Isolation and purification of an extracellular keratinase of Trichophyton mentagrophytes.

The keratinolytic activities of dermatophytes which parasitize skin, nails, and hair have been described previously (1, 3, 6, 8); however, the isolation of a keratinase from these organisms has not yet been reported. Trichophyton mentagrophytes var. granulosum (T. granulosum Sabouraud, 1909) was grown in a keratin medium composed of (per liter): horse hair, 2.5 g; glucose, 0.9 g; MgSO4.7H20, 0.6 g; thiamine, 0.01 g; pyridoxine 0.01 g; and inositol, 0.05 g in 0.028 M phosphate buffer (pH 7.8). Media ingredients were sterilized at 121 C and 15 psi for 20 min. After inoculation, the cultures were allowed to stand for 5 days at 25 C and then were shaken for 7 days. At the end of the growth period, the mycelium and residual hair were removed from the culture fluid by filtration. Keratinolytic activity was assayed by a modification of the method of J. J. Noval and W. J. Nickerson (4). The substrate was white guinea pig hair, Hartley strain, extracted with chloroform-methanol and air-dried. The unsterilized hair (1 to 3 mm long, 50 mg) was suspended in 0.028 M phosphate buffer containing 1 mm MgSO4 (pH 8.0) to which 45 ,ug of enzyme material was added; the final volume was 5 ml. Each assay included the following controls: (i) enzyme material in buffer solution, and (ii) hair in buffer solution. The reaction vessels, all tubes in duplicate, were incubated at 37 C for 1 hr and then were immersed in ice water for 5 min. The remaining hair was removed by filtration. Corrected absorbance values at 280 nm were con verted to keratinase units (1 KU = 0.100 corrected absorbance), and the specific activity was expressed as KU per milligram of protein. Protein contents of solutions were determined spectrophotometrically from the absorbance at 280 and at 260 nm according to the method of Layne (2). The purification of the keratinase is summarized in Table 1. The culture filtrate was passed through a diethylaminoethyl (DEAE) cellulose column (Whatman DEI) and the effluent, adjusted to pH 6.5, was transferred onto a carboxymethyl (CM) cellulose column (What-