P nitrophenyl β d glucopyranoside pnpg

The strains and plasmids used in this study were listed in additional file (Additional file 1: Table S1). E. coli Trans5α (TransGen, Beijing, China) was used for plasmid construction. E. coli BL21(DE3) and E. coli BL21(DE3)pLysS (TransGen, Beijing, China) were used as the hosts for Bgl1A(A24S/F297Y) production. Ampicillin, chloramphenicol, and IPTG were purchased from Sangon Biotech (Shanghai, China). p-Nitrophenyl β-D-glucopyranoside (pNPG) was from Sigma-Aldrich (St. Louis, MO, USA). The insoluble microcrystalline cellulose with an average particle size of 25 μm was acquired from Aladdin Chemistry (Shanghai, China). Ni²⁺-charged chelating sepharose fast flow was purchased from GE Healthcare (Uppsala, Sweden). All other chemicals were of analytical grade unless otherwise specified. Standard TB medium (per liter contains 4 g glycerol, 24 g yeast extract, 12 g peptone, 17 mM KH₂PO₄, and 72 mM K₂HPO₄) was used as culture medium in 250-mL Erlenmeyer flasks. Modified TB medium (per liter contains 15 g glycerol) was employed in high cell density culture (HCDC), the feeding solutions contained 45 g tryptone, 45 g yeast extract, and 500 g glycerol per liter.

The supernatants collected via centrifugation (10,000×g for 10 min at 4 °C) were used for enzyme and secreted protein concentration assays. The FPase and CMCase activities were measured via the DNS method using glucose as a standard. One unit represents the amount of enzyme that formed 1 µmol of reducing sugar per minute during the hydrolysis reaction. The pNPCase and pNPGase activities were measured against p-nitrophenol-d-cellbioside (pNPC) and p-nitrophenyl β-d-glucopyranoside (pNPG) (Sigma-Aldrich, St. Louis, USA), respectively. One unit of pNPCase and pNPGase activity was defined as 1 μmol of p-nitrophenol released per minute during the hydrolysis reaction. Xylanase I and II activities were determined by xylan degradation at pH values of 3.7 and 5.0 [29 (link)], respectively. One unit of xylanase activity is defined as releasing 1 μmol of xylose reducing sugar equivalents per minute under the defined assay conditions. Protein concentration was determined using the Modified Lowry Protein Assay Kit (Sangon Biotech, Shanghai, China). All experiments were performed in three biological replicates. SDS-PAGE electrophoresis was carried out with 12% polyacrylamide separating gel.

The filter paper (FP), EG, CBH, and BGL activities of T. reesei were measured as described before (Ghose, 1987 (link)) by using Whatman No. 1 FP (Whatman, UK), CMC–Na (Sigma, USA), p-nitrophenyl-β-D-cellobioside (pNPC; Sigma, USA), and p-nitrophenyl-β-D-glucopyranoside (PNPG; Sigma, USA) as the substrates, respectively. One unit of enzyme activity was defined as the amount of enzymes releasing 1 mole of reducing sugar (or p-nitrophenol in the CBH and BGL assays) per minute under the assay conditions. Renaturing SDS–PAGE electrophoresis was performed in 12% polyacrylamide separating gel containing 0.1% SDS with 0.3% CMC–Na as the substrate; this procedure was performed in a Mini-PROTEAN tetra electrophoresis cell (Bio-Rad Laboratories, Milan, Italy).

β-glucosidase activity was measured by determining the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (pNPG) (Sigma, USA) using the initial rate of colored reaction product accumulation as described by Korotkova et al.^{36 (link)}. A 20 μl aliquot of the acquired enzyme was mixed with 180 μL of 5 mM pNPG substrate in 50 mM sodium citrate buffer (pH 7.0). After incubation at 50 °C for 10 min, the reaction was terminated by adding 100 μl of ice-cold 0.5 M Na₂CO₃. The release of p-nitrophenol (pNP) via enzymatic hydrolysis was indicated by the appearance of a yellow color and monitored with an xMark Microplate Spectrophotometer (Bio-Rad, Canada) at 405 nm. One unit (U) of β-glucosidase activity was defined as the amount of enzyme that released 1 μmol of pNP per minute from the substrate.
Protein content was measured with a BCA protein assay kit (Pierce, Rockford, IL, USA) using bovine serum albumin (BSA) as the standard. All enzyme activities are expressed as units per milligram of protein.
All experiments were performed with three replicates and repeated at least thrice.

Cells were grown in Stainer medium supplemented with 0.2% (wt/vol) glucose or 0.4% (wt/vol) Avicel. Cells of the middle exponential phase were gathered through centrifugation at 5,000 × g for 10 min. For intact cell samples, cell pellets were washed with Na₂HPO₄-KH₂PO₄ buffer (50 mM, pH 6.8) and resuspended in the same buffer. For cell extract samples, cell pellets were washed and resuspended with Na₂HPO₄-KH₂PO₄ buffer containing 2% (vol/vol) Triton X-100. Then the mixture was incubated at 4°C for 4 h to make sure that all the proteins were released into the buffer. Sodium carboxymethyl cellulose (CMC-Na) and p-nitrophenyl β-D-glucopyranoside (pNPG) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as substrates to measure endo-glucanase and β-glucosidase activities, respectively, according to previously described methods (Ji et al., 2014 (link)). Protein concentrations were quantified as described by Bradford (1976) (link), and all the enzymatic assays were carried out in triplicate.

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.

Flavonol standards (HPLC ≥ 95%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Acetonitrile and methanol of chromatographic grade for liquid chromatography-mass spectrometry (LC-MS), as well as α-glucosidase from Saccharomyces cerevisiae (23 U/mg), acarbose, and p-nitrophenyl-β-d-glucopyranoside (PNPG), were obtained from Sigma-Aldrich (St. Louis, MO, USA). Other solvents were all of analytical grade purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).