determine the base sequence. As a result of searching for homology with known sequences, both strains were identified as Bacillus sp. Was found to belong to.
All of these 4 strains of the genus Bacillus are recognized as new strains and have been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. Each deposit number is FERM P-19080 for the Bacillus sp. 330K strain. The 350K strain is FERM P-19081, the 360K strain is FERM P-19082, and the 860K strain is FERM P-19083.
[0016]
Next, a method for producing a polymeric cyclodextran in which 10 to 12 molecules of glucose according to the present invention form a cyclic structure by α-1,6 bonds will be described.
After inoculating the aforementioned Bacillus strain having the ability to produce polymer cyclodextran into a medium containing the above-mentioned dextran and culturing according to the method described above, the target polymer cyclodextran is produced, followed by high performance liquid chromatography. Using graph PU-980 (manufactured by JASCO Corporation) and Amide-80 column (4.6 mm × 25 cm) (manufactured by Tosoh Corporation), elution was performed with 55% acetonitrile at a flow rate of 0.6 ml / min. Sort and fractionate one by one. 10 μL to 50 μL of each fraction was taken and again flowed at 55% acetonitrile using a high performance liquid chromatograph PU-980 (manufactured by JASCO Corporation) and Amide-80 column (4.6 mm × 25 cm) (manufactured by Tosoh Corporation). 1 ml / min and a spray evaporation light scattering detector SEDEX55 (manufactured by SIDERE) using analysis software BOWIN (manufactured by JASCO Corporation), and the target oligosaccharide (a, b, c, d, Analyze which fraction contains e, f). Acetonitrile is removed by a centrifugal concentrator for each of the fractions containing a, b, c, d, e, and f shown in FIG. It is then lyophilized. In addition, the manufacturing method of the above-described polymer cyclodextran according to the present invention is an example, and the present invention is not limited to this method.
[0017]
Polymeric cyclodextran CI-10, CI-11, CI-12, CI-13, CI-14, CI-15 and CI-16 according to the present invention have an inhibitory action on dextransucrase activity and glucosyltransferase activity. Therefore, the substance can be contained as an active ingredient and used as an anti-cariogenic agent. For example, under the conditions of pH 5.5 to 7.0 and temperature 30 to 37 ° C., the mutans chain which is a caries bacterium in the presence of 5% sucrose at a concentration of 0.2 mM or more of high molecular cyclodextran Staphylococcal glucosyltransferase inhibits the action of synthesizing glucan causing plaque.
Furthermore, as a result of an inclusion test for Victoria Blue, which is a basic dye, CI-10 has an effect of including Victoria Blue effectively and stably among the above-described polymer cyclodextran. Moreover, any of the polymer cyclodextrans according to the present invention is water-soluble. For inclusion ability of CI-10, Victoria Blue blue amber at a concentration of 0.032 mM in 100 mM sodium phosphate buffer (pH 7.0) at 25 ° C. at a concentration of 0.6 mM or higher. Can be suppressed.
Therefore, the said high molecular cyclodextran can be applied to foodstuffs, a pharmaceutical, and a chemical product, and can be utilized for inclusion of a specific component.
[0018]
【Example】
EXAMPLES Next, although an Example demonstrates this invention in detail, this invention is not limited by these.
Example 1
(1) In a sugar factory, intermediate products produced in the raw sugar production process and soil in the sugar factory were collected.
(2) 5 g of the above sample was suspended in 45 ml of sterilized raw buried saline, and the suspension was appropriately diluted. 1 liter of this diluted solution contains 2 g of blue dextran (Pharmacia), 5 g of yeast extract (Bact), 10 g of tryptone (Bact), 10 g of NaCl, 15 g of agar (Bact), and has a pH of 7 A plate medium adjusted to 0.0 was smeared with a sterilized congeal rod and cultured at 30 ° C. overnight for 2 days. From among the colonies grown on the plate, colonies forming halos due to degradation of blue dextran were dextran. The primary screening was performed as a nuclease producing strain.
[0019]
The colony obtained above is picked up with a platinum loop, smeared again on the above-mentioned 0.2% blue dextran plate medium, and further cultured twice at 30 ° C. for 1 to 2 days. Purely isolated.
To a test tube, add a medium containing 2% agar to a medium containing 0.5% dextran T500 (Pharmacia), autoclav it at 121 ° C. for 20 minutes, and then leave it diagonally and harden at room temperature. A storage medium prepared as described above. The strain obtained above was streaked on this storage medium using a platinum loop. This was cultured at 30 ° C. for 2 days to obtain a stock strain.
One platinum loop of this stock strain was inoculated into a 5 mL liquid medium containing the above-mentioned 0.5% dextran T500 (manufactured by Pharmacia) in a test tube and autoclaved at 121 ° C. for 20 minutes. The cells were cultured at 24 ° C. for 24 hours.
[0020]
Next, a liquid containing 5 g of dextran T500 (manufactured by Pharmacia), 5 g of yeast extract (manufactured by Bacto), 10 g of tryptone (manufactured by Bacto), and 10 g of NaCl in 1 liter, and having a pH adjusted to 7.0. After inoculating the medium and shaking culture at 30 ° C. overnight to 2 days, the cells were collected by centrifugation (20 minutes at 10,000 rpm), and the obtained cells were frozen at −80 ° C. Surfactant B-PER^TM  Reagent (manufactured by PIERCE) 3 ml was added to lyse, and this was used as a crude enzyme solution.
To this crude enzyme solution, 1.6 g of dextran T40 (Pharmacia) and a final concentration of 20 mM were added so that the final concentration was 4%.₂ ・ 2H₂ 0.12 g of O and 80 mM sodium acetate buffer (pH 5.5) were added to adjust the total volume to 40 ml, and the reaction was performed while gently shaking at 40 ° C. for 24-48 hours.
[0021]
Next, the reaction was terminated by heating at 100 ° C. for 10 minutes, followed by cooling and centrifugation at 8,000 rpm for 20 minutes to remove the precipitate. HBDase was added to the centrifuged supernatant, and the reaction was carried out in 20 mM sodium acetate buffer (pH 5.5) at 30 ° C. for 12 hours to decompose all remaining linear oligosaccharides or dextran to glucose. Here, HBDase may be obtained by culturing Sphingobacteria sp. V-54 (FERM P-16086) described in JP-A-10-229876, or Escherichia coli described in JP-A-2001-054382. It may be obtained by culturing an E. coli transformant introduced with a multi-branched dextran hydrolase gene such as JM109 (pKK223-3-3) (FERM P-17510). This was heated at 100 ° C. for 10 minutes, cooled, and centrifuged at 8,000 rpm for 20 minutes to remove the precipitate.
[0022]
The centrifugal supernatant thus obtained was passed through a reverse phase column Sep-Pak C18 cartridge (manufactured by Waters) and washed with water to remove non-adsorbed linear oligosaccharide or dextran. Subsequently, the fraction adsorbed on the column was eluted with 20% ethanol. The ethanol was removed from this by a centrifugal evaporator, dried, dissolved in 50% acetonitrile, and centrifuged at 15,000 rpm for 10 minutes to remove dust.
[0023]
Using the high-performance liquid chromatograph PU-980 (manufactured by JASCO Corporation) and Amide-80 column (4.6 mm × 25 cm) (manufactured by Tosoh Corporation), the reaction solution obtained by the above method was flowed at a flow rate of 1 ml / min with 55% acetonitrile. And a spray evaporation light scattering detector SEDEX55 (manufactured by SIDERE) and analysis software BOWIN (manufactured by JASCO), and the oligosaccharide peak that is not decomposed by HBDase is behind the retention time at which CI-9 elutes. Obtained by selecting the ones to be detected.
As a result, the obtained high-molecular cyclodextran producing strains are Bacillus SP 330K, 350K, 360K, and 860K.
[0024]
Example 2
For the 4 strains obtained in Example 1, colony morphology on agar plate medium, cell morphology by microscopic observation, Gram staining, presence or absence of spores, presence or absence of movement by flagella and catalase, oxidase, glucose oxidation fermentation (OF ) Etc. were observed and tested. As a result, all were classified and identified as belonging to Bacillus strains. Table 1 shows the results of the first stage test for bacterial identification.
[0025]
[Table 1]
Table 1

[0026]
Each of the above four strains was inoculated in LB Broth (Becton Dickinson, NJ, USA) + agar medium and cultured at 30 ° C. for 1 day. Genomic DNA was extracted from the cells collected from the culture. For extraction of genomic DNA, PrepMan^TMMethod (Applied Biosystems, CA, USA) was used.
Using the extracted DNA as a template, a region of about 500 bp on the 5 'end side of 16S ribosomal RNA gene (16S rDNA) was amplified by PCR, and the base sequence was sequenced and used for analysis. For purification of PCR products and cycle sequencing, MicroSeq^TM  A 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems, CA, USA) was used.
GeneAmp PCR System 9600 (Applied Biosystems, CA, USA) was used as the thermal cycler, and ABI PRISM 377 DNA Sequencer (Applied Biosystems, CA, USA) was used as the DNA sequencer.
In addition, the procedures from genomic DNA extraction to cycle sequence were in accordance with Applied Biosystems protocol (P / N4308132 Rev. A).
[0027]
Using the obtained 16S rDNA base sequence, homology search and generation of a phylogenetic tree were carried out to examine the related species and belonging taxonomic groups of the specimens. For the homology search and phylogenetic tree creation, MicroSeq^TM  Microbial Identification System Software V. 1.4.1 and MicroSeq^TM  Bacterial 500 Library v. 0023 (Applied Biosystems, CA, USA) was used. MicroSeq^TM  When the homology rate in Bacterial 500 Library was 97% or less, a homology search was performed on a DNA base sequence database (GenBank) using BLAST.
In the sequence listing, SEQ ID NO: 1 shows the base sequence of 16S rDNA of the Bacillus sp. 330K strain, SEQ ID NO: 2 shows the base sequence of 16S rDNA of the 350K strain, SEQ ID NO: 3 shows the base sequence of 16S rDNA of the 360K strain, SEQ ID NO: 4 shows the base sequence of 16S rDNA of the 860K strain.
[0028]
The 16S rDNA base sequences of the Bacillus sp 350K, 360K and 860K strains are identical to each other, and the 16S rDNA base sequence of the Bacillus sp 330K strain is the same as that of the 350K, 360K and 860K strains. There was 99.1% homology.
As a result of analysis using MicroSeq, the partial base sequence of 16S rDNA of Bacillus sp. 330K strain was 92.91%, showing the highest homology to 16S rDNA of Bacillus licheniformis. Further, the partial base sequences of 16S rDNA of Bacillus sp 350K, 360K and 860K were 93.10%, which showed the highest homology to 16S rDNA of Bacillus licheniformis.
[0029]
As a result of homology search against GenBank using BLAST, 16S rDNA of Bacillus sp. 330K strain was 98.1%, 16S rDNA of 350K strain, 360K strain and 860K strain was 98.7%, Bacillus sp. . It showed the highest homology to the MN-003 strain.
On the other hand, in the molecular phylogenetic tree, as shown in FIG. 2, the Bacillus sp. 330K strain, 350K strain, 360K strain, and 860K strain formed clusters with 16S rDNA of Bacillus soleronius. Not so much related species were shown.
Based on the above results, it is considered that no nucleotide sequence that matches 16S rDNA of Bacillus sp. 330K strain, 350K strain, 360K strain, and 860K strain is registered at present. These four strains are all Bacillus sp. It was concluded that it was appropriate to belong to
[0030]
Example 3
In this example, polymer cyclodextran was produced using 4 strains of Bacillus sp. 330K strain, 350K strain, 360K strain and 860K strain.
First, 5 mL of a liquid medium containing 0.5% dextran T500 (Pharmacia) was put in a test tube and sterilized at 121 ° C. for 20 minutes by inoculating 1 platinum ear of each strain with shaking. The cells were cultured at 30 ° C. for 24 hours.
Next, 500 ml of 100 ml of a liquid medium containing 0.5% dextran T500 (Pharmacia), 0.5% yeast extract (Bact), 1.0% tryptone (Bact), 1.0% NaCl Into an Erlenmeyer flask, autoclaved at 121 ° C. for 20 minutes, 1 ml of the above seed culture was added, and cultured with shaking at 30 ° C. for 24-48 hours.
[0031]
After completion of the culture, the cells collected by centrifugation at 10,000 rpm for 20 minutes were frozen at −80 ° C., and the surfactant B-PER was added thereto.^TM  Reagent (manufactured by PIERCE) 3 ml was added to lyse, and this was used as a crude enzyme solution.
To this crude enzyme solution, 4% dextran T-40 (manufactured by Pharmacia), 20 mM CaCl₂ 80 mM sodium acetate buffer (pH 5.5) was added to adjust the total volume to 40 ml, and the reaction was carried out with gentle shaking at 40 ° C. for 24-48 hours.
[0032]
Next, the reaction was terminated by heating at 100 ° C. for 10 minutes, followed by cooling and centrifugation at 8,000 rpm for 20 minutes to remove the precipitate.
HBDase was added to the centrifuged supernatant, and the reaction was carried out in 20 mM sodium acetate buffer (pH 5.5) at 30 ° C. for 24 hours to decompose all remaining linear oligosaccharides or dextran to glucose. Subsequently, after heating at 100 degreeC for 10 minutes, it cooled and centrifuged at 8,000 rpm for 20 minutes, and the deposit was removed.
[0033]
The centrifugal supernatant thus obtained was passed through a reverse phase column Sep-Pak C18 cartridge (manufactured by Waters) and washed with water to remove non-adsorbed linear oligosaccharide or dextran. Further, the fraction adsorbed on the column was eluted with 20% ethanol.
The fraction containing cyclodextran was flowed at a flow rate of 0.6 ml / min with 55% acetonitrile using a high performance liquid chromatograph PU-980 (manufactured by JASCO Corporation) and an Amide-80 column (4.6 mm × 25 cm) (manufactured by Tosoh Corporation). The eluate was fractionated into 0.6 ml fractions.
10 μL to 50 μL of each fraction was taken and again flowed at 55% acetonitrile using a high performance liquid chromatograph PU-980 (manufactured by JASCO Corporation) and Amide-80 column (4.6 mm × 25 cm) (manufactured by Tosoh Corporation). 1 ml / min and a spray evaporation light scattering detector SEDEX55 (manufactured by SIDERE) using analysis software BOWIN (manufactured by JASCO Corporation), and the target oligosaccharide (a, b, c, d, The fraction containing e, f) was analyzed.
Each of the fractions containing a, b, c, d, e, and f shown in FIG. 1 was lyophilized after removing acetonitrile with a centrifugal concentrator.
[0034]
The structure of the oligosaccharide thus obtained is¹³Analysis by C NMR. Specifically, to obtain a cyclic oligosaccharide solution having a concentration of 0.5 to 1.4 mg / 0.6 ml, heavy water (D₂ O) (manufactured by Aldrich) and 0.025% 3- (trimethylsilyl) -1-propanesulfonic acid (DSS) was added as an indicator, and completely decoupled at 298 K using a 600 MHz NMR apparatus (DRX600) (manufactured by Bruker). In the condition of the ring¹³C one-dimensional spectrum was measured.
The same conditions apply to CI-8 produced by the Bacillus circulans T-3040 strain, which has already been structurally analyzed as a target and has been clarified to have a structure in which eight glucoses are linked in a cyclic manner with α-1,6 bonds. so¹³Analysis by C NMR. As a result, as shown in FIG. 3, among the oligosaccharides produced by the four strains according to the present invention, b, c, d, e, and f are α-1,6-linked glucose exactly the same as CI-8. Only a signal indicating was shown. For peak a, the signal was disturbed because the yield was low and there was a lot of impurities, but it was thought that only the signal indicating α-1,6-linked glucose was the only signal.
[0035]
Next, mass spectrometry of the oligosaccharide obtained above was performed. 0.1 μg each of lyophilized preparations a, b, c, d, e, f, 5 μL of H₂ Measurement was performed by secondary ion mass spectrometry (SIMS) using HX-110A / 110A (manufactured by JEOL) using glycerol as a matrix. The measurement was performed in positive ion mode and negative ion mode. The result measured in positive ion mode is shown in FIG.
Cyclodextran is more H than linear isomaltooligosaccharides composed of the same glucose molecules.₂ The molecular weight of each cyclodextran is 1134, CI-8 is 1296, CI-8 is 1296, CI-9 is 1458, CI-10 is 1620, CI-11 is 1782, and CI-12 is 1944. Calculated.
The estimated molecular weights of oligosaccharides a, b, c, d, e, and f by mass spectrometry are a for CI-7, b for CI-8, c for CI-9, d for CI-10, e for CI-11, f matched the molecular weight of CI-12.
[0036]
Said¹³As a result of C NMR analysis and mass spectrometry, all of the oligosaccharides a, b, c, d, e, and f produced from dextran as a raw material by the Bacillus sp. 330K, 350K, 360K and 860K strains It was found that glucose is a cyclic isomaltooligosaccharide in which glucose is linked cyclically by α-1,6 bonds, that is, cyclodextran, and the molecular formula thereof is shown as follows.
a: C₄₂H₇₀O₃₅
b: C₄₈H₈₀O₄₀
c: C₅₄H₉₀O₄₅
d: C₆₀H₁₀₀ O₅₀
e: C₆₆H₁₁₀ O₅₅
f: C₇₂H₁₂₀ O₆₀
[0037]
The above four strains mainly produce cyclodextran of CI-10, CI-11 and CI-12 to which 10-12 molecules of glucose are bound, but in small amounts, CI-7, CI-8, CI- 9 will also be produced. Furthermore, as described above, there is a possibility that high molecular cyclic oligosaccharides such as CI-13 to CI-16 are produced after the peak f.
From the above results, it was revealed that the Bacillus sp. 330K strain, the 350K strain, the 360K strain and the 860K strain are novel strains of the genus Bacillus that mainly produce high molecular cyclodextran.
[0038]
Example 4
Next, the function of the polymer cyclodextran (CI) according to the present invention was examined. First, the anti-caries ability of CI was examined.
The high molecular weight cyclodextran CI-10 and CI-11 obtained in Example 3 were measured for the inhibitory effect on dextransclase derived from Leuconostoc mesenteroides. Specifically, in 20 μl of the reaction solution, CI was added to 20 mM acetate buffer (pH 5.2) containing 5% sucrose at a final concentration to a final concentration of 0 to 1 mM, dextransclase was added, and the enzyme was incubated at 30 ° C. for 10 minutes. After the reaction was performed, the amount of fructose produced by the decomposition of sucrose was measured as the amount of reducing sugar by the Nelson somology method. As the dextransclase derived from Leuconostoc mesenteroides used here, an enzyme obtained by culturing Leuconostoc mesenteroides NRRL B-512F strain in a medium to which sucrose was added was used.
FIG. 5 shows the results of a dextransclase inhibition test using CI-10, CI-11, and CI-8 as a control. As is clear from FIG. 5, CI-10 and CI-11, like CI-8, were found to have an anti-cariogenic property with an inhibition of about 50% at a concentration of 1 mM. In addition, it can be estimated from the structure of CI-12 or more that dextransclase activity is inhibited similarly to other CIs.
[0039]
Next, the inhibitory effect of CI-10 and CI-11 on the glucosyltransferase (GTF) derived from Streptococcus mutans was examined. That is, inhibition of GTF activity by CI is achieved by adding CI to 20 mM acetate buffer (pH 5.5) or 20 mM sodium phosphate buffer (pH 7.0) containing sucrose at a final concentration of 5% in 20 μl of reaction solution. After adding GTF and performing an enzyme reaction at 37 ° C. for 18 hours, the resulting insoluble glucan is collected by centrifugation, and the amount of the glucan is quantified by the phenol-sulfuric acid method. Was measured. The GTF derived from Streptococcus mutans used here was obtained by culturing Streptococcus mutans.
The results of the glucosyltransferase inhibition test with CI-10, CI-11 and CI-8 as a control are shown in FIG. As is clear from FIG. 6, CI-10 and CI-11 are also considered to have an anti-cariogenic property in the same manner as CI-8, with a decrease in the amount of glucan produced. In addition, about CI-12 or more, it can be presumed that there is GTF activity inhibition similarly to other CIs from its structure.
[0040]
Furthermore, the inclusion test with respect to Victoria Blue of CI-10, CI-11, and CI-12 was done. That is, in a reaction solution of 50 μl, a final concentration of 0.032 mM Victoria Blue B (manufactured by Wako), 100 mM sodium phosphate buffer (pH 7.0), 1.2 mM CI, cyclodextrin (CD) or glucose (Glc ), And the absorbance at 620 nm was measured while keeping at room temperature.
As a result, as shown in FIG. 7, CI-7, CI-8, CI-9, and CI-11, CI-12, and α-CD used as controls were not added to the reaction solution with or without glucose added. As in the case of the reaction solution, a decrease in absorbance at 620 nm was observed, and no remarkable Victoria Blue B inclusion ability was found. However, the reaction solution to which CI-10 was added showed an absorbance of about 50% after 22 hours from the start of the reaction. This is a higher value than β-CD and γ-CD, and CI-10 is considered to effectively include Victoria Blue.
[0041]
The reason why the inclusion ability of low molecular cyclodextran such as CI-7, CI-8, CI-9 is low is that the ring diameter is larger than that of cyclodextrins having the same number of glucose molecules, and the structure is shallow. It is considered that the power to hold the substance is weak.
On the other hand, as a reason why CI-10 having a large aperture has a high inclusion ability, it can be estimated that the molecule has become large and thus has a twisted structure. Since CI-11 and CI-12 can be presumed to form a kinked structure such as the figure 8, it is expected that a substance having an inclusion ability will be found in the future. That is, as described above, the four strains according to the present invention may produce high molecular CIs CI-13 to CI-16, and these substances can also be expected to have inclusion ability.
[0042]
The change in absorption spectrum was measured by adding Victoria Blue to CI-10, CI-11 and CI-12. That is, Victoria Blue B (manufactured by Wako Co., Ltd.) having a final concentration of 0.032 mM, 100 mM sodium phosphate buffer (pH 7.0), 1.2 mM CI, or CD or glucose was added to 50 μl of the reaction solution, and 6 at room temperature. After the time was maintained, the change in the absorption spectrum from 300 nm to 800 nm was measured using a spectrophotometer UV / VIS Spectrophotometer V-550 (manufactured by JASCO Corporation).
The result is shown in FIG. From this figure, the reaction solution added with CI-10 showing high clathrate ability for Victoria Blue shows a remarkable change in the shape of the absorption spectrum, confirming that CI-10 effectively claws Victoria Blue. Was thought to be.
[0043]
【The invention's effect】
The present invention provides a novel polymeric cyclodextran. This substance can be obtained by culturing the Bacillus sp. Strains 330K, 350K, 360K and 860K which are genus Bacillus.
Since these strains produce mainly cyclodextran having a higher molecular weight than conventional cyclodextran-producing strains, these strains are suitable for the production of high-molecular cyclodextran.
[0044]
Moreover, the polymer cyclodextran produced by the Bacillus strain of the present invention can be used as an anti-cariogenic agent. Among the polymer cyclodextran, CI-10 has inclusion ability and high water solubility, so that it can be applied to foods, pharmaceuticals, chemical products and the like. Since the compound that CI-10 is included in is also included in γ-CD, it is possible for CI-10 to effectively include substances known to be included in γ-CD. There is sex. In addition, since the high-molecular cyclodextran is very water-soluble compared to cyclodextrins, it can be expected that the solubilization effect by inclusion of insoluble substances is higher than that of cyclodextrins.
[0045]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is an HPLC analysis diagram showing oligosaccharides produced by (a) Bacillus sp. 330K strain, (b) 350K strain, (c) 360K strain and (d) 860K strain that are not degraded by HBDase. is there.
FIG. 2 shows molecular phylogenetic trees of (a) Bacillus sp. 330K strain, (b) 350K strain, (c) 360K strain, and (d) 860K strain.
FIG. 3 shows oligosaccharides a, b, c, d, e, f and known cyclodextran CI- produced by Bacillus sp. 330K strain, 350K strain, 360K strain, 360K strain and 860K strain that are not degraded by HBDase. About 8¹³It is a figure which shows the result of C NMR analysis.
FIG. 4 is a diagram showing the results of mass spectrometry of oligosaccharides a, b, c, d, e, and f that are not decomposed by HBDase produced by the Bacillus sp. 330K, 350K, 360K, and 860K strains. ((a) to (f)).
FIG. 5 is a graph comparing the inhibitory effect of high molecular weight cyclodextran CI-10 and CI-11 on dextransclase activity with known cyclodextran CI-8.
FIG. 6 is a diagram comparing the GTF inhibitory action of high-molecular cyclodextran CI-10 and CI-11 with known cyclodextran CI-8. (a) shows the results at pH 5.5, and (b) shows the results at pH 7.0.
FIG. 7: Cyclodextran CI-7, CI-8, CI-9 and α-CD, β-CD of known cyclodextran CI-7, CI-8, CI-12, and macromolecule cyclodextran CI-10, CI-11, CI-12 It is a figure compared with γ-CD and glucose.
FIG. 8 shows absorption spectra of a solution obtained by adding a polymer cyclodextran CI-10, CI-11 or CI-12 to Victoria Blue, and known cyclodextran CI-7, CI-8, CI-9 and α-CD. It is the figure compared with the case where (beta)-(beta) -CD, (gamma) -CD, and glucose are added.

Claims (7)

A polymer cyclodextran in which 10 to 12 glucose molecules form a cyclic structure by α-1,6 bonds. Bacillus sp. 330K strain (FERM P-19080), 350K strain (FERM P-19081), 360K strain (FERM P-19082) or 860K strain (FERM P-19083) is cultured in a medium containing dextran, The method for producing a polymer cyclodextran according to claim 1, wherein the polymer cyclodextran according to claim 1 is collected from the culture. Bacillus sp 330K strain (FERM P-19080), the 350K strain (FERM P-19081), and the same 360K strain (FERM P-19082) or the 860K strain (FERM P-19083) were cultured in medium containing dextran The method for producing a polymer cyclodextran according to claim 1, wherein a product obtained by lysing the obtained microorganism is reacted with dextran, and the polymer cyclodextran according to claim 1 is collected from the reaction product.   Bacillus sp. 330K strain (FERM P-19080), 350K strain (FERM P-19081), 360K strain (FERM P-19082) belonging to the genus Bacillus and having the ability to produce the high-molecular cyclodextran according to claim 1. ) Or 860K strain (FERM P-19083).   An anti-cariogenic agent comprising the polymeric cyclodextran according to claim 1 as an active ingredient.   A water-soluble cyclic oligosaccharide composition having an inclusion ability containing, as an active ingredient, a high-molecular cyclodextran in which 10 molecules of glucose form a cyclic structure by α-1,6 bonds.   A food or drink comprising the polymeric cyclodextran according to claim 1 as an active ingredient.

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