Yuki NISHIGORI
Expression, purification and characterization of HN-tagged haloalkylphosphorus hydrolase .
Yoshio KERAˇ¤Shouji TAKAHASHIˇ¤Katsumasa ABE
The chlorinated organophosphate triester, tris(2-chloroethyl) phosphate (TCEP), has been widely used as a flame retardant and a plasticizer. Although it has been detected in various environments, especially in landfill leachates with a high concentration, there has been no information on the microbial degradation of TCEP. Recently, carcinogenicity of TCEP has been reported and thus raises concern about its harmful effect on organisms including human. Our laboratory isolated a TCEP-degrading bacterium, Sphingobium sp. strain TCM1, using TCEP as the sole phosphorus source, and the TCEP-degrading enzyme (TCM1 haloalkylphosphorus hydrolase; cHAD) was purified, characterized and cloned. In the future, it is necessary to clarify the structure and reaction mechanism of HAD to develop more efficient degradation technique. In this study, we have constructed E. coli expression system for HN-tagged HAD from Sphingomonas sp. TDK1 and purified recombinant enzyme.
The gene encoding HN-tagged HAD was amplified by PCR using the primers and genomic DNA of Sphingobium sp. strain TCM1 as a template. The resulting PCR product was ligated into the pET-25b, and expressed in E. coli BL21 (DE3). The crude extract from E. coli transformant cells showed TDCPP-degrading activity. HN-tagged HAD was purified from E. coli BL21 (DE3) using TALON column and treated with thrombin to remove the HN tag. Purified enzyme appeared as a single band on SDS-PAGE. The optimum pH and temperature of recombinant dHAD were 9 and 25ˇëC, respectively. The recombinant HAD was inhibited by 2-mercaptoethanol and dithiothreitol, whereas N-ethylmaleimide did not affect the enzyme. TCM-HAD was moderately inhibited by Ca2+, Cu2+ and Fe2+, while the enzyme was moderately activated by Co2+, Zn2+ and Mg2+. The enzyme was inhibited by chelating agents, dithiothreitol and 2-mercaptoethanol. The enzyme hydrolyzed trihaloalkyl phosphate and triaryl phosphates but not trialkyl phosphates,
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