CHARACTERIZATION OF NOVEL HALOALKANE DEHALOGENASE ISOLATED FROM PSYCHROPHILIC BACTERIUM MARINOBACTER SP. ELB17

Lukáš Chrást, Radka Chaloupková, Jiří Damborský

Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic

Haloalkane dehalogenases (EC 3.8.1.5) catalyse hydrolytic conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols and halide anions. Since the first member of the family was isolated [1], haloalkane dehalogenases became widely studied due to their potential application in biocatalysis, biosensing of environmental pollutants and biodegradation of toxic compounds. Searching for new enzymes has attracted the interest of many enzymologist and companies which look for biocatalysts suitable for biotechnological and pharmaceutical applications. Extremophilic organisms offer opportunities for finding novel enzymes exhibiting unique properties. The principal advantage of exploitation of such enzymes is their high catalytic efficiency under extreme conditions.

This study is focused on cloning, expression, purification and biochemical characterization of novel haloalkane dehalogenase DmxA isolated from psychrophilic bacterium Marinobacter sp. ELB17. Synthetic dmxA gene in pMA vector (Mr. Gene, Germany) was subcloned into pET21b expression vector. DmxA was expressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metalloaffinity chromatography. Correct folding and thermostability of DmxA was assessed by circular dichroism spectroscopy. Compared to other haloalkane dehalogenases, DmxA exhibited, paradoxically, the highest melting temperature (T_m = 65.9 ± 0.1 °C). Substrate specificity of the enzyme was measured with thirty different halogenated substrates. DmxA was the most active towards 1,3-dibromopropane, 1-bromo-3-chloropropane and 4-bromobutyronitrile. Temperature and pH profiles of DmxA were determined with 1,3-dibromopropane by activity measurement. Maximal activity was detected at 55 °C and at pH 9.0. Steady-state kinetic parameters of DmxA were measured with 1,3-dibromopropane and 1-chlorobutane. The complex kinetic mechanism of the enzyme was determined, involving cooperativity and substrate inhibition. Enantioselectivity of DmxA was tested towards selected β-substituted bromoalkanes and brominated esters. High enantioselectivity of DmxA was observed in the reaction with ethyl-2-bromobutyrate (E > 200), methyl-2-bromobutyrate (E > 200) and 2-bromopentane (E = 100). The unique properties, as high thermostability and enantioselectivity, make DmxA suitable catalysts for practical application in biosynthesis of optically pure compounds.

This work was supported by the European Regional Development Fund (CZ.1.05/2.1.00/01.0001 and CZ.1.05/1.1.00/02.0123), the Grant Agency of the Czech Republic (203/08/0114 and P207/12/0775) and the Grant Agency of the Czech Academy of Sciences (IAA401630901).

[1] Keuning S., Janssen D. B., Witholt B. (1985): Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. J. Bacteriol. 163: 635-639.