Con_2026_3.Biotech_16_242

The gene encoding a hypothetical YqiA/YcfP family alpha/beta-fold hydrolase from Pseudomonas sivasensis R11S16 was cloned, heterologously expressed in Escherichia coli, and purified as a histidine-tagged recombinant enzyme (PsEst(yfh)). The enzyme, with a molecular weight of about 23 kDa, exhibited typical esterase activity against short-chain p-nitrophenyl esters (C(4) > C(8) > C(2) > C(10)). PsEst(yfh) exhibited the highest activity at 20 degreesC and pH 9, indicating its cold-active and alkaline-tolerant nature. The half-life of PsEst(yfh) at 30 degreesC and 40 degreesC 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 Ni^2, Cu^2, and Ca^2, 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 (K (m) ) and the maximum velocity (V (max) ) of PsEst(yfh) were determined from a Lineweaver-Burk plot to be 129.97 microM and 33.3 micromol/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 alpha/beta-fold hydrolase gene from P. sivasensis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-026-04884-y.