Family 17 and 28 Carbohydrate-Binding Modules Discriminated Different Cell-Wall Sites in Sweet Potato Roots

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.

Section: Resultsmentioning

confidence: 99%

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

Gourlay

Arantes

Saddler

2012

“…It is important to note that the dried cellulosic samples have a completely different supramolecular structure and substrate reactivity from hydrated cellulosic samples [7,10]; additionally, many experiments use cellulose-surface probing molecules (for example, dinitrogen, D 2 O) that are small relative to the enzymes and a true probe should have similar size to cellulases. Several fusion proteins containing a fluorescent protein and a carbohydrate-binding module (CBM) have been used to qualitatively visualize the polysaccharide recognition on cellulose [11,12] but not quantitatively. Specifically, a quantitative measurement for determining cellulose accessibility to cellulase (CAC) was established by using a non-hydrolytic fusion protein containing a green fluorescent protein (GFP) and CBM3, which was cloned from the cipA gene in Clostridium thermocellum , called GC3 [8].…”

Section: Introductionmentioning

confidence: 99%

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

Gao

You

Renneckar

et al. 2014

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.

“…Type B CBMs are reported to bind both cello-oligomers and non-crystalline/amorphous cellulose, covering a broad range of polymeric structural diversity and suggesting recognition processes may involve interactions beyond the primary binding site. Interestingly, CBMs from families 17 and 28 appear to bind non-crystalline cellulose with high and low binding affinities, as determined from isothermal titration calorimetry (ITC) data, and the two families do not compete with each other for carbohydrate binding sites [ 13 , 33 ]. We further explore both the concept of bi-directional binding and the high/low binding affinity phenomena of family 17 and 28 CBMs on non-crystalline cellulose by modeling representative Type B CBMs bound with a model non-crystalline substrate in multiple orientations.…”

Section: Resultsmentioning

confidence: 99%

Cellulose-specific Type B carbohydrate binding modules: understanding oligomeric and non-crystalline substrate recognition mechanisms

Kognole

Payne

2018

BackgroundEffective enzymatic degradation of crystalline polysaccharides requires a synergistic cocktail of hydrolytic enzymes tailored to the wide-ranging degree of substrate crystallinity. To accomplish this type of targeted carbohydrate recognition, nature produces multi-modular enzymes, having at least one catalytic domain appended to one or more carbohydrate binding modules (CBMs). The Type B CBM categorization encompasses several families (i.e., protein folds) of CBMs that are generally thought to selectively bind oligomeric polysaccharides; however, a subset of cellulose-specific CBM families (17 and 28) appear to bind non-crystalline cellulose more tightly than oligomers and in a manner that discriminates between surface topology.ResultsTo provide insight into this unexplained phenomenon, we investigated the molecular-level origins of oligomeric and non-crystalline carbohydrate recognition in cellulose-specific Type B CBMs using molecular dynamics (MD) simulation and free energy calculations. Examining two CBMs from three different families (4, 17, and 28), we describe how protein–ligand dynamics contribute to observed variations in binding affinity of oligomers within the same CBM family. Comparisons across the three CBM families identified factors leading to modified functionality prohibiting competitive binding, despite similarity in sequence and specificity. Using free energy perturbation with Hamiltonian replica exchange MD, we also examined the hypothesis that the open topology of the binding grooves in families 17 and 28 necessitates tight binding of an oligomer, while the more confined family 4 binding groove does not require the same degree of tight binding. Finally, we elucidated the mechanisms of non-crystalline carbohydrate recognition by modeling CBMs complexed with a partially decrystallized cellulose substrate. Molecular simulation provided structural and dynamic data for direct comparison to oligomeric modes of carbohydrate recognition, and umbrella sampling MD was used to determine ligand binding free energy. Comparing both protein–carbohydrate interactions and ligand binding free energies, which were in good agreement with experimental values, we confirmed the hypothesis that family 17 and 28 CBMs bind non-crystalline cellulose and oligomers with different affinities (i.e., high and low).ConclusionsOur study provides an unprecedented level of insight into the complex solid and soluble carbohydrate substrate recognition mechanisms of Type B CBMs, the findings of which hold considerable promise for enhancing lignocellulosic biomass conversion technology and development of plant cell wall probes.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1321-7) contains supplementary material, which is available to authorized users.