Expression and properties of three novel fungal lipases/sterol esterases predicted in silico: comparison with other enzymes of the Candida rugosa-like family.
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.
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040, Madrid, Spain.
María Eugenia
ME
Vaquero
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040, Madrid, Spain.
Alicia
A
Prieto
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040, Madrid, Spain. jbarriuso@cib.csic.es.
Jorge
J
Barriuso
Department of Environmental Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040, Madrid, Spain. mjmartinez@cib.csic.es.
María Jesús
MJ
Martínez
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D019295:Computational Biology; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000235:genetics* / Q000378:metabolism*; D055156:Ophiostoma / Q000201:enzymology; D002787:Sterol Esterase / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D002787:Sterol Esterase; D008049:Lipase
Genome mining; Lipases; Polycaprolactone; Sterol esterases; β-Sitostanol esters