The application of the recombinant α-L-arabinofuranosidase (AbfB) from A. niger that selectively removed arabinose side chains from oat spelt arabinoxylans and arabinoglucuronoxylans extracted from bagasse and bamboo feedstocks resulted into decreased solubility of the xylans. The reduction in the solubility of arabinoglucuronoxylans extracted from bagasse (Fig. 5.3b and 5.8b) and bamboo (Fig. 5.3d and 5.8d) was reflected by the increase in the viscosity and the precipitation upon selective removal of their arabinose side chains. Similar increase in viscosity and the precipitation of xylans were observed with selective removal of the arabinose side chains from the oat spelt xylan by the recombinant AbfB (Figs. 5.3a and 5.8a). However, the results showed that the selective removal of arabinose side chains from arabinoglucuronoxylan extracted from P. patula by the recombinant AbfB neither increased the viscosity (Fig. 5.3e) nor resulted into formation of xylan hydrogel precipitates (Fig. 5.8e).
The observed changes in viscosity and the precipitation of the oat spelt, bagasse and bamboo xylans could suggest that the recombinant AbfB selective removal of the arabinose side chains from these substrates created linear regions in the xylan main chain that resulted in increased attractive forces between and within the xylans. Kabel et al. (2007) suggested that the presence of a critical linear length of at least 15 xylose units on the main xylan chain to be the prerequisite for the formation and precipitation of the xylan hydrogels. Therefore, the unsubstituted regions of the xylans as a result of side chain removal are the driving force for increased viscosity and xylan hydrogel precipitation because the steric hindrance within and between the xylans is reduced, leading to precipitation of the xylan fractions (Ebringerová and Heinze, 2000). Kabel et al. (2007) also indicated that different xylan conformations arise from existence of varying degree of attractive forces within and between the xylan as a result of different degrees of xylan substitution and substitution pattern. Furthermore, Höije et al. (2008) reported that arabinoxylans precipitated into insoluble hydrogels when the degree of arabinose substitution, i.e. arabinose to xylose ratio, was lower than 0.3. In view of such precipitation conditions, the results in this study suggest that the selective removal of arabinose side groups from arabinoglucuronoxylans extracted
123 from P. patula by the recombinant AbfB did not create regions of unsubstituted xylose within the main xylan chain with required critical length for precipitation and the formation of hydrogels to occur. Therefore, the similarities in the viscosity changes and xylan precipitation due to the selective removal of the arabinose from arabinoglucuronoxylans extracted from bagasse and bamboo by the recombinant AbfB may be explained by the similarities in the degree and pattern of arabinose substitution to that of the oat spelt xylan (Chapter 3). For instance, the xylan extracted from bamboo had arabinose to xylose ratio of 1:7.6, which was similar to the arabinose to xylan ratio of oat spelt xylan (Table 5.1). On the other hand, the arabinose to xylose ratio of the P. patula xylan was 1:4 (Table 5.1) suggesting that the
P. patula xylan was heavily substituted with arabinose side chains compared to the
xylan extracted from bamboo.
The selective removal of the MeGlcA side chains did not result in formation of xylan hydrogel precipitates of the water soluble arabinoglucuronoxylans from bagasse, bamboo and P. patula as well as of the 4-O-methylglucuronoxylan extracted from E.
grandis but of only the commercially obtained birch glucuronoxylan (Fig. 5.8f). The
failure of the purified AguA to reduce the solubility of the arabinoglucuronoxylans extracted from bagasse, bamboo and P. patula and glucuronoxylan extracted from E.
grandis upon the selective removal of the MeGlcA side chain, could similarly, be
attributed to the differences in the degree of MeGlcA substitution and pattern and the presence of other side groups between the xylans. The chemical composition of the xylans extracted from bagasse, bamboo and E. grandis showed MeGlcA to xylose ratio ranging from 1:5.3- 1:1.75, which is higher than the MeGlcA to xylose ratio of almost 1:9 observed for the birch xylan. In addition, the birch xylans were deacetylated and substituted with only MeGlcA side chains (Chapter 3). In contrast, the chemical composition of the E. grandis glucuronoxylan showed evidence of acetylation despite alkaline hydrolysis and that of arabinoglucuronoxylans from bagasse, bamboo and P. patula showed high degree of arabinose side chains substitution (Chapter 3).
The purified AguA removed MeGlcA side groups to higher levels (50% of the available MeGlcA) that led to precipitation of the mainly from deacetylated glucuronoxylans compared to the corresponding acetylated glucuronoxylans (10% of
124 the available MeGlcA (Tenkanen and Siika-aho, 2000). However, it should be noted that the substrate specificity of the purified AguA reported by Tenkanen and Siika- aho (2000) is rather confusing because the purified AguA failed to increase the removal of MeGlcA upon removal of the acetyl groups by esterases, yet was able to release high amounts of MeGlcA from the water soluble deacetylated birch xylan. Therefore, the limited removal of the MeGlcA side chain by the AguA from the acetylated substrates cannot be attributed to the presence of the acetyl group. On the other hand, the observation that the purified AguA had high preference for the removal of MeGlcA from water soluble arabinoglucuronoxylans such as those extracted from bagasse (Fig. 5.1b) compared to birch glucuronoxylan and those extracted from E. grandis (Fig. 5.2) was in agreement with that of Tenkanen and Siika-aho (2000). In this study the purified AguA removed 50% more MeGlcA from arabinoglucuronoxylans (Fig 5.1b) compared to glucuronoxylans of birch and E.
grandis (Fig. 5.2). The results of Tenkanen and Siika-aho (2000) showed that the
purified AguA was capable of removing almost all (96%) MeGlcA side chains from arabinoglucuronoxylans compared to 50% degree of MeGlcA removal from deacetylated birch glucuronoxylan. Tenkanen and Siika-aho (2000) indicated that the action of the purified AguA was affected substantially by the degree and nature of substitution of the xylans. However, Tenkanen and Siika-aho (2000) neither reported on the changes in viscosity nor precipitation associated with the selective removal of the MeGlcA from the arabinoglucuronoxylans after hydrolysis with the purified AguA.
5.3.2 Role of recombinant AbfB and purified AguA in xylan processing
The formation of xylan hydrogels precipitates from water soluble xylan extracted from oat spelt, bagasse and bamboo upon selective hydrolysis by the recombinant AbfB and of birch xylan by the purified AguA is a clear indication that the recombinant AbfB and the purified AguA have potential application in production of high value speciality coatings and encapsulation matrices for use as speciality carriers or entrapments matrices of bioactive or immobilised biological compounds. These products have potential novel applications beyond the pulp and paper industries including biomedical engineering, pharmaceutical, food and cosmetic industries. This is because the xylans with reduced solubility are known to possess increased binding power that promote fibre to fibre bonding and improve the surface properties as
125 demonstrated with other types of xylans by Dahlman et al. (2003), Köhnke et al. (2008), Danielsson, (2007) and Kabel et al. (2007), Dahlman et al. (2003), Köhnke et al. (2008), Danielsson, (2007) and Kabel et al. (2007).
The ability to alter the viscosity of the xylans by the selective removal of the side chains is of special interest in the production of speciality thickening, antifoamimg and emulsifying agents. The rheological properties of xylans are limited but so far have been studied in relation to bread making (Rattan et al., 1994). Ebringerová et al. (2005) indicated that the high intrinsic viscosity is an indication of the emulsifying effect of the xylans. Therefore, the differences in the change of viscosity of the xylan that occurred with the selective removal of the arabinose side chains by the AbfB from bamboo, bagasse and oat spelt xylans and that of xylan extracted from P. patula could form the basis for choosing the appropriate xylan sources for special end uses as demonstrated in the improvement bread quality by Biliaderis et al. (1995). The results in Figures 5.3a, 5.3b and 5.3d showed that the oat spelt, bagasse and bamboo xylans respectively, selectively hydrolysed by the recombinant AbfB had higher apparent viscosity relative to the corresponding untreated xylans. However, the apparent viscosity of both the enzyme treated and untreated xylans reduced with increased shear rate. Ebringerová et al. (2005) associated such effect to the thixotropic and shear thinning behaviour of less substituted xylan when in aqueous solutions compared to only the shear thinning property of highly substituted xylans.
5.3.3 Synergetic effect of recombinant AbfB and purified AguA on removal of side groups and precipitation of polymeric xylans
Both the recombinant AbfB and the purified AguA present potential biological tools for functionalisation of xylans that could lead to production of different novel products. However, the activity of the recombinant AbfB and purified AguA but in this study depended on the structural properties of the xylans, thus the type, degree and pattern of side chain substitution on the main xylan chain. Therefore, the synergetic effect of the recombinant AbfB and AguA and other side chain removing enzymes may be necessary to enhance the change in viscosity and precipitation of the arabinoglucuronoxylans extracted from bagasse, bamboo and P. patula. The results showed that there was a more positive synergetic effect between the recombinant
126 AbfB and purified AguA for the removal of arabinose side chains from the arabinoglucuronoxylans extracted from bagasse, bamboo and P. patula than the removal of MeGlcA side chains from these xylans (Figs. 5.1a and 5.1b). For example, the simultaneous application of recombinant AbfB and purified AguA enzymes (i.e. the recombinant AbfB and purified AguA enzymes were added at the same time in the reaction mixture) on xylan extracted from bagasse by the Hoije method (Chapter 3) increased the arabinose removal by 21% (Fig. 5.1a) whereas, the removal of MeGlcA increased by only 13% (Fig. 5.1b). Furthermore, the hydrolysis by combined application of the recombinant AbfB and the purified AguA enzymes of xylan extracted from bamboo by the Hoije method (Chapter 3) increased the arabinose removal by 33% (Fig. 5.1b) but reduced the removal of the MeGlcA by the purified AguA by 13% (Fig. 5.1b).
The limited or lack of increase in the degree of removal of MeGlcA side chains by the purified AguA from the bagasse, bamboo and P. patula arabinoglucuronoxylans during combined application with the recombinant AbfB could probably be associated with the problems of restricted access or lack of recognition of the glucuronic side chains on the main xylan chain due to conformational changes caused by the removal of the arabinose side chains. The lack of synergistic effect between the AbfB and purified AguA on the removal of glucuronic side chains from arabinoglucuronoxylans was earlier observed by Tenkanen and Siika-aho (2000) during hydrolysis of xylans extracted from spruce wood and spruce pulp whereby, the increase in the removal of arabinose side groups was possible but not the increase in the removal of MeGlcA. Furthermore, Tenkanen and Siika-aho (2000) also observed reduced increase in the removal of MeGlcA from acetylated birch xylan by the AguA when working in concert with acetyl xylan esterases that removed the acetyl groups in acetylated birch xylan. It appears that the action of the AguA is not only hindered by the presence of the other side chains (Tenkanen and Siika-aho, 2000) but also by the conformation changes in structure of the substrate as a result of the action of other side chain removing enzymes. Such changes could affect the AguA’s substrate accessibility and recognition. It is reported that the molecular sizes of the AguA, which is as high as 180 kDa (Castanares et al. 1995, Tenkanen and Siika-aho, 2000; De Wet and Prior, 2004) could be restrictive to access its substrates. In addition, most α-D- glucuronidases are known to possess complex substrate recognition systems, which
127 could be affected by changes in xylan chain length, the structural arrangement and the position of the MeGlcA they attack on the xylan main chain and the presence or absence of other functional groups (Siika-aho et al., 1994; Tenkanen and Siika-aho, 2000; Nagy et al., 2003; Biely, 2003; De Wet and Prior, 2004). However, the AguA with polymeric xylan substrate specificity hydrolyzes the α-1, 2-glycosidic bond between the MeGlcA and single xylose moiety located at the non-reducing end of xylooligosaccharides (Siika-aho et al., 1994; Biely, 2003). Therefore, the lack of reduction of water solubility of the arabinoglucuronoxylans could be attributed to the change in substrate specificity on heterosubstituted xylans. Relatively, the recombinant AbfB would be a preferred enzyme for the precipitation of the arabinoglucuronoxylans, probably because of its inherent broad but restricted substrate specificity.