JPH0262237B2 - - Google Patents
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- Publication number
- JPH0262237B2 JPH0262237B2 JP14766685A JP14766685A JPH0262237B2 JP H0262237 B2 JPH0262237 B2 JP H0262237B2 JP 14766685 A JP14766685 A JP 14766685A JP 14766685 A JP14766685 A JP 14766685A JP H0262237 B2 JPH0262237 B2 JP H0262237B2
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- approximately
- activity
- hours
- determined
- 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.)
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- 102000004190 Enzymes Human genes 0.000 claims description 68
- 108090000790 Enzymes Proteins 0.000 claims description 68
- 230000000694 effects Effects 0.000 claims description 29
- 102000004400 Aminopeptidases Human genes 0.000 claims description 12
- 108090000915 Aminopeptidases Proteins 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 12
- 238000001962 electrophoresis Methods 0.000 claims description 10
- 238000004587 chromatography analysis Methods 0.000 claims description 9
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 7
- 241000589500 Thermus aquaticus Species 0.000 claims description 7
- 238000002523 gelfiltration Methods 0.000 claims description 7
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 239000002738 chelating agent Substances 0.000 claims description 4
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 4
- 239000003112 inhibitor Substances 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 4
- 238000005185 salting out Methods 0.000 claims description 3
- 238000002525 ultrasonication Methods 0.000 claims description 3
- 229940088598 enzyme Drugs 0.000 description 45
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000010586 diagram Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 7
- 239000008363 phosphate buffer Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 238000010828 elution Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- PIFJAFRUVWZRKR-QMMMGPOBSA-N Val-Gly-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O PIFJAFRUVWZRKR-QMMMGPOBSA-N 0.000 description 4
- 238000005194 fractionation Methods 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000011780 sodium chloride Substances 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
- 101710099484 Aminopeptidase T Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 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
- YLEARPUNMCCKMP-DOFZRALJSA-N N-arachidonoylglycine Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)NCC(O)=O YLEARPUNMCCKMP-DOFZRALJSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- -1 polypeptone Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】
〔産業上の利用分野〕
この発明は耐熱性の新規アミノペプチダーゼ及
びその精造方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention relates to a new heat-stable aminopeptidase and a method for purifying the same.
〔発明が解決しようとする問題点〕
アミノペプチダーゼはチーズをはじめとした食
品の製造において、呈味アミノ酸の生成、苦味ペ
プチドの分解などのプロセスにおいて重要な役割
りを果している。このようなプロセスにおいてバ
イオリアクターとしてアミノペプチダーゼを利用
するには、熱などの環境要因に対して安定である
ことが必要であるが、公知のアミノペプチダーゼ
は熱的安定性が不十分である。[Problems to be Solved by the Invention] Aminopeptidases play an important role in processes such as production of taste amino acids and decomposition of bitter peptides in the production of foods such as cheese. The use of aminopeptidases as bioreactors in such processes requires stability against environmental factors such as heat, but known aminopeptidases have insufficient thermal stability.
そこでこの出願の発明者は熱的安定性が優れ、
食品の製造プロセスに有利に用いることができる
新規アミノペプチダーゼを検索すべく研究を重ね
た結果、高度好熱性細菌、サーマス・アクアテイ
カス（Thermus aquaticus）YT−１（ATCC
25104）の菌体から耐熱性に優れるアミノペプチ
ダーゼを製造することを見い出し、この知見に基
づいてこの発明を完成した。
Therefore, the inventor of this application has excellent thermal stability,
As a result of repeated research to search for novel aminopeptidases that can be advantageously used in food manufacturing processes, the highly thermophilic bacterium Thermus aquaticus YT-1 (ATCC
We discovered that an aminopeptidase with excellent heat resistance can be produced from the bacterial cells of B. 25104), and based on this knowledge, we completed this invention.
この発明は、第１に、サーマス・アクアテイカ
ス（Thermus aquaticus）YT−１（ATCC
25104）の菌体から取り出され、下記の性質を有
する新規アミノペプチダーゼに関する。 This invention first relates to Thermus aquaticus YT-1 (ATCC
25104) and has the following properties.
(a) この酵素の活性の至適PHは8.5〜9.0である。(a) The optimum pH for the activity of this enzyme is 8.5-9.0.
(b) この酵素の活性の至適温度は75℃〜80℃であ
る。(b) The optimum temperature for the activity of this enzyme is 75°C to 80°C.
(c) この酵素の熱安定性は、Ala−２−NAを基
質として、80℃で酵素濃度20μｇ／ml、PH7.2で
は、５時間後で約90％、20時間後で約60％の残
存活性を示す。(c) The thermostability of this enzyme is approximately 90% after 5 hours and approximately 60% after 20 hours using Ala-2-NA as a substrate at 80℃, enzyme concentration of 20μg/ml, and pH 7.2. Indicates residual activity.
(d) この酵素は、精製工程中の粗酵素では更に高
い熱安定性を有する。(d) The enzyme has higher thermostability than the crude enzyme during the purification process.
(e) この酵素は、金属キレート剤によつて完全に
失活し、SH阻害剤によつてかなり阻害される。(e) The enzyme is completely inactivated by metal chelators and significantly inhibited by SH inhibitors.
(f) この酵素は、EDTAで処理した後、Co2+に
より賦活する。(f) The enzyme is activated with Co 2+ after treatment with EDTA.
(g) この酵素の分子量はゲル過法で約10万８千
であり、SDS電気泳動法で約４万８千のサブユ
ニツトからなる二量体酵素である。(g) The molecular weight of this enzyme is approximately 108,000 as determined by gel filtration, and it is a dimeric enzyme consisting of approximately 48,000 subunits as determined by SDS electrophoresis.
(h) この酵素は広い基質特異性を示す。(h) This enzyme exhibits broad substrate specificity.
この発明は、第２に、サーマス・アクアテイカ
ス（Thermus aquaticus）YT−１（ATCC
25104）を超音波処理で粉砕抽出し、その後、80
％飽和硫安による塩析、DEAEセフアセル及びハ
イドロキシアパタイトクロマトグラフイ、ゲル
過、デイスク電気泳動など適宜既知の手法を組み
合わせることにより上述の(a)〜(h)の性質を有する
新規アミノペプチダーゼを製造する方法に関す
る。 Second, this invention relates to Thermus aquaticus YT-1 (ATCC
25104) was crushed and extracted using ultrasonication, and then 80
A novel aminopeptidase having the above-mentioned properties (a) to (h) is produced by appropriately combining known methods such as salting out with % saturated ammonium sulfate, DEAE cephacel and hydroxyapatite chromatography, gel filtration, and disk electrophoresis. Regarding the method.
次にこの発明を実施例により詳しく説明する。 Next, this invention will be explained in detail with reference to examples.
Ａ 最初に高度好熱性細菌、サーマス・アクアテ
イカス（Thermus aquaticus）YT−１の菌体
より新規アミノペプチダーゼを製造する過程に
ついて説明する。A First, we will explain the process of producing a novel aminopeptidase from the cells of the highly thermophilic bacterium Thermus aquaticus YT-1.
(1) 菌の培養及び保存：サーマス・アクアテイ
カスYT−１の培養は、グルコース、ポリペ
プトン、イーストエキストラクトの混合培地
PH7.6を用いて70〜80℃で行い。定常期の菌
体を凍結保存した。 (1) Culture and preservation of bacteria: Thermus aquaticus YT-1 is cultured in a mixed medium of glucose, polypeptone, and yeast extract.
Perform at 70-80 °C using PH7.6. The cells in the stationary phase were stored frozen.
(2) 菌体抽出粗酵素の調製：培養後、凍結保存
してある菌体を0.05Mリン酸緩衝液をPH7.0
に懸濁（10％Ｗ／Ｖ）した後、超音波処理を
行なつた。その後、遠心分離（35000×ｇ、
20分）で得られた上清液を、同上緩衝液で透
析し、粗酵素液とした。 (2) Preparation of bacterial cell extraction crude enzyme: After culturing, freeze-preserved bacterial cells are mixed with 0.05M phosphate buffer at pH 7.0.
(10% W/V) and then subjected to ultrasonication. After that, centrifugation (35000 x g,
The supernatant obtained in 20 minutes) was dialyzed against the same buffer solution to obtain a crude enzyme solution.
(3) 硫安塩析：上記粗酵素液に硫安を80％飽和
となるように加え、30分放置後、遠心分離
（10000×ｇ、30分）を行い、沈殿を0.01Mリ
ン酸緩衝液PH7.0に懸濁し、同緩衝液で透析
を行なつた。 (3) Ammonium sulfate salting out: Add ammonium sulfate to the above crude enzyme solution to 80% saturation, leave it for 30 minutes, centrifuge (10000 x g, 30 minutes), and collect the precipitate in 0.01M phosphate buffer PH7. 0, and dialyzed against the same buffer.
(4) DEAEセフアセルクロマトグラフイによる
分画：硫安沈殿画分を、0.01Mリン酸緩衝液
PH7.0で平衡化したDEAEセフアセルカラム
（20×500mm）に吸着させた。0.1〜0.6Mの
NaCl濃度勾配で１の同緩衝液で溶出した。
その結果を第１図に示す。なお第１図におい
て、溶出速度50ml／Ｈ、10ml／画分、反応条
件は0.05Mトリス塩酸緩衝液PH9.0を使用し、
80℃、30分である。第１図より、Al2−２−
NAとVal−Gly−Glyとの活性画分は一致し
ていることが分かる。No.33〜46を活性画分と
した。 (4) Fractionation by DEAE-cephacel chromatography: The ammonium sulfate precipitated fraction was added to 0.01M phosphate buffer.
It was adsorbed onto a DEAE Sephacel column (20 x 500 mm) equilibrated at pH 7.0. 0.1~0.6M
It was eluted with 1 part of the same buffer using a NaCl concentration gradient.
The results are shown in FIG. In Figure 1, the elution rate is 50 ml/H, 10 ml/fraction, and the reaction conditions are 0.05 M Tris-HCl buffer pH 9.0.
80℃, 30 minutes. From Figure 1, Al 2 -2-
It can be seen that the active fractions of NA and Val-Gly-Gly are the same. Nos. 33 to 46 were designated as active fractions.
(5) ハイドロキシアパタイトクロマトグラフイ
による分画：(4)の画分の1/2を同上緩衝液で
透析した後、カラム（15×150mm）に吸着さ
せ、0.01〜0.4Mのリン酸緩衝液PH7.0、400ml
で溶出した結果を第２図に示す。なお第２図
において、溶出速度10ml／Ｈ、５ml／画分、
反応条件は第１図と同一、No.28〜39を活性画
分とした。 (5) Fractionation by hydroxyapatite chromatography: After dialyzing 1/2 of the fraction in (4) with the same buffer, adsorb it on a column (15 x 150 mm) and add 0.01 to 0.4 M phosphate buffer. PH7.0, 400ml
The results of the elution are shown in Figure 2. In addition, in Fig. 2, the elution rate is 10 ml/H, 5 ml/fraction,
The reaction conditions were the same as in FIG. 1, and Nos. 28 to 39 were used as active fractions.
(6) セフアデツクス．G150ゲル過による分
画：(5)の活性画分を濃縮後、0.05Mリン酸緩
衝液PH7.0で平衡化を行なつた。試料をカラ
ム（φ20×550mm）にのせ、先の緩衝液で溶
出した。その結果を第３図に示す。なお第３
図において、溶出速度10ml、５ml画分、反応
条件は第１図と同一であり、No.19〜25を活性
画分とした。 (6) Safety index. Fractionation by G150 gel filtration: After concentrating the active fraction of (5), equilibration was performed with 0.05M phosphate buffer PH7.0. The sample was placed on a column (φ20 x 550 mm) and eluted with the above buffer. The results are shown in FIG. Furthermore, the third
In the figure, the elution rate of 10 ml, 5 ml fractions, and reaction conditions were the same as in Figure 1, and Nos. 19 to 25 were designated as active fractions.
(7) DEAEセフアセルによる再クロマトグラフ
イー：セフアデツクス．G150における活性
画分を0.01Mリン酸緩衝液PHで7.0で透析後、
カラム（15×120mm）に吸着させ、同上緩衝
液で0.1M〜0.4MのNaCl 400mlで溶出させ
た。その結果を第４図に示す。なお第４図に
おいて、溶出速度20ml／Ｈ、５ml／画分、反
応条件は第１図と同一であり、画分No.36〜39
を集め、活性画分とした。 (7) Rechromatography using DEAE Sephacel: Sephadec. After dialyzing the active fraction in G150 with 0.01M phosphate buffer pH 7.0,
It was adsorbed onto a column (15 x 120 mm) and eluted with 400 ml of 0.1M to 0.4M NaCl in the same buffer. The results are shown in FIG. In Fig. 4, the elution rate is 20 ml/H, 5 ml/fraction, and the reaction conditions are the same as in Fig. 1.
were collected and used as the active fraction.
(8) デイスク電気泳動による分画及び精製の確
認：(7)の活性画分を濃縮した後、7.5％ポリ
アクリルアミドゲル（PH8.0）を用い、１本
のゲル当り、たん白質200μｇをのせ、２ｍ
Ａ／１本で約２時、泳動を行い、１本は染色
し、他は２mmごとにスライスし、0.05Mリン
酸緩衝液PH7.0で抽出を行つた。染色したゲ
ルは第５図のバンドを示し、第５図の↓の部
分と酵素活性が一致した。この活性画分を集
め、さらに泳動を行ない、酵素の精製を確認
した。第６図は精製した酵素の活性を示す。
この画分を酵素標品とした。酵素の製造過程
を第７図の図表に示す。新規アミノペプチダ
ーゼは粗酵素液と比較して比活性において
Al2−２−NAでは約600倍、Val−Gly−Gly
では約150培に精製され、収率は各々、34.5
％、7.9％であつた。 (8) Confirmation of fractionation and purification by disc electrophoresis: After concentrating the active fraction from (7), use a 7.5% polyacrylamide gel (PH8.0) and place 200 μg of protein on each gel. , 2m
A/1 tube was run for about 2 hours, one tube was stained, the others were sliced every 2 mm, and extracted with 0.05M phosphate buffer PH7.0. The stained gel showed the bands shown in Figure 5, and the enzyme activity coincided with the ↓ part in Figure 5. The active fractions were collected and subjected to further electrophoresis to confirm the purification of the enzyme. Figure 6 shows the activity of the purified enzyme.
This fraction was used as an enzyme standard. The enzyme manufacturing process is shown in the diagram of FIG. The new aminopeptidase has a higher specific activity compared to the crude enzyme solution.
Approximately 600 times greater for Al 2 −2−NA, Val−Gly−Gly
It was purified to about 150 cultures, and the yield was 34.5 each.
%, 7.9%.
Ｂ こうして製造された新規アミノペプチダーゼ
の性質を調べた実験結果について次に説明す
る。B Next, the results of experiments investigating the properties of the novel aminopeptidase produced in this way will be explained.
(1) 第８図は酵素活性と温度との関係について
示している。この酵素は高い温度で活性があ
り、至適活性温度は75℃〜80℃である。反応
条件は、Al2−２−NA及びVal−Gly−Gly
を基質としてPH9.0で30分行なつた。 (1) Figure 8 shows the relationship between enzyme activity and temperature. This enzyme is active at high temperatures, and its optimal activity temperature is 75°C to 80°C. The reaction conditions were Al2-2 -NA and Val-Gly-Gly
was used as a substrate at pH 9.0 for 30 minutes.
(2) 第９図は、酵素標品及び粗酵素に対する温
度の影響結果を示している。酵素標品を80℃
の温度で保持した場合、酵素濃度20μｇ／ml
PH7.2では、20時間で約60％、40時間でも約
20％の残存活性を有する。また粗酵素では、
先と同一の条件で20時間約70％、40時間でも
約50％の残存活性を示す。非常に熱に安定で
ある。反応条件は、Leu−４−NAを基質と
して70℃で30分行なつた。 (2) Figure 9 shows the effects of temperature on enzyme preparations and crude enzymes. Enzyme preparation at 80℃
Enzyme concentration is 20 μg/ml when kept at a temperature of
At PH7.2, it is about 60% after 20 hours, and about 60% after 40 hours.
Has 20% residual activity. In addition, for crude enzyme,
Under the same conditions as before, it shows about 70% residual activity after 20 hours and about 50% after 40 hours. Very heat stable. The reaction conditions were as follows: Leu-4-NA was used as a substrate at 70°C for 30 minutes.
(3) 第１０図は酵素活性とPHの関係について示
している。酵素の至適活性とPHは8.5〜9.0で
ある。反応条件はAla−２−NA及びVal−
Gly−Glyを基質として70℃で30分行なつた。 (3) Figure 10 shows the relationship between enzyme activity and PH. The optimal activity and pH of the enzyme is 8.5-9.0. The reaction conditions were Ala-2-NA and Val-
The experiment was carried out at 70°C for 30 minutes using Gly-Gly as a substrate.
(4) 第１１図、第１２図及び第１３図は酵素活
性に対する金属イオン及び試薬の影響結果を
示す図表である。この酵素は、EDTAで処
理した後に透析で除去した場合、Co2+によ
つて賦活化する。金属キレート剤によつて完
全に失活し、SH阻害剤によつてかなり阻害
される。また有機溶媒、各種変性剤によつて
も阻害される。反応条件は、Ala−２−NA
及びVal−Gly−Glyを基質としてPH8.5、70
℃で30分行つた。 (4) Figures 11, 12, and 13 are charts showing the effects of metal ions and reagents on enzyme activity. This enzyme is activated by Co 2+ when treated with EDTA and removed by dialysis. It is completely inactivated by metal chelators and significantly inhibited by SH inhibitors. It is also inhibited by organic solvents and various modifiers. The reaction conditions are Ala-2-NA
and PH8.5, 70 using Val-Gly-Gly as a substrate.
℃ for 30 minutes.
(5) 第１４図及び第１５図は酵素の分子量を示
す図表である。この酵素はゲル過法では、
分子量は約10万８千である。SDS電気泳動法
では、分子量は約４万８千である。この事よ
り、約４万８千のサブユニツトからなる二量
体酵素である。 (5) Figures 14 and 15 are charts showing the molecular weight of enzymes. By gel filtration method, this enzyme is
The molecular weight is approximately 108,000. According to SDS electrophoresis, the molecular weight is approximately 48,000. Therefore, it is a dimeric enzyme consisting of approximately 48,000 subunits.
(6) 第１６図は、酵素の基質特異性を調べた実
験の結果を示す図表である。非常に広い基質
特異性を持つ酵素である。反応条件は、PH
8.5で70℃ 30分である。 (6) Figure 16 is a chart showing the results of an experiment investigating the substrate specificity of enzymes. It is an enzyme with extremely broad substrate specificity. The reaction conditions were PH
8.5 and 70℃ for 30 minutes.
第１図はDEAEセフアセルクロマトグラフイに
よる硫安塩析沈殿フラクシヨンのクロマトグラフ
イの結果を示す図、第２図はDEAEセフアセルク
ロマトグラフイによつて得られた酵素フラクシヨ
ンのハイドロキシアパタイトクロマトグラフイの
結果を示す図、第３図はセフアデツクス、Ｇ−
150ゲル過の結果を示す図、第４図はDEAEセ
フアセルによる再クロマトグラフイの結果を示す
図、第５図はデイスク電気泳動後染色したゲルの
バンドを示す図、第６図はゲルスライスの加水分
解活性画分とデイスク電気泳動パターンが一致す
る事を示す図、第７図は新規アミノ−ペプチダー
ゼの製造過程を表わす図表、第８図はこの酵素の
活性と温度の関係を示す図、第９図は酵素活性へ
の加熱の影響を示す図、第１０図は酵素活性とPH
の関係を示す図、第１１図は酵素活性への金属イ
オンの影響を示す図表、第１２図及び第１図は酵
素活性への種々の試薬の影響を示す図表、第１４
図は、酵素の分子量を示す図、第１５図は、酵素
のサブユニツトの分子量を示す図、第１６図は
種々の合成ペプチドに対するこの酵素の相対活性
を示す図表である。
Figure 1 shows the results of chromatography of the ammonium sulfate salt precipitation precipitate fraction by DEAE Cephacel chromatography, and Figure 2 shows the hydroxyapatite chromatography of the enzyme fraction obtained by DEAE Cephacel chromatography. Figure 3 shows the results of Sephadex, G-
Figure 4 shows the results of re-chromatography using DEAE Sephacel, Figure 5 shows the bands of the gel stained after disk electrophoresis, and Figure 6 shows the results of gel slices. Figure 7 shows the agreement between the hydrolyzed active fraction and disk electrophoresis pattern. Figure 7 is a diagram showing the manufacturing process of the new amino-peptidase. Figure 8 is a diagram showing the relationship between the activity of this enzyme and temperature. Figure 9 shows the effect of heating on enzyme activity, and Figure 10 shows enzyme activity and pH.
Figure 11 is a diagram showing the influence of metal ions on enzyme activity, Figure 12 and Figure 1 are diagrams showing the influence of various reagents on enzyme activity, and Figure 14 is a diagram showing the influence of various reagents on enzyme activity.
Figure 15 is a diagram showing the molecular weight of the enzyme, Figure 15 is a diagram showing the molecular weight of the subunits of the enzyme, and Figure 16 is a diagram showing the relative activity of this enzyme with respect to various synthetic peptides.
Claims (1)
aquaticus）YT−１（ATCC 25104）の菌体から
取り出され、下記の性質を有する新規アミノペプ
チダーゼ。 (a) この酵素の活性の至適PHは8.5〜9.0である。 (b) この酵素の活性の至適温度は75℃〜80℃であ
る。 (c) この酵素の熱安定性は、Ala−２−NAを基
質として、80℃で酵素濃度20μｇ／ml、PH7.2で
は、５時間後で約90％、20時間後で約60％の残
存活性を示す。 (d) この酵素は、精製工程中の粗酵素では更に高
い熱安定性を有する。 (e) この酵素は、金属キレート剤によつて完全に
失活し、SH阻害剤によつてかなり阻害される。 (f) この酵素は、EDTAで処理した後、Co2+に
より賦活する。 (g) この酵素の分子量はゲル過法で約10万８千
であり、SDS電気泳動法で約４万８千のサブユ
ニツトからなる二量体酵素である。 (h) この酵素は広い基質特異性を示す。 ２ サーマス・アクアテイカス（Thermus
aquaticus）YT−１（ATCC 25104）を超音波処
理で破砕抽出し、その後、80％飽和硫安による塩
析、DEAEセフアセル及びハイドロキシアパタイ
トクロマトグラフイ、ゲル過、デイスク電気泳
動などの酵素精製に用いられる通常の操作を適宜
組み合わせることにより下記の性質を有する新規
アミノペプチダーゼを製造する方法。 (a) この酵素の活性の至適PHは8.5〜9.0である。 (b) この酵素の活性の至適温度は75℃〜80℃であ
る。 (c) この酵素の熱安定性は、Ala−２−NAを基
質として、80℃で酵素濃度20μｇ／ml、PH7.2で
は、５時間後で約90％、20時間後で約60％の残
存活性を示す。 (d) この酵素は、精製工程中の粗酵素では更に高
い熱安定性を有する。 (e) この酵素は、金属キレート剤によつて完全に
失活し、SH阻害剤によつてかなり阻害される。 (f) この酵素は、EDTAで処理した後、Co2+に
より賦活する。 (g) この酵素の分子量はゲル過法で約10万８千
であり、SDS電気泳動法で約４万８千のサブユ
ニツトからなる二量体酵素である。 (h) この酵素は広い基質特異性を示す。[Claims] 1. Thermus aquaticus
A novel aminopeptidase isolated from the bacterial cells of YT-1 (ATCC 25104) and having the following properties. (a) The optimum pH for the activity of this enzyme is 8.5-9.0. (b) The optimum temperature for the activity of this enzyme is 75°C to 80°C. (c) The thermostability of this enzyme is approximately 90% after 5 hours and approximately 60% after 20 hours using Ala-2-NA as a substrate at 80℃, enzyme concentration of 20μg/ml, and pH 7.2. Indicates residual activity. (d) The enzyme has higher thermostability than the crude enzyme during the purification process. (e) The enzyme is completely inactivated by metal chelators and significantly inhibited by SH inhibitors. (f) The enzyme is activated with Co 2+ after treatment with EDTA. (g) The molecular weight of this enzyme is approximately 108,000 as determined by gel filtration, and it is a dimeric enzyme consisting of approximately 48,000 subunits as determined by SDS electrophoresis. (h) This enzyme exhibits broad substrate specificity. 2 Thermus aquaticus
aquaticus) YT-1 (ATCC 25104) is crushed and extracted by ultrasonication, and then used for enzyme purification such as salting out with 80% saturated ammonium sulfate, DEAE cephacel and hydroxyapatite chromatography, gel filtration, and disk electrophoresis. A method for producing a novel aminopeptidase having the following properties by appropriately combining conventional operations. (a) The optimum pH for the activity of this enzyme is 8.5-9.0. (b) The optimum temperature for the activity of this enzyme is 75°C to 80°C. (c) The thermostability of this enzyme is approximately 90% after 5 hours and approximately 60% after 20 hours using Ala-2-NA as a substrate at 80℃, enzyme concentration of 20μg/ml, and pH 7.2. Indicates residual activity. (d) The enzyme has higher thermostability than the crude enzyme during the purification process. (e) The enzyme is completely inactivated by metal chelators and significantly inhibited by SH inhibitors. (f) The enzyme is activated with Co 2+ after treatment with EDTA. (g) The molecular weight of this enzyme is approximately 108,000 as determined by gel filtration, and it is a dimeric enzyme consisting of approximately 48,000 subunits as determined by SDS electrophoresis. (h) This enzyme exhibits broad substrate specificity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14766685A JPS6211092A (en) | 1985-07-06 | 1985-07-06 | Novel aminopeptidase and method of purifying same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14766685A JPS6211092A (en) | 1985-07-06 | 1985-07-06 | Novel aminopeptidase and method of purifying same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6211092A JPS6211092A (en) | 1987-01-20 |
| JPH0262237B2 true JPH0262237B2 (en) | 1990-12-25 |
Family
ID=15435524
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14766685A Granted JPS6211092A (en) | 1985-07-06 | 1985-07-06 | Novel aminopeptidase and method of purifying same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6211092A (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 (3)
| 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 |
| EP0946106B1 (en) * | 1996-12-23 | 2002-05-29 | Dsm N.V. | Method for producing a protein hydrolysate |
| KR100365838B1 (en) * | 2000-03-24 | 2002-12-26 | 한국생명공학연구원 | A New thermostable D-stereospecific dipeptidase from Brevibacillus borstelensis BCS-1 and its use as a biocatalyst for the synthesis of peptides containing D-amino acids |
-
1985
- 1985-07-06 JP JP14766685A patent/JPS6211092A/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 |
|---|---|
| JPS6211092A (en) | 1987-01-20 |
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