Purification And Immobilization Of Esterase B1 From Engineered Bacteria

Recombinant plasmid pRL-B1 was constructed from detoxifying gene B1 of pesticide resistant Culex pipiens quinquefasciatus and from plasmid pRL-439 containing the strong promoter PpsbA. The engineered bacteria can degrade organophosphorous pesticides, organic chloride pesticides and several other pesticides. The engineered strain E.Coli DH5 α/pRL-B1 was cultured in LB medium(containing100μg/mL Amp) at 37℃, 180rpm, 16 hours with 5% inoculation. The bacterial pellet was centrifuged at 5000rpm,sonicated and centrifuged at 8000rpm for 20 mimutes at 4℃in Buffer A. The soluble protein was purified with 40%⁸0% ammonium sulphate precipitation.Esterase B1 was purified from engineered bacteria by (NH4)2SO4 precipitation, DEAE-Sepharose CL-6B,Sephadex G-150 gel filtration with 33.3 fold purification,17.9% recovery and 74.7U/mg specific activity. The purified enzyme was homogenous on polyacrylamide gel electrophoresis.The molecular weight of the enzyme estimated with SDS-PAGE was 56KD,The optimal condition for activity were pH7.0,temperature 37℃,Mcihaelis constant of the enzyme was 1.35×10^ï¹£3mmol/L and Vmax=2.7×10⁴ u/mL with α-NA as the substrate.The enzyme was stable over the range of pH6⁹ ,the activity was inhibited by SDS and stimulated by Mg²⁺. Degradation of 1605 by esterase B1 is 91.5% in 90 minutes. The optimum factors of immobilized enzymatic reaction and the degradation of 1605 by immobilized esterase B1 were studied for the immobilizing conditions of esterase B1. The results indicated that the optimum immobilizing conditions of...