Optimized production, purification and biochemical characterization of a novel glycerophosphodiesterase from a hyperthermophilic archaeon Pyrococcus abyssi

Organophosphate (OP) based pesticides, insecticides and nerve agents can cause nervous, respiratory, reproductive and hepatic problems in living organisms by serving as inhibitors of acetylcholinesterase. Recently, glycerophosphodiesterases (GDPDs) emerged as useful tools in degradation of organophosphate containing pesticides and nerve agents. The present work aims at recombinant production of a thermostable GDPD from Pyrococcus abyssi (Pa-GDPD) in E. coli under optimized cultural conditions. The actual molecular weight of the soluble recombinant enzyme was estimated to be ∼45 kDa with the help of gel filtration chromatography. Moreover, the optimum temperature and pH for Pa-GDPD’s activity were found to be 70 °C and 7.0, respectively. Activity of Pa-GDPD was found to be stimulated by Mn^2 + followed by Fe²⁺ and Mg²⁺. The recombinant enzyme exhibited its hydrolytic activity towards glycerophosphodiester as well as bis (p-nitrophenyl) phosphate (BpNPP) at elevated temperatures (50–90 °C). K_m, V_max and K_cat for BpNPP hydrolysis were determined to be 1 ± 0.145 mM, 88 ± 2.538 U/mg and 2.654 $\times$ 10³ min⁻¹, respectively. Thermoactivity, broad substrate spectrum and stability in organic solvents make recombinant Pa-GDPD an ideal candidate for organophosphate bioremediation.