Characterization and synergistic interactions of Fibrobacter succinogenes glycoside hydrolases

Abstract

The objectives of this study were to characterize Fibrobacter succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by F. succinogenes, Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.

FIG. 1.

Cellulases used in this study and their domain structures. GH, glycoside hydrolase; pentagons, His₆ tags from vectors. BTDs were divided into two groups (I and II) based on their sequence similarity.

FIG. 2.

Effect of chloride on cellobiosidase activity of the native ClCBase (open triangles) and rcClCBase (filled triangles). The enzyme assays were performed with p-nitrophenyl cellobioside as the substrate in 10 mM imidazole buffer (pH 6.5) at various concentrations of chloride ions. The bars represent the standard errors (n = 3). Values for 100% activity of the native ClCBase and rcClCBase were 13.8 and 12.0 U/mg, respectively.

FIG. 3.

Reducing sugar produced (glucose equivalents) from ball-milled cellulose by binary mixtures of Cel9B and Cel51A after 2 h of incubation at 37°C. Purified rcCel9B and rcCel51A were each diluted to equal ball-milled cellulase activities and combined at different ratios, as indicated, in the assay mixture to maintain a constant sum of individual activities. The mixture that showed the highest degree of synergism (Cel9B/Cel51A = 40:60) contained 2.9 pmol of Cel9B and 17.1 pmol of Cel51A in a 100-μl reaction mixture.

FIG. 4.

Reducing sugar produced (glucose equivalents) (A) and degree of synergism (B) during the hydrolysis of ball-milled cellulose (0.5%) by different combinations of cellulases. The compositions of the mixtures (in pmol/100 μl) are indicated in parentheses. The cellulase mixture “all set 1” contains 2 pmol Cel9B, 8 pmol Cel51A, 30 pmol ClCBase, 30pmol Cel8B, and 10 pmol Cel5H (per 100 μl). “All set 2” contains 1 pmol Cel9B, 4 pmol Cel51A, 15 pmol ClCBase, 15 pmol Cel8B, and 5 pmol Cel5H (per 100 μl).