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

Epoxide hydrolase (EC ) 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 frommethyl jasmonate induced leaf tissue, and the5¢-RACE technique. The cDNA encodes a soluble protein containing 318 amino acid residues. | Eur. J. Biochem. 269 5295-5302 2002 FEBS 2002 doi Brassicanapus soluble epoxide hydrolase BNSEH1 Cloning and characterization of the recombinant enzyme expressed in Pichia pastoris Stefan Bellevik1 Jiaming Zhang2 and Johan Meijer1 1 Department of Plant Biology Genetics Center Swedish University of Agricultural Sciences Uppsala Sweden 2National Biotechnology Laboratory of Tropical Crops Chinese Academy of Tropical Agricultural Sciences Chengxi Haikou China Epoxide hydrolase EC 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 cisstilbene oxide but much higher using trans-stilbene oxide as substrate with Vmax of pmol-min-mg 1 Km of 11 M and kcat of s 1. The optimum temperature of the recombinant enzyme was 55 C 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 modification in this important oilcrop. Keywords epoxide hydrolase oilseed rape yeast recombinant .

TÀI LIỆU LIÊN QUAN
Đã phát hiện trình chặn quảng cáo AdBlock
Trang web này phụ thuộc vào doanh thu từ số lần hiển thị quảng cáo để tồn tại. Vui lòng tắt trình chặn quảng cáo của bạn hoặc tạm dừng tính năng chặn quảng cáo cho trang web này.