S-selective hydroxynitrile lyase from a plant Baliospermum montanum: Molecular characterization of recombinant enzyme
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
2011-05-20
发表期刊JOURNAL OF BIOTECHNOLOGY
ISSN0168-1656
卷号153期号:3-4页码:100-110
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.
关键词S-selective Hydroxynitrile Lyase Substrate Specificity Characterization Molecular Cloning Homology Modeling Alanine Scanning
资助信息Ministry of Education, Culture, Sport, Science and Technology (MEXT) of Japan; Japan Society for the Promotion of Sciences[JSPS 20380053]; Institute for Fermentation, Osaka, Japan
学科领域Biotechnology & Applied Microbiology
DOI10.1016/j.jbiotec.2011.02.004
收录类别SCI
语种英语
WOS研究方向Biotechnology & Applied Microbiology
WOS类目Biotechnology & Applied Microbiology
WOS记录号WOS:000290513700004
引用统计
被引频次:21[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://ir.kib.ac.cn/handle/151853/5235
专题资源植物与生物技术所级重点实验室
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
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Dadashipour, Mohammad,Yamazaki, Mizue,Momonoi, Kazumi,et al. S-selective hydroxynitrile lyase from a plant Baliospermum montanum: Molecular characterization of recombinant enzyme[J]. JOURNAL OF BIOTECHNOLOGY,2011,153(3-4):100-110.
APA Dadashipour, Mohammad.,Yamazaki, Mizue.,Momonoi, Kazumi.,Tamura, Ken'ichirou.,Fuhshuku, Ken-ichi.,...&Asano, Yasuhisa.(2011).S-selective hydroxynitrile lyase from a plant Baliospermum montanum: Molecular characterization of recombinant enzyme.JOURNAL OF BIOTECHNOLOGY,153(3-4),100-110.
MLA Dadashipour, Mohammad,et al."S-selective hydroxynitrile lyase from a plant Baliospermum montanum: Molecular characterization of recombinant enzyme".JOURNAL OF BIOTECHNOLOGY 153.3-4(2011):100-110.
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