Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus

Abstract

Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70 degrees C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.

FIG. 1

Zymogram analysis of proteases excreted by S. albidoflavus. Track 1, crude culture supernatant; track 2, supernatant treated with 10 mM EDTA; track 3, supernatant treated with 10 mM EDTA plus 1 mM PMSF.

FIG. 2

SDS-PAGE of purified keratinase. Lane 1, molecular mass marker proteins (α₂ macroglobulin, 170 kDa; β-galactosidase, 116.4 kDa; fructose-6-phosphate kinase, 85.2 kDa; glutamate dehydrogenase, 55.6 kDa; aldolase, 39.2 kDa; triosephosphate isomerase, 26.6 kDa; trypsin inhibitor, 20.1 kDa; lysozyme, 14.3 kDa); lane 2, crude enzyme preparation; lane 3, purified keratinase.

FIG. 3

Alignment of the N-terminal sequences of S. albidoflavus serine protease (S.albid.prot.), SGPB, SFase-2, SGPA, and SGPD. Boldface type indicates residues that are different. The data for SGPB were obtained from reference , the data for SFase-2 were obtained from reference , the data for SGPA were obtained from reference , and the data for SGPD were obtained from reference .

FIG. 4

Feather meal hydrolysis with SAKase and SGPB. The experimental conditions were as follows: 50°C; 20 mM Tris HCl (pH 8.5); 5% (wt/vol) feather meal; constant agitation at 900 rpm; 50 keratinolytic activity units/ml was added each 4 h until the residual dry weight (measured after three washes) was constant.