Plant thioesterase having preferential hydrolase activity toward C12 acyl-acp substrate
AssigneeCalgene, Inc.
InventorsHuw M. Davies, Michael R. Pollard
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Abstract
This invention relates to a novel class of plant enzymes, thioesterases having preferential hydrolase activity toward one or more medium-chain acyl-ACP substrates. Purification, especially the removal of substantially all other plant proteins, and use of the plant thioesterase is provided, including the use of the protein as a tool in gene isolation for biotechnological applications and a description of various uses for such nucleic acid sequences thereto. The enzyme is preferrably obtained from Umbellularia californica and has preferential hydrolase activity toward C12 acyl-ACP substrate.
Claims11
A plant thioesterase being substantially purified from other proteins of said plant and having preferential hydrolase activity toward C12 acyl-ACP substrate.
A plant thioesterase protein characterized as:
- (i) free from intact cells of said plant;
- (ii) capable of catalyzing the production of laurate and ACP from lauroyl-ACP;
- (iii) having preferential activity toward a lauroyl-ACP substrate as compared to a palmitoyl-ACP substrate;
- (iv) having preferential activity toward a lauroyl-ACP substrate as compared to a lauroyl-CoA substrate;
- and
- (v) an active thiol type esterase.
A C12-preferring acyl-ACP thioesterase obtainable from a plant, wherein said thioesterase is substantially free from a long chain acyl-ACP thioesterase of said plant.
An Umbellularia californica medium-chain acyl-ACP thioesterase wherein said thioesterase is substantially free from other fatty acid synthetase proteins of said Umbellularia californica, said medium-chain thioesterase being characterized as capable of being purified by:
- (i) preparing a crude extract from ground Umbellularia californica immature embryos;
- (ii) subjecting said crude extract to ammonium sulfate fractionation;
- (iii) recovering and resuspending the pellet from said ammonium sulfate fractionation;
- (iv) subjecting said resuspended pellet to hydroxyapatite column chromatography and collecting the eluted thioesterase active fractions;
- (v) applying said active fractions to a cation exchange column and collecting the eluted fractions having thioesterase activity;
- (iv) applying said eluted fractions having thioesterase activity to an immobilized ACP column;
- and
- (vii) recovering the C12 preferring acyl-ACP thioesterase fractions eluted from said ACP column.
Dependencies are inferred from the claim text.