Prions-infectious agents involved in transmissible spongiform encephalopathies-normally survive proteolytic and mild protein-destructive processes. Using bacterial keratinase produced by Bacillus licheniformis strain PWD-1, we tested conditions to accomplish the full degradation of prion protein (PrP) in brain-stem tissue from animals with bovine spongiform encephalopathy and scrapie. The detection of PrPSc, the disease-associated isoform of PrP, in homogenates was done by Western blotting and various antibodies. The results indicated that only in the presence of detergents did heat pretreatment at >100 degrees C allow the extensive enzymatic breakdown of PrPSc to a state where it is immunochemically undetectable. Proteinase K and 2 other subtilisin proteases, but not trypsin and pepsin, were also effective. This enzymatic process could lead to the development of a method for the decontamination of medical and laboratory equipment. The ultimate effectiveness of this method of prion inactivation has to be tested in mouse bioassays.

To increase the production of keratinase, stable strains of Bacillus licheniformis carrying multiple keratinase gene copies in the chromosome were developed. Integrative vectors carrying kerA with or without P43-promoter were constructed and subcloned into B. licheniformis T399D and Bacillus subtilis DB104. In T399D, multiple copies of kerA integration into the chromosome were identified and determined by Southern blot. The optimal integration of kerA was found in the range of 3-5 copies. Higher integration of gene copies (>5) caused reduced processing and secretion of the extracellular keratinase. In DB104, kerA was cloned in the plasmid, not integrated into the chromosome. The strong constitutive promoter P43 not only increased the keratinase production in plasmid-based expression in DB104 but also improved the enzyme yield of the integrants of T399D. New strains were able to enhance cell growth and enzyme yield at higher concentrations of medium substrate. When they were grown in either soy or feather medium, the keratinase activity was stable and improved by about 4-6 times.

Immobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties. Conventional purification followed by chemical immobilization is a laborious and costly process. A new genetic construct was developed to produce the keratinase-streptavidin fusion protein. Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step. The method was tested in both the Bacillus subtilis and Escherichia coli systems. In B. subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium. In E. coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract. The overall efficiencies were approximately the same, 24-28%, using both systems.