\

Phytochemistry\ Vol[ 38\ No[ 0\ pp[ 1825\ 0887

0887 Elsevier Science Ltd[ All rights reserved
Printed in Great Britain
99208311:87:,see front matter

Pergamon
PII] S99208311"86#998568

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[ 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 07[ 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 0\ 1[
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 2[ Peroxidase can con!
tribute to deteriorating changes in ~avour\ texture\
colour and nutrition in improperly processed fruits
and vegetables 3[ In orange\ the level of peroxidase
in the juice is associated with loss of ~avour quality
4[ 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
5\ 6[ Renaturation of peroxidase following heat inac!
tivation has also been reported for some vegetable and
fruit extracts\ e[g[ kohlrabi 7\ Brussels sprouts and
cabbage 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

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

29

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 08[ 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
19[
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[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 from
our sources[ For example\ kiwifruit Mr 39k31 k 04\
papaya fruit Mr 30 k43 k 10\ peanuts Mr 39 k31 k
11\ and horseradish peroxidase C Mr 33 k 12[

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
07[ 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

21

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 05[
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 609) 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 13[ The higher heat stability of
the puri_ed mango A0 isoenzyme was attributed to a

23

E[ CLEMENTE

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

greater proportion of hydroxy amino acids 05[ 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 07[ 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> 14[ 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 h0# 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 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 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 16[

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