FIG 4.

Sequential BC addition in endpoint assays. The initial 20-h BC (1 mg) digestion was followed by the addition of fresh BC (1 mg) or buffer to the reaction mixtures. The reaction mixtures were then incubated for another 20 h, and the amount of soluble sugar measured in the reaction mixtures is presented (normalized such that the value of 100 represents the amount of sugar produced after the first overnight incubation with the enzyme). Both enzymes showed enhanced activity when fresh BC was added. The double amount of soluble sugar produced by TfCel9A after the second addition of BC implies that the enzyme was largely dissociated from the original BC fraction, while in the case of TfCel48A, some enzyme may be deactivated by irreversible unproductive binding to the original substrate.