PURIFICATION AND CHARACTERIZATION OF A FATTY ACYL-ESTER HYDROLASE FROM POST-GERMINATED SUNFLOWER SEEDS

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.