产酸克雷伯菌中水解酶的表达及其在果蔬表面多菌灵去除中的应用

为研究酶法清除果蔬表面多菌灵残留效果,从多菌灵污染的土壤中筛选多菌灵降解细菌,并从中克隆多菌灵水解酶,表达纯化后制备为酶制剂,催化果蔬表面多菌灵降解。实验结果表明:通过筛选,从土壤中得到了一株可利用多菌灵为唯一碳源的产酸克雷伯菌Klebsiella oxytoca KO-1。以基因组为模板使用简并引物PCR扩增,得到了一段837 bp的水解酶编码基因。将该基因连接至pET-28a并转化至大肠杆菌BL21中,重组菌成功表达出了28 kDa的水解酶KY-1。使用镍柱蛋白纯化法,成功实现了KY-1的纯化并制备了KY-1酶水剂。经液质检测,KY-1将多菌灵催化为2-氨基苯并咪唑,其纯化蛋白酶活达到36.8 U/mg。将KY-1酶水剂喷洒至果蔬表面后,成功提高了黄瓜、草莓、西红柿和土豆表面多菌灵残留的降解率,最高可达37.3%。本研究成功筛选了一株多菌灵降解菌,并制备了可用于果蔬表面多菌灵降解的酶制剂,为酶法清除农药残留奠定了基础。

Expression of a Novel Hydrolase from Klebsiella oxytoca for Carbendazim Residue Degradation on the Surface of Fruit and Vegetable

In order to explore enzymatic method for pesticide residue degradation on surface of fruits,a novel carbendazim degradation bacteria was isolated and the carbendazim hydrolase coding gene was amplified.The results showed that a novel Klebsiella oxytoca KO-1 which was able to survive with carbendazim as the sole carbon source was screened and identified. Moreover,a 837bp hydrolase coding gene was amplified by PCR using degenerate primers,and hydrolase KY-1 that catalyzing carbendazim degradation was expressed and purified in E.coli BL 21.KY-1successfully catalyzed carbendazim into 2-aminobenzimidazole depends on HPLC-MS analysis results,and the relative enzymatic activity was 36.8U/mg.The enzyme preparation of KY-1 successfully accelerated carbendazim degradation rate on the surface of cucumber,potato,tomato and strawberry,and the highest acceleration rate was 37.3%.In conclusion,a carbendazim degradation bacterium was isolated and the enzyme preparation was prepared.Moreover,this work lay the foundation for the enzymatic restoration of pesticide contaminated fruits.