Bradford kit

β-glucosidase activity was measured using 5 mM _p-nitrophenyl-β- _D-glucopyranoside (pNPG) (Sigma-Aldrich, St. Louis, MO, USA) in 5 mM sodium citrate buffer, pH 4.8 as substrate. Fifty microliter substrate and 100 μl sample were incubated at 50°C for 30 min, 150 μl of 10% (wt/vol) Na₂CO₃ was added to terminate the reaction. Absorbance was read at 420 nm in a microplate using a plate reader (Infinite 200 NanoQuant, Tecan, Zurich, Switzerland)._p-nitrophenol was used to prepare a standard curve. One unit (IU) of enzyme activity was defined as the amount of enzyme needed to hydrolyze 1 μmol pNPG in 1 min.
Protein quantification was measured using Bradford with bovine serum albumin (BSA) as standard (Bradford kit, Sangon Biotech, Shanghai, China).

Western blot analysis was performed to verify the expression level of methyl-accepting chemotaxis protein (McpC), a chemoreceptor in chemotaxis, of P. larvae after the treatment with N-MRJP2. The total amount of protein was extracted from cells, and the concentration was determined by Bradford Kit (Sangon Biotech, Shanghai, China). Equal amounts of proteins were then separated by stacking (4%) and separating (12%) SDS-PAGE, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Invitrogen). After being blocked with 5% skimmed milk in Tris-buffered saline with Tween (TBST) 20 for 1 h, the membrane was washed and incubated with a primary antibody of McpC (Signalway antibody, ShenYang, China) at a dilution of 1:2000, then washed with TBST three times and incubated with secondary antibodies (goat anti-mouse, Invitrogen) at room temperature for 1 h. The blots were visualized using the Chemic Doc XRS imaging system (Bio-Rad) in the presence of enhanced chemiluminescence (Pierce, Thermo Fisher). The targeted protein band was quantified by NIH Image J software and normalized by Sodium Potassium ATPase, which was used as a loading control and its antibody was purchased from Abcam (Cambridge, MA, USA).

The fermentation broth was collected via centrifugation, and the aliquots of the supernatant were diluted to measure enzyme activity. To examine β-glucosidase activity, pNPG and cellobiose (Sigma, USA) were used as substrates. We conducted the enzyme reaction in acetate buffer (pH 4.8) at 50 °C for a total of 30 min, after which we added 10 % Na₂CO₃ to stop the reaction. pNP release was measured and the absorbance was read at 405 nm. The glucose level was measured with the Biosensor. One enzyme activity unit represents the amount of enzyme required to either produce one µmol glucose, or pNP per minute under the above condition. To measure the FPase activity, the enzyme reaction was conducted in 0.2 mol/L acetate buffer (pH 4.8) at 50 °C for a total of 60 min with 0.05 g Whatman No. 1 paper as the substrate. DNS method was used to quantify the released reducing sugars. A protein concentration assay was performed using a Bradford kit (Sangon Biotech, Shanghai, China). Three biological triplicates were performed throughout all described experiments. Both equal quality and volume of culture supernatants were performed for SDS-PAGE analysis, and the predicted β-glucosidase bands were excised for MALDI-TOF–MS identification. We measured the released sugars within the broth supernatants via LC-10 AD HPLC (Shimadzu, Japan) by a BioRad Aminex HPX-42A carbohydrate column (Bio-Rad, USA).

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Hyaluronic acid (HA), laminin (LN), collagen III (Col III) and collagen IV (Col IV) ELISA kits were obtained from Abbott Laboratories, USA. A Bradford kit was bought from Sangon Biotech, Shanghai, China. Sirius red and hematoxylin-eosin (HE), protease inhibitor cocktail, 3-[(3-Cholamidopropyl) dimethylammonio]-propanesulfonate (CHAPS), glycine, ammonium persulfate (APS), TEMED, and protease inhibitor cocktail were obtained from Sigma Chemical (St. Louis, MO, USA) Dithiothreitol (DTT), glycine, Immobilized pH gradient (IPG) strips, IPG buffer, dry strip cover fluid and other reagents that were used in two-dimensional gel electrophoresis were acquired from Bio-Rad (Hercules, CA, USA).

Supernatants were collected by centrifugation for removing mycelia and residual medium. The concentration of total extracellular protein was measured by a Bradford Kit (Sangon, Shang Hai, China), according to the manufacturer’s instructions. FPA and CMCase activities were assayed with filter paper Whatman No. 1 (Shanghai, China) and CMC-NA (Sigma, USA) as the substrates, respectively. The enzyme reactions were performed in 0.2 M acetate buffer (pH 4.8) at 50°C for 60 and 30 min, respectively, using DNS method to quantify the released reducing sugar. Xylanase activity were assayed according to the method described by Sun et al. [56 (link)]. The pNPCase and pNPGase activities were measured in the above-used acetate buffer at 30°C for 30 min with pNPC and pNPG (Sigma, USA) as substrates. One enzyme activity unit was defined as the amount of enzyme required for producing 1 μmol glucose or pNP per minute under the assayed conditions. Three biological triplicates were performed in all analyses.