Gly pro amc

The oats were provided by Lvba Grain and Oil Group Co., Ltd. (Zhangjiakou, Hebei, China). Flavourzyme, papain, neutrase, and alcalase were purchased from Pangbo Biological Engineering Co., Ltd. (Nanning, Guangxi, China). The 2,4,6-trinitrobenzene sulfonic acid (TNBS) was purchased from Huaxia Reagent Co., Ltd. (Chengdu, Sichuan, China). Pepsin (from porcine gastric mucosa, 3200 U/mg), pancreatic (from porcine pancreas, 3 × USP specifications), Tris-HCl, human DPP-IV, acetonitrile (ACN), and formic acid (FA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid was purchased from Acro Biosystems Co., Ltd. (Beijing, China). L-Leu and HEPES were purchased from Solarbio Technology Co., Ltd. (Beijing, China). Gly-Pro-AMC was purchased from AAT Bioquest (Sunnyvale, CA, USA). All of the other chemicals were of analytical grade.

DPP4 activity was measured using glycylprolyl-7-amino-4-methylcoumarin (Gly-Pro-AMC) (AAT Bioquest Inc., CA, USA) as the fluorogenic substrate following the instructions of the manufacturer (44 (link)). In brief, biofilm cell pellets were washed once and resuspended with 10 mM Tris buffer (pH 7.6). The resuspensions were mixed with 100 μM Gly-Pro-AMC solution at a 1:1 ratio in a 96-well plate. After 40 min of incubation at 37°C, the fluorescence intensity (FI) of the mixture was measured in a fluorimeter (SpectraMax M2; Molecular Devices, Sunnyvale, CA) with 380-nm excitation and 500-nm emission wavelength. A background control group with substrate but without sample was included to indicate background FI, which was subsequently subtracted from sample FI readings.