Purification and properties of a β-galactosidase from carambola fruit with significant activity towards cell wall polysaccharides

Article abstract of DOI:10.1016/j.phytochem.2004.11.005

β-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. β-galactosidase I, II, III and IV. This β-galactosid

Full text of DOI:10.1016/j.phytochem.2004.11.005

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 153–163
www.elsevier.com/locate/phytochem
Purification and properties of a b-galactosidase from carambola
fruit with significant activity towards cell wall polysaccharides
Sumathi Balasubramaniam, Heng Chin Lee, Hamid Lazan, Roohaida Othman,
*
Zainon Mohd. Ali
Faculty of Science and Technology, School of BioSciences and Biotechnology, Universiti Kebangsaan Malaysia, 43000 Bangi, Selangor, Malaysia
Received 1 July 2004; received in revised form 9 November 2004
Available online 19 December 2004
Abstract
b-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combina-
tion of ion exchange and gel filtration chromatography into four isoforms, viz. b-galactosidase I, II, III and IV. This b-galactosid-
ases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. b-Galactosidase I, the predominant isoform, was
purified to electrophoretic homogeneity; analysis of the protein by SDS–PAGE revealed two subunits with molecular masses of 48
and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with
the deduced amino acid sequence of certain plant b-galactosidases, thus, explaining the observed low similarity between the two
subunits. b-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the
potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified b-galactosidase I was substantially
active in hydrolyzing (1 ! 4)b-linked spruce and a mixture of (1 ! 3)b- and (1 ! 6)b-linked gum arabic galactans. This isoform
also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses,
reflecting in part changes that occur during ripening.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Averrhoa carambola L.; Oxalidaceae; Fruit; b-Galactosidase; Enzyme purification and properties; Cell wall modification
1. Introduction
1998) and carambola (Chin et al., 1999). Apart from
pectic polysaccharides, modification of hemicelluloses
may also contribute to softening-related wall disassem-
bly during ripening (Knee, 1973; Huber, 1983; McCol-
lum et al., 1989; Ali et al., 1998; Rose et al., 1998;
Chin et al., 1999).
Evidence indicates that polygalacturonase (PG) may
not be the key enzyme involved in cell wall disassembly
even in tomato fruits that are known to contain high lev-
els of endo-PG activities (Smith et al., 1988; Giovannoni
et al., 1992). In tomato as well as in many other fruits,
substantial pectin modifications can proceed in the
apparent absence of any significant endo-PG activities
(McCollum et al., 1989; Giovannoni et al., 1992; Cheng
and Huber, 1996). Furthermore, in tropical fruits
Firmness loss during fruit ripening is contributed
mainly by the action of a variety of cell wall modifying
enzymes and proteins (Rose and Bennett, 1999;
Cosgrove, 2001). Amongst the wall polysaccharides,
pectin modification as characterized by increased solu-
bility and depolymerization have been widely studied
in fruits such as tomato (Huber, 1983), muskmelon
(McCollum et al., 1989), kiwifruit (Redgwell et al.,
1992), melon (Rose et al., 1998), papaya (Ali et al.,
*
Corresponding author. Tel.: +60 389 21 5995; fax: +60 389 25
2698.
E-mail address: zainon@pkrisc.cc.ukm.my (Z.Mohd. Ali).
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.11.005

154
S. Balasubramaniam et al. / Phytochemistry 66 (2005) 153–163
including carambola that differ markedly in their firm-
ness loss rates, the polygalacturonase activities were
attributed predominantly to exo-PG (Ali et al., 2004a).
Thus, PG alone might not be pivotal to wall modifica-
tions and fruit softening. Other wall modifying enzymes,
notably pectin methylesterase, endo-(1 ! 4)b-glucanase,
xyloglucan endotransglucosylase/hydrolase and b-galac-
tosidase/galactanase, might be involved in affecting an
overall wall disassembly and firmness loss during ripen-
ing (Rose and Bennett, 1999; Ali et al., 2004a).
fects of using ammonium sulphate precipitation as the
first purification step were carried out before attempting
further purification protocols. It was found that a 2:1 ra-
tio being fresh tissue weight (ꢀ1 kg) to extraction buffer
volume was suitable and able to retain almost 95% of
b-galactosidase activity in the supernatant as well as
yielding a total workable extraction volume of about
1.5 l. Increasing the ratio to 3:1 in order to decrease
the extraction volume, however, reduced the extractabil-
ity of the enzyme in the supernatant to only 77%.
Ammonium sulphate precipitation of the crude extract
followed by dialysis was attempted as an initial purifica-
tion step to remove other protein as well as to reduce
sample volume for subsequent chromatographic proce-
dures. Almost 90% of the b-galactosidase activity was
loss at this step. As an alternative, the crude extract
was instead dialysed extensively. Dialysis appeared to
retain about 75% of the total b-galactosidase activity
in the crude extract. Significant loss of activity at this
stage might be due to instability of the crude enzyme
caused by long standing during dialysis. This step was
however essential for sample to be fractionated using
ion exchange chromatography.
Typical results for separation and purification of
b-galactosidase isoforms are presented in Fig. 1 and
summarized in Table 1. Supernatant extracted from
1.2 kg of ripe carambola tissues comprising 10.8 lkat
of total b-galactosidase was dialysed and then purified
through a series of cation exchange (CM Sepharose fast
flow), anion exchange (DEAE-Sepharose fast flow) and
gel filtration (Sephacryl S-200) chromatography. Four
isoforms of b-galactosidase were resolved. Fractionation
of crude extract from ripe fruits on a CM-Sepharose col-
umn at pH 5.0 (Fig. 1(a)) was able to remove significant
amount of unbound protein, as well as to separate two
peaks of b-galactosidase activity (peaks A and B) bound
to the column, with stepwise elution using 0.05 and 0.5
M NaCl. One of the peak (peak A) was resolved into
two activity peaks – the unbound b-galactosidase I
(peak AI) and bound b-galactosidase II (peak AII) –
when passed through a DEAE-Sepharose column at
pH 7.2 (Fig. 1(b)). The second peak (peak B) was also
resolved into two activity peaks, comprising the un-
bound b-galactosidase III (peak BI) and bound b-galac-
tosidase IV (peak BII) on the same anion exchange
column (Fig. 1(c)). Each of the b-galactosidase I and
b-galactosidase III peak was fractionated further by
gel filtration chromatography to remove contaminating
proteins, and both were individually eluted as a single
peak through a Sephacryl S-200 HR column (Fig. 1(d)
and (e)). In all cases, although significant amount of
other proteins were successfully separated from the
b-galactosidase isoforms, only the b-galactosidase I
peak was particularly enriched in specific activity (Table
1 and Fig. 1). This predominant isoform was purified up
to 95-fold (specific activity 5.5 lkat mgꢁ1) with a recov-
Ripening associated b-galactosidases have been puri-
fied and partially characterized from fruits such as to-
mato (Pressey, 1983; Carey et al., 1995), apple (Dick
et al., 1990; Ross et al., 1994), muskmelon (Ranwala
et al., 1992), avocado (deVeau et al., 1993), kiwifruit
(Ross et al., 1993), persimmon (Kang et al., 1994), man-
go (Ali et al., 1995), Japanese pear (Kitagawa et al.,
1995) and papaya (Ali et al., 1998). b-Galactosidases
from these fruit sources occur in multi-forms, seemingly
encoded by multi-gene families; however, not all mem-
bers of the gene family were ripening related (Smith
and Gross, 2000; Trainotti et al., 2001). Evidence indi-
cates that some of the major b-galactosidase isoforms
are capable to modify pectins that were either in al-
ready-soluble forms (Ranwala et al., 1992; deVeau et
al., 1993) or in still structurally attached forms (Carring-
ton and Pressey, 1996), apart from ability to also modify
alkali-soluble hemicelluloses (Ali et al., 1998; Soh,
2002). The mechanism by which b-galactosidase affects
modification of both pectin and hemicellulose is unclear.
In carambola (Averrhoa carambola L., family Oxalid-
aceae) – a non-climacteric (OÕHare, 1997) and slow-soft-
ening economically important tropical fruit (Ali et al.,
2004a) – ripening related pectin and hemicellulose mod-
ifications were accompanied by increased activity of a
number of wall degrading enzymes including pectin
methylesterase and b-galactosidase (Chin et al., 1999;
Ali et al., 2004a). Moreover, accelerated firmness loss
during subsequent ripening of chilled-injured carambola
at ambient temperature correlated positively with an en-
hanced increase in activity of both enzymes (Ali et al.,
2004b). Thus, it seems, pectin methylesterase and
b-galactosidase might contribute significantly to wall
modifications during ripening of the fruit. In the present
paper, we report the purification and characterization of
a b-galactosidase isoform from ripe carambola fruit that
appeared to have the capability to markedly modify
both pectins and hemicelluloses.
2. Results and discussion
2.1. Purification of b-galactosidase
Initial studies to determine the appropriate amount
of extraction buffer to fresh tissue ratio as well as the ef-

S. Balasubramaniam et al. / Phytochemistry 66 (2005) 153–163
155
Fig. 1. Elution profile of carambola b-galactosidase activity (d) and absorbance at 280 nm (—) on (a) CM-Sepharose fast flow, (b) DEAE-Sepharose
fast flow for peak A, (c) DEAE-Sepharose fast flow for peak B, (d) Sephacryl S-200 for peak AI (b-galactosidase I) and (e) Sephacryl S-200 for peak
BI (b-galactosidase III). A fraction (6.2 ml) containing highest specific activity and constituting part of the activity peak of b-galactosidase I as shown
in (d), was used in the in vitro wall modification studies.
ery of 12% from the extractable activity. In contrast, iso-
forms II, III and IV were only purified up to 0.9-, 1.90-
and 0.06-fold, respectively (Table 1).
Analysis on native polyacrylamide gels showed that
b-galactosidase I migrated as a single protein band, sug-
gesting that it has been purified to homogeneity (Fig.
2(a)). Activities of the potentially significant wall
degrading enzymes, polygalacturonase and pectin
methylesterase, assayed by the methods described in
Lazan et al. (1995), were not detectable in the purified
b-galactosidase I fractions. The other isoforms were only
partially purified as a number of protein bands were de-
tected on the gel (data not shown). Further purification
could not be carried out for these isoforms due to the
low levels of protein as well as activity in the fractions.
2.2. Properties of b-galactosidase
The apparent native molecular sizes of b-galactosi-
dase I, II, III and IV estimated by chromatography on

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S. Balasubramaniam et al. / Phytochemistry 66 (2005) 153–163
Table 1
Purification of b-galactosidase from ripe carambola
Purification step
Total protein (mg)
Total activity (nkat)
Specific activity (nkat mgꢁ1
)
Recovery (%)
Purification (fold)
Crude extract
Dialysis
186
173
10813
7969
58
46
100
74
1.0
0.8
CM-Sepharose
Peak A
Peak B
26
36
6287
1494
242
42
58
14
4.2
0.7
DEAE-Sepharose
Peak AI (b-Gal I)
Peak AII (b-Gal II)
Peak BI (b-Gal III)
Peak BII (b-Gal IV)
3.6
3.5
3.7
8.3
5171
175
243
27
1436
50
48
25
1.6
2.3
0.3
0.9
1.1
0.06
66
3.3
Sephacryl S-200
b-Gal I
b-Gal III
0.24
0.82
1328
91
5533
111
12
95
0.8
1.9
Fig. 2. Fractionation on native (a) and SDS–PAGE (b), and isoelectric focusing (c) of purified b-galactosidase I from ripe carambola fruit, and
concanavalin A Sepharose profile of purified b-galactosidase I activity (d) and absorbance (—) at 280 nm (d).
a Sephadex G-200 column were 84, 77, 58 and 130 kDa,
respectively. These values were generally comparable
with the apparent molecular sizes of b-galactosidase
from other fruits such as tomato, 62 kDa (Pressey,
1983), kiwifruit, 60 kDa (Ross et al., 1993), apple, 59
kDa (Ross et al., 1994), pepper, 51 kDa (Biles et al.,

S. Balasubramaniam et al. / Phytochemistry 66 (2005) 153–163
157
1997) and papaya, 55 and 67 kDa (Ali et al., 1998),
whereas the native molecular mass of b-galactosidase
IV was comparable with the estimated 118 kDa-size of
persimmon fruit b-galactosidase (Kang et al., 1994).
Purified b-galactosidase I as analysed by SDS–
PAGE, appeared to comprise two polypeptides with
the size of 48 and 36 kDa, suggesting that the native pro-
tein is an aggregate of two non-identical subunits (Fig.
2(b)). Presence of two non-identical subunits of b-galac-
tosidase was also observed in persimmon fruit (Kang et
al., 1994) and mung bean seedlings (Li et al., 2001),
whilst the existence of several polypeptides of b-galacto-
sidase – some of the enzymes appeared to be dimers –
has been shown in other fruits such as apples, tomato,
Japanese pear and papaya (Ross et al., 1994; Carey
et al., 1995; Kitagawa et al., 1995; Ali et al., 1998). Car-
ambola b-galactosidase I was shown to have a glycopro-
tein property as it was bound to an affinity concanavalin
A Sepharose column and can be eluted with 0.1 M glu-
cose (Fig. 2(d)). The possibility that some b-galactosi-
dase proteins might be glycosylated had been reported
in mango and papaya as well as in mung bean seedling
(Ali et al., 1995, 1998; Li et al., 2001).
As determined by isoelectric focusing, native b-galac-
tosidase I was shown to have a pI of 7.2 (Fig. 2(c)). pI
values in the neutral or basic pH range had also been ob-
served in b-galactosidase from other fruits such as kiwi-
fruit, tomatoes, red pepper and papaya (Ross et al.,
1993; Carey et al., 1995; Biles et al., 1997; Ng, 1998),
suggesting that certain b-galactosidase isoforms may
bind ionically to the acidic cell wall. A number of b-
galactosidase isoforms, however, appeared to have pI
values in the acidic ranges such as those from avocado,
mature green pepper and papaya (deVeau et al., 1993;
Biles et al., 1997; Ng, 1998).
Analysis of the first 30 N-terminal amino acid se-
quence of the two subunits of b-galactosidase I was pre-
sented in Fig. 3. BLAST searches of the 48-kDa
polypeptide indicated that its N-terminal amino acid se-
quence shared high similarities with the deduced amino
acid sequence of many of the published plant b-galacto-
sidases, such as those of strawberry (Accession No.
AJ278704, 82%), Arabidopsis thaliana (AJ270304,
80%), tomato (AF154423, 80%), mango (AF004812,
80%), papaya (AF064786, 80%), sand pear (AB046543,
80%), chick pea (AJ011010, 76%) and hot chilli
(AY029226, 76%) fruits (Fig. 3(a)). The 36-kDa poly-
peptide showed significant similarity only with a limited
number of b-galactosidases, particularly that of the
putative strawberry (AJ278704, 85%) protein (Fig.
3(b)). The N-terminal amino acid sequence of the two
subunits, however, differed significantly, exhibiting
about 20% similarity (Fig. 3(c)). This low degree of sim-
ilarity between the component polypeptides of b-galac-
tosidase I was ascribed to the fact that significant
homologies between the 48-kDa subunit and that of
the known b-galactosidases occurred near the N-termi-
nus, whereas for the 36-kDa subunit, homologies oc-
curred near the middle of the proteins sequence (Fig.
3(a) and (b)). It is unclear as whether the polypeptides
that constitute the functional b-galactosidase I protein
are encoded by different genes or as evidence of high se-
quence similarity with the strawberry protein seems to
suggest, by a single gene (Fig. 3(a) and (b)). Overall,
our results imply that the putative products of the
carambola b-galactosidase I gene/genes may have to un-
dergo certain modifications either at the post-transcrip-
tional or post-translational stages, as had been reported
for persimmon b-galactosidase (Kang et al., 1994). Plant
b-galactosidases may contain more than one glycosyla-
tion domains (Trainotti et al., 2001), and in the case of
the b-galactosidase I, one of the potential N-glycosyla-
tion sites seemed to reside within the larger-size subunit
(Fig. 3). This result corroborates the evidence that b-
galactosidase I was possibly a glycoprotein (Fig. 2(d)).
The apparent Km values for b-galactosidase I, II, III
and IV against p-nitrophenyl b-D-galactopyranoside
(PNPG) estimated from Lineweaver–Burk plots were
21.7, 4.2, 3.3 and 2.0 mM, respectively. The Vmax values
for the respective isoforms were 5.7, 31.6, 33.6 and 5.5
nkat mgꢁ1 protein. All isoforms lost 50% of its enzy-
matic activity at 65 ꢁC, comparable with the results re-
ported for tomato (Pressey, 1983), mango (Ali et al.,
1995), and papaya (Ali et al., 1998). Isoforms of b-galac-
tosidase were optimally active at acidic pH ranging from
pH 3.0–4.0. b-Galactosidases isolated from muskmelon,
kiwifruit and papaya also seemed to be optimally active
at acidic pH range (Ranwala et al., 1992; Ross et al.,
1993; Ali et al., 1998).
Specificity studies of b-galactosidase isoforms to-
wards some of the synthetic and endogenous substrates
showed that the purified b-galactosidase I, and the semi-
purified b-galactosidase II and III had no a-galactosi-
dase, a-arabinosidase, b-glucosidase and a-mannosidase
activity, and none of the four isoforms was able to de-
grade birchwood xylan. b-Galactosidase I was found
to be substantially active in hydrolyzing the endogenous
wall polymers (1 ! 4)b-linked spruce galactan at a rate
twice as rapid as that against the (1 ! 3)b- and
(1 ! 6)b-linked gum arabic galactan. To a limited ex-
tent, the enzyme also was capable to degrade arabinoga-
lactan. Purified b-galactosidases from ripe mango and
papaya fruits were also capable to hydrolyse both spruce
and gum arabic galactans (Ali et al., 1995, 1998). How-
ever, unlike the isoform I of carambola, all the three
b-galactosidases of papaya showed significantly higher
activity against galactan with the (1 ! 3)b- and
(1 ! 6)b-linkages than with the (1 ! 4)b-linkages (Ali
et al., 1998). Since galactans and arabinogalactans might
exist as rhamnogalacturonan 1 (RG1) side-chains or as
glycan cross-links that inter-connect RG1 and xyloglu-
can-cellulose microfibrils networks (Keegstra et al.,

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S. Balasubramaniam et al. / Phytochemistry 66 (2005) 153–163
Fig. 3. Alignment of the N-terminal amino acid sequence of the (a) 48-kDa and (b) 36-kDa subunits of carambola b-galactosidase I with the deduced
amino acid sequence of selected plant b-galactosidases, and (c) sequence alignment of the two subunits of b-galactosidase I. Accession numbers for
the selected strawberry CAC44501, Arabidopsis thaliana CAB64744 and AAD21482, and tomato AAF70824 b-galactosidases are AJ278704,
AJ270304, A006587 and AF154423, respectively. Identical amino acid residues and conservative substitutions are shaded as dark and grey areas,
respectively. Putative Asn glycosylation site (N–X–S) that reside in the 48-kDa-polypeptide sequence is indicated in (c).
1973; Carpita and McCaan, 2000), thus, functional
capacity as b-galactanases probably suggests that the
carambola enzyme have the potential to modify the cell
wall.
(CSP) and carbohydrates (CSC), were increasingly solu-
bilized (Table 2) and depolymerized (Fig. 4(a) and (b)).
Likewise, alkaline-soluble hemicelluloses were depoly-
merized with ripening (Fig. 4(c)). Results of incubating
the purified b-galactosidase I with unripe fruitÕs CW
preparation that contains pectins that are still structur-
ally attached to the wall network or with the hemicellu-
lose fractions extractable in alkali showed that the
isoform was capable of solubilizing (Table 2) as well
as downsizing the pectins (4d and 4e) and the hemicellu-
loses (Fig. 4(f)). The extent of the modifications ap-
peared comparable with the changes that occur in situ
during normal ripening, thus, suggesting the potential
significance of this isoform in softening-related wall dis-
2.3. Significance of b-galactosidase I to carambola
fruit softening
In order to evaluate the functional significance of b-
galactosidase I as a wall-modifying enzyme, in vitro wall
modification studies were carried out as a comparison to
changes that occur during ripening. As ripening pro-
gressed and fruit firmness decreased, pectins as reflected
by changes in the levels of chelator-soluble polyuronides
Table 2
Changes in tissue firmness (Newton) and levels of chelator-soluble polyuronides (CSP) and carbohydrates (CSC) in ripening carambola fruit and in
vitro pectin solubilisation from unripe fruitÕs cell walls by purified b-galactosidase I after 22 h incubation
Sample
Texture
CSP (N) (lg/mg EIR)
CSC (lg/mg EIR)
Unripe fruit (day 0)
Half ripe fruit (day 12)
35
21
9
3
43 0.4
66 0.7
83 0.1
41 0.4
98 0.4
47 0.3
72 0.6
100 0.7
50 0.4
122 2.9
2
0.2
Ripe fruit (day 24)
Unripe tissue + boiled b-galactosidase I
Unripe tissue + active b-galactosidase I
–
–
Values are means of 4 fruits or analyses SE.

S. Balasubramaniam et al. / Phytochemistry 66 (2005) 153–163
159
Fig. 4. Sephacryl S-500 profiles of EDTA-soluble polyuronides and carbohydrates of ripening carambola (a) and (b) and of unripe fruitÕs cell wall
upon incubation with purified b-galactosidase I for 22 h (d) and (e), and the profiles for 4 M alkali-soluble hemicellulose of ripening carambola (c)
and of unripe fruitÕs hemicellulose fraction upon incubation with the purified enzyme (f). Elution volume for dextran blue 2000 (Vd) and glucose (Vg)
was shown. Ripening stage, day 0 (Ã), day 12 (e) and day 24 (j), and active enzyme (d), boiled enzyme (s).
assembly during ripening (Table 2 and Fig. 4; Chin
et al., 1999). Besides carambola, b-galactosidases from
ripening papaya were also capable to modify both the
structurally attached pectins as well as the alkaline-solu-
ble hemicelluloses (Ali et al., 1998; Soh, 2002). The
mechanism by which b-galactosidase with b-galactanase
activity can substantially affect modification of the wall
polysaccharides is unclear and need further investiga-
tion. Ability of b-galactosidases to hydrolyse synthetic
PNPG substrate suggests that they might have an exo-
acting activity. However, the ability of the carambola
enzyme to significantly affect modification of a more

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S. Balasubramaniam et al. / Phytochemistry 66 (2005) 153–163
complex and structurally intact cell wall components
could not discount the possibility that the enzyme might
also function as an endo-glycanase.
the pooled fractions of peak AI were precipitated with
85% ammonium sulphate. Pellet was redissolved in
0.05 M Tris–HCL, pH 7.2, containing 1 mM DTT and
then passed through a Sephacryl S-200 HR (Pharmacia)
column (2.5 cm · 40 cm), previously equilibrated with
the same buffer. Fractions of 6.2 ml were collected at a
flow rate of 40 ml hꢁ1. One prominent activity peak,
designated as b-galactosidase I, with a corresponding
single protein peak was obtained.
3. Experimental
3.1. Plant material
Carambola fruit (A. carambola L. cv. B10) of uniform
size were harvested at commercial maturity stage (Day
0: stage 2, light green) from a private farm in Semenyih,
Selangor, Malaysia. Fruits were rinsed with water,
soaked for 5 min in 0.02% banlate, air-dried and left
to ripen (Day 24: stage 6, orange) at ambient tempera-
ture. The ripe fruits were peeled, cut into 1 cm3 cubes,
frozen in liquid nitrogen and then stored at ꢁ80 ꢁC until
required.
Two b-galactosidase activity peaks were also ob-
tained from peak B, namely peak BI and peak BII
(b-galactosidase IV) from the DEAE-Sepharose column.
Fractions from peak BI having high specific activity
were pooled and precipitated with 85% ammonium sul-
phate. Pellet was dissolved in 0.05 M Tris–HCl, pH 7.2,
containing 1 mM DTT and loaded onto a Sephacryl
S-200 HR (Pharmacia) column (2.5 cm · 40 cm), previ-
ously equilibrated with the same buffer. A prominent
b-galactosidase activity peak (b-galactosidase III) was
recovered. Peak fractions of b-galactosidase I, II, III
and IV with high specific activity were used in physical
characterization and kinetic studies of the enzymes,
whilst a fraction with the highest specific activity and
exhibiting only b-galactosidase I band on native gel
was used in in vitro cell wall modification studies.
3.2. Purification of b-galactosidase
Purification was carried out at 4 ꢁC. Enzyme was ex-
tracted according to the methods described in Chin et al.
(1999). About 1.2 kg of frozen tissue was blended to fine
powder and homogenized in 2:1 ratio of cold extraction
buffer (0.1 M sodium citrate, pH 4.6, containing 1 M
NaCl, 13 mM EDTA, 10 mM b-mercaptoethanol and
1% (w/v) soluble polyvinylpyrrolidone (PVP-40)) using
3.3. Assay of b-galactosidase
a blender (Edmund Buhler 7400, Tubingen, Germany).
¨
b-Galactosidase was assayed according to the meth-
ods described in Lazan et al. (1995). The assay mixture
consisted of 0.52 ml 0.1 M sodium citrate, pH 4.1, 0.4
ml 0.1% (w/v) BSA and 0.4 ml 13 mM p-nitrophenyl-
The homogenate was kept on ice for 30 min with occa-
sional stirring before centrifuging at 17,000g (Sorvall
Superspeed RC-5B, SS34 rotor) for 30 min. The super-
natant was filtered through two layers of nylon cloth.
The filtrate was then dialyzed overnight against 0.01
M acetate buffer, pH 5.0, containing 10 mM b-mercap-
toethanol with twice changes of fresh buffer. Following
dialysis, the sample was loaded onto a CM-Sepharose
fast flow (Pharmacia) column (5.0 cm · 10.0 cm), previ-
ously equilibrated with the same acetate buffer contain-
ing 1 mM DTT. Fractions (8.0 ml) were collected at a
flow rate of 75 ml hꢁ1. No b-galactosidase activity was
detected in the unbound fractions. Bound proteins were
further eluted stepwisely using 0.05 and 0.5 M NaCl.
b-Galactosidase activity was detected in both the 0.05
M NaCl (peak A) and 0.5 M NaCl eluents (peak B).
Peaks A and B were pooled separately and dialysed
overnight against 0.01 M Tris–HCl, pH 7.2, containing
10 mM b-mercaptoethanol. The respective peaks were
then loaded separately onto a DEAE-Sepharose fast
flow (Pharmacia) column (2.5 cm · 15.0 cm) previously
equilibrated with the same buffer containing 1 mM
DTT. Fractions (7.2 ml) were collected at a flow rate
of 55 ml hꢁ1. From peak A, two peaks having b-galacto-
sidase activity were recovered namely, peak AI,
obtained in the unbound fraction, and peak AII
(b-galactosidase II), eluted in 0.2 M NaCl. Proteins in
b-D-galactopyranoside (PNPG) as substrate. The reac-
tion mixture was preincubated at 37 ꢁC for 10 min
before addition of 0.08 ml enzyme. The reaction was fur-
ther incubated for 15 min before addition of 2 ml 0.2 M
Na2CO3. Boiled enzyme was used as a control in the as-
say mixture. The amount of p-nitrophenol formed was
determined spectrophotometrically through absorbance
at 415 nm. Enzyme activity was expressed as nmol
p-nitrophenol formed per second (nkat).
3.4. Protein determination
Samples from crude extracts were precipitated with
an equal volume of 10% (w/v) TCA, rinsed twice with
5% TCA and redissolved in 0.1 M NaOH prior to
Bradford (1976) assay, using bovine serum albumin
(BSA) as a standard. Protein content of chromato-
graphic fractions was estimated from the absorbance
at 280 nm.
3.5. Molecular mass estimation
The apparent molecular mass of native b-galactosi-
dase protein was estimated using a Sephadex G-200

S. Balasubramaniam et al. / Phytochemistry 66 (2005) 153–163
161
(Pharmacia) column (2.5 cm · 40 cm) calibrated with 5
mg mlꢁ1 of ribonuclease A (13.7 kDa), chymotrypsino-
gen (25 kDa), ovalbumin (43 kDa), albumin (67 kDa),
aldolase (158 kDa) and catalase (232 kDa) and 1
mg mlꢁ1 of blue dextran (200 kDa) [Sigma High Molec-
ular weight Calibration kit]. The column was equili-
brated with 50 mM potassium phosphate, pH 7.0,
containing 10 M NaCl and 0.02% (w/v) sodium azide
annopyranoside, and b-galactosidase activity and absor-
bance at 280 nm were monitored.
3.9. Kinetics and substrate specificity studies
Values of Km and Vmax, optimal pH and temperature,
heat stability as well as studies on the ability of isolated
b-galactosidase isoforms to hydrolysed synthetic and
endogenous substrates were done as described in Ali
et al. (1998).
and eluted at a flow rate of 26 ml hꢁ1
.
3.6. Gel electrophoresis
3.10. Extraction, analysis and in vitro modification of
pectin and hemicellulose by b-galactosidase I
Polyacrylamide gel electrophoresis was done accord-
ing to the methods of Laemmli (1970) for SDS–PAGE
and Reisfeld et al. (1962) for native-PAGE using 12%
and 15% acrylamide, respectively. About 2 lg of
b-galactosidase protein was loaded for native-PAGE
whereas 1.5 lg for SDS–PAGE. Analytical isoelectric
focusing was performed using precast Ampholine PAG
plates (Pharmacia, LKB, Uppsala, Sweden) and stained
with Coomassie brilliant blue R-250 exactly according
to the manufacturerÕs instructions.
Soluble pectins were extracted from ethanol insoluble
residues (EIR) by the methods described in Chin et al.
(1999). Briefly, 100 mg of EIR from day 0, 12 or 24 fruit
was suspended (37 ꢁC, 8 h) in 10 ml of 30 mM NaOAc,
pH 5.0, containing 10 mM EDTA. The supernatant was
resolved by centrifugation (Biofuge 17RS Heraeus Sep-
atech) at 2000g for 30 min. Amounts of uronic acid
(Blumenkrantz and Asboe-Hansen, 1973) and carbohy-
drate (Dubois et al., 1956) were estimated.
3.7. N-terminal sequencing
Cell wall preparation from day 0, 12 or 24 fruit was
done according to the protocols as described earlier
(Chin et al., 1999). For hemicellulose extraction, about
500 mg of the wall material was incubated for 8 h in 7
ml 4 M NaOH containing 0.24 M NaBH4. This proce-
dure was repeated after filtration, and the combined fil-
trate was neutralized with concentrated acetic acid. The
extracts were then dialysed against tap water for 12 h
followed by 10% methanol and distilled water for 24
h, respectively. The amount of extractable hemicellulose
(as glucose equivalent) was determined (Dubois et al.,
1956).
For analysis of ripening-related pectin depolymeriza-
tion, about 800 lg CSP was loaded onto a Sephacryl
S-500 column (1.6 cm · 50 cm) previously equilibrated
with 80 mM NaOAc, pH 5.0, containing 10 mM EDTA
and 50 mM NaCl. Fractions (1.8 ml) were collected at a
flow rate of 20 ml hꢁ1 and levels of uronic acid and car-
bohydrate were determined. For hemicellulose depoly-
merization analysis, about 800 lg of alkaline-soluble
hemicellulose was loaded onto a Sephacryl S-500 (1.6
cm x 45.0 cm) column previously equilibrated with 15
mM sodium citrate phosphate pH 5.5, containing 100
mM NaCl, and 2 ml fractions were collected at a flow
rate of 20 ml hꢁ1. The levels of hemicellulose in each
fraction were determined (Dubois et al., 1956).
Amino acid sequencing of 48 and 36 kDa subunits of
b-galactosidase I was performed as described in Matsu-
daira (1987). About 20 lg of purified b-galactosidase I
protein was run on SDS–PAGE and the resolved pro-
teins were electroblotted onto polyvinylidene difluoride
(PVDF) membrane (Immobilon, Millipore, Bedford,
MA) in 10 mM 3-[cyclohexylamino]-1-propanesul-
phonic acid buffer containing 10% methanol (pH 11).
The PVDF membrane was stained with 0.1% Coomassie
brilliant blue R-250 in 50% methanol, destained in acetic
acid and methanol (1:5) mixture, rinsed in deionized
water and then air-dried. Protein bands were excised
and submitted to Microchemical Facility, The Babra-
ham Institute of Animal Physiology and Genetics, Cam-
bridge, UK for N-terminal sequencing. Sequence
comparison and alignment were performed using the
BLAST searches (NCBI) and BCM Search Launcher
respectively.
3.8. Glycoprotein property
b-Galactosidase I was ascertained for its glycoprotein
properties using a Con-A Sepharose (Pharmacia) col-
umn (1.5 cm · 3.0 cm). Purified b-galactosidase I frac-
tions from Sephacryl S-200 column was dialysed
extensively against 0.02 M Tris–HCl, pH 7.4, containing
0.5 M NaCl, 1 mM CaCl2 and 1 mM MnCl2 before
being loaded onto the affinity column previously equili-
brated with the same buffer, and fractions were collected
at a flow rate of 55 ml hꢁ1. Bound proteins were eluted
with buffer containing 0.1 M glucose or 0.1 M methylm-
Modification of pectins and hemicelluloses by puri-
fied b-galactosidase I was analysed by the protocols de-
scribed in Ali et al. (1998). For pectin, about 15 mg EIR
from unripe (stage 2) fruit was incubated (37 ꢁC, 22 h)
with about 6 nkat purified b-galactosidase I, 5 ml 30
mM sodium acetate, pH 5.0, containing 10 mM EDTA
and 2 drops of toluene. After stopping the reaction by

162
S. Balasubramaniam et al. / Phytochemistry 66 (2005) 153–163
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boiling (5 min), the supernatant was recovered by centri-
fugation (MSE superspeed at 2000g for 30 min). Levels
of uronic acid and carbohydrate were determined, and
gel filtration analysis was performed as described above.
For hemicellulose modification, about 800 lg of the un-
ripe fruit alkaline-soluble hemicellulose in 2.4 ml buffer
was incubated (37 ꢁC, 22 h) with about 6 nkat of the
purified enzyme in the presence of 1–2 drops of toluene
and the reaction was stopped by boiling (5 min). Hemi-
Chin, L.H., Ali, Z.M., Lazan, H., 1999. Cell wall modifications,
degrading enzymes and softening of carambola fruit during
ripening. Journal of Experimental Botany 50, 767–775.
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Dubois, M.K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956.
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Acknowledgement
Huber, D.J., 1983. Polyuronide degradation and hemicellulose mod-
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We thank the Ministry of Science, Technology and
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