JP3878293B2 - Sucrose fatty acid esterase - Google Patents

Description

【００６９】
この結果、本酵素はショ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）を分解した。糖エステルであっても、水に溶解しないショ糖脂肪酸ポリエステルにはほとんど作用しなかった。また、エステラーゼの基質である酢酸エチルはこの反応条件下では水溶性であるが、本酵素はほとんど作用しなかった。尚、データは示していないが、エステラーゼ及びリパーゼの活性測定用基質として広く使用されているパラニトロフェニルアセテートにも、本酵素はほとんど活性を示さなかった。さらに、表１に示すように、水溶性のトリアセチンならびに非水溶性のトリブチン及びオリーブ油などのトリグリセリドに対してほとんど活性を示さなかったことから、本酵素はいわゆるリパーゼと称される酵素には属さないと考えられる。このように、得られた酵素は水溶性の糖類エステルに特異的に作用するものであることがわかった。
【００７０】
▲４▼ショ糖脂肪酸エステルの分解反応
１．に記載の粗酵素を用いて、ショ糖脂肪酸エステルの分解反応を確認した。ショ糖脂肪酸エステルとしては、主成分としてショ糖モノステアレートを含有するDKエステルSS（第一工業製薬社製）を用いた。5重量／容量％のDKエステルSS水溶液50μl、0.1 Mリン酸緩衝液（pH 7）、粗酵素液10μl（0.6単位）、0〜100μlのジメチルスルホキシド（最終濃度0、5、10、25及び50容量％）及び適宜の量の水からなる総容量200μlの反応液をエッペンドルフチューブに入れ、53℃にて一夜反応させた後、エタノール800μlを加えて反応を停止した。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。10,000 rpmにて５分間遠心した後、得られた上清10μlを、薄層クロマトグラフィー分析に供した。薄層クロマトグラフィーには、シリカゲルプレート60（メルク社製）を使用し、展開溶剤としてクロロホルム／メタノール／酢酸／水＝80／10／8／2の混合溶剤を用い、発色は20容量％の硫酸を噴霧した後オーブンで加熱して行った。この結果を図５に示す。図５において反応基質であるショ糖モノステアレートは主として、おそらくエステル化部位が相違する３種の置換位置変異体を表す、３つのスポットとして示される。それぞれのRf値は、およそ0.21、0.18及び0.14であった。本酵素を添加して反応を行うと、ジメチルスルホキシド濃度25容量％以下では（図５、レーンｆ〜ｉ）、これら３つのスポットのうちRf値が低い２つのスポット（Rf値：0.18及び0.14）が選択的に消失しており、従って、本酵素によってエステル化部位特異的にショ糖モノステアレートが分解され、エステルの置換位置が限定されたショ糖脂肪酸モノエステルが得られることが明らかになった。
【００７１】
反応液中にジメチルスルホキシドが50容量％存在すると、分解反応は進行しなかった（同、レーンｊ）。酵素を添加しない場合は、いずれのジメチルスルホキシド濃度においても分解反応は認められていない（同、レーンａ〜ｅ）。
【００７２】
▲５▼ショ糖脂肪酸エステルの合成反応
１．に記載の粗酵素を用いて、様々なショ糖脂肪酸エステルの合成反応を行った。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▲４▼にて前記したと同様の薄層クロマトグラフィー分析に供した。ここで展開溶剤は、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。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６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）。図６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６、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、上記▲４▼の分解反応において本酵素が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種であることに基づくと、これらの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。
【００７３】
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。展開溶剤は、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７に示す。図７に明示されるように、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。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８に示す。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。従って、本発明の酵素存在下に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。
【００７４】
３．ショ糖脂肪酸エステラーゼ遺伝子のクローニング
イデオネラ エス・ピー No.13菌株由来のショ糖脂肪酸エステラーゼ遺伝子のクローニングをショットガン法を用いて以下の通りに行った。すなわち、sarkosyl法［Morikawaら、J.Ferment.Bioeng.、74巻、255〜261頁(1992)］で調製したイデオネラ エス・ピー No.13菌株の染色体ＤＮＡを、制限酵素EcoRIで切断し、pBR322ベクターのEcoRI部位に連結した。Wizard Kit（プロメガ社製）を用い、製造業者のプロトコルに従ってゲノミックライブラリーを作成した。得られたゲノミックライブラリーでEpicurian coli ABLE K [lac(LacZw ^- ),Kan ^- ,McrA ^- ,McrCB ^- ,McrF ^- ,Mrr ^- ,HsdRk ^- Mk ^- [F'proAB,lacI ^q Z Δ M15,Tn10(Tet ^r )]]（ストラタジーン社製）を、LederbergとCohen（Lederberg,E.M.及びCohen,S.N.、J.Bacteriol.119巻、1072〜1074頁(1974)）の方法に従って形質転換した。この宿主細胞は、通常用いられる宿主細胞E. coli JM109などよりもベクターのコピー数を1/10に低減するものである。この形質転換体を、アンピシリン50μg/ml及びショ糖脂肪酸エステル（DKエステルSS）2g/L及び1.5重量／容量％の寒天を含むL培地に塗布した。37℃にて一夜培養した後、室温下に１週間放置した。約5000の形質転換体コロニーについて、その周辺に生じる、エステラーゼの作用によって形成される白いハローを指標として、目的のクローンを探索した。しかして、微弱であるがハローを形成する11の陽性クローンが得られた。
【００７５】
この陽性クローンから、約10 kbのEcoRI−EcoRI断片を取り出し、サブクローニングに供した。サブクローニングにおいて、E. coli JM109 [recA1,endA1,gryA96,thi,hsdR17,supE44,relA1, Δ (Lac-proAB),F'(traD36,proAB ⁺ ,lacI ^q ,lacZ Δ M15)]を宿主細胞として用い、ベクターとしてはpUC18を用いた。次いで前記の約10 kbのEcoRI−EcoRI断片を、BamHIで消化した。切断部位は、図９、ａに示す通りであり、得られる４つの断片の各々について上記寒天法にてエステラーゼ活性を確認したところ、EcoRI−EcoRI断片の5'上流域に位置する、3.3 kbのEcoRI−BamHI（図９、ｂ）断片に高い活性が認められた。この活性は、EcoRI−EcoRI断片よりも高いものであった。このようにして得られたEcoRI−BamHI断片の塩基配列を、Dideoxy法に従って、Auto Read Sequencing Kit（ファルマシア社製）を用い、ALFexpress装置で分析した。かくして得られた塩基配列は、配列番号：１に示される通りであった。
【００７６】
次いで、得られた塩基配列のうち、エステラーゼの構造遺伝子を特定するために、当該ＤＮＡの上流及び下流領域の削除を行い、削除に伴うエステラーゼ活性の変動に基づき、1613塩基対の断片（図９、ｃ）にまで活性断片を限定した。このように領域を限定していくに伴って、コロニー周囲のハローの直径は大きくなったので、得られた断片により発現されるエステラーゼ活性が強くなっていることが示唆された。
【００７７】
この1613塩基対の断片の塩基配列を調べると、1416塩基対の読み取り枠が存在していることが示された。また、この断片をプロモーターに対して逆向きになるようにpUC19ベクターに挿入すると、エステラーゼ活性は著しく低下したので、この遺伝子の向きが図９、ｃに示すとおりであることが示唆された。
【００７８】
図９、ｃに示すように、得られたエステラーゼＤＮＡをコードする1613塩基対の断片の読み取り枠において、▲印に示す４箇所の開始コドンの配列が認められた。正確な開始コドンを確認するために、この断片の5'上流側の配列を欠失させた。図９、ｄに示すように２番目のATG配列の下流まで削除したところエステラーゼ活性が認められなくなったので、開始コドンは１または２番目のATG配列であると考えられた。さらにシャイン・ダルガーノ配列を検索すると、１番目のATG配列（配列番号：１の第689〜691位）上流には見出されず、２番目のATG配列（配列番号：１の第710〜712位）の上流（同、第697〜703位）に当該配列の存在が確認された。従って、２番目のATG配列が開始コドンであると考えた。また、終止コドンの下流には、ターミネーター配列（配列番号：１の第2134〜2149位）が見出された。
【００７９】
上記の結果より、イデオネラ エス・ピー No.13の有するショ糖脂肪酸エステラーゼの構造遺伝子は、1416塩基対からなり、酵素タンパク質を構成するアミノ酸残基数は471（配列番号：２参照）、そしてタンパク質の分子量は48,758ダルトンと推定された。これは１．において前記したイデオネラ エス・ピー No.13由来のエステラーゼタンパク質のSDS電気泳動上の見かけの分子量約48,000ダルトンとほぼ一致していた。
【００８０】
このタンパク質のアミノ酸配列のアミノ末端部分には、塩基性アミノ酸に富む配列の次に非極性アミノ酸に富む配列が続いており、およそ２０のアミノ酸残基からなるシグナルペプチドが存在していると推定された。
【００８１】
また、アミノ酸配列のほぼ中央部（配列番号：１のアミノ酸残基第189〜193位）に、エステラーゼ類及びリパーゼ類のタンパク質の活性中心に共通のアミノ酸配列「-Gly-Xaa-Ser-Xaa-Gly-」（配列番号：４）が見出された。このなかのセリン残基をシステイン残基に置換すべく、配列番号：１の塩基配列第1340〜1342位のTCGを、TGCにする変異を導入し、このＤＮＡがコードするタンパク質を発現させたところ、そのエステラーゼ活性は著しく低いことが示された。従って、この５つのアミノ酸残基からなる配列が活性中心であることが確認された。
【００８２】
このようにして明らかになったイデオネラ エス・ピー由来のエステラーゼの配列について、既知の配列との相同性をDNASIS（日立社製）のソフトを用いて解析した。一般に真核細胞性物のエステラーゼ類及びリパーゼ類の配列は高い相同性を呈することが見出されている［Cygler,M.ら、Protein Science、2巻、366〜382頁(1993)］が、細菌のリパーゼについてはあまり互いに相同性がないことが報告されている（Jeager,K.E.ら、Bacterial lipase, FEMS Microbiology Reviews、15巻、29〜63頁(1994)）。配列を検索した結果、本ショ糖脂肪酸エステラーゼタンパク質と高い相同性を示すものは見出されなかった。
【００８３】
４．ショ糖脂肪酸エステラーゼ遺伝子を含む形質転換体におけるタンパク質発現
前記３．の工程によって調製した、配列番号：１に示すＤＮＡの第658〜2276位の部分をpUC18ベクターのマルチクローニングサイトに挿入し、これを用いて宿主細胞の大腸菌（E.coli JM109）を形質転換した。前記と同様に、LederbergとCohenの方法に従って形質転換を行った。
【００８４】
形質転換した大腸菌をアンピシリン50μg/ml及びショ糖脂肪酸エステル（DKエステルSS）2 g/Lを含むL寒天培地に塗布し、37℃で一夜培養した。ハローを形成している１つのコロニーを選択し、アンピシリン50μg/mlを含む5 mlのL培地に植菌し、37℃で振盪培養した。培養開始２時間後に、最終濃度が0.1 mMとなるようにIPTG（イソプロピルチオガラクトシド）を添加し、誘導発現を行った。さらに３時間振盪培養して培養を終了した。培養物は10,000 rpmにて２分遠心分離して集菌した。集菌した菌体を50 mMリン酸緩衝液pH 7.0に懸濁し、超音波破砕した。破砕液を上記の寒天法によって活性測定したところ、培養液1 ml当たり0.25単位の酵素活性が検出された。
【００８５】
【発明の効果】
本発明のショ糖脂肪酸エステラーゼによって、特殊な誘導体などの原料を用いる必要なく、また副産物の少ない温和な条件下でショ糖と脂肪酸から直接ショ糖脂肪酸エステルを合成することが可能になるという効果が奏される。特にかかる反応において生成するショ糖脂肪酸エステルがモノエステルであって、ジエステル以上のポリエステルが合成されないので、本発明によって、選択的にショ糖脂肪酸モノエステルを製造する上で非常に有用な製造方法が提供されるのである。また、かかるショ糖脂肪酸モノエステルが、特にショ糖が位置選択的にエステル化された結果得られるものであるので、置換位置を限定したショ糖脂肪酸モノエステルの製造において、本発明が貢献するとことろは大である。
【００８６】
また、本発明によりショ糖脂肪酸エステラーゼタンパク質の遺伝子の構造が解明されたので、この遺伝子の塩基配列を改変して、さらにショ糖脂肪酸エステル合成に有利な酵素を作り出すことが可能になる。例えば、プロテアーゼにおいて実施されているように、酵素タンパク質自体の分解を抑制するような改変を行なって、より安定な酵素を作り出すことができると予測される。このような酵素の組換え体を適切な宿主細胞において発現させることによって、大量の酵素生産が可能になると考えられる。
【００８７】
また、このショ糖脂肪酸エステラーゼによるショ糖脂肪酸エステルの分解反応の部位特異性を利用して、化学的に合成したショ糖脂肪酸エステルに含まれる３種のモノエステル異性体のうちの２つを選択的に分解し、特定のエステル化部位を有する高純度のモノエステル異性体を得ることができる。このような高純度ショ糖脂肪酸エステルは、従来知られているショ糖脂肪酸エステルの用途のみならず、さらに広範な用途において有用であることが期待される。
【００８８】
【配列表】

【図面の簡単な説明】
【図１】本発明のショ糖脂肪酸エステラーゼの製造方法の１工程である高性能陰イオン交換カラムからの、タンパク質溶出の態様を示す図である。
【図２】本発明のショ糖脂肪酸エステラーゼの製造方法の１工程であるゲル濾過カラムからの、タンパク質溶出の態様を示す図である。
【図３】本発明のショ糖脂肪酸エステラーゼの活性のpH依存性を示すグラフである。
【図４】本発明のショ糖脂肪酸エステラーゼの温度依存性を示すグラフである。
【図５】本発明のショ糖脂肪酸エステラーゼによるショ糖脂肪酸エステルの分解の態様を表す図である。
【図６】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７】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８】図７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。
【図９】本発明のショ糖脂肪酸エステラーゼ遺伝子の制限酵素地図を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a sucrose fatty acid esterase, and more particularly to an enzyme protein involved in the synthesis / decomposition reaction of a sucrose fatty acid ester using sucrose and a fatty acid as substrates, and a DNA encoding them.
[0002]
[Prior art]
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)] uses a highly reactive fatty acid trichloroethyl ester as a raw material, and selectively esterifies the 1 'position of sucrose by using protease as a catalyst [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)].
[0010]
Also, Fregapane et al. [Fregapane et al., "Enzymatic solvent-free synthesis of sugar fatty acid ester",Enzyme Microb. Technol., 13, pp. 796-800 (1991)] improve the solubility of sugar in fatty acid methyl by using sugar added with acetone (isopropylidene derivative) as a raw material, and further, commercially available immobilized lipase. Monosaccharides are mainly esterified by using an 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",J. Am. Oil Chem. Soc.71, pp.711-714 (1994)].
[0011]
Also, Klibanov et al. [Klibanov et al., "Lipase-catalyzed acylation of sugars solubilized in hydrophobic solvents by complexation",Biotechnol. Bioeng., 42, pp. 788-791 (1993)] improves the solubility of saccharides in solvents by using a phenyl borate ester of sugar as a raw material, and further uses lipoprotein lipase as a catalyst. Synthesizes acrylates [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 Ljunger et al., "Lipase catalyzed acylation of glucose",Biotechnol. Letters, 11, pp.1167-1172 (1994), etc.].
[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]
[Problems to be solved by the invention]
Thus, a sucrose fatty acid esterase protein involved in the synthesis / degradation reaction of sucrose fatty acid ester using sucrose and fatty acid as substrates has not been known so far. The present invention has been made in view of the present situation. The following sucrose fatty acid esterase proteins or mutants having the activity or active fragments thereof, DNA encoding the same, vectors containing the DNA, and such vectors It aims at providing the transformant containing this. Furthermore, the protein of the present invention provides a preferable method for producing a high-purity sucrose fatty acid ester at an industrial level by using an easily available substrate and utilizing a highly safe biological method.
[0016]
[Means for Solving the Problems]
The present invention has been accomplished as a result of intensive studies in order to achieve the above object in the synthesis of such sucrose fatty acid esters, and is characterized by the following (1) to (13).
[0017]
(1) A sucrose fatty acid esterase protein involved in the synthesis / decomposition reaction of a sucrose fatty acid ester using sucrose and a fatty acid as substrates, or a variant having the activity or an active fragment thereof.
[0018]
(2) Sucrose comprising the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in this sequence, and using sucrose and a fatty acid as substrates A protein having sucrose fatty acid esterase activity involved in the synthesis / decomposition reaction of fatty acid ester.
[0019]
(3) A method for producing sucrose fatty acid esterase involved in the synthesis / degradation reaction of sucrose fatty acid ester using sucrose and fatty acid as a substrate, cultivating Ideoneella sp. A method for producing a sucrose fatty acid esterase protein or an active fragment thereof, comprising the step of:
[0020]
(4) A protein having a sucrose fatty acid esterase activity or an active fragment thereof, which is involved in the synthesis / decomposition reaction of a sucrose fatty acid ester using sucrose and a fatty acid as a substrate, produced by the production method of (3).
[0021]
(5) DNA encoding the protein of (2) above.
[0022]
(6) Synthesis of a sucrose fatty acid ester that hybridizes with DNA comprising the base sequence represented by SEQ ID NO: 1 or DNA comprising this base sequence under stringent conditions and uses sucrose and a fatty acid as substrates. DNA encoding a protein having sucrose fatty acid esterase activity involved in a degradation reaction.
[0023]
(7) A protein having a sucrose fatty acid esterase activity involved in a synthesis / degradation reaction of a sucrose fatty acid ester using sucrose and a fatty acid as a substrate, encoded by the DNA of (6).
[0024]
(8) A recombinant vector containing the DNA of (6).
[0025]
(9) A transformant in which the recombinant vector of (8) is contained in a host cell.
[0026]
(10) A recombinant protein produced by the transformant of (9) above and having a sucrose fatty acid esterase activity involved in a synthesis / decomposition reaction of a sucrose fatty acid ester using sucrose and a fatty acid as substrates.
[0027]
(11) A method for producing a sucrose fatty acid esterase involved in a synthesis / decomposition reaction of a sucrose fatty acid ester using sucrose and a fatty acid as a substrate, wherein the transformant of (9) is cultured, A method for producing a sucrose fatty acid esterase protein or an active fragment thereof, comprising a step of obtaining a liquid.
[0028]
(12) A protein having sucrose fatty acid esterase activity according to the above (1), (2), (4), (7) or (10), a mutant having the activity, or an active fragment thereof, in sucrose and a fatty acid The manufacturing method of sucrose fatty acid ester including the process of making it act.
[0029]
(13) The sucrose fatty acid esterase activity of (1), (2), (4), (7) or (10) is added to a mixture of a plurality of sucrose fatty acid monoester substitution position isomers synthesized by a chemical method. A method for producing a sucrose fatty acid monoester having a limited substitution position by allowing an active protein or a mutant having the activity or an active fragment thereof to act.
[0030]
DETAILED DESCRIPTION OF THE INVENTION
The sucrose fatty acid esterase protein involved in the synthesis / decomposition reaction of sucrose fatty acid ester using sucrose and fatty acid as substrates, or a variant having the activity thereof or an active fragment thereof (hereinafter referred to as “sucrose fatty acid esterase”) Is preferably derived from the strain of Ideoneella sp. No. 13 (Accession number: FERM P-16206, contracted to Biotechnology Institute of Industrial Technology, April 24, 1997). It is not limited to bacteria. Furthermore, the sucrose fatty acid esterase is preferably produced by a production method including a step of culturing Ideoneella sp. Moreover, in this manufacturing method, the process of obtaining the sucrose fatty acid esterase active fraction fractionated by the refinement | purification process which combined the anion exchange column chromatography and the gel filtration column chromatography may be included as needed.
[0031]
The present invention also provides a method for producing sucrose fatty acid esterase, which comprises a step of culturing Ideoneella sp. No. 13 to obtain a culture supernatant thereof. This production method can also include a step of obtaining a sucrose fatty acid esterase activity fraction fractionated by a purification step combining anion exchange column chromatography and gel filtration column chromatography.
[0032]
Here, Ideoneella sp. May be cultured in L medium, L / 10 medium, other broth medium, various known complex media, and the like. As a carbon source, sucrose fatty acid ester, acetic acid, lactic acid, malic acid, pyruvic acid, succinic acid, D-fructose, D-glucose, glycerol, maltose and the like can be added. In particular, in view of production and secretion amount of sucrose fatty acid esterase, L medium or L / 10 medium supplemented with sucrose fatty acid ester and / or glucose is preferable as the medium. Using this medium, the cells are cultured preferably aerobically. The culture time, temperature, pH and the like can be appropriately selected according to the medium and the amount of bacteria, but when culturing using the above-mentioned preferable medium, it is preferably 30 to 37 ° C, particularly preferably about 37 ° C. OD of medium at around neutral pH₆₆₀It is good to carry out until it becomes about 2-4.
[0033]
After completion of the culture, a culture supernatant containing sucrose fatty acid esterase is obtained by centrifugation or filtration. Thereafter, if necessary, the enzyme is roughly extracted by, for example, ammonium sulfate fractionation, ultrafiltration, etc., and the resulting crude enzyme solution may be appropriately purified by column chromatography. Purification is preferably carried out in combination with an anion exchange column and a gel filtration column. In addition to these, for example, cation exchange columns, affinity columns, and reverse phase columns can be used to inactivate and denature enzyme proteins. As long as it does not occur, sucrose fatty acid esterase can be purified by appropriately performing a purification operation using the enzyme activity as an index.
[0034]
The sucrose fatty acid esterase of the present invention preferably comprises the amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in this sequence, It is a protein having sucrose fatty acid esterase activity involved in the synthesis / decomposition reaction of sucrose fatty acid esters using sucrose and fatty acids as substrates. Furthermore, the synthesis / synthesis of sucrose fatty acid ester which hybridizes with DNA comprising the base sequence shown in SEQ ID NO: 3 or DNA comprising this base sequence under stringent conditions and uses sucrose and fatty acid as substrates. A protein having sucrose fatty acid esterase activity encoded by a DNA encoding a protein having sucrose fatty acid esterase activity involved in the degradation reaction is also preferred.
[0035]
The present invention also comprises an amino acid sequence represented by SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in this sequence, and sucrose and fatty acids as substrates. Provided is a DNA encoding a protein having sucrose fatty acid esterase activity involved in the synthesis / degradation reaction of sucrose fatty acid ester. Synthesis / decomposition of DNA consisting of the base sequence shown in SEQ ID NO: 3 or sucrose fatty acid ester that hybridizes with DNA consisting of this base sequence under stringent conditions and uses sucrose and fatty acids as substrates. A DNA encoding a protein having sucrose fatty acid esterase activity involved in the reaction is also contemplated by the present invention.
[0036]
Furthermore, the present invention provides a recombinant vector containing any of the above DNAs. As the vector into which DNA is inserted in this recombinant vector, various commonly used vectors can be used, for example, plasmids such as pBR322, PUC19, pUB110, pET series, RSF1010, phages such as λgt, etc. And is appropriately selected according to the infectivity to cells transformed with this vector.
[0037]
The present invention also provides a transformant in which the above recombinant vector is contained in a host cell. The host cell can be variously transformed into E. coli, yeast, Bacillus subtilis, and other animal cells as long as it can be transformed with the above-described vector to produce and secrete the desired sucrose fatty acid esterase. E. coli is preferably used.
[0038]
The transformation is accomplished by introducing a vector into the host cell, which is well known to those skilled in the art, for example, calcium chloride method, electroporation method and the like.
[0039]
A recombinant protein having sucrose fatty acid esterase activity produced by such a transformant is provided by the present invention. Here, in the sucrose fatty acid esterase of the present invention, DNA encoding the sequence is chemically synthesized, inserted downstream of an appropriate promoter in the vector, introduced into the host cell, and this transformant is introduced. It can also be produced by culturing. In addition, DNA encoding the protein is isolated from the cDNA library of an appropriate fungus, and a desired mutation is introduced into the DNA by site-directed mutagenesis to produce a DNA encoding the modified protein. It is also possible to make it. For example, by applying a method for producing a stable enzyme by modification that suppresses the degradation of the enzyme protein itself, as practiced in protease recombinants, by modifying the base sequence of the DNA of the present invention, Furthermore, it becomes possible to produce an enzyme advantageous for sucrose fatty acid ester synthesis. It is expected that the enzyme can be mass-produced by expressing such a recombinant in an appropriate host cell.
[0040]
According to the present invention, there is provided a method for producing sucrose fatty acid esterase, the method comprising culturing the transformant to obtain a culture supernatant. Subsequently, a sucrose fatty acid esterase active fraction is isolated from the obtained culture supernatant as necessary. The method for this isolation is not particularly limited, and after concentration and crude purification by ammonium sulfate fractionation, ultrafiltration, etc. according to protein purification from the above-mentioned Ideone sp. It may be purified by a graphic operation. Here, when Escherichia coli is used as the host cell constituting the transformant, the culture supernatant contains a larger amount of the target protein and the amount of impurities than the culture supernatant of Ideone sp. This makes it possible to obtain the desired protein more easily and in large quantities.
[0041]
It is also effective to connect the sucrose fatty acid esterase of the present invention to a suitable other protein and produce it in the form of a stable fusion protein in the host cell. The expressed fusion protein is advantageous in that it can be purified by utilizing the antigenic properties of the connected protein portion, and can be obtained, for example, after such purification. The target protein can also be isolated by cleaving the fusion protein in a site-specific manner using an appropriate proteolytic enzyme or a chemical cleavage method corresponding to the peptide sequence.
[0042]
Furthermore, this invention provides the manufacturing method of sucrose fatty acid ester including the process which makes sucrose fatty acid esterase described so far act on sucrose and a fatty acid.
[0043]
The raw material fatty acid is not particularly limited, such as the number of carbon atoms and the degree of unsaturation, and various fatty acids can be used. As shown in the following examples, when sucrose is esterified with a saturated saturated fatty acid having 8 to 14 carbon atoms using the sucrose fatty acid esterase of the present invention, sucrose fatty acid monoesters that are two ester-substituted positional isomers Esters have been produced.
[0044]
In the esterification, when a fatty acid having a large number of carbon atoms and hardly soluble in water is used, it is preferable to appropriately add a substance for increasing the water solubility of the fatty acid to the reaction solution. An example of such a substance is dimethyl sulfoxide, but in view of the influence on the enzyme activity, the upper limit of the addition amount should be about 25% by volume. The pH of the reaction solution is preferably adjusted to 6-9, particularly preferably about 7-8. In addition, to the reaction mixture, additives such as protease inhibitors and stabilizers that can prevent the degradation of sucrose fatty acid esterase in order to maintain the stability of the enzyme are added to the extent that they do not affect the esterase activity. Also good. The enzyme reaction solution contains, for example, an appropriate amount of fatty acid, sucrose, enzyme solution and dimethyl sulfoxide in a phosphate buffer (pH 6 to 8) of about 50 mM.
[0045]
The reaction solution is preferably incubated at 30 to 80 ° C, particularly preferably at about 40 to 55 ° C for an appropriate time. Next, the reaction solution is extracted with a solvent selected according to the philicity of the obtained sucrose fatty acid ester. For example, when an ester of a fatty acid having 8 to 14 carbon atoms is obtained, solvent extraction may be performed using a mixed solvent of methanol / chloroform equivalents, isobutanol and the like.
[0046]
In one embodiment of the present invention, there is provided a method for producing a sucrose fatty acid ester, wherein the product obtained by the production method is a sucrose fatty acid monoester and does not contain a polyester of a diester or higher. . As described above, the present invention makes it possible to synthesize sucrose fatty acid esters with a limited number of ester substitutions, which has been difficult with conventional chemical synthesis methods.
[0047]
Furthermore, the esterification site | part of sucrose is limited in the said method, The manufacture of the sucrose fatty acid monoester which contains ester only in a specific substitution position is enabled by this invention.
[0048]
Moreover, since the decomposition reaction of sucrose fatty acid ester by this sucrose fatty acid esterase has site specificity, using this, among the plural types of monoester isomers contained in chemically synthesized sucrose fatty acid ester High purity having a specific esterification site, selectively decomposing only the specific substitution position isomers, leaving only the sucrose fatty acid ester resistant to the sucrose fatty acid esterase of the present invention undegraded The monoester substituted regioisomers can be prepared. Such high-purity sucrose fatty acid esters are expected to be useful not only for conventionally known uses of sucrose fatty acid esters but also for a wider range of uses.
[0049]
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further, this invention should not be limitedly interpreted by these Examples.
[0050]
【Example】
1.Ideonera SP No.13 (Trust number: FERM P-16206 ) Purification of sucrose fatty acid esterase from strains
First, for the purpose of purifying sucrose fatty acid esterase, various strains isolated from activated sludge, soil, etc. of a wastewater treatment plant were isolated. The medium contains L + SE medium (peptone 10 g / l, yeast extract 5 g / l, NaCl 5 g / l and sucrose fatty acid ester (DK ester SS, manufactured by Daiichi Kogyo Seiyaku) 10 g / l , PH 7) or L / 10 + SE medium (containing peptone 1 g / l, yeast extract 0.5 g / l, NaCl 0.5 g / l, sucrose fatty acid ester 10 g / l, pH 7), solid To the medium, 15 g / l of agar was further added to any of the above media. Of the 84 isolates isolated, a strain that highly expresses sucrose fatty acid esterase activity and secretes a large amount of this enzyme in the culture medium was isolated, and Ideoneella sp (Ideonella sp.) No.13 This Ideoneella sp. No. 13 strain was deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology, and was deposited with the accession number: FERM P-16206 on April 24, 1997.
[0051]
Here, as an index of sucrose fatty acid esterase activity, the determination result of sucrose fatty acid ester resolution by sucrose fatty acid ester agar method or titration method was adopted. Specifically, these methods are as follows.
[0052]
(a) Sucrose fatty acid ester agar method
A 1.5 wt / vol% agar solution containing 0.2 wt / vol% sucrose fatty acid ester and 50 mM phosphate buffer at pH 7 is solidified in a Petri dish to a thickness of 5-7 mm, and the agar has a diameter. A 4 mm hole was made and 20 μl of the sample solution was injected. The enzyme reaction was allowed to stand overnight at 37 ° C., and the diameter of a cloudy circle (halo) formed around the pores was measured.
[0053]
(b) Titration method
Enzymatic reaction containing 5 ml of 5% w / v sucrose fatty acid ester aqueous solution, 4 ml of McIlvaine buffer (adjusted to pH 8 by mixing 0.2 M disodium hydrogen phosphate and 0.1 M citric acid) and 1 ml enzyme sample solution The solution was incubated at 37 ° C. or 60 ° C. for 30 minutes. After completion of the reaction, 10 ml of a mixture of acetone / ethanol = 50/50 (volume%) and 10 ml of 0.05 N NaOH were added to stop the reaction, and another mixture of acetone / ethanol = 50/50 (volume%) was added to 10 ml. ml was added. The free fatty acid produced by the enzyme reaction was back titrated with 0.05 N hydrochloric acid using phenolphthalein as an indicator. Under this condition, the amount of enzyme that liberates 1 μmol of fatty acid per minute was defined as 1 unit.
[0054]
There is a linear correlation between the halo diameter (mm) measured by the sucrose fatty acid ester agar method (a) and the enzyme activity (unit, logarithm) measured by the titration method (b). It was shown that there is.
[0055]
In order to examine a preferable medium for production and secretion of sucrose fatty acid esterase by Ideonella sp. No. 13 strain selected using such a method, this bacterium was cultured using various media and carbon sources. That is, L medium (containing peptone 10 g / l, yeast extract 5 g / l and NaCl 5 g / l, pH 7), L / 10 medium (peptone 1 g / l, yeast extract 0.5 g / l, NaCl 0.5 Contains g / l, pH 7) as well as minimal medium ((NH_Four)₂SO_Four 5g / l, K₂HPO_Four 0.5 g / l and MgSO_Four・ 7H₂Three types of medium containing O 0.25 g / l and pH 7) were used as the basic medium, and sucrose fatty acid esters and / or glucose (0.5 or 1% by weight / volume) were added as the carbon source. After shaking culture at 37 ° C. for 2 days in these media, the sucrose fatty acid esterase activity of the culture supernatant is measured by the sucrose fatty acid ester agar method or titration method, and the cell growth is determined by OD of the culture solution.₆₀₀More estimated.
[0056]
As a result, Ideoneella sp. No. 13 strain grows well in L / 10 medium supplemented with carbon source, L medium and L medium supplemented with carbon source, and produces and secretes a large amount of sucrose fatty acid esterase. It became clear that. Ideone SSP No. using L medium (hereinafter referred to as “L + Glu 1% medium”) containing 1% by weight / volume of glucose, which is considered to express the highest sucrose fatty acid esterase activity. It was decided to culture .13 strains.
[0057]
In order to obtain a large amount of sucrose fatty acid esterase protein, Ideoneella sp. No. 13 strain was cultured in a large volume. First, Ideone sp. No. 13 strain was cultured in advance in a test tube in 5 ml of an L / 10 medium containing 1% by weight / volume of glucose overnight at 37 ° C. overnight. Next, 1 L of L + Glu 1% medium supplemented with this culture solution was cultured with shaking overnight at 37 ° C. in a 2 L Sakaguchi flask to prepare a preculture solution. This preculture is added to 50 L of L + Glu 1% medium, and the aeration rate is 50 L / min at 37 ° C. in a 100 L capacity jar fermenter (type MPF, manufactured by Marubishi Bio Engine). 1 vvm) and cultured for 48 hours.
[0058]
The culture broth thus obtained was removed from the bacterial cell components with a Sharpless continuous centrifuge, and the supernatant was reduced to 1/10 volume with an ultrafiltration concentrator (Asahi Kasei Co., Ltd., Microza UF SPL-1053) at 4 ° C. Until concentrated. Subsequently, ammonium sulfate was added to the concentrate so as to be 60 wt / vol% saturation, and the mixture was allowed to stand overnight, and the resulting precipitate was collected by centrifugation. This precipitate was dissolved in 20 mM Tris-HCl buffer having a pH of 7.5, and desalted by dialysis in the same buffer. The desalted enzyme solution was freeze-dried to obtain a powdery crude enzyme. The obtained crude enzyme was found to be about 3000 units / g when sucrose fatty acid esterase activity was measured by a titration method. Using this crude enzyme, sucrose fatty acid esterase protein was purified according to the following steps. In the purification step, the sucrose fatty acid esterase activity was measured using the sucrose fatty acid ester agar method or titration method as an index.
[0059]
(1) First anion exchange column: DEAE cellulose (DE52, manufactured by Whatman) φ5.7 × 14 cm packed in the column was equilibrated with 20 mM Tris-HCl buffer (pH 8), and a protein sample solution (2 g Protein). In the same buffer, protein was eluted with a linear concentration gradient of 0 to 0.5 M NaCl, and fractions exhibiting sucrose fatty acid esterase activity were collected.
[0060]
(2) Second anion exchange column: Hitrap Q (Pharmacia, 1 ml) with the active fraction of (1) in the same buffer system as in (1), and sucrose fatty acid esterase Was purified. The column was equipped with an FPLC system (Pharmacia), and the protein was eluted by applying a linear concentration gradient over 50 minutes at a flow rate of 1 ml / min. The enzyme activity eluted at a NaCl concentration of about 0.25 M.
[0061]
(3) Third anion exchange column: Using the same buffer as in (1), the active fraction obtained in (2) on the high performance anion exchange column Mono Q (Pharmacia, 1 ml) Further purified. The column was equipped with an FPLC system (Pharmacia), and the protein was eluted by applying a linear concentration gradient over 30 minutes at a flow rate of 1 ml / min. The desired esterase activity eluted at a NaCl concentration around 0.2 M. The elution profile is shown in FIG. An arrow is attached to the peak of the esterase activity.
[0062]
(4) Gel filtration column: Using 50 mM phosphate buffer (pH 7.2) containing 150 mM NaCl, the active fraction obtained in (3) is gel filtration column Supedex 200 HR10 / 30 (manufactured by Pharmacia) The active fraction was collected by eluting at a flow rate of 0.2 ml / min. The elution profile from this column is shown in FIG.
[0063]
By these steps, sucrose fatty acid esterase could be purified to a state close to a single band on SDS electrophoresis. The apparent molecular weight of the protein corresponding to the main band on electrophoresis was about 48,000 daltons. A portion corresponding to this protein band was cut out from the gel after SDS electrophoresis, and an analysis of the N-terminal amino acid sequence was attempted, but the sequence could not be determined. This was probably because the N-terminus of the protein was blocked.
[0064]
2.Identification of sucrose fatty acid esterase
In order to enzymatically identify the sucrose fatty acid esterase possessed by Ideoneella sp. No. 13, experiments were conducted to examine the following characteristics.
[0065]
(1) Temperature dependence: Using the sucrose fatty acid esterase crude enzyme described in 1), the temperature dependence of the sucrose fatty acid ester decomposition reaction was examined. The enzyme activity was measured using 2 units of crude enzyme by the titration method. The results are shown in FIG. 3, and it is clear that the activity is the highest at 60 ° C. and the activity is also at 70-80 ° C.
[0066]
(2) pH dependence: The pH dependency of the sucrose fatty acid ester decomposition reaction was examined using the crude enzyme described in 1. above. The enzyme activity was measured using the above-mentioned McIlvaine buffer at pH 4-8, and Clark-Lubs buffer ((i) 0.1 M boric acid + 0.1 M KCl, (ii) 0.1 M at pH 9-10). According to the titration method, 2 units of crude enzyme were used except that NaOH (adjusted to a predetermined pH with (i) + (ii)) was used. The results are shown in FIG. 4, and it is clear that the optimum pH of this enzyme activity is 8, and it has high activity at a pH in the range of 6-8.
[0067]
(3) Substrate specificity: Degradation activity of typical ester compounds that can generally serve as substrates for esterases or lipases. The crude enzyme described in 1. The reaction conditions are essentially 1. This is the same as the titration method described in 1. The reaction solution of pH 8 McIlvaine buffer, 4.75 ml water, 0.25 g of each substrate and 2 units (1 ml) of crude enzyme was reacted at 60 ° C for 30 minutes. Each degradation product was quantified. The obtained results are shown in Table 1.
[0068]
[Table 1]

[0069]
As a result, this enzyme decomposed Tween 80 (polyoxyethylene sorbitan fatty acid ester) which is an ester derivative of saccharides in addition to sucrose fatty acid monoester. Even sugar esters had little effect on sucrose fatty acid polyesters that were not soluble in water. In addition, ethyl acetate, which is a substrate for esterase, is water-soluble under the reaction conditions, but the enzyme hardly acted. Although the data is not shown, this enzyme showed almost no activity even for paranitrophenyl acetate which is widely used as a substrate for measuring the activity of esterase and lipase. Furthermore, as shown in Table 1, since this enzyme showed little activity against triglycerides such as water-soluble triacetin and water-insoluble tributin and olive oil, this enzyme does not belong to an enzyme called so-called lipase. it is conceivable that. Thus, the obtained enzyme was found to act specifically on the water-soluble saccharide ester.
[0070]
(4) Decomposition reaction of sucrose fatty acid ester
1. The decomposition reaction of sucrose fatty acid ester was confirmed using the crude enzyme described in 1. As the sucrose fatty acid ester, DK ester SS (Daiichi Kogyo Seiyaku Co., Ltd.) containing sucrose monostearate as a main component was used. 50 μl of 5% w / v DK ester SS aqueous solution, 0.1 M phosphate buffer (pH 7), 10 μl of crude enzyme solution (0.6 units), 0-100 μl of dimethyl sulfoxide (final concentrations 0, 5, 10, 25 and 50) The reaction solution having a total volume of 200 μl consisting of a volume%) and an appropriate amount of water was placed in an Eppendorf tube and reacted at 53 ° C. overnight, and then 800 μl of ethanol was added to stop the reaction. As a control group, the same operation was performed with a reaction solution not containing the crude enzyme solution. After centrifugation at 10,000 rpm for 5 minutes, 10 μl of the obtained supernatant was subjected to thin layer chromatography analysis. For thin layer chromatography, silica gel plate 60 (manufactured by Merck & Co., Inc.) is used, and a mixed solvent of chloroform / methanol / acetic acid / water = 80/10/8/2 is used as a developing solvent. After spraying, it was performed by heating in an oven. The result is shown in FIG. In FIG. 5, the reaction substrate, sucrose monostearate, is shown primarily as three spots, representing the three substitution position variants that probably differ in the esterification site. The respective Rf values were approximately 0.21, 0.18 and 0.14. When the reaction is carried out with the addition of this enzyme, two spots with low Rf values (Rf values: 0.18 and 0.14) among these three spots when the dimethyl sulfoxide concentration is 25% by volume or less (FIG. 5, lanes fi). Therefore, it is clear that sucrose monostearate is decomposed by the enzyme in an esterification site-specific manner, and a sucrose fatty acid monoester having a limited ester substitution position is obtained. It was.
[0071]
When 50% by volume of dimethyl sulfoxide was present in the reaction solution, the decomposition reaction did not proceed (the same, lane j). When no enzyme was added, no degradation reaction was observed at any dimethyl sulfoxide concentration (same lanes a to e).
[0072]
(5) Synthesis reaction of sucrose fatty acid ester
1. Using the crude enzyme described in 1., various sucrose fatty acid esters were synthesized. 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 were added to the reaction solution to perform solvent extraction, and 40 μl of the lower chloroform layer was subjected to the same thin layer chromatography analysis as described in (4) above. Here, as a developing solvent, a mixed solvent of chloroform / methanol / acetic acid / water = 70/20/8/2 was used. As a control group, the same operation was performed with a reaction solution not containing the crude enzyme solution. The result is shown in FIG. 6 (in the figure, the presence of a weak spot was marked by handwriting to indicate the presence). In FIG. 6, 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 the fatty acids of each carbon number are indicated by arrows. Two of each of the reaction products shown are generated (Figure 6, lanes b, d and f). Here, the Rf value of the product increases as the carbon number of the fatty acid increases, and this corresponds to the fact that the present enzyme decomposes the two sucrose fatty acid ester isomers in the decomposition reaction of (4) above. Based on the fact that there are two kinds of reaction products, it is considered that two kinds of substitution position isomers of sucrose fatty acid monoester were generated by these esterase reactions.
[0073]
Furthermore, in order to confirm the formation of esters in the reaction under the same conditions, the products synthesized by reacting sucrose and lauric acid were examined. Reaction conditions are the same as above, and thin-layer chromatography analysis is performed to compare the extract after completion of the reaction with a standard sample (DK ester S-L18A, manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) containing sucrose monolaurate. did. As the developing solvent, a mixed solvent of chloroform / methanol / acetic acid / water = 70/20/8/2 was used. The result is shown in FIG. As clearly shown in FIG. 7, a product having an Rf value (0.40 and 0.33) similar to that of the standard sucrose monolaurate was obtained by the reaction between 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. 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. The retention times of the main peaks a and b were consistent 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. Accordingly, 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.
[0074]
3.Cloning of sucrose fatty acid esterase gene
Cloning of the sucrose fatty acid esterase gene derived from Ideoneella sp. No. 13 was performed as follows using the shotgun method. That is, the sarkosyl method [Morikawa et al.J. Ferment.Bioeng.74, pp. 255-261 (1992)], the chromosomal DNA of Ideoneella sp.EcoCut with RI and pBR322 vectorEcoLigated to the RI site. A genomic library was created using Wizard Kit (Promega) according to the manufacturer's protocol. With the resulting genomic libraryEpicurian coli ABLE K [lac (LacZw ^- ), Kan ^- , McrA ^- , McrCB ^- , McrF ^- , Mrr ^- , HsdRk ^- Mk ^- [F'proAB, lacI ^q Z Δ M15, Tn10 (Tet ^r )]] (Stratagene) was transformed according to the method of Lederberg and Cohen (Lederberg, E.M. and Cohen, S.N., J. Bacteriol. 119, 1072-1074 (1974)). This host cell is a commonly used host cellE. coli The number of vector copies is reduced to 1/10 compared to JM109. This transformant was applied to L medium containing 50 μg / ml of ampicillin, 2 g / L of sucrose fatty acid ester (DK ester SS) and 1.5% weight / volume agar. After overnight culture at 37 ° C., it was left at room temperature for 1 week. About 5,000 transformant colonies were searched for the target clone using white halo formed by the action of esterase in the vicinity as an index. Thus, 11 positive clones that were weak but formed a halo were obtained.
[0075]
About 10 kb from this positive cloneEcoRI−EcoThe RI fragment was removed and subjected to subcloning. In subcloning,E. coli JM109 [recA1, endA1, gryA96, thi, hsdR17, supE44, relA1, Δ (Lac-proAB), F '(traD36, proAB ⁺ , lacI ^q , lacZ Δ M15)] As a host cell and pUC18 as a vector. Then about 10 kbEcoRI−EcoRI fragment,BamDigested with HI. The cleavage site is as shown in FIG. 9, a, and when the esterase activity was confirmed by the agar method for each of the four fragments obtained,EcoRI−Eco3.3 kb, located 5 'upstream of the RI fragmentEcoRI−BamHigh activity was observed in the HI (FIG. 9, b) fragment. This activity isEcoRI−EcoIt was higher than the RI fragment. Obtained in this wayEcoRI−BamThe base sequence of the HI fragment was analyzed with an ALFexpress apparatus using an Auto Read Sequencing Kit (Pharmacia) according to the Dideoxy method. The base sequence thus obtained was as shown in SEQ ID NO: 1.
[0076]
Next, in order to specify the structural gene of esterase in the obtained base sequence, the upstream and downstream regions of the DNA were deleted, and based on the fluctuation of esterase activity accompanying the deletion, a fragment of 1613 base pairs (FIG. 9). The active fragment was limited to c). As the area was limited in this way, the diameter of the halo around the colony increased, suggesting that the esterase activity expressed by the obtained fragment was enhanced.
[0077]
Examination of the base sequence of this 1613 base pair fragment indicated that an open reading frame of 1416 base pairs was present. In addition, when this fragment was inserted into the pUC19 vector in the opposite direction to the promoter, the esterase activity was significantly reduced, suggesting that the orientation of this gene is as shown in FIG.
[0078]
As shown in FIGS. 9 and c, in the open reading frame of the 1613 base pair fragment encoding the obtained esterase DNA, sequences of four initiation codons indicated by ▲ were observed. In order to confirm the correct start codon, the sequence 5 ′ upstream of this fragment was deleted. As shown in FIG. 9 and d, since the esterase activity was not observed when it was deleted to the downstream of the second ATG sequence, the start codon was considered to be the first or second ATG sequence. Furthermore, when the Shine-Dalgarno sequence was searched, it was not found upstream of the first ATG sequence (positions 689 to 691 of SEQ ID NO: 1), but the second ATG sequence (positions 710 to 712 of SEQ ID NO: 1) The presence of the sequence was confirmed upstream (positions 697 to 703). Therefore, the second ATG sequence was considered the start codon. Further, a terminator sequence (positions 2134 to 2149 in SEQ ID NO: 1) was found downstream of the stop codon.
[0079]
From the above results, the structural gene of sucrose fatty acid esterase possessed by Ideoneella sp. No. 13 consists of 1416 base pairs, the number of amino acid residues constituting the enzyme protein is 471 (see SEQ ID NO: 2), and the protein The molecular weight of was estimated to be 48,758 daltons. This is 1. In FIG. 4, the apparent molecular weight of the esterase protein derived from Ideoneella sp.
[0080]
The amino terminal portion of the amino acid sequence of this protein is followed by a sequence rich in non-polar amino acids followed by a sequence rich in basic amino acids, and it is estimated that a signal peptide consisting of about 20 amino acid residues exists. It was.
[0081]
In addition, the amino acid sequence “-Gly-Xaa-Ser-Xaa-”, which is common to the active centers of the proteins of esterases and lipases, is located in the almost central part of the amino acid sequence (amino acid residues 189 to 193 of SEQ ID NO: 1). "Gly-" (SEQ ID NO: 4) was found. In order to replace the serine residue in this with a cysteine residue, the mutation which makes TGC the TCG of the 1340th to 1342th base sequence of SEQ ID NO: 1 was introduced, and the protein encoded by this DNA was expressed. Its esterase activity was shown to be significantly lower. Therefore, it was confirmed that the sequence consisting of these five amino acid residues is the active center.
[0082]
The homology with the known sequence of the esterase sequence derived from Ideoneella sp. Thus clarified was analyzed using DNASIS (manufactured by Hitachi). In general, the sequences of eukaryotic esterases and lipases have been found to exhibit high homology [Cygler, M. et al.Protein Science2, 366-382 (1993)] have been reported to be less homologous to bacterial lipases (Jeager, KE et al., Bacterial lipase, FEMS Microbiology Reviews, 15, 29-63). (1994)). As a result of searching the sequence, no one showing high homology with the present sucrose fatty acid esterase protein was found.
[0083]
4).Protein expression in transformants containing sucrose fatty acid esterase gene
3 above. The portion from position 658 to 2276 of the DNA shown in SEQ ID NO: 1 prepared by the above step was inserted into the multi-cloning site of the pUC18 vector, and this was used for E. coli (E.coli JM109) was transformed. In the same manner as described above, transformation was performed according to the method of Lederberg and Cohen.
[0084]
The transformed E. coli was applied to an L agar medium containing 50 μg / ml of ampicillin and 2 g / L of sucrose fatty acid ester (DK ester SS), and cultured overnight at 37 ° C. One colony forming a halo was selected, inoculated into 5 ml of L medium containing 50 μg / ml of ampicillin, and cultured at 37 ° C. with shaking. Two hours after the start of the culture, IPTG (isopropylthiogalactoside) was added so that the final concentration was 0.1 mM, and induced expression was performed. The culture was further terminated by shaking for 3 hours. The culture was collected by centrifugation at 10,000 rpm for 2 minutes. The collected cells were suspended in 50 mM phosphate buffer pH 7.0 and sonicated. When the activity of the crushed liquid was measured by the agar method described above, 0.25 units of enzyme activity was detected per 1 ml of the culture solution.
[0085]
【The invention's effect】
The sucrose fatty acid esterase of the present invention has the effect that it is possible to synthesize sucrose fatty acid esters directly from sucrose and fatty acids under mild conditions with little by-products and without the use of raw materials such as special derivatives. Played. In particular, since the sucrose fatty acid ester produced in such a reaction is a monoester, and a polyester higher than a diester is not synthesized, the present invention provides a very useful production method for selectively producing a sucrose fatty acid monoester. It is provided. In addition, since the sucrose fatty acid monoester is obtained as a result of regioselective esterification of sucrose in particular, the present invention contributes to the production of sucrose fatty acid monoester with limited substitution positions. Roh is big.
[0086]
Further, since the structure of the sucrose fatty acid esterase protein gene has been elucidated by the present invention, it is possible to modify the base sequence of this gene to further produce an enzyme advantageous for sucrose fatty acid ester synthesis. For example, it is expected that a more stable enzyme can be created by performing modifications that inhibit the degradation of the enzyme protein itself, as is done in proteases. It is considered that a large amount of enzyme can be produced by expressing such a recombinant enzyme in an appropriate host cell.
[0087]
In addition, using the site specificity of the sucrose fatty acid ester degradation reaction by this sucrose fatty acid esterase, two of the three monoester isomers contained in the chemically synthesized sucrose fatty acid ester are selected. It is possible to obtain a highly pure monoester isomer having a specific esterification site. Such high-purity sucrose fatty acid esters are expected to be useful not only for conventionally known uses of sucrose fatty acid esters but also for a wider range of uses.
[0088]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a view showing a mode of protein elution from a high-performance anion exchange column, which is one step of the method for producing sucrose fatty acid esterase of the present invention.
FIG. 2 is a diagram showing an embodiment of protein elution from a gel filtration column, which is one step in the method for producing sucrose fatty acid esterase of the present invention.
FIG. 3 is a graph showing the pH dependence of the activity of the sucrose fatty acid esterase of the present invention.
FIG. 4 is a graph showing the temperature dependence of the sucrose fatty acid esterase of the present invention.
FIG. 5 is a view showing an aspect of decomposition of sucrose fatty acid ester by sucrose fatty acid esterase of the present invention.
FIG. 6 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. 7 is a diagram showing an embodiment of synthesis of sucrose laurate by sucrose fatty acid esterase of the present invention.
8 is a diagram showing separation of sucrose laurate obtained in FIG. 7 by high performance liquid chromatography.
FIG. 9 shows a restriction enzyme map of the sucrose fatty acid esterase gene of the present invention.

Claims (18)

Sucrose fatty acid esterase involved in the synthesis / degradation reaction of sucrose fatty acid ester using sucrose and fatty acid as substrate, and derived from Ideonella sp. No. 13 (FERM P-16206) strain protein. The following amino acid sequence:
Met-Arg-Phe-Leu-Arg-Pro-Leu-Ser-Leu-Leu-Pro-Leu-Thr-Ala-Ala-Leu-
Val-Leu-Ala-Gly-Cys-Gly-Gly-Gly-Ser-Glu-Asp-Ala-Pro-Pro-Ala-Arg-
Gly-Thr-Ile-Met-Ser-Ala-Leu-Ala-Asp-Ala-Gln-Leu-Asn-Pro-Asn-Gln-
Thr-Trp-Pro-Ile-Pro-Ala-Ala-Gln-Ile-Asp-Ala-Gly-Thr-Ser-Ala-Ser-
Gly-Val-Gln-Ala-Leu-Thr-Gly-Ala-Ala-Thr-Cys-Asp-Val-His-Ile-Ala-
Tyr-Val-Leu-Tyr-Met-Thr-Arg-Asp-Pro-Lys-Gly-Glu-Ala-Ala-Thr-Ala-
Ser-Thr-Ala-Val-Phe-Val-Pro-Ser-Gly-Thr-Asn-Ala-Ala-Cys-Thr-Gly-
Ser-Arg-Pro-Val-Val-Leu-Tyr-Ala-His-Gly-Thr-Thr-Thr-Glu-Lys-Ala-
Phe-Asn-Met-Ala-Asp-Ile-Ala-Asn-Asn-Gln-Glu-Gly-Ser-Leu-Thr-Ala-
Ala-Met-Phe-Ala-Ala-Gln-Gly-Tyr-Ile-Val-Val-Ala-Pro-Asn-Tyr-Leu-
Gly-Tyr-Asp-Leu-Ser-Ser-Leu-Ser-Tyr-His-Pro-Tyr-Leu-Asn-Ala-Glu-
Ala-Glu-Ala-Val-Asp-Met-Val-Asp-Gly-Leu-Arg-Ala-Ala-Lys-Ser-Tyr-
Leu-Thr-Ala-Ser-Gly-Leu-Gly-Thr-Pro-Ser-Ala-Gln-Leu-Phe-Val-Thr-
Gly-Tyr-Ser-Glu-Gly-Gly-His-Val-Ala-Met-Ala-Thr-Gln-Lys-Val-Leu-
Glu-Arg-Asp-Tyr-Ala-Ser-Glu-Phe-Thr-Val-Thr-Ala-Ala-Ala-Pro-Met-
Ser-Gly-Pro-Tyr-Asn-Leu-Thr-Lys-Phe-Val-Gly-Gln-Ile-Met-Ala-Ser-
Pro-Asp-Tyr-Cys-Ala-Ser-Val-Gly-Ser-Thr-Asp-Pro-Asn-Cys-Ala-Val-
Asn-Val-Gly-Ala-Thr-Leu-Phe-Thr-Pro-Leu-Leu-Leu-Thr-Ser-Trp-Gln-
Lys-Ala-Tyr-Gly-Ile-Tyr-Gly-Ser-Ala-Ser-Asp-Val-Tyr-Gln-Ala-Ala-
Tyr-Ala-Gly-Phe-Met-Glu-Thr-Leu-Leu-Pro-Ser-Asn-Ser-Ser-Val-Asp-
Asp-Leu-Val-Ala-Ala-Gly-Lys-Leu-Pro-Ala-Asp-Pro-Thr-Ala-Arg-Leu-
Leu-Phe-Gly-Thr-Gly-Gly-Met-Ile-Asn-Glu-Ser-Phe-Arg-Thr-Ala-Ala-
Leu-Ala-Asn-Ala-Ser-Ser-Gly-Leu-Met-Gly-Ala-Ala-Ala-Thr-Asn-Thr-
Leu-Leu-Gly-Trp-Lys-Pro-Lys-Asn-His-Met-Ala-Met-Cys-Tyr-Ser-Ser-
Ala-Asp-Pro-Thr-Val-Tyr-Gly-Tyr-Asn-Thr-Thr-Asp-Ala-Gln-Ala-Asp-
Phe-Tyr-Ser-Ser-Gly-Val-Thr-Val-Pro-Ala-Leu-Asn-Leu-Arg-Gly-Asp-
Leu-Ala-Thr-Ile-Gln-Thr-Gln-Phe-Gly-Ala-Ala-Thr-Ala-Gln-Leu-Ala-
Gly-Ala-Phe-Gln-Gln-Thr-Tyr-Ser-Leu-Ser-Val-Pro-Gln-Asn-Gln-Gly-
Asn-Glu-His-Ser-Asn-Ala-Ala-Pro-Phe-Cys-Ala-Ala-Phe-Val-Arg-Gly-
Tyr-Phe-Ala-Asn-Phe-Ala-Gln
Or a sucrose fatty acid ester comprising a sucrose and a fatty acid as a substrate, wherein the amino acid sequence is represented by the following formula or an amino acid sequence in which one or several amino acids are deleted, substituted and / or added: A protein having sucrose fatty acid esterase activity involved in the degradation reaction. A method for producing a sucrose fatty acid esterase involved in the synthesis / degradation reaction of a sucrose fatty acid ester using sucrose and a fatty acid as a substrate, which comprises Ideonella sp. No. 13 ) were cultured, and, to obtain the culture supernatant, the manufacturing method of the sucrose fatty acid esterase protein comprising the step. The method according to claim 3, further comprising a step of obtaining a fraction of sucrose fatty acid esterase activity separated by a purification step combining anion exchange column chromatography and / or gel filtration column chromatography. Claim 3 or 4 produced by the method described in, and sucrose and proteins with sucrose fatty acid esterase activity involved the fatty acid synthesis / degradation reaction of sucrose fatty acid ester as a substrate. A DNA encoding the protein according to claim 2 . The following base sequence:
5'-ATGCGTTTCC TGCGCCCATT GTCTTTGCTG CCGCTGACTG CGGCCCTTGT GTTGGCCGGT
TGTGGTGGTG GCAGCGAGGA TGCCCCGCCG GCTCGGGGAA CCATCATGTC TGCGCTGGCG
GATGCCCAGC TCAACCCCAA CCAGACATGG CCCATCCCTG CGGCCCAGAT CGACGCCGGA
ACCTCGGCGT CCGGCGTTCA GGCGCTGACC GGTGCTGCGA CCTGTGATGT GCACATCGCC
TACGTGCTGT ACATGACGCG TGACCCGAAG GGTGAGGCGG CCACCGCCTC CACCGCGGTG
TTCGTGCCTT CCGGCACCAA CGCGGCCTGC ACGGGCTCTC GGCCGGTGGT GCTGTATGCG
CACGGCACGA CGACCGAGAA GGCCTTCAAC ATGGCCGACA TCGCCAACAA CCAGGAAGGC
AGCCTGACCG CTGCCATGTT CGCCGCGCAA GGCTACATCG TCGTCGCGCC GAACTACCTG
GGTTACGACC TTTCCAGCCT GAGCTACCAC CCCTACCTCA ACGCCGAAGC CGAGGCGGTG
GACATGGTCG ATGGTCTGCG TGCCGCCAAG AGCTATCTGA CGGCGTCCGG TCTGGGCACG
CCCTCGGCCC AGCTGTTCGT GACCGGCTAT TCGGAAGGTG GCCATGTGGC CATGGCCACC
CAGAAGGTGC TCGAGCGCGA CTATGCGAGC GAGTTCACCG TGACGGCCGC CGCGCCCATG
TCTGGTCCTT ATAACCTGAC CAAGTTCGTT GGCCAGATCA TGGCCAGCCC AGACTATTGC
GCCAGTGTCG GCTCCACCGA CCCGAACTGC GCGGTCAATG TGGGCGCTAC GCTGTTTACT
CCGCTACTGC TCACCAGCTG GCAGAAAGCC TACGGTATCT ACGGCTCGGC CTCGGACGTG
TATCAGGCGG CCTACGCCGG GTTCATGGAG ACGCTGCTGC CTAGCAACAG CTCCGTGGAC
GACTTGGTCG CAGCAGGCAA GTTGCCGGCC GATCCGACAG CCCGCCTGCT GTTCGGCACC
GGTGGCATGA TCAATGAAAG CTTCCGCACT GCAGCCTTGG CCAATGCGTC CAGCGGCTTG
ATGGGGGCGG CTGCCACCAA TACCTTGCTG GGGTGGAAGC CCAAGAACCA CATGGCGATG
TGCTACTCGT CCGCAGACCC GACCGTGTAT GGCTATAACA CGACGGATGC GCAGGCCGAC
TTTTACAGCA GTGGCGTGAC CGTGCCTGCT CTGAACCTGC GGGGAGACTT AGCCACGATT
CAAACTCAGT TCGGCGCGGC GACGGCGCAA CTGGCAGGTG CTTTCCAACA GACCTATTCG
CTGAGCGTGC CGCAGAACCA GGGCAACGAG CATAGCAACG CCGCTCCCTT CTGCGCGGCC
TTCGTCCGCG GCTACTTCGC GAACTTCGCC CAGTGA-3 '
In indicated by Do nucleotide sequence Ri, and sucrose and fatty acids encoding a protein having sucrose fatty acid esterase activity involved in the synthesis / degradation reaction of sucrose fatty acid ester as a substrate DNA. 7. encoded by DNA according to, and sucrose and proteins with sucrose fatty acid esterase activity involved the fatty acid synthesis / degradation reaction of sucrose fatty acid ester as a substrate. A recombinant vector containing the DNA according to claim 6 or 7 . A host cell transformed with the recombinant vector according to claim 9 . The host cell according to claim 10 , which is Escherichia coli. Claim 10 or 11 produced by the host cell according to, and sucrose and recombinant proteins with sucrose fatty acid esterase activity involved the fatty acid synthesis / degradation reaction of sucrose fatty acid ester as a substrate. A method for producing a sucrose fatty acid esterase involved in a synthesis / decomposition reaction of a sucrose fatty acid ester using sucrose and a fatty acid as a substrate, culturing the host cell according to claim 10 or 11 , and a culture solution thereof A method for producing a sucrose fatty acid esterase protein, comprising a step of obtaining a supernatant. The method according to claim 13, further comprising a step of obtaining a fraction of sucrose fatty acid esterase activity separated by a purification step combining anion exchange column chromatography and / or gel filtration column chromatography. A method for producing a sucrose fatty acid ester, comprising a step of allowing a protein having sucrose fatty acid esterase activity according to any one of claims 1, 2, 5 , 8 or 12 to act on sucrose and a fatty acid. 16. The process according to claim 15 , wherein a product free of polyesters above the diester is obtained , i.e. a product of sucrose fatty acid monoester. The method according to claim 15 or 16 , wherein two kinds of sucrose fatty acid monoester substituted regioisomers are produced. A plurality of sucrose fatty acid monoester substituted regioisomer mixture synthesized by chemical methods, Ru allowed to act for a protein having the sucrose fatty acid esterase activity of any of claims 1, 2, 5, 8 or 12 A method for producing a sucrose fatty acid monoester having a limited substitution position , comprising a step .

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