Partial purification and enzymatic characterization of avocado(Persea americana Mill, cv. Hass) lipoxygenase

Daniel A. Jacobo-Velázquez a, Carmen Hernández-Brenes b, Luis Cisneros-Zevallos a, Jorge Benavides b,*

a Department of Horticultural Sciences, Texas A&M University, HFSB Building, Room 202, MS 2133, College Station, TX 77843-2133, United Statesb Centro de Biotecnología, Departamento de Biotecnología e Ingeniería de Alimentos, Tecnológico de Monterrey, Campus Monterrey, Av. Eugenio Garza Sada 2501 Sur,Monterrey, NL 64849, Mexico

a r t i c l e i n f o

Article history:Received 5 October 2009Accepted 21 January 2010

Keywords:Hass avocadoLipoxygenasePartial purificationEnzymatic characterizationb-carotene co-oxidation

a b s t r a c t

Detrimental effects on minimally processed fruits and vegetables induced by increased lipoxygenase(LOX) activity are a major concern in the food industry. Partial purification and enzymatic characteriza-tion of lipoxygenase (LOX) from avocado fruit (Persea americana Mill, cv. Hass) is presented. Avocado LOXwas partially purified by fractional precipitation with ammonium sulfate, followed by anion exchangeadsorption. Substrate specificity and effects of temperature, pH and calcium ion concentration on LOXactivity were determined. Likewise, the in vitro ability of avocado LOX to co-oxidize b-carotene wasdetermined. LOX showed a higher substrate affinity for linolenic than for linoleic acid. Optimum activitywas reached at 40 !C, pH 6.5, and 0.20 mM of calcium ion concentration. Avocado LOX showed b-caroteneco-oxidation capability. Considering the optimums and operational ranges established for each studiedfactor, strategies concerning the inactivation of LOX may be proposed to reduce the detrimental effectson minimally processed avocado products. Additionally, the co-oxidation capability of avocado LOXmay be of interest for the avocado industry due to its potential application as a bleaching agent.

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1. Introduction

Lipoxygenases (LOX, linoleate:oxygen reductase, EC 1.13.11.12)enzymatic activity has a significant detrimental effect on the orga-noleptic and nutritional qualities of minimally processed fruits andvegetables (Robinson, Wu, Domoney, & Casey, 1995). These groupof enzymes catalyze the oxidation of polyunsaturated fatty acids(PUFA) promoting the initiation and propagation of the free radicalchain reaction (Whitaker, 1991). LOX is segregated from its sub-strates (PUFAs) in the plant cells due to compartmentalization.While LOX is mainly located in the cytoplasm and organelles suchas chloroplasts, mitochondria and vacuoles (Baysal & Demirdöven,2007), PUFAs are localized in the plasma membrane and oleosomes(Taiz & Zeiger, 2006). When wounding takes place during fruit andvegetable processing, enzymes and substrates start to interact as aresult of decompartmentalization. In addition, when plants aresubjected to wounding stress biochemical routs directed to thesynthesis of jasmonic acid (JA), a molecule that functions as geneactivator under stress conditions, are triggered (Wasternack & Par-thier, 1997). As a result of the activation of these biochemicalroutes, enzymatic release of PUFAs from the cell membrane has

been observed (Wasternack et al., 2006), which ultimately pro-motes the interaction between LOX and its substrate.

In vegetal tissues, the major substrates for LOX are linoleic andlinolenic acid, which are methylene-interrupted cis,cis-pentadienemoieties (Baysal & Demirdöven, 2007). In these PUFAs the methy-lene group is situated at the x8 carbon and located between twodouble bonds (Robinson et al., 1995; Whitaker, 1991). Substratespecificity depends on the source of the enzyme. Additionally,although some LOX isozymes can oxidize PUFAs linked to a glycerolmoiety some LOX can only utilize free moieties (Boyington, Gaffney,& Amzel, 1993). These PUFAs are liberated from glycerides, glyco-lipids and phospholipids by the enzymatic action of lipases andhydrolases. LOX, in the presence of oxygen, transform linoleic andlinolenic acid into hydroperoxides. Some LOX isoenzymes insertoxygen in carbon-9 and others in carbon-13, and both quiral config-urations (R and S) can occur. As a result 13S-ROOH, 13R-ROOH, 9S-ROOH and 9R-ROOH hydroperoxides from linoleic and linolenicacid can be synthesized (Whitaker, 1991). All these compoundsthen become substrates for hydroperoxide lyase and other cis–transisomerases, double-bond reducing and alcohol dehydrogenasesoriginating aldehydes and alcohols responsible for off-flavors(Hatanaka, Kajiwara, & Sekiya, 1987; Whitaker, 1991). Likewise,hydroperoxide cyclase can transform linoleic and linolenic acidhydroperoxides into 12-oxo-phytodienoic acid and by a series ofsubsequent reactions JA is synthesized (Delker et al., 2006).

0963-9969/$ - see front matter " 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodres.2010.01.021

* Corresponding author. Tel./fax: +52 818 358 2000x4820.E-mail address: jorben@itesm.mx (J. Benavides).

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All these reactions can result in the organoleptic degradation ofminimally processed fruits and vegetables. Additionally, it is wellknown that some LOX isoenzymes are capable of co-oxidizingcarotenoids and other nutrients in fruits and vegetables (Klein,Grossman, King, Cohen, & Pinsky, 1984; Robinson et al., 1995).LOX isoenzymes can be classified according to their optimum pHvalue of activity, their activation or inhibition by calcium ion andtheir capacity to co-oxidize carotenoids. LOX I shows its optimumactivity at alkaline pH values (9.0–9.5) and has low carotenoid co-oxidation activity; LOX II shows its optimum activity at acidic pHvalues (5.5–6.5), is activated by calcium and has high carotenoidco-oxidation activity; while LOX III shows activity at neutral pH,is inhibited by calcium and has a high capacity to co-oxidizecarotenoids (Boyes, Perera, & Young, 1992; Robinson et al., 1995;Baysal & Demirdöven, 2007). Although the activity of LOX to co-oxidize carotenoids is well known, the mechanism of the bleachingreaction is not totally understood. It has been proposed that an en-zyme-bound hydroperoxide complex can be the oxidizing specie(Klein et al., 1984). Likewise, the hydroperoxides formed by the di-rect action of LOX on PUFAs in the presence of oxygen can directlyattack carotenoids (Robinson et al., 1995). These hydroperoxidesmay also have a detrimental effect on other food components suchas proteins, modifying their native structure by oxidizing thiolsgroups, consequently affecting their functionality (Nicolas & Dra-pron, 1983). These reactions catalyzed by LOX have captured theattention of food technologists and nowadays the enzyme is usedas a processing aid additive in cereal products. For instance, en-zyme-active soya flour (containing LOX) is used as a bleachingagent in flour influencing dough and bread color (Faubion & Hose-ney, 1981). In addition, this food additive is used to catalyze thecoupled oxidation of gluten proteins leading to an increase indough relaxation time and also increasing the volume of bread(Nicolas & Potus, 1994). A proposed mechanism of action that re-lates biochemical events in the plant tissue with the impact ofLOX on the quality of foods are summarized in Fig. 1. In addition,the industrial applications of the reaction intermediates are alsoincluded.

Avocado (Persea americana Mill.) processing and commerciali-zation are activities of major socioeconomic relevance for Mexico,the country that occupies the first place in the production of thishorticultural crop worldwide (FAOSTAT, 2008). Due to the higheconomic importance that avocado has to Mexico, the food indus-try is showing a remarkable interest in processing and enhancingthe value of this crop. However, avocado products are quiteunstable due to the presence of oxidative enzymes such aspolyphenoloxidase (PPO) and LOX (Elez-Martínez, Soliva-Fortuny,Gorinstein, & Martín-Belloso, 2005). Inhibition of avocado LOX ispossible by using phenolic compounds such as epicatechin (Mar-cus, Prusky, & Jacoby, 1988; Prunsky, Kobiler, Jacoby, Sims, & Mid-land, 1985). However, this approach to inactivate LOX may also bedetrimental for avocado products since epicatequin is substrate forPPO, increasing the rate of browning. Characterization of avocadoLOX offers the potential of increasing scientific knowledge thatcan aid in the establishment of optimum processing and storageconditions at which the detrimental effects of this enzyme areminimized, preventing product organoleptic changes and nutri-tional quality losses. The determination of other catalytic proper-ties of avocado LOX such as the ability to co-oxidize carotenoidsmay also help to promote the use of the avocado enzyme as ableaching agent in the food industry. Additionally, the enzymaticcharacterization of avocado LOX may lead into finding alternativeuses for LOX as a food ingredient or additive.

The objective of the present project was to partially purify andcharacterize avocado fruit (Persea americana Mill, cv. Hass) LOX en-zyme including substrate affinity, optimum temperature and pH,and the effect of calcium ion concentration on activity. In addition,

the in vitro carotenoid co-oxidation capability of avocado LOX wasevaluated.

2. Materials and methods

2.1. Fruit samples and chemicals

Avocados (Persea americana Mill, cv. Hass) used in the studywere obtained from a local market. Mature Hass avocados were se-lected for the experiments based on their skin appearance (com-plete change from green to black), fruit palpability (slightly yieldto gentle pressure) and flesh appearance (green/yellowish richcreamy flesh with no black spots). Ammonium sulfate, linoleicand linolenic acids, calcium acetate, Tris–HCl, Triton X-100, NaOHwere purchased from Fisher Scientific Inc. (Winnipeg, MB, Canada).EDTA, Tween-20, Tween-80, Bradford reagent, bovine serum albu-min (BSA), b-carotene and other chemicals and reagents were ob-tained from Sigma Chemical Co. (St. Louis, MO, USA). Dowex# 1-X8anion exchange resin from Dow Chemical Co. was obtained fromSigma Chemical Co. (St. Louis, MO, USA). Membrane for dialysiswas obtained from Spectrum# Laboratories Inc. (Rancho Domin-guez, CA, USA).

2.2. Preparation of crude lipoxygenase extract

Crude extracts were prepared from a subsample (30 g) obtainedfrom a composite of three individual fruits (excluding peel and seedtissues). Samples were homogenized with 60 ml of a sodium phos-phate buffer solution (5 mM, pH 6.5) in the presence of 6 mM EDTAand 1% Triton X-100. Homogenates were filtered through two lay-ers of cheesecloth and centrifuged at 20,000g for 15 min. EDTAwas used during extraction as a reversible inhibitor of proteasesin order to minimize LOX activity losses. Supernatants were col-lected, pooled and used as the enzyme crude extract (LOXce). Allextraction and purification procedures were performed at 0–4 !C.

2.3. Partial purification of lipoxygenase

LOXce was partially purified by stepwise precipitation withammonium sulfate, followed by anion exchange adsorption. Frac-tional precipitation of LOX was carried out in order to determinethe saturation point for the enzyme. LOXce was brought to differ-ent ammonium sulfate concentrations (10–80% saturation) and al-lowed to stand for one hour at 4 !C. Precipitated fractions werecollected by centrifugation (20,000g for 15 min) and re-suspendedin 10 ml of sodium phosphate buffer (5 mM, pH 6.5). LOX activityof each fraction was determined. Fractions with significant LOXactivity were identified and the saturation cut for avocado (Perseaamericana Mill, cv. Hass) LOX was determined. LOX saturation cutfraction (20–50% (NH4)2SO4) was dialyzed overnight against200 ml of sodium phosphate buffer (5 mM, pH 6.5) using a14,700 MWCO membrane. The retentate was stirred for 20 minin the presence of 5 g Dowex# 1-X8 anion exchange resin. Thesupernatant (not adsorbed fraction; Snaf) was collected. Adsorbedcompounds were desorbed from the resin using a buffer with high-er ionic strength. 20 ml of sodium phosphate buffer solution(100 mM, pH 6.5) were added to the resin and stirred for 20 min.Adsorbed fraction (Saf) was collected. LOX activity of both fractions(Snaf and Saf) was determined. Snaf was used in all subsequentexperimental stages as a partially purified LOX solution.

2.4. Lipoxygenase activity assay

LOX activity determinations were assayed using the method de-scribed by Daglia, Aceti, Giorgetti, Papetti, and Gazzani (2005).

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Substrate assay stock solutions were prepared by mixing either lin-oleic or linolenic acid (19 ll) with Tween-20 (20 ll) and nanopurewater (5 ml) and clarified by adding 1 N NaOH solution (0.5 ml).The pH was kept constant using 50 mM citrate phosphate bufferfor pH 4–5, 50 mM sodium phosphate buffer for pH 5.5–6.5,50 mM Tris–HCl buffer for pH 7–9, and sodium hydroxide–sodiumborate buffer for pH 10. Before the assay, substrate assay stocksolution (110 ll) was diluted using buffer at different pH values(from 4 to 10) and calcium acetate concentrations (from 0 to1 mM) to give a 4 ml final volume. Reaction was started by adding5 ll of LOX extracts into a microplate well containing 200 ll ofsubstrate solution; 96 well microplates were used for LOX activitydeterminations. Spectrophotometric determinations were col-lected at 234 nm every 30 s for 10 min using a plate reader withstirring and temperature control (Synergy HT, Bio-Tek Instru-ments, Inc., Winooski, VT). Prior to LOX extract addition; micro-plates with substrate solution were equilibrated to assaytemperature in an incubator for 10 min. Plate reader was alsopre-heated until assay temperature was reached in order to assurethermal equilibrium. After microplate pre-incubation time LOX ex-tract was promptly added. Microplate was then swiftly placed inthe plate reader to start spectrophotometric determinations.

Enzyme activities (DA/min) were estimated by the initial velocitiesmethod from the linear portion of the curves. One unit of activitywas defined as an increase in absorbance of 0.001 min!1. A wellcontaining substrate solution (200 ll) and nanopure water (5 ll)was used as blank. Each sample was read against an assay solutionwithout substrate. All determinations were carried out at least intriplicate.

2.5. Substrate specificity and effects of temperature, pH and calciumion concentration on lipoxygenase activity

Activity assays using either linoleic or linolenic acid were car-ried out in order to determine LOX substrate specificity. LOX activ-ity at different substrate concentrations (0–3 mM) wasdetermined. Reaction conditions were kept constant at 25 !C, pH7 and 0 mM Ca+2. LOX kinetic constants (KM and Vmax) were calcu-lated using the Lineweaver–Burk (L–B), Hanes (HA) and Eadie–Hof-stee (E–H) linearization methods. A substrate concentration of3 mM was then used at subsequent experimental stages. Effectsof temperature (20–75 !C), pH (4–10) and calcium ion concentra-tion (0–1 mM) on LOX activities were also determined. One exper-imental factor was varied at a time in the previously mentioned

Fig. 1. Mechanism of action and impact of lipoxygenase (LOX) on foods quality. Once decompartmentalization occurs LOX and its substrates (polyunsaturated fatty acids;PUFAs) interact promoting the initiation and propagation of the free radical chain reaction. Some intermediates have industrial applications.

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order. The optimum parameters for temperature, pH and Ca+2 con-centration estimated at each experimental stage were then used insubsequent experimental stages.

2.6. In vitro carotenoid co-oxidation activity assay

LOX b-carotene co-oxidation activity was measured using themethod described by Daglia et al. (2005) adapted for 96 wellmicroplates. b-carotene (1 mg) was dissolved in chloroform(1 ml) containing Tween-80 (40 ll), evaporated to dryness andthen dissolved in 1 mM EDTA (10 ml) to obtain a b-carotene solu-tion of 0.19 mM. The assay solution contained in the microplatewell was a mixture of substrate solution (200 ll) and b-carotenesolution (3.5 ll), giving a b-carotene concentration of 3.2 lM inthe reaction mixture. Partially purified LOX solution (5 ll) wasadded at last in order to initiate the reaction. A blank without b-carotene was also measured. Carotenoid co-oxidation activitywas determined by calculating the relative b-carotene degradationpercentage according to Daglia et al. (2005). The following equa-tion was used

relative b-carotene degradation percentage

¼ 100! ðAt=AoÞ % 100 ð1Þ

where Ao = absorbance of the sample – absorbance of blank sampleat time 0 and At = absorbance of the sample – absorbance of blank attime t.

2.7. Protein quantification

Protein concentration was determined using the Bradfordmethod (Bradford, 1976) adapted for 96 well microplates. Briefly,250 ll of Bradford reagent and 5 ll of sample were poured in amicroplate well. The microplate was incubated at room tempera-ture (20–25 !C) for 10 min. Spectrophotometric determinationswere collected at 595 nm using a plate reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski, VT). Total protein concentrationwas estimated using a bovine serum albumin standard curve. Alldeterminations were carried out at least in triplicate.

2.8. Statistical analysis

Results reported are the average of three independent determi-nations ± standard error of the mean. Linear regression, and analy-sis of variance were conducted using JMP software (SAS, Cary, NC),with mean separation performed using the LSD test (p < 0.05).

3. Results and discussion

3.1. Partial purification of lipoxygenase

LOX was partially purified from avocado by means of stepwiseprecipitation using ammonium sulfate and anionic adsorption.Experiments that involved the fractional precipitation of LOX fromthe avocado crude extract showed that ammonium sulfate frac-tions that precipitated at 20%, 30%, 40% and 50% saturation allowedthe recovery of most of LOX activity. Fig. 2 shows the relative activ-ity (%) recovered in each fraction. These results agree with thoseobtained for LOX from other plant sources. Bean (Phaseolus vulgariscv. Haricot) LOX was found to precipitate at 20–50% ammoniumsulfate saturation (Kermasha & Metche, 2006). Galliard and Phillips(1971) reported that LOX from potato tubers (Solanum tuberosumcv. Majestic) precipitated almost entirely at 50% ammonium sul-fate saturation. Based on the relative activity obtained from eachfraction, a 20–50% saturation cut was selected in order to obtainan avocado LOX enriched fraction using stepwise precipitation.

Anionic adsorption was selected as a subsequent stage for thepartial purification process. Both fractions (non adsorbed and ad-sorbed) were analyzed for LOX activity. Adsorption was carriedout at pH 6.5 (pH value naturally found in avocado pulp). LOXactivity was detected only in the unabsorbed fraction, indicatinga positive electrochemical charge of LOX at pH 6.5. Marcus et al.(1988) reported an isoelectric point (pI) of 5.9 for avocado (Perseaamericana Mill, cv. Fuerte) LOX. Other authors have reported thatthe pI of LOX from other species is located above 6.5, which seemsto be the case for avocado LOX from the cultivar Hass. Daglia et al.(2005) reported that LOX from chicory (Cichorium intybus) has a pIof 6.85, while Garbe, Barbosa de Almeida, Nagel, Wackerbauer, andTressl (2006) reported a value of 6.7 for LOX extracted from barley(Hordeum vulgare).

Table 1 summarizes the partial purification process of avocadoLOX. Interestingly, overall yield of the partial purification process(obtained as function of the total LOX activity of the crude extract)was estimated to be 100% (Table 1). This only can be explained interms of the elimination of potential LOX inhibitors during the an-ionic adsorption. Some low molecular weight compounds, particu-larly phenolics such as epicatechin, have been found to be LOXinhibitors (Oszmianski & Lee, 1990; Prunsky et al., 1985). Phenoliccompounds usually are attached to proteins either by hydrogenbonding, hydrophobic or ionic interactions, difficulting their elim-ination from crude extracts (Loomis, 1974). It is well known thatphenolic compounds are found in avocado (Ramírez-Martínez &Luh, 1973; Torres, Mau-Lastovicka, & Rezaaiyan, 1987). Since pKavalues of the COOH groups in most phenolic acids are located be-tween 2.5 and 4.5 at pH 6.5, they have negative electrochemicalcharge, being able to interact with the anionic adsorption resin.This explains the elimination of these potential LOX inhibitors dur-ing the anionic adsorption stage, ultimately increasing the overallyield of the process. Although the purification process used just al-lowed a 3.4 purification factor (in reference to the total proteincontent) it resulted in a 100% overall yield and the elimination ofpotential LOX inhibitors.

3.2. Substrate specificity

Linoleic and linolenic acid were used as LOX substrates and theirconcentration was varied in the reaction solution in order to estab-lish the initial velocities. The values of both Michaelis–Menten

Fig. 2. Lipoxygenase (LOX) relative activity (%) in ammonium sulfate fractions.Fractionated precipitation of proteins using ammonium sulfate was conducted at4 !C. A saturation cut of 20–50% ammonium sulfate was determined for avocado(cv. Hass) LOX, achieving a 93% recovery yield from the total activity. Relativeactivity (%) represents the perceptual relation that exists between the LOX activityobtained at a particular (NH4)2SO4 saturation fraction and that obtained in thefraction where the higher LOX activity was obtained (30% (NH4)2SO4).

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parameters, Km and Vmax, for each type of substrate were calculatedusing the L–B, HA and E–H linearization methods. Both substrateswere effectively oxidized by the enzyme. Although a higher valueof Vmax was observed for linoleic acid, the lower Km was achievedwhen linolenic acid was used as a substrate. Therefore, the Vmax/Km ratio was selected as a criterion for substrate specificity. The ob-tained data is shown in Table 2. Although Vmax and Km values calcu-lated from the three linearization methods used (L–B, HA and E–H)were not identical; no large differences were observed neither forlinoleic nor linolenic acid. L–B linearization is known to renderthe least accurate estimation of Michaelis–Menten parametersfrom the three methods used (Leskovac, 2003). Experimental valuesobtained at low substrate concentrations are overemphasizedwhen the L–B method is used. Since at low substrate concentrationsexperimental error may have a particularly significant impact oninitial velocity determination, inaccurate Vmax estimations are oftenrendered by this method. On the other hand, HA linearization is re-garded as the most accurate of the three methods used (Leskovac,2003). The mean Vmax/Km values from L–B and HA linearizationshad a difference of 11.1% and 13.7% for linoleic and linolenic acid,respectively, demonstrating that regardless experimental errorboth methods render similar estimates. Kinetic parameters esti-mated using E–H linearization were similar to those obtained withthe other two methods. Coefficient of determination (R2) for L–B,HA and E–H linearizations for linoleic acid were 0.98, 0.96 and0.86, respectively. In the case of linolenic acid R2 for L–B, HA andE–H linearizations were 0.98, 0.99 and 0.88, respectively. R2 valuesindicate that all three linearization methods properly fit the exper-imental data. Marcus et al. (1988) reported a Km of 0.072 mM foravocado fruit (cv. Fuerte) LOX when linoleic acid was used as sub-strate. This Km value is considerably lower than the one obtainedin the present study for avocado fruit (cv. Hass) LOX (Table 2). Suchdissimilitude may be attributed to differences in the cultivar, thepartial purification method and the enzymatic assay conditionsused.

Regardless of the linearization method used (L–B, HA or E–H) itcan be concluded, based on the Vmax/Km ratio, that avocado LOX hasa higher specificity for linolenic acid. Several authors have reportedsubstrate specificity of LOX from other plant sources. Daglia et al.(2005) reported that LOX extracted from chicory (Cichorium intybuscv. silvestre) has lower specificity for linolenic acid, achieving only50% of the activity with linoleic acid. In LOX extracted from otherspecies, such as the one from banana leaves, the specificity for lin-oleic and linolenic acid is similar (Kuo et al. (2006)). The obtainedresults regarding avocado (cv. Hass) LOX substrate specificity maybe associated to the relative concentration of linoleic and linolenicacid in the fruit. Avocado is richer in linoleic acid (USDA NationalNutrient Database., 2009), and therefore, as part of its structuralevolution, avocado LOX may have changed in order to compensatethe lower concentration of linolenic acid by increasing itsspecificity.

3.3. Effects of temperature, pH and calcium ion concentration onavocado lipoxygenase activity

Optimum temperature and pH of avocado LOX was estimatedfor both, linoleic and linolenic acid. Additionally, since it has beenreported that the activity of some LOX isoenzymes is calciumdependant (Boyes et al., 1992; Robinson et al., 1995; Baysal &Demirdöven, 2007), the effect of calcium ion concentration onactivity was also determined. Fig. 3 shows the effect of tempera-ture on avocado LOX activity. The optimum temperature for theoxidation of both substrates was found to occur at 40 !C. For bothsubstrates, LOX activity decreases considerably at temperaturesbelow 30 !C and above 40 !C. Kuo et al. (2006) also reported a tem-perature optimum of 40 !C for banana leaf (Musa acuminata) LOX,

Table 1Avocado (cv. Hass) lipoxygenase (LOX) partial purification. LOX partial purification was achieved using consecutively protein stepwise precipitation with ammonium sulfate(saturation cut 20–50% ammonium sulfate saturation) and anion exchange adsorption.

Step Total activity % 10!6 (U) Protein (mg/ml) Specific activity % 10!6 (U/mg) Yielda (%) Purification factorb

Crude extract 8.57 ± 0.42 20.0 ± 0.4 0.43 ± 0.02 100 –Precipitation 7.96 ± 0.17 10.8 ± 0.5 0.74 ± 0.02 93 1.7Adsorption 8.55 ± 0.38 5.8 ± 0.2 1.46 ± 0.06 100 3.4

Temperature was kept constant at 4 !C.a Yield (%) for each step was obtained considering the total activity (U) in the LOX crude extract (LOXce).b Purification factor for each step was estimated as the ratio of its specific activity and the specific activity of LOXce.

Table 2Kinetic parameters for the oxidation of polyunsaturated fatty acids by avocado (cv.Hass) lipoxygenase (LOX) estimated using Lineweaver–Burk (L–B), Hanes (HA) andEadie–Hofstee (E–H) linearization methods.

Kinetic parameter Linearization method Substrate

Linoleic acid Linolenic acid

Vmax (mM/min) L–B 12.52 ± 1.17 10.63 ± 0.10HA 11.98 ± 1.40 9.97 ± 0.01E–H 12.24 ± 0.74 10.19 ± 0.07

km (mM) L–B 0.89 ± 0.20 0.21 ± 0.02HA 0.80 ± 0.21 0.17 ± 0.02E–H 0.84 ± 0.08 0.18 ± 0.01

Vmax/km (min!1) L–B 14.07 ± 2.43 50.62 ± 5.26HA 12.51 ± 1.15 58.64 ± 5.45E–H 14.75 ± 0.92 59.03 ± 3.37

Temperature, pH and calcium ion concentration were wept constant at 25 !C, 7.0,and 0 mM, respectively. Coefficient of determination (R2) for L–B, HA and E–Hlinearizations for linoleic acid were 0.98, 0.96 and 0.86, respectively. R2 for L–B, HAand E–H linearizations for linolenic acid were 0.98, 0.99 and 0.88, respectively.

Fig. 3. Effects of temperature on avocado (cv. Hass) lipoxygenase activity. Twodifferent substrates (linoleic (d) and linolenic (s) acid) were used. pH and calciumconcentration were kept constant at 7.0 and 0.0 mM, respectively. Substrateconcentration was kept constant at 3.0 mM. Local optimum (100% relative activity;78.3 % 106 U/ml) was found at 40 !C for linolenic acid.

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while Daglia et al. (2005) reported an optimum of 38.5 for LOX ex-tracted from chicory (Cichorium intybus cv. silvestre). On the otherhand, Kuribayashi et al. (2002) reported a considerably lower opti-mum (25 !C) for LOX extracted from oyster mushroom (Pleurotusostreatus). These and other temperature optimums reported in lit-erature match the average temperatures at which each sourcegrows. Given that avocado grows in tropical and semitropical cli-mates, with high humidity and temperature, a temperature opti-mum for LOX activity between 30 !C and 40 !C, as the one foundin the present work, was expected. Since thermal processing orlong exposure to high temperatures is likely to promote undesir-able organoleptic changes in avocado pulp, the use of high hydro-static pressure pasteurization is an effective strategy to partiallyinactivate LOX (Palou et al., 2000). However, the residual activityof LOX in hyperbaric processed avocado products may affect theshelf-life. Therefore, a viable strategy for reducing LOX activitycould involve maintaining the pressurized product under an oxy-gen depleted atmosphere at low temperatures (<20 !C).

Fig. 4 shows the effect of pH on avocado LOX activity. The opti-mum pH occurs between 6.0 and 6.5 for linoleic acid, while for lin-olenic acid is 6.5, matching the pH naturally found in avocado pulp.The optimum pH of LOX enzyme from other sources has been esti-mated to be between a wide pH range (5.5–9.5). Optimum pH isknown to be related to the type of LOX isoenzyme. Marcus et al.(1988) reported a pH optimum of 7.1 for avocado cv. Fuerte LOX.In the particular case of avocado cv. Hass LOX, it seems to have anarrow range of functional pH, remaining almost inactive belowpH 5 and above pH 7 (Fig. 4), mainly when linoleic acid is usedas substrate. Since linoleic acid is in a considerably higher concen-tration in avocado than linolenic acid, the reduction (<5) of pH mayrepresent an effective strategy for significantly reducing the detri-mental effects of LOX on the product.

Calcium ion concentration was found to have significant influ-ence (p < 0.05) on avocado LOX activity, as can be seen in Fig. 5. Avo-cado LOX is active at low calcium concentrations, independently ofthe substrate. An activity peak can be observed when calcium con-centration is 0.2 mM. The promptness of the PUFAs oxidation pro-cess that the avocado pulp undergoes once the fruit is wounded(and therefore exposed to the oxygen) may be explained in termsof the optimums found herein. Since the pH optimum match thecondition naturally found in avocado and the temperature rangeof activity is wide (30 –40 !C for linoleic acid), the action of LOXover PUFAs once in contact with oxygen is immediate, generating

off-flavors. LOX initially produced flavors are desirable and charac-teristic of some products. However, further oxidation and advanceof the reaction with time leads to the formation of the off-flavors.During storage, these reactions in combination with the activityof PPO (which generates colored compounds causing the browningof the product) are the two major problems concerning the loss ofsensorial quality of minimally processed avocado pulp (Elez-Martí-nez et al., 2005).

3.4. Carotene co-oxidation by avocado lipoxygenase

The capacity of avocado LOX for the co-oxidation of carotenoidswas studied at two different temperatures (20 !C and 40 !C). b-car-otene was used as a model during this experimental stage, and theresults are presented in Fig. 6. Carotene co-oxidation capabilitiesof avocado LOX were observed when either linoleic or linolenicacid were used as substrates. However, co-oxidation activity isconsiderably higher when linolenic acid is used. For that particularsubstrate, temperature seems to have significant effect on the

Fig. 4. Effects of pH on avocado (cv. Hass) lipoxygenase activity. Two differentsubstrates (linoleic (d) and linolenic (s) acid) were used. Temperature and calciumconcentration were kept constant at 40 !C and 0.0 mM, respectively. Substrateconcentration was kept constant at 3.0 mM. Local optimum (100% relative activity;125.2 % 106 U/ml) was found at 40 !C and pH 6.5 for linolenic acid.

Fig. 5. Effects of calcium concentration on avocado (cv. Hass) lipoxygenase activity.Two different substrates (linoleic (d) and linolenic (s) acid) were used. Temper-ature and pH were kept constant at their optimum values (40 !C and 6.5 !C,respectively). Substrate concentration was kept constant at 3.0 mM. Local optimum(100% relative activity; 152.5 % 106 U/ml) was found at 40 !C, pH 6.5 and 0.2 mMcalcium concentration for linolenic acid.

Fig. 6. b-carotene co-oxidation activity of avocado (cv. Hass) lipoxygenase. Capacityof LOX extracted from avocado to co-oxidate b-carotene was studied using twosubstrates and temperatures: linoleic 20 !C (5), linoleic 40 !C (s), linolenic 20 !C(.), linolenic 40 !C (d). Substrate concentration, pH, calcium concentration werekept constant at 3.0, 6.5 mM and 0.0 mM, respectively.

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co-oxidation velocity. b-carotene degradation reached 40%approximately at 250 s at 40 !C, while at 20 !C degradation is con-siderably lower (<20%). The results presented herein confirm that,regardless of substrate and temperature, avocado LOX has indeedcarotene co-oxidation capability. Avocado is rich in linoleic acid,and to a lesser degree in linolenic acid (USDA National NutrientDatabase, 2009). Therefore, total or partial inactivation of the en-zyme may help to preserve the nutritional quality of avocado pulp.These findings also demonstrate the potential industrial applica-tion of avocado (cv. Hass) LOX as bleaching agent. The partial puri-fication process developed in the present work may be easilyscaled up in order to obtain LOX enzymatic extracts suitable forsuch application. The use of avocado fruit as source of enzymeswith potential application in food industry as aid additives mayimpulse the production and processing of this fruit of economicalrelevance for México and other countries.

4. Conclusion

In the present study lipoxygenase from avocado (cv. Hass) hasbeen partially purified and the effects of experimental parameterson enzymatic activities were studied, indicating that it appear to bea LOX II-like isoenzyme with optimum activity at 40 !C, pH 6.5,0.2 mM calcium ion concentration, and capable of co-oxidizingcarotenoids. The results presented herein can help the avocadoindustry to propose strategies concerning the inactivation of theenzyme. By inactivating the enzyme an enhancement of the sen-sory shelf-life and a higher retention of carotenoids in minimallyprocessed avocado products can be achieved. Additionally, theco-oxidation capability of avocado LOX may be of interest for theavocado industry since it can be used as a bleaching agent in flourand other products.

Acknowledgment

The authors would like to thank the TAMU-CONACYT ResearchInitiative (0020CONA81) for its economical support. Jacobo-Veláz-quez also acknowledges the scholarship from the Consejo Nacionalde Ciencia y Tecnología (CONACYT, México).

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