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.

https://www.mdpi.com/2076-2607/10/1/93/pdf?version=1641018406

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

Keratinolytic bacteria were isolated from poultry sludge samples using chicken feathers formulated media, and the identities of potent isolates were confirmed through 16S rRNA gene sequencing. Fermentation process conditions were optimized, and the amino acid profile of the feather hydrolysate was determined. Ten proteolytic bacteria were obtained from 20 isolates recovered from the samples with halo zone diameters on skimmed milk agar ranging from 15.5 ± 0.71 mm for PSW-15 to 28 ± 1.41 mm for PSW-08. The proteolytic bacteria showed percentage chicken feathers degradation that ranged from 29% for PSW-11 to 84% for PSW-14, and keratinase activity ranged from 99.99 ± 5.14 U/mL for PSW-15 to 761.82 ± 27.42 U/mL for PSW-14. The most potent isolates coded as PSW-14 and PSW-16 were identified as Chryseobacterium cucumeris FHN1 and Sphingobacterium multivorum HNFx, nucleotide sequences were submitted to the GenBank with accession numbers MW165487 and MW165488, respectively. C. cucumeris FHN1 showed the maximum keratinase activity of 1030.90 ± 15.42 U/mL after 72 h, at optimized fermentation conditions of initial medium pH 5–6, inoculum size (4%, v/v), chicken feather (1%, w/v), and incubation temperature (25 °C). The amino acid analysis showed serine (5.19%), aspartic acid (4.75%), glutamic acid (5.86%), and proline (6.08%) were relatively high in concentration and these amino acids possess polar, anionic and non-polar imino side chain characteristics. The chicken feathers-degrading capacity and the amino acid composition of the feather hydrolysate underpin C. cucumeris FHN1 dexterity with keratinous biomass valorization into valuable products with biotechnological application potentials.

Chryseobacterium cucumeris FHN1 keratinolytic enzyme valorized chicken feathers to amino acids with polar, anionic and non-polar imino side chain characteristics

The keratinolytic bacteria isolated from soil samples which containing the degrading feathers was investigated. Thirteen bacterial isolates were selected and were subjected to preliminary screening through protease assay using Milk Agar Medium. Six isolates were detected positive for protease activity and were further characterized via biochemical and microscopic assays. Isolates were grown in basal medium containing feathers as sole nutrient source and the degree of feather degradation was monitored. Two isolates exhibited conspicuous keratinase activity. DNA from these two candidate organisms were isolated and subjected to PCR using 16S rRNA specific primers. PCR products were sequenced and analysis revealed that both of them belong to Bacillus cereus species. Isolation of potential keratinolytic microorganisms could have potential biotechnological used especially in processes which involved keratin hyrdolysis.

I. D. Ambita

Papers

Isolation and characterization of keratinolytic bacteria from soil samples of poultry waste dumping sites.