Jahng D, Wood T K
Department of Chemical and Biochemical Engineering, University of California, Irvine 92717-2575.
Appl Environ Microbiol. 1994 Jul;60(7):2473-82. doi: 10.1128/aem.60.7.2473-2482.1994.
Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b can degrade many halogenated aliphatic compounds that are found in contaminated soil and groundwater. This enzyme oxidizes the most frequently detected pollutant, trichloroethylene (TCE), at least 50 times faster than other enzymes. However, slow growth of the strain, strong competition between TCE and methane for sMMO, and repression of the smmo locus by low concentrations of copper ions limit the use of this bacterium. To overcome these obstacles, the 5.5-kb smmo locus of M. trichosporium OB3b was cloned into a wide-host-range vector (to form pSMMO20), and this plasmid was electroporated into five Pseudomonas strains. The best TCE degradation results were obtained with Pseudomonas putida F1/pSMMO20. The plasmid was maintained stably, and all five of the sMMO proteins (alpha, beta, and gamma hydroxylase proteins, reductase, and component B) were observed clearly by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. TCE degradation rates were quantified for P. putida F1/pSMMO20 with a gas chromatograph (Vmax = 5 nmol per min per mg of protein), and the recombinant strain mineralized 55% of the TCE (10 microM) as indicated by measuring chloride ion concentrations with a chloride ion-specific electrode. The maximum TCE degradation rate obtained with the recombinant strain was lower than that of M. trichosporium OB3b but greater than other TCE-degrading recombinants and most well-studied pseudomonads. In addition, this recombinant strain mineralizes chloroform (a specific substrate for sMMO), grows much faster than M. trichosporium OB3b, and degrades TCE without competitive inhibition from the growth substrate.
来自甲基弯曲菌OB3b的可溶性甲烷单加氧酶（sMMO）能够降解污染土壤和地下水中发现的许多卤代脂肪族化合物。这种酶氧化最常检测到的污染物三氯乙烯（TCE）的速度比其他酶至少快50倍。然而，该菌株生长缓慢、TCE与甲烷对sMMO的强烈竞争以及低浓度铜离子对smmo基因座的抑制作用限制了这种细菌的应用。为克服这些障碍，将甲基弯曲菌OB3b的5.5 kb smmo基因座克隆到一个广宿主范围载体中（构建成pSMMO20），并将该质粒电穿孔导入5株假单胞菌菌株。恶臭假单胞菌F1/pSMMO20获得了最佳的TCE降解结果。该质粒稳定维持，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质免疫印迹法均清晰观察到所有5种sMMO蛋白（α、β和γ羟化酶蛋白、还原酶和组分B）。用气相色谱仪对恶臭假单胞菌F1/pSMMO20的TCE降解速率进行了定量（Vmax = 5 nmol每分钟每毫克蛋白），通过氯离子特异性电极测量氯离子浓度表明，该重组菌株使55%的TCE（10 μM）矿化。重组菌株获得的最大TCE降解速率低于甲基弯曲菌OB3b，但高于其他降解TCE的重组体以及大多数经过充分研究的假单胞菌。此外，该重组菌株能使氯仿（sMMO的特异性底物）矿化，生长速度比甲基弯曲菌OB3b快得多，并且降解TCE时不受生长底物的竞争性抑制。
