Identification of Two Functionally Different Classes of Exocellulases

Barr, Brian K.; Hsieh, Y. L.; Ganem, Bruce; Wilson, David B.

doi:10.1021/bi9520388

Cited by 251 publications

(189 citation statements)

References 28 publications

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“…As expected, no hydrolysis of pNP-cellobioside or pNP-cellotrioside could be detected (Fig. 3), supporting the hypothesis that cellobiose cannot be released from the nonreducing ends of these substrates [9,10]. Such an exo-processive mode of action suggests that Cel48C hydrolyses polysaccharides and cellodextrins from the reducing end of the sugar chain [9,10].…”

Section: Mode Of Action Of Cel48csupporting

confidence: 82%

Exo‐mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP‐23

Sánchez

Pastor

Dı́az

2003

European Journal of Biochemistry

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Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose-binding module, and a putative fibronectin-III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45°C and pH 6.0 on acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, pNPglycosides or 4-methylumbeliferyl a-D-glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP-23 would contribute to widen up its range of action on natural cellulosic substrates.

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Section: Mode Of Action Of Cel48csupporting

confidence: 82%

Exo‐mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP‐23

Sánchez

Pastor

Dı́az

2003

European Journal of Biochemistry

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“…Finally, both enzymes examined in this study have been characterized as cellobiohydrolases (44), which historically has implied that they are able to processively decrystallize and hydrolyze a cellulose chain. However, recent high-speed atomic force microscopy (HS-AFM) measurements indicate that whereas TrCel7A performs the expected processive function (36,45), TrCel6A is not observed to processively translate down the microfibril.…”

Section: Discussionmentioning

confidence: 99%

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Payne

Resch

Chen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

158

147

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Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls. biofuels | cellulase | post-translational modification | carbohydrate recognition

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“…Cellobiohydrolases (also called 1,4-b-D-glucan cellobiohydrolases; exoglucanases; CBH; EC 3.2.1.91) are thought to be processive enzymes and initiate activity from the end of the cellulose chains. T. reesei produces CBH I (Cel7A) and CBH II (Cel6A), which act specifically on the reducing and non-reducing chain ends, respectively (Barr et al, 1996). T. reesei Cel7A comprises nearly 60% of the total protein secreted by the fungus (Nummi et al, 1983).…”

Section: Introductionmentioning

confidence: 99%

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

Jeoh¹,

Ishizawa²,

Davis³

et al. 2007

Biotech & Bioengineering

459

318

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Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.

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Identification of Two Functionally Different Classes of Exocellulases

Cited by 251 publications

References 28 publications

Exo‐mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP‐23

Exo‐mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP‐23

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

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