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.

Progress in Microbial Degradation of Feather Waste

“…The mutants exhibited higher feather degradation rate by 15% than the wild type strain. In addition, the mutants showed higher keratinolytic activity and sulfide yield than the wild type strain (de Paiva et al, 2018). Mutation using ultraviolet irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatment or N-methyl-N′-nitro-N-nitrosoguanidine treatment alone was carried out on B. subtilis .…”

Structure, Application, and Biochemistry of Microbial Keratinases

“…Second, a medium for the microorganism growth needs to be set up. As the media for enriching bacteria or fungi are different, a suitable medium is important in the screening (De Azeredo et al, 2006;Fakhfakh et al, 2011;Mazotto et al, 2013;Parrado et al, 2014;Arokiyaraj et al, 2019). In addition, other parameters such as cultural time and temperature should be considered based on the experimental objectives.…”

“…Interestingly, a study showed that glucose and ammonium nitrate are not good sources for growing Stenotrophomonas maltophilia to degrade feathers (Qu et al, 2018). Accumulated studies suggest that different microorganisms require various fermentation conditions for large-scale production, indicating that careful exploratory studies are necessary prior to the large-scale production of the enzymes (Jagadeesan et al, 2020;de Menezes et al, 2021;Deniz et al, 2021;Sharma and Kango, 2021). It was demonstrated that the solidstate fermentation increased keratinase production compared with the commonly used submerged fermentation (Inácio et al, 2018).…”

“…As keratin does not accumulate in nature, microorganisms are playing the major role in its degradation and recycling. Therefore, keratinous wastes threatening the environment can be converted into value-added products by using microbial treatment (de Menezes et al, 2021;Nnolim and Nwodo, 2021). Extensive studies have been carried out to search suitable microorganisms and obtain optimized processes to make full use of keratinous wastes (Gradišar et al, 2000;Sangali and Brandelli, 2000;Kim et al, 2001;Rai and Mukherjee, 2011).…”

“…Keratin is also classified as hard keratin and soft keratin based on the content of cysteine (Jin et al, 2017). As basic units of keratin are polypeptides, keratinases play a major role in keratin degradation by breaking the disulfide bonds and peptidic bonds (Fraser and Parry, 2008;Hassan et al, 2020;de Menezes et al, 2021). One of the features of keratinases is that they are able to cleave a sequence with hydrophobic residues at the P1 position (Brandelli et al, 2010).…”