Fig. 1

Purification and analyses of rCel48S_CD proteins. a Purification of the rCel48S_CD protein by gel filtration. After Ni²⁺-affinity purification, denatured rCel48S_CD proteins were dialyzed against 10 mM Tris–HCl buffer (pH 8.0) containing 100 mM NaCl for 4 h at 25 °C with two buffer changes, and were further purified using a Superdex 200 column (GE healthcare). Two peaks were detected with retention volumes of approximately 50 and 80 mL (fraction-50 and fraction-80, respectively). The relative abundances of fraction-50 or fraction-80 were determined by calculating the proportion of the areas of corresponding peaks in the total area, i.e., the relative abundance of fraction-50 is Area_fraction-50/(Area_fraction-50 + Area_fraction-80). The activities of each fraction are shown below the chromatogram. b SDS-PAGE analysis. Lane 1, rCel48S_CD protein after Ni²⁺-affinity purification; lane 2, fraction-50; lane 3, fraction-80. M, protein standards. c CD analyses. CD spectra are given as the standard mean residue ellipticity units (MREs). d TIF analyses. The emission spectra from 314 to 490 nm were detected after excitation at 295 nm. The absorption spectrum of fraction-50 is red-shifted slightly and has reduced slope with that of fraction-80, indicating difference in the secondary structures [55]