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

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 K_m of 0.11 ± 0.01 mM, k_cat 27.4 ± 0.77, and 2.4 × 10⁵ k_cat/K_m. 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.