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

Abstract

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 K_m of 6.52 ± 0.31 mM and V_max 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.