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, O2. 3 9 ti N is isH. D. isH. MD isH. D. E.O 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 468OOO 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. 10-500863 55 with the enzyme prepared from Bacillus licheniformis (patent reference 2). PWD-1 (see Examples 7 and 8). Further, it was surprisingly Further, U.S. Pat. No. 6,613,505 (patent reference 3) dis found that the protein was digested without a thermal treat closes that the keratinase derived from Bacillus licheniformis ment described in U.S. Pat. No. 6,613,505 (see Examples 7 PWD-1 is used indigesting a pathogenic prion protein highly and 8). resistant to denaturation and degradation. However, to reduce 60 In comparison with the enzyme prepared from Bacillus or digest the pathogenic prion protein by the method dis thermoproteolyticus Rokko, it was found that the enzyme closed in U.S. Pat. No. 6,613,505, two of treatment step, that which may be used in the present invention exhibited an is, a heat treatment as a pretreatment, and an enzyme treat extremely high activity of digesting a pathogenic prion pro ment, are necessary. In this method, an apparatus for heating tein (see Examples 9 to 11). Further, it was surprisingly found is necessary, and thus it is not easy to carry out the method in 65 that the protein exhibited an excellent activity of digesting a common facilities without Such an apparatus for heating. pathogenic prion protein regardless of the presence of sodium Further, the two-step procedures are complicated. dodecyl sulfate (see Examples 9 to 11). US 7,776,579 B2 3 4 Further, the present inventors provided an agent for digest 10 a method for detoxifying a pathogenic prion protein, ing a protein highly resistant to denaturation and degradation comprising the step of bringing a Subject which may be and an agent for detoxifying a pathogenic prion protein, con contaminated with a pathogenic prion protein into contact taining the newly found enzyme as an active ingredient, and with the enzyme of 1 to 5 or the agent of 8, without further found a method for digesting a protein highly resistant 5 preheating the Subject; to denaturation and degradation (particularly a pathogenic 11 a method for detoxifying a pathogenic prion protein, prion protein) and a method for detoxifying a pathogenic comprising the step of bringing a Subject which may be prion protein, using the enzyme or the agent. contaminated with a pathogenic prion protein into contact The present invention relates to: with the enzyme of 1 to 5 or the agent of 8, without 1 an agent for digesting a protein highly resistant to dena 10 preheating the subject at 90° C. or more; and turation and degradation, comprising as an active ingredi 12 use of the enzyme of 1 to 5, in the manufacture of an ent an enzyme exhibiting an activity of digesting a protein agent for detoxifying a pathogenic prion protein. highly resistant to denaturation and degradation and hav ing the following properties: BRIEF DESCRIPTION OF DRAWINGS (a) activity and Substrate specificity: hydrolyzing a peptide 15 bond of a protein highly resistant to denaturation and deg FIG. 1 is a graph showing the optimum pH and stable pH of radation, a purified enzyme used in the present invention at 37°C. (b) molecular weight: 31,000 (determined by an SDS-poly FIG. 2 is a graph showing the optimum temperature of a acrylamide gel electrophoresis using a homogeneous gel purified enzyme used in the present invention at pH 9.0. having a gel concentration of 12%), FIG. 3 shows the results in which the mouse pathogenic (c) isoelectric point: p19.3 (determined by polyacrylamide prion protein was digested with a purified enzyme used in the gel isoelectric focusing electrophoresis), present invention. (d) optimum pH: pH 9.0 to 10.0, and FIG. 4 shows the results in which the mouse pathogenic (e) optimum temperature for activity: 60 to 70° C.; prion protein was digested with enzyme composition A used 2 the agent of 1, wherein the enzyme has the following 25 in the present invention. property: FIG. 5 shows the results in which the sheep pathogenic (g) exhibiting an activity of 2 U/g or more as the activity of prion protein was digested with enzyme composition A used digesting a protein highly resistant to denaturation and in the present invention. degradation (determined as an activity of digesting keratin FIG. 6 shows the results in which the mouse pathogenic aZure; 30 prion protein was digested with enzyme composition A used 3 the agent of 1 or 2, wherein the enzyme has the fol in the present invention. lowing property: FIG. 7 shows the results in which the hamster pathogenic (h) derived from a microorganism belonging to genus Bacil prion protein (strain Sc237) was digested with enzyme com lus, position A' used in the present invention or thermoase for 4 an agent for digesting a protein highly resistant to dena 35 comparison. turation and degradation, comprising as an active ingredi FIG. 8 shows the results in which the hamster pathogenic ent an enzyme selected from the group consisting of prion protein (strain Sc237) was digested in the presence of (X) an enzyme comprising the amino acid sequence of SEQ SDS with enzyme composition A' used in the present inven ID NO: 2; tion or thermoase for comparison. (Y) a modified enzyme exhibiting an activity of digesting a 40 protein highly resistant to denaturation and degradation, FIG. 9 shows the results in which the hamster pathogenic and comprising an amino acid sequence in which one or prion protein (strain Sc237) stuck on polystyrene was plural amino acids are deleted, substituted, or added in the digested with enzyme composition A' used in the present amino acid sequence of SEQID NO: 2; and invention or thermoase for comparison. (Z) a homologous enzyme exhibiting an activity of digesting 45 a protein highly resistant to denaturation and degradation, BEST MODE FOR CARRYING OUT THE and comprising an amino acid sequence having an 85% or INVENTION more homology with the amino acid sequence of SEQID NO: 2; The present invention will be explained in detail hereinaf 5the agent of 1 to 4, wherein the protein highly resistant 50 ter. to denaturation and degradation is a pathogenic prion pro The enzyme used in the present invention exhibits an activ tein; ity of digesting a protein highly resistant to denaturation and 6 a method for digesting a protein highly resistant to dena degradation, that is, a hydrolytic activity of peptide bonds in turation and degradation, comprising the step of bringing the protein highly resistant to denaturation and degradation. the protein highly resistant to denaturation and degradation 55 The term “protein highly resistant to denaturation and deg into contact with the agent or enzyme of 1 to 5; radation' as used herein means a protein which is not easily 7 use of the enzyme of 1 to 5, in the manufacture of an digested with a commonly used protease Such as proteinase K agent for digesting a protein highly resistant to denatur or trypsin. More particularly, it means a protein which is not ation and degradation; completely digested when treated with 1 ug/mL of proteinase 8 an agent for detoxifying a pathogenic prion protein in a 60 K at 37° C. for 1 hour. As the protein highly resistant to Subject which may be contaminated with a pathogenic denaturation and degradation, there may be mentioned, for prion protein, comprising as an active ingredient the example, a pathogenic prion protein, keratin, collagen, or enzyme of 1 to 5: elastin. 9 a method for detoxifying a pathogenic prion protein, The term "pathogenic prion protein’ as used herein means comprising the step of bringing a Subject which may be 65 a protein which is involved in the onset of, for example, contaminated with a pathogenic prion protein into contact scrapie, CJD., or BSE, more particularly, a prion protein con with the enzyme of 1 to 5 or the agent of 8: formationally changed from a normal prion generally located US 7,776,579 B2 5 6 in the brain. The pathogenic prion protein includes a protein tin derived from wool). The keratin azure is widely used as a derived from, for example, a human, hamster, mouse, bovine, Substance for measuring an activity (i.e., a keratinase activity) or sheep. of digesting keratin, a protein highly resistant to denaturation The normal prion protein and the pathogenic prion protein and degradation, because an azo-pigment-bound amino acid have the same amino acid sequence, but different tertial struc 5 or an azo-pigment-bound peptide released by digesting pep tures. In the normal prion protein, the content of the C-helix tide bonds in keratin can be spectroscopically measured. structure in which the polypeptide chain of the prion protein The “activity of digesting a pathogenic prion protein’ as takes a spiral form is high, and the content of the B-sheet used herein means a hydrolytic activity of peptide bonds in structure in which the polypeptide chain takes a plane form is the pathogenic prion protein. A degree or strength of the low. In construct, the pathogenic prion protein contains a high 10 “activity of digesting a pathogenic prion protein’ may be content of the B-sheet structure (Pan, PNAS, 90, 10962, judged, for example, by analyzing a digestion of the patho 1993). In addition, each prion protein derived from the above genic prion protein contained in a Suspension containing 1% animals has a high homology among the amino acid of a brain tissue derived from a mouse Suffering from scrapie. sequences thereof, and shows the same property in which a More particularly, a brain tissue derived from a mouse conformational change of the normal prion protein causes the 15 infected with the pathogenic prion protein is homogenized to change to the pathogenic prion protein highly resistant to prepare a homogeneous Suspension, and the Suspension is denaturation and degradation. treated with an enzyme or enzyme composition to be judged. The pathogenic prion protein considered to be a pathogen Proteins contained in the reaction mixture are separated by of the above diseases is extremely stable when subjected to a electrophoresis, and the prion protein is detected by Western general sterilizing treatment such as boiling, and thus exhibits blotting. When no band is detected, the result shows that the little or no loss of infectivity by such a sterilizing treatment. enzyme or enzyme composition to be judged exhibits an Further, while the normal prion protein is easily digested, and extremely high activity of digesting a pathogenic prion pro thus a half life thereof in the body is approximately 2 hours, tein. When a protein band resistant to the protease is detected the pathogenic prion protein has a half life of 24 hours or and the band is thin, the result shows that the enzyme or more, and is highly resistant to digestion. When the digest 25 enzyme composition exhibits a moderate activity of digesting ibilities of the normal and pathogenic prion proteins to con a pathogenic prion protein. When the protein band is dense, ventional proteases such as commercially available protein the result shows that the enzyme or enzyme composition ase K were evaluated, it was reported that the normal prion exhibits a low activity of digesting a pathogenic prion protein. protein was easily digested and was sensitive, but the patho genic prion protein exhibited a low digestibility and was 30 Protein Exhibiting an Activity of Digesting a Protein Highly highly resistant to digestion (Prusiner, Science, 252, 1515, Resistant to Denaturation and Degradation (Particularly a 1991). It is considered that the difference in digestibility is Pathogenic Prion Protein) due to the difference in the tertial structures as described In the present invention, for example, an enzyme having above. the following properties may be used. The normal prion protein may be distinguished from the 35 pathogenic prion protein, for example, by utilizing the differ (a) Activity and Substrate Specificity ence in digestibility to a protease. For example, a tissue The enzyme hydrolyzes one or more peptide bonds of a derived from an animal which may be contaminated with the protein, particularly one or more peptide bonds of a protein pathogenic prion protein is homogenized to prepare a homo highly resistant to denaturation and degradation (such as a geneous Suspension. The Suspension is treated with a com 40 pathogenic prion protein). As to the Substrate specificity, the monly used protease Such as proteinase K, and analyzed by enzyme exhibits high activities of digesting casein, collagen, Western blotting (Burnette, Anal. Biochem., 112, 195, 1981) elastin, and keratin, as well as the pathogenic prion protein. to detect the prion protein. When no band is detected, it may (b) Molecular Weight be judged that the tissue contains only the normal prion pro The molecular weight determined by an SDS-polyacryla tein. When a protein band resistant to the protease is detected, 45 it may be judged that the tissue contains the pathogenic prion mide gel electrophoresis using a 12% homogeneous gel (i.e., protein. a homogeneous gel in which a concentration of polyacryla The term “activity of digesting a protein highly resistant to mide is 12%) is approximately 31,000. denaturation and degradation” as used herein means a hydro The molecular weight determined by an SDS-polyacryla lytic activity of peptide bonds in the protein highly resistant to 50 mide gel electrophoresis using a 15% homogeneous gel (i.e., denaturation and degradation. As a unit of the “activity of a homogeneous gel in which a concentration of polyacryla digesting a protein highly resistant to denaturation and deg mide is 15%; such as a gel manufactured by ATTO) is radation', two different units are used herein. In the first unit, approximately 26,000. “1 unit of an enzyme is defined as an amount of the enzyme (c) Isoelectric Point which can generate a product corresponding to 1 umol of 55 The isoelectric point (pl) determined by a polyacrylamide glycine per minute, when a suspension containing 0.5% (as a gel isoelectric focusing electrophoresis is approximately 9.3. final concentration) of keratin powder (derived from human hair; Nacalai Tesque) is treated with the enzyme at pH 8.0 and (d) Optimum pH and Stable pH 60° C. for 1 hour. In the second unit, “1 unit of an enzyme is The optimum pH, evaluated by an activity of digesting defined as 0.001 of an amount of absorbance changed, when 60 keratinazure as an index, is approximately pH 9.0 to 10.0. The a suspension containing 0.8% (as a final concentration) of enzyme exhibits a stable activity at approximately pH 7.0 to keratinazure (Sigma) is treated with the enzyme at pH 8.0 and 12.0, and a high activity at approximately pH 8.0 to 10.5. 37°C. for 16 hours, and an amount of a pigment released to a Supernatant of the reaction mixture perminute is measured at (e) Optimum Temperature for Activity an absorbance of 595 nm. 65 The optimum temperature for activity, evaluated by an In this connection, the keratin azure is a compound in activity of digesting keratin azure as an index, is approxi which an azo pigment is bound to keratin (for example, kera mately 60 to 70° C. US 7,776,579 B2 7 8 (f) Deactivating pH mentioned, for example, a FLAG tag, a hexa-histidine tag, a The enzyme was inactivated at approximately pH 5 or less, hemagglutinin tag, or a myc epitope. when evaluated by an activity of digesting keratinaZure as an As the partner for fusion, there may be mentioned, for index. example, a polypeptide for purification for example, glu In Table 1, the above properties of the enzyme which may 5 tathione S-transferase (GST) or a fragment thereof, a be used in the present invention are shown incomparison with polypeptide for detection for example, hemagglutinin or those of a known protease exhibiting an activity of digesting B-galactosidase Opeptide (LacZC), or a fragment thereof, or a protein highly resistant to denaturation and degradation a polypeptide for expression (for example, a signal sequence). (keratinase derived from Bacillus licheniformis PWD-1). In the above fusion polypeptide, an amino acid sequence 10 which can be specifically digested with a protease Such as thrombin or factor Xa may be optionally inserted between the TABLE 1. polypeptide consisting of the amino acid sequence of SEQID Enzyme used in the NO: 2 and the marker sequence or the partner for fusion. present invention Known protease The term “modified enzyme” as used herein means a pro Activity and substrate specificity --- -- 15 tein comprising an amino acid sequence in which one or pathogenic prion protein plural (for example, one or several) amino acids are deleted, casein -- -- substituted, or added in the amino acid sequence of SEQID collagen -- -- NO: 2, and exhibiting an activity of digesting a protein highly elastin -- -- keratin -- -- resistant to denaturation and degradation (particularly a Molecular weight 31,000 33,000 pathogenic prion protein). In this connection, the number of Isoelectric point 9.3 7.25 amino acids to be modified, such as “deleted, substituted, or Optimum pH 9.0 to 10.O 7.5 added, is preferably 1 to 30, more preferably 1 to 10, most Optimum temperature 60 to 70° C. 50° C. preferably 1 to 6. The “modified enzyme includes a protein comprising an In another embodiment of the present invention, an enzyme 25 amino acid sequence in which one or plural (for example, one comprising the amino acid sequence of SEQID NO: 2, or a or several) amino acids are conservatively Substituted in the modified or homologous enzyme thereof may be used. amino acid sequence of SEQ ID NO: 2, and exhibiting an The “enzyme comprising the amino acid sequence of SEQ activity of digesting a protein highly resistant to denaturation ID NO: 2 includes, for example, and degradation (particularly a pathogenic prion protein). 30 The term "conservative substitution' as used herein means an enzyme consisting of the amino acid sequence of SEQID that one or plural amino acid residues contained in a protein NO: 2; are replaced with differentamino acids having similar chemi cal properties so that the activities of the protein are not a fusion enzyme consisting of an amino acid sequence in Substantially changed. As the conservative substitution, there which an appropriate marker sequence is added to the N-ter 35 may be mentioned, for example, a Substitution of a hydropho minus and/or the C-terminus of the polypeptide consisting of bic residue for another hydrophobic residue, or a substitution the amino acid sequence of SEQID NO: 2, and exhibiting an of a polar residue for another polar residue having the same activity of digesting a protein highly resistant to denaturation charge. Amino acids which have similar chemical properties and degradation (particularly a pathogenic prion protein); and can be conservatively substituted with each other are a fusion enzyme consisting of the polypeptide consisting of 40 known to those skilled in the art. the amino acid sequence of SEQID NO: 2 and a partner for More particularly, as nonpolar (hydrophobic) amino acids, fusion, and exhibiting an activity of digesting a protein highly there may be mentioned, for example, alanine, Valine, isoleu resistant to denaturation and degradation (particularly a cine, leucine, proline, tryptophan, phenylalanine, or methion pathogenic prion protein); and ine. As polar (neutral) amino acids, there may be mentioned, 45 for example, glycine, serine, threonine, tyrosine, glutamine, an enzyme consisting of an amino acid sequence in which a asparagine, or cysteine. As basic amino acids having a posi presequence (signal sequence) or a fragment thereof is added tive charge, there may be mentioned, for example, arginine, to the N-terminus of the amino acid sequence of SEQID NO: histidine, or lysine. As acidic amino acids having a negative 2. In addition, a fusion enzyme consisting of an amino acid charge, there may be mentioned, for example, aspartic acid or sequence in which an appropriate marker sequence and/oran 50 glutamic acid. appropriate partner for fusion is further added to the amino The term "homologous protein’ as used herein means a acid sequence in which a presequence is added to the N-ter protein comprising an amino acid sequence having an 85% or minus of the amino acid sequence of SEQ ID NO: 2, is more (preferably 90% or more, more preferably 95% or more, included in the “enzyme comprising the amino acid sequence still further preferably 98% or more, most preferably 99% or of SEQ ID NO. 2”. 55 more) homology with the amino acid sequence of SEQ ID As the presequence, a naturally-occurring presequence or NO: 2, and exhibiting an activity of digesting a protein highly an artificially designed sequence may be used. As the natu resistant to denaturation and degradation (particularly a rally-occurring presequence, not only a presequence derived pathogenic prion protein). The term "homology' as used from Bacillus licheniformis (particularly a presequence of a herein means a value obtained by a known program for a Bacillus licheniformis derived enzyme capable of digesting a 60 homology search, BLAST (Basic local alignment search tool; protein highly resistant to denaturation and degradation), but Altschul, S. F. et al., J. Mol. Biol., 215, 403-410, 1990; also a presequence derived from organisms other than Bacil obtained from National Center for Biotechnology Informa lus licheniformis, may be used. tion). As the marker sequence, for example, a sequence for easily The enzyme comprising the amino acid sequence of SEQ carrying out a confirmation of polypeptide expression, a con 65 ID NO: 2, or the modified or homologous enzyme thereof firmation of intracellular localization thereof, or a purifica exhibits an activity of digesting a protein highly resistant to tion thereof may be used. As the sequence, there may be denaturation and degradation (particularly a pathogenic prion US 7,776,579 B2 9 10 protein) of preferably 2U/g or more, more preferably 2 to 500 The above Supernatant or concentrated Supernatant is fil U/g, still further preferably 10 to 500U/g, most preferably 20 tered with a microfilter membrane (pore size-approximately to 500 U/g, as an activity of digesting keratin azure. When an 0.45 Lm) to remove microorganisms. Ammonium sulfate is activity of digesting keratin powder is used as an index, it is added to the resulting sterile filtrate, to a final concentration of preferably 1 U/g or more, more preferably 1 to 5000 U/g, 1 mol/L, and a buffer agent (Tris-HCl) is further added to pH most preferably 5 to 3000 U/g. 8.5 and a final concentration of 50 mmol/L. For a further An origin of the enzyme used in the present invention is not purification by a hydrophobic chromatography, the prepared particularly limited, so long as it is an enzyme having the Solution is adsorbed to a phenyl Sepharose column, and above-mentioned physical and chemical properties, an eluted by a linear gradient with ammonium sulfate (1 mol/L to enzyme comprising the amino acid sequence of SEQID NO: 10 O mol/L) in a Tris-HCl buffer, to obtain a fraction containing 2, or a modified or homologous enzyme thereof. For example, the enzyme used in the present invention. The fraction is enzymes derived from animals, plants, or microorganisms concentrated 20 to 30-fold with an ultrafilter (5,000 to may be used. An enzyme produced by an microorganism 10,000-molecular-weight cutoff), and a gel filtration chroma belonging to genus Bacillus is preferable, an enzyme pro tography is carried out, for example, using Superdex 75 duced by Bacillus licheniformis is more preferable, and an 15 (Pharmacia) gel. The concentrated solution is developed enzyme produced by Bacillus licheniformis MSK-103 through the gel with a phosphate buffer (0.025 mol/L, pH 7.0) (FERM BP-08487) is most preferable. Further, a mutant containing 0.1 mol/L Sodium chloride as an eluent, to obtain derived from the microorganisms may be used. the enzyme used in the present invention. According to the above purification procedures, the enzyme used in the present Deposit of Microorganism invention can be purified as a band by an electrophoretic Bacillus licheniformis MSK-103 (FERM BP-08487) was analysis. domestically deposited in the International Patent Organism Polynucleotide Encoding Protein having an Activity of Depositary National Institute of Advanced Industrial Science Digesting a Protein Highly Resistant to Denaturation and and Technology (Address: AIST Tsukuba Central 6, 1-1, Degradation (Particularly a Pathogenic Prion Protein) Higashi 1-chome Tukuba-shi, Ibaraki-ken 305-8566 Japan) 25 The polynucleotide encoding the enzyme used in the on Oct. 16, 2002, and was transferred to an international present invention may be obtained, for example, by the fol deposit on Sep. 16, 2003. The international deposit number (a lowing procedures. When a certain amino acid sequence is number in parenthesis following the international deposit given, a nucleotide sequence encoding the amino acid number is a domestic deposit number) is FERM BP-08487 sequence can be easily determined. Therefore, those skilled FERMP-19068). 30 in the art can select various nucleotide sequences encoding As the enzyme used in the present invention, Subtilisins the enzyme used in the present invention. The term “poly may be used, and subtilisin DY (WO98/30682) is preferable. nucleotide' as used herein includes DNA and RNA, prefer The enzyme used in the present invention may be obtained ably DNA. by isolating and purifying the enzyme of interest from a The polynucleotide encoding the enzyme used in the microorganism, for example, as described in Example 1. 35 present invention may be typically selected from the group Alternatively, it may be obtained by expressing a polynucle consisting of: otide encoding the protein of interest in an appropriate host by (i) a polynucleotide comprising the nucleotide sequence of genetic engineering techniques, and isolating and purifying SEQID NO: 1 (preferably a polynucleotide consisting of the produced protein, as described below. 40 the nucleotide sequence of SEQID NO: 1); To obtain the enzyme used in the present invention from a (ii) a polynucleotide comprising a nucleotide sequence in microorganism producing the enzyme, the microorganism which one or plural (for example, one or several) nucle may be cultivated under the conditions suitable for the micro otides are deleted, substituted, or added in the nucleotide organism, and the obtained broth, Supernatant, or microor sequence of SEQID NO: 1, and encoding a protein exhib ganism may be treated by known separation and purification 45 iting an activity of digesting a protein highly resistant to techniques. Hereinafter procedures of the microorganism denaturation and degradation (particularly a pathogenic cultivation and the protein purification will be explained in prion protein); and accordance with an embodiment using Bacillus licheniformis (iii) a polynucleotide hybridizing under Stringent conditions MSK-103 (FERM BP-08487) as the microorganism produc to a polynucleotide consisting of the nucleotide sequence ing the enzyme used in the present invention. 50 of SEQID NO: 1, and encoding a protein highly resistant A culture medium 1% polypeptone, 0.2% yeast extract, to denaturation and degradation (particularly a pathogenic and 0.1% magnesium sulfate heptahydrate (pH 7.0) is auto prion protein). claved by a conventional method, and the medium is inocu In the polynucleotide described in the above item (ii), the lated with Bacillus licheniformis MSK-103 (FERM number of nucleotides to be deleted, substituted, or added is, BP-08487). A cultivation is carried out at 37-50° C. under 55 for example, 1 to 50, preferably 1 to 30, more preferably 1 to aeration and agitation for 24-72 hours. The resulting broth is 18, most preferably 1 to 9. centrifuged at approximately 3000 G to obtain a supernatant The term “under stringent conditions in the above item containing the enzyme used in the present invention. If nec (iii) means the conditions in which a probe comprising the essary, the supernatant is concentrated 2 to 50-fold with an nucleotide sequence of SEQ ID NO: 1 is hybridized to a ultrafilter (5,000 to 30,000-molecular-weight cutoff) to 60 polynucleotide encoding the above-mentioned homologous obtain a concentrated Supernatant containing the enzyme protein, but the probe is not hybridized to that encoding used in the present invention. keratinase derived from Bacillus licheniformis PWD-1 (U.S. The above Supernatant or the above concentrated Superna Pat. No. 6,613.505) or a protease (such as thermoase) derived tant contains various Substances other than the enzyme used from Bacillus thermoproteolyticus Rokko. in the present invention, and thus the enzyme used in the 65 More particularly, in accordance with a protocol attached present invention may be further purified, for example, by the to an ECL direct DNA/RNA labeling and detection system following procedures. (Amersham), after a polynucleotide to be tested is prehybrid US 7,776,579 B2 11 12 ized at 42°C. for an hour, a labeled probe having the full isolated enzyme used in the present invention. The transfor length of the nucleotide sequence of SEQID NO: 1 is added, mant can be cultivated under the conditions commonly used and hybridization is carried out at 42°C. for 15 hours. After in the cultivation thereof. Further, after the cultivation, the the hybridization, a washing treatment with 0.4 or lessxSSC enzyme of interest can be collected in accordance with an (1xSSC: 15 mmol/L sodium citrate, 150 mmol/L sodium ordinary method in the art. chloride) containing 0.4% SDS and 6 mol/L urea at 42°C. for The optimum process for producing the enzyme used in the 20 minutes is repeated twice, and a washing treatment with present invention may be carried out by using preferably a 5xSSC at room temperature for 10 minutes is carried out microorganism belonging to genus Bacillus, more preferably twice. Bacillus licheniformis, most preferably Bacillus lichenifor The polynucleotide encoding the enzyme used in the 10 mis MSK-103 (FERM BP-08487) or a mutant thereof. present invention includes a naturally-occurring polynucle otide. Further, the whole can be synthesized. Furthermore, the Enzyme Composition, and Agent for Digesting a Protein synthesis may be carried out using part of the naturally Highly Resistant to Denaturation and Degradation and Agent occurring polynucleotide. Typically, the polynucleotide may for Detoxifying a Pathogenic Prion Protein be obtained by Screening a genomic library derived from 15 The enzyme composition or enzyme agent used in the Bacillus licheniformis MSK-103 (FERM BP-08487) in present invention comprises at least an enzyme (hereinafter accordance with an ordinary method commonly used in referred to as “enzyme used in the present invention') genetic engineering, for example, using an appropriate DNA selected from the group consisting of the enzyme having the probe designed on the basis of information of a partial amino above-mentioned physical and chemical properties (includ acid sequence. ing the enzyme obtained by a microorganism); the enzyme comprising the amino acid sequence of SEQID NO: 2, and Expression Vector and Transformed Microorganism the modified or homologous enzyme thereof, and the enzyme The enzyme comprising the amino acid sequence of SEQ by cultivating the above-mentioned host cell. ID NO: 2, or the modified or homologous enzyme thereof, The enzyme composition used in the present invention is which may be used in the present invention, may be produced 25 not particularly limited, so long as it contains as an active by an expression vector comprising a nucleotide sequence ingredient the enzyme used in the present invention. The encoding the enzyme so that the nucleotide sequence may be enzyme composition may be produced by mixing the active replicated and the enzyme may be expressed. The expression ingredient with a carrier or diluent commonly used in prepar vector can be constructed on the basis of a self-replicating ing an enzyme composition, Such as fillers (for example, vector (Such as a plasmid), which exists as an extrachromo 30 Somal element and can replicate independently of the repli lactose, sodium chloride, or Sorbitol), Surfactants, or antisep cation of chromosomes. Alternatively, the expression vector tics, in a desired form Such as powder or liquid. may be a vector which is integrated into the genome of the The content of the enzyme in the enzyme composition is host microorganism and replicated together with chromo not particularly limited, so long as an activity thereof is Suf ficient for the purpose. The content may be 0.01 to 99% by somes, when the host is transformed with the vector. The 35 construction of the vector can be carried out by ordinary weight, preferably 0.1 to 80% by weight. procedures or methods commonly used in genetic engineer With respect to an activity of digesting a protein highly ing. resistant to denaturation and degradation, it is preferable that To express a protein having a desired activity by transform the enzyme composition exhibits 2 U/g or more (more pref ing a host microorganism with the expression vector, it is 40 erably 2 to 500 U/g, still further preferably 10 to 500 U/g, preferable that the expression vector contains, for example, a most preferably 20 to 500 U/g) as an activity of digesting polynucleotide capable of controlling the expression, or a keratin azure, or 1 U/g or more (more preferably 1 to 5000 genetic marker to select transformants, in addition to the U/g, most preferably 5 to 3000U/g) as an activity of digesting polynucleotide encoding the enzyme used in the present keratin powder. The amount of the enzyme is sufficient to invention. The polynucleotide capable of controlling the 45 digest a pathogenic prion protein contained in 1 mL of a 1% expression includes, for example, a promoter, a terminator, or Suspension containing a brain tissue derived from a mouse a polynucleotide encoding a signal peptide. The promoter is Suffering from Scrapie. not particularly limited, so long as it shows a transcriptional In addition to the enzyme used in the present invention, the activity in a host microorganism. The promoter can be enzyme composition used in the present invention may fur obtained as a polynucleotide which controls the expression of 50 ther contain at least one of enzymes other than the enzyme a gene encoding a protein the same as or different from that used in the present invention, Such as a protease (for example, derived from the host microorganism. The genetic marker can keratinase), lipase, cellulase, or Xylanase. The use of the be appropriately selected in accordance with the method for enzymes other than the enzyme used in the present invention selecting a transformant. As the genetic marker, for example, is expected to develop the efficiency indigesting a pathogenic a drug resistance gene or a gene complementing an aux 55 prion protein, in comparison with the enzyme composition otrophic mutation can be used. containing the enzyme used in the present invention alone. The enzyme used in the present invention may be prepared The enzyme used in the present invention exhibits an activ by a microorganism transformed with the above expression ity of digesting a protein highly resistant to denaturation and vector. A host-vector system which can be used in the present degradation (particularly a pathogenic prion protein). There invention is not particularly limited. For example, a system 60 fore, the enzyme used in the present invention, or the enzyme utilizing E. coli, Actinomycetes, yeasts, or filamentous fungi, composition used in the present invention containing the or a system for the expression of a fusion protein using Such enzyme is useful as an active ingredient for an agent for a microorganism can be used. Transformation of a microor digesting a protein highly resistant to denaturation and deg ganism with the expression vector can be carried out in accor radation (particularly a pathogenic prion protein), or as an dance with an ordinary method. 65 active ingredient for an agent for detoxifying a pathogenic The transformant is cultivated in an appropriate medium, prion proteinina Subject which may be contaminated with the and the resulting host cells or culture is used to obtain the pathogenic prion protein. US 7,776,579 B2 13 14 The agent of the present invention may contain as an active thus it is preferable that the subject to be treated is used ingredient the enzyme used in the present invention alone, or without preheating (for example, at approximately 100° C. or together with an appropriate carrier and/or diluent. As the more, at 95°C. or more, at 90° C. or more, or at 80° C. or carrier or diluent, a conventional carrier or diluent which does more) before contact with the enzyme used in the present not Suppress or inhibit an activity of the enzyme used in the invention. When the preheating is not carried out, an addi present invention, such as fillers (for example, lactose, tional apparatus for heating is not necessary, and procedures Sodium chloride, Sodium sulfate, or Sorbitol), Surfactants, or can be simplified. antiseptics, can be used. The procedure of bringing the enzyme or enzyme compo While the form of the agent of the present invention is not sition used in the present invention into contact with the particularly limited, a foaming agent, which may be rapidly 10 subject to be treated is not particularly limited and may be dissolved in water while foaming, is preferable. The formu appropriately selected in accordance with the Subject to be lation and preparation of the foaming agent are not particu treated, so long as a protein highly resistant to denaturation larly limited, but a conventional method may be used. The and degradation (particularly a pathogenic prion protein), foaming agent may be prepared, for example, by mixing which may be contained in the subject to be treated, may be Sodium bicarbonate, sodium percarbonate, or the like with an 15 digested by the activity of the enzyme used in the present acid, Such as citric acid, malic acid, or Succinic acid, or by invention, i.e., an activity of digesting a protein highly resis further adding thereto a mobilization agent Such as silicic tant to denaturation and degradation (particularly a patho anhydride or other binders. genic prion protein). For example, when the subject to be treated is feed which Method for Digesting a Protein Highly Resistant to Denatur may contain a pathogenic prion protein, the contact may be ation and Degradation (Particularly a Pathogenic Prion Pro carried out, for example, by uniformly mixing the enzyme or tein) enzyme composition used in the present invention with the The enzyme or enzyme composition used in the present feed, or by spraying the feed with an aqueous solution con invention may be used alone, or in the form of the above taining the enzyme used in the present invention. mentioned agent of the present invention, to digest a protein 25 When the subject to be treated is an instrument on which a highly resistant to denaturation and degradation (particularly Surface may be contaminated with a pathogenic prion protein, a pathogenic prion protein), or to detoxify a pathogenic prion there may be mentioned, for example, a method of immersing protein in a subject which may be contaminated with the the instrument in an aqueous solution containing the enzyme pathogenic prion protein. used in the present invention, a method of spraying the instru Therefore, the present invention includes a method for 30 ment with an aqueous solution containing the enzyme used in digesting a protein highly resistant to denaturation and deg the present invention, or a method of Washing the Surface of radation (particularly a pathogenic prion protein), using the the instrument with a washing tool (for example, a cloth, enzyme or enzyme composition used in the present invention, sponge, or brush) having an aqueous Solution containing the and a method for detoxifying a pathogenic prion protein in a enzyme used in the present invention. Subject which may be contaminated with the pathogenic 35 When the subject to be treated is a facility in which a prion protein, using the enzyme or enzyme composition used pathogenic prion protein may be present, the contact may be in the present invention. carried out, for example, by spraying an aqueous solution The method of the present invention for digesting a protein containing the enzyme used in the present invention. highly resistant to denaturation and degradation comprises at It is preferable that the contact of the subject to be treated least the step of bringing the enzyme or enzyme composition 40 with the enzyme or enzyme composition used in the present used in the present invention into contact with a protein highly invention is carried out under the conditions in which the resistant to denaturation and degradation (particularly a enzyme used in the present invention may exhibit a sufficient pathogenic prion protein) or a subject to be digested which activity of digesting a protein highly resistant to denaturation may contain the same. The method of the present invention and degradation (particularly a pathogenic prion protein). For for detoxifying a pathogenic prion protein comprises at least 45 example, pH 7 to 12 is preferable. The contact may be carried the step of bringing the enzyme or enzyme composition used out preferably at 20 to 80°C., more preferably 40 to 80° C. in the present invention into contact with a Subject to be The content of the enzyme used may be appropriately detoxified which may be contaminated with a pathogenic selected in accordance with the content of a protein highly prion protein. resistant to denaturation and degradation (particularly a As the subject to be digested or the subject to be detoxified 50 pathogenic prion protein) in the Subject to be treated. For (hereinafter collectively and simply referred to as “subject to example, to digest a pathogenic prion protein contained in 1 be treated'), there may be mentioned, for example, feed mL of a 1% Suspension containing a brain tissue derived from which may contain a pathogenic prion protein (for example, a mouse Suffering from scrapie, it is preferable to use the meat and bone meal, or compost), instruments or equipment enzyme composition containing 0.5 to 10 ug of the enzyme on which Surfaces may be contaminated with a pathogenic 55 used in the present invention; the enzyme composition con prion protein (for example, instruments or equipment for taining 2 U/g or more (more preferably 2 to 500 U/g, still slaughter, examination, or operations), or facilities in which a further preferably 10 to 500 U/g, most preferably 20 to 500 pathogenic prion protein may be present (for example, a U/g), as an activity of digesting keratin azure, of the enzyme slaughterhouse, a cowshed where BSE was present, or a labo used in the present invention; or the enzyme composition ratory for infection). 60 containing 1 U/g or more (more preferably 1 to 5000 U/g, The subject to be treated may be used without preheating or most preferably 5 to 3000 U/g), as an activity of digesting with preheating (for example, at approximately 100° C. or keratin powder, of the enzyme used in the present invention. more, preferably at 95°C. or more, more preferably at 90° C. When the content of the enzyme is less than 0.5 lug, or the or more, most preferably at 80° C. or more) before contact activity is less than 2 U/g or more (as an activity of digesting with the enzyme used in the present invention. According to 65 keratin azure) or less than 1 U/g (as an activity of digesting the method of the present invention, a Sufficient digestion or keratin powder), a complete digestion of the above content of detoxification can be carried out without the preheating, and the pathogenic prion protein becomes difficult. When the US 7,776,579 B2 15 16 content of the enzyme is more than 10 ug, or the activity is Concentration of the substrate: 0.5% more than 500U/g or more (as an activity of digesting keratin pH: 9.0 azure) or more than 5000 U/g (as an activity of digesting keratin powder) to completely digest the above content of the Temperature: 60° C. pathogenic prion protein, it is not practically preferable from the viewpoint of production costs. TABLE 2

EXAMPLES Substrate Digestion activity (U)

10 The present invention now will be further illustrated by, but casein 326677 is by no means limited to, the following Examples. collagen 36958 Example 1 elastin 1OSO1 keratin 7187 Preparation of Purified Enzyme 15 In this example, cultivation and purification were carried out to obtain a purified enzyme used in the present invention (2) Molecular Weight as follows. An SDS-polyacrylamide gel electrophoresis using a 12% Culture medium A1% polypeptone (Wako Pure Chemical homogeneous gel (Tefco) was carried out to determine a Industries), 0.2% yeast extract (Difco), and 0.1% magnesium molecular weight of the purified enzyme prepared in sulfate heptahydrate (Wako Pure Chemical Industries)(pH Example 1. As a result, the molecular weight of the enzyme 7.0) was autoclaved by a conventional method, and the capable of digesting a pathogenic prion protein was approxi medium A (200 mL) was inoculated with Bacillus lichenifor mately 31,000. mis MSK-103 (FERM BP-08487). A cultivation was carried 25 out at 37° C. under aeration and agitation for 72 hours. The Another SDS-polyacrylamide gel electrophoresis using a resulting broth was centrifuged at 3000 G for 20 minutes to 15% homogeneous gel (ATTO) was carried out to determine obtain a Supernatant containing an enzyme used in the present a molecular weight of the purified enzyme prepared in invention. Example 1. As a result, the molecular weight of the enzyme The supernatant was concentrated 20-fold with an ultrafil 30 capable of digesting a pathogenic prion protein was approxi ter (5,000-molecular-weight cutoff) to obtain a concentrated mately 26,000. In this connection, Protein Molecular Weight Supernatant containing the enzyme used in the present inven Standard (Bio-Rad) was used as a standard marker in this tion. The concentrated Supernatant was filtered with a micro example. filter membrane (pore size 0.45um) to remove microorgan isms. To the resulting sterile filtrate, ammonium sulfate was 35 (3) Isoelectric Point added to a final concentration of 1 mol/L, and a buffer agent A polyacrylamide gel isoelectric focusing electrophoresis (Tris-HCl) was further added to pH 8.5 and a final concen using an LKB electrophoresis system was carried out to deter tration of 50 mmol/L. For a further purification by a hydro phobic chromatography, the prepared solution was adsorbed mine an isoelectric point (pl) of the purified enzyme prepared to a phenyl Sepharose column, and eluted by a linear gradient 40 in Example 1. As a result, the isoelectric point of the enzyme with ammonium sulfate (1 mol/L to 0 mol/L) in a Tris-HCl capable of digesting a pathogenic prion protein was 9.3. In buffer, to obtain a fraction containing the enzyme used in the this connection, each isoelectric point of standard samples present invention. The fraction was concentrated 20-fold with used in this example is shown in Table 3. an ultrafilter (5,000-molecular-weight cutoff), and a gel fil tration chromatography was carried out using Superdex 75 45 TABLE 3 (Pharmacia) gel. The concentrated Solution was developed through the gel with a phosphate buffer (0.025 mol/L, pH 7.0) Standard sample Isoelectric point containing 0.1 mol/L Sodium chloride as an eluent, to obtain trypsinogen 9.30 the enzyme used in the present invention. As a result, 201g of lentil lectin basic band 8.65 50 purified enzyme used in the present invention was obtained. lentil lectin neutral band 8.45 lentil lectin acidic band 8.15 Example 2 horse myoglobin basic band 7.35 horse myoglobin acidic band 6.85 Confirmation of Physical and Chemical Properties of human carbonic anhydrase B 6.55 the Enzyme 55 horse carbonic anhydrase B 5.8O B-lactoglobulin A S.2O soybean trypsin inhibitor 4.SS (1) Activity and Substrate Specificity amyloglucosidase 3.SO Activities of the purified enzyme obtained in Example 1 to various Substrates (casein, collagen, elastin, and keratin) were 60 examined. The results are shown in Table 2. (4) Optimum pH and Stable pH As shown in Table 2, the enzyme exhibited a high activity of digesting each Substrate, particularly keratin. In this con The optimum pH at 37° C. of purified enzyme prepared in nection, the “1 unit (U) of digestion activities compared in Example 1, determined by an activity of digesting keratina Table 2 is defined as an amount of the enzyme which can 65 Zure (Sigma) as an index, was pH 9.0 to 10.0, as shown in FIG. develop ninhydrin corresponding to 1 Limol of glycine per 1. The stable pH at 37° C. of the enzyme was pH 7.0 to 12.0, minute, under the following conditions: preferably pH 8.0 to 10.5, as shown in FIG. 1. US 7,776,579 B2 17 18 (5) Optimum Temperature for 30 seconds, at 48° C. for 30 seconds and at 68° C. for 2 The optimum temperature at pH 9.0 (i.e., optimum pH), minutes 30 times, to amplify the DNA of interest. Primer determined by an activity of digesting keratin azure as an PDE-2 for partial fragment amplification: index, was 60 to 70° C., as shown in FIG. 2. 5'-agagcgg.cggaaaagtggac-3' (SEQ ID NO: 3) Primer PDE-5 for partial fragment amplification: Example 3 5'-cctg.cgccaggagccatgac-3' (SEQID NO: 4) As a result, a fragment of approximately 700 bp was ampli Cloning of Enzyme Gene and Determination of fied. The amplified DNA fragment was used as a probe to Amino Acid Sequence Thereof clone the full-length of the gene of interest from a genomic 10 library derived from Bacillus licheniformis MSK-103 In this example, a gene encoding the purified enzyme pre (FERM BP-08487) as follows. pared in Example 1 was cloned, and the nucleotide sequence Genomic DNA derived from Bacillus licheniformis MSK of the gene was determined to confirm the amino acid 103 (FERM BP-08487) was partially digested with restric sequence of the enzyme. tion enzyme Saul IIIA1, and the fragments were ligated into an To purify the enzyme, the culture medium A (see Example 15 EMBLIII vector (Stratagene). A commercially available 1) was inoculated with Bacillus licheniformis MSK-103 packaging kit (MaxPlax Lambda packaging extract; Epicen (FERMBP-08487). A cultivation was carried out at 37°C. for tre technologies) was used to form phage particles containing 3 days, and the resulting broth was centrifuged to obtain a the constructs. The obtained phage library was screened by Supernatant. The Supernatant was concentrated approxi using a commercially available screening kit (DIG high prime mately 20-fold with Pelicon XL (cut 5000; Millipore), and DNA labeling and detection starter kit; Roche) to obtain 100 was adjusted to a solution containing 1 mol/L magnesium positive clones from approximately 10000 plaques. DNAS sulfate and 0.05 mol/L Tris-HCl (pH8.5). The prepared solu were purified from 10 positive clones, an SphI fragment of tion was applied to a phenyl Sepharose column (Phenyl approximately 4.1 kb, which was contained in 4 positive Sepharose FF; low sub, 26x300mm; Amersham Bioscience), clones thereamong, was subcloned into puC119 to construct and eluted by a linear concentration gradient with ammonium 25 pUC-PDE4. The size of the SphI fragment accorded with a sulfate (1 mol/L to 0 mol/L) in a 0.05 mol/L Tris-HCl buffer result of a Southern analysis of Bacillus licheniformis MSK (pH8.5), to obtain a fraction eluted with 0 mol/L of ammo 103 (FERM BP-08487) using the PCR product as a probe. nium sulfate. The fraction was concentrated with Pellicon XL The plasmid puC-PDE4 was used to determine the DNA (cut 5000) followed by Ultrafree 15 (UcutS000; Millipore). sequence thereof by a shotgun sequence method using a DNA The concentrated solution was applied to Superdex 30 sequencer (model 3730XL: Applied Biosystems). As a result, (Superdex75pg; 16x600 mm; Amersham Bioscience), and the plasmid contained the full-length of the gene encoding the eluted with a phosphate buffer (0.05 mol/L, pH 7.0) contain enzyme capable of digesting an abnormal prion, and the ing 0.1 mol/L Sodium chloride to obtain a fraction having a nucleotide sequence of the coding region was that of SEQID molecular weight of approximately 31 kDa. It was confirmed NO: 1. by SDS-PAGE that the fraction contained, as a single sub 35 As a result, it was confirmed that the amino acid sequence stance, a protein having a molecular weight of approximately of the purified enzyme obtained in Example 1 is completely 31 kDa. identical to that of subtilisin DY (WO98/30682). Further, the The purified protein was subjected to SDS-PAGE, and second highest homology was 81% in a kerA gene derived transferred to a polyvinylidene difluoride (PVDF) membrane from Bacillus licheniformis (by a BLAST search). (Immobilon PSQ: Millipore), to blot the protein on the PVDF 40 membrane. The PVDF membrane was washed with water and Example 4 air-dried, and then was used to analyze the amino acid sequence of the protein by a protein sequencer (Model 492; Preparation of Enzyme Composition Applied Biosystems). As a result, the following amino acid sequence was obtained: 45 To obtain an enzyme composition used in the present invention, the culture medium A (200 mL) described in N-terminal amino acid: AQTVPYGIPLI (the sequence con Example 1 was inoculated with Bacillus licheniformis MSK sisting of the 1st to 11th amino acids in the amino acid 103 (FERM BP-08487). A cultivation was carried out at 37° sequence of SEQID NO: 2) C. under aeration and agitation for 48 hours. The obtained It was found that the obtained amino acid sequence is the 50 broth was centrifuged at 3000G for 30 minutes to obtain a same sequence as those of keratinase derived from Bacillus Supernatant containing the enzyme used in the present inven licheniformis PWD-1 Lin, X. et. al., Appl. Environ. Micro tion. The supernatant was concentrated 30-fold with an ultra biol (1995) 61, 1469-1474 and subtilisin carlsberg derived filter (5,000-molecular-weight cutoff) to obtain a concen from Bacillus licheniformis Jacobs, M. et. al., Nucleic Acid trated Supernatant. The concentrated Supernatant was filtered Res. (1985) 13, 8913-8926. Next, a partial fragment was 55 with a microfilter (pore size=0.45um) to remove microorgan amplified by PCR, and was used as a probe to clone a gene of isms. As a result, a solution of enzyme composition A con interest as follows. taining the enzyme used in the present invention was Genomic DNA derived from Bacillus licheniformis MSK obtained. The enzyme composition A exhibited an activity of 103 (FERM BP-08487) was prepared in accordance with a digesting keratinaZure, and the activity was 285 U/g. The method of Wilson et al. Wilson, C. R., J. Bacteriol. (1985) 60 Solution of the enzyme composition A was lyophilized to 163, 445-453. A PCR using the genome DNA as a template obtain powder of enzyme composition A. and a combination of the following primers was carried out to Culture medium B 0.01% yeast extract (Difco), 1% amplify a partial fragment of a gene encoding the enzyme feather meal (ITOCHU FEED MILLS CO., LTD), 0.01% capable of digesting an abnormal prion. The PCR was carried magnesium chloride (Wako Pure Chemical Industries), out by using Takara Taq (Takara Bio) as an enzyme for PCR, 65 0.04% dipotassium hydrogen phosphate (Wako Pure Chemi and by performing a heat denaturation at 94°C. for a minute, cal Industries), 0.03% potassium dihydrogen phosphate followed by repeating a cycle consisting of reactions at 94°C. (Wako Pure Chemical Industries), 0.05% sodium chloride US 7,776,579 B2 19 20 (Wako Pure Chemical Industries), and 0.05% ammonium obtained rabbit antiserum was subjected to a protein A col chloride (Wako Pure Chemical Industries)(pH 7.0) was auto umn to purify the antibody of interest. The antibody reacts to claved, and the medium B (40 mL) was inoculated with not only sheep prion protein but also hamster, mouse, and Bacillus licheniformis PWD-1 (ATCC-53757). A cultivation bovine prion proteins. was carried out at 37° C. under aeration and agitation for 48 5 The results are shown in FIG. 3. When using proteinase K hours. The resulting broth was centrifuged at 3000 G for 30 as a standard, or Subtilisin carlsberg as a commercially avail minutes to obtain a Supernatant. The Supernatant was concen able protease, bands resistant to proteases, which indicated trated 18-fold with an ultrafilter (5,000-molecular-weight the presence of the pathogenic prion protein, were detected cutoff) to obtain a concentrated Supernatant. The concen even at the concentration of 1 g/mL. The molecular weight trated supernatant was filtered with a microfilter (pore 10 of the band not digested at all was 32 kDa, and those of size 0.45um) to remove microorganisms. As a result, a solu partially-digested bands (three bands) were 30 kDa, 25-26 tion of enzyme composition B for comparison was obtained. kDa, and 20-21 kDa. In contrast, when using the purified enzyme used in the present invention, a band was detected at Example 5 0.2 Lug/mL, but almost all the pathogenic prion proteins con 15 tained in the 1% brain homogenate were digested at 1 ug/mL. Digestion of Mouse Pathogenic Prion Protein with Purified Enzyme Example 6 In this example, the purified enzyme used in the present Digestion of Mouse Pathogenic Prion Protein with invention prepared in accordance with the method described Enzyme Composition in Example 1, and a commercially available protease (Subtili sin carlsberg; Sigma) were used to evaluate an activity of In this example, the solution of enzyme composition A digesting a pathogenic prion protein. In this connection, an used in the present invention prepared in accordance with the method described in Example 4 was used to evaluate an activity of an enzyme preparation (proteinase K: Wako Pure activity of digesting mouse pathogenic prion protein. In this Chemical Industries) was used as a standard. 25 As a substrate used in this example, the brain derived from connection, an activity of an enzyme preparation (proteinase a mouse infected with the pathogenic prion protein CLINI K; Wako Pure Chemical Industries) was used as a standard. CAL AND DIAGNOSTIC LABORATORY IMMUNOL As a substrate, the same substrate used in Example 5 (i.e., 1% brain homogenate derived from a mouse infected with the OGY (USA), American Society for Microbiology (Asm), pathogenic prion protein) was used. March in 1995, p. 172-176 was used to prepare a 5% homo 30 genate 2% N-Sodium lauroyl sarcosinate, and 10 mmol/L An enzyme reaction was carried out by mixing the 1% Tris-HCl buffer (pH 7.5), and the 5% homogenate was brain homogenate with an equal Volume of the enzyme com diluted to a final concentration of 1% with 50 mmol/L Tris position solution or the enzyme preparation Solution, and incubating each mixture at 37° C. for 1 hour. The concentra HC1 buffer (pH 8.3). tions of the enzyme preparation were 50 ug/mL, 25 ug/mL, An enzyme reaction was carried out by mixing the 1% 35 brain homogenate with an equal Volume of the purified 12.5ug/mL, and 6.25 g/mL, as a final concentration. As to enzyme solution, the commercially available protease solu the enzyme composition A, the original Solution was diluted to 1, /2, /4, /s, and /16. The diluted solutions exhibited 285 tion, or the enzyme preparation Solution, and incubating each U/g, 143 U/g, 71 U/g, 36 U/g, and 18 U/g as an activity of mixture at 37°C. for 1 hour. The concentrations of the puri digesting keratin azure, respectively. fied enzyme, the commercially available protease, and the 40 enzyme preparation were 1 g/mL and 0.2 ug/mL, as a final An aliquot of each reaction mixture after the enzyme reac concentration in each reaction mixture during the enzyme tion was used to detect the pathogenic prion protein in accor reaction. dance with the method described in Example 5. An aliquot of each reaction mixture after the enzyme reac The results are shown in FIG. 4. When using proteinase K tion was used to carry out a sodium dodecyl sulfate-polyacry 45 as a standard, bands resistant to proteases, which indicated lamide gel electrophoresis (SDS-PAGE) using a electro the presence of the pathogenic prion protein, were detected phoresis system (ATTO) and an SDS-polyacrylamide gel even at a high concentration of 50 g/mL. In contrast, when (10% gel: ATTO). Proteins in the polyacrylamide gel after using the enzyme composition used in the present invention, SDS-PAGE were transferred to a polyvinylidene difluoride a band was slightly detected at 18 U/g as an activity of (PVDF) membrane (Millipore) by a blotting system (ATTO) 50 digesting keratin azure (diluted to /16; a 1.875-fold concen in accordance with a protocol attached thereto. The patho trated solution of the broth), but the pathogenic prion protein genic prion protein bound to the PVDF membrane was was completely digested at 36 U/g (diluted to /s; a 3.75-fold labeled by an antibody-antigen method using an anti-prion concentrated solution of the broth) or more. protein rabbit antibody as the first antibody, and a horserad Example 7 ish-peroxidase-labeled anti-rabbit-IgG goat antibody 55 (Zymed) as the second antibody. The pathogenic prion pro Digestion of Sheep Pathogenic Prion Protein with tein was detected by a commercially available labeling and Enzyme Composition detecting kit (ECL+Plus Western Blotting Detection System; Amersham BioScience) in accordance with a protocol In this example, the solution of enzyme composition A attached thereto. 60 used in the present invention and the solution of enzyme In this connection, the anti-prion-protein rabbit antibody composition B (containing keratinase derived from Bacillus used as the first antibody was prepared as follows. A peptide licheniformis PWD-1) for comparison, each being prepared (PrP94-112) consisting of 20 amino acids in which cystein in accordance with the method described in Example 4, were (CyS) was added to an N-terminal sequence of a core frag used to evaluate an activity of digesting sheep pathogenic ment P27-30 of sheep scrapie prion protein was synthesized, 65 prion protein. In this connection, an activity of an enzyme and a rabbit was immunized with the peptide conjugated to preparation (proteinase K: Wako Pure Chemical Industries) Keyhole limpet hemocyanin (KLH) as an immunogen. The was used as a standard. US 7,776,579 B2 21 22 As a substrate used in this example, the brain derived from a sheep infected with the pathogenic prion protein was used to TABLE 4 prepare a 5% homogenate 2% N-Sodium lauroyl sarcosinate, and 10 mmol/L Tris-HCl buffer (pH 7.5), and the 5% homo Enzyme composition Strain Medium genate was diluted to a final concentration of 1% with 50 FERMBP-08487 mmol/L Tris-HCl buffer (pH 8.3). PWD-1 PWD-1 An enzyme reaction was carried out by mixing the 1% DSM-8782 brain homogenate with an equal Volume of the enzyme com DSM-8782 position solution or the enzyme preparation Solution, and incubating each mixture at 37°C. for 1 hour. The concentra 10 tions of the enzyme preparation were 50 g/mL, 10 g/mL, 2 The resulting enzyme composition A used in the present ug/mL, and 0.4 g/mL, as a final concentration. As to the invention and four enzyme compositions B to E for compari enzyme composition A used in the present invention, the son were used to compare the activity of digesting mouse original solution was diluted to 1, /2, 4, and /s. The diluted pathogenic prion protein, in accordance with the procedures solutions exhibited 285 U/g, 143 U/g, 71 U/g, and 36 U/gas 15 described in Example 7, except that a final concentration of an activity of digesting keratin azure, respectively. As to the each enzyme composition was concentrated 18-fold with enzyme composition B for comparison, the original Solution respect to each Supernatant. was diluted to 1, /2, /4, and /8. The diluted solutions exhibited The results are shown in FIG. 6. As shown in FIG. 6, the 37 U/g, 19 U/g, 9 U/g, and 5 U/g. pathogenic prion protein was not digested by the enzyme An aliquot of each reaction mixture after the enzyme reac compositions B to E for comparison, but was completely tion was used to detect the pathogenic prion protein in accor digested by the enzyme composition A used in the present dance with the method described in Example 5. invention. The results are shown in FIG. 5. When using proteinase K as a standard, bands resistant to proteases, which indicated Example 9 the presence of the pathogenic prion protein, were detected at 25 the concentrations of 10 ug/mL or less. When using the Comparative Test to Thermoase (1) enzyme composition B for comparison, the pathogenic prion protein was not digested at any concentration. In contrast, The enzyme composition A' used in the present invention when using the enzyme composition A used in the present prepared in accordance with the procedures described in invention, the pathogenic prion protein was almost com 30 Example 4, and thermoase (DAIWA KASEI K.K.), as an pletely digested at any concentration. In addition, it was enzyme for comparison, derived from Bacillus thermopro found that the enzyme of the present invention can digest a teolyticus Rokko disclosed in WO02/053723 were used to pathogenic prion protein derived from a different species and evaluate the activity of digesting hamster pathogenic prion having a different amino acid sequence with a minor varia protein (strain Sc237). tion. 35 As a substrate used in this example, the brain derived from a mouse infected with hamster-type pathogenic prion protein Example 8 (strain Sc237) was used to prepare a 1% brain homogenate 50 mmol/L Tris-HCl buffer (pH 8.3). The Sc237-type Digestion of Mouse Pathogenic Prion Protein with pathogenic prion protein was accumulated in the brain of the Enzyme Composition 40 OUS. Each enzyme solution was prepared by dissolving the Bacillus licheniformis PWD-1 was cultivated in accor enzyme composition A' or thermoase in a 50 mmol/L Tris dance with the method described in Example 4 for preparing HCl buffer (pH8.3). The concentrations of each solution were the enzyme composition A used in the present invention (i.e., 4, 8, 16, and 32 U/mL (as a final concentration) as an activity using the medium A), and an enzyme composition C for 45 of digesting keratin powder. comparison was prepared in accordance with the method The enzyme reaction was carried out by mixing the 1% described in Example 4 for preparing the enzyme composi brain homogenate with an equal Volume of each enzyme tion A. solution, and incubating the mixture at 37°C. for 20 hours. Further, Bacillus licheniformis DSM-8782 was cultivated 50 An aliquot of each reaction mixture after the enzyme reac in accordance with the method described in Example 4 for tion was used to detect the pathogenic prion protein in accor preparing the enzyme composition A used in the present dance with the method described in Example 5. invention (i.e., using the medium A), and an enzyme compo The results are shown in FIG. 7. When using the enzyme sition D for comparison was prepared in accordance with the Solution containing thermoase, bands resistant to proteases, method described in Example 4 for preparing the enzyme 55 which indicated the presence of the pathogenic prion protein, composition A. were detected at any concentration. In contrast, when using Furthermore, Bacillus licheniformis DSM-8782 was culti the enzyme composition used in the present invention, the vated in accordance with the method described in Example 4 pathogenic prion protein was completely digested below the for preparing the enzyme composition B for comparison (i.e., levels of detection by Western blotting, at any concentration. using the medium B), and an enzyme composition E for 60 comparison was prepared in accordance with the method Example 10 described in Example 4 for preparing the enzyme composi tion B. Comparative Test to Thermoase (2) In Table 4, the relationships of the enzyme compositions to the strains and the media are shown. The medium B is a 65 WO02/053723 discloses that an activity of thermoase in medium for inducing keratinase Japanese Unexamined digesting a protein (a pathogenic prion protein derived from Patent Publication (Kokai) No. 6-46871). BSE) is increased in the presence of sodium dodecyl sulfate US 7,776,579 B2 23 24 (SDS). In this example, an activity of digesting hamster activity of digesting keratin powder) with 50 mmol/L Tris pathogenic prion protein was evaluated under Such condi HCl buffer (pH8.3) to prepare a washing solution A (7.5 tions. U/mL) and washing solution B (15 U/mL), respectively. The enzyme composition A' used in the present invention The enzyme reaction was carried out by adding 100 prepared in accordance with the procedures described in 5 LL/well of each washing Solution, and incubating the plate at Example 4, and the thermoase solution used in Example 9 37° C. for 1 hour under shaking at 100 rpm. Each washing were used to evaluate the activity of digestinghamster patho solution was removed, and each well was washed twice with genic prion protein (strain Sc237). approximately 300 uL of PBS. As a substrate used in this example, the brain derived from A denature treatment was carried out by adding 100 a mouse infected with hamster-type pathogenic prion protein 10 uL/well of 6 mol/L guanidine hydrochloride (Wako Pure (strain Sc237) was used to prepare a 1% brain homogenate Chemical Industries) and allowing the plate to stand at room 50 mmol/L Tris-HCl buffer (pH 8.3) containing 0.1, 1, or 4% temperature for 1 hour. Each well was washed three times SDS (final concentrations of SDS in the following reac with approximately 300 uL of PBS to remove guanidine tion=0.05, 0.5, or 2%). The Sc237-type pathogenic prion hydrochloride. Blocking was carried out by adding 300 protein was accumulated in the brain of the mouse. 15 uL/well of 5% skimmed milk (Amersham) and allowing the The enzyme reaction was carried out by mixing the 1% plate to stand at room temperature for 1 hour. Each well was brain homogenate with an equal Volume of each enzyme washed twice with approximately 300 uL of 0.05% Tween solution, and incubating the mixture at 37° C. for 20 hours. 2O-PBS. The concentration of each solution was 4 U/mL (as a final The pathogenic prion protein bound to the microplate was concentration) as an activity of digesting keratin powder. 2O labeled by an antibody-antigen method using an anti-prion An aliquot of each reaction mixture after the enzyme reac protein mouse antibody (3F4; Chemicon International) as the tion was used to detect the pathogenic prion protein in accor first antibody, and a horseradish-peroxidase-labeled anti dance with the method described in Example 5. mouse-IgG goat antibody (Zymed) as the second antibody. The results are shown in FIG. 8. In FIG. 8, lane 1 is The prion protein remaining in each well of the microplate thermoase (4 U/mL, 0.05% SDS), lane 2 is thermoase (4 25 was detected by a luminescent reaction using a commercially U/mL: 0.5% SDS), lane 3 is thermoase (4 U/mL:2% SDS), available labeling and detecting kit (Super Signal West Dura; and lane 4 is the enzyme composition A' Solution (4 U/mL, Amersham BioScience) in accordance with a protocol 2% SDS). attached thereto. The amount of luminescence was recorded When using the enzyme solution containing thermoase, by a light capture (AE-6962: ATTO), and an image analysis bands resistant to proteases, which indicated the presence of 30 was carried out by a software for image analysis (CS Ana the pathogenic prion protein, were detected even at the final lyzer; ATTO). concentration of 2% SDS. In contrast, when using the enzyme The results are shown in FIG. 9. When using the enzyme composition used in the present invention, the pathogenic composition used in the present invention, a residual rate of prion protein was completely digested below the levels of the pathogenic prion protein was less than 10% at the con detection by Western blotting, at any concentration. 35 centration of 7.5 U/mL, and the rate was less than approxi As described above, it is found that the enzyme used in the mately 1% at the concentration of 15 U/mL. In contrast, when present invention exhibits an excellent activity of digesting a using thermoase, the rate was 40% or more at any concentra pathogenic prion protein even in the presence of SDS, in tion. As a result, it was found that the enzyme used in the comparison with thermoase. present invention exhibits an excellent activity of washing 40 Example 11 away a pathogenic prion protein. Washing Model Test Using Microplate INDUSTRIAL APPLICABILITY To evaluate the effects on washing instruments contami- 45 The enzyme used in the present invention exhibits an excel nated with a pathogenic prion protein, a model test was car lent activity of digesting a protein highly resistant to denatur ried out as follows. ation and degradation (particularly a pathogenic prion pro The enzyme composition A' used in the present invention tein) in comparison with known proteases. Therefore, prepared in accordance with the procedures described in according to the enzyme used in the present invention or the Example 4, and the thermoase solution used in Example 9 50 enzyme composition used in the present invention containing were used to evaluate the activity of digestinghamster patho the enzyme, a pathogenic prion protein can be efficiently genic prion protein (strain Sc237) stuck on polystyrene. digested. Further, the enzyme used in the present invention As a substrate used in this example, the brain derived from can be produced at a low cost. a mouse infected with a hamster-type pathogenic prion pro According to the enzyme or the enzyme composition, con tein (strain Sc237) was used to prepare a 1% brain homoge- 55 tamination in a Subject which may be contaminated with a nate 50 mmol/L Tris-HCl buffer (pH 8.3). The Sc237-type pathogenic prion protein can be removed. The enzyme is pathogenic prion protein was accumulated in the brain of the useful as an active ingredient for an agent of the present mouse. As a control, a normal brain not infected with the invention for digesting or detoxifying a pathogenic prion abnormal prion protein was used to prepare a 1% brain homo protein. genate 50 mmol/L Tris-HCl buffer (pH 8.3). 60 Each 1% brain homogenate (25 uL/well) of normal or FREE TEST INSEQUENCE LISTING Sc237-infected hamster was added to a polystyrene micro plate (IMMUNO MODULE: Nunc), and the plate was com Features of Artificial Sequence' are described in the pletely dried at room temperature for one day. numeric identifier <223> in the Sequence Listing. More par To carry out a washing treatment of the resulting micro- 65 ticularly, the nucleotide sequence of SEQID NO:3 is primer plate using enzyme solutions, the enzyme composition A'and PDE-2, and the nucleotide sequence of SEQ ID NO. 4 is thermoase were diluted to 7.5 U/mL and 15 U/mL (as an primer PDE-5. US 7,776,579 B2 25 26 Although the present invention has been described with modifications obvious to those skilled in the art are possible reference to specific embodiments, various changes and without departing from the scope of the appended claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 4

<21 Os SEQ ID NO 1 &211s LENGTH: 825 &212s. TYPE: DNA <213> ORGANISM; Bacillus Licheniformis 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (825)

<4 OOs SEQUENCE: 1 gcc caa aca gtt cot tac ggc atc cc.g. citc at C aag gct gac aaa gtg 48 Ala Glin Thr Val Pro Tyr Gly Ile Pro Lieu. Ile Lys Ala Asp Llys Val 1. 5 1O 15

cag gcc caa ggit tat aaa ggg gca aat gtc. aaa gtC ggt atc att gat 96 Glin Ala Glin Gly Tyr Lys Gly Ala Asn. Wall Lys Val Gly Ile Ile Asp 2O 25 3 O

acg gga atc gct tcg tct cat aca gac titg aag gta gtC gec gga gca 144 Thr Gly Ile Ala Ser Ser His Thr Asp Lieu Lys Val Val Gly Gly Ala 35 4 O 45

agc titt gta tot ggit gala agt tat aat acg gaC ggit aac gga cac ggc 192 Ser Phe Val Ser Gly Glu Ser Tyr Asn Thr Asp Gly Asn Gly His Gly SO 55 60

aca Cat gtt gcc gga aca gtg gcg gcg Ctt gaC aat aca aca ggc gtt 24 O Thr His Val Ala Gly Thr Val Ala Ala Lieu. Asp ASn Thir Thr Gly Val 65 70 7s 8O

tta ggc gtt gca ccg aac gtc. tcc ctic tac gcg att aag gtg ttgaat 288 Lieu. Gly Val Ala Pro Asn Val Ser Lieu. Tyr Ala Ile Llys Val Lieu. Asn 85 90 95

tda agc gga agc gga aca tac agc gca atc gt C agc gga att gag tig 336 Ser Ser Gly Ser Gly Thr Tyr Ser Ala Ile Val Ser Gly Ile Glu Trp 1OO 105 110

gcc aca caa aac ggc ctg gat gtc atc aac atg agc ct c gC gga cca 384 Ala Thr Glin Asn Gly Lieu. Asp Val Ile Asn Met Ser Lieu. Gly Gly Pro 115 12O 125

to C ggc tica act gcg ctgaaa cag gct gtg gat aaa gCa tat gcc agc 432 Ser Gly Ser Thr Ala Lieu Lys Glin Ala Val Asp Lys Ala Tyr Ala Ser 13 O 135 14 O

gga att gtc gta gtg gca gca gcg ggg aac agc gga tot t cc ggc agc 48O Gly Ile Val Val Val Ala Ala Ala Gly Asn. Ser Gly Ser Ser Gly Ser 145 15 O 155 16 O

caa aac aca atc ggc tat cog gca aaa tat gac toc gtc atc gcc gtc 528 Gln Asn. Thir Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val 1.65 17 O 17s

ggit gcg gtt gac agc aac aaa aac aga gct tca tt C to C agc gtc ggc 576 Gly Ala Val Asp Ser Asn Lys Asn Arg Ala Ser Phe Ser Ser Val Gly 18O 185 190

to a gag Ctt gala gt C atg gCt cct ggc gtc agc gta tac agc acia tat 624 Ser Glu Lieu. Glu Val Met Ala Pro Gly Val Ser Val Tyr Ser Thr Tyr 195 2 OO 2O5

cct tct aac acg tac aca toa ttgaac giga act tca atg gct tcg cct 672 Pro Ser Asn Thr Tyr Thr Ser Lieu. Asn Gly. Thir Ser Met Ala Ser Pro 210 215 22 O

cat gta gcg gga gca gca gcc titg atc titg tog aaa tac Cct acg Ctt 72O His Val Ala Gly Ala Ala Ala Lieu. Ile Lieu. Ser Lys Tyr Pro Thr Lieu. 225 23 O 235 24 O US 7,776,579 B2 27 28

- Continued toa gct to c caa gtt cgc aac cgc citc. to a agc act gcg act aat ttg 768 Ser Ala Ser Glin Wall Arg Asn Arg Luell Ser Ser Thir Ala Thir Asn Luell 245 250 255 gga gat to c ttic tact tac ggc a.a.a. 999 Ctg atc. aat gta gala gct gcc 816 Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Luell Ile Asn Wall Glu Ala Ala 26 O 265 27 O gct Cala taa 825 Ala Glin

<210s, SEQ ID NO 2 &211s LENGTH: 274 212. TYPE : PRT &213s ORGANISM: Bacillus Licheniformis

<4 OOs, SEQUENCE: 2

Ala Glin. Thir Wall Pro Gly Ile Pro Luell Ile Ala Asp Lys Wall 1. 15

Glin Ala Glin Gly Tyr Gly Ala Asn Wall Lys Wall Gly Ile Ile Asp 2O 25

Thir Gly Ile Ala Ser Ser His Thir Asp Luell Lys Wall Wall Gly Gly Ala 35 4 O 45

Ser Phe Wall Ser Gly Glu Ser Asn Thir Asp Gly Asn Gly His Gly SO 55 6 O

Thir His Wall Ala Gly Thir Wall Ala Ala Luell Asp Asn Thir Thir Gly Wall 65 70

Lell Gly Wall Ala Pro Asn Wall Ser Luell Tyr Ala Ile Wall Luell Asn 85 90 95

Ser Ser Gly Ser Gly Thir Ser Ala Ile Wall Ser Gly Ile Glu Trp 1OO 105 11 O

Ala Thir Glin Asin Gly Lell Asp Wall Ile Asn Met Ser Lell Gly Gly Pro 115 12 O 125

Ser Gly Ser Thir Ala Lell Lys Glin Ala Wall Asp Lys Ala Ala Ser 13 O 135 14 O

Gly Ile Wall Wall Wall Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Ser 145 150 155 160

Glin Asn Thir Ile Gly Pro Ala Tyr Asp Ser Wall Ile Ala Wall 1.65 17O

Gly Ala Wall Asp Ser Asn Asn Arg Ala Ser Phe Ser Ser Wall Gly 18O 185 19 O

Ser Glu Luell Glu Wall Met Ala Pro Gly Wall Ser Wall Tyr Ser Thir 195

Pro Ser Asn Thr Tyr Thir Ser Luell Asn Gly Thir Ser Met Ala Ser Pro 21 O 215 22O

His Wall Ala Gly Ala Ala Ala Luell Ile Luell Ser Pro Thir Luell 225 23 O 235 24 O

Ser Ala Ser Glin Wall Arg Asn Arg Luell Ser Ser Thir Ala Thir Asn Luell 245 250 255

Gly Asp Ser Phe Tyr Gly Gly Luell Ile Asn Wall Glu Ala Ala 26 O 265 27 O

Ala Glin

SEQ ID NO 3 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically - synthesized Primer PDE-2 US 7,776,579 B2 29 30

- Continued

<4 OOs, SEQUENCE: 3 agagcggcgg aaaagttggac

SEQ ID NO 4 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically-synthesized Primer PDE-5

<4 OOs, SEQUENCE: 4 Cctg.cgc.cag gagccatgac

The invention claimed is: 4. The method according to claim 1, wherein the enzyme is 1. A method for detoxifying a pathogenic prion protein, selected from the group consisting of comprising the step of bringing a surface which may be (X) an enzyme comprising the amino acid sequence of contaminated with a pathogenic prion protein into contact SEQ ID NO. 2; with an enzyme exhibiting an activity of digesting a protein (Y) a modified enzyme exhibiting an activity of digesting a highly resistant to denaturation and degradation and having protein highly resistant to denaturation and degradation, the following properties: 25 and comprising an amino acid sequence in which one or (a) activity and Substrate specificity: hydrolyzing a peptide plural amino acids are deleted, Substituted, or added in bond of a protein highly resistant to denaturation and the amino acid sequence of SEQID NO: 2; and degradation; (Z) a homologous enzyme exhibiting an activity of digest (b) molecular weight: 31,000 (determined by an SDS ing a protein highly resistant to denaturation and degra polyacrylamide gel electrophoresis using a homoge 30 dation, and comprising an amino acid sequence having neous gel having a gel concentration of 12%); an 85% or more homology with the amino acid sequence (c) isoelectric point: p19.3 (determined by polyacrylamide of SEQID NO: 2. gel isoelectric focusing electrophoresis); 5. The method according to claim 1, wherein the protein (d) optimum pH: pH 9.0 to 10.0; and highly resistant to denaturation and degradation is a patho (e) optimum temperature for activity: 60 to 70° C., 35 genic prion protein. wherein the contacting step is carried out without preheat ing the Subject, and said enzyme is produced by Bacillus 6. The method according to claim3, wherein the contacting licheniformis MSK-103 (FERM BP-08487). step is carried out without preheating the subject at 90° C. or 2. The method according to claim 1, wherein the contacting O. step is carried out without preheating the subject at 90° C. or 40 7. The method according to claim 4, wherein the contacting O. step is carried out without preheating the subject at 90° C. or 3. The method according to claim 1, wherein the enzyme O. has the following property: 8. The method according to claim 5, wherein the contacting (g) exhibiting an activity of 2 U/g or more as the activity of step is carried out without preheating the subject at 90° C. or digesting a protein highly resistant to denaturation and 45 O. degradation (determined as an activity of digesting kera tin azure). UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 7,776,579 B2 Page 1 of 1 APPLICATIONNO. : 10/532605 DATED : August 17, 2010 INVENTOR(S) : Takehiro Miwa et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

In claim 1 at column 29, line 37: Delete “subject and insert-surface--

In claim 2 at column 29, line 40: Delete “subject and insert-surface--

In claim 6 at column 30, line 37: Delete “subject and insert-surface--

In claim 7 at column 30, line 40: Delete “subject and insert-surface--

In claim 8 at column 30, line 43: Delete “subject and insert-surface--

Signed and Sealed this Twenty-eighth Day of December, 2010

David J. Kappos Director of the United States Patent and Trademark Office