Phytochemistry\ Vol[ 38\ No[ 0\ pp[ 1825\ 0887 0887 Elsevier Science Ltd[ All rights reserved\ Pergamon Printed in Great Britain

99208311:87:,see front matterPII] S99208311"86#998568

PURIFICATION AND THERMOSTABILITY OF ISOPEROXIDASEFROM ORANGES

E[ CLEMENTE

Laboratorio de Bioquimica de Alimentos!DQI!UEM Av[ Colombo 4689!Maringa!76919!899!PR!Brazil

"Received in revised from 19 October 0886#

Key Word Index*Citrus sinenis^ Rutaceae^ oranges^ puri_cation^ thermostability^ iso!peroxidase[

Abstract*Soluble and ionically bound peroxidase were extracted from oranges "citrus sinenis "L[# Osbeckcultivar Large Valencia\ Small Sweet and Navel[ Cationic and anionic isoperoxidase were obtained by Rotoforand ion!exchange chromatography[ The pI for the soluble isoenzymes "3[4 to 8[9# was measured using a surfaceelectrode and the Mr "11 k to 33 k# was estimated by gel!_ltration[ The puri_ed isoperoxidases "C0 and C1#were more heat stable than the peroxidase present in the crude extract[ The inactivation of orange peroxidaseactivity in a mixture or individually in a puri_ed state\ was found to be non!linear with heating time[ 0887Elsevier Science Ltd[ All rights reserved

INTRODUCTION

Peroxidase "EC 0[00[0[6# are part of a large group ofenzymes known collectively as oxidoreductases 0\ 1[Extensive studies describe the action of peroxidase onsubstances which yield bright colours on oxidation\but peroxidase can promote a large variety of reac!tions and therefore can exhibit a degree of versatilityunsurpassed by any other enzyme[ The quality ofextracted citrus juices depends on enzymic reactionsthat occur not only in the fruit during the developmentperiod\ but also in the juice during processing[ Theformation of o}!~avours in canned fruit and veg!etables has been associated with residual peroxidaseactivity following processing 2[ Peroxidase can con!tribute to deteriorating changes in ~avour\ texture\colour and nutrition in improperly processed fruitsand vegetables 3[ In orange\ the level of peroxidasein the juice is associated with loss of ~avour quality4[ The heat inactivation of peroxidase is non!linear\a large decrease in activity is observed during theinitial stages of a given heating process\ but the rateof inactivation then changes to a much slower process5\ 6[ Renaturation of peroxidase following heat inac!tivation has also been reported for some vegetable andfruit extracts\ e[g[ kohlrabi 7\ Brussels sprouts andcabbage 8\ grapes 09\ 00\ avocados 01\ apples 02\pears 03\ kiwifruit 04\ mango 05[

The HTST "High Temperature Short Time# treat!ments commonly used commercially in fruit and veg!etable processing are less e}ective for irreversible per!oxidase inactivation than the traditional\ more pro!longed methods 06[ Inactivation of peroxidase

18

activity in plant materials is generally found to bea non!linear process against heating time\ which isthought to be due to the presence of separate iso!peroxidases\ with di}erent heat stabilities 07[ Inplant extracts\ peroxidase activity has been found inboth soluble and bound states which di}er withrespect to heat stability and regeneration properties[

Commercial fruit juice production includes somealbedo and peel of the fruit in the juice\ which willmake a large contribution to the total peroxidaseactivity[ In the present research\ we report a study ofthe consideration of the heat stability of orange juice\albedo and peel crude extracts and thermal stabilityof orange isoperoxidases as individual puri_ed iso!peroxidases[

RESULTS AND DISCUSSION

The crude extracts obtained from the three cultivars"Large Valencia from Brazil*CLV\ Navel fromSpain*CN and Small Sweet from Spain*CSS# wereassayed by isoelectric focusing "IEF# and both anionicand cationic isoperoxidase were present in the solublefractions from juice and ~avedo[ The fraction fromthe albedo contained only anionic isoenzymes andthe fraction from the peel contained more isoenzymesthan the other fractions "Fig[ 0#[ Similar numbers ofperoxidase isoenzymes were found in the cultivarCLV\ CN\ CSS\ and the POD activity in the di}erentfractions of that cultivar decreased in the followingorder ~avedo soluble peroxidase "FSP#\ albedo sol!uble peroxidase "ASP#\ juice soluble peroxidase "JSP#[

E[ CLEMENTE29

Fig[ 0[ IEF patterns of orange extract obtained on polyacrylamide gels "stained for peroxidase activity with o!dianisidine#["CLV*Cultivar Large Valencia\ CN*Cultivar Navel and CSS*Cultivar Small Sweet#[ JSP*Juice solube peroxidase\JIP*Juice ionically bound peroxidase\ ASP*Albedo soluble peroxidase\ AIP*Albedo ionically bound peroxidase\ FSP*

~avedo soluble peroxidase and FIP*~avedo ionically bound peroxidase[

The enzymic activity in the crude extracts fordi}erent fractions of the three cultivars are shown inTable 0[

From the results shown in Table 0 it can be seenthat the greatest peroxidase activity was detected inthe peel fractions\ probably due to the number ofisoenzymes\ which is highest in that fraction[ The ripe!ness of the fruit can also be a factor for that\ oncethe peroxidase enzymes are involved in physiologicprocesses[ Also it has been suggested that the numberof peroxidase isoenzymes from the same kind of fruitmay di}er\ depending on ecological and environ!mental di}erences\ as well as di}erences in variety\and also di}erences in stage of maturity and detection

Table 0[ Peroxidase activity in crude extracts from three oranges cultivars

Cultivar

CLV CN CSS

Samples DOD Sd2 DOD Sd2 DOD Sd2

Juice soluble peroxidase "JSP# 0[16 9[91 9[77 9[92 9[36 9[91Juice ionically bound peroxidase "JIP# 9[00 9[90 9[09 9[90 9[14 9[92Albedo soluble peroxidase "ASP# 1[19 9[91 9[27 9[90 9[37 9[91Albedo ionically bound peroxidase "AIP# 9[25 9[91 9[09 9[91 9[09 9[91Flavedo soluble peroxidase "FSP# 09[19 9[90 6[59 9[99 3[69 9[90Flavedo ionically bound peroxidase "FIP# 7[39 9[99 1[07 9[90 0[42 9[99

"n 2#\ n number of measurements\ CLV*Cultivar Large Valencia\ CN*Cultivar Navel\ CSS*Cultivar SmallSweet\ Sd*standard deviation\ DOD*change in A "359 nm:min[ml#

techniques 08[ The relationship between the ripenessand peroxidase activity indicates the involvement ofperoxidase in post!harvest physiology\ which is inagreement with the reported studies on citrus fruits19[

Numerous studies have suggested that isoenzymesmay be artifacts which arise during the course of puri!_cation[ However\ in the present work peroxidase iso!enzymes from orange are unlikely to be artifacts\ asthese isoenzymes were identi_ed before puri_cationsteps using the Rotofor and ~at bed IEF\ and theirquantity did not seem to change during puri_cation[In order to investigate the e}ect of heat on individualorange peroxidases\ isoperoxidases were separated by

Puri_cation and thermostability of isoperoxidase from oranges 20

Fig[ 1[ Anionic!exchange chromatography of the fractions contained the cationic isoenzymes "C0 and C1# and anionicisoenzymes "A0 and A3# on FPLC "mono Q column#[

a series of chromatographic methods[ The fractionscollected after preparative isoelectric focusing "PIF#\were applied for gel _ltration on Sephacryl S!099 HRand then on ion!exchange chromatography on FPLC\resulting in a separation of distinct peaks "Fig[ 1#[The distribution of a number of isoperoxidases in thepeaks\ was determined by isoelectric focusing "IEF#[

The A1\ A2 and A6 were isolated by PIF and gel_ltration on Sephacryl S!099HR and Sephadex G!49were used to eliminate contaminants[ This step wascarried out after the _rst series of PIF and gel _ltrationseries[ In Fig[ 2 are shown the results of PIF for theisolated isoenzymes\ and in Fig[ 3 the protein silverstain for the same fractions[

The pI for the isoenzymes was measured using asurface electrode[ In addition\ a narrow strip offocused gel was cut into 4 mm pieces parallel with theelectrode strips[ Each piece was soaked in 1 ml ofwater for 1 h before pH of each result was measured[The Mr of the puri_ed isoenzymes was estimated fol!lowing the Pharmacia gel _ltration calibration kitinstruction manual by using gel!_ltration "SephacrylS!099 HR\ a crossed linked dextran#[ The eqn of thecalibration curve of the standards Mr used for thedetermination of Mrs of puri_ed isoenzymes wasY 1[23419[3422X\ where Y Kav elution par!ameter and X log "Mr#\ the elution parameter is"VeVo#:"VtVo#[ The equation above was ob!tained by regression analysis of the data "Table 1#[

The values obtained for Mrs of the anionic iso!enzymes were constant with the Mr of peroxidase fromour sources[ For example\ kiwifruit Mr 39k31 k 04\papaya fruit Mr 30 k43 k 10\ peanuts Mr 39 k31 k11\ and horseradish peroxidase C Mr 33 k 12[

Table 1[ pIs and Mrs of puri_ed orange ispperoxidases

Isoperoxidase pI Mr k

A0 "JSP# 3[4 33A1 "JSP# 4[9 11A2 "JSP# 4[2 29A3 "JSP# 4[5 15C0 "JSP# 8[9 15C1 "JSP# 7[9 32A0 "ASP# 3[4 33A0 "FSP# 3[4 33A3 "FSP# 4[5 15C0 "FSP# 8[9 13C1 "FSP# 7[9 39

A*anionic isoenzymes^ C*cationic isoenzymes

The Mrs of puri_ed orange cationic isoenzymesdetermined by the gel _ltration technique\ were foundto be similar to those enzymes in mango\ where thecationic isoperoxidase had a Mr value less than 29 k07[ However two anionic isoperoxidase identi_ed inthis work were shown to have Mr less 29 k which islow for peroxidase generally[

Inactivation plots for peroxidase activity present incrude soluble extracts from orange juice\ albedo andpeel are shown in Fig[ 4[ For all three extracts theloss of peroxidase activity was non!linear with heatingtime[ The non!linearity for heat inactivation in thecrude extracts\ may be due to the presence of a numberof isoperoxidases with di}ering thermostabilities[

The peroxidase activity and the number of iso!enzymes in the ~avedo fraction were higher than in

E[ CLEMENTE21

Fig[ 2[ Isoelectric focusing of the puri_ed oranges peroxidases obtained from the soluble fractions of the juice "JSP#\ albedo"ASP# and ~avedo "FSP#\ stained for peroxidase activity with o!dianisidine[

Fig[ 3[ Protein silver stain of puri_ed orange isoperoxidases[

the other fractions "JSP and ASP#\ and the higher heatstability of the soluble peroxidase from the ~avedofraction\ could be due to the presence of isoenzymeswith di}erent degrees of heat stability which do notappear in the other fractions[ In mango crude extractsthe initial rapid loss of enzymic activity may be dueto the inactivation of heat labile isoperoxidases\ andthe further smaller losses may be due to the presenceof more thermostable isoperoxidase 05[

The puri_ed isoperoxidases obtained from orangejuice "A0\ A3\ C0 and C1# lost about 69)\ 79) and04) of their original activity when exposed to 69> for

49 s "Figs 5 and 6# compared with a loss of about 74)when present as a mixture in the soluble crude extract[

The A0 "ASP# and A0 "FSP# "Fig[ 5# showed simi!larity with the A0 isoperoxidase obtained from thejuice fraction when heated at 69>[ After heating at 69>puri_ed C0 and C1 "obtained from the fraction JSPand FSP# isoperoxidase were shown to possess greaterthermostability "Fig[ 6#\ when compared with the pur!i_ed anionic isoenzymes[ This indicated that the cat!ionic isoenzymes were responsible for the higher PODstability[

The enzymic activity of puri_ed orange iso!

Puri_cation and thermostability of isoperoxidase from oranges 22

Fig[ 4[ Heat inactivation "at 69># for the crude extracts of orange peroxidase\ JSP*Juice soluble peroxidase\ ASP*Albedosoluble peroxidase and FSP*Flavedo soluble peroxidase "cultivar Large Valencia#[

Fig[ 5[ Isoenzyme activity ") of original# versus time in sec "at 69>#[

peroxidases did not regenerate substantially during a29 min period after heat treatment[ The isoperoxidases"A0\ A3\ C0 and C1# showed about 609) regain inenzymic activity at 29> when held for 39 min[

The b!secondary structure in protein is consideredthe most stable when there is a high content of hyd!roxy amino acids 13[ The higher heat stability ofthe puri_ed mango A0 isoenzyme was attributed to a

E[ CLEMENTE23

Fig[ 6[ Isoenzyme activity ") of original# versus time in sec "at 69>#[

greater proportion of hydroxy amino acids 05[ Thenon!linear inactivation of a single puri_ed isoenzymefrom oranges may happen because of mic!roheterogeneity due to the presence of variableamounts of covalently bound neutral carbohydratewhich will not a}ect the isoelectric point but mighta}ect the thermostability[ The possible e}ect of coval!ently bound carbohydrates on thermostability of per!oxidase has not yet been proven[

EXPERIMENTAL

Material

Fresh ripe oranges "Citrus sinenis "L[# Osbeck# cul!tivars Large Valencia\ Small Sweet and Navel werepurchased in a local supermarket[ Sephacryl S!099HR\ Sephadex G!64\ Ampholine Carrier Ampholyteand all other materials and equipment for analyticalisoelectric focusing and FPLC was used as suppliedby LKB Instruments Ltd[ Rotofor "preparative iso!electric focusing# and silver stain kit were used assupplied by BioRad[ o!Dianisidine was obtained fromKoch Light^ Bovine serum albumin was from Sigmaand Pentosanese from Novo Products[ All otherChemicals where available in Analar grade\ wereobtained from BDH[

Sample preparation

Orange fruit were washed in tap H1O and peeled[The juice sacs were carefully separated from thealbedo\ seeds and central placenta or core\ 499 g sam!ple of the juice sacs were then homogenized in 499 mlof 9[0 M Na!Pi bu}er at pH 5 using a liquidizer[ 0)"w:v# insoluble polyvinylpolypyrrolidone was addedto the extract bu}er to improve the stability of theenzyme by removing phenolic compounds 07[ Thehomogenized suspension was _ltered through a dou!ble layer of muslin cloth and the resultant _ltratewas centrifuged at 06\999 for 19 min at 3>[ Thesupernatant was collected and stored at 07> anddesignated juice soluble peroxidase "JSP#[ For extrac!tion of the soluble peroxidase from albedo and from~avedo\ _rst the albedo was carefully separated fromthe endocarp and ~avedo[ Then 09 g of albedo andalso 09 g of ~avedo were cut into small pieces\ andeach sample was homogenized for 0 min in 099 ml of9[0 M Na!Pi bu}er at pH 5 containing 0) "v:v# ofpentosanase\ using a Waring blender[ Then the sameprocess was used for _ltration and centrifugation toobtain the soluble fraction from the juice^ two frac!tions were obtained and were designated albedo sol!uble peroxidase "ASP# and ~avedo soluble peroxidase"FSP# respectively[ The preparation of the fractionswith ionically bound peroxidase forms\ was carriedout from each residue remaining after the extraction

Puri_cation and thermostability of isoperoxidase from oranges 24

of the soluble fractions[ The residue was washed twiceto remove any traces of soluble peroxidase activity\by resuspending in 099 ml of 9[0 M Na!Pi bu}er pH5[ Each suspension was centrifuged at 06\999 andthe supernatant was discarded[ Then the washed resi!due was resuspended in 099 ml 0 M NaCl in Tris:HClbu}er "pH 6[4#\ the suspension centrifuged at 06\999 for 19 min at 3> and the supernatant ~uids collectedand stored at 07>[ Those fractions were designated{juice ionically bound peroxidase| "JIP#\ {albedo ion!ically bound peroxidase| "AIP# and {~avedo ionicallybound peroxidase| "FIP#[

Analysis

Peroxidase activity was assayed with the orto!dianisidine method at pH 5 at 14> 14[ The reactionmixture contained 1[6 ml of 9[92) H1O1 in 9[0 M Na!Pi bu}er at pH 5 and 9[1 ml of the peroxidase extract[The enzymatic reaction was initiated by the additionof 9[0 ml\ 0) "w:v# o!dianisidine and the initial changein A was recorded at 359 nm at 14> using a Pye UnicamSP 7!199 UV:VIS spectrophotometer for a period of0 min[ Each sample was assayed in triplicate[

Preparative isoelectric focusin "PIF#

All extracts were dialysed overnight in deionisedH1O\ to remove the excess of small Mr compounds\with changes of deionised H1O each 1 h for a periodof 09 h\ prior to analysis in Rotofor[ PIF was carriedout using a Rotofor unit equipped with a BioRadMultitemp thermostatic circulator[ following that\ thepH gradient was measured in the 19 fractions orig!inated from Rotofor and then applied to gel _ltration[

Gel _ltration

The enzyme soln "09 ml# collected after gel _ltrationin Sephacryl S!099 HR "column 05 mm i[d[69 cm\eluent 9[0 M Na!Pi at pH 5\ ~ow rate 29 ml h0# wasdialysed against 01[4 mM Tris:HCl bu}er "pH 6[4#overnight and then applied to a FPLC!LCC499"Mono!Q column 4:4#[ Fractions were eluted withbu}er "9[914 M Tris:HCl bu}er pH 6[4# up to 9[4 MNaCl[ Isoelectric focusing "IEF# was carried out usingan Ultrophor Electrofocusing unit equipped with anLKB Multi!Temp thermostatic circulator[ The pHgradient in the gel was measured by surface electrode[The focused gels were stained for peroxidase activitywith o!dianisidine 15[

Heat inactivation studies

Micro tubes "0 cm length and 0 mm inside diam[#were _lled with 49 ml of POD samples using a syringe[The tubes were sealed at both ends\ and plunged in awater bath at 69> for a period of 09\ 19\ 29\ 39\ 59\ 79and 019 s[ Once the required heating time was reachedthe micro tubes were immediately transferred in trip!

licate to a water bath at 9> and the activity of eachsample was then measured[

Total protein content of the crude and puri_edextracts was estimated by dye binding 16[

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