Keratinolytic activity of Bacillus subtilis LFB-FIOCRUZ 1266 enhanced by whole-cell mutagenesis

Abstract

Discarded feathers represent an important residue from the poultry industry and are a rich source of keratin. Bacillus subtilis LFB-FIOCRUZ 1266, previously isolated from industrial poultry wastes, was used in this work and, through random mutation using ethyl methanesulfonate, ten strains were selected based on the size of their degradation halos. The feather degradation was increased to 115% and all selected mutants showed 1.4- to 2.4-fold increase in keratinolytic activity compared to their wild-type counterparts. The protein concentrations in the culture supernatants increased approximately 2.5 times, as a result of feather degradation. The mutants produced more sulfide than the wild-type bacteria that produced 0.45 µg/ml, while mutant D8 produced 1.45 µg/ml. The best pH for enzyme production and feather degradation was pH 8. Zymography showed differences in the intensity and molecular mass of some bands. The peptidase activity of the enzyme blend was predominantly inhibited by PMSF and EDTA, suggesting the presence of serine peptidases. HPTLC analysis evidenced few differences in band intensities of the amino acid profiles produced by the mutant peptidase activities. The mutants showed an increase in keratinolytic and peptidase activities, demonstrating their biotechnological potential to recycle feather and help to reduce the environmental impact.

Keywords: Bacillus; Biodegradation; Keratin; Mutagenesis; Peptidase.

Fig. 1

Comparative studies of feather degradation in percentage (a), keratinolytic activity (b), protein concentration (c) and gelatino lytic activity (d) of the selected mutant strains from Bacillus subtilis LFB-FIOCRUZ 1266 and the wild type (WT) cultured in feather medium at pH 8.0 and 28 °C

Fig. 2

Sulfide concentrations in the culture supernatants of B. subtilis LFB-FIOCRUZ 1266 mutants and wild-type strains grown in feather medium

Fig. 3

Zymographic analyses of the enzymatic extracts of strains of B. subtilisLFB-FIOCRUZ 1266 wild type and mutants using keratin (a), gelatin (b), BSA (c), casein (d) and hemoglobin (e) as substrates. The values on the left are the molecular weight pattern

Fig. 4

Determination of the keratinase classes of the wild-type strain LFB-FIOCRUZ 1266 and its mutants. The gels were incubated with the following inhibitors: PMSF, phenanthroline, E-64 and EDTA

Fig. 5

HPTLC of feather hydrolysates obtained by microbial degradation of feathers by the wild-type (LFB-FIOCRUZ 1266) and mutant B. subtilis strains