S-selective hydroxynitrile lyase from a plant Baliospermum montanum: molecular characterization of recombinant enzyme

Mohammad Dadashipour, Mizue Yamazaki, Kazumi Momonoi, Ken'ichirou Tamura, Ken-Ichi Fuhshuku, Yurina Kanase, Etsuzoh Uchimura, Guan Kaiyun, Yasuhisa Asano

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

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. k(cat)/K(m) ratio for aromatic aldehydes was much larger than those of aliphatic aldehydes and ketones. It was strongly inhibited by AgNO₃, 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 °C, being active at 0-65 °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.

Original languageEnglish
Pages (from-to)100-10
Number of pages11
JournalJournal of Biotechnology
Volume153
Issue number3-4
DOIs
Publication statusPublished - 20 May 2011
Externally publishedYes

Bibliographical note

Copyright © 2011 Elsevier B.V. All rights reserved.

Keywords

  • Aldehyde-Lyases/chemistry
  • Benzaldehydes
  • Cloning, Molecular
  • Enzyme Stability
  • Escherichia coli/genetics
  • Euphorbiaceae/enzymology
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Nitriles/metabolism
  • Plant Proteins/chemistry
  • Recombinant Proteins/chemistry
  • Stereoisomerism
  • Substrate Specificity
  • Temperature

Fingerprint Dive into the research topics of 'S-selective hydroxynitrile lyase from a plant Baliospermum montanum: molecular characterization of recombinant enzyme'. Together they form a unique fingerprint.

Cite this