Mycobiology.  2007 Dec;35(4):219-225.

Purification and Characterization of a Keratinase from a Feather-Degrading Fungus, Aspergillus flavus Strain K-03

• Kim JD

Abstract

A keratinolytic enzyme secreted by Aspergillus flavus K-03 cultured in feather meal basal medium (FMBM) containing 2% (w/v) chicken feather was purified and characterized. Keratinolytic enzyme secretion was the maximal at day 16 of the incubation period at pH 8 and 28degrees C. No relationship was detected between enzyme yield and increase of fungal biomass. The fraction obtained at 80% ammonium sulfate saturation showed 2.39-fold purification and was further purified by gel filtration in Sephadex G-100 followed by ion exchange chromatography on DEAE-Sephadex A-50, yielding an active protein peak showing 11.53-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms indicated that the purified keratinase is a monomeric enzyme with 31 kDa molecular weight. The extracellular keratinase of A. flavus was active in a board range of pH (7~10) and temperature (30degrees C~70degrees C) profiles with the optimal for keratinase activity at pH 8 and 45degrees C. The keratinase activity was totally inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, and ethylenediaminetetraacetate (EDTA) while no reduction of activity by the addition of dithiothreitol (DTT) was observed. N-terminal amino acid sequences were up to 80% homologous with the fungal subtilisins produced by Fusarium culmorum. Therefore, on the basis of these characteristics, the keratinase of A. flavus K-03 is determined to be subtilisins-like.

Keyword

Aspergillus flavus; Keratinase; Subtilisins; Serine protease

MeSH Terms

Amino Acid Sequence
Ammonium Sulfate
Animals
Aspergillus flavus*
Aspergillus*
Biomass
Chickens
Chromatography, Gel
Chromatography, Ion Exchange
Dithiothreitol
Electrophoresis, Polyacrylamide Gel
Feathers
Fungi*
Fusarium
Hydrogen-Ion Concentration
Iodoacetic Acid
Meals
Molecular Weight
Phenylmethylsulfonyl Fluoride
Protease Inhibitors
Serine Proteases
Sodium Dodecyl Sulfate
Subtilisin
Subtilisins
Ammonium Sulfate
Dithiothreitol
Iodoacetic Acid
Phenylmethylsulfonyl Fluoride
Protease Inhibitors
Serine Proteases
Sodium Dodecyl Sulfate
Subtilisin
Subtilisins