2.4 Enzyme purification
The culture supernatant was filtered with 0.45 μm filters before being
loaded on the column. Purification of the rAGL was performed with the
AKTA Prime Plus (GEHealthcare Bio-science, Uppsala, Sweden) using a 5-mL
high affinity Ni-Charged resin FF prepacked column (Genscript, Nanjing,
China) [44]. After exchanging with sodium acetate
buffer (100 mM, pH 4.0) and concentrated using an
Amicon® Ultra-15 Centrifugal Filter Unit with an
Ultracel-30 membrane (Merck Millipore Ltd., Ireland), the elute solution
containing AGL was collected and stored at 4 °C for further experiments.
The molecular weight of rAGL and protein purity were determined by 8%
(w/v) SDS-PAGE. The protein concentration was determined using the
Bradford method [45].