Abstract
The yeast Candida deformans CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water. To clone the gene encoding this extracellular lipase, Saccharomyces cerevisiae was transformed with C. deformans genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B. Three members of a lipase gene family (CdLIP1, CdLIP2 and CdLIP3) were cloned and characterized. Each deduced lipase sequence has a Gly-His-Ser-Leu-Gly-(Gly/Ala)-Ala conserved motif, eight cysteine residues and encodes an N-terminal signal sequence. MALDI-TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the CdLIP1 gene encoded the extracellular lipase. Recombinant expression studies confirmed that the cloned genes encoded functional lipases. The three lipases are very similar to lipases from the related species Yarrowia lipolytica. Significant homologies were also found with several yeast and fungal lipases. As C. deformans CBS 2071 was previously considered to be synonymous with Y. lipolytica, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5'-end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region has diverged sufficiently to suggest that C. deformans is a separate species. The nucleotide sequences of the CdLIP1, CdLIP2 and CdLIP3 genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively.
Highlights
Lipases or triacylglycerol lipases (E.C. 3.1.1.3) are enzymes capable of catalysing the hydrolysis of the ester bond between fatty acids and glycerol
Preliminary experiments showed that S. cerevisiae strains OL1, W303-1a and LL-20, unlike C. deformans, did not form a halo in this assay and did not secrete detectable lipases
This suggested that the lipase plate assay could be useful in cloning the lipase gene from C. deformans through the ability of its DNA sequences to complement S. cerevisiae strains
Summary
Lipases or triacylglycerol lipases (E.C. 3.1.1.3) are enzymes capable of catalysing the hydrolysis of the ester bond between fatty acids and glycerol. Triacylglycerols (triglycerides), having very low solubility in water, are their preferential substrates. Under natural conditions, they catalyse the hydrolysis of ester bonds at the interface between an insoluble substrate phase and the aqueous phase in which the enzyme is dissolved. They catalyse the hydrolysis of ester bonds at the interface between an insoluble substrate phase and the aqueous phase in which the enzyme is dissolved They differ from esterases, which hydrolyse only soluble esters of fatty acids with short carbon chains. In nearly anhydrous organic solvents, esterification and transesterification reactions are generally favoured while, in the presence of water, the reaction equilibrium is shifted towards hydrolysis and ester synthesis is limited
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