Binding of cellulose binding modules reveal differences between cellulose substrates

Abstract

The interaction between cellulase enzymes and their substrates is of central importance to several technological and scientific challenges. Here we report that the binding of cellulose binding modules (CBM) from Trichoderma reesei cellulases Cel6A and Cel7A show a major difference in how they interact with substrates originating from wood compared to bacterial cellulose. We found that the CBM from TrCel7A recognizes the two substrates differently and as a consequence shows an unexpected way of binding. We show that the substrate has a large impact on the exchange rate of the studied CBM, and moreover, CBM-TrCel7A seems to have an additional mode of binding on wood derived cellulose but not on cellulose originating from bacterial source. This mode is not seen in double CBM (DCBM) constructs comprising both CBM-TrCel7A and CBM-TrCel6A. The linker length of DCBMs affects the binding properties, and slows down the exchange rates of the proteins and thus, can be used to analyze the differences between the single CBM. These results have impact on the cellulase research and offer new understanding on how these industrially relevant enzymes act.

Figure 1. The initial slopes of the binding isotherms for CBM-Cel7A, CBM-Cel6A, DCBM-12, DCBM-24, and DCBM-48 on CNF and BMCC.

(A) CBM-Cel7A and CBM-Cel6A, DCBM-12, DCBM-24, and DCBM-48 on BMCC, (B) CBM-Cel7A and CBM-Cel6A, DCBM-12, DCBM-24, and DCBM-48 on CNF. Violet diamond: CBM-CeI7A, orange triangle: CBM-Cel6A, red circle: DCBM-12, green triangle: DCBM-24, and blue square: DCBM-48.

Figure 2. Binding isotherms for CBM-Cel7A, CBM-Cel6A, DCBM-12, DCBM-24, and DCBM-48 with high protein concentrations on CNF and BMCC.

(A) CBM-Cel7A and CBM-Cel6A on CNF, (B) DCBM-12, −24, −48 on CNF, (C) CBM-Cel7A and CBM-Cel6A on BMCC, and (D) DCBM-12, −24, −48 on BMCC. Violet diamond: CBM-CeI7A, orange triangle: CBM-Cel6A, red circle: DCBM-12, green triangle: DCBM-24, and blue square: DCBM-48.

Figure 3. The exchange of ³H-labelled proteins from substrate surface with the non-labelled protein in the solution during time.

(A) Exchange of ³H-labelled CBM-Cel7A and CBM-Cel6A from BMCC surface, (B) Exchange of ³H-labelled DCBM-12, DCBM-24, and DCBM-48 from BMCC surface, (C) Exchange of ³H-labelled CBM-Cel7A and CBM-Cel6A from CNF surface, and (D) Exchange of ³H-labelled DCBM-12, DCBM-24, and DCBM-48 from CNF surface. Violet diamond: CBM-CeI7A, orange triangle: CBM-Cel6A, red circle: DCBM-12, green triangle: DCBM-24, and blue square: DCBM-48.