Production of oxidative and hydrolytic enzymes by Hornodermoporus martius LBM 224 under solid-state fermentation using lignocellulosic waste as substrate

ACOSTA, GABRIELA ALEJANDRA; BENITEZ, SILVANA FLORENCIA; FONSECA, MARIA ISABEL; ZAPATA, PEDRO DARIO

Los Cocos

Congreso; XVII Congreso Argentino de Microbiología General; 2022

Sociedad Argentina de Microbiología General (SAMIGE)

Fungi are known to secrete numerous enzymes of biotechnologial interest. In this sense, the Agaricomycete Hornodermoporus martius LBM 224 has shown the ability to secrete various enzyme complexes. The aim of this work was to produce oxidative and hydrolytic enzymes under solid-state fermentation (SSF) using lignocellulosic waste as substrate.Enzyme production was carried out under SSF using sugarcane bagasse (SCB), a previously reported inexpensive substrate, which was generated by agroforestry industries of Misiones, Argentina. The substrate was sampled from a sugarcane mill at San Javier, Misiones, Argentina. Initial moisture content was adjusted to 75 % w/w either with distilled water or Czapek medium. SCB (5 g; 40-mesh) was inoculated with five agar plugs from MEA (Malt Extract Agar) plates and incubated at 28 ± 1 °C for 18 days under static conditions. After incubation, enzymes were extracted by adding 50 mL of distilled water and shaking at 150 rpm for 60 min at 28 ± 1 °C. The extract was filtered with filter paper and centrifuged at 4400 x g for 10 min. Endoxylanase and endoglucanase activity were assayed using beechwood xylan and carboxymethylcellulose as substrate, respectively. Reducing sugars obtained were determined by the 3,5-dinitrosalycillyc acid (DNS) method. For both enzymes, activity was expressed in units (U), defined as the amount of enzyme needed to produce 1 μmol of reducing sugars per min at 50 °C. β-Glucosidase and Cellobiohydrolase activity were determined using p-nitrophenyl-β-D-glucopyranoside and p-nitrophenyl-β-D-cellobioside as substrate, respectively. The amount of p-nitrophenol released was measured at 405 nm after addition of Na2CO3. Enzyme activity was expressed in U, defined as the amount of enzyme necessary to release 1 μmol of p-nitrophenol per minute at 50 °C.Amylase activity was determined using soluble starch as substrate. Reducing sugars obtained were determined by DNS method. Amylase activity was expressed in U, defined as the amount of enzyme needed to produce 1 μmol of reducing sugar per min at 50 °C.Laccase activity was measured using 5 mM of DMP. The absorbance increase was monitored at 469 nm (E469 = 27.5mM−1 cm−1). Lac activity was expressed in U, defined as the amount of enzyme needed to produce 1 µmol product per min at 30 °C. Higher enzymatic production was obtained when SCB initial moisture was adjusted with Czapek medium. Endoxylanase (803.00 ± 33.91 U/L) and Endoglucanase (729.27 ± 90.97 U/L) presented the highest yields, followed by Amylase (532.60 ± 55.85 U/L) and Laccase (470.40 ± 49.79 U/L). β-Glucosidase (7.50 ± 0.37 U/L) and Cellobiohydrolase (5.32 ± 0.10 U/L) showed the lower yields. These results suggest that SCB is a suitable substrate to produce oxidative and hydrolytic enzymes from H. martius LBM 224, with potential biotechnological applications.