Ligninolytic enzyme activity

The activities of MnP, Laccase and LiP in the samples were determined according to the methods described by Wariishi et al. (1992), Johannes and Majcherczyk (2000) and Tien and Kirk (1988), respectively. MnP activity was determined by oxidation of 2, 6-dimethoxyphenol (2, 6-DMP). 100 µL of sample, manganese sulphate solution and 2, 6-DMP solution were added to 600 µL malonate buffer solution and 100 µL of hydrogen peroxide solution was added (total volume 1000 µL) to start the oxidation reaction. Laccase activity was determined by oxidation of 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). 100 µL ABTS solution was added to 800 µL sodium acetate buffer and 100 µL sample were added to start the oxidation reaction. LiP activity was determined by oxidation of veratryl alcohol. 100 µL veratryl alcohol solution and 100 µL sample were added to 700 µL sodium acetate buffer and 100 µL hydrogen peroxide solution was added to start the oxidation reaction. The oxidation of 2, 6-DMP by the MnP, ABTS by the laccase and veratryl alcohol by the LiP was followed spectroscopically at 469 nm, 420 nm and 310 nm, respectively, by a spectrophotometer for 3 min at room temperature. The enzyme activity was calculated by the Beer-Lambert law and one unit was defined as producing 1 µmol min-1 product under the assay conditions.

Ref.

Johannes and Majcherczyk. 2000. J. Biotechnol. 78: 193-199

Tien and Kirk. 1988. Methods Enzymol. 161: 238-249

Wariishi et al. 1992. J. Biol. Chem. 267: 23688-23695

Cellulolytic enzyme activity (FPA)

The fungus was grown and maintained on 2% MEA at room temperature one agar plug with mycelium was inoculated in 10 mL of Mandels' medium [urea 0.3 g, (NH4)2SO4 1.4 g, KH2PO4 2 g, CaCl2 0.3g, MgSO4 0.3 g, yeast extract 0.25 g, peptone 0.75 g, FeSO4·7H2O 5 mg, CoCl2·6H2O 36 mg, MnSO4·H2O 1.8 mg, ZnSO4·7H2O 2.5 mg L-1] containing 5% (w/v) avicel as an energy source and substrate. In small-scale cultivation, the avicel (0.5 g) and culture medium (10 mL) were dispensed into each 50 mL sterilized conical tube (SPL, Korea). The culture medium was inoculated with the appropriate fungal culture and the tubes were sealed with silistoppers. The tubes were placed at a 50° angle on a rotary shaker. Cultivation was performed with agitation at 150 rpm for 7 days. For miniaturized enzyme assay (micro-assay), FPase assay for total cellulase activity was performed using filter paper. A volume of 25 μL of enzyme were incubated at 50 °C for 30 min. After incubation, 150 μL of DNS reagent was added and boiled for 5 min. Following the color development, 33 μL of each samplewas then diluted with 165 μL of distilled water in a 96-well microtiter plate. The absorbance was measured at 540 nm. One unit per mL of FPase activity was described as the amount of enzyme required to release 1 μmol of glucose equivalents per milliliter of culture medium per minute.

Ref.

Lee et al. 2011. J. Microbiol. Methods. 86: 124-127