Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry

ABSTRACT 1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (k cat = 0.005 s−1) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.

The Evaluation of Genotoxicity Using DNA Repairing Test for Chemicals Produced in Chlorination and Ozonation Processes

The umu-test which can detect the induction of DNA repair is applied in order to analyze the genotoxicity of by-products of chlorination and ozonation. In this research work, the genotoxicities of 37 comnercial chemicals which are expected to be involved in the by-products of chlorination and ozonation processes are checked and evaluated by the umu-test. The genotoxicities of the following organic halogenated compounds are clearly detected: Without microsomal activation; m-dichlorobenzene, 1,2,4-trichlorobenzene and bromoform: With microsomal activation; m-dichlorobenzene, dichloroacetic acid, trichloroacetic acid and chloral are detected. From the results on some compounds which are expected to be produced by ozonation, formaldehyde and ionone show positive genotoxicities without microsomal activation, and 5 compounds have positive genotoxicities with microsomal activation; formaldehyde, furfrol, carvone, glyoxal and acrolein. The effects of the concentration on genotoxicities of those chemicals are discussed and compared with the results obtained in other bacterial assays. Some of the selected organic compounds, chloroform and so on, are identified positive genotoxic, which were reported not to be mutagenic in other bacterial assays. As the Quantitative evaluation for genotoxicity on chemical dose, the time of DNA repairing on damaged spots by SOS genes and the induction rate of umu gene are experimentally evaluated.