Parametrically optimized feather degradation by Bacillus velezensis NCIM 5802 and delineation of keratin hydrolysis by multi-scale analysis for poultry waste management

Abstract

Enormous amounts of keratinaceous waste make a significant and unexploited protein reserve that can be utilized through bioconversion into high-value products using microbial keratinases. This study was intended to assess the keratinase production from a newly isolated B. velezensis NCIM 5802 that can proficiently hydrolyze chicken feathers. Incubation parameters used to produce keratinase enzyme were optimized through the Response Surface Methodology (RSM) with chicken feathers as substrate. Optimization elevated the keratinase production and feather degradation by 4.92-folds (109.7 U/mL) and 2.5 folds (95.8%), respectively. Time-course profile revealed a direct correlation among bacterial growth, feather degradation, keratinase production and amino acid generation. Biochemical properties of the keratinase were evaluated, where it showed optimal activity at 60 °C and pH 10.0. The keratinase was inhibited by EDTA and PMSF, indicating it to be a serine–metalloprotease. Zymography revealed the presence of four distinct keratinases (Mr ~ 100, 62.5, 36.5 and 25 kDa) indicating its multiple forms. NMR and mass spectroscopic studies confirmed the presence of 18 free amino acids in the feather hydrolysates. Changes in feather keratin brought about by the keratinase action were studied by X-ray diffraction (XRD) and spectroscopic (FTIR, Raman) analyses, which showed a decrease in the total crystallinity index (TCI) (1.00–0.63) and confirmed the degradation of its crystalline domain. Scanning electron microscopy (SEM) revealed the sequential structural changes occurring in the feather keratin during degradation. Present study explored the use of keratinolytic potential of the newly isolated B. velezensis NCIM 5802 in chicken feather degradation and also, unraveled the underlying keratin hydrolysis mechanism through various analyses.

Response surface methodology based optimization of keratinase from Bacillus velezensis strain ZBE1 and nanoparticle synthesis, biological and molecular characterization

“…However, in acidic region not significant production was observed, this might be due to low pH value, acidic conditions may have altered ionization state of amino acid leading to decrease in activity. Most of the literatures reported maximum keratinase production in alkaline conditions due to increase in hydrolysis resulted by breakage of di-sulfide bonds and soluble proteins are liberated ( Peng et al, 2019 , Sharma et al, 2022 , Nnolim and Nwodo, 2020 ). Among the studied temperature range of 30–50 °C, a temperature of 35 °C was established to be most favorable for keratinase production (16.15 U/mL) with protein of 0.6 mg/mL ( Fig.…”

“… Khalid et al, (2021) reported two industrial important enzymes; β-glucanase and protease production and optimization from Bacillus velezensis strain. A study reported feather degradation ability by Bacillus velezensis aiding in keratinase production for poultry waste management ( Sharma et al, 2022 ) Nevertheless, to the best of our understanding, very scare literatures are existing on keratinolytic Bacillus velezensis strain.…”

“…The accumulation of non-degradable recalcitrant form of the keratinous wastes threatens the ecological balance ( Nnolim and Nwodo, 2020 ). Among listed keratinous wastes, avian feathers wastes are massively produced worldwide ( Sharma et al, 2022 ). Around 5–7% of the overall weight of a chicken consists of feathers ( Nnolim and Nwodo, 2020 ).…”

“…This rigid structure provides keratins, the strength to resist for the action of hydrolytic enzymes such as, common proteases, papain, trypsin, pepsin etc. ( Sharma et al, 2022 ) and chemical treatments (acidic, alkaline, reducing agents) ( Hassan et al, 2020 ). This situation leads to dumping of the waste in the environment through landfills causing harmful effects to the living beings ( Kaewsalud et al, 2021 ) or burning of feather causing huge toxic pollution ( Prajapati et al, 2021 ) or incineration ( Shalaby et al, 2021 ).…”

“…Additional uses of keratinase encompass development of thermo-bioplastics, compostable films, detergents, prion protein degradation, nail treatments etc. ( Prajapati et al, 2021 , Sharma et al, 2022 ). Although several literatures exists, attempts are necessary to increase the applications of keratinase in various areas by focused research in isolating potent strains and structural and biochemical characterization of keratinase ( Li, 2021 ).…”