Modification of solubility properties with respect to viscosity and precipitation during hydrolysis of xylans by the recombinant AbfB and purified AguA was influenced by the enzyme xylan specific dosage, hydrolysis time and temperature. Therefore, customised removal of the side chains and manipulation of conditions for selective hydrolysis of the xylans by the recombinant AbfB and purified AguA could possibly enable the xylans to possess structures that would enhance precipitation and formation of hydrogels to suit special end uses. This idea is based on the results that showed that the viscosity of xylans extracted from bamboo increased by increasing the recombinant AbfB xylan specific dosage from 180.0 to 540.0 nkat g-1 substrate (Fig. 5.4a). Similarly, the viscosity of the oat spelt xylan decreased by increasing the temperature from 40 to 60ºC (Fig. 5.4b).
The differences in the viscosity changes of the polymeric xylan treated with the recombinant AbfB under the given hydrolysis condition could be attributed to the increased degree of removal of the arabinose side groups from the xylans at 40ºC and at higher recombinant AbfB xylan specific dosage (720.0 nkat g-1substrate) compared to the release of arabinose at 60ºC and at lower AbfB xylan specific dosage (180.0 nkat g-1substrate). However, the hydrolysis time and arabinose removal from oat spelt xylan by the recombinant AbfB had a non-linear relationship, which yielded a significant negative correlation (r) of -0.544) at p< 0.05 (Fig. 5.5) with viscosity. Therefore,
128 factors that influence degree of side chain removal would also impact on the viscosity properties of the xylans.
However, the negative relationship between viscosity and degree of arabinose removal suggests that although the viscosity of the oat spelt xylan increased with application of the recombinant AbfB (Fig. 5.3 a), such viscosity is not sustained with time, hence the observed decreasing trend displayed in particular with increased hydrolysis time (Fig. 5.7a-c). The decrease in viscosity of the oat spelt xylan with time could be as a result of phase separation due to aggregation of the xylan hydrogel precipitates. Such behaviour could possibly be explained by the thixotropic and shear thinning behaviour that prevail with less substituted xylans as reported by Ebringerová et al. (2005). The surface plots for viscosity fitted with the second order polynomial as a function of time and temperature and AbfB xylan specific dosage (nkat g-1substrate) showed that the viscosity decreased to the lowest value of 2.03 mPa.s (measured at shear rate of 10.3 s-1) at hydrolysis time of 9 h and temperature of 45.8ºC with recombinant AbfB xylan specific dosage of ≈ 400.0 nkat g-1substrate (Fig. 5.7a-c). However, the results showed that beyond 10 h an increase in viscosity of up to ≥ 4 mPa.s prevailed (Figs. 5.7a and 5.7b). Therefore, depending on the hydrolysis time and degree of arabinose removal from the oat spelt xylan at which the viscosity is measured, an increasing or decreasing trend of the change in viscosity could prevail.
The removal of arabinose side chains increased with increase in recombinant AbfB enzyme xylan specific dosage during hydrolysis of oat spelt xylan (Fig. 5.6). However, the degree of precipitation of the oat spelt xylan did not increase any further with increase in the recombinant AbfB xylan specific dosage after release of 10% of the available arabinose (Fig. 5.6). The levelling off of the precipitation of the oat spelt xylan with increased arabinose removal was probably a consequence of the AbfB continued removal of the arabinose from xylan that had already precipitated from the solution (Fig. 5.8a) rather than from unmodified xylan still present in the solution. Similarly, the degree of purified AguA removal of the MeGlcA from birch xylan increased 5 times by increasing the purified AguA xylan specific dosage from 2500 to 8000 nkat g-1substrate. In addition, the precipitated birch xylan hydrogels were visible in the reaction mixture in which birch xylans were treated with purified AguA at xylan specific dosage of 2500 nkat g-1substrate (Fig. 5.8g). However, the volume of
129 the birch xylan hydrogel precipitates was not significantly different for that of the birch xylans treated with the purified AguA at xylan specific dosage 5000 and 8000 nkat g-1substrate (Fig. 5.8g). Notably, the maximum degree of removal of the MeGlcA of 2.5% of the available MeGlcA from the birch xylans was reached with purified AguA xylan specific dosage of 5000 nkat g-1substrate (Fig. 5.8f). The release of MeGlcA side chains from the birch xylan by the AguA obtained in this study constituted 5% of MeGlcA side chain removal reported for birch xylan by Puls (1992) and Tenkanen and Siika-aho (2000). The large difference between the results of this study those results may be attributed to the differences in the hydrolysis conditions and possibly the methods for quantifying the MeGlcA, which used glucuronic acid as the standard sugar.
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5.4
CONCLUSION
Application of the recombinant α-L-Arabinofuranosidase (AbfB) from A. niger and purified α-D-glucuronidase (AguA) from S. commune offer a potential novel technique for decreasing solubility and formation of insoluble hydrogels from bamboo, E. grandis and bagasse polymeric xylans. Such technique is required in lignocellulosic processing for production of speciality coatings, additives and encapsulation matrix. The recombinant AbfB and purified AguA both individually and in combination, selectively removed arabinose and MeGlcA side chains respectively, from polymeric xylans pre-extracted from wood and grass sources that led to varying degrees of viscosity change and precipitation. The combined application of the recombinant AbfB and purified AguA increased the removal of arabinose side chains from arabinoglucuronoxylans but not the removal of MeGlcA side chains by the purified AguA. The reduction in the solubility state of the xylans was attributed to both the degree of side chain removal and change in substitution pattern that was dependant on the structural properties of the xylan, enzyme xylan specific dosage, hydrolysis time and temperature. Therefore, optimisation of the enzyme xylan specific dosage, hydrolysis time and temperature during hydrolysis of xylans by the recombinant AbfB and AguA for specific xylan is required for customised removal of the side chains for precipitation and hydrogel formation to ensure efficient use of both the enzymes and the xylan.
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