INVESTIGATION on ISOLATED and PURIFIED LIPOXYGENASE from AVOCADO in the PRESENCE of LINOLENIC ACID Lachezar Manovski, Vera Semedzieva, Lubov Yotova

Journal ofLachezar Chemical Manovski, Technology Vera and Semedzieva, Metallurgy, Lubov 50, 3, Yotova 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, Received 03 February 2015 University of Chemical Technology and Metallurgy Accepted 27 March 2015 8 Kl. Ohridski, 1756 Sofia, Bulgaria E-mail: [email protected]

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 60.7287 U/ml in the presence of a substrate at concentration of 3 mM. pH and temperature optimums are 6,5 and 40 oC. 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,

INTRODUCTION Linoleic and linolenic acid are the major polyunsaturated fatty acids in plant tissues, and the insertion Lipoxygenase (LOX) is an enzyme found in many of oxygen takes place at either the 9th or 13th position plants and animals, which catalyses the oxygenation of to generate the corresponding 9- or 13-hydroperoxides. polyunsaturated fatty acids (PUFA) to form fatty acid While most LOXs so far characterized are soluble cy- hydroperoxides. The latter are present in a wide range of tosolic enzymes, some are chloroplastic, mitochondrial, biological organs and tissues, but are particularly abun- or located in the vacuoles. In soybean, lipoxygenases dant in grain legume seeds (beans and peas) and potato have been identified with involvement in nitrogen and tubers [1]. The first lipoxygenase isolated in 1947 by assimilated partitioning and appear to be regulated in Theorell et al. [17], was that of soy bean. Lipoxygenase response to plant nitrogen status in both tissue-specific from different sources, catalyses oxygenation at differ- and developmentally controlled patterns [1 - 4]. ent points along the carbon chain, which is referred to Avocado (Persea americana Mill.) processing and as “positional” or “regio” specificity. Such specificity commercialization are activities of major socioeconomic has significant implications for the metabolism of the relevance for Mexico, the country that occupies the resultant hydroperoxides into a number of important first place in the production of this horticultural crop secondary metabolites [2, 3]. worldwide [6]. Due to the high economic importance 249 Journal of Chemical Technology and Metallurgy, 50, 3, 2015 that avocado has to Mexico, the food industry is showing changes and nutritional quality losses are prevented. The a remarkable interest in processing and enhancing the determination of other catalytic properties of avocado value of this crop. However, avocado products are quite LOX, such as its ability to co-oxidize carotenoids, may unstable, due to the presence of oxidative enzymes such help to promote the use of the avocado enzyme as a as polyphenoloxidase (PPO) and LOX [5]. Inhibition of bleaching agent in the food industry. avocado LOX is possible by using phenolic compounds Additionally, the enzymatic characterization of such as epicatechin [7]. However, this approach to inacti- avocado LOX may lead to finding alternative uses for vate LOX may also be detrimental for avocado products, LOX as a food ingredient or an additive [8]. since epicatequin is a substrate for PPO, increasing the In vegetal tissues, the major substrates for LOX rate of browning. Characterization of avocado LOX are linoleic and linolenic acid, which are methylene- offers the potential of increasing scientific knowledge interrupted cis, cis-pentadiene moieties [10]. In these that can aid in the establishment of processing and stor- PUFAs the methylene group is situated at the x8 carbon age conditions at which the detrimental effects of this and located between two double bonds [9]. Substrate enzyme are minimized and the products organoleptic specificity depends on the source of the enzyme.

Fig. 1. Mechanism of action and impact of lipoxygenase (LOX) on foods quality. 250 Lachezar Manovski, Vera Semedzieva, Lubov Yotova

A proposed mechanism of action that relates bio- the peptide nitrogen [s] with copper (II) ions under chemical events in the plant tissue with the impact of alkaline conditions and the subsequent reduction of the LOX on the quality of foods are summarized in Fig. 1. Folin-Ciocalteu phosphomolybdic phosphotungstic acid In addition, the industrial applications of the reaction to heteropolymolybdenum blue by the copper-catalyzed intermediates are also included. oxidation of aromatic acids [16]. The aim of this study was to isolate and characterize lipoxygenase from avocado, setting basic enzyme param- Enzyme activity eters like pH and temperature optimum, Km and Vmax. Activity assays using linolenic acid were carried out in order to determine LOX substrate specificity. LOX EXPERIMENTAL activity at different substrate concentrations (2.5 mM; Method for isolation and purification of Lipoxyge- 3 mM) was determined. Reaction conditions were kept nase from Avocado (Persea Americana Mill.) constant at 25°C, pH 6.5 (0,1 M acetic buffer), λ 234 Avocado is peeled (stripped bark and stone), and nm. Absorbance was measured for 10 minutes at an then cut into small particles. The sliced fruit is subjected interval of 1 minute. to a degreasing treatment by the following steps: l Degreasing with acetone under constant stirring Kinetics of enzyme reaction for 2 hours; the procedure is repeated after filtration and LOX kinetic constants (KM and Vmax) were calcu- replacement of the solvent; lated using the Lineweaver-Burk (L-B) method. Linolenic acid was used as LOX substrate and its l Skimmed avocado is extracted with water (the ratio of water:avocado is 10:1) with constant agitation concentration in the reaction solution was varied in in a shaker at room temperature for 1 hour; order to establish the initial velocities. The values of both Michaelis-Menten parameters, Km and Vmax, for l The extract is clarified by centrifugation for 15 each type of substrate were calculated using the L-B min at 3500 rev min-1; linealization method [11]. l The pH of the clear extract is adjusted to 4.5 with The experimental values obtained at low substrate 1 M HCl, and the precipitate is discarded. The clear frac- concentrations, are overestimated when the L-B method tion is treated with NaOH (2M) to pH 8 and allowed to is used. Since at low substrate concentrations experimen- stand for 12 hours at 4°C for precipitating herbal salts; tal error may have a particularly significant impact on -1 l After centrifugation for 15 min at 3500 rev min , initial velocity determination, inaccurate Vmax estima- the precipitate is separated, the supernatant is satu- tions are often rendered by this method. rated with (NH4)2SO4 to a final concentration of 40 g

(NH4)2SO4 in 100 ml of solution, and the active fraction pH optimum is precipitated; The optimum pH occurs between 6.0 and 6.5 for l The latter is redissolved in a small amount of linoleic acid, while for linolenic acid it is 6.5, matching distilled water and heated to 63°C for 5 minutes to co- the pH naturally found in avocado pulp. The optimum pH agulate the albumin; of LOX enzyme from other sources has been estimated The precipitate is collected by centrifugation (3500 to be within a wide pH range 5.5 - 9.5. Determination rev min-1, 15 min) and the supernatant is again saturated of the pH optimum of the enzyme was done in the with (NH4)2SO4, while maintaining the fraction between presence of 0.1 M acetate buffer at different pHs of the 35 % and 50 % saturation; solutions (5 - 9). l The resulting solution is further purified, using ultra centrifugation tubes Sartorius Vivaspin 6 with a Temperature optimum pore size of 100 kDa. Effects of temperature (20 - 75°C) on LOX activities were also determined. Given that avocado grows in Determination of protein content (Method of Lowry) tropical and semitropical climates, with high humidity The principle behind the Lowry method of deter- and temperature, a temperature optimum for LOX ac- mining protein concentrations lies in the reactivity of tivity between 30°C and 40°C, as the one found in the 251 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 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).

Fig. 4. pH optimum of the enzyme. 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.

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 Fig. 2. Activity of lipoxygenase in the presence of linoleic kinetic parameters Vmax and Km. The latter is associ- acid at a concentration 3 mM. 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 parameters are as follows: Km = 0.0248.10-6 M, Vmax = 0.745.10-6 M mg-1min-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 lipoxygenases from different plant sources show that the Fig. 3. Kinetics and kinetic parameters of the reaction enzyme isolated from avocados showed greater affinity catalyzed by lipoxygenase in the presence of linoleic acid with the substrate than that isolated from tomato (Km = -5 -5 at different concentrations (0,5.10 – 1.67.10 ). 0.015.10-4 M) [13]. 252 Lachezar Manovski, Vera Semedzieva, Lubov Yotova

Table 1. Enzyme parameters on lipoxygenase from avocado.

Protein Activity, U/ml Kinetic parameters Temperature content, pH optimum -1 optimum -6 Vmax mg ml S = 3 мМ Кm, 10 M -6 -1 -1 ,10 M mg min 0,1482 60,7287 0,0248 0,745 6,5 40°C

After testing the activity of lipoxygenase from In: G.R. Fenwick, C. Hedley, R.L. Richards, S. Khokhar avocado in the presence of 0.1 M acetate buffer with (eds.), The significance of plant lipoxygenases to the different pH we have found that the optimum of the agrifood industry in agrifood quality: an interdiscipli- enzyme was 6.5. nary approach. The Royal Society of Chemistry, 1996. When comparing to the optimum for the lipoxy- p. 127-130. genase isolated from other plant sources it is found 3. G.A.Veldink, M.P. Hilbers, W.F. Nieuwenhuizen, that the optimum pH of lipoxygenases of tomato and J.F.G.Vliegenthart, In: A.F. Rowley, K. Kühn, T. Schewe avocado are close 6 - 6.5, [13]. The pH optimum of the (eds.), Plant lipoxygenase: structure and mechanism soy lipoxygenases is higher (pH = 8) [14], than that of in eicosanoids and related compounds in plants and the avocado lipoxygenase. animals, Portland Press, 1998, p. 69-95. From research on the activity of lipoxygenase from 4. J.R. Whitaker, Handbook of food enzymology, avocado at different temperatures, we found that the NewYork, Marcel Dekker Inc., 2002. optimum of the enzyme is at 40°C. 5. P. Elez-Martínez, R.C. Soliva-Fortuny, S. Gorinstein, According to literature data, the temperature op- O. Martín-Belloso. Natural antioxidants preserve the timum of lipoxygenases from broccoli and asparagus lipid oxidative stability of minimally processed avocado is at a lower temperature compared to that of avocado purée. Journal of Food Science, 70, 5, 2005, 325-329. lipoxygenase [12], while the temperature optimum of the 6. FAOSTAT, FAO Statistical databases, agricultural data, lipoxygenases from soy and avocado are at 40°C [14]. 2008, http://faostat.fao.org. 7. L. Marcus, D. Prusky, B. Jacoby, Purification and char- CONCLUSIONS acterization of avocado lipoxygenase, Phytochemistry, Llipoxygenase from avocado was isolated, purified 27, 2, 1988, 323-327. and characterized in the laboratory. The antioxidant ef- 8. D. A. Jacobo-Velázquez, C. Hernández-Brenes, L. fectiveness of the contained in the avocado unsaturated Cisneros-Zevallos, J. Benavides, Partial purification fatty acids, enzymes, vitamins, etc., and the isolated and enzymatic characterization of avocado (Persea lipoxygenase in particular may find application in medi- americana Mill, cv. Hass) lipoxygenase, Food Research cine, as part of an anti-inflammatory agent, in the textile International, 43, 2010, 1079-1085. industry for the bleaching of fabrics, in the production 9. D.S. Robinson, Z. Wu, C. Domoney, R. Casey, of wine, for the detection of mycotoxins, but for that the Lipoxygenases and the quality of foods. Food Chemistry, enzyme has to be immobilized. 54, 1, 1995, 33-43. 10. T. Baysal, A. Demirdöven, Lipoxygenase in fruits Acknowledgements and vegetables: A review, Enzyme and Microbial The work is financially supported by Erasmus Mun- Technology, 40, 2007, 491-496. dus 2013, 2016 EU Project. 11. E.F. Morales-Blancas, V.E. Chandia, L. Cisneros- Zevallos, Thermal Inactivation Kinetics of Peroxidase REFERENCE and Lipoxygenase from Broccoli, Green Asparagus and Carrots, J. Food Sci., 67, 1, 2002. 1. R. Casey, Lipoxygenases. In: R. Casey, P.R. Shrewy 12. E. Koch, B.M. Meier, H.G. Eiben, A. Slusarenko, A (eds.), Seed proteins. London, Chapman and Hall, 1998. Lipoxygenase from Leaves of Tomato (Lycopersicon 2. R. Casey, C. Domoney, C. Forster, D. Robinson, Z. Wu,. esculentum Mill.) Is Induced in Response to Plant

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