M. Teissere et al., PURIFICATION AND CHARACTERIZATION OF A FATTY ACYL-ESTER HYDROLASE FROM POST-GERMINATED SUNFLOWER SEEDS, Biochimica et biophysica acta, L. Lipids and lipid metabolism, 1255(2), 1995, pp. 105-112
Fatty acyl-ester hydrolase was not detectable in dry sunflower seeds u
sing various p-nitrophenyl-acyl-esters, 1,2-O-didodecyl-rac-glycero-3-
glutaric acid-resorufin ester or emulsified sunflower oil as substrate
. After imbibition of the seeds, acyl-ester hydrolase activity slowly
developed in cotyledon extracts and was maximal after 5 days. No activ
ity was directly measurable on oil bodies. The enzyme was purified 615
-fold to apparent homogeneity, as determined by performing SDS-PAGE el
ectrophoresis, and biochemically characterized. With p-nitrophenyl-cap
rylate the optimum pH was around 8.0. The purification procedure invol
ved an acetone powder from 5-day dark-germinated seedlings, chloroform
-butanol extraction and three chromatography steps. We obtained 35 mu
g of pure enzyme from 250 g of fresh cotyledons with an activity yield
of around 7%. It should be possible to subsequently improve this low
recovery as we gave priority here, in the first instance, to purity at
the expense of the yield. The enzyme consisted of one glycosylated po
lypeptide chain with a molecular mass of approx. 45 kDa and, as far as
we could tell, it did not seem to require metal ions to be fully acti
ve, as it was not inhibited by EDTA or o-phenanthroline and not activa
ted by various mono or bivalent metal ions. The amino acid composition
showed the presence of four cysteine and four tryptophan residues. Th
e enzyme was partially inhibited by dithiothreitol, DTNB and PCMB. The
fact that high inhibition was observed in the presence of PMSF indica
tes that a serine residue may possibly be involved in the catalytic me
chanism. The hydrophobicity index was about 53.6% which places this en
zyme in the class of the soluble proteins in good agreement with the f
act that it was mainly present in the soluble part of the crude extrac
t. Partial characterization of glycan chains, using antiglycan antibod
ies, showed the presence of complex Asn-linked glycans. The enzyme was
active on purified sunflower glycerol derivatives. It was also able t
o hydrolyze monooleyl and dioleyl glycerols, as well as phosphatidylch
oline, but not cholesteryl esters. Using p-nitrophenyl-acyl-esters as
substrate, the highest activity was observed with middle-chain derivat
ives (C6 and C8). Its maximum activity was about 0.015 units mg(-1) wi
th sunflower oil. It was not activated by an organic solvent such as i
sooctane. This enzyme probably is involved in acyl-ester hydrolysis wh
ich follows triacylglycerol mobilization by true lipases.