JPH0646953B2 - Process for producing higher N-acetyl chitooligosaccharide - Google Patents
Process for producing higher N-acetyl chitooligosaccharideInfo
- Publication number
- JPH0646953B2 JPH0646953B2 JP27095687A JP27095687A JPH0646953B2 JP H0646953 B2 JPH0646953 B2 JP H0646953B2 JP 27095687 A JP27095687 A JP 27095687A JP 27095687 A JP27095687 A JP 27095687A JP H0646953 B2 JPH0646953 B2 JP H0646953B2
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- acetylchitooligosaccharide
- lysozyme
- acetylchitooligosaccharides
- sugars
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】 産業上の利用分野 本発明は、医薬品として有用な高級Ｎ−アセチルキトオ
リゴ糖の製造法に関する。TECHNICAL FIELD The present invention relates to a method for producing a higher N-acetylchitooligosaccharide useful as a pharmaceutical.
高級Ｎ−アセチルキトオリゴ糖は、ペンタオース、ヘキ
サオース、ペプタオースなど５糖以上のＮ−アセチルキ
トオリゴ糖を指し、臨床検査におけるリゾチーム活性の
測定基質として有用で、また、Ｎ−アセチルヘキサオー
スは、免疫機能増強作用、抗腫瘍効果などがあり、生理
活性物質としても有用である。Higher N-acetylchitooligosaccharides indicate penta-ose, hexaose, peptaose, and other pentasaccharide N-acetylchitooligosaccharides, which are useful as substrates for measuring lysozyme activity in clinical tests, and N-acetylhexaose It has a function-enhancing effect and an antitumor effect, and is useful as a physiologically active substance.
従来の技術 従来、Ｎ−アセチルキトオリゴ糖の製造法としては、酸
によるキチンの限定加水分解［バイオケミカ・バイオフ
ィジカ・アクタ（Bio-chem. Biophys.Acta) 83.245-25
5 (1964)］、キチナーゼなどの酵素によるキチンの加水
分解［ネイチャー（Neture） 200. 1128 (1963)］、キ
チナーゼ、リゾチームなどの糖転移反応を利用した製造
方法（特開昭62-146598）などが知られている。Conventional Technology Conventionally, as a method for producing N-acetylchitooligosaccharide, limited hydrolysis of chitin with acid [Bio-chem. Biophys. Acta] 83 .245-25
5 (1964)], hydrolysis of chitin by an enzyme such as chitinase [Nature (Neture) 200 .1128 (1963)], a production method using a glycosyl transfer reaction of chitinase, lysozyme, etc. (JP-A-62-146598), etc. It has been known.
しかしながら、酸によるキチンの限定加水分解では、２
〜４糖の低級Ｎ−アセチルキトオリゴ糖を比較的高い収
率で得ることができるが、５等以上の高級Ｎ−アセチル
キトオリゴ糖の収率は非常に低い。However, in the limited hydrolysis of chitin with acid, 2
It is possible to obtain lower N-acetylchitooligosaccharides having 4 to 4 sugars in a relatively high yield, but the yield of higher N-acetylchitooligosaccharides of 5 or more is very low.
またキチナーゼなどの酵素によるキチンの加水分解で
は、５糖以上のＮ−アセチルキトオリゴ糖を特異的に生
産する酸素は発見されていない。リゾチームの糖転移反
応では、リゾチームが２〜４糖の低級Ｎ−アセチルキト
オリゴ糖に対し、糖転移活性が弱いため、５糖以上の高
級Ｎ−アセチルキトオリゴ糖の生成量はきわめて少な
い。本発明者らは、キチナーゼの糖転移反応がリゾチー
ムの糖転移反応より強いことを見出し、キチナーゼを用
いた高級Ｎ−アセチルキトオリゴ糖の効率的な生産を開
示した（特開昭62-146598）。しかし、この方法は、３
糖以上のＮ−アセチルキトオリゴ糖に作用し５糖以上の
Ｎ−アセチルキトオリゴ糖を生成するもので、２糖以上
のＮ−アセチルキトビオースにはほとんど作用しない欠
点がある。In addition, in the hydrolysis of chitin with an enzyme such as chitinase, oxygen that specifically produces N-acetylchitooligosaccharide having 5 or more sugars has not been found. In the transglycosylation reaction of lysozyme, lysozyme has a weak transglycosylation activity with respect to lower N-acetylchitooligosaccharides having 2 to 4 sugars, and thus the production amount of higher N-acetylchitooligosaccharides having 5 or more sugars is extremely small. The present inventors have found that the transglycosylation reaction of chitinase is stronger than the transglycosylation reaction of lysozyme, and disclosed efficient production of higher N-acetylchitooligosaccharides using chitinase (JP-A-62-146598). . However, this method
It acts on N-acetylchitooligosaccharides having sugars or more to produce N-acetylchitooligosaccharides having pentasaccharides or more, and has a drawback that it hardly acts on N-acetylchitobiose having two or more sugars.
本発明は、これらの欠点を解決するため種々研究した結
果、２〜４糖の低級Ｎ−アセチルキトオリゴ糖を基質と
し、リゾチームを親水性有機溶媒存在下で作用させると
縮合反応あるいは転移反応により効率的に５糖以上のＮ
−アセチルキトオリゴ糖が生成されるという知見に基い
ている。As a result of various studies to solve these drawbacks, the present invention uses a lower N-acetylchitooligosaccharide having 2 to 4 sugars as a substrate and causes lysozyme to act in the presence of a hydrophilic organic solvent to cause a condensation reaction or a transfer reaction. Efficiently N of 5 sugars or more
-Based on the finding that acetylchitooligosaccharides are produced.
発明が解決しようとする問題点 従って、本発明の目的は、２〜４糖の低級Ｎ−アセチル
キトオリゴ糖を用いて効率的に５糖以上の高級Ｎ−アセ
チルキトオリゴ糖を生産する改良された製造法を提供す
ることにある。DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention Accordingly, the object of the present invention is to improve production of a higher N-acetylchitooligosaccharide having 5 or more sugars efficiently by using a lower N-acetylchitooligosaccharide having 2 to 4 sugars. To provide a manufacturing method.
問題点を解決するための手段 本発明は、低級Ｎ−アセチルキトオリゴ糖を基質とし、
リゾチームを親水性有機溶媒存在下で作用させることを
特徴とする高級Ｎ−アセチルキトオリゴ糖の製造法を提
供する。Means for Solving the Problems The present invention uses a lower N-acetylchitooligosaccharide as a substrate,
Disclosed is a method for producing a higher N-acetylchitooligosaccharide, which comprises allowing lysozyme to act in the presence of a hydrophilic organic solvent.
本発明に使用する低級Ｎ−アセチルキトオリゴ糖は、例
えば、カニ、エビの甲殻から調製したキチンを酸や酵素
で加水分解して得たものを用いることができる。実用的
な調製法の一例を示すと、カニの甲殻を希塩酸処理によ
って脱灰後、希アルカリ処理で除蛋白して得たキチンに
濃塩酸を加え、３０〜５０℃で１〜６時間部分加水分解
する。得られた分解物を水酸化ナトリウムで中和後、残
渣を吸引濾過して除去し、瀘液を活性炭カラムに展開
し、エタノール直線濃度勾配法で溶出し、Ｎ−アセチル
キトビオースからＮ−アセチルキトテトラオースの低級
Ｎ−アセチルキトオリゴ糖をそれぞれ分離することがで
きる。また、これらの低級Ｎ−アセチルキトオリゴ糖の
混合物でも本発明に用いることができる。As the lower N-acetylchitooligosaccharide used in the present invention, for example, one obtained by hydrolyzing chitin prepared from crab or shrimp shells with an acid or an enzyme can be used. As an example of a practical preparation method, after decalcifying crab shells by dilute hydrochloric acid treatment and deproteinizing with dilute alkali treatment, concentrated hydrochloric acid was added to chitin, and the mixture was partially hydrolyzed at 30 to 50 ° C for 1 to 6 hours. Disassemble. After the obtained decomposed product was neutralized with sodium hydroxide, the residue was removed by suction filtration, the filtrate was developed on an activated carbon column, and eluted with a linear concentration gradient method of ethanol, and N-acetylchitobiose was added to give N-. The lower N-acetylchitooligosaccharides of acetylchitotetraose can be isolated respectively. Also, a mixture of these lower N-acetylchitooligosaccharides can be used in the present invention.
本発明に用いるリゾチームは、市販の卵白リゾチームな
どを用いることができ、反応溶液中の親水性有機溶媒
は、メタノール、エタノール、ｎ−プロパノール、イソ
プロパノール、ｎ−ブタノール、イソブタノール、ジメ
チルスルホキサイド、ジオキサン、ホルムアミド、アセ
トンのうち１種類上を用いることができ、親水性有機溶
媒は２０〜８０％、好ましくは３０〜７０％の濃度にな
るように水または緩衝液を加え調整する。反応に用いる
低級Ｎ−アセチルキトオリゴ糖の量は、反応溶液の飽和
濃度付近が好ましく、またリゾチームの量は、用いた低
級Ｎ−アセチルキトオリゴ糖１mgに対し、リゾチーム活
性として1,000 〜10,000Ｕが好ましい（リゾチーム活
性：M.Iuteusの細胞壁を基質とし、３５℃、pH 6.2で５
４０nmにおける吸光度を１分間に0.001 減少させる酵素
量を１Ｕとする）。As the lysozyme used in the present invention, commercially available egg white lysozyme or the like can be used, and the hydrophilic organic solvent in the reaction solution is methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, dimethyl sulfoxide, One or more of dioxane, formamide, and acetone can be used, and the hydrophilic organic solvent is adjusted to have a concentration of 20 to 80%, preferably 30 to 70% by adding water or a buffer solution. The amount of lower N-acetylchitooligosaccharide used in the reaction is preferably around the saturated concentration of the reaction solution, and the amount of lysozyme is 1,000 to 10,000 U as lysozyme activity per 1 mg of lower N-acetylchitooligosaccharide used. Preferred (lysozyme activity: M. Iuteus cell wall as a substrate, at 35 ° C, pH 6.2, 5
The amount of enzyme that reduces the absorbance at 40 nm by 0.001 per minute is 1 U).
反応時間は、２０〜６０℃、好ましくは３０〜５０℃
で、反応 pＨは４〜７が好ましい。反応時間は酵素量、
反応温度、 pＨによって異なるが、通常は１２〜１２０
時間で目的とする反応生成物が最大値となる。The reaction time is 20 to 60 ° C, preferably 30 to 50 ° C
Then, the reaction pH is preferably 4 to 7. The reaction time is the amount of enzyme,
It depends on the reaction temperature and pH, but usually 12 to 120
The desired reaction product reaches a maximum value with time.
目的とする５糖以上のＮ−アセチルキトオリゴ糖は、非
常に溶解度が低く、反応液中に析出されるので、反応の
進行状況が確認でき、分離と回収とが容易である。加熱
などによって酵素反応を停止した後、例えば、遠心分
離、濾過等によって回収できる。回収された生成物は、
５糖以上のＮ−アセチルキトオリゴ糖を多く含む混合物
である。The target N-acetylchitooligosaccharide having 5 or more sugars has very low solubility and is deposited in the reaction solution, so that the progress of the reaction can be confirmed and the separation and recovery are easy. After stopping the enzymatic reaction by heating or the like, it can be recovered by, for example, centrifugation, filtration or the like. The recovered product is
It is a mixture containing a large amount of N-acetylchitooligosaccharides having 5 or more sugars.
このようにして得られる反応混合物からの５糖以上のＮ
−アセチルキトオリゴ糖の分離、精製は、周知の分離精
製手段により行なうことができる。例えば、活性炭に反
応混合物を吸着させ、アルコール濃度勾配法により分離
溶出する方法や、ゲル濾過、高速液体クロマトグラフィ
ー等の手段により分離、精製することができる。N of 5 or more sugars from the reaction mixture thus obtained
Separation and purification of acetylchitooligosaccharide can be performed by a known separation and purification means. For example, the reaction mixture can be adsorbed on activated carbon and separated and eluted by an alcohol concentration gradient method, or can be separated and purified by means such as gel filtration and high performance liquid chromatography.
次に、本発明の実施例についてさらに具体的に説明する
が、これらの実施例によって本発明が何ら限定されるも
のではない。Next, examples of the present invention will be described more specifically, but the present invention is not limited to these examples.
実施例１ Ｎ−アセチルキトビオース（１g ）をの0.1 Ｍ酢酸緩衝
液（ pＨ 5.5；７ml）のメタノール（７ml）混合した溶
液に溶解し、４０℃に保った後、市販の卵白リゾチーム
（６回結晶、生化学工業社製）を2.5 ×１０６Ｕ添加し
て４０℃で９６時間反応させた。反応終了後、１００℃
で１０分間加熱し反応を停止させた。生成した不溶物を
遠心分離によって回収し、ついで少量の熱水に懸濁し、
再び遠心分離によって分離した。分離した不溶物を凍結
乾燥機を用いて乾燥し、白色粉末（２００mg）を得た。
この白色粉末の生成物組成を、高速液体クロマトグラフ
ィーによって確認したところ、Ｎ−アセチルキトオクタ
オース１６％、Ｎ−アセチルキトヘプタオーク３５％、
Ｎ−アセチルキトヘキサオース２１％、Ｎ−アセチルキ
トペンタオース７％が含まれていた。Example 1 N-Acetylchitobiose (1 g) was dissolved in a solution of 0.1 M acetate buffer (pH 5.5; 7 ml) in methanol (7 ml) and kept at 40 ° C., and then commercially available egg white lysozyme (6 2.5 × 10 6 U of a recrystallized product, manufactured by Seikagaku Corporation was added and reacted at 40 ° C. for 96 hours. After the reaction, 100 ℃
The reaction was stopped by heating for 10 minutes. The insoluble matter formed is recovered by centrifugation, then suspended in a small amount of hot water,
Separated again by centrifugation. The separated insoluble material was dried using a freeze dryer to obtain a white powder (200 mg).
The product composition of this white powder was confirmed by high performance liquid chromatography to find that N-acetylchitooctaose 16%, N-acetylchitoheptaoque 35%,
21% of N-acetylchitohexaose and 7% of N-acetylchitopentaose were contained.
実施例２ Ｎ−アセチルキトトリオース（６００mg）を0.3 Ｍ酢酸
緩衝液（ pＨ 5.5；１０ml）とメタノール（１０ml）と
水（１０ml）とを混合した溶液に溶解し、４０℃に保っ
た後、市販の卵白リゾチーム（６回結晶、生化学工業社
製）を2.5 ×１０６Ｕ添加して、４０℃で７２時間反応
させた。反応終了後、１００℃で１０分間加熱し、反応
を停止させた。生成した不溶物を遠心分離によって回収
し、ついで少量の熱水に懸濁し、再び遠心分離によって
分離した。分離した不溶物を凍結乾燥機を用いて乾燥
し、白色粉末（１２９mg）を得た。Example 2 N-acetylchitotriose (600 mg) was dissolved in a mixed solution of 0.3 M acetate buffer (pH 5.5; 10 ml), methanol (10 ml) and water (10 ml), and the mixture was kept at 40 ° C. 2.5 × 10 6 U of commercially available egg white lysozyme (6 times crystal, manufactured by Seikagaku Corporation) was added and reacted at 40 ° C. for 72 hours. After completion of the reaction, the reaction was stopped by heating at 100 ° C for 10 minutes. The produced insoluble matter was recovered by centrifugation, then suspended in a small amount of hot water, and again separated by centrifugation. The separated insoluble material was dried using a freeze dryer to obtain a white powder (129 mg).
この白色粉末の生成物組成を、高速液体クロマトグラフ
ィーによって確認したところ、Ｎ−アセチルキトオクタ
オース１８％、Ｎ−アセチルキトヘプタオース３９％、
Ｎ−アセチルキトヘキサオース１８％、Ｎ−アセチルキ
トペンタオース５％が含まれていた。When the product composition of this white powder was confirmed by high performance liquid chromatography, N-acetylchitooctaose 18%, N-acetylchitoheptaose 39%,
18% of N-acetylchitohexaose and 5% of N-acetylchitopentaose were contained.
発明の効果 本発明により、５糖以上の高級Ｎ−アセチルキトオリゴ
糖を効率的に製造することができる。EFFECTS OF THE INVENTION According to the present invention, higher N-acetylchitooligosaccharides having 5 or more sugars can be efficiently produced.
Claims (2)
し、リゾチームを親水性有機溶媒の存在下で作用させる
ことを特徴とする高級Ｎ−アセチルキトオリゴ糖の製造
法。1. A method for producing a higher N-acetylchitooligosaccharide, which comprises using a lower N-acetylchitooligosaccharide as a substrate and allowing lysozyme to act in the presence of a hydrophilic organic solvent.
ノール、ｎ−プロパノール、イソプロパノール、ｎ−ブ
タノール、イソブタノール、ジメチルスルホキサイド、
ジオキサン、ホルムアミド、アセトンのうち１種類以上
を用いる特許請求の範囲第１項記載の方法。2. As a hydrophilic organic solvent, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, dimethyl sulfoxide,
The method according to claim 1, wherein at least one of dioxane, formamide, and acetone is used.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27095687A JPH0646953B2 (en) | 1987-10-27 | 1987-10-27 | Process for producing higher N-acetyl chitooligosaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27095687A JPH0646953B2 (en) | 1987-10-27 | 1987-10-27 | Process for producing higher N-acetyl chitooligosaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01112995A JPH01112995A (en) | 1989-05-01 |
| JPH0646953B2 true JPH0646953B2 (en) | 1994-06-22 |
Family
ID=17493363
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP27095687A Expired - Lifetime JPH0646953B2 (en) | 1987-10-27 | 1987-10-27 | Process for producing higher N-acetyl chitooligosaccharide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0646953B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5553295B2 (en) * | 2009-02-04 | 2014-07-16 | 焼津水産化学工業株式会社 | Method for producing higher N-acetylchitooligosaccharide |
-
1987
- 1987-10-27 JP JP27095687A patent/JPH0646953B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01112995A (en) | 1989-05-01 |
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