Chungjatupornchai Wipa, Fa-Aroonsawat Sirirat
Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
J Microbiol Biotechnol. 2008 May;18(5):946-51.
The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency (Vmax/Km) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a lowcost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.
编码来源于黄杆菌属、能够降解多种有机磷酸酯农药的有机磷水解酶（OPH）的opd基因，在光合自养蓝细菌的典型代表集胞藻PCC7942中进行了表面表达和细胞内表达。使用截短的冰核蛋白作为锚定基序，将OPH展示在蓝细菌细胞表面。通过共聚焦激光扫描显微镜观察到的免疫染色和OPH活性分析证实，一小部分OPH展示在蓝细菌细胞的最外表面；然而，蛋白酶K和溶菌酶处理表明，相当一部分OPH被埋在细胞壁中。蓝细菌外膜充当底物（对氧磷）扩散屏障，影响全细胞生物降解效率。冻融处理后，细胞内表达OPH的透化全细胞表现出比表面表达OPH的细胞高14倍的生物转化效率（Vmax/Km）。由于蓝细菌生长要求简单且维护成本低，在蓝细菌中表达OPH可能会导致开发出一种低成本、低维护的生物催化剂，用于有机磷酸酯农药的解毒。
