Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis

Abstract

BackgroundCellulose amorphogenesis, described as the non-hydrolytic “opening up” or disruption of a cellulosic substrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosic biomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play a major role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of this process.ResultsIn this work, techniques for measuring changes in cellulose accessibility are reviewed and a new quantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to cellulose accessibility were successfully quantified using this technique.ConclusionsThe adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick, accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs used for adsorption studies to include those specific for such compounds as xylan or mannan should also allow for the accurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomass matrix.

The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- and Swollenin-induced Deconstruction of Lignocellulosic Substrates

“…CBM Adsorption-CBM adsorption was carried out as described previously (19). Briefly, 250 g of CBM (as determined by absorption at 280 nm) was added to 2.5 mg (dry weight) of pulp in 1 ml of 50 mM potassium phosphate (pH 7.0).…”

“…To determine whether the reduction in fiber length over the course of hydrolysis corresponded with specific changes in the surface morphology of the cellulose, a previously described cellulose substructure-specific CBM technique was used (19). A Type B CBM (CBM44), which contains a cleft-shaped binding site that preferentially binds to amorphous regions of cellulose (30), was used as a probe to determine the amount of accessible amorphous cellulose, whereas a Type A CBM (CBM2a), with a planar, hydrophobic binding face that preferentially binds to crystalline cellulose regions (29), was used as a probe to determine the amount of accessible crystalline cellulose on the dissolving pulp fibers.…”

“…Recent work has shown that Swollenin targets the less-ordered regions of the cellulose, rather than directly disrupting the more crystalline regions of the substrate (8,19), and enhances the solubilization of the hemicellulosic fraction of steam-pretreated corn stover when it synergistically interacted with family 10 and 11 xylanases, promoting a 3-fold increase in xylose released by these hydrolases (8). It appears that, although Swollenin does to some extent enhance the disruption of well structured components of cellulosic substrates, its primary role is facilitating the disruption of the less-ordered regions of pretreated lignocellulosic substrates (8,19).…”

“…Swollenin, in particular, has been shown to effectively swell cotton fibers (9), disperse filter paper squares (12), roughen filter paper fibers (12), reduce the particle size of a number of model cellulosic substrates (12), enhance the accessibility of cotton fibers (19), and enhance hydrolysis (8,20). Recent work has shown that Swollenin targets the less-ordered regions of the cellulose, rather than directly disrupting the more crystalline regions of the substrate (8,19), and enhances the solubilization of the hemicellulosic fraction of steam-pretreated corn stover when it synergistically interacted with family 10 and 11 xylanases, promoting a 3-fold increase in xylose released by these hydrolases (8).…”

A Mini-review on the Applications of Cellulose-Binding Domains in Lignocellulosic Material Utilizations

“…Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibers during amorphogenesis (Gourlay et al 2012). The extents of phosphoric acid-induced and swollenin-induced changes to cellulose accessibility were successfully quantified using this technique.…”