JPS6123994B2 - - Google Patents
Info
- Publication number
- JPS6123994B2 JPS6123994B2 JP3683482A JP3683482A JPS6123994B2 JP S6123994 B2 JPS6123994 B2 JP S6123994B2 JP 3683482 A JP3683482 A JP 3683482A JP 3683482 A JP3683482 A JP 3683482A JP S6123994 B2 JPS6123994 B2 JP S6123994B2
- Authority
- JP
- Japan
- Prior art keywords
- aqualysin
- heat
- stable
- enzyme
- thermus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 108091005804 Peptidases Proteins 0.000 claims description 16
- 241000589596 Thermus Species 0.000 claims description 10
- 239000004365 Protease Substances 0.000 claims description 9
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 9
- 241000589500 Thermus aquaticus Species 0.000 claims description 9
- 102000011632 Caseins Human genes 0.000 claims description 8
- 108010076119 Caseins Proteins 0.000 claims description 8
- 239000005018 casein Substances 0.000 claims description 8
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 8
- 235000021240 caseins Nutrition 0.000 claims description 8
- 102000035195 Peptidases Human genes 0.000 claims description 7
- MUCZHBLJLSDCSD-UHFFFAOYSA-N diisopropyl fluorophosphate Chemical compound CC(C)OP(F)(=O)OC(C)C MUCZHBLJLSDCSD-UHFFFAOYSA-N 0.000 claims description 6
- 229960005051 fluostigmine Drugs 0.000 claims description 6
- 235000018102 proteins Nutrition 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 239000003112 inhibitor Substances 0.000 claims description 3
- 150000002500 ions Chemical class 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 3
- 108010038807 Oligopeptides Proteins 0.000 claims description 2
- 102000015636 Oligopeptides Human genes 0.000 claims description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 2
- 239000003381 stabilizer Substances 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 1
- 229910021645 metal ion Inorganic materials 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 description 23
- 108090000790 Enzymes Proteins 0.000 description 23
- 229940088598 enzyme Drugs 0.000 description 23
- 239000000243 solution Substances 0.000 description 13
- 239000000872 buffer Substances 0.000 description 9
- 239000008363 phosphate buffer Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000589501 Thermus caldophilus Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 108010092440 caldolysin Proteins 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000003846 Carbonic anhydrases Human genes 0.000 description 1
- 108090000209 Carbonic anhydrases Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000868182 Thermus thermophilus HB8 Species 0.000 description 1
- 241000006364 Torula Species 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
本発明はサーマス（Thermus）属に属する菌
株が産生する高度に耐熱性を有する蛋白分解酵素
アクアライシンに関する。
従来、耐熱性を有する蛋白分解酵素の生産菌と
しては、ストレプトミセス属（Streptomyces
属）、バチルス属（Bacillus属）、トルーラ属
（Torula属）、およびサーマスＴ−351（Thermus
Ｔ−351）〔欧州特許24182号〕等が報告されてい
る。又、本発明者者等は、すでに、栃木県奥鬼怒
の温泉源から分離したサーマス・カルドフイラス
GK−24（Thermus caldophilus GK−24）の生
産する耐熱性蛋白分解酵素について報告（日本農
芸化学会 昭和53年度大会講演要旨集 65ペー
ジ、生化学第51巻 749ページ（昭和54年）、日本
農芸化学会 昭和55年度大会講演要旨集 269ペ
ージ、日本農芸化学会昭和56年度大会講演要旨集
201ページ）してきたが、今回さらに優秀な菌
株を求めて、公知の保存菌株も含めて、鋭意に検
索を続けたところサーマス・アクアテイカス
ATCC25104（Thermus aquaticus
ATCC25104）が、従来のものとは全く異なる高
度に耐熱性を有する蛋白分解酵素を産生すること
を見出した。
サーマス・アクアテイカスATCC25104の産生
する耐熱性蛋白分解酵素は培養条件の違いにより
２種類存在し、培養開始後約１日で産生され、次
に示す理化学的性質を有するものを本発明者等は
アクアライシンと命名した。
耐熱性蛋白分解酵素アクアライシンは以下に
示す理化学的性質を有する。
(1) 作用：カゼイン等の蛋白質に作用してトリク
ロル酢酸溶液に可溶性のオリゴペプチドあるい
はアミノ酸を生ずる。
(2) 至適PH：各種PHの0.6％カゼイン基質500μ
に本酵素液100μを70℃、10分間反応させ
た。緩衝液はPH３〜６に対しては50mMクエン
酸−クエン酸ナトリウム緩衝液、PH６〜８に対
しては同濃度のリン酸緩衝液、PH8.5〜10.5に
対しては同濃度のホウ酸緩衝液をそれぞれ使用
した。その結果、至適PHは9.4以上である。（第
１図）
(3) PH安定性：各種PHの緩衝液に本酵素を溶解
し、60℃、１時間処理した。緩衝液はPH３、PH
４は50mMクエン酸−クエン酸ナトリウム緩衝
液、PH６、PH7.3は同濃度のリン酸緩衝液、PH
9.4、PH10.5は同濃度のホウ酸緩衝液を使用し
た。PH処理後酵素液を50mMリン酸緩衝液（PH
7.3）で希釈し、0.6％カゼイン基質（PH7.3）と
70℃、１時間反応させた。その結果、PH7.3以
上では安定であるが、PH６以下では不安定であ
る。（第２図）
(4) 至適温度：本酵素（50mMホウ酸緩衝液、PH
9.4に溶解）100μと0.6％カゼイン基質（PH
9.4）500μを各種温度で10分間反応させた。
その結果至適温度は70℃付近に存在する。（第
３図）
(5) 熱安定性：本酵素液（50mMホウ酸緩衝液、
PH9.4に溶解）を70℃及び80℃で各時間処理
し、その後0.6％カゼイン基質（PH9.4）と70
℃、１時間反応させ活性を測定した。その結果
70℃、２時間で約90％残存し、80℃、30分では
約15％残存する。（第４図）
(6) 分子量：本酵素を5mMのDFP（ジイソプロ
ピルフルオロリン酸）と50℃、30分処理、ある
いは115mMの塩酸と室温で数十分処理して失
活させたのち、同量の以下の組成物、すなわち
62mMトリス塩酸、2.3％ドデシル硫酸ナトリ
ウム（SDS）、５％β−メルカプトエタノー
ル、10％グリセロール、（PH6.8）を加えて、
100℃、３分間の処理をし、レムリ（Laemli）
の方法〔ネイチヤー（Nature）第227巻680〜
685頁（1970）〕に従つてSDSポリアクリルアミ
ドゲル電気泳動を行つた。標準蛋白としては牛
血清アルブミン（分子量67000）、卵白アルブミ
ン（分子量43000）カルボニツク・アンヒドラ
ーゼ（分子量30000）、大豆トリプシンインヒビ
ター（分子量20100）を使用した。
その結果、本酵素の分子量は約28500であ
る。
(7) 電気泳動：本酵素を前記(6)項と同様の方法に
よりSDSポリアクリルアミドゲル電気泳動を行
つた。本酵素は電気泳動的に単一バンドが得ら
れる。
(8) 阻害剤：本酵素液を各種阻害剤を含む50mM
ホウ酸緩衝液（PH9.4）とともに50℃、30分間
処理したのち、0.6％カゼイン基質（PH9.4）と
70℃、１時間反応させ残存する活性を測定し
た。本酵素はDFP（ジイソプロピルフルオロ
リン酸）により著しく阻害される。（第１表）
The present invention relates to a highly thermostable proteolytic enzyme aqualysin produced by a strain belonging to the genus Thermus. Conventionally, the genus Streptomyces has been used as a heat-resistant proteolytic enzyme producing bacterium.
), Bacillus spp., Torula spp., and Thermus T-351
T-351) [European Patent No. 24182] etc. have been reported. In addition, the present inventors have already discovered Thermus caldophyllus isolated from the hot spring source in Okukinu, Tochigi Prefecture.
Report on the heat-stable protease produced by GK-24 (Thermus caldophilus GK-24) (Japan Agricultural Chemistry Society 1978 Conference Abstracts, page 65, Biochemistry Vol. 51, page 749 (1978), Japan Agriculture Collection of lecture abstracts for the 1981 conference of the Chemical Society, 269 pages, collection of lecture abstracts for the 1981 conference of the Japanese Society of Agricultural Chemistry
(page 201), but this time, in search of an even better strain, I continued my search, including known preserved strains, and found Thermus aquaticus.
ATCC25104 (Thermus aquaticus
ATCC25104) was found to produce a highly thermostable protease that is completely different from conventional ones. There are two types of thermostable proteolytic enzymes produced by Thermus aquaticus ATCC25104 depending on the culture conditions. It was named. The heat-stable protease Aqualysin has the following physical and chemical properties. (1) Action: Acts on proteins such as casein to produce oligopeptides or amino acids that are soluble in trichloroacetic acid solution. (2) Optimum PH: 500μ of 0.6% casein substrate with various PHs
100μ of this enzyme solution was reacted at 70°C for 10 minutes. The buffer solution is 50mM citric acid-sodium citrate buffer for pH 3 to 6, phosphate buffer of the same concentration for pH 6 to 8, and borate buffer of the same concentration for pH 8.5 to 10.5. Each liquid was used. As a result, the optimal pH is 9.4 or higher. (Figure 1) (3) PH stability: This enzyme was dissolved in buffer solutions of various PH and treated at 60°C for 1 hour. Buffer solution is PH3, PH
4 is 50mM citric acid-sodium citrate buffer, PH6, PH7.3 is phosphate buffer of the same concentration, PH
9.4 and PH10.5, boric acid buffer solutions of the same concentration were used. After PH treatment, the enzyme solution was diluted with 50mM phosphate buffer (PH
7.3) with 0.6% casein substrate (PH7.3)
The reaction was carried out at 70°C for 1 hour. As a result, it is stable at pH 7.3 or higher, but unstable at pH 6 or lower. (Figure 2) (4) Optimal temperature: This enzyme (50mM borate buffer, pH
9.4) 100μ and 0.6% casein substrate (PH
9.4) 500μ was reacted for 10 minutes at various temperatures.
As a result, the optimum temperature exists around 70℃. (Figure 3) (5) Thermostability: This enzyme solution (50mM borate buffer,
(dissolved in PH9.4) at 70°C and 80°C for each time, then treated with 0.6% casein substrate (PH9.4) at 70°C.
The reaction was carried out at 100°C for 1 hour and the activity was measured. the result
About 90% remains after 2 hours at 70°C, and about 15% remains after 30 minutes at 80°C. (Figure 4) (6) Molecular weight: This enzyme was inactivated by treatment with 5mM DFP (diisopropylfluorophosphate) at 50℃ for 30 minutes, or with 115mM hydrochloric acid at room temperature for several minutes. The following compositions in amounts viz.
Add 62mM Tris-HCl, 2.3% sodium dodecyl sulfate (SDS), 5% β-mercaptoethanol, 10% glycerol, (PH6.8),
Treated at 100°C for 3 minutes, Laemli
Method [Nature Vol. 227 680~
685 (1970)]. As standard proteins, bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight 43,000), carbonic anhydrase (molecular weight 30,000), and soybean trypsin inhibitor (molecular weight 20,100) were used. As a result, the molecular weight of this enzyme is approximately 28,500. (7) Electrophoresis: This enzyme was subjected to SDS polyacrylamide gel electrophoresis in the same manner as in section (6) above. This enzyme yields a single band electrophoretically. (8) Inhibitor: 50mM of this enzyme solution containing various inhibitors.
After treatment with borate buffer (PH9.4) at 50℃ for 30 minutes, 0.6% casein substrate (PH9.4) was added.
The reaction was carried out at 70°C for 1 hour and the remaining activity was measured. This enzyme is significantly inhibited by DFP (diisopropylfluorophosphate). (Table 1)
【表】
以上の理化学的性質より、本酵素はアルカリ
性のセリン酵素であると考えられる。
(9) 安定化剤：本酵素液を1mM濃度のCa2+
（CaCl2）を含む50mMグリシン−水酸化ナトリ
ウム緩衝液（PH9.5）とともに80℃で、各時間
処理したのち、残存する活性を測定した。第５
図に示すように、本酵素はCa2+イオンによつ
て安定化される。
以上の性質を有する本酵素は、ソリユブルプロ
テイン（Soluble Protein：食品添加物）の製
造、ペプチツドの合成、洗剤用添加物、バイオリ
アクター等への利用が期待される。
耐熱性蛋白分解酵素アクアライシンは、本発
明者等がすでに報告しているサーマス・カルドフ
イラスGK−24の産生する耐熱性蛋白分解酵素お
よびサーマスＴ−351の産生する耐熱性蛋白分解
酵素カルドリシン（CALDOLYSIN）〔欧州特許
24182号参照〕とは第２表に示すように、各種性
質の全く異なる新規酵素である。
本発明の耐熱性蛋白分解酵素アクアライシン
は、サーマス属に属するアクアライシン生産微
生物を好気的に培養し、培養物からアクアライシ
ン円取得することを特徴とする方法によつて製
造することができる。
本発明において使用するアクアライシン生産
微生物は、アクアライシンを生産することがで
きるサーマス属に属するすべての菌株、突然変異
株、変種を含む。その好ましい菌株はサーマス・
アクアテイカスATCC25104（Thermus
aquaticus ATCC25104）である。
サーマス・アクアテイカスATCC25104の培養
は、本菌株が生育することのできる通常の[Table] Based on the above physical and chemical properties, this enzyme is considered to be an alkaline serine enzyme. (9) Stabilizer: Add this enzyme solution to 1mM Ca 2+
After treatment with 50 mM glycine-sodium hydroxide buffer (PH9.5) containing (CaCl 2 ) at 80° C. for each time, the remaining activity was measured. Fifth
As shown in the figure, this enzyme is stabilized by Ca 2+ ions. This enzyme with the above properties is expected to be used in the production of soluble proteins (food additives), peptide synthesis, detergent additives, bioreactors, etc. The heat-stable protease aqualysin is a heat-stable protease produced by Thermus caldophilus GK-24 and caldolysin (CALDOLYSIN) produced by Thermus T-351, which the present inventors have already reported. [European patent
24182] is a new enzyme with completely different properties, as shown in Table 2. The heat-stable protease aqualysin of the present invention can be produced by a method characterized by aerobically cultivating an aqualysin-producing microorganism belonging to the genus Thermus and obtaining aqualysin circles from the culture. . Aqualysin-producing microorganisms used in the present invention include all strains, mutant strains, and variants belonging to the genus Thermus that are capable of producing aquarysin. The preferred strain is Thermus
Aquaticus ATCC25104 (Thermus
aquaticus ATCC25104). Culture of Thermus aquaticus ATCC25104 is carried out in a normal manner in which this strain can grow.
【表】
栄養源を含有する培地中で行うことができる。例
えば、ポリペプトン、酵母エキス、グルコース、
無機塩類などを含む培地が用いられる。ビタミ
ン、アミノ酸は要求しない。培養は固体培養、液
体培養、いずれでもよいが、通常液体培養の方が
工業的に有利である。又、本菌株は好気性である
ため、液体培養においては通気撹拌を必須とす
る。
培養温度は約40〜79℃の間ならば生育できる
が、より好ましくは70℃前後である。培地PHは７
〜８の間で生育可能である。PH６以下、9.5以上
では生育しない。培養時間は約１日で活性が最大
に達する。さらに培養を延長すると、３日以降に
生産されれる蛋白分解酵素も存在するが、これは
アクアライシンとは別のものである。
このようにして耐熱性蛋白分解酵素アクアライ
シンが培地中に生成される。生成されたアクア
ライシンの採取、精製は公知の方法の組合せに
よつて行うことができる。例えば、培養液から遠
心分離又は瀘過によつて菌体を除去したのち、瀘
液又は上清を硫安塩析し、沈澱を10mMリン酸緩
衝液（PH6.0）に溶解する。本溶液を透析脱塩
し、CMセルロースカラムに吸着させる。溶離
は、0.1M塩化カリウムを含む。10mMリン酸緩衝
液（PH6.0で行なわれる。溶離したアクアライシ
ンを透析、濃縮、凍結乾燥することにより、
SDSポリカリウアミドゲル電気泳動的に単一バン
ドを与えるアクアライシン粉末が得られる。
次に本酵素の活性測定法について述べる。
酵素液100μと0.6％カゼイン溶液（50mMホ
ウ酸緩衝液、PH9.4に溶解）500μを混合し、70
℃、30分間反応させる。反応終了後、５％トリク
ロル酢酸溶液を500μ加え、沈澱を遠心により
除き、上清の280nmの吸収の増加を測定する。１
分間に280nmの吸収を0.001上昇させる酵素力を
0.5単位とする。
以下試験例、実施例により詳しく説明する。
試験例 １
サーマス属に属する保存菌株を用いて、耐熱性
蛋白分解酵素の生産能を調べた。ポリペプトン
0.8％、酵母エキス0.4％、CaSO4・2H2O60mg／
、MgSO4・7H2O100mg／、NaNO3689mg／
、FeCl3・5H2O0.4mg／、MnSO4・5H2O3.16
mg／、ZnSO4・7H2O0.5mg／、H3BO30.5mg／
、PH7.2の組成の培地 各々200mlを500ml容坂
口フラスコに入れ、各種保存菌株を接種して、70
℃にて各時間培養し、培養液中の酵素活性を測定
した。その結果第３表に示す。表から明らかなよ
うに、サーマス・アクアテイカスATCC25104以
外にもサーマス・サーモフイラスATCC27634、
サーマス・カルドフイラスFERM−Ｐ No.5723
に若干ではあるが本酵素活性生産能のあることが
わかる。[Table] Can be carried out in a medium containing a nutrient source. For example, polypeptone, yeast extract, glucose,
A medium containing inorganic salts and the like is used. Does not require vitamins or amino acids. The culture may be either solid culture or liquid culture, but liquid culture is usually industrially more advantageous. Furthermore, since this strain is aerobic, aeration and stirring are essential in liquid culture. The culture can be grown at a culture temperature of about 40 to 79°C, more preferably around 70°C. Medium pH is 7
It is possible to grow between . It will not grow at pH below 6 or above 9.5. The culture reaches its maximum activity in about 1 day. If the culture is extended further, there is also a protease produced after 3 days, but this is different from aqualysin. In this way, the heat-stable proteolytic enzyme aqualysin is produced in the medium. The produced Aqualysin can be collected and purified by a combination of known methods. For example, after removing bacterial cells from the culture medium by centrifugation or filtration, the filtrate or supernatant is salted out with ammonium sulfate, and the precipitate is dissolved in 10 mM phosphate buffer (PH6.0). This solution is desalted by dialysis and adsorbed onto a CM cellulose column. Elution includes 0.1M potassium chloride. This is done in 10mM phosphate buffer (PH6.0).The eluted aqualysin is dialyzed, concentrated, and lyophilized.
Aqualysin powder is obtained which gives a single band on SDS polypotassium gel electrophoresis. Next, a method for measuring the activity of this enzyme will be described. Mix 100μ of enzyme solution and 500μ of 0.6% casein solution (dissolved in 50mM borate buffer, PH9.4),
Incubate for 30 minutes at ℃. After the reaction is complete, add 500μ of 5% trichloroacetic acid solution, remove the precipitate by centrifugation, and measure the increase in absorption of the supernatant at 280 nm. 1
Enzyme power to increase absorption at 280nm by 0.001 per minute
0.5 unit. This will be explained in detail below using test examples and examples. Test Example 1 Using preserved strains belonging to the genus Thermus, the ability to produce heat-stable proteolytic enzymes was investigated. Polypeptone
0.8%, yeast extract 0.4%, CaSO 4 2H 2 O 60mg/
, MgSO 4 7H 2 O 100mg/, NaNO 3 689mg/
, FeCl 3・5H 2 O0.4mg/, MnSO 4・5H 2 O3.16
mg/, ZnSO 4・7H 2 O 0.5 mg/, H 3 BO 3 0.5 mg/
, 200 ml of each medium with a composition of PH 7.2 was placed in a 500 ml Sakaguchi flask, inoculated with various stock strains, and
The cells were cultured at ℃ for various hours, and the enzyme activity in the culture solution was measured. The results are shown in Table 3. As is clear from the table, in addition to Thermus Aquaticus ATCC25104, Thermus Thermophilus ATCC27634,
Thermus cardofilas FERM-P No.5723
It can be seen that there is a slight ability to produce this enzyme activity.
【表】
実施例 １
以下の組成の種培地200mlを含む500ml容坂口フ
ラスコにサーマス・アクアテイカスATCC25104
の一白金耳を接種し75℃で１日振とう培養した。
ポリペプトン 0.8％
酵母エキス 0.4％
無機塩類：CaSO4・2H2O 60mg／
MgSO4・7H2O 100
NaNO3 689
FeCl3・5H2O 0.4
MnSO4・5H2O 3.16
ZnSO4・7H2O 0.5
H3BO3 0.5
PH 7.5
上記培地組成と同一の培地15の入つたジヤー
フアーメンターに種培養液を１％接種し、65℃で
24時間通気撹拌したところ、800u／mlのアクア
ライシンを含む培養液が得られた。
実施例 ２
実施例１で得られた培養液を遠心分離によつて
菌体を除き、上清920mlに硫酸アンモニウムを100
％飽和になるように加えた。冷却下、遠心分離に
より沈澱を採取し、これを10mMリン酸緩衝液
（PH6.0）に溶解し、同じ緩衝液に透析した。透析
液を10mMリン酸緩衝液（PH6.0）で平衡化した
CMセルロースカラム（径2.8cm、高さ2.76cm）に
吸着させた。0.1M塩化カリウムを含む10mMリン
酸緩衝液（PH6.0）で溶離を行い、活性画分を集
めた。活性画分を透析脱塩、濃縮、凍結乾燥して
62900u／mgのアクアライシン粉末を得た。こ
のアクアライシン粉末はポリアクリルアミドゲ
ルデイスク電気泳動的に単一であつた。精製の要
約を第４表に示す。[Table] Example 1 Thermus aquaticus ATCC25104 was placed in a 500 ml Sakaguchi flask containing 200 ml of seed medium with the following composition.
One platinum loopful of this was inoculated and cultured with shaking at 75°C for 1 day. Polypeptone 0.8% Yeast extract 0.4% Inorganic salts: CaSO 4・2H 2 O 60mg/ MgSO 4・7H 2 O 100 NaNO 3 689 FeCl 3・5H 2 O 0.4 MnSO 4・5H 2 O 3.16 ZnSO 4・7H 2 O 0.5 H 3 BO 3 0.5 PH 7.5 Inoculate 1% of the seed culture into a jar fermenter containing medium 15 with the same medium composition as above, and inoculate at 65℃.
When the mixture was aerated and stirred for 24 hours, a culture solution containing 800 u/ml of aquarysin was obtained. Example 2 The culture solution obtained in Example 1 was centrifuged to remove bacterial cells, and 920 ml of the supernatant was mixed with 100 ml of ammonium sulfate.
% saturation. The precipitate was collected by centrifugation under cooling, dissolved in 10 mM phosphate buffer (PH6.0), and dialyzed against the same buffer. Dialysate was equilibrated with 10mM phosphate buffer (PH6.0)
It was adsorbed onto a CM cellulose column (diameter 2.8 cm, height 2.76 cm). Elution was performed with 10 mM phosphate buffer (PH6.0) containing 0.1 M potassium chloride, and active fractions were collected. The active fraction was desalted by dialysis, concentrated, and lyophilized.
Aqualysin powder of 62900u/mg was obtained. This Aqualysin powder was uniform in polyacrylamide gel disc electrophoresis. A summary of the purification is shown in Table 4.
第１図は本発明の酵素の至適PHを示す図であ
り、第２図は同じくPH安定性を、第３図は至適温
度を、第４図は熱安定性をそれぞれ示す図であ
る。第５図はCa2+イオンの熱安定性に対する影
響を示す図である。
Figure 1 is a diagram showing the optimal pH of the enzyme of the present invention, Figure 2 is a diagram showing the PH stability, Figure 3 is the optimal temperature, and Figure 4 is a diagram showing the thermostability. . FIG. 5 is a diagram showing the influence of Ca 2+ ions on thermal stability.
Claims (1)
素アクアライシン。 (1) 作用：カゼイン等の蛋白質に作用してトリク
ロル酢酸溶液に可溶性のオリゴペプチドあるい
はアミノ酸を生ずる。 (2) 至適PH：PH9.4以上 (3) PH安定性：PH7.3以上で安定、PH６以下では
不安定。 (4) 至適温度：70℃付近 (5) 熱安定性：70℃、２時間で約90％残存し、80
℃、30分では約15％残存する（Ca2+不存在
下）。 (6) 分子量：約28500（SDSポリアクリルアミド
ゲル電気泳動法による。） (7) 阻害剤：DFP（ジイソプロピルフルオロリ
ン酸）により著しく阻害される。 (8) 安定化剤：２価金属イオン、例えばCa2+イ
オンにより安定化される。 ２ サーマス属に属する菌株を培養し培養物から
耐熱性蛋白分解酵素アクアライシンを取得する
ことを特徴とする耐熱性蛋白分解酵素アクアライ
シンの製造方法。 ３ サーマス属に属する菌株がサーマス・アクア
テイカスATCC25104である特許請求の範囲第２
項記載の耐熱性蛋白分解酵素アクアライシンの
製造法。[Scope of Claims] 1. A heat-stable proteolytic enzyme aqualysin having the following physical and chemical properties. (1) Action: Acts on proteins such as casein to produce oligopeptides or amino acids that are soluble in trichloroacetic acid solution. (2) Optimal PH: PH9.4 or higher (3) PH stability: Stable at PH7.3 or higher, unstable at PH6 or lower. (4) Optimum temperature: Around 70℃ (5) Thermal stability: Approximately 90% remains after 2 hours at 70℃, 80%
After 30 minutes at ℃, approximately 15% remains (in the absence of Ca 2+ ). (6) Molecular weight: Approximately 28,500 (according to SDS polyacrylamide gel electrophoresis) (7) Inhibitor: Significantly inhibited by DFP (diisopropylfluorophosphate). (8) Stabilizer: Stabilized by divalent metal ions, such as Ca 2+ ions. 2. A method for producing a heat-stable protease aqualysin, which comprises culturing a strain belonging to the genus Thermus and obtaining the heat-stable protease aqualysin from the culture. 3 Claim 2 in which the strain belonging to the genus Thermus is Thermus aquaticus ATCC25104
A method for producing the heat-stable proteolytic enzyme aqualysin as described in .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3683482A JPS58152482A (en) | 1982-03-08 | 1982-03-08 | Heat-resistant proteolytic enzyme aqualysin i and preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3683482A JPS58152482A (en) | 1982-03-08 | 1982-03-08 | Heat-resistant proteolytic enzyme aqualysin i and preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58152482A JPS58152482A (en) | 1983-09-10 |
| JPS6123994B2 true JPS6123994B2 (en) | 1986-06-09 |
Family
ID=12480768
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3683482A Granted JPS58152482A (en) | 1982-03-08 | 1982-03-08 | Heat-resistant proteolytic enzyme aqualysin i and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58152482A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994010290A1 (en) * | 1992-10-30 | 1994-05-11 | Gomei Kaisha Nakamura Sangyo | Thermophilic cellulose-decomposing bacterium and utilization thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2639803B2 (en) * | 1987-03-02 | 1997-08-13 | オリエンタル酵母工業株式会社 | Production of thermostable isocitrate dehydrogenase |
-
1982
- 1982-03-08 JP JP3683482A patent/JPS58152482A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994010290A1 (en) * | 1992-10-30 | 1994-05-11 | Gomei Kaisha Nakamura Sangyo | Thermophilic cellulose-decomposing bacterium and utilization thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58152482A (en) | 1983-09-10 |
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