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题名: S-selective hydroxynitrile lyase from a plant Baliospermum montanum: Molecular characterization of recombinant enzyme
作者: Dadashipour, Mohammad1, 2; Yamazaki, Mizue1, 2; Momonoi, Kazumi3; Tamura, Ken'ichirou1, 2; Fuhshuku, Ken-ichi1, 2; Kanase, Yurina1, 2; Uchimura, Etsuzoh4; Guan Kaiyun5; Asano, Yasuhisa1, 2
刊名: JOURNAL OF BIOTECHNOLOGY
关键词: S-selective hydroxynitrile lyase ; Substrate specificity ; Characterization ; Molecular cloning ; Homology modeling ; Alanine scanning
英文摘要: A novel S-hydroxynitrile lyase (HNL) was purified from leaves of a plant, Baliospermum montanum, by ammonium sulfate fractionation and column chromatographies. Full-length cDNA and genomic DNA were cloned and sequenced. The latter contained two introns and one ORF encoding a 263-residue protein (subunit: 29.5 kDa). The hnl gene was expressed in Escherichia coli and the enzyme was characterized including detailed kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. The enzyme exhibited the highest specific activity (178 U/mg), k(cat) (98/s) and k(cat)/K(m) ratio for piperonal. kcat/Km ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO(3), PMSF, phenol and methyl ethyl ketone, showed an optimum at pH 5, while having activity at range of 4-6.5. It exhibited stability at wide pH range 2.4-11, the highest activity at 20 degrees C, being active at 0-65 degrees C. The enzyme showed variations in residues involved in substrate pocket and substrate entrance channel compared to other S-selective HNLs, based on a model was built. C-terminal short truncations provided more enzyme production. Gel filtration revealed a 60-65 kDa molecular mass for this non-FAD enzyme and its C-terminally truncated forms using three buffer compositions, indicating dimeric structures. (C) 2011 Elsevier B. V. All rights reserved.
出版日期: 2011-05-20
卷号: 153, 期号:3-4, 页码:100-110
语种: 英语
收录类别: SCI
学科分类: Biotechnology & Applied Microbiology
ISSN号: 0168-1656
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.kib.ac.cn/handle/151853/5235
Appears in Collections:资源植物与生物技术所级重点实验室_期刊论文

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作者单位: 1.Toyama Prefectural Univ, Biotechnol Res Ctr, Toyama 9390398, Japan
2.Toyama Prefectural Univ, Dept Biotechnol, Toyama 9390398, Japan
3.Toyama Agr Res Ctr, Toyama 9398153, Japan
4.Bot Gardens Toyama, Toyama 9392713, Japan
5.Chinese Acad Sci, Kunming Inst Bot, Kunming Bot Garden, Kunming 650204, Yunnan, Peoples R China

Recommended Citation:
Dadashipour, M; Yamazaki, M; Momonoi, K; Tamura, K; Fuhshuku, K; Kanase, Y; Uchimura, E; Guan, KY; Asano, Y.S-selective hydroxynitrile lyase from a plant Baliospermum montanum: Molecular characterization of recombinant enzyme,JOURNAL OF BIOTECHNOLOGY,2011,153(3-4 ):100-110
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