Aims: The purpose of this paper was to screen candidate bacterial strains for the production of proteases suitable for application to the degradation of pathogenic forms of prion protein (PrPSc). This paper describes the biochemical characteristics and proteolytic activity of the isolated protease.
Methods and Results: After screening more than 200 bacterial proteases for keratinolytic activity, we identified a Bacillus stain that produced a protease exhibiting high‐degradation activity against a scrapie PrPSc. Sequence analysis indicated that this serine‐protease belonged to the Subtilisin family and had optimum pH and temperature ranges of 9–10 and 60–70°C. Western blotting analysis revealed that the protease was also capable of decomposing bovine spongiform encephalopathy‐infected brain homogenate. In addition, the protease was demonstrated to degrade dried PrPSc that had become firmly attached to a plastic surface considerably more effectively than proteinase K or PWD‐1, a previously reported keratinase.
Conclusions: These results indicate that the isolated protease exhibited higher activity for PrPSc degradation compared with other proteases examined.
Significance and Impact of the Study: This protease could be used under moderate conditions for the decontamination of precision instruments that are susceptible to PrPSc contamination.
Batch culture experiments showed that permeabilized cells and membranes of Ruminococcus albus and Fibrobacter succinogenes, acid-intolerant cellulolytic bacteria, have only one-fourth to one-fifth as much H ؉-ATPase as Megasphaera elsdenii and Streptococcus bovis, which are relatively acid tolerant. Even in the cells grown in continuous culture at pH 7.0, the acid-intolerant bacteria contained less than half as much H ؉-ATPase as the acid-tolerant bacteria. The amounts of H ؉-ATPase in the acid-tolerant bacteria were increased by more than twofold when the cells were grown at the lowest pH permitting growth, whereas little increase was observed in the case of the acid-intolerant bacteria. These results indicate that the acid-intolerant bacteria not only contain smaller amounts of H ؉-ATPase at neutral pH but also have a lower capacity to enhance the level of H ؉-ATPase in response to low pH than the acid-tolerant bacteria. In addition, the H ؉-ATPases of the acid-intolerant bacteria were more sensitive to low pH than those of the acid-tolerant bacteria, although the optimal pHs were similar.
The capacity of ruminal bacteria to regulate H(+)-ATPase synthesis in response to reduced pH was investigated to explain acid tolerance. The activity of H(+)-ATPase in Streptococcus bovis, an acid-tolerant bacterium, was 2.2-fold higher at pH 4.5 than at pH 5.5. The increase in the amount of H(+)-ATPase protein was similar, suggesting that the increase in H(+)-ATPase activity is owing to the increase in H(+)-ATPase synthesis. The level of atp-mRNA at pH 4.5 was 2.5-fold higher than at pH 5.5, indicating that H(+)-ATPase synthesis is regulated at the transcriptional level, responding to low pH. In Ruminococcus albus, an acid-sensitive bacterium, H(+)-ATPase activity, the amount of H(+)-ATPase protein, and the level of atp-mRNA at pH 7.0 were similar to the values at pH 6.0, the lowest pH permitting growth. This result suggests that R. albus is incapable of enhancing H(+)-ATPase synthesis at low pH. Thus, acid tolerance appeared to be related to the capacity to augment the synthesis of H(+)-ATPase responding to low pH.
Prions, infectious agents causing transmissible spongiform encephalopathy, retain infectivity even after undergoing routine sterilization processes. We found that MSK103 protease, identified in our previous study, effectively reduces infectivity and the level of misfolded isoform of the prion protein in scrapie-infected brain homogenates in the presence of SDS. The treatment therefore can be applied to the decontamination of thermolabile instruments.
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