Processing of soybean products by semipurified plant and microbial α-galactosidases

Article abstract of DOI:10.1021/jf0617162

Galactooligosaccharides (GO) are responsible for intestinal disturbances following ingestion of legume-derived products. Enzymatic reduction of GO level in these products is highly desirable to improve their acceptance. For this purpose, plant and microbial semipurified α-galactosidases were used for GO hydrolysis in soybean flour and soy molasses. α-Galactosidases from soybean germinating seeds, Aspergillus terreus, and Penicillium griseoroseum presented maximal activities at pH 4.0-5.0 and 45-65°C. The K _M,app values determined for raffinose by the soybean, A. terreus, and P. griseoroseum α-galactosidases were 3.44, 19.39, and 20.67 mM, respectively. The enzymes were completely inhibited by Ag⁺ and Hg²⁺, whereas only soybean enzyme was inhibited by galactose. A. terreus α-galactosidase was more thermostable than the enzymes from the other two sources. This enzyme maintained about 100% of its original activity after 3 h at 60°C. The microbial a-galactosidases were more efficient for reducing GO in soybean flour and soy molasses than soybean enzyme.

Full text of DOI:10.1021/jf0617162

10184 J. Agric. Food Chem. 2006, 54, 10184−10190
Processing of Soybean Products by Semipurified Plant and
Microbial -Galactosidases
r
DANIEL L. FALKOSKI, VALEÄ RIA M. GUIMARA˜ ES, CARINA M. CALLEGARI,
ANGEÄ LICA P. REIS, EVERALDO G. DE BARROS, AND SEBASTIA˜ O T. DE REZENDE*
BIOAGRO, Universidade Federal de Vic¸osa, Vic¸osa, MG 36.570-000, Brazil
Galactooligosaccharides (GO) are responsible for intestinal disturbances following ingestion of legume-
derived products. Enzymatic reduction of GO level in these products is highly desirable to improve
their acceptance. For this purpose, plant and microbial semipurified R-galactosidases were used for
GO hydrolysis in soybean flour and soy molasses. R-Galactosidases from soybean germinating seeds,
Aspergillus terreus, and Penicillium griseoroseum presented maximal activities at pH 4.0-5.0 and
45-65 °C. The KM,app values determined for raffinose by the soybean, A. terreus, and P. griseoroseum
R-galactosidases were 3.44, 19.39, and 20.67 mM, respectively. The enzymes were completely
inhibited by Ag+ and Hg2+, whereas only soybean enzyme was inhibited by galactose. A. terreus
R-galactosidase was more thermostable than the enzymes from the other two sources. This enzyme
maintained about 100% of its original activity after 3 h at 60 °C. The microbial R-galactosidases were
more efficient for reducing GO in soybean flour and soy molasses than soybean enzyme.
KEYWORDS: Soybean; r-galactosidase; galactooligosaccharides; Aspergillus; Penicillium
INTRODUCTION
present stability over a wide range of pH values and tempera-
tures (8).
Human consumption of soy products is increasing, not only
because of their high nutritional value but also in view of their
health-promoting effects, such as reduction in cardiovascular
disease, osteoporosis, and cancer risks (1). The consumption is
limited partially due to the presence of R-galactooligosaccharides
(GO) such as melibiose, raffinose, stachyose, and verbascose
in soybean. Because humans and monogastric animals lack the
enzyme R-galactosidase (EC 3.2.1.22, R-D-galactoside galac-
tohydrolase), necessary to hydrolyze the R-linkages in these
sugars, they pass on intact into the large intestine, where
anaerobic microorganisms ferment them and cause gastrointes-
tinal disturbances (2). Many attempts have been proposed to
reduce soy GO contents through dehulling, soaking, cooking,
γ irradiation, aqueous or alcoholic extraction, germination, and
fermentation (3, 4). However, the enzymatic processing of soy-
derived products seems to be the most rational and effective
alternative. The nutritional value of soy foods could be upgraded
by procedures using microbial or plant R-galactosidases to
hydrolyze the R-galactosides prior to consumption (5, 6). The
use of plant R-galactosidases for GO reduction in soy products,
particularly from soybean sources, is advantageous because this
enzyme is uniquely suited for the high-protein and strongly
buffered environment of soybean products (7). On the other
hand, fungal R-galactosidases are generally secreted in the
culture facilitating the purification process. In addition, they
In the present investigation we describe the purification and
characterization of R-galactosidases from soybean seeds and
from the fungi Aspergillus terreus and Penicillium griseoroseum
and the hydrolysis of soybean flour and soy molasses by these
enzymes. Our long-term goal is to select efficient R-galactosi-
dase sources for technological use.
MATERIALS AND METHODS
Enzyme Sources. Soybean seeds (Glycine max L. Merr. cv.
Monarca) were provided by the soybean breeding program of BIO-
AGRO, Federal University of Vic¸osa, MG, Brazil. A. terreus was
obtained from the Andre´ Toselo Tropical Research Foundation,
Campinas, SP, Brazil. P. griseoroseum was obtained from the Fungal
Collection of the Microbiology Department of the Federal University
of Vic¸osa, MG, Brazil.
r-Galactosidase Production. Soybean seeds were germinated on
water-soaked filter paper for 60 h at 27 °C and then kept at -20 °C.
The pregerminated seeds (800 g of fresh weight) were ground in a
blender and resuspended in 1.7 L of solution containing 0.1 M citric
acid and 0.05 M sodium phosphate, pH 5.0, and kept under agitation
for 1 h at 4 °C. The suspension was filtered through cheesecloth, and
the filtrate was centrifuged at 15300g for 40 min at 4 °C. The
supernatant was used as a crude enzyme preparation.
The stock cultures of A. terreus and P. griseoroseum were maintained
on potato dextrose agar medium (PDA). For enzyme production, spores
(107/mL) were transferred to 1.0 L of liquid medium containing (in
g/L) KH2PO4, 7.0; K2HPO4, 2.0; MgSO4‚7H2O, 0.1; (NH4)2SO4, 1.0;
yeast extract, 0.6; and wheat straw or locust bean gum, 10, for A. terreus
and P. griseoroseum, respectively. The wheat straw was obtained from
a local market, and locust bean gum was purchased from Sigma
* Corresponding author [telephone (+5531)3899-2374; fax (+5531)-
3899-2864; e-mail srezende@ufv.br].
10.1021/jf0617162 CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/05/2006

Processing of Soybean Products
J. Agric. Food Chem., Vol. 54, No. 26, 2006 10185
Chemical Co. (St. Louis, MO). Cultures were incubated under constant
agitation at 120 rpm for 168 h at 28 °C (A. terreus) or for 132 h at 28
°C (P. griseoroseum). Culture filtrates were collected by filtration
through filter paper, concentrated by lyophilization, and kept at -20
°C until use.
(KM,app) and Vmax,app for pNPGal, raffinose, and melibiose hydrolysis
were calculated by using the Michaelis-Menten plot. The substrate
concentrations ranged from 0.01 to 2.00 mM in the case of pNPGal,
from 0.1 to 40.0 mM for raffinose, and from 0.05 to 60.0 mM for
melibiose.
r-Galactosidase Purification. The crude soybean extract was kept
at -20 °C for 24 h, thawed, and centrifuged at 15300g for 35 min at
4 °C. The pH of the supernatant was lowered to 4.0 with citric acid.
The solution was stirred for 30 min at 4 °C and centrifuged as described
above. The pH of the supernatant was adjusted to 5.0 with a NaOH
saturated solution and then submitted to fractionation with 20-50%
(NH4)2SO4 (7). Protein precipitated at 50% saturation, which contained
the R-galactosidase activity, was resuspended in 25 mM sodium acetate
buffer, pH 5.0, and loaded onto a Sephadex G-100 (Amersham
Biosciences, Uppsala, Sweden) column (95 × 2.5 cm) equilibrated with
25 mM sodium acetate buffer, pH 5.0. The proteins were eluted with
the same buffer, and the fractions containing R-galactosidase activity
were pooled and chromatographed on a CM-Sepharose Fast Flow
(Amersham Biosciences) column (14 × 2.9 cm) equilibrated with 50
mM sodium acetate buffer, pH 5.0. The proteins were eluted with a
linear gradient consisting of 200 mL of 50 mM sodium acetate buffer,
pH 5.0, and 200 mL of the same buffer containing 0.8 M NaCl. Active
protein fractions were pooled and concentrated by the use of an Amicon
ultrafiltration cell model 8400 (Bedford, MA) with a 10 kDa molecular
cutoff PM 10 Amicon membrane.
The A. terreus culture supernatant was concentrated by lyophilization
and chromatographed on a Sephacryl S-200 (Amersham Biosciences)
column (100 × 2.5 cm), equilibrated and eluted with 25 mM sodium
acetate buffer, pH 5.0. The active protein fraction was concentrated by
ultrafiltration using a PM 10 Amicon membrane filter. The concentrated
fraction was loaded on a phenyl-Sepharose (Amersham Biosciences)
column (15 × 2.9 cm) equilibrated with 25 mM sodium acetate buffer,
pH 5.0, containing 1 M (NH4)2SO4, and eluted by a negative linear
gradient consisting of 40 mL of the equilibration buffer and 40 mL of
the equilibration buffer without ammonium sulfate. Active protein
fractions were pooled and concentrated by ultrafiltration using a PM
10 Amicon membrane filter.
The P. griseoroseum culture supernatant was submitted to fraction-
ation with 40-100% (NH4)2SO4. Protein precipitated at 100% saturation
containing R-galactosidase activity was resuspended in 25 mM sodium
acetate buffer, pH 5.0, and loaded onto a Sephacryl S-200 column (100
× 2.5 cm) equilibrated with 25 mM sodium acetate buffer and eluted
with the same buffer. Active protein fractions were pooled and
concentrated by ultrafiltration using a PM 10 Amicon membrane filter.
All chromatography procedures were performed at 4 °C.
r-Galactosidase Assay. R-Galactosidase was assayed as described
previously (5). The reaction mixture contained 650 µL of 0.1 M sodium
acetate buffer, pH 5.0, 100 µL of enzyme solution, and 250 µL of 2
mM p-nitrophenyl-R-D-galactopyranoside (FNPGal) or other synthetic
substrates. The reaction was run for 20 min at 40 °C and ended by the
addition of 1 mL of 0.5 M sodium carbonate. The amount of
p-nitrophenol (pNP) released was determined at 410 nm. This procedure
was defined as the standard assay.
Substrate Specificity. Enzymatic assays were performed with
various synthetic and natural substrates. The reaction mixtures contained
650 µL of 0.1 M sodium acetate buffer, pH 5.0, 100 µL of enzyme
solutions, and 250 µL of synthetic substrates (2 mM), or maltose,
melibiose, lactose, and sucrose (16 mM), or raffinose and stachyose
(28 mM). The activities were measured under standard assay conditions
at 40 °C.
Effect of Ions, Simple Sugars, and Reducing Agents. The enzyme
samples were preincubated with each of the compounds (10 mM) in
0.1 M sodium acetate buffer, pH 5.0, for 20 min at 40 °C. After
preincubation, the effect of ions, simple sugars, and reducing agents
on the enzyme activity was determined according to the standard assay.
Treatment of Soy Products with r-Galactosidase. Commercial
defatted flour (Bunge Alimentos, RS, Brazil) (2 g) was mixed with
water 1:10 (w/v) and was added with 8 units of R-galactosidases purified
from soybean seeds, A. terreus, or P. griseoroseum. The mixtures were
incubated for 0, 4, 6, and 8 h under agitation (100 rpm) at 40 °C. Each
reaction mixture was lyophilized, and the soluble sugars were extracted
from 30 mg of dried powder with 80% aqueous ethanol (v/v) (5). The
solvent was evaporated at 45 °C, and the sugars were resuspended in
80% ethanol (1.2 mL) and analyzed by HPLC as described below.
Soybean molasses was mixed with water (1:5 w/v), and a 10 g sample
was incubated with 32 units of each R-galactosidase at 40 °C for several
periods of time. The GO hydrolysis efficiency was estimated by the
production of reducing sugars (10). One enzyme unit was defined as
the amount of enzyme that released 1 µmol of product per minute under
the assay conditions.
Determination of GO Content. Sugars were analyzed by HPLC
on a Shimadzu series 10A chromatograph (Kyoto, Japan), using the
analytical column Supelcosil LC-NH2 25 cm × 4.6 mm (Supelco,
Bellefonte, PA), eluted with an acetonitrile/water isocratic mixture (80:
20 v/v) at 35 °C, at a flow rate of 1 mL/min. The sugars eluted were
monitored by a refractive index detector model 6A from Shimadzu.
They were automatically identified and quantified by comparison with
retention times and known concentrations of the sugar standards sucrose,
raffinose, and stachyose, which were purchased from Sigma Chemical
Co. (6).
Protein Determination. The protein concentration in the enzymatic
extract was determined according to the Coomassie Blue binding
method with bovine serum albumin (BSA) as standard (12).
RESULTS AND DISCUSSION
The R-galactosidases from soybean germinating seeds and
those secreted by the fungi A. terreus and P. griseoroseum were
characterized in this work. R-Galactosidase from soybean
germinating seeds have been proposed to reduce GOs in
soybean-derived products (5). Although A. terreus and P.
griseoroseum are not food grade fungi, A. terreus is a source
of pharmaceutical products (13), and P. griseoroseum is a
potential source of pectinolytic enzymes for the food industry
(14). Enzyme production was done when R-galactosidase
activity was maximum. In soybean germinating seeds this
happened 60 h after imbibition (5, 6). For A. terreus and P.
griseoroseum maximum activity was reached after 168 and 132
h of culturing, respectively. These time points were previously
determined in our laboratory (data not shown).
Fractionation of crude extract from germinating soybean seeds
on gel filtration and ion exchange columns resulted in a 19.7-
fold purification with a 36.6% recovery of the enzyme activity
(Table 1). The R-galactosidases secreted by A. terreus were
partially purified using gel filtration and hydrophobic chroma-
tography procedures, with a purification factor of 20.2 and
The activities against melibiose, maltose, and lactose were evaluated
according to the glucose-oxidase method (9). When sucrose, raffinose,
and stachyose were used as substrate, the production of reducing sugars
was determined using the 3,5-dinitrosalicylate reagent (10).
Effect of pH and Temperature. The effect of pH on R-galactosidase
activities was determined within the pH range of 3.0-7.0 using
McIlvaine buffer (citric acid/sodium phosphate) at 40 °C (11) under
otherwise standard enzyme assay conditions. The optimum temperature
was determined within the temperature range of 35-70 °C, at pH 5.0.
Thermal stability was evaluated by preincubating 100 µL of enzyme
solutions with 650 µL of 0.1 M sodium acetate buffer, pH 5.0, for
different times and temperatures. Soybean and P. griseoroseum enzymes
were preincubated at 40, 45, and 50 °C for 0-10 and 0-5 h,
respectively. A. terreus enzyme was preincubated at 55, 60, and 65 °C
for 0-36 h. After preincubation, 250 µL of 2 mM pNPGal was added,
and the enzyme activity was determined.
Determination of Kinetic Parameters. Kinetic experiments were
performed at 40 °C and pH 5.0. The Michaelis-Menten constant

10186 J. Agric. Food Chem., Vol. 54, No. 26, 2006
Falkoski et al.
Table 1. Summary of the Steps for
R-Galactosidase Purification from Germinating Soybean Seeds Var. Monarca
total protein
total activity
specific activity
purification
(fold)
recovery
(%)
-
1
-
-1
purification step
(mg)
(mM min
)
(mM min 1 mg
)
crude extracta
13300.80
9036.71
5018.22
990.00
516.94
246.84
366.0
312.6
295.3
170.3
155.6
134.1
0.03
0.04
0.06
0.17
0.30
0.54
1.00
1.25
2.14
6.25
10.94
19.74
100
cryoprecipitationb
85.4
80.7
46.5
42.5
36.6
acid precipitationc
(NH4)2SO4 (20−50%)
Sephadex G-100
CM-Sepharose
a Crude extract was prepared from 800 g of pregerminated soybean seeds with 1.7 L of buffer solution. b Cryoprecipitation: crude soybean extract was kept at
for 24 h and then thawed, and the precipitate was removed by centrifugation at 15300g for 35 min at 4
C. c Acid precipitation: the pH of the enzyme solution was lowered
to 4.0 with citric acid, and the solution was stirred for 30 min at 4 C and centrifuged as described above.
−20 °C
°
°
Table 2. Summary of the Steps for
R-Galactosidase Purification from Aspergillus terreus
total protein
(mg)
total activity
specific activity
purification
(fold)
recovery
(%)
-
1
-
-1
purification step
(mM min
)
(mM min 1 mg
)
crude extracta
Sephacryl S-200
phenyl Sepharose
16.1
0.69
0.11
53.6
9.75
7.40
3.33
14.1
67.3
1.00
4.20
20.20
100
18.2
13.8
a The purification process started from 1 L of culture medium.
Table 3. Summary of the Steps for
R-Galactosidase Purification from Penicillium griseoroseum
total protein
(mg)
total activity
specific activity
purification
(fold)
recovery
(%)
-
1
-
-1
purification step
crude extracta
(mM min
)
(mM min 1 mg
)
70.0
27.0
1.94
170
140
108
2.02
5.67
55.0
1.00
2.81
27.2
100
82.2
63.1
(NH4)2SO4 (40−100%)
Sephacryl S-200
a The purification process started from 1 L of culture medium.
recovery of 13.8% (Table 2). P. griseoroseum R-galactosidase
was purified 27-fold with 63% recovery of the enzyme activity
(Table 3).
at 40 °C, respectively (Figure 2A). These values decreased to
60 and 33%, respectively, when incubation was performed at
45 °C. The half-life (t1/2) of this enzyme at 45 °C was 6 h and
36 min. At 50 °C, 91% of its original activity was lost after 30
min of incubation. These t1/2 values were higher than those
reported for soybean R-galactosidases extracted from other
varieties (5, 6).
Maximum R-galactosidase activities were detected at pH 5.0
(soybean), 4.0 (A. terreus), and 5.0 (P. griseoroseum) (Figure
1A). Similar optimum pH values were verified for R-galactosi-
dases of other fungi, such as Thermomyces lanuginosus (15),
Aspergillus niger (16), and Aspergillus oryzae (17) and for other
soybean varieties (5, 6). Optima pH values for crude extracel-
lular R-galactosidase from A. niger, A. oryzae, A. awamori, and
Rhizopus oligosporus grown in different carbon sources were
also in the acidic range (18).
The temperature effect on the R-galactosidase activities is
shown in Figure 1B. For R-galactosidases of germinating
soybean seeds, A. terreus, and P. griseoroseum, highest enzyme
activities were detected at 50, 65, and 45 °C, respectively.
Optimal temperature values in the range of 50-60 °C were
observed for crude extracellular R-galactosidases produced by
fungi from four different species (18).
The A. terreus R-galactosidase was more thermostable than
the enzymes from the other two sources (Figure 2). No
reduction in A. terreus R-galactosidase activity was observed
after a 24 h period at 55 °C. This enzyme maintained about
100 and 40% of its original activity after 3 and 36 h at 60 °C,
respectively (Figure 2B). The enzyme showed significant
stability for a wide range of temperatures, which is desirable
for industrial applications. Four forms of purified A. niger ATCC
46890 R-galactosidases were identified, which retained about
95% of the original activity after incubation at 40 °C for 21 h
(16).
No loss in enzyme activity was observed after incubation of
P. griseoroseum R-galactosidase for 5 h at 40 °C. The enzyme
maintained about 65% of its original activity after 2 h at 45 °C,
but about 90% of the activity was lost during an incubation
period of 30 min at 50 °C (Figure 2C). The t1/2 of P.
griseoroseum R-galactosidase at 45 °C was 3 h and 27 min.
This t1/2 value was lower than that reported for R-galactosidase
derived from Bacillus stearothermophilus NCIM 5146 (19).
The KM,app values for pNPGal, melibiose, and raffinose are
displayed in Table 4. The KM values determined in this study,
using pNPGal as substrate, were similar to those obtained for
R-galactosidases of germinating Cyamopsis tetragonolobus
seeds (20), Trichoderma reesei RUT C-30 (21), and Penicillium
species 23 (22). The KM values determined with melibiose were
close to that of R-gal I of A. niger (16), but lower than the KM
value observed for R-galactosidase of germinating soybean seeds
of the vareity Doko (5). The KM values determined for
R-galactosidases of A. terreus and P. griseoroseum with
raffinose, 19.39 and 20.67 mM, respectively, are close to that
obtained for R-galactosidase of B. stearothermophilus NUB3621
(23). However, a lower KM value, 3.44 mM, was determined,
with raffinose, for soybean R-galactosidase (Table 4). Lower
KM values indicate that the enzyme has more affinity for the
substrate and that the ES complex formation is not a limiting
step for the reaction. In practical terms KM is an important
R-Galactosidase from germinating soybean seeds retained 90
and 70% of its original activity after incubation for 4 and 10 h

Processing of Soybean Products
J. Agric. Food Chem., Vol. 54, No. 26, 2006 10187
Figure 1. Effects of pH (A) and temperature (B) on the activity of soybean
(
1
), Aspergillus terreus (b), and Penicillium griseoroseum (O) R-galac-
tosidase.
parameter; however, it is not the only characteristic determining
enzyme efficiency.
Under the experimental conditions used, the soybean R-ga-
lactosidase was more effective in the hydrolysis of pNPRGal,
followed by the GO raffinose, stachyose, and melibiose (Table
5). On the other hand, the substrate stachyose was more
efficiently hydrolyzed than raffinose by enzyme preparations
from A. terreus and P. griseoroseum. The more effective
hydrolysis of the synthetic substrate FNPRGal compared to the
natural oligosaccharide hydrolysis was also observed for grape
flesh R-galactosidase (24). The three preparations containing
R-galactosidase activity hydrolyzed raffinose more efficiently
than melibiose. Lactose and synthetic substrates containing
â-linkages or containing xylose, mannose, and glucose residues
were poorly hydrolyzed by the enzyme preparations.
The microbial enzyme preparations contained other enzymes
such as invertase, as they were able to hydrolyze sucrose (Table
5). The presence of invertase activity in the R-galactosidase
preparations can contribute to the complete hydrolysis of the
oligosaccharides, because these GOs are substrates for both
enzymes.
Figure 2. Thermal stability of the
R-galactosidases from (A) soybean,
(B) Aspergillus terreus, and (C) Penicillium griseoroseum. The enzyme
samples were preincubated for several periods at different temperatures
and assayed as described for the standard assay. The activity at 0 min
of preincubation was considered to be 100%.
Table 4. KM,app Values Determined by the Michaelis
−Menten Plot for
R
-Galactosidases from Soybean Seeds, Aspergillus terreus, and
Penicillium griseoroseum
KM,app (mM)
substrate
soybean
A. terreus
P. griseoroseum
pNPGal
melibiose
raffinose
0.47
1.47
3.44
1.89
0.45
19.39
1.32
2.06
20.67
The R-galactosidases differed in sensitivity to simple sugars
and mono- and bivalent ions (Table 6). The enzymes were little
or not at all inhibited by EDTA, Mg(II), iodoacetamide, Na(I),
K(I), Ca(II), 2-mercaptoethanol, maltose, lactose, sucrose,
D-glucose, and D-mannose. These results suggest that R-galac-
tosidase is not a metalloenzyme and that sulfhydryl groups do
not take part in the catalysis. This agrees with results reported
for R-galactosidase isolated from Penicillium sp. 23 (22), grape
flesh R-galactosidase (24), and soybean R-galactosidase (5, 6).
The enzymes from soybean, A. terreus, and P. griseoroseum
were completely inhibited by Ag(I) and Hg(II), whereas A.
terreus and P. griseoroseum enzymes were completely inhibited

10188 J. Agric. Food Chem., Vol. 54, No. 26, 2006
Falkoski et al.
Table 5. Hydrolysis of Several Synthetic and Natural Substrates by
Table 7. Hydrolysis of Oligosaccharides Present in Fatfree Soybean
Semipurified
R
-Galactosidases from Soybean Seeds, Aspergillus
Flour at Different Incubation Times by
R-Galactosidase from Soybean
terreus, and Penicillium griseoroseum
Var. Monarca, Aspergillus terreus, and Penicillium griseoroseum
hydrolytic activitya (%)
± SD
GO enzymatic hydrolysis by
R
-galactosidasea (%)
P. griseoroseum
soybean A. terreus
concen-
tration
(mM)
time
(h)
raffinose
stachyose
raffinose
stachyose
raffinose
0
stachyose
substrate
soybean
A. terreus
P. griseoroseum
0
4
6
8
0
0
0
0
0
pNP
R
Gal
Gal
Glc
Xyl
0.5
0.5
0.5
0.5
0.5
0.5
7.0
4.0
7.0
4.0
4.0
4.0
100
4.53
0
±
±
0.01
0.01
100
0
±
0.03
100
0
3.30
0
±
0.05
0.07
71.4
78.1
100
±
±
3.9
0.4
34.7
51.8
53.1
±
±
±
1.4
8.12
2.67
100
100
100
100
100
100
72.7
73.7
75.5
±
±
±
2.6
2.7
3.2
100
100
100
ï
NP
â
R
R
R
pNP
pNP
pNP
pNP
0
±
0
0
0
9.64
0
±
0.02
Man
0
0
a The results were calculated on the basis of the chromatograms obtained from
the GO analyses by HPLC. The assays were triplicate and represent the mean
obtained values.
â
Gal
0
raffinose
maltose
stachyose
melibiose
lactose
30.60
18.12
11.74
±
±
±
0.01
0.05
0.02
78.57
±
0.06
60.65
10.60
78.65
16.15
0
±
±
±
±
0.03
6.07
85.71
15.36
0
±
±
±
0.05
0.01
0.07
0.03
0.02
0.01
7.68
2.30
0
±
±
0.05
0.04
inhibition is likely to be an advantage for industrial applications
of the A. terreus and P. griseoroseum R-galactosidases.
The data presented for R-galactosidase activity, GO, and
reducing sugar determinations are means of triplicate assays in
which the standard deviation values were smaller than 10%.
Despite the different procedures proposed for GO reduction
in leguminous products, enzyme processing has proved to be
more effective in terms of time and cost. It has been proposed
that the use of crude or semipurified R-galactosidase is more
cost-effective than other types of procedures (30, 31). Hydrolysis
time will depend on the amount of enzyme used during
processing. The ability of soybean, A. terreus, and P. griseo-
roseum R-galactosidases to hydrolyze oligosaccharides in fatfree
soybean flour was demonstrated (Table 7). Soybean flour (2
g) incubated with the soybean, A. terreus, and P. griseoroseum
enzymes (8 units) for a period of 4 h promoted reductions of
71, 100, and 73% in the raffinose content, respectively, whereas
stachyose was reduced by 35, 100, and 100%, respectively. No
oligosaccharide hydrolysis was detected in control samples in
which the enzyme extracts had been replaced by water. Using
a much higher R-galactosidase amount, GOs present in pinto
bean flour (4 g) were completely eliminated after 2 h of
incubation with 40 mL of crude extracellular fungal enzyme
(60 units mL-1) (18). The extracellular R-galactosidase of
Debaryomyces hansenii UFV-1 completely hydrolyzed soy milk
GO in a 4 h treatment (26). Our results, especially with the
microbial enzymes, indicate that the genes encoding R-galac-
tosidases in these microorganisms can be potentially superex-
pressed in heterologous systems for optimum industrial use.
Soy molasses was also subjected to treatment with the
R-galactosidases, and GO hydrolysis was evaluated by the
production of reducing sugars (Figure 3). No oligosaccharide
hydrolysis was detected in the control, in which the enzyme
extracts had been replaced by water. The preparations containing
microbial enzymes were more effective in hydrolyzing GO in
soy molasses, as was also the case when soybean flour was
used as the GO source.
sucrose
35.71
±
0.03
16.30
±
0.05
a Relative activities were calculated in relation to the
was considered to be 100%.
FNPRGal activity, which
Table 6. Effect of Sugars, Salts, Sodium Dodecyl Sulfate,
Iodoacetamide, EDTA, and 2-Mercaptoethanol on -Galctosidases
from Soybean, Aspergillus terreus, and Penicillium griseoroseum
R
R
-galactosidase activityb (%)
A. terreus P. griseoroseum
100 1.21 100 2.91
± SD
effectora
soybean
100 0.05
0.02
±
±
±
lactose
maltose
melibiose
raffinose
89.51
91.33
74.14
81.73
90.05
53.53
87.69
98.72
94.40
97.21
94.15
49.36
96.25
59.16
96.48
94.20
98.66
95.88
0
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
102.5
92.80
39.70
80.60
90.50
107.2
95.73
116.5
105.1
105.0
105.6
46.90
109.9
0
±
±
±
±
±
±
±
±
±
±
±
±
±
1.09
0.7
88.00
94.00
58.00
93.00
94.00
100.0
88.00
103.0
96.00
101.0
98.00
0
±
±
±
±
±
±
±
±
±
±
±
1.55
0.02
0.01
0.05
0.02
0.01
0.04
0.07
0.01
0.02
0.04
0.01
0.02
0.01
0.02
0.03
0.03
0.03
3.78
3.56
0.80
1.25
2.34
3.81
2.80
2.05
2.08
1.47
1.03
1.34
0.67
1.45
0.85
2.90
2.57
1.02
1.25
2.30
2.79
D
-mannose
-galactose
D
stachyose
sucrose
D-glucose
EDTA
MgCl2
SDS
CaCl2
CuSO4
KCl
NaCl
2-mercaptoethanol
iodoacetamide
HgCl2
94.00
11.00
102.0
±
±
±
2.52
1.88
1.21
105.7
124.8
107.8
104.3
0
±
±
±
±
1.80
0.82
2.35
1.27
96.0
95.0
98.87
±
±
±
4.31
1.68
0.56
0
0
AgNO3
0
0
a The final concentration of all effectors was 1 mM. b Relative activities were
calculated in relation to the NPGal activity, which was considered to be 100%.
F
by Cu(II) and SDS, respectively. Reduction in R-galactosidase
activity by Cu(II) and Ag(I) was reported for R-galactosidases
from Torulaspora delbrueckii IFO 1255 (25), B. stearothermo-
philus NCIM 5146 (19), and D. hansenii UFV-1 (26). Participa-
tion of carboxyl and/or histidine imidazolium groups in the
catalytic action is presumed on the basis of the inhibitory effect
(27). The ionic detergent SDS is an extremely effective
denaturing agent for proteins; in its presence most proteins lose
their functions either completely or partially with the disruption
of tertiary and quaternary structures (28, 29). The enzymes were
partially inhibited by melibiose, raffinose, and stachyose.
Soybean R-galactosidase was inhibited by D-galactose, an end
product from GO hydrolysis. Galactose is a competitive inhibitor
of purified soybean R-galactosidase (5). The microbial enzymes
were not affected by this sugar (Table 6). This lack of galactose
As the enzyme preparations from A. terreus and P. griseo-
roseum showed invertase activity, it is possible that invertase
and R-galactosidase acted synergistically, especially against
stachyose, which was fully hydrolyzed in all treatment periods
(Table 7). Complete GO hydrolysis can be achieved by either
R-galactosidase, invertase, or both. Whereas R-galactosidase
hydrolyzes the R-1,6 linkage of raffinose and produces galactose
and sucrose, invertase hydrolyzes the â-1,2 linkage and produces
melibiose and fructose (5). Breaking down sucrose into glucose
and fructose will also increase sweetness and increase the rate
of Maillard browning reactions to darken the products. Although
more research should be done, our results indicate that the

Processing of Soybean Products
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The R-galactosidases described in this work are of interest
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Received for review June 19, 2006. Revised manuscript received
October 19, 2006. Accepted October 30, 2006. We thank FAPEMIG
for financial support and CAPES for providing scholarships.
JF0617162