RESUMEN
Lipases are currently the subject of intensive studies due to their large range of industrial applications. The Lip2p lipase from the yeast Yarrowia lipolytica (YlLIP2) was recently shown to be a good candidate for different biotechnological applications. Using a combination of comparative genomics approaches based on sequence similarity, synteny conservation, and phylogeny, we constructed the evolutionary scenario of the lipase family for six species of the Yarrowia clade. RNA-seq based transcriptome analysis revealed the primary role of LIP2 homologues in the assimilation of different substrates. Once identified, these YlLIP2 homologues were expressed in Y. lipolytica. The lipase Lip2a from Candida phangngensis was shown to naturally present better activity and enantioselectivity than YlLip2. Enantioselectivity was further improved by site-directed mutagenesis targeted to the substrate binding site. The mono-substituted variant V232S displayed enantioselectivity greater than 200 and a 2.5 fold increase in velocity. A double-substituted variant 97A-V232F presented reversed enantioselectivity, with a total preference for the R-enantiomer.
Asunto(s)
Evolución Molecular , Proteínas Fúngicas/genética , Lipasa/genética , Filogenia , Transcriptoma , Yarrowia/genética , Secuencia de Aminoácidos , Sitios de Unión , Butiratos/química , Candida/enzimología , Candida/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expresión Génica , Hidrólisis , Microbiología Industrial , Lipasa/química , Lipasa/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Estereoisomerismo , Especificidad por Sustrato , Sintenía , Yarrowia/clasificación , Yarrowia/enzimologíaRESUMEN
The production of Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) rich in cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) was studied using lipase-catalysed hydrolysis of a mixture of ethyl esters from tuna oil. Lipases from Yarrowia lipolytica (YLL2), Thermomyces lanuginosus (TLL) and Candida rugosa (CRL1, CRL3 and CRL4) were tested. C. rugosa lipases discriminated esters on the basis of their chain length, with less affinity for γ-linolenate, 11-eicosenoate, arachidonate, EPA, DPA and DHA ethyl esters. However, YLL2 and TLL improved discrimination towards DHA, as enzyme selectivity was shown to be mainly based on the position of the double bond closest to the carboxylic group. From the point of view of kinetics, purity and yield, YLL2 was the most effective lipase for DHA purification. Using this enzyme in an open reactor process resulted in the highest concentrations of DHA ethyl ester (77%) and ω-3 esters (81%) with a recovery of 94% and 77% respectively.