Identification and characterization of a novel alkalistable and salt‐tolerant esterase from the deep‐sea hydrothermal vent of the East Pacific Rise

A novel esterase gene selected from metagenomic sequences of deep‐sea hydrothermal vents was successfully expressed in Escherichia coli. The recombinant protein (est‐OKK), which belongs to the lipolytic enzyme family V, exhibited high activity toward pNP‐esters with short acyl chains and especially p‐nitrophenyl butyrate. Site‐mutagenesis results confirmed that est‐OKK contains the nonclassical catalytic tetrad predicted by alignment and computational modeling. The est‐OKK protein is a moderately thermophilic enzyme that is relatively thermostable, and highly salt‐tolerant, which remained stable in 3 mol/L NaCl for 6 hr. The est‐OKK protein showed the considerable alkalistability, displayed optimal activity at pH 9.0 and maintained approximately 70% of its residual activity after incubation at pH 10 for 4 hr. Furthermore, the est‐OKK activity was strongly resistant to a variety of metal ions such as Co(2+), Zn(2+), Fe(2+), Na(+), and K(+); nonionic detergents such as Tween‐20, Tween‐80; and organic solvents such as acetone and isopropanol. Taken together, the novel esterase with unique characteristics may give us a new insight into the family V of lipolytic enzymes, and could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions or food and pharmaceutical industries.