Les galactofuranoconjugués, bien que xénobiotiques chez les mammifères, sont des constituants cruciaux de la paroi cellulaire de nombreux micro-organismes pathogènes. La présence de ces motifs font des galactofuranoconjugués des cibles de choix pour le développement d’outils de lutte ou de diagnostic contre ces micro-organismes et leurs maladies associées. Au cours de ces travaux de thèse, une nouvelle stratégie de synthèse utilisant une α-Larabinofuranosidase, capable de reconnaître un mime du motif D-galactofuranose a été utilisée, la CtAraf51 de Ruminiclostridium thermocellum. Des premiers travaux de mutagénèse ont été entrepris, afin d’améliorer l’affinité de l’enzyme pour le motif non naturel D-Galf, sur trois résidus acides aminés de la poche catalytique, identifiés par des études de modélisation. La création de banques de mutants, le criblage de leur activité et la détermination des paramètres cinétiques nous ont permis d’identifier plusieurs mutants à fort potentiel pour le développement d’une néogalactufuranosidase. Dans le but d’étendre l’activité de l’enzyme à d’autres réactions que l’hydrolyse, l’autocondensation et la transglycosylation, un second axe de recherche a été exploré. La mise au point d’une réaction standard à partir du mutant thioglycoligase et le crible d’accepteurs nucléophiles ont permis d’identifier de nouvelles réactions de thioligation et d’acylation. Enfin les deux axes de recherche ont été mis en commun et ont permis de synthétiser de nouveaux S- et O-galactofuranoconjugués de manière efficace par voie biocatalytique. / Galactofuranoconjugates, while xenobiotic in mammals, are crucial constituents on the cell walls of pathogenic microorganisms. The presence of these patterns, in these microorganisms, makes them molecular targets for the development of tools to fight or prevent the associated diseases. This work describes a new strategy to access galactofuranoconjugates mimetic, using an α-Larabinofuranosidase, the CtAraf51 from Ruminiclostridium Thermocellum. Initial mutagenesis work was undertaken, to improve the affinity of the enzyme for the unnatural DGalf motif. Three amino acid residues in the catalytic pocket were identified by modeling studies and subsequently mutated. The screening of activity of the resulting mutants and the determination of kinetic parameters allowed us to identify several mutants with high potential for the development of a néogalactufuranosidase. A second area of research has been explored with the aim to extend the activity of the enzyme to others reactions than hydrolysis, self-condensation and transglycosylation. The optimization of standard condition from the thioglycoligase mutant and the screening of nucleophilic acceptors has led to the identification of new thioligation and acylation reactions. Finally, the two previous researches were combined in order to synthesize new S- and Ogalactofuranoconjugates in an efficient way.

<p>To ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. &alpha / -L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones.</p>

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.

Hemicellulases are key components in the degradation of plant biomass and carbon flow in nature. Thermotoga hypogea is a bacterium that can grow anaerobically at 90°C. It utilizes carbohydrates and peptides as energy and carbon sources. Three hemicellulytic enzymes: β-xylosidase, α-L-arabinofuranosidase and acetylesterase were investigated. Xylan and xylose were the best substrates for the growth as well as for yielding high activity for all three enzymes in the cells. Glucose grown cells possessed the least amount of enzyme activity for all three enzymes. More than 87% ± 3.0 of β-xylosidase and α-L-arabinofuranosidase activities and 34% ± 11 of acetylesterase activity were associated with the cells. Arabinofuranosidase and acetylesterase were partially purified but β-xylosidase was purified to homogeneity using the Fast Performance Liquid Chromatography system. The latter enzyme has an apparent molecular mass of 75 kDa demonstrated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a nondenatured weight of 130 kDa estimated by Gel-filtration. Its optimal temperature and pH-value for activity were 70°C and 6.0, respectively. The purified enzyme had a half life of 22 min at 70°C and pH 6.0. Among all tested substrates, the purified enzyme had specific activities of 44, 32, 4.5, 1.71 U/mg on p-nitrophenyl-β-xylopyranoside (pNβxp), 4-nitrophenyl-β-D-glucopyranoside (pNβgp), 4-nitrophenyl-α-L-arabinofuranoside (pNαLaf) and 4-nitrophenyl-α-D-xylopyranoside (pNαxp) respectively. The apparent Km of the xylosidase with pNβxp, was 2.6 mM and Vmax was 196 U/mg and for pNβgp the Km and Vmax values were 0.31 mM and 24 U/mg respectively. Based on N-terminal analysis, xylosidase showed high homology with Family 3 β-glucosidases.

<p>To ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. &alpha / -L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones.</p>

Magister Scientiae - MSc / To ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. α-L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones. / South Africa

Élargir le répertoire de composés accessibles dans le domaine des Glycosciences est d’un intérêt majeur pour la communauté des biologistes du fait que ces composés, oligosaccharides et glyco-conjugués, sont impliqués dans diverses fonctions biologiques, aussi bien au niveau structurel, qu’énergétique voire même signalétique jouant un rôle primordial dans les interactions inter- ou intracellulaires. L’assemblage, la modification ou la déconstruction de ces glyco-structures complexes est possible grâce à l’action d’enzymes, parmi lesquelles l’on retrouve les CAZymes (Carbohydrate Active enZymes). Ces enzymes font partie du répertoire de la base de données CAZy, incluant les Glycoside Hydrolases (GHs) qui représentent le groupe le plus important et ayant pour fonction biologique principale l’hydrolyse des liens glycosidiques. Cependant, un certain nombre de GHs possède aussi la capacité de catalyser des réactions de synthèse (transglycosylation) en tant qu’activité secondaire mineure, voire en tant qu’activité principale pour un nombre restreint d’entre elles, qui sont alors appelées transglycosylases. Sachant que ces deux types de comportements peuvent se retrouver au sein d’une même famille de GH (donc étroitement liés sur le plan évolutif), la découverte et la compréhension des déterminants moléculaires qui ont été développés par les GHs au cours de leur évolution pour permettre cette partition d’activité, entre hydrolyse et transglycosylation, est d’une importance capitale pour le domaine de la synthèse chimio-enzymatique et des Glycosciences de manière plus générale.Ce travail de thèse décrit une proposition de synthèse pour apporter une réponse à cette question fondamentale via une revue critique de la littérature sur le sujet. Sur le plan expérimental, a été réalisée l’évolution moléculaire d’une enzyme spécifique des pentoses, l’α-L-arabinofuranosidase de Thermobacillus xylanilyticus (TxAbf) de la famille GH51, vers les premières transarabinofuranosylases de type ‘non-Leloir’. Cette évolution itérative a été développée en utilisant un panel d’outils d’ingénierie enzymatique combinant des approches aléatoire, semi-rationnelle, de prédiction in silico suivie de recombinaison dans un processus d’évolution dirigée global. Une analyse fine des mutants générés sur le plan mécanistique en lien avec la partition hydrolyse/transglycosylation mène à des conclusions en accord avec la proposition de synthèse issue de la revue de la littérature sur le sujet. Sur un plan plus appliqué, ces nouveaux biocatalyseurs ont ensuite été mis en oeuvre dans des voies de synthèse chimio-enzymatiques pour la préparation de composés furanosylés de structure contrôlée. Le transfert d’L-arabinofuranosyles permet la génération d’arabinoxylo-oligosaccharides (AXOS) ainsi que la conception d’oligosaccharides non naturels, tel que des galactofuranoxylo-oligosaccharides ou des arabinofuranogluco-oligosaccharides. Dans son ensemble, ce travail de recherche constitue les premières étapes clés du développement de méthodes de synthèse chimio-enzymatique plus élaborées pour la conception d’arabinoxylanes artificiels. Dans le contexte actuel de transition vers une bio-économie, reposant sur des concepts tels que ceux de la bioraffinerie ou de la chimie verte, nous espérons que les outils de glycosynthèse développés au cours de ces travaux trouveront leur application dans la valorisation des pentoses issus de la biomasse. La synthèse à-façon d’arabinoxylooligo- et polysaccharides présente nombre de valorisations possibles allant de la préparation de prébiotiques à la conception de matériaux bio-inspirés en passant par la synthèse de modèles de parois végétales. / Widening the spectrum of available compounds in the field of Glycosciences is of utmost importance for the entire biology community, because carbohydrates are determinants of a myriad of life-sustaining or threatening processes. The assembly, modification or deconstruction of complex carbohydrate-based structures mainly involves the action of enzymes, among which one can identify Carbohydrate Active enZymes (CAZymes). These enzymes form part of the CAZy database repertoire and include Glycoside Hydrolases (GHs), which are the biggest group of CAZymes, whose main role is to hydrolyze glycosidic linkages. However, some GHs also display the ability to perform synthesis (transglycosylation), an activity that mostly manifests itself as a minor one alongside hydrolysis, but which is the only activity displayed by a rather select group of GHs that are often called transglycosylases. Understanding how transglycosylases have resulted from the process of evolution is both intringuing and crucial, because it holds the key to the creation of tailored glycosynthetic enzymes that will revolutionize the field of glycosciences.In this thesis, an extensive review of relevant scientific literature that treats the different aspects of GH-catalyzed transglycosylation and glycosynthesis is presented, along with experimental results of work that has been performed on a family GH-51 α-L-arabinofuranosidase, a pentose-acting enzyme from Thermobacillus xylanilyticus (TxAbf). The conclusions of the literature are presented in the form of a hypothesis, which describes the molecular basis of the hydrolysis/transglycosylation partition and thus provides a proposal on how to engineer dominant transglycosylation activity in a GH. Afterwards, using a directed evolution approach, including random mutagenesis, semi-rational approaches, in silico predictions and recombination it has been experimentally possible to create the very first ‘non-Leloir’ transarabinofuranosylases. The mechanistic analysis of the resultant TxAbf mutants notably focusing on the hydrolysis/transglycosylation partition reveals that the results obtained are consistent with the initial hypothesis that was formulated on the basis of the literature review.To demonstrate the applicative value of the experimental work performed in this study, the TxAbf mutants were used to develop a chemo-enzymatic methodology that has procured a panel of well-defined furanosylated compounds. Enzyme-catalyzed transfer of arabinofuranosyl moities can be used to generate arabinoxylo-oligosaccharides (AXOS), but the design of non-natural oligosaccharides, such as galactofuranoxylo-oligosaccharides or arabinofuranogluco-oligosaccharides is also possible. Overall, the work presented constitutes the first steps towards the development of more sophiscated methodologies that will procure the means to synthesize artificial arabinoxylans, with a first proof of concept being presented at the very end of this manuscript.In the present context of the bioeconomy transition, which relies on technologies such as biorefining and green chemistry, it is expected that the glycosynthetic tools that have been developed in this work will be useful for the conversion of pentose sugars obtained from biomass. The synthesis of tailor-made arabinoxylo-oligo- and polysaccharides may lead to a variety of potential applications including the production of prebiotics, surfactants or bio-inspired materials and, more fundamentally, the synthesis of artificial models of plant cell wall.

Thesis (PhD (Process Engineering))--University of Stellenbosch, 2010. / 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. / AFRIKAANSE OPSOMMING: Daar is ‘n toenemende belangstelling om spesialiteit biopolimere uit xilaan ontwikkel, en op soortgelyke wyse as hoë molekulêre massa polisakkariede soos stysel en sellulose te benut. Die behoefte om xilaan biodegradeerbare polimere as ‘n alternatief tot sellulose en stysel te gebruik neem toe omdat laasgenoemde baie ander kompeterende gebruike het. Anders as sellulose en stysel is uit xilaan heteropolimere met ‘n hoë graad van substitusie in die hoofketling met sygroepe en lae molekulêre massas, en raak daarom nie geredelik onoplosbaar om hidrojel en biofilms te vorm nie. Gevolglik is xilaan nie geskik vir toepassings van stysel en sellulose as spesialiteit biodegradeerbare bymiddels en bedekkings in die voedsel-, farmaseutiese-, pulp en papier-, tekstiel-, en vele ander industrieë nie. Hierdie studie is uitgevoer om ‘n ensiemtegnologie te ontwikkel gebaseer op rekombinante α-L-arabinofuranosidase en gesuiwerde α-D-glukuronidase met polimeriese xilaan substraat spesifisiteit, vir beheerde vermindering van die oplosbaarheid van wateroplosbare polimeriese xilaan wat lei tot die vorming van onoplosbare nanohidrojels. Alhoewel xilaan volop beskikbaar is, word ‘n groot deel daarvan tans vermors in afvalstrome uit lignosellulose prosessering, primêr verpulping, met min vooruitsigte vir herwinning en toevoeging van waarde. Lignosellulose materiaal wat in Suid-Afrika geproduseer word, insluitend Eucalyptus grandis (E. grandis), Pinus patula (P. patula), Bambusa balcooa (bamboes) en suikerriet (Saccharum officinarum L) (bagasse), is ondersoek as bronne van wateroplosbare xilaan vir ensiem modifikasie. Twee gematigde, lae temperatuur alkali-metodes (‘nalkali lading van < 14% en temperatuur van < 80°C), een met ultrasuiwering aangedui as Hoije en die ander met etanolpresipitasie aangedui as Lopez metode, is evalueer vir selektiewe ekstraksie van wateroplosbare xilaan vanuit die genoemde lignosellulose materiale. Die wateroplosbare xilaan is ge-ekshaheer vanuit P. patula, bagasse, E. grandis en bamboes met die Hoije metode met ekstraksie doeltreffendhede van 71.0, 66.0, 35.0, en 20.0%, onderskeidelik. Met die Lopez metode is xilaan vanuit bagasse en E. grandis geëkstraheer met ekstraksie doeltreffendhede van 28.0% en 12.0%, onderskeidelik. Die xilaan wat vanuit P. patula, bamboes, en bagasse ekstraheer is, is as arabinoglukuronoxilaan geïdentifiseer, wat met arabinose en 4-O-metiel-D glukuronsuur sykettings vervang is, terwyl die xilaan wat vanuit E. grandis ekstraheer is as 4-O-metiel--D-glukuronoxilaan (glukuronoxilaan), met substitusie met MeGlcA en asetiel-groepe op die hoof xilaan-ketting (ruggraat) is. Die glukuronoxilaan het verder spore van arabinose en rhaminose funksionele groepe bevat. Die geëkstraheerde xilaan fraksies het grade van polimerisasie > 10 gehad en was wateroplosbaar, wat die vereiste eienskappe van die xilaan vir doelgemaakte ensiem modifikasies bevredig het. Die selektiewe verwydering van die arabinose, MeGlcA, en asetiel-groepe om xilose eenhede sonder substitusie in polimeriese xilaan te vorm, wat intra- en inter-polimeer binding veroorsaak, word beskou as die belangrikste proses vir die vermindering van die oplosbaarheid van wateroplosbare xilaan. Die α-L-arabinofuranosidase van Aspergillus niger (AbfB) en α-D-glukuronidase van Schizophyllum commune (AguA) is spsialiteutsensieme wat tot dusver is met die vermoë om selektief die arabinose en MeGlcA sykettings, onderskeidelik, vanaf wateroplosbare xilaan te verwyder. Grootskaalse toepassing van die AbfB en AguA ensieme, vir die vermindering van die oplosbaarheid van wateroplosbare xilaan , sal ekstrasellulêre produksie deur mikrobes in groot hoeveelhede en vry van kontaminasie van die xilaan hoofketting degraderende ensieme insluitend die endo-1,4--xilanase vereis. Selektiewe produksie van die AbfB vry van xilanase aktiwiteit is verkry deur kultivering van rekombinante A. niger D15 [abfB], met transkipsie van die abfB-geen beheer deur die gliseraldehied-3-fosfaat dehidrogenase promotor (gpdp) en glukoamilase termineerder (glaAT). Die rekombinante AbfB ensiem is ekstrasellulêr geproduseer in 125 mL skudflesse en ‘n10 L bioreaktor fermentasiekulture met volumetriese aktiwiteite van tot 10.0 en 8.0 nkat mL-1, onderskeidelik, teen para-nitrofenol arabinofuranosied (pNPA). Die uitskeiding van die rekombinante AbfB was groei geassosieerd en het daarom tot 2.5 keer toegeneem met die byvoeging van gekonsentreerde mielieweekvloeistof as ‘n addisionele bron van stikstof in die 2 x minimale standaard kwekingsmedium. Die biomassa spesifieke aktiwiteit van die rekombinante AbfB teen die pNPA substraat was ongeveer 366 nkat g-1 (droë massa basis). Die rekombinante AbfB het ‘n enkele suiwer spesie band getoon op 10% SDS-PAGE gevlek met Coomassie blou en het ‘n beraamde molekulêre massa van 67 kDa gehad. Die rekombinante AbfB het verder optimale aktiwiteit by 40-55°C en pH 3.0-5.0 getoon en was stabiel onder kweking-, storing-, en bedryfstoestande by temperature tussen 30-60°C en pH 3.0-6.0. Die rekombinante AbfB het ook wye substraatspesifisiteit getoon om arabinose sy-groepe selektief te verwyder vanaf lae viskositeit koring-en hawerbiopolimere, lariks arabinogalaktaan, onvertakte arabinaan, en arabinoglukuronoxilaan biopolimere, geëkstraheer vanaf bagasse, bamboes en P.patula wat in Suid-Afrika aangetief word. Die rekombinante AbfB kon xilaan, ge-ekshaheer vanaf bagasse, bamboes en hawer onoplosbaar maak, maar die xilaan geëkstraheer vanaf P. patula nie. Meer as 95% van die aktiwiteit van die rekombinante AbfB teen die pNPA kon hersirkuleer word na selektiewe hidrolise van die xilaan by 40°C vir 16 h. Aan die ander kant kon die gesuiwerde AguA-ensiem slegs berkehout glukuronoxilaan onoplosbaar maak, maar nie glukuronoxilaan wat vanaf E. grandis geëkstraheer is of arabinoglukuronoxilaan wat vanaf bagasse, bamboes en P. patula geëkstraheer is nie. Die sinergistiese aksie van die rekombinante AbfB en die gesuiwerde AguA het die verwydering van die arabinose sykettings vanaf bagassexilaan met 22% vermeerder en met 33% in die geval van bamboesxilaan. Die verwydering van MeGlcA sykettings vanaf bagassexilaan is met slegs 5% vermeerder, terwyl dit met 13% verminder het in die geval van bamboesxilaan. Die selektiewe verwydering van die arabinose sykettings vanaf xilaan van hawer, bagasse, en bamboes deur die rekombinante AbfB het hoër skynbare viskositeit gehad relatief tot die ooreenstemmende onbehandelde xilaan . Die skynbare viskositeit van beide die behandelde en onbehandelde xilaan het egter verminder met toenemende skuiftempo. Die viskositeit het ‘n algehele negatiewe korrelasie met arabinose syketting verwydering gehad en het ‘n minimum van 2.03 mPa.s bereik vir hidrolise van hawerxilaan wat uitgevoer is vir 9.0 h by ‘n temperatuur van 45.8°C met rekombinante AbfB xilaan met ‘n spesifieke dosering van 400.0 nkat g-1substraat. Die wysiging van die viskositeit van die xilaan deur die selektiewe verwydering van die sykettings is van besondere belang in die produksie van spesialiteit emulsifisering, verdikking- en skuimweermiddels. Die optimale waardes vir hidrolisetyd, ensiemdosering en temperatuur vir maksimum graad van arabinose syketting verwydering vanaf hawerxilaan met die rekombinante AbfB, en van MeGlcA syketting verwydering vanaf berkehout xilaan met die gesuiwerde AguA, is vasgestel deur middel van die Box-Benhken responsie oppervlak metode. Die eksperimentele gebied het die xilaanspesifieke dosering met die rekombinante AbfB tussen 18.0 en 540.0 nkat g-1substraat en vir die gesuiwerde AguA xilaan tussen 2.0 en 18.0 μkat g-1substraat by temperature tussen 30 en 50°C en hidrolisetye tussen 1 en 16 h gedek. Die temperatuur, ensiem xilaan spesifieke dosering en hidrolise tyd het elk ‘n beduidende invloed (p<0.05) gehad op beide die selektiewe verwydering van arabinose vanaf hawerxilaan met die rekombinante AbfB en die selektiewe verwydering van MeGlcA vanaf berkehout xilaan met die gesuiwerde AguA. Die interaksie van hierdie hidroliseparameters was egter net beduidend (p<0.05) in die geval van arabinose syketting verwydering vanaf hawer xilaan met die rekombinante AbfB. Die optimale waardes vir die hidrolise tyd, temperatuur, en xilaan spesifieke dosering is beraam om gelyk aan 14-16 h, 38-45°C, en 607.0 nkat g-1substraat, onderskeidelik, te wees vir maksimale verwydering van 43% van die beskikbare arabinose in die hawer xilaan met die rekombinante AbfB. Die optimale waardes vir die hidrolise tyd, temperatuur en xilaan spesifieke dosering vir maksimale verwydering van 0.5% van die beskikbare MeGlcA sykettings vanaf die berkehout xilaan met die gesuiwerde AguA is beraam om gelyk aan 11 h, 38°C, en 18.0 μkat g-1substraat, onderskeidelik, te wees. Die optimale waardes van die hidrolise parameters, vir beide die verwydering van die arabinose vanaf hawer xilaan met die rekombinante AbfB en van MeGlcA sykettings vanaf berkehout met die gesuiwerde AguA, kon voorspel word deur gebruik te maak van kwadratiese modelle wat die responsie-oppervlak grafieke met regressie koeffisiënte > 0.9 gepas het. Die effek van in situ selektiewe verwydering van arabinose en MeGlcA sykettings met rekombinante AbfB en gesuiwerde AguA, onderskeidelik, vanaf wateroplosbare xilaan op hulle presipitasie en adsorpsie op katoen lint is ondersoek. Die katoenlint is behandel met xilaan ge-ekstraheer vanuit bagasse, bamboes, P. patula, en E. grandis deur gebruik te maak van die Hoije metode in die teenwoordigheid van die rekombinante AbfB, AguA, en ‘n mengsel van die twee ensieme. Die effek van in situ selektiewe hidrolise, deur die ensieme van model xilaan insluitende berkehout, hawer en H2O2-gebleikte bagasse en E. grandis xilaan jel, op hulle adsorpsie op katoen lint is gebruik vir verwysingsdoeleindes. Die gesuiwerde AguA het die adsorpsie van arabinoglukuronoxilaan , wat vanuit bagasse, bamboes en P. patula ekstraheer is deur middel van die Hoije metode, op katoenlint die meeste laat toeneem in vergelyking met die effek van die rekombinante AbfB en die mengsel van die rekombinante AbfB en die gesuiwerde AguA. Die gesuiwerde AguA het die adsorpsie van die xilaan wat vanuit bagasse en E. grandis ekstraheer is met 334 en 29%, onderskeidelik, laat toeneem, maar het die adsorpsie van E. grandis xilaanjel en H2O2 gebleikte bagasse xilaan met 31 en 6%, onderskeidelik, laat afneem. Op ‘n soortgelyke wyse het die teenwoordigheid van die rekombinante AbfB die adsorpsie van die bamboes, P. Patula en hawer xilaan met 31, 44, en 900%, onderskeidelik, laat toeneem, maar die adsorpsie van die xilaan ekstraheer vanuit bagasse en die H2O2 gebleikte bagasse xilaan met 13 en 30%, onderskeidelik, laat afneem. Verskillende xilaan-sellulose interaksies en water adsorpsie kapasiteite van die katoen lint is opgemerk met die in situ modifikasie en adsorpsie van die xilaan ekstraheer vanuit die bagasse, bamboes, E. grandis en P. patula in die teenwoordigheid van die rekombinante AbfB en gesuiwerde AguA. Die ensiem bygestaande adsorpsie van xilaan kon daarom gebruik word om die funksionele eienskappe van die sellulose materiaal aan te pas of te verbeter. Die wekverrigting van ensimaties gevormde xilaan nanohidrojels as enkapsuleringmatrikse vir stadige vrystelling van bioaktiewe middels is geevalueer. Onoplosbare xilaan nanohidrojels wat gevorm is deur selektiewe verwydering van arabinose sykettings vanaf wateroplosbare hawer xilaan met die rekombinante AfbA, is gekarakteriseer vir partikelgrootteverspreiding, oppervlaklading (zeta potensiaal), morfologiese stabiliteit, en die vermoë om die ramenas peroksidase te enkapsuleer en stadig vry te stel. Die ensimaties gevormde hawer xilaan hidrojels het ‘n sferiese vorm gehad met partikelgroottes wat gewissel het van 18 nm tot > 10 000 nm. Die xilaan nanohidrojels het ‘n negatiewe zeta potensiaal van tot -19 mV getoon, en het self-vormings gedrag vir partikels ten toon gestel indien dit by xilaankonsentrasies hoër as 1.5% (m/v) en hidrolise tye langer as 17 h gevorm is. Die xilaan konsentrasie het beide die partikelgrootte en die zeta potensiaal van die hawerxilaan nanohidrojels beduidend (P < 0.05) beïnvloed terwyl die rekombinante AbfB hidrolise tyd beduidend (P < 0.05) was op die zeta potensiaal. Die hawer xilaan nanohidrojels, het die ramenasperoksidase ensiem suksesvol enkapsuleer, beide gedurende en na die vorming van die hawer xilaan nanohidrojels en die vrystelling van die geënkapsuleerde ramenas peroksidase in aktiewe vorm is volgehou vir ‘n periode van 180 min. Die ensieme wat die syketting van die xilaan verwyder het, het dus ‘n rol in die voorbereiding van biodegadeerbare nano-enkapsulasie geedskap. In die geheel veskaf die rekombinante AbfB en gesuiwerde AguA ‘n nuwe stel manier voor om wateroplosbare xilaan te funksionaliseer om as spesialiteit bymiddels, bedekking, en inplanting of enkapsulasiematrikse gebruik te word met ‘n verminderde impak op die omgewing. Dit sal prosessering bevorder en die produkspektrum van lignosellulose materiale uitbrei.

The world stocks of fossil fuels are dwindling and may be all but out before the end of the century. Despite this there is increasing demand for them to be used for transport, and the ever increasing green house gases which their use produces. Renewable and less environmentally damaging forms of fuel are needed. Biofuels, particularly bioethanol, are a possibility to subsidise or replace fossil fuels altogether. Ethanol produced from fermentation of starch sugars from corn are already in wide use. As this bioethanol is currently produced from crops such as corn and sugar cane, that puts fuel crops in direct competition for space and resources with food crops. This has led to increases in food prices and the search for more arable land. Hydrolysis of lignocellulosic biomass, a waste by-product of many industries, to produce the sugars necessary for ethanol production would ease many of the problems with current biofuels. Degradation of lignocellulose is not simple and requires expensive chemical pre-treatments and large quantities of enzymes usually from fungal species making it about 10 times more expensive to produce than corn starch bioethanol. The production of a consolidated bioprocessor, an organism able to degrade, metabolise and ferment cellulosic material to produce ethanol or other useful products would greatly reduce the cost currently associated with lignocellulosic biofuel. Cellulomonas fimi ATCC 484 is an actinomycete soil bacterium able to degrade efficiently cellulosic material. The US Department of Energy (DOE) released the genome sequence at the start of 2012. In this thesis the released genome has been searched, for genes annotated as encoding polysaccharide degrading enzymes as well as for metabolic pathways. Over 100 genes predicted to code for polysaccharide hydrolysing enzymes were identified. Fifteen of these genes have been cloned as BioBricks, the standard synthetic biology functional unit, expressed in E. coli and C. freundii and assayed for endo β-1,4-glucanase activity using RBB-CMC, endo β-1,4-xylanase activity using RBB-xylan, β-D-xylosidase activity using ONPX, β-D-cellobiohydrolase activity using ONPC and α-L-arabinofuranosidase activity using PNPA. Eleven enzymes not previously reported from C. fimi were identified as active on a substrate with the strongest activities being for 2 arabinofuranosidases (AfsA+B), 4 β-xylosidases (BxyC, BxyF, CelE and XynH), an endoglucanase (CelA), and 2 multifunctional enzymes CelD and XynF, active as cellobiohydrolases, xylosidases and endoxylanases. Four enzymes were purified from E. coli cell lysates and characterised. It was found that AfsB has an optimum activity at pH 6.5 and 45ºC, BxyF has optimum activity at pH 6.0 and 45ºC and XynH has optimum activity at pH 9.0 and 80ºC. XynF exhibited different optima for the 3 substrates with pH 6.0 and 60ºC for ONPC, pH 4.5 and 50ºC for ONPX and pH 5.5 and 40ºC for RBB-xylan. Searching the genome and screening genes for activities will help genome annotation in the future by increasing the number of positively annotated genes in the databases. The BioBrick format is well suited for rapid cloning and expression of genes to be classified. Searching and screening the genome has also given insights into the complex and large network of enzymes required to fully hydrolyse and metabolise the sugars released from lignocellulose. These enzymes are spread across many different glycosyl hydrolase families conferring different catalytic activities. The characterisation of these novel enzymes points towards a system adapted to not only a broad specificity of substrate but also environmental factors such as high temperature and pH. Genomic analysis revealed gene clusters and traits which could be used in the design of a synthetic cellulolytic network, or for the conversion of C. fimi into a consolidated bioprocessor itself.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: The euascomycetous fungus Aureobasidium pullulans produces xylanolytic accessory enzymes, including an α-L-arabinofuranosidase. The deduced amino acid sequence of the abfA gene encoding α-L-arabinofuranosidase was 69-76% identical to family 54 glycoside hydrolases. The abfA gene encoded a 498 amino acid polypeptide including a signal peptide consisting of 20 amino acids. The mature protein had a calculated molecular weight of 49.9 kDa. One putative N-glycosylation site was found and an iso-electric point of 4.97 was calculated. A. pullulans AbfA was also found to consist of an N-terminal catalytic domain (residues 1-317) and a C-terminal arabinose-binding domain (residues 318-442). The abfA gene from the colour-variant strain of A. pullulans, NRRL Y-2311-1, was recently transferred in Saccharomyces cerevisiae Y294. The yeast culture was grown on synthetic defined medium and α-L-arabinofuranosidase was expressed successfully, secreted from the cells and was purified from the supernatant in a single step using gel filtration. It had an apparent mobility of 52.7 kDa on SDS-PAGE and 36 kDa estimated by gel filtration. The heterologous enzyme was characterized according to pH and temperature dependence and stability, apparent mobility and kinetic properties. The temperature optimum of the recombinant α–L-arabinofuranosidase was 55 °C and it was stable over 3 h at 40 °C. The enzyme displayed optimum activity between pH 4 and 4.5 and was stable at pH 4 over 3 h. Kinetic analysis on p-nitrophenyl-α-arabinofuranoside yielded a Km of 1.43 mM and a Vmax of 23.7 U/mg. Product inhibition was observed and a Ki of 28 ± 3 mM was determined during assaying in the presence of arabinose. A specific activity of 3.85 ± 0.008 U/mg was determined on p-nitrophenyl-α-L-arabinofuranoside and no activity was found on chromogenic substrates which contained a β-linked arabinofuranosyl. The enzyme showed low activity against the 1,5-α-L-arabino-oligosaccharides and cleaved arabinose from corn fibre, oat spelt arabinoxylan and to a lesser degree wheat arabinoxylan. No release of arabinose was observed from larch wood arabinogalactan, α-1,5-debranched arabinan and lignin-arabinose substrates. Linkage preference showed less activity against α-1,5-linked than α-1,2 or α-1,3-linked arabinofuranosyl subunits. Synergism between α–L-arabinofuranosidase and endo-β-1,4-xylanase occurred when measuring the increase in arabinose during wheat arabinoxylan degradation. A. pullulans NRRL Y-2311-1 was grown on synthetic defined medium and native α-L-arabinofuranosidase was expressed and secreted into the culture medium. The native enzyme was partially purified from the supernatant in two steps using gel filtration. The native α–L-arabinofuranosidase had an apparent mobility of 51.5 kDa on SDS-PAGE, displayed optimum activity at 50°C and pH 3. Kinetic analysis on p-nitrophenyl-α-arabinofuranoside gave a Km of 8.33 mM and a Vmax of 1.54 U/mg, and the enzyme showed slight activity against 1,5-α-L-arabinotriose. The properties of the native enzyme were similar to that of the heterologous α–L-arabinofuranosidase. Hydrolysis of sugar cane bagasse by heterologous α–L-arabinofuranosidase and xylanase revealed that pre-treatment with liquid ammonium was more effective in releasing component sugars than a pre-treatment with water at 140º C. A three-dimensional homology model of the heterologous α–L-arabinofuranosidase was constructed using the solved crystal structure of arabinofuranosidase (AkabfB) from Aspergillus kawachii, which was 71 % identical. / AFRIKAANSE OPSOMMING: Die euaskomisetiese swam Aureobasidium pullulans produseer xilanolitiese ensieme, insluitend ‘n α-L-arabinofuranosidase. Die afgeleide aminosuurvolgorde van die abfA geen wat α-L-arabinofuranosidase enkodeer, was 69-76% identies aan familie 54 glikosied hidrolase. Die abfA geen enkodeer ‘n 498 aminosure polipeptied, insluitend ‘n sein peptied bestaande uit 20 aminosure. Die volwasse proteïen het ‘n berekende molekulêre gewig van 49.9 kDa. Een moontlike N-glikosilasie plek is gevind en ‘n iso-elektriese punt van 4.97 is bereken. A. pullulans AbfA bestaan uit ‘n N-terminaal katalitiese gebied (residu 1-317) en ‘n C-terminaal arabinose-bindingsgebied (residu 318-442). Die abfA geen van die kleur-variante ras van A. pullulans, NRRL Y-2311-1, is onlangs na Saccharomyces cerevisiae Y294 oorgedra. Die giskultuur is op ‘n sinteties gedefinieërde medium gegroei en α-L-arabinofuranosidase was suksesvol uitgedruk, uitgedra van af die selle en van uit die supernatant gesuiwer in ‘n enkele stap deur gel filtrasie te gebruik. Dit het ‘n berekende mobiliteit van 52.7 kDa op SDS-PAGE en 36 kDa geskat deur middel van gel filtrasie. Die heteroloë ensiem is gekarakteriseer volgens pH en temperatuurafhanklikheid en stabiliteit, klaarblyklike mobiliteit en kinetiese eienskappe. Die temperatuur optimale van α–L-arabinofuranosidase was 55 °C en dit was stabiel oor 3 ure teen 40 °C. Die ensiem het optimale aktiwiteit tussen pH 4 en 4.5 getoon en was stabiel teen pH4 en oor 3 ure. Kinetiese analiese op p-nitrofeniel-α-L-arabinofuranosied het ‘n Km van 1.43 gelewer en ‘n Vmax van 23.7 U/mg. Produkinhibisie is opgelet en ‘n Ki van 28 ± 3 mM is vasgestel gedurende toetsing in die teenwoordigheid van arabinose. ‘n Spesifieke aktiwiteit van 3.85 ± 0.008 U/mg is vasgestel op p-nitrofeniel-α-L-arabinofuranosied en geen aktiwiteit was gevind op chromogeniese substrate wat ‘n β-verbinding arabinofuranosiel bevat het nie. Die ensiem het ‘n lae aktiwiteit getoon teen die 1,5-α-L-arabino-oligosakkaried en het die arabinose van mielievesel, hawerspelt arabinoxilaan en tot ‘n mindere mate koring arabinoxilaan geskei. Geen vrystelling van arabinose is van af lorkehout arabinogalactan, α-1,5-onvertakte arabinan en lignin-arabinose substrate nie opgemerk. Verbindingsvoorkeure het minder aktiwiteit teen α-1,5-verbindings as α-1,2 of α-1,3- verbindings arabinofuranosiel subeenhede getoon. Sinergisme tussen α–L-arabinofuranosidase en endo-β-1,4-xilanase het plaasgevind met die bepaling van meer arabinose gedurende koring arabinoxilaan degradasie. A. pullulans NRRL Y-2311-1 is op sinteties gedefinieerde medium gegroei en α-L-arabinofuranosidase was uitgedruk en uitgeskei in die kultuur medium. Die inheemse ensiem was gedeeltelik gesuiwer van die supernatant in twee stappe met die gebruik van gel filtrasie. Die inheemse α–L-arabinofuranosidase het ‘n klaarblyklike mobiliteit van 51.5 kDa op SDS-PAGE, het optimum aktiwiteit vertoon by 50°C en pH 3. Kinetiese analiese op p-nitrofeniel-α-arabinofuranosiede het ‘n Km van 8.33 mM en ‘n Vmax van 1.54 U/mg, en die ensiem het effense aktiwiteit teen 1,5-α-L-arabinotrios getoon. Die eienskappe van die inheemse ensiem was soortgelyk aan die van die heteroloë α–L-arabinofuranosidase. Hidroliese van suikerrietbagasse met heteroloë α–L-arabinofuranosidase en xilanase het aan die lig gebring dat vooraf behandeling met vloeistof ammonium meer effektief is in die vrystelling van komponent suikers as ‘n vooraf behandeling met water teen 140º C. ‘n Driedimensionele homologiese model van die heteroloë α–L-arabinofuranosidase is gekonstrueer deur die gebruik van die verklaarde kristal struktuur van arabinofuranosidase (AkabfB) van Aspergillus kawachii, wat 71% identies was.