1National Centre for Biomolecular research, Faculty of Science - Masaryk University, Kotlářská 2, Brno, 611 37, Czech Republic
2Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan
Haloalkane dehalogenases are bacterial enzymes catalyzing cleavage of the carbon-halogen bond of halogenated aliphatic compounds by a hydrolytic mechanism. Improvement of cataytic properties of these environmentally important enzymes can be reached by application of non-recombinant directed evolution techniqes [1,2] or recombining several homologous genes .
Four haloalkane dehalogenase genes of different origin were used to construct a hybrid gene: dhlA cloned from Xanthobacter autotrophicus GJ10 , linB from Sphingomonas paucimobilis UT26 , dhaA from Rhodococcus rhodochrous NCIMB13064  and dhmA from Mycobacterium avium N85 . The technique called Degenerate Oligonucleotide Gene Shuffling was used for in vitro recombination of four different genes . Altogether twelve hybrid genes were constructed using one pair of degenerate oligonucleotides.
For preliminary characterization, hybrid proteins were expressed in Escherichia coli BL21(DE3) and tested in the resting cells assay for activity towards six halogenated aliphatic compounds. Four out of twelve hybrid proteins keep good expression and ten proteins showed obvious catalytic activity. Comparison of relative activities determined for the hybrid enzymes with the activities of wild type enzymes suggests that constructs do not possess novel substrate specificities.
All hybrid genes were cloned to pET-32(a) vector to support high level of expression an stability of hybrid proteins. His-taggged tail was introduced to C-terminus of hybrid proteins. All hybrid haloalkane dehalogenases were successfuy expressed in fusion with thioredoxin in host cells E. coli HB101. Optimization of purification conditions on Ni-NTA agarose and cleavage of hybrid protein-thioredoxin complexes is under progress.
1. K. A. Gray, T. H. Richardson, K. Kretz, J. M. Short, F. Bartnek, L. Knowles, L. Kann, P. E. Swanson, & D. E. Robertson, Adv. Synth. Catal., 343 (2001) 607-617.