JP3607789B2 - α-1,3-Multi-branched dextran hydrolase, process for producing the same, and process for producing cyclic isomaltoligosaccharide - Google Patents

Description

【００２３】

【００２４】

【００２５】
スフィンゴバクテリウム・エスピー．Ｖ−５４菌株は、上記の諸性質さらに、菌体内の脂質としてスフィンゴリピドを持つこと、主要なキノンがグラム陰性菌にもかかわらずメナキノンであることよりスフィンゴバクテリウム属に属する細菌であると同定した。更に、種の同定のために、バージェイズ・マニュアル・オブ・システィマティック・バクテリオロジーに基づき、種々検討したが、種の確定までには至らなかったため、スフィンゴバクテリウム属に属する１菌株として分類同定した。
【００２６】
なお、スフィンゴバクテリウム・エスピー．Ｖ−５４は、平成９年２月１８日、工業技術院生命工学工業技術研究所にＦＥＲＭ Ｐ−１６０８６として寄託されている。
この酵素の製造法において使用される微生物としては、上記菌株のほかスフィンゴバクテリウム（Ｓｐｈｉｎｇｏｂａｃｔｅｒｉｕｍ）属に属し、α−１，３−の多分岐デキストランから著量のグルコースを生成するものであれば、如何なるものでもよい。
【００２７】
本発明方法における菌株の培養は、原則的には一般微生物の好気的培養で採用される方法と同じであるが、通常は、液体培地による振盪培養法または通気撹拌培養法等が用いられる。
【００２８】
培地としては、適当な窒素源（例えば、ペプトン、ポリペプトン、バクトトリプトン等のカゼイン分解物あるいはソイトン等の大豆蛋白質分解物等）、炭素源（例えばグルコース、グリセリン等の糖質類）、そして必要により、酵母エキス、リボフラビン等のビタミン類及びリン酸塩、マグネシウム塩、塩化ナトリウム、微量金属等のミネラル類、そして本酵素の誘導基質となるデキストラン等を含有したものが用いられる。ｐＨは、本菌が成育するｐＨ域ならばいずれでもよく、通常は、６〜８の範囲が好ましい。
【００２９】
培養条件は、例えば、通常２０〜４０℃で、１６時間〜４日間振盪培養または通気撹拌培養を行なう。
以上の如くして得た培養物を用いて例えば、以下に示す酵素精製工程により該多分岐デキストラン水解酵素を得る。
【００３０】
上記培養物から該酵素を得るには、遠心分離、膜濃縮等により集菌した菌体を更に超音波処理あるいは界面活性剤処理して溶菌し、遠心分離して細胞残渣を除去する。さらに、必要により、通常用いられる酵素の精製法により上記粗酵素液から精製標品を得る。
【００３１】
本酵素の精製法は、通常の酵素分離方法であれば如何なる方法でもよいが、本製造法の場合、菌体を超音波処理あるいは界面活性剤処理して溶菌し、遠心分離して細胞残渣を除去した上清液を実施例に示した精製方法に従って処理することにより、高純度の多分岐デキストラン水解酵素標品を得ることが出来る。尚、本製造法は、これらの精製法に限定されるものではない。さらに、本酵素を環状イソマルトオリゴ糖生成反応液に作用させ、反応液中に存在する、分岐オリゴ糖特に分岐環状オリゴ糖をグルコースと環状イソマルトオリゴ糖に水解した後に環状オリゴ糖を分離精製する。尚、本製造法の場合、精製酵素を用いずに、先に述べた細胞残渣を除去した上清液を用いても精製酵素と同等の効率で分岐環状イソマルトオリゴ糖を水解することができる。反応液より該オリゴ糖を分離精製する手段としては、オリゴ糖精製法であれば如何なる方法でもよい。例えば、冷却処理、有機溶媒添加処理、活性炭処理、あるいは特異的に環状オリゴ糖を吸着するカラムクロマトグラフィー、活性炭カラムクロマトグラフィー等の公知方法を単独もしくは適宜組合わせて分離精製することができる。
【００３２】
【実施例】
以下に、実施例を挙げて本発明を更に具体的に説明する。
【００３３】
〔実施例１〕
１％デキストラン４０（名糖産業社製）、１％ペプトン（極東製薬工業社製）、０．５％ ＮａＣｌ及び０．１％イーストエキス（ディフコ社製）からなる液体培地（水道水使用、ｐＨ ７．０）１００ｍｌを５００ｍｌ容坂口フラスコに入れ、１２０℃で２０分間、殺菌処理を行なった。これに、スフィンゴバクテリウム・エスピー．Ｖ−５４菌株（ＦＥＲＭ Ｐ− １６０８６）保存スラントより１白金耳接種し、３０℃で１日間振盪培養した。本培養液を１０ｍｌずつ上記と同様の培地組成と殺滅菌条件により調製した２Ｌの培地を含有する３Ｌ容ミニジャーファーメンター８基に接種し、３０℃、０．５ｖｖｍ、３００ｒｐｍの条件で３日間通気撹拌培養を行ない、培養終了後、培養液１６Ｌを遠心分離処理により集菌し、得られた約６００ｍｌの菌体懸濁液を２００ｍｌずつ超音波処理により破砕し、遠心分離して細胞残渣を除去した。得られた上清液約６００ｍｌを粗酵素液として用いて、多分岐デキストラン水解酵素の精製を行なった。
【００３４】
先ず、本酵素液に対して、終濃度１．０Ｍになるように硫安を加え、遠心分離により不溶物を除去した。上澄液を以下の操作による分取用ＨＰＬＣで精製した。上澄液を１００ｍｌずつ１．０Ｍ硫安含有の１００ｍＭリン酸緩衝液（ｐＨ７．０）で平衡化したＴＳＫｇｅｌ Ｐｈｅｎｙｌ５ＰＷカラムにアプライし、同緩衝液で洗浄後、１．０〜０Ｍの硫安濃度勾配により吸着蛋白質を溶出した。６回の操作により集めた活性画分約５００ｍｌを限外濾過で約２０ｍｌに濃縮した後、１０ｍＭリン酸緩衝液（ｐＨ７．０）約５００ｍｌを加えて塩濃度を稀釈した。再度同希釈液を、限外濾過で約１００ｍｌに濃縮した。濃縮液を１０ｍＭリン酸緩衝液（ｐＨ７．０）で平衡化したＴＳＫｇｅｌ ＤＥＡＥ５ＰＷカラムにアプライし、同緩衝液で非吸着蛋白質を洗浄した。本条件で、該酵素は全て非吸着画分に溶出していたので、非吸着画分約２００ｍｌを約２０ｍｌにまで濃縮後、１０ｍＭリン酸緩衝液（ｐＨ７．５）を１８０ｍｌ加えることにより、ｐＨを７．０から７．５に変更した。
【００３５】
本酵素液を再度、約１００ｍｌにまで限外濃縮した後、濃縮液を１０ｍＭリン酸緩衝液（ｐＨ７．５）で平衡化したＴＳＫｇｅｌ ＤＥＡＥ５ＰＷカラムに再度アプライした。同緩衝液で非吸着蛋白質を洗浄した後、０〜０．５ＭのＮａＣｌ濃度勾配により吸着蛋白質を溶出した。
【００３６】
活性は吸着溶出画分に認められたので、活性画分４０ｍｌを集め、更に本酵素液を１．５ｍｌまで限外濃縮した。２００ｍＭ ＮａＣｌ含有の１００ｍＭリン酸緩衝液（ｐＨ７．０）で平衡化したＴＳＫｇｅｌ Ｇ３０００ＳＷカラムに、０．３ｍｌずつ酵素溶液をアプライし、同緩衝液により溶出した。本操作を残り１．２ｍｌについて同様に行なった（０．３ｍｌずつ４回）。得られた活性画分を集めてＳＤＳ ＰＡＧＥに供したところ、シングルバンドを示したことから、他の夾雑蛋白質は除去されたと判断し精製を終了した。本精製酵素をα−１，３−多分岐デキストランとインキュベーションしたところ、多分岐デキストランから著量のグルコースの生成が確認された。
【００３７】
〔実施例２〕
バチルス・エスピー（Ｂａｃｉｌｌｕｓ ｓｐ．）Ｔ−３０４０株（ＦＥＲＭ ＢＰ−４１３２）の培養液３００Ｌをマイクローザを用いた膜処理により除菌し、得られた除菌液を更に分子量６０００カットのフォロファイバーにより６．３Ｌにまで濃縮した後、濃縮液を９００ｍｌずつ−２０℃に凍結保存した。濃縮液の一部を、デキストラン（名糖産業社製）１００ｇを１０ｍＭリン酸緩衝液（ｐＨ６．５）１０Ｌに溶解した水溶液と混合し、４０℃で４８時間インキュベーションを行なった。活性炭を加えて煮沸することにより反応を停止させ、かつ未反応のデキストランを吸着させた。活性炭を除去した上清液を脱イオン水で平衡化した活性炭カラムにアプライし、脱イオン水で洗浄後、エタノール濃度勾配により吸着したオリゴ糖を溶出した。環状オリゴ糖画分を集め、濃縮した後、脱イオン水で平衡化したＯＤＳカラムにアプライした。脱イオン水で洗浄後、エタノール濃度勾配によりオリゴ糖を溶出し、各環状イソマルトオリゴ糖画分を集め、凍結乾燥した。サイクロイソマルトノナオースの画分に、分岐環状イソマルトオリゴ糖が約５％程度混入していた。
【００３８】
〔実施例３〕
実施例２で得られた分岐環状オリゴ糖の混入したサイクロイソマルトノナオース標品の４％水溶液を調製し、この水溶液０．５ｍｌを、実施例１の菌体の超音波処理破砕液より調製した粗酵素液０．１ｍｌ、１００ｍＭリン酸緩衝液（ｐＨ７．０）０．４ｍｌと混和し、４０℃で２４時間インキュベーションを行なった。本処理により、通常のエキソ型酵素であるグルコアミラーゼ、グルコデキストラナーゼでは殆ど水解できなかった分岐環状オリゴ糖をグルコースと環状オリゴ糖に水解できた。
【００３９】
【発明の効果】
本発明により、新規なα−１，３−多分岐デキストラン水解酵素、その製造法及び環状イソマルトオリゴ糖の製造法を提供した。本酵素は、環状イソマルトオリゴ糖の分離精製時に混入している既存のエキソ型グルコアミラーゼ、グルコデキストラナーゼ等では殆ど水解されない分岐環状イソマルトオリゴ糖の分岐鎖を特異的に水解でき、該環状オリゴ糖の精製を更に簡略化でき且つ環状イソマルトオリゴ糖の純度の向上に寄与することができた。従って、本発明は、産業上極めて有意義である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel α-1,3-multibranched dextran hydrolase, a method for producing the same, and a method for producing a cyclic isomaltoligosaccharide.
[0002]
[Prior art]
Cyclic isomaltoligosaccharides are novel cyclic oligosaccharides obtained only by culturing microorganisms isolated from soil by the present inventors in a dextran-containing medium. Since this cyclic isomaltoligosaccharide forms an inclusion compound with a low molecular weight compound like cyclodextrin, it is a useful sugar expected to be used for pharmaceuticals, foods and the like (Japanese Patent Laid-Open No. Hei 6-197783). Publication). Further, since this cyclic isomaltoligosaccharide has an action of specifically inhibiting glucan synthase produced by a microorganism belonging to the genus Streptococcus known as a causative agent of caries, an anti-cariogenic agent Application to this area is also expected (Japanese Patent Laid-Open No. 8-59484). As a method for producing this cyclic isomaltoligosaccharide, a method for efficiently producing the oligosaccharide using cyclic isomaltoligosaccharide synthase, which is an enzyme for synthesizing cyclic isomaltoligosaccharide from dextran, has been developed (JP-A-7- No. 8276).
[0003]
On the other hand, as a method for purifying this cyclic isomaltoligosaccharide, a method for specifically removing linear isomaltoligosaccharide mixed as an impurity in the crude cyclic isomaltoligosaccharide using glucodextranase has been developed. A patent application has been filed for this method, the enzyme gene, and a method for producing the enzyme (Japanese Patent Application No. 8-230128). However, at the time of separation and purification of the cyclic isomaltoligosaccharide, branched oligosaccharides and branched cyclic oligosaccharides are mixed in in addition to the linear isomaltoligosaccharides, so removal of these oligosaccharides becomes an important problem. Yes. These oligosaccharides are hardly degradable oligosaccharides that are hardly hydrolyzed by the above glucodextranase. If the oligosaccharide can be hydrolyzed specifically, purification of the cyclic oligosaccharide is further simplified and cyclic It is possible to improve the purity of isomaltoligosaccharide.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to find a novel enzyme having an action of specifically hydrolyzing a branched chain of a branched oligosaccharide without acting on the cyclic oligosaccharide, and using the enzyme production method and the enzyme, a cyclic isomaltoligo It is to further simplify the purification of the sugar and improve the purity of the cyclic isomaltoligosaccharide.
[0005]
[Means for Solving the Problems]
Therefore, the present inventors have conducted extensive studies to obtain a novel enzyme having an action of specifically hydrolyzing a branched chain of a branched oligosaccharide without acting on the above-mentioned cyclic oligosaccharide. As a result, α-1 , 3-Sphingobacterium sp. Which is an assimilating bacterium of multi-branched dextran. (Sphingobacterium sp.) V-54 strain was found to contain an enzyme having the above properties, and the enzyme was separated and purified from the culture solution to clarify its properties and use this enzyme. The present invention was completed by confirming that the branched chain of the branched cyclic oligosaccharide was efficiently hydrolyzed.
[0006]
That is, the present invention is an α-1,3-multibranched dextran hydrolase having the following physicochemical properties:
(1) Action It acts on a multi-branched dextran having an α-1,3-linked branch to produce glucose.
(2) Optimum pH and stable pH range The optimum pH is around 7.0, and the stable pH range is 5.5 to 8.0.
[0007]
(3) Substrate-specificity When glucodisaccharide is used as a substrate, it works well in the order of α-1,2>α-1,4>α-1,3> α-1,6 bonds. In the case of polysaccharides, it works best on dextran and then on α-1,3-multi-branched dextran, but hardly on mutan, which is an α-1,3-glucan.
[0008]
(4) Inhibition and activation Hg ²⁺ , Cu ²⁺ , Ni ²⁺ and Fe ²⁺ are inhibited by 90% or more, and Ca ²⁺ is activated by about 30%.
(5) The molecular weight gel filtration method is about 90,000, and the SDS PAGE method is about 80,000.
[0009]
(6) Range of optimum temperature of action It has a suitable temperature in the vicinity of 45 ° C.
(7) Deactivation conditions due to temperature, etc. Approximately 30% by treatment at 45 ° C. for 15 minutes, almost deactivated by treatment at 50 ° C. for 15 minutes.
[0010]
Furthermore, the present invention is characterized in that a microorganism belonging to the genus Sphingobacteria and capable of producing α-1,3-multibranched dextran hydrolase is cultured in a medium containing dextran, and the enzyme is collected from the culture. This is a production method of α-1,3-multi-branched dextran hydrolase.
[0011]
Furthermore, the present invention is a method for producing a cyclic isomaltoligosaccharide characterized in that a cyclic isomaltoligosaccharide is produced by allowing α-1,3-multibranched dextran hydrolase to act on a branched cyclic isomaltoligosaccharide.
[0012]
The α-1,3-multibranched dextran hydrolase used in the production of the cyclic isomaltoligosaccharide is a culture of microorganisms belonging to the genus Sphingobacteria that produce the enzyme in addition to the purified enzyme and crude enzyme. Any of a liquid, a microbial cell, or a microbial cell processed material may be sufficient.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
First, the physicochemical properties of this enzyme are described below.
(1) Action It acts on a multi-branched dextran having an α-1,3-linked branch to produce glucose.
(2) Optimum pH and stable pH range The optimum pH is around 7.0, and the stable pH range is 5.5 to 8.0.
[0014]
(3) Substrate-specificity When glucodisaccharide is used as a substrate, it works well in the order of α-1,2>α-1,4>α-1,3> α-1,6 bonds. In the case of polysaccharides, it works best on dextran and then on α-1,3-multi-branched dextran, but hardly on mutan, which is an α-1,3-glucan.
[0015]
(4) Inhibition and activation Hg ²⁺ , Cu ²⁺ , Ni ²⁺ and Fe ²⁺ are inhibited by 90% or more, and Ca ²⁺ is activated by about 30%.
(5) The molecular weight gel filtration method is about 90,000, and the SDS PAGE method is about 80,000.
[0016]
(6) Range of optimum temperature of action It has a suitable temperature in the vicinity of 45 ° C.
(7) Deactivation conditions due to temperature, etc. Approximately 30% by treatment at 45 ° C. for 15 minutes, almost deactivated by treatment at 50 ° C. for 15 minutes.
[0017]
As described in detail above, this enzyme is a known polysaccharide hydrolase, α-amylase (EC 3.2.1.1), glucoamylase (EC 3.2.1.3), dextranase (EC 3). 2.11.1), glucodextranase (EC 3.2.1.70) and endo α-1,3-glucanase (mutanase) (EC 3.2.1.59) differ in their properties. This is a completely new enzyme in that it produces a significant amount of glucose from α-1,3-multi-branched dextran, which these enzymes can hardly act on.
[0018]
This α-1,3-multibranched dextran hydrolase is converted into a branched cyclic isomaltooligosaccharide, for example, a cyclic isomaltoligosaccharide in which glucose is branched by one molecule α-1,3-linkage, 3-O-α-D-glucosyl. Cycloisomaltoheptaose, 3-O-α-D-glucosyl cycloisomaltooctaose, 3-O-α-D-glucosyl cycloisomaltononaose, for example, at a temperature of 10 to 50 ° C., preferably 40 At a pH of 5.5 to 8.5, preferably at a pH of 7.0 for 30 minutes to 48 hours, preferably 24 hours as it is or with stirring, the branched cyclic isomaltoligosaccharide to the cyclic isomaltoligosaccharide, for example, Can produce cyclo isomalt heptaose, cyclo isomalt octaose, cyclo isomalt nonaose, etc. it can.
[0019]
By using this α-1,3-multi-branched dextran hydrolase during the production of cyclic isomaltoligosaccharides, it is possible to use a non-degradable branched oligosaccharide or branched cyclic isomaltoligosaccharide mixed in addition to the cyclic isomaltoligosaccharide, in particular a branched ring The branched chain of isomaltoligosaccharide can be hydrolyzed specifically, the purification of the cyclic isomaltoligosaccharide can be further simplified, and the purity thereof can be improved.
[0020]
The produced cyclic isomaltoligosaccharide has an inclusion action like cyclodextrin, and is useful for stabilizing, solubilizing, etc., a substance having a size that could not be included by cyclodextrin. Furthermore, cyclic isomaltoligosaccharides are composed of α-1,6-linkages between glucose residues, and are not easily hydrolyzed by saccharide-degrading enzymes such as α-amylase present in large amounts in the living body. . Therefore, this cyclic isomaltooligosaccharide is much more stable than cyclodextrin, and when this cyclic oligosaccharide is used, it can form a more stable inclusion compound than conventional cyclodextrins. It can be said that the cyclic oligosaccharide is a highly useful substance.
[0021]
Next, a method for producing this α-1,3-multibranched dextran hydrolase will be described. Examples of microorganisms used in this enzyme production method include wild strain V-54 obtained by the present inventors from soil.
The mycological properties of this V-54 strain are shown below.
[0022]

[0023]

[0024]

[0025]
Sphingobacterium sp. The V-54 strain is identified as a bacterium belonging to the genus Sphingobacteria because it has sphingolipids as lipids in the cells and the main quinone is menaquinone despite the fact that it is a gram-negative bacterium. did. Furthermore, in order to identify the species, various studies were made based on the Barjays Manual of Systematic Bacteriology, but since the species was not finalized, it was classified and identified as one strain belonging to the genus Sphingobacteria. did.
[0026]
Sphingobacterium sp. V-54 was deposited as FERM P-16086 on February 18, 1997 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.
As a microorganism used in the method for producing this enzyme, if it belongs to the genus Sphingobacterium in addition to the above strain and produces a significant amount of glucose from α-1,3-multi-branched dextran, It can be anything.
[0027]
The culture of the strain in the method of the present invention is basically the same as the method employed in the aerobic culture of general microorganisms, but usually the shaking culture method or the aeration and stirring culture method using a liquid medium is used.
[0028]
As a medium, an appropriate nitrogen source (for example, casein degradation products such as peptone, polypeptone, and bacttotryptone, or soy protein degradation products such as soyton), carbon sources (for example, carbohydrates such as glucose and glycerin), and necessary Thus, yeast extract, vitamins such as riboflavin, and minerals such as phosphates, magnesium salts, sodium chloride, trace metals, and dextran as an induction substrate for the enzyme are used. The pH may be any pH range as long as the present bacterium grows, and a range of 6 to 8 is usually preferable.
[0029]
The culture conditions are, for example, usually 20 to 40 ° C., shaking culture or aeration and agitation culture for 16 hours to 4 days.
Using the culture obtained as described above, for example, the multibranched dextran hydrolase is obtained by the following enzyme purification step.
[0030]
In order to obtain the enzyme from the culture, the cells collected by centrifugation, membrane concentration, etc. are further sonicated or treated with a surfactant to lyse, and centrifuged to remove cell debris. Furthermore, if necessary, a purified sample is obtained from the crude enzyme solution by a commonly used enzyme purification method.
[0031]
The enzyme may be purified by any method as long as it is a normal enzyme separation method. In the case of this production method, the cells are lysed by sonication or surfactant treatment, and centrifuged to remove cell debris. By processing the removed supernatant according to the purification method shown in the Examples, a highly pure multi-branched dextran hydrolase preparation can be obtained. In addition, this manufacturing method is not limited to these purification methods. Further, the present enzyme is allowed to act on the cyclic isomaltoligosaccharide production reaction solution, and the branched oligosaccharide, particularly the branched cyclic oligosaccharide present in the reaction solution is hydrolyzed into glucose and cyclic isomaltoligosaccharide, and then the cyclic oligosaccharide is separated and purified. In the case of this production method, the branched cyclic isomaltoligosaccharide can be hydrolyzed with the same efficiency as that of the purified enzyme without using the purified enzyme and using the supernatant liquid from which the cell residue described above is removed. As a means for separating and purifying the oligosaccharide from the reaction solution, any method may be used as long as it is an oligosaccharide purification method. For example, a known method such as cooling treatment, organic solvent addition treatment, activated carbon treatment, column chromatography that specifically adsorbs cyclic oligosaccharides, activated carbon column chromatography or the like can be separated and purified alone or in appropriate combination.
[0032]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0033]
[Example 1]
1% dextran 40 (manufactured by Meito Sangyo Co., Ltd.), 1% peptone (manufactured by Far East Pharmaceutical Co., Ltd.), 0.5% NaCl and 0.1% yeast extract (manufactured by Difco), liquid medium (using tap water, pH 7.0) 100 ml was placed in a 500 ml Sakaguchi flask and sterilized at 120 ° C. for 20 minutes. To this, Sphingobacterium sp. One platinum loop was inoculated from a storage slant of V-54 strain (FERM P-16086) and cultured with shaking at 30 ° C for 1 day. 10 ml each of the main culture solution was inoculated into 8 3 L mini jar fermenters containing 2 L medium prepared by the same medium composition and sterilization conditions as described above, and the conditions were 30 ° C., 0.5 vvm, 300 rpm for 3 days. Aeration and agitation culture is performed, and after completion of the culture, 16 L of the culture broth is collected by centrifugation. The resulting suspension of about 600 ml of cells is crushed by ultrasonic treatment 200 ml at a time and centrifuged to remove cell residues. Removed. About 600 ml of the obtained supernatant was used as a crude enzyme solution, and the multibranched dextran hydrolase was purified.
[0034]
First, ammonium sulfate was added to the enzyme solution to a final concentration of 1.0 M, and insoluble matters were removed by centrifugation. The supernatant was purified by preparative HPLC by the following procedure. The supernatant was applied to a TSKgel Phenyl5PW column equilibrated with 100 mM phosphate buffer (pH 7.0) containing 1.0 M ammonium sulfate in 100 ml portions, washed with the same buffer solution, and then washed with 1.0 to 0 M ammonium sulfate concentration gradient. Adsorbed protein was eluted. About 500 ml of the active fraction collected by the 6 operations was concentrated to about 20 ml by ultrafiltration, and about 500 ml of 10 mM phosphate buffer (pH 7.0) was added to dilute the salt concentration. The diluted solution was concentrated again to about 100 ml by ultrafiltration. The concentrated solution was applied to a TSKgel DEAE5PW column equilibrated with 10 mM phosphate buffer (pH 7.0), and non-adsorbed protein was washed with the same buffer. Under this condition, all of the enzyme was eluted in the non-adsorbed fraction, so about 200 ml of the non-adsorbed fraction was concentrated to about 20 ml, and then 180 ml of 10 mM phosphate buffer (pH 7.5) was added to adjust the pH. Was changed from 7.0 to 7.5.
[0035]
The enzyme solution was ultra-concentrated again to about 100 ml, and then the concentrate was reapplied to a TSKgel DEAE5PW column equilibrated with 10 mM phosphate buffer (pH 7.5). After washing the non-adsorbed protein with the same buffer, the adsorbed protein was eluted with a NaCl concentration gradient of 0 to 0.5M.
[0036]
Since activity was observed in the adsorbed elution fraction, 40 ml of the active fraction was collected, and the enzyme solution was further concentrated to 1.5 ml. The enzyme solution was applied in an amount of 0.3 ml onto a TSKgel G3000SW column equilibrated with a 100 mM phosphate buffer (pH 7.0) containing 200 mM NaCl, and eluted with the same buffer. This operation was performed in the same manner for the remaining 1.2 ml (0.3 ml 4 times). The obtained active fractions were collected and subjected to SDS PAGE, which showed a single band. Therefore, it was judged that other contaminating proteins had been removed, and purification was completed. When this purified enzyme was incubated with α-1,3-multibranched dextran, production of a significant amount of glucose was confirmed from the multibranched dextran.
[0037]
[Example 2]
300 L of the culture solution of Bacillus sp. T-3040 strain (FERM BP-4132) was sterilized by membrane treatment using a microzer, and the obtained sterilized solution was further removed with a 6000 cut molecular weight follower fiber. After concentrating to 6.3 L, 900 ml of the concentrated solution was stored frozen at -20 ° C. A part of the concentrated solution was mixed with an aqueous solution obtained by dissolving 100 g of dextran (manufactured by Meito Sangyo Co., Ltd.) in 10 L of 10 mM phosphate buffer (pH 6.5), and incubated at 40 ° C. for 48 hours. The reaction was stopped by adding activated carbon and boiling, and unreacted dextran was adsorbed. The supernatant liquid from which the activated carbon was removed was applied to an activated carbon column equilibrated with deionized water, washed with deionized water, and then the oligosaccharide adsorbed by an ethanol concentration gradient was eluted. The cyclic oligosaccharide fractions were collected, concentrated, and applied to an ODS column equilibrated with deionized water. After washing with deionized water, oligosaccharides were eluted with an ethanol concentration gradient, and each cyclic isomaltoligosaccharide fraction was collected and lyophilized. About 5% of the branched cyclic isomaltoligosaccharide was mixed in the cycloisomaltnonaose fraction.
[0038]
Example 3
A 4% aqueous solution of cycloisomaltononaose preparation mixed with the branched cyclic oligosaccharide obtained in Example 2 was prepared, and 0.5 ml of this aqueous solution was prepared from the sonicated disruption solution of the bacterial cells of Example 1. The crude enzyme solution (0.1 ml) and 100 mM phosphate buffer solution (pH 7.0) (0.4 ml) were mixed and incubated at 40 ° C. for 24 hours. By this treatment, branched cyclic oligosaccharides that could hardly be hydrolyzed by normal exo-type enzymes glucoamylase and glucodextranase could be hydrolyzed into glucose and cyclic oligosaccharides.
[0039]
【The invention's effect】
According to the present invention, a novel α-1,3-multibranched dextran hydrolase, a method for producing the same, and a method for producing a cyclic isomaltoligosaccharide have been provided. This enzyme can specifically hydrolyze the branched chain of a branched cyclic isomalto-oligosaccharide that is hardly hydrolyzed by existing exo-type glucoamylase, glucodextranase, etc. that are mixed during the separation and purification of the cyclic isomaltoligosaccharide. It was possible to further simplify the purification of the sugar and contribute to the improvement of the purity of the cyclic isomaltoligosaccharide. Therefore, the present invention is extremely significant in industry.

Claims (3)

An α-1,3-multibranched dextran hydrolase having the following physicochemical properties:
(1) Action It acts on a multi-branched dextran having an α-1,3-linked branch to produce glucose.
(2) Optimum pH and stable pH range The optimum pH is 7.0 , and the stable pH range is 5.5 to 8.0.
(3) Substrate-specificity When glucodisaccharide is used as a substrate, it works well in the order of α-1,2>α-1,4>α-1,3> α-1,6 binding. In the case of polysaccharides, it works best on dextran and then on α-1,3-multibranched dextran, but has little effect on mu-1, which is an α-1,3-glucan.
(4) Inhibition and activation
Hg ^2+, Cu ^2+, inhibited more than 90% Ni ²⁺ and Fe ^2+, it is 30% activated Ca ^2+.
(5) is 90,000 in molecular weight gel filtration, which is 80,000 in SDS PAGE method.
(6) Optimum temperature range
Has a suitable temperature at 45 ° C.
(7) conditions of deactivation due to temperature
It is almost inactivated by treatment at 45 ° C for 15 minutes and 30 % at 50 ° C for 15 minutes. A microorganism belonging to the genus Sphingobacterium and having the ability to produce α-1,3-multibranched dextran hydrolase according to claim 1 is cultured in a medium containing dextran, and the enzyme is collected from the culture. The method for producing an α-1,3-multibranched dextran hydrolase according to claim 1, which is characterized by the following. A method for producing a cyclic isomaltoligosaccharide, characterized in that the α-1,3-multibranched dextran hydrolase according to claim 1 is allowed to act on a branched cyclic isomaltoligosaccharide to produce a cyclic isomaltoligosaccharide.