Efficient Mining and Characterization of Two Novel Keratinases from Metagenomic Database

Keratin is a fibrous structural protein found in various natural materials such as hair, feathers, and nails. Its high stability and cross-linked structure make it resistant to degradation by common proteases, leading to the accumulation of keratinous waste in various industries. In this study, we developed and validated an effective bioinformatics-driven strategy for mining novel keratinase genes from the Esmatlas (ESM Metagenomic Atlas) macrogenomic database. Two candidate genes, <i>ker820</i> and <i>ker907</i>, were identified through sequence alignment, structural modeling, and phylogenetic analysis, and were subsequently heterologously expressed in <i>Escherichia coli</i> Rosetta (DE3) with the assistance of a solubility-enhancing chaperone system. Both enzymes belong to the Peptidase S8 family. Enzymatic characterization revealed that GST-tagged ker820 and ker907 exhibited strong keratinolytic activity, with optimal conditions at pH 9.0 and temperatures of 60 °C and 50 °C, respectively. Both enzymes showed significant degradation of feather and cat-hair keratin. Kinetic analysis showed favorable catalytic parameters, including <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><msub><mi>K</mi><mi>m</mi></msub></semantics></math></inline-formula> values of 9.81 mg/mL (ker820) and 5.25 mg/mL (ker907), and <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><msub><mi>V</mi><mo movablelimits="true" form="prefix">max</mo></msub></semantics></math></inline-formula> values of 120.99 U/mg (ker820) and 89.52 U/mg (ker907). Stability tests indicated that GST-ker820 retained 70% activity at 60 °C for 120 min, while both enzymes remained stable at 4 °C for up to 10 days. These results demonstrate the high catalytic capacity, thermal stability, and substrate specificity of the enzymes, supporting their classification as active keratinases. This study introduces a promising strategy for efficiently discovering novel functional enzymes using an integrated computational and experimental approach. Beyond keratinases, this methodology could be extended to screen for enzymes with potential applications in environmental remediation.