ABSTRACT
AIMS: This study was developed to purify and to characterize a keratinolytic protease from the bacterium Microbacterium sp. strain kr10. METHODS AND RESULTS: Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-100 and Q-Sepharose columns. The purification was about 255-fold, with a yield of 34%, as determined with azocasein as substrate. The molecular weight of the enzyme was estimated as 42,000 Da by SDS-PAGE. The enzyme had pH and temperature optima of 7.5 and 50 degrees C respectively. This keratinase was inhibited by EDTA and 1,10-phenanthroline, and analysis of metal content indicates that Zn(2+) and Mg(2+) are present. A 2(2) factorial design was developed to investigate the effect of keratinase and mercaptoacetate concentration on feather keratinolysis. Statistical analysis showed that both variables have a significant effect on hydrolysis of keratin. CONCLUSIONS: A new keratinase produced by Microbacterium sp. was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This keratinolytic enzyme offers an interesting potential for the hydrolysis of keratin wastes to be used as feed supplement or bioconversion to added-value products.
Subject(s)
Feathers/microbiology , Industrial Microbiology , Mycobacterium/enzymology , Peptide Hydrolases/isolation & purification , Animals , Chromatography, Liquid , Edetic Acid/pharmacology , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Feathers/metabolism , Hydrogen-Ion Concentration , Keratins/metabolism , Magnesium/analysis , Molecular Weight , Peptide Hydrolases/chemistry , Peptide Hydrolases/pharmacology , Phenanthrolines/pharmacology , Temperature , Thioglycolates/pharmacology , Zinc/analysisABSTRACT
AIMS: To characterize a new feather-degrading bacterium. METHODS AND RESULTS: The strain kr10 producing a high keratinolytic activity when cultured on native feather broth was identified as Microbacterium sp., based on phenotypical characteristics and 16S rDNA sequence. The bacterium presented optimum growth and feather-degrading activity at pH 7.0 and 30 degrees C. Complete feather degradation was achieved during cultivation. The keratinase was partially purified by gel filtration chromatography. It was optimally active at pH 7.0 and 55 degrees C. The enzyme was inhibited by 1,10-phenanthroline, EDTA, p-chloromercuribenzoic acid, 2-mercaptoethanol and metal ions like Hg(2+), Cu(2+) and Zn(2+). SIGNIFICANCE AND IMPACT OF THE STUDY: A new Microbacterium sp. strain was characterized presenting high feather-degrading activity, which appears to be associated to a metalloprotease-type keratinase. This micro-organism has enormous potential for use in biotechnological processes involving keratin hydrolysis.