Purification, characterization and N-terminal sequence analysis of betel leaf (Piper betle) invertase

Article abstract of DOI:10.1002/jccs.201190042

The present study is the first report describing the purification, enzymatic properties and N-terminal amino acid sequence of a native invertase in betel leaf. The invertase was purified as a monomeric glycoprotein of molecular mass (Mr) 68 kDa. The enzyme was capable to attack β-fructofuranoside linkages from the fructose end of sucrose, raffinose and stachyose indicating it as an authentic β-D-fructofurano-sidase with high specificity for sucrose (Km 4.83 mM). The maximum activity was detected at pH 5.2 and 37 °C. Glucose and fructose showed typical inhibitory effect on the enzyme activity where as lectin was found to be effective activators of the enzyme. Significant inhibition by heavy metal ion Hg²⁺ and sulf-hydryl group modifying agents suggesting that free sulfhydryl group containing amino acid, cysteine is necessary for the catalytic activity of the invertase. A BLAST search of the N-terminal amino acid sequence of betel leaf invertase showed significant homology with the homologous invertases in database.

Full text of DOI:10.1002/jccs.201190042

Journal of the Chinese Chemical Society, 2011, 58, 389-397
389
Purification, Characterization and N-terminal Sequence Analysis of Betel
Leaf (Piper betle) Invertase
Md. Murad Hossain,†* Farzana Pervin and Nurul Absar
Department of Biochemistry and Molecular Biology, Faculty of Science, University of Rajshahi,
Rajshahi-6205, Bangladesh
Received May 14, 2010; Accepted February 21, 2011; Published Online March 7, 2011
The present study is the first report describing the purification, enzymatic properties and N-terminal
amino acid sequence of a native invertase in betel leaf. The invertase was purified as a monomeric glyco-
protein of molecular mass (Mr) 68 kDa. The enzyme was capable to attack b-fructofuranoside linkages
from the fructose end of sucrose, raffinose and stachyose indicating it as an authentic b-D-fructofurano-
sidase with high specificity for sucrose (Km 4.83 mM). The maximum activity was detected at pH 5.2 and
37 ºC. Glucose and fructose showed typical inhibitory effect on the enzyme activity where as lectin was
found to be effective activators of the enzyme. Significant inhibition by heavy metal ion Hg2+ and sulf-
hydryl group modifying agents suggesting that free sulfhydryl group containing amino acid, cysteine is
necessary for the catalytic activity of the invertase. A BLAST search of the N-terminal amino acid se-
quence of betel leaf invertase showed significant homology with the homologous invertases in database.
Keywords: Betel leaf; b-D-fructofuranosidase; Invertase; N-terminal sequence; Sucrose.
INTRODUCTION
Invertase (b-D-fructofuranosidase; EC 3.2.1.26) cat-
trast, alkaline invertases are thought to be nonglycosylated
cytoplasmic polypeptides15,16 and may regulate hexose and
sucrose levels in cytoplasm.17,18 However, alkaline and
acid invertases are also reported to be present in the same
tissue.18-20
alyzes the irreversible hydrolysis of sucrose to produce in-
vert sugars, a 1:1 mixture of dextrarotatory D-glucose and
levorotatory D-fructose.1,2 In plants, due to the central role
of sucrose in assimilate transport, invertase is considered a
key enzyme for carbohydrate metabolism, translocation
and storage.3 Invertases may be distinguished based on
their intracellular location, their isoelectric points and pH
optima. Distinct isoforms of invertases exist in the vacuole,
cytoplasm and apoplasm of plant cells. The acid isoforms
(pH optima 4.5-5.5) are present in the vacuole and apo-
plast, whereas the neutral and alkaline isoforms (pH optima
8.0) are located in the cytoplasm.4-7 Acid invertases are
usually N-linked glycoproteins.8,9 Soluble acid invertases
are involved in growth and differentiation,10 and sucrose
metabolism and storage in the vacuole.11-13 Cell wall bound
acidic invertases in apoplast are thought to play important
role in photosynthetic assimilates uptake by maintaining a
sucrose concentration gradient between photosynthetic
leaf tissue and heterotrophic parts of the plant.14 In con-
High acid invertase activity is predominant in imma-
ture plant organs and declines with the accumulation of su-
crose as the organ matures where as alkaline invertases are
exclusively present in mature tissues.21-23 Though a nega-
tive correlation was found between sucrose storage and
invertase activity,24-26 however, this finding is inconsistent
with the observation that leaves of temperature Gramineae
such as Lolium temulentum, oat and barley store large
amounts of sucrose in the presence of substantial intracel-
lular invertase activity.27-29 Thus it is thought that plant
leaves either employ regulatory mechanisms to modulate
invertase activity or there is compartmentalization of su-
crose to keep away from invertase activity.30
Betel leaf is a tropical herb belonging to the pepper
family of plants named Piper betle and locally called Pan.
The leaf is usually bit sour in taste but is popularly used as
* Corresponding author. E-mail: mmhossaink140@yahoo.com
† Present address: Department of Cell Biology, Institute of Nephrology, Niigata University, 1-757 Asahimachi-dori, Niigata 951-8510,
Japan

390 J. Chin. Chem. Soc., Vol. 58, No. 3, 2011
Hossain et al.
folk medicine. It consists of a lot of nutrients including pro-
tein, carbohydrate, vitamins, minerals, essential oil and
phenol like chavicol which has powerful antiseptic proper-
ties. Betel leaf showed decreasing acid invertase activity
with maturity though significant extent of acid invertase
activity was detected in mature leaf and almost no activity
detected in ripened leaf (data not shown). However, it is not
clear the role of invertases in different maturity stages of
leaf tissues. Betel leaf thus can be used as a model to know
the distinctive function of invertases in different maturity
stages. The high abundance and low price of betel leaf in
South and South-East Asia raises the prospect of commer-
cial production of invertases which can be economically
advantageous in industrial processes such as production of
syrup, non-crystallizable invert sugars and soft-centered
chocolates. So far, betel leaf has not been studied to analyze
the feasibility of its use as a possible cost effective in-
vertase source. Thus, in this study, we aim to investigate the
biochemical, biophysical and kinetic properties of betel
leaf invertase in order to justify the possibility of industrial
usage of betel leaf invertase.
second peak containing invertase activity (Fig. 1d). The
homogeneity of the invertase was judged by SDS-PAGE
and the purity was raised to 146 fold with a yield of 2.35%
(Table 1). The relative molecular mass was 72 kDa and 68
kDa, respectively, as estimated by gel filtration chromatog-
raphy and SDS-PAGE (Fig. 2). These results indicate that
the purified invertase remains as a monomer at its func-
tional state. The sugar content of 19.4% was determined by
phenol-sulphuric acid method indicating the glycoprotein
nature of the purified invertase.
Substrate specificity and kinetic parameters
Betel leaf invertase showed hydrolyzing capability
toward sucrose, raffinose and stachyose. Though the en-
zyme hydrolyzed raffinose and stachyose, the respective
activities toward raffinose and stachyose were about 25.3%
and 16.4% of the activity toward sucrose.
Betel leaf invertase was shown to follow the Michae-
lis-Menten kinetics in sucrose cleavage reaction. And its
Km value for sucrose as determined by the Lineweaver-
Burk double reciprocal plot (Fig. 3) was calculated to be
4.83 mM.
RESULTS
pH and temperature dependent activity profile
The sucrose cleavage activities of betel leaf invertase
were found to be maximum in the acidic pH range and
showed remarkably less activity as the pH increases toward
neutral or basic ranges. The optimum activity was detected
at pH 5.2 and fell sharply at a higher pH values implying
that the enzyme is an acid invertase (Fig. 4).
Purification and characterization of soluble invertase
isozyme from betel leaf
Betel leaf contained at least two soluble invertase
isozymes which were separated by ammonium sulfate frac-
tionation of the crude extract followed by anion exchange
chromatography on DEAE-Sephacel column. An immedi-
ately eluted nonbound protein fraction and the fraction
eluted with 0.25 M NaCl in 20 mM acetic acid-sodium ace-
tate buffer, pH 5.0 (AS-5.0) from the DEAE-Sephacel col-
umn possess the invertase activity (Fig. 1a). Only the
DEAE-Sephacel bound invertase fraction was used in this
study for further purification. The method was accom-
plished by loading the enzyme active fraction to the CM-
Sepharose column and eluted as nonbound fraction con-
taining enzyme activity, while two other fractions with no
invertase activity were eluted with 0.15 M and 0.35 M
NaCl (Fig. 1b). The partially purified invertase was loaded
on Mono Q chromatography and the bound active enzyme
was eluted from the column with 0.25-0.35 M NaCl of a
linear gradient of 0 to 0.5 M NaCl in AS-5.0 (Fig. 1c). The
active invertase was further purified by gel filtration on
HiLoad 16/60 Superdex 200 column. The elution profile of
gel filtration column produced three peaks with only the
The effect of temperature on betel leaf invertase ac-
tivities were analyzed at temperatures ranging from 20 to
80 °C. The highest level of invertase activities were found
at a temperature range of 30 to 40 °C with the optimum at
37 °C. Invertase activities were decreased gradually with
further increase in temperature and at 55 °C there caused a
sudden drop in activities where no activity was observed
above 75 °C (Fig. 4).
Effect of metal ions and chemical modification agents
on invertase activity
Betel leaf invertase activities were abruptly ceased by
heavy metal HgCl2. The invertase activity was lost by
92.6% in the presence of 1 mM HgCl2 and was completely
inhibited in presence of 5 mM HgCl2. ZnSO4, AgNO3 and
CuCl2 also exerted inhibitory effect on betel leaf invertase
by reducing the activity to 70-90% in the presence of 5 mM

Purification of Betel Leaf Invertase
J. Chin. Chem. Soc., Vol. 58, No. 3, 2011 391
Fig. 1. Purification of betel leaf invertase by column chromatography. The columns were equilibrated with 20 mM acetic
acid-sodium acetate, pH 5.0 (AS-5.0). (a) Anion-exchange chromatography on DEAE-Sephacel column. Nonbound
protein was eluted with column equilibrium buffer AS-5.0 and column bound proteins were stepwise eluted with 0.1
M, 0.2 M, 0.25 M and 0.3 M NaCl in equilibrium buffer. The nonbound protein and protein eluted with 0.25 M NaCl
exhibit invertase activity. (b) Cation-exchange chromatography on CM-Sepharose column. The invertase obtained
from DEAE-column with 0.25 M NaCl was eluted as nonbound protein with AS-5.0 from the CM-Sepharose column
and the bound proteins possess no invertase activity were stepwise eluted with 0.15 M and 0.3 M NaCl in buffer
AS-5.0. (c) Mono Q column chromatography of invertase eluted from CM-Sepharose column. The nonbound protein
fraction showed no invertase activity. Bound proteins containing invertase activity were eluted with a linear gradient
of 0-0.5 M NaCl in AS-5.0. (d) Gel filtration chromatography on HiLoad 16/60 Superdex 200 column. The elution
profile of invertase obtained from Mono Q column chromatography on gel filtration column produced three peaks
with only the second peak containing invertase activity. The both end arrows indicate the protein of the correspond-
ing peak fractions possess invertase activity.
of these salts. Manganese salt did not affect betel leaf
invertase activity. On the contrary, invertase activity was
increased moderately by KCl while MgCl2 and CaCl2 stim-
ulated invertase activity about 2.0-2.5 fold as compared to
that in their absence. The presence of 5 mM MgCl2 caused
maximum increased in betel leaf invertase activity up to
249.6% (Table 2). The effect of metal ions on invertase ac-
tivity varies greatly depending on invertase source. Thus
the difference in metal ion inhibition and enhancement may
be due to difference in active site structure.
(0.5 mM) and 5,5-dithiol-bis(2-nitrobenzoic acid) (DTNB)
(0.5 mM), invertase activity was decreased by 60%, 72%
and 37% respectively (Table 2).
Protein and lectin effect on invertase activity
A prominent feature of higher plant invertases is their
in vitro activation by various proteins. To analyze the effect
of proteins on betel leaf invertase activity, six different pro-
teins and lectins were used. All the tested proteins served as
activator of betel leaf invertase activity but lectin such as
concanavalin A and ricin were found to be better activators
than other proteins examined and showed a good agree-
In the presence of cysteine modifying agents, iodo-
acetamide (5 mM), p-chloromercuribenzoic acid (PCMB)

392 J. Chin. Chem. Soc., Vol. 58, No. 3, 2011
Hossain et al.
Table 1. Purification of betel leaf invertase
Specific activity
(Units/mg)
Purification
fold
Purification steps
Protein (mg) Activity (Units)
Yield (%)
Crude extract
(NH4)2SO4 ppt.
DEAE-Sephacel
CM-Sepharose
Mono Q
1236
336
21
807
644
149
51
0.65
1.92
100
79.80
18.46
6.32
1
2.95
7.10
10.92
28.02
50.00
146.15
2.8
0.8
0.2
18.21
32.50
95.00
26
3.22
Superdex S-200
19
2.35
ment with Ricinus communis leaf invertase37 as well as rice
acid invertase.38 Ricin (0.1 mM) was the best activator
which enhanced the invertase activity about 145%. Con-
canavaline A (0.1 mM) enhanced the invetase activity by
88% where other proteins BSA(0.2 mM), urease (0.5 mM),
alkaline phosphatase (0.5 mM) and b-galactosidase (0.5
mM) enhanced the activity by 30%, 40%, 44% and 42%
respectively (Table 3).
PAQNWMNDPXGPMXXNGVYHLFYQY (Fig. 5). The
amino acids of position 14, 28, 32 and 33 were not possible
to be identified. The N-terminal sequence homology was
compared with homologous proteins. Using N-terminal
amino acid sequence as a query, a BLAST search found
previously reported invertases in data base as homologous
proteins to betel leaf invertase.
DISCUSSION
Effect of end-products on betel leaf invertase activity
Betel leaf invertase was inhibited by the end prod-
ucts, glucose and fructose. Invertase activities were de-
creased with the increase in concentration of glucose and
fructose suggesting that glucose and fructose may function
as competitive inhibitors of invertase. At a concentration of
10 mM glucose and 10 mM fructose, the enzyme activity
was inhibited by 28% and 39% respectively (Table 4). Thus
fructose appeared as more effective inhibitor than glucose.
Betel leaf contained at least two invertase, one of
which can be grouped as acid invertase due to its high ac-
tivity at low pH buffer and low activity at high pH buffer.
The presence of multiple invertase isozymes was also re-
ported in barley leaves,11 mung bean39 and Japanese pear
fruit.40 The acid invetase was purified to 146 fold with a
yield of 2.35%. The gel filtration and SDS-PAGE esti-
mated similar molecular weight (Mr) 72 kDa and 68 kDa,
respectively, indicating that the invertase is a monomer in
the functional state. Betel leaf invertase showed hydrolyz-
ing capability toward sucrose, raffinose and stachyose but
N-terminal amino acid sequence
The N-terminal amino acid sequence of betel leaf
invertase was identified as SVGANPVLSVKRTXYHFQ
9
8
7
6
5
4
3
2
1
I
M
116
97
66
45
31
21
14.4
6. 5
-1
0
1
2
3
4
5
6
7
8
9
Fig. 2. SDS-PAGE pattern of betel leaf invertase. The
invertase was analyzed on a Polyacrylamide
gel (0.1% SDS): Lane I, purified invertase and
lane M, molecular mass standard proteins.
1/[S]
Fig. 3. Lineweaver-Burk plot of hydrolysis of sucrose
by purified betel leaf invertase.

Purification of Betel Leaf Invertase
J. Chin. Chem. Soc., Vol. 58, No. 3, 2011 393
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
2
4
6
8
10
0
20
40
60
80
100
pH
Temperature (ºC)
Fig. 4. Effect of pH and temperature on the purified betel leaf invertase activity. Relative activity versus pH (left). Relative
activity is expressed as the percentage of the maximum activity at pH 5.2. Relative activity versus temperature
(right). Relative activity is shown as the percentage of the maximum activity at 37 °C.
not lactose, maltose, trehalose, cellobiose, melezitose or
inulin which are saccharides without b-fructofuranosyl
residue. This substrate specificity suggests that the enzyme
is a b-fructofuranosidase which can be able to attack the
b-fructofuranosides from the fructose end. The Km value
of betel leaf invertase against sucrose was determined to be
4.83 mM. Invertases are reported to have Km values rang-
ing 2.0-6.6 mM38,40-43 using sucrose as substrate. Km value
of invertase from Thermotoga neapolitana was 14 mM2
where as the Km for sugarcane acid invertase was reported
exceptionally higher, 160.6 mM.5
Plant invertases characteristically inhibited by heavy
metals and reagents that react with sulfhydryl (–SH)
Table 2. Effect of metal ions and chemical modification agents
on invertase activity
Table 3. Protein and lectin effect on invertase activity
Effectors
Concentration (mM)
Relative activity (%)
Concentration Maximum activation
Effectors
None
--
1
100
7.4
(mM)*
(%)
HgCl2
Control
--
--
30
40
44
42
88
145
5
0.1
BSA
0.2
0.5
0.5
0.5
0.1
0.1
ZnSO4
AgNO3
CuCl2
1
17.2
11.4
32.3
27.1
41.5
16.5
100.1
102.3
115.2
120.8
120.4
230.9
167.5
249.6
75.4
40.1
55.5
27.9
79.2
63.1
Urease
5
Alkaline phosphatase
b-Galactosidase
Concanavalin A
Ricin
1
5
1
5
*Minimum concentration of effector which produced maximum
activation.
MnCl2
KCl
1
5
1
5
Table 4. Effect of end products (glucose and fructose) on
invertase activity
CaCl2
1
5
MgCl2
Iodoacetamide
PCMB
DTNB
1
End product
Glucose
Concentration (mM)
Relative activity (%)
5
1
5
93.7
86.8
72.2
88.6
79.4
61.5
1
5
10
1
0.1
0.5
0.1
0.5
Fructose
5
10

394 J. Chin. Chem. Soc., Vol. 58, No. 3, 2011
Hossain et al.
P. betle
(1-43)
-SVGANPVLSVKRTXYHFQPAQNWMNDPXGPMXXNGVYHLFYQY
-SHQDLNQPYRTRTGYHFQPLKNWMNDPNGPMIYKGIYHLFYQY
-GVHASEDLQPYRTAFHFQPLKNWMNDPNGPMYFNGVYHLFYQY
ARSPPTNQPYR-RTAYHFQPRKNWINDPNGPMLYKGVYHLFYQY
-SLFPATSEQPYRTAFHFQPPQNWMNDPNGPMCYNGVYHLFYQY
-PTEADTANHARRTAYHFQPAKNWQNDPNGPMYHNGMYHLFYQY
-NVKNVHVKNVHRTGYHFQPPKNWINDPNGPMYYNGVYHLFYQY
-YPWTNAMLSWQRTSYHFQPEKNWMNDPNGPLFHMGWYHLFYQY
-SVGAENVKQVHRTGYHFQPKQNWINDPNGPMYYKGVYHLFYQY
(28-70)
(22-64)
(29-71)
(10-52)
(29-71)
(40-82)
(46-88)
(49-91)
A. thaliana
H. annuus
B. vulgaris
C. intybus
O. sativa
N. tabacum
C. canephora
D. carota
Fig. 5. Alignment of N-terminal sequences of betel leaf invertase and homologous invertases in NCBI data base. The N-ter-
minal sequences of following invertases have been used for the multisequence alignment: P. betle, from Piper betle;
A. Thaliana, from Arabidopsis thaliana (accession BAA89048); H. annus, from Helianthus annuus (accession
AAY85659); B. vulgaris, from Beta vulgaris (accession CAD19322); C. intybus from Cichorium intybus (accession
CAA72009); O. sativa, from Oryza sativa subsp. Indica (accession AAD38399); N. tabacum, from Nicotiana
tabacum (accession CAA57428); C. canephora, from Coffea canephora (accession ABI17894); D. carota, from
Daucus carota (accession CAA49162). The numerical in the parenthesis indicates the position of the amino acids in
the protein sequences. The amino acids of position 14, 28, 32 and 33 were not possible to be identified and were
shown as “X”.
group.19,44-47 Betel leaf invertase activities were abruptly
ceased in presence of 5 mM HgCl2 suggesting that sulf-
hydryl group containing amino acid such as cysteine might
be essential for the enzyme’s catalytic activity. Inhibition
of invertase activity by HgCl2 was also reported for in-
vertases from Thermotoga neapolitana,2 sugarcane48 and
bamboo cell.49 Cysteine modifying agents iodoacetamide,
PCMB and DTNB inhibited betel leaf invertase activity.
Consistent with the inhibition by HgCl2, cysteine modify-
ing agents also significantly diminished invertase activity
implying the importance of free sulfhydryl group contain-
ing amino acid e.g. cysteine for the catalytic activity of the
enzyme. The necessity of free sulfhydryl group was also re-
ported for invertase from T. neapolitana,2 Ricinus com-
munis50 and Aspergillus niger.51 However, the necessity of
disulfur bond was noticed for invertase from Lactobacillus
reuteri CRL 1100.52
ably a complex with other proteins or enzymes under phys-
iological conditions. End product inhibition of invertase is
typical in many species.4,48,54,55 Betel leaf invertase was
also inhibited by the end products glucose and fructose.
This product inhibition may be important control point of
cytoplasmic sucrose metabolism. A deficiency of glucose
and fructose may increase sucrose cleavage by activating
invertases. However, an excess of cytoplasmic glucose and
fructose than their demand may decrease sucrose break-
down directly through inhibition of invertases.
The N-terminal sequence of betel leaf invertase was
determined which shares a very high sequence similarity
with the N-terminal sequences of homologous invertases in
the data base e.g. 72% similarity with Daucus carota in-
vertase (NCBI accession CAA49162), 65% with Nicotiana
tabacum and Helianthus annuus invertase (NCBI acces-
sion CAA57428 and AAY85659), 63% with Cichorium
intybus invertase (accession CAA72009), 60% similarity
with Coffea canephora (accession ABI17894) and Oryza
sativa subsp. Indica (accession AAD38399) invertase,
58% with Arabidopsis thaliana invertase (accession
BAA89048) and 56% with Beta vulgaris invertase (acces-
sion CAD19322). This high overall sequence similarity
with homologous invertases implies that the betel leaf
invertase is an authentic member of invertase family. Clon-
ing and expression of recombinant betel leaf invertase will
explore further insight into the structure-function relation-
Betel leaf invertase activity was stimulated by vari-
ous proteins and lectins. Higher plant invertase usually be-
comes activated by various proteins in vitro. Though in-
vertase activation by various proteins was detected in vitro,
it is thought to be important also for in vivo invertase activ-
ity. Plant acid invertases together with many soluble pro-
teins are reported to localize in vacuole7,53 and protein-
invertase interaction can therefore take place in vivo.
Higher plant acid invertases typically bind to proteins in vi-
tro suggesting that the functional state of invertase is prob-

Purification of Betel Leaf Invertase
J. Chin. Chem. Soc., Vol. 58, No. 3, 2011 395
ship of the enzyme.
tein was removed and the active bound protein was eluted
with a linear gradient of 0.0 to 0.5 M NaCl in AS-5.0 at a
flow rate of 30 mL/h. The active fraction, for further purifi-
cation, was concentrated in an Amicon cell (YM-10 mem-
brane; 10,000 MW) and loaded onto a HiLoad 16/60
Superdex 200 (GE Healthcare) column pre-equilibrated
with AS-5.0. The proteins were eluted with the same buffer
at a flow rate of 20 mL/h and fractions of 2 mL were col-
lected. The active fraction containing invertase activity
was checked for purity by SDS-PAGE as the method of
Laemmli.31 The purified enzyme was stored frozen for fur-
ther characterization. The molecular mass (Mr) of the in-
vertase was determined by gel filtration using the same col-
umn and conditions as for purification. The apparent Mr of
the betel leaf invertase was estimated by comparing its
migration with protein marker on SDS-PAGE in the ab-
sence and presence of 2-mercaptoethanol.
This study revealed that the functional properties of
betel leaf invertase show a good agreement with most
invertases studied to date. Thus due to low price and avail-
ability of betel leaf in all seasons, betel leaf invertase can be
used commercially. Industries based on the production of
invertase from betel leaves can be set up in South Asia spe-
cially in Bangladesh where a significant amount of betel
leaves are produced every year.
EXPERIMENTAL
Collection of betel leaves
A variety of betel leaf locally called DOGA was col-
lected from the area, Mohonpur in the district of Rajshahi,
Bangladesh. After collection, the leaves were cleaned and
used for this study.
Purification of invertase enzyme from betel leaf
Betel leaves were cut into small pieces and grinded in
a mortar and pestle with AS-5.0 buffer and finally crushed
into paste using homogenizer. The temperature was main-
tained at 4 °C for all purification steps. The homogenate
was filtered through four layers of cheesecloth and the fil-
trate was centrifuged at 15,000 rpm for 30 minutes. The
clear supernatant was used as crude enzyme extract. Solid
ammonium sulfate was added slowly to the crude extract
and the precipitate formed between 20 and 80% salt satura-
tion was collected by centrifugation at 15,000 rpm for 30
minutes. The precipitate was resuspended in a small vol-
ume of AS-5.0, dialyzed against buffer AS-5.0, centrifuged
at 15,000 rpm for 15 minutes and the supernatant was ad-
sorbed to DEAE-Sephacel column (2.6 × 15 cm) pre-equil-
ibrated with buffer AS-5.0. The column was washed with
the equilibrium buffer for complete elution of non-bound
protein and then the column bound proteins were eluted
with 0.1 M, 0.2 M, 0.25 M and 0.3 M NaCl in buffer AS-5.0
at a flow rate of 30 mL/h. The non-bound protein and the
protein fraction eluted with 0.25 M NaCl exhibits invertase
activity. However, this study focuses on DEAE-Sephacel
bound active invertase which was then applied to CM-
Sepharose cation exchange column (2.6 × 15 cm) pre-
equilibrated with AS-5.0. The active invertase eluted from
the column as non-bound protein and the bound proteins
containing no invertase activity were eluted with 0.15 M
and 0.3 M NaCl in AS-5.0 at a flow rate of 30 mL/h.
Invertase obtained from CM-Sepharose column was ad-
sorbed to a Mono Q column (GE Healthcare; 0.5 × 5.0 cm)
pre-equilibrated with AS-5.0. The inactive non-bound pro-
Total sugar content of invertase
The amount of total sugar was measured by the phe-
nol sulfuric acid method with D-glucose as standard.32
Invertase activity assay
The reaction mixture contained 100 mM acetic acid-
sodium acetate buffer (pH 5), 10 mM sucrose and an appro-
priate volume of enzyme. The mixture was incubated at 37
ºC for 30 minutes and the reaction was stopped by adding
500 mM Tris-HCl buffer (pH 8.5). The invertase activity
was determined by measuring the amount of reducing
sugar formed.33 One unit of invertase activity was defined
as the amount of enzyme required for liberating 1 mmole of
reducing sugar from the breakdown of sucrose per minute
at 37 ºC.
Substrate specificity and enzyme kinetics
The enzyme mixture containing 10 mM of each sub-
strate (cellobiose, lactose, maltose, raffinose, stachyose,
sucrose, trehalose, melezitose and inulin) was incubated at
37 ºC for 30 min and the activity was determined. Michae-
lis-Menten constant (Km) and maximum velocity (Vmax)
were determined as a function of the sucrose concentration
at 37 ºC. Km and Vmax were calculated from the slope and
intercept of the regression line of Lineweaver-Burk plots.34
Effect of pH and temperature on invertase activity
The invertase activity was measured in various buff-
ers of different pH ranging from 2.0 to 9.0. The reaction
mixture was prepared using Glycine-HCl buffer (pH 2-3),
acetate buffer (pH 4-6), phosphate buffer (pH 6.5-7.5) and
Tris-HCl buffer (pH 8-9). The effect of temperature on en-
zyme activity was tested in the range of 20-80 °C using ace-

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tate buffer, pH 5.2.
Effect of metal ions and chemical modification agents
on invertase activity
The effects of various metal ions on invertase activity
were examined. The reaction mixture was incubated for 30
min at 37 ºC with one of the following compounds in ace-
tate buffer, pH-5.2: KCl, MnCl2, MgCl2, HgCl2, CaCl2,
CuCl2, ZnSO4, AgNO3, iodoacetamide, PCMB and DTNB.
The residual activity was measured.
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Effect of end products on invertase activity
The invertase activity was measured against sucrose
in the presence of its reaction products, glucose and fruc-
tose. The enzyme mixture containing 1, 5 and 10 mM of
each of glucose and fructose was incubated at 37 ºC for 30
min. To assess the effect of glucose on invertase activity,
the fructose production was measured by the enzyme cou-
pling method using fructose dehydrogenase.35 To study the
effect of fructose on invertase activity, production of glu-
cose was determined by glucose test kit (Wako Pure Chem-
ical Co., Japan) according to manufacturer’s protocol.
N-terminal amino acid sequence analysis
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fied protein band was transferred from SDS-PAGE gel
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bilon PVDF Transfer Membrane, Millipore Corp., Bed-
ford, MA) and was stained with Commassie Brilliant Blue
R-250 according to Matsudaira.36 The PVDF membrane
was then dried, excised and sequenced by automated Edman
degradation using Beckman LF3000 Protein Sequencer as
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ACKNOWLEDGMENT
The authors thank to Dr. Fuzio Kawasaki, Dept. of
Bioscience and Biotechnology, Kyushu University, Japan
for kindly performing the N-terminal sequence analysis.
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