Recognition and Hydrolysis of Noncrystalline Cellulose

Cellulase Cel5A from alkalophilic Bacillus sp. 1139 contains a family 17 carbohydrate-binding module ( Bsp CBM17) and a family 28 CBM ( Bsp CBM28) in tandem. The two modules have significantly similar amino acid sequences, but amino acid residues essential for binding are not conserved. Bsp CBM28 was obtained as a discrete polypeptide by engineering the cel5A gene. Bsp CBM17 could not be obtained as a discrete polypeptide, so a family 17 CBM from endoglucanase Cel5A of Clostridium cellulovorans , Cc CBM17, was used to compare the binding characteristics of the two families of CBM. Both Cc CBM17 and Bsp CBM28 recognized two classes of binding sites on amorphous cellulose: a high affinity site ( K a ∼1 × 10 6 m −1 ) and a low affinity site ( K a ∼2 × 10 4 m −1 ). They did not compete for binding to the high affinity sites, suggesting that they bound at different sites on the cellulose. A polypeptide, Bsp CBM17/CBM28, comprising the tandem CBMs from Cel5A, bound to amorphous cellulose with a significantly higher affinity than the sum of the affinities of Cc CBM17 and Bsp CBM28, indicating cooperativity between the linked CBMs. Cel5A mutants were constructed that were defective in one or both of the CBMs. The mutants differed from the wild-type enzyme in the amounts and sizes of the soluble products produced from amorphous cellulose. This suggests that either the CBMs can modify the action of the catalytic module of Cel5A or that they target the enzyme to areas of the cellulose that differ in susceptibility to hydrolysis.