JP2018191566A - Method for producing sugar carboxylic acid - Google Patents
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本発明は、還元末端にグルコース残基を有する重合度２以上の澱粉分解物又は転移反応物の還元末端側のアルデヒド基が酸化された糖カルボン酸、その塩類及びそのラクトンを製造する方法に関する。 The present invention relates to a method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at a reducing end of 2 or more or a transfer reaction product is oxidized, a salt thereof, and a lactone thereof.
グルコースの還元末端を酸化することで得られるアルドン酸の一つであるグルコン酸は、単糖でありながらビフィズス菌増殖選択性を持つ機能性等を有しているだけでなく、カルシウムなどの無機カチオンと安定な塩を形成する特徴を持つことから、ミネラル補強剤として利用されている。しかしながら溶液安定性が悪く、高濃度下で保存すると析出してしまう欠点があった。 Gluconic acid, which is one of the aldonic acids obtained by oxidizing the reducing end of glucose, is not only a monosaccharide but also has functionality such as bifidobacteria growth selectivity, as well as inorganic substances such as calcium. Since it has a characteristic of forming a stable salt with a cation, it is used as a mineral reinforcing agent. However, the solution stability is poor, and there is a drawback that it is precipitated when stored at a high concentration.
これら欠点を補う素材として、グルコン酸の非還元末端側にグルコースが結合したマルトビオン酸などの糖カルボン酸が挙げられる。糖カルボン酸であるマルトビオン酸においても無機カチオンと安定な塩を形成するが、溶解性が良好であり、高濃度条件で保存しても析出しない特徴を有している。このように二糖類以上の糖質の還元末端を酸化することで、多くの機能性物質が得られることが期待される。 As a material to compensate for these drawbacks, sugar carboxylic acids such as maltobionic acid in which glucose is bonded to the non-reducing terminal side of gluconic acid can be mentioned. Maltobionic acid, which is a sugar carboxylic acid, also forms a stable salt with an inorganic cation, but has good solubility and does not precipitate even when stored under high concentration conditions. In this way, it is expected that many functional substances can be obtained by oxidizing the reducing end of a saccharide higher than a disaccharide.
特許文献１及び２には、重合度２のマルトース、ラクトースやセロビオースなどを酸化する手法として、アシネトバクター属、ブルクホルデリア属、グルコノバクター属やアセトバクター属などの微生物を用いた方法が開示されている。また、重合度４以上の澱粉分解物を酸化する酵素的な手法として、Ｍｉｃｒｏｄｏｃｈｉｕｍ属に属する微生物由来の糖質酸化酵素製剤や、Ａｃｒｅｍｏｎｉｕｍ属に属する微生物由来の糖質酸化酵素製剤を用いる手法が知られている。 Patent Documents 1 and 2 disclose methods using microorganisms such as Acinetobacter genus, Burkholderia genus, Gluconobacter genus and Acetobacter genus as a technique for oxidizing maltose, lactose, cellobiose and the like having a polymerization degree of 2. ing. In addition, as an enzymatic method for oxidizing starch degradation products having a polymerization degree of 4 or more, a method using a saccharide oxidase preparation derived from a microorganism belonging to the genus Microdochium or a saccharide oxidase preparation derived from a microorganism belonging to the genus Acremonium is known. It has been.
特許文献３〜５の糖質酸化酵素は、糖質を酸化する反応で副生成分として過酸化水素を生成する。過酸化水素は、殺菌や漂白剤として使用されるなど、タンパク質を変性させる力があり、糖質酸化時に副生する過酸化水素が、糖質酸化酵素を変性失活させてしまう。このため、糖質酸化酵素を用いて工業的に安定且つ効率的に重合度２以上の澱粉分解物および転移反応物を酸化するためには、過酸化水素の速やかな分解が必要となる。 The saccharide oxidases of Patent Documents 3 to 5 generate hydrogen peroxide as a by-product by a reaction that oxidizes saccharides. Hydrogen peroxide has the power to denature proteins, such as being used as a bactericidal or bleaching agent, and hydrogen peroxide produced as a by-product during oxidization of carbohydrates denatures and deactivates carbohydrate oxidase. For this reason, in order to oxidize starch degradation products and transfer reaction products having a polymerization degree of 2 or more industrially and efficiently using carbohydrate oxidase, it is necessary to rapidly decompose hydrogen peroxide.
特許文献６や７のグルコースオキシダーゼ製剤においても、グルコースをグルコン酸へ酸化する過程で過酸化水素が発生する。副生する過酸化水素を速やかに分解する生産技術としてカタラーゼ製剤を使用することが記載されている。 In the glucose oxidase preparations of Patent Documents 6 and 7, hydrogen peroxide is generated in the process of oxidizing glucose to gluconic acid. It is described that a catalase preparation is used as a production technique for rapidly decomposing hydrogen peroxide produced as a by-product.
重合度２以上の澱粉分解物や転移反応物を酸化する場合においても、糖質酸化酵素と一緒に、カタラーゼ製剤を添加すると、糖質酸化時に副生する過酸化水素を速やかに分解することが出来る。しかしながら、原因は不明ではあるが、重合度２以上の酸化物を高収率で安定して生産することができない。 Even when starch degradation products and transfer products with a polymerization degree of 2 or more are oxidized, adding a catalase preparation together with a carbohydrate oxidase can quickly decompose hydrogen peroxide produced as a by-product during carbohydrate oxidation. I can do it. However, although the cause is unknown, an oxide having a degree of polymerization of 2 or more cannot be stably produced in a high yield.
本発明は、以上の実情に鑑みてなされたものであり、酸化反応で副生する過酸化水素を速やかに分解するカタラーゼ製剤を用い、かつ、高収率で、重合度２以上の澱粉分解酸化物或いは転移反応酸化物で糖カルボン酸を工業的に生産する方法を提供することを目的とする。 The present invention has been made in view of the above circumstances, and uses a catalase preparation that quickly decomposes hydrogen peroxide produced as a by-product in an oxidation reaction, and has high yield and has a degree of polymerization of 2 or more. It is an object of the present invention to provide a method for industrially producing a sugar carboxylic acid using a product or a transfer reaction oxide.
本発明者らは、原料となる重合度２以上の澱粉分解物や転移反応物が、カタラーゼ製剤中に含まれる夾雑酵素であるα−グルコシダーゼやグルコアミラーゼ等の澱粉分解酵素により加水分解されることが、重合度２以上の糖酸化物の生産を不安定化あせる原因であることを発見した。酵素法によるグルコン酸製造では、原料であるグルコースは単糖であるためカタラーゼ製剤中の夾雑酵素による加水分解を考慮する必要がないこととは対照的である。この発見に基づき、夾雑酵素である澱粉分解酵素を所要以下となるように調製したカラターゼ製剤を用いることで、重合度２以上の澱粉分解物或いはその転移反応物の酸化物を安定的に得る方法を見出し、本発明を完成するに至った。より具体的には、本発明は以下のようなものを提供する。 The inventors of the present invention are that a starch degradation product or transfer reaction product having a polymerization degree of 2 or more as a raw material is hydrolyzed by a starch degrading enzyme such as α-glucosidase or glucoamylase, which is a contaminating enzyme contained in a catalase preparation. Has been found to be the cause of destabilizing the production of sugar oxides having a degree of polymerization of 2 or more. In contrast to the production of gluconic acid by an enzymatic method, the raw material glucose is a monosaccharide, so that it is not necessary to consider hydrolysis by a contaminating enzyme in the catalase preparation. Based on this discovery, a method of stably obtaining a starch degradation product having a polymerization degree of 2 or more or an oxide of its transfer reaction product by using a calatase preparation prepared so that the amylolytic enzyme, which is a contaminating enzyme, is less than required. As a result, the present invention has been completed. More specifically, the present invention provides the following.
（１） 還元末端にグルコース残基を有する重合度２以上の澱粉分解物又は転移反応物の還元末端側のアルデヒド基が酸化された糖カルボン酸の製造方法であって、
糖質酸化時に過酸化水素を副生する糖質酸化酵素剤を、カタラーゼ製剤中のカタラーゼ活性（Ａ）に対する糖化活性（Ｂ）の含有比率（Ｂ／Ａ）が０．００００２以上０．００５以下であるカタラーゼ製剤の存在下、前記澱粉分解物或いは転移反応物を含む原料基質に作用させる工程を含み、
前記カタラーゼ製剤は、その糖化活性が前記原料基質中の還元糖量（ｗｔ％）に対して０．９ｕ／ｇ以下である量で存在する方法。
(1) A method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at a reducing end of 2 or more is oxidized,
Carbohydrate oxidase that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) of 0.00002 to 0.005 in catalase preparation to catalase activity (A). In the presence of a catalase preparation, which comprises the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.
（２） 還元末端にグルコース残基を有する重合度２以上の澱粉分解物又は転移反応物の還元末端側のアルデヒド基が酸化された糖カルボン酸の製造方法であって、
糖質酸化時に過酸化水素を副生する糖質酸化酵素剤を、カタラーゼ製剤中のカタラーゼ活性（Ａ）に対する糖化活性（Ｂ）の含有比率（Ｂ／Ａ）が０．００５以下でありかつ糖化活性（Ｂ）が０．１ｕ／ｍｌ以上であるカタラーゼ製剤の存在下、前記澱粉分解物或いは転移反応物を含む原料基質に作用させる工程を含み、
前記カタラーゼ製剤は、その糖化活性が前記原料基質中の還元糖量（ｗｔ％）に対して０．９ｕ／ｇ以下である量で存在する方法。
(2) A method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at the reducing end of 2 or more is oxidized,
A saccharide oxidase agent that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) to catalase activity (A) in the catalase preparation of 0.005 or less and saccharification Including the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product in the presence of a catalase preparation having an activity (B) of 0.1 u / ml or more,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.
（３） 前記糖質酸化酵素剤の添加量は、前記原料基質中の還元糖量（ｗｔ％）に対して１ｕ／ｇ以上３０ｕ／ｇ以下である（１）又は（２）記載の方法。 (3) The method according to (1) or (2), wherein the amount of the carbohydrate oxidase agent added is 1 u / g or more and 30 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.
（４） カタラーゼ製剤は、そのカタラーゼ活性が前記原料基質中の還元糖量（ｗｔ％）に対して４０ｕ／ｇ以上１０００ｕ／ｇ以下である量で存在する（１）から（３）いずれか記載の方法。 (4) The catalase preparation is present in an amount such that the catalase activity is 40 u / g or more and 1000 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate (1) to (3) the method of.
（５） 前記カタラーゼ製剤は、その糖化活性が前記原料基質中の還元糖量（ｗｔ％）に対して０．００００８ｕ／ｇ以上である量で存在する（１）から（４）いずれか記載の方法。 (5) The catalase preparation is present in any amount (1) to (4), wherein the saccharification activity is 0.00008 u / g or more with respect to the amount of reducing sugar (wt%) in the raw material substrate. Method.
（６） 前記作用工程における前記澱粉分解物或いは転移反応物の濃度が１０％（ｗ／ｗ）以上である（１）から（５）のいずれかに記載の製造方法。 (6) The production method according to any one of (1) to (5), wherein the concentration of the starch degradation product or transfer reaction product in the action step is 10% (w / w) or more.
本発明によれば、食品、医薬や工業分野等において、ミネラル成分を可溶させる素材等として有用である糖カルボン酸を収率よく製造することができる。 According to the present invention, sugar carboxylic acids that are useful as materials for solubilizing mineral components and the like in foods, medicines, and industrial fields can be produced with high yield.
以下、本発明の具体的な実施形態について詳細に説明するが、本発明は以下の実施形態に何ら限定されるものではなく、本発明の目的の範囲内において、適宜変更を加えて実施することができる。なお、説明が重複する箇所については、適宜説明を省略する場合があるが、発明の要旨を限定するものではない。 Hereinafter, specific embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and may be implemented with appropriate modifications within the scope of the object of the present invention. Can do. In addition, although description may be abbreviate | omitted suitably about the location where description overlaps, the summary of invention is not limited.
本発明の一実施形態は、糖質酸化時に過酸化水素を副生する糖質酸化酵素剤を、カタラーゼ製剤中のカタラーゼ活性（Ａ）に対する糖化活性（Ｂ）の含有比率（Ｂ／Ａ）が０．００００２以上０．００５以下であるカタラーゼ製剤の存在下、前記澱粉分解物或いは転移反応物を含む原料基質に作用させる工程を含み、
前記カタラーゼ製剤は、その糖化活性が前記原料基質中の還元糖量（ｗｔ％）に対して０．９ｕ／ｇ以下である量で存在する方法である。
In one embodiment of the present invention, a saccharide oxidase agent that produces hydrogen peroxide as a by-product during oxidization of a saccharide has a content ratio (B / A) of saccharification activity (B) to catalase activity (A) in the catalase preparation. In the presence of a catalase preparation of 0.00002 or more and 0.005 or less, comprising a step of acting on a raw material substrate containing the starch degradation product or transfer reaction product,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.
本発明の別の実施形態は、還元末端にグルコース残基を有する重合度２以上の澱粉分解物又は転移反応物の還元末端側のアルデヒド基が酸化された糖カルボン酸の製造方法であって、
糖質酸化時に過酸化水素を副生する糖質酸化酵素剤を、カタラーゼ製剤中のカタラーゼ活性（Ａ）に対する糖化活性（Ｂ）の含有比率（Ｂ／Ａ）が０．００５以下でありかつ糖化活性（Ｂ）が０．１ｕ／ｍｌ以上であるカタラーゼ製剤の存在下、前記澱粉分解物或いは転移反応物を含む原料基質に作用させる工程を含み、
前記カタラーゼ製剤は、その糖化活性が前記原料基質中の還元糖量（ｗｔ％）に対して０．９ｕ／ｇ以下である量で存在する方法である。
Another embodiment of the present invention is a method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a degree of polymerization of 2 or more having a glucose residue at the reducing end is oxidized,
A saccharide oxidase agent that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) to catalase activity (A) in the catalase preparation of 0.005 or less and saccharification Including the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product in the presence of a catalase preparation having an activity (B) of 0.1 u / ml or more,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.
例えば、還元末端にグルコース残基を有する澱粉分解物である、重合度２のマルトースを原料として場合、糖質酸化時に過酸化水素を副生する糖質酸化酵素剤を作用させることにより、マルトビオン酸と過酸化水素が生成するが、副生する過酸化水素が、酸化酵素を変性失活させてしまう。このため、高収率でマルトビオン酸を製造するためには、過酸化水素を速やかに分解するカタラーゼ製剤を添加する必要がある。 For example, when maltose having a polymerization degree of 2, which is a starch degradation product having a glucose residue at the reducing end, is used as a raw material, maltobionic acid can be obtained by acting a sugar oxidase agent that produces hydrogen peroxide as a by-product during sugar oxidation. Hydrogen peroxide is produced, but the by-produced hydrogen peroxide denatures and deactivates the oxidase. For this reason, in order to produce maltobionic acid in a high yield, it is necessary to add a catalase preparation that rapidly decomposes hydrogen peroxide.
（カタラーゼ製剤）
本発明で言うカタラーゼ製剤とは、Ａｓｐｅｒｇｉｌｌｕｓ属や、Ｍｉｃｒｏｃｏｃｃｕｓ属などの微生物由来のカタラーゼ製剤などが挙げられ、具体的には、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｒ又はＭｉｃｒｏｃｏｃｃｕｓ ｌｙｓｏｄｅｉｋｔｉｃｕｓ由来のカタラーゼ製剤が挙げられる。また、副活性としてカタラーゼ活性を有する市販のグルコースオキシダーゼ製剤を選択して用いることも含まれる。
(Catalase preparation)
Examples of the catalase preparation referred to in the present invention include catalase preparations derived from microorganisms such as the genus Aspergillus and Micrococcus, and specific examples include a catalase preparation derived from Aspergillus nigr or Micrococcus lysodeikticus. It also includes selecting and using a commercially available glucose oxidase preparation having catalase activity as a side activity.
本発明のカタラーゼ製剤中には、グルコアミラーゼやα‐グルコシダーゼなどの糖化活性を持つ夾雑酵素が多く混在していると、糖カルボン酸の原料となる還元末端にグルコース残基を有する重合度２以上の澱粉分解物又は転移反応物や、これら原料を酸化した糖カルボン酸が分解されてしまい、安定的な品質の糖カルボン酸の製造が不可能となる。 In the catalase preparation of the present invention, when there are many contaminating enzymes having saccharification activity such as glucoamylase and α-glucosidase, the degree of polymerization is 2 or more having a glucose residue at the reducing end as a raw material for sugar carboxylic acid. The starch degradation product or transfer reaction product, and the sugar carboxylic acid obtained by oxidizing these raw materials are decomposed, making it impossible to produce a sugar carboxylic acid having a stable quality.
このため本発明では、マルトビオン酸等の糖カルボン酸を高収率で安定生産するために、カタラーゼ製剤中のカタラーゼ活性（Ａ）に対する糖化活性の含有比率（Ｂ／Ａ）が０．００５以下であるカタラーゼ製剤を用いる。好ましくは、Ｂ／Ａは、０．００４５以下、０．００３以下、０．００２以下、０．００１５以下、０．００１以下、０．０００５以下、０．０００４以下である。 Therefore, in the present invention, in order to stably produce a sugar carboxylic acid such as maltobionic acid at a high yield, the content ratio (B / A) of saccharification activity to catalase activity (A) in the catalase preparation is 0.005 or less. Some catalase preparations are used. Preferably, B / A is 0.0045 or less, 0.003 or less, 0.002 or less, 0.0015 or less, 0.001 or less, 0.0005 or less, or 0.0004 or less.
本発明で用いるカタラーゼ製剤は、工業的に糖カルボン酸を製造する観点で、試薬のように高度に精製されたカタラーゼ酵素のみからなるものではなく、むしろ許容範囲内での夾雑酵素を含む。具体的に、Ｂ／Ａは０．００００２以上であることが好ましく、具体的には０．０００１以上、０．０００２以上、０．０００３以上、０．０００４以上であってよい。カタラーゼ製剤がこの程度の比率で糖化活性を有していても、糖化反応に比べて糖質酸化の主反応が速やかに進むため、収率低下につながりにくい。 The catalase preparation used in the present invention does not consist only of a highly purified catalase enzyme like a reagent from the viewpoint of industrially producing a sugar carboxylic acid, but rather contains a contaminating enzyme within an acceptable range. Specifically, B / A is preferably 0.00002 or more, and specifically may be 0.0001 or more, 0.0002 or more, 0.0003 or more, or 0.0004 or more. Even if the catalase preparation has saccharification activity at such a ratio, the main reaction of saccharide oxidation proceeds more rapidly than the saccharification reaction, so that the yield is unlikely to decrease.
本発明においては、試験例４の比較例５に示したようにカタラーゼ製剤中のカタラーゼ活性（Ａ）に対する糖化活性（Ｂ）の含有比率（Ｂ／Ａ）が０．００５以下であっても、原料糖質に対してカタラーゼ製剤を多めに添加すると、カタラーゼ製剤中の夾雑酵素が原料基質を加水分解し、マルトビオン酸等の糖カルボン酸が想定している組成のものが得られない場合がある。このためカタラーゼ製剤中の糖化活性が、原料基質中の還元糖量（固形分当たりｗｔ％）に対して０．９ｕ／ｇ以下（好ましくは、０．８ｕ／ｇ以下、０．７ｕ／ｇ以下、０．６５ｕ／ｇ以下）となるようにカタラーゼ製剤を作用させる必要がある。 In the present invention, as shown in Comparative Example 5 of Test Example 4, even if the content ratio (B / A) of saccharification activity (B) to catalase activity (A) in the catalase preparation is 0.005 or less, If a large amount of catalase preparation is added to the raw sugar, the contaminating enzyme in the catalase preparation hydrolyzes the raw material substrate, and the composition of the sugar carboxylic acid such as maltobionic acid may not be obtained. . Therefore, the saccharification activity in the catalase preparation is 0.9 u / g or less (preferably 0.8 u / g or less, 0.7 u / g or less) with respect to the amount of reducing sugar (wt% per solid content) in the raw material substrate. , 0.65 u / g or less), it is necessary to make the catalase preparation act.
カタラーゼ製剤中のカタラーゼ活性（Ａ）は、５０００ｕ／ｍｌ以上であることが好ましく、具体的には１００００ｕ／ｍｌ以上、１５０００ｕ／ｍｌ以上、２００００ｕ／ｍｌ以上、２２５００ｕ／ｍｌ以上であってよい。高いカタラーゼ活性を有すると、糖化活性がある程度高くても、収率に与える影響を小さくとどめやすい。 The catalase activity (A) in the catalase preparation is preferably 5000 u / ml or more, specifically 10000 u / ml or more, 15000 u / ml or more, 20000 u / ml or more, 22500 u / ml or more. When it has high catalase activity, even if saccharification activity is high to some extent, it is easy to keep the influence on the yield small.
カタラーゼ製剤中のカタラーゼ活性（Ａ）は、５０００００ｕ／ｍｌ以下であることが好ましく、具体的には、２５０００００ｕ／ｍｌ以下、１５００００ｕ／ｍｌ以下、１０００００ｕ／ｍｌ以下、７５０００ｕ／ｍｌ以下であってよい。本発明で用いられるカタラーゼ製剤は糖化活性が低いので、過大なカタラーゼ活性を有しなくても、収率に与える影響を小さくとどめやすい。 The catalase activity (A) in the catalase preparation is preferably 500000 u / ml or less, and specifically may be 2500000 u / ml, 150,000 u / ml, 100000 u / ml or less, 75000 u / ml or less. Since the catalase preparation used in the present invention has a low saccharification activity, even if it does not have an excessive catalase activity, the influence on the yield can be kept small.
カタラーゼ製剤中の糖化活性（Ｂ）は、２５０ｕ／ｍｌ以下であることが好ましく、具体的には、１００ｕ／ｍｌ以下、５０ｕ／ｍｌ以下、３０ｕ／ｍｌ以下、２５ｕ／ｍｌ以下であってよい。 The saccharification activity (B) in the catalase preparation is preferably 250 u / ml or less, specifically, 100 u / ml or less, 50 u / ml or less, 30 u / ml or less, or 25 u / ml or less.
他方、カタラーゼ製剤中の糖化活性（Ｂ）は、許容範囲内で有してよく、０．１ｕ／ｍｌ以上であることが好ましく、具体的には０．５ｕ／ｍｌ以上、１．０ｕ／ｍｌ以上、１．５ｕ／ｍｌ以上、２．０ｕ／ｍｌ以上であってよい。この程度の糖化活性が存在しても、糖化反応に比べて糖質酸化の主反応が速やかに進むため、収率低下につながりにくい。 On the other hand, the saccharification activity (B) in the catalase preparation may be within an acceptable range, and is preferably 0.1 u / ml or more, specifically 0.5 u / ml or more, 1.0 u / ml. As described above, it may be 1.5 u / ml or more and 2.0 u / ml or more. Even if this level of saccharification activity is present, the main reaction of carbohydrate oxidation proceeds more rapidly than the saccharification reaction, so that it is difficult to lead to a decrease in yield.
カタラーゼ製剤中の糖化活性は、許容範囲内で有してよく、具体的には原料基質中の還元糖量（固形分当たりｗｔ％）に対して、０．００００８ｕ／ｇ以上、好ましくは０．０００５ｕ／ｇ以上、０．００１ｕ／ｇ以上、０．００１５ｕ／ｇ以上であってよい。この程度の糖化活性が存在しても、糖化反応に比べて糖質酸化の主反応が速やかに進むため、収率低下につながりにくい。 The saccharification activity in the catalase preparation may be within an acceptable range. Specifically, it is 0.00008 u / g or more with respect to the amount of reducing sugar (wt% per solid content) in the raw material substrate, preferably 0.8. It may be 0005 u / g or more, 0.001 u / g or more, 0.0015 u / g or more. Even if this level of saccharification activity is present, the main reaction of carbohydrate oxidation proceeds more rapidly than the saccharification reaction, so that it is difficult to lead to a decrease in yield.
また、マルトビオン酸等の糖カルボン酸製造にあたり、前記カタラーゼ製剤は、原料基質中の還元糖量（固形分当たり）に対して４０ｕ／ｇ以上１０００ｕ／ｇ以下で存在するのが好ましく、より好ましくは、６０ｕ／ｇ以上５００ｕ／ｇ以下で存在する。本発明では、カタラーゼ製剤中の糖化活性が低く抑えられているため、過酸化水素による糖質酸化酵素の分解を抑制するのに十分な量のカタラーゼ製剤を使っても収率低下を招きにくい。また、糖化活性による原料となる重合度２以上の澱粉分解物や転移反応物の分解が抑制され、ある程度の時間をかけて糖質酸化反応を行っても収率低下を招きにくいので、過剰なカタラーゼ活性を必要としない。 Further, in the production of sugar carboxylic acids such as maltobionic acid, the catalase preparation is preferably present at 40 u / g or more and 1000 u / g or less with respect to the amount of reducing sugar (per solid content) in the raw material substrate, more preferably , 60 u / g or more and 500 u / g or less. In the present invention, since the saccharification activity in the catalase preparation is kept low, even if a sufficient amount of the catalase preparation is used to suppress the degradation of the carbohydrate oxidase by hydrogen peroxide, the yield is unlikely to decrease. In addition, degradation of starch degradation products and transfer reaction products having a polymerization degree of 2 or more, which is a raw material due to saccharification activity, is suppressed, and it is difficult to cause a decrease in yield even if a saccharide oxidation reaction is performed over a certain period of time. Catalase activity is not required.
本発明のカタラーゼ製剤中のカタラーゼ活性は、次のようにして測定する。
酵素反応後の残存過酸化水素をチオ硫酸ナトリウムで滴定する方法に従う（小崎道雄監修「酵素利用ハンドブック」、地人書館昭和６０年版、ｐ４０４〜４１０）。すなわち、市販の３０重量％過酸化水素を５０ｍＭリン酸緩衝液（ｐＨ７．０）で８００倍に希釈した基質溶液５ｍｌを容器にとり、３０℃の恒温水槽に１５分入れ恒温とする。これに３０℃に保温した検体酵素液１ｍｌを加え、正確に５分後に０．５Ｎ硫酸２ｍｌを加えよく振り混ぜ酵素作用を止める。これに１０重量％ヨウ化カリウム溶液１ｍｌと１％モリブデン酸アンモニウム１滴及び指示薬として０．５％デンプン試薬５滴を加え、この溶液を撹拌しながら、０．００５Ｎチオ硫酸ナトリウム溶液（定量用）で滴定し、ブランクは試料の代わりに水１ｍｌを添加し、ブランクの値から検体の値を差し引いてカタラーゼ作用によって分解された過酸化水素の量を算出し、標準曲線から検体酵素液のカタラーゼ活性を求める。なお、１Ｕは１分間に１μｍｏｌの過酸化水素を分解する活性を示している。
カタラーゼ活性（Ｕ／ｍｌ）＝Ａ×ｎ
ｎ：希釈倍率
Ａ：標準曲線のグラフよりｙ＝（Ｔ0−ＴS）×２４.１８／Ｔ0×２．５×ｆのｘ軸の値Ａを求める
ｆ：０．００５Ｎチオ硫酸ナトリウムのファクター
Ｔ0：ブランクの滴定値（ｍｌ）
ＴS：サンプルの滴定値（ｍｌ）
２４.１８／Ｔ0：初発基質濃度による活性測定変化に対する補正値
２．５：０．００５Ｎチオ硫酸ナトリウム溶液１ｍｌは過酸化水素２．５μｍｏｌに相当
The catalase activity in the catalase preparation of the present invention is measured as follows.
Follow the method of titrating the residual hydrogen peroxide after the enzyme reaction with sodium thiosulfate (supervised by Michio Kosaki “Enzyme Use Handbook”, Jinjinshokan 1985 edition, p404-410). That is, 5 ml of a substrate solution obtained by diluting commercially available 30% by weight hydrogen peroxide 800-fold with 50 mM phosphate buffer (pH 7.0) is placed in a container and placed in a constant temperature water bath at 30 ° C. for 15 minutes to obtain a constant temperature. Add 1 ml of the sample enzyme solution kept at 30 ° C., and after 5 minutes, add 2 ml of 0.5N sulfuric acid and shake well to stop the enzyme action. To this was added 1 ml of 10 wt% potassium iodide solution, 1 drop of 1% ammonium molybdate and 5 drops of 0.5% starch reagent as an indicator, and while stirring this solution, 0.005N sodium thiosulfate solution (for determination) In the blank, 1 ml of water is added instead of the sample, the amount of hydrogen peroxide decomposed by the catalase action is calculated by subtracting the value of the sample from the blank value, and the catalase activity of the sample enzyme solution from the standard curve Ask for. In addition, 1U has shown the activity which decomposes | disassembles 1 micromol hydrogen peroxide in 1 minute.
Catalase activity (U / ml) = A × n
n: Dilution ratio A: Obtain the value A on the x-axis of y = (T 0 −T S ) × 24.18 / T 0 × 2.5 × f from the standard curve graph. f: 0.005N sodium thiosulfate factor T 0: blank titration value (ml)
T S : Titration value of sample (ml)
24.18 / T 0 : Correction value for change in activity measurement due to initial substrate concentration 2.5: 1 ml of 0.005N sodium thiosulfate solution corresponds to 2.5 μmol of hydrogen peroxide
本発明で定義する糖化活性とは、グルコアミラーゼ活性とα−グルコシダーゼ活性により澱粉分解物が加水分解されグルコースを遊離する力であり、本発明の糖化活性は、基質の４−ニトロフェニルβ−マルトシド（Ｇ２−β−ＰＮＰ）より、１分間に１μｍｏｌのＰＮＰを遊離する活性を１Ｕと定義することができる。 The saccharification activity defined in the present invention is a force that hydrolyzes the starch degradation product by glucoamylase activity and α-glucosidase activity to release glucose, and the saccharification activity of the present invention is the substrate 4-nitrophenyl β-maltoside. From (G2-β-PNP), the activity of releasing 1 μmol of PNP per minute can be defined as 1U.
カタラーゼ製剤中の糖化活性は、カタラーゼ製剤を４−ニトロフェニルβ−マルトシドと反応させて４−ニトロフェニルβ−グルコシドを生成させ、それをβ-グルコシダーゼによって分解して４−ニトロフェノールを生成させ、４−ニトロフェノールを定量することにより測定される。具体的には、キッコーマン社製の糖化力測定キット或いは糖化力分別定量キットなどを利用して、カタラーゼ製剤中の糖化活性を測定する。 The saccharification activity in the catalase preparation is obtained by reacting the catalase preparation with 4-nitrophenyl β-maltoside to produce 4-nitrophenyl β-glucoside, which is decomposed by β-glucosidase to produce 4-nitrophenol, It is measured by quantifying 4-nitrophenol. Specifically, the saccharification activity in the catalase preparation is measured using a saccharification power measurement kit or a saccharification power fractionation determination kit manufactured by Kikkoman Corporation.
（キッコーマン社製の糖化力測定キットを使用した糖化力活性の測定）
キッコーマン社製の糖化力測定キットを使用する場合、４−ニトロフェニルβ−マルトシドを含有する基質溶液０．５ｍｌにβ−グルコシダーゼを含有する酵素溶液０．５ｍｌを混ぜ、３７℃で５分間予備加温を行った後、測定試料０．１ｍｌを加え、混合して３７℃で１０分間反応させる。反応停止は、炭酸ナトリウムを含有する酵素停止液２ｍｌを加え混合する。反応終了後の液を波長400nmで定量することにより糖化力を測定し、以下の計算式より活性を算出する。
糖化力活性 （Ｕ／ｍｌ）＝（Ｅｓ−Ｅｂ）× ０．１７１×ｎ
Ｅｓ：測定試料の吸光度
Ｅｂ：ブランクの吸光度
ｎ：酵素液の希釈倍率
(Measurement of saccharification activity using a saccharification test kit manufactured by Kikkoman)
When using a kit for measuring saccharification power manufactured by Kikkoman, 0.5 ml of a substrate solution containing 4-nitrophenyl β-maltoside is mixed with 0.5 ml of an enzyme solution containing β-glucosidase and preliminarily added at 37 ° C. for 5 minutes. After performing the temperature, 0.1 ml of a measurement sample is added, mixed, and reacted at 37 ° C. for 10 minutes. To stop the reaction, 2 ml of enzyme stop solution containing sodium carbonate is added and mixed. The saccharification power is measured by quantifying the liquid after the reaction at a wavelength of 400 nm, and the activity is calculated from the following formula.
Saccharification activity (U / ml) = (Es−Eb) × 0.171 × n
Es: Absorbance of measurement sample Eb: Absorbance of blank n: Dilution ratio of enzyme solution
（糖質酸化酵素製剤）
本発明で言う糖質酸化酵素製剤とは、還元末端にグルコース残基を有する重合度２以上の糖質を酸化し、副生成分として過酸化水素を発生するものをいう。Ｍｉｃｒｏｄｏｃｈｉｕｍ属に属する微生物由来の糖質酸化酵素製剤や、Ａｃｒｅｍｏｎｉｕｍ属に属する微生物由来の糖質酸化酵素製剤などが挙げられ、具体的には、Ａｃｒｅｍｏｎｉｕｍ ｃｈｒｙｓｏｇｅｎｕｍに由来する糖質酸化酵素などが挙げられる。
(Sugar oxidase preparation)
The saccharide oxidase preparation referred to in the present invention means a substance that oxidizes a saccharide having a glucose residue at the reducing end and having a polymerization degree of 2 or more to generate hydrogen peroxide as a by-product. Examples include saccharide oxidase preparations derived from microorganisms belonging to the genus Microdocium, saccharide oxidase preparations derived from microorganisms belonging to the genus Acremonium, and specifically, saccharide oxidases derived from Acremonium chrysogenum and the like.
マルトビオン酸等の糖カルボン酸製造にあたり糖質酸化酵素は、原料基質中の還元糖量（ｗｔ％）に対して１ｕ／ｇ以上３０ｕ／ｇ以下が好ましく、より好ましくは、２ｕ／ｇ以上２０ｕ／ｇ以下で作用させる。本発明では、カタラーゼ製剤による過酸化水素の分解が十分になされるので、副生される過酸化水素の増加にかかわらず、糖質酸化反応を十分な速度で行うことができる。また、糖化活性による原料となる重合度２以上の澱粉分解物や転移反応物の分解が抑制され、ある程度の時間をかけて糖質酸化反応を行っても収率低下を招きにくいので、過剰な量の糖質酸化酵素を必要としない。 In producing a sugar carboxylic acid such as maltobionic acid, the sugar oxidase is preferably from 1 u / g to 30 u / g, more preferably from 2 u / g to 20 u / g, based on the amount of reducing sugar (wt%) in the raw material substrate. Act at g or less. In the present invention, since the decomposition of hydrogen peroxide by the catalase preparation is sufficiently performed, the carbohydrate oxidation reaction can be performed at a sufficient rate regardless of the increase of hydrogen peroxide produced as a by-product. In addition, degradation of starch degradation products and transfer reaction products having a polymerization degree of 2 or more, which is a raw material due to saccharification activity, is suppressed, and it is difficult to cause a decrease in yield even if a saccharide oxidation reaction is performed over a certain period of time. Does not require the amount of carbohydrate oxidase.
本発明の糖質酸化酵素の酵素活性は、次のようにして測定する。
０．１５％（ｗ／ｖ）フェノール及び０．１５％（ｗ／ｖ）トリトンＸ−１００を含む０．１Ｍリン酸一カリウム−水酸化ナトリウム緩衝液（ｐＨ７．０）２ｍｌ、１０％マルトース一水和物溶液０．５ｍｌ、２５Ｕ／ｍｌペルオキシダーゼ溶液０．５ｍｌ、及び０．４％（ｗ／ｖ）４−アミノアンチピリン溶液０．１ｍｌを混合し、３７℃で１０分保温後、酵素溶液０．１ｍｌを添加し、反応を開始した。酵素反応進行と共に、波長５００ｎｍにおける吸光度の増加を測定することにより糖質酸化活性を測定した。なお、ブンランクには０．１Ｍリン酸緩衝液（ｐＨ７．０）を使用し、１分間に１μｍｏｌのマルトース一水和物を酸化するのに必要な酵素量を１単位とし、以下の計算式より活性を算出する。
マルトース酸化活性 （Ｕ／ｍｌ）
＝｛（Ａ５−Ａ２）−（Ａｂ５−Ａｂ２）｝× ２．２１８ ×ｎ
Ａ２， Ａ５ ： 反応開始後、２分後および５ 分後の吸光度 (検体)
Ａｂ２， Ａｂ５ ： 反応開始後、２ 分後および5 分後の吸光度 (ブランク)
ｎ：酵素液の希釈倍率
The enzyme activity of the carbohydrate oxidase of the present invention is measured as follows.
0.1 ml monopotassium phosphate-sodium hydroxide buffer solution (pH 7.0) containing 0.15% (w / v) phenol and 0.15% (w / v) Triton X-100, 10% maltose Mix 0.5 ml of hydrate solution, 0.5 ml of 25 U / ml peroxidase solution, and 0.1 ml of 0.4% (w / v) 4-aminoantipyrine solution, and incubate at 37 ° C. for 10 minutes. .1 ml was added to start the reaction. The carbohydrate oxidation activity was measured by measuring the increase in absorbance at a wavelength of 500 nm as the enzyme reaction progressed. For Bunrank, 0.1M phosphate buffer (pH 7.0) was used, and the amount of enzyme required to oxidize 1 μmol of maltose monohydrate per minute was defined as 1 unit. Calculate activity.
Maltose oxidation activity (U / ml)
= {(A5-A2)-(Ab5-Ab2)} * 2.218 * n
A2, A5: Absorbance after 2 minutes and 5 minutes after starting the reaction (sample)
Ab2, Ab5: Absorbance 2 minutes and 5 minutes after the start of the reaction (blank)
n: dilution rate of enzyme solution
（原料糖質）
本発明において原料に用いる糖質は、還元末端にグルコース残基を有する重合度２以上の澱粉分解物或いは転移反応物であり、マルトース、イソマルトース、マルトトリオース、イソマルトトリオース、マルトテトラオース、マルトヘキサオース、パノース、マルトオリゴ糖、イソマルトオリゴ糖、水飴、粉飴、デキストリン、分岐デキストリン、イソマルトデキストリン等が挙げられる。
(Raw sugar)
The carbohydrate used as a raw material in the present invention is a starch degradation product or transfer reaction product having a glucose residue at the reducing end and having a polymerization degree of 2 or more, and maltose, isomaltose, maltotriose, isomaltotriose, maltotetraose. , Maltohexaose, panose, malto-oligosaccharide, isomalto-oligosaccharide, starch syrup, powder cake, dextrin, branched dextrin, isomaltodextrin and the like.
糖カルボン酸生産時の原料糖質の濃度は、精製工程での濃縮等を考慮すると１０〜５０（ｗｔ）％が好ましく、２０〜４０（ｗｔ）％がより好ましい。なお、本明細書において、「（ｗｔ）％」は、対象成分の含有量（質量）を意味し、ここでは、液体中における糖質の含有量を意味する。 The concentration of the raw sugar during the production of the sugar carboxylic acid is preferably 10 to 50 (wt)%, more preferably 20 to 40 (wt)% in consideration of the concentration in the purification step. In the present specification, “(wt)%” means the content (mass) of the target component, and here, the content of the carbohydrate in the liquid.
（反応温度と反応ｐＨ）
糖質酸化酵素とカタラーゼの反応工程での反応温度は、例えば２０〜６０℃程度の条件下で行うのが好ましく、より好ましくは、２５〜４０℃の範囲である。
(Reaction temperature and reaction pH)
The reaction temperature in the reaction step between the carbohydrate oxidase and catalase is preferably, for example, about 20 to 60 ° C, and more preferably 25 to 40 ° C.
反応ｐＨは４〜１０程度の条件下で行うのが好ましく、より好ましくはｐＨ５〜８の範囲である。また、糖カルボン酸が生成する過程で、反応ｐＨが低下するため、反応液に中和剤を添加調整する必要がある。中和剤としては、水酸化ナトリウム、水酸化カリウム、炭酸カルシウム、炭酸マグネシウム、炭酸ナトリウム、炭酸水素ナトリウム、酸化カルシウムなどが挙げられる。 The reaction pH is preferably about 4 to 10 and more preferably in the range of pH 5 to 8. Moreover, since reaction pH falls in the process in which sugar carboxylic acid produces | generates, it is necessary to add and adjust a neutralizing agent to a reaction liquid. Examples of the neutralizing agent include sodium hydroxide, potassium hydroxide, calcium carbonate, magnesium carbonate, sodium carbonate, sodium hydrogen carbonate, calcium oxide and the like.
また、本発明の酸化反応では、酸素が必要となるため、空気や酸素を通気撹拌することで、反応溶液中の溶存酸素量を維持することが好ましい。 Moreover, since oxygen is required in the oxidation reaction of the present invention, it is preferable to maintain the amount of dissolved oxygen in the reaction solution by aeration and stirring of air or oxygen.
（糖カルボン酸）
本発明方法を使用して調製される糖カルボン酸は、重合度２以上、好ましくは重合度４以上の澱粉分解物又は転移反応物の還元末端側のアルデヒド基が酸化されたものであれば、特に限定されない。澱粉分解物又は転移反応物の重合度は、例えば、２〜１００、好ましくは４〜１００等であってもよい。より具体的には、糖カルボン酸は、より具体的には、マルトデキストリン酸化物、粉飴酸化物、水飴酸化物、マルトヘキサオン酸、マルトテトラオン酸、マルトトリオン酸、マルトビオン酸、イソマルトデキストリン酸化物、パノース酸化物、イソマルトトリオン酸、イソマルトビオン酸、ニゲロビオン酸、コージビオン酸などが挙げられる。これらのうち、糖カルボン酸は、遊離の酸であってもよく、ラクトンであってもよく、その塩類であってもよい。
(Sugar carboxylic acid)
The sugar carboxylic acid prepared by using the method of the present invention is a starch decomposition product having a polymerization degree of 2 or more, preferably 4 or more, or an aldehyde group on the reducing end side of the transfer reaction product is oxidized. There is no particular limitation. The degree of polymerization of the starch decomposition product or transfer reaction product may be, for example, 2 to 100, preferably 4 to 100 or the like. More specifically, the sugar carboxylic acid is more specifically maltodextrin oxide, powdered oxide, starch oxalate, maltohexanoic acid, maltotetraonic acid, maltotriionic acid, maltobionic acid, isomalt. Examples thereof include dextrin oxide, panose oxide, isomaltorionic acid, isomaltionic acid, nigerovionic acid, and cordibionic acid. Among these, the sugar carboxylic acid may be a free acid, a lactone, or a salt thereof.
糖カルボン酸の塩としては、特に限定されないが、カルシウム塩、マグネシウム塩、カリウム塩、ナトリウム塩、亜鉛塩、鉄塩、銅塩等が挙げられる。 Although it does not specifically limit as salt of sugar carboxylic acid, Calcium salt, magnesium salt, potassium salt, sodium salt, zinc salt, iron salt, copper salt etc. are mentioned.
糖カルボン酸への酸化反応は、還元糖量の減少から確認することができ、例えばネルソン・ソモギ法による比色定量法を用いることが出来る。 The oxidation reaction to the sugar carboxylic acid can be confirmed from the decrease in the amount of reducing sugar, and for example, a colorimetric determination method by the Nelson-Somogi method can be used.
また、ＨＰＬＣにより原料糖質や糖カルボン酸を分析することで確認することも可能である。例えば、マルトースを原料に酸化反応を行った後、ＨＰＡＥＤ−ＰＡＤ法（パルスドアンペロメトリー検出器、ＣａｒｂｏＰａｃ ＰＡ１カラム）により、溶出：３５℃、１．０ｍｌ／ｍｉｎ、水酸化ナトリウム濃度：１００ｍＭ、酢酸ナトリウム濃度：０分−０ｍＭ、２分−０ｍＭ、２０分−２０ｍＭの条件で測定すれば、マルトース、マルトビオン酸を定量することが可能である。 Moreover, it can also confirm by analyzing raw material saccharide | sugar and sugar carboxylic acid by HPLC. For example, after an oxidation reaction using maltose as a raw material, elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, acetic acid by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column) Sodium concentration: Maltose and maltobionic acid can be quantified by measurement under conditions of 0 min-0 mM, 2 min-0 mM, and 20 min-20 mM.
本発明方法を使用して調製した糖カルボン酸は、飲食物や化粧品、医薬品、化成品等へ使用することが可能である。 The sugar carboxylic acid prepared using the method of the present invention can be used for foods and drinks, cosmetics, pharmaceuticals, chemical products and the like.
（試験例１） カタラーゼ製剤Ａによるマルトビオン酸生産への影響
バッフル付きの三角フラスコへ、マルトース（和光純薬製）９ｇを蒸留水へ溶解させ３０ｇ（３０ｗｔ％）とした後、炭酸カルシウム（和光純薬製）１．５ｇと、Ａｃｒｅｍｏｎｉｕｍ ｃｈｒｙｓｏｇｅｎｕｍ由来糖質酸化酵素製剤（糖質酸化活性３００ｕ／ｍｌ）１２０μｌ（３６ｕ、４ｕ／ｇ基質）と、Ａｓｐｅｒｇｉｌｌｕｓ属由来のカタラーゼ製剤Ａ（カタラーゼ活性７５０６０ｕ／ｍｌ、糖化活性２４．８ｕ／ｍｌ、糖化活性／カタラーゼ活性比＝０．０００３３）０〜４５００Ｕ（０〜５００ｕ／ｇ基質）を加え、３０℃、２００ｒｐｍで振盪反応を行いマルトビオン酸の生産効率への影響を評価した。
(Test Example 1) Effect of catalase preparation A on maltobionic acid production 9 g of maltose (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water in an Erlenmeyer flask with a baffle to make 30 g (30 wt%), and then calcium carbonate (Wako Pure) 1.5 g of sugar oxidase preparation (sugar oxidase activity 300 u / ml) from Acremonium chrysogenum (36 u, 4 u / g substrate), catalase preparation A (catalase activity 75060 u / ml) derived from the genus Aspergillus, Saccharification activity 24.8u / ml, Saccharification activity / catalase activity ratio = 0.00033) Add 0-4500U (0-500u / g substrate), shake reaction at 30 ° C, 200rpm and effect on production efficiency of maltobionic acid Evaluated.
反応組成中のマルトビオン酸量は、ＨＰＡＥＤ−ＰＡＤ法（パルスドアンペロメトリー検出器、ＣａｒｂｏＰａｃ ＰＡ１カラム）により、溶出：３５℃、１．０ｍｌ／ｍｉｎ、水酸化ナトリウム濃度：１００ｍＭ、酢酸ナトリウム濃度：０分−０ｍＭ、２分−０ｍＭ、２０分−２０ｍＭの条件で分析した。 The amount of maltobionic acid in the reaction composition was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: 0. Analysis was performed under conditions of min-0 mM, 2 min-0 mM, and 20 min-20 mM.
評価の結果、比較例１のカタラーゼ無添加では、マルトビオン酸への変換率は２５％に留まった。これに対し、所定のカタラーゼ製剤を適量で用いた実施例１〜６では、マルトビオン酸へ９０％以上変換することができた。 As a result of the evaluation, when no catalase was added in Comparative Example 1, the conversion rate to maltobionic acid remained at 25%. On the other hand, in Examples 1 to 6 using a predetermined catalase preparation in an appropriate amount, 90% or more could be converted to maltobionic acid.
（試験例２） カタラーゼ製剤Ａによるデキストリン酸化物生産への影響
バッフル付きの三角フラスコへ、デキストリン（商品名ＮＳＤ７００，サンエイ糖化社製）９ｇを蒸留水へ溶解させ３０ｇ（３０ｗｔ％）とした後、炭酸カルシウム（和光純薬製）０．７５ｇ、Ａｃｒｅｍｏｎｉｕｍ ｃｈｒｙｓｏｇｅｎｕｍ由来糖質酸化酵素製剤（糖質酸化活性３００ｕ／ｍｌ）６０μｌ（１８ｕ、２ｕ／ｇ基質）と、Ａｓｐｅｒｇｉｌｌｕｓ属由来のカタラーゼ製剤Ａ（カタラーゼ活性７５０６０ｕ／ｍｌ、糖化活性２４．８ｕ／ｍｌ、糖化活性／カタラーゼ活性比＝０．０００３３）０〜９０００Ｕ（０〜３００ｕ／ｇ基質）を加え、３０℃、２００ｒｐｍで振盪反応を行いデキストリンの酸化効率への影響を評価した。酸化率は、反応液の還元糖量をネルソン・ソモギ法で定量し、次式により変換率を算出した。
（反応開始前還元糖量−反応液還元糖量）／反応開始前還元糖量×１００＝酸化率（％）
(Test Example 2) Effect on Catalyst Formulation A on Dextrin Oxide Production After 9 g of dextrin (trade name NSD700, manufactured by Sanei Saccharification Co., Ltd.) was dissolved in distilled water in an Erlenmeyer flask with baffle to make 30 g (30 wt%), 0.75 g of calcium carbonate (manufactured by Wako Pure Chemical Industries), 60 μl (18 u, 2 u / g substrate) of saccharide oxidase preparation (carbohydrate oxidative activity 300 u / ml) derived from Acremonium chrysogenum, and catalase preparation A (catalase activity derived from Aspergillus genus) 75060 u / ml, saccharification activity 24.8 u / ml, saccharification activity / catalase activity ratio = 0.00033) 0-9000 U (0-300 u / g substrate) was added, and the dextrin oxidation efficiency was carried out at 30 ° C. and 200 rpm. The impact on was evaluated. For the oxidation rate, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)
評価の結果、比較例２のカタラーゼ無添加では、副生した過酸化水素量により糖質酸化酵素が変性失活されたため３４％の酸化率に留まった。所定のカタラーゼ製剤を適量で用いた実施例７〜１１では、９０％以上を酸化することができた。 As a result of the evaluation, when no catalase of Comparative Example 2 was added, the carbohydrate oxidase was denatured and inactivated by the amount of hydrogen peroxide produced as a by-product, so that the oxidation rate remained at 34%. In Examples 7 to 11 using a predetermined catalase preparation in an appropriate amount, 90% or more could be oxidized.
（試験例３） カタラーゼ製剤Ｂによるデキストリン酸化物生産への影響
バッフル付きの三角フラスコへ、デキストリン（商品名ＮＳＤ７００，サンエイ糖化社製）９ｇを蒸留水へ溶解させ３０ｇ（３０ｗｔ％）とした後、炭酸カルシウム（和光純薬製）０．７５ｇ、Ａｃｒｅｍｏｎｉｕｍ ｃｈｒｙｓｏｇｅｎｕｍ由来糖質酸化酵素製剤（糖質酸化活性３００ｕ／ｍｌ）６０μｌ（１８ｕ、２ｕ／ｇ基質）と、Ａｓｐｅｒｇｉｌｌｕｓ属由来のカタラーゼ製剤Ｂ（カタラーゼ活性２７４００ｕ／ｍｌ、糖化活性２４４ｕ／ｍｌ、糖化活性／カタラーゼ活性比＝０．００８９）を表３の記載量を加え、３０℃、２００ｒｐｍで振盪反応を行いデキストリンの酸化効率への影響を評価した。酸化率は、反応液の還元糖量をネルソン・ソモギ法で定量し、次式により変換率を算出した。
（反応開始前還元糖量−反応液還元糖量）／反応開始前還元糖量×１００＝酸化率（％）
(Test Example 3) Effect of catalase preparation B on dextrin oxide production In a conical flask with a baffle, 9 g of dextrin (trade name NSD700, manufactured by Sanei Saccharification Co., Ltd.) was dissolved in distilled water to make 30 g (30 wt%). 0.75 g of calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), sugar oxidase preparation derived from Acremonium chrysogenum (carbohydrate oxidizing activity 300 u / ml) 60 μl (18 u, 2 u / g substrate), and catalase preparation B (catalase activity derived from Aspergillus genus) 27400 u / ml, saccharification activity 244 u / ml, saccharification activity / catalase activity ratio = 0.0089) were added in the amounts shown in Table 3, and the shaking reaction was performed at 30 ° C. and 200 rpm to evaluate the effect on dextrin oxidation efficiency. For the oxidation rate, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)
また、反応生成物の糖組成は、ＨＰＡＥＤ−ＰＡＤ法（パルスドアンペロメトリー検出器、ＣａｒｂｏＰａｃ ＰＡ１カラム）により、溶出：３５℃、１．０ｍｌ／ｍｉｎ、水酸化ナトリウム濃度：１００ｍＭ、酢酸ナトリウム濃度：０分−０ｍＭ、５分−０ｍＭ、５５分−４０ｍＭの条件で分析した。 Further, the sugar composition of the reaction product was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: The analysis was performed under the conditions of 0 min-0 mM, 5 min-0 mM, 55 min-40 mM.
評価の結果、糖化活性が原料基質の還元糖あたり０．９ｕ／ｇ以下でも、糖化活性／カタラーゼ活性比＝０．００５以上となる比較例３は、酸化率８割以下であり、且つ組成中の加水分解により生成したグルコースが酸化されたグルコン酸量が９％程度となっており、原料のデキストリンが加水分解による影響を受けていることが分かる。 As a result of the evaluation, even if the saccharification activity is 0.9 u / g or less per reducing sugar of the raw material substrate, Comparative Example 3 in which the saccharification activity / catalase activity ratio is 0.005 or more has an oxidation rate of 80% or less and is in the composition The amount of gluconic acid obtained by oxidation of glucose produced by hydrolysis of 9 is about 9%, which indicates that the dextrin as a raw material is affected by hydrolysis.
また、糖化活性が原料基質の還元糖あたり０．９ｕ／ｇ以上で、糖化活性／カタラーゼ活性比＝０．００５以上となる比較例４は、酸化率は７割以下であり、且つ組成中の加水分解により生成したグルコースが酸化されたグルコン酸量が１６％程度と、原料のデキストリンが加水分解による影響を受けていることが分かる。 Further, in Comparative Example 4 in which the saccharification activity is 0.9 u / g or more per reducing sugar of the raw material substrate and the saccharification activity / catalase activity ratio is 0.005 or more, the oxidation rate is 70% or less, and It can be seen that the amount of gluconic acid in which glucose produced by hydrolysis is oxidized is about 16%, and the dextrin as a raw material is affected by hydrolysis.
（試験例４） カタラーゼ製剤Ｃによるデキストリン酸化物生産への影響
バッフル付きの三角フラスコへ、デキストリン（商品名ＮＳＤ７００，サンエイ糖化社製）９ｇを蒸留水へ溶解させ３０ｇ（３０ｗｔ％）とした後、炭酸カルシウム（和光純薬製）０．７５ｇ、Ａｃｒｅｍｏｎｉｕｍ ｃｈｒｙｓｏｇｅｎｕｍ由来糖質酸化酵素製剤（糖質酸化活性３００ｕ／ｍｌ）と、Ａｓｐｅｒｇｉｌｌｕｓ属由来のカタラーゼ製剤Ｃ（カタラーゼ活性５２０００ｕ／ｍｌ、糖化活性６４ｕ／ｍｌ、糖化活性／カタラーゼ活性比＝０．００１２）を表４の記載量を加え、３０℃、２００ｒｐｍで振盪反応を行いデキストリンの酸化効率への影響を評価した。酸化率は、反応液の還元糖量をネルソン・ソモギ法で定量し、次式により変換率を算出した。
（反応開始前還元糖量−反応液還元糖量）／反応開始前還元糖量×１００＝酸化率（％）
(Test Example 4) Effect of catalase preparation C on dextrin oxide production In a conical flask with a baffle, 9 g of dextrin (trade name NSD700, manufactured by Sanei Saccharification Co., Ltd.) was dissolved in distilled water to make 30 g (30 wt%). Calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) 0.75 g, Acrenium chrysogenum-derived carbohydrate oxidase preparation (carbohydrate oxidation activity 300 u / ml), Aspergillus-derived catalase preparation C (catalase activity 52000 u / ml, saccharification activity 64 u / ml) The ratio of saccharification activity / catalase activity = 0.0012) was added in the amount shown in Table 4, and a shaking reaction was performed at 30 ° C and 200 rpm to evaluate the effect on dextrin oxidation efficiency. For the oxidation rate, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)
また、反応生成物の糖組成は、ＨＰＡＥＤ−ＰＡＤ法（パルスドアンペロメトリー検出器、ＣａｒｂｏＰａｃ ＰＡ１カラム）により、溶出：３５℃、１．０ｍｌ／ｍｉｎ、水酸化ナトリウム濃度：１００ｍＭ、酢酸ナトリウム濃度：０分−０ｍＭ、５分−０ｍＭ、５５分−４０ｍＭの条件で分析した。 Further, the sugar composition of the reaction product was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: The analysis was performed under the conditions of 0 min-0 mM, 5 min-0 mM, 55 min-40 mM.
評価の結果、糖化活性／カタラーゼ活性比＝０．００５以下でも、糖化活性が原料基質の還元糖あたり０．９ｕ／ｇ以上となる比較例５は、酸化率は７割以下であり、且つ組成中の加水分解により生成したグルコースが酸化されたグルコン酸量が１９％程度となっており、原料のデキストリンが加水分解による影響を受けていることが分かる。一方、糖化活性が原料基質の還元糖あたり０．９ｕ／ｇ以下である実施例１２では、酸化率は９割以上であり組成中のグルコン酸量も７％以下と、大きく低分子化することなく、酸化反応が進んだ。 As a result of the evaluation, even when the ratio of saccharification activity / catalase activity = 0.005 or less, Comparative Example 5 in which the saccharification activity is 0.9 u / g or more per reducing sugar of the raw material substrate has an oxidation rate of 70% or less and a composition The amount of gluconic acid obtained by oxidizing glucose produced by hydrolysis in the medium is about 19%, which indicates that the dextrin as a raw material is affected by hydrolysis. On the other hand, in Example 12, in which the saccharification activity is 0.9 u / g or less per reducing sugar of the raw material substrate, the oxidation rate is 90% or more and the amount of gluconic acid in the composition is 7% or less, and the molecular weight is greatly reduced. There was no oxidation reaction.
（試験例５） カタラーゼ製剤Ｃによるマルトビオン酸生産への影響
バッフル付きの三角フラスコへ、３０（ｗｔ）％ハイマルトース溶液３０ｇ、炭酸カルシウム（和光純薬製）０．７５ｇ、Ａｃｒｅｍｏｎｉｕｍ ｃｈｒｙｓｏｇｅｎｕｍ由来糖質酸化酵素製剤（糖質酸化活性３００ｕ／ｍｌ）と、Ａｓｐｅｒｇｉｌｌｕｓ属由来のカタラーゼ製剤Ｃ（カタラーゼ活性５２０００ｕ／ｍｌ、糖化活性６４ｕ／ｍｌ、糖化活性／カタラーゼ活性比＝０．００１２）を表５の記載量を加え、３０℃、２００ｒｐｍで振盪反応を行いマルトビオン酸の生産効率への影響を評価した。
(Test Example 5) Influence of catalase preparation C on maltobionic acid production To a conical flask with a baffle, 30 g of 30 (wt)% hymaltose solution, 0.75 g of calcium carbonate (manufactured by Wako Pure Chemical Industries), saccharide oxidation from Acremonium chrysogenum Table 5 shows enzyme preparation (sugar oxidation activity 300 u / ml) and catalase preparation C derived from the genus Aspergillus (catalase activity 52000 u / ml, saccharification activity 64 u / ml, saccharification activity / catalase activity ratio = 0.0012) And the shaking reaction was carried out at 30 ° C. and 200 rpm to evaluate the influence on the production efficiency of maltobionic acid.
反応組成中のマルトビオン酸量は、ＨＰＡＥＤ−ＰＡＤ法（パルスドアンペロメトリー検出器、ＣａｒｂｏＰａｃ ＰＡ１カラム）により、溶出：３５℃、１．０ｍｌ／ｍｉｎ、水酸化ナトリウム濃度：１００ｍＭ、酢酸ナトリウム濃度：０分−０ｍＭ、２分−０ｍＭ、２０分−２０ｍＭの条件で分析した。 The amount of maltobionic acid in the reaction composition was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: 0. Analysis was performed under conditions of min-0 mM, 2 min-0 mM, and 20 min-20 mM.
評価の結果、糖化活性／カタラーゼ活性比＝０．００５以下でも、糖化活性が原料基質の還元糖あたり０．９ｕ／ｇ以上となる比較例６は、原料のマルトースが加水分解されたことで、マルトビオン酸への変換量は８割以下であったの対して、糖化活性が原料基質の還元糖あたり０．９ｕ／ｇ以下で実施例１３では、殆ど夾雑酵素による加水分解の影響を受けることなく、９８％以上がマルトビオン酸へ変換された。 As a result of evaluation, Comparative Example 6 in which the saccharification activity is 0.9 u / g or more per reducing sugar of the raw material substrate even when the saccharification activity / catalase activity ratio is 0.005 or less is that maltose of the raw material is hydrolyzed, While the conversion amount to maltobionic acid was 80% or less, the saccharification activity was 0.9 u / g or less per reducing sugar of the raw material substrate, and in Example 13, there was almost no influence of hydrolysis by contaminating enzymes. 98% or more was converted to maltobionic acid.
（試験例６） カタラーゼ製剤Ｄによるデキストリン酸化物生産への影響
バッフル付きの三角フラスコへ、デキストリン（商品名ＮＳＤ７００，サンエイ糖化社製）９ｇを蒸留水へ溶解させ３０ｇ（３０ｗｔ％）とした後、炭酸カルシウム（和光純薬製）０．７５ｇ、Ａｃｒｅｍｏｎｉｕｍ ｃｈｒｙｓｏｇｅｎｕｍ由来糖質酸化酵素製剤（糖質酸化活性３００ｕ／ｍｌ）６０μｌ（１８ｕ、２ｕ／ｇ基質）と、Ａｓｐｅｒｇｉｌｌｕｓ属由来のカタラーゼ製剤Ｄ（カタラーゼ活性５４４００ｕ／ｍｌ、糖化活性２４２ｕ／ｍｌ、糖化活性／カタラーゼ活性比＝０．００４４）を表６の記載量を加え、３０℃、２００ｒｐｍで振盪反応を行いデキストリンの酸化効率への影響を評価した。酸化率は、反応液の還元糖量をネルソン・ソモギ法で定量し、次式により変換率を算出した。
（反応開始前還元糖量−反応液還元糖量）／反応開始前還元糖量×１００＝酸化率（％）
(Test Example 6) Influence on dextrin oxide production by catalase preparation D In an Erlenmeyer flask with baffle, 9 g of dextrin (trade name NSD700, manufactured by Sanei Saccharification Co., Ltd.) was dissolved in distilled water to make 30 g (30 wt%). 0.75 g of calcium carbonate (manufactured by Wako Pure Chemical Industries), sugar oxidase preparation from Acrenium chrysogenum (sugar oxidation activity 300 u / ml) 60 μl (18 u, 2 u / g substrate), and catalase preparation D (catalase activity from Aspergillus genus) 54400 u / ml, saccharification activity 242 u / ml, saccharification activity / catalase activity ratio = 0.0044) were added in the amounts shown in Table 6, and the shaking reaction was performed at 30 ° C. and 200 rpm to evaluate the effect on dextrin oxidation efficiency. For the oxidation rate, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)
また、反応生成物の糖組成は、ＨＰＡＥＤ−ＰＡＤ法（パルスドアンペロメトリー検出器、ＣａｒｂｏＰａｃ ＰＡ１カラム）により、溶出：３５℃、１．０ｍｌ／ｍｉｎ、水酸化ナトリウム濃度：１００ｍＭ、酢酸ナトリウム濃度：０分−０ｍＭ、５分−０ｍＭ、４０分−４０ｍＭの条件で分析した。 Further, the sugar composition of the reaction product was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: The analysis was performed under the conditions of 0 min-0 mM, 5 min-0 mM, and 40 min-40 mM.
評価の結果、糖化活性／カタラーゼ活性比＝０．００５以下でも、糖化活性が原料基質の還元糖あたり０．９ｕ／ｇ以上となる、比較例７は、酸化率は７割以下であり、且つ組成中の加水分解により生成したグルコースが酸化されたグルコン酸量が２６％となっており、原料のデキストリンが加水分解による影響を受けていることが分かる。一方、糖化活性が原料基質の還元糖あたり０．９ｕ／ｇ以下である実施例１４では、酸化率は９割以上であり組成中のグルコン酸量も８％以下と、大きく低分子化することなく、酸化反応が進んだ。 As a result of the evaluation, even when the saccharification activity / catalase activity ratio is 0.005 or less, the saccharification activity is 0.9 u / g or more per reducing sugar of the raw material substrate. In Comparative Example 7, the oxidation rate is 70% or less, and The amount of gluconic acid obtained by oxidizing glucose produced by hydrolysis in the composition is 26%, which indicates that the dextrin as a raw material is affected by hydrolysis. On the other hand, in Example 14 where the saccharification activity is 0.9 u / g or less per reducing sugar of the raw material substrate, the oxidation rate is 90% or more, and the amount of gluconic acid in the composition is 8% or less, which greatly reduces the molecular weight. There was no oxidation reaction.
（試験例７）
（実施例１５）
マルトース ７０．３ｗｔ％に加えて、グルコース １．２ｗｔ％、マルトトリオース１５．０ｗｔ％及びマルトテトラオース（重合度４）以上のマルトオリゴ糖１３．５ｗｔ％を含むハイマルトース水飴（Ｂｘ．７５％、サンエイ糖化製）８８０ｇに蒸留水１３２０ｇを加え、３０％ｗｔとなるように溶解させた後、炭酸カルシウム（和光純薬製）８６ｇ、Ａｃｒｅｍｏｎｉｕｍ ｃｈｒｙｓｏｇｅｎｕｍ由来糖質酸化酵素製剤（糖質酸化活性３００ｕ／ｍｌ）４．４ｍｌ（１３２０ｕ、２ｕ／ｇ基質）と、Ａｓｐｅｒｇｉｌｌｕｓ属由来のカタラーゼ製剤Ａ（カタラーゼ活性７５０６０ｕ／ｍｌ、糖化活性２４．８ｕ／ｍｌ、糖化活性／カタラーゼ活性比＝０．０００３３）０．６６ｍ（ｌ４９５００Ｕ、７０ｕ／ｇ基質）を加え、３５℃、５００ｒｐｍ、空気通気２Ｌ／分で通気攪拌をおこなった。反応開始から１２時間後に、糖質酸化酵素剤１．１ｍｌ（３３０ｕ、０．５ｕ／ｇ基質）とカタラーゼ製剤０．２２ｍｌ（１６５００ｕ、２５ｕ／ｇ基質）を追加添加し酸化反応を行った。
(Test Example 7)
(Example 15)
In addition to maltose 70.3 wt%, glucose maltose (Bx.75%, containing 1.2 wt% glucose, 15.0 wt% maltotriose and 13.5 wt% malto-oligosaccharide (degree of polymerization 4) or higher) After adding 1320 g of distilled water to 880 g of Sanei Saccharification Co., Ltd. and dissolving to 30% wt, 86 g of calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.), sugar oxidase preparation derived from Acremonium chrysogenum (carbohydrate oxidative activity 300 u / ml) ) 4.4 ml (1320u, 2u / g substrate) and catalase preparation A derived from the genus Aspergillus (catalase activity 75060u / ml, saccharification activity 24.8u / ml, saccharification activity / catalase activity ratio = 0.00033) 0.66m (149500 U, 70 u / g substrate) 00rpm, it was subjected to aeration-agitation in the air ventilation 2L / minute. Twelve hours after the start of the reaction, 1.1 ml (330 u, 0.5 u / g substrate) of a carbohydrate oxidase agent and 0.22 ml of a catalase preparation (16500 u, 25 u / g substrate) were additionally added to carry out an oxidation reaction.
酸化反応の推移は、反応液の還元糖量をネルソン・ソモギ法で定量し、次式により変換率を算出した。
（反応開始前還元糖量−反応液還元糖量）／反応開始前還元糖量×１００＝酸化率（％）
For the transition of the oxidation reaction, the amount of reducing sugar in the reaction solution was quantified by the Nelson-Somogi method, and the conversion rate was calculated by the following formula.
(Reducing sugar amount before reaction start-reducing sugar amount in reaction solution) / Reducing sugar amount before reaction start × 100 = oxidation rate (%)
また、反応生成物の糖組成は、ＨＰＡＥＤ−ＰＡＤ法（パルスドアンペロメトリー検出器、ＣａｒｂｏＰａｃ ＰＡ１カラム）により、溶出：３５℃、１．０ｍｌ／ｍｉｎ、水酸化ナトリウム濃度：１００ｍＭ、酢酸ナトリウム濃度：０分−０ｍＭ、２分−０ｍＭ、２０分−２０ｍＭの条件で分析した。 Further, the sugar composition of the reaction product was determined by HPAED-PAD method (pulsed amperometry detector, CarboPac PA1 column), elution: 35 ° C., 1.0 ml / min, sodium hydroxide concentration: 100 mM, sodium acetate concentration: The analysis was performed under the conditions of 0 min-0 mM, 2 min-0 mM, and 20 min-20 mM.
糖化活性／カタラーゼ活性比＝０．００５以下（０．０００３３）且つ糖化活性が原料基質の還元糖あたり０．９ｕ／ｇ以下（０．０６６）の実施例１５について、表７に酸化反応開始時から４５時間までの経過時における酸化率を示す。表７に示すとおり、反応４５時間後には１００％酸化され、この時の生成物は、カルシウム４．１ｗｔ％を含む、マルトビオン酸 ６７．４ｗｔ％に加えて、グルコン酸 １．２ｗｔ％、マルトトリオン酸１４．４ｗｔ％及びマルトテトラオン酸（重合度４）以上のマルトオリゴ糖酸１２．９ｗｔ％で構成されており、糖化活性による加水分解等の影響がないことが確認された。 Table 7 shows the saccharification activity / catalase activity ratio = 0.005 or less (0.00033) and the saccharification activity is 0.9 u / g or less (0.066) per reducing sugar of the raw material substrate. The oxidation rate at the time from 45 hours to 45 hours is shown. As shown in Table 7, after 45 hours of reaction, the product was 100% oxidized. The product at this time contained calcium 4.1 wt%, maltobionic acid 67.4 wt%, gluconic acid 1.2 wt%, maltotrione It is composed of 14.4 wt% acid and 12.9 wt% malto-oligosaccharide acid (polymerization degree 4) or more, and it was confirmed that there is no influence of hydrolysis or the like due to saccharification activity.
Claims (6)
糖質酸化時に過酸化水素を副生する糖質酸化酵素剤を、カタラーゼ製剤中のカタラーゼ活性（Ａ）に対する糖化活性（Ｂ）の含有比率（Ｂ／Ａ）が０．００００２以上０．００５以下であるカタラーゼ製剤の存在下、前記澱粉分解物或いは転移反応物を含む原料基質に作用させる工程を含み、
前記カタラーゼ製剤は、その糖化活性が前記原料基質中の還元糖量（ｗｔ％）に対して０．９ｕ／ｇ以下である量で存在する方法。 A method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at the reducing end of 2 or more is oxidized,
Carbohydrate oxidase that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) of 0.00002 to 0.005 in catalase preparation to catalase activity (A). In the presence of a catalase preparation, which comprises the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.
糖質酸化時に過酸化水素を副生する糖質酸化酵素剤を、カタラーゼ製剤中のカタラーゼ活性（Ａ）に対する糖化活性（Ｂ）の含有比率（Ｂ／Ａ）が０．００５以下でありかつ糖化活性（Ｂ）が０．１ｕ／ｍｌ以上であるカタラーゼ製剤の存在下、前記澱粉分解物或いは転移反応物を含む原料基質に作用させる工程を含み、
前記カタラーゼ製剤は、その糖化活性が前記原料基質中の還元糖量（ｗｔ％）に対して０．９ｕ／ｇ以下である量で存在する方法。 A method for producing a sugar carboxylic acid in which an aldehyde group on the reducing end side of a starch degradation product or transfer reaction product having a glucose residue at the reducing end of 2 or more is oxidized,
A saccharide oxidase agent that produces hydrogen peroxide as a by-product during oxidization of saccharides has a saccharification activity (B) content ratio (B / A) to catalase activity (A) in the catalase preparation of 0.005 or less and saccharification Including the step of acting on a raw material substrate containing the starch degradation product or transfer reaction product in the presence of a catalase preparation having an activity (B) of 0.1 u / ml or more,
The catalase preparation is a method in which the saccharification activity is present in an amount of 0.9 u / g or less with respect to the amount of reducing sugar (wt%) in the raw material substrate.
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