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<p>The gene encoding a hypothetical YqiA/YcfP family α/β-fold hydrolase from <i>Pseudomonas sivasensis</i> R11S16 was cloned, heterologously expressed in <i>Escherichia coli</i>, and purified as a histidine-tagged recombinant enzyme (PsEst<sub>yfh</sub>). The enzyme, with a molecular weight of about 23&#xa0;kDa, exhibited typical esterase activity against short-chain <i>p</i>-nitrophenyl esters (C<sub>4</sub> &gt; C<sub>8</sub> &gt; C<sub>2</sub> &gt; C<sub>10</sub>). PsEst<sub>yfh</sub> exhibited the highest activity at 20&#xa0;°C and pH 9, indicating its cold-active and alkaline-tolerant nature. The half-life of PsEst<sub>yfh</sub> at 30&#xa0;°C and 40&#xa0;°C was nearly 6&#xa0;h and 2&#xa0;h, respectively. The enzyme retained about 70–95% of its activity after 1&#xa0;h of incubation over the pH range of 5–10 and remained remarkably stable, with ~ 50% relative activity even in the presence of 16% NaCl. In the presence of 1 mM Ni²⁺, Cu²⁺, and Ca²⁺, the enzyme activity increased by 26.74%, 24.20%, and 20.84%, respectively. The enzyme retained more than 80% of its activity in the presence of 10% (v/v) methanol, acetone, chloroform, and butanol. The Michaelis constant (<i>K</i><sub><i>m</i></sub>) and the maximum velocity (<i>V</i><sub><i>max</i></sub>) of PsEst<sub>yfh</sub> were determined from a Lineweaver-Burk plot to be 129.97 µM and 33.3 µmol/min, respectively, with <i>p</i>-NPB as the substrate. Importantly, PsEstyfh achieved over 95% reduction in azithromycin’s antimicrobial activity. Furthermore, ecotoxicological assessment indicated that the resulting transformation products did not exert significant toxic effects on <i>Lemna minor</i>. To our knowledge, this study represents the first report describing the recombinant production, functional characterization, and biotechnological potential of an esterolytic enzyme encoded by a hypothetical YqiA/YcfP family α/β-fold hydrolase gene from <i>P. sivasensis</i>.</p>

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Heterologous expression of a cold-active small esterolytic enzyme from Pseudomonas sivasensis R11S16 and its potential for azithromycin removal

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  • 发表日期：

    2026年6月1日

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  • Gizem Çon,
  • Emre Karakaya,
  • Hayrettin Saygin,
  • Salih Saricaoğlu,
  • Serpil Könen Adıgüzel,
  • Ali Osman Adıgüzel

摘要

The gene encoding a hypothetical YqiA/YcfP family α/β-fold hydrolase from Pseudomonas sivasensis R11S16 was cloned, heterologously expressed in Escherichia coli, and purified as a histidine-tagged recombinant enzyme (PsEstyfh). The enzyme, with a molecular weight of about 23 kDa, exhibited typical esterase activity against short-chain p-nitrophenyl esters (C4 > C8 > C2 > C10). PsEstyfh exhibited the highest activity at 20 °C and pH 9, indicating its cold-active and alkaline-tolerant nature. The half-life of PsEstyfh at 30 °C and 40 °C was nearly 6 h and 2 h, respectively. The enzyme retained about 70–95% of its activity after 1 h of incubation over the pH range of 5–10 and remained remarkably stable, with ~ 50% relative activity even in the presence of 16% NaCl. In the presence of 1 mM Ni²⁺, Cu²⁺, and Ca²⁺, the enzyme activity increased by 26.74%, 24.20%, and 20.84%, respectively. The enzyme retained more than 80% of its activity in the presence of 10% (v/v) methanol, acetone, chloroform, and butanol. The Michaelis constant (Km) and the maximum velocity (Vmax) of PsEstyfh were determined from a Lineweaver-Burk plot to be 129.97 µM and 33.3 µmol/min, respectively, with p-NPB as the substrate. Importantly, PsEstyfh achieved over 95% reduction in azithromycin’s antimicrobial activity. Furthermore, ecotoxicological assessment indicated that the resulting transformation products did not exert significant toxic effects on Lemna minor. To our knowledge, this study represents the first report describing the recombinant production, functional characterization, and biotechnological potential of an esterolytic enzyme encoded by a hypothetical YqiA/YcfP family α/β-fold hydrolase gene from P. sivasensis.