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<p>Protease enzymes are widely used in industrial applications, often requiring resistance to alkaline and high-temperature conditions while maintaining activity in organic solvents. Discovering thermotolerant proteases from thermophilic organisms is crucial for such applications. This study aimed to identify a novel thermotolerant protease among 201 thermophilic strains isolated from hot springs in Aydın province. <i>Geobacillus thermoleovorans</i> HBB208 was identified as the most efficient protease producer, exhibiting a 3.1 (D/d) ratio on skim milk agar. The protease purified via ammonium sulfate precipitation, hydrophobic interaction, and ion-exchange chromatography, resulting in a 70.2-fold purification. SDS-PAGE and zymogram analyses confirmed the molecular weight of approximately 33.5&#xa0;kDa and proteolytic activity. The enzyme showed optimal activity at pH 8.0 and 70&#xa0;°C, and retained 50% activity after 30&#xa0;min at 87.3&#xa0;°C in the presence of 10&#xa0;mM Ca<sup>2</sup>⁺, indicating remarkable thermostability. Kinetic analysis using casein as substrate yielded a <i>K</i><sub>m</sub> of 0.11 ± 0.01&#xa0;mM, <i>k</i><sub>cat</sub> 27.4 ± 0.77, and 2.4 × 10<sup>5</sup> <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>. The enzyme was stable in the presence of various organic solvents and detergents and displayed broad substrate specificity. These findings suggest that HBB208pro metalloprotease enzyme is a promising candidate for biotechnological and industrial applications requiring extreme operational conditions.</p>

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Purification and characterization of an extremely thermostable metalloprotease from Geobacillus thermoleovorans HBB208

  • 期刊论文

  • 发表日期：

    2025年8月29日

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  • Sezgin Karaman,
  • Kubilay Metin

摘要

Protease enzymes are widely used in industrial applications, often requiring resistance to alkaline and high-temperature conditions while maintaining activity in organic solvents. Discovering thermotolerant proteases from thermophilic organisms is crucial for such applications. This study aimed to identify a novel thermotolerant protease among 201 thermophilic strains isolated from hot springs in Aydın province. Geobacillus thermoleovorans HBB208 was identified as the most efficient protease producer, exhibiting a 3.1 (D/d) ratio on skim milk agar. The protease purified via ammonium sulfate precipitation, hydrophobic interaction, and ion-exchange chromatography, resulting in a 70.2-fold purification. SDS-PAGE and zymogram analyses confirmed the molecular weight of approximately 33.5 kDa and proteolytic activity. The enzyme showed optimal activity at pH 8.0 and 70 °C, and retained 50% activity after 30 min at 87.3 °C in the presence of 10 mM Ca2⁺, indicating remarkable thermostability. Kinetic analysis using casein as substrate yielded a Km of 0.11 ± 0.01 mM, kcat 27.4 ± 0.77, and 2.4 × 105 kcat/Km. The enzyme was stable in the presence of various organic solvents and detergents and displayed broad substrate specificity. These findings suggest that HBB208pro metalloprotease enzyme is a promising candidate for biotechnological and industrial applications requiring extreme operational conditions.