Fig 1.
Multiple sequence alignment of heparinases I from F. heparinum (FhHep-I), B. cellulosilyticus (BcHep-I), B. thetaiotaomicron (BtHep-I: PDB ID: 3ikw), B. eggerthii, and B. stercoris (BsHep-I).
The residues of BcHep-I and the nonconserved residues of other Hep-Is were shown and it was generated with JalView (2.11.1.2).
Fig 2.
Effect of induction temperature on the activity (A) and the expression (B) (SDS-PAGE image of the BcHep-I at different temperatures.
M: protein molecular weight marker, lane 1~6: supernatant after the sonication of the IPTG induced E. coli at 15, 20, 25, 30, 35, and 37°C; lane 7~12: precipitate after the sonicated of the IPTG induced E. coli at 15, 20, 25, 30, 35, and 37°C; BcHep-I bands were marked with solid red wireframe) of BcHep-I. Effect of the induction time on the activity of BcHep-I (C). SDS-PAGE of the purified Hep-I (D) (M: protein molecular weight marker, lane 1: the purified BcHep-I).
Fig 3.
Effects of pH (A), temperature (B), metal ions and EDTA (C), and (D) substrate concentration (D) on the activity of the Hep-I.
The enzyme activity was measured according to the enzyme assay and every experiment was repeated three times.
Table 1.
Comparison of the activity, half-life and kinetic parameters of heparinases I from different organisms.
Fig 4.
The thermostability of BcHep-I at 30 and 37°C (A) and the storage stability at 4°C (B).
The enzyme activity was determined according to the enzyme assay and every experiment was repeated three times.
Fig 5.
Homology modeling of BcHep-I based on the crystal structure of 3ikw and molecular docking with heparin.
(A: the homology model of BcHep-I; B: Ramachandran plot of BcHep-I, C: The superimposition of the template (BtHep-I, 3ikw, red) and BcHep-I (blue), D: molecular docking of BcHep-I with heparin). The images were produced with PyMOL (2.5) and AutoDockTools (1.5.7).