title: 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.
abstract: 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.
authors.affiliation: Département de Phytologie, Université Laval, Sainte-Foy, Que., Canada G1K 7P4. baehyeunjong@hotmail.com
authors.forename: Hyeun-Jong
authors.initials: HJ
authors.lastname: Bae
authors.forename: Ginette
authors.initials: G
authors.lastname: Turcotte
authors.forename: Yoon
authors.initials: Y
authors.lastname: Soo Kim
authors.forename: Louis-Philippe
authors.initials: LP
authors.lastname: Vézina
authors.forename: Serge
authors.initials: S
authors.lastname: Laberge
mesh_terms: D001665:Binding Sites; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D017979:Gold Colloid; D006868:Hydrolysis; D000455:Medicago sativa / Q000382:microbiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D045857:Ruminococcus / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D017979:Gold Colloid; D002482:Cellulose; C407024:endoglucanase Cel5A; D002480:Cellulase