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<p>The carboxypeptidase gene from <i>Thermococcus siculi</i> belonging to family metalloprotease was cloned and expressed in <i>Escherichia coli.</i> The gene was expressed utilizing&#xa0;<i>Escherichia coli</i>&#xa0;expression system that showed leaky expression. The recombinant protein was partially soluble and active. The soluble fraction was purified to apparent homogeneity by heat treatment at 85 ℃ for 25 min. 34 mg/L of highly purified enzyme, displaying improved stability and catalytic efficiency was obtained. Recombinant carboxypeptidase exhibited optimal activity at 80 ℃ and pH 7.5. The enzyme specific activity was 1470U/mL and was metal ion dependent. In silico analysis using I-TASSER predicted the carboxypeptidase to be a metalloprotease which requires zinc ions as a co-factor. The predicted active site residues of the Carboxypeptidase by I-TASSER are HIS-269, GLU-299, and TYR-423. This result was further confirmed by Prankweb, Gene ontology analysis suggested its role in catalyzing the C-terminal cleavage of proteins, releasing single amino acids, dipeptides, or tripeptides. Molecular docking showed a high binding affinity of the substrate Hippuryl-L-Arginine with the Carboxypeptidase of <i>Thermococcus siculi, Homo sapiens,</i> and <i>Bubalus bubalis.</i> Activity and stability at high temperatures makes recombinant carboxypeptidase an ideal candidate for various biotechnological applications.</p>

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Recombinant production and characterization of novel carboxypeptidase from thermophilic archaeon Thermococcus siculi

  • 期刊论文

  • 发表日期：

    2025年6月11日

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  • Saima Iftikhar,
  • Maida Wahid,
  • Muhammad Shahid Nadeem,
  • Farheen Aslam,
  • Khushi Muhammad

摘要

The carboxypeptidase gene from Thermococcus siculi belonging to family metalloprotease was cloned and expressed in Escherichia coli. The gene was expressed utilizing Escherichia coli expression system that showed leaky expression. The recombinant protein was partially soluble and active. The soluble fraction was purified to apparent homogeneity by heat treatment at 85 ℃ for 25 min. 34 mg/L of highly purified enzyme, displaying improved stability and catalytic efficiency was obtained. Recombinant carboxypeptidase exhibited optimal activity at 80 ℃ and pH 7.5. The enzyme specific activity was 1470U/mL and was metal ion dependent. In silico analysis using I-TASSER predicted the carboxypeptidase to be a metalloprotease which requires zinc ions as a co-factor. The predicted active site residues of the Carboxypeptidase by I-TASSER are HIS-269, GLU-299, and TYR-423. This result was further confirmed by Prankweb, Gene ontology analysis suggested its role in catalyzing the C-terminal cleavage of proteins, releasing single amino acids, dipeptides, or tripeptides. Molecular docking showed a high binding affinity of the substrate Hippuryl-L-Arginine with the Carboxypeptidase of Thermococcus siculi, Homo sapiens, and Bubalus bubalis. Activity and stability at high temperatures makes recombinant carboxypeptidase an ideal candidate for various biotechnological applications.