JPH07322883A - Recombinant type enzyme, its production and use thereof - Google Patents

Recombinant type enzyme, its production and use thereof

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Publication number
JPH07322883A
JPH07322883A JP7058258A JP5825895A JPH07322883A JP H07322883 A JPH07322883 A JP H07322883A JP 7058258 A JP7058258 A JP 7058258A JP 5825895 A JP5825895 A JP 5825895A JP H07322883 A JPH07322883 A JP H07322883A
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JP
Japan
Prior art keywords
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arg
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Prior art date
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Application number
JP7058258A
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Japanese (ja)
Other versions
JP3557272B2 (en
Inventor
Michio Kubota
倫夫 久保田
Keiji Tsusaki
桂二 津▲さき▼
Kazuhiko Maruta
和彦 丸田
Toshiyuki Sugimoto
利行 杉本
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hayashibara Seibutsu Kagaku Kenkyujo KK
Original Assignee
Hayashibara Biochemical Laboratories Co Ltd
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Priority to JP05825895A priority Critical patent/JP3557272B2/en
Publication of JPH07322883A publication Critical patent/JPH07322883A/en
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Publication of JP3557272B2 publication Critical patent/JP3557272B2/en
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

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  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

PURPOSE:To obtain a recombinant type enzyme capable of producing a glucide, useful for sweetening a food and drink without any anxiety about coloring arid deteriorating and having a trehalose structure from a starch saccharide. CONSTITUTION:This recombinant type enzyme is capable of producing a nonreducing glucide having a trehalose structure at the terminal obtained from a reducing starch saccharide having >=3 glucose polymerization degree and has the following physico-chemical properties: (1) about 76000 to 87000Da molecular weight measured by a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; (2) about 3.6-4.6 isoelectric point measured by an isoelectric focusing; (3) 35-40 deg.C optimum temperature; (4) 6.4-7.2 optimum pH and (5) stable to about 35-40'C by incubation at pH 7.0 for 60min and stable at about pH 5.5-11.0 by incubation at 25 deg.C for 16hr. The recombinant type enzyme has, e.g. an amino acid sequence expressed by the formula and is obtained from a culture of a transformant BMT7, etc., prepared by using a chromosomal DNA of Rhizobium sp. M-11 (FERM BP-4130), etc.

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【産業上の利用分野】この発明は、グルコース重合度３
以上の還元性澱粉糖から末端にトレハロース構造を有す
る非還元性糖質を生成する新規な組換え型酵素とその製
造方法並びに用途に関する。This invention relates to a glucose polymerization degree of 3
The present invention relates to a novel recombinant enzyme for producing a non-reducing sugar having a trehalose structure at the terminal from the reducing starch sugar, a method for producing the same, and an application thereof.

【０００２】[0002]

【従来の技術】トレハロースは、グルコース２分子が還
元性基同士結合した二糖類であり、天然には細菌、真
菌、藻類、昆虫などに微量存在する。トレハロースは分
子中に還元性基を持たないので、アミノ酸類の存在下で
加熱しても褐変反応を起こすことがなく、着色や変質の
懸念なく飲食物を甘味付けできる利点がある。しかしな
がら、従来の製造方法では所望量を入手するのが難し
く、実際に飲食物の甘味付けに使われることは殆ど無か
った。BACKGROUND OF THE INVENTION Trehalose is a disaccharide in which two glucose molecules are linked to each other by reducing groups, and is naturally present in trace amounts in bacteria, fungi, algae, insects and the like. Since trehalose does not have a reducing group in the molecule, it does not cause a browning reaction even when heated in the presence of amino acids, and has an advantage that foods and drinks can be sweetened without fear of coloring or deterioration. However, it is difficult to obtain a desired amount by the conventional production method, and it has hardly been actually used for sweetening foods and drinks.

【０００３】これまでの製造方法は、微生物の菌体を利
用する方法と、糖質に複合酵素系を作用させる方法とに
大別される。前者の方法は、特開昭５０−１５４４８５
号公報などにも見られるように、細菌、酵母などの微生
物を栄養培地で増殖させ、培養物中の菌体からトレハロ
ースを採取するものである。一方、後者の方法は、特開
昭５８−２１６６９５号公報などにも見られるように、
基質にマルトースを使用し、これにマルトース・フォス
フォリラーゼとトレハロース・フォスフォリラーゼから
なる複合酵素系を作用させ、生成したトレハロースを系
外に取出すものである。前者の方法は、微生物そのもの
の増殖は比較的容易なものの、トレハロースを菌体から
採取するのに一連の繁雑な工程を要し、しかも、菌体に
含まれるトレハロースが１５％（ｗ／ｗ）と僅少である
という問題があった。後者の方法は、トレハロースその
ものの分離は比較的容易なものの、反応自体が２種類の
酵素による平衡反応であり、しかも、その平衡が常時グ
ルコース燐酸側に傾いていることから、基質を高濃度に
して反応させ、トレハロースの収量を上げることが原理
的に難しかった。The conventional production methods are roughly classified into a method of utilizing microbial cells and a method of allowing a complex enzyme system to act on sugars. The former method is disclosed in JP-A-50-154485.
As can be seen in the publications, etc., microorganisms such as bacteria and yeast are grown in a nutrient medium, and trehalose is collected from the bacterial cells in the culture. On the other hand, the latter method, as seen in JP-A-58-216695,
Maltose is used as a substrate, and a complex enzyme system composed of maltose phosphorylase and trehalose phosphorylase is allowed to act on this, and the produced trehalose is taken out of the system. Although the former method is relatively easy for the microorganisms to grow, it requires a series of complicated steps to collect trehalose from the cells, and the trehalose contained in the cells is 15% (w / w). There was a problem that it was scarce. In the latter method, although trehalose itself is relatively easy to separate, the reaction itself is an equilibrium reaction by two kinds of enzymes, and the equilibrium is always inclined to the glucose phosphate side. In principle, it was difficult to increase the yield of trehalose by reacting with the above reaction.

【０００４】斯かる状況に鑑み、本発明者が、澱粉糖か
らトレハロース構造を有する糖質を生成する酵素につき
鋭意検索したところ、リゾビウム・スピーシーズＭ−１
１やアルスロバクター・スピーシーズＱ３６などの微生
物が、グルコース重合度３以上の還元性澱粉糖から末端
にトレハロース構造を有する非還元性糖質を生成すると
いう、従来未知の全く新規な酵素を産生することが判明
した。この知見とあい前後して、この非還元性糖質は、
同じくリゾビウム・スピーシーズＭ−１１やアルスロバ
クター・スピーシーズＱ３６が産生する別の酵素によ
り、ほぼ定量的にトレハロースとグルコース及び／又は
マルトオリゴ糖に加水分解されることが判明した。これ
ら酵素を併用することにより、澱粉を原料に所望量のト
レハロースが比較的容易に得られることとなり、トレハ
ロースに係わる前記課題は悉く解決されていくものと期
待される。しかしながら、リゾビウム・スピーシーズＭ
−１１もアルスロバクター・スピーシーズＱ３６も当該
酵素の産生能が充分でなく、トレハロースや末端にトレ
ハロース構造を有する非還元性糖質を大規模に製造しよ
うとすると、微生物を大量に培養しなければならないと
いう問題がある。In view of such a situation, the present inventor conducted an intensive search for an enzyme that produces a sugar having a trehalose structure from starch sugar, and found that Rhizobium species M-1
Microorganisms such as No. 1 and Arthrobacter species Q36 produce a completely unknown enzyme which is unknown in the past, which produces a non-reducing sugar having a trehalose structure at the end from a reducing starch sugar having a glucose polymerization degree of 3 or more. It has been found. Around this finding, this non-reducing sugar is
Similarly, it was revealed that another enzyme produced by Rhizobium species M-11 and Arthrobacter species Q36 hydrolyzes trehalose and glucose and / or maltooligosaccharides almost quantitatively. By using these enzymes in combination, a desired amount of trehalose can be obtained relatively easily from starch as a raw material, and it is expected that the above-mentioned problems relating to trehalose will be solved completely. However, Rhizobium species M
Neither -11 nor Arthrobacter sp. Q36 has a sufficient ability to produce the enzyme, and if a large-scale production of trehalose or a non-reducing sugar having a trehalose structure at the end is attempted, a large amount of microorganism must be cultured. There is a problem of not becoming.

【０００５】一方、昨今の組換えＤＮＡ技術の進歩には
目覚しいものがある。今日では、全アミノ酸配列が解明
されていない酵素であっても、これをコードする遺伝子
を単離し、その塩基配列を解明できれば、その酵素をコ
ードするＤＮＡを含む組換えＤＮＡを作製し、これを微
生物や動植物の細胞に導入して得られる形質転換体を培
養することにより、比較的容易に所望量の酵素が取得で
きるようになった。斯かる状況に鑑み、両酵素をコード
する遺伝子を突き止め、その塩基配列を解明するのが急
務となっている。On the other hand, recent advances in recombinant DNA technology are remarkable. Nowadays, even for an enzyme whose entire amino acid sequence has not been elucidated, the gene encoding it can be isolated, and if its nucleotide sequence can be elucidated, recombinant DNA containing the DNA encoding that enzyme can be prepared and By culturing a transformant obtained by introducing it into cells of microorganisms and animals and plants, it has become possible to obtain a desired amount of enzyme relatively easily. In view of such a situation, it is urgent to find out the genes encoding both enzymes and elucidate their nucleotide sequences.

【０００６】[0006]

【発明が解決しようとする課題】この発明の目的は、組
換えＤＮＡ技術を応用して斯かる酵素を創製することに
ある。An object of the present invention is to apply such recombinant DNA technology to create such an enzyme.

【０００７】この発明の別の目的は、その創製された酵
素の製造方法を提供することにある。Another object of the present invention is to provide a method for producing the created enzyme.

【０００８】この発明のさらに別の目的は、その創製さ
れた酵素を使用する還元性澱粉糖の変換方法を提供する
ことにある。Yet another object of the present invention is to provide a method for converting reducing starch sugar using the created enzyme.

【０００９】[0009]

【課題を解決するための手段】この発明は、前記第一の
課題を、グルコース重合度３以上の還元性澱粉糖から末
端にトレハロース構造を有する非還元性糖質を生成する
組換え型酵素により解決するものである。Means for Solving the Problems The present invention is directed to the above-mentioned first object by a recombinant enzyme for producing a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. It is a solution.

【００１０】この発明は、前記第二の課題を、その組換
え型酵素を産生する形質転換体を培養し、培養物から組
換え型酵素を採取する組換え型酵素の製造方法により解
決するものである。The present invention solves the above-mentioned second problem by a method for producing a recombinant enzyme by culturing a transformant producing the recombinant enzyme and collecting the recombinant enzyme from the culture. Is.

【００１１】この発明は、前記第三の課題を、グルコー
ス重合度３以上の還元性澱粉糖に組換え型酵素を作用さ
せて該澱粉糖から末端にトレハロース構造を有する非還
元性糖質を生成させる工程を含んでなる還元性澱粉糖の
変換方法により解決するものである。The present invention is directed to the third object, wherein a recombinant enzyme is allowed to act on a reducing starch sugar having a glucose polymerization degree of 3 or more to produce a non-reducing sugar having a trehalose structure at the terminal from the starch sugar. This is solved by a method for converting reducing starch sugar, which comprises the step of:

【００１２】[0012]

【作用】この発明による組換え型酵素は、グルコース重
合度３以上の還元性澱粉糖に作用して末端にトレハロー
ス構造を有する非還元性糖質を生成する。The recombinant enzyme according to the present invention acts on a reducing starch sugar having a glucose polymerization degree of 3 or more to produce a non-reducing sugar having a trehalose structure at the terminal.

【００１３】この発明の製造方法にしたがって形質転換
体を培養すれば、所望量の組換え型酵素が比較的容易に
得られる。By culturing the transformant according to the production method of the present invention, a desired amount of the recombinant enzyme can be obtained relatively easily.

【００１４】この発明の変換方法により、グルコース重
合度３以上の還元性澱粉糖は、末端にトレハロース構造
を有する非還元性糖質に変換される。By the conversion method of the present invention, the reducing starch sugar having a glucose polymerization degree of 3 or more is converted into a non-reducing sugar having a trehalose structure at the terminal.

【００１５】この発明は、グルコース重合度３以上の還
元性澱粉糖から末端にトレハロース構造を有する非還元
性糖質を生成する、従来未知の全く新規な酵素の発見に
基づくものである。斯かる酵素はリゾビウム・スピーシ
ーズＭ−１１やアルスロバクター・スピーシーズＱ３６
の培養物から得ることができ（以下、それぞれ「酵素Ｍ
−１１」又は「酵素Ｑ３６」と云う。）、本発明者がカ
ラムクロマトグラフィーを中心とする種々の精製方法を
組合せてこの酵素を単離し、その性質・性状を調べたと
ころ、その本質はポリペプチドであり、次のような理化
学的性質を有することが判明した。 （１） 作用 グルコース重合度３以上の還元性澱粉糖から末端にトレ
ハロース構造を有する非還元性糖質を生成する。 （２） 分子量 約７６，０００乃至８７，０００ダルトン（ＳＤＳ−ポ
リアクリルアミドゲル電気泳動） （３） 等電点 約３．６乃至４．６（等電点電気泳動） （４） 至適温度 ｐＨ７．０で６０分間インキュベートすると、３５乃至
４０℃付近に至適温度を示す。 （５） 至適ｐＨ ４０℃で６０分間インキュベートすると、ｐＨ６．４乃
至７．２付近に至適ｐＨを示す。 （６） 熱安定性 ｐＨ７．０で６０分間インキュベートすると、３５乃至
４０℃付近まで安定である。 （７） ｐＨ安定性 ２５℃で１６時間インキュベートすると、ｐＨ５．５乃
至１１．０付近まで安定である。The present invention is based on the discovery of a novel enzyme, which has not been heretofore known, that produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. Such enzymes are Rhizobium species M-11 and Arthrobacter species Q36.
Can be obtained from the cultures of
-11 "or" enzyme Q36 ". ), The present inventor isolated this enzyme by combining various purification methods centered on column chromatography, and investigated the properties and properties of the enzyme. As a result, the essence was a polypeptide, and the following physicochemical properties Was found to have. (1) Action A non-reducing sugar having a trehalose structure at its end is produced from a reducing starch sugar having a glucose polymerization degree of 3 or more. (2) Molecular weight about 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis) (3) Isoelectric point about 3.6 to 4.6 (isoelectric focusing) (4) Optimum temperature pH7 When it is incubated at 0.0 for 60 minutes, it shows an optimum temperature around 35 to 40 ° C. (5) Optimum pH When incubated at 40 ° C. for 60 minutes, the optimum pH is shown in the vicinity of pH 6.4 to 7.2. (6) Thermostability When incubated at pH 7.0 for 60 minutes, it is stable up to around 35 to 40 ° C. (7) pH stability When incubated at 25 ° C for 16 hours, it is stable up to about pH 5.5 to 11.0.

【００１６】次に、これら理化学的性質を解明すべく行
なった実験について説明する。Next, experiments conducted to clarify these physicochemical properties will be described.

【００１７】[0017]

【実験例１ 精製酵素の調製】[Experimental Example 1 Preparation of purified enzyme]

【００１８】[0018]

【実験例１−１ 酵素Ｍ−１１の精製】５００ｍｌ容三
角フラスコにマルトース２．０％（ｗ／ｖ）、ペプトン
０．５％（ｗ／ｖ）、酵母エキス０．１％（ｗ／ｖ）、
燐酸水素二ナトリウム０．１％（ｗ／ｖ）及び燐酸二水
素カリウム０．１％（ｗ／ｖ）を含む液体培地（ｐＨ
７．０）を１００ｍｌずつとり、１２０℃で２０分間オ
ートクレーブして滅菌した。冷却後、三角フラスコ内の
液体培地にリゾビウム・スピーシーズＭ−１１を植菌
し、回転振盪下、２７℃で２４時間種培養した。別途、
３０ｌ容ジャーファーメンタに上記と同組成の液体培地
を２０ｌとり、滅菌後、上記で得た種培養液を１％（ｖ
／ｖ）接種し、液体培地をｐＨ６乃至８に保ちつつ、３
０℃で２４時間通気撹拌培養した。[Experimental Example 1-1 Purification of Enzyme M-11] Maltose 2.0% (w / v), peptone 0.5% (w / v), yeast extract 0.1% (w / v) were added to a 500 ml Erlenmeyer flask. ),
Liquid medium containing 0.1% (w / v) disodium hydrogen phosphate and 0.1% (w / v) potassium dihydrogen phosphate (pH)
Each 100 ml of 7.0) was sterilized by autoclaving at 120 ° C. for 20 minutes. After cooling, Rhizobium species M-11 was inoculated into the liquid medium in the Erlenmeyer flask, and seed culture was carried out at 27 ° C. for 24 hours under rotary shaking. Separately
20 liters of a liquid medium having the same composition as described above was placed in a 30-liter jar fermenter, and after sterilization, the seed culture solution obtained above was added to 1% (v
/ V) inoculate and maintain the liquid medium at pH 6 to 8
The culture was performed at 0 ° C. for 24 hours with aeration and stirring.

【００１９】次に、上記で得た培養物約１８ｌを超高圧
菌体破砕装置にとり、菌体を破砕後、遠心分離により採
取した上清約１６ｌに硫酸アンモニウムを２０％飽和に
なるように加え、４℃で１時間静置後、遠心分離により
沈澱部を除去した。得られた上清に６０％飽和になるよ
うに硫酸アンモニウムを加え、４℃で２４時間静置後、
沈澱部を遠心分離により採取し、最少量の１０ｍＭ燐酸
緩衝液（ｐＨ７．０）に溶解し、１０ｍＭ燐酸緩衝液
（ｐＨ７．０）に対して２４時間透析後、遠心分離によ
り不溶物を除去した。得られた上清を予め１０ｍＭ燐酸
緩衝液（ｐＨ７．０）により平衡化させておいた東ソー
製イオン交換クロマトグラフィー用カラム『ＤＥＡＥ−
トヨパール』に負荷し、０Ｍから０．５Ｍに上昇する塩
化ナトリウムの濃度勾配下、カラムに１０ｍＭ燐酸緩衝
液（ｐＨ７．０）を通液した。溶出液より酵素を含む画
分を採取し、２Ｍ硫酸アンモニウムを含む５０ｍＭ燐酸
緩衝液（ｐＨ７．０）に対して１０時間透析後、遠心分
離により不溶物を除去した。その後、上清を予め２Ｍ硫
酸アンモニウムを含む５０ｍＭ燐酸緩衝液（ｐＨ７．
０）により平衡化させておいた東ソー製疎水クロマトグ
ラフィー用カラム『ブチルトヨパール』に負荷し、２Ｍ
から０Ｍに低下する硫酸アンモニウムの濃度勾配下、カ
ラムに５０ｍＭ燐酸緩衝液（ｐＨ７．０）を通液した。
溶出液から酵素を含む画分を採取し、予め５０ｍＭ燐酸
緩衝液（ｐＨ７．０）により平衡化させておいた東ソー
製ゲル濾過カラムクロマトグラフィー用カラム『トヨパ
ールＨＷ−５５』に負荷し、カラムに５０ｍＭ燐酸緩衝
液（ｐＨ７．０）を通液し、溶出液から酵素を含む画分
を採取した。このようにして精製した酵素Ｍ−１１の比
活性は約１９５単位／ｍｇ蛋白質であり、収量は培養物
１ｌ当たり約２２０単位であった。Next, about 18 liters of the above-obtained culture was placed in an ultrahigh-pressure cell crushing device, and after crushing the cells, about 16 liters of the supernatant collected by centrifugation was added with ammonium sulfate to 20% saturation, After standing at 4 ° C for 1 hour, the precipitate was removed by centrifugation. Ammonium sulfate was added to the resulting supernatant to 60% saturation, and the mixture was allowed to stand at 4 ° C. for 24 hours,
The precipitate was collected by centrifugation, dissolved in a minimum amount of 10 mM phosphate buffer (pH 7.0), dialyzed against 10 mM phosphate buffer (pH 7.0) for 24 hours, and then insoluble matter was removed by centrifugation. . The obtained supernatant was previously equilibrated with a 10 mM phosphate buffer (pH 7.0), a column for ion exchange chromatography "DEAE-" manufactured by Tosoh Corporation.
Toyopearl ”was loaded, and 10 mM phosphate buffer (pH 7.0) was passed through the column under a concentration gradient of sodium chloride increasing from 0 M to 0.5 M. Fractions containing the enzyme were collected from the eluate, dialyzed against 50 mM phosphate buffer (pH 7.0) containing 2 M ammonium sulfate for 10 hours, and then insoluble matters were removed by centrifugation. Then, the supernatant was previously added with 50 mM phosphate buffer (pH 7.
0M) and equilibrated with Tosoh's hydrophobic chromatography column "Butyl Toyopearl" and loaded with 2M
A 50 mM phosphate buffer solution (pH 7.0) was passed through the column under a concentration gradient of ammonium sulfate from 0 to 0 M.
The enzyme-containing fraction was collected from the eluate, and loaded on Toso gel filtration column chromatography column "Toyopearl HW-55", which had been equilibrated with 50 mM phosphate buffer (pH 7.0) in advance, and loaded on the column. A 50 mM phosphate buffer solution (pH 7.0) was passed through, and an enzyme-containing fraction was collected from the eluate. The specific activity of enzyme M-11 thus purified was about 195 units / mg protein and the yield was about 220 units per liter of culture.

【００２０】なお、この発明を通じて、酵素の活性は次
の方法により測定した活性値（単位）で表示する。すな
わち、マルトペンタオースを１．２５％（ｗ／ｖ）含む
５０ｍＭ燐酸緩衝液（ｐＨ７．０）を４ｍｌとり、これ
に酵素液を１ｍｌ加え、４０℃で６０分間インキュベー
トして反応させた後、反応液を１００℃で１０分間加熱
して反応を停止させる。反応液を蒸留水で１０倍希釈し
た後、ソモギ・ネルソン法により還元力を測定する。当
該酵素の１単位とは、上記条件下において、１分間にマ
ルトペンタオース１μｍｏｌに相当する還元力を低下さ
せる酵素の量と定義する。Throughout the present invention, the activity of the enzyme is expressed as an activity value (unit) measured by the following method. That is, 4 ml of 50 mM phosphate buffer solution (pH 7.0) containing 1.25% (w / v) maltopentaose was taken, 1 ml of the enzyme solution was added thereto, and the mixture was incubated at 40 ° C. for 60 minutes to react, The reaction solution is heated at 100 ° C. for 10 minutes to stop the reaction. After diluting the reaction solution 10 times with distilled water, the reducing power is measured by the Somogyi-Nelson method. One unit of the enzyme is defined as the amount of the enzyme that reduces the reducing power corresponding to 1 μmol of maltopentaose per minute under the above conditions.

【００２１】[0021]

【実験例１−２ 酵素Ｑ３６の精製】実験例１−１と同
様にアルスロバクター・スピーシーズＱ３６を培養し、
培養物を処理したところ、比活性約２００単位／ｍｇ蛋
白質の精製酵素Ｑ３６が、培養物１ｌ当たり、約２９５
単位の収量で得られた。[Experimental Example 1-2 Purification of Enzyme Q36] Arthrobacter species Q36 was cultured in the same manner as in Experimental Example 1-1,
When the culture was treated, the purified enzyme Q36 having a specific activity of about 200 units / mg protein was treated at about 295 liters of culture per liter.
Obtained in unit yield.

【００２２】[0022]

【実験例２ 酵素の理化学的性質】[Experimental Example 2 Physicochemical properties of enzyme]

【００２３】[0023]

【実験例２−１ 作用】基質としてグルコース、マルト
ース、マルトトリオース、マルトテトラオース、マルト
ペンタオース、マルトヘキサオース又はマルトヘプタオ
ースを２０％（ｗ／ｖ）含む５０ｍＭ燐酸緩衝液（ｐＨ
７．０）に実験例１で得た精製酵素Ｍ−１１又は精製酵
素Ｑ３６を基質１ｇ当たり２単位加え、４０℃で４８時
間反応させた。常法により反応物を脱塩した後、和光純
薬製高速液体クロマトグラフィー用カラム『ＷＢ−Ｔ−
３３０』に負荷し、溶出液の糖濃度を東ソー製示差屈折
計『ＲＩ−８０１２型』でモニターしながら、室温下に
てカラムに蒸留水を０．５ｍｌ／分の流速で通液するこ
とにより、反応物に含まれる糖質を分離した。表１及び
表２に、それぞれ、酵素Ｍ−１１または酵素Ｑ３６を加
えた場合の反応物の糖組成を示す。 なお、表中の糖質
Ｐ１乃至Ｐ５は、反応により生成した糖質をグルコース
重合度の小さい順に命名したものである。[Experimental Example 2-1] Action 50 mM phosphate buffer solution (pH: 20% (w / v) containing glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose or maltoheptaose as a substrate (pH)
To 7.0), 2 units of the purified enzyme M-11 or purified enzyme Q36 obtained in Experimental Example 1 was added per 1 g of the substrate, and the mixture was reacted at 40 ° C. for 48 hours. After desalting the reaction product by a conventional method, a column for high performance liquid chromatography “WB-T-” manufactured by Wako Pure Chemical Industries, Ltd.
330 ", and the sugar concentration of the eluate was monitored with a Tosoh differential refractometer" RI-8012 type ", and distilled water was passed through the column at a flow rate of 0.5 ml / min at room temperature. The sugar contained in the reaction product was separated. Tables 1 and 2 show the sugar composition of the reaction product when the enzyme M-11 or the enzyme Q36 was added, respectively. The sugars P1 to P5 in the table are the sugars produced by the reaction, which are named in ascending order of glucose polymerization degree.

【００２４】[0024]

【表１】 [Table 1]

【００２５】[0025]

【表２】 [Table 2]

【００２６】表１及び表２の結果から明らかなように、
酵素Ｍ−１１及び酵素Ｑ３６は、マルトトリオース、マ
ルトテトラオース、マルトペンタオース、マルトヘキサ
オース及びマルトヘプタオースなどのグルコース重合度
が３以上の還元性澱粉糖からは新たな糖質を生成するけ
れども、グルコース重合度が３を下回るグルコースやマ
ルトースからは新たな糖質を生成しない。また、反応に
より生成した糖質はそれぞれ糖質Ｐ１乃至Ｐ５のみであ
り、糖質Ｐ２乃至Ｐ５の含量は固形分当たり８５％以上
と著しく高かった。As is clear from the results shown in Tables 1 and 2,
Enzymes M-11 and Q36 form new sugars from reducing starch sugars having a degree of glucose polymerization of 3 or more, such as maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. However, no new sugar is produced from glucose or maltose having a glucose polymerization degree of less than 3. The sugars produced by the reaction were only the sugars P1 to P5, respectively, and the contents of the sugars P2 to P5 were remarkably high at 85% or more per solid content.

【００２７】次に、糖質Ｐ１乃至Ｐ５を分離すべく、東
京有機化学工業製強酸性カチオン交換樹脂『ＸＴ−１０
１６（Ｎａ+型）』を内径２．０ｃｍ、長さ１ｍのジャ
ケット付きステンレス製カラム３本に充填し、これらカ
ラムを直列に連結した。そして、カラム内の温度を５５
℃に保ちつつ、カラムに糖質Ｐ１乃至Ｐ５のいずれかを
含む前記反応物を別々に負荷した後、カラムに５５℃の
蒸留水をＳＶ０．１３の流速で通液した。溶出液の糖組
成を調べ、糖質Ｐ１乃至Ｐ５のいずれかを固形分で９７
％以上含む画分を採取し、真空乾燥により粉末化した。
このようにして精製した糖質Ｐ１乃至Ｐ５の還元力をソ
モギ・ネルソン法により調べたところ、いずれの糖質に
も実質的な還元力は認められなかった。Next, in order to separate the sugars P1 to P5, a strongly acidic cation exchange resin "XT-10 manufactured by Tokyo Organic Chemical Industry" is used.
16 (Na + type) ”was packed in three jacketed stainless steel columns having an inner diameter of 2.0 cm and a length of 1 m, and these columns were connected in series. Then, the temperature in the column is set to 55
While maintaining the temperature at 0 ° C., the reaction product containing any of the sugars P1 to P5 was separately loaded onto the column, and then distilled water at 55 ° C. was passed through the column at a flow rate of SV 0.13. Examine the sugar composition of the eluate and find out whether any of the sugars P1 to P5 has a solid content of 97.
Fractions containing more than 100% were collected and pulverized by vacuum drying.
When the reducing powers of the thus purified sugars P1 to P5 were examined by the Somogyi-Nelson method, no substantial reducing power was observed for any of the sugars.

【００２８】さらに、糖質Ｐ１乃至Ｐ５を同定すべく、
これら糖質のいずれかを５０ｍｇとり、５０ｍＭ酢酸緩
衝液（ｐＨ４．５）１ｍｌに溶解後、グルコアミラーゼ
を１単位加え、４０℃で６時間インキュベートした。表
１及び表２に示す反応物の糖組成を高速液体クロマトグ
ラフィーにより分析したところ、それぞれ表３及び表４
に示すように、全ての反応物からグルコースとトレハロ
ースが検出された。同様にして、糖質Ｐ１乃至Ｐ５にβ
−アミラーゼを作用させたところ、糖質Ｐ１及びＰ２が
β−アミラーゼの作用を受けなかったのに対して、糖質
Ｐ３は１分子のマルトースと糖質Ｐ１を、糖質Ｐ４は１
分子のマルトースと糖質Ｐ２を、また、糖質Ｐ５は２分
子のマルトースと糖質Ｐ１を与えた。Further, in order to identify the sugars P1 to P5,
50 mg of any of these sugars was taken, dissolved in 1 ml of 50 mM acetate buffer (pH 4.5), 1 unit of glucoamylase was added, and the mixture was incubated at 40 ° C. for 6 hours. When the sugar compositions of the reaction products shown in Table 1 and Table 2 were analyzed by high performance liquid chromatography, Table 3 and Table 4 respectively.
As shown in, glucose and trehalose were detected in all the reaction products. Similarly, β for sugars P1 to P5
-When amylase was allowed to act, the sugars P1 and P2 were not affected by β-amylase, while the sugar P3 contained one molecule of maltose and sugar P1, and the sugar P4 contained 1 molecule.
Molecule maltose and saccharide P2 were given, and saccharide P5 gave two molecules of maltose and saccharide P1.

【００２９】[0029]

【表３】 [Table 3]

【００３０】[0030]

【表４】 [Table 4]

【００３１】表３及び表４の結果は、糖質Ｐ１乃至Ｐ５
が１分子のトレハロースと１乃至５分子のグルコースに
より構成されることを強く示唆している。また、グルコ
アミラーゼがマルトオリゴ糖におけるα−１，４結合及
びα−１，６結合に特異的に切断することと、β−アミ
ラーゼがマルトオリゴ糖におけるα−１，４結合をその
末端よりマルトース単位で切断することから、糖質Ｐ１
乃至Ｐ５は、グルコース又はグルコース重合度が２乃至
５のマルトオリゴ糖の末端にトレハロース残基が１個結
合した構造を有していると推定される。The results of Tables 3 and 4 show that the sugars P1 to P5
Strongly suggest that it is composed of 1 molecule of trehalose and 1 to 5 molecules of glucose. Further, glucoamylase specifically cleaves α-1,4 bond and α-1,6 bond in maltooligosaccharide, and β-amylase cuts α-1,4 bond in maltooligosaccharide from its end in maltose units. Since it is cleaved, carbohydrate P1
It is assumed that each of P5 to P5 has a structure in which one trehalose residue is bound to the terminal of glucose or a maltooligosaccharide having a glucose polymerization degree of 2 to 5.

【００３２】以上の結果を総合的に判断すると、糖質Ｐ
１乃至Ｐ５は、それぞれ、α−グルコシルトレハロー
ス、α−マルトシルトレハロース、α−マルトトリオシ
ルトレハロース、α−マルトテトラオシルトレハロース
又はα−マルトペンタオシルトレハロースと同定され、
このことは、当該酵素にグルコース重合度３以上の還元
性澱粉糖から末端にトレハロース構造を有する非還元性
糖質を生成する作用のあることを裏付けている。Judging from the above results, the sugar P
1 to P5 were respectively identified as α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltotetraosyltrehalose or α-maltopentaosyltrehalose,
This confirms that the enzyme has an action of producing a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more.

【００３３】[0033]

【実験例２−２ 分子量】ユー・ケー・レムリが『ネー
チャー』、第２２７巻、第６８０〜６８５頁（１９７０
年）に報告している方法に準じて精製酵素をＳＤＳ−ポ
リアクリルアミドゲル電気泳動したところ、酵素Ｍ−１
１、酵素Ｑ３６とも、分子量約７６，０００乃至８７，
０００ダルトンに相当する位置に単一バンドが観察され
た。なお、このときの分子量マーカは、ミオシン（２０
０，０００ダルトン）、β−ガラクトシダーゼ（１１
６，２５０ダルトン）、フォスフォリラーゼＢ（９７，
４００ダルトン）、血清アルブミン（６６，２００ダル
トン）及びオボアルブミン（４５，０００ダルトン）で
あった。[Experimental Example 2-2 Molecular Weight] U.K. Laemli, "Nature", Vol. 227, pp. 680-685 (1970)
SDS-polyacrylamide gel electrophoresis of the purified enzyme according to the method reported in
1. The enzyme Q36 has a molecular weight of about 76,000 to 87,
A single band was observed at a position corresponding to 000 Daltons. The molecular weight marker used at this time was myosin (20
10,000 Daltons), β-galactosidase (11
6,250 daltons), phosphorylase B (97,
400 daltons), serum albumin (66,200 daltons) and ovalbumin (45,000 daltons).

【００３４】[0034]

【実験例２−３ 等電点】等電点電気泳動法により測定
したところ、酵素Ｍ−１１、酵素Ｑ３６とも、約３．６
乃至４．６に等電点を示した。[Experimental Example 2-3 Isoelectric point] As measured by an isoelectric focusing method, both of the enzyme M-11 and the enzyme Q36 were about 3.6.
The isoelectric points are shown in FIGS.

【００３５】[0035]

【実験例２−４ 至適温度】常法により、５０ｍＭ燐酸
緩衝液（ｐＨ７．０）中で６０分間インキュベートする
条件で試験したところ、図１又は図２に示すように、酵
素Ｍ−１１、酵素Ｑ３６とも、３５乃至４０℃付近に至
適温度を示した。[Experimental Example 2-4 Optimum temperature] A test was carried out by a conventional method under the condition of incubating in 50 mM phosphate buffer (pH 7.0) for 60 minutes, and as shown in FIG. 1 or 2, the enzyme M-11, The enzyme Q36 also showed an optimum temperature around 35 to 40 ° C.

【００３６】[0036]

【実験例２−５ 至適ｐＨ】常法により、ｐＨの相違す
る５０ｍＭ酢酸緩衝液、燐酸緩衝液又は炭酸ナトリウム
−炭酸水素ナトリウム緩衝液中、４０℃で６０分間イン
キュベートする条件で試験したところ、図３又は図４に
示すように、酵素Ｍ−１１、酵素Ｑ３６とも、ｐＨ６．
４乃至７．２付近に至適ｐＨを示した。[Experimental Example 2-5 Optimum pH] The test was carried out by an ordinary method under the conditions of incubating at 40 ° C. for 60 minutes in 50 mM acetate buffer, phosphate buffer or sodium carbonate-sodium hydrogen carbonate buffer having different pH. As shown in FIG. 3 or FIG. 4, both the enzyme M-11 and the enzyme Q36 had a pH of 6.
The optimum pH was shown in the vicinity of 4 to 7.2.

【００３７】[0037]

【実験例２−６ 熱安定性】常法により、５０ｍＭ燐酸
緩衝液（ｐＨ７．０）中で６０分間インキュベートする
条件で試験したところ、図５又は図６に示すように、酵
素Ｍ−１１、酵素Ｑ３６とも、３５乃至４０℃付近まで
安定であった。[Experimental Example 2-6 Thermostability] When tested by a conventional method under the condition of incubating in 50 mM phosphate buffer (pH 7.0) for 60 minutes, as shown in FIG. 5 or 6, the enzyme M-11, Both enzyme Q36 was stable up to around 35 to 40 ° C.

【００３８】[0038]

【実験例２−７ ｐＨ安定性】常法により、ｐＨの相違
する５０ｍＭ酢酸緩衝液、燐酸緩衝液又は炭酸ナトリウ
ム−炭酸水素ナトリウム緩衝液中、２５℃で１６時間イ
ンキュベートする条件で試験したところ、図７又は図８
に示すように、酵素Ｍ−１１、酵素Ｑ３６とも、ｐＨ
５．５乃至１１．０付近まで安定であった。[Experimental Example 2-7 pH stability] According to a conventional method, a test was carried out under the conditions of incubating at 25 ° C. for 16 hours in 50 mM acetate buffer, phosphate buffer or sodium carbonate-sodium hydrogen carbonate buffer having different pH. 7 or 8
As shown in, both the enzyme M-11 and the enzyme Q36 have pH
It was stable up to around 5.5 to 11.0.

【００３９】[0039]

【実験例２−８ Ｎ末端アミノ酸配列】常法により、ア
プライッド・バイオシステム製気相プロテイン・シーケ
ンサ『４７０Ａ型』を使用して分析したところ、酵素Ｍ
−１１は、Ｎ末端に配列表における配列番号７に示すア
ミノ酸配列を有していることが判明した。[Experimental example 2-8 N-terminal amino acid sequence] When analyzed using a gas phase protein sequencer "470A type" manufactured by Applied Biosystem by a conventional method, the enzyme M
-11 was found to have the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing at the N-terminus.

【００４０】同様に分析したところ、酵素Ｑ３６は、Ｎ
末端に配列表における配列番号８に示すアミノ酸配列を
有していることが判明した。When analyzed in the same manner, the enzyme Q36 was
It was found to have the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing at the end.

【００４１】[0041]

【実験例２−９ 部分アミノ酸配列】実験例１−１で得
た精製酵素Ｍ−１１を適量とり、１０ｍＭトリス−塩酸
緩衝液（ｐＨ９．０）に対して４℃で１８時間透析後、
１０ｍＭトリス−塩酸緩衝液（ｐＨ９．０）を加えて酵
素濃度を約１ｍｇ／ｍｌとした。この溶液を約１ｍｌと
り、リジルエンドペプチダーゼを１０μｇ加え、３０℃
で２２時間インキュベートして酵素を部分加水分解し
た。加水分解物を、予め１６％（ｖ／ｖ）水性アセトニ
トリルを含む０．１％（ｖ／ｖ）トリフルオロ酢酸によ
り平衡化させておいた資生堂製逆相高速液体クロマトグ
ラフィー用カラム『カプセルパックＣ１８』に負荷し、
次いで、１６％（ｖ／ｖ）から６４％（ｖ／ｖ）に上昇
するアセトニトリルの濃度勾配下、カラムに０．１％
（ｖ／ｖ）トリフルオロ酢酸を０．９ｍｌ／分の流速で
通液した。そして、通液開始から約２８分後又は約４０
分後に溶出したペプチド断片（以下、それぞれ「ペプチ
ド断片Ａ」又は「ペプチド断片Ｂ」と云う。）を含む画
分を別々に採取し、真空乾燥後、５０％（ｖ／ｖ）水性
アセトニトリルを含む０．１％（ｖ／ｖ）トリフルオロ
酢酸に溶解した。以後、実験例２−８と同様に分析した
ところ、ペプチド断片Ａ及びＢは、配列表における配列
番号９及び１０に示すアミノ酸配列を有していることが
判明した[Experimental Example 2-9 Partial Amino Acid Sequence] An appropriate amount of the purified enzyme M-11 obtained in Experimental Example 1-1 was taken and dialyzed against 10 mM Tris-hydrochloric acid buffer solution (pH 9.0) at 4 ° C. for 18 hours.
10 mM Tris-hydrochloric acid buffer (pH 9.0) was added to make the enzyme concentration about 1 mg / ml. Take about 1 ml of this solution, add 10 μg of lysyl endopeptidase, and add 30 μC
The enzyme was partially hydrolyzed by incubating for 22 hours. The hydrolyzate was previously equilibrated with 0.1% (v / v) trifluoroacetic acid containing 16% (v / v) aqueous acetonitrile. Shiseido reverse phase high performance liquid chromatography column "Capsule Pack C18". ],
Then, under a gradient of acetonitrile increasing from 16% (v / v) to 64% (v / v), 0.1% was applied to the column.
(V / v) trifluoroacetic acid was passed through at a flow rate of 0.9 ml / min. And, about 28 minutes after the start of liquid flow or about 40 minutes
Fractions containing the peptide fragments (hereinafter referred to as "peptide fragment A" or "peptide fragment B", respectively) that were eluted after 50 minutes were collected separately, dried under vacuum, and then containing 50% (v / v) aqueous acetonitrile. It was dissolved in 0.1% (v / v) trifluoroacetic acid. After that, the same analysis as in Experimental Example 2-8 revealed that the peptide fragments A and B had the amino acid sequences shown in SEQ ID NOs: 9 and 10 in the sequence listing.

【００４２】別途、実験例１−２で得た精製酵素Ｑ３６
を上記と同様にして部分加水分解し、予め２４％（ｖ／
ｖ）水性アセトニトリルを含む０．１％（ｖ／ｖ）トリ
フルオロ酢酸により平衡化させておいた日本ミリポア・
リミテッド製逆相高速液体クロマトグラフィー用カラム
『マイクロボンダパックＣ１８』に負荷し、２４％（ｖ
／ｖ）から４４％（ｖ／ｖ）に上昇する水性アセトニト
リルの濃度勾配下、カラムに０．１％（ｖ／ｖ）トリフ
ルオロ酢酸を０．９ｍｌ／分の流速で通液した。そし
て、通液開始から約２２分後又は約４０分後に溶出した
ペプチド断片（以下、それぞれ「ペプチド断片Ｃ」又は
「ペプチド断片Ｄ」と云う。）を含む画分を採取し、真
空乾燥後、５０％（ｖ／ｖ）水性アセトニトリルを含む
０．１％（ｖ／ｖ）トリフルオロ酢酸に溶解した。以
後、上記と同様に分析したところ、ペプチド断片Ｃ及び
Ｄは、配列表における配列番号１１及び１２に示すアミ
ノ酸配列を有していることが判明した。Separately, purified enzyme Q36 obtained in Experimental Example 1-2
Is partially hydrolyzed in the same manner as above, and 24% (v /
v) Japan Millipore, equilibrated with 0.1% (v / v) trifluoroacetic acid containing aqueous acetonitrile.
Loaded on a limited-phase reversed-phase high-performance liquid chromatography column "Microbonder Pack C18" at 24% (v
/ V) to 0.1% (v / v) trifluoroacetic acid at a flow rate of 0.9 ml / min under a concentration gradient of aqueous acetonitrile increasing from 44% (v / v) to 44% (v / v). Then, a fraction containing a peptide fragment (hereinafter referred to as “peptide fragment C” or “peptide fragment D”, respectively) that was eluted about 22 minutes or about 40 minutes after the start of the passage of the liquid was collected, vacuum-dried, It was dissolved in 0.1% (v / v) trifluoroacetic acid containing 50% (v / v) aqueous acetonitrile. Subsequent analysis as described above revealed that peptide fragments C and D had the amino acid sequences shown in SEQ ID NOs: 11 and 12 in the sequence listing.

【００４３】以上のような理化学的性質を有する酵素は
未だ知られておらず、新規物質であると判断される。な
お、リゾビウム・スピーシーズＭ−１１は岡山県岡山市
の土壌から分離され、平成４年１２月２４日以降、茨城
県つくば市東１丁目１番３号にある通商産業省、工業技
術院、生命工学工業技術研究所、特許微生物寄託センタ
ーに寄託番号『ＦＥＲＭ ＢＰ−４１３０』で寄託され
ている。一方、アルスロバクター・スピーシーズＱ３６
は岡山県総社市の土壌から分離されたものであり、平成
５年６月３日以降、同センターに寄託番号『ＦＥＲＭ
ＢＰ−４３１６』で寄託されている。同じ出願人による
特願平５−３４９２１６号明細書には、酵素の性質・性
状とともに、両微生物の菌学的性質が詳細に開示されて
いる。The enzyme having the above physicochemical properties has not been known yet, and it is judged to be a novel substance. Rhizobium species M-11 was separated from the soil in Okayama City, Okayama Prefecture, and from December 24, 1992, the Ministry of International Trade and Industry, the Agency of Industrial Science and Technology, the Ministry of International Trade and Industry, located at 1-3-1 East, Tsukuba City, Ibaraki Prefecture It has been deposited under the deposit number "FERM BP-4130" at the National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary Center. On the other hand, Arthrobacter Species Q36
Is separated from the soil of Soja City, Okayama Prefecture, and after June 3, 1993, the deposit number "FERM
BP-4316 ”. Japanese Patent Application No. 5-349216 filed by the same applicant discloses in detail the properties and characteristics of the enzyme as well as the mycological properties of both microorganisms.

【００４４】そこで、本発明者が、実験例２−９で明ら
かにした酵素Ｍ−１１の部分アミノ酸配列に基づき化学
合成したオリゴヌクレオチドをプローブに使用し、リゾ
ビウム・スピーシーズＭ−１１の染色体ＤＮＡを鋭意検
索した結果、配列表における配列番号３に示す塩基配列
を有する２，３１６塩基対からなるＤＮＡ断片が得られ
た。そして、その塩基配列を解読したところ、酵素Ｍ−
１１は７７２個のアミノ酸からなる配列表における配列
番号１に示すアミノ酸配列を有していることが判明し
た。Therefore, the present inventor used an oligonucleotide chemically synthesized based on the partial amino acid sequence of the enzyme M-11 clarified in Experimental Example 2-9 as a probe to obtain a chromosomal DNA of Rhizobium species M-11. As a result of diligent search, a DNA fragment consisting of 2,316 base pairs having the base sequence shown in SEQ ID NO: 3 in the sequence listing was obtained. Then, when the nucleotide sequence was decoded, the enzyme M-
11 was found to have the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing consisting of 772 amino acids.

【００４５】一方、酵素Ｑ３６の部分アミノ酸配列に基
づき化学合成したオリゴヌクレオチドをプローブにし、
アルスロバクター・スピーシーズＱ３６の染色体ＤＮＡ
を同様に検索したところ、配列表における配列番号４に
示す塩基配列を有する２，３２５塩基対からなるＤＮＡ
断片が得られた。この塩基配列を解読したところ、酵素
Ｑ３６は７７５個のアミノ酸からなり、配列表における
配列番号２に示すアミノ酸配列を有していることが判明
した。On the other hand, an oligonucleotide chemically synthesized based on the partial amino acid sequence of the enzyme Q36 was used as a probe,
Chromosomal DNA of Arthrobacter species Q36
Was similarly searched, DNA consisting of 2,325 base pairs having the base sequence shown in SEQ ID NO: 4 in the sequence listing.
Fragments were obtained. When the nucleotide sequence was decoded, it was revealed that the enzyme Q36 was composed of 775 amino acids and had the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

【００４６】配列表における配列番号１乃至４に示す塩
基配列及びアミノ酸配列を解明するに到った一連の工程
を要約すると、次のようになる。 （１） 供与体微生物の培養物から当該酵素を分離し、
高度に精製した。精製酵素をプロテアーゼにより部分加
水分解後、加水分解物から２種類のペプチド断片を単離
し、そのアミノ酸配列を決定した。 （２） 別途、供与体微生物の菌体より染色体ＤＮＡを
分離し、精製後、制限酵素により部分的に切断して約
３，０００乃至７，０００塩基対からなるＤＮＡ断片を
採取した。ＤＮＡリガーゼにより、このＤＮＡ断片を予
め制限酵素で切断しておいたプラスミドベクターに連結
し、組換えＤＮＡを作製した。 （３） 大腸菌に組換えＤＮＡを導入して形質転換体を
作製し、前記部分アミノ酸配列に基づき化学合成したオ
リゴヌクレオチドをプローブとするコロニーハイブリダ
イゼーションにより当該酵素をコードするＤＮＡを含む
形質転換体を選択した。 （４） 形質転換体から組換えＤＮＡを採取し、プライ
マーとともにアニーリング後、ＤＮＡポリメラーゼを作
用させてプライマーを伸長し、得られた相補鎖ＤＮＡを
ジデオキシ・チェーン・ターミネータ法により分析して
塩基配列を決定した。そして、その塩基配列から推定さ
れるアミノ酸配列と前記部分アミノ酸配列とを比較し、
その塩基配列が当該酵素をコードしていることを確認し
た。The series of steps leading to the elucidation of the nucleotide sequences and amino acid sequences shown in SEQ ID NOS: 1 to 4 in the Sequence Listing is summarized as follows. (1) separating the enzyme from the culture of the donor microorganism,
Highly purified. After partially hydrolyzing the purified enzyme with a protease, two types of peptide fragments were isolated from the hydrolyzate, and the amino acid sequences thereof were determined. (2) Separately, chromosomal DNA was isolated from the microorganism of the donor microorganism, purified, and partially digested with a restriction enzyme to collect a DNA fragment of about 3,000 to 7,000 base pairs. This DNA fragment was ligated with a plasmid vector that had been cleaved in advance with a restriction enzyme with a DNA ligase to prepare a recombinant DNA. (3) A transformant is prepared by introducing recombinant DNA into Escherichia coli and performing colony hybridization using an oligonucleotide chemically synthesized based on the partial amino acid sequence as a probe to obtain a transformant containing the DNA encoding the enzyme. Selected. (4) Recombinant DNA was collected from the transformant, annealed with the primer, extended with the action of DNA polymerase, and the resulting complementary strand DNA was analyzed by the dideoxy chain terminator method to determine the nucleotide sequence. Were determined. Then, the amino acid sequence deduced from the base sequence and the partial amino acid sequence are compared,
It was confirmed that the base sequence encoded the enzyme.

【００４７】次の実験例３乃至６では、上記の工程
（２）乃至（４）を具体的に説明するが、これら実施例
で用いる手法自体は斯界において公知のものであり、例
えば、ジェー・サムブルック等『モレキュラー・クロー
ニング・ア・ラボラトリー・マニュアル』、第２版、１
９８９年、コールド・スプリング・ハーバー・ラボラト
リー・プレス発行などにも詳述されている。In the following Experimental Examples 3 to 6, the above steps (2) to (4) will be specifically described. The techniques themselves used in these Examples are well known in the art, for example, J. Sambrook et al. "Molecular Cloning a Laboratory Manual", 2nd Edition, 1
It was also described in detail in Cold Spring Harbor Laboratory Press published in 989.

【００４８】[0048]

【実験例３ リゾビウム・スピーシーズＭ−１１に由来
するＤＮＡを含む組換えＤＮＡと形質転換体の調製】Experimental Example 3 Preparation of Recombinant DNA Containing DNA Derived from Rhizobium species M-11 and Transformant

【００４９】[0049]

【実験例３−１ 染色体ＤＮＡの調製】リゾビウム・ス
ピーシーズＭ−１１をバクト・ニュートリエント・ブロ
ス培地（ｐＨ７．０）に植菌し、２７℃で２４時間回転
振盪培養した。遠心分離により培養物から菌体を分離
し、ＴＥＳ緩衝液（ｐＨ８．０）に浮遊させ、リゾチー
ムを０．０５％（ｗ／ｖ）加えた後、３７℃で３０分間
インキュベートした。処理物を−８０℃で１時間凍結
後、ＴＳＳ緩衝液（ｐＨ９．０）を加えて６０℃に加温
し、ＴＥＳ緩衝液／フェノール混液を加え、氷冷後、遠
心分離により上清を採取した。この上清に２倍容の冷エ
タノールを加え、沈澱した粗染色体ＤＮＡを採取し、Ｓ
ＳＣ緩衝液（ｐＨ７．１）に溶解後、リボヌクレアーゼ
とプロテアーゼをそれぞれ７．５μｇ又は１２５μｇ加
え、３７℃で１時間インキュベートして反応させた。そ
の後、反応物にクロロフォルム／イソアミルアルコール
混液を加えて染色体ＤＮＡを抽出し、冷エタノールを加
え、生成した染色体ＤＮＡを含む沈澱を採取した。この
ようにして得た精製染色体ＤＮＡを濃度約１ｍｇ／ｍｌ
になるようにＳＳＣ緩衝液（ｐＨ７．１）に溶解し、溶
液を−８０℃で凍結した。[Experimental example 3-1 Preparation of chromosomal DNA] Rhizobium species M-11 was inoculated into Bact Nutrient Broth medium (pH 7.0), and cultivated at 27 ° C for 24 hours with shaking under rotation. The cells were separated from the culture by centrifugation, suspended in TES buffer (pH 8.0), 0.05% (w / v) of lysozyme was added, and the mixture was incubated at 37 ° C for 30 minutes. After freezing the treated product for 1 hour at -80 ° C, add TSS buffer (pH 9.0) and heat to 60 ° C, add TES buffer / phenol mixture, and after ice cooling, collect the supernatant by centrifugation. did. To this supernatant was added 2 volumes of cold ethanol, and the precipitated crude chromosomal DNA was collected.
After dissolution in SC buffer (pH 7.1), ribonuclease and protease were added at 7.5 μg or 125 μg, respectively, and incubated at 37 ° C. for 1 hour to react. Then, a chloroform / isoamyl alcohol mixed solution was added to the reaction product to extract chromosomal DNA, cold ethanol was added, and a precipitate containing the generated chromosomal DNA was collected. The purified chromosomal DNA thus obtained had a concentration of about 1 mg / ml.
Was dissolved in SSC buffer (pH 7.1) so that the resulting solution was frozen at -80 ° C.

【００５０】[0050]

【実験例３−２ 組換えＤＮＡ ｐＢＭＴ７と形質転換
体ＢＭＴ７の調製】実験例３−１で得た精製染色体ＤＮ
Ａ溶液を約１ｍｌとり、これに制限酵素Ｓａｕ ３ＡＩ
を約３５単位加え、３７℃で約２０分間反応させて染色
体ＤＮＡを部分切断した後、蔗糖密度勾配超遠心法によ
り約３，０００乃至７，０００塩基対からなるＤＮＡ断
片を採取した。別途、プラスミドベクターＢｌｕｅｓｃ
ｒｉｐｔ ＩＩ ＳＫ（＋）を１μｇとり、常法により
制限酵素Ｂａｍ ＨＩを作用させて完全に切断した後、
上記で得たＤＮＡ断片１０μｇとＴ４ ＤＮＡリガーゼ
を２単位加え、４℃で一夜静置することによりＤＮＡ断
片をベクター断片に連結した。そして、得られた組換え
ＤＮＡに東洋紡績製コンピテントセル『Ｅｐｉｃｕｒｉ
ａｎ Ｃｏｌｉ ＸＬＩ−Ｂｌｕｅ』を３０μｌ加え、
氷冷下に３０分間静置後、４２℃に加温し、ＳＯＣブロ
スを加えて３７℃で１時間インキュベートすることによ
り、組換えＤＮＡを大腸菌に導入した。[Experimental Example 3-2 Preparation of recombinant DNA pBMT7 and transformant BMT7] Purified chromosome DN obtained in Experimental Example 3-1
Approximately 1 ml of solution A is taken and the restriction enzyme Sau 3AI
Approximately 35 units were added and reacted at 37 ° C. for about 20 minutes to partially cut the chromosomal DNA, and then a DNA fragment consisting of about 3,000 to 7,000 base pairs was collected by the sucrose density gradient ultracentrifugation method. Separately, plasmid vector Bluesc
After taking 1 μg of ript II SK (+) and completely digesting it with a restriction enzyme Bam HI by a conventional method,
The DNA fragment was ligated to the vector fragment by adding 10 μg of the DNA fragment obtained above and 2 units of T4 DNA ligase and allowing to stand at 4 ° C. overnight. Then, the obtained recombinant DNA is added to Toyobo's competent cell "Epicuri
30 μl of “An coli XLI-Blue”,
After standing for 30 minutes under ice-cooling, the mixture was heated to 42 ° C, SOC broth was added, and the mixture was incubated at 37 ° C for 1 hour to introduce the recombinant DNA into Escherichia coli.

【００５１】次に、上記で得た形質転換体を５−ブロモ
−４−クロロ−３−インドリル−β−ガラクトシド５０
μｇ／ｍｌを含む寒天平板培地（ｐＨ７．０）に植菌
し、３７℃で１８時間培養後、培地上にナイロン膜を載
置し、培地上に形成された約４，４００個のコロニーを
ナイロン膜に固定した。別途、常法により、配列表にお
ける配列番号９に示すアミノ酸配列における第１７乃至
２１番目のＰｒｏ−Ｇｌｕ−Ｔｒｐ−Ｇｌｕ−Ｌｙｓで
表される配列に基づき５′−ＣＣＮＧＡＲＴＧＧＧＡＲ
ＡＡ−３′で表される塩基配列のプローブ１を化学合成
し、同位体32Ｐで標識後、前記ナイロン膜上に固定した
形質転換体のコロニーにハイブリダイズさせ、顕著な会
合が認められた９種類の形質転換体を選択した。Next, the transformant obtained above was treated with 5-bromo-4-chloro-3-indolyl-β-galactoside 50.
After inoculating to an agar plate medium (pH 7.0) containing μg / ml and culturing at 37 ° C. for 18 hours, a nylon membrane was placed on the medium, and about 4,400 colonies formed on the medium were placed. It was fixed on a nylon membrane. Separately, based on the sequence represented by the 17th to 21st Pro-Glu-Trp-Glu-Lys in the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, 5'-CCNGARTGGGAR was separately prepared.
A probe 1 having a base sequence represented by AA-3 ′ was chemically synthesized, labeled with the isotope 32 P, and then hybridized to the transformant colonies immobilized on the nylon membrane, and a remarkable association was observed. Nine types of transformants were selected.

【００５２】常法により、これら９種類の形質転換体か
ら組換えＤＮＡを採取し、配列表における配列番号１０
に示すアミノ酸配列における第１６乃至２０番目のＴｈ
ｒ−Ｇｌｕ−Ｐｈｅ−Ｔｒｐ−Ａｓｐで表される配列に
基づき化学合成した５′−ＡＣＮＧＡＲＴＴＹＴＧＧＧ
Ａ−３′で表される塩基配列のプローブ２をイー・エム
・サザーン『ジャーナル・オブ・モレキュラー・バイオ
ロジー』、第９８巻、第５０３〜５１７頁（１９７５
年）に記載されている方法に準じてハイブリダイズさ
せ、プローブ２と顕著な会合を示した組換えＤＮＡを選
択した。以上のようにして選択した組換えＤＮＡと形質
転換体を、それぞれ、『ｐＢＭＴ７』又は『ＢＭＴ７』
と命名した。Recombinant DNA was collected from these 9 kinds of transformants by the conventional method, and the SEQ ID NO: 10 in the sequence listing was collected.
16th to 20th Th in the amino acid sequence shown in
5'-ACNGARTTYTGGG chemically synthesized based on the sequence represented by r-Glu-Phe-Trp-Asp
The probe 2 having the nucleotide sequence represented by A-3 'was prepared by EM Southern, "Journal of Molecular Biology", Vol. 98, pp. 503-517 (1975).
Y.) and hybridized according to the method described in (1), and a recombinant DNA showing a remarkable association with probe 2 was selected. Recombinant DNA and transformants selected as described above were respectively designated as "pBMT7" or "BMT7".
I named it.

【００５３】上記で得た形質転換体ＢＭＴ７をアンピシ
リン１００μｇ／ｍｌを含むＬ−ブロス培地（ｐＨ７．
０）に植菌し、３７℃で２４時間回転振盪培養した。培
養終了後、遠心分離により培養物から菌体を採取し、通
常一般のアルカリ法により組換えＤＮＡを菌体外に溶出
させた。処理物を常法により精製し、分析したところ、
組換えＤＮＡ ｐＢＭＴ７は約９，３００塩基対からな
り、図９に示す制限酵素地図で表される構造を有してい
た。図９に示すように、酵素Ｍ−１１をコードする２，
３１６塩基対からなるＤＮＡは、制限酵素Ｐｓｔ Ｉに
よる切断部位付近の下流に位置していることが判明し
た。The transformant BMT7 obtained above was transformed into L-broth medium containing 100 μg / ml of ampicillin (pH 7.
0) and cultured at 37 ° C. for 24 hours under rotary shaking. After completion of the culture, cells were collected from the culture by centrifugation and the recombinant DNA was eluted out of the cells by a usual alkaline method. The processed product was purified by a conventional method and analyzed,
Recombinant DNA pBMT7 consisted of about 9,300 base pairs and had the structure represented by the restriction enzyme map shown in FIG. As shown in FIG. 9, 2, which encodes the enzyme M-11
It was revealed that the DNA consisting of 316 base pairs is located downstream of the cleavage site by the restriction enzyme PstI.

【００５４】[0054]

【実験例３−３ 形質転換体による酵素の産生】マルト
ース２．０％（ｗ／ｖ）、ペプトン０．５％（ｗ／
ｖ）、酵母エキス０．１％（ｗ／ｖ）、燐酸水素二ナト
リウム０．１％（ｗ／ｖ）、燐酸二水素カリウム０．１
％（ｗ／ｖ）を含む液体培地をｐＨ７．０に調整し、ア
ンピシリンを５０μｇ／ｍｌ加え、１２０℃で２０分間
加熱滅菌し、冷却後、実験例３−２で得た形質転換体Ｂ
ＭＴ７を植菌し、３７℃で２４時間回転振盪培養した。
培養物を超音波処理して菌体を破砕し、遠心分離により
不溶物を除去後、上清中の酵素活性を測定したところ、
培養物１ｌ当たりに換算して、約３，０００単位の酵素
が産生していた。[Experimental example 3-3 Production of enzyme by transformant] Maltose 2.0% (w / v), peptone 0.5% (w / v)
v), yeast extract 0.1% (w / v), disodium hydrogen phosphate 0.1% (w / v), potassium dihydrogen phosphate 0.1
% (W / v) -containing liquid medium was adjusted to pH 7.0, ampicillin was added at 50 μg / ml, and the mixture was sterilized by heating at 120 ° C. for 20 minutes and cooled, and then the transformant B obtained in Experimental Example 3-2 was obtained.
MT7 was inoculated and cultivated at 37 ° C. for 24 hours with rotary shaking.
When the culture was sonicated to disrupt the cells and insoluble matter was removed by centrifugation, the enzyme activity in the supernatant was measured,
About 3,000 units of enzyme were produced per 1 liter of culture.

【００５５】別途、対照として、大腸菌ＸＬＩ−Ｂｌｕ
ｅ株及びリゾビウム・スピーシーズＭ−１１をアンピシ
リン無含有の同じ液体培地に植菌し、リゾビウム・スピ
ーシーズＭ−１１の場合、培養温度を３０℃に設定した
以外は上記と同様に培養・処理した。処理物の活性を測
定したところ、リゾビウム・スピーシーズＭ−１１によ
る酵素の産生は培養物１ｌ当たり約１，５００単位と、
形質転換体ＢＭＴ７と比較して有意に低いものであっ
た。なお、宿主に使用した大腸菌ＸＬＩ−Ｂｌｕｅ株
は、当該酵素を全く産生しなかった。Separately, as a control, Escherichia coli XLI-Blue
The e strain and Rhizobium species M-11 were inoculated into the same liquid medium containing no ampicillin, and in the case of Rhizobium species M-11, the culture and treatment were performed in the same manner as above except that the culture temperature was set to 30 ° C. When the activity of the treated product was measured, the production of the enzyme by Rhizobium species M-11 was about 1,500 units per liter of the culture,
It was significantly lower than that of the transformant BMT7. The Escherichia coli XLI-Blue strain used as a host did not produce the enzyme at all.

【００５６】その後、形質転換体ＢＭＴ７が産生した酵
素を実験例１−１と同様に精製し、その性質・性状を調
べたところ、組換え型酵素はＳＤＳ−ポリアクリルアミ
ドゲル電気泳動で分子量値約７６，０００乃至８７，０
００ダルトンを、また、等電点電気泳動で約３．６乃至
４．６に等電点を示すなど、実験例２で得られた酵素Ｍ
−１１のものと同様の理化学的性質を有することが判明
した。このことは、組換えＤＮＡ技術によっても当該酵
素を製造でき、且つ、酵素の生産性も有意に向上するこ
とを示唆している。Then, the enzyme produced by the transformant BMT7 was purified in the same manner as in Experimental Example 1-1, and its properties and properties were examined. The recombinant enzyme was found to have a molecular weight value of about 76,000 to 87,0
The enzyme M obtained in Experimental Example 2 had an isoelectric point of 00 Dalton and an isoelectric point of about 3.6 to 4.6 by isoelectric focusing.
It was found to have physicochemical properties similar to those of -11. This suggests that the enzyme can be produced by the recombinant DNA technique, and the productivity of the enzyme is significantly improved.

【００５７】[0057]

【実験例４ リゾビウム・スピーシーズＭ
−１１に由来する相補鎖ＤＮＡの調製とその塩基配列、
アミノ酸配列の決定】実験例３−２で得た組換えＤＮＡ
ｐＢＭＴ７を、常法に従って、各種制限酵素で分解
し、Ｂｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）にサブク
ローニングして、塩基配列決定用ＤＮＡとした。これら
塩基配列決定用ＤＮＡを２μｇとり、これに２Ｍ水酸化
ナトリウム水溶液を加えて変性させた後、適量の冷エタ
ノールを加え、生成したテンプレートＤＮＡを含む沈澱
を採取し、真空乾燥した。このテンプレートＤＮＡに化
学合成した５′−ＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＴ
−３′で表される塩基配列のプライマー１を５０ｐｍｏ
ｌ／ｍｌと、２０ｍＭ塩化マグネシウムと５０ｍＭ塩化
ナトリウムを含む４０ｍＭトリス−塩酸緩衝液（ｐＨ
７．５）を１０μｌ加え、６５℃で２分間インキュベー
トしてアニーリングした後、ｄＡＴＰ、ｄＧＴＰ及びｄ
ＴＴＰをそれぞれ７．５μＭ含む水溶液を２μｌと、
［α−32Ｐ］ｄＣＴＰ（２ｍＣｉ／ｍｌ）を０．５μｌ
と、０．１Ｍジチオスレイトールを１μｌと、１．５単
位／ｍｌのＴ７ ＤＮＡポリメラーゼを２μｌ加え、２
５℃で５分間インキュベートすることによりプライマー
１を５′末端から３′末端に向かって伸長させ、相補鎖
ＤＮＡを生成させた。[Experimental Example 4 Rhizobium species M
Preparation of complementary strand DNA derived from -11 and its nucleotide sequence,
Determination of amino acid sequence] Recombinant DNA obtained in Experimental Example 3-2
pBMT7 was digested with various restriction enzymes and subcloned into Bluescript II SK (+) according to a conventional method to obtain a DNA for nucleotide sequencing. After taking 2 μg of these DNAs for determining a base sequence and denaturing them by adding 2M aqueous sodium hydroxide solution, an appropriate amount of cold ethanol was added, and the resulting precipitate containing the template DNA was collected and vacuum dried. 5'-GTAAAACGACGGCCAGT chemically synthesized on this template DNA
-50 'for primer 1 having the base sequence represented by -3'
1 / ml, 40 mM Tris-hydrochloric acid buffer solution (pH containing 20 mM magnesium chloride and 50 mM sodium chloride)
7.5) was added and incubated at 65 ° C. for 2 minutes to anneal, followed by dATP, dGTP and d.
2 μl of an aqueous solution containing 7.5 μM each of TTP,
0.5 μl of [α- 32 P] dCTP (2 mCi / ml)
And 1 μl of 0.1 M dithiothreitol and 2 μl of 1.5 units / ml of T7 DNA polymerase were added, and 2
Primer 1 was extended from the 5'end to the 3'end by incubating at 5 ° C for 5 minutes to generate complementary strand DNA.

【００５８】次に、上記で得た相補鎖ＤＮＡを含む反応
物を四等分し、それぞれにｄｄＡＴＰ、ｄｄＣＴＰ、ｄ
ｄＧＴＰ及びｄｄＴＴＰのいずれかを８μＭと８０μＭ
ｄＮＴＰを含む５０ｍＭ塩化ナトリウム水溶液を２．
５μｌ加え、３７℃で５分間インキュベートして反応さ
せ、２０ｍＭ ＥＤＴＡ、０．０５％（ｗ／ｖ）ブロム
フェノールブルー及び０．０５％（ｗ／ｖ）キシレンシ
アノールを含む９５％（ｖ／ｖ）水性ホルムアミド溶液
を４μｌ加えて反応を停止させた。反応物を沸騰水浴中
で３分間加熱後、６％（ｗ／ｖ）ポリアクリルアミドゲ
ル上にとり、約２，０００Ｖの定電圧を印加しながら電
気泳動してＤＮＡ断片を分離し、次いで、常法によりゲ
ルを固定し、乾燥させた後、オートラジオグラフィーし
た。Next, the reaction product containing the complementary strand DNA obtained above was divided into four equal parts, and ddATP, ddCTP and d
Either 8 μM or 80 μM of dGTP or ddTTP
A 50 mM sodium chloride aqueous solution containing dNTP was added to 2.
5 μl was added, and the mixture was incubated at 37 ° C. for 5 minutes to react, and 95% (v / v) containing 20 mM EDTA, 0.05% (w / v) bromphenol blue and 0.05% (w / v) xylene cyanol. ) The reaction was stopped by adding 4 μl of an aqueous formamide solution. After heating the reaction product in a boiling water bath for 3 minutes, the reaction product was loaded on a 6% (w / v) polyacrylamide gel and electrophoresed while applying a constant voltage of about 2,000 V to separate the DNA fragments. The gel was fixed by, dried and autoradiographed.

【００５９】ラジオグラム上に分離したＤＮＡ断片を解
析した結果、相補鎖ＤＮＡは配列表における配列番号５
に示す２，９３６塩基対からなる塩基配列を含んでいる
ことが判明した。この塩基配列から推定されるアミノ酸
配列は配列表における配列番号５に併記したとおりであ
り、このアミノ酸配列と配列表における配列番号７、９
又は１０に示す酵素Ｍ−１１のＮ末端アミノ酸配列、部
分アミノ酸配列を比較したところ、配列番号７のＮ末端
アミノ酸配列は配列表における配列番号５における第１
乃至２０番目の配列に、また、配列番号９又は１０の部
分アミノ酸配列は配列表における配列番号５における第
４８６乃至５０６番目又は第６０６乃至６２６番目の配
列に一致した。これは、酵素Ｍ−１１が配列表における
配列番号１に示すアミノ酸配列を有するものであり、リ
ゾビウム・スピーシーズＭ−１１においては、酵素Ｍ−
１１が配列表における配列番号３に示す塩基配列のＤＮ
Ａによりコードされていることを示している。As a result of analyzing the separated DNA fragments on the radiogram, the complementary strand DNA was found to be SEQ ID NO: 5 in the sequence listing.
It was found to contain the base sequence consisting of 2,936 base pairs shown in. The amino acid sequence deduced from this base sequence is as described in SEQ ID NO: 5 in the sequence listing, and this amino acid sequence and SEQ ID NOs: 7 and 9 in the sequence listing are shown.
Or the N-terminal amino acid sequence of the enzyme M-11 shown in 10 or 10 was compared, and the N-terminal amino acid sequence of SEQ ID NO: 7 was the first in SEQ ID NO: 5 in the sequence listing
To the 20th sequence, and the partial amino acid sequence of SEQ ID NO: 9 or 10 matched with the 486th to 506th sequences or the 606th to 626th sequences of SEQ ID NO: 5 in the sequence listing. This is because the enzyme M-11 has the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and in Rhizobium species M-11, the enzyme M-
11 is DN of the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing
It is shown to be coded by A.

【００６０】[0060]

【実験例５ アルスロバクター・スピーシ
ーズＱ３６に由来するＤＮＡを含む組換えＤＮＡと形質
転換体の調製】Experimental Example 5 Preparation of Recombinant DNA Containing DNA Derived from Arthrobacter species Q36 and Transformant

【００６１】[0061]

【実験例５−１ 染色体ＤＮＡの調製】実験例３−１と
同様にしてアルスロバクター・スピーシーズＱ３６から
染色体ＤＮＡを分離・精製し、濃度約１ｍｇ／ｍｌにな
るようにＳＳＣ緩衝液（ｐＨ７．１）に溶解し、−８０
℃で凍結した。[Experimental example 5-1 Preparation of chromosomal DNA] Chromosomal DNA was isolated and purified from Arthrobacter species Q36 in the same manner as in Experimental example 3-1, and the SSC buffer (pH 7. Dissolved in 1), -80
Frozen at ℃.

【００６２】[0062]

【実験例５−２ 組換えＤＮＡ ｐＢＱＴ１３と形質転
換体ＢＱＴ１３の調製】実験例５−１で得た精製染色体
ＤＮＡ溶液を実験例３−２と同様に部分切断した後、蔗
糖密度勾配超遠心法により約３，０００乃至６，０００
塩基対からなるＤＮＡ断片を採取した。その後、Ｔ４
ＤＮＡリガーゼを使用し、このＤＮＡ断片を実験例３−
２と同様に制限酵素Ｂａｍ ＨＩによるベクターＢｌｕ
ｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）の消化物に連結し、
得られた組換えＤＮＡを大腸菌ＸＬＩ−Ｂｌｕｅ株に導
入した。得られた形質転換体を実験例３−２と同様に５
−ブロモ−４−クロロ−３−インドリル−β−ガラクト
シドを含む寒天平板培地で培養し、生成した約４，５０
０個のコロニーをナイロン膜上に固定する一方、配列表
における配列番号１２に示すアミノ酸配列における第１
１乃至１６番目のＰｈｅ−Ａｓｐ−Ｖａｌ−Ａｓｐ−Ｔ
ｒｐ−Ａｓｐで表される配列に基づき５′−ＴＴＹＧＡ
ＹＧＴＮＧＡＹＴＧＧＧＡ−３′で表される塩基配列の
プローブ３を化学合成し、同位体32Ｐで標識後、前記ナ
イロン膜上に固定した形質転換体のコロニーにハイブリ
ダイズさせ、顕著な会合が認められた８種類の形質転換
体を選択した。[Experimental Example 5-2 Preparation of Recombinant DNA pBQT13 and Transformant BQT13] The purified chromosomal DNA solution obtained in Experimental Example 5-1 was partially cleaved in the same manner as in Experimental Example 3-2, and then sucrose density gradient ultracentrifugation About 3,000 to 6,000
A DNA fragment consisting of base pairs was collected. Then T4
Using DNA ligase, this DNA fragment was used in Experimental Example 3-
Vector Blu with restriction enzyme Bam HI as in 2
ligation to digest of escript II SK (+),
The obtained recombinant DNA was introduced into Escherichia coli XLI-Blue strain. The resulting transformant was treated with 5 as in Experimental Example 3-2.
About 4,50 produced by culturing on an agar plate medium containing -bromo-4-chloro-3-indolyl-β-galactoside
While fixing 0 colonies on the nylon membrane, the first in the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing was fixed.
1 to 16th Phe-Asp-Val-Asp-T
5'-TTYGA based on the sequence represented by rp-Asp
A probe 3 having a nucleotide sequence represented by YGTNGAYTGGGA-3 'was chemically synthesized, labeled with the isotope 32 P, and then hybridized with the colonies of the transformant immobilized on the nylon membrane, and a remarkable association was observed. Eight types of transformants were selected.

【００６３】実験例３−２と同様にして、これら８種類
の形質転換体から組換えＤＮＡを採取し、配列表におけ
る配列番号１１に示すアミノ酸配列における第１６乃至
２０番目のＴｈｒ−Ｇｌｕ−Ｐｈｅ−Ｔｒｐ−Ａｓｐで
表される配列に基づき化学合成した５′−ＡＣＮＧＡＲ
ＴＴＹＴＧＧＧＡ−３′で表される塩基配列のプローブ
４をハイブリダイズさせ、顕著な会合を示した組換えＤ
ＮＡを選択した。以上のようにして選択した組換えＤＮ
Ａと形質転換体を、それぞれ、『ｐＢＱＴ１３』又は
『ＢＱＴ１３』と命名した。Recombinant DNAs were collected from these eight types of transformants in the same manner as in Experimental Example 3-2, and the 16th to 20th Thr-Glu-Phe in the amino acid sequence shown in SEQ ID NO: 11 in the Sequence Listing were collected. 5'-ACNGAR chemically synthesized based on the sequence represented by -Trp-Asp
Recombinant D hybridized with probe 4 having the nucleotide sequence represented by TTYTGGGGA-3 ′ and showing a remarkable association.
NA was selected. Recombinant DN selected as described above
A and the transformant were designated as "pBQT13" or "BQT13", respectively.

【００６４】その後、この形質転換体ＢＱＴ１３をアン
ピシリンを含むＬ−ブロス培地で実験例３−２と同様に
培養し、培養物より採取した菌体から組換えＤＮＡを溶
出させ、精製し、分析したところ、組換えＤＮＡ ｐＢ
ＱＴ１３は約７，２００塩基対からなり、図１０に示す
制限酵素地図で表される構造を有していた。図１０に示
すように、酵素Ｑ３６をコードする２，３２５塩基対か
らなるＤＮＡは、制限酵素Ｘｍｎ Ｉによる切断部位付
近の下流に位置していることが判明した。Thereafter, this transformant BQT13 was cultured in L-broth medium containing ampicillin in the same manner as in Experimental Example 3-2, and recombinant DNA was eluted from the cells collected from the culture, purified, and analyzed. By the way, recombinant DNA pB
QT13 consisted of about 7,200 base pairs and had the structure represented by the restriction enzyme map shown in FIG. As shown in FIG. 10, it was revealed that the DNA composed of 2,325 base pairs encoding the enzyme Q36 was located downstream near the cleavage site by the restriction enzyme Xmn I.

【００６５】[0065]

【実験例５−３ 形質転換体ＢＱＴ１３による酵素の産
生】マルトース２．０％（ｗ／ｖ）、ペプトン０．５％
（ｗ／ｖ）、酵母エキス０．１％（ｗ／ｖ）、燐酸水素
二ナトリウム０．１％（ｗ／ｖ）、燐酸二水素カリウム
０．１％（ｗ／ｖ）を含む液体培地をｐＨ７．０に調整
し、アンピシリンを５０μｇ／ｍｌ加え、１２０℃で２
０分間加熱滅菌し、冷却後、実験例５−２で得た形質転
換体ＢＱＴ１３を植菌し、３７℃で２４時間回転振盪培
養した。培養物を超音波処理して菌体を破砕し、遠心分
離により不溶物を除去後、上清中の酵素活性を測定した
ところ、培養物１ｌ当たりに換算して、約２，４５０単
位の酵素が産生していた。[Experimental Example 5-3 Production of enzyme by transformant BQT13] Maltose 2.0% (w / v), peptone 0.5%
(W / v), yeast extract 0.1% (w / v), disodium hydrogen phosphate 0.1% (w / v), potassium dihydrogen phosphate 0.1% (w / v). Adjust the pH to 7.0, add 50 μg / ml of ampicillin, and add 2 at 120 ° C.
After heat sterilization for 0 minutes and cooling, the transformant BQT13 obtained in Experimental Example 5-2 was inoculated and cultivated at 37 ° C. for 24 hours with rotary shaking. The culture was sonicated to disrupt the cells, the insoluble matter was removed by centrifugation, and the enzyme activity in the supernatant was measured. As a result, about 2450 units of enzyme were calculated per 1 liter of culture. Was produced.

【００６６】別途、対照として、大腸菌ＸＬＩ−Ｂｌｕ
ｅ株及びアルスロバクター・スピーシーズＱ３６をアン
ピシリン無含有の同じ組成の液体培地に植菌し、アルス
ロバクター・スピーシーズＱ３６の場合、培養温度を３
０℃に設定した以外は上記と同様に培養・処理した。処
理物の活性を測定したところ、アルスロバクター・スピ
ーシーズＱ３６による酵素の産生は培養物１ｌ当たり約
１，２００単位と、形質転換体ＢＱＴ１３と比較して有
意に低いものであった。なお、宿主に使用した大腸菌Ｘ
ＬＩ−Ｂｌｕｅ株は、当該酵素を産生しなかった。Separately, as a control, Escherichia coli XLI-Blu was used.
strain e and Arthrobacter species Q36 were inoculated into a liquid medium of the same composition containing no ampicillin, and in the case of Arthrobacter species Q36, the culture temperature was 3
Culture and treatment were performed in the same manner as above except that the temperature was set to 0 ° C. When the activity of the treated product was measured, the production of the enzyme by Arthrobacter species Q36 was about 1,200 units per liter of the culture, which was significantly lower than that of the transformant BQT13. E. coli X used as a host
The LI-Blue strain did not produce the enzyme.

【００６７】その後、形質転換体ＢＱＴ１３が産生した
酵素を実験例１−１と同様に精製し、その性質・性状を
調べたところ、ＳＤＳ−ポリアクリルアミドゲル電気泳
動で分子量約７６，０００乃至８７，０００ダルトン
を、また、等電点電気泳動で約３．６乃至４．６に等電
点を示すなど、実験例２で得られた酵素Ｑ３６のものと
同様の理化学的性質を有することが判明した。このこと
は、組換えＤＮＡ技術によっても当該酵素を製造でき、
且つ、酵素の生産性も有意に向上することを示唆してい
る。Then, the enzyme produced by the transformant BQT13 was purified in the same manner as in Experimental Example 1-1, and its properties and properties were examined. As a result, it was confirmed by SDS-polyacrylamide gel electrophoresis that the molecular weight was about 76,000 to 87, 000 Dalton, and has an physicochemical property similar to that of the enzyme Q36 obtained in Experimental Example 2, such as having an isoelectric point of about 3.6 to 4.6 by isoelectric focusing. did. This means that the enzyme can also be produced by recombinant DNA technology,
Moreover, it is suggested that the productivity of the enzyme is significantly improved.

【００６８】[0068]

【実験例６ アルスロバクター・スピーシ
ーズＱ３６に由来する相補鎖ＤＮＡの調製とその塩基配
列、アミノ酸配列の決定】実験例５−２で得た組換えＤ
ＮＡ ｐＢＱＴ１３を実験例４と同様に処理してテンプ
レートＤＮＡとし、これをプライマー１とともにアニー
リング後、Ｔ７ＤＮＡポリメラーゼを作用させてプライ
マー１を５′末端から３′末端に向かって伸長させ、相
補鎖ＤＮＡを生成させた。実験例４と同様に、この相補
鎖ＤＮＡにジデオキシ・チェーン・ターミネータ法を適
用し、ラジオグラム上に分離したＤＮＡ断片を解析した
結果、相補鎖ＤＮＡは配列表における配列番号６に示す
３，０７３塩基対からなる塩基配列を含んでいることが
判明した。この塩基配列から推定されるアミノ酸配列は
配列表における配列番号６に併記したとおりであり、こ
のアミノ酸配列と配列表における配列番号８、１１又は
１２に示すＮ末端アミノ酸配列、部分アミノ酸配列を比
較したところ、配列番号８のＮ末端アミノ酸配列は配列
表における配列番号６における第１乃至２０番目の配列
に、また、配列番号１１又は１２の部分アミノ酸配列は
配列表における配列番号６における第６０６乃至６２５
番目又は第１１０乃至１２９番目の配列に一致した。こ
れは、酵素Ｑ３６が配列表における配列番号２に示すア
ミノ酸配列を有するものであり、アルスロバクター・ス
ピーシーズＱ３６においては、酵素Ｑ３６が配列表にお
ける配列番号４に示す塩基配列のＤＮＡによりコードさ
れていることを示している。[Experimental Example 6 Preparation of complementary strand DNA derived from Arthrobacter species Q36 and determination of its nucleotide sequence and amino acid sequence] Recombinant D obtained in Experimental Example 5-2
NA pBQT13 was treated in the same manner as in Experimental Example 4 to obtain a template DNA, which was annealed together with Primer 1 and then allowed to act on T7 DNA polymerase to extend Primer 1 from the 5 ′ end to the 3 ′ end to obtain a complementary strand DNA. Was generated. As in Experimental Example 4, the dideoxy chain terminator method was applied to the complementary strand DNA, and the separated DNA fragment was analyzed on the radiogram. As a result, the complementary strand DNA was 3,073 shown in SEQ ID NO: 6 in the sequence listing. It was found to contain a base sequence consisting of base pairs. The amino acid sequence deduced from this base sequence is as described in SEQ ID NO: 6 in the sequence listing, and this amino acid sequence was compared with the N-terminal amino acid sequence and partial amino acid sequence shown in SEQ ID NO: 8, 11 or 12 in the sequence listing. By the way, the N-terminal amino acid sequence of SEQ ID NO: 8 is the 1st to 20th sequence in SEQ ID NO: 6 in the sequence listing, and the partial amino acid sequence of SEQ ID NO: 11 or 12 is the 606th to 625th sequence in SEQ ID NO: 6 in the sequence listing.
The sequence matched the 1st or 110th to 129th sequences. This is because the enzyme Q36 has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and in Arthrobacter species Q36, the enzyme Q36 is encoded by the DNA of the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing. It indicates that

【００６９】以上説明したように、グルコース重合度３
以上の還元性澱粉糖から末端にトレハロース構造を有す
る非還元性糖質を生成する酵素は、本発明者が長年に亙
る研究の一成果として見出されたものであり、従来公知
の酵素には見られない独特の理化学的性質を具備してい
る。この発明は、組換えＤＮＡ技術を応用することによ
り、この酵素を創製しようというものである。以下、実
施例等を参照しながら、この発明の組換え型酵素とその
製造方法並びに用途につき、具体的に説明する。As explained above, the degree of glucose polymerization is 3
The enzyme that produces a non-reducing sugar having a trehalose structure at the end from the reducing starch sugar is one that the present inventor has discovered as a result of many years of research, and is not known as a conventionally known enzyme. It has unique physicochemical properties that cannot be seen. The present invention aims to create this enzyme by applying recombinant DNA technology. Hereinafter, the recombinant enzyme of the present invention, a method for producing the same, and uses thereof will be specifically described with reference to Examples and the like.

【００７０】この発明でいう組換え型酵素とは、組換え
ＤＮＡ技術により創製され、グルコース重合度３以上の
還元性澱粉糖から末端にトレハロース構造を有する非還
元性糖質を生成する酵素全般を意味する。この発明の組
換え型酵素は、通常、解明されたアミノ酸配列を有して
おり、その一例として、例えば、配列表における配列番
号１又は２に示すアミノ酸配列かそれに相同的なアミノ
酸配列が挙げられる。配列表における配列番号１又は２
のアミノ酸配列に相同的なアミノ酸配列を有する変異体
は、所期の酵素作用を実質的に変えることなく、配列表
における配列番号１又は２に示すアミノ酸配列における
構成アミノ酸の１個又は２個以上を他のアミノ酸で置換
することにより得ることができる。なお、同じＤＮＡで
あっても、それを導入する宿主や、そのＤＮＡを含む形
質転換体の培養に使用する栄養培地の成分・組成、培養
温度・ｐＨなどに依っては、宿主内酵素によるＤＮＡ発
現後の修飾などにより、所期の酵素反応は保持している
ものの、配列表における配列番号１又は２に示すアミノ
酸配列におけるＮ末端付近のアミノ酸が１個又は２個以
上欠失したり、Ｎ末端に１個又は２個以上のアミノ酸が
新たに付加した変異体の産生することがある。斯かる変
異体も、それがグルコース重合度３以上の還元性澱粉糖
から末端にトレハロース構造を有する非還元性糖質を生
成するかぎり、当然、この発明の組換え型酵素に包含さ
れる。The term "recombinant enzyme" as used in the present invention means any enzyme which is created by recombinant DNA technology and produces a non-reducing sugar having a trehalose structure at the end from a reducing starch sugar having a glucose polymerization degree of 3 or more. means. The recombinant enzyme of the present invention usually has an elucidated amino acid sequence, and examples thereof include the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing or an amino acid sequence homologous thereto. . SEQ ID NO: 1 or 2 in the sequence listing
A variant having an amino acid sequence homologous to the amino acid sequence of is one or more of the constituent amino acids in the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing without substantially changing the intended enzyme action. Can be obtained by substituting for another amino acid. Even with the same DNA, depending on the host into which it is introduced, the components / composition of the nutrient medium used for culturing the transformant containing the DNA, the culture temperature / pH, etc., the DNA produced by the enzyme in the host Although the desired enzymatic reaction is retained due to modification after expression, one or more amino acids near the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing may be deleted, or N A mutant having one or more amino acids newly added to the end may be produced. Such a mutant is naturally included in the recombinant enzyme of the present invention as long as it produces a non-reducing sugar having a trehalose structure at the end from a reducing starch sugar having a glucose polymerization degree of 3 or more.

【００７１】この発明による組換え型酵素は、特定のＤ
ＮＡを含む形質転換体の培養物から採取することができ
る。この発明で使用する形質転換体は、例えば、配列表
における配列番号３又は４に示す塩基配列かそれに相同
的な塩基配列又はそれらに相補的な塩基配列のＤＮＡを
適宜宿主に導入することにより得ることができる。な
お、上記塩基配列は、遺伝子コードの縮重を利用して、
コードするアミノ酸配列を変えることなく、塩基の１個
又は２個以上を他の塩基で置き換えてもよい。また、Ｄ
ＮＡが宿主中で実際に当該酵素の産生を発現するため
に、当該酵素又はその相同変異体をコードする塩基配列
における塩基の１個又は２個以上を他の塩基で適宜置換
し得ることはいうまでもない。The recombinant enzyme according to the present invention has a specific D
It can be collected from the culture of the transformant containing NA. The transformant used in the present invention can be obtained, for example, by appropriately introducing into a host a DNA having the nucleotide sequence shown in SEQ ID NO: 3 or 4 or a nucleotide sequence homologous thereto or a nucleotide sequence complementary thereto. be able to. In addition, the above nucleotide sequence utilizes the degeneracy of the genetic code to
One or two or more of the bases may be replaced with another base without changing the encoded amino acid sequence. Also, D
It is said that one or two or more of the bases in the base sequence encoding the enzyme or a homologous variant thereof may be appropriately replaced with another base in order for NA to actually express the production of the enzyme in the host. There is no end.

【００７２】この発明で使用するＤＮＡは、それが前述
のような配列を有するかぎり、それが天然に由来するも
のか人為的に合成されたものであるかは問わない。天然
の給源としては、例えば、リゾビウム・スピーシーズＭ
−１１（ＦＥＲＭ ＢＰ−４１３０）、アルスロバクタ
ー・スピーシーズＱ３６（ＦＥＲＭ ＢＰ−４３１
６）、ブレビバクテリウム・ヘロボルム（ＡＴＣＣ１１
８２２）、フラボバクテリウム・アクアチレ（ＩＦＯ３
７７２）、ミクロコッカス・ルテウス（ＩＦＯ３０６
４）、ミクロコッカス・ロゼウス（ＡＴＣＣ１８６）、
クルトバクテリウム・シトレウム（ＩＦＯ１５２３
１）、マイコバクテリウム・スメグマチス（ＡＴＣＣ１
９４２０）及びテラバクター・ツメスセンス（ＩＦＯ１
２９６０）を含むリゾビウム属、アルスロバクター属、
ブレビバクテリウム属、フラボバクテリウム属、ミクロ
コッカス属、クルトバクテリウム属、マイコバクテリウ
ム属、テラバクター属の微生物が挙げられ、これら微生
物の菌体からはこの発明のＤＮＡを含む遺伝子が得られ
る。すなわち、斯かる微生物を栄養培地に植菌し、好気
的条件下で約１乃至３日間培養後、培養物から菌体を採
取し、リゾチームやβ−グルカナーゼなどの細胞壁溶解
酵素や超音波で処理することにより、当該ＤＮＡを含む
遺伝子を菌体外に溶出させる。このとき、細胞壁溶解酵
素にプロテアーゼなどの蛋白質加水分解酵素を併用した
り、菌体を超音波処理する際、ＳＤＳなどの界面活性剤
を共存させたり凍結融解してもよい。斯くして得られる
処理物に、例えば、フェノール抽出、アルコール沈澱、
遠心分離、プロテアーゼ処理、リボヌクレアーゼ処理な
どの斯界における通常一般の方法を適用すれば目的のＤ
ＮＡが得られる。一方、ＤＮＡを人為的に合成するに
は、例えば、配列表における配列番号３又は４に示す塩
基配列に基づいて化学合成するか、配列表における配列
番号１又は２に示すアミノ酸配列をコードするＤＮＡを
自律複製可能な適宜ベクターに挿入して組換えＤＮＡと
し、これを適宜宿主に導入して得られる形質転換体を培
養し、培養物から菌体を分離し、その菌体から当該ＤＮ
Ａを含むプラスミドを採取すればよい。The DNA used in the present invention may be of natural origin or artificially synthesized as long as it has the above-mentioned sequence. As a natural source, for example, Rhizobium species M
-11 (FERM BP-4130), Arthrobacter Species Q36 (FERM BP-431)
6), Brevibacterium heroborum (ATCC11
822), Flavobacterium aquatile (IFO3
772), Micrococcus luteus (IFO306
4), Micrococcus roseus (ATCC186),
Kurtobacter citrium (IFO1523
1), Mycobacterium smegmatis (ATCC1
9420) and Terrabacter tumescens (IFO1
2960) including Rhizobium, Arthrobacter,
The microorganisms of the genus Brevibacterium, the genus Flavobacterium, the genus Micrococcus, the genus Curtobacterium, the genus Mycobacterium, and the genus Terrabaca are mentioned, and the gene containing the DNA of the present invention can be obtained from the cells of these microorganisms. That is, such a microorganism is inoculated into a nutrient medium and cultured under aerobic conditions for about 1 to 3 days, and then the bacterial cells are collected from the culture and treated with a cell wall lytic enzyme such as lysozyme or β-glucanase or ultrasonic waves. By the treatment, the gene containing the DNA is eluted outside the cell. At this time, a protein hydrolase such as a protease may be used in combination with the cell wall lysing enzyme, or a surfactant such as SDS may coexist or freeze-thaw when the bacterial cells are subjected to ultrasonic treatment. The treated product thus obtained may be subjected to, for example, phenol extraction, alcohol precipitation,
If a general method generally used in this field such as centrifugation, protease treatment, ribonuclease treatment is applied, the desired D
NA is obtained. On the other hand, for artificially synthesizing DNA, for example, it is chemically synthesized based on the nucleotide sequence shown in SEQ ID NO: 3 or 4 in the sequence listing, or DNA encoding the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing. Is inserted into an appropriate vector capable of autonomous replication to form a recombinant DNA, the transformant is appropriately introduced into the host, the resulting transformant is cultured, and the bacterium is separated from the culture.
A plasmid containing A may be collected.

【００７３】斯かるＤＮＡは、通常、組換えＤＮＡの形
態で宿主に導入される。組換えＤＮＡは、通常、ＤＮＡ
と自律複製可能なベクターを含んでなり、ＤＮＡが入手
できれば、通常一般の組換えＤＮＡ技術により比較的容
易に調製することができる。斯かるベクターの例として
は、ｐＢＲ３２２、ｐＵＣ１８、Ｂｌｕｅｓｃｒｉｐｔ
ＩＩ ＳＫ（＋）、ｐＵＢ１１０、ｐＴＺ４、ｐＣ１
９４、ｐＨＶ１４、ＴＲｐ７、ＹＥｐ７、ｐＢＳ７など
のプラスミドベクターやλｇｔ・λＣ、λｇｔ・λＢ、
ρ１１、φ１、φ１０５などのファージベクターが挙げ
られ、このうち、この発明のＤＮＡを大腸菌で発現させ
るにはｐＢＲ３２２、ｐＵＣ１８、Ｂｌｕｅｓｃｒｉｐ
ｔ ＩＩ ＳＫ（＋）、λｇｔ・λＣ及びλｇｔ・λＢ
が好適であり、一方、枯草菌で発現させるにはｐＵＢ１
１０、ｐＴＺ４、ｐＣ１９４、ρ１１、φ１及びφ１０
５が好適である。ｐＨＶ１４、ＴＲｐ７、ＹＥｐ７及び
ｐＢＳ７は、組換えＤＮＡを２種以上の宿主内で増殖さ
せる場合に有用である。Such DNA is usually introduced into the host in the form of recombinant DNA. Recombinant DNA is usually DNA
If a DNA is available, which comprises a vector capable of autonomous replication and can be prepared relatively easily by a general recombinant DNA technique. Examples of such vectors include pBR322, pUC18, Bluescript.
II SK (+), pUB110, pTZ4, pC1
94, pHV14, TRp7, YEp7, pBS7 and other plasmid vectors, λgt · λC, λgt · λB,
Phage vectors such as ρ11, φ1 and φ105 can be mentioned. Among them, pBR322, pUC18 and Bluescript can be used to express the DNA of the present invention in E. coli.
t II SK (+), λgt · λC and λgt · λB
Is preferred, while pUB1 for expression in B. subtilis
10, pTZ4, pC194, ρ11, φ1 and φ10
5 is suitable. pHV14, TRp7, YEp7 and pBS7 are useful when growing recombinant DNA in more than one host.

【００７４】ＤＮＡを斯かるベクターに挿入するには、
斯界において通常一般の方法が採用される。具体的に
は、先ず、ＤＮＡを含む遺伝子と自律複製可能なベクタ
ーとを制限酵素及び／又は超音波により切断し、次に、
生成したＤＮＡ断片とベクター断片とを連結する。遺伝
子及びベクターの切断にヌクレオチドに特異的に作用す
る制限酵素、とりわけ、ＩＩ型の制限酵素、詳細には、
Ｓａｕ ３ＡＩ、ＥｃｏＲＩ、Ｈｉｎｄ ＩＩＩ、Ｂａ
ｍ ＨＩ、Ｓａｌ Ｉ、Ｘｂａ Ｉ、ＳａｃＩ、Ｐｓｔ
Ｉなどを使用すれば、ＤＮＡ断片とベクター断片を連
結するのが容易となる。ＤＮＡ断片とベクター断片を連
結するには、必要に応じて、両者をアニーリングした
後、生体内又は生体外でＤＮＡリガーゼを作用させれば
よい。斯くして得られる組換えＤＮＡは、適宜宿主に導
入して形質転換体とし、これを培養することにより無限
に複製可能である。To insert the DNA into such a vector,
Commonly used methods are commonly used in the art. Specifically, first, a gene containing DNA and a vector capable of autonomous replication are cleaved with a restriction enzyme and / or ultrasonic waves, and then,
The generated DNA fragment and the vector fragment are ligated. Restriction enzymes that act specifically on nucleotides to cleave genes and vectors, especially type II restriction enzymes, in particular
Sau 3AI, EcoRI, Hind III, Ba
m HI, Sal I, Xba I, SacI, Pst
When I or the like is used, it becomes easy to ligate the DNA fragment and the vector fragment. In order to ligate the DNA fragment and the vector fragment, if necessary, both may be annealed and then a DNA ligase is allowed to act in vivo or in vitro. The recombinant DNA thus obtained can be infinitely replicated by appropriately introducing it into a host to obtain a transformant and culturing the transformant.

【００７５】このようにして得られる組換えＤＮＡは、
大腸菌、枯草菌、放線菌、酵母を始めとする適宜の宿主
微生物に導入することができる。宿主が大腸菌の場合に
は、宿主を組換えＤＮＡとカルシウムイオンの存在下で
培養すればよく、一方、宿主が枯草菌の場合には、コン
ピテントセル法やプロトプラスト法を適用すればよい。
形質転換体をクローニングするには、コロニーハイブリ
ダイゼーション法を適用するか、グルコース重合度３以
上の還元性澱粉糖を含む栄養培地で培養し、該澱粉糖よ
り末端にトレハロース構造を有する非還元性糖質を生成
するものを選択すればよい。The recombinant DNA thus obtained is
It can be introduced into an appropriate host microorganism such as Escherichia coli, Bacillus subtilis, actinomycete and yeast. When the host is E. coli, the host may be cultured in the presence of recombinant DNA and calcium ions, while when the host is Bacillus subtilis, the competent cell method or the protoplast method may be applied.
In order to clone the transformant, a colony hybridization method is applied, or the transformant is cultured in a nutrient medium containing a reducing starch sugar having a degree of glucose polymerization of 3 or more, and a non-reducing sugar having a trehalose structure at the end of the starch sugar. You can select the one that produces quality.

【００７６】斯くして得られる形質転換体は、栄養培地
で培養すると、菌体内外に当該酵素を産生する。栄養培
地には、通常、炭素源、窒素源、ミネラル、さらには、
必要に応じて、アミノ酸やビタミンなどの微量栄養素を
補足した通常一般の液体培地が使用され、個々の炭素源
としては、例えば、澱粉、澱粉加水分解物、グルコー
ス、果糖、蔗糖などの糖質が、また、窒素源としては、
例えば、アンモニア若しくはアンモニウム塩、尿素、硝
酸塩、ペプトン、酵母エキス、脱脂大豆、コーンスティ
ープリカー、肉エキスなどの含窒素無機乃至有機物が挙
げられる。形質転換体を斯かる栄養培地に植菌し、栄養
培地を温度２５乃至６５℃、ｐＨ２乃至８に保ちつつ、
通気撹拌などによる好気的条件下で約１乃至６日間培養
すれば、当該酵素を含む培養物が得られる。この培養物
は酵素剤としてそのまま使用可能ではあるが、通常は使
用に先立ち、必要に応じて、超音波や細胞壁溶解酵素に
より菌体を破砕した後、濾過、遠心分離などにより酵素
を菌体又は菌体破砕物から分離し、精製する。精製には
酵素を精製するための通常一般の方法が採用でき、例え
ば、菌体又は菌体破砕物を除去した培養物に濃縮、塩
析、透析、分別沈澱、ゲル濾過クロマトグラフィー、イ
オン交換クロマトグラフィー、疎水クロマトグラフィ
ー、アフィニティークロマトグラフィー、ゲル電気泳
動、等電点電気泳動などの１種若しくは２種以上を適宜
組合せて適用すればよい。When the transformant thus obtained is cultured in a nutrient medium, the enzyme is produced inside and outside the cells. The nutrient medium usually contains carbon sources, nitrogen sources, minerals,
If necessary, a general liquid medium supplemented with micronutrients such as amino acids and vitamins is usually used, and examples of individual carbon sources include starch, starch hydrolysates, glucose, fructose, and sugars such as sucrose. Also, as a nitrogen source,
Examples thereof include nitrogen-containing inorganic or organic substances such as ammonia or ammonium salt, urea, nitrate, peptone, yeast extract, defatted soybean, corn steep liquor and meat extract. The transformant was inoculated into such a nutrient medium, and while maintaining the nutrient medium at a temperature of 25 to 65 ° C. and a pH of 2 to 8,
Culturing under aerobic conditions such as aeration and stirring for about 1 to 6 days gives a culture containing the enzyme. This culture can be used as an enzyme preparation as it is, but usually before use, if necessary, the cells are disrupted by ultrasonic waves or cell wall lysing enzyme, and then the enzyme is removed by filtration or centrifugation. It is separated from the disrupted cells and purified. For purification, a generally-used general method for purifying an enzyme can be adopted, and examples thereof include concentration, salting out, dialysis, fractional precipitation, gel filtration chromatography, and ion exchange chromatography in a culture in which cells or disrupted cells are removed. One or a combination of two or more types such as chromatography, hydrophobic chromatography, affinity chromatography, gel electrophoresis, isoelectric focusing may be applied.

【００７７】前述のとおり、この発明による組換え型酵
素は、グルコース重合度３以上の還元性澱粉糖から末端
にトレハロース構造を有する非還元性糖質を生成すると
いう、従来の酵素に見られない顕著な性質を有する。生
成した非還元性糖質は温和で上品な甘味に加えて適度の
粘度と保湿性を有し、そして、何よりも、分子中に還元
性基を有しないので、着色や変質の懸念なく飲食物を甘
味付けできるという大きな利点がある。当該組換え型酵
素のこの性質を利用することにより、従来、還元性故に
敬遠されがちであった種々の澱粉糖を、還元性を有しな
いか還元性が顕著に低下した、扱い易い、有用な糖質に
変換できることとなる。As described above, the recombinant enzyme according to the present invention is not found in conventional enzymes which produce a non-reducing sugar having a trehalose structure at the end from a reducing starch sugar having a glucose polymerization degree of 3 or more. It has outstanding properties. The produced non-reducing sugar has mild viscosity and sweetness, moderate viscosity and moisturizing property, and above all, it has no reducing group in the molecule, so there is no fear of coloring or deterioration. Has the great advantage that it can be sweetened. By utilizing this property of the recombinant enzyme, various starch sugars, which have been conventionally shunned due to their reducibility, have no reducibility or have remarkably reduced reducibility, are easy to handle, and are useful. It can be converted to sugar.

【００７８】斯かる変換方法につきさらに説明すると、
この発明による組換え型酵素の基質には、通常、澱粉、
又はアミロペクチン、アミロースなどの澱粉質を酸及び
／又はアミラーゼによって部分的に加水分解して得られ
る還元性澱粉加水分解物が用いられる。斯かる澱粉加水
分解物は斯界における通常一般の方法により得ることが
でき、通常、マルトトリオース、マルトテトラオース、
マルトペンタオース、マルトヘキサオース、マルトヘプ
タオースなどのグルコース重合度３以上のマルトオリゴ
糖の１種若しくは２種以上を含んでなる。アミラーゼ研
究会編『ハンドブック・オブ・アミレーシーズ・アンド
・リレイテッド・エンザイムズ』、１９８８年、パーガ
モン・プレス発行に記載されているα−アミラーゼ、マ
ルトテトラオース生成アミラーゼ、マルトペンタオース
生成アミラーゼ及びマルトヘキサオース生成アミラーゼ
は、この発明で使用する還元性澱粉糖の調製に特に有用
であり、これらアミラーゼのいずれかを使用することに
より、グルコース重合度３以上の還元性澱粉糖を豊富に
含む澱粉糖混合物が容易且つ効率的に得られる。なお、
このとき、必要に応じて、プルラナーゼやイソアミラー
ゼなどの澱粉枝切酵素を併用すれば、当該組換え型酵素
の基質となり得る還元性澱粉糖の収量を上げることがで
きる。To further explain such a conversion method,
The substrate for the recombinant enzyme according to the present invention is usually starch,
Alternatively, a reducing starch hydrolyzate obtained by partially hydrolyzing a starch substance such as amylopectin and amylose with an acid and / or amylase is used. Such a starch hydrolyzate can be obtained by a method commonly used in the art, usually maltotriose, maltotetraose,
It comprises one or more malto-oligosaccharides having a glucose polymerization degree of 3 or more, such as maltopentaose, maltohexaose and maltoheptaose. "Handbook of Amylases and Related Enzymes" edited by Amylase Research Group, 1988, published by Pergamon Press, α-amylase, maltotetraose-producing amylase, maltopentaose-forming amylase and maltohexaose. The produced amylase is particularly useful for the preparation of the reducing starch sugar used in the present invention, and by using any of these amylases, a starch sugar mixture rich in reducing starch sugar with a glucose polymerization degree of 3 or more can be obtained. Obtained easily and efficiently. In addition,
At this time, if necessary, a starch debranching enzyme such as pullulanase or isoamylase may be used in combination to increase the yield of reducing starch sugar that can serve as a substrate for the recombinant enzyme.

【００７９】この発明による変換方法においては、通
常、基質として上記したような還元性澱粉糖の１種又は
２種以上を含む水溶液にこの発明による組換え型酵素を
共存せしめ、水溶液を所定の温度、ｐＨに保ちつつ、所
望量の非還元性糖質が生成するまで反応させる。反応は
０．１％（ｗ／ｗ）程度の基質濃度下でも進行するが、
この発明による変換方法を大規模に実施する場合には、
より高濃度の２％（ｗ／ｗ）以上、望ましくは、５乃至
５０％（ｗ／ｗ）とするのがよい。反応時の温度とｐＨ
は組換え型酵素が失活することなく基質に効率的に作用
するレベルに設定され、温度は５５℃付近まで、望まし
くは、約４０乃至５５℃に、また、ｐＨは５乃至１０、
望ましくは、約６乃至８の範囲に設定される。組換え型
酵素の量と反応時間は、反応の進行具合に依って適宜に
設定する。斯かる反応によりグルコース重合度３以上の
還元性澱粉糖の還元力は顕著に低下し、マルトペンタオ
ースの場合、還元力はもとの約７％程度にまで低下す
る。In the conversion method according to the present invention, the recombinant enzyme according to the present invention is usually allowed to coexist in an aqueous solution containing one or more reducing starch sugars as described above as a substrate, and the aqueous solution is heated to a predetermined temperature. While maintaining the pH, the reaction is continued until a desired amount of non-reducing sugar is produced. The reaction proceeds even under a substrate concentration of about 0.1% (w / w),
When implementing the conversion method according to the present invention on a large scale,
A higher concentration of 2% (w / w) or more, preferably 5 to 50% (w / w) is preferable. Reaction temperature and pH
Is set to a level at which the recombinant enzyme efficiently acts on the substrate without deactivation, the temperature is up to around 55 ° C., preferably about 40 to 55 ° C., and the pH is 5 to 10,
Desirably, it is set in the range of about 6 to 8. The amount of the recombinant enzyme and the reaction time are appropriately set depending on the progress of the reaction. Due to such a reaction, the reducing power of the reducing starch sugar having a glucose polymerization degree of 3 or more is remarkably reduced, and in the case of maltopentaose, the reducing power is reduced to about 7% of the original amount.

【００８０】この発明の変換方法により得られた反応物
はそのまま使用可能であるが、通常、使用に先立ち精製
する。すなわち、濾過・遠心分離などにより反応物から
不溶物を除去し、活性炭により脱色した後、イオン交換
樹脂により脱塩・精製し、濃縮してシロップ状物とす
る。用途に依っては、このシロップ状物を真空乾燥、噴
霧乾燥などにより固状物としてもよい。実質的に非還元
性糖質のみからなる製品を得るには、上記シロップ状物
にイオン交換樹脂、活性炭、シリカゲルなどによる糖質
を分離するための種々のクロマトグラフィー、アルコー
ル、アセトンなどによる分別沈澱、膜濾過、酵母による
発酵、アルカリによる還元性糖質の分解除去などの１種
若しくは２種以上を適用する。大量の反応物を処理する
には、例えば、特開昭５８−２３７９９号公報や特開昭
５８−７２５９８号公報に開示されている強酸性カチオ
ン交換樹脂を使用する固定床方式、移動床方式又は擬似
移動床方式のイオン交換クロマトグラフィーが有用であ
り、これらの方法によるときには、非還元性糖質の含量
が高い製品を大量且つ効率的に得ることができる。The reaction product obtained by the conversion method of the present invention can be used as it is, but usually, it is purified before use. That is, insoluble matter is removed from the reaction product by filtration, centrifugation, etc., decolorized with activated carbon, desalted and purified with an ion exchange resin, and concentrated to give a syrup-like product. Depending on the application, this syrup-like product may be vacuum-dried, spray-dried or the like to be a solid product. In order to obtain a product consisting essentially of non-reducing sugars, various syrup-like substances such as ion exchange resin, activated carbon, silica gel, etc. for various sugar separations, alcohol, acetone, etc. , One or more of membrane filtration, fermentation with yeast, decomposition and removal of reducing sugars with alkali, etc. are applied. To treat a large amount of reactants, for example, a fixed bed system using a strongly acidic cation exchange resin disclosed in JP-A-58-23799 and JP-A-58-72598, a moving bed system, or Pseudo moving bed ion exchange chromatography is useful, and by these methods, a large amount of products having a high content of non-reducing sugars can be efficiently obtained.

【００８１】斯くして得られる非還元性糖質は、糖質甘
味剤の還元性を嫌う種々の物品に広範な用途を有し、例
えば、食品、化粧品、医薬品の甘味剤、呈味改善剤、品
質改善剤、安定剤、賦形剤として極めて有用である。加
えて、当該非還元性糖質は、グルコアミラーゼ、α−グ
ルコシダーゼ、あるいは、特願平５−３４０３４３号明
細書に開示されているトレハロース遊離酵素を作用させ
ると、ほぼ定量的にトレハロースを与えることから、従
来、大量入手が難しかったトレハロースを製造するため
の中間体としても有用である。The non-reducing saccharide thus obtained has a wide range of uses in various articles in which the reducibility of a sugar sweetener is disliked. For example, sweeteners for foods, cosmetics, pharmaceuticals, and taste improvers. It is extremely useful as a quality improver, stabilizer and excipient. In addition, the non-reducing sugar gives trehalose almost quantitatively when glucoamylase, α-glucosidase, or trehalose-releasing enzyme disclosed in Japanese Patent Application No. 5-340343 is acted on. Therefore, it is also useful as an intermediate for producing trehalose, which has been difficult to obtain in large quantities in the past.

【００８２】以下、２〜３の実施例により、この発明に
よる組換え型酵素の製造方法と還元性澱粉糖の変換方法
を具体的に説明する。Hereinafter, the method for producing the recombinant enzyme and the method for converting reducing starch sugar according to the present invention will be specifically described with reference to a few examples.

【００８３】[0083]

【実施例Ａ−１ 組換え型酵素の製造】５００ｍｌ容三
角フラスコにマルトース２．０％（ｗ／ｖ）、ペプトン
０．５％（ｗ／ｖ）、酵母エキス０．１％（ｗ／ｖ）、
燐酸水素二ナトリウム０．１％（ｗ／ｖ）、燐酸二水素
カリウム０．１％（ｗ／ｖ）を含む液体培地（ｐＨ７．
０）を１００ｍｌずつとり、アンピシリンを５０μｇ／
ｍｌ加えた後、１２０℃で２０分間オートクレーブして
加熱滅菌した。冷却後、三角フラスコ内の液体培地に実
験例３−２の方法で得た形質転換体ＢＭＴ７を植菌し、
回転振盪下、２７℃で２４時間種培養した。別途、上記
と同組成の液体培地を１８ｌとり、アンピシリンを５０
μｇ／ｍｌ加え、１２０℃で２０分間加熱滅菌し、冷却
後、上記で得た種培養液を１％（ｖ／ｖ）接種し、３７
℃で２４時間通気撹拌培養した。培養物を超音波処理し
て菌体を破砕し、遠心分離により不溶物を除去後、上清
中の酵素活性を測定したところ、培養物１ｌ当たりに換
算して、約３，０００単位の酵素が産生していた。この
上清を実験例１−１の方法により精製したところ、比活
性約２００単位／ｍｇ蛋白質の組換え型酵素を１ｍｌ当
たり約１３５単位含む水溶液が約５０ｍｌ得られた。Example A-1 Production of Recombinant Enzyme In a 500 ml Erlenmeyer flask, maltose 2.0% (w / v), peptone 0.5% (w / v), yeast extract 0.1% (w / v). ),
Liquid medium containing 0.1% (w / v) disodium hydrogen phosphate and 0.1% (w / v) potassium dihydrogen phosphate (pH 7.
0) in 100 ml aliquots and ampicillin at 50 μg /
After adding ml, the mixture was autoclaved at 120 ° C. for 20 minutes for heat sterilization. After cooling, the transformant BMT7 obtained by the method of Experimental Example 3-2 was inoculated into the liquid medium in the Erlenmeyer flask,
Seed culture was carried out at 27 ° C. for 24 hours under rotary shaking. Separately, take 18 liters of liquid medium having the same composition as above and add 50 ml of ampicillin.
μg / ml was added, and the mixture was sterilized by heating at 120 ° C. for 20 minutes, cooled, and inoculated with 1% (v / v) of the seed culture solution obtained above.
The culture was performed at 24 ° C. for 24 hours with aeration and stirring. The culture was sonicated to disrupt the cells, the insoluble matter was removed by centrifugation, and the enzyme activity in the supernatant was measured. As a result, about 3,000 units of enzyme were calculated per 1 liter of culture. Was produced. When this supernatant was purified by the method of Experimental Example 1-1, about 50 ml of an aqueous solution containing about 135 units / ml of the recombinant enzyme having a specific activity of about 200 units / mg protein was obtained.

【００８４】[0084]

【実施例Ａ−２ 組換え型酵素の製造】実験例５−２の
方法により得た形質転換体ＢＱＴ１３を実施例Ａ−１と
同様に培養し、培養物を超音波処理して菌体を破砕し、
遠心分離により不溶物を除去後、上清中の酵素活性を測
定したところ、培養物１ｌ当たりに換算して、約２，４
５０単位の酵素が産生していた。この上清を実施例１−
１の方法により精製したところ、比活性約２００単位／
ｍｇ蛋白質の組換え型酵素を１ｍｌ当たり約１２０単位
含む水溶液が約４５ｍｌ得られた。Example A-2 Production of Recombinant Enzyme The transformant BQT13 obtained by the method of Experimental Example 5-2 was cultured in the same manner as in Example A-1, and the culture was sonicated to remove the bacterial cells. Crush,
After removing the insoluble matter by centrifugation, the enzyme activity in the supernatant was measured, and it was converted to about 2,4 per 1 l of the culture.
50 units of enzyme were produced. This supernatant was used in Example 1-
Purified by the method 1, the specific activity is about 200 units /
About 45 ml of an aqueous solution containing about 120 units of recombinant enzyme of mg protein per ml was obtained.

【００８５】[0085]

【実施例Ｂ−１ 組換え型酵素による澱粉加水分解物の
変換】馬鈴薯澱粉を濃度６％（ｗ／ｗ）になるよう水中
に懸濁し、１２０℃で１０分間オートクレーブして糊化
した。糊化液を５０℃に急冷し、ｐＨを約４．５に調整
後、林原生物化学研究所製イソアミラーゼ剤を澱粉固形
分１ｇ当たり２，５００単位加え、５０℃で２０時間反
応させた。反応物をｐＨ６．０に調整し、１２０℃で１
０分間オートクレーブして酵素を失活させた後、４５℃
に急冷し、ノボ・ノルディスク・インダストリー製α−
アミラーゼ剤『ターマミル６０Ｌ』を澱粉固形分１ｇ当
たり１５０単位加え、４５℃で２４時間反応させてマル
トトリオース、マルトテトラオース、マルトペンタオー
スなどのグルコース重合度３以上の還元性澱粉糖を含む
反応物を得た。この反応物を１２０℃で２０分間オート
クレーブして酵素を失活させ、４５℃まで急冷後、実施
例Ａ−１で得た組換え型酵素を澱粉糖固形分１ｇ当たり
１単位加え、４５℃で９６時間反応させた。反応物を９
６℃で１０分間加熱して酵素を失活させ、冷却し、濾過
後、常法にしたがって濾液を活性炭により脱色し、イオ
ン交換樹脂により脱塩・精製し、濃縮して濃度約７０％
（ｗ／ｗ）のシロップ状物を澱粉固形分当たり約９１％
の収率で得た。[Example B-1 Conversion of starch hydrolyzate by recombinant enzyme] Potato starch was suspended in water to a concentration of 6% (w / w) and autoclaved at 120 ° C for 10 minutes to gelatinize it. The gelatinization liquid was rapidly cooled to 50 ° C. and the pH was adjusted to about 4.5. Then, 2,500 units of an isoamylase agent manufactured by Hayashibara Biochemical Laboratories was added to 1 g of starch solid content, and the mixture was reacted at 50 ° C. for 20 hours. The reaction was adjusted to pH 6.0 and 1 at 120 ° C.
After deactivating the enzyme by autoclaving for 0 minutes, 45 ° C
Rapidly cooled to Novo Nordisk Industry α-
Addition of 150 units of amylase agent "Termamyl 60L" per 1 g of starch solid content and reaction at 45 ° C for 24 hours to contain reducing starch sugars such as maltotriose, maltotetraose, maltopentaose and the like having a degree of glucose polymerization of 3 or more. I got a thing. The reaction product was autoclaved at 120 ° C. for 20 minutes to inactivate the enzyme, and after rapidly cooling to 45 ° C., 1 unit of the recombinant enzyme obtained in Example A-1 was added per 1 g of starch sugar solid content, and the mixture was heated at 45 ° C. The reaction was carried out for 96 hours. 9 reactants
The enzyme is inactivated by heating at 6 ° C for 10 minutes, cooled, filtered, and then the filtrate is decolorized with activated carbon according to a conventional method, desalted and purified with an ion exchange resin, and concentrated to a concentration of about 70%.
About 91% of (w / w) syrup based on starch solids
It was obtained in a yield of.

【００８６】実験例２−１の方法によりこのシロップ状
物を分析したところ、ＤＥ値は１８．７、主成分とし
て、固形分当たりα−グルコシルトレハロースを８．４
％、α−マルトシルトレハロースを５．６％、α−マル
トリオシルトレハロースを３７．９％含み、前記還元性
糖質の殆どが対応する非還元性糖質に変換されていた。
温和で上品な甘味に加えて適度の粘性と保湿性を有する
本品は、食品、化粧品、医薬品の甘味剤、呈味改善剤、
品質改善剤、安定剤、賦形剤として有用である。また、
本品は非還元性糖質の含量が高いので、トレハロースを
製造するための中間体としても有用である。When this syrup-like substance was analyzed by the method of Experimental Example 2-1, the DE value was 18.7, and α-glucosyltrehalose per solid content was 8.4 as the main component.
%, Α-maltosyltrehalose was included in 5.6%, and α-maltriosyltrehalose was included in 37.9%, and most of the reducing sugars were converted into corresponding non-reducing sugars.
In addition to mild and elegant sweetness, this product has moderate viscosity and moisturizing properties, and is a sweetener for foods, cosmetics and pharmaceuticals, a taste improver,
It is useful as a quality improver, stabilizer and excipient. Also,
Since this product has a high content of non-reducing sugar, it is also useful as an intermediate for producing trehalose.

【００８７】[0087]

【実施例Ｂ−２ 組換え型酵素による澱粉加水分解物の
変換】トウモロコシ澱粉を３３％（ｗ／ｗ）になるよう
水中に懸濁し、炭酸カルシウムを０．１％（ｗ／ｗ）加
えた。懸濁液にノボ・ノルディスク・インダストリー製
α−アミラーゼ剤『ターマミル６０Ｌ』を澱粉固形分当
たり０．２％加え、９５℃で１５分間反応させて澱粉を
液化した。液化物を１２０℃で１０分間オートクレーブ
して酵素を失活させ、５５℃に急冷後、特開昭６３−２
４０７８４号公報に開示されているシュードモナス・ス
ツッツェリ由来のマルトテトラオース生成アミラーゼを
澱粉固形分１ｇ当たり５単位加え、５５℃で６時間反応
させた。その後、反応物に上田化学製α−アミラーゼ剤
『α−アミラーゼ２Ａ』を澱粉固形分１ｇ当たり３０単
位加え、６５℃で４時間反応させてマルトトリオース、
マルトテトラオース、マルトペンタオースなどのグルコ
ース重合度３以上の還元性澱粉糖を固形分当たり約５０
％含む反応物を得た。この反応物を１２０℃で１０分間
オートクレーブして酵素を失活させ、４５℃に急冷後、
ｐＨ６．５に調整し、実施例Ａ−１の方法で得た組換え
型酵素を澱粉糖固形分１ｇ当たり２単位加え、４５℃で
６４時間反応させた。反応物を９５℃で１０分間加熱し
て酵素を失活させ、冷却し、濾過後、常法にしたがって
活性炭により脱色し、イオン交換樹脂により脱塩・精製
し、濃縮して濃度約７０％（ｗ／ｗ）のシロップ状物を
澱粉固形分当たり約９０％の収量で得た。Example B-2 Conversion of Starch Hydrolyzate by Recombinant Enzyme Corn starch was suspended in water to 33% (w / w), and calcium carbonate was added at 0.1% (w / w). . An α-amylase agent “Termamyl 60L” manufactured by Novo Nordisk Industries Ltd. was added to the suspension in an amount of 0.2% per starch solid content, and the mixture was reacted at 95 ° C. for 15 minutes to liquefy the starch. The liquefaction product was autoclaved at 120 ° C. for 10 minutes to inactivate the enzyme and then rapidly cooled to 55 ° C.
The maltotetraose-producing amylase derived from Pseudomonas stutzeri disclosed in Japanese Patent No. 40784 was added in an amount of 5 units per 1 g of the starch solid content and reacted at 55 ° C. for 6 hours. Then, 30 units of the α-amylase agent “α-amylase 2A” manufactured by Ueda Chemical Co., Ltd. per 1 g of starch solid content was added to the reaction product, and the mixture was reacted at 65 ° C. for 4 hours to give maltotriose,
Approximately 50 units of reducing starch sugar with a degree of glucose polymerization of 3 or more such as maltotetraose and maltopentaose per solid content.
% Containing reactant was obtained. This reaction product was autoclaved at 120 ° C. for 10 minutes to inactivate the enzyme and then rapidly cooled to 45 ° C.
The pH was adjusted to 6.5, and 2 units of the recombinant enzyme obtained by the method of Example A-1 was added to 1 g of the starch sugar solid content, and the mixture was reacted at 45 ° C. for 64 hours. The reaction product is heated at 95 ° C for 10 minutes to inactivate the enzyme, cooled, filtered, decolorized with activated carbon according to a conventional method, desalted and purified with an ion exchange resin, and concentrated to a concentration of about 70% ( A syrup (w / w) was obtained in a yield of about 90% per starch solids.

【００８８】実験例２−１の方法によりこのシロップ状
物を分析したところ、ＤＥ値は１０．５、主成分とし
て、固形分当たりα−グルコシルトレハロースを３．８
％、α−マルトシルトレハロースを４３．８％、α−マ
ルトリオシルトレハロースを１．２％含み、前記還元性
澱粉糖の殆どが対応する非還元性糖質に変換されてい
た。温和で上品な甘味に加えて適度の粘性と保湿性を有
する本品は、食品、化粧品、医薬品の甘味剤、呈味改善
剤、品質改善剤、安定剤、賦形剤として有用である。ま
た、本品は非還元性糖質の含量が高いので、トレハロー
スを製造するための中間体としても有用である。When this syrup was analyzed by the method of Experimental Example 2-1, the DE value was 10.5, and α-glucosyltrehalose per solid content was 3.8 as the main component.
%, Α-maltosyltrehalose was 43.8%, and α-maltriosyltrehalose was 1.2%, and most of the reducing starch sugar was converted to the corresponding non-reducing sugar. The product, which has a moderate viscosity and a moderate sweetness, as well as an appropriate viscosity and moisturizing property, is useful as a sweetener, a taste improver, a quality improver, a stabilizer and an excipient for foods, cosmetics and pharmaceuticals. Further, since this product has a high content of non-reducing sugar, it is also useful as an intermediate for producing trehalose.

【００８９】[0089]

【実施例Ｂ−３ 組換え型酵素によるマルトペンタオー
スの変換】林原生物化学研究所製高純度マルトペンタオ
ースを２０％（ｗ／ｗ）になるよう水中に溶解し、ｐＨ
６．５に調整後、実施例Ａ−１の方法で得た組換え型酵
素をマルトペンタオース１ｇ当たり１単位加え、４５℃
で４８時間反応させた。反応物を９５℃で１０分間加熱
して酵素を失活させ、冷却し、濾過し、濃縮後、実験例
２−１の方法により分析したところ、マルトペンタオー
スの約９２％がα−マルトトリオシルトレハロースに変
換されていた。[Example B-3 Conversion of Maltopentaose with Recombinant Enzyme] High-purity maltopentaose manufactured by Hayashibara Biochemical Laboratories was dissolved in water to 20% (w / w), and pH was adjusted.
After adjusting to 6.5, 1 unit of the recombinant enzyme obtained by the method of Example A-1 was added per 1 g of maltopentaose, and the mixture was added at 45 ° C.
And reacted for 48 hours. The reaction was heated at 95 ° C for 10 minutes to inactivate the enzyme, cooled, filtered, concentrated, and analyzed by the method of Experimental Example 2-1 to find that about 92% of maltopentaose was α-maltotrio. It had been converted to siltrehalose.

【００９０】別途、内径５．４ｃｍ、長さ５ｍのジャケ
ット付きステンレス製カラム４本に東京有機化学工業製
強酸性カチオン交換樹脂『ＸＴ−１０１６（Ｎａ
+型）』を均一に充填し、カラムを直列に連結して全長
を２０ｍとした。カラム内温度を５５℃に保ちつつ上記
反応物を樹脂に対して約５％（ｖ／ｖ）の割合で負荷
し、次いで、カラムに５５℃の温水をＳＶ０．１３の流
速で通液して糖質成分を溶出させた。溶出液の糖組成を
分析し、当該非還元性糖質の含量が高い画分を採取し、
これを濃縮し、真空乾燥し、破砕して固状物を固形分当
たり約５５％の収量で得た。Separately, four stainless steel columns with a jacket having an inner diameter of 5.4 cm and a length of 5 m were attached to a strongly acidic cation exchange resin “XT-1016 (Na
+ Type) ”was uniformly packed and columns were connected in series to make the total length 20 m. While maintaining the temperature in the column at 55 ° C, the above reaction product was loaded at a ratio of about 5% (v / v) to the resin, and then warm water at 55 ° C was passed through the column at a flow rate of SV 0.13. The sugar component was eluted. Analyzing the sugar composition of the eluate, collecting the fraction with a high content of the non-reducing sugar,
It was concentrated, dried in vacuo and crushed to give a solid in about 55% yield per solids.

【００９１】実験例２−１の方法により分析したとこ
ろ、この固状物のＤＥ値は約０．２未満であり、固形分
当たりα−マルトトリオシルトレハロースを９９．０％
含んでいた。吸湿性低く、極めて低い還元性とかすかな
甘味を有する本品は、食品、化粧品、医薬品の甘味剤、
呈味改善剤、品質改善剤、安定剤、賦形剤として有用で
ある。また、本品は非還元性糖質の含量が高いので、ト
レハロースを製造するための中間体としても有用であ
る。When analyzed by the method of Experimental Example 2-1, the DE value of this solid was less than about 0.2, and 99.0% of α-maltotriosyltrehalose was contained per solid.
Included. This product, which has low hygroscopicity, extremely low reducing property and faint sweetness, is a sweetener for foods, cosmetics and pharmaceuticals.
It is useful as a taste improver, quality improver, stabilizer and excipient. Further, since this product has a high content of non-reducing sugar, it is also useful as an intermediate for producing trehalose.

【００９２】[0092]

【実施例Ｂ−４ 組換え型酵素による澱粉加水分解物の
変換】松谷化学工業製澱粉加水分解物『パインデックス
＃４』を４０％（ｗ／ｗ）となるよう水中に溶解し、溶
液を４５℃、ｐＨ６．５に調整後、実施例Ａ−１の方法
で得た組換え型酵素を澱粉加水分解物１ｇ当たり１単位
加えて９６時間反応させ、末端にトレハロース構造を有
する非還元性糖質を含む反応物を得た。次いで、この反
応物を１００℃で１０分間加熱して酵素を失活させ、濃
度約２０％（ｗ／ｗ）まで濃縮し、５５℃に冷却後、ｐ
Ｈ４．５に調整してナガセ生化学工業製グルコアミラー
ゼ剤『グルコチーム』を糖質固形分１ｇ当たり１０単位
加え、４０時間反応させた。反応物を１００℃で１０分
間加熱して酵素を失活させ、冷却し、濾過後、常法にし
たがって活性炭により脱色し、イオン交換樹脂により脱
塩・精製し、濃縮してトレハロースを固形分当たり約２
９．７％含む濃度約６０％（ｗ／ｗ）のシロップ状物を
得た。[Example B-4 Conversion of starch hydrolyzate by recombinant enzyme] Starch hydrolyzate "Paindex # 4" manufactured by Matsutani Chemical Industry was dissolved in water to 40% (w / w), and the solution was After adjusting the pH to 45 ° C at 45 ° C, 1 unit of the recombinant enzyme obtained by the method of Example A-1 was added per 1 g of starch hydrolyzate and reacted for 96 hours to give a non-reducing sugar having a trehalose structure at the end. A quality containing reaction product was obtained. Then, this reaction product was heated at 100 ° C. for 10 minutes to inactivate the enzyme, concentrated to a concentration of about 20% (w / w), cooled to 55 ° C., and then p
After adjusting to H4.5, 10 units of glucoamylase agent "glucozyme" manufactured by Nagase Seikagaku Co., Ltd. was added per 1 g of the sugar solid content and reacted for 40 hours. The reaction product was heated at 100 ° C for 10 minutes to inactivate the enzyme, cooled, filtered, decolorized with activated carbon according to a conventional method, desalted and purified with an ion exchange resin, and concentrated to give trehalose per solid content. About 2
A syrup having a concentration of about 60% (w / w) containing 9.7% was obtained.

【００９３】このシロップ状物を強酸性カチオン交換樹
脂としてオルガノ製『ＣＧ６０００（Ｎａ+型）』を使
用した以外実施例Ｂ−３と同様に分画し、トレハロース
を固形分当たり約９０％含む画分を採取した。この画分
を濃度約７５％（ｗ／ｗ）に濃縮後、助晶罐にとり、種
晶としてトレハロース含水結晶を糖質固形分当たり約２
％加え、緩やかに撹拌しながら助晶して結晶化度約４５
％のマスキットを得た。このマスキットを約１５０ｋｇ
／ｃｍ2の圧力下、噴霧乾燥塔の上部に設けた噴霧ノズ
ルより噴霧乾燥塔下方に向かって噴霧する一方、約８５
℃の温風を噴霧乾燥塔の上部から下方に向かって送風し
つつ、噴霧乾燥塔の底部に設けたベルトコンベア上に蓄
積した結晶性粉状物を噴霧乾燥塔外に徐々に搬出した。
その後、粉状物を熟成塔に移し、約４０℃の温風を送風
しながら１０時間熟成して結晶化と乾燥を完了した。This syrup was fractionated in the same manner as in Example B-3 except that "CG6000 (Na + type)" manufactured by Organo was used as the strongly acidic cation exchange resin, and the trehalose content was about 90% based on the solid content. Minutes were collected. After concentrating this fraction to a concentration of about 75% (w / w), it was transferred to a supporting crystal can and trehalose hydrous crystals as seed crystals were added at about 2 per solid sugar content.
%, And the crystallinity of about 45
I got a% mask kit. About 150 kg of this mass kit
Under a pressure of / cm 2 while spraying downward from the spray drying tower through a spray nozzle provided at the top of the spray drying tower,
While blowing warm air of ℃ from the upper part of the spray drying tower downward, the crystalline powder material accumulated on the belt conveyor provided at the bottom of the spray drying tower was gradually carried out of the spray drying tower.
Then, the powdery matter was transferred to an aging tower and aged for 10 hours while blowing warm air of about 40 ° C. to complete crystallization and drying.

【００９４】吸湿性なく、温和で上品な甘味を有する本
品は、食品、化粧品、医薬品、飼料の甘味剤、呈味改善
剤、品質改善剤、安定剤、賦形剤として有用である。The product, which is not hygroscopic and has mild and elegant sweetness, is useful as a sweetener, taste improver, quality improver, stabilizer, excipient for foods, cosmetics, pharmaceuticals, and feeds.

【００９５】[0095]

【実施例Ｂ−５ 組換え型酵素による澱粉加水分解物の
変換】タピオカ澱粉を３４％（ｗ／ｗ）になるように水
中に懸濁し、炭酸カルシウムを０．１％（ｗ／ｗ）加え
た。懸濁液にノボ・ノルディスク・インダストリー製α
−アミラーゼ剤『ターマミル６０Ｌ』を澱粉固形分当た
り０．２％加え、９５℃で１５分間反応させて澱粉を液
化した。液化物を１２０℃で１０分間オートクレーブし
て酵素を失活させ、５５℃に急冷し、ｐＨ５．２に調整
後、上田化学製α−アミラーゼ剤『α−アミラーゼ２
Ａ』と林原生物化学研究所製イソアミラーゼ剤を澱粉固
形分１ｇ当たりそれぞれ１０単位又は５００単位加え、
５５℃で２０時間反応させてマルトトリオース、マルト
テトラオース、マルトペンタオース、マルトヘキサオー
スなどのグルコース重合度３以上の還元性澱粉糖を固形
分当たり約６０％含むＤＥ約２９の反応物を得た。この
反応物を１２０℃で１０分間オートクレーブして酵素を
失活させ、４５℃に急冷し、ｐＨ６．５に調整後、実施
例Ａ−２の方法で得た組換え型酵素を澱粉糖固形分１ｇ
当たり２単位加え、４５℃で６４時間反応させた。反応
物を９５℃で１０分間加熱して酵素を失活させ、冷却
し、濾過後、常法にしたがって活性炭により脱色し、イ
オン交換樹脂により脱塩・精製し、濃縮して濃度約７０
％（ｗ／ｗ）のシロップ状物を澱粉固形分当たり約９０
％の収量で得た。Example B-5 Conversion of Starch Hydrolyzate by Recombinant Enzyme Tapioca starch was suspended in water to 34% (w / w) and calcium carbonate was added at 0.1% (w / w). It was Suspension made by Novo Nordisk Industries α
-The amylase agent "Termamyl 60L" was added at 0.2% per starch solid content, and the mixture was reacted at 95 ° C for 15 minutes to liquefy the starch. The liquefaction product was autoclaved at 120 ° C. for 10 minutes to inactivate the enzyme, rapidly cooled to 55 ° C. and adjusted to pH 5.2, and then α-amylase agent “α-amylase 2 manufactured by Ueda Chemical Co.
A ”and Hayashibara Biochemical Research Institute's isoamylase agent were added in an amount of 10 units or 500 units per 1 g of starch solids, respectively.
By reacting at 55 ° C. for 20 hours, a reaction product of DE 29 containing malttriose, maltotetraose, maltopentaose, maltohexaose and the like reducing starch sugar having a glucose polymerization degree of 3 or more at about 60% based on the solid content is obtained. Obtained. This reaction product was autoclaved at 120 ° C. for 10 minutes to inactivate the enzyme, rapidly cooled to 45 ° C. and adjusted to pH 6.5, and then the recombinant enzyme obtained by the method of Example A-2 was added to starch sugar solids. 1 g
2 units per unit was added and reacted at 45 ° C. for 64 hours. The reaction product is heated at 95 ° C. for 10 minutes to inactivate the enzyme, cooled, filtered, decolorized with activated carbon according to a conventional method, desalted and purified with an ion exchange resin, and concentrated to a concentration of about 70.
% (W / w) of syrup about 90% starch solids
Obtained in% yield.

【００９６】実験例２−１の方法によりこのシロップ状
物を分析したところ、ＤＥ値は１５．８、主成分とし
て、固形分当たりα−グルコシルトレハロースを５．８
％、α−マルトシルトレハロースを８．５％、α−マル
トトリオシルトレハロースを１３．１％、α−マルトテ
トラオシルトレハロースを１８．９％、α−マルトペン
タオシルトレハロースを３．６％含み、前記還元性澱粉
糖の殆どが対応する非還元性糖質に変換されていた。温
和で上品な甘味に加えて適度の粘性と保湿性を有する本
品は、食品、化粧品、医薬品の甘味剤、呈味改善剤、品
質改善剤、安定剤、賦形剤として有用である。本品は非
還元性糖質の含量が高いので、トレハロースを製造する
ための中間体としても有用である。When this syrup was analyzed by the method of Experimental Example 2-1, the DE value was 15.8, and as the main component, α-glucosyltrehalose per solid content was 5.8.
%, Α-maltosyltrehalose 8.5%, α-maltotriosyltrehalose 13.1%, α-maltotetraosyltrehalose 18.9%, α-maltopentaosyltrehalose 3.6%. Including, most of the reducing starch sugar was converted to the corresponding non-reducing sugar. The product, which has a moderate viscosity and a moderate sweetness, as well as an appropriate viscosity and moisturizing property, is useful as a sweetener, a taste improver, a quality improver, a stabilizer and an excipient for foods, cosmetics and pharmaceuticals. Since this product has a high content of non-reducing sugar, it is also useful as an intermediate for producing trehalose.

【００９７】[0097]

【実施例Ｂ−６ 組換え型酵素による澱粉加水分解部の
変換】実施例Ｂ−２と同様に液化トウモロコシ澱粉にマ
ルトテトラオース生成アミラーゼとα−アミラーゼを逐
次反応させ、マルトトリオース、マルトテトラオース、
マルトペンタオースなどのグルコース重合度３以上の還
元性澱粉糖を固形分当たり約５０％含む反応物を得た。
この反応物を１２０℃で１０分間オートクレーブして酵
素を失活させ、４５℃に急冷後、ｐＨ６．５に調整し、
実施例Ａ−２の方法で得た組換え型酵素を澱粉糖固形分
１ｇ当たり２単位加え、４５℃で６４時間反応させた。
反応物を９５℃で１０分間加熱して酵素を失活させ、冷
却し、濾過後、常法にしたがって活性炭により脱色し、
イオン交換樹脂により脱塩・精製し、濃縮して濃度約７
０％（ｗ／ｗ）のシロップ状物を澱粉固形分当たり約９
０％の収量で得た。Example B-6 Conversion of Starch Hydrolyzed Portion by Recombinant Enzyme In the same manner as in Example B-2, liquefied corn starch was sequentially reacted with maltotetraose-forming amylase and α-amylase to give maltotriose and maltotetra. Aus,
A reaction product containing about 50% of reducing starch sugar having a glucose polymerization degree of 3 or more such as maltopentaose based on the solid content was obtained.
This reaction product was autoclaved at 120 ° C. for 10 minutes to inactivate the enzyme, rapidly cooled to 45 ° C., and then adjusted to pH 6.5,
2 units of the recombinant enzyme obtained by the method of Example A-2 was added per 1 g of starch sugar solid content, and the mixture was reacted at 45 ° C. for 64 hours.
The reaction was heated at 95 ° C for 10 minutes to inactivate the enzyme, cooled, filtered, and then decolorized with activated carbon according to a conventional method,
Deionized and purified by ion exchange resin and concentrated to a concentration of about 7
About 0% (w / w) syrup was added to the starch solids content of about 9
Obtained in a yield of 0%.

【００９８】実験例２−１の方法によりこのシロップ状
物を分析したところ、ＤＥ値は１０．３、主成分とし
て、固形分当たりα−グルコシルトレハロースを３．６
％、α−マルトシルトレハロースを４４．０％、α−マ
ルトリオシルトレハロースを１．０％含み、前記還元性
澱粉糖の殆どが対応する非還元性糖質に変換されてい
た。温和で上品な甘味に加えて適度の粘性と保湿性を有
する本品は、食品、化粧品、医薬品の甘味剤、呈味改善
剤、品質改善剤、安定剤、賦形剤として有用である。ま
た、本品は非還元性糖質の含量が高いので、トレハロー
スを製造するための中間体としても有用である。When this syrup was analyzed by the method of Experimental Example 2-1, the DE value was 10.3, and α-glucosyltrehalose per solid content as a main component was 3.6.
%, Α-maltosyltrehalose 44.0%, α-maltriosyltrehalose 1.0%, and most of the reducing starch sugar was converted to the corresponding non-reducing sugar. The product, which has a moderate viscosity and a moderate sweetness, as well as an appropriate viscosity and moisturizing property, is useful as a sweetener, a taste improver, a quality improver, a stabilizer and an excipient for foods, cosmetics and pharmaceuticals. Further, since this product has a high content of non-reducing sugar, it is also useful as an intermediate for producing trehalose.

【００９９】[0099]

【発明の効果】以上説明したように、この発明は、グル
コース重合度３以上の還元性澱粉糖から末端にトレハロ
ース構造を有する非還元性糖質を生成する、従来未知の
全く新規な酵素の発見に基づくものである。この発明
は、組換えＤＮＡ技術により斯かる酵素を大規模且つ効
率的に生産する道を拓くものである。この発明による組
換え型酵素を使用する変換方法により、還元性澱粉糖は
効率的に対応する非還元性糖質に変換され、その生成し
た非還元性糖質は温和で上品な甘味に加えて適度の粘性
と保湿性を有し、しかも、分子中に還元性基を有しない
ので、着色や変質の懸念なく飲食物を甘味付けできる実
益がある。加えて、この発明による組換え型酵素は全ア
ミノ酸配列までが明らかにされた酵素であり、飲食物等
への配合使用を前提とするトレハロースや末端にトレハ
ロース構造を有する非還元性糖質の製造に安心して使用
し得るものである。INDUSTRIAL APPLICABILITY As described above, the present invention is a discovery of a novel enzyme which has not been previously known and produces a non-reducing sugar having a trehalose structure at the terminal from a reducing starch sugar having a glucose polymerization degree of 3 or more. It is based on. This invention paves the way for large-scale and efficient production of such enzymes by recombinant DNA technology. By the conversion method using the recombinant enzyme according to the present invention, the reducing starch sugar is efficiently converted into the corresponding non-reducing sugar, and the produced non-reducing sugar has a mild and elegant sweetness. Since it has appropriate viscosity and moisturizing property and does not have a reducing group in the molecule, there is a practical advantage that foods and drinks can be sweetened without fear of coloring or deterioration. In addition, the recombinant enzyme according to the present invention is an enzyme whose entire amino acid sequence has been clarified, and is used for the production of trehalose or a non-reducing sugar having a trehalose structure at the end, which is premised on the compounding use in food and drink. It can be used with confidence.

【０１００】この発明は斯くも顕著な作用効果を奏する
意義のある発明であり、斯界に貢献すること誠に多大な
発明であると言える。[0100] The present invention is an invention having a significant effect of producing such remarkable effects, and it can be said that it is a great invention to contribute to the field.

【０１０１】[0101]

【配列表】[Sequence list]

配列番号：1 配列の長さ：772 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ポリペプチド 配列 Met Arg Thr Pro Ala Ser Thr Tyr Arg Leu Gln Ile Arg Arg Gly Phe Thr 1 5 10 15 Leu Phe Asp Ala Ala Glu Thr Val Pro Tyr Leu Lys Ser Leu Gly Val Asp 20 25 30 Trp Ile Tyr Leu Ser Pro Ile Leu Lys Ala Glu Ser Gly Ser Asp His Gly 35 40 45 50 Tyr Asp Val Thr Asp Pro Ala Val Val Asp Pro Glu Arg Gly Gly Pro Glu 55 60 65 Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Gly Ala Gly Met Gly Val Leu 70 75 80 85 Ile Asp Ile Val Pro Asn His Val Gly Val Ala Ser Pro Pro Gln Asn Pro 90 95 100 Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg Gly Ser Pro Tyr Ala Val Ala 105 110 115 Phe Asp Val Asp Trp Asp Leu Ala Gly Gly Arg Ile Arg Ile Pro Val Leu 120 125 130 135 Gly Ser Asp Asp Asp Leu Asp Gln Leu Glu Ile Lys Asp Gly Glu Leu Arg 140 145 150 Tyr Tyr Asp His Arg Phe Pro Leu Ala Glu Gly Ser Tyr Arg Asp Gly Asp 155 160 165 170 Ser Pro Gln Asp Val His Gly Arg Gln His Tyr Glu Leu Ile Gly Trp Arg 175 180 185 Arg Ala Asp Asn Glu Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu 190 195 200 Ala Gly Ile Arg Val Glu Val Pro Pro Val Phe Asp Glu Ala His Gln Glu 205 210 215 220 Val Val Arg Trp Phe Arg Ala Gly Leu Ala Asp Gly Leu Arg Ile Asp His 225 230 235 Pro Asp Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val 240 245 250 255 Thr Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu Ala 275 280 285 Asp Val Asp Arg Val Phe Val Asp Pro Arg Gly Gln Val Pro Leu Asp Arg 290 295 300 305 Leu Asp Ala Arg Leu Arg Gly Gly Ala Pro Ala Asp Tyr Glu Asp Met Ile 310 315 320 Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His Ser Glu Ile Leu 325 330 335 340 Arg Leu Ala Arg Leu Val Pro Glu Gln Thr Gly Ile Pro Gly Glu Ala Ala 345 350 355 Ala Asp Ala Ile Ala Glu Ile Ile Ala Ala Phe Pro Val Tyr Arg Ser Tyr 360 365 370 Leu Pro Glu Gly Ala Glu Ile Leu Lys Glu Ala Cys Asp Leu Ala Ala Arg 375 380 385 390 Arg Arg Pro Glu Leu Gly Gln Thr Val Gln Leu Leu Gln Pro Leu Leu Leu 395 400 405 Asp Thr Asp Leu Glu Ile Ser Arg Arg Phe Gln Gln Thr Ser Gly Met Val 410 415 420 425 Met Ala Lys Gly Val Glu Asp Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly 430 435 440 Thr Leu Thr Glu Val Gly Ala Asp Pro Thr Glu Phe Ser Leu Glu Pro Glu 445 450 455 Glu Phe His Val Arg Met Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met 460 465 470 475 Thr Thr Leu Ser Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg 480 485 490 Ile Ser Val Ile Ala Glu Val Ala Pro Glu Trp Glu Lys Ala Leu Asp Arg 495 500 505 510 Leu Asn Thr Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Thr Leu Leu Trp 515 520 525 Gln Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Ser Tyr 530 535 540 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Ser Trp Thr Asp Pro 545 550 555 560 Asp Pro Ala Phe Glu Glu Ala Leu Ser Ala Val Val Asp Ser Ala Phe Asp 565 570 575 Asn Pro Glu Val Arg Ala Glu Leu Glu Ala Leu Val Gly Leu Leu Ala Pro 580 585 590 595 His Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln Leu Thr Met Pro 600 605 610 Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp Asp Arg Ser Leu Thr 615 620 625 Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Ala Glu Arg Ile Arg Ala Leu 630 635 640 645 Asp Gln Leu Asp Ala Gly His Arg Pro Asp Ser Phe Gln Asp Glu Ala Val 650 655 660 Lys Leu Leu Val Thr Ser Arg Ala Leu Arg Leu Arg Arg Asn Arg Pro Glu 665 670 675 680 Leu Phe Thr Gly Tyr Arg Pro Val His Ala Arg Gly Pro Ala Ala Gly His 685 690 695 Leu Val Ala Phe Asp Arg Gly Ala Gly Gly Val Leu Ala Leu Ala Thr Arg 700 705 710 Leu Pro Tyr Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu 715 720 725 730 Leu Glu Ala Ala Met Thr Asp Glu Leu Thr Gly Ser Thr Phe Gly Pro Gly 735 740 745 Pro Ala Ala Leu Ser Glu Val Phe Arg Ala Tyr Pro Val Ala Leu Leu Val 750 755 760 765 Pro Ala Thr Gly Gly Lys Ser 770  SEQ ID NO: 1 Sequence Length: 772 Sequence Type: Amino Acid Topology: Linear Sequence Type: Polypeptide Sequence Met Arg Thr Pro Ala Ser Thr Tyr Arg Leu Gln Ile Arg Arg Gly Phe Thr 1 5 10 15 Leu Phe Asp Ala Ala Glu Thr Val Pro Tyr Leu Lys Ser Leu Gly Val Asp 20 25 30 Trp Ile Tyr Leu Ser Pro Ile Leu Lys Ala Glu Ser Gly Ser Asp His Gly 35 40 45 50 Tyr Asp Val Thr Asp Pro Ala Val Val Asp Pro Glu Arg Gly Gly Pro Glu 55 60 65 Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Gly Ala Gly Met Gly Val Leu 70 75 80 85 Ile Asp Ile Val Pro Asn His Val Gly Val Ala Ser Pro Pro Gln Asn Pro 90 95 100 Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg Gly Ser Pro Tyr Ala Val Ala 105 110 115 Phe Asp Val Asp Trp Asp Leu Ala Gly Gly Arg Ile Arg Ile Pro Val Leu 120 125 130 135 Gly Ser Asp Asp Asp Leu Asp Gln Leu Glu Ile Lys Asp Gly Glu Leu Arg 140 145 150 Tyr Tyr Asp His Arg Phe Pro Leu Ala Glu Gly Ser Tyr Arg Asp Gly Asp 155 160 165 170 Ser Pro Gln Asp Val His Gly Arg Gln His Tyr Glu Leu Ile Gly Trp Arg 175 180 185 Arg Ala Asp Asn Glu Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu 190 195 200 Ala Gly Ile Arg Val Glu Val Pro Pro Val Phe Asp Glu Ala His Gln Glu 205 210 215 220 Val Val Arg Trp Phe Arg Ala Gly Leu Ala Asp Gly Leu Arg Ile Asp His 225 230 235 Pro Asp Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val 240 245 250 255 Thr Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu Ala 275 280 285 Asp Val Asp Arg Val Phe Val Asp Pro Arg Gly Gln Val Pro Leu Asp Arg 290 295 300 305 Leu Asp Ala Arg Leu Arg Gly Gly Ala Pro Ala Asp Tyr Glu Asp Met Ile 310 315 320 Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His Ser Glu Ile Leu 325 330 335 340 Arg Leu Ala Arg Leu Val Pro Glu Gln Thr Gly Ile Pro Gly Glu Ala Ala 345 350 355 Ala Asp Ala Ile Ala Glu Ile Ile Ala Ala Phe Pro Val Tyr Arg Ser Tyr 360 365 370 Leu Pro Glu Gly Ala Glu Ile Leu Lys Glu Ala Cys Asp Leu Ala Ala Arg 375 380 385 390 Arg Arg Pro Glu Leu Gly Gln Thr Val Gln Leu Leu Gln Pro Leu Leu Leu 395 400 405 Asp Thr Asp Leu Glu Ile Ser Arg Arg Phe Gln Gln Thr Ser Gly Met Val 410 415 420 425 Met Ala Lys Gly Val Glu Asp Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly 430 435 440 Thr Leu Thr Glu Val Gly Ala Asp Pro Thr Glu Phe Ser Leu Glu Pro Glu 445 450 455 Glu Phe His Val Arg Met Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met 460 465 470 475 Thr Thr Leu Ser Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg 480 485 490 Ile Ser Val Ile Ala Glu Val Ala Pro Glu Trp Glu Lys Ala Leu Asp Arg 495 500 505 510 Leu Asn Thr Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Thr Leu Leu Trp 515 520 525 Gln Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Ser Tyr 530 535 540 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Ser Trp Thr Asp Pro 545 550 555 560 Asp Pro Ala Phe Glu Glu Ala Leu Ser Ala Val Val Asp Ser Ala Phe Asp 565 570 575 Asn Pro Glu Val Arg Ala Glu Leu Glu Ala Leu Val Gly Leu Leu Ala Pro 580 585 590 595 His Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln Leu Thr Met Pro 600 605 610 Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp Asp Arg Ser Leu Thr 615 620 625 Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Ala Glu Arg Ile Arg Ala Leu 630 635 640 645 Asp Gln Leu Asp Ala Gly His Arg Pro Asp Ser Phe Gln Asp Glu Ala Val 650 655 660 Lys Leu Leu Val Thr Ser Arg Ala Leu Arg Leu Arg Arg Asn Arg Pro Glu 665 670 675 680 Leu Phe Thr Gly Tyr Arg Pro Val His Ala Arg Gly Pro Ala Ala Gly His 685 690 695 Leu Val Ala Phe Asp Arg Gly Ala Gly Gly Val Leu Ala Leu Ala Thr Arg 700 705 710 Leu Pro Tyr Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu 715 720 725 730 Leu Glu Ala Ala Met Thr Asp Glu Leu Thr Gly Ser Thr Phe Gly Pro Gly 735 740 745 Pro Ala Ala Leu Ser Glu Val Phe Arg Ala Tyr Pro Val Ala Leu Leu Val 750 755 760 765 Pro Ala Thr Gly Gly Lys Ser 770

【０１０２】配列番号：2 配列の長さ：775 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ポリペプチド 配列 Met Arg Thr Pro Val Ser Thr Tyr Arg Leu Gln Ile Arg Lys Gly Phe Thr 1 5 10 15 Leu Phe Asp Ala Ala Lys Thr Val Pro Tyr Leu His Ser Leu Gly Val Asp 20 25 30 Trp Val Tyr Leu Ser Pro Val Leu Thr Ala Glu Gln Gly Ser Asp His Gly 35 40 45 50 Tyr Asp Val Thr Asp Pro Ser Ala Val Asp Pro Glu Arg Gly Gly Pro Glu 55 60 65 Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Ala Ala Gly Met Gly Val Leu 70 75 80 85 Ile Asp Ile Val Pro Asn His Val Gly Val Ala Thr Pro Ala Gln Asn Pro 90 95 100 Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg Gln Ser Arg Tyr Ala Glu Ala 105 110 115 Phe Asp Val Asp Trp Asp Leu Ala Gly Gly Arg Ile Arg Leu Pro Val Leu 120 125 130 135 Gly Ser Asp Asp Asp Leu Asp Gln Leu Glu Ile Arg Asp Gly Glu Leu Arg 140 145 150 Tyr Tyr Asp His Arg Phe Pro Leu Ala Glu Gly Thr Tyr Ala Glu Gly Asp 155 160 165 170 Ala Pro Arg Asp Val His Ala Arg Gln His Tyr Glu Leu Ile Gly Trp Arg 175 180 185 Arg Ala Asp Asn Glu Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu 190 195 200 Ala Gly Val Arg Val Glu Ile Pro Ala Val Phe Asp Glu Ala His Gln Glu 205 210 215 220 Val Val Arg Trp Phe Arg Glu Asp Leu Ala Asp Gly Leu Arg Ile Asp His 225 230 235 Pro Asp Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val 240 245 250 255 Thr Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu Ala 275 280 285 Asp Val Asp Arg Val Leu Val Asp Pro Arg Gly Gln Glu Pro Leu Asp Arg 290 295 300 305 Leu Asp Ala Ser Leu Arg Gly Gly Glu Pro Ala Asp Tyr Gln Asp Met Ile 310 315 320 Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His Ser Glu Ile Leu 325 330 335 340 Arg Leu Ala Arg Leu Val Pro Gly Asp Ala Asn Val Ser Ile Asp Ala Gly 345 350 355 Ala Asp Ala Leu Ala Glu Ile Ile Ala Ala Phe Pro Val Tyr Arg Thr Tyr 360 365 370 Leu Pro Glu Gly Ala Glu Val Leu Lys Glu Ala Cys Glu Leu Ala Ala Arg 375 380 385 390 Arg Arg Pro Glu Leu Asp Gln Ala Ile Gln Ala Leu Gln Pro Leu Leu Leu 395 400 405 Asp Thr Asp Leu Glu Leu Ala Arg Arg Phe Gln Gln Thr Ser Gly Met Val 410 415 420 425 Met Ala Lys Gly Val Glu Asp Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly 430 435 440 Thr Leu Thr Glu Val Gly Ala Asp Pro Thr Glu Phe Ala Val Glu Pro Asp 445 450 455 Glu Phe His Ala Arg Leu Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met 460 465 470 475 Thr Thr Leu Ser Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg 480 485 490 Ile Ser Val Ile Ser Glu Val Ala Gly Asp Trp Glu Lys Ala Leu Asn Arg 495 500 505 510 Leu Arg Asp Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Ala Leu Leu Trp 515 520 525 Gln Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Tyr Tyr 530 535 540 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Asn Trp Thr Asp Pro 545 550 555 560 Ala Pro Ala Phe Glu Glu Lys Leu Lys Ala Ala Val Asp Ala Val Phe Asp 565 570 575 Asn Pro Ala Val Gln Ala Glu Val Glu Ala Leu Val Glu Leu Leu Glu Pro 580 585 590 595 Tyr Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln Leu Thr Met Pro 600 605 610 Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp Asp Arg Ser Leu Thr 615 620 625 Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Asp Asp Arg Arg Ala Ala Leu 630 635 640 645 Glu Gln Leu Asp Ala Gly Asp Leu Pro Ala Ser Phe Thr Asp Glu Arg Thr 650 655 660 Lys Leu Leu Val Thr Ser Arg Ala Leu Arg Leu Arg Arg Asp Arg Pro Glu 665 670 675 680 Leu Phe Thr Gly Tyr Arg Pro Val Leu Ala Ser Gly Pro Ala Ala Gly His 685 690 695 Leu Leu Ala Phe Asp Arg Gly Thr Ala Ala Ala Pro Gly Ala Leu Thr Leu 700 705 710 Ala Thr Arg Leu Pro Tyr Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr 715 720 725 730 Ala Val Glu Leu Asn Thr Ala Met Lys Asp Glu Leu Thr Gly Ala Gly Phe 735 740 745 Gly Pro Gly Ala Val Lys Ile Ala Asp Ile Phe Arg Ser Phe Pro Val Ala 750 755 760 765 Leu Leu Val Pro Gln Thr Gly Gly Glu Ser 770 775SEQ ID NO: 2 Sequence Length: 775 Sequence Type: Amino Acid Topology: Linear Sequence Type: Polypeptide Sequence Met Arg Thr Pro Val Ser Thr Tyr Arg Leu Gln Ile Arg Lys Gly Phe Thr 1 5 10 15 Leu Phe Asp Ala Ala Lys Thr Val Pro Tyr Leu His Ser Leu Gly Val Asp 20 25 30 Trp Val Tyr Leu Ser Pro Val Leu Thr Ala Glu Gln Gly Ser Asp His Gly 35 40 45 50 Tyr Asp Val Thr Asp Pro Ser Ala Val Asp Pro Glu Arg Gly Gly Pro Glu 55 60 65 Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Ala Ala Gly Met Gly Val Leu 70 75 80 85 Ile Asp Ile Val Pro Asn His Val Gly Val Ala Thr Pro Ala Gln Asn Pro 90 95 100 Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg Gln Ser Arg Tyr Ala Glu Ala 105 110 115 Phe Asp Val Asp Trp Asp Leu Ala Gly Gly Arg Ile Arg Leu Pro Val Leu 120 125 130 135 Gly Ser Asp Asp Asp Leu Asp Gln Leu Glu Ile Arg Asp Gly Glu Leu Arg 140 145 150 Tyr Tyr Asp His Arg Phe Pro Leu Ala Glu Gly Thr Tyr Ala Glu Gly Asp 155 160 165 170 Ala Pro Arg Asp Val His Ala Arg Gln His Tyr G lu Leu Ile Gly Trp Arg 175 180 185 Arg Ala Asp Asn Glu Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu 190 195 200 Ala Gly Val Arg Val Glu Ile Pro Ala Val Phe Asp Glu Ala His Gln Glu 205 210 215 220 Val Val Arg Trp Phe Arg Glu Asp Leu Ala Asp Gly Leu Arg Ile Asp His 225 230 235 Pro Asp Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val 240 245 250 255 Thr Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu Ala 275 280 285 Asp Val Asp Arg Val Leu Val Asp Pro Arg Gly Gln Glu Pro Leu Asp Arg 290 295 300 305 Leu Asp Ala Ser Leu Arg Gly Gly Glu Pro Ala Asp Tyr Gln Asp Met Ile 310 315 320 Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His Ser Glu Ile Leu 325 330 335 340 Arg Leu Ala Arg Leu Val Pro Gly Asp Ala Asn Val Ser Ile Asp Ala Gly 345 350 355 Ala Asp Ala Leu Ala Glu Ile Ile Ala Ala Phe Pro Val Tyr Arg Thr Tyr 360 365 370 Leu Pro Glu Gly Ala Glu Val Leu Lys Glu Ala Cys Glu Leu Ala Ala Arg 3 75 380 385 390 Arg Arg Pro Glu Leu Asp Gln Ala Ile Gln Ala Leu Gln Pro Leu Leu Leu 395 400 405 Asp Thr Asp Leu Glu Leu Ala Arg Arg Phe Gln Gln Thr Ser Gly Met Val 410 415 420 425 Met Ala Lys Gly Val Glu Asp Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly 430 435 440 Thr Leu Thr Glu Val Gly Ala Asp Pro Thr Glu Phe Ala Val Glu Pro Asp 445 450 455 Glu Phe His Ala Arg Leu Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met 460 465 470 475 Thr Thr Leu Ser Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg 480 485 490 Ile Ser Val Ile Ser Glu Val Ala Gly Asp Trp Glu Lys Ala Leu Asn Arg 495 500 505 510 Leu Arg Asp Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Ala Leu Leu Trp 515 520 525 Gln Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Tyr Tyr 530 535 540 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Asn Trp Thr Asp Pro 545 550 555 560 Ala Pro Ala Phe Glu Glu Lys Leu Lys Ala Ala Val Asp Ala Val Phe Asp 565 570 575 Asn Pro Ala Val Gln Ala Glu Val Glu Ala Leu Val Glu Leu Leu Glu Pro 580 585 590 595 Tyr G ly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln Leu Thr Met Pro 600 605 610 Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp Asp Arg Ser Leu Thr 615 620 625 Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Asp Asp Arg Arg Ala Ala Leu 630 635 640 645 Glu Gln Leu Asp Ala Gly Asp Leu Pro Ala Ser Phe Thr Asp Glu Arg Thr 650 655 660 Lys Leu Leu Val Thr Ser Arg Ala Leu Arg Leu Arg Arg Asp Arg Pro Glu 665 670 675 680 Leu Phe Thr Gly Tyr Arg Pro Val Leu Ala Ser Gly Pro Ala Ala Gly His 685 690 695 Leu Leu Ala Phe Asp Arg Gly Thr Ala Ala Ala Pro Gly Ala Leu Thr Leu 700 705 710 Ala Thr Arg Leu Pro Tyr Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr 715 720 725 730 Ala Val Glu Leu Asn Thr Ala Met Lys Asp Glu Leu Thr Gly Ala Gly Phe 735 740 745 Gly Pro Gly Ala Val Lys Ile Ala Asp Ile Phe Arg Ser Phe Pro Val Ala 750 755 760 765 Leu Leu Val Pro Gln Thr Gly Gly Glu Ser 770 775

【０１０３】配列番号：3 配列の長さ：2316 配列の型：核酸 トポロジー：直鎖状 配列 ATGAGGACAC CCGCCTCGAC CTACCGGCTG CAGATCAGGC GGGGTTTCAC GCTGTTTGAT 60 GCCGCCGAGA CCGTGCCCTA CCTGAAGTCA CTCGGGGTGG ACTGGATCTA CCTGTCGCCC 120 ATCCTGAAGG CAGAGAGCGG CTCCGACCAC GGCTATGACG TCACCGATCC CGCCGTAGTG 180 GACCCGGAGC GCGGCGGCCC TGAAGGGCTG GCCGCGGTGT CCAAGGCGGC CCGCGGTGCC 240 GGCATGGGCG TGCTGATCGA CATCGTGCCG AACCACGTGG GCGTGGCGTC GCCGCCGCAG 300 AACCCGTGGT GGTGGTCGCT GCTCAAGGAA GGGCGCGGGT CGCCCTACGC CGTGGCGTTC 360 GACGTCGACT GGGACCTGGC GGGGGGCCGC ATCCGGATCC CCGTCCTGGG CAGCGACGAC 420 GATCTGGACC AGCTCGAAAT CAAGGACGGC GAGCTGCGGT ACTACGACCA CCGCTTCCCG 480 CTGGCCGAGG GCAGCTACCG GGACGGCGAC TCCCCGCAGG ACGTCCACGG CCGGCAGCAC 540 TACGAACTCA TCGGCTGGCG GCGCGCCGAC AATGAACTGA ACTACCGCCG GTTCTTCGCG 600 GTGAACACGC TCGCCGGCAT CCGGGTGGAG GTGCCGCCGG TCTTCGATGA AGCGCACCAG 660 GAGGTGGTGC GCTGGTTCCG TGCGGGGCTC GCCGACGGGC TGCGGATCGA CCACCCGGAC 720 GGCCTGGCCG ATCCCGAGGG GTATTTGAAG CGGCTCCGTG AGGTCACCGG GGGCGCGTAC 780 CTGCTCATCG AAAAGATCCT CGAGCCGGGC GAACAGTTGC CGGCCAGCTT CGAGTGCGAA 840 GGCACCACCG GCTACGACGC CCTCGCGGAT GTCGACAGGG TCTTCGTGGA CCCGCGGGGA 900 CAGGTGCCGC TGGACCGTCT GGACGCACGG CTGCGCGGCG GTGCGCCGGC CGACTACGAG 960 GACATGATCC GCGGGACCAA GCGCCGGATC ACCGACGGCA TCCTGCACTC CGAGATCCTG 1020 CGCCTTGCCA GGCTGGTGCC CGAGCAGACC GGAATTCCCG GGGAGGCGGC CGCGGATGCG 1080 ATCGCGGAGA TCATCGCGGC CTTCCCGGTC TACCGGTCCT ATCTTCCCGA GGGCGCGGAG 1140 ATCCTGAAGG AGGCCTGCGA CCTCGCCGCG CGGAGGCGTC CGGAACTGGG CCAGACCGTC 1200 CAGCTGCTGC AGCCGCTGCT GCTGGATACC GACCTCGAGA TTTCCCGCAG GTTCCAGCAG 1260 ACCTCGGGAA TGGTCATGGC CAAAGGCGTG GAGGACACCG CGTTCTTCCG CTACAACCGG 1320 CTGGGAACGC TCACCGAGGT GGGCGCCGAC CCCACCGAGT TCTCGCTGGA ACCGGAGGAG 1380 TTTCACGTCC GGATGGCCCG CCGGCAGGCC GAACTCCCGC TCTCCATGAC CACCCTGAGC 1440 ACGCACGACA CCAAGCGCAG CGAGGACACC CGGGCCCGGA TCTCGGTGAT CGCCGAGGTC 1500 GCGCCTGAAT GGGAAAAGGC CCTGGACAGG CTGAACACCC TCGCTCCGCT GCCGGACGGC 1560 CCGCTCTCCA CGCTGCTCTG GCAGGCGATT GCGGGGGCAT GGCCGGCCAG CCGGGAACGC 1620 CTTCAGTCCT ACGCCCTGAA AGCGGCGCGC GAAGCCGGGA ACTCGACCAG CTGGACCGAT 1680 CCGGACCCGG CATTCGAGGA GGCACTTTCC GCCGTCGTCG ACTCCGCCTT CGACAATCCG 1740 GAGGTGCGTG CGGAACTTGA GGCCCTGGTG GGCCTCCTTG CGCCGCACGG TGCGTCCAAC 1800 TCGCTCGCGG CAAAGCTTGT CCAGCTGACC ATGCCGGGCG TTCCGGACGT GTACCAGGGC 1860 ACCGAGTTCT GGGACAGGTC GCTGACCGAT CCGGACAACC GGCGCCCCTT CAGCTTCGCC 1920 GAACGGATTA GGGCCTTGGA CCAGTTGGAC GCCGGCCACC GTCCGGACTC CTTCCAGGAC 1980 GAGGCGGTCA AGCTGCTGGT CACCTCGAGG GCGCTGCGGC TGCGGCGGAA CCGGCCCGAG 2040 CTCTTCACCG GCTACCGCCC CGTGCATGCC AGGGGCCCCG CCGCCGGGCA CCTGGTGGCG 2100 TTCGACCGCG GCGCCGGGGG AGTGCTGGCG CTTGCCACCC GGCTCCCCTA CGGGCTGGAA 2160 CAGTCGGGCG GCTGGCGGGA CACCGCCGTC GAGCTTGAAG CCGCCATGAC GGACGAACTG 2220 ACCGGCTCCA CTTTCGGGCC GGGACCGGCG GCGCTGTCAG AAGTCTTCCG GGCCTACCCG 2280 GTGGCCTTGT TGGTCCCCGC GACAGGAGGC AAGTCA 2316[0103] SEQ ID NO: 3 sequence Length: type 2316 sequence: nucleic acid Topology: linear sequence ATGAGGACAC CCGCCTCGAC CTACCGGCTG CAGATCAGGC GGGGTTTCAC GCTGTTTGAT 60 GCCGCCGAGA CCGTGCCCTA CCTGAAGTCA CTCGGGGTGG ACTGGATCTA CCTGTCGCCC 120 ATCCTGAAGG CAGAGAGCGG CTCCGACCAC GGCTATGACG TCACCGATCC CGCCGTAGTG 180 GACCCGGAGC GCGGCGGCCC TGAAGGGCTG GCCGCGGTGT CCAAGGCGGC CCGCGGTGCC 240 GGCATGGGCG TGCTGATCGA CATCGTGCCG AACCACGTGG GCGTGGCGTC GCCGCCGCAG 300 AACCCGTGGT GGTGGTCGCT GCTCAAGGAA GGGCGCGGGT CGCCCTACGC CGTGGCGTTC 360 GACGTCGACT GGGACCTGGC GGGGGGCCGC ATCCGGATCC CCGTCCTGGG CAGCGACGAC 420 GATCTGGACC AGCTCGAAAT CAAGGACGGC GAGCTGCGGT ACTACGACCA CCGCTTCCCG 480 CTGGCCGAGG GCAGCTACCG GGACGGCGAC TCCCCGCAGG ACGTCCACGG CCGGCAGCAC 540 TACGAACTCA TCGGCTGGCG GCGCGCCGAC AATGAACTGA ACTACCGCCG GTTCTTCGCG 600 GTGAACACGC TCGCCGGCAT CCGGGTGGAG GTGCCGCCGG TCTTCGATGA AGCGCACCAG 660 GAGGTGGTGC GCTGGTTCCG TGCGGGGCTC GCCGACGGGC TGCGGATCGA CCACCCGGAC 720 GGCCTGGCCG ATCCCGAGGG GTATTTGAAG CGGCTCCGTG AG GTCACCGG GGGCGCGTAC 780 CTGCTCATCG AAAAGATCCT CGAGCCGGGC GAACAGTTGC CGGCCAGCTT CGAGTGCGAA 840 GGCACCACCG GCTACGACGC CCTCGCGGAT GTCGACAGGG TCTTCGTGGA CCCGCGGGGA 900 CAGGTGCCGC TGGACCGTCT GGACGCACGG CTGCGCGGCG GTGCGCCGGC CGACTACGAG 960 GACATGATCC GCGGGACCAA GCGCCGGATC ACCGACGGCA TCCTGCACTC CGAGATCCTG 1020 CGCCTTGCCA GGCTGGTGCC CGAGCAGACC GGAATTCCCG GGGAGGCGGC CGCGGATGCG 1080 ATCGCGGAGA TCATCGCGGC CTTCCCGGTC TACCGGTCCT ATCTTCCCGA GGGCGCGGAG 1140 ATCCTGAAGG AGGCCTGCGA CCTCGCCGCG CGGAGGCGTC CGGAACTGGG CCAGACCGTC 1200 CAGCTGCTGC AGCCGCTGCT GCTGGATACC GACCTCGAGA TTTCCCGCAG GTTCCAGCAG 1260 ACCTCGGGAA TGGTCATGGC CAAAGGCGTG GAGGACACCG CGTTCTTCCG CTACAACCGG 1320 CTGGGAACGC TCACCGAGGT GGGCGCCGAC CCCACCGAGT TCTCGCTGGA ACCGGAGGAG 1380 TTTCACGTCC GGATGGCCCG CCGGCAGGCC GAACTCCCGC TCTCCATGAC CACCCTGAGC 1440 ACGCACGACA CCAAGCGCAG CGAGGACACC CGGGCCCGGA TCTCGGTGAT CGCCGAGGTC 1500 GCGCCTGAAT GGGAAAAGGC CCTGGACAGG CTGAACACCC TCGCTCCGCT GCCGGACGGC 1560 CCGCTCTCCA CGCTGCTCTG GCAGGCGATT GCGGGGGCAT GGCCGGCCAG C CGGGAACGC 1620 CTTCAGTCCT ACGCCCTGAA AGCGGCGCGC GAAGCCGGGA ACTCGACCAG CTGGACCGAT 1680 CCGGACCCGG CATTCGAGGA GGCACTTTCC GCCGTCGTCG ACTCCGCCTT CGACAATCCG 1740 GAGGTGCGTG CGGAACTTGA GGCCCTGGTG GGCCTCCTTG CGCCGCACGG TGCGTCCAAC 1800 TCGCTCGCGG CAAAGCTTGT CCAGCTGACC ATGCCGGGCG TTCCGGACGT GTACCAGGGC 1860 ACCGAGTTCT GGGACAGGTC GCTGACCGAT CCGGACAACC GGCGCCCCTT CAGCTTCGCC 1920 GAACGGATTA GGGCCTTGGA CCAGTTGGAC GCCGGCCACC GTCCGGACTC CTTCCAGGAC 1980 GAGGCGGTCA AGCTGCTGGT CACCTCGAGG GCGCTGCGGC TGCGGCGGAA CCGGCCCGAG 2040 CTCTTCACCG GCTACCGCCC CGTGCATGCC AGGGGCCCCG CCGCCGGGCA CCTGGTGGCG 2100 TTCGACCGCG GCGCCGGGGG AGTGCTGGCG CTTGCCACCC GGCTCCCCTA CGGGCTGGAA 2160 CAGTCGGGCG GCTGGCGGGA CACCGCCGTC GAGCTTGAAG CCGCCATGAC GGACGAACTG 2220 ACCGGCTCCA CTTTCGGGCC GGGACCGGCG GCGCTGTCAG AAGTCTTCCG GGCCTACCCG 2280 GTGGCCTTGT TGGTCCCCGC GACAGGAGGC AAGTCA 2316

【０１０４】配列番号：4 配列の長さ：2325 配列の型：核酸 トポロジー：直鎖状 配列 ATGAGAACGC CAGTCTCCAC GTACAGGCTG CAGATCAGGA AGGGATTCAC ACTCTTCGAC 60 GCGGCCAAAA CCGTTCCGTA CCTGCACTCG CTCGGCGTCG ACTGGGTCTA CCTTTCTCCG 120 GTCCTGACTG CCGAGCAGGG CTCCGACCAC GGGTACGACG TCACCGATCC CTCCGCCGTC 180 GACCCCGAAC GCGGCGGGCC GGAGGGCCTC GCGGCGGTTT CCAAGGCGGC CCGCGCCGCG 240 GGCATGGGCG TGCTGATCGA CATCGTGCCC AACCACGTGG GCGTCGCGAC GCCGGCGCAG 300 AACCCCTGGT GGTGGTCGCT GCTCAAGGAG GGACGCCAGT CCCGTTACGC GGAGGCGTTC 360 GACGTCGATT GGGACCTCGC CGGGGGACGC ATCCGGCTGC CGGTGCTCGG CAGCGACGAT 420 GACCTCGACC AGCTCGAAAT CAGGGACGGG GAGCTGCGGT ACTACGACCA CCGATTCCCG 480 CTCGCCGAGG GAACCTACGC CGAAGGCGAC GCCCCGCGGG ATGTCCACGC CCGGCAGCAC 540 TACGAGCTCA TCGGCTGGCG CCGCGCGGAC AACGAGCTGA ACTACCGCCG CTTTTTCGCG 600 GTGAACACGC TCGCCGGCGT CCGCGTGGAA ATCCCCGCCG TCTTCGACGA GGCACACCAG 660 GAGGTGGTGC GCTGGTTCCG CGAGGACCTT GCGGACGGCC TGCGGATCGA CCACCCGGAC 720 GGCCTCGCTG ACCCCGAGGG GTACCTGAAG CGACTCCGGG AAGTCACCGG CGGCGCTTAC 780 CTGCTGATCG AAAAGATCCT GGAGCCGGGG GAGCAGCTGC CCGCCAGCTT CGAGTGTGAA 840 GGCACCACAG GCTACGACGC CCTCGCCGAC GTCGACCGGG TTCTCGTGGA CCCGCGCGGC 900 CAGGAACCGC TGGACCGGCT TGACGCGTCC CTGCGTGGCG GCGAGCCCGC CGACTACCAG 960 GACATGATCC GCGGAACCAA GCGCCGGATC ACCGACGGTA TCCTGCACTC GGAGATCCTG 1020 CGGCTGGCCC GGCTGGTTCC GGGCGACGCC AACGTTTCAA TCGACGCCGG AGCCGACGCT 1080 CTCGCCGAAA TCATCGCCGC CTTCCCGGTC TACCGCACCT ACCTGCCGGA GGGCGCCGAG 1140 GTCCTGAAGG AGGCGTGCGA GCTTGCCGCG CGTAGGCGGC CGGAACTCGA CCAGGCCATC 1200 CAGGCTCTGC AGCCGCTGCT GCTGGACACG GACCTCGAGC TTGCCCGGCG CTTCCAGCAG 1260 ACCTCGGGCA TGGTCATGGC CAAGGGCGTG GAGGACACCG CGTTCTTCCG CTACAACCGC 1320 CTGGGCACCC TCACGGAAGT GGGCGCCGAC CCCACCGAGT TCGCCGTGGA GCCGGACGAG 1380 TTCCACGCCC GGCTGGCACG CCGGCAGGCC GAGCTTCCGC TGTCCATGAC GACGCTGAGC 1440 ACGCACGACA CCAAGCGCAG CGAGGACACC CGAGCAAGGA TTTCGGTCAT TTCCGAGGTT 1500 GCGGGTGACT GGGAAAAGGC CTTGAACCGG CTGCGCGACC TGGCCCCGCT GCCGGACGGC 1560 CCGCTGTCCG CGCTGCTCTG GCAGGCCATT GCCGGCGCCT GGCCCGCCAG CCGGGAACGC 1620 CTGCAGTACT ACGCGCTGAA GGCCGCGCGT GAAGCGGGGA ACTCGACCAA CTGGACCGAT 1680 CCGGCCCCCG CGTTCGAGGA GAAGCTGAAG GCCGCGGTCG ACGCCGTGTT CGACAATCCC 1740 GCCGTGCAGG CCGAGGTGGA AGCCCTCGTC GAGCTCCTGG AGCCGTACGG AGCTTCGAAC 1800 TCCCTCGCCG CCAAGCTCGT GCAGCTGACC ATGCCCGGCG TCCCGGACGT CTACCAGGGC 1860 ACGGAGTTCT GGGACCGGTC GCTGACGGAC CCGGACAACC GGCGGCCGTT CAGCTTCGAC 1920 GACCGCCGCG CCGCGCTGGA GCAGCTGGAT GCCGGCGACC TTCCCGCGTC ATTTACCGAT 1980 GAGCGGACGA AGCTGCTAGT GACGTCGCGC GCGCTGCGGC TGCGCCGGGA CCGTCCGGAG 2040 CTGTTCACGG GGTACCGGCC GGTCCTGGCC AGCGGGCCCG CCGCCGGGCA CCTGCTCGCG 2100 TTCGACCGCG GCACCGCGGC GGCGCCGGGT GCATTGACCC TCGCCACGCG GCTTCCCTAC 2160 GGGCTGGAAC AGTCGGGTGG ATGGCGGGAC ACCGCCGTCG AACTTAACAC CGCCATGAAA 2220 GACGAACTGA CCGGTGCCGG CTTCGGACCG GGGGCAGTGA AGATCGCCGA CATCTTCCGG 2280 TCGTTCCCCG TTGCGCTGCT GGTGCCGCAG ACAGGAGGAG AGTCA 2325SEQ ID NO: 4 Sequence length: 2325 Sequence type: Nucleic acid Topology: Linear sequence ATGAGAACGC CAGTCTCCAC GTACAGGCTG CAGATCAGGA AGGGATTCAC ACTCTTCGAC 60 GCGGCCAAAA CCGTTCCGTA CCTGCCCCCGGCGCGTCGCCCT 120GTCCTGCCCG 120GC CCGCGCCGCG 240 GGCATGGGCG TGCTGATCGA CATCGTGCCC AACCACGTGG GCGTCGCGAC GCCGGCGCAG 300 AACCCCTGGT GGTGGTCGCT GCTCAAGGAG GGACGCCAGT CCCGTTACGC GGAGGCGTTC 360 GACGTCGATT GGGACCTCGC CGGGGGACGC ATCCGGCTGC CGGTGCTCGG CAGCGACGAT 420 GACCTCGACC AGCTCGAAAT CAGGGACGGG GAGCTGCGGT ACTACGACCA CCGATTCCCG 480 CTCGCCGAGG GAACCTACGC CGAAGGCGAC GCCCCGCGGG ATGTCCACGC CCGGCAGCAC 540 TACGAGCTCA TCGGCTGGCG CCGCGCGGAC AACGAGCTGA ACTACCGCCG CTTTTTCGCG 600 GTGAACACGC TCGCCGGCGT CCGCGTGGAA ATCCCCGCCG TCTTCGACGA GGCACACCAG 660 GAGGTGGTGC GCTGGTTCCG CGAGGACCTT GCGGACGGCC TGCGGATCGA CCACCCGGAC 720 GGCCTCGCTG ACCCCGAGGG GTACCTGAAG CGACTCCGGG AA GTCACCGG CGGCGCTTAC 780 CTGCTGATCG AAAAGATCCT GGAGCCGGGG GAGCAGCTGC CCGCCAGCTT CGAGTGTGAA 840 GGCACCACAG GCTACGACGC CCTCGCCGAC GTCGACCGGG TTCTCGTGGA CCCGCGCGGC 900 CAGGAACCGC TGGACCGGCT TGACGCGTCC CTGCGTGGCG GCGAGCCCGC CGACTACCAG 960 GACATGATCC GCGGAACCAA GCGCCGGATC ACCGACGGTA TCCTGCACTC GGAGATCCTG 1020 CGGCTGGCCC GGCTGGTTCC GGGCGACGCC AACGTTTCAA TCGACGCCGG AGCCGACGCT 1080 CTCGCCGAAA TCATCGCCGC CTTCCCGGTC TACCGCACCT ACCTGCCGGA GGGCGCCGAG 1140 GTCCTGAAGG AGGCGTGCGA GCTTGCCGCG CGTAGGCGGC CGGAACTCGA CCAGGCCATC 1200 CAGGCTCTGC AGCCGCTGCT GCTGGACACG GACCTCGAGC TTGCCCGGCG CTTCCAGCAG 1260 ACCTCGGGCA TGGTCATGGC CAAGGGCGTG GAGGACACCG CGTTCTTCCG CTACAACCGC 1320 CTGGGCACCC TCACGGAAGT GGGCGCCGAC CCCACCGAGT TCGCCGTGGA GCCGGACGAG 1380 TTCCACGCCC GGCTGGCACG CCGGCAGGCC GAGCTTCCGC TGTCCATGAC GACGCTGAGC 1440 ACGCACGACA CCAAGCGCAG CGAGGACACC CGAGCAAGGA TTTCGGTCAT TTCCGAGGTT 1500 GCGGGTGACT GGGAAAAGGC CTTGAACCGG CTGCGCGACC TGGCCCCGCT GCCGGACGGC 1560 CCGCTGTCCG CGCTGCTCTG GCAGGCCATT GCCGGCGCCT GGCCCGCCAG C CGGGAACGC 1620 CTGCAGTACT ACGCGCTGAA GGCCGCGCGT GAAGCGGGGA ACTCGACCAA CTGGACCGAT 1680 CCGGCCCCCG CGTTCGAGGA GAAGCTGAAG GCCGCGGTCG ACGCCGTGTT CGACAATCCC 1740 GCCGTGCAGG CCGAGGTGGA AGCCCTCGTC GAGCTCCTGG AGCCGTACGG AGCTTCGAAC 1800 TCCCTCGCCG CCAAGCTCGT GCAGCTGACC ATGCCCGGCG TCCCGGACGT CTACCAGGGC 1860 ACGGAGTTCT GGGACCGGTC GCTGACGGAC CCGGACAACC GGCGGCCGTT CAGCTTCGAC 1920 GACCGCCGCG CCGCGCTGGA GCAGCTGGAT GCCGGCGACC TTCCCGCGTC ATTTACCGAT 1980 GAGCGGACGA AGCTGCTAGT GACGTCGCGC GCGCTGCGGC TGCGCCGGGA CCGTCCGGAG 2040 CTGTTCACGG GGTACCGGCC GGTCCTGGCC AGCGGGCCCG CCGCCGGGCA CCTGCTCGCG 2100 TTCGACCGCG GCACCGCGGC GGCGCCGGGT GCATTGACCC TCGCCACGCG GCTTCCCTAC 2160 GGGCTGGAAC AGTCGGGTGG ATGGCGGGAC ACCGCCGTCG AACTTAACAC CGCCATGAAA 2220 GACGAACTGA CCGGTGCCGG CTTCGGACCG GGGGCAGTGA AGATCGCCGA CATCTTCCGG 2280 TCGTTCCCCG TTGCGCTGCT GGTGCCGCAG ACAGGAGGAG AGTCA 2325

【０１０５】配列番号：5 配列の長さ：2936 配列の型：核酸 鎖の数：二本鎖 トポロジー：直鎖状 配列の種類：Genomic DNA 配列の特徴 起源 生物名：リゾビウム・スピーシーズ(Rhizobium sp.) 株名：M-11(FERM BP-4130) 配列の特徴 特徴を表わす記号：5´UTR 存在位置：1..564 特徴を決定した方法：E 特徴を表わす記号：mat peptide 存在位置：565..2880 特徴を決定した方法：S 特徴を表わす記号：3´UTR 存在位置：2881..2936 特徴を決定した方法：E 配列 CGTGCTCTAC TTCAACGCGC ACGACGGCGA CGTCGTGTTC AAGCTCCCGT CGGATGAATA 60 CGCCCCGGCC TGGGACGTCA TCATCGACAC CGCCGGCGCG GGTGCCGATT CCGAACCCGT 120 GCAGGCTGGC GGCAAACTCA CCGTGGCAGC GAAATCGCTC GTGGTGCTCC GTGCCCACAG 180 CGCCCCGGAG GAGGAACCGG ACCACTCGGT GGCCGCCTCC CTCGCAGCGC TGACGCAGAC 240 TGCGACCGCC GAAACCGCGG CGCTCACCGC CCCCACCGTT CCGGAGCCGA GGAAGACCAA 300 GAAGGCAGCG CCGAAGCCGG AAGAGGAGGC TCCCGACGAG GCGGCGCCGA AGCCGGAAGA 360 GAAGGCTCCC GACGAGGCGG CGGCGAAGCC GGAAGAGGCT GCTTCCGACG AGGCGGCGGC 420 GAAGCCGGAA GAGAAGGCTC CCGACGAGGC GGCGGCGAAG CCGGAAGAGG CTGCTTCCGA 480 CGAGGCGGCG GCGAAGCCCG CGGGGAAGGC AGCGGCCAAA ACGGCCGGCA GGCGAGCGCC 540 AGGCAAGCAG GGCGGGACGG GCTC 564 ATG AGG ACA CCC GCC TCG ACC TAC CGG CTG CAG ATC AGG CGG GGT TTC 612 Met Arg Thr Pro Ala Ser Thr Tyr Arg Leu Gln Ile Arg Arg Gly Phe 1 5 10 15 ACG CTG TTT GAT GCC GCC GAG ACC GTG CCC TAC CTG AAG TCA CTC GGG 660 Thr Leu Phe Asp Ala Ala Glu Thr Val Pro Tyr Leu Lys Ser Leu Gly 20 25 30 GTG GAC TGG ATC TAC CTG TCG CCC ATC CTG AAG GCA GAG AGC GGC TCC 708 Val Asp Trp Ile Tyr Leu Ser Pro Ile Leu Lys Ala Glu Ser Gly Ser 35 40 45 GAC CAC GGC TAT GAC GTC ACC GAT CCC GCC GTA GTG GAC CCG GAG CGC 756 Asp His Gly Tyr Asp Val Thr Asp Pro Ala Val Val Asp Pro Glu Arg 50 55 60 GGC GGC CCT GAA GGG CTG GCC GCG GTG TCC AAG GCG GCC CGC GGT GCC 804 Gly Gly Pro Glu Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Gly Ala 65 70 75 80 GGC ATG GGC GTG CTG ATC GAC ATC GTG CCG AAC CAC GTG GGC GTG GCG 852 Gly Met Gly Val Leu Ile Asp Ile Val Pro Asn His Val Gly Val Ala 85 90 95 TCG CCG CCG CAG AAC CCG TGG TGG TGG TCG CTG CTC AAG GAA GGG CGC 900 Ser Pro Pro Gln Asn Pro Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg 100 105 110 GGG TCG CCC TAC GCC GTG GCG TTC GAC GTC GAC TGG GAC CTG GCG GGG 948 Gly Ser Pro Tyr Ala Val Ala Phe Asp Val Asp Trp Asp Leu Ala Gly 115 120 125 GGC CGC ATC CGG ATC CCC GTC CTG GGC AGC GAC GAC GAT CTG GAC CAG 996 Gly Arg Ile Arg Ile Pro Val Leu Gly Ser Asp Asp Asp Leu Asp Gln 130 135 140 CTC GAA ATC AAG GAC GGC GAG CTG CGG TAC TAC GAC CAC CGC TTC CCG 1044 Leu Glu Ile Lys Asp Gly Glu Leu Arg Tyr Tyr Asp His Arg Phe Pro 145 150 155 160 CTG GCC GAG GGC AGC TAC CGG GAC GGC GAC TCC CCG CAG GAC GTC CAC 1092 Leu Ala Glu Gly Ser Tyr Arg Asp Gly Asp Ser Pro Gln Asp Val His 165 170 175 GGC CGG CAG CAC TAC GAA CTC ATC GGC TGG CGG CGC GCC GAC AAT GAA 1140 Gly Arg Gln His Tyr Glu Leu Ile Gly Trp Arg Arg Ala Asp Asn Glu 180 185 190 CTG AAC TAC CGC CGG TTC TTC GCG GTG AAC ACG CTC GCC GGC ATC CGG 1188 Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu Ala Gly Ile Arg 195 200 205 GTG GAG GTG CCG CCG GTC TTC GAT GAA GCG CAC CAG GAG GTG GTG CGC 1236 Val Glu Val Pro Pro Val Phe Asp Glu Ala His Gln Glu Val Val Arg 210 215 220 TGG TTC CGT GCG GGG CTC GCC GAC GGG CTG CGG ATC GAC CAC CCG GAC 1284 Trp Phe Arg Ala Gly Leu Ala Asp Gly Leu Arg Ile Asp His Pro Asp 225 230 235 240 GGC CTG GCC GAT CCC GAG GGG TAT TTG AAG CGG CTC CGT GAG GTC ACC 1332 Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val Thr 245 250 255 GGG GGC GCG TAC CTG CTC ATC GAA AAG ATC CTC GAG CCG GGC GAA CAG 1380 Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 TTG CCG GCC AGC TTC GAG TGC GAA GGC ACC ACC GGC TAC GAC GCC CTC 1428 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu 275 280 285 GCG GAT GTC GAC AGG GTC TTC GTG GAC CCG CGG GGA CAG GTG CCG CTG 1476 Ala Asp Val Asp Arg Val Phe Val Asp Pro Arg Gly Gln Val Pro Leu 290 295 300 GAC CGT CTG GAC GCA CGG CTG CGC GGC GGT GCG CCG GCC GAC TAC GAG 1524 Asp Arg Leu Asp Ala Arg Leu Arg Gly Gly Ala Pro Ala Asp Tyr Glu 305 310 315 320 GAC ATG ATC CGC GGG ACC AAG CGC CGG ATC ACC GAC GGC ATC CTG CAC 1572 Asp Met Ile Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His 325 330 335 TCC GAG ATC CTG CGC CTT GCC AGG CTG GTG CCC GAG CAG ACC GGA ATT 1620 Ser Glu Ile Leu Arg Leu Ala Arg Leu Val Pro Glu Gln Thr Gly Ile 340 345 350 CCC GGG GAG GCG GCC GCG GAT GCG ATC GCG GAG ATC ATC GCG GCC TTC 1668 Pro Gly Glu Ala Ala Ala Asp Ala Ile Ala Glu Ile Ile Ala Ala Phe 355 360 365 CCG GTC TAC CGG TCC TAT CTT CCC GAG GGC GCG GAG ATC CTG AAG GAG 1716 Pro Val Tyr Arg Ser Tyr Leu Pro Glu Gly Ala Glu Ile Leu Lys Glu 370 375 380 GCC TGC GAC CTC GCC GCG CGG AGG CGT CCG GAA CTG GGC CAG ACC GTC 1764 Ala Cys Asp Leu Ala Ala Arg Arg Arg Pro Glu Leu Gly Gln Thr Val 385 390 395 400 CAG CTG CTG CAG CCG CTG CTG CTG GAT ACC GAC CTC GAG ATT TCC CGC 1812 Gln Leu Leu Gln Pro Leu Leu Leu Asp Thr Asp Leu Glu Ile Ser Arg 405 410 415 AGG TTC CAG CAG ACC TCG GGA ATG GTC ATG GCC AAA GGC GTG GAG GAC 1860 Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu Asp 420 425 430 ACC GCG TTC TTC CGC TAC AAC CGG CTG GGA ACG CTC ACC GAG GTG GGC 1908 Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly Thr Leu Thr Glu Val Gly 435 440 445 GCC GAC CCC ACC GAG TTC TCG CTG GAA CCG GAG GAG TTT CAC GTC CGG 1956 Ala Asp Pro Thr Glu Phe Ser Leu Glu Pro Glu Glu Phe His Val Arg 450 455 460 ATG GCC CGC CGG CAG GCC GAA CTC CCG CTC TCC ATG ACC ACC CTG AGC 2004 Met Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met Thr Thr Leu Ser 465 470 475 480 ACG CAC GAC ACC AAG CGC AGC GAG GAC ACC CGG GCC CGG ATC TCG GTG 2052 Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val 485 490 495 ATC GCC GAG GTC GCG CCT GAA TGG GAA AAG GCC CTG GAC AGG CTG AAC 2100 Ile Ala Glu Val Ala Pro Glu Trp Glu Lys Ala Leu Asp Arg Leu Asn 500 505 510 ACC CTC GCT CCG CTG CCG GAC GGC CCG CTC TCC ACG CTG CTC TGG CAG 2148 Thr Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Thr Leu Leu Trp Gln 515 520 525 GCG ATT GCG GGG GCA TGG CCG GCC AGC CGG GAA CGC CTT CAG TCC TAC 2196 Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Ser Tyr 530 535 540 GCC CTG AAA GCG GCG CGC GAA GCC GGG AAC TCG ACC AGC TGG ACC GAT 2244 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Ser Trp Thr Asp 545 550 555 560 CCG GAC CCG GCA TTC GAG GAG GCA CTT TCC GCC GTC GTC GAC TCC GCC 2292 Pro Asp Pro Ala Phe Glu Glu Ala Leu Ser Ala Val Val Asp Ser Ala 565 570 575 TTC GAC AAT CCG GAG GTG CGT GCG GAA CTT GAG GCC CTG GTG GGC CTC 2340 Phe Asp Asn Pro Glu Val Arg Ala Glu Leu Glu Ala Leu Val Gly Leu 580 585 590 CTT GCG CCG CAC GGT GCG TCC AAC TCG CTC GCG GCA AAG CTT GTC CAG 2388 Leu Ala Pro His Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln 595 600 605 CTG ACC ATG CCG GGC GTT CCG GAC GTG TAC CAG GGC ACC GAG TTC TGG 2436 Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp 610 615 620 GAC AGG TCG CTG ACC GAT CCG GAC AAC CGG CGC CCC TTC AGC TTC GCC 2484 Asp Arg Ser Leu Thr Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Ala 625 630 635 640 GAA CGG ATT AGG GCC TTG GAC CAG TTG GAC GCC GGC CAC CGT CCG GAC 2532 Glu Arg Ile Arg Ala Leu Asp Gln Leu Asp Ala Gly His Arg Pro Asp 645 650 655 TCC TTC CAG GAC GAG GCG GTC AAG CTG CTG GTC ACC TCG AGG GCG CTG 2580 Ser Phe Gln Asp Glu Ala Val Lys Leu Leu Val Thr Ser Arg Ala Leu 660 665 670 CGG CTG CGG CGG AAC CGG CCC GAG CTC TTC ACC GGC TAC CGC CCC GTG 2628 Arg Leu Arg Arg Asn Arg Pro Glu Leu Phe Thr Gly Tyr Arg Pro Val 675 680 685 CAT GCC AGG GGC CCC GCC GCC GGG CAC CTG GTG GCG TTC GAC CGC GGC 2676 His Ala Arg Gly Pro Ala Ala Gly His Leu Val Ala Phe Asp Arg Gly 690 695 700 GCC GGG GGA GTG CTG GCG CTT GCC ACC CGG CTC CCC TAC GGG CTG GAA 2724 Ala Gly Gly Val Leu Ala Leu Ala Thr Arg Leu Pro Tyr Gly Leu Glu 705 710 715 720 CAG TCG GGC GGC TGG CGG GAC ACC GCC GTC GAG CTT GAA GCC GCC ATG 2772 Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu Leu Glu Ala Ala Met 725 730 735 ACG GAC GAA CTG ACC GGC TCC ACT TTC GGG CCG GGA CCG GCG GCG CTG 2820 Thr Asp Glu Leu Thr Gly Ser Thr Phe Gly Pro Gly Pro Ala Ala Leu 740 745 750 TCA GAA GTC TTC CGG GCC TAC CCG GTG GCC TTG TTG GTC CCC GCG ACA 2868 Ser Glu Val Phe Arg Ala Tyr Pro Val Ala Leu Leu Val Pro Ala Thr 755 760 765 GGA GGC AAG TCA 2880 Gly Gly Lys Ser 770 TGACGCAGCC CAACGATGCG GCCAAGCCGG TGCAGGGAGC GGGGCGCTTC GATATC 2936SEQ ID NO: 5 Sequence length: 2936 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Genomic DNA Sequence features Origin Biological name: Rhizobium sp. ) Strain name: M-11 (FERM BP-4130) Sequence features Characteristic signature: 5'UTR Location: 1..564 Method of determining feature: E Characteristic signature: mat peptide Location: 565. .2880 Characteristic determination method: S Characteristic symbol: 3'UTR Location: 2881..2936 Characteristic determination method: E Sequence CGTGCTCTAC TTCAACGCGC ACGACGGCGACGCGGCTCGCAGGAGAGCGC CCCGGACCCACGCCGCACGACGGC120C GTGGTGCTCC GTGCCCACAG 180 CGCCCCGGAG GAGGAACCGG ACCACTCGGT GGCCGCCTCC CTCGCAGCGC TGACGCAGAC 240 TGCGACCGCC GAAACCGCGG CGCTCACCGC CCCCACCGTT CCGGAGCCGA GGAAGACCAA 300 GAAGGCAGGA CCGAAGCCGG AAGAGGAGGGGCGCGCCGAGA CCC GACGAGGCGG CGGCGAAGCC GGAAGAGGCT GCTTCCGACG AGGCGGCGGC 420 GAAGCCGGAA GAGAAGGCTC CCGACGAGGC GGCGGCGAAG CCGGAAGAGG CTGCTTCCGA 480 CGAGGCGGCG GCGAAGCCCG CGGGGAAGGC AGCGGCCAAA ACGGCCGGCA GGCGAGCGCC 540 AGGCAAGCAG GGCGGGACGG GCTC 564 ATG AGG ACA CCC GCC TCG ACC TAC CGG CTG CAG ATC AGG CGG GGT TTC 612 Met Arg Thr Pro Ala Ser Thr Tyr Arg Leu Gln Ile Arg Arg Gly Phe 1 5 10 15 ACG CTG TTT GAT GCC GCC GAG ACC GTG CCC TAC CTG AAG TCA CTC GGG 660 Thr Leu Phe Asp Ala Ala Glu Thr Val Pro Tyr Leu Lys Ser Leu Gly 20 25 30 GTG GAC TGG ATC TAC CTG TCG CCC ATC CTG AAG GCA GAG AGC GGC TCC 708 Val Asp Trp Ile Tyr Leu Ser Pro Ile Leu Lys Ala Glu Ser Gly Ser 35 40 45 GAC CAC GGC TAT GAC GTC ACC GAT CCC GCC GTA GTG GAC CCG GAG CGC 756 Asp His Gly Tyr Asp Val Thr Asp Pro Ala Val Val Asp Pro Glu Arg 50 55 60 GGC GGC CCT GAA GGG CTG GCC GCG GTG TCC AAG GCG GCC CGC GGT GCC 804 Gly Gly Pro Glu Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Gly Ala 65 70 75 80 GGC ATG GGC GTG CTG ATC GAC ATC GTG CCG AAC CAC GTG GGC G TG GCG 852 Gly Met Gly Val Leu Ile Asp Ile Val Pro Asn His Val Gly Val Ala 85 90 95 TCG CCG CCG CAG AAC CCG TGG TGG TGG TCG CTG CTC AAG GAA GGG CGC 900 Ser Pro Pro Gln Asn Pro Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg 100 105 110 GGG TCG CCC TAC GCC GTG GCG TTC GAC GTC GAC TGG GAC CTG GCG GGG 948 Gly Ser Pro Tyr Ala Val Ala Phe Asp Val Asp Trp Asp Leu Ala Gly 115 120 125 GGC CGC ATC CGG ATC CCC GTC CTG GGC AGC GAC GAC GAT CTG GAC CAG 996 Gly Arg Ile Arg Ile Pro Val Leu Gly Ser Asp Asp Asp Leu Asp Gln 130 135 140 CTC GAA ATC AAG GAC GGC GAG CTG CGG TAC TAC GAC CAC CGC TTC CCG 1044 Leu Glu Ile Lys Asp Gly Glu Leu Arg Tyr Tyr Asp His Arg Phe Pro 145 150 155 160 CTG GCC GAG GGC AGC TAC CGG GAC GGC GAC TCC CCG CAG GAC GTC CAC 1092 Leu Ala Glu Gly Ser Tyr Arg Asp Gly Asp Ser Pro Gln Asp Val His 165 170 175 GGC CGG CAG CAC TAC GAA CTC ATC GGC TGG CGG CGC GCC GAC AAT GAA 1140 Gly Arg Gln His Tyr Glu Leu Ile Gly Trp Arg Arg Ala Asp Asn Glu 180 185 190 CTG AAC TAC CGC CGG TTC TTC GCG GTG AAC ACG C TC GCC GGC ATC CGG 1188 Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu Ala Gly Ile Arg 195 200 205 GTG GAG GTG CCG CCG GTC TTC GAT GAA GCG CAC CAG GAG GTG GTG CGC 1236 Val Glu Val Pro Pro Val Phe Asp Glu Ala His Gln Glu Val Val Arg 210 215 220 TGG TTC CGT GCG GGG CTC GCC GAC GGG CTG CGG ATC GAC CAC CCG GAC 1284 Trp Phe Arg Ala Gly Leu Ala Asp Gly Leu Arg Ile Asp His Pro Asp 225 230 235 240 GGC CTG GCC GAT CCC GAG GGG TAT TTG AAG CGG CTC CGT GAG GTC ACC 1332 Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val Thr 245 250 255 GGG GGC GCG TAC CTG CTC ATC GAA AAG ATC CTC GAG CCG GGC GAA CAG 1380 Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 TTG CCG GCC AGC TTC GAG TGC GAA GGC ACC ACC GGC TAC GAC GCC CTC 1428 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu 275 280 285 GCG GAT GTC GAC AGG GTC TTC GTG GAC CCG CGG GGA CAG GTG CCG CTG 1476 Ala Asp Val Asp Arg Val Phe Val Asp Pro Arg Gly Gln Val Pro Leu 290 295 300 GAC CGT CTG GAC GCA CGG CT G CGC GGC GGT GCG CCG GCC GAC TAC GAG 1524 Asp Arg Leu Asp Ala Arg Leu Arg Gly Gly Ala Pro Ala Asp Tyr Glu 305 310 315 320 GAC ATG ATC CGC GGG ACC AAG CGC CGG ATC ACC GAC GGC ATC CTG CAC 1572 Asp Met Ile Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His 325 330 335 TCC GAG ATC CTG CGC CTT GCC AGG CTG GTG CCC GAG CAG ACC GGA ATT 1620 Ser Glu Ile Leu Arg Leu Ala Arg Leu Val Pro Glu Gln Thr Gly Ile 340 345 350 CCC GGG GAG GCG GCC GCG GAT GCG ATC GCG GAG ATC ATC GCG GCC TTC 1668 Pro Gly Glu Ala Ala Ala Asp Ala Ile Ala Glu Ile Ile Ala Ala Phe 355 360 365 CCG GTC TAC CGG TCC TAT CTT CCC GAG GGC GCG GAG ATC CTG AAG GAG 1716 Pro Val Tyr Arg Ser Tyr Leu Pro Glu Gly Ala Glu Ile Leu Lys Glu 370 375 380 GCC TGC GAC CTC GCC GCG CGG AGG CGT CCG GAA CTG GGC CAG ACC GTC 1764 Ala Cys Asp Leu Ala Ala Arg Arg Arg Pro Glu Leu Gly Gln Thr Val 385 390 395 400 CAG CTG CTG CAG CCG CTG CTG CTG GAT ACC GAC CTC GAG ATT TCC CGC 1812 Gln Leu Leu Gln Pro Leu Leu Leu Asp Thr Asp Leu Glu Ile Ser Arg 405 410 415 AGG TTC CAG CAG ACC TCG GGA ATG GTC ATG GCC AAA GGC GTG GAG GAC 1860 Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu Asp 420 425 430 ACC GCG TTC TTC CGC TAC AAC CGG CTG GGA ACG CTC ACC GAG GTG GGC 1908 Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly Thr Leu Thr Glu Val Gly 435 440 445 GCC GAC CCC ACC GAG TTC TCG CTG GAA CCG GAG GAG TTT CAC GTC CGG 1956 Ala Asp Pro Thr Glu Phe Ser Leu Glu Pro Glu Glu Phe His Val Arg 450 455 460 ATG GCC CGC CGG CAG GCC GAA CTC CCG CTC TCC ATG ACC ACC CTG AGC 2004 Met Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met Thr Thr Leu Ser 465 475 480 480 ACG CAC GAC ACC AAG CGC AGC GAG GAC ACC CGG GCC CGG ATC TCG GTG 2052 Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val 485 490 495 ATC GCC GAG GTC GCG CCT GAA TGG GAA AAG GCC CTG GAC AGG CTG AAC 2100 Ile Ala Glu Val Ala Pro Glu Trp Glu Lys Ala Leu Asp Arg Leu Asn 500 505 510 ACC CTC GCT CCG CTG CCG GAC GGC CCG CTC TCC ACG CTG CTC TGG CAG 2148 Thr Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Thr Leu Leu Trp Gln 515 520 525 GCG ATT GCG GGG GCA TGG CCG GCC AGC CGG GAA CGC CTT CAG TCC TAC 2196 Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Ser Tyr 530 535 540 GCC CTG AAA GCG GCG CGC GAA GCC GGG AAC TCG ACC AGC TGG ACC GAT 2244 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Ser Trp Thr Asp 545 550 555 560 CCG GAC CCG GCA TTC GAG GAG GCA CTT TCC GCC GTC GTC GAC TCC GCC 2292 Pro Asp Pro Ala Phe Glu Glu Ala Leu Ser Ala Val Val Asp Ser Ala 565 570 575 TTC GAC AAT CCG GAG GTG CGT GCG GAA CTT GAG GCC CTG GTG GGC CTC 2340 Phe Asp Asn Pro Glu Val Arg Ala Glu Leu Glu Ala Leu Val Gly Leu 580 585 590 CTT GCG CCG CAC GGT GCG TCC AAC TCG CTC GCG GCA AAG CTT GTC CAG 2388 Leu Ala Pro His Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln 595 600 605 CTG ACC ATG CCG GGC GTT CCG GAC GTG TAC CAG GGC ACC GAG TTC TGG 2436 Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp 610 615 620 GAC AGG TCG CTG ACC GAT CCG GAC AAC CGG CGC CCC TTC AGC TTC GCC 2484 Asp Arg Ser Leu Thr Asp Pro Asp Asn Arg A rg Pro Phe Ser Phe Ala 625 630 635 640 GAA CGG ATT AGG GCC TTG GAC CAG TTG GAC GCC GGC CAC CGT CCG GAC 2532 Glu Arg Ile Arg Ala Leu Asp Gln Leu Asp Ala Gly His Arg Pro Asp 645 650 655 TCC TTC CAG GAC GAG GCG GTC AAG CTG CTG GTC ACC TCG AGG GCG CTG 2580 Ser Phe Gln Asp Glu Ala Val Lys Leu Leu Val Thr Ser Arg Ala Leu 660 665 670 CGG CTG CGG CGG AAC CGG CCC GAG CTC TTC ACC GGC TAC CGC CCC GTG 2628 Arg Leu Arg Arg Asn Arg Pro Glu Leu Phe Thr Gly Tyr Arg Pro Val 675 680 685 CAT GCC AGG GGC CCC GCC GCC GGG CAC CTG GTG GCG TTC GAC CGC GGC 2676 His Ala Arg Gly Pro Ala Ala Gly His Leu Val Ala Phe Asp Arg Gly 690 695 700 GCC GGG GGA GTG CTG GCG CTT GCC ACC CGG CTC CCC TAC GGG CTG GAA 2724 Ala Gly Gly Val Leu Ala Leu Ala Thr Arg Leu Pro Tyr Gly Leu Glu 705 710 715 720 CAG TCG GGC GGC TGG CGG GAC ACC GCC GTC GAG CTT GAA GCC GCC ATG 2772 Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu Leu Glu Ala Ala Met 725 730 735 ACG GAC GAA CTG ACC GGC TCC ACT TTC GGG CCG GGA CCG GCG GCG CTG 2820 Thr Asp Glu Leu Th r Gly Ser Thr Phe Gly Pro Gly Pro Ala Ala Leu 740 745 750 TCA GAA GTC TTC CGG GCC TAC CCG GTG GCC TTG TTG GTC CCC GCG ACA 2868 Ser Glu Val Phe Arg Ala Tyr Pro Val Ala Leu Leu Val Pro Ala Thr 755 760 765 GGA GGC AAG TCA 2880 Gly Gly Lys Ser 770 TGACGCAGCC CAACGATGCG GCCAAGCCGG TGCAGGGAGC GGGGCGCTTC GATATC 2936

【０１０６】配列番号：6 配列の長さ：3084 配列の型：核酸 鎖の数：二本鎖 トポロジー：直鎖状 配列の種類：Genomic DNA 配列の特徴 起源 生物名：アルスロバクター・スピーシーズ(Arthrobacte
r sp.) 株名：Q36(FERM BP-4316) 配列の特徴 特徴を表わす記号：5´UTR 存在位置：1..677 特徴を決定した方法：E 特徴を表わす記号：mat peptide 存在位置：678..3002 特徴を決定した方法：S 特徴を表わす記号：3´UTR 存在位置：3003..3073 特徴を決定した方法：E 配列 GATCCGGACG GCAACCTCAT GTCCCCGGAG GACTGGGACA GCGGCTTCGG CCGTTCGGTG 60 GGCATGTTCC TCAACGGCGA CGGCATCCAG GGCCACGATG ACCGCGGCCG CCGCATCACG 120 GACGTGAACT TCCTGCTGTA CTTCAACGCC CACGACGGCG ACGTCGAGTT CACGCTGCCG 180 CCGGACGAAT ACGCCCCGGC CTGGGACGTC ATCATCGACA CCGCCGGTGA AGGGGCCGAC 240 TCCAAGCCCG CGGACGCCGG AACCATCCTG TCCGTTGCGG CCAAGTCGCT GGTTGTGCTT 300 CGCGCCCACA GCGCACCGGA GGAGGAGCCT GACCATTCCG TGGCTGCTTC CCTGGCTGCA 360 CTGACGCAGA CCGCCACCGC CGAGACGGCG GCGCTCACAG CTCCTGCCGT TCCCGAGCCG 420 GCCAAGACGA AGAAGCCGGC CGCTGACCCG GTTGCTGAAC CGGCCGACCC GCCGGTTGCT 480 GACCCGGCCG ACCCGGTTGC TGACCCGGTT GCTGACCCGG CGCCGGAACC GGCTGCGGAG 540 CCTGCGAAAT CCGCAGCGGA ACCTGGTGCG GAGCCTGCGA AGGACCCGGA GGAGCAGCCG 600 GCGGAAAAGC CGGCGCGCAA GCCTGCGGCA AAGCGCGGCG GCCACCTGAG GGCGGTCAAG 660 CCCGCTGGGG AGGACGC 677 ATG AGA ACG CCA GTC TCC ACG TAC AGG CTG CAG ATC AGG AAG GGA TTC 725 Met Arg Thr Pro Val Ser Thr Tyr Arg Leu Gln Ile Arg Lys Gly Phe 1 5 10 15 ACA CTC TTC GAC GCG GCC AAA ACC GTT CCG TAC CTG CAC TCG CTC GGC 773 Thr Leu Phe Asp Ala Ala Lys Thr Val Pro Tyr Leu His Ser Leu Gly 20 25 30 GTC GAC TGG GTC TAC CTT TCT CCG GTC CTG ACT GCC GAG CAG GGC TCC 821 Val Asp Trp Val Tyr Leu Ser Pro Val Leu Thr Ala Glu Gln Gly Ser 35 40 45 GAC CAC GGG TAC GAC GTC ACC GAT CCC TCC GCC GTC GAC CCC GAA CGC 869 Asp His Gly Tyr Asp Val Thr Asp Pro Ser Ala Val Asp Pro Glu Arg 50 55 60 GGC GGG CCG GAG GGC CTC GCG GCG GTT TCC AAG GCG GCC CGC GCC GCG 917 Gly Gly Pro Glu Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Ala Ala 65 70 75 80 GGC ATG GGC GTG CTG ATC GAC ATC GTG CCC AAC CAC GTG GGC GTC GCG 965 Gly Met Gly Val Leu Ile Asp Ile Val Pro Asn His Val Gly Val Ala 85 90 95 ACG CCG GCG CAG AAC CCC TGG TGG TGG TCG CTG CTC AAG GAG GGA CGC 1013 Thr Pro Ala Gln Asn Pro Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg 100 105 110 CAG TCC CGT TAC GCG GAG GCG TTC GAC GTC GAT TGG GAC CTC GCC GGG 1061 Gln Ser Arg Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu Ala Gly 115 120 125 GGA CGC ATC CGG CTG CCG GTG CTC GGC AGC GAC GAT GAC CTC GAC CAG 1109 Gly Arg Ile Arg Leu Pro Val Leu Gly Ser Asp Asp Asp Leu Asp Gln 130 135 140 CTC GAA ATC AGG GAC GGG GAG CTG CGG TAC TAC GAC CAC CGA TTC CCG 1157 Leu Glu Ile Arg Asp Gly Glu Leu Arg Tyr Tyr Asp His Arg Phe Pro 145 150 155 160 CTC GCC GAG GGA ACC TAC GCC GAA GGC GAC GCC CCG CGG GAT GTC CAC 1205 Leu Ala Glu Gly Thr Tyr Ala Glu Gly Asp Ala Pro Arg Asp Val His 165 170 175 GCC CGG CAG CAC TAC GAG CTC ATC GGC TGG CGC CGC GCG GAC AAC GAG 1253 Ala Arg Gln His Tyr Glu Leu Ile Gly Trp Arg Arg Ala Asp Asn Glu 180 185 190 CTG AAC TAC CGC CGC TTT TTC GCG GTG AAC ACG CTC GCC GGC GTC CGC 1301 Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu Ala Gly Val Arg 195 200 205 GTG GAA ATC CCC GCC GTC TTC GAC GAG GCA CAC CAG GAG GTG GTG CGC 1349 Val Glu Ile Pro Ala Val Phe Asp Glu Ala His Gln Glu Val Val Arg 210 215 220 TGG TTC CGC GAG GAC CTT GCG GAC GGC CTG CGG ATC GAC CAC CCG GAC 1397 Trp Phe Arg Glu Asp Leu Ala Asp Gly Leu Arg Ile Asp His Pro Asp 225 230 235 240 GGC CTC GCT GAC CCC GAG GGG TAC CTG AAG CGA CTC CGG GAA GTC ACC 1445 Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val Thr 245 250 255 GGC GGC GCT TAC CTG CTG ATC GAA AAG ATC CTG GAG CCG GGG GAG CAG 1493 Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 CTG CCC GCC AGC TTC GAG TGT GAA GGC ACC ACA GGC TAC GAC GCC CTC 1541 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu 275 280 285 GCC GAC GTC GAC CGG GTT CTC GTG GAC CCG CGC GGC CAG GAA CCG CTG 1589 Ala Asp Val Asp Arg Val Leu Val Asp Pro Arg Gly Gln Glu Pro Leu 290 295 300 GAC CGG CTT GAC GCG TCC CTG CGT GGC GGC GAG CCC GCC GAC TAC CAG 1637 Asp Arg Leu Asp Ala Ser Leu Arg Gly Gly Glu Pro Ala Asp Tyr Gln 305 310 315 320 GAC ATG ATC CGC GGA ACC AAG CGC CGG ATC ACC GAC GGT ATC CTG CAC 1685 Asp Met Ile Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His 325 330 335 TCG GAG ATC CTG CGG CTG GCC CGG CTG GTT CCG GGC GAC GCC AAC GTT 1733 Ser Glu Ile Leu Arg Leu Ala Arg Leu Val Pro Gly Asp Ala Asn Val 340 345 350 TCA ATC GAC GCC GGA GCC GAC GCT CTC GCC GAA ATC ATC GCC GCC TTC 1781 Ser Ile Asp Ala Gly Ala Asp Ala Leu Ala Glu Ile Ile Ala Ala Phe 355 360 365 CCG GTC TAC CGC ACC TAC CTG CCG GAG GGC GCC GAG GTC CTG AAG GAG 1829 Pro Val Tyr Arg Thr Tyr Leu Pro Glu Gly Ala Glu Val Leu Lys Glu 370 375 380 GCG TGC GAG CTT GCC GCG CGT AGG CGG CCG GAA CTC GAC CAG GCC ATC 1877 Ala Cys Glu Leu Ala Ala Arg Arg Arg Pro Glu Leu Asp Gln Ala Ile 385 390 395 400 CAG GCT CTG CAG CCG CTG CTG CTG GAC ACG GAC CTC GAG CTT GCC CGG 1925 Gln Ala Leu Gln Pro Leu Leu Leu Asp Thr Asp Leu Glu Leu Ala Arg 405 410 415 CGC TTC CAG CAG ACC TCG GGC ATG GTC ATG GCC AAG GGC GTG GAG GAC 1973 Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu Asp 420 425 430 ACC GCG TTC TTC CGC TAC AAC CGC CTG GGC ACC CTC ACG GAA GTG GGC 2021 Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly Thr Leu Thr Glu Val Gly 435 440 445 GCC GAC CCC ACC GAG TTC GCC GTG GAG CCG GAC GAG TTC CAC GCC CGG 2069 Ala Asp Pro Thr Glu Phe Ala Val Glu Pro Asp Glu Phe His Ala Arg 450 455 460 CTG GCA CGC CGG CAG GCC GAG CTT CCG CTG TCC ATG ACG ACG CTG AGC 2117 Leu Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met Thr Thr Leu Ser 465 470 475 480 ACG CAC GAC ACC AAG CGC AGC GAG GAC ACC CGA GCA AGG ATT TCG GTC 2165 Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val 485 490 495 ATT TCC GAG GTT GCG GGT GAC TGG GAA AAG GCC TTG AAC CGG CTG CGC 2213 Ile Ser Glu Val Ala Gly Asp Trp Glu Lys Ala Leu Asn Arg Leu Arg 500 505 510 GAC CTG GCC CCG CTG CCG GAC GGC CCG CTG TCC GCG CTG CTC TGG CAG 2261 Asp Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Ala Leu Leu Trp Gln 515 520 525 GCC ATT GCC GGC GCC TGG CCC GCC AGC CGG GAA CGC CTG CAG TAC TAC 2309 Ala Ile Ala Gly Ala Trp Pro Ala Ser Arg Glu Arg Leu Gln Tyr Tyr 530 535 540 GCG CTG AAG GCC GCG CGT GAA GCG GGG AAC TCG ACC AAC TGG ACC GAT 2357 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Asn Trp Thr Asp 545 550 555 560 CCG GCC CCC GCG TTC GAG GAG AAG CTG AAG GCC GCG GTC GAC GCC GTG 2405 Pro Ala Pro Ala Phe Glu Glu Lys Leu Lys Ala Ala Val Asp Ala Val 565 570 575 TTC GAC AAT CCC GCC GTG CAG GCC GAG GTG GAA GCC CTC GTC GAG CTC 2453 Phe Asp Asn Pro Ala Val Gln Ala Glu Val Glu Ala Leu Val Glu Leu 580 585 590 CTG GAG CCG TAC GGA GCT TCG AAC TCC CTC GCC GCC AAG CTC GTG CAG 2501 Leu Glu Pro Tyr Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln 595 600 605 CTG ACC ATG CCC GGC GTC CCG GAC GTC TAC CAG GGC ACG GAG TTC TGG 2549 Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp 610 615 620 GAC CGG TCG CTG ACG GAC CCG GAC AAC CGG CGG CCG TTC AGC TTC GAC 2597 Asp Arg Ser Leu Thr Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Asp 625 630 635 640 GAC CGC CGC GCC GCG CTG GAG CAG CTG GAT GCC GGC GAC CTT CCC GCG 2645 Asp Arg Arg Ala Ala Leu Glu Gln Leu Asp Ala Gly Asp Leu Pro Ala 645 650 655 TCA TTT ACC GAT GAG CGG ACG AAG CTG CTA GTG ACG TCG CGC GCG CTG 2693 Ser Phe Thr Asp Glu Arg Thr Lys Leu Leu Val Thr Ser Arg Ala Leu 660 665 670 CGG CTG CGC CGG GAC CGT CCG GAG CTG TTC ACG GGG TAC CGG CCG GTC 2741 Arg Leu Arg Arg Asp Arg Pro Glu Leu Phe Thr Gly Tyr Arg Pro Val 675 680 685 CTG GCC AGC GGG CCC GCC GCC GGG CAC CTG CTC GCG TTC GAC CGC GGC 2789 Leu Ala Ser Gly Pro Ala Ala Gly His Leu Leu Ala Phe Asp Arg Gly 690 695 700 ACC GCG GCG GCG CCG GGT GCA TTG ACC CTC GCC ACG CGG CTT CCC TAC 2837 Thr Ala Ala Ala Pro Gly Ala Leu Thr Leu Ala Thr Arg Leu Pro Tyr 705 710 715 720 GGG CTG GAA CAG TCG GGT GGA TGG CGG GAC ACC GCC GTC GAA CTT AAC 2885 Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu Leu Asn 725 730 735 ACC GCC ATG AAA GAC GAA CTG ACC GGT GCC GGC TTC GGA CCG GGG GCA 2933 Thr Ala Met Lys Asp Glu Leu Thr Gly Ala Gly Phe Gly Pro Gly Ala 740 745 750 GTG AAG ATC GCC GAC ATC TTC CGG TCG TTC CCC GTT GCG CTG CTG GTG 2981 Val Lys Ile Ala Asp Ile Phe Arg Ser Phe Pro Val Ala Leu Leu Val 755 760 765 CCG CAG ACA GGA GGA GAG TCA 3002 Pro Gln Thr Gly Gly Glu Ser 770 775 TGACGCACAC CTACCCGCGG GAAGCCGCGA AACCCGTCCT GGGCCCCGCA CGCTACGACG 3063 TCTGGGCGCC C 3073SEQ ID NO: 6 Sequence length: 3084 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Sequence features Origin Biological name: Arthrobacter species (Arthrobacte species)
r sp.) Strain name: Q36 (FERM BP-4316) Sequence features Characteristic signature: 5'UTR Location: 1..677 Method of determining feature: E Characteristic signature: mat peptide Location: 678 ..3002 Method of determining features: S Characteristic symbol: 3'UTR Location: 3003..3073 Method of determining features: E array GATCCGGACG GCAACCTCAT GTCCCCGGAG GACTGGGACA GCGGCTTCGG CCGTTCGGTG 60 GGCATGTTCCTCCAAACGGCGACGGGCATCCAGGAGGGCCGCTCATCGACCGTG CACGACGGCG ACGTCGAGTT CACGCTGCCG 180 CCGGACGAAT ACGCCCCGGC CTGGGACGTC ATCATCGACA CCGCCGGTGA AGGGGCCGAC 240 TCCAAGCCCG CGGACGCCGG AACCATCCTG TCCGTTGCGG CCAAGTCGCT GGTTGTGCTT 300 CGCGCCCACA GCGCACCGGA GGAGGAGCCT GACCATTCCG TGGCTGCTTC CCTGGCTGCA 360 CTGACGCAGA CCGCCACCGC CGAGACGGCG GCGCTCACAG CTCCTGCCGT TCCCGAGCCG 420 GCCAAGACGA AGAAGCCGGC CGCTGACCCG GTTGCTGAAC CGGCCGACCC GCCGGTTGCT 480 GACCCGGCCG ACCCGGTTGC TGACCCGGTT GCTGACCCGG CGCCGGAACC GGCTGCGGAG 540 CCTGCGAAAT CCGCAGCGGA ACCTGGTGCG GAGCC TGCGA AGGACCCGGA GGAGCAGCCG 600 GCGGAAAAGC CGGCGCGCAA GCCTGCGGCA AAGCGCGGCG GCCACCTGAG GGCGGTCAAG 660 CCCGCTGGGG AGGACGC 677 ATG AGA ACG CCA GTC TCC ACG TAC AGG CTG CAg ATG Cle Gru Tg Arle Glu TTC Arg 15 ACA CTC TTC GAC GCG GCC AAA ACC GTT CCG TAC CTG CAC TCG CTC GGC 773 Thr Leu Phe Asp Ala Ala Lys Thr Val Pro Tyr Leu His Ser Leu Gly 20 25 30 GTC GAC TGG GTC TAC CTT TCT CCG GTC CTG ACT GCC GAG CAG GGC TCC 821 Val Asp Trp Val Tyr Leu Ser Pro Val Leu Thr Ala Glu Gln Gly Ser 35 40 45 GAC CAC GGG TAC GAC GTC ACC GAT CCC TCC GCC GTC GAC CCC GAA CGC 869 Asp His Gly Tyr Asp Val Thr Asp Pro Ser Ala Val Asp Pro Glu Arg 50 55 60 GGC GGG CCG GAG GGC CTC GCG GCG GTT TCC AAG GCG GCC CGC GCC GCG 917 Gly Gly Pro Glu Gly Leu Ala Ala Val Ser Lys Ala Ala Arg Ala Ala 65 70 75 80 GGC ATG GGC GTG CTG ATC GAC ATC GTG CCC AAC CAC GTG GGC GTC GCG 965 Gly Met Gly Val Leu Ile Asp Ile Val Pro Asn His Val Gly Val Ala 85 90 95 ACG CCG GCG CAG AAC CCC T GG TGG TGG TCG CTG CTC AAG GAG GGA CGC 1013 Thr Pro Ala Gln Asn Pro Trp Trp Trp Ser Leu Leu Lys Glu Gly Arg 100 105 110 CAG TCC CGT TAC GCG GAG GCG TTC GAC GTC GAT TGG GAC CTC GCC GGG 1061 Gln Ser Arg Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu Ala Gly 115 120 125 GGA CGC ATC CGG CTG CCG GTG CTC GGC AGC GAC GAT GAC CTC GAC CAG 1109 Gly Arg Ile Arg Leu Pro Val Leu Gly Ser Asp Asp Asp Leu Asp Gln 130 135 140 CTC GAA ATC AGG GAC GGG GAG CTG CGG TAC TAC GAC CAC CGA TTC CCG 1157 Leu Glu Ile Arg Asp Gly Glu Leu Arg Tyr Tyr Asp His Arg Phe Pro 145 150 155 160 CTC GCC GAG GGA ACC TAC GCC GAA GGC GAC GCC CCG CGG GAT GTC CAC 1205 Leu Ala Glu Gly Thr Tyr Ala Glu Gly Asp Ala Pro Arg Asp Val His 165 170 175 GCC CGG CAG CAC TAC GAG CTC ATC GGC TGG CGC CGC GCG GAC AAC GAG 1253 Ala Arg Gln His Tyr Glu Leu Ile Gly Trp Arg Arg Ala Asp Asn Glu 180 185 190 CTG AAC TAC CGC CGC TTT TTC GCG GTG AAC ACG CTC GCC GGC GTC CGC 1301 Leu Asn Tyr Arg Arg Phe Phe Ala Val Asn Thr Leu Ala Gly Val Arg 195 200 205 GTG GA A ATC CCC GCC GTC TTC GAC GAG GCA CAC CAG GAG GTG GTG CGC 1349 Val Glu Ile Pro Ala Val Phe Asp Glu Ala His Gln Glu Val Val Arg 210 215 220 TGG TTC CGC GAG GAC CTT GCG GAC GGC CTG CGG ATC GAC CAC CCG GAC 1397 Trp Phe Arg Glu Asp Leu Ala Asp Gly Leu Arg Ile Asp His Pro Asp 225 230 235 240 GGC CTC GCT GAC CCC GAG GGG TAC CTG AAG CGA CTC CGG GAA GTC ACC 1445 Gly Leu Ala Asp Pro Glu Gly Tyr Leu Lys Arg Leu Arg Glu Val Thr 245 250 255 GGC GGC GCT TAC CTG CTG ATC GAA AAG ATC CTG GAG CCG GGG GAG CAG 1493 Gly Gly Ala Tyr Leu Leu Ile Glu Lys Ile Leu Glu Pro Gly Glu Gln 260 265 270 CTG CCC GCC AGC TTC GAG TGT GAA GGC ACC ACA GGC TAC GAC GCC CTC 1541 Leu Pro Ala Ser Phe Glu Cys Glu Gly Thr Thr Gly Tyr Asp Ala Leu 275 280 285 GCC GAC GTC GAC CGG GTT CTC GTG GAC CCG CGC GGC CAG GAA CCG CTG 1589 Ala Asp Val Asp Arg Val Leu Val Asp Pro Arg Gly Gln Glu Pro Leu 290 295 300 GAC CGG CTT GAC GCG TCC CTG CGT GGC GGC GAG CCC GCC GAC TAC CAG 1637 Asp Arg Leu Asp Ala Ser Leu Arg Gly Gly Glu Pro Ala Asp Tyr Gln 305 310 315 320 GAC ATG ATC CGC GGA ACC AAG CGC CGG ATC ACC GAC GGT ATC CTG CAC 1685 Asp Met Ile Arg Gly Thr Lys Arg Arg Ile Thr Asp Gly Ile Leu His 325 330 335 TCG GAG ATC CTG CGG CTG GCC CGG CTG GTT CCG GGC GAC GCC AAC GTT 1733 Ser Glu Ile Leu Arg Leu Ala Arg Leu Val Pro Gly Asp Ala Asn Val 340 345 350 TCA ATC GAC GCC GGA GCC GAC GCT CTC GCC GAA ATC ATC GCC GCC TTC 1781 Ser Ile Asp Ala Gly Ala Asp Ala Leu Ala Glu Ile Ile Ala Ala Phe 355 360 365 CCG GTC TAC CGC ACC TAC CTG CCG GAG GGC GCC GAG GTC CTG AAG GAG 1829 Pro Val Tyr Arg Thr Tyr Leu Pro Glu Gly Ala Glu Val Leu Lys Glu 370 375 380 GCG TGC GAG CTT GCC GCG CGT AGG CGG CCG GAA CTC GAC CAG GCC ATC 1877 Ala Cys Glu Leu Ala Ala Arg Arg Arg Pro Glu Leu Asp Gln Ala Ile 385 390 395 400 CAG GCT CTG CAG CCG CTG CTG CTG GAC ACG GAC CTC GAG CTT GCC CGG 1925 Gln Ala Leu Gln Pro Leu Leu Leu Asp Thr Asp Leu Glu Leu Ala Arg 405 410 415 CGC TTC CAG CAG ACC TCG GGC ATG GTC ATG GCC AAG GGC GTG GAG GAC 1973 Arg Phe Gln Gln Thr Ser Gly Met Val Met Ala Lys Gly Val Glu Asp 420 425 430 ACC GCG TTC TTC CGC TAC AAC CGC CTG GGC ACC CTC ACG GAA GTG GGC 2021 Thr Ala Phe Phe Arg Tyr Asn Arg Leu Gly Thr Leu Thr Glu Val Gly 435 440 445 GCC GAC CCC ACC GAG TTC GCC GTG GAG CCG GAC GAG TTC CAC GCC CGG 2069 Ala Asp Pro Thr Glu Phe Ala Val Glu Pro Asp Glu Phe His Ala Arg 450 455 460 CTG GCA CGC CGG CAG GCC GAG CTT CCG CTG TCC ATG ACG ACG CTG AGC 2117 Leu Ala Arg Arg Gln Ala Glu Leu Pro Leu Ser Met Thr Thr Leu Ser 465 470 475 480 ACG CAC GAC ACC AAG CGC AGC GAG GAC ACC CGA GCA AGG ATT TCG GTC 2165 Thr His Asp Thr Lys Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val 485 490 495 ATT TCC GAG GTT GCG GGT GAC TGG GAA AAG GCC TTG AAC CGG CTG CGC 2213 Ile Ser Glu Val Ala Gly Asp Trp Glu Lys Ala Leu Asn Arg Leu Arg 500 505 510 GAC CTG GCC CCG CTG CCG GAC GGC CCG CTG TCC GCG CTG CTC TGG CAG 2261 Asp Leu Ala Pro Leu Pro Asp Gly Pro Leu Ser Ala Leu Leu Trp Gln 515 520 525 GCC ATT GCC GGC GCC TGG CCC GCC AGC CGG GAA CGC CTG CAG TAC TAC 2309 Ala Ile Ala Gly Ala T rp Pro Ala Ser Arg Glu Arg Leu Gln Tyr Tyr 530 535 540 GCG CTG AAG GCC GCG CGT GAA GCG GGG AAC TCG ACC AAC TGG ACC GAT 2357 Ala Leu Lys Ala Ala Arg Glu Ala Gly Asn Ser Thr Asn Trp Thr Asp 545 550 555 560 CCG GCC CCC GCG TTC GAG GAG AAG CTG AAG GCC GCG GTC GAC GCC GTG 2405 Pro Ala Pro Ala Phe Glu Glu Lys Leu Lys Ala Ala Val Asp Ala Val 565 570 575 TTC GAC AAT CCC GCC GTG CAG GCC GAG GTG GAA GCC CTC GTC GAG CTC 2453 Phe Asp Asn Pro Ala Val Gln Ala Glu Val Glu Ala Leu Val Glu Leu 580 585 590 CTG GAG CCG TAC GGA GCT TCG AAC TCC CTC GCC GCC AAG CTC GTG CAG 2501 Leu Glu Pro Tyr Gly Ala Ser Asn Ser Leu Ala Ala Lys Leu Val Gln 595 600 605 CTG ACC ATG CCC GGC GTC CCG GAC GTC TAC CAG GGC ACG GAG TTC TGG 2549 Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu Phe Trp 610 615 620 GAC CGG TCG CTG ACG GAC CCG GAC AAC CGG CGG CCG TTC AGC TTC GAC 2597 Asp Arg Ser Leu Thr Asp Pro Asp Asn Arg Arg Pro Phe Ser Phe Asp 625 630 635 640 GAC CGC CGC GCC GCG CTG GAG CAG CTG GAT GCC GGC GAC CTT CCC GCG 264 5 Asp Arg Arg Ala Ala Leu Glu Gln Leu Asp Ala Gly Asp Leu Pro Ala 645 650 655 TCA TTT ACC GAT GAG CGG ACG AAG CTG CTA GTG ACG TCG CGC GCG CTG 2693 Ser Phe Thr Asp Glu Arg Thr Lys Leu Leu Val Thr Ser Arg Ala Leu 660 665 670 CGG CTG CGC CGG GAC CGT CCG GAG CTG TTC ACG GGG TAC CGG CCG GTC 2741 Arg Leu Arg Arg Asp Arg Pro Glu Leu Phe Thr Gly Tyr Arg Pro Val 675 680 685 CTG GCC AGC GGG CCC GCC GCC GGG CAC CTG CTC GCG TTC GAC CGC GGC 2789 Leu Ala Ser Gly Pro Ala Ala Gly His Leu Leu Ala Phe Asp Arg Gly 690 695 700 ACC GCG GCG GCG CCG GGT GCA TTG ACC CTC GCC ACG CGG CTT CCC TAC 2837 Thr Ala Ala Ala Ala Pro Gly Ala Leu Thr Leu Ala Thr Arg Leu Pro Tyr 705 710 715 720 GGG CTG GAA CAG TCG GGT GGA TGG CGG GAC ACC GCC GTC GAA CTT AAC 2885 Gly Leu Glu Gln Ser Gly Gly Trp Arg Asp Thr Ala Val Glu Leu Asn 725 730 735 ACC GCC ATG AAA GAC GAA CTG ACC GGT GCC GGC TTC GGA CCG GGG GCA 2933 Thr Ala Met Lys Asp Glu Leu Thr Gly Ala Gly Phe Gly Pro Gly Ala 740 745 750 GTG AAG ATC GCC GAC ATC TTC CGG TCG TTC CCC GTT GCG CTG CTG GTG 2981 Val Lys Ile Ala Asp Ile Phe Arg Ser Phe Pro Val Ala Leu Leu Val 755 760 765 CCG CAG ACA GGA GGA GAG TCA 3002 Pro Gln Thr Gly Gly Glu Ser 770 775 TGACGCACCC CTAC CCGCGG GAAGCCGCGA AACCCGTCCT CG 3073

【０１０７】配列番号：7 配列の長さ：20 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：Ｎ末端フラグメント 配列 Met Arg Thr Pro Ala Ser Thr Tyr Arg Leu Gln Ile Arg Arg Gly Phe Thr 1 5 10 15 Leu Phe Asp 20SEQ ID NO: 7 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence Met Arg Thr Pro Ala Ser Thr Tyr Arg Leu Gln Ile Arg Arg Gly Phe Thr 1 5 10 15 Leu Phe Asp 20

【０１０８】配列番号：8 配列の長さ：20 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：Ｎ末端フラグメント 配列 Met Arg Thr Pro Val Ser Thr Tyr Arg Leu Gln Ile Arg Lys Gly Phe Thr 1 5 10 15 Leu Phe Asp 20SEQ ID NO: 8 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence Met Arg Thr Pro Val Ser Thr Tyr Arg Leu Gln Ile Arg Lys Gly Phe Thr 1 5 10 15 Leu Phe Asp 20

【０１０９】配列番号：9 配列の長さ：21 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント 配列 Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val Ile Ala Glu Val Ala Pro 1 5 10 15 Glu Trp Glu Lys 20SEQ ID NO: 9 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence Arg Ser Glu Asp Thr Arg Ala Arg Ile Ser Val Ile Ala Glu Val Ala Pro 1 5 10 15 Glu Trp Glu Lys 20

【０１１０】配列番号：10 配列の長さ：21 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント 配列 Leu Val Gln Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu 1 5 10 15 Phe Trp Asp Arg 20SEQ ID NO: 10 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence Leu Val Gln Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu 1 5 10 15 Phe Trp Asp Arg 20

【０１１１】配列番号：11 配列の長さ：20 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント 配列 Leu Val Gln Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu 1 5 10 15 Phe Trp Asp 20SEQ ID NO: 11 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence Leu Val Gln Leu Thr Met Pro Gly Val Pro Asp Val Tyr Gln Gly Thr Glu 1 5 10 15 Phe Trp Asp 20

【０１１２】配列番号：12 配列の長さ：20 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド フラグメント型：中間部フラグメント 配列 Glu Gly Arg Gln Ser Arg Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu 1 5 10 15 Ala Gly Gly 20SEQ ID NO: 12 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment Sequence Glu Gly Arg Gln Ser Arg Tyr Ala Glu Ala Phe Asp Val Asp Trp Asp Leu 1 5 10 15 Ala Gly Gly 20

【図面の簡単な説明】[Brief description of drawings]

【図１】酵素Ｍ−１１の至適温度を示す図である。FIG. 1 is a diagram showing the optimum temperature of enzyme M-11.

【図２】酵素Ｑ３６の至適温度を示す図である。FIG. 2 is a diagram showing the optimum temperature of enzyme Q36.

【図３】酵素Ｍ−１１の至適ｐＨを示す図である。FIG. 3 is a graph showing the optimum pH of enzyme M-11.

【図４】酵素Ｑ３６の至適ｐＨを示す図である。FIG. 4 is a diagram showing the optimum pH of enzyme Q36.

【図５】酵素Ｍ−１１の熱安定性を示す図である。FIG. 5 is a diagram showing thermostability of enzyme M-11.

【図６】酵素Ｑ３６の熱安定性を示す図である。FIG. 6 shows the thermostability of enzyme Q36.

【図７】酵素Ｍ−１１のｐＨ安定性を示す図である。FIG. 7 is a graph showing pH stability of enzyme M-11.

【図８】酵素Ｑ３６のｐＨ安定性を示す図である。FIG. 8 is a graph showing pH stability of enzyme Q36.

【図９】この発明による組換えＤＮＡであるｐＢＭＴ７
の制限酵素地図である。なお、図中、太線表示部は、酵
素をコードするＤＮＡを示す。FIG. 9: Recombinant DNA pBMT7 according to the present invention
Is a restriction enzyme map of. It should be noted that in the figure, the bold-lined portion indicates the DNA encoding the enzyme.

【図１０】この発明による組換えＤＮＡであるｐＢＱＴ
１３の制限酵素地図である。なお、図中、太線表示部
は、酵素をコードするＤＮＡを示す。FIG. 10: Recombinant DNA pBQT according to the present invention
13 is a restriction enzyme map of 13. It should be noted that in the figure, the bold-lined portion indicates the DNA encoding the enzyme.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 //(Ｃ１２Ｎ 15/09 ＺＮＡ Ｃ１２Ｒ 1:41） (Ｃ１２Ｎ 9/24 Ｃ１２Ｒ 1:19） Ｃ１２Ｒ 1:41） ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification number Internal reference number FI Technical display area // (C12N 15/09 ZNA C12R 1:41) (C12N 9/24 C12R 1:19) C12R 1 : 41)

Claims (22)

【特許請求の範囲】[Claims] 【請求項１】 グルコース重合度３以上の還元性澱粉糖
から末端にトレハロース構造を有する非還元性糖質を生
成する組換え型酵素。1. A recombinant enzyme that produces a non-reducing sugar having a trehalose structure at the end from a reducing starch sugar having a glucose polymerization degree of 3 or more. 【請求項２】 下記の理化学的性質を有する請求項１に
記載の組換え型酵素。 （１） 分子量 約７６，０００乃至８７，０００ダルトン（ＳＤＳ−ポ
リアクリルアミドゲル電気泳動） （２） 等電点 約３．６乃至４．６（等電点電気泳動）2. The recombinant enzyme according to claim 1, which has the following physicochemical properties. (1) Molecular weight of about 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point of about 3.6 to 4.6 (isoelectric focusing) 【請求項３】 配列表における配列番号１又は２に示す
アミノ酸配列かそれに相同的なアミノ酸配列を有する請
求項１又は２に記載の組換え型酵素。3. The recombinant enzyme according to claim 1 or 2, which has the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing or an amino acid sequence homologous thereto. 【請求項４】 請求項１に記載の組換え型酵素を産生す
る形質転換体を培養し、培養物から組換え型酵素を採取
する組換え型酵素の製造方法。4. A method for producing a recombinant enzyme, which comprises culturing the transformant producing the recombinant enzyme according to claim 1 and collecting the recombinant enzyme from the culture. 【請求項５】 組換え型酵素が下記の理化学的性質を有
する請求項４に記載の組換え型酵素の製造方法。 （１） 分子量 約７６，０００乃至８７，０００ダルトン（ＳＤＳ−ポ
リアクリルアミドゲル電気泳動） （２） 等電点 約３．６乃至４．６（等電点電気泳動）5. The method for producing a recombinant enzyme according to claim 4, wherein the recombinant enzyme has the following physicochemical properties. (1) Molecular weight of about 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point of about 3.6 to 4.6 (isoelectric focusing) 【請求項６】 組換え型酵素が配列表における配列番号
１又は２に示すアミノ酸配列かそれに相同的なアミノ酸
配列を有する請求項４又は５に記載の組換え型酵素の製
造方法。6. The method for producing a recombinant enzyme according to claim 4 or 5, wherein the recombinant enzyme has the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing or an amino acid sequence homologous thereto. 【請求項７】 形質転換体が、グルコース重合度３以上
の還元性澱粉糖から末端にトレハロース構造を有する非
還元性糖質を生成する酵素をコードするＤＮＡと自律複
製可能なベクターを含む組換えＤＮＡを適宜宿主に導入
してなる請求項４、５又は６に記載の組換え型酵素の製
造方法。7. A recombinant containing a vector capable of autonomous replication and a DNA encoding an enzyme that produces a non-reducing sugar having a trehalose structure at the end from a reducing starch sugar having a glucose polymerization degree of 3 or more. The method for producing a recombinant enzyme according to claim 4, 5 or 6, which comprises appropriately introducing DNA into a host. 【請求項８】 ＤＮＡが配列表における配列番号３又は
４に示す塩基配列かそれに相同的な塩基配列又はそれら
に相補的な塩基配列を有する請求項７に記載の組換え型
酵素の製造方法。8. The method for producing a recombinant enzyme according to claim 7, wherein the DNA has a base sequence shown in SEQ ID NO: 3 or 4 in the sequence listing, a base sequence homologous thereto, or a base sequence complementary thereto. 【請求項９】 ＤＮＡが、遺伝子コードの縮重に基づ
き、配列表における配列番号１又は２に示すアミノ酸配
列を変えることなく、配列表における配列番号３又は４
に示す塩基配列における塩基の１個又は２個以上を他の
塩基で置換したものである請求項７又は８に記載の組換
え型酵素の製造方法。9. A DNA having SEQ ID NO: 3 or 4 in the Sequence Listing without changing the amino acid sequence shown in the SEQ ID NO: 1 or 2 in the Sequence Listing based on the degeneracy of the genetic code.
The method for producing a recombinant enzyme according to claim 7 or 8, wherein one or more of the bases in the base sequence shown in (1) is replaced with another base. 【請求項１０】 ＤＮＡが配列表における配列番号５又
は６に示す塩基配列を有する請求項７、８又は９に記載
の組換え型酵素の製造方法。10. The method for producing a recombinant enzyme according to claim 7, 8 or 9, wherein the DNA has the base sequence shown in SEQ ID NO: 5 or 6. 【請求項１１】 ＤＮＡがリゾビウム属、アルスロバク
ター属、ブレビバクテリウム属、フラボバクテリウム
属、ミクロコッカス属、クルトバクテリウム属、マイコ
バクテリウム属又はテラバクター属の微生物に由来する
請求項７、８、９又は１０に記載の組換え型酵素の製造
方法。11. The method according to claim 7, wherein the DNA is derived from a microorganism of the genus Rhizobium, Arthrobacter, Brevibacterium, Flavobacterium, Micrococcus, Curtobacterium, Mycobacterium or Terrabactor. The method for producing the recombinant enzyme according to 8, 9, or 10. 【請求項１２】 宿主が大腸菌である請求項７、８、
９、１０又は１１に記載の組換え型酵素の製造方法。12. The method according to claim 7, wherein the host is Escherichia coli.
The method for producing the recombinant enzyme according to 9, 10, or 11. 【請求項１３】 自律複製可能なベクターがプラスミド
ベクターＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）であ
る請求項７、８、９、１０、１１又は１２に記載の組換
え型酵素の製造方法。13. The method for producing the recombinant enzyme according to claim 7, 8, 9, 10, 11 or 12, wherein the vector capable of autonomous replication is a plasmid vector Bluescript II SK (+). 【請求項１４】 形質転換体を炭素源及び窒素源を含む
ｐＨ２乃至８の液体培地に植菌し、温度２５乃至６５℃
で１乃至６日間培養する請求項４、５、６、７、８、
９、１０、１１、１２又は１３に記載の組換え型酵素の
製造方法。14. The transformant is inoculated into a liquid medium having a carbon source and a nitrogen source and having a pH of 2 to 8, and the temperature is 25 to 65 ° C.
Culturing for 1 to 6 days at 4, 5, 6, 7, 8,
The method for producing the recombinant enzyme according to 9, 10, 11, 12 or 13. 【請求項１５】 培養物中の組換え型酵素を遠心分離、
濾過、濃縮、塩析、透析、イオン交換クロマトグラフィ
ー、ゲル濾過クロマトグラフィー、疎水クロマトグラフ
ィー、アフィニティークロマトグラフィー、ゲル電気泳
動及び／又は等電点電気泳動により採取する請求項４、
５、６、７、８、９、１０、１１、１２、１３又は１４
に記載の組換え型酵素の製造方法。15. Centrifuging the recombinant enzyme in the culture,
5. Collection by filtration, concentration, salting out, dialysis, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, gel electrophoresis and / or isoelectric focusing.
5, 6, 7, 8, 9, 10, 11, 12, 13 or 14
The method for producing the recombinant enzyme according to 1. 【請求項１６】 グルコース重合度３以上の還元性澱粉
糖に請求項１に記載の組換え型酵素を作用させて該澱粉
糖から末端にトレハロース構造を有する非還元性糖質を
生成させる工程を含んでなる還元性澱粉糖の変換方法。16. A step of reacting a reducing starch sugar having a glucose polymerization degree of 3 or more with the recombinant enzyme according to claim 1 to produce a non-reducing sugar having a trehalose structure at the terminal from the starch sugar. A method for converting reducing starch sugars comprising. 【請求項１７】 組換え型酵素が下記の理化学的性質を
有する請求項１６に記載の還元性澱粉糖の変換方法。 （１） 分子量 約７６，０００乃至８７，０００ダルトン（ＳＤＳ−ポ
リアクリルアミドゲル電気泳動） （２） 等電点 約３．６乃至４．６（等電点電気泳動）17. The method for converting reducing starch sugar according to claim 16, wherein the recombinant enzyme has the following physicochemical properties. (1) Molecular weight of about 76,000 to 87,000 daltons (SDS-polyacrylamide gel electrophoresis) (2) Isoelectric point of about 3.6 to 4.6 (isoelectric focusing) 【請求項１８】 組換え型酵素が配列表における配列番
号１又は２に示すアミノ酸配列かそれに相同的なアミノ
酸配列を有する請求項１６又は１７に記載の還元性澱粉
糖の変換方法。18. The method for converting reducing starch sugar according to claim 16 or 17, wherein the recombinant enzyme has the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing or an amino acid sequence homologous thereto. 【請求項１９】 還元性澱粉糖が澱粉又は澱粉質を酸及
び／又はアミラーゼにより加水分解して得られたもので
ある請求項１６、１７又は１８に記載の還元性澱粉糖の
変換方法。19. The method for converting reducing starch sugar according to claim 16, 17 or 18, wherein the reducing starch sugar is obtained by hydrolyzing starch or starchy substance with acid and / or amylase. 【請求項２０】 還元性澱粉糖がマルトトリオース、マ
ルトテトラオース、マルトペンタオース、マルトヘキサ
オース及び／又はマルトヘプタオースである請求項１
６、１７、１８又は１９に記載の還元性澱粉糖の変換方
法。20. The reducing starch sugar is maltotriose, maltotetraose, maltopentaose, maltohexaose and / or maltoheptaose.
6. The method for converting reducing starch sugar according to 6, 17, 18 or 19. 【請求項２１】 還元性澱粉糖の濃度が５０％（ｗ／
ｗ）以下の水溶液中に組換え型酵素を共存せしめ、温度
４０乃至５５℃、ｐＨ５乃至１０で作用させる請求項１
６、１７、１８、１９又は２０に記載の還元性澱粉糖の
変換方法。21. The concentration of reducing starch sugar is 50% (w /
w) The recombinant enzyme is allowed to coexist in the following aqueous solution and is allowed to act at a temperature of 40 to 55 ° C and a pH of 5 to 10.
The method for converting reducing starch sugar according to 6, 17, 18, 19 or 20. 【請求項２２】 非還元性糖質がα−グルコシルトレハ
ロース、α−マルトシルトレハロース、α−マルトトリ
オシルトレハロース、α−マルトテトラオシルトレハロ
ース又はα−マルトペンタオシルトレハロースである請
求項１６、１７、１８、１９、２０又は２１に記載の還
元性澱粉糖の変換方法。22. The non-reducing sugar is α-glucosyltrehalose, α-maltosyltrehalose, α-maltotriosyltrehalose, α-maltotetraosyltrehalose or α-maltopentaosyltrehalose, 16. The method for converting reducing starch sugar according to 17, 18, 19, 20 or 21.
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US6497862B2 (en) 2000-03-02 2002-12-24 Kabushiki Kaisha Hayashibara Composition for inhibiting body odor and uses thereof
US6555526B2 (en) 2000-09-14 2003-04-29 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Pharmaceutical composition for ophthalmic use
US6641853B1 (en) 1999-01-11 2003-11-04 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenyuojo Inhibitory agent for protein denaturation, kneaded meat with suppressed freezing-denaturation, process thereof, and process of fish and meat paste products
US7186535B1 (en) 1998-09-11 2007-03-06 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes
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US7186535B1 (en) 1998-09-11 2007-03-06 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes
US7582463B2 (en) 1998-09-11 2009-09-01 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes
US7575900B2 (en) 1998-09-11 2009-08-18 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Non-reducing saccharide-forming enzyme, trehalose-releasing enzyme, and process for producing saccharides using the enzymes
US6641853B1 (en) 1999-01-11 2003-11-04 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenyuojo Inhibitory agent for protein denaturation, kneaded meat with suppressed freezing-denaturation, process thereof, and process of fish and meat paste products
US7060310B2 (en) 1999-01-11 2006-06-13 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenyuojo Process for producing a kneaded meat
US6497862B2 (en) 2000-03-02 2002-12-24 Kabushiki Kaisha Hayashibara Composition for inhibiting body odor and uses thereof
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US7732425B2 (en) 2000-09-14 2010-06-08 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Pharmaceutical composition for ophthalmic use
US7214667B2 (en) 2000-12-22 2007-05-08 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenyujo Drugs against articular failure
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JP4822207B2 (en) * 2004-02-03 2011-11-24 株式会社林原生物化学研究所 Cosmetics
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EP3404110A1 (en) 2011-09-21 2018-11-21 Hayashibara Co., Ltd. Particulate composition comprising crystalline alpha, alpha-trehalose dihydrate

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