文献类型： 外文期刊

作者： Bellevik, S ¹ ; Zhang, JM ² ; Meijer, J ² ;

作者机构： 1.Swedish Univ Agr Sci, Genet Ctr, Dept Plant Biol, SE-75007 Uppsala, Sweden

2.Swedish Univ Agr Sci, Genet Ctr, Dept Plant Biol, SE-75007 Uppsala, Sweden; Chinese Acad Trop Agr Sci, Natl Biotechnol Lab Trop Crops, Chengxi, Haikou, Peoples R China

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.