JP2011177118A - Transferase, method for producing saccharide, method for producing glucoside and method for producing transferase - Google Patents

Transferase, method for producing saccharide, method for producing glucoside and method for producing transferase Download PDF

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JP2011177118A
JP2011177118A JP2010045270A JP2010045270A JP2011177118A JP 2011177118 A JP2011177118 A JP 2011177118A JP 2010045270 A JP2010045270 A JP 2010045270A JP 2010045270 A JP2010045270 A JP 2010045270A JP 2011177118 A JP2011177118 A JP 2011177118A
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transferase
enzyme
producing
reaction
saccharide
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JP4830031B2 (en
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Nozomi Yasutake
望 安武
Kei Nomura
圭 野村
Atsushi Terada
敦 寺田
Taisuke Nakanishi
泰介 中西
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Showa Sangyo Co Ltd
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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
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a transferase having high activities at a high temperature, and to provide a method for producing saccharide by using the transferase. <P>SOLUTION: The transferase can produce a transglucosylated product having α-1, 2-bond or α-1, 3-bond at a high temperature because the transferase has a high residual activities even at the high temperature of 50-65°C. The risk of contamination with various bacteria at the production process of the saccharide is small compared to the conventional transferase because the high-temperature treatment can be carried out. Further, the risk of forming an insoluble product is small because the reaction rate is high, the charged amount per unit volume can be increased, and macromolecular components are hardly assembled. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

本発明は転移酵素に関する。より詳しくは、α−グルカン、α−グルコオリゴ糖、グルコースのいずれか１種以上を含む糖液に作用して、α１，２グルコシド結合、α１，３グルコシド結合を有する糖質を製造可能な耐熱性転移酵素に関する。   The present invention relates to a transferase. More specifically, heat resistance capable of producing a carbohydrate having an α1,2 glucoside bond or an α1,3 glucoside bond by acting on a sugar solution containing at least one of α-glucan, α-glucooligosaccharide, and glucose. Related to transferase.

従来、安価な澱粉を原料にした糖質として、澱粉を軽度に分解したデキストリンや、さらに分解の程度を高くしたマルトオリゴ糖、イソマルトオリゴ糖が知られている。α−１，４グルコシド結合（以後、α１，４結合と記載）を主成分とするデキストリンやマルトオリゴ糖は低甘味性や保湿性が高いなどの特徴を利用して甘味料や各種たれ、ソースなど食品に広く利用されている。
また、α−１，６グルコシド結合（以後、α１，６結合と記載）を分子内に有するイソマルトオリゴ糖は低甘味性や保湿性などのマルトオリゴ糖の特徴に加え、ビフィズス菌の選択増殖活性や虫歯になりにくいなどの生理効果が知られており、その特徴を生かして飲料や菓子など幅広く食品に応用されている。このように食品物性の改良および生理機能の付与を目的として幅広く利用されている澱粉由来の糖質は、その殆どがα１，４、α１，６結合で構成されている。 Conventionally, dextrins obtained by mildly degrading starch, maltooligosaccharides and isomaltoligosaccharides having a higher degree of degradation are known as saccharides made from inexpensive starch. Dextrins and maltooligosaccharides mainly composed of α-1,4 glucoside bonds (hereinafter referred to as α1,4 bonds) are sweeteners, various sauces, sauces, etc. by utilizing characteristics such as low sweetness and high moisture retention. Widely used in food.
In addition to the characteristics of maltooligosaccharides such as low sweetness and moisturizing properties, isomaltoligosaccharides having α-1,6 glucoside bonds (hereinafter referred to as α1,6 bonds) in the molecule, and selective growth activity of bifidobacteria Physiological effects such as resistance to caries are known, and their characteristics are utilized in a wide range of beverages and confectionery. As described above, most of the starch-derived carbohydrates widely used for the purpose of improving food properties and imparting physiological functions are composed of α1,4 and α1,6 bonds.

一方、α−１，２、α−１，３グルコシド結合（以後、α１，２結合、α１，３結合と記載）を有する糖質についてはその効率的な調製方法に関する報告がまだ少なく、前述ほど広範囲な食品への利用には至っていないが、最近になってこのような結合様式を含有するオリゴ糖が様々な機能性を有することが報告され、新規な澱粉由来のオリゴ糖としての可能性が注目されている。
α１，２結合を有する糖質については、還元力が弱くメイラード反応性が弱い、う蝕原性菌によって酸醗酵されないなどの機能性（非特許文献１）が報告されている。 On the other hand, with regard to carbohydrates having α-1,2, α-1,3 glucoside bonds (hereinafter referred to as α1,2 bonds, α1,3 bonds), there are still few reports on their efficient preparation methods. Although it has not been used for a wide range of foods, recently, it has been reported that oligosaccharides containing such a binding mode have various functions, and there is a possibility as novel oligosaccharides derived from starch. Attention has been paid.
With regard to carbohydrates having α1,2 bonds, functionality (Non-patent Document 1) has been reported, such as low reducing power and weak Maillard reactivity, and non-acid fermentation by cariogenic bacteria.

α１，３結合を有する糖質に特徴的な機能としては、食塩を含む食品の”塩かど”を緩和し、低食塩下での嗜好性を改良する食品風味改良剤（特許文献１）や、食品用の色素退色防止効果（特許文献２）などの物性改良剤としての用途のほか、生理機能として、ラクトバチルス属由来の菌と併用することによりインターロイキン１２の産生を誘導する免疫賦活効果（特許文献３）やニゲロオリゴ糖を与えることにより、病原菌に暴露された植物が、病原菌に対する自己免疫作用として、抗菌作用を有するファイトアレキシンを植物体内に誘導蓄積させる機能（特許文献４）などが報告されている。このような様々な機能性を有するα１，２、α１，３結合を有する糖質の製造方法として、以下の例が挙げられる。   As a characteristic function of carbohydrates having α1,3 bonds, a food flavor improver (patent document 1) that relaxes “salt corners” of foods containing salt and improves palatability under low salt, In addition to its use as a physical property improving agent such as a pigment fading prevention effect for food (Patent Document 2), as a physiological function, an immunostimulatory effect that induces the production of interleukin 12 when used in combination with bacteria derived from Lactobacillus genus Patent Document 3) and a function that allows plants exposed to pathogenic bacteria to induce and accumulate phytoalexin having antibacterial activity in the plant body as an autoimmune action against the pathogenic bacteria by giving Nigero-oligosaccharide (Patent Document 4) Has been. Examples of methods for producing carbohydrates having α1,2 and α1,3 bonds having such various functions include the following.

α１，２結合を有する糖質の製造法としては、β−Ｄ−グルコース１−リン酸とグルコースにコージビオースホスホリラーゼを作用させて、オリゴ糖を製造する方法（特許文献５）が開示されている。本酵素は産業への応用に十分な耐熱性を有しているが、反応液中には、目的とするオリゴ糖以外にもグルコース、リン酸が多く残存しており、目的とするオリゴ糖を得るためには、高度に精製する必要があるため実用化は考えられていない。   As a method for producing a carbohydrate having an α1,2 bond, there is disclosed a method for producing an oligosaccharide by reacting β-D-glucose 1-phosphate and glucose with cordobiose phosphorylase (Patent Document 5). Yes. Although this enzyme has sufficient heat resistance for industrial application, in the reaction solution, a lot of glucose and phosphoric acid remain in addition to the target oligosaccharide. In order to obtain it, since it needs to refine | purify highly, utilization is not considered.

α１，３結合を有する糖質の製造法としては、Ａｃｒｅｍｏｎｉｕｍ ｓｐ．由来の真菌を培養して得られるα−グルコシダーゼの転移反応により、オリゴ糖を製造する方法（特許文献６）が開示されている。本酵素は確かに特異な基質特異性を有しているが、酵素の熱安定性が低く、５５℃で３０分の処理により約６５％に低下する。他にもα−１，３結合を生成しα１，６結合を生成しないα−グルコシダーゼがＡｃｒｅｍｏｎｉｕｍ由来の真菌に知られている（特許文献７）。しかし、酵素の熱安定性が低く、６０℃で１５分の処理により約２０％に低下する。また、上記のＡｃｒｅｍｏｎｉｕｍ ｓｐ．由来の酵素とシクロデキストリン生成酵素を用いて重合度１１〜３５のグルカンを製造する手法が知られている（特許文献８）。   As a method for producing a carbohydrate having an α1,3 bond, Acremonium sp. A method (Patent Document 6) for producing an oligosaccharide by a transfer reaction of α-glucosidase obtained by culturing a derived fungus is disclosed. Although the enzyme has a specific substrate specificity, the enzyme has low thermal stability, and is reduced to about 65% by treatment at 55 ° C. for 30 minutes. In addition, α-glucosidase that generates α-1,3 bonds and does not generate α1,6 bonds is known to fungi derived from Acremonium (Patent Document 7). However, the thermal stability of the enzyme is low, and it is reduced to about 20% by treatment at 60 ° C. for 15 minutes. In addition, the above-mentioned Acremonium sp. A technique for producing glucan having a degree of polymerization of 11 to 35 using an enzyme derived from the enzyme and a cyclodextrin producing enzyme is known (Patent Document 8).

α１，２結合、α１，３結合を有する糖質の製造法としては、Ｐａｅｃｉｌｏｍｙｃｅｓ ｌｉｌａｃｉｎｕｓ由来の真菌を培養して得られるα−グルコシダーゼの転移反応により、オリゴ糖を製造する方法（特許文献９）が開示されている。本酵素は確かに特異な基質特異性を有しているが、酵素の熱安定性が低く、６５℃で３０分の処理により５０％に低下する。   As a method for producing a carbohydrate having an α1,2 bond or an α1,3 bond, there is a method for producing an oligosaccharide by a transfer reaction of α-glucosidase obtained by culturing a fungus derived from Paecilomyces lilacinus (Patent Document 9). It is disclosed. Although this enzyme has a specific substrate specificity, the enzyme has low thermal stability and is reduced to 50% by treatment at 65 ° C. for 30 minutes.

Ａｓｐｅｒｇｉｌｌｕｓ属のαグルコシダーゼは、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ（非特許文献２）、Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ（非特許文献３）、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｄｕｌａｎｓ（非特許文献４）などにその存在が知られている。しかしいずれの酵素もα１，４結合、α１，６結合の転移活性が強く、α１，２結合、α１，３結合を優先的に生成する性質を持つ酵素は知られていない。   The presence of Aspergillus genus α-glucosidase is known in Aspergillus niger (Non-patent document 2), Aspergillus oryzae (Non-patent document 3), Aspergillus nidulans (Non-patent document 4), and the like. However, any of the enzymes has a strong transfer activity of α1,4 bond and α1,6 bond, and no enzyme having the property of preferentially generating α1,2 bond and α1,3 bond is known.

特開平１０−２１０９４９号公報JP-A-10-210949 特開２０００−１８９１０１号公報JP 2000-189101 A 特開平１１−２２８４２５号公報JP-A-11-228425 特開平１０−３６２１０号公報JP-A-10-36210 特開平１０−３０４８８２号公報JP-A-10-304882 特開平７−５９５５９号公報JP-A-7-59559 特許第４１９７６０４号公報Japanese Patent No. 4197604 特許第４３９７９６５号公報Japanese Patent No. 4397965 特開２００３−１６９６６５号公報JP 2003-169665 A

日本応用糖質科学会２００１年度大会講演要旨集 第４８巻 ４１３ページ、２００１年Abstracts of Annual Meeting of the Society of Applied Glycoscience 2001, Vol. 48, p. 413, 2001 Ｃａｒｂｏｈｙｄｒａｔｅ Ｒｅｓｅａｒｃｈ 第１８５巻 １４７ページ １９８９年Carbohydrate Research Vol. 185, page 147, 1989 Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ 第１９６巻 ２６５ページ １９５１年The Journal of Biological Chemistry, Vol. 196, p. 265, 1951 Ａｐｐｌｉｅｄ ａｎｄ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ 第６８巻 １２５０ページ ２００２年Applied and Environmental Microbiology Vol. 68, page 1250 2002

このように、従来の方法は生成物の精製負荷が高いことや、生成に用いる酵素の耐熱性に問題がある。特に耐熱性については、種々の分解度の澱粉分解物に酵素を作用させてα１，２結合、α１，３結合を有する糖質を製造すること考えると、６５℃で安定な酵素反応が望まれる。
これに関して、『酵素応用の知識』 初版 ８０乃至１２９ページ １９８６年 「糖質関連酵素とその応用」の「糖質関連酵素」の項において、「工業的な糖化条件では、５５℃以下では雑菌汚染の危険性が伴い、糖化反応中にｐＨが低下する。」と記載されているように、澱粉を原料とし、長時間にわたる酵素反応の場合、５５℃以下の温度の反応条件では、雑菌汚染により反応液がｐＨ低下し、反応途中で酵素失活することが懸念され、リゾチーム等の添加による雑菌汚染防止や反応液のｐＨ調整を必要とする場合もある。 As described above, the conventional method has a problem of high product purification load and the heat resistance of the enzyme used for the production. In particular, regarding heat resistance, when an enzyme is allowed to act on starch degradation products having various degrees of degradation to produce carbohydrates having α1,2 and α1,3 bonds, a stable enzyme reaction at 65 ° C. is desired. .
In this regard, “Knowledge of Enzyme Application”, first edition, pages 80 to 129, 1986 “Sugar-related enzymes and their applications”, “Sugar-related enzymes” In the case of an enzymatic reaction using starch as a raw material for a long period of time and a reaction condition at a temperature of 55 ° C. or less, it is caused by contamination with various bacteria. There is a concern that the pH of the reaction solution is lowered and the enzyme is deactivated in the middle of the reaction. In some cases, it is necessary to prevent contamination of bacteria by adding lysozyme or the like and to adjust the pH of the reaction solution.

また、澱粉を原料として製造される澱粉部分分解物、例えば、澱粉液化物、各種デキストリン、各種マルトオリゴ糖などの分解度の指標は、一般的に、固形物当りの還元力の大きさで表しており、デキストロース・エクイバレント（Ｄｅｘｔｒｏｓｅ ｅｑｕｉｖａｌｅｎｔ、以後ＤＥと記載）という数値で示される。この値の小さいものは分子が大きく高粘度で、反応温度が低い場合には、高分子成分が会合し、不溶化物の生成も懸念される。
なお、反応温度の高温化は、基質と生産物の溶解度を上げて単位体積当たりの仕込量を多くすることができる。酵素反応速度が早くなり反応時間の短縮化ができる等の利点があり、コスト的にも有効である。 In addition, an index of the degree of degradation of a partially decomposed starch produced using starch as a raw material, for example, starch liquefied product, various dextrins, various maltooligosaccharides, etc. is generally expressed by the magnitude of reducing power per solid matter. It is indicated by a numerical value called dextrose equivalence (hereinafter referred to as DE). If this value is small, the molecule is large and the viscosity is high, and if the reaction temperature is low, the polymer components associate and there is a concern about the formation of insolubilized products.
Note that increasing the reaction temperature can increase the solubility of the substrate and the product to increase the amount charged per unit volume. There are advantages such that the enzyme reaction rate is increased and the reaction time can be shortened, which is also effective in terms of cost.

そのため、６５℃で十分な安定性を持つ耐熱性酵素を用いた、α１，２結合、α１，３結合を含有する糖質の新規製造法が望まれている。そこで、本発明は、澱粉分解物に作用してα１，２結合、α１，３結合を生成する、６５℃で十分な安定性を持つ耐熱性酵素、及びそれを用いた、α１，２結合、α１，３結合を含有する糖質の新規製造法を提供することを主目的とする。   Therefore, a new method for producing a carbohydrate containing α1,2 bond and α1,3 bond using a thermostable enzyme having sufficient stability at 65 ° C. is desired. Therefore, the present invention is a thermostable enzyme having sufficient stability at 65 ° C. that acts on a starch degradation product to produce α1,2 bond and α1,3 bond, and α1,2 bond using the same. The main object is to provide a novel process for producing carbohydrates containing α1,3 bonds.

本発明者らは上記課題を解決するため、種々の分解度の澱粉分解物に作用し、α１，２結合、α１，３結合を生成する産業上有意な耐熱性を持つ酵素の実現に期待を込めて、この酵素を生産する微生物を広く検索してきた。
その結果、Ａｓｐｅｒｇｉｌｌｕｓ属の菌株、特に、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ属菌株またはＡｓｐｅｒｇｉｌｌｕｓ ａｗａｍｏｒｉ属菌株が、６５℃で十分な耐熱性を持つ新規酵素を産生することを見出した。本菌株を適当な条件で培養し、得られた酵素を、澱粉分解物を基質として反応させると、α１，２結合、α１，３結合を有する糖転移物を生成する活性を持つことを見出し、これらの知見に基づいて本発明を完成した。 In order to solve the above-mentioned problems, the present inventors have high expectations for the realization of an industrially significant heat-resistant enzyme that acts on starch degradation products having various degrees of degradation and generates α1,2 bonds and α1,3 bonds. Therefore, we have searched extensively for microorganisms that produce this enzyme.
As a result, it was found that strains of the genus Aspergillus, particularly Aspergillus niger or Aspergillus awamori, produce a novel enzyme having sufficient heat resistance at 65 ° C. When this strain is cultured under appropriate conditions, and the resulting enzyme is reacted with a starch degradation product as a substrate, it has been found to have an activity of producing a glycosylated product having α1,2 bonds and α1,3 bonds, The present invention has been completed based on these findings.

係る知見に基づいて成された本発明は、α−グルカンと、α−グルコオリゴ糖と、グルコースとからなる群より選択されるいずれか１種以上の糖類に作用して、α１，２結合を有する糖質と、α１，３結合を有する糖質を生成し、６５℃以上の温度で酵素反応可能な耐熱性のＡｓｐｅｒｇｉｌｌｕｓ属由来の転移酵素を提供することである。
本発明の転移酵素は、耐熱性が５０〜６５℃のいずれかの温度でｐＨ５．０、３０分間保持において、９０％以上の残存活性を有するものが含まれる。
また、本発明の転移酵素は、下記の理化学的性質を有するものが含まれる。
（１）作用：マルトース、ニゲロース、コージビオース、マルトオリゴ糖などα−グルコオリゴ糖および、アミロース、可溶性澱粉などのα−グルカンの非還元末端側のα−グルコシド結合を分解し、グルコースを遊離する。また、スクロースを分解する活性を持つ。更に、α−グルカン、α−グルコオリゴ糖、グルコースから選ばれる少なくとも一種を含む糖液に作用して、α１，２グルコシド結合、α１，３結合グルコシドを有する糖質を産生する。
（２）分子量：ＳＤＳ−ゲル電気泳動法により、４８，０００、５９，０００ダルトン。
（３）等電点：アンフォライン含有電気泳動法により、ｐＩ４．９〜５．５
（４）至適温度：ｐＨ４．０、１０分間反応で、６５℃
（５）至適ｐＨ：５０℃、１０分間反応で、ｐＨ３．５
（６）温度安定性：ｐＨ４．０、３０分間保持で、６５℃では初期活性の９０％以上の残存
（７）ｐＨ安定性：４℃、２４時間保持で、ｐＨ３．０〜５．０
本発明の転移酵素は、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ属菌株またはＡｓｐｅｒｇｉｌｌｕｓ ａｗａｍｏｒｉ属菌株のいずれか一方から生産することもできる。
本発明の転移酵素は、酵素をコードするアミノ酸配列に、配列Ｉ：ＬＬＶＥＹＱＴＤＥＲＬＨＶＭＩＹＤＡＤＥＥＶＹＱＶＰＥＳＶＬＰＲ、配列ＩＩ：ＴＷＬＰＤＤＰＹＶＹＧＬＧＥＨＳＤＰＭＲ、配列ＩＩＩ：ＩＰＬＥＴＭＷＴＤＩＤＹＭＤＫＲ、配列ＩＶ：ＶＦＴＬＤＰＱＲ、配列Ｖ：ＷＡＳＬＧＡＦＹＴＦＹＲ、とからなる配列群より選択されるいずれか１以上の配列を含有するものがある。
本発明は、上記いずれかの転移酵素を、α−グルカン、α−グルコオリゴ糖、グルコースから選ばれる少なくとも一種を含む糖に作用させ、α１，２結合を有する糖質と、α１，３結合を有する糖質を生成した後、前記糖質を採取して糖質を製造することもできる。
また、本発明は、澱粉部分分解物に、上記のいずれかの転移酵素を作用させ、α１，２結合を有する糖質と、α１，３結合を有する糖質を生成した後、前記糖質を採取することもできる。
前記澱粉部分分解物は、澱粉質を前記転移酵素以外の酵素又は酸によって部分的に加水分解して得られるものである。
更に、受容体糖分子に直鎖オリゴ糖を転移させる不均一化反応（Ｄｉｓｐｒｏｐｏｒｔｉｏｎａｔｉｏｎ反応）を触媒する酵素、及び／又は枝切酵素、及び／又はグルコアミラーゼを作用させることも可能である。
本発明によれば、食物繊維含有量が３０％以上とすることができる。更に、糖質を採取するに際し、塩型強酸性カチオン交換樹脂を用いるカラムクロマトグラフィーを用いることもできる。
前記転移酵素を前記糖又は前記澱粉分解物に作用させる工程は、６０〜８０℃で行うことが好ましい。
また、上記いずれかの転移酵素を、α−グルカン、α−グルコオリゴ糖、グルコースから選ばれる少なくとも一種を含む糖液とグルコースの誘導体に作用して、配糖体を採取することもできる。
本発明は、上記いずれかの転移酵素産生能を有するＡｓｐｅｒｇｉｌｌｕｓに属する微生物を栄養培地中で培養して、当該転移酵素を生成させ、得られる培養物から当該転移酵素を採取することが可能である。 The present invention based on such knowledge has an α1,2 bond by acting on any one or more saccharides selected from the group consisting of α-glucan, α-glucooligosaccharide, and glucose. It is to provide a heat-resistant Aspergillus-derived transferase capable of producing a saccharide and a saccharide having an α1,3 bond and capable of performing an enzyme reaction at a temperature of 65 ° C. or higher.
The transferase of the present invention includes those having a residual activity of 90% or more when kept at pH 5.0 for 30 minutes at any temperature of 50 to 65 ° C.
The transferase of the present invention includes those having the following physicochemical properties.
(1) Action: Decomposes α-glucooligosaccharides such as maltose, nigerose, cordobiose and maltooligosaccharide, and α-glucoside bonds on the non-reducing terminal side of α-glucans such as amylose and soluble starch, and releases glucose. In addition, it has an activity of decomposing sucrose. Furthermore, it acts on a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide and glucose to produce a carbohydrate having an α1,2 glucoside bond and an α1,3 linked glucoside.
(2) Molecular weight: 48,000, 59,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point: pI of 4.9 to 5.5 by an ampholine-containing electrophoresis method
(4) Optimum temperature: pH 4.0, reaction at 10 minutes, 65 ° C
(5) Optimal pH: 50 ° C., 10 minutes reaction, pH 3.5
(6) Temperature stability: pH 4.0, held for 30 minutes, remaining at 90% or more of initial activity at 65 ° C. (7) pH stability: pH 3.0-5.0 at 4 ° C., held for 24 hours
The transferase of the present invention can also be produced from either Aspergillus niger or Aspergillus awamori.
The transfer enzyme of the present invention has an amino acid sequence encoding the enzyme, the sequence I: LLVEYQTDERLHVMIYDADEVYQVPESVLPR, the sequence II: TWLPDDPYVYGLGEHSDPMR, the sequence III: IPLETMWTDIDYMDKR, the sequence IV: VFLDDPQR, the sequence V: WASL, and the sequence V: WAY. Or contain one or more sequences.
In the present invention, any of the above transferases is allowed to act on a sugar containing at least one selected from α-glucan, α-glucooligosaccharide, and glucose, and has a carbohydrate having an α1,2 bond and an α1,3 bond. After the saccharide is produced, the saccharide can be collected to produce the saccharide.
In addition, the present invention allows any of the above transferases to act on a partially decomposed starch to produce a saccharide having an α1,2 bond and a saccharide having an α1,3 bond, It can also be collected.
The starch partial decomposition product is obtained by partially hydrolyzing starch with an enzyme or acid other than the transferase.
Furthermore, an enzyme that catalyzes a heterogeneous reaction (Disproportionation reaction) that transfers a linear oligosaccharide to a receptor sugar molecule, and / or a debranching enzyme, and / or a glucoamylase can be allowed to act.
According to the present invention, the dietary fiber content can be 30% or more. Furthermore, when collecting carbohydrates, column chromatography using a salt-type strongly acidic cation exchange resin can also be used.
The step of allowing the transferase to act on the sugar or the starch degradation product is preferably performed at 60 to 80 ° C.
Alternatively, any of the above transferases can act on a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide, and glucose and a glucose derivative to collect a glycoside.
In the present invention, any of the above-mentioned microorganisms belonging to Aspergillus having the ability to produce transferase can be cultured in a nutrient medium to produce the transferase, and the transferase can be collected from the resulting culture. .

本発明の転移酵素は、５０〜６５℃の高温でも転移酵素の残存活性が高いので、α１，２結合、α１，３結合を有する糖転移物を高温で製造することができる。リン酸等の余分な副生成物も生成されないから、糖質を高度に精製する必要が無い。   Since the transfer enzyme of the present invention has a high residual activity of the transfer enzyme even at a high temperature of 50 to 65 ° C., a glycosyltransferase having α1,2 bond and α1,3 bond can be produced at high temperature. Since no extra by-products such as phosphoric acid are produced, it is not necessary to highly refine the carbohydrate.

本発明の転移酵素の電気泳動写真（ａ）と、推測されるアミノ酸配列（ｂ）Electrophoresis photograph (a) of the transferase of the present invention and deduced amino acid sequence (b) 本発明の転移酵素の一例、温度と酵素活性との関係を示すグラフ。The graph which shows an example of the transfer enzyme of this invention, the relationship between temperature and enzyme activity. 本発明の転移酵素の一例、ｐＨと酵素活性との関係を示すグラフ。The graph which shows an example of the transfer enzyme of this invention, and the relationship between pH and enzyme activity. 本発明の転移酵素の一例、温度安定性のグラフExample of transferase of the present invention, temperature stability graph 本発明の転移酵素の一例、ｐＨ安定性のグラフExample of transferase of the present invention, pH stability graph 本発明の転移酵素を用いてマルトースを原料とした反応の一例、反応液の重合度分布を示すＨＰＬＣチャート（ａ）、反応液２〜３糖中の糖構造異性体を示すＨＰＬＣチャート（ｂ）。An example of a reaction using maltose as a raw material using the transferase of the present invention, an HPLC chart (a) showing the polymerization degree distribution of the reaction solution, and an HPLC chart (b) showing saccharide structural isomers in the reaction solution 2-3 sugars . 本発明の転移酵素を用いてマルトースを原料とした反応の一例、重合度分布の経時変化を示すグラフ（ａ）、α１，２結合オリゴ糖、α１，３結合オリゴ糖、Ｐｒｏｓｋｙ値の経時変化を示すグラフ（ｂ）An example of a reaction using maltose as a raw material using the transferase of the present invention, graph (a) showing a change in polymerization degree over time, α1,2-linked oligosaccharide, α1,3-linked oligosaccharide, and change in Prosky value over time Graph (b) 本発明の転移酵素により生成された糖反応液のＨＰＬＣチャート。The HPLC chart of the sugar reaction liquid produced | generated by the transferase of this invention.

＜本発明の酵素の説明＞
本発明は、このように、α−グルカン、α−グルコオリゴ糖、グルコースから選ばれる少なくとも一種を含む澱粉分解物等の糖原料に作用してα１，２結合、α１，３結合を有する糖質を生成する特性を持った、耐熱性のＡｓｐｅｒｇｉｌｌｕｓ属の転移酵素と、本酵素を用いた糖転移物の製造法に関するものである。
本発明の転移酵素は、後述するようにαグルコシド結合を加水分解する酵素であるから、以下本発明の転移酵素をα−グルコシダーゼ、又は本酵素とも称する。 <Description of the enzyme of the present invention>
In this way, the present invention acts on a sugar raw material such as a starch degradation product containing at least one selected from α-glucan, α-glucooligosaccharide, and glucose to thereby provide a saccharide having α1,2 bond and α1,3 bond. The present invention relates to a thermostable Aspergillus transferase having the properties to be produced, and a method for producing a glycosyltransferase using the enzyme.
Since the transferase of the present invention is an enzyme that hydrolyzes an α-glucoside bond as described later, the transferase of the present invention is hereinafter also referred to as α-glucosidase or the present enzyme.

なお、本発明においてα１，２結合を含む糖質とは、分子内にα１，２結合を一箇所以上含む２糖以上の糖質を意味し、α１，２結合のみからなるオリゴ糖の他、α１，２結合とそれ以外の結合とからなるオリゴ糖も含む。
具体的には、２糖であるコージビオース［Ｏ−α−Ｄ−グルコピラノシル−（１→２）−Ｏ−Ｄ−グルコピラノース］］、三糖類であるコージビオシルグルコース［Ｏ−α−Ｄ−グルコピラノシル−（１→２）−Ｏ−α−Ｄ−グルコピラノシル−（１→４）−Ｄ−グルコピラノース］などが例示できる。また、メチル化分析法（Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｃｈｅｍｉｓｔｒｙ 第５５巻 ２０５ページ １９６４年）により、３糖以上の成分についても、その構造中に１，２結合が含有されるか判断できる。 In the present invention, a saccharide containing an α1,2 bond means a saccharide having two or more saccharides containing at least one α1,2 bond in the molecule, in addition to an oligosaccharide consisting of only an α1,2 bond, Also included are oligosaccharides composed of α1,2 bonds and other bonds.
Specifically, cordobiose [O-α-D-glucopyranosyl- (1 → 2) -OD-glucopyranose]], which is a disaccharide, and cordobiosyl glucose [O-α-D-glucopyranosyl, which is a trisaccharide. -(1 → 2) -O-α-D-glucopyranosyl- (1 → 4) -D-glucopyranose] and the like. In addition, it can be determined by the methylation analysis method (Journal of Biochemistry Vol. 55, p. 205, 1964) whether or not a 1,2-bond is contained in the structure of a trisaccharide or higher component.

本発明においてα１，３結合を含む糖質とは、分子内にα１，３結合を一箇所以上含むことを意味し、α１，３結合のみからなるオリゴ糖の他、α１，３結合とそれ以外の結合とからなるオリゴ糖も含む。具体的には、二糖であるニゲロース［Ｏ−α−Ｄ−グルコピラノシル−（１→３）−Ｏ−Ｄ−グルコピラノース］のほか、三糖類であるニゲロシルグルコース［Ｏ−α−Ｄ−グルコピラノシル−（１→３）−Ｏ−α−Ｄ−グルコピラノシル−（１→４）−Ｄ−グルコピラノース］、ニゲロトリオース［Ｏ−α−Ｄ−グルコピラノシル−（１→３）−Ｏ−α−Ｄ−グルコピラノシル−（１→３）−Ｄ−グルコピラノース］などが例示できる。また、メチル化分析法により、３糖以上の成分についても、その構造中に１，３結合が含有されるか判断できる。   In the present invention, a carbohydrate containing an α1,3 bond means that the molecule contains one or more α1,3 bonds. In addition to an oligosaccharide consisting of only an α1,3 bond, an α1,3 bond and others Also included are oligosaccharides consisting of Specifically, in addition to nigerose [O-α-D-glucopyranosyl- (1 → 3) -OD-glucopyranose], which is a disaccharide, nigerosyl glucose [O-α-D-glucopyranosyl, which is a trisaccharide. -(1 → 3) -O-α-D-glucopyranosyl- (1 → 4) -D-glucopyranose], nigerotriose [O-α-D-glucopyranosyl- (1 → 3) -O-α-D -Glucopyranosyl- (1 → 3) -D-glucopyranose] and the like. In addition, it can be determined by methylation analysis whether a 1,3 bond is contained in the structure of a trisaccharide or higher component.

３糖以上の成分でその構造中に１，２結合や１，３結合が含有されるかの判定には、酵素−ＨＰＬＣ法（Ｊｏｕｒｎａｌ ｏｆ ＡＯＡＣ Ｉｎｔｅｒｎａｔｉｏｎａｌ 第８５巻 ４３５ページ ２００２年、もしくは、栄養表示基準における栄養性分等の分析方法について 平成１１年４月２６日衛新第１３号）で算出される食物繊維含有量（Ｐｒｏｓｋｙ値）によっても判断することができる。   To determine whether the structure contains more than 3 sugars and includes 1,2 bonds or 1,3 bonds, the enzyme-HPLC method (Journal of AOAC International Vol. 85, page 435, 2002, or nutrition labeling About the analysis method of nutritional content etc. in a reference | standard, it can judge also by dietary fiber content (Prosky value) calculated by April 26, 1999 in Eshin No. 13).

澱粉糖におけるグルコース間の結合様式は、α１，４結合、α１，６結合であり、通常の澱粉糖には１，２結合や１，３結合は殆ど存在しない。上記の酵素−ＨＰＬＣ法を用いた分析例より、澱粉を酸焙焼すると、１，２結合及び／又は１，３結合（α、βの結合様式は不明）の含有量が増加し、Ｐｒｏｓｋｙ値が上昇することが知られている（Ｊｏｕｒｎａｌ ｏｆ Ａｐｐｌｉｅｄ Ｇｌｙｃｏｓｃｉｅｎｃｅ 第４９巻 ４７９ページ ２００２年）が、本発明では転移酵素によりα１，２結合およびα１，３結合の糖質を顕著に増加させるのである。   The binding mode between glucose in starch sugar is α1,4 bond and α1,6 bond, and normal starch sugar has almost no 1,2 bond or 1,3 bond. From the analysis example using the enzyme-HPLC method described above, when starch is acid roasted, the content of 1,2 bonds and / or 1,3 bonds (α and β binding modes are unknown) increases, and the Prosky value (Journal of Applied Glycoscience 49: 479, 2002), however, in the present invention, α1,2 and α1,3 linked carbohydrates are significantly increased by transferase.

本発明における転移酵素は６５℃では９０％以上の残存活性を持ち、α−グルカン、マルトオリゴ糖、グルコースから選ばれる少なくとも一種を含む糖液に作用して、α１，２結合、α１，３結合を含む糖質を生成する作用を示す酵素であればよく、
糸状菌（Ａｂｓｉｄｉａ、Ａｃｒｅｍｏｎｉｕｍ、Ａｃｔｉｎｏｍａｄｕｒａ、Ａｌｔｅｒｎａｒｉａ、Ａｓｐｅｒｇｉｌｌｕｓ、Ｃｈａｅｔｏｍｉｕｍ、Ｃｏｐｒｉｎｕｓ、Ｃｏｒｉｏｌｕｓ、Ｇｅｏｔｒｉｃｈｕｍ、Ｈｕｍｉｃｏｌａ、Ｍｏｎａｓｃｕｓ、Ｍｏｒｔｉｅｒｅｌｌａ、Ｍｕｃｏｒ、Ｎｏｃａｒｄｉｏｐｓｉｓ、Ｏｉｄｉｏｄｅｎｄｒｏｎ、Ｐｅｎｉｃｉｌｌｉｕｍ、Ｒｈｉｚｏｍｕｃｏｒ、Ｒｈｉｚｏｐｕｓ、Ｔｒｉｃｈｏｄｅｒｍａ、Ｖｅｒｔｉｃｉｌｌｉｕｍ）
担子菌（Ｃｏｌｉｏｌｕｓ、Ｃｏｒｔｉｃｉｕｍ、Ｃｙａｔｈｕｓ、Ｉｒｐｅｘｓ、Ｐｏｌｙｐｏｒｕｓ、Ｐｙｃｎｏｐｏｒｕｓ、Ｔｒａｍｅｔｅｓ）、
細菌（Ａｅｒｏｍｏｎａｓ、Ａｇｒｏｂａｃｔｅｒｉｕｍ、Ａｌｃａｌｉｇｅｎｅｓ、Ａｇｒｏｂａｃｔｅｒｉｕｍ、Ａｌｔｅｒｏｍｏｎａｓ、Ａｒｔｈｒｏｂａｃｔｅｒ、Ｂａｃｉｌｌｕｓ、Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ、Ｃｈｒｏｍｏｂａｃｔｅｒｉｕｍ、Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ、Ｃｒｙｐｎｏｈｅｃｔｒｉａ、Ｅｒｗｉｎｉａ、Ｅｓｃｈｅｒｉｃｈｉａ、Ｆｌａｖｏｂａｃｔｅｒｉｕｍ、Ｋｌｅｂｓｉｅｌｌａ、Ｌａｃｔｏｂａｃｉｌｌｕｓ、Ｌａｃｔｏｃｏｃｃｕｓ、Ｌｅｕｃｏｎｏｓｔｏｃ、Ｍｉｃｒｏｂａｃｔｅｒｉｕｍ、Ｍｉｃｒｏｃｏｃｃｕｓ、Ｐｉｍｅｌｏｂａｃｔｅｒ、Ｐｌｅｓｉｏｍｏｎａｓ、Ｐｒｏｔａｍｉｎｏｂａｃｔｅｒ、Ｐｓｅｕｄｏｍｏｎａｓ、Ｓｅｒｒａｔｉａ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ、Ｓｔｒｅｐｔｏｖｅｒｔｉｃｉｌｌｉｕｍ、Ｓｕｌｆｏｌｏｂｕｓ、Ｔｈｅｒｍｕｓ、Ｘａｎｔｈｏｍｏｎａｓ）
放線菌（Ａｃｔｉｎｏｍａｄｕｒａ、Ａｃｔｉｎｏｍｙｃｅｓ、Ａｃｔｉｎｏｐｌａｎｅｓ、Ａｍｙｃｏｌａｔｏｐｓｉｓ、Ｅｕｐｅｎｉｃｉｌｌｉｕｍ、Ｎｏｃａｒｄｉｏｐｓｉｓ、Ｓｔｒｅｐｔｏｍｙｃｅｓ、Ｔｈｅｒｍｏｍｏｎｏｓｐｏｒａ）
酵母（Ａｕｒｅｏｂａｓｉｄｉｕｍ、Ｃａｎｄｉｄａ、Ｉｒｐｅｘ、Ｋｌｕｙｖｅｒｏｍｙｃｅｓ、Ｐｙｃｎｏｐｏｒｕｓ、Ｓａｃｃｈａｒｏｍｙｃｅｓ、Ｔｒｉｃｈｏｓｐｏｒｏｎ）など食品製造にて使用例のある株が望ましい。 The transferase in the present invention has a residual activity of 90% or more at 65 ° C. and acts on a sugar solution containing at least one selected from α-glucan, maltooligosaccharide, and glucose, and has α1,2 bond and α1,3 bond. Any enzyme that has the action of producing carbohydrates can be included.
Filamentous fungi (Absidia, Acremonium, Actinomadura, Alternaria, Aspergillus, Chaetomium, Coprinus, Coriolus, Geotrichum, Humicola, Monascus, Mortierella, Mucor, Nocardiopsis, Oidiodendron, Penicillium, Rhizomucor, Rhizopus, Trichoderma, Verticillium)
Basidiomycetes (Coliolus, Corticium, Cyathus, Irpex, Polyporus, Pynoporus, Trametes),
Bacteria (Aeromonas, Agrobacterium, Alcaligenes, Agrobacterium, Alteromonas, Arthrobacter, Bacillus, Brevibacterium, Chromobacterium, Corynebacterium, Crypnohectria, Erwinia, Escherichia, Flavobacterium, Klebsiella, Lactobacillus, Lactococcus, Leuconostoc, Microbacterium, Micrococcus, Pimelobacter, Plesiomonas, Protaminobacter, Pseudomonas, Serratia, Streptococcus , Streptoverticillium, Sulfolobus, Thermus, Xanthomonas)
Actinomydra (Actinomadra, Actinomyces, Actinoplanes, Amycolatopsis, Eupenicillium, Nocardiopsis, Streptomyces, Thermomonospora)
A strain having an example of use in food production such as yeast (Aureobasidium, Candida, Irpex, Kluyveromyces, Pycnoporus, Saccharomyces, Trichosporon) is desirable.

中でもＡｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ属菌株またはＡｓｐｅｒｇｉｌｌｕｓ ａｗａｍｏｒｉ属菌株等のＡｓｐｅｒｇｉｌｌｕｓ属が好ましく、それらの中でも、
Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ（ＡＴＣＣ １０２５４）、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ ｖａｎ Ｔｉｅｇｈｅｍ ｖａｒ． ｎｉｇｅｒ ｆｓｐ． ｈｅｎｎｅｂｅｒｇｉｉ Ｂｌｏｃｈｗｉｔｚ ｅｘ Ａｌ−Ｍｕｓａｌｌａｍ（ＮＢＲＣ ４０４３）、Ａｓｐｅｒｇｉｌｌｕｓ ａｗａｍｏｒｉ（ＡＴＣＣ １４３３１）などが好適な菌株として挙げられる。
本発明においてＡｓｐｅｒｇｉｌｌｕｓ属の菌を用いて、目的とする転移酵素を得るに際しては、その培養には、公知の手法が適宜に採用され、例えば液体培養及び固体培養の何れもが任意に用いられ得るものである。
本発明で使用する微生物は野生株に限らず、上記野生株を紫外線、エックス線、放射線、各種薬品［ＮＴＧ（Ｎ−メチル−Ｎ’−ニトロ−Ｎ−ニトロソグアニジン）、ＥＭＳ（エチルメタンスルホネート）等］などを用いる人工的変異手段で変異した変異株も、α１，２結合、α１，３結合を有する糖質を産生する耐熱性の転移酵素である限り、使用できる。 Among them, Aspergillus genus such as Aspergillus niger genus strain or Aspergillus awamori genus strain is preferable, among them,
Aspergillus niger (ATCC 10254), Aspergillus niger van Tieghem var. niger fsp. Henbergii Blochwitz ex Al-Musallalam (NBRC 4043), Aspergillus awamori (ATCC 14331), etc. are mentioned as suitable strains.
In the present invention, when obtaining a target transferase using a bacterium belonging to the genus Aspergillus, a known technique is appropriately employed for the culture, and for example, both liquid culture and solid culture can be arbitrarily used. Is.
Microorganisms used in the present invention are not limited to wild strains, and the above wild strains may be ultraviolet rays, X-rays, radiation, various chemicals [NTG (N-methyl-N′-nitro-N-nitrosoguanidine), EMS (ethyl methanesulfonate), etc. Mutants that have been mutated by artificial mutation means such as] can be used as long as they are thermostable transferases that produce carbohydrates having α1,2 linkages or α1,3 linkages.

Ａｓｐｅｒｇｉｌｌｕｓ属の菌を用いた培養に際して用いられる培地の炭素源としては、例えば、グルコース、フルクトース、ショ糖、乳糖、澱粉、グリセリン、デキストリン、レシチン等が、単独で又は組み合わせて用いられ、また、窒素源としては、有機及び無機の窒素源の何れもが利用可能であり、そのうち、有機窒素源としては、例えば、ペプトン、酵母エキス、大豆、きなこ、米ぬか、コーンスティープリカー、肉エキス、カゼイン、アミノ酸等が用いられ、一方、無機窒素源としては、硫酸アンモニウム、硝酸アンモニウム、リン酸‐アンモニウム、リン酸二アンモニウム、塩化アンモニウム等が用いられることとなる。更に、そのような培地に添加される無機塩や微量栄養素としては、例えば、ナトリウム、マグネシウム、カリウム、鉄、亜鉛、カルシウム、マンガンの塩類の他、ビタミン等を挙げることが出来る。また、上記の各種成分を含有する培地成分として小麦ふすま等の天然物を用いることも可能である。   As the carbon source of the medium used in the culture using Aspergillus bacteria, for example, glucose, fructose, sucrose, lactose, starch, glycerin, dextrin, lecithin and the like are used alone or in combination, and nitrogen As the source, both organic and inorganic nitrogen sources can be used. Among them, examples of the organic nitrogen source include peptone, yeast extract, soybean, kinako, rice bran, corn steep liquor, meat extract, casein, amino acid. On the other hand, ammonium sulfate, ammonium nitrate, phosphate-ammonium phosphate, diammonium phosphate, ammonium chloride, etc. will be used as the inorganic nitrogen source. Furthermore, examples of inorganic salts and micronutrients added to such a medium include sodium, magnesium, potassium, iron, zinc, calcium, manganese salts, vitamins, and the like. Moreover, it is also possible to use natural products, such as wheat bran, as a culture medium component containing said various components.

Ａｓｐｅｒｇｉｌｌｕｓ属の菌を用いた培養は、一般に１０〜４０℃の温度で行なわれるが、好ましくは２５〜３０℃の培養温度が有利に採用され、更に、培地ｐＨは２．５〜８．０であれば良い。そして、必要な培養期間は、菌体濃度、培地ｐＨ、培地温度、培地の構成等によって異なるが、通常、４日〜９日程度であり、目的物である転移酵素が最大に達した頃に、その培養が停止される。   Cultivation using Aspergillus bacteria is generally performed at a temperature of 10 to 40 ° C., preferably a culture temperature of 25 to 30 ° C. is advantageously employed, and the medium pH is 2.5 to 8.0. I just need it. The required culture period varies depending on the cell concentration, medium pH, medium temperature, medium composition, etc., but is usually about 4 to 9 days, and when the target transferase has reached its maximum. The culture is stopped.

このようにして、微生物を培養した後、本発明の酵素を回収する。本酵素の活性は、培養物の菌体と培地の両方に認められ、公知の方法によって精製して利用することができる。一例として、培養液の処理物を濃縮した粗酵素標品を透析後、東ソー（株）社製ゲル「ＴＯＹＯＰＥＡＲＬ ＤＥＡＥ−６５０Ｍ」などを用いた陰イオン交換カラムクロマトグラフィー、続いて、ＧＥヘルスケア・ジャパン（株）社製カラム「ＨｉＬｏａｄ １６／６０ Ｓｕｐｅｒｄｅｘ ２００ｐｇ」、「ＨｉＬｏａｄ １６／６０ Ｓｕｐｅｒｄｅｘ ７５ｐｇ」を連結させたゲル瀘過クロマトグラフィー、次に、ＧＥヘルスケア・ジャパン（株）社製カラム「ＭｏｎｏＰ ５／２００ ＧＬ」を用いた等電点クロマトグラフィーを行うことで、電気泳動的に単一な酵素を得ることができる。   Thus, after culturing the microorganism, the enzyme of the present invention is recovered. The activity of this enzyme is found in both the cells of the culture and the medium, and can be purified and used by a known method. As an example, after dialysis of a crude enzyme preparation obtained by concentrating a treated product of a culture solution, anion exchange column chromatography using a gel “TOYOPEARL DEAE-650M” manufactured by Tosoh Corporation, followed by GE Healthcare Gel filtration chromatography in which columns “HiLoad 16/60 Superdex 200 pg” and “HiLoad 16/60 Superdex 75 pg” manufactured by Japan Co., Ltd. are linked, and then column “MonoP” manufactured by GE Healthcare Japan Co., Ltd. By performing isoelectric focusing using “5/200 GL”, a single enzyme can be obtained electrophoretically.

Ａｓｐｅｒｇｉｌｌｕｓ由来の酵素を用いた場合の酵素化学的性質は以下のとおりである。
（１）作用
本酵素は基質の非還元末端のα−グルコシド結合をエキソ型に切断するα−グルコシダーゼで、α１，４結合以外にα１，２結合、α１，３結合の加水分解を行う一方、糖供与体からのグルコース残基を糖受容体のグルコース残基の２，３，４位いずれかの水酸基に転移する糖転移反応も行う。
α−グルコシル基が４位の水酸基に転移した転移生成物は、本酵素によって再び分解されるため、最終的に転移生成物としてα１，２、α１，３結合を有する糖質が蓄積される。また、グルコース骨格を持つ誘導体、ＯＨ基を有する化合物を受容体とした場合でも、糖転移活性も持つ。従って、α−グルカン、α−グルコオリゴ糖、グルコース等に、グルコース誘導体を加えた基質に作用して、配糖体を採取することができる。基質としては、澱粉、アミロース、アミロペクチン，グリコーゲン、デキストリン、などのα−グルカン、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、マルトオリゴ糖、コージビオース、ニゲロースなどα−グルコオリゴ糖、グルコースを使用することができる。 Enzymatic chemistry when Aspergillus-derived enzyme is used is as follows.
(1) Action This enzyme is an α-glucosidase that cleaves the α-glucoside bond at the non-reducing end of the substrate into an exo-type, and hydrolyzes α1,2 and α1,3 bonds in addition to α1,4 bonds, A transglycosylation reaction is also performed in which the glucose residue from the sugar donor is transferred to any one of the 2, 3, and 4 hydroxyl groups of the glucose residue of the sugar acceptor.
Since the transfer product in which the α-glucosyl group is transferred to the hydroxyl group at the 4-position is decomposed again by the present enzyme, a saccharide having α1,2, α1,3 bonds is finally accumulated as the transfer product. In addition, even when a derivative having a glucose skeleton or a compound having an OH group is used as a receptor, it has transglycosylation activity. Accordingly, glycosides can be collected by acting on a substrate obtained by adding a glucose derivative to α-glucan, α-glucooligosaccharide, glucose and the like. As the substrate, α-glucan such as starch, amylose, amylopectin, glycogen, dextrin, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltooligosaccharide, cordobiose, α-glucooligosaccharide such as nigerose, Glucose can be used.

（２）分子量
Ｎａｔｉｖｅ-ゲル電気泳動（ＧＥヘルスケア・ジャパン（株）社製、ＰｈａｓｔＳｙｓｔｅｍ）より、分子量９７，０００ダルトンであった。
ＳＤＳ-ゲル電気泳動（ＧＥヘルスケア・ジャパン（株）社製、ＰｈａｓｔＳｙｓｔｅｍ）より、分子量４８，０００、５９，０００ダルトンの２本のバンドが見られた。
（３）等電点
本酵素を等電点既知の標準タンパク質とともに等電点電気泳動（ＧＥヘルスケア・ジャパン（株）社製、ＰｈａｓｔＳｙｓｔｅｍ）を行った結果、本酵素の等電点はｐＩ：４．９〜５．５であり、中心値が５．２であった。
（４）至適温度
ｐＨ４．０、１０分間の反応で、６５℃であった。
（５）至適ｐＨ
５０℃、１０分間の反応で、ｐＨ３．５であった。
（６）温度安定性
６５℃、３０分の処理で、初期活性の９０％以上残存した。
（７）ｐＨ安定性
４℃、２４時間の保存で、ｐＨ３．０〜５．０であった。 (2) Molecular weight The molecular weight was 97,000 daltons from Native-gel electrophoresis (manufactured by GE Healthcare Japan Co., Ltd., PhasSystem).
Two bands with molecular weights of 48,000 and 59,000 daltons were observed from SDS-gel electrophoresis (manufactured by GE Healthcare Japan Co., Ltd., PhasSystem).
(3) Isoelectric point As a result of performing isoelectric focusing (manufactured by GE Healthcare Japan Co., Ltd., Phase System) together with a standard protein whose isoelectric point is known, the isoelectric point of the enzyme is pI: It was 4.9 to 5.5, and the center value was 5.2.
(4) Optimum temperature It was 65 degreeC by reaction for pH 4.0 and 10 minutes.
(5) Optimum pH
The pH was 3.5 after reaction at 50 ° C. for 10 minutes.
(6) Temperature stability 90% or more of the initial activity remained after treatment at 65 ° C. for 30 minutes.
(7) pH stability The pH was 3.0 to 5.0 after storage at 4 ° C for 24 hours.

＜糖質製造方法の具体例＞
以下に本発明の酵素利用法の具体例を説明するが、本発明の酵素利用法は特に限定されるものではない。
菌体及び培養液から取得した本発明の転移酵素を、基質であるα−グルカン、α−グルコオリゴ糖、グルコースから選ばれる少なくとも一種からなる糖原料に作用させることにより、α１，２、α１，３結合を有する糖質を取得することができる。
α１，２結合、α１，３結合を含有する糖質を効率よく生産することのできる 基質としては、澱粉、アミロース、アミロペクチン，グリコーゲン、デキストリン、などのα−グルカン、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、マルトオリゴ糖、コージビオース、ニゲロースなどα−グルコオリゴ糖、グルコースを使用することができる。 <Specific examples of sugar production methods>
Although the specific example of the enzyme utilization method of this invention is demonstrated below, the enzyme utilization method of this invention is not specifically limited.
By causing the transferase of the present invention obtained from the microbial cells and the culture solution to act on a sugar raw material consisting of at least one selected from α-glucan, α-glucooligosaccharide and glucose as substrates, α1,2, α1,3 A carbohydrate having a bond can be obtained.
As a substrate capable of efficiently producing carbohydrates containing α1,2 bond and α1,3 bond, α-glucan such as starch, amylose, amylopectin, glycogen, dextrin, maltose, maltotriose, maltotetra Α-Gluco-oligosaccharides such as oose, maltopentaose, maltohexaose, malto-oligosaccharide, cordobiose, nigerose, and glucose can be used.

また、澱粉、アミロペクチン、アミロースなどの澱粉質をアミラーゼ又は酸などによって部分的に加水分解して得られる澱粉分解物を用いてもよい。
澱粉を部分的に加水分解するアミラーゼとしては、例えば、Ｈａｎｄｂｏｏｋ ｏｆ Ａｍｙｌａｓｅｓ ａｎｄ Ｒｅｌａｔｅｄ Ｅｎｚｙｍｅｓ （パーガモン・プレス社 東京 １９８８年）に記載されている、α−アミラーゼ、マルトペンタオース生成アミラーゼ、マルトヘキサオース生成アミラーセなどが用いられる。これらアミラーゼとプルラナーゼ及びイソアミラーゼなどの枝切酵素を併用することも有利に実施できる。 Moreover, you may use the starch degradation product obtained by partially hydrolyzing starch substances, such as starch, amylopectin, and amylose, by amylase or an acid.
Examples of the amylase that partially hydrolyzes starch include α-amylase, maltopentaose-producing amylase, maltohexaose-producing amylase described in Handbook of Amylases and Related Enzymes (Pergamon Press, Tokyo 1988). Etc. are used. A combination of these amylases and debranching enzymes such as pullulanase and isoamylase can also be advantageously carried out.

グルコースを基質として用いた場合、他の基質の場合と異なり，加水分解反応の逆反応である縮合反応によって，α１，２結合、α１，３結合を含有する糖質を生産することができるが，その収率は転移反応に比べて低いため、経済性の点からマルトース以上の糖質が望ましい。
α−グルコシル基の受容体としては、非還元末端にグルコース残基を有していればよく、具体的にはグルコース、マルトース、マルトトリオース、コージビオース、コージトリオース、コージビオシルグルコース、ニゲロース、ニゲロトリオース、ニゲロシルグルコース、イソマルトース、イソマルトトリオース、パノース、セロビオース、ソホロース、ラミナリビオース、ゲンチオビオース、トレハロース、スクロースなどが挙げられる。また、グルコースの誘導体やＯＨ基を持つ化合物も受容体となりうる。 When glucose is used as a substrate, unlike other substrates, carbohydrates containing α1,2 bonds and α1,3 bonds can be produced by a condensation reaction that is the reverse reaction of the hydrolysis reaction. Since the yield is lower than that of the transfer reaction, carbohydrates higher than maltose are desirable from the viewpoint of economy.
As an α-glucosyl group receptor, it is only necessary to have a glucose residue at the non-reducing end. Specifically, glucose, maltose, maltotriose, cordierbiose, cordierioose, cordierbiosylglucose, nigerose, Nigerotriose, nigerosyl glucose, isomaltose, isomaltotriose, panose, cellobiose, sophorose, laminaribiose, gentiobiose, trehalose, sucrose and the like. A glucose derivative or a compound having an OH group can also serve as a receptor.

本発明のα１，２結合、α１，３結合を生成する酵素を基質に作用させる際に、α−アミラーゼ、β−アミラーゼ、グルコアミラーゼ、α−グルコシダーゼ、などで加水分解したり、ブランチングエンザイム、グルコシルトランスフェラーゼ、不均一化反応を触媒する酵素｛サイクロデキストリン・グルカノトランスフェラーゼ（以後ＣＧＴａｓｅと記載）、アミロマルターゼ｝などを作用させて、分子量や、甘味性、還元力などを調整したり、粘性を低下させたりすることも可能である。
これらの酵素の中でも、５０℃〜６５℃でも失活しない耐熱性酵素を選択すれば、本発明の転移酵素と一緒に作用させる際に、５０℃を超える高温で処理できる。
これらの酵素は、α１，２結合、α１，３結合を生成する転移酵素と同時に作用させても良いし、別々に反応させても良い。他の酵素が澱粉分解酵素の場合、本発明の転移酵素と同時に作用させると、澱粉部分分解物の生成と、当該分解物からの糖生成とが同時に進行する。また、得られたα１，２結合、α１，３結合を有する糖質を水素添加し糖アルコールにして、還元力を消滅せしめることなどの更なる加工処理を施すことも随意である。 When an enzyme that generates α1,2 bond or α1,3 bond of the present invention is allowed to act on a substrate, it can be hydrolyzed with α-amylase, β-amylase, glucoamylase, α-glucosidase, etc., blanching enzyme, Glucosyltransferase, an enzyme that catalyzes a heterogeneous reaction {cyclodextrin / glucanotransferase (hereinafter referred to as CGTase), amylomaltase}, etc. are allowed to act to adjust molecular weight, sweetness, reducing power, etc. It can also be lowered.
Among these enzymes, if a thermostable enzyme that does not inactivate at 50 ° C. to 65 ° C. is selected, it can be treated at a high temperature exceeding 50 ° C. when acting together with the transferase of the present invention.
These enzymes may be allowed to act simultaneously with transferases that generate α1,2 bonds and α1,3 bonds, or may be reacted separately. When the other enzyme is a starch degrading enzyme, when it is caused to act simultaneously with the transferase of the present invention, the production of a partially decomposed starch and the production of sugar from the decomposed product proceed simultaneously. In addition, it is optional to carry out further processing such as hydrogenation of the obtained saccharides having α1,2 bonds and α1,3 bonds to form sugar alcohols to eliminate the reducing power.

反応における基質濃度は、反応液中に溶解する濃度であればよく、１〜８０質量％の範囲、より効率よく糖質を得るためには３０〜６０質量％の条件で行うのが望ましい。
反応に用いられる温度としては酵素が反応液中で安定である温度域ならばよく、６０〜８０℃、望ましくは６０℃から７０℃で行うのが適当である。反応に用いられるｐＨは、通常ｐＨ３．５〜７．０、望ましくはｐＨ４．０〜６．０で行うのが適当である。
反応に用いる酵素濃度は濃いほうが反応時間の短縮が図れて都合がよい。酵素濃度が薄いとα１，２結合、α１，３結合を有する糖質の収率が悪くなる。 The substrate concentration in the reaction may be a concentration that dissolves in the reaction solution, and is preferably in the range of 1 to 80% by mass, and more preferably 30 to 60% by mass in order to obtain a carbohydrate more efficiently.
The temperature used in the reaction may be a temperature range in which the enzyme is stable in the reaction solution, and it is appropriate to carry out at 60 to 80 ° C., preferably 60 to 70 ° C. The pH used in the reaction is usually from 3.5 to 7.0, preferably from 4.0 to 6.0.
A higher enzyme concentration used for the reaction is advantageous because it shortens the reaction time. If the enzyme concentration is low, the yield of saccharides having α1,2 bonds and α1,3 bonds will deteriorate.

α１，２結合、α１，３結合を持つ糖質を含有する反応液は、常法により、瀘過、遠心分離などして不溶物を除去した後、活性炭で脱色、Ｈ型、ＯＨ型イオン交換樹脂で脱塩し、濃縮し、シラップ状製品とする。更に、乾燥して粉末状製品にすることも随意である。
必要ならば、更に、高度な精製をすることも随意である。例えば、イオン交換カラムクロマトグラフィーによる分画、活性炭カラムクロマトグラフィーによる分画することにより、高純度化することもできる。 Reaction solutions containing carbohydrates with α1,2 bonds and α1,3 bonds are filtered, centrifuged, etc. to remove insoluble matters, then decolorized with activated carbon, H-type, OH-type ion exchange Desalinate with resin and concentrate to syrup product. Furthermore, it is optional to dry it into a powdered product.
If necessary, further advanced purification is optional. For example, it can be highly purified by fractionation by ion exchange column chromatography or fractionation by activated carbon column chromatography.

イオン交換カラムクロマトグラフィーとしては、特開昭５８−２３７９９号公報、特開昭５８−７２５９８号公報などに開示されている塩型強酸性カチオン交換樹脂を用いるカチオン交換樹脂を用いるカラムクロマトグラフィーにより、夾雑糖類を除去して高含有画分を採取する方法が有利に実施できる。この際、固定床方式、移動床方式、疑似移動床方式のいずれの方式を採用することも随意である。   As ion exchange column chromatography, column chromatography using a cation exchange resin using a salt-type strongly acidic cation exchange resin disclosed in JP-A-58-23799, JP-A-58-72598, and the like, A method of removing contaminating saccharides and collecting a high-content fraction can be advantageously carried out. At this time, it is optional to adopt any of a fixed floor method, a moving floor method, and a simulated moving floor method.

以下に実施例を挙げて本発明を更に詳細に説明するが、本発明はこれらの実施例により限定されるものではない。   The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

＜分析方法＞
本酵素の活性はマルトースを基質とした加水分解活性にて評価した。
本酵素の分解活性として、マルトースを基質とした加水分解活性は、以下のように測定した。２０ｍＭ マルトース １００μＬに対して、２００ｍＭ酢酸緩衝液（ｐＨ４．０）を１６μＬ、酵素溶液を８４μＬ加え、５０℃で１０分間反応させた後、沸騰浴で５分間処理することで反応を停止した。反応液に生成したグルコース量をグルコースCIIテストワコー（和光純薬工業製）にて測定した。１分間に１μｍｏｌのマルトースを分解する酵素量を１Ｕと定義した。 <Analysis method>
The activity of this enzyme was evaluated by hydrolysis activity using maltose as a substrate.
As the degradation activity of this enzyme, the hydrolysis activity using maltose as a substrate was measured as follows. To 100 μL of 20 mM maltose, 16 μL of 200 mM acetate buffer (pH 4.0) and 84 μL of enzyme solution were added and reacted at 50 ° C. for 10 minutes. Then, the reaction was stopped by treating in a boiling bath for 5 minutes. The amount of glucose produced in the reaction solution was measured with Glucose CII Test Wako (manufactured by Wako Pure Chemical Industries). The amount of enzyme that decomposes 1 μmol of maltose per minute was defined as 1 U.

本酵素に各種基質を作用させた反応液の組成分析は、以下の分析法を用いて実施した。
反応液の重合度分布は以下の条件でゲルろ過カラムによる分析にて行った。
ＨＰＬＣ測定条件 ： カラム；三菱化学ＣＫ０４Ｓ、
カラム温度 ： ６５℃、
移動層組成 ： Ｄ．Ｗ．（蒸留水）
検出 ： ＲＩ（示差屈折検出機）
流速 ： ０．３５ｍＬ／分 The composition analysis of the reaction solution in which various substrates were allowed to act on this enzyme was performed using the following analysis method.
The polymerization degree distribution of the reaction solution was analyzed by gel filtration column under the following conditions.
HPLC measurement conditions: Column; Mitsubishi Chemical CK04S,
Column temperature: 65 ° C
Moving bed composition: D.D. W. (Distilled water)
Detection: RI (differential refraction detector)
Flow rate: 0.35 mL / min

得られた組成物の２、３糖成分における、α１，２結合を有するオリゴ糖（コージビオース、コージビオシルグルコース）、α１，３結合を有するオリゴ糖（ニゲロース、ニゲロシルグルコース、ニゲロトリオース）は、糖の還元末端を標識化するりん酸−フェニルヒドラジン法（特許第２８４６０５９号公報）にて分析を行った。
各ピークは、あらかじめ同条件で分析した標準糖の溶出時間と比較して同定し、ピーク面積より生成量を算出した。 Among the 2, 3 sugar components of the resulting composition, oligosaccharides having α1,2 bonds (Codybiose, Codybiosylglucose), oligosaccharides having α1,3 bonds (Nigerose, Nigerosylglucose, Nigerotriose) are Analysis was carried out by the phosphoric acid-phenylhydrazine method for labeling the reducing end of sugar (Japanese Patent No. 2846059).
Each peak was identified by comparing with the elution time of a standard sugar analyzed in advance under the same conditions, and the production amount was calculated from the peak area.

なお、２糖類の標準糖は市販試薬を用い、３糖類の標準糖には市販試薬と以下の手法で得られる組成物を用いた。ニゲロトリオース、ニゲロシルグルコースはＢｉｏｃｈｉｍｉｃａ ｅｔ Ｂｉｏｐｈｙｓｉｃａ Ａｃｔａ 第１７００巻 １８９ページ ２００４年に記載された手法に従い調製し標品とした。コージビオシルグルコース、ニゲロシルグルコースは特開２００３−１６９６６５号公報に記載された手法に従い調製し標品とした。
具体的な分析方法は、以下のように行った。
ＨＰＬＣ測定条件 ： カラム： Ｕｎｉｓｏｎ ＵＫ−Ａｍｉｎｏ ２５０×４．６ｍｍ （インタクト（株）社製）
カラム温度 ： ３５℃ Commercially available reagents were used as standard sugars for disaccharides, and commercially available reagents and compositions obtained by the following methods were used as standard sugars for trisaccharides. Nigerotriose and nigerosyl glucose were prepared according to the procedure described in Biochimica et Biophysica Acta, Vol. 1700, page 189, 2004, and used as preparations. Codybiosyl glucose and nigerosyl glucose were prepared according to the method described in JP-A No. 2003-169665 and used as preparations.
The specific analysis method was performed as follows.
HPLC measurement conditions: Column: Unison UK-Amino 250 × 4.6 mm (manufactured by Intact Corporation)
Column temperature: 35 ° C

検出機 ：蛍光検出器 Ｅｘ ３３０ｎｍ、Ｅｍ ４７０ｎｍ
流速 ：溶離液：１．０ｍＬ／分、反応液：０．４ｍＬ／分
得られた組成物全体において、１，２結合、１，３結合の糖質が含有されるか評価するためには、メチル化分析法（Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｃｈｅｍｉｓｔｒｙ 第５５巻 ２０５ページ １９６４年）を行った。
得られた組成物の３糖以上の成分に１，２結合、１，３結合の糖質が含有されるか評価するために、酵素−ＨＰＬＣ法（Ｊｏｕｒｎａｌ ｏｆ ＡＯＡＣ Ｉｎｔｅｒｎａｔｉｏｎａｌ 第８５巻 ４３５ページ ２００２年、もしくは、栄養表示基準における栄養成分等の分析方法について 平成１１年４月２６日衛新第１３号）を行った。本分析法で検出される３糖以上の成分を食物繊維含有量（Ｐｒｏｓｋｙ値）とした。
Ｊｏｕｒｎａｌ ｏｆ Ａｐｐｌｉｅｄ Ｇｌｙｃｏｓｃｉｅｎｃｅ 第４９巻 ４７９ページ ２００２年の分析例では、αグルカンの分子中に１，２結合、１，３結合が存在すると糖質のＰｒｏｓｋｙ値が高くなることが知られている。そのため、この分析法で検出される３糖以上の成分には１，２結合、１，３結合が存在すると考えられる。 Detector: Fluorescence detector Ex 330nm, Em 470nm
Flow rate: Eluent: 1.0 mL / min, Reaction solution: 0.4 mL / min In order to evaluate whether or not 1,2-bonded and 1,3-bonded saccharides are contained in the whole composition obtained, A methylation analysis method (Journal of Biochemistry, Vol. 55, p. 205, 1964) was performed.
In order to evaluate whether or not 1,3-linked and 1,3-linked saccharides are contained in the three or more saccharide components of the obtained composition, the enzyme-HPLC method (Journal of AOAC International Vol. 85, page 435, 2002) Alternatively, the analysis method for nutritional components and the like in the nutrition labeling standards was conducted on April 26, 1999, in the No. 13 of Shinsei. A component of 3 or more sugars detected by this analysis method was defined as dietary fiber content (Prosky value).
Journal of Applied Glycoscience Vol. 49, page 479 It is known from the 2002 analysis example that the presence of 1,2 and 1,3 bonds in the α-glucan molecule increases the Prosky value of carbohydrates. Therefore, it is considered that 1,2-bond and 1,3-bond exist in components of 3 or more sugars detected by this analysis method.

＜参考例：低いＤＥ成分の老化性と反応温度の関係＞
コーンスターチを常法によりαアミラーゼを用いて液化させ、濃度３０％、ＤＥ１０の澱粉液化液を得た。次いで、この澱粉液化液を５０、５５、６０、６５、７０℃の各温度に保存して反応を継続させＤＥ２８まで分解した。なお、ＤＥは粉糖関連工業分析法、（株）食品化学新聞社に記載された手法にて測定した。
反応終了時に塩酸を添加し、反応液をｐＨ４．０に調整して８０℃に１時間保存することによりαアミラーゼを失活させた。得られた各反応液中の異物を除去するため、φ９ｃｍのヌッチェにろ布を張り、ろ過助剤（セライトＫＣ５８０、米国、セライト社製）を２０ｇ重層してケーキを作成し反応液を通過させた。得られたサンプルの分解性、性状を評価するため、濁度（分光光度計ＵＶ−１２００、島津製作所、１ｃｍ石英セル）を測定した。その結果を下記表４に記載する。 <Reference example: Relationship between aging of low DE component and reaction temperature>
Corn starch was liquefied using α-amylase by a conventional method to obtain a starch liquefaction solution having a concentration of 30% and DE10. Subsequently, this starch liquefaction liquid was stored at each temperature of 50, 55, 60, 65, and 70 ° C., and the reaction was continued to decompose to DE28. In addition, DE was measured by the method described in the powdered sugar-related industrial analysis method and the Food Chemical Newspaper Co., Ltd.
Hydrochloric acid was added at the end of the reaction, and the reaction solution was adjusted to pH 4.0 and stored at 80 ° C. for 1 hour to inactivate α-amylase. In order to remove foreign substances in each of the obtained reaction liquids, a filter cloth is put on a 9 cm Nutsche, and 20 g of a filter aid (Celite KC580, manufactured by Celite, USA) is overlaid to form a cake and let the reaction liquid pass. It was. Turbidity (spectrophotometer UV-1200, Shimadzu Corporation, 1 cm quartz cell) was measured in order to evaluate the decomposability and properties of the obtained sample. The results are listed in Table 4 below.

表４に示すように、６５℃以上の反応温度であれば濁度に大きな変化はみられない。６０℃では濁度に変化が確認され、５５℃以下の反応では大幅な濁度の上昇がみられた。反応温度が低下するに従い、澱粉液化液中の分子量が大きく結晶性の高い成分が析出し、酵素が作用できなくなったため、濁度の上昇がみられたと考えられる。以上より、低いＤＥ成分（特にＤＥ０〜２０）での酵素反応では、６５℃以上の反応温度で行うのが望ましいことがわかる。   As shown in Table 4, there is no significant change in turbidity at a reaction temperature of 65 ° C. or higher. A change in turbidity was confirmed at 60 ° C., and a significant increase in turbidity was observed in reactions below 55 ° C. As the reaction temperature decreased, it was considered that the turbidity was increased because the components having high molecular weight and high crystallinity in the starch liquefaction liquid were precipitated and the enzyme could not act. From the above, it can be seen that an enzyme reaction with a low DE component (particularly DE 0 to 20) is preferably performed at a reaction temperature of 65 ° C. or higher.

＜実施例１：ＡＴＣＣ１０２５４株由来転移酵素の生産＞
培地として、澱粉：２％、ペプトン：０．２５％、酵母エキス：０．２５％、大豆粉：１％、リン酸一カリウム：０．０３％、硫酸マグネシウム：０．０１％、塩化カルシウム：０．０１％、及び塩化ナトリウム：０．０１％を含み、ｐＨ＝６．５としたものを準備し、その１００ｍＬを、５００ｍＬ容の三角フラスコに入れて、蒸気滅菌した後、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ ＡＴＣＣ １０２５４株を植菌し、３０℃の温度で３日間、振とう培養を行なった。
上記の種を滅菌水で８０倍に希釈し、その１０ｍＬを、５００ｍＬの三角フラスコ内に収容した、オートクレーブ滅菌した１０ｇの小麦ふすまに移植せしめ、水分が均一になるようによく攪拌した後、３０℃で４日間、固体培養を行なった。培養終了後、１００ｇの滅菌水で小麦ふすまを洗浄し、１６，０００×ｇ， ３０分、４℃で遠心して不溶物を除いた。酵素活性を上記＜分析方法＞に記載したマルトース分解活性にて評価した結果、培地原料１ｇあたり５３Ｕの活性を得た。 <Example 1: Production of transferase from ATCC 10254 strain>
As media, starch: 2%, peptone: 0.25%, yeast extract: 0.25%, soybean flour: 1%, monopotassium phosphate: 0.03%, magnesium sulfate: 0.01%, calcium chloride: 0.01% and sodium chloride: 0.01% containing pH = 6.5 was prepared, and 100 mL of the solution was placed in a 500 mL Erlenmeyer flask and steam sterilized, and then the Aspergillus niger ATCC 10254 The strain was inoculated and cultured with shaking at a temperature of 30 ° C. for 3 days.
The above seed was diluted 80-fold with sterilized water, 10 mL of the seed was transplanted into 10 g of wheat bran sterilized by autoclaving contained in a 500 mL Erlenmeyer flask, and stirred well so that the water content became uniform. Solid culture was carried out at 4 ° C. for 4 days. After completion of the culture, wheat bran was washed with 100 g of sterilized water and centrifuged at 16,000 × g for 30 minutes at 4 ° C. to remove insoluble matters. As a result of evaluating the enzyme activity by the maltose decomposition activity described in the above <Analysis method>, 53 U of activity was obtained per 1 g of medium raw material.

＜実施例２：ＡＴＣＣ１０２５４株由来転移酵素の精製＞
実施例１の手法にて得られる培養液１．８Ｌ、９，７００Ｕを濃縮し、以下の４段階のクロマト分離を行った。
（１）陰イオン交換クロマトグラフィー（１回目）：培養液を限外ろ過により２０ｍＭ酢酸ナトリウム緩衝液ｐＨ５．５に置換し、０．２μｍのフィルターを通過したものを酵素原液として用いた。分離樹脂はＴＯＹＯＰＥＡＲＬ ＤＥＡＥ−６５０Ｍ（東ソー（株）社製）を用い、樹脂量（以後ＣＶと記載）は１００ｍＬとした。
初発の緩衝液として２０ｍＭ 酢酸ナトリム緩衝液ｐＨ５．５を用いて、０→０．４Ｍ 塩化ナトリウムの直線的濃度勾配（８ＣＶ）で溶出し、上記＜分析方法＞で記載したマルトース分解活性を含む画分を回収した。 <Example 2: Purification of ATCC 10254 strain-derived transferase>
The culture solutions 1.8 L and 9,700 U obtained by the method of Example 1 were concentrated, and the following four stages of chromatographic separation were performed.
(1) Anion exchange chromatography (first time): The culture solution was replaced with 20 mM sodium acetate buffer pH 5.5 by ultrafiltration, and the solution passed through a 0.2 μm filter was used as the enzyme stock solution. The separation resin was TOYOPEARL DEAE-650M (manufactured by Tosoh Corporation), and the amount of resin (hereinafter referred to as CV) was 100 mL.
Elution with a linear concentration gradient (8 CV) of 0 to 0.4 M sodium chloride using 20 mM sodium acetate buffer pH 5.5 as the initial buffer, and containing the maltose-degrading activity described in <Analysis method> above Minutes were collected.

（２）陰イオン交換クロマトグラフィー（２回目）： （１）で得られたフラクションを回収し、限外ろ過により（１）と同じ組成の初発緩衝液に置換し、再度陽イオン交換樹脂による分離を行った。ＣＶ＝２５ｍＬ、０→０．２５Ｍ 塩化ナトリウムの直線的濃度勾配（１０ＣＶ）の直線的濃度勾配で溶出し、マルトース分解活性を含む画分を回収した。   (2) Anion exchange chromatography (second time): The fraction obtained in (1) is collected, replaced by the initial buffer solution having the same composition as (1) by ultrafiltration, and again separated by cation exchange resin. Went. Elution was performed with a linear concentration gradient of CV = 25 mL, 0 → 0.25 M sodium chloride (10 CV), and fractions containing maltose-degrading activity were collected.

（３）ゲルろ過クロマトグラフィー： 上記（２）の工程で得られたフラクションを回収し、次にゲルろ過クロマトグラフィーによる精製を行った。分離はＨｉＬｏａｄ １６／６０ Ｓｕｐｅｒｄｅｘ ２００ｐｇ（ＧＥヘルスケア・ジャパン（株）社製）を２本と、ＨｉＬｏａｄ １６／６０ Ｓｕｐｅｒｄｅｘ ７５ｐｇ（ＧＥヘルスケア・ジャパン（株）社製）１本を連結したカラムで行った。サンプルは限外ろ過によって０．２Ｍの塩化ナトリウムを含む２０ｍＭ 酢酸緩衝液ｐＨ５．５ に置換すると同時に、１ｍＬまで濃縮した。同じ組成のバッファーで平衡化したカラムに供して、得られたフラクションから、マルトース分解活性を含む画分を回収した。   (3) Gel filtration chromatography: The fraction obtained in the step (2) was collected, and then purified by gel filtration chromatography. Separation is a column that connects two HiLoad 16/60 Superdex 200pg (manufactured by GE Healthcare Japan) and one HiLoad 16/60 Superdex 75pg (manufactured by GE Healthcare Japan). went. The sample was replaced with 20 mM acetate buffer pH 5.5 containing 0.2 M sodium chloride by ultrafiltration and simultaneously concentrated to 1 mL. It was applied to a column equilibrated with a buffer having the same composition, and a fraction containing maltose-degrading activity was recovered from the obtained fraction.

（４）等電点クロマトグラフィー（クロマトフォーカシング）： 上記（３）の工程で得られたフラクションを回収し、クロマトフォーカシングを行った。カラムはＭｏｎｏＰ ５／２００ ＧＬ（ＧＥヘルスケア・ジャパン（株）社製）を使用した。初発バッファーは０．０２５Ｍ ヒスチジン−塩酸緩衝液ｐＨ５．５、溶出バッファーはＰｏｌｙｂｕｆｆｅｒ７４−塩酸緩衝液ｐＨ３．５（ＧＥヘルスケア・ジャパン（株）社製）を使用してｐＨ５．５から３．５の勾配によるクロマトグラフィーを行い、２Ｕのマルトース分解活性を含む画分を回収した。
得られた画分はＮａｔｉｖｅ−ポリアクリルアミドゲルゲル電気泳動（ＧＥヘルスケア・ジャパン（株）社製、ＰｈａｓｔＳｙｓｔｅｍ、以後Ｎａｔｉｖｅ−ＰＡＧＥと記載）にて純度を評価した結果を図１ａ（左方）に示す。９７，０００ダルトンの単一バンドであった。 (4) Isoelectric focusing (chromatographic focusing): The fraction obtained in the step (3) was collected and chromatofocused. As the column, MonoP 5/200 GL (manufactured by GE Healthcare Japan Co., Ltd.) was used. The initial buffer is 0.025M histidine-hydrochloric acid buffer pH 5.5, and the elution buffer is Polybuffer 74-hydrochloric acid buffer pH 3.5 (manufactured by GE Healthcare Japan Co., Ltd.) with a pH of 5.5 to 3.5. Chromatographic chromatography was performed and fractions containing 2 U of maltose-degrading activity were collected.
The obtained fraction was evaluated for purity by Native-polyacrylamide gel gel electrophoresis (GE Healthcare Japan, Inc., PhasSystem, hereinafter referred to as Native-PAGE), and the results are shown in FIG. 1a (left). . It was a single band of 97,000 daltons.

＜実施例３：ＡＴＣＣ１０２５４株由来転移酵素の性質＞
実施例２の方法で得られた酵素をＳＤＳ−ポリアクリルアミドゲル電気泳動（ＧＥヘルスケア・ジャパン（株）社製、ＰｈａｓｔＳｙｓｔｅｍ、以後ＳＤＳ−ＰＡＧＥと記載））にて分析した結果を図１ａ（右方）に示す。分子量４８，０００、５９，０００の２本が検出された。Ｎａｔｉｖｅ−ＰＡＧＥによる結果と比較すると、本酵素はヘテロダイマーの構造を持つと考えられた。
また、等電点電気泳動（ＧＥヘルスケア・ジャパン（株）社製、ＰｈａｓｔＳｙｓｔｅｍ）にて分析し、等電点は約４．９〜５．５であった（中心値が５．２）。蛋白質が糖鎖等の修飾を受けているために、広い分布を示すと考えられる。 <Example 3: Properties of transferase from ATCC 10254 strain>
The result obtained by analyzing the enzyme obtained by the method of Example 2 by SDS-polyacrylamide gel electrophoresis (manufactured by GE Healthcare Japan Ltd., “PhastSystem”, hereinafter referred to as “SDS-PAGE”) is shown in FIG. Method). Two molecules having molecular weights of 48,000 and 59,000 were detected. Compared with the results by Native-PAGE, this enzyme was considered to have a heterodimeric structure.
Moreover, it analyzed by isoelectric focusing (The GE Healthcare Japan Co., Ltd. make, PhastSystem), and the isoelectric point was about 4.9-5.5 (center value is 5.2). Since proteins are modified with sugar chains and the like, they are considered to show a wide distribution.

＜最適温度、最適ｐＨ＞
本酵素活性に対する温度、ｐＨの影響を上記＜分析方法＞に記載したマルトース分解活性の測定方法に準じて調べた。ｐＨの影響の評価では酢酸緩衝液の代わりにブリトン−ロビンソン緩衝液を用いて実施した。
その結果を図２（温度の影響）、図３（ｐＨの影響）に示した。酵素の至適温度はｐＨ４．０、１０分間反応で６５℃、至適ｐＨは５０℃、１０分間反応で３．５であった。 <Optimum temperature and pH>
The effects of temperature and pH on the enzyme activity were examined according to the method for measuring maltose decomposition activity described in <Analysis method> above. The evaluation of the influence of pH was carried out using Briton-Robinson buffer instead of acetate buffer.
The results are shown in FIG. 2 (effect of temperature) and FIG. 3 (effect of pH). The optimum temperature of the enzyme was pH 4.0 at a reaction of pH 4.0 for 10 minutes, and the optimum pH was 3.5 at a reaction of 50 ° C. for 10 minutes.

＜温度安定性、ｐＨ安定性＞
本酵素活性に対する温度、ｐＨの安定性を上記＜分析方法＞に記載したマルトース分解活性の測定方法に準じて調べた。ｐＨの安定性の評価では酢酸緩衝液の代わりにブリトン−ロビンソン緩衝液を用いて実施した。
温度安定性は酵素溶液（２０ｍＭ酢酸緩衝液、ｐＨ４．０）を各温度に３０分間保持し、氷水にて冷却後、残存する酵素活性を評価した。ｐＨ安定性は酵素溶液（各ｐＨの２０ｍＭブリトン−ロビンソン緩衝液）を４℃、２４時間保持し、ｐＨを４．０に調整した後、残存する酵素活性を評価した。
それぞれの結果を図４（温度安定性）、図５（ｐＨ安定性）に示した。本酵素の温度安定性は６５℃では初期活性の９５％以上残存し、７０℃では９０％以上残存していた。ｐＨ安定性は３．０〜５．０の範囲であった。
＜基質特異性＞
本酵素の基質特異性を５０℃、ｐＨ４．０の条件にて評価した。下記表５に示す。 <Temperature stability, pH stability>
The stability of temperature and pH with respect to the enzyme activity was examined according to the method for measuring maltose degrading activity described in <Analysis method> above. The pH stability was evaluated using Briton-Robinson buffer instead of acetate buffer.
For temperature stability, an enzyme solution (20 mM acetate buffer, pH 4.0) was held at each temperature for 30 minutes, cooled with ice water, and then the remaining enzyme activity was evaluated. The pH stability was determined by maintaining the enzyme solution (20 mM Briton-Robinson buffer solution at each pH) at 4 ° C. for 24 hours, adjusting the pH to 4.0, and then evaluating the remaining enzyme activity.
The respective results are shown in FIG. 4 (temperature stability) and FIG. 5 (pH stability). As for the temperature stability of this enzyme, 95% or more of the initial activity remained at 65 ° C, and 90% or more remained at 70 ° C. The pH stability was in the range of 3.0 to 5.0.
<Substrate specificity>
The substrate specificity of this enzyme was evaluated under the conditions of 50 ° C. and pH 4.0. It shows in Table 5 below.

マルトースや、コージビオース、ニゲロース、スクロースなどの２糖類、および、重合度２〜７のマルトオリゴ糖、アミロース、可溶性澱粉などには良好に作用してグルコースを生成する。特にα１，３結合を有する２糖類であるニゲロースが最も良好な基質である。
重合度２〜４のイソマルトオリゴ糖、パノース、αサイクロデキストリン、アミロース、グリコーゲンについては作用性が低下する。パラニトロフェニルα−グルコシド、メチルα―グルコシド、γ−シクロデキストリン、についてはわずかに反応がみられた。トレハロース、β−サイクロデキストリンには全く反応がみられなかった。 It works well on maltose, disaccharides such as cordobiose, nigerose and sucrose, and malto-oligosaccharides having a polymerization degree of 2 to 7, amylose, soluble starch and the like to produce glucose. Nigerose, which is a disaccharide having an α1,3 bond, is the best substrate.
With respect to isomaltoligosaccharides having a degree of polymerization of 2 to 4, panose, α-cyclodextrin, amylose and glycogen, the activity is lowered. A slight reaction was observed for paranitrophenyl α-glucoside, methyl α-glucoside, and γ-cyclodextrin. No reaction was observed with trehalose or β-cyclodextrin.

＜金属イオンの影響＞
本酵素の各金属イオンの影響を上記＜分析方法＞に記載したマルトース分解活性の測定方法に準じて実施した。各種イオンを反応系に添加して測定した結果を下記表６に示す。
本酵素は亜鉛イオン、銅イオンによりその活性を阻害される。ＥＤＴＡは本酵素のマルトース分解活性には影響を与えなかった。 <Influence of metal ions>
The influence of each metal ion of this enzyme was carried out according to the method for measuring maltose degrading activity described in <Analysis method> above. The results obtained by adding various ions to the reaction system are shown in Table 6 below.
The activity of this enzyme is inhibited by zinc ions and copper ions. EDTA did not affect the maltose degradation activity of this enzyme.

＜アミノ酸配列＞
本酵素に含まれるアミノ酸配列を以下の方法で分析した。ＳＤＳ−ＰＡＧＥのゲルから、タンパク質のバンドを切り出し、還元アルキル化処理でタンパク質分子中のＳ−Ｓ結合を切断した後、トリプシン消化によってペプチド断片化して分析サンプルを調製した。ＬＣ／ＭＳ（高速液体クロマトグラフ質量分析計、Ａｇｉｌｅｎｔ １１００シリーズ、アジレント・テクノロジー(株）社製）に供した。Aspergillus niger CBS 513.88株の配列と比較した結果、分子量５９，０００のバンドからは以下の配列が含有されると予測された。
配列Ｉ：ＬＬＶＥＹＱＴ ＤＥＲＬＨＶＭＩＹＤＡＤＥＥＶＹＱＶＰＥＳＶＬＰＲ、
配列ＩＩ：ＴＷＬＰＤＤＰＹＶＹＧＬＧＥＨＳＤＰＭＲ、
配列ＩＩＩ：ＩＰＬＥＴＭＷＴＤＩＤＹＭＤＫＲ、
配列ＩＶ：ＶＦＴＬＤＰＱＲ
また、分子量４８，０００のバンドからは以下の配列が含有されると予測された。
配列Ｖ：ＷＡＳＬＧＡＦＹＴＦＹＲ <Amino acid sequence>
The amino acid sequence contained in this enzyme was analyzed by the following method. A protein band was cut out from the SDS-PAGE gel, the SS bond in the protein molecule was cut by reductive alkylation treatment, and then peptide fragmentation was performed by trypsin digestion to prepare an analytical sample. LC / MS (high performance liquid chromatograph mass spectrometer, Agilent 1100 series, manufactured by Agilent Technologies) was used. As a result of comparison with the sequence of Aspergillus niger CBS 513.88 strain, it was predicted that the following sequence was contained from the band having a molecular weight of 59,000.
Sequence I: LLVEYQT DERLHVMIYDADEVYQVPESVLPR,
Sequence II: TWLPDDPYVYGLGEHSDPMR,
Sequence III: IPLETMWTDIDYMDKR,
Sequence IV: VFTLDPQR
Moreover, it was estimated that the following sequences were contained from the band of molecular weight 48,000.
Sequence V: WASLGAFYTFYR

上記配列と、既知の酵素アミノ酸配列との比較結果を図１ｂに示す。Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒの生産するα−グルコシダーゼは複数種あることが予測されている（Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ 第２５巻 第２２１ページ ２００７年）。 図１ｂに記載したアミノ酸配列はそのうち１種（Ｇｅｎｅ ａｇｄＢ、Ａｃｃｅｓｓｉｏｎ ｎｏ．Ａｎ０１ｇ１０９３０）である。
本発明の転移酵素は図１ｂの下線部で示した配列、すなわち配列番号６４−９５、１４６−１６５、２９５−３１０、３１２−３１９、６１９−６３０の５箇所でＧｅｎｅ ａｇｄＢの配列と一致が見られるため、Ｇｅｎｅ ａｇｄＢである可能性が高い。
Ｇｅｎｅ ａｇｄＢには、本発明の転移酵素のような、基質特性、転移特性、耐熱特性等があるということは今まで全く見出されていない。 The comparison result between the above sequence and the known enzyme amino acid sequence is shown in FIG. 1b. It is predicted that there are multiple types of α-glucosidase produced by Aspergillus niger (Nature Biotechnology, Vol. 25, p. 221, 2007). The amino acid sequence described in FIG. 1b is one of them (Gene agdB, Accession no. An01g10930).
The transferase of the present invention is identical with the sequence of Gene agdB at the five positions of the underlined portion in FIG. 1b, ie, SEQ ID NOs: 64-95, 146-165, 295-310, 312-319, and 619-630. Therefore, there is a high possibility of being Gene agdB.
Until now, it has not been found that Gene agdB has substrate characteristics, transfer characteristics, heat resistance characteristics and the like, like the transferase of the present invention.

比較配列として、既知のα１，４、α１，６結合オリゴ糖生成酵素（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来α−グルコシダーゼ、Ｇｅｎｅ ａｇｄＡ、Ａｃｃｅｓｓｉｏｎ ｎｏ．：ａｎｇ＿Ａｎ０４ｇ０６９２０、Ａｇｒｉｃｕｌｔｕｒａｌ ａｎｄ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ 第５５巻 ２３２７ページ １９９１年）、 α１，３、α１，６結合オリゴ糖生成酵素（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｊｏｈｎｓｏｎｉｉ由来α−グルコシダーゼ、Ｇｅｎｅ ｌｊａｇ３１、Ｂｉｏｃｈｉｍｉｅ 第９１巻 １４３４ページ ２００９年）、 α１，２、α１，３結合オリゴ糖生成酵素（ソバ由来α−グルコシダーゼ 特開２００２−６５２７３号公報、Ａｇｒｉｃｕｌｔｕｒａｌ ａｎｄ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ 第３２巻 ９２９ページ １９６８年）、とのアミノ酸配列の比較を行ったところ、これらの酵素には本発明の転移酵素と一致する配列は存在しなかった。また、上記酵素の耐熱性は、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来の酵素では６５℃、１５分で４０％以下に低下する。 Ｌａｃｔｏｂａｃｉｌｌｕｓ ｊｏｈｎｓｏｎｉｉ由来の酵素では５５℃、１０分で２０％以下に低下する。ソバ由来の酵素では６５℃、１０分で活性は残存していない。以上より耐熱性が劣っていた。   As a comparative sequence, known α1,4, α1,6-linked oligosaccharide-producing enzyme (α-glucosidase derived from Aspergillus niger, Gene agdA, Accession no .: ang_An04g06920, Agricultural and Biological Chemistry, Vol. 1, Volume 91, p. 3, α1,6-linked oligosaccharide-forming enzyme (Lactobacillus johnsonii-derived α-glucosidase, Gene ljag31, Biochimie 91, 1434, 2009), α1,2, α1,3-linked oligosaccharide-forming enzyme (buckwheat-derived α-glucosidase) No. 2002-65273, Agricultural and Biological Chemistry 32, page 929 (1968), amino acid sequences were compared with those of these enzymes, and there was no sequence corresponding to the transferase of the present invention. In addition, the heat resistance of the enzyme is lowered to 40% or less at 65 ° C. for 15 minutes with an enzyme derived from Aspergillus niger. The enzyme derived from Lactobacillus johnsonii decreases to 20% or less at 55 ° C. for 10 minutes. In the buckwheat-derived enzyme, no activity remains at 65 ° C. for 10 minutes. From the above, heat resistance was inferior.

α１，３結合オリゴ糖生成酵素：Ａｃｒｅｍｏｎｉｕｍ ｓｐ由来α−グルコシダーゼ（特開平７−５９５５９及び特開平１１−９２７６号公報に記載、株名：Ｓ４Ｇ１３）は、配列III,IV,Vと類似する配列を有していたが、各配列III,IV,Vにおいて、本発明の転移酵素と２個の差異があった。このαグルコシダーゼは、α１，３結合、α１，４結合を有する糖質を生成するが、α１，２結合転移は殆ど見られず、しかも上述したように耐熱性が劣る（５５℃、３０分で活性が６５％に低下）。   α1,3-linked oligosaccharide-producing enzyme: Acrenium sp-derived α-glucosidase (described in JP-A-7-59559 and JP-A-11-9276, strain name: S4G13) has a sequence similar to sequences III, IV, V However, each sequence III, IV, V had two differences from the transferase of the present invention. This α-glucosidase produces carbohydrates having α1,3 bonds and α1,4 bonds, but almost no α1,2 bond transition is observed, and the heat resistance is poor as described above (at 55 ° C. for 30 minutes). Activity reduced to 65%).

他のα１，３結合オリゴ糖生成酵素：Ａｃｒｅｍｏｎｉｕｍ ｉｍｐｌｉｃａｔｕｍ由来α−グルコシダーゼ（Ｂｉｏｃｈｉｍｉｃａ ｅｔ Ｂｉｏｐｈｙｓｉｃａ Ａｃｔａ 第１７００巻 第１８９ページ ２００４年、及び特開２００４−１７３６５０号広報）では、配列III,IV,Vと類似する配列を有していたが、配列III,IVにおいて本発明の転移酵素と２個の差異、配列Vにおいて１個の差異があった。このαグルコシダーゼは、α１，３結合、α１，４結合を有する糖質を生成するが、α１，２結合転移は殆ど見られず、耐熱性が劣る（６０℃、１５分で活性がなくなると記載）。
従って、上記配列I〜Vのいずれか１以上と一致する物が、耐熱性を備え、かつ、新規の糖組成物を生産可能なことが分かる。なお、α１，２、α１，３結合オリゴ糖生成酵素（Ｐａｅｃｉｌｏｍｙｃｅｓ ｌｉｌａｃｉｎｕｓ由来α―グルコシダーゼ 特開２００３−１６９６６５号公報）の配列は公開されていなかった。 Other α1,3-linked oligosaccharide-producing enzymes: α-glucosidase derived from Acremonium implicatum (Biochimica et Biophysica Acta Vol. 1700, page 189 2004 and JP 2004-173650 A), similar to sequences III, IV, V However, there were two differences in the sequences III and IV from the transferase of the present invention and one difference in the sequence V. This α-glucosidase produces carbohydrates with α1,3 bonds and α1,4 bonds, but there is almost no α1,2 bond transition, and heat resistance is poor (described as having no activity at 60 ° C for 15 minutes) ).
Therefore, it can be seen that a substance matching any one or more of the above sequences I to V has heat resistance and can produce a novel sugar composition. The sequence of α1,2 and α1,3-linked oligosaccharide-producing enzyme (α-glucosidase derived from Paecilomyces lilacinus, JP-A No. 2003-169665) was not disclosed.

＜実施例４：精製酵素によるマルトースの転移物＞
実施例２の方法で得られた精製酵素、０．５ｍＬ（０．７Ｕ／ｍＬ）を、４５％マルトース１ｍＬに作用させ（最終濃度３０％）、６５℃で反応を開始した。
所定時間反応後に沸騰浴槽で１０分間加熱し、酵素を失活させた。
この反応液中の重合度分布を上記＜分析方法＞に記載したゲルろ過クロマトグラフィーで分析した。また、２糖、３糖の異性体の組成を＜分析方法＞に記載したポストカラム法にて分析し、２種のＨＰＬＣより各成分の組成を算出した。反応開始後４８時間における２種のＨＰＬＣ分析におけるクロマトグラムを図６ａ、図６ｂに示す。 <Example 4: Transfer product of maltose by purified enzyme>
The purified enzyme obtained by the method of Example 2, 0.5 mL (0.7 U / mL) was allowed to act on 1 mL of 45% maltose (final concentration 30%), and the reaction was started at 65 ° C.
After the reaction for a predetermined time, the enzyme was inactivated by heating in a boiling bath for 10 minutes.
The polymerization degree distribution in the reaction solution was analyzed by gel filtration chromatography described in <Analysis method> above. Further, the composition of isomers of disaccharides and trisaccharides was analyzed by the post-column method described in <Analysis Method>, and the composition of each component was calculated from two types of HPLC. Chromatograms of two types of HPLC analysis 48 hours after the start of the reaction are shown in FIGS. 6a and 6b.

反応開始後４８時間での分子量分布は、単糖（ＤＰ１）２５．２％、２糖（ＤＰ２）２９．５％、３糖（ＤＰ３）２０．８％、４糖以上（ＤＰ４＋）２４．５％となった。２、３糖中の異性体分析より、α１，２結合を有する低分子糖質（以後、α１，２結合オリゴ糖と記載）が１５．６％、α１，３結合を有する低分子糖質（以後、α１，３結合オリゴ糖と記載）が１４．９％となった。コージオリゴ糖のうち二糖類であるコージビオースが１１．２％、三糖類であるコージビオシルグルコースが４．４％得られた。ニゲロオリゴ糖のうち二糖類であるニゲロースが８．５％、三糖類であるニゲロトリオースが２．５％、ニゲロシルグルコースが３．９％であった。２糖、３糖成分中のα１，２結合オリゴ糖とα１，３結合オリゴ糖の割合の合計は６１％となっていた。
また、上記＜分析方法＞に記載した手法に従い、Ｐｒｏｓｋｙ値を算出した結果、３１％となった。反応液では３糖以上（ＤＰ３＋）の成分は４５．３％となることより、ＤＰ３＋成分中の６６％はα１，２、α１，３結合を持つ糖質が含有されると考えられた。 The molecular weight distribution at 48 hours after the start of the reaction was as follows: monosaccharide (DP1) 25.2%, disaccharide (DP2) 29.5%, trisaccharide (DP3) 20.8%, tetrasaccharide or more (DP4 +) 24.5 %. Analysis of isomers in 2,3 sugars revealed that 15.6% of low molecular weight carbohydrates having α1,2 bonds (hereinafter referred to as α1,2 linked oligosaccharides) and low molecular weight carbohydrates having α1,3 bonds ( Hereinafter, α1,3-linked oligosaccharide) was 14.9%. Among cordierigosaccharides, 11.2% of cordobiose, a disaccharide, and 4.4% of cordobiosyl glucose, a trisaccharide were obtained. Among the nigerooligosaccharides, the disaccharide nigerose was 8.5%, the trisaccharide nigerotriose was 2.5%, and the nigerosyl glucose was 3.9%. The total ratio of α1,2-linked oligosaccharide and α1,3-linked oligosaccharide in the disaccharide and trisaccharide components was 61%.
Further, the Prosky value was calculated according to the method described in <Analysis Method> above, and was 31%. In the reaction solution, the component of 3 or more sugars (DP3 +) was 45.3%, so 66% of the DP3 + component was considered to contain carbohydrates having α1,2 and α1,3 bonds.

反応開後の７２時間までの経時変化を図７ａ、図７ｂに示す。重合度（ＤＰ）分布では、基質であるマルトースが減少と共に３糖成分が生成し、続いてさらに重合度が増加していく。ＤＰ３、ＤＰ４＋成分の合成と共に、α１，２結合オリゴ糖、α１，３結合オリゴ糖の増加がみられ、同様にＰｒｏｓｋｙ値も増加する。そのため、α１，２結合、α１，３結合を有する糖質の生成量は反応時間と共に転移物として蓄積してくることがわかる。   Changes with time up to 72 hours after the reaction is opened are shown in FIGS. 7a and 7b. In the degree of polymerization (DP) distribution, the maltose as a substrate decreases and a trisaccharide component is generated, and then the degree of polymerization further increases. As the DP3 and DP4 + components are synthesized, α1,2 linked oligosaccharides and α1,3 linked oligosaccharides are increased, and the Prosky value is also increased. Therefore, it can be seen that the amount of carbohydrates having α1,2 bonds and α1,3 bonds accumulates as transfer products with the reaction time.

＜実施例５：粗酵素によるマルトースの転移物＞
実施例１の方法で得られた粗酵素、０．５ｍＬ（４．０Ｕ／ｍＬ）を、４５％マルトース１ｍＬに作用させ（最終濃度３０％）、６５℃で反応を開始した。反応開始７２時間後に沸騰浴槽で１０分間加熱し、酵素を失活させた。
この反応液中の重合度分布、２糖、３糖の異性体分析を実施例４と同様の手法を用いて行った。
その結果を下記表７に示す。実施例４と同様にα１，２結合オリゴ糖、α１，３結合オリゴ糖の生成がみられ、反応液の２糖、３糖成分中のα１，２結合オリゴ糖とα１，３結合オリゴ糖の割合の合計は５２％となっていた。
また、Ｐｒｏｓｋｙ値を算出した結果、３１％となり、反応液３糖以上の成分中の６１％はα１，２、α１，３結合を持つ糖質が含有されると考えられた。 <Example 5: Transfer product of maltose by crude enzyme>
The crude enzyme, 0.5 mL (4.0 U / mL) obtained by the method of Example 1 was allowed to act on 1 mL of 45% maltose (final concentration 30%), and the reaction was started at 65 ° C. The enzyme was inactivated by heating for 10 minutes in a boiling bath 72 hours after the start of the reaction.
The polymerization degree distribution in this reaction solution and the isomer analysis of disaccharides and trisaccharides were performed using the same method as in Example 4.
The results are shown in Table 7 below. In the same manner as in Example 4, the production of α1,2 linked oligosaccharides and α1,3 linked oligosaccharides was observed, and α1,2 linked oligosaccharides and α1,3 linked oligosaccharides in the disaccharide and trisaccharide components of the reaction solution were observed. The total percentage was 52%.
As a result of calculating the Prosky value, it was 31%, and it was considered that 61% of the components of the reaction solution having 3 or more sugars contained carbohydrates having α1,2, α1,3 bonds.

＜実施例６：他のＡｓｐｅｒｇｉｌｌｕs属由来転移酵素の調製＞
Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ ｖａｎ Ｔｉｅｇｈｅｍ ｖａｒ． ｎｉｇｅｒ ｆｓｐ． ｈｅｎｎｅｂｅｒｇｉｉ Ｂｌｏｃｈｗｉｔｚ ｅｘ Ａｌ−Ｍｕｓａｌｌａｍ（ＮＢＲＣ ４０４３）、Ａｓｐｅｒｇｉｌｌｕｓ ａｗａｍｏｒｉ（ＡＴＣＣ１４３３１）を用いた以外は、実施例１と同様に液体培地による前培養、固層培地による本培養を行い、培養１ｇ当たり、４１Ｕ、３５Ｕの活性を持つ培養抽出液を得た。
それぞれの抽出液より、実施例２の方法に準じて、２回の陰イオン交換クロマトグラフィー、ゲルろ過クロマトグラフィーを行い、部分精製酵素を得た。これらの酵素の性質を実施例３にて記載した手法に従い評価した。ＡＴＣＣ１０２５４株の場合とともに表８にまとめた。 <Example 6: Preparation of other Aspergillus-derived transferases>
Aspergillus niger van Tieghem var. niger fsp. Henbergii Blochwitz ex Al-Musalllam (NBRC 4043) and Aspergillus awamori (ATCC14331) were used in the same manner as in Example 1 except that a preculture using a liquid medium and a main culture using a solid layer medium were performed. A culture extract having the following activity was obtained.
From each extract, the anion exchange chromatography and the gel filtration chromatography were performed twice according to the method of Example 2 to obtain a partially purified enzyme. The properties of these enzymes were evaluated according to the procedure described in Example 3. The results are shown in Table 8 together with ATCC10254 strain.

これらの部分精製酵素を用いて、実施例５に従って、マルトースを基質とした場合の転移反応を行い、その生成物の組成を分析した結果、ＡＴＣＣ１０２５４由来の酵素の場合と同様にα１，２結合、α１，３結合を有する糖質が生成することを確認した。
＜実施例７：精製酵素によるマルトペンタオースの転移物＞
実施例２の方法で得られた精製酵素、０．５ｍＬ（０．７Ｕ／ｍＬ）を、４５％マルトペンタオース１ｍＬに作用させ（最終濃度３０％）、６５℃で反応を開始した。反応開始７２時間後に沸騰浴槽で１０分間加熱し、酵素を失活させた。この反応液中の重合度分布、２糖、３糖の異性体分析を実施例４と同様な手法を用いて行い、各成分の組成分析を行った。分析結果を表９に示す。
実施例４同様にα１，２結合オリゴ糖、α１，３結合オリゴ糖の生成がみられ、反応液の２糖、３糖成分中のα１，２結合オリゴ糖とα１，３結合オリゴ糖の割合の合計は５４％となっていた。また、Ｐｒｏｓｋｙ値を算出した結果、４４％となり、反応液３糖以上の成分中の７５％はα１，２、α１，３結合を持つ糖質が含有されると考えられた。 Using these partially purified enzymes, according to Example 5, a transfer reaction was performed when maltose was used as a substrate, and the composition of the product was analyzed. As a result, α1,2 bond as in the case of the enzyme derived from ATCC10254, It was confirmed that carbohydrates having α1,3 bonds were produced.
<Example 7: Transfer product of maltopentaose by purified enzyme>
The purified enzyme 0.5 mL (0.7 U / mL) obtained by the method of Example 2 was allowed to act on 1 mL of 45% maltopentaose (final concentration 30%), and the reaction was started at 65 ° C. The enzyme was inactivated by heating for 10 minutes in a boiling bath 72 hours after the start of the reaction. The polymerization degree distribution in this reaction solution, disaccharide, and trisaccharide isomer analysis were performed using the same method as in Example 4, and the composition analysis of each component was performed. The analysis results are shown in Table 9.
The production of α1,2 linked oligosaccharide and α1,3 linked oligosaccharide was observed in the same manner as in Example 4, and the ratio of α1,2 linked oligosaccharide to α1,3 linked oligosaccharide in the disaccharide and trisaccharide components of the reaction solution. The total was 54%. As a result of calculating the Prosky value, it was 44%, and it was considered that 75% of the components of the reaction solution having 3 or more sugars contained saccharides having α1,2, α1,3 bonds.

＜実施例８：精製酵素による液化液の転移物、αアミラーゼ、枝切酵素との同時反応＞
高分子のαグルカンを基質とした転移反応は以下のように行った。ＤＥ１０の３０％（Ｗ／Ｗ）コーンスターチ液化液１００ｇをｐＨ６．０に調整した。液温６５℃にて、実施例２の方法で得られた精製酵素を０．７Ｕ／ｇＤＳ（ＤＳ：Ｄｒｙ Ｓｏｌｉｄ ｂａｓｉｓ、乾燥固形物基準）、αアミラーゼ（ターマミル１２０Ｌ、ノボザイムズ社製）０．０１％／ｇＤＳ、枝切酵素（クライスターゼＰＬＦ、天野エンザイム社製）０．１％／ｇＤＳを添加し、７２時間反応した。
反応開始７２時間後に沸騰浴槽で１０分間加熱し、酵素を失活させた。この反応液中の重合度分布、２糖、３糖の異性体分析を実施例４と同様な手法を用いて行い、各成分の組成分析を行った。分析結果を表１０に示す。
実施例４と同様にα１，２結合オリゴ糖、α１，３結合オリゴ糖の生成がみられ、反応液の２糖、３糖成分中のα１，２結合オリゴ糖とα１，３結合オリゴ糖の割合の合計は５７％となっていた。
また、Ｐｒｏｓｋｙ値を算出した結果、５５％となり、反応液３糖以上の成分中の７７％はα１，２、α１，３結合を持つ糖質が含有されると考えられた。 <Example 8: Simultaneous reaction of liquefied liquid transferred with purified enzyme, α-amylase, and debranching enzyme>
The transfer reaction using a polymeric α-glucan as a substrate was carried out as follows. 100 g of 30% (W / W) corn starch liquefied liquid of DE10 was adjusted to pH 6.0. At a liquid temperature of 65 ° C., the purified enzyme obtained by the method of Example 2 was 0.7 U / gDS (DS: Dry Solid basis, dry solid standard), α-amylase (Termamyl Mill 120L, Novozymes) 0.01 % / GDS and debranching enzyme (Chrytase PLF, Amano Enzyme) 0.1% / gDS were added and reacted for 72 hours.
The enzyme was inactivated by heating for 10 minutes in a boiling bath 72 hours after the start of the reaction. The polymerization degree distribution in this reaction solution, disaccharide, and trisaccharide isomer analysis were performed using the same method as in Example 4, and the composition analysis of each component was performed. The analysis results are shown in Table 10.
In the same manner as in Example 4, the production of α1,2 linked oligosaccharides and α1,3 linked oligosaccharides was observed, and α1,2 linked oligosaccharides and α1,3 linked oligosaccharides in the disaccharide and trisaccharide components of the reaction solution were observed. The total percentage was 57%.
As a result of calculating the Prosky value, it was 55%, and it was considered that 77% of the components of the reaction solution having 3 or more sugars contained carbohydrates having α1,2, α1,3 bonds.

＜実施例９：粗酵素による液化液の転移物、αアミラーゼ、枝切酵素との同時反応、ゲルろ過カラムによる分離＞
高分子のαグルカンを基質とした転移反応は以下のように行った。ＤＥ１０の３０％（Ｗ／Ｗ）コーンスターチ液化液１００ｇをｐＨ６．０に調整した。液温６５℃にて、実施例１の方法で得られた粗酵素を４．０Ｕ／ｇＤＳ、αアミラーゼ（ターマミル１２０Ｌ、ノボザイムズ社製）０．０１％／ｇＤＳ、枝切酵素（クライスターゼＰＬＦ、天野エンザイム社製）０．１％／ｇＤＳを量添加し、７２時間反応した。
反応開始７２時間後に沸騰浴槽で１０分間加熱し、酵素を失活させた。この反応液中の重合度分布、２糖、３糖の異性体分析を実施例４と同様な手法を用いて行い、各成分の組成分析を行った。分析結果を表１１に示す。
実施例４と同様にα１，２結合オリゴ糖、α１，３結合オリゴ糖の生成がみられ、反応液の２糖、３糖成分中のα１，２結合オリゴ糖とα１，３結合オリゴ糖の割合の合計は５３％となっていた。
また、Ｐｒｏｓｋｙ値を算出した結果、５５％となり、反応液３糖以上の成分中の７４％はα１，２、α１，３結合を持つ糖質が含有されると考えられた。 <Example 9: Transfer product of liquefied liquid by crude enzyme, simultaneous reaction with α-amylase and debranching enzyme, separation by gel filtration column>
The transfer reaction using a polymeric α-glucan as a substrate was carried out as follows. 100 g of 30% (W / W) corn starch liquefied liquid of DE10 was adjusted to pH 6.0. The crude enzyme obtained by the method of Example 1 at a liquid temperature of 65 ° C. was 4.0 U / gDS, α-amylase (Termamyl 200L, Novozymes) 0.01% / gDS, debranching enzyme (Chrytase PLF, (Amano Enzyme) 0.1% / gDS was added in an amount and reacted for 72 hours.
The enzyme was inactivated by heating for 10 minutes in a boiling bath 72 hours after the start of the reaction. The polymerization degree distribution in this reaction solution, disaccharide, and trisaccharide isomer analysis were performed using the same method as in Example 4, and the composition analysis of each component was performed. The analysis results are shown in Table 11.
In the same manner as in Example 4, the production of α1,2 linked oligosaccharides and α1,3 linked oligosaccharides was observed, and α1,2 linked oligosaccharides and α1,3 linked oligosaccharides in the disaccharide and trisaccharide components of the reaction solution were observed. The total percentage was 53%.
As a result of calculating the Prosky value, it was 55%, and it was considered that 74% of the components of the reaction solution having 3 or more sugars contained carbohydrates having α1,2 and α1,3 bonds.

Ｐｒｏｓｋｙ値の測定で得たサンプルを精製・濃縮し、固形分１ｇの処理物を得た。サンプルをゲルろ過樹脂Ｂｉｏ−Ｇｅｌ Ｐ２（バイオラッド社製）を充填したゲルろ過カラム（φ６ｃｍ×１００ｃｍ）に添加してクロマト分離を行い、３糖以上の成分を回収した。常法により精製、濃縮、凍結乾燥をして０．２ｇの分画物を得た。   The sample obtained by measuring the Prosky value was purified and concentrated to obtain a processed product having a solid content of 1 g. The sample was added to a gel filtration column (φ6 cm × 100 cm) packed with gel filtration resin Bio-Gel P2 (Bio-Rad), and chromatographic separation was performed to recover a trisaccharide or higher component. Purification, concentration, and lyophilization were performed by a conventional method to obtain 0.2 g of a fraction.

分画したサンプルに含有する結合様式を確認するため、＜分析方法＞に記載したメチル化分析をおこなった。結果を下記表１２に示す。Ｐｒｏｓｋｙ値として算出される３糖以上の成分中には１，２結合、１，３結合が含有されることが分かる。   In order to confirm the binding mode contained in the fractionated sample, the methylation analysis described in <Analysis method> was performed. The results are shown in Table 12 below. It can be seen that 1,3-bond and 1,3-bond are contained in the components of 3 or more sugars calculated as the Prosky value.

＜実施例１０：粗酵素（ＡＴＣＣ１４３３１由来）による液化液の転移物、αアミラーゼ、枝切酵素との同時反応、擬似移動層クロマト分離装置による分離＞
実施例６の方法で得られたＡｓｐｅｒｇｉｌｌｕｓ ａｗａｍｏｒｉ（ＡＴＣＣ１４３３１）由来の粗酵素を用い、実施例９に記載した手法で高分子のαグルカンを基質にした反応を実施した。この反応液中の重合度分布、２糖、３糖の異性体分析を実施例４と同様な手法を用いて行い、各成分の組成分析を行った。分析結果を表１３に示す。
実施例４と同様にα１，２結合オリゴ糖、α１，３結合オリゴ糖の生成がみられ反応液の２糖、３糖成分中のα１，２結合オリゴ糖とα１，３結合オリゴ糖の割合の合計４４％となっていた。また、Ｐｒｏｓｋｙ値を算出した結果、５５％となり、反応液３糖以上の成分中の６５％はα１，２、α１，３結合を持つ糖質が含有されると考えられた。 <Example 10: Transfer of liquefied liquid by crude enzyme (derived from ATCC 14331), simultaneous reaction with α-amylase, debranching enzyme, separation by simulated moving bed chromatographic separation apparatus>
Using the crude enzyme derived from Aspergillus awamori (ATCC 14331) obtained by the method of Example 6, a reaction using a polymeric α-glucan as a substrate was performed by the method described in Example 9. The polymerization degree distribution in this reaction solution, disaccharide, and trisaccharide isomer analysis were performed using the same method as in Example 4, and the composition analysis of each component was performed. The analysis results are shown in Table 13.
In the same manner as in Example 4, α1,2-linked oligosaccharide and α1,3-linked oligosaccharide were produced, and the ratio of α1,2-linked oligosaccharide and α1,3-linked oligosaccharide in the disaccharide and trisaccharide components of the reaction solution The total was 44%. As a result of calculating the Prosky value, it was 55%, and it was considered that 65% of the components of the reaction solution having 3 or more sugars contained carbohydrates having α1,2, α1,3 bonds.

得られた反応液ＤＳ３０％、５０ｋｇをｐＨ５．０に調製し、αアミラーゼ（ターマミル１２０Ｌ、ノボザイムズ社製）を３％／ｇＤＳ、グルコアミラーゼ（ＡＭＧ３００、ノボザイムズ社製）を８％／ｇＤＳ添加して、６０℃で１時間反応した。酵素分解後のサンプルを精製・濃縮し、固形分１０ｋｇの処理物を得た。
酵素処理物は塩型強酸性カチオン交換樹脂（ＦＸ１０４０オルガノ社製）を充填した連続式クロマト分離装置（トレソーネ、オルガノ社製）により分離を行い、３糖以上の成分を分画し、常法により精製、濃縮、噴霧乾燥を行い、固形分２ｋｇの分画物を得た。分画したサンプルの糖組成はＤＰ１、ＤＰ２、ＤＰ３、ＤＰ４＋が、０．０、０．０、２７．１、７２．９％となった。また、＜分析方法＞に記載した手法に従い、Ｐｒｏｓｋｙ値を算出した結果、９３％となった。
得られたサンプルに含有する結合様式を確認するため、実施例９と同様にメチル化分析を行った。結果上記表１４に示す。クロマト分画物には１，２結合、１，３結合が存在することがわかる。 The obtained reaction solution DS 30%, 50 kg was adjusted to pH 5.0, α-amylase (Termamyl 120L, Novozymes) 3% / gDS, glucoamylase (AMG300, Novozymes) 8% / gDS was added. , Reacted at 60 ° C. for 1 hour. The sample after enzymatic degradation was purified and concentrated to obtain a processed product having a solid content of 10 kg.
The enzyme-treated product is separated by a continuous chromatographic separation device (Tresone, manufactured by Organo) filled with a salt-type strongly acidic cation exchange resin (manufactured by FX1040 Organo) to fractionate components of three or more sugars by a conventional method. Purification, concentration, and spray drying were performed to obtain a fraction having a solid content of 2 kg. The sugar composition of the fractionated sample was DP1, DP2, DP3, DP4 + of 0.0, 0.0, 27.1, 72.9%. Further, according to the method described in <Analysis method>, the Prosky value was calculated to be 93%.
In order to confirm the binding mode contained in the obtained sample, methylation analysis was performed in the same manner as in Example 9. Results are shown in Table 14 above. It can be seen that 1,2 bonds and 1,3 bonds exist in the chromatographic fraction.

＜実施例１１：粗酵素による液化液の転移物、枝切酵素、ＣＧＴａｓｅとの同時反応＞
高分子のαグルカンを基質とし、ＣＧＴａｓｅと組み合わせた転移反応は以下のように行った。ＤＥ１０の３０％（Ｗ／Ｗ）コーンスターチ液化液１００ｇをｐＨ６．０に調整した。液温60℃にて実施例１で得られる粗酵素４．０Ｕ／ｇＤＳと、枝切酵素（クライスラーゼＰＬＦ、天野エンザイム社製）を０．２％／ｇＤＳと、ＣＧＴａｓｅ（コンチザイム、天野エンザイム社製）を０．８％／ｇＤＳ所定量添加し、７２時間反応した。
反応終了時に沸騰浴槽で１０分間加熱し、酵素を失活させた。この反応液中の重合度分布、２糖、３糖の異性体分析を実施例４と同様な手法を用いて行い、各成分の組成分析を行った。分析結果を表１５に示す。
実施例４と同様にα１，２結合オリゴ糖、α１，３結合オリゴ糖の生成がみられ、反応液の２糖、３糖成分中のα１，２結合オリゴ糖とα１，３結合オリゴ糖の割合の合計は５６％となっていた。
また、Ｐｒｏｓｋｙ値を算出した結果、７４％となり、反応液３糖以上の成分中の７９％はα１，２、α１，３結合を持つ糖質が含有されると考えられた。実施例９〜１０における分析と同様に、構成分子中に１，２、１，３結合が生じていると推定される。 <Example 11: Transfer of liquefied liquid with crude enzyme, debranching enzyme, and simultaneous reaction with CGTase>
A transfer reaction in which a polymer α-glucan was used as a substrate and combined with CGTase was performed as follows. 100 g of 30% (W / W) corn starch liquefied liquid of DE10 was adjusted to pH 6.0. The crude enzyme 4.0 U / gDS obtained in Example 1 at a liquid temperature of 60 ° C., the debranching enzyme (Chrysase PLF, Amano Enzyme) 0.2% / gDS, and CGTase (Contizyme Amano Enzyme) A predetermined amount of 0.8% / gDS was added and reacted for 72 hours.
At the end of the reaction, it was heated in a boiling bath for 10 minutes to deactivate the enzyme. The polymerization degree distribution in this reaction solution, disaccharide, and trisaccharide isomer analysis were performed using the same method as in Example 4, and the composition analysis of each component was performed. The analysis results are shown in Table 15.
In the same manner as in Example 4, the production of α1,2 linked oligosaccharides and α1,3 linked oligosaccharides was observed, and α1,2 linked oligosaccharides and α1,3 linked oligosaccharides in the disaccharide and trisaccharide components of the reaction solution were observed. The total percentage was 56%.
As a result of calculating the Prosky value, it was 74%, and it was considered that 79% of the components of the reaction solution having 3 or more sugars contained carbohydrates having α1,2, α1,3 bonds. Similar to the analysis in Examples 9 to 10, it is presumed that 1, 2, 1, 3 bonds are formed in the constituent molecules.

＜実施例１２：精製酵素によるグルコース誘導体の反応＞
糖供与体として３０％マルトース、受容体としてＮ−アセチルグルコサミン（ＧｌｃＮＡｃ）３％を含む、２０ｍＭ 酢酸緩衝液ｐＨ６．０、１０ｇに対して、実施例３で調製した精製酵素を０．７Ｕ／ｇＤＳ作用させ、６５℃、２４時間反応させた。反応終了時に沸騰浴槽で１０分間加熱して酵素を失活させて、下記条件の ＨＰＬＣ分析を行った。 <Example 12: Reaction of glucose derivative with purified enzyme>
The purified enzyme prepared in Example 3 was added at 0.7 U / g DS to 10 g of 20 mM acetate buffer pH 6.0 containing 30% maltose as a sugar donor and 3% of N-acetylglucosamine (GlcNAc) as an acceptor. And allowed to react at 65 ° C. for 24 hours. At the end of the reaction, the enzyme was inactivated by heating in a boiling bath for 10 minutes, and HPLC analysis was performed under the following conditions.

カラム：Ｕｎｉｓｏｎ ＵＫ−Ａｍｉｎｏ ３ｕｍ ２５０×４．６ｍｍ
カラム温度：４０℃
溶離液：７５％アセトニトリル
流速：１．０ｍｌ／分
検出機：ＵＶ（２８０ｎｍ） Column: Unison UK-Amino 3um 250x4.6mm
Column temperature: 40 ° C
Eluent: 75% acetonitrile Flow rate: 1.0 ml / min Detector: UV (280 nm)

ＨＰＬＣ分析のチャートを図８に示す。溶出時間４．７分付近のＧｌｃＮＡｃのピークに対して、溶出時間６．０分、７．５分に新たなピークが生じ、重合物が形成されたことがわかる。チャートの面積比より、ＧｌｃＮＡｃの２５％が配糖化されていることが判明した。また、ＧｌｃＮＡｃのみを基質として酵素を作用させた場合にはこのような重合物はみられなかった。   A chart of the HPLC analysis is shown in FIG. It can be seen that a new peak was formed at the elution time of 6.0 minutes and 7.5 minutes with respect to the GlcNAc peak at an elution time of about 4.7 minutes, and a polymer was formed. From the area ratio of the chart, it was found that 25% of GlcNAc was glycosylated. Moreover, such a polymer was not observed when the enzyme was allowed to act using only GlcNAc as a substrate.

本発明の転移酵素は、α１，２、１，３結合の糖質の生成能が高く、耐熱性も高いことから、工業的に利用する糖質製造酵素としての価値を有する。
本発明の転移酵素を用いて生成された糖質は、飲料や菓子等の食品だけでなく、医薬、化粧品、家畜飼料等広い分野で添加剤として使用することができる。また、その糖質は、水に分散しやすく、飲料や食品等の添加剤として利用価値が高い、水溶性食物繊維としての利用も可能である。 Since the transfer enzyme of the present invention has a high ability to produce α1,2,1,3-linked saccharides and has high heat resistance, it has value as a saccharide-producing enzyme for industrial use.
The saccharide produced using the transferase of the present invention can be used as an additive in a wide range of fields such as pharmaceuticals, cosmetics and livestock feed as well as foods such as beverages and confectionery. Moreover, the carbohydrate is easily dispersible in water, and can be used as a water-soluble dietary fiber that has high utility value as an additive for beverages and foods.

Claims (14)

α−グルカンと、α−グルコオリゴ糖と、グルコースとからなる群より選択されるいずれか１種以上の糖類に作用して、α１，２グルコシド結合を有する糖質と、α１，３グルコシド結合を有する糖質を生成し、６５℃以上の温度で酵素反応可能な耐熱性のＡｓｐｅｒｇｉｌｌｕｓ属由来の転移酵素。   It acts on any one or more saccharides selected from the group consisting of α-glucan, α-glucooligosaccharide and glucose, and has α1,3 glucoside bond and α1,3 glucoside bond A thermostable Aspergillus-derived transferase that produces carbohydrates and is capable of enzymatic reaction at a temperature of 65 ° C. or higher. 耐熱性が５０〜６５℃のいずれかの温度でｐＨ５．０、３０分間保持において、９０％以上の残存活性を有することを特徴とする請求項１記載の転移酵素。   The transferase according to claim 1, wherein the transferase has a residual activity of 90% or more when kept at pH 5.0 for 30 minutes at any temperature of 50 to 65 ° C. 下記の理化学的性質を有する請求項１又は請求項２のいずれか１項記載の転移酵素。
（１）作用：マルトース、ニゲロース、コージビオース、マルトオリゴ糖などα−グルコオリゴ糖および、アミロース、可溶性澱粉などのα−グルカンの非還元末端側のα−グルコシド結合を分解し、グルコースを遊離する。また、スクロースを分解する活性を持つ。更に、α−グルカン、α−グルコオリゴ糖、グルコースから選ばれる少なくとも一種を含む糖液に作用して、α１，２グルコシド結合、α１，３結合グルコシドを有する糖質を産生する。
（２）分子量：ＳＤＳ−ゲル電気泳動法により、４８，０００、５９，０００ダルトン。
（３）等電点：アンフォライン含有電気泳動法により、ｐＩ４．９〜５．５（中心値が５．２）
（４）至適温度：ｐＨ４．０、１０分間反応で、６５℃
（５）至適ｐＨ：５０℃、１０分間反応で、ｐＨ３．５
（６）温度安定性：ｐＨ４．０、３０分間保持で、６５℃では初期活性の９０％以上の残存
（７）ｐＨ安定性：４℃、２４時間保持で、ｐＨ３．０〜５．５ The transferase according to any one of claims 1 and 2, which has the following physicochemical properties.
(1) Action: Decomposes α-glucooligosaccharides such as maltose, nigerose, cordobiose and maltooligosaccharide, and α-glucoside bonds on the non-reducing terminal side of α-glucans such as amylose and soluble starch, and releases glucose. In addition, it has an activity of decomposing sucrose. Furthermore, it acts on a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide and glucose to produce a carbohydrate having an α1,2 glucoside bond and an α1,3 linked glucoside.
(2) Molecular weight: 48,000, 59,000 daltons by SDS-gel electrophoresis.
(3) Isoelectric point: pI of 4.9 to 5.5 (center value is 5.2) according to an ampholine-containing electrophoresis method
(4) Optimum temperature: pH 4.0, reaction at 10 minutes, 65 ° C
(5) Optimal pH: 50 ° C., 10 minutes reaction, pH 3.5
(6) Temperature stability: pH 4.0, maintained for 30 minutes, remaining at 90% or more of initial activity at 65 ° C. (7) pH stability: pH 3.0-5.5 at 4 ° C., maintained for 24 hours Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ属菌株またはＡｓｐｅｒｇｉｌｌｕｓ ａｗａｍｏｒｉ属菌株のいずれか一方から生産される請求項１乃至請求項３のいずれか１項記載の転移酵素。   The transferase according to any one of claims 1 to 3, which is produced from any one of the genus Aspergillus niger or the genus Aspergillus awamori. 酵素をコードするアミノ酸配列に、
配列Ｉ：ＬＬＶＥＹＱＴＤＥＲＬＨＶＭＩＹＤＡＤＥＥＶＹＱＶＰＥＳＶＬＰＲ、
配列ＩＩ：ＴＷＬＰＤＤＰＹＶＹＧＬＧＥＨＳＤＰＭＲ、
配列ＩＩＩ：ＩＰＬＥＴＭＷＴＤＩＤＹＭＤＫＲ、
配列ＩＶ：ＶＦＴＬＤＰＱＲ、
配列Ｖ：ＷＡＳＬＧＡＦＹＴＦＹＲ、
とからなる配列群より選択されるいずれか１以上の配列を含有することを特徴とする請求項１乃至請求項４のいずれか１項記載の転移酵素。 In the amino acid sequence encoding the enzyme,
Sequence I: LLVEYQTDERLHVMIYDADEVYQVPESVLPR,
Sequence II: TWLPDDPYVYGLGEHSDPMR,
Sequence III: IPLETMWTDIDYMDKR,
Sequence IV: VFTLDPQR,
Sequence V: WASLGAFYTFYR,
The transferase according to any one of claims 1 to 4, which comprises any one or more sequences selected from a sequence group consisting of: 請求項１乃至請求項５のいずれか１項記載の転移酵素と他酵素を、α−グルカン、α−グルコオリゴ糖、グルコースから選ばれる少なくとも一種を含む糖に作用させ、α１，２グルコシド結合を有する糖質と、α１，３グルコシド結合を有する糖質を生成した後、前記糖質を採取する糖質の製造方法。   The transferase and the other enzyme according to any one of claims 1 to 5 are allowed to act on a sugar containing at least one selected from α-glucan, α-glucooligosaccharide and glucose, and have an α1,2 glucoside bond. A method for producing a saccharide, wherein a saccharide and a saccharide having an α1,3 glucoside bond are produced, and then the saccharide is collected. 澱粉部分分解物に、請求項１乃至５のいずれか１項記載の転移酵素を作用させ、
α１，２グルコシド結合を有する糖質と、α１，３グルコシド結合を有する糖質を生成した後、前記糖質を採取する糖質の製造方法。 The transferase according to any one of claims 1 to 5 is allowed to act on a partially decomposed starch,
A method for producing a carbohydrate, wherein a carbohydrate having an α1,2 glucoside bond and a carbohydrate having an α1,3 glucoside bond are produced, and then the carbohydrate is collected. 前記澱粉部分分解物が、澱粉質を前記転移酵素以外の酵素又は酸によって部分的に加水分解して得られる請求項７記載の糖質の製造方法。   The method for producing a saccharide according to claim 7, wherein the partially decomposed starch is obtained by partially hydrolyzing starch with an enzyme or an acid other than the transferase. 請求項６記載の他酵素が、受容体糖分子に直鎖オリゴ糖を転移させる不均一化反応（Ｄｉｓｐｒｏｐｏｒｔｉｏｎａｔｉｏｎ反応）を触媒する酵素、及び／又は枝切酵素、及び／又はグルコアミラーゼである糖質の製造方法。   The carbohydrate according to claim 6, wherein the other enzyme is an enzyme that catalyzes a heterogeneous reaction (Disproportionation reaction) that transfers a linear oligosaccharide to a receptor sugar molecule, and / or a debranching enzyme, and / or a glucoamylase. Manufacturing method. 食物繊維含有量が全糖中の３０％以上となる請求項６乃至請求項９のいずれか１項記載の糖質の製造方法。   The method for producing a carbohydrate according to any one of claims 6 to 9, wherein the dietary fiber content is 30% or more of the total sugar. 前記糖質を採取するに際し、塩型強酸性カチオン交換樹脂を用いるカラムクロマトグラフィーを用いることを特徴とする、請求項６乃至請求項１０のいずれか１項記載の糖質の製造方法。   The method for producing a saccharide according to any one of claims 6 to 10, wherein column chromatography using a salt-type strongly acidic cation exchange resin is used when collecting the saccharide. 前記転移酵素を前記糖又は前記澱粉部分分解物に作用させる工程を、６０〜８０℃で行う請求項６乃至請求項１１のいずれか１項記載の糖質の製造方法。   The method for producing a saccharide according to any one of claims 6 to 11, wherein the step of allowing the transferase to act on the sugar or the partially decomposed starch is performed at 60 to 80 ° C. 請求項１乃至請求項５のいずれか１項記載の転移酵素を、α−グルカン、α−グルコオリゴ糖、グルコースから選ばれる少なくとも一種を含む糖液とグルコースの誘導体に作用して、配糖体を採取することを特徴とする、配糖体の製造方法   The transferase according to any one of claims 1 to 5 is allowed to act on a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide, and glucose and a derivative of glucose, whereby a glycoside is obtained. A method for producing a glycoside characterized by collecting the glycoside 請求項１乃至５のいずれかに記載の転移酵素産生能を有するＡｓｐｅｒｇｉｌｌｕｓ属に属する微生物を栄養培地中で培養して、当該転移酵素を生成させ、得られる培養物から当該転移酵素を採取することを特徴とする、転移酵素の製造方法   A microorganism belonging to the genus Aspergillus having the ability to produce transferase according to any one of claims 1 to 5 is cultured in a nutrient medium to produce the transferase, and the transferase is collected from the obtained culture. A method for producing a transferase, characterized in that
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