Doctoral Degrees (Chemical Engineering)

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Browsing Doctoral Degrees (Chemical Engineering) by Author "Chimphango, Annie Fabian Abel"

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    Development of enzyme technology for modification of functional properties of xylan biopolymers
    (Stellenbosch : University of Stellenbosch, 2010-12) Chimphango, Annie Fabian Abel; Gorgens, Johann F.; Van Zyl, Willem Heber; University of Stellenbosch. Faculty of Engineering. Dept. of Process Engineering.
    ENGLISH ABSTRACT: There is growing interest to utilise xylan as speciality biopolymers in similar ways as high molecular weight polysaccharides such as starch and cellulose. The need to utilise xylan as alternative to cellulose and starch has increased because the cellulose and starch have many other competing uses. Unlike cellulose and starch, xylans are heteropolymers with higher degree of substitution and are of lower molecular mass and therefore, do not readily become insoluble to form hydrogels and biofilms. Consequently, xylans do not suit applications of starch and cellulose as speciality biodegradable additives and coatings in the food, pharmaceutical, pulp and paper and textile and many other industries. This study was conducted to develop an enzyme technology, based on recombinant α-L-arabinofuranosidase and purified α-D-glucuronidase with polymeric xylan substrate specificity, for controlled reduction of the solubility of water soluble polymeric xylan, leading to formation of insoluble nanohydrogels. Although xylan is available in abundance, a large proportion of it is currently wasted in lignocellulose process waste streams with little prospects for recovery and addition of value. Lignocellulosic materials including Eucalyptus grandis, Pinus patula, Bambusa balcooa (bamboo) and sugarcane (Saccharum officinarum L) bagasse (bagasse) found in South Africa were investigated as sources of water soluble xylan for enzyme modification. Two mild alkali-low temperature methods (alkali charge of < 14% and temperature of < 80ºC), one with ultrapurification denoted as the Hoije and the other with ethanol precipitation, denoted as Lopez method, were evaluated for their selective extraction of water soluble xylans from the specified lignocellulosic materials. The water soluble xylans were extracted from P. patula, bagasse, E. grandis and bamboo by the Hoije method with extraction efficiencies of 71.0, 66.0, 35.0 and 20.0% respectively. Using the Lopez method, the xylans from bagasse and E. grandis were extracted with extraction efficiencies of 28.0 and 12.0% respectively. The xylans extracted from P. patula, bamboo and bagasse were identified as arabinoglucuronoxylans, which were substituted with arabinose and 4-O-methyl-D- glucuronic acid (MeGlcA) side chains, whereas, the xylan extracted from E. grandis were identified as 4-O-methyl-β-D-glucuronoxylan (glucuronoxylan) substituted with MeGlcA groups on the main xylan chain. In addition, the glucuronoxylans contained some traces of arabinose and rhaminose sugar residues. The extracted xylan fractions had degree of polymerisation (DP) of > 10 and were water soluble, which suited the required properties of xylans for customised enzyme modification. The selective removal of the arabinose, MeGlcA and acetyl groups to create linear regions of xylose units in xylans that causes intra and inter-polymer bonding is considered to be the key process for reducing the solubility of water soluble xylans. The α-L-arabinofuranosidase of Aspergillus niger (AbfB) and α-D-glucuronidase of Schizophyllum commune (AguA) are special enzymes so far identified with the ability to selectively remove arabinose and MeGlcA side chains respectively, from water soluble xylans. Large scale application of the AbfB and AguA for reducing solubility of the water soluble xylans would require their extracellular production in large quantities and free of contamination from the xylan main chain degrading enzymes including the endo-1,4-β -xylanase. Selective production of the AbfB free of xylanase activity was achieved in recombinant A. niger D15 [abfB] strain under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP) and glucoamylase terminator (glaAT). The recombinant AbfB was secreted extracellulary in 125 mL shake flasks and 10 L bioreactor fermentation cultures with volumetric activities of up to 10.0 and 8.0 nkat mL-1 respectively, against para-nitrophenol arabinofuranoside (pNPA). The secretion of the recombinant AbfB was growth associated and therefore, increased up to 2.5 times with addition of concentrate corn steep liquor (CCSL) as an additional source of nitrogen in the 2 x minimal standard cultivation media. The biomass specific activity of the recombinant AbfB against the pNPA substrate was approximately 366 nkat g-1 (dry weight basis). The recombinant AbfB displayed a single pure species band on 10% SDS-PAGE stained with Coomassie blue and had an estimated molecular mass of 67 kDa. In addition, the recombinant AbfB showed optimal activity at 40-55ºC and pH 3.0-5.0 and was stable under cultivation, storage and operating conditions at temperatures between 30-60ºC and pH 3.0-6.0. Furthermore, the recombinant AbfB showed broad substrate specificity selectively removing arabinose side groups from low viscosity wheat and oat spelt arabinoxylans, larchwood arabinogalactan, debranched arabinan and arabiglucuronoxylans extracted from bagasse, bamboo and P. patula found in South Africa,. The recombinant AbfB was able to precipitate xylans extracted from bagasse, bamboo and oat spelt but not from P. patula. Over 95% of the activity of the recombinant AbfB against the pNPA was recyclable after selective hydrolysis of the xylan at 40ºC for 16 h. On the other hand, the purified AguA enzyme could only precipitate the birch glucuronoxylan but not the glucuronoxylan extracted from E. grandis and arabinoglucuronoxylans extracted from bagasse, bamboo and P. patula. The synergetic action of the recombinant AbfB and the purified AguA increased the removal of the arabinose side chains from bagasse xylan by 22% and from bamboo xylan by 33%, whereas, the removal of the MeGlcA side chains from bagasse xylan increased by only 5% and that from bamboo xylan decreased by 13%. The selective removal of the arabinose side chains from oat spelt, bagasse and bamboo xylans by the recombinant AbfB had higher apparent viscosity relative the corresponding untreated xylans. However, the apparent viscosity of both the treated and untreated xylans reduced with increased shear rate. The viscosity had an overall negative correlation with arabinose side chain removal reaching a minimum of 2.03 mPa.s for hydrolysis of oat spelt xylan that was performed for 9.0 h at a temperature of 45.8ºC with recombinant AbfB xylan specific dosage of 400.0 nkat g-1substrate . The alteration of the viscosity of the xylans by the selective removal of the side chains is of special interest in the production of speciality emulsifying, thickening and antifoaming agents. The optimal values for hydrolysis time, enzyme dosage and temperature for maximum degree of removal of arabinose side chains from oat spelt xylan by the recombinant AbfB and of the removal of MeGlcA side chains from birch xylan by the purified AguA were determined by the Box-Benhken response surface method (RSM). The experimental region covered the xylan specific dosage for the recombinant AbfB between 18.0 and 540.0 nkatg-1substrate and for the purified AguA xylan between 2.0 and 18.0 μkatg-1substrate at temperatures between 30 and 50ºC and hydrolysis time between 1 and 16 h. The temperature, enzyme xylan specific dosage and hydrolysis time had significant effect (p<0.05) on both the selective removal of arabinose from oat spelt xylan by the recombinant AbfB and the selective removal of MeGlcA from birch xylan by the purified AguA. However, the interaction of these hydrolysis parameters were significant (p<0.05) on only the removal of arabinose side chains from oat spelt xylan by the recombinant AbfB. The optimal values for hydrolysis time, temperature and xylan specific dosage were estimated to be 14-16 h, 38-45ºC and 607.0 nkatg-1substrate respectively, for maximum removal of 43% of the available arabinose in oat spelt xylan by the recombinant AbfB. Whereas, the optimal values for hydrolysis time, temperature and xylan specific dosage for maximum removal of 0.5% of the available MeGlcA side chains from the birch xylan by the purified AguA were estimated to be 11 h, 38ºC and 18.0 μkatg-1substrate respectively. The optimal values of the hydrolysis parameters for both the removal of the arabinose from oat spelt xylan by the recombinant AbfB and of MeGlcA side chains from birch by the purified AguA could be predicted using quadratic models that fitted the response surface plots with regression coefficients of > 0.9. The effects of in situ selective removal of arabinose and MeGlcA side chains by AbfB and AguA respectively, from water soluble xylans, on their precipitation and adsorption onto cotton lint were investigated. The cotton lint was treated with xylans extracted from bagasse, bamboo, P. patula and E. grandis using the Hoije method in the presence of the recombinant AbfB, AguA and the cocktail of the two enzymes. The effects of in situ selective hydrolysis of model xylans including birch, oat spelt and H2O2 bleached bagasse and E. grandis xylan gel by the enzymes on their adsorption onto cotton lint were used for reference purposes. The purified AguA increased the adsorption of arabinoglucuronoxylans extracted from bagasse bamboo and P. Patula using the Hoije method onto cotton lint the most compared to the effect of the recombinant AbfB and the cocktail of the recombinant AbfB and purified AguA. The purified AguA increased the adsorption of the xylans extracted from bagasse and E. grandis xylans by 334 and 29% respectively, but decreased that of E. grandis xylan gel and H2O2 bleached bagasse xylan by 31 and 6% respectively. Similarly, the presence of the recombinant AbfB increased the adsorption of the bamboo, P. Patula and oat spelt xylans by 31, 44 and 900% respectively, but decreased the adsorption of the xylan extracted from bagasse and the H2O2 bleached bagasse xylan by 13 and 30% respectively. Furthermore, different xylan-cellulose interactions and water adsorption capacities of the cotton lint were observed with the in situ modification and adsorption of the xylans extracted from bagasse, bamboo, E. grandis and P. patula in the presence of the recombinant AbfB and purified AguA. Therefore, the enzyme aided adsorption of xylans could be used to alter or improve functional properties of cellulosic materials. The performance of enzymatically formed xylan nanohydrogels as encapsulation matrices for slow delivery of bioactive agents was evaluated. Insoluble xylan nanohydrogels formed by selective removal of arabinose side chains from water soluble oat spelt xylan by the recombinant AbfB were characterized for particle size distribution, surface charge (zeta potential), morphology stability and ability to encapsulate and slowly release the HRP. The enzymatically formed oat spelt xylan hydrogels were spherical in shape with particle sizes ranging from 18 nm to > 10 000 nm. The xylan nanohydrogels exhibited a negative zeta potential of up to -19 mV and displayed self assembling behaviour when formed at xylan concentrations of higher than 1.5% (w/v) and hydrolysis time beyond 17 h. The xylan concentration significantly (P < 0.05) influenced both the particle size and zeta potential of the oat spelt xylan nanohydrogels whereas the recombinant AbfB hydrolysis time was significant (P < 0.05) on the zeta potential. The oat spelt xylan nanohydrogels successfully encapsulated the HRP enzyme both during and after formation of the oat spelt xylan nanohydrogels and the release of the encapsulated HRP in active form, was sustained for a period of 180 min. Therefore, the xylan side chain removing enzymes have a role in preparation of biodegradable nanoencapsulation devices. Overall, the AbfB and AguA have presented a novel tool for functionalising water soluble xylans to be used as speciality additives, coating and implantation or encapsulation matrices, with reduced impact on the environment. This will advance processing and expand the product spectrum of lignocellulosic materials.