Overexpression of Keratinase Candidate from Bacillus velezensis LPL061 in Escherichia coli BL21(DE3)

Proteases have diverse industrial applications including keratinase, which degrade keratin-rich substrates, such as hair, nails, feathers, and skin. This study aimed to express a protease of Bacillus velezensis LPL061 (WP_003155195.1) in Escherichia coli BL21(DE3) and to evaluate its keratinolytic as a new source of keratinase. Two gene constructs were designed, one containing the propeptide domain (kerfull) and one without it (kerhalf). Expression was induced with 0.05 mM IPTG at 20°C for 20 h. KerFull was partially purified using sequential ultrafiltration. SDS-PAGE analysis showed that KerHalf as a 32.2 kDa protein, while KerFull appeared as a 38 kDa fusion and a 28.5 kDa mature form. QTOF-MS confirmed the amino acid sequence. Only KerFull exhibited proteolytic and keratinolytic, highlighting the importance of the I9 domain for proper folding and enzyme activation. The partially purified mature KerFull protein (P50 fraction) retained activity despite low yield. KerFull showed a broad pH stability (6−11) with optimum at pH 9 and active over a wide temperature range (37–70℃), with an optimum at 60℃. Protein remained stable at 20–40℃ and pH 8. Specific activity reached 16.67 U/mg in the crude and 34.14 U/mg of P50 fraction. When combined with DTT, KerFull effectively degraded chicken feather barbules within 4 h at 37 °C. These findings suggest that one of the proteases from B. velezensis LPL061 (WP_003155195.1) is a robust keratinase candidate, offering broad operational pH and temperature, and efficient keratin degradation even at low concentrations, making it a promising candidate for industrial keratinase applications.