Classifications

• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
  • D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
  • D21H17/20—Macromolecular organic compounds
  • D21H17/21—Macromolecular organic compounds of natural origin; Derivatives thereof
  • D21H17/24—Polysaccharides
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D105/00—Coating compositions based on polysaccharides or on their derivatives, not provided for in groups C09D101/00 or C09D103/00
  • C09D105/14—Hemicellulose; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01055—Alpha-N-arabinofuranosidase (3.2.1.55)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01139—Alpha-glucuronidase (3.2.1.139)
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
  • D21C9/001—Modification of pulp properties
  • D21C9/002—Modification of pulp properties by chemical means; preparation of dewatered pulp, e.g. in sheet or bulk form, containing special additives
  • D21C9/005—Modification of pulp properties by chemical means; preparation of dewatered pulp, e.g. in sheet or bulk form, containing special additives organic compounds
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
  • D21H11/00—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only
  • D21H11/16—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only modified by a particular after-treatment
  • D21H11/20—Chemically or biochemically modified fibres
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

• 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.
• 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.
• 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.
• feruloyl and p-coumaroyl residues which are ester or ether linked to lignin via glucuronic acid and arabinose side groups, respectively.
• 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].
• 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.
• 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.
• adsorbing xylan onto a substrate 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 ⁇ -D-glucuronidase and ⁇ -L- arabinofuranosidase;
• 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 ⁇ -D-glucuronidase and ⁇ -L- arabinofuranosidase.
• Acetyl groups may also be selectively removed from the xylan with acetyl xylan esterase.
• the ⁇ -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 ⁇ -L-arabinofuranosidase enzyme loading may be about 2.5 to about 5 mL.g "1 and the ⁇ -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 ⁇ -D-glucuronidase may have a specific activity of about 300 nKat mg "1 .
• 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.
• 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;
• 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.
• 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
• Figure 3 is a graph showing the production characteristics of AbfB by recombinant A. niger
• 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.
• 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;
• BM Hoije method
• PP Pinus patula
• Sigma commercial oatspelt xylan
• 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
• 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
• 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.
• FIG. 22 shows the characterisation of xylan by (A) 1 H-NMR and (B) 13 C-NMR analyses of birch xylan, (C) 1 H-NMR and (D) 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.
• 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 ⁇ -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 ⁇ -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 ⁇ -glu and
• Figure 28 shows a graph of arabinose removed from oatspelt xylan after ⁇ -L- arabinofuranosidase (AbfB) hydrolysis at varying xylan specific dosage, hydrolysis time (h) and temperature (°C);
• AbfB ⁇ -L- arabinofuranosidase
• 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.
• Figure 35 shows xylose removed, glucuronic acid released and adsorbed onto cotton lint from
• A birch xylan modified with ⁇ -D-glucuronidase ( ⁇ -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 ⁇ -glu cocktail (AG), ⁇ -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
• 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 ⁇ -L-arabinofuranosidase and o-D-glucuronidase (AguA) cocktail, and [5] ⁇ -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 ⁇ -D-glucuronidase (AguA), [4] ⁇ -L-arabinofuranosidase and ⁇ -D- glucuronidase (AguA) cocktail, and [5] ⁇ -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.
• a method of modifying xylan so that it can be adsorbed onto other substrates 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 ⁇ -L-arabinofuranosidases (AbfB) (EC 3.2.1.55) and/or ⁇ -D-glucuronidases (AguA) (EC 3.2.1.131-).
• AbfB ⁇ -L-arabinofuranosidases
• AguA ⁇ -D-glucuronidases
• 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.
• the modified xylan which is less substituted, can be used in pulping so as to increase binding properties of pulp fibres.
• the modified xylan can be adsorbed onto a surface of a substrate, so as to form a coating on the substrate.
• 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 ⁇ -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 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 ⁇ -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 ⁇ -L-arabinofuranosidase enzyme loading can be about 2 to about 10 mL.g '1 and the ⁇ -L-arabinofuranosidase can have a volumetric activity of about 18 nKat mL "1 .
• the ⁇ -D-glucuronidase enzyme loading can be about 0.2 mL.g "1 and the ⁇ -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.
• 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.
• the modified xylan 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.
• 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.
• 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.
• 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 ⁇ -L-arabinofuranosidase (AbfB) with volumetric activity of 18 nKat ml_ "1 on p-Nitrophenyl Arabinofuranoside (p-NPA) produced inhouse using recombinant Aspergillus niger D15.
• a recombinant ⁇ -L-Arabinofuranosidase 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.
• gpd P glyceraldehyde-3-phosphate dehydrogenase promoter
• glaA T glucoamylase terminator
• 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.
• 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].
• 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.
• 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.
• 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.
• A. niger D15[abfB] The morphological and growth characteristics of A. niger D15[abfB] were monitored visually and under electronic microscopic. Mycelia and pellets from bioreactor and terminated shake flasks cultures, were observed under a microscope (100 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.
• 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.
• BIOFLO 1 10 modular bench top fermentation system New Brunswick Scientific company, Inc, USA
• 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 ⁇ 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 ⁇ 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 ( ⁇ 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.
• MW AbfB molecular weight
• 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 BenchMarkTM 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.
• 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 ⁇ reaction mixture was prepared which consisted of 25 ⁇ _ 5mM pNPA in 0.05M citrate buffer (pH 5.0), 25 //L deionised water (dH 2 0), 25 ⁇ _ of appropriately diluted supernatant and 25 ⁇ . 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 ⁇ . 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.
• 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.
• 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.
• BSA bovine serum albumin
• 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.
• 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 ⁇ ) were diluted 5 times and centrifuged at 10 000 rpm for 5 min at 4°C followed by filtration using filters with 0.22 ⁇ 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).
• HPAEC-PAD pulsed electrochemical detection
• ED40 electrochemical detector
• 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.
• 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 ⁇ birch xylan (1 % w/v) and 150 ⁇ L ⁇ ⁇ -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 ⁇ . 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 ⁇ -L-arabinofuranosidase and ⁇ -D-glucuronidase in glass bottles.
• the reaction mixture contained 0.2 g cotton lint, 1000 ⁇ xylan (1 % w/v),
• the adsorption mixture contained ⁇ -L- arabinofuranosidase (AbfB) (18.0 nkat mL "1 ), and ⁇ -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.
• 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.
• the xylan adsorption reaction mixture filtrates were centrifuged at 10,000 rpm for 5 min at 4 °C followed by filtration using 0.22 ⁇ 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.
• HPAEC-PAD Densham
• 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
• Eucalyptus Eucalyptus Mild alkali (Hoije et al., 4-O-MeglcA This study
• Eucalyptus (Eucalyptus nd 4-O-MeglcA Donated by Arlene grandis) gel from pulp (ES) Bayley, SAPPI
• 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 Whybosch University forest plantations in the Western Cape City of South Africa.
• the giant bamboo stems (one and half year plant) were supplied from mature plantations located in Paarl in the Western Cape City 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.
• mc moisture content
• 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.
• ASTM stackable sieves
• 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.
• NREL LAP National Renewable Energy Laboratory Analytical Procedure
• 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.
• 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).
• HPAEC-PAD pulsed amperometric detection
• ED40 electrochemical detector
• 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 ⁇ 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 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.
• 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.
• 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 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.
• 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 CarbopacTM 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.
• DP degree of polymerisation
• 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 ⁇ _) 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.
• 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 ⁇ pore size filter before subjecting it to HPAEC-PAD (Dionex) on Carbopac PA 10 column.
• HPAEC-PAD Density Polymerization-PAD
• 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.
• 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
• 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.
• 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 ⁇ 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.
• the degree of selective removal of arabinose and 4-O-methyl glucuronic acid (4-O-MeglCA) side groups by ⁇ -L-Arabinofuranosidase (AbfB) of Aspergillus niger and ⁇ -D-glucuronidase of Schizophyllum commune ( ⁇ -glu) respectively was determined using xylan derived from Eucalyptus grandis, Pinus patula, Bambusa balcooa, and bagasse found in South Africa.
• the AbfB and ⁇ -glu from A.niger and S. ses 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.
• a xylan solution (1 % w/v) prepared from 4-O-MeglcA substituted substrates was treated with ⁇ -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 ⁇ -glu in 1000 ⁇ .
• 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.
• HPAEC-PAD 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 ).
• Oatspelt xylan was incubated with AbfB in 400 //L reaction mixtures containing 200 ⁇ _ 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.
• Optimal set points for time, temperature, and enzyme dosage for the AbfB removal of arabinose from oatspelt xylan and ⁇ -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.
• x coded value for variable.
• X natural value
• ⁇ scaling factor (half the range of the independent variables which constituted Time, temperature, and enzyme xylan specific dosage).
• 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
• 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 ⁇ birch xylan (1 % w/v) and 150 ⁇ 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 ⁇ . 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 ⁇ -L-Arabinofuranosidase and ⁇ -D-glucuronidase onto cotton lint with pre-extracted xylan was performed in glass bottles.
• the reaction mixture contained 0.2 g cotton lint, 1000 ⁇ xylan (1 % w/v), 40 ⁇ AguA ( 900 nKat mL “1 ), 100 ⁇ 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.
• 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.
• the xylan adsorption reaction mixture filtrates were centrifuged at 10,000 rpm for 5 min at 4°C followed by filtration using 0.22 ⁇ 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.
• 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.
• 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).
• 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).
• 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).
• 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.
• 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 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).
• 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 ⁇ 20 - 22 , 30 - 40, 50-60, 140 - 160 and 170 - 190 ppm, respectively ( Figures 16A-D spectra 3).
• 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).
• 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).
• commercial oatspelt xylan is reported to have 10% arabinose (Sigma), this study showed arabinose content of 7.4 % (Table 7).
• 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.
• 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.
• ⁇ -L-Arabinofuranosidase 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 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 ( ⁇ -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 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).
• 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 " .
• the optimal set points for ⁇ -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 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.
• AbfB arabinose removal to a maximum of 4.7% arabinose 42700 ig g "1 substrate or 42.7% available arabinose
• the largest significant effect on the arabinose removal was xylan concentration rather than AbfB volumetric activity (Figure 33).
• Glucuronic acid release f f(Time,Tem fjfTime.Agu f(Temp,AguA dose)
• 1 time or xylan loading
• 2 temperature
• 3 enzyme xylan specific dosage
• L linear
• Q quadratic
• R linear regression coefficient
• 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.
• Table 12 Cotton lint xylan specific weight gain after adsorption of pre-extracted xylan
• the prefix A, AG and G denote modification of xylan by ⁇ -L- arabinofuranosidase, cocktail of cr-L- arabinofuranosidase and ⁇ - D-glucuronidase and ⁇ -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
• 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).
• 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).
• 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 cotton lint treated with birch xylan (1 % w/v) in the presence of a-D-glucuronidase (BCXE) and in the absence of ⁇ -AguA (BCX) at birch xylan loading of 3.75 mL g "1 showed specific xylan weight gain of 16% and 7%, respectively.
• 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).
• 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).
• 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).
• 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.
• 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 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 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.
• EH E. grandis xylan
• more xylose 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.
• SES unmodified E. grandis xylan gel
• GES AguA modified ES gel
• the signals between 6 88-89ppm and around 65 ppm are assigned to crystalline cellulose G4 and C6 respectively, whereas the signals between ⁇ 83-84 ppm and 61 -62 ppm are assigned to amorphous region of cellulose C4 and C6, respectively.
• 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 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.
• 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.
• 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.
• 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 can be reintroduced together with the AbfB or AguA at the wet end processing stages.
• 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.
• 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.
• xylan 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.
• 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.

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