Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis

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.

Perspectives on Converting Keratin-Containing Wastes Into Biofertilizers for Sustainable Agriculture

“…As a protease usually prefers to a cleavage site formed by specific residues, different types of proteases are needed to degrade keratin into amino acids completely (Qiu et al, 2020 ). Diverse proteases from different microorganisms have been identified with the molecular weights at the range from 20 to 130 kDa (Kanoksilapatham and Intagun, 2017 ; Sharma and Devi, 2018 ; Pinski et al, 2020 ; Qiu et al, 2020 ) and preference to various pHs, temperatures, and substrates (Tatineni et al, 2007 ; Tiwary and Gupta, 2010b ; Rai and Mukherjee, 2011 ; Khodayari and Kafilzadeh, 2018 ; Arokiyaraj et al, 2019 ; Nnolim et al, 2020a ; Almahasheer et al, 2022 ). Even the structures of the keratinases within the same protease family exhibit different folding and contain different structural elements ( Figure 3 ).…”

Thermoactive metallo-keratinase from Bacillus sp. NFH5: Characterization, structural elucidation, and potential application as detergent additive

“…Among bacterial species, Bacillus licheniformis keratinases have been the most extensively studied and used, as bioadditives, in commercial keratinase-based formulations [ 7 ]. More recently, however, keratinases from other Bacillus species showing high levels of variability have grown in importance, owing to their ability to degrade keratin within short timelines [ 14 ]. The uniqueness of keratinases from different bacterial sources could be due to variations in protein sequence identity, molecular configuration, and the nature of conserved residues.…”

Chicken Feather Waste Valorization Into Nutritive Protein Hydrolysate: Role of Novel Thermostable Keratinase From Bacillus pacificus RSA27

“…Diverse groups of microorganisms are reported to produce keratinases (Kalia and Purohit, 2008 ), such as numerous Bacillus sp. (Gupta and Singh, 2013 ; Gupta et al, 2015 ; Dong et al, 2017 ; Hamiche et al, 2019 ; Nnolim et al, 2020b ; Almahasheer et al, 2022 ; Devi et al, 2022 ; Mazotto et al, 2022 ), Aspergillus oryzae (Farag and Hassan, 2004 ), Paecilomyces marquandii and Doratomyces microsporum (Gradisar et al, 2005 ), Streptomyces sp. (Tatineni et al, 2008 ), Stenotrophomonas maltophilia (Cao et al, 2009 ), Myrothecium verrucaria (Moreira-Gasparin et al, 2009 ), Thermoactinomyces sp.…”