Fig. 2: Characterization of metformin hydrolase.

a Co-purification analysis of MetCaCb. MBP-tagged MetCa with or without tag-free MetCb were expressed in E. coli cells and purified using a dextrin column. The asterisk indicates the position of MBP-tagged MetCa, and the triangle indicates the position of tag-free MetCb. The metformin hydrolase (MH) activity is indicated at the bottom. Uncropped image of the gel can be found in source data file. The result shown is from a representative experiment of more than three times. b The size exclusion chromatography (SEC) analysis of the His-tagged MetCaCb complex. The activities of each fraction were indicated by guanylurea production within 60 min in 1-mL reaction mixture containing 5 μl of enzyme, 20 mM metformin and 0.1 mM Ni²⁺. An active fraction was analyzed by SDS-PAGE (insert). The result shown is from a representative experiment of more than ten times. Uncropped image of the gel can be found in source data file. c Determination of the molecular weight of the active MetCaCb complex by SEC coupled with MALS. The red line represents the experimentally determined molecular weight. d Guanylurea production at various temperatures and pH 9.0. e Guanylurea production from metformin at various pH values. f Guanylurea production at various metformin concentrations was determined at pH 9.0 and pH 10.0. g Guanylurea production at various 1-methylbiguanide concentrations was determined at pH 9.0. All assays were performed in triplicate of the same preparation, and independent enzyme preparations exhibited consistent results. Data are presented as mean values ± SD. Source data are provided as a Source Data file.