Chapter 1. General Introduction
1. Carbohydrates
1.3 Family 43 glycoside hydrolases
1.3.1 α-L-Arabinofuranosidases
The most recent classification scheme based on amino acid sequences, primary structure similarities and hydrophobic cluster analysis has classified α-L- arabinofuranosidases (α-L-Araf) into five glycosyl hydrolase (GHs) families i.e.
GH30, GH43, GH51, GH54 and GH62 (Zhou et al., 2012; Cartmell et al., 2011;
Sørensen et al., 2006; Guais et al., 2010; Hashimoto et al., 2010). The α-L- arabinofuranoside arabinofuranohydrolases (α-L-Araf, EC 3.2.1.55) are the enzymes involved in the hydrolysis of L-arabinosyl linkages. These enzymes have been purified from several bacteria, fungi and plants (Numan and Bhosle, 2005). They form an array of GHs required for the complete degradation of arabinose containing polysaccharides (Saha, 2000; Saha, 2003). The action of these enzymes accelerates the hydrolysis of the glycosidic bonds by more than 1000 fold, making them one of the most efficient catalysts known (Saha, 2000). Such enzymatic hydrolysis release soluble substrates which are utilized by both prokaryotic and eukaryotic microorganisms (Bayer et al., 2000). The α-L-Araf specifically catalyze the hydrolysis of terminal non-reducing- α-L-1,2-, α-L-1,3-, and α-L-1,5-arabinofuranosyl residues from different oligosaccharides and polysaccharides (Numan and Bhosle, 2005). The α-L-Arafs do not distinguish between the saccharides linked to the arabinofuranosyl moiety, which enables them to exhibit wide substrate specificity (Saha, 2003).
Effective hydrolysis of α-L-arabinofuranosyl residues from various homohemicellulosic polysaccharides (branched arabinans, debranched arabinans), heteropolysaccharides (arabinogalactans, arabinoxylans, arabinoxyloglucans, glucuronoarabinoxylans, etc.), pectic and different glycoconjugates is carried out by α-L-Araf (Cartmell et al., 2011; Mckee et al. 2012).
1.3.1.1 Biochemical properties of α-L-arabinofuranosidases
The biochemical properties of α-L-arabinofuranosidases (α-L-Araf) from various sources have been characterized (Numan and Bhosle, 2005). The molecular size of the α-L-Araf varies and it can be as low as 30 kDa to as high as 495 kDa, although the usual size ranges between 30-80 kDa (Saha, 2003; Numan and Bhosle, 2005). The α-L-Araf shows optimum pH range of 5-7 but there are few exceptions (few fungal α-L-Araf). Similarly, the optimum temperature range is very wide (50- 90°C) (Cartmell et al., 2011; McKee et al., 2012). The activities of α-L-Araf are affected by metal ions, ionic and nonionic detergents, chelating and reducing agents depending on the enzyme and concentration of the agent used (Numan and Bhosle, 2005). In some cases, metal ions such as Ag+, Cu2+, Hg2+, Zn2+, Cd2+ and Co2+
displayed inhibitory effect on α-L-Araf (Shin et al., 2003; Khandeparkar et al., 2008).
1.3.1.2 Molecular cloning of α-L-arabinofuranosidases
Some α-L-Araf has been studied at the molecular level. The genes coding for these enzymes (α-L-Araf) were identified, cloned and expressed in different bacterial and fungal systems. A few genes of α-L arabinofuranosidase have been sequenced and the evolutionary relationship among some of the sequenced proteins has been reported using the phylogenetic analysis (Degrassi et al., 2003). The gene sequencing results as well as the crystal structure studies reported previously indicated the presence of CBMs in some of the reported enzymes. The CBMs may take part in increasing the efficiency of the enzyme function (Numan and Bhosle, 2005). However, the possible roles of α-L-Araf in the release of arabinofuranosyl residues is not yet clear (Numan and Bhosle, 2005).The α-L-Araf (AkAbfB) from Aspergillus kawachii was reported to contain an arabinose-binding domain or module (ABD) which showed a number of
distinct characteristics that are different from those of carbohydrate-binding module (Shallom and Shoham, 2003).
1.3.1.3 Applications α-L-arabinofuranosidases of family 43 glycoside hydrolase α-L-Arabinofuranosidases (α-L-Araf) have been used synergistically along with xylanase for complete degradation of hemicellulose to pentose and hexose sugars for ethanol production and in the paper and pulp industry (Numan and Bhosle, 2005).
α-L-Arabinofuranosidases have several applications which are described in sub sections 1.3.1.3.1, 1.3.1.3.2 and 1.3.1.3.3.
1.3.1.3.1 α-L-Arabinofuranosidase in acetic acid production and quality of bread Pentose sugars (soluble pentosans) such as arabinose and xylose is important functional ingredient in bread and their positive role in bread texture and staling is well known (Gobbetti et al., 2000; Devesa et al., 2003). Wheat flour added to the dough may be moderately hydrolysed by wheat flour degrading enzymes and especially, by exogenous enzymes, such as xylan degrading system including α-L- arabinofuranosidase (Saha, 2000). The pentose sugars released by α-L-Araf can be easily converted to acetic acid to be used as raw material for textile finishing agents (Saha, 2003).
1.3.1.3.2 α-L-Arabinofuranosidase in pulp and paper industry
Several commercial xylanase preparations are available for the treatment of pulp (Gubitz et al., 1997). Application of α-L-arabinofuranosidase further enhances the delignification of pulp as the enzyme acts to release arabinose side chain that retard the action of other bleaching enzymes (Numan and Bhosle, 2006). The removal
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).