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

Transcription

1 Journal of Lachezar Chemical Manovski, Technology Vera and Semedzieva, Metallurgy, Lubov 50, 3, Yotova 2015, 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 l_manovski@mail.com Received 03 February 2015 Accepted 27 March 2015 ABSTRACT Lipoxygenase (LOX) is an enzyme that is found in many plants and animals, which catalyses the oxygenation of polyunsaturated 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 U/ml in the presence 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: M; Vmax = M/mg min. Keywords: linolenic acid, enzyme activity, kinetics, lipoxygenase, 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 abundant 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 different 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 polyunsaturated 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 cytosolic 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 249

2 Journal of Chemical Technology and Metallurgy, 50, 3, 2015 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 inactivate 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 storage 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 methyleneinterrupted 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. 250

3 Lachezar Manovski, Vera Semedzieva, Lubov Yotova A proposed mechanism of action that relates biochemical 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 parameters like ph and temperature optimum, Km and Vmax. EXPERIMENTAL Method for isolation and purification of Lipoxygenase 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: Degreasing with acetone under constant stirring for 2 hours; the procedure is repeated after filtration and replacement of the solvent; 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; The extract is clarified by centrifugation for 15 min at 3500 rev min -1 ; The ph of the clear extract is adjusted to 4.5 with 1 M HCl, and the precipitate is discarded. The clear fraction is treated with NaOH (2M) to ph 8 and allowed to stand for 12 hours at 4 C for precipitating herbal salts; After centrifugation for 15 min at 3500 rev min -1, the precipitate is separated, the supernatant is saturated with (NH 4 ) 2 SO 4 to a final concentration of 40 g (NH 4 ) 2 SO 4 in 100 ml of solution, and the active fraction is precipitated; The latter is redissolved in a small amount of distilled water and heated to 63 C for 5 minutes to coagulate the albumin; The precipitate is collected by centrifugation (3500 rev min -1, 15 min) and the supernatant is again saturated with (NH 4 ) 2 SO 4, while maintaining the fraction between 35 % and 50 % saturation; 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 determining 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 calculated 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 experimental error may have a particularly significant impact on initial velocity determination, inaccurate Vmax estimations 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 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 activities were also determined. Given that avocado grows in tropical and semitropical climates, with high humidity and temperature, a temperature optimum for LOX activity between 30 C and 40 C, as the one found in the 251

4 Journal of Chemical Technology and Metallurgy, 50, 3, 2015 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 effective 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 Fig. 4. ph optimum of the enzyme. 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 U ml -1. 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, ). 252 Fig. 5. Temperature optimum of the enzyme. 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 activity of the lipoxygenase from asparagus [11,12]. Enzyme-catalyzed reactions are described by the kinetic parameters Vmax and Km. The latter is associated 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 parameters are as follows: Km = M, Vmax = M mg -1 min -1. From these data it can be concluded that the relationship of the enzyme to the substrate is high. For comparison, the kinetic parameters of the lipoxygenases from different plant sources show that the enzyme isolated from avocados showed greater affinity with the substrate than that isolated from tomato (Km = M) [13].

5 Lachezar Manovski, Vera Semedzieva, Lubov Yotova Table 1. Enzyme parameters on lipoxygenase from avocado. Protein content, mg ml -1 Activity, U/ml S = 3 мм Кm, 10-6 M Kinetic parameters Vmax,10-6 M mg -1 min -1 ph optimum Temperature optimum 0, ,7287 0,0248 0,745 6,5 40 C 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 lipoxygenase 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 optimum 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 effectiveness of the contained in the avocado unsaturated fatty acids, enzymes, vitamins, etc., and the isolated lipoxygenase in particular may find application in medicine, 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 Mundus 2013, 2016 EU Project. REFERENCE 1. R. Casey, Lipoxygenases. In: R. Casey, P.R. Shrewy (eds.), Seed proteins. London, Chapman and Hall, R. Casey, C. Domoney, C. Forster, D. Robinson, Z. Wu,. In: G.R. Fenwick, C. Hedley, R.L. Richards, S. Khokhar (eds.), The significance of plant lipoxygenases to the agrifood industry in agrifood quality: an interdisciplinary approach. The Royal Society of Chemistry, p G.A.Veldink, M.P. Hilbers, W.F. Nieuwenhuizen, J.F.G.Vliegenthart, In: A.F. Rowley, K. Kühn, T. Schewe (eds.), Plant lipoxygenase: structure and mechanism in eicosanoids and related compounds in plants and animals, Portland Press, 1998, p J.R. Whitaker, Handbook of food enzymology, NewYork, Marcel Dekker Inc., P. Elez-Martínez, R.C. Soliva-Fortuny, S. Gorinstein, O. Martín-Belloso. Natural antioxidants preserve the lipid oxidative stability of minimally processed avocado purée. Journal of Food Science, 70, 5, 2005, FAOSTAT, FAO Statistical databases, agricultural data, 2008, 7. L. Marcus, D. Prusky, B. Jacoby, Purification and characterization of avocado lipoxygenase, Phytochemistry, 27, 2, 1988, D. A. Jacobo-Velázquez, C. Hernández-Brenes, L. Cisneros-Zevallos, J. Benavides, Partial purification and enzymatic characterization of avocado (Persea americana Mill, cv. Hass) lipoxygenase, Food Research International, 43, 2010, D.S. Robinson, Z. Wu, C. Domoney, R. Casey, Lipoxygenases and the quality of foods. Food Chemistry, 54, 1, 1995, T. Baysal, A. Demirdöven, Lipoxygenase in fruits and vegetables: A review, Enzyme and Microbial Technology, 40, 2007, E.F. Morales-Blancas, V.E. Chandia, L. Cisneros- Zevallos, Thermal Inactivation Kinetics of Peroxidase and Lipoxygenase from Broccoli, Green Asparagus and Carrots, J. Food Sci., 67, 1, E. Koch, B.M. Meier, H.G. Eiben, A. Slusarenko, A Lipoxygenase from Leaves of Tomato (Lycopersicon esculentum Mill.) Is Induced in Response to Plant 253

6 Journal of Chemical Technology and Metallurgy, 50, 3, 2015 Pathogenic Pseudomonads, Plant Physiol., 99, 1992, E. Yilmaz, Kinetic Studies With Crude Tomato Lipoxygenase, Türk. J. Agric. For., 25, 2001, G.E. Sellhorn, B.N. Webb, C.H. Kang, H.D. Grimes, Biochemical Characterization, Kinetic Analysis and Molecular Modeling of Recombinant Vegetative Lipoxygenases from Soybean, Intern. J. Biology, 3, 1, H. Lineweaver, D. Burk, The Determination of Enzyme Dissociation Constants, J. Amer. Chem. Soc., 56, 3, 1934, O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 1, 1951, PMID H. Theorell, T. Hollman, Crystalline lipoxidas, Acta. Chem. Scandinavica,1, 1947,