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

文献类型： 外文期刊

第一作者： Bellevik, S

作者： Bellevik, S;Zhang, JM;Meijer, J

作者机构：

关键词： epoxide hydrolase;oilseed rape;yeast;recombinant enzyme;stilbene oxide

期刊名称：EUROPEAN JOURNAL OF BIOCHEMISTRY （ 影响因子：3.579； ）

ISSN： 0014-2956

年卷期： 2002 年 269 卷 21 期

页码：

收录情况： SCI

摘要： 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.

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