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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
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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
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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.
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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.
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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
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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).
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