Optimization of lipase-catalyzed enantioselective production of 1-phenyl 1-propanol using response surface methodology

Article abstract of DOI:10.1080/10826068.2010.525433

Optically active 1-phenyl 1-propanol is used as a chiral building block and synthetic intermediate in the pharmaceutical industries. In this study, the enantioselective production of 1-phenyl 1-propanol was investigated systematically using response surface methodology (RSM). Before RSM was applied, the effects of the enzyme source, the type of acyl donor, and the type of solvent on the kinetic resolution of 1-phenyl 1-propanol were studied. The best results were obtained with Candida antartica lipase (commercially available as Novozym 435), vinyl laurate as the acyl donor, and isooctane as the solvent. In the RSM, substrate concentration, molar ratio of acyl donor to the substrate, amount of enzyme, temperature, and stirring rate were chosen as independent variables. The predicted optimum conditions for a higher enantiomeric excess (ee) were as follows: substrate concentration, 233mM; molar ratio of acyl donor to substrate, 1.5; enzyme amount, 116mg; temperature, 47°C; and stirring rate, 161rpm. A verification experiment conducted at these optimized conditions for maximum ee yielded 91% for 3hr, which is higher than the predicted value of 83%. The effect of microwave on the ee was also investigated and ee reached 87% at only 5min. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10826068.2010.525433

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OPTIMIZATION OF LIPASE-CATALYZED
ENANTIOSELECTIVE PRODUCTION OF 1-
PHENYL 1-PROPANOL USING RESPONSE
SURFACE METHODOLOGY
Aslı Soyer a , Emine Bayraktar a & Ülkü Mehmetoglu a
a Faculty of Engineering, Department of Chemical Engineering,
Ankara University, Ankara, Turkey
Version of record first published: 24 Nov 2010.
To cite this article: Aslı Soyer, Emine Bayraktar & Ülkü Mehmetoglu (2010): OPTIMIZATION OF LIPASE-
CATALYZED ENANTIOSELECTIVE PRODUCTION OF 1-PHENYL 1-PROPANOL USING RESPONSE SURFACE
METHODOLOGY, Preparative Biochemistry and Biotechnology, 40:4, 389-404
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Preparative Biochemistry & Biotechnology, 40:389–404, 2010
Copyright # 2010 Crown copyright
ISSN: 1082-6068 print/1532-2297 online
DOI: 10.1080/10826068.2010.525433
OPTIMIZATION OF LIPASE-CATALYZED ENANTIOSELECTIVE
PRODUCTION OF 1-PHENYL 1-PROPANOL USING RESPONSE
SURFACE METHODOLOGY
¨
Aslı Soyer, Emine Bayraktar, and Ulku¨ Mehmetoglu
Faculty of Engineering, Department of Chemical Engineering,
Ankara University, Ankara, Turkey
& Optically active 1-phenyl 1-propanol is used as a chiral building block and synthetic inter-
mediate in the pharmaceutical industries. In this study, the enantioselective production of 1-phenyl
1-propanol was investigated systematically using response surface methodology (RSM). Before RSM
was applied, the effects of the enzyme source, the type of acyl donor, and the type of solvent on the
kinetic resolution of 1-phenyl 1-propanol were studied. The best results were obtained with Candida
antartica lipase (commercially available as Novozym 435), vinyl laurate as the acyl donor, and
isooctane as the solvent. In the RSM, substrate concentration, molar ratio of acyl donor to the
substrate, amount of enzyme, temperature, and stirring rate were chosen as independent variables.
The predicted optimum conditions for a higher enantiomeric excess (ee) were as follows: substrate
concentration, 233 mM; molar ratio of acyl donor to substrate, 1.5; enzyme amount, 116 mg; tem-
perature, 47ꢀC; and stirring rate, 161 rpm. A verification experiment conducted at these optimized
conditions for maximum ee yielded 91% for 3 hr, which is higher than the predicted value of 83%.
The effect of microwave on the ee was also investigated and ee reached 87% at only 5 min.
Keywords 1-phenyl 1-propanol, enantiomeric ratio, enantioselectivity, kinetic
resolution, lipase, response surface methodology
INTRODUCTION
The different forms of enantiomeric products can cause quite different
biological effects. Chiral drugs, agrochemicals, food additives, and fra-
grances are classes of compounds with high economic and scientific
potential. Therefore, during the last decade there has been increased
interest in using enzymes for asymmetric synthesis and kinetic resolution
to obtain pure enantiomers. Lipases are frequently used both in kinetic
resolutions of racemates and in asymmetrizations of prochiral and meso
Address correspondence to Emine Bayraktar, Faculty of Engineering, Department of Chemical
Engineering, Ankara University, 06100 Tandogan, Ankara, Turkey. E-mail: bayrakta@eng.ankara.edu.tr

390
A. Soyer et al.
compounds.[1] Lipase activity and selectivity are strongly influenced by the
medium used for the desired reaction.[2] Lipases successfully resolve chiral
secondary alcohols.
Chiral secondary alcohols are biologically active naturally occurring
compounds, and are important intermediates for halides, amines, esters,
and ethers. They are also chiral auxiliaries in the syntheses of some drugs
and insecticides.[3–11]
The kinetic resolution is dependent on the different rates of two enan-
tiomers. The theoretical yield of kinetic resolution of racemate is 50%. It
was reported that secondary alcohol is a sluggish reactant; therefore, to
obtain an efficient kinetic resolution, the irreversible condition must be
provided.[5]
In order to improve the enantioselectivity of the kinetic resolution of
alcohols, the combinations of the lipase, solvent, and acyl donor are impor-
tant. Lipases from Pseudomonas cepacia, Candida rugosa, and Candida antarc-
tica have been used in secondary alcohol resolution.[12–15] Vinyl esters are
commonly used as acyl donors for irreversible acylation. Hirose et al.[12]
demonstrated the effective kinetic resolution of 2-phenyl-1-propanol by
using an acyl donor, vinyl 3-phenylbutanoate. Isopropyl ether was used as
a solvent and Pseudomonas cepacia lipase as an enzyme. They obtained a high
ee of 85% and enantiomeric ratio (E) of 98. It was also reported that vinyl
3-phenylpropanoate was effective for the resolution of 2-phenyl-1-propanol.
Suan and Sarmidi[13] investigated the enzyme activity and enantioselec-
tivity by acyl length of fatty acid from C12 to C18 in the enantioselective
esterification of (R,S)-1-phenylethanol. They reported that the carbon num-
ber of fatty acids did not influence the enantioselectivities of the enzymes.
The solvent effect is important for the application of lipase in the race-
mate resolution. The solvent hydrophobicity, characterized by log P value,
had a certain effect on the activity of the catalyst. Log P above 2 has been
suggested as the best value. However, it was reported that there was no sat-
isfactory correlation between log P of a solvent and enantioselectivity.[5]
Frings et al.[16] reported that no dependency of enantioselectivity on log
P could be detected in the kinetic resolution of 1-phenylethanol.
Temperature control is regarded as a simple and applicable method for
the lipase catalyzed kinetic resolution of alcohols. Sakai[17] demonstrated
that the low-temperature method was easily applied for the kinetic resol-
ution of primary and secondary alcohols. For example, in the resolution
of solketal, while a low enantiomeric ratio (E ¼ 16) was obtained at 23ꢀC,
a high E (E ¼ 55) was obtained at ꢁ40ꢀC.
Immobilization of the enzyme was also affected enantioselectivity of
secondary alcohol. Ghanem and Schuring[7] investigated the effect of
immobilized lipase in sol-gel on the kinetic resolution secondary alcohol.
They found that immobilization increased the enantioselectivity.

Enantioselective Production of 1-Phenyl 1-Propanol
391
Recently, microwave irradiation on the organic synthesis has been
applied. It has provided a powerful method to enhance the reaction rate
and to improve yields.[18,19]
As can be seen, many parameters affect the enantioselectivity of
enzymes. In the literature, most studies on secondary alcohols are based
on 1-phenyl ethanol. The aim of this work is to investigate the enantioselec-
tive kinetic resolution of a racemic secondary alcohol, 1-phenyl 1-propanol,
systemically. This study includes statistical optimization of some process
parameters that have been reported to play a very significant role in chan-
ging the enantioselective resolution of racemates, namely, the substrate
concentration, the molar ratio of acyl donor to the substrate, the amount
of enzyme, and the stirring rate. The effect of microwave irritation on enan-
tioselectivity was also investigated under optimum conditions.
EXPERIMENTAL
Enzyme and Chemicals
Candida antartica (Novozym 435) and Rhizomucor meihei (Lipozyme
RMIM) were obtained from Novozymes. Lipases from P. cepacia, P. stutzeri,
P. fluorescens, and Amona lipase PS-CI were purchased from Aldrich
(Germany) and Mucor mihei lipase from Sigma (Germany). (R,S)-1--
Phenyl-1-propanol, isopropenyl acetate, and vinyl acetate were purchased
from Aldrich (Germany); vinyl laurate and vinyl butyrate from Fluka
(Japan); and heptane, toluene, isooctane, tetrahydrofuran (THF), hexane,
and thin-layer chromatography (TLC) sheets from Merck (Germany). The
˚
molecular sieve (4 A) was purchased from Sigma (Germany). All chemicals
were analytical grade.
Resolution of (R,S)-1-Phenyl 1-Propanol
The desired amount of (R,S)-1-phenyl-1-propanol was dissolved in 3 mL
˚
of solvent. The desired amount of acyl donors, molecular sieves (4 A), and
desired amount of enzyme were added to the reaction medium. The trans-
esterification reaction was performed in a 10-mL bottle with a screw cap on
an orbital shaker at the desired stirring rate and temperature. The progress
of the reaction was monitored by thin-layer chromatography (TLC), and it
was finished when the conversion reached about 50%. TLC was carried out
on precoated aluminum sheets 60 F254 (Merck) using ethyl acetate:hexane
(1:3) solution. The spots were visualized with ultraviolet (UV) light
(k ¼ 254 nm).

392
A. Soyer et al.
Microwave Irradiation Experiments
The microwave enzyme reactor was configured with a glass reactor;
233 mM (R,S)-1-phenyl-1-propanol, 200 mM vinyl laurate, 116 mg Novozym
435, and 3 mL solvent were used in microwave irradiation experiments. The
reaction mixture was placed into a 5-mL glass reactor with an air cooling
system. The transesterification reaction was performed in a microwave
synthesis system (Biotage initiator, 400 W magnetron).
HPLC Analysis
The reaction progress, monitored by TLC, was stopped when the con-
version reached about 50%. After the solvent was evaporated using a rotary
evaporator, the substrates were analyzed with high-performance liquid
chromatography (HPLC) using a Chiralcel-OB column. The mobile phase
was hexane=2-propanol (97=3) with 0.8 mL=min flow rate at column tem-
perature 30ꢀC, and the compounds were detected at 254 nm with a diode
array detector. The conversion and the enantiomeric excess (ee) of the
substrate were calculated using the equations x ¼ [1 – (CSþCR)=
(CS0 þ CR0)]100 and ee ¼ [(CS – CR)=(CS þ CR)]100 (for CS > CR), respect-
ively. Here, CS0 and CR0 are the initial substrate concentrations and CS
and CR the substrate concentrations at certain times of S and R form,
respectively.
The ‘‘Design Expert’’ (version 6.01, Stat. Ease, Inc., Minneapolis, MN)
was used for regression and graphical analyses of the data obtained.
RESULTS AND DISCUSSION
Lipase-catalyzed enantioselective transesterification of 1-phenyl-1-
propanol was selected as a model system for the enzymatic resolution of
secondary alcohols. The enantioselectivity of kinetic resolution of alcohol
is considerably influenced by the source of enzyme, and type of acyl donor
and solvent. In the preliminary experiments, the effects of enzyme source,
and type of acyl donor and solvent on the kinetic resolution of (R,S)-1-
phenyl-1-propanol were investigated. Six commercial lipases were screened
with vinyl laurate at 40ꢀC in isooctane.
The enzymes used were Mucor miehei lipase, Pseudomonas cepacia lipase,
Pseudomonas stutzeri lipase, Pseudomonas fluorescens lipase, Lipozyme RMIM,
and Novozym 435. All of these enzymes were capable of catalyzing the reac-
tion. (R)-1-phenyl-1-propanol was the fastest reacting enantiomer, yielding
the (R)-acetate and leaving (S)-1-phenyl-1-propanol as enantiomerically
pure unreacted enantiomer (Figure 1).

Enantioselective Production of 1-Phenyl 1-Propanol
393
FIGURE 1 Transesterification reaction of (R,S)-1-phenyl-1-propanol.
As can be seen from Table 1, the highest ee for substrate was obtained
with Novozym 435. All the enzymes except for Mucor miehei lipase are immo-
bilized enzymes. While the minimum ee value was obtained with Mucor mie-
hei lipase, ee increased with the use of Lipozyme TL IM, which is
immobilized lipase produced from Mucor miehei. Immobilization of an
enzyme is known to affect the enzyme conformation, rigidity, and reactivity.
In an organic solvent, lipase molecules usually aggregate, which reduces
the activity, but when the lipases are immobilized, they can be highly
dispersed.[16,20]
The enantioselectivity of the reaction using Novozym 435 was much bet-
ter than that of the reaction with other enzymes. This commercial lipase is
in immobilized form on acrylic resin. Novozym 435 has been proven to be
an efficient biocatalyst for esterification and transesterification of second-
ary alcohol.[11,13] After lipases were screened, the effect of solvent was inves-
tigated for the reaction with vinyl laurate as an acyl donor and Novozym
435. In all cases, the (R)-alcohol was more rapidly reacting enantiomer,
yielding the (R)-ester in high enanatiomeric ester. Thus (S)-alcohol was
obtained in enantiomerically pure form.
The influence of organic solvents on the enzymatic reaction is different.
The hydrophobicity of the solvents can be expressed in terms of log P.
The enzymes can show higher activity with the nonpolar (log P > 4) and
TABLE 1 The Effect of Enzyme Type on ee
Type of Enzyme
ee (%)
Time (hr)
Mucor meihei
Pseudomanas cepacia
Pseudomanas stutzeri
Pseudomanas floresans
Rhizomucor meihei (Lipozyme RM IM)
Candida antartica (Novozym 435)
2
25
38
9
10
71
0.5
0.5
0.5
0.5
0.5
0.5
Note. Resolution was carried with alcohol (0.5 mmol), vinyl laurate (1 mmol), enzyme (0.1 g),
molecular sieve (0.1 g), isooctane (3 mL), temperature (40ꢀC), stirring rate (150 rpm).

394
A. Soyer et al.
TABLE 2 The Effect of Solvent Type on ee
Solvent
ee (%)
Time (hr)
THF
15
70
68
63
71
4
4
3.5
1
Toluene
Hexane
Heptane
Isooctane
0.5
Note. Resolution was carried with alcohol (0.5 mmol), vinyl laurate (1 mmol), Novozym 435
(0.1 g), molecular sieve (0.1 g), solvent (3 mL), temperature (40ꢀC), stirring rate (150 rpm).
midpolar solvents (2 < log P < 4), whereas they have the lowest activity with
the polar solvents (log P < 2).[21] Therefore, in order to investigate the sol-
vent effect on the enantioselectivity of enzyme, solvents having different log
P were used. For this aim, THF (log P 0.5), toluene (log P 2.5), hexane (log
P 3.5), heptanes (log P 4), and isooctane (log P 4.5) were used. The results
are summarized in Table 2. While the enantioselectivity was the lowest with
THF, ee values showed almost no change when used with other solvents
having up to log P 2.5. At the same time, hydrophobic solvents give a lower
reaction time than hydrophilic ones. This can be due to the hydrophobic
properties of solvents. Hydrophobicity increases the transesterification
reaction rate but it does not affect the enantioselectivity.
Afterward, the effect of acyl donor on enantioselectivity for transester-
ification was investigated by using ethyl acetate, vinyl acetate, isopropenyl
acetate, vinyl butyrate, and vinyl laurate, which have different carbon num-
bers. As shown in Table 3, a vinyl group seems to affect the ee value. Isopro-
penyl acetate and ethyl acetate do not show any enantioselectivities.
Similarly, to solve the reversibility problem of esterification and transester-
ification, Reetz[22] used vinyl acetate as an acylating agent, with the reac-
tions being irreversible because of the formation of acetaldehyde.
In our study, an acyl donor having a vinyl group gave a high ee. The
maximum ee obtained was 71%, with vinyl laurate. If we look at the acyl
TABLE 3 The Effect of Acyl Donor Type on ee
Acyl Donor
ee (%)
Time (hr)
Ethyl acetate (C4)
Vinyl acetate (C4)
Izoprophenyl acetate (C5)
Vinyl butyrate (C6)
Vinyl laurate (C14)
3
19
0
26
71
0.5
0.5
0.5
0.5
0.5
Note. Resolution was carried with alcohol (0.5 mmol), vinyl laurate (1 mmol), Novozym 435
(0.1 g), molecular sieve (0.1 g), iso-octane (3 mL), temperature (40ꢀC), stirring rate (150 rpm).

Enantioselective Production of 1-Phenyl 1-Propanol
395
donors from the point of view of carbon number, ee increased with increas-
ing chain length of the acyl donor having a vinyl group. In the literature,
Suan and Sarmidi[13] investigated the effect of chain length of fatty acids
on the esterification reaction of (R,S)-1-phenyl ethanol. Lauric, myristic,
palmitic, and stearic acids were used as acylating agents. The authors
reported that although the carbon number of fatty acids affected the reac-
tion rate, it did not influence the enantioselectivities of enzymes. However,
our results are similar to those reported by Ottoson and Hult,[23] that the
enantioselectivity of C. antartica lipase B was strongly influenced by the
achiral chain length of vinyl ester in the transesterification of 3-methyl-2-
butanol.
After determining the source of enzyme, solvent, and acyl donor, we
investigated the other reaction conditions using RSM. The classical method
of parameter optimization involves changing one variable at a time, keep-
ing the others at fixed levels. Being single-dimensional, this laborious
and time-consuming method often does not guarantee determination of
the optimal conditions. Previous experiments helped us to identify the five
experimental variables markedly influencing the enantioselective pro-
duction of 1-phenyl-1-propanol: concentration of alcohol, molar ratio of
acyl donor to the alcohol, amount of enzyme, temperature, and stirring
rate.
The range and levels of the variables investigated in this study, which
were chosen based on previous studies, are given in Table 4. In the
regression equation, the test variables were coded according to the
equation:[24,25]
ðXi ꢁ XiꢂÞ
xi ¼
ð1Þ
DXi
where xi is the coded value of the ith independent variable, Xi is the
uncoded value of the ith independent variable, Xiꢂ is the uncoded value
TABLE 4 Experimental Range and Levels of the Independent Variable
Range and Levels
Variables
ꢁ2.38
ꢁ1
0
1
2.38
Alcohol concentration (mM), X1
Acyl donor=alcohol (mol=mol), X2
Enzyme (mg), X3
8, 34
0, 81
40, 54
16, 22
31, 08
100
1, 5
75
30
100
167
2
100
40
233
2, 5
125
50
325
3, 19
159, 46
63, 78
268, 92
Temperature (ꢀC), X4
Stirring rate (rpm), X5
150
200

396
A. Soyer et al.
TABLE 5 Full Factorial Central Composite Design Matrix of Five Variables and Natural Units Along
with Observed Responses (ee %)
X1
(Alcohol
Concentration,
mM)
X2
(Acyl Donor=
Alcohol,
mol=mol)
X3
X4
X5
(Stirring
Rate, RPM) Response (ee %)
(Enzyme, (Temperature,
mg)
Experimental
Number
ꢀC)
1
2
3
4
5
6
7
8
9
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
100.00
233.00
8.34
1.50
1.50
2.50
2.50
1.50
1.50
2.50
2.50
1.50
1.50
2.50
2.50
1.50
1.50
2.50
2.50
1.50
1.50
2.50
2.50
1.50
1.50
2.50
2.50
1.50
1.50
2.50
2.50
1.50
1.50
2.50
2.50
2.00
2.00
0.81
3.19
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
2.00
75.00
75.00
75.00
30.00
30.00
30.00
30.00
30.00
30.00
30.00
30.00
50.00
50.00
50.00
50.00
50.00
50.00
50.00
50.00
30.00
30.00
30.00
30.00
30.00
30.00
30.00
30.00
50.00
50.00
50.00
50.00
50.00
50.00
50.00
50.00
40.00
40.00
40.00
40.00
40.00
40.00
16.22
63.78
40.00
40.00
40.00
40.00
40.00
40.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
200.00
150.00
150.00
150.00
150.00
150.00
150.00
150.00
150.00
31.08
31
55
42
32
41
59
63
31
36
74
75
58
31
89
33
53
57
58
51
52
24
68
27
72
52
70
60
54
33
79
26
74
77
34
62
72
14
43
38
30
38
42
79
71
77
65
75.00
125.00
125.00
125.00
125.00
75.00
75.00
75.00
75.00
125.00
125.00
125.00
125.00
75.00
75.00
75.00
75.00
125.00
125.00
125.00
125.00
75.00
75.00
75.00
75.00
125.00
125.00
125.00
125.00
100.00
100.00
100.00
100.00
40.54
159.46
100.00
100.00
100.00
100.00
100.00
100.00
100.00
100.00
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
324.66
166.50
166.50
166.50
166.50
166.50
166.50
166.50
166.50
166.50
166.50
166.50
166.50
268.92
150.00
150.00
150.00
150.00
(Continued )

Enantioselective Production of 1-Phenyl 1-Propanol
397
TABLE 5 Continued
X1
X2
(Alcohol
Concentration,
(Acyl Donor=
Alcohol,
mol=mol)
X3
X4
X5
(Stirring
Rate, RPM) Response (ee %)
(Enzyme, (Temperature,
mg)
Experimental
Number
mM)
ꢀC)
47
48
49
50
166.50
166.50
166.50
166.50
2.00
2.00
2.00
2.00
100.00
100.00
100.00
100.00
40.00
40.00
40.00
40.00
150.00
150.00
150.00
150.00
68
70
70
65
of the ith independent variable at the center point, and DXi is the step
change value. A 25 full factorial composite for five independent variables
was used in this study. The full factorial composite design consists of a com-
plete 2k factorial design, where k is the number of test variables, n0 center
points (n0 ꢃ 1), and two axial points on the axis of each design variable at a
distance of a (¼2k=4, (¼2.378 for k ¼ 5) from the design center. The total
number of design points is N ¼ 2k þ 2k þ n0 (50 experiments for 5
independent variables).
A second-order polynomial model was fitted to obtain the ee value of
the transesterification reaction of 1-phenyl-1-propanol. The experiments
performed and the results obtained under the operational conditions
employed are listed in Table 5.
The application of RSM yielded the following regression equation,
which is an empirical relationship between the ee value and the experi-
mental parameters in coded units:
y ¼ 71:41 þ 7:93x1 ꢁ 1:37x2 ꢁ 2:21x3 þ 3:22x4 þ 0:80x5 ꢁ 8:69x12
þ 0:065x22 ꢁ 3:03x32 ꢁ 5:77x42 ꢁ 4:71x52 ꢁ 7:19x1x2 þ 5:91x1x3
þ 4:55x1x4 þ 2:09x1x5 ꢁ 1:47x2x3 þ 0:66x2x4 ꢁ 0:78x2x5
ꢁ 1:78x3x4 ꢁ 1:84x3x5 ꢁ 0:84x4x5
ð2Þ
The results of the second-order response surface model fitting in the
form of analysis of variance (ANOVA) are given in Table 6. The Fisher F-test
with a very low probability value (Pmodel > F < 0.0001) demonstrates a very
high significance for the regression model and confirms the adequacy of
the quadratic model. This result indicates that it is statistically significant
at 99.999% confidence level. The significance of each coefficient was
determined by F-value and Prop > F-value (Table 6). Values of ‘‘Prop > F 00
less than 0.0500 indicate that the model terms are significant. In this case,
the coefficients of x1, x12, x1x2, and x1x3 are significant model terms. The
first order and the quadratic main effects of alcohol concentration, x1x12
(Prop > F50.0001), are the most significant. The interaction terms

398
A. Soyer et al.
TABLE 6 Analysis of Variance (ANOVA) for the Quadratic Model
Coefficient Estimate
as Coded Value
Sum of
Squares
Mean
Square
Source
df
F-Value
Prob > F
Intercept
x1
x2
x3
x4
71.41
7.93
ꢁ1.37
ꢁ2.21
3.32
2723.94
80.96
212.42
478.56
27.50
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
20
29
22
7
2723.94
80.96
212.42
478.56
27.50
23.19
0.69
1.81
4.07
0.23
35.69
1.969E-003
4.34
15. 74
10.49
13.95
9.50
5.59
1.19
0.59
0.12
0.17
0.86
0.93
0.19
<0.0001
0.4132
0.1891
0.0529
0.6321
<0.0001
0.9649
0.0461
0.0004
0.0030
0.0008
0.0045
0.0249
0.2835
0.4495
0.7344
0.6864
0.3602
0.3439
0.6629
<0.0001
x5
0.80
x12
ꢁ8.69
0.065
ꢁ3.03
ꢁ5.77
ꢁ4.71
ꢁ7.16
5.91
4192.41
0.23
4192.41
0.23
x22
x32
509.86
1849.48
1231.93
1638.78
1116.28
657.03
140.28
69.03
13.78
19.53
101.53
108.78
22.78
509.86
1849.48
1231.93
1638.78
1116.28
657.03
140.28
69.03
x42
x52
x1x2
x1x3
x1x4
x1x5
x2x3
x2x4
x2x5
x3x4
x3x5
x4x5
Model
Residual
Lack of fit
Pure error
Cor total
4.53
2.09
ꢁ1.47
0.66
13.78
19.53
ꢁ0.78
ꢁ1.78
ꢁ1.84
ꢁ0.84
101.53
108.78
22.78
683.62
117.47
146.58
25.98
13,672.47
3406.65
3224.77
181.88
17,079.12
5.82
5.64
0.0126
49
Note. R-squared ¼ 0.8005, adeq. precision ¼ 10.519.
between alcohol concentration and acyl donor=alcohol concentration
(Prop > F ¼ 0.0008) and alcohol concentration and amount of enzyme
(Prop > F ¼ 0.0045) are also more significant than the others.
The response surfaces obtained according to the RSM analysis for
each ee value are shown in Figures 2–6. Response surface plots provide
a method to predict the ee value for different values of the test variables.
When the response surface is examined, it is clear that the interaction
between alcohol concentration and acyl donor=alcohol is more effective
(Figure 2). ee increased with increasing 1-phenyl 1-propanol concen-
tration from 10 to 250 mM, and above this alcohol concentration ee
decreased. The effect of molar ratio of acyl donor=alcohol is dominant
especially at high alcohol concentration. The ee value reached 91% at
250 mM alcohol concentration. This result can be explained as follows.
In the transesterification reaction, firstly, the enzyme is acylated and
then transesterification occurs.[13] Therefore, the amount of acyl donor
is important. The depletion of acyl donor on the active site of the

Enantioselective Production of 1-Phenyl 1-Propanol
399
FIGURE 2 Response surface plot showing the mutual effect of acyl donor=alcohol (mol=mol) and alco-
hol concentration when the other variables are held at zero level.
FIGURE 3 Response surface plot showing the mutual effect of amount of enzyme and alcohol concen-
tration when the other variables are held at zero level.

400
A. Soyer et al.
FIGURE 4 Response surface plot showing the mutual effect of temperature and alcohol concentration
when the other variables are held at zero level.
FIGURE 5 Response surface plot showing the mutual effect of stirring rate and alcohol concentration
when the other variables are held at zero level.

Enantioselective Production of 1-Phenyl 1-Propanol
401
FIGURE 6 Response surface plot showing the mutual effect of stirring rate and temperature when the
other variables are held at zero level.
enzyme may be prevented by increasing the molar ratio of the acyl
donor=alcohol. Moreover, an increase in alcohol concentration may pre-
vent the acyl donor reaching the active site. The molar ratio of acyl
donor=alcohol and alcohol concentration interact with each other. The
contour plots help us to identify the types of interactions between test
variables. An elliptical or saddle nature of the contour plots indicates
the significance of the interactions between the corresponding variables.
A similar interaction between alcohol concentration and amount of
enzyme is shown in Figure 3. The ee value increases with amount of
enzyme up to 120 mg; above this value it remains almost unchanged
(Figure 3). Because additional mass transfer limitation can occur with
increasing catalyst loading in liquid–solid (catalyst) reaction or the
amount of enzyme can be more than the substrate concentration, no
free substrate was available to bind the excess enzyme.
The variations in temperature and alcohol concentration with ee can be
seen in Figure 4. The ee value increased with increasing temperature up to
47ꢀC. The effect of temperature on the enantioselectivity can differ with
the enzyme used and type of reaction. The effect of stirring rate on the
ee is shown in Figures 5 and 6. Because of the mass transfer effects, the

402
A. Soyer et al.
enantioselectivity of the enzyme was affected by the stirring rate. It
increased with stirring rate between 30 and 175 rpm; above this stirring rate
it decreased. This decrease may be due to conformational changes or
attrition of the immobilized structure of the enzyme.
The maximum predicted ee value is indicated by the surface confined
in the smallest ellipse in the contour diagrams temperature versus the alco-
hol concentration (Figure 4) and in the contour diagrams stirring rate ver-
sus to alcohol concentration (Figure 5). The temperature has no
interaction with stirring rate as proved by the relatively circular nature of
the contour curves (Figure 6) and shows the optimal stirring rate around
150–200 rpm and optimal temperature around 45ꢀC.
Microsoft Excel software was used to solve the regression equation
[Eq. (2)]. The optimum values for the test variables for the maximum
ee value (83%) were alcohol concentration 233 mM, acyl donor=
alcohol concentration 1.5, amount of enzyme 116 mg, temperature 47ꢀC,
and stirring rate 161 rpm. In order to verify these results, enantio-
merically pure production of 1-phenyl-1-propanol was carried out at the
optimum condition and ee was obtained as 91% for 3 hr. The maximum
ee value predicted by the equation (83%) agrees with this experimental
value.
Finally, the effect of microwave irradiation on the transesterification of
1-phenyl 1-propanol was investigated using conditions that were optimal
except for temperature. The temperature was kept at 60ꢀC under micro-
wave with an air compressor system, and 87% ee was obtained at a con-
siderably short reaction time (5 min). Microwave irradiation increased
the rate of the lipase-catalyzed reaction. In the literature, there are limited
studies on microwave assisted enantioselective synthesis.[11,18,19,26] There
are contradictory statements about the effect of microwave irradiation in
the literature. Unlike our study, Leadbeater et al.[26] reported no differ-
ences between the effect of microwave heating and conventional heating
on the conversion and reaction rate of lipase-catalyzed transesterification
reaction. Souze et al.[19] studied on the kinetic resolution of rac-1-pheny-
lethanol with microwave irradiation. They reported that microwave
irradiation displayed no changes in both activity and selectivity of the
immobilized lipases according to the conventional heating at the same
temperature. On the other hand, Lin and Lin[18] demonstrated the reac-
tion rate and enantioselectivities of lipase catalyzed reaction significantly
enhanced under microwave irradiation. Similarly, Bachu et al.[11] also
obtained high conversion under the microwave irradiation. Therefore,
the effect of microwave irradiation on the enzyme-catalyzed reaction in
organic solvents must be investigated particularly. Our studies have contin-
ued in this direction.

Enantioselective Production of 1-Phenyl 1-Propanol
CONCLUSION
403
The kinetic resolution of rac-1-phenyl-1-propanol was carried out success-
fully with a transesterification reaction. Enantiomerically pure secondary alco-
hols are important synthetic intermediates and chiral auxiliaries. For this
resolution, the best results were obtained using Novozym 435, vinyl laurate,
and isooctane. The methodology of response surface optimization was shown
to be very useful for the determination of relevant variables for process opti-
mization. This made it possible to consider a large number of variables and
avoid the loss of information that might be essential in the optimization of
the process. It was possible to determine optimal operating conditions for
high ee value by using the method of experimental factorial design and
response surface analysis. In order to verify the results of RSM, experiments
were carried out and ee was found to be 91% at the optimum conditions for
three hours. Moreover, at these optimum conditions (except temperature),
microwave irritation was applied and 87% ee was obtained at only 5 min.
ACKNOWLEDGMENT
The authors gratefully acknowledge the financial support given to
this work by Ankara University, Institute of Biotechnology (project
2001K120140-162).
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