Optimization, Isolation and Characterization of a Keratinolytic Protease from Human Hair-Degrading Bacterium Alcaligenes Faecalis Strain TPB18

M. O. EZENWALI

Alcaligenes faecalis strain TPB18 and AIR10, Keratin, Keratinase, Co-factors, Optimum pH, Optimum Temperature, 16SrRNA gene amplification, Phylogenetic tree, DNA Sequence.

Microbial keratinases are a group of highly specific proteases that catalyze the hydrolytic cleavage of peptide bonds in keratin, a structural fibrous polypeptide/protein highly resistant to most proteolytic degradation/attack because of its complex bonding structure stabilized by disulfide and hydrogen bonds, an attribute that also made it one of the most world environmental richest proteinous solid waste (keratinaceous waste). In this present research, an extracellular keratinase identified by genomics, proteomics, and biochemical methods was isolated from soil Alcaligenes faecalis (TPB18), purified and then applied in the degradation of defatted human hair. The keratinase activities were determined by measuring the concentrations of both free amino acids and soluble peptides/proteins using Ninhydrin and Lowry methods respectively. This heat-stable keratinase exhibited keratinolytic activities at temperatures ranging from 0 to 90oC. The optimum pH, temperature, and the best co-factors for this isolated human hair keratinase activity using Lowry and Ninhydrin methods were found to be at pH {(12.5) and (5.5, 9, 10)}, temperature of {(37 °C) and (37-60 °C)} and co-factors of {(Co2+, Al3+, Hg2+) and (Co2+, Hg2+, Cu2+)} respectively. The optimal incubation time and the percentage degree of human hair degradation were found to be 85% on day 5. The phylogenetic tree identified two out of the four isolates, isolate-3 and -4 with 98.72% and 96.01% pairwise identities to Alcaligenes faecalis strain AIR10 and TPB18 with NCBI accession numbers of MG835355.1 and MW475277.1 respectively. An end-point polymerase chain reaction gel image test showed 16SrRNA gene amplification for both Alcaligenes faecalis (TPB18) and (AIR10) at about 1.5kb