HUNGARIAN JOURNAL OF
INDUSTRY AND CHEMISTRY

Vol. 45(2) pp. 35–39 (2017)

hjic.mk.uni-pannon.hu

DOI: 10.1515/hjic-2017-0018

EFFECT OF CHAIN LENGTH AND ORDER OF THE ALCOHOL ON
ENZYME ACTIVITY DURING ENZYMATIC ESTERIFICATION IN
ORGANIC MEDIA

ZSÓFIA MÁRKUS, KATALIN BÉLAFI-BAKÓ, GÁBOR TÓTH, NÁNDOR NEMESTÓTHY AND LÁSZLÓ
GUBICZA*

Research Institute on Bioengineering, Membrane Technology and Energetics,
University of Pannonia, Egyetem u. 10, Veszprém, 8200, Hungary

Esters of short chain acids and alcohols are found in nature as compounds of flavors. Lately the method for
their manufacture has been the enzymatic esterification in non-conventional media. Although several reactions
have been studied in various media (organic solvents, ionic liquids, supercritical fluids, solvent-free systems),
there has been no systematic investigation to clarify the effects of chain length and order of alcohols on the ac-
tivity of the enzyme. In this work acetic acid was used as an acyl donor and the roles of the linear and branched
chains of C2-C8 primary, secondary and tertiary alcohols on the activity of Novozym 435, the widely used lipase
preparation were studied. Both the length of the carbon chain and the order of the alcohol were found to strong-
ly influence the activity of the enzyme using the same operational parameters for the reactions. As a result of
this project general conclusions were made with regard to the characters of alcohols affecting the reaction rates,
which can be applied to other similar reactions.

Keywords: enzymatic esterification, non-conventional media, effect of alcohol chain lengths,
lipase activity

1. Introduction

Enzyme technology provides a promising solution for
the biosynthesis of natural flavor esters, since several
enzymes are able to catalyze the synthesis of aroma
compounds from precursor molecules [1]. Nowadays
most flavor compounds are manufactured by conven-
tional methods: chemical synthesis or recovery from
natural sources. Esters produced chemically are quite
common, but their method of production is not consid-
ered environmentally safe and cannot be classed as
“natural”. Recently interest has been growing in the
production of these components by biotransformation,
that is the manufacture of natural flavor esters by using
natural raw materials. Lipases belong to the most di-
verse class of enzymes, they catalyze various reactions
due to their wide spectrum of industrial applications.
Lipase enzymes have been applied in many industrial
sectors, e.g. the food and pharmaceutical industries, in
the production of biological detergents (esters of carbo-
hydrates), moreover in the manufacture of certain cos-
metics and fragrances. Recently interest has grown in
the production of natural flavor esters by the biosynthe-
sis of short chain acids and alcohols [2,3].

Several similar reactions have been studied, most
of which focused on the synthesis of acetates, like ethyl

*Correspondence: gubiczal@almos.uni-pannon.hu

[4], butyl [5], hexyl [3], cinnamyl [6], and benzyl [7]
acetates.

During the investigation of the reactions‘ parame-
ters the roles of temperature, the molar ratio of acid to
alcohol and the amount of enzyme were described in
almost every paper. From these data the optimal values
of these parameters could be estimated. Numerous reac-
tions were carried out in organic solvents [8, 9], ionic
liquids [10, 11], supercritical fluids (mainly in super-
critical carbon dioxide) [12], solvent-free systems [13],
in addition to in the gas phase [14]. Since these reac-
tions can be conducted in non-aqueous media, the water
content, to be more precise the water activity of the re-
action mixture plays an extremely important role in
terms of the reaction rate. For the operation of the en-
zyme lipase it is necessary to provide a minimal amount
of water. On the other hand it is an equilibrium reaction,
thus the greater excess of water shifts the reaction to-
wards hydrolysis, decreasing the conversion rate signif-
icantly. The investigation of the effect of water content,
or at least an intention to adjust the initial water content
by a constant value is missing in several papers. The
water content during the reaction continuously changes
due to the production of water in the esterification. This
effect can be neglected during the investigation of the
initial reaction rates, but in terms of the development of
continuous production it should be taken into account.
A number of methods are known to maintain water con-
tent/activity. From a practical point of view membrane
separation processes, like pervaporation provide attrac-
tive procedures [15].

MÁRKUS, BÉLAFI-BAKÓ, TÓTH, NEMESTÓTHY AND GUBICZA

Hungarian Journal of Industry and Chemistry

Beyond the parameters mentioned and the water
content, only a limited attention was paid to the role of
substrates present in the reaction. As pointed out earlier,
acetic acid was the acidic component used in most cas-
es. The alcohols applied, however, were much more
diverse: linear and branched chains, aromatic and dif-
ferent orders of alcohols were investigated. Neverthe-
less our group has not found a single study in the litera-
ture where systematic research was conducted to assess
the role of alcohols on enzyme activity.

The effect of the chain length of various alcohols
was investigated in reactions catalyzed by enzymes in
non-conventional media. Romero and colleagues con-
ducted experiments in supercritical carbon dioxide us-
ing four different alcohols: propanol, butanol, pentanol
and octanol. It was found that higher degrees of conver-
sion could be obtained by applying longer chain alco-
hols. Enzymes have a higher affinity towards longer
chain alcohols, the difference, however, was small [16].

In another paper Romero used butyric acid as an
acyl donor and the enzyme Novozym 435. An insignifi-
cant difference was observed in terms of the reaction
rate or conversion during esterification when the four
primary alcohols (propanol, butanol, hexanol and oc-
tanol) were applied. Applying secondary alcohols, e.g.
2-hexanol, however, yielded higher reaction rate and
conversion than was the case with 2-butanol [17].

Pan et al. studied how the chain length of alcohol
compounds can influence the resolution reaction of
mandelic acid. Using methanol, ethanol, butanol, hep-
tanol and octanol it was found that the highest degree of
enantioselectivity could be obtained by ethanol. Moreo-
ver the reaction was described as following Michaelis-
Menten kinetics in all cases and the inhibition constant
increased as the carbon chain go longer [18].

Varma and Madras investigated the esterification
of propionic acid and three different alcohols by the
enzyme Novozym 435 in supercritical carbon dioxide.
Primary (isobutanol, isoamyl alcohol) and secondary
alcohols (isopropyl alcohol) were used, as well. Based
on the measurements, it was concluded that enzymatic
esterification was faster with primary alcohols than with
secondary alcohols, moreover a greater degree of con-
version was achieved with isobutanol than with isoamyl
alcohol [19].

Therefore the aim of this paper was to study the
role of alcohols by the preparation of a given enzyme
whilst maintaining the operation parameters as con-
stants. The esterification primary, secondary and tertiary
alcohols of C2-C8 carbon chain lengths were investigat-
ed to be able to draw general conclusions concerning
the role of alcohol structure on reaction rate. For the
measurements a popular immobilized lipase enzyme
preparation, Novozym 435

®
, was used.

2. Experimental

2.1. Chemicals and Enzymes

All chemicals: acetic acid, ethanol, 1-propanol. 1-
butanol and n-hexane (Merck), 1-pentanol, 2-pentanol
tert-butanol (2-methylpropan-2-ol) and 1-hexanol (Sig-
ma-Aldrich); 1-heptanol (BDH Chemicals); 1-octanol
and isobutanol (2-methylpropan-1-ol) (Spektrum-3D);
isoamyl alcohol (3-methylbutan-1-ol) (Molar Chemi-
cals); and 2-propanol, 2-butanol, tert-amyl alcohol (2-
methylbutan-2-ol) and toluene (Reanal) were of analyti-
cal grade. The water content of the chemicals varied
greatly, that is why they were dewatered over a 3Å mo-
lecular sieve in the form of beads (Sigma-Aldrich).

The enzyme used was Novozym 435
®

from Can-
dida antarctica lipase B, immobilized on a macroporous
acrylic resin with a water content of 1-2% w/w, which
was kindly provided as a gift by Novo Nordisk A/S,
Denmark. According to their commercial product man-
ual, its catalytic activity was 7000 PLU/g (propyl laurate
units/gram).

2.2. Reaction and Analysis

Reactions were carried out in 50 mL Erlenmeyer flasks
on a laboratory incubator shaker (IKA incubator shaker,
KS 4000i) at 150 rpm and 50

o
C. The typical reaction

mixture contained acetic acid (0.5 mmol), alcohol (3.0
mmol), Novozym 435

®
lipase (60 mg) and n-hexane (20

mL). The reaction was commenced by adding the en-
zyme.

The gas chromatography (GC) analyses for the de-
termination of ester concentrations were conducted by a
HP 5890 A gas chromatograph, with an HP-FFAP col-
umn (Macherey-Nagel), split: 70 kPa, N2: 19 cm

3
/min,

using a flame ionization detector (FID). Toluene was
used as an internal standard, the changes in ester yield
were followed during the reaction. Samples were taken
after reaction times of 0.5, 1.0, 2.0 4.0 and 6.0 min. The
water contents of the reaction mixtures were determined
by a Mettler DL35 Karl Fisher titrator.

3. Results and discussion

Although several publications have presented results on
the production of flavor esters, the optimal initial condi-
tions suggested were quite different and a high degree
of deviation was found among data in the literature.
Firstly the average of the literature data was used for
our preliminary experiments. Based on these figures the
following initial parameters were applied: in the reac-
tion mixture the molar ratio of acetic acid to alcohol
was 1:6, and 20 ml of n-hexane, 20 mmol of toluene and
60 mg of the enzyme Novozym 435 were added to it.

ENZYMATIC ESTERIFICATION

45(2) pp. 35–39 (2017)

3.1. The effect of water content

Experiments had to be conducted to determine the cor-
rect water content since it could not be found in the lit-
erature. The esterification of acetic acid and isoamyl
alcohol – a reaction that has been quite frequently stud-
ied – was investigated under the conditions mentioned
earlier. During the measurements completely dried reac-
tion mixtures (0 % w/w water content) were used and
others adjusted the initial water content to the levels of
0.3, 0.5 and 0.7 % w/w by adding water. As can be seen
from the data of Table 1, the reaction was extremely
slow in the case of completely dried solvents and rea-
gents – as was expected. The reaction rate began to rise
when the water content grew slowly (and approached
the optimal value) due to the water forming in the reac-
tion.

Based on the experimental results an initial water
content of 0.3 % w/w was applied to further measure-
ments since this water concentration provided the high-
est yield. The yields of esterification after a reaction
time of 4 h were presented in Fig.1, where the meas-
urements were taken under the conditions given earlier,
with an adjusted and the same initial water contents.
The yields of esterification were sufficiently high to
observe the differences caused by the different struc-
tures of alcohols, but saturation levels were not reached
and the distinct amounts of water formed during the
reaction did not affect such a tendency either.

3.2. Primary alcohols

Our study involved linear and branched alcohols
with a chain length of C2-C8. As can be seen from Fig.1
for primary alcohols, the yield increased as the length of
the carbon chain grew. The effect of chain length was
investigated by Romero using propanol, butanol, hexa-
nol as well as octanol and a similar conclusion was
drawn: acetic acid conversion was greater with alcohols
of longer chain lengths, thus the yields of esterification
were higher, as well [16].

As far as branched primary alcohols were con-
cerned, the opposite trend was observed: the yield of
esterification decreased as the chain length increased. At
the beginning of the reactions the differences were only
minor: only a difference of 3 % was observed in the
yields in the cases of alcohols consisting of a carbon
chain of 4 or 5. The behavior of isooctanol was espe-

cially interesting, because a significant drop in yield
was observed compared to the other linear, 8-carbon-
chain alcohols – a far smaller amount of ester was
formed in the reaction mixture.

3.3. Secondary alcohols

In this work three secondary alcohols: 2-propanol,
2-butanol and 2-pentanol were used. As presented in
Table 2, an increase in ester yields was observed as the
carbon chain length of secondary alcohols grew, as
well. The values, however were not as high as for pri-
mary alcohols. Neji et al. observed a similar behavior
when butanol and 2-butanol were used in the esterifica-
tion reaction [20]. Although both alcohols could per-
form esterification, yields of esterification were 50 %
lower for secondary alcohols.

3.4. Tertiary alcohols

Among tertiary alcohols tert-amyl alcohol and tert-
butanol were used, however, the enzyme was not able to
convert them into esters using acetic acid. From the
literature, Stavarache et al. applied tertiary alcohols for
transesterification in the production of biodiesel [21].
Similarly no activity was observed during their experi-
ments, not even when using ultrasonic radiation.

3.5. Discussion of the experimental results

Although our experimental results did not reveal entire-
ly general conclusions, which are valid in all cases, ob-
vious relationships could be formulated for certain

Figure 1. Ester yields during the reactions of acetic
acid and primary alcohols after a reaction time of 4 h

31,4

37,3

46,2

76,1

49,6

73,6

53,9

63,2

76,4

7,2

Y
ie

ld
[

%
]

Table 1. The effect of water content on ester yield in
the esterification of acetic acid and isoamyl alcohol

Time (h)
0.0% 0.3% 0.5% 0.7%

Ester yield (%)

0 0.0 0.0 0.0 0.0

0.5 3.2 16.7 18.5 20.5

1 8.3 28.8 33.4 30.7

2 15.8 50.2 48.3 46.7

4 24.2 73.5 68.2 60.3

6 33.1 85.4 79.7 68.5

Table 2. Ester yields using secondary alcohols

Secondary alcohol
Ester yield after 4 h

(%)

2-propanol
2-butanol

35.8
44.8

2-pentanol 50.5

MÁRKUS, BÉLAFI-BAKÓ, TÓTH, NEMESTÓTHY AND GUBICZA

Hungarian Journal of Industry and Chemistry

groups of alcohols. It is certain that for alcohols with a
linear carbon chain of C2-C8 in length, the conversion
rate increases proportionally to the lengthening of the
carbon chains. As for branched alcohols, the opposite
tendency can be observed: yields were found to de-
crease as the carbon chain grew.

The relationship is more obvious when the order of
alcohols is taken into consideration. Yields of esterifica-
tion decreased in the following order: primary alcohol >
secondary alcohol > tertiary alcohol (noting that tertiary
alcohols did not react at all under the conditions used by
Novozym 435). By applying other enzymes, e.g. car-
boxylesterase from Bacillus licheniformis, small de-
grees of conversion were measured, but remained close
to the limit of detection [22]. In an attempt to justify
such behavior, it can be assumed that access of the hy-
droxyl group of the alcohol to the active centre of the
enzyme is severely sterically hindered in the case of
secondary and especially tertiary alcohols, which cause
enzyme activity to decline or even cease.

4. Conclusion

The expected reaction rate produced by a given enzyme
can be predicted according to characteristics of the al-
cohol used, namely carbon chain length, linear or
branched, and the order in production of flavor esters by
enzymatic esterification of natural acids and alcohols.
The expected ester yield of the esterification reaction
using acetic acid as an acyl donor depends on certain
characteristics of the alcohol according to a well-
defined tendency. It can be assumed that a similar ten-
dency (though distinct in terms of rate) could be ob-
served for other enzymes regarding the effect of the
alcohol. This should be studied separately to decide
whether a similar tendency could be detected if various
acids are used with the same alcohol.

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