JPH0387178A - Novel endodextrase, its production and production of isomaltooligosaccharide using the same enzyme - Google Patents
Novel endodextrase, its production and production of isomaltooligosaccharide using the same enzymeInfo
- Publication number
- JPH0387178A JPH0387178A JP1224161A JP22416189A JPH0387178A JP H0387178 A JPH0387178 A JP H0387178A JP 1224161 A JP1224161 A JP 1224161A JP 22416189 A JP22416189 A JP 22416189A JP H0387178 A JPH0387178 A JP H0387178A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- endodextranase
- dextran
- culture
- isomaltotriose
- 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.)
- Granted
Links
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
[産業上の利用分野]
本発明は新規エンドデキストラナーゼ、その製造方法及
び本酵素を用いたイソマルトオリゴ糖の製造方法に関す
る。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel endodextranase, a method for producing the same, and a method for producing isomalto-oligosaccharide using the present enzyme.
[従来の技術]
近年、各種オリゴ糖が食品工業界等で機能性食品素材と
して注目されている。これらのオリゴ糖の一種であるイ
ソマルトトリオースは、難う触性甘味剤、腸内のビフィ
ズス菌増殖因子、あるいは研究用試薬、また、保d性が
極めて高いので有効な保湿剤などとしての用途が期待出
来、その効率的な製造法の開発が甲まれている。[Prior Art] In recent years, various oligosaccharides have attracted attention as functional food materials in the food industry. Isomaltotriose, a type of these oligosaccharides, is used as a difficult-to-tactile sweetener, a growth factor for bifidobacteria in the intestines, a research reagent, and as an effective moisturizer due to its extremely high retention properties. The development of an efficient manufacturing method is highly anticipated.
イソマルトトリオースを製造する方法としては、従来よ
りデキストランを鉱酸で部分加水分解する方法が知られ
ているが、この方法ではイソマルトトリオースが選択的
に取得できず、その収率も極めて低い。また、精製工程
も煩雑であり、工業的方法としては適していなかった。As a method for producing isomaltotriose, a method of partially hydrolyzing dextran with mineral acid has been known, but with this method, isomaltotriose cannot be obtained selectively and the yield is extremely low. low. Furthermore, the purification process was complicated and was not suitable as an industrial method.
他の方法として、デキストランを加水分解し、主として
イソマルトトリオースを生成するデキストラナーゼを利
用した、酵素法によるイソマルトトリオースの製造も検
討されている。As another method, production of isomaltotriose by an enzymatic method using dextranase, which hydrolyzes dextran and mainly produces isomaltotriose, is also being considered.
デキストランを加水分解し、主としてイソマルトトリオ
ースを生成するデキストラナーゼとしては、ブレビバク
テリウム・フスクム(Brevibact。Brevibacterium fuscum (Brevibact) is an example of a dextranase that hydrolyzes dextran to mainly produce isomaltotriose.
erium fuscum var、 dextran
lyticum)が生産する細菌性エキソ型酵素[Bi
ochim、 肋泄□ Acta世、61〜70 f1
974)]が知られている。しがしながら、ブレビバク
テリウム起源のデキストラナーゼは、精製工程の煩雑さ
に加えて、酵素の回収率ならびに精製酵素の示す比活性
も低く、工業的生産には適していなかった、。erium fuscum var, dextran
Bacterial exo-enzyme [Bi
ochim, rib excretion □ Acta, 61-70 f1
974)] is known. However, dextranase originating from Brevibacterium was not suitable for industrial production due to the complicated purification process and low enzyme recovery rate and low specific activity of the purified enzyme.
[発明が解決しようとする課題]
本発明は、上記従来技術の問題点に鑑みてなされたちの
であり、その目的は、ブレビバクテリウム起源でない新
たなエンドデキストラナーゼ生産菌を利用することによ
り高力価の新規なエンドデキストラナーゼを提供するこ
と、」1記エンドデキストラナーゼ生産菌を用いてエン
ドデキストラナーゼを高収率で工業的に製造する方法を
提供すること、更に、上記エンドデキストラナーゼを用
いてイソマルトトリオースを主体としたイソマルトオリ
ゴ糖を効率的に製造する方法を提供することにある。[Problems to be Solved by the Invention] The present invention has been made in view of the problems of the prior art described above, and its purpose is to achieve high levels of endodextranase production by utilizing a new endodextranase-producing bacterium that is not derived from Brevibacterium. To provide a novel endodextranase with a high potency, 1) to provide a method for industrially producing endodextranase at a high yield using the endodextranase-producing bacterium; An object of the present invention is to provide a method for efficiently producing isomaltooligosaccharides mainly composed of isomaltotriose using dextranase.
[課題を解決するための手段]
本発明者らは、デキストランに作用し、王としてイソマ
ルトトリオースを生成する酵素を生産する微生物の検索
を行った結果、糸状菌フザリウム(Fusariuml
属に属するl菌株が、デキストランに作用して主として
イソマルトトリオースを生成する酵素を、効率よく菌体
外に大量生産することを認め、本発明を完成するに至っ
た。[Means for Solving the Problems] The present inventors conducted a search for microorganisms that produce an enzyme that acts on dextran and produces isomaltotriose as the king, and found that the filamentous fungus Fusarium
The present invention was completed based on the recognition that a strain belonging to the genus I can efficiently produce an enzyme that mainly produces isomaltotriose by acting on dextran in large quantities outside the bacterial body.
本菌株の菌学的性質を以下に述べる。The mycological properties of this strain are described below.
(1)各種培養培地における生育状態
■麦芽寒天培地
本菌の麦芽寒天培地上での生育速度は、25°C13日
間ではコロニーの直径が0.5〜1.5 cmに達し、
7日間では2.0〜3.0 cmに達する。性状は白色
・ビロード状であり、分生子の形成は良好である。(1) Growth status on various culture media ■ Malt agar medium The growth rate of this fungus on malt agar medium is that the colony diameter reaches 0.5 to 1.5 cm in 13 days at 25°C.
It reaches 2.0-3.0 cm in 7 days. It is white and velvety in appearance, and conidia are well formed.
■ポテト・スクロース寒天培地
本閑のポテト・スクロース寒天培地上での生育速度は、
25℃、3日間ではコロニーの直径が1.0〜2.0
cmに達し、7日間では3.0〜5.0cmに達する。■Potato sucrose agar medium Honkan's growth rate on potato sucrose agar medium is
At 25℃ for 3 days, the colony diameter was 1.0-2.0.
cm, and reaches 3.0 to 5.0 cm in 7 days.
性状は白色・ビロード状であり、分生子の形成は良好で
ある。It is white and velvety in appearance, and conidia are well formed.
■オートミール寒天培地
本閑のオートミール寒天培地上での生育速度は、25°
C13日間ではコロニーの直径が05〜1 cmに達し
、7日間では1.5〜2.0cmに達する。性状は白色
・ビロード状であり、分生子の形成は少ない。■Oatmeal agar medium Honkan's growth rate on oatmeal agar medium is 25°
The colony diameter reaches 0.5-1 cm in 13 days and 1.5-2.0 cm in 7 days. It is white and velvety in appearance, with few conidia formed.
(2)生理的性質
■生育pH
pl+4.5〜8.0のpl+領域で生育てきるが、生
育に至適なpHは5.0〜6.5である。(2) Physiological properties ■Growth pH It grows in the pl+ range of pl+4.5 to 8.0, but the optimum pH for growth is 5.0 to 6.5.
■15〜45℃の温度域で生育できるが、生育に至適な
温度は20〜30℃である。■It can grow in a temperature range of 15 to 45°C, but the optimal temperature for growth is 20 to 30°C.
■その他の卯著な特徴
デキストラン含有培地での培養ににす、エンドデキスト
ラナーゼを菌体外に誘導生産する。■Other notable features Endodextranase is induced and produced extracellularly when cultured in a dextran-containing medium.
(3)形態的特徴 ■分生子柄 分生子は側生である。(3) Morphological characteristics ■ Conidiophore Conidia are lateral.
■フィアライド モノフィアライドである。■Fear Ride It is a monophial ride.
■分生子
大型分生子と小型分生子を有する。大型分生子は25〜
32×4〜5ILmで、腕曲しており、ショルダーを有
する。小型分生子は5〜7×2〜3μmである6大を分
生子と小型分生子は0〜3個の隔壁を有する。■Conidia Contains large conidia and small conidia. Large conidia are 25~
It measures 32 x 4 to 5 ILm, has bent arms, and has shoulders. Small conidia have six large conidia that are 5-7 x 2-3 μm and small conidia have 0 to 3 septa.
以上の所見をもとに、「菌類図鑑上・下」、宇田用俊−
1椿啓介ばか著、講談社、1977年刊行などにより分
類し、本菌株をFusarium sp、と同定した。Based on the above findings, Illustrated Encyclopedia of Fungi Part 1 and Part 2, Yoshitoshi Uda
This strain was classified as Fusarium sp., written by Keisuke Tsubaki, published in 1977 by Kodansha, etc.
なお、上記菌株は、工業技術院微生物工業技術研究所に
、徹工研菌寄第10976号(FERMP−10976
)として寄託されている。The above strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology under the contract No. 10976 (FERMP-10976).
) has been deposited as.
本発明におけるエンドデキストラナーゼの製造方法の骨
子とするところは、」1記のような糸状菌フザリウム(
Fusariuml属に属するエンドデキストラナーゼ
生産菌またはその突然変異株を培養培地に接種し、所定
の培養条件下で培養した後、当該培養物から菌体を除去
し、簡便な分離・精製法により高力価のデキストラナー
ゼを高収率で採取するようにしたことにある。The gist of the method for producing endodextranase in the present invention is that the filamentous fungus Fusarium (
Endodextranase-producing bacteria belonging to the genus Fusarium or their mutant strains are inoculated into a culture medium and cultured under predetermined culture conditions.The cells are removed from the culture, and high-density The reason is that dextranase with a high titer can be collected at a high yield.
以下に、本発明で得られた新規エンドデキストラナーゼ
の諸性質について記・1反する。The properties of the novel endodextranase obtained by the present invention are described below.
(1)作用
本酵素はデキストランに作…し、主としてαイソマルト
トリオースを生成する。(1) Action This enzyme produces dextran and mainly produces α-isomaltotriose.
(2)基質特異性
本酵素は主として細菌性多糖デキストランを加水分解す
る。一方、可溶性デンプン、アミロペクチン、プルラン
、カードラン、ラミナリン、キシランなどには作用を示
さない。(2) Substrate specificity This enzyme mainly hydrolyzes bacterial polysaccharide dextran. On the other hand, it has no effect on soluble starch, amylopectin, pullulan, curdlan, laminarin, xylan, etc.
(3)至適pH及び安定pH範囲
本酵素はpH4,0〜11.5の広範囲で作用し、至適
pHは6.4付近にある( 0.25%ファルマシア社
製デキストランT2O00に30”C,7分間作用させ
た)。(3) Optimal pH and stable pH range This enzyme acts over a wide range of pH 4.0 to 11.5, and the optimal pH is around 6.4 (0.25% Pharmacia Dextran T2O00 at 30"C) , allowed to act for 7 minutes).
5 mM KCl−NaOH緩衝液中で、本酵素はpH
11,0においても約40%の相対活性発現を示した。In 5 mM KCl-NaOH buffer, the enzyme was adjusted to pH
11.0 also showed a relative activity expression of about 40%.
したがって、本酵素が高アルカリ領域においても高活性
を発現することが確認された。また、本酵素はpH4,
5〜10.0の広範囲で安定である(各種pHの25
mM McT]vaine緩衝液中で4℃、24時間放
置後、それぞれの残存活性を測定した)。Therefore, it was confirmed that this enzyme expresses high activity even in a highly alkaline region. In addition, this enzyme has a pH of 4,
Stable over a wide range of pH 5 to 10.0 (25
After standing in mM McT]vaine buffer at 4°C for 24 hours, the residual activity of each was measured).
(4)作用温度範囲及び至適作用温度
本酵素はほぼ20〜55℃の範囲で作用し、至適作用温
度は35℃付近である(0.25%デキストランT2O
00、pH6,4,5分間反応)。(4) Action temperature range and optimal action temperature This enzyme acts in the range of approximately 20 to 55°C, and the optimal action temperature is around 35°C (0.25% dextran T2O
00, pH 6, 4, 5 minute reaction).
(5)温度安定性
本酵素をpH6,4で、10分分間様温度で加熱処理後
、それぞれの残存活性を測定した。その結集、45℃ま
では100%、50”Cで約25%の残存活性を示し、
55℃でほぼ完全に失活した。(5) Temperature stability After heat treatment of the enzyme at pH 6.4 for 10 minutes, the residual activity of each enzyme was measured. Its concentration shows 100% residual activity up to 45°C and about 25% residual activity at 50"C.
It was almost completely inactivated at 55°C.
(6)酵素力価の測定法
エンドデキストラナーゼ活性測定における反応組成は1
%デキストラン丁2000溶液0.5 ml、25 m
MMcilvaine緩衝液(pH6,4) 1.0
mlおよび酵素溶液0.5 mlからなり、30℃で一
定時間反応させる。(6) Enzyme titer measurement method The reaction composition for endodextranase activity measurement is 1
% Dextran D2000 solution 0.5 ml, 25 m
MMcilvaine buffer (pH 6,4) 1.0
ml and 0.5 ml of enzyme solution, and reacted at 30°C for a certain period of time.
反応混液1.Omlにつき、酵素反応により生成された
還元糖をSomogyi−Nelson法を用いて定量
する。Reaction mixture 1. The reducing sugar produced by the enzymatic reaction is quantified per Oml using the Somogyi-Nelson method.
この条件下で1分間にILLmolのグルコースに相当
する還元力を生成せしめる酵素量を1単位と定義する。Under these conditions, the amount of enzyme that generates a reducing power equivalent to ILL mol of glucose per minute is defined as 1 unit.
(7)エンドデキストラナーゼの精製方法 0
Fusarium sp、を液体培地にて25°C,5
日間ジャーファメンターを用いて培養し、その培養液3
℃を高速冷却遠心(8,000X H)にかけ、除菌す
る。4℃で、」二清液に冷エタノールを加え、30〜6
0%濃度で沈澱する画分を高速冷却遠心(8,000x
g )にかけて集めた。次いて、このエタノール沈澱
物を直ちに、25 mM McIlvajne緩衝液(
pH6,4)に溶解し、同」二緩衝液で充分に平衡化し
たDEAE−1−ヨバール650Sを充填したカラム(
4,4X 55 cm)にのせた。100 mM Mc
Ilvaine緩衝液(pH6,])にて溶出される活
性画分を集めた。この両分を濃縮・透析後、25mM
McTlvaine緩衝液(pH6,4)で充分に平衡
化したバイオゲルP−100を充填したカラム(+、8
x 1’20 cm )にかけ、ゲル濾過を行った。(7) Endodextranase purification method 0 Fusarium sp. in a liquid medium at 25°C, 5
Culture using a jar fermenter for 1 day, and use the culture solution 3
℃ and high-speed refrigerated centrifugation (8,000×H) to sterilize. At 4°C, add cold ethanol to the two-seed liquid and incubate at 30-6
The fraction precipitated at 0% concentration was subjected to high-speed refrigerated centrifugation (8,000x
g) and collected. The ethanol precipitate was then immediately dissolved in 25 mM McIlvajne buffer (
A column packed with DEAE-1-Yobal 650S dissolved in pH 6.4 and sufficiently equilibrated with the same buffer (pH 6.4) was used.
4.4 x 55 cm). 100mM Mc
The active fraction eluted with Ilvaine buffer (pH 6) was collected. After concentrating and dialyzing both of these, 25mM
A column (+, 8
x 1'20 cm) and gel filtration was performed.
得られた単一の活性画分を集め、濃縮し、高度に精製さ
れたエンドデキストラナーゼを得た。The resulting single active fraction was collected and concentrated to obtain highly purified endodextranase.
(8)分子量
5DS−ポリアクリルアミドゲル電気泳動(SOSPA
GE)法により推定されたエンドデキストラナ1
ゼの分子量は約88.000である。(8) Molecular weight 5DS-polyacrylamide gel electrophoresis (SOSPA)
The molecular weight of endodextranase estimated by the GE method is approximately 88,000.
(9)等電点(pT)
1、、 K r3社製等電点雷気泳動袈置(110ml
用カラム)により測定された精製エンドデキストラナー
ゼの等電点は4.50である。(9) Isoelectric point (pT) 1, Isoelectric focusing lightning phoresis rack (110ml) manufactured by Kr3
The isoelectric point of purified endodextranase, measured using a column for sterilization, is 4.50.
(10)阻害及び活性化
5 mMの水銀、銀、鉄、銅などの重金属イオンにより
、またこれと同一濃度のN−ブロモスクシンイミドなど
により該酵素の活性が阻害される。(10) Inhibition and Activation The activity of the enzyme is inhibited by 5 mM of heavy metal ions such as mercury, silver, iron, copper, etc., and by the same concentration of N-bromosuccinimide.
一方、5 mMのカルシウムイオンにより該酵素の活性
が賦活される。On the other hand, the activity of the enzyme is activated by 5 mM calcium ions.
(11)アミノ酸組成(モル比%)
アルギニン4.24、リジン4.31. ヒスチジン2
、■5、フェニルアラニン4.04、チロシン373、
ロイシン6.82、インロイシン5,87、メチオニン
047、バリン7.56、アラニン9.16、グリシン
8.69、プロリン6.34、グルタミン酸8.08、
セリン6.5+、スレオニン7.31.アスパラギン酸
12.26、トリプトファン1.94、シスチン0.5
2次に、当該エンドデキストラナーゼを生産せし2
める為の培養方法について述べる。(11) Amino acid composition (mole ratio %) Arginine 4.24, Lysine 4.31. histidine 2
,■5, Phenylalanine 4.04, Tyrosine 373,
Leucine 6.82, inleucine 5.87, methionine 047, valine 7.56, alanine 9.16, glycine 8.69, proline 6.34, glutamic acid 8.08,
Serine 6.5+, threonine 7.31. Aspartic acid 12.26, tryptophan 1.94, cystine 0.5
2 Next, a culture method for producing the endodextranase will be described.
デキストラナーゼを製造するためには、前記した糸状菌
FusΩriom sp などの糸状菌フザリウム(
Fusariuml属に属するエンドデキストラナーゼ
生産菌またはその突然変異株を、培養培地に接種して培
養する。水閘の培養方法については、液体培養おJ:び
固形倍谷のいずれにおいてb可能であるが、工業的生産
には好気的に液体培養するのが好ましい。In order to produce dextranase, the filamentous fungus Fusarium (such as the above-mentioned filamentous fungus FusΩriom sp.
An endodextranase-producing bacterium belonging to the genus Fusarium or a mutant strain thereof is inoculated into a culture medium and cultured. Regarding the cultivation method of water spores, either liquid culture or solid-state culture is possible, but aerobic liquid culture is preferable for industrial production.
当該菌の培地の栄養源は、一般に微生物培養に用いられ
るものを使用することが可能である。また、細菌性多糖
デキストランを唯一の炭素源に用いると、当該エンドデ
キストラナーゼを誘導生成せしめることができる。As the nutrient source for the culture medium of the bacteria, those commonly used for culturing microorganisms can be used. Furthermore, when bacterial polysaccharide dextran is used as the sole carbon source, the endodextranase can be induced to be produced.
窒素源としては、有機窒素含有物として各種アミノ酸、
肉エキス、ペプトン、マルトエキス、尿素などが、また
、無機窒素化合物としては塩化アンモニウム、リン酸ア
ンモニウム、硝酸アンモニウム、硫酸アンモニウムなど
が単独または組合わせて用いることができる。その他、
無機塩類、ビ 3
クミン類などを適時添加するとよい。As nitrogen sources, various amino acids, organic nitrogen-containing substances,
Meat extract, peptone, malt extract, urea, etc. can be used, and as inorganic nitrogen compounds, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonium sulfate, etc. can be used alone or in combination. others,
It is recommended to add inorganic salts, bicumins, etc. as appropriate.
培養温度は20〜30°Cが適しており、より好ましく
は24〜27℃がよい。培養開始時のpHは4.5〜8
.0が適しているが、より好ましくはpH5,0〜6.
5がよい。培養日数は5〜7日間が好ましい。A suitable culture temperature is 20 to 30°C, more preferably 24 to 27°C. pH at the start of culture is 4.5-8
.. pH 0 is suitable, but pH 5.0 to 6.0 is more preferable.
5 is good. The number of days for culturing is preferably 5 to 7 days.
上記のような培養条件下で、充分にデキストラナーゼを
産生せしめた培養物から、遠心分離により菌体を除去す
れば、粗酵素液が得られる。A crude enzyme solution can be obtained by removing bacterial cells by centrifugation from a culture that has sufficiently produced dextranase under the above culture conditions.
このようにして得られた粗酵素液は、そのままでも製品
として充分に使用可能であるが、さらに粗酵素液を公知
の分離・精製方法、例えば限外濾過膜濃縮法、エタノー
ルなどによる有機溶媒沈澱法、硫酸アンモニウムなどに
よる塩析法、カラムクロマト分画法、アフィニティーク
ロマト分画法、等電点電気泳動法などを、単独あるいは
適宜組合わせることによって、当該エンドデキストラナ
ーゼの精製酵素液を採取することが可能である。次にそ
の一例を示す。The crude enzyme solution obtained in this way can be used as a product as it is, but the crude enzyme solution can be further processed by known separation and purification methods, such as ultrafiltration membrane concentration method, organic solvent precipitation with ethanol, etc. A purified enzyme solution of the endodextranase is collected by a method such as salting out method using ammonium sulfate, column chromatography fractionation method, affinity chromatography fractionation method, isoelectric focusing method, etc. alone or in an appropriate combination. Is possible. An example is shown below.
まず、培養液中の菌体等を遠心分離で除去し、無細胞上
清液を得る。この上清液を粗酵素として4
利用することも可能である。この」二清液に冷エタノー
ルを加え、30〜60%濃度で沈澱する両分を25mM
Mcrlvaine緩衝液(pH6,4)に溶解し、
r] E A Eトヨパール650Sカラムにのセる。First, bacterial cells and the like in the culture solution are removed by centrifugation to obtain a cell-free supernatant. It is also possible to use this supernatant as crude enzyme. Add cold ethanol to this two-separate solution to precipitate at a concentration of 30-60% to 25mM.
Dissolved in Mcrlvaine buffer (pH 6,4),
r] E A E Toyopearl 650S column.
IF]lImMMcI]vainc緩衝液(pH6,1
)で溶出される活性画分をバイオゲルP−100カラム
でゲル濾過を行い、精製エンドデキストラナーゼを得る
。IF]lImMMcI]vainc buffer (pH 6,1
) is subjected to gel filtration using a Biogel P-100 column to obtain purified endodextranase.
本発明のイソフル1−オリゴ糖の製造方決は、こうして
得られたエンドデキストラナーゼを、デキストランに作
用させて、イソマルトトリオースを主体とするイソマル
トオリゴ糖を生成させることを特徴としている。この場
合、エンドデキストラナーゼは、前記のようにして培養
された培養物として、または培養物から調製された粗酵
素液として、さらには精製された酵素として、デキスト
ランに作用させることができる。The method for producing isoflu-1-oligosaccharide of the present invention is characterized in that the endodextranase thus obtained is allowed to act on dextran to produce isomalto-oligosaccharide mainly composed of isomaltotriose. In this case, endodextranase can act on dextran as a culture cultured as described above, as a crude enzyme solution prepared from the culture, or further as a purified enzyme.
このようにして生成されたエンドデキストラナーゼを用
い、イソマルトトリオースを製造するための酵素反応は
、好ましくは30〜50℃、p+(5,0〜8,0、反
応時間は4〜18時間、デキストランにこ度は 5
2〜8%で行うとよい。反応液中の生成糖組成はイソマ
ルトテトラオース以上のもの約30%、イソマルトトリ
オース約60%、イソマルトース約7%、グルコース約
3%であった。The enzyme reaction for producing isomaltotriose using the endodextranase thus produced is preferably carried out at 30-50°C, p+ (5,0-8,0, reaction time 4-18 The reaction time and dextran concentration are preferably 52 to 8%.The composition of the produced sugars in the reaction solution is approximately 30% isomaltotetraose or higher, approximately 60% isomaltotriose, approximately 7% isomaltose, Glucose was about 3%.
また、予めデキストランT2O00を希塩酸などで部分
加水分解したものに、本精製エンドデキストラナーゼを
作用させると、短時間内にイソマルトトリオースを主体
としたイソマルトオリゴ糖を効率よく生成・取得できる
ことが判明した。Additionally, if the purified endodextranase is applied to dextran T2O00 which has been partially hydrolyzed with dilute hydrochloric acid etc., isomalto-oligosaccharides mainly consisting of isomaltotriose can be efficiently produced and obtained within a short period of time. found.
[実施例]
次に、以下の実施例にJ:す、木兄IVlを具体的に説
明する。[Example] Next, J:Su, Kinai IVl will be specifically explained in the following example.
実施例1 粗酵素液の調製1
10% デキストラン(8糖70) 、0.2%NH4
N0.、O1%KH□PO4,0,05%ugso4
・7H□0.0.04%(:aC1z2H20,0,3
% ポリペプトン、0.3% イーストエクストラクト
からなる培養培地(pH5,513℃を、 5Cのジャ
ーファーメンタ−に仕込み、糸状菌Fusarium
sp、を1.1 vvll、110 rrlmにて25
℃、5日間培養した。培養後、培養液を高速冷却連続遠
心6
分離にかけて菌体を除去し、無細胞上清液を得た。この
」二清液は5.2Qt位/mlのデキストラナーゼを含
有していた。本活性は前述したブレビバクテリウム属の
生産するデキストラナーゼ(42単位/mll と比較
して明らかに高い。Example 1 Preparation of crude enzyme solution 1 10% dextran (octasaccharide 70), 0.2% NH4
N0. , O1%KH□PO4,0,05%ugso4
・7H□0.0.04% (:aC1z2H20,0,3
A culture medium (pH 5, 513°C) consisting of % polypeptone and 0.3% yeast extract was placed in a jar fermenter at 5C.
sp, 1.1 vvll, 25 at 110 rrlm
The cells were cultured at ℃ for 5 days. After culturing, the culture solution was subjected to high-speed cooling continuous centrifugation to remove bacterial cells and obtain a cell-free supernatant. This second serum liquid contained dextranase at approximately 5.2 Qt/ml. This activity is clearly higher than the dextranase (42 units/ml) produced by Brevibacterium mentioned above.
実施例2 粗酵素液の調製2
実施例1で得られた除菌液を30〜60%濃度の冷エタ
ノールで処理し、生した沈澱を25 mMMcI 1v
ai ne緩衝液(pH6,41に溶解せしめ、粗酵素
液50m1を得た。この酵素液は58.1単位/mlの
エンドデキストラナーゼ活性を含自していた。Example 2 Preparation of crude enzyme solution 2 The sterilizing solution obtained in Example 1 was treated with cold ethanol at a concentration of 30-60%, and the resulting precipitate was treated with 25 mM McI 1v.
aine buffer (pH 6.41) to obtain 50 ml of a crude enzyme solution. This enzyme solution contained 58.1 units/ml of endodextranase activity.
実施例3 酵素の精製
実施例2で得られた+11Ql素液のうち、48.4
mlを25 mM McTIvainc i3衝TJA
I (I’ll [i、 41 て充分にit’衡化し
たDEAE−1−ヨパール650Sを充填したカラム(
4,4x 55 cml にのせ、本酵素を吸着せしめ
た後、Inn mM McTIvainc緩fIi液(
pHli、Il にて溶出。Example 3 Enzyme Purification Of the +11Ql stock solution obtained in Example 2, 48.4
ml of 25 mM McTIvainc i3-TJA
I'll [i, 41 A column packed with DEAE-1-Yopal 650S that has been sufficiently equilibrated with
After adsorbing the enzyme on 4.4x 55 cml, InnmM McTIvainc weak flI solution (
Elute at pHli, Il.
される活性画分を集めた。この活性画分を濃縮・這析後
、25 mM Mcllvair+a 緩衝液(pI)
ら4)で充分に平衡化したバイオゲルP−100を充填
したカラ7
ム(1,8x 120 cmlにのぜ、ゲル濾過を行
った。The active fractions were collected. After concentrating and separating this active fraction, 25 mM Mcllvair+a buffer (pI)
Gel filtration was performed using a column (1.8 x 120 cml) filled with Biogel P-100, which had been sufficiently equilibrated as described in et al. 4).
得られた単一の活性画分を集め、コロジオンバッグで濃
縮し、精製エンドデキストラナーゼを得た。本精製酵素
について、ポリアクリルアミドゲル電気泳動(PAGE
)およびSO3−ポリアクリルアミドゲル電気泳動(S
DS−PAGE)を行ったところ、それぞれ単一のタン
パク質染色バンドのみが認められた。これらの精製操作
によって、本エンドデキストラナーゼが電気泳動的に均
一な標品にまで精製されたことが明らかとなった。The resulting single active fraction was collected and concentrated in a collodion bag to obtain purified endodextranase. This purified enzyme was analyzed by polyacrylamide gel electrophoresis (PAGE).
) and SO3-polyacrylamide gel electrophoresis (S
When DS-PAGE) was performed, only a single protein staining band was observed in each case. Through these purification operations, it was revealed that the present endodextranase was purified to an electrophoretically homogeneous standard.
以上の酵素精製過程に伴う総タンパク質量、酵素の総活
性量、活性回収率、比活性等の変化を第1表に示ず。こ
の表に示されるように、3℃の培養土清液より、本酵素
の精製標品16.Omgを活性回収率86.7%で得た
。本精製酵素標品の力価(比活性)は152単位/mg
タンパク質であった。また、本精製酵素は前記の性質を
有していた。Table 1 does not show changes in the total protein amount, total enzyme activity amount, activity recovery rate, specific activity, etc. due to the above enzyme purification process. As shown in this table, 16. Omg was obtained with an activity recovery of 86.7%. The titer (specific activity) of this purified enzyme preparation is 152 units/mg
It was protein. Moreover, this purified enzyme had the above-mentioned properties.
(以下余白)
8
実施例4
10αのデキストランT2O00(ファルマシア社製)
を12.5 mM Mcllvaine緩衝液(pH6
,4125(] mmに溶解し、Fusarium s
p、起源のエンドデキストラナーゼを基質1mg当り0
.085単位添加し、35℃で30分間、1.3,6.
18.27時間それぞれ反応を行った。反応液中の生成
糖組成の経時的な変化を高速液体クロマトグラフィーに
より分析した結果を第1図に示す。反応時間30分にお
ける生成糖組成は、イソマルトテトラオース以上のもの
約47.8%、イソマルトトリオース約51%、イソマ
ルトース約1%、グルコース約0.2%であった。さら
に、反応時間6時間における生成糖組成は、イソマルト
テトラオース以上のもの約33%、イソマルトトリオス
約61%、イソマルトース約4%、グルコース約2%の
分布を示した。これ以後、反応時間が経過するにつれイ
ソマルトトリオースの収率は若干低下する傾向がみられ
た。なお、高速液体クロマトグラフィーの条件は以下の
如くである。(Left below) 8 Example 4 10α dextran T2O00 (manufactured by Pharmacia)
in 12.5 mM Mcllvaine buffer (pH 6).
, 4125 (] mm, Fusarium s
p, the original endodextranase per mg of substrate
.. 085 units were added and heated at 35°C for 30 minutes, 1.3, 6.
Reactions were carried out for 18.27 hours, respectively. Figure 1 shows the results of analysis of changes in the composition of sugar produced in the reaction solution over time by high performance liquid chromatography. The composition of the produced sugar at a reaction time of 30 minutes was approximately 47.8% isomaltotetraose or higher, approximately 51% isomaltotriose, approximately 1% isomaltose, and approximately 0.2% glucose. Further, the composition of the produced sugar after a reaction time of 6 hours showed a distribution of about 33% isomaltotetraose or higher, about 61% isomaltotriose, about 4% isomaltose, and about 2% glucose. Thereafter, the yield of isomaltotriose tended to decrease slightly as the reaction time progressed. Note that the conditions for high performance liquid chromatography are as follows.
カラム: Am1nex HPX−42A(7,8mm
φx 300 mm10
移動相:脱イオン水
流速−0,6ml/min
カラム圧: 30 kg/cm”
カラム温度ニア0℃
検出器:示差屈折計、■品性製作所製、商品名rRID
−6AJ
分岐オリゴ糖はデキストランの酸による部分加水分解物
を標準物質として、ペーパークロマトグラフィーおよび
高速液体クロマトグラフィーにより構造確認を行ったと
ころ、すべてα−1,6−グルコシド結合からなるイソ
マルトオリゴ糖であった。Column: Am1nex HPX-42A (7,8mm
φx 300 mm10 Mobile phase: Deionized water flow rate -0.6 ml/min Column pressure: 30 kg/cm" Column temperature near 0°C Detector: Differential refractometer, manufactured by Kansei Seisakusho, product name rRID
-6AJ The structure of the branched oligosaccharide was confirmed by paper chromatography and high performance liquid chromatography using the acid partial hydrolyzate of dextran as a standard substance, and it was found that all of the branched oligosaccharides were isomalto-oligosaccharides consisting of α-1,6-glucosidic bonds. there were.
実施例5
デキストランT2O00(ファルマシア社製) 5.0
gを0.2N塩酸80 mlに溶解し、95°Cで4
時間加水分解した後に2N水酸化ナトリウムを添加して
、pH6,1に調整した。次いで、脱イオン水を加えて
、全量を125 mlとした。上記したデキストランの
部分加水分解物1mg当り精製エンドデキストラナーゼ
0.085単位を添加し、35℃で30分間、■、3.
6.18時間それぞれ反応を行った。反応液中の生成糖
組成の経時的な変化を高速液体クロマトグラフィーによ
り分析した。反応時間30分における生成糖組成は、イ
ソマルトテトラオース以上のもの約19%、イソマルト
トリオース約51%、イソマルトース約15%、グルコ
ース約15%であった。さらに、反応時間3時間におけ
る生成糖組成は、イソマルトテトラオース以上のもの約
8%、イソマルトトリオース約60%、イソマルトース
約15%、グルコース約17%の分布を示した。Example 5 Dextran T2O00 (manufactured by Pharmacia) 5.0
Dissolve g in 80 ml of 0.2N hydrochloric acid and incubate at 95°C for 4 hours.
After hydrolysis for a period of time, 2N sodium hydroxide was added to adjust the pH to 6.1. Deionized water was then added to bring the total volume to 125 ml. 1. Add 0.085 units of purified endodextranase per 1 mg of the above-mentioned dextran partial hydrolyzate and heat at 35°C for 30 minutes.
Reactions were carried out for 6.18 hours. Changes in the sugar composition produced in the reaction solution over time were analyzed using high performance liquid chromatography. The composition of the produced sugar at a reaction time of 30 minutes was about 19% isomaltotetraose or higher, about 51% isomaltotriose, about 15% isomaltose, and about 15% glucose. Furthermore, the composition of the produced sugar after a reaction time of 3 hours showed a distribution of about 8% isomaltotetraose or higher, about 60% isomaltotriose, about 15% isomaltose, and about 17% glucose.
なお、高速液体クロマトグラフィーの実施条件は実施例
4の場合と同一である。Note that the conditions for performing high performance liquid chromatography are the same as in Example 4.
実施例6
1O%シヨ糖、0.05%MgSO4・7H70,0,
2%に2HP04.1%ポリペプトンからなる液体培地
(pH7,0,121に、う蝕原性連鎖球菌(Stre
tococcusmutansl を接種し、37℃
にて5日間静置培養する。培養液中に生成された不溶性
物質を濾別し、脱イオン水にて充分洗浄した。残渣の懸
濁液(約80 mllをロータリーエバポレーターを用
いて減圧ン り
濃縮しく60”C1,凍結乾燥を施した。これら一連の
操作を経て、1℃の培養液より不溶性グルカンの凍結乾
燥標品的1.2gを得た。Example 6 10% sucrose, 0.05% MgSO4.7H70.0,
A liquid medium (pH 7.0, 121) consisting of 2% 2HP, 4.1% polypeptone, and cariogenic streptococci (Streptococcus
tococcus mutansl and incubated at 37°C.
Culture for 5 days. Insoluble substances produced in the culture solution were filtered off and thoroughly washed with deionized water. The suspension of the residue (approximately 80 ml) was concentrated under reduced pressure using a rotary evaporator and lyophilized to 60"C1. Through this series of operations, a lyophilized sample of insoluble glucan was obtained from the 1°C culture solution. 1.2 g of target was obtained.
上記不溶性グルカンを02%含有する25 mMMcT
lvaine緩衝液(pH6,412,4mlに、Fu
sariumsp 起源の精製エンドデキストラナー
ゼ(8液(2,5単位)を100μm加え、37℃で反
応させた。この反応条件下で経時的に生成される還元糖
をSomogyi−Nelson法を用いて定量した。25 mM McT containing 0.2% of the above insoluble glucan
lvaine buffer (pH 6,412, 4 ml, Fu
100 μm of purified endodextranase (8 liquids (2.5 units)) originating from Sarium sp was added and reacted at 37°C. Reducing sugars produced over time under this reaction condition were quantified using the Somogyi-Nelson method. did.
ま゛た、不溶性グルカン中の全糖をフェノール硫酸法で
定量し、不溶性グルカンの分解率を算出した。Additionally, the total sugar in the insoluble glucan was determined by the phenol-sulfuric acid method, and the decomposition rate of the insoluble glucan was calculated.
得られた結果を総合すると、Fusarium sp、
起源のエンドデキストラナーゼは、S L r e f
、o c o c c u smutansの産生する
不溶性グルカンを約25%分解することが確認された。Combining the obtained results, Fusarium sp.
The endodextranase of origin is S L r e f
It was confirmed that about 25% of the insoluble glucan produced by O. c.
[発明の効果]
以上説明したように、本発明の一つによれば、糸状菌F
usarium sp、の産生する高力価の新規エンド
デキストラナーゼを提供することができる。[Effect of the invention] As explained above, according to one aspect of the present invention, filamentous fungi F
A novel high-titer endodextranase produced by P. usarium sp.
また、本発明のもう一つによれば、糸状菌フザ3
リウム(Fusariuml属に属するエンドデキスト
ラナーゼ生産菌またはその突然変異株を培養して、培養
物中からエンドデキストラナーゼを高収率で工業的に得
ることができる。According to another aspect of the present invention, an endodextranase-producing bacterium belonging to the genus Fusarium (Fusarium genus) or a mutant strain thereof is cultured to produce endodextranase from the culture in high yield. can be obtained industrially.
更に、本発明のもう一つによれば、上記エンドデキスト
ラナーゼをデキストランに作用させて、イソマルトトリ
オースを主体とするイソマルトオリゴ糖を効率的に生成
させることができる。Furthermore, according to another aspect of the present invention, isomalto-oligosaccharides mainly composed of isomaltotriose can be efficiently produced by allowing the endodextranase to act on dextran.
このイソマルトオリゴ糖は、難う触性甘味料、保況剤、
または腸内のビフィズス菌を活性化する因子などとして
、食品工業、化粧品工業等で有効に利用し得るちのであ
る。This isomalto-oligosaccharide is used as a hard-to-tactile sweetener, preservative,
It can also be effectively used in the food industry, cosmetics industry, etc. as a factor that activates bifidobacteria in the intestine.
更に、本発明のエンドデキストラナーゼは、5tre
tococcus mutansの産生する不溶性グル
カン分解能力をも有するので、歯磨き剤、義歯洗浄剤、
口臭除去剤などの口腔衛生関連商品への利用も可能であ
る。Furthermore, the endodextranase of the present invention has 5tre
It also has the ability to decompose insoluble glucans produced by tococcus mutans, so it can be used in toothpastes, denture cleaners,
It can also be used in oral hygiene-related products such as bad breath removers.
第1図は2%デキストランT2O00溶液を基質として
、精製エンドデキストラナーゼを作用させた 4
場合の生成糖組成の経時変化を示す図である。FIG. 1 is a graph showing changes over time in the sugar composition produced when purified endodextranase was allowed to act on a 2% dextran T2O00 solution as a substrate.
Claims (1)
エンドデキストラナーゼ。 (1)作用 本酵素はデキストランをエンド様式で加水分解し、主と
してα−イソマルトトリオースを生成する。 (2)基質特異性 本酵素は主として細菌性多糖デキストランを加水分解す
る。 (3)至適pH及び安定pH範囲 本酵素はpH4.0〜11.5の広範囲で作用し、至適
pHは6.4付近にある。また、pH4.5〜10.0
の範囲で安定である。 (4)作用温度範囲及び至適作用温度 本酵素は温度20〜55℃の範囲で作用し、至適作用温
度は35℃付近である。 (5)温度安定性 本酵素をpH6.4で、10分間各種温度で加熱処理す
ると、45℃までは100%、50℃で約25%の残存
活性を示し、55℃でほぼ完全に失活する。 (6)分子量 本酵素の分子量は約88,000である。 (7)等電点(p¥I¥) 本酵素の等電点は4.50である。 (8)アミノ酸組成(モル比%) アルギニン4.24、リジン4.31、ヒスチジン2.
15、フェニルアラニン4.04、チロシン3.73、
ロイシン6.82、イソロイシン5.87、メチオニン
0.47、バリン7.56、アラニン9.16、グリシ
ン8.69、プロリン6.34、グルタミン酸8.08
、セリン6.51、スレオニン7.31、アスパラギン
酸12.26、トリプトファン1.94、シスチン0.
522、糸状菌フザリウム(¥Fusarium¥)属
に属するエンドデキストラナーゼ生産菌またはその突然
変異株を培養培地に接種して、所定の培養条件下で培養
し、該培養物からエンドデキストラナーゼを採取するこ
とを特徴とするエンドデキストラナーゼの製造方法。 3、請求項2記載の方法によって得られたエンドデキス
トラナーゼを、デキストランに作用させて、イソマルト
トリオースを主体とするイソマルトオリゴ糖を生成させ
ることを特徴とするイソマルトオリゴ糖の製造方法。 4、前記エンドデキストラナーゼを、この酵素を含む培
養物、この培養物から調製された粗酵素液、または精製
酵素として作用させる請求項3記載のイソマルトオリゴ
糖の製造方法。[Claims] 1. A novel endodextranase characterized by having the following physicochemical properties. (1) Action This enzyme hydrolyzes dextran in an endo-mode to mainly produce α-isomaltotriose. (2) Substrate specificity This enzyme mainly hydrolyzes bacterial polysaccharide dextran. (3) Optimal pH and stable pH range This enzyme acts over a wide range of pH 4.0 to 11.5, and the optimal pH is around 6.4. Also, pH 4.5 to 10.0
It is stable within the range of . (4) Action temperature range and optimum action temperature This enzyme acts in a temperature range of 20 to 55°C, and the optimum action temperature is around 35°C. (5) Temperature stability When this enzyme is heated at pH 6.4 for 10 minutes at various temperatures, it shows 100% residual activity up to 45°C, about 25% at 50°C, and is almost completely inactivated at 55°C. do. (6) Molecular weight The molecular weight of this enzyme is approximately 88,000. (7) Isoelectric point (p\I\) The isoelectric point of this enzyme is 4.50. (8) Amino acid composition (mole ratio %) Arginine 4.24, Lysine 4.31, Histidine 2.
15, phenylalanine 4.04, tyrosine 3.73,
Leucine 6.82, Isoleucine 5.87, Methionine 0.47, Valine 7.56, Alanine 9.16, Glycine 8.69, Proline 6.34, Glutamic acid 8.08
, serine 6.51, threonine 7.31, aspartic acid 12.26, tryptophan 1.94, cystine 0.
522, an endodextranase-producing bacterium belonging to the filamentous fungus Fusarium genus or a mutant strain thereof is inoculated into a culture medium, cultured under predetermined culture conditions, and endodextranase is extracted from the culture. A method for producing endodextranase, which comprises collecting the endodextranase. 3. A method for producing isomalto-oligosaccharides, which comprises allowing the endodextranase obtained by the method according to claim 2 to act on dextran to produce isomalto-oligosaccharides mainly composed of isomaltotriose. 4. The method for producing isomalto-oligosaccharide according to claim 3, wherein the endodextranase is made to act as a culture containing this enzyme, a crude enzyme solution prepared from this culture, or a purified enzyme.
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JP1224161A JP2959777B2 (en) | 1989-08-30 | 1989-08-30 | Novel endodextranase, method for producing the same, and method for producing isomaltooligosaccharide using the enzyme |
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JP1224161A JP2959777B2 (en) | 1989-08-30 | 1989-08-30 | Novel endodextranase, method for producing the same, and method for producing isomaltooligosaccharide using the enzyme |
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JPH0387178A true JPH0387178A (en) | 1991-04-11 |
JP2959777B2 JP2959777B2 (en) | 1999-10-06 |
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JP1224161A Expired - Fee Related JP2959777B2 (en) | 1989-08-30 | 1989-08-30 | Novel endodextranase, method for producing the same, and method for producing isomaltooligosaccharide using the enzyme |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107674839A (en) * | 2017-10-31 | 2018-02-09 | 广西鼎乐生物科技有限公司 | A kind of method of Fusarium solani and its fermenting and producing dextranase |
-
1989
- 1989-08-30 JP JP1224161A patent/JP2959777B2/en not_active Expired - Fee Related
Cited By (2)
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CN107674839A (en) * | 2017-10-31 | 2018-02-09 | 广西鼎乐生物科技有限公司 | A kind of method of Fusarium solani and its fermenting and producing dextranase |
CN107674839B (en) * | 2017-10-31 | 2020-08-25 | 广西鼎乐生物科技有限公司 | Fusarium solani and method for producing dextranase by fermenting fusarium solani |
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JP2959777B2 (en) | 1999-10-06 |
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