Cel6B of Thermobifidus fusca and a Cel5‐CBM6 of Ruminococcus albus containing a cellulose binding site show synergistic effect on hydrolysis of native plant cellulose

Hydrolysis of cellulose requires two different types of cellulases: exo‐ and endocellulase. Here, we investigated for the hydrolysis of cellulose by two types of cellulases, an endoglucanase (Cel5) from Ruminococcus albus fused with the xylanase A cellulose binding domain II (CBM6) of Clostridium stercorarium and Thermobifidus fusca E3, an exoglucanase (Cel6B). Cel5‐CBM6 or Cel6B showed a linear relationship between the production of soluble sugars and the incubation time when native alfalfa cellulose was used as a substrate. Cel5‐CBM6 produces more soluble sugars than Cel6B and the hydrolysis of cellulose by a mixture of the two enzymes produces substantially more (22%) soluble sugars than the total amount produced by these enzymes individually. Although Cel5‐CBM6 solubilized high quantities of sugars from alfalfa cellulose, it did not significantly decrease its crystallinity, while Cel6B decreased the crystallinity of cellulose by 34%. When the two cellulases were combined, a decrease of more than 50% in the content of crystalline cellulose was observed. The enzyme–gold labeling experiments revealed that both enzymes showed a high affinity for all substrates. Furthermore, simultaneous visualization of the enzyme‐binding sites revealed the preferred substrates in native lignocellulosic material. When plant cellulose was pre‐incubated with Cel5‐CBM6, density of the gold labeling greatly increased suggesting that preliminary exposure of lignocellulosic material to Cel5‐CBM6 may have enhanced the accessibility of the substrate to Cel5‐CBM6 and Cel6B. This result provides a plausible explanation for the observed endo/exo cellulase synergism during hydrolysis.