JP3882961B2 - Microorganisms producing sucrose fatty acid ester synthase - Google Patents

Description

【００２４】
【表２】

【００２５】
なお、蔗糖モノステアレートを主成分とするＤＫエステルＳＳ（商品名；第一工業製薬株式会社製）を50g/lの水溶液とし、これを別途に高圧蒸気滅菌または濾過滅菌したものを、「蔗糖脂肪酸エステル(SE)」として各培地に添加した。
そして、固体培地とする場合には、15g/lの寒天をさらに加えた。
【００２６】
各試料を滅菌水に懸濁した後、寒天培地に塗布し、30℃または37℃で、菌体の十分な増殖が目視で確認できるまで培養を継続した。 培地中の蔗糖脂肪酸エステル(SE)は、培養当初は培地に溶解して透明であるが、分解が進行すると、高級脂肪酸を遊離して白濁する。 菌体の周囲に形成される白いハロー（白濁円）の有無に基づいて、蔗糖脂肪酸エステル(SE)を分解しているSE分解菌を選抜した。
具体的には、以下に記載のSE寒天法または滴定法によった。
【００２７】
［SE寒天法］
SE寒天法は、0.2％の蔗糖脂肪酸エステル(SE)、pH７の50mMリン酸緩衝液を含む1.5％の寒天に、直径４mmの円形の穴を開け、異なる濃度の酵素液20μlをそこに注入し、37℃で一夜静置した後、白濁円の直径(mm)を測定した（図１参照）。
【００２８】
［滴定法］
滴定法は、まず、（５mlの５％蔗糖脂肪酸エステル(SE)水溶液、４mlのMcIlvanine緩衝液(pH 8)、１mlの様々な濃度の酵素液からなる）反応液を、37℃または60℃で30分間反応させる。 次いで、アセトン／エタノール＝50%/50%の混液を10ml、そして、0.05規定水酸化ナトリウムを10ml加えて反応を停止し、さらにアセトン／エタノール＝50%/50%の混液を10mlを加えた後、フェノールフタレインを指示薬として0.05規定の塩酸で遊離脂肪酸を逆滴定した。 そして、この条件下で、１分間に１μmolの脂肪酸を遊離するために必要な酵素量を１単位と定義した。
【００２９】
なお、SE寒天法で測定した白濁円の直径(mm)と、滴定法で測定した酵素活性（単位/ml）との間には、図２に示したように、片対数グラフにて直線関係が得られる。 この関係式を利用することで、両者のデータの互換が図れる。
【００３０】
ここまでの分離手順で、計44株が、コロニー周辺にハローを形成した。
【００３１】
また、L+SE寒天培地よりも、L/10+SE寒天培地の方でハローを形成した菌が圧倒的に多かった。 これは、分離源となった工場廃水（汚泥）や土壌が、栄養分の希薄な環境であるため、栄養価が希薄なL/10+SE寒天培地でよく生育したものと思われる。
【００３２】
そして、高い分解活性を示した菌の多くは、蔗糖脂肪酸エステル製造工場の排水および廃水処理場から分離された菌であった。 これは、常に蔗糖脂肪酸エステル(SE)に曝され続け、比較的温度が安定している分離源の環境が、SE分解菌の生育に適合したものと考えられる。
【００３３】
なお、分解活性は高くないものの、土壌試料からもSE分解菌は分離されており、このことは、SE分解菌が自然界に比較的広範に分布していることを示唆するものである。
【００３４】
次に、分離株を、L/10+SE液体培地に接種し、充分に増殖するまで、１〜３日間、30℃または37℃で、振とう培養し、培養液の遠心上清の酵素活性をSE寒天法で測定した。 その結果、84株の内、８株がSE寒天上で８mm以上の直径の白濁円を形成した。 これらの菌株は、顕微鏡観察により、その菌体の大きさから、いずれも細菌であると認められた。 ８株の内、最も活性の高かった分離株、SE分解菌 No.13株を選択し、そのハロー形成の状態を図３に示した。
【００３５】
なお、リパーゼの産生能を備えたArthrobacter属細菌（４株）についてもSE寒天法を適用したが、いずれの株によっても、直径８mmを超える白濁円の形成には至らなかった。
【００３６】
実施例２： SE 分解菌 No.13 株の同定
実施例１にて単離されたNo.13株の菌学的位置づけを決定すべく、No.13株の形態的性質、培地における生育状態、生理学的性質およびNo.13株が保有する遺伝子について分析（解析）を行った。
【００３７】
［No.13株の形態的、培養的性質］
図４および５は、（SEを含む／含まない）寒天培地上で生育したNo.13株の写真であり、コロニーの色は白色乃至クリーム色であった。 Leptothrix属（鞘皮）細菌はマンガンを含む寒天培地にて、マンガンを酸化して酸化マンガンを生成し、コロニーが黒色になるものが多いが、マンガンを含む寒天培地にNo.13株を接種してもコロニーは黒色は呈さず、また、鞘皮細菌によく見られる歪つな形状のコロニーも形成されなかった。 液体培養した菌体を集菌したところ、薄いピンク色乃至普通のピンク色を呈していた。 これに対して、蔗糖脂肪酸エステル(SE)を含む培地では、コロニー周辺に、SEの分解に起因する白いハローが形成されていた。
【００３８】
図６は、No.13株の光学顕微鏡写真（倍率：1000倍）であり、この細菌は、幅0.5μm×長さ1.5μm〜2.0μmの桿菌で、運動性が認められた。 培養条件によっては、菌体の長さが５μmに至ることがあり、２〜５個の菌が、鎖状に連なることもある。 しかしながら、鞘皮細菌に見られる鞘状物は認められなかった。
【００３９】
［No.13株の生理学的性質］
No.13株の生理学的性質を決定すべく、api 20E キット(bio Merieux)およびapi 20NE キット(bio Merieux)を用い、各キットに添付のプロトコールに従った。
【００４０】
炭素源利用能力の判定
炭素源利用能力を判定するために、No.13株が良好に生育したPTYP基本培地（表３に組成を示した）に、濾過滅菌した炭素源を１g/lまたは２g/l添加した培地を用いた。
【００４１】
【表３】

【００４２】
なお、炭素源にカルボン酸を用いる場合、水酸化ナトリウムでpHを７に調整した上でカルボン酸を添加した。 グルコース１g/lを含むPTYP基本培地に、１白金耳のNo.13株を接種し、これを37℃で、一夜振とう培養して前培養液を得た。
この前培養液を、所定の炭素源を含む前出のPTYP基本培地に0.6体積％接種し、好気的に、37℃で、２日間振とう培養した。 菌体の生育を、培養液に関して、600nmの吸光度で測定した。
【００４３】
各種炭素源を添加したPTYP培地で好気培養した時の吸光度を、炭素源を添加しなかった培地で培養した場合の吸光度と比較し、炭素源の資化性（利用能力）を判定した。 その結果を、表４に示した。
【００４４】
【表４】

【００４５】
api 20 kitによる判定結果と、後述する16S-rRNA遺伝子塩基配列の相同性比較において、高い相同性を示した細菌の炭素源利用能力に関する比較を、表５に示した。
【００４６】
【表５】

【００４７】
表５の結果から、Rubribivax gelatinosusとは、クエン酸とグリセリン、メタノール、エタノール、マンノースの利用性において相違が認められる。
【００４８】
Leptothrix cholodniiは、種々の有機酸や糖類を資化しているのに対し、No.13株は利用できない有機酸や糖類が散見される。
【００４９】
Ideonella属細菌は、１属１種(イデオネラ デクロラタンス(Ideonella dechloratans))が報告されている[Malmqvist et al., "Ideonella dechloratans gen.nov., sp.nov., a New Bacterium Capable of Growing Anaerobically with Chlorate as an Electron Acceptor", System. Appl. Microbiol. 17, 58-64 (1994)］のみで、炭素源資化性に関するデータも少ないが、報告されている範囲内ではNo.13株の炭素源資化性データと一致している。
【００５０】
また、活性汚泥からよく分離されるBrachymonas denitrificansは、No.13株とはグルコース、フルクトース、グリセリンの利用性が異なるのに加え、Brachymonas denitrificansは運動性が無い点でNo.13株の性状とは一致しない。
【００５１】
嫌気条件下での生育の判定
グルコース１g/lを含むPTYP基本培地に、表６に記載の電子受容体を10mMまたは20mM添加して培地を調製した。 電子受容体を含まない培地で、好気的に、37℃で、一夜振とう培養した前培養液を、電子受容体を含む培地に、0.6体積％接種し、嫌気ボックス中（窒素ガス、水素ガス、炭酸ガスの90：５：５の体積比率の混合ガス雰囲気）、蛍光灯照射下、30℃で７日間静置培養した。 菌体の生育を、培養液に関して、600nmの吸光度で測定した。 その結果を、以下の表６に示した。
【００５２】
【表６】

【００５３】
電子受容体としてKNO₃を添加した場合にのみ、無添加条件のものと比較して吸光度の有意な増加が観察された。 好気的に培養した場合と比べれば、増殖効果は極めて悪いが、No.13株には微弱ながら硝酸呼吸能力のあることが確認された。 後述する通り、No.13株が、硝酸塩を亜硝酸塩に還元する能力を有していることも、この結果を支持している。
【００５４】
16S-rRNA遺伝子塩基配列の比較においてNo.13株との間で高い相同性が認められたIdeonella属細菌は、硝酸塩と塩素酸塩を電子受容体として、嫌気条件下で生育できることが知られているのに対し、No.13株は硝酸塩の利用しか認められなかった。 No.13株の硝酸呼吸能力が微弱であることは、絶対好気性であるLeptothrix属細菌の挙動と似ている。
【００５５】
［No.13株が保有する遺伝子の解析］
No.13株の細菌学的位置づけを遺伝子工学的側面から特徴付けるために、No.13株の16S-rRNA遺伝子のクローニング、次いで、その塩基配列の決定（解析）を行った。
【００５６】
No.13 株が保有する遺伝子のクローニング
まず、鋳型ＤＮＡには、Sarkosyl法[Morikawa et al., "Isolation of a new surfactin producer Bacillus pumilus A-1, and cloning and nucleotide sequence of regulator gene, psf-1", J. Ferment. Bioeng., 74, 255-261 (1992)]で調製したNo.13株染色体DNAを充てた。
【００５７】
そして、上流側プライマーに、5'-AAGAGTTTGATCATGGC-3'(配列番号：１）を、そして、下流側プライマーに、5'-GGTTACCTTGTTACGACTT-3'(配列番号：２）のプライマーを用い、Vent DNA ポリメラーゼ（東洋紡社製）でPCR増幅した。
【００５８】
PCR反応の条件は、添付のカタログの記載に従った。 PCR増幅断片をプラスミドベクターpUC18のSmaI部位にクローニングした。
【００５９】
宿主菌に E. coli JM109[recA1, endA1, gryA96, thi, hsdR17, supE44, relA1, △(lac-proAB), F'(traD36, proAB⁺, lac1^y, lacZ △ M15)]を用いた。 QIAGEN KIT(フナコシ）で組み換えプラスミドの大量調製を行った。 大腸菌の形質転換は、Lederberg とCohen[Lederberg et al., "Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid", J. Bacteriol., 119, 1072-1074 (1992)]の方法によった。 また、必要に応じて、適当な制限酵素部位を利用してサブクローニングを行った。 さらに、培地は、Ｌ培地（組成を表７に示した）を用いた。
【００６０】
【表７】

【００６１】
なお、抗生物質を添加する場合、50μg/mlアンピシリンを用いた。
【００６２】
No.13 株が保有する遺伝子の解析
No.13株のクローンが保有する遺伝子の塩基配列を、自動解析装置(Auto Read Sequencing Kit)(Pharmacia社製）とALFexpress(Pharmacia社製）装置を用いて、ジデオキシ法によって解析した。 なお、解析ソフトウェアには、DNASIS(Hitachi)を使用した。 その解析結果を、配列番号：３として示した。
【００６３】
微生物分類の重要な指標の一つである、16S-rRNA遺伝子の塩基配列（配列番号：３）を基に、他の微生物が保有する16S-rRNA遺伝子との相同性について検索を行い、その結果を、以下の表８に示した。
【００６４】
【表８】

【００６５】
配列番号：３から明らかなように、この配列には、Proteobacteriaに特徴的な塩基配列が含まれている。 Proteobacteriaは、紅色細菌とその類縁細菌の系統分類名であり、さらに、α、β、γ、δのサブクラスに細分される。 表８に記した微生物の大半は、Proteobacteriaβサブクラスに分類される菌であり、No.13株も、Proteobacteriaβサブクラスに属すると考えられる。
【００６６】
16S-rRNAの塩基配列を基に作成したSphaerotilus-Leptothrixグループの系統図[Ghiorse et al., "Phylogeny of Sphaerotilus-Leptothrix group inferred from morphological comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence analysis", Int. J. Syst. Bacteriol., 46, pp.173-182 (1996)]によれば、No.13株と相同性の高いLeptothrix discophora、Sphaerotilus natans、Rubribivax gelatinosusはクラスターとなっており、ProteobacteriaβサブクラスのRubribivax サブディビィジョンに位置している。
【００６７】
そして、前出のGhiorseらの文献によれば、Ideonella dechloratansは、ProteobacteriaβサブクラスのComamonasグループに分類されている。 99.2％という最も高い一致率を示したIdeonella 属は、活性汚泥から分離された細菌で、塩素酸塩を電子受容体として嫌気条件下で生育することを特徴とした菌である。 また、Rubribivax 属、Rhodocyclus属細菌は光合成細菌であり、嫌気・光条件下で生育できることを特徴としている。Leptothrix属、Sphaerotilus属細菌は鞘皮細菌と呼ばれ、菌体の周囲に長い筒状の鞘を形成することを特徴としている。
【００６８】
これに対して、No.13株は、嫌気条件下でほとんど生育せず、また、表５に記載の炭素源資化性でもクエン酸とグリセリンの利用性が異なるなど、No.13株が Rubribivax 属細菌とは考えにくい。 なお、Ideonella 属細菌については、これまでに報告された内容から検討してみると、シトクロムＣオキシダーゼが陽性であること、プロテアーゼとエステラーゼを生産すること、およびグルコースを発酵／酸化しない点においてNo.13株との間で一致が認められる。
【００６９】
以上述べた方法によって得られたデータを、以下にまとめた。
【００７０】

No.13 株の同定
No.13株の16S-rRNA遺伝子の塩基配列を比較したところ、Ideonella 属細菌、Rubribivax 属（光合成）細菌、 Leptothrix属（鞘皮）細菌の16S-rRNA遺伝子の塩基配列と95％以上の高い一致をみた。
【００７１】
光合成細菌の特徴である嫌気・光条件での生育が極めて悪いこと、クエン酸とグリセリンの利用能力が異なることから、No.13株は、Rubribivax 属細菌ではないと判断した。
【００７２】
Leptothrix属細菌の中には、鞘を形成せず、マンガンを酸化しない変異株も知られているが、No.13株が鞘皮細菌の特徴である鞘を菌体の周囲に形成しないこと、マンガン塩を酸化して黒色の酸化マンガンを培地中に形成しないこと、そして利用できない糖類が比較的多いことから、No.13株がLeptothrix属細菌である可能性も否定された。
【００７３】
そして、Ideonella 属細菌が、塩素酸塩と硝酸塩を電子受容体として嫌気で生育できるのに対し、No.13株は微弱ながら硝酸塩のみを利用して嫌気で生育した。
【００７４】
また、Ideonella 属細菌について報告された範囲内で検討したところ［前出の、Malmqvist 等の文献］、炭素源の資化性と生理学的性質の一致をみている。
さらに、16S-rRNA遺伝子の塩基配列は、99.2%という極めて高い一致率を示しており、No.13株はIdeonella 属に最も近縁にある細菌であると判断し、No.13株をIdeonella sp. No.13と命名した。
【００７５】
実施例３： SE 分解菌 No.13 株による酵素生産
実施例１で単離したNo.13株に関して、その酵素（蔗糖脂肪酸エステル(SE)の合成酵素）の生成能力を以下の手順に従って評価した。
【００７６】
(1) 培地の調製ならびに検討
L培地（組成を下記表９を示した）、L/10培地（組成を下記表10に示した）、および最少培地（組成を下記表11を示した）の３種類の培地を基本培地とし、炭素源として、蔗糖脂肪酸エステル(SE)またはグルコース(Glu)を添加した培地を調製した。
【００７７】
【表９】

【００７８】
【表１０】

【００７９】
【表１１】

【００８０】
No.13株の培養は500ml容の坂口フラスコで行った。 L/10＋１％グルコース培地で培養した前培養液0.5mlを100mlの各培地に接種し、37℃で２日間振とうした。
【００８１】
そして、No.13株の培地および炭素源が与える影響について検討した。 つまり、前出のL/10培地、Ｌ培地および最少培地に、炭素源として蔗糖脂肪酸エステル(SE)またはグルコース(Glu)を添加し、 図７に示した培地の栄養条件下でNo.13株を培養し、菌体増殖とエステラーゼ活性を調べた。 炭素源を添加したL/10培地、Ｌ培地で菌体は良好に増殖し、高いエステラーゼ活性が認められた。
【００８２】
最少培地での菌体の増殖は芳しくなかった。 誘導剤として用いた蔗糖脂肪酸エステル(SE)は、その使用の有無にかかわらず、No.13株はエステラーゼを生産しているいことが判明した。 これらの結果を受けて、エステラーゼ生産量の多かったＬ＋Glu１％培地を、以後の実験に用いた。
【００８３】
(2) No.13 株の培養
まず、前培養を、L/10＋１％グルコース培地で、37℃で、一夜振とう培養して行った。 L培地よりもL/10培地の方が、培養初期の増殖が良好なため、前培養にはL/10培地を用いた。 本培養には、５ｌ容量のミニジャーファーメンター（マルビシ バイオ エンジ製）を用い、培地は、L＋１％グルコース培地とし、培地量2.5l、温度37℃、通気量1.25l/分(0.5VVM)、接種量50ml(2%)の条件下で培養を行った。 消泡剤としてアデカノール103を必要時に添加した。
【００８４】
図８に、No.13株をミニジャーファーメンターで培養した時の培養経過図を示した。 それによると、約10時間後の誘導期の後、対数増殖し、その後、吸光度は徐々に減少した。 目視観察によれば、栄養的に貧弱な培地であるL/10＋Glu１％培地を用いた方が、誘導期は短かった。 そして、培養液上清のエステラーゼ活性は、菌体増殖に伴って増加し、増殖が停止した後も、徐々に増加した。
最終的に、2.6単位/mlのエステラーゼを生産した。
【００８５】
一方、培養液のpHは培養の進行に従って上昇し、培養開始時に7.3であったのが、終了時には8.79になった。 培養終了時の培養液はアンモニア様の臭気を呈しており、アミン系化合物が生成され、その結果、pHがかように上昇したものと考えられる。 高いpHによる、菌体増殖および酵素生産の阻害を懸念して、pHコントローラーを用い、塩酸によりpHを7.6に調整して同様の培養実験を行ったが、菌体増殖および酵素生産にはほとんど変化はなかった。
【００８６】
(3) 粗酵素の調製
まず、No.13株の前々培養を、試験管を用いて、５mlのL/10＋１％グルコース培地で、37℃で、一夜振とう培養して行った。 前培養は、２l容量の坂口フラスコを用い、前々培養液の全量を、１lのL＋１％グルコース培地で、37℃で、１日振とう培養して行った。 本培養には、100ｌ容量のジャーファーメンター（マルビシ バイオ エンジ製、タイプMPF)を用い、培地は、L＋１％グルコース培地とし、培地量50l、温度37℃、通気量50l/分(1VVM)、接種量１l(2%)、培養時間48時間の条件下で培養を行った。
【００８７】
100 l容量のジャーファーメンターで培養した培養液を、シャープレス連続遠心機で除菌した。 得られた上清液を、４℃で、限外濾過濃縮器（旭化成、マイクローザUF SPL-1053)で1/10量にまで濃縮した。 濃縮液に、pH7.5の20mMトリス塩酸緩衝液に溶解し、同緩衝液中で透析、脱塩した。 得られた粗酵素液を凍結乾燥して、粗酵素粉末とした。
【００８８】
そして、実施例１に記載のSE寒天法および滴定法で酵素活性を測定したところ、2000単位／ｇの酵素活性であった。
【００８９】
次に、No.13株培養液上清を濃縮、硫安塩析、透析脱塩した粗酵素について、蔗糖脂肪酸エステル分解の温度特性とpH特性を、前出の滴定法によって分析し、その結果を、それぞれ図９と図10に示した。 pH４〜pH８のpH域ではMcIlvaine緩衝液（リン酸水素２ナトリウムとクエン酸を含む）を、また、pH９〜pH10のpH域ではClark-Lubs緩衝液（ホウ酸、塩化カリウムおよび水酸化ナトリウムを含む）を用いた。
【００９０】
図９のグラフから明らかなように、蔗糖脂肪酸エステル(SE)分解の至適温度は60℃であり、70℃と80℃でも活性が認められた。 一方、図10のグラフから明らかなように、至適pHは８で、pH６〜pH８で高い活性を示した。
【００９１】
そして、エステラーゼまたはリパーゼの基質となり得る代表的なエステル化合物の分解活性を調べた。 以下の表12に、蔗糖脂肪酸モノエステルに対する分解活性を100としたときの相対活性を示した。
【００９２】
【表１２】

【００９３】
４mlのMcIlvaine緩衝液（pH８）、4.75mlの水、0.25ｇの基質（エステル化合物）、１mlの粗酵素液からなる反応液を、60℃で、30分振とうして反応させた後、滴定法による活性測定と同様にして酵素の相対活性を求めた。 蔗糖脂肪酸モノエステル以外に、糖類のエステル誘導体であるTween-80（ポリオキシエチレンソルビタン脂肪酸エステル）に対しても分解活性が認められた。 この粗酵素は、同じ糖エステルでも、水に溶解しない蔗糖脂肪酸ポリエステルにはほとんど作用しなかった。
【００９４】
また、エステラーゼの基質である（活性測定条件下では水溶性を呈する）酢酸エチルにもほとんど作用しなかった。 さらに、エステラーゼとリパーゼの活性測定基質として広く使用されているPNPA（パラニトロフェニルアセテート）にもほとんど作用しなかった（データ示さず）。 そして、トリグリセライドについても、水溶性のトリアセチン、および、非水溶性のトリブチリンとオリーブ油に対してもほとんど作用しなかったことから、本酵素はいわゆるリパーゼと呼ばれる酵素ではないものと思われる。 よって、水溶性の糖類エステルに特異的に作用する特殊なエステラーゼである可能性が高く、今後、さらに多くの基質について調査すればその傾向はさらに明確になるであると考えられる。
【００９５】
実施例４： SE 分解菌 No.13 株産生酵素による蔗糖脂肪酸エステルの合成
実施例３(3)で得られた粗酵素を用いて、様々なショ糖脂肪酸エステルの合成反応を行った。
【００９６】
オクタン酸（C8）、ラウリン酸（C12）またはミリスチン酸（C14）のうちいずれかの脂肪酸10 mg、50重量／容量％ショ糖を含む50 mMリン酸緩衝液（pH 7）100μl、粗酵素液20μl（3単位）及びジメチルスルホキシド20μl（13.3容量％）を含む反応液をエッペンドルフチューブに入れ、45℃にて20時間反応させた。
次いで、反応液に400μlのメタノール、400μlのクロロホルム及び100μlの水を加えて溶媒抽出し、下層のクロロホルム層40μlを、シリカゲルプレート60（メルク社製）に適用して、薄層クロマトグラフィー分析に供した。 この際の展開溶剤として、クロロホルム／メタノール／酢酸／水＝70／20／８／２の混合溶剤を用いた。 また、対照群として、粗酵素液を含有しない反応液で同様の操作を行った。 この結果を図11に示す（図中、微弱なスポットが存在していた箇所に、手書きにてマークしてその存在を示した）。
【００９７】
図11において、酵素存在下に反応を行うと、各炭素数の脂肪酸について鎖長に応じたRf値（C8：0.19及び0.15、C12：0.22及び0.18ならびにC14：0.25及び0.20）を有する矢印で示される各々２種の反応産物が生成されている（図11、レーンｂ、ｄ及びｆ）。 ここで、脂肪酸の炭素数が大きくなるほど生成物のRf値は大きくなっており、また、本酵素が２つのショ糖脂肪酸エステル異性体を分解したことに対応するように反応産物が２種であるとの知見からして、これらエステラーゼ反応によってショ糖脂肪酸モノエステルの置換位置異性体２種が生成されたと考えられる。
【００９８】
さらに、同様の条件下での反応におけるエステルの生成を確認するために、ショ糖とラウリン酸を反応させて合成される産物について検討した。 反応条件は、上記と同様であり、反応終了後の抽出物を、ショ糖モノラウレートを含む標準試料（DKエステルS-L18A、第一工業製薬社製）と比較すべく、薄層クロマトグラフィー分析した。 展開溶剤として、クロロホルム／メタノール／酢酸／水＝70／20／8／2の混合溶剤を用いた。 この結果を図12に示す。
【００９９】
図12に明示したように、本酵素を用いたショ糖とラウリン酸との反応によって、標品のショ糖モノラウレートと同様のRf値（0.40及び0.33）を有する産物が得られた。 次いで、この反応産物を高速液体クロマトグラフィー質量分析（LC-MS）に供した。 LC-MS装置として、高速液体クロマトグラフィー部分には、カラム Cosmosil 5C18-AR（φ4.6 mm×150 mm、ナカライテスク社製）を装備したLC10AT（島津社製）を用いた。 使用溶離液は、10 mMの酢酸アンモニウム水溶液（溶離液Ａ）と10 mM酢酸アンモニウムのメタノール溶液（溶離液Ｂ）を用い、流速 0.7 ml/分にて溶離液Ａ100％から溶離液Ｂ100％までの直線濃度勾配とした。
【０１００】
質量分析部分には、Finigan Mat LCQ装置（Finigan社製）を用いて、イオン化はESI法により、また検出は負イオンにて行った。 得られた液体クロマトグラフィーの結果を図13に示す。
【０１０１】
図13において、主たるピークａ及びｂの保持時間は、市販のショ糖モノラウレートの標品の保持時間と一致していた。 そして、双方のピークのマススペクトルにより、ショ糖モノラウレートの分子量524.6に対して、-H(523.6)、+CH3CO0(583.6)及び+CF3COO(637.6)の質量数にピークが確認された。 また、市販の標品のマススペクトルにおいても同様の質量数のピークが確認された。
【０１０２】
このように、本発明の酵素存在下で、ショ糖とラウリン酸とを反応させると、ショ糖モノラウレートの２種類のエステル置換位置異性体が得られることが明らかになった。
【０１０３】
【発明の効果】
このように、本発明によると、所期の目的であった、蔗糖と脂肪酸から直接に蔗糖脂肪酸エステル(SE)を合成する酵素と、これを産生する新規の微生物が提供される。
【０１０４】
この酵素の発現に関与する遺伝子の解析がなされている現状では、遺伝子に変更を加えて改変を施し、蔗糖脂肪酸エステル(SE)の合成により適した酵素を作り出すことも可能となる。 すでにプロテアーゼにおいては、構造的に改変を加えて分解活性を抑え、水が多量にある反応系においても高い合成力を備えた酵素が作り出されており、また、エステラーゼとプロテアーゼの反応機構は同じであるため、合成力の高いエステラーゼを作り出すことは、当該技術分野での技術水準からすれば、さほど困難でないと予想される。 同時に、合成反応の条件を最適化することで、反応率を着実に改善することも当然に期待できる。
【０１０５】
つまり、遺伝子レベルでの改変、酵素の改変／修飾と反応条件の検討ならびに改善によって、蔗糖脂肪酸エステル(SE)の収率の改善が大いに期待できるのである。
【０１０６】
【配列表】

【０１０７】

【０１０８】

【０１０９】

【０１１０】

【０１１１】

【０１１２】

【図面の簡単な説明】
【図１】 SE寒天法にて培地上に形成されたハローの状態を示す写真である。
【図２】 SE寒天法による白濁円の直径(mm)と、滴定法で測定した酵素活性（単位/ml）との間の相関を示すグラフである。
【図３】 SE分解菌 No.13株によるハロー形成の状態を示す写真である。
【図４】 SEを含む寒天培地上で生育したNo.13株によるハロー形成を示す写真である。
【図５】 SEを含まない寒天培地上で生育したNo.13株によるハロー形成を示す写真である。
【図６】 No.13株の光学顕微鏡写真（倍率：1000倍）である。
【図７】 三種の培地でのNo.13株の生育状況を示すグラフである。
【図８】 No.13株をミニジャーファーメンターで培養した時の培養経過図を示すグラフである。
【図９】 粗酵素の温度特性を示すグラフである。
【図１０】 粗酵素のpH特性を示すグラフである。
【図１１】 本発明のショ糖脂肪酸エステラーゼによる種々のショ糖脂肪酸エステルの合成の態様を表す図である。
【図１２】 本発明のショ糖脂肪酸エステラーゼによるショ糖ラウレートの合成の態様を表す図である。
【図１３】 図１２において得られたショ糖ラウレートの高速液体クロマトグラフィーにおける分離を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an isolated novel microorganism that produces an enzyme involved in the synthesis of sucrose fatty acid ester (SE).
[0002]
[Background Art and Problems to be Solved by the Invention]
Sucrose fatty acid esters are a type of surfactant that has been confirmed to be highly safe for the human body and ecosystem, and to date, have been mass-produced industrially on a commercial basis and used as emulsifying and dispersing agents for food products. Widely used, including applications.
[0003]
The conventional production method of this sucrose fatty acid ester is mainly a chemical method, but on the other hand, it is also true that there is a long-awaited voice for a synthesis method by an enzymatic method.
[0004]
This is because the following advantages can be expected according to the enzymatic method as compared with the chemical method. Ie;
(1) Since the enzyme reaction proceeds under normal temperature, normal pressure, and neutral pH conditions, there is no need for acid, alkali, and pressure resistant equipment, and the reaction temperature is lower than the chemical method, so the temperature is adjusted. The equipment burden required for this is reduced and its management becomes easy.
[0005]
(2) Since an enzyme is a protein, it is possible to realize a method for producing SE that is non-toxic and biodegradable and has little influence on the environment and ecosystem.
[0006]
(3) The enzymatic reaction has high reaction specificity, and the reaction proceeds at room temperature and neutral pH. Therefore, side reactions are unlikely to occur, and thus a product with high purity is easily obtained. Further, the raw material is hardly changed during the reaction, and the unreacted raw material can be used for the next reaction, so that the raw material can be effectively used.
[0007]
(4) According to the enzymatic reaction, a position-specific reaction is possible, and by utilizing this, only the hydroxyl group at a specific position of sucrose is esterified, and the target modified product (product) has high purity. Obtainable.
[0008]
In recent years, the availability of immobilized lipase agents (lipozymes, novozymes, etc.) suitable for ester synthesis has increased, and interest in enzymatic ester synthesis has increased. In particular, saccharides have many esterifiable hydroxyl groups. Because of this, regioselective (site-specific) esterification has been the subject of research by many scientists.
[0009]
For example, Klibanov et al. [Klibanov et al., “Protease-catalyzed Regioselective esterification of sugars and related compunds in anhydrous dimethylformamide”, J. Am. Chem. Soc., 110, pp. 584-589 (1988)] By using highly reactive fatty acid trichloroethyl ester and protease as a catalyst, the 1 'position of sucrose is selectively esterified. [Patil et al., "Enzymatic synthesis of a sucrose-containing linear polyester in nearly anhydrous organic media", Biotechnol. Bioeng., 37, pp. 639-646 (1991); Chan et al., "A regioselective, chemoenzymatic synthesis of sucrose-1'-methacrylate ", Biocatalysis, 8, pp.163-169 (1993); and Soedjak et al.," Enzymatic transesterification of sugars in anhydrous pyridine, Biocatalysis, 11, pp.241-248 (1994). See also literature].
[0010]
In addition, Fregapane et al. [Fregapane et al., “Enzymatic solvent-free synthesis of sugar fatty acid ester”, Enzyme Microb. Technol., 13, pp. 796-800 (1991)] is a sugar added with acetone (isopropylidene). Derivatives) are used as raw materials to improve the solubility of sugars in fatty acid methyl, and furthermore, monosaccharides are mainly esterified by using a commercially available immobilized lipase agent as a catalyst. [Fregapane et al., "Facile chemo-enzymatic synthesis of monosaccharide fatty acid esters", Biocatalysis, 11, pp.9-18 (1994); and Sarney et al., "Chemo-enzymatic synthesis of disaccharide fatty acid esters", See also J. Am. Oil Chem. Soc., 71, pp.711-714 (1994)].
[0011]
Klibanov et al. [Klibanov et al., “Lipase-catalyzed acylation of sugars solubilized in hydrophobic solvents by complexation”, Biotechnol. Bioeng., 42, pp. 788-791 (1993)] By using it for improving the solubility of saccharides in a solvent, sucrose acrylate is synthesized by using lipoprotein lipase as a catalyst. [Schlotterbeck et al., "Lipase-catalyzed monoacylation of fructose", Biotechnol. Letters, 15, pp.61-64 (1993) and Oguntimein et al., "Lipase catalyzed synthesis of sugar ester in organic solvents", Biotechnol. Letters , 15, pp.175-180 (1993) etc.].
[0012]
Furthermore, as an example of directly reacting a monosaccharide and a fatty acid without using a special derivative, it has been reported that fructose or glucose is esterified using a commercially available immobilized enzyme agent (lipozyme, novozyme, etc.) as a catalyst. . [Khaled et al., "Fructose oleate synthesis in a fixed catalyst bed reactor", Biotechnol. Letters, 13, pp.167-172 (1991); Coulon et al., "Comparison of direct esterification of fructose by Candida antartica lipase" , Biotechnol. Letters, 17, pp.183-186 (1995); and references such as Ljunger et al., "Lipase catalyzed acylation of glucose", Biotechnol. Letters, 11, pp.1167-1172 (1994) Wanna]
[0013]
In this way, it was possible to synthesize monosaccharide esters using sugar and fatty acids as raw materials, whereas sucrose esters could only be synthesized by enzymatic methods using special derivatives as raw materials. It was the actual situation. The use of such special derivatives is disadvantageous for industrial production of sucrose esters, and this is one of the reasons why industrial production by enzymatic methods has not yet been achieved.
[0014]
In addition, the enzymes used for synthesis are all commercially available lipase agents or protease agents, and their activity against sucrose fatty acid esters is not satisfactory. In fact, of the 13 types of commercially available lipase agents, only two types of lipase MY (name sugar industry) and lipase OF (name sugar industry) have been found to have a degradation activity on sucrose fatty acid esters. Even the decomposition activity was far from practical level.
[0015]
[Means for Solving the Problems]
In view of the problems in the prior art described above, the present invention searches for enzymes having high activity against sucrose fatty acid ester (SE) and microorganisms that produce them from the natural world, and sucrose on an industrial scale by an enzymatic method using them. As a result of intensive studies aimed at synthesizing fatty acid esters (SE), the present invention has been completed.
[0016]
That is, the gist of the present invention is an isolated novel microorganism belonging to the genus Ideoneella that produces an enzyme that synthesizes a sucrose fatty acid ester from sucrose and a fatty acid, and uses thereof.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
First, since the esterification reaction is an equilibrium reaction, the present inventors have recognized that an enzyme exhibiting a high degradation activity for SE exhibits a high synthetic activity (depending on the reaction conditions) due to changes in the reaction conditions. And searched for microorganisms having high fatty acid ester degrading activity (hereinafter simply referred to as “SE-degrading bacteria”). Microbial samples were soil samples (84 strains) around sucrose fatty acid ester manufacturing factories and research institutions such as universities. And 8 strains which exhibited comparatively high activity were selected, and 1 strain which showed the highest degradation activity was selected from these.
[0018]
This strain was named Ideoneella sp. No.13 (Ideonella sp. No.13 (hereinafter referred to as “No.13 strain”)), and on April 24, 1997, the applicant of the present application was Deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3-1 Higashimachi, Tsukuba City, where the accession number FERM P-16206 has been assigned. The microorganism itself is of course intended to its mutants and progeny that have substantially inherited the ability of the microorganism.
[0019]
And when this No.13 strain | stump | stock was cultured and a crude enzyme was prepared, No.13 strain | stump | stock produced esterase with the proliferation of a microbial cell in the L culture medium which added glucose 1% [production amount: 2.6. Unit / ml]. In addition, the synthesis of sucrose fatty acid esters in a reaction system using this esterase was confirmed.
[0020]
【Example】
The present invention will be specifically described below, but the present invention should not be construed in a limited manner based on the disclosure of the examples.
[0021]
Example 1: SE Isolation of degrading bacteria
First, the soil of the applicant's sucrose fatty acid ester production plant (427 Shimohiyoshi, Gokaso-cho, Kanzaki-gun, Shiga Prefecture), the wastewater from the wastewater treatment plant attached to the site, activated sludge, and the Osaka University Faculty of Engineering (Suita, Osaka Prefecture) Ichiyamadaoka 2-1) A total of 84 separation samples were arbitrarily collected from the premises and nearby soil.
[0022]
These samples were inoculated in both L + SE liquid medium (composition shown in Table 1 below) and L / 10 + SE medium (composition shown in Table 2 below).
[0023]
[Table 1]

[0024]
[Table 2]

[0025]
In addition, DK ester SS (trade name; manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) containing sucrose monostearate as a main component was made into a 50 g / l aqueous solution, which was separately sterilized by high-pressure steam sterilization or filtration. Fatty acid ester (SE) "was added to each medium.
When a solid medium was used, 15 g / l agar was further added.
[0026]
Each sample was suspended in sterilized water and then applied to an agar medium, and the culture was continued at 30 ° C. or 37 ° C. until sufficient growth of the bacterial cells could be visually confirmed. The sucrose fatty acid ester (SE) in the medium is transparent when dissolved in the medium at the beginning of the culture. However, when the decomposition proceeds, higher fatty acids are released and become cloudy. Based on the presence or absence of a white halo (cloudy circle) formed around the cells, SE-degrading bacteria that decompose sucrose fatty acid esters (SE) were selected.
Specifically, the SE agar method or titration method described below was used.
[0027]
[SE agar method]
The SE agar method is a 1.5% agar containing 0.2% sucrose fatty acid ester (SE) and pH 7 50 mM phosphate buffer. A 4 mm diameter circular hole is drilled and 20 μl of enzyme solution of different concentration is injected into it. After standing at 37 ° C. overnight, the diameter (mm) of the cloudy circle was measured (see FIG. 1).
[0028]
[Titration method]
In the titration method, first, a reaction solution (comprising 5 ml of 5% sucrose fatty acid ester (SE) aqueous solution, 4 ml of McIlvanine buffer (pH 8), 1 ml of various concentrations of enzyme solution) at 37 ° C. or 60 ° C. Incubate for 30 minutes. Next, 10 ml of a mixture of acetone / ethanol = 50% / 50% and 10 ml of 0.05N sodium hydroxide were added to stop the reaction, and 10 ml of a mixture of acetone / ethanol = 50% / 50% was added. Then, free fatty acid was back titrated with 0.05 N hydrochloric acid using phenolphthalein as an indicator. Under this condition, the amount of enzyme required to release 1 μmol of fatty acid per minute was defined as 1 unit.
[0029]
In addition, as shown in FIG. 2, there is a linear relationship between the diameter (mm) of the cloudy circle measured by the SE agar method and the enzyme activity (unit / ml) measured by the titration method. Is obtained. By using this relational expression, the data of both can be compatible.
[0030]
In the separation procedure so far, a total of 44 strains formed halos around the colonies.
[0031]
In addition, the number of bacteria that formed halos in the L / 10 + SE agar medium was overwhelmingly higher than that in the L + SE agar medium. This is probably because the factory wastewater (sludge) and soil that became the source of separation were in a nutrient-poor environment, so they grew well on the L / 10 + SE agar medium with a low nutrient value.
[0032]
Many of the bacteria that showed high degrading activity were isolated from waste water and wastewater treatment plants of sucrose fatty acid ester manufacturing plants. It is considered that the environment of the separation source, which is constantly exposed to sucrose fatty acid ester (SE) and is relatively stable in temperature, is suitable for the growth of SE-degrading bacteria.
[0033]
Although the degradation activity is not high, SE-degrading bacteria are isolated from soil samples, which suggests that SE-degrading bacteria are relatively widely distributed in nature.
[0034]
Next, the isolate is inoculated into an L / 10 + SE liquid medium and cultured for 1 to 3 days with shaking at 30 ° C. or 37 ° C. until it fully grows. Was measured by SE agar method. As a result, 8 strains among 84 strains formed cloudy circles with a diameter of 8 mm or more on SE agar. These strains were confirmed to be bacteria from the size of the cells by microscopic observation. Among the 8 strains, the isolate with the highest activity, SE-degrading bacterium No. 13, was selected, and the state of halo formation is shown in FIG.
[0035]
The SE agar method was applied to Arthrobacter bacteria (4 strains) having lipase production ability, but none of the strains resulted in the formation of a cloudy circle having a diameter of more than 8 mm.
[0036]
Example 2: SE Degrading bacteria No.13 Strain identification
In order to determine the mycological positioning of the No. 13 strain isolated in Example 1, the morphological properties of the No. 13 strain, the growth state in the medium, the physiological properties, and the genes possessed by the No. 13 strain Analysis (analysis) was performed.
[0037]
[Morphological and cultural properties of No.13 strain]
4 and 5 are photographs of No. 13 strain grown on an agar medium (with / without SE), and the color of the colony was white to cream. Leptothrix genus (scabbard) bacterium oxidizes manganese in an agar medium containing manganese to produce manganese oxide, and many colonies turn black, but inoculate No. 13 strain on the agar medium containing manganese. However, the colonies did not appear black, and the distorted colonies often found in scabs were not formed. When the cells cultured in liquid culture were collected, they showed a light pink color to a normal pink color. In contrast, in the medium containing sucrose fatty acid ester (SE), a white halo due to the degradation of SE was formed around the colony.
[0038]
FIG. 6 is an optical micrograph of the No. 13 strain (magnification: 1000 times). This bacterium is a gonococcus having a width of 0.5 μm × length of 1.5 μm to 2.0 μm, and motility was observed. Depending on the culture conditions, the length of the cells may reach 5 μm, and 2 to 5 bacteria may be chained. However, no pods found in scabs were observed.
[0039]
[Physiological properties of No.13 strain]
In order to determine the physiological properties of No. 13 strain, api 20E kit (bio Merieux) and api 20NE kit (bio Merieux) were used, and the protocol attached to each kit was followed.
[0040]
Determination of carbon source utilization capacity
In order to determine the carbon source utilization capacity, a medium in which 1 g / l or 2 g / l of a carbon source sterilized by filtration was added to a PTYP basic medium (the composition is shown in Table 3) on which No. 13 strain grew well. Using.
[0041]
[Table 3]

[0042]
In addition, when using carboxylic acid for a carbon source, after adjusting pH to 7 with sodium hydroxide, carboxylic acid was added. A PTYP basic medium containing 1 g / l of glucose was inoculated with No. 13 strain of 1 platinum loop, and this was shaken overnight at 37 ° C. to obtain a precultured solution.
This preculture was inoculated at 0.6% by volume into the above-mentioned PTYP basic medium containing a predetermined carbon source and aerobically cultured at 37 ° C. with shaking for 2 days. The growth of the cells was measured with respect to the culture solution at an absorbance of 600 nm.
[0043]
The absorbance at the time of aerobic culture in a PTYP medium to which various carbon sources were added was compared with the absorbance at the time of culturing in a medium to which no carbon source was added, and the assimilation (utilization ability) of the carbon source was determined. The results are shown in Table 4.
[0044]
[Table 4]

[0045]
Table 5 shows a comparison of the carbon source utilization ability of bacteria showing high homology in the homology comparison between the determination result by api 20 kit and the 16S-rRNA gene base sequence described later.
[0046]
[Table 5]

[0047]
From the results in Table 5, there are differences in the availability of citric acid and glycerin, methanol, ethanol, and mannose from Rubribivax gelatinosus.
[0048]
Leptothrix cholodnii assimilate various organic acids and saccharides, while organic acids and saccharides that can not be used by No.13 strain are found.
[0049]
One genus of Ideonella (Ideonella dechloratans) has been reported [Malmqvist et al., "Ideonella dechloratans gen. Nov., Sp. Nov., A New Bacterium Capable of Growing Anaerobically with Chlorate as an Electron Acceptor ", System. Appl. Microbiol. 17, 58-64 (1994)], and there are few data on carbon source assimilation. Is consistent with the chemical data.
[0050]
In addition, Brachymonas denitrificans, which is well separated from activated sludge, differs from No. 13 strain in the utilization of glucose, fructose and glycerin, and Brachymonas denitrificans is not motility and is characterized by No. 13 strain It does not match.
[0051]
Determination of growth under anaerobic conditions
A medium was prepared by adding 10 mM or 20 mM of the electron acceptor shown in Table 6 to a PTYP basic medium containing 1 g / l of glucose. Inoculate 0.6% by volume of the pre-cultured solution aerobically cultured at 37 ° C with shaking overnight at 37 ° C in a medium containing no electron acceptor, and in an anaerobic box (nitrogen gas, hydrogen Gas and carbon dioxide 90: 5: 5 volume ratio mixed gas atmosphere) under fluorescent lamp irradiation, static culture was performed at 30 ° C. for 7 days. The growth of the cells was measured with respect to the culture solution at an absorbance of 600 nm. The results are shown in Table 6 below.
[0052]
[Table 6]

[0053]
KNO as electron acceptor_ThreeA significant increase in absorbance was observed only when was added compared to that without the addition. Compared with aerobic culture, the growth effect was extremely poor, but it was confirmed that No. 13 strain was weak but capable of nitrate respiration. As will be described later, the fact that No. 13 strain has the ability to reduce nitrate to nitrite also supports this result.
[0054]
It is known that the bacteria of the genus Ideonella, which showed high homology with No.13 strain in comparison of 16S-rRNA gene base sequences, can grow under anaerobic conditions using nitrate and chlorate as electron acceptors. In contrast, No. 13 strain was only allowed to use nitrate. The weak nitrate respiration ability of No.13 strain is similar to the behavior of Leptothrix spp.
[0055]
[Analysis of genes held by No.13 strain]
In order to characterize the bacteriological position of the No. 13 strain from the viewpoint of genetic engineering, the 16S-rRNA gene of the No. 13 strain was cloned, and then its base sequence was determined (analyzed).
[0056]
No.13 Cloning of genes held by the strain
First, the template DNA was subjected to the Sarkosyl method [Morikawa et al., “Isolation of a new surfactin producer Bacillus pumilus A-1, and cloning and nucleotide sequence of regulator gene, psf-1”, J. Ferment. Bioeng., 74 , 255-261 (1992)].
[0057]
Then, 5′-AAGAGTTTGATCATGGC-3 ′ (SEQ ID NO: 1) is used as the upstream primer, and 5′-GGTTACCTTGTTACGACTT-3 ′ (SEQ ID NO: 2) is used as the downstream primer, and Vent DNA polymerase PCR amplification was carried out (Toyobo Co., Ltd.).
[0058]
The conditions for the PCR reaction were as described in the attached catalog. The PCR amplified fragment was cloned into the SmaI site of plasmid vector pUC18.
[0059]
E. coli JM109 [recA1, endA1, gryA96, thi, hsdR17, supE44, relA1, △ (lac-proAB), F '(traD36, proAB⁺, lac1^y, lacZ △ M15)]. Recombinant plasmids were prepared in large quantities with QIAGEN KIT (Funakoshi). E. coli was transformed by the method of Lederberg and Cohen [Lederberg et al., “Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid”, J. Bacteriol., 119, 1072-1074 (1992)]. Further, subcloning was performed using an appropriate restriction enzyme site as required. Furthermore, L medium (the composition is shown in Table 7) was used as the medium.
[0060]
[Table 7]

[0061]
When adding antibiotics, 50 μg / ml ampicillin was used.
[0062]
No.13 Analysis of genes held by strains
The base sequence of the gene possessed by the No. 13 clone was analyzed by the dideoxy method using an autoanalyzer (Auto Read Sequencing Kit) (Pharmacia) and ALFexpress (Pharmacia). DNASIS (Hitachi) was used as the analysis software. The analysis result is shown as SEQ ID NO: 3.
[0063]
Based on the 16S-rRNA gene base sequence (SEQ ID NO: 3), which is one of the important indicators of microbial classification, we searched for homology with 16S-rRNA genes possessed by other microorganisms. Is shown in Table 8 below.
[0064]
[Table 8]

[0065]
As is apparent from SEQ ID NO: 3, this sequence contains a base sequence characteristic of Proteobacteria. Proteobacteria is a phylogenetic name of red bacteria and related bacteria, and is subdivided into subclasses of α, β, γ, and δ. Most of the microorganisms listed in Table 8 are bacteria classified into the Proteobacteria β subclass, and the No. 13 strain is also considered to belong to the Proteobacteria β subclass.
[0066]
Phylogeny of Sphaerotilus-Leptothrix group inferred from morphological comparisons, genomic fingerprinting, and 16S ribosomal DNA sequence analysis ", Int. J According to Syst. Bacteriol., 46, pp.173-182 (1996)], Leptothrix discophora, Sphaerotilus natans, and Rubribivax gelatinosus, which are highly homologous to the No. 13 strain, are clustered, and the Proteobacteria β subclass Rubribivax subdivi Located in the region.
[0067]
And according to the above-mentioned literature by Ghiorse et al., Ideonella dechloratans is classified into the Comamonas group of the Proteobacteria β subclass. The genus Ideonella, which showed the highest concordance rate of 99.2%, is a bacterium isolated from activated sludge and characterized by growing under anaerobic conditions using chlorate as an electron acceptor. Also, the genus Rubribivax and Rhodocyclus are photosynthetic bacteria and are characterized by being able to grow under anaerobic and light conditions. The bacteria of the genus Leptothrix and Sphaerotilus are called sheath-skin bacteria and are characterized by the formation of a long tubular sheath around the cells.
[0068]
In contrast, the No. 13 strain hardly grows under anaerobic conditions, and the utilization of citric acid and glycerin is different even though the carbon source utilization as shown in Table 5 is different. It is difficult to consider it as a genus bacteria. In addition, regarding the bacteria of the genus Ideonella, considering the contents reported so far, it is No. in terms of being positive for cytochrome C oxidase, producing protease and esterase, and not fermenting / oxidizing glucose. There is agreement between the 13 strains.
[0069]
The data obtained by the method described above is summarized below.
[0070]

No.13 Strain identification
Comparing the base sequence of 16S-rRNA gene of No.13 strain, it is highly consistent with the base sequence of 16S-rRNA gene of Ideonella genus bacteria, Rubribivax genus (photosynthesis) bacterium, Leptothrix genus (scabbard) bacterium more than 95% I saw.
[0071]
Since the growth under anaerobic and light conditions, which are the characteristics of photosynthetic bacteria, is extremely poor, and the ability to use citrate and glycerin is different, the No. 13 strain was judged not to be a genus Rubribivax.
[0072]
Among Leptothrix bacteria, mutants that do not form pods and do not oxidize manganese are known, but the No. 13 strain does not form the pods that are characteristic of deciduous bacteria around the cells, The possibility that the No. 13 strain is a Leptothrix genus bacterium was denied because the manganese salt was not oxidized to form black manganese oxide in the medium and there were relatively many sugars that could not be used.
[0073]
And Ideonella genus bacteria can grow anaerobically using chlorate and nitrate as electron acceptors, whereas No. 13 strain was weak but grew anaerobically using only nitrate.
[0074]
In addition, when we examined within the reported range of bacteria of the genus Ideonella [supra, literature by Malmqvist et al.], We found that the assimilation and physiological properties of the carbon source are consistent.
Furthermore, the base sequence of the 16S-rRNA gene showed an extremely high coincidence rate of 99.2%, and the No.13 strain was judged to be the closest bacterium to the genus Ideonella. It was named No.13.
[0075]
Example 3: SE Degrading bacteria No.13 Enzyme production by strains
Regarding the No. 13 strain isolated in Example 1, the ability to produce the enzyme (sucrose fatty acid ester (SE) synthase) was evaluated according to the following procedure.
[0076]
(1) Preparation and examination of medium
Three types of medium, L medium (composition shown in Table 9 below), L / 10 medium (composition shown in Table 10 below), and minimal medium (composition shown in Table 11 below) were used as the basic medium. A medium supplemented with sucrose fatty acid ester (SE) or glucose (Glu) as a carbon source was prepared.
[0077]
[Table 9]

[0078]
[Table 10]

[0079]
[Table 11]

[0080]
The No. 13 strain was cultured in a 500 ml Sakaguchi flask. 100 ml of each medium was inoculated with 0.5 ml of the preculture solution cultured in L / 10 + 1% glucose medium and shaken at 37 ° C. for 2 days.
[0081]
And the influence which the culture medium and carbon source of No.13 stock give was examined. That is, sucrose fatty acid ester (SE) or glucose (Glu) was added as a carbon source to the L / 10 medium, L medium and minimal medium described above, and the No. 13 strain was obtained under the nutrient conditions of the medium shown in FIG. Were cultured and examined for cell growth and esterase activity. The cells grew well on the L / 10 medium and L medium supplemented with a carbon source, and high esterase activity was observed.
[0082]
The growth of the cells in the minimal medium was not good. The sucrose fatty acid ester (SE) used as the inducer was found to be capable of producing esterase regardless of whether or not it was used. Based on these results, L + Glu 1% medium, which produced a large amount of esterase, was used in the subsequent experiments.
[0083]
(2) No.13 Strain culture
First, pre-culture was performed in an L / 10 + 1% glucose medium at 37 ° C. with shaking overnight. L / 10 medium was used for pre-culture because L / 10 medium had better growth at the initial stage of culture than L medium. For the main culture, a 5-liter mini jar fermenter (manufactured by Marubishi Bio-Engine) is used, and the medium is L + 1% glucose medium, the volume of the medium is 2.5 l, the temperature is 37 ° C., the aeration rate is 1.25 l / min (0.5 VVM), Cultivation was performed under conditions of an inoculum of 50 ml (2%). Adecanol 103 was added as needed as an antifoam.
[0084]
FIG. 8 shows a culture progress chart when No. 13 strain was cultured in a mini jar fermenter. According to it, after an induction period of about 10 hours, logarithmic growth, after which the absorbance gradually decreased. According to visual observation, the induction period was shorter when the L / 10 + Glu 1% medium, which is a nutritionally poor medium, was used. Then, the esterase activity of the culture supernatant increased with the growth of the cells and gradually increased after the growth stopped.
Finally, 2.6 units / ml of esterase was produced.
[0085]
On the other hand, the pH of the culture broth increased with the progress of the culture, and was 7.3 at the start of the culture to 8.79 at the end. The culture solution at the end of the culture has an ammonia-like odor, and an amine compound is produced, and as a result, the pH is considered to have increased. Concerned about the inhibition of cell growth and enzyme production due to high pH, the same culture experiment was conducted using a pH controller and adjusting the pH to 7.6 with hydrochloric acid, but there was little change in cell growth and enzyme production. There was no.
[0086]
(3) Preparation of crude enzyme
First, pre-culture of No. 13 strain was performed by shaking culture overnight at 37 ° C. in 5 ml of L / 10 + 1% glucose medium using a test tube. Pre-culture was performed using a 2 l Sakaguchi flask, and the whole culture solution was cultured in 1 l L + 1% glucose medium at 37 ° C. with shaking for 1 day. For the main culture, a 100 liter jar fermenter (manufactured by Marubishi Bio-Engineering, type MPF) is used. Cultivation was carried out under conditions of an amount of 1 l (2%) and a culture time of 48 hours.
[0087]
The culture solution cultured in a 100 l jar fermenter was sterilized with a sharpless continuous centrifuge. The obtained supernatant was concentrated to 1/10 volume with an ultrafiltration concentrator (Asahi Kasei, Microza UF SPL-1053) at 4 ° C. The concentrated solution was dissolved in 20 mM Tris-HCl buffer with pH 7.5, dialyzed and desalted in the same buffer. The obtained crude enzyme solution was freeze-dried to obtain a crude enzyme powder.
[0088]
When the enzyme activity was measured by the SE agar method and titration method described in Example 1, the enzyme activity was 2000 units / g.
[0089]
Next, the temperature and pH characteristics of sucrose fatty acid ester degradation were analyzed by the above-mentioned titration method for the crude enzyme obtained by concentrating the supernatant of No.13 culture solution, salting out with ammonium sulfate, and desalting with dialysis. These are shown in FIGS. 9 and 10, respectively. McIlvaine buffer (containing disodium hydrogen phosphate and citric acid) in the pH range of pH 4 to pH 8, and Clark-Lubs buffer (containing boric acid, potassium chloride and sodium hydroxide) in the pH range of pH 9 to pH 10 ) Was used.
[0090]
As is apparent from the graph of FIG. 9, the optimum temperature for sucrose fatty acid ester (SE) decomposition was 60 ° C., and activity was also observed at 70 ° C. and 80 ° C. On the other hand, as apparent from the graph of FIG. 10, the optimum pH was 8, and high activity was exhibited at pH 6 to pH 8.
[0091]
Then, the degradation activity of typical ester compounds that can be substrates for esterase or lipase was examined. Table 12 below shows the relative activity when the decomposition activity for sucrose fatty acid monoester is defined as 100.
[0092]
[Table 12]

[0093]
A reaction solution consisting of 4 ml of McIlvaine buffer (pH 8), 4.75 ml of water, 0.25 g of substrate (ester compound) and 1 ml of crude enzyme solution was reacted at 60 ° C. with shaking for 30 minutes, followed by titration. The relative activity of the enzyme was determined in the same manner as the activity measurement by the method. In addition to sucrose fatty acid monoesters, degradation activity was also observed for Tween-80 (polyoxyethylene sorbitan fatty acid ester), which is an ester derivative of saccharides. This crude enzyme had little effect on sucrose fatty acid polyesters that were not soluble in water, even with the same sugar ester.
[0094]
In addition, it hardly affected ethyl acetate which is a substrate for esterase (which exhibits water solubility under the activity measurement conditions). Furthermore, PNPA (paranitrophenyl acetate), which is widely used as a substrate for measuring the activity of esterases and lipases, hardly acted (data not shown). Triglyceride has little effect on water-soluble triacetin and water-insoluble tributyrin and olive oil, so this enzyme is considered not to be an enzyme called so-called lipase. Therefore, there is a high possibility that it is a special esterase that specifically acts on water-soluble saccharide esters, and if more substrates are investigated in the future, the tendency will become clearer.
[0095]
Example 4: SE Degrading bacteria No.13 Synthesis of sucrose fatty acid esters by strain-producing enzymes
Using the crude enzyme obtained in Example 3 (3), various sucrose fatty acid ester synthesis reactions were carried out.
[0096]
Fatty acid 10 mg of either octanoic acid (C8), lauric acid (C12) or myristic acid (C14), 50 mM phosphate buffer (pH 7) 100 μl containing 50% w / v sucrose, crude enzyme solution A reaction solution containing 20 μl (3 units) and dimethyl sulfoxide 20 μl (13.3% by volume) was placed in an Eppendorf tube and reacted at 45 ° C. for 20 hours.
Next, 400 μl of methanol, 400 μl of chloroform and 100 μl of water are added to the reaction solution to perform solvent extraction, and 40 μl of the lower chloroform layer is applied to silica gel plate 60 (Merck) for thin layer chromatography analysis. did. As a developing solvent at this time, a mixed solvent of chloroform / methanol / acetic acid / water = 70/20/8/2 was used. Further, as a control group, the same operation was performed with a reaction solution not containing a crude enzyme solution. The result is shown in FIG. 11 (in the figure, the presence of a weak spot was marked by handwriting to indicate its presence).
[0097]
In FIG. 11, when the reaction is carried out in the presence of an enzyme, the Rf values (C8: 0.19 and 0.15, C12: 0.22 and 0.18, and C14: 0.25 and 0.20) corresponding to the chain length of each carbon number fatty acid are indicated by arrows. Two kinds of reaction products are generated (FIG. 11, lanes b, d and f). Here, as the carbon number of the fatty acid increases, the Rf value of the product increases, and there are two reaction products corresponding to the fact that this enzyme decomposes two sucrose fatty acid ester isomers. From these findings, it is considered that two kinds of substitution position isomers of sucrose fatty acid monoesters were generated by these esterase reactions.
[0098]
Furthermore, in order to confirm the production | generation of ester in reaction on the same conditions, the product synthesize | combined by making sucrose and lauric acid react was examined. The reaction conditions are the same as above, and the extract after completion of the reaction is compared with a standard sample containing sucrose monolaurate (DK ester S-L18A, manufactured by Daiichi Kogyo Seiyaku Co., Ltd.). analyzed. As a developing solvent, a mixed solvent of chloroform / methanol / acetic acid / water = 70/20/8/2 was used. The results are shown in FIG.
[0099]
As clearly shown in FIG. 12, a product having the same Rf value (0.40 and 0.33) as that of the standard sucrose monolaurate was obtained by the reaction of sucrose and lauric acid using this enzyme. The reaction product was then subjected to high performance liquid chromatography mass spectrometry (LC-MS). As the LC-MS apparatus, LC10AT (manufactured by Shimadzu Corporation) equipped with a column Cosmosil 5C18-AR (φ4.6 mm × 150 mm, manufactured by Nacalai Tesque) was used for the high performance liquid chromatography part. The eluent used was a 10 mM ammonium acetate aqueous solution (eluent A) and a 10 mM ammonium acetate methanol solution (eluent B), and the eluent A from 100% to eluent B 100% at a flow rate of 0.7 ml / min. A linear concentration gradient was used.
[0100]
For the mass spectrometric part, a Finigan Mat LCQ apparatus (manufactured by Finigan) was used, ionization was performed by the ESI method, and detection was performed by negative ions. The obtained liquid chromatography results are shown in FIG.
[0101]
In FIG. 13, the retention times of the main peaks a and b coincided with the retention times of the commercial sucrose monolaurate preparation. From the mass spectra of both peaks, peaks were confirmed in the mass numbers of -H (523.6), + CH3CO0 (583.6) and + CF3COO (637.6) with respect to the molecular weight of 524.6 of sucrose monolaurate. Moreover, the peak of the same mass number was confirmed also in the mass spectrum of the commercial sample.
[0102]
Thus, it has been clarified that when sucrose and lauric acid are reacted in the presence of the enzyme of the present invention, two types of ester substitution position isomers of sucrose monolaurate are obtained.
[0103]
【The invention's effect】
Thus, according to the present invention, there are provided an enzyme for synthesizing a sucrose fatty acid ester (SE) directly from sucrose and a fatty acid, which was the intended purpose, and a novel microorganism that produces the enzyme.
[0104]
At present, the genes involved in the expression of the enzyme are analyzed, and it is possible to modify the gene and modify it to produce a suitable enzyme by synthesizing sucrose fatty acid ester (SE). Already, proteases have been structurally modified to suppress degradation activity, and enzymes with high synthesizing power have been created even in reaction systems with large amounts of water. The reaction mechanism of esterases and proteases is the same. Therefore, it is expected that it is not so difficult to produce esterase having high synthetic ability from the technical level in the technical field. At the same time, it can naturally be expected to steadily improve the reaction rate by optimizing the conditions of the synthesis reaction.
[0105]
In other words, the improvement in the yield of sucrose fatty acid ester (SE) can be greatly expected by the modification at the gene level, the modification / modification of the enzyme and the examination and improvement of the reaction conditions.
[0106]
[Sequence Listing]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[Brief description of the drawings]
FIG. 1 is a photograph showing the state of halo formed on a medium by SE agar method.
FIG. 2 is a graph showing a correlation between the diameter (mm) of a cloudy circle by SE agar method and the enzyme activity (unit / ml) measured by a titration method.
FIG. 3 is a photograph showing the state of halo formation by SE-degrading bacteria No. 13 strain.
FIG. 4 is a photograph showing halo formation by No. 13 strain grown on an agar medium containing SE.
FIG. 5 is a photograph showing halo formation by No. 13 strain grown on an agar medium not containing SE.
6 is an optical micrograph (magnification: 1000 times) of No. 13 strain.
FIG. 7 is a graph showing the growth status of No. 13 strain in three types of media.
FIG. 8 is a graph showing a culture progress chart when No. 13 strain was cultured in a mini jar fermenter.
FIG. 9 is a graph showing temperature characteristics of a crude enzyme.
FIG. 10 is a graph showing pH characteristics of a crude enzyme.
FIG. 11 is a diagram showing a mode of synthesis of various sucrose fatty acid esters by the sucrose fatty acid esterase of the present invention.
FIG. 12 is a diagram showing an embodiment of the synthesis of sucrose laurate by the sucrose fatty acid esterase of the present invention.
FIG. 13 is a diagram showing separation of sucrose laurate obtained in FIG. 12 by high performance liquid chromatography.

Claims (1)

Ideonera bacterium No. 13 that produces an enzyme that synthesizes sucrose fatty acid esters from sucrose and fatty acids (Ideonella sp. No. 13 (FERM P - 16206 ))) .

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