P nitrophenyl β d cellobioside pnpc

For the enzyme activity assay, all strains were incubated in the enzyme production medium. Fresh spores were inoculated at 10⁸/mL into liquid MMS medium containing 2% glucose and incubated at 30°C and 200 rpm for 24 h. Next, the mycelia were collected by vacuum pump filtration. Exactly 0.5 g mycelium was transferred to a 50 mL measure of enzyme-producing medium and incubated for 6 days in a 300 mL bottle at 30°C and 200 rpm. Fermentation broths were sampled and measured every 24 h from 72 to 144 h. For the filter paper activity (FPA), endoglucanase, cellobiohydrolase, β-glucosidase, and amylase activity assays, Whatman™ 1 filter paper, sodium carboxymethyl cellulose (CMC-Na, Sigma), p-nitrophenyl-β-D-cellobioside (pNPC, Sigma), p-nitrophenyl-β-D-glucopyranoside (pNPG, Sigma), and starch (Sigma) were used as substrates, respectively (Chen et al., 2013 (link)). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol glucose or p-nitrophenyl (Sigma) per minute under the assayed conditions (Wood and Bhat, 1988 (link)). Three biological triplicates were performed, and the mean values and corresponding standard deviations were calculated.

Trichoderma reesei Δtku70 (ATCC MYA-256), a nonhomologous end joining pathway-deficient strain, was used as the parent strain in this study [52 (link)]. All the other strains constructed in this study are listed in Additional file 8: Table S1. The E. coli strain GB05-dir was used for constructing all the plasmids [53 (link)].
Wheat bran was kindly provided by Longlive Bio-Technology Co., Ltd. (Yucheng, Shandong, China). The p-nitrophenyl-β-d-cellobioside (pNPC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Uridine, PEG6000, sorbitol, and lactose were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). KOD FX DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) was used for all polymerase chain reaction amplifications. RNAiso™ reagent, PrimeScript^® RT reagent Kit With gDNA Eraser (Perfect Real Time) and SYBR^® Premix Ex Tag™ (Tli RNase H Plus) were purchased from Takara Bio Inc. (Shiga, Japan). The DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics, Mannheim, Germany) was used for Southern blotting analysis. The restriction enzymes Ase I and Xho I used for genome digestion were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). All other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

An international unit of enzymatic activity is defined as the amount of enzyme required to produce 1 μmol of product per minute. 1 mM p-nitrophenyl-β-D-glucopyranoside (p-NPG), 1 mM p-nitrophenyl-β-D-xylopyranoside, and 4 mM p-nitrophenyl-β-D-cellobioside (p-NPC) (Sigma–Aldrich, St. Louis, EUA) were the substrates for the activity determining reaction for β-glucosidase, β-xylosidase and cellobiohydrolase, respectively. After 10 min for β-glucosidase and β-xylosidase and 30 min for cellobiohydrolase activity measurement at 50°C, the reactions (comprised of 80 μL of the substrate and 20 μL of the enzyme) were stopped by addition of 1 mol/L sodium carbonate. Absorbance at 400 nm was used to estimate p-NP concentration release (Bussamra et al., 2015 (link)).
The total cellulose activity measured according to the filter paper units (FPU) was performed by reducing the NREL methods (Adney and Baker, 2008 ) 10 times. The sugar release was quantified following the methods suggested by Miller (1959 (link)).

The filter paper activity (FPA), cellobiohydrolase (CBH), β-glucosidase (BGL), and endoglucanase (EG) activities were measured using Whatman no. 1 paper (Whatman, UK), p-nitrophenyl-β-D-cellobioside (pNPC; Sigma, USA), p-nitrophenyl-β-D-glucopyranoside (pNPG; Sigma, USA) and CMC-Na (Sigma, USA) as substrates respectively by the methods described previously [57 ]. One unit of enzyme activity was defined as the amount of enzyme required to liberate one micromole reducing sugars (FPA, EG) or p-nitrophenol (CBH, BGL) per minute under the assay conditions. SDS-PAGE electrophoresis was performed in 12% polyacrylamide separating gel with equal volume of culture supernatants boiled for 10 min for degeneration. The specific cellulase bands were excised for MALDI-TOF–MS identification.

For enzyme activities assay, strains P. oxalicum 114-2, ΔPohtf1 and CPohtf1 were fermented in the cellulase production medium under the conditions described above for 6 days. Fermentation broths were sampled and measured every 24 h from day 3 to day 6. Whatman^TM 1 filter papers, sodium carboxymethyl cellulose (CMC-Na, Sigma), p-nitrophenyl-β-D-cellobioside (pNPC, Sigma), and p-nitrophenyl-β-D-glucopyranoside (pNPG, Sigma) were used as substrates for filter paper activity (FPA), endoglucanase, cellobiohydrolase, and beta-glucosidase activities assays, respectively (Chen et al., 2013 (link)). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol glucose or p-nitrophenyl (pNP, Sigma) per minute under the assayed conditions.

The activities of cellobiohydrolases (pNPCase) and β-glucosidases (pNPGase) were determined by measuring the amount of released p-nitrophenol using p-nitrophenyl-β-d-cellobioside (pNPC; Sigma) and p-nitrophenyl-β-d-glucopyranoside (pNPG; Sigma) as substrates, respectively. The filter paper activity (FPA) was determined by measuring the released reducing sugar with Whatman No. 1 filter paper as the substrate. The pNPC and pNPG activity assays were performed in 200 μl reaction mixtures containing 50 μl of culture supernatant and 50 μl of the respective substrate plus 100 μl of 50 mM sodium acetate buffer (pH 4.8) and then incubated at 45 °C for 30 min. One unit (U) of pNPCase or pNPGase activity was defined as the conversion of 1 μmol of substrate per minute under the test conditions. The FPA assay was performed at 50 °C in 200 μl reaction mixture including 50 μl of appropriately diluted culture supernatant and 150 μl 50 mM sodium acetate buffer (pH 4.8). One unit (U) of FPA was defined as the release of 1 μmol reducing sugar per minute under the test conditions. Total secreted proteins were determined using the Bradford protein assay with bovine serum albumin (BSA) as a standard. At least two biological replicates were carried out for each experiment.