Purification and thermostability of isoperoxidase from oranges

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Phytochemistry\ Vol[ 38\ No[ 0\ pp[ 18Ð25\ 0887 Þ 0887 Elsevier Science Ltd[ All rights reserved Printed in Great Britain 9920Ð8311:87:,Ðsee front matter

Pergamon PII] S9920Ð8311"86#99856Ð8

PURIFICATION AND THERMOSTABILITY OF ISOPEROXIDASE FROM 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[# Osbeck cultivar Large Valencia\ Small Sweet and Navel[ Cationic and anionic isoperoxidase were obtained by Rotofor and ion!exchange chromatography[ The pI for the soluble isoenzymes "3[4 to 8[9# was measured using a surface electrode 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 peroxidase activity in a mixture or individually in a puri_ed state\ was found to be non!linear with heating time[ Þ 0887 Elsevier Science Ltd[ All rights reserved

activity in plant materials is generally found to be a non!linear process against heating time\ which is thought to be due to the presence of separate iso! peroxidases\ with di}erent heat stabilities ð07Ł[ In plant extracts\ peroxidase activity has been found in both soluble and bound states which di}er with respect to heat stability and regeneration properties[ Commercial fruit juice production includes some albedo and peel of the fruit in the juice\ which will make a large contribution to the total peroxidase activity[ In the present research\ we report a study of the consideration of the heat stability of orange juice\ albedo and peel crude extracts and thermal stability of orange isoperoxidases as individual puri_ed iso! peroxidases[

INTRODUCTION

Peroxidase "EC 0[00[0[6# are part of a large group of enzymes known collectively as oxidoreductases ð0\ 1Ł[ Extensive studies describe the action of peroxidase on substances which yield bright colours on oxidation\ but peroxidase can promote a large variety of reac! tions and therefore can exhibit a degree of versatility unsurpassed by any other enzyme[ The quality of extracted citrus juices depends on enzymic reactions that occur not only in the fruit during the development period\ but also in the juice during processing[ The formation of o}!~avours in canned fruit and veg! etables has been associated with residual peroxidase activity following processing ð2Ł[ Peroxidase can con! tribute to deteriorating changes in ~avour\ texture\ colour and nutrition in improperly processed fruits and vegetables ð3Ł[ In orange\ the level of peroxidase in the juice is associated with loss of ~avour quality ð4Ł[ The heat inactivation of peroxidase is non!linear\ a large decrease in activity is observed during the initial stages of a given heating process\ but the rate of inactivation then changes to a much slower process ð5\ 6Ł[ Renaturation of peroxidase following heat inac! tivation has also been reported for some vegetable and fruit extracts\ e[g[ kohlrabi ð7Ł\ Brussels sprouts and cabbage ð8Ł\ grapes ð09\ 00Ł\ avocados ð01Ł\ apples ð02Ł\ pears ð03Ł\ kiwifruit ð04Ł\ mango ð05Ł[ 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 ð06Ł[ Inactivation of peroxidase

RESULTS AND DISCUSSION

The crude extracts obtained from the three cultivars "Large Valencia from Brazil*CLV\ Navel from Spain*CN and Small Sweet from Spain*CSS# were assayed by isoelectric focusing "IEF# and both anionic and cationic isoperoxidase were present in the soluble fractions from juice and ~avedo[ The fraction from the albedo contained only anionic isoenzymes and the fraction from the peel contained more isoenzymes than the other fractions "Fig[ 0#[ Similar numbers of peroxidase isoenzymes were found in the cultivar CLV\ CN\ CSS\ and the POD activity in the di}erent fractions of that cultivar decreased in the following order ~avedo soluble peroxidase "FSP#\ albedo sol! uble peroxidase "ASP#\ juice soluble peroxidase "JSP#[ 18

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E[ CLEMENTE

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 for di}erent fractions of the three cultivars are shown in Table 0[ From the results shown in Table 0 it can be seen that the greatest peroxidase activity was detected in the peel fractions\ probably due to the number of isoenzymes\ which is highest in that fraction[ The ripe! ness of the fruit can also be a factor for that\ once the peroxidase enzymes are involved in physiologic processes[ Also it has been suggested that the number of peroxidase isoenzymes from the same kind of fruit may 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

techniques ð08Ł[ The relationship between the ripeness and peroxidase activity indicates the involvement of peroxidase in post!harvest physiology\ which is in agreement with the reported studies on citrus fruits ð19Ł[ Numerous studies have suggested that isoenzymes may 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\ as these isoenzymes were identi_ed before puri_cation steps using the Rotofor and ~at bed IEF\ and their quantity did not seem to change during puri_cation[ In order to investigate the e}ect of heat on individual orange peroxidases\ isoperoxidases were separated by

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# Juice ionically bound peroxidase "JIP# Albedo soluble peroxidase "ASP# Albedo ionically bound peroxidase "AIP# Flavedo soluble peroxidase "FSP# Flavedo ionically bound peroxidase "FIP#

0[16 9[00 1[19 9[25 09[19 7[39

9[91 9[90 9[91 9[91 9[90 9[99

9[77 9[09 9[27 9[09 6[59 1[07

9[92 9[90 9[90 9[91 9[99 9[90

9[36 9[14 9[37 9[09 3[69 0[42

9[91 9[92 9[91 9[91 9[90 9[99

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

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 anionic isoenzymes "A0 and A3# on FPLC "mono Q column#[

a series of chromatographic methods[ The fractions collected after preparative isoelectric focusing "PIF#\ were applied for gel _ltration on Sephacryl S!099 HR and then on ion!exchange chromatography on FPLC\ resulting in a separation of distinct peaks "Fig[ 1#[ The distribution of a number of isoperoxidases in the peaks\ 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!49 were used to eliminate contaminants[ This step was carried out after the _rst series of PIF and gel _ltration series[ In Fig[ 2 are shown the results of PIF for the isolated isoenzymes\ and in Fig[ 3 the protein silver stain for the same fractions[ The pI for the isoenzymes was measured using a surface electrode[ In addition\ a narrow strip of focused gel was cut into 4 mm pieces parallel with the electrode strips[ Each piece was soaked in 1 ml of water 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 kit instruction manual by using gel!_ltration "Sephacryl S!099 HR\ a crossed linked dextran#[ The eqn of the calibration curve of the standards Mr used for the determination of Mrs of puri_ed isoenzymes was Y  1[2341Ð9[3422X\ where Y  Kav elution par! ameter and X  log "Mr#\ the elution parameter is "Ve−Vo#:"Vt−Vo#[ 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 from our sources[ For example\ kiwifruit Mr 39kÐ31 k ð04Ł\ papaya fruit Mr 30 kÐ43 k ð10Ł\ peanuts Mr 39 kÐ31 k ð11Ł\ and horseradish peroxidase C Mr 33 k ð12Ł[

Table 1[ pIs and Mrs of puri_ed orange ispperoxidases Isoperoxidase

pI

Mr k

A0 "JSP# A1 "JSP# A2 "JSP# A3 "JSP# C0 "JSP# C1 "JSP# A0 "ASP# A0 "FSP# A3 "FSP# C0 "FSP# C1 "FSP#

3[4 4[9 4[2 4[5 8[9 7[9 3[4 3[4 4[5 8[9 7[9

33 11 29 15 15 32 33 33 15 13 39

A*anionic isoenzymes^ C*cationic isoenzymes

The Mrs of puri_ed orange cationic isoenzymes determined by the gel _ltration technique\ were found to be similar to those enzymes in mango\ where the cationic isoperoxidase had a Mr value less than 29 k ð07Ł[ However two anionic isoperoxidase identi_ed in this work were shown to have Mr less 29 k which is low for peroxidase generally[ Inactivation plots for peroxidase activity present in crude soluble extracts from orange juice\ albedo and peel are shown in Fig[ 4[ For all three extracts the loss of peroxidase activity was non!linear with heating time[ The non!linearity for heat inactivation in the crude extracts\ may be due to the presence of a number of isoperoxidases with di}ering thermostabilities[ The peroxidase activity and the number of iso! enzymes in the ~avedo fraction were higher than in

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E[ CLEMENTE

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 heat stability of the soluble peroxidase from the ~avedo fraction\ could be due to the presence of isoenzymes with di}erent degrees of heat stability which do not appear in the other fractions[ In mango crude extracts the initial rapid loss of enzymic activity may be due to the inactivation of heat labile isoperoxidases\ and the further smaller losses may be due to the presence of more thermostable isoperoxidase ð05Ł[ The puri_ed isoperoxidases obtained from orange juice "A0\ A3\ C0 and C1# lost about 69)\ 79) and 04) 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 the juice fraction when heated at 69>[ After heating at 69> puri_ed C0 and C1 "obtained from the fraction JSP and FSP# isoperoxidase were shown to possess greater thermostability "Fig[ 6#\ when compared with the pur! i_ed anionic isoenzymes[ This indicated that the cat! ionic isoenzymes were responsible for the higher POD stability[ 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*Albedo soluble 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 a 29 min period after heat treatment[ The isoperoxidases "A0\ A3\ C0 and C1# showed about 6Ð09) regain in enzymic activity at 29> when held for 39 min[

The b!secondary structure in protein is considered the most stable when there is a high content of hyd! roxy amino acids ð13Ł[ The higher heat stability of the puri_ed mango A0 isoenzyme was attributed to a

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E[ CLEMENTE

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

greater proportion of hydroxy amino acids ð05Ł[ The non!linear inactivation of a single puri_ed isoenzyme from oranges may happen because of mic! roheterogeneity due to the presence of variable amounts of covalently bound neutral carbohydrate which will not a}ect the isoelectric point but might a}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 were purchased in a local supermarket[ Sephacryl S!099 HR\ Sephadex G!64\ Ampholine Carrier Ampholyte and all other materials and equipment for analytical isoelectric focusing and FPLC was used as supplied by LKB Instruments Ltd[ Rotofor "preparative iso! electric focusing# and silver stain kit were used as supplied by BioRad[ o!Dianisidine was obtained from Koch Light^ Bovine serum albumin was from Sigma and Pentosanese from Novo Products[ All other Chemicals where available in Analar grade\ were obtained from BDH[

Sample preparation Orange fruit were washed in tap H1O and peeled[ The juice sacs were carefully separated from the albedo\ seeds and central placenta or core\ 499 g sam! ple of the juice sacs were then homogenized in 499 ml of 9[0 M Na!Pi bu}er at pH 5 using a liquidizer[ 0) "w:v# insoluble polyvinylpolypyrrolidone was added to the extract bu}er to improve the stability of the enzyme by removing phenolic compounds ð07Ł[ The homogenized suspension was _ltered through a dou! ble layer of muslin cloth and the resultant _ltrate was centrifuged at 06\999 ` for 19 min at 3>[ The supernatant was collected and stored at −07> and designated juice soluble peroxidase "JSP#[ For extrac! tion of the soluble peroxidase from albedo and from ~avedo\ _rst the albedo was carefully separated from the endocarp and ~avedo[ Then 09 g of albedo and also 09 g of ~avedo were cut into small pieces\ and each sample was homogenized for 0 min in 099 ml of 9[0 M Na!Pi bu}er at pH 5 containing 0) "v:v# of pentosanase\ using a Waring blender[ Then the same process was used for _ltration and centrifugation to obtain 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 fractions with ionically bound peroxidase forms\ was carried out from each residue remaining after the extraction

Puri_cation and thermostability of isoperoxidase from oranges

of the soluble fractions[ The residue was washed twice to remove any traces of soluble peroxidase activity\ by resuspending in 099 ml of 9[0 M Na!Pi bu}er pH 5[ Each suspension was centrifuged at 06\999 ` and the supernatant was discarded[ Then the washed resi! due was resuspended in 099 ml 0 M NaCl in Tris:HCl bu}er "pH 6[4#\ the suspension centrifuged at 06\999 ` for 19 min at 3> and the supernatant ~uids collected and stored at −07>[ Those fractions were designated {juice ionically bound peroxidase| "JIP#\ {albedo ion! ically bound peroxidase| "AIP# and {~avedo ionically bound peroxidase| "FIP#[ Analysis Peroxidase activity was assayed with the orto! dianisidine method at pH 5 at 14> ð14Ł[ The reaction mixture 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 addition of 9[0 ml\ 0) "w:v# o!dianisidine and the initial change in A was recorded at 359 nm at 14> using a Pye Unicam SP 7!199 UV:VIS spectrophotometer for a period of 0 min[ Each sample was assayed in triplicate[ Preparative isoelectric focusin` "PIF# All extracts were dialysed overnight in deionised H1O\ to remove the excess of small Mr compounds\ with changes of deionised H1O each 1 h for a period of 09 h\ prior to analysis in Rotofor[ PIF was carried out using a Rotofor unit equipped with a BioRad Multitemp thermostatic circulator[ following that\ the pH 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 _ltration in Sephacryl S!099 HR "column 05 mm i[d[×69 cm\ eluent 9[0 M Na!Pi at pH 5\ ~ow rate 29 ml h−0# was dialysed 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 with bu}er "9[914 M Tris:HCl bu}er pH 6[4# up to 9[4 M NaCl[ Isoelectric focusing "IEF# was carried out using an Ultrophor Electrofocusing unit equipped with an LKB Multi!Temp thermostatic circulator[ The pH gradient in the gel was measured by surface electrode[ The focused gels were stained for peroxidase activity with o!dianisidine ð15Ł[ 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 a water bath at 69> for a period of 09\ 19\ 29\ 39\ 59\ 79 and 019 s[ Once the required heating time was reached the micro tubes were immediately transferred in trip!

24

licate to a water bath at 9> and the activity of each sample was then measured[ Total protein content of the crude and puri_ed extracts was estimated by dye binding ð16Ł[

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