Purification and characterization of a xylanase from
Transcription
Purification and characterization of a xylanase from
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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Process Biochemistry 43 (2008) 49–55 www.elsevier.com/locate/procbio Purification and characterization of a xylanase from Aspergillus carneus M34 and its potential use in photoprotectant preparation Hsin-Yu Fang a,c, Shin-Min Chang a, Cheng-Hang Lan b, Tony J. Fang c,* a Department of Food Nutrition, Chung-Hwa University of Medical Technology, Tainan Hsien, Taiwan, ROC Department of Occupational Safety and Health, Chung-Hwa University of Medical Technology, Tainan Hsien, Taiwan, ROC c Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, ROC b Received 31 May 2007; received in revised form 9 October 2007; accepted 18 October 2007 Abstract An extracellular xylanase was purified to homogeneity from a culture of Aspergillus carneus M34. In contrast to xylanases from other microorganisms, only a low-molecular weight xylanase, approximately 18.8 kDa with a pI value of 7.7–7.9, was purified in this investigation. The optimum temperature and pH of this purified xylanase activity were 50 8C and 6, respectively. The xylanase was more stable under alkaline conditions and retained more than 50% activity after 12 h incubation at pH 7–9. Considering of its characteristics and N-terminal sequence, this xylanase was concluded as a new one belonging to the group I of family 11 endoxylanases. In addition, hemicellulose of coba husk was selected as the substrate for xylooligosaccharide preparation owing to its higher specificity for this xylanase. Feruloyl xylooligosaccharides were separated and shown potential antioxidative capacity in a cell model of ultraviolet B (UVB)-induced oxidative damage to keratinocyte xb-2. # 2007 Elsevier Ltd. All rights reserved. Keywords: Aspergillus carneus; Xylanase purification; Characterization; Feruloyl xylooligosaccharide; Antioxidative capacity 1. Introduction Heteroxylan, the major component of hemicellulose, is an important biomass reservoir in the plant cell wall [1]. As hemicellulose is a recyclable material, xylanolytic enzymes from microorganisms have been intensively investigated over the past few decades. Xylanases (1,4-b-D-xylan xylanohydrolase; E.C 3.2.1.8), which cleave internal xylosidic linkages on the backbone and initiate the depolymerization of heteroxylan, have received much attention recently owing to their potential uses in different field applications, such as pulp bleaching [2], improving the nutritional properties of animal feedstuffs [3], and preparation of xylooligosaccharides [4]. Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is an abundant phenolic acid that is present in the plant cell wall and has an important role in linkage of hemicellulosic polysaccharides with other cell wall components [5]. Ferulic acid has several potential industrial and medical applications, such as a topical protective agent against UV-radiation-induced skin * Corresponding author. Tel.: +886 4 22861505; fax: +886 4 22876211. E-mail address: [email protected] (T.J. Fang). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.10.015 damage [6], an adjuvant in chemo- and radiotherapy to reduce side effects, and as an anti-inflammatory agent [7]. Moreover, ferulic acid-esterified xylooligosaccharides had greater antioxidative capacity than ferulic acid in inhibiting the peroxidation of low-density lipoprotein (LDL) [8]. This demonstrates that feruloyl xylooligosaccharides have potential use in atherosclerosis prevention or other applications involving its antioxidative capacity. Agricultural wastes containing hemicelluloses were globally generated. The application of agro-industrial residues in biotechnology bioprocesses not only provides an alternative substrate but also helps to solve some of the pollution problems caused by their accumulation [9]. Based on the regard, bioconversion of agricultural wastes to feruloyl xylooligosaccharides preparation by microbial xylanases has considerable promise. However, xylanases from various microorganisms have different physicochemical properties, modes of action, and substrate specificity [10]. Therefore, characterization of the physicochemical properties of xylanase and identification of the particular substrate for this xylanase are necessary. Xylanases produced from various microorganisms have been reported [11]. Filamentous fungi such as Aspergillus spp. and Trichoderma spp. are of particular interest, because they Author's personal copy 50 H.-Y. Fang et al. / Process Biochemistry 43 (2008) 49–55 can excrete higher levels of xylanase than yeast and bacteria [12]. Aspergillus carneus M34, originally isolated by our laboratory from soil and identified by the Bioresources Collection and Research Center (BCRC, Taiwan), is a xylanase- and phytase-producing strain. Its xylanase had broad pH stability and showed higher specificity for agricultural waste of coba husk and corn cob compared with commercial xylan [13]. To fully understand the industrial potential of xylanase from this strain, purification and characterization of the extracellular xylanase were attempted. In addition, the availability of feruloyl oligosaccharides from agricultural wastes owing to the actions of purified xylanase and their antioxidative capacity were evaluated in this work using the skin cell model of UVB-induced oxidative damage. albumin (BSA) as a standard. Proteins in the column effluents were monitored by measuring the absorbance at 280 nm. 2.4. SDS-PAGE, zymogram and pI Denaturing sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) with a 12% gel was performed according to the method of Laemmli [15]. The gel was stained with Coomassie blue R250 or silver stain. Zymograms were obtained using 12% acrylamide gels that contained 0.5% birch wood xylan. After electrophoresis, the gel was soaked with 1% Triton-X100 and washed with 20 mM dithiothreitol in 20 mM citrate buffer (pH 4.8). The gel was stained for xylanase activity by 0.1% Congo red solution for 30 min at room temperature and then washed with 1 M NaCl. Clear bands that contrast with the background were visualized. The isoelectric point (pI) was estimated by two-dimensional electrophoresis. In the first dimension, protein was separated by native charge using a 3–10 IPG strip (Bio-Rad) equipped with a protein IEF system (BioRad). Separation in the second dimension was by molecule weight using 12% SDS-PAGE as described above. 2. Materials and methods 2.5. Effect of pH and temperature on enzyme activity and stability 2.1. Preparation of the crude enzyme Aspergillus carneus M34 was maintained at 28 8C on PD (potato dextrose) agar. Conidia were inoculated in 50 ml medium supplemented with 1% oatspelt xylan in 250-ml Erlenmeyer flasks. The pH was adjusted to pH 5.2 with 2N HCl. The cultures were incubated at 35 8C, 112 rpm for 3 days. Cultured media were centrifuged at 4 8C, 10,000 g for 30 min, and the supernatants were then filtered through a 0.45-mm membrane (ADVANTEC MFS, Inc. USA). The resulting filtrate was treated as crude enzyme for purification. 2.2. Xylanase purification All purification steps were carried out at 4 8C, unless otherwise specified. The crude enzyme solutions were precipitated by 20–50% ammonium sulfate. After centrifugation (12,000 g, 60 min, 4 8C), the pellets were dissolved in 20 mM Tris–HCl buffer (pH 8.0) and then ultra-filtrated (Amicon, YM 10) using a stir cell. The concentrated samples were slowly loaded (0.5 ml/min) onto a DEAE-Sepharose CL-6B (2.6 cm 10 cm) column that was pre-equilibrated with the same buffer. The unbound proteins were washed with the same buffer and then eluted with NaCl (0–0.4 M) in the same buffer by a stepwise gradient at a flow rate of 1 ml/min. The fractions with xylanase activity were pooled. Further purification was conducted by Sephacryl S-200 column (1.6 cm 80 cm) that was pre-equilibrated with 20 mM Tris–HCl buffer pH 8.0. After elution with the same buffer containing 150 mM NaCl at a flow rate of 0.5 ml/min, a pure xylanase sample was obtained. The purified xylanase was stored at 4 8C for further characterization experiments. The effect of temperature on the xylanase activity was determined by performing a standard assay at 10 8C intervals from 30 8C to 80 8C. The effect of pH was evaluated under standard assay conditions with 1% birch wood xylan dissolved in different 20 mM pH buffers, including glycine–HCl buffer, acetate buffer, phosphate buffer, Tris–HCl buffer, and glycine–NaOH buffer with pH values of 3–4, 4–6, 6–8, 8–9, and 10, respectively [16]. In a thermal inactivation test, the enzyme solution was separately incubated at 50 8C, 55 8C, and 60 8C and then withdrawn at set times, before the residual enzyme activity was measured as described above. In the pH stability test, the enzyme was placed in the above buffers with different pH levels and incubated at room temperature for 12 h, and the residual enzyme activity was then assayed as described above. All of these assays were repeated three times and results are expressed as relative percentages compared with the highest value. 2.6. Substrate specificity Agricultural waste, including rice straw, rice bran, wheat bran, corn cob, coba husk, and sugarcane bagasse, was collected from local farms or traditional markets in Taiwan, Republic of China. Hemicelluloses from agricultural waste were prepared according to the method of Chen and Anderson [17]. Soluble commercial xylan (beech wood xylan, birch wood xylan, and oat-spelt xylan purchase from Sigma Chemical Co., St. Louis, MO) was prepared according to the method of Ryan et al. [18]. Commercial xylan and agricultural hemicelluloses at 1% concentration were prepared and used as substrates for the enzyme assay using standard assay conditions with 0.5 U purified xylanase (2.04 mg in citrate buffer, pH 4.8). Data were expressed as relative percentages compared with the highest level for commercial xylan. 2.3. Enzyme activity and protein assay Xylanase activity was routinely assayed by measuring the reducing sugars that were released after incubation properly diluted enzyme solution with 1% birch wood xylan in 0.2 M citrate buffer pH 4.8 at 50 8C for 15 min. The amounts of reducing sugars were determined by the dinitrosalicylic acid (DNS) method using xylose (Sigma) as a standard [14]. One unit (U) of xylanase activity was defined as the amount of enzyme that released 1 mmole reducing sugar equivalent to xylose per minute under the standard assay conditions. a-L-Arabinofuranosidase, b-xylosidase and b-galactosidase activities of the purified xylanase were measured using 0.5 ml 2 mM 4-nitrophenyl a-L-arabinofuranoside, p-nitrophenyl b-D-xylopyranoside and p-nitrophenyl b-D-galactopyranoside, respectively (Sigma, St. Louis, MO, USA) in 20 mM acetate buffer (pH 5) at 50 8C for 10 min. The reaction was then stopped with 1 ml 0.2 M Na2CO3 and the amount of p-nitrophenol released was quantified by the absorbance of 408 nm using a spectrophotometer (HelIOS, Spectronic Unicam, Germany). One unit of enzyme activity was defined as the activity producing 1 mmole of p-nitrophenol per minute. Protein quantification was determined using the bicinchoninic acid (BCA) protein assay kit (Sigma) with bovine serum 2.7. Analysis of hydrolyzed products Hydrolysis of 1% beech wood xylan, corn cob and coba husk hemicelluloses were performed by incubating the substrates with purified enzyme (0.5 U) under the standard assay conditions (50 8C, pH 4.8). Aliquots were collected at different time points and products were analyzed by thin-layer chromatography (TLC). Thin-layer chromatography was developed using ethyl acetate: acetic acid: formic acid: H2O (9:3:1:1, v/v/v/v) as the mobile phase and xylose, xylobiose and xylotriose (Wako) as standards. The hydrolysis products were detected by spraying with a 1:1 (v/v) mixture of 0.2% (w/v) orcinol in sulfuric acid/methanol (10:90, v/v) after developed. 2.8. Degree of coba husk hydrolysis Coba husk hemicellulose solution at 1% concentration (in 20 mM sodium acetate buffer, pH 6) with different enzyme activities (1–40 U/100 mg) were incubated at 50 8C for 30 min. After inactivation (10 min at 100 8C), the Author's personal copy H.-Y. Fang et al. / Process Biochemistry 43 (2008) 49–55 51 Table 1 Summary of the purification of xylanase from Aspergillus carneus M34 Purification step Total protein (mg) Total activity (U) Specific activity (U/mg) Purification (fold) Yield (%) Culture filtrate Ammonium sulfate DEAE-Sepharose Sephacryl S-200HR 184.11 29.1 9.7 3.19 3043.2 2492.0 1875.9 784.5 16.53 85.64 193.4 245.94 1 5.2 11.70 14.88 100 81.9 61.6 25.8 hydrolyzate was centrifuged (10,000 g for 30 min) and filtrated using a 0.45 mm membrane. The amounts of reducing sugars were analyzed by the DNS method and the degree of solubilization was expressed as the percentage of reducing sugars released from the substrate. 2.9. Enzymatic hydrolyzate preparation and feruloyl oligosaccharides fractionation Hydrolysis of coba husk hemicellulose (5 g) in 20 mM sodium acetate buffer (500 ml, pH 6) was performed using purified xylanase (1200 U) for 30 min at 50 8C with constant stirring. After inactivation (10 min at 100 8C), the hydrolyzate was centrifuged (at 10,000 g for 60 min) and filtrated through a 0.45-mm membrane. The supernatant solution was lyophilized and re-suspended in distilled water. The concentrated solution was applied to an open column (40 cm 2.5 cm diameter) packed with Amberlite XAD-2 (previously washed with methanol and water). Elution was successively performed with 2 volumes of distilled water, 50% (v/v) methanol/water and methanol. The absorbance of the eluent was monitored at 320 nm to detect feruloyl oligosaccharides. The fraction with the highest absorbance was concentrated and lyophilized. Feruloyl oligosaccharides were identified by paper chromatography using filter paper (No. 50, ADVANTEC) and the descending method with nbutanol/acetic acid/water (12:3:5) as the mobile phase. The separated feruloyl oligosaccharides were located by UV radiation (before and after exposure to NH3), and the spots were visualized using an oxalate/aniline reagent (two volumes of 2% aniline in ethanol and three volumes of 2.5% oxalic acid) by heating in an oven at 105 8C for 10–20 min [19]. 2.10. Mouse keratinocyte cultures Keratinocytes xb-2 (BCRC 60546) were cultured in complete growth medium (cMEM) supplemented with 10% fetal bovine serum (ICN Biomedical), 10 mg/ml insulin, 10 mg/ml hydrocortisone, 100 units/ml penicillin, 100 mg/ml streptomycin, 0.05 mg/ml fungizone, 10 ng/ml epidermal growth factor (EGF), and bovine pituitary extract (all from Sigma Co.) in a humidified atmosphere with CO2 (5% v/v) at 37 8C. Keratinocytes xb-2 were seeded in 6well plates at a density of 105 cells/ml and grown near to confluence for 4 days. color change produced is directly proportionate to the number of lyzed cells. The level of extracellular LDH was used for evaluation of the cytotoxicity of test compounds. For further measurement of cytotoxicity, the intracellular LDH levels were also measured and used as indicators of cell viability. After 24 h, cells were collected and washed with PBS. After sonication and centrifugation at 800 g for 10 min, the level of intracellular LDH was measured. The mean OD490 of the untreated control tissues was set to represent 100% viability, and results were expressed as percentages of the untreated control. All assays were repeated three times. 3. Results 3.1. Purification, molecule weight, pI and N-terminal sequence of xylanase An extracellular xylanase was purified from the culture filtrate of A. carneus M34 grown on oat-spelt xylan. A summary of a representative purification protocol is shown in Table 1. In purification, xylanase activity was found in the unbound eluent after ion-exchange chromatography (pH 8). There was a minimum peak observed in the 0.2 M NaCl elution step (Fig. 1). This peak is likely to be due to unspecific binding owing to its low specific activity. The unbound portion with xylanase activity were pooled and concentrated for further purification. After gel filtration, only a single protein peak that coincided with the peak of enzyme activity was obtained. This protocol overall afforded a 14.88-fold purification of the xylanase from the culture supernatant with a yield of 25.8% of the activity and 1.73% retention of total protein. This purified enzyme seemed to be homogeneous because it migrated as a single band on SDS-PAGE. The molecular weight of this purified xylanase was estimated to be 2.11. UVB source and treatment UVB irradiation was performed with UVB fluorescent lamps (MODEL UVM-57, UVP Inc., USA) that emit 280–320 nm UV radiation, with a peak at 313 nm. Light irradiance was determined using a UV radiometer (USB 4000, Ocean Optic Inc., USA). Prior to UVB radiation, vitamin C (Sigma) and feruloyl oligosaccharides were separately exposed to keratinocyte cells for 10 min, washed and covered with the vehicle (phosphate buffered saline (PBS) pH 7.0), and then irradiated with 120 mJ/cm2 for 45 s, or not irradiated as a control. After UVB irradiation, the cells were replaced in fresh cMEM and incubated at 37 8C for specified time intervals. 2.12. Cytotoxicity and cell viability Extracellular and intracellular lactate dehydrogenase (LDH) activities were measured using the In Vitro Toxicology Assay Kit for Lactate Dehydrogenase (Sigma). It is based on the reduction of NAD by LDH and measurement of the resulting color changes owing to the conversion of tetrazolium salt into the formazan product. The optical measurement of the Fig. 1. Elution profile of xylanase activity (^) and absorbance at 280 nm (&) in a DEAE-Sepharose CL-6B column equilibrated with 20 mM Tris–HCl buffer (pH 8.0) and then eluted with NaCl (0–0.4 M) in the same buffer by a stepwise gradient. Author's personal copy 52 H.-Y. Fang et al. / Process Biochemistry 43 (2008) 49–55 Table 2 Relative activities of purified xylanase towards different substrates Fig. 2. (A) SDS-PAGE profiles with silver stain of different stages of xylanase purification. S: protein marker, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 37 kDa, 50 kDa, 75 kDa, 100 kDa, 150 kDa and 250 kDa; C: crude extract; A: ammonium sulfate precipitation and ultrafiltration; D: after DEAE-Sepharose; G: after gel filtration. (B) Coomassie brilliant blue staining and zymogram analysis of purified xylanase. 18.8 kDa by SDS-PAGE (Fig. 2A). Zymogram revealed the presence of a zone of hydrolysis that corresponded with the Coomassie stained band of purified xylanase (Fig. 2B). Its purity was further confirmed by 2D electrophoresis, and the isoelectric point (pI) of this enzyme was estimated to be 7.7– 7.9. The first 10 N-terminal residues of this xylanase were identified as NH2-S-T-P-S-S-T-G-W-Y-N by N-terminal amino acid sequencing (Fig. 3). Substrate (1%, w/v) Relative activity (%) Beech wood xylan Soluble beech wood xylan Birch wood xylan Soluble birch wood xylan Oat-spelt xylan Soluble oat-spelt xylan Carboxylmethyl cellulose Cellulose Avicel Corn starch Dextrin Corn cob Coba husk Rice straw Sugarcane baggase Rice bran Wheat bran Peanut shell p-Nitrophenyl b-D-xylopyranoside p-Nitrophenyl b-D-glucopyranoside 4-Nitrophenyl a-L-arabinofuranoside Polygalacturonate 100 116.4 4.4 88.3 1.3 94.9 1.2 50.1 3.6 71.9 4.8 N.D N.D N.D N.D N.D 73.4 2.0 64.5 1.0 50.3 0.5 44.4 1.2 26.1 1.4 32.8 0.2 36.4 1.9 N.D N.D N.D N.D 3.2. Influence of pH and temperature on activity and stability Studies on the effects of temperature and pH on purified xylanase showed that the optimum pH was 6 and that the xylanase had high stability under alkaline conditions (pH 7–10) after 1 h incubation. Moreover, more than 50% activity was maintained after 12 h incubation at pH 7–9 (Fig. 4A and B). Fig. 3. N-terminal sequence of Aspergillus carneus M34 xylanase and other homologous fungal xylanases. Fig. 4. Optimal pH and pH stability test of A. carneus M34 xylanase. (A) the optimum pH of xylanase was measured under standard assay conditions (50 8C, 15 min) in each buffer (20 mM). The buffers used were glycine–HCl buffer (pH 3–4), acetate buffer (pH 4–6), phosphate buffer (pH 6–8), Tris–HCl buffer (pH 8–9), and glycine–NaOH buffer (pH 10). (B) The pH stability of xylanase was measured by incubation of purified xylanase in the buffers mentioned above for 1 h and 12 h at room temperature and then detected under standard assay conditions. Author's personal copy H.-Y. Fang et al. / Process Biochemistry 43 (2008) 49–55 53 Fig. 5. Optimal temperature and thermal stability test (A), and thermal inactivation test (B) of purified xylanase from A. carneus M34. The optimum temperature for this xylanase activity was 50 8C (Fig. 5A). Thermal stability testing showed that the activity reduced markedly when the temperature increased to more than 50 8C. The half-lives (t1/2) of the xylanase inactivation at 50 8C, 55 8C, and 60 8C were approximately >60 min, 7.5 min, and 4.5 min, respectively (Fig. 5B). still the main products and no xylose was observed, indicating that it is a typical endo-acting xylanase (Fig. 6). 3.4. Feruloyl xylooligosaccharides preparation from coba husk The hydrolytic property of this xylanase on various substrates was examined. This xylanase was specific to xylan-containing substrates and shown greater activities with the soluble fraction of commercial xylan (Table 2). In addition to commercial xylan, hemicelluloses of corn cob and coba husk were found to be excellent substrates for this enzyme degradation. The purified xylanase had no carboxymethylcellulase (CMCase), cellulase, a-L-arabinofuranosidase, b-galactosidase or b-xylosidase activities. These results indicate that this xylanase is a true xylanase. The mode of action of the purified xylanase was shown by TLC analysis of hydrolyzates of beech wood xylan. After 30 min, the enzyme liberated mainly xylotriose and xylotetraose. After prolonged incubation, the level of xylotetraose gradually reduced and the xylobiose level increased markedly. Xylobiose and xylotriose were the main products after 3 h digestion. After 24 h incubation, xylobiose and xylotriose were Measurement of the degree of coba husk hemicellulose hydrolysis with different xylanase concentrations (1–40 U/ 100 mg) showed the maximum solubilization of 33% to be obtained with 24 U/ml xylanase; further solubilization was not observed with increased xylanase levels. Based on this ratio, mini-scale preparation of enzymatic hydrolyzates from 5 g coba husk hemicellulose was prepared. The enzyme hydrolyzate was applied to an Amberlite XAD-2 column, which is a polymeric adsorbent binding aromatic compound [20]. Feruloyl xylooligosaccharides eluted in the fraction of 50% (v/v) methanol/water were monitored by their absorbance at 320 nm. The separated compounds fluoresced blue in ultraviolet radiation and their color changed green on exposure to NH3, indicating they were feruloyl oligosaccharides [21]. The sugar moieties of feruloyl oligosaccharides were reddish in color with oxalate/aniline staining that demonstrated they were pentoses and their DP (degree of polymerization) was greater than 2. Only about 10% (0.48 g) of the total feruloyl oligosaccharide level was obtained using this protocol. Fig. 6. Hydrolysis products analysis of purified xylanase toward beechwood xylan at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h and hemicellulose of coba husk (A, B) and corn cob (C, D) at 6 h and 24 h by thin-layer chromatography (TLC) analysis. S: standards. Fig. 7. Protective effects of vitamin C and feruloyl xylooligosaccharides (FX) on UVB-induced oxidation. ( ) Extracellular LDH activity measured 24 h after exposure to UVB; ( ) Intracellular LDH activity after 24 h incubation with no irradiation. 3.3. Substrate specificity and hydrolysis pattern Author's personal copy 54 H.-Y. Fang et al. / Process Biochemistry 43 (2008) 49–55 3.5. Cytotoxicity of feruloyl oligosaccharides The cytotoxicity of feruloyl xylooligosaccharides was evaluated by the extracellular LDH assay. Vitamin C and feruloyl xylooligosaccharides had similar effects on UVBirradiated cells, having no cytotoxic effects but a protective effect on the skin cells, as shown by the extracellular LDH activity results 24 h after UVB irradiation. Related 93.17 3.46% cellular LDH activities to control cells (without UVB irradiation) were observed at 24 h after exposed to UVB irradiation (Fig. 7). 4. Discussion Unlike other microorganisms that produce multiple xylanases [22], only one xylanase was purified from this strain by a four-step process. Xylanase activity was found in the unbound fraction after DEAE-Sepharose CL-6B anion exchange chromatography (pH 8) and was shown to have purity by SDS-PAGE identification. This procedure not only accelerates the enzyme purification but also reflects the fact that most extracellular proteins produced by this strain are acidic. In our previous work, the crude xylanase from this strain was found to be stable at pH 3–10 (12), but the pure xylanase was found to be stable at pH 7–10. This suggests acidic proteins produced from this strain should relate to stabilize the activity of crude xylanase under acid conditions. This purified enzyme had a lower molecular weight than most xylanases [23]. Apart from its low-molecular weight, this xylanase is similar to Aspergillus niger [24], which has a particularly alkaline pI. The xylanase in the present study, which is similar to the xylanase from alkalophilic thermophilic Bacillus sp. (NCIM 59), has a near neutral optimum pH but is stable under alkaline conditions. Similar to the Bacillus sp. xylanase (NCIM 59), this might lead to pH-induced structural changes and also affect the ionization of key residues in the active site (NCIM 59) [25]. The low-molecular weight and basic pI of this xylanase indicate that it may belong to the glycoside hydrolase family 11 [1]. BLAST analysis and alignment of its N-terminal sequence using the NCBI database found it to be highly homologous (more than 80% sequence identity) to the family 11 xylanases produced by Emericella nidulans (CAA90073), Aspergillus oryzae (BAB20794), Aspergillus niger (AAS46914) and Aspergillus cf. niger (AAS67299) (Fig. 3). Furthermore, this enzyme shows no CMCase, cellulase, a-L-arabinofuranosidase, b-galactosidase or b-xylosidase activities and there was no xylose produced in hydrolysis tests. These characteristics differ from those of other family 10 xylanases, which typically have lower substrate specificities (frequently having endoglucanase activity) [26]. Considering of its characteristics and N-terminal sequence similarity, xylanase produced by the newly isolated A. carneus M34 should be one of family 11 glycoside hydrolases. A lot of family 11 xylanases had been purified and sequenced. Most xylanases from fungi origins were further subdivided into five groups by computer analysis of their protein sequences. There are two groups related to enzymes of 20 kDa, group I with basic pI and group II with acid pI [27]. The near 20 kDa molecular weight and N-terminal sequence of the enzyme in this work is highly similar to xylanases from E. nidulans and A. oryzae. However, this xylanase shown basic pI that in contrast to acid pI (6.4) of E. nidulans [27], and were especially stable at alkaline pH that in contrast to pH 4–8 of A. oryzae [28]. Apparently, the characteristic difference between them should cause by their amino acids composition and structural difference. Therefore, this xylanase should be a new family 11 xylanase and can be categorized into the group I of family 11 endoxylanases. When beech wood xylan was treated with the purified enzyme, the main hydrolysis products xylobiose and xylotriose were observed after 24 h incubation at 50 8C. The enzyme hydrolysis of coba husk or corn cob hemicellulose also had the same profile. Xylotriose is the smallest oligomer hydrolyzed by most of known xylanases [10]. The xylotetraose level decreased gradually, no xylose was produced, and xylobiose was the main product. This indicates that xylotetraose was converted into xylobiose, which is involved in the glycosyl transfer reaction. As there was no evidence of xylose production, the purified xylanase can potentially be used in xylooligosaccharides preparation. Xylooligosaccharides can be utilized selectively by the beneficial intestinal microflora, Bifidobacterium spp., are expected to be used as valuable food additive [29]. Xylooligosaccharides prepared from cheaper agricultural waste, such as wheat bran and corn cob, by xylanase hydrolysis have been reported [30,31]. Coba husk, which is a common agricultural waste in Taiwan, was found to have relatively high substrate specificity toward this xylanase among the tested agricultural wastes, and was selected for xylooligosaccharides preparation in this study. UVB is a strong oxidative stress that can stimulate the production of reactive oxygen species (ROS) in keratinocytes [32]. Vitamin C is known to be a predominant antioxidant in skin because it can protect porcine skin from UVB- and UVAphototoxic injury and the effects of sunburn [33]. From Fig. 7, antioxidative capacity of feruloyl oligosaccharides prepared from hemicellulose, similar to vitamin C, was evidenced with cell model of UVB-induced oxidative damage to keratinocyte xb-2. In addition to the antioxidative capacity of feruloyl xylooligosaccharides, the bifidogenic capacity was also found similar to arabinoxylan oligosaccharides [34]. Thus, the application of feruloyl xylooligosaccharides seems to be more benefit than xylooligosaccharides. Preparations of feruloyl xylooligosaccharides with endoxylanase were reported by hydrolyzing the insoluble dietary fiber of cereals [20,35]. As the purified xylanases had greater specificity with the xylan, to use hemicellulose for feruloyl xylooligosaccharides preparation by xylanase degradation seems more reasonable. Since the ferulic acid contents in various hemicelluloses of agricultural wastes are still unknown, the related efficiency between insoluble dietary fiber and hemicellulose in feruloyl oligosaccharide preparation by enzyme degradation needs further investigation. 5. Conclusion In this work, we purified a novel low-molecular weight and alkaline-tolerant xylanase from A. carneus M34. A specific Author's personal copy H.-Y. Fang et al. / Process Biochemistry 43 (2008) 49–55 substrate of coba husk was found and used as the substrate for feruloyl oligosaccharide preparation. Feruloyl oligosaccharides prepared from the hydrolyzate showed potential antioxidative capacity, as illustrated by the model of UVB-induced oxidative damage to keratinocyte xb-2 cells. These results demonstrate the feasibility of use of this enzyme in potent antioxidant preparation. Further investigations on their photoprotective capacity as reactive oxygen species (ROS) scavengers and production of increasing yield of feruloyl oligosaccharides are still needed. Acknowledgements We gratefully acknowledge the support for this research by the National Science Council, Taiwan, and Republic of China (NSC 92-2313-B-005-060). References [1] Wong KKY, Tan LUL, Sadder JN. Multiplicity of beta-1, 4-xylanases in microorganisms: functions and applications. Microbiol Rev 1988;52:305– 17. [2] Sandrim VC, Rizzatti ACS, Terenzi HF, Jorge JA, Milagres AMF, Polizeli MLTM. Purification and biochemical characterization of two xylanases produced by Aspergillus caespitosus and their potential for kraft pulp bleaching. Process Biochem 2005;40:1823–8. [3] Tengerdy RP, Szakacs G. Bioconversion of lignocellulose in solid substrate fermentation. Biochem Eng J 2003;13:169–79. [4] Ai Z, Jiang ZQ, Li L, Deng W, Kusakabe I, Li HH. Immobilization of Streptomyces olivaceoviridis E-86 xylanase on Eudragit S-100 for xylooligosaccharide production. Process Biochem 2005;40:2707–14. [5] Ishii T. Structure and functions of feruloylated oligosaccharides. Plant Sci 1997;127:111–27. [6] Saija A, Tomaino A, Lo Cascio R, Trombetta D, Proteggente A, De Pasquale A, et al. Ferulic and caffeic acids as potential protective agents against photooxidative skin damage. J Sci Food Agric 1999;79:476–80. [7] Graf E. Antioxidant potential of ferulic acid. Free Radic Bio Med 1992;13:435–48. [8] Katapodis P, Vardakou M, Kalogeris M, Kekos D, Macris BJ, Christakopoulos P. Enzymic production of a feruloylated oligosaccharide with antioxidant activity from wheat flour arabinoxylan. Eur J Nutr 2003;42:55–60. [9] Botella C, Diaz A, Ory I, Webb C, Blandino A. Xylanase and pectinase production by Aspergillus awamori on grape pomace in solid state fermentation. Process Biochem 2007;42:98–101. [10] Kulkarni N, Shendye A, Rao M. Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 1999;23:411–56. [11] Sunna A, Antranikian G. Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol 1997;17:39–67. [12] Haltrich D, Nidetzky B, Kulbe KD, Steiner W, Zupancic S. Production of fungal xylanases. Bioresour Technol 1996;58:137–61. [13] Fang H-Y, Chang S-M, Lan C-H, Fang TJ. Production, Optimization growth conditions and properties of the xylanase from Aspergillus carneus M34. J Mol Catal B-Enzym 2007;49:36–42. [14] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–8. [15] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. 55 [16] Mckenzie HA. pH and Buffers. Contents 20. In: Dawson RMC, Elliott DC, Elliott WH, Jones KM, editors. Data for Biochemical Research. Oxford: Clarendon Press; 1978. p. 476–508. [17] Chen WP, Anderson AW. Extraction of hemicellulose from rye grass straw for the production of glucose isomerase and use of resuiting straw residue for animal. Feed Biotechnol Bioeng 1980;22:519–31. [18] Ryan SE, Nolan K, Thompson R, Gubitz GM, Savage A, Tuohy MG. Purification and characterization of a new low molecule weight endoxylanase from Penicillium capsulatum. Enzyme Microb Technol 2003;33:775–85. [19] Yuan XP, Wang J, Yao H, Venant N. Separation and identification of endoxylanases from Bacillus subtilis and their actions on wheat bran insoluble dietary fibre. Process Biochem 2005;40:2339–43. [20] Saulnier L, Vigouroux J, Thibault JF. Isolation and partial characterization of feruloylated oligosaccharides from maize bran. Carbohydr Res 1995;272:241–53. [21] Harris PJ, Hartley RD. Detection of bound ferulic acid in cell walls of the Gramineae by ultraviolet fluorescence microscopy. Nature 1976;259:508– 10. [22] Saha BC, Bothast RJ. Enzymology of xylan degradation. In: Imam SH, Greene RV, Zaidi BR, editors. Biopolymers: utilizing nature’s advanced materials. Washington, DC: American Chemical Society; 1999. p. 167– 94. [23] Polizeli ML, Rizzatti AC, Monti R, Terenzi HF, Jorge JA, Amorim DS. Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 2005;67:577–91. [24] Frederick MM, Kiang C, Frederick JR, Reilly PJ. Purification and characterization of endo-xylanases from Aspergillus niger. I. Two isozymes active on xylan backbones near branch points. Biotechnol Bioeng 1985;27:525–32. [25] Nath D, Rao M. pH dependent conformational and structural changes of xylanase from an alkalophilic and thermophilic Bacillus sp (NCIM 59). Enzyme Microb Technol 2001;28:397–403. [26] Collins T, Meuwis MA, Stals I, Claeyssens M, Feller G, Gerday C. A novel family 8 xylanase, functional and physicochemical characterization. J Biol Chem 2002;277:35133–9. [27] Sapag A, Wouters J, Lambert C, Ioannes P, Eyzaguirre J, Depiereux E. The endoxylanases from family 11: computer analysis of protein sequences reveals important structural and phylogenetic relationships. J Biotechnol 2002;95:109–31. [28] Kimura T, Suzuki H, Furuhashi H, Aburatani T, Morimoto K, Karita S, et al. Molecular cloning, overexpression, and purification of a major xylanase from Aspergillus oryzae. Biosci Biotechnol Biochem 2000;64: 2734–8. [29] Vazquez MJ, Alonso JL, Dominguez H, Parajo JC. Xylooligosaccharides: manufacture and application. Trend Food Sci Technol 2000;11: 387–93. [30] Okazaki M, Fugikawa S, Matsumoto N. Effects of xylooligosaccharide on growth of Bifidobacteria. J Jan Soc Nutr Food Sci 1990;43:395–401. [31] Yamada H, Itoh K, Morishita Y, Taniguchi H. Structure and properties of oligosaccharides from wheat bran. Cereal Foods World 1993;38:490–2. [32] Heck DE, Vetrano AM, Mariano TM, Laskin JD. UVB light stimulates production of reactive oxygen species: unexpected role for catalase. J Biol Chem 2003;278:22432–6. [33] Darr D, Combs S, Dunston S, Manning T, Pinnell S. Topical vitamin C protects porcine skin from ultraviolet radiation-induced damage. Br J Dermatol 1992;127:247–53. [34] Yuan XP, Wang J, Yao H. Feruloyloligosaccharides stimulate the growth of Bifidobacterium bifidum. Anaerobe 2005;11:225–9. [35] Lequart C, Nuzillard JM, Kurek B, Debeire P. Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides. Carbohydr Res 1999;319:102–11.