JPH08322554A - Aspergillus-fumigatus mutant bacteria and its production enzyme, and manufacture of chitosan-oligosaccharide using said bacteria or production enzyme - Google Patents
Aspergillus-fumigatus mutant bacteria and its production enzyme, and manufacture of chitosan-oligosaccharide using said bacteria or production enzymeInfo
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- JPH08322554A JPH08322554A JP8061793A JP6179396A JPH08322554A JP H08322554 A JPH08322554 A JP H08322554A JP 8061793 A JP8061793 A JP 8061793A JP 6179396 A JP6179396 A JP 6179396A JP H08322554 A JPH08322554 A JP H08322554A
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- Prior art keywords
- chitosan
- oligosaccharide
- mycelium
- producing
- aspergillus fumigatus
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
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- Polymers & Plastics (AREA)
- Molecular Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、アスペルギルスフ
ミガーツス突然変異菌および当該突然変異菌を用いたキ
トサン−オリゴ糖の製造方法に関し、より詳細には、ア
スペルギルスフミガーツス突然変異菌が生産するキトサ
ン−オリゴ糖分解酵素を用い、天然資源としてカニやエ
ビなどの甲殻類に大量に含まれるキトサンを有効利用す
る、キトサンからキトサン−オリゴ糖(Chitosa
n−Oligosaccharide)を製造する方法
に関する。TECHNICAL FIELD The present invention relates to an Aspergillus fumigatus mutant bacterium and a method for producing chitosan-oligosaccharide using the mutant bacterium, and more specifically, it is produced by an Aspergillus fumigatus mutant bacterium. A chitosan-oligosaccharide degrading enzyme is used to effectively utilize chitosan, which is contained in a large amount in crustaceans such as crabs and shrimps, as a natural resource. From chitosan to chitosan-oligosaccharide (Chitosa)
n-Oligosaccharide).
【0002】[0002]
【従来の技術】キチン、すなわちβ−1,4−ポリ−N
−アセチル−D−グルコサミン(β−1,4−poly
−N−acetyl−D−glucosamine)
は、節足動物、菌類等の主要多糖類であり、天然では蛋
白質と結合して糖蛋白の形で存在する。この糖蛋白から
アルカリで蛋白質を除去し、キチンを得ることができ
る。キチンは塩酸で加水分解すれば単糖類であるD−グ
ルコサミン塩酸塩となるが、濃アルカリ溶液で加熱し、
脱アセチル化すればキトサンを得ることができる。Chitin, namely β-1,4-poly-N
-Acetyl-D-glucosamine (β-1,4-poly)
-N-acetyl-D-glucosamine)
Is a major polysaccharide such as arthropods and fungi, and naturally exists in the form of glycoprotein by binding to a protein. Chitin can be obtained by removing the protein from this glycoprotein with an alkali. If chitin is hydrolyzed with hydrochloric acid, it becomes D-glucosamine hydrochloride which is a monosaccharide, but it is heated in a concentrated alkaline solution,
Chitosan can be obtained by deacetylation.
【0003】キトサンは、D−グルコサミン、すなわち
2−アミノ−2−デオキシ−D−グルコースがβ−1,
4結合で縮重合した塩基性多糖類である。カニやエビ等
の甲殻類に含有されるキチンから、濃アルカリ溶液中で
の加熱や脱アセチル化を経て得るため、キトサンの分子
量はキチン分子量よりやや小さくなる。Chitosan is composed of D-glucosamine, that is, 2-amino-2-deoxy-D-glucose is β-1,
It is a basic polysaccharide that is polycondensed with 4 bonds. Since chitin contained in crustaceans such as crab and shrimp is obtained through heating and deacetylation in a concentrated alkaline solution, the molecular weight of chitosan is slightly smaller than the molecular weight of chitin.
【0004】キトサンは分子内に反応性の遊離アミノ基
を有し、天然高分子凝集剤として廃水処理に利用され、
また、化学的にも高分子材料として興味が持たれ、その
利用研究が活発に行われている。Chitosan has a reactive free amino group in the molecule and is used as a natural polymer flocculant in wastewater treatment.
Also, as a polymer material, it is of interest chemically, and its use research is actively conducted.
【0005】ここに、「キトサン−オリゴ糖」とは、D
−グルコサミンがβ−1,4結合で2乃至10個結合し
たキトサン由来のオリゴ糖をいう。キチン由来のオリゴ
糖を脱N−アセチル化して得たキトサンを、部分加水分
解して得ることができる。Here, "chitosan-oligosaccharide" means D
-It means an oligosaccharide derived from chitosan in which 2 to 10 glucosamines are linked by β-1,4 bond. Chitosan obtained by de-N-acetylating oligosaccharide derived from chitin can be obtained by partial hydrolysis.
【0006】キトサン−オリゴ糖は、医薬、食品、化粧
品、農業等の分野において、抗腫瘍性や生理活性機能を
有することが知られている。キトサン自体は水に不溶で
あるが、キトサン−オリゴ糖は水に可溶であり、キトサ
ン特有の苦味と渋味がなく、しかも優れた生理活性機能
を有する。Chitosan-oligosaccharides are known to have antitumor properties and physiologically active functions in the fields of medicine, food, cosmetics, agriculture and the like. Chitosan itself is insoluble in water, but chitosan-oligosaccharide is soluble in water, has no bitterness and astringency peculiar to chitosan, and has an excellent physiological activity function.
【0007】例えば、N−アセチル−D−グルコサミン
が6つ結合したN−アセチルキトヘキサオーズ(Glc
NAc6)やD−グルコサミンが6つ結合したキトヘキ
サオーズ(GlcN6)が、マウスの移植腫瘍に対する
腫瘍転移抑制効果、免疫増強作用及び感染防御作用を有
するとの報告がある(Carbo−hyd,Res.,
151,403(1986))。For example, N-acetyl-chitohexaose (Glc) in which six N-acetyl-D-glucosamines are bonded.
It has been reported that chitohexaose (GlcN6) having six NAc6) and D-glucosamine bound thereto has a tumor metastasis-suppressing effect, an immunopotentiating effect and an infection-preventing effect on a transplanted tumor of mice (Carbo-hyd, Res.,
151, 403 (1986)).
【0008】また、植物には、病虫害に対する自己防御
能力の復活作用と植物細胞の活性化を誘導し、農業分野
に活用できる。また、植物病原菌の増殖抑制作用があ
り、土壌改良剤あるいは天然性農薬として開発が期待さ
れている(日本化学工業44.10.30〜63.(1
991))。[0008] In addition, the plant can be utilized in the agricultural field by inducing a revival action of self-defense ability against pest damage and activation of plant cells. Further, it has an effect of inhibiting the growth of plant pathogens and is expected to be developed as a soil conditioner or a natural pesticide (Nippon Kagaku Kogyo 44.10.30-63. (1
991)).
【0009】さらに、ホットケーキシロップ、ヨーグル
ト、飲料、漬け物食品などの食品保存剤や低カロリー、
低甘味度の新規糖質素材として開発も期待されている
(日本化学工業44.10.30〜63.(199
1))。Furthermore, food preservatives such as pancake syrup, yogurt, beverages, pickled foods and low calories,
It is expected to be developed as a novel sugar material having a low degree of sweetness (Nippon Kagaku Kogyo 44.10.30-63.
1)).
【0010】また、酵素に関しては、食品分野では機能
性食品の重要性と共に新規な酵素の市場開発の加速化が
予測されている。実際日本だけでも数百億ウォンの各種
オリゴ糖が既に商業化されている。他の分野でも、例え
ば、アメリカで研究中のコレステロールレダクターゼを
利用した摂取抑制研究で、商業化により年1億ドル以上
の新しい市場が形成される可能性がある(日本化学工業
44.10.30〜63.(1991))。Regarding enzymes, in the food field, it is expected that the market development of new enzymes will be accelerated along with the importance of functional foods. In fact, several tens of billion won of various oligosaccharides have already been commercialized in Japan alone. In other fields as well, for example, intake suppression research using cholesterol reductase, which is under study in the United States, may create a new market of more than 100 million dollars per year by commercialization (Nippon Kagaku Kogyo 44.10.30). ~ 63. (1991)).
【0011】一方、このように優れた生理活性を有する
キトサンーオリゴ糖の製造方法としては、(1)酸加水
分解法、(2)酵素分解法、(3)糖転移反応利用法等
が知られている。On the other hand, as methods for producing chitosan-oligosaccharides having such excellent physiological activity, (1) acid hydrolysis method, (2) enzymatic decomposition method, (3) glycosyl transfer reaction utilization method, etc. are known. There is.
【0012】(1)酸加水分解法 ラプリ(Rupley)が1964年度に、酸加水分解
によりキチンを分解し、その生成物を活性炭−ゼオライ
トカラムを利用して単離し、単糖から5糖のN−アセチ
ルキトペンタオーズまでを得ている。また、最近ではH
PLC用カラムを併用してキチン−オリゴ糖が製造され
ている(Biochem.Biophys Acta.
83,245.1964))。さらに、特開昭61−2
1102号公報には、酸加水分解の改良法であって、キ
トサンに10規定以上の濃い塩酸を、キトサン重量の5
乃至30倍量加えて温度60乃至100℃、1乃至4時
間で部分加水分解させて高重合度のオリゴ糖を得たこと
が開示されている。しかし、酸加水分解法は大量の酸
と、加熱を要し製造原価が上昇し、廃液処理も必要とな
り2次的な環境汚染誘発の可能性が生ずる点で問題とな
る。(1) Acid Hydrolysis Method In 1964, Rupley decomposed chitin by acid hydrolysis and isolated the product using an activated carbon-zeolite column. -Acetylchitopentaose is obtained. Also, recently H
Chitin-oligosaccharides have been produced in combination with a column for PLC (Biochem. Biophys Acta.
83, 245.1964)). Furthermore, JP-A-61-2
Japanese Patent Laid-Open No. 1102 is an improved method of acid hydrolysis, wherein chitosan is added with concentrated hydrochloric acid of 10 normal or more, and chitosan is added in an amount of 5% by weight.
It is disclosed that an oligosaccharide having a high degree of polymerization was obtained by adding an amount of 30 to 30 times and partial hydrolysis at a temperature of 60 to 100 ° C. for 1 to 4 hours. However, the acid hydrolysis method is problematic in that it requires a large amount of acid and heating, the manufacturing cost rises, waste liquid treatment is required, and secondary environmental pollution may occur.
【0013】(2)酵素分解法 海水で分離された中温性細菌ビブリオアングィラルム
(Vibrio anguillarum)E−383
aをキチンを含有する液体培地(表−1の培地A)で3
5℃、16日間振盪培養し、N,N’−アセチルキトビ
オース(GlcNAc2)が得られている(Agri
c.Biol.Chem.53.1537〜1541,
(1989))。しかし、当該ビブリオアングィラルム
E−383aを利用する方法は、加水分解率が53%と
低く、16日間という長時間の培養を要する点で問題が
ある。(2) Enzymatic Degradation Method Vibrio anguillarum E-383, a mesophilic bacterium isolated in seawater
a is 3 in liquid medium containing chitin (medium A in Table 1)
After shaking culture at 5 ° C. for 16 days, N, N′-acetylchitobiose (GlcNAc2) has been obtained (Agri).
c. Biol. Chem. 53.1537-1541,
(1989)). However, the method using Vibrio anguillarum E-383a has a problem that the hydrolysis rate is as low as 53% and that a long culture time of 16 days is required.
【0014】温泉で分離した好熱性細菌であるバシラス
リケニホルミスX−7uを利用し、液体培地(表−1の
培地B)で50℃で3.5日間の回転振盪培養により、
N,N’−アセチルキトトリオース(GlcNAc3)
が得られている(日本農芸化学会誌63,7,1199
〜1205,1989)。しかし、当該バシラスリケニ
ホルミスX−7uを利用する方法は、加水分解率が72
%と比較的高く、得られたキチン−オリゴ糖も3糖であ
るが、3.5日間も回転振盪しなければならない点で問
題がある。Utilizing Bacillus licheniformis X-7u, which is a thermophilic bacterium isolated in a hot spring, by rotary shaking culture at 50 ° C. for 3.5 days in a liquid medium (medium B in Table 1),
N, N'-acetylchitotriose (GlcNAc3)
Has been obtained (Journal of the Japanese Society of Agricultural Chemistry 63, 7, 1199)
~ 1205,1989). However, the method utilizing Bacillus licheniformis X-7u has a hydrolysis rate of 72.
%, Which is relatively high, and the obtained chitin-oligosaccharide is also trisaccharide, but there is a problem in that it must be shaken by rotation for 3.5 days.
【0015】また、バシラス属No.7−Mに由来する
キトサン分解酵素であるキトサナーゼを利用し、2糖か
ら5糖までのキトサン−オリゴ糖が得られている(Ag
ric.Biol.Chem.51.(1989))。
しかし、そのほとんどは単糖である。In addition, Bacillus No. Utilizing chitosanase, which is a chitosan-degrading enzyme derived from 7-M, chitosan-oligosaccharides from disaccharides to pentasaccharides have been obtained (Ag
ric. Biol. Chem. 51. (1989)).
However, most of them are monosaccharides.
【0016】また、土壌で分離されたキトサン分解菌シ
ュードモナス属(Pseudomonas spp.)
を培地(表−1の培地C)中で、温度25℃で3乃至1
0日間培養し、種子菌を接種し26乃至28℃で培養し
たところ、その二日後から培地内にキトサナーゼを分泌
し、培地内酵素量のピークは4日目であったと報告され
ている(Agric.Biol.Chem.54.1
2,3341〜3343,1990)。しかし、土壌で
分離された当該シュードモナス属(Pseudomon
as spp.)を利用する方法では、加水分解率と生
産物が不明である。Also, a chitosan-degrading bacterium isolated from soil, Pseudomonas spp.
In a medium (medium C in Table 1) at a temperature of 25 ° C. for 3 to 1
After culturing for 0 days, inoculating seed bacteria and culturing at 26 to 28 ° C., it was reported that two days after that, chitosanase was secreted into the medium, and the peak of the amount of enzyme in the medium was on the 4th day (Agric). Biol Chem. 54.1
2, 3341-3433, 1990). However, the Pseudomonas genus (Pseudomon) isolated in soil
as spp. ), The hydrolysis rate and product are unknown.
【0017】(3)糖転移反応利用法 ノカディア・オリエンタルリス(Nocarda or
ientalis)またはトリコドマ・レーシ(Tri
choderma reesi)由来のキチナーゼを利
用し、硝酸緩衝液中でN−アセチルキトテトラオーズ
(4糖)(5乃至10重量%)を基質として糖転移反応
により6糖の白色沈澱を得、一方、5糖を基質として上
記と同様に操作して7糖を得ている。しかし、当該方法
は基質濃度、反応温度及びpHの影響を強く受けるが加
水分解率は低く34%である。このため、既存のキチン
−オリゴ糖の4糖と2糖とを要し、また、加水分解率も
低いという欠点がある。(3) Utilization of glycosyl transfer reaction Nocardia orientalis (Nocarda or)
ientalis) or Trichodoma lactis (Tri
A white precipitate of hexasaccharide was obtained by a transglycosylation reaction using a chitinase derived from choderma reesi) in a nitrate buffer with N-acetylchitotetraose (tetrasaccharide) (5 to 10% by weight) as a substrate, while Using the sugar as a substrate, the same procedure as above was carried out to obtain a hepta sugar. However, although the method is strongly influenced by the substrate concentration, reaction temperature and pH, the hydrolysis rate is low and is 34%. For this reason, the existing chitin-oligosaccharide tetrasaccharide and disaccharide are required, and the hydrolysis rate is low.
【0018】上記問題を解決すべく、効率が高く、所望
の重合度を有するキトサン−オリゴ糖の製造方法の開発
が熱望されている。[0018] In order to solve the above problems, it has been earnestly desired to develop a method for producing a chitosan-oligosaccharide having a high efficiency and a desired degree of polymerization.
【0019】[0019]
【課題を解決するための手段】本発明者らは、前記従来
技術の問題点を解決するため鋭意研究した結果、アスペ
ルギルスフミガーツス突然変異菌が生産するキトサンを
分解する酵素または前記アスペルギルスフミガーツス突
然変異菌自体を利用することにより、加水分解率が高
く、経済的かつ環境汚染を起こさずキトサン−オリゴ糖
を製造できることを見いだし、本発明を完成するに至っ
た。Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems of the prior art, the present inventors have found that an enzyme that decomposes chitosan produced by an Aspergillus fumigatus mutant or the above Aspergillus fumigatus. The inventors have found that the use of the mutated bacterium itself can produce chitosan-oligosaccharide with a high hydrolysis rate, economically and without causing environmental pollution, and completed the present invention.
【0020】すなわち本発明は、キトサンをキトサン−
オリゴ糖に分解する性質を持つ韓国科学技術院遺伝工学
研究所に受託番号KCTC0139BP号で寄託された
アスペルギルスフミガーツス突然変異菌を提供するもの
である。また、キトサンをキトサン−オリゴ糖に分解す
る前記アスペルギルスフミガーツス突然変異菌が生産す
る酵素を提供するものである。また、前記アスペルギル
スフミガーツス突然変異菌を培養して菌糸体を収穫し、
得られた菌糸体をキトサンと反応させることを特徴とす
るキトサン−オリゴ糖の製造方法を提供するものであ
る。以下、本発明を詳細に説明する。That is, in the present invention, chitosan is
The Aspergillus fumigatus mutant strain deposited under the deposit number KCTC0139BP at the Institute of Genetic Engineering, Korea Institute of Science and Technology, which has the property of degrading into oligosaccharides, is provided. The present invention also provides an enzyme produced by the Aspergillus fumigatus mutant bacterium that decomposes chitosan into chitosan-oligosaccharide. In addition, the Aspergillus fumigatus mutant bacterium is cultured to collect mycelium,
The present invention provides a method for producing a chitosan-oligosaccharide, which comprises reacting the obtained mycelium with chitosan. Hereinafter, the present invention will be described in detail.
【0021】[0021]
【発明の実施の形態】本発明は、アスペルギルスフミガ
ーツス突然変異菌を培養し、培養により生産された菌糸
体を回収し、その菌糸体をキトサンと50乃至60℃、
特に好ましくは60℃で、10乃至60分、特に好まし
くは30分間反応させることによりキトサン−オリゴ糖
を製造する。このため酸加水分解法のように、塩酸の使
用や加熱が不要である。また、このように培養された菌
糸体を使用するため、酵素を用いる過程で、塩析等の操
作が不要となり、かつ簡単で短い時間に高い加水分解率
でキトサン−オリゴ糖の製造ができる。BEST MODE FOR CARRYING OUT THE INVENTION The present invention comprises culturing an Aspergillus fumigatus mutant bacterium, collecting the mycelium produced by the culturing, and collecting the mycelium with chitosan at 50 to 60 ° C.
The chitosan-oligosaccharide is produced by reacting at 60 ° C. for 10 to 60 minutes, particularly preferably 30 minutes. Therefore, unlike the acid hydrolysis method, the use of hydrochloric acid or heating is unnecessary. Further, since the mycelium thus cultivated is used, an operation such as salting out is unnecessary in the process of using the enzyme, and the chitosan-oligosaccharide can be easily produced at a high hydrolysis rate in a short time.
【0022】本発明の、キトサン−オリゴ糖の製造方法
において、アスペルギルスフミガーツス突然変異菌の培
養のための培地は、蒸留水1リットルに対し、キトサン
5乃至15g、酵母エキス0.25乃至1.0g、硝酸
アンモニウム0.5乃至2.0g、塩化ナトリウム0.
25乃至1.5g、リン酸第2カリウム0.5乃至2.
0g、リン酸第2ナトリウム2.0乃至5g、硫酸マグ
ネシウム0.25乃至1.0g、塩化カルシウム0.0
2乃至0.1g、寒天10乃至20g、硝酸10mlか
らなることが好ましく、前記アスペルギルスフミガーツ
ス突然変異菌の培養は、液体培地で温度20乃至30
℃、70乃至150rpmで3乃至6日間の振盪培養で
あることが好ましい。In the method for producing chitosan-oligosaccharide of the present invention, the medium for culturing the Aspergillus fumigatus mutant strain is 5 to 15 g of chitosan and 0.25 to 1 of yeast extract per 1 liter of distilled water. 0.0 g, ammonium nitrate 0.5 to 2.0 g, sodium chloride 0.
25 to 1.5 g, dibasic potassium phosphate 0.5 to 2.
0 g, dibasic sodium phosphate 2.0 to 5 g, magnesium sulfate 0.25 to 1.0 g, calcium chloride 0.0
2 to 0.1 g, 10 to 20 g of agar, and 10 ml of nitric acid are preferable, and the Aspergillus fumigatus mutant is cultured in a liquid medium at a temperature of 20 to 30.
The shaking culture is preferably carried out at 70 ° C. and 70 to 150 rpm for 3 to 6 days.
【0023】前記菌糸体とキトサンとの反応は、温度範
囲50乃至60℃、pH4.0乃至5.0、反応時間1
0乃至60分間で行ない、キトサンの濃度は、1乃至5
重量%であることが好ましい。The reaction between the mycelium and chitosan is carried out at a temperature range of 50 to 60 ° C., a pH of 4.0 to 5.0, and a reaction time of 1
It takes 0 to 60 minutes, and the concentration of chitosan is 1 to 5
It is preferably in the weight%.
【0024】本発明のアスペルギルスフミガーツス突然
変異菌の培養は、表−1に示す培地Dを用いることが好
ましい。For culturing the Aspergillus fumigatus mutant of the present invention, it is preferable to use the medium D shown in Table 1.
【0025】(酵素生産菌の分離)本発明による酵素生
産菌は、キトサンを含有する培地(表−1の培地D)で
キトサン分解機能を調べ、キトサン加水分解率が最も高
い菌株を選別した。選別された菌の特徴を表−2に示
す。(Separation of Enzyme-Producing Bacteria) With respect to the enzyme-producing bacterium according to the present invention, a chitosan-degrading function was examined in a medium containing chitosan (medium D in Table 1), and a strain having the highest hydrolysis rate of chitosan was selected. The characteristics of the selected bacteria are shown in Table-2.
【0026】(菌学的性質)選別された菌をスライド培
養し、光学顕微鏡で形態的性質を観察した。菌糸の太さ
は4〜6μmであり、その一部が肥大する。分生胞子柄
の太さは6〜9μm、長さは300〜500μmであ
り、菌糸から垂直に分枝する。分生子表面は滑らかであ
る。ツァペック寒天平板培地での集落の色は、初期は黄
褐色であったが、培養期間の経過と共にだんだん褐色に
変わった。The Fungi(Ai−nswort
h, G.C.Sprrw, F.K. and Su
ssman A.S.:The Fungi, Vol
4A. Academic Press. New
York, pp 45〜68(1973))及びTh
e Genus Aspergillus(Pape
r, K.B.and Fennell, D.I.:
The Genus Aspergillus, Ro
bert E.Kriege Pub.Co.Hunt
ingt−on. New York, pp 13〜
577(1973))等の分類書及びユビキノンシステ
ムの結果から、アスペルギルスフミガーツスと確認し、
アスペルギルスフミガーツスKB−1と命名した。(Mycological properties) The selected bacteria were slide-cultured and observed for their morphological properties with an optical microscope. The hypha has a thickness of 4 to 6 μm, and a part of it thickens. The thickness of the conidia spores is 6 to 9 μm, the length is 300 to 500 μm, and they branch vertically from the hyphae. The conidium surface is smooth. The color of the colony on the Czapek agar plate medium was initially yellowish brown, but gradually changed to brown over the course of the culture period. The Fungi (Ai-nsworth
h, G. C. Sprrw, F.F. K. and Su
ssman A. S. : The Fungi, Vol
4A. Academic Press. New
York, pp 45-68 (1973)) and Th.
e Genus Aspergillus (Pape
r, K. B. and Fennell, D.M. I. :
The Genus Aspergillus, Ro
bert E. Kriege Pub. Co. Hunt
ingt-on. New York, pp 13-
577 (1973)), etc., and the results of the ubiquinone system, confirming that Aspergillus fumigatus,
It was named Aspergillus fumigatus KB-1.
【0027】(寄託)本発明者は、本発明のアスペルギ
ルスフミガーツスKB−1を1994年12月15日付
けで韓国科学技術院遺伝工学研究所に寄託し、受託番号
は第KCTC0139BPである。(Deposit) The present inventor has deposited the Aspergillus fumigatus KB-1 of the present invention with the Institute of Genetic Engineering, Korea Institute of Science and Technology on December 15, 1994, and the deposit number is KCTC0139BP.
【0028】アスペルギルスフミガーツスKB−1を保
管する場合は、ポテトデキストロース液体培地(pot
ato dextrose broth)100mlに
アスペルギルスフミガーツスKB−1を接種し25℃、
120rpmで5日間振盪培養し、得た菌浮遊液を無菌
的に6枚のチーズクロースで濾して菌糸を除去し、次い
で1,500rpmで遠心分離し分生胞子を得、これを
保管する。When Aspergillus fumigatus KB-1 is stored, potato dextrose liquid medium (pot) is used.
100 ml of ato dextrose broth was inoculated with Aspergillus fumigatus KB-1 at 25 ° C,
After culturing with shaking at 120 rpm for 5 days, the obtained bacterial suspension is aseptically filtered with 6 pieces of cheese cloth to remove mycelium, and then centrifuged at 1,500 rpm to obtain conidia and stored.
【0029】アスペルギルスフミガーツスKB−1の上
記分生胞子を使用する場合は、滅菌蒸留水で洗滌し分生
胞子浮遊液とし、その培養は、表−1の培地Dの組成の
内、寒天を除去した液体培地で25℃、120rpm、
5日間振盪培養したものを使用する。When the above-mentioned conidia of Aspergillus fumigatus KB-1 are used, they are washed with sterilized distilled water to prepare a conidial suspension, and the culture is carried out by using agar in the composition of medium D in Table-1. Liquid culture medium from which is removed at 25 ° C., 120 rpm,
What was shake-cultured for 5 days is used.
【0030】[0030]
【実施例】以下、実施例により本発明を具体的に説明す
るが、本発明はこれらに限定されるものではない。な
お、濃度に関する%は、重量%を示す。EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited thereto. In addition,% regarding a density | concentration shows weight%.
【0031】(実施例1:酵素活性に及ぼす温度の影
響)アスペルギルスフミガーツスKB−1の菌糸体とキ
トサンとを添加した後、30乃至80℃まで10℃間隔
ごとに、各温度の酵素活性を測定した。その結果を図2
に示す。最適温度は60℃であり、50℃で87%程酵
素活性を見せたが40℃以下、または70乃至80℃で
は酵素活性が急激に減少した。Example 1 Effect of Temperature on Enzyme Activity After adding mycelium of Aspergillus fumigatus KB-1 and chitosan, the enzyme activity at each temperature was increased from 30 to 80 ° C. at 10 ° C. intervals. Was measured. The result is shown in Figure 2.
Shown in The optimum temperature was 60 ° C., and the enzyme activity was about 87% at 50 ° C., but the enzyme activity decreased sharply at 40 ° C. or lower, or at 70 to 80 ° C.
【0032】(実施例2:酵素活性に及ぼすpHの影
響)反応溶液のpHを0.5ずつ変化させ、各pHごと
の酵素活性を調べた。結果を図3に示す。アスペルギル
スフミガーツスKB−1が生産する酵素の最適pHは
4.0乃至5.0であり、pH3.5以下またはpH
6.5以上では酵素活性が殆どなかった。(Example 2: Effect of pH on enzyme activity) The pH of the reaction solution was changed by 0.5, and the enzyme activity at each pH was examined. The results are shown in Fig. 3. The optimum pH of the enzyme produced by Aspergillus fumigatus KB-1 is 4.0 to 5.0, pH 3.5 or lower or pH
At 6.5 or more, there was almost no enzyme activity.
【0033】(実施例3:キトサン濃度のキトサン−オ
リゴ糖製造への影響)キトサンの濃度を1乃至8%まで
1%ずつ変化させたキトサン溶液をキトサンを1%硝酸
で溶解して作製した。キトサンが5乃至8%の場合は、
温度60℃で溶解させた。各キトサン溶液50mlに、
予め培養したアスペルギルスフミガーツスKB−1の菌
糸体を混合し、60℃水浴槽で30分間ゆっくりと振盪
し反応させた。酵素と反応せずに残存するキトサンは、
同量のエタノールを添加し遠心分離により沈澱を除去し
た。この上清をキトサン−オリゴ糖に換算した。結果を
図4に示す。キトサンの濃度が、1乃至3%の範囲で
は、95%以上がキトサン−オリゴ糖に分解され、キト
サン濃度が5%でも74%が分解された。しかしそれ以
上のキトサン濃度では、キトサン−オリゴ糖の生産が大
きく減少した。(Example 3: Influence of chitosan concentration on chitosan-oligosaccharide production) A chitosan solution in which the chitosan concentration was changed from 1 to 8% in 1% steps was prepared by dissolving chitosan in 1% nitric acid. If chitosan is 5-8%,
It was melted at a temperature of 60 ° C. 50 ml of each chitosan solution,
The pre-cultured mycelium of Aspergillus fumigatus KB-1 was mixed and allowed to react by gently shaking in a 60 ° C water bath for 30 minutes. Chitosan remaining without reacting with the enzyme is
The same amount of ethanol was added and the precipitate was removed by centrifugation. This supernatant was converted to chitosan-oligosaccharide. FIG. 4 shows the results. In the chitosan concentration range of 1 to 3%, 95% or more was decomposed into chitosan-oligosaccharide, and 74% was decomposed even when the chitosan concentration was 5%. However, at higher chitosan concentrations, chitosan-oligosaccharide production was greatly reduced.
【0034】実施例3で生産されたキトサン−オリゴ糖
を、高速液体クロマトグラフィーにより分析した。結果
を図5に示す。キトサン濃度が1%の場合、生産される
キトサン−オリゴ糖は2糖以下が43%、3糖以上が5
7%であった(図5B)。キトサン濃度が3%またはそ
れ以上の場合には、2糖が22%、3糖が34%、4糖
が44%であった(図5C)。本発明に使用したアスペ
ルギルスフミガーツスKB−1菌の菌糸体を利用して生
産するキトサン−オリゴ糖は、酸加水分解に比べて単糖
の生産が殆どなく、キトサン濃度が3%では、78%が
3糖乃至6糖のキトサン−オリゴ糖であった。The chitosan-oligosaccharide produced in Example 3 was analyzed by high performance liquid chromatography. Results are shown in FIG. When the concentration of chitosan is 1%, the produced chitosan-oligosaccharide has 43% disaccharide or less and 5% disaccharide or more.
It was 7% (FIG. 5B). When the chitosan concentration was 3% or higher, disaccharide was 22%, trisaccharide was 34%, and tetrasaccharide was 44% (FIG. 5C). The chitosan-oligosaccharide produced by using the mycelium of Aspergillus fumigatus KB-1 used in the present invention produces almost no monosaccharide as compared with acid hydrolysis, and at a chitosan concentration of 3%, 78 % Was chitosan-oligosaccharide with 3 to 6 sugars.
【0035】[0035]
【表1】 [Table 1]
【0036】[0036]
【表2】 [Table 2]
【0037】[0037]
【発明の効果】本発明のアスペルギルスフミガーツス突
然変異菌は、キトサンのキトサン−オリゴ糖への分解に
優れる酵素を生産し、当該菌の菌糸体を使用し、キトサ
ンを分解すればキトサン−オリゴ糖の加水分解率が高
く、3乃至5%の高基質濃度でも大部分が3糖以上であ
った。本発明により、工程が簡単で経済的かつ環境汚染
問題を起こさずにキトサンからキトサン−オリゴ糖を製
造する方法が提供される。The Aspergillus fumigatus mutant bacterium of the present invention produces an enzyme excellent in the decomposition of chitosan into chitosan-oligosaccharide, and the mycelium of the bacterium is used to decompose chitosan-oligosaccharide. The sugar hydrolysis rate was high, and most of the sugars were 3 sugars or more even at a high substrate concentration of 3 to 5%. The present invention provides a method for producing chitosan-oligosaccharide from chitosan which is simple in process, economical, and does not cause environmental pollution problems.
【図1】図1は、アスペルギルスフミガーツスKB−1
のユビキノンシステムの薄層クロマトグラムを示す写真
である。FIG. 1 is an Aspergillus fumigatus KB-1.
2 is a photograph showing a thin-layer chromatogram of the ubiquinone system of FIG.
【図2】図2は、酵素活性と温度(横軸)との関係を示
す。FIG. 2 shows the relationship between enzyme activity and temperature (horizontal axis).
【図3】図3は、酵素活性とpH(横軸)との関係を示
す。FIG. 3 shows the relationship between enzyme activity and pH (horizontal axis).
【図4】図4は、キトサン濃度(横軸)とキトサン−オ
リゴ糖の生産との関係を示す。FIG. 4 shows the relationship between chitosan concentration (horizontal axis) and chitosan-oligosaccharide production.
【図5】図5Aは、(Glc1)、(Glc2)、(G
lc3)、(Glc4)、(Glc5)、(Glc6)
のHPLCのスタンダードを示す。図5Bは、キトサン
濃度1%のときの生産されたキトサンーオリゴ糖のHP
LCを示す。図5Cは、キトサン濃度3%のときの生産
されたキトサンーオリゴ糖のHPLCを示す。FIG. 5A shows (Glc1), (Glc2), and (Glc).
lc3), (Glc4), (Glc5), (Glc6)
Shows the HPLC standard. FIG. 5B shows the HP of the produced chitosan-oligosaccharide at a chitosan concentration of 1%.
LC is shown. FIG. 5C shows HPLC of chitosan-oligosaccharide produced at a chitosan concentration of 3%.
フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 FI 技術表示箇所 A01N 63/04 A01N 63/04 A A23L 3/3562 A23L 3/3562 A61K 31/73 ABD A61K 31/73 ABD ADU ADU ADX ADX (C12N 1/14 C12R 1:68) (C12N 9/24 C12R 1:68) (C12P 19/26 C12R 1:68) Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical display area A01N 63/04 A01N 63/04 A A23L 3/3562 A23L 3/3562 A61K 31/73 ABD A61K 31/73 ABD ADU ADU ADX ADX (C12N 1/14 C12R 1:68) (C12N 9/24 C12R 1:68) (C12P 19/26 C12R 1:68)
Claims (9)
る性質を持つ韓国科学技術院遺伝工学研究所において受
託番号KCTC0139BP号で寄託されたアスペルギ
ルスフミガーツス突然変異菌。1. An Aspergillus fumigatus mutant strain deposited under accession number KCTC0139BP at the Institute of Genetic Engineering, Korea Institute of Science and Technology, which has the property of degrading chitosan into chitosan-oligosaccharide.
る請求項1記載のアスペルギルスフミガーツス突然変異
菌が生産する酵素。2. The enzyme produced by the Aspergillus fumigatus mutant strain according to claim 1, which decomposes chitosan into chitosan-oligosaccharide.
ツス突然変異菌を培養して菌糸体を収穫し、得られた菌
糸体をキトサンと反応させることを特徴とするキトサン
−オリゴ糖の製造方法。3. A method for producing chitosan-oligosaccharide, which comprises culturing the Aspergillus fumigatus mutant bacterium according to claim 1 to harvest mycelium, and reacting the obtained mycelium with chitosan.
の培養が、蒸留水1リットルに対し、キトサン5乃至1
5g、酵母エキス0.25乃至1.0g、硝酸アンモニ
ウム0.5乃至2.0g、塩化ナトリウム0.25乃至
1.5g、リン酸第2カリウム0.5乃至2.0g、リ
ン酸第2ナトリウム2.0乃至5g、硫酸マグネシウム
0.25乃至1.0g、塩化カルシウム0.02乃至
0.1g、寒天10乃至20g、硝酸10mlからなる
培地で行われることを特徴とする請求項3記載のキトサ
ン−オリゴ糖の製造方法。4. The cultivation of Aspergillus fumigatus mutant bacterium in chitosan 5 to 1 per 1 liter of distilled water.
5 g, yeast extract 0.25 to 1.0 g, ammonium nitrate 0.5 to 2.0 g, sodium chloride 0.25 to 1.5 g, dibasic potassium phosphate 0.5 to 2.0 g, dibasic sodium phosphate 2 4. Chitosan according to claim 3, characterized in that it is carried out in a medium consisting of 0.0 to 5 g, magnesium sulfate 0.25 to 1.0 g, calcium chloride 0.02 to 0.1 g, agar 10 to 20 g, and nitric acid 10 ml. A method for producing an oligosaccharide.
の培養が液体培地で、温度20乃至30℃、70乃至1
50rpmで3乃至6日間の振盪培養であることを特徴
とする請求項3または4記載のキトサン−オリゴ糖の製
造方法。5. A culture of Aspergillus fumigatus mutant in a liquid medium at a temperature of 20 to 30 ° C. and 70 to 1
The method for producing a chitosan-oligosaccharide according to claim 3 or 4, wherein the shaking culture is performed at 50 rpm for 3 to 6 days.
乃至60℃で行われることを特徴とする請求項3乃至5
記載のキトサン−オリゴ糖の製造方法。6. The reaction between mycelium and chitosan is carried out at a temperature of 50.
6. The method according to claim 3, wherein the temperature is 60 to 60 ° C.
A method for producing the described chitosan-oligosaccharide.
0乃至5.0で行われることを特徴とする請求項3乃至
6記載のキトサン−オリゴ糖の製造方法。7. The reaction between mycelium and chitosan is carried out at pH 4.
The method for producing a chitosan-oligosaccharide according to claim 3, wherein the method is performed at 0 to 5.0.
乃至60分間であることを特徴とする請求項3乃至7記
載のキトサン−オリゴ糖の製造方法。8. The reaction time between the mycelium and chitosan is 10
8. The method for producing chitosan-oligosaccharide according to claim 3, wherein the method is for 60 to 60 minutes.
トサンの濃度が、1乃至5重量%であることを特徴とす
る請求項3乃至8記載のキトサン−オリゴ糖の製造方
法。9. The method for producing a chitosan-oligosaccharide according to claim 3, wherein the concentration of chitosan in the reaction between the mycelium and chitosan is 1 to 5% by weight.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1019950003713A KR0140430B1 (en) | 1995-02-24 | 1995-02-24 | Aspergillus fumigatus mutant strain and producing method of chitosan oligosaccharide |
KR1995/P/3713 | 1995-02-24 |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH08322554A true JPH08322554A (en) | 1996-12-10 |
JP2797081B2 JP2797081B2 (en) | 1998-09-17 |
Family
ID=19408765
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JP8061793A Expired - Fee Related JP2797081B2 (en) | 1995-02-24 | 1996-02-23 | Aspergillus fumigatus mutant bacterium and method for producing chitosan-oligosaccharide using the bacterium or enzyme producing the bacterium |
Country Status (2)
Country | Link |
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JP (1) | JP2797081B2 (en) |
KR (1) | KR0140430B1 (en) |
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-
1995
- 1995-02-24 KR KR1019950003713A patent/KR0140430B1/en not_active IP Right Cessation
-
1996
- 1996-02-23 JP JP8061793A patent/JP2797081B2/en not_active Expired - Fee Related
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JP2797081B2 (en) | 1998-09-17 |
KR960031592A (en) | 1996-09-17 |
KR0140430B1 (en) | 1998-07-01 |
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