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 [3].
Four
haloalkane dehalogenase genes of different origin were used to construct a
hybrid gene: dhlA cloned from Xanthobacter autotrophicus GJ10
[4], linB from Sphingomonas paucimobilis UT26 [5], dhaA
from Rhodococcus rhodochrous NCIMB13064 [6] and dhmA from Mycobacterium
avium N85 [7]. The technique called Degenerate Oligonucleotide Gene
Shuffling was used for in vitro recombination of four different genes
[8]. 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.