β-hexosaminidase, an enzyme from ripening bell capsicum (Capsicum annuum var. variata)

Article abstract of DOI:10.1016/S0031-9422(02)00181-4

β-Hexosaminidase activity increased significantly during fruit development and ripening of bell capsicum (Capsicuum annuum var. variata). Three isoforms of β-hexosaminidase from bell capsicum could be resolved upon ion exchange chromatography with step wi

Full text of DOI:10.1016/S0031-9422(02)00181-4

Phytochemistry 61 (2002) 295–300
www.elsevier.com/locate/phytochem
b-Hexosaminidase, an enzyme from ripening bell
capsicum (Capsicum annuum var. variata)
B.H. Jagadeesh*, T.N. Prabha
Biochemistry and Nutrition Department, Central Food Technological Research Institute, Mysore, 570013, India
Received 8 January 2002; received in revised form 26 April 2002
Abstract
b-Hexosaminidase activity increased significantly during fruit development and ripening of bell capsicum (Capsicuum annuum
var. variata). Three isoforms of b-hexosaminidase from bell capsicum could be resolved upon ion exchange chromatography with
step wise gradient (0.10, 0.15 and 0.20 M NaCl) having an abundance of 38, 47 and 15% for isoforms I, II and III respectively.
Isoforms I and II were further purified on gel permeation chromatography. The pH optimum for these two isoforms was around 5.
Isoform II exhibited higher thermal stability. Hg2+ and Zn2+ inhibited both, but isoform I showed a much higher inhibition by
Cu2+ also. The Km for isoforms I and II with pnp-b-d-N-acetyl glucosamine pyranoside was 3.00 and 1.75 mM, respectively. Iso-
form II on SDS–PAGE was found to be a monomer with a relative molecular mass of 85 kD. This isoform (the most major)
appeared to be electrophoretically homogeneous. b-Hexosaminidase is novel in the context of fruit ripening. This enzyme has not
been reported from fruits and studied hitherto.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Capsicum annum var. variata; Solanaceae; Bell capsicum; b-Hexosaminidase
1. Introduction
osaminidase in bell capsicum and other fruits tested (in our
laboratory) and its increasing activity during fruit devel-
opment followed by a further increase during ripening was
noted to be striking. It was thought important to study this
enzyme in the context of fruit ripening and to look into the
nature of this enzyme in bell capsicum for a further study
on its role in development and fruit ripening. This is the
first report on b-hexosaminidase purification in fruit sys-
tems. Among other higher plant systems, the enzyme was
purified and studied only in two tissues viz.: cabbage and
lettuce (Chang et al., 1998; Pocsi et al., 1990). This enzyme
is not well established and reported from higher plants.
b-Hexosaminidase, so far has been studied only in
microbial and animal systems, where amino sugars are
commonly present (Rankema et al., 1995; Sakamoto et
al., 1998). Recently some microbial systems were repor-
ted to have a high activity of b-hexosaminidase, which
were purified and studied (Keyhani and Roseman,
1996). b-Hexosaminidase is implicated in signal trans-
duction, cell division and cell integrity in animal systems
(Sokamoto et al., 1998; Roseman, 1991). In microbial
system, this enzyme is known to play an important role
in host-pathogen interaction (Chitlaru and Roseman,
1996; Roseman, 1991). Among higher plants, tomato
fruit and leaves were shown to contain free N-glycans,
which were absent in roots and stems (Priem et al., 1993).
Recently the profile of different glycosidases was
checked in solanaceous fruits, interestingly b-hex-
osaminidase activity was found to be significantly higher
than other glycosidases. Such a high activity of b-hex-
2. Results and discussion
Fig. 1 shows an increased b-hexosaminidase activity
during developmental stages followed by a significant
rise in activity during ripening in bell capsicum. The pur-
ification data of b-hexosaminidase are detailed in Table 1.
The purification profile of the same on DEAE-cellulose
followed by Sephadex G-200 are depicted in Figs. 2 and 3,
respectively. Ion exchange chromatography resolved the
b-hexosaminidase activity into three distinct fractions
* Corresponding author. Tel.: +91-821-514-876; fax: +91-821-
517-233.
E-mail address: bhjagadeesh@yahoo.com (B.H. Jagadeesh).
0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00181-4

296
B.H. Jagadeesh, T.N. Prabha / Phytochemistry 61(2002) 295–300
for b-hexosaminidase activity was 4.60 and 5.00 for
isoforms I and II, respectively, their optimum tempera-
ture being 37–47 ꢀC for isoform I and 47 ꢀC for isoform
II. b-Hexosaminidase II retained 100% activity at 57 ꢀC
for 15 min, while b-hexosaminidase I retained 71% of
the activity under the same conditions. The Km for pnp-
b-d-N-acetyl glucosamine pyranoside was 3.00 and 1.75
mM for isoforms I and II, respectively. Among the
metal ions tested for inhibitory action (Table 3), Hg2+
and Zn2+showed higher inhibition, and the percentage
inhibition being more for isoform I compared to iso-
form II. Also, Cu2+ showed a high inhibition for iso-
form I. At 1 mM concentration of Hg2+, Cu2+ and
Zn2+, the enzyme showed 3, 9 and 14% residual activity
over control for isoform I, while it was 5, 78 and 38%
for isoform II. EDTA at 1 mM concentration exhibited
40% more activity for isoforms II. The substrate speci-
ficity of isoform I and II for pnp-b-d-N-acetyl galacto-
samine pyranoside was much less when compared to
pnp-b-d-N-acetyl glucosamine pyranoside. The purified
isoform II was checked for other glycosidase activities
with pnp-substrates such as a- and b-d-glucopyrano-
side, a- and b-d-galactopyranoside, a-mannopyranoside
and b-d-N-acetyl galactosamino pyranoside. This
enzyme did not show any activity with these substrates
except for b-d-N-acetyl galactosamino pyranoside. When
compared to the b-d-N-acetyl glucosamino pyrano-
side(100%), enzyme with b-d-N-acetyl galactosamino
pyranoside exhibited 33.5% activity.
Fig. 1. b-Hexosaminidase activity during fruit development (A) and
ripening (B) in bell capsicum.
The electrophoretic profiles (native and SDS–PAGE)
for post ion exchange chromatographic fractions (a and
b) are depicted in Fig. 4. The purified enzyme (isoform
II) on non-denatured SDS–PAGE was homogeneous
(Fig. 4c). This isoform showed a single band on SDS–
PAGE with a relative molecular mass of 85 kD (Fig. 4d)
and appeared to be a monomer.
b-Hexosaminidase enzyme from cabbage was recently
reported to contain three subunits of 51, 57 and 64 kD on
SDS–PAGE. Its Km for pnp-b-d-N-acetylglucosamine
was 0.94 mM, with an optimum pH of 4 and optimum
elutable with 0.10, 0.15 and 0.20 M NaCl with a relative
abundance of 38, 47 and 15% respectively. Based on
elution profile, they were designated as isoform I, II and
III. Further studies were conducted with isoforms I and
II, after gel permeation chromatography (Fig. 3). The
purity of isoforms I and II increased by 9 and 10 fold
respectively with a recovery of 10 and 11%.
Some enzymatic properties of these isoforms
are consolidated in Table 2. The optimum pH
Table 1
Purification profile of b-hexosaminidase from bell capsicum
Purification steps
Total protein
(mg)
Total
activitya (EU)
Specific activity
(EU/mg protein)
Purification
fold
Recovery
(%)
1. Crude extraction
256.00
136.40
268.92
174.54
1.05
1.27
1.00
1.20
100
2. Ammonium sulfate precipitation (35–70%)
64.9
3. DEAE-cellulose chromatography
Isoform I
Isoform II
Isoform II
14.32
15.25
8.00
53.50
43.92
17.70
3.75
2.88
2.21
3.57
2.74
2.10
19.89
16.33
6.58
4. Gel permeation chromatography
Isoform I
Isoform II
3.01
2.85
30.46
26.56
10.11
9.31
9.62
8.87
11.32
9.87
a
1EU is equivalent to 1 mmol pnp released minÀ1
.

B.H. Jagadeesh, T.N. Prabha / Phytochemistry 61(2002) 295–300
297
Fig. 2. Elution profiles of b-hexosaminidase isoforms from bell capsicum on DEAE-cellulose column equilibrated with double distilled water and
eluted with increasing concentrations of 0.05, 0.10, 0.15, 0.20, and 0.25 M NaCl; pH 6.8, flow rate 0.66 ml minÀ1. Isoforms I, II and III eluted with
0.10, 0.15 and 0.20 M NaCl, respectively. Arrows indicate the change of NaCl gradient ( –ꢁ– is absorbance and -Á- enzyme activity).
Table 2
Enzymatic properties of b-hexosaminidase isoforms from bell capsicum
Properties
Isoform I
Isoform II
1. Elution on IEC
2. Abundance
0.10 M NaCl
38%
4.60
37–47 ꢀC
70%
Hg2+, Zn2+ and Cu2+
3.00 mM
10.11
0.15 M NaCl
47%
5.00
3. pH optimum
4. Temperature optimum
5. Thermal stability at 57 ꢀC for 15 min
6. Inhibition
47 ꢀC
100%
Hg2+ and Zn2+
1.75 mM
9.31
7. Km for pnp-b-d-N-acetyl glucosamine pyranoside
8. Specific activity (EU/mg protein) pnp-b-d-N-acetyl glucosamine
temperature of 60 ꢀC (Chang et al., 1998). b-Hex-
osaminidase from lettuce was partially purified where its
relative molecular mass was shown to be 69 kD on
SDS–PAGE (Pocsi et al., 1990).
The abundant activity of b-hexosaminidase in bell
capsicum and tomato when compared to other fruits is
noted here (Table 4). This is the first report on b-hex-
osaminidase and its purification in fruits and also this
enzyme is not established in higher plant systems.
Though the functional role of b-hexosaminidase in sig-
nal transduction, cell multiplication, cellular integrity
and host-pathogen interaction is studied in some animal
and microbial systems, the implication of this enzyme in
higher plants, particularly in fruits, is not known, which
deserves further attention on the study of this enzyme in
fruits in the context of fruit ripening. The increased
activity of b-hexosaminidase during ripening is promi-
nent. Besides, it was noticed in our earlier experiments
(data not shown) that some new b-hexosaminidase band
were developed during ripening. This also coincides
with the increased b-hexosaminidase activity during
ripening, when compared to developmental stages. It is
thought that b-hexosaminidase may have an important
function in fruit ripening by way of deglycosylation and
generating free N-glycans. Free N-glycans were shown to
be involved in fruit ripening in tomato (Priem et al., 1993).

298
B.H. Jagadeesh, T.N. Prabha / Phytochemistry 61(2002) 295–300
Fig. 3. Elution profiles of b-hexosaminidase isoforms from bell capsicum on Sephadex G-200 ( –ꢁ– is absorbance and -Á- enzyme activity). (A)
Isoform I and (B) Isoform II.
Table 3
Effect of divalent metal ions on b-hexosaminidase activity
Table 4
Comparison of b-hexosaminidase activity in some important fruits
Inhibitors (1 mM)
Activity retained (%)ÆS.D.
Fruits
EUa
Isoform I
Isoform II
1. Tomato
2. Capsicum
3. Papaya
4. Banana
5. Mango
42.30
38.38
18.19
3.75
Control
Cu2+
Fe2+
Hg2+
Mg2+
Ca2+
Mn2+
Zn2+
EDTA
100.00 (Æ0.19)
9.00 (Æ0.34)
100.00 (Æ0.30)
78.00 (Æ0.51)
119.35 (Æ0.63)
5.20 (Æ0.31)
90.16 (Æ0.54)
2.78 (Æ0.30)
1.05
a
109.5 (Æ0.48)
110.80 (Æ0.86)
86.57 (Æ0.81)
14.57 (Æ0.33)
115.18 (Æ0.88)
67.33 (Æ0.40)
84.00 (Æ0.36)
101.45 (Æ0.61)
38.71 (Æ0.25)
139.96 (Æ0.81)
EU/g acetone powder.
(I) 8-d, 2.5–3.5 g; (II) 16-d, 8.5–9.5 g; (III) 24-d, 60–62 g;
and (IV) 32-d, 65–67 g. The four stages of ripening
chosen were based on days after harvest. (I) Mature
dark green, 3-d; (II) light green, 7-d; (III) orange green,
14-d and (IV) red ripe stage, 21-d. Acetone dried pow-
ders were prepared from the respective sample taken for
the experiment. Acetone dried powders were also pre-
pared from tomato, papaya, banana and mango from
their climacteric stage. Post climacteric bell capsicum
served as the source of enzyme for purification and
characterization.
3. Experimental
3.1. Materials
Freshly harvested bell capsicum was collected from a
local farm. The four different developmental stages were
selected based on size/weight and days (d) after fruit set.

B.H. Jagadeesh, T.N. Prabha / Phytochemistry 61(2002) 295–300
299
Fig. 4. Native (A and C) and SDS–PAGE (B and D) of b-hexosaminidase isoforms from bell capsicum. Lanes are as follows: 1, crude; 2, unbound
fraction; 3, 0.05 M NaCl eluted; 4, Isoform I; 5, Isoform II; 6, Isoform III and 7, molecular mass standards (50 mg of protein was loaded per track
for A and B and 10 mg were loaded for C and D).
3.2. Extraction, purification and electrophoresis
fractions were designated as isoforms I, II and III. Iso-
forms I and II were suitably concentrated and further
subjected to gel permeation chromatography using
Sephadex G-200 (1.6Â140 cm). The post gel filtration
chromatographic fractions were used for further studies.
Native and SDS–PAGE were carried out according to
the method of Laemmli (1970), and the proteins were
visualized with silver staining according to the method
(Porro et al., 1982).
Acetone dried powder (10 g) was extracted (Â3) with
0.05 M potassium phosphate buffer (pH: 6.60) containing
0.5 M soꢀdium chloride and kept overnight for extrac-
tion at 4 C. The enzyme extracts were pooled, filtered,
clarified and dialyzed against double distilled water. The
clear fraction was subjected to two-step ammonium
sulphate precipitation (0–35% and 5–70% saturation).
The fraction (35–70%) rich in b-hexosaminidase activity
was subjected to ion exchange column chromatography
(4Â49 cm) on DEAE-cellulose after dialyzing against
double distilled water (3Â). The fractions were eluted with
gradients of 0.05–0.25 M NaCl. The active fractions were
collected individually and dialyzed. Distinctly separable
3.3. Enzyme assay
The enzymeꢀ/substrate incubation was carried out for
15 min at 37 C and the reaction mixture consisted of
1.25 mM pnp-b-d-N-acetyl glucosamine pyranoside,

300
B.H. Jagadeesh, T.N. Prabha / Phytochemistry 61(2002) 295–300
100 mM sodium acetate buffer (pH: 5.00) and suitable
aliquot of the enzyme. The activity was determined by
measuring the liberated p-nitrophenol at 405 nm after
addition of 500 mM sodium bicarbonate to the reaction
mixture. One unit of the enzyme is defined as the
amount of enzyme required to liberate one micro mol
(mmol) of p-nitrophenol per minute. The specific activity
was expressed as enzyme units per mg protein. Protein
determination was done by the method of Sedmak and
Grossberg (1977).
Chang, C.T., Young, F.P., Chang, M.H., Sung, H.Y., 1998. Purifica-
tion and properties of b-N-acetylhexosaminidase from cabbage.
Biochem. Mol. Biol. Int 45, 371–380.
Keyhani, N.O., Roseman, S., 1996. The chitin catabolic cascade in
murine bacterium Vibrio furnissi: molecular cloning, isolation and
characterization of a periplasmic b-N-acetyl-d-glucosaminidase. J.
Biol. Chem. 52, 33425–33432.
Laemmli, U.K., 1970. Cleavage of structural protein during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
Porro, M., Viti, S., Antoni, G., Saletti, M., 1982. Ultra sensitive silver
staining method for the detection of protein on polyacrylamide gels
and immunoprecipitates on agarose gels. Anal. Biochem. 127, 316–
321.
Posci, I., Kiss, L., Nanasi, P., 1990. Studies on N-acetyl-b-d-hex-
osaminidase B from germinating Lupinus luteus L. seeds. I. Pur-
ification and characterization. Biochem. Biophys. Acta. 31, 110–
118.
3.4. Enzyme properties
The experimental conditions for the study of various
enzymic properties such has optimum pH, optimum tem-
perature, thermal stability, Km and divalent metal ion
inhibition have been detailed previously (Priya Sethu and
Prabha, 1997; Suvarnalatha and Prabha, 1999).
Priem, B., Gitti, R., Bush, C.A., Gross, K.C., 1993. Structure of ten
free N-glycans in ripening tomato fruit. Plant Physiol 102, 445–458.
Priya, Sethu, Prabha, T.N., 1997. a-Mannosidase from Capsicum
annum. Phytochemistry 44, 383–387.
Rankema, G.H., Boot, R.G., Muijsers, A.O., Danker-Koopman,
W.E., Johannes, M.F.G., 1995. Purification and characterization of
human chitotriosidase, a novel member of the chitinase family pro-
teins. J. Biol. Chem. 270, 2198–2202.
Acknowledgements
Roseman, S., 1991. Chitin utilization by marine bacteria: a physi-
ological function for bacterial adhesion to immobilized carbohy-
drates. J. Biol. Chem. 26, 24260–24267.
B.H.J. thanks CSIR, New Delhi, India for a Senior
Research Fellowship.
Sakamoto, Y., Kurimura, Y., Tsuji, Y., Yasunobu, T., 1998. A novel
fungal endo-b-N-acetyl-d-glucosaminidase that specifically acts on
plant glycoproteins. Biosci. Biotechnol. Biochem. 62, 1344–1350.
Sedmak, J.J., Grossberg, S.E., 1977. A rapid, sensitive and versatile
assay for protein using Coomassie brilliant blue G250. Anal. Bio-
chem. 79, 544–553.
References
Chitlaru, E., Roseman, S., 1996. Molecular cloning and characteriza-
tion of a novel b-N-acetyl-d-glucosaminidase from Vibrio furnissii.
J. Biol. Chem. 52, 33433–33439.
Suvarnalatha, G., Prabha, T.N., 1999. a-Mannosidase from Lyco-
persicum esculentum II. Phytochemistry 50, 1111–1115.