Potential capacity of lacases produced by Phlebia brevispora BAFC 633 in the degradation of chlorpyrifos

ALAN ROLANDO AYALA SCHIMPF; RODRÍGUEZ, MARÍA DANIELA; FONSECA, MARÍA ISABEL; VILLALBA, LAURA LIDIA; ZAPATA, PEDRO DARÍO

Encuentro; Tercer Encuentro & Primer Worshop de la Red Argentina de Tecnología Enzimática; 2021

Agriculture in the province of Misiones (Argentina) constitutes the main productive activity within the agricultural sector, which implies the use and application of a large amount of agrochemicals. Among the most commonly used agrochemicals are organophosphates such as chlorpyrifos, a pesticide that applied in significant quantities, produces a negative impact on environmental quality by polluting of soils, surface and underground waters, causing in turn, the poisoning of any kind of living beings, due to its high toxicity. The monitoring of this pollutant is becoming a mandatory parameter to assess the performance of agricultural practices. In this sense, it has been shown that the ligninolytic enzymes produced by white rot fungi have the ability to degrade / mineralize toxic substances; both structurally diverse xenobiotics and persistent pollutants in the environment, as well as toxic compounds of a phenolic nature and low molecular weight substances. Within these enzymes, laccases have a predominant role; these belong to the protein family of multi-copper oxidases. Although its catalytic action consists of the oxidation of p-diphenols in the presence of oxygen, the specificity of the substrate that can be oxidized is quite wide and varies with the source of the enzyme. This non-specificity character allows it to have important biotechnological applications, which have been widely reported in various fields, including the degradation of recalcitrant organic pollutants such as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAH), pesticides, dyes, and in the manufacture of biosensors for the determination of aromatic compounds. The objective of the work was to evaluate the potential capacity of the laccases produced by the white rot fungus Phlebia brevispora BAFC 633 to dedrade the pesticide chlorpyrifos. P. brevispora was activated in MEA medium (12.7 g/L malt extract and 20 g/L agar) in a Petri dish for 6 days at 28°C. The enzymatic production was carried out under submerged fermentation in 4 erlenmeyers of 250 mL: To obtain the inoculum, three blocks (Ø 5 mm) of young mycelium were cut and cultivated in ME medium (12.7 g/L of malt extract and 5 g/L of soluble corn extract) with the addition of sulfate of 0.5 mM copper for 10 days at 28°C. The assay was carried out in 100 mL erlenmeyers containing 35 mL of enzymatic extract with laccase 1 U/mL and chlorpyrifos 16 mg/L reaching the final volume in sodium acetate buffer pH 3.6 0.05 M, at 110 rpm and 28ºC, covered with aluminum foil to protect them from light. The samples consisted of 1.5 mL taken at 0, 14, 22 and 40 hours after starting the experiment. The assay was carried out in duplicate. Controls consisted of enzyme extract alone and chlorpyrifos alone under the same conditions. Enzyme activity measurements were made at the beginning of the assay and at each sample collection. Chlorpyrifos were determined spectrophotometrically at 297 nm (maximum absorption peak of chlorpyrifos obtained in a previous test) and concentration was determined byusing a calibration curve. Laccase activity was carried out using the kinetic technique with 2,6 dimethoxyphenol (DMP) 5 mM as substrate in 0.1 mM sodium acetate buffer at pH 3.6. The change in absorbance was monitored at 469 nm (E469 = 27.5 mM-1 cm-1) in a spectrophotometer. The enzymatic activity was expressed in enzyme units (U), where 1 U is equivalent to 1 μM/min of product at 30°C. Enzyme activity was determined in duplicate. The initial laccase activity of the control corresponding to the enzymatic extract at the beginning of the assay (t = 0) was 736 U/L (σ 175), decreasing during the subsequent times until reaching 415 U/L (σ 6) at 40 h. The activity of the extract in the presence of chlorpyrifos remained constant during the different times with an average value of 381 U/L (σ 28). Chlorpyrifos degradation in the control treatments was 0%, keeping the absorbance values constant during the experiment. The degradation rate obtained in the experimental treatments of the pesticide.