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

Abstract

Microbial keratinases exhibit a momentous role in converting keratin biowastes into exceedingly valuable protein supplements. This study reports a novel, highly stable keratinase from Bacillus pacificus RSA27 for the production of pure peptides rich in essential amino acids from chicken feathers. Purified keratinase showed a specific activity of 38.73 U/mg, 2.58-fold purification, and molecular weight of 36 kDa. Kinetic studies using a chicken feather as substrate report K _m and V _max values of 5.69 mg/ml and 142.40 μg/ml/min, respectively, suggesting significant enzyme-substrate affinity/biocatalysis. Identification and in silico structural-functional analysis of keratinase discovered the presence of distinct amino acid residues and their positions. Besides, keratinase possesses a high-affinity calcium-binding site (Asp¹²⁸, Leu¹⁶², Asn¹⁶⁴, Ile¹⁶⁶, and Val¹⁶⁸) and a catalytic triad of Asp¹¹⁹, His¹⁵¹, and Ser³⁰⁸, known attributes of serine protease (subtilisin family). Furthermore, a scale-up to 5 L fermenter revealed complete feather hydrolysis (94.5%) within 24 h with high activity (789 U/ml) and total amino acid of 153.97 μmol/ml. Finally, cytotoxicity evaluation of protein hydrolysate resulted in negligible cytotoxic effects (1.02%) on the mammalian hepatoblastoma cell line, signifying its potential biotechnological applications.

Keywords: amino acid production; characterization; keratinolytic protease; purification; scale-up; structural modeling.

Figure 1

Chicken feather degradation by RSA27. (A) RSA27 efficacy with respect to time at 180 rpm and 37°C. 0 days: control, 1 day: RSA27 initiated feather degradation after 24 h, 2 days: complete degradation of feathers after 48 h. (B) SEM of feather degradation at 0 h (intact feather: control), 24 h (degradation of feather barbs), and 48 h (calamus degradation leading to complete degradation of the feather). (C) A residual dry weight of feathers after 12, 24, 36, and 48 h.

Figure 2

SDS-PAGE of purified keratinase. M, protein marker; KE, keratinase.

Figure 3

Effect of temperature and pH on activity and stability of keratinase. (A,B) The optimal activity of keratinase was observed at pH 9 and 60°C. (C,D) Keratinase exhibits stability within wide pH (3–12) and temperature (20–80°C) range.

Figure 4

Effect of various metal ions/inhibitors/surfactants on the keratinolytic activity of the enzyme from Bacillus pacificus RSA27. (A,B) Effect of metal ions and inhibitors (2 and 5 mM). (C) Effect of surfactants (1% solution with water).

Figure 5

Enzyme kinetics (Michaelis-Menton plot) for keratinase using a chicken feather as substrate (R² = 0.9902).

Figure 6

Weblogo for conserved domain analysis of keratinase. Blue, green, and black colored are hydrophilic, neutral, and hydrophobic residues, respectively. The relative frequency of each residue is symbolized by height of symbol within stack and overall height of stack demarcates degree of conservation.

Figure 7

Modeled structures of keratinase and model-template alignment. (A) 3D structure of keratinase modeled using Swiss-model. The green ball here represents calcium ions. (B) 3D structure of keratinase modeled using Phyre2. (C) In silico analysis of Ca1 binding site. Two-dimensional view of interactive residues of keratinase with Ca1 (Asp¹²⁸, Leu¹⁶², Asn¹⁶⁴, Ile¹⁶⁶, and Val¹⁶⁸) along with the distance between target atom and Ca1. (D) In vitro effect of CaCl₂ (5 mM) on keratinase activity at different temperatures (20–80°C). (E) Encircled (green) are missing/dissimilar residues of keratinase in comparison to template and red marked are the predicted Ca1-binding sites of keratinase.