CARRILLO W*, TUBÓN J, VILCACUNDO R

Vol 9, Issue 3, 2016 Online - 2455-3891 Print - 0974-2441 Reserch Article ISOLATION OF HEN EGG WHITE LYSOZYME BY CATION EXCHANGE CHROMATOGRAPHY, ANALYSIS OF ITS DIGESTIBILITY AND EVALUATION OF THE INHIBITION LIPID PEROXIDATION IN THE ZEBRAFISH MODEL CARRILLO W*, TUBÓN J, VILCACUNDO R Lortory Functionl Foods, Fculty of Foods Science nd Engineering, Technicl University of Amto, Av. Los Chsquis y Rio Pymino, Cmpus Huchi, CP 1801334, Amto-Ecudor. Emil: wi.crrillo@ut.edu.ec ABSTRACT Received: 29 Ferury 2016, Revised nd Accepted: 10 Mrch 2016 Ojective: The im of this study ws to seprte nd identify lysozyme using ction exchnge chromtogrphy, evlute the protein digestiility nd nlyze the inhiition lipid peroxidtion in the zerfish model. Methods: Hen egg white lysozyme ws isolte with ction exchnge chromtogrphy. Residul murmidse ctivity ws evluted with the spectrophotometric method. Isolte lysozyme (ILZ) nd hydrolystes were nlyzed with sodium dodecyl sulfte-polycrylmide gel electrophoresis (SDS-PAGE) electrophoresis nd reversed-phse high-performnce liquid chromtogrphy (RP-HPLC). Results: To identify the protein, the smple ws isolted with ctionic exchnge chromtogrphy, smple ws nlyzed using RP-HPLC nd SDS-PAGE confirming tht the frction ws hen lysozyme egg white. The enzymtic ctivity of the isolted protein ws norml compred to the commercil lysozyme ctivity. Hydrolystes hd no murmidse ctivity nd were le to inhiit lipid peroxidtion in zerfish lrve. Conclusions: Ction exchnge chromtogrphy is good method to ILZ from egg white. Hydrolystes of lysozyme were effective to inhiit lipid peroxidtion in zerfish model. Keywords: Lysozyme, Ction exchnge chromtogrphy, Enzymtic hydrolysis, Murmidse ctivity. INTRODUCTION Lysozyme (EC 3.2.17, N-cetyl-murmic-hydrolse) is gloulr sic protein found in nture nd is chrcterized y its high enzymtic ctivity. It ws first discovered in nsl mucous y Alexnder Fleming, who nmed it Lysozyme s he oserved its lytic ctivity towrd cteril cocci [1]. The egg lumen is known to hve n exceptionlly high mount of lysozyme, normlly referred to s hen s egg lysozyme, representing 3.5% of the egg white protein content [2-5]. The lysozyme is sic protein consisting of 129 mino cids nd moleculr weight of 14.3 kd. This enzyme cts y lysing the cell wlls of certin Grm-positive cteri such s lctic cid cteri nd Clostridium sp. y splitting β (1-4) linkges etween N-cetylmurmic cid nd N-cetylglucosmine of the peptidoglycn of cteril cell wlls [6-9]. Lysozyme hs een ssocited with mny iologicl ctivities such s nticteril, ntivirl, immunomodulting, immunostimulting, ntioxidnt, ntivirl, nd ntitumorl ctivities mong others [10-17]. Clssicl seprtion methods of proteins re sed on the slting out of solution or precipittion with lcohol. These techniques hve een extended y the ion exchnge (IEX) chromtogrphy nd memrne seprtion in recent yers. Presently, reserchers re looking for seprtion methods, which will e chep, esy, nontoxic, nd mintining the highest iologicl ctivity of isolted proteins [18-21]. The im of the study ws to isolte lysozyme (ILZ) using ction exchnge chromtogrphy, evlute its digestiility nd then mesure their murmidse ctivity nd ility to inhiit lipid peroxidtion in zerfish lrve. METHODS Lysozyme nd mterils Lysozyme (L2879, chloride form from chicken egg white Grde VI, 40000 units/mg protein, EC 3.2.1.17) nd pepsin crystlline (4500 units/mg otined from porcine stomch mucus nd Micrococcus lysodeikticus) were purchsed from Sigm Chemicl Co. (Sint Louis, MO, USA). Isoltion of proteins from egg white Aout 500 ml of egg white treted with ethnol 30% nd djusted ph t 5.8 to seprte the mucine. The solution ws centrifuged t 4.000 rpm for 30 minutes t 4 C. The superntnt ws discrded, nd the precipitte ws djusted t ph 7.4 with 1 M NOH nd suject to IEX chromtogrphy. IEX chromtogrphy Proteins form egg white were seprted y IEX s descried y Recio nd Visser [22] with some modifictions using fst protein liquid chromtogrphy (FPLC) system with ction exchnge column HiLodTM 26/10 SP-Sephrose Fst Flow (Phrmci) [23]. Solvent A ws 10 mm mmonium hydrogen cronte cidified to ph 7 with formic cid, nd solvent B ws 3 M of mmoni solution. The effluent ws monitored t 280 nm. Ech chromtogrphic run ws repeted 10 times, nd the collected frctions were pooled, frozen, nd lyophilized. Following these frctions were nlyzed with reversedphse high-performnce liquid chromtogrphy (RP-HPLC) nd sodium dodecyl sulfte-polycrylmide gel electrophoresis (SDS-PAGE). Enzymtic hydrolysis of lysozyme Commercil, ILZ nd hydrolystes were initilly dissolved t 5 mg/ml in potssium phosphte uffer 10 mm (ph 1.5). 1 ml of this lysozyme solution ws mixed with 50 ml of pepsin solution of 200 U/mg (5 mg/ml in solution of 0.035 M NCl, ph 2.0) to otin n enzymeto-sustrte rtio of 1:20 w/w. This mixture ws incuted t 37 C for 1 hr. The rection ws stopped y heting t 80 C for 15 minutes, nd the ph ws djusted t 7.0 y ddition of 1 M NOH [7]. SDS-PAGE nlysis The smples were dissolved in 10 mm Tris-HCl uffer, ph 8, 2.5% SDS, nd 10 mm ethylenediminetetrcetic cid (non-reducing conditions) or the sme uffer contining 5% -mercptoethnol (reducing conditions), nd heted t 95 C for 10 minutes [24]. Anlysis y SDS-

PAGE used PhstSystem Electrophoresis pprtus, precst 20% homogeneous gels nd PhstGel SDS Buffer Strips (GE HelthCre, Brcelon, Spin), following the electrophoretic nd silver stining conditions of the mnufcturer. RP-HPLC nlysis Lysozyme hydrolystes, t concentrtion of 2.0 mg/ml, were nlyzed using Hi-Pore RP-318 (250 mm 4.6 mm i.d.) column (Wters, Milford, MA) in Wters 600 HPLC system. Solvent A ws 0.37% (v/v) trifluorocetic cid (Schrlu Chemie, Brcelon, Spin) in doule-distilled wter, nd solvent B ws 0.27% (v/v) trifluorocetic cid in HPLC-grde cetonitrile (L-Scn, Gliwice, Polnd). The chromtogrphic conditions were s in Mrtos et l. [25]. Detection ws t 220 nm, nd dt were processed using Empower 2 Softwre (Wters). Murmidse ctivity ssy The lytic ctivity of lysozyme ws determined y monitoring the decrese in turidity of suspension of M. lysodeikticus cell spectrophotometriclly t 450 nm t 25 C, ccording to Shugr s method [26]. One unit of lysozyme ws defined s decrese in the sornce t 450 nm of 0.001 minutes 1. The murmidse ctivity of ech smple ws ssyed in triplicte. Thiorituric cid rective sustnces (TBARS) The thiorituric cid rective species method ws used s descried y Westerfield, 1995 [27]. The zerfish colony ws estlished in the lortory, in n environmentl growth or glss qurium, provided with internl filter nd ertor ctivted cron for wter oxygention. The popultion of nimls ws fed three times dy with food chips for fish. Adult fish were kept on 16 hrs light nd 10 hrs drk cycles. Emryos were otined y photo-induced spwning over green plnts nd were cultured t 28 C in fish tnk wter. Lrve of 5 dpf were then incuted in 24-well pltes, 30 lrve per well for ech 100 mg/ml of lysozymes nd hydrolystes. Lipid peroxidtion ws initited y dding 1 ml 500 µm H 2 O 2 nd incuted for 24 hrs t 28 C. Groups from 30 lrve/well in qurium wter were used s controls. Then, it ws removed the H 2 O 2, nd it ws dded 500 µl of Tween 0.1%. Ech group ws mixed nd homogenize with T25 ultr turrx IKA fter the sornce ws mesured t 532 nm, nd the decrese of sornce indictes n increse of ntioxidnt ctivity. Vlues of ntioxidnt ctivity were expressed s the percentge inhiition of lipid peroxidtion in lrve homogente s follows: The totl ntioxidnt ctivity % inhiition of lipid peroxidtion=[(a As)/A 100] where A = sornce of control nd As= sornce of smple. The test lrvl ws monitored using microscope with Motic Moticm 580, 5 MP. determined y the nlysis of vrince (ANOVA). The post hoc nlysis ws performed y the Tukey test. All tests were considered significnt t p<0.05. Sttisticl nlysis ws performed using the softwre pckge Prism 4 for Windows, version 4.3 (GrphPd Softwre Inc., www.grphpd.com). RESULTS Hen egg white lysozyme is protein with high isoelectric point (pi=10.7) in physiologicl conditions. At ph 7.0, lysozyme hs positive chrge ut t ph >10.7 hs negtive chrge. The egg white hs different proteins such s ovlumin (pi=4.5), ovotrnsferrin (pi=6.0), nd ovomucin (pi=4.1); these proteins hve n isoelectric point under 7.0; therefore, they hve net negtive chrge. These chrge differences ccording to pi llow seprting the white egg through ctionic exchnge chromtogrphy [19]. This fct cn e oserved in Fig. 1. At the eginning of the chromtogrm, pek with high sorption cn e oserved t the wvelength used (220 nm), this pek reltes to proteins with negtive chrge nture, s these proteins re unle to join to the negtive chrge of the column, resulting in eing eluted with the solvent. While lysozyme ws found retined in the column y chrge ffinity, the percentge of mmonic concentrtion ws incresed to grdully increse ph, when ph overcome the pi of lysozyme, lysozyme chnges the chrge to ecome negtively chrged nd eing eluded from the column, this fct is reflected t the second pek of the chromtogrm with n sorption t the wvelength of 220 nm. To determine if lysozyme ws effectively seprted, the collected FPLC frction ws suject to RP-HPLC with the im of confirming with its time retined compred to the time retined of stndrd of commercil lysozyme (CLZ), to confirm whether or not effectively is lysozyme. Fig. 2 shows 38 minutes retined time for stndrd lysozyme t the concentrtion of 2 mg/ml; it ws then oserved the sme retined time for the frction otined through fst protein liquid chromtogrphy (FPLC) (Fig. 2). Both CLZ nd the frction of FPLC were suject to hydrolysis with pepsin t ph 1.2; nd it ws oserved the sme ehvior for oth of them. Pepsin hydrolyzes protein completely t this ph. The products of the hydrolysis re identicl in the CLZ nd the isolted lysozyme (Fig. 2c nd d). The frction otined through FPLC ws nlyzed y SDS-PAGE. In Fig. 3, it is shown tht lne 2 reflects the CLZ, lne 3 to the purified FPLC lysozyme, oth nds eing t the sme height of retin in the gel. Using dt of lne 1, which corresponds to the moleculr weight mrker, we cn see tht oth nds re t 14.400 D. Confirming the moleculr weight of the purified protein in the FPLC otined frction. In lnes numer 4 nd 5, two nds elow 14.400 D cn e oserved; Test of toxicity in the model of zerfish Zerfish of the AB strin (wild-type, wt) emryos were otined from nturl spwnings. Emryos were rised nd fish were mintined s descried y Westerfield [27]. After collection nd disinfection, eggs were rered in 24-well micropltes with 1 ml of wter. Fish emryo toxicity (FET) test The ssy ws sed on the OECD drft guideline on FET test [28] nd is descried in detil y Domingues et l. [29]. 10 eggs per tretment (3 replictes) were selected nd distriuted in 24-well micropltes. The test strted with newly fertilized eggs exposed to the nominl concentrtions of 0; 2.5; 5; 25; 50; 100 mg/ml of lysozymes nd hydrolystes run for 2 dys. Emryos were oserved t 24 nd 48 hrs under stereomicroscope (mgnifiction used for oservtions ws 40). The following prmeters were evluted s n endpoint nd considered lethl if one of them is detected egg cogultion, lck of development somites, lck of til detchment, nd lck of hertet [30]. Sttisticl nlysis Results re presented s mens±stndrd devition from three replictes of ech experiment. Differences etween men vlues were Fig. 1: Seprtion of lysozyme of egg white y ction exchnge chromtogrphy using mmoni 3 M 346

those lnes relte to peptides resulting of the product of the hydrolysis with pepsin. No intct lysozyme cn e oserved t this hydrolysis ph. We finlly evlute the enzymtic ctivity of the lysozyme of white egg or lso clled murmidse ctivity. White egg lysozyme hs the cpcity of hydrolyzing the wlls of cteri Grm-positive. For this test, lyophilized wlls of Micrococcus lysodeikticus ATCC 4698 were used, the decrese of oservnce t 450 nm ws mesured. In Fig. 4, it cn e seen tht the FPLC purified lysozyme presented 97% of murmidse ctivity, the CLZ hydrolyzed only kept 1.7% of sid ctivity nd the FPLC purified lysozyme hydrolyzed presented only kept 2.0% of sid enzymtic ctivity, this dt indicte tht the ctive site of lysozyme ws reduced to smll frgments, which hve no murmidse ctivity. These results re in greement with different studies where hs een descried hydrolyzed lysozyme with no murmidse ctivity [4,31]. In Fig. 5, it cn e seen tht CLZ nd the isolted lysozyme through chromtogrphy were only le to inhiit the lipid peroxidtion in zerfish lrve 21% nd 23%, respectively, wheres hydrolyzed presented high ctivity, eing le to inhiit 82% for the hydrolyzed of CLZ nd 84% for the isolted hydrolyzed lysozyme through ctionic exchnge chromtogrphy. Both hydrolyzed were effective, nd there re not meningful differences when they re nlyzed sttisticlly. The zerfish lrve fter 24 hrs of the ssy when were exmined no show ovious morphologicl normlities s crooked odies, spinl deformities, nd not exhiit ny significnt effects on the growth of the ody zerfish lrve (Fig. 5). As shown in Fig. 6, the mortlity of the Zerfish egg when they were treted with different concentrtions of lysozymes nd hydrolystes. No significnt effect on the survivl rte ws oserved for ll tretments groups. When the morphologies of these lysozymes treted were exmined, no show ovious morphologicl normlities such s egg cogultion, lck of development somites, lck of til detchment, nd lck of hertet. The emryos were norml (Fig. 6). DISCUSSION Hen egg is one of the most common foods tht induce hypersensitive rections in young children. Egg white contins more thn 20 kinds of proteins. Ovomucoid (Gl d 1), ovlumin (Gl d 2), ovotrnsferrin (Gl d 3), nd lysozyme (Gl d 4) hve een identified s min llergens in the egg white. Hen egg white lysozyme is potent llergen nmed Gl d4 with resistnt t hydrolysis with pepsin. It is known tht lysozyme c Fig. 2: Reversed-phse high-performnce liquid chromtogrphy of lysozyme, () commercil lysozyme (CLZ), () isolte lysozyme (ILZ), (c) hydrolyste of CLZ, (d) hydrolyste of ILZ d Fig. 4: Residul murmidse ctivity of commercil lysozyme (CLZ), isolte lysozyme (ILZ), hydrolyste of CLZ nd hydrolyste of ILZ. The decrese in turidity of cell suspension of Micrococcus lysodeikticus ws determined t 450 nm in phosphte uffer t ph 6.24 nd 25 C. Dt were nlyzed y nlysis of vrince (GrphPd Prism), nd the mens were seprted y Tukey s multiple rnge test. The significnce ws defined t p 0.05 Fig. 3: Sodium dodecyl sulfte-polycrylmide gel electrophoresis nlysis of lysozyme. Lne 1: Weight moleculr, Lne 2: Commercil lysozyme (CLZ), Lne 3: Isolte lysozyme (ILZ), Lne 4: Hydrolyste of CLZ, Lne 5: Hydrolyste of ILZ. Coomssiestined polycrylmide gel Fig. 5: () Thiorituric cid rective results of lysozyme. Dt re expressed s % thiorituric cid rective sustnces inhiition compred to negtive control (error rs expressed s ±SD). n=30 zerfish lrve. Commercil lysozyme (CLZ), isolte lysozyme (ILZ), hydrolyste of CLZ nd hydrolyste of ILZ, () Morphologies of zerfish lrve fter incution with lysozymes 347

Fig. 6: () Survivl rte of zerfish emryos exposed with vrious concentrtions of lysozymes for 48 hrs, () photogrphy of zerfish egg fter 48 hrs incution with lysozymes hs resistnce to the hydrolysis with pepsin, ut it hs een recently descried tht lysozyme t ph 1.2 hs totl susceptiility to the hydrolysis with pepsin [7,32-34]. In this study, hen egg white lysozyme ws hydrolyzed with pepsin t low ph in simulted gstric fluid (SGF). The reltionship etween llergenicity nd stility to digestion nd the most pproprite experimentl conditions for mesurement of stility hve een the suject of some discussion. The first report y Astwood et l. [31] demonstrted tht mny nimls nd plnt food llergens displyed resistnce to pepsin digestion in vitro, wheres other common plnt proteins elieved not to e llergenic were digested rpidly (within 30 seconds). The hydrolysis in vitro with pepsin ws ssyed in SGF 0.35 M NCl. 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