Enzymatic degradation of prion protein by keratinase producing proteolytic micro-organisms

Doctoral thesis English OPEN
Okoroma, Emeka A.

Prions are highly resistant to common proteases and conventional sterilisation processes. Consequently, prion infectivity is destroyed by methods such as incineration, alkaline and thermal hydrolysis. These harsh, destructive and potentially hazardous methods are unsuitable for processing specified risk materials (SRM) and animal by-products, and in the decontamination of medical and laboratory devices, and prion contaminated environments. Thus an environmentally friendly, enzymatic degradation and \ud decontamination process is a highly desirable alternative. The structural similarity of prion and feather keratin suggests that feather degrading microorganisms have the potential to degrade prions. The objective of this research was to isolate and characterise microbial keratinase and to investigate its ability to degrade ME7 scrapie prion. Thirty two microbial strains were isolated on feather meal agar from primary effluent and farmyard wastes. One of the isolates, a Grams positive bacterium, demonstrated ignificant keratinolytic activity (11.00 ± 0.71 U/ml), and was investigated further. The isolate was identified by 16S rDNA and designated as Bacillus licheniformis N22, and was deposited in National Collection of Industrial Food and Marine Bacteria (NCIMB). The growth conditions for optimum \ud keratinase synthesis in a minimal growth medium (MGM) were found to be pH 8.5, 50°C, 1.1 % (w/v) feather meal substrate and at incubation time of 32h. The molecular weight of purified keratinase was �28 KDa as measured by SDS-PAGE and confirmed by MALDITOF-MS. Optimum keratinase activity was obtained at pH 8.5 and 50°C. This keratinase \ud fully degraded recalcitrant melanised feather in 48h, and also digested ME7 scrapie prion at 65°C in 2h to levels of PrPSc undetectable by western blot analysis. In a remarkable synergistic enzymatic preparation composed of keratinase and biosurfactant derived from Pseudomonas aeruginosa NCIMB 8626, ME7 scrapie prion was degraded to undetectable levels at 65°C in 10 min. Interestingly biosurfactant alone showed no detectable activity on ME7 scrapie prion. Time-course degradation analysis showed progressive attenuation of PrPSc signal at 50°C over time. Test of residual infectivity by standard cell culture assay \ud showed that this enzymatic method completely destroyed standard sheep scrapie prion (SSBP/1) at 65°C in 1h. The mean survival time of mice challenged with enzyme-\ud digested inocula significantly increased from 278 ± 9 days to 334 ± 42 days compared to those inoculated intraperitoneally with neat ME7 scrapie (p = 0.008 at 95% confidence interval). Furthermore, 47% of all the mice in enzyme-digested group lacked detectable levels of PrPSc . These results suggest a substantial reduction in the infectious titre or complete destruction of ME7 prion infectivity by the enzymatic preparation. Therefore, this mild enzymatic treatment method has potential applications for prion decontamination.
  • References (7)

    1. Schmidt WF: Innovative feather utilization strategies. In Proceeding poultry waste management conference. 19-22 Oct. 1998, Springdale, Arkansas; 1998.276-282 2. United Kingdom Food and Drink Processing Mass Balance [http://www.massbalance.org/downloads/projectfiles/2182-00335.pdf] 3. Shih JCH: Recent developments in poultry waste digestion and feather utilization. Poult Sci 1993, 72:1617-1620.

    4. Onifade AA, Al-Sane NA, Al-Musallam AA, Al-Zarban S: A review: Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresource Technol 1998, 66:1-11.

    5. Wang X, Parsons CM: Effect of processing systems on protein quality of feather meal and hog hair meal. Poult Sci 1997, 76:491-496.

    6. Fraser RDB, MacRae TP, Rogers GE: Keratins: their composition, structure and biosynthesis. Springfield: Charles C. Thomas Publishers; 1972.

    7. Alexander P, Hudson R F: Wool, its chemistry and physics. New York: Reinhold Publishing Co; 1954.

    8. Fuchs E: Keratins and the skin. Ann Rev Cell Dev 1995, 11:123-153.

    9. Parry DAT, North ACT: Hard a-keratin intermediate filament chains: substructure of the N- and C-terminal domains and the predicted structure and function of the Cterminal domains of type I and type II chains. J Struct Biol 1998, 122:67-75, 10. Noval JJ, Nickerson WJ: Decomposition of native keratin by Streptomyces fradiae. J Bacteriol 1959, 77:251-263.

  • Metrics
    0
    views in OpenAIRE
    0
    views in local repository
    299
    downloads in local repository

    The information is available from the following content providers:

    From Number Of Views Number Of Downloads
    Middlesex University Research Repository - IRUS-UK 0 299
Share - Bookmark