ASSESSMENT OF OXIDATIVE AND HYDROLYTIC ABILITY OF Hornodermoporus martius LBM 224 FROM MISIONES (ARGENTINA)

ACOSTA GA; SVIERCZ FA; BENITEZ SF; FONSECA MI, ; ZAPATA PD

Simposio; LVI SAIB Meeting ? XV SAMIGE Meeting; 2020

The discovery and production of new enzymes from diverse natural sources is crucial and has enabled large economic benefits. White rot fungi (WRF) are capable of producing different enzymes suitable for biotechnological applications. Misiones rainforest is one of the most biodiverse systems on the planet representing a potential source of WRF enzyme producers. Hornodermoporus martius LBM 224 is a WRF isolated from the Misiones rainforest. The aim of this work was to determine the ability of this strain to secrete hydrolytic and oxidative enzymes in solid media. Qualitative enzyme determination was evaluated at days 7 and 14 of incubation with different substrates in solid media. Laccase (Lac) activity was determined on MEA (malt extract 12.7 g/L, agar 17 g/L) using the 2,6-dimethoxyphenol (DMP) as substrate following two methodologies: as medium supplement (1 mM) and as reveal solution (1 g/L in sodium acetate buffer 0.1 M pH 3.6). Lac activity was also revealed by adding an alcoholic guaiacol solution (12.4 g/L in 96 % v/v) or 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (1.2 mM). Peroxidase (POX) activity was revealed by adding a solution containing 0.7 mM H2O2 to the ABTS plates and was also evidenced by pouring equal parts of 0.4 % v/v H2O2 and 1 % w/v pyrogallol to MEA plates. Manganese peroxidase (MnP) activity was revealed on MEA plates supplemented with 1 mM MnCl2·4H2O. Endo-1,4-β-glucanase (CMCase) activity was evaluated on MEA plates supplemented with 1 g/L carboxymethylcellulose (CMC) and was revealed with a 3 g/L Congo Red solution. β?glucosidase (BGL) and cellobiohydrolase (CBH) activity were detected on agar plates containing 1 g/L CMC, 1 g/L yeast extract, 15 g/L agar, supplemented with 0.1 mM 4-methylumbelliferyl glucoside (Mu-g) substrate for BGL activity, and 0.1 mM 4-methyl umbelliferyl cellobioside (Mu-c) substrate for CBH activity. These plates were revealed on UV-light. Amylase activity was determined on MEA plates containing 1 g/L soluble starch and revealed with a 1 % v/v Lugol solution. Lipolytic activity was revealed on agar plates containing 5 g/L meat peptone, 3 g/L yeast extract, 15 g/L agar, and 1 % v/v tributyrin. Lac activity was detected on day 7 using both DMP techniques, ABTS and guaiacol as substrate. On day 14, Lac production was evidenced with both DMP techniques and ABTS. POX activity was only detected on plates revealed with ABTS+H2O2 on day 7. CMCase, BGL, CBH and amylase activities were observed in both times tested. MnP and lipolytic activity could not be detected by the technique used. The present methodology represents a simple and economic approach for the screening of a variety of extracellular enzymes. The oxidative and hydrolytic enzymes secreted by H. martius LBM 224 have a potential application in diverse biotechnological processes.