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

Abstract

In recent times, robust green technological developments have advanced the goal of a circular economy by minimizing waste generation. The study was undertaken to explore the keratinolytic activity of chicken feather-degrading bacteria from South African soil. Isolates coded as SSN-01 and HSN-01 were identified as Bacillus sp. NFH5 and Bacillus sp. FHNM and their sequences were deposited in GenBank, with accession numbers MW165830.1 and MW165831.1, respectively. Extracellular enzyme production and thiol group generation by Bacillus sp. NFH5 peaked at 120 h with 1879.09 ± 88.70 U/mL and 9.49 ± 0.78 mM, respectively. Glutamic acid (4.44%), aspartic acid (3.50%), arginine (3.23%), glycine (2.61%), serine (2.08%), and proline (2.08%) were relatively higher in concentration. Keratinase (KerBAN) activity was highest at pH 8.0 and 90 °C but was inhibited by both EDTA and 1,10-phenanthroline. In addition, the keratinase-encoding gene (kerBAN) accessioned OK033360 had 362 amino acid residues, with molecular weight and theoretical isoelectric point of 39 kDa and 8.81, respectively. Findings from this study highlight the significance of Bacillus sp. NFH5 in the bio-recycling of recalcitrant keratinous wastes to protein hydrolysates - potential dietary supplements for livestock feeds. The properties of KerBAN underscore its application potential in green biotechnological processes.

Keywords: Amino acids; Bio-recycling; Keratinolytic protease; Livestock feed; Protein hydrolysate.

Fig. 1

Time-course profile of keratinase production by Bacillus sp. NFH5 in optimized medium conditions. Each point represents the average and standard deviation of triplicate experiments.

Fig. 2

Amino acid composition of feather hydrolysate from Bacillus sp. NFH5 fermentation medium.

Fig. 3

Effects of assay pH and temperature on KerBAN activity. Each point represents the average and standard deviation of triplicate experiments.

Fig. 4

The effect of laundry detergents on KerBAN stability. Each bar represents the average and standard deviation of triplicate experiments.

Fig. 5

Homology three-dimensional (3D) structure of keratinase (KerBAN) from Bacillus sp. NFH5 generated by Swiss-Modelling using PDB ID: 1thm as a template (a); and active site prediction by GASS-WEB (b).

Fig. 6

(A) QMEAN PDB structure of KerBAN showing Z-score values. (B) Ramachandran plot of the observed conformations. The numbers of highly preferred residues (green crosses; 93.227%), preferred residues (brown triangles; 3.984%), and questionable residues (red circles; 2.789%) are colour-coded for convenience.