Extracellular Enzyme production
The production of amylase was estimated by inoculating the fungi on glucose yeast extract peptone (GYP) agar medium (glucose: 1g, yeast extract: 0.1g, agar: 15 g, distilled water: 1000mL and pH 6) that contain 1% soluble starch for 5 days. The plates with fungal colony were flooded with 1% iodine and 2% potassium iodide as indicator .The appearance of clear zone surrounding the colony was considered as a positive result for amylase enzyme (Rajput et al., 2016).
The glucose yeast extract peptone (GYP) agar media supplemented by 0.5% CMC (carboxy methyl cellulose) was utilized to test cellulose production. Plates were incubated for five days at 28°C then flooded with a solution of 1% congo red dye for twenty minutes and destained with1N NaCl solution for 15 minutes. The presence of cellulase enzyme was marked by the appearance of light yellow area around the fungal colonies (Rajput et al., 2016).
Tyrosinase production was assessed using Tyrosine agar medium (Peptone 0.5% Beef extract 0.3% Agar 2% L-tyrosine 0.5%) and adjusted at pH 7. Brown color zone was observed around the colonies after incubation indicate the production of tyrosinase enzyme (Majumdar et al., 2018).
Protease production was screened by growing the endophytic fungi on glucose yeast extract peptone (GYP) agar media amended with 1% casein and pH 6.5.Plates were incubated for 5 days. A clear zone around colony indicated a positive result. Plates were flooded with Frazier’s reagent to increase the clearity of the zones (Rajput et al., 2016).
For production of Chitinase, fungi were cultured in colloidal chitin prepared according to Rodriguez-Kabana et al. (1984), by partial hydrolysis using 10 N HCl for 1.5 h at room temperature, then washed the colloidal chitin several times using large volumes of tap water and then washed with distilled water for five to seven times to adjust the pH. Media of chitin agar was prepared by the following ingredients (yeast extract, 1.5 g; chitin, 2.0 g; agar, 20 g; distilled water, 1 L). Plates were inoculated with test cultures and then incubated at 26°C up to 72 h. The appearance of the clear zone surrounding the culture showed chitinase activity.
Lignin degrading enzyme production
With slight modifications, lignin degrading enzymes were tested according to (Atalla et al., 2010). Using the cork borer 6mm mycelial plugs discs were cut from the edge of 6 days old fungal cultures. Fungal plugs were inoculated on Boyd and Kohlmeyer (B;K) medium (glucose-10 g, peptone-2 g, yeast extract-1 g, agar-18 g, distilled water-1000 ml and pH-6.0) amended with 4 mM guaiacol.The petriplates were incubated at 25±2°C for two weeks after wrapping them with black polythene bags .Oxidation of guaiacol leads to formation of reddish brown color under and around the fungal colony. Endophytic fungi inoculated on guaiacol free media were used as control.
The isolates were inoculated on glucose yeast extract peptone (GYP) agar media supplemented with 0.005%1-naphthol.The change in color to blue indicated positive laccase enzyme due to oxidation of 1-naphthol by endophytic fungi(Sathish et al.,2012).
Extracellular Enzyme production