CONICET | Buscador de Institutos y Recursos Humanos

Recently microbial lipases have gained attention due to their varied characteristics andbiotechnological applications. In this sense, tests previously carried out by the working groupshowed that three strains of Penicillium citrinum (LBM150, LBM151, and LBM154) isolated inMisiones showed lipolytic activity. However, there is limited information about the molecular andstructural features of these enzymes. Therefore, the objective of this work was the geneticidentification, three-dimensional modeling and in silico characterization of P. citrinum lipases.Genes belonging to the lipase enzyme were searched for by mapping to the P. citrinum genomereference (scaffold JCM22607 GenBank BCKA01000001.1) using conserved sequences andBLASTn. For the prediction of the gene sequences that code for lipases, the reads were subjected toan identity search using complete and partial nucleotide sequences of lipases reported in the NCBI(https://www.ncbi.nlm.nih.gov/genbank) that contain the conserved lipase sites characteristic oftheir catalytic domain. The nucleotide sequences obtained were translated using the ExPASyprogram (http://web.expassy.org/translate/). They were then analyzed in silico using bioinformaticstools available online. Signal peptide identification was carried out using SignalP(http://www.cbs.dtu.dk/services/SignalP/), and putative glycosylation sites were identified usingNetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc-1.0/output.php). Theoretical properties of theprotein were obtained with the ProtParam software (http://web.expasy.org/protparam/). Molecularweights and isoelectric points (p/I) were calculated in ExPAS and ProtParam and phylogeneticanalysis was performed using the maximum likelihood algorithm with 1000 replicates (Bootstrap)using MEGA 6 software. Subsequently, 3D modeling was performed using the Phyre 2.0 online serverfrom the Structural Bioinformatics Group (Imperial College, London) obtaining, in each case, themost appropriate template to be used according to coverage and identity. To assess the validity andquality of the structures obtained, the MolProbity online server was used. The identification andcharacterization of active sites of each enzyme were done using DogSiteScorer (ProteinPlus). Ineach case, the "Drug Score" value obtained was prioritized, which establishes the present potentialof each pocket to interact with possible ligands. In turn, the amino acids present in each potentialinteraction site were recorded, taking into account the volume, surface, and depth of the pockets. Atotal of five lipases (Lip1, Lip2, Lip3, Lip4 and Lip5) were found in the P. citrinum genome scaffoldand are therefore plausible candidates to explain the extracellular lipolytic activity measured instrains LBM150, LBM151, and LBM154 from P. citrinum. The molecular weight of the annotatedlipases ranged from 20 to 60 kDa. The phylogenetic study revealed that the sequences under studyare related to lipases from different species within the same Penicillium genus, such as P. citrinum(Lip1), P. rolfsii (Lip2) and P. brasilianum (Lip4), with accession number KAJ5226974.1,KAF3387092.1, and OOQ87380.1, respectively. Amino acid sequence alignment with other lipasesrevealed the presence of the consensus sequence GHSLG (Lip1), GESAG (Lip2 and Lip3), and GDSAG(Lip5). The lipase engineering database (LED) revealed that the lipases found belong to the GX andGGGX classes. Domain and motif identification confirmed the domain of lipase 3 and COesterase,specific for triacylglycerols and carboxylic esters, respectively. Also by predicting cell location, it was determined that Lip1, Lip2, Lip3, and Lip5 present signal peptides. All proteins were modeled with90% coverage and 100% confidence with each chosen template using Phyre2. The highest identityvalues were obtained for Lip1 (69%) and Lip5 (47%), positioning themselves as the best temperatelipases from P. camemberti (PDBID: 1TIA) and Aspergillus niger (PDBID: 1UKC) respectively. TheMolProbity score revealed that all the structures had an acceptable level of crystallographic quality(

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