BACKGROUND
proteases form a major part of the industrial enzyme market, and are applied to detergents as well as food, leather, and fabric processing, and are also used as catalysts in organic synthesis and as therapeutics  <cit> .
with the annual protease sales of about $ <dig> – <dig>  billion, these enzymes account for 60% of the total enzyme market.
detergent proteases, particularly alkaline proteases, with an annual market of about $ <dig> billion, account for the largest part of protease application.
subtilisin carlsberg and related subtilisin serine proteases represent the first generation of detergent proteases with an optimal ph of 9– <dig>  the second-generation detergent proteases, having a higher optimal ph of 10– <dig> and greater temperature stability, are produced from alkalophilic strains of bacillus.
the third generation consists of detergent proteases in which the active sites have been modified or various attempts have been made to enhance their stability through site-directed mutagenesis and protein engineering  <cit> .
proteolytic enzymes from microorganisms are the most widely exploited enzymes in the detergent industries  <cit> .
these enzymes are incorporated into detergent formulations to obtain certain characteristics, such as activity and stability in high alkaline ph range and high temperatures, compatibility with compounds used in detergents such as surfactants, perfumes, and bleaches, and hydrolysis specificity towards other proteins  <cit> .
therefore, there is always a need for newer hyper-stable alkaline proteases with potential application in detergent formulations.
more recently, studies on enzymatic degradation of prion protein have highlighted the ability of some proteases to digest this class of protein  <cit> .
prion diseases are fatal neurodegenerative disorders that include creutzfeldt-jacob disease , gerstmann-sträussler-scheinker syndrome, fatal familial insomnia, and kuru in humans, bovine spongiform encephalopathy  in cattle, and scrapie in sheep  <cit> .
their central feature is the posttranslational conversion of host-encoded, cellular prion protein  to an abnormal isoform, known as prpsc.
this transition appears to involve only a conformational change to a predominantly β-pleated structure, and confers prpsc with partial resistance to proteolytic degradation and detergent insolubility  <cit> .
prpsc is largely unaffected by standard methods of sterilization; thus, contaminated neurosurgical instruments are the major cause of human transmissible spongiform encephalopathies , in addition to exposure to infectious materials through the use of human cadaveric-derived pituitary hormones as well as dural and cornea homotransplants  <cit> .
the results of numerous studies designed to define conditions for the inactivation of prpsc concluded that protein denaturants, such as sodium dodecyl sulfate   <cit> , are effective in reducing infectivity, but that complete inactivation requires extremely intense conditions, such as 1 h of autoclaving at 134°c or treatment with 2 n naoh or sodium hypochlorite for 1 h  <cit> .
however, such intense conditions cannot be applied to certain equipments such as endoscope.
hence, validated methods to inactivate prpsc are highly desirable.
thermococcus kodakarensis is a hyperthermophilic archaeon, which grows most optimally at 90°c  <cit> .
its genome contains three genes encoding subtilisin-like serine proteases: tk-subtilisin   <cit> , tk-sp   <cit> , and tk- <dig> .
of these homologs, tk-subtilisin and tk-sp have been structurally and functionally well studied  <cit> .
tk-subtilisin and tk-sp are highly thermostable enzymes with optimal temperature for activity in the range of 90–100°c and show broad substrate specificity.
tk-subtilisin and tk-sp exhibit the ph dependence and temperature dependence of the activities.
tk-sp exhibits  <dig> to 70% of the maximal activity at a neutral ph range between  <dig>  and  <dig>  at 20°c.
it exhibits  <dig> to 50% of the maximal activity at between  <dig> and 60°c  <cit> .
in particular, tk-sp is highly resistant to heat, 5% sds, 8 m urea, 10% triton x- <dig>  or 10 mm edta; however, unlike tk-subtilisin and bacterial subtilisin, tk-sp does not require ca2+ for folding  <cit> .
thus, tk-sp has a great potential for technological applications.
in the present study, to examine the activity of tk-sp in the presence of  <dig> surfactants  and edta at high temperatures, tk-sp was overproduced in e. coli and purified, and subsequently analyzed for its relative activity at 80°c and 90°c using azocasein as the substrate.
we found that tk-sp could maintain its proteolytic activity in the presence of surfactants, suggesting that this enzyme could be a candidate for industrial applications in detergent formulation.
in addition, to determine whether tk-sp is capable of degrading prpsc, prpsc  accumulated in scrapie-infected mouse brain homogenate  was incubated with tk-sp and then detected by western-blot analysis.
we observed that tk-sp can disrupt prpsc to a level undetectable by western-blot analysis, suggesting that this enzyme has a remarkable characteristic, considering its potential application for the inactivation of prpsc.
RESULTS
effect of surfactants and edta on tk-sp activity
some studies have suggested that an effective detergent protease must be compatible and stable with commonly used detergent compounds, such as surfactants, which might be present in the formulation  <cit> .
to analyze the effects of surfactants on the stability and enzymatic activity of tk-sp,  <dig> nm tk-sp was incubated in 50 mm tris–hcl  containing  <dig>  or 1%  surfactants for 20 min at 80°c or 90°c using azocasein as the substrate.
while tk-sp is known to exhibit its highest activity at 100°c , its relative activities at 90°c or 80°c are  <dig> and 80%, respectively  <cit> .
one unit of enzymatic activity is defined as the amount of enzyme that increases the a <dig> value of the assay reaction mixture by  <dig>  in 1 min  <cit> .
the results of our analysis are summarized in figures  <dig> and  <dig>  the data show that tk-sp retained its activity in the presence of surfactants tested at 80°c and 90°c.
furthermore, tk-sp was found to be highly stable in the presence of both  <dig>  and 1%  nonionic surfactants.
particularly, tk-sp retained more than 100% of its activity in the presence of four of the nonionic surfactants, namely, emulgen  <dig>  emulgen ls- <dig>  emulgen pp- <dig>  and rheodol tw-0120 v. however, the color of the reaction mixture changed to white after the addition of the cationic surfactant, quartamin 60 w, indicating that tk-sp was less stable in the presence of quartamin 60 w. furthermore, although tk-sp was stable in the presence of  <dig> %  amphitol 20y-b, the presence of 1%  amphitol 20y-b caused strong inhibitions, resulting in tk-sp retaining only 50% of its activity.
in contrast, the activities in the presence of 1% sanisol c and amphitol 20 n were higher than those at the concentration of  <dig> %.
this may relate to the critical micelle concentration of the surfactants.
in the presence of anionic surfactants, tk-sp was unstable, losing up to 80% of its activity.
on the other hand, a previous study analyzed the activity of tk-sp at 55°c using n-succinyl-ala-ala-pro-phe-p-nitroanilide  as the substrate, and found that the enzyme is highly resistant to 5%  sds  <cit> .
this discrepancy in the findings may be due to the temperature of the reaction.
in addition, the concentration of tk-sp analyzed in the previous study was ten-fold higher than that in this study, suggesting that high concentration of tk-sp may increase the stability.
thus, from these results, it can be concluded that tk-sp is more stable in the presence of nonionic surfactants, suggesting that intramolecular interactions of ion pairs of tk-sp may contribute to its stability.
the improvement in the activity of tk-sp in the presence of nonionic surfactants might be due to the stimulation of conformational changes in tk-sp or the substrate, leading to activity gain.
chelating agents, such as edta, which function as water softeners and assist in the removal of stains, are valuable components of most of the detergents  <cit> .
as tk-sp has been found to exhibit activity at 80°c in the presence of  <dig> %  edta  <cit> , to analyze the stability and activity of tk-sp against different concentrations of edta,  <dig> nm tk-sp was incubated in 50 mm tris–hcl  containing  <dig> ,  <dig> ,  <dig> ,  <dig> , or 1%  edta for 20 min at 80°c using azocasein as the substrate.
as can be seen in figure  <dig>  the tk-sp activity decreased with the increasing concentration of edta.
several reports have shown that the active structure of serine proteases contains ca2+-binding site, and that the removal of ca2+ from the strong binding site is associated with a significant reduction in thermal stability  <cit> .
our results are in agreement with the previous findings showing that two ca2+ ions bind to the β-jelly roll domain of tk-sp  <cit>  and that tk-sp requires ca2+ ions for maximal stability  <cit> .
in the present study, tk-sp retained 80% of its activity in the presence of  <dig> %  edta , suggesting that this enzyme will be effective in the presence of  <dig> %  edta, which is a commonly used concentration of edta in detergents.
to analyze the compatibility and activity of tk-sp in the presence of both surfactants and edta, the enzyme was incubated in the presence of four  <dig> %  nonionic surfactants and  <dig> %  edta at 80°c.
as shown in figure  <dig>  tk-sp was highly stable in the presence of all of the nonionic surfactants, retaining up to 80% of its activity.
among the additives examined, the best result with respect to tk-sp retaining almost its entire initial activity was observed in the presence of emulgen ls- <dig> and rheodol tw-0120 v. previous reports have shown that stability of protease could be achieved either through site-directed mutagenesis or protein engineering; however, the protease, tk-sp, examined in the present study has been noted to show inherent stability in the presence of surfactants and edta.
in addition, tk-sp has also been observed to exhibit activity at high temperature, suggesting that this enzyme meets the standard requirements of the washer-disinfector , which recommend that a suitable temperature of thermal disinfection for medical instruments should be in the range of 80 ~ 95°c  <cit> .
considering the above-mentioned characteristics, it can be concluded that tk-sp is a potential candidate for industrial applications.
digestion of infectious mbh with tk-sp
our western-blot analysis revealed that tk-sp can reduce the levels of saf- <dig> immunoreactive material .
the western blots probed with anti-prion antibody, saf- <dig>  showed three characteristic anti-prion antibody reactive bands corresponding to the unglycosylated, monoglycosylated, and diglycosylated forms of prpsc.
we found that 1%  sds was relatively poor at eliminating prpsc with little or no effect, while  <dig>  mg/ml  tk-sp produced complete digestion.
furthermore, a combination of  <dig>  mg/ml  tk-sp and 1%  sds was noted to digest infectious mbh, suggesting that tk-sp has the ability to digest prpsc in the absence or presence of sds.
sds is known to be a strong denaturant of proteins.
it has the ability to unfold most of the proteins through the interaction between the charged head group of sds and the positively charged amino acid side chains of proteins, and that between the alkyl chain of sds and the nonpolar parts on the surface as well as in the interior of proteins  <cit> .
as sds-stable enzymes have been rarely reported, it is important to remark the capability of tk-sp to inactivate prpsc, as observed in this study, and its ability to retain only 20% of its activity in the presence of sds .
our results also suggest that tk-sp may have potential application as a detergent additive to decrease the infectivity of prpsc.
here, the concentration of tk-sp  in figure  <dig> was higher than that  of figures  <dig> and  <dig>  to determine the minimum amount of tk-sp for the complete digestion of prion protein in this condition, further quantitative analysis is necessary.
previous reports have shown that alkaline ph is critical for effective digestion of prpsc by subtilisin-type proteases; at ph  <dig>  stable β-sheet structures may be significantly reduced or eliminated, allowing greater access or susceptibility to proteases  <cit> .
the ability of the enzymes to inactivate prions requires them to function at ph  <dig>  a condition in which the conformation is accessible and susceptible to the protease.
tk-sp has been found to exhibit high activity at a wide range of ph between  <dig>  and  <dig> ; additionally, the enzyme has been noted to be stable even at a ph of  <dig>  <cit> .
in the present study, digestion of infectious mbh was assessed at a ph of  <dig> , and tk-sp showed complete inactivation of prpsc, suggesting that the enzyme can be useful for the inactivation of prpsc not only in alkaline ph, but also in neutral ph.
thus, the remarkable characteristic of the ability of tk-sp to degrade prpsc completely indicates its potential application for the digestion of prpsc.
CONCLUSIONS
in the present study, tk-sp, a subtilisin homolog from thermococcus kodakarensis, exhibited more than 100% of its activity and was highly stable in the presence of  <dig> %  nonionic surfactants, but lost up to 20% of its activity in the presence of  <dig> %  edta at 80°c.
however, in the presence of  <dig> %  nonionic surfactants, such as emulgen ls- <dig> or rheodol tw-0120 v, and  <dig> %  edta, tk-sp retained almost its entire initial activity.
furthermore, the enzyme showed inherent stability in the presence of both surfactants and edta without site-directed mutagenesis or protein engineering.
moreover, tk-sp was able to reduce the level of prpsc as detected by western-blot analysis.
thus, tk-sp can possibly be used as a detergent adjunct for industrial and healthcare applications.

 proteases form a major part of the industrial enzyme market, and are applied to detergents as well as food, leather, and fabric processing, and are also used as catalysts in organic synthesis and as therapeutics. with the annual protease sales of about $ – billion, these enzymes account for 60% of the total enzyme market. detergent proteases, particularly alkaline proteases, with an annual market of about $ billion, account for the largest part of protease application. subtilisin carlsberg and related subtilisin serine proteases represent the first generation of detergent proteases with an optimal ph of 9– the second-generation detergent proteases, having a higher optimal ph of 10– and greater temperature stability, are produced from alkalophilic strains of bacillus. the third generation consists of detergent proteases in which the active sites have been modified or various attempts have been made to enhance their stability through site-directed mutagenesis and protein engineering. proteolytic enzymes from microorganisms are the most widely exploited enzymes in the detergent industries. these enzymes are incorporated into detergent formulations to obtain certain characteristics, such as activity and stability in high alkaline ph range and high temperatures, compatibility with compounds used in detergents such as surfactants, perfumes, and bleaches, and hydrolysis specificity towards other proteins. therefore, there is always a need for newer hyper-stable alkaline proteases with potential application in detergent formulations. more recently, studies on enzymatic degradation of prion protein have highlighted the ability of some proteases to digest this class of protein. prion diseases are fatal neurodegenerative disorders that include creutzfeldt-jacob disease, gerstmann-sträussler-scheinker syndrome, fatal familial insomnia, and kuru in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep. their central feature is the posttranslational conversion of host-encoded, cellular prion protein to an abnormal isoform, known as prpsc. this transition appears to involve only a conformational change to a predominantly β-pleated structure, and confers prpsc with partial resistance to proteolytic degradation and detergent insolubility. prpsc is largely unaffected by standard methods of sterilization; thus, contaminated neurosurgical instruments are the major cause of human transmissible spongiform encephalopathies, in addition to exposure to infectious materials through the use of human cadaveric-derived pituitary hormones as well as dural and cornea homotransplants. the results of numerous studies designed to define conditions for the inactivation of prpsc concluded that protein denaturants, such as sodium dodecyl sulfate, are effective in reducing infectivity, but that complete inactivation requires extremely intense conditions, such as 1 h of autoclaving at 134°c or treatment with 2 n naoh or sodium hypochlorite for 1 h. however, such intense conditions cannot be applied to certain equipments such as endoscope. hence, validated methods to inactivate prpsc are highly desirable. thermococcus kodakarensis is a hyperthermophilic archaeon, which grows most optimally at 90°c. its genome contains three genes encoding subtilisin-like serine proteases: tk-subtilisin, tk-sp, and tk-. of these homologs, tk-subtilisin and tk-sp have been structurally and functionally well studied. tk-subtilisin and tk-sp are highly thermostable enzymes with optimal temperature for activity in the range of 90–100°c and show broad substrate specificity. tk-subtilisin and tk-sp exhibit the ph dependence and temperature dependence of the activities. tk-sp exhibits to 70% of the maximal activity at a neutral ph range between and at 20°c. it exhibits to 50% of the maximal activity at between and 60°c. in particular, tk-sp is highly resistant to heat, 5% sds, 8 m urea, 10% triton x- or 10 mm edta; however, unlike tk-subtilisin and bacterial subtilisin, tk-sp does not require ca2+ for folding. thus, tk-sp has a great potential for technological applications. in the present study, to examine the activity of tk-sp in the presence of surfactants and edta at high temperatures, tk-sp was overproduced in e. coli and purified, and subsequently analyzed for its relative activity at 80°c and 90°c using azocasein as the substrate. we found that tk-sp could maintain its proteolytic activity in the presence of surfactants, suggesting that this enzyme could be a candidate for industrial applications in detergent formulation. in addition, to determine whether tk-sp is capable of degrading prpsc, prpsc accumulated in scrapie-infected mouse brain homogenate was incubated with tk-sp and then detected by western-blot analysis. we observed that tk-sp can disrupt prpsc to a level undetectable by western-blot analysis, suggesting that this enzyme has a remarkable characteristic, considering its potential application for the inactivation of prpsc.  effect of surfactants and edta on tk-sp activity some studies have suggested that an effective detergent protease must be compatible and stable with commonly used detergent compounds, such as surfactants, which might be present in the formulation. to analyze the effects of surfactants on the stability and enzymatic activity of tk-sp, nm tk-sp was incubated in 50 mm tris–hcl containing or 1% surfactants for 20 min at 80°c or 90°c using azocasein as the substrate. while tk-sp is known to exhibit its highest activity at 100°c, its relative activities at 90°c or 80°c are and 80%, respectively. one unit of enzymatic activity is defined as the amount of enzyme that increases the a value of the assay reaction mixture by in 1 min. the results of our analysis are summarized in figures  and the data show that tk-sp retained its activity in the presence of surfactants tested at 80°c and 90°c. furthermore, tk-sp was found to be highly stable in the presence of both and 1% nonionic surfactants. particularly, tk-sp retained more than 100% of its activity in the presence of four of the nonionic surfactants, namely, emulgen emulgen ls- emulgen pp- and rheodol tw-0120 v. however, the color of the reaction mixture changed to white after the addition of the cationic surfactant, quartamin 60 w, indicating that tk-sp was less stable in the presence of quartamin 60 w. furthermore, although tk-sp was stable in the presence of % amphitol 20y-b, the presence of 1% amphitol 20y-b caused strong inhibitions, resulting in tk-sp retaining only 50% of its activity. in contrast, the activities in the presence of 1% sanisol c and amphitol 20 n were higher than those at the concentration of %. this may relate to the critical micelle concentration of the surfactants. in the presence of anionic surfactants, tk-sp was unstable, losing up to 80% of its activity. on the other hand, a previous study analyzed the activity of tk-sp at 55°c using n-succinyl-ala-ala-pro-phe-p-nitroanilide as the substrate, and found that the enzyme is highly resistant to 5% sds. this discrepancy in the findings may be due to the temperature of the reaction. in addition, the concentration of tk-sp analyzed in the previous study was ten-fold higher than that in this study, suggesting that high concentration of tk-sp may increase the stability. thus, from these results, it can be concluded that tk-sp is more stable in the presence of nonionic surfactants, suggesting that intramolecular interactions of ion pairs of tk-sp may contribute to its stability. the improvement in the activity of tk-sp in the presence of nonionic surfactants might be due to the stimulation of conformational changes in tk-sp or the substrate, leading to activity gain. chelating agents, such as edta, which function as water softeners and assist in the removal of stains, are valuable components of most of the detergents. as tk-sp has been found to exhibit activity at 80°c in the presence of % edta, to analyze the stability and activity of tk-sp against different concentrations of edta, nm tk-sp was incubated in 50 mm tris–hcl containing,,,, or 1% edta for 20 min at 80°c using azocasein as the substrate. as can be seen in figure  the tk-sp activity decreased with the increasing concentration of edta. several reports have shown that the active structure of serine proteases contains ca2+-binding site, and that the removal of ca2+ from the strong binding site is associated with a significant reduction in thermal stability. our results are in agreement with the previous findings showing that two ca2+ ions bind to the β-jelly roll domain of tk-sp and that tk-sp requires ca2+ ions for maximal stability. in the present study, tk-sp retained 80% of its activity in the presence of % edta, suggesting that this enzyme will be effective in the presence of % edta, which is a commonly used concentration of edta in detergents. to analyze the compatibility and activity of tk-sp in the presence of both surfactants and edta, the enzyme was incubated in the presence of four % nonionic surfactants and % edta at 80°c. as shown in figure  tk-sp was highly stable in the presence of all of the nonionic surfactants, retaining up to 80% of its activity. among the additives examined, the best result with respect to tk-sp retaining almost its entire initial activity was observed in the presence of emulgen ls- and rheodol tw-0120 v. previous reports have shown that stability of protease could be achieved either through site-directed mutagenesis or protein engineering; however, the protease, tk-sp, examined in the present study has been noted to show inherent stability in the presence of surfactants and edta. in addition, tk-sp has also been observed to exhibit activity at high temperature, suggesting that this enzyme meets the standard requirements of the washer-disinfector, which recommend that a suitable temperature of thermal disinfection for medical instruments should be in the range of 80 ~ 95°c. considering the above-mentioned characteristics, it can be concluded that tk-sp is a potential candidate for industrial applications. digestion of infectious mbh with tk-sp our western-blot analysis revealed that tk-sp can reduce the levels of saf- immunoreactive material. the western blots probed with anti-prion antibody, saf- showed three characteristic anti-prion antibody reactive bands corresponding to the unglycosylated, monoglycosylated, and diglycosylated forms of prpsc. we found that 1% sds was relatively poor at eliminating prpsc with little or no effect, while  mg/ml tk-sp produced complete digestion. furthermore, a combination of  mg/ml tk-sp and 1% sds was noted to digest infectious mbh, suggesting that tk-sp has the ability to digest prpsc in the absence or presence of sds. sds is known to be a strong denaturant of proteins. it has the ability to unfold most of the proteins through the interaction between the charged head group of sds and the positively charged amino acid side chains of proteins, and that between the alkyl chain of sds and the nonpolar parts on the surface as well as in the interior of proteins. as sds-stable enzymes have been rarely reported, it is important to remark the capability of tk-sp to inactivate prpsc, as observed in this study, and its ability to retain only 20% of its activity in the presence of sds. our results also suggest that tk-sp may have potential application as a detergent additive to decrease the infectivity of prpsc. here, the concentration of tk-sp in figure  was higher than that of figures  and to determine the minimum amount of tk-sp for the complete digestion of prion protein in this condition, further quantitative analysis is necessary. previous reports have shown that alkaline ph is critical for effective digestion of prpsc by subtilisin-type proteases; at ph  stable β-sheet structures may be significantly reduced or eliminated, allowing greater access or susceptibility to proteases. the ability of the enzymes to inactivate prions requires them to function at ph  a condition in which the conformation is accessible and susceptible to the protease. tk-sp has been found to exhibit high activity at a wide range of ph between and; additionally, the enzyme has been noted to be stable even at a ph of. in the present study, digestion of infectious mbh was assessed at a ph of, and tk-sp showed complete inactivation of prpsc, suggesting that the enzyme can be useful for the inactivation of prpsc not only in alkaline ph, but also in neutral ph. thus, the remarkable characteristic of the ability of tk-sp to degrade prpsc completely indicates its potential application for the digestion of prpsc.  in the present study, tk-sp, a subtilisin homolog from thermococcus kodakarensis, exhibited more than 100% of its activity and was highly stable in the presence of % nonionic surfactants, but lost up to 20% of its activity in the presence of % edta at 80°c. however, in the presence of % nonionic surfactants, such as emulgen ls- or rheodol tw-0120 v, and % edta, tk-sp retained almost its entire initial activity. furthermore, the enzyme showed inherent stability in the presence of both surfactants and edta without site-directed mutagenesis or protein engineering. moreover, tk-sp was able to reduce the level of prpsc as detected by western-blot analysis. thus, tk-sp can possibly be used as a detergent adjunct for industrial and healthcare applications. 
tk-sp was observed to maintain its proteolytic activity with nonionic surfactants and edta at 80°c. it has been known that the hyper-stable protease, tk-sp, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. in this work, the enzymatic activity of tk-sp was measured in the presence of detergents and edta. we optimized the condition in which tk-sp functions efficiently, and demonstrated that the enzyme is highly stable in the presence of % nonionic surfactants and % edta, retaining up to 80% of its activity. our results indicate that tk-sp has a great potential for technological applications, such as thermo-stable detergent additives. tk-sp is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon thermococcus kodakarensis. additionally, we also found that tk-sp can degrade prpsc to a level undetectable by western-blot analysis.
 tk-sp is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon thermococcus kodakarensis. it has been known that the hyper-stable protease, tk-sp, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. in this work, the enzymatic activity of tk-sp was measured in the presence of detergents and edta. in addition, we focused to demonstrate that tk-sp could degrade the abnormal prion protein, a protease-resistant isoform of normal prion protein.  tk-sp was observed to maintain its proteolytic activity with nonionic surfactants and edta at 80°c. we optimized the condition in which tk-sp functions efficiently, and demonstrated that the enzyme is highly stable in the presence of % nonionic surfactants and % edta, retaining up to 80% of its activity. additionally, we also found that tk-sp can degrade prpsc to a level undetectable by western-blot analysis.  our results indicate that tk-sp has a great potential for technological applications, such as thermo-stable detergent additives. in addition, it is also suggested that tk-sp-containing detergents can be developed to decrease the secondary infection risks of transmissible spongiform encephalopathies. serine proteasehyperthermophilic archaeonsubtilisindetergent compatibilitypriontransmissible spongiform encephalopathies degradationdecontamination 