Fig 1.
Fluorescent haloes of lipase-producing fungi, on rhodamine B with 1% (v/v) olive oil agar medium, were visible under UV light at 365 nm.
Ten lipase-producing fungi (TN10, M45, AS15, AI16, R22, CBS, F6, P63, C58, and V35) were obtained showing variable lipase activities. After incubation for 5 days at 25°C plates were subjected to UV irradiation and photographed.
Fig 2.
Time course of the Fusarium annulatum Bugnicourt strain CBS.
Biomass (dry weight of mycelia) (π) and lipolytic enzyme activity using TC8 (●) or egg PC (◯) as the substrate on optimized culture medium. Cultures were performed in 200 mL Erlenmeyer flasks of optimized liquid medium A containing: 15 g/L casein peptone, 5 g/L yeast extract, 1.75 g/L KH2PO4, 0.5 g/L MgSO4, and 1% olive oil and incubated, at 25°C, under agitation of 160 rpm. Cell growth was performed by measuring the dry matter. Lipase or phospholipase activity was determined in culture filtrates obtained after removal of cells by centrifugation, as described in the Methods section. Each point represents the mean (n = 3) ± standard deviation.
Table 1.
Morphological features of Fusarium annulatum isolate CBS, causing fruit rot in cantaloupes, compared to previous descriptions of Fusarium annulatum by Bugnicourt (1952), Nelson et al. [38] and Yilmaz et al. [39].
Fig 3.
Colony of strain CBS isolated from soil-borne fungi of the olive tree, Olea europaea cv. Chemlali.
(A) Upper view of a colony on PDA. (B) reverse view of colony on PDA.
Table 2.
Blast search for gene sequences of the Fusarium annulatum Bugnicourt strain CBS, comparted to the reference sequences obtained from type culture material.
Fig 4.
Molecular identification of the strain CBS.
Multilocus phylogenetic analysis using combined sequences from the ITS, TEF1-α, and RPB2 gene regions showing the position of Fusarium annulatum Bugnicourt strain CBS (in bold) within the cluster comprising Fusarium species. Isolate CBS in bold was sequenced in this study. The sequences of Neofusicoccum parvum strain CCF216T (GenBank accession no.s: KC507814, KC507811, KC507805) were used as root, and the root position of the neighbor-joining tree was estimated using this strain as the outgroup. Distances and clustering were calculated using the neighbor-joining method. The tree topology of the neighbor-joining data was evaluated by Bootstrap analysis with 100 re-samplings. Bar, 0.05 substitutions per nucleotide position. Numbers at nodes (>50%) indicate support for the internal branches within the tree obtained by bootstrap analysis (percentages of 100 bootstraps). GeneBank accession numbers are presented in parentheses. T: Type strain, same formatting.
Fig 5.
Purification and electrophoretic analysis of FAL.
(A) Chromatogram profile of FAL purification on a HiTrap™ Q-Sepharose FF column. Adsorbed proteins were eluted with a linear NaCl gradient of 0 to 0.5 M NaCl in buffer A. FAL activity was measured, as described in Material and Methods (Section 4.5), using TC8 as the substrate. (B) SDS-PAGE (12%) analysis of eluted proteins. Lane 1, molecular mass marker; lane 2, resuspended pellets after ammonium sulphate (80%) precipitation; line 3, fraction obtained after gel filtration chromatography on Superdex® 200 Increase 10/300 GL column; lines 4 and 5, purified fractions (7 μg) from HiTrap™ Q-Sepharose FF column. (C) FAL activity staining with MUF-butyrate.
Table 3.
Flow sheet for the purification of the lipase FAL from Fusarium annulatum Bugnicourt strain CBS.
Fig 6.
Effects of pH on the activity (A) and stability (B) of the purified FAL using TC8 or egg PC at 40°C under standard conditions. The pH profile was determined in different buffers by varying the pH values from 4 to 12. The pH stability of the FAL was determined by incubating the enzyme at different pH values, ranging from 4 to 12, for 1 h at 40°C and the residual activity was measured at pH 9 on TC8 or pH 11 on egg PC, also at 40°C. The maximum activity on TC8, obtained at pH 9, or on egg PC, at pH 11, was considered as 100%. Each point represents the mean of three independent experiments.
Fig 7.
Effects of temperature on the activity and stability of the purified FAL using TC8 or egg PC at 40°C under standard conditions.
(A) Enzymatic activity at various temperatures (from 25°C to 55°C) was determined using TC8 or egg PC at pH 9 under standard conditions. For stability of FAL on TC8 (B) at pH 9 or egg PC (C) at pH 11, the activity was measured after incubation of the enzyme for the indicated time at various temperatures under standard conditions. Each point represents the mean of three independent experiments.
Table 4.
Effects of some selected metal ions, inhibitors and chemical reagents on the purified FAL from Fusarium annulatum Bugnicourt strain CBS.
The enzyme assay was performed after pre-incubation of the enzyme with each tested chemical compound, for 1 h at 40°C. The non-treated and dialyzed enzyme was considered as 100% for the metal ion assay. The lipase activity measured in the absence of any inhibitor or reducing agent was taken as the control and considered as 100%. Residual activity was measured at pH 9 and 40°C, using TC8 as a substrate.
Fig 8.
Influence of Ca2+ (CaCl2) and NaTDC on FAL activity.
(A) Effect of the concentration of Ca2+ on FAL activity. Enzyme activity was measured at increasing concentrations of Ca2+. TC8 emulsions as substrate for lipase activity in the presence of 2 mM NaTDC, and PC as substrate for PLA1 activity in the presence of 4 mM NaTDC. (B) Effect of increasing concentrations of bile salt (NaTDC) on lipase activity in the presence of 2 mM CaCl2 and phospholipase activity in the presence of 4 mM CaCl2, using TC8 emulsion and phosphatidylcholine as substrates, respectively. The star indicates the FAL activity measured in the absence of CaCl2 and in the presence of 10 mM EDTA. Each point represents the mean of three independent experiments.
Fig 9.
Effect of Orlistat on the FAL activities.
DMSO (blue lines) or Orlistat in DMSO (40 μM, final concentration, red curves) was injected into the reaction medium at 8 min (A) and 8 min (B) after starting lipolysis with FAL on TC8 and on egg PC, respectively. Lipase and PLA1 activities were measured, at pH 9 and 40°C and at pH 11 and 45°C, using TC8 and egg PC as substrates, respectively. Curves are representative of three independent experiments.
Fig 10.
(A) Chain length selectivity of FAL. The specific activity was measured under standards conditions using TC2, TC4, TC8, olive oil (OO), or egg PC as substrate, as described in Material and Methods. (B) Kinetic recordings of coated sn-EOPC or sn-OEPC lipolysis by FAL. Variations, with time, of the absorbance at 272 nm were recorded for 10 min, for stabilization, and then for 40 min after FAL injection (0.65 μg per well).
Fig 11.
Effect of organic solvents on the activity and stability of FAL, GZEL, and Lipolase®.
The effect of organic solvents was determined by incubating each enzyme with the solvent [25% (v/v) final concentration] for 24 h. The residual lipase activities were determined under the same conditions, using TC8 as the substrate at 40°C and pH 9, as described in the Materials and Methods, and then expressed as a percentage of the activity level in the absence of organic solvents. The activity of the enzyme without any organic solvent was taken as 100%. Each point represents the mean of three independent experiments. Vertical bars indicate standard error of the mean (n = 3). a–d Means in indicator enzymes with different lowercase letters differed significantly (P < 0.05).
Table 5.
Effects of various laboratory detergent additives on the stability of FAL compared to GZEL and Lipolase®.
Table 6.
Effects of various commercialized detergent additives on the stability of FAL compared to GZEL and Lipolase®.
Fig 12.
Stability and compatibility of the tested lipases (FAL, GZEL, and Lipolase®) in the presence of commercial laundry detergents.
Lipases were incubated with laundry detergents (7 mg/mL) for 60 min at 40°C. The enzyme activity of the control sample, without additive and incubated under similar conditions, was taken as 100%. Each point represents the mean of three independent experiments. Vertical bars indicate the standard error of the mean (n = 3). a–b Means in indicator enzymes with different lowercase letters differed significantly (P < 0.05).
Fig 13.
Evaluation assay of used lipases on olive oil removal from cotton fabric with detergents.
Effect of FAL, GZEL, and Lipolase® on the removal of olive oil from cotton fabric with various liquid and solid laundry detergents. a–b Means for all columns of each parameter with different lower-case letters differed significantly (P < 0.05). Values represent the means of 3 independent replicates and the ± SE is shown.
Fig 14.
Wash performance test on oil removal with lipases used.
Wash performance test on oil removal with a commercial detergent in the presence of FAL, GZEL, or Lipolase®. The washing performance analysis test of the lipase was conducted with the commercial detergent Class (7 mg/mL) using cotton cloth stained with tomato sauce, ketchup, or egg yolk. The oil-stained cloth was rinsed with tap water, washed with Class (7 mg/mL), washed with Class supplemented with GZEL (500 U/mL), washed with Ariel supplemented with Lipolase® (500 U/mL), or washed with Ariel supplemented with FAL (500 U/mL).