Abstract
In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40–45 °C, pH 8–9, feather concentration 0.5–1%, and using white chicken feather as keratin substrate for 72 h. Enhancement of keratinase activity through physical mutagen UV radiation and/or chemical mutagen ethyl methanesulfonate (EMS) resulted in five mutants with 1.51–3.73-fold increased activity over the wild type. When compared with the wild type, scanning electron microscopy validated the mutants’ effectiveness in feather degradation. Bacterial isolates are classified as members of the S8 family peptidase Bacillus cereus group based on sequence analysis of the 16S rRNA and keratinase genes. Interestingly, keratinase KerS gene shared 95.5–100% identity to keratinase, thermitase alkaline serine protease, and thermophilic serine protease of the B. cereus group. D137N substitution was observed in the keratinase KerS gene of the mutant strain S13 (KerS13uv+ems), and also seven substitution variations in KerS26 and KerS26uv of strain S26 and its mutant S26uv. Functional analysis revealed that the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad of KerS gene was not affected by the predicted substitutions. Prediction of physicochemical properties of KerS gene showed instability index between 17.5–19.3 and aliphatic index between 74.7–75.7, which imply keratinase stability and significant thermostability. The docking studies revealed the impact of substitutions on the superimposed structure and an increase in binding of mutant D137N of KerS13uv+ems (affinity: −7.17; S score: −6.54 kcal/mol) and seven mutants of KerS26uv (affinity: −7.43; S score: −7.17 kcal/mol) compared to the wild predicted structure (affinity: −6.57; S score: −6.68 kcal/mol). Together, the keratinolytic activity, similarity to thermostable keratinases, and binding affinity suggest that keratinases KerS13uv+ems and KerS26uv could be used for feather processing in the industry.
Highlights
Feathers are considered a byproduct of poultry production; untreated feather waste is a source of many pathogenic microorganisms and pollutants [1]
Novel feather degrading keratinase isolated from different keratinolytic bacteria was characterized in vitro and in silico
Physical and chemical mutagenesis resulted in efficient mutants with high keratinase activity and remarkable feather hydrolysis compared with the wild type
Summary
Feathers are considered a byproduct of poultry production; untreated feather waste is a source of many pathogenic microorganisms and pollutants [1]. 90% keratin which is highly disulfide-bonded and resistant to degradation when treated with various proteases such as papain, pepsin, and trypsin [2–4]. Despite their rigid structure, keratinase degrades feather keratin more efficiently than other proteases [4,5]. Keratinases belong to the subtilisin group, serine protease (S8 family) [6,7]. In silico analysis was used to identify various bacterial keratinases. Amino acid sequences alignment indicated that the two keratinases of B. pumilus strain C4 are subtilisin-like serine proteases belonging to the protease S8 family [10]
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