Fig. 3.

Cellulosome complex assembly demonstration. (A) Flow diagram of cellulosome complex formation. (B) The SEC chromatogram represents the CipA:DocT complex colored in cyan and the corresponding complex verified on 4 to 15% SDS–PAGE. (C) The chromatogram in pink denotes the unbound OlpB and DocT complex. (D) Native PAGE analysis revealed the complex formation of CipA:OlpB. Each protein band was identified using mass spectrometry. (E–I) Enzymatic assays of purified cellulosome complex contain CipA and five types of enzymes. The CipA alone and cellulosome complex were purified using cellulose. All enzyme assays were performed at 40 °C at 1,200 rpm. (E) EG activity assay using dye CMC as substrate. (F) CBH activity assay using PASC as substrate. (G) β-Glucosidase assay using pNPG as substrate. (H and I) The H₂O₂ release was measured using the Amplex Red/HRP assay kit and excitation/emission was measured at 530/590 nm using a fluorescence spectrophotometer. The assay was performed using cellobiose (H), and ascorbate (I) as an electron donor.