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

Bae, H.J.; Turcotte, G.; Kim, Y.S.; Vezina, L.P.; Laberge, S.

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

2004

Bae, H.J. | Turcotte, G. | Kim, Y.S. | Vezina, L.P. | Laberge, S.

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.

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