0.22 μm pes membrane

Aspergillus niger O1 and transformants were cultured in fermentation medium and samples were collected at 2, 4, and 6 days of cultures. The samples were centrifuged and the supernatants were filtered through 0.22 μm PES membrane (Millipore). The protein concentration in the culture supernatant was determined using a Bio-Rad protein assay kit according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, United States). Bovine serum albumin (BSA) was used as the standard, and the absorbance was measured at 595 nm. The supernatant of 6-day cultures was assayed by SDS-PAGE. The subsequent LC-MS/MS analyses and identification of proteins in the secretome were performed as described previously (Guo et al., 2021 (link)).

A. niger O1 was cultured in fermentation medium and the cultures of 2 days, 4 days and 6 days were collected. The cultures were centrifuged and then filtered through a 0.22-μm PES membrane (Millipore). Protein concentrations of the culture supernatants were determined using Bradford assay (Bio-Rad Laboratories, CA) with bovine serum albumin (BSA) as standard according to the manufacturer’s instructions. The supernatants of 2 days, 4 days, 6 days cultures were assayed by SDS-PAGE. Then, the SDS-PAGE gel was cut into small pieces. In-gel protein digestion was performed following the previously published protocol [42 (link)]. The LC-MS/MS analysis was performed with an Eksigent Nano LC coupled to Triple-TOF 5600 mass spectrometer (SCIEX, USA) with a nano-electrospray ionization source [43 (link)]. The top 40 precursor ions with the most intensity were fragmented with 22 s of dynamic exclusion time. The WIFF files from shotgun data acquisition were searched against the Uniprot database of Aspergillus niger (strain CBS 513.88/FGSC A1513, 2020.04, 14084 entries) in MaxQuant (ver. 1.6.3.4) search engine [44 (link)]. Trypsin was set as the specific enzyme, and up to two missed cleavages per peptide were allowed. Precursor ion mass tolerance was set to 20 ppm and fragment ion tolerance was 0.05 Da. For protein identification, the peptide false discovery rates (FDR) were set at 1%.

Sample supernatants were thawed at room temperature and filtered with a 0.22 μm PES membrane (Millipore). Lactate concentrations were measured by high-performance liquid chromatography (Prominence HPLC, Shimadzu). One milliliter of each sample supernatant was placed with a syringe in the sampler holder and 20 μL of the sample was manually injected into the column. The column used was a Fast Fruit Juice (Waters) and H₃PO₄ 5 mM at 0.8 mL/min was used as the eluent. The temperature of the column was maintained at 55°C with a water bath and the separated compound was detected with a refractive index detector (IOTA). Lactate known concentrations were analyzed by HPLC to produce a standard calibration curve in the same operated conditions as for sample supernatants. ${\text{NH}}_4^{+}$ concentrations were measured by using the Nessler colorimetric method. One milliliter of each sample supernatant was mixed with 1 ml of arabic gum (0.5% (w/v) and 100 μL of Nessler reagent (potasium iodomercurate and ammonia). Absorbance was read at 400 nm in a spectrophotometer (UviLine9400, SECOMAM, France) after an incubation time of 5 min. ${\text{NH}}_4^{+}$ known concentrations were analyzed as well by the Nessler colorimetric method to produce a standard calibration curve in the same operated conditions as for sample supernatants.