Significantly improved equilibrium yield of long-chain alkyl glucosides via reverse hydrolysis in a water-poor system using cross-linked almond meal as a cheap and robust biocatalyst

Article abstract of DOI:10.1016/S1872-2067(11)60333-1

An array of ten β-D-glucopyranosides with varied alkyl chain lengths were enzymatically synthesized. It was found that for longer alkyl chains a lower initial rate and final yield of glucoside was obtained except for methyl glucoside because of the severe toxicity of methanol to the enzyme. From a thermodynamics point of view, the equilibrium constant and Gibbs free energy variation of the glucoside syntheses were systematically investigated. To improve the final yields of the glucosides containing long alkyl chains the equilibrium of the enzymatic glucoside synthesis was altered. The equilibrium yield of decyl β-D-glucoside increased from 1.9 to 6.1 when the water content was reduced from 10 to 5 (v/v) using tert-butanol as a cosolvent and 0.10 mol/L of glucose as a substrate. As for the other longer alkyl chain glucosides, heptyl β-D-glucoside was found to have significant surface activity as well.

Full text of DOI:10.1016/S1872-2067(11)60333-1

CHINESE JOURNAL OF CATALYSIS
Volume 33, Issue 2, 2012
Online English edition of the Chinese language journal
Cite this article as: Chin. J. Catal., 2012, 33: 275–280.
ARTICLE
Significantly Improved Equilibrium Yield of Long-Chain Alkyl
Glucosides via Reverse Hydrolysis in a Water-Poor System
Using Cross-Linked Almond Meal as a Cheap and Robust
Biocatalyst
WANG Qinqqin1, YU Huilei1,a, ZHAO Na2, LI Chunxiu1, SHANG Yazhuo2, LIU Honglai2, XU Jianhe1,b
1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
2Department of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China
Abstract: An array of ten ꢀ-D-glucopyranosides with varied alkyl chain lengths were enzymatically synthesized. It was found that for
longer alkyl chains a lower initial rate and final yield of glucoside was obtained except for methyl glucoside because of the severe toxicity of
methanol to the enzyme. From a thermodynamics point of view, the equilibrium constant and Gibbs free energy variation of the glucoside
syntheses were systematically investigated. To improve the final yields of the glucosides containing long alkyl chains the equilibrium of the
enzymatic glucoside synthesis was altered. The equilibrium yield of decyl ꢀ-D-glucoside increased from 1.9% to 6.1% when the water
content was reduced from 10% to 5% (v/v) using tert-butanol as a cosolvent and 0.10 mol/L of glucose as a substrate. As for the other longer
alkyl chain glucosides, heptyl ꢀ-D-glucoside was found to have significant surface activity as well.
Key words: alkyl ꢀ-D-glucosides; decyl ꢀ-D-glucoside; enzymatic synthesis; reverse hydrolysis; water-poor system; equilibrium yield
As a type of non-ionic surfactant, alkyl ꢀ-D-glucosides are
non-toxic and biodegradable [1] and have antimicrobial prop-
erties [2,3]. They are widely used as biocompatible detergents
in foods, detergents, and in the pharmaceutical industry [4,5].
Traditionally, alkyl ꢀ-D-glucosides have been produced by a
chemical process but this leads to a mixture of products [6]. To
obtain more defined compounds several protection and de-
protection steps for the reactive hydroxyl groups and the acti-
vation of anomeric carbon are needed [7]. Recently, much work
on the environmentally benign enzymatic synthesis of alky1
glucosides has appeared in the literature. The synthesis can be
achieved through two pathways, the first of which is reverse
hydrolysis (a thermodynamic approach) and the other is
transglycosylation (a kinetic approach) [8]. Vulfson et al. [9]
used almond ꢀ-glucosidase to catalyze the alkyl ꢀ-glucoside
synthesis in a two-phase system of water/organic solvent. Vic
et al. [8] synthesized alkyl ꢀ-D-glucosides from D-glucose and
alcohols by reverse hydrolysis using the commercially avail-
able almond ꢀ-D-glucosidase in a homogeneous medium of
acetonitrile-water (9:1, v/v). Thereafter, more reports on the
synthesis of alkyl ꢀ-glucosides have been published. Among
them, hexyl [10–20] and octyl [12–24] ꢀ-D-glucosides were
more extensively studied by comparison with the other gluco-
sides containing long alkyl chains. However, the equilibrium
constants and Gibbs free energies of glucosides with various
alkyl chain lengths have never been reported or compared
systematically in terms of thermodynamics.
Not all alkyl ꢀ-D-glucosides have obvious surfactant prop-
erties. Only octyl, nonyl, and decyl ꢀ-D-glucosides show sig-
nificant surface activity among all the reported ꢀ-D-glucosides.
The enzymatic synthesis of glucosides containing long alkyl
chains has not been reported much perhaps because of their
extremely low yields [10,14,17–19,25]. It is especially difficult
to synthesize decyl ꢀ-D-glucoside containing a highly hydro-
Received date 9 September 2011. Accepted 26 October 2011.
aCorresponding author. Tel/Fax: +86-21-6425-0840; Email: huileiyu@ecust.edu.cn
bCorresponding author. Tel/Fax: +86-21-6425-0840; Email: jianhexu@ecust.edu.cn
This work was supported by the National Natural Science Foundation of China (20902023, 31071604, and 21173079), the Fundamental Research Funds for the Cen-
tral Universities, the Foundation of Education Ministry (20090074120014), and the National Key Technology R&D Program (2009BADB1B0301).
Copyright © 2012, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Published by Elsevier BV. All rights reserved.
DOI: 10.1016/S1872-2067(11)60333-1

WANG Qinqqin et al. / Chinese Journal of Catalysis, 2012, 33: 275–280
phobic chain enzymatically using a general glucosidase. The
reported yield of decyl glucoside was 0.8% even after 14 d of
reaction by the reverse-hydrolysis pathway [16].
propyl, hexyl, or octyl glucoside was dissolved in a mixture of
18 ml of the corresponding alcohol and 2 ml of water con-
taining 60 mg almond meal. The reaction was performed in a
100-ml flask that was constantly shaken at 180 r/min at 50 °C.
After the mixture was incubated for 72 and 144 h respectively,
30 mg almond meal was supplemented. The reverse hydrolysis
of the propyl, hexyl, and octyl glucosides was performed as in
the n-alkyl ꢀ-D-glucosides synthesis.
In our work, various alkyl ꢀ-D-glucosides were synthesized
by the direct condensation of glucose with corresponding al-
cohols using almond meal as a cheap biocatalyst. Meanwhile, a
method to tune the enzymatic synthesis of alkyl ꢀ-D-glucosides
was found by a systematic investigation into the reaction ki-
netics of ten n-alkyl ꢀ-D-glucosides. Furthermore, important
factors that affect the equilibrium yield were also identified and
optimized to improve the final yield of surface active
ꢀ-D-glucosides containing long alkyl chains.
1.3 HPLC analysis
Glucose as well as methyl, ethyl, propyl, butyl, pentyl, and
hexyl glucosides were determined by HPLC (Agilent Tech-
nologies 1200 Series) using an Aminex HPX-87H Column
(Bio-Rad, ꢁ300 mm × 7.8 mm, 9 ꢀm). They were eluted with 5
mmol/L H2SO4 at a flow rate of 0.6 ml/min at 65 °C and de-
tected using a refractive index detector.
Heptyl, octyl, nonyl, and decyl glucosides were determined
by HPLC (Agilent Technologies 1200 Series) using an Agilent
XDB-C18 Column (ꢁ4.6 mm × 150 mm, 5 μm). They were
eluted with MeOH-H2O (2:1, v/v) at a flow rate of 0.8 ml/min
at 30 °C and detected using a refractive index detector.
1 Experimental
Fresh almonds were peeled and powdered, washed three
times with ethyl acetate and twice with acetone. The powder
was dried in a vacuum desiccator and stored at 4 °C. A powder
of the dry almond meal between 40 and 200 mesh was used in
this work. The hydrolytic activity of the almond meal was
assayed as reported previously [18]. The almond meal was
further cross-linked with 30 mmol/L glutaraldehyde for 1 h
[26] to improve the biocatalyst’s stability.
Ten home-made n-alkyl ꢀ-D-glucopyranosides (from methyl
to decyl) were used as standards for high performance liquid
chromatography (HPLC) analysis. All other chemicals were
obtained commercially and were reagent grade or better.
1.4 Optimization of the decyl ꢀ-D-glucoside synthesis
The effects of water content, solvent content, glucose con-
centration, pH, and temperature on the enzymatic synthesis of
decyl glucoside were systematically investigated.
1.1 Enzymatic synthesis of alkyl ꢀ-D-glucosides
To choose the most suitable water content for the reaction
system, the effect of water content on the initial rate and final
yield were determined. Various amounts of water including
5%, 8%, 10%, 15%, 20%, 30%, and 50% were added to the
reaction system (5 ml), in which deionized water was used
instead of a potassium phosphate buffer.
To determine the best solvent, the synthesized decyl gluco-
side was measured using a standard assay after being incubated
in a decanol solution with different solvents (1:1, v/v) at 50 °C
for 48 h.
In a typical experiment, an alkyl alcohol was used as the
substrate and the medium. The reaction mixture was composed
of 18 ml of primary alcohol and 2 ml of water containing 0.25
mol/L glucose (Scheme 1). After 60 mg almond meal was
added, the mixture was incubated in a 100-ml flask and con-
stantly shaken at 180 r/min at 50 °C. At certain intervals, 50 ꢀl
of the mixture was withdrawn and analyzed by HPLC.
For the synthesis of methyl ꢀ-D-glucoside, 2 ml of methanol,
16 ml of acetonitrile, and 2 ml of water containing 0.25 mol/L
glucose were mixed.
To determine a suitable glucose concentration, various
concentrations of glucose (0.05, 0.10, 0.25, and 0.75 mol/L)
were used for the synthesis of decyl glucoside.
1.2 Equilibrium between glucoside hydrolysis and
reverse-hydrolysis
1.5 Characterization of the alkyl ꢀ-D-glucosides
For the hydrolytic reaction of glucosides, 0.25 mol/L of
Optical rotations were measured using an Autopol I Po-
OH
H
OH
H
H
Almond meal
50 oC, 7 d
H
O
O
+
HO
HO
OH
n-1
HO
HO
H2O
+
OH
ꢀ
O
H
(n = 1 10)
OH
H
OH
n-1
H
H
H
H
Scheme 1. Enzymatic synthesis of alkyl ꢀ-glucosides by reverse hydrolysis.

WANG Qinqqin et al. / Chinese Journal of Catalysis, 2012, 33: 275–280
larimeter (Rudolph Research Analytical, Inc. USA). For sur-
mond glucosidase.
face tension measurements [27], the surfactant was dissolved in
double-distilled water. Surface tension was measured at 25 °C
with a DCA315 system (Thermo-Cahn Instruments, Inc. USA).
Before the measurement, the equipment was calibrated using
double-distilled water at 25 °C.
The purity of all these alkyl glucosides was detected by
HPLC in Table
1 while propyl ꢀ-D-glucoside, butyl
ꢀ-D-glucoside, and pentyl ꢀ-D-glucoside were also character-
ized by 1H NMR as follows.
Propyl ꢀ-D-glucoside. 1H NMR (500 MHz, D2O): ꢂ
0.85–0.87 (m, 3H), 1.54–1.62 (m, 2H), 3.19–3.22 (m, 1H),
3.30–3.34 (m, 1H), 3.38–3.45 (m, 2H), 3.56–3.61 (m, 1H),
3.64–3.68 (m, 1H), 3.80–3.88 (m, 2H), 4.41 (d, J = 8.0 Hz, 1H).
Butyl ꢀ-D-glucoside. 1H NMR (500 MHz, D2O): ꢂ 0.84–0.87
(m, 3H), 1.28–1.36 (m, 2H), 1.53–1.58 (m, 2H), 3.18–3.22 (m,
1H), 3.28–3.34 (m, 1H), 3.38–3.45 (m, 2H), 3.60–3.68 (m,
2H), 3.85–3.90 (m, 2H), 4.40 (d, J = 8.0 Hz, 1H).
A plot of surface tension versus concentration was used to
determine the critical micelle concentration (cmc). The free
energy associated with the micelle formation per mole of
monomer unit (G) was evaluated according to the equation
0
[28]: ꢁGm = RTln(cmc).
2 Results and discussion
Pentyl ꢀ-D-glucoside. 1H NMR (500 MHz, D2O): ꢂ
0.83–0.85 (m, 3H), 1.27–1.29 (m, 4H), 1.57–1.58 (m, 2H),
3.19–3.22 (m, 1H), 3.31–3.35 (m, 1H), 3.38–3.45 (m, 2H),
3.60–3.68 (m, 2H), 3.85–3.89 (m, 2H), 4.40 (d, J = 8.0 Hz, 1H).
2.1 Synthesis of alkyl ꢀ-D-glucosides
Synthetic reaction profiles for the ten n-alkyl ꢀ-D-glucosides
(C1–C10) are shown in Fig. 1. Their final yields (Table 1) were
significantly affected by the alkyl chain lengths of the alcohols
that functioned as glycosidyl acceptors [29]. Longer alkyl
chains led to lower reaction rates and final yields with the
exception of the synthesis of methyl ꢀ-D-glucoside. Both the
rate and the yield for the synthesis of methyl ꢀ-D-glucoside
were lower than that of the ethyl, propyl, and butyl glucosides.
This may be due to the severe toxicity of methanol to the al-
2.2 Equilibrium between glucoside hydrolysis and reverse
hydrolysis
The yield of alkyl ꢀ-D-glucoside is controlled by the ther-
modynamic equilibrium and the biocatalyst activity [16]. To
verify the equilibrium yield of alkyl ꢀ-D-glucoside, both the
hydrolysis and reverse-hydrolysis of propyl, hexyl, and octyl
glucosides were performed (Fig. 2). On this basis, 30 mg al-
mond meal was added twice at 72 and 144 h to avoid the effect
of biocatalyst activity loss. With an increase in the alkyl chain
length the yield of n-alkyl ꢀ-D-glucosides synthesis decreased
sharply while the yield and rate of glucoside hydrolysis in-
creased greatly. The equilibrium yield did not change after the
addition of almond meal. Therefore, the final yield of alkyl
ꢀ-D-glucosides was affected not by the residual activity of the
biocatalyst but by the thermodynamic equilibrium.
70
(2)
(3)
60
50
40
30
20
10
(4)
(1)
(5)
Table 1 Thermodynamic data for the synthesis of alkyl-ꢀ-D-glucosides
Product
Yield (%)
27.6
62.5
58.7
36.1
13.7
9.17
4.57
4.05
K
1.03
ꢁGmꢂ/(kJ/mol)
–0.073
0.609
1.02
0
10
Methyl ꢀ-D-glucoside
Ethyl ꢀ-D-glucoside
Propyl ꢀ-D-glucoside
Butyl ꢀ-D-glucoside
Pentyl ꢀ-D-glucoside
Hexyl ꢀ-D-glucoside
Heptyl ꢀ-D-glucoside
Octyl ꢀ-D-glucoside
Nonyl ꢀ-D-glucoside
Decyl ꢀ-D-glucoside
(6)
(7)
0.797
0.685
0.326
0.107
0.079
0.042
0.041
0.033
0.023
8
6
4
2
0
3.01
6.01
6.82
8.51
8.58
9.20
10.1
(8)
(9)
2.92
1.93
(10)
Yield = CP/CG0, where CP is the final concentration of glucoside and CG0
is the initial concentration of glucose.
0
24
48
72
96
Time (h)
120 144 168
K is the equilibrium constants of ten n-alkyl ꢀ-D-glucosides; K =
CPCW/(CGCA), where CP is the final concentration of glucoside, CW is the
final concentration of water, CG is the final concentration of glucose, and
CA is the final concentration of n-alcohol.
Fig. 1. Progress curves for n-alkyl ꢀ-D-glucosides synthesis (C1–C10). (1)
Methyl glucoside; (2) Ethyl glucoside; (3) Propyl glucoside; (4) Butyl
glucoside; (5) Pentyl glucoside; (6) Hexyl glucoside; (7) Heptyl glucoside;
(8) Octyl glucoside; (9) Nonyl glucoside; (10) Decyl glucoside.
ꢁGmꢂ = –RTlnK, where the temperature was 50 °C.

WANG Qinqqin et al. / Chinese Journal of Catalysis, 2012, 33: 275–280
1.2
6
5
4
3
2
1
0
100
80
60
40
20
Propyl glucoside
1.0
0.8
0.6
0.4
0.2
0.0
Initial rate over 24 h
Yield at 120 h
0
100
Hexyl glucoside
Octyl glucoside
80
60
40
20
0
10
20
30
40
50
Water content (%, v/v)
Fig. 3. Synthetic yield of decyl ꢀ-D-glucoside as a function of water
content. Reaction conditions: immobilized biocatalyst 60 mg, reaction
system 5 ml, glucose 0.25 mol/L, 50 °C.
0
100
80
60
40
20
0
ꢀ-D-glucoside increased significantly from 1.9% to 5.4% when
the water content was reduced from 10% to 5% (v/v).
A cosolvent was added to the reaction system to decrease the
amount of required alcohol and to increase the solubility of the
hydrophobic substrate. Meanwhile, a suitable organic cosol-
vent should not affect the activity of the enzyme and should be
easily removed and reused [8,22–24]. The influence of diox-
ane, tert-butanol, acetone, and acetonitrile on the reaction was
investigated. The highest yield at 48 h was obtained in a
tert-butanol system, as shown in Fig. 4.
The glucose concentration is also an important factor that
affects the equilibrium of the reaction. Generally, in a reversi-
ble-hydrolysis synthesis reaction the product concentration
increases with an increase in the glucose concentration [21].
However, the yield of decyl glucoside formation was the low-
est at the highest glucose concentration (0.75 mol/L) used in
this study (Fig. 5). The separate phase became more increas-
0
24
48
72
Time (h)
96
120 144 168
Fig. 2. Reaction profiles of glucoside hydrolysis and reverse hydrolysis
glucoside synthesis. Open symbols represent the glucoside hydrolysis
and the filled symbols represent the reverse hydrolysis while the arrow-
head represents the extra addition of enzyme (30 mg each).
Moreover, the equilibrium constants and Gibbs free energy
of ten n-alkyl ꢀ-D-glucosides were calculated and are shown in
Table 1. The equilibrium constant decreased and the Gibbs free
energy increased greatly when the alkyl chain became longer.
The obtained values differed from the constants reported by
Panintrarux et al. [29] as the enzymatic synthesis of alkyl
glycosides in this study was carried out in monophasic systems
instead of biphasic systems.
Longer alkyl chains resulted in lower final equilibrium
yields and higher Gibbs free energies were required. To im-
prove the final yield of the glucosides with long alkyl chains,
the reaction system was optimized for the reduction of the
Gibbs free energy.
2.5
2.0
1.5
1.0
0.5
0.0
2.3 Optimized synthesis of decyl glucoside with
cross-linked almond meal
t-Butanol
Water content is a critical factor that influences the activity
of the catalyst and the equilibrium of the reaction [17–20]. At a
higher water content (5%–15%, v/v), a faster initial rate was
observed (Fig. 3). With further increase in water content (above
5%, v/v) the conversion of glucose decreased gradually. By
optimizing the water content the equilibrium yield of decyl
Control Dioxane Acetonitrile Acetone
Cosolvent
Fig. 4. Effect of various cosolvents on the enzymatic synthesis of decyl
ꢀ-D-glucoside. Reaction conditions: cosolvent to decanol volume ratio
1:1, 0.25 mol/L glucose, 5% water, reaction system 5 ml, 50 °C, 48 h.
The control represents the reaction system without any cosolvent.

WANG Qinqqin et al. / Chinese Journal of Catalysis, 2012, 33: 275–280
scribe heptyl ꢀ-D-glucoside having significant surface activity
10
8
in addition to octyl and decyl ꢀ-D-glucoside with longer alkyl
chains. The cmc of heptyl ꢀ-D-glucoside is lower than that of
many commonly used surfactants.
(3)
(2)
(4)
6
3 Conclusions
4
From a thermodynamic point of view, we investigated the
equilibrium constant and Gibbs free energy of glucosides
synthesis. The final yield of alkyl glucosides was not deter-
mined by the residual activity of the biocatalyst but by the
thermodynamic equilibrium and the equilibrium yield was
significantly affected by the alkyl chain length. Moreover, to
enhance the synthesis yield of the glucoside containing a long
alkyl chain we optimized the important factors that affect the
equilibrium yield systematically. The surface activity of the
alkyl glucosides was measured and we found that heptyl
ꢀ-D-glucoside has significant surface activity.
(1)
2
0
0
24
48
72
Time (h)
96
120
Fig. 5. Effect of glucose concentration on the enzymatic synthesis of
decyl glucoside. Reactions were performed in a 20-ml water-decanol
system with 5% water and 60 mg/ml cross-linked almond meal at 50 °C.
Initial concentration of glucose (mol/L): (1) 0.05; (2) 0.1; (3) 0.25; (4)
0.75.
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[ꢃ]D
Lit. [ꢃ]D
cmcc (mmol/L)
Lit. cmc (mmol/L)
ꢁGm0/(kJ/mol)
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ꢀ33.9 [18]
—
—
—
—
—
—
—
40
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10
2
—
—
—
—
—
—
—
—
—
—
—
—
—
ꢀ7.89
ꢀ9.70
ꢀ11.4
ꢀ15.4
ꢀ31.7 [18]
—
23.0 [32]
—
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—
aThe glucosides were dissolved in water.
bThe glucosides were dissolved in dichloromethane.
cThe measurement temperature was 25 °C

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