JPS63164885A - Production of polygalactosamine-decomposition enzyme - Google Patents
Production of polygalactosamine-decomposition enzymeInfo
- Publication number
- JPS63164885A JPS63164885A JP30858086A JP30858086A JPS63164885A JP S63164885 A JPS63164885 A JP S63164885A JP 30858086 A JP30858086 A JP 30858086A JP 30858086 A JP30858086 A JP 30858086A JP S63164885 A JPS63164885 A JP S63164885A
- Authority
- JP
- Japan
- Prior art keywords
- polygalactosamine
- enzyme
- decomposition enzyme
- pseudomonas
- range
- 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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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
本発明は新規なポリガラクトサミン分解酵素(ポリガラ
クトサミニダーゼ)を製造する方法に関するものである
。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a novel polygalactosamine-degrading enzyme (polygalactosaminidase).
更に詳細には1本発明は、ポリガラクトサミンを加水分
解してオリゴガラクトサミンを生成する新規なポリガラ
クトサミン分解酵素を製造する方法に関するものである
。More specifically, the present invention relates to a method for producing a novel polygalactosamine-degrading enzyme that hydrolyzes polygalactosamine to produce oligogalactosamine.
一般に、微生物の生産するポリガラクトサミン(ポリα
−1,4ガラクトサミノガラクタン)としては、pFl
ol (特公昭56−12639)及びPF102 (
特願昭61−134799)が認知されている程度であ
る。In general, polygalactosamine (poly-alpha) produced by microorganisms is
-1,4 galactosaminogalactan), pFl
ol (Special Publication No. 56-12639) and PF102 (
Japanese Patent Application No. 61-134799) is only recognized.
近年、微生物、植物あるいは動物の生産する多糖あるい
はそれらのオリゴ糖が種々の生理活性を有することが知
られるようになり、多糖又はそれらのオリゴネΔに関心
が高まっている。In recent years, it has become known that polysaccharides or their oligosaccharides produced by microorganisms, plants, or animals have various physiological activities, and interest in polysaccharides or their oligones Δ is increasing.
また、ポリガラクトサミンの類似多糖として知られるポ
リグルコサミン(キトサン)においてもキチン、キトサ
ン及びそのオリゴ糖が抗腫瘍活性を有する事が発見され
ている。さらにポリガラクトサミン自身においても同様
な生理活性の在ることが見出され(石谷幸菩他日本生化
学会講演要旨集 871.1986)、そのオリゴ糖の
生理活性にも関心が高まっている。生理活性以外の分野
にもポリガラクトサミン、オリゴガラクトサミンが有用
になる可能性があり、特にオリゴマーは、用途分野や作
用面でポリマーにない特性を発揮するものと期待され、
注目されている。It has also been discovered that chitin, chitosan, and their oligosaccharides have antitumor activity in polyglucosamine (chitosan), which is known as a polysaccharide similar to polygalactosamine. Furthermore, it has been discovered that polygalactosamine itself has a similar physiological activity (Yukibo Ishitani et al. Japanese Biochemical Society Abstracts 871.1986), and there is growing interest in the physiological activity of its oligosaccharides. Polygalactosamine and oligogalactosamine may be useful in fields other than bioactivity, and oligomers in particular are expected to exhibit properties that polymers do not have in terms of application and action.
Attention has been paid.
ポリガラクトサミンを酸又はアルカリによって加水分解
することによりオリゴ糖を得ることは可能であるが、オ
リゴマーの収率は非常に悪い、 131えば塩酸によっ
てポリガラクトサミンを加水分解する時、ランダムな分
解の結果、得られるオリゴ糖の量は七ノーガラクトサミ
ン、ジ−ガラクトサミン、トリーガラクトサミン、テト
ラ−ガラクトサミン、ペンタ−ガラクトサミンの順であ
り1重合度が大きい程その収量は激減するということに
なる。Although it is possible to obtain oligosaccharides by hydrolyzing polygalactosamine with acids or alkalis, the yield of oligomers is very poor.131 For example, when hydrolyzing polygalactosamine with hydrochloric acid, as a result of random decomposition, The amount of oligosaccharides obtained is in the order of heptanogalactosamine, di-galactosamine, trigalactosamine, tetra-galactosamine, and penta-galactosamine, and the higher the degree of polymerization, the sharply reduced the yield.
そこで、ポリガラクトサミンを分解して1重合度が大き
く、種々の重合度のオリゴ糖を得ることのできるポリガ
ラクトサミン分解酵素が必要とされるのである。Therefore, there is a need for a polygalactosamine-degrading enzyme that can decompose polygalactosamine to obtain oligosaccharides with a large degree of polymerization and various degrees of polymerization.
本発明者らは、広範な微生物についてはポリガラクトサ
ミン分解菌を検索した結果、シュードモナス属に属する
細菌が、新規なポリガラクトサミン分解酵素を生産する
ことを見出し本発明を完成した。The present inventors searched a wide range of microorganisms for polygalactosamine-degrading bacteria, and as a result found that bacteria belonging to the genus Pseudomonas produce a novel polygalactosamine-degrading enzyme, and completed the present invention.
本発明は新規なポリガラクトサミン分解酵素の製造法に
関する。The present invention relates to a novel method for producing a polygalactosamine degrading enzyme.
本発明における使用菌はシュードモナス属に属し、ポリ
ガラクトサミン分解酵素を生産する菌であればいかなる
菌株でもよいが、本発明者らが土壌より分離したシュー
ドモナスsp H881が有利に使用される。シュード
モナスsp H881の菌学的性質は下記のとうりであ
る。The bacteria used in the present invention belong to the genus Pseudomonas and may be any strain as long as it produces a polygalactosamine-degrading enzyme, but Pseudomonas sp H881, which the present inventors isolated from soil, is advantageously used. The mycological properties of Pseudomonas sp H881 are as follows.
(a)形態
顕微鏡的観察(肉汁寒天培地30℃、16時間培養)(
1)細胞の大きさ:0.3〜0.6 X 1.0〜2.
0ミクロンの桿菌
(2)細胞の多形性:認められない
(3)運動性:極鞭毛を有し、運動性有り(4)胞子の
有無:形成せず。(a) Morphological microscopic observation (cultured on broth agar medium at 30°C for 16 hours) (
1) Cell size: 0.3-0.6 x 1.0-2.
0 micron bacillus (2) Cell pleomorphism: Not observed (3) Motility: Possesses polar flagella and is motile (4) Presence or absence of spores: Not formed.
(5)ダラム染色性:陰性
(6)抗酸性:陰性
(b)各種培地における生育状態
(1)肉汁寒天平板培養:30℃、24時間でうす黄茶
色のコロニー、表面円滑で光沢を有し半ないし不透明で
ある6色素の生成はない。(5) Durham staining: Negative (6) Acid-fastness: Negative (b) Growth status in various media (1) Broth agar plate culture: Light yellow-brown colonies after 24 hours at 30°C, with smooth and glossy surface. There is no production of 6 pigments which are semi to opaque.
(2)肉汁寒天斜面培養:よく生育する。(2) Broth agar slant culture: Grows well.
(3)肉汁液体培養:培養液表面に厚膜状に生育、液内
には中程度に生育。(3) Meat juice liquid culture: Growth in a thick film on the surface of the culture medium, and moderate growth within the liquid.
(4)肉汁ゼラチン穿刺培養二表面に生育し、層状に液
化する。(4) Meat juice gelatin puncture culture Grows on the surface and liquefies in layers.
(5)リドマスミルク:アルカリ性、完全に液化する。(5) Lidomus milk: alkaline, completely liquefied.
(c)生理的性質
(1)硝酸塩の還元:陰性
(2)脱窒反応:陰性
(3) MRテスト:陽性
(4) vpテスト:陰性
(5)インドールの生成:lta性
(6)硫化水素の生成:陰性
(7)澱粉の加水分解:陰性
(8)クエン酸の利用:コーザー、クリステンセンの両
培地で利用する。(c) Physiological properties (1) Nitrate reduction: negative (2) Denitrification reaction: negative (3) MR test: positive (4) VP test: negative (5) Indole formation: lta (6) Hydrogen sulfide Production: Negative (7) Starch hydrolysis: Negative (8) Utilization of citric acid: Used in both Koser and Christensen's media.
(9)無機窒素の利用:硝酸塩、アンモニアとも利用す
る。(9) Use of inorganic nitrogen: Nitrate and ammonia are also used.
(10)色素の生成: KingA ;陰性、King
B ;弱い青蛍光の色素を生成、 F agar ;弱
い青蛍光の色素を生成、P agar ;陰性
(11)ウレアーゼ:陽性
(12)オキシダーゼ:陽性
(13)カタラーゼ:陽性
(14)生育の範囲:生¥fpH5〜9、至適温度30
〜40℃(15)酸素に対する態度:好気性
(16) O−Fテスト二酸化
(17)カゼインの分解:陽性
(1g) DNAの分解:陽性
(19)耐塩性:2%食塩;陽性、5%食塩;陰性(2
0)糖類から酸及びガスの生成
酸の生成 ガスの生成
(1)L−アラビノース 十 −(2)D
−キシロース 十 −(3)D−グ
ルコース 十 −(4)D−マンノ
ース + −(5)D−フラクトー
ス −−
(6)D−ガラクトース 十 −(7)マ
ルトース − −(8)シューク
ロース 十 −(9)ラクトース
− −(10)トレハロース 十
−(11)D−ソルビトール −−
(12)D−マンニトール −−
(13)イノシトール −−
(14)グリセリン −−
(15)デンプン −−
(d)その他の性質
(1)窒素源欠乏培地で菌体内にポリ−β−ハイドロキ
シブチル酸エステル(PH8,)を蓄積する。(10) Pigment production: KingA; negative, King
B: produces a weak blue fluorescent dye, Fagar: produces a weak blue fluorescent dye, Pagar: negative (11) urease: positive (12) oxidase: positive (13) catalase: positive (14) Growth range: Raw¥fpH5-9, optimum temperature 30
~40℃ (15) Attitude towards oxygen: aerobic (16) O-F test dioxide (17) Casein degradation: positive (1 g) DNA degradation: positive (19) Salt tolerance: 2% salt; positive, 5% Salt; negative (2
0) Generation of acid and gas from sugars Generation of acid Generation of gas (1) L-arabinose 10 - (2) D
-xylose 10 -(3) D-glucose 10 -(4) D-mannose + -(5) D-fructose -- (6) D-galactose 10 -(7) Maltose - -(8) Sucrose 10 -( 9) Lactose
- - (10) Trehalose - (11) D-sorbitol - - (12) D-mannitol - - (13) Inositol - - (14) Glycerin - (15) Starch - (d) Other properties (1) ) Accumulate poly-β-hydroxybutyric acid ester (PH8,) within the bacterial cells in a nitrogen source-deficient medium.
(2)アルギニン、ベタインを唯一の炭素源として生育
し、アルギニンデヒドロラーゼ活性を持たない。(2) It grows using arginine and betaine as the sole carbon source and does not have arginine dehydrolase activity.
(3)脂肪酸(ツイーン80.60.20)を分解する
。(3) Decompose fatty acids (Tween 80.60.20).
(4) 40℃で生育する。4℃で生育不能。(4) Grows at 40℃. Unable to grow at 4℃.
上述の新規なポリガラクトサミン分解酵素生産能を有す
る本省の分類学的性質を、「バージェズ・マニュアル・
オブ・デターミイティブ・バクテリオロジー」第8版(
1974年)及び「バージェズ・マニュアル・オブ・シ
ステマティソク・バクテリオロジー」第1巻(1984
年)の分類と対比すると、本省はグロスファクターを要
求せず、P)IBを蓄積し、アルギニン、ベタインを唯
一の炭素源として生(f L 、アルギニン・デヒドロ
ラーゼ陰性、脱窒反応陰性、40°Cで生育可能からセ
クション2(あるいはRNAグループ2)のP、cep
acia、 P41adioli+P、margina
teの煩縁菌と思われるがP、cepaciaとは硝酸
塩の還元陽性、炭素源の資化性ではD(−)−1−レバ
ロース、マルトース、ラクトース、マレイン酸において
異なる。P、gladioliとは、マルトース、ラク
トース、マレイン酸、m−ハイドロキシブチル酸エステ
ルの資化性の結果が異なる。The taxonomic properties of the above-mentioned novel polygalactosamine-degrading enzyme producing ability are summarized in the ``Burges Manual.''
of Deterministic Bacteriology” 8th edition (
1974) and Burgess Manual of Systematic Bacteriology, Volume 1 (1984).
In contrast to the classification of P) which accumulates IB and uses arginine and betaine as the sole carbon source, the Ministry does not require a gross factor (P), and the classification of arginine dehydrolase negative, denitrification reaction negative, 40 P, cep in section 2 (or RNA group 2) because it can grow at °C.
acia, P41adioli+P, margina
Although it is thought to be a related fungus to P. cepacia, it differs from P. cepacia in that it is positive for nitrate reduction and assimilates carbon sources in D(-)-1-levalose, maltose, lactose, and maleic acid. P. gladioli differs from P. gladioli in terms of assimilation of maltose, lactose, maleic acid, and m-hydroxybutyric acid ester.
p、IIIarginateとは、m−ハイドロキシブ
チル酸エステルの結果が異なる。また、P、cepac
ia 。The results for m-hydroxybutyric acid ester are different from p,IIIarginate. Also, P, cepac
ia.
P、marginateは、非蛍光性色素を生成するが
本省はKing B、 F agar及びし−グルタミ
ン酸、L−アルギニン、L−スレオニン、L−ヒスチジ
ンを唯一の炭素源とした時弱い蛍光色素(青白蛍光)は
生成するが非蛍光性色素の生成は種々の培地条件におい
ても認められない。これらの結果から、本省はP、ce
pacia、 P、gladioli、 P、marg
inateとは異なる5peciesである。P,marginate produces a non-fluorescent dye, but the Ministry of Health, Labor and Welfare has determined that when King B, Fagar, glutamic acid, L-arginine, L-threonine, and L-histidine are used as the sole carbon source, weak fluorescent dyes (blue-white fluorescence) are produced. ) is produced, but no non-fluorescent dye is observed under various culture conditions. Based on these results, the Ministry has decided to
pacia, P, gladioli, P, marg
5 pecies different from inate.
本省の生理学的諸性質で特徴的なことは、O−Fテスト
において単糖のみならずフル1−−ス、シュークロース
、ラクトース、セルビオースなどの二糖類からも酸を生
成することである。この性質はPseudomonas
属、低温性のP、fragi、 P。A characteristic feature of the physiological properties of this plant is that in the O-F test, acids are produced not only from monosaccharides but also from disaccharides such as flu-1-saccharide, sucrose, lactose, and cellbiose. This property is similar to Pseudomonas
Genus, psychrotrophic P, fragi, P.
taetrolens(いずれもセクション5 ) P
、1undensisと似ているが生育温度で違いがあ
る。taetrolens (both section 5) P
, 1undensis, but there are differences in growth temperature.
以上の結果より本省はPseudomonasの新菌種
と認められ、本省をシュードモナスsp 888]と命
名し、通商産業省工業技術院微生物工業技術研究所に、
微工研菌寄第8955号(PERM P−8955)と
して寄託されている。Based on the above results, the Ministry recognized this as a new species of Pseudomonas, named it Pseudomonas sp 888], and submitted it to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.
It has been deposited as PERM P-8955.
ポリガラクトサミン分解酵素生産菌の培養培地としては
、炭素源、窒素源、無機物、その他の栄養素を程よく含
有する培地ならば、合成培地あるいは天然培地のいずれ
でも使用可能である。該培養培地の好適な例としては、
ポリガラクトサミン0.25%、グルコース0.25%
、酵母エキス0.05%。As a culture medium for polygalactosamine-degrading enzyme-producing bacteria, either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances, and other nutrients. Suitable examples of the culture medium include:
Polygalactosamine 0.25%, glucose 0.25%
, yeast extract 0.05%.
ポリペプトン0.05%、pH7,0の例が挙げられる
。An example is polypeptone 0.05%, pH 7.0.
培養温度は20〜40℃、好ましくは30〜38℃の範
囲、培養開始pHは6〜8、好ましくは7付近で35〜
72時間振盪又は深部攪拌培養すれば、培養液中にポリ
ガラクトサミン分解酵素が得られる。 そして。The culture temperature is in the range of 20-40°C, preferably 30-38°C, and the culture starting pH is 6-8, preferably around 7 and 35-38°C.
If cultured with shaking or deep stirring for 72 hours, a polygalactosamine-degrading enzyme can be obtained in the culture solution. and.
ポリガラクトサミン分解酵素は必要に応じて単離精製さ
れる。例えば、培養濾液をエタノール沈殿法によって粗
酵素を分離し、これを水性媒質に溶解し、セファデック
スG−50ゲル濾過、CM−セファデックスC−25イ
オン交換クロマトグラフイー、フェニル−セファロース
CL−4B疎水クロマトグラフィー等の処理により精製
されたポリガラクトサミン分解酵素が得られる。Polygalactosamine degrading enzymes are isolated and purified as necessary. For example, the crude enzyme is separated from the culture filtrate by ethanol precipitation, dissolved in an aqueous medium, subjected to Sephadex G-50 gel filtration, CM-Sephadex C-25 ion exchange chromatography, and phenyl-Sepharose CL-4B. A purified polygalactosamine-degrading enzyme is obtained by treatment such as hydrophobic chromatography.
実施例1において得られたポリガラクトサミン分解酵素
の理化学的性質は下記のとうりである。The physicochemical properties of the polygalactosamine degrading enzyme obtained in Example 1 are as follows.
(1)作用及び基質特異性
本酵素は重合度n=5(ペンタ−ガラクトサミン)以上
のオリゴ及びポリガラクトサミン(α−1,4ポリガラ
クトサミン)に作用してオリゴガラクトサミンを生成す
る。(1) Action and substrate specificity This enzyme acts on oligo and polygalactosamine (α-1,4 polygalactosamine) with a degree of polymerization n=5 (penta-galactosamine) or higher to produce oligogalactosamine.
その他の多糖類、澱粉(α−1,4グルカン)、グリコ
ーゲン(α−1,4グルカン)、プルラン(α−1,4
グルカン)、デキストラン(α−1,6グルカン)、ラ
ミナラン(β−1,3グルカン)、カルボキシルセルロ
ース(β−1,4−グルカン)、キトサン(β−1,4
ゲルコサミノグルカン)、エチレングリコールキチン(
β−1,4N−アセチルゲルコサミノグルカン)、Ps
eudomonas 5olanacaaru11のポ
リN−アセチルガラクトサミノガラクタン(ポリβ−1
,3N−アセチルガラクトサミノガラクタン)(Y、A
kiyama、、 et、 al、。Other polysaccharides, starch (α-1,4 glucan), glycogen (α-1,4 glucan), pullulan (α-1,4
glucan), dextran (α-1,6 glucan), laminaran (β-1,3 glucan), carboxylcellulose (β-1,4-glucan), chitosan (β-1,4
gelcosaminoglucan), ethylene glycol chitin (
β-1,4N-acetylgelcosaminoglucan), Ps
poly N-acetylgalactosaminogalactan (poly β-1
, 3N-acetylgalactosaminogalactan) (Y, A
kiyama,, et, al.
Agric、 Biol、 Chem、+ 50(3)
747.1986)などには全く作用しない。Agric, Biol, Chem, +50 (3)
747.1986) etc. has no effect at all.
また、重合度n=4(テトラ−ガラクトサミン)以下の
α−1,4ガラクトサミノオリゴ糖にも作用しない。Moreover, it does not act on α-1,4 galactosaminooligosaccharide having a degree of polymerization n=4 (tetra-galactosamine) or less.
(2)至適pH及び安定pH範囲
クエン酸リン酸緩衝液を用いた場合、至適pHは4.5
〜7.0である。(第1図)また、安定pH範囲は第2
図に示すとと<pi(4,5〜8.0である。この測定
は37℃で1時間放置した酵素の残存活性を相対値で示
した。(2) Optimal pH and stable pH range When using citrate phosphate buffer, the optimal pH is 4.5
~7.0. (Figure 1) Also, the stable pH range is
As shown in the figure, it is <pi (4.5 to 8.0). This measurement shows the residual activity of the enzyme after being left at 37° C. for 1 hour as a relative value.
(3)酵素活性の測定法
酵素活性は基質にPaecilomyces I−1菌
の生産するPFIOI又はPF102 (その主構成糖
はα−1,4ガラクトサミノガラクタン)を用いた。こ
の0.5%10.1モル酢酸緩衝液pH6,0溶液0.
5+nQに酵素溶液0 、5+nQを加え、37℃、1
0分間反応させ、生じる還元力をSomogyi−Ne
lson法で測定した。なお酵素単位は1分間当りに1
μモルのガラクトサミンに相当する還元力を増加させる
活性を1単位とした。(3) Enzyme activity measurement method Enzyme activity was determined using PFIOI or PF102 produced by Paecilomyces I-1 bacteria (the main constituent sugar being α-1,4 galactosaminogalactan). This 0.5% 10.1M acetate buffer pH 6.0 solution.
Add enzyme solution 0 and 5+nQ to 5+nQ, and incubate at 37℃ for 1
React for 0 minutes, and reduce the resulting reducing power with Somogyi-Ne
It was measured by the lson method. The enzyme unit is 1 per minute.
The activity that increases the reducing power corresponding to μmol of galactosamine was defined as one unit.
(4)作用適温及び温度安定性の範囲 20〜70℃の範囲で測定した結果を第3図に示した。(4) Range of suitable operating temperature and temperature stability The results of measurements in the range of 20 to 70°C are shown in Figure 3.
この酵素の至適温度は55°Cであり、それ以上で急激
に低下する。The optimum temperature for this enzyme is 55°C, and the temperature decreases rapidly above that temperature.
つぎに温度安定性についてみたものが第4図である。p
H6,0の条件で各温度で0〜80分間保った時の残存
活性を示した。50℃、1時間で70%の活性が残存し
ている。Next, Figure 4 shows the temperature stability. p
The residual activity when kept at each temperature for 0 to 80 minutes under H6.0 conditions is shown. 70% activity remains after 1 hour at 50°C.
(5)金属イオン等の影響
各種金属イオン及び阻害剤L mM(PCMBのみ0.
1mM)を含む溶液中に37℃、1時間放置後、残存酵
素活性を測定し、相対値で示した。(表−1)表−1,
金属等の影響
阻害物 残存活性(%) 阻害物 残存活性(%
)無添加 100
にC196NaC197
CaC1,98LiC1100
BaC1□’ 99 MnC1,103
CoC1□88 NiC1,90CdC1
z 98 5nC1226FeC
1,5FeCl36
ZnC1292HgC1,0
Pb(CH,Coo)295 NH4Cl
98(Ni+4)、So、 100
CuSO429tris 1)
97 SDS 2) 4NBS
3) 88 EDTA 4
) 99MIA 5) 100
PCMB 6) 93■)トリス(ヒド
ロキシル)アミノメタン2) ドデシル硫酸ナトリウム
3) N−ブロモコハク酸イミド
4)エチレンジアミン四酢酸二ナトリウム5) モノヨ
ード酢酸
6)パラオキシ安息香酸第二水銀
以上の結果から、このポリガラクトサミン分解酵素はス
ズ、鉄、銅、無機水銀及びSO5により阻害される。(5) Effects of metal ions, etc. Various metal ions and inhibitors LmM (PCMB only 0.
After standing in a solution containing 1mM) at 37°C for 1 hour, the residual enzyme activity was measured and expressed as a relative value. (Table-1) Table-1,
Inhibitors such as metals Residual activity (%) Inhibitors Residual activity (%)
) No additives 100 to C196NaC197 CaC1,98LiC1100 BaC1□' 99 MnC1,103
CoC1□88 NiC1,90CdC1
z 98 5nC1226FeC
1,5FeCl36 ZnC1292HgC1,0 Pb(CH,Coo)295 NH4Cl
98 (Ni+4), So, 100
CuSO429tris 1)
97 SDS 2) 4NBS
3) 88 EDTA 4
) 99MIA 5) 100
PCMB 6) 93■) Tris(hydroxyl)aminomethane 2) Sodium dodecyl sulfate 3) N-bromosuccinimide 4) Disodium ethylenediaminetetraacetate 5) Monoiodoacetic acid 6) From the above results, it was found that this poly Galactosamine degrading enzymes are inhibited by tin, iron, copper, inorganic mercury and SO5.
(6)酵素の精製法 本酵素の単離、精製は常法に従って行うことができる。(6) Enzyme purification method Isolation and purification of this enzyme can be performed according to conventional methods.
エタノールによる沈殿物をセファデックスG−50カラ
ムクロマトグラフイー(第5図)、Cトセファデックス
C−25カラムクロマトグラフィー(第6図)、フェニ
ル−セファロース4Bカラムクロマトグラフイー(第7
図)などのfi!’A手段又はこれらの組合せにより精
製される。The ethanol precipitate was subjected to Sephadex G-50 column chromatography (Figure 5), Sephadex C-25 column chromatography (Figure 6), and phenyl-Sepharose 4B column chromatography (Figure 7).
fi! 'Purified by means A or a combination thereof.
(7)分子量
本酵素の分子量はポリアクリルアミドゲルスラブ電気泳
動法により測定すると、31,000と計算される。結
果は第8図に示した。(7) Molecular Weight The molecular weight of this enzyme is calculated to be 31,000 when measured by polyacrylamide gel slab electrophoresis. The results are shown in Figure 8.
(8)ポリアクリルアミドゲル電気泳動精製酵素を常法
に従って、7.5%のポリアクリルアミドゲル(pt(
8,6)電気泳動にかけた。結果は第9図に示すとうり
単一のバンドが認められた。(8) Polyacrylamide gel electrophoresis The purified enzyme was purified using a 7.5% polyacrylamide gel (pt(
8,6) Subjected to electrophoresis. As shown in FIG. 9, a single band was observed.
(9)等電点
常法によりシュークロース密度勾配の等電点電気泳動を
行った。結果は第10図に示すようにこの酵素の等電点
はpI=6.7である。(9) Isoelectric focusing of a sucrose density gradient was performed using a conventional isoelectric focusing method. As shown in FIG. 10, the isoelectric point of this enzyme is pI=6.7.
本酵素は、その作用及び部質特異性において従来全く知
られていない新規酵素である。This enzyme is a novel enzyme whose action and site specificity are completely unknown.
次に本発明の実施例を示す。Next, examples of the present invention will be shown.
実施例1
シュードモナスsp 11881、FERM P−89
55を500mΩ三角フラスコ中で、グルコース0.5
%、酵母エキス0.05%、ポリペプトン0.05%の
組成を有する種培地100mGに植菌し、30℃で20
時間培養する。Example 1 Pseudomonas sp 11881, FERM P-89
55 in a 500 mΩ Erlenmeyer flask with 0.5 glucose
%, yeast extract 0.05%, and polypeptone 0.05%.
Incubate for hours.
得られた種培養液を3[)Qのジャーファーメンタ−中
で、ポリガラクトサミン(PF102) 0.25%、
グルコース0.25%、酵母エキス0.05%、ポリペ
プトン0.05%の酵素生産培地18111に植菌し、
30℃で48時間通気(18Q /分)攪拌(200r
pm)培養する。The obtained seed culture solution was mixed with 0.25% polygalactosamine (PF102) in a 3[)Q jar fermenter.
Inoculated into enzyme production medium 18111 containing 0.25% glucose, 0.05% yeast extract, and 0.05% polypeptone,
Aeration (18Q/min) stirring (200r) at 30℃ for 48 hours
pm) Cultivate.
得られた培養液を遠心分離(14,000rρm) し
て、菌体を除き、得られた培養濾液(酵素活性0.00
35U/ml、総置性63U/1.8 Q )に冷却し
たエタノールを60%濃度まで加えて、タンパク質を沈
殿させ、この沈殿タンパク質を遠心して、溶液から分離
する。The obtained culture solution was centrifuged (14,000 rpm) to remove bacterial cells, and the obtained culture filtrate (enzyme activity 0.00
The proteins are precipitated by adding chilled ethanol to 60% concentration (35 U/ml, total concentration 63 U/1.8 Q), and the precipitated proteins are separated from the solution by centrifugation.
得られたタンパク質を0.1モル酢酸緩衝液(ρ115
.0)で平衡化したCトセファデックスC−25カラム
(2,5X60cm)に吸着させ、0〜0.5モル食塩
の濃度勾配を有する同緩衝液を用いて溶出させる。The obtained protein was dissolved in 0.1 molar acetate buffer (ρ115
.. The sample is adsorbed onto a C-tocephadex C-25 column (2.5 x 60 cm) equilibrated with 0) and eluted using the same buffer with a concentration gradient of 0 to 0.5 molar saline.
溶出した酵素活性区分を集め、限外濾過装置(分子量1
万保持)を使って濃縮する。次に、2モル食塩を含む0
.1モル酢酸緩衝液(pH6,0)溶液とし、同緩衝液
で平衡化したセファデックスG−50カラム(5X 9
0cm)クロマトグラフィーにかける。次いで、活性区
分の食塩濃度を4モルにまで高め、同様な溶液で平衡化
したフェニル−セファロースCL−/IBカラム(2,
5X 20cm)に吸着させ1食塩の逆5i度勾配を持
つ0.1モル酢酸緩衝液で溶出して精製ポリガラクトサ
ミン分解酵素50mg (収率23.1%、比活性52
μg galN+/min/mHprotein)を得
た。The eluted enzyme activity fraction was collected and filtered using an ultrafiltration device (molecular weight 1
Concentrate using 1,000,000 ml of water. Next, 0 containing 2 molar salt
.. A Sephadex G-50 column (5X 9
0 cm) chromatography. The salt concentration in the active section was then increased to 4 molar, and a phenyl-Sepharose CL-/IB column (2,
50 mg of purified polygalactosamine-degrading enzyme (yield: 23.1%, specific activity: 52).
μg galN+/min/mH protein) was obtained.
第1図は本酵素についての至適pi(を、第2図は安定
pHを、第3図には作用適温を、第4図は温度安定性を
、第5図にはセファデックスG−50のゲル濾過パター
ンを、第6図はCトセファデツクスC−25のパターン
を、第7図にはフェニル−セファロースCL−4Bのカ
ラムクロマトのパターンを、第・ 8図はポリアクリ
ルアミドゲルスラブ電気泳動の結果を、第9図はポリア
クリルアミドゲルデスク電気泳動の結果を、さらに第1
0図には等電点電気泳動の結果を、それぞれ示す図であ
る。
代理人 弁理士 戸 1)親 男
第 1 図
x 10 ’
3ト
pH
第 2 図
。L −−−m=
温度(0C)
jifr Pit (min)
第 5 図
7つクシヨシ程(818mQ)
第 6 図
ワラクシコレ叡 (618mλ)
第 7 図
フラクシヨンU (各10mffi)
第 8 図
Rm
第 9 図
第 10 図Figure 1 shows the optimum pi for this enzyme, Figure 2 shows the stable pH, Figure 3 shows the optimum temperature for action, Figure 4 shows the temperature stability, and Figure 5 shows Sephadex G-50. Figure 6 shows the pattern of C-tosephadex C-25, Figure 7 shows the column chromatography pattern of Phenyl-Sepharose CL-4B, and Figures 8 and 8 show the results of polyacrylamide gel slab electrophoresis. , Figure 9 shows the results of polyacrylamide gel desk electrophoresis, and the first
Figure 0 shows the results of isoelectric focusing, respectively. Agent Patent Attorney Door 1) Parent Male Diagram 1 x 10' 3 to pH Diagram 2. L ---m= Temperature (0C) jifr Pit (min) Fig. 5 7 comb length (818mQ) Fig. 6 Warakushikorei (618mλ) Fig. 7 Fraction U (each 10mffi) Fig. 8 Rm Fig. 9 10 Figure
Claims (1)
解酵素生産菌を培養し、培養物よりポリガラクトサミン
分解酵素を採取することを特徴とする、ポリガラクトサ
ミン分解酵素の製造法。(1) A method for producing a polygalactosamine-degrading enzyme, which comprises culturing a polygalactosamine-degrading enzyme-producing bacterium belonging to the genus Pseudomonas, and collecting the polygalactosamine-degrading enzyme from the culture.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP30858086A JPH0236232B2 (en) | 1986-12-26 | 1986-12-26 | HORIGARAKUTOSAMINBUNKAIKOSONOSEIZOHO |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP30858086A JPH0236232B2 (en) | 1986-12-26 | 1986-12-26 | HORIGARAKUTOSAMINBUNKAIKOSONOSEIZOHO |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS63164885A true JPS63164885A (en) | 1988-07-08 |
JPH0236232B2 JPH0236232B2 (en) | 1990-08-16 |
Family
ID=17982740
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP30858086A Expired - Lifetime JPH0236232B2 (en) | 1986-12-26 | 1986-12-26 | HORIGARAKUTOSAMINBUNKAIKOSONOSEIZOHO |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0236232B2 (en) |
-
1986
- 1986-12-26 JP JP30858086A patent/JPH0236232B2/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
JPH0236232B2 (en) | 1990-08-16 |
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