Stability and recovery ofPenicillium funiculosum cellulase under use conditions

The stability ofPenicillium funiculosum cellulase has been investigated under the conditions used for cellulose hydrolysis. Fifty five percent of filter paper activity (FPA) was inactivated on incubation at 50°C for 24 h, whereas there was no loss in endoglucanase and β-glucosidase activity. The addition of 2% polyethylene glycol (PEG) during incubation stabilized the FPA. The influence of pH during fermentation on the thermal stability of the enzyme is discussed. The recovery of enzymes after hydrolysis of bagasse at 50°C was between 8 and 14%. Under the optimal conditions of elution, the recovery of enzyme was 35% (1). Increasing the enzyme to the substrate ratio fivefold and presence of PEG during hydrolysis resulted in 80, 83, and 95% recovery of β-glucosidase, FPA, and endoglucanase activity, respectively. Index Entries: Stability; recovery of cellulase P.funiculosum.     

Biorefining of Waste Paper Biomass: Increasing the Concentration of Glucose by Optimising Enzymatic Hydrolysis

Waste copier paper is a potential substrate for the production of glucose relevant for manufacture of platform chemicals and intermediates, being composed of 51 % glucan. The yield and concentration of glucose arising from the enzymatic saccharification of solid ink-free copier paper as cellulosic substrate was studied using a range of commercial cellulase preparations. The results show that in all cellulase preparations examined, maximum hydrolysis was only achieved with the addition of beta-glucosidase, despite its presence in the enzyme mixtures. With the use of Accellerase® (cellulase), high substrate loading decreased conversion yield. However, this was overcome if the enzyme was added between 12.5 and 20 FPU g substrate^?1. Furthermore, this reaction condition facilitated continual stirring and enabled sequential additions (up to 50 % w/v) of paper to be made to the hydrolysis reaction, degrading nearly all (99 %) of the cellulose fibres and increasing the final concentration of glucose whilst simultaneously making high substrate concentrations achievable. Under optimal conditions (50 °C, pH 5.0, 72 h), digestions facilitate the production of glucose to much improved concentrations of up to 1.33 mol l^?1.     

Isolation and properties of a thermostable endoglucanase from a thermophilic mutant strain ofTielavia terrestris

A heat-stable enzyme was isolated from the cellulase complex of a thermophilic strain of the micromyceteThielavia terrestris. The purified enzyme exhibited both endoglucanase and xylanase activities and had a mol mass of 69,000 Daltons and an isoelectric point of 6.4. When the cells were grown at 48°C, the initial activity of the purified enzyme using carboxymethylcellulose as a substrate was 150 nkat/mg and the Michaelis constant was 6.6 g/L. The heat stability of the enzyme was high, losing only 20% of the initial activity after a 6-h incubation at 65 °C. When cultures were grown on microcrystalline cellulose and xylose was added after 48 h of growth, endoglucanase and xylanase activities were more than doubled. Similar increases in these activities were observed by growing the cultures on straw.     

Characterization and performance of immobilized amylase and cellulase

The performance of cellulase and amylase immobilized on siliceous supports was investigated. Enzyme uptake onto the support depended on the enzyme source and immobilization conditions. For amylase, the uptake ranged between 20 and 60%, and for cellulase, 7–10%. Immobilized amylase performance was assessed by batch kinetics in 100–300 g/L of corn flour at 65°C. Depending on the substrate and enzyme loading, between 40 and 60% starch conversion was obtained. Immobilized amylase was more stable than soluble amylase. Enzyme samples were preincubated in a water bath at various temperatures, then tested for activity. At 105°C, soluble amylase lost ∼55% of its activity, compared with ∼30% loss for immobilized amylase. The performance of immobilized cellulase was evaluated from batch kinetics in 10 g/L of substrate (shredded wastepaper) at 55°C. Significant hydrolysis of the wastepaper was also observed, indicating that immobilization does not preclude access to and hydrolysis of insoluble cellulose.     

Effect of alkaline pretreatment on delignification of wheat straw

This study was conducted to analyse structural changes through scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) after alkaline pretreatment of wheat straw for optimum steaming period. During the study, 2 mm size of substrate was soaked in 2.5% NaOH for 1 h at room temperature and then autoclaved at 121°C for various steaming time (30, 60, 90 and 120 min). Results revealed that residence time of 90 min at 121°C has strong effect on substrate, achieving a maximum cellulose content of 83%, delignification of 81% and hemicellulose content of 10.5%. Further SEM and FTIR spectroscopy confirmed structural modification caused by alkaline pretreatment in substrate. Maximum saccharification yield of 52.93% was achieved with 0.5% enzyme concentration using 2.5% substrate concentration for 8 h of incubation at 50°C. This result indicates that the above-mentioned pretreatment conditions create accessible areas for enzymatic hydrolysis.     

Effect of ethanediamine on bio-polishing of cotton fabrics with cellulase

n-Propylamine and n-butylamine showed an inhibitory effect on cellulase A and cellulase B, while ethanediamine displayed a positive effect on both of these cellulases. Relative filter paper activity and relative CMCase activity of cellulase A and cellulase B measured at 50 °C were increased by 16.0 and 25.2 %, and 18.9 and 13.9 %, respectively, by the appearance of ethanediamine at a certain concentration. Also the addition of ethanediamine maintained the thermal stability of cellulase A and B at 65 °C to some extent and showed a stronger stabilizing effect on cellulase A than cellulase B. Third, the addition of ethanediamine within a certain concentration range enhanced the bio-polishing effect of cotton fabric enzymatic treatment at 50 °C to some extent, obtaining a close bio-polishing effect of cotton fabrics treated at 50 °C; the addition of ethanediamine saved some of the dose of cellulase A and B. Last but not least, the appearance of ethanediamine broadened the operating temperature of cellulase A to 65 °C, and it had a less positive effect on cellulase B at 65 °C.     

To understand the superior hydrolytic activity after polymorphic conversion from cellulose I to II from the adsorption behaviors of enzymes

The role of the cellulose ultrastructure on the relationship between cellulase binding and activity is not clear yet. In this article, a quartz crystal microbalance with dissipation (QCM-D) was employed to monitor the interactions between a given cellulase and the cellulose substrates with varied polymorphs of pure cellulose I and II and the intermediate state (I/II). Initially, cellulose nanocrystals (CNCs) with polymorphs of cellulose I, I/II and II were prepared and spin-coated on QCM sensors. The cellulose substrates’ crystallinity degree was examined by XRD, and morphology was detected by AFM. Then, a commercial cellulase from Trichoderma reesei was used to test the adsorption and hydrolysis of cellulose substrates with polymorphs of I, I/II and II, respectively. The results revealed that in the enzyme adsorption and desorption process at a temperature of 15 °C, CNC-II had the lowest adsorption capacity with a total adsorption mass of 179 ng cm^?2 but the highest reversible binding ratio of 33.7%; for comparison, the values were 235 ng cm^?2 versus 25.6% and 207 ng cm^?2 versus 26.9% for CNC-I and -I/II, respectively. And the conformation of adlayers on CNC-I, -I/II and -II derived from the QCM data became softer and softer in turn. On the other hand, CNC-II exhibited the best enzymatic hydrolytic ability among three substrates when enzymatic hydrolysis experiments were conducted at 45 °C. The results indicated that polymorphic conversion from I to II changes the affinity between the enzyme and cellulose surface; CNC-II has the lowest affinity to the enzyme, but the softer conformation of the adsorbed enzyme layer, and the more reversible adsorption may facilitate its hydrolytic activity. This article gives a perspective from the adsorption dynamics and conformation of the adsorbed enzyme layer, helping to understand the superior hydrolytic activity of cellulose with polymorph II. Thus, there is a potential of polymorphic conversion in the reduction of enzyme dosage and cost in the enzymatic hydrolysis process.     

Hydrolysis of Ammonia-pretreated Sugar Cane Bagasse with Cellulase, β-Glucosidase, and Hemicellulase Preparations

Sugar cane bagasse consists of hemicellulose (24%) and cellulose (38%), and bioconversion of both fractions to ethanol should be considered for a viable process. We have evaluated the hydrolysis of pretreated bagasse with combinations of cellulase, β-glucosidase, and hemicellulase. Ground bagasse was pretreated either by the AFEX process (2NH₃: 1 biomass, 100 °C, 30 min) or with NH₄OH (0.5 g NH₄OH of a 28% [v/v] per gram dry biomass; 160 °C, 60 min), and composition analysis showed that the glucan and xylan fractions remained largely intact. The enzyme activities of four commercial xylanase preparations and supernatants of four laboratory-grown fungi were determined and evaluated for their ability to boost xylan hydrolysis when added to cellulase and β-glucosidase (10 filter paper units [FPU]: 20 cellobiase units [CBU]/g glucan). At 1% glucan loading, the commercial enzyme preparations (added at 10% or 50% levels of total protein in the enzyme preparations) boosted xylan and glucan hydrolysis in both pretreated bagasse samples. Xylanase addition at 10% protein level also improved hydrolysis of xylan and glucan fractions up to 10% glucan loading (28% solids loading). Significant xylanase activity in enzyme cocktails appears to be required for improving hydrolysis of both glucan and xylan fractions of ammonia pretreated sugar cane bagasse.     

Crude Cellulase from Oil Palm Empty Fruit Bunch by Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2 for Fermentable Sugars Production

Cellulase is an enzyme that converts the polymer structure of polysaccharides into fermentable sugars. The high market demand for this enzyme together with the variety of applications in the industry has brought the research on cellulase into focus. In this study, crude cellulase was produced from oil palm empty fruit bunch (OPEFB) pretreated with 2 % NaOH with autoclave, which was composed of 59.7 % cellulose, 21.6 % hemicellulose, and 12.3 % lignin using Trichoderma asperellum UPM1 and Aspergillus fumigatus UPM2. Approximately 0.8 U/ml of FPase, 24.7 U/ml of CMCase and 5.0 U/ml of β-glucosidase were produced by T. asperellum UPM1 at a temperature of 35 °C and at an initial pH of 7.0. A 1.7 U/ml of FPase, 24.2 U/ml of CMCase, and 1.1 U/ml of β-glucosidase were produced by A. fumigatus UPM2 at a temperature of 45 °C and at initial pH of 6.0. The crude cellulase was best produced at 1 % of substrate concentration for both T. asperellum UPM1 and A. fumigatus UPM2. The hydrolysis percentage of pretreated OPEFB using 5 % of crude cellulase concentration from T. asperellum UPM1 and A. fumigatus UPM2 were 3.33 % and 19.11 %, with the reducing sugars concentration of 1.47 and 8.63 g/l, respectively.     

Lactose-inducted production of a complete lignocellulolytic enzyme system by a novel bacterium <Emphasis Type="Italic">Bacillus</Emphasis> sp. BS-5 and its application for saccharification of alkali-pretreated corn cob

In this study, with combined carboxymethyl cellulose agar plate, xylan agar plate and filter paper hydrolysis assay, a novel cellulase and xylanase-producing strain identified as Bacillus sp. was isolated. Using lactose as the only carbon source, a complete and balanced lignocellulolytic enzyme system containing at least endoglucanase (9.6 U/ml), exoglucanase (0.8 U/ml), Fpase (1.4 U/ml), xylanase (3.8 U/ml) and β-glucosidase (1.2 U/ml) was produced. Interestingly, a zymogram of the crude culture supernatant displayed a multifunctional lignocellulolytic enzyme system including at least four bonds with both endoglucanase activity and xylanase activity at 21.2, 23.8, 28.9 and 31.2 kDa, respectively, indicating that these enzymes might be bifunctional. More gratifyingly, according to the binding affinity analysis and scanning electron microscopy, the crude enzyme complex produced by strain BS-5 was capable of hydrolyzing not only pure insoluble polysaccharides, but also agricultural residues such as corn cob. At 5% substrate concentration and 20 FPU/g enzyme loading, the reducing sugar was 350.8 mg/g of alkali-pretreated corn cob after 72 h enzymatic hydrolysis. These results suggested that this strain could be a good candidate for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail for the saccharification of lignocellulosic biomass.     

Stabilization of the Cellulase Enzyme Complex as Enzyme Nanoparticle

The native Celluclast BG cellulase enzyme complex consists of different enzymes which can also degrade great substrate molecules as native celluloses. This enzyme complex has been covered by a very thin, a few nanometers thick, polymer layer, in order to improve its stability. It has been proved that the polymer layer around the enzyme molecules does not hinder the digestion as great substrates as crystalline cellulose polymer. The stability of the prepared enzyme nanoparticles (PE) could significantly be increased comparing to that of the native one what was proved by results of the total cellulose activity measured. The pretreated enzyme complex holds its activity often a few magnitudes of orders longer in time than that of the native enzyme complex (enzyme without pretreatment). It retains its activity at least ten times longer than that of the native one, at a temperature range between 20 and 37?°C. The pretreated enzyme complex can have about 50?% of its original activity during 12?h of incubation at even 80?°C, while the native cellulase one totally lost it during 6?h incubation time. The activity of PE has not been significantly reduced even at extreme pH values, namely in the pH range of 1.5 to 12.     

Application of Endo-β-1,4,d-mannanase and Cellulase for the Release of Mannooligosaccharides from Steam-Pretreated Spent Coffee Ground

Spent coffee ground (SCG), a present waste stream from instant coffee production, represents a potential feedstock for mannooligosaccharides (MOS) production. MOS can be used in nutraceutical products for humans/animals or added to instant coffee, increasing process yield and improving product health properties. The SCG was evaluated for MOS production by steam pretreatment and enzymatic hydrolysis with a recombinant mannanase and a commercial cellulase cocktail (Acremonium, Bioshigen Co. Ltd, Japan). The mannanase was produced using a recombinant strain of Yarrowia lipolytica, used to produce and secrete endo-1,4-β,d-mannanase from Aspergillus aculeatus in bioreactor cultures. Endo-1,4-β,d-mannanase was produced with an activity of 183.5 U/mL and 0.23 mg protein/mL. The enzyme had an optimum temperature of 80 °C, and the activity in the supernatant was improved by 150 % by supplementation with 0.2 % sodium benzoate and 35 % sorbitol as a preservative and stabiliser, respectively. The steam pretreatment of SCG improved the enzymatic digestibility of SCG, thus reducing the required enzyme dosage for MOS release. Combined enzymatic hydrolysis of untreated or steam-pretreated (150, 190 and 200 °C for 10 min) SCG with mannanase and cellulase cocktail resulted in 36–57 % (based on mannan content) of MOS production with a degree of polymerization of up to 6. The untreated material required at least 1 % of both mannanase and cellulase loading. The optimum mannanase and cellulase loadings for pretreated SCG hydrolysis were between 0.3 and 1 and 0.4 and 0.8 %, respectively. Statistical analysis suggested additive effect between cellulase cocktail and mannanase on MOS release, with no indication of synergism observed.     

Efficient Production of γ-GABA Using Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A cellulase-producing bacterium, designated as strain AK9, was isolated from a hot spring of Tatta Pani, Azad Kashmir, Pakistan. The bacterium was identified as Bacillus amyloliquefaciens through 16S rRNA sequencing. Cellulase from strain AK9 was able to liberate glucose from soluble cellulose and carboxymethyl cellulose (CMC). Enzyme was purified through size exclusion chromatography and a single band of ～47 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified with recovery of 35.5%, 3.6-fold purity with specific activity of 31 U mg^?1. The purified cellulase retained its activity over a wide range of temperature (50–70 °C) and pH (3–7) with maximum stability at 60 °C and pH 5.0. The activity inhibited by ethylenediaminetetraacetic acid (EDTA), suggested that it was metalloenzyme. Diethyl pyrocarbonate (DEPC) and β-mercaptoethanol significantly inhibited cellulase activity that revealed the essentiality of histidine residues and disulfide bonds for its catalytic function. It was stable in non-ionic surfactants, in the presence of various metal ions, and in water-insoluble organic solvents. Approximately 9.1% of reducing sugar was released after enzymatic saccharification of DAP-pretreated agro-residue, compared to a very low percentage by autohydrolysis treatment. Hence, it is concluded that cellulase from B. amyloliquefaciens AK9 can potentially be used in bioconversion of lignocellulosic biomass to fermentable sugars.     

Protein Extraction and Enzymatic Hydrolysis of Ammonia-Treated Cassava Leaves (Manihot esculenta Crantz)

In the present work, cassava leaves were treated with 0.5 kg ammonia/kg dry matter at 78 °C and 30% moisture content in a 2-kg reactor. Protein extraction was carried out with a calcium hydroxide solution (pH 10) for 30 min at several temperatures (30 °C, 45 °C, 60 °C, 75 °C, and 90 °C) and solid/liquid ratios (1:10 and 1:15) in a thermostatized bath. Soluble protein content of the extracts was determined by Lowry’s method. Dry substrate concentrations of 5%, 7.5%, and 10% and enzyme doses of 2 and 5 IU/g dry matter were used for the enzymatic hydrolysis in an orbital incubator at 50 °C and 100 rpm. Both cellulase and xylanase were used. Reducing sugars produced were determined with the dinitrosalicylic acid method. The highest protein extraction yield for the ammonia-treated leaves was 29.10%, which was 50% higher than with the untreated leaves (20%), and was obtained at 90 °C with a 1:10 solid/liquid ratio. The concentrate had a protein content of 36.35% and the amino acid profile was suitable for swine and poultry. The highest sugar yield was 54.72% with respect to theoretical and was obtained with 5% solids and an enzyme dose of 5 IU/g dry matter. This yield was 3.4 times higher than the yield of the untreated leaves (16.13%). These results indicate that cassava leaves have a great potential for animal feeding and ethanol production. Both protein extraction and sugar yields may be enhanced by optimizing the ammonia treatment.     

Chemical physics: The standing of a mature discipline

Background

The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex? Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities.

Results

Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross-linking. The V_max/K_m values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity.

Conclusion

A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes.     

Bioconversion potential of Trichoderma viride HN1 cellulase for a lignocellulosic biomass Saccharum spontaneum

The industrialisation of lignocellulose conversion is impeded by expensive cellulase enzymes required for saccharification in bioethanol production. Current research undertakes cellulase production from pretreated Saccharum spontaneum through Trichoderma viride HN1 under submerged fermentation conditions. Pretreatment of substrate with 2% NaOH resulted in 88% delignification. Maximum cellulase production (2603 ± 16.39 U/mL/min carboxymethyl cellulase and 1393 ± 25.55 U/mL/min FPase) was achieved at 6% substrate at pH 5.0, with 5% inoculum, incubated at 35°C for 120 h of fermentation period. Addition of surfactant, Tween 80 and metal ion Mn⁺², significantly enhanced cellulase yield. This study accounts proficient cellulase yield through process optimisation by exploiting cheaper substrate to escalate their commercial endeavour.     

Production and Optimization of Physicochemical Parameters of Cellulase Using Untreated Orange Waste by Newly Isolated <Emphasis Type="Italic">Emericella variecolor</Emphasis> NS3

Cellulase enzymes have versatile industrial applications. This study was directed towards the isolation, production, and characterization of cellulase enzyme system. Among the five isolated fungal cultures, Emericella variecolor NS3 showed maximum cellulase production using untreated orange peel waste as substrate using solid-state fermentation (SSF). Maximum enzyme production of 31 IU/gds (per gram of dry substrate) was noticed at 6.0 g concentration of orange peel. Further, 50 °C was recorded as the optimum temperature for cellulase activity and the thermal stability for 240 min was observed at this temperature. In addition, the crude enzyme was stable at pH 5.0 and held its complete relative activity in presence of Mn²⁺ and Fe³⁺. This study explored the production of crude enzyme system using biological waste with future potential for research and industrial applications.     

β-d-Galactosidase from Enterobacter cloacae: Production and Some Physicochemical Properties

A bacterial strain isolated from soil and identified as Enterobacter cloacae had been found to be capable of producing both intra and extracellular β-d-galactosidase.The intracellular enzyme was thermostable and its optimum temperature, pH and time for enzyme—substrate reaction were found to be 50?°C, 9.0 and 5 min respectively, using ONPG as substrate. The maximum β-galactosidase production in shake flask was achieved at 30?°C, pH 7.0, incubation time 72 h using 50 ml medium in 250 ml Erlenmeyer flask. Only Mg²⁺ stimulated the activity of enzyme. Cetyl trimethyl ammonium bromide showed stimulatory effect on catalytic activity of the enzyme whereas EDTA inhibited enzyme activity. The enzyme retained its activity upto 55?°C after incubating at that temperature for 1 h.The maximum activity of crude intracellular enzyme was 14.35 IU/mg of protein. The K _m and V _max values of β-galactosidase using ONPG as substrate at 50?°C were 2.805 mM and 37.45?×?10^?3?mM/min/mg, respectively.     

Highly Thermostable and pH-Stable Cellulases from Aspergillus niger NS-2: Properties and Application for Cellulose Hydrolysis

Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9?±?20.1 U/g, FPase 101.1?±?3.5 U/g and β-glucosidase 99?±?4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0–9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92–98 %.     

Application of Pigeon Pea (Cajanus cajan) Stalks as Raw Material for Xylooligosaccharides Production

Pigeon pea (Cajanus cajan) is a perennial plant widely cultivated in tropical and subtropical regions of many countries. The present studies aimed to produce xylooligosaccharides (XOS) from pigeon pea stalks in order to do value addition. The chemical analysis of stalks revealed 18.33?±?1.40 % hemicelluloses in addition to cellulose, protein, and lignin. Sodium hydroxide coupled with steam application enabled almost 96 % recovery of original xylan, present in the pigeon pea stalks. Enzymatic hydrolysis of xylan led to production of XOS namely, xylobiose and xylotriose. Response surface model indicated a maximum yield of xylobiose (0.502 mg/ml) under the hydrolysis conditions of pH 4.91, temperature at 48.11 °C, enzyme dose at 11.01 U, and incubation time at 15.65 h. The ideal conditions for higher xylotriose yield (0.204 mg/ml) were pH 5.44, temperature at 39.29 °C, enzyme dose at 3.23 U, and incubation time at 15.26 h. The present investigation was successful in assessing the prospect of using pigeon pea stalks as a raw material for xylan extraction vis-à-vis XOS production.