ABSTRACT
Transmissible spongiform encephalopathies are caused by accumulation of highly resistant misfolded amyloid prion protein PrPres and can be initiated by penetration of such pathogen molecules from infected tissue to intact organism. Decontamination of animal meal containing amyloid prion protein is proposed thanks to the use of proteolytic enzymes secreted by thermophilic bacteria Thermoanaerobacter, Thermosipho, and Thermococcus subsp. and mesophilic soil bacteria Streptomyces subsp. Keratins alpha and beta, which resemble amyloid structures, were used as the substrates for the screening for microorganisms able to grow on keratins and producing efficient proteases specific for hydrolysis of beta-sheeted proteic structures, hence amyloids. Secretion of keratin-degrading proteases was evidenced by a zymogram method. Enzymes from thermophilic strains VC13, VC15, and S290 and Streptomyces subsp. S6 were strongly active against amyloid recombinant ovine prion protein and animal meal proteins. The studied proteases displayed broad primary specificities hydrolyzing low molecular mass peptide model substrates. Strong amyloidolytic activity of detected proteases was confirmed by experiments of hydrolysis of PrPres in SAFs produced from brain homogenates of mice infected with the 6PB1 BSE strain. The proteases from Thermoanaerobacter subsp. S290 and Streptomyces subsp. S6 are the best candidates for neutralization/elimination of amyloids in meat and bone meal and other protein-containing substances and materials.
Subject(s)
Amyloid/metabolism , Bacteria/enzymology , Meat , Minerals/metabolism , Peptide Hydrolases/metabolism , Prions/metabolism , Animals , Biological Products , Brain Chemistry , Cattle , Encephalopathy, Bovine Spongiform/metabolism , Hydrolysis , Keratins/metabolism , Mice , Streptomyces/enzymology , Thermococcus/enzymologyABSTRACT
Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion-exchange chromatography and reversed-phase high-performance liquid chromatography (HPLC) showed the presence of two fractions of beta-lactoglobulin but only one of alpha-lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation-mass spectrometry (ESI-MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine beta-lactoglobulin. Amino acid compositions of the two variants of beta-lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of beta-lactoglobulin A compared to beta-lactoglobulin B at pH 2, and indicated high instability of ovine alpha-lactalbumin at this pH.
