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<p>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 <i>Geobacillus thermoleovorans</i> HBB208 was cloned and heterologously expressed in <i>Escherichia coli</i> BL21(DE3), yielding a soluble recombinant enzyme. The purified mature enzyme (GtRS1pro; ~34.6&#xa0;kDa) contains the conserved HEXXH + E catalytic motif, a Zn<sup>2+</sup>-centered active site, and multiple Ca<sup>2+</sup>-binding sites characteristic of M4 proteases. GtRS1pro exhibited optimal activity at pH 8.0 and 70&#xa0;°C, with a high catalytic efficiency (<i>k</i><sub><i>cat</i></sub>/<i>K</i><sub><i>m</i></sub>) of 2.25 × 10<sup>6</sup> M<sup>− 1</sup> s<sup>− 1</sup>. The enzyme showed remarkable thermostability (T<sub>50</sub> = 84.6&#xa0;°C) and retained 99% residual activity after 1&#xa0;h at 70&#xa0;°C in the presence of 10 mM Ca<sup>2+</sup>. 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.</p>

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

  • 期刊论文

  • 发表日期：

    2026年7月30日

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  • Sezgin Karaman,
  • Kubilay Metin

摘要

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 (kcat/Km) 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.