Characterization of a Novel GH3 β-Glucosidase with Alkali-Tolerant and Halophilic Properties from Pontixanthobacter luteolus SW-109.

β-Glucosidases are in high demand for industrial processes such as lignocellulosic saccharification and high-salt food fermentation. However, conventional enzymes often lose activity under high-salt and alkaline conditions, which constrains their broader applicability. In this study, a glycoside hydrolase family 3 (GH3) β-glucosidase (designated PlGH3) was cloned from Pontixanthobacter luteolus SW-109, and was heterologously expressed in Escherichia coli, followed by systematic characterization of its enzymatic properties and structural simulation. PlGH3 showed an optimal pH of 9.0 and optimal temperature of 50 °C, the kinetic parameters of PlGH3 toward p-nitrophenyl-β-D-glucopyranoside (pNPG) were determined as Kₘ = 0.63 mM and Vₘₐₓ = 3.00 U/mg. PlGH3 still maintained 40%-70% activity in alkaline environment for 24 h. Ca²⁺, Mg²⁺, and Mn²⁺ improved its activity by 120%-140%. Notably, high salt strongly enhanced PlGH3's activity, 4 M NaCl increased enzyme activity by ~ 2.5-fold. It also exhibited ≥ 80% stability in most organic solvents and detergents. Local enrichment of acidic residues on the PlGH3 surface could generate a negative electrostatic potential, which enables adaptation to high-salt and alkaline environments, thereby sustaining the enzyme's catalytic activity under such extreme conditions. Our study enriches marine extremophilic β-glucosidase resources and provides a promising candidate for high-salt/alkaline processes.