Enzymatic Hydrolysis of Black Liquor Xylan by a Novel Xylose-Tolerant, Thermostable β-Xylosidase from a Tropical Strain of Aureobasidium pullulans CBS 135684

Article abstract of DOI:10.1007/s12010-017-2598-x

From three cell-associated β-xylosidases produced by Aureobasidium pullulans CBS 135684, the principal enzyme was enriched to apparent homogeneity and found to be active at high temperatures (60–70?°C) over a pH range of 5–9 with a specific activity of 163.3?units (U) mg^?1. The enzyme was thermostable, retaining over 80% of its initial activity after a 12-h incubation at 60?°C, with half-lives of 38, 22, and 10?h at 60, 65, and 70?°C, respectively. Moreover, it was tolerant to xylose inhibition with a K_i value of 18?mM. The K_m and V_max values against p-nitrophenyl-β-d-xylopyranoside were 5.57?±?0.27?mM and 137.0?±?4.8?μmol?min^?1?mg^?1 protein, respectively. When combining this β-xylosidase with xylanase from the same A. pullulans strain, the rate of black liquor xylan hydrolysis was significantly improved by up to 1.6-fold. The maximum xylose yield (0.812?±?0.015?g?g^?1 dry weight) was obtained from a reaction mixture containing 10% (w/v) black liquor xylan, 6?U?g^?1 β-xylosidase and 16?U?g^?1 xylanase after incubation for 4?h at 70?°C and pH 6.0.

Full text of DOI:10.1007/s12010-017-2598-x

Appl Biochem Biotechnol
DOI 10.1007/s12010-017-2598-x
Enzymatic Hydrolysis of Black Liquor Xylan by a Novel
Xylose-Tolerant, Thermostable β-Xylosidase
from a Tropical Strain of Aureobasidium pullulans
CBS 135684
Wichanee Bankeeree1 & Rinji Akada2
&
Pongtharin Lotrakul1 & Hunsa Punnapayak1
&
Sehanat Prasongsuk1
Received: 30 May 2017 /Accepted: 6 September 2017
#
Springer Science+Business Media, LLC 2017
Abstract From three cell-associated β-xylosidases produced by Aureobasidium pullulans
CBS 135684, the principal enzyme was enriched to apparent homogeneity and found to be
active at high temperatures (60–70 °C) over a pH range of 5–9 with a specific activity of
163.3 units (U) mg−1. The enzyme was thermostable, retaining over 80% of its initial
activity after a 12-h incubation at 60 °C, with half-lives of 38, 22, and 10 h at 60, 65, and
70 °C, respectively. Moreover, it was tolerant to xylose inhibition with a Ki value of
18 mM. The Km and Vmax values against p-nitrophenyl-β-d-xylopyranoside were
5.57 0.27 mM and 137.0 4.8 μmol min−1 mg−1 protein, respectively. When combining
this β-xylosidase with xylanase from the same A. pullulans strain, the rate of black liquor
xylan hydrolysis was significantly improved by up to 1.6-fold. The maximum xylose yield
(0.812 0.015 g g−1 dry weight) was obtained from a reaction mixture containing 10% (w/
v) black liquor xylan, 6 U g−1 β-xylosidase and 16 U g−1 xylanase after incubation for 4 h
at 70 °C and pH 6.0.
.
.
.
Keywords Black yeast Thermoactive enzyme Xylose tolerant Xylan hydrolysis
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-017-
2598-x) contains supplementary material, which is available to authorized users.
* Sehanat Prasongsuk
sehanat.p@chula.ac.th
1
Plant Biomass Utilization Research Unit, Department of Botany, Faculty of Science, Chulalongkorn
University, Bangkok 10330, Thailand
2
Department of Applied Chemistry, Yamaguchi University Graduate School of Science and
Technology for Innovation, Ube 755-8611, Japan

Appl Biochem Biotechnol
Introduction
Hemicellulose is the most abundant heteropolysaccharide in plant biomass and is com-
prised of xylan, β-(1,4)-linked xylopyranose residues, as the backbone polymer with
various substituents, including acetyl, arabinosyl, and glucuronyl residues. The complete
hydrolysis of hemicellulose yields a mixture of monosaccharides, including xylose and
arabinose, which can be used as substrates in the food, fuel, and chemical industries [1].
Hemicellulose can be extracted from any plant biomass, but recently, those prepared
from agricultural residues have gained more attention due to their cheap cost and
abundance. Most studies have focused on the extraction of xylan from corn cobs, rice
hulls, cereal straw, and other feedstock [2]. On the contrary, xylan recovery from waste
water of biomass-related industries such as pulp and paper mills, especially the black
liquor, has rarely been reported in spite of its high potential [3]. Approximately 8600 L
of black liquor are generated every day from Eucalyptus grandis kraft pulping process by
a commercial pulp and paper company in Thailand. The amount of xylan extracted from
this black liquor was about 13 g L−1 or 95% of xylan recovery from the pulp feedstock
[4]. The degradation of hemicellulose requires the synergistic action of several enzymes.
Among these, xylanase (1,4-β-D-xylan xylanohydrolase, EC 3.2.1.8) and β-xylosidase
(1,4-β-D-xylan xylanohydrolase, EC 3.2.1.37) are the most well studied. Most xylanases
randomly hydrolyze glycosidic linkages along the xylan chain to release
xylooligosaccharides (XOs) and xylobioses that are subsequently hydrolyzed by β-
xylosidase to xylose monomers. The key enzyme in the complete degradation is β-
xylosidase, since xylosyl residues and the reducing end product can inhibit xylanase.
From a biotechnological viewpoint, the combination of β-xylosidase with other
hemicellulose-degrading enzymes has opened new possibilities for a number of applica-
tions, including in the pulp and paper, food, beverage, feed, textile, and pharmaceutical
industries [5]. For such applications, a large quantity of the enzyme possessing specific
properties, including thermostability and tolerance to elevated concentration of substrate,
product, and other additives, is required [6].
Among several microorganisms producing xylan-degrading enzymes, Aureobasidium
pullulans has been recognized as one of the best producers with xylanase and β-
xylosidase activity as high as 373.0 U mL−1 and 10.0 U mg−1 cell dry weight, respec-
tively [7–9], compared to the extracellular enzymes from other fungi including Penicil-
lium janczewskii (151.1 and 1.9 U mL−1, respectively) [10] and Aspergillus niger (91.5
and 2.1 U mL−1, respectively) [11]. In 2013, A. pullulans CBS 135684 isolated from
Thailand was found to produce a cellulase-free xylanase that was active at relatively high
temperatures (70–80 °C) over a wide optimal pH range from 4 to 10 [9]. These
properties are relatively uncommon in the xylanases from previously reported A.
pullulans strains and other typical industrial yeast species. Since this strain also exhibited
β-xylosidase activity, it was of interest to investigate whether β-xylosidase also pos-
sessed such remarkable properties similar to the xylanase, and whether it could be used
in any xylose-based applications.
Therefore, the goals of this study were to enrich and characterize the β-xylosidase from A.
pullulans CBS 135684 and to explore the potential of using this enzyme in the saccharification
of xylan extracted from the black liquor effluent of the pulp and paper industry.

Appl Biochem Biotechnol
Materials and Methods
Microorganism
Aureobasidium pullulans CBS 135684 was obtained from the fungal culture collection of the
Plant Biomass Utilization Research Unit, Department of Botany, Faculty of Science,
Chulalongkorn University, Bangkok, Thailand. The yeast was grown in yeast malt (YM) agar
medium at room temperature (28 2 °C) for 2 days and short-term stock cultures were stored
at 4 °C. For long-term storage, the strain was stored at − 20 °C in YM broth containing 20% (v/
v) glycerol.
Production of β-Xylosidase
The seed culture was prepared by growing A. pullulans in YM broth at room temperature with
agitation at 150 rpm for 72 h. The inoculum was adjusted to 2.5 × 106 cells mL-1 and then
100 μl was transferred into a 250-mL Erlenmeyer flask containing 100 mL of production
medium [12]. The incubation was continued under the same condition for 5 days, when A.
pullulans cells were then harvested by centrifugation at 9000×g for 10 min and freeze-dried at
− 60 °C for 6 h. The lyophilized cells (1 g) were ground, suspended in 100 mL of sterile
distilled water and centrifuged at 5000×g for 10 min for debris removal with harvesting of the
supernatant as the crude enzyme.
Enzyme and Protein Assays
For determination of the β-xylosidase activity, the reaction mixture consisting of 0.1 mL of
appropriately diluted crude enzyme solution (see BProduction of β-Xylosidase^) or purified
enzyme solution (1 mg protein per mL), 0.2 mL of 5 mM p-nitrophenyl-β-D-xylopyranoside
(pNPX; Sigma, USA) in deionized water, and 0.7 mL of 50 mM acetate buffer (pH 6.0) was
incubated at 70 °C for 10 min. The reaction was terminated by adding 1.0 mL of 0.5 M
Na2CO3, and the absorbance at 410 nm was measured. One unit (U) of β-xylosidase activity
was defined as the amount of enzyme that liberated 1 μmol of p-nitrophenol (pNP) from
pNPX per min. Protein concentrations were measured by the method of Lowry et al. [13] using
bovine serum albumin (Sigma, USA) as the standard.
Enzyme Enrichment and SDS-PAGE Analysis
Enrichment of the assumed principal β-xylosidase was performed at 4 °C. The crude
enzyme preparation (see BProduction of β-Xylosidase^) was concentrated tenfold by
ultrafiltration (10 kDa MW membrane cut-off, Amicon, Beverly, MA, USA) prior to
precipitation using ammonium sulfate (40–80% saturation). After recovery by centrifu-
gation at 10,000×g for 20 min, the pellet was dissolved in 20 mM Tris-HCl buffer (pH
8.0) and dialyzed for 16 h against the same buffer. The concentrated enzyme (40 mg
protein mL−1) was applied to a pre-equilibrated DEAE Sepharose Fast Flow column
(7 mm × 25 mm, GE Healthcare, Buckinghamshire, UK) at a flow rate of 1.0 mL min−1
The adsorbed proteins were eluted with a linear gradient of 0–1.0 M NaCl in 50 mL of
.

Appl Biochem Biotechnol
the same buffer. Five-milliliter fractions were collected and analyzed for β-xylosidase
activity and protein content. Fractions that represented the major portion of β-xylosidase
activity were pooled and dialyzed against 50 mM acetate buffer (pH 4.0). The concen-
trated enzyme solution (18 mg protein mL−1) was then purified through a pre-
equilibrated SP Sepharose Fast Flow column (7 mm × 25 mm, GE Healthcare) at a flow
rate of 1.0 mL min−1. Elution was performed with a linear gradient of 0–1.0 M NaCl in
50 mL of the same buffer and collected in 5-mL fractions. Fractions with β-xylosidase
activity were pooled, dialyzed against 50 mM acetate buffer (pH 6.0), and applied to a
pre-equilibrated Sephacryl-S100 gel filtration column (30 cm × 5 cm Sephacryl S-100
HR, Sigma, USA). Elution was performed at a flow rate of 0.5 mL min−1 collecting
3-mL fractions that were assayed for β-xylosidase activity and protein content as
described above. Protein profiles of the collected fractions were determined by resolution
through SDS-PAGE (12.5% (w/v) resolving gel in 1.5 M Tris-HCl, pH 8.8), run at
20 mA for 2 h, and stained with Coomassie Brilliant Blue R-250 [14].
Determination of β-Xylosidase Properties
Effects of pH and Temperature on the Enzyme Activity
Effects of temperature and pH on the enriched β-xylosidase activity were determined by
incubating the reaction mixture (1 U mL−1 enzyme and 5 mM pNPX, total volume 1 mL) at
various temperatures (40–80 °C) in 50 mM sodium acetate (pH 3.0–6.0), sodium phosphate
(pH 6.0–9.0), or glycine (pH 9.0–11.0) reaction buffer for 10 min. The relative activity was
calculated as the percentage of the maximal activity.
Effect of Temperature on the Enzyme Stability
Enzyme thermostability was assessed by measuring the residual β-xylosidase activity after
incubation at different temperatures from 50 to 70 °C in the absence of pNPX for a 6–4-h
period. The initial activity of the enriched β-xylosidase at 1 U mL−1 was set as 100% relative
activity.
Effects of Different Ions and Organic Solvents on the Enzyme Activity
To investigate the effects of different cations and a divalent cation chelator on the
enzyme activity, CaCl2, CoCl2, CuCl2, FeSO4·7H2O, KCl, MgCl2, MnSO4, NaCl,
ZnCl2, and EDTA were separately added to the reaction mixture at 1 mM prior to
the enzyme assay under the optimal conditions (see BEffects of pH and Temperature
on the Enzyme Activity^). The hydrolytic activity of β-xylosidase was also deter-
mined in the presence of some organic solvents including, primary alcohols (C1–6),
secondary alcohols (2-propanol and 2-butanol), tertiary alcohol (2-methyl-2-propanol),
acetone, and chloroform at final concentrations of 10–30% (v/v) under the optimal
condition (see BEffects of pH and Temperature on the Enzyme Activity^). The relative
activity was calculated as a percentage of the enzyme activity without the aforemen-
tioned chemicals added.

Appl Biochem Biotechnol
Substrate Specificity
Substrate specificity was determined by incubating the enriched enzyme (1 μg protein per
reaction) in the presence of various individual substrates, including pNPX, cellobiose,
carboxymethyl cellulose (CMC), beechwood xylan, oat spelt xylan, and commercial
xylooligosaccharides (X2–4), at different final concentrations from 0 to 1 mM. The assay
was performed under the optimal condition and the activity was measured by quantification of
the released pNP (for pNPX), xylose (for xylooligosaccharides and xylan), and glucose (for
cellobiose and CMC). The amount of released reducing sugars was determined by the 3,5-
dinitrosalicylic acid (DNS) method [15]. One U was defined as the amount of enzyme that
liberated 1 μmol of each product per min. A 100% relative activity was set as that when
xylobiose was used as the substrate. The hydrolytic products were qualitatively analyzed by
thin-layer chromatography (TLC) on silica gel plates (60F 254, 0.2 mm, Merck, Germany)
[16]. The unit activity was also used to calculate the kinetic values of the enzyme, in terms of
the Michaelis constant (Km) and maximum velocity (Vmax), by fitting the initial hydrolysis
rates to the Lineweaver-Burk equation.
Monosaccharide Inhibition
The effects of various monosaccharides on the enzyme activity were studied using 1 mM
pNPX as the substrate in the presence of xylose, glucose, arabinose, mannose, or xylitol at
different final concentrations (1–70 mM). The Ki value of inhibition was defined as the amount
of a monosaccharide required to inhibit 50% of the β-xylosidase activity, and was calculated
from the Dixon plot. Kinetic constants were determined from the reactions that were per-
formed with 5 mM or 10 mM monosaccharide at varying pNPX concentrations from 0 to
1 mM under optimal conditions to further confirm the type of inhibition.
Synergy of Xylanase and β-Xylosidase on Black Liquor Xylan Hydrolysis
The xylan used in this study was extracted from the black liquor of a commercial pulp and
paper plant (Double A (1991) Public Co., Ltd., Thailand) as described by Bankeeree et al. [4].
In order to enhance the xylan degradation, response surface methodology (RSM) was used to
determine the optimum condition, in terms of the incubation time and amount of β-xylosidase
and extracellular xylanase from A. pullulans CBS 135684. A Box-Behnken design matrix with
three factors at three different levels and three replicates at the center point was used for fitting
the second-order response surface [17]. The reaction was performed with 10% (v/v) black
liquor xylan (Fluka, USA) in 50 mM acetate buffer (pH 6.0) at 70 °C. The amount of xylose
from each reaction was taken as the response and analyzed by HPLC using a RP-C18 column
(Nucleodur 100-5 C18 ec, 250 × 4, Macherey Nagel). The column was eluted with 70:30 (v/v)
acetonitrile: water at 1.0 mL min−1 and detection was performed using a differential refrac-
tometer R-401. Statistical analysis of the data was performed using the design package Design-
Expert software (version 8.0.7.1, Stat-Ease, Inc., Minneapolis, USA) to evaluate the analysis
of variance (ANOVA) and to determine the significance of each term in the equations fit. In
order to verify the accuracy of the predicted model, a repeated experiment was performed
using the optimized amount of each enzyme.

Appl Biochem Biotechnol
Other Data Analysis
Statistical differences among the means of data (n = 3) were calculated when appropriate using
one-way analysis of variance (ANOVA) and either Duncan’s multiple range test (DMRT) or
Student’s t test (2 tailed) with the SPSS 17.0 software package (SPSS Inc., Chicago, U.S.A.).
Differences at P ≤ 0.05 were considered significant.
Results and Discussion
β-Xylosidase Production
Although A. pullulans is well known as an excellent xylanase producer, there have been only a
few reports on its β-xylosidase, where most of them have shown that the enzymes were cell
associated [18, 19]. Similar to those reports, the β-xylosidase activity of A. pullulans CBS
135684 was only detected only in the cell extract. After cultivation for 5 days in production
medium with corncob as the sole carbon source, the maximum activity of this enzyme was
2.36 0.01 U mg−1 cell dry weight (DW), some 1.5-fold higher than that when commercial
beechwood xylan was used (1.58 0.02 U mg−1 cell DW). The maximum activity of β-
xylosidase from A. pullulans CBS 135684 was higher than those previously reported from A.
pullulans CBS 58475 (0.9 U mg−1) [20] and Penicillium janczewskii (1.9 U mg−1) [21].
β-Xylosidase Enrichment and SDS-PAGE Analysis
The likely principal cell-associated β-xylosidase was enriched to apparent homogeneity, with
the summary of the enrichment steps shown in Table 1 and Fig. 1. The DEAE-Sepharose
column chromatography yielded two separate peaks of β-xylosidase activity (Fig. 1a). The
unbound enzyme peak, which represented the major portion (~ 70%) of β-xylosidase activity,
was further fractionated using SP Sepharose Fast Flow column chromatography, where the
bound fraction yielded two separate peaks of enzyme activity upon subsequent elution with
NaCl (Fig. 1b). The enzyme from the major peak (~ 50% total activity) was subsequently
fractionated over Sephacryl S-100 gel filtration chromatography to yield a single peak of β-
Table 1 Enrichment of the (principal) cell-associated β-xylosidase from A. pullulans CBS 135684
Enrichment step
Total
protein (mg)
Total
Specific activity
(U mg−1 protein)
Purification (fold) Yield (%)
activity (U)a
Cell extract
Ultrafiltration
(NH4)2SO4
1054 13.79 5258 24.87 4.97 0.05
1.00
1.09
1.22
100.00
91.58
67.48
885 11.35
583 9.64
4815 20.14 5.42 0.06
3548 13.59 6.05 0.08
precipitation
(50–80% saturation)
DEAE-Sepharose
SP Sepharose
Fast Flow
84 2.94
18 0.34
2052 18.46 24.18 0.70
4.87
19.84
39.03
33.95
1785 9.84
98.59 1.55
Sephacryl S-100
6
0.12
985 2.63
163.32 2.70
32.86
18.73
a 1 U is the amount of enzyme required to release 1 μmol p-nitrophenol equivalent per min under the assay
conditions. Experiments were performed in triplicate. Data are shown as the mean SD (error bars), derived from
triplicate experiments

Appl Biochem Biotechnol
Fig. 1 Sequential purification steps of β-xylosidase from A. pullulans CBS 135684. The amounts of protein (- -
-) and β-xylosidase activity (—) in fractions eluted from a DEAE Sepharose, b SP Sepharose Fast Flow, and c
Sephacryl S-100 gel filtration column chromatography were profiled. The proteins that adsorbed to DEAE
Sepharose or SP Sepharose Fast Flow column chromatography were eluted with a linear gradient of 0–1.0 mol L−1
NaCl (·····) in the corresponding buffer. SDS-PAGE analysis d of the enriched β-xylosidase from A. pullulans
CBS 135684 was performed on a 10% (w/v) gel in 1.5 M Tris-HCl (pH 8.8) and stained with Coomassie Brilliant
Blue R-250. Lane M: Blueye prestained protein ladder (GeneDireX, Taiwan). Lane 1: the enriched β-xylosidase
from DEAE Sepharose column (~ 30 μg protein). Lane 2: the enriched β-xylosidase from SP Sepharose Fast
Flow column (~ 30 μg protein). Lane 3: the enriched β-xylosidase from Sephacryl S-100 gel filtration column
chromatography (~ 30 μg protein)
xylosidase activity (Fig. 1c). These results indicated that A. pullulans CBS 135684 potentially
produced at least three cell-associated β-xylosidases, which, if so, is the first report of multiple
forms of β-xylosidase in A. pullulans. However, their genetic and post-translational relation-
ship to each other remains unknown. The overall percent recovery of this (potentially
principal) enzyme was approximately 18.7% with a 32.9-fold enrichment and a specific
activity of 163.3 U mg−1. The final enriched enzyme showed a single band of ~ 172 kDa
on one-dimensional SDS-PAGE resolution with Coomassie Brilliant Blue staining (Fig. 1d),
which was similar to the size estimated from the Sephacryl S-100 gel filtration chromatogra-
phy (data not shown). Differences in size between this enriched β-xylosidase from A.
pullulans CBS 135684 and those previously reported from other strains of A. pullulans
(88.5 kDa) [18–20] implied that it might be a distinct enzyme.
Effects of the pH and Temperature on the β-Xylosidase Activity
The effects of pH and temperature on activity of the enriched β-xylosidase are summarized in
Fig. 2a. The maximum β-xylosidase activity was found at 70 °C and pH 6.0, while it was also
active over a relatively wide pH range (5.0–9.0) with more than 80% of the maximum activity
at the same temperature. This preference for a mildly acidic to mildly basic condition makes
the enzyme strikingly different from the previously reported Aureobasidium β-xylosidases that

Appl Biochem Biotechnol
Fig. 2 Effect of the a pH and temperature on the enzyme activity and b the thermostability profile of the
enriched β-xylosidase from A. pullulans CBS 135684. a The β-xylosidase activity was assayed by incubating the
purified enzyme with 5 mM pNPX in 50 mM sodium acetate (pH 3.0–6.0; - - -), phosphate (pH 6.0–10.0; —), or
glycine (pH 10.0–11.0; ·····) buffer at 40 °C (■), 50 °C (♦), 60 °C (▲), 70 °C (●), or 80 °C (○) for 10 mins.
Relative activity was calculated as a percentage of the maximum activity (50 mM sodium acetate buffer (pH 6.0),
70 °C). b The thermostability profile of the enriched β-xylosidase was investigated by separate preincubation in
50 mM sodium acetate buffer (pH 6.0) at 50 °C (♦), 55 °C (◊), 60 °C (▲), 65 °C (Δ), or 70 °C (●) without the
substrate for 48 h. The residual β-xylosidase activity was then calculated as a percentage of the initial activity.
Data are shown as the mean SD (error bars), derived from triplicate experiments
were active at a moderately acidic pH range (3.5–4.5) [19, 20]. The A. pullulans CBS 135684
β-xylosidase activity at each pH was significantly increased when the incubation temperature
was increased from 40 °C to the optimal at 70 °C. However, a significantly lower enzyme
activity was observed at 80 °C (e.g., 12.08 0.42% at pH 6.0), which might be caused by
protein denaturation. A fungal β-xylosidase that is active in an alkaline environment and a
high temperature are uncommon (Table 2). With its broad optimum pH and thermophilic
properties, this enzyme is likely to be applicable in several industrial processes, and especially
in the bioconversion of lignocellulosic materials as this is optimally performed at a high pH
and temperature.
Effect of Temperature on the β-Xylosidase Stability
The enriched β-xylosidase was found to be relatively stable at 50–60 °C with more than 80% of
its original activity remaining after 18 h incubation at these temperatures (Fig. 2b). The half-life
of the purified enzyme at 70 °C, the optimum temperature of enzyme activity, was approxi-
mately 10 h. In comparison, the previously reported β-xylosidases from the thermophilic
bacterium Geobacillus thermodenitrificans [21] and the thermotolerant fungi Aspergillus
fumigatus [31] were clearly less stable, with a half-life at 70 °C of less than 3 h in both cases.
Effects of Different Ions and Organic Solvents on the β-Xylosidase Activity
The enriched enzyme activity was not significantly affected by the presence of any of the
tested ions at 1 mM, except for Cu2+ that reduced the residual enzyme activity to 80.8 1.4%
(Table 3). The sensitivity of β-xylosidase to Cu2+ is relatively common for this enzyme from
fungi, including those from Fusarium proliferatum [25] and Paecilomyces thermophila [32].
The presence of EDTA did not significantly affect the β-xylosidase activity, suggesting that
divalent cations were not essential for the catalytic activity of this enzyme.

Appl Biochem Biotechnol
Table 2 Properties of the enriched (principal) β-xylosidase from A. pullulans CBS 135684 and those reported
from other fungal strains
Species
MW Optimum activity Specific
Km
Vmax
Ki
(mM)
Reference
(kDa)
activity
(mM) (μmol min−1 mg−1
)
(U mg−1
)
pH Temperature
(°C)
Aureobasidium
pullulans
CBS 135684
Aureobasidium
pullulans
ATCC 20524
Aspergillus
japonicas
95.0 6.0 70
88.5 3.5 70
120.0 4.0 70
163.3
288.0
5.6
3.5
137.0
263.0
24.0 This
study
–
[19]
42.6
386.7
150.0
53.0
19.6
11.6
1.9
0.3
6.1
1.0
0.8
1.4
2.5
3.4
0.5
4.5
98.0
1364.0
250.0
75.0
13.0
37.3
33.2
0.8
2.9
–
[22]
[23]
[24]
[25]
[26]
Aspergillus niger 111.0 5.0 60
GS1
Aspergillus
84.7 4.5 55
91.2 4.5 60
2.7
5.0
–
oryzaea
Fusarium
proliferatum
Humicola grisea 87.0 6.5 55
var. thermoidea
Humicola
83.2 6.0 60
100.0 6.0 65
144.0 2.5 60
52.3 7.0 55
29.0 [27]
6.0 [10]
32.1 [28]
insolens Y1a
Penicillium
janczewskii
Penicillium
sclerotiorum
Paecilomyces
thermophila
J18a
31.1
37.9
90.2
–
[29]
Trichoderma
reesei
Talaromyces
amestolkiae
80.0 3.5 60
200.0 3.0 70
16.0
47.1
0.5
0.2
12.0
52.0
1.4
1.7
[22]
[30]
a Recombinant enzyme
Since organic solvents have been used for solubilizing hydrophobic substrates in enzymatic
reactions, their effects on the activity of the enriched β-xylosidase were also determined in this
study. The addition of alcohols significantly decreased the β-xylosidase activity in a
concentration-dependent manner (P ≤ 0.05) and also with the increasing alkyl chain length of
the alcohol (Table 4), the latter of which might be due to the increasing hydrophobicity with
increasing alkyl chain length [33]. However, this enzyme seemed to be particularly tolerant to
methanol and ethanol, since it was not significantly inhibited by both alcohols at 10% (v/v) and
retained more than 80% of its initial activity in the presence of 30% (v/v) of either alcohol. This
level of tolerance was higher than that previously reported for the β-xylosidase from the
thermophilic fungus Sporotrichum thermophile [34] and the thermophilic bacterium
Geobacillus thermodenitrificans [21], where the presence of 10% (v/v) methanol or ethanol
caused a 30% decrease in their activity. On the other hand, the CBS 135684 β-xylosidase was
highly sensitive to 2-methyl-2-propanol, with a more than 50% reduction of its initial activity at
a concentration as low as 10% (v/v). Similar to most enzymes, the β-xylosidase was sensitive to
the denaturation caused by acetone and chloroform, with up to 12 and 24% of the enzyme
activity being lost in the presence of 10% (v/v) acetone and chloroform, respectively.

Appl Biochem Biotechnol
Table 3 Effects of different cations (at 1 mM) on the activity of the (principal) β-xylosidase from A. pullulans
CBS 135684
Additive
Relative activity (%)*
None
EDTA
KCl
100.0 1.4b
104.5 2.7b
101.2 7.1b
100.7 2.3b
107.2 8.7b
100.2 8.7b
80.8 1.4a
NaCl
CaCl2
CoCl2
CuSO4
FeSO4
LiCl2
MgCl2
MnSO4
ZnCl2
105.9 5.9b
102.1 2.5b
104.5 3.7b
103.3 4.9b
103.4 2.2b
Means with different superscript letters are significantly different (ANOVA and DMRT, P ≤ 0.05)
*100% relative activity corresponded to activity of the β-xylosidase under the standard assay condition without
ion/EDTA added. Data are shown as the mean SD (error bars), derived from triplicate experiments
β-Xylosidase Substrate Specificity
Substrate specificity of the enriched β-xylosidase was tested with synthetic substrates, natural
polymers, and XOs, with the results summarized in Table 5. Compared to xylobiose, the
enzyme exhibited a significantly higher activity towards pNPX (114%) but noticeably less
towards xylotriose (85%) and xylotetraose (71%). The enzyme was also able to hydrolyze
cellobiose, although at a much lower amount (21%). No hydrolytic activity was detected when
polymeric glucans, both natural beechwood xylan and synthetic CMC, were used as its
Table 4 Effect of organic solvents on the activity of the (principal) β-xylosidase from A. pullulans CBS 135684
Additive
Relative activity (%)*
10% (v/v)
20% (v/v)
30% (v/v)
None
Methanol
Ethanol
Propanol
Butanol
Pentanol
Hexanol
2-Propanol
2-Butanol
2-Methyl-2-propanol
Acetone
100.0 1.4ef
103.0 3.3f
101.5 4.3ef
94.8 3.3de
76.4 1.3c
73.7 5.9bc
68.5 5.9b
99.9 3.5ef
88.5 5.9d
30.2 3.2a
88.1 2.2d
76.7 2.3c
100.0 1.4H
99.4 4.2H
92.3 2.9G
60.0 2.0E
26.1 1.3C
24.9 1.0C
20.0 1.9B
72.0 2.3F
56.2 1.0DE
9.4 0.4A
100.0 1.4z
95.0 2.9y
83.5 1.0x
41.7 2.0u
19.7 1.2r
15.8 1.2q
13.7 2.0q
50.3 1.8v
29.3 0.9s
ND
70.2 2.4F
53.7 5.9D
57.3 2.2w
38.6 1.1t
Chloroform
Means with different superscript letters (a–f, A–H, and q–z for data in the first to the third column, respectively)
in the same column are significantly different (ANOVA and DMRT, P ≤ 0.05)
ND not detectable
*100% relative activity corresponded to activity of the β-xylosidase under standard assay conditions without any
solvent added. Data are shown as the mean SD (error bars), derived from triplicate experiments

Appl Biochem Biotechnol
Table 5 Substrate specificity and kinetic analysis of the (principal) β-xylosidase from A. pullulans CBS 135684
against synthetic and natural substrates
Substrates
Relative activity (%) at 1 mM of Products
substrate
Km*
(mM)
Vmax*
(μmol min−1 mg−1
)
p-Nitrophenyl
xylopyranoside
Xylobiose (X2)
114.4 2.9e
100.0 4.1d
85.2 3.4c
70.8 2.3b
p-Nitrophenol 5.67
136.99 4.81
95.50 0.96
41.30 0.74
21.30 0.25
0.27
0.11
0.13
0.28
X1, X2
4.60
6.97
8.26
–
Xylotriose (X3)
X1, X2, X3
Xylotetraose (X4)
X1, X2, X3,
X4
–
Beech wood xylan
Cellobiose (C2)
ND
–
20.94 2.5a
Glucose (C1) 12.64
0.34
6.97 0.14
Carboxymethyl
cellulose
ND
–
–
–
ND not detectable
Means with different superscript letters are significantly different (ANOVA and DMRT, P ≤ 0.05)
*Kinetic values were calculated from reactions performed with different concentrations of substrate (0–1 mM) in
50 mM sodium acetate buffer (pH 6.0) at 70 °C for 10 min. Data are shown as the mean SD (error bars),
derived from triplicate experiments
substrate. A similar β-D-glucosidase activity of β-xylosidase has been reported previously for
the β-xylosidase from Penicillium janczewskii (1.3% of β-xylosidase activity) [35] and
Aspergillus japonicus (31.6% of β-xylosidase activity) [22].
The deduced Michaelis-Menten kinetic constants for Km and Vmax against xylobiose for the
enriched A. pullulans CBS 135684 β-xylosidase were 4.60 0.11 mM and 95.5 0.96 μmol
xylose min−1 mg−1 protein, respectively. Thus, the enzyme bound natural xylobiose more
tightly but hydrolyzed it less efficiently than the synthetic pNPX (5.57 0.27 mM and
136.99 4.81 μmol pNP min−1 mg−1 proteins, respectively). The Vmax value against pNPX
of A. pullulans CBS 135684 β-xylosidase was much lower than that reported previously for
another A. pullulans strain, but higher than those of several filamentous fungi (Table 2). The
Km value of the enzyme increased almost 1.8-fold as the chain length of the substrate increased
from X2 to X4, suggesting a reduced binding affinity and so a decreased catalytic rate with
longer chain substrates. Xylose was detected from the TLC analysis as an initial product of the
enzymatic hydrolysis in all the XOs in this study (Supplementary data 1), which indicates that
the enriched β-xylosidase from A. pullulans CBS 135684 is an exo-cutting enzyme.
Monosaccharide Inhibition of β-Xylosidase
Xylose as the final product is a well-known inhibitor of many microbial β-xylosidases [10]. The
presence of the monosaccharides used in this study including glucose, arabinose, mannose, and
xylitol at different concentrations from 1 to 70 mM did not significantly inhibit the β-xylosidase
activity (Supplementary data 2). In contrast, the enzyme activity gradually decreased with increasing
xylose concentrations and was completely inhibited at 55 mM xylose. The plot of the reciprocal of
the reaction rate (1/V) against the xylose concentration was used to estimate the inhibition constant
(Ki) between xylose and the enzyme (Fig. 3a). The calculated value of Ki from this Dixon plot was
18.20 0.16 mM, which was 1.8- to 9.1-fold higher than those previously reported for other β-

Appl Biochem Biotechnol
Fig. 3 Effect of the xylose concentration on the activity of the enriched β-xylosidase from A. pullulans CBS
135684, as shown by a Dixon and b Lineweaver-Burk plots. a The Dixon plot was constructed by plotting xylose
concentrations (0–25 mM) against the reciprocal of the initial rate when reactions were conducted for 10 min
with 5 mM p-NPX in 50 mM of sodium acetate buffer (pH 6.0). b The Lineweaver-Burk plot was constructed by
plotting the reciprocal of the xylose concentrations at 10 mM (▲), 20 mM (■), and 0 mM (●) against the initial
rate when reactions were conducted for 10 mins with 0–0.8 mM p-NPX in 50 mM of sodium acetate buffer (pH
6.0). Data are shown as the mean SD (error bars), derived from triplicate experiments
xylosidases of 2–10 mM [10, 22]. Therefore, with a lower sensitivity to xylose inhibition, this
enzyme has a great potential in the conversion of hemicellulose in many fields. At 5 and 10 mM,
xylose altered the Vmax (24.39 0.85 and 8.70 0.76 μmol min−1 mg−1, respectively) but did not
significantly modify the Km (5.70 0.35 and 5.82 0.10 mM, respectively) when PNX was used as
the substrate (Fig. 3b). This suggests non-competitive inhibition, where xylose might bind with the
enzyme at sites other than the active site used in catalysis and interferes with the catalytic hydrolysis
without affecting the substrate binding [36].
Synergy of Xylanase and β-Xylosidase on Xylan Hydrolysis
The xylan used in this study was extracted from black liquor effluent released during the kraft
pulping process of Eucalyptus grandis wood from a commercial company in Thailand. Hemicel-
luloses in wood chips are partially depolymerized during chemical pulping resulting in dissolved
short-chain xylans in the effluent [37]. Therefore, black liquor xylan is an attractive substrate for
xylose and XOs production compared with commercial long-chained xylans due to its low cost,
suitable structure for degradation, and environmental advantage of waste treatment. In preliminary
experiments, the effect of the crude β-xylosidase preparation from A. pullulans CBS 135684
(Production of β-Xylosidase) on xylan hydrolysis was investigated using 10% (w/v) black liquor
xylan, due to its similar properties to those of the purified enzyme (Supplementary data 3). Since the
β-xylosidase preferably hydrolyzed X2–4, the crude xylanase from A. pullulans CBS 135684 was
used in combination with the crude β-xylosidase to hydrolyze oligoxylans longer than X4 and
enhance the xylose yield. At 5 U g−1 xylanase, the amount of reducing sugar released from the
reactions with and without 5 U g−1 β-xylosidase was 0.22 0.03 and 0.36 0.02 g g−1 DW of
xylan, respectively. Thus, a 1.6-fold increase in xylan production yield was obtained by the co-
digestion compared to that with the xylanase alone. In an attempt to optimize the condition for xylan
hydrolysis, the effect of the xylanase and β-xylosidase concentrations on the xylose yield at different
incubation times were investigated using a sequential univariate approach to select the operational
ranges of variables (data not shown). Subsequently, a RSM using a Box-Behnken design was
applied within these ranges of parameters to obtain the maximum response. The maximum amount
of xylose produced, 0.812 0.015 g g−1 DW of xylan (trial number 14 in Table 6), was very close

Appl Biochem Biotechnol
Table 6 Box-Behnken design matrix of black liquor xylan hydrolysis by xylanase and β-xylosidase from A.
pullulans CBS 135684
Trial
Variable factor
Xylose yield
Predicted value
(m g−1
(mg g−1 DW xylan)*
)
Xylanase (U g−1
)
β-Xylosidase (U g−1
)
Time (h)
1
2
3
4
5
6
7
8
10 (− 1)
22 (+ 1)
10 (− 1)
22 (+ 1)
10 (− 1)
22 (+ 1)
10 (− 1)
22 (+ 1)
16 (0)
16 (0)
16 (0)
16 (0)
16 (0)
3 (− 1)
3 (− 1)
9 (+ 1)
9 (+ 1)
6 (0)
6 (0)
6 (0)
6 (0)
3 (− 1)
9 (+ 1)
3 (− 1)
9 (+ 1)
6 (0)
4 (0)
4 (0)
4 (0)
4 (0)
2 (− 1)
2 (− 1)
6 (+ 1)
6 (+ 1)
2 (− 1)
2 (− 1)
6 (+ 1)
6 (+ 1)
4 (0)
0.57 0.03
0.57 0.02
0.56 0.03
0.58 0.01
0.54 0.01
0.56 0.03
0.69 0.02
0.70 0.01
0.49 0.02
0.43 0.01
0.57 0.02
0.64 0.03
0.80 0.02
0.81 0.02
0.81 0.02
0.55 0.01
0.56 0.01
0.55 0.01
0.58 0.01
0.54 0.01
0.56 0.01
0.69 0.01
0.70 0.01
0.47 0.01
0.41 0.01
0.55 0.01
0.63 0.01
0.81 0.01
0.81 0.01
0.81 0.01
9
10
11
12
13
14
15
16 (0)
16 (0)
6 (0)
6 (0)
4 (0)
4 (0)
Numbers in parenthesis (− 1, 0, or + 1) are the coded value (low, normal or high). The second-order regression
equation, providing the xylose yield as a function of the variables. This model was significant, based on the F
value (328.63) and the model terms value of Prob > F (0.001)
* Data are shown as the mean SD (error bars), derived from triplicate experiments
to the predicted value of 0.808 0.009 g g−1 DW (Table 6). Comparison of the predicted and
experimental values revealed a good correspondence between them, with a coefficient of determi-
nation (R2) of 0.95. Three-dimensional response plots and their corresponding contour plots (Fig. 4)
were drawn on the basis of the model equation to investigate the interaction among the variables and
to determine the optimum concentration of each compound for xylan hydrolysis. Canonical analysis
revealed the maximum xylose yield of 0.812 0.015 g g−1 DW with 16 U g−1 xylanase and 6 U g−1
β-xylosidase 6 U g−1 after incubation at 70 °C for 4.5 h. The validity of the predicted results by the
regression model was confirmed by a repeated experiment under the same (predicted to be optimal)
concentrations, where three replications gave an average yield of xylose (0.809 0.018 g g−1 DW)
that was close to the predicted value (0.808 0.009 g g−1 DW). The synergistic effect of selected
variables corresponded to a 2.25-fold increase in the production yield compared to the preliminary
experiment. In addition, the production yield of xylose in this study was higher than those previous
Fig. 4 The response surface and contour plots showing the interactions between the different concentrations of
xylanase and β-xylosidase and reaction time on the xylose concentration. The experiment was performed
according to the central composite design

Appl Biochem Biotechnol
reports of xylan hydrolysis by the co-incubation with xylanase and β-xylosidase from Penicillium
janczewskii (0.22 g g−1 after 24 h) [10], and the co-action of the xylanase from Anoxybacillus
flavithermus and β-xylosidase from Sulfolobus solfataricus (0.63 g g−1 after 4 h) [38].
Conclusion
The major cell-associated β-xylosidase from the Thai A. pullulans strain CBS 135684 was
enriched to apparent homogeneity, characterized and found to exhibit several interesting
properties, including being active and stable over a high temperature (60–70 °C), catalyzed
well at mild alkali pHs and was tolerant to xylose and alcohols. The maximum xylose yield
from black liquor xylan hydrolysis with crude xylanase (16 U g−1) and β-xylosidase (6 U g−1)
was over 80% of the theoretical yield under a high concentration of xylan (10%, w/v) at an
elevated temperature (70 °C) after incubation for 4.5 h. In conclusion, the β-xylosidase from
A. pullulans CBS 135684 is a prospective enzyme for a wide range of applications, especially
for the bioconversion of hemicellulose.
Acknowledgements This research was performed under the Core-to-Core Program, which was financially
supported by the Japan Society for the Promotion of Science (JSPS), National Research Council of Thailand
(NRCT), Vietnam Ministry of Science and Technology (MOST), the National University of Laos, Beuth
University of Applied Sciences, and Brawijaya University. In addition, financial support from the Rachadapisek
Somphot Endowment under Outstanding Research Performance Program and the Rachadapisek Sompote Fund
for Postdoctoral Fellowship, Chulalongkorn University, are acknowledged.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
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