High-level expression and characterization of a thermostable thermolysin-like metalloprotease with industrial...

Proteases with high thermostability and catalytic efficiency are highly sought after for industrial applications, particularly within the M4 family of thermolysin-like metalloproteases. In this study, a thermostable thermolysin-like metalloprotease from Geobacillus thermoleovorans HBB208 was cloned and heterologously expressed in Escherichia coli BL21(DE3), yielding a soluble recombinant enzyme. The purified mature enzyme (GtRS1pro; ~34.6 kDa) contains the conserved HEXXH + E catalytic motif, a Zn²⁺-centered active site, and multiple Ca²⁺-binding sites characteristic of M4 proteases. GtRS1pro exhibited optimal activity at pH 8.0 and 70 °C, with a high catalytic efficiency (k_cat/K_m) of 2.25 × 10⁶ M^- 1 s^- 1. The enzyme showed remarkable thermostability (T₅₀ = 84.6 °C) and retained 99% residual activity after 1 h at 70 °C in the presence of 10 mM Ca²⁺. Functional assays demonstrated efficient hydrolysis of protein-rich substrates, including meat, collagen, and keratin. These properties position GtRS1pro as a robust and industrially relevant biocatalyst for high-temperature proteolysis and biowaste valorization, with potential applications in food processing and nutraceutical production.