[目的] Rhodococcus sp.T1是一株高效的精恶唑禾草灵(FE)降解菌株,T1菌株可以断裂FE的酯键将其转化为精恶唑禾草灵酸(FA).对T1菌株胞内精恶唑禾草灵酯酶的酶学性质进行研究并对其进行分离纯化,以期为生物修复精恶唑禾草灵污染提供更多的理论依据.[方法]以酯酶的通用底物乙酸-1-萘酯作为定量测定胞内酯酶活力的底物,分析温度和pH值对酯酶活力和稳定性的影响,同时探讨金属离子对酯酶活力的影响;通过硫酸铵沉淀、疏水层析、DEAE离子交换层析和Superdex-200凝胶层析柱组合技术对精恶唑禾草灵酯酶进行分离纯化,计算了纯化过程中酯酶的比活力、纯化倍数和回收率.[结果]胞内酯酶最适反应pH值为8.0,在pH 4.0~10.0内处理24 h活性稳定;最适反应温度为42 ℃,在温度50 ℃以下处理30 min活性稳定;1.0 mmol？L-1 Ag+对酯酶活力有强烈的抑制作用.纯化后的酯酶比活力从0.058 U？mg-1提高到21.5 U？mg-1,纯化倍数为369.5倍,回收率为3%.纯化后的粗酶经SDS-PAGE电泳后至少有4条明显的蛋白条带,通过酶谱确定精恶唑禾草灵酯酶条带的相对分子质量为42.3×103.[结论]精恶唑禾草灵酯酶具有较好的酸碱稳定性和热稳定性,在精恶唑禾草灵污染土壤修复中可能具有较好的应用潜力.[Objectives] Rhodococcus sp.T1 was an efficient fenoxaprop-p-ethyl(FE)degrading strain and could convert FE to fenoxaprop acid(FA)through hydrolyzing the ester bond of FE. In order to provide more technical support for the bioremediation of FE pollution, the enzymatic characteristics and purification of the intracellular fenoxaprop-p-ethyl esterase from strain T1 were investigated in this study. [Methods]The effects of temperature and pH on the activity and stability of esterase and the effect of metal ions on the activity of esterase were determined by using 1-acetoxynaphthalene, the general substrate of esterase, as the substrates for quantitatively detecting the activities of esterase. Afterwards, the esterase was separated and purified by the combination of ammonium sulfate precipitation, hydrophobic chromatography, DEAE ion-exchange chromatography and Superdex-200 gel chromatography. The specific activity, purification fold and recovery of the esterase were calculated during the processes of purification. The molecular weight of the esterase was determined by using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and zymogram. [Results]The esterase was stable from pH 4.0 to pH 10.0 for 24 h with the optimum pH of 8.0. It was stable at temperature below 50 ℃with the optimum of 42 ℃. The activity of the esterase was seriously inhibited by 1.0 mmol？L-1 Ag+. The specific activity of the purified esterase was increased to 21.5 U？mg-1 from 0.058 U？mg-1 in the crude enzyme extract. The esterase was purified 369.5-fold and a recovery rate of 3% was achieved. At least four protein bands were observed in the purified fenoxaprop-p-ethyl esterase. The esterase protein presented as a band with a molecular weight of about 42.3×10-3 as confirmed by zymogram. [Conclusions]The fenoxaprop-p-ethyl esterase exhibits broader pH stability and temperature stability. Therefore, it could be potentially applied in remediation of FE-contaminated soil
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