Optimized synthesis of lipase-catalyzed hexyl acetate in n-hexane by response surface methodology

Article abstract of DOI:10.1021/jf001050q

Hexyl acetate, a short-chain ester with fruity odor, is a significant green note flavor compound and widely used in the food industry. The ability for immobilized lipase from Mucor miehei (Lipozyme IM-77) to catalyze the transesterification of hexanol with triacetin was investigated in this study. Response surface methodology and five-level-five-factor central composite rotatable design were adopted to evaluate the effects of synthesis variables, such as reaction time (2-10 h), temperature (25-65°C), enzyme amount (10-50%; 0.024-0.118 BAUN), substrate molar ratio of triacetin to hexanol (1:1 to 3:1), and added water content (0-20%) on percentage molar conversion of hexyl acetate. The results showed that reaction temperature and substrate molar ratio were the most important parameters and that added water content had less of an effect on percent molar conversion. On the basis of canonical analysis, optimum synthesis conditions were as follows: reaction time, 7.7 h; temperature, 52.6°C; enzyme amount, 37.1% (0.089 BAUN); substrate molar ratio, 2.7:1; and added water, 12.5%. The predicted value was 88.9% molar conversion, and the actual experimental value was 86.6% molar conversion.

Full text of DOI:10.1021/jf001050q

J. Agric. Food Chem. 2001, 49, 1203−1207
1203
Op tim ized Syn th esis of Lip a se-Ca ta lyzed Hexyl Aceta te in n -Hexa n e
by Resp on se Su r fa ce Meth od ology
Chwen-J en Shieh* and Shu-Wei Chang
Department of Food Engineering, Dayeh University, 112 Shan-J iau Road, Da-Tsuen,
Changhua 51505, Taiwan
Hexyl acetate, a short-chain ester with fruity odor, is a significant green note flavor compound and
widely used in the food industry. The ability for immobilized lipase from Mucor miehei (Lipozyme
IM-77) to catalyze the transesterification of hexanol with triacetin was investigated in this study.
Response surface methodology and five-level-five-factor central composite rotatable design were
adopted to evaluate the effects of synthesis variables, such as reaction time (2-10 h), temperature
(25-65 °C), enzyme amount (10-50%; 0.024-0.118 BAUN), substrate molar ratio of triacetin to
hexanol (1:1 to 3:1), and added water content (0-20%) on percentage molar conversion of hexyl
acetate. The results showed that reaction temperature and substrate molar ratio were the most
important parameters and that added water content had less of an effect on percent molar conversion.
On the basis of canonical analysis, optimum synthesis conditions were as follows: reaction time,
7.7 h; temperature, 52.6 °C; enzyme amount, 37.1% (0.089 BAUN); substrate molar ratio, 2.7:1;
and added water, 12.5%. The predicted value was 88.9% molar conversion, and the actual
experimental value was 86.6% molar conversion.
Keyw or d s: Biosynthesis; contour plots; hexyl acetate; lipase; optimization; response surface
INTRODUCTION
memory, acyl donors, etc. (5). Response surface meth-
odology (RSM) and central composite rotatable design
(CCRD) are useful statistical techniques for investiga-
tion of the complex processes and have been successfully
applied for optimizing of ester production by lipase (6,
7).
Esters of short-chain alcohols derived from short-
chain carboxylic acids (acetates, propionates, butyrates,
etc.) represent an important class of flavoring materials,
responsible for the fruity odors of many foods and
fragrances (1). For instance, hexyl acetate is an ex-
tremely aromatic compound with green note flavor and
widely used in the food industry. The present worldwide
market for natural “green notes” is estimated to be 5-10
metric tons each year at a current market price of 2500-
6000 U.S.$/kg. Traditionally, it has been isolated from
natural sources or produced by chemical synthesis.
There is a growing demand for natural flavors contain-
ing green notes represented by C-6 alcohol (hexanol)
derivatives (2); however, this steadily growing demand
has left these compounds in increasingly short supply
(3). Therefore, the biosynthesis of such esters by lipase-
catalyzed chemical reactions under mild conditions has
become of much current commercial interest. An opti-
mized enzymatic synthesis of the hexyl ester improves
the conversion yield and reduces the costs of production
in most favorable conditions. This should benefit manu-
facturers and be more appealing to the consumers.
The importance of the enzymatic synthesis with
lipases (triacylglycerol hydrolases EC 3.1.1.3) as cata-
lysts to produce a number of commercially important
flavor esters by esterification in nonaqueous organic
solvents has been emphasized in several works (4). The
lipase-catalyzed esterification reactions for esters are
reviewed, in which the parameters affecting the activi-
ties of lipase on esterification reaction include reaction
time, synthesis temperature, added water content, pH
Carvalho et al. reported that hexyl acetate was
synthesized by the cutinase-catalyzed transesterifica-
tion reaction of butyl acetate with hexanol in a reversed
micelles system, and the optimization of the transes-
terification was described using RSM (8). Bourg-Garros
et al. synthesized (Z)-3-hexen-1-yl butyrate by direct
esterification using lipases Mucor miehei (Lipozyme IM)
and Candida antarctica (Novozym 435) in hexane with
high yield (>90%). In the absence of solvent at 60 °C,
Novozym 435-catalyzed esterification afforded a yield
of 80% (2). Bourg-Garros et al. investigated the use of
immobilized lipase from C. antarctica (Novozym 435)
to synthesize (Z)-3-hexen-1-yl acetate by direct esteri-
fication in n-hexane and a solvent-free medium. The
ester yield reached 94% in <27 h in n-hexane at 70 °C
in the presence of 2% (w/w reactants) of the lipase and
1.5 mol L-1 of substrate (3).
The present work focuses on the reaction parameters
that affect lipase from M. miehei (Lipozyme IM-77)-
catalyzed the transesterification of hexyl acetate using
triacetin as acyl donor in n-hexane. Our purposes were
to better understand relationships between the factors
(reaction time, temperature, enzyme amount, substrate
molar ratio, and added water content) and the response
(percent molar conversion) and to determine the optimal
conditions for hexyl acetate synthesis using CCRD and
RSM analyses.
MATERIALS AND METHODS
* Author to whom correspondence should be addressed
(telephone 886-4-853-0421; fax 886-4-853-4845; e-mail cjshieh@
mail.dyu.edu.tw).
Ma ter ia ls. Immobilized lipase (triacylglycerol hydrolase,
EC 3.1.1.3; Lipozyme IM-77, 7.7 BAUN/g, water ) 5.4% w/w)
10.1021/jf001050q CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/01/2001

1204 J. Agric. Food Chem., Vol. 49, No. 3, 2001
Shieh and Chang
Ta ble 1. Cen tr a l Com p osite Rota ta ble Secon d -Or d er Design , Exp er im en ta l Da ta , a n d P r ed icted Va lu es for
F ive-Level-F ive-F a ctor Resp on se Su r fa ce An a lysis
enzyme
substrate
molar ratio
(triacetin/hexanol) x4
added H2O
(% by wt of
hexanol) x5
treat-
menta
time (h)
temp (°C)
(%c by wt of
hexanol) x3
obsd yield (% molar
predicted valued
(% molar conversion)
x1
x2
conversion) Y
1
2
3
4
5
6
7
8
9
-1 (4)b
1 (8)
-1 (4)
1 (8)
-1 (4)
1 (8)
-1 (4)
1 (8)
-1 (4)
1 (8)
-1 (4)
1 (8)
-1 (4)
1 (8)
-1 (4)
1 (8)
-2 (2)
2 (10)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
0 (6)
-1 (35)
-1 (35)
1 (55)
1 (55)
-1 (35)
-1 (35)
1 (55)
1 (55)
-1 (35)
-1 (35)
1 (55)
1 (55)
-1 (35)
-1 (35)
1 (55)
1 (55)
0 (45)
0 (45)
-2 (25)
2 (65)
0 (45)
0 (45)
0 (45)
0 (45)
0 (45)
0 (45)
0 (45)
0 (45)
0 (45)
0 (45)
0 (45)
0 (45)
-1 (20)
-1 (20)
-1 (20)
-1 (20)
1 (40)
1 (40)
1 (40)
1 (40)
-1 (20)
-1 (20)
-1 (20)
-1 (20)
1 (40)
1 (40)
1 (40)
1 (40)
0 (30)
0 (30)
0 (30)
0 (30)
-2 (10)
2 (50)
0 (30)
0 (30)
0 (30)
0 (30)
0 (30)
0 (30)
0 (30)
0 (30)
0 (30)
0 (30)
-1 (1.5:1)
-1 (1.5:1)
-1 (1.5:1)
-1 (1.5:1)
-1 (1.5:1)
-1 (1.5:1)
-1 (1.5:1)
-1 (1.5:1)
1 (2.5:1)
1 (2.5:1)
1 (2.5:1)
1 (2.5:1)
1 (2.5:1)
1 (2.5:1)
1 (2.5:1)
1 (2.5:1)
0 (2:1)
0 (2:1)
0 (2:1)
0 (2:1)
0 (2:1)
0 (2:1)
-2 (1:1)
2 (3:1)
1 (15)
-1 (5)
-1 (5)
1 (15)
-1 (5)
1 (15)
1 (15)
-1 (5)
-1 (5)
1 (15)
1 (15)
-1 (5)
1 (15)
-1 (5)
-1 (5)
1 (15)
0 (10)
0 (10)
0 (10)
0 (10)
0 (10)
0 (10)
0 (10)
0 (10)
-2 (0)
2 (20)
0 (10)
0 (10)
0 (10)
0 (10)
0 (10)
0 (10)
25.714
38.820
26.329
31.083
33.568
44.509
32.913
42.200
35.206
54.355
55.099
63.278
49.527
60.940
60.564
78.836
43.898
79.511
42.206
67.729
50.714
73.284
32.813
80.877
63.716
70.695
70.965
70.339
72.598
72.207
71.941
72.061
26.646
41.026
28.830
36.256
34.514
48.127
36.826
47.387
35.838
57.660
58.699
68.151
51.572
64.258
64.177
85.121
41.326
69.047
40.223
56.675
45.621
65.340
27.094
73.560
58.596
62.778
73.858
73.858
73.858
73.858
73.858
73.858
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
0 (2:1)
0 (2:1)
0 (2:1)
0 (2:1)
0 (2:1)
0 (2:1)
0 (2:1)
0 (2:1)
a
b
The treatments were run in a random order. Numbers in parentheses represent actual experimental amounts. c The activity of 10%
d
Lipozyme IM-77 is 0.024 BAUN. The predicted values are derived from eq 2.
from M. miehei (presently named Rhizomucor miehei) sup-
ported on macroporous weak anionic resin beads was pur-
chased from Novo Nordisk Bioindustrials, Inc. (Bagsvaerd,
Denmark). Hexanol (98% pure), triacetin (99% pure), and
tributyrin (99% pure) were purchased from Sigma Chemical
Co. (St. Louis, MO). A 4 Å molecular sieve was purchased from
Davison Chemical (Baltimore, MD), and n-hexane was ob-
tained from Merck Chemical Co. (Darmstadt, Germany). All
other chemicals were of analytical reagent grade.
Exp er im en ta l Design . A five-level-five-factor CCRD was
employed in this study, requiring 32 experiments (7, 9). The
fractional factorial design consisted of 16 factorial points, 10
axial points (2 axial points on the axis of each design variable
at a distance of 2 from the design center), and 6 center points.
The variables and their levels selected for the study of hexyl
acetate synthesis were as follows: time, 2-10 h; temperature,
25-65 °C; enzyme amount, 10-50% by weight of hexanol
(0.024-0.118 BAUN); substrate molar ratio, 3:1 to 1:1, triace-
tin/hexanol; and amount of added water, 0-20% by weight of
hexanol. Table 1 shows the independent factors (xi), levels, and
experimental design in terms of coded and uncoded.
by injecting a 1 µL aliquot in a splitless mode into a Hewlett-
Packard 4890 gas chromatograph (Avondale, PA) equipped
with a flame ionization detector. A DB-5 fused-silica capillary
column (30 m × 0.32 mm i.d.; film thickness ) 1 µm; J &W
Scientific, Folsom, CA) was used. Injector and detector tem-
peratures were set at 280 and 300 °C, respectively. Oven
temperature was maintained at 50 °C for 2 min, elevated to
200 °C at a rate of 50 °C/min, held for 4 min, and then
increased up to 300 °C at a rate of 70 °C/min. Nitrogen was
used as carrier gas. The percentage yield (percent molar
conversion) was defined as (millimoles of hexyl acetate/
millimoles of initial hexanol) × 100% and was estimated using
peak area integrated by on-line software Hewlett-Packard
3365 Series II ChemStation (Avondale, PA).
Sta tistica l An a lysis. The experimental data (Table 1) were
analyzed by the response surface regression (RSREG) proce-
dure to fit the following second-order polynomial equation (10)
5
5
4
5
Y ) âk0
+
âkixi +
â
kiixi2
+
â
kijxixj
(1)
∑
∑
∑ ∑
i)1
i)1
i)1 j)i+1
Ester ifica tion Meth od . All materials were dehydrated by
4 Å molecular sieve for 24 h. Hexyl acetate synthesis was
carried out in screw-capped test tubes (16 × 125 mm). Hexanol
(100 mM, 30.66 mg) and different molar ratios of triacetin were
added to 3 mL of n-hexane, followed by different amounts of
added water and enzyme. The mixtures of hexanol, triacetin,
and Lipozyme IM-77 were stirred in an orbital shaking water
bath (200 rpm) at different reaction temperatures and reaction
times (Table 1).
where Y is response (percent molar conversion); âk0, âki, âkii
,
and âkij are constant coefficients; and xi are the uncoded
independent variables. Canonical analysis was one part of the
RSREG SAS output and the RIDGE MAX option was used to
compute the estimated ridge of maximum response for in-
creasing radii from the center of the original design. Holding
one of three variables constant created individual contour
plots.
Extr a ction a n d An a lysis. The enzyme and any residual
water were removed by passing reaction media through an
anhydrous sodium sulfate column. Before sample analysis, the
reactant was taken to mix with an equal volume of an internal
standard solution (50 mM tributyrin). Then analysis was done
RESULTS AND DISCUSSION
RSM is a useful statistical technique for investigating
complex synthesis processes, especially for the optimum

Optimization of Hexyl Acetate Synthesis
J. Agric. Food Chem., Vol. 49, No. 3, 2001 1205
Ta ble 2. An a lysis of Va r ia n ce for J oin t Test
degrees of sum of
factor
freedom squares
prob > Fa
time (x1)
temp (x2)
enzyme amount (x3)
substrate molar ratio (x4)
added water content (x5)
6
6
6
6
6
1829.115
1890.785
1214.255
4608.530
385.045
0.0155
0.0138
0.0561
0.0004
0.5034b
a
b
Prob > F ) level of significance. Not significant at p ) 0.1.
F igu r e 1. Time course of transesterification of hexanol with
triacetin by Lipozyme IM-77. The reaction was carried out at
45 °C in 3 mL of hexane containing 100 mM hexanol, substrate
molar ratio of 2:1 (triacetin/hexanol), 30% (weight of hexanol)
Lipozyme IM-77, and without added water. The activity of 10%
Lipozyme IM-77 is 0.024 BAUN.
synthesis of lipase-catalyzed reaction. Compared with
one-factor-at-a-time design, which has been adopted
most often in the literature, the five-level-five-factor
CCRD employed in this study was more efficient in
reducing the experimental runs and time for investigat-
ing the optimal condition of short-chain ester synthesis.
The time course for the transesterification of hexanol
with triacetin by Lipozyme IM-77 is shown in Figure 1.
The percent molar conversion of hexyl acetate increased
up to 80% at 9 h; therefore, the range of reaction time
from 2 to 10 h was chosen in this experiment. The
selection of reaction time range needs to be extremely
precise in the CCRD; otherwise, the optimal condition
of synthesis cannot be found within the experimental
region through the analyses of statistics and contour
plots. Also, as shown by Figure 1 the conversion was
>80% after 9 h, so the commercially immobilized
Lipozyme IM-77 was added in the reaction system
directly and the pH was not controlled to increase the
yield during the reaction. Therefore, the ionization of
the amino acid residues from the enzyme protein
hydration of pH control did not affect the esterification
reaction in this study.
The RSREG procedure for SAS was employed to fit
the second-order polynomial eq 1 to the experimental
dataspercent molar conversions (Table 1). Among the
various treatments, the greatest molar conversion (80.9%)
was treatment 24 (6 h, 45 °C, 30% enzyme, molar ratio
3:1, and 10% added water), and the smallest conversion
(only 25.7%) was treatment 1 (4 h, 35 °C, 20% enzyme,
substrate molar ratio 1.5:1, and 15% added water). From
the SAS output of RSREG, the second-order polynomial
eq 1 is
F igu r e 2. Response surface plot showing the effect of enzyme
amount, reaction time, and their mutual interaction on hexyl
acetate synthesis. Other synthesis parameters (reaction tem-
perature, substrate molar ratio, and added water content) are
constant at zero level.
With very small P value (0.0015) from the analysis of
variance (ANOVA) and a satisfactory coefficient of
determination (R2 ) 0.921), the second-order polynomial
model (eq 2) was highly significant and adequate to
represent the actual relationship between the response
(percent molar conversion) and the significant variables.
Furthermore, the overall effect of the five synthesis
variables on the percent molar conversion of hexyl
acetate was further analyzed by a joint test (Table 2).
The results revealed that the time (x1), temperature (x2),
enzyme amount (x3), and substrate molar ratio (x4) were
the important factors, exerting a statistically significant
overall effect (p < 0.1) on the response molar conversion
of hexyl acetate; and water content (x5) had a less
significant effect (p > 0.1) on this reaction. The impor-
tance of control of the water content in lipase-catalyzed
hexanol esterification has been frequently emphasized
in the literature (11). However, water content in the
experimental scale (0-20%, w/w) of this study was the
least important factor compared to the other reaction
parameters from the statistical analysis. Besides, the
reaction was done by transesterification in which tri-
acetin, not acetic acid by direct esterification, was
employed as acyl donor. No additional esterification
water was produced along the conversion. Therefore,
added water content was constant at zero level (10%)
in the following discussion.
Y ) -242.247 + 16.898x1 + 4.617x2 + 2.700x3 +
2
61.965x4 + 1.548x5 - 1.167x1 - 0.044x2x1 -
2
2
0.064x2 + 0.015x3x1 + 0.003x3x2 - 0.046x3
+
Enzyme amount and reaction time were investigated
in the range of 10-50% and 2-10 h, respectively. Figure
2 shows the effect of enzyme amount, reaction time, and
their mutual interaction on hexyl acetate synthesis at
45 °C, substrate molar ratio 2:1, and added water
2
1.183x4x1 + 0.848x4x2 + 0.134x4x3 - 23.531x4
-
0.025x5x1 - 0.000014x5x2 + 0.007x5x3 + 0.613x5x4 -
2
0.132x5 (2)

1206 J. Agric. Food Chem., Vol. 49, No. 3, 2001
Shieh and Chang
F igu r e 3. Response surface plot showing the effect of reaction
temperature, enzyme amount, and their mutual interaction
on hexyl acetate synthesis. Other synthesis parameters (reac-
tion time, substrate molar ratio, and added water content) are
constant at zero level.
F igu r e 4. Response surface plot showing the effect of sub-
strate molar ratio, reaction temperature, and their mutual
interaction on hexyl acetate synthesis. Other synthesis pa-
rameters (reaction time, enzyme amount, and added water
content) are constant at zero level.
amount of 10%. At the lowest reaction time (2 h) with
the lowest enzyme amount (10%), molar conversion was
only 10%. A reaction with an enzyme amount of 30%
and a reaction time of 8 h led to the maximum molar
conversion (>70%). The effect of varying enzyme amount
and reaction temperature on esterification at constant
reaction time (6 h), substrate molar ratio (2:1), and
added water content (10%) is show in Figure 3. At any
given temperature from 25 to 65 °C, an increase of
enzyme amount led to higher yields. A reaction with a
moderate reaction temperature (55 °C) and the highest
enzyme amount favored maximal yield, and an increase
of temperature up to 65 °C resulted in less esterification
at any given enzyme amount because of the inhibition
of the enzyme by temperatures >55 °C, indicating that
the optimal temperature for lipase IM-77 was ∼55 °C.
Figure 4 represents the effect of varying substrate molar
ratio and reaction temperature on esterification at 6 h,
45 °C, and 10% added water amount. At low substrate
molar ratio (1.0) and any reaction temperature, the yield
was low (e20%). A reaction with high substrate molar
ratio (2.5-3.0) and high reaction temperature (55 °C)
produced maximal molar conversion (∼80%). An in-
crease in substrate molar ratio up to 3.0 resulted in less
molar conversion at any given temperature. The reason
high substrate molar ratio decreased the yield was that
lipase was inhibited by the accumulation of acetic acid
from the release of triacetin.
F igu r e 5. Contour plots of percent molar conversion of hexyl
acetate at an added water content of 10%. Enzyme amount
was by weight of hexanol. The numbers inside the contour
plots indicate molar conversions at given reaction conditions.
The relationships between reaction factors and re-
sponse can be better understood by examining the
planned series of contour plots (Figure 5) generated from
the predicted model (eq 2) by holding constant the
enzyme amount (10, 20, and 30%) and substrate molar
ratio (2:1, 2.5:1, and 3:1). Figure 5A-C represent the
same substrate molar ratio (2:1), and Figure 5A,D,G
represent the same enzyme amount (10%). Such an
application could be adopted to study the synthesis
variables simultaneously in a five-dimensional space.
Reaction time (x1) and temperature (x2) were the most
important variables for hexyl acetate synthesis with
small p values (see Table 2) and considered to be
indicators of effectiveness and economical performance.
The amount of added water content was kept (10%) with
less significant effect on response in the optimization
studies. In general, all nine contour plots in Figure 5
exhibited similar behaviors in that predicted molar
conversion increased with the reaction time; however,
it decreased over 8 h. Likewise, a reaction temperature
of ∼55 °C gave a higher percent molar conversion than
45 and 65 °C. The reaction of 30% enzyme and a
substrate molar ratio of 2.5:1 (Figure 5F) possessed
more predicted molar conversion than the others.
The optimum synthesis of hexyl acetate was deter-
mined by the ridge maximum analysis and the canonical
analysis (10, 12). The method of ridge analysis computes
the estimated ridge of maximum response for increasing

Optimization of Hexyl Acetate Synthesis
J. Agric. Food Chem., Vol. 49, No. 3, 2001 1207
Ta ble 3. Estim a ted Rid ge of Ma xim u m Resp on se for
Va r ia ble P er cen t Mola r Con ver sion
) 53 °C, 37.1% or 0.089 BAUN enzyme amount, 2.7:1
substrate molar ratio, and 12.5% added water content)
was recommended as the optimization for hexyl acetate
synthesis with 88.9% molar conversion in this study.
The adequacy of the predicted model here was exam-
ined by additional independent experiments at the
suggested optimal synthesis conditions. The predicted
value was 88.9% obtained by canonical analysis, and
the actual value was 86.6 ( 2.9%. Chi-square test (p
value ) 0.991, degrees of freedom ) 7) indicated that
observed values were significantly the same as the
predicted values and the generated model adequately
predicted the percent molar conversion (13). Thus, the
optimization of lipase-catalyzed synthesis for hexyl
acetate by Lipozyme IM-77 was successfully developed
by CCRD and RSM.
estimated
coded responses (%
radius conversion)
x4
x1
(h)
x2
(°C)
x3
(%)
(triacetin/
hexanol)
x5
(%)
SE
0
73.858
79.236
83.393
86.368
88.191
88.888
3.292 6.000 45.000 30.000
3.257 6.369 46.240 31.344
3.260 6.728 47.673 32.729
3.612 7.070 49.213 34.134
4.629 7.391 50.810 35.542
6.384 7.690 52.433 36.944
2.000
2.151
2.296
2.437
2.575
2.710
10.000
10.208
10.553
11.037
11.659
12.414
0.2
0.4
0.6
0.8
1.0
LITERATURE CITED
(1) Bauer, K.; Garbe, D.; Surburg, H. Common Fragrance
and Flavor Materials; VCH Publishers: New York,
1990.
(2) Bourg-Garros, S.; Razafindramboa, N.; Pavia, A. A.
Synthesis of (Z)-3-hexen-1-yl Butyrate in Hexane and
Solvent-Free Medium Using Mucor miehei and Candida
antarctica Lipases. J . Am. Oil Chem. Soc. 1997, 74,
1471-1475.
(3) Bourg-Garros, S.; Razafindramboa, N.; Pavia, A. A.
Optimization of Lipase-Catalyzed Synthesis of (Z)-3-
hexen-1-yl Acetate by Direct Esterification in Hexane
and Solvent-Free medium. Enzyme Microb. Technol.
1998, 22, 240-245.
(4) Chatterjee, T.; Bhattacharyya, D. K. Synthesis of Monot-
erpene Esters by Alcoholysis Reaction with Mucor
miehei Lipase in a Solvent-Free System. J . Am. Oil
Chem. Soc. 1998, 75, 651-655.
F igu r e 6. Contour plots showing response behavior of reac-
tion time and temperature of the optimum synthesis condition
at the stationary point (enzyme amount, 37.1%; molar ratio,
2.7:1; added water content, 12.5%) suggested by canonical
analysis.
(5) Ahmad, R. W.; Anderson, W. A.; Moo-Young, M. Ester
Synthesis in Lipase-Catalyzed Reactions. Enzyme Mi-
crob. Technol. 1998, 23, 438-450.
(6) Krishna Hari, S.; Manohar, B.; Divakar, S.; Karanth,
N. G. Lipase-Catalyzed Synthesis of Isoamyl Butyrate:
Optimization by Response Surface Methodology. J . Am.
Oil Chem. Soc. 1999, 76, 1483-1488.
(7) Shieh, C.-J .; Lou, Y.-H. Five-Factor Response Surface
Optimization of the Enzymatic Synthesis of Citronellyl
Butyrate by Lipase IM77 from Mucor miehei. J . Am. Oil
Chem. Soc. 2000, 77, 521-526.
radii from the center of the original design. The ridge
maximum analysis (Table 3) indicated that maximum
molar conversion was 88.9 ( 6.4% at 7.7 h, 52.4 °C,
36.9% enzyme amount, 2.7:1 substrate molar ratio, and
12.4% added water content at the distance of the coded
radius of 1.0. The optimum point was also determined
by canonical analysis. The stationary point (reaction
time ) 7.7 h, reaction temperature ) 52.6 °C, 37.1% or
0.089 BAUN enzyme amount, substrate molar ratio )
2.7:1, and added water content ) 12.5%), values of
variables at which the first derivative of the response
was zero, was located exactly in the experimental region
with the predicted value of 88.9%. The canonical analy-
sis based on the stationary point resulted in the equa-
tion
(8) Carvalho, C. M. L.; Serralheiro, M. L. M.; Cabral, J . M.
S.; Aires-Barros, M. R. Application of Factorial Design
to the Study of Transesterification Reactions Using
Cutinase in AOT-Reversed Micelles. Enzyme Microb.
Technol. 1996, 21, 117-123.
(9) Cochran, W. G.; Cox, G. M. Experimental Designs;
Wiley: New York, 1992.
(10) SAS. SAS User’s Guide; SAS Institute Inc.: Cary, NC,
1990.
(11) Linko, Y.-Y.; Lamsa, M.; Huhtala, A.; Linko, P. Lipase-
Catalyzed Transesterification of Rapeseed Oil and 2-Eth-
yl-1-hexanol. J . Am. Oil Chem. Soc. 1994, 71, 1411-
1414.
(12) Shieh, C.-J .; Lai, Y.-F. Application of Response Surface
Methodology to the Study of Methyl Glucoside Polyester
Synthesis Parameters in a Solvent-Free System. J .
Agric. Food Chem. 2000, 48, 1124-1128.
(13) Ott, L. An Introduction to Statistical Methods and Data
Analysis; PWS-Kent Publishing: Boston, MA, 1988.
2
2
Y ) 88.897 - 11.466W1 - 16.451W2
-
2
2
2
18.312W3 - 19.139W4 - 33.791W5 (3)
where W1, W2, W3, W4, and W5 are eigenvalues based
on coded data and Y is the molar conversion of hexyl
acetate (percent). All eigenvalues were negative, indi-
cating that the predicted response surface of the sta-
tionary point is shaped like a maximum. The response
behavior of reaction time and synthesis temperature
(Figure 6) was followed while the other reaction param-
eters were held constant at the suggested optimum
point. The maximum value (88.5%) was predicted to be
near a combination of 7.7 h and 53 °C. Because results
from both maximum ridge analysis and canonical
analysis indicated similar conclusions, the reaction
condition (reaction time ) 7.7 h, synthesis temperature
Received for review August 24, 2000. Revised manuscript
received J anuary 2, 2001. Accepted J anuary 4, 2001. This
research was supported by the National Science Council (NSC-
89-2313-B-212-005), Taiwan, Republic of China.
J F001050Q