6
F. WANG ET AL.
78.1, 77.9, 75.1, 72.1, 71.6, 62.7, and 36.4. ESI-MS (m/z):
13, and 14, were examined. To a mixture of 1.76 mL
of t-butanol and 0.04 mL of 50 mM sodium phosphate
buffer (pH 7.0), 6 mmol of the glucosyl acceptor and 5
1
323.1 (MþNaþ). 6: H NMR (400 MHz, MeOD) d (ppm)
3.13–3.22 (dd, 3H), 3.79–3.84 (d, 2H), 5.85 (d, J = 2.7
Hz, 1H), 7.37 (ddd, J = 8.0, 1.2, 0.4 Hz, 2H), 8.16 (ddd, J
= 8.0, 2.1, 0.4 Hz, 2H). 13C NMR d (ppm) 101.2(C1),
157.7, 144.1, 71.2, 117.4, 77.6, 77.0, 74.2, 125.7, and
62.1. ESI-MS (m/z): 324.1 (MþNaþ). 15: 1H NMR (400
MHz, MeOD) d (ppm) 3.17 (dd, 3H), 3.53 (dt, J = 10.3,
6.6 Hz, 1H), 3.79–3.83 (d, 2H), 4.67–4.68 (s, 2H),
7.28–7.47 (m, 5H). 13C NMR d (ppm) 103.5(C1), 136.5,
128.9, 128.5, 77, 62.1, 77.6, 127.9, 73.9, 70.0, and 71.2.
ESI-MS (m/z): 293.1 (MþNaþ). 16: 1H NMR (400 MHz,
MeOD) d (ppm) 3.13–3.21 (dd, 3H), 3.53 (dt, J =
10.3Hz, 1H), 3.79–3.84 (d, 2H), 5.08 (d, J = 2.7 Hz, 1H),
6.81–6.92 (ddd, 4H). 13C NMR d (ppm) 101.234(C1),
151.937, 77.0, 150.3, 115.44, 77.6, 116.1, 62.1, 71.2, and
74.2. ESI-MS (m/z): 295.1 (MþNaþ). 19: 1H NMR(400
MHz, MeOD) d(ppm) 1.21–1.62 (m, 10H), 1.68 (ttt, J =
10.3, 5.9, 2.8 Hz, 1H), 3.13–3.21 (dd, 3H), 3.53 (dt, J =
10.3, 6.6 Hz, 1H), 3.71–3.75 (d, 2H), 3.79–3.83 (d, 2H),
4.47 (d, J = 2.7 Hz, 1H). 13C NMR d (ppm) 104.7(C1),
38.9, 77.6, 77.0, 73.9, 71.2, 70.0, 62.1, 26.3, 28.3, and
25.3. 20: 1H NMR (400 MHz, MeOD) d (ppm) 1.37
(dquint, J = 12.7, 2.8 Hz, 1H), 1.35–1.76 (m, 9H),
3.13–3.21 (dd, 3H), 3.47–3.74 (m, 2H), 3.79–3.83 (d, 2H),
4.48 (d, J = 2.7 Hz, 1H). 13C NMR d (ppm) 102.8 (C1),
77.6, 77, 74.1, 72.5, 71.2, 62.1, 31.6, 25.3, and 23.4. ESI-
MS (m/z): 285.1 (MþNaþ).
U
of immobilized b-glucosidase were added.
ꢁ
Incubation was carried out on a rotary shaker at 50 C
and 600 rpm. In the control experiment, 50 lL of 50
mM sodium phosphate buffer (pH 7.0) was used
instead of a glucosyl acceptor. After 24 h of incuba-
tion, the enzyme was separated and its activity was
determined. The residual b-glucosidase activity was
expressed as % residual activity compared to the ini-
tial activity of the enzyme.
Results and discussion
Influence of EP113/S immobilization on the
activity and stability of b-glucosidase from
bitter almonds
Supports containing epoxy groups can generate multi-
point covalent attachment with different nucleophiles
located on the surfaces of enzyme molecules (e.g.
amino, thiol, and hydroxyl groups) (Mateo et al.
2007a). The multipoint covalent linkages between, for
example, b-glucosidase and EP113/S beads, involve
most of the subunits to increase the overall rigidity of
the protein, thus enhancing the stability of the
enzyme (Fernandez-Lafuente 2009). Immobilization of
enzymes is often accompanied by a partial loss of
activity, which can often be minimized by loading the
active sites with a reactant or a reactant mimic to pre-
vent unfavorable modification of the active sites
(Gupta, 1993). All b-glucosidases require a b-glycoside
(i.e. glucoside and to a much lesser extent, fucoside
and galactoside) as the substrate (Esen 1993). In this
study, in order to maintain the active conformation of
bitter almond b-glucosidase during immobilization on
EP113/S beads, cellobiose, a disaccharide consisting of
two b-glucose molecules linked by a b(1 ! 4) bond,
was added to the enzyme solution. This incubation
resulted in the blocking of the substrate-binding
active sites of the enzyme with cellobiose, resulting in
the protection of the active sites from chemical modi-
fication by the epoxy groups on EP113/S beads (Roig
et al. 1986; Mateo et al. 2007b); further, the active
conformation was fixed by multi-subunit immobiliza-
tion of enzymes. Upon the application of this strategy,
the specific activity of immobilized b-glucosidase was
found to be 1.82 U/mg protein, which is 84.25% of
the specific activity of the free enzyme but higher
than that obtained in the case without cellobiose add-
ition (1.23 U/mg protein). A similar strategy was
employed for the immobilization of A. oryzae
Evaluation of the influence of various non-
aqueous media on the synthesis of 5
The influence of various non-aqueous media on the
synthesis of 5, catalyzed using immobilized b-glucosi-
dase, was evaluated using the following procedure. To
a medium containing a mixture of 50 mM sodium
phosphate buffer (pH 8.0) and an organic solvent, 6
mmol 4 and 3 mmol 3 were added. The reaction was
initiated by the addition of 5 U of immobilized bꢁ-gluco-
sidase and carried out on a rotary shaker at 50 C and
600 rpm. At the end of the reaction period, the mixture
was filtered through a 0.22-lm membrane to remove
theꢁimmobilized enzyme. The filtrate was evaporated at
80 C in vacuum. 5 in the dried mixture was extracted
using acetone and purified on a silica gel column.
Evaluation of stability of the immobilized
b-glucosidase in the presence of various
glucosyl acceptors
The stabilities of the immobilized enzymes in t-butanol
solutions of the glucosyl acceptors, 7, 9, 10, 11, 12,