Patent Application
20130233501
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.
The principal components of plant biomass are lignin, cellulose and hemicellulose. Cellulose in the plant cell wall is present in close association with hemicellulose in a matrix encrusted by lignin. One of the major hemicelluloses in most plant cell walls is xylan. Xylan is a polymer consisting of a 1,4-β-linked D-xylose backbone chain substituted with L-arabinose, D-glucuronic acid or both and various short oligosaccharide chains consisting of D-xylose, -L-arabinose, galactose, glucuronic acid, and O-acetyl groups. Depending on the plant biomass source, morphological plant part and physiological stages of plant maturity, the xylan contained therein may also contain feruloyl and p-coumaroyl residues which are ester or ether linked to lignin via glucuronic acid and arabinose side groups, respectively.
During conventional kraft pulping of lignocellulosic materials, high alkalinity [pH 12-14] and high temperature [165-170° C.] conditions are used to selectively remove lignin while preserving the cellulosic component [Söjström, 1993, Wood Chemistry: Fundamentals and Applications. 2nd edition. Academic Press Limited, London, UK]. However, because xylan is soluble, an undesirable side effect of this process is that xylan is removed along with the lignin, resulting in reduced pulp yields and reduced fibre strength.
South Africa uses about 10 million tons of wood annually for kraft pulping. Wood for kraft pulping contains approximately 25% xylan, and about 1.25 million tons of the 2.5 million tons of xylan subjected to kraft processing are estimated to go to waste. Not only is this a waste of a valuable resource, but it is also an environmental hazard, as the degraded xylans release organic acids and chromophogenic compounds which, when coupled with large amounts of chemical discharges from the kraft process, increase the COD loading of waste water streams.
Many chemical methods for insolubilizing xylan include the use of alkaline hydrolysis [Söjström, 1993, supra] and desolvation steps, while physical treatments include exposing the xylan to supercritically heated (e.g. up to 170° C.) and compressed fluid for long periods (>3 h) using carbon as an antisolvent. These methods, however, result in random desubstitution and undesirable depolymerisation of the xylan. Under such conditions, xylan and cellulose losses of up to 50% occur, mainly due to deacetylation, depolymerisation and formation of chromophogenic compounds and lignin-xylan complexes (LCC) [Söjström, 1993, supra]. Sternemalm et al. [2008, Carbohydr. Res. 343: 753-757] used oxalic acid to selectively remove arabinose side groups from oatspelt xylan, but this process caused a reduction in the molecular weight of the xylan from 305 kDa to 169 kDa, with only an associated 6% decrease in arabinose degree of substitution. Although additives such as clay, derivatised starch and cellulosic products are often added to increase yield and improve the pulp fibre function, higher loading of these additives is associated with retention, dusting and fibre-to-fibre contact problems. Inter-fibre bonding is consequently weakened, further reducing paper strength. The use of commercially available cellulose and starch in the pulp and paper making industry must also compete with that of industries associated with food, pharmaceutical, textiles and biofuels production.
Accordingly, there is a need for modifying xylan so that it can be beneficially used, and in particular so that it can be adsorbed or coated onto substrates.
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:
The xylan may be modified in the presence of the substrate, or may be modified in the absence of the substrate and brought into contact with the substrate after it has been modified.
The xylan may be modified by selectively removing only glucuronic acid side chains with α-D-glucuronidase, by selectively removing only arabinose side chains with α-L-arabinofuranosidase, or by selectively removing both glucuronic acid and arabinose side chains with α-D-glucuronidase and α-L-arabinofuranosidase. Acetyl groups may also be selectively removed from the xylan with acetyl xylan esterase.
The α-L-arabinofuranosidase may be recombinantly produced.
The xylan modification step is preferably carried out without the main xylan chain being damaged or degraded.
The xylan source may be sugarcane bagasse, bamboo, eucalyptus, pine, oatspelt and birch.
The substrate may be a cellulosic substrate, such as pulp, or may be a non-cellulosic substrate.
The modified xylan may form a coating on the substrate.
A sufficient number of side chains may be removed from the xylan so as to reduce the water solubility of the modified xylan.
The xylan may be modified at a temperature of from about 30° C. to about 50° C., and more preferably at from about 35° C. to about 45° C. or at from about 40° C. to about 50° C. The xylan may be contacted with the enzyme for between about 9 and about 18 hours, and the pH may be from about pH4 to about pH6.
The xylan to enzyme ratio may be about 5:2, and the xylan loading may be from about 12.5 mL·g−1 to about 25 mL·g−1. The α-L-arabinofuranosidase enzyme loading may be about 2.5 to about 5 mL·g−1 and the α-L-arabinofuranosidase may have a volumetric activity of about 18.0 nKat mL−1. The α-D-glucuronidase enzyme loading may be about 0.2 mL·g−1 and the α-D-glucuronidase may have a specific activity of about 300 nKat mg−1.
According to a second embodiment of the invention, there is provided a product comprising xylan adsorbed onto a substrate by a method substantially as described above. The product preferably comprises a higher amount of adsorbed xylan than a product containing adsorbed xylan which has not been modified by removal of some of its arabinose or glucoronic acid side chains.
The product may be a cellulosic product, such as pulp, paper, cardboard, packaging or a timber product.
The substrate may be coated with the modified xylan.
According to a third embodiment of the invention, there is provided a pulping process which includes a xylan adsorption step, the xylan adsorption step comprising the steps of:
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.
A method of modifying xylan so that it can be adsorbed onto other substrates is described herein. The method includes the step of modifying water-soluble xylan containing arabinose and/or glucuronic acid side chains by selectively removing some of these side chains with an enzyme which has selective hydrolytic activity for these side chains, and in particular with α-L-arabinofuranosidases (AbfB) (EC 3.2.1.55) and/or α-D-glucuronidases (AguA) (EC 3.2:1.131-). If the xylan also contains acetyl side chains, then an acetyl xylan esterase (EC 3.1.1.72) can also be used to selectively remove these groups. The modified (desubstituted) xylan, which is insoluble in water, is then allowed to adsorb onto the substrate. For example, the modified xylan, which is less substituted, can be used in pulping so as to increase binding properties of pulp fibres. Alternatively, the modified xylan can be adsorbed onto a surface of a substrate, so as to form a coating on the substrate.
Depending on the source of the xylan, it may first be necessary to extract the xylan from a lignocellulosic material, such as oatspelt, birch, bagasse (BH), bamboo (BM), Pinus patula (PP) or Eucalyptus grandis (EH). H2O2 bleached bagasse xylan (BB) or E. grandis xylan gel (ES) can also be used. The xylan can be extracted by a mild alkali extraction method, which does not damage the main xylan chain.
The xylan should be modified with one or more enzymes which do not degrade the main xylan chain but which are able to remove a sufficient number of arabinose or glucuronic acid side chains, typically non-terminal side chains, from the xylan. Suitable enzymes for use in this method are α-L-arabinofuranosidases (AbfB) (EC 3.2.1.55) and/or α-D-glucuronidases (AguA) (EC 3.2.1.131-), which have been found to selectively remove arabinose and glucuronic acid, respectively, without depolymerising the xylan main chain. The applicant has found that these enzymes, on their own or in combination, are able to effectively remove a sufficient number of the arabinose and/or glucuronic acid side chains, on an industrial scale, so as to decrease water solubility of the xylan and hence to induce xylan precipitation and allow adsorption of the xylan onto other materials or substrates. In some xylans, acetyl groups are attached to the glucuronic acid side chains and should also be removed during modification of the xylan using acetyl xylan esterases (EC 3.1.1.72).
The enzymes should be purified or prepared recombinantly so that they aren't contaminated with other enzymes which could break down the main xylan chain, such as xylanase. For this reason, the α-L-arabinofuranosidase which was used in the examples below was recombinantly produced.
The xylan is generally modified at a temperature of from about 30° C. to about 50° C., and more preferably at from about 35° C. to about 45° C. or at from about 40° C. to about 50° C., and can be contacted with the enzyme for between about 9 and about 18 hours, and the pH may be from about pH4 to about pH6.
The extracted xylan to enzyme ratio is typically about 5:2, and the xylan loading can be from about 12.5 mL·g−1 to about 25 mL·g−1. The α-L-arabinofuranosidase enzyme loading can be about 2 to about 10 mL·g−1 and the α-L-arabinofuranosidase can have a volumetric activity of about 18 nKat mL−1. The α-D-glucuronidase enzyme loading can be about 0.2 mL·g−1 and the α-D-glucuronidase can have a specific activity of about 300 nKat mg−1.
The xylan can be modified in situ in the presence of the substrate, such as when it is used as an additive for pulp, or alternatively may be modified in the absence of the substrate and then brought into contact with the substrate, such as when it is used to form a coating on the substrate.
The substrates onto which the modified xylan can be adsorbed include cellulosic materials such as pulp, paper, cardboard, packaging, textiles and timber products. The substrate can also be a non-cellulosic material, such as metal, mica, magnetic material, pharmaceutical capsules or tablets and the like. When used as an additive, e.g. in the pulping process, adsorption of the xylan to the pulp can result in improved pulp yield, improved pulp bonding properties, improved pulp and paper strength and so forth. Alternatively, when used as a coating material, adsorption of the modified xylan to the substrate can alter the surface properties of these substrates, allowing them to have different uses. For example, the modified xylan may form a hydrogel which encapsulates or entraps a bioactive molecule, such as a bactericide, and this hydrogel may adsorb, onto a surface of a substrate to impart antibacterial properties to the substrate.
The steps of modifying the xylan and adsorbing it onto a pulp substrate can be incorporated into existing wet end pulping processes without too much difficulty, such as between the filtering and bleaching steps, during washing post bleaching, at the drying stage or at the paper fining stage. Typical pulping processes include kraft, alkaline and sodaAQ processes.
In the examples described below, the effects of α-L-arabinofuranosidase (AbfB) and α-D-glucuronidase (AguA) on adsorption onto cotton lint of xylan derived from oatspelt, birch, bagasse (BH), bamboo (BM), Pinus patula (PP) and Eucalyptus grandis (EH), and of H2O2 bleached bagasse xylan (BB) and E. grandis xylan gel (ES) were investigated. The adsorption of oatspelt xylan (1% w/v) onto the cotton lint increased by 33% and 900% (9-fold) in the presence of AbfB at xylan loading of 25 mL g−1 and 12.5 mL g−1 cotton lint, which corresponded to 30% and 50% release of the available arabinose, respectively. The adsorption of BH, BM and P. Patula in the presence of AguA increased by 29, 82 and 112%, respectively, whereas in the presence of AbfB, the adsorption of BH decreased by 13% but that of the BM and P. patula xylan increased by 31% and 44%, respectively. A cocktail of AbfB and AguA increased adsorption of the BH, BM and P. patula xylan more than that of AbfB but such increase was lower than the increase which occurred in the presence of AguA. The AguA aided xylan adsorption was more effective for birch, P. patula and E. grandis than bagasse and bamboo and for mild alkali extracted bagasse and E. grandis xylan than H2O2 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.
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.
Mild alkali extracted xylan from Saccharum officinarum L (sugarcane) bagasse (BH), Bambusidae balcooa [giant bamboo (BM)], Eucalyptus grandis, [Eucalyptus (EH)], Pinus patula [Pine (PP)] (extracted using a mild alkali method described by Höije et al. [2005 Carbohydr. Polym. 61: 266-275]), commercial oatspelt xylan (Sigma), birch xylan (Roth), mild alkali H2O2 bleached xylan from bagasse (BB) (donated by Prof. A. M. F. Milagres, University of Sáo Paulo, Brazil) and xylan gel from E. grandis (ES) (donated by SAPPI—Pretoria, South Africa) were used in the experiments.
Xylan solutions (1% w/v) were prepared according to de Wet et al. [2008, Appl. Microbiol. Biotechnol. 77: 975-983]. Non absorbent cotton lint (Grade 1, Cotton King) was used as a cellulosic fibre source. The arabinose side chains were moved by crude α-L-arabinofuranosidase (AbfB) with volumetric activity of 18 nKat mL−1 on p-Nitrophenyl Arabinofuranoside (p-NPA) produced inhouse using recombinant Aspergillus niger D15. The glucuronic acid side chains were removed by α-D-glucuronidase (AguA) with specific activity=300 nKat mg−1 that was purified from wild type Schizophyllum commune (VTT-D-88362-ATCC 38548, donated by Prof. Matti Siika-aho of VTT Biotechnology Institute in Finland).
A recombinant α-L-Arabinofuranosidase (AbfB) was produced by cloning the AbfB gene from A. niger into the protease deficient and medium non-acidifying strain A. niger D15, under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP) of A. nidulans and the glucoamylase terminator (glaAT) of Aspergillus awamori (A. awamori). The growth characteristics of the A. niger D15[AnabfB] strain and production levels of AbfB were studied in shake flasks and a bioreactor using defined standard media (2×MM media) and cornsteep liquor enriched media. The protein profiles, substrate specificity, substrate dependency, optimal pH and temperature, stability in application and storage, and recyclability were assessed as described below.
The genotypes of the bacterial and fungal strains as well as the plasmids used are summarised in Table 1. Recombinant plasmids were constructed and amplified in E. coli DH5α. E. coli was cultivated at 37° C. in LB medium (1% yeast extract, 1% tryptone and 0.5% NaCl) on a rotary shaker at 100 rpm, supplemented with 100 μg/L ampicillin. The A. niger fungal strains were maintained at 30° C. in minimal media (MM) and on spore plates according to the procedure of Rose and Van Zyl [2002 Appl. Microbiol. Biotechnol. 58: 461-468]. The media in which the A. niger D15 parental strain was maintained was supplemented with 0.01 M uridine. Transformants were prepared according to the procedure of Rose and Van Zyl [2002 Appl. Microbiol. Biotechnol. 58: 461-468] and selected on MM lacking casamino acids and uridine. Transformants were cultivated in Erlenmeyer shake flasks (125 mL) containing 30 mL of double strength traditional minimal medium (2×MM) containing 10% glucose. The medium was inoculated to a final spore concentration of 1×106 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.
Aspergillus niger
A. niger D15
Bioeng. 76: 164-174 ATCC
A. niger van Tieghem
A. niger D15[pGTP]*
A. niger D15 with gpdP-glaAT
Biotechnol. J. 2: 167-175
A. niger D15[abfB]*
A. niger D15 with gpdP-abfB-glaAT
E. coli DH5α
Curr. Genet. 43: 439-446
Biotechnol. J. 2: 167-175
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 (>1×106). To prepare the rice sporulation medium, tastic rice (20 g) was placed in Erlenmeyer shake flasks (250 mL) to which 8 mL of 0.1% (w/v) urea were added. The rice was then autoclaved at 121° C. for 15 min. Spores were added to each flask and incubated at 30° C. for a period of 3-6 days or until 80% of the rice surface was observed to be covered with spores. The spores were harvested into a 0.9% NaCl solution (approximately 50 mL for each flask) and stored in Schott bottles at 4° C.
A. niger spores were inoculated in 2×MM medium enriched with concentrated corn steep liquor (CCSL) which was prepared according to a modified version of the protocol of Gurlal et al. [2006, CSIR/BIO/IR/PPD/2006/0023/B, CSIR, pp 1-8]. The CCSL was donated by Mr. Hough Joubert of African Products-Belleville, South Africa. The CCSL with initial pH of pH 3.87 was sterilised (121° C., 15 min, 1 bar) and filtered (0.22 μm pore size) before being added to the standard medium at 1%, 2%, and 10% (w/v). A. niger was cultivated in the respective media under standard A. niger cultivation conditions. Culture samples were taken at 24 h intervals for 7 days and the AbfB activity determined. Unless otherwise stated, the CCSL optimised medium was used in the subsequent cultivations of A. niger.
Cloning of pGTP and pGTP-AbfB Plasmid Constructs
Standard protocols such as those described by Sambrook et al. [1989, Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.] were followed for all DNA manipulations. The method used to construct the plasmids and the fungal strains was substantially according to that described by Rose and Van Zyl [2002 Appl. Microbiol. Biotechnol. 58: 461-468]. Briefly, the pyrG gene was amplified from pBS-pyrGamdS [Plüddennann and Van Zyl, 2003 Curr. Genet. 43: 439-446] by standard PCR techniques and cloned into the EcoRI site of plasmid pGT [Rose and Van Zyl, 2002, Appl. Microbiol. Biotechnol. 58: 461-468] to generate pGTP. For the production of recombinant α-L-arabinofuranosidase (AbfB), the abfB gene was amplified from the genome of A. niger 10864 using standard PCR methods. The abfB was then cloned into the NotI site of pGTP, generating pGTP-AbfB (
Cultivation of A. niger D15[abfB]
The morphological and growth characteristics of A. niger D15[abfB] were monitored visually and under electronic microscopic. Mycelia and pellets from bioreactor and terminated shake flasks cultures, were observed under a microscope (100× magnification) prior to filtering through Mira cloth mesh. The biomass was washed with deionised water (dH2O) before being transferred into pre-weighed aluminum foils for drying in an oven at 60° C. until a constant weight was obtained. The biomass concentration was calculated as an average dry weight of biomass (dry wt) per unit volume of the culture (g L−1).
The pellet formation was achieved by inoculating the medium (either 2×MM or CCSL (2% w/v) enriched 2×MM medium), with 1×106 spores mL−1 and incubating the shake flasks at 30° C. on a shaker (120 rpm). The cultivation medium had an initial pH of 5.5-6.0 and 5.0 for 2×MM and CCSL enriched, respectively. Mycelial production of AbfB by A. niger D15[abfB] was carried out in a 14 L capacity stirred tank bioreactor (BIOFLO 110 modular bench top fermentation system, New Brunswick Scientific company, Inc, USA) containing 8 L of optimised medium enriched with CCSL (2% w/v) and a spore inoculum of 1×106 spores mL−1 maintained at 30° C. Mass transfer was achieved using a single 3 bladed pitched impeller (30×20 mm) at an agitation speed regulated between 350-700 rpm depending on the level of the dissolved oxygen (DO) in the bioreactor. The DO was maintained above the critical point (>20%) with air and oxygen supplied through a sparger and the levels regulated by a rotameter at 0.5 vessel volume per min (0.5 VVM). Sampling was done through a port installed with a 0.2 μm air filter connected to a syringe for suction into JA 20 centrifuge bottles every hour for the initial 24 h and thereafter every 3-4 h for determination of extracellular AbfB activity, biomass growth and substrate concentration. Foaming was controlled by the addition of 0.1% (v/v) of antifoam A (30% aqueous emulsion with emulsifiers, A 5758, Sigma). Excess foam was collected in a foam trap. The cultures were terminated after 144 h.
The enzyme supernatant harvested from bioreactor fermentation cultures was filtered through Mira cloth. The filtrate was centrifuged (Beckman, J2-21 centrifuge) at 12 000 rpm for 10 min at 4° C. The resulting supernatant was filtered through 0.2 μm filters and concentrated using an Amicon system, Diaflo Ultrafilter PM 10 concentrator, (Amicon Division, W.R. Grace & Co., USA) or a Millpore Minitan ultrafiltration system (Millpore Corporation) with MWCO of 10 kDa. The choice of the ultrafiltration system was based on the initial sample volume. Concentration of crude enzyme samples was carried out by lyophilising the enzyme supernatant in Virtis freeze dryer. The concentrated enzyme supernatant was subjected to ammonium sulfate fractionation at a percentage saturation of 60 and 80% while mixing at 200-250 rpm for 4 h at 4° C. The protein mixture was centrifuged at 1200 rpm for 1 h at 4° C. The pellet obtained at the desired saturation level was re-suspended in 5 mL of Milli-Q water and dialysed against 2 L of 10 mM acetate buffer (pH 5.0) at 4° C. overnight. The desalted concentrated enzyme supernatant was subjected to a single step partial purification in a fast performance gel filtration chromatography (ÄKTA purifier system, Amersham Pharmacia Biotech) installed with UNICORN computer control system (version 3.2). The protein sample (0.3 mL) was applied to a Superdex 75 HR 10/30 size exclusion column. The protein was eluted with 0.05 M acetate buffer pH 4.0 containing 0.4 M NaCl at a flow rate of 0.5 mL min−1. Fractions demonstrating AbfB activity were pooled and concentrated using the Amicon system before molecular and kinetic analysis.
Enzyme preparations were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [1970, Nat. 227: 680-685]. The resolved proteins were initially stained with Coomassie Brilliant Blue R250 and subsequently with silver stain (Bio-Rad kit). The molecular weight (MW) of the resolved protein was estimated from the gel using BenchMark™ prestained protein marker (Invitrogen and Prestained protein ladder (Fermentas). The theoretical MW and isoelectric point (pl) of the AbfB was estimated from the original protein sequence (protein length of 499 bases) using DNAMAN sequence analysis computer software.
Determination of α-L-Arabinofuranosidase Activity of AbfB Using pNPA
α-L-arabinofuranosidase activity of AbfB (AbfB activity) was determined using a colorimetric assay in which the release of p-Nitrophenol from p-Nitrophenyl-α-arabinofuranoside (C11H13NO7, pNPA, Sigma) was measured. A 100 μL reaction mixture was prepared which consisted of 25 μL 5 mM pNPA in 0.05M citrate buffer (pH 5.0), 25 μL deionised water (dH2O), 25 μL of appropriately diluted supernatant and 25 μL 0.05M citrate buffer (pH 5.0). The reaction was incubated at 40° C. for 10 min and was terminated by the addition of 100 μL saturated sodium tetraborate (Na2B4O7, 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, C6H5NO3, Sigma) as standard under similar assay conditions. The AbfB activity was calculated as the amount of enzyme capable of releasing 1 nmole of pNP from the pNPA substrate per mL per second (μkat/mL, conversion factor: 1 Unit=0.0167 μkat). Unless stated otherwise, all assays were conducted in triplicate. Commercial α-L-arabinofuranosidase from A. niger (E-AFASE, Megazyme) was used as a positive control.
Recombinant AbfB was characterised with respect to optimal pH and temperature under the assay conditions described above. The pH optimum and pH stability of AbfB was determined using pNPA desolved in McIlvaine's buffer [McIlvaine, 1921, J. Biol. Chem. 49: 183-186]. Optimal temperature conditions for and temperature stability of the recombinant AbfB was determined using pNPA in 0.05 M citrate buffer at pH 5.0. The effect of substrate concentration on AbfB activity was then determined by using pNPA prepared at varying concentrations in 0.05M citrate buffer pH 5.0. The assays were conducted under AbfB assay conditions as described above. The saturation kinetic properties, maximum activity (Vmax) 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−1sec−1) presented as nkat mL−1. Residual sugar concentration in the cultures was determined using the phenol-sulfuric acid assay [Dubois et al., 1956, Annal. Chem. 28 (3):350-356]. The quantity of the respective sugars was calculated from a standard curve plot of absorbance as a function of the sugar concentration. All reactions were performed in triplicate. Total protein in enzyme preparations was determined by the Bradford method [Bradford, 1976, Anal. Biochem. 72: 248-254] using the Biorad protein assay kit. The quantity of protein was determined using bovine serum albumin (BSA) as standard.
Crude and partially purified forms of recombinant AbfB were assessed for activity against pNPA and polymeric substrates. The substrates tested included: oatspelt xylan (10:15:75 (arabinose:glucose:xylose); Sigma), low viscosity wheat arabinoxylan (37:61:2, arabinose:xylose:other sugars, Megazyme), corn fibre xylan (30% arabinose), donated by Dr. Shin Li, NRL, Preoria, Ill., U.S.A), larchwood arabinogalactan (15:85, arabinose:galactose, Megazyme), debrached arabinan (sugar beet), (88:4:2:6) (arabinose:galactose: rhaminose:galactouronic acidUA) (Megazyme) and mild alkali extracted xylan from bagasse, pine (Pinus patula), and bamboo (Bambusidae balcooa). A solution containing 1% (w/v) of the substrate was prepared. Substrates with limited solubility in water were first dissolved in ethanol (0.1 g in 0.8 mL 99% ethanol), 9 mL of dH2O was added, heated to 70-100° C. while stirring for 10 min, after cooling to room temperature, the volume was adjusted to 10 mL). The reaction mixture with a final volume of 5 mL, contained 2.5 mL of the substrate, 1.5 mL 0.05 M citrate buffer (pH 5.0), and 1 mL AbfB of known enzyme activity. The reaction was performed in a water bath at 40° C. for 16-24 h. Termination of the reaction was achieved by placing the reaction test tubes on ice. The enzymatic hydrolysates (200 μL) were diluted 5 times and centrifuged at 10 000 rpm for 5 min at 4° C. followed by filtration using filters with 0.22 μm pore size. Samples were stored at −20° C. prior to sugar analysis. The sugar analysis was performed using a high pH anion exchange chromatography coupled with pulsed electrochemical detection (HPAEC-PAD) (Dionex) equipped with a gradient pump GP 50, a Carbopac PA 10 (4 mm×250 mm) column, and electrochemical detector (ED40) for pulsed amperometric detection (PAD). The column was eluted with helium degassed 250 mM NaOH and Milli-Q water in 1.5:98.5 ratio at elution rate of 1 mL min−1. The PEAKNET software package was used for data acquisition and analysis of sugar concentration. Quantity of the respective sugars was determined from a standard curve plot of the respective analytical grade sugars (arabinose, rhaminose, galactose, glucose, mannose, and xylose). The quantity of the sugars was expressed on oven dry substrate weight. Residual activity in the AbfB xylan hydrolysate was assessed using the AbfB standard assay.
Enzyme Aided Xylan Adsorption onto Cellulosic Material
Cotton lint samples (1 g) were autoclaved in 100 ml Schott bottles at 121° C. for 15 min. After cooling to room temperature, the oatspelt xylan solution (1% w/v) 5, 12.5, 15.0 and 25 mL and AbfB (18.0 nKat mL−1) were added in a fixed xylan to enzyme ratio of 5:2. The xylan adsorption mixtures contained no enzyme while the positive control mixtures contained the enzyme but in absence of the cotton lint. The adsorptions were performed in 0.05 M citrate buffer pH 5.0 at 40° C. for 24 h in a 40 mL reaction volume. All reactions were terminated by placing the bottles in water containing ice. Cotton lint samples weighing 0.2 g were placed in 15 mL glass bottles in two sets of three and a control. The bottles were autoclaved at 121° C. for 15 min. Upon cooling to room temperature, 750 μL birch xylan (1% w/v) and 150 μL α-D-glucuronidase (AguA) (900 nKat mL−1) were added to one set. Subsequently, 0.05 M acetate buffer (pH 4.8) was added to make a total reaction mixture volume of 1650 μL. The negative and positive controls consisted of the birch xylan in the absence of AguA and in the absence of cotton lint, respectively. Cotton lint (0.2 g) was treated in the adsorption mixture prepared from mild alkali pre-extracted bagasse, bamboo and P. patula xylan in presence of a combined cocktail of α-L-arabinofuranosidase and α-D-glucuronidase in glass bottles. The reaction mixture contained 0.2 g cotton lint, 1000 μL xylan (1% w/v), The adsorption mixture contained α-L-arabinofuranosidase (AbfB) (18.0 nkat mL−1), and α-D-glucuronidase (900 nKat mL−1) and 0.05 M acetate buffer pH 4.8. The reaction was performed at 40° C. in a water bath for 16 h.
Treated cotton lint samples were vacuum-filtered and subsequently washed by suspending them in 50-100 mL Milli-Q H2O while agitating for 1 h to disengage xylan precipitates loosely absorbed to the cotton lint. The Milli-Q H2O 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 μm pore size filters. The filtrates were analysed for L-arabinose and α-D glucuronic acid release using HPAEC-PAD (Dionex) on the Carbopac PA 10 column. L-arabinose (Merck) and glucuronic (Sigma) acid were used for plotting standard curves. Samples from xylan adsorption mixture filtrates were subjected to a phenol-sulfuric assay described by Dubois [1956, Annal. Chem. 28 (3):350-356]. Analytical grade of xylose sugar (Merck) was used as a standard. Precipitation efficiency was defined as the amount of xylan removed from the adsorption reaction mixture as a percentage of the initial amount of xylan in the adsorption mixture (xylan in this case was measured in the form of xylose sugar). Samples of xylan treated cotton lint weighing 0.05 g were hydrolysed in 0.5 mL of 72% H2SO4 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 dH2O. The samples were autoclaved at 121° C. for 1 h. The hydrolysates were vacuum filtered using glass microfibre filters followed by filtration using 0.22 μm pore size filter discs before sugar analysis. The xylose content in the cotton lint was determined using HPAEC-PAD (Dionex) on Carbopac PA 10 column. The efficiency of xylan adsorption onto the cotton lint due to enzymatic treatment of the xylan during the adsorption was defined as the difference between the amount of xylose released from the hydrolysate of cotton lint treated in the presence and absence of the enzymes corrected for any xylose that pre-existed in the untreated cotton lint. Synergetic effect was defined as the difference between the amount of xylose released from acid hydrolysate of cotton lint treated in xylan adsorption mixtures in the presence of a cocktail of AbfB and AguA and the amount of xylose released from the acid hydrolysate of cotton lint treated with the same type of xylan but in the presence of AbfB or AguA. Synergetic effect was expressed as a percentage of the amount of xylose in the hydrolysate of cotton lint treated with xylan in the presence of either AbfB or AguA.
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 13C using a 6 mm T3 probe with a probe temperature of 25° C. Dry cotton lint samples were loaded to fill 6 mm zirconium oxide rotors. Spectra were recorded using cross-polarisation and magic angle spinning (CP/MAS). The speed of rotation was 5 kHz, the proton 90° pulse was 5 μs, the contact pulse 1500 μs and the delay between repetitions 5 sec. Chemical shifts were determined relative to TMS by setting the downfield peak of an external adamantane reference to 38.3 ppm.
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.
The selectivity and effectiveness of the two mild alkali xylan extraction methods which were adopted from Höije et al. [2005, Carbohydr. Polym. 61: 266-275] and De Lopez et al. [1996, Biomass and Bioenergy, 10: (4): 201-211] were assessed for potential use in integrated production of substantially pure xylan biopolymers and pulp production from the feedstock commonly found in South Africa. The assessment was based on five factors as follows: (1) xylan extraction efficiency, (2) degree of polymerization and substitution, (3) chemical composition of the extracted xylan, (4) purity of the extracted xylan, and (5) the structural integrity and chemical composition of the raw material post xylan extraction. A summary of the xylan substrates used for evaluating enzymatic substrate specificity and degree of removal of xylan side chains is shown in Table 2.
officinarum L) Bagasse (BH)
officinarum L) Bagasse (BL)
officinarum L) bagasse (BB
balcoa) (BM or Bam)
Eucalyptus (Eucalyptus
grandis) (EH)
Eucalyptus (Eucalyptus
grandis) (EL)
Eucalyptus (Eucalyptus
grandis) gel from pulp (ES)
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 H2O2 bleached bagasse xylans (donated by Prof. A. M. F. Milagres, University of Sao Paulo, Brazil) were used as reference xylans.
The feedstock materials were prepared for analysis according to TAPPI test methods (TAPPI, T264 cm-97 (2002-2003)), and NREL Laboratory Analytical Procedures (NREL LAP) [Hammes et al., 2005, Laboratory Analytical Procedure (LAP), version 2005, NREL Biomass Program. National Bioenergy Center]. Chips derived from the various feedstocks were dried to a moisture content (mc) of ≈10%, and subsequently conditioned to a relative humidity of 55% at 20° C. for at least 24 h prior to further size reduction. The chips were successively reduced in size by Condux hammer-mill, a Retch, and a Wiley laboratory mill and fractionated by sieving using stackable sieves (ASTM) of 850 μm/20 mesh size, 425 μm/40 mesh size, and 250 μm/60 mesh size with a lid and pan. The particulates that passed through 425 μm/40 mesh size but were retained on a 250 μm/60 mesh sieve were collected for chemical composition analyses and those retained on the 425 μm/40 mesh were used for xylan extraction. The moisture content of the feedstock was determined using National Renewable Energy Laboratory Analytical Procedure (NREL LAP) for determination of total solids in biomass [Hammes et al., 2005, Laboratory Analytical Procedure (LAP), NREL Biomass Program. National Bioenergy Center]. The percent moisture content was calculated as a % of oven dry (o.d) weight biomass.
Extractives were determined in two sequential steps, starting with cyclohexane/ethanol (2:1) followed by hot water extraction, using soxhlet apparatus. Both extractions were done according to TAPPI Test Method T 264 om-88, and NREL LAP methods [Sluiter et al., 2005, Analytical Procedure (LAP), version 2006. NREL Biomass Program. National Bioenergy Center]. The extractives were quantified on a moisture free basis.
Klason lignin (acid insoluble) content of the feedstock was determined following a NREL LAP method for determination of structural carbohydrates and lignin in biomass [Sluiter et al., 2005, Analytical Procedure (LAP), version 2006. NREL Biomass Program. National Bioenergy Center] and TAPPI test procedures (T249 cm-85). The Klason lignin was calculated on o.d. mass.
Seifert cellulose content was determined according to the analytical methods outlined by Browning [1967, Methods of wood chemistry, Vol II. Interscience publishers], and Fengel and Wegner [1989, Wood Chemistry, Ultrastructure, Reactions. Walter de Gruyter, Berlin, Germany]. Extractive free material weighing 1.1 g oven dry was treated with a mixture of acetyl acetone (6 ml), dioxane (2 ml) and 32% HCl (2 ml) in round bottom flasks followed by incubation in a boiling water bath for 30 min. The treated samples were transferred quantitatively into pre-weighed sinter glass crucibles for vacuum filtration and washing. The residues were successively washed with 100 mL each of methanol, cyclo-dioxane, warm water (80° C.), methanol, and diethyl ether and subsequently dried at 105° C. for 2 h. The Seifert cellulose content was defined as the weight of the dried residue presented as a percentage of the extractive free material.
Monomeric sugar composition of the acid hydrolysate was analysed after storage at −20° C. for at least 24 h. The analysis was performed in high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD, (Dionex) that was equipped with a gradient pump GP 50, a Carbopac PA 10 (4 mm×250 mm) column, and electrochemical detector (ED40). The data acquisition and analysis were performed using PEAKNET software package. The eluents were 250 mM NaOH and Milli-Q-water in the ratio of 1.5:98.5 at a flow rate of 1 mL min−1. Sodium acetate (1M NaOAc) eluent was used when acid sugars (glucuronic/methyl glucuronic acid) were analysed. The samples were filtered on 0.22 μm pore size filters before analysis on HPAEC-PAD. The quantity of the sugars was determined from standard plot of the respective analytical grade sugars (arabinose, rhaminose, galactose, glucose, mannose, xylose, and glucuronic acid). The amount of sugar was presented as a percentage relative to the oven dry (o.d) weight of the substrate. The pentosan content in the feedstocks was determined according to TAPPI standard methods for measuring pentosans in wood and pulp (T223 cm-84). The xylan content was calculated from a standard plot prepared from xylose (analytical grade) using equation 3.1.1 as percentage of the o.d. biomass [Xyl=xyl×cf, where: Xyl=Xylan content (mg), xyl=Xylose content (mg), cf=Correction factor (0.88)].
The ash content was determined by a thermogravimetric method. Lignocellulosic samples (0.5 g) were incinerated in a Muffle furnace at 575±25° C. for 4 h or until a constant weight was obtained. Ash content was calculated as a percentage of the initial o.d. biomass.
Extraction of xylan from the feedstock was performed using the two mild alkali extraction methods described above. The Hoije method involved post xylan extraction ultrapurification using membrane dialysis (MWCO 12-14 kDa) whereas the Lopez method involved fractionation of the hydrolysates by ethanol precipitation. In both methods, xylan extraction was performed without prior removal of solvent and hot water extractives. The extracts were concentrated before ultrapurification or fractionation to a third of the initial volume using a rotary evaporator (Rotavapor Büchi R-124, Switzerland) under vacuum at 40° C. The extraction efficiency was defined as the yield of xylan per theoretical content of pentosans in the material. The Lopez method was limited to extraction of xylan from E. grandis and bagasse only.
The structure and chemical composition of the feedstocks for xylan extraction were analysed pre- and post-xylan extraction by solid state 13C-Nuclear Magnetic Resonance Cross-Polarisation/Magic Angle Spinning (13C-NMR CP/MAS) on a Varian VNMRS 500 wide bore solid state NMR spectrometer with an operating frequency of 125 MHz for 13C, using a 6 mm T3 probe with a probe temperature of 25° C. Dry samples were loaded into 6 mm zirconium oxide rotors for analysis. Spectra were recorded using cross-polarisation and magic angle spinning (CP/MAS). The speed of rotation was 5 kHz, the proton 90° pulse was 5 μs, the contact pulse 1500 μs and the delay between repetitions 5 sec. Chemical shifts were determined relative to TMS by setting the downfield peak of an external adamantane reference to 38.3 ppm. The carbon resonances in the solid state NMR spectra were assigned according to Larsson et al. [1999, Solid State Nuclear Magnetic Resonance 15: 31-40]; Renard and Jarvis [1999, Plant Physiology 119: 1315-1322], Maunu [2002, Progress in Nuclear Magnetic Response Spectroscopy 40: 151-174], Lahaye et al. [2003, Carbohydrate Research 338: 1559-1569]; Atalla and Isogai [2005, Recent developments in spectroscopic and chemical characterisation of cellulose. In Dumitriu, S. (ed.) Marcel Dekker. New York. pp 123-157], Virkki et al. [2005, Carbohydrate Polymers 59: 357-366], Geng et al. [2006, International Journal of Polymer Characterization 11: 209-226], and Oliveira et al. [2008, Chemical composition and lignin structural features of banana plant leaf sheath and rachis. In Hu, T. Q. (ed) Chapter 10: 171-188].
The extracted xylan samples were analysed using solid state 13C-Nuclear Magnetic Resonance Cross-Polarisation/Magic Angle Spinning (13C-NMR CP/MAS) and Liquid 13C and 1H NMR and Fourier Transform Infrared (FTIR) spectroscopy. The xylan samples were subjected to a 13C and a 1H NMR run either on a Varian Inova 400 or 600 NMR spectrometer. 13C NMR spectra were collected using a 1.3 s acquisition time and 1 s pulse delay at 25° C. The 13C spectra were collected overnight (minimum 19000 scans). 1H NMR spectra were collected after filtration of the sample with a 4.8 s acquisition time at 50° C. 1H spectra were collected with 64 scans and pre-saturation of the HDO peak. The 13C and 1H NMR spectra were interpreted according to assignment of characteristic signals of related feedstock presented by Ebringerová et al. [1998, Carbohydrate Polymers 37: 231-239], Vignon and Gey [1998, Carbohydrate Research 307: 107-111], Renard and Jarvis [1999, Plant Physiology 119: 1315-1322], Teleman et al. [2002, Carbohydrate Research 337: 373-377], Gröndahl at al. [2003, Carbohydr. Polym. 53: 359-366], Lahaye at al. [2003, Carbohydrate Research 338: 1559-1569], Sun et al. [2004, Carbohydrate Research 339: 291-300; Polym. Degrad. and Stability 84: 331-337; Carbohydrate Polymers 56: 195-204], Sims and Newman [2006, Carbohydrate Polymers 63: 379-384]; Habibi and Vignon [2005, Carbohydrate Research 340: 1431-1436], Pinto et al. [2005, Carbohydrate Polymers 60: 489-497], Geng et al. [2006, International Journal of Polymer Characterization 11: 209-226], Maunu [2008. 13C 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 (4×250 mm) and a guard column, and electrochemical detector (ED40) for pulsed amperometric detection (PAD). The PA 100 column separates monomers and oligomers up to a degree of polymerisation (DP) 10 which usually elutes within a retention time of 25 min. The HPAEC PA100 column bases its separation on DP and degree of substitution, thus the longer the retention time, the higher the DP or degree of substitution (Combined CarboPac manual pp 52-56). Samples (10 μL) were injected into the column and were eluted with helium degassed 0.25 M NaOH, Milli-Q H2O, 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 H2O2 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% H2SO4 was added. The mixture was incubated at 30° C. in a water bath for 1 h. De-ionized water (30 mL) was added followed by autoclaving at 121° C. for 1 h. The samples were cooled to room temperature before filtering. The filter cake was dried at 105° C. for residual Klason lignin determination. The liquid fraction was filtered through a 0.22 μm pore size filter before subjecting it to HPAEC-PAD (Dionex) on Carbopac PA 10 column. The monomeric sugars were quantified from standard plots of analytical grade arabinose, rhaminose, galactose, glucose, xylose, and mannose). The total neutral sugar content of the samples was presented relative to the initial xylan o.d mass.
Uronic acid content of the xylan samples and the feedstocks were quantified using chromatographic and colorimetric methods. In the chromatographic method, a two step acid hydrolysis method adopted from Prof. A. M. F. Milagres of University of Sáo Paulo, Brazil (Personal communication, 2007) was used. Xylan samples (150 mg o.d mass) were hydrolysed in 0.75 mL of 72% (w/w) H2SO4 in McCartney bottles. The mixture was incubated at 45° C. for 7 min in a water bath after which 22.5 mL of distilled water were added. The bottles were loosely covered and autoclaved at 121° C. for 30 min. After cooling to room temperature, the liquid fraction was separated by vacuum filtering through glass micro fibre filters (GF/A—Whatman). The liquid fraction was further filtered through a 0.22 μm filter and kept frozen overnight at −20° C. before analysing for glucuronic acid content using HPAEC-PAD (Dionex) on Carbopac PA 10 column. Quantification of uronic acid was based on standard plots for glucuronic acid (Sigma). Uronic acid losses during autoclaving were accounted for by autoclaved glucuronic acid at 121° C. for 1 h in 4% H2SO4. 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 α-L-Arabinofuranosidase (AbfB) of Aspergillus niger and α-D-glucuronidase of Schizophyllum commune (α-glu) respectively was determined using xylan derived from Eucalyptus grandis, Pinus patula, Bambusa balcooa, and bagasse found in South Africa.
The AbfB and α-glu from A. niger and S. commune were assessed in individual application and in synergy for selective removal of arabinose and 4-O-MeglcA side chains respectively from xylan derived from hardwood, softwood and grass (including cereals) sources with the aim of developing a controlled enzymatic technology for diversification of the xylan functional properties. Therefore, the effect of hydrolysis time, temperature and enzyme xylan specific dosage on the removal of arabinose and 4-O-MeglcA side chains, and the subsequent modification of viscosity, solubility, precipitation and aggregation of the xylan were examined. Xylan samples substituted with arabinose and/or 4-O-methyl glucuronic acid (4-O-MeglcA) side chains are shown in Table 2. Oatspelt xylan (Sigma) and birch xylan (Roth) were utilised as model xylans. Xylan solution (1% w/v) for each material was prepared in de-ionized water (dH2O). The xylan that showed limited solubility in water was prepared by first dissolving in ethanol and subsequently heated according to de Wet et al. [2008, Appl. Microbiol. Biotechnol. 77: 975-983]. Xylan solutions were made in bulk and stored in vials at −20° C. Oatspelt xylan (Sigma) with a sugar composition of 10:15:75 (arabinose:glucose: xylose) and birch xylan (Roth) with sugar composition of 8.3:1.4:89.3 (4-O-MeglcA: glucose, and xylose) [Kormelink and Voragen, 1993, Carbohydr. Res. 249: 345-353] made in similar way were used as model xylan. The enzymes used were crude α-L-arabinofuranosidase (AbfB) produced by recombinant Aspergillus niger D15 produced inhouse with volumetric activity of 18.0 nKat mL−1 on p-Nitrophenyl Arabinofuranoside (p-NPA) and α-D-glucuronidase (α-glu) with specific activity=300 nKat mg−1 purified from wild type Schizophyllum commune (VTT-D-88362-ATCC 38548 (donated by Prof. Matti Siika-aho of VTT Biotechnology institute in Finland). These enzymes were used for the selective removal of L-arabinose and 4-O-methyl-D-glucuronic acid/D-glucuronic acid side groups, respectively. The enzyme aliquots were stored at 4° C.
Xylan known to be substituted with arabinose (Table 2) was treated with AbfB in a reaction volume of 5 mL containing 2.5 mL xylan, 1.0 mL enzyme supernatant and 1.5 mL 0.05 M citrate buffer pH 5.0. The hydrolysis was performed at 40° C. for 16 h. A xylan solution (1% w/v) prepared from 4-O-MeglcA substituted substrates was treated with α-glu (9000 nKat g−1) in 5 mL reaction volumes consisting of 2.5 mL of the substrate and made up to 5 mL with 0.05 M acetate buffer, pH 4.8. The reactions proceeded for 16 h at 40° C. Xylan solutions (1% w/v) prepared from substrates substituted with both arabinose and 4-O-MeglcA side chains (Table 2) were simultaneously treated with AbfB and α-glu in 1000 μL reaction mixtures containing 500 μL of the respective substrates, AbfB 540.0 nKat g−1 and α-glu 9000.00 nKat g−1 in 0.05 M acetate buffer pH 4.8. The reactions were performed in a water bath set at 40° C. for 24 h. The synergetic effect was calculated based on the difference between the amount of the specific sugar released due to individual enzyme action and combined enzyme action.
Effects of hydrolysis parameters (time, temperature and enzyme xylan specific dosage) on arabinose removal and viscosity were determined using a three factor Box-Behnken statistical design with 3 central points in 15 runs (Statistica 7.0 software programme, (StatSoft, Inc., 1984-2005) as shown in Table 3. Time, temperature, and enzyme xylan specific dosage constituted the independent variable whereas degree of side chain removal and viscosity change formed the dependent variables.
Arabinose and 4-O-MeglcA side chains sugar release were analysed using (HPAEC-PAD) on Carbopac PA 10 column eluted with helium degassed Mill-Q H2O, 250 mM NaOH and 1M NaOAc (for acid sugars only). L(+) arabinose and D-glucuronic acid were used as standard sugars. Insolubilization, precipitation and aggregation of the xylan hydrogels were confirmed by visual inspection (photographs taken) and quantified by measuring viscosity using Rheometer (MCR501). The degree of xylan precipitation was quantified by determining residual xylose in solution using phenol-sulphuric assay for total sugar [Dubois of al., 1956, Annal. Chem. 28 (3):350-356].
A three factor Box-Behnken statistical design experiment with 3 central points making a total of 15 runs was run in duplicates: Statistica 7.0 software programme, (StatSoft, Inc., 1984-2005) was used for designing and analysisbased on response surface method (RSM) as shown in Table 4. Regression and ANOVA analyses were performed to determine the size and significance of individual and interaction effects of the hydrolysis parameters on viscosity. Optimal conditions were determined using the desirability function. The response surface plots were fitted with a second order polynomial as follows:
Z=β
0+β1x1+β11x12+β2x2+β22x22+β3x3+β33x32+ε
where: Z=Viscosity (mPa·s), β0+β1 . . . βn=linear regression coefficient, β11 . . . βnn=Quadratic regression coefficient, ε=Error, and X1, X2, X3=Hydrolysis time, temperature, and enzyme xylan specific dosage.
The optimal combination levels of hydrolysis parameters were determined: time, temperature, and dosage of recombinant α-L-arabinofuranosidase and α-D-glucuronidase for removal of arabinose and 4-O-methyl glucuronic acid side chains, respectively.
The effect of xylan loading, enzyme loading, hydrolysis time, and temperature, and their interaction on AbfB removal of arabinose and α-glu removal of 4-O-MeglcA side chains from oatspelt and birch xylan, respectively, were determined by using Response Surface Methodology (RSM). Statistica 7.0 software programme, (StatSoft, Inc., 1984-2005) was used for designing and analysing the experiments. Statistical analysis included regression and ANOVA analyses. Pareto Chart plots were used to present size and significance of effects while the desirability and profiling function was used to determine optimal set point for the hydrolysis parameters. The oatspelt xylan (1% w/v) was prepared according to de Wet et al. [2008, Appl. Microbiol. Biotechnol. 77: 975-983]. The solutions were made in bulk and stored in vials at −20° C. Crude α-L-arabinofuranosidase (AbfB) produced inhouse using recombinant Aspergillus niger D15 (volumetric activity of 18 nKat mL−1 on p-NPA) and α-D-glucuronidase (α-glu) purified from wild type Schizophyllum commune (VTT-D-88362-ATCC 38548) with specific activity of 300 nKat mg−1 (donated by Prof. Matti Siika-aho of VTT Biotechnology Institute in Finland), were used for the selective removal of L-arabinose and 4-O-methyl-D-glucuronic acid/D-glucuronic acid side groups, respectively. The enzyme aliquots were stored at 4° C. In addition L (+)-arabinose (Merck) and D-Glucuronic acid (Sigma) were used as standard sugars.
Oatspelt xylan was incubated with AbfB in 400 μL reaction mixtures containing 200 μL of the substrate, with AbfB xylan specific dosage ranging from 44.0-140.0 nKat substrates (g)−1 in 0.05 M citrate buffer pH 5. The reactions were performed in a water bath set at 40° C. for durations of 2, 4, 8 and 16 h. The effect of temperature was assessed in 5 mL reaction volumes into which 2500 mL of substrate was added and incubated with AbfB of xylan specific dosage of 180 and 720 nKat g substrate−1 at 40 and 60° C. for 4 and 16 h. The reactions were stopped by boiling for 10 min or by immediately placing the samples on ice. The hydrolysates were analysed for arabinose release.
Optimal set points for time, temperature, and enzyme dosage for the AbfB removal of arabinose from oatspelt xylan and α-glu removal of 4-O-MeglcA from birch xylan were determined in a three factor Box-Behnken statistical design with 3 central points making a total of 15 runs in duplicates. The hydrolysis parameters were each tested at two levels and middle point with the highest, middle and lowest levels denoted as 1, 0, and −1 respectively. Temperature was tested at 30° C. and 50° C., time at 1 h and 16 h, and enzyme dosage for AbfB was 180 nKat g−1 and 540 nKat g substrate−1 while that of α-glu was 2 000 nKat g−1 and 18 000 nKat g−1. The central points for temperature and time were 40° C., and 8.5 h, while for AbfB and α-glu xylan specific dosages were 360000 nKat g−1 and 11000 nKat g−1, respectively. The full experimental set up with runs presented in random order for arabinose removal from oatspelt xylan and 4-O-MeglcA from birch xylan are presented in Tables 3 and 4, respectively. The variables were coded according to the equation:
where: xi=coded value for variable, Xi=natural value, ΔXi=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 H2O, 250 mM NaOH and 1M NaOAc (for acid sugars only). L (+) arabinose and D-glucuronic acid were used as standard sugars.
The response surface plot was fitted with a second order polynomial which included both linear and quadratic interactions as follows:
Z=β
0+β1x1+β11x12+β2x2+β22x22+β3x3+β33x32+β112x12x2+β113x12x3+β122x1x22+β133x1x32+β223x22x3+β233x2x32β1122x12x22+β2233x22x32+ε
where: Z=Response (degree of side chain removal), β0+β1 . . . βn . . . =linear regression coefficient, β11 . . . βnn . . . =Quadratic regression coefficient, X1, X2, X3=Hydrolysis time, temperature, and enzyme xylan specific dosage or xylan and enzyme loading, ε=Error.
Individual and synergetic effects of applying recombinant α-L-arabinofuranosidase and α-D-glucuronidase on the adsorption capacity of xylan derived from Eucalyptus grandis, Pinus patula, Bambusa balcooa, and bagasse onto cellulosic material was assessed. The individual and synergetic effects of selective removal of L-arabinose and 4-O-methyl-D-glucuronic acid/D-glucuronic acid side groups by recombinant AbfB and wild purified AguA, respectively, on adsorption of xylan extracted from Saccharum officinarum L (sugarcane) bagasse (BH), Bambusidae balcoa (giant bamboo) (BM), Eucalyptus grandis, [Eucalyptus (EH)], and Pinus patula [Pine (PP)] using mild alkali method were investigated onto non-absorbent cotton lint (Grade 1, Cotton King). Crude α-L-arabinofuranosidase (AbfB) produced by recombinant Aspergillus niger D15 with volumetric activity of 18.0 nKat mL−1 on p-NPA produced inhouse and α-D-glucuronidase (α-glu) with specific activity=300 nKat mg−1 purified from wild type Schizophyllum commune (VTT-D-88362-ATCC 38548 (donated by Prof. Matti Siika-aho of VTT Biotechnology institute in Finland) were added to xylan solutions (1% w/v) prepared according to de Wet et al. [2008, Appl. Microbiol. Biotechnol. 77: 975-983]. Solutions prepared from commercial oatspelt xylan (Sigma) and birch xylan (Roth) were used as model xylan. To broaden the substrate base, mild alkali H2O2 bleached xylan from bagasse (BB) (donated by Prof. A. M. F. Milagres, University of Sao Paulo, Brazil), and processed xylan gel from E. grandis (ES) (donated by SAPPI—Pretoria, South Africa) were used as reference xylan in assessing effect of different xylan extraction protocols.
Samples of cotton lint (1 g) were autoclaved in 100 ml Schott bottles at 121° C. for 15 min. After cooling to room temperature, varying amounts of oatspelt xylan 5, 12.5, 15.0 and 25 mL with AbfB (18.0 nKat mL−1) in a fixed xylan:enzyme ratio of 5:2 and without AbfB were added. The adsorption treatments were performed in 0.05 M citrate buffer pH 5.0 in a total reaction mixture volume of 40 mL. The reaction mixture was incubated at 40° C. in a water bath for 24 h. The control sample contained treated xylan without cotton lint. All reactions were terminated by placing the bottles in water containing ice.
Cotton lint samples weighing 0.2 g were placed in 15 mL glass bottles in two sets of three and a control. The bottles were autoclaved at 121° C. for 15 min. Upon cooling to room temperature, 750 μL birch xylan (1% w/v) and 150 μL AguA (900 nKat mL−1) were added to one set. Subsequently, 0.05 M acetate buffer (pH 4.8) was added to make a total reaction mixture volume of 1650 μL. In addition, the same amount of birch was treated with AguA in separate glass bottles in the absence of cotton lint.
Adsorption of xylan treated by both α-L-Arabinofuranosidase and α-D-glucuronidase onto cotton lint with pre-extracted xylan was performed in glass bottles. The reaction mixture contained 0.2 g cotton lint, 1000 μL xylan (1% w/v), 40 μL AguA (900 nKat mL−1), 100 μL AbfB (18.0 nkat mL−1), and 0.05 M acetate buffer pH 4.8. The reaction was performed at 40° C. in a water bath for 16 h according to the experimental set up shown in Table 6.
Treated cotton lint samples were vacuum filtered from the adsorption mixture and subsequently transferred into a Schott bottle in which the cotton lint was washed by suspending it in 50-100 mL Milli-Q H2O while agitating for 1 h to disengage and remove xylan precipitates loosely absorbed to the cotton lint. The Milli-Q H2O 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.
Eucalyptus H1
Eucalyptus H2
Eucalyptus gel S1
Eucalyptus gelS2
The xylan adsorption reaction mixture filtrates were centrifuged at 10,000 rpm for 5 min at 4° C. followed by filtration using 0.22 μm pore size filters. The filtrates were analysed for L-arabinose and α-D glucuronic acid release using HPAEC-PAD (Dionex) on Carbopac PA 10 column. L-Arabinose (Merck) and glucuronic (Sigma) acid were used for plotting standard curves. Samples from xylan adsorption mixture filtrates were subjected to phenol-sulfuric assay (200 μL filtrate sample, 200 μL (5%) phenol and 800 μL concentrated sulfuric acid, absorbance was measured at 490 nm in a spectrophotometer) to determine changes in xylan content of the reaction after adsorption onto the cotton lint. Analytical grade of xylose sugar (Merck) was used as a standard. Precipitation efficiency was defined as the amount of xylan removed from the adsorption reaction mixture as a percentage of the initial amount of xylan in the adsorption mixture (xylan in this case was measured in the form of xylose sugar).
Samples of xylan treated cotton lint weighing 0.050 g were hydrolysed in 0.5 mL of 72% H2SO4 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 dH2O. The samples were autoclaved at 121° C. for 1 h. The hydrolysates were vacuum filtered using glass microfibre filters followed by filtration using 0.22 μm pore size filter discs before sugar analysis. The xylose content in the cotton lint was determined using HPAEC-PAD (Dionex) on Carbopac PA 10 column. The efficiency of xylan adsorption onto the cotton lint due to enzymatic treatment of the xylan during the adsorption was defined as the difference between the amount of xylose released from cotton lint treated in xylan adsorption mixture in the presence of the enzymes and the amount of xylose released from cotton lint treated in unmodified xylan, corrected for any xylose that pre-existed in the untreated cotton lint. Synergetic effect was defined as the difference between the amount of xylose released from acid hydrolysate of cotton lint treated in xylan adsorption mixtures in the presence of AbfB and AguA cocktail and the amount of xylose released from the acid hydrolysate of cotton lint treated with the same type xylan but in the presence of either AbfB or AguA individually. Synergetic effect was expressed as a percentage of the amount of xylose in the hydrolysate of cotton lint treated with xylan in the presence of either AbfB or AguA.
Structural changes of the dried cotton lint samples as a result of xylan adsorption were analysed using solid state (CP/MAS) NMR on a Varian VNMRS 500 wide bore solid state NMR spectrometer with an operating frequency of 125 MHz for 13C using a 6 mm T3 probe with a probe temperature of 25° C. Dry cotton lint samples were loaded to fill 6 mm zirconium oxide rotors. Spectra were recorded using cross-polarisation and magic angle spinning (CP/MAS). The speed of rotation was 5 kHz, the proton 90° pulse was 5 μs, the contact pulse 1500 μs and the delay between repetitions 5 sec. Chemical shifts were determined relative to TMS by setting the downfield peak of an external adamantane reference to 38.3 ppm.
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 6th day of incubation (
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 (
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. (
The dependency of crude and partially purified AbfB activity on pNPA concentration is presented in
Extracting and Characterising Xylan from Lignocellulosic Materials
The chemical composition of bagasse, pine (Pinus patula), and bamboo (Bambusidae balcooa) is presented in
The solid state 13C-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] (
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 (
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 H2O2 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 (
Eucalyptus
grandis Hoije
Pinus patula
adenotes mean of at least three samples with standard deviation < 0.1,
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% H2SO4) hydrolysis yielded between 16 and 55% insoluble residues (
The 1H NMR and 13C NMR spectra of the reference xylan from birch, H2O2 bleached bagasse, and oatspelt xylan displayed characteristic signals for proton and carbon resonances (
The proton spectra of xylan extracted from E. grandis by Lopez method (EU-L) and Hoije method (EU-H) displayed signals at δ 4.48, 3.96/3.99, 3.63/3.68, and 3.50/3.52 ppm arising from xylose units substituted with 4-O-methylglucuronic acid (
The characteristic carbon resonances from the five carbons of (1→4) linked β-D-xylopyranosyl residues between δ 103 and 62 ppm were reflected in the 13C NMR spectra of the extracted xylan fractions (
The FTIR spectra of the extracted xylan fractions displayed characteristic bands for xylan residues which included β-glycosidic linkages reflected at ≈897 cm−1 (
Controlled Enzymatic Removal of Side Chains from Lignocellulosic Feedstocks
The recombinant α-L-Arabinofuranosidase (AbfB) produced by the Aspergillus niger released arabinose from oatspelt xylan and mild alkali extracted xylan from bagasse Höije (BH), H2O2 bleached bagasse (BB), bamboo (BM) and Pinus patula (PP). Arabinose of about 15 mg g−1 substrate and 14 mg g−1 substrate (representing 20% and 9% of available arabinose) was removed from oatspelt xylan and BH, respectively, whereas about 5 mg g−1 substrate (3% available arabinose) was released from AbfB treated bamboo (BM) xylan fractions (
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 (
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 (
The hydrolysis parameters having significant effects on removal of 4-O-MeglcA from birch xylan by α-glu were in descending magnitude, from linear effects from α-glu xylan specific dosage [α-glu, nKat/g (L)], temperature [Temp (L)], and the quadratic effect of temperature [Temp (Q)] (
A maximum of ≈4% arabinose (substrate basis or 40.% available arabinose) was achievable with combinations of xylan concentrations between 3 and 6 mg mL−1 and AbfB volumetric activity of >10 nkat mL−1 (
Enzyme Aided Adsorption of Xylan onto Cellulosic Material
In the presence of AbfB, cotton lint treated with 25 mL (25CXE), 15 mL (15CXE), 12.5 mL (12.5CXE) and 5 mL (5CXE) oatspelt xylan (1% w/v) mixture had a specific xylan weight gain of approximately 23, 27, 26, and 42%, respectively (Table 11). The specific xylan weight gain of the corresponding cotton lint treated with oatspelt xylan in the absence of AbfB in 25 mL (25CX) and 12.5 mL (12.5 CX) was approximately 18 and 27%, respectively (Table 11). The cotton lint treated with birch xylan in the presence (BCXE) and absence (BCX) of α-D-glucuronidase (AguA) had a specific weight gain of 16% and 7%, respectively (Table 11). The treatment of cotton lint with xylan from bagasse extracted by the Hoije method (ABH), H2O2 bleached bagasse xylan (ABB), bamboo (ABM) and P. patula (AP) [both extracted by the Hoije method] in the presence of AbfB gave a cotton lint xylan specific weight gain of 31.0, 11.0, 16.5 and 10.0%, respectively (Table 12). On the other hand, treatment of cotton lint with bagasse extracted by the Hoije method (GBH), H2O2 bleached bagasse xylan (GBB), bamboo (GBM), P. patula (GP), and E. grandis (GEH), all extracted by the Hoije method, and E. grandis xylan gel (GES) in the presence of α-D-glucuronidase (AguA) gave a cotton lint xylan specific weight gain of 28.5, 17.0, 30.0, 32.0, 15 and 25%, respectively (Table 12). Treating the cotton lint in the absence of AguA with xylan from E. grandis extracted by the Hoije method (SEH) and E. grandis gel (SES) resulted in a cotton lint xylan specific weight gain of 3 and 40%, respectively (Table 12).
9476.69
b
0
b
0
b
0
29.009
<0.01
3989.39
b
1
b
1
19.942
<0.01
−3145.29
b
4
b
4
−5.341
<0.01
2071.60
b
3
b
3
7.322
<0.01
−4178.84
b
1122
b
1122
b
1122
−3.548
<0.01
−1461.58
b
122
b
122
b
122
−5.166
<0.01
1323.20
b
112
b
112
b
112
3.8189
<0.01
−553.82
b
12
b
12
b
12
−2.768
<0.01
437.94
b
13
b
13
b
13
2.189
<0.05
b
113
b
113
b
113
278.0162
b
0
b
0
b
0
13.9314
<0.01
<0.01
−70.9028
b
22
b
2
−3.9416
<0.01
66.164
b
3
b
3
3.8280
<0.01
−38.087
b
2
b
2
−2.2040
<0.05
−71.385
b
122
b
122
b
122
−2.9210
<0.01
3.993
b
0
11.615
<0.01
<0.01
0.899
b
1
5.228
<0.01
−0.835
b
11
−3.674
<0.01
Pinus patula
Eucalyptus grandis (EH)
Eucalyptus grandis gel
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], H2O2 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 H2O2 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 (
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 (
The solid state 13C—(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 (
Effect of Enzymatic Treatment on Adsorption of Xylan onto Cotton Lint-Xylan Specific Weight Gain
The specific xylan weight gain of the cotton lint after being treated with oatspelt xylan in the presence of AbfB at xylan (1% w/v) loading of 5 mL g−1 (5CXE) was 42%, whereas at 12.5 mL g−1 (12.5CXE) and 25 mL g−1 (25CXE) xylan loading, the specific xylan weight gain was 26 and 22%, respectively. The corresponding cotton lint treated with oatspelt xylan in absence of AbfB at 12.5 mL g−1 (12.5 CX) and 25 mL g−1 (25CX) xylan loading had a specific xylan weight gain of approximately 27 and 18%, respectively. The cotton lint treated with birch xylan (1% w/v) in the presence of α-D-glucuronidase (BCXE) and in the absence of α-AguA (BCX) at birch xylan loading of 3.75 mL g−1 showed specific xylan weight gain of 16% and 7%, respectively. In addition, the cotton lint treated with xylan (1% w/v) from bagasse (BH), bamboo (BM) and P. patula (P) and the H2O2 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 H2O2 bleached bagasse (BB) was the highest (19%) in the presence of AbfB/AguA cocktail (AGBB). However, the specific xylan weight gain (11%) of the cotton lint when treated with BB in the presence of AbfB was lower than that of the (ABB) cotton lint treated with unmodified BB xylan (SBB). Similarly, a lower xylan specific weight gain prevailed with the cotton lint treated in E. grandis gel xylan in the presence of AguA (GES) than with the gel in the absence of the AguA (SES).
Effect of Enzymatic Treatment on Adsorption of Xylan onto Cotton Lint-Post Xylan Adsorption Sugar Profile
The acid hydrolysate of the cotton lint treated with oatspelt xylan in the presence of AbfB yielded 8 and 4% xylose at xylan dosage levels of 25 mL g−1 (25CXE) and 12.5 mL g−1 (12.5 CXE), respectively. About 6% and 0.4% xylose were obtained from the corresponding cotton lint treated with oatspelt xylan in the absence of AbfB at the same dosage levels (25CX and 12.5 CX, respectively). The sugar profile of the adsorption mixture showed that xylose removal of about 64% occurred in the reaction mixtures in which the cotton lint was treated with oatspelt xylan in the presence of AbfB at both xylan loading of 25 mL (25CXE) and 12.5 mL (12.5 CXE). The removal of the xylose from the reaction mixtures corresponded to arabinose release of about 30 and 50% from 25CXE and 12.5 CXE in the oatspelt xylan adsorption mixtures, respectively. In the absence of the cotton lint, the precipitation efficiency of the oatspelt xylan of 25 and 42% at xylan occurred in the presence of the AbfB at equivalent xylan loading of 25 mL g−1 (25XYE) and 12 mL g−1 (12XYE), which corresponded to 60% release of the available arabinose in both cases. Traces of arabinose were detected in 25CX, 25XY, 12.5CX and 12.5 XY reaction mixtures in which AbfB was absent.
The acid hydrolysate of cotton lint acid treated with birch xylan in the presence of AguA (BCXE) yielded 1.21% xylose whereas cotton lint treated with the corresponding unmodified birch xylan (BCX) yielded 0.77% xylose. In the AguA treated birch xylan adsorption mixture, xylose removal of 32% occurred, which corresponded to 7.87% release removal of the available glucuronic acid. The xylose removal in the corresponding birch xylan mixture in which AguA was absent was 28%, in which 2% glucuronic acid was detected.
The xylose present in the acid hydrolysate of the cotton lint treated with mild alkali extracted xylan in the presence of AbfB, AguA and a cocktail of the two enzymes. The xylose content of the cotton lint treated with the mild alkali extracted bagasse xylan (BH) was in the following order of magnitude GBH>AGBH>SBH>ABH, whereas the order of magnitude of the xylose present in the hydrolysate of the cotton lint treated with H2O2 bleached bagasse (BB) was SBB>GBB>AGBB>ABB. The xylose contents of the cotton lint treated with BM (1.8%) and P. Patula (1.3%) xylan in the presence of AguA (GBM and GP, respectively) were over 100% higher than of cotton lint treated without the AguA (SBM and SP, respectively). About 2.3% xylose was detected in the acid hydrolysate of cotton lint treated with E. grandis xylan (EH) in the presence of AguA and <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.
13C-(CP/MAS) NMR Characterization of Cotton Lint Structural Changes after Treatment with Enzymatically Modified Polymeric Xylan
The solid state 13C—(CP/MAS) NMR spectra resonances in
α-L-Arabinofuranosidase with polymeric substrate specificity increased adsorption of oatspelt xylan onto cotton lint by up to 33% and 900% in the presence of the AbfB at oatspelt xylan loading of 25 mL g−1 (25CXE) and 12.5 mL g−1 (12.5CXE), respectively. The results indicate that oatspelt xylan adsorption in the presence of the AbfB was more efficient at the lower xylan loading than at the higher xylan loading, indicating an optimal xylan loading for obtaining maximum AbfB aided oatspelt xylan adsorption. Since xylan adsorption is influenced by a decrease in the degree of xylan substitution, the increased efficiency in AbfB aided oatspelt xylan adsorption at the lower xylan loading (12.5 mL g−1) is proposed to be due to a higher efficiency in the removal of the arabinose side chains by the AbfB than at higher xylan loading (25 mL, g−1). The removal of the side chain is proposed to have provided an increased effective surface area available for adsorption onto the cotton lint fibres.
The results show that in the presence of the AbfB about 50% of the available arabinose was released at the xylan loading of 12.5 mL g−1 compared to 30% obtained at xylan loading of 25 mL g−1. In addition, a corresponding precipitation efficiency of 42% of the oatspelt xylan occurred at xylan loading of 12.5 mL g−1 in the absence of the cotton lint (12.5XYE) compared to the 25% precipitation that occurred at xylan loading of 25.0 mL g−1 (25XYE). However, at both xylan loadings, the xylan to enzyme ratio was 5:2 and the precipitation corresponded to the release of 60% of the available arabinose.
The removal of glucuronic acid side chains by the AguA provided BH, BM and P. patula xylan with higher binding power to the cotton lint than the removal of the arabinose side groups. The AguA was over 2.5 times more effective in increasing adsorption of BH, BM and P. patula xylan onto the cotton lint than AbfB. The combined application of the AbfB and AguA in a cocktail in BH, BM and P. patula xylan adsorption mixtures had an added advantage over the use of AbfB but not over AguA. Thus a higher degree of adsorption prevailed in the presence of the cocktail of AguA and AbfB than in the presence of AbfB but not in the presence of AguA.
The AbfB and AguA were more effective in increasing adsorption of xylan from wood sources than grasses. Furthermore, the effect of AbfB and AguA was higher on xylan substituted with a single type of side than when substituted with both arabinose and glucuronic acid side chains. E. grandis was noted as the preferred xylan source, although among the grass sources, bamboo would be the preferred choice over bagasse.
The presence of AbfB and AguA in bagasse (BH), H2O2 bleached bagasse (BB), bamboo (BM) and P. patula (P) xylan adsorption mixtures altered the xylan properties in a manner that allowed introduction of varying hydration properties and conformations or hydrogen bonding on the cotton lint fibre surface. The specific xylan weight gain of the cotton lint was the highest for cotton lint that was treated with xylan in the presence of AguA. The increase in the specific xylan weight gain could not be matched with the actual weight of the xylan that adsorbed on the cotton lint. The xylan structure is held together by the presence of moisture, which is known to increase with high content of arabinose side groups. Therefore, the higher xylan specific weight gain in the presence of AguA, rather than AbfB, is proposed to be associated with the higher arabinose content of the AguA treated xylan that adsorbed onto the cotton lint. The AbfB removal of the arabinose groups is proposed to have reduced the hydration capacity of the adsorbed xylan. Furthermore, the solid state 13C-(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 13C-(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
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010/03273 | Jun 2010 | ZA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2011/001250 | 6/7/2011 | WO | 00 | 5/17/2013 |
