State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
J Agric Food Chem. 2010 Jun 9;58(11):6762-6. doi: 10.1021/jf101105s.
In this work, Stenotrophomonas sp. strain YC-1, a native soil bacterium that produces methyl parathion hydrolase (MPH), was genetically engineered to possess a broader substrate range for organophosphates (OPs). A surface anchor system derived from the truncated ice nucleation protein (INPNC) from Pseudomonas syringae was used to target organophosphorus hydrolase (OPH) onto the cell surface of strain YC-1, reducing the potential substrate uptake limitation. The surface localization of INPNC-OPH was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. No growth inhibition was observed for the engineered strain, and suspended cultures retained almost 100% activity over a period of 2 weeks. Concomitant expression of OPH in strain YC-1 resulted in a recombinant strain capable of simultaneously degrading diethyl and dimethyl OPs. A mixture of six OP pesticides (0.2 mM each) could be degraded completely within 5 h. The broader substrate specificity in combination with the rapid degradation rate makes this engineered strain a promising candidate for in situ remediation of OP-contaminated sites.
在这项工作中，产甲基对硫磷水解酶（MPH）的土著土壤细菌 Stenotrophomonas sp. strain YC-1 经过基因工程改造，具有更广泛的有机磷（OP）底物范围。一种源自丁香假单胞菌截断冰核蛋白（INPNC）的表面锚定系统被用于将有机磷水解酶（OPH）靶向到 YC-1 菌株的细胞表面，减少潜在的底物摄取限制。通过细胞分级分离、Western blot、蛋白酶可及性和免疫荧光显微镜证实了 INPNC-OPH 的表面定位。工程菌株没有观察到生长抑制，悬浮培养物在两周的时间内保留了近 100%的活性。在 YC-1 菌株中同时表达 OPH 导致能够同时降解二乙基和二甲基 OP 的重组菌株。六种 OP 农药（每种 0.2 mM）的混合物可以在 5 小时内完全降解。这种更广泛的底物特异性与快速降解速率相结合，使该工程菌株成为原位修复 OP 污染场所的有前途的候选者。
