JP5152831B2 - Abnormal prion-degrading enzyme inhibitor and its use - Google Patents
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- 230000002159 abnormal effect Effects 0.000 title claims description 41
- 239000002532 enzyme inhibitor Substances 0.000 title claims description 20
- 229940125532 enzyme inhibitor Drugs 0.000 title claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 230000000694 effects Effects 0.000 claims description 17
- 240000002234 Allium sativum Species 0.000 claims description 15
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- 238000000034 method Methods 0.000 claims description 13
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- 239000003112 inhibitor Substances 0.000 claims description 11
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- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 7
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 4
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 claims description 4
- 230000017854 proteolysis Effects 0.000 claims description 4
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- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims 4
- 238000006731 degradation reaction Methods 0.000 claims 4
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- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims 1
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- 239000006000 Garlic extract Substances 0.000 description 23
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- 108090000790 Enzymes Proteins 0.000 description 16
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 14
- 239000000126 substance Substances 0.000 description 12
- 241001494479 Pecora Species 0.000 description 11
- 210000000936 intestine Anatomy 0.000 description 9
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- 239000000203 mixture Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- MUCZHBLJLSDCSD-UHFFFAOYSA-N diisopropyl fluorophosphate Chemical compound CC(C)OP(F)(=O)OC(C)C MUCZHBLJLSDCSD-UHFFFAOYSA-N 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 229960005051 fluostigmine Drugs 0.000 description 4
- 235000013305 food Nutrition 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
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- 101000807635 Gallus gallus Ribonuclease UK114 Proteins 0.000 description 2
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- 230000000593 degrading effect Effects 0.000 description 2
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- 241000283690 Bos taurus Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
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- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Inorganic materials [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
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Description
本発明は、異常プリオン分解酵素阻害物質及びその用途に関する。 The present invention relates to an abnormal prion-degrading enzyme inhibitor and its use.
わが国において海綿状脳症（ＢＳＥ）に罹患した牛が平成１９年２月までに３２頭確認され、プリオン病発症機序の解明及び治療・予防法の開発が緊急課題となっている。一方、本発明者らは、ラット肝臓から進化的に高度に保存された新規なタンパク質を世界に先駆けて発見し、これを過塩素酸可溶性タンパク質（ＰＳＰ）と名付けた（非特許文献１）。ＰＳＰの立体構造は、異常プリオンタンパク質と極めて類似した立体構造を示す（非特許文献２）。また、プロテイナーゼＫや熱に対する耐性も異常プリオンタンパク質と同様の性質を示したことから、ＰＳＰは異常プリオンタンパク質のモデルとなりうるとの着想に至った。ＰＳＰを基質として異常プリオンタンパク質を分解する酵素をスクリーニングした結果、ある種の放線菌（ストレプトミセス・エスピー９９−ＧＰ−２Ｄ−５株（ＦＥＲＭ Ｐ−１９３３６））が分泌する酵素（プリオナーゼＴＭ）が異常プリオンタンパク質を分解することを見出した（特許文献１及び非特許文献３）。
In Japan, 32 cattle affected by spongiform encephalopathy (BSE) have been confirmed by February 2007, and elucidation of the onset mechanism of prion disease and development of treatment / prevention methods are urgent issues. On the other hand, the present inventors have pioneered a novel protein that is evolutionarily highly conserved from the rat liver and named it a perchloric acid-soluble protein (PSP) (Non-patent Document 1). The three-dimensional structure of PSP shows a three-dimensional structure very similar to the abnormal prion protein (Non-patent Document 2). In addition, the proteinase K and heat resistance showed the same properties as the abnormal prion protein, leading to the idea that PSP can be a model for an abnormal prion protein. As a result of screening an enzyme that degrades abnormal prion protein using PSP as a substrate, an enzyme (prionase TM ) secreted by a certain actinomycetes (Streptomyces sp. 99-GP-2D-5 strain (FERM P-19336)) It has been found that the abnormal prion protein is degraded (
一方、ソーセージの皮は天然ケーシングとして羊腸から生成される。ＥＵではＢＳＥが羊へ感染することが実験的に確認されている（非特許文献４）。自然界でＢＳＥ罹患羊が確認された場合、食の安全に深刻な影響を与えることになる（非特許文献５）。本発明者らは、既にプリオナーゼの性質を明らかにするとともに、本酵素を天然ケーシング加工過程に導入し、天然ケーシングの浄化法について検討した（特許文献２）。スクレイピー由来の異常プリオンタンパク質を混在させたモデルケーシングをプリオナーゼで処理すると、わずか１５分で異常プリオンタンパク質が検出限界以下にまで浄化された。また、プリオナーゼは羊腸の異常プリオンタンパク質を浄化できるだけでなく、羊腸をコラーゲンのみの理想的なケーシングに仕上げる酵素であることが明らかにされた。 On the other hand, sausage skin is produced from the sheep intestine as a natural casing. In the EU, it has been experimentally confirmed that BSE infects sheep (Non-patent Document 4). When BSE-affected sheep is confirmed in nature, it will have a serious impact on food safety (Non-patent Document 5). The present inventors have already clarified the properties of prionase, introduced the enzyme into the natural casing processing process, and studied a method for purifying the natural casing (Patent Document 2). When the model casing mixed with scrapie-derived abnormal prion protein was treated with prionase, the abnormal prion protein was purified to below the detection limit in only 15 minutes. In addition, it has been clarified that prionase is an enzyme that not only purifies abnormal prion protein in the sheep intestine but also finishes the sheep intestine into an ideal casing made only of collagen.
過去にオーストラリアで同様の酵素が見出され、ソーセージの製造過程に応用されたが、酵素の活性があまりに強力であったため、加工の過程でケーシングが破損したという経緯が報告されている。プリオナーゼは強力な酵素であることから、ソーセージ製造にプリオナーゼを応用した場合にもケーシングが破損することは十分に懸念される。従って、プリオナーゼを用いる際には、阻害剤を併せて使用することで、ソーセージの安全性及び品質を改善しつつ、ケーシングの破損を予防することが可能となる。プリオナーゼはセリン性プロテアーゼであり、既知の阻害剤としてフェニルメチルスルホニルフルオリド（ＰＭＳＦ）やフルオロリン酸ジイソプロピル（ＤＦＰ）が知られているが、これらの阻害剤は毒性があり、食品に使用することは不可能である。そこで、本発明者らは、食品に由来するプリオナーゼ阻害物質のスクリーニングを行った。その結果、ニンニク抽出液にプリオナーゼ阻害効果があることを明らかにし、その活性本体は低分子の物質であると報告した（非特許文献６）。 In the past, a similar enzyme was found in Australia and applied to the sausage production process, but the activity of the enzyme was so strong that it was reported that the casing was damaged during processing. Since prionase is a powerful enzyme, there is a sufficient concern that the casing will be damaged even when prionase is applied to sausage production. Therefore, when using prionase, it is possible to prevent breakage of the casing while improving the safety and quality of the sausage by using the inhibitor together. Prionase is a serine protease. Phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP) are known as known inhibitors, but these inhibitors are toxic and should be used in foods. Is impossible. Therefore, the present inventors screened a prionase inhibitor derived from food. As a result, it was clarified that the garlic extract had a prionase inhibitory effect, and the active body was reported to be a low-molecular substance (Non-patent Document 6).
本発明の課題は、食品由来の異常プリオン分解酵素阻害物質を提供することである。
前記課題を解決するため、本発明者らが更に検討を重ねたところ、ニンニク抽出液のプリオナーゼ阻害効果の活性本体は先に予想していた低分子の物質ではなく、タンパク質であることを見出すとともに、当該物質を単離・精製することに成功し、本発明を完成するに至った。
An object of the present invention is to provide a food-derived abnormal prion-degrading enzyme inhibitor.
In order to solve the above-mentioned problems, the present inventors have further studied and found that the active body of the garlic extract's prionase inhibitory effect is not a low-molecular substance previously predicted but a protein. The present inventors have succeeded in isolating and purifying the substance and completed the present invention.
（１）ニンニクから抽出され、異常プリオン分解酵素プリオナーゼによるタンパク質分解を阻害する活性を有し、１００℃で２０分間加熱しても当該活性を保持しているが、プロテイナーゼＫ処理により失活することを特徴とする異常プリオン分解酵素阻害物質。
（２）ニンニク水抽出液に８０％飽和になるように硫安を加え、遠心分離後、沈殿を水に再溶解し、一晩透析後、１００℃で２０分間加熱した後、再び遠心分離し、上清を回収後、Ｎａｔｉｖｅ−ＰＡＧＥ法で分離することにより得られる前記（１）に記載の異常プリオン分解酵素阻害物質。
（３）ＳＤＳ−ＰＡＧＥ法にて分子量約１２，０００を示す前記（１）又は（２）に記載の異常プリオン分解酵素阻害物質。
（４）次のアミノ酸配列：
ＧＮＩＬＭＮＤＥＧＬＹＡＧＱＳＬ
で示されるＮ末端アミノ酸配列を含むタンパク質である前記（１）〜（３）のいずれかに記載の異常プリオン分解酵素阻害物質。
（５）前記（１）〜（４）のいずれかに記載の異常プリオン分解酵素阻害物質を含有する異常プリオン分解酵素阻害剤。
（６）ニンニク水抽出液、又はニンニク水抽出物の水溶液に７５〜８５％飽和になるように硫安を加え、遠心分離後、回収した沈殿を水に再溶解し、透析後、８０〜１００℃で加熱した後、再び遠心分離して得られる上清又はその処理物を含有する異常プリオン分解酵素阻害剤。
（７）前記（５）又は（６）記載の異常プリオン分解酵素阻害剤及び異常プリオン分解酵素を用いる羊腸又は羊腸から製造される天然ケーシングの浄化方法。
(1) Extracted from garlic, has activity to inhibit proteolysis by abnormal prionase prionase, and retains the activity even when heated at 100 ° C. for 20 minutes, but is inactivated by proteinase K treatment An abnormal prion-degrading enzyme inhibitor characterized by
(2) Ammonium sulfate was added to the garlic water extract so as to be 80% saturated, and after centrifugation, the precipitate was redissolved in water, dialyzed overnight, heated at 100 ° C. for 20 minutes, and then centrifuged again. The abnormal prion-degrading enzyme inhibitor according to (1) above, which is obtained by collecting the supernatant and then separating it by the Native-PAGE method.
(3) The abnormal prion-degrading enzyme inhibitor according to (1) or (2), which shows a molecular weight of about 12,000 by SDS-PAGE.
(4) The following amino acid sequence:
GNILMNDEGLYAGQSL
The abnormal prion-degrading enzyme inhibitor according to any one of (1) to (3), which is a protein comprising the N-terminal amino acid sequence represented by
(5) An abnormal prion degrading enzyme inhibitor containing the abnormal prion degrading enzyme inhibitor according to any one of (1) to (4).
(6) Ammonium sulfate is added to an aqueous solution of garlic water extract or garlic water extract so as to be 75 to 85% saturated, and after centrifugation, the recovered precipitate is redissolved in water, and after dialysis, 80 to 100 ° C. An abnormal prion-degrading enzyme inhibitor containing a supernatant obtained by heating again with the following centrifugation or a processed product thereof.
(7) A method for purifying a natural casing produced from a sheep intestine or a sheep intestine using the abnormal prion-degrading enzyme inhibitor and the abnormal prion-degrading enzyme according to (5) or (6).
本発明の異常プリオン分解酵素阻害物質はニンニク由来であるので、これを用いることにより、フェニルメチルスルホニルフルオリド（ＰＭＳＦ）やフルオロリン酸ジイソプロピル（ＤＦＰ）のように毒性を有する阻害剤を用いることなく、羊腸又は羊腸から製造される天然ケーシングを浄化することができ、安全でかつ高品質のソーセージの製造が可能になる。 Since the abnormal prion-degrading enzyme inhibitor of the present invention is derived from garlic, it can be used without using a toxic inhibitor such as phenylmethylsulfonyl fluoride (PMSF) or diisopropyl fluorophosphate (DFP). In addition, it is possible to purify the sheep intestine or the natural casing produced from the sheep intestine, and it is possible to produce a safe and high quality sausage.
本発明の異常プリオン分解酵素阻害物質は、ニンニク抽出物から、異常プリオン分解酵素プリオナーゼによるタンパク質分解を阻害する活性を有し、１００℃で２０分間加熱しても当該活性を保持しているが、プロテイナーゼＫ処理により失活する物質を分離することにより得ることができる。 The abnormal prion-degrading enzyme inhibitor of the present invention has an activity of inhibiting proteolysis by the abnormal prion-degrading enzyme prionase from the garlic extract, and retains the activity even when heated at 100 ° C. for 20 minutes. It can be obtained by separating a substance that is inactivated by proteinase K treatment.
抽出溶媒としては、好ましくは水、リン酸緩衝液が用いられ、抽出温度は、通常４〜２０℃であり、抽出時間は、通常１０〜２０分である。本発明の物質は、１００℃で２０分間加熱しても、異常プリオン分解酵素プリオナーゼによるタンパク質分解を阻害する活性を保持しているので、抽出溶媒として、熱水を用いることもできる。 As the extraction solvent, water or a phosphate buffer is preferably used, the extraction temperature is usually 4 to 20 ° C., and the extraction time is usually 10 to 20 minutes. Since the substance of the present invention retains the activity of inhibiting the proteolysis by the abnormal prionase prionase even when heated at 100 ° C. for 20 minutes, hot water can also be used as the extraction solvent.
本発明の異常プリオン分解酵素阻害物質により酵素活性が阻害される異常プリオン分解酵素としては、例えば特開２００５−３４１５２号公報（特許文献１）に記載のストレプトミセス・エスピー９９−ＧＰ−２Ｄ−５株（ＦＥＲＭ Ｐ−１９３３６）が分泌する酵素（プリオナーゼＴＭ）が挙げられる。 Examples of the abnormal prion-degrading enzyme whose enzyme activity is inhibited by the abnormal prion-degrading enzyme inhibitor of the present invention include, for example, Streptomyces sp. 99-GP-2D-5 described in JP-A-2005-34152 (Patent Document 1). An enzyme (prionase TM ) secreted by a strain (FERM P-19336) is mentioned.
本発明の物質を含有するニンニク水抽出液、ニンニク水抽出物の水溶液などの水溶液に７５〜８５％飽和になるように硫安を加え、遠心分離後、回収した沈殿を水に再溶解し、透析後、８０〜１００℃で１０〜６０分間、好ましくは２０分間加熱した後、再び遠心分離し、上清を回収後、Ｎａｔｉｖｅ−ＰＡＧＥ法で精製・分離することにより本発明の物質を得ることができる。 Add ammonium sulfate to an aqueous solution such as a garlic water extract or an aqueous solution of the garlic water extract containing the substance of the present invention so as to be 75 to 85% saturated, and after centrifugation, the recovered precipitate is redissolved in water and dialyzed Thereafter, after heating at 80 to 100 ° C. for 10 to 60 minutes, preferably 20 minutes, centrifugation is performed again, and after collecting the supernatant, the substance of the present invention can be obtained by purification and separation by the Native-PAGE method. it can.
本発明の異常プリオン分解酵素阻害剤には、前記のようにして精製・分離した本発明の物質の他、前記上清又はこれをクロマトグラフィー等の各種精製手段で処理して得られる処理物を用いることもできる。 In addition to the substance of the present invention purified and separated as described above, the abnormal prion-degrading enzyme inhibitor of the present invention includes the supernatant or a treatment product obtained by treating this with various purification means such as chromatography. It can also be used.
本発明の物質は、異常プリオン分解酵素阻害作用を有し、異常プリオン分解酵素を用いる羊腸又は羊腸から製造される天然ケーシングの浄化方法に用いることにより、ソーセージの安全性及び品質を改善しつつ、ケーシングの破損を予防することが可能となる。この際の使用方法としては、特開２００６−１８０８２６号公報（特許文献２）に記載されているように、異常プリオンを有し得る天然ケーシングに異常プリオン分解酵素を加え、該分解酵素の活動に有効な温度でインキュベートし、インキュベート終了後に本発明の異常プリオン分解酵素阻害物質を阻害剤として添加して反応を停止させる方法が挙げられる。 The substance of the present invention has an abnormal prion-degrading enzyme inhibitory action, and improves the safety and quality of sausage by using it in a method for purifying a natural casing produced from the sheep intestine or sheep intestine using the abnormal prion-degrading enzyme. It becomes possible to prevent damage to the casing. As used in this case, as described in JP-A-2006-180826 (Patent Document 2), an abnormal prion-degrading enzyme is added to a natural casing that may have an abnormal prion, and the activity of the decomposing enzyme is thereby increased. There is a method of incubating at an effective temperature and stopping the reaction by adding the abnormal prion-degrading enzyme inhibitor of the present invention as an inhibitor after completion of the incubation.
以下、実施例を挙げて本発明を具体的に説明するが、本発明の範囲は以下の実施例に限定されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, the scope of the present invention is not limited to a following example.
（実施例１）
（１）ニンニク抽出液の調製
ニンニク球根をその５倍重量の水でホモジナイズし、１４，０００×ｇで２０分間遠心分離して上清を得、サンプルとした。
Example 1
(1) Preparation of garlic extract A garlic bulb was homogenized with 5 times its weight of water and centrifuged at 14,000 × g for 20 minutes to obtain a supernatant, which was used as a sample.
（２）ニンニク抽出液のプリオナーゼ阻害活性
非特許文献１(J. Biol. Chem., 270, 30060-30067 (1995))記載の方法に準じて豚肝臓より抽出した過塩素酸可溶性タンパク質（ＰＳＰ）にプリオナーゼを添加し、各倍率に希釈したニンニク抽出液又はフェニルメチルスルホニルフルオリド（ＰＭＳＦ）存在下において６０℃で３０分間加熱した。その後、抗ＰＳＰ抗体（Comp Biochem Physiol B Biochem Mol Biol. 2003 Apr; 134(4): 571-8）を用いたウェスタンブロッティング法でＰＳＰの検出を行った。
ニンニク抽出液はＰＭＳＦと同様にプリオナーゼ阻害効果を示した。また、その阻害効果はニンニク抽出液の濃度依存的であった（図１）。
(2) Prionase inhibitory activity of garlic extract Perchlorate soluble protein (PSP) extracted from pig liver according to the method described in Non-Patent Document 1 (J. Biol. Chem., 270, 30060-30067 (1995)) Prionase was added to the mixture, and the mixture was heated at 60 ° C. for 30 minutes in the presence of garlic extract or phenylmethylsulfonyl fluoride (PMSF) diluted to each magnification. Thereafter, PSP was detected by Western blotting using an anti-PSP antibody (Comp Biochem Physiol B Biochem Mol Biol. 2003 Apr; 134 (4): 571-8).
The garlic extract showed a prionase inhibitory effect similar to PMSF. The inhibitory effect was dependent on the concentration of garlic extract (FIG. 1).
（３）プロテイナーゼＫ処理がニンニク抽出液のプリオナーゼ阻害活性に及ぼす効果
前記ＰＳＰにプリオナーゼを添加し、ニンニク抽出液及びタンパク質分解酵素であるプロテイナーゼＫの存在下、６０℃で３０分間加熱した。その後、前記抗ＰＳＰ抗体を用いたウェスタンブロッティング法でＰＳＰの検出を行った。
その結果、ニンニク抽出液からプリオナーゼ阻害活性が失われた（図２）。これらの結果は、ニンニク抽出液中の阻害物質がタンパク質であることを示唆している。
(3) Effect of proteinase K treatment on prionase inhibitory activity of garlic extract Prionase was added to the PSP, and heated at 60 ° C. for 30 minutes in the presence of garlic extract and proteinase K, which is a proteolytic enzyme. Thereafter, PSP was detected by Western blotting using the anti-PSP antibody.
As a result, the prionase inhibitory activity was lost from the garlic extract (FIG. 2). These results suggest that the inhibitory substance in the garlic extract is a protein.
（４）ニンニク抽出液のプリオナーゼ阻害活性に及ぼす加熱の効果
ニンニク抽出液を１００℃で２０分間加熱した後、１４，０００×ｇで２０分間遠心分離し、沈殿を除去した。更に前記ＰＳＰにプリオナーゼを添加し、加熱又は非加熱のニンニク抽出液存在下で３０分間加熱した。最後に前記抗ＰＳＰ抗体を用いたウェスタンブロッティング法でＰＳＰの検出を行った。
ニンニク抽出液を１００℃で２０分間加熱してもニンニク抽出液はプリオナーゼに対する活性阻害効果を保持していた（図３）。これらの結果は、ニンニク抽出液中の阻害物質は耐熱性を有するタンパク質であることを示唆している。
(4) Effect of heating on prionase inhibitory activity of garlic extract The garlic extract was heated at 100 ° C. for 20 minutes, and then centrifuged at 14,000 × g for 20 minutes to remove precipitates. Further, prionase was added to the PSP, and the mixture was heated for 30 minutes in the presence of a heated or non-heated garlic extract. Finally, PSP was detected by Western blotting using the anti-PSP antibody.
Even when the garlic extract was heated at 100 ° C. for 20 minutes, the garlic extract retained the activity-inhibiting effect on prionase (FIG. 3). These results suggest that the inhibitor in the garlic extract is a protein with heat resistance.
（５）プリオナーゼ阻害物質の精製
以下のようにして、プリオナーゼ阻害物質を精製した。
先ず、ニンニク水抽出液に８０％飽和になるように硫安を加え、１時間静置した。次に１４，０００×ｇで２０分間遠心分離後、沈殿を回収して水に再溶解した。一晩透析後、１４，０００×ｇで２０分間遠心分離後、上清を回収した。最後に、１００℃で２０分間加熱した後、１４，０００×ｇで２０分間遠心分離し、上清を回収後、Ｎａｔｉｖｅ−ＰＡＧＥ法で分離して精製標品とした。本精製標品は、濃度依存的にプリオナーゼを阻害した（図４）。
(5) Purification of Prionase Inhibiting Substance The prionase inhibiting substance was purified as follows.
First, ammonium sulfate was added to the garlic water extract so as to be 80% saturation, and the mixture was allowed to stand for 1 hour. Next, after centrifugation at 14,000 × g for 20 minutes, the precipitate was recovered and redissolved in water. After dialysis overnight, the supernatant was collected after centrifugation at 14,000 × g for 20 minutes. Finally, after heating at 100 ° C. for 20 minutes, the mixture was centrifuged at 14,000 × g for 20 minutes, and the supernatant was collected and separated by the Native-PAGE method to obtain a purified sample. This purified preparation inhibited prionase in a concentration-dependent manner (FIG. 4).
また、精製標品をＳＤＳ−ＰＡＧＥに供した結果、分子量約１２，０００のタンパク質が検出された（図５）。本タンパク質のＮ末端アミノ酸配列を解析した結果、ＧＮＩＬＭＮＤＥＧＬＹＡＧＱＳＬ（配列番号１）であることが明らかにされた。更に、データベースによる解析を行った結果、同様のアミノ酸配列を有するタンパク質は見出されなかったことから本阻害物質は新規なタンパク質であることが明らかにされた。 Further, as a result of subjecting the purified sample to SDS-PAGE, a protein having a molecular weight of about 12,000 was detected (FIG. 5). As a result of analyzing the N-terminal amino acid sequence of this protein, it was revealed that it was GNILMNDEGLYAGQSL (SEQ ID NO: 1). Furthermore, as a result of analysis using a database, no protein having the same amino acid sequence was found. Thus, it was revealed that this inhibitor is a novel protein.
本発明の異常プリオン分解酵素阻害物質は、安全でかつ高品質のソーセージの製造に利用される。 The abnormal prion-degrading enzyme inhibitor of the present invention is used for the production of safe and high-quality sausages.
Claims (6)
ＧＮＩＬＭＮＤＥＧＬＹＡＧＱＳＬ
で示されるＮ末端アミノ酸配列を含むタンパク質からなる請求項１又は２記載の異常プリオン分解酵素阻害剤。 The following amino acid sequence:
GNILMNDEGLYAGQSL
Abnormal prion degradation inhibitor according to claim 1 or 2, wherein consists protein comprising the N-terminal amino acid sequence shown in.
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