Expression and properties of three novel fungal lipases/sterol esterases predicted in silico: comparison with other enzymes of the Candida rugosa-like family.
María Eugenia Vaquero, Alicia Prieto, Jorge Barriuso, María Jesús Martínez
Lipases from the Candida rugosa-like family are enzymes with great biotechnological interest. In a previous work, several enzymes from this family were identified by in silico mining of fungal genomes. Here, we describe the cloning, expression, and characterization of putative lipases from the genomes of Nectria haematococca, Trichoderma reesei, and Aspergillus niger and compared their catalytic properties with those of OPE, a well-characterized sterol esterase/lipase from Ophiostoma piceae. All of them hydrolyzed p-nitrophenol esters and triglycerides with different efficiency, but their activity against sterol esters was dissimilar, and the enzyme from A. niger was unable of hydrolyzing these substrates while OPE showed the best k cat values, which in general leads to an improved catalytic efficiency. Similarly, OPE was the best catalyst in the synthesis of β-sitostanyl oleate, followed by the commercial CRL from C. rugosa, while the A. niger enzyme was unable to produce this compound. When the enzymes were evaluated for caprolactone oligomerization, the A. niger enzyme gave similar results than CRL, being OPE slightly more efficient. The expression of the putative selected proteins allowed their functional validation, suggesting that the hydrophobicity of the lid region may be an important factor, although the enzymatic efficiency is also influenced by other parameters, as the aggregation state and the size and morphology of the tunnel, where substrate recognition and catalysis takes place.
