INVESTIGATION ON ISOLATED AND PURIFIED LIPOXYGENASE FROM AVOCADO IN THE PRESENCE OF LINOLENIC ACID

1. Lachezar Manovski, Vera Semedzieva, Lubov Yotova
249
Journal of Chemical Technology and Metallurgy, 50, 3, 2015, 249-254
INVESTIGATION ON ISOLATED AND PURIFIED LIPOXYGENASE
FROM AVOCADO IN THE PRESENCE OF LINOLENIC ACID
Lachezar Manovski, Vera Semedzieva, Lubov Yotova
Department of Biotechnology,
University of Chemical Technology and Metallurgy
8 Kl. Ohridski, 1756 Sofia, Bulgaria
E-mail: l_manovski@mail.com
ABSTRACT
Lipoxygenase (LOX) is an enzyme that is found in many plants and animals, which catalyses the oxygenation of polyun-
saturated fatty acids (PUFA) to form fatty acid hydroperoxides. Linoleic and linolenic acid are the major polyunsaturated
fatty acids in plant tissues, and insertion of the oxygen takes place at either the 9th or the 13th position to generate the
corresponding 9- or 13-hydroperoxides.
Characterization of avocado LOX offers the potential of increasing scientific knowledge that can aid in the establishment
of optimum processing and storage conditions at which the detrimental effects of this enzyme are minimized, preventing
product organoleptic changes and nutritional quality losses. The determination of other catalytic properties of avocado
LOX such as the ability to co-oxidize carotenoids may also help to promote the use of the avocado enzyme as a bleaching
agent in the food industry. In the laboratory, lipoxygenase from avocado was isolated, purified and characterized.
After processing of the results from the experiments, the value for the enzyme activity is 60.7287 U/ml in the pres-
ence of a substrate at concentration of 3 mM. pH and temperature optimums are 6,5 and 40 o
C. The kinetic parameters
obtained in coordinates Lineweaver–Burk at different concentrations of substrate, have values as follows: 0.0248.10-6
M;
Vmax = 0.745. 10-6
M/mg min.
Keywords: linolenic acid, enzyme activity, kinetics, lipoxygenase,
Received 03 February 2015
Accepted 27 March 2015
INTRODUCTION
Lipoxygenase (LOX) is an enzyme found in many
plants and animals, which catalyses the oxygenation of
polyunsaturated fatty acids (PUFA) to form fatty acid
hydroperoxides. The latter are present in a wide range of
biological organs and tissues, but are particularly abun-
dant in grain legume seeds (beans and peas) and potato
tubers [1]. The first lipoxygenase isolated in 1947 by
Theorell et al. [17], was that of soy bean. Lipoxygenase
from different sources, catalyses oxygenation at differ-
ent points along the carbon chain, which is referred to
as “positional” or “regio” specificity. Such specificity
has significant implications for the metabolism of the
resultant hydroperoxides into a number of important
secondary metabolites [2, 3].
Linoleic and linolenic acid are the major polyun-
saturated fatty acids in plant tissues, and the insertion
of oxygen takes place at either the 9th or 13th position
to generate the corresponding 9- or 13-hydroperoxides.
While most LOXs so far characterized are soluble cy-
tosolic enzymes, some are chloroplastic, mitochondrial,
or located in the vacuoles. In soybean, lipoxygenases
have been identified with involvement in nitrogen and
assimilated partitioning and appear to be regulated in
response to plant nitrogen status in both tissue-specific
and developmentally controlled patterns [1 - 4].
Avocado (Persea americana Mill.) processing and
commercialization are activities of major socioeconomic
relevance for Mexico, the country that occupies the
first place in the production of this horticultural crop
worldwide [6]. Due to the high economic importance

2. Journal of Chemical Technology and Metallurgy, 50, 3, 2015
250
that avocado has to Mexico, the food industry is showing
a remarkable interest in processing and enhancing the
value of this crop. However, avocado products are quite
unstable, due to the presence of oxidative enzymes such
as polyphenoloxidase (PPO) and LOX [5]. Inhibition of
avocado LOX is possible by using phenolic compounds
such as epicatechin [7]. However, this approach to inacti-
vate LOX may also be detrimental for avocado products,
since epicatequin is a substrate for PPO, increasing the
rate of browning. Characterization of avocado LOX
offers the potential of increasing scientific knowledge
that can aid in the establishment of processing and stor-
age conditions at which the detrimental effects of this
enzyme are minimized and the products organoleptic
changes and nutritional quality losses are prevented. The
determination of other catalytic properties of avocado
LOX, such as its ability to co-oxidize carotenoids, may
help to promote the use of the avocado enzyme as a
bleaching agent in the food industry.
Additionally, the enzymatic characterization of
avocado LOX may lead to finding alternative uses for
LOX as a food ingredient or an additive [8].
In vegetal tissues, the major substrates for LOX
are linoleic and linolenic acid, which are methylene-
interrupted cis, cis-pentadiene moieties [10]. In these
PUFAs the methylene group is situated at the x8 carbon
and located between two double bonds [9]. Substrate
specificity depends on the source of the enzyme.
Fig. 1. Mechanism of action and impact of lipoxygenase (LOX) on foods quality.

3. Lachezar Manovski, Vera Semedzieva, Lubov Yotova
251
A proposed mechanism of action that relates bio-
chemical events in the plant tissue with the impact of
LOX on the quality of foods are summarized in Fig. 1.
In addition, the industrial applications of the reaction
intermediates are also included.
The aim of this study was to isolate and characterize
lipoxygenase from avocado, setting basic enzyme param-
eters like pH and temperature optimum, Km and Vmax.
EXPERIMENTAL
Method for isolation and purification of Lipoxyge-
nase from Avocado (Persea Americana Mill.)
Avocado is peeled (stripped bark and stone), and
then cut into small particles. The sliced fruit is subjected
to a degreasing treatment by the following steps:
l Degreasing with acetone under constant stirring
for 2 hours; the procedure is repeated after filtration and
replacement of the solvent;
l Skimmed avocado is extracted with water (the
ratio of water:avocado is 10:1) with constant agitation
in a shaker at room temperature for 1 hour;
l The extract is clarified by centrifugation for 15
min at 3500 rev min-1
;
l The pH of the clear extract is adjusted to 4.5 with
1 M HCl, and the precipitate is discarded. The clear frac-
tion is treated with NaOH (2M) to pH 8 and allowed to
stand for 12 hours at 4°C for precipitating herbal salts;
l After centrifugation for 15 min at 3500 rev min-1
,
the precipitate is separated, the supernatant is satu-
rated with (NH4
)2
SO4
to a final concentration of 40 g
(NH4
)2
SO4
in 100 ml of solution, and the active fraction
is precipitated;
l The latter is redissolved in a small amount of
distilled water and heated to 63°C for 5 minutes to co-
agulate the albumin;
The precipitate is collected by centrifugation (3500
rev min-1
, 15 min) and the supernatant is again saturated
with (NH4
)2
SO4
, while maintaining the fraction between
35 % and 50 % saturation;
l The resulting solution is further purified, using
ultra centrifugation tubes Sartorius Vivaspin 6 with a
pore size of 100 kDa.
Determination of protein content (Method of Lowry)
The principle behind the Lowry method of deter-
mining protein concentrations lies in the reactivity of
the peptide nitrogen [s] with copper (II) ions under
alkaline conditions and the subsequent reduction of the
Folin-Ciocalteu phosphomolybdic phosphotungstic acid
to heteropolymolybdenum blue by the copper-catalyzed
oxidation of aromatic acids [16].
Enzyme activity
Activity assays using linolenic acid were carried out
in order to determine LOX substrate specificity. LOX
activity at different substrate concentrations (2.5 mM;
3 mM) was determined. Reaction conditions were kept
constant at 25°C, pH 6.5 (0,1 M acetic buffer), λ 234
nm. Absorbance was measured for 10 minutes at an
interval of 1 minute.
Kinetics of enzyme reaction
LOX kinetic constants (KM and Vmax) were calcu-
lated using the Lineweaver-Burk (L-B) method.
Linolenic acid was used as LOX substrate and its
concentration in the reaction solution was varied in
order to establish the initial velocities. The values of
both Michaelis-Menten parameters, Km and Vmax, for
each type of substrate were calculated using the L-B
linealization method [11].
The experimental values obtained at low substrate
concentrations, are overestimated when the L-B method
is used. Since at low substrate concentrations experimen-
tal error may have a particularly significant impact on
initial velocity determination, inaccurate Vmax estima-
tions are often rendered by this method.
pH optimum
The optimum pH occurs between 6.0 and 6.5 for
linoleic acid, while for linolenic acid it is 6.5, matching
the pH naturally found in avocado pulp.The optimum pH
of LOX enzyme from other sources has been estimated
to be within a wide pH range 5.5 - 9.5. Determination
of the pH optimum of the enzyme was done in the
presence of 0.1 M acetate buffer at different pHs of the
solutions (5 - 9).
Temperature optimum
Effects of temperature (20 - 75°C) on LOX activi-
ties were also determined. Given that avocado grows in
tropical and semitropical climates, with high humidity
and temperature, a temperature optimum for LOX ac-
tivity between 30°C and 40°C, as the one found in the

4. Journal of Chemical Technology and Metallurgy, 50, 3, 2015
252
present work, was expected. Since thermal processing or
long exposure to high temperatures is likely to promote
undesirable organoleptic changes in avocado pulp, the
use of high hydrostatic pressure pasteurization is an ef-
fective strategy to partially inactivate LOX. However,
the residual activity of LOX in hyperbaric processed
avocado products may affect the shelf-life. Therefore, a
viable strategy for reducing LOX activity could involve
maintaining the pressurized product under an oxygen
depleted atmosphere at low temperatures (< 20°C).
RESULTS AND DISCUSSION
After carrying out a series of experiments in order
to characterize the lipoxygenase isolated from the fruit
of the tropical plant Persea Americana Mill we found
that the curve of enzyme activity had the form shown
in Fig. 2. From the obtained curves it appears that at a
concentration of the substrate of 3 mM, the activity of
the lipoxygenase, isolated from avocado is high - 60.73
U ml-1
.
In comparison to lipoxygenase isolated from various
plant sources, the activity of the lipoxygenase avocado is
higher than lipoxygenase isolated from tomato, broccoli
and asparagus. Lipoxygenase activities from broccoli
and tomatoes are close in value, but lower than the activ-
ity of the lipoxygenase from asparagus [11,12].
Enzyme-catalyzed reactions are described by the
kinetic parameters Vmax and Km. The latter is associ-
ated with the stability of the enzyme-substrate (ES)
complex, and the degree of conversion to the product
of the reaction. The purpose of enzyme kinetics is to
determine Km and Vmax.
After processing of the results of the equation and
linearization the received values for the kinetic param-
eters are as follows:
Km = 0.0248.10-6
M, Vmax = 0.745.10-6
M mg-1
min-1
.
From these data it can be concluded that the relation-
ship of the enzyme to the substrate is high.
For comparison, the kinetic parameters of the li-
poxygenases from different plant sources show that the
enzyme isolated from avocados showed greater affinity
with the substrate than that isolated from tomato (Km =
0.015.10-4
M) [13].
Fig. 2. Activity of lipoxygenase in the presence of linoleic
acid at a concentration 3 mM.
Fig. 3. Kinetics and kinetic parameters of the reaction
catalyzed by lipoxygenase in the presence of linoleic acid
at different concentrations (0,5.10-5
– 1.67.10-5
).
Fig. 4. pH optimum of the enzyme.
Fig. 5. Temperature optimum of the enzyme.

5. Lachezar Manovski, Vera Semedzieva, Lubov Yotova
253
After testing the activity of lipoxygenase from
avocado in the presence of 0.1 M acetate buffer with
different pH we have found that the optimum of the
enzyme was 6.5.
When comparing to the optimum for the lipoxy-
genase isolated from other plant sources it is found
that the optimum pH of lipoxygenases of tomato and
avocado are close 6 - 6.5, [13]. The pH optimum of the
soy lipoxygenases is higher (pH = 8) [14], than that of
the avocado lipoxygenase.
From research on the activity of lipoxygenase from
avocado at different temperatures, we found that the
optimum of the enzyme is at 40°C.
According to literature data, the temperature op-
timum of lipoxygenases from broccoli and asparagus
is at a lower temperature compared to that of avocado
lipoxygenase [12], while the temperature optimum of the
lipoxygenases from soy and avocado are at 40°C [14].
CONCLUSIONS
Llipoxygenase from avocado was isolated, purified
and characterized in the laboratory. The antioxidant ef-
fectiveness of the contained in the avocado unsaturated
fatty acids, enzymes, vitamins, etc., and the isolated
lipoxygenase in particular may find application in medi-
cine, as part of an anti-inflammatory agent, in the textile
industry for the bleaching of fabrics, in the production
of wine, for the detection of mycotoxins, but for that the
enzyme has to be immobilized.
Acknowledgements
The work is financially supported by Erasmus Mun-
dus 2013, 2016 EU Project.
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Table 1. Enzyme parameters on lipoxygenase from avocado.
Protein
content,
mg ml-1
Activity, U/ml Kinetic parameters
pH optimum
Temperature
optimum
S = 3 мМ Кm, 10-6
M
Vmax
,10-6
M mg-1
min-1
0,1482 60,7287 0,0248 0,745 6,5 40°C

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