工程菌酯酶B1的纯化及性质研究

Purification and Properties of Esterase B1 from Engineered Bacteria

工程菌酯酶B1降解酶经硫酸铵分级沉淀、DEAE SepharoseCL 6B离子交换柱层析、SephadexG 1 5 0凝胶过滤 ,得到了分离纯化 ,SDS PAGE鉴定为单一组分。经 1 2 .5 %SDS PAGE法测得分子量约为 5 6KD ,提纯倍数为33.3,收率为 1 7.9%,比活力为 74 .7U/mg。该酶的最佳作用条件是 37℃ ,pH =7.0 ,该酶作用于α 乙酸萘酯的Km为1 .35× 1 0 -3 mM ,Vmax为 2 .7× 1 0 4μ/ml。酶在pH6～ 9范围内较稳定 ,重金属Cu2 + 对该酶具有明显的促进作用 ,而SDS对酶具有抑制作用 ,对有机磷农药对硫磷 (1 6 0 5 )的降解率在 90min内达 91 .5 %。

Esterase B1 was purified from engineered bacteria by (NH 4) 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 -3 mM and Vmax=2.7×10 4 μ/ml with α-NA as the substrate.The enzyme was stable over the range of pH6～9 ,the activity was inhibited by SDS and stimulated by Cu 2+. Degradation of 1605 by esterase B1 is 91.5% in 90 minutes.