New insights into enzymatic hydrolysis of heterogeneous cellulose by using carbohydrate-binding module 3 containing GFP and carbohydrate-binding module 17 containing CFP

Abstract

BackgroundThe in-depth understanding of the enzymatic hydrolysis of cellulose with heterogeneous morphology (that is, crystalline versus amorphous) may help develop better cellulase cocktail mixtures and biomass pretreatment, wherein cost-effective release of soluble sugars from solid cellulosic materials remains the largest obstacle to the economic viability of second generation biorefineries.ResultsIn addition to the previously developed non-hydrolytic fusion protein, GC3, containing a green fluorescent protein (GFP) and a family 3 carbohydrate-binding module (CBM3) that can bind both surfaces of amorphous and crystalline celluloses, we developed a new protein probe, CC17, which contained a mono-cherry fluorescent protein (CFP) and a family 17 carbohydrate-binding module (CBM17) that can bind only amorphous cellulose surfaces. Via these two probes, the surface accessibilities of amorphous and crystalline celluloses were determined quantitatively. Our results for the enzymatic hydrolysis of microcrystalline cellulose (Avicel) suggested that: 1) easily accessible amorphous cellulose on the surface of Avicel is preferentially hydrolyzed at the very early period of hydrolysis (that is, several minutes with a cellulose conversion of 2.8%); 2) further hydrolysis of Avicel is a typical layer-by-layer mechanism, that is, amorphous and crystalline cellulose regions were hydrolyzed simultaneously; and 3) most amorphous cellulose within the interior of the Avicel particles cannot be accessed by cellulase.ConclusionsThe crystallinity index (CrI), reflecting a mass-average (three-dimensional) cellulose characteristic, did not represent the key substrate surface (two-dimensional) characteristic related to enzymatic hydrolysis.

Modification of paper properties using carbohydrate-binding module 3 from the Clostridium thermocellum CipA scaffolding protein produced in Pichia pastoris: elucidation of the glycosylation effect

“…Pinto et al (2006) reported that the distribution of glycosylated fungal CBMs (labeled with FITC) on Whatman CF11 fibers (crystalline cellulose) was not uniform, but concentrated instead around the fiber's extremities, which are characterized by disordered packing of the microfibrils (non-uniformity) that have a higher surface area available for CBM adsorption. Since CBM3 can bind amorphous and crystalline cellulose (Gao et al 2014), although with different affinities (Hong et al 2008), it is able to recognize both cellulose surfaces in E. globulus fibers. Our results may also indicate a higher concentration of adsorbing sites for CBM3 on E. globulus fibers extremities or higher affinity of CBM3 for these places.…”

Functional and structural characterization of a potent GH74 endo‐xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation

“…Indeed, a particular challenge in CBM characterization is the difficulty in obtaining soluble recombinant production in E. coli as a result of improper protein folding [29]. Recombinant CBM-fusion technology has been used extensively for various applications [30], and we were particularly inspired by the specific use of CBM-GFP fusion constructs to facilitate the production and purification of CBMs and CBM-tagged fusion proteins [31][32][33][34][35] and to make probes for the determination of surface accessibility of cellulose [36,37]. Hence, both CBMs were fused to an N-terminal superfolder GFP (sfGFP) domain (Fig.…”

Cellulase biocatalysis: key influencing factors and mode of action

“…Some other reports have claimed that there is no direct effect of CrI on the enzymatic hydrolysis rate (Gao et al 2014;Grethlein 1985). Gao et al (2014) hypothesized that, CrI could represent overall 3D properties of cellulose substrate rather than 2D surface properties, which is strongly related to enzymatic hydrolysis.…”

“…Gao et al (2014) hypothesized that, CrI could represent overall 3D properties of cellulose substrate rather than 2D surface properties, which is strongly related to enzymatic hydrolysis. It was also argued that the CrI varies with calculation approach, measurement techniques, source of substrate and sample drying techniques.…”