JP3637088B2 - Method for producing hetero-oligosaccharide trisaccharide - Google Patents
Method for producing hetero-oligosaccharide trisaccharide Download PDFInfo
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- JP3637088B2 JP3637088B2 JP33511294A JP33511294A JP3637088B2 JP 3637088 B2 JP3637088 B2 JP 3637088B2 JP 33511294 A JP33511294 A JP 33511294A JP 33511294 A JP33511294 A JP 33511294A JP 3637088 B2 JP3637088 B2 JP 3637088B2
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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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Description
【0001】
【産業上の利用分野】
本発明は、ヘテロオリゴ糖3糖類の製造方法に関し、詳しくはヘテロオリゴ糖にタカアミラーゼを作用させてマルトースの非還元性末端の4位に1分子のα−グルコシル基以外の糖質が結合しているヘテロオリゴ糖3糖類を製造する方法に関する。
【0002】
【従来の技術および発明が解決しようとする課題】
医療現場で各種の検査が行われるようになり、特に膵液や尿などの体液に含有されるα−アミラーゼの活性測定は重要な検査事項になっており、疾病診断の一助となっている。
近年、α−アミラーゼの活性測定用の基質として、追随酵素を使用しない、高感度の基質が開発されている。その構造は、マルトースの還元性末端にフェニル基,ナフチル基またはそれらの誘導体をアグリコンとして結合させたマルトース誘導体の非還元性末端の4位をグルコース以外の糖質で修飾したものである。
一般に、上記化合物の製造方法は、まずマルトースに種々の酵素の転移作用を利用して、非還元性末端の4位を1分子のα−グルコシル基以外の糖質で修飾した後、還元性末端を化学合成によりフェニル基,ナフチル基などで結合させる方法である。
【0003】
しかし、上記製造方法では、例えば乳糖存在下、β−ガラクトシダーゼの糖転移作用を用いた場合、マルトースへのガラクトシル基転移生成物と乳糖へのガラクトシル基転移生成物が、その分画のときに溶出時間が似通っているため、分画が困難であるという欠点がある。同様のことがメリビオース存在下にα−ガラクトシダーゼを作用させた場合、α−マンノビオース存在下にα−マンノシダーゼを作用させた場合、β−マンノビオース存在下にβ−マンノシダーゼを作用させた場合にも生じる。
そこで、本発明者らは上記した従来のマルトースの非還元性末端の4位を1分子のα−グルコシル基以外の糖質で修飾した3糖類(以下、ヘテロオリゴ糖3糖類と称する。)の製造法の欠点を解消し、効率良く、低コストでヘテロオリゴ糖3糖類を製造する方法を確立すべく検討を重ねた。
【0004】
その結果、マルトトリオース以上のマルトオリゴ糖(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等)を用いて、既知の手法により糖転移酵素を作用させ、マルトオリゴ糖の非還元性末端の4位を1分子のα−グルコシル基以外の糖質で修飾したヘテロオリゴ糖を調製し、これにタカアミラーゼを作用させてヘテロオリゴ糖3糖類のみを効率よく製造する方法を完成した。
元来、タカアミラーゼをマルトオリゴ糖に作用させた場合、最終産物はマルトースとグルコースである。しかし、マルトオリゴ糖の非還元性末端の4位を1分子のα−グルコシル基以外の糖質で修飾したヘテロオリゴ糖にタカアミラーゼを作用させた場合、その最終産物はヘテロオリゴ糖3糖類とマルトースおよびグルコースであるという新知見に基づき、本発明を完成したのである。
【0005】
【課題を解決するための手段】
すなわち、本発明は下記の一般式(I)で表されるヘテロオリゴ糖(式中、Rはα−グルコシル基以外の1分子の糖質を示し、nは1〜5の整数を示す。)
【0006】
【化3】
【0007】
にタカアミラーゼを作用させることを特徴とする下記の一般式(II)で表されるヘテロオリゴ糖3糖類(式中、Rはα−グルコシル基以外の1分子の糖質を示す。)の製造方法を提供するものである。
【0008】
【化4】
【0009】
本発明に用いるヘテロオリゴ糖は、マルトトリオース以上のマルトオリゴ糖とα−グルコシル基以外の糖質を糖転移酵素の存在下に反応させて、マルトオリゴ糖の非還元性末端の4位に1分子のα−グルコシル基以外の糖質を結合させて調製することができる。
マルトオリゴ糖としては、マルトトリオース,マルトテトラオース,マルトペンタオース,マルトヘキサオース等が用いられ、特にマルトトリオース,マルトテトラオースが好ましい。
α−グルコシル基以外の糖質(すなわち基質)としては、乳糖,メリビオース,セロビオース,α−マンノビオース等が用いられ、特に乳糖が好ましい。
また、糖転移酵素としては、基質に応じてβ−ガラクトシダーゼ,α−ガラクトシダーゼ,β−グルコシダーゼ,α−マンノシダーゼ等が用いられる。
この反応は、マルトオリゴ糖と基質を含む反応系の濃度が20〜60%、その構成比、すなわちマルトオリゴ糖:α−グルコシル基以外の糖質=1:0.5〜8:1(モル比)、pH3〜9、温度30〜60℃、時間0.5〜24時間の条件で実施される。また、反応生成物であるヘテロオリゴ糖は高速液体クロマトグラフィーなどの精製手段により分画され、容易に分取することができる。
【0010】
このようにして調製されたヘテロオリゴ糖は、還元性末端にマルトトリオース以上のマルトオリゴ糖部分を有し、非還元性末端の4位に1分子のα−グルコシル基以外の糖質が結合していればよく、これらの混合物であってもよい。
また、ヘテロオリゴ糖中の非還元性末端の4位に存在するα−グルコシル基以外の糖質としては、ガラクトシル基,マンノシル基,N−アセチルグルコサミニル基,β−グルコシル基等があり、特にガラクトシル基が好ましい。
【0011】
本発明に用いるタカアミラーゼは、還元性末端のマルトオリゴ糖部分がマルトトリオース以上であるヘテロオリゴ糖に作用させたとき、ヘテロオリゴ糖3糖類を生成する酵素であり、アスペルギルス・オリーゼを起源とするものである。なお、アスペルギルス・オリーゼ由来の酵素(商品名:タカジアスターゼ、三共株式会社製)も同様に使用可能である。
【0012】
本発明の反応系において、ヘテロオリゴ糖溶液(水溶液または懸濁液)は、ヘテロオリゴ糖の濃度が約1〜50%(w/w)、好ましくは30〜40(w/w)であることが望ましいが、これらに限定されない。
また、反応液のpHは3〜10、好ましくは4〜9、温度は20〜70℃、好ましくは30〜60℃である。使用酵素量は反応時間と密接な関係があるので、通常は5〜100時間で反応が終了するような酵素量とすればよい。なお、目的とするヘテロオリゴ糖3糖類の収率は基質などに影響されるが、通常5〜30%である。なお、反応に際してグルコアミラーゼを併用することにより、生成したマルトースをグルコースに分解させて分画を容易にすることができる。
【0013】
上記の反応終了後、反応液を加熱して酵素を失活させた後、反応液を高速液体クロマトグラフィー(HPLC)などの精製手段により、目的とするヘテロオリゴ糖3糖類を分取する。この場合のHPLCの分析条件を例示すると下記の通りである。
【0014】
カラム: TOSOH TSK-gel Amide-80 (250 ×4.6mm) 東ソー(株)製
溶媒 : 61%アセトニトリル
流速 : 1.0ml/min.
温度 : 35℃
検出器: 示差屈折計
【0015】
このようにして得られたヘテロオリゴ糖3糖類は、さらに還元性末端を化学合成によりフェニル基,ナフチル基などで修飾してα−アミラーゼ活性測定用の基質として利用される。
【0016】
【実施例】
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。
実施例1
43 −O−β−ガラクトシル−マルトトリオース(以下、Gal-G3と記す。)3gを25mM酢酸緩衝液(pH5.2)10mlに溶解させた後、アスペルギルス・オリーゼ由来の酵素(商品名:タカヂアスターゼ、三共株式会社製)を90単位加え、50℃にて反応させた。反応開始後、0,2,3,4,24時間経過した反応液の糖組成を、高速液体クロマトグラフィーにて分析した。その結果を第1表に示した。表から明らかなように、Gal-G3が分解され、Gal-G2とグルコースが生成していた。
【0017】
【表1】
【0018】
実施例2
44 −O−β−ガラクトシル−マルトテトラオース(以下、Gal-G4と記す。)3gを25mM酢酸緩衝液(pH5.2)10mlに溶解させた後、アスペルギルス・オリーゼ由来の酵素(商品名:タカヂアスターゼ、三共株式会社製)より既知の精製法により得られたタカアミラーゼを135単位加え、50℃で1.5時間反応させた。
上記反応液の糖組成を、高速液体クロマトグラフィーにて分析し、その結果を第2表に示した。表から明らかなように、Gal-G4が分解され、Gal-G2とマルトースが生成していた。
【0019】
【表2】
【0020】
実施例3
43 −O−β−N−アセチル−グルコサミニル−マルトトリオース(以下、GlcNAc-G3 と記す。)50mgを25mM酢酸緩衝液(pH5.2)500μlに溶解させた後、アスペルギルス・オリーゼ由来の酵素(商品名:タカヂアスターゼ、三共株式会社製)を5単位加え、50℃で反応させた。反応開始後、24時間経過した反応液の糖組成を高速液体クロマトグラフィーにて分析した結果、GlcNAc-G3 が分解され、GlcNAc-G2 とグルコースが生成していた。
【0021】
実施例4
44 −O−β−マンノシル−マルトテトラオース(以下、Man-G4と記す。)100mgを25mM酢酸緩衝液(pH5.2)1mlに溶解させた後、アスペルギルス・オリーゼ由来の酵素(商品名:タカヂアスターゼ、三共株式会社製)より既知の精製法により得られたタカアミラーゼを10単位加え、50℃にて24時間反応させた。
上記反応液の糖組成を高速液体クロマトグラフィーにて分析した結果、Man-G4が分解され、Man-G2とマルトースが生成していた。
【0022】
実施例5
(1)ヘテロオリゴ糖の調製
マルトオリゴ糖混合品(マルトトリオース50%、マルトテトラオース30%およびマルトペンタオース20%)10gと乳糖6gを50mM酢酸緩衝液(pH6.0)20mlに溶解させた後、バチルス・サーキュランス由来のβ−ガラクトシダーゼ(商品名:ビオラクタ、大和化成株式会社製)を156単位加え、40℃にて3時間反応させた。
【0023】
反応終了後、酵素を熱失活させた溶液に水70mlを加え、リゾプス属由来のグルコアミラーゼ(商品名:グルクザイム、天野製薬株式会社製)を400単位加え、40℃で17時間反応させて未反応のマルトオリゴ糖をグルコースに分解した。
反応終了後、酵素を熱失活させた溶液を高速液体クロマトグラフィーにかけて3糖類以下のものを除き、転移生成物であるヘテロオリゴ糖2.0g(4糖類60%、5糖類26%、6糖類14%)を得た。
【0024】
(2)ヘテロオリゴ糖3糖類の調製
上記の方法により得られた転移生成物2.0gを20mM酢酸緩衝液(pH5.0)18mlに溶解させ、アスペルギルス・オリーゼ由来の酵素(商品名:タカヂアスターゼ、三共株式会社製)を100単位、リゾプス属由来のグルコアミラーゼ(商品名:グルクザイム、天野製薬株式会社製)を100単位加え、40℃にて17時間反応させた。
反応終了後、酵素を熱失活させた溶液を高速液体クロマトグラフィーにかけてヘテロオリゴ糖3糖類画分1.2gを得た。反応液のHPLCを図1に示す。この場合のHPLCの分析条件は下記の通りである。
【0025】
カラム: 島津 Shim-pack SCR-101C (300 ×7.9mm)
溶媒 : 水
流速 : 0.5ml/min.
温度 : 80℃
検出器: 示差屈折計
【0026】
(3)構造解析
得られた3糖類画分は、ペニシリウム・マルチカラー由来のβ−ガラクトシダーゼ(ケイアイ化成株式会社製)の加水分解反応により、マルトースとガラクトースが1:1のモル比で生成した。
また、13C−NMR分析では、マルトースの非還元性末端のグルコシル基の4位炭素に由来するシグナルが低磁場側に大きくシフトしていることより、マルトースの非還元性末端のグルコシル基の4位の水酸基が結合に関与していることが確認された。
以上の結果より、得られた3糖類画分は、マルトースの非還元末端側グルコシル基の4位に1分子のガラクトシル基がβ結合したガラクトシル・マルトースであると確認された。
【0027】
【発明の効果】
本発明によれば、タカアミラーゼを用いて、マルトースの非還元末端側グルコシル基の4位にα−グルコシル基以外の1分子の糖質が結合したヘテロオリゴ糖を効率よく得ることができる。
【図面の簡単な説明】
【図1】 実施例5の反応液の高速液体クロマトグラフィーである。[0001]
[Industrial application fields]
The present invention relates to a method for producing a hetero-oligosaccharide trisaccharide, and more specifically, takaamylase is allowed to act on the hetero-oligosaccharide so that a carbohydrate other than one molecule of α-glucosyl group is bonded to the 4-position of the non-reducing end of maltose. The present invention relates to a method for producing a hetero-oligosaccharide trisaccharide.
[0002]
[Background Art and Problems to be Solved by the Invention]
Various tests have been carried out at medical sites, and in particular, measuring the activity of α-amylase contained in body fluids such as pancreatic juice and urine has become an important test item, and has been an aid for disease diagnosis.
In recent years, a highly sensitive substrate that does not use a following enzyme has been developed as a substrate for measuring the activity of α-amylase. The structure is obtained by modifying the 4-position of the non-reducing end of a maltose derivative in which a phenyl group, a naphthyl group or a derivative thereof is bonded as an aglycon to the reducing end of maltose with a sugar other than glucose.
In general, the above-mentioned compound production method is based on the use of the transfer action of various enzymes to maltose to modify the 4-position of the non-reducing end with a saccharide other than one molecule of α-glucosyl group, and then the reducing end. Is bonded by a phenyl group, a naphthyl group or the like by chemical synthesis.
[0003]
However, in the above production method, for example, when the transglycosylation action of β-galactosidase is used in the presence of lactose, the galactosyl group transfer product to maltose and the galactosyl group transfer product to lactose are eluted at the time of fractionation. Since the time is similar, there is a drawback that fractionation is difficult. The same occurs when α-galactosidase is allowed to act in the presence of melibiose, when α-mannosidase is allowed to act in the presence of α-mannobiose, and when β-mannosidase is allowed to act in the presence of β-mannobiose.
Therefore, the present inventors produce a trisaccharide (hereinafter referred to as a hetero-oligosaccharide trisaccharide) in which the non-reducing terminal position of the above-described conventional maltose is modified with a saccharide other than one molecule of α-glucosyl group. In order to eliminate the shortcomings of the method and to establish a method for producing heterooligosaccharide trisaccharides efficiently and at low cost, investigations were repeated.
[0004]
As a result, using a malto-oligosaccharide (maltotriose, maltotetraose, maltopentaose, etc.) higher than maltotriose, a glycosyltransferase was allowed to act by a known method, and the 4-position of the non-reducing end of the maltooligosaccharide was A hetero-oligosaccharide modified with a saccharide other than one molecule of α-glucosyl group was prepared, and Taka-amylase was allowed to act on the hetero-oligosaccharide to produce only the hetero-oligosaccharide trisaccharide.
Originally, when Taka-amylase is allowed to act on maltooligosaccharides, the final products are maltose and glucose. However, when Takaamylase is allowed to act on a hetero-oligosaccharide in which the 4-position of the non-reducing end of maltooligosaccharide is modified with a saccharide other than one α-glucosyl group, the final product is a hetero-oligosaccharide trisaccharide, maltose and glucose. The present invention has been completed based on the new finding that
[0005]
[Means for Solving the Problems]
That is, the present invention is a hetero-oligosaccharide represented by the following general formula (I) (wherein R represents one molecule of carbohydrate other than the α-glucosyl group, and n represents an integer of 1 to 5).
[0006]
[Chemical 3]
[0007]
A method for producing a hetero-oligosaccharide trisaccharide represented by the following general formula (II) (wherein R represents a saccharide of one molecule other than the α-glucosyl group), which is characterized in that Takaamylase is allowed to act on Is to provide.
[0008]
[Formula 4]
[0009]
The hetero-oligosaccharide used in the present invention reacts with a malto-oligosaccharide of maltotriose or higher and a saccharide other than an α-glucosyl group in the presence of a glycosyltransferase, so that one molecule is located at the 4-position of the non-reducing end of the maltooligosaccharide. It can be prepared by binding a carbohydrate other than the α-glucosyl group.
As maltooligosaccharide, maltotriose, maltotetraose, maltopentaose, maltohexaose, etc. are used, and maltotriose and maltotetraose are particularly preferable.
Lactose, melibiose, cellobiose, α-mannobiose, and the like are used as carbohydrates other than the α-glucosyl group (that is, the substrate), and lactose is particularly preferable.
As the glycosyltransferase, β-galactosidase, α-galactosidase, β-glucosidase, α-mannosidase and the like are used depending on the substrate.
In this reaction, the concentration of the reaction system containing malto-oligosaccharide and substrate is 20 to 60%, and the composition ratio thereof, that is, carbohydrate other than malto-oligosaccharide: α-glucosyl group = 1: 0.5 to 8: 1 (molar ratio). , PH 3-9, temperature 30-60 ° C., time 0.5-24 hours. In addition, the hetero-oligosaccharide as a reaction product is fractionated by a purification means such as high performance liquid chromatography and can be easily fractionated.
[0010]
The thus prepared hetero-oligosaccharide has a malto-oligosaccharide moiety of maltotriose or higher at the reducing end, and a carbohydrate other than one molecule of α-glucosyl group is bonded to the 4-position of the non-reducing end. Any mixture of these may be used.
In addition, saccharides other than the α-glucosyl group present at the 4-position of the non-reducing end in the hetero-oligosaccharide include galactosyl group, mannosyl group, N-acetylglucosaminyl group, β-glucosyl group, etc. A galactosyl group is preferred.
[0011]
The Taka-amylase used in the present invention is an enzyme that produces a hetero-oligosaccharide trisaccharide when acting on a hetero-oligosaccharide having a malto-oligosaccharide moiety at the reducing end that is maltotriose or higher, and originates from Aspergillus oryzae. is there. An enzyme derived from Aspergillus oryzae (trade name: Takadiastase, manufactured by Sankyo Co., Ltd.) can also be used in the same manner.
[0012]
In the reaction system of the present invention, the heterooligosaccharide solution (aqueous solution or suspension) has a heterooligosaccharide concentration of about 1 to 50% (w / w), preferably 30 to 40 (w / w). However, it is not limited to these.
The pH of the reaction solution is 3 to 10, preferably 4 to 9, and the temperature is 20 to 70 ° C, preferably 30 to 60 ° C. Since the amount of enzyme used is closely related to the reaction time, the amount of enzyme is usually such that the reaction is completed in 5 to 100 hours. In addition, although the yield of the target hetero oligosaccharide trisaccharide is influenced by a substrate etc., it is 5-30% normally. In addition, by using glucoamylase together in the reaction, the produced maltose can be decomposed into glucose to facilitate fractionation.
[0013]
After completion of the above reaction, the reaction solution is heated to inactivate the enzyme, and then the desired hetero-oligosaccharide trisaccharide is fractionated by a purification means such as high performance liquid chromatography (HPLC). Examples of HPLC analysis conditions in this case are as follows.
[0014]
Column: TOSOH TSK-gel Amide-80 (250 × 4.6 mm) Tosoh Corporation solvent: 61% acetonitrile flow rate: 1.0 ml / min.
Temperature: 35 ° C
Detector: Differential refractometer [0015]
The thus obtained hetero-oligosaccharide trisaccharide is further used as a substrate for measuring α-amylase activity by modifying the reducing end with a phenyl group, a naphthyl group or the like by chemical synthesis.
[0016]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.
Example 1
After dissolving 3 g of 4 3 -O-β-galactosyl-maltotriose (hereinafter referred to as Gal-G 3 ) in 10 ml of 25 mM acetate buffer (pH 5.2), an enzyme derived from Aspergillus oryzae (trade name) : Takadiastase (manufactured by Sankyo Co., Ltd.) was added in 90 units and reacted at 50 ° C. After the start of the reaction, the sugar composition of the reaction solution after 0, 2, 3, 4 and 24 hours was analyzed by high performance liquid chromatography. The results are shown in Table 1. As is apparent from the table, Gal-G 3 was decomposed to produce Gal-G 2 and glucose.
[0017]
[Table 1]
[0018]
Example 2
3 g of 4 4 -O-β-galactosyl-maltotetraose (hereinafter referred to as Gal-G 4 ) was dissolved in 10 ml of 25 mM acetate buffer (pH 5.2), and then an enzyme derived from Aspergillus oryzae (trade name) : Takadiastase (manufactured by Sankyo Co., Ltd.) and 135 units of Takaamylase obtained by a known purification method were added and reacted at 50 ° C. for 1.5 hours.
The sugar composition of the reaction solution was analyzed by high performance liquid chromatography, and the results are shown in Table 2. As is apparent from the table, Gal-G 4 was decomposed to produce Gal-G 2 and maltose.
[0019]
[Table 2]
[0020]
Example 3
50 mg of 4 3 -O-β-N-acetyl-glucosaminyl-maltotriose (hereinafter referred to as GlcNAc-G 3 ) was dissolved in 500 μl of 25 mM acetate buffer (pH 5.2), and then derived from Aspergillus oryzae. Five units of enzyme (trade name: Takadia Astase, Sankyo Co., Ltd.) were added and reacted at 50 ° C. As a result of analyzing the sugar composition of the reaction solution after 24 hours from the start of the reaction by high performance liquid chromatography, GlcNAc-G 3 was decomposed and GlcNAc-G 2 and glucose were produced.
[0021]
Example 4
After dissolving 100 mg of 4 4 -O-β-mannosyl-maltotetraose (hereinafter referred to as Man-G 4 ) in 1 ml of 25 mM acetate buffer (pH 5.2), an enzyme derived from Aspergillus oryzae (trade name) 10 units of Taka-amylase obtained by a known purification method from Takadiastase (manufactured by Sankyo Co., Ltd.) and reacted at 50 ° C. for 24 hours.
As a result of analyzing the sugar composition of the reaction solution by high performance liquid chromatography, Man-G 4 was decomposed and Man-G 2 and maltose were produced.
[0022]
Example 5
(1) Preparation of hetero-oligosaccharide 10 g of maltooligosaccharide mixture (maltotriose 50%, maltotetraose 30% and maltopentaose 20%) and 6 g of lactose were dissolved in 20 ml of 50 mM acetate buffer (pH 6.0). 156 units of β-galactosidase derived from Bacillus circulans (trade name: Biolacta, manufactured by Daiwa Kasei Co., Ltd.) was added and reacted at 40 ° C. for 3 hours.
[0023]
After completion of the reaction, 70 ml of water was added to the solution in which the enzyme was heat-inactivated, and 400 units of Rhizopus-derived glucoamylase (trade name: Gluczyme, Amano Pharmaceutical Co., Ltd.) was added and reacted at 40 ° C. for 17 hours. The malto-oligosaccharide of the reaction was broken down into glucose.
After completion of the reaction, the enzyme-heat-inactivated solution was subjected to high performance liquid chromatography to remove trisaccharide or less, and 2.0 g of a hetero-oligosaccharide as a transfer product (60% tetrasaccharide, 26% 5 saccharide, 14 saccharide 14 %).
[0024]
(2) Preparation of hetero-oligosaccharide trisaccharide 2.0 g of the transfer product obtained by the above method is dissolved in 18 ml of 20 mM acetate buffer (pH 5.0), and an enzyme derived from Aspergillus oryzae (trade name: Takadiastase, Sankyo) 100 units) and 100 units of Rhizopus-derived glucoamylase (trade name: Gluczyme, Amano Pharmaceutical Co., Ltd.) were added and reacted at 40 ° C. for 17 hours.
After completion of the reaction, the enzyme-thermally inactivated solution was subjected to high performance liquid chromatography to obtain 1.2 g of a heterooligosaccharide trisaccharide fraction. The HPLC of the reaction solution is shown in FIG. The HPLC analysis conditions in this case are as follows.
[0025]
Column: Shimadzu Shim-pack SCR-101C (300 x 7.9mm)
Solvent: Water flow rate: 0.5 ml / min.
Temperature: 80 ° C
Detector: Differential refractometer [0026]
(3) Structural analysis The obtained trisaccharide fraction was produced by a hydrolysis reaction of penicillium multicolor-derived β-galactosidase (manufactured by KAI Kasei Co., Ltd.), and maltose and galactose were produced at a molar ratio of 1: 1.
In the 13 C-NMR analysis, the signal derived from the 4-position carbon of the glucosyl group at the non-reducing end of maltose is greatly shifted to the low magnetic field side, so that 4 of the glucosyl group at the non-reducing end of maltose It was confirmed that the hydroxyl group at the position was involved in the bond.
From the above results, it was confirmed that the obtained trisaccharide fraction was galactosyl maltose in which one molecule of galactosyl group was β-bonded at the 4-position of the non-reducing terminal glucosyl group of maltose.
[0027]
【The invention's effect】
According to the present invention, a hetero-oligosaccharide in which one molecule of a carbohydrate other than the α-glucosyl group is bonded to the 4-position of the non-reducing terminal glucosyl group of maltose can be efficiently obtained using takaamylase.
[Brief description of the drawings]
1 is a high performance liquid chromatography of the reaction solution of Example 5. FIG.
Claims (2)
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JP33511294A JP3637088B2 (en) | 1994-12-21 | 1994-12-21 | Method for producing hetero-oligosaccharide trisaccharide |
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JP33511294A JP3637088B2 (en) | 1994-12-21 | 1994-12-21 | Method for producing hetero-oligosaccharide trisaccharide |
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JPH08173180A JPH08173180A (en) | 1996-07-09 |
JP3637088B2 true JP3637088B2 (en) | 2005-04-06 |
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JP33511294A Expired - Lifetime JP3637088B2 (en) | 1994-12-21 | 1994-12-21 | Method for producing hetero-oligosaccharide trisaccharide |
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