Synthesis of (Z)-3-hexen-1-yl butyrate in hexane and solvent-free medium using Mucor miehei and Candida antarctica lipases

Article abstract of DOI:10.1007/s11746-997-0256-0

(Z)-3-Hexen-1 -yl butyrate is an important flavor and fragrance compound as it represents the model of a natural herbaceous (green) note. Two immobilized lipases from Mucor miehei (Lipozym IM) and from Candida antarctica (Novozym 435) were investigated for their use in the synthesis of (Z)-3-hexen-1-yl butyrate by direct esterification in n-hexane. To determine optimal conditions for esterification, we examined the following parameters: temperature, amount of lipase, acid/alcohol ratio, and absence of solvent. In n-hexane, bioconversion yields reached 95 (after 4 h) and 92% (after 6 h) for, respectively, Lipozym IM [17 (w/w reactants)] and Novozym 435 [2% (w/w reactants)]. In the absence of solvent, at 60 °C, Novozym 435-catalyzed esterification afforded the title compound in 80% yield. Up to 250 g (in hexane) and 160 g (without solvent) of ester were easily prepared, in a single operation, at a laboratory scale, in few hours, using 2% (w/w reactants) lipase.

Full text of DOI:10.1007/s11746-997-0256-0

Synthesis of (Z)-3-Hexen-1-yl Butyrate in Hexane
and Solvent-Free Medium Using Mucor miehei
and Candida antarctica Lipases
S. Bourg-Garros, N. Razafindramboa1, and A.A. Pavia2,*
Laboratoire d’Agrobiologie et Chimie du végétal, Faculté des Sciences, 84000 Avignon, France
ABSTRACT: (Z)-3-Hexen-1-yl butyrate is an important flavor would make food industry less dependent on seasonal, cli-
and fragrance compound as it represents the model of a natural
herbaceous (green) note. Two immobilized lipases from Mucor
miehei (Lipozym IM) and from Candida antarctica (Novozym
435) were investigated for their use in the synthesis of (Z)-3-
hexen-1-yl butyrate by direct esterification in n-hexane. To de-
termine optimal conditions for esterification, we examined the
following parameters: temperature, amount of lipase, acid/alco-
hol ratio, and absence of solvent. In n-hexane, bioconversion
yields reached 95 (after 4 h) and 92% (after 6 h) for, respec-
tively, Lipozym IM [17 (w/w reactants)] and Novozym 435 [2%
(w/w reactants)]. In the absence of solvent, at 60°C, Novozym
matic, and geographical variations.
The present work deals with the biosynthesis of (Z)-3-
hexen-1-yl butyrate as a model compound catalyzed by two
commercial immobilized lipases from Mucor miehei and
from Candida antarctica. Our purpose was to determine best
experimental conditions and procedure to produce rather
large amounts of C6 alcohol-derived esters (150 to 250 g), at
a laboratory scale, in a single operation as an intermediate
step to pilot scale, our ultimate goal being the industrial pro-
duction from natural acids and alcohols of esters considered
435-catalyzed esterification afforded the title compound in 80% to be natural (15–17). The impact of several parameters on
yield. Up to 250 g (in hexane) and 160 g (without solvent) of
ester were easily prepared, in a single operation, at a laboratory
scale, in few hours, using 2% (w/w reactants) lipase.
JAOCS 74, 1471–1475 (1997).
the bioreaction was carefully investigated and optimized, i.e.,
the nature and the amount of enzyme, the substrate concen-
tration, the temperature, and the acid/alcohol ratio. We also
investigated C. antarctica-catalyzed synthesis of the title
compound in the absence of solvent.
KEY WORDS: Biosynthesis, Candida antarctica, direct esteri-
fication, n-hexane, (Z)-3-hexen-1-yl butyrate, lipases, Mucor
miehei, solvent-free medium.
MATERIALS AND METHODS
Materials. Lipases from M. miehei immobilized on macro-
Low-molecular-weight esters derived from short-chain car- porous anion exchange resin Lipozym IM [5–6 Batch Acidol-
boxylic acids (acetates, propionates, butyrates, …) represent ysis Units NOVO (BAUN)·g−1, cloned into Aspergillus
an important class of flavoring materials. They are responsi- oryzae] and C. antarctica immobilized on acrylic resin
ble for the fruity odors of many foods and fragrances (1,2). In Novozym 435 [7000 Propyl Laurate Units (PLU)·g−1, cloned
foodstuff industries, flavor and fragrance loss due to various into A. oryzae] were purchased from Novo Nordisk Bioindus-
reasons including manufacturing processes must be compen- trials, Inc. (Bagsvaerd, Denmark). (Z)-3-Hexen-1-ol (98%
sated for by the addition of aromas. Moreover, there is a pure) was provided by the Company Naturex (Montfavet,
growing demand for natural flavors containing green notes France). Butyric acid was purchased from Aldrich (Stein-
represented by C-6 alcohols derivatives. Hence, the biologi- heim, Germany).
cal production of such esters is of much current commercial
Esterification method. Ester synthesis was carried out in a
interest. In this context, the use of immobilized lipases work- 50-mL two-necked round flask equipped with a condenser
ing in nonaqueous media (3–14) has potential since esters topped with a calcium chloride guard tube. For the scale-up,
produced from natural acids and alcohols may be considered we used either a 500-mL or 2000-mL three-necked round
as natural. This mode of production is of great interest, as it flask equipped with a mechanical stirrer. (i) With Lipozym
IM: To dry n-hexane (20 mL) were successively added alco-
1Present address: Laboratoire de Chimie Bioorganique et des Systèmes hol (0.5 M), butyric acid (0.75 M), and M. miehei lipase [17%
Moléculaires Vectoriels, Faculté des Sciences, 33, rue Louis Pasteur, 84000
(w/w reactants)]. The reaction mixture was magnetically
Avignon, France.
stirred (250 rpm) in a thermostated oil bath at 40°C. (ii) With
Novozym 435: Alcohol (1.5 M), butyric acid (1.5 M), and C.
antarctica lipase [2% (w/w reactants)] were added to 20 mL
of hexane. The suspension was incubated in a thermostated
2Present address: Laboratoire de Chimie Biomoléculaire, Université de
Montpellier 2, CC 020, 34095 Montpellier Cedex 05.
*To whom correspondence should be addressed.
E-mail: aapavia@univ-montp2.fr.
Copyright © 1997 by AOCS Press
1471
JAOCS, Vol. 74, no. 11 (1997)

1472
S. BOURG-GARROS ET AL.
oil bath at 70˚C and magnetically stirred at 250 rpm (18). (iii)
Esterification in a solvent-free medium was carried out with
the same apparatus. The mixture of alcohol (0.12 mole), bu-
tyric acid (0.12 mole), and Novozym 435 [2% (w/w reac-
tants)] was incubated at 60°C and magnetically stirred at 250
rpm.
Chromatography analyses. Aliquots were periodically
withdrawn and analyzed on a Hewlett-Packard gas chromato-
graph model HP 5890 (Palo Alto, CA) equipped with a capil-
lary column HP INNO WAX (0.4 µm, 50 m × 0.2 mm).
Split/splitless injector and mass spectrometer detector were
set at 250 and 280°C, respectively. Helium was used as car-
rier at a flow rate of 15 mL·min−1. The temperature gradient
was the following: 10°C/min from 50 to 150°C, followed by
15°C/min from 150 to 230°C. (Z)-3-Hexen-1-ol and (Z)-3-
hexen-1-yl butyrate were quantitated relative to ( ) linalol as
internal standard.
Time (min)
FIG. 2. Effect of butyric acid/(Z)-3-hexen-1-ol molar ratio on the rate of
formation of (Z)-3-hexen-1-yl butyrate in hexane (20 mL) in the pres-
ence of Mucor miehei lipase [17% (w/w reactants)], 40°C; (+) = 0.75
mol·L−1 acid/0.5 mol·L−1 alcohol (R = 1.5); (●) = 0.5 mol·L−1 acid/0.5
mol·L−1 alcohol (R = 1); (×) = 0.75 mol·L−1 acid/0.75 mol·L−1 alcohol
(R = 1); (▲) = 1.5 mol·L−1 acid/1 mol·L−1 alcohol (R = 1.5).
RESULTS AND DISCUSSION
involved (9,12,19,21–23). In our hands, the minimum con-
Mucor miehei lipase-mediated synthesis. The effect of tem- centration of Lipozym IM necessary to achieve maximum
perature on the synthesis of (Z)-3-hexen-1-yl butyrate by di- conversion yield (92% in 15 h) was 4% (w/w reactants).
rect esterification is shown in Figure 1A. In accordance with
Two approaches were followed in the study of the effect
previous reports (4,19,20), the optimal temperature for of substrate concentration. First, we evaluated the effect of
Lipozym IM was found to be 40˚C. In that condition, 92% increasing the concentration of one of the substrates while
conversion yield was observed in 3 h compared with 87% in keeping the other constant, then the effect of increasing the
6.5 h at 25°C.
concentration of both substrates while maintaining the same
The amount of lipase is a crucial economic factor for any molar ratio. The effects of substrate molar ratio on both the
bioconversion process. The concentrations of lipase reported reaction rate and amount of ester formed are presented in Fig-
are often too high to meet industrial requirements (21). En- ure 2, for three different situations: #1, 0.75 M acid/0.5 M al-
zyme concentrations of 38 and 93% (w/w reactants) have cohol (R1 = 1.5); #2, 0.5 M acid/0.5 M alcohol (R2 = 1); #3,
been reported (7,10). Having as an ultimate goal the develop- 0.75 M acid/0.75 M alcohol (R3 = 1).
ment of a large-scale process, we assessed the effect of vary-
As seen in Figure 2, maximum conversion yield (94 to
ing enzyme concentration on the yield of (Z)-3-hexen-1-yl 95%) and esterification rate (4 h) were obtained with reaction
butyrate by direct esterification (Fig. 1B). As already noted, conditions #1 [R1 = 1.5; 0.5 M (Z)-3-hexen-1-ol]. Reactions
the rate of esterification was related to the amount of enzyme #2 and #3 led to maximum conversion yields of, respectively,
85% (4 h) and 83% (4.5 h). As expected, reaction rate was de-
pendent on substrate concentration. For 0.5 M alcohol, an ex-
cess of butyric acid increased both the rate of esterification
and maximum conversion yield (#1). The inhibitory effect of
butyric acid on Lipozym IM activity was not detected as pre-
viously reported (4,6,21). One possible explanation may arise
from the nature of the support to which the enzyme is linked.
Several reports have emphasized the role that the support may
play on the efficiency of the enzyme (4,5,24–27). Several pa-
A
rameters, including the hydrophilic-lipophilic balance (24),
the accessibility to the catalytic site, the particle size, pore
size, etc. (3,15,24), govern the enzyme activity. In the case of
a celite-adsorbed M. miehei lipase, Manjón et al. (4) noted a
significant decrease of lipase activity when butyric acid con-
B
Time (min)
Time (min)
FIG. 1. (A) Effect of reaction temperature on the rate of esterification of
(Z)-3-hexen-1-ol with butyric acid catalyzed by the lipase of Mucor centration exceeded 0.4 mol·L−1. According to these authors,
miehei [Lipozym IM 17% (w/w reactants)] in hexane (20 mL), 0.75
this behavior should be ascribed to a decrease of the pH of
the enzyme aqueous environment. Others have reported (11)
that the effectiveness of the lipase from C. cylindraceae im-
mobilized on nylon was not affected by butyric acid concen-
tration since the rate of esterification increased steadily up to
mol·L−1 acid/0.5 mol·L−1 alcohol; (x) = 40°C; (+) = 25°C. Lipozym IM:
Novo Nordisk, Bagsvaerd, Denmark. (B) Effect of the concentration of
Lipozym IM [% (w/w reactants)] on the rate of formation of (Z)-3-hexen-
1-yl butyrate in hexane (20 mL), 40°C, 0.75 mol·L−1 acid/0.5 mol·L−1
alcohol; (+) = 17%; (●) = 13%; (×) = 4%.
JAOCS, Vol. 74, no. 11 (1997)

SYNTHESES OF (Z)-3-HEXEN-1-YL BUTYRATE
1473
2 mol·L−1. By keeping constant the butyric acid/(Z)-3-hexen-
1-ol molar ratio (R = 1), an increase of the concentration of
both substrates affected the rate of the reaction. Similarly, a
twofold increase of substrate concentrations in the optimal
molar ratio situation R′ = 1.5 resulted in a reduction of the
maximum conversion yield from 95 to 85% (for 4 h reaction).
These findings are consistent with published results
(4,12,19,21) dealing with M. miehei lipase (Lipozym IM).
The loss of effectiveness resulting from an increase of sub-
strate concentration may be due either to a substrate inhibi-
tion or, more likely, to a modification of the polarity of the
medium. Zaks and Klibanov (28) demonstrated that it is the
water bound to the enzyme which determines the catalytic ac-
tivity rather than the total water content. It is generally ac-
cepted that the more polar the solvent, the more water will be
held in solution instead of bound to the enzyme, with a con-
comitant decrease of both the stability and activity (28). Best
solvents for lipase-mediated esterifications are those which
do not withdraw water from the enzyme water monolayer.
These are characterized by high log P values (log P ≥ 4) (29).
Candida antarctica lipase-mediated biosynthesis. In view
of the excellent results obtained in our laboratory in the acy-
lation of (Z)-3-hexen-1-ol by acetic acid using the enzyme of
C. antarctica (18), we decided to investigate the Novozym
435-mediated esterification of this alcohol. In using the same
conditions as those determined for Lipozym IM [17% (w/w
reactants)] with Novozym 435, maximum conversion yield
(96%) was obtained in 20 min whereas in the same conditions
only 7% of ester was produced with Lipozym IM. With the
latter, it took 130 min to reach the plateau (92%). These find-
ings substantiate the observations reported by Claon and
Akoh (8) and others (20,30).
For safety, economic, technological and environmental
reasons, enzyme-mediated direct esterification in a solvent-
free medium is of utmost importance especially in the food
industry. In the last 6 yr, lipase-mediated direct esterification
and transesterification have motivated research on M. miehei
(9,23,25,31), C. cylindraceae (11,25), and C. antarctica
(13,14,32,33) lipases. For instance, lipase from C. antarctica
was reported (32) to mediate the transesterification synthesis
of geranyl and β-citronellyl acetate in 94% yield (in 10 h)
compared to 74% (in 6 h) for the direct esterification of β-cit-
ronellol by acetic acid (13). However, geranyl butyrate was
produced in almost quantitative yield in 7 h at 60°C from a
mixture of acid, alcohol (molar ratio 0.87), and 500 mg of C.
antarctica lipase, i.e., 7% (w/w reactants) (14). In our experi-
ments, the formation of (Z)-3-hexen-1-yl butyrate was slower
(6 h) and the procedure less efficient (80% yield) in a solvent-
free medium than in hexane (92% yield in 2 h).
B
A
Time (min)
Time (min)
FIG. 3. (A) Effect of butyric acid/(Z)-3-hexen-1-ol molar ratio on the rate
of formation of (Z)-3-hexen-1-yl butyrate in hexane (20 mL) in the pres-
ence of Candida antarctica lipase [2% (w/w reactants)], 70°C; (▲) = 1.5
mol·L−1 acid/1.5 mol·L−1 alcohol (R = 1); (●) = 1.725 mol·L−1 acid/1.5
mol·L−1 alcohol (R = 1.15); (+) = 1.875 mol·L−1 acid/1.5 mol·L−1 alco-
hol (R = 1.25); (×) = 2.25 mol·L−1 acid/1.5 mol·L−1 alcohol (R = 1.5). (B)
Effect of butyric acid/(Z)-3-hexen-1-ol molar ratio on the rate of forma-
tion of (Z)-3-hexen-1-yl butyrate in a solvent-free medium in the pres-
ence of C. antarctica lipase [2% (w/w reactants)], 60°C; (▲) = 0.12 mol
acid/0.12 mol alcohol (R = 1); (●) = 0.138 mol acid/0.12 mol alcohol
(R = 1.15); (+) = 0.15 mol acid/0.12 mol alcohol (R = 1.25); (×) = 0.18
mol acid/0.12 mol alcohol (R = 1.5).
reactants) of lipase (Fig. 3A), best results (92 to 95% yield in
less than 3 h) were obtained for molar ratios R > 1 (ii, iii, iv)
compared to 88% within the same time when R = 1 (i). In that
respect, Novozym 435 behaved similarly to Lipozym IM ex-
cept that: (i) maximum conversion yield was slightly superior;
(ii) neither the diminution of conversion yield nor the increase
of reaction rate was observed even when the concentration in
butyric acid reached 2.25 mol·L−1. Similar observations have
been reported for the lipase of C. cylindraceae immobilized
on nylon (11). (iii) In contrast to what happened when acetic
acid was the acylating reagent (18), conversion yield increased
from 90 to 95% yield when butyric acid concentration in-
creased from 1.5 to 2.25 mol·L−1. (iv) This study allowed us to
establish the superiority of the lipase of C. antarctica over that
of M. miehei for the direct esterification of (Z)-3-hexen-1-ol.
Working with only 2% (w/w reactants) enzyme, conversion
yield reached 85% within 1 h compared to 30% using 17%
(w/w reactants) of Lipozym IM.
Without solvent, whatever the acid/alcohol molar ratio, maxi-
mum conversion yield was 82% (Fig. 3B). The latter was reached
after 8 h (R = 1), 5 h (R = 1.15; R = 1.25), and 6 h (R = 1.5)
Very few reports deal with the extrapolation of a biosyn-
thetic process from the laboratory (≈20 mL) to semiprepara-
tive (100 to 200 mL) (10,34) and/or to large scale (1 to 5 L)
(22). We investigated the effect of the reaction volume by
multiplying by 12.5 (250 mL) and 50 (1000 mL) all parame-
ters, standard experiments being those performed in a 20-mL
volume. To achieve such experiments, we used a “reflux” ro-
tary evaporator equipped with a graduated tube instead of the
conventional receiving flask. The advantages of using such
apparatus are: (i) It ensures a gentle although efficient disper-
sion of the immobilized lipase. Thus we noted an increase of
the lipase lifetime. The enzyme could be reused several times
To determine the influence of the acid/alcohol molar ratio
on the course of the direct esterification of (Z)-3-hexen-1-ol in
hexane, we investigated the following situations: (i) 1.5 mol·L−
1 acid, 1.5 mol·L−1 alcohol, R = 1; (ii) 1.725 mol·L−1 acid, 1.5
mol·L−1 alcohol, R = 1.15; (iii) 1.875 mol·L−1 acid, 1.5 mol·L−
1 alcohol, R = 1.25; (iv) 2.25 mol·L−1 acid, 1.5 mol·L−1 alco-
hol, R = 1.5. In hexane, at 70°C, in the presence of 2% (w/w
JAOCS, Vol. 74, no. 11 (1997)

1474
S. BOURG-GARROS ET AL.
without a significant loss of efficiency (Bourg-Garros, S., N. published by Claon and Akoh (8). It is noteworthy that sub-
Razafindramboa, and A.A. Pavia, unpublished results). This strate concentrations employed in this work are 6 to 15 times
is probably due to a lower degree of breakdown of the solid higher than those usually reported in the literature (4,7,8).
material compared with that observed when the medium is
From the comparative study of the ability of two commer-
magnetically stirred. (ii) It represents an intermediate step be- cial immobilized lipases from M. miehei (Lipozym IM) and
tween the laboratory and the pilot-scale which was our next C. antarctica (Novozym 435) to catalyze direct esterification
goal, especially since the apparatus to be used at the pilot of (Z)-3-hexen-1-ol by butyric acid, we established the supe-
plant was a rotary-type device.
riority of Novozym 435 in terms of both reaction rate and
In contrast to results reported by Langrand et al. (10) deal- conversion yield. Best results were obtained in hexane
ing with Rhizopus arrhizus-mediated esterification of geran- whereas solvent-free medium biosyntheses proved less effi-
iol, a multifold increase of reaction volume was not accom- cient and were slower. It is noteworthy that experimental con-
panied by a diminution of the conversion yield (Fig. 4A). The ditions set up in this work are significantly different from
latter was 91, 90, and 94%, respectively, for 20-, 250-, and those reported to date in the literature. In hexane, substrate
1000-mL experiments. When the same approach was applied concentrations were approximately 15 times higher than those
to a solvent-free medium system, using the same apparatus, reported whereas the amount of enzyme was five times
we obtained similar results except that the maximum conver- smaller (8). Without solvent, our experiments were performed
sion yield did not exceed 77% (5 to 6 h) (Fig. 4B). From these with amount of substrate 4 to 40 times higher and amount of
results, we may anticipate that the extrapolation of such ex- lipase 3.5 times smaller (14). We were able to extrapolate ex-
perimental conditions and procedures to large-scale prepara- perimental condition and parameters to large-scale laboratory
tion should be straightforward.
syntheses: 250 g of (Z)-3-hexen-1-yl butyrate was easily pre-
The ability of C. antarctica lipase [10% (w/w reactants)] pared in one run, in a few hours with a yield of >90%.
to synthesize other flavor esters was demonstrated at the 500-
mL scale in hexane using equimolar concentrations (1.5
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[Received April 7, 1997; accepted June 22, 1997]
JAOCS, Vol. 74, no. 11 (1997)