JP3609102B2 - Novel protease, process for producing the same and novel microorganism of the genus Alteromonas - Google Patents
Novel protease, process for producing the same and novel microorganism of the genus Alteromonas Download PDFInfo
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Description
【0001】
【産業上の利用分野】
本発明は、海洋汚損生物の一種であるムラサキイガイを除去することを目的とした、ムラサキイガイ足糸分解能を有するプロテアーゼ、その製造法及び該プロテアーゼ生産能を有する新規細菌、Alteromonas peptidysinに関する。
【0002】
【従来の技術】
ムラサキイガイ等の海洋付着生物は、船底や養殖施設、発電所の取水路等に付着し、様々な被害を与えている。そのため、これらの汚損生物の除去は、従来、水圧や人力等による物理的手段、あるいは亜硫化銅や有機錫化合物等を使った化学的除去法が行われてきた。しかし、これらの方法は、人的危険を伴う多大な労力と出費を必要とし、更には、環境を汚染するという欠点を有していた。
【0003】
【発明が解決しようとする課題】
本発明は、ムラサキイガイ足糸の分解除去が目的であり、それを達成すべく鋭意研究を行い本発明を完成するに至った。
【0004】
【課題を解決するための手段】
海洋付着生物は色々な様式で付着しているが、その中でムラサキイガイは、L−ドーパ、4−ヒドロキシプロリンを含むフェノール性タンパクとコラーゲン等からなる足糸で付着している。
本発明者らは、ムラサキイガイ足糸を基質として海洋より細菌を採取し、その中からムラサキイガイ足糸だけを栄養源として生育できる細菌、即ちムラサキイガイ足糸分解能を有する新規な細菌を分離した。更に、本新規細菌の産生するムラサキイガイ足糸を分解できる3種のプロテアーゼを単離した。
1.本細菌のスクリーニングは以下の通り行った。
(1)一次スクリーニング
一次スクリーニング培地は洗浄前処理した足糸を濾過海水に0.5 %(W/V) 入れ、加熱殺菌したもので、試験管に入れた本培地10mlに 100μl の採取した海水を添加し、20℃で10日間振とう培養した。濁度を測定し、OD660 >0.3 まで増殖した分解活性の高いものを選択した。この増殖した培養液をマリンアガー2216培地に100 μl塗布し菌株を単離した。
(2) 二次スクリーニング
二次スクリーニングにより得られた海洋細菌をマリンブロス2216培地でOD660 =1.0 まで培養した。培養液を10,000回転/分で15分遠心し、その上清を得た。この上清200 μlと0.2 μ以下の大きさに粉末篩分した足糸10mgを50mMのトリスバッファー(pH7.5 )中で30℃、1時間反応させ、しかる後、100 ℃、10分の加熱により反応を停止させた。反応液を10,000回転/分で5分遠心し、遠心上清中の遊離したペプチドをニンヒドリン法により定量した。なお、1酵素単位(U)は上記実験条件下で1分間に足糸から遊離されるヒドロキシプロリンの1μmol に相当する440nm における吸光度を与える場合の活性度として表した。また、一次スクリーニング培地に、マリンブロス2216培地でOD660 =1.0 に培養した一次スクリーニングで得られた海洋細菌を100 μl接種し、20℃、15日間培養後、基質乾燥重量を測定した。これら2つの結果を併せて2次スクリーニングとし、最も分解活性の高い菌を選択した。
2.二次スクリーニングにより選択した本発明の細菌の性状は以下の通りである。
(1)形態的性状
a.全長1μm、全幅300 〜400nm の桿菌で、走査型電子顕微鏡下で図1に示されるもの。
【0005】
b.長さ約2μmの鞭毛を一本持ち、それは極毛である。
c.運動性:あり
(2)生理学的性状
a.グラム染色: グラム陰性である。
b.糖発酵: 発酵しない。
【0006】
c.菌体色素: 茶色の色素を産生する。
d.好塩性: 1〜10%のNaCl濃度で生育する。
e.ゼラチン分解:分解する。
f.DNA分解: 分解する。
g.GC含量: 42.4%
3.本発明の新規細菌の分類上の位置の決定は、次の通り行った。
(1)属の決定
細菌は上記の形態学的及び生理学的性状よりアルテロモナス (Alteromonas)属と同定した。
(2)種の決定
アルテロモナス属にはバージェイ式分類法により11種が知られている。バージェイ式分類法により本細菌の分類を試みたところ、公知種とは一致しなかった。公知11種の中でバージェイ式分類法で一番近縁であったA.luteoviolacea と本種の性状を表1及び表2に示す。
【0007】
表1及び表2において、記号「+」は90〜100 %の菌株で陽性であること、記号「d」は10〜89%の菌株で陽性であること、記号「−」は10%未満の菌株で陽性であるか、全く認められないこと、記号「St」は桿菌を表す。
【0008】
【表1】
【0009】
【表2】
a.本細菌は40℃で生育するが、A.luteoviolacea は生育しない。
b.本細菌はコハク酸、フマル酸、ペラルゴン酸、カプリン酸、L−ロイシン、L−イソロイシン、L−バリンを利用できるが、A.luteoviolacea は利用できない。
【0010】
また、本種とA.luteoviolacea 及び対照としてA.haloplanktis、E.coli、B.subtilisとのDNAの相同性を検討した結果を表3に示す。
【0011】
【表3】
a.本細菌とA.luteoviolacea とのDNAの相同性は5〜10%である。
b.本細菌とE.coli及びB.subtilisとのDNAの相同性は1%以下である。
以上の性状の相違から、本細菌はアルテロモナス属の新種であると結論し、Alteromonas peptidysinと命名した。
【0012】
本細菌は、工業技術院微生物工業技術研究所に微工研菌寄第12875号として寄託されている。
本発明は、また、下記の理化学的性質を有する3種のプロテアーゼをも提供するものである。
A.下記の理化学的性質を有するプロテアーゼ(以下「Enz−F20 」という。)
(1) 作用:ムラサキイガイ足糸分解能を有する。
(2)基質特異性:
(a)配列表の配列番号1で示される過ギ酸酸化ウシインシュリンB鎖における3番目のAsn と4番目のGln の間の結合、4番目のGln と5番目のHis の間の結合、9番目のSer と10番目のHis の間の結合、14番目のAla と15番目のLeu の間の結合、15番目のLeu と16番目のTyr の間の結合、20番目のGly と21番目のGlu の間の結合、24番目のPhe と25番目のPhe の間の結合及び29番目のLys と30番目のAla の間の結合を切断する(図2参照)。
【0013】
(b)配列表の配列番号2で示されるデカペプチドにおける8番目のThr と9番目のL−ドーパ(Dopa)の間の結合、9番目のL−ドーパ(Dopa)と10番目のLys の間の結合を切断する(図3参照)。
(3)至適pH: 9.5〜10
(4)至適温度:50〜55℃
(5)至適NaCl濃度:5%
(6)分子量:44,000
(7)阻害剤:フェニルメタンスルホニルフルオリドにより阻害される。
B.下記の理化学的性質を有するプロテアーゼ(以下「Enz−F30 」という。)
(1) 作用:ムラサキイガイ足糸分解能を有する。
(2)基質特異性:配列表の配列番号2で示されるデカペプチドにおける9番目のL−ドーパ(Dopa)と10番目のLys の間の結合を切断する(図3参照)。
(3)至適pH: 8.5〜9
(4)至適温度:60〜65℃
(5)分子量:40,000
(6)阻害剤:p−クロロメルクリ安息香酸により阻害される。
C.下記の理化学的性質を有するプロテアーゼ(以下「Enz−F15 」という。)
(1) 作用:ムラサキイガイ足糸分解能を有する。
(2)基質特異性:配列表の配列番号2で示されるデカペプチドにおける8番目のThr と9番目のL−ドーパ(Dopa)の間の結合を切断する(図3参照)。
(3)至適pH: 8〜8.5
(4)至適温度:45〜50℃
(5)分子量:22,000
(6)阻害剤:エチレンジアミン四酢酸により阻害される。
【0014】
本発明のプロテアーゼは、例えば、該プロテアーゼ生産能を有するアルテロモナス属細菌を培地に培養して、培養物中に該プロテアーゼを生成蓄積させ、該培養物からこれを採取することにより製造することができる。
ここで用いられるアルテロモナス属細菌としては、例えば前述したAlteromonas peptidysinが挙げられる。
【0015】
前記製造法における好ましい条件を以下に示す。
(1)培地: マリンブロス2216(DIFCO) 又はこれに類するもの
(2)培養温度: 10〜40℃(至適温度、30℃)
(3)培養pH: 6〜11(至適pH、pH7)
(4) 培養NaCl濃度: 1〜10%(至適NaCl濃度、5%)
(5)培養時間: 6時間以上(至適培養時間、48時間)
(6)培養回転数: 60〜120 回転/分(至適回転数、100 回転/分)
【0016】
【実施例】
以下、実施例により本発明を更に具体的に説明するが、本発明の範囲はこれらの実施例に限定されるものではない。
(実施例1)A.peptidysinの産生するムラサキイガイ足糸分解能を有する新規プロテアーゼの製造例
100ml の一次スクリーニング培地(滅菌海水に0.5 %(W/V )の殺菌済の足糸を入れた培地)に、マリンブロス2216培地でOD660 =1.0 まで培養したA.peptidysinを100 μl 添加し、25℃で一週間振とう培養した。培養液を10,000回転/分で15分遠心分離し、その上清を得た。足糸分解活性を測定したところ、乾燥重量で基質残存率が36.4%、足糸分解比活性が1,600 U/mgを示した。足糸分解比活性の値は、市販、既存のプロテアーゼの値を大きく上回るものであった(図4に示す)。
【0017】
(実施例2)A.peptidysinの産生するムラサキイガイ足糸分解能を有する新規プロテアーゼの製造例
2Lの三角フラスコ内の400ml のマリンブロス2216培地でA.peptidysinを2日間培養した。10,000回転/分、15分の遠心分離でその上清を得た。これに硫酸アンモニウムを添加し、その40〜60%画分を得た。この沈澱を0.02Mのトリスバッファー(pH7.2 )に溶解し、超純水で十分透析した。凍結乾燥後、セファクリルS−100HRでのゲルクロマトグラフィー、DEAEクロマトグラフィー、CMクロマトグラフィー、HPLC(ゲルクロマトグラフィー)により足糸分解能を有するプロテアーゼの精製を行った。
【0018】
その結果、3種の新規プロテアーゼ、Enz−F15 、Enz−F20 及びEnz−F30 を得た。
【0019】
【発明の効果】
本発明によれば、ムラサキイガイ足糸分解能を有し、海洋汚損生物であるムラサキイガイの除去に有用な新規プロテアーゼを提供することができる。
【0020】
【配列表】
【0021】
【0022】
【図面の簡単な説明】
【図1】Alteromonas peptidysinの生物形態を示す電子顕微鏡写真である。
【図2】本発明のプロテアーゼの過ギ酸酸化ウシインシュリンB鎖に対する特異性を示す図である。
【図3】本発明のプロテアーゼのデカペプチドに対する特異性を示す図である。
【図4】ムラサキイガイ足糸分解活性の比較を示す図である。[0001]
[Industrial application fields]
The present invention relates to a protease having the ability to remove blue mussel foot thread, a method for producing the same, and a novel bacterium having the ability to produce the protease, Alteromonas peptidysin, for the purpose of removing blue mussels, a kind of marine fouling organisms.
[0002]
[Prior art]
Marine-adherent organisms such as blue mussels adhere to the bottom of the ship, aquaculture facilities, intakes of power plants, etc. and cause various damages. Therefore, removal of these fouling organisms has conventionally been carried out by physical means such as water pressure or human power, or chemical removal methods using copper sulfite, organic tin compounds, or the like. However, these methods require a great deal of labor and expense with human danger and have the disadvantage of polluting the environment.
[0003]
[Problems to be solved by the invention]
The object of the present invention is to disassemble and remove the mussel gait, and to accomplish this, the present invention has been completed through extensive research.
[0004]
[Means for Solving the Problems]
Marine adhering organisms are attached in various ways, and among them, mussels are attached with a foot thread composed of L-dopa, a phenolic protein containing 4-hydroxyproline, collagen, and the like.
The present inventors collected bacteria from the ocean using the mussel gait as a substrate, and isolated a bacterium that can grow using only the mussel gait as a nutrient source, that is, a novel bacterium having a mussel gait resolution. Furthermore, three types of protease capable of degrading mussel foot thread produced by the novel bacterium were isolated.
1. The bacteria were screened as follows.
(1) Primary screening The primary screening medium is prepared by adding pre-washed foot threads to filtered seawater at 0.5% (W / V) and sterilizing by heating. 100 μl of seawater collected in 10 ml of the main medium in a test tube And cultured with shaking at 20 ° C. for 10 days. The turbidity was measured, and the one with high degradation activity grown to OD660> 0.3 was selected. 100 μl of this grown culture was applied to Marine Agar 2216 medium to isolate the strain.
(2) Secondary screening Marine bacteria obtained by secondary screening were cultured in Marine Broth 2216 medium until OD660 = 1.0. The culture solution was centrifuged at 10,000 rpm for 15 minutes to obtain the supernatant. 200 μl of this supernatant and 10 mg of foot thread sieved to a size of 0.2 μm or less were reacted in 50 mM Tris buffer (pH 7.5) at 30 ° C. for 1 hour, and then at 100 ° C. for 10 minutes. The reaction was stopped by heating. The reaction solution was centrifuged at 10,000 rpm for 5 minutes, and the released peptide in the centrifuged supernatant was quantified by the ninhydrin method. One enzyme unit (U) was expressed as the activity when giving absorbance at 440 nm corresponding to 1 μmol of hydroxyproline released from the foot thread per minute under the above experimental conditions. In addition, 100 μl of the marine bacteria obtained by the primary screening cultured in the marine broth 2216 medium at OD660 = 1.0 was inoculated into the primary screening medium, cultured at 20 ° C. for 15 days, and then the substrate dry weight was measured. These two results were combined for the secondary screening, and the bacteria with the highest degradation activity were selected.
2. The properties of the bacterium of the present invention selected by the secondary screening are as follows.
(1) Morphological properties a. Aspergillus having a total length of 1 μm and a total width of 300 to 400 nm, shown in FIG. 1 under a scanning electron microscope.
[0005]
b. It has one flagellum about 2 μm in length and it is polar hair.
c. Motility: Yes (2) Physiological properties a. Gram staining: Gram negative.
b. Sugar fermentation: No fermentation.
[0006]
c. Bacterial pigment: Produces brown pigment.
d. Salty: grows at a NaCl concentration of 1-10%.
e. Gelatin degradation: Decomposes.
f. DNA degradation: Degraded.
g. GC content: 42.4%
3. The classification position of the novel bacteria of the present invention was determined as follows.
(1) Determination of genus Bacteria were identified as the genus Alteromonas from the above morphological and physiological characteristics.
(2) Determination of species Eleven species of Alteromonas are known by the Barjay method. Attempts to classify the bacteria using the Barjay method did not match the known species. Among the 11 known types, A. was the closest related to the Barjay classification. Table 1 and Table 2 show the properties of luteoviolacea and this species.
[0007]
In Tables 1 and 2, the symbol “+” is positive in 90-100% strains, the symbol “d” is positive in 10-89% strains, and the symbol “−” is less than 10%. It is positive in the strain or not recognized at all, and the symbol “St” represents Neisseria gonorrhoeae.
[0008]
[Table 1]
[0009]
[Table 2]
a. The bacterium grows at 40 ° C. luteobiolacea does not grow.
b. The bacterium can use succinic acid, fumaric acid, pelargonic acid, capric acid, L-leucine, L-isoleucine, and L-valine. luteviolacea is not available.
[0010]
In addition, this species and A. luteviolacea and A. as a control. halloplantis, E.H. coli, B.I. Table 3 shows the results of examining the DNA homology with subtilis.
[0011]
[Table 3]
a. The bacteria and A. The DNA homology with luteobiolacea is 5-10%.
b. This bacterium and E. coli E. coli and B.I. DNA homology with subtilis is 1% or less.
From the above differences in properties, it was concluded that this bacterium is a new species of the genus Alteromonas and was named Alteromonas peptidinsin.
[0012]
This bacterium has been deposited at the National Institute of Advanced Industrial Science and Technology as No. 12875
The present invention also provides three proteases having the following physicochemical properties.
A. Protease having the following physicochemical properties (hereinafter referred to as “Enz-F20”)
(1) Action: It has mussel gait resolution.
(2) Substrate specificity:
(A) a bond between the third Asn and the fourth Gln in the formic acid oxidized bovine insulin B chain represented by SEQ ID NO: 1 in the sequence listing, a bond between the fourth Gln and the fifth His, the ninth Bond between Ser and 10th His, bond between 14th Ala and 15th Leu, bond between 15th Leu and 16th Tyr, 20th Gly and 21st Glu And the bond between the 24th Phe and the 25th Phe and the bond between the 29th Lys and the 30th Ala (see FIG. 2).
[0013]
(B) Binding between the 8th Thr and the 9th L-dopa (Dopa) in the decapeptide represented by SEQ ID NO: 2 in the sequence listing, between the 9th L-dopa (Dopa) and the 10th Lys Is cut (see FIG. 3).
(3) Optimum pH: 9.5-10
(4) Optimal temperature: 50-55 ° C
(5) Optimal NaCl concentration: 5%
(6) Molecular weight: 44,000
(7) Inhibitor: Inhibited by phenylmethanesulfonyl fluoride.
B. Protease having the following physicochemical properties (hereinafter referred to as “Enz-F30”)
(1) Action: It has mussel gait resolution.
(2) Substrate specificity: cleaving the bond between the 9th L-dopa (Dopa) and the 10th Lys in the decapeptide represented by SEQ ID NO: 2 in the sequence listing (see FIG. 3).
(3) Optimum pH: 8.5-9
(4) Optimal temperature: 60-65 ° C
(5) Molecular weight: 40,000
(6) Inhibitor: Inhibited by p-chloromercuribenzoic acid.
C. Protease having the following physicochemical properties (hereinafter referred to as “Enz-F15”)
(1) Action: It has mussel gait resolution.
(2) Substrate specificity: cleaving the bond between the 8th Thr and the 9th L-dopa (Dopa) in the decapeptide shown in SEQ ID NO: 2 in the sequence listing (see FIG. 3).
(3) Optimal pH: 8 to 8.5
(4) Optimal temperature: 45-50 ° C
(5) Molecular weight: 22,000
(6) Inhibitor: Inhibited by ethylenediaminetetraacetic acid.
[0014]
The protease of the present invention can be produced, for example, by culturing an Alteromonas bacterium having the ability to produce the protease in a medium, producing and accumulating the protease in the culture, and collecting the protease from the culture. .
Examples of the bacteria belonging to the genus Alteromonas used here include the aforementioned Alteromonas peptidysin.
[0015]
Preferred conditions in the production method are shown below.
(1) Medium: Marine Broth 2216 (DIFCO) or similar (2) Culture temperature: 10-40 ° C (optimum temperature, 30 ° C)
(3) Culture pH: 6-11 (optimum pH, pH 7)
(4) Cultured NaCl concentration: 1 to 10% (optimal NaCl concentration, 5%)
(5) Culture time: 6 hours or more (optimum culture time, 48 hours)
(6) Culture rotation speed: 60-120 rotations / minute (optimum rotation speed, 100 rotations / minute)
[0016]
【Example】
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.
Example 1 Example of production of a novel protease having the resolution of mussel mussel produced by peptidysin 100 ml of primary screening medium (medium containing 0.5% (W / V) sterilized foot thread in sterilized seawater) and marine broth 2216 medium Cultured to OD660 = 1.0 in A. 100 μl of peptidin was added and cultured with shaking at 25 ° C. for one week. The culture solution was centrifuged at 10,000 rpm for 15 minutes to obtain the supernatant. As a result of measuring the foot fissure degradation activity, the substrate residual rate was 36.4% and the foot fissure degradation specific activity was 1,600 U / mg by dry weight. The value of the specific activity of foot fissure was significantly higher than that of commercially available and existing proteases (shown in FIG. 4).
[0017]
Example 2 Example 1 Production of a novel protease having the resolution of mussel pods produced by peptidysin In a 2 L Erlenmeyer flask with 400 ml of marine broth 2216 medium Peptidysin was cultured for 2 days. The supernatant was obtained by centrifugation at 10,000 rpm for 15 minutes. Ammonium sulfate was added thereto to obtain a 40-60% fraction thereof. This precipitate was dissolved in 0.02 M Tris buffer (pH 7.2) and sufficiently dialyzed against ultrapure water. After lyophilization, a protease having a foot resolution was purified by gel chromatography using Sephacryl S-100HR, DEAE chromatography, CM chromatography, and HPLC (gel chromatography).
[0018]
As a result, three novel proteases, Enz-F15, Enz-F20 and Enz-F30 were obtained.
[0019]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the novel protease which has a blue mussel leg string resolution | decomposability and is useful for removal of the blue mussel which is a marine fouling organism can be provided.
[0020]
[Sequence Listing]
[0021]
[0022]
[Brief description of the drawings]
FIG. 1 is an electron micrograph showing the biological form of Alteromonas peptidysin.
FIG. 2 shows the specificity of the protease of the present invention for the formic acid oxidized bovine insulin B chain.
FIG. 3 shows the specificity of the protease of the present invention for decapeptide.
FIG. 4 is a diagram showing a comparison of mussel octopus legolytic activity.
Claims (5)
(1) 作用:ムラサキイガイ足糸分解能を有する。
(2)基質特異性:
(a)配列表の配列番号1で示される過ギ酸酸化ウシインシュリンB鎖における3番目のAsn と4番目のGln の間の結合、4番目のGln と5番目のHis の間の結合、9番目のSer と10番目のHis の間の結合、14番目のAla と15番目のLeu の間の結合、15番目のLeu と16番目のTyr の間の結合、20番目のGly と21番目のGlu の間の結合、24番目のPhe と25番目のPhe の間の結合及び29番目のLys と30番目のAla の間の結合を切断する。
(b)配列表の配列番号2で示されるデカペプチドにおける8番目のThr と9番目のL−ドーパの間の結合、9番目のL−ドーパと10番目のLys の間の結合を切断する。
(3)至適pH: 9.5〜10
(4)至適温度:50〜55℃
(5)至適NaCl濃度:5%
(6)分子量:44,000
(7)阻害剤:フェニルメタンスルホニルフルオリドにより阻害される。A protease having the following physicochemical properties.
(1) Action: Has mussel gait resolution.
(2) Substrate specificity:
(A) a bond between the third Asn and the fourth Gln in the formic acid oxidized bovine insulin B chain represented by SEQ ID NO: 1 in the sequence listing, a bond between the fourth Gln and the fifth His, the ninth Bond between Ser and 10th His, bond between 14th Ala and 15th Leu, bond between 15th Leu and 16th Tyr, 20th Gly and 21st Glu The bond between the 24th Phe and the 25th Phe and the bond between the 29th Lys and the 30th Ala.
(B) The bond between the 8th Thr and the 9th L-dopa and the bond between the 9th L-dopa and the 10th Lys in the decapeptide represented by SEQ ID NO: 2 in the sequence listing are cleaved.
(3) Optimum pH: 9.5-10
(4) Optimal temperature: 50-55 ° C
(5) Optimal NaCl concentration: 5%
(6) Molecular weight: 44,000
(7) Inhibitor: Inhibited by phenylmethanesulfonyl fluoride.
(1) 作用:ムラサキイガイ足糸分解能を有する。
(2)基質特異性:配列表の配列番号2で示されるデカペプチドにおける9番目のL−ドーパと10番目のLys の間の結合を切断する。
(3)至適pH: 8.5〜9
(4)至適温度:60〜65℃
(5)分子量:40,000
(6)阻害剤:p−クロロメルクリ安息香酸により阻害される。A protease having the following physicochemical properties.
(1) Action: It has mussel gait resolution.
(2) Substrate specificity: cleaving the bond between the 9th L-dopa and the 10th Lys in the decapeptide represented by SEQ ID NO: 2 in the sequence listing.
(3) Optimum pH: 8.5-9
(4) Optimal temperature: 60-65 ° C
(5) Molecular weight: 40,000
(6) Inhibitor: Inhibited by p-chloromercuribenzoic acid.
(1) 作用:ムラサキイガイ足糸分解能を有する。
(2)基質特異性:配列表の配列番号2で示されるデカペプチドにおける8番目のThr と9番目のL−ドーパの間の結合を切断する。
(3)至適pH: 8〜8.5
(4)至適温度:45〜50℃
(5)分子量:22,000
(6)阻害剤:エチレンジアミン四酢酸により阻害される。A protease having the following physicochemical properties.
(1) Action: Has mussel gait resolution.
(2) Substrate specificity: cleaving the bond between the 8th Thr and the 9th L-dopa in the decapeptide represented by SEQ ID NO: 2 in the sequence listing.
(3) Optimal pH: 8 to 8.5
(4) Optimal temperature: 45-50 ° C
(5) Molecular weight: 22,000
(6) Inhibitor: Inhibited by ethylenediaminetetraacetic acid.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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JP7500093A JP3609102B2 (en) | 1992-04-03 | 1993-03-10 | Novel protease, process for producing the same and novel microorganism of the genus Alteromonas |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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JP10910892 | 1992-04-03 | ||
JP4-109108 | 1992-04-03 | ||
JP7500093A JP3609102B2 (en) | 1992-04-03 | 1993-03-10 | Novel protease, process for producing the same and novel microorganism of the genus Alteromonas |
Publications (2)
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JPH0638749A JPH0638749A (en) | 1994-02-15 |
JP3609102B2 true JP3609102B2 (en) | 2005-01-12 |
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Families Citing this family (2)
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US5919689A (en) * | 1996-10-29 | 1999-07-06 | Selvig; Thomas Allan | Marine antifouling methods and compositions |
JP2005289910A (en) * | 2004-03-31 | 2005-10-20 | Chugoku Electric Power Co Inc:The | Method for floating micro substance and method for floating jellyfish decomposing micro substance |
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