Brassica napus soluble epoxide hydrolase (BNSEH1) -: Cloning and characterization of the recombinant enzyme expressed in Pichia pastoris

Epoxide hydrolase (EC 3. 3.2.3) in plants is involved in the metabolism of epoxy fatty acids and in mediating defence responses. We report the cloning of a full-length epoxide hydrolase cDNA (BNSEH1) from oilseed rape ( Brassica napus) obtained by screening of a cDNA library prepared from methyl jasmonate induced leaf tissue, and the 5'-RACE technique. The cDNA encodes a soluble protein containing 318 amino acid residues. The identity on the protein level is 85% to an Arabidopsis soluble epoxide hydrolase (sEH) and 50-60% to sEHs cloned from other plants. A 5 x His tag was added to the N-terminus of the BNSEH1 and the construct was over-expressed in the yeast Pichia pastoris. The recombinant protein was recovered at high levels after Ni-agarose chromatography of lysed cell extracts, had a molecular mass of 37 kDa on SDS/PAGE and cross-reacted on Western blots with antibodies raised to a sEH from Arabidopsis thaliana. BNSEH1 was shown to be a monomer by gel filtration analysis. The activity was low towards cis stilbene oxide but much higher using trans-stilbene oxide as substrate with V-max of 0.47 mumol.min.mg(-1), K-m of 11 muM and k(cat) of 0.3 s(-1). The optimum temperature of the recombinant enzyme was 55 degreesC and the optimum pH 6-7 for trans-stilbene oxide hydrolysis. The isolation of BNSEH1 will facilitate metabolic engineering of epoxy fatty acid metabolism for functional studies of resistance and seed oil modi cation in this important oilcrop.