Aims: The aim of this study was to develop a method for microbial degradation of indigenous keratin wastes and to compare it with a method of alkaline hydrolysis. Methods and Results: Native sheep skin and wool were chosen as a model mixture of collagen and keratin wastes discarded by the leather and fur industries. Suitable conditions were found for hydrolysis of this mixture by four newly isolated thermoactinomycete strains. Another set of experiments was carried out using alkaline hydrolysis of keratin wastes. It was shown that microbial hydrolysates contained predominantly low molecular peptides and amino acids, including essential ones, while the alkaline hydrolysis produced predominantly peptides of higher molecular weight. Conclusion: A simple and a low-cost method was proposed for rapid and effective biodegradation of keratin wastes using Thermoactinomyces strains. Significance and Impact of the Study: The proposed method could find application in agriculture for preparing mixtures containing valuable peptides and amino acids.
The keratinase production by the thermophilic actinomycete strain Thermoactinomyces candidus was induced by sheep wool as the sole source of carbon and nitrogen in the cultivation medium. For complete digestion of wool by the above strain, both keratinolytic serine proteinase and cellular reduction of disulfide bonds were involved. Evidence was presented that substrate induction was a major regulatory mechanism and the keratinase biosynthesis was not completely repressed by addition of other carbon (glucose) and nitrogen (NH4C1) sources. The enzyme was purified 62-fold by diethylaminoethyl-anion exchange and Sephadex G-75 gel permeation chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the purified keratinase is a monomeric enzyme with a molecular mass of 30 kDa. The pH and temperature optima were determined to be 8.6 and 70 degrees C, respectively. The purified thermophilic keratinase catalyses the hydrolysis of a broad range of substrates and displays higher proteolytic activity against native keratins than other proteinases. Ca2+ was found to have a stabilizing effect on the enzyme activity at elevated temperatures.
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.
The effect of adding alkaline hydrolysate of sheep's wool waste on the chemical and microbiological properties of a park soil (Sofia, Bulgaria) has been assessed in a 9 month laboratory experiment. The waste product contained 75-80% water-soluble materials: peptides, amino acids, salts, dyes, lipids, some carbohydrates, potassium ions, and it seemed likely that the hydrolysate obtained could be used as a harmless and valuable fertilizer in agriculture. It was demonstrated that the organic material positively influenced microbial soil populations and ryegrass growth. As the remaining partially degraded keratin is highly dispersed it should act as a slow release fertilizer thus feeding plants additionally. The results suggest that the wool hydrolysate is beneficial for improving soil characteristics and could successfully be used as alternative biofertilizer. The authors hope that by utilization of the keratin wastes, the environment around leather and fur plants could be influenced positively and that the expense of removing the wastes to controlled landfill sites and part of the expenses for sustaining the latter will be saved.
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