Patent Publication Number: US-2013233501-A1

Title: Modification of xylan

Description:
FIELD OF THE INVENTION 
     This invention describes a method for enzymatically modifying xylan so that it can be adsorbed or coated onto a substrate, a product comprising modified xylan adsorbed or coated onto the substrate and a pulping process incorporating the method. 
     BACKGROUND TO THE INVENTION 
     The principal components of plant biomass are lignin, cellulose and hemicellulose. Cellulose in the plant cell wall is present in close association with hemicellulose in a matrix encrusted by lignin. One of the major hemicelluloses in most plant cell walls is xylan. Xylan is a polymer consisting of a 1,4-β-linked D-xylose backbone chain substituted with L-arabinose, D-glucuronic acid or both and various short oligosaccharide chains consisting of D-xylose, -L-arabinose, galactose, glucuronic acid, and O-acetyl groups. Depending on the plant biomass source, morphological plant part and physiological stages of plant maturity, the xylan contained therein may also contain feruloyl and p-coumaroyl residues which are ester or ether linked to lignin via glucuronic acid and arabinose side groups, respectively. 
     During conventional kraft pulping of lignocellulosic materials, high alkalinity [pH 12-14] and high temperature [165-170° C.] conditions are used to selectively remove lignin while preserving the cellulosic component [Söjström, 1993, Wood Chemistry: Fundamentals and Applications. 2 nd  edition. Academic Press Limited, London, UK]. However, because xylan is soluble, an undesirable side effect of this process is that xylan is removed along with the lignin, resulting in reduced pulp yields and reduced fibre strength. 
     South Africa uses about 10 million tons of wood annually for kraft pulping. Wood for kraft pulping contains approximately 25% xylan, and about 1.25 million tons of the 2.5 million tons of xylan subjected to kraft processing are estimated to go to waste. Not only is this a waste of a valuable resource, but it is also an environmental hazard, as the degraded xylans release organic acids and chromophogenic compounds which, when coupled with large amounts of chemical discharges from the kraft process, increase the COD loading of waste water streams. 
     Many chemical methods for insolubilizing xylan include the use of alkaline hydrolysis [Söjström, 1993, supra] and desolvation steps, while physical treatments include exposing the xylan to supercritically heated (e.g. up to 170° C.) and compressed fluid for long periods (&gt;3 h) using carbon as an antisolvent. These methods, however, result in random desubstitution and undesirable depolymerisation of the xylan. Under such conditions, xylan and cellulose losses of up to 50% occur, mainly due to deacetylation, depolymerisation and formation of chromophogenic compounds and lignin-xylan complexes (LCC) [Söjström, 1993, supra]. Sternemalm et al. [2008 , Carbohydr. Res.  343: 753-757] used oxalic acid to selectively remove arabinose side groups from oatspelt xylan, but this process caused a reduction in the molecular weight of the xylan from 305 kDa to 169 kDa, with only an associated 6% decrease in arabinose degree of substitution. Although additives such as clay, derivatised starch and cellulosic products are often added to increase yield and improve the pulp fibre function, higher loading of these additives is associated with retention, dusting and fibre-to-fibre contact problems. Inter-fibre bonding is consequently weakened, further reducing paper strength. The use of commercially available cellulose and starch in the pulp and paper making industry must also compete with that of industries associated with food, pharmaceutical, textiles and biofuels production. 
     Accordingly, there is a need for modifying xylan so that it can be beneficially used, and in particular so that it can be adsorbed or coated onto substrates. 
     SUMMARY OF THE INVENTION 
     According to a first embodiment of the invention, there is provided a method of adsorbing xylan onto a substrate, the method comprising the steps of:
         enzymatically modifying xylan which contains glucuronic acid and/or arabinose side chains so that it has reduced solubility in water compared to naturally occurring xylan, by selectively removing glucuronic acid and/or arabinose side chains with one or both of α-D-glucuronidase and α-L-arabinofuranosidase; and   allowing the modified xylan to adsorb onto the substrate.       

     The xylan may be modified in the presence of the substrate, or may be modified in the absence of the substrate and brought into contact with the substrate after it has been modified. 
     The xylan may be modified by selectively removing only glucuronic acid side chains with α-D-glucuronidase, by selectively removing only arabinose side chains with α-L-arabinofuranosidase, or by selectively removing both glucuronic acid and arabinose side chains with α-D-glucuronidase and α-L-arabinofuranosidase. Acetyl groups may also be selectively removed from the xylan with acetyl xylan esterase. 
     The α-L-arabinofuranosidase may be recombinantly produced. 
     The xylan modification step is preferably carried out without the main xylan chain being damaged or degraded. 
     The xylan source may be sugarcane bagasse, bamboo, eucalyptus, pine, oatspelt and birch. 
     The substrate may be a cellulosic substrate, such as pulp, or may be a non-cellulosic substrate. 
     The modified xylan may form a coating on the substrate. 
     A sufficient number of side chains may be removed from the xylan so as to reduce the water solubility of the modified xylan. 
     The xylan may be modified at a temperature of from about 30° C. to about 50° C., and more preferably at from about 35° C. to about 45° C. or at from about 40° C. to about 50° C. The xylan may be contacted with the enzyme for between about 9 and about 18 hours, and the pH may be from about pH4 to about pH6. 
     The xylan to enzyme ratio may be about 5:2, and the xylan loading may be from about 12.5 mL·g −1  to about 25 mL·g −1 . The α-L-arabinofuranosidase enzyme loading may be about 2.5 to about 5 mL·g −1  and the α-L-arabinofuranosidase may have a volumetric activity of about 18.0 nKat mL −1 . The α-D-glucuronidase enzyme loading may be about 0.2 mL·g −1  and the α-D-glucuronidase may have a specific activity of about 300 nKat mg −1 . 
     According to a second embodiment of the invention, there is provided a product comprising xylan adsorbed onto a substrate by a method substantially as described above. The product preferably comprises a higher amount of adsorbed xylan than a product containing adsorbed xylan which has not been modified by removal of some of its arabinose or glucoronic acid side chains. 
     The product may be a cellulosic product, such as pulp, paper, cardboard, packaging or a timber product. 
     The substrate may be coated with the modified xylan. 
     According to a third embodiment of the invention, there is provided a pulping process which includes a xylan adsorption step, the xylan adsorption step comprising the steps of:
         adding xylan which contains glucuronic acid and/or arabinose side chains to a pulp corn position;   enzymatically modifying the xylan so that it has reduced solubility in water compared to naturally occurring xylan by selectively removing the glucuronic acid and/or arabinose side chains with one or both of α-D-glucuronidase and α-L-arabinofuranosidase; and   allowing the modified xylan to adsorb onto the pulp in the pulp composition.       

     The xylan adsorption step may be performed at any one of the following stages in the pulping process: between the filtering and bleaching steps, during washing post bleaching, at the drying stage and at the paper fining stage. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  shows a schematic representation of plasmid pGTP-AbfB: the abfB gene was cloned into the NotI site of pGTP, with the transcriptional control of abfB directed by the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd P ) of  A. nidulans  and glucoamylase terminator (glaA T ) of  A. awamori;    
         FIG. 2  is a graph showing the production characteristics of AbfB by recombinant  A. niger  D15[abfB] in shake flasks in pellet form; 
         FIG. 3  is a graph showing the production characteristics of AbfB by recombinant  A. niger  D15[abfB] in shake flasks produced in 2×MM media (std), 1% concentrate corn steep liquor enriched media (CCSL1), 2% concentrate corn steep liquor enriched media (CCSL2) and 10% concentrate corn steep liquor enriched media (CCSL10), vertical bars denote 0.95 confidence intervals; 
         FIG. 4  is a graph showing the production of AbfB in 10 L BIOFLO 110 bioreactor (mycelial morphology) indicating release of extracellular AbfB activity; 
         FIG. 5  shows the mycelial morphological changes of  A. niger  D15[abfB] at different time intervals during cultivation; 
         FIG. 6  shows graphs of the relative enzyme activity (%) of recombinant AbfB produced by  A. niger  D15[abfB] over a range of (A) pH and (B) temperature using p-NPA as substrate (vertical bars denote standard deviation); 
         FIG. 7  shows graphs of (A) the optical density at 405 nm of a  A. niger  D15[abfB] culture over time at various pH levels, and (B) the relative enzyme activity (%) over time at various temperatures. AbfB assays were conducted under standard conditions (Vertical bars denote 0.95 confidence intervals, p&lt;0.01); 
         FIG. 8  shows (A) a bar graph of the arabinose release (% oatspelt xylan) activity of AbfB on mild alkaline extracted xylan substrate from bagasse (BagH), H 2 O 2  bleached bagasse (BagB), bamboo (Barn), Oatspelt (OT) and  Pinus patula  (Pine). Means with different letters are significantly different (p&lt;0.05); and (B) a bar graph of the arabinose release (% available) activity of AbfB on mild alkaline extracted xylan substrate from Wheat (WAX), Corn (CAX), Debrached arabinan (DAA) and Larchwood arabino galactan (LAG). Means with different letters are significantly different (p&lt;0.05); 
         FIG. 9  shows a graph of the remaining relative activity (%) of the recombinant AbfB after storage at 26, 30 and 37° C.; 
         FIG. 10  shows a graph of the AbfB recovery (residual activity %) of AbfB after hydrolysis of xylan from. H 2 O 2  bleached bagasse (BagBraz), mild alkaline extracted xylan from bagasse using the Höije et al. [2005,  Carbohydr. Polym.  61: 266-275] method (BH) and using the De Lopez et al. [1996,  Biomass and Bioenergy,  10: (4): 201-211] method (BL) and mild alkaline extracted xylan using the Höije method from bamboo (BM) and  Pinus patula  (PP) and commercial oatspelt xylan (Sigma) at 40° C. for 16 h; 
         FIG. 11  shows a graph of the enzyme activity of AbfB in the presence of varying concentrations of pNPA substrate, demonstrating the effect of p-NPA concentration on reaction rate by AbfB produced in (A) a bioreactor on CCSL enriched medium and (B) in shake flasks on CCSL enriched medium. Dotted lines show values derived for Vmax and Km; 
         FIG. 12  shows a graph of the enzyme activity of AbfB in the presence of varying concentrations of pNPA substrate, demonstrating the effect of p-NPA concentration on reaction rate by (A) partially purified AbfB produced in a bioreactor on enriched medium and (B) by AbfB produced in standard 2×MM medium. Dotted lines show values derived for Vmax and Km; 
         FIG. 13  is a comparison of silver stained (Biorad) 10% SDS-Page of crude AbfB profile in lane (a) produced in CCSL enriched medium in a 10 L bioreactor diluted 20×, (b) CCSL enriched medium in a bioreactor at 10× dilution, (c) in standard medium in shake flask at 20× dilution, (d) in standard medium in shake flask at 10× dilution, (e) AkTA partially purified AbfB produced in CCSL enriched medium in fermenters at 20× dilution, and (f) AkTA partially purified AbfB produced in CCSL enriched medium in fermenters at 10× dilution AbfB produced in standard 2×MM medium; 
         FIG. 14  shows (A) a bar graph of the content (% DW biomass) of extractives and ash of bagasse, pine ( Pinus patula ), and bamboo ( Bambusidae balcooa ) and (B) (% DW biomass) of bagasse, pine ( Pinus patula ), and bamboo ( Bambusidae balcooa ); 
         FIG. 15  shows (A) a bar graph of the content (% DW biomass) of cellulose and pentosan of bagasse, pine ( Pinus patula ) and bamboo ( Bambusidae balcooa ); and (B) the xylan yield (% pentosan) extracted using ultrapurification and ethanol precipitation protocols from bagasse, pine ( Pinus patula ) and bamboo ( Bambusidae balcooa ); 
         FIG. 16  shows solid state  13 C-CPMAS NMR spectra showing the effect of mild alkali xylan extraction on the integrity of cellulosic fibres in (A)  Pinus patula , (B) Bagasse, (C)  Eucalyptus grandis  and (D) giant bamboo. The spectra 1, 2, and 3 denote: raw material, extractive free material, and post xylan extracted material and ** denotes peaks for resonances of carbon in glucose units of less ordered cellulose; 
         FIG. 17  shows a comparison of neutral sugar composition of lignocellulosic materials before (EF) and after xylan extraction (Pxyl) for (A)  Pinus patula  (Pine), (B) bagasse (Bag), (C)  Eucalyptus grandis  (EU) and (D) bamboo (BM); 
         FIG. 18  shows the elution profiles of xylan on HPAEC-PAD (Dionex) CarboPac P10 column from (A) monomeric sugars, (B) xylitol), (C) birch xylan (Roth), and (D) Oatspelt xylan; 
         FIG. 19  shows the elution profiles of xylan on HPAEC-PAD (Dionex) CarboPac column P10 from (A) mild alkali extracted H 2 O 2  bleached bagasse (Bag B), (B) mild alkali extracted ultra purified bagasse (Bag H) and (C) mild alkali extracted ethanol precipitated bagasse (Bag L); 
         FIG. 20  shows the elution profiles of xylan on HPAEC-PAD (Dionex) CarboPac column P10 from (A)  Eucalyptus grandis  H [EU H] and (B)  Eucalyptus grandis , L [EU L], (C) bamboo and (D)  Pinus patula;    
         FIG. 21  shows a bar graph of the insoluble fraction obtained after 72% acid hydrolysis of mild alkali extracted xylan H 2 O 2  bleached bagasse (Bag B), ultrapurified bagasse (Bag H), ethanol precipitated bagasse (Bag L), bamboo, ultrapurified  E. grandis  (EU H), ethanol precipitated  E. grandis  (EU L), and  P. patula  (Pine) referenced to Birch xylan (Roth); 
         FIG. 22  shows the characterisation of xylan by (A)  1 H-NMR and (B)  13 C-NMR analyses of birch xylan, (C)  1 H-NMR and (D)  13 C-NMR analyses of H 2 O 2  bleached bagasse (Bag B), and (E)  1 H-NMR and (F)  13 C-NMR analyses of oatspelt xylan. Me denotes Methyl group from glucuronic acid and Ac=acetyl group, Ar=arabinose group; 
         FIG. 23  shows the characterisation of xylan by (A)  1 H-NMR and (B)  13 C-NMR analyses of bagasse, (C)  1 H-NMR and (D)  13 C-NMR analyses of  E. grandis  xylan: In the spectra (1)=Lopez extracted xylan (Bag L or EU L), and (2) Hoije ultapurified xylan (Bag H or EU H); 
         FIG. 24  shows the characterisation of xylan by (A)  1 H-NMR and (B)  13 C-NMR analyses of bamboo, (C) 1 H-NMR and (D) 13 C-NMR analyses of  P. patula  xylan; 
         FIG. 25  shows the FTIR spectra of xylan extracted from different types of lignocellulosic materials from bottom (iv) birch**, (F) ethanol precipitated bagasse [Bag L] (2), (E) ultrapurified bagasse [Bag H] (1), (D) oatspelt xylan*, (C) bamboo, (B) ethanol precipitated  E. grandis  [EU L] (2), (B) ultrapurified  E. grandis  [EU H] (1) and (A)  P. patula;    
         FIG. 26  shows the removal of (A) arabinose and (B) 4-O-MeglcA by AbfB and α-glu respectively from oatspelt/birch, mild alkali pre-extracted bagasse Höije (BH), H 2 O 2  bleached bagasse (BB), bamboo (BM), and  Pinus patula  (PP) xylan, and by α-glu from mild alkali pre-extracted  Eucalyptus grandi , (EH),  Eucalyptus grandis  gel (ES) extracted from pulp, (C) removal of arabinose by AbfB in combination with α-glu and (D) of 4-O-MeglcA by α-glu in combination with AbfB. AbfB xylan specific dosage=720.0 nKat g −1 , Temp=40° C., time=16 h pH 5.0 and α-glu xylan specific dose=9000 nKat g −1  substrate, Temp=40° C., time=16 h and pH 4.8; 
         FIG. 27  shows a graph of arabinose removal from oatspelt xylan after α-L-arabinofuranosidase (AbfB) hydrolysis at varying AbfB xylan specific dosage (nKat g −1  substrate) and hydrolysis time (h) (hydrolysis was performed at 40° C., and pH 5); 
         FIG. 28  shows a graph of arabinose removed from oatspelt xylan after α-L-arabinofuranosidase (AbfB) hydrolysis at varying xylan specific dosage, hydrolysis time (h) and temperature (° C.); 
         FIG. 29  shows response surface plots arabinose removal as a function of (A) time (h) and temperature (° C.) at 42.70 nKat g −1  substrate and (B) temperature (° C.) and enzyme dose (nKat g −1  substrate at 10.8 h) and (C) time (h) and AbfB xylan specific dosage (nKat g −1  substrate) at 35.9° C., and response surface plots of glucuronic acid removal as a function of (D) time (h) and temperature (° C.) at 16500 nKat g −1  substrate, (E) temperature (° C.) and enzyme dose (nKat g′ substrate) at 9 h, and (F) time (h) and enzyme dose (nKat g substrate) at 33.5° C.; 
         FIG. 30  shows interaction effects between time, temperature, and enzyme dose on [A] arabinose [B] glucuronic acid removal. First columns from top showing interaction between temperature (Temp) and time, enzyme dose (AbfB/α-glu) and time, and enzyme dose (AbfB/α-glu) and temperature. In second columns top right cell shows the size and significance of the treatment and interaction effects as measured by the size of bar graph. The t (1, 14)  values are indicated at the end of each bar graph in the respective Pareto chart. The vertical dotted line in the Pareto chart is a measure of statistical significance at p=0.05; 
         FIG. 31  shows a surface plot of the effect of arabinose removal as a function of xylan concentration (μg mL −1 ) and enzyme (AbfB) dose (nkat mL −1 ); 
         FIG. 32  shows a desirability plot of the effect of arabinose removal as a function of xylan concentration (μg mL −1 ) and enzyme (AbfB) dose (nkat mL −1 ); 
         FIG. 33  shows a Pareto chart of the size and the significance of the treatment and interaction effect, the t (1,9)  values are indicated at the end of each bar graph, the vertical dotted line in the Pareto chart is a measure of statistical significance at p=0.05, hydrolysis was performed at 40° C. for 16 h at pH 5.0; 
         FIG. 34  shows (A) AbfB modified oatspelt xylan adsorbed onto cotton lint at a dosage level of 12.5 mL (12.5 CXE) and 25 mL (25 CXE) and unmodified oatspelt xylan at a dosage level of 12.5 mL (12.5CX) and 25.0 mL (25CX), and (B) comparison of xylose removed and arabinose released during adsorption of xylan on cotton lint from unmodified xylan at a dosage level of 12.5 mL (12.5CX) and 25 mL (25CX) and from AbfB modified xylan at a dosage level of 12.5 mL (12.5 CXE) and 25 mL (25 CXE) and Xylose removed and arabinose released from untreated in absence of cotton lint xylan at a dosage level of 12.5 mL (12.5 XY) and 25 mL (25XY) and AbfB modified xylan in the absence of cotton lint at a dosage level of 12.5 mL (12.5XYE) and 25 mL (25XYE); 
         FIG. 35  shows xylose removed, glucuronic acid released and adsorbed onto cotton lint from (A) birch xylan modified with α-D-glucuronidase (α-glu) (BCXE) and unmodified birch xylan (BCX), (B) adsorbed onto cotton lint from pre-extracted xylan from bagasse (BH), H 2 O 2  bleached bagasse (BB), bamboo (BM),  P. patula  (P),  E. grandis  (EH) and  E. grandis  gel (ES) modified by AbfB (A) AbfB and α-glu cocktail (AG), α-glu (G) and unmodified (S); 
         FIG. 36  shows  13 C CPMAS-NMR spectra for cotton lint treated with oatspelt xylan (left) and birch xylan (right). [1] denotes untreated cotton [2] treated with xylan in unmodified form(S), [3] treated with xylan modified with α-L-arabinofuranosidase (AbfB) or α-D-glucuronidase (AguA); 
         FIG. 37  shows  13 C CPMAS-NMR spectra for cotton lint treated with mild alkali extracted xylan from (top)-Bagasse (BH), -Bagasse (BB); and -Bamboo (BM). [1] denotes untreated cotton [2] treated with xylan in unmodified form(S), [3] treated with xylan modified with α-D-glucuronidase [4] modified with α-L-arabinofuranosidase and α-D-glucuronidase (AguA) cocktail, and [5] α-L-arabinofuranosidase (AbfB); 
         FIG. 38  shows  13 C CPMAS-NMR spectra for cotton lint treated with mild alkali extracted xylan from (A)  P. patula  (P), (B)  E. grandis  (EH) and (C)  E. grandis  gel (ES), [1] denotes untreated cotton [2] treated with unmodified form(S), [3] treated with xylan/xylan gel modified with α-D-glucuronidase (AguA), [4] α-L-arabinofuranosidase and α-D-glucuronidase (AguA) cocktail, and [5] α-L-arabinofuranosidase (AbfB); 
         FIG. 39  shows an illustration of the integration of xylan extraction and re-introduction of the xylan in the presence of enzymes to allow adsorption onto pulp fibres during the kraft pulp and paper making process. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A method of modifying xylan so that it can be adsorbed onto other substrates is described herein. The method includes the step of modifying water-soluble xylan containing arabinose and/or glucuronic acid side chains by selectively removing some of these side chains with an enzyme which has selective hydrolytic activity for these side chains, and in particular with α-L-arabinofuranosidases (AbfB) (EC 3.2.1.55) and/or α-D-glucuronidases (AguA) (EC 3.2:1.131-). If the xylan also contains acetyl side chains, then an acetyl xylan esterase (EC 3.1.1.72) can also be used to selectively remove these groups. The modified (desubstituted) xylan, which is insoluble in water, is then allowed to adsorb onto the substrate. For example, the modified xylan, which is less substituted, can be used in pulping so as to increase binding properties of pulp fibres. Alternatively, the modified xylan can be adsorbed onto a surface of a substrate, so as to form a coating on the substrate. 
     Depending on the source of the xylan, it may first be necessary to extract the xylan from a lignocellulosic material, such as oatspelt, birch, bagasse (BH), bamboo (BM),  Pinus patula  (PP) or  Eucalyptus grandis  (EH). H 2 O 2  bleached bagasse xylan (BB) or  E. grandis  xylan gel (ES) can also be used. The xylan can be extracted by a mild alkali extraction method, which does not damage the main xylan chain. 
     The xylan should be modified with one or more enzymes which do not degrade the main xylan chain but which are able to remove a sufficient number of arabinose or glucuronic acid side chains, typically non-terminal side chains, from the xylan. Suitable enzymes for use in this method are α-L-arabinofuranosidases (AbfB) (EC 3.2.1.55) and/or α-D-glucuronidases (AguA) (EC 3.2.1.131-), which have been found to selectively remove arabinose and glucuronic acid, respectively, without depolymerising the xylan main chain. The applicant has found that these enzymes, on their own or in combination, are able to effectively remove a sufficient number of the arabinose and/or glucuronic acid side chains, on an industrial scale, so as to decrease water solubility of the xylan and hence to induce xylan precipitation and allow adsorption of the xylan onto other materials or substrates. In some xylans, acetyl groups are attached to the glucuronic acid side chains and should also be removed during modification of the xylan using acetyl xylan esterases (EC 3.1.1.72). 
     The enzymes should be purified or prepared recombinantly so that they aren&#39;t contaminated with other enzymes which could break down the main xylan chain, such as xylanase. For this reason, the α-L-arabinofuranosidase which was used in the examples below was recombinantly produced. 
     The xylan is generally modified at a temperature of from about 30° C. to about 50° C., and more preferably at from about 35° C. to about 45° C. or at from about 40° C. to about 50° C., and can be contacted with the enzyme for between about 9 and about 18 hours, and the pH may be from about pH4 to about pH6. 
     The extracted xylan to enzyme ratio is typically about 5:2, and the xylan loading can be from about 12.5 mL·g −1  to about 25 mL·g −1 . The α-L-arabinofuranosidase enzyme loading can be about 2 to about 10 mL·g −1  and the α-L-arabinofuranosidase can have a volumetric activity of about 18 nKat mL −1 . The α-D-glucuronidase enzyme loading can be about 0.2 mL·g −1  and the α-D-glucuronidase can have a specific activity of about 300 nKat mg −1 . 
     The xylan can be modified in situ in the presence of the substrate, such as when it is used as an additive for pulp, or alternatively may be modified in the absence of the substrate and then brought into contact with the substrate, such as when it is used to form a coating on the substrate. 
     The substrates onto which the modified xylan can be adsorbed include cellulosic materials such as pulp, paper, cardboard, packaging, textiles and timber products. The substrate can also be a non-cellulosic material, such as metal, mica, magnetic material, pharmaceutical capsules or tablets and the like. When used as an additive, e.g. in the pulping process, adsorption of the xylan to the pulp can result in improved pulp yield, improved pulp bonding properties, improved pulp and paper strength and so forth. Alternatively, when used as a coating material, adsorption of the modified xylan to the substrate can alter the surface properties of these substrates, allowing them to have different uses. For example, the modified xylan may form a hydrogel which encapsulates or entraps a bioactive molecule, such as a bactericide, and this hydrogel may adsorb, onto a surface of a substrate to impart antibacterial properties to the substrate. 
     The steps of modifying the xylan and adsorbing it onto a pulp substrate can be incorporated into existing wet end pulping processes without too much difficulty, such as between the filtering and bleaching steps, during washing post bleaching, at the drying stage or at the paper fining stage. Typical pulping processes include kraft, alkaline and sodaAQ processes. 
     In the examples described below, the effects of α-L-arabinofuranosidase (AbfB) and α-D-glucuronidase (AguA) on adsorption onto cotton lint of xylan derived from oatspelt, birch, bagasse (BH), bamboo (BM),  Pinus patula  (PP) and  Eucalyptus grandis  (EH), and of H 2 O 2  bleached bagasse xylan (BB) and  E. grandis  xylan gel (ES) were investigated. The adsorption of oatspelt xylan (1% w/v) onto the cotton lint increased by 33% and 900% (9-fold) in the presence of AbfB at xylan loading of 25 mL g −1  and 12.5 mL g −1  cotton lint, which corresponded to 30% and 50% release of the available arabinose, respectively. The adsorption of BH, BM and  P. Patula  in the presence of AguA increased by 29, 82 and 112%, respectively, whereas in the presence of AbfB, the adsorption of BH decreased by 13% but that of the BM and  P. patula  xylan increased by 31% and 44%, respectively. A cocktail of AbfB and AguA increased adsorption of the BH, BM and  P. patula  xylan more than that of AbfB but such increase was lower than the increase which occurred in the presence of AguA. The AguA aided xylan adsorption was more effective for birch,  P. patula  and  E. grandis  than bagasse and bamboo and for mild alkali extracted bagasse and  E. grandis  xylan than H 2 O 2  bleached bagasse xylan and  E. grandis  xylan gel. The removal of the glucuronic acid by AguA imparted xylan with more binding Power towards the cotton lint than the removal of arabinose side groups by the AbfB. 
     EXAMPLES 
     The invention will now be described in more detail by way of the following non-limiting examples. Although only cotton lint is used as a substrate onto which the modified xylan can be adsorbed, it will be apparent to a person skilled in the art that the modified xylan could also be adsorbed onto other substrates. 
     Material and Methods 
     Materials 
     Mild alkali extracted xylan from  Saccharum officinarum  L (sugarcane) bagasse (BH),  Bambusidae balcooa  [giant bamboo (BM)],  Eucalyptus grandis , [Eucalyptus (EH)],  Pinus patula  [Pine (PP)] (extracted using a mild alkali method described by Höije et al. [2005  Carbohydr. Polym.  61: 266-275]), commercial oatspelt xylan (Sigma), birch xylan (Roth), mild alkali H 2 O 2  bleached xylan from bagasse (BB) (donated by Prof. A. M. F. Milagres, University of Sáo Paulo, Brazil) and xylan gel from  E. grandis  (ES) (donated by SAPPI—Pretoria, South Africa) were used in the experiments. 
     Xylan solutions (1% w/v) were prepared according to de Wet et al. [2008, Appl. Microbiol. Biotechnol. 77: 975-983]. Non absorbent cotton lint (Grade 1, Cotton King) was used as a cellulosic fibre source. The arabinose side chains were moved by crude α-L-arabinofuranosidase (AbfB) with volumetric activity of 18 nKat mL −1  on p-Nitrophenyl Arabinofuranoside (p-NPA) produced inhouse using recombinant  Aspergillus niger  D15. The glucuronic acid side chains were removed by α-D-glucuronidase (AguA) with specific activity=300 nKat mg −1  that was purified from wild type  Schizophyllum commune  (VTT-D-88362-ATCC 38548, donated by Prof. Matti Siika-aho of VTT Biotechnology Institute in Finland). 
     Production of Recombinant α-L-Arabinofuranosidase (AbfB) 
     A recombinant α-L-Arabinofuranosidase (AbfB) was produced by cloning the AbfB gene from  A. niger  into the protease deficient and medium non-acidifying strain  A. niger  D15, under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd P ) of  A. nidulans  and the glucoamylase terminator (glaA T ) of  Aspergillus awamori  ( A. awamori ). The growth characteristics of the  A. niger  D15[AnabfB] strain and production levels of AbfB were studied in shake flasks and a bioreactor using defined standard media (2×MM media) and cornsteep liquor enriched media. The protein profiles, substrate specificity, substrate dependency, optimal pH and temperature, stability in application and storage, and recyclability were assessed as described below. 
     Cultivation of Bacterial and Fungal Strains 
     The genotypes of the bacterial and fungal strains as well as the plasmids used are summarised in Table 1. Recombinant plasmids were constructed and amplified in  E. coli  DH5α.  E. coli  was cultivated at 37° C. in LB medium (1% yeast extract, 1% tryptone and 0.5% NaCl) on a rotary shaker at 100 rpm, supplemented with 100 μg/L ampicillin. The  A. niger  fungal strains were maintained at 30° C. in minimal media (MM) and on spore plates according to the procedure of Rose and Van Zyl [2002  Appl. Microbiol. Biotechnol.  58: 461-468]. The media in which the  A. niger  D15 parental strain was maintained was supplemented with 0.01 M uridine. Transformants were prepared according to the procedure of Rose and Van Zyl [2002  Appl. Microbiol. Biotechnol.  58: 461-468] and selected on MM lacking casamino acids and uridine. Transformants were cultivated in Erlenmeyer shake flasks (125 mL) containing 30 mL of double strength traditional minimal medium (2×MM) containing 10% glucose. The medium was inoculated to a final spore concentration of 1×10 6  spores ml −1 . The  A. niger  strains were cultivated at 30° C. on a rotary shaker (New Brunswick Scientific, Edison, W. J., and U.S.A) at 120 rpm. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Genotype and sources of the strains and plasmids used in transforming 
               
               
                 
                   Aspergillus niger 
                 
               
            
           
           
               
               
               
            
               
                   
                 Genotype 
                 Source 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                 Strain: 
                   
                   
               
               
                   A. niger  D15 
                 pyrG prtT phmA (nonacidifying) 
                 Wiebe et al., 2001,  Biotechnol.   
               
               
                   
                   
                   Bioeng.  76: 164-174 ATCC 
               
               
                   
                   
                 9029 
               
               
                   A. niger  van Tieghem 
                 Wild-type 
                 ATCC 10864 
               
               
                   A. niger  D15[pGTP]* 
                   A. niger  D15 with gpdP-glaAT 
                 Rose and Van Zyl, 2008,  The Open   
               
               
                   
                   
                   Biotechnol. J.  2: 167-175 
               
               
                   A. niger  D15[abfB]* 
                   A. niger  D15 with gpdP-abfB-glaAT 
                 This study 
               
               
                   E. coli  DH5α 
                 supE44 ΔlacU169 (ø/80lacZΔM15) 
                 Sambrook et al., 1989, Molecular 
               
               
                   
                 hsdR17 recA1 endA1 gyrA96 thi-1 
                 cloning: a laboratory manual. Cold 
               
               
                   
                 relA1 
                 Spring Harbor Laboratory, Cold 
               
               
                   
                   
                 Spring Harbor, NY 
               
               
                 Plasmids: 
               
               
                 pBS-pyrGamdS 
                 bla pyrG amdS 
                 Plüddemann and Van Zyl, 2003, 
               
               
                   
                   
                   Curr. Genet.  43: 439-446 
               
               
                 pGTP 
                 bla gpdP-glaAT pyrG 
                 Rose and Van Zyl, 2008,  The Open   
               
               
                   
                   
                   Biotechnol. J.  2: 167-175 
               
               
                 pGTP-abfB 
                 bla gpdP-abfB-glaAT pyrG 
                 This study 
               
               
                   
               
               
                 *plasmids were integrated 
               
            
           
         
       
     
     Spores produced from  A. niger  D15 [abfB] plates were re-inoculated on rice (Tastic rice) sporulation medium to produce spores at a relatively higher concentration (&gt;1×10 6 ). To prepare the rice sporulation medium, tastic rice (20 g) was placed in Erlenmeyer shake flasks (250 mL) to which 8 mL of 0.1% (w/v) urea were added. The rice was then autoclaved at 121° C. for 15 min. Spores were added to each flask and incubated at 30° C. for a period of 3-6 days or until 80% of the rice surface was observed to be covered with spores. The spores were harvested into a 0.9% NaCl solution (approximately 50 mL for each flask) and stored in Schott bottles at 4° C. 
       A. niger  spores were inoculated in 2×MM medium enriched with concentrated corn steep liquor (CCSL) which was prepared according to a modified version of the protocol of Gurlal et al. [2006, CSIR/BIO/IR/PPD/2006/0023/B, CSIR, pp 1-8]. The CCSL was donated by Mr. Hough Joubert of African Products-Belleville, South Africa. The CCSL with initial pH of pH 3.87 was sterilised (121° C., 15 min, 1 bar) and filtered (0.22 μm pore size) before being added to the standard medium at 1%, 2%, and 10% (w/v).  A. niger  was cultivated in the respective media under standard  A. niger  cultivation conditions. Culture samples were taken at 24 h intervals for 7 days and the AbfB activity determined. Unless otherwise stated, the CCSL optimised medium was used in the subsequent cultivations of  A. niger.    
     Cloning of pGTP and pGTP-AbfB Plasmid Constructs 
     Standard protocols such as those described by Sambrook et al. [1989, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.] were followed for all DNA manipulations. The method used to construct the plasmids and the fungal strains was substantially according to that described by Rose and Van Zyl [2002 Appl. Microbiol. Biotechnol. 58: 461-468]. Briefly, the pyrG gene was amplified from pBS-pyrGamdS [Plüddennann and Van Zyl, 2003  Curr. Genet.  43: 439-446] by standard PCR techniques and cloned into the EcoRI site of plasmid pGT [Rose and Van Zyl, 2002 , Appl. Microbiol. Biotechnol.  58: 461-468] to generate pGTP. For the production of recombinant α-L-arabinofuranosidase (AbfB), the abfB gene was amplified from the genome of  A. niger  10864 using standard PCR methods. The abfB was then cloned into the NotI site of pGTP, generating pGTP-AbfB ( FIG. 1 ) in which the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd P ) of  A. nidulans  and the glucoamylase terminator (glaA T ) of  A. awamori  were located upstream and downstream of the abfB gene respectively. Plasmids pGTP and pGTP-AbfB were then integrated into the genome of  A. niger  D15 in multiple sites according to standard techniques. A total of 100 putative  A. niger  D15[abfB] transformants were prepared and screened for extracellular AbfB activity. The selected  A. niger  D15[abfB] transformants were then cultivated in shake flasks in at least three replicates (with 3 repetitions) to evaluate stability in cultivation conditions with respect to extracellular AbfB production. 
     Cultivation of  A. niger  D15[abfB] 
     The morphological and growth characteristics of  A. niger  D15[abfB] were monitored visually and under electronic microscopic. Mycelia and pellets from bioreactor and terminated shake flasks cultures, were observed under a microscope (100× magnification) prior to filtering through Mira cloth mesh. The biomass was washed with deionised water (dH 2 O) before being transferred into pre-weighed aluminum foils for drying in an oven at 60° C. until a constant weight was obtained. The biomass concentration was calculated as an average dry weight of biomass (dry wt) per unit volume of the culture (g L −1 ). 
     The pellet formation was achieved by inoculating the medium (either 2×MM or CCSL (2% w/v) enriched 2×MM medium), with 1×10 6  spores mL −1  and incubating the shake flasks at 30° C. on a shaker (120 rpm). The cultivation medium had an initial pH of 5.5-6.0 and 5.0 for 2×MM and CCSL enriched, respectively. Mycelial production of AbfB by  A. niger  D15[abfB] was carried out in a 14 L capacity stirred tank bioreactor (BIOFLO 110 modular bench top fermentation system, New Brunswick Scientific company, Inc, USA) containing 8 L of optimised medium enriched with CCSL (2% w/v) and a spore inoculum of 1×10 6  spores mL −1  maintained at 30° C. Mass transfer was achieved using a single 3 bladed pitched impeller (30×20 mm) at an agitation speed regulated between 350-700 rpm depending on the level of the dissolved oxygen (DO) in the bioreactor. The DO was maintained above the critical point (&gt;20%) with air and oxygen supplied through a sparger and the levels regulated by a rotameter at 0.5 vessel volume per min (0.5 VVM). Sampling was done through a port installed with a 0.2 μm air filter connected to a syringe for suction into JA 20 centrifuge bottles every hour for the initial 24 h and thereafter every 3-4 h for determination of extracellular AbfB activity, biomass growth and substrate concentration. Foaming was controlled by the addition of 0.1% (v/v) of antifoam A (30% aqueous emulsion with emulsifiers, A 5758, Sigma). Excess foam was collected in a foam trap. The cultures were terminated after 144 h. 
     Fractionation and Partial Purification of AbfB Enzyme Preparations 
     The enzyme supernatant harvested from bioreactor fermentation cultures was filtered through Mira cloth. The filtrate was centrifuged (Beckman, J2-21 centrifuge) at 12 000 rpm for 10 min at 4° C. The resulting supernatant was filtered through 0.2 μm filters and concentrated using an Amicon system, Diaflo Ultrafilter PM 10 concentrator, (Amicon Division, W.R. Grace &amp; Co., USA) or a Millpore Minitan ultrafiltration system (Millpore Corporation) with MWCO of 10 kDa. The choice of the ultrafiltration system was based on the initial sample volume. Concentration of crude enzyme samples was carried out by lyophilising the enzyme supernatant in Virtis freeze dryer. The concentrated enzyme supernatant was subjected to ammonium sulfate fractionation at a percentage saturation of 60 and 80% while mixing at 200-250 rpm for 4 h at 4° C. The protein mixture was centrifuged at 1200 rpm for 1 h at 4° C. The pellet obtained at the desired saturation level was re-suspended in 5 mL of Milli-Q water and dialysed against 2 L of 10 mM acetate buffer (pH 5.0) at 4° C. overnight. The desalted concentrated enzyme supernatant was subjected to a single step partial purification in a fast performance gel filtration chromatography (ÄKTA purifier system, Amersham Pharmacia Biotech) installed with UNICORN computer control system (version 3.2). The protein sample (0.3 mL) was applied to a Superdex 75 HR 10/30 size exclusion column. The protein was eluted with 0.05 M acetate buffer pH 4.0 containing 0.4 M NaCl at a flow rate of 0.5 mL min −1 . Fractions demonstrating AbfB activity were pooled and concentrated using the Amicon system before molecular and kinetic analysis. 
     AbfB Characterisation 
     Determination of AbfB Molecular Weight (MW) 
     Enzyme preparations were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [1970, Nat. 227: 680-685]. The resolved proteins were initially stained with Coomassie Brilliant Blue R250 and subsequently with silver stain (Bio-Rad kit). The molecular weight (MW) of the resolved protein was estimated from the gel using BenchMark™ prestained protein marker (Invitrogen and Prestained protein ladder (Fermentas). The theoretical MW and isoelectric point (pl) of the AbfB was estimated from the original protein sequence (protein length of 499 bases) using DNAMAN sequence analysis computer software. 
     Determination of α-L-Arabinofuranosidase Activity of AbfB Using pNPA 
     α-L-arabinofuranosidase activity of AbfB (AbfB activity) was determined using a colorimetric assay in which the release of p-Nitrophenol from p-Nitrophenyl-α-arabinofuranoside (C 11 H 13 NO 7 , pNPA, Sigma) was measured. A 100 μL reaction mixture was prepared which consisted of 25 μL 5 mM pNPA in 0.05M citrate buffer (pH 5.0), 25 μL deionised water (dH 2 O), 25 μL of appropriately diluted supernatant and 25 μL 0.05M citrate buffer (pH 5.0). The reaction was incubated at 40° C. for 10 min and was terminated by the addition of 100 μL saturated sodium tetraborate (Na 2 B 4 O 7 , Sigma-Aldrich). The mixture was diluted five times before measuring the absorbance at 405 nm wavelength using a spectrophotometer. The AbfB activity was determined from a standard curve using p-Nitrophenol (pNP, C 6 H 5 NO 3 , Sigma) as standard under similar assay conditions. The AbfB activity was calculated as the amount of enzyme capable of releasing 1 nmole of pNP from the pNPA substrate per mL per second (μkat/mL, conversion factor: 1 Unit=0.0167 μkat). Unless stated otherwise, all assays were conducted in triplicate. Commercial α-L-arabinofuranosidase from  A. niger  (E-AFASE, Megazyme) was used as a positive control. 
     Determination of pH and Temperature Optima for AbfB Activity 
     Recombinant AbfB was characterised with respect to optimal pH and temperature under the assay conditions described above. The pH optimum and pH stability of AbfB was determined using pNPA desolved in McIlvaine&#39;s buffer [McIlvaine, 1921,  J. Biol. Chem.  49: 183-186]. Optimal temperature conditions for and temperature stability of the recombinant AbfB was determined using pNPA in 0.05 M citrate buffer at pH 5.0. The effect of substrate concentration on AbfB activity was then determined by using pNPA prepared at varying concentrations in 0.05M citrate buffer pH 5.0. The assays were conducted under AbfB assay conditions as described above. The saturation kinetic properties, maximum activity (V max ) and Michael Menten constant (Km) of partially purified and crude AbfB, were estimated from the plot of release of p-NP as a function of substrate concentration. 
     Determination of α-L-Arabinofuranosidase Activity of AbfB Using Xylan 
     Endo-xylanase (Xyln) activity of the AbfB preparations was assessed by measuring the release of reducing sugars from birchwood xylan (Roth, Germany) using dinitrosalicylic acid (DNS) [Miller, 1959,  Anal. Chem.  31: 426-428] according to the assay protocol described by Bailey et al. [1992 , J. Biotechnol.  23: 257-270] and using xylose (Merck) as standard. Xylan activity was calculated as the amount of enzyme required to release nmoles of xylose per unit volume per second (nmoles mL −1 sec −1 ) presented as nkat mL −1 . Residual sugar concentration in the cultures was determined using the phenol-sulfuric acid assay [Dubois et al., 1956 , Annal. Chem.  28 (3):350-356]. The quantity of the respective sugars was calculated from a standard curve plot of absorbance as a function of the sugar concentration. All reactions were performed in triplicate. Total protein in enzyme preparations was determined by the Bradford method [Bradford, 1976 , Anal. Biochem.  72: 248-254] using the Biorad protein assay kit. The quantity of protein was determined using bovine serum albumin (BSA) as standard. 
     Crude and partially purified forms of recombinant AbfB were assessed for activity against pNPA and polymeric substrates. The substrates tested included: oatspelt xylan (10:15:75 (arabinose:glucose:xylose); Sigma), low viscosity wheat arabinoxylan (37:61:2, arabinose:xylose:other sugars, Megazyme), corn fibre xylan (30% arabinose), donated by Dr. Shin Li, NRL, Preoria, Ill., U.S.A), larchwood arabinogalactan (15:85, arabinose:galactose, Megazyme), debrached arabinan (sugar beet), (88:4:2:6) (arabinose:galactose: rhaminose:galactouronic acidUA) (Megazyme) and mild alkali extracted xylan from bagasse, pine ( Pinus patula ), and bamboo ( Bambusidae balcooa ). A solution containing 1% (w/v) of the substrate was prepared. Substrates with limited solubility in water were first dissolved in ethanol (0.1 g in 0.8 mL 99% ethanol), 9 mL of dH 2 O was added, heated to 70-100° C. while stirring for 10 min, after cooling to room temperature, the volume was adjusted to 10 mL). The reaction mixture with a final volume of 5 mL, contained 2.5 mL of the substrate, 1.5 mL 0.05 M citrate buffer (pH 5.0), and 1 mL AbfB of known enzyme activity. The reaction was performed in a water bath at 40° C. for 16-24 h. Termination of the reaction was achieved by placing the reaction test tubes on ice. The enzymatic hydrolysates (200 μL) were diluted 5 times and centrifuged at 10 000 rpm for 5 min at 4° C. followed by filtration using filters with 0.22 μm pore size. Samples were stored at −20° C. prior to sugar analysis. The sugar analysis was performed using a high pH anion exchange chromatography coupled with pulsed electrochemical detection (HPAEC-PAD) (Dionex) equipped with a gradient pump GP 50, a Carbopac PA 10 (4 mm×250 mm) column, and electrochemical detector (ED40) for pulsed amperometric detection (PAD). The column was eluted with helium degassed 250 mM NaOH and Milli-Q water in 1.5:98.5 ratio at elution rate of 1 mL min −1 . The PEAKNET software package was used for data acquisition and analysis of sugar concentration. Quantity of the respective sugars was determined from a standard curve plot of the respective analytical grade sugars (arabinose, rhaminose, galactose, glucose, mannose, and xylose). The quantity of the sugars was expressed on oven dry substrate weight. Residual activity in the AbfB xylan hydrolysate was assessed using the AbfB standard assay. 
     Enzyme Aided Xylan Adsorption onto Cellulosic Material 
     Cotton lint samples (1 g) were autoclaved in 100 ml Schott bottles at 121° C. for 15 min. After cooling to room temperature, the oatspelt xylan solution (1% w/v) 5, 12.5, 15.0 and 25 mL and AbfB (18.0 nKat mL −1 ) were added in a fixed xylan to enzyme ratio of 5:2. The xylan adsorption mixtures contained no enzyme while the positive control mixtures contained the enzyme but in absence of the cotton lint. The adsorptions were performed in 0.05 M citrate buffer pH 5.0 at 40° C. for 24 h in a 40 mL reaction volume. All reactions were terminated by placing the bottles in water containing ice. Cotton lint samples weighing 0.2 g were placed in 15 mL glass bottles in two sets of three and a control. The bottles were autoclaved at 121° C. for 15 min. Upon cooling to room temperature, 750 μL birch xylan (1% w/v) and 150 μL α-D-glucuronidase (AguA) (900 nKat mL −1 ) were added to one set. Subsequently, 0.05 M acetate buffer (pH 4.8) was added to make a total reaction mixture volume of 1650 μL. The negative and positive controls consisted of the birch xylan in the absence of AguA and in the absence of cotton lint, respectively. Cotton lint (0.2 g) was treated in the adsorption mixture prepared from mild alkali pre-extracted bagasse, bamboo and  P. patula  xylan in presence of a combined cocktail of α-L-arabinofuranosidase and α-D-glucuronidase in glass bottles. The reaction mixture contained 0.2 g cotton lint, 1000 μL xylan (1% w/v), The adsorption mixture contained α-L-arabinofuranosidase (AbfB) (18.0 nkat mL −1 ), and α-D-glucuronidase (900 nKat mL −1 ) and 0.05 M acetate buffer pH 4.8. The reaction was performed at 40° C. in a water bath for 16 h. 
     Post Xylan Adsorption Cotton Lint Analysis 
     Treated cotton lint samples were vacuum-filtered and subsequently washed by suspending them in 50-100 mL Milli-Q H 2 O while agitating for 1 h to disengage xylan precipitates loosely absorbed to the cotton lint. The Milli-Q H 2 O was changed 3 times during the washing process. The samples were, after the final wash, vacuum filtered in pre-weighed filter papers and placed in pre-weighed foils for drying overnight at 30° C. to a constant weight. The amount of xylan adsorbed onto the cotton lint was defined as the difference between the initial weight of the cotton lint and the weight after the xylan treatment presented as a percentage of the initial weight of the cotton lint. Xylan specific weight gain for the cotton lint was defined as the weight gained by the cotton lint as a percentage of the initial amount of xylan in the reaction mixture. The calculations were corrected for moisture content of the starting materials and cotton lint losses during post treatment. The individual enzyme effect on xylan adsorption onto cotton lint was defined as the difference in weight gain between cotton lint treated with unmodified xylan and cotton lint treated in presence of the xylan. 
     Carbohydrate Composition Adsorption Mixture and Cotton Lint 
     The xylan adsorption reaction mixture filtrates were centrifuged at 10,000 rpm for 5 min at 4° C. followed by filtration using 0.22 μm pore size filters. The filtrates were analysed for L-arabinose and α-D glucuronic acid release using HPAEC-PAD (Dionex) on the Carbopac PA 10 column. L-arabinose (Merck) and glucuronic (Sigma) acid were used for plotting standard curves. Samples from xylan adsorption mixture filtrates were subjected to a phenol-sulfuric assay described by Dubois [1956,  Annal. Chem.  28 (3):350-356]. Analytical grade of xylose sugar (Merck) was used as a standard. Precipitation efficiency was defined as the amount of xylan removed from the adsorption reaction mixture as a percentage of the initial amount of xylan in the adsorption mixture (xylan in this case was measured in the form of xylose sugar). Samples of xylan treated cotton lint weighing 0.05 g were hydrolysed in 0.5 mL of 72% H 2 SO 4  in McCartney bottles followed by incubation in a water bath at 30° C. for 1 h. The reaction mixture was diluted to 4% by addition of 15 mL dH 2 O. The samples were autoclaved at 121° C. for 1 h. The hydrolysates were vacuum filtered using glass microfibre filters followed by filtration using 0.22 μm pore size filter discs before sugar analysis. The xylose content in the cotton lint was determined using HPAEC-PAD (Dionex) on Carbopac PA 10 column. The efficiency of xylan adsorption onto the cotton lint due to enzymatic treatment of the xylan during the adsorption was defined as the difference between the amount of xylose released from the hydrolysate of cotton lint treated in the presence and absence of the enzymes corrected for any xylose that pre-existed in the untreated cotton lint. Synergetic effect was defined as the difference between the amount of xylose released from acid hydrolysate of cotton lint treated in xylan adsorption mixtures in the presence of a cocktail of AbfB and AguA and the amount of xylose released from the acid hydrolysate of cotton lint treated with the same type of xylan but in the presence of AbfB or AguA. Synergetic effect was expressed as a percentage of the amount of xylose in the hydrolysate of cotton lint treated with xylan in the presence of either AbfB or AguA. 
     Cotton Lint Solid State (CP/MAS) NMR Analysis 
     Structural changes of the dried cotton lint samples as a result of xylan adsorption were analysed using solid state (CP/MAS) NMR on a Varian VNMRS 500 wide bore solid state NMR spectrometer with an operating frequency of 125 MHz for  13 C using a 6 mm T3 probe with a probe temperature of 25° C. Dry cotton lint samples were loaded to fill 6 mm zirconium oxide rotors. Spectra were recorded using cross-polarisation and magic angle spinning (CP/MAS). The speed of rotation was 5 kHz, the proton 90° pulse was 5 μs, the contact pulse 1500 μs and the delay between repetitions 5 sec. Chemical shifts were determined relative to TMS by setting the downfield peak of an external adamantane reference to 38.3 ppm. 
     Statistical Analysis 
     Unless stated otherwise, samples were conducted in triplicates. Analysis of variance (ANOVA) including sample means and standard deviations were performed using Microsoft Excel and Statistica 2007. 
     Optimisation of Xylan Extraction 
     The selectivity and effectiveness of the two mild alkali xylan extraction methods which were adopted from Höije et al. [2005 , Carbohydr. Polym.  61: 266-275] and De Lopez et al. [1996 , Biomass and Bioenergy,  10: (4): 201-211] were assessed for potential use in integrated production of substantially pure xylan biopolymers and pulp production from the feedstock commonly found in South Africa. The assessment was based on five factors as follows: (1) xylan extraction efficiency, (2) degree of polymerization and substitution, (3) chemical composition of the extracted xylan, (4) purity of the extracted xylan, and (5) the structural integrity and chemical composition of the raw material post xylan extraction. A summary of the xylan substrates used for evaluating enzymatic substrate specificity and degree of removal of xylan side chains is shown in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Xylan substrates used for evaluating enzymatic substrate specificity and degree of 
               
               
                 removal of xylan side chains 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Type of side 
                   
               
               
                 Xylan 
                 Method of extraction 
                 chain 
                 Source 
               
               
                   
               
               
                 Sugarcane ( Saccharum   
                 Mild alkali (Höije et 
                 Arabinose and 
                 This study 
               
               
                   officinarum  L) Bagasse (BH) 
                 al., 2005) 
                 4-O-MeglcA 
               
               
                 Sugarcane ( Saccharum   
                 Mild alkali (de Lopéz et 
                 Arabinose and 
                 This study 
               
               
                   officinarum  L) Bagasse (BL) 
                 al., 1996) 
                 4-O-MeglcA 
               
               
                 Sugarcane ( Saccharum   
                 Mild alkali extracted 
                 Arabinose and 
                 Donated by Prof. 
               
               
                   officinarum  L) bagasse (BB 
                 and H 2 O 2  bleached 
                 4-O-MeglcA 
                 A. M. F. Milagres, 
               
               
                 or BagBraz) 
                   
                   
                 University of Sáo Paulo, 
               
               
                   
                   
                   
                 Brazil 
               
               
                 Giant bamboo ( Bambusidae   
                 Mild alkali (Höije et 
                 Arabinose and 
                 This study 
               
               
                   balcoa ) (BM or Bam) 
                 al., 2005) 
                 4-O-MeglcA 
               
               
                   Eucalyptus  ( Eucalyptus   
                 Mild alkali (Höije et al., 
                 4-O-MeglcA 
                 This study 
               
               
                   grandis ) (EH) 
                 2005) 
               
               
                   Eucalyptus  ( Eucalyptus   
                 Mild alkali (de Lopéz et 
                 4-O-MeglcA 
                 This study 
               
               
                   grandis ) (EL) 
                 al., 1996) 
               
               
                   Eucalyptus  ( Eucalyptus   
                 nd 
                 4-O-MeglcA 
                 Donated by Arlene 
               
               
                   grandis ) gel from pulp (ES) 
                   
                   
                 Bayley, SAPPI 
               
               
                   
                   
                   
                 Technology Centre, 
               
               
                   
                   
                   
                 Pretoria, South Africa 
               
               
                 Pine ( Pinus patula ) (PP) 
                 Höije et al. (2005) 
               
               
                 Oat spelt xylan 
                 nd 
                 10% Arabinose, 
                 Sigma 
               
               
                 Birch xylan 
                 nd 
                 8-10% MeglcA 
                 Roth 
               
               
                   
               
               
                 Note: 
               
               
                 nd denotes not disclosed 
               
            
           
         
       
     
     Raw Material Characterisation 
     The feedstock used included Eucalyptus ( Eucalyptus grandis ), pine ( Pinus patula ), giant bamboo ( Bambusa balcooa ) and sugarcane ( Saccharum officinarum  L) bagasse. The  E. grandis  chips were supplied by The Transvaal Wattle Cooperatives from Piet Retief, Mpumalanga Province, and the  P. patula  trees were harvested from Stellenbosch University forest plantations in the Western Cape Province of South Africa. The giant bamboo stems (one and half year plant) were supplied from mature plantations located in Paarl in the Western Cape Province of South Africa. The bagasse was a by-product from the sugar processing industry and was donated by TBS Company located in the Nkomazi region of the South-Eastern Lowveld of Mpumalanga province in South Africa. Oatspelt xylan (Sigma), birch xylan (Roth), and mild alkali extracted H 2 O 2  bleached bagasse xylans (donated by Prof. A. M. F. Milagres, University of Sao Paulo, Brazil) were used as reference xylans. 
     The feedstock materials were prepared for analysis according to TAPPI test methods (TAPPI, T264 cm-97 (2002-2003)), and NREL Laboratory Analytical Procedures (NREL LAP) [Hammes et al., 2005, Laboratory Analytical Procedure (LAP), version 2005, NREL Biomass Program. National Bioenergy Center]. Chips derived from the various feedstocks were dried to a moisture content (mc) of ≈10%, and subsequently conditioned to a relative humidity of 55% at 20° C. for at least 24 h prior to further size reduction. The chips were successively reduced in size by Condux hammer-mill, a Retch, and a Wiley laboratory mill and fractionated by sieving using stackable sieves (ASTM) of 850 μm/20 mesh size, 425 μm/40 mesh size, and 250 μm/60 mesh size with a lid and pan. The particulates that passed through 425 μm/40 mesh size but were retained on a 250 μm/60 mesh sieve were collected for chemical composition analyses and those retained on the 425 μm/40 mesh were used for xylan extraction. The moisture content of the feedstock was determined using National Renewable Energy Laboratory Analytical Procedure (NREL LAP) for determination of total solids in biomass [Hammes et al., 2005, Laboratory Analytical Procedure (LAP), NREL Biomass Program. National Bioenergy Center]. The percent moisture content was calculated as a % of oven dry (o.d) weight biomass. 
     Extractives were determined in two sequential steps, starting with cyclohexane/ethanol (2:1) followed by hot water extraction, using soxhlet apparatus. Both extractions were done according to TAPPI Test Method T 264 om-88, and NREL LAP methods [Sluiter et al., 2005, Analytical Procedure (LAP), version 2006. NREL Biomass Program. National Bioenergy Center]. The extractives were quantified on a moisture free basis. 
     Klason lignin (acid insoluble) content of the feedstock was determined following a NREL LAP method for determination of structural carbohydrates and lignin in biomass [Sluiter et al., 2005, Analytical Procedure (LAP), version 2006. NREL Biomass Program. National Bioenergy Center] and TAPPI test procedures (T249 cm-85). The Klason lignin was calculated on o.d. mass. 
     Seifert cellulose content was determined according to the analytical methods outlined by Browning [1967, Methods of wood chemistry, Vol II. Interscience publishers], and Fengel and Wegner [1989, Wood Chemistry, Ultrastructure, Reactions. Walter de Gruyter, Berlin, Germany]. Extractive free material weighing 1.1 g oven dry was treated with a mixture of acetyl acetone (6 ml), dioxane (2 ml) and 32% HCl (2 ml) in round bottom flasks followed by incubation in a boiling water bath for 30 min. The treated samples were transferred quantitatively into pre-weighed sinter glass crucibles for vacuum filtration and washing. The residues were successively washed with 100 mL each of methanol, cyclo-dioxane, warm water (80° C.), methanol, and diethyl ether and subsequently dried at 105° C. for 2 h. The Seifert cellulose content was defined as the weight of the dried residue presented as a percentage of the extractive free material. 
     Monomeric sugar composition of the acid hydrolysate was analysed after storage at −20° C. for at least 24 h. The analysis was performed in high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD, (Dionex) that was equipped with a gradient pump GP 50, a Carbopac PA 10 (4 mm×250 mm) column, and electrochemical detector (ED40). The data acquisition and analysis were performed using PEAKNET software package. The eluents were 250 mM NaOH and Milli-Q-water in the ratio of 1.5:98.5 at a flow rate of 1 mL min −1 . Sodium acetate (1M NaOAc) eluent was used when acid sugars (glucuronic/methyl glucuronic acid) were analysed. The samples were filtered on 0.22 μm pore size filters before analysis on HPAEC-PAD. The quantity of the sugars was determined from standard plot of the respective analytical grade sugars (arabinose, rhaminose, galactose, glucose, mannose, xylose, and glucuronic acid). The amount of sugar was presented as a percentage relative to the oven dry (o.d) weight of the substrate. The pentosan content in the feedstocks was determined according to TAPPI standard methods for measuring pentosans in wood and pulp (T223 cm-84). The xylan content was calculated from a standard plot prepared from xylose (analytical grade) using equation 3.1.1 as percentage of the o.d. biomass [Xyl=xyl×cf, where: Xyl=Xylan content (mg), xyl=Xylose content (mg), cf=Correction factor (0.88)]. 
     The ash content was determined by a thermogravimetric method. Lignocellulosic samples (0.5 g) were incinerated in a Muffle furnace at 575±25° C. for 4 h or until a constant weight was obtained. Ash content was calculated as a percentage of the initial o.d. biomass. 
     Xylan Extraction and Characterisation 
     Extraction of xylan from the feedstock was performed using the two mild alkali extraction methods described above. The Hoije method involved post xylan extraction ultrapurification using membrane dialysis (MWCO 12-14 kDa) whereas the Lopez method involved fractionation of the hydrolysates by ethanol precipitation. In both methods, xylan extraction was performed without prior removal of solvent and hot water extractives. The extracts were concentrated before ultrapurification or fractionation to a third of the initial volume using a rotary evaporator (Rotavapor Büchi R-124, Switzerland) under vacuum at 40° C. The extraction efficiency was defined as the yield of xylan per theoretical content of pentosans in the material. The Lopez method was limited to extraction of xylan from  E. grandis  and bagasse only. 
     The structure and chemical composition of the feedstocks for xylan extraction were analysed pre- and post-xylan extraction by solid state  13 C-Nuclear Magnetic Resonance Cross-Polarisation/Magic Angle Spinning ( 13 C-NMR CP/MAS) on a Varian VNMRS 500 wide bore solid state NMR spectrometer with an operating frequency of 125 MHz for  13 C, using a 6 mm T3 probe with a probe temperature of 25° C. Dry samples were loaded into 6 mm zirconium oxide rotors for analysis. Spectra were recorded using cross-polarisation and magic angle spinning (CP/MAS). The speed of rotation was 5 kHz, the proton 90° pulse was 5 μs, the contact pulse 1500 μs and the delay between repetitions 5 sec. Chemical shifts were determined relative to TMS by setting the downfield peak of an external adamantane reference to 38.3 ppm. The carbon resonances in the solid state NMR spectra were assigned according to Larsson et al. [1999 , Solid State Nuclear Magnetic Resonance  15: 31-40]; Renard and Jarvis [1999 , Plant Physiology  119: 1315-1322], Maunu [2002 , Progress in Nuclear Magnetic Response Spectroscopy  40: 151-174], Lahaye et al. [2003 , Carbohydrate Research  338: 1559-1569]; Atalla and Isogai [2005, Recent developments in spectroscopic and chemical characterisation of cellulose. In Dumitriu, S. (ed.) Marcel Dekker. New York. pp 123-157], Virkki et al. [2005 , Carbohydrate Polymers  59: 357-366], Geng et al. [2006 , International Journal of Polymer Characterization  11: 209-226], and Oliveira et al. [2008, Chemical composition and lignin structural features of banana plant leaf sheath and rachis. In Hu, T. Q. (ed) Chapter 10: 171-188]. 
     The extracted xylan samples were analysed using solid state  13 C-Nuclear Magnetic Resonance Cross-Polarisation/Magic Angle Spinning ( 13 C-NMR CP/MAS) and Liquid  13 C and  1 H NMR and Fourier Transform Infrared (FTIR) spectroscopy. The xylan samples were subjected to a  13 C and a  1 H NMR run either on a Varian Inova 400 or 600 NMR spectrometer.  13 C NMR spectra were collected using a 1.3 s acquisition time and 1 s pulse delay at 25° C. The  13 C spectra were collected overnight (minimum 19000 scans).  1 H NMR spectra were collected after filtration of the sample with a 4.8 s acquisition time at 50° C.  1 H spectra were collected with 64 scans and pre-saturation of the HDO peak. The  13 C and  1 H NMR spectra were interpreted according to assignment of characteristic signals of related feedstock presented by Ebringerová et al. [1998 , Carbohydrate Polymers  37: 231-239], Vignon and Gey [1998 , Carbohydrate Research  307: 107-111], Renard and Jarvis [1999 , Plant Physiology  119: 1315-1322], Teleman et al. [2002 , Carbohydrate Research  337: 373-377], Gröndahl at al. [2003 , Carbohydr. Polym.  53: 359-366], Lahaye at al. [2003 , Carbohydrate Research  338: 1559-1569], Sun et al. [2004 , Carbohydrate Research  339: 291-300 ; Polym. Degrad. and Stability  84: 331-337 ; Carbohydrate Polymers  56: 195-204], Sims and Newman [2006 , Carbohydrate Polymers  63: 379-384]; Habibi and Vignon [2005 , Carbohydrate Research  340: 1431-1436], Pinto et al. [2005 , Carbohydrate Polymers  60: 489-497], Geng et al. [2006 , International Journal of Polymer Characterization  11: 209-226], Maunu [2008.  13 C CPMAS NMR Studies of wood, cellulose fibers, and derivatives. In Hu, T. Q. (ed)], Shao et al. [2008, Wood Science Technology 42: 439-451]. 
     In FTIR spectroscopy, dry solid samples of the xylan were recorded on a Nexus 670 spectrometer from Thermo Nicolet with the Smart Golden Gate ATR accessory installed. This single-reflection accessory features a diamond ATR crystal bonded to a tungsten carbide support equipped with ZnSe focusing lenses. The spectra were collected over the spectral range of 4000 to 650 cm −1  using 16 scans at 6 cm −1  resolution and were calibrated against a previously recorded background. Thermo Nicolet&#39;s OMNIC® Software was used for collecting and processing of the infrared spectra. The spectra signals for FTIR were interpreted according to characteristic bands presented in Fengel and Wegener (1989); Sun et al (2004); Xu et al (2000), Sims and Newman (2006). 
     The degree of polymerization of the extracted xylan fractions was evaluated on HPAEC (Dionex) using a Carbopac™ PA100 column (4×250 mm) and a guard column, and electrochemical detector (ED40) for pulsed amperometric detection (PAD). The PA 100 column separates monomers and oligomers up to a degree of polymerisation (DP) 10 which usually elutes within a retention time of 25 min. The HPAEC PA100 column bases its separation on DP and degree of substitution, thus the longer the retention time, the higher the DP or degree of substitution (Combined CarboPac manual pp 52-56). Samples (10 μL) were injected into the column and were eluted with helium degassed 0.25 M NaOH, Milli-Q H 2 O, and 1 M NaOAc at a flow rate of 1 mLmin −1 . Elution profiles of the samples were referenced to elution profiles of monomeric sugars (arabinose, rhaminose, galactose, glucose, xylose and mannose), and polymeric xylan (birch, and oatspelt xylan) and H 2 O 2  bleached bagasse. Samples with less intense peaks&lt;20 nC or no peaks eluting within the 25 min retention time were considered polymeric with DP&gt;10 sugar units. 
     The composition of neutral sugars in the extracted xylan samples were determined on HPAEC-PAD (Dionex) on Carbopac PA 10 column after mild acid hydrolysis described by Yang et al. [2005, LWT 38: 677-682]. Samples (0.1 g) were placed in Schott bottles (50 mL) into which 1 mL 72% H 2 SO 4  was added. The mixture was incubated at 30° C. in a water bath for 1 h. De-ionized water (30 mL) was added followed by autoclaving at 121° C. for 1 h. The samples were cooled to room temperature before filtering. The filter cake was dried at 105° C. for residual Klason lignin determination. The liquid fraction was filtered through a 0.22 μm pore size filter before subjecting it to HPAEC-PAD (Dionex) on Carbopac PA 10 column. The monomeric sugars were quantified from standard plots of analytical grade arabinose, rhaminose, galactose, glucose, xylose, and mannose). The total neutral sugar content of the samples was presented relative to the initial xylan o.d mass. 
     Determination of Uronic Acid Composition 
     Uronic acid content of the xylan samples and the feedstocks were quantified using chromatographic and colorimetric methods. In the chromatographic method, a two step acid hydrolysis method adopted from Prof. A. M. F. Milagres of University of Sáo Paulo, Brazil (Personal communication, 2007) was used. Xylan samples (150 mg o.d mass) were hydrolysed in 0.75 mL of 72% (w/w) H 2 SO 4  in McCartney bottles. The mixture was incubated at 45° C. for 7 min in a water bath after which 22.5 mL of distilled water were added. The bottles were loosely covered and autoclaved at 121° C. for 30 min. After cooling to room temperature, the liquid fraction was separated by vacuum filtering through glass micro fibre filters (GF/A—Whatman). The liquid fraction was further filtered through a 0.22 μm filter and kept frozen overnight at −20° C. before analysing for glucuronic acid content using HPAEC-PAD (Dionex) on Carbopac PA 10 column. Quantification of uronic acid was based on standard plots for glucuronic acid (Sigma). Uronic acid losses during autoclaving were accounted for by autoclaved glucuronic acid at 121° C. for 1 h in 4% H 2 SO 4 . In the colorimetric method, carbazole-sulfuric assay adopted from Li et al. [2007 , Carbohydr. Res.  342 (11): 1442-1449] was used. Total uronic acid concentration was determined from standard curve plot for D-galacturonic acid (Merck) and in both methods uronic acid content was presented as percentage of the initial xylan amount. 
     Removal of Xylan Side Chains 
     The degree of selective removal of arabinose and 4-O-methyl glucuronic acid (4-O-MeglCA) side groups by α-L-Arabinofuranosidase (AbfB) of  Aspergillus niger  and α-D-glucuronidase of  Schizophyllum commune  (α-glu) respectively was determined using xylan derived from  Eucalyptus grandis, Pinus patula, Bambusa balcooa , and bagasse found in South Africa. 
     The AbfB and α-glu from  A. niger  and  S. commune  were assessed in individual application and in synergy for selective removal of arabinose and 4-O-MeglcA side chains respectively from xylan derived from hardwood, softwood and grass (including cereals) sources with the aim of developing a controlled enzymatic technology for diversification of the xylan functional properties. Therefore, the effect of hydrolysis time, temperature and enzyme xylan specific dosage on the removal of arabinose and 4-O-MeglcA side chains, and the subsequent modification of viscosity, solubility, precipitation and aggregation of the xylan were examined. Xylan samples substituted with arabinose and/or 4-O-methyl glucuronic acid (4-O-MeglcA) side chains are shown in Table 2. Oatspelt xylan (Sigma) and birch xylan (Roth) were utilised as model xylans. Xylan solution (1% w/v) for each material was prepared in de-ionized water (dH 2 O). The xylan that showed limited solubility in water was prepared by first dissolving in ethanol and subsequently heated according to de Wet et al. [2008 , Appl. Microbiol. Biotechnol.  77: 975-983]. Xylan solutions were made in bulk and stored in vials at −20° C. Oatspelt xylan (Sigma) with a sugar composition of 10:15:75 (arabinose:glucose: xylose) and birch xylan (Roth) with sugar composition of 8.3:1.4:89.3 (4-O-MeglcA: glucose, and xylose) [Kormelink and Voragen, 1993 , Carbohydr. Res.  249: 345-353] made in similar way were used as model xylan. The enzymes used were crude α-L-arabinofuranosidase (AbfB) produced by recombinant  Aspergillus niger  D15 produced inhouse with volumetric activity of 18.0 nKat mL −1  on p-Nitrophenyl Arabinofuranoside (p-NPA) and α-D-glucuronidase (α-glu) with specific activity=300 nKat mg −1  purified from wild type  Schizophyllum commune  (VTT-D-88362-ATCC 38548 (donated by Prof. Matti Siika-aho of VTT Biotechnology institute in Finland). These enzymes were used for the selective removal of L-arabinose and 4-O-methyl-D-glucuronic acid/D-glucuronic acid side groups, respectively. The enzyme aliquots were stored at 4° C. 
     Xylan known to be substituted with arabinose (Table 2) was treated with AbfB in a reaction volume of 5 mL containing 2.5 mL xylan, 1.0 mL enzyme supernatant and 1.5 mL 0.05 M citrate buffer pH 5.0. The hydrolysis was performed at 40° C. for 16 h. A xylan solution (1% w/v) prepared from 4-O-MeglcA substituted substrates was treated with α-glu (9000 nKat g −1 ) in 5 mL reaction volumes consisting of 2.5 mL of the substrate and made up to 5 mL with 0.05 M acetate buffer, pH 4.8. The reactions proceeded for 16 h at 40° C. Xylan solutions (1% w/v) prepared from substrates substituted with both arabinose and 4-O-MeglcA side chains (Table 2) were simultaneously treated with AbfB and α-glu in 1000 μL reaction mixtures containing 500 μL of the respective substrates, AbfB 540.0 nKat g −1  and α-glu 9000.00 nKat g −1  in 0.05 M acetate buffer pH 4.8. The reactions were performed in a water bath set at 40° C. for 24 h. The synergetic effect was calculated based on the difference between the amount of the specific sugar released due to individual enzyme action and combined enzyme action. 
     Effects of hydrolysis parameters (time, temperature and enzyme xylan specific dosage) on arabinose removal and viscosity were determined using a three factor Box-Behnken statistical design with 3 central points in 15 runs (Statistica 7.0 software programme, (StatSoft, Inc., 1984-2005) as shown in Table 3. Time, temperature, and enzyme xylan specific dosage constituted the independent variable whereas degree of side chain removal and viscosity change formed the dependent variables. 
     Arabinose and 4-O-MeglcA side chains sugar release were analysed using (HPAEC-PAD) on Carbopac PA 10 column eluted with helium degassed Mill-Q H 2 O, 250 mM NaOH and 1M NaOAc (for acid sugars only). L(+) arabinose and D-glucuronic acid were used as standard sugars. Insolubilization, precipitation and aggregation of the xylan hydrogels were confirmed by visual inspection (photographs taken) and quantified by measuring viscosity using Rheometer (MCR501). The degree of xylan precipitation was quantified by determining residual xylose in solution using phenol-sulphuric assay for total sugar [Dubois of al., 1956 , Annal. Chem.  28 (3):350-356]. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Box-Behnken statistical design for determining effects 
               
               
                 of hydrolysis parameters on removal of arabinose side 
               
               
                 chains from oat spelt xylan by recombinant AbfB 
               
            
           
           
               
               
            
               
                   
                 Natural variables 
               
            
           
           
               
               
               
               
               
            
               
                 Treatment run 
                 Coded variables 
                 Time 
                 Temperature 
                 Enzyme 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Random order 
                 X1 
                 X2 
                 X3 
                 (h) 
                 (° C.) 
                 dose (nkat/g) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 9 
                 0 
                 −1 
                 −1 
                 8.5 
                 30 
                 180 
               
               
                 3 
                 −1 
                 1 
                 0 
                 1 
                 50 
                 360 
               
               
                 10 
                 0 
                 1 
                 −1 
                 8.5 
                 50 
                 180 
               
               
                 6 
                 1 
                 0 
                 −1 
                 16 
                 40 
                 360 
               
               
                 11 
                 0 
                 −1 
                 1 
                 8.5 
                 30 
                 180 
               
               
                 4 
                 1 
                 1 
                 0 
                 16 
                 50 
                 540 
               
               
                 7 
                 −1 
                 0 
                 1 
                 1 
                 40 
                 360 
               
               
                 14 
                 0 
                 0 
                 0 
                 8.5 
                 40 
                 540 
               
               
                 12 
                 0 
                 1 
                 1 
                 8.5 
                 50 
                 360 
               
               
                 15 
                 0 
                 0 
                 0 
                 8.5 
                 40 
                 540 
               
               
                 5 
                 −1 
                 0 
                 −1 
                 1 
                 40 
                 360 
               
               
                 2 
                 1 
                 −1 
                 0 
                 16 
                 30 
                 180 
               
               
                 13 
                 0 
                 0 
                 0 
                 8.5 
                 40 
                 360 
               
               
                 8 
                 1 
                 0 
                 1 
                 16 
                 40 
                 360 
               
               
                 1 
                 −1 
                 −1 
                 0 
                 1 
                 30 
                 540 
               
               
                   
               
               
                 Design summary: 3 factor Box-Behnken design, number of factors independent variables: 3, number of runs (cases, experiments): 15 and number of blocks: 1 
               
            
           
         
       
     
     A three factor Box-Behnken statistical design experiment with 3 central points making a total of 15 runs was run in duplicates: Statistica 7.0 software programme, (StatSoft, Inc., 1984-2005) was used for designing and analysisbased on response surface method (RSM) as shown in Table 4. Regression and ANOVA analyses were performed to determine the size and significance of individual and interaction effects of the hydrolysis parameters on viscosity. Optimal conditions were determined using the desirability function. The response surface plots were fitted with a second order polynomial as follows: 
         Z=β   0 +β 1 x 1 +β 11 x 1   2 +β 2 x 2 +β 22 x 2   2 +β 3 x 3 +β 33 x 3   2 +ε
 
     where: Z=Viscosity (mPa·s), β 0 +β 1  . . . β n =linear regression coefficient, β 11  . . . β nn =Quadratic regression coefficient, ε=Error, and X 1 , X 2 , X 3 =Hydrolysis time, temperature, and enzyme xylan specific dosage. 
     Optimal Conditions for Side Chain Removal 
     The optimal combination levels of hydrolysis parameters were determined: time, temperature, and dosage of recombinant α-L-arabinofuranosidase and α-D-glucuronidase for removal of arabinose and 4-O-methyl glucuronic acid side chains, respectively. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Box-Behnken experimental set up for selective removal 
               
               
                 of MeGlcA side chains from birch xylan by purified AguA 
               
            
           
           
               
               
            
               
                   
                 Natural variables 
               
            
           
           
               
               
               
            
               
                   
                 Enzyme 
                   
               
               
                   
                 dose [μkat 
               
            
           
           
               
               
               
               
               
            
               
                 Treatment Run 
                 Coded variables 
                 Substrate 
                 Time 
                 Temperature 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 (Random order) 
                 X1 
                 X2 
                 X3 
                 (g) −1 ] 
                 (h) 
                 (° C.) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 6 
                 1 
                 0 
                 −1 
                 18 
                 8.5 
                 30 
               
               
                 15 
                 0 
                 0 
                 0 
                 10 
                 8.5 
                 40 
               
               
                 1 
                 −1 
                 −1 
                 0 
                 2 
                 1.0 
                 40 
               
               
                 14 
                 0 
                 0 
                 0 
                 10 
                 8.5 
                 40 
               
               
                 8 
                 1 
                 0 
                 1 
                 18 
                 8.5 
                 50 
               
               
                 5 
                 −1 
                 0 
                 −1 
                 2 
                 8.5 
                 30 
               
               
                 2 
                 1 
                 −1 
                 0 
                 18 
                 1.0 
                 40 
               
               
                 13 
                 0 
                 0 
                 0 
                 10 
                 8.5 
                 40 
               
               
                 11 
                 0 
                 −1 
                 1 
                 10 
                 1.0 
                 50 
               
               
                 9 
                 0 
                 −1 
                 −1 
                 10 
                 1.0 
                 30 
               
               
                 3 
                 −1 
                 1 
                 0 
                 2 
                 16.0 
                 40 
               
               
                 10 
                 0 
                 1 
                 −1 
                 10 
                 16.0 
                 30 
               
               
                 12 
                 0 
                 1 
                 1 
                 10 
                 16.0 
                 50 
               
               
                 4 
                 1 
                 1 
                 0 
                 18 
                 16.0 
                 40 
               
               
                 7 
                 −1 
                 0 
                 1 
                 2 
                 8.5 
                 50 
               
               
                   
               
               
                 Design summary: 3 factor Box-Behnken design, number of factors independent variables: 3, number of runs (cases, experiments): 15 and number of blocks: 1 
               
            
           
         
       
     
     The effect of xylan loading, enzyme loading, hydrolysis time, and temperature, and their interaction on AbfB removal of arabinose and α-glu removal of 4-O-MeglcA side chains from oatspelt and birch xylan, respectively, were determined by using Response Surface Methodology (RSM). Statistica 7.0 software programme, (StatSoft, Inc., 1984-2005) was used for designing and analysing the experiments. Statistical analysis included regression and ANOVA analyses. Pareto Chart plots were used to present size and significance of effects while the desirability and profiling function was used to determine optimal set point for the hydrolysis parameters. The oatspelt xylan (1% w/v) was prepared according to de Wet et al. [2008 , Appl. Microbiol. Biotechnol.  77: 975-983]. The solutions were made in bulk and stored in vials at −20° C. Crude α-L-arabinofuranosidase (AbfB) produced inhouse using recombinant  Aspergillus niger  D15 (volumetric activity of 18 nKat mL −1  on p-NPA) and α-D-glucuronidase (α-glu) purified from wild type  Schizophyllum commune  (VTT-D-88362-ATCC 38548) with specific activity of 300 nKat mg −1  (donated by Prof. Matti Siika-aho of VTT Biotechnology Institute in Finland), were used for the selective removal of L-arabinose and 4-O-methyl-D-glucuronic acid/D-glucuronic acid side groups, respectively. The enzyme aliquots were stored at 4° C. In addition L (+)-arabinose (Merck) and D-Glucuronic acid (Sigma) were used as standard sugars. 
     Oatspelt xylan was incubated with AbfB in 400 μL reaction mixtures containing 200 μL of the substrate, with AbfB xylan specific dosage ranging from 44.0-140.0 nKat substrates (g) −1  in 0.05 M citrate buffer pH 5. The reactions were performed in a water bath set at 40° C. for durations of 2, 4, 8 and 16 h. The effect of temperature was assessed in 5 mL reaction volumes into which 2500 mL of substrate was added and incubated with AbfB of xylan specific dosage of 180 and 720 nKat g substrate −1  at 40 and 60° C. for 4 and 16 h. The reactions were stopped by boiling for 10 min or by immediately placing the samples on ice. The hydrolysates were analysed for arabinose release. 
     Optimization of Hydrolysis Parameters 
     Optimal set points for time, temperature, and enzyme dosage for the AbfB removal of arabinose from oatspelt xylan and α-glu removal of 4-O-MeglcA from birch xylan were determined in a three factor Box-Behnken statistical design with 3 central points making a total of 15 runs in duplicates. The hydrolysis parameters were each tested at two levels and middle point with the highest, middle and lowest levels denoted as 1, 0, and −1 respectively. Temperature was tested at 30° C. and 50° C., time at 1 h and 16 h, and enzyme dosage for AbfB was 180 nKat g −1  and 540 nKat g substrate −1  while that of α-glu was 2 000 nKat g −1  and 18 000 nKat g −1 . The central points for temperature and time were 40° C., and 8.5 h, while for AbfB and α-glu xylan specific dosages were 360000 nKat g −1  and 11000 nKat g −1 , respectively. The full experimental set up with runs presented in random order for arabinose removal from oatspelt xylan and 4-O-MeglcA from birch xylan are presented in Tables 3 and 4, respectively. The variables were coded according to the equation: 
     
       
         
           
             
               
                 x 
                 i 
               
               = 
               
                 ( 
                 
                   
                     
                       X 
                       i 
                     
                     - 
                     
                       
                         X 
                         _ 
                       
                       i 
                     
                   
                   
                     Δ 
                      
                     
                         
                     
                      
                     
                       X 
                       i 
                     
                   
                 
                 ) 
               
             
             , 
           
         
       
     
     where: x i =coded value for variable, X i =natural value, ΔX i =scaling factor (half the range of the independent variables which constituted Time, temperature, and enzyme xylan specific dosage). 
     The effect of substrate and enzyme loading on arabinose removal was assessed in a two factors cube plus star central composite statistical design experiment consisting of 10 runs. The experiments were performed in a total reaction mixture of 8 mL which contained oatspelt xylan (1% w/v) treated with varying enzyme dosage (nKat g −1 ) according to the experimental design in Table 5. The reaction was done in a water bath set at 40° C. for 16 h. The hydrolysed samples were analysed for arabinose removal. 
     Arabinose and 4-O-MeglcA side chains were analysed using (HPAEC-PAD) on Carbopac PA 10 column eluted with helium degassed Mill-Q H 2 O, 250 mM NaOH and 1M NaOAc (for acid sugars only). L (+) arabinose and D-glucuronic acid were used as standard sugars. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Central composite design for assessing effect of oat spelt 
               
               
                 xylan concentration and enzyme activity on selective 
               
               
                 removal of arabinose side chain from oat spelt xylan by the 
               
               
                 recombinant AbfB 
               
            
           
           
               
               
            
               
                   
                 Natural variables 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 xylan 
                 *Enzyme 
               
               
                 Treatment run 
                 Coded variables 
                 concentration 
                 dosage 
               
            
           
           
               
               
               
               
               
            
               
                 Random order 
                 A 
                 B 
                 (μg mL −1 ) 
                 (nkat mL −1 ) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                  1 
                 −1.00000 
                 −1.00000 
                 1562.50 
                 9.0 
               
               
                  6 
                 1.41421 
                 0.00000 
                 7220.30 
                 22.5 
               
               
                 10 (C) 
                 0.00000 
                 0.00000 
                 3906.30 
                 22.5 
               
               
                  4 
                 1.00000 
                 1.00000 
                 6250.00 
                 36.0 
               
               
                  3 
                 1.00000 
                 −1.00000 
                 6250.00 
                 9.0 
               
               
                  8 
                 0.00000 
                 1.41421 
                 3906.30 
                 41.0 
               
               
                  5 
                 −1.41421 
                 0.00000 
                 592.20 
                 22.5 
               
               
                  7 
                 0.00000 
                 −1.41421 
                 3906.30 
                 3.4 
               
               
                  9 (C) 
                 0.00000 
                 0.00000 
                 3906.30 
                 22.5 
               
               
                  2 
                 −1.00000 
                 1.00000 
                 1562.50 
                 36.0 
               
               
                   
               
               
                 Design summary: 2** (2) central composite, nc = 4, ns = 4, n0 = 2, Runs = 10 * values rounded off to significant numbers, C = central point run 
               
            
           
         
       
     
     The response surface plot was fitted with a second order polynomial which included both linear and quadratic interactions as follows: 
         Z=β   0 +β 1 x 1 +β 11 x 1   2 +β 2 x 2 +β 22 x 2   2 +β 3 x 3 +β 33 x 3   2 +β 112 x 1   2 x 2 +β 113 x 1   2 x 3 +β 122 x 1 x 2   2 +β 133 x 1 x 3   2 +β 223 x 2   2 x 3 +β 233 x 2 x 3   2 β 1122 x 1   2 x 2   2 +β 2233 x 2   2 x 3   2 +ε
 
     where: Z=Response (degree of side chain removal), β 0 +β 1  . . . β n  . . . =linear regression coefficient, β 11  . . . β nn  . . . =Quadratic regression coefficient, X 1 , X 2 , X 3 =Hydrolysis time, temperature, and enzyme xylan specific dosage or xylan and enzyme loading, ε=Error. 
     Enzyme Aided Adsorption of Xylan on Cellulosic Material 
     Individual and synergetic effects of applying recombinant α-L-arabinofuranosidase and α-D-glucuronidase on the adsorption capacity of xylan derived from  Eucalyptus grandis, Pinus patula, Bambusa balcooa , and bagasse onto cellulosic material was assessed. The individual and synergetic effects of selective removal of L-arabinose and 4-O-methyl-D-glucuronic acid/D-glucuronic acid side groups by recombinant AbfB and wild purified AguA, respectively, on adsorption of xylan extracted from  Saccharum officinarum  L (sugarcane) bagasse (BH),  Bambusidae balcoa  (giant bamboo) (BM),  Eucalyptus grandis , [Eucalyptus (EH)], and  Pinus patula  [Pine (PP)] using mild alkali method were investigated onto non-absorbent cotton lint (Grade 1, Cotton King). Crude α-L-arabinofuranosidase (AbfB) produced by recombinant  Aspergillus niger  D15 with volumetric activity of 18.0 nKat mL −1  on p-NPA produced inhouse and α-D-glucuronidase (α-glu) with specific activity=300 nKat mg −1  purified from wild type  Schizophyllum commune  (VTT-D-88362-ATCC 38548 (donated by Prof. Matti Siika-aho of VTT Biotechnology institute in Finland) were added to xylan solutions (1% w/v) prepared according to de Wet et al. [2008 , Appl. Microbiol. Biotechnol.  77: 975-983]. Solutions prepared from commercial oatspelt xylan (Sigma) and birch xylan (Roth) were used as model xylan. To broaden the substrate base, mild alkali H 2 O 2  bleached xylan from bagasse (BB) (donated by Prof. A. M. F. Milagres, University of Sao Paulo, Brazil), and processed xylan gel from  E. grandis  (ES) (donated by SAPPI—Pretoria, South Africa) were used as reference xylan in assessing effect of different xylan extraction protocols. 
     Samples of cotton lint (1 g) were autoclaved in 100 ml Schott bottles at 121° C. for 15 min. After cooling to room temperature, varying amounts of oatspelt xylan 5, 12.5, 15.0 and 25 mL with AbfB (18.0 nKat mL −1 ) in a fixed xylan:enzyme ratio of 5:2 and without AbfB were added. The adsorption treatments were performed in 0.05 M citrate buffer pH 5.0 in a total reaction mixture volume of 40 mL. The reaction mixture was incubated at 40° C. in a water bath for 24 h. The control sample contained treated xylan without cotton lint. All reactions were terminated by placing the bottles in water containing ice. 
     Cotton lint samples weighing 0.2 g were placed in 15 mL glass bottles in two sets of three and a control. The bottles were autoclaved at 121° C. for 15 min. Upon cooling to room temperature, 750 μL birch xylan (1% w/v) and 150 μL AguA (900 nKat mL −1 ) were added to one set. Subsequently, 0.05 M acetate buffer (pH 4.8) was added to make a total reaction mixture volume of 1650 μL. In addition, the same amount of birch was treated with AguA in separate glass bottles in the absence of cotton lint. 
     Adsorption of xylan treated by both α-L-Arabinofuranosidase and α-D-glucuronidase onto cotton lint with pre-extracted xylan was performed in glass bottles. The reaction mixture contained 0.2 g cotton lint, 1000 μL xylan (1% w/v), 40 μL AguA (900 nKat mL −1 ), 100 μL AbfB (18.0 nkat mL −1 ), and 0.05 M acetate buffer pH 4.8. The reaction was performed at 40° C. in a water bath for 16 h according to the experimental set up shown in Table 6. 
     Treated cotton lint samples were vacuum filtered from the adsorption mixture and subsequently transferred into a Schott bottle in which the cotton lint was washed by suspending it in 50-100 mL Milli-Q H 2 O while agitating for 1 h to disengage and remove xylan precipitates loosely absorbed to the cotton lint. The Milli-Q H 2 O was changed 3 times during the washing process. The samples were, after the final wash, vacuum filtered in pre-weighed filter papers and placed in pre-weighed foils for drying overnight at 30° C. to a constant weight. The amount of xylan adsorbed onto the cotton lint was defined as the difference between the initial weight of the cotton lint and the weight after the xylan treatment presented as a percentage of the initial weight of the cotton lint. Xylan specific weight gain for the cotton lint was defined as the weight gained by the cotton lint as a percentage of the theoretical initial amount of xylan in the reaction mixture. The calculations were corrected for moisture content of the starting materials and loss of material during post treatment. The individual enzyme effect on xylan adsorption onto cotton lint was defined as the difference in weight gain between cotton lint treated with unmodified xylan and cotton lint treated in enzymatically modified xylan adsorption mixtures. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Experimental set up for treatment of cotton lint with xylan in the 
               
               
                 presence of α-L-arabinofuranosidase (AbfB), α-D-glucuronidase 
               
               
                 (AguA) and their cocktail (AG) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 AbfB and 
               
               
                   
                   
                 Agua (900 
                 AbfB (18.0 
                 AguA 
               
               
                   
                   
                 nkat mL −1 ) 
                 nkat mL −1 ) 
                 100 μL + 
               
               
                 Xylan type* 
                 Control** 
                 40 μL*** 
                 100 μL)**** 
                 40 μL***** 
               
               
                   
               
               
                 Bagasse H1 
                 SBH1 
                 GBH1 
                 ABH1 
                 AGBH1 
               
               
                 Bagasse H2 
                 SBH2 
                 GBH2 
                 ABH2 
                 AGBH2 
               
               
                 BagasseBraz1 
                 SBB1 
                 GBB1 
                 ABB1 
                 AGBB1 
               
               
                 BagasseBraz2 
                 SBB2 
                 GBB2 
                 ABB2 
                 AGBB2 
               
               
                 Bamboo1 
                 SBM1 
                 GBM1 
                 ABM1 
                 AGBM1 
               
               
                 Bamboo2 
                 SBM2 
                 GBM2 
                 ABM2 
                 AGBM2 
               
               
                 Pine 1 
                 SP1 
                 GP1 
                 AP1 
                 AGP1 
               
               
                 Pine 2 
                 SP2 
                 GP2 
                 AP2 
                 AGP2 
               
               
                   Eucalyptus  H1 
                 SEH1 
                 GEH1 
               
               
                   Eucalyptus  H2 
                 SEH2 
                 GEH2 
               
               
                   Eucalyptus  gel S1 
                 SES1 
                 GES1 
               
               
                   Eucalyptus  gelS2 
                 SES2 
                 GES2 
               
               
                   
               
               
                 *Xylan type prepared in duplicates (1 and 2), The BH, BB, BM, P, EH and ES denote mild alkali extracted xylan from bagasse, H 2 O 2  bleached bagasse, mild alkali extracted bamboo,  Pinus patula ,  Eucalyptus grandis  and  Eucalyptus grandis  gel respectively. 
               
               
                 **Cotton lint treated with unmodified xylan (S), 
               
               
                 ***Cotton lint treated with xylan in presence of purified α-glucuronidase (G), 
               
               
                 ****Cotton lint treated with xylan in presence of recombinant α-arabinofuranosidase (A), 
               
               
                 *****Cotton lint treated with xylan in presence a cocktail of purified α-glucuronidase and recombinant α-arabinofuranosidase(AG). 
               
            
           
         
       
     
     The xylan adsorption reaction mixture filtrates were centrifuged at 10,000 rpm for 5 min at 4° C. followed by filtration using 0.22 μm pore size filters. The filtrates were analysed for L-arabinose and α-D glucuronic acid release using HPAEC-PAD (Dionex) on Carbopac PA 10 column. L-Arabinose (Merck) and glucuronic (Sigma) acid were used for plotting standard curves. Samples from xylan adsorption mixture filtrates were subjected to phenol-sulfuric assay (200 μL filtrate sample, 200 μL (5%) phenol and 800 μL concentrated sulfuric acid, absorbance was measured at 490 nm in a spectrophotometer) to determine changes in xylan content of the reaction after adsorption onto the cotton lint. Analytical grade of xylose sugar (Merck) was used as a standard. Precipitation efficiency was defined as the amount of xylan removed from the adsorption reaction mixture as a percentage of the initial amount of xylan in the adsorption mixture (xylan in this case was measured in the form of xylose sugar). 
     Samples of xylan treated cotton lint weighing 0.050 g were hydrolysed in 0.5 mL of 72% H 2 SO 4  in McCartney bottles followed by incubation in a water bath at 30° C. for 1 h. The reaction mixture was diluted to 4% by addition of 15 mL dH 2 O. The samples were autoclaved at 121° C. for 1 h. The hydrolysates were vacuum filtered using glass microfibre filters followed by filtration using 0.22 μm pore size filter discs before sugar analysis. The xylose content in the cotton lint was determined using HPAEC-PAD (Dionex) on Carbopac PA 10 column. The efficiency of xylan adsorption onto the cotton lint due to enzymatic treatment of the xylan during the adsorption was defined as the difference between the amount of xylose released from cotton lint treated in xylan adsorption mixture in the presence of the enzymes and the amount of xylose released from cotton lint treated in unmodified xylan, corrected for any xylose that pre-existed in the untreated cotton lint. Synergetic effect was defined as the difference between the amount of xylose released from acid hydrolysate of cotton lint treated in xylan adsorption mixtures in the presence of AbfB and AguA cocktail and the amount of xylose released from the acid hydrolysate of cotton lint treated with the same type xylan but in the presence of either AbfB or AguA individually. Synergetic effect was expressed as a percentage of the amount of xylose in the hydrolysate of cotton lint treated with xylan in the presence of either AbfB or AguA. 
     Structural changes of the dried cotton lint samples as a result of xylan adsorption were analysed using solid state (CP/MAS) NMR on a Varian VNMRS 500 wide bore solid state NMR spectrometer with an operating frequency of 125 MHz for  13 C using a 6 mm T3 probe with a probe temperature of 25° C. Dry cotton lint samples were loaded to fill 6 mm zirconium oxide rotors. Spectra were recorded using cross-polarisation and magic angle spinning (CP/MAS). The speed of rotation was 5 kHz, the proton 90° pulse was 5 μs, the contact pulse 1500 μs and the delay between repetitions 5 sec. Chemical shifts were determined relative to TMS by setting the downfield peak of an external adamantane reference to 38.3 ppm. 
     Results 
     Production and Application of Side Chain Removing Enzymes 
       A. niger  D15 was transformed with pGTP-abfB. The recombinant AbfB was expressed extracellulary in pellet and mycelial formation by  A. niger  cultivated in shake flasks and a bioreactor, respectively. The volumetric activity of the AbfB in the shake flasks reached a maximum of 10 nkat mL −1  on the 6 th  day of incubation ( FIG. 2 ). The volumetric activity of AbfB in CCSL in 1%, 2%, and 10% CCSL enriched media were by the second day 1.8, 2.2 and 2.6 times the volumetric activity in 2×MM, respectively. The Abfb activity in 2×MM with 10% CCSL and 2% CCSL reached a maximum activity of 10.0 nkat mL −1  a day earlier than in the 2×MM media ( FIG. 3 ). Notably the AbfB activity in 2×MM with 2% and 10% CCSL were not significantly different (p&lt;0.05) during the incubation period. The AbfB produced in the bioreactor in 2×MM with 2% CCSL had a maximum volumetric activity of approximately 8 nkat mL −1  which was achieved after 36 h of incubation ( FIG. 4 ). In crude form, the specific activity of the recombinant AbfB was 18 nkat mg −1 . The AbfB production increased with biomass growth. The biomass growth corresponded with a decrease in glucose concentration ( FIG. 4 ). The biomass concentration of the  A. niger  grown on 2×MM 2% CCSL enriched medium in the bioreactor reached 32 g L −1  ( FIG. 4 ). During the incubation period, the morphology of  A. niger  was observed to change from pellets to an extensive network of mycelia ( FIG. 5 ). The mycellium showed signs of cell lysis after 144 h of incubation which corresponded to a fall in biomass concentration and depletion of glucose in the bioreactor ( FIG. 4 ). 
     The optimal pH of AbfB ranged from pH 3.0 to pH 5.0 and was stable over pH 3.0 to pH 6.0 ( FIGS. 6A and 7A ). The recombinant AbfB displayed an optimal temperature of 40° C. and demonstrated stability at temperatures up to 60° C. for at least 30 min ( FIGS. 6B and 7B ). The AbfB was relatively more stable when incubated at 40° C. for 1 h but lost 95% of the activity within 5 min when incubated at 80° C. The AbfB released from mild alkaline extracted xylan from bagasse (BagH), H 2 O 2  bleached bagasse (BagB), bamboo (Barn) and  Pinus patula  (Pine) 40, 25, 23 and 28% arabinose, respectively, of the arabinose released from oatspelt xylan ( FIG. 8A ) by the AbfB. Arabinose from low viscosity wheat arabinoxylan (WAX), corn fibre xylan (CAX), debranched arabinan (DAA) and larchwood arabinogalactan xylan (LAG), the AbfB released approximately 20.0, 2.0, 7.0 and 7.0% of the available arabinose ( FIG. 8B ). 
     The relative residual activity against pNPA of AbfB, initially stored at 4° C., increased by 13% within 24 h of storage at 26, 30 and 37° C. ( FIG. 9 ). The AbfB activity at the three storage temperatures remained stable up to 72 h and no significant differences (p&lt;0.05) in the AbfB activities were observed for the entire storage period ( FIG. 9 ). The residual AbfB activity in the reaction mixture after hydrolysis of xylan from bagasse (BagH), H 2 O 2  bleached bagasse (BagBraz), bamboo (Barn) and  P. patula  (Pine) was more than 95% after 16 h of hydrolysis ( FIG. 10 ). The action of the AbfB on the commercial substrates induced visible precipitation of the xylan polymers. 
     The dependency of crude and partially purified AbfB activity on pNPA concentration is presented in  FIGS. 11 and 12 . The initial specific activity against pNPA of crude AbfB obtained from bioreactor on 2×MM with 2% CCSL (w/v) ( FIG. 11A ), from shake flasks on 2×MM with 2% CCSL (w/v) ( FIG. 11B ) and on 2×MM medium ( FIG. 12B ) increased linearly with increase in pNPA concentration, reaching a maximum velocity (V max ) at 40 mM pNPA. The Km value (the amount of substrate to get the maximum velocity) was estimated at pNPA concentration of 10 mM. The velocity of partially purified AbfB by ÄKTA purifying process increased linearly with increase in pNPA concentration up to 10 mM and remained constant up to 40 mM. However a second phase increase emerged beyond 40 mM ( FIG. 12A ). The molecular weight and isoelectric point (pl) of the AbfB enzyme estimated from abfB gene sequence using DNAMAN sequence analysis software were 52.5 kDa and pl 4.04, respectively, whereas the estimated molecular weight from silver stained (Biorad) 10% SDS-PAGE was 67 kDa ( FIG. 13 ). Multiple protein species were visualised in the 10% SDS-PAGE of AbfB produced in bioreactor using CCSL enriched medium and in the ÄKTA partially purified AbfB, whereas single protein species was present in the SDS-PAGE of the AbfB produced in shake flasks both in 2×MM and CCSL enriched media ( FIG. 13 ). 
     Extracting and Characterising Xylan from Lignocellulosic Materials 
     The chemical composition of bagasse, pine ( Pinus patula ), and bamboo ( Bambusidae balcooa ) is presented in  FIGS. 14 and 15 . Bagasse had the highest ash (8.6%) and solvent extractives (6.2%) ( FIG. 14A ), lignin (30.0%) ( FIG. 14B ), cellulose (53.80%) and pentosans (22.00%) ( FIG. 15A ). Both  E. grandis  and  P. patula  had ash and extractive contents of less than 3% ( FIG. 14A ). However,  P. patula  displayed the lowest pentosan level (8.49%) ( FIG. 15A ). The cellulose level in  E. grandis  and bamboo was in the range of 40-43% ( FIG. 15A ) whereas the lignin content was about 23% ( FIG. 14B ). The extraction of xylan from  P. patula , bagasse,  E. grandis , and bamboo by the Hoije method gave extraction efficiencies of 71.20, 65.50, 35.20, and 20.20%, respectively, whereas extraction of xylan from bagasse and  E. grandis  using the Lopez method gave extraction efficiencies of 28.00 and 12.00%, respectively. 
     The solid state  13 C-CP/MAS NMR spectra for unprocessed, extractive free, and xylan extracted residue of  P. patula , bagasse,  E. grandis , and bamboo materials displayed characteristic signals originated from the six carbon resonances of the anhydrous glucose ring in cellulose which were assigned according to Atalla and Isogai [2005, Recent developments in spectroscopic and chemical characterisation of cellulose. In Dumitriu, S. (ed.) Marcel Dekker. New York. pp 123-157] ( FIGS. 16A-D  spectra 1). Beginning with the upfield of the spectra, the C6 of the primary alcohol group emerged at chemical shift (δ) 60-70 ppm and the resonances for a cluster of C2, C3, and C5 of the ring carbons other than those anchoring the glycosidic linkage were displayed at δ 70-81 ppm, the C4 resonances at δ 81-93 ppm, and the C1 at δ 102-108 ppm. In addition, typical doublets were displayed in C4 and C6 resonances (upper field) that represent less ordered (armophous) cellulose and in the ordered (crystalline) cellulose (downfield) (Atalla and Isogai, 2005) in spectra of all the lignocellulosic feedstock materials ( FIGS. 16A-D ). However, the doublets for C6 in the spectra of  P. patula  ( FIG. 16A  spectra 1) were more resolved than in bagasse ( FIG. 16B ),  E. grandis  ( FIG. 16C ) and bamboo ( FIG. 16D ). Furthermore, characteristic signals for acetyl groups (at δ 20-22 ppm, aliphatic groups at δ 30-40 ppm, methyl (CH 3 ) arising from lignin residues at δ 50-60 ppm, C1 of arabinose residues at δ 110-120 ppm, aromatic compounds from lignin residues at δ 140-160 ppm, and C6 of uronic acid residues or carbonyl groups at δ 170-190 ppm were in accordance with Liitia et al. [2001, Holzforschung 55: 503-510]; Maunu [2002, Progress in Nuclear Magnetic Response Spectroscopy 40: 151-174]; Lahaye et al. [2003 , Carbohydrate Research  338: 1559-1569]; Oliveira et al. [2008, Chemical composition and lignin structural features of banana plant leaf sheath and rachis. In Hu, T. Q. (ed). Chapter 10: 171-188] identified in the spectra of the unprocessed raw materials ( FIGS. 16A-D  spectra 1). The  13 C-CP/MAS NMR spectra of the feedstock in which extractives were removed showed changes in line and splitting pattern of signals in the upfield of C4 and C6 s and the resonances between δ 81-93; 60-70 and 20-22 ppm, respectively ( FIGS. 16A-D  spectra 2). Whereas the  13 C-CP/MAS NMR spectra for feedstock from which xylan was removed showed disappearance or reduction in intensity of signals emanating from acetyl, aliphatic, methyl, aromatic, C6 of uronic/carbonyl groups at δ 20-22, 30-40, 50-60, 140-160 and 170-190 ppm, respectively ( FIGS. 16A-D  spectra 3). The  13 C-CP/MAS NMR spectra for bagasse from which xylan was removed displayed sharpened signals particularly in resonances between δ 30 and 40 ppm which originate from aliphatic groups (Oliveira, 2008) and complete disappearance of reasonances arising from methyl groups at δ 40-50 ppm ( FIG. 16B  spectra 3). Whereas in  13 C-CP/MAS NMR spectra of  P. patula, E. grandis , and bamboo, a reduced intensity of the signal for methyl group was notable ( FIGS. 16A , C, D spectra 3, respectively). 
     The initial glucose levels in extractive free bagasse, bamboo,  P. patula  and  E. grandis  were 68.0, 66.0, 61.0, and 59.0%, respectively. Upon xylan extraction using the Hoije method the glucose proportion in the feedstocks increased to 75.0, 76.0, 65.0, and 79.0%, respectively, while xylose concentration decreased from 14.0 to 10.0%, 27.0 to 25%, 35 to 19%, and 30.0 to 22.0% in  P. patula , bagasse,  E. grandis  and bamboo, respectively ( FIGS. 17A-D ). Furthermore the xylan extraction corresponded to a decrease in arabinose, and galactose content in all the feedstock ( FIGS. 17A-D ). The concentration of mannose (16.0%), which was detectable only in  P. patula  feedstock, increased to 18% in the xylan extracted residue ( FIG. 17A ). The presence of uronic acids was detectable in all the four lignocellulosic feedstock materials (Table 7). The highest and lowest uronic acids content were found in  E. grandis  and bagasse feedstocks, respectively (Table 7). 
     The elution profiles of the extracted xylan fractions were referenced to the elution profiles of the monomeric sugars (arabinose, rhaminose, galactose, glucose, xylose, and mannose), xylitol sugar, birch xylan, oatspelt xylan, and H 2 O 2  bleached bagasse (Bag B). The HPAEC-PAD (Dionex) chromatogram showed that the monomeric sugars including the xylitol eluted on CarboPac PA 100 column within a retention time of 5 min ( FIGS. 18A  and B). Between 0 and 3 min, oatspelt xylan elution profile showed a high intensity peak with detector response &gt;300 nC which corresponded to retention time for xylitol ( FIG. 18D ). Otherwise, between 3 and 6 min the chromatogram for both birch xylan and the oatspelt xylan displayed only low intensity peaks (detector response of &lt;20 nC) ( FIGS. 18C  and D, respectively). Whereas, the chromatogram for H 2 O 2  bleached bagasse xylan (Bag B) displayed multiple peaks of high intensities with detector response of over 100 nC appearing at retention time between 2 and 30 min ( FIG. 19A ). The chromatograms of both Hoije and Lopez extracted xylan displayed low intensity peaks (&lt;20 nC) within 25 min retention time ( FIGS. 19B  and C). Among the extrcated xyla samples, the peak corresponding to xylitol was present in the chromatogram of xylan from  E. grandis  ( FIG. 20A ), bamboo ( FIG. 20C ) and  P. patula  ( FIG. 20D ) extracted by the Hoije method. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Profile of neutral sugars and uronic acid of pre-extracted xylan 
               
            
           
           
               
               
            
               
                   
                 Total uronic acid (%) a   
               
            
           
           
               
               
               
               
            
               
                   
                 Total neutral sugar composition of xylan (%) a   
                 Raw 
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Xylan type 
                 Arabinose 
                 Galactose 
                 Glucose 
                 Rhaminose 
                 Xylose 
                 material* 
                 Xylan 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Bagasse 
                 17.44 
                 3.4 
                 8.2 
                 — 
                 70.97 
                 6.02 
                 8.5 
               
               
                 Hojie (BH) 
               
               
                 Bamboo (BM) 
                 10.50 
                 2.86 
                 7.09 
                 — 
                 79.54 
                 6.72 
                 11.20 
               
               
                 
                   Eucalyptus 
                 
                 0.26 
                 4.45 
                 2.28 
                 0.92 
                 92.09 
                 10.34 
                 12.83 
               
               
                   grandis  Hoije 
               
               
                 (EH) 
               
               
                 
                   Pinus patula 
                 
                 15.53 
                 9.98 
                 13.23 
                 — 
                 61.26 
                 8.68 
                 11.54 
               
               
                 Birch 
                 — 
                 0.43 
                 19.53 
                 — 
                 80.03 
                 — 
                 10 
               
               
                 Oatspelt** 
                 7.4 
                 1.35 
                 4 
                 — 
                 87.24 
                 — 
                 — 
               
               
                 xylan 
               
               
                   
               
               
                 Note: 
               
               
                   a denotes mean of at least three samples with standard deviation &lt; 0.1, 
               
               
                 *percentage of extractive free raw material 
               
               
                 **Normal composition of Oatspelt xylan (Sigma) is 10:75:15 for arabinose:xylose:glucose 
               
            
           
         
       
     
     The xylose content of xylan from  E. grandis  extracted by Hoije method (EU H), bamboo, bagasse extracted by Hoije method (Bag H), and  P. Patula  was 92.00, 79.50, 71.00 and 61.30%, respectively, whereas, the xylose content in birch and oatspelt xylan was 80.00 and 87.20%, respectively (Table 7). The proportion of arabinose in Bag H,  P. patula , and bamboo xylan fractions was 17.45, 15.50, and 10.50%, respectively (Table 7). Although commercial oatspelt xylan is reported to have 10% arabinose (Sigma), this study showed arabinose content of 7.4% (Table 7). About 2.30% glucose was present in EU H xylan fractions, whereas 13.20% glucose was present in  P. patula  xylan fractions (Table 7). In addition, the EU-H contained 4.45% galactose and traces of rhaminose and arabinose (Table 7). Total uronic acid content of EU H, bamboo, and  P. Patula  xylan was 12.83, 11.20 and 11.54%, whereas bagasse xylan contained 8.5% (Table 7). 
     The xylan fractions extracted by the Hoije method when subjected to mild acid (72% H 2 SO 4 ) hydrolysis yielded between 16 and 55% insoluble residues ( FIG. 21 ). The highest acid insoluble residues 55% were obtained from xylan extracted from  P. patula  using the Hoije method. The acid insoluble residues from bagasse xylan extracted by the Lopez method (Bag L) were 16%, whereas the reference material, birch xylan, had 3.5% ( FIG. 21 ). 
     Structural Characteristics of the Extracted Xylan 
     The  1 H NMR and  13 C NMR spectra of the reference xylan from birch, H 2 O 2  bleached bagasse, and oatspelt xylan displayed characteristic signals for proton and carbon resonances ( FIGS. 22A-D ). The  1 H NMR spectra of the extracted xylan displayed characteristic proton signals from xylose, 4-O-methylglucuronic acid, and arabinose units at chemical shifts (6) between 3.3 to 5.7 ppm ( FIGS. 22-24 ). In the  1 H NMR the proton signals for xylose in the xylan fractions from bagasse extracted by Hoije (Bag H) and by Lopez (Bag L) methods were displayed at δ 4.44/4.45, 3.50, 3.67 and 4.01 ppm ( FIG. 23A ). According to Vignon and Gey [1998 , Carbohydrate Research  307: 107-111] such signals correspond to H1, H3, H4, and H5 of xylose unit substituted with 4-O-methylglucuronic acid linked at 0-2, respectively. In addition, the proton spectra for Lopez extracted bagasse (Bag L) and Hoije extracted bagasse (Bag H) displayed proton resonances from D-xylopyranosyl units residues substituted with 4-O-methylglucuronic acid at O-2 and acetyl group at O-3 at δ 4.72, 3.76/3.75, and 3.97/3.96 ppm that correspond to H1, H2 and H4 of the β-D-xylopyranosyl units, respectively ( FIG. 23A ). 
     The proton spectra of xylan extracted from  E. grandis  by Lopez method (EU-L) and Hoije method (EU-H) displayed signals at δ 4.48, 3.96/3.99, 3.63/3.68, and 3.50/3.52 ppm arising from xylose units substituted with 4-O-methylglucuronic acid ( FIG. 23C ). According to Sims and Newman (2006) such chemical shifts could originate from D-xylopyranosyl units residues substituted with 4-O-methylglucuronic acid at O-2 and acetyl group at O-3. In the same spectra, proton signals originating from H1 and H3 of the 4-O-methylglucuronic acid residues were evident at δ 5.48/5.49/5.46 ppm, and between 1.06 and 1.54 ppm δ 5.16 and 3.63 ppm ( FIG. 23C ). In the proton spectra of bamboo, and  P. patula , signals for the 4-O-methylglucuronic acid residues were displayed at 5.47/5.48 ppm ( FIGS. 23A  and C). Such signals could appear at δ 5.16 and 3.63 ppm [Sun et al., 2004, Carbohydrate Research 339: 291-300, Polym. Degrad. and Stability 84: 331-337, Carbohydrate Polymers 56: 195-204; Ebringerová et al., 1998 , Carbohydrate Polymers  37: 231-239]. The proton spectra of the extracted xylan further displayed characteristic signals originating from C-2 linked arabinose to xylose units [Höije et al., 2005 , Carbohydr. Polym.  61: 266-275; Ebringerová et al., 1998 , Carbohydrate Polymers  37: 231-239]. In the proton spectra for Bag H and Bag L, the C2 linked arabinose were identified at δ 5.58, 5.60, 4.29/4.30 ppm ( FIGS. 23A  and C). The presence of arabinose in the proton spectra of bamboo and  P. patula  xylan were in accordance with Ebringerováet al. (1998) and Vignon and Gey, (1998) identified inter alia, at δ 5.58/5.59 ppm and 5.47/5.48 ppm, respectively ( FIGS. 24A  and C). Arabinose signals were present in the proton spectra of EU-L and EU-H at δ between 3.83 and 3.85 ppm ( FIG. 23C ) whereas the presence of O-2 linked acetyl groups which based on Shao et al. [2008, Wood Science Technology 42: 439-451]; Höije et al. (2005) Sun et al. (2004); Ebringerová et al. (1998); Vignon and Gey (1998) supra were identified at δ 4-2.4 ppm ( FIGS. 22-24 ). In addition, broad signals associated with aromatic or phenolic compounds originating from lignin residues (Höije et al., 2005 supra; Xu et al., 2006, Carbohydrate Research and Oliveira et al., 2008, Chemical composition and lignin structural features of banana plant leaf sheath and rachis. In Hu, T. Q. (ed). Chapter 10: 171-188] were displayed between δ 6.5 and 7.9 ppm in the proton spectra of the extracted xylan ( FIGS. 23 and 24 ). 
     The characteristic carbon resonances from the five carbons of (1→4) linked β-D-xylopyranosyl residues between δ 103 and 62 ppm were reflected in the  13 C NMR spectra of the extracted xylan fractions ( FIGS. 23 and 24 ). In the carbon spectra of  E. grandis  xylan fractions, the resonance originating from C1 of xylose units with C2-linked arabinose groups appeared at δ≈102.33 ppm while those from C1, C2, C3, C4, and C5 of arabinofuranosyl residues displayed at δ≈108, 81.7, 78, 85.5, and 62 ppm ( FIG. 23D ). In the spectra the arabinose associated carbon signals in EU H and EU L were seen at d 61.81/61.59 ppm ( FIG. 23D ). In Bag L and Bag H the arabinose signals identified based on Vignon and Gey (1998) supra were displayed at δ 112.50/111.56 and 89.31 ppm which correspond to C1 and C2 of C-2 linked arabinose residues, respectively ( FIG. 23B ). The C1, C2, and C4 resonances belonging to arabinofuranosyl residues mono substituted xylose units at O-3 (Ebringerová et al., 1998, supra) were present in the  13 C NMR spectra of both bamboo and  P. patula  at δ 108.60/108.33 ppm, 81.71/81.42 ppm, and 85.72/85.43 ppm respectively ( FIGS. 24B  and D). The presence of 4-O-methylglucuronic acid residues in the extracted xylan fractions was evident from characteristic carbon signals that originate from C1, C4, C6 and C5 at δ between 97 and 100 ppm, 83 and 84 ppm, 179-172 ppm, and 59 and 61 ppm, respectively (Habibi and Vignon, 2005, Carbohydrate Research 340: 1431-1436; Xu et al., 2000, supra; Ebringerová et al., 1998, supra) ( FIGS. 23 and 24 ). The presence of acetyl, phenolic, and aromatic groups arising from lignin compounds, and hexose sugars were identified in the  13 C NMR spectra of the extracted xylan fractions. In addition, acetyl groups in all the xylan fractions were identified between δ 21-24 ppm ( FIGS. 23 and 24 ). However, absence of the acetyl signals was apparent in  13 C NMR spectra of birch xylan ( FIG. 22B ) and H 2 O 2  bleached bagasse (Bag B) ( FIG. 22D ). The methoxyl groups signifying presence of lignin compounds [Ebringerová et al., 1998 supra, Sun et al., 2004, supra, Xu et al., 2006 supra; Maunu, 2008,  13 C CPMAS NMR Studies of wood, cellulose fibers, and derivatives. In Hu, T. Q. (ed)]; were identified in the  13 C NMR spectra at d 56.62/56.58 ppm ( FIGS. 23 and 24 ). The lignin compounds particularly those linked to arabinosyl side chains through ferulic acid bridges were reflected by carbon signals at δ 140-160 ppm and 116.6-117.08 ppm ( FIGS. 23 and 24 ). Other lignin compounds associated with ferulic or p-coumaric acids groups and of the —CH 3 — in Ar—COCH 3  (Maunu, 2002 , Progress in Nuclear Magnetic Response Spectroscopy  40: 151-174; Sun et al. 2004, supra) were seen at δ 26-49 ppm at δ 115.38 and δ 17.55/17.69 ppm in Bag L ( FIG. 23B  spectra 1) bamboo and  P. Patula  xylan fractions ( FIGS. 24B  and D). The presence hexose sugars such as galactose or glucose [Sun et al., 2004, supra] was evident in the  13 C NMR spectra, in particular of EU H and EU L at δ between 69 and 71 ppm ( FIG. 23B ). 
     The FTIR spectra of the extracted xylan fractions displayed characteristic bands for xylan residues which included β-glycosidic linkages reflected at ≈897 cm −1  ( FIG. 25 ). However, such signal was absent in the FTIR spectra of the extracted xylan from  P. Patula . In addition the spectra of the extracted xylan displayed signals in the band region between 1600 and 1200 cm −1  ( FIG. 25 ), which according to Fengel and Wegener [1989, Wood Chemistry, Ultrastructure, Reactions. Walter de Gruyter, Berlin, Germany] is a region associated with aromatic compounds that originate from lignin fractions. Bands arising from syringyl ring breathing with C Ar —OCH 3 , and methoxyl groups in lignin were reflected at 1329 cm −1  and 1591-1595, and 1460-1461 cm −1  in the spectra of  E. grandis  and bamboo ( FIG. 25 ). The bagasse xylan fractions, Bag H and Bag L contained signals of varying intensities in the 1600-1200 cm −1  wavelength region and spectra for Bag L reflected a relatively strong intensity band for C—H stretching vibrations at 2919 cm −1  ( FIG. 25 ). The FTIR spectra for EU L displayed two intense signals related to lignin compounds at 1591 and 1379 cm −1  while in EU H spectra, multiple bands of lesser intensity in the 1600-1200 cm −1  region in particular at 1595, 1461, and 1329 cm −1  were seen ( FIG. 25 ). 
     Controlled Enzymatic Removal of Side Chains from Lignocellulosic Feedstocks 
     The recombinant α-L-Arabinofuranosidase (AbfB) produced by the  Aspergillus niger  released arabinose from oatspelt xylan and mild alkali extracted xylan from bagasse Höije (BH), H 2 O 2  bleached bagasse (BB), bamboo (BM) and  Pinus patula  (PP). Arabinose of about 15 mg g −1  substrate and 14 mg g −1  substrate (representing 20% and 9% of available arabinose) was removed from oatspelt xylan and BH, respectively, whereas about 5 mg g −1  substrate (3% available arabinose) was released from AbfB treated bamboo (BM) xylan fractions ( FIG. 26A ). The purified α-glucuronidase (α-glu) from  Schizophyllum commune  removed 1.2 mg g −1  4-O-MeglCA (1.3% available uronic acid) from birch xylan, whereas about 1.6 mg g −1  4-O-MeglCA (2% available uronic acids) was released from the BH xylan fractions ( FIG. 26B ). The proportion of 4-O-MeglCA removed from  Eucalyptus grandis  xylan extracted by the Hoije method (EH) and from  Eucalyptus grandis  xylan gel (ES) was about 1.3 mg g substrate −1  ( FIG. 26B ). The lowest α-glu removal of 4-O-MeglCA was &lt;0.6 mg g substrate −1  from H 2 O 2  bleached bagasse (BB) ( FIG. 26B ). The synergistic effect of AbfB and α-glu (AG) released 3% arabinose (on substrate basis) from BH whereas 2.4% was released with individual application of AbfB ( FIG. 26C ). The synergistic effect of AbfB and α-glu on the release of glucuronic acid from BH was about 0.18% while individual application of α-glu released 0.16% 4-O-MeglCA ( FIG. 26C ). The AG removed 0.13% and 0.08% MeglCA from  P. patula  (PP) and Bamboo (BM) respectively while the individual application of the α-glu released about 0.1% and 0.09% MeglCA, respectively ( FIG. 26C ). 
     Effect of Process Parameters on Enzymatic Xylan Side Chain Removal 
     A maximum of 27% arabinose was liberated from oatspelt xylan treated with AbfB for 8 h at AbfB xylan specific dosage of 1 400 nKat g −1  ( FIG. 27 ). Equal amount of arabinose was released by treating the oatspelt xylan with AbfB at xylan specific dosage of about 700 nKat g −1  for 16 h ( FIG. 27 ). In addition, the change in the amount of arabinose released at hydrolysis time ≧8 h and AbfB xylan specific dosage ≧350 nKat g −1  was not significant (p&lt;0.05) ( FIG. 27 ). Arabinose was not detectable when oatspelt xylan was treated with AbfB at specific dosage of 180 nKat g substrate −1  at 60° C. for hydrolysis performed both for 4 h and 16 h ( FIG. 28 ). On the other hand, oatspelt xylan treated at the same AbfB dosage level at 40° C. for 4 h, released about 4% arabinose ( FIG. 28 ). Increasing the AbfB xylan specific dosage to 720 nKat substrate g −1  at 60° C. released about 1.1% arabinose when hydrolysis was performed at both 4 and 16 h. The same AbfB dosage level released 14% arabinose from the oatspelt xylan when the hydrolysis was performed at 40° C. for similar duration ( FIG. 28 ). Consequently, precipitated hydrogels not visible in oatspelt xylan were treated with AbfB at 60° C. 
     Determination of Optimal Conditions for Removal of Side Chains 
     The response surface plots for arabinose removal reflected both linear and quadratic relationships between temperature, time, and AbfB xylan specific dosage in relation to arabinose removal. A maximum of 12 mg g −1  substrate of arabinose was liberated from oatspelt xylan at hydrolysis time ranging from 10.8 to 18 h; temperature from 35.9 to 44.5° C. and AbfB xylan specific dosage from 427 to 725 nKat g substrate −1  ( FIGS. 29A-C ). Similarly, the response surface plots for removal of glucuronic acid (4-O-MeglcA) from birch xylan α-glu reflected both linear and quadratic relationship with hydrolysis time, temperature, and α-glu xylan specific dosage. A maximum of 350 μg g −1  substrate of 4-O-MeglcA was removed from birch by α-glu at xylan specific dosage between 16500 and 18000 nkat g substrate −1  when hydrolysis was performed for durations between 9 and 10.2 h at temperatures between 33.5 and 42° C. ( FIG. 29D-F ). As shown in the Pareto chart, the effect of the hydrolysis parameters and their interaction on removal of arabinose reflected significant quadratic effects (p&lt;0.05) in descending magnitude of temperature, time, and AbfB xylan specific dosage level ( FIG. 30A ). Furthermore, the Pareto charts showed significant interaction effects (p&lt;0.05) mainly between linear and quadratic effects of time and temperature which in descending magnitude were time (L) by temperature (Q), Time (Q) by temperature (L), time (Q) by temperature (Q), and time (L) by temperature (L) (the L denotes linear and Q Quadratic relationships) ( FIG. 30A ). 
     The hydrolysis parameters having significant effects on removal of 4-O-MeglcA from birch xylan by α-glu were in descending magnitude, from linear effects from α-glu xylan specific dosage [α-glu, nKat/g (L)], temperature [Temp (L)], and the quadratic effect of temperature [Temp (Q)] ( FIG. 30B , Pareto chart). The only significant interaction effect on the removal of 4-O-MeglcA from birch xylan by α-glu was from the linear effect of hydrolysis time and the quadratic effect of temperature [time (L) by temperature (Q)] ( FIG. 30B , Pareto chart). The desirability contour plots showed optimal set points for AbfB removal of arabinose from oatspelt xylan ( FIG. 30A ) to fall between 10.8 h and 18 h, 35.9° C. and 44.5° C., and enzyme dosage level between 427.0 and 725.0 nKat g substrate −1 . In contrast, the optimal set points for α-glu removal of 4-O-MeglcA were between 9 h and 10.2 h, 33.5 and 42° C., and 16500 and 18000 nKat g substrate −1 . The regression coefficients of the variable in the second-order polynomial model fitted to the response surface plots for AbfB removal of arabinose as a function of time, temperature and enzyme dose gave a regression coefficient (R 2 ) of 0.99 (adjusted R 2 =0.97) (Table 8) while that of 4-O-MeglcA removal gave R 2  of 0.90 (R 2  adjusted=0.81) (Table 9). 
     A maximum of ≈4% arabinose (substrate basis or 40.% available arabinose) was achievable with combinations of xylan concentrations between 3 and 6 mg mL −1  and AbfB volumetric activity of &gt;10 nkat mL −1  ( FIG. 31 ). The desirability contour plot ( FIG. 32 ) located the optimal xylan concentration and volumetric activity of the AbfB to be 5563.3 μg mL −1  and 27.2 nKat respectively, for hydrolysis performed at 40° C. for 16 h. At such optimal conditions, AbfB arabinose removal to a maximum of 4.7% arabinose (42700 μg g −1  substrate or 42.7% available arabinose) was achieved from oatspelt xylan. The largest significant effect on the arabinose removal was xylan concentration rather than AbfB volumetric activity ( FIG. 33 ). The regression coefficients in the fitted model for the arabinose removal response surface plot as a function of xylan and enzyme concentration gave R 2  of 0.89 (adjusted R 2 =0.79) (Table 10). 
     Enzyme Aided Adsorption of Xylan onto Cellulosic Material 
     In the presence of AbfB, cotton lint treated with 25 mL (25CXE), 15 mL (15CXE), 12.5 mL (12.5CXE) and 5 mL (5CXE) oatspelt xylan (1% w/v) mixture had a specific xylan weight gain of approximately 23, 27, 26, and 42%, respectively (Table 11). The specific xylan weight gain of the corresponding cotton lint treated with oatspelt xylan in the absence of AbfB in 25 mL (25CX) and 12.5 mL (12.5 CX) was approximately 18 and 27%, respectively (Table 11). The cotton lint treated with birch xylan in the presence (BCXE) and absence (BCX) of α-D-glucuronidase (AguA) had a specific weight gain of 16% and 7%, respectively (Table 11). The treatment of cotton lint with xylan from bagasse extracted by the Hoije method (ABH), H 2 O 2  bleached bagasse xylan (ABB), bamboo (ABM) and  P. patula  (AP) [both extracted by the Hoije method] in the presence of AbfB gave a cotton lint xylan specific weight gain of 31.0, 11.0, 16.5 and 10.0%, respectively (Table 12). On the other hand, treatment of cotton lint with bagasse extracted by the Hoije method (GBH), H 2 O 2  bleached bagasse xylan (GBB), bamboo (GBM),  P. patula  (GP), and  E. grandis  (GEH), all extracted by the Hoije method, and  E. grandis  xylan gel (GES) in the presence of α-D-glucuronidase (AguA) gave a cotton lint xylan specific weight gain of 28.5, 17.0, 30.0, 32.0, 15 and 25%, respectively (Table 12). Treating the cotton lint in the absence of AguA with xylan from  E. grandis  extracted by the Hoije method (SEH) and  E. grandis  gel (SES) resulted in a cotton lint xylan specific weight gain of 3 and 40%, respectively (Table 12). 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Regression coefficients for arabinose release as a function 
               
               
                 of the hydrolysis parameters (coded values) 
               
            
           
           
               
               
               
               
            
               
                 Hydrolysis parameter and relationship 
                   
                 Coefficients in arabinose 
                   
               
               
                 Arabinose release = f (time, 
                   
                 release equations f(x, y) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 temperature, specific AbfB level) 
                 Regression 
                 f(time, 
                 f(time, 
                 f(temp, 
                   
                 Significance 
               
               
                 R 2  = 0.99 (R 2  adjusted = 0.97) 
                 coefficient 
                 temp) 
                 AbfB ) 
                 AbfB; 
                 t(14) 
                 level (p) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Mean/interaction 
                 
                   9476.69 
                 
                 
                   b 
                   0 
                 
                 
                   b 
                   0 
                 
                 
                   b 
                   0 
                 
                 
                   29.009 
                 
                 
                   &lt;0.01 
                 
               
               
                 Main effects 
               
               
                 (1)Time(h)(L) 
                 
                   3989.39 
                 
                 
                   b 
                   1 
                 
                 
                   b 
                   1 
                 
                   
                 
                   19.942 
                 
                 
                   &lt;0.01 
                 
               
               
                 Enzyme dose (nKat/g substrate)(Q) 
                 
                   −3145.29 
                 
                   
                 
                   b 
                   4 
                 
                 
                   b 
                   4 
                 
                 
                   −5.341 
                 
                 
                   &lt;0.01 
                 
               
               
                 (3)Enzyme dose (nKat/g substrate)(L) 
                 
                   2071.60 
                 
                   
                 
                   b 
                   3 
                 
                 
                   b 
                   3 
                 
                 
                   7.322 
                 
                 
                   &lt;0.01 
                 
               
               
                 (2)Temp (° C.)(L) 
                 −540.89 
                 b 3   
                   
                 b 1   
                 −1.912 
                 0.076 
               
               
                 Temp (° C.)(Q) 
                 −405.20 
                 b 4   
                   
                 b 2   
                 −0.641 
                 0.532 
               
               
                 Time (h)(Q) 
                 5.18 
                 b 2   
                 b 2   
                   
                 0.0100 
                 0.992 
               
               
                 Interactions 
               
               
                 1Q by 2Q 
                 
                   −4178.84 
                 
                 
                   b 
                   1122 
                 
                 
                   b 
                   1122 
                 
                 
                   b 
                   1122 
                 
                 
                   −3.548 
                 
                 
                   &lt;0.01 
                 
               
               
                 1L by 2Q 
                 
                   −1461.58 
                 
                 
                   b 
                   122 
                 
                 
                   b 
                   122 
                 
                 
                   b 
                   122 
                 
                 
                   −5.166 
                 
                 
                   &lt;0.01 
                 
               
               
                 1Q by 2L 
                 
                   1323.20 
                 
                 
                   b 
                   112 
                 
                 
                   b 
                   112 
                 
                 
                   b 
                   112 
                 
                 
                   3.8189 
                 
                 
                   &lt;0.01 
                 
               
               
                 1L by 2L 
                 
                   −553.82 
                 
                 
                   b 
                   12 
                 
                 
                   b 
                   12 
                 
                 
                   b 
                   12 
                 
                 
                   −2.768 
                 
                 
                   &lt;0.01 
                 
               
               
                 1L by 3L 
                 
                   437.94 
                 
                 
                   b 
                   13 
                 
                 
                   b 
                   13 
                 
                 
                   b 
                   13 
                 
                 
                   2.189 
                 
                 
                   &lt;0.05 
                 
               
               
                 1Q by 3L 
                 −352.67 
                 
                   b 
                   113 
                 
                 
                   b 
                   113 
                 
                 
                   b 
                   113 
                 
                 −1.018 
                 0.326 
               
               
                 Error 
                 37.84 
               
               
                   
               
               
                 Note: 
               
               
                 1 = time, 2 = temperature, 3 = enzyme xylan specific dosage, L = linear, Q = quadratic, R 2  = linear regression coefficient, b i  . . . ni = model regression coefficient, letters and numbers in bold = significance 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Regression coefficients for glucuronic acid release as a 
               
               
                 function of the hydrolysis parameters (coded variable) 
               
            
           
           
               
            
               
                 Hydrolysis parameter and 
               
               
                 relationship 
               
            
           
           
               
               
               
               
            
               
                 Glucuronic acid release = f 
                   
                 Coefficient in glucuronic acid 
                   
               
               
                 (time, temperature, specific 
                   
                 release equations f(x, y) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 AbfB level) R 2  = 0.90 (R 2   
                 Regression 
                 f(Time, 
                 f(Time, 
                 f(Temp, 
                   
                   
               
               
                 adjusted = 0.81) 
                 coefficient 
                 Temp) 
                 AguA dose) 
                 AguA dose) 
                 t(17) 
                 p 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Mean/interaction 
                 
                   278.0162 
                 
                 
                   b 
                   0 
                 
                 
                   b 
                   0 
                 
                 
                   b 
                   0 
                 
                 
                   13.9314 
                 
                 
                   &lt;0.01 
                 
               
               
                 Main effects 
                   
                   
                   
                   
                   
                 
                   &lt;0.01 
                 
               
               
                 Temp ° C. (Q) 
                 
                   −70.9028 
                 
                 
                   b 
                   22 
                 
                   
                 
                   b 
                   2 
                 
                 
                   −3.9416 
                 
                 
                   &lt;0.01 
                 
               
               
                 (3)α-glu (nKat/g substrate) (L) 
                 
                   66.164 
                 
                   
                 
                   b 
                   3 
                 
                 
                   b 
                   3 
                 
                 
                   3.8280 
                 
                 
                   &lt;0.01 
                 
               
               
                 (2)Temp ° C. (L) 
                 
                   −38.087 
                 
                 
                   b 
                   2 
                 
                   
                 
                   b 
                   2 
                 
                 
                   −2.2040 
                 
                 
                   &lt;0.05 
                 
               
               
                 (1)Time(h)(L) 
                 35.295 
                 b 1   
                 b 1   
                   
                 2.0420 
                 0.057 
               
               
                 α-glu (nKat/g substrate) (Q) 
                 −10.911 
                   
                 b 3   
                 b 3   
                 −0.6070 
                 0.552 
               
               
                 Time (h) (Q) 
                 −26.958 
                 b 22   
                 b 22   
                   
                 −1.4990 
                 0.152 
               
               
                 Interactions 
               
               
                 1L by 2Q 
                 
                   −71.385 
                 
                 
                   b 
                   122 
                 
                 
                   b 
                   122 
                 
                 
                   b 
                   122 
                 
                 
                   −2.9210 
                 
                 
                   &lt;0.01 
                 
               
               
                 1L by 2L 
                 −21.584 
                 b 12   
                 b 12   
                 b 12   
                 −1.2490 
                 0.229 
               
               
                 1Q by 2L 
                 −21.151 
                 b 112   
                 b 112   
                 b 112   
                 −0.8650 
                 0.399 
               
               
                 1Q by 3L 
                 −16.121 
                 b 113   
                 b 113   
                 b 113   
                 −0.6600 
                 0.518 
               
               
                 1L by 3L 
                 27.506 
                 b 13   
                 b 13   
                 b 13   
                 1.5920 
                 0.139 
               
               
                 2L by 3L 
                 9.056 
                 b 23   
                 b 23   
                 b 23   
                 0.5240 
                 0.607 
               
               
                   
               
               
                 Note: 
               
               
                 1 = time or xylan loading, 2 = temperature, 3 = enzyme xylan specific dosage, L = linear, Q = quadratic, R 2  = linear regression coefficient, b i  . . . b n  = model regression coefficient, letters and numbers in bold = significance. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Regression coefficients for arabinose release as a function of 
               
               
                 the hydrolysis parameters (coded variables) 
               
            
           
           
               
               
               
               
               
            
               
                 Arabinose release = 
                   
                   
                   
                   
               
               
                 f(AbfB, xylan loading) 
                   
                   
                   
                 Significance 
               
               
                 R 2  = 0.88 
                   
                 f(AbfB, 
                   
                 level 
               
               
                 (R 2  adjusted = 0.79) 
                   
                 Xylan) 
                 t(5) 
                 (p) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Mean/interaction 
                 
                   3.993 
                 
                 
                   b 
                   0 
                 
                 
                   11.615 
                 
                 
                   &lt;0.01 
                 
               
               
                 Main effects 
                   
                   
                   
                 
                   &lt;0.01 
                 
               
               
                 (1)Xylan (g)(L) 
                 
                   0.899 
                 
                 
                   b 
                   1 
                 
                 
                   5.228 
                 
                 
                   &lt;0.01 
                 
               
               
                 Xylan (g)(Q) 
                 
                   −0.835 
                 
                 
                   b 
                   11 
                 
                 
                   −3.674 
                 
                 
                   &lt;0.01 
                 
               
               
                 AbfB dose (nKat mL −1 )(Q) 
                 −0.314 
                 b 22   
                 −1.380 
                 0.226 
               
               
                 Interaction 
               
               
                 1L by 2L 
                 0.221 
                 b 12   
                 0.909 
                 0.405 
               
               
                   
               
               
                 Note: 
               
               
                 1 = xylan loading, 
               
               
                 2 = enzyme xylan specific dosage, 
               
               
                 L = linear, 
               
               
                 Q = quadratic, 
               
               
                 R 2  = linear regression coefficient, 
               
               
                 b i  . . . n i  = model regression coefficient, 
               
               
                 letters and numbers in bold = significance 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Cotton lint xylan specific weight gain after 
               
               
                 commercial xylan adsorption 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 *Cotton lint 
                 **Specific 
               
               
                 Xylan Type 
                   
                 weight gain 
                 weight gain 
               
               
                 (commercial sources) 
                 Treatment 
                 (%) 
                 (%) 
               
               
                   
               
               
                 Oatspelt (Sigma) 
                 25CXE 
                 5.69 ± 0.22 
                 22.74 ± 0.88 
               
               
                   
                 25CX 
                 4.38 ± 1.26 
                 17.52 ± 5.03 
               
               
                   
                 15CXE 
                 3.99 ± 0.0  
                 26.6 ± 0.0 
               
               
                   
                 15CX 
                 nd 
                 nd 
               
               
                   
                 12.5CXE 
                 3.29 ± 0.08 
                 26.28 ± 0.62 
               
               
                   
                 12.5CX 
                 3.38 ± 0.04 
                 27.04 ± 0.34 
               
               
                   
                  5CXE 
                 2.11 ± 0.36 
                  42.3 ± 7.21 
               
               
                   
                  5CX 
                 nd 
                 nd 
               
               
                 Birch (Roth) 
                 BCXE 
                 0.62 ± 0.33 
                 16.44 ± 8.88 
               
               
                   
                 BCX 
                 0.08 ± 0.14 
                 6.67 ± 0.0 
               
               
                   
               
               
                 Note: 
               
               
                 CXE = Cotton treated with AbfB modified oatspelt xylan, 
               
               
                 BCXE = Cotton treated with AguA modified xylan from birch, 
               
               
                 CX and BCX = cotton treated with unmodified xylan from oatspelt xylan and birch xylan, respectively. 
               
               
                 *Weight gain as a percentage of initial cotton lint weight, 
               
               
                 **Weight gain as a percentage of theoretical amount of xylan in the reaction mixture 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Cotton lint xylan specific weight gain after adsorption of 
               
               
                 pre-extracted xylan 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 *Cotton lint 
                 **Specific 
               
               
                   
                   
                 weight gain 
                 weight 
               
               
                 Xylan Type 
                 Treatment 
                 (%) 
                 gain (%) 
               
               
                   
               
               
                 Mild alkali extracted xylan 
                   
                   
                   
               
               
                 from grasses 
               
               
                 Bagasse (BH) 
                 ABH 
                 1.55 ± 0.14 
                   31 ± 2.83 
               
               
                   
                 AGBH 
                 1.68 ± 0.39 
                 33.5 ± 7.78 
               
               
                   
                 GBH 
                 1.43 ± 0.04 
                 28.5 ± 0.71 
               
               
                   
                 SBH 
                 0.20 ± 0.0  
                   4 ± 0.0 
               
               
                 H 2 O 2  bleached Bagasse 
                 ABB 
                 0.55 ± 0.0  
                  11 ± 0.0 
               
               
                 (BB) 
                 AGBB 
                 0.95 ± 0.0  
                  19 ± 0.0 
               
               
                   
                 GBB 
                 0.85 ± 0.07 
                   17 ± 1.41 
               
               
                   
                 SBB 
                 0.72 ± 0.18 
                 14.5 ± 1.41 
               
               
                 Bamboo ( B. balcoa ) (BM) 
                 ABM 
                 0.82 ± 0.11 
                 16.5 ± 2.12 
               
               
                   
                 AGBM 
                 0.82 ± 0.11 
                 0.82 ± 0.11 
               
               
                   
                 GBM 
                 1.10 ± 0.14 
                 22.0 ± 2.83 
               
               
                   
                 SBM 
                 0.00 ± 0.0  
                 30.0 ± 1.41 
               
               
                 Mild alkali extracted xylan 
               
               
                 from softwood 
               
               
                 
                   Pinus patula 
                 
                 AP 
                 0.50 ± 0.07 
                   10 ± 1.41 
               
               
                   
                 AGP 
                 0.62 ± 00   
                 12.5 ± 2.83 
               
               
                   
                 GP 
                 1.60 ± 0.49 
                   32 ± 2.83 
               
               
                   
                 SP 
                  0.12 ± 0.049 
                  2.5 ± 0.090 
               
               
                 Mild alkali extracted xylan 
               
               
                 from hardwood 
               
               
                   Eucalyptus grandis  (EH) 
                 GEH 
                 0.75 ± 0.0  
                  15 ± 0.0 
               
               
                   
                 S-EH 
                 0.15 ± 0.11 
                   3 ± 0.12 
               
               
                 Processed xylan from 
               
               
                 hardwood 
               
               
                   Eucalyptus grandis  gel 
                 GES 
                 1.25 ± 0.0  
                  25 ± 0.0 
               
               
                 (SAPPI) (ES) 
                 SES 
                 2.00 ± 0.11 
                   40 ± 2.12 
               
               
                   
               
               
                 The prefix A, AG and G denote modification of xylan by α-L-arabinofuranosidase, cocktail of α-L-arabinofuranosidase and α-D-glucuronidase and α-D-glucuronidase. 
               
               
                 Prefix S denotes treatment with untreated xylan. 
               
               
                 The BH, BB, BM, P, EH and ES denote xylan from mild alkali extracted from bagasse, H 2 O 2  bleached bagasse, mild alkali extracted bamboo,  Pinus patula ,  Eucalyptus grandis  and  Eucalyptus grandis  gel, respectively. 
               
               
                 *Weight gain as a percentage of initial cotton lint weight, 
               
               
                 **Weight gain as a percentage of theoretical amount of xylan in the reaction mixture 
               
            
           
         
       
     
     In synergistic effect assessment, the cotton lint treated in the presence of a cocktail of AbfB and AguA with xylan substituted with both arabinose and glucuronic acid side chains from bagasse (AGBH) [extracted by Hoije method], H 2 O 2  bleached bagasse xylan (AGBB), bamboo (AGBM) and  P. patula  (AGP) [both extracted by Hoije method] gave cotton lint xylan a specific weight gain of 34.0, 19.0, 22 and 13% (Table 12). The order of magnitude of the cotton lint xylan specific weight gain for bagasse extracted by the Hoije method was AGBH&gt;, ABH&gt;GBH&gt;SBH (BH), whereas for H 2 O 2  bleached bagasse was (BB) AGBB&gt;GBB&gt;SBB&gt;ABB (Table 12). The specific xylan weight again of the cotton lint after treatment with bamboo (BM) and  P. patula  was highest with presence of AguA (GBM/GP) followed by the cocktail (AGBM/AGP), then AbfB (ABM/AP) and was lowest for cotton lint treated in unmodified xylan (SBM/SP (Table 12). 
     About 8% and 4% of the initial xylose content was released upon acid hydrolysis of the cotton lint treated with oatspelt xylan in the presence of AbfB at a dosage level of 25 mL (25CXE) and 12.5 mL (12.5 CXE), whereas about 6% and 0.4% was released from cotton lint treated with corresponding oatspelt xylan at a dosage level (25CX) and (12.5 CX) in the absence of AbfB, respectively ( FIG. 34A ). About 64% of the xylose initially present was removed from the oatspelt xylan reaction mixtures in which the cotton lint was treated in the presence of AbfB (25CXE and 12.5 CXE) ( FIG. 34B ). The removal of the xylose in the reaction mixtures corresponded to arabinose release of about 30 and 50% in 25CXE and 12.5 CXE oatspelt xylan mixtures, whereas 60% of the available arabinose was released in the corresponding reaction mixtures of AbfB treated oatspelt xylan in the absence of the cotton lints ( FIG. 34B ). The acid hydrolysate of cotton lint acid treated with birch xylan in the presence of AguA (BCXE) released 1.21% xylose, whereas 0.77% xylose was released from cotton lint treated with the corresponding unmodified birch xylan (BCX) ( FIG. 35A ). The xylose removal from the birch xylan reaction mixture was about 32%, which corresponded to 7.87% release of the available glucuronic acid ( FIG. 35A ). 
     The order of magnitude of xylose released from acid hydrolysis of cotton lint treated with mild alkali pre-extracted xylan from bagasse (BH) xylan in the presence and absence of AbfB, AguA, and a cocktail (AG) was GBH&gt;AGBH&gt;SBH&gt;ABH, whereas in the BB treated cotton lint acid hydrolysate it was SBB&gt;GBB&gt;AGBB&gt;ABB ( FIG. 35B ). In cotton lint treated with BM and  P. patula  xylan in the presence of AguA, the xylose content was 1.8 and 1.3%, respectively, and in the absence of AguA was 0.97 and 0.63% ( FIG. 35B ). About 2.3% xylose was detected in the acid hydrolysate of cotton lint treated with  E. grandis  (EH) in the presence of α-D-glucuronidase (AguA), whereas in the acid hydrolysed cotton lint treated with the corresponding unmodified xylan, the xylose detected was &lt;1.0%. In contrast, the cotton lint treated with unmodified  E. grandis  xylan gel (SES) released 5.7%, but only 4.0% xylose was detected in the cotton lint treated with  E. grandis  xylan gel in the presence of AguA (GES) ( FIG. 35B ). 
     Structural Analysis of Xylan Treated Cotton Lint 
     The solid state  13 C—(CP/MAS) NMR of the xylan treated cotton lints reflected changes in carbon characteristic chemical shifts, relative intensities, and line shapes of carbon resonances from the sugar units making up the cellulosic component of the cotton lint ( FIGS. 36 to 38 ). The spectra resonances assigned according to Atalla and Isogai (2005) and Nuopponen et al. (2006) showed carbon resonances of the glucose units between chemical shifts (δ) 60 and 70 ppm for C6 of primary alcohol group, between 70 and 81 ppm for C2, C3, and C5 ring carbons other than those anchoring glycosidic bonds and the region between 81-93 ppm associated with C4 and between δ 102 and 108 ppm for anomeric carbon (C1) ( FIGS. 36 to 38 ). The signals between δ 88-89 ppm and around 65 ppm are assigned to crystalline cellulose C4 and C6 respectively, whereas the signals between δ 83-84 ppm and 61-62 ppm are assigned to amorphous region of cellulose C4 and C6 respectively (Nuopponen et al., 2006). The changes in cotton lint treated with birch and oatspelt xylan occurred at C1, C4, C3, C5, and C6 of the cellulose backbone in the presence of AbfB and AguA, respectively ( FIG. 36 ). The changes were in the form of line shape and splitting pattern, magnitude of resonance intensity, emergence of new signals, and change in position of characteristic signals chemical shifts. The spectra for cotton lint treated with oatspelt xylan in the presence of AbfB (25CXE) ( FIG. 36 ) show different splitting pattern of C1, C3 and C5 resonances at δ 105.62/104.32, 72.57, and 71.27 ppm, respectively ( FIG. 36  spectra 3) from the untreated cotton lint ( FIG. 36  spectra 1). In the spectra of the cotton lint treated with oatspelt xylan, the chemical shift of C4 and C6 upfield resonances in the amorphous region of the cellulose appeared at δ 83.59 ppm and 61.9 ppm, respectively whereas such carbon reasonances appeared at δ 83.91 ppm and 62.2 ppm, respectively, in the untreated cotton lint ( FIG. 36 ). Similarly, the spectra of cotton lint treated with birch xylan in the presence of AguA (BCXE) displayed changes in line shapes and a shift in the signal for C4 dowfield (the crystalline region) from δ 88.77 ppm in the untreated cotton lint ( FIG. 36  spectra 1) to 89.09 ppm ( FIG. 36  spectra 3). Low intensity signals appeared at δ 95.57 and 96.87 ppm in  13 C-(CP/MAS) NMR spectra of cotton lint treated with mild alkali extracted xylan from bagasse (BH) in presence of α-glucuronidase and (GBH) and α-L-arabinofuranosidase (ABH), respectively ( FIG. 37  spectra 3 and 5, respectively). In all the spectra, the individual resonance intensities were presented relative to the signal for C2 (δ 74-75 ppm). The solid state  13 C-(CP/MAS) NMR spectra of cotton lint treated with mild alkali extracted xylan from  P. patula  (P),  E. grandis  (EH) and  E. grandis  xylan gel (ES), irrespective of the presence of enzymes or not, showed splitting of C1 signals in triplets ( FIG. 38  spectra 2 and 3). 
     Effect of Enzymatic Treatment on Adsorption of Xylan onto Cotton Lint-Xylan Specific Weight Gain 
     The specific xylan weight gain of the cotton lint after being treated with oatspelt xylan in the presence of AbfB at xylan (1% w/v) loading of 5 mL g −1  (5CXE) was 42%, whereas at 12.5 mL g −1  (12.5CXE) and 25 mL g −1  (25CXE) xylan loading, the specific xylan weight gain was 26 and 22%, respectively. The corresponding cotton lint treated with oatspelt xylan in absence of AbfB at 12.5 mL g −1  (12.5 CX) and 25 mL g −1  (25CX) xylan loading had a specific xylan weight gain of approximately 27 and 18%, respectively. The cotton lint treated with birch xylan (1% w/v) in the presence of α-D-glucuronidase (BCXE) and in the absence of α-AguA (BCX) at birch xylan loading of 3.75 mL g −1  showed specific xylan weight gain of 16% and 7%, respectively. In addition, the cotton lint treated with xylan (1% w/v) from bagasse (BH), bamboo (BM) and  P. patula  (P) and the H 2 O 2  bleached bagasse (BB) xylan in the presence of AbfB, and the cocktail of AbfB and AguA at xylan loading of 5 mL g −1 . The magnitude of the xylan specific weight gain when the treatment cotton lint treated with the BH xylan in presence of AbfB (ABH), AguA (GBH) and cocktail (AGBH) was in the following order of magnitude: AGBH&gt;ABH&gt;GBH&gt;SBH. Treatment of the cotton lint with bamboo and  P. patula  xylan resulted in the highest specific xylan weight gains being obtained in the presence of AguA followed by the presence of a cocktail, whereas the specific xylan weight gain of the cotton lint treated with H 2 O 2  bleached bagasse (BB) was the highest (19%) in the presence of AbfB/AguA cocktail (AGBB). However, the specific xylan weight gain (11%) of the cotton lint when treated with BB in the presence of AbfB was lower than that of the (ABB) cotton lint treated with unmodified BB xylan (SBB). Similarly, a lower xylan specific weight gain prevailed with the cotton lint treated in  E. grandis  gel xylan in the presence of AguA (GES) than with the gel in the absence of the AguA (SES). 
     Effect of Enzymatic Treatment on Adsorption of Xylan onto Cotton Lint-Post Xylan Adsorption Sugar Profile 
     The acid hydrolysate of the cotton lint treated with oatspelt xylan in the presence of AbfB yielded 8 and 4% xylose at xylan dosage levels of 25 mL g −1  (25CXE) and 12.5 mL g −1  (12.5 CXE), respectively. About 6% and 0.4% xylose were obtained from the corresponding cotton lint treated with oatspelt xylan in the absence of AbfB at the same dosage levels (25CX and 12.5 CX, respectively). The sugar profile of the adsorption mixture showed that xylose removal of about 64% occurred in the reaction mixtures in which the cotton lint was treated with oatspelt xylan in the presence of AbfB at both xylan loading of 25 mL (25CXE) and 12.5 mL (12.5 CXE). The removal of the xylose from the reaction mixtures corresponded to arabinose release of about 30 and 50% from 25CXE and 12.5 CXE in the oatspelt xylan adsorption mixtures, respectively. In the absence of the cotton lint, the precipitation efficiency of the oatspelt xylan of 25 and 42% at xylan occurred in the presence of the AbfB at equivalent xylan loading of 25 mL g −1  (25XYE) and 12 mL g −1  (12XYE), which corresponded to 60% release of the available arabinose in both cases. Traces of arabinose were detected in 25CX, 25XY, 12.5CX and 12.5 XY reaction mixtures in which AbfB was absent. 
     The acid hydrolysate of cotton lint acid treated with birch xylan in the presence of AguA (BCXE) yielded 1.21% xylose whereas cotton lint treated with the corresponding unmodified birch xylan (BCX) yielded 0.77% xylose. In the AguA treated birch xylan adsorption mixture, xylose removal of 32% occurred, which corresponded to 7.87% release removal of the available glucuronic acid. The xylose removal in the corresponding birch xylan mixture in which AguA was absent was 28%, in which 2% glucuronic acid was detected. 
     The xylose present in the acid hydrolysate of the cotton lint treated with mild alkali extracted xylan in the presence of AbfB, AguA and a cocktail of the two enzymes. The xylose content of the cotton lint treated with the mild alkali extracted bagasse xylan (BH) was in the following order of magnitude GBH&gt;AGBH&gt;SBH&gt;ABH, whereas the order of magnitude of the xylose present in the hydrolysate of the cotton lint treated with H 2 O 2  bleached bagasse (BB) was SBB&gt;GBB&gt;AGBB&gt;ABB. The xylose contents of the cotton lint treated with BM (1.8%) and  P. Patula  (1.3%) xylan in the presence of AguA (GBM and GP, respectively) were over 100% higher than of cotton lint treated without the AguA (SBM and SP, respectively). About 2.3% xylose was detected in the acid hydrolysate of cotton lint treated with  E. grandis  xylan (EH) in the presence of AguA and &lt;1.0% in hydrolysate of cotton lint treated with unmodified EH. In contrast, more xylose (5.7%) was present in the hydrolysate of the cotton lint treated with unmodified  E. grandis  xylan gel (SES) than the one treated with AguA modified ES gel (GES) which only yielded 4.0% xylose. 
       13 C-(CP/MAS) NMR Characterization of Cotton Lint Structural Changes after Treatment with Enzymatically Modified Polymeric Xylan 
     The solid state  13 C—(CP/MAS) NMR spectra resonances in  FIGS. 36 to 38  were assigned according to Atalla and Isogai (2005) and Larsson et al (1999) who showed that carbon resonances of the glucose units between chemical shifts (δ) 60 and 70 ppm are associated with C6 of primary alcohol group, between 70 and 81 ppm with C2, C3, and C5 ring carbons other than those anchoring glycosidic bonds and the region between 81-93 ppm associated with C4 and between δ 102 and 108 ppm the anomeric carbon (C1). The signals between δ 88-89 ppm and around 65 ppm are assigned to crystalline cellulose C4 and C6 respectively, whereas the signals between δ 83-84 ppm and 61-62 ppm are assigned to amorphous region of cellulose C4 and C6, respectively. The solid state  13 C-(CP/MAS) NMR spectra of the cotton lint treated with oatspelt and birch xylans in the presence or absence of AbfB (25CXE) and AguA (BCXE), respectively, reflected changes in splitting patterns of the anomeric carbon (C1) resonances between δ 102 and 108 ppm. Such differences in the splitting pattern also occurred between the solid state  13 C-(CP/MAS) NMR spectra of the cotton lints treated with BH, BB, BM, and ES xylans in the presence and absence of the AbfB and AguA. In all the solid state  13 C-(CP/MAS) NMR spectra, the enzyme effected differences were evident from changes that occurred in line sharpening and intensity of C4 and C6 upfield resonances in the amorphous region of the cellulose of the cotton lint treated with the xylan in the presence of the enzymes at δ 83-84 ppm and 61-62 ppm. In these spectra, the individual resonance intensities were presented relative to the signal for C2 (δ 74-75 ppm). The effect of treating the cotton lint with mild alkali xylan in presence of different enzyme combinations. The  13 C-(CP/MAS) NMR spectra of cotton lint treated with mild alkali extracted xylan from bagasse (BH) in presence of α-glucuronidase (GBH) and α-L-arabinofuranosidase (ABH) displayed low intensity signals at d 95.57 and 96.87 ppm, respectively which were not present in the cotton lint treated with BH xylan in the absence or in a cocktail of the enzymes). Signals of carbon resonances with similar chemical shifts were present in the solid state  13 C-(CP/MAS) NMR spectra of cotton lint treated with BB xylan in presence of a cocktail of AbfB and AguA. There were differences in the splitting pattern and line sharpness of the signals for the six glucose carbon resonances (C1-C6) evident in the solid state  13 C-(CP/MAS) NMR spectra of cotton lint treated with mild  P. patula  and BB xylan in presence of AbfB and AguA. However, no such differences displayed in the solid state  13 C—(CP/MAS) NMR spectra of the cotton lint treated with  P. patula  and BB xylan in presence of AguA and a cocktail of AbfB and AguA. 
     Discussion 
     α-L-Arabinofuranosidase with polymeric substrate specificity increased adsorption of oatspelt xylan onto cotton lint by up to 33% and 900% in the presence of the AbfB at oatspelt xylan loading of 25 mL g −1  (25CXE) and 12.5 mL g −1  (12.5CXE), respectively. The results indicate that oatspelt xylan adsorption in the presence of the AbfB was more efficient at the lower xylan loading than at the higher xylan loading, indicating an optimal xylan loading for obtaining maximum AbfB aided oatspelt xylan adsorption. Since xylan adsorption is influenced by a decrease in the degree of xylan substitution, the increased efficiency in AbfB aided oatspelt xylan adsorption at the lower xylan loading (12.5 mL g −1 ) is proposed to be due to a higher efficiency in the removal of the arabinose side chains by the AbfB than at higher xylan loading (25 mL, g −1 ). The removal of the side chain is proposed to have provided an increased effective surface area available for adsorption onto the cotton lint fibres. 
     The results show that in the presence of the AbfB about 50% of the available arabinose was released at the xylan loading of 12.5 mL g −1  compared to 30% obtained at xylan loading of 25 mL g −1 . In addition, a corresponding precipitation efficiency of 42% of the oatspelt xylan occurred at xylan loading of 12.5 mL g −1  in the absence of the cotton lint (12.5XYE) compared to the 25% precipitation that occurred at xylan loading of 25.0 mL g −1  (25XYE). However, at both xylan loadings, the xylan to enzyme ratio was 5:2 and the precipitation corresponded to the release of 60% of the available arabinose. 
     The removal of glucuronic acid side chains by the AguA provided BH, BM and  P. patula  xylan with higher binding power to the cotton lint than the removal of the arabinose side groups. The AguA was over 2.5 times more effective in increasing adsorption of BH, BM and  P. patula  xylan onto the cotton lint than AbfB. The combined application of the AbfB and AguA in a cocktail in BH, BM and  P. patula  xylan adsorption mixtures had an added advantage over the use of AbfB but not over AguA. Thus a higher degree of adsorption prevailed in the presence of the cocktail of AguA and AbfB than in the presence of AbfB but not in the presence of AguA. 
     The AbfB and AguA were more effective in increasing adsorption of xylan from wood sources than grasses. Furthermore, the effect of AbfB and AguA was higher on xylan substituted with a single type of side than when substituted with both arabinose and glucuronic acid side chains.  E. grandis  was noted as the preferred xylan source, although among the grass sources, bamboo would be the preferred choice over bagasse. 
     The presence of AbfB and AguA in bagasse (BH), H 2 O 2  bleached bagasse (BB), bamboo (BM) and  P. patula  (P) xylan adsorption mixtures altered the xylan properties in a manner that allowed introduction of varying hydration properties and conformations or hydrogen bonding on the cotton lint fibre surface. The specific xylan weight gain of the cotton lint was the highest for cotton lint that was treated with xylan in the presence of AguA. The increase in the specific xylan weight gain could not be matched with the actual weight of the xylan that adsorbed on the cotton lint. The xylan structure is held together by the presence of moisture, which is known to increase with high content of arabinose side groups. Therefore, the higher xylan specific weight gain in the presence of AguA, rather than AbfB, is proposed to be associated with the higher arabinose content of the AguA treated xylan that adsorbed onto the cotton lint. The AbfB removal of the arabinose groups is proposed to have reduced the hydration capacity of the adsorbed xylan. Furthermore, the solid state  13 C-(CP/MAS) NMR spectra of the cotton lint treated in bagasse xylan (BH and BB) in the presence of AguA showed a new carbon signal at δ 96.57 ppm, whereas in the presence of AbfB a new signal appeared at δ 95.57 ppm. Such signals are associated with the presence of xylan substituted with arabinose and glucuronic side groups. In addition, changes in the splitting pattern and intensities for C1, C4 and C6 carbon resonance were observed in the solid state  13 C-(CP/MAS) NMR spectra of cotton lint treated with bagasse and  P. patula  xylan in the presence of AbfB and AguA, indicating varying structural changes emanating from the changes in the purity of the cellulose and the possible xylan-cellulose interactions. 
     Industrial scale application of AguA and/or AbfB as bio-based catalysts in xylan adsorption on cellulosic surfaces can be readily integrated in the conventional kraft pulping process as shown in  FIG. 39 . Importantly, the invention provides for the method of modifying xylan to be capable of being carried out in the substantial absence of other hydrolytic enzymes, such as xylanase. The conditions under which the AbfB and AguA increased xylan adsorption in this study, i.e. pH4.8-pH5.0 at 40° C., are conducive to incorporating the enzyme aided xylan adsorption process at the wet end fibre processing stage where the temperature and pH of the cellulosic fibres can easily be adjusted to be within the acceptable range for optimal functioning of the enzymes. The observation that  E. grandis, P. patula  and bamboo feedstocks are particularly suitable sources for enzyme aided xylan adsorption makes it feasible for integration with the kraft pulping process, because such feedstocks are commonly used raw materials for pulp and paper making. Xylan fractions with desirable physico chemical properties can be pre-extracted from  E. grandis, P. patula  and bamboo and can be re-introduced together with the AbfB or AguA at the wet end processing stages. In enzyme aided xylan adsorption, four possible stages are proposed, i.e. (1) introducing a xylan adsorption reactor between filtering and bleaching, (2) during washing post bleaching, (3) at the drying stage and (4) at the paper fining stage. At all the stages the pH and temperature of the pulp slurry may be adjusted to between pH 4 and pH 6 and 40-50° C. for the enzymes to operate optimally. The whole procedure will require minimum alterations to the current kraft pulping processes. 
     In South Africa, potential sources of xylan include  E. grandis , softwood  P. patula , sugarcane process residues (bagasse) and bamboo, which form a commercial source of raw materials for the pulp and paper industry. The available plantations for these feedstocks suggest sustainable supply of raw materials for integrated commercial production of xylan biopolymers, conventional pulp and timber products. Furthermore, methods optimised for selective isolation of xylan in relatively pure and less degraded form, while preserving the structural integrity of the remaining cellulosic component, are of particular interest to allow co-production of xylan with multiple streams of other value added products including co-production with pulp, timber, and bio-energy. Such an approach provides maximum economic value to be obtained from the raw materials.