Substrate Report for: Ethyl-oleate

The inhibition of recombinant CpLIP2 lipase/acyltransferase from Candida parapsiolosis was considered a key model for novel antifungal drug discovery and a potential therapeutic target for candidiasis. Lipases have identified recently as potent virulence factors in C. parapsilosis and some other yeasts. The inhibition effects of orlistat and four flavonols (galangin, kaempferol, quercetin and myricetin) characterized by an increasing degree of hydroxylation in B-ring, were investigated using ethyl oleate hydrolysis as the model reaction. Orlistat and kaempferol (14 microM) strongly inhibited CpLIP2 catalytic activity within 1 min of pre-incubation, by 90% and 80%, respectively. The relative potency of flavonols as inhibitors was: kaempferol > quercetin > myricetin > galangin. The results suggested that orlistat bound to the catalytic site while kaempferol interacted with W294 on the protein lid. A static mechanism of interactions between flavonols and CpLIP2 lipase was confirmed by fluorescence quenching analyses, indicating that the interactions were mainly driven by hydrophobic bonds and electrostatic forces. From the Lehrer equation, fractions of tryptophan accessibility to the quencher were evaluated, and a relationship with the calculated number of binding sites was suggested.

Lipases produced by solid-state fermentation were used directly as biocatalysts for continuous synthesis of ethyl oleate in a continuously stirred tank reactor. The effect of biocatalyst reutilisation, molar ratio of substrates, agitation rate and feed rate on the esterification of oleic acid with ethanol were investigated. The catalyst maintained 90% conversion for four batch cycles with a 1:2 molar ratio (oleic acid:ethanol). Mechanical agitation at 200 and 300rpm during 12h of continuous reaction did not affect the biocatalytic conversion, allowing substrate conversions greater than 90% that were obtained with 50mM oleic acid at a molar ratio of 1:2 during 14h reaction. In contrast, substrate conversion was 70% with 100mM oleic acid at a flow rate of 2mL/min during 25h of reaction. These results are promising and offer a technical alternative for the development of accessible biocatalysts that can be used in continuous operations.

With 31% of identity with the Lipase A of Candida antarctica (CaLA), the promising lipase/acyltransferase from Candida parapsilosis (CpLIP2) can be classified in the original CaLA-like superfamily. Contrary to CaLA, CpLIP2 has the exceptional property to catalyze acyltransfer reactions preferentially to hydrolysis even in aqueous media with high thermodynamic activity of water (aw > 0.9). Two new enzymes, CtroL4 from Candida tropicalis and AflaL0 from Aspergillus flavus, homologous to the CaLA-like superfamily proteins were obtained by heterologous production and used for comparative functional characterization in aqueous media. The ability of the lipases to catalyze acyltransfer reaction in water was correlated with their degree of homology with CpLIP2, indicating a global sequence/function relationship that could be very useful for the selection of new biocatalysts of high industrial interest. The four enzymes exhibited different substrate specificity profiles, considering the length and the carbon chain unsaturation degree of the acyl group in the donor ester, and the class and the position of the hydroxyl group of the acyl accepting alcohol. Within the lipases sequences, peculiar variability in the (putative) substrate binding site was observed and will be further investigated for the elucidation of structure/function relationships.