Abstract
The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrPSc signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrPSc at 65°C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50°C over 2 h revealed the progressive attenuation of PrPSc intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrPSc infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1) (p-value = 0.008 at 95% confidence interval). This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions.
Highlights
Alkaline hydrolysis is unsuitable for application in the rendering of recoverable Specific Risk Materials (SRM) and carcases of prion infected tissue as it could result in end products that are extremely degraded and contain high concentration of salt residue, diminishing its commercial value [20]
This paper reports the degradation of scrapie prion under mild digestion conditions by a novel enzymatic formulation which comprises keratinase and biosurfactant isolated from bacterial sources
In vitro Degradation of ME7 Scrapie ME7 scrapie brain homogenate (10 ml) digested with the enzymatic formulation (EF+BS) at 65uC for 1 h resulted in undetectable level of PrPSc as determined by Western Blot analysis (Fig. 1; Lane 3)
Summary
Common prion decontamination methods includes porous load autoclaving at 134uC for 18 min and immersing contaminated material in 1 M NaOH and/or 20,000 ppm NaOCl for 1 h at 20uC [1], [7]. These methods result in irreversible damage to medical devices [8], and may be ineffective in destroying prion infectivity [5], [6], [9]. Alkaline hydrolysis is unsuitable for application in the rendering of recoverable Specific Risk Materials (SRM) and carcases of prion infected tissue as it could result in end products that are extremely degraded and contain high concentration of salt residue, diminishing its commercial value [20]
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