Chapter 7 SAQ 7.1 Identify each of the following statements as true or false If false give a reason for your response 1)D-xylose is a sugar commonly found in microbial polysaccharides 2) Pyruvate ketals contribute to the cationic nature of exopolysaccharides 3)The presence of acetate in exopolysaccharides increases their lipophilicity 4) Exopolysaccharides are not produced by yeasts and filamentous fungi 5)An exopolysaccharide containing a high content of D-ght ic acid will tend to bind cations 7.3 Exopolysaccharide structure As with all polysaccharides, microbial exopolysaccharides can be divided into homopolysaccharide and heteropolysaccha Homopolysaccharides contain only one type of monosaccharide, whereas heteropolysaccharides contain more than one Many are neutral glucans, being composed of the monosaccharide component D-glucose. 7.3.1 Homopolysaccharide types of homopolysaccharide are known(Figure e polyanionic homopolymers and, unlike the glucans, also contain acyl groups 1)Single linkage type. Several of these are neutral glucans, eg curdlan. Others Side chain type. Scleroglycan is typical and possesses tetrasaccharide repeating units due to a 1, 6p-D-glucosyl side-chain on every third main chain residue 3) Branched types. These are dextrans which are composed entirely of a-linked glucosyl residues. In some dextrans the linkage is almost entirely 1-6, but up to 50% of the glucose residues may be linked1→2,1→3or1→4
198 Chapter 7 Identify each of the following statements as true or false. If false give a reason for your response. 1) D-xylose is a sugar commonly found in microbial polysaccharides. 2) Pyruvate ketals contribute to the cationic nature of exopolysaccharides. 3) The presence of acetate in exopolysaccharides inmases their lipophilicity. 4) Exopolysaccharides are not produced by yeasts and filamentous fungi. 5) An exopolysaccharide containing a high content of D-glucuronic acid will tend to bind cations. 7.3 Exopolysaccharide structure As with all polysaccharides, microbial exopolysaccharides can be divided into homoplysaccharides and heteropolysaccharides. Homopolysaccharides contain only one type of monosaccharide, whereas heteropolysaccharides contain more than one. Many are neutral glucans, being composed of the monosaccharide component D-glucose. 7.3.1 Homopolysaccharides Three main types of homopolysaccharides are known (Figure 7.2). 1) Single linkage type. Several of these are neutral glucans, eg curdlan. Others are polyanionic homopolymers and, unlike the glucans, also contain acyl groups. 2) Side chain type. Weroglycan is typical and possesses tetrasaccharide repeatingunits due to a 1,6-~-Dglucosyl side-chain on every third main chain residue. 3) Branched types. These are dextrans which are composed entirely of a-linked glucosyl residues. In some dextrans the linkage is almost entirely 1 + 6, but up to 50% of the glucose residues may be linked 1 4 2,1+ 3 or 1 + 4
Production and applications of microbial exopolysaccharides CH,OH CH2OH CH,OH b)βDgu(1→3)-BDu(1→3)-Dgu(1→3)阝-Dgu B-D-glu OOOooO6 ooooooooooo。 igure 7. 2 Three main types of homopolysaccharide. a) Single linkage type, eg curdlan. b) Side chain type, eg scleroglycan. c)Branched chain type, eg dextran. The figure also show various ways of illustrating exopolysaccharide structure 7.3.2 Heteropolysaccharides repeat units Microbial heteropolysaccharides are almost entirely all composed of repeating units of between two and eight monosaccharides. The units often contain D-glucuronicacid and short side-chains of one to four residues are com everal different side-chains are found in some heteropolysaccharides. The structure of xanthan illustrates these points and is shown in Figure 7.3. A few bacterial alginates are exceptions
Production and applications of microbial exopolysaccharides 199 Figure 7.2 Three main types of hornopolysaccharides. a) Single linkage type, eg curdJan. b) Side chain type, eg scleroglycan. c) Branched chain type, eg dextran. The figure also show various ways of illustrating exopolysaccharide structure. 7.3.2 Heteropolysaccharides Microbial heteropolysaccharides are almost entirely all composed of repeating units of between two and eight monosaccharides. The units often contain D-glucuronic acid and short sidechains of one to four residues are common. Several different sidechains are found in some heternplysaccharide. The structure of xanthan illustrates these points and is shown in Figure 7.3. A few bacterid alginates are exceptions. repeat units
Chapter 7 B1 -G-G-G- presence or absence give rise to different repeat units( Pyr not present with 2x A G= D Ac=acetate 4, 6-2 pyr Pyr= pyruvate ooo6o9oa。9o9。0 sntermsolecuyar Figure 7. 3 The structure of xanthan illustrated in three ways(a, b, c). SAQ 7.2 Complete the illustration of the repeat unit in xanthan by adding bonds and ups to the molecule(similar to the illustration in Figure 7.2a). Refer to Figures 7.1 and 7. 3 for chemical structures ∏ Examine Figure 7.3 and give two reasons why xanthan has an anionic natur The anionic nature of xanthan arises from the presence of glucuronic adid and pyruvate
200 Chapter 7 v I Figure 7.3 The structure of xanthan illustrated in three ways (a, b, c). Complete the illustration of the repeat unit in xanthan by adding bonds and groups to the molecule (similar to the illustration in Figure 7.2a). Refer to Figures 7.1 and 7.3 for chemical structures. n Examine Figure 7.3 and give two reasons why xanthan has an anionic MM. The anionic nature of xanthan arises from the presence of glucuronic acid and pyruvate
Production and applications of microbial exopolysaccharides The unique physical properties of microbial exopolysaccharides(considered in Section s molecular 7.7), which determine their commercial importance, arises from their molecular informaton conformation. This, in turn, is determined by the primary structure and from associations between molecules in solution ond angles For most exopolysaccharides their shape is determined by the angle of bonds which governs the relative orientations of adjacent sugar residues in the chain. However, the range of relative orientations of adjacent sugar molecules is limited by stericinteractions between molecules along the chain ∏ Which group substituents influence inter-atomic electrostatic repulsion in a glyc The carbonyl( Coo)group, which carries a full charge, will have the most pronounced effect. Oxygen atoms of hydroxyl groups carry a partial negative charge and, therefore each oth helical Exopolysaccharides in solution have an ordered helical conformation, which may be conformation single, double or triple; forexample, xanthan forms a double or triple helix(Figure 7.3c) These are stabilised by intermolecular hydrogen bonds. The helical conformation makes the exopolysaccharide semirigid and the molecules can move large volumes of solution. These volumes overlap even at low concentrations of exopolysaccharide, giving rise to relatively high viscosities. intermolecular The intermolecular interactions stabilise the helices and greatly influence the properties racbons of exopolysaccharides in solution, ie solubility, viscosity and gel-formation. a strong interaction or good-fit between molecules will lead to insolubility, whereas poor interaction will lead to solubility of exopolysaccharides. The interactions between molecules is influenced by the presence of side-chains. For example, cellulose is insoluble but introduction of a three monosaccharide side- chain into the cellulose chain gives the soluble xanthan. Small changes in the structure of the side-chains can alter the molecular interactions and thus properties of the exopolysaccharide These changes may be brought about by choice of production organism; conditions of fermentation chemical modification(post fermentation); nzymatic modification(post fermentation) 7.4 Medium composition for exopolysaccharide production Many different types of carbon substrate can be converted by micro-organisms to exopolysaccharides, these include · carbohydrates; · amino acids
Production and applications of microbial exopolysaccharides 201 molecular mformatial bond angles held conformatian inbrmdecular inbractions The unique physical properties of microbial exopolysaccharides (considered in Section 7.7), which determine their commercial importance, arises from their molecular conformation. This, in turn, is determined by the primary structure and from associations between molecules in solution. For most exopolysaccharides their shape is determined by the angle of bonds which governs the relative orientations of adjacent sugar residues in the chain. However, the range of relative orientations of adjacent sugar molecules is limited by steric interactions between molecules along the chain. Which group substituents influence inter-atomic electrostatic repulsion in a n glycosyl chain? The carbonyl (COO-) group, which carries a full charge, will have the most pronounced effect. Oxygen atoms of hydroxyl pups carry a partial negative charge and, therefore, repel each other. Exopolysaccharides in solution have an ordered helical conformation, which may be single, double or triple; for example, xanthan forms a double or triple helix Figure 7.3~). These are stabilised by intermolecular hydrogen bonds. The helical confoxmation makes the exopolysaccharide semirigid and the molecules can move large volumes of solution. These volumes overlap even at low concentrations of exopolysaccharide, giving rise to relatively high viscosities. The intermolecular interactions stabilise the helices and greatly influence the properties of exopolysaccharides in solution, ie solubility, viscosity and gel-formation. A strong interaction or 'good-fit' between molecules will lead to insolubility, whereas poor interaction will lead to solubility of exopolysaccharides. The interactions between molecules is influenced by the presence of side-chains. For example, cellulose is insoluble but introduction of a three monosaccharide side-chain into the cellulose chain gives the soluble xanthan. Small changes in the structure of the side-chains can alter the molecular interactions and thus properties of the exopolysaccharide. These changes may be brought about by: choice of production organism; conditions of fermentation; chemical modification (post fermentation); enzymatic modification (post fermentation). 7.4 Medium composition for exopolysaccharide production Many different types of carbon substrate can be converted by micmrganisms to exopolysaccharides, these include: carbohydrates; a aminoacids;
Chapter 7 fatty acids; certain central metabolites(such as TCA cycle intermediates) carbohydrates Carbohydrates are the most widely utilised carbon substrate by exopolysaccharide producing micro-organisms and are used as substrate for commercial production. The structure of the exopolysaccharide is generally independent of the carbon substrate However, choice of carbon substrate can influence both the quantity produced and the extent of acylation of exopolysaccharides. The bacteria that produce dextran are unusually specific in their carbon substrate requirement for exopolysaccharide production: they synthesise dextran only when grown on sucrose and are apparently unable to synthesise the polymer when grown on other substrates, such as glucose ∏ Why do you think carbohydrates are the most widely utilised carbon substrate for commercial production? Carbohydrates are relatively cheap, available in large quantities and utilisable source of carbon and energy for most micro-organisms. These cons are particularly important for those exopolysaccharides produced on a Utilisable nitrogen sources for exopolysaccharide producing organisms include ammonium salts; · amino acid · nitrate dinitrogen(nitrogen gas) Ammonium salts or amino acids are by far the most commonly used nitrogen sources in production media. Nitrate is rarely used. Although most nitrogen fixing micro-organisms do produce exopolysaccharide, their growth and the quantity of the polymer produced is often improved if a fixed source of nitrogen, such as Nh, is cations Most micro-organisms require various cations for optimal growth, in particular K and Ca. Other cations(trace elements) are required in smaller quantities and, in some culture media, may be present as components of other ingredients Phosphate is the major anionic requirement of micro-organisms K has a role in substrate uptake and during efficient exopolysaccharide synthesis, adequate supplies of this ion is essential for ensuring sufficient intracellular carbon substrate is maintained. Other ions, such as phosphate and magnesium, have roles in the acylation of exopolysaccharides and influence their physical properties
202 Chapter 7 0 hydrocarbons; fattyacids; certain central metabolites (such as TCA cycle intermediates). Carbohydrates are the mast widely utilised &n substrate by exopolysamharide producing micro-organisms and are used as substrate for commercial production. The structure of the exopolysaccharide is generally independent of the carbon substrate. However, choice of carbon substrate can influence both the quantity produced and the extent of acylation of exopolysaccharides. The bacteria that produce dextran are unusually specific in their carbon substrate requirement for exopolysaccharide production: they synthesise dextran only when grown on sucrose and are apparently unable to synthesise the polymer when grown on other substrates, such as glucose. Why do you think carbohydrates are the most widely utilised carbon substrate n for commercial production? cartmhydrates Carbohydrates are relatively cheap, available in large quantities and are readily utilisable source of carbon and energy for most miao-organisms. These considerations are particularly important for those exopolysaccharides produd on a large (bulk chemical) scale. nitrogen sources Utilisable nitrogen sources for exopolysaccharide producing organisms include: ammoniumsalts; 0 aminoacids; nitrate; dinitrogen (nitrogen gas). Ammonium salts or amino acids are by far the most commonly used nitrogen sources in production media. Nitrate is rarely used. Although most nitrogen fixing micmrganisms do produce exopolysaccharide, their growth and the quantity of the polymer produced is often improved if a fixed source of nitrogen, such as M, is supplied. Most micro-organisms require various cations for optimal growth, in particular K', Mg", Fe2+ and Ca2+. Other cations (trace elements) are required in smaller quantities and, in some culture media, may be present as components of other inments. Phosphate is the major anionic requirement of micro-organisms. K+ has a role in substrate uptake and during efficient exopolysaccharide synthesis, adequate supplies of this ion is essential for ensuring sufficient intracellular carbon substrate is maintained. Other ions, such as phosphate and magnesium, have roles in the acylation of exopolysaccharides and influence their physical properties. cations