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-/?- 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 [Sojstrom, 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 [Sojstrom, 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 (>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) [Sojstrom, 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 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 σ-D-glucuronidase and σ-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 a-D- glucuronidase, by selectively removing only arabinose side chains with a-L-arabinofuranosidase, or by selectively removing both glucuronic acid and arabinose side chains with σ-D-glucuronidase and σ-L- arabinofuranosidase. Acetyl groups may also be selectively removed from the xylan with acetyl xylan esterase. The σ-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 σ-L-arabinofuranosidase enzyme loading may be about 2.5 to about 5 mL.g ^"1 and the σ-L-arabinofuranosidase may have a volumetric activity of about 18.0 nKat mL ^" . The a-D- glucuronidase enzyme loading may be about 0.2 mL.g ^"1 and the σ-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 composition;

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 σ-D-glucuronidase and σ-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 Figure 1 shows a schematic representation of plasmid pGTP-AbfB: the abfB gene was cloned into the Not\ 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; Figure 2 is a graph showing the production characteristics of AbfB by recombinant A. niger

D15[abfB] in shake flasks in pellet form;

Figure 3 is a graph showing the production characteristics of AbfB by recombinant A. niger

D15[abfB] in shake flasks produced in 2 x 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;

Figure 4 is a graph showing the production of AbfB in 10 L BIOFLO 110 bioreactor (mycelial morphology) indicating release of extracellular AbfB activity;

Figure s shows the mycelial morphological changes of A. niger D15[abfB] at different time intervals during cultivation; Figure 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);

Figure 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<0.01 );

Figure 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 0 ₂ bleached bagasse (BagB), bamboo (Bam), Oatspelt (OT) and Pinus patula (Pine). Means with different letters are significantly different (p<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<0.05);

Figure 9 shows a graph of the remaining relative activity (%) of the recombinant AbfB after storage at 26, 30 and 37°C;

Figure 10 shows a graph of the AbfB recovery (residual activity %) of AbfB after hydrolysis of xylan from H ₂ 0 ₂ bleached bagasse (BagBraz), mild alkaline extracted xylan from bagasse using the Hoije et al. [2005, Carbohydr. Polym. 61 : 266-275] method (BH) and using the De Lopez et al. [1996, Biomass and Bioenergy, 10: (4) : 201 -21 1] method (BL) and mild alkaline extracted xylan using the Hoije method from bamboo (BM) and Pinus patula (PP) and commercial oatspelt xylan (Sigma) at 40°C for 16 h;

Figure 11 shows a graph of the enzyme activity of AbfB in the presence of varying concentrations of pNPA substrate, demonstrating the effect of -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;

Figure 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 x MM medium. Dotted lines show values derived for Vmax and Km;

Figure 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 20x, (b) CCSL enriched medium in a bioreactor at 10x dilution, (c) in standard medium in shake flask at 20x dilution, (d) in standard medium in shake flask at 10x dilution, (e) AkTA partially purified AbfB produced in CCSL enriched medium in fermenters at 20x dilution, and (f) AkTA partially purified AbfB produced in CCSL enriched medium in fermenters at 10x dilution AbfB produced in standard 2 x MM medium;

Figure 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);

Figure 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);

Figure 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;

Figure 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);

Figure 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;

Figure 19 shows the elution profiles of xylan on HPAEC-PAD (Dionex) CarboPac column P10 from (A) mild alkali extracted H ₂ 0 ₂ bleached bagasse (Bag B), (B) mild alkali extracted ultra purified bagasse (Bag H) and (C) mild alkali extracted ethanol precipitated bagasse (Bag L);

Figure 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; Figure 21 shows a bar graph of the insoluble fraction obtained after 72% acid hydrolysis of mild alkali extracted xylan H ₂ 0 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); Figure 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) ³ C-NMR analyses of H ₂ 0 ₂ bleached bagasse (Bag B), and (E) ¹ H-NMR and (F) ³ C-NMR analyses of oatspelt xylan. Me denotes Methyl group from glucuronic acid and Ac = acetyl group, Ar = arabinose group;

Figure 23 shows the characterisation of xylan by (A) 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);

Figure 24 shows the characterisation of xylan by (A) ¹ H-NMR and (B) ¹³ C-NMR analyses of bamboo, (C) ¹ H-NM R and (D) ¹³ C-NMR analyses of P. patula xylan; Figure 25 shows the FTIR spectra of xylan extracted from different types of Iignocellulosic 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;

Figure 26 shows the removal of (A) arabinose and (B) 4-O-MeglcA by AbfB and σ-glu respectively from oatspelt/birch, mild alkali pre-extracted bagasse Hoije (BH), H ₂ 0 ₂ bleached bagasse (BB), bamboo(BM), and Pinus patula (PP) xylan, and by σ-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 σ-glu and

(D) of 4-O-MeglcA by σ-glu in combination with AbfB. AbfB xylan specific dosage = 720.0 nKat g ^"1 , Temp = 40°C , time = 16 h pH 5.0 and σ-glu xylan specific dose = 9000 nKat g ^"1 substrate, Temp = 40°C , time = 16 h and pH 4.8; Figure 27 shows a graph of arabinose removal from oatspelt xylan after ct-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);

Figure 28 shows a graph of arabinose removed from oatspelt xylan after σ-L- arabinofuranosidase (AbfB) hydrolysis at varying xylan specific dosage, hydrolysis time (h) and temperature (°C);

Figure 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 ^" 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 ^"1 substrate) at 9 h, and (F) time (h) and enzyme dose (nKat g ^"1 substrate) at 33.5 °C; 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 /a-glu) and time, and enzyme dose (AbfB /o-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 ₍ , ) 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; shows a surface plot of the effect of arabinose removal as a function of xylan concentration ( g mL ^"1 ) and enzyme (AbfB) dose (nkat mL ^"1 ); shows a desirability plot of the effect of arabinose removal as a function of xylan concentration (jjg mL ^"1 ) and enzyme (AbfB) dose (nkat mL ^"1 ); shows a Pareto chart of the size and the significance of the treatment and interaction effect, the 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; 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 12.5mL (12.5CX) and 25 mL (25CX) and from AbfB modified xylan a t 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);

Figure 35 shows xylose removed, glucuronic acid released and adsorbed onto cotton lint from

(A) birch xylan modified with σ-D-glucuronidase (σ-glu) (BCXE) and unmodified birch xylan (BCX), (B) adsorbed onto cotton lint from pre-extracted xylan from bagasse (BH), H ₂ 0 ₂ bleached bagasse (BB), bamboo (BM), P. patula (P), E. grandis (EH) and E. grandis gel (ES) modified by AbfB (A) AbfB and σ-glu cocktail (AG), α-glu (G) and unmodified (S);

Figure 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 σ-L-arabinofuranosidase (AbfB) or σ-D- glucuronidase (AguA);

Figure 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 o-D-glucuronidase [4] modified with σ-L-arabinofuranosidase and o-D-glucuronidase (AguA) cocktail, and [5] σ-L-arabinofuranosidase (AbfB);

Figure 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 σ-D-glucuronidase (AguA), [4] σ-L-arabinofuranosidase and σ-D- glucuronidase (AguA) cocktail, and [5] σ-L-arabinofuranosidase (AbfB);

Figure 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 σ-L-arabinofuranosidases (AbfB) (EC 3.2.1.55) and/or σ-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 0 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 o-L- arabinofuranosidases (AbfB) (EC 3.2.1.55) and/or σ-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't contaminated with other enzymes which could break down the main xylan chain, such as xylanase. For this reason, the σ-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 ^" . The σ-L-arabinofuranosidase enzyme loading can be about 2 to about 10 mL.g ^'1 and the σ-L-arabinofuranosidase can have a volumetric activity of about 18 nKat mL ^"1 . The σ-D-glucuronidase enzyme loading can be about 0.2 mL.g ^"1 and the σ-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 σ-L-arabinofuranosidase (AbfB) and σ-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 0 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 25mL g ^"1 and 12.5 ml_ g ^" 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 0 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 Hoije et al. [2005 Carbohydr. Polym. 61 : 266-275]), commercial oatspelt xylan (Sigma), birch xylan (Roth), mild alkali H ₂ 0 ₂ bleached xylan from bagasse (BB) (donated by Prof. A.M.F. Milagres, University of Sao 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 σ-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 σ-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 σ-L-arabinofuranosidase (AbfB)

A recombinant σ-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 x 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 DH5a. E. coli was cultivated at 37 ^° C in LB medium (1 % yeast extract, 1 % tryptone and 0.5% NaCI) on a rotary shaker at 100 rpm, supplemented with l OOyi/g/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 Όλ 5 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 x MM) containing 10% glucose. The medium was inoculated to a final spore concentration of 1x10 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 prtTphmA (nonacidifying) Wiebe er a/., 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 D 15 with gpdP-abfB-glaA T This study

E. coli DH5a supE44 MacU169 (0/8OlacZAM15) Sam brook ef a/., 1989, Molecular hsdR17 recA1 endA1 gyrA96 thi-1 cloning: a laboratory manual. Cold relA 1 Spring Harbor Laboratory, Cold

Spring Harbor, NY

Plasmids:

pBS-pyrGamdS bla pyrG amdS Pliiddemann 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 (> 1x10 ⁶ ). To prepare the rice sporulation medium, tastic rice (20g) 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% NaCI solution (approximately 50 mL for each flask) and stored in Schott bottles at 4°C.

A. niger spores were inoculated in 2 x 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 /vm 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 ef al. [1989, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY] 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 [Plijddemann 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 σ-L-arabinofuranosidase (AbfB), the abfB gene was amplified from the genome of A. niger 10864 using standard PCR methods. The ^' aWB was then cloned into the Not\ site of pGTP, generating pGTP-AbfB (Figure 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 upsteam 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[abfB1

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 x magnification) prior to filtering through Mira cloth mesh. The biomass was washed with deionised water (dH ₂ 0) 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 ^" ).

The pellet formation was achieved by inoculating the medium (either 2 x MM or CCSL (2% w/v) enriched 2 x MM medium), with 1x10 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 x 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 1 10 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 1x10 ⁶ spores mL ^"1 maintained at 30°C. Mass transfer was achieved using a single 3 bladed pitched impeller (30 X 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 (>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 WM). Sampling was done through a port installed with a 0.2 μιτι 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 μνη filters and concentrated using an Amicon system, Diaflo Uitrafilter PM 10 concentrator, (Amicon Division, W.R. Grace & 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 (ΑΚΤΑ purifier system, Amersham Pharmacia Biotech) installed with UNICORN computer control system (version 3.2). The protein sample (0.3ml_) 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 NaCI 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 (pi) of the AbfB was estimated from the original protein sequence (protein length of 499 bases) using DNAMAN sequence analysis computer software.

Determination of g-L-arabinofuranosidase activity of AbfB using pNPA g-L-arabinofuranosidase activity of AbfB (AbfB activity) was determined using a colorimetric assay in which the release of p-Nitrophenol from p-Nitrophenyl-g-arabinofuranoside (CnH ₁₃ N0 ₇ pNPA, Sigma) was measured. A 100 μΐ reaction mixture was prepared which consisted of 25 μΙ_ 5mM pNPA in 0.05M citrate buffer (pH 5.0), 25 //L deionised water (dH ₂ 0), 25 μΙ_ of appropriately diluted supernatant and 25 μΐ. 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 μΥ. saturated sodium tetraborate (Na2B ₄ 0 _7i 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 N0 _3i 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 ( ykat mL, conversion factor: 1 Unit = 0.0167 vkat). Unless stated otherwise, all assays were conducted in triplicate. Commercial cr-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 Mcllvaine's buffer [Mcllvaine, 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 g-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 ?NPA 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, IL, 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 0 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 μΐ) were diluted 5 times and centrifuged at 10 000 rpm for 5 min at 4°C followed by filtration using filters with 0.22 μνη 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 X 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 μΐ birch xylan (1 % w/v) and 150 μL· σ-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 μΐ. 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 σ-L-arabinofuranosidase and σ-D-glucuronidase in glass bottles. The reaction mixture contained 0.2 g cotton lint, 1000 μΐ xylan (1 % w/v), The adsorption mixture contained σ-L- arabinofuranosidase (AbfB) (18.0 nkat mL ^"1 ), and σ-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 0 while agitating for 1 h to disengage xylan precipitates loosely absorbed to the cotton lint. The Milli-Q H ₂ 0 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 μτη pore size filters. The filtrates were analysed for L-arabinose and a- 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 S0 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 0. 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 /vm 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 preexisted 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 6mm 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 /vs, the contact pulse 1500 //s 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 Hoije 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 deg

removal of xylan side chains

Xylan Method of extraction Type of side Source

chain

Sugarcane (Saccharum Mild alkali (Hoije et Arabinose and This study

officinarum L) Bagasse (BH) al.,2005) 4-O-MeglcA

Sugarcane (Saccharum Mild alkali (de Lopez 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 0 ₂ bleached 4-O-MeglcA A.M.F. Milagres, or BagBraz) University of Sao Paulo,

Brazil

Giant bamboo (Bambusidae Mild alkali (Hoije et Arabinose and This study

balcoa) (BM or Bam) al.,2005) 4-O-MeglcA

Eucalyptus (Eucalyptus Mild alkali (Hoije et al., 4-O-MeglcA This study

grand is) (EH) 2005)

Eucalyptus (Eucalyptus Mild alkali (de Lopez 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) Hoije 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 0 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 ef 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 //m/20 mesh size, 425 m/40 mesh size, and 250 /vm/60 mesh size with a lid and pan. The particulates that passed through 425 ym/40 mesh size but were retained on a 250 /vm/60 mesh sieve were collected for chemical composition analyses and those retained on the 425 /vm/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 ef 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 ef 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, Ultrastucture, 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% HQ (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 x 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 (1 M NaOAc) eluent was used when acid sugars (glucuronic/ methyl glucuronic acid) were analysed. The samples were filtered on 0.22 μτη 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 x 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 Buchi 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 6mm 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 yt/s, the contact pulse 1500 με 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 ef 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 ef 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 ef 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 ( ³ 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 ³ 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. H spectra were collected with 64 scans and pre-saturation of the HDO peak. The ¹³ C and H NMR spectra were interpreted according to assignment of characteristic signals of related feedstock presented by Ebringerova 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], Grondahl ef al. [2003, Carbohydr. Polym. 53: 359-366], Lahaye ef al. [2003, Carbohydrate Research 338: 1559- 1569], Sun ef 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. 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'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 x 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 μΙ_) were injected into the column and were eluted with helium degassed 0.25 M NaOH, Milli-Q H ₂ 0, 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 0 ₂ bleached bagasse. Samples with less intense peaks < 20 nC or no peaks eluting within the 25 min retention time were considered polymeric with DP>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 S0 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 μη\ 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 Q.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 Sao 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 S0 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 μτη 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 S0 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 σ-L-Arabinofuranosidase (AbfB) of Aspergillus niger and σ-D-glucuronidase of Schizophyllum commune (σ-glu) respectively was determined using xylan derived from Eucalyptus grandis, Pinus patula, Bambusa balcooa, and bagasse found in South Africa.

The AbfB and σ-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 0). The xylan that showed limited solubility in water was prepared by first dissolving in ethanol and subsequently heated according to de Wet ef 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 cr-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 σ-D-glucuronidase (a-glu) with specific activity = 300 nKat mg ^"1 purified from wild type Schizop yllum 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 σ-glu (9000 nKat g ^" ) 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 σ-glu in 1000 μ\. reaction mixtures containing 500 μL· of the respective substrates, AbfB 540.0 nKat g ^"1 and a- 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 0, 250mM NaOH and 1 M 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 et al, 956, 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

Treatment run Coded variables Natural variables

Random order X1 X2 X3 Time (h) Temperature (°C) Enzyme 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): 5 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 = βθ + β\Χ\ + β\ \Χ\ ² + βΐΧ2 + βΐΐΧΙ ¹ + βΐΧί + /?33Χ3 ² + 8

where: Ζ = Viscosity (mPa.s), β _{0 +} β _\ ? _η = linear regression coefficient, /?n ? _nn = Quadratic regression coefficient, e= Error, and Χί, X2, X3= 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 σ-L-arabinofuranosidase and σ-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 MeGIcA side chains from birch xylan by purified AguA

Treatment Run Coded variables Natural variables

(Random order)

X1 X2 X3 Enzyme dose f/ kat Time (h) Temperature (°C)

Substrate (g) ^"1 ]

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 σ-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 σ-L-arabinofuranosidase (AbfB) produced inhouse using recombinant Aspergillus niger D15 (volumetric activity of 18 nKat mL ^"1 on p-NPA) and a-D- glucuronidase (σ-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 //L reaction mixtures containing 200 μΙ_ of the substrate, with AbfB xylan specific dosage ranging from 44.0-140.0 nKat substrates (g) ^" 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 σ-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 σ-glu was 2 000 nKat g ^" and 18 000 nKat g ^"1 . The central points for temperature and time were 40 °C, and 8.5 h, while for AbfB and σ-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:

where: x,= coded value for variable.X, = natural value, ΔΧ;= 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 0, 250mM NaOH and 1 M NaOAc (for acid sugars only). L (+) arabinose and D-glucuronic acid were used as standard sugars. 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

Treatment run Coded variables Natural variables

Random order A B xylan concentration Enzyme dosage

(pg 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, nO =2, Runs= 0 "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 = ? _{0 η} Χ _{ί 2} Χ ₂ + /?3 ^Χ 3 + ¾3· ^¾ '3 ^~ * ^~ Al2 l ⁺ βΐ \ϊ ^Χ 1 ⁺ /¾ 33 ^Χ 1 3 ⁺

where: Ζ= Response (degree of side chain removal), β _{0 +} β _\ β _η .... = linear regression coefficient,

Quadratic regression coefficient, X _{1 (} X ₂ , X ₃ = Hydrolysis time, temperature, and enzyme xylan specific dosage or xylan and enzyme loading, e= Error.

Enzyme aided adsorption of xylan on cellulosic material

Individual and synergetic effects of applying recombinant σ-L-arabinofuranosidase and σ-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 extractedfrom Saccharum ofHcinarum 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 a-L- arabinofuranosidase (AbfB) produced by recombinant Aspergillus niger D15 with volumetric activity of 18.0 nKat mL ^"1 on p-NPA produced inhouse and σ-D-glucuronidase (σ-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 a/. [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 0 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 μΐ birch xylan (1 % w/v) and 150 μΐ 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 μΐ. 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 σ-L-Arabinofuranosidase and σ-D-glucuronidase onto cotton lint with pre-extracted xylan was performed in glass bottles. The reaction mixture contained 0.2 g cotton lint, 1000 μΐ xylan (1 % w/v), 40 μΐ AguA ( 900 nKat mL ^"1 ), 100 μΐ 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 0 while agitating for 1 h to disengage and remove xylan precipitates loosely absorbed to the cotton lint. The Milli-Q H ₂ 0 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 σ-L- arabinofuranosidase (AbfB), σ-D-glucuronidase (AguA) and their cocktail (AG)

Xylan type Control Agua (900 nkat AbfB (18.0 nkat mL ^" AbfB and j guA mL ^"1 ) 40 μΐ " ¹ ) 100 //L) μL + 40 //L

Bagasse H1 SBH1 GBH1 ABH1 AGBH1

Bagasse H2 SBH2 GBH2 ABH2 AGBH2

BagasseBrazl SBB1 GBB1 ABB1 AGBB1

BagasseBraz2 SBB2 GBB2 ABB2 AGBB2

Bambool 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 gelS 2 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 σ-glucuronidase (G), **** Cotton lint treated with xylan in presence of recombinant a- arabinofuranosidase (A), ***** Cotton lint treated with xylan in presence a cocktail of purified σ-glucuronidase and recombinant a-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 μτη pore size filters. The filtrates were analysed for L-arabinose and a- 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 μΙ_ filtrate sample, 200 μΙ_ (5%) phenol and 800 μΐ 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 S0 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 0. 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 μιη 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 6mm 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 με, the contact pulse 1500 //s 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 (Figure 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 x MM, respectively. The Abfb activity in 2 x MM with 10% CCSL and 2% CCSL reached a maximum activity of 10.0 nkat mL ^"1 a day earlier than in the 2 x MM media (Figure 3). Notably the AbfB activity in 2 x MM with 2% and 10% CCSL were not significantly different (p<0.05) during the incubation period. The AbfB produced in the bioreactor in 2 x MM with 2% CCSL had a maximum volumetric activity of approximately 8 nkat ml_ ^"1 which was achieved after 36 h of incubation (Figure 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 (Figure 4). The biomass concentration of the A. niger grown on 2 x MM 2% CCSL enriched medium in the bioreactor reached 32 g L ^"1 (Figure 4). During the incubation period, the morphology of A. niger was observed to change from pellets to an extensive network of mycelia (Figure 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 (Figure 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 (Figures 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 (Figures 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 0 ₂ bleached bagasse (BagB), bamboo (Bam) and Pinus patula (Pine) 40, 25, 23 and 28% arabinose, respectively, of the arabinose released from oatspelt xylan (Figure 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 (Figure 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 (Figure 9). The AbfB activity at the three storage temperatures remained stable up to 72 h and no significant differences (p< 0.05) in the AbfB activities were observed for the entire storage period (Figure 9). The residual AbfB activity in the reaction mixture after hydrolysis of xylan from bagasse (BagH), H ₂ 0 ₂ bleached bagasse (BagBraz), bamboo (Bam) and P. patula (Pine) was more than 95% after 16 h of hydrolysis (Figure 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 Figures 11 and 12. The initial specific activity against pNPA of crude AbfB obtained from bioreactor on 2 x MM with 2% CCSL (w/v) (Figure 11 A) , from shake flasks on 2 x MM with 2% CCSL (w/v) (Figure 11B) and on 2 x MM medium (Figure 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 10mM. The velocity of partially purified AbfB by AKTA 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 (Figure 12A). The molecular weight and isoelectric point (pi) of the AbfB enzyme estimated from abfB gene sequence using DNAMAN sequence analysis software were 52.5 kDa and pi 4.04, respectively, whereas the estimated molecular weight from silver stained (Biorad) 10% SDS-PAGE was 67 kDa (Figure 13). Multiple protein species were visualised in the 10% SDS-PAGE of AbfB produced in bioreactor using CCSL enriched medium and in the AKTA partially purified AbfB, whereas single protein species was present in the SDS-PAGE of the AbfB produced in shake flasks both in 2 x MM and CCSL enriched media (Figure 13).

Extracting and characterising xylan from lignocelluiosic materials

The chemical composition of bagasse, pine {Pinus patula), and bamboo (Bambusidae balcooa) is presented in Figures 14 and 15. Bagasse had the highest ash (8.6%) and solvent extractives (6.2%) (Figure 14A), lignin (30.0%) (Figure 14B), cellulose (53.80%) and pentosans (22.00%) (Figure 15A). Both E. grandis and P. patula had ash and extractive contents of less than 3% (Figure 14A). However, P. patula displayed the lowest pentosan level (8.49%) (Figure 15A). The cellulose level in E.grandis and bamboo was in the range of 40-43% (Figure 15A) whereas the lignin content was about 23 % (Figure 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 ³ 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] (Figures 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 6 70-81 ppm, the C4 resonances at 6 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 lignocelluiosic feedstock materials (Figures 16A-D). However, the doublets for C6 in the spectra of P. patula (Figure 16A spectra 1) were more resolved than in bagasse (Figure 16B), E. grandis (Figure 16C) and bamboo (Figure 16D). Furthermore, characteristic signals for acetyl groups (at δ 20 - 22 ppm, aliphatic groups at 6 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 ei 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 (Figures 16A-D spectra 1). The 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 6 81-93; 60-70 and 20-22 ppm, respectively (Figures 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 (Figures 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 6 40-50 ppm (Figure 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 (Figures 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 (Figures 17A-D). Furthermore the xylan extraction corresponded to a decrease in arabinose, and galactose content in all the feedstock (Figures 17A-D). The concentration of mannose (16.0%), which was detectable only in P. patula feedstock, increased to 18% in the xylan extracted residue (Figure 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 0 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 (Figures 18A and B). Between 0 and 3 min, oatspelt xylan elution profile showed a high intensity peak with detector response > 300 nC which corresponded to retention time for xylitol (Figure 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 < 20 nC) (Figures 18C and D, respectively). Whereas, the chromatogram for H ₂ 0 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 (Figure 19A). The chromatograms of both Hoije and Lopez extracted xylan displayed low intensity peaks (<20 nC) within 25 min retention time (Figures 19B and C). Among the extrcated xyla samples, the peak corresponding to xylitol was present in the chromatogram of xylan from E. grandis (Figure 20A), bamboo (Figure 20C) and P. patula (Figure 20D) extracted by the Hoije method. Table 7 Profile of neutral sugars and uronic acid of pre-extracted xylan

Total neutral sugar composition of xylan (%, Total uronic acid

(%f

Xylan type Arabinose Galactose Glucose Rhaminose Xylose Raw Xylan material*

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 1 1.20

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 1 1.54 Birch 0.43 19.53 80.03 10

Oatspelt ^" 7.4 1.35 4 87.24

xylan

Note: ³ denotes mean of at least three samples with standard deviation < 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 S0 ₄ ) hydrolysis yielded between 16 and 55% insoluble residues (Figure 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% (Figure 21). Structural characteristics of the extracted xylan

The ¹ H NMR and ¹³ C NMR spectra of the reference xylan from birch, H ₂ 0 ₂ bleached bagasse, and oatspelt xylan displayed characteristic signals for proton and carbon resonances (Figures 22A-D). The ¹ H NMR spectra of the extracted xylan displayed characteristic proton signals from xylose, 4-0- methylglucuronic acid, and arabinose units at chemical shifts (6) between 3.3 to 5.7 ppm (Figures 22- 24). In the 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 (Figure 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 0-2 and acetyl group at 0-3 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 (Figure 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 4-0- methylglucuronic acid (Figure 23C). According to Sims and Newman (2006) such chemical shifts could originate from D-xylopyranosyl units residues substituted with 4-O- methylglucuronic acid at 0-2 and acetyl group at 0-3. 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 (Figure 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 (Figures 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; Ebringerova 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 [Hoije ef al., 2005, Carbohydr. Polym. 61 : 266-275; Ebringerova 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 (Figures 23A and C). The presence of arabinose in the proton spectra of bamboo and P. patula xylan were in accordance with Ebringerova et al. (1998) and Vignon and Gey, (1998) identified inter alia, at δ 5.58/5.59 ppm and 5.47/5.48 ppm, respectively (Figures 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 (Figure 23C) whereas the presence of O- 2 linked acetyl groups which based on Shao ef al. [2008, Wood Science Technology 42: 439-451]; Hoije et al.(2005) Sun et al. (2004); Ebringerova et al. (1998); Vignon and Gey (1998) supra were identified at δ 4-2.4 ppm (Figures 22-24). In addition, broad signals associated with aromatic or phenolic compounds originating from lignin residues (Hoije er a/., 2005 supra; Xu et al., 2006, Carbohydrate Research and Oliveira ef 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 (Figures 23 and 24). The characteristic carbon resonances from the five carbons of (1→4) linked β-Ό- xylopyranosyl residues between δ 103 and 62 ppm were reflected in the ¹³ C NMR spectra of the extracted xylan fractions (Figures 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 = 08, 81.7, 78, 85.5, and 62 ppm (Figure 23D). In the spectra the arabinose associated carbon signals in EU H and EU L were seen at δ 61.81/61.59 ppm (Figure 23D). In Bag L and Bag H the arabinose signals identified based on Vignon and Gey (1998) supra were displayed at δ 112.50/ 1 1.56 and 89.31 ppm which correspond to C1 and C2 of C-2 linked arabinose residues, respectively (Figure 23B). The C1 , C2, and C4 resonances belonging to arabinofuranosyl residues mono substituted xylose units at 0-3 (Ebringerova 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 (Figures 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; Ebringerova et al.,1998, supra) (Figures 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 (Figures 23 and 24). However, absence of the acetyl signals was apparent in ¹³ C NMR spectra of birch xylan (Figure 22B) and H ₂ 0 ₂ bleached bagasse (Bag B) (Figure 22D). The methoxyl groups signifying presence of lignin compounds [Ebringerova et al., 1998 supra, Sun et al., 2004, supra, Xu et al., 2006 supra; Maunu, 2008, ³ C CPMAS NMR Studies of wood, cellulose fibers, and derivatives. In Hu, T.Q. (ed)]; were identified in the ¹³ C NMR spectra at δ 56.62/ 56.58 ppm (Figures 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- 1 17.08 ppm (Figures 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 £17.55/17.69 ppm in Bag L (Figure 23B spectra 1) bamboo and P. Patula xylan fractions (Figures 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 (Figure 23B).

The FTIR spectra of the extracted xylan fractions displayed characteristic bands for xylan residues which included ?-glycosidic linkages reflected at «897 cm ^"1 (Figure 25). However, such signal was absent in the FTI 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 (Figure 25), which according to Fengel and Wegener [1989, Wood Chemistry, Ultrastucture, 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 _A rOCH _3i and methoxyl groups in lignin were reflected at 1329 cm ^"1 and 1591-1595, and 1460-1461 cm ^"1 in the spectra of £. grandis and bamboo (Figure 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 (Figure 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 (Figure 25).

Controlled enzymatic removal of side chains from lignocellulosic feedstocks

The recombinant σ-L-Arabinofuranosidase (AbfB) produced by the Aspergillus niger released arabinose from oatspelt xylan and mild alkali extracted xylan from bagasse Hoije (BH), H ₂ 0 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 ^" substrate (3% available arabinose) was released from AbfB treated bamboo (BM) xylan fractions (Figure 26A). The purified o-glucuronidase (σ-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 (Figure 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 (Figure 26B). The lowest σ-glu removal of 4-O-MeglCA was < 0.6 mg g substrate ^"1 from H ₂ 0 ₂ bleached bagasse (BB) (Figure 26B). The synergistic effect of AbfB and σ-glu (AG) released 3% arabinose (on substrate basis) from BH whereas 2.4% was released with individual application of AbfB (Figure 26C). The synergistic effect of AbfB and σ-glu on the release of glucuronic acid from BH was about 0.18% while individual application of σ-glu released 0.16 % 4-O-MeglCA (Figure 26C). The AG removed 0.13% and 0.08 % MegICA from P.patula (PP) and Bamboo (BM) respectively while the individual application of the σ-glu released about 0.1 % and 0.09% MegICA, respectively (Figure 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 (Figure 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 (Figure 27). In addition, the change in the amount of arabinose released at hydrolysis time _£h and AbfB xylan specific dosage _S50 nKat g ^"1 was not significant (p<0.05J (Figure 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 (Figure 28). On the other hand, oatspelt xylan treated at the same AbfB dosage level at 40°C for 4h, released about 4% arabinose (Figure 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 (Figure 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 (Figures 29A-C). Similarly, the response surface plots for removal of glucuronic acid (4-O-MeglcA) from birch xylan σ-glu reflected both linear and quadratic relationship with hydrolysis time, temperature, and σ-glu xylan specific dosage. A maximum of 350 pg g ^"1 substrate of 4-O-MeglcA was removed from birch by σ-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 (Figure 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 < 0.05) in descending magnitude of temperature, time, and AbfB xylan specific dosage level (Figure 30A). Furthermore, the Pareto charts showed significant interaction effects (p < 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) (Figure 30A). The hydrolysis parameters having significant effects on removal of 4-O-MeglcA from birch xylan by a- glu were in descending magnitude, from linear effects from o-glu xylan specific dosage [σ-glu, nKat/g (L)], temperature [Temp (L)], and the quadratic effect of temperature [Temp (Q)] (Figure 30B, Pareto chart). The only significant interaction effect on the removal of 4-O-MeglcA from birch xylan by σ-glu was from the linear effect of hydrolysis time and the quadratic effect of temperature [ time (L) by temperature (Q)] (Figure 30B, Pareto chart). The desirability contour plots showed optimal set points for AbfB removal of arabinose from oatspelt xylan (Figure 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 ^" . In contrast, the optimal set points for σ-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 ^" and AbfB volumetric activity of >10 nkat mL ^"1 (Figure 31). The desirability contour plot (Figure 32) located the optimal xylan concentration and volumetric activity of the AbfB to be 5563.3 /yg mL ^"1 and 27.2 nKat mL ^'1 , 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 ig 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 (Figure 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 σ-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 0 ₂ bleached bagasse xylan (ABB), bamboo (ABM) and P.patula (APJ [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 0 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 σ-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 Regression Coefficients in arabinose release equations t(14) Significance level (p) Arabinose release = f (time, temperature, coefficient f(*,y)

specific AbfB level )

R* = 0.99 ( Re adjusted =0.97) /(time, temp) /(time, AbfB ) /(temp, AbfB;

Mean/interaction 9476.69 bo bo bo 29.009 <0.01

Main effects

(1 )Time (h)(L) 3989.39 bi b ₁ 19.942 <0.01

Enzyme dose (nKat/g substrate)(Q) -3145.29 b ₄ b ₄ -5.341 <0.01

(3)Enzyme dose (nKat/g substrate)(L) 2071.60 b ₃ b ₃ 7.322 <0.01

(2)Temp (°C)(L) -540.89 b ₃ i -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

1 Q by 2Q -4178.84 hi 122 bl122 bl122 -3.548 <0.01

1 l_ by 2Q -1461.58 bi22 bl22 bl22 -5.166 <0.01

1 Q by 2L 1323.20 b ₁₁₂ bl12 bl12 3.8189 <p.oi

1 L by 2L -553.82 l2 bl2 bl2 -2.768 <0.01

1 L by 3L 437.94 b ₁₃ bi ₃ bl3 2.189 <0.05

1 Q by 3L -352.67 b ₁₁₃ bii _{3 l} 1 ₃ -1.018 0.326

Error 37.84

Note: 1 = time, 2 = temperature, 3 = enzyme xyian specific dosage, L = linear, Q = quadratic, R ² = linear regression coefficient, bj..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 Regression Coefficients in glucuronic acid release equations t(17)

relationship coefficient f(*,y)

Glucuronic acid release =f f(Time,Tem fjfTime.Agu f(Temp,AguA dose)

(time, temperature, specific P) A dose)

AbfB level) R ² = 0.90 ( R ²

adjusted =0.81)

Mean /interaction 278.0162 bo bo bo 13.9314 <0.01

Main effects <0.01

Temp °C (Q) -70.9028 b ₂₂ b ₂ -3.9416 <0.01

(3)a-glu (nKat/g substrate) (L) 66.164 b _{3 3} 3.8280 <0.01

(2)Temp°C (L) -38.087 b ₂ b ₂ -2.2040 <0.05

(1)Time (h)(L) 35.295 bi bi 2.0420 0.057 σ-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

1 L by 2Q -71.385 bl22 bi ₂₂ bl22 -2.9210 <0.01

1 L by 2L -21.584 b _l2 bl2 b ₁₂ -1.2490 0.229

1 Q by 2L -21.151 l12 bl12 l12 -0.8650 0.399

1Q by 3L -16.121 t>1l3 bn3 bn3 -0.6600 0.518

1 L by 3L 27.506 l3 bl3 bl3 1.5920 0.139

2L by 3L 9.056 b ₂ 3 b ₂ 3 b∑3 0.5240 0.607

Note: 1 =time or xylan loading, 2 = temperature, 3 = enzyme xylan specific dosage, L = linear, Q = quadratic, R = linear regression coefficient, model regression coefficient, letters and numbers in bold = significance.

Regression coefficients for arabinose release as a function of the hydrolysis parameters (coded variables)

Arabinose release = f( AbfB, xylan loading) {AbfB, Xylan) Significance level t(5)

R ² = 0.88 (R ² adjusted =0.79) (p)

Mean/interaction 3.993 bo 11.615 <0.01

Main effects <0.01

(1 )Xylan (g)(L) 0.899 bi 5.228 <0.01

Xylan (g)(Q) -0.835 bn -3.674 <0.01

AbfB dose (nKat mL ^~ )(Q) -0.314 b22 -1.380 0.226

Interaction

1 L by 2L 0.221 bl2 0.909 0.405

Note: 1 = xylan loading, 2 = enzyme xylan specific dosage, L = linear, Q = quadratic, R^ = linear regression coefficient, b,...ni = model regression coefficient, letters and numbers in bold = significance

Table 11 Cotton lint xylan specific weight gain after commercial xylan adsorption

Xylan Type "Cotton lint weight gain **Specific weight gain

Treatment

(commercial sources) (%) (%)

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 weight gain "^Specific 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 (BB) ABB 0.55 ±0.0 11 ±0.0

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 (SAPPI) (ES) GES 1.25 ±0.0 25 ±0.0

SES 2.00 ±0.11 40 ±2.12

The prefix A, AG and G denote modification of xylan by σ-L- arabinofuranosidase, cocktail of cr-L- arabinofuranosidase and σ- D-glucuronidase and σ-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 0 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 0 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>, ABH> GBH> SBH (BH), whereas for H ₂ 0 ₂ bleached bagasse was (BB) AGBB> GBB> SBB> 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 (Figure 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) (Figure 34B). The removal of the xylose in the rection 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 (Figure 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) (Figure 35A). The xylose removal from the birch xylan reaction mixture was about 32%, which corresponded to 7.87% release of the available glucuronic acid (Figure 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> AGBH> SBH> ABH, whereas in the BB treated cotton lint acid hydrolysate it was SBB> GBB> AGBB> ABB (Figure 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% (Figure 35B). About 2.3% xylose was detected in the acid hydrolysate of cotton lint treated with E. grandis (EH) in the presence of σ-D-glucuronidase (AguA), whereas in the acid hydrolysed cotton lint treated with the corresponding unmodified xylan, the xylose detected was < 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) (Figure 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 (Figures 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 ) (Figures 36 to 38). The signals between δ 88-89ppm 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 (Figure 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) (Figure 36) show different splitting pattern of C1 , C3 and C5 resonances at δ 105.62/104.32, 72.57, and 71.27 ppm, respectively (Figure 36 spectra 3) from the untreated cotton lint (Figure 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 (Figure 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 (Figure 36 spectra 1) to 89.09 ppm (Figure 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 σ-glucuronidase and (GBH) and σ-L-arabinofuranosidase (ABH), respectively (Figure 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 ³ 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 (Figure 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 ^" (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 a-D-glucuronidase (BCXE) and in the absence of σ-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 0 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>ABH>GBH>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 0 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 ^" (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> AGBH> SBH> ABH, whereas the order of magnitude of the xylose present in the hydrolysate of the cotton lint treated with H ₂ 0 ₂ bleached bagasse (BB) was SBB> GBB> AGBB> ABB. The xylose contents of the cotton lint treated with BM (1.8%) and P. Patula f1.3%) 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 < 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. 3C-(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 Figures 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 6 88-89ppm and around 65 ppm are assigned to crystalline cellulose G4 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 ³ 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 ³ C-(CP/MAS) NMR spectra of cotton lint treated with mild alkali extracted xylan from bagasse (BH) in presence of a-glucuronidase (GBH) and cr-L- arabinofuranosidase (ABH) displayed low intensity signals at δ 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 andAguA.

Discussion σ-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 0 ₂ bleached bagasse (BB), bamboo (BM) and P. patula (P) xylan adsorption mixtures altered the 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 Figure 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 reintroduced 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.