Extended Data Fig. 2: Separation and purification of enzymes responsible for acarbose degradation in K. grimontii TD1.
From: Inactivation of the antidiabetic drug acarbose by human intestinal microbial-mediated degradation
a, Separation of total proteins of the K. grimontii TD1 cell lysate using HitrapTM Q HP by FPLC. The fractions 16, 17 and 18 showed catalytic activity towards acarbose. b, Separation of the protein mixture of the above three fractions (16–18) using HitrapTM SP HP by FPLC. The resulting fractions 27‒31 showed catalytic activity towards acarbose. c, A further separation of the protein mixture of the above fractions 27‒31 using Hiload 16/600 Superdex 200. The resulting fractions 28 and 29 showed catalytic activity towards acarbose. Each fraction was incubated with acarbose at 37 °C for 6 h and the concentrations of residual acarbose in the reaction mixture were assayed by HPLC. d, SDS-PAGE analysis of protein fractions from K. grimontii TD1 for testing acarbose catalytic activity. The fractions 27‒32 from size exclusion chromatography (c) were analysed by SDS-PAGE. Among them, the factions 28 and 29 exhibited obvious activity towards acarbose degradation. The red asterisks indicated the protein components with specific high content in these two factions. The marked band in fraction 28 was then used for the proteomic assay. e, Confirmation of the in vivo activity of Apg in E. coli. The E. coli strain that expressed apg was cultivated at 37 °C for 4 h with the supplementation of 1.8 mg/ml of acarbose. The initial (0 h) and residual (4 h) acarbose concentration were determined. Data are represented as the mean ± s. d. (n = 3). Error bars show s. d. Statistical analysis was performed by a two-tailed Student’s t-tes.
