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<p>An intracellular salt-tolerant esterase, Est40, was purified from the halophilic <i>Vreelandella</i> sp. CH40 from Chaka Salt Lake, China. Purification via two-step ammonium sulfate precipitation, dialysis, and Sephadex G-75 chromatography achieved a 7.41-fold increase, 87.23 U/mg specific activity and 23.12% recovery. Est40 had a molecular weight of ~ 30&#xa0;kDa and preferentially hydrolyzed short-chain <i>p</i>-nitrophenyl esters, particularly <i>p</i>-NPC<sub>2</sub>, with a <i>K</i><sub>m</sub> of 6.52 ± 0.31 mM and <i>V</i><sub>max</sub> of 220.91 ± 3.87 µmol/min. Est40 exhibited notable tolerance to NaCl (0-3.5&#xa0;M), with maximum activity at 0&#xa0;M and over 20% activity at 3.5&#xa0;M. As a weakly alkaline enzyme, it maintained over 60% activity at pH 7.5–8.7 and was most active at pH 8.5. Est40 was highly thermostable, retaining 80% activity at 70 ℃ and 45% at 80 ℃ after 2&#xa0;h incubation. Maximal efficiency and stability were observed at 50 ℃. DMSO enhanced the enzyme activity, and over 50% activity was retained in 30% (v/v) acetonitrile, ethanol, isopropanol and isooctane. Fe²⁺, Mg²⁺, K⁺, and Li⁺ slightly promoted activity, whereas Cu²⁺, Mn²⁺, and Zn²⁺ significantly inhibited it. Despite inhibition by surfactants and metal chelators, Est40 retained a residual activity of 30-70%. Est40 belongs to the α/β hydrolase superfamily, featuring the pentapeptide motif GFSQG and catalytic triad composed of Ser119, His202, Asp171. Molecular docking confirmed strong binding affinity for <i>p-</i>NPC<sub>2</sub>. The salt tolerance, thermostability and organic solvent resistance of Est40 make it promising for food fermentation and environmental remediation applications.</p>

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Optimization, purification and characterization of an intracellular salt-tolerant esterase Est40 from Vreelandella sp. CH40

  • 期刊论文

  • 发表日期：

    2025年7月9日

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  • Jiaxuan Lv,
  • Liang Zhang,
  • Derui Zhu,
  • Qifu Long,
  • Guoping Shen,
  • Jiangwa Xing

摘要

An intracellular salt-tolerant esterase, Est40, was purified from the halophilic Vreelandella sp. CH40 from Chaka Salt Lake, China. Purification via two-step ammonium sulfate precipitation, dialysis, and Sephadex G-75 chromatography achieved a 7.41-fold increase, 87.23 U/mg specific activity and 23.12% recovery. Est40 had a molecular weight of ~ 30 kDa and preferentially hydrolyzed short-chain p-nitrophenyl esters, particularly p-NPC2, with a Km of 6.52 ± 0.31 mM and Vmax of 220.91 ± 3.87 µmol/min. Est40 exhibited notable tolerance to NaCl (0-3.5 M), with maximum activity at 0 M and over 20% activity at 3.5 M. As a weakly alkaline enzyme, it maintained over 60% activity at pH 7.5–8.7 and was most active at pH 8.5. Est40 was highly thermostable, retaining 80% activity at 70 ℃ and 45% at 80 ℃ after 2 h incubation. Maximal efficiency and stability were observed at 50 ℃. DMSO enhanced the enzyme activity, and over 50% activity was retained in 30% (v/v) acetonitrile, ethanol, isopropanol and isooctane. Fe²⁺, Mg²⁺, K⁺, and Li⁺ slightly promoted activity, whereas Cu²⁺, Mn²⁺, and Zn²⁺ significantly inhibited it. Despite inhibition by surfactants and metal chelators, Est40 retained a residual activity of 30-70%. Est40 belongs to the α/β hydrolase superfamily, featuring the pentapeptide motif GFSQG and catalytic triad composed of Ser119, His202, Asp171. Molecular docking confirmed strong binding affinity for p-NPC2. The salt tolerance, thermostability and organic solvent resistance of Est40 make it promising for food fermentation and environmental remediation applications.