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

Authors

Sezgin Karaman, Kubilay Metin

Published in

Folia microbiologica. Jul 30, 2026. Epub Jul 30, 2026.

Abstract

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.

PMID:
42527817
Bibliographic data and abstract were imported from PubMed on 30 Jul 2026.

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