Chapter 1. General Introduction 1
1.3 Carbohydrate binding modules
Initially the non-catalytic polysaccharide-recognizing modules of glycoside hydrolases were described as cellulose-binding domains (CBDs) because the first described module showed affinity towards crystalline cellulose. Later on more diverse carbohydrate binding domains were identified and then the term, carbohydrate binding module (CBM) was adopted as they reflected the diverse ligand specificities.
(Tomme et al., 1988; Van Tilbeurgh et al., 1986). CBMs have an ability to fold and express independently of their associated catalytic modules. The well-defined function of this non-catalytic module is to increase the effective concentration of substrate in the vicinity of associated catalytic module by bringing the catalytic modules in close proximity to the substrate and thus to improve its performance (Shoseyov et al., 2006a). CBMs can be situated at either C- or N- terminal and occasionally occupy central position sandwiched by catalytic modules. It usually contains up to 200 amino acids and is found as single, double or multiple modules in one protein.
1.3.1 Classification of carbohydrate binding modules
Carbohydrate binding modules (CBMs) are currently grouped into 69 families based on the sequence similarity (http://www.cazy.org/Carbohydrate-Binding- Modules.html). The structure fold of proteins is better conserved than their sequences;
so CBMs are further subdivided into three types based on mechanism of ligand binding and binding site topology (Boraston et al., 2004; Hashimoto, 2006). The Type A CBMs are described as ‘surface binding’ CBMs. They bind highly crystalline polysaccharides such as chitin or cellulose. The binding site architecture of type ‘A’
CBMs, is flat or platform-like formed by aromatic amino acid to accommodate flat surfaces of insoluble polysaccharides (chitin or cellulose) (Nagy et al., 1998). The type ‘B’ (glycan chain-binding), CBMs show affinity towards soluble polysaccharides. Binding site is situated in the grooves formed by the aromatic amino acid like Type A, in a planer, twisted or sandwich form to accommodate the ligand molecule. Type ‘C’ (small sugar binding), binds the smaller oligosaccharides by recognizing the terminal sugars of these molecules. The binding site is formed by the grooves as in case of type B but is smaller.
Another classification on the basis of ‘fold similarity’ was suggested (Czjzek et al., 2001; Sunna et al., 2001). In this approach 22 different families were classified into seven ‘fold families’. The seven different folds include the β-sandwich fold, the β-trefoil fold, oligonucleotide/oligosaccharides binding fold (OB), the hevein fold, the knottin fold (cysteine knot), the hevein-like fold and a unique fold (Boraston et al., 2004). The most abundant fold in terms of total number is the β-sandwich fold. This fold comprises two β-sheets, each consisting of three to six antiparallel β-strands. The second most frequent fold is β-trefoil fold. It is present in ricin toxin B-chain. This
fold contains 12 strands of β-sheet, forming six hairpin turns. (Rutenber and Robertus, 1991, Murzin et al., 1992).
1.3.2 Functions of carbohydrate binding modules
Apart from the increasing effective concentration by bringing the biocatalyst into prolonged and close vicinity with its substrate, the other well described function of CBMs are as follows:
a) Substrate Binding and Selectivity: CBMs show affinity towards insoluble as well as soluble polysaccharides. CBMs also functions as selectively targeting the hydrolytic enzymes towards a specific substrate with in the dense plant cell wall (McLean et al., 2002).
b) Nonhydrolytic Substrate Disruption: CBMs are also known to disrupt the substrate without possesing any hydrolytic activity (Southall et al., 1999). The interesting application associated with this property is removal of dental plaque polysaccharides (mainly fructan and glucan), by applying CBM (Fuglsang and Tsuchiya, 2011).
c) Surface/Interfacial Modifications: CBMs also showed the phenomenon by which it is capable of changing the surface of the polysaccharides. It was shown that on treatment of cotton fibres with CBM alters their affinity for dyeing (Paulo et al, 1999).
1.3.3 Applications of carbohydrate binding modules
Three basic features of CBMs have made them perfect candidates for several 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 of CBMs can be controlled by modifying the structure by site directed mutagenesis and therefore the right solution can be adopted for a particular task (Abbott et al., 2009).
The important application of CBMs includes, construction of an expression vector containing a CBM as a fusion tag (Novy et al., 1997). CBMs are used in immobilized affinity ligand technology for purification of biomolecules (Greenwood et al., 1992). The high thermo-stability of CBMs play significant role in separating different carbohydrate analyte (cello- and xylo-oligomers) by retaining them at higher temperatures for better separation of oligosaccharides (Johansson et al., 2006). CBMs are also used in making biosensors such as CBM of Cellulomonas fimi was used for glucose sensing in bioreactor (Levy and Shoseyov, 2002; Phelps et al., 1995). Whole- cell immobilization by cellulosic material was first demonstrated when an E. coli surface anchored CBM, derived from Cellulomonas fimi, was attached to cellulose (Francisco et al., 1993; Wang et al., 2001). The cell immobilization technique using CBM, was also explored for bioremediation purpose such as detoxification of nerve gas by immobilizing the organo-phosphorus hydrolase as well as removal of heavy metal contamination from polluted air (Shoseyov et al., 2006; Wang et al., 2002).
1.3.4 Carbohydrate Binding Modules of family 6
The overall status of the family 6 CBM is shown in the Fig. 1.17. The snapshot from the CAZy database is displaying the current information related to CBM6 viz. the affinity shown by different characterized members, the dominant 3-D structure topology and proper indexing of present members across the different strata of hierarchy (www.cazy.org/CBM6.html). The family 6 CBM shows diverse binding affinities with different polysaccharides therefore it is interesting to investigate the
protein-carbohydrate interaction and structural aspect of the protein which makes family 6 flexible enough to interact with soluble as well as insoluble polysaccharide having different monomeric units.
Fig. 1.17 A snapshot from CAZy database displaying basic information and all members of different classes (and their respective numbers) from CBM6.