US007776579B2 (12) United States Patent (10) Patent No.: US 7,776,579 B2 Miwa et al. (45) Date of Patent: Aug. 17, 2010 (54) METHOD OF DEGRADING HARDLY FOREIGN PATENT DOCUMENTS DEGRADABLE PROTEIN CN 12014.89 12/1998 (75) Inventors: Takehiro Miwa, Kanagawa (JP); Koji E. o: E. Nishizawa, Saitama (JP); Yoshie WO WO95/33056 12/1995 Hayashi, Saitama (JP); Manabu WO WO98, 20115 A1 5, 1998 Watanabe, Kanagawa (JP); Yuichi WO WO 98.30682 A1 * T 1998 Murayama, Ibaraki (JP); Miyako WO WO O2/O53723 A2 7, 2002 Yoshioka, Ibaraki (JP); Katsuhiro WO WO 02/083O82 A1 10, 2002 Miura, Ibaraki (JP) OTHER PUBLICATIONS (73) Assignee: Meiji Seika Kaisha, Ltd., Tokyo (JP) Genov et al., Biochem J, 1982, vol. 207, p. 193-200.* Lin et al. "Nucleotide Sequence and Expression of Kera, the Gene (*) Notice: Subject to any disclaimer, the term of this Encoding a Keartinolytic Protease of Bacillus licheniformis PWD patent is extended or adjusted under 35 I”, Applied and Environmental Microbiology, Washington, D.C., U.S.C. 154(b) by 513 days. US, vol. 61, No. 4, Apr. 1995, pp. 1469-1474, XP002042752. Yoshioka et al. “Characterization of a Proteolytic Enzyme Derived (21) Appl. No.: 10/532,605 From a Bacillus Strain That Effectively Degrades Prion Protein', Journal of Applied Microbiology Feb. 2007, vol. 102, No. Feb. 2007, (22) PCT Filed: Oct. 24, 2003 pp. 509-515, XP002422549. Linet al., App. Env, Microbiol. 1992, vol. 58, No. 10, pp. 3271-3275. (86) PCT NO.: PCT/UP03/13658 Nature, Aug. 11, 1994, pp. 471-474, vol. 370. S371 (c)(1), k cited. by examiner (2), (4) Date: Aug. 18, 2005 Primary Examiner L. Blaine Lankford Assistant Examiner—Kade Ariani (87) PCT Pub. No.: WO2004/042049 (74) Attorney, Agent, or Firm Sughrue Mion, PLLC PCT Pub. Date: May 21, 2004 (57) ABSTRACT (65) Prior Publication Data Disclosed is an agent for digesting a protein highly resistant to US 2006/0134092 A1 Jun. 22, 2006 denaturation and degradation, comprising as an active ingre s dient an enzyme exhibiting an activity of digesting a protein (30) Foreign Application Priority Data highly resistant to denaturation and degradation and having the following properties: Oct. 24, 2002 (JP) ............................. 2002-309248 (a) activity and Substrate specificity: hydrolyzing a peptide (51) Int. Cl. Ra protein highly resistant to denaturation and deg CI2N 9/56 (2006.01) (b) molecular weight: 31,000 (determined by SDS-polyacry (52) U.S. Cl. ........................ 435/222; 435/183; 435/212 lamide gel electrophoresis using a homogeneous gel hav (58) Field of Classification Search ............... ... None ing a gel concentration of 12%); See application file for complete search history. (c) isoelectric point: p19.3 (determined by polyacrylamide (56) References Cited gel isoelectric focusing electrophoresis); (d) optimum pH: pH 9.0 to 10.0; and U.S. PATENT DOCUMENTS (e) optimum temperature for activity: 60 to 70° C. 5,981,255 A 1 1/1999 Miyota et al. 6,613,505 B2 9, 2003 Shih 8 Claims, 6 Drawing Sheets U.S. Patent Aug. 17, 2010 Sheet 1 of 6 US 7,776,579 B2 100.0 1V O O O. O 1N O 1 2 3 4 5 6 7 8 9 10 1 12 13 14 Temperature (C) U.S. Patent Aug. 17, 2010 Sheet 2 of 6 US 7,776,579 B2 F I G. 3 h H. h g O A O Hl (t D Hl. s D : x - N N N N N - 0.2 0.2 1 0.2 (Aug/mL)l F I G. 4 Enzyme composition A Proteinase K -1N-N -1N-N 2 4 8 16 50 25 12.5 6.25 (pug-1mL) (dilution) -C 32kD - 19kD U.S. Patent Aug. 17, 2010 Sheet 3 of 6 US 7,776,579 B2 F I. G. 5 Enzyme Enzyme Proteinase K composition B composition A --1N N---N-N 4 2 0.4 2 O 5 (dilution) (11 g/mL) F I G. 6 O () O 8 8 O O O it it t g g O2, 2.O 3 9 ti N is isH. D. isH. MD isH. D. OE. OE. O O O s 1 U as a 2 -C 32kD - 19kD U.S. Patent Aug. 17, 2010 Sheet 4 of 6 US 7,776,579 B2 Enzyme composition A' Thermoase M.W. -N- (-1^ N marker 4 8 16 32 4 8 16 32 (U/mL) i 25 20 U.S. Patent Aug. 17, 2010 Sheet 5 of 6 US 7,776,579 B2 F I. G. 8 U.S. Patent Aug. 17, 2010 Sheet 6 of 6 US 7,776,579 B2 F I. G. 9 100 468 OOO 2 O O 7.5 Enzyme 15 7.5 15 composition A' (U/mL) Thermoase (U/mL) US 7,776,579 B2 1. 2 METHOD OF DEGRADINGHARDLY Furthermore, International Publication No. 02/053723 DEGRADABLE PROTEIN (patent reference 4) discloses that a heat-resistant protease is used in digesting a pathogenic prion protein. However, it TECHNICAL FIELD discloses that when a pathogenic prion protein was digested by a protease derived from Bacillus thermoproteolytics The present invention relates to an agent for digesting a Rokko described in Examples thereof, the pathogenic prion protein highly resistant to denaturation and degradation (par protein was not sufficiently digested with the protease alone, ticularly a pathogenic prion protein) and a method for digest but was sufficiently digested with the protease in the presence ing the protein. of Sodium dodecyl Sulfate. In addition, a neutral Salt is nec 10 essary to activate the protease. Further, the protease requires Background Art a metal ion, and thus when a chelating agent is present in a reaction, the activity is remarkably decreased. A pathogenic prion protein seems to be involved in Such (non-patent reference 1) Nature, (Great Britain), 1994, Vol. diseases as scrapie in sheep or mice, Creutzfeldt-Jakob dis 370, p. 471 ease (CJD) in humans, and bovine spongiform encephalopa 15 (patent reference 1) Japanese Unexamined Patent Publication thy (BSE: popularly known as mad cow disease) in cattle give (Kokai) No. 6-46871 rise to nervous symptoms such as dysstasia or dysbasia. It is (patent reference 2) Unexamined International Publication noted that human consumption of beef infected with the (Kohyo) No. 10-500863 pathogenic prion protein may cause a variant Creutzfeldt (patent reference 3) U.S. Pat. No. 6,613.505 Jakob disease (VCJD) by infection. In particular, BSE is an (patent reference 4) International Publication No. 02/053723 extremely serious disease in the light of a safe supply of beef for human consumption. DISCLOSURE OF THE INVENTION Such diseases may develop when the pathogenic prion protein transferred into the human body from the outside An object of the present invention is to provide an enzyme causes a conformational change of a normal prion protein 25 produced at a low cost and exhibiting a high activity of digest generally located in the brain Nature, (Great Britain), 1994, ing a protein highly resistant to denaturation and degradation Vol. 370, p. 471 (non-patent reference 1). To prevent the (particularly a pathogenic prion protein) in comparison with development of disease by an infection of the pathogenic known proteases; an agent for digesting a protein highly prion protein, it is necessary to digest and detoxify the patho resistant to denaturation and degradation and an agent for 30 detoxifying a pathogenic prion protein, containing the genic prion protein as a cause thereof to the extent that the enzyme as an active ingredient; and a method for digesting a disease does not develop. protein highly resistant to denaturation and degradation (par However, the pathogenic prion protein is believed to be ticularly a pathogenic prion protein) and a method for detoxi extremely stable when subjected to a commonly used steril fying a pathogenic prion protein, using the enzyme or the izing treatment (Such as boiling) and exhibits little or no loss 35 agent. of infectivity by the sterilizing treatment. Further, although The present inventors found an enzyme exhibiting an the pathogen is a protein, it is not difficult to digest the extremely high activity of digesting a protein strongly resis pathogen completely with a conventional protease. Under tant to denaturation and degradation (particularly a patho these circumstances, a method for digesting the pathogenic genic prion protein) derived from a microorganism belonging prion protein efficiently and a method for preventing the 40 to genus Bacillus, in comparison with enzymes known to diseases from developing by infection are desired. digest a protein highly resistant to denaturation and degrada As a method for digesting a protein highly resistant to tion. denaturation and degradation Such as a pathogenic prion pro The enzyme which may be used in the present invention tein, for example, Japanese Unexamined Patent Publication exhibited excellent properties, as shown in Examples (Kokai) No. 6-46871 (patent reference 1) discloses a method 45 described below, in comparison with the above-mentioned for digesting keratin-containing proteins highly resistant to enzymes previously reported to be used in digesting a patho conventional proteases, using keratinase, a protease, derived genic prion protein, for example, the enzyme (keratinase) from Bacillus licheniformis PWD-1. The publication dis prepared from Bacillus licheniformis PWD-1 disclosed in closes that keratinase is used in digesting keratin-containing U.S. Pat. No. 6,613,505, and the enzyme prepared from proteins (for example, animal hair, human hair, or feathers), 50 Bacillus thermoproteolyticus Rokko disclosed in Interna but neither discloses nor Suggests any effects of the keratinase tional Publication No. 02/053723. on a pathogenic prion protein. Particularly, it was found that the enzyme which may be In this connection, a DNA encoding the keratinase derived used in the present invention exhibited an extremely high from Bacillus licheniformis PWD-1 was obtained Unexam activity of digesting a pathogenic prion protein in comparison ined International Publication (Kohyo) No.