Efficient synthesis of vitamin e intermediate by lipase-catalyzed regioselective transesterification

Article abstract of DOI:10.1016/j.molcatb.2014.05.003

Trimethylhydroquinone-1-monoacetate (TMHQ-1-MA) is a valuable synthetic intermediate for vitamin E acetate. Immobilized Lipozyme RM IM from Mucor miehei was shown to be the best biocatalyst for the production of TMHQ-1-MA through regioselective transesterification between trimethylhydroquinone diacetate (TMHQ-DA) and alcohol. The effects of lipase-catalyzed reaction conditions including solvent, acyl receptor, substrate mole ratio, reaction temperature and agitation speed were investigated. The optimum conditions for Lipozyme RM IM catalyzed regioselective transesterification were achieved at a substrate mole ratio of 1:1, an agitation of 200 rpm at 50 °C in MTBE/n-hexane (3:7). Under the above conditions, Lipozyme RM IM exhibited high substrate tolerance (substrate concentrations of 1.06 M). Recycling experiments demonstrated that Lipozyme RM IM was quite steady under the reaction conditions. The analysis of kinetic experiment showed that the enzymatic reaction obeys the Ping-Pong bi-bi mechanism with n-butanol inhibition.

Full text of DOI:10.1016/j.molcatb.2014.05.003

Journal of Molecular Catalysis B: Enzymatic 106 (2014) 90–94
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Efficient synthesis of vitamin E intermediate by lipase-catalyzed
regioselective transesterification
Chun Wei, Xian-Feng Fu, Zhao Wang, Xin-Jun Yu, Yin-Jun Zhang, Jian-Yong Zheng∗
College of Biological and Environmental Engineering, Zhejiang University of Technology, No.18, Chaowang Road, Hang Zhou 310032, People’s Republic of
China
a r t i c l e i n f o
a b s t r a c t
Article history:
Trimethylhydroquinone-1-monoacetate (TMHQ-1-MA) is a valuable synthetic intermediate for vitamin
E acetate. Immobilized Lipozyme RM IM from Mucor miehei was shown to be the best biocatalyst for the
production of TMHQ-1-MA through regioselective transesterification between trimethylhydroquinone
diacetate (TMHQ-DA) and alcohol. The effects of lipase-catalyzed reaction conditions including solvent,
acyl receptor, substrate mole ratio, reaction temperature and agitation speed were investigated. The
optimum conditions for Lipozyme RM IM catalyzed regioselective transesterification were achieved at
a substrate mole ratio of 1:1, an agitation of 200 rpm at 50 ◦C in MTBE/n-hexane (3:7). Under the above
conditions, Lipozyme RM IM exhibited high substrate tolerance (substrate concentrations of 1.06 M).
Recycling experiments demonstrated that Lipozyme RM IM was quite steady under the reaction condi-
tions. The analysis of kinetic experiment showed that the enzymatic reaction obeys the Ping–Pong bi–bi
mechanism with n-butanol inhibition.
Received 21 February 2014
Received in revised form 8 May 2014
Accepted 8 May 2014
Available online 20 May 2014
Keywords:
Lipase
TMHQ-1-MA
Vitamin E
Regioselective transesterification
High substrate concentration
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
less expensive ␣-isophorone via ketoisophorone and trimethylhy-
droquinone diacetate (TMHQ-DA), the latter having to undergo an
Vitamin E (␣-tocopherol), is a kind of effective chain-breaking
lipid-soluble antioxidant that prevents the propagation of free
radical reaction [1,2]. In addition, vitamin E has also been known as
an essential nutrient for growth and reproduction with functions of
maintaining normal permeability, enhancing skin blood capillary
resistance, improving blood circulation and resisting aging [3].
Due to its powerful functions, vitamin E has been widely applied
in forage, pharmaceutical, food and cosmetic with increasing
demands [4].
The major commercial form of vitamin E is its acetate deriva-
tive. Trimethylhydroquinone-1-monoacetate (TMHQ-1-MA) is a
valuable synthetic intermediate for vitamin E acetate [5]. Nowa-
days, industrial syntheses of (all-rac)-␣-tocopherol are based on
the condensation of trimethylhydroquinone (TMHQ) with iso-
phytol, phytol or a derivative thereof, followed by acylation to
the commercial form [6,7]. TMHQ is normally obtained from
2,3,6-trimethylphenol which is expensive. Alternatively, (all-rac)-
␣-tocopherol acetate can be synthesized by using TMHQ-1-MA as
an aromatic starting material instead of TMHQ. The TMHQ-1-MA
used in this alternative synthesis can be obtained from the much
absolutely regioselectively converted into TMHQ-1-MA which is
difficult to achieve by chemical methods. Compared with TMHQ,
the condensation reaction performed by starting from TMHQ-1-MA
would be advantageous, since the synthesis of tocopheryl acetate is
then achieved in a direct manner, which improves atom economy
and reduces reaction steps [4].
Recently, the enzymatic synthesis of vitamin E intermediate has
attracted considerable interest due to its high regioselectivity, mild
operating conditions, and environmentally friendly nature [8–10].
It has now been reported that TMHQ-1-MA can be prepared from
TMHQ-DA by a selective enzymatic hydrolysis using a lipase such as
Thermomyces lanuginosus lipase (TLL) and Pseudomonas fluorescens
lipase (PFL) in water saturated solvent with high regioselectivity
and conversion [4,5]. However, from an economic point of view,
the bottlenecks for lipase catalytic TMHQ-1-MA production in the
reported patent are the relatively low substrate concentration and
its low reaction rate.
Herein our aim in this paper was to develop an efficient bio-
catalytic process for synthesis of TMHQ-1-MA. Lipase-catalyzed
regioselective transesterification between TMHQ-DA and short
chain alcohol in organic solvent was used as a model reac-
tion (Scheme 1). Subsequently, the effect of reaction conditions
including nature of biocatalyst, solvent, acyl receptor, substrate
mole ratio, reaction temperature, agitation speed and substrate
∗
Corresponding author. Tel.: +86 571 88871072; fax: +86 571 88320781.
E-mail addresses: zhengjy@126.com, zjy821212@zjut.edu.cn (J.-Y. Zheng).
http://dx.doi.org/10.1016/j.molcatb.2014.05.003
1381-1177/© 2014 Elsevier B.V. All rights reserved.

C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 90–94
91
O
HO
AcO
AcO
Lipase
Org. solvent
ROH
+
+
+
CH3
RO
OAc
TMHQ-4-MA
OAc
TMHQ-DA
OH
TMHQ-1-MA
Alcohol
Ester
Scheme 1. General reaction of transesterification between TMHQ-DA and an alcohol.
Table 1
concentration were systematically examined to deduce mechanism
and kinetics. Besides, enzyme reusability was investigated.
Performance of seven commercial available lipases for the transesterification of
TMHQ-DAa.
Entry
Enzyme
Yield (%)b
1ꢀ-Regioselectivity (%)
2. Materials and methods
1
2
3
4
5
6
7
Lipozyme RM IM
Lipase PS IM
Lipozyme 435
Lipozyme TL IM
Lipase AK
99.14
35.1
32.54
16.36
17.20
0.69
–
100
100
77.68
100
100
100
–
2.1. Enzymes
Lipozyme 435 (CAL-B, lipase
B from Candida Antarctica
Lipase AY
Lipase A
immobilized on macroporous polyacrylate resin, 10,000 U/g),
Lipozyme RM IM (RML, Mucor miehei immobilized on ionic resin,
20,000 U/g), Lipozyme TL IM (TLL, T. lanuginosus immobilized on
silica, 50,000 U/g) were supplied by Novozymes A/S (Bagsvaerd,
Denmark); Lipase PS IM (PCL, Pseudomonas cepacia lipase immo-
bilized on diatomaceous earth, 30,000 U/g) was purchased from
Wako Pure Chemical Industries Ltd. (Osaka, Japan); Lipase AK (PFL,
P. fluorescens lipase, 20,000 U/g), Lipase A (ANL, Aspergillus niger
lipase, 120,000 U/g), and Lipase AY (CRL, Candida rugosa lipase,
700,000 U/g) were obtained from Sigma-Aldrich (shanghai) Trading
Co. Ltd. (Shanghai, China).
aThe reactions were carried out in 10 ml MTBE contained 0.424 mmol TMHQ-DA and
0.848 mmol n-butanol at 200 rpm and 30 ◦C by adding the lipases with the amount
of lipase activity (100 U/ml), and the reaction was stopped at 6 h.
bThe yields of TMHQ-1-MA were determined by GC.
3. Results and discussion
3.1. Enzyme screening
Seven commercially available lipases, including four immobi-
lized enzymes and three enzyme powders, were evaluated for their
capacity for TMHQ-1-MA production by regioselective transesteri-
fication between TMHQ-DA and short chain alcohol. Fortunately,
all tested lipases displayed absolute 1ꢀ-regioselectivities towards
TMHQ-DA except for Lipozyme 435. The 1ꢀ-regioselectivity of
Lipozyme 435 was only 77.68%. Yang et al. reported that excellent
selectivity was observed in the butanoylation of arbutin catalyzed
by Novozym 435, Lipozyme TL IM or Lipase PS IM [11]. Therefore,
activity was the only factor to be considered. As shown in Table 1, a
general regularity that immobilized lipases showed higher activity
towards TMHQ-DA than lipase powders was observed. One of the
possible reasons is that native enzymes are likely to aggregate in
apoloar solvent. Among these enzymes, Lipozyme RM IM exhibited
highest activity (99.14%) and no activity was detected of Lipase A
(ANL) during the transesterification process. Thus, Lipozyme RM
IM was selected as the best biocatalyst for TMHQ-1-MA synthesis
from TMHQ-DA.
2.2. Chemicals
TMHQ-DA was kindly provided by Zhejiang Medicine Co. Ltd
(Zhejiang, China). The other chemicals used herein were of ana-
lytical grade and purchased from local suppliers. All solvents and
˚
reactants were pretreated by 4 A molecular sieves.
2.3. Experimental setup
2.3.1. General procedure for lipase catalyzed transesterification of
TMHQ-DA
Transesterification reaction was carried out in a 50 ml capped
vial by adding a certain quantities of TMHQ-DA (0.42–10.59 mmol)
and alcohol (0.42–10.59 mmol) with 0.05 g lipase in 10 ml organic
solvent. The reaction mixtures were shaken in the C76 water bath
shaker at an agitation speed range of 100–300 rpm and a tem-
perature range of 20–60 ◦C. All experiments were conducted in
triplicate.
3.2. Effect of solvent
2.3.2. Purification of product
As reaction medium, organic solvent has a direct effect on
enzymatic activity. It has been reported that a minimum quan-
tity of water is essential surrounding the immobilized lipase for
maintaining the enzyme activity. Therefore, hydrophobic solvents
are more preferred as compared to hydrophilic solvents since
the latter causes striping of the essential water layer around the
enzyme, which is necessary for enzyme activity [12–14]. Herein, the
influence of various solvents on the enzymatic reaction was inves-
tigated. It was observed that both yield and initial rate were low in
polar solvents (Log P < 2), while MTBE was an exception, solvents
with Log P values in the range of 2–4 were found to be favourable
for the synthesis of TMHQ-1-MA. Similar results were obtained as
reported in earlier literatures that the transesterification yield was
relatively high in non-polar solvents (Log P > 2) instead of in polar
solvents (Log P < 2) [15,16]. n-Hexane having Log P value of 3.764
was found to be the best single solvent in the present study, offering
the maximum yield compared with others (Table 2).
After the reaction, the immobilized enzyme was separated by fil-
tration. The filtrate was concentrated in a rotary evaporator under
reduced pressure at 95 ◦C for 20 min. Then, the product was pre-
cipitated by adding ethanol (approximately 10 times the volume of
the residue), followed by dropwise adding deionized water at 4 ◦C.
2.4. Analytical methods
Analysis was done by gas chromatography (Agilent 6890N, Agi-
lent Technology, Avondal, PA, USA) equipped with a flame ioniza-
tion detector (FID) and column HP-5 (30 m × 0.32 mm × 0.25 ␮m,
Agilent Technologies, USA). The carrier gas was nitrogen with an
inlet flow of 1 ml/min and a split ratio of 1:20. The column temper-
ature was held at 100 ◦C for 2 min, increased to 180 ◦C at 10 ◦C/min,
and maintained for 5 min. The retention times of TMHQ-1-MA and
TMHQ-DA were 12.3 and 13.6 min, respectively.

92
C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 90–94
Table 2
Table 3
Influence of solvents on the transesterification of TMHQ-DAa.
Effect of substrate mole ratio on transesterification of TMHQ-DAa.
Solvent
Log Pb
Solubility
Initial rate
Yield
(%)
Entry
Substrate mole ratiob
Yield (%)
(10−1 g/ml)c
(10−1 mM/min)d
1
2
3
4
1:1
1:2
1:3
1:4
93.06
89.18
84.46
79.13
Acetonitrile
THF
tert-Butanol
MTBE
Dichloromethane
Toluene
Cyclohexane
Hexane
−0.33
5.88
4.27
0.56
0.97
6.05
2.73
0.12
0.07
0.07
0.21
0.07
0.14
0.64
10.38
0.53
12.18
11.85
16.64
10.60
22.80
0.19
1.00
4.19
77.96
6.33
79.67
78.51
90.55
76.26
97.61
0.47
0.58
1.29
1.41
2.72
3.16
3.76
4.37
–
a
The reactions were carried out in 10 ml MTBE/n-hexane (3:7) contained
0.424 mmol TMHQ-DA and 0.424–1.696 mmol n-butanol, 0.05 g Lipozyme RM IM
at 200 rpm and 30 ◦C for 30 min.
b
Mole ratio of TMHQ-DA to n-butanol.
Isooctane
MTBE/hexane (3:7)
above results, n-butanol was chosen as the optimum alcohol in the
following work.
a
The reactions were carried out in 10 ml of different solvents contained
0.424 mmol TMHQ-DA and 0.848 mmol n-butanol, 0.05 g Lipozyme RM IM at
200 rpm and 30 ◦C, 1 h (reaction was stopped at 1 h).
3.4. Effect of mole ratio of TMHQ-DA to n-butanol
b
Data of Log P at 25 ◦C was cited from SciFinder Scholar.
c
Solubility of TMHQ-DA in different solvents was determined at 30 ◦C.
d
TMHQ-1-MA synthesis was notably influenced by the addition
of n-butanol. The effect of the substrate mole ratio on transesterifi-
cation reaction was studied using different mole ratios of TMHQ-DA
to n-butanol in the range of 1:1–1:4, while keeping the moles of
TMHQ-DA constant. It was observed that the maximum yield and
reaction rate were obtained at a mole ratio 1:1 of TMHQ-DA:n-
butanol (Table 3). By further increasing substrate mole ratio from
1:2 to 1:4, a decrease in yield and reaction rate was followed.
This could be attributed to the inhibitory effect of n-butanol on
the Lipozyme RM IM. The enzyme is hydrophobic in nature and n-
butanol contains the hydrophobic tail and polar head. There may
be a hydrophobic–hydrophobic interaction between the enzyme
and n-butanol. This interaction increases the residence time of
n-butanol with enzyme which would lead to partial dehydration
which may destabilize the native conformation of enzyme lead-
ing to inhibitory effect caused by n-butanol [17]. Therefore, it can
be concluded that n-butanol at high concentration interacts with
enzyme to form dead-end inhibitory complex. A molar ratio of 1:1
was considered as the optimum substrate ratio.
Initial reaction rate was determined according to the yield of product within
10%.
In consideration of TMHQ-DA solubility, yield and solvent tox-
icity, subsequently, the effect of mixed solvents of MTBE and
n-hexane on transesterification of TMHQ-DA was examined. Dif-
ferent volume ratios of MTBE and n-hexane were carried out under
otherwise similar conditions, demonstrating that 3:7 of MTBE and
n-hexane was the best choice with highest yield (97.61%) and initial
rate (2.28 mM/min) for the present study. Therefore, 3:7 of MTBE
and n-hexane was used as a solvent for further studies.
3.3. Effect of acyl receptor
Lipozyme RM IM catalyzed regioselective transesterification of
TMHQ-DA was studied with different alcohols such as methanol,
ethanol, n-propanol, 2-propanol and n-butanol, keeping all other
parameters constant. As shown in Fig. 1, the transesterification
between TMHQ-DA and n-butanol has the highest yield in the
first 60 min. Compared with primary alcohol, the reaction yield
achieved with 2-propanol had a remarkable decrease. A further
study showed that the variation in yield have been associated with
the change of chain length and type of alcohol during esterifica-
tion and transesterification reactions catalyzed by lipase, but not
caused by the denaturation effect of Lipozyme RM IM. Based on the
3.5. Effect of temperature
The reaction temperature is an important parameter that affects
enzyme-catalyzed reactions. Herein, the influence of reaction tem-
perature on transesterification reaction was investigated in the
range of 20–60 ◦C. In general, an elevated temperature within a
certain range could accelerate initial reaction rate and improve
yield. Conversely, the treatment at high temperature may dis-
rupt enzyme tertiary structure, making it inactivation. Similar
results were achieved in the present study (Fig. 2). The initial rate
increased proportionally from 2.381 to 4.254 mM/min in the range
of 20–50 ◦C. However, with further increase of temperature to 60 ◦C,
only a slight enhance of initial rate and yield were observed. In con-
sideration of the volatility of solvent (Boiling point of MTBE: 55.2 ◦C)
and enzyme stability, thus 50 ◦C was selected in the following work.
100
80
60
40
20
0
3.6. Effect of agitation speed
Agitation speed influenced diffusion and partition of substrate
and product in reaction system. The effect of agitation speed on
Lipozyme RM IM catalyzed transesterification of TMHQ-DA was
studied in the range of 100–300 rpm (figure not shown). An increase
in agitation speed from 100 to 200 rpm resulted in a remarkable
increase in reaction rate due to the decrease of mass transfer resis-
tance. However, there was no significant change in reaction rate
up to 200 rpm. This can be attributed to the cause that a certain
amount of catalyst particles were thrown out of the liquid phase
on the reactor wall above 200 rpm. Therefore, an agitation speed of
200 rpm was chosen.
0
30
60
90
120
150
180
Time (min)
Fig. 1. Effect of different acyl receptors on transesterification of TMHQ-DA. Reaction
conditions: TMHQ-DA 0.424 mmol, acyl receptor 0.848 mmol, MTBE:n-hexane (3:7)
10 ml, Lipozyme RM IM 0.005 g/ml, temperature 30 ◦C, speed of agitation 200 rpm.
Symbols: methanol (ꢀ); ethanol (᭹); n-propanol (ꢁ); 2-propanol (ꢂ); n-butanol (ꢃ).

C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 90–94
93
9.6
9.2
8.8
8.4
8.0
7.6
7.2
100
80
60
40
20
0
100
90
5
4
3
2
80
70
20
30
40
50
)
60
Temperature (
60
0
5
10
15
20
Batch
0
20
40
60
80
100
120
Time (min)
Fig. 4. Effect of reusability of biocatalyst on transesterification of TMHQ-DA. Reac-
tion conditions: TMHQ-DA 2.12 mmol, n-butanol 2.12 mmol, MTBE:n-hexane (3:7)
10 ml, Lipozyme RM IM 0.005 g/ml, temperature 50 ◦C, speed of agitation 200 rpm,
reaction time 3 h. Symbols: yield (ꢀ); initial reaction rate (᭹).
Fig. 2. Effect of temperature on transesterification of TMHQ-DA and initial reaction
rate. Reaction conditions: TMHQ-DA 0.424 mmol, n-butanol 0.424 mmol, MTBE:n-
hexane (3:7) 10 ml, Lipozyme RM IM 0.005 g/ml, speed of agitation 200 rpm.
Symbols: 20 ◦C (ꢀ); 30 ◦C (᭹); 40 ◦C (ꢁ); 50 ◦C (ꢂ); 60 ◦C (ꢃ).
substrate loading, no drop of initial rate was observed, thus indicat-
ing that Lipozyme RM IM was tolerant against high concentrations
of both substrate and product.
3.7. Effect of substrate concentration
3.8. Reusability of the enzyme
A high substrate concentration would be beneficial for the prac-
tical application of a biocatalytic process because it could improve
the space-time yield and reduce the cost of product isolation to
a large extent [18]. Therefore, it was of significance to optimize
the substrate concentration to maximize the value of the certain
enzyme. Herein, the effect of substrate concentration on yield as
well as initial reaction rate was investigated at a fixed amount of
enzyme (0.005 g/ml). As shown in Fig. 3, the product formation
was significantly increased from 41.2 mM to 1.02 M when substrate
concentration was raised from 42.4 mM to 1.06 M after 12 h. More-
over, the initial reaction rate increased proportionally to substrate
concentration in the range of 42.4–211.9 mM. By further increasing
The reusability of enzyme is another essential factor to reduce
cost for industrial production. Catalyst reusability studies were car-
ried out to evaluate the stability of Lipozyme RM IM under the
optimum reaction conditions. To evaluate the reusability of biocat-
alyst, Lipozyme RM IM was isolated from organic solvent after each
reaction by filtration and washed with n-hexane for twice, then
reused. As shown in Fig. 4, the yield of product was remaining after
20 cycles of reuse. And the initial reaction rate was not decrease
until the 15th repeating circle. The result suggested that Lipozyme
RM IM was quite stable in present system and had great potential
for practical application for TMHQ-1-MA production.
3.9. Kinetic study
1000
800
To analysis the reaction mechanism of Lipozyme RM IM cat-
alyzed transesterification between TMHQ-DA and n-butanol, the
effect of concentration of both substrates (TMHQ-DA and n-
butanol) on initial reaction rate was investigated under earlier
optimized parameters. The Lineweaver–Burk plots of reciprocal
rate versus reciprocal concentration of TMHQ-DA (A) were illus-
trated in Fig. 5. When n-butanol (B) was at low concentrations
(25–55 mM), the initial reaction rate increased with the increase
of the concentration of n-butanol and reached the maximum at
55 mM. Therefore, it can be concluded that no inhibition by n-
butanol in the range of 25–55 mM. As expected, there was no
evidence of inhibition by TMHQ-DA at any concentration tested
which agreed with the former conclusion. A series of parallel lines
without common intersection given in Fig. 5 demonstrated that
the transesterification of TMHQ-DA and n-butanol was correspond-
ing with Ping–Pong bi–bi mechanism [10,19–23]. However, further
increase in n-butanol concentration (above 55 mM) resulted in the
initial reaction rate to fall and the slops of Lineweaver–Burk dou-
ble inversion plot to rise (data not shown), which implied that
n-butanol at higher concentration reacted with the enzyme to
form dead end inhibitory complex. Hence, a sequential mechanism
called Ping–Pong bi–bi mechanism with n-butanol inhibition was
ruled out.
600
400
200
0
10
8
6
4
0
250
500
750
1000
Substrate concentration (mM)
0
2
4
6
8
10
12
Time (h)
Fig. 3. Effect of substrate concentration on transesterification of TMHQ-DA. Reac-
tion conditions: TMHQ-DA:n-butanol (1:1), MTBE:n-hexane (3:7) 10 ml, Lipozyme
RM IM 0.005 g/ml, temperature 50 ◦C, speed of agitation 200 rpm. Symbols: 42.4 mM
(ꢀ); 84.7 mM (᭹); 127.1 mM (ꢁ); 169.5 mM (ꢂ); 211.9 mM (ꢃ); 254.2 mM (ꢄ);
296.6 mM (ꢅ); 339.0 mM
635.6 mM (×); 762.7 mM (*); 847.5 mM ( ); 1059.3 mM (|).
(
); 381.4 mM (ꢀ); 423.7 mM
(
); 508.5 mM (+);

94
C. Wei et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 90–94
Table 4
Values of the kinetic parameter for Ping–Pong bi–bi mechanism with n-butanol
inhibition.
0.13
0.12
0.11
0.10
0.09
Kinetic parameter
Value
Apparent vm (mM/min)
Apparent KmA (mM)
Apparent KmB (mM)
Apparent Ki (mM)
17.23
59.79
9.82
90.60
proposed model for Lipozyme RM IM catalyzed transesterification
of TMHQ-DA and n-butanol is valid.
4. Conclusion
A new biocatalytic process for the production of TMHQ-1-MA
has been developed. The optimum conditions for Lipozyme RM IM
catalyzed regioselective transesterification between TMHQ-DA and
n-butanol were achieved at a substrate mole ratio of 1:1, an agita-
tion of 200 rpm at 50 ◦C in MTBE/n-hexane (3:7). Under the above
conditions, the substrate loading was increased as high as 1.06 M
with 99% yield. Furthermore, it was demonstrated that Lipozyme
RM IM was active after 20 cylces. Meanwhile, based on the ini-
tial rate data and concentration profiles, a kinetic model called
Ping–Pong bi–bi mechanism with n-butanol inhibition was pro-
posed. The kinetic parameters were evaluated by using non-linear
regression analysis with excellent correlation coefficient.
0.008
0.010
0.012
0.014
0.016
1/TMHQ-DA (1/mM)
Fig. 5. Lineweaver–Burk double inversion plot for different concentrations of
n-butanol. Reaction conditions: MTBE: n-hexane (3:7) 10 ml, Lipozyme RM IM
0.005 g/ml, temperature 50 ◦C, speed of agitation 200 rpm. Symbols: 55 mM (ꢀ);
45 mM (᭹); 35 mM (ꢁ); 25 mM (ꢂ).
KmA
k1
k-1
KmB
k3
k2
k4
E'B
+
+
B
E
+
B
A
EA
E'
E
k-3
Acknowledgements
The authors are thankful to Zhejiang Medicine Co. Ltd for
supplying TMHQ-DA. This research was financially supported by
the Natural Science Foundation of Zhejiang Province, China (nos.
LQ13C010002, LY12B06010).
Ki
EiB
Scheme 2. Ping–Pong bi–bi mechanism with single substrate inhibition.
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By analogy to the classical Ping–Pong bi–bi mechanism
with alcohol inhibition by lipase catalyzed transesterification
(Scheme 2), it is assumed that TMHQ-DA [A] first binds to the free
enzyme [E] and forms a noncovalent enzyme-substrate complex
[EA], which releases the first product and modified enzyme [Eꢀ].
The second substrate, n-butanol [B], reacts with Eꢀ to give the com-
plex EꢀB and gives the product and free enzyme [E]. Along with
this, B also forms the dead-end complex [EiB] by binding to the free
enzyme [E]. The rate equation for the Ping–Pong bi–bi mechanism
with alcohol inhibitory is as follows:
vm[A][B]
mB[A] + KmA[B](1 + [B]/Ki) + [A][B]
v =
K
where KmA is the Michaelis constant for TMHQ-DA, KmB is the
Michaelis constant for n-butanol, and Ki is the inhibition constant
due to n-butanol. vand vm are the initial reaction rate and maximum
reaction rate, respectively.
To verify the application of Ping–Pong bi–bi mechanism, the
same data were analyzed by non-linear regression using the Soft-
ware Package Polymath 6.0. The kinetic parameters determined
by Polymath 6.0 were given in Table 4, which showed excellent
correlation coefficient (data not shown). This suggests that the
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