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. 2018 Jan 25;9(2):62.
doi: 10.3390/genes9020062.

Fungal Screening on Olive Oil for Extracellular Triacylglycerol Lipases: Selection of a Trichoderma Harzianum Strain and Genome Wide Search for the Genes

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Free PMC article

Fungal Screening on Olive Oil for Extracellular Triacylglycerol Lipases: Selection of a Trichoderma Harzianum Strain and Genome Wide Search for the Genes

Miguel Angel Canseco-Pérez et al. Genes (Basel). .
Free PMC article

Abstract

A lipolytic screening with fungal strains isolated from lignocellulosic waste collected in banana plantation dumps was carried out. A Trichoderma harzianum strain (B13-1) showed good extracellular lipolytic activity (205 UmL-1). Subsequently, functional screening of the lipolytic activity on Rhodamine B enriched with olive oil as the only carbon source was performed. The successful growth of the strain allows us to suggest that a true lipase is responsible for the lipolytic activity in the B13-1 strain. In order to identify the gene(s) encoding the protein responsible for the lipolytic activity, in silico identification and characterization of triacylglycerol lipases from T. harzianum is reported for the first time. A survey in the genome of this fungus retrieved 50 lipases; however, bioinformatic analyses and putative functional descriptions in different databases allowed us to choose seven lipases as candidates. Suitability of the bioinformatic screening to select the candidates was confirmed by reverse transcription polymerase chain reaction (RT-PCR). The gene codifying 526309 was expressed when the fungus grew in a medium with olive oil as carbon source. This protein shares homology with commercial lipases, making it a candidate for further applications. The success in identifying a lipase gene inducible with olive oil and the suitability of the functional screening and bioinformatic survey carried out herein, support the premise that the strategy can be used in other microorganisms with sequenced genomes to search for true lipases, or other enzymes belonging to large protein families.

Keywords: enzymes; lipases; olive oil induced lipases; protein bioinformatic analyses; triacylglycerol lipases; true lipases.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Extracellular lipolytic activity measured with p-nitrophenol palmitate. Black bars show the activity in the strain B13-1; gray bars show the activity in the strain B13-3. The data show the standard deviation of three independent samples.
Figure 2
Figure 2
Phylogenetic tree of fungal lipases. The tree was constructed with 35 characterized fungal lipases (accessions correspond to GenBank unless another source is specified) and 15 putative triacylglycerol lipases from T. harzianum. (*) after the accession numbers are lipases which have been characterized; unlabeled proteins correspond to hypothetical, uncharacterized, predicted lipases. The tree was generated by MAFFT software using the neighbor-joining method [55] with 500 bootstrap re-samplings. Clusters IA, IB and II as described by Yadav et al. [6]. Highlighted in purple letters, the Rhizomocur miehei lipase-like group and in olive green letters, the Candida rugosa lipase-like group, according Schmidt-Dannert [44]. Clade I and Clade II, are consistent with Gupta et al. [76]. T. harzianum triacylglycerol lipases from this study, highlighted in bold red letters.
Figure 3
Figure 3
Reverse transcription polymerase chain reaction (RT-PCR) analysis of selected putative extracellular triacylglycerollipases from Trichoderma harzianum in medium without (B) or with 1% (v/v) olive oil (C) as carbon source. Lane (M) 1 Kb plus DNA Ladder (ThermoFisher, Carlsbad, CA, USA). The number of ID at genome portal of each candidate lipase corresponds to: (1) 77338; (2) 78181; (3) 514252; (4) 526309; (5) 514427; and (6) 87496. Lane (7) Elongation factor 1 (400 bp), as positive control of PCR. Panel (A) corresponds to PCR on genomic DNA, to test the primers. Base pair (bp).
Figure 4
Figure 4
Structural-based multi-alignment of T. harzianum 526309, 551811 and 87496 with 2VEO lipase from C. antarctica (CALA). Blue line highlights the pentapeptide; orange line, the lid domain; green triangles, the catalytic triad; and yellow triangles, the oxyanion. Alpha helices and beta sheets are indicated at the top. Identical (bold white letters on red background) and similar amino acids (standard red letters) are shown.

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