DIRECTED EVOLUTION OF HALOALKANE DEHALOGENASE TOWARDS HIGHER ACTIVITY IN BUFFER CONTAINING DIMETHYL SULFOXIDE

 

T. Koudeláková1, R. Chaloupková1, M. Pavlová1, C. Zimmer2, U.T. Bornscheuer2 and J. Damborský1

 

1Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, 62500 Brno, Czech Republic

2Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, Ernst Moritz Arndt-University, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany

jiri@chemi.muni.cz

 

Haloalkane dehalogenases (EC 3.8.1.5) are bacterial proteins which structurally belong to the superfamily of α/β-hydrolases. Haloalkane dehalogenases convert a broad range of halogenated hydrocarbons into corresponding alcohols by the hydrolytic cleavage of the carbon-halogen bond. Improvement of enzyme stability in organic co-solvents would make these enzymes more suitable for applications in biodegradation and decontamination of toxic compounds. In this study, the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 [1] was subjected to the random mutagenesis with the aim to obtain variants with improved activity in buffer containing organic co-solvent dimethyl sulfoxide. Dehalogenase gene was mutated at an average rate of 1.5 nucleotide substitutions per gene using error-prone polymerase chain reaction. Mutated genes were heterologously expressed in the autolysis strain Escherichia coli XJb. Modified pH indicator assay [2] enabled screening of seven thousand colonies of the mutant library for activity with 1,2-dibromoethane in the buffer containing 42% dimethyl sulfoxide. Four variants showing enhanced activity were detected: D73G+E98V, L95V+A172V, F144I+C176G, and V245L. These positive variants were purified to homogeneity by affinity chromatography and characterized by spectroscopic analysis and steady-state kinetics. Circular dichroism spectroscopy revealed that all four enzymes possessed proper folding. During thermal denaturation, all mutant enzymes, except the variant F144I+C176G, exhibited improved thermostability in comparison with the wild-type enzyme. Activity assays with the substrates 1,2-dibromoethane and 1-iodohexane showed that the mutant L95V+A172V possessed increased activity in the presence of 40% dimethyl sulfoxide, compared to the wild-type enzyme. Structure-function relationships are currently being analyzed for this mutant.

 

Ernst Moritz Arndt-University and FEMS are acknowledged for financial support.

 

References

1.     A. N. Kulakova, M. J. Larkin, L. A. Kulakov, Microbiology, 143, (1997), 109-115.

2.     P. Holloway, J. T. Trevors, H. Lee, J. Microbiol. Methods, 32, (1998), 31-36.