Cellulase Production and Enzymatic Characterization

Cellulase was produced in a 500 mL flask that contained 100 mL of fluid medium through a two-step cultivation procedure. Strains were first grown at 30°C in 100 mL of medium that contained 2 g of glucose as a carbon source and were then regulated at pH 5.5 and 200 rpm for 20 hours. The cultures were collected through vacuum drum filtration during this second step, and 0.5 g vegetative mycelia was added to 100 mL of Vogel’s medium that contained 2% cellulose as carbon source or wheat bran medium at an initial pH of 5.5 at 30°C and 200 rpm. Culture supernatants (crude enzyme) were diluted with sodium acetate buffer solution (SABF, 0.2 M, pH 4.8). Enzymatic hydrolyses of the polysaccharides were also performed in SABF (0.2 M, pH 4.8). The filter paper enzyme (FPA), endoglucanase (CMCase), xylanase, and amylase activities of the culture supernatants (diluted samples) were assayed using a DNS reagent (10 g 3, 5-dinitrosalicylic acid, 20 g sodium hydroxide, 200 g sodium potassium tartrate, 2.0 g redistilled phenol, and 0.50 g sodium sulfite anhydrous per 1000 mL DNS reagent) against Whatman No. 1 filter paper, carboxymethylcellulose sodium salt (CMC-Na), xylan (from beechwood), and soluble starch. CMC-Na, xylan, or starch was dissolved in SABF to a final concentration of 1% (mass/volume percent, m/v %), and then the mixture was left overnight and was shaken well before using. The following components were added in a 2.0 mL reaction mixture: 0.5 mL diluted culture supernatants and 1.5 mL CMC-Na, xylan, or starch solution for CMCase, xylanase, or amylase activity assays, respectively; and 2.0 mL diluted culture supernatants and 50 mg Whatman No. 1 filter paper for FPA assay into 25 mL colorimetric tube. The mixture was mixed gently and the reaction mixture was incubated for FPA measurement in a 50°C water bath for 1 hour, for CMCase and xylanase activity measurements at 50°C for 30 min, and for amylase activity measurement at 40°C for 10 min. Three milliliters of DNS reagent were then added to stop the reaction. A blank tube (with boiled crude enzyme) was used as control to correct any reducing sugar present in the crude enzyme samples. The tubes were placed in boiling water for 10 min, 20 mL distilled water was added, 200 μL of reaction mixture was pipetted, and the absorbance was determined at 540 nm. The cellobiohydrolase (pNPCase) and β-glucosidase (pNPGase) activities were measured by using 4-Nitrophenyl β-D-cellobioside (pNPC) and 4-Nitrophenyl β-D-glucopyranoside (pNPG) as substrates, respectively. The pNPC or pNPG was dissolved in SABF to a final concentration of 1 mg/mL. Moreover, 50 μL of pNPC solution (containing 1 mg/mL D-Glucono-δ-lactone) or 50 μL of pNPG solution and 100 μL of diluted culture supernatants were mixed, and then the mixtures were incubated in a 50°C water bath for 30 min. The reaction was stopped by adding 0.15 mL of sodium carbonate solution (10%, m/v), then 200 μL of these reaction mixtures was pipetted, and the absorbance was measured at 420 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of glycoside bonds of the substrate per minute under defined assay conditions. Independent triplicate cultures were sampled and analyzed.
The total protein was determined using a Bradford assay kit according to the instructions of the manufacturer.