JP2008290959A - Abnormal prion-decomposing enzyme inhibitory substance and use of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an abnormal prion-decomposing enzyme inhibitory substance derived from a food stuff. <P>SOLUTION: This abnormal pion-decomposing enzyme inhibitory substance is characterized by being extracted from garlic, having activity of inhibiting the protein decomposition by the abnormal prion-decomposing enzyme, prionase, holding the activity even by heating at 100°C for 20 min, but inactivated by a treatment with a proteinase K, and the prion-decomposing enzyme inhibitor containing the above substance and a method for cleaning sheep intestine or natural casings produced from the sheep intestine by using the above substance and the abnormal prion-decomposing enzyme are also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an abnormal prion-degrading enzyme inhibitor and its use.

In Japan, 32 cattle affected with 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 (Patent Document 1 and Non-Patent Document 3).

  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.

  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).

JP-A-2005-34152 JP 2006-180826 A Oka T, Tsuji H, Noda C, Sakai K, Hong YM, Suzuki I, Munoz S, Natori Y., Isolation and characterization of a novel perchloric acid-soluble protein inhibiting cell-free protein synthesis., J. Biol. Chem. , 270, 30060-30067 (1995). Carugo KD, Saraste M, Oka T., Crystallization and preliminary X-ray diffraction studies of perchloric acid soluble protein (PSP) from rat liver., Acta Cryst., D55, 667-668 (1999). Hui Z, Doi H, Kanouchi H, Matsuura Y, Mohri S, Nonomura Y, Oka T., Alkaline serine protease produced by Streptomyces sp. Degrades PrP (Sc)., Biochem. Biophys. Res. Commun., 321, 45- 50 (2004). Foster JD, Bruce M, McConnell I, Chree A, Fraser H., Detection of BSE infectivity in brain and spleen of experimentally infected sheep., Vet. Rec., 138, 546-548 (1996). Declan Butler., A wolf in sheep's clothing., Nature, 414, 576-577 (2001). Proceedings of the 2006 Annual Meeting of the Japan Society of Nutrition and Food Science Kyushu / Okinawa Branch and Agricultural Chemical Society West Japan Branch

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) 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).

  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.

  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.

  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.

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.

  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.

  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.

  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.

  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.

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) 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) 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) 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) 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).

  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.

  The abnormal prion-degrading enzyme inhibitor of the present invention is used for the production of safe and high-quality sausages.

It is a figure which shows the effect of the garlic extract on prionase activity. It is a figure which shows the effect which proteinase K processing has on the prionase inhibitory activity of a garlic extract. It is a figure which shows the effect of the heating which acts on the prionase inhibitory activity of a garlic extract. It is a figure which shows the prionase inhibitory activity of a garlic extract refinement | purification sample. It is a figure which shows the result of SDS-PAGE of a garlic extract refinement | purification standard.

Claims (7)

  It is extracted from garlic, has an 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. Abnormal prion-degrading enzyme inhibitor.   Add ammonium sulfate to the garlic water extract to 80% saturation, centrifuge and then redissolve the precipitate in water. After dialysis overnight, heat at 100 ° C. for 20 minutes and then centrifuge again. The abnormal prion-degrading enzyme inhibitor according to claim 1, which is obtained by separation by the Native-PAGE method after recovery.   The abnormal prion-degrading enzyme inhibitor according to claim 1 or 2, which exhibits a molecular weight of about 12,000 by SDS-PAGE. The following amino acid sequence:
GNILMNDEGLYAGQSL
The abnormal prion-degrading enzyme inhibitor according to any one of claims 1 to 3, which is a protein comprising an N-terminal amino acid sequence represented by:   An abnormal prion-degrading enzyme inhibitor comprising the abnormal prion-degrading enzyme inhibitor according to any one of claims 1 to 4.   Ammonium sulfate was added to a garlic water extract or an aqueous solution of garlic water extract so as to become 75 to 85% saturation, and after centrifugation, the recovered precipitate was redissolved in water, and after dialysis, heated at 80 to 100 ° C. Thereafter, an abnormal prion-degrading enzyme inhibitor containing a supernatant obtained by centrifugation again or a processed product thereof.   The purification method of the natural casing manufactured from the sheep intestine using the abnormal prion-degrading enzyme inhibitor and abnormal prion-degrading enzyme of Claim 5 or 6.

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