Cel9D, an Atypical 1,4-β- d -Glucan Glucohydrolase from Fibrobacter succinogenes : Characteristics, Catalytic Residues, and Synergistic Interactions with Other Cellulases

Abstract

The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5-to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to ␤-134-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased k cat /K m values for cello-oligosaccharides with higher degrees of polymerization. The k cat /K m value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-␤-D-glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.Plant material is now viewed as the bioenergy feedstock of the future. Cellulose, which accounts for the bulk of plant materials, is recalcitrant, which necessitates a combination of the classes of cellulases for biodegradation. These include endoglucanases (EC 3.2.1.4) that cut randomly at internal amorphous sites in the cellulose chain; exoglucanases (EC 3.2.1.91 and EC 3.2.1.74) that act processively on the reducing or nonreducing ends of cellulose chains, releasing either cellobiose or glucose as major products; and ␤-glucosidases (EC 3.2.1.21) that hydrolyze soluble cellodextrins and cellobiose to glucose (18).Fibrobacter succinogenes is a highly cellulolytic bacterium that is closely related to the Cytophaga-Flavobacterium-Bacteroides group (8). It is commonly found in the rumen of ruminant animals and appears to be one of the most active rumen cellulose degraders (4). Recently, the genome was sequenced (21) and a number of cellulases (24) and cellulose binding proteins (12) were identified. We reported synergistic interactions among five cellulases of this organism (24). Although these enzymes had degrees of synergism (DoS) of up to 3.7 and the combination of Cel9B, Ce51A, and Cel8B gave the highest activity, the extent of hydrolysis of cellulose was ...

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

“…Located between the two modules are three carbohydrate-binding modules (CBMs). GH9 family enzymes have an (␣/␣) 6 -barrel fold, and this family of enzymes is known to contain members that exhibit different enzymatic activities, including endoglucanase (45), cellobiohydrolase (15), 1,4-␤-D-glucan glucohydrolase (43), ␤-glucosidase (40), and exo-␤-glucosaminidase (23). The GH5 family of enzymes have an (␤/␣) 8 -barrel fold, and this family also encodes diverse enzymatic activities, including endoglucanase (16), mannanase (20), mannosidase (20), xylanase (14), chitosanase (50), glucosidase (11), licheninase (25), fucosidase (61), galactanase (44), endoglycoceramidase (9), and mannan transglycosylase (6).…”

Biochemical and Domain Analyses of FSUAxe6B, a Modular Acetyl Xylan Esterase, Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85

“…Since the initial report of a C-terminal basic domain (BTD), specific to enzymes in F. succinogenes (32), many BTDs in this strain have been reported (22,37,38). To date, the role of the BTDs remain unknown.…”

A 3-D Model of a Perennial Ryegrass Primary Cell Wall and Its Enzymatic Degradation

“…Our simulations involving three characterised enzymes from F. succinogenes S85 showed synergies between the endoglucanase Cel51A and the exocellulase Cel9D, in terms of increased activity on glucosidic bonds. Qi et al [69] found Cel51A and Cel9D showed in vitro synergies when degrading crystalline cellulose, compared with Cel51A alone. Our simulation result cannot be compared quantitatively with this result since the substrate being degraded differed.…”

“…The first two enzymes were Cel51A and Cel9D, which have been characterised and shown to act synergistically [69,70]. Cel51A is an endoglucanase that acts on cellulose, but also (1,3;1,4)-β-glucans and xyloglucans mainly producing oligosaccharides with a DP of 2-5.…”