EVALUATION OF CHROMATE REDUCTASE ACTIVITY IN PENICILLIUM BRASILIANUM LBM 260

TATARIN, ANA SILVIA; ARANGUIZ, CAMILA; SADAÑOSKI, MARCELA ALEJANDRA; POLTI, MARTA ALEJANDRA; FONSECA, MARÍA ISABEL

Congreso; XVIII Congreso Argentino de Microbiología General; 2022

Chromium (Cr) is one of the most toxic metals that cause pollution of soil and groundwater. As a metal, Cr is non-biodegradable and hence of major concern. However, it can be transformed from its highly toxic state of Cr(VI) to a less-toxic state, Cr(III), through a bioreduction process mediated by intracellular and extracellular fungal enzymes. In this sense, it is important to find a fungal for biotransformation of Cr(VI) into a less toxic form through extracellular and intracellular enzymes. The aim of this work was to determine extracellular and intracellular chromate reductase (ChrR) activity in Penicillium brasilianum LBM 260. Fungal was cultured in 100 mL Erlenmeyer flasks containing 20 mL modified minimal Lee medium amended with 200 mg/L of Cr(VI). The flasks were inoculated with 1 mL of 2.4 x 104 mL fungal spore suspension. Inoculated Erlenmeyer flasks were incubated at 28 ± 1°C in static conditions. Destructive samples were taken out at 4, 8 and 12 d. Controls of culture were performed: biotic free-Cr(VI) controls and abiotic controls with Cr(VI). After period of incubation, samples were centrifuged at 6000 rpm for 10 min to separate the supernatant of the mycelium. This supernatant acts as a source of extracellular enzyme. For ChrR intracellular activity, mycelia were disrupted with liquid nitrogen, resuspended in 50 μL of bidistilled water and the obtained products were centrifuged at 6000 rpm for 30 min. The obtained supernatant acts as an extract to intracellular activity. The reaction system for the determination of ChrR consisted of 700 μL of K2HPO4 buffer (0.02 M pH 7) added with 250 μL of supernatant and 50 μL of NADH (2.5 mM). ChrR was measured at 37∘C for 5 min. After the remaining Cr(VI) concentration was quantified colorimetrically by diphenylcarbazide method with an UV-spectrophotometer at 543 nm. Specific activity was defined as unit chromate reductase activity per minute per mg protein concentration. The protein concentration was determined by Lowry’s method using bovine serum albumin as protein standard. Besides, non-specific reduction controls for reaction were carried out.P. brasilianum LBM 260 exhibited extracellular and intracellular production of ChrR, significantly higher in the extracellular fraction at all tested samples. The highest extracellular ChrR activity was observed at 4 d with a value of 4490.17 ± 490.42 U/mL, in Cr presence, a 5-fold rise in activity was detected with respect to the activity in Cr absence. The intracellular ChrR activity was significantly higher at 8 d with a value of 363.72 ± 47.90 U/mL being 3-fold rise in activity with respect to the activity at other tested samples. Nonspecific reduction was discarded with four controls of reaction.The ChrR activity of the extracellular fraction in Cr absence in the culture medium conditions showed lower activity compared to the fraction in culture medium Cr supplemented, suggesting that extracellular ChrR production was induced in presence of Cr.