Purification and Characterization of A Novel (R)-Hydroxynitrile Lyase fromEriobotrya japonica(Loquat)

Abstract

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.

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

“…First, mandelonitrile lyase (EC 4.1.2.10) catalyzes the natural substrate (R)-or (S)-mandelonitrile and is found in X. americana (S stereo selective), 3) Pr. subfamily (R stereo selective), 11,16) E. japonica (R), 22) and M. americana (R). Secondly, hydroxymandelonitrile lyase (EC 4.1.2.11) utilizing (S)-4-hydroxymandelonitrile as a natural substrate has been reported only for Sorghum bicolor.…”

“…20) We have discovered seven new HNLs by screening for new HNLs in several cyanogenic plants available in Japan and its vicinity. 21) Among these, HNLs from Eriobotrya japonica (EjHNL), 22,23) the Maloideae subfamily of the Rosaceae family, and from Passiflora edulis (PeHNL) 24) of Passifloraceae were purified to homogeneity and their enzymatic properties were characterized. Pr.…”

Synthesis of (R)-β-nitro alcohols catalyzed by R-selective hydroxynitrile lyase from Arabidopsis thaliana in the aqueous–organic biphasic system

“…S-selective HNL (S-HNL) activity was found in a homogenate of the leaves of Baliospermum montanum, while R-selective HNLs (R-HNLs) were detected in the leaves and seeds of Passiflora edulis, and the seeds of Eriobotrya japonica, Prunus mume, Prunus persica, Chaenomles sinensis and Sorbus aucuparia. The HNLs from E. japonica (EjHNL) (Ueatrongchit et al, 2008(Ueatrongchit et al, , 2009, P. edulis (PeHNL) (Ueatrongchit et al, 2010), P. mume (PmHNL) (Fukuta et al, 2011;Nanda et al, 2005Nanda et al, , 2006, and B. montanum (BmHNL) (Dadashipour et al, 2011) have been purified to homogeneity and their enzymatic properties characterized. PmHNL shows excellent potential for the asymmetric synthesis of cyanohydrins in organic solvents.…”

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

“…Structure Specific activity (U mg −1 ) 1 min under standard conditions of the assay (Ueatrongchit et al, 2008). Decomposition activity of S-selective BmHNL was about 5 times higher than the synthetic activity.…”

“…The temperature stability was determined following 60 min of incubation of the same amount of the enzyme at temperature range 0-70 • C (pH 6.0). The same amount of the enzyme also was incubated for 60 min in various pH values (3-8 in citrate-phosphate and 8-11 in glycine-NaOH buffers, 20 • C), then the mixture used for activity assay (Ueatrongchit et al, 2008).…”