Introduction

The major constituents of plant cell wall (PCW) are the polysaccharides that accumulate to form a network. These polysaccharides are cellulose, hemicellulose and pectin (Fontes & Gilbert, 2010). The PCW is able to fulfil its structural role by providing strength and protection to the cell owing to the presence of cellulose, hemicellulose and lignin (Vincken et al., 2003). PCW has several functional roles such as cell-cell adhesion, cell signalling, wall porosity, pollen tube growth, leaf abscission.

The pectin component of the cell wall has been credited to play these roles (Ridley et al., 2001). Structural role of pectin in promoting upright growth of plants has also been reported (Matsunaga et al., 2004). Pectin is a naturally occurring flexible and complex polysaccharide present in the middle lamella of plant cell wall and plant-derived food products. Pectin is present throughout the plant kingdom. More than 80% of the total pectin consists of D-galactopyranosyluronic acid (DGalp A), and the rest is rhamnose (Rhap), galactose (Galp), xylose (Xylp) and arabinose (Araf) (Ridley et al., 2001).

Citrus and apple are a major source of commercial production of pectin for industrial applications. Pectin is used in food industry for its stabilizing and gelling properties.

Traditionally, it is used as gelling agents in jellies, jams and marmalades. Pectin is also used in confectionery, acidified milk products and bakery fillings (May, 1990; Rolin, 2002). Pectin is categorized into three different kinds of polymers based on the covalent bonding of galacturonic acid. The primary structural elements of pectin are homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II), which are discussed in chapter 1, section 1.2.

Degradation of plant cell wall makes a reservoir of nutrients available for recycling. Nature has bestowed a diverse group of microorganisms with enzymes to breakdown the plant cell wall polysaccharides (Ochiai et al., 2007, Pages et al., 2003 and McKie et al., 2001). When pectin comes under microbial attack both glycoside hydrolases, polysaccharide lyases and pectin esterases are recruited. Glycoside hydrolases cleave the glycosidic bonds via an acid-base catalytic mechanism (Koshland et al., 1953). Polysaccharide lyases cleave their substrates via a β- elimination mechanism, generating a double bond between C-4 and C-5 in the residue at the non-reducing end (Moran et al., 1968). Glycoside hydrolases and polysaccharide lyases have been classified into different families based on sequence similarity (Lombard et al., 2014). Many of the plant cell wall polysaccharide degrading enzymes are modular in nature and have one or more specialized substrate binding module(s) referred to as Carbohydrate Binding Module(s) (CBM) in addition to a catalytic module (Duan et al., 2016).

Pectin methylesterases (PMEs; EC: 3.1.1.11) are a group of enzymes, belonging to family 8 carbohydrate esterase (CE8) categorized under pectin esterase

superfamily (CAZy database, www.cazy.org). PME hydrolyses methyl esters at the C- 6 position of D-Galp A residues in HG and releases pectate and the methanol (Fraeye et al., 2010). The de-esterified pectate is further degraded by polygalacturonase, pectate lyase and rhamnogalacturonan lyase (Kashyap et al., 2001), which are produced by different organisms such as bacteria, fungi and plants, as well as insects (Jiang et al., 2013). In plants, pectin methylesterase used for ripening by destabilization in cell wall metabolism (Frenkel et al., 1998). Plant-derived PMEs work under alkaline conditions (pH 7.0–9.0) (Michelis, 2001; Duvetter et al., 2006), whereas fungal, e.g. Aspergillus niger, PMEs work under acidic conditions (Limberg et al., 2000; Kim et al., 2013; Kent et al., 2016). Phytopathogenic micro-organisms can degrade plant cell walls through its PME during the plant invasion; therefore, this enzyme is known to be a virulence factor of phytopathogens (Michelis, 2001).

The modular protein, Cthe_2949 (GenBank accession number ABN54147.1 and Uniprot ID A3DJL8) of Clostridium thermocellum ATCC 27405 (renamed as Ruminiclostridium thermocellum) is a modular carbohydrate-active enzyme. Its nucleotide and protein sequences were retrieved from the CAZy database and analyzed by different computational approaches including the BLAST tool. The protein sequence, Cthe_2949 contained 567 amino acids. BLAST analysis revealed that the protein, Cthe_2949 contained a N-terminal putative pectin methylesterase (PME) module (CtPME) belonging to carbohydrate esterase family 8 (CE8), linker and X-157 (Unknown function module) and followed by a Dockerin at C-terminal.

The molecular architecture of CE8 protein sequence was drawn by using the DOG2.0 software and presented in Fig. 2.1. The Conserved Domains Database was referred for determining the regions of conserved domains (http://www.ncbi.nlm.nih.

gov/cdd/). The CE8 protein sequence was analysed by InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) to identify various domains. The N-terminal contains the signal peptide was predicted between 1 to 30 amino acids by SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP-3.0/). Towards the N-terminal, downstream of the signal peptide, a stretch of 300 amino acids, the catalytic module CtPME showed similarity to the super family of pectin methyl esterases (Fig. 2.1). A short stretch of 22 amino acids (331-353) linker region connects another module spanning from amino acids, from 354 to 494, an un known module named CtX157.

This is followed a type I dockerin from 503 to 567 amino acids and a Ca²⁺ ion binding site from 509 to 551 amino acid residues at the C-terminal. The interaction between enzymes borne Dockerin modules and the Cohesin modules of a scaffold protein gives rise to the celluosomal complex (Fontes et al., 2010). The presence of a putative Dockerin I at C-terminal and the subcellular localisation score predicted by PSORT server indicated that the protein encoded by sequence Cthe_2429 is an extracellular enzyme and probably integrates as a component of the C. thermocellum cellulosome.

Fig. 2.1 Molecular architecture of protein Cthe_2949 showing CE8 and derivative domains.

In the present study the genes encoding CE8 and its truncated derivatives CtPME-catalytic, CtX157 (Unknown) and CtPME-X157 (CtPMEf) were cloned. All the proteins were expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography (IMAC) for further biochemical, functional and structural characterization.