Evidence that linker sequences and cellulose-binding domains enhance the activity of hemicellulases against complex substrates

Abstract

Xylanase A (XYLA) and arabinofuranosidase C (XYLC) from Pseudomonas fluorescens subsp. cellulosa are modular enzymes consisting of discrete cellulose-binding domains (CBDs) and catalytic domains joined by serine-rich linker sequences. To evaluate the role of the CBDs and interdomain regions, the capacity of full-length and truncated derivatives of the two enzymes, lacking either the linker sequences or CBDs, to hydrolyse a range of substrates, and bind to cellulose, was determined. Removal of the CBDs did not affect either the activity of XYLA or XYLC against soluble arabinoxylan. Similarly, deletion of the linker sequences did not alter the affinity of the enzymes for cellulose or their activity against soluble substrates, even when bound to cellulose via the CBDs. Truncated derivatives of XYLA lacking either the linker sequences or the CBD were less active against xylan contained in cellulose-hemicellulose complexes, compared with the full-length xylanase. Similarly, removal of the CBD from XYLC diminished the activity of the enzyme (XYLC''') against plant-cell-wall material containing highly substituted arabinoxylan. The role of CBDs and linker sequences in the catalytic activity of hemicellulases against the plant cell wall is discussed.

Substrate Recognition of a Family 10 Xylanase from Streptomyces olivaceoviridis E-86: A Study by Site-directed Mutagenesis to Make an Hindrance around the Entrance toward the Substrate-binding Cleft

“…It was demonstrated that the truncated derivatives of a xylanase lacking either the linker sequences or the cellulose binding module were less active against xylan contained in cellulose hemicellulose complexes, compared with the full length xylanase. 32) The data suggested the cellulose binding module restricts substrate availability by anchoring the enzyme to a fixed location within the macromolecule in the hydrolysis of integral xylan when the linker sequence has been truncated.…”

Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase: cloning, expression and binding studies

“…Hemicellulase catalytic modules are also connected to CBMs [16][17][18], and it has been shown that the hemicellulase activity on complex substrates (such as cellulose\hemicellulose complexes) is enhanced by the presence of a CBM [4,19]. The role of the binding modules with affinity for cellulose could be to participate Abbreviations used : AE, affinity electrophoresis ; C 6 , cellohexaose ; CBM, carbohydrate-binding module ; CenC, Cellulomonas fimi endoglucanase C ; CMC, carboxymethylcellulose ; PASC, phosphoric-acid-swollen cellulose ; X 6 , xylohexaose.…”

“…in the dissociation of cell-wall matrix polysaccharides such as xylans that are in close contact with microfibrils, or to mediate an intimate contact between the enzyme and the plant cell wall [4,19]. In most cases CBMs have been observed to bind to cellulose in both the crystalline and amorphous forms.…”

“…Typically these enzymes are composed of catalytic modules joined to one or more auxiliary domains by recognizable linker sequences [2,4]. The most common auxiliary domains in hemicellulases (such as xylanases, mannanases, xylosidases and acetylesterases) and cellulases are the carbohydrate-binding modules (CBMs) [3,5], most of which bind preferentially to cellulose.…”

“…Many of the modules in this classification system are, however, not functionally characterized, and their precise roles in hemicellulase action are not yet fully understood [4,[6][7][8].…”