Decomposition of extremely hard-to-degrade animal proteins by thermophilic bacteria

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 102, No. 2, 73–81. 2006 DOI: 10.1263/jbb.102.73

© 2006, The Society for Biotechnology, Japan

REVIEW Decomposition of Extremely Hard-to-Degrade Animal Proteins by Thermophilic Bacteria Yasunori Suzuki,1 Yoshiyuki Tsujimoto,1 Hiroshi Matsui,1 and Kunihiko Watanabe1* Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522, Japan1 Received 2 March 2006/Accepted 15 May 2006

Hard-to-degrade animal proteins are ubiquitously present throughout animal bodies. Enormous numbers of these proteins generated in the meat industry are converted to industrial wastes, the disposal of which is tremendously difficult. Most hard-to-degrade animal proteins are currently disposed of by incineration; however, this method has ecological disadvantages in terms of an apparent energy loss and the production of a large amount of carbon dioxide. As a result, an innovative solution to these problems has been sought. In this review, we focus on the degradation of three hard-to-degrade animal proteins (extracellular matrix proteins, collagen in particular, keratin, and prion proteins) and discuss the decomposing capability of thermophilic bacteria. These proteins are strongly resistant to proteinases because of their structural features; therefore, new approaches employing bacterial proteases with strong activity and broad specificity are required for practical application. [Key words: protein degradation, thermophile, extracellular matrix protein, collagen, keratin, prion, protease, keratinase, collagenase]

teins are strongly resistant to proteinases because of their rigid structures; therefore, the practical application of new approaches employing bacterial proteases with strong activity and broad specificity is required. In this review, we focus on the degradation of EMPs, KRTs, and prion proteins and discuss the decomposing capability of thermophilic bacteria. Thermophilic bacteria are used in the decomposition of these hard-to-degrade animal proteins because in the elevated temperature range where thermophilic bacteria grow, such proteins tend to gain plasticity, resulting in more susceptibility to protease attack (9). However, the temperature range (over approximately 80°C) suitable for growing extremely thermophilic bacteria too rapidly induces thermal denaturation of the proteins (10). In addition, moderately thermophilic bacteria that show an optimum temperature for growing below about 80°C are superior to extremely thermophilic bacteria in terms of the energy cost required to maintain high temperature for bacterial growth. Moreover, thermophilic bacteria possess significant merits in pathogenity. Microbes with strong proteolytic activity are often significantly related to pathogenity in humans, other animals, and insect hosts, as seen in the case of collagens (11, 12). Strong proteolytic proteases assist such microbes to invade the tissues of animal bodies through extracellular matrix including collagen, and as a result, bacterial toxins can spread over the cells. However, there have been no reports of the pathogenicity of obligate thermophiles in a meso-

Insoluble and hard-to-degrade animal proteins are ubiquitously present throughout animal bodies. Enormous numbers of these proteins are generated in the meat industry in a mixture of bones, organs, and hard tissues, finally being converted to industrial wastes, the disposal of which is tremendously difficult (1). Most hard-to-degrade animal proteins are currently disposed of by incineration (1–3). This method, however, has ecological disadvantages in terms of an apparent energy loss and the production of a large amount of carbon dioxide. Thus, an innovative solution to these problems is urgently needed. In terms of quantity, the major hard-to-degrade animal proteins are extracellular matrix proteins (EMPs; see the next section for details). A large number of keratins (KRTs) are also generated, mainly from poultry processing and the leather industry (4, 5). Compared with these two proteins, prion proteins are produced in much smaller amounts but pose more serious problems because they have highly aggregated, hard-to-degrade amyloid isoforms that cause bovine spongiform encephalopathy (BSE). Prion proteins, which have not been fully characterized, recently have attracted general attention due to their serious pathogenity in meat. Prion proteins show extraordinary resistance to most physical and chemical methods used for the inactivation of conventional pathogens (6–8). EMPs, KRTs, and prion pro* Corresponding author. e-mail: [email protected] phone/fax: +81-(0)75-703-5667 73

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philic temperature range, which suggests that thermophiles have a great advantage in terms of their safe use. For the above reasons, it should be noted that characteristic proteases produced from thermophilic bacteria take precedence over other proteases from mesophilic and psychrophilic ones. This review deals in detail with the employment of thermophilic bacteria for the decomposition of EMPs, KRTs, and prion proteins. I. DECOMPOSITION OF EXTRACELLULAR MATRIX PROTEINS (EMPs) Animal tissues consist of an extracellular space in addition to cells themselves. The extracellular space is filled with various species of macromolecules composed of an extracellular matrix that occupies the major part of the tissue volume (Fig. 1). The main components of the extracellular matrix are usually a variety of proteins and polysaccharides that maintain or protect the shapes of cells and tissues over the long term (13). The polysaccharide chains are called glycosaminoglycans, which are mostly found covalently bound to proteins in the form of proteoglycans. The matrix proteins are classified into two functional types: structural and adhesive. The structural proteins (e.g., collagen and elastin) provide tensile strength and/or rubber-like resilience to the matrix itself, while the adhesive proteins (e.g., fibronectin and laminin) allow cells to attach to the appropriate part of the extracellular matrix. Among these proteins,

FIG. 1. Schematic representation for the structure of extracellular matrix proteins in a tissue. The grey portion shows the extracellular matrix area containing water molecules, a whole bunch of proteoglycans such as hyaluronan, chondroitin sulfate, dermatan sulfate, heparan sulfate and keratan sulfate, and other minor components. Collagen fibrils (thick lines) composed of three wound polypeptides are located in the outside of cells with elastins (cross-linked lines).

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collagens are found in all animals and are the most abundant proteins. They are, in fact, the major components of skin and bone in mammals, constituting over 25% of their total protein mass (11, 14). Consequently, collagens appear to be one of the biggest protein sources on earth. In this section, the main focus is on the decomposition of collagens in EMPs. Until recently, mammalian EMPs containing collagens were thought to be inert due to their major but static role of maintaining the physical structure of tissues. However, it is becoming more apparent that EMPs play a much more dynamic and complicated role in regulating cell functions, such as development, migration, proliferation, shape maintenance, and protection. As a result, interest in the medical application of collagens and their fragments has increased steadily (15). The structure of collagen is unique compared with those of soluble animal proteins (16). In primary sequence, glycine and proline occur more frequently than any other amino acids, and the two amino acids account for nearly 50% of all amino acids in mature collagens due to numerous repetitions of a tripeptide unit -Gly-Pro-X- in the sequences of collagens. Proline residues are often posttranslationally modified to hydroxyprolines as well as lysine residues. In the tertiary structure, collagens consist of fibrils composed of laterally aggregated, polarized tropocollagen molecules (molecular weight approximately 300,000). Each rod-like tropocollagen unit consists of three helically wound polypeptide α-chains, causing the collagens’ stiff structure and insolubility, which make degradation difficult. Thus far, a limited number of microorganisms that can decompose collagen have been reported (11, 17). It should be noted that most such microorganisms are mesophiles and tend to be pathogenic to other organisms (11, 12, 18), which is reasonable since these pathogens always try to invade and devour the higher eukaryotic cells by injuring the collagencontaining extracellular matrix under parasitic conditions. Under normal circumstances, it is very difficult to deal with such pathogens in practical applications, e.g., the cultivation of the microorganisms and the purification of collagenolytic proteases. On the other hand, there are rare pathogenic strains of thermophiles, even those that decompose collagen. Thermophiles, which show optimal growth in an elevated temperature range, essentially live in an environment that is very different from that of mesophiles. In this sense, thermophilic bacteria which degrade collagens have an advantage over mesophilic ones. Only a few collagen-degrading thermophiles have been reported (11, 19, 20). One interesting thermophile, Alicyclobacillus sendaiensis NTAP-1, was isolated from soil in Sendai, Japan (20). This strain preferentially grows in the acidic range of pH 2.5–6.5 and produces a very characteristic collagenolytic protease (ScpA) belonging to a family of serine-carboxy proteases and showing unique cleavage sites toward collagens, with an optimum pH of 3.9 for catalytic activity (19, 21). The tertiary structure of the collagenolytic protease was revealed by X-ray crystallography, and extensive analyses were done (22). Although there were no strain employed for application among acidic and thermophilic bacilli, the screening of this strain and its series of intensive studies tell us the possibility of acidic thermophiles that can

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preferentially function within a specific pH range (21). In addition, Geobacillus collagenovorans MO-1 has been reported to efficiently degrade collagens in a neutral pH range (20). This strain can optimally grow at temperatures between 50–70°C and is peculiar in producing two types of collagen-degrading enzymes (12). One is a collagenolytic protease which acts on macromolecular collagens and is characterized by a very high molecular mass (210 kDa) consisting of two identical 105 kDa subunits. The other enzymes are two isozymes of oligopeptidases that recognize the collagen-specific tripeptide unit, Gly-Pro-X, contained in collagen and cleave the peptide bond proceeding to Gly (12). The collagenolytic protease has high activity toward types I and IV collagens and gelatin. Type I collagen is known as the most major form of fibrillar collagen, while type IV collagen is non-fibrillar but is consisted of a basement membrane-associated network. However, a peptide substrate for typical collagenases (4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz-peptide]) is inert for the enzyme. The enzyme belongs to a serine protease family, whereas the majority of collagenases belong to the family of zinc metalloproteases (11, 19). Furthermore, the collagenolytic protease is not only secreted into the culture but also attached to the cells as a cell-surface proteinase (unpublished result). Similar cell-surface proteinases have been reported for gram-negative bacteria, such as lactobacilli (23). Their common feature is that the C-terminal segment functions as an anchor to the outer membrane or cell wall, which allows the application of the mechanisms to display some proteins on the cell surface (24). On the other hand, two isozymes of Pz-peptide-hydrolyzing oligopeptidases can recognize the collagen-specific sequence but can hydrolyze fewer than 13-residue oligopeptides (12). Therefore, these two Pz-peptidases do not directly digest collagens but cleave peptides generated from collagens by collagenolytic protease. Interestingly, there is only 22% identity in the primary structure between two Pz-peptidases in spite of their similar enzymatic functions. They share no antigenic determinants, so each protein is independently translated from a distinct gene. Moreover, the finding that bacteria with one set of two Pz-peptidase homologues are apparently mesophilic pathogens such as the Bacillus anthrasis strain Ames and B. cereus ATCC 10987 gives some clues to the pathogenity of bacteria (12). It is plausible that the Pz-peptidase homologues are integrated into a mechanism in which pathogenic microorganisms attach to the skin of host cells, decompose collagens, and finally display pathogenity. In the collagen-degrading mechanism in strain MO-1, macromolecular collagens are initially degraded by extracellular collagenolytic protease, and then the resulting oligopeptides are transported to the inside of cells to be further hydrolyzed by the two Pz-peptidases (Fig. 2). The transportation of oligopeptides into cells most likely occurs with the oligopeptide-binding protein or receptor (25, 26). In addition, a shorter form of prolyl peptides generated by collagen digestion, which are essentially resistant to further enzymatic hydrolysis, can be cleaved by using an aminopeptidase with wide substrate specificity produced in a different strain (27). Another potential application of collagenolytic proteases is the use of collagen-binding domains contained in bac-

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FIG. 2. Putative degradation mechanism of collagen in Geobacillus collagenovorans MO-1. Collagenolytic protease is located extracellularly and on the cell surface while two Pz-peptidases are cytosolic. The end products are supposed to be dipeptides and tripeptides.

terial collagenolytic proteases as a drug delivery system. For example, a fusion protein carrying the epidermal growth factor (EGF) at the N-terminal of collagen-binding domain derived from C. hystolyticum collagenase, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 d, whereas unfused EGF was not detectable 24 h after injection. This is the result that the collagenbinding domain functioned for a drug delivery system as an anchoring unit by recognizing and binding collagens. Essentially, these domains show looser specificity in collagen species compared with those of mammalian collagenolytic proteases (28). The looser specificity may restrict the use of bacterial collagen-binding domains in targeting a specific organs and/or tissues. In contrast with the fact that mesophilic collagenolytic proteases from Streptomyces parvulum subsp. citrinum and Clostridium histolyticum were easily inactivated by 5 min incubation at 60°C (20, 21, 29), the collagenolytic protease from A. sendaiensis and three collagen-degrading enzymes from G. collagenovorans MO-1 were by far more resistant to thermal denaturation (20, 21); i.e., they were active after 30 min treatment at pH 7.5 and 60°C, which indicates that their thermostability is greatly advantageous for industries manufacturing collagen or EMP-derived products. Among EMP-derived products, several EMP fragments have potent antiangiogenic properties that are only detected after proteolytic cleavage of the fragments from their parental molecules (15, 30). These cryptic endogenous inhibitors of angiogenesis specifically inhibit endothelial cell prolifer-

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ation and migration in vitro and also have impressive antitumor activity in vivo (31–33). Endostatin (31) and tumstatin (32, 34) represent non-collagenous domains of specific collagen molecules. The two fragments are derived from the α1 chain of collagen XVIII and the α3 chain of collagen IV and are 20 kDa and 26 kDa in molecular mass, respectively. These globular domains are released from a collagen-containing vascular basement membrane by the action of proteolytic enzymes, such as elastase, cathepsin L, or matrix metalloprotease-9, in a protease-sensitive region. It is highly possible that many other EMP-derived angiogenesis inhibitors will be found. However, further investigations of these EMP-derived angiogenesis inhibitors, as well as endostatin and tumstatin, are needed in order to confirm antitumor effects and reveal the mechanism that allows the collagen fragments to work on tumor endothelial cells. A critical problem is how to prepare large quantities of pharmaceutical grade-EMP fragments. An alternative method for peptide fragment preparation is organic synthesis. The development of a more efficient and cheaper method using thermophilic and collagenolytic proteases instead of mammalian collagenolytic proteases has been attempted; however, the application of thermophilic enzymes to the preparation of peptide fragments has not yet been achieved since there is no example overcoming practical difficulties. II. DECOMPOSITION OF KERATIN PROTEINS Keratins (KRTs) are the most abundant proteins in mammalian epithelial cells and the major components of skin, nail, hair, feather, and wool. Worldwide, more than 10,000 t of KRT is generated as a waste by-product at poultry processing plants annually (35). Two types of KRTs, α-KRTs and β-KRTs, consist of tightly packed protein chains in α-helices and β-sheets, respectively (36). Furthermore, KRT filament structures are stabilized by their high degree of cross-linking of disulfide bonds, hydrophobic interactions, and hydrogen bonds (37). Due to their extremely rigid structures, KRTs are insoluble and hard to degrade. On the other hand, since poultry feathers contain a great deal of potentially useful proteins and amino acids, KRTs, to some extent, have been used in cheap animal feedstuffs as feather meal. However, the conversion of feathers to feather meal results in the loss of nutritionally essential amino acids, such as methionine, lysine, and tryptophane, and the formation of non-nutritive amino acids, such as lysinoalanine and lanthionine (38). This is because the process in conversion of feathers to feather meal includes severe treatments under high temperature and pressure. Therefore, an alternative method of efficiently degrading KRTs without this conventional defect has been sought. Although a limited number of potentially useful microorganisms have been found, such microorganisms must have the capacity to utilize KRT for growth, and specific and strong keratinases must be able to degrade the KRT. For more practical use bearing commercial benefits, the development of a durable and fast KRT-degrading system using suitable microorganisms is essential. The keratinolytic activity produced by thermophilic microorganisms is of greatest importance. Most of the keratinolytic proteases reported previously, with the exception of

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keratinases from yeasts, belong to a family of serine-proteases (39), although they are secreted extracellularly and/or in the outer-membrane-bound form. This finding is in contrast with the fact that the majority of collagenolytic proteases are zinc-metallopeptidases. In this situation, only a few cases for KRT degradation by thermophilic bacteria have been reported. One case of the use of Thermoactinomyces candidus for the complete digestion of wool KRT has been reported (40). This thermophilic actinomycete strain was isolated from soil, and the keratinolytic activity was induced by the addition of KRT into the medium. The keratinase molecule produced by the strain was monomeric, with an apparent molecular mass of 30 kDa. The inducible nature of the excreted keratinase allows the assumption that its primary function is to degrade keratinaceous substrates as a nutrient source for actinomycetes. Interestingly, elastase, specifically degrading elastin can play a role of keratinase in a keratinolytic fungus Trichophyton rubrum (41). The high thermostability of the enzyme and the strain tested allows performance of the process at 65–70°C. A series of studies done with two species of Fervidobacter strains provides another example (42–45). Fervidobacterium pennivorans, isolated from a hot spring of the Azore islands in the Atlantic Ocean, belongs to the anaerobic Thermotogales and grows optimally at 70°C and pH 6.5 (42). This extreme thermophile is the first known to be able to degrade native feathers at high temperatures. The strain produces an extracellular subtilisin-like serine protease, Fervidolysin, which is composed of a signal peptide and a proteolytic part containing a catalytic region (45). Crystal structure analysis on Fervidolysin at 1.7 Å resolution suggests a very characteristic functional relationship to the fibronectin-like domains of the human pro-matrix metalloprotease-2 (proMMP-2), which degrades fibrous polymeric substrate gelatin (46). Another native-feather-degrading Fervidobacter species is Fervidobacterium islandicum AW-1, isolated from a geothermal hot stream in Indonesia (43). The strain grows anaerobically at 40–80°C and at pH 5–9, whereas the keratinolytic protease that is homomultimeric membrane-bound (>200 kDa; 97 kDa subunits) shows optimal protease activity at 100°C and pH 9 and has a half-life of 90 min at 100°C. These characteristics are in contrast to those of Fervidolysin. From the same region where F. pennivorans was isolated, Thermoanaerobacter keratinophilus was isolated by the same group (44). The properties of its keratinolytic protease are similar to those of Fervidolysin; however, no further details have been reported. Some reports have described thermoactive keratinolytic proteases produced by mesophilic microorganisms (47, 48). These enzymes show keratinolytic activity at temperatures above 70°C, whereas most of the other keratinases from mesophilic bacteria and fungi are active at a rather alkaline pH but show optimal activity at lower temperatures (47, 48). Thermoactive keratinases from mesophiles seem to be useful; however, it takes several days to degrade native KRT completely; therefore, keratinolytic activity that is more resistant to higher temperatures is required for practical applications. Moreover, because mesophilic bacteria and dermatophytes producing strong keratinases are mostly pathogenic, they are undesirable for application (49).

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For screening of KRT-degrading microorganisms, the isolation of those possessing higher disulfide reductase activity is significant because the use of reducing agents, such as dithiothreitol and 2-mercaptothethanol, has remarkably improved KRT degradation (43, 50). Furthermore, the reduction of disulfide bridges to thiol in KRT degradation was observed and has become a powerful tool in several mesophiles (51–56). Therefore, a thermophilic bacterium possessing the extracellular activities of a strong keratinolytic protease and disulfide reductase seems to have potential for the degradation of native KRT. There is only one report related to thermostable disulfide reductase for thioredoxin produced by a thermophile (57). Unfortunately, there is no report dealing with an extracellular disulfide reductase from thermophile with a broad specificity. In evaluating keratinolytic activity, it is very difficult to judge whether one thermophilic candidate is superior to another because there is no standard for differentiating keratinolytic activity and substrate specificity. One of the main reasons is that KRT substrates are a lot of variety, KRT azure (or azokeratin), chicken feather, sheep wool, feather meal, cow horn, and human hair in native or milled (or powder) form. In addition, the strength of keratinolytic activity has been determined and compared for whole cells or subcellular fractions (extracellular, cellular [membrane and wall], and intracellular) with various systems. The cases with whole cells show that it takes 2 to 7 d to digest 1% (w/v) KRT completely (42, 44, 55). Enzyme activity with subcellular fractions are described in units defined as the amount of enzyme causing an increase of absorbance at 440 nm by cleaving azokeratin (51), resulting in an increase in absorbance at 280 nm per min (43, 44) or at 660 nm by Folin-Ciocalteu reagent (55, 58) per proper time. Furthermore, in some cases, the increase of thiol groups derived from disulfide reduction in KRT molecules was chased (40, 51, 55, 56). These different factors have resulted in confusion as to which microorganism is the most practical candidate for KRT degradation. It was very recently reported that bacterial keratinase has the potential to replace sodium sulfide in the dehairing process (59). Enzymatic dehairing in tanneries has been envisaged as an alternative to sulfides because treatment with extremely toxic sodium sulfide causes an obnoxious odor and pollution. Keratinolytic proteases from thermophiles may clear properties such as strong activity, alkaline pH optima, and low collagenolytic activity, which are the requirements of the tanning industry. It may be the best way to focus the employment of keratinolytic thermophiles on a definitive issue. III. DECOMPOSITION OF PRION PROTEINS Prion proteins (PrP) are causative agents of transmissible spongiform encephalopathy (TSE), a fatal and transmissible neurodegenerative disease. BSE in cow, scrapie in sheep and goat, chronic wasting disease (CWD) in elk and deer, transmissible mink encephalopathy (TME) in mink and Kuru and Creutzfeldt-jakob disease (CJD) in human are types of TSE (60). On the basis of solid scientific data, BSE has been confirmed to be propagated by inappropriate treat-

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ment and the use of animal wastes for feed (61). The agent responsible for BSE could cross the species barrier, even to humans (62–64). Thus, it is believed that the variant CJD (vCJD), which is a new type of CJD, could be a consequence of exposure of humans to BSE (65–67). These diseases are characterized by the accumulation of an abnormal isoform of PrP, represented as PrPSc (68). Since many studies have shown that PrPSc is an essential component of the transmission agent of TSE diseases (69–72), the inactivation of PrPSc is important to prevent the propagation of these diseases. However, PrPSc is highly resistant to conventional sterilization methods for viruses or bacteria, including the use of organic solvents and detergents or autoclaving at 121°C (6, 7, 73). Enzymatic degradation of PrPSc may be effective for inactivation and therefore useful in practical applications such as the decontamination of meat and bone meal or medical apparatus. Although the precise molecular mechanism remains poorly understood, it appears that PrPSc is transformed in conformation from normal PrP (PrPC) (74, 75). Despite the fact that PrPC and PrPSc have the same primary structure, their molecular properties are extremely distinct (72, 76, 77). PrPC is soluble and degraded completely by such proteases as proteinase K, whereas PrPSc forms insoluble aggregates consisting of rigid amyloid and is resistant to proteinase K, although the enzyme removes ~67 amino acid residues (6–7 kDa) from the N-terminus of PrPC (76, 78, 79). The molecular masses of the two proteins PrPC and PrPSc are 33 to 35 kDa and 27 to 30 kDa, respectively. The difference leads to the fact that the remaining region of PrPSc has a much higher β-sheet content than PrPC has (75, 80–82). Such differences in structure are the key to the proteinase resistance of PrPSc. Recent advanced studies have shown that PrPSc is degraded by some microbial proteinases through the use of the denaturing pretreatment or condition. For example, a keratinase produced by Bacillus licheniformis PWD-1 was effective for full digestion of PrPSc in infected cow or sheep brain stem (83). In this study, however, pre-heating above 115°C for 40 min in the presence of the detergent N-lauroylsacrosine was essential for the full digestion of PrPSc by the enzyme. Furthermore, proteinase K, Alcalase, and subtilisin Carlsburg, all serine proteinases, were also able to fully degrade PrPSc under the same conditions, including pre-heating and the presence of the detergent. Alternatively, an alkaline condition also enhances the digestion of PrPSc by the proteinases (84). In strong basic pH range (above pH 12), BSE-infected PrP (probably with more PrPSc included) in mouse brain homogenate was efficiently digested by a subtilisin-like proteinase from an alkalophilic bacterium. The results demonstrate that these proteases are effective for the degradation and inactivation of PrPSc and that the proteolytic susceptibility of PrPSc depends on how many deaggregated and denatured states are caused by heating in the presence of detergent or at an alkaline pH. Thus, the search continues for proteolytic enzymes more specific for degradation of the PrPSc without denaturing treatment. The fact that proteinase-resistant PrPSc contains more β-sheet structure contents allows us to think of β-KRT, contained in feather (85, 86). Because of its rigid fibrous struc-

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TABLE 1. Proteinases effective to PrPSc degradation Enzyme

Source microorganism

Keratinase

Bacillus licheniformis PWD-1

Alcalase, subtilisin Carlsburg

Bacillus licheniformis (commercially available) Tritirachium album (commercially available) Bacillus sp. (commercially available) Streptomyces sp. 99-GP-2D-5 Thermus sp. 16132 Thermus sp. 15573 Thermosipho subsp. VC15 Thermococcus subsp. VC13 Thermoanaerobacter subsp. S290 Streptomyces galbus var. achromogenes 695-206

Proteinase K Properase, protease M, purafect Ox, purafect Alkaline proteinase Protease E Protease F Keratinolytic enzyme Keratinolytic enzyme Keratinolytic enzyme Kertinolytic enzyme

Enzyme reaction Temperature pH (°C) 50 7.2

ture, β-KRT is hard to degrade with ordinary proteinases. Therefore, in evaluating the ability to digest the β-sheetstructured protein, feather KRT could be used as a substrate to substitute for the abnormal prion proteins for proteinase. Anaerobic thermophiles were subjected to screening for growth on the KRTs and the activity of proteinases specifically acting on β-KRT together with the genus Streptomyces (87). Three anaerobic thermophiles belonging to the Thermoanaerobacter, Thermosipho, and Thermococcus subspecies were found to hydrolyze efficiently the thermally denatured amyloid recombinant prion and were selected as candidates for decontaminating amyloid aggregates in animal wastes. Moreover, the Thermoanaerobacter subspecies S290 was shown to hydrolyze the PrPSc deposits in mouse brain without denaturing treatment (87). Although these enzymes degraded PrPSc at 60–80°C, such a moderately thermophilic range of reaction temperature would not be crucial for the deaggregation of PrPSc. Some proteinases still required a detergent in addition to pre-treating above 100°C in order to extensively break down PrPSc to a state in which it is immunochemically undetectable (83). These results indicate that proteinases from these screened thermophiles have substrate specificity preferentially hydrolyzing proteins rich in the β-sheet structure and that the eminent specificity of a proteinase with more activity enables the exclusion of requirements for denaturation and deaggregation. Therefore, the preference of the proteinases for the β-sheet structure is an important element in the degradation of PrPSc without a denaturing state. The proteinases introduced here are summarized in Table 1. Also listed in Table 1 is an alkaline serine proteinase from Streptomyces sp. 99-GP-2D-5, which degrades the scrapie prion included in hamster brain (88). In the upper part of the table, proteinases degrading PrPSc and requiring detergent or alkaline conditions for the function are shown, while those able to degrade PrPSc without denaturing treatments are shown in the lower part. Although there have been no reports dealing with the enzymatic degradation of PrPSc among mesophiles, the proteinases degrading PrPSc

Pre-treatment

Reduction of infectivity

Ref

35

7.2

50

7.2

60

12.0

Heating at 115°C with N-lauroylsacrosine Heating at 115°C with N-lauroylsacrosine Heating at 115°C with N-lauroylsacrosine Nothing

No data

83

No data

83

No data

83

Effective

84

60 80 80 80 80 60

11.0 7.0 7.0 7.2 7.2 7.2

Nothing Nothing Nothing Nothing Nothing Nothing

No data Ineffective Ineffective No data No data No data

88 84 84 87 87 87

30

7.2

Nothing

No data

87

without detergent or under a non-alkaline condition are found in thermophiles and the genus of Streptomyces. Further intense research of microorganisms producing unique proteinases would increase the number of reports regarding the enzymatic degradation of PrPSc. In terms of practical application, the degradation and inactivation of PrPSc and the subsequent decontamination of animal waste and medical apparatus are the most crucial issues. As described above, several microbial enzymes significantly degraded PrPSc, which was definitively shown by Western-blotting analysis. However, the degradation of PrPSc does not always result in the inactivation of PrPSc, as indicated by the data given in Table 1. In fact, protease E and protease F produced by the Thermus species digested PrPSc at a neutral pH, but these enzymes failed to reduce the titration of infectivity (84). Only when PrPSc was denatured or deaggregated by heating, detergents, or at an alkaline pH prior to enzyme reaction could proteinase reduce its infectivity (83, 84, 89). Jackson et al. reported that, after preheating at 100°C for 15 min in the presence of 2% SDS, mouse PrP infected with human vCJD was not fully degraded by proteinase K, whereas, in succession to proteinase K, the addition of pronase produced by Streptomyces griseus succeeded in eliminating the signal of PrPSc as well as its infectivity (89). In most studies employing conventional microbial enzymes from mesophiles, the denaturing treatment was carried out prior to enzyme digestion because the enzymes are unstable under PrPSc-denaturing conditions. In contrast, the employment of enzymes from thermophiles is advantageous for the decontamination of PrPSc even if they are not specific for a β-sheet structure because thermophilic enzymes are stable against higher temperatures, detergents, and other denaturing conditions (90) and, therefore, enzyme degradation can be carried out together with denaturing treatment. However, the simplest and best way to employ proteinases that are extremely stable and preferentially hydrolyze β-sheet-structured proteins would be by a onestep procedure without denaturing treatment. In conclusion, screening for thermophilic microorganisms producing en-

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zymes with the two unique features (stability and preference for β-sheet-structured proteins) would dramatically advance the procedures for decontaminating materials and apparatus containing infectious prion proteins.

9. 10.

IV. FUTURE PERSPECTIVE In addition to the hard-to-degrade animal proteins introduced here, there are still many unknown forms of animal proteins whose products are multifunctional as bioactive agents. The study of such molecules allows us to foresee the possibility of a new approach to using thermophilic degradative enzymes as decomposing pathways or mechanisms. For example, amyloid β peptide is the main component of senile plaques, and its fibrillar accumulation in brain results in Alzheimer’s disease. A similar form of aggregates occurs in some other neurodegenerative diseases, such as BSE, Huntington’s disease, and Parkinson’s disease (91). These fibrillar aggregates are synthesized in brain since they are hard-to-degrade animal proteins and an unbalance or defect arises in the catabolism of these degradation mechanisms. However, little is known about how those proteins are degraded and cleared according to normal catabolism. The development of rapid and efficient degradation of amyloid β peptides with the use of thermophilic proteases will help to advance therapeutics designed to prevent or treat these diseases (92). In conclusion, it is necessary to pursue a greater variety of thermophilic microorganisms producing characteristic proteases with broad substrate specificity and thermostable activity in order to establish the utility of thermophilic microorganisms for the degradation of hard-to-degrade animal proteins.

11. 12.

13. 14. 15. 16. 17. 18. 19.

20.

REFERENCES 1. Deydier, E., Guilet, R., Sarda, S., and Sharrock, P.: Physical and chemical characterization of crude meat and bone meal combustion residue: “waste or raw material?”. J. Hazard Mater., 121, 141–148 (2005). 2. Paisley, L. G. and Hostrup-Pedersen, J.: A quantitative assessment of the BSE risk associated with fly ash and slag from the incineration of meat-and-bone meal in a gas-fired power plant in Denmark. Prev. Vet. Med., 68, 263–275 (2005). 3. Haruta, S., Nakayama, T., Nakamura, K., Hemmi, H., Ishii, M., Igarashi, Y., and Nishino, T.: Microbial diversity in biodegradation and reutilization processes of garbage. J. Biosci. Bioeng., 99, 1–11 (2005). 4. Barone, J. R. and Schmidt, W. F.: Effect of formic acid exposure on keratin fiber derived from poultry feather biomass. Bioresour. Technol., 97, 233–242 (2006). 5. Balint, B., Bagi, Z., Toth, A., Rakhely, G., Perei, K., and Kovacs, K. L.: Utilization of keratin-containing biowaste to produce biohydrogen. Appl. Microbiol. Biotechnol., 69, 404– 410 (2005). 6. Taylor, D. M.: Inactivation of BSE agent. Dev. Biol. Stand., 75, 97–102 (1991). 7. Taylor, D. M., Woodgate, S. L., and Atkinson, M. J.: Inactivation of the bovine spongiform encephalopathy agent by rendering procedures. Vet. Rec., 137, 605–610 (1995). 8. Kuczius, T., Buschmann, A., Zhang, W., Karch, H., Becker, K., Peters, G., and Groschup, M. H.: Cellular prion

21.

22.

23.

24. 25. 26.

79

protein acquires resistance to proteolytic degradation following copper ion binding. Biol. Chem., 385, 739–747 (2004). Sugiyama, S., Hirota, A., Okada, C., Yorita, T., Sato, K., and Ohtsuki, K.: Effect of kiwifruit juice on beef collagen. J. Nutr. Sci. Vitaminol., 51, 27–33 (2005). van der Plancken, I., van Remoortere, M., Indrawati, I., van Loey, A., and Hendrickx, M. E.: Heat-induced changes in the susceptibility of egg white proteins to enzymatic hydrolysis: a kinetic study. J. Agric. Food Chem., 51, 3819–3823 (2003). Watanabe, K.: Collagenolytic proteases from bacteria. Appl. Microbiol. Biotechnol., 63, 520–526 (2004). Miyake, R., Shigeri, Y., Tatsu, Y., Yumoto, N., Umekawa, M., Tsujimoto, Y., Matsui, H., and Watanabe, K.: Two thimet oligopeptidase-like Pz peptidases produced by a collagen-degrading thermophile Geobacillus collagenovorans MO-1. J. Bacteriol., 187, 4140–4148 (2005). Huang, S. and Ingber, D. E.: The structural and mechanical complexity of cell-growth control. Nat. Cell Biol., 1, E131– 138 (1999). Linsenmeyer, T. F.: Collagen, p. 7–44. In Hay, E. D. (ed.), Cell biology of extracellular matrix. Plenum, New York (1991). Clamp, A. R. and Jayson, G. C.: The clinical potential of antiangiogenic fragments of extracellular matrix proteins. Br. J. Cancer, 93, 967–972 (2005). Nimni, M. E.: Collagen: structure, function, and metabolism in normal and fibrotic tissues. Semin. Arthritis Rheum., 13, 1–86 (1983). Peterkofsky, B.: Bacterial collagenase. Methods Enzymol., 82, 453–471 (1982). Harrington, D. J.: Bacterial collagenases and collagen degrading enzymes and their potential role in human disease. Infect. Immun., 64, 1885–1891 (1996). Tsuruoka, N., Isono, Y., Shida, O., Hemmi, H., Nakayama, T., and Nishino, T.: Alicyclobacillus sendaiensis sp. nov., a novel acidophilic, slightly thermophilic species isolated from soil in Sendai, Japan. Int. J. Syst. Evol. Microbiol., 53, 1081– 1084 (2003). Okamoto, M., Yonejima, Y., Tsujimoto, Y., Suzuki, Y., and Watanabe, K.: A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1. Appl. Microbiol. Biotechnol., 57, 103–108 (2001) Tsuruoka, N., Nakayama, T., Ashida, M., Hemmi, H., Nakao, M., Minakata, H., Oyama, H., Oda, K., and Nishino, T.: Collagenolytic serine-carboxy proteinase from Alicyclobacillus sendaiensis strain NTSP-1: purification, characterization, gene cloning and heterologous expression. Appl. Environ. Microbiol., 69, 162–169 (2003). Wlodawer, A., Li, M., Gustchina, A., Tsuruoka, N., Ashida, M., Minakata, H., Oyama, H., Oda, K., Nishino, T., and Nakayama, T.: Crystallographic and biochemical investigations of kumamolisin-As, a serine-carboxyl peptidase with collagenase activity. J. Biol. Chem., 279, 21500–21510 (2004). Germond, J. E., Delley, M., Gilbert, C., and Atlan, D.: Determination of the domain of the Lactobacillus delbrueckii subsp. bulgaricus cell surface proteinase PrtB involved in attachment to the cell wall after heterologous expression of the prtB gene in Lactococcus lactis. Appl. Environ. Microbiol., 69, 3377–3384 (2003). Ueda, M. and Tanaka, A.: Cell surface engineering of yeast: construction of arming yeast with biocatalyst. J. Biosci. Bioeng., 90, 125–136 (2000). Monnnet, V.: Bacterial oligopeptide-binding proteins. Cell Mol. Life Sci., 60, 2100–2114 (2003). Garault, P., Le Bars, D., Besset, C., and Monnet, V.: Three oligopeptide-binding proteins are involved in the oligopeptide transport of Streptococcus thermophilus. J. Biol. Chem., 277,

80

J. BIOSCI. BIOENG.,

SUZUKI ET AL.

32–39 (2002). 27. Murai, A., Tsujimoto, Y., Matsui, H., and Watanabe, K.: An Aneurinibacillus sp. strain AM-1 produces a proline-specific aminopeptidase useful for collagen degradation. J. Appl. Microbiol., 96, 810–818 (2004). 28. Nishi, N., Matsushita, O., Yuube, K., Miyanaka, H., Okabe, A., and Wada, F.: Collagen-binding growth factors: production and characterization of functional fusion proteins having a collagen-binding domain. Proc. Natl. Acad. Sci. USA, 95, 7018–7023 (1998). 29. Nakayama, T., Tsuruoka, N., Akai, M., and Nishino, T.: Thermostable collagenolytic activity of a novel thermophilic isolate, Bacillus sp. strain NTAP-1. J. Biosci. Bioeng., 89, 612–614 (2000). 30. Nyberg, P., Xie, L., and Kalluri, R.: Endogenous inhibitors of angiogenesis. Cancer Res., 65, 3967–3979 (2005). 31. O’Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J.: Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell, 88, 277–285 (1997). 32. Hamano, Y., Zeisberg, M., Sugimoto, H., Lively, J. C., Maeshima, Y., Yang, C., Hynes, R. O., Werb, Z., Sudhakar, A., and Kalluri, R.: Physiological levels of tumstatin, a fragment of collagen IV α3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via αV β3 integrin. Cancer Cell, 3, 589–601 (2003). 33. Chang, J. H., Javier, J. A., Chang, G. Y., Oliveira, H. B., and Azar, D. T.: Functional characterization of neostatins, the MMP-derived, enzymatic cleavage products of type XVIII collagen. FEBS Lett., 579, 3601–3606 (2005). 34. Maeshima, Y., Manfredi, M., Reimer, C., Holthaus, K. A., Hopfer, H., Chandamuri, B. R., Kharbanda, S., and Kalluri, R.: Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin. J. Biol. Chem., 276, 15240–15248 (2001). 35. Williams, C. M., Lee, C. G., Garlich, J. D., and Shih, J. C. H.: Evaluation of a bacterial feather fermentation product, feather-lysate, as a feed protein. Poultry Sci., 70, 85–94 (1991). 36. Parry, D. A. D. and North, A. C. T.: Hard α-keratin intermediate filament chains: substructure of the N-and C-terminal domains and the predicted structure and function of the C-terminal domains of type I and type II chains. J. Struct. Biol., 122, 279–290 (1998). 37. Fuchs, E.: Keratins and the skin. Annu. Rev. Cell Dev., 11, 123–153 (1995). 38. Dalev, P., Ivanov, I., and Liubomirova, A.: Enzymic modification of feather keratin hydrolyzates with lysine aimed at increasing the biological value. J. Sci. Food Agric., 73, 242– 244 (1997). 39. Lin, X., Tang, J., Koelsch, G., Monod, M., and Foundling, S.: Recombinant canditropsin, an extracellular aspartic protease from yeast Candida tropicalis. Escherichia coli expression, purification, zymogen activation, and enzymic properties. J. Biol. Chem., 268, 20143–20147 (1993). 40. Ignatova, Z., Gousterova, A., Spassov, G., and Nedkov, P.: Isolation and partial characterization of extracellular keratinase from a wool degrading thermophilic actinomycete strain Thermoactinomyces candidus. Can. J. Microbiol., 45, 217– 222 (1999). 41. Asahi, M., Lindquist, R., Fukuyama, K., Apodaca, G., Epstein, W. L., and McKerrow, J. H.: Purification and characterization major extracellular proteinases from Trichophyton rubrum. Biochem. J., 232, 139–144 (1985). 42. Friedlich, A. B. and Antranikian, G.: Keratin degradation by Fervidobacterium pennivorans, a novel thermophilic anaerobic species of the order Thermotogales. Appl. Environ. Microbiol., 62, 2875–2882 (1996). 43. Nam, G. W., Lee, D. W., Lee, H. S., Lee, N. J., Kim, B. C.,

44.

45.

46.

47.

48.

49.

50. 51.

52. 53. 54. 55.

56.

57. 58. 59.

60. 61.

Choe, E. A., Hwang, J. K., Suhartono, M. T., and Pyun, Y. R.: Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch. Microbiol., 178, 538–547 (2002). Riessen, S. and Antranikian, G.: Isolation of Thermoanaerobacter keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity. Extremophile, 5, 399–408 (2001). Kluskens, L. D., Voorhorst, W. G., Siezen, R. J., Schwerdtfeger, R. M., Antranikian, G., van der Oost, J., and de Vos, W. M.: Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans. Extremophiles, 6, 185–194 (2002). Kim, J. S., Kluskens, L. D., de Vos, W. M., Huber, R., and van der Oost, J.: Crystal structure of fervidolysin from Fervidobacterium pennivorans, a keratinolytic enzyme related to subtilisin. J. Mol. Biol., 335, 787–797 (2004). Dozie, I. N. S., Okeke, C. N., and Unaeze, N. C.: A thermostable, alkaline-active keratinolytic proteinases from Chrysosporium keratinophilum. World J. Microbiol. Biotechnol., 10, 563–567 (1994). Takami, H., Nakamura, S., Aono, R., and Horikoshi, K.: Degradation of human hair by a thermostable alkaline protease from alkalophilic Bacillus sp. no. AH-101. Biosci. Biotechnol. Biochem., 56, 1667–1669 (1992). Gradisar, H., Friedrich, J., Krizaj, I., and Jerala, R.: Similarities and specificities of fungal keratinolytic proteases: comparison of keratinases of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Appl. Environ. Microbiol., 71, 3420–3426 (2005). Böckle, B., Gaulunsky, B., and Müller, R.: Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530. Appl. Environ. Microbiol., 61, 3705–3710 (1995). Riffel, A., Lucas, F., Heeb, P., and Brandelli, A.: Characterization of a new keratinolytic bacterium that completely degrades native feather keratin. Arch. Microbiol., 179, 258–265 (2003). Böckle, B. and Müller, R.: Reduction of disulfide bonds by Streptomyces pactum during growth on chicken feathers. Appl. Environ. Microbiol., 63, 790–792 (1997). Kunert, J. and Stransky, Z.: Thiosulfate production from cysteine by the keratinophilic prokaryote Streptomyces fradiae. Arch. Microbiol., 150, 600–601 (1988). Santos, R. M. D. B., Firmino, A. A., de Sa, C. M., and Felix, C. R.: Keratinolytic activity of Aspergillus fumigatus fresenius. Curr. Microbiol., 33, 364–370 (1996). Yamamura, S., Morita, Y., Hasan, Q., Rao, S. R., Murakami, Y., Yokoyama, K., and Tamiya, E.: Characterization of a new keratin-degrading bacterium isolated from deer fur. J. Biosci. Bioeng., 93, 595–600 (2002). Yamamura, S., Morita, Y., Hasan, Q., Yokoyama, K., and Tamiya, E.: Keratin degradation: a cooperative action of two enzymes from Stenotrophomonas sp. Biochem. Biophys. Res. Commun., 294, 1138–1143 (2002). Erratum: 295, 1034 (2002). Jeon, S. J. and Ishikawa, K.: Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1. Eur. J. Biochem., 269, 5423–5430 (2002). Ledoux, M. and Lamy, F.: Determination of proteins and sulfobetaine with the Folin-phenol reagent. Anal. Biochem., 157, 28–31 (1986). Macedo, A. J,, da Silva, W. O., Gava, R., Driemeier, D., Henriques, J. A., and Termignoni, C.: Novel keratinase from Bacillus subtilis S14 exhibiting remarkable dehairing capabilities. Appl. Environ. Microbiol., 71, 594–596 (2005). Johnson, R. T.: Prion diseases. Lancet Neurol., 4, 635–642 (2005). Anderson, R. M., Donnelly, C. A., Ferguson, N. M., Woolhouse, M. E., Watt, C. J., Udy, H. J., MaWhinney, S.,

VOL. 102, 2006

62. 63.

64.

65.

66.

67.

68. 69. 70. 71. 72.

73. 74.

75.

76.

77.

BREAKDOWN OF HARD-TO-DEGRADE PROTEINS BY THERMOPHILES

Dunstan, S. P., Southwood, T. R., Wilesmith, J. W., Ryan, J. B., Hoinville, L. J., Hillerton, J. E., Austin, A. R., and Wells, G. A.: Transmission dynamics and epidemiology of BSE in British cattle. Nature, 382, 779–788 (1996). Foster, J. D., Hope, J., and Fraser, H.: Transmission of bovine spongiform encephalopathy to sheep and goats. Vet. Rec., 133, 339–341 (1993). Bruce, M., Chree, A., McConnell, I., Foster, J., Pearson, G., and Fraser, H.: Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Philos. Trans. R. Soc. Lond. B Biol. Sci., 343, 405–411 (1994). Maignien, T., Lasmezas, C. I., Beringue, V., Dormont, D., and Deslys, J. P.: Pathogenesis of the oral route of infection of mice with scrapie and bovine spongiform encephalopathy agents. J. Gen. Virol., 80, 3035–3042 (1999). Bruce, M. E., Will, R. G., Ironside, J. W., McConnell, I., Drummond, D., Suttie, A., McCardle, L., Chree, A., Hope, J., Birkett, C., Cousens, S., Fraser, H., and Bostock, C. J.: Transmission to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature, 389, 498–501 (1997). Hill, A. F., Desbruslais, M., Joiner, S., Sidle, K. C., Gowland, I., Collinge, J., Doey, L. J., and Lantos, P.: The same prion strain causes vCJD and BSE. Nature, 389, 448– 450 (1997). Will, R. G., Ironside, J. W., Zeidler, M., Cousens, S. N., Estibeiro, K., Alperovitch, A., Poser, S., Pocchiari, M., Hofman, A., and Smith, P. G.: A new variant of CreutzfeldtJakob disease in the UK. Lancet, 347, 921–925 (1996). Priola, S. A.: Prion protein diversity and disease in the transmissible spongiform encephalopathies. Adv. Protein Chem., 57, 1–27 (2001). Prüsiner, S. B.: Novel proteinaceous infectious particles cause scrapie. Science, 216, 136–144 (1982). Bolton, D. C., McKinley, M. P., and Prüsiner, S. B.: Identification of a protein that purifies with the scrapie prion. Science, 218, 1309–1311 (1982). Weissmann, C., Enari, M., Klohn, P. C., Rossi, D., and Flechsig, E.: Transmission of prions. Proc. Natl. Acad. Sci. USA, 99, 16378–16383 (2002). Oesch, B., Westaway, D., Walchli, M., McKinley, M. P., Kent, S. B., Aebersold, R., Barry, R. A., Tempst, P., Teplow, D. B., and Hood, L. E.: A cellular gene encodes scrapie PrP 27–30 protein. Cell, 40, 735–746 (1985). Taylor, D. M.: Resistance of transmissible spongiform encephalopathy agents to decontamination. Contrib. Microbiol., 11, 136–145 (2004). Kocisko, D. A., Come, J. H., Priola, S. A., Chesebro, B., Raymond, G. J., Lansbury, P. T., and Caughey, B.: Cellfree formation of protease-resistant prion protein. Nature, 370, 471–474 (1994). Pan, K. M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., and Prüsiner, S. B.: Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins. Proc. Natl. Acad. Sci. USA, 90, 10962–10966 (1993). Hope, J., Morton, L. J., Farquhar, C. F., Multhaup, G., Beyreuther, K., and Kimberlin, R. H.: The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP). EMBO J., 5, 2591– 2597 (1986). Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., and Prüsiner, S. B.: Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry, 32, 1991–2002 (1993).

81

78. Meyer, R. K., McKinley, M. P., Bowman, K. A., Braunfeld, M. B., Barry, R. A., and Prüsiner, S. B.: Separation and properties of cellular and scrapie prion proteins. Proc. Natl. Acad. Sci. USA, 83, 2310–2314 (1986). 79. Rubenstein, R., Kascsak, R. J., Merz, P. A., Papini, M. C., Carp, R. I., Robakis, N. K., and Wisniewski, H. M.: Detection of scrapie-associated fibril (SAF) proteins using antiSAF antibody in non-purified tissue preparations. J. Gen. Virol., 67, 671–681 (1986). 80. Safar, J., Roller, P. P., Gajdusek, D. C., and Gibbs, C. J., Jr.: Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein. J. Biol. Chem., 268, 20276– 20284 (1993). 81. Safar, J., Roller, P. P., Gajdusek, D. C., and Gibbs, C. J., Jr.: Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity. Protein Sci., 2, 2206–2216 (1993). 82. Caughey, B. W., Dong, A., Bhat, K. S., Ernst, D., Hayes, S. F., and Caughey, W. S.: Secondary structure analysis of the scrapie-associated protein PrP 27–30 in water by infrared spectroscopy. Biochemistry, 30, 7672–7680 (1991). 83. Langeveld, J. P., Wang, J. J., Van de Wiel, D. F., Shih, G. C., Garssen, G. J., Bossers, A., and Shih, J. C.: Enzymatic degradation of prion protein in brain stem from infected cattle and sheep. J. Infect. Dis., 188, 1782–1789 (2003). 84. McLeod, A. H., Murdoch, H., Dickinson, J., Dennis, M. J., Hall, G. A., Buswell, C. M., Carr, J., Taylor, D. M., Sutton, J. M., and Raven, N. D.: Proteolytic inactivation of the bovine spongiform encephalopathy agent. Biochem. Biophys. Res. Commun., 317, 1165–1170 (2004). 85. Arai, K. M., Takahashi, R., Yokote, Y., and Akahane, K.: Amino-acid sequence of feather keratin from fowl. Eur. J. Biochem., 132, 501–507 (1983). 86. Tsuboi, M., Kaneuchi, F., Ikeda, T., and Akahane, K.: Infrared and Raman microscopy of fowl feather barbs. Can. J. Chem., 69, 1752–1757 (1991). 87. Tsiroulnikov, K., Rezai, H., Bonch-Osmolovskaya, E., Nedkov, P., Gousterova, A., Cueff, V., Godfroy, A., Barbier, G., Metro, F., Chobert, J. M., Clayette, P., Dormont, D., Grosclaude, J., and Haertle, T.: Hydrolysis of the amyloid prion protein and nonpathogenic meat and bone meal by anaerobic thermophilic prokaryotes and streptomyces subspecies. J. Agric. Food Chem., 52, 6353–6360 (2004). 88. Hui, Z., Doi, H., Kanouchi, H., Matsuura, Y., Mohri, S., Nonomura, Y., and Oka, T.: Alkaline serine protease produced by Streptomyces sp. degrades PrP(Sc). Biochem. Biophys. Res. Commun., 321, 45–50 (2004). 89. Jackson, G. S., McKintosh, E., Flechsig, E., Prodromidou, K., Hirsch, P., Linehan, J., Brandner, S., Clarke, A. R., Weissmann, C., and Collinge, J.: An enzyme-detergent method for effective prion decontamination of surgical steel. J. Gen. Virol., 86, 869–878 (2005). 90. Vieille, C. and Zeikus, G. J.: Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev., 65, 1–43 (2001). 91. Irie, K., Murakami, K., Masuda, Y., Morimoto, A., Ohigashi, H., Ohashi, R., Takegoshi, K., Nagao, M., Shimizu, T., and Shirasawa, T.: Structure of β-amyloid fibrils and its relevance to their neurotoxicity: implications for the pathogenesis of Alzheimer’s disease. J. Biosci. Bioeng., 99, 437–447 (2005). 92. Shirotani, K., Tsubuki, S., Iwata, N., Takaki, Y., Harigaya, W., Maruyama, K., Kiryu-Seo, S., Kiyama, H., Iwata, H., Tomita, T., Iwatsubo, T., and Saido, T. C.: Neprilysin degrades both amyloid β peptides 1–40 and 1–42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases. J. Biol. Chem., 276, 21895–21901 (2001).