Purification and characterization of a novel (R)-hydroxynitrile lyase from Eriobotrya japonica (Loquat).
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.
Ueatrongchit
Techawaree
T
Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand.
Kayo
Ai
A
Komeda
Hidenobu
H
Asano
Yasuhisa
Y
H-Kittikun
Aran
A
D000446:Aldehyde-Lyases / Q000302:isolation & purification* / Q000378:metabolism*; D002384:Catalysis; D031983:Eriobotrya / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D009570:Nitriles / Q000737:chemistry; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D009570:Nitriles; C430593:cyanohydrin; D000446:Aldehyde-Lyases