Plant Physiol. (1990) 92, 679-683
Received for publication June 5, 1989
and in revised form September 19, 1989
0032-0889/90/92/0679/05/$01 .00/0
Purification and Partial Characterization of a
Fructanase which Hydrolyzes Natural Polysaccharides from
Sugarcane Juice1
M. Estrella Legaz*, Luisa Martin, Mercedes M. Pedrosa, Carlos Vicente, Roberto de Armas, Maritza Martinez,
Isabel Medina, and Carlos W. Rodriguez
Laboratory of Plant Physiology, Faculty of Biology, Complutense University, 28040 Madrid, Spain (M.E.L., L.M.,
M.M.P., C.V.), and Department of Plant Physiology, Faculty of Biology, La Havanne University, Cuba (R.d.A., M.M.,
I.M., C.W.R.)
ABSTRACT
Substrates Preparation
A new sugarcane (Saccharum officinarum L.) fructanase which
hydrolyzes both high molecular weight polysaccharides
IFructose4:Galactitol5l,, (SP) and moderate-sized carbohydrates
IFructose2:Galactitol33l,, (MMWC) has been purified from sugarcane juice. The Km, value has been estimated to be 33.7 micrograms per milliliter and 20 micrograms per milliliter for SP and
MMWC, respectively. The optimal pH and temperature values are
6.0 and 300C, respectively. Purified protein has a pi value of 6.35
and a molecular weight of 13.2 kilodaltons. Fructanase activity
appears to be Mn2 -dependent.
Stalks from 11 month-old plants were mechanically crushed
immediately after being cut, and the crude juice was brought
to 5% (w/v) with trichloroacetic acid and centrifuged at
20,000g for 30 min at room temperature. The pellet was
discarded, and the supernatant was adjusted to pH 8.0 by
adding a saturated solution of ammonium carbonate. The
juice was recentrifuged at 20,000g for 15 min at room temperature. The supernatant was filtered through Whatman No.
4 paper. Sodium azide was added to the filtrate to obtain a
final concentration of 0.02% (w/v).
This clarified juice was then filtered through a column of
Sephadex G- 10 (15 x 2.5 cm) preequilibrated saturated ammonium carbonate containing 0.02% sodium azide. Elution
was carried out with distilled water. Fractions (1 mL) 1 to 20
were discarded. Fractions 20 to 32 mL were collected and
filtered through a Sephadex G-50 column (30 x 2.5 cm)
preequilibrated as above. Fractions 40 to 70 contained the SP
fraction, whereas MMWC eluted in fractions 70 to 120 mL.
HPLC analyses showed the absence of sucrose and monosaccharides in fractions 40 to 120.
Normal carbohydrate metabolism in sugarcane produces a
heterogeneous pool of soluble polysaccharides. This pool includes arabino-galactans (11), starch-like glucose polymers (6)
that contain some 1,6-glucans (7), and 1,4-glucans (12). Recently, heteropolymers containing both fructose and galacitol
have been described (R de Armas, unpublished data).
Mechanical injury and some storage conditions result in an
accumulation of these polysaccharides in sugar cane juice.
Glucans, coloidal fructans, and soluble MMWCs,2 such as
sarkaran (4), have been found after stalk deterioration. The
amount of total polysaccharides in sugarcane juice increases
with mechanical injury, the age of plants, and oxygen content
(15, 16). Varietal differences in soluble carbohydrates have
also been reported (13).
No information is available on the synthesis and breakdown
of these carbohydrates. Thus, we undertook an effort to purify
and characterize the enzyme which uses the polysaccharides
present in sugarcane as substrates.
Enzyme Extraction and Partial Purification
Crude extracts from crushed stalks (100 mL) were brought
to 30, 50, 70, and 90% (w/v) with ammonium sulfate and
stored for 4 h at 2°C. Suspensions were centrifuged at 27,000g
for 1 h at 2°C, and the pellet was resuspended in 100 mL of
1 mM sodium citrate buffer (pH 6.0). The supernatants and
precipitates were dialyzed against 5.0 L of 1 mm sodium
citrate containing 0.02% sodium azide (w/v) for 60 h at 4°C.
The supernatant from 90% saturation with ammonium sulfate contained the highest hydrolase activity. Thus, this fraction was adsorbed on calcium phosphate gel (75 mg dry per
mg protein), and the protein was desorbed with increasing
concentrations of citrate buffer (pH 6.0), from 1 to 70 mM (5
mm increments). The highest specific activity was obtained in
the fraction desorbed with 10 mM citrate.
The 10 mm citrate fraction was then dialyzed against distilled water overnight at 4°C and was lyophilized. The residue
was resuspended in 4.0 mL distilled water and electrofocused.
Electrofocusing was performed on an LKB 8100-1 column,
110 mL volume. Gradients were prepared with sucrose (den-
MATERIAL AND METHODS
Plant Material
Sugarcane (Saccharum officinarum L.) var. C-734-72, field
grown, was used throughout this work.
'This work
Universities.
was
supported by La Havanne and Complutense
2Abbreviations: MMWC, carbohydrates of midmol wt; SP, high
mol wt soluble polysaccharides.
679
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Plant Physiol. Vol. 92, 1990
LEGAZ ET AL.
680
sity) and Servalyt 3- 10 (pH) at 1% (w/v). Electrofocusing was
developed at 1,000 V for 48 h at 10°C. Three-mL fractions
were recovered and dialyzed against 5.0 L 1 mm sodium
citrate buffer (pH 6.0), containing 0.02% sodium azide (w/v)
for 4 d before protein measurement.
Enzyme Assay
Hydrolase activity was measured in reaction mixtures containing 3.0 jg protein, 20 ,imol sodium citrate (pH 6.0), 5.0
,imol Mn2" (as MnCl2), and 2.0j,g MMWC or 8.0,ug SP in a
final volume of 1.3 mL. Reactions were carried out for 30
min at 30°C and stopped by adding sufficient 2 N NaOH to
give a pH of 8.0. Blanks contained no substrates or protein.
Fructose produced during the reaction was determined by
reaction with dinitrosalicylic reagent and the developed color
was measured at 540 nm (14). Absorbance was transformed
by using a straight line calibration made with known concentrations of fructose. Protein was measured by the method of
Lowry et al. (10) using bovine serum albumin as a standard.
One unit of activity was defined as 1.0 ,imol of fructose
produced per mg protein per min. Mn2" was substituted, as
indicated, by other cations.
Determination of Mol Wt
Approximately 10 jAg of purified fructanase was chromatographed on a 30 x 7.8 cm PWSX GO209 HPLC column
packed with G5000 PWXL (3), equilibrated with 10 mm
sodium citrate buffer (pH 6.0), using a Spectra Physics SP8800
liquid chromatograph equipped with a SP 4290 computer.
Blue dextran was used to measure the void volume. Tyroglobulin (669 kD), apoferritin (443 kD), bovine serum albumin (66 kD), alcohol dehydrogenase (40 kD), carbonic anhydrase (29 kD), and Cyt c (12.4 kD), from Sigma Chemical
Co., were used as mol wt markers. The elution volume of
fructanase and standards were determined by absorbance at
280 nm. Mol wt of fructanase was determined by plotting log
mol wt versus log ( Vel /V), where VO is the void volume of the
column and Ve is the elution volume of a protein.
Analysis of Sucrose and Monosaccharides by HPLC and
GLC
SP and MMWC as well as reaction mixtures were lyophilized; the residues were extracted with 80% cold ethanol and
stored at -1I3C for 14 h. Then, the precipitate was
discarded and the supernatant heated at 60°C for 20 min.
One mL of 80% (v/v) cold ethanol was added to 1.0 mL of
clear supernatant and heated three times as above. After
heating, the suspension was centrifuged at 3000g for 15 min.
The precipitate was discarded and the supernatant dried in
air flow (8). The residues were resuspended in 0.5 mL acetonitrile:water (80:20 v/v) and filtered through Millipore GS
filters (0.22,um pore diameter).
HPLC analysis was performed in a Varian 5060 liquid
chromatograph equipped with a Vista CDS 401 computer,
according to Legaz et al. (9). Chromatographic conditions
were as follows: column, MicroPak-NH2 10 P/N (30 cm x 3
mm i.d.); loading, 10 ,uL; mobile phase, acetonitrile:water
(80:20, v/v); flow rate, 1.3 mL min-'; temperature, 20°C;
pressure, 90 atm; detector, UV set at 195 nm; absorbance
range, 0.05; attenuation, 64; internal standard, 2.0 mg mL-'
ribose.
Alternatively, residues were dissolved with 1.0 mL pyridine
and derivatized with 1% trimethylchlorosilane according to
Bandurski and Ehmann (1) to be analyzed by GLC. Chromatographic conditions were as follows: column (1.5 m x 2
mm i.d.) packed with 3% OV17 on Chromosorb W HP, 80
to 100 mesh, particle diameter, 0.165 mm; temperature of
the detector, 350°C; temperature of the injector, 250°C; hydrogen flow rate, 30 mL min-'; air flow rate, 500 mL min-';
nitrogen (as carrier gas) flow rate, 20 mL min-'. Column
temperature was programmed as follows: 2 min at 150°C,
temperature was increased to reach 180°C at 5°C min-' intervals; 2 min at 180°C; then, temperature was newly increased
to reach 280°C at 10°C min-' intervals. Ten mg ribose was
added to the samples, as internal standard, before derivatization. As external standards, arabitol, galactitol, mannitol,
ribitol, D-arabinose, D-fructose, D-galactose, D-glucose, Dmannose, D-rhamnose, sucrose, and D-xylose were used at a
concentration of 2.0 mg mLU' for HPLC or 10 mg mLU' for
GLC. These standards were provided by Sigma Chemical Co.
were
RESULTS
The enzyme has been purified 171-fold with an overall
yield of 16.3% (Table I). This enzyme preparation was used
for kinetic analysis.
The kinetics of the saturation of the enzyme by SP yielded
a sigmoidal rather than hyperbolic curve (Fig. IA). The ap-
Table I. Purification of a Fructanase from Sugarcane Juice
Step
Clarified juice
Supernatant from 90%
saturation with ammonium sulfate
Eluate with 10 mm citrate
buffer from calcium
phosphate gel
Electrofocusing at pH 6.35
Total
Specific Activity
Total Activity
MMWC
SP
Yield
MMWC
SP
Purification
Volume
Protein
Protein
mL
mLg1
mg
100
120
0.583
0.014
58.30
1.68
0.024
0.83
0.011
0.23
1.40
1.39
0.64
0.39
100
99.3
100
60.4
34.58
20.91
30
0.020
0.60
1.94
0.43
1.16
0.26
82.8
40.3
80.83
39.1
3
0.043
0.13
3.74
1.88
0.37
0.24
26.4
38.2
MMWC
SP
MMWC
-fold
%
units
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Copyright © 1990 American Society of Plant Biologists. All rights reserved.
SP
155.8
171.0
FRUCTANASE IN SUGAR CANE JUICE
681
Table II. Effect of Several Cations on Hydrolase Activity
Substrate
SP
Cation (5 mM)
None
Mn2+
Mg2+
Ca2+
Ba2+, Zn2+, Cd2+,
Hg2+ or C02+
%
activity
units
0.0
1.88
0.64
0.22
0.0
0.0
100
34
11.7
0.0
Specific
activity
%
activity
units
0.0
3.74
1.38
0.75
0.0
100
36.9
20
0.0
0.0
uz
Figure 1. SP saturation curve of fructanase at 300C and pH 6.0.
Values are the average of four determinations. Inset A, Hill replot of
data. The nH value was estimated to be 2.5 from this plot. Inset B1
Hanes replot of data. The Km value was estimated to be 33.7 ,g mL1
from this plot.
A
MMWC
Specific
activity
._
n
E
0 y=2.38x - 0.35; r2 0.86
1.2
pH
'0.8
0
E
,0.
10
0
0°
8
w-0.4
-0.2
6
L .0.
E 0
,
7 0.4
0 0
og ES] *
0.8 /
0
0.5
B y= 0.0013x +0.26
0 00
7
0.1
0
0
1.0
00
030 0
00
~~~0.2
~~~~
2
0
as
~~~~SV
.
0
s
2
Figure 3. pH response of fructanase using MMWC (0) and SP (U)
substrates, respectively. Values are the average of four determinations.
0
1.5
2.0
ES]
2
1
2.5
3.0
3
3.5
[SI
6/
4.0
50
pig/ml
Figure 2. MMWC saturation curve of fructanase at 300C and pH
6.0. Values are the average of four replicates. Inset A, Hill replot of
data. The nH value was estimated to be 2.38 from this plot. Inset B,
Hanes replot of data. The Km value was estimated to be 20 ug mL-'
from this plot.
parent Km value for SP was 33.7 Ag mL-', as deduced from
the Hanes plot (Fig. 1B). The interaction coefficient of the
enzyme with the substrate is approximately 2.0, as determined
by a Hill plot (Fig. IC). Such a value agrees with an allosteric
behaviour for this hydrolase.
The kinetics of saturation of the enzyme by MMWC gave
a similar sigmoidal relationship (Fig. 2A) with an apparent
Km value of 20 ,ug mL-' MMWC (Fig. 2B) and an interaction
coefficient near 2.0 (Fig. 2C).
Enzyme activity is clearly dependent on the presence of
Mn2+ in the reaction mixtures (Table II). Mg2' partially
>2
-
0
0
10
20 30 40 50 60
Temperature (°C)
70
Figure 4. Temperature response of fructanase using MMWC (@) and
SP (U) as substrates, respectively. Values are the average of four
determinations.
substitutes Mn2+ whereas other cations were ineffective. The
optimum pH value for hydrolase activity was approximately
6.0 (Fig. 3). Enzyme activity was only 23% of its maximal
value with MMWC as a substrate and 7% with SP when
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Copyright © 1990 American Society of Plant Biologists. All rights reserved.
682
Plant Physiol. Vol. 92, 1990
LEGAZ ET AL.
r
3.4
-
I
I
3.0
pH
1;2
SA:0.05
pI 8.6
1 000 o
OODP000000
0o
-
SA:162 units
2.6
2.2
0
p-.22 0
~
1.8
i
V
1.44 3
*
000
6-
D
0
01. 6
00
~~SA=1.62units
l2
oo
oo
0
.2
pH
0
° SA-0.03
*
4
l
p1:8
0
~
~
~ 20 3
2002
4
2
4
0
4
5
m
00000
05
I
C
D
f
d
E
c
0
9
18
27
36 45 54
63 72
81
90
99
ml
Figure 5. Electrofocusing determination of pi value of sugar cane
fructanase using (A) clarified crude juice and (B) partially purified
fructanase.
assayed at pH 7.0. The optimum reaction temperature was
30°C (Fig. 4). the enzyme retained only a 21% of its maximal
activity against MMWC and 17% against SP at 40°C and was
completely inactive at 70°C.
Electrofocusing of partially purified fructanase showed that
the protein focused at pH 6.35 (Fig. 5). Purified native fructanase migrated as a monomer in G5000 PWXL column with
a Ve/Vo value of 0.275. Values of Ve Vo ratio for the standards
were 0.093 for tyroglobulin, 0.127 for apoferritin, 0.165 for
bovine serum albumin, 0.189 for alcohol dehydrogenase,
0.233 for carbonic anhydrase, and 0.296 for Cy c. The apparent mol wt of fructanase was then 13.2 kD. A slightly active
isoform with a pl value of about 8.5 copurified with the main
form during electrofocusing.
HPLC analysis of the MMWC and SP preparations confirmed that neither contained sucrose nor monosaccharides
(Fig. 6, C and E). The hydrolysis of SP and MMWC results
in free fructose and galactitol (Fig. 6, D and F). The presence
of both hexitol and ketose has also been confirmed, as trimethylsilil derivatives, by GLC (Fig. 7).
DISCUSSION
This work demonstrates the presence in mature stalks of
sugar cane of an enzyme that hydrolyzes the heterogeneous
polysaccharides produced in these tissues. The end-products
of enzyme hydrolysis are fructose and galactitol, as reported
by R de Armas et al. (unpublished data) and confirmed here
by HPLC and GLC (Figs. 6 and 7). Polyols, including galactitol, have never been reported in sugarcane juice (5). Fermented sugarcane juice contains manitol, ethanol, and lactic
acid (17).
The fructanase described in this work behaves as an allosteric enzyme. Its affinity for MMWC is 1.7 times higher than
for SP (Figs. 1 and 2). This may be related to the structural
U,
s
iI
IV
r
I
I
I
I
3
6
9
12
a
15
3
6
.
.
.
9
12
15
c
F
E
-.0
o
U,
.0
to
r
s
s
0
r
d
v
________________________
p
pA
4
8 12 16 20 0 4 8 12 16 20
minutes
Figure 6. HPLC analysis of substrates and reaction mixtures. A,
Chromatographic trace of standards fructose (f) and galactitol (d); B,
chromatographic trace of mannose (m) and galactitol (d); C, chromatographic trace of MMWC before and D, after hydrolysis for 60
min at 300C by a purified fructanase; E, chromatographic trace of SP
before and F, after hydrolysis for 60 min at 300C by a purified
fructanase. Arrows indicate remaining, partially depolymerized substrates after reaction. S, solvent; R, ribose as intemal standard.
differences in the polymerization degree of polysaccharides.
MMWC is thought to be a IFructose2:Galactitol3lI polymer,
while SP has been described as a IFructose4:Galactitol5l.
polysaccharide (R de Armas et al., unpublished data). Although acidic hydrolysis of MMWC and SP produces more
free galactitol than fructose, their enzymatic hydrolysis produces three times more fructose than the polyol. This could
imply that the bond joining the polyol in the polysaccharide
sequence is more resistant to enzymatic hydrolysis than the
glycosidic bond between fructose units (2).
There is no evidence about the physiological role of polysaccharides in sugar cane. Valdes et al. (16) hypothesized that
their synthesis is a response to mechanical injuries to parenchymatous cells of stalks. In any event, one enzyme seems to
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FRUCTANASE IN SUGAR CANE JUICE
3.
4.
5.
6.
7.
8.
9.
Figure 7. GLC analysis of trimethylsilil derivatives of standards (A)
and reaction mixture after hydrolysis of MMWC for 60 min at 300C
by a purified fructanase (B). In (C) samples used in (B) were loaded
with 10 mg galactitol before derivatization to confirm the chemical
nature of the peak which elutes at 1960C. a, arabinose; x, xilose; r,
ribose; f, fructose; g, glucose; d, galactitol; s, sucrose. Ribose was
used in (B) and (C) as internal standard.
be enough to hydrolyze both SP and MMWC, in spite of their
biochemical origin.
10.
11.
12.
13.
14.
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