Improved octyl glucoside synthesis using immobilized β-glucosidase on PA-M with reduced glucose surplus inhibition

Article abstract of DOI:10.1080/10242422.2017.1345886

A β-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH 6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobilization. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on dynamic and Dixon plot analyses, the initial reaction velocity (V₀) increased approximately three-fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dissociation constants for free and immobilized enzyme were 219 mM and 116 mM, respectively. A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336 h of reaction, the OG yield and the conversion rate of glucose reached 134 mM and 59.6%, respectively. Compared to the batch operation, the fed-bath operation increased the OG yield and the conversion rate of glucose by 340% and 381%, respectively.

Full text of DOI:10.1080/10242422.2017.1345886

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Improved octyl glucoside synthesis using
immobilized β-glucosidase on PA-M with reduced
glucose surplus inhibition
Feng Wang, Yong Ma, Yan-Hua Liu, Xuan Zhang, Fuming Zhang & Robert J.
Linhardt
To cite this article: Feng Wang, Yong Ma, Yan-Hua Liu, Xuan Zhang, Fuming Zhang &
Robert J. Linhardt (2017): Improved octyl glucoside synthesis using immobilized β-glucosidase
on PA-M with reduced glucose surplus inhibition, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2017.1345886
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1345886
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Date: 07 August 2017, At: 20:58

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
https://doi.org/10.1080/10242422.2017.1345886
RESEARCH ARTICLE
Improved octyl glucoside synthesis using immobilized b-glucosidase on
PA-M with reduced glucose surplus inhibition
a,bꢀ
a
a
a
cꢀ and Robert J. Linhardtcꢀ
ꢀ
ꢀ
ꢀ
Feng Wang , Yong Ma , Yan-Hua Liu , Xuan Zhang , Fuming Zhang
a
b
School of Chemical and Material Engineering, Jiangnan University, Wuxi, China; The Key Laboratory of Food Colloids and
c
Biotechnology of Ministry of Education, Jiangnan University, Wuxi, China; Department of Chemistry and Chemical Biology,
Departments of Chemical and Biological Engineering, Biology and Biomedical Engineering, Center for Biotechnology and
Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA
ABSTRACT
ARTICLE HISTORY
A b-glucosidase extracted from bitter almond (Prunus dulcis var. amara) was immobilized on
polyamine microspheres (PA-M) for catalytic octyl glucoside (OG) synthesis from glucose and
octanol through reversed hydrolysis. The immobilization increased the activity of enzyme at pH
Received 8 January 2017
Revised 21 March 2017
Accepted 9 June 2017
6.0–7.0, and the optimal reaction temperature for immobilized enzyme was identical to the free
enzyme. The thermal stability and solvent tolerance of enzyme were increased by its immobiliza-
tion. In the co-solvent system using 10% t-butyl alcohol and 10% (v/v) water, the yield of OG
was increased by 1.7-fold compared to the yield from the system without co-solvent. Based on
KEYWORDS
Octyl glucoside;
b-glucosidase; PA-M;
immobilization; glucose
inhibition; fed-batch
dynamic and Dixon plot analyses, the initial reaction velocity (V ) increased approximately three-
0
fold on immobilization and the OG synthesis was inhibited by surplus glucose. The inhibition dis-
sociation constants for free and immobilized enzyme were 219mM and 116 mM, respectively.
A fed-batch mode was applied in the OG synthesis to minimize substrate inhibition. After 336 h
of reaction, the OG yield and the conversion rate of glucose reached 134 mM and 59.6%,
respectively. Compared to the batch operation, the fed-bath operation increased the OG yield
and the conversion rate of glucose by 340% and 381%, respectively.
Introduction
a short alcohol (usually butanol) producing the corre-
sponding alkyl glycoside, which is subsequently transa-
cetalated with a longer chain alcohol. Unfortunately,
chemical procedures for the preparation of pure glyco-
sides require multiple protection and deprotection
steps increasing the cost of producing the long-chain
alkylglucosides (Ducret et al. 2006). An alternative to
chemical syntheses of glycosides is enzymatic synthe-
sis relying on glycosidases that use mild reaction con-
ditions and are regio- and stereo-selective (Rather and
Mishra 2013).
Alkyl glycosides belong to a family of low toxic bio-
degradable nonionic surfactants with properties of
detergency, foaming, wetting and emulsification. They
have potential applications in the food, cosmetic,
pharmaceutical and detergent industries (Bhatia et al.
2002; Dembitsky 2004; Lalitha et al. 2015). Short-chain
alkyl glucosides show a low level of surface activity,
while the long-chain alkyl glucosides, such as octyl
glucoside (OG), show significant surface activity (Lopez
et al. 2001). OG has been used for solubilization
b-Glucosidase (b-D-glucoside glucohydrolase, EC
(
(
Rosevear et al. 1980), reconstitution of carrier
Newman et al. 1981) and crystallization of membrane
3.2.1.21) is capable of catalysing the hydrolysis of
b-glucosidic linkages in disaccharides and oligosac-
proteins (Prive 2007).
charides and other glycoconjugates (Chang et al.
Chemical methods for long-chain alkyl glycoside
synthesis mainly rely on Fischer glycosylation and
transacetalization (Corma et al. 1998). In the Fischer
glycosylation, alcohol reacts directly with the sugar in
the presence of catalysts to produce alkyl glycoside. In
transacetalization, the carbohydrate is first coupled to
2
013). b-Glucosidase can also be used in the synthesis
of various glucosides by reverse enzymatic catalysis
(Figure 1) (Van Rantwijk et al. 1999; Yu et al. 2008).
The synthesis of alkyl glycosides catalysed by glycosi-
dases can be carried out through the reversed hydroly-
sis and transglycosylation (Van Rantwijk et al. 1999).
CONTACT Feng Wang
These authors contributed equally to this work.
fwang@jiangnan.edu.cn
School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China
ꢀ
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

2
F. WANG ET AL.
CH OH
2
CH OH
CH OH
2
2
O
O
OH
O
OH
OH
O
Octanol
OH
+
H O
2
O
C
O
OH
OH
OH
OH
OH
OH
Active site
of β-glucosidase
Hydrolysis
Glucose surplus inhibition
CH OH
2
O
OH
OH
CH OH
2
O
OH
OH
OH
CH OH
2
O
C
O
O
OH
OH
OH
O
Active site
of β-glucosidase
OH
OH
Figure 1. Reaction scheme of the conversion of glucose to octyl-b-glycoside (OG) in octanol (reversed hydrolysis) catalysed by
b-glycosidase. Conversion of OG to glucose with water (hydrolysis reaction) was a side reaction. The secondary side reaction is the
glucose inhibition on the OG synthesis.
Transglycosylation can give higher yields than the
reversed hydrolysis does. However, reversed hydrolysis
is more facile and economic by using monosaccharides
and alcohols as the reactants or substrates and
demands less complicated downstream processing for
separating the by-products (Yang et al. 2015).
Glucosidases have been applied in the enzymatic
synthesis of OG. Ducret et al. reported 13 glycosidases
for the synthesis of OG and found almond b-glucosi-
dase was less inhibited by high concentration of glu-
cose (Ducret et al. 2002b). The rate of reaction and the
percent conversion could be enhanced by using co-
solvents, especially N,N-dimethylformamide (DMF)
improved by its immobilization to Eupergit C resins
(Tu et al. 2006). Polyethylene imine (PEI) was used to
enhance the stability of b-glucosidase immobilized on
agarose matrix (Vieira et al. 2011). Polyamine micro-
spheres (PA-M), developed in our laboratory from PEI
through emulsion polymerization, are microspheres
with a high density of ionizable amino groups avail-
able to load enzymes through the electrostatic interac-
tions (Wang et al. 2012, 2014). In this study, a
b-glucosidase extracted from bitter almond was immo-
bilized on PA-M. We measured the kinetic characteris-
tics of this immobilized b-glucosidase for OG synthesis,
thermal stability and solvent tolerance. A fed-batch
mode was designed and investigated to reduce glu-
cose inhibition of enzymatic OG synthesis.
(
Ducret et al. 2002a). The synthesis of OG was also
examined through transglycosylation with almond
b-glucosidase (Mladenoska 2016). The conversion rate
reached 38%, which was higher than that (15.2%)
obtained in reverse hydrolysis. However, the p-nitro-
phenyl-b-glucoside glucose donor used in transglyco-
sylation is more expensive than the final product OG,
thus, hampering its application.
Compared to the soluble enzymes, immobilized
enzymes have the advantages of reusability and
increased stability against inactivation (Wang et al.
Materials and methods
Materials
Bitter almond (Prunus dulcis var. amara) used for b-glu-
cosidase extraction was a gift from Zhangye Chinese
Medicine Technology Co., Ltd., Gangsu, China.
4-Nitrophenyl b-D-glucopyranoside (pNPG), p-nitrophe-
nol (pNP), dimethyl sulphoxide (DMSO), DMF, t-butyl
alcohol (t-BA), dimethoxyethane (DME), acetonitrile,
DEAE52-cellulose, SephadexG-100, polyethyleneimine
(Mw ¼70,000 Da; 50 wt% solution in water) were pur-
chased from Sigma-Aldrich, Shanghai Trading Co. Ltd.
(Shanghai, China). Glutaraldehyde solution (25 wt%),
liquid paraffin, Span-80 were obtained from
2
014). Various immobilization methods, such as linkage
to solid supports, entrapment in hydrogels and micro-
encapsulation, have been used for glucosidases from
different sources (Ducret et al. 2006; Synowiecki and
Wolosowska 2006; Wei et al. 2013). Enzyme stability is
typically improved through immobilization. For
example, the thermal stability of b-glucosidase

BIOCATALYSIS AND BIOTRANSFORMATION
3
Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China).
or immobilized enzyme (20 mg) was allowed to react
with 300 lL pNPG (10 mM dissolved in KPB) for 120 s
using 50 lL of buffer as a control. The absorbance at
4
05 nm was measured with a microplate reader
Extraction and purification of b-glucosidase
TM
(Thermo Scientific , Waltham, MA). One unit of b-glu-
b-Glucosidase was extracted and purified from the
cosidase hydrolysis activity was defined as the amount
of enzyme that released 1.0 lmol pNP/min at 50 C.
ꢁ
fresh bitter almonds according to
a
previously
described method (Yu et al. 2007). The fresh almonds
were first peeled and powdered, washed three times
with ethyl acetate, and two times with acetone at
room temperature to remove lipid and then dried in a
vacuum oven at 4 C. Next, 1 g of defatted almond
powder was soaked in 25 mL sodium phosphate buffer
Immobilization of b-glucosidase on PA-M
microspheres
ꢁ
The preparation of the PA-M microspheres was con-
ducted according to the method reported in our previ-
ous studies (Wang et al. 2012, 2014). PA-M (50 mg)
was incubated with 2 mL b-glucosidase buffer solution
(10 mg/mL) on a rotary shaker with an agitation speed
(
1
50 mM, pH 7) for 2 h and centrifugation (10,000ꢂg,
0 min). The supernatant was added with finely
ground ammonium sulphate crystals to achieve 30%
saturation to remove the impurity by centrifugation at
ꢁ
of 500 rpm at 25 C for 4 h. The adsorbed enzyme was
1
0,000ꢂg for 10 min. The precipitate was discarded
separated by filtration and washed three-times with
the same buffer to remove unbound enzyme. The
b-glucosidase adsorbed PA-M was put into a 2 mL of
0.1 wt% glutaraldehyde solution (pH 7.5) and incu-
bated for one hour on a rotary shaker (300 rpm, 25 C).
The immobilized b-glucosidase was collected by filtra-
and the supernatant was collected. By adding ground
ammonium sulphate crystal, the saturation level of
ammonium sulphate in the supernatant was increased
to 70% to precipitate the crude enzyme. After centrifu-
gation (10,000ꢂg for 10 min), the crude enzyme was
collected and reconstituted in 50 mM sodium phos-
phate solution of pH 7 at the ratio of 1:1 (w/w). The
ammonium sulphate in the enzyme solution was
ꢁ
tion and washed four-times with distilled water and
ꢁ
stored at 4 C. The coupling capacity of b-glucosidase
on PA-M was 147 mg/g, and the specific activity of the
immobilized enzyme was 2.01 U/mg protein.
ꢁ
removed by dialysis at 4 C overnight against the same
buffer.
The immobilization yield was defined as follows:
ꢀ
ꢁ
The enzyme solution obtained after dialysis was
applied to a DEAE52-cellulose column (1.0 cm ꢂ20 cm)
pre-equilibrated with 10 mM Tris–HCl buffer of pH 7.
The elution was conducted with NaCl gradient from
aimm
afree
Immobilization yield % ¼
ꢂ 100
where aimm is the specific activity of immobilized
enzyme (U/mg protein) and afree is the specific activity
of the initial enzyme preparation (U/mg protein).
The enzyme recovery of the process was calculated
as the ratio of the total amount of bound enzyme to
the total amount of enzyme initially present in the
immobilization mixture.
0
M to 0.15 M at a flow rate of 0.5 mL/min.
Fractions containing b-glucosidase activity were col-
lected and concentrated by ultrafiltration with
V
R
Amicon Ultra-15 centrifugal filter devices (Millipore of
0 kDa). The concentrated enzyme was further purified
1
on a Sephadex G100 column (2.0 cm ꢂ100 cm) eluted
with 10 mM pH 7.4 Tris–HCl buffer at a flow rate of
0
.3 mL/min. The fractions possessing the highest activ-
Determination of enzyme kinetics parameters
ꢁ
ity of b-glucosidase were pooled and stored at 4 C.
Protein concentration was determined by the Bradford
method (Bradford 1976) using bovine serum albumin
as a standard and the enzyme purity and molecular
weight were measured by SDS-PAGE (Laemmli 1970).
The hydrolysis activity and kinetics of immobilized
b-glucosidase were determined using pNPG as the
ꢁ
substrate at 50 C, pH 7.0. The Michaelis–Menten kin-
etic parameters of both free and immobilized b-gluco-
sidase were determined from a Lineweaver–Burk plot.
In the b-glucosidase catalysed synthesis of OG, an
excess amount of octanol was used compared to the
amount of glucose, the kinetics of glucose inhibition
on the OG synthesis could be described using the
reduced Haldane equation (Sonnad and Goudar 2004).
The kinetic parameters, inhibition dissociation constant
b-glucosidase activity assay
The hydrolytic activity of b-glucosidase was deter-
mined by measuring the release of p-nitrophenol
(
NPG) from p-nitrophenyl b-D-glucopyranoside (pNPG).
ꢁ
All samples were assayed at 50 C in potassium phos-
phate buffer (KPB, 50 mM, pH 7.0). Free enzyme (50 lL)
(K ), maximal reaction velocity (Vmax
)
and the
SI

4
F. WANG ET AL.
Michaelis–Menten constant (K ), were determined
from Dixon plots.
buffer (pH 6.0, 50 mM). The reaction, performed at
50 C, was initiated by adding 5 U of enzyme. After
m
ꢁ
4
1
8 h of reaction, the reaction mixture was added to
0 mL methanol and filtered through a 0.22 lm mem-
Influences of temperature and pH on the activity
of immobilized b-glucosidase
brane. The amount of OG in the filtrate was deter-
mined by HPLC.
The effect of temperature on the activity of b-glucosi-
dase was determined at a pH 5.5 over temperatures
ranging from 40 to 70 C. The effect of pH on b-gluco-
sidase activity was determined in 50 mM citric acid–ci-
trate buffer (pH 4.0–6.0) and 50 mM sodium phosphate
Influence of co-solvent addition on the OG
synthesis catalysed by the immobilized enzyme
ꢁ
In a 2 mL of the reaction mixture, 0.5 mmol glucose
and 0.2 mL of sodium phosphate buffer (pH 6.0,
50 mM), either DMSO, DMF, t-BA, DME, or acetonitrile
were added together with octanol. The concentration
of co-solvent was 2%, 5%, 10%, 20% and 30% (v/v
total volume) with 15% water in octanol solution.
Immobilized b-glucosidase (5 U) was added to initiate
(
pH 6.0–8.0) buffer. The relative activity (%) was calcu-
lated by comparing the activity of every sample with
the maximum activity (defined as 100%). The data
shown in the figures are average values and standard
deviations of triplicated experiments.
ꢁ
the reaction at 50 C on a shaker with an agitation
Evaluation of thermal stability and solvent
tolerance of b-glucosidase
speed of 200 rpm. After 48 h, the reaction mixture was
added with 10 mL methanol and was filtered with
0.22-lm membrane. The amount of OG in the filtrate
was measured and the concentration of OG in the
reaction medium was calculated.
The thermal stabilities of the free and immobilized
b-glucosidase were tested by incubating the enzymes
ꢁ
at 30–100 C for 48 h in sodium phosphate buffer of
pH 5.5. The enzymes were cooled in an ice bath and
the residual activities were measured. The initial activ-
ity of the enzyme was defined as 100%.
Influence of water content on the synthesis of OG
The solvent tolerance of b-glucosidase was exam-
ined by incubating the enzyme at 50 C in different
co-solvent–octanol–water buffer systems. The water
content in all tested systems was set at 15% (v/v total
volume). The concentration of co-solvents, namely
DMSO, DMF, t-BA, DME, and acetonitrile, was 2%, 5%,
Glucose (0.5 mmol) and 0.1 mL t-BA were added to
1.9 mL mixture of sodium phosphate buffer (pH 6.0,
50 mM) and octanol. The effect of water content was
investigated at the levels of 5%, 10%, 15%, and 20%
(v/v total volume). The reaction was initiated by add-
ꢁ
ꢁ
ing 5 U of enzyme and carried out at 50 C for 48 h on
1
0%, 20% and 30% (v/v total volume), respectively.
a shaker at a speed of 200 rpm. After the reaction,
10 mL of methanol was added to the reaction mixture,
which was then filtered through a 0.22-lm membrane
and the amount of OG in the filtrate was determined.
Correspondingly, the final concentration of octanol
was 83%, 80%, 75%, 65% and 55%, respectively. In the
testing of the solvent tolerance of the free enzyme,
5
U b-glucosidase was added into a 1 mL of co-solvent–
octanol–water buffer with various content of co-solv-
ent. After incubation for 48 h, 50 lL of enzyme solution
was dawn, and the activity of enzyme was determined
with pNPG as the substrate. For testing of the
solvent tolerance of the immobilized enzyme, 20 mg
immobilized enzyme was incubated in 1 mL of
co-solvent–octanol–water buffer for 48 h. After that,
the enzyme was separated and the activity was
assayed. b-Glucosidase activity was expressed as %
residual activity compared to the initial activity of the
enzyme.
Synthesis of OG in a fed-batch mode
Based on the optimized reaction conditions of the
batch operation, a glass reactor was designed for OG
synthesis in fed-batch mode. The reactor with a work-
ing volume of 100 mL was comprised of a neck jack-
eted glass cylinder with three outlet ports, one for
feeding the glucose powder, one connecting stirrer,
and another for sampling. The temperature was main-
ꢁ
tained at 50 C by circulating hot water through outer
jacket. The reactor (with a volume of 80 mL) was filled
with 64 mL of n-octanol, 12 mL of sodium phosphate
buffer (50 mM, pH 6.0), 4 mL t-BA, 225 mM glucose and
General procedure for enzymatic glucosylation
200 U of enzyme. The glucose powder was fed every
D-Glucose (0.5 mmol), octanol (10 mmol) and 0.1 mL
t-BA were added into 0.3 mL of sodium phosphate
6 h to keep the glucose concentration in the reactor
nearly constant.

BIOCATALYSIS AND BIOTRANSFORMATION
5
The feeding speed of glucose was determined
according to following equation:
14 carbon signals, including the carbon signals from
1
3
glucose and octanol. C NMR ((MeOD): d 13.94 (C-8),
2.08 (C-7), 25.53 (C-3), 28.69 (C-4), 28.88 (C-5), 29.27
2
dðVrCSÞ
¼
FC ꢃ V M
(1)
0
0
S0
r
t
(C-2), 31.25 (C-6), 61.10 (C-6 ), 68.53 (C-1), 70.10 (C-4 ),
dt
0
0
0
0
73.43 (C-2 ), 76.79 (C-3 , C-5 ), and 102.83 (C-1 ). 22.08,
where V is the volume of reaction mixture in the
r
2
5.53, 28.69, 28.88, 29.27, and 31.25 for CH -alkyl chain
2
reactor (80 mL); C is the glucose concentration in the
s
and 13.94 for alkyl CH . The peak at 102.83 (doublet)
3
reactor (mM); CS0 is glucose concentration in the feed-
ings (5.55 mmol/g); F is the feeding speed of ground
can be assigned to anomeric carbon. Mass spectra
were recorded on a Bruker APEXIII 7.0TESL A FTMS
D-glucose powder (g); M is the consumption rate of
t
þ
(
Billerica, MA) using ESI mode: m/z 315 (M þ Na ) was
glucose in the reactor (mM/h); t is the reaction time
detected for the molecular ions of OG.
(
h), assuming the addition of glucose powder had no
effect on the volume of the reaction mixture and the
glucose concentration remains constant upon reaching
a pseudo-steady state. Therefore, Equation (1) can be
reduced to be Equation (2).
Results and discussions
b-Glucosidase purification and immobilization
b-Glucosidase was extracted and purified from the fresh
bitter almonds by two steps of ammonium sulphate pre-
cipitation, two steps of chromatography (DEAE column
and Sephadex G100 column) and ultrafiltration. The
overall b-glucosidase extraction and purification yield
from bitter almonds was 0.456 U/g dry bitter almonds.
The molecular weight of purified b-glycosidase was esti-
mated to be 68 kDa by PAGE analysis (Figure 2).
F ¼ VrMt
(2)
Samples were taken at regular intervals for the esti-
mation of residual glucose and the yield of OG.
OG determination by HPLC
The quantity of OG was measured by HPLC equipped
with an ODS column (YMC-Pack ODS-AQ Ø250 mm ꢂ
PEI contains high density of amino functional groups
and has been used in the immobilization of enzymes as
the coating or building material of carriers for enzyme
4
.6 mm, 5 lm) and a refractive-index detector (model
RID-10A; Shimadzu, Kyoto, Japan). HPLC was carried
out at the room temperature using a methanol–water
mixture (80:20, v/v) as the mobile phase at a flow rate
of 1.0 mL/min. The analytical errors were less than 1%.
(Brady and Jordaan 2009). Microspheres of PA-M synthe-
sized by cross-linking PEI through emulsion
Purification of OG (Ismail et al. 1999)
After evaporation of the t-TA and water under vac-
uum, OG in the reaction mixture was purified on a sil-
ica gel liquid chromatography column (70 ꢂ 3 cm,
Merck, Kenilworth, NJ) using a dichloromethane–me-
thanol solution (8:2, v/v) as the mobile phase at a flow
rate of 4 mL/min.
Structure determination of OG by NMR and mass
spectroscopy
1
H NMR of OG was recorded on Bruker FT DRX 500
(
500 MHz). The chemical shifts were determined in
parts per million (ppm) referenced to the appropriate
solvent peak, methanol-d (MeOD), or to 0.0 ppm. The
Figure 2. SDS-PAGE of b-glycosidase isolated from bitter
almond during the process of purification (the lane on the far
left is marker; lane 1, the crude extract; lane 2, the extract
treated with 30% ammonium sulphate; lane 3, the crude
enzyme precipitated by increasing the saturation level of
ammonium sulphate in supernatant from 30% to 70%; lane 4,
the enzyme obtained after purifying the crude enzyme on a
DEAE52-cellulose column; lane 5, the final pure enzyme
obtained using Sephadex G100 column purification).
1
doublet at 4.24 (J ¼ 7.79 Hz) in the H NMR of purified
OG confirmed b-configuration at the anomeric centre.
1
H NMR (MeOD): d 4.24 (d, J ¼ 7.79 Hz, 1H), 3.93–3.74
(
m, 3H), 3.59 (dd, J ¼ 16.1, 7.4 Hz, 1H), 3.48 (d,
J ¼ 8.3 Hz, 2H), 3.37 (s, 1H), 3.30 (t, J ¼ 8.0 Hz, 1H), 1.65
(
(
s, 2H), 1.31 (d, J ¼ 13.0 Hz, 10H), and 0.88
13
d, J ¼ 11.5 Hz, 3H). The C NMR spectrum recorded

6
F. WANG ET AL.
(
A)
(B)
1
00
100
90
80
70
60
50
40
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
Free enzyme
Immobilized derivative
30
20
10
0
Free enzyme
Immobilized derivative
40
45
50
55
60
65
70
4.0
4.5
5.0
5.5
pH
6.0
6.5
7.0
Temperature °C
Figure 3. The hydrolysis activities of free b-glucosidase and the PA-M immobilized b-glucosidase under various values of pH (A)
and temperature (B).
polymerization have the diameter distribution in the
range of 0.37–4.29 lm (Figure S1). Analysis of the sur-
face charge of PA-M using a ZetaPlus zeta potential ana-
lyser (Brookhaven Instruments Corporation, Holtsville,
NY) demonstrated the isoelectric point (pI) of the micro-
spheres was 10.6. Element analysis using a Vario EL III
elemental analyser (Elemental Analysis, Langenselbold,
Germany) demonstrated the N content of the micro-
spheres was 16.58 ± 0.15% (wt%). Enzyme was first incu-
bated with PA-M at neutral pH to be physically
adsorbed on the microspheres. Electrostatic attraction
between the amino groups of PA-M and the charged
b-glucosidase (pI 4.3) together with hydrogen bonds
and Van der Waals forces drive this adsorption. The
adsorbed enzyme was then treated with glutaraldehyde
under mild conditions. This results in cross-linking
between the amino groups of the enzyme and the
amino groups of the support establishing multi-point
covalent enzyme-support attachment. Under the given
conditions of immobilization, the immobilization yield
reached 84.8% and the enzyme recovery was 58.4%.
higher relative activity than the free enzyme. This may
be due to the protonation of amino groups of PA-M con-
structing suitable microenvironments around the bound
enzyme molecules (Akinc et al. 2005).
The effect of temperature, on the activity of the free
and immobilized b-glucosidases, was examined at pH
ꢁ
ꢁ
6.0 over temperatures ranging from 40 C to 70 C
(Figure 3(B)). The activities of both free and immobilized
enzymes increased with increased temperatures below
ꢁ
6
0 C. The activities of both enzymes reached a max-
ꢁ
imum at 60 C. Thus, the optimal temperature for b-glu-
cosidase was unchanged after immobilization. The
immobilized enzyme retained about 70% of its initial
activity, while the free enzyme only retained about 40%
ꢁ
of its initial activity at 70 C (Figure 3(B)). This suggests
that immobilization protects b-glucosidase from the
thermal deactivation. Besides, the enhancing enzyme
activity, high temperatures may also thermodynamically
increase the reaction velocity.
The slopes of the temperature–activity profiles
(
Figure 4), within the region where temperature
dependent denaturization did not occur (Figure 3(B)),
were used to calculate the activation energy of the
free and immobilized enzyme mediated reaction using
the Arrhenius equation (Wang et al. 2014). The activa-
tion energy was 3.45 kJ/mol for the free enzyme and
3.06 kJ/mol for the immobilized derivative. The slightly
reduced activation energy of the reaction catalysed by
immobilized b-glucosidase implied that the active con-
formations of the enzyme molecules were probably
maintained during the immobilization.
Optimal pH and temperature for the hydrolysis
activity of the free and immobilized b-glucosidase
The activity of b-glucosidase is in direct relationship
with the specific protonation status of catalytic amino
acids (glutamate or aspartate) in the active sites of the
enzyme, which is influenced by pH (Woodward and
Arnold 1981). The pH-activity profiles of the free b-glu-
ꢁ
cosidase and the immobilized b-glucosidase at 50 C are
shown in Figure 3(A). The relative activity of both free
and immobilized enzyme increased with increasing pH
and reaching an optimum value at pH 6.0. These activ-
ities sharply decreased when the pH exceeded 7.0. The
relative activities of enzyme decreased after immobiliza-
tion in the pH range of 4.0–6.5. However, in the range of
pH 6.0–7.0, the immobilized enzyme maintained a
Thermal stability and organic solvents tolerance
of b-glucosidase
Next, the thermal stability and organic solvent toler-
ance of b-glucosidase were investigated. The

BIOCATALYSIS AND BIOTRANSFORMATION
7
5
5
4
4
3
3
2
2
1
1
.5
.0
.5
.0
.5
.0
.5
.0
.5
.0
challenging due to the poor miscibility of octanol in
water and the low solubility of glucose in organic
media. One approach for promoting long-chain alkyl
glucoside synthesis is to increase the miscibility of gly-
cone and aglycone in the reaction media by adding a
water-miscible solvent as the co-solvent. For instance,
Ducret et al. found the reaction rate and the yield of
OG synthesis, catalysed by immobilized almond b-glu-
cosidase, could be enhanced by using acetonitrile and
DMF co-solvents (Ducret et al. 2002a). However, a lim-
iting factor for application of co-solvent in enzyme-
mediated alkyl glucoside synthesis is the low activity
and stability of enzymes in non-aqueous environments
due to the decreased water activity (Ducret et al.
Free enzyme
The immobilized enzyme
3.00
3.05
3.10
3.15
3.20
/T (K−1
1
)
2
002a). The tolerance of b-glucosidase to the different
Figure 4. Arrhenius plots for the free enzyme (
w
) and the
contents of co-solvents were tested here by determin-
ing the activity of enzyme after incubating the enzyme
in the co-solvent–octanol–water system at 50 C for
immobilized derivative (ꢄ). The slope of a logarithmic
Arrhenius plot is related to the activation energy (Ea) for the
molecule by the relationship: slope ¼ –Ea/R, where R (8.314 J/
mol/K) is the gas constant.
ꢁ
4
8 h and the results are presented in Figure 6. The
selected co-solvents, DMSO, DMF, t-BA, and DME, are
organic co-solvents that have been reported to be
widely used in the non-aqueous system of enzymatic
catalysis for enhancing the miscibility of solvents and
buffers (Karagulyan et al. 2008). Free enzyme could
maintain 54% of its activity after incubating in the
pure octanol (Figure 6). Enzyme activity decreased as
the concentration of added co-solvents increased.
DMSO and DMF addition had the greatest negative
impact on the activity of the free b-glucosidase. The
free enzyme lost all of its activity, in a co-solvent–octa-
nol–water buffer system, when the concentration of
DMSO and DMF addition was >2% (v/v). The highest
value of residual relative activity (48%) for the free
enzyme was observed in the case of t-BA addition.
The immobilization with PA-M as the support signifi-
cantly increased the stability of b-glucosidase in the
presence of co-solvent based on the results in Figure
1
00
Free enzyme
Immobilized enzyme
8
6
4
2
0
0
0
0
0
2
0
30
40
50
60
70
80
90
100
T (°C)
Figure 5. Thermal stability of the free and immobilized
b-glucosidase.
immobilized enzyme retained about 40% of its initial
activity after incubating at 80 C for 48 h while the free
6(B). The highest residual activity of the immobilized
ꢁ
enzyme was demonstrated in the system using t-BA as
the co-solvent. Approximately 80% residual relative
activity could be retained after 48 h of incubation in
the medium containing 2–10% t-BA. It is believed the
polarity of organic solvent used in bio-catalysis has a
direct relation with the activity of enzyme, and the
logarithm of the partition coefficient, log P, has been
proposed as a quantitative measure of solvent polarity
b-glucosidase almost lost all of its activity (Figure 5).
The results suggest that the thermal stability of b-glu-
cosidase increased on immobilization. Other carriers
for b-glucosidase immobilization, such as chitosan and
Eupergit C resin, were also found to enhance enzyme
thermal stability (Tu et al. 2006).
The commonly used glycone in alkyl glucoside syn-
thesis, catalysed by glycosidase through reversed
hydrolysis, is carbohydrate like glucose, which is very
soluble in water but nearly insoluble in organic sol-
vents, especially for long-chain alkyl glucoside synthe-
sis. In contrast, aglucones, such as octanol used in OG
synthesis, are poorly soluble in water. Thus, the
enzymatic synthesis of OG using reversed hydrolysis is
(
(
Dordick and Isermann 2009). Hydrophilic solvent
log P < 2) can strip away the water associated with
the enzyme resulting in catalytic deactivation. The
value of log P of octanol is 2.9 and other co-solvents
show much lower log P values, i.e. –1.3 (DMSO), –1.0
(DMF), –0.33 (acetonitrile), –0.2 (DME), and 0.584(t-BA),
respectively. Owing to their low values of log P, the

8
F. WANG ET AL.
(
A) 60
(B) 100
90
1
00% Octanol
DMSO
DMF
t-BA
DME
Acetonitrile
55
50
45
40
35
30
25
20
15
10
5
0
DMSO
DMF
t-BA
DME
Acetonitrile
80
7
6
0
0
1
00% Octanol
50
40
30
20
10
0
0
2
5
10
20
30
0
2
5
10
20
30
Co-solvent content (% v/v total volume)
Co-solvent content (% v/v total volume)
Figure 6. Organic solvent tolerance of the free (A) and immobilized b-glucosidase (B).
synthesis, reducing the yield by 83% and 73%, respect-
ively. The OG synthesis was terminated when the con-
centration of DMF or DMSO was higher than 2%. The
yields of OG synthesized by immobilized b-glucosidase
were improved by t-BA and DME in addition over the
concentration range of 2–10% and 2–20%, respect-
ively. The most pronounced effect was found in the
case of 10% t-BA addition. In this case, the yield of OG
increased by 1.7-fold compared to the yield obtained
in the synthesis conducted in reaction media not con-
taining co-solvent. The application of co-solvent to
construct low water environments, facilitating the syn-
thesis of glycosides catalysed by glycosidases through
reversed hydrolysis, has attracted considerable interest
in recent years. For instance, Ducret et al. found add-
ing 20% DMF in the reaction media of OG synthesis
catalysed by almond b-glucosidase can increase OG
yield by 2.5-fold (Ducret et al. 2002a).
5
4
4
3
3
2
2
1
1
0
5
0
5
0
5
0
5
0
5
0
1
00% Octanol
DMSO
DMF
t-BA
DME
Acetonitrile
0
2
5
10
20
30
Concentration of co-solvent (% v/v)
Figure 7. Effect of co-solvent content on the OG yield in the
synthesis catalysed by immobilized b-glucosidase.
activity of b-glucosidase was reduced maximally in the
media containing DMSO or DMF as the co-solvent. The
log P value of t-BA was the highest among the co-sol-
vents tested which accounts for the highest activities
of the free enzyme and the immobilized derivative in
the system with t-BA as the co-solvent.
Influence of water content on OG synthesis by the
immobilized enzyme
In the synthesis of alkyl glucosides catalysed by glyco-
sidases through reversed hydrolysis, water acts as a
competing nucleophile and causes parasitic hydrolysis
of the product (Van Rantwijk et al. 1999). It is clear
that a lower water concentration in media favours the
equilibrium yield of glycoside product. While applica-
tion of co-solvent can improve the miscibility of agly-
cone and glycone and decrease the water demand for
the solubility of glucose in the medium for OG synthe-
sis, a minimum amount of water is necessary to main-
tain the enzyme activity (Vic et al. 1995). In addition,
the position of the reaction equilibrium greatly
depends on water content of the medium. The yields
of OG synthesis catalysed by b-glucosidase
The effect of different co-solvents addition on OG
synthesis
The use of an organic co-solvent in the enzymatic syn-
thesis of alkyl glucosides allows the reaction equilib-
rium to shift towards the synthesis reaction (Figure 1)
rather than towards hydrolysis reaction, as a result of
decreasing the water concentration (Selisko et al.
990). The effect of the addition of different co-
solvents on OG synthesis is shown in Figure 7. The
addition DMF and DMSO (2%) greatly impaired the OG
1

BIOCATALYSIS AND BIOTRANSFORMATION
9
(
A) 50
(B) 25
45
40
35
30
25
20
15
10
5
0
Free enzyme
Immobilized enzyme
Free enzyme
Immobilized enzyme
20
15
10
5
0
5
10
15
20
25
30
5
10
15
20
25
30
Water content (%)
Water content (%)
Figure 8. Influence of various water contents in the medium on the yield of OG synthesis catalysed by PA-M immobilized b-gluco-
sidase (the initial concentration of glucose was 0.25 mM).
immobilized on PA-M in the media of different water
content were presented in Figure 8. The OG yields and
the conversion rates of glucose can be increased by
more than three-fold after the immobilization of b-glu-
cosidase. The maximal value of OG yield in the synthe-
sis catalysed by the immobilized enzyme was obtained
in the medium of 10% (v/v) water content, which was
less than that (15%) for obtaining the maximal yield
using the free enzyme. This may be because the
immobilization increased the organic solvent tolerance
of b-glucosidase reducing the amount of water
needed for the maintenance of enzymatic activity dur-
ing the OG synthesis. Reducing the water content fur-
ther to 5% and 10%, the yield and the conversion rate
of OG synthesis did not increase because the activity
of enzyme is optimal at certain water content and the
less water content may be unfavourable for the
enzyme to maintain its proper active configuration.
Changing the water content can alter the water activ-
ity in the medium of the water immiscible solvent
favouring the synthesis of alkyl glucosides (De Roode
et al. 2001). However, the alkyl glucosides are surface
active substances which can interfere with the inter-
action between water and water in the reaction media
increased initial glucose concentrations on the OG syn-
thesis reaction yield and the conversion rate, catalysed
by the immobilized b-glucosidase and the free enzyme
ꢁ
at 50 C in the media with 10% t-BA as the co-solvent,
is presented in Figure 9. The conversion rate of glu-
cose to OG was reduced at increased glucose concen-
trations (Figure 9). The maximal yield of OG catalysed
by the immobilized enzyme, about 40.8 mM, was
obtained when the content of glucose in the medium
was 250 mM. This value of yield was 2.9-fold higher
than the highest value of yield catalysed by the free
enzyme obtained at the glucose concentration of
150 mM. The yield of OG decreased as the glucose
concentration >250 mM in the case of immobilized
enzyme and >150 mM when the free enzyme was
used in the OG synthesis. These results suggest that
surplus glucose inhibited OG synthesis.
Glucose inhibition dynamic analysis on the OG
synthesis catalysed by b-glucosidase
Dynamic analysis (Figure 10) was conducted to under-
stand the relationship between the initial reaction vel-
ocity (V ) and the glucose concentration in OG
0
(
Mathlouthi 2001), thus, how to control the water
synthesis catalysed by free and immobilized b-glucosi-
dase at 50 C. In non-aqueous medium, containing
10% t-BA as the co-solvent, the V value of OG synthe-
0
ꢁ
activity during the reaction is the main objective of
our future research.
sis increased with increased glucose concentration and
reached at the maximum value at approximately
Influence of glucose concentration on the OG
synthesis catalysed by immobilized b-glucosidase
200 mM of glucose. The maximum value of V0
increased about three-fold by enzyme immobilization,
from 0.40 mM/h to 1.23 mM/h (Figure 10(A)). The
velocity of OG synthesis decreased when the glucose
concentration increased to >200 mM. The Dixon plots
of the initial reaction velocity against the
Based on the nature of reversed hydrolysis, the yield
of OG should be increased with increased glucose con-
centration, pushing the equilibrium towards product
formation (Panintrarux et al. 1995). The effect of

1
0
F. WANG ET AL.
(A) 45
(B) 40
40
35
30
25
20
15
10
5
0
35
30
25
20
15
10
5
0
Immobilized enzyme
Free enzyme
Immobilized enzyme
Free enzyme
0
100
200
300
400
500
0
100
200
300
400
500
Initial concentration of glucose (mM)
Initial concentration of glucose (mM)
ꢁ
Figure 9. OG synthesis using immobilized b-glucosidase at different initial concentration of glucose at 50 C with 10% t-BA as the
co-solvent.
(
A) 1.4
(B)
6
5
4
3
2
1
0
Immobilzed enzyme
Free enzyme
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Immobilized enzyme
Free enzyme
0
100
200
300
400
500
−300 −200 −100
0
100 200 300 400 500
Cs
Cs (mM)
Figure 10. Influence of various glucose concentrations on the initial reaction velocity (A) and Dixon plots (B) of the OG synthesis
by the free b-glucosidase and immobilized derivative on PA-M.
concentration of glucose clearly indicated that the V0
of OG was inhibited at high glucose concentrations
b-glucosidase to glucose increased after enzyme
immobilization. The results of simulation with Haldane
equation showed the optimal glucose concentration
(
Figure 10(B)), and that the inhibition was predomin-
antly non-competitive (Stambaugh and Post 1966).
According to the Haldane equation, the predicted
for obtaining maximal V using the free enzyme and
0
the immobilized derivative was 180 mM and 225 mM,
respectively. The immobilization reduced the effect of
substrate inhibition on the OG synthesis catalysed by
b-glucosidase. The substrate inhibition is non-competi-
tive inhibition which occurs in about 20% of all known
enzymes (Ekinci and Senturk 2010). This is in agree-
ment with a recent report describing a weak glucose
inhibition on almond b-glucosidase (Reshmi and
Sugunan 2013). In this study, an obvious glucose
inhibition was found in the OG synthesis catalysed by
the b-glucosidase extracted from bitter almonds.
maximal value of V appears at 225 mM glucose con-
0
centration for the immobilized enzyme and 180 mM
glucose concentration for the free enzyme. The value
is close to the glucose concentration, 250 mM (the
immobilized enzyme) and 150 mM (the free enzyme),
respectively, used for obtaining the highest yield of
OG (Figure 9). Based on the Dixon plots (Figure 10(B)),
the value of inhibition dissociation constant (K ) was
SI
2
19 mM for the free enzyme and 116 mM for the
immobilized enzyme. Hence, the affinity of

BIOCATALYSIS AND BIOTRANSFORMATION
11
OG synthesis in the fed-batch mode
When the fed batch operation reached a pseudo-
steady state, the glucose concentration remains con-
stant and close to zero. Combining Equations (2) and
The glucose concentration should be kept at a reason-
able level during the whole reaction period to elimin-
ate/reduce the substrate inhibition and maintain the
reaction velocity as high as possible. Strategies to over-
come substrate inhibition include feeding of substrate
to maintain its concentration below a certain limit (fed-
batch reaction) or application of a continuous stirred
tank reactor (CSTR) to minimize substrate concentration
in the reactor (Bommarius and Riebel-Bommarius 2004).
However, using a CSTR can cause catalyst loss in the
outflow. In this study, the fed-batch reaction mode was
used to control the glucose concentration in reactor to
minimize the glucose inhibition. According to the
results of batch reaction of OG synthesis, the reaction
velocity of OG synthesis catalysed by the immobilized
enzyme and the free enzyme reached maximal value of
(
4), Equation (5) was obtained for determining the
feeding rate of glucose.
F ¼ 80 ꢂ M0 expðꢃAtÞ
(5)
The results of fed-batch reaction of OG synthesis
catalysed by b-glucosidase immobilized on PA-M are
shown in Figure 11(A). The fed-batch reaction lasted
for 288 h, after which it was changed to a batch reac-
tion. In the course of the fed-batch reaction, the con-
centration of glucose in the reactor could be
maintained at approximately 225 mM with the regular
addition of fine glucose powder. A 225 mM of glucose
concentration in the reaction should be optimal for
achieving the maximum reaction velocity of OG syn-
thesis (Figure 10). At the end of 336 h of reaction, the
OG yield and the conversion rate of glucose were
1
.23mM/h and 0.40 mM/h at 225 mM and 180 mM of
glucose concentration. Ground powder of glucose was
fed in every 6 h to maintain the glucose concentration
in the reactor to obtain a high reaction velocity. The
feeding rate of glucose was determined by its con-
sumption rate during reaction. The loss rate of enzyme
activity in the longer term of incubation was also con-
sidered in determining the feeding rate of glucose. In a
batch mode, the immobilized b-glucosidase lost about
1
34 mM and 59.6%, respectively. Compared to the
batch reaction, the fed-bath reaction was able to
increase the OG yield and the conversion rate of glu-
cose by 340% and 381%, respectively. In the case with
free enzyme, a fed-batch reaction was carried out for
72 h, after that it was changed to a batch reaction.
According to Figure 10(B), a 180 mM of glucose con-
centration in the reaction should be optimal for
achieving the maximum reaction velocity of OG syn-
thesis. At the end of 96 h of reaction, the OG yield and
the conversion rate of glucose were 43.58 mM and
5
7.4% of its initial activity in 306 h while the free
enzyme maintained 27.3% of its initial activity in 48h
data not shown). The half-life value of the immobilized
(
enzyme and the free enzyme was 357h and 25.6h sep-
2
0.7% (Figure 11(B)). Compared to the batch reaction,
arately. The deactivation constant (K ) for the immobi-
d
the fed-bath reaction was able to increase the OG
yield and the conversion rate of glucose by about
2
–1
lized enzyme and the free enzyme was 0.0028h and
.027 h , respectively. Therefore, the activity of immobi-
–1
0
88% and 246.7%. Unlike in the case with the immobi-
lized enzyme at t time of reaction can be determined
by calculation based on the equation:
lized enzyme, the batch reaction carried out after the
fed-batch reaction with the free enzyme produced a
small amount of OG. This may be due to the lower
stability of the free enzyme caused by denaturation
after 72 h of fed-batch reaction. Hence, the immobiliza-
tion on PA-M can significantly increase the output of
the OG synthesis catalysed by the b-glucosidase
through enhanced stability of the enzyme.
The yield and the conversion rate of OG synthesis
mediated by the almond b-glucosidase immobilized
on PA-M using reversed hydrolysis in fed-batch mode
of reaction was higher than that obtained using trans-
glycosylation catalysed by almond b-glucosidase
adsorbed onto Celite R-640 or by yeast Pichia etchellsii
displaying cell wall bound b-glucosidase using pNPG
as glycone (Basso et al. 2002; Rather et al. 2010). The
fed-batch mode of reaction is simple and easy to be
scaled up. Compared to batch operation, fed-batch
operation increases both the yield and conversion rate
CE ¼ CE expðꢃAtÞ
(3)
0
where C is the activity of enzyme at t time, C is the
E
E0
–
1
initial activity of enzyme. A is 0.0028 h in the case of
immobilized enzyme and 0.027 h in the case of free
enzyme.
–
1
Assuming the glucose consumption rate (M ) was in
t
linear relation to the activity change of the immobi-
lized enzyme, the change of M with the reaction time
t
can be determined by Equation (4):
Mt ¼ M0 expðꢃAtÞ
(4)
where M is the consumption rate of glucose at the
t
reaction time of t (mM/h), M is the maximal consump-
0
tion rate of glucose, which value equals to the value
of Vmax (1.23 mM/h for immobilized enzyme and
0
.40 mM/h for the free enzyme).

1
2
F. WANG ET AL.
300
250
200
150
100
50
0
(
A) 160
100
90
80
70
60
140
120
100
80
60
40
20
0
50
40
30
20
10
0
OG yield
Conversion rate
Glucose concentration
0
40
80
120
160
200
240
280
320
360
Time (h)
(
B) 100
100
90
80
70
60
50
40
30
20
10
0
250
200
150
100
50
90
80
70
60
50
40
30
20
10
0
OG yield
Conversion rate
Concentration of glucose
0
0
20
40
60
80
100
Time (h)
Figure 11. Time course of OG synthesis catalysed by the b-glucosidase immobilized on PA-M (A) and the free enzyme (B) in the
fed-batch reaction mode.
of OG synthesis by dilution of glucose inhibition. That
will lower down-stream processing cost due to higher
product concentration.
reaction velocity of OG synthesis as high as possible
during the OG synthesis. The OG yield and the conver-
sion rate of glucose increased 340% and 381% by
using a fed-batch reaction.
Conclusions
Disclosure statement
The OG synthesis mediated by b-glucosidase using
reversed hydrolysis can be promoted by the immobil-
ization. The OG yield was increased by 1.7-fold when
The authors have no conflict of interest and were solely
responsible for the writing and content of the paper.
1
0% t-BA was used as co-solvent in the reaction
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