3.3 Purification of rAGL and kinetic analysis
N -terminal his-tagged rAGL in culture supernatant was purified by Ni-NTA affinity chromatography. The details of purification are shown in Table 1. At 37 °C and pH 4.0, the specific activity of rAGL is 49.83 U/ mg. In SDS-PAGE, rAGL with an N -terminal 6-histidine showed a band of roughly 100 kDa (Fig. 3).
The kinetic parameters of rAGL were analyzed using maltose as the substrate at 37 °C and pH 4.0 (Supplementary Figure 2). As a result, the kinetic constants Km , Vmaxand Kcat of the rAGL were found to be 61 mM, 184.64 U/mg, and 146.70 1/s, respectively.
Table 1
Figure 3