5096 Bi et al.
Asian J. Chem.
TABLE-1
EFFECT OF SOLVENTS ON ENZYMATIC
SYNTHESIS OF SALIDROSIDE
company. Tyrosol was from Aladdin. All other chemicals
were also from commercial sources and of the highest purity
available.
Solvents
Acetonitrile
V0 (mM/h)
1.3
Time (h)
60
Yield (%)
3.3
Determination of β-glycosidase activity:The glucosylation
activity of the Malus pumila seed meal was determined as
described by our previous report with a specific activity of
23.4 U/g12.
Acetone
1.2
60
6.8
THF
0.4
72
5.0
tert-Butanol
0.7
72
9.3
DMSO
0.1
48
2.1
General procedure for enzymatic synthesis of salidroside:
In a typical experiment, the mixture (2.0 mL) of ionic liquid,
phosphate buffer (100 mM, pH 6.0) and dioxane containing
0.5 mmol D-glucose, 3.0 mmol tyrosol and 6.0 U Malus pumila
seed meal were incubated in a 10 mL Erlenmeyer shaking
flask capped with a septum under predetermined reaction condi-
tions (50 ºC, 200 rpm). Aliquots were withdrawn at specified
time intervals from the reaction mixture and then diluted 50
times with corresponding mobile phase prior to HPLC analysis.
HPLC analysis: The reaction mixture was analyzed by
RP-HPLC on a 4.6 mm × 250 mm (5 µm) Zorbax SB-C18
column (Agilent Technologies Industries Co., Ltd., USA) using
an Agilent G1311A pump and a UV detector at 275 nm. The
mobile phase is a mixture of water and methanol at a flow rate
of 1.0 mL/min. The volumetric ratio of water to methanol and
the retention time for salidroside were 40/60 and 2.47 min.
DMF
0.1
48
1.0
Dioxane
1.4
72
12.0
7.6
Dioxane-C4MIm·Br
Dioxane-C4MIm·Cl
Dioxane-C4MIm·I
Dioxane-C4MIm·NO3
Dioxane-C6Py·Tf2N
Dioxane-C4MIm·BF4
Dioxane-C6MIm·PF6
Dioxane-C4MIm·C8SO4
1.0
56
1.0
56
6.1
1.2
72
12.4
8.2
0.9
72
0.7
48
4.3
1.4
72
14.8
13.9
10.2
1.4
72
0.7
72
Reaction conditions: 6.0 U Malus pumila seed meal, 0.5 mmol D-
glucose, 2.0 mmol tyrosol, 2.0 mL organic solvent or dioxane-IL (1 %,
v/v) containing 0.2 mL phosphate buffer (100 mM, pH 6.2), 50 ºC, 200
rpm
of ionic liquid content on both of the initial reaction rate and
yield was investigated.As shown in Fig. 1, the enzyme activity
was enhanced as the ionic liquid content increased from 0.5-
5.0 %. The maximal yield of 16.2 % was observed at 5.0 % of
the ionic liquid content and was not further improved by
increasing the ionic liquid content (5.0-11.0 %). High ionic
strength and the severe dehydration of the enzyme caused by
high concentration of hydrophilic C4MIm·BF4 might inactivate
the enzyme3,4, thus leading to poor results. Besides, high
viscosity of the reaction mixture resulting from the high ionic
liquid concentration can also be proposed as another reason
for this phenomenon14.
RESULTS AND DISCUSSION
Screening the reaction solvent: The nature of the solvent
such as polarity, hydrophobicity, dielectric constant and
concentration will govern the hydration water of the enzyme
molecular, protein conformation change and solubility of the
substrate and product, thus affecting the activity, stability and
specificity of the enzyme1,13
.
As shown in Table-1, among the several pure organic
solvents tested, the β-glycosidase from Malus pumila seed
meal displayed the highest yield in dioxane (12.0 %), moder-
ate yields in THF (5.0 %) and acetone (6.8 %) and lower yields
in other solvents (1.0-3.3 %). However, the synthetic activity
of β-glycosidase was changed remarkably in co-solvent
systems with different ionic liquids as additives than that
observed in neat dioxane, suggesting that the nature of the
anion in ionic liquid exerts an unexpected effect on the perfor-
mance of the enzyme. With respect to the initial reaction rates
and yields (4.3-10.2 %), β-glycosidase showed low or moderate
activities in dioxane-ionic liquid cosolvents containing Tf2N¯,
Cl¯, Br¯, NO3¯ and C8SO4¯ anions, which seem to have nega-
tive effect on the enzyme. For ionic liquid based on I¯, BF4¯
and PF6¯ anions, the higher synthetic activities were obtained
with excellent yields (12.4-13.9 %).
1.8
18
16
14
12
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
10
8
0
2
4
6
IL content (%, v/v)
8
10
The difference in the effects that the various ionic liquids
had on the β-glycosidase may be attributable to several reasons,
such as the hydrogen-bond basicity, hydrophilic or hydrophobic
property, nucleophilicity of the anion and the viscosity of the
ionic liquid2,3. In this case, the C4MIm·BF4 was considered as
the most suitable ionic liquid for the salidroside preparation
in cosolvent systems.
: Initial reaction rate;
: Yield
Fig. 1. Effect of ionic liquid content on enzymatic synthesis of salidroside.
Reaction conditions: 0.5 mmol D-glucose, 2.0 mmol tyrosol, 6.0 U
Malus pumila seed meal, 2.0 mL solvent containing 0.2 mL
phosphate buffer (100 mM, pH 6.2) and various amounts of
C4MIm·BF4, 50 ºC, 200 rpm
The effect of the buffer pH on synthetic activity in the
ionic liquid-containing cosolvent system is shown in Fig. 2a.
The initial rate and yield were clearly boosted in the pH range
of 5.6-6.0 and then decreased at pH 6.0-6.6. The highest yield
was achieved at pH 6.0 (18.3%). Understandably, buffer
Optimization of enzymatic synthesis of salidroside: For a
better understanding of the characteristics of the β-glycosidase
in the C4MIm·BF4-containing system, several crucial factors
influencing the reaction were also studied. Firstly, the impact