P nitrophenyl palmitate p npp

The substrate for the catalytic tests was p-nitrophenyl palmitate (p-NPP) purchased from Sigma-Aldrich. This substrate was dissolved in 1% isopropanol, 2% Triton X-100 as emulsifier, and 50 mmol·L⁻¹ Tris buffer pH 8.0 [29 ]. For the immobilized enzyme 0.7 g of microspheres was used. A negative control without the enzyme was also studied, and the reaction mixture in each set was incubated for 60 min, stopping the reaction by removing the immobilized enzyme using a sieve. The assays were performed in triplicate.

Graphene flakes were purchased from Sigma-Aldrich (St. Louis, MO, USA), and used without further purification. Candida rugosa lipase (lyophilized powder, Type VII, 700 U/mg solid) and p-nitrophenylpalmitate (p-NPP) were also purchased from Sigma-Aldrich. Phosphate buffer solution (PBS, 0.1 M, pH = 7.0), which was prepared by mixing standard stock solution of 0.1 M KH₂PO₄ and 0.1 M K₂HPO₄, was used as the supporting electrolyte. Unless otherwise stated, reagents were of analytical grade and used as received.

Lipolytic activity was determined according to Kotogán et al. [45 ], with slight modifications. A crude enzymatic extract was obtained after inoculation of two plugs (5 mm of diameter) in 50 mL of PDB medium. p-nitrophenyl palmitate (pNPP; Sigma-Aldrich, Lisbon, Portugal) was used as substrate. A stock solution (3 mM) was prepared in 25% dimethyl sulfoxide and 0.5% of Triton X-100 and completed with potassium phosphate buffer (pH 6.8). A volume of 50 µL of buffered pNPP solution was added to 150 µL of crude extract, and incubated at the test temperature for 0 min, 45 min and 90 min. The reaction was stopped by adding 50 µL of 0.1 M sodium carbonate. The released p-nitrophenol (pNP) was measured at 405 nm in 96-well plates using a microplate reader (Biotek, Synergy HT, Winooski, VE, USA). The blank sample was treated as all the samples, replacing the enzymatic crude extract by ultrapure water. The molar absorption coefficient of pNP (ε = 1.2475 × 10⁴ M⁻¹·cm⁻¹) was estimated from the absorbance of pNP standard solutions measured at 405 nm. One enzymatic unit was defined as the amount of the enzyme that releases 1 mmol of pNP per minute.
The quantification of the lipolytic activity was made at 25 °C, 30 °C, 37 °C, 45 °C since higher temperatures lead to spontaneous substrate degradation. Data is presented as mean of three replicates, normalized for mycelium dry weight.

Lipolytic activity assay was conducted according to the method described by Martínez and co-workers [37 (link)] with some modifications. A. baumannii was grown to the mid-logarithmic phase (OD600 ∼ 0.6). The adjusted cultures were supplemented with the studied extracts and then mixed with an equal volume of the substrate solution. The latter consisted of 2 mM p-nitrophenyl palmitate (pNPP) (Sigma-Aldrich) in 50 mM Tris-HCl (pH 7.2, Sigma-Aldrich) containing 2% ACN (Sigma-Aldrich). The final concentration of the extracts in the assay mixture was 512 µg/mL for both BR and CC and 256 µg/mL for the other 4 species. An aliquot of the reaction mixture was transferred to a flat-bottomed 96-well plate and the p-nitrophenol (pNP) released after pNPP hydrolysis was determined by measuring the absorbance at 410 nm both at time zero and after 3 h of incubation at 37 °C. Samples incubated with the equivalent amounts of DMSO were used as controls.