Role Of Carbohydrate-binding Modules Research Articles

Role of carbohydrate binding modules, CBM3A and CBM3B in stability and catalysis by a β-1,4 endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC 27405

A recombinant β-1,4 endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC27405 was explored for biochemical properties and the role of its associated CBMs in catalysis. The gene expressing full-length multi-modular β-1,4-endoglucanase (AtGH9C-CBM3A-CBM3B) and its truncated derivatives (AtGH9C-CBM3A, AtGH9C, CBM3A and CBM3B) were independently cloned and expressed in Escherichia coli BL21(DE3) cells and purified. AtGH9C-CBM3A-CBM3B showed maximal activity at 55 °C and pH 7.5. AtGH9C-CBM3A-CBM3B exhibited highest activity against carboxy methyl cellulose (58.8 U/mg) followed by lichenan (44.5 U/mg), β-glucan (36.2 U/mg) and hydroxy ethyl cellulose (17.9 U/mg). Catalytic module, AtGH9C showed insignificant activity against the substrates, signifying the essential requirement of CBMs in catalysis. AtGH9C-CBM3A-CBM3B displayed stability in pH range, 6.0–9.0 and thermostability up to 60 °C for 90 min with unfolding transition midpoint (Tm) of 65 °C. The generation of cellotetraose and other higher oligosaccharides by AtGH9C-CBM3A-CBM3B confirmed it as an endo-β-1,4-glucanase. AtGH9C activity was partially recovered by the addition of equimolar concentration of CBM3A, CBM3B or CBM3A + CBM3B by 47 %, 13 % or 50 %, respectively. Moreover, the associated CBMs imparted thermostability to the catalytic module, AtGH9C. These results showed that the physical association of AtGH9C with its associated CBMs and the cross-talk between CBMs are necessary for AtGH9C-CBM3A-CBM3B in effective cellulose catalysis.

Reduced type-A carbohydrate-binding module interactions to cellulose I leads to improved endocellulase activity.

Dissociation of nonproductively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective biomass conversion to fermentable sugars. However, the role of carbohydrate-binding modules (CBMs) in enabling nonproductive enzyme binding is not well understood. Here, we examine the subtle interplay of CBM binding and cellulose hydrolysis activity for three models type-A CBMs (Families 1, 3a, and 64) tethered to multifunctional endoglucanase (CelE) on two distinct cellulose allomorphs (i.e., cellulose I and III). We generated a smalllibrary of mutant CBMs with varying cellulose affinity, as determined by equilibrium binding assays, followed by monitoring cellulose hydrolysis activity of CelE-CBM fusion constructs. Finally, kinetic binding assays using quartz crystal microbalance with dissipationwere employed to measure CBM adsorption and desorption rate constants and , respectively, towards nanocrystalline cellulose derived from both allomorphs. Overall, our results indicate that reduced CBM equilibrium binding affinity towards cellulose I alone, resulting from increased desorption rates ( ) and reduced effective adsorption rates ( ), is correlated to overall improved endocellulase activity. Future studies could employ similar approaches to unravel the role of CBMs in nonproductive enzyme binding and develop improved cellulolytic enzymes for industrial applications.

Elucidation of a Unique Pattern and the Role of Carbohydrate Binding Module of an Alginate Lyase.

Alginate oligosaccharides with different degrees of polymerization (DPs) possess diverse physiological activities. Therefore, in recent years, increasing attention has been drawn to the use of enzymes for the preparation of alginate oligosaccharides for food and industrial applications. Previously, we identified and characterized a novel bifunctional alginate lyase Aly7A, which can specifically release trisaccharide from three different substrate types with a unique degradation pattern. Herein, we investigated its degradation pattern by modular truncation and molecular docking. The results suggested that Aly7A adopted a unique action mode towards different substrates with the substrate chain sliding into the binding pocket of the catalytic domain to position the next trisaccharide for cleavage. Deletion of the Aly7A carbohydrate binding module (CBM) domain resulted in a complex distribution of degradation products and no preference for trisaccharide formation, indicating that the CBM may act as a “controller” during the trisaccharide release process. This study further testifies CBM as a regulator of product distribution and provides new insights into well-defined generation of alginate oligosaccharides with associated CBMs.

Carbohydrate binding modules enhance cellulose enzymatic hydrolysis by increasing access of cellulases to the substrate

Plant biomass is a low-cost and abundant source of carbohydrates for production of fuels, “green” chemicals and materials. Currently, biochemical conversion of the biomass into sugars via enzymatic hydrolysis is the most viable technology. Here, the role of carbohydrate binding modules (CBMs) in the disruption of insoluble polysaccharide structures and their capacity to enhance cellulase-promoted lignocellulosic biomass hydrolysis was investigated. We show that CBM addition promotes generation of additional reducing ends in the insoluble substrate by cellulases. On the contrary, bovine serum albumin (BSA), widely used in prevention of a non-specific protein binding, causes an increase in soluble reducing-end production, when applied jointly with cellulases. We demonstrate that binding of CBMs to cellulose is non-homogeneous, irreversible and leads to its amorphisation. Our results also reveal effects of CBM-promoted amorphogenesis on cellulose hydrolysis by cellulases.

Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 103 -104 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc.

Cellulase–lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme–lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase–lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM–cellulose interaction were also shown to contribute to lignin-binding.

The role of carbohydrate binding module (CBM) at high substrate consistency: Comparison of Trichoderma reesei and Thermoascus aurantiacus Cel7A (CBHI) and Cel5A (EGII)

The role of CBM in two fungal model cellulase systems, consisting of Cel7A and Cel5A, from Trichoderma reesei and Thermoascus aurantiacus, were compared in the hydrolysis of various substrates. For comparison, family-1 CBM's were introduced to the T. aurantiacus and removed from the T. reesei enzymes. Especially at high dry matter consistencies of lignocellulosic substrates, pretreated wheat straw and spruce, the T. aurantiacus enzymes lacking CBM outperformed the enzymes carrying the CBM. In these conditions, the CBM-less enzymes from both organisms obviously recognized and bound to the substrate at higher probability than in dilute systems. Avoiding the unproductive binding to lignin caused by the CBMs obviously enhanced the hydrolytic performance. The lignin binding effect was, however, not entirely caused by the CBM, but also by the different structures and affinities of the core enzymes to lignin. Due to decreased binding, the CBM-less enzymes would allow reuse, potentially decreasing hydrolysis costs.

The thermostable β-1,3-1,4-glucanase from Clostridium thermocellum improves the nutritive value of highly viscous barley-based diets for broilers

1. Microbial β-1,3-1,4-glucanases improve the nutritive value of barley-based diets for poultry by effectively decreasing the degree of polymerisation of the anti-nutritive soluble β-glucans. Glycoside hydrolases (GHs) acting on recalcitrant polysaccharides display a modular architecture comprising a catalytic domain linked to one or more non-catalytic Carbohydrate-Binding Modules (CBMs). 2. GHs and CBMs have been classified in different families based on primary structure similarity (see CAZy webpage at http://www.cazy.org). The role of CBMs is to anchor the appended GHs into their target substrates, therefore eliciting the efficient hydrolysis of structural carbohydrates. 3. Here we describe the biochemical properties of the family 16 GH from Clostridium thermocellum, termed CtGlc16A. CtGlc16A is a thermostable enzyme that specifically acts on β-1,3-1,4-glucans with a remarkable catalytic activity (38000 U/mg protein). 4. CtGlc16A, individually or fused to the family 11 β-glucan-binding domain of cellulase CtLic26A-Cel5E of C. thermocellum, was used to supplement a highly viscous barley-based diet for broilers. 5. The data showed that birds fed on diets supplemented with the recombinant enzymes displayed an improved performance when compared with birds given diets not supplemented with exogenous enzymes. However, inclusion of the non-catalytic CBMs had no influence on the capacity of CtGlc16A to reduce the anti-nutritive effects of soluble β-1,3-1,4-glucans. 6. The data suggest that at elevated dosage rates, CBMs might be unable to potentiate the catalytic activity of appended catalytic domains; this effect may only be revealed when feed enzymes are incorporated at low levels.

The role of carbohydrate-binding module (CBM) repeat of a multimodular xylanase (XynX) from Clostridium thermocellum in cellulose and xylan binding

A non-cellulosomal xylanase from Clostridium thermocellum, XynX, consists of a family-22 carbohydratebinding module (CBM22), a family-10 glycoside hydrolase (GH10) catalytic module, two family-9 carbohydrate-binding modules (CBM9-I and CBM9-II), and an S-layer homology (SLH) module. E. coli BL21(DE3) (pKM29), a transformant carrying xynX', produced several truncated forms of the enzyme. Among them, three major active species were purified by SDS-PAGE, activity staining, gel-slicing, and diffusion from the gel. The truncated xylanases were different from each other only in their C-terminal regions. In addition to the CBM22 and GH10 catalytic modules, XynX(1) had the CBM9-I and most of the CBM9-II, XynX(2) had the CBM9-I and about 40% of the CBM9-II, and XynX(3) had about 75% of the CBM9-I. The truncated xylanases showed higher binding capacities toward Avicel than those toward insoluble xylan. XynX(1) showed a higher affinity toward Avicel (70.5%) than XynX(2) (46.0%) and XynX(3) (42.1%); however, there were no significant differences in the affinities toward insoluble xylan. It is suggested that the CBM9 repeat, especially CBM9-II, of XynX plays a role in xylan degradation in nature by strengthening cellulose binding rather than by enhancing xylan binding.

Family 6 Carbohydrate Binding Modules in β-Agarases Display Exquisite Selectivity for the Non-reducing Termini of Agarose Chains

Carbohydrate recognition is central to the biological and industrial exploitation of plant structural polysaccharides. These insoluble polymers are recalcitrant to microbial degradation, and enzymes that catalyze this process generally contain non-catalytic carbohydrate binding modules (CBMs) that potentiate activity by increasing substrate binding. Agarose, a repeat of the disaccharide 3,6-anhydro-alpha-L-galactose-(1,3)-beta-D-galactopyranose-(1,4), is the dominant matrix polysaccharide in marine algae, yet the role of CBMs in the hydrolysis of this important polymer has not previously been explored. Here we show that family 6 CBMs, present in two different beta-agarases, bind specifically to the non-reducing end of agarose chains, recognizing only the first repeat of the disaccharide. The crystal structure of one of these modules Aga16B-CBM6-2, in complex with neoagarohexaose, reveals the mechanism by which the protein displays exquisite specificity, targeting the equatorial O4 and the axial O3 of the anhydro-L-galactose. Targeting of the CBM6 to the non-reducing end of agarose chains may direct the appended catalytic modules to areas of the plant cell wall attacked by beta-agarases where the matrix polysaccharide is likely to be more amenable to further enzymic hydrolysis.