Purification and characterization of a novel (R)-hydroxynitrile lyase from Eriobotrya japonica (Loquat)

Biosci Biotechnol Biochem. 2008 Jun;72(6):1513-22. doi: 10.1271/bbb.80023. Epub 2008 Jun 7.


A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30-80% (NH(4))(2)SO(4) fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.3 kDa as determined by SDS-gel electrophoresis. The N-terminal sequence is reported. The enzyme was a flavoprotein containing FAD as a prosthetic group, and it exhibited a K(m) of 161 microM and a k(cat)/K(m) of 348 s(-1) mM(-1) for mandelonitrile. The optimum pH and temperature were pH 5.5 and 40 degrees C respectively. The enzyme showed excellent stability with regard to pH and temperature. Metal ions were not required for its activity, while activity was significantly inhibited by CuSO(4), HgCl(2), AgNO(3), FeCl(3), beta-mercaptoethanol, iodoacetic acid, phenylmethylsulfonylfluoride, and diethylpyrocarbonate. The specificity constant (k(cat)/K(m)) of the enzyme was investigated for the first time using various aldehydes as substrates. The enzyme was active toward aromatic and aliphatic aldehydes, and showed a preference for smaller substrates over bulky one.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde-Lyases / isolation & purification*
  • Aldehyde-Lyases / metabolism*
  • Catalysis
  • Eriobotrya / enzymology*
  • Hydrogen-Ion Concentration
  • Molecular Structure
  • Nitriles / chemistry
  • Stereoisomerism
  • Substrate Specificity
  • Temperature


  • Nitriles
  • cyanohydrin
  • Aldehyde-Lyases