Purification and characterization of a soluble lipolytic acylhydrolase from Cowpea (Vigna unguiculata L.) leaves.
With the use of [14C]monogalactosyl diacylglycerol as substrate for enzymatic test, a lipolytic acylhydrolase (EC 3.1.1.26) was purified 263-fold with a yield of 2.0% from soluble leaf extract of Vigna unguiculata L. cv. EPACE-1. The procedure involved ammonium sulfate precipitation, Q-Sepharose Fast Flow chromatography, gel filtration on Sephacryl 300 HR and chromatofocusing on Mono-P, followed by a semi-preparative electrophoresis on polyacrylamide gel. The purified enzyme had a molecular mass of about 80 kDa, as determined by gel filtration. On SDS-PAGE, it showed a single band corresponding to a molecular mass of 40 kDa. The isoelectric point of the enzyme was estimated to be 5.0-5.1 by isoelectric focusing and chromatofocusing. The Km value was 0.119 mM for monogalactosyl-diacylglycerol. The hydrolytic activity of the enzyme on different substrates was determined: the relative rates were digalactosyl-diacylglycerol > monogalactosyl-diacylglycerol > phosphatidylcholine > phosphatidylglycerol. For all substrates, the products of hydrolysis were free fatty acids. Triacylglycerols were not hydrolysed. The enzyme was activated by calcium but was not calcium-dependent. Experiments concerning the enzyme stability as affected by temperature and pH demonstrated that it was quite stable.
Sahsah
Y
Y
Laboratoire Biochimie et Physiologie de l'Adaptation Végétale, Université Paris 7 Denis Diderot, France.
Pham Thi
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AT
Roy-Macauley
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H
d'Arcy-Lameta
A
A
Repellin
A
A
Zuily-Fodil
Y
Y
D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D004795:Enzyme Stability; D010936:Plant Extracts / Q000302:isolation & purification; D018515:Plant Leaves / Q000201:enzymology*; D013379:Substrate Specificity
D010936:Plant Extracts; D002265:Carboxylic Ester Hydrolases; C019262:galactolipase