Katsue MIURA
Expression, affinity purification, and characterization of HN-tagged haloalkylphosphorus hydrolase.
Yoshio KERAˇ¤Shouji TAKAHASHIˇ¤Katsumasa ABE
Tris (1,3-dichloro-2-propyl) phosphate (TDCPP), a typical chlorinated organophosphate triesters, has been used worldwide as flame-retardants and plasticizers, and thus resulting in its dispersion into the environment. Since this compound is reported to possess carcinogenicity, mutagenicity and neurotoxicity, persistence of TDCPP in the environment has been concerned. Recently, TDCPP-degrading bacterium Sphingomonas sp. strain TDK1 was isolated, and the TDCPP-degrading enzyme (Haloalkylphosphotriester degrading enzyme; HAD) 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.
The gene encoding HN-tagged HAD was amplified by PCR using the primers and genomic DNA of Sphingomonas sp. TDK1 as a template. The resulting PCR product was ligated into the pET25b, 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 9.5 and 35ˇëC, respectively. The recombinant HAD was inhibited by 2-mercaptoethanol and dithiothreitol, whereas N-ethylmaleimide did not affect the enzyme. The enzyme activities were increased by the addition of divalent cations, whereas chelating agents, such as EDTA and EGTA, did not affect the enzyme.
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