Chapter 1. General Introduction
1. Carbohydrates
1.4 Carbohydrate binding modules
of lignin from semi-bleached kraft pulp was improved when the pulp was treated with α-L-arabinofuranosidase from Bacills stearothermophilus L1 together with xylanase (Dhiman et al., 2000; Numan and Bhosle, 2006). The enzyme acted synergistically with a thermophilic xylanase in the delignification process, releasing 19.2% of lignin (Dhiman et al., 2000; Numan and Bhosle, 2006). The delignification obtained using the combined enzyme treatment is more than the sum of the amounts obtained using the enzymes separately (Gubitz et al., 1997).
1.3.1.3.3 L-Arabinose as antiglycemic agent
L-Arabinose released from arabinose containing polysaccharides by α-L-Araf can be used as food additive because of its sweet taste and low uptake due to its poor absorption by the human body (Numan and Bhosle, 2006). It has been reported that L- arabinose selectively inhibits intestinal sucrase in a competitive manner and thus reduces the glycemic response after sucrose ingestion in animals so α-L-Araf can be used as prebiotics (Saha, 2000; Numan and Bhosle, 2006).
defined as independently folding modules, occurring alongside the carbohydrate- active enzymes. The CBMs are the non-catalytic modules known to help or bring the catalytic modules in close proximity to its substrates and also some CBMs are known to stabilize the structure of catalytic modules and increase its thermal stability (Boraston et al., 2004; Henshaw et al., 2006; Dvortsov et al., 2009). The CBMs may be found to contain up to 200 amino acids and can be found attached as single, double or triple domain in one protein, located at both C- or N-terminal within the parental protein (Shoseyov et al., 2006). Similar to the catalytic modules of glycoside hydrolases, CBMs have been grouped into 64 families according to the data in Carbohydrate Active enZYmes (CaZy) database, based upon amino acid sequence similarity (http://www.cazy.org/Carbohydrate-Binding-Modules.html).
1.4.1 Binding site architecture of carbohydrate binding modules
The sequence-based carbohydrate binding module (CBM) families have been further subdivided into three types based on mechanism of ligand binding and binding site topology (Fig. 1.19). The type A, CBMs bind to insoluble crystalline cellulose or chitin and have flat platform-like binding sites (Boraston et al., 2004;
Abbott et al., 2009; Abbott et al., 2012,). The planar arrangement of aromatic residues of CBMs within the binding site, allows it to rest on the flat hydrophobic surface of a crystalline ligand (Fig. 1.19A). The type B, CBMs have a binding site that exists as an extended groove, within the groove there are multiple sugar sub sites (Fig. 1.19B).
These CBMs bind chains of soluble polysaccharides and both polar and nonpolar interactions drive specificity of the binding. The type C, CBMs, considered lectin- like, have a shallow binding pocket ideal for binding mono-, di- or tri-saccharides (Fig. 1.19C). As with the Type B CBMs both polar and apolar interactions drive
specificity; however, the hydrogen bonding interactions are more dominant in type C CBMs (Boraston et al. 2004).
Aromatic amino acids
Aromatic amino acids Aromatic amino acids
A B C
Type A Type B Type C
Fig. 1.19 Three types of binding site architecture and ligand binding in CBMs.
Aromatic amino acids involved in binding are shown in stick A) Type A, cellulose binding HjCBM1 from Hypocrea jecorina (PDB code 1CBH), B) Type B, CtCBM6 from Clostridium thermocellum in complex with xylopentaose (PDB code 1UXX), C) Type C, SlCBM13 from Streptococcus lividans in complex with xylopentaose (PDB code 1MC9).
1.4.2 Carbohydrate binding module clans based on fold of their 3-dimensional structure
Currently seven families of folds are reported for CBMs in the CAZy database (Cantarel et al. 2008). The seven different folds include the β-sandwich fold, the β-trefoil fold, the oligonucleotide-carbohydrate binding fold (OB), the knottin fold, the hevein fold, the hevein-like fold and a unique fold (http://www.cazy.org/CBM6.html). The most frequent is the β-sandwich fold which is common to plant legume lectins and animal galectins such as CBM6s reported by van Beuren et al. (2005) and Vandermarliere et al. (2009). The second most common fold is the β-trefoil fold reported in CBM6s from Clostridium cellulolyticum and Clostridium thermocellum by Abbott et al. (2009) and Czjzek et al. (2001), respectively.
1.4.3 Functions of carbohydrate binding modules
Carbohydrate binding modules (CBMs) increase the ability of glycoside hydrolases to efficiently degrade the polysaccharide substrates. This occurs through two different roles that CBMs play in polysaccharide breakdown, i) they localize the soluble enzyme to its target substrate (Shallom and Shoham, 2003), ii) they are associated with a catalytic domain and contain multiple clefts to attach with substrate, thereby enhancing the catalytic activity (Henshaw et al., 2004; Hashimoto et al., 2006) iii) they can act as fusion proteins capable of binding to a cellulose matrix and consequently used in a protein purification (Shpigel et al., 1998).
The proximity effect describes the binding of the CBM that brings the catalytic module in close proximity (or association) to the substrate and maintains it for a prolonged period. This effect is seen primarily on insoluble substrates such as cellulose and xylan (Tomme et al. 1988; Boraston et al. 2004). The CBMs bring about the close proximity of catalytic enzyme to its target substrates by two effects as described in sub sections 1.4.3.1 and 1.4.3.2.
1.4.3.1 Targeting effect of carbohydrate binding modules
The targeting effect is observed where CBMs (associated with catalytic module) specifically bind to a substrate thereby bringing the substrate in close proximity of the enzyme (Shallom and Shoham, 2003). For example, there are many cellulose specific CBMs, however, type A cellulose binding CBMs bind crystalline cellulose, whereas, type B cellulose binding CBMs bind non-crystalline components of cellulose. Thus, two different binding mechanisms are driving the recognition of different components of cellulose substructure. This targeting effect would drive
hydrolysis in specific regions of cellulose, rather than just bringing the catalytic modules into proximity as described above (Carrard et al. 2000; Boraston et al. 2004).
1.4.3.2 Disruptive effect of carbohydrate binding modules
The disruptive effectmay arise due to disruption of the polysaccharide fibres due to CBM permeation within the fibres. It was suggested that the CBMs bind and disrupt the crystalline cellulose (Din et al. 1994) or chitin (Vaaje-Kolstad et al. 2005) allowing the release of any non-covalently attached fibres thereby exposing the sites for polysaccharide hydrolysis.
1.4.4 Carbohydrate binding modules and multi-valency
Sometimes, more than one CBM may be found within a glycoside hydrolase.
CBMs can occur side by side with one another within the enzyme though this is not always the case as the CBMs may also be separated by other modules. More than one CBM from the same family may occur in an enzyme, however, CBMs from different families may also occur within the same enzyme. CBM present side by side may show increased affinity for ligand over that of the individual CBMs though this is not always true (Tomme et al. 1998; Boraston et al. 2004; Abbott et al. 2009).
Multivalent binding (two side by side CBMs binding to substrate) can help maintain the CBMs proximity to the carbohydrate surface or fine tune targeting.
1.4.5 Family 6 carbohydrate binding module
Family 6 carbohydrate binding module (CBM6s) are different from other CBM families in that, these modules are known to contain multiple distinct ligand binding sites. The CBMs of family 6 are known to contain modules of diverse
specificity and variation in the location of substrate binding site with respect to their 3-dimensional structure (Henshaw et al., 2004).This variation in ligand recognition is exemplified in CBM family 6 (CBM6), which contains proteins that recognize xylan (Cezjek et al., 2004), cellulose (β-1,4-linked glucose homopolymer) (Henshaw et al., 2004) , laminarin (β -1,3-linked glucose homopolymer), and β-1,4- and β -1,3-mixed linked β -glucans such as lichenan (Pires et al., 2004; Boraston et al., 2004). In all crystal structures of CBM6, characterized to date, the conserved amino acid residues viz. Tyr-33, Trp-92 and Asn-120 have been reported to play a critical role in ligand binding (Pires et al., 2004; Fontes et al., 2004; Michel et al., 2009). The studies on CBMs that recognize the branched hemicelluloses xylan and galactomannan indicated that the side chains of decorated polysaccharides are usually solvent exposed and do not restrict ligand binding or represent the determinants of specificity (Hashimoto et al., 2006; Abbott et al., 2009; Montanier et al., 2011).
1.4.5.1 Type A family 6 carbohydrate binding module
Type A family 6 carbohydrate binding module(CBM6A) generally binds to crystalline cellulose and contains a planar hydrophobic ligand binding surface (Boraston et al., 2004). CBM families, such as 1, 2A, 3A, 5 and 10 are known to contain “Type A” modules which bind crystalline polysaccharides. The ligand specificity of different members of this family is highly conserved even though they are present along with different glycoside hydrolases such as cellulases (Pires et al., 2004; Henshaw et al., 2004), xylanases (Michel et al., 2009), mannanase (Boraston et al., 2003), acetyl xylan esterase and arabinofuranosidases (Fontes and Gilbert, 2010).
1.4.5.2 Type B family 6 carbohydrate binding module
Type B family 6 carbohydrate binding modules (CBM6B) are believed to be evolved from 11 different families. They bind to individual polysaccharide chains and accommodate their target ligands in a cleft of varying depth and can also have multiple clefts for binding (Czjzek et al., 2001; vanBueren et al., 2005). One of the features that distinguish Type B CBMs from lectins is the mechanism of ligand recognition. Each binding site in lectins recognizes one or two sugars through an extensive network of hydrogen bonds, while Type B CBMs generally accommodate four to six sugars, with specificity conferred primarily by the conformation of the ligand, which reflects the topology of the binding site.
1.4.5.3 Type C family 6 carbohydrate binding module
Type C family 6 carbohydrate binding module (CBM6C) are commonly referred to as small sugar-binding CBMs and known to exist in CBM families 9, 13, 14, 18 and 32 (Boraston et al., 2004). They have lectin-like properties and bind to mono-, di- as well as tri-saccharides. There are CBM6Cs that recognize crystalline cellulose, non-crystalline cellulose, chitin, β-1,3-glucans and β-1,3-1,4-mixed linkage glucans, xylan, mannan, galactan and starch, while some CBMs display ‘lectin-like’
specificity and bind to a variety of cell-surface glycans (Abbott et al., 2004; Abbott et al., 2009).
1.4.6 Applications of carbohydrate binding modules
Three basic features have led to CBMs being perfect candidates for many applications: (i) CBMs are usually independently folding units and therefore can function autonomously in chimeric proteins; (ii) the attachment matrices are abundant and inexpensive and have excellent chemical and physical properties; and (iii) the
binding specificities can be controlled by modifying the structure using tools such as site directed mutagenesis and therefore the right solution can be adopted for a particular problem (Abbott et al., 2009).
1.4.6.1 Bioprocessing
Bio-specific affinity purification (affinity chromatography) has become one of the most rapidly developing divisions of immobilized affinity ligand technology.
Many protein entities have been expressed when fused to CBMs, establishing CBMs as high-capacity purification tags for the isolation of biologically active target peptides at relatively low cost (Shosheyov et al., 2006; Bolam et al., 2004; Abbott et al., 2009). A high-level production of a cellulose binding domain (CBDCex) of cellulase (Cex) from Cellulomonas fimi expressed in E. coil, served as affinity tag in a novel secretion-affinity fusion system for purification of recombinant exoglucanase (Hasenwinkle et al., 1997). Also, a strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli for purification of recombinant proteins was developed (Kavoosi et al., 2007).
1.4.6.2 Cell immobilization using carbohydrate binding modules
Surface-exposed CBMs can be an efficient means of whole-cell immobilization. Whole-cell immobilization by cellulosic material was first demonstrated when an E. coli surface anchored CBM, derived from Cellulomonas fimi, was attached to cellulose (Saha, 2000). The cells bound tightly to cellulose at a wide range of pH and the extent of immobilization was dependent on the surface of exposed CBM (Saha, 2000; Numan and Bhosle, 2006).
1.4.6.3 Bio-engineering of carbohydrate binding modules for different applications The potential of carbohydrate binding modules (CBMs) for modifying the characteristics of several enzymes has been reported. The basic approach in CBM engineering was to replace or add a CBM in order to improve hydrolytic activity.
Addition of a CBM derived from cellobiohydrolase II of Trichoderma reesei to Trichoderma harzianum chitinase resulted in increased hydrolytic activity of insoluble substrates (Shoseyov et al., 2006). The replacement of the CBM of endo- 1,4-β-glucanase from Bacillus subtilis with the CBM of exoglucanase I (Texl) from Trichoderma viride conferred higher binding with enhanced hydrolytic activity on the microcrystalline cellulose (Shoseyov et al., 2006). In addition, the hybrid enzyme was more resistant to the tryptic digestion (Shoseyov et al., 2006).