Fig. 1
Expression of endogenous protein and biochemical characterization of recombinant NmPin. a MALDI-TOF spectrum of recombinant NmPin. b Upper illustration, representative SDS-PAGE gel of N. maritimus cell lysates (Coomassie staining). Cells were treated with different salt concentrations before fractionation. Lower illustration, single Western blot experiment of endogenous NmPin using α-NmPin antibody. The corresponding fractions and salt conditions are annotated below. c Absorption curves revealing the time resolved cleavage of the peptide Suc-A-R-P-F-pNA in the presence of different NmPin concentrations in a protease-coupled isomerase assay. The amount of NmPin added to the reaction is indicated by a color gradient from 0 μM (bright blue) to 2 μM (dark blue). A steeper initial slope represents a faster isomerization. Insert: plot of catalytic constants kcat derived from absorption curves by bi-exponential curve fits against the corresponding NmPin concentrations. d Diagram of kcat/KM values for various substrates comprising the scaffold Suc-A-X-P-F-pNA measured for NmPin (blue, left scale), CsPinA (light blue, left scale) and Par14 (black, right scale) [4]. The residue X is specified on the x-axis of the diagram. Data are presented as means ± standard deviation from six (two for CsPin) independent measurements with different enzyme concentrations