HK1086857B - Method of degrading hardly degradable protein - Google Patents

Description

Method for decomposing hardly decomposable protein

Technical Field

The present invention relates to a degradation agent for hardly degradable proteins (particularly, pathogenic prion proteins) and a method for degrading the hardly degradable proteins.

Background

Sheep or mouse scrapie, Cretzfeldt-Jakobsideal (CJD) in humans, or bovine spongiform encephalopathy in cattle (BSE); commonly known as mad cow disease ] and other diseases are manifested by nervous symptoms such as standing, walking difficulty and the like, which are possibly related to pathogenic prion protein. It is currently indicated that: humans are infected with beef contaminated with pathogenic prion protein and may develop variant Creutzfeldt-Jakobsease (vCJD). Therefore, BSE is a very serious disease from the viewpoint of securing the supply of safe edible meat.

It is considered that these diseases are caused by the change in the three-dimensional structure of normal prion protein which is usually present in the brain due to pathogenic prion protein which penetrates from the outside to the inside of the body [ "Nature", (uk), 1994, volume 370, page 471 (non-patent document 1) ]. Thus, to prevent these diseases from developing due to infection with pathogenic prion protein, it is necessary to degrade the pathogenic prion protein that is the cause of the disease to a non-pathogenic degree and to detoxify it.

However, the pathogenic prion protein is a protein that is extremely stable to ordinary sterilization treatment (e.g., boiling), and the infection ability of the pathogenic prion protein is hardly reduced by such sterilization treatment. In addition, although the pathogen is a protein, it is difficult to completely decompose the pathogen by using a conventional protease even when the pathogen is decomposed by using the protease. Therefore, it is desired to develop a method for effectively decomposing pathogenic prion protein and reducing the incidence of infection.

As a method for degrading a protein which is hardly degradable such as a pathogenic prion protein, Japanese patent application laid-open No. 6-46871 (patent document 1) proposes a method of using a keratinase derived from Bacillus licheniformis (PWD-1) strain as an enzyme for degrading a protein containing a keratin which is hardly degradable by a conventional protease. However, in the above-mentioned publication, the above-mentioned keratinase is used for decomposing keratin-containing proteins (for example, animal hair, human hair, bird feathers, etc.), and there is no mention as to whether it is effective for pathogenic prion proteins.

DNA of keratinase derived from Bacillus licheniformis PWD-1 strain has been obtained so far [ Japanese patent publication No. Hei 10-500863 (patent document 2) ].

It has also been proposed that: the above keratinase derived from Bacillus licheniformis PWD-1 strain is used to decompose a refractory pathogenic prion protein [ U.S. Pat. No. 6,613,505 (patent document 3) ]. However, in the method described in the above patent specification, in order to reduce or decompose pathogenic prion protein, two levels of treatment, that is, heat treatment as a pretreatment and then enzyme treatment, are required. In this case, since equipment for heat treatment is required, it is difficult to perform the method described in the above patent specification without providing such equipment. In addition, since two-step processing is necessary, there is also a problem of poor operability.

International publication No. 02/053723 (patent document 4) proposes the use of a thermostable protease for decomposing a pathogenic prion protein. However, this manual describes: when pathogenic prion protein is decomposed using the protease derived from Bacillus thermoproteolyticus Rokko strain described in the examples of the above International publication manual, the protease alone cannot be decomposed sufficiently, and only when sodium lauryl sulfate is present in the reaction mixture, the protease can be decomposed sufficiently. In addition, since neutral salts are required for activating the above enzymes and the enzymes have properties requiring metal ions, there is a problem that the enzyme activity is significantly reduced when the enzymes coexist with a substance having a chelating effect.

(non-patent document 1) "Nature", (uk), 1994, volume 370, page 471

(patent document 1) Japanese patent application laid-open No. 6-46871

(patent document 2) Japanese Kohyo publication Hei 10-500863

(patent document 3) specification of U.S. Pat. No. 6,613,505

(patent document 4) International publication No. 02/053723 Manual

Disclosure of Invention

The purpose of the present invention is to provide an enzyme which exhibits a decomposing activity for a highly recalcitrant protein (particularly, a pathogenic prion protein) as compared with a known protease and can be produced at a low cost; and a hardly decomposable protein-decomposing agent and a pathogenic prion protein-detoxifying agent each containing the enzyme as an active ingredient, and also to provide a method for decomposing a hardly decomposable protein (particularly a pathogenic prion protein) and a method for detoxifying a pathogenic prion protein using the same.

The present inventors have found an enzyme having an extremely high degradation activity for a protein having poor degradability (particularly, a pathogenic prion protein) as compared with a known enzyme that has been reported to degrade a protein having poor degradability from a microorganism belonging to the genus Bacillus.

As shown in the examples described later, the enzymes of the present invention have far superior properties to those reported for the decomposition of pathogenic prion proteins, such as the enzyme prepared from Bacillus licheniformis PWD-1 strain (keratinase) reported in the specification of U.S. Pat. No. 6,613,505 and the enzyme prepared from Bacillus thermoproteolyticus Rokko strain reported in the International publication No. 02/053723.

It was found in particular that: the enzyme used in the present invention has much higher pathogenic prion proteolytic activity than the enzyme prepared from Bacillus licheniformis PWD-1 strain (see examples 7 and 8). Even more surprisingly, it was found that the treatment can be carried out without the heat treatment described in the above patent specification (cf. examples 7 and 8).

As a result of comparison with an enzyme prepared from Bacillus thermoproteolyticus Rokko strain, it was found that: the enzymes used in the present invention have much higher pathogenic prion proteolytic activity (see examples 9-11). Even more surprising is the discovery that: the enzyme used in the present invention has excellent pathogenic prion proteolytic activity regardless of the presence of sodium lauryl sulfate (see examples 9-11).

The present invention also provides a hardly decomposable protein degrading agent and a pathogenic prion protein detoxifying agent each containing the enzyme thus found as an active ingredient, and also provides a method for degrading a hardly decomposable protein (particularly a pathogenic prion protein) using the hardly decomposable protein degrading agent and the pathogenic prion protein detoxifying agent.

That is, the present invention includes the following inventions.

A hardly decomposable protein degrading agent comprising, as an active ingredient, an enzyme having a hardly decomposable protein degrading activity, which has:

(a) activity and substrate specificity: hydrolyzing peptide bonds of the hardly degradable protein;

(b) molecular weight: 31,000 (SDS-polyacrylamide gel electrophoresis using a homogeneous gel with a gel concentration of 12%);

(c) isoelectric point: pI9.3 (polyacrylamide gel isoelectric point electrophoresis);

(d) optimum pH: pH9.0-10.0;

(e) optimum temperature for activity: 60-70 ℃.

The hardly decomposable protein degrading agent according to [1], wherein the enzyme further has the following property (g):

(g) the hardly decomposable proteolytic activity is 2 units/g or more (keratin azurin decomposing activity is used as an index).

The hardly decomposable protein degrading agent according to [1] or [2], wherein the enzyme further has the following property (h):

(h) a microorganism belonging to the genus Bacillus.

A hardly decomposable protein degrading agent comprising an enzyme selected from the group consisting of:

(X) an enzyme comprising the amino acid sequence shown in SEQ ID NO. 2;

(Y) a modified enzyme having a hardly decomposable protease activity, which comprises an amino acid sequence having 1 or more amino acid deletions, substitutions or additions in the amino acid sequence represented by SEQ ID NO. 2; and

(Z) a homologous enzyme having an amino acid sequence having 85% or more homology with the amino acid sequence shown in SEQ ID NO.2 and having a hardly decomposable proteolytic activity.

[1] The hardly decomposable protein decomposing agent according to any of [4], wherein the hardly decomposable protein is a pathogenic prion protein.

A method for decomposing a protein having poor degradability, which comprises the step of contacting the enzyme of [1] to [5] or a protein degradation agent having poor degradability with the protein having poor degradability.

[1] [5] use of the enzyme of [5] for producing a hardly decomposable protein decomposing agent.

An antidote for pathogenic prion protein, which comprises the enzymes of [1] to [5] as an active ingredient, against a treatment target substance which is likely to be contaminated with the pathogenic prion protein.

A method for detoxifying a pathogenic prion protein, comprising the step of contacting a subject to be treated which has a possibility of being contaminated with a pathogenic prion protein with the enzyme of [1] to [5] or the pathogenic prion protein detoxification agent of [ 8].

A method for detoxifying a pathogenic prion protein, comprising the step of bringing an object to be treated into contact with the enzymes of [1] to [5] or the pathogenic prion protein detoxification agent of [8] without heat-treating the object to be treated beforehand, said object possibly being contaminated with the pathogenic prion protein.

A method for detoxifying a pathogenic prion protein, comprising the step of bringing an object to be treated into contact with the enzymes [1] to [5] or the pathogenic prion protein detoxification agents [8] without previously subjecting the object to a heating treatment at 90 ℃ or higher, said heating treatment being likely to cause contamination with the pathogenic prion protein.

[1] Use of the enzyme of [5] for the preparation of an antidote for pathogenic prion proteins.

Brief Description of Drawings

FIG. 1 is a graph showing the optimum pH and the stable pH range at 37 ℃ of a purified enzyme to be used in the present invention.

FIG. 2 is a graph showing the optimum temperature range of the purified enzyme used in the present invention at pH 9.0.

FIG. 3 is a diagram showing the degradation of mouse-derived pathogenic prion protein by the purified enzyme used in the present invention.

FIG. 4 shows the degradation of mouse-derived pathogenic prion protein by the enzyme composition A used in the present invention.

FIG. 5 shows the degradation of sheep-derived pathogenic prion protein by the enzyme composition A used in the present invention.

FIG. 6 shows the degradation of mouse-derived pathogenic prion protein by the enzyme composition A used in the present invention.

FIG. 7 shows that the enzyme composition A' used in the present invention or comparative thermoase decomposed hamster pathogenic prion protein (Sc237 strain).

FIG. 8 shows the degradation of hamster pathogenic prion protein (Sc237 strain) by the enzyme composition A' used in the present invention or by a comparative thermoase in the presence of SDS.

FIG. 9 shows the decomposition of hamster pathogenic prion protein (Sc237 strain) immobilized on polystyrene by the enzyme composition A' used in the present invention or by comparative thermoase.

Best Mode for Carrying Out The Invention

The present invention is described in detail below.

The enzyme used in the present invention has a protein decomposition activity which is difficult to decompose, that is, an activity of hydrolyzing a peptide bond of a protein difficult to decompose.

In the present specification, the term "protein which is hardly degraded" means a protein which is not easily degraded by a usual protease (e.g., proteinase K or trypsin), and more specifically, means a protein which is not completely degraded by a reaction with proteinase K at a concentration of 1. mu.g/mL for 1 hour at 37 ℃. Examples of the above-mentioned hardly degradable protein include pathogenic prion protein, keratin, collagen, elastin and the like.

In the present specification, the term "pathogenic prion protein" refers to a protein involved in the onset of scrapie, CJD, BSE, or the like, and specifically refers to a prion protein in which the steric structure of a normal prion protein normally present in the brain is changed. Examples of the source of the inhibitor include human, hamster, mouse, cow, and sheep.

Although the amino acid sequences of the normal prion protein and the pathogenic prion protein are identical, the proteins differ in their steric structure. Specifically, among normal prion proteins, prion proteins have a high content of alpha-helices in which polypeptides are in a helical form, and a low content of beta-sheets in which polypeptides are in a planar fold. While the pathogenic prion proteins have a high beta sheet content (Pan, PNAS, 90, 10962, 1993). In addition, the above-mentioned animal-derived prion proteins have high amino acid sequence homology, and the properties of pathogenic prion proteins that are hardly degradable by changing the steric structure of normal prion proteins are also the same.

The pathogenic prion protein, which is considered to be a causative agent of the above-mentioned diseases, is a protein that is extremely stable after ordinary sterilization treatment such as boiling, and the infection ability thereof is hardly reduced by the sterilization treatment. In addition, normal prion protein is easily decomposed and has a half-life in vivo of about 2 hours, whereas pathogenic prion protein has a half-life in vivo of 24 hours or more and is a protein that is very difficult to decompose. Actually, when the degradability of the protease is evaluated by using a commercially available protease such as proteinase K, it is shown that: while normal prion proteins are easily decomposed and sensitive, pathogenic prion proteins are resistant and refractory to decomposition with low degradability (pruiner, Science, 252, 1515, 1991). The reason why the degree of difficulty in decomposition thereof differs is the difference in the aforementioned three-dimensional structure.

The method for discriminating between a normal prion protein and a pathogenic prion protein is, for example, the method utilizing the difference in the protease decomposition properties described above. That is, tissues from animals that may be infected with pathogenic prion protein are ground, homogenized, made into a suspension, treated with a conventional protease (e.g., proteinase K, etc.), and then assayed for the presence of prion protein by Western blotting (Burnette, anal. biochem., 112, 195, 1981). In this case, if no protein is detected, it indicates that only a normal prion protein is present, and if a protein band showing protease resistance is detected, it indicates that a pathogenic prion protein is present.

In the present specification, the "protein degradation activity which is hardly degradable" refers to an activity of hydrolyzing a peptide bond of a protein which is hardly degradable. In the present specification, the unit of "hardly decomposable proteolytic activity" is 2 units. Unit 1 was prepared by mixing the enzyme with keratin powder at a final concentration of 0.5% (from human hair; the suspension of Nacalai Tesque was allowed to act for 1 hour, and the amount of the enzyme which produced a decomposition product corresponding to 1. mu. mol of glycine per 1 minute in the measurement system was defined as "1 unit". The unit 2 was prepared by allowing the enzyme to act on a suspension of azurin [ Sigma ] having a final concentration of 0.8% at pH 8.0 and 37 ℃ for 16 hours, measuring the absorbance at 595nm in the measurement system, and defining the change in absorbance of 0.001 by the amount of the dye released into the reaction mixture every 1 minute as "1 unit".

Keratin azure is a compound in which an azo dye is bound to keratin derived from, for example, wool, and an amino acid to which an azo dye is bound or a peptide to which an azo dye is bound, which is released by cleaving a peptide bond of keratin, can be quantified by spectroscopic optics, and therefore, it is often used as a compound which is a substrate for measuring the decomposition activity of keratin (i.e., the activity of keratin enzyme), which is one of hardly decomposable proteins.

As used herein, the term "pathogenic prion proteolytic activity" refers to an activity of hydrolyzing peptide bonds of a pathogenic prion protein. The "pathogenic prion protein degradation activity" can be determined by, for example, using as an index the degradation of 1% of pathogenic prion protein contained in a brain tissue suspension of a mouse infected with scrapie, and determining the degree (intensity) of the activity.

More specifically, brain tissue from a mouse infected with a pathogenic prion protein is ground, homogenized, a suspension is prepared, and treated with an enzyme or enzyme composition to be determined. The protein contained in the treated product was separated by electrophoresis, and the presence of prion protein was detected by western blotting. In this case, if any protein is not detected, it is indicated that the enzyme or enzyme composition to be determined has a very strong pathogenic prion proteolytic activity. Similarly, when a protein band showing protease resistance is detected, if the protein band is shallow, it indicates that the protein band has a certain level of pathogenic prion proteolytic activity; if the protein band is deep, the pathogenic prion proteolytic activity is weak.

Protein having activity of degrading hardly degradable protein (particularly pathogenic prion protein)

The enzyme used in the present invention may have the following physicochemical properties.

(a) Activity and substrate specificity

The peptide bond of a hydrolyzed protein, particularly a peptide bond of a protein which is hardly decomposable (for example, a pathogenic prion protein). With respect to substrate specificity, in addition to the pathogenic prion protein, it has high lytic activity against casein, collagen, elastin, and keratin.

(b) Molecular weight

SDS-polyacrylamide gel electrophoresis was performed using a 12% homogeneous gel (i.e., a homogeneous gel having a polyacrylamide concentration of 12%), and the molecular weight was determined to be about 31,000.

SDS-polyacrylamide gel electrophoresis using a 15% homogeneous gel (i.e., a homogeneous gel having a polyacrylamide concentration of 15%; manufactured by ATTO, for example) showed a molecular weight of about 26,000.

(c) Isoelectric point

The isoelectric point (p I) of the polyacrylamide gel was about 9.3 as determined by isoelectric electrophoresis.

(d) Optimum pH and stable pH

The optimum pH for evaluation using the decomposition activity of azurin as an index is about 9.0 to 10.0. Has stable activity at a pH in the range of about 7.0 to 12.0, and has high activity at a pH in the range of about 8.0 to 10.5.

(e) Optimum temperature of activity

The optimum temperature for the activity to be evaluated using the decomposition activity of azurin as an index is about 60 to 70 ℃.

(f) Inactivating pH

In the evaluation using the decomposition activity of azurin as an index, the inactivation is performed at a pH of about 5 or less.

These properties of the enzyme used in the present invention were compared with those of a known protease having a hardly decomposable proteolytic activity (keratinase derived from Bacillus licheniformis PWD-1 strain), and the results are shown in Table 1.

TABLE 1

In another embodiment of the present invention, an enzyme having an amino acid sequence shown in SEQ ID NO.2 and a modified enzyme thereof or a homologous enzyme thereof can be used.

"an enzyme having an amino acid sequence shown by SEQ ID No. 2" includes an enzyme consisting of an amino acid sequence shown by SEQ ID No. 2; a fusion enzyme having an amino acid sequence such as an appropriate tag sequence added to the N-terminus and/or C-terminus of a polypeptide comprising the amino acid sequence shown in SEQ ID NO.2 and having a protein (particularly a pathogenic prion protein) degradation activity which is hardly degraded; a fusion polypeptide consisting of a fusion polypeptide comprising a polypeptide having an amino acid sequence represented by SEQ ID NO.2 and a fusion partner, and having a protein (particularly a pathogenic prion protein) degradation activity which is hardly degradable; or an enzyme having an amino acid sequence in which a pre-sequence (signal sequence) or a part thereof is added to the N-terminus of the amino acid sequence shown in SEQ ID NO. 2. Furthermore, a fusion enzyme obtained by further adding an appropriate tag sequence and/or fusion partner to the amino acid sequence having the N-terminus of the amino acid sequence shown in SEQ ID NO.2 to which the pre-sequence is added is also included in the scope of "an enzyme having the amino acid sequence shown in SEQ ID NO. 2".

The above-mentioned pro-sequence may be a natural pro-sequence or an artificially designed pro-sequence. As the native pro-sequence, not only a pro-sequence derived from Bacillus licheniformis (particularly a pro-sequence derived from a non-decomposable protease derived from Bacillus licheniformis) but also a pro-sequence derived from an organism other than Bacillus licheniformis can be used.

For example, a sequence for confirming the expression of the polypeptide, the presence in the cell, or the convenience of purification may be used as the tag sequence, and for example, a FLAG tag, a hexahistidine tag, a hemagglutinin tag, a myc epitope, or the like may be used.

For example, a polypeptide for purification [ e.g., all or part of glutathione S-transferase (GST) ], a polypeptide for detection [ e.g., all or part of hemagglutinin or β -galactosidase α peptide (LacZ α) ], a polypeptide for expression (e.g., a signal peptide), and the like can be used as the fusion partner.

In the above fusion polypeptide, an amino acid sequence specifically digestible by protease (e.g., thrombin or factor Xa) can be appropriately introduced between the polypeptide having the amino acid sequence shown in SEQ ID NO.2 and the above marker sequence or the fusion partner.

The "modified enzyme" in the present specification is a protein having an amino acid sequence in which 1 or more (for example, 1 or several) amino acids are deleted, substituted or added from the amino acid sequence shown in SEQ ID NO.2, and having a degradation activity of a protein which is hardly degraded (particularly, a pathogenic prion protein). Among them, the number of amino acids involved in the mutation such as "deletion, substitution or addition" is preferably 1 to 30, more preferably 1 to 10, and still more preferably 1 to 6.

The above-mentioned modified enzyme also includes a protein having an amino acid sequence in which 1 or more (for example, 1 or several) amino acid residues are conservatively substituted in the amino acid sequence shown in SEQ ID NO.2, and having a degradation activity of a protein which is hardly degradable (particularly, a pathogenic prion protein). Wherein "conservative substitution" means that the activity of the protein is not substantially changed and 1 or several amino acid residues are substituted with another chemically similar amino acid. For example, the hydrophobic residue may be substituted with another hydrophobic residue, or the polar residue may be substituted with another polar residue having the same charge. Functionally similar amino acids that may be subject to such conservative substitutions with respect to each other are well known in the art.

Specifically, examples of the nonpolar (hydrophobic) amino acid include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of the polar (neutral) amino acid include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the positively charged (basic) amino acid include asparagine, histidine, and lysine. Examples of the negatively charged (acidic) amino acid include aspartic acid and glutamic acid.

The "homologous protein" in the present invention is a protein having an amino acid sequence having a homology of 85% or more (preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, particularly preferably 99% or more) with the amino acid sequence shown in SEQ ID NO.2, and having a degradation activity of a protein which is hardly degradable (particularly a pathogenic prion protein). The homology values indicated here are those calculated using the BLAST method (Basic local alignment search tool; Altschul, S.F., et al, J.mol.biol., 215, 403-.

The enzyme having an amino acid sequence represented by SEQ ID NO.2, a modified enzyme thereof, or a homologous enzyme thereof has a decomposition activity for a hardly decomposable protein (particularly a pathogenic prion protein), and preferably has a decomposition activity for a hardly decomposable protein (particularly a pathogenic prion protein) of 2 units/g or more (more preferably 2 to 500 units/g, still more preferably 10 to 500 units/g, and particularly preferably 20 to 500 units/g) as an index for a keratinocyte decomposition activity. When the activity of degrading keratin powder is used as an index, it is preferable that the protein has a degradation activity of hardly degradable proteins (particularly, pathogenic prion proteins) of 1 unit/g or more (more preferably 1 to 5000 units/g, still more preferably 5 to 3000 units/g).

The enzyme used in the present invention is not particularly limited as long as it exhibits the above-mentioned various physicochemical properties, or an enzyme having an amino acid sequence represented by SEQ ID NO.2, or a modified enzyme thereof or a homologous enzyme thereof, and the origin thereof is, for example, an enzyme derived from an animal, a plant or a microorganism. The source thereof is preferably an enzyme produced by a microorganism belonging to the genus Bacillus, more preferably an enzyme produced by Bacillus licheniformis, particularly preferably an enzyme produced by Bacillus licheniformis MSK-103 strain (FERM BP-08487). Further, a mutant strain thereof may be used.

Preservation of microorganisms

Bacillus licheniformis MSK-103 strain (FERM BP-08487) was deposited at the International patent organism depositary, national institute of Integrated Industrial and technology, national institute of advanced independent Engineers, national institute of advanced Industrial science and technology, 2002, 10/16.2002 (address: 305: 8566, 1 st Central office, 1 st Summit, 1 st site, east 1 st site, on Tobo, Tokyo, Ichwa, Japan), and was transferred to international deposit at 2003, 9/16.9/16. The international deposit number (domestic deposit number within the international deposit number [ ]) is FERM BP-08487[ FERM P-19068].

Specific examples of the enzyme to be used in the present invention include subtilisin enzymes, and subtilisin DY (International publication No. 98/30682 handbook) is particularly preferable.

The enzyme used in the present invention can be obtained by, for example, isolation and purification from a microorganism as described in example 1. As described later, the protein can also be obtained by expressing a polynucleotide encoding the protein used in the present invention in an appropriate host by gene recombination techniques, and isolating and purifying the produced protein.

In order to obtain the enzyme used in the present invention from the microorganism producing the enzyme used in the present invention, the enzyme used in the present invention can be prepared from the supernatant, cell body or culture (culture solution) containing the enzyme used in the present invention by a known protein purification method by culturing under conditions suitable for the microorganism. Hereinafter, the procedure of culturing the microorganism and purifying the protein will be described by taking Bacillus licheniformis MSK-103 strain (FERM BP-08487) as an example of the microorganism producing the enzyme used in the present invention.

According to a conventional method, a medium containing 1% polypeptone, 0.2% yeast extract and 0.1% magnesium sulfate 7 hydrate (pH7.0) was heat-sterilized, and then Bacillus licheniformis MSK-103 strain (FERM BP-08487) was inoculated into the medium and cultured at 37 to 50 ℃ for 24 to 72 hours with aeration and agitation. The resulting culture broth was centrifuged (about 3000G) by a centrifuge to obtain a culture supernatant containing the enzyme used in the present invention. Then, as necessary, the culture supernatant was concentrated to 2 to 50 times with an ultrafiltration membrane having a molecular weight cut-off of 5,000-30,000 to obtain a culture supernatant concentrate containing the enzyme used in the present invention.

Since the culture supernatant or the culture supernatant concentrate contains many impurities in addition to the enzyme to be used in the present invention, the enzyme to be used in the present invention can be purified, for example, by the following procedure.

The culture supernatant or the culture supernatant concentrate is subjected to filtration sterilization with a microfiltration membrane having a pore size of about 0.45. mu.m. Ammonium sulfate was added to the sterilized filtrate to give a final concentration of 1mol/L, and the mixture was prepared with a buffer (Tris-hydrochloric acid buffer) to give a final concentration of 50mmol/L and pH 8.5. Then, in order to carry out hydrophobic chromatography, the preparation solution is adsorbed on a phenyl agarose column, and a Tris-hydrochloric acid buffer solution containing ammonium sulfate at a concentration of 1mol/L to 0mol/L is subjected to linear concentration gradient elution to obtain a fraction containing the enzyme used in the present invention. The fraction was concentrated to 20-30 times with an ultrafiltration membrane having a molecular weight cut-off of 5,000-10,000, and then subjected to gel filtration chromatography for further purification. Gel filtration chromatography may use, for example, Sephadex75(Pharmacia) gel. The enzyme used in the present invention was obtained by passing the above concentrated solution through the gel using a phosphate buffer solution (0.025mol/L, pH7.0) containing 0.1mol/L sodium chloride as an eluent. By the above purification operation, one band in electrophoresis can be purified.

Polynucleotide encoding protein having activity of degrading hardly degradable protein (particularly pathogenic prion protein)

The polynucleotide encoding the enzyme used in the present invention can be obtained, for example, by the following steps. When the amino acid sequence of the protein is determined, the nucleotide sequence encoding the amino acid sequence can be easily determined, and various nucleotide sequences encoding the enzyme used in the present invention can be selected. In the present specification, the term "polynucleotide" includes both DNA and RNA, preferably DNA.

Typical polynucleotides encoding enzymes used in the present invention may be selected from the following:

(i) a polynucleotide comprising a base sequence represented by SEQ ID NO.1 (preferably a polynucleotide comprising a base sequence represented by SEQ ID NO. 1);

(ii) a polynucleotide which comprises a base sequence having 1 or more (1 or several) bases deleted, substituted or added from the base sequence represented by SEQ ID NO.1 and which encodes a protein having a protein hardly decomposable (particularly, pathogenic prion protein) decomposing activity; or

(iii) A polynucleotide which hybridizes with a polynucleotide having a nucleotide sequence represented by SEQ ID NO.1 under stringent conditions and encodes a protein having a protein hardly decomposable (particularly, a pathogenic prion protein) decomposing activity.

The number of nucleotides that can be deleted, substituted or added in the polynucleotide (ii) is specifically 1 to 50, preferably 1 to 30, more preferably 1 to 18, and still more preferably 1 to 9.

Wherein the "stringent conditions" in the above-mentioned (iii) mean conditions under which a probe controlled to have a base sequence represented by SEQ ID NO.1 hybridizes with a polynucleotide encoding a homologous protein, but the probe does not hybridize with keratinase derived from Bacillus licheniformis PWD-1 strain (specification of U.S. Pat. No. 6,613,505) or protease derived from Bacillus thermoproteolyticus Rokko [ e.g., Thermoase ].

More specifically, for example, the following conditions are present: for example, using the full length of the base sequence shown in SEQ ID NO.1 having a label as a probe, prehybridization is carried out at 42 ℃ for 1 hour, then the probe is incorporated, hybridization is carried out at 42 ℃ for 15 hours, washing is carried out at 42 ℃ for 20 minutes with SSC (1-fold concentration SSC; 15mmol/L trisodium citrate, 150mmol/L sodium chloride) at a concentration of 0.4-fold or less to which 0.4% SDS and 6mol/L urea are added, 2 times, followed by washing with SSC at 5-fold concentration at room temperature for 10 minutes, and 2 times, according to the protocol attached to the ECL direct DNA/RNA labeling detection System (Amersham).

The polynucleotide encoding the enzyme used in the present invention may be naturally-derived, may be completely synthesized, or may be synthesized using a part of naturally-derived polynucleotides. A typical method for obtaining the above-mentioned polynucleotides is a method generally used in the field of genetic engineering, for example, a method of screening from a genomic library of Bacillus licheniformis MSK-103 strain (FERM BP-08487) using an appropriate DNA probe prepared based on partial amino acid sequence information.

Expression vector and transformed microorganism

The enzyme containing the amino acid sequence shown in SEQ ID NO.2 or a modified enzyme or a homologous enzyme thereof used in the present invention can be produced by an expression vector which can replicate the base sequence encoding the above enzyme in a host microorganism and can contain the protein encoded by the base sequence in an expressible state. The expression vector may be constructed on the basis of an autonomously replicating vector, i.e., a vector which exists as an independent body other than a chromosome and whose replication is independent of chromosomal replication, such as a plasmid. Furthermore, when introduced into a host microorganism, the above expression vector is integrated into the genome of the host microorganism, and can be replicated together with the integrated chromosome. The steps and methods for constructing the above expression vector may employ those commonly used in the field of genetic engineering.

In order to introduce the expression vector into a host microorganism and express a protein having a desired activity, it is preferable to include a nucleotide sequence for controlling the expression thereof, a gene marker for selecting a transformant, and the like, in addition to the polynucleotide encoding the enzyme used in the present invention. The base sequence for controlling expression includes a promoter, a terminator, a base sequence encoding a signal peptide, and the like. The promoter is not particularly limited as long as it shows transcriptional activity in the host microorganism, and may be any nucleotide sequence that controls the expression of a gene encoding a protein in a microorganism of the same species as or different species from the host microorganism. The gene marker may be appropriately selected according to the method of selecting a transformant, and for example, a gene encoding drug resistance or a gene complementary to an auxotrophy may be used.

The enzyme used in the present invention can be prepared by a microorganism transformed with the above-mentioned expression vector. The host-vector system is not particularly limited, and for example, a system using Escherichia coli, actinomycetes, yeast, filamentous fungi, or the like, or an expression system for expressing a fusion protein fused with another protein using the above-mentioned microorganism, or the like can be used. In addition, transformation of the microorganism with the expression vector can be carried out according to a method commonly used in the art.

The transformant is cultured in an appropriate medium, and the enzyme used in the present invention can be isolated from the host cell or the culture. The culture of the transformant and the conditions thereof may be substantially the same as those commonly used for the microorganism to be used. The method for recovering the target enzyme after culturing the transformant may be a method commonly used in the art.

The optimum method for producing the enzyme to be used in the present invention is preferably a method using a microorganism of the genus Bacillus, more preferably a method using Bacillus licheniformis, and particularly preferably a method using Bacillus licheniformis MSK-103 strain (FERM BP-08487) and a mutant strain thereof.

Enzyme composition, hardly decomposable protein decomposing agent and pathogenic prion protein detoxifying agent

The enzyme preparation used in the present invention includes any one or more of an enzyme having the above-mentioned physicochemical properties, an enzyme obtained by culturing a microorganism, an enzyme having an amino acid sequence represented by SEQ ID NO.2 or a modified enzyme or a homologous enzyme thereof, or an enzyme obtained by culturing the above-mentioned host cell (hereinafter, one or more of these enzymes will be referred to as "enzyme used in the present invention").

The enzyme composition used in the present invention is not particularly limited as long as it contains the enzyme used in the present invention as an active ingredient, and can be prepared by mixing with a commonly used carrier and/or diluent, for example, an excipient (for example, lactose, sodium chloride, sorbitol, or the like), a surfactant, a preservative, or the like. The enzyme composition used in the present invention may be appropriately prepared in an appropriate form, for example, a powder or a liquid, as required.

The enzyme content in the enzyme composition is only required depending on the purpose of use. The enzyme activity may be sufficiently exhibited, and is not particularly limited, and may be contained in an amount of, for example, 0.01 to 99% by weight, preferably 0.1 to 80% by weight.

Regarding the activity of degrading a protein which is hardly degradable, it is preferable to contain the enzyme used in the present invention in an amount of activity (indicated by a keratin degrading activity) of 2 units/g or more (more preferably 2 to 500 units/g, still more preferably 10 to 500 units/g, particularly preferably 20 to 500 units/g) or in an amount of activity (indicated by a keratin powder degrading activity) of 1 unit/g or more (more preferably 1 to 5000 units/g, still more preferably 5 to 3000 units/g). The amount of the enzyme was an amount sufficient for decomposing 1mL of 1% of pathogenic prion protein contained in brain tissue suspension of mice infected with scrapie.

The enzyme composition used in the present invention may contain, in addition to the enzyme used in the present invention, an enzyme other than the enzyme used in the present invention, for example, at least one of a protease (e.g., keratinase), a lipase, a cellobiase, or a xylanase. By using an enzyme other than the enzyme used in the present invention in combination, it is expected that the degradation efficiency of pathogenic prion protein will be further improved as compared with an enzyme composition containing the enzyme used in the present invention alone.

The enzyme used in the present invention has an activity of degrading a protein which is hardly degradable (particularly, a pathogenic prion protein). Therefore, the enzyme used in the present invention or the enzyme composition used in the present invention containing the enzyme can be used as an active ingredient of a degradation agent for hardly degradable proteins (particularly, pathogenic prion proteins) or an active ingredient of an antidote for pathogenic prion proteins against a treatment target substance that may be contaminated with the pathogenic prion proteins.

The lytic agent or antidote of the present invention may contain the enzyme used in the present invention as an active ingredient alone or in combination with a suitable carrier and/or diluent. The carrier or diluent may be any carrier or diluent as long as it does not inhibit or inhibit the enzymatic activity of the enzyme used in the present invention as an active ingredient, and a commonly used carrier or diluent, for example, an excipient (e.g., lactose, sodium chloride, sodium sulfate, sorbitol, or the like), a surfactant, or a preservative may be used as the carrier or diluent for the protein-decomposing agent or the detoxifying agent.

The shape of the disintegrator or antidote of the present invention is not particularly limited, and a foaming preparation which can be rapidly dissolved while foaming when the preparation is put into water is preferred. The mixing and preparation method of the foaming agent is not particularly limited, and a known method can be used. For example, a preparation in which an acid such as citric acid, malic acid or succinic acid is mixed with sodium bicarbonate or sodium percarbonate, or a preparation in which a flow agent such as silicic anhydride or other binder is further mixed with these mixed materials can be used.

Method for decomposing protein (especially pathogenic prion protein) with difficult decomposition

The enzyme or enzyme composition used in the present invention can be used alone or in the form of the lytic agent or antidote of the present invention to decompose a protein which is hardly decomposable (particularly, a pathogenic prion protein), or to detoxify a pathogenic prion protein in a treatment target which may be contaminated with a pathogenic prion protein.

That is, the present invention includes both a method of degrading a protein that is hardly decomposable (particularly, a pathogenic prion protein) by using the enzyme or the enzyme composition used in the present invention, and a method of detoxifying a pathogenic prion protein in a treatment target that may be contaminated with the pathogenic prion protein by using the enzyme or the enzyme composition used in the present invention.

The degradation method of the present invention includes at least a step of bringing an enzyme or an enzyme composition used in the present invention into contact with a protein that is hardly degradable (particularly, a pathogenic prion protein) or a degradation target that may contain the protein. The detoxification method of the present invention also includes at least a step of bringing the enzyme or the enzyme composition used in the present invention into contact with a detoxification treatment target that may be contaminated with pathogenic prion protein.

The object to be decomposed or detoxified (hereinafter, simply referred to as "object to be treated") may be, for example, a feed (for example, meat and bone meal, compost, or the like) which may contain a pathogenic prion protein, an instrument (for example, slaughter instrument, examination instrument, surgical instrument, or the like) which may have a pathogenic prion protein attached to the surface, or a place (for example, slaughterhouse, kennel in which BSE has occurred, infection laboratory facility, or the like) in which a pathogenic prion protein may be present.

The object to be treated may be used without heat treatment (for example, about 100 ℃ C. or higher, preferably 95 ℃ C. or higher, more preferably 90 ℃ C. or higher, and particularly preferably 80 ℃ C. or higher) before being brought into contact with the enzyme used in the present invention, or may be used after heat treatment. In the method of the present invention, since sufficient decomposition or detoxification can be achieved without performing heat treatment, it is preferable to use the enzyme used in the present invention without subjecting the object to heat treatment (for example, about 100 ℃ or higher, 95 ℃ or higher, 90 ℃ or higher, or 80 ℃ or higher). Without performing the heat treatment, a special heat treatment apparatus is not required, and the operation process can be simplified.

The method for bringing the enzyme or the enzyme composition used in the present invention into contact with the object to be treated is not particularly limited as long as it is a method for degrading a protein that is hardly degradable (particularly, a pathogenic prion protein) that may be contained in the object to be treated by the activity of degrading a protein that is hardly degradable (particularly, a pathogenic prion protein) by the enzyme used in the present invention, and it can be appropriately selected depending on the object to be treated.

For example, when the object to be treated is a feed which may contain a pathogenic prion protein, the contact can be achieved by, for example, uniformly mixing the enzyme or the enzyme composition used in the present invention with the feed or by sprinkling an aqueous solution containing the enzyme used in the present invention on the feed.

When the object to be treated is an instrument having pathogenic prion protein attached to its surface, for example, a method of immersing the instrument in an aqueous solution containing the enzyme used in the present invention, a method of spreading an aqueous solution containing the enzyme used in the present invention on the instrument, or a method of wiping the surface of the instrument with a cleaning implement (e.g., a cloth, a brush, or the like) containing an aqueous solution containing the enzyme used in the present invention.

When the object to be treated is a place where there is a possibility of pathogenic prion protein, for example, a method of spraying an aqueous solution containing the enzyme used in the present invention may be employed.

In the method of the present invention, when the enzyme or the enzyme composition used in the present invention is brought into contact with the object to be treated, the enzyme or the enzyme composition used in the present invention is preferably brought into contact under conditions such that the enzyme used in the present invention can sufficiently exhibit the activity of degrading proteins which are hardly degradable (particularly, pathogenic prion proteins). For example, it is preferable that the pH is in the range of 7 to 12. Preferably the temperature is in the range of 20-80 c, more preferably 40-80 c.

The amount of the protein to be used may be determined appropriately depending on the amount of the protein which is hardly decomposable (particularly, pathogenic prion protein) contained in the object to be treated. For example, in order to degrade 1mL of 1% of pathogenic prion protein contained in brain tissue fluid of mice infected with scrapie, it is preferable to use 0.5 to 10. mu.g of the enzyme used in the present invention, or in order to have a hardly degradable proteolytic activity, it is preferable to use an enzyme composition used in the present invention having an activity (as an index of a keratin azurin degradation activity) of 2 units/g or more (more preferably 2 to 500 units/g, still more preferably 10 to 500 units/g, and particularly preferably 20 to 500 units/g) or an activity (as an index of a keratin powder degradation activity) of 1 unit/g or more (more preferably 1 to 5000 units/g, and still more preferably 5 to 3000 units/g). When the amount of the enzyme used in the present invention is less than 0.5. mu.g, or the activity of degrading hardly degradable proteins contained in the enzyme composition used in the present invention is less than 2 units/g (as an index of the activity of degrading azurin) or less than 1 unit/g (as an index of the activity of degrading keratin powder), it may be difficult to completely degrade the pathogenic prion protein in the above-mentioned amount. In addition, it is not practical from the viewpoint of production cost to use an enzyme composition in which the amount of the enzyme exceeds 10. mu.g, or the amount of the hardly decomposable protein decomposing activity exceeds 500 units/g (as an index of the keratin decomposing activity), or the amount exceeds 5000 units/g (as an index of the keratin powder decomposing activity) in order to completely decompose the pathogenic prion protein in the above-mentioned amount.

Examples

The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

Example 1: preparation of purified enzyme

In this example, in order to obtain the purified enzyme used in the present invention, cultivation and purification were performed according to the following procedures.

First, a medium A [ 1% polypeptone (Wako pure chemical industries), 0.2% yeast extract (Difco), and 0.1% magnesium sulfate 7 hydrate (Wako pure chemical industries) (pH7.0) ] was heat-sterilized according to a conventional method, and then the medium A (200mL) was inoculated with Bacillus licheniformis MSK-103 strain (FERM BP-08487) and cultured at 37 ℃ for 72 hours with aeration and agitation. The resulting culture broth was centrifuged (about 3000G for 20 minutes) by a centrifuge to obtain a culture supernatant containing the enzyme used in the present invention.

Subsequently, the mixture was concentrated to 20 times with an ultrafiltration membrane having a molecular weight cut-off of 5,000 to obtain a culture supernatant concentrate containing the enzyme used in the present invention. The culture supernatant concentrate was subjected to filtration sterilization with a microfiltration membrane having a pore size of 0.45. mu.m. Ammonium sulfate was added to the sterilized filtrate to a final concentration of 1mol/L, and the mixture was further prepared with a buffer (Tris-hydrochloric acid buffer) to a final concentration of 50mmol/L and pH 8.5. Then, in order to carry out hydrophobic chromatography, the preparation solution is adsorbed on a phenyl agarose column, and a Tris-hydrochloric acid buffer solution containing ammonium sulfate at a concentration of 1mol/L to 0mol/L is subjected to linear concentration gradient elution to obtain a fraction containing the enzyme used in the present invention. The fraction was concentrated to 20 times with an ultrafiltration membrane having a molecular weight cut-off of 5,000, followed by gel filtration chromatography. Gel filtration chromatography may be performed using Sephadex75(Pharmacia) gel. The enzyme used in the present invention was obtained by passing the above concentrated solution through the gel using a phosphate buffer solution (0.025mol/L, pH7.0) containing 0.1mol/L sodium chloride as an eluent. By the above purification operation, the purified enzyme (20. mu.g) used in the present invention was obtained.

Example 2: verification of physicochemical Properties of enzyme

(1) Activity and substrate specificity

The activities of the purified enzymes obtained in example 1 on various substrates (casein, collagen, elastin, and keratin) were investigated. The results are shown in Table 2.

As shown in Table 2, high decomposing activity was exhibited for any substrate, particularly for keratin. The decomposition activities in Table 2 were compared under the conditions of a substrate concentration of 0.5%, pH9.0 and 60 ℃ with the amount of enzyme having a ninhydrin color developing ability equivalent to 1. mu. mol of glycine per 1 minute as 1 unit (U).

TABLE 2

(2) Molecular weight

In order to determine the molecular weight of the purified enzyme obtained in example 1, SDS-polyacrylamide gel electrophoresis using 12% homogeneous gel (Tefco) was performed. The molecular weight of the pathogenic prion proteolytic enzyme was calculated to be about 31,000.

In addition, SDS-polyacrylamide gel electrophoresis of the purified enzyme obtained in example 1 was carried out using another gel (15% homogeneous gel; ATTO) to calculate the molecular weight of the pathogenic prion proteolytic enzyme to be about 26,000.

(3) Isoelectric point

In order to determine the isoelectric point (pI) of the purified enzyme obtained in example 1, polyacrylamide gel isoelectric electrophoresis was performed using an LKB amphoteric isoelectric point electrophoresis apparatus. As a result, the isoelectric point of the pathogenic prion protease was calculated to be 9.3. The isoelectric points of the standard samples used in this experiment are shown in table 3.

TABLE 3

(4) Optimum pH and stable pH

The optimum pH of the purified enzyme obtained in example 1 at 37 ℃ was measured using the decomposition activity of azurin (Sigma) as an index, and the results are shown in FIG. 1, where the pH was 9.0 to 10.0. Further, the stable pH at 37 ℃ is, as shown in FIG. 1, 7.0 to 12.0, preferably 8.0 to 10.5.

(5) Optimum temperature

The optimum temperature of the purified enzyme obtained in example 1 was measured at an optimum pH of 9.0 using the decomposition activity of azurin as an index, and as a result, the optimum temperature was 60-70 ℃ as shown in FIG. 2.

Example 3: cloning of enzyme genes and determination of amino acid sequences

In this example, in order to determine the amino acid sequence of the purified enzyme obtained in example 1, the gene encoding the enzyme was cloned, and the base sequence thereof was determined to confirm the amino acid sequence.

For purification of the above enzyme, Bacillus licheniformis MSK-103 strain (FERM BP-08487) was inoculated into Medium A (see example 1), cultured at 37 ℃ for 3 days, and centrifuged to obtain a culture supernatant. The supernatant was concentrated to about 20-fold with pellicon XL (cut-off 5000; Millipore) to prepare a solution containing 1mol/L magnesium sulfate and 0.05mol/L Tris-hydrochloric acid (pH 8.5). The resulting solution was passed through a Phenyl Sepharose FF (Phenyl Sepharose FF; sub, 26X 300 mm; Amersham Bioscience) and eluted with 0.05mol/L Tris-hydrochloric acid (pH8.5) containing a linear concentration gradient of ammonium sulfate of 1mol/L to 0mol/L, and fractions eluted with a concentration of 0mol/L ammonium sulfate were collected. The fraction was concentrated with pellicon XL (cut-off 5000), followed by Ultrafree-15 (cut-off 5000; Millipore), and eluted through a Superdex (Superdex75 pg; 16X 600 mm; Amersham Bioscience) column with 0.05mol/L phosphate buffer (pH7.0) containing 0.1mol/L sodium chloride to recover a fraction having a molecular weight of about 31 kDa. The fraction was subjected to SDS-PAGE, and as a result, it contained a single protein having a molecular weight of about 31 kDa.

The purified protein was subjected to SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon PSQ; Millipore), washed with water, and air-dried. The amino acid sequence thereof was analyzed by a protein sequencer (Applied Biosystems) of type 492. As a result, the amino acid sequences shown below were obtained.

N-terminal amino acid sequence: AQTVPYGIPLI (amino acid sequence No. 1-11 in the amino acid sequence shown in SEQ ID NO. 2)

This sequence was confirmed to be identical to that of keratinase [ Lin, X. et al, appl. environ. Microbiol (1995)61, 1469-8924 ] from Bacillus licheniformis and subtilisin Carlsberg [ Jacobs, M. et al, Nucleic Acid Res. (1985)13, 8913-8926] from Bacillus licheniformis. A part of the fragment was amplified by PCR, and the gene was cloned using it as a probe.

Genomic DNA of Bacillus licheniformis MSK-103 strain (FERM BP-08487) was prepared according to Wilson et al (Wilson, C.R., J.Bacteriol. (1985)163, 445-453). Using this genomic DNA as a template, a partial fragment of the abnormal prion protease gene was amplified by PCR using a primer set containing the following sequences. The enzyme for PCR was heat-denatured at 94 ℃ for 1 minute using Takara Taq (Takara Taq; Takara Bio), and then subjected to 30 cycles of steps of 94 ℃ for 30 seconds, 48 ℃ for 30 seconds and 68 ℃ for 2 minutes to amplify the target DNA.

Primer PDE-2 for amplification of partial fragments: 5'-agagcggcggaaaagtggac-3' (SEQ ID NO.3)

Primer PDE-5 for amplification of partial fragments: 5'-cctgcgccaggagccatgac-3' (SEQ ID NO.4)

As a result, a fragment of about 700bp was amplified. The full-length target gene was cloned from the genomic library of Bacillus licheniformis MSK-103 strain (FERM BP-08487) using this fragment as a probe.

First, genomic DNA of Bacillus licheniformis MSK-103 strain (FERM BP-08487) was partially digested with restriction enzyme SauIIIA1, and the digested fragments were ligated with EMBLIII vector (Stratagene) to form phage particles using a commercially available packaging kit (MaxPlax Lambda packaging extract; Epicentre technologies). Screening was performed from this phage library using a commercially available screening kit (DIG advanced primer DNA labeling and detection primer kit; Roche) to obtain 100 positive clones from about 10000 plaques. Among them, DNA was purified from 10 positive phages, and an about 4.1kb SphI fragment, which was contained in common in 4 positive clones, was subcloned into pUC119 (hereinafter referred to as pUC-PDE 4). The size of the SphI fragment was consistent with the results obtained by southern blot analysis of Bacillus licheniformis MSK-103 strain (FERM BP-08487) using the PCR product as a probe. The plasmid pUC-PDE4 was used to determine the sequence by shotgun sequencing using a 3730XL (applied biosystems) DNA sequencer. As a result, the entire length of the gene containing the abnormal prion protease, and the nucleotide sequence of the translated region thereof was represented by SEQ ID NO. 1.

The results confirmed that: the amino acid sequence of the purified enzyme obtained in example 1 was completely identical to that of subtilisin DY (International publication No. 98/30682 manual). In addition, the kerA gene from B.licheniformis was found to be the highest in homology of 81% (by BLAST search).

Example 4: preparation of enzyme compositions

To obtain the enzyme composition used in the present invention, Bacillus licheniformis MSK-103 strain (FERM BP-08487) was inoculated into the medium A (200mL) described in example 1 and cultured at 37 ℃ for 48 hours with aeration and agitation. The resulting culture broth was centrifuged (about 3000G, 30 minutes) by a centrifuge to obtain a culture supernatant containing the enzyme used in the present invention. Subsequently, the mixture was concentrated to 30 times with an ultrafiltration membrane having a molecular weight cut-off of 5,000 to obtain a culture supernatant concentrate. The culture supernatant concentrate was subjected to filtration sterilization with a microfiltration membrane having a pore size of 0.45. mu.m, to obtain an enzyme composition solution A containing the enzyme used in the present invention. The enzymatic composition A had a keratinocyte decomposition activity of 285 units/g. Subsequently, the enzyme composition solution A was freeze-dried to obtain an enzyme composition powder A'.

On the other hand, medium B [ 0.01% yeast extract (Difco), 1% fish meal (Yiteng faithful feed), 0.01% magnesium chloride (Wako pure chemical industries), 0.04% dipotassium hydrogenphosphate (Wako pure chemical industries), 0.03% potassium dihydrogenphosphate (Wako pure chemical industries), 0.05% sodium chloride (Wako pure chemical industries), and 0.05% ammonium chloride (Wako pure chemical industries) (pH7.0) ] was heat-sterilized, and then Bacillus licheniformis PWD-1 strain (ATCC-53757) was inoculated into this medium B (40mL) and cultured for 48 hours at 37 ℃ with aeration and agitation. The resulting culture broth was centrifuged (about 3000G, 30 minutes) by a centrifuge to obtain a culture supernatant. Subsequently, the mixture was concentrated to 18 times with an ultrafiltration membrane having a molecular weight cut-off of 5,000 to obtain a culture supernatant concentrate. This culture supernatant concentrate was subjected to filtration sterilization with a microfiltration membrane having a pore size of 0.45 μm to obtain an enzyme composition solution B for comparison.

Example 5: use of purified enzymes to break down pathogenic prion protein from mice

In this example, the purified enzyme used in the present invention prepared by the method described in example 1 and a commercially available protease [ subtilisin Carlsberg; aigma ], enzyme-labeled standard [ proteinase K; and Wako pure chemical industries ] the ability of the enzyme used in the present invention to decompose pathogenic prion protein was evaluated on the basis of the ability to decompose the enzyme.

In this example, a 5% homogenate of 2% N-lauryl sarcosine sodium, 10mol/L-Tris-HCl buffer (pH7.5) was prepared from the brain of a mouse [ "CLINICAL ANDDIAGNOSTIC LABORATORY IMMUNOLOGY, (USA) America Society for microbiology (Asm), 1995.3, page 172-176 ] infected with pathogenic prion protein, and the homogenate was diluted with 50mmol/L Tris-HCl buffer (pH8.3) to a final concentration of 1%.

The enzymatic reaction proceeds as follows: the 1% brain homogenate was mixed with an equal volume of purified enzyme solution, commercially available protease solution or enzyme standard solution, and incubated at 37 ℃ for 1 hour. The concentrations of the purified enzyme, the commercially available protease and the enzyme-labeled reagent were graded into two grades of 1. mu.g/mL and 0.2. mu.g/mL, as measured according to the final concentration of the reaction mixture in the enzymatic reaction.

A part of the reaction mixture after completion of the enzymatic reaction was electrophoresed using an electrophoresis apparatus (ATTO Co.) and SDS-polyacrylamide gel (10% gel; ATTO Co.) [ sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ]. The proteins in the polyacrylamide gel after SDS-PAGE were transferred to a polyvinylidene fluoride (PVDF) membrane (Milipore) by a transfer device (ATTO Co.) according to the attached instructions. The antigen-antibody method is used for labeling the pathogenic prion protein combined with the membrane by taking a rabbit anti-prion protein antibody as a first antibody and a goat anti-rabbit IgG-antibody (Zymed) labeled by horseradish peroxidase as a second antibody. Thereafter, detection of pathogenic prion protein was performed by a commercially available labeling and detection system (ECL + Plus Western blot detection System; Amersham Bioscience) according to the attached instructions.

The rabbit anti-prion protein antibody used as the primary antibody can be prepared as follows: a synthetic peptide (PrP94-112) comprising 20 amino acids, wherein glycine (Cys) is added to the N-terminal sequence of the core fragment P27-30 of scrapie prion protein, a Keyhole Limpet Hemocyanin (KLH) peptide is added to the peptide as an immunogen, and the resulting antiserum is purified using a protein A column. The resulting antibodies reacted not only with sheep prion protein but also with various prion proteins of hamsters, mice and cattle.

The results are shown in FIG. 3. As shown in FIG. 3, a protease-resistant band showing the presence of a pathogenic prion protein was detected at a concentration of 1. mu.g/mL for protein K as a standard or for subtilisin, a commercially available protease. As for the molecular weights of these bands, the molecular weight of the band which is not decomposed at all was 32kDa, and the partially decomposed bands (3 bands) were 30kDa, 25-26kDa and 20-21kDa, respectively. In the purified enzyme used in the present invention, a band was detected at 0.2. mu.g/mL, but at a concentration of 1. mu.g/mL, almost all of the pathogenic prion protein contained in 1% of the brain homogenate was decomposed.

Example 6: use of enzyme compositions for the breakdown of pathogenic prion proteins from mice

The ability of the enzyme composition solution a used in the present invention prepared by the method described in example 4 to decompose pathogenic prion protein derived from mice was evaluated. In this case, the decomposition ability of enzyme-labeled standard (proteinase K; Wako pure chemical industries, Ltd.) was used as a standard. In addition, the same substrate as used in example 5 (i.e. 1% brain homogenate from mice infected with pathogenic prion protein) was used.

The enzymatic reaction proceeds as follows: the above 1% brain homogenate was mixed with an enzyme composition solution or an enzyme standard solution of equal volume and incubated at 37 ℃ for 1 hour. The concentrations of the enzyme standards were classified into four grades of 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL and 6.25. mu.g/mL, and the enzyme composition A used a solution of the above composition diluted to 1, 1/2, 1/4, 1/8 and 1/16. The decomposition activity of each dilution of the above composition on the keratin azurin was 285 units/g, 143 units/g, 71 units/g, 36 units/g and 18 units/g, respectively.

A portion of the reaction mixture after completion of the enzymatic reaction was subjected to detection of pathogenic prion protein according to the method described in example 5.

The results are shown in FIG. 4. As shown in FIG. 4, the band of the protease-resistant protein showing the presence of the pathogenic prion protein was detected even at a high concentration of 50. mu.g/mL in the reference proteinase K. In the enzyme composition used in the present invention, a slight band was detected in the enzyme composition having a keratinocyte decomposition activity of 18 units/g (1/16 dilution; 1.875-fold concentrate of culture supernatant), and the pathogenic prion protein was completely decomposed in the enzyme composition having a keratinocyte decomposition activity of 36 units/g (1/8 dilution; 3.75-fold concentrate of culture supernatant) or more.

Example 7: use of enzyme compositions for the breakdown of sheep-derived pathogenic prion protein

Using the enzyme composition solution A used in the present invention prepared as described in example 4 and the enzyme composition solution B for comparison (containing keratinase derived from Bacillus licheniformis PWD-1), the ability to decompose pathogenic prion protein derived from sheep was evaluated based on the ability to decompose enzyme standards (proteinase K; and Wako pure chemical industries).

In this example, a 5% homogenate of 2% N-lauryl sarcosine sodium and 10mol/L-Tris-HCl buffer (pH7.5) was prepared from sheep brains infected with pathogenic prion protein as a substrate, and the homogenate was diluted with 50mmol/L Tris-HCl buffer (pH8.3) to a final concentration of 1%.

The enzymatic reaction proceeds as follows: the above 1% brain homogenate was mixed with an enzyme composition solution or an enzyme standard solution of equal volume and incubated at 37 ℃ for 1 hour. The concentrations of the enzyme standards were divided into 50. mu.g/mL, 10. mu.g/mL, 2. mu.g/mL, and 0.4. mu.g/mL, based on the final concentration. On the other hand, the enzyme composition A used in the present invention used solutions having a decomposition activity for the keratin azurin of 285 units/g, 143 units/g, 71 units/g and 36 units/g (dilution ratios of 1, 1/2, 1/4 and 1/8); the enzyme composition solution B for comparison used solutions having a decomposition activity of the keratin azurin of 37 units/g, 19 units/g, 9 units/g and 5 units/g (dilution magnification of 1, 1/2, 1/4 and 1/8).

A portion of the reaction mixture after completion of the enzymatic reaction was subjected to detection of pathogenic prion protein according to the method described in example 5.

The results are shown in FIG. 5. As shown in FIG. 5, a protease-resistant protein band showing the presence of a pathogenic prion protein was detected at a concentration of 10. mu.g/mL or less in the reference proteinase K. The comparative enzyme composition solution B did not decompose pathogenic prion protein at any concentration. In the enzyme composition A used in the present invention, the pathogenic prion protein is decomposed almost completely at any concentration. In addition, when the source of the pathogenic prion protein is different and the amino acid sequence is slightly different, the enzyme also has the capability of decomposing the pathogenic prion protein.

Example 8: use of enzyme compositions for the breakdown of pathogenic prion proteins from mice

After culturing the Bacillus licheniformis PWD-1 strain in the same manner as described in example 4 for the preparation of the enzyme composition A used in the present invention (i.e., using medium A), the comparative enzyme composition C was prepared in the same manner as described in example 4 for the preparation of the enzyme composition A.

After culturing the Bacillus licheniformis DSM-8782 strain in the same manner as described in example 4 for the preparation of the enzyme composition A used in the present invention (i.e., using the medium A), a comparative enzyme composition D was prepared in the same manner as described in example 4 for the preparation of the enzyme composition A.

The comparative enzyme composition E was prepared by culturing the Bacillus licheniformis DSM-8782 strain in the same manner as described in example 4 for the preparation of comparative enzyme composition B (i.e.using medium B), and then in the same manner as described in example 4 for the preparation of enzyme composition B.

Table 4 shows the relationship between the enzyme compositions and the strains and the culture media. The medium B is a keratinase-inducing medium (Japanese patent application laid-open No. 6-46871).

TABLE 4

The ability to decompose pathogenic prion protein from mice was compared for the enzyme composition a used in the present invention and the 4 enzyme compositions B to E used for comparison prepared as described above, according to the procedure of example 7, except that the final concentration of each enzyme composition was 18 times the concentration of the culture supernatant.

The results are shown in FIG. 6. As shown in FIG. 6, the enzyme compositions B-E used for comparison were not able to decompose pathogenic prion protein. However, the enzyme composition A used in the present invention can completely decompose it.

Example 9: comparative experiment with Thermoase (1)

The ability of an enzyme thermoase (Kagaku corporation) derived from Bacillus thermoproteolyticus Rokko strain, which is a strain described in International publication No. 02/053723 as an enzyme for comparison, to decompose hamster pathogenic prion protein (Sc237 strain) was evaluated using the enzyme composition A' used in the present invention prepared by the method described in example 4.

A1% brain homogenate [50mmol/L-Tris-HCl buffer (pH8.3) ] was prepared from mouse brains infected with hamster pathogenic prion protein (Sc237 strain) and having Sc237 strain pathogenic prion protein accumulated in the brains, and used as a substrate.

The enzyme solutions were prepared by dissolving the enzyme composition A' and the thermoase in 50mmol/L-Tris-HCl buffer (pH8.3), respectively. The concentration of the enzyme composition solution or the enzyme solution is indicated by the keratin powder decomposition activity, and the final concentration is 4, 8, 16 or 32 units/mL.

The enzymatic reaction proceeds as follows: the above 1% brain homogenate was mixed with an equal volume of an enzyme composition solution or an enzyme solution, and incubated at 37 ℃ for 20 hours.

A portion of the reaction mixture after completion of the enzymatic reaction was subjected to detection of pathogenic prion protein according to the method described in example 5.

The results are shown in FIG. 7. As shown in FIG. 7, a protease-resistant protein band showing the presence of pathogenic prion protein was detected in the thermolase enzyme solution at any enzyme concentration. On the other hand, it was found that the pathogenic prion protein was completely decomposed at all concentrations in the enzyme composition used in the present invention to a level that could not be detected by western blotting.

Example 10: comparative experiment with Thermoase (2)

International publication No. 02/053723 discloses: since thermolase has an increased activity of decomposing proteins (BSE-derived pathogenic prion proteins) in the presence of Sodium Dodecyl Sulfate (SDS), the ability to decompose hamster-derived pathogenic prion proteins (Sc237 strain) was evaluated under such conditions.

The ability of the enzyme composition a' used in the present invention prepared by the method described in example 4 to decompose hamster pathogenic prion protein (Sc237 strain) was evaluated using the same thermolase solution as used in example 9.

A 1% brain homogenate was prepared from mouse brains infected with hamster pathogenic prion protein (Sc237 strain) and having Sc237 strain pathogenic prion protein accumulated in the brain [50mmol/L-Tris-HCl buffer (ph8.3) containing SDS (final SDS concentration 0.05, 0.5, or 2%) was added at a ratio of 0.1, 1, or 4% ], and this was used as a substrate.

The enzymatic reaction proceeds as follows: the above 1% brain homogenate was mixed with an equal volume of an enzyme composition solution or an enzyme solution, and incubated at 37 ℃ for 20 hours. The concentration of the enzyme composition solution or the enzyme solution was 4 units/mL as a final concentration, using the keratin powder decomposition activity as an index.

A portion of the reaction mixture after completion of the enzymatic reaction was subjected to detection of pathogenic prion protein according to the method described in example 5.

The results are shown in FIG. 8. In FIG. 8, lane 1 shows the results for thermoase (4U/mL; 0.05% SDS), lane 2 shows the results for thermoase (4U/mL; 0.5% SDS), lane 3 shows the results for thermoase (4U/mL; 2% SDS), and lane 4 shows the results for enzyme composition A' solution (4U/mL; 2% SDS).

As shown in FIG. 8, a protease-resistant protein band showing the presence of pathogenic prion protein was detected in the thermolase enzyme solution at an enzyme concentration of 4 units/mL with a final SDS concentration of 2%. The enzyme compositions used in the present invention, however, completely decomposed pathogenic prion protein at all concentrations to levels undetectable by western blotting.

The following results are obtained: in the presence of SDS, the enzyme has an excellent effect of degrading pathogenic prion protein as compared to thermoase.

Example 11: washing model test Using microtiter plates

In order to examine the washing effect on instruments contaminated with pathogenic prion protein, a model test system was set up and the following experiment was performed.

The ability to decompose hamster pathogenic protein (Sc237 strain) strongly bound to polystyrene was evaluated using the enzyme composition a' solution used in the present invention prepared in the method described in example 4 and the same thermoase solution as used in example 9.

A1% brain homogenate [50mmol/L-Tris-HCl buffer (pH8.3) ] was prepared from mouse brains infected with hamster pathogenic prion protein (Sc237 strain) and having Sc237 strain pathogenic prion protein accumulated in the brains, and used as a substrate. In addition, normal brain not infected with abnormal prion protein was used as a control to prepare 1% brain homogenate [50mmol/L-Tris-HCl buffer (pH8.3) ].

The normal or Sc 237-infected hamster 1% brain homogenate thus prepared was applied to polystyrene-made microplates (IMMUNO MODULE; Nunc) at each 25. mu.L/well, and dried at room temperature for one day to completely dry it.

In order to wash the microplate prepared as described above with the enzyme solution, the enzyme composition A' and the thermolase were diluted with 50mmol/L-Tris-HCl buffer (pH8.3) to 7.5 and 15 units/mL using the keratin powder decomposition activity as an index, to prepare a washing solution A (7.5 units/mL) and a washing solution B (15 units/mL), respectively.

For the enzymatic reaction, the washing solutions were injected at a rate of 100. mu.L/well, shaken at 100rpm, and incubated at 37 ℃ for 1 hour. Thereafter, the washing solution was removed, and washed 2 times with about 300. mu.L of PBS.

6mol/L guanidine hydrochloride (Wako pure chemical industries, Ltd.) was injected into 100. mu.L/well, and the mixture was allowed to stand at room temperature for 1 hour to denature the guanidine hydrochloride. This was followed by 3 washes with about 300 μ L PBS to remove guanidine hydrochloride. Subsequently, 5% skim milk (Amersham) was injected at a rate of 300. mu.L/well, and the mixture was allowed to stand at room temperature for 1 hour for blocking. Then with about 300 u L0.05% Tween 20-PBS washing 2 times.

The pathogenic prion protein bound to the membrane microplate is labeled by an antigen-antibody method with a mouse anti-prion protein antibody (3F 4; chemical International) as a first antibody and a goat anti-mouse IgG-antibody (Zymed) labeled with horseradish peroxidase as a second antibody. Thereafter, prion protein remaining in the wells of the microplate was subjected to luminescence reaction by a commercially available labeling and detection system (Super Signal West Dura; Amersham Bioscience) according to the attached instructions. The light emission amount was photographed by a photographic device (light capture AE-6962 type; ATTO), and image analysis was performed by image analysis software (CS Analyzer; ATTO).

The results are shown in FIG. 9. As shown in FIG. 9, the remaining rate of pathogenic prion protein in the enzyme composition used in the present invention was less than 10% at a concentration of 7.5 units/mL; at a concentration of 15 units/mL, the residual rate of pathogenic prion protein is less than about 1%. While at any concentration, the residual rate of the original prion protein is 40% or more, indicating that the pathogenic prion protein of the invention has high washing capacity.

Industrial applicability

The enzyme used in the present invention has a higher degradation activity for hardly degradable proteins, particularly pathogenic prion proteins, than known proteases. Therefore, the enzyme used in the present invention or the enzyme composition used in the present invention containing the enzyme can efficiently decompose pathogenic prion protein. The enzymes used in the present invention can also be produced at low cost.

Therefore, by using the enzyme or the enzyme composition, contamination of the object to be treated which may be contaminated with the pathogenic prion protein can be removed. The enzyme is effective as an active ingredient of the pathogenic prion protein degradation agent or the contamination removal agent of the present invention.

Sequence Listing independent text

The following description of "artificial sequence" is described in the numerical symbol "223" of the sequence listing. Specifically, the base sequence represented by SEQ ID NO.3 of the sequence Listing is primer-PDE-2, and the base sequence represented by SEQ ID NO.4 of the sequence Listing is primer-PDE-5.

The present invention has been described above based on specific embodiments, and modifications and improvements that can be inferred by those skilled in the art are also included in the scope of the present invention.

Claims (7)

1. Use of an enzyme derived from Bacillus licheniformis MSK-103 strain FERM BP-08487 having recalcitrant proteolytic activity and having the following properties in the preparation of a pathogenic prion protein degradation agent:

(a) activity and substrate specificity: hydrolyzing peptide bonds of the hardly degradable protein;

(b) molecular weight: 31,000 as determined by SDS-polyacrylamide gel electrophoresis using a homogeneous gel having a gel concentration of 12%;

(c) isoelectric point: pI9.3, which is determined by polyacrylamide gel isoelectric electrophoresis;

(d) optimum pH: pH of 9.0-10.0, and determination with keratin azurin decomposition activity as index;

(e) optimum temperature for activity: measuring the decomposition activity of azurin at 60-70 deg.C;

(f) and (3) stabilizing pH: the pH value is 7.0-12.0,

wherein the protein difficult to decompose is a protein which is not completely decomposed after being reacted with proteinase K with a concentration of 1 mug/mL for 1 hour at 37 ℃,

the N-terminal amino acid sequence of the enzyme is AQTVPYGIPLI,

the enzyme was obtained by the following procedure:

concentrating culture supernatant of Bacillus licheniformis MSK-103 strain with registration number of FERM BP-08487 with ultrafiltration membrane having molecular weight cutoff of 5,000 to 20 times, filtering the concentrated culture supernatant with precision filter membrane having pore diameter of 0.45 μm to remove bacteria,

adding ammonium sulfate to a final concentration of 1mol/L, then preparing with Tris-hydrochloric acid buffer solution to a final concentration of 50mmol/L and pH8.5,

adsorbing the above preparation solution to a phenyl-agarose column, performing linear concentration gradient elution with a Tris-hydrochloric acid buffer solution containing ammonium sulfate at a concentration of 1-0 mol/L, collecting fractions containing the enzyme, and

the fraction was concentrated to 20 times with an ultrafiltration membrane having a molecular weight cut-off of 5,000, and then subjected to gel filtration chromatography using Sephadex75 gel, and the above concentrate was passed through the gel using 0.025mol/L phosphate buffer solution containing 0.1mol/L sodium chloride and pH7.0 as an eluent, to thereby obtain the enzyme.

2. The use of claim 1, wherein the enzyme further has the following properties (g):

(g) the hardly decomposable proteolytic activity is an activity of 2 units/g or more, and is measured using the keratinocyte decomposition activity as an index.

3. A method for decomposing a pathogenic prion protein, comprising the step of contacting the pathogenic prion protein with an enzyme or a pathogenic prion protein decomposing agent as recited in claim 1 or 2.

4. Use of the enzyme according to claim 1 or 2 for the preparation of an antidote for pathogenic prion proteins.

5. A method for detoxifying a pathogenic prion protein, comprising the step of contacting a treatment target that has a possibility of being contaminated with a pathogenic prion protein with an enzyme according to claim 1 or 2, or with a pathogenic prion protein detoxification agent according to claim 4.

6. A method for detoxifying a pathogenic prion protein, comprising the step of bringing a treatment target into contact with the enzyme according to claim 1 or 2 or the pathogenic prion protein detoxification agent according to claim 4, without previously heat-treating the treatment target which may be contaminated with the pathogenic prion protein.

7. A method for detoxifying a pathogenic prion protein, comprising the step of bringing a subject to be treated into contact with the enzyme according to claim 1 or 2 or the pathogenic prion protein detoxification agent according to claim 4, without previously subjecting the subject to a heating treatment at 90 ℃ or higher, the subject possibly being contaminated with the pathogenic prion protein.