Chapter 1. General Introduction
1.4 Oligosaccharides production by acceptor reaction
were remarkably improved by conjugation with dextran (Liu et al., 2012). In comparison with PPI, mixture and conjugates had more compacted tertiary conformation but exhibited better thermal stability, protein solubility, emulsifying and foaming properties (Liu et al., 2012). Dextran based nanoparticles find application in specific drug targeting where dextran is used as polymer coating, which can be functionalized (McBain et al., 2007). Biosensor analysis based on carboxymethylated dextran (CM-dextran) surfaces has provided reliable platform for the rapid determination of a wide range of compounds relevant to food safety and quality (Situa et al., 2008). Dextrans are used as a stabilizing coating for protecting metal nanoparticles against oxidation (Bautista et al., 2005). Dextran coating on biomaterials are being done to prevent undesirable protein absorption to improve their biocompatibility (Sengupta et al., 2006). Dextrans are used in nanotechnology as tool for antigen delivery in vaccination (Sahoo et al.,2007).
accept glucose at the active-site of glucansucrases to give a series of isomaltodextrin
“acceptor products” presenting an isomaltosyl residue at their non-reducing end (Robyt and Walseth, 1978). Synthesis progresses by successive transfers of glucosyl units to oligosaccharides, which are alternately product and substrate. These acceptor reaction products are synthesized in decreasing amounts, as the size of the products increase (Robyt and Eklund, 1983; Fu and Robyt, 1990b; Su and Robyt, 1993). The major product is the first oligosaccharide homologue, e.g. isomaltose from glucose, isomaltotriose from isomaltose and panose from maltose. Fructose and cellobiose are the exceptional acceptor molecules which give only one product. Fructose gives the disaccharide leucrose, and lactose gives a trisaccharide, with glucose attached to the C2-OH of the reducing-end, glucose moiety (Robyt and Eklund, 1983). Cellobiose, an analogue of lactose, gave a series of isomaltodextrins attached to the C2-OH of the reducing-end glucose moiety (Robyt and Eklund, 1983).
1.4.1 Acceptor molecules
The acceptor efficiency of various sugar molecules and their corresponding acceptor reaction products have been studied (Robyt and Walseth, 1978; Robyt and Eklund, 1983). The efficiency of the acceptor molecules vary depending upon their ability to compete with glucan synthesis or their effect on the reaction velocity (Sidebotham, 1974). Acceptors such as maltose and isomaltose are classified as strong acceptors (Koepsell et al.,1952; Robyt and Walseth, 1978; Robyt and Eklund, 1983).
They have activator effect on the reaction velocity and strongly inhibit the yield of glucan synthesis (Robyt and Elkund, 1983). On the other hand weak acceptors like fructose or melibiose, have an inhibitory effect on the reaction (Koepsell et al., 1953) therefore the yield of oligosaccharides is low (Robyt and Elkund, 1983). The yield,
branching and length of the acceptor reaction products can be controlled by changing the acceptor molecule concentration in the reaction mixture of dextransucrase along with sucrose. The yield of oligosaccharides decreases with increasing the ratio of sucrose concentration to maltose concentration (Monchois et al.,1998b). However, by increasing the sucrose/maltose ratio (S/M), it is possible to catalyze the synthesis of oligosaccharides of increasing degree of polymerization (Vasileva et al., 2010). For an S/M ratio of 7, both linear oligosaccharides (only composed of α-(1→6) linkages and a maltose residue at the reducing end) and branched oligosaccharides were produced (Vasileva et al., 2010).
1.4.2 Kinetics and mechanism of acceptor reaction
Various mechanisms have been proposed to explain the kinetics of acceptor reaction of dextransucrase. Oligosaccharides may be synthesized by a nucleophilic attack of the hydroxyl group located at the non-reducing end of the acceptor to the C- 1 of one of the two glucosyl residues involved in the two covalent glucosyl-enzyme complexes. This mechanism for oligosaccharide production is in accordance with the glucan biosynthesis mechanism proposed by Robyt et al., 1974. There have been dispute for the acceptor binding site in the dextransucrase. Many studies established the fact that the acceptor binding site is really unique and is distinguished from the two active sites for glucan biosynthesis (Tanrivseven and Robyt, 1992; Su and Robyt, 1994). However, till date no direct evidence is available explaining the existence of a separate acceptor binding site. Moreover, one of the two sucrose binding sites may also be an acceptor binding site (Germaine and Schachtele, 1976; Kobayashi and Matsuda, 1978). Heincke et al., 1999 explained the phenomenon of substrate inhibition and its elimination in the presence of acceptors on the basis of the
mechanistic model. Heincke et al., 1999 also proposed the existence of a separate acceptor binding site between the two active sites for dextran biosynthesis. It is assumed that occupation of the acceptor site by sucrose or an acceptor molecule causes hindrance to the dextran formation reaction. Both sucrose and acceptor molecule compete for the acceptor binding site (Heincke et al., 1999). Therefore, increasing acceptor concentration with respect to sucrose enhances oligosaccharide production (Heincke et al.,1999).
1.4.3 Prebiotic potential of gluco-oligosaccharides
Non digestible oligosaccharides (NDOs) are oligomeric carbohydrates, the osidic bond of which is in a spatial configuration that allows resistance to hydrolytic activities of intestinal digestive enzymes (Roberfroid, 2000). These NDOs possess physiological and physiochemical properties and they functions as dietary fibers and as a prebiotics (Grootaert et al.,2007). The ability of a probiotic lactic acid bacterium to survive in the gastrointestinal tract was promoted by oligosaccharides facilitating the metabolism and growth of LAB (Salminen et al., 1998). The dietary fiber, mainly oligosaccharides and polysaccharides in the colon may act as prebiotics (Ziemer and Gibson, 1998; Fooks et al., 1999). The maltose acceptor reaction has been used to produce these non-digestible glucooligosaccharides (Remaud-Simeon et al., 1994).
These glucooligosaccharides are presently marketed for human nutrition and dermocosmetic applications. The production of glucooligosaccharides having α- (1→3) branched linkages has been done using glucosyltransferases from Leuconostoc mesenteroides NRRL B-1355 and NRRL B-742 (Remaud-Smeon et al., 1999; Cote and Sheng, 2006). Kim et al., 2009 studied the production of linear isomalto- oligosaccharides (IMO) with DP2 - DP10 using engineered fusion enzyme (DXSR) of
endo-dextranase and only α-(1→6) glucan synthesizing dextransucrase using sucrose as sole substrate. Leuconostoc mesenteroides NRRL B-1299 dextransucrase synthesizes specifically α-(1→2) linear and branched glucooligosaccharides which are highly resistant to glycolytic digestive enzymes and can be fermented by beneficial species of the intestinal microflora (Ramaud-Simeon et al., 1994; Dols et al., 1998). Branched oligosaccharides produced by GTFs coming from Leuconostoc mesenteroides M2860 were readily catabolized by lactobacilli but not by Escherichia coli and Listeria innocua strains, pointing toward their application in intestinal microflora modification. Thermo acid-stable oligosaccharides (TASO) were produced from Leuconostoc mesenteroides B-512 FMCM (Seo et al., 2007). These resistant oligosaccharides inhibited the growth of Streptococcus sobrinus. However, it stimulated the growth of probiotic organisms such as Bifidobaterium sp (Seo et al., 2007). Gluco-oligosaccharides are known to selectively stimulate the growth of bifidobacteria. In particular, long-chain glucooligosaccharides with a degree of polymerization of 3 or higher are preferred to short-chain oligosaccharides because of the longer persistence in the colon (Chung and Day, 2002). These probiotic strains get advantage over other harmful bacteria in the sense that they can ferment these oligosaccharides. Short chain fatty acids like propionic acids are formed as the results of the fermentation of these oligosaccharides which play decisive role in the prevention of colon cancer (Cummings, 1981).