JP4395243B2 - Polygalacturonase - Google Patents

Description

【００３４】
（１２）界面活性剤の影響
各種界面活性剤を０．２％（ｗ／ｖ）になるように添加した反応系において、酵素の反応性を調べた結果、高濃度の界面活性剤の存在下においても本酵素は、対象に比べ８０％以上の活性を発現した（表２）。
【００３５】
【表２】

【００３６】
（１３）金属塩の影響
各種金属塩を標準反応系に１mM添加し、酵素活性に与える影響を調べた結果、本酵素は塩化カルシウム、塩化マグネシウム、塩化マンガン、塩化第１鉄、塩化第２鉄により、対照に比べ１１６〜１３５％と活性化された。一方、塩化亜鉛、塩化銅により約９５％、８５％とそれぞれ阻害を受けた。次いで塩化ニッケル、塩化コバルトによって約７０％、６０％の阻害を受けた（表３）。
【００３７】
【表３】

【００３８】
上述したポリガラクツロナーゼ産生菌、例えばBacillus属に属する微生物、好ましくはBacillus sp. KSM-P358株(FERM P-17560)等から、例えばショットガン法、ＰＣＲ法で目的とする遺伝子をクローニングする方法等により、本発明のポリガラクツロナーゼをコードする遺伝子をクローニングすることができる。
【００３９】
本発明のポリガラクツロナーゼ遺伝子は、例えば、配列番号１に示すアミノ酸配列、又は該アミノ酸配列の１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列をコードするものが好ましい。ここで、該アミノ酸配列中のアミノ酸の欠失、置換又は付加（以下、変異ということがある）は、ポリガラクツロナーゼ活性を失わない限り、その変異アミノ酸の数及び部位は制限されるものではない。また、配列番号１のアミノ酸配列におけるＮ末端には、１〜数個のアミノ酸が付加又は欠失していてもよい。
【００４０】
配列番号１に示すアミノ酸番号１〜９５３の間で、他の酵素との相同性を検討すると、いずれもエキソ型のポリガラクツロナーゼであるErwinia chrysanthemi PehX(He and Collmer, J. Bacteriol., 172, 4988 4995, 1990)、Ralstonia Solanacearum PehB(Huahg and Allen, J. Bacteriol., 179, 7369-7378, 1997)とそれぞれ２９．１％、２８．５％の相同性を示すのみであり、配列番号１に示すアミノ酸からなるポリガラクツロナーゼは、これまでに報告のない新規な配列を有する酵素である。
【００４１】
本発明ポリガラクツロナーゼ遺伝子は、上記のように配列番号１のアミノ酸配列又はその前記変異体をコードするものが好ましいが、特に配列番号２で示される塩基配列又は該塩基配列の１若しくは数個の塩基が欠失、置換若しくは付加された塩基配列を有するものが好ましい。
【００４２】
ポリガラクツロナーゼ遺伝子を含む組換えベクターを作製するには、目的とする宿主内で遺伝子を発現するのに適した任意のベクターにポリガラクツロナーゼ遺伝子を組込めばよい。斯かるベクターとしては、大腸菌を宿主とする場合、ｐＵＣ１８、ｐＵＣ１９、ｐＢＲ３２２等が挙げられ、枯草菌を宿主とする場合、ｐＵＢ１１０等が挙げられる。
【００４３】
かくして得られた組換えベクターを用いて宿主を形質転換するには、常法、例えばプロトプラス法、コンピテントセル法、エレクトロポレーション法等により行われる。宿主としては微生物が好ましく、Bacillus属（枯草菌）、Streptomyces属（放線菌）等のグラム陽性菌；Escherichia coli（大腸菌）等のグラム陰性菌；Saccharomyces属（酵母）、Aspergillus属（カビ）等の真菌等が挙げられる。
【００４４】
得られた形質転換体を培養し、当該培養液からポリガラクツロナーゼを採取すれば、ポリガラクツロナーゼを得ることができる。培養は微生物の資化可能な炭素源、窒素源その他の必須栄養素を含む培地に接種し、常法に従って行えばよい。かくして得られた培養液から、一般に知られている酵素の採取及び精製方法に準じた方法により、ポリガラクツロナーゼを得ることができる。
【００４５】
【実施例】
実施例１ ポリガラクツロナーゼ生産菌のスクリーニング
日本各地の土壌を滅菌水に懸濁したものを８０℃、２０分間熱処理し、下記組成の寒天平板培地に塗布した。３０℃の培養器で３〜５日間静置培養し、菌の生育後、冷蔵庫で冷却した。生育した菌の周辺にペクチンの分解に伴う溶解斑が検出されたものについて選抜し、シングルコロニー化を繰り返し、ポリガラクツロン酸分解酵素の生産能を検定した。このようにして得られた多くの菌株は、主にペクチン酸リアーゼを生産したが、その中でポリガラクツロナーゼ生産菌としてBacillus sp. KSM-P358株を得た。
【００４６】
【表４】

【００４７】
実施例２ Bacillus sp. KSM-P358株によるポリガラクツロナーゼの生産上述のスクリーニングにより得られたBacillus sp. KSM-P358株の培養は、５００mL容坂口フラスコに５０mLの培地を加え、３０℃、２日間好気的に行った。培地組成は、０．５％（ｗ／ｖ）ペクチン、１．５％ポリペプトンＳ、０．５％酵母エキス、１．０％魚肉エキス、０．１％リン酸２水素カリウム、０．０２％硫酸マグネシウム７水塩、５０mMトリス−塩酸緩衝液（pH８．６、別滅菌）であった。培養液中に生産されるポリガラクツロナーゼ活性は、２mMＥＤＴＡを添加しペクチン酸リアーゼ活性を完全に失活させて測定を行った。この測定法により、培養液あたり２００〜４００Ｕ／Ｌの生産量を得た。
【００４８】
実施例３ ポリガラクツロナーゼの精製
Bacillus sp. KSM-P358株の培養液を遠心分離（８０００×ｇ、１５分間、４℃）し上清液（２Ｌ）を得た。これを透析膜に入れポリエチレングリコール２００００（和光純薬）をまぶして内液の濃縮を行った。更に限外濾過用モジュール（ＡＩＰ１０１０：旭化成）により濃縮、脱塩を行った。得られた濃縮液（１００mL）は１mMジチオスレイトールを含む２０mMトリス−塩酸緩衝液（pH７．０）にて平衡化しておいたＤＥＡＥトヨパール６５０Ｍカラム（３×１５cm：東ソー）に添着した。約５００mLの平衡化緩衝液を用いて非吸着タンパク質を洗浄溶出させた後、０から０．２Ｍ塩化ナトリウムを含む緩衝液（５００mLずつ）を用い、濃度勾配溶出法により吸着タンパク質の溶出を行った。約０．１Ｍの塩化ナトリウム濃度付近にポリガラクツロナーゼ活性が溶出された。次いで、ＤＥＡＥトヨパール６５０Ｍカラムで同様の精製操作を行い、ペクチン酸リアーゼ活性を含まないポリガラクツロナーゼ画分を得た（１００mL）。本活性画分を限外濾過（ＹＭ１０メンブレン：アミコン）により濃縮し、０．１Ｍ塩化ナトリウム及び１mMジチオスレイトールを含む２０mMトリス−塩酸緩衝液（pH７．０）にて平衡化しておいたトヨパールＨＷ５５カラム（１．５×６５cm：東ソー）へ供した。同緩衝液にて溶出されたポリガラクツロナーゼ活性画分を集めた（１０mL、４２Ｕ、１４mgタンパク質）。
上記精製操作により得られたポリガラクツロナーゼ画分は、前述の酵素学的性質を示した。
【００４９】
実施例４ Bacillus sp. KSM-P358株染色体ＤＮＡの調製
Bacillus sp. KSM-P358株をポリペプトンＳ（日本製薬製）主成分とする液体培地にて、３０℃で一晩振盪培養し、種培養とした。この種培養液を同様の培地に接種し、約８時間振盪培養したものを主培養とした。主培養液から遠心分離にて回収した菌体から、SaitoとMiuraの方法（Biochim. Biophys. Acta, 72, 619-629, 1963）にて染色体ＤＮＡを調製した。
【００５０】
実施例５ Bacillus sp. KSM-P358ポリガラクツロナーゼの部分アミノ酸配列
１）Ｎ末端アミノ酸配列の決定
Bacillus sp. KSM-P358株の培養液から各種クロマトグラフィーによって精製した酵素を、プロテインシークエンサー476A（PEアプライドバイオシステム社製）に供し、Ｎ末端アミノ酸配列を９残基決定した。得られた配列は、Lys-Ser-Glu-Gly-(Pro or Ser or Ala)-Pro-Asn-Ala-Proであった。
【００５１】
２）リジルエンドペプチダーゼ分解による中間アミノ酸配列の決定
精製酵素のＳＤＳ−ＰＡＧＥ（ソディウムドデシル硫酸−ポリアクリルアミドゲル電気泳動）を行った１２．５％のポリアクリルアミドゲルから回収した酵素に対して、重量比で約１０００分の１のリジルエンドペプチダーゼ（和光純薬工業製）を添加し、６０mMのトリス−塩酸緩衝液（pH９）中にて２５℃で１５時間恒温した。UL TRAFREE MCスピンカラム(0.45μｍＰＶＤＦ膜、ミリポア社製)にて回収した試料をＳＤＳ−ＰＡＧＥに供した後、Milliblot type I(ミリポア社製)を用いてＰＶＤＦ膜ヘエレクトロプロティングし、転写後のＰＶＤＦ膜をクマシーブリリアントブルーにて染色した。染色後のＰＶＤＦ膜から染色されたタンパク質バンドを切り出し、プロテインシークエンサー476Aに供した。リジルエンドペプチダーゼ部分分解物から得られたアミノ酸配列(中間アミノ酸配列Ａ)は、Asn-Ile-Leu-Phe-Arg-Asn-Asn-Ala-Leu-Glu-Glyであった。
【００５２】
３）Ｖ８プロテアーゼ分解による中間アミノ酸配列の決定
精製試料に０．０１％となるようにＳＤＳを加え１００℃にて１０分間煮沸したものに、重量比で約６０分の１のＶ８プロテアーゼ(ベーリンガーマンハイム社製)を加え、110mMのトリス−塩酸緩衝液(pH8.5)中にて、１０℃で１５時間恒温した。回収した試料をＳＤＳ−ＰＡＧＥに供し、エレクトロプロティング後のＰＶＤＦ膜からタンパク質バンドを切り出し、プロテインシークエンサー476Aに供した。Ｖ８プロテアーゼ部分分解物から得られたアミノ酸配列(中間アミノ酸配列Ｂ)は、Phe-Glu-(Thr or Asn or Arg)-Ala-Arg-Pro-Tyr-(Gly or Pro)-(Ala or Ile)-Gly-Ala-Proであった。
【００５３】
実施例６ Ｐ３５８ポリガラクツロナーゼ遺伝子のクローニング
精製酵素のＮ末端アミノ酸配列から配列番号３に示した混合塩基を用いたプライマーを作製した。同様に、リジルエンドペクチダーゼ部分分解物から得られた中間アミノ酸配列Ａをもとに、配列番号４、５に示したプライマー、Ｖ８プロテアーゼ部分分解物のアミノ酸配列Ｂから配列番号６に示したプライマーを作製した。
これらのプライマーを使用し、実施例４で調製したBacillus sp. KSM-P358株染色体ＤＮＡを鋳型とするＰＣＲを実施した。配列番号３、４のプライマーの組合せと配列番号５、６のプライマーの組合せにて、それぞれ約１．３kbp、１．１kbpの増幅断片が得られ、塩基配列の解析の結果、推定されるアミノ酸配列がそれぞれのアミノ酸配列と一致したことから、ＰＣＲ増幅断片は目的とするKSM-P358ポリガラクツロナーゼ遺伝子の一部であることが判明した。
これらＰＣＲ増幅断片の塩基配列をもとに配列番号７、８に示すプライマーを作製し、これらのプライマーと染色体ＤＮＡを制限酵素EcoR Iで切断したのちに、T4 DNA Ligaseにて自己閉環させたものを鋳型ＤＮＡとして、inverse ＰＣＲを実施した。このようにして得られた約３．０kbpの増幅断片の塩基配列の解析を行い、決定した塩基配列から配列番号９、１０に示すプライマーを作製し、これらとBacillus sp. KSM-P358株染色体ＤＮＡを鋳型とするＰＣＲを行い、全長約４．５kbpのP358ポリガラクツロナーゼ遺伝子断片を取得した。
【００５４】
実施例７ Ｐ３５８ポリガラクツロナーゼ遺伝子塩基配列の決定
実施例６で調製したＰＣＲ増幅断片０．５〜１μｇを鋳型として、BigDye Terminator Cycle Sequencing Kit(PEアプライドバイオシステム社製)を用いてシークエンス解析試料を調製し、３７７ＤＮＡシークエンサー(PEアプライドバイオシステム社製)にてＰ３５８ポリガラクツロナーゼ遺伝子の塩基配列を決定した（配列番号１３）。その結果、Ｐ３５８ポリガラクツロナーゼは配列番号１３の塩基番号５４０〜３４７９に示すように２９４０残基の塩基配列によりコードされ、９８０アミノ酸残基より構成される酵素であることが判明した。
さらに、アミノ酸配列中には、精製酵素から決定したＮ末端アミノ酸配列とプロテアーゼ部分分解物から決定した中間アミノ酸配列が確認された。Ｎ末端アミノ酸配列から終止コドン(TAA)までの９５３アミノ酸からなるポリガラクツロナーゼの分子量は、１０３，８１０ Ｄａと推定された。
【００５５】
実施例８ Ｐ３５８ポリガラクツロナーゼ発現プラスミドの作製
実施例７で決定したＰ３５８ポリガラクツロナーゼ遺伝子の塩基配列を基に、配列番号１１、１２に示す配列のプライマーを作製し、実施例４にて調製したBacillus sp. KSM-P358株染色体ＤＮＡを鋳型としてＰＣＲを実施し、Ｐ３５８ポリガラクツロナーゼをコードするＤＮＡ断片を増幅した。
この際、使用するプライマーにはプラスミドベクターpHY300PLK(ヤクルト社製)のマルチクローニングサイトに効率よくクローニングすることが可能となるように、制限酵素SalI、XbaIの認識配列を付加させた。
プラスミドベクターpHY300PLK及びP358ポリガラクツロナーゼ遺伝子のＰＣＲ増幅断片を制限酵素SalI、XbaIにて消化した後、GFX PCR DNA and Gel Band Purification Kit(ファルマシア社製)を用いた精製を行った。
精製試料を重量比で１：１となるように混合した後、T4 DNA Ligaseにより結合させ、この結合サンプルを用いて、E. coli HB101コンピテントセル(宝酒造社製)の形質転換を行った。得られた形質転換体の中から任意の形質転換体を選別し、選別した形質転換体からプラスミドの調製を行い、このプラスミドを制限酵素SalI、XbaIにて消化した。アガロース電気泳動にてＰＣＲ増幅断片の挿入が確認されたプラスミドをpHYK358PECとした（図６）。
【００５６】
実施例９ pHYK358PECを用いた枯草菌形質転換体によるポリガラクツロナーゼの生産
プラスミドpHYK358PECを用い、プロトプラスト法(Chang and Choen. Mol. Gen. Genet., 168, 111-115, 1978)にてBacillus subtilis ISW1214株(ヤクルト社製)の形質転換を行った。得られた形質転換体を、１５μｇ／mLのテトラサイクリンを添加したLB培地に接種し、３０℃で一晩振盪培養を行い種培養とした。この種培養液をポリペプトンＳあるいはコーンスティープリカー、マルトースを主成分とする主培養培地４０mLに１％（v/v)になるように接種し、３０℃、１２０rpmで４日間、振盪培養を行った。組換えポリガラクツロナーゼの生産量は、この培養条件で約１０００〜３０００Ｕ／Ｌであった。
【００５７】
実施例１０ 組換えポリガラクツロナーゼの特性
実施例９で得られた培養液上清を陰イオン交換樹脂に吸着させ、塩化ナトリウム濃度勾配にて溶出させて精製し、この精製した組換え酵素の性質について検討した結果、以下に示すように、その性質は野生型のP358ポリガラクツロナーゼの性質とよく一致した。
【００５８】
１）最適反応ｐＨ
標準酵素活性測定法のかわりに、５０mMトリス−塩酸緩衝液(pH7〜9.5)、５０mMグリシリン−水酸化ナトリウム緩衝液(pH9〜11)を用い、最適反応pHを調べたところ、本酵素はトリス-塩酸緩衝液中(pH8.0)で最も高い反応速度を示した。
【００５９】
２）分子量
電気泳動用標準タンパク質(バイオラッド社製)を用いてＳＤＳ−ＰＡＧＥ(12.5％アクリルアミドゲル)にて分子量を求めた結果、本酵素の分子量は約１０５kDaであると推定された。
【００６０】
３）プロトペクチナーゼ活性
３０cmしつけ糸(金鈴印)１本(25mg)を基質に用い、０．４mM塩化カルシウムを含む５０mMグリシン−水酸化ナトリウム緩衝液(pH10)を酵素反応液として、これに０．２Uの酵素(ポリガラクツロナーゼ活性)を加え最終液量を２．０mLとした。３０℃で１時間の反応を行った後、上清に遊離したペクチン質をオルシノール塩酸法により測定した。結果、上記条件において０．２Uのポリガラクツロナーゼあたり約４０μｇのペクチンを遊離することが認められた。
【００６１】
【発明の効果】
本発明のポリガラクツロナーゼは、ポリガラクツロン酸に対しエキソ的に作用するが最適反応pHをpH８付近に有し、アルカリ性領域においても活性を維持すること、またプロトペクチナーゼ活性を示すことから衣料用洗剤酵素、繊維処理用酵素として有用である。また、本発明のポリガラクツロナーゼ遺伝子を用いることにより、当該ポリガラクツロナーゼを単一且つ大量に生産することが可能となる。
【００６２】
【配列表】

【００６３】

【００６４】

【００６５】

【００６６】

【００６７】

【００６８】

【００６９】

【００７０】

【００７１】

【００７２】

【００７３】

【００７４】

【図面の簡単な説明】
【図１】本発明のポリガラクツロナーゼ活性に及ぼすpHの影響を示す図である。
【図２】本発明のプロトペクチナーゼ活性に及ぼすpHの影響を示す図である。
【図３】本発明のポリガラクツロナーゼ活性に及ぼす温度の影響を示す図である。
【図４】本発明のポリガラクツロナーゼ安定性に及ぼすpHの影響を示す図である。
【図５】本発明のポリガラクツロナーゼ安定性に及ぼす温度の影響を示す図である。
【図６】ポリガラクツロナーゼ遺伝子(Bacillus sp.KSM-P358株由来)のプラスミドベクター(pHY300PLK)への導入と構築したポリガラクツロナーゼ発現分泌ベクターpHYK358PECを示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a polygalacturonase useful as a cleaning agent, a fiber treatment agent and the like, and a gene encoding the same.
[0002]
[Prior art and problems to be solved by the invention]
Polygalacturonase is generally called pectinase, and is a hydrolase that degrades polygalacturonic acid (pectinic acid) and pectin. Such polygalacturonase has been used mainly as an enzyme for the food industry because it has an optimum reaction pH of acid-weak acidity. Recently, it has been used as a detergent enzyme for clothing. (JP-A-60-226599, JP-B-6-39596, WO98 / 06809, etc.).
[0003]
When polygalacturonase is used in a laundry detergent, it is necessary that the enzyme acts in the alkaline region and is stable against detergent components such as surfactants and chelating agents. In addition, since the pectic substance on cotton fibers exists as protopectin, polygalacturonase having protopectinase activity that acts on insoluble protopectin to release pectin in the alkaline region is necessary.
[0004]
Conventionally, as an alkali-optimal polygalacturonase,Bacillus An endo-type polygalacturonase produced by sp. P-4-N strain (Japanese Examined Patent Publication No. 48-6557) has been reported. Such an enzyme requires a calcium ion in the reaction and has a low chelate resistance. Therefore, application to detergent is difficult. Recently, utilization of pectate lyase having protopectinase activity as a detergent is also considered (Japanese Patent Laid-Open No. 11-140589).
However, an enzyme having a protopectinase activity, which is a polygalacturonase acting in the alkaline region, has never been known so far.
[0005]
An object of the present invention is to provide a polygalacturonase that is stable to detergent components such as surfactants and chelating agents, acts in an alkaline region and has protopectinase activity, a gene encoding the same, and a gene engineering technique for the polygalacturonase. The object is to provide a method for efficiently mass-producing galacturonase.
[0006]
[Means for Solving the Problems]
The present inventors, among enzymes produced by microorganisms in the soil, act in an alkaline region, are stable to detergent components such as surfactants and chelating agent resistance, and have polyprotolactinase activity In addition, the inventors have succeeded in cloning a gene encoding the polygalacturonase and establishing a production technique by gene recombination.
[0007]
That is, the present invention provides a polygalacturonase having a protopectinase activity of 30% or more of the maximum protopectinase activity at least at pH 8 to 11, and a microorganism producing the polygalacturonase.
[0008]
Furthermore, the present invention provides a gene encoding the polygalacturonase, a recombinant vector containing the gene, a transformant containing the recombinant vector, and a method for producing polygalacturonase using these. is there.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The polygalacturonase of the present invention has protopectinase activity, that is, protopectin degradation activity at least at pH 8-11. The protopectin-degrading activity is that which decomposes and solubilizes the polygalacturonic acid portion of protopectin (A type).
The protopectinase activity was determined by using a damped yarn (substrate) as a buffer solution (Mccle Bain buffer (pH 5 to 7), Tris-HCl buffer (pH 7 to 9) and glycine-sodium hydroxide (pH 9 to 11), After putting into a reaction solution containing calcium chloride and polygalacturonase and reacting for a predetermined temperature and time, the pectin liberated in the supernatant was measured by the amount of produced pectin obtained by measuring by the orcinol hydrochloric acid method. The ratio of the amount of produced pectin at each pH to the amount of produced pectin at the optimum reaction pH, ie, the relative ratio to the maximum protopectinase activity was evaluated.
[0010]
In addition, it has polygalacturonase activity, that is, polygalacturonic acid hydrolysis activity even in the alkaline region. The polygalacturonase activity is exo and produces digalacturonic acid. Thus, the polygalacturonase of the present invention acts on both protopectin, which is an insoluble natural pectin, and polygalacturonic acid, which is a part thereof, in the alkaline region to produce digalacturonic acid. It is effective in removing dirt adhering to protopectin and food spills and stains of foods with high insoluble pectin content such as ketchup and jam.
[0011]
The polygalacturonase of the present invention is further stable against surfactants and chelating agents. For example, an activity of 80% or more is retained in a liquid containing 0.2% sodium dodecyl sulfate. In addition, it retains 80% activity in a solution containing 2 mM EDTA.
[0012]
The polygalacturonase of the present invention preferably further has the following enzymological properties.
(1) Action: Acts on polygalacturonic acid (pectinic acid), pectin and protopectin, and exohydrolyzes α-1,4 bonds of polygalacturonic acid to produce digalacturonic acid.
(2) Optimum reaction pH: around pH 8 (Tris-HCl buffer)
(3) Optimum reaction temperature: about 50 ° C. (Tris-HCl buffer, pH 8.0)
(4) pH stability: pH 7 to 11.5 (30 ° C., 30 minutes treatment)
(5) Heat resistance: stable up to about 50 ° C. (Tris-HCl buffer, pH 8.0, treated for 30 minutes)
(6) Molecular weight: about 105000 (gel filtration method)
(7) Isoelectric point: around pH 4.6 (isoelectric focusing method)
[0013]
The polygalacturonase of the present invention is produced, for example, by culturing a polygalacturonase-producing bacterium having protopectinase activity and collecting it from the culture.
[0014]
As the fungus producing the polygalacturonase of the present invention,BacillusExamples include bacteria belonging to the genus, such as the KSM-P358 strain. The KSM-P358 strain has the following mycological properties.
[0015]
A Morphological properties
(A) Shape and size of cells: Neisseria gonorrhoeae (0.6 to 0.8 × 2.4 to 3.2 μm)
(B) Polymorphism: None
(C) Mobility: Yes
(D) Spore (size, shape, position, presence / absence of swelling): elliptical, 0.6-0.8 × 1.2-1.6 μm, quasi-end, with swelling
(E) Gram staining: indefinite (does not grow on CVT agar medium)
(F) Antiacid: negative
(G) Growth on a broth agar medium: white, all-round colonies formed
[0016]
B Physiological properties
(A) Reduction of nitrate: +
(B) Denitrification reaction: −
(C) MR test:-
(D) VP test:-
(E) Indole production: −
(F) Production of hydrogen sulfide: −
(G) Starch hydrolysis:-
(H) Gelatin hydrolysis: +
(I) Casein hydrolysis: +
(J) Use of citric acid:-
(K) Use of inorganic nitrogen: −
(L) Urease: +
(M) Oxidase: +
(N) Catalase: +
(O) Litmus milk: no change in color
(P) Growth temperature range: 18-41 ° C
(Q) Growth pH range: pH 7-9
(R) Growth under anaerobic conditions: no growth
(S) OF test:-
(T) Gas production from glucose: −
(U) Resistance to sodium chloride: 2% does not grow
(V) Acid production from sugars: Acid production from the following sugars was not observed. Galactose, xylose, arabinose, sucrose, glucose, mannitol, mannose, inositol, sorbitol, trehalose, lactose, glycerin, maltose, fructose, raffinose, melibiose, soluble starch
[0017]
As a result of comparative examination according to the description of “Bergey's Manual of Systematic Bacteriology” (Williams & Wilkins, 1984) about the morphology and physiological properties of KSM-P358 strain,Bacillus brevisIt was considered to be a close relative of the species. But its nature is knownBacillus brevisDoes not match otherBacillusBecause it does not match the properties of the genus bacteria,BacillusAs a genus bacterium, this strain was transferred to the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.Bacillus sp. KSM-P358; FERM P-17560).
[0018]
In order to produce the polygalacturonase of the present invention using a polygalacturonase producing bacterium such as KSM-P358 strain, the strain is inoculated into a medium containing an anabolic carbon source, a nitrogen source, and other essential nutrients, What is necessary is just to culture by shaking or aeration stirring according to a conventional method. The pH of the medium is preferably adjusted to 7-9.
[0019]
The collection and purification of polygalacturonase from the thus obtained culture can be performed according to a general method. That is, the cells can be removed from the culture by centrifugation or filtration, and the target enzyme can be concentrated from the obtained culture supernatant by conventional means. The enzyme solution or dry powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method.
[0020]
Bacillus The polygalacturonase derived from sp. KSM-P358 has the following enzymatic properties.
[0021]
The polygalacturonase activity was measured as follows.
[Standard enzyme activity assay]
In a test tube, 0.2 mL of 0.5 M Tris-HCl buffer (pH 8.0), 0.2 mL of 1% (w / v) polygalacturonic acid (ICN biomedical; lot 14482, pH 6.0 with sodium hydroxide solution). 8), 0.5 mL of deionized water was added, and the temperature was kept constant at 30 ° C. for 5 minutes. To this was added 0.1 mL of an appropriately diluted enzyme solution (dilution was performed with deionized water), allowed to react for 20 minutes, 1 mL of dinitrosalicylic acid reagent was added, and coloring of reducing sugar was performed in boiling water for 5 minutes. went. After quenching in ice water, 4 mL of deionized water was added and the absorbance at 535 nm was measured to determine the amount of reducing sugar produced. In addition, after adding a dinitrosalicylic acid reagent to the reaction liquid processed without adding an enzyme liquid, the blank added the enzyme liquid and prepared the same color. One unit (1 U) of enzyme was defined as an amount that produces 1 μmol of reducing sugar equivalent to D-galacturonic acid per minute under the above reaction conditions.
[0022]
(1) Substrate specificity
The reaction rate was examined by a standard activity measurement method using pectin (28, 67, 93%) having a different esterification degree as a substrate instead of polygalacturonic acid. Pectins with 28% esterification (Sigma; lot74H1092) decomposed at a reaction rate of about 50% for pectin with 70% esterification (Sigma; lot74H1093), but with an esterification degree of 93%. No degradation activity was observed for pectin (Sigma; lot25H0123) under these conditions.
Next, one 30 cm basting thread (gold bell) (about 25 mg) was used as a substrate, and a commercial garment detergent solution [0.067% (w / v)] and enzyme (0.2 U) were added at 30 ° C. The reaction was performed for 1 hour. The reaction solution (2 mL) was centrifuged, and pectin released into the supernatant was measured by the orcinol hydrochloric acid method. As a result, about 45 μg of pectin release was observed in the reaction solution. It acted on protopectin on the surface of cotton fibers and had protopectinase activity belonging to type A.
[0023]
(2) Substrate decomposition mode
0.05 U of enzyme was added to a reaction solution consisting of 50 mM Tris-HCl buffer (pH 7.5), 0.2% polygalacturonic acid, and 0.2 mM calcium chloride to make a total volume of 0.25 mL. About 10 μL of the solution reacted at 30 ° C. for 30 minutes was spotted on a thin-layer chromatographic plate (kiesel gel 60: Merck), and in a solvent system of n-butanol: acetic acid: water = 5: 2: 3 (v / v). Deployed. Anisaldehyde-sulfuric acid solution was used for detection of the reaction product. After spraying on the plate, the color was developed in a dryer at 100 ° C. for 10 minutes. As a result, only digalacturonic acid was detected as a reaction product, and the enzyme was an exo-type polygalacturonase.
In addition, 2 U of enzyme was added to a reaction solution consisting of 2 g of damped yarn, 0.067% commercial laundry detergent, 10 mM glycine-sodium hydroxide buffer (pH 10.5) to make a total volume of 20 mL. The reaction was allowed for 8 hours. Sampling was performed over time and thin layer chromatography was performed by the above method. As a result, monogalacturonic acid and digalacturon were detected 1 hour after the reaction. For this reason, it also acted on the exo-type against protopectin.
[0024]
(3) Optimum reaction pH for polygalacturonase activity
The optimum reaction pH was examined using each buffer solution (100 mM) of McKlvain buffer (pH 5-7), Tris-hydrochloric acid buffer (pH 7-9), and glycine-sodium hydroxide buffer (pH 9-11). As a result, this enzyme showed the highest reaction rate in Tris-HCl buffer at pH 8.0, and showed an activity of 50% or more of the maximum activity in a wide range of pH 5-9 (FIG. 1).
[0025]
(4) Optimum reaction pH for protopectinase activity
Using a 30 cm braid (gold bell mark) (25 mg) as a substrate, Mr. Mcclebain buffer (pH 5-7), Tris-HCl buffer (pH 7-9), glycine-sodium hydroxide buffer (pH 9-11) Polygalacturonase (0.2 U) was added to each buffer solution (50 mM) and a reaction solution (2 mL) containing 0.4 mM calcium chloride, reacted at 30 ° C. for 1 hour, and released into the supernatant. Pectin was measured by the orcinol hydrochloric acid method. As a result, this enzyme showed the highest reaction rate in Tris-HCl buffer at pH 8.0. Moreover, the activity of 30% or more of the maximum activity was exhibited in a wide range of pH 5 to 11 (FIG. 2).
[0026]
(5) Optimal reaction temperature
The enzyme reaction was carried out at a temperature of 10 ° C. to 80 ° C. in 100 mM Tris-HCl buffer (pH 8.0), and the optimum reaction temperature was examined. The activity was 50% or more of the maximum activity in the range of -60 ° C. Moreover, when 1 mM calcium chloride was added to the reaction system, the optimum reaction temperature shifted to around 55 ° C. and the reaction rate on the high temperature side was increased. That is, the activity was 50% or more of the maximum activity in the range of 40 ° C to 70 ° C (Fig. 3).
[0027]
(6) Stable pH range
In each buffer solution (50 mM) of McKlvain buffer (pH 2 to 8), glycine-sodium hydroxide buffer (pH 7 to 11.5), potassium chloride-sodium hydroxide buffer (pH 10 to 12.5) The enzyme was added and incubated at 30 ° C. for 30 minutes, and the residual activity was measured. As a result, the residual enzyme activity in glycine-sodium hydroxide buffer (pH 9.5) was 100%. The remaining activity was 80% or more in the range of 11.5 (FIG. 4). Note that the addition of 1 mM calcium chloride in each treatment did not affect the increase in enzyme stability.
[0028]
(7) Heat resistance
This enzyme was added to 50 mM Tris-HCl buffer (pH 8.0), and the residual activity was measured after incubation at 5 ° C. to 80 ° C. for 30 minutes. This enzyme was very stable up to 50 ° C. under these conditions, and rapidly deactivated at 60 ° C. or higher (FIG. 5). It should be noted that the addition of 1 mM calcium chloride at each temperature did not affect the enzyme stability.
[0029]
(8) Molecular weight
a. Gel filtration method: The present enzyme was placed on a Toyopearl HW55 column (1.5 × 65 cm) equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 100 mM sodium chloride, and eluted at a flow rate of about 36 mL / h. It was. Catalase (232000), aldolase (158000), bovine serum albumin (67000), and ovalbumin (43000) were used as standard proteins, and a calibration curve was prepared from the respective eluate amounts and molecular weights to determine the molecular weight of the enzyme. It was estimated to be about 105000. b. SDS-polyacrylamide electrophoresis: Enzyme activity fractions eluted by gel filtration were concentrated and subjected to SDS-electrophoresis using 7.5% acrylamide gel. Using myosin (200000), β-galactosidase (116200), phosphorylase (97400) and bovine serum albumin (67000) as standard proteins, a calibration curve was prepared from the respective mobility and molecular weight, and the molecular weight of the enzyme was determined. About 103,000.
[0030]
(9) Isoelectric point
The enzyme was subjected to isoelectric focusing using PAG-Plate (Pharmacia; pH 3.5 to 9.5). After the electrophoresed gel was immersed in 10 mM phosphate buffer (pH 7.0), an agar plate containing polygalacturonic acid [1.0% polygalacturonic acid, 0.1% potassium monohydrogen phosphate, 1.0% Sodium chloride, 50 mM Tris-HCl buffer (pH 7.5, 1.0% agar)]. After incubating at 37 ° C. for 3 hours, the gel was removed and 1% cetyltrimethylammonium bromide solution was poured. The isoelectric point of the enzyme is around pH 4.6 according to the calibration curve obtained from the protein mobility of the part where dissolution spots with activity occurred after about 10 minutes and the isoelectric point and mobility of the standard protein (BioRad). Met.
[0031]
(10) Amino terminal sequence
After SDS-electrophoresis of this enzyme, the protein was blotted onto an immobilon membrane (Millipore) and stained with a Coomassie brilliant blue solution. The protein band portion having a molecular weight of around 103000 was cut out and cut, and then the amino terminal sequence was determined using a protein sequencer (476A type: Applied Biosystem). As a result, the amino terminal sequence of the enzyme up to the ninth position was Lys-Ser-Glu-Gly- (Pro or Ser or Ala) -Pro-Asn-Ala-Pro.
[0032]
(11) Effects of various compounds
The effect of various compounds on the activity of this enzyme was determined by adding each compound to the reaction system to a predetermined concentration and measuring the activity. As a result, this enzyme was found to be p-chloromercuribenzoic acid (0.5 mM). ) About 60% and citric acid (5 mM) about 35%. EDTA (2, 5 mM) was inhibited by about 20% and 75%, and EGTA (5 mM) was inhibited by about 65%, but they were not completely inactivated and showed chelator resistance (Table 1).
[0033]
[Table 1]

[0034]
(12) Influence of surfactant
As a result of examining the reactivity of the enzyme in a reaction system in which various surfactants were added to 0.2% (w / v), the enzyme was not targeted even in the presence of a high concentration of surfactant. More than 80% of the activity was expressed (Table 2).
[0035]
[Table 2]

[0036]
(13) Effects of metal salts
As a result of adding 1 mM of various metal salts to the standard reaction system and investigating the influence on the enzyme activity, this enzyme is calcium chloride, magnesium chloride, manganese chloride, ferrous chloride, and ferric chloride. It was activated with 135%. On the other hand, it was inhibited by zinc chloride and copper chloride at about 95% and 85%, respectively. Next, nickel chloride and cobalt chloride were inhibited by about 70% and 60% (Table 3).
[0037]
[Table 3]

[0038]
Polygalacturonase producing bacteria as described above, for exampleBacillusMicroorganism belonging to the genus, preferablyBacillus The gene encoding the polygalacturonase of the present invention can be cloned from sp. KSM-P358 strain (FERM P-17560) or the like by, for example, a method of cloning a target gene by the shotgun method or PCR method. it can.
[0039]
The polygalacturonase gene of the present invention preferably encodes the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added. Here, deletion, substitution or addition of amino acids in the amino acid sequence (hereinafter sometimes referred to as mutation) does not limit the number and sites of the mutant amino acids unless the polygalacturonase activity is lost. Absent. Moreover, 1 to several amino acids may be added or deleted at the N-terminus in the amino acid sequence of SEQ ID NO: 1.
[0040]
When homology with other enzymes is examined between amino acid numbers 1 to 953 shown in SEQ ID NO: 1, all are exo-type polygalacturonases.Erwinia chrysanthemi PehX (He and Collmer, J. Bacteriol., 172, 4988 4995, 1990),Ralstonia Solanacearum PehB (Huahg and Allen, J. Bacteriol., 179, 7369-7378, 1997) and only 29.1% and 28.5% of homology, respectively, and a polygalacturo consisting of the amino acid shown in SEQ ID NO: 1. A enzyme is an enzyme having a novel sequence that has not been reported so far.
[0041]
As described above, the polygalacturonase gene of the present invention preferably encodes the amino acid sequence of SEQ ID NO: 1 or a variant thereof, and in particular, the base sequence shown by SEQ ID NO: 2 or one or several of the base sequences Those having a base sequence in which is deleted, substituted or added are preferred.
[0042]
In order to produce a recombinant vector containing a polygalacturonase gene, the polygalacturonase gene may be incorporated into any vector suitable for expressing the gene in the target host. Examples of such vectors include pUC18, pUC19, pBR322 and the like when Escherichia coli is used as a host, and pUB110 and the like when Bacillus subtilis is used as a host.
[0043]
In order to transform a host using the recombinant vector thus obtained, a conventional method such as a protoplus method, a competent cell method, an electroporation method or the like is performed. The host is preferably a microorganism,BacillusGenus (Bacillus subtilis),StreptomycesGram-positive bacteria such as genus (actinomycetes);Escherichia coliGram-negative bacteria such as (E. coli);SaccharomycesGenus (yeast),AspergillusExamples include fungi such as genus (mold).
[0044]
By culturing the obtained transformant and collecting polygalacturonase from the culture solution, polygalacturonase can be obtained. The culture may be carried out by inoculating a medium containing a carbon source, a nitrogen source and other essential nutrients that can assimilate microorganisms, and following a conventional method. Polygalacturonase can be obtained from the culture solution thus obtained by a method according to a generally known method for collecting and purifying an enzyme.
[0045]
【Example】
Example 1 Screening for Polygalacturonase-Producing Bacteria
A suspension of soil from various parts of Japan in sterile water was heat-treated at 80 ° C. for 20 minutes and applied to an agar plate medium having the following composition. The culture was allowed to stand for 3 to 5 days in a 30 ° C. incubator. Those in which lysis spots associated with the degradation of pectin were detected around the grown bacteria were selected, repeated single colonization, and tested for the ability to produce polygalacturonic acid-degrading enzyme. Many of the strains thus obtained mainly produced pectate lyase, among which polygalacturonase producing bacteriaBacillus sp. KSM-P358 strain was obtained.
[0046]
[Table 4]

[0047]
Example 2Bacillus Production of polygalacturonase by sp. KSM-P358 Strain obtained by the above screeningBacillus The sp. KSM-P358 strain was cultured aerobically at 30 ° C. for 2 days by adding 50 mL of medium to a 500 mL Sakaguchi flask. Medium composition is 0.5% (w / v) pectin, 1.5% polypeptone S, 0.5% yeast extract, 1.0% fish meat extract, 0.1% potassium dihydrogen phosphate, 0.02% Magnesium sulfate heptahydrate, 50 mM Tris-HCl buffer (pH 8.6, separately sterilized). The polygalacturonase activity produced in the culture was measured by adding 2 mM EDTA to completely deactivate the pectate lyase activity. By this measurement method, a production amount of 200 to 400 U / L was obtained per culture solution.
[0048]
Example 3 Purification of polygalacturonase
Bacillus The culture solution of sp. KSM-P358 strain was centrifuged (8000 × g, 15 minutes, 4 ° C.) to obtain a supernatant (2 L). This was put in a dialysis membrane and covered with polyethylene glycol 20000 (Wako Pure Chemical Industries) to concentrate the internal solution. Further, concentration and desalting were carried out using an ultrafiltration module (AIP1010: Asahi Kasei). The obtained concentrated solution (100 mL) was attached to a DEAE Toyopearl 650M column (3 × 15 cm: Tosoh) equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 1 mM dithiothreitol. The non-adsorbed protein was washed and eluted using about 500 mL of equilibration buffer, and then the adsorbed protein was eluted by a concentration gradient elution method using a buffer solution (500 mL each) containing 0 to 0.2 M sodium chloride. . Polygalacturonase activity was eluted in the vicinity of a sodium chloride concentration of about 0.1M. Subsequently, the same purification operation was performed on a DEAE Toyopearl 650M column to obtain a polygalacturonase fraction containing no pectate lyase activity (100 mL). This active fraction was concentrated by ultrafiltration (YM10 membrane: Amicon) and equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 0.1 M sodium chloride and 1 mM dithiothreitol. It applied to the column (1.5x65cm: Tosoh). The polygalacturonase active fraction eluted with the same buffer was collected (10 mL, 42 U, 14 mg protein).
The polygalacturonase fraction obtained by the above purification operation exhibited the aforementioned enzymatic properties.
[0049]
Example 4Bacillus sp. Preparation of chromosomal DNA of KSM-P358 strain
Bacillus The sp. KSM-P358 strain was shaken and cultured overnight at 30 ° C. in a liquid medium containing polypeptone S (manufactured by Nippon Pharmaceutical) as a seed culture. This seed culture was inoculated into the same medium and shake-cultured for about 8 hours as the main culture. Chromosomal DNA was prepared from the cells collected by centrifugation from the main culture solution by the method of Saito and Miura (Biochim. Biophys. Acta, 72, 619-629, 1963).
[0050]
Example 5Bacillus sp. Partial amino acid sequence of KSM-P358 polygalacturonase
1) Determination of N-terminal amino acid sequence
Bacillus The enzyme purified by various chromatography from the culture solution of sp. KSM-P358 strain was applied to a protein sequencer 476A (PE Applied Biosystems), and 9 residues of the N-terminal amino acid sequence were determined. The sequence obtained was Lys-Ser-Glu-Gly- (Pro or Ser or Ala) -Pro-Asn-Ala-Pro.
[0051]
2) Determination of intermediate amino acid sequence by lysyl endopeptidase degradation
The enzyme recovered from 12.5% polyacrylamide gel subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of the purified enzyme was approximately 1/1000 by weight of lysyl endopeptidase (sum). (Manufactured by Kosei Pharmaceutical Co., Ltd.) was added, and the mixture was incubated at 25 ° C. for 15 hours in 60 mM Tris-HCl buffer (pH 9). Samples collected on a UL TRAFREE MC spin column (0.45 μm PVDF membrane, manufactured by Millipore) were subjected to SDS-PAGE, and then electro-plotted to a PVDF membrane using Milliblot type I (Millipore). PVDF membrane was stained with Coomassie Brilliant Blue. The stained protein band was cut out from the stained PVDF membrane and subjected to a protein sequencer 476A. The amino acid sequence (intermediate amino acid sequence A) obtained from the lysyl endopeptidase partial degradation product was Asn-Ile-Leu-Phe-Arg-Asn-Asn-Ala-Leu-Glu-Gly.
[0052]
3) Determination of intermediate amino acid sequence by V8 protease degradation
SDS was added to the purified sample to 0.01% and the mixture was boiled at 100 ° C. for 10 minutes, to which about 1/60 V8 protease (manufactured by Boehringer Mannheim) was added, and 110 mM Tris-HCl was added. Incubate in buffer (pH 8.5) at 10 ° C. for 15 hours. The collected sample was subjected to SDS-PAGE, and a protein band was cut out from the PVDF membrane after electro-plotting and subjected to a protein sequencer 476A. The amino acid sequence (intermediate amino acid sequence B) obtained from the V8 protease partial degradation product is Phe-Glu- (Thr or Asn or Arg) -Ala-Arg-Pro-Tyr- (Gly or Pro)-(Ala or Ile) -It was Gly-Ala-Pro.
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Example 6 Cloning of P358 Polygalacturonase Gene
A primer using the mixed base shown in SEQ ID NO: 3 was prepared from the N-terminal amino acid sequence of the purified enzyme. Similarly, based on the intermediate amino acid sequence A obtained from the lysyl endopeptidase partial degradation product, primers shown in SEQ ID NOs: 4 and 5, and primers shown in SEQ ID NO: 6 from amino acid sequence B of the V8 protease partial degradation product Was made.
Prepared in Example 4 using these primersBacillus PCR was performed using sp. KSM-P358 strain chromosomal DNA as a template. Amplified fragments of about 1.3 kbp and 1.1 kbp are obtained by the combination of the primers of SEQ ID NOs: 3 and 4 and the primers of SEQ ID NOs: 5 and 6, respectively. Was consistent with each amino acid sequence, and thus the PCR amplified fragment was found to be a part of the target KSM-P358 polygalacturonase gene.
Primers shown in SEQ ID NOs: 7 and 8 are prepared based on the base sequences of these PCR amplified fragments, and these primers and chromosomal DNA are used as restriction enzymes.EcoInverse PCR was performed using the template DNA after self-closing with T4 DNA Ligase after cutting with RI. The base sequence of the amplified fragment of about 3.0 kbp thus obtained was analyzed, and the primers shown in SEQ ID NOs: 9 and 10 were prepared from the determined base sequence.Bacillus PCR using sp. KSM-P358 strain chromosomal DNA as a template was performed to obtain a P358 polygalacturonase gene fragment having a total length of about 4.5 kbp.
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Example 7 Determination of P358 polygalacturonase gene base sequence
A sequence analysis sample was prepared using BigDye Terminator Cycle Sequencing Kit (manufactured by PE Applied Biosystem) using 0.5-1 μg of the PCR amplified fragment prepared in Example 6 as a template, and 377 DNA Sequencer (manufactured by PE Applied Biosystem). ) To determine the base sequence of the P358 polygalacturonase gene (SEQ ID NO: 13). As a result, it was revealed that P358 polygalacturonase is an enzyme encoded by a base sequence of 2940 residues as shown in base numbers 540 to 3479 of SEQ ID NO: 13, and composed of 980 amino acid residues.
Furthermore, in the amino acid sequence, the N-terminal amino acid sequence determined from the purified enzyme and the intermediate amino acid sequence determined from the protease partial degradation product were confirmed. The molecular weight of polygalacturonase consisting of 953 amino acids from the N-terminal amino acid sequence to the stop codon (TAA) was estimated to be 103,810 Da.
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Example 8 Construction of P358 Polygalacturonase Expression Plasmid
Based on the base sequence of the P358 polygalacturonase gene determined in Example 7, primers having the sequences shown in SEQ ID NOs: 11 and 12 were prepared and prepared in Example 4.Bacillus PCR was performed using the chromosomal DNA of sp. KSM-P358 strain as a template, and a DNA fragment encoding P358 polygalacturonase was amplified.
In this case, the primers used are restriction enzymes so that they can be efficiently cloned into the multi-cloning site of the plasmid vector pHY300PLK (manufactured by Yakult).SalI,XbaI recognition sequence was added.
Restriction enzymes for PCR-amplified fragments of plasmid vectors pHY300PLK and P358 polygalacturonase geneSalI,XbaAfter digestion with I, purification was performed using GFX PCR DNA and Gel Band Purification Kit (Pharmacia).
After the purified sample was mixed at a weight ratio of 1: 1, it was bound with T4 DNA Ligase, and this bound sample was used.E.coli HB101 competent cells (Takara Shuzo) were transformed. Arbitrary transformants are selected from the obtained transformants, and a plasmid is prepared from the selected transformants.SalI,XbaDigested with I. The plasmid in which the insertion of the PCR amplified fragment was confirmed by agarose electrophoresis was designated as pHYK358PEC (FIG. 6).
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Example 9 Production of polygalacturonase by Bacillus subtilis transformant using pHYK358PEC
Using plasmid pHYK358PEC and protoplast method (Chang and Choen. Mol. Gen. Genet., 168, 111-115, 1978)Bacillus subtilis ISW1214 strain (manufactured by Yakult) was transformed. The obtained transformant was inoculated into LB medium supplemented with 15 μg / mL tetracycline, and cultured overnight at 30 ° C. as a seed culture. This seed culture solution was inoculated to 40 mL of a main culture medium mainly composed of Polypeptone S or corn steep liquor and maltose so as to be 1% (v / v), followed by shaking culture at 30 ° C. and 120 rpm for 4 days. . The production amount of recombinant polygalacturonase was about 1000 to 3000 U / L under these culture conditions.
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Example 10 Properties of recombinant polygalacturonase
The culture supernatant obtained in Example 9 was adsorbed on an anion exchange resin, purified by eluting with a sodium chloride concentration gradient, and the properties of the purified recombinant enzyme were examined. The properties were in good agreement with those of wild-type P358 polygalacturonase.
[0058]
1) Optimum reaction pH
In place of the standard enzyme activity measurement method, 50 mM Tris-HCl buffer (pH 7 to 9.5) and 50 mM glycillin-sodium hydroxide buffer (pH 9 to 11) were used to examine the optimum reaction pH. The highest reaction rate was obtained in hydrochloric acid buffer (pH 8.0).
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2) Molecular weight
As a result of obtaining the molecular weight by SDS-PAGE (12.5% acrylamide gel) using a standard protein for electrophoresis (manufactured by Bio-Rad), the molecular weight of the enzyme was estimated to be about 105 kDa.
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3) Protopectinase activity
One 30cm braid (gold bell) (25mg) was used as a substrate, 50mM glycine-sodium hydroxide buffer (pH 10) containing 0.4mM calcium chloride as an enzyme reaction solution, and 0.2U enzyme (polygala) Cutulonase activity) was added to make the final volume 2.0 mL. After the reaction at 30 ° C. for 1 hour, the pectic substance released to the supernatant was measured by the orcinol hydrochloric acid method. As a result, it was confirmed that about 40 μg of pectin was released per 0.2 U of polygalacturonase under the above conditions.
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【The invention's effect】
The polygalacturonase of the present invention acts exogenously on polygalacturonic acid but has an optimum reaction pH near pH 8, maintains activity even in an alkaline region, and exhibits protopectinase activity. It is useful as a detergent enzyme and a fiber processing enzyme. Further, by using the polygalacturonase gene of the present invention, it becomes possible to produce the polygalacturonase in a single and large amount.
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[Brief description of the drawings]
FIG. 1 is a graph showing the effect of pH on polygalacturonase activity of the present invention.
FIG. 2 is a graph showing the influence of pH on the protopectinase activity of the present invention.
FIG. 3 is a graph showing the influence of temperature on the polygalacturonase activity of the present invention.
FIG. 4 is a graph showing the effect of pH on the stability of polygalacturonase of the present invention.
FIG. 5 is a graph showing the effect of temperature on the stability of polygalacturonase of the present invention.
FIG. 6: Polygalacturonase gene (Bacillus FIG. 2 is a view showing a polygalacturonase expression / secretion vector pHYK358PEC constructed by introducing a sp. KSM-P358 strain) into a plasmid vector (pHY300PLK).

Claims (9)

A polygalacturonase derived from a bacterium belonging to the genus Bacillus having the following enzymological properties.
(1) Action: Acts on polygalacturonic acid (pectinic acid), pectin and protopectin, and exohydrolyzes α-1,4 bonds of polygalacturonic acid to produce digalacturonic acid.
(2) Optimal reaction pH: around pH 8 (Tris-HCl buffer)
It has a protopectinase activity of 30% or more of the protopectinase activity at the optimum reaction pH at pH 8-11.
(3) Optimal reaction temperature: about 50 ° C. (Tris-HCl buffer, pH 8.0)
(4) pH stability: pH 7 to 11.5 (30 ° C., 30 minutes treatment)
(5) Heat resistance: stable up to about 50 ° C. (Tris-HCl buffer, pH 8.0, treated for 30 minutes)
(6) Molecular weight: about 105000 (gel filtration method)
(7) Isoelectric point: around pH 4.6 (isoelectric focusing method)   The polygalacturonase according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added. The polygalacturonase according to claim 1 or 2, which is derived from Bacillus sp. KSM-P358 (FERM P-17560).   Bacillus sp. KSM-P358 (Bacillus sp. KSM-P358; FERM P-17560).   A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or a protein consisting of an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added, and having polygalacturonase activity.   The base sequence shown in SEQ ID NO: 2 or a base sequence encoding a protein having polygalacturonase activity in which one or several bases of the base sequence are deleted, substituted or added. gene.   A recombinant vector containing the gene according to claim 5 or 6.   A transformant comprising the recombinant vector according to claim 7.   A method for producing polygalacturonase, comprising culturing the transformant according to claim 8.

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