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Production of xylooligosaccharides by xylanase from Pichia stipitis based on xylan preparation from triploid Populas tomentosa
Bioresource Technology 102 (2011) 7171–7176
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Production of xylooligosaccharides by xylanase from Pichia stipitis based on xylan preparation from triploid Populas tomentosa Haiyan Yang a, Kun Wang a,b, Xianliang Song a, Feng Xu a,⇑ a b
Institute of Biomass Chemistry and Technology, College of Material Science and Technology, Beijing Forestry University, Beijing, 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, 510640, China
a r t i c l e
i n f o
Article history: Received 28 January 2011 Received in revised form 31 March 2011 Accepted 31 March 2011 Available online 5 April 2011 Keywords: Enzymatic hydrolysis Pichia stipitis xylanase Triploid Populas tomentosa Xylan preparation Xylooligosaccharides (XOS)
a b s t r a c t Xylooligosaccharides (XOS) with DP 2–4 are important synbiotics used as food ingredients based on its prebiotic characteristics. In this work, the production of XOS from lignocellulosic material was performed by combined chemical-enzymatic methods. Xylan was prepared from triploid Populas tomentosa, and bioconverted into XOS by crude xylanase solution obtained from Pichia stipitis. The effects of reaction time, temperature, enzyme dosage, and pH value on the production of XOS were fully evaluated. Under the optimal condition (25 U g 1 substrate, pH 5.4 and 50 °C), 36.8% of the xylan preparation was converted to XOS, equivalent to 3.95 mg/mL of the hydrolyzate. Xylobiose, xylotriose and xylotetrose were analyzed to be the main products of the enzymatic hydrolyzate, which together accounted for over 95% of the released oligosaccharides. Meanwhile, the effect of sonication pretreatment on the conversion efficiency of the xylan preparation was also investigated. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Increased scientific and commercial interest in the development of viable biorefining strategies converting renewable raw materials into conventional or new chemical products is driven by environmental concerns, depleting petroleum resources, and public awareness (Koutinas et al., 2007). The bioconversion of lignocellulosic biomass to biofuel or biomaterials through a ‘carbohydrate platform’ is of current interest. Lignocellulosic feedstock is mainly composed of three biopolymers (i.e., cellulose, hemicelluloses, and lignin), and its effective utilization necessitates the development of cost-effective process technologies. Marketable high-value products should be one of the major goals of optimizing a biomassto-ethanol process. Xylan is the most common polysaccharide hemicelluloses and is considered to be the second most abundant biopolymer in the plant kingdom. Recently, the manufacture of Xylooligosaccharides (XOS) from lignocellulosic materials as novel sweeteners and functional foods attracts growing interest. Oligosaccharides are generally defined as saccharides containing between 2 and 10 sugar moieties (Wang et al., 2009). As one of the non-digestible oligosaccharides (NDOs), XOS exhibit many excellent functional properties, including non-toxicity, non-metabolism by human digestive system, promotion of bowel function, calcium absorption, lipid metabolism, and growth of beneficial intestinal bacteria, and ⇑ Corresponding author. Tel./fax: +86 10 6233 6972. E-mail address: [email protected] (F. Xu). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.110
reduction of colon cancer by forming short-chain fatty acids. Besides, the modified XOS could be employed as thermoplastic biomaterials for water-soluble films, coatings and capsules. In last decade, the demand of commercial XOS products dramatically increased in Japan, North America and Europe for promoting health by supporting the improvement of the diet (Vázquez et al., 2000). An in vitro evaluation of commercial prebiotic oligosaccharides concluded that XOS increased the number Bifidobacteria, with comparative advantage respect to other oligosaccharides (Rycroft et al., 2001). Loo et al. (1999) reported that for food-related applications, the preferred DP range of XOS is 2–4. In 2005, Zhang et al. (2005) prepared xylobiose and xylotriose from XOS by gel permeation chromatography, and found that xylotriose gave the best effect on the proliferation of Bifidobacterium adolescent. The production of XOS was carried out in two stages: fractionation of xylan and following acid or enzymatic hydrolysis. Because xylan is stable in alkaline media, it can be obtained by dissolving in caustic liquors and then precipitating with organic solvents. Then enzymatic hydrolysis was extensively employed to degrade xylan to produce XOS, due to less undesirable by-products and specialized equipments. During this bioconversion process, the branch structure of natural xylan requires the coordination of several hydrolases to breakdown. 1,4-b-D-xylanase xylanohydrolase (known as endoxylanases) hydrolyzes 1,4-b-D-xylosidic bonds within the b-(1,4)-linked D-xylosyl backbone of xylan randomly and liberate b-anomeric XOS (Belkacemi and Hamoudi, 2003). Meanwhile, enzyme complexes with low exo-xylanase and/or b-xylosidase activity are desired, in order to avoid the production
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of xylose (Vázquez et al., 2000). According to the previous literatures, the enzymatic hydrolysis efficiency is closely related to the substrates structure and environmental issues, such as enzyme specificity, pH value, incubation time and temperature (Aachary and Prapulla, 2009; Akpinar et al., 2009). In present study, we optimized the enzymatic hydrolysis condition of xylan preparation to produce low-DP XOS using endoxylanase from Pichia Stipitis. Moreover, sonication could significantly affect the molecular properties of the water-soluble xylan. The influence on the following enzymatic hydrolysis after sonicating in different media was also discussed.
methods (Aachary and Prapulla, 2008). Briefly, 50 mg xylan preparation was dissolved in 1.0 mL 50 mM sodium acetate buffer (pH = 4.8), containing 0.5 mL crude enzyme solution. After incubation at 50 °C for 20 min, the liberated reducing sugars were determined by 3,5-Dinitrosalicylic acid (DNS) method (Miller, 1959). One unit of xylanase activity was defined as the amount of enzyme required to produce 1 lmol xylose per minute. The xylanase obtained by submerged fermentation of P. stipitis strain contained 10 U mL 1 of endo-xylanase. All enzyme assays were done in duplicate. 2.3. Production of XOS from xylan
2. Methods 2.1. Xylan preparation and characterization The chips of triploid Populas tomentosa Carr., 3 years old, were obtained from Shandong Province, China. After being ground to pass a 60–80 mesh sieve, the powder was dewaxed with ethanol/ toluene (1:2, v/v) for 8 h, and then totally delignified by sodium chlorite under the acidic condition. The xylan preparation used for P. Stipitis culture and XOS production were obtained according to the method described elsewhere with little modification (Sun et al., 1998). 3% KOH solution was chosen with acceptable yield and minor structure changes. The monosaccharide and uronic acid components of the xylan preparation were determined by highperformance anion exchange chromatography (HPAEC) system. The sugars and uronic acid were liberated by hydrolysis with 6% H2SO4 at 105 °C for 2.5 h. The hydrolyzate was diluted 50-folds, filtrated and then injected into the HPAEC system equipped with a CarbopacTM PA-20 column (4 250 mm, Dionex). The average molecular weight (Mw) was determined by gel permeation chromatography (GPC) performed on an Aglient 1200 Series system equipped with refractive index detector (RID). A PL aquagel-OH mix column (300 7.5 mm, Polymer Laboratories Ltd.) was used at a flow rate of 0.5 mL min 1 and a column temperature of 30 °C. Date was calibrated with PL pullulan polysaccharide standards (Mw 738, 12200, 100000, and 1600000, Polymer Laboratories Ltd.). The eluent was 0.02 M NaCl in 0.005 M sodium phosphate buffer, pH 7.5. Xylan was dissolved in the eluent at a concentration of 1%. Liquid 1H and 13C NMR spectra were recorded on a Bruker AVIII 400 MHz spectrometer operating in the FT mode at 100.6 MHz. The sample (20 mg for 1H, 80 mg for 13C) was dissolved in 1 mL D2O. A 30° pulse flipping angle, a 9.2 ls pulse width, a 1.36 s acquisition time, and 2 s relation delay time were used. HSQC spectrum was also obtained on the same instrument after 128 scans with 20 mg sample dissolved in 1 mL D2O. The spectral widths were 2200 and 15400 Hz for the 1H- and 13C- dimensions, respectively. The number of collected complex points was 1024 for 1H-dimension with a relaxation delay of 1.5 s. The number of scans was 128, and 256 time increments were always recorded in 13 C-dimension. The 1JCH used was 146 Hz. Prior to Fourier transformation, the data matrixes were zero filled up to 1024 points in the 13 C-dimension.
For evaluating the effect of various reaction parameters on the XOS production, the enzymatic reaction was carried out in a shaking water bath using the crude endo-xylanase solution produced above. The effects of temperature (40–50 °C), enzyme concentration (15–45 U g 1 substrate), reaction time (2–24 h) and pH value (3.6–6.0) on the yield of XOS were investigated. The samples were withdrawn periodically followed by deactivation of the enzymes in boiling water for 5 min and centrifugation at 10, 000g for 10 min. The released monosaccharides were analyzed by HPAEC system. All enzymatic hydrolysis experiments were performed in duplicate, and average values and corresponding derivations are illustrated in Fig. 1. 2.4. Ultrasonic treatment An ultrasound cleaning bath with 200 W ultrasonic power was used in this study. 0.1 g xylan preparation was suspended and sonicated in 5 mL water, 1% and 2% NaOH at 25 °C for 30 min, respectively. Then, the solution was instantly adjusted to the optimal pH value by addition of acetic acid, and hydrolyzed under the optimized condition. 2.5. Analysis of XOS According to a previous research, HPAEC analysis was found to be robust and additionally the precision, which was suited for the mapping and characterization of oligosaccharides (Grey et al., 2009). In this work, XOS were estimated by the Dionex ICS 3000 system equipped with an AS50 autosampler. A CarbopacTM PA-100 column (4 250 mm, Dionex) with guard PA-100 column (4 50 mm, Dionex) was used at a flow rate of 0.4 mL min 1 and the column temperature was 30 °C. XOS was separated in 0–80 mM NaAc gradient in a 100 mM NaOH isocratic (carbonate free and purged with nitrogen) for 15 min, followed by a 80–300 mM NaAc gradient in 100 mM NaOH for 10 min, then a 10 min elution with 100 mM NaOH was used to re-equilibrate the column before the next injection. The formed XOS was quantified by comparing the peak area with that of the authoritative xylose, xylobiose, xylotriose, xylotetrose, xylopentose and xylohexose (Aldrich-Chemical Co. Ltd.), and were expressed as mg L 1 of hydrolyzate. Error bars show the standard deviation of triplicate measurements.
2.2. Microorganism xylanase production and enzyme assay
3. Results and discussion
Xylanase was produced by P. stipitis strain under submerged fermentation condition using the xylan preparation at the concentration of 5% (wt) as the inducer. Cultivation was performed at 30 °C for 6 days. The crude enzymatic solution was recovered by filtration with filter paper and stored in 4 °C refrigerator without further purification. The xylanase activity was assayed by using the oat spelt xylan as the substrate based on previously reported
3.1. Physicochemical properties of the xylan preparation The structure of the XOS was partly depended on the xylan-structure of the original feedstock. Feruloylated arabinoxylooligosaccharides can be collected from microwave-assisted autohydrolysis maize bran heteroxylan (Rose and Inglett, 2010). Kabel et al. (2002) released XOS substituted with different side
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Fig. 1. Effect of reaction parameters on the yield of XOS, (a) temperature, (b) enzyme concentration, and (c) pH.
chains (arabinose, glucuronic acid and acetyl) from four xylan rich materials by hydrothermally treatment. In addition, Puls and Schuseil (1993) reported that the nature, number and distribution of substituents along the xylan main-chain have a remarkable impact on the types of sugar fragments released by the random action of xylanase. The monosaccharide composition of the xylan preparation was obtained by the analysis of acid hydrolyzate, xylose (75.8%) and glucuronic acid (18.5%) were determined as the main components; meanwhile, rhamnose (1.2%), arabinose (1.2%), galactose (2.3%), and glucose (1.1%) were detected in minor amounts. This result indicated that the main structure of this alkali-soluble xylan preparation was glucuronoxylan, which was a typical hemicelluloses existed in hardwood (Ebringerová et al., 2005). Moreover, the average molecular weight (MW = 42400 g mol 1, Table 1) of the xylan preparation was estimated by GPC in aqueous medium, and deduced the degree of polymerization (DP) under the given condition was more than 200. Furthermore, the low polydispersity value (1.4) illuminated the relatively homogeneous structure of the isolated xylan preparation. The properties of the xylan preparation were closely characterized by employing FT-IR (Supporting materials Fig. S1) and NMR
spectroscopy (Supporting materials Figs. S2 and S3). As shown in the FT-IR spectrum, a board stretching intense characteristic peak is shown at 3424 cm 1 for the hydroxyl groups, and a prominent CAH stretching band is observed at 2926 cm 1. In the fingerprint region (1000–1700 cm 1), many sharp and discrete absorption bands due to the various functional groups are observed. The distinct absorbance peak at 1617 cm 1 is probably due to the absorbed water in the xylan preparation, and the peak at 1411 cm 1 is resulted from uronic acids (Marchessault and Liang, 1962). Three absorption bands at 1114, 1088 and 1045 cm 1 are attributed to the C-O stretching. The sharp peak at 896 cm 1 is the characteristic of b-linkage (Pandey, 1999). In the 1H NMR spectrum of the xylan preparation (Supporting materials Fig. S2), the chemical shifts at 4.42 and 5.24 ppm are assigned to the b-anomeric proton of xylose and a-anomeric proton of glucuronic acid, respectively. Signals appear at 3.27, 3.50, 3.74 and 4.06/ 3.36 ppm corresponding to the protons of b-D-xylose. The 4-Omethyl group of the glucuronic acid is evidenced by the sharp singlet at d 3.42 ppm. The marked signals in the 13C NMR spectrum (Supporting materials Fig. S3) at 102.17 (C-1), 73.15 (C-2), 74.61 (C-3), 76.08 (C-4) and 63.32 (C-5) ppm confirm the 1,4-b-glycosidic
Table 1 The yield of xylose and xylooligosaccharides, and the average molecular weight of the hydrolyzate before and after the 14 h enzymatic hydrolysis under the optimum condition. Without Sonication
Xylose Xylobiose Xylotriose Xylotetrose Xylopentose Xylohexose Yieldb(%) Mw A b
Water
1% NaOH
2% NaOH
0
14
0
14
0
14
0
14
53.1 18 9.5 9.6 5.1 4.8 1.1 42400
0.2 27.5 39.1 31.7 1.1 0.4 36.8 13400
4.7 4.4 26.7 31 17.4 15.9 0.7 39300
0.2 30.5 32.9 33.9 1.6 0.9 37.3 14100
NDa 20.1 19.7 29.5 15.4 15.3 0.5 34700
ND 28.7 34 35.9 1 0.4 43.8 15100
2.7 41 15.6 26.3 14.3 ND 0.7 32900
0.2 27.1 36.4 35.3 0.8 0.2 38.9 15500
Not detectable. The yield was expressed as weight (%) based on xylose content of substrate.
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linkages of the xylan backbone. Signals observed at 97.58 (C-1), 71.65 (C-2), 82.56 (C-4), 72.28 (C-5), 176.95 (C-6) and 59.70 (C-OMe) ppm are contributed to the 4-O-methyl-D-glucuronic acid residues. The obtained chemical shifts in the NMR spectra were in accord with the values already published for hardwood 4-O-methylglucuronoxylan (Teleman et al., 2000). The HSQC spectrum was also obtained and assigned to provide more information and confirm the main structure (Supporting materials Fig. S4). Based on the above results, the xylan preparation from triploid P. tomentosa consists of a substituted b-(1 ? 4)-linked-D-xylopranosy backbone, with 4-O-methyl-a-D-glucuronic acid decorated as side-chain at approximately every 5.7 xylose residues (mole ratio). Minor amounts of rhamnose, arabinose, galactose and glucose were released mainly from the side chains of the main xylan backbone, and minimum amounts were liberated from the non-xylan hemicellulosic polymers. It could be expected that this xylan preparation is potentially an appropriate starting material for the production of XOS. 3.2. Effect of reaction parameters on the production of XOS As we know, the activity of enzyme is variously in different substrates and medium conditions. The study herein described the effect of different reaction parameters on the production of XOS. The time course combined with temperature study had been carried out for 24 h in a shaking water bath, and samples were withdrawn from 2–24 h with designed intervals. Enzymatic hydrolysis was performed by mixing 0.15 mL crude xylanase solution from P. stipitis with 0.1 g xylan preparation in 5 mL 50 mM sodium acetate buffer (pH 4.8) and in the temperature range of 40–50 °C. The yield of XOS as function of hydrolysis time at 40, 48 and 50 °C, respectively, are shown in Fig. 1a (Supporting materials Fig. S5a). The yield of XOS exhibited a near-linear increase in the first 10 h, and increased slightly between 10 and 14 h, and leveled off beyond 14 h hydrolysis. This phenomenon was quite consistent with the assays reported by Ihsanawati et al., 2005 and Boraston et al., 2002. They suggested that the end-products in the hydrolyzate could compete with the substrates for binding to both of the ancillary and catalytic modules
of the enzyme, consequently reduce the enzyme activities and hydrolysis rate. Besides, increasing incubation temperature from 40 to 50 °C slightly improved the yield of XOS, indicating the positive impact of temperature on the enzymatic activities. In addition, a slightly lower yield of XOS at 50 °C than 48 °C after 24 h incubation was observed, which was probably due to the weak thermal stability of the xylanase. Díaz et al. (2004) suggested that mesophilic xylanase was no so active or stable at higher temperature, the activity of the xylanase decreased 80–90% as the incubation temperature increased to 60 °C. However, the proper increment of reaction temperature could improve the initial enzymatic hydrolysis rate. Thereby, 14 h was considered as the optimal hydrolysis time for maximum XOS production and 50 °C was selected as the optimum temperature for the following experiments. The effect of varying enzyme loading on the weight basis (15–35 U g 1 substrate) was evaluated and is shown in Fig. 1b. The hydrolysis rate was increased with increasing xylanase dosage; however, the yield of XOS did not exhibit a corresponding change as increasing the enzyme dosage from 25 to 35 U g 1 substrate. This result can be attributed to the potential problems of end-product inhibition, which were more significant at lower enzyme loadings than that at larger loadings of these biocatalysts (Belkacemi and Hamoudi, 2003). Boraston et al. (2002) also dictated that the binding ability of enzyme protein to xylan chain would reach a plateau at a certain enzyme/xylan ratio. Based on above data, 25 U g 1 substrate was selected as the sufficient enzyme dosage. In addition, initial pH value of the buffer also had effect on the xylanase activity. Yan et al. (2009) suggested that the activity of xylanase from Streptomyces matensis was stable over the range of pH 4.5–8. The high activity could remain at pH range of 5.5–8.0 with the maximum at pH 7.0. The selection of optimal pH value was taken out in the range of 3.6–6.0, and the curves of XOS yield versus reaction time in pH 6.0, 5.4, 4.8, 3.9 and 3.6 are shown in Fig. 1c, respectively. (Other curves are illustrated in the Supporting materials Fig. S5b). Apparently, no distinct changes were observed between pH 4.0–6.0, and the maximum yield of XOS was obtained at pH 5.4. However, the yield of
Fig. 2. Yield and relative concentration of XOS obtained by enzymatic hydrolysis under the optimum condition, and the molecular weight of the hydrolysate during reaction.
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XOS dropped 1.5–2 times under the acidic condition (pH < 4). The loss of enzyme activity was probably attributed to the ionization of the general base catalyst under the acidic condition (Tull and Withers, 1994). Based on above results, enzyme loading at 25 U g 1 substrate, pH 5.4 and 50 °C was selected as the optimal condition for XOS production. Fig. 2 shows the yield and relative concentration of XOS, and the Mw of hydrolyzate during the enzymatic hydrolysis process under the optimal condition. The hydrolysis rate was significantly increased in the first 2 h, and the final conversion 36.8% of xylan preparation was obtained after 14 h enzymatic hydrolysis. The Mw of hydrolyzate showed totally opposite trends, and obviously decreased from 42400 to 13400 g mol 1. These phenomena indicated that the b-1,4-endoxylanase cleaved the internal glycosidic linkages of the xylan backbone, resulting in the decreased DP of the substrate and large amounts of XOS released in the incunabular solution (Squina et al., 2009; Yan et al., 2009). In despite of prolonging incubation time the smaller molecules could not be further degraded probably due to the loss of enzyme activity. Besides, the aggregated portion of xylan limited the penetration of enzyme and remained intact (Puls and Schuseil, 1993). In present study, most of xylopentose and xylohexose were consumed and yielded xylobiose, xylotriose and xylotetrose as the major products in the hydrolyzate. Similarly, it was found that the xylanase of family 8 was more active on the xylan substrate with longer chain, principally producing xylotriose and xylotetrose (Collins et al., 2002). Compared with other substrates (Aachary and Prapulla, 2009; Akpinar et al., 2009; Squina et al., 2009; Yan et al., 2009), much more xylotetrose was obtained by hydrolysis of the xylan preparation from triploid P. tomentosa. It was confirmed that the original structural feature of the substrate was essential for the polymerization degree and structure of XOS products. The physiochemical properties analysis suggested that the xylan preparation from triploid P. tomentosa was punctuated with 4-O-methyl-a-D-glucuronic acid spaced every 5.7 xylose units. These branches interfered with the interaction between enzyme proteins and polysaccharides, leading to production of xylotetrose and some uronic acid containing oligomers. Besides, the previous research suggested that the degradation of xylan to xylose requires the synergistic action of b-xylosidase and b-1,4-endoxylanase (Ines et al., 2009). The trace amount of xylose in the hydrolyzate indicated the low activity of exo-xylanase and/or b-xylosidase in the P. stipitis xylanase.
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3.3. Effect of sonication on the enzymatic hydrolysis Sonication is usually applied to degrade polysaccharides from macromolecular to smaller molecular by mechanic wave energy and the reduction and oxidation reactions from cavitation (Luche, 1997). Ebringerová et al., 1997 sonicated the xylan in neutral aqueous medium at a high sonication power and/or a long irradiation time caused no significant in its sugar composition, primary structure and viscoelastic properties, whereas obviously affected the molecular properties of the water-soluble fraction. The solvent characteristics had a considerable effect on the ultrasonic degradation reactions (Mason and Lorimer, 1988). Chang and Yang (2006) comparably studied the sonication of xylan in acid and alkaline media and found that alkaline solution was better medium than acid solution for xylan sonication. In acidic condition, larger molecules of xylan were observed due to the aggregation of smaller size molecules. However, large polymer molecules were significantly degraded by ultrasonic wave in alkaline circumstance. Thereby, this technologic process was applied prior to the enzymatic hydrolysis to improve the XOS production in the present study. In this investigation, the medium employed in sonication pretreatment of the xylan preparation was water, 1% and 2% NaOH solution, and correspondingly, the Mw was obviously decreased from 42400 to 39300, 34700 and 32900 g mol 1 after sonicating at room temperature for 30 min (Table 1).The degradation of macromolecule was further confirmed by the shift to the smaller molecular region in the molecular weight distribution curves (Fig. 3). The ultrasonic wave provides the kinetic energy of svibration sufficient to break the bond of longer chains, and generates more homogeneous products. This result agreed with the previous reports on the dextran (Lorimer et al., 1995) and xylan (Chang and Yang, 2006) treated with or without sonication. It is interesting to note that the obtained solution contained relatively much lower amount of xylose, and the distribution of the components was obviously transferred from monosaccharide to oligosaccharides. Although the sonication process had a significant effect on larger molecules, it could lead the aggregation between smaller size molecules to form larger molecule products. Concerning the ultrasonic effect on the following enzymatic hydrolysis (the detailed data were listed in the Supporting materials Table S1–S3), no significant differences were observed in yield of XOS and Mw from sonicated-xylan in water. In comparison,
Fig. 3. Molecular weight distribution of the xylan preparation without and with sonicated in different media.
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the sonicated-xylan in alkaline solution released larger amounts of XOS during the enzymatic hydrolysis. It is probably due to the fact that the alkaline solution could increase the swelling ability of xylan and thus increase the hydrodynamic volume of xylan (Hromádková et al., 1999). It is worthy to note that sonication pretreatment in 1% NaOH was more efficient than 2% NaOH for improving the bioconversion of xylan to XOS, although the initial Mw of the starting material was slightly higher in the lower alkali concentration solution. The probable explanation could be the weakly self-association of proteins in strong ionic medium suggested by Plesinak et al., 1996 4. Conclusion On the basis of results obtained, the optimal conversion of xylan preparation into XOS was achieved with 25 U g 1 substrate and pH 5.4 at 50 °C. At this condition, 36.8% of the xylan was conversed to XOS equivalent to 3.95 mg mL 1 of XOS. When the substrate was pretreated with sonication, the conversion of XOS was increased to 43.8%. The main hydrolysis products were xylobiose, xylotriose and xylotetrose, which together accounted for over 95% of the total oligosaccharides. The results obtained in this work provide a possibility for taking full advantage of hemicelluloses conversion into high-valued products. Acknowledgements This work was supported by the grants from the State Forestry Administration (200804015), Natural Science Foundation of China (No. 30930073, 30710103906), China Ministry of Education (No. 1 1 1), and Ministry of Science and Technology (9732010CB732204). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.03.110. References Aachary, A.A., Prapulla, S.G., 2009. Value addition to corncob: Production and characterization of xylooligosaccharides from alkali pretreated ligninsaccharide complex using Aspergillus oryzae MTCC 5154. Bioresour. Technol. 100, 991–995. Aachary, A.A., Prapulla, S.G., 2008. Corncob-induced endo-1, 4-b-D-xylanase of Aspergillus oryzae MTCC 5154: Production and characterization of xylobiose from glucuronoxylan. J. Agric. Food. Chem. 56, 3981–3988. Akpinar, O., Erdogan, K., Bostanci, S., 2009. Enzymatic production of xylooligosaccharide from selected agricultural wastes. Food Bioprod. Process 87, 145–151. Belkacemi, K., Hamoudi, S., 2003. Enzymatic hydrolysis of dissolved corn stalk hemicelluloses: reaction kinetics and modeling. J. Chem. Technol. Biotechnol. 78, 802–808. Boraston, A.B., McLean, B.W., AnsonLi, G.C., Warren, R.A.J., Kilburn, D.G., 2002. Cooperative binding of triplicate carbohydrate-binding modules from a thermophilic xylanase. Mol. Microbiol. 43, 187–194. Chang, Y.H., Yang, J.C., 2006. Molecular mass distribution and degradation rate of xylan sonicated in acid and alkaline media. Food Hydrocolloid 26, 348–356. Collins, T., Meuwis, M.A., Stals, I., Claeyssens, M., Feller, G., Gerday, C., 2002. A novel family 8 xylanase, functioal and physicochemical characterization. J. Biol. Chem. 277, 35133–35139. Díaz, M., Rodriguez, S., Fernández-Abalos, J.M., Rivas, J.D.L., Ruiz-Arribas, A., Shnyrov, V.L., Santamaría, R.I., 2004. Single mutatuions of residues outside the active center of the xylanase Xysl from Streptomyces halstedii JM8 affect its activity. FEMS Microbiol. Lett. 240, 237–243.
Ebringerová, A., Hromádková, Z., Heinze, T., 1997. The effect of ultrasound on the structure and properties of the water-soluble corn hull heteroxylans. Ultrason. Sonochem. 4, 305–309. Ebringerová, A., Hromádková, Z., Heinze, T., 2005. Hemicelluose. Adv. Polym. Sci. 186, 1–67. Grey, C., Edebrink, P., Krook, M., Jacobsson, S.P., 2009. Development of a high performance anion exchange chromatography analysis for mapping of oligosaccharides. J. Chromatogr. B. 877, 1827–1832. Hromádková, Z., Kováciková, J., Ebringerová, A., 1999. Study of the classical and ultrasound-assisted extraction of the corn cob xylan. Ind. Crop. Prod. 9, 101– 109. Ihsanawati, Kumasaka, T., Kaneko, T., Morokuma, C., Yatsunami, R., Sato, T., Nakamura, S., Tanaka, N., 2005. Structural basis of the substrate subsite and the highly thermal stability of xylanase 10B from Thermotoga maritime MSB8. Proteins 61, 999–1009. Ines, M.A., Guerfali, M., Gargouri, A., Belghith, H., 2009. Production of xylooligosaccharides from argo-industrial residues using immobilized Talaromyces thermophilus xylanase. J. Mol. Catal. B: Enzymatic 59, 145–152. Kabel, M.A., Carvalheiro, F., Garrote, G., Avgerinos, E., Koukios, E., Parajó, J.C., Gírio, F.M., Schols, H.A., Voragen, A.G.J., 2002. Hydrothermally treated xylan rich byproducts yield different classes of xylo-oligosaccharides. Carbohydr. Polym. 50, 47–56. Koutinas, A.A., Wang, R.H., Webb, C., 2007. The biochemurgist-bioconversion of agricultural raw materials for chemical production. Biofuel. Bioprod. Bior. 1, 24– 38. Loo, J.V., Cummings, J., Delzenne, N., Englyst, H., Frank, A., Hopkin, M., Van Vliet, T., Van den Heuvel, E., 1999. Functional food properties of non-digestible oligosaccharides: a consensus report from the ENDO project (DGXII-AIRIICT94–1095). Brit. J. Nutr. 81, 121–132. Lorimer, J.P., Mason, T.J., Cuthbert, T.C., Brookfield, E.A., 1995. Effect of ultrasound on the degradation of aqueous native dextran. Ultrason. Sonochem. 2, S55–S57. Luche, J.L., 1997. A few questions on the sonochemistry of solutions. Ultrason. Sonochem. 4, 211–215. Marchessault, R.H., Liang, C.Y., 1962. The infrared spectra of crystalline polysaccharides. VIII. Xylans. J. Polym. Sci. 59, 357–378. Mason, T.J., Lorimer, J.P., 1988. Polymers. In: Mason, T.J., Lorimer, J.P. (Eds.), Sonochmistry: theory, Application and uses of Ultrasound in Chemistry. Ellis Horwood, London, pp. 99–138. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar analysis chemistry. Anal. Chem. 31, 426–428. Pandey, K.K., 1999. A study of chemical structure of soft and hardwood and wood polymers by FTIR spectroscopy. J. Appl. Polym. Sci. 71, 1975–1989. Plesinak, L.A., Wakarchuk, W.W., McIntosh, L.P., 1996. Secondary structure and NMR assignments of Bacillus circulans xylanase. Protein Sci. 5, 1118–1135. Puls, J., Schuseil, J., 1993. Chemistry of hemicelluloses: relationship between hemicellulose Structure and enzymes required for hydrolysis. In: Coughlan, M.P., Hazlewood, G.P. (Eds.), Hemicellulose and Hemicellulases. Porland Press, London, UK, pp. 1–27. Rose, D.J., Inglett, G.E., 2010. Production of feruloylated arabinoxylooligosaccharides from maize (Zea mays) bran by microwave-assisted autohydrolysis. Food Chem. 119, 1613–1618. Rycroft, C.E., Jones, M.R., Gibson, G.R., Rastall, R.A., 2001. A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. J. Appl. Microbiol. 91, 878–887. Squina, F.M., Mort, A.J., Decker, S.R., Prade, R.A., 2009. Xylan decomposition by Aspergillus clavatus endo-xylanase. Protein Expres. Purif. 68, 65–71. Sun, R.C., Fang, J.M., Goodwin, A., Lawther, J.M., Bolton, A.J., 1998. Fraction and characterization of polysaccharides from abaca fiber. Carbohydr. Polym. 37, 351–359. Teleman, A., Lundqvist, J., Tjerneld, F., Stalbrand, H., Dahlman, O., 2000. Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy. Carbohydr. Polym. 329, 807–815. Tull, D., Withers, S.G., 1994. Mechanisms of cellulase and xylanase-a detailed kinetic study of the exo-beta-1, 4-glycanase from Cellulomonas fimi. Biochem. 33, 6363–6370. Vázquez, M.J., Alonso, J.L., Domínguez, H., Parajó, J.C., 2000. Xylooligosaccharides: manufacture and applications. Trends Food Sci. Technol. 11, 387–393. Wang, J., Sun, B.G., Cao, Y.P., Tian, Y., Wang, C.T., 2009. Enzymatic preparation of wheat bran xylooligosaccharides and their stability during pasteurization and autoclave sterilization at low pH. Carbohydr. Polym. 77, 816–821. Yan, Q.J., Hao, S.S., Jiang, Z.Q., Zhai, Q., Chen, W.W., 2009. Properties of a xylanase from Streptomyces matensis being suitable for xylooligosaccharides production. J. Mol. Catal. B: Enzymatic 58, 72–77. Zhang, J.H., Xu, Y., Yong, Q., Bu, X.L., Yu, S.Y., 2005. Preparative separation of xylobiose and xylotriose and their in vitro application in fermentation by bifidobacteria. Chem. Ind. For. Prod. 25, 15–18.
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Production of xylooligosaccharides by xylanase from Pichia stipitis based on xylan preparation from triploid Populas tomentosa Haiyan Yang a, Kun Wang a,b, Xianliang Song a, Feng Xu a,⇑ a b
Institute of Biomass Chemistry and Technology, College of Material Science and Technology, Beijing Forestry University, Beijing, 100083, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, 510640, China
a r t i c l e
i n f o
Article history: Received 28 January 2011 Received in revised form 31 March 2011 Accepted 31 March 2011 Available online 5 April 2011 Keywords: Enzymatic hydrolysis Pichia stipitis xylanase Triploid Populas tomentosa Xylan preparation Xylooligosaccharides (XOS)
a b s t r a c t Xylooligosaccharides (XOS) with DP 2–4 are important synbiotics used as food ingredients based on its prebiotic characteristics. In this work, the production of XOS from lignocellulosic material was performed by combined chemical-enzymatic methods. Xylan was prepared from triploid Populas tomentosa, and bioconverted into XOS by crude xylanase solution obtained from Pichia stipitis. The effects of reaction time, temperature, enzyme dosage, and pH value on the production of XOS were fully evaluated. Under the optimal condition (25 U g 1 substrate, pH 5.4 and 50 °C), 36.8% of the xylan preparation was converted to XOS, equivalent to 3.95 mg/mL of the hydrolyzate. Xylobiose, xylotriose and xylotetrose were analyzed to be the main products of the enzymatic hydrolyzate, which together accounted for over 95% of the released oligosaccharides. Meanwhile, the effect of sonication pretreatment on the conversion efficiency of the xylan preparation was also investigated. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Increased scientific and commercial interest in the development of viable biorefining strategies converting renewable raw materials into conventional or new chemical products is driven by environmental concerns, depleting petroleum resources, and public awareness (Koutinas et al., 2007). The bioconversion of lignocellulosic biomass to biofuel or biomaterials through a ‘carbohydrate platform’ is of current interest. Lignocellulosic feedstock is mainly composed of three biopolymers (i.e., cellulose, hemicelluloses, and lignin), and its effective utilization necessitates the development of cost-effective process technologies. Marketable high-value products should be one of the major goals of optimizing a biomassto-ethanol process. Xylan is the most common polysaccharide hemicelluloses and is considered to be the second most abundant biopolymer in the plant kingdom. Recently, the manufacture of Xylooligosaccharides (XOS) from lignocellulosic materials as novel sweeteners and functional foods attracts growing interest. Oligosaccharides are generally defined as saccharides containing between 2 and 10 sugar moieties (Wang et al., 2009). As one of the non-digestible oligosaccharides (NDOs), XOS exhibit many excellent functional properties, including non-toxicity, non-metabolism by human digestive system, promotion of bowel function, calcium absorption, lipid metabolism, and growth of beneficial intestinal bacteria, and ⇑ Corresponding author. Tel./fax: +86 10 6233 6972. E-mail address: [email protected] (F. Xu). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.110
reduction of colon cancer by forming short-chain fatty acids. Besides, the modified XOS could be employed as thermoplastic biomaterials for water-soluble films, coatings and capsules. In last decade, the demand of commercial XOS products dramatically increased in Japan, North America and Europe for promoting health by supporting the improvement of the diet (Vázquez et al., 2000). An in vitro evaluation of commercial prebiotic oligosaccharides concluded that XOS increased the number Bifidobacteria, with comparative advantage respect to other oligosaccharides (Rycroft et al., 2001). Loo et al. (1999) reported that for food-related applications, the preferred DP range of XOS is 2–4. In 2005, Zhang et al. (2005) prepared xylobiose and xylotriose from XOS by gel permeation chromatography, and found that xylotriose gave the best effect on the proliferation of Bifidobacterium adolescent. The production of XOS was carried out in two stages: fractionation of xylan and following acid or enzymatic hydrolysis. Because xylan is stable in alkaline media, it can be obtained by dissolving in caustic liquors and then precipitating with organic solvents. Then enzymatic hydrolysis was extensively employed to degrade xylan to produce XOS, due to less undesirable by-products and specialized equipments. During this bioconversion process, the branch structure of natural xylan requires the coordination of several hydrolases to breakdown. 1,4-b-D-xylanase xylanohydrolase (known as endoxylanases) hydrolyzes 1,4-b-D-xylosidic bonds within the b-(1,4)-linked D-xylosyl backbone of xylan randomly and liberate b-anomeric XOS (Belkacemi and Hamoudi, 2003). Meanwhile, enzyme complexes with low exo-xylanase and/or b-xylosidase activity are desired, in order to avoid the production
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of xylose (Vázquez et al., 2000). According to the previous literatures, the enzymatic hydrolysis efficiency is closely related to the substrates structure and environmental issues, such as enzyme specificity, pH value, incubation time and temperature (Aachary and Prapulla, 2009; Akpinar et al., 2009). In present study, we optimized the enzymatic hydrolysis condition of xylan preparation to produce low-DP XOS using endoxylanase from Pichia Stipitis. Moreover, sonication could significantly affect the molecular properties of the water-soluble xylan. The influence on the following enzymatic hydrolysis after sonicating in different media was also discussed.
methods (Aachary and Prapulla, 2008). Briefly, 50 mg xylan preparation was dissolved in 1.0 mL 50 mM sodium acetate buffer (pH = 4.8), containing 0.5 mL crude enzyme solution. After incubation at 50 °C for 20 min, the liberated reducing sugars were determined by 3,5-Dinitrosalicylic acid (DNS) method (Miller, 1959). One unit of xylanase activity was defined as the amount of enzyme required to produce 1 lmol xylose per minute. The xylanase obtained by submerged fermentation of P. stipitis strain contained 10 U mL 1 of endo-xylanase. All enzyme assays were done in duplicate. 2.3. Production of XOS from xylan
2. Methods 2.1. Xylan preparation and characterization The chips of triploid Populas tomentosa Carr., 3 years old, were obtained from Shandong Province, China. After being ground to pass a 60–80 mesh sieve, the powder was dewaxed with ethanol/ toluene (1:2, v/v) for 8 h, and then totally delignified by sodium chlorite under the acidic condition. The xylan preparation used for P. Stipitis culture and XOS production were obtained according to the method described elsewhere with little modification (Sun et al., 1998). 3% KOH solution was chosen with acceptable yield and minor structure changes. The monosaccharide and uronic acid components of the xylan preparation were determined by highperformance anion exchange chromatography (HPAEC) system. The sugars and uronic acid were liberated by hydrolysis with 6% H2SO4 at 105 °C for 2.5 h. The hydrolyzate was diluted 50-folds, filtrated and then injected into the HPAEC system equipped with a CarbopacTM PA-20 column (4 250 mm, Dionex). The average molecular weight (Mw) was determined by gel permeation chromatography (GPC) performed on an Aglient 1200 Series system equipped with refractive index detector (RID). A PL aquagel-OH mix column (300 7.5 mm, Polymer Laboratories Ltd.) was used at a flow rate of 0.5 mL min 1 and a column temperature of 30 °C. Date was calibrated with PL pullulan polysaccharide standards (Mw 738, 12200, 100000, and 1600000, Polymer Laboratories Ltd.). The eluent was 0.02 M NaCl in 0.005 M sodium phosphate buffer, pH 7.5. Xylan was dissolved in the eluent at a concentration of 1%. Liquid 1H and 13C NMR spectra were recorded on a Bruker AVIII 400 MHz spectrometer operating in the FT mode at 100.6 MHz. The sample (20 mg for 1H, 80 mg for 13C) was dissolved in 1 mL D2O. A 30° pulse flipping angle, a 9.2 ls pulse width, a 1.36 s acquisition time, and 2 s relation delay time were used. HSQC spectrum was also obtained on the same instrument after 128 scans with 20 mg sample dissolved in 1 mL D2O. The spectral widths were 2200 and 15400 Hz for the 1H- and 13C- dimensions, respectively. The number of collected complex points was 1024 for 1H-dimension with a relaxation delay of 1.5 s. The number of scans was 128, and 256 time increments were always recorded in 13 C-dimension. The 1JCH used was 146 Hz. Prior to Fourier transformation, the data matrixes were zero filled up to 1024 points in the 13 C-dimension.
For evaluating the effect of various reaction parameters on the XOS production, the enzymatic reaction was carried out in a shaking water bath using the crude endo-xylanase solution produced above. The effects of temperature (40–50 °C), enzyme concentration (15–45 U g 1 substrate), reaction time (2–24 h) and pH value (3.6–6.0) on the yield of XOS were investigated. The samples were withdrawn periodically followed by deactivation of the enzymes in boiling water for 5 min and centrifugation at 10, 000g for 10 min. The released monosaccharides were analyzed by HPAEC system. All enzymatic hydrolysis experiments were performed in duplicate, and average values and corresponding derivations are illustrated in Fig. 1. 2.4. Ultrasonic treatment An ultrasound cleaning bath with 200 W ultrasonic power was used in this study. 0.1 g xylan preparation was suspended and sonicated in 5 mL water, 1% and 2% NaOH at 25 °C for 30 min, respectively. Then, the solution was instantly adjusted to the optimal pH value by addition of acetic acid, and hydrolyzed under the optimized condition. 2.5. Analysis of XOS According to a previous research, HPAEC analysis was found to be robust and additionally the precision, which was suited for the mapping and characterization of oligosaccharides (Grey et al., 2009). In this work, XOS were estimated by the Dionex ICS 3000 system equipped with an AS50 autosampler. A CarbopacTM PA-100 column (4 250 mm, Dionex) with guard PA-100 column (4 50 mm, Dionex) was used at a flow rate of 0.4 mL min 1 and the column temperature was 30 °C. XOS was separated in 0–80 mM NaAc gradient in a 100 mM NaOH isocratic (carbonate free and purged with nitrogen) for 15 min, followed by a 80–300 mM NaAc gradient in 100 mM NaOH for 10 min, then a 10 min elution with 100 mM NaOH was used to re-equilibrate the column before the next injection. The formed XOS was quantified by comparing the peak area with that of the authoritative xylose, xylobiose, xylotriose, xylotetrose, xylopentose and xylohexose (Aldrich-Chemical Co. Ltd.), and were expressed as mg L 1 of hydrolyzate. Error bars show the standard deviation of triplicate measurements.
2.2. Microorganism xylanase production and enzyme assay
3. Results and discussion
Xylanase was produced by P. stipitis strain under submerged fermentation condition using the xylan preparation at the concentration of 5% (wt) as the inducer. Cultivation was performed at 30 °C for 6 days. The crude enzymatic solution was recovered by filtration with filter paper and stored in 4 °C refrigerator without further purification. The xylanase activity was assayed by using the oat spelt xylan as the substrate based on previously reported
3.1. Physicochemical properties of the xylan preparation The structure of the XOS was partly depended on the xylan-structure of the original feedstock. Feruloylated arabinoxylooligosaccharides can be collected from microwave-assisted autohydrolysis maize bran heteroxylan (Rose and Inglett, 2010). Kabel et al. (2002) released XOS substituted with different side
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Fig. 1. Effect of reaction parameters on the yield of XOS, (a) temperature, (b) enzyme concentration, and (c) pH.
chains (arabinose, glucuronic acid and acetyl) from four xylan rich materials by hydrothermally treatment. In addition, Puls and Schuseil (1993) reported that the nature, number and distribution of substituents along the xylan main-chain have a remarkable impact on the types of sugar fragments released by the random action of xylanase. The monosaccharide composition of the xylan preparation was obtained by the analysis of acid hydrolyzate, xylose (75.8%) and glucuronic acid (18.5%) were determined as the main components; meanwhile, rhamnose (1.2%), arabinose (1.2%), galactose (2.3%), and glucose (1.1%) were detected in minor amounts. This result indicated that the main structure of this alkali-soluble xylan preparation was glucuronoxylan, which was a typical hemicelluloses existed in hardwood (Ebringerová et al., 2005). Moreover, the average molecular weight (MW = 42400 g mol 1, Table 1) of the xylan preparation was estimated by GPC in aqueous medium, and deduced the degree of polymerization (DP) under the given condition was more than 200. Furthermore, the low polydispersity value (1.4) illuminated the relatively homogeneous structure of the isolated xylan preparation. The properties of the xylan preparation were closely characterized by employing FT-IR (Supporting materials Fig. S1) and NMR
spectroscopy (Supporting materials Figs. S2 and S3). As shown in the FT-IR spectrum, a board stretching intense characteristic peak is shown at 3424 cm 1 for the hydroxyl groups, and a prominent CAH stretching band is observed at 2926 cm 1. In the fingerprint region (1000–1700 cm 1), many sharp and discrete absorption bands due to the various functional groups are observed. The distinct absorbance peak at 1617 cm 1 is probably due to the absorbed water in the xylan preparation, and the peak at 1411 cm 1 is resulted from uronic acids (Marchessault and Liang, 1962). Three absorption bands at 1114, 1088 and 1045 cm 1 are attributed to the C-O stretching. The sharp peak at 896 cm 1 is the characteristic of b-linkage (Pandey, 1999). In the 1H NMR spectrum of the xylan preparation (Supporting materials Fig. S2), the chemical shifts at 4.42 and 5.24 ppm are assigned to the b-anomeric proton of xylose and a-anomeric proton of glucuronic acid, respectively. Signals appear at 3.27, 3.50, 3.74 and 4.06/ 3.36 ppm corresponding to the protons of b-D-xylose. The 4-Omethyl group of the glucuronic acid is evidenced by the sharp singlet at d 3.42 ppm. The marked signals in the 13C NMR spectrum (Supporting materials Fig. S3) at 102.17 (C-1), 73.15 (C-2), 74.61 (C-3), 76.08 (C-4) and 63.32 (C-5) ppm confirm the 1,4-b-glycosidic
Table 1 The yield of xylose and xylooligosaccharides, and the average molecular weight of the hydrolyzate before and after the 14 h enzymatic hydrolysis under the optimum condition. Without Sonication
Xylose Xylobiose Xylotriose Xylotetrose Xylopentose Xylohexose Yieldb(%) Mw A b
Water
1% NaOH
2% NaOH
0
14
0
14
0
14
0
14
53.1 18 9.5 9.6 5.1 4.8 1.1 42400
0.2 27.5 39.1 31.7 1.1 0.4 36.8 13400
4.7 4.4 26.7 31 17.4 15.9 0.7 39300
0.2 30.5 32.9 33.9 1.6 0.9 37.3 14100
NDa 20.1 19.7 29.5 15.4 15.3 0.5 34700
ND 28.7 34 35.9 1 0.4 43.8 15100
2.7 41 15.6 26.3 14.3 ND 0.7 32900
0.2 27.1 36.4 35.3 0.8 0.2 38.9 15500
Not detectable. The yield was expressed as weight (%) based on xylose content of substrate.
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linkages of the xylan backbone. Signals observed at 97.58 (C-1), 71.65 (C-2), 82.56 (C-4), 72.28 (C-5), 176.95 (C-6) and 59.70 (C-OMe) ppm are contributed to the 4-O-methyl-D-glucuronic acid residues. The obtained chemical shifts in the NMR spectra were in accord with the values already published for hardwood 4-O-methylglucuronoxylan (Teleman et al., 2000). The HSQC spectrum was also obtained and assigned to provide more information and confirm the main structure (Supporting materials Fig. S4). Based on the above results, the xylan preparation from triploid P. tomentosa consists of a substituted b-(1 ? 4)-linked-D-xylopranosy backbone, with 4-O-methyl-a-D-glucuronic acid decorated as side-chain at approximately every 5.7 xylose residues (mole ratio). Minor amounts of rhamnose, arabinose, galactose and glucose were released mainly from the side chains of the main xylan backbone, and minimum amounts were liberated from the non-xylan hemicellulosic polymers. It could be expected that this xylan preparation is potentially an appropriate starting material for the production of XOS. 3.2. Effect of reaction parameters on the production of XOS As we know, the activity of enzyme is variously in different substrates and medium conditions. The study herein described the effect of different reaction parameters on the production of XOS. The time course combined with temperature study had been carried out for 24 h in a shaking water bath, and samples were withdrawn from 2–24 h with designed intervals. Enzymatic hydrolysis was performed by mixing 0.15 mL crude xylanase solution from P. stipitis with 0.1 g xylan preparation in 5 mL 50 mM sodium acetate buffer (pH 4.8) and in the temperature range of 40–50 °C. The yield of XOS as function of hydrolysis time at 40, 48 and 50 °C, respectively, are shown in Fig. 1a (Supporting materials Fig. S5a). The yield of XOS exhibited a near-linear increase in the first 10 h, and increased slightly between 10 and 14 h, and leveled off beyond 14 h hydrolysis. This phenomenon was quite consistent with the assays reported by Ihsanawati et al., 2005 and Boraston et al., 2002. They suggested that the end-products in the hydrolyzate could compete with the substrates for binding to both of the ancillary and catalytic modules
of the enzyme, consequently reduce the enzyme activities and hydrolysis rate. Besides, increasing incubation temperature from 40 to 50 °C slightly improved the yield of XOS, indicating the positive impact of temperature on the enzymatic activities. In addition, a slightly lower yield of XOS at 50 °C than 48 °C after 24 h incubation was observed, which was probably due to the weak thermal stability of the xylanase. Díaz et al. (2004) suggested that mesophilic xylanase was no so active or stable at higher temperature, the activity of the xylanase decreased 80–90% as the incubation temperature increased to 60 °C. However, the proper increment of reaction temperature could improve the initial enzymatic hydrolysis rate. Thereby, 14 h was considered as the optimal hydrolysis time for maximum XOS production and 50 °C was selected as the optimum temperature for the following experiments. The effect of varying enzyme loading on the weight basis (15–35 U g 1 substrate) was evaluated and is shown in Fig. 1b. The hydrolysis rate was increased with increasing xylanase dosage; however, the yield of XOS did not exhibit a corresponding change as increasing the enzyme dosage from 25 to 35 U g 1 substrate. This result can be attributed to the potential problems of end-product inhibition, which were more significant at lower enzyme loadings than that at larger loadings of these biocatalysts (Belkacemi and Hamoudi, 2003). Boraston et al. (2002) also dictated that the binding ability of enzyme protein to xylan chain would reach a plateau at a certain enzyme/xylan ratio. Based on above data, 25 U g 1 substrate was selected as the sufficient enzyme dosage. In addition, initial pH value of the buffer also had effect on the xylanase activity. Yan et al. (2009) suggested that the activity of xylanase from Streptomyces matensis was stable over the range of pH 4.5–8. The high activity could remain at pH range of 5.5–8.0 with the maximum at pH 7.0. The selection of optimal pH value was taken out in the range of 3.6–6.0, and the curves of XOS yield versus reaction time in pH 6.0, 5.4, 4.8, 3.9 and 3.6 are shown in Fig. 1c, respectively. (Other curves are illustrated in the Supporting materials Fig. S5b). Apparently, no distinct changes were observed between pH 4.0–6.0, and the maximum yield of XOS was obtained at pH 5.4. However, the yield of
Fig. 2. Yield and relative concentration of XOS obtained by enzymatic hydrolysis under the optimum condition, and the molecular weight of the hydrolysate during reaction.
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XOS dropped 1.5–2 times under the acidic condition (pH < 4). The loss of enzyme activity was probably attributed to the ionization of the general base catalyst under the acidic condition (Tull and Withers, 1994). Based on above results, enzyme loading at 25 U g 1 substrate, pH 5.4 and 50 °C was selected as the optimal condition for XOS production. Fig. 2 shows the yield and relative concentration of XOS, and the Mw of hydrolyzate during the enzymatic hydrolysis process under the optimal condition. The hydrolysis rate was significantly increased in the first 2 h, and the final conversion 36.8% of xylan preparation was obtained after 14 h enzymatic hydrolysis. The Mw of hydrolyzate showed totally opposite trends, and obviously decreased from 42400 to 13400 g mol 1. These phenomena indicated that the b-1,4-endoxylanase cleaved the internal glycosidic linkages of the xylan backbone, resulting in the decreased DP of the substrate and large amounts of XOS released in the incunabular solution (Squina et al., 2009; Yan et al., 2009). In despite of prolonging incubation time the smaller molecules could not be further degraded probably due to the loss of enzyme activity. Besides, the aggregated portion of xylan limited the penetration of enzyme and remained intact (Puls and Schuseil, 1993). In present study, most of xylopentose and xylohexose were consumed and yielded xylobiose, xylotriose and xylotetrose as the major products in the hydrolyzate. Similarly, it was found that the xylanase of family 8 was more active on the xylan substrate with longer chain, principally producing xylotriose and xylotetrose (Collins et al., 2002). Compared with other substrates (Aachary and Prapulla, 2009; Akpinar et al., 2009; Squina et al., 2009; Yan et al., 2009), much more xylotetrose was obtained by hydrolysis of the xylan preparation from triploid P. tomentosa. It was confirmed that the original structural feature of the substrate was essential for the polymerization degree and structure of XOS products. The physiochemical properties analysis suggested that the xylan preparation from triploid P. tomentosa was punctuated with 4-O-methyl-a-D-glucuronic acid spaced every 5.7 xylose units. These branches interfered with the interaction between enzyme proteins and polysaccharides, leading to production of xylotetrose and some uronic acid containing oligomers. Besides, the previous research suggested that the degradation of xylan to xylose requires the synergistic action of b-xylosidase and b-1,4-endoxylanase (Ines et al., 2009). The trace amount of xylose in the hydrolyzate indicated the low activity of exo-xylanase and/or b-xylosidase in the P. stipitis xylanase.
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3.3. Effect of sonication on the enzymatic hydrolysis Sonication is usually applied to degrade polysaccharides from macromolecular to smaller molecular by mechanic wave energy and the reduction and oxidation reactions from cavitation (Luche, 1997). Ebringerová et al., 1997 sonicated the xylan in neutral aqueous medium at a high sonication power and/or a long irradiation time caused no significant in its sugar composition, primary structure and viscoelastic properties, whereas obviously affected the molecular properties of the water-soluble fraction. The solvent characteristics had a considerable effect on the ultrasonic degradation reactions (Mason and Lorimer, 1988). Chang and Yang (2006) comparably studied the sonication of xylan in acid and alkaline media and found that alkaline solution was better medium than acid solution for xylan sonication. In acidic condition, larger molecules of xylan were observed due to the aggregation of smaller size molecules. However, large polymer molecules were significantly degraded by ultrasonic wave in alkaline circumstance. Thereby, this technologic process was applied prior to the enzymatic hydrolysis to improve the XOS production in the present study. In this investigation, the medium employed in sonication pretreatment of the xylan preparation was water, 1% and 2% NaOH solution, and correspondingly, the Mw was obviously decreased from 42400 to 39300, 34700 and 32900 g mol 1 after sonicating at room temperature for 30 min (Table 1).The degradation of macromolecule was further confirmed by the shift to the smaller molecular region in the molecular weight distribution curves (Fig. 3). The ultrasonic wave provides the kinetic energy of svibration sufficient to break the bond of longer chains, and generates more homogeneous products. This result agreed with the previous reports on the dextran (Lorimer et al., 1995) and xylan (Chang and Yang, 2006) treated with or without sonication. It is interesting to note that the obtained solution contained relatively much lower amount of xylose, and the distribution of the components was obviously transferred from monosaccharide to oligosaccharides. Although the sonication process had a significant effect on larger molecules, it could lead the aggregation between smaller size molecules to form larger molecule products. Concerning the ultrasonic effect on the following enzymatic hydrolysis (the detailed data were listed in the Supporting materials Table S1–S3), no significant differences were observed in yield of XOS and Mw from sonicated-xylan in water. In comparison,
Fig. 3. Molecular weight distribution of the xylan preparation without and with sonicated in different media.
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the sonicated-xylan in alkaline solution released larger amounts of XOS during the enzymatic hydrolysis. It is probably due to the fact that the alkaline solution could increase the swelling ability of xylan and thus increase the hydrodynamic volume of xylan (Hromádková et al., 1999). It is worthy to note that sonication pretreatment in 1% NaOH was more efficient than 2% NaOH for improving the bioconversion of xylan to XOS, although the initial Mw of the starting material was slightly higher in the lower alkali concentration solution. The probable explanation could be the weakly self-association of proteins in strong ionic medium suggested by Plesinak et al., 1996 4. Conclusion On the basis of results obtained, the optimal conversion of xylan preparation into XOS was achieved with 25 U g 1 substrate and pH 5.4 at 50 °C. At this condition, 36.8% of the xylan was conversed to XOS equivalent to 3.95 mg mL 1 of XOS. When the substrate was pretreated with sonication, the conversion of XOS was increased to 43.8%. The main hydrolysis products were xylobiose, xylotriose and xylotetrose, which together accounted for over 95% of the total oligosaccharides. The results obtained in this work provide a possibility for taking full advantage of hemicelluloses conversion into high-valued products. Acknowledgements This work was supported by the grants from the State Forestry Administration (200804015), Natural Science Foundation of China (No. 30930073, 30710103906), China Ministry of Education (No. 1 1 1), and Ministry of Science and Technology (9732010CB732204). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.03.110. References Aachary, A.A., Prapulla, S.G., 2009. Value addition to corncob: Production and characterization of xylooligosaccharides from alkali pretreated ligninsaccharide complex using Aspergillus oryzae MTCC 5154. Bioresour. Technol. 100, 991–995. Aachary, A.A., Prapulla, S.G., 2008. Corncob-induced endo-1, 4-b-D-xylanase of Aspergillus oryzae MTCC 5154: Production and characterization of xylobiose from glucuronoxylan. J. Agric. Food. Chem. 56, 3981–3988. Akpinar, O., Erdogan, K., Bostanci, S., 2009. Enzymatic production of xylooligosaccharide from selected agricultural wastes. Food Bioprod. Process 87, 145–151. Belkacemi, K., Hamoudi, S., 2003. Enzymatic hydrolysis of dissolved corn stalk hemicelluloses: reaction kinetics and modeling. J. Chem. Technol. Biotechnol. 78, 802–808. Boraston, A.B., McLean, B.W., AnsonLi, G.C., Warren, R.A.J., Kilburn, D.G., 2002. Cooperative binding of triplicate carbohydrate-binding modules from a thermophilic xylanase. Mol. Microbiol. 43, 187–194. Chang, Y.H., Yang, J.C., 2006. Molecular mass distribution and degradation rate of xylan sonicated in acid and alkaline media. Food Hydrocolloid 26, 348–356. Collins, T., Meuwis, M.A., Stals, I., Claeyssens, M., Feller, G., Gerday, C., 2002. A novel family 8 xylanase, functioal and physicochemical characterization. J. Biol. Chem. 277, 35133–35139. Díaz, M., Rodriguez, S., Fernández-Abalos, J.M., Rivas, J.D.L., Ruiz-Arribas, A., Shnyrov, V.L., Santamaría, R.I., 2004. Single mutatuions of residues outside the active center of the xylanase Xysl from Streptomyces halstedii JM8 affect its activity. FEMS Microbiol. Lett. 240, 237–243.
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