A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

Article abstract of DOI:10.1016/j.procbio.2011.10.023

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.

Full text of DOI:10.1016/j.procbio.2011.10.023

Process Biochemistry 47 (2012) 127–132
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
A glucose-tolerant ␤-glucosidase from Prunus domestica seeds: Purification and
characterization
Lei Chena, Ning Lib,∗∗, Min-Hua Zongb,∗
a College of Biosciences and Bioengineering, South China University of Technology, Guangzhou 510006, China
b State Key Laboratory of Pulp and Paper Engineering, College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 June 2011
Received in revised form 21 October 2011
Accepted 21 October 2011
Available online 25 October 2011
A glucose-tolerant ␤-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds
by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-
exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE
and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an
optimum activity at pH 5.5 and 55 ◦C. It is stable at temperatures up to 45 ◦C and in a broad pH range. Its
activity was completely inhibited by 5 mM of Ag+ and Hg2+. The enzyme hydrolyzed both p-nitrophenyl
␤-d-glucopyranoside with a Km of 3.09 mM and a Vmax of 122.1 ␮mol/min mg and p-nitrophenyl ␤-d-
fucopyranoside with a Km of 1.65 mM and a Vmax of 217.6 ␮mol/min mg, while cellobiose was not a
substrate. Glucono-␦-lactone and glucose competitively inhibited the enzyme with Ki values of 0.033
and 468 mM, respectively.
Keywords:
␤-Glucosidase
Kinetics
Prunus domestica
Purification
Seeds
© 2011 Elsevier Ltd. All rights reserved.
Substrate specificity
1. Introduction
While most microbial ␤-glucosidases are inhibited by glucose
[11,12], ␤-glucosidases of plant origin, especially those from plant
␤-Glucosidases (EC 3.2.1.21) catalyze the hydrolysis of gly-
cosidic linkages in aryl and alkyl ␤-d-glucopyranosides as well
as diglucosides and oligosaccharides. In plants, ␤-glucosidases
play an important role in diverse fundamental biological pro-
cesses including defense against pathogens and herbivores, scent
release, phytohormone conjugate activation and the degrada-
tion of cell wall oligosaccharides during germination [1]. In the
last two decades, ␤-glucosidases have attracted attention due to
their applications in food detoxification, biomass conversion and
flavor enhancement in wines and beverages [2–4]. In addition, ␤-
glucosidases can be used for the synthesis of various glycosides
[5–7] that are of commercial interest to the food, cosmetic, phar-
maceutical and detergent industries. For example, alkyl glycosides,
one of the most promising nonionic surfactants, have a variety of
unique features such as a good surfactant performance, low toxic-
ity and good biodegradability [8,9]. Furthermore, many glycosides
possess a number of biological activities including antioxidant, low-
ering of the blood glucose levels, protecting the membrane from
freeze–thaw damage and inhibiting tyrosinase [10].
seeds, are very useful for glycosides synthesis [13] or biomass
conversion [3], because these enzymes are tolerant to high concen-
trations of glucose [14]. Large amounts of fruit seeds are annually
disposed of as waste in the food processing industry [15], and the
use of the glycosidases from this industrial waste as biocatalysts
would provide an added value for these byproducts. The develop-
ment of a green enzymatic process for alkyl glycosides synthesis
could greatly reduce the production costs. Recently, we found a
novel ␤-glucosidase with a high activity from a group of fruit seeds
[16]. The crude enzyme from prune seeds was capable of catalyzing
the synthesis of a variety of alkyl ␤-d-glucopyranosides via reverse
hydrolysis with satisfactory yields. Prunes are a functional food
containing phenolic compounds, dietary fiber and potassium and
are thus beneficial for cardiovascular and bowel health. The chem-
icals present in prunes have been previously examined [17,18];
however, there are no reports of functional enzymes in prune seeds.
To better understand the characteristics of this novel ␤-glucosidase
and extend the knowledge about its potential roles, we purified and
characterized a prune seed ␤-glucosidase.
2. Materials and methods
∗
Corresponding author. Tel.: +86 20 8711 1452; fax: +86 20 2223 6669.
Corresponding author. Tel.: +86 20 2223 6669; fax: +86 20 2223 6669.
E-mail addresses: lining@scut.edu.cn (N. Li), btmhzong@scut.edu.cn
2.1. Materials
∗∗
Prune seeds were obtained from Yikang Food Processing Co. (Guangzhou, China).
(M.-H. Zong).
p-Nitrophenyl ␤-d-glucopyranoside (pNPG), 2-mercaptoethanol, esculin, arbutin
1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2011.10.023

128
L. Chen et al. / Process Biochemistry 47 (2012) 127–132
and glucono-␦-lactone were purchased from Sigma (St. Louis, MO, USA). Molec-
ular weight marker proteins for SDS-PAGE were obtained from TaKaRa (Dalian,
China). The gel filtration calibration kit (high molecular weight) for molecular
weight determination was purchased from GE Healthcare (USA). p-Nitrophenyl
␤-d-fucopyranoside (pNPF), p-nitrophenyl ␤-d-galactopyranoside, p-nitrophenyl
␤-d-xylopyranoside and p-nitrophenyl ␤-d-glucuronide were purchased from TCI
(Japan). Salicin and salicyl alcohol were obtained from Aladdin (Shanghai, China).
All other reagents were obtained from commercial sources and were of analytical
grade.
669,000). The subunit molecular mass was determined by sodium dodecylsulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) as described below.
2.5. Electrophoresis
2.5.1. SDS-PAGE
For the determination of the subunit molecular weight, SDS-PAGE was per-
formed on a 12.5% polyacrylamide gel in a Mini-PROTEAN® Tetra Cell (Bio-Rad, USA)
as described by Laemmli [19]. A mixture of molecular weight marker proteins was
used as a reference: lysozyme (Mw 14,300), trypsin inhibitor (Mw 20,100), carbonic
anhydrase (Mw 29,000), ovalbumin (Mw 44,287), bovine serum albumin (Mw 66,409)
and phosphorylase B (Mw 97,200).
2.2. Enzyme extraction and purification
Seeds were washed with deionized water, air-dried, shelled and powdered. The
meal was defatted three times with cold acetone (−20 ◦C), and then air-dried and
stored at 4 ◦C. The crude enzyme extract was prepared as follows: 20 g of defatted
prune seed meal was added to 50 mL of phosphate buffer (50 mM, pH 7.4) con-
taining 1 mM EDTA, 1 mM phenylmethanesulfonylfluoride and 100 mM NaCl. The
suspension was combined with quartz sand and milled using a mortar and pestle.
The extraction was performed for 1 h at 4 ◦C with gentle stirring after addition of
950 mL of phosphate buffer (50 mM, pH 7.4). The supernatant was collected as the
crude enzyme extract after centrifugation at 10,000 × g (4 ◦C) for 15 min.
Purification of the enzyme was carried out on an ÄKTA Purifier 100 (GE Health-
care, Piscataway, NJ, USA) at 20 ◦C.
Finely ground (NH4)2SO4 was slowly added to the crude enzyme extract to a 50%
saturation level, and after centrifugation at 14,000 × g for 15 min, the supernatant
was concentrated by ultrafiltration using a 30 kDa cutoff membrane (Pellicon XL
Biomax 30 50 cm2) in a LabscaleTM TFF System (Millipore, Bedford, MA, USA).
The enzyme solution was again centrifuged at 14,000 × g and then submitted to
a Phenyl Sepharose CL-4B hydrophobic chromatography column (1.6 cm × 20 cm,
GE Healthcare Amersham Biosciences) pre-equilibrated with 2 M (NH4)2SO4 dis-
solved in 50 mM phosphate buffer (pH 7.0). A stepwise elution was carried out with
decreasing concentrations of (NH4)2SO4 (2, 0.5 and 0 M) dissolved in 50 mM phos-
phate buffer (pH 7.0). Active fractions were eluted with 50 mM phosphate buffer (pH
7.0), pooled and concentrated with 10 kDa cutoff microcentrifuge filters (Sartorius,
Göttingen, Germany).
2.5.2. Native PAGE
For the ␤-glucosidase activity staining, native PAGE was performed on a 8.5%
polyacrylamide gel with Tris–glycine buffer (pH 8.8). After electrophoresis, an activ-
ity staining was carried out as described by Kwon et al. [20]. The gel was soaked in
0.2 M sodium acetate buffer (pH 5.0) for 10 min to exchange the buffer, and was
stained with 0.2 M sodium acetate buffer (pH 5.0) containing 0.1% (w/v) esculin and
0.03% (w/v) ferric chloride. The gel was incubated at 50 ◦C for several minutes until
a black band appeared.
2.5.3. IEF
Isoelectric focusing (IEF) was carried out on 24 cm ImmobilineTM Dry Strips
(pH 3–10, GE Healthcare) using an Ettan IPGphor 3 (GE Healthcare, USA), as recom-
mended by the manufacturer.
2.6. Effect of pH
The effect of pH on the activity of ␤-glucosidase was assessed in a buffer of
50 mM citric acid–Na2HPO4 (pH 4.0–8.0). To characterize the pH stability, the activ-
ity was measured after the enzyme (10 ␮L, 0.34 mg/mL) was incubated in the buffer
for 24 h at 25 ◦C and the designated pH.
2.7. Effect of temperature
The concentrated active fractions were dialyzed overnight at 4 ◦C in 20 mM
Tris–HCl buffer (pH 7.8) to exchange the buffer and remove any remaining
(NH4)2SO4 in the enzyme solution. The enzyme solution was loaded onto a Mono
QTM 5/50 GL anion exchange chromatography column (GE Healthcare Amersham
Biosciences, USA) pre-equilibrated with 20 mM Tris–HCl buffer (pH 7.8). A stepwise
elution was performed with increasing concentrations of NaCl (0.1, 0.15 and 0.2 M)
dissolved in 20 mM Tris–HCl buffer (pH 7.8). The active fractions were eluted with
0.2 M NaCl.
To determine the optimum temperature, the enzyme (10 ␮L, 0.34 mg/mL) was
preincubated for 3 min at the given temperature, and the activity was determined in
a temperature range of 25–65 ◦C. To characterize the thermal stability, the activity
was measured after incubating the purified enzyme (10 ␮L, 0.34 mg/mL) in 50 mM
citric acid–Na2HPO4 buffer (pH 5.5) at the designated temperature for specific inter-
vals (15–480 min).
2.8. Effects of metal ions and chemical reagents
2.3. ˇ-Glucosidase activity assay
The purified ␤-glucosidase was preincubated for 1 h at 4 ◦C in the presence of
different metal ions, and the activity was measured using the standard assay. The
effect of chemical reagents on the activity was examined by adding them directly
to the reaction mixture, followed by measuring the activity. The relative activity
was defined as the ratio of the enzyme activity in the presence of the tested ions or
reagents to that in the absence of metal ions and chemical reagents.
The pNPG solution was added to 50 mM citric acid–Na2HPO4 buffer (pH 5.5) con-
taining an appropriate amount of enzyme (10 ␮L, 0.34 mg/mL) at 45 ◦C to initiate the
reaction. If not otherwise specified, the concentration of pNPG was 1.0 mM in the
reaction mixture, and the total volume was 3.0 mL. The reaction was quenched after
3 min by the addition of 3 mL of Na2CO3 solution (1 M) and the absorbance at 402 nm
was recorded by a UV–VIS spectrophotometer (Shimadzu 2550, Japan). A reaction
mixture without the enzyme was used as a control. One unit of ␤-glucosidase activ-
ity was defined as the amount of enzyme that releases 1.0 ␮mol of p-nitrophenol
per minute under the above-mentioned conditions. For substrates that cannot be
assayed spectrophotometrically, the initial hydrolytic rate was determined by mea-
suring the release of the aglycon or glucose with HPLC. For the hydrolysis of arbutin,
the initial rate of release of hydroquinol was assayed by HPLC (Waters 1525, USA)
using an XBridgeTM C18 column (4.6 mm × 250 mm, 5 ␮m, Waters) and detection
at 280 nm. The mobile phase was a mixture of water and methanol (70/30, v/v)
with a flow rate of 1 mL/min, and the retention times of arbutin and hydroquinol
were 2.92 and 3.56 min, respectively. For the hydrolysis of salicin, the initial rate of
release of salicyl alcohol was assayed on the same C18 column as described above
with detection at 220 nm. The mobile phase was a mixture of water and methanol
(80/20, v/v) with a flow rate of 1 mL/min, and the retention times of salicin and sal-
icyl alcohol were 6.70 and 8.77 min, respectively. For the hydrolysis of cellobiose
and esculetin, the initial rate of the release of glucose was assayed on an Aminex®
HPX-87H ion exclusion column (BIO-RAD, USA) by HPLC (Waters 600, USA) with
a refractive index detector. The mobile phase was 5 mM sulfuric acid with a flow
rate of 0.5 mL/min, and the retention times of cellobiose and glucose were 9.71 and
11.99 min, respectively. The detection limits of cellobiose and glucose were 0.02 and
0.05 mM, respectively.
2.9. Kinetic parameters and inhibition constants
The kinetic studies of enzyme hydrolysis were performed in 50 mM citric
acid–Na2HPO4 buffer (pH 5.5) at 45 ◦
C with pNPG (0.37–8.98 mM) and pNPF
(0.35–8.37 mM) as the substrates. The Km and Vmax values were calculated from
Hanes–Woolf plots. The enzyme inhibition by glucose (49.7–497 mM) or glucono-
␦-lactone (0.2–3.0 mM) was studied in 50 mM citric acid–Na2HPO4 buffer (pH 5.5)
at 45 ◦C with pNPG as the substrate. The inhibition constants (Ki) were calculated
from Dixon plots [12].
2.10. Substrate specificity
To initiate the reaction, the purified enzyme (10 ␮L, 0.34 mg/mL) was added to
3 mL of citric acid–Na2HPO4 buffer (50 mM, pH 5.5) at 45 ◦C and containing each
tested substrate at a 2 mM final concentration.
3. Results and discussion
3.1. Purification
A three-step procedure was designed to purify the prune seed ␤-
glucosidase. The crude extract was first brought to 50% (NH4)2SO4
saturation to precipitate a large amount of undesired proteins. After
centrifugation, the supernatant was submitted to hydrophobic
interaction chromatography on a Phenyl Sepharose CL-4B column
that was pre-equilibrated with 2 M (NH4)2SO4 (Fig. 1A). A relatively
2.4. Molecular mass determination
The molecular mass of the native protein was determined by gel permeation
chromatography on a Superdex 200 PG column (1.2 cm × 82 cm) with a flow rate
of 1 mL/min and a mobile phase of 50 mM phosphate buffer (pH 7.0) containing
0.15 M NaCl. The column was calibrated with ovalbumin (Mw 44,000), conalbumin
(Mw 75,000), aldolase (Mw 158,000), ferritin (Mw 440,000) and thyroglobulin (Mw

L. Chen et al. / Process Biochemistry 47 (2012) 127–132
129
electrophoretically homogeneous. The subunit molecular mass of
this enzyme estimated by SDS-PAGE was approximately 61 kDa,
which is similar to several ␤-glucosidases from other plant sources
such as Prunus serotina Ehrh. (black cherry) seeds [21], apple seeds
[15], rye seedlings [22] and Citrus sinensis [23]. The molecular mass
of the native protein was estimated to be 54 kDa by gel permeation
chromatography, suggesting that ␤-glucosidase I is a monomer in
solution. The ␤-glucosidase isoenzymes, PH I and PH IIb, from black
cherry seeds were also reported to be monomeric glycoproteins
[24].
Native PAGE of the purified ␤-glucosidase I remained homo-
geneous, because a single band was observed upon staining with
Coomassie Brilliant Blue. In addition, the purified enzyme had
a single band at the corresponding position after staining for
␤-glucosidase activity with esculin (Fig. 2B). The pI value of ␤-
glucosidase I was determined to be 5.0 by IEF (data not shown),
which is higher than those (4.55 and 4.6, respectively) of ␤-
glucosidases from sweet almond [25] and Prunus avium L. [26],
but lower than those (5.8–6.4) of ␤-glucosidases from black cherry
seeds [24].
120
100
80
60
40
20
0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
A
2 M (NH4)2SO4
0.5 M (NH4)2SO
4 0 M (NH4)2SO4
0
50
100
150
200
250
Elution volume (mL)
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
B
I
80
60
40
20
0
0.2 M NaCl
As shown in Fig. 3, the optimum pH of ␤-glucosidase I is 5.5.
Similar to ␤-glucosidases from other plant sources such as apple
seeds [15] and soybean okra [27], ␤-glucosidase I was stable over a
broad pH range. For example, it retained approximately 90% of the
original activity after incubation at pH 8.0 for 24 h.
0.15 M NaCl
II
0.1 M NaCl
As shown in Fig. 4A, the optimum temperature of ␤-glucosidase
I is 55 ◦C, which is slightly lower than that (60 ◦C) of almond ␤-
glucosidase [15]. However, ␤-glucosidase I was not stable at the
optimum temperature; the half-life was approximately 23 min
(Fig. 4B). The enzyme was fairly stable at 45 ◦C, as it still maintained
76% initial activity after 480 min of incubation at this temperature.
Furthermore, it had excellent storage stability, keeping almost 100%
initial activity after storage for 15 days at 4 ◦C (data not shown).
The effects of various cations and chemical reagents on the activ-
ity of ␤-glucosidase I are shown in Table 2. Mn2+, Mg2+, Co2+ and
Ca2+ exerted little effect on the enzyme activity at 5 mM concen-
tration, while the activity was inhibited by 50–60% in the presence
of 5 mM Cu2+ and Zn2+. And the enzyme activity was not affected
by the chelating agent EDTA, implying that divalent cations are
not essential for the enzyme activity. Like other ␤-glucosidases
[14,15,28,29], ␤-glucosidase I was significantly inhibited by the
sulfhydryl oxidant Hg2+ and Ag+ metals ions. However, marginal
inhibition of the enzyme activity was caused by thiol-reducing
agents 2-mercaptoethanol at 40 mM and DTT at 5 mM (Table 2).
Lucas et al. proposed that the inactivation by sulfhydryl oxidant
metals might be ascribed to nonspecific salt formation rather than
complex formation with thiol groups and/or oxidation of thiol
groups [11]. So, thiol groups might be inessential for catalytic activ-
ity of ␤-glucosidase I. The detergents SDS and Tween 80 only had
negligible effect on the enzyme activity, but the enzyme lost 35%
initial activity in the presence of 1% (v/v) Triton X-100.
0
20
40
60
80
100
Elution volume (mL)
Fig. 1. Purification of ␤-glucosidase. (A) Hydrophobic interaction chromatography
on a Phenyl Sepharose CL-4B column. (B) Anion exchange chromatography on a
Mono QTM 5/50 GL column. The absorbance at 280 nm is shown as open symbols,
while the activity is shown as closed symbols.
high salt concentration was used because the main active protein
was precipitated at approximately 60% (NH4)2SO4 saturation. A
stepwise elution was used with decreasing (NH4)2SO4 concentra-
tions. The active fractions were collected upon elution with 50 mM
phosphate buffer (pH 7.0).
Next, anion exchange chromatography on a Mono QTM 5/50 GL
column was conducted, resulting in the separation of major (I) and
minor (II) peaks of activity (Fig. 1B). A stepwise elution, rather than
a continuous gradient elution, was chosen for the purification, as
this process minimizes the separation time and the consumption of
buffer and may be more practical for industrial scale-up. The entire
purification process is summarized in Table 1. The major active frac-
tion of ␤-glucosidase I was purified 59-fold to a specific activity of
23.1 U/mg protein with a yield of 5.4%.
3.3. Inhibitory constants
3.2. General properties
Glucose and glucono-␦-lactone are the two competitive
inhibitors of ␤-glucosidase. Therefore, high resistance to glu-
cose inhibition is one of the most important features of ideal
The purified ␤-glucosidase I gave a single major band on
SDS-PAGE (Fig. 2A), thus indicating that the obtained enzyme is
Table 1
Purification of ␤-glucosidase I from prune seeds.
Step
Total activity (U)
Total protein (mg)
Specific activity (U/mg)
Activity recovery (%)
Purification factor
Crude extract
1474
1382
179.6
80.3
3772
318.5
14.9
3.5
0.4
4.3
12.1
23.1
100
93.8
12.2
5.4
1.0
11.1
31.0
59.2
(NH4)2SO4 precipitation
Phenyl Sepharose CL-4B
Mono QTM 5/50 GL

130
L. Chen et al. / Process Biochemistry 47 (2012) 127–132
Fig. 2. Electrophoresis. (A) SDS-PAGE analysis (12.5% polyacrylamide) of protein samples from each step of the purification. The protein was stained with Coomassie Brilliant
Blue R-250: lane 1, molecular weight markers; lane 2, crude protein extract; lane 3, the supernatant after (NH4)2SO4 precipitation and ultrafiltration; lane 4, the active
peak from Phenyl Sepharose CL-4B; lane 5, the active peak I from Mono QTM 5/50 GL. (B) Native PAGE analysis (8.5% polyacrylamide) of the purified ␤-glucosidase I: lane 1,
␤-glucosidase activity stained with esculin; lane 2, protein stained with Coomassie Brilliant Blue R-250.
␤-glucosidases for industrial applications. Most microbial ␤-
glucosidases that have been tested are strongly inhibited by glucose
with Ki values of 1.3–11 mM [11,12,30], although there are several
reports on highly glucose-tolerant ␤-glucosidases from microbial
sources, such as Candida peltata (Ki = 1.4 M) [31] and Aspergillus
oryzae (Ki = 1.36 M) [29]. Hence, the effect of the two competi-
tive inhibitors on ␤-glucosidase I was evaluated with pNPG as
a substrate (Fig. 5). Lineweaver–Burk plots revealed that glucose
and glucono-␦-lactone followed competitive inhibition (data not
shown). Dixon plots were used to determine Ki values of 468
and 0.033 mM for glucose and glucono-␦-lactone, respectively.
Glucono-␦-lactone significantly inhibits ␤-glucosidase I, while glu-
cose does not, ␤-Glucosidases from black cherry seeds [21] and
sweet almond [25] were also inhibited by glucono-␦-lactone with
Ki values of 1.3 and 0.16 mM, respectively. However, 200 mM of
fructose, galactose, arabinose or xylose did not display the inhibi-
tion effect on ␤-glucosidase I (data not shown), which indicates
that this sugar-tolerant enzyme may be promising for industrial
applications.
3.4. Substrate specificity
The initial hydrolysis rates of various substrates and rela-
tive activities of ␤-glucosidase I are shown in Table 3. Similar
A
100
90
80
70
60
50
40
30
20
20
30
40
50
60
70
Temperature (ºC)
100
80
60
40
20
0
B
100
80
60
40
20
0
40 oC
45 oC
50 oC
55 oC
60 oC
0
100
200
300
400
500
4
5
6
7
8
pH
Time (min)
Fig. 3. Effect of pH on the hydrolysis of pNPG catalyzed by ␤-glucosidase I. Symbols:
activity (ꢀ); stability (ꢀ). The activity was measured in the pH range of 4.0–8.0. To
Fig. 4. Effect of temperature on the hydrolysis of pNPG catalyzed by ␤-glucosidase
I. (A) Activity; (B) stability. The activity was measured in the temperature range
of 25–65 ◦C. To characterize the thermal stability, the activity was measured after
incubating at the designated temperature and pH 5.5 for the designated time.
characterize pH stability, the activity was measured after incubating for 24 h at 25 ◦
and the designated pH.
C

L. Chen et al. / Process Biochemistry 47 (2012) 127–132
131
Table 2
Table 3
Effects of metal ions and reagents on the activity of ␤-glucosidase I.
The activity of ␤-glucosidase I on various substrates.a
Relative activity (%)
Substrate
Initial hydrolysis
Relative
rate (␮mol/min)
activity (%)
Control
100
pNPG
pNPF
0.261
0.633 0.007
100
Metal ions
Mn2+
Mg2+
242.5 2.1
19.5 0.7
2.3 0.0
nd
8.4 0.3
22.6 0.0
44.8 1.1
nd
5 mM
5 mM
5 mM
5 mM
1 mM
5 mM
1 mM
5 mM
1 mM
5 mM
1 mM
5 mM
105.3 0.3
97.0 0.2
92.2 0.1
103.3 0.1
63.2 0.2
46.3 0.2
64.2 1.3
41.1 0.8
27.2 0.3
0
p-Nitrophenyl ␤-d-galactopyranoside 0.051 0.001
p-Nitrophenyl ␤-d-xylopyranoside
p-Nitrophenyl ␤-d-glucuronide
Arbutinc
0.006 0.000
Co2+
ndb
Ca2+
Cu2+
0.022 0.001
0.059 0.000
0.117 0.003
nd
Salicinc
Esculetin
Zn2+
Hg2+
Ag+
Cellobiose (2 and 10 mM)c
a
Conditions: 50 mM phosphate buffer (pH 7.0), 45 ◦C, 2 mM substrate.
No activity detected.
Reaction for 1 h.
b
c
0
0
Reagents
2-Mercaptoethanol
DTTa
to ␤-glucosidases from other sources [11,12], ␤-glucosidase I
was also able to hydrolyze ␤-galactopyranoside (19.5%) and ␤-
xylopyranoside (2.3%). The enzyme had maximal hydrolytic activity
with pNPF as a substrate, which was approximately 2.4-fold higher
than that of pNPG at 2 mM. A similar phenomenon was also
observed in the cases of vanilla bean and rice ␤-glucosidases
[14,32]. Moderate activity was found in the enzymatic hydrolysis of
esculetin (44.8%). Arbutin and salicin were hydrolyzed at very slow
rates by ␤-glucosidase I (8.4% and 22.6%, respectively). No hydrol-
ysis occurred with p-nitrophenyl ␤-d-glucuronide and cellobiose
40 mM
5 mM
5 mM
5% (v/v)
1% (v/v)
5 mM
88.3 0.2
91.7 0.1
101.2 1.5
96.1 0.7
65.1 2.2
105.8 0.1
SDS
Tween 80
Triton X-100
EDTA
a
Dithiothreitol.
A
25
20
15
10
5
A
0.16
100
80
60
40
20
0
0.14
0.12
0.10
0.08
0.06
0.04
60
45
30
15
0
0
100 200 300 400 500
Glucose concentration (mM)
Ki = 468 mM
0
-2
0
2
4
6
8
10
-500 -400 -300 -200 -100
0
100 200 300 400 500
0.02
[S] (mM)
Glucose concentration (mM)
0
2
4
6
8
10
Concentration of pNPG (mM)
B
300
250
200
150
100
50
B
0.35
0.30
0.25
0.20
0.15
0.10
0.05
25
20
15
10
5
Ki= 0.033 mM
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0
-2
0
2
4
6
8
10
Glucono-
δ-lactone concentration (mM)
[S] (mM]
Fig. 5. Inhibitory kinetics. (A) A dixon plot of the inhibitory effect of glucose on pNPG
hydrolysis [pNPG: 1.37 mM (ꢁ) and 5.46 mM (ꢂ)]. (B) A dixon plot of the inhibitory
effect of glucono-␦-lactone on pNPG hydrolysis [pNPG: 1.37 mM (ꢁ) and 4.10 mM
(ꢂ)].
0
2
4
6
8
10
Concentration of pNPF (mM)
Fig. 6. Dependence of the ␤-glucosidase I activity on substrate concentrations. (A)
pNPG; (B) pNPF. Michaelis–Menten curves are shown with Hanes–Woolf plots inset.

132
L. Chen et al. / Process Biochemistry 47 (2012) 127–132
as the substrates. Previously, it was reported that ␤-glucosidases
from black cherry seeds [21,33], vanilla bean [14], maize [34] and
Arabidopsis thaliana [35] were also unable to catalyze the hydrolysis
of cellobiose. Prune seed ␤-glucosidase I was similar in substrate
specificity to vanilla bean ␤-glucosidase [14].
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Aspergillus niger. J Agric Food Chem 1998;46:431–7.
3.5. Kinetics studies
In the study of substrate specificity, both pNPG and pNPF were
found to be good substrates of ␤-glucosidase I. The effect of the sub-
strate concentration on the enzyme activity was evaluated (Fig. 6).
According to the Hanes–Woolf plot, the Km value was 3.09 mM and
the Vmax was 122.1 ␮mol/(min mg protein) with pNPG as the sub-
strate. The kinetic parameters of ␤-glucosidase I are similar to those
of ␤-glucosidases from other plants. For example, the Km and Vmax
values of almond ␤-glucosidase were reported to be 2.24 mM and
588 ␮mol/(min mg protein), respectively [36], and corn stover ␤-
glucosidase had a Km of 2.3 mM and a Vmax of 18.6 ␮mol/(min mg
protein) [3]. As shown in Fig. 6B, pNPF did not inhibit the activity of
␤-glucosidase I with concentrations up to 8.4 mM, and Km and Vmax
values of 1.65 mM and 217.6 ␮mol/(min mg protein), respectively,
were obtained with this compound. The lower Km and higher Vmax
suggest that the enzyme has a higher catalytic efficiency with pNPF
than with pNPG.
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4. Conclusions
␤-glucosidase I from prune seeds has potential in industrial
applications (the glycosides synthesis, etc.) because of its excellent
properties, such as being highly tolerant to glucose and stable in a
broad pH range. Indeed, we recently synthesized a group of alkyl
glycosides with satisfactory yields using the crude enzyme (in the
form of prune seed meal) [16]. In addition, this enzyme can be har-
vested from waste fruit seeds that are produced by food processing
industries, thus possessing the advantages of low cost and avail-
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presumed to be unrelated to the degradation of polysaccharides.
Like other ␤-glucosidases in Prunus plant seeds [37], it might be
involved in cyanogenesis as a chemical defense against herbivores
and microorganisms.
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okara shows specificity toward glucosyl Isoflavones.
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Acknowledgements
[29] Riou C, Salmon JM, Vallier MJ, Gunata Z, Barre P. Purification, char-
This research was financially supported by the National Nat-
ural Science Foundation of China (20906032, 20876059 and
21072065), the Major State Basic Research Development Program
‘973’ (2010CB732201), and the Research Fund for the Doctoral Pro-
gram of Higher Education (20090172110019).
acterization, and substrate specificity of
a novel highly glucose-tolerant
␤-glucosidase from Aspergillus oryzae. Appl Environ Microbiol 1998;64:
3607–14.
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tubingensis CBS 643.92: purification and characterization of four ␤-
glucosidases and their differentiation with respect to substrate specificity,
glucose inhibition and acid tolerance. Appl Microbiol Biotechnol 2001;55:
157–63.
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glucose-tolerant novel ␤-glucosidase from Candida peltata. Appl Environ
Microbiol 1996;62:3165–70.
[32] Seshadri S, Akiyama T, Opassiri R, Kuaprasert B, Cairns JK. Structural and
enzymatic characterization of Os3BGlu6, a rice ␤-glucosidase hydrolyzing
hydrophobic glycosides and (1-3)- and (1-2)-linked disaccharides. Plant Physiol
2009;151:47–58.
[33] Kuroki GW, Poulton JE. Isolation and characterization of multiple forms of
prunasin hydrolase from black cherry (Prunus serotina Ehrh.) seeds. Arch
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