Extracellular α-galactosidase from Debaryomyces hansenii UFV-1 and its use in the hydrolysis of raffinose oligosaccharides

Article abstract of DOI:10.1021/jf0526442

Raffinose oligosaccharides (RO) are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. ROs are hydrolyzed by α-galactosidases that cleave α-1,6-linkages of α-galactoside residues. The objectives of this study were

Full text of DOI:10.1021/jf0526442

J. Agric. Food Chem. 2006, 54, 2385−2391 2385
Extracellular
r-Galactosidase from Debaryomyces hansenii
UFV-1 and Its Use in the Hydrolysis of Raffinose
Oligosaccharides
†
†
†
POLLYANNA A. VIANA, SEBASTI A˜ O T. DE REZENDE, VIRG IÄ NIA M. MARQUES,
†
†
†
LARISSA M. TREVIZANO, FL AÄ VIA M. L. PASSOS, MARIA G. A. OLIVEIRA,
MARCELO P. BEMQUERER, JAMIL S. OLIVEIRA, AND VAL EÄ RIA M. GUIMAR A˜ ES*,†
‡
‡
BIOAGRO, Universidade Federal de Vi c¸ osa, Vi c¸ osa, MG, 36570-000, and Departamento de
Bioqu ´ı mica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG,
3
1270-901, Brazil
Raffinose oligosaccharides (RO) are the factors primarily responsible for flatulence upon ingestion
of soybean-derived products. ROs are hydrolyzed by R-galactosidases that cleave R-1,6-linkages of
R-galactoside residues. The objectives of this study were the purification and characterization of
extracellular R-galactosidase from Debaryomyces hansenii UFV-1. The enzyme purified by gel filtration
r
and anion exchange chromatographies presented an M value of 60 kDa and the N-terminal amino
acid sequence YENGLNLVPQMGWN. The K values for hydrolysis of pNPRGal, melibiose, stachyose,
m
and raffinose were 0.30, 2.01, 9.66, and 16 mM, respectively. The R-galactosidase presented absolute
specificity for galactose in the R-position, hydrolyzing pNPGal, stachyose, raffinose, melibiose, and
polymers. The enzyme was noncompetitively inhibited by galactose (K
1.2 mM). Enzyme treatments of soy milk for 4 h at 60 °C reduced the amounts of stachyose and
raffinose by 100%.
i
) 2.7 mM) and melibiose (K
i
)
KEYWORDS: r-Galactosidase; Debaryomyces hansenii UFV-1; raffinose oligosaccharides; characteriza-
tion; flatulence
INTRODUCTION
activity. In addition to melibiose, galactose has been shown to
induce enzyme synthesis and results from our laboratory suggest
that galactose could induce intra- and extracellular R-galactosi-
dases in Debaryomyces hansenii UFV-1. It is the most frequent
yeast species in protein-rich fermented products such as sausages
and cheeses (12).
In the present study, we report the purification and charac-
terization of the R-galactosidase secreted by D. hansenii UFV-1
when grown on galactose. The hydrolytic properties and
substrate specificities of this R-galactosidase were studied to
elucidate its possible applications.
The enzyme R-galactosidase (R-D-galactoside galactohydro-
lase EC 3.2.1.22) catalyzes hydrolysis of R-1,6-linked R-ga-
lactoside residues from simple oligosaccharides such as meli-
biose, raffinose, and stachyose and from polymeric galacto-
mannans (1). R-Galactosidase is widely distributed in micro-
organisms, plants, and animals (2-4). Purification and charac-
terization of R-galactosidases from several sources have been
reported, and according to their source, their properties differ
markedly (5-8).
This enzyme is of particular interest in view of its biotech-
nological applications. Microbial or plant R-galactosidases are
added to soybean meal, soy milk, and molasses to hydrolyze
the raffinose oligosaccharides (ROs) to digestible carbohydrates
and thereby moderate the flatulence-causing property of soybean
products (9, 10). Enzymatic hydrolysis of hemicelluloses is of
interest for the pulp and paper industry, and R-galactosidase-
modified galactomannan has been used to improve the gelling
properties of polysaccharide (11).
MATERIALS AND METHODS
Microorganism. The yeast strain used in this study was isolated
from a dairy environment in Minas Gerais, Brazil, and has been
maintained in the culture collection of the Laboratory of Microorganism
Physiology, BIOAGRO, UFV, Brazil. This yeast was identified by the
Institute of Yeasts Identification, Centraalbureau voor Schimmelcultures,
Utrecht, The Netherlands, as D. hansenii (Zopf) Lodder & Kreger-van
Rij var. fabryi Nakase & Suzuki. In this work, it is designated as D.
hansenii UFV-1.
Several yeast strains are known to assimilate melibiose as a
nutrient, so they possibly have significant R-galactosidase
Conditions for Enzyme Production. D. hansenii UFV-1 stored at
-
80 °C in glycerol and YPD medium (1% yeast extract, 2% peptone,
*
To whom correspondence should be addressed. Tel: (5531)3899-2374.
and 2% glucose) was streaked on a YPD agar surface (1.5% agar) and
maintained in an incubation chamber at 30 °C for 36 h. The yeast was
then activated in YPD liquid medium and incubated at 30 °C, 200 rpm,
Fax: (5531)3899-2373. E-mail: vmonteze@ufv.br.
†
Universidade Federal de Vi c¸ osa.
Universidade Federal de Minas Gerais.
‡
1
0.1021/jf0526442 CCC: $33.50
© 2006 American Chemical Society
Published on Web 02/17/2006

2386 J. Agric. Food Chem., Vol. 54, No. 6, 2006
Viana et al.
Table 1. Summary of D. hansenii UFV-1 Extracellular
R-Galactosidase Purification
purification
step
volume
(mL)
protein
(mg)
activity
(U/mL)
total
activitya (U)
specific activity
(U/mg protein)
purification
factor
recovery
(%)
lyophilized extract
Sephadex G-150
DEAE-Sepharose
102.0
190.0
17.0
7.45
0.57
0.26
0.87
0.31
3.04
88.7
58.9
51.7
11.9
103.0
199.0
1.00
8.65
16.70
100.0
66.4
58.3
a
One unit (U) of enzyme activity is defined as the amount of enzyme that released 1
µmol of pNP per minute.
for 12-15 h. The cells obtained after centrifugation (4000g for 5 min
at 4 °C) were inoculated in mineral medium containing 0.62 g/L KH
PO , 2.0 g/L K HPO , 1.0 g/L (NH SO , 0.1 g/L MgSO ‚7H O, and
.0 g/L yeast extract with galactose as the carbon source. After
incubation at 30 °C, 200 rpm for 31 h, the biomass was separated by
centrifugation and the supernatant containing R-galactosidase was
lyophilized.
Michaelis-Menten plot. The substrate concentrations expressed in mM
were 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.75, 0.9, and
1.0 for pNPRGal; 1.25, 2.5, 5, 10, 20, 30, 40, 60, 70, and 80 for
raffinose; 2.5, 5, 8, 10, 15, 20, 25, and 30 for stachyose; and 0.15, 0.3,
0.625, 1.25, 2.5, 5.0, 7.5, 10, 15, 20, 30, and 40 for melibiose. The
2
-
4
2
4
4
)
2
4
4
2
5
i
inhibition constants (K ) for galactose and melibiose were calculated
by the Dixon plot. The pNPRGal concentrations were 0.05, 0.1, 0.2,
0.3, 0.4, 0.5, and 1.0 mM. The concentrations of inhibitor galactose
were 0.5, 1.0, and 2.0 mM, and the melibiose concentrations were 0.25,
0.50, and 1.0 mM.
r-Galactosidase Purification. The lyophilized enzymatic sample
resuspended in 25 mM sodium acetate buffer was subjected to gel
filtration chromatography in a Sephadex G-150 column (87.5 cm ×
2
.5 cm) equilibrated with 25 mM sodium acetate buffer, pH 5.5. The
Substrate Specificity. Enzymatic assays were performed with
various synthetic, natural, and polymeric substrates. The reaction
mixtures contained 650 µL of 0.1 M sodium acetate buffer, pH 5.0, 70
µL of enzyme solution (0.75 µg protein/mL), and 250 µL of synthetic
substrates (2 mM), or lactose, maltose, gentiobiose, stachyose, and
sucrose (10 mM), or raffinose (15 mM), or melibiose (2 mM), or locust
bean gum and guar gum solutions (1%). The activities were measured
under standard assay conditions at 60 °C. The data presented for all
enzyme activity determinations are mean values ( SD of three
measurements.
Effect of Ions, Simple Sugars, and Reducing Agents. The effect
of ions, simple sugars, and reducing agents on the enzyme activity was
assayed by the standard methods; 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 60 °C. The data presented for all enzyme activity
determinations are mean values ( SD of triplicate assays.
N-Terminal Amino Acid Sequence Analysis. After blotting onto
PVDF membrane, the N-terminal amino acid sequences of the R-ga-
lactosidase were determined by automated Edman degradation, using
an automatic protein sequencer (PPSQ-21/23). Similarity searches were
preformed using BLASTp software.
Enzymatic Hydrolysis of Oligosaccharides Present in Soy Milk.
Soy milk was prepared from dry seeds (50 g). The seeds were chopped
up, homogenized in 400 mL of water at 80 °C, incubated for 10 min
at 85 °C, and filtered through cheesecloth. Soy milk samples (5 mL)
were then incubated with either water or 10.5 U of purified R-galac-
tosidase for 0, 2, 4, and 6 h under shaking (100 rpm) at 60 °C. The
reaction mixtures were dried, and the soluble sugars from 20 to 30 mg
of lyophilized samples were extracted with 80% aqueous ethanol (v/v)
proteins were eluted at a flow rate of 20 mL/h, and 3.3 mL fractions
were collected. Fractions containing R-galactosidase activity were
pooled and subjected to ion exchange chromatography in a DEAE-
Sepharose column (14.5 cm × 1.9 cm) equilibrated with 0.1 M sodium
acetate buffer, pH 5.5. The proteins were eluted at a flow rate of 40
mL/h, with a linear increasing gradient of NaCl (0-1.0 M) in 0.1 M
sodium acetate buffer. All purification procedures were performed at
4
°C. The active fraction was pooled and analyzed for purity by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Enzyme Assay. R-Galactosidase was assayed in a reaction system
containing 650 µL of 0.1 M sodium acetate buffer, pH 5.0, 100 µL of
enzyme solution (0.75 µg protein/mL), and 250 µL of 2 mM
p-nitrophenyl-R-D-galactopyranoside (pNPRGal) or other synthetic
substrates. The reaction was run for 15 min at 60 or at 40 °C for the
purification assays and ended with 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.
The activities against melibiose, maltose, gentiobiose, and lactose
were evaluated by the glucose-oxidase method (13). When sucrose,
raffinose, and stachyose were used as the substrate, the production of
reducing sugar was determined using the 3,5-dinitrosalicylate reagent
(14). One enzyme unit (U) was defined as the amount of enzyme that
released 1 µmol of product per min under assay conditions.
Determination of Protein Concentration. The protein concentration
in the enzymatic extract was determined by the Coomassie Blue binding
method with bovine serum albumin (BSA) as the standard (15).
Determination of Molecular Mass. The enzyme molecular mass
was estimated by SDS-PAGE using a 12.5% polyacrylamide gel (16).
After electrophoresis, the gel was silver-stained (17). The molecular
mass standards (Pharmacia) were as follows: BSA, 66.0 kDa;
ovalbumin, 45.0 kDa; glyceraldehyde-3-phosphate dehydrogenase, 36.0
kDa; carbonic anhydrase, 29.0 kDa; trypsinogen, 24.0 kDa; trypsin
inhibitor, 20.1 kDa; and R-lactalbumin, 14.2 kDa.
Effect of pH and Temperature. The pH effect of the R-galactosi-
dase activity was investigated at different pH levels (from 3.0 to 8.0)
by Mcllvaine buffer (citric acid/sodium phosphate) at 60 °C (18). The
pH stability of R-galactosidase was also determined by incubating 15
µL of enzyme solution (15.0 µg protein/mL) with 285 µL of above-
mentioned buffer at 60 °C for 30 min. After incubation, 100 µL of the
mixture was used for determination of the residual activity, according
to the standard assay, using pNPRGal as the substrate.
(9). The solvent was evaporated at 50 °C, and the sugars were
resuspended in 1 mL of 80% ethanol and analyzed by high-performance
liquid chromatography (HPLC) on a Shimadzu series 10A chromato-
graph. An analytical column [aminopropil (NH )] was used for this
2
purpose, eluted with an acetonitrile-water isocratic mixture (80:20 v/v)
at 35 °C at a flow rate of 1 mL/min. The individual sugars were
automatically identified and quantified by comparison with the retention
times and standard sugar concentrations. Gentiobiose was used as an
internal standard as it does not interfere with the other sugars and is
not found in soybean seeds.
RESULTS AND DISCUSSION
The optimum temperature was determined within a temperature range
of 25-80 °C at pH 5.0. Thermal stability was investigated by incubating
Galactose-grown D. hansenii UFV-1 produced high levels
of intracellular and extracellular R-galactosidases, similar to
other species such as Debaryomyces castellii IFO 1359, De-
baryomyces nepalensis IFO 1428, and other yeasts (19). Results
of the purification of extracellular R-galactosidase from D.
hansenii UFV-1 are summarized in Table 1. The concentrated
culture supernatant was subjected to gel filtration chromatog-
raphy resulting in the separation of one protein fraction with
1
00 µL of enzyme solution (0.75 µg protein/mL) and 650 µL of 0.1 M
sodium acetate buffer, pH 5.0, at various temperatures (40, 50, 55, 60,
5, and 70 °C) for 0-48 h. After incubation, 250 µL of 2 mM pNPRGal
6
was added, and the remaining activity was measured. Results of the
analyses are presented as means ( SD for three measurements.
Determination of Kinetic Parameters. The Michaelis-Menten
m
constant (K ) and Vmax for substrate hydrolysis were calculated by the

Extracellular
R
-Galactosidase from D. hansenii UFV-1
J. Agric. Food Chem., Vol. 54, No. 6, 2006 2387
(6), respectively. The N-terminal amino acid sequence of D.
hansenii UFV-1 R-galactosidase was determined as YENGLN-
LVPQMGWN (Table 2). Although this region does not appear
to be conserved among the known R-galactosidases, the N-
terminal amino acid sequence of D. hansenii UFV-1 R-galac-
tosidase shared a high similarity with other microbial R-galac-
tosidases, which belong to the glycoside hydrolase family 27
in comparison with the sequences available in the protein
database (Table 2). The alignment of the N-terminal amino acid
sequence of D. hansenii UFV-1 R-galactosidase (14 amino acid
residues) with the sequence of D. hansenii CBS767 R-galac-
tosidase showed two nonconservative changes, a substitution
of N for G at residue six and a substitution of V for T at residue
eight. However, the N and V residues presented in D. hansenii
UFV-1 were conserved in other microbial R-galactosidases, as
observed in the sequence of the Magnaporthe grisea 70-15
R-galactosidase, which showed the perfect match to the last 12
amino acids of the protein under study (Table 2).
Substantial activity against pNPRGal was observed for
enzyme preparation within a temperature range of 40-65 °C
and pH range of 3.5-6.0 (Figure 3). The enzyme achieved
maximal substrate hydrolysis at a temperature of 60 °C (Figure
3
B). The optimum pH for the enzyme was 5.0 (Figure 3A).
These optimum pH and temperature values are close to those
determined for hydrolysis of pNPRGal by four R-galactosidases
from Aspergillus niger ATCC 46890 (1), Torulaspora del-
brueckii IFO 1255 (31), and Thermomyces lanuginosus IMI
1
58749 (6).
Figure 1. Elution profile of the
on a (A) Sephadex G-150 column and (B) DEAE-Sepharose column.
-Galactosidase activity, ; protein, ; and gradient of NaCl,
R-galactosidase from D. hansenii UFV-1
The Km and Vmax values were calculated by the Michaelis-
Menten plot for pNPRGal, melibiose, raffinose, and stachyose
R
0
b
−.
(Table 3). The Km value for pNPRGal is comparable to the
one determined for hydrolysis of the same substrate by
R-galactosidase purified from Aspergillus fumigatus (21) and
Ganoderma lucidum (32). The Km values for raffinose and
stachyose were lower than those determined for hydrolysis of
the same substrates by the enzyme of T. delbrueckii IFO 1255
(31). For the natural substrates in use, the lowest Km and Vmax
values were calculated for melibiose. Results indicate that D.
hansenii UFV-1 R-galactosidase presents a higher affinity for
melibiose and that the complex ES formation is not the limiting
step for the reaction. The ratio Vmax/Km showed that substrate
pNPRGal was used more efficiently by the enzyme, followed
by stachyose, raffinose, and melibiose. The purified R-galac-
tosidase was thermostable. No loss in enzyme activity was
observed after incubation for 24 h at 40 °C. The enzyme
maintained about 91% of its original activity after 3 h at 60 °C,
but 40% of the activity was lost following 15 min of incubation
at 70 °C (Figure 4). The half-life of D. hansenii UFV-1
R-galactosidase at 50, 55, 60, 65, and 70 °C was 821, 647, 373,
Figure 2. SDS−PAGE (12.5%) of D. hansenii UFV-1 R-galactosidase
purification steps. Lane 1, molecular mass standards; lane 2, lyophilized
extract fraction; lane 3, Sephadex G-150 fraction; and lane 4, DEAE-
Sepharose fraction. Protein gel was stained with silver.
R-galactosidase activity (Figure 1A). This step resulted in
considerable specific activity enrichment (Table 1). The final
step was carried out by ion exchange chromatography. The
active fractions were eluted from 0.45 to 0.6 M NaCl and
appeared as a single activity peak (Figure 1B). This procedure
resulted in a purification factor of 16.7 with a recovery level of
the original R-galactosidase activity of 58.3% (Table 1). Similar
results were reported for purification of extracellular Saccha-
romyces carlsbergensis R-galactosidase (2). No invertase activity
was detected in the final enzyme preparation.
The electrophoretic profile of the enzyme in SDS-PAGE
confirmed the presence of a single protein band, with an
estimated molecular mass of 60 kDa (Figure 2). In previous
reports, purified R-galactosidases from Penicillium purpuroge-
num, Aspergillus fumigatus, and Thermomyces lanuginosus IMI
1
80, and 34.6 min, respectively. These t1/2 values were higher
than those reported for soybean R-galactosidases (9, 10) but
lower than those reported for R-galactosidase from Bacillus
stearothermophilus NCIM 5146 (8).
The D. hansenii UFV-1 R-galactosidase retained about 82%
of its activity after incubation for 30 min at 60 °C in a pH range
of 4.0-7.6, but 90% of the activity was lost after incubation in
the same conditions at pH 3.0 (Figure 5). The enzyme showed
significant stability for a wide range of pH and temperature
levels, which is desirable for industrial applications.
The activity of R-galactosidase against several substrates is
shown in Table 4. Under the experimental conditions, this
enzyme was the most effective to hydrolyze pNPRGal, followed
by stachyose, raffinose, and melibiose, as expressed in the Vmax/
Km ratio (Table 3). Lactose and synthetic substrates containing
1
58749 presented Mr values of 67 (20), 54.7 (21), and 57 kDa

2388 J. Agric. Food Chem., Vol. 54, No. 6, 2006
Viana et al.
Table 2. Comparison of D. hansenii UFV-1
R-Galactosidase N-Terminal Sequence to Other Microbial R
-Galactosidasesa
a
Amino acid residues conserved in all sequences are printed in bold.
Figure 3. pH (A) and temperature (B) influences on the activity of
R-galactosidase from D. hansenii UFV-1.
Table 3.
K
m
, Vmax, and Vmax/K
m
Values Determined by
Michaelis
−Menten Plot
a
(min-1)
20.3
0.01
substrates
pNP Gal
melibiose
stachyose
raffinose
K
m
(mM)
V
max
V
max/K
m
R
0.30
2.01
9.66
6.09
0.02
8.18
5.99
0.85
0.37
16.0
a
Vmax is expressed in mM pNP/min for pNPRGal, mM glucose/min for melibiose,
and mM reducing sugar/min for stachyose and rafinose.
â-linkages or containing xylose, arabinose, mannose, and
glucose residues were not hydrolyzed by the enzyme. Thus, the
enzyme presents specificity not only for anomeric carbon but
it also seems to be regiospecific for the galactoside configura-
tion, in contradistinction to some promiscuous glycosidases
reported in the literature (33). The enzyme exhibited the ability
to hydrolyze polymers such as locust bean gum and guar gum,
suggesting its potential industrial application for an improvement
of the gelling properties of polysaccharide. On the other hand,
Trichoderma reesei R-galactosidase and A. niger R-galactosidase
showed high specificity for oligosaccharides and low activity
for polymeric substrates (34). The aforementioned R-galactosi-
dases from Penicillium simplicissimum seem to have different
substrate specificities. One form of the enzyme is able to
hydrolyze galactomannan, while the other acts more specifically
on small oligosaccharides (35). Extracellular R-galactosidase
from D. castellii IFO 1359 (19) and rice R-galactosidase (36)
Figure 4. Temperature influences on the stability of the
R
-galactosidase
from D. hansenii UFV-1. Enzyme preparations were preincubated for 48
h at temperatures of 40 ( ), 50 ( ), 55 ( ), 60 ( ), 65 ( ), and 70
).
b
1
O
3
9
°C
(
0
The D. hansenii UFV-1 R-galactosidase showed distinct
sensitivities to simple sugars and mono- and bivalent ions
(Figure 6). The enzyme presented very low or no inhibition by
EDTA, Mg(II), iodoacetamide, Na(I), SDS, K(I), Ca(II), â-mer-
captoethanol, raffinose, maltose, sucrose, D-glucose, lactose,
gentiobiose, stachyose, and D-mannose. The enzyme was
completely inhibited by Cu(II) and Ag(I) and was partially
inhibited by D-galactose and melibiose. Reduction in the
3
hydrolyzed galactomanno-oligosaccharides such as Gal Man3
3
(
6 -R-D-galactopyranosyl-1,4-â-D-mannotriose).

Extracellular
R
-Galactosidase from D. hansenii UFV-1
J. Agric. Food Chem., Vol. 54, No. 6, 2006 2389
Figure 5. pH influences on the activity
-galactosidase from D. hansenii UFV-1.
(
b
)
and stability
(
O
)
of
Figure 6. Effect of EDTA (2), MgCl
2
(3), iodoacetamide (4), AgNO
(9), CaCl (10), -mercaptoethanol
11), raffinose (12), maltose (13), sucrose (14), melibiose (15), -glucose
16), -galactose (17), lactose (18), gentiobiose (19), stachyose (20), and
-galactosidase from D. hansenii UFV-1 and without
3
(5),
R
NaCl (6), SDS (7), KCl (8), CuSO
4
2
â
(
(
D
Table 4. Hydrolysis of Several Substrates with
D. hansenii UFV-1
R
-Galactosidase from
D
mannose (21) on
R
effectors (1). The final concentration of all effectors was 2 mM.
substrate
concentrationa
activity (U/mL) ± SD
b
pNP
pNP
pNP
pNP
pNP
pNP
oNP
oNP
R
Gal
Gal
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
10
10
15
2
3.39
0.0
0.0
0.0
0.0
0.0
0.0
0.0
±
0.15
b
â
b
R
Glc
b
â
R
R
Xil
b
Man
b
Ara
b
â
â
Gal
Glc
b
sucrose
0.0
stachyose
raffinose
melibiose
gentiobiose
maltose
3.84
2.78
0.002
0.0
0.0
0.0
±
±
0.02
0.05
10
10
10
1
lactose
locust bean gum
guar gum
0.64
0.56
±
±
0.01
0.01
1
Figure 7. HPLC analysis of hydrolysis products of ROs present in soy
a
Concentrations in mM, except the locust bean gum and guar gum substrates
%). b pNP
Gal, para-nitrophenyl- -galactopyranoside; pNP Glc, para-nitrophen-
yl- -glucopiranoside; pNP X, para-nitrophenyl- -xylopyranoside; pNP A, para-
nitrophenyl- -arabinopyranoside; pNP M, para-nitrophenyl- -mannopyranoside;
oNP Glc, ortho-nitrophenyl- -glucopyranoside; and oNP Gal, ortho-nitrophenyl-
-galactopyranoside.
milk by extracellular
R-galactosidase from D. hansenii UFV-1. (A) Soy
(
â
â-D
R
milk before enzyme treatment and (B) after 4 h of enzyme treatment.
R-
D
â
â-D
R
R
-D
R
R-D
The potential of D. hansenii UFV-1 R-galactosidase to
hydrolyze the oligosaccharides present in soybean aqueous
extract (soy milk) was demonstrated (Figure 7). Sucrose,
raffinose, and stachyose were present in the reaction mixture at
the concentrations 3.69, 1.33, and 2.70% (w/v), respectively
â
â
-D
â
â
-D
R-galactosidase activity by Cu(II) and Ag(I) was reported for
R-galactosidases purified from T. delbrueckii IFO 1255 (31)
and B. stearothermophilus NCIM 5146 (8). Participation of
carboxyl and/or histidine imidazolium groups in the catalytic
action is supposed on the basis of the inhibitory effect (37). It
has been suggested that R-galactosidase is not a metalloenzyme
and that the sulfhydryl groups do not take part in catalysis, since
there is no enzyme inhibition under EDTA or iodoacetamide
treatment, respectively. This is in agreement with results reported
for R-galactosidase isolated from Penicillium sp. 23 (38) and
for soybean R-galactosidase (10).
Galactose and melibiose inhibition were found to be non-
competitive, and the Ki values were 2.7 and 1.2 mM, respec-
tively, as determined by the Dixon plot, in opposition to data
found in the literature for most R-galactosidases that are
competitively inhibited by D-galactose (9, 39).
(Figure 7A). After 2 h of incubation with the enzyme, a
reduction of 24 and 100% was observed in the amount of
raffinose and stachyose, respectively. The difference in hy-
drolysis may be due to accumulation of raffinose, which is
formed after stachyose hydrolysis. However, when the enzyme
was incubated with soy milk under the same conditions for a
period of 4 h (Figure 7B), raffinose and stachyose were
completely hydrolyzed and the sucrose concentration rose to
6
.53% (w/v). No oligosaccharide hydrolysis was detected in
control tubes where the enzyme extracts had been replaced by
water. As the enzyme preparation showed no invertase activity,
our results indicate that D. hansenii UFV-1 R-galactosidase acts
on the oligosaccharides present in soy milk. The ability of the
enzyme to hydrolyze stachyose and raffinose is of particular
interest for biotechnological applications. Several food scientists
have suggested the possibility of improving the nutritional value
of soy milk and soybean flour by reducing the RO content.
Microbial R-galactosidases were used to degrade RO in soy milk
The data presented for all R-galactosidase activity determina-
tions are mean values of triplicate assays in which the standard
deviations values were always smaller than 10%.

2390 J. Agric. Food Chem., Vol. 54, No. 6, 2006
Viana et al.
(
40-42). Nevertheless, no reliable, inexpensive, and efficient
(13) Stemberg, D.; Vijaykumar, P.; Reese, E. T. â-glucosidase:
Microbiology of production and effect on enzymatic hydrolysis
of cellulose. Can. J. Microbiol. 1970, 23, 139-147.
enzymic process with native or recombinant yeast enzymes is
available so far. The potential enzymes suggested for this
purpose are generally of microbial origin and do not have the
GRAS (generally recognized as safe) status. D. hansenii is the
most frequent yeast species found in protein-rich fermented
products, such as sausages and cheeses (43). There should be
no restriction regarding safeness for the use of this microorgan-
ism in food processing.
(
14) Miller, G. L. Use of dinitrosalicylic acid reagent for determination
of reducing sugar. Anal. Chem. 1956, 31, 426-428.
15) Bradford, M. M. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 1976, 72, 248-254.
16) Laemmli, U. K. Cleavage of structural proteins during the
assembly of head of bacteriophage T4. Nature 1970, 227, 680-
683.
(
(
As the D. hansenii UFV-1 R-galactosidase shows stability at
the pH range of soy milk (6.0-6.5) and at the temperature of
6
treatment, during the cooling step, at approximately 60 °C. The
properties of R-galactosidase described here, especially the broad
pH and temperature stability and the specificity for ROs, suggest
its application to remove flatulence-inducing compounds from
soy milk.
(17) Blum, H.; Beier, H.; Gross, H. Improved silver staining of plant
0 °C, the enzyme could be applied after the soy milk thermal
proteins, RNA and DNA in polyacrylamide gels. Electrophoresis
1
987, 8, 93-99.
(18) Mcllvaine, T. C. A buffer solution for colorimetric comparison.
J. Biol. Biochem. 1921, 49, 183-186.
(
19) Yoshida, S.; Tan, C. H.; Shimokawa, T.; Turakainen, H.;
Kusakabe, I. Substrate specificities of R-galactosidases from
yeasts. Biosci., Biotechnol., Biochem. 1997, 61, 359-361.
20) Shibuya, H.; Kobayashi, H.; Park, G. G.; Komatsu, Y.; Sato,
T.; Kaneko, R.; Nagasaki, H.; Yoshida, S.; Kasamo, K.;
Kusakabe, I. Purification and some properties of R-galactosidase
from Penicillium purpurogenum. Biosci., Biotechnol., Biochem.
(
LITERATURE CITED
(
(
(
(
(
(
(
1) Ademark, P.; Larsson, M.; Tjerneld, F.; Stalbrand, H. Multiple
R-galactosidases from Aspergillus niger: Purification, charac-
terization and substrate specificities. Enzyme Microbiol. Technol.
1
995, 59, 2333-2335.
(
21) De Rezende, S. T.; Felix, C. R. Production and characterization
of raffinose-hydrolyzing and invertase activities of Aspergillus
fumigatus. Folia Microbiol. 1999, 44, 191-195.
2
001, 29, 441-448.
2) Lazo, P. S.; Ochoa, A. G.; Gasc o´ n, S. R-Galactosidase from
Saccharomyces carlsbergensis: Cellular localization and puri-
fication of the external enzyme. Eur. J. Biochem. 1976, 77, 375-
(
22) Dean, R. A.; Talbot, N. J.; Ebbole, D. J.; Farman, M. L.; Mitchell,
T. K.; Orbach, M. J.; Thon, M.; Kulkarni, R.; Xu, J. R.; Pan, H.
The genome sequence of the rice blast fungus Magnaporthe
grisea. Nature 2005, 434, 980-986.
3
82.
3) Zapater, I. G.; Ullah, A. H. J.; Wodzinski, R. J. Extracellular
R-galactosidase (EC 3.2.1.22) from Aspergillus ficuum NRRL
(
23) Dujon, B.; Sherman, D.; Fischer, G.; Durrens, P.; Casaregola,
S.; Lafontaine, I.; De Montigny, J.; Marck, C.; Neuveglise, C.;
Talla, E. Genome evolution in yeasts. Nature 2004, 430, 35-
3
135: Purification and characterization. Prep. Biochem. 1990,
2
0, 263-296.
4) Dey, P. M.; Patel, S.; Brownleader, M. D. Induction of
R-galactosidase in Penicillium ochrochlorn by guar (Cyanopsis
tetragonobola) gum. Biotechnol. Appl. Biochem. 1993, 17, 361-
4
4.
(
(
(
24) Oda, Y.; Fukunaga, M. Isolation and characterization of MELt
gene from Torulaspora delbrueckii IFO 1255. Yeast 1999, 15,
3
71.
1
797-1801.
5) Kotwal, S. M.; Gote, M. M.; Sainkar, S. R.; Khan, M. I.; Khire,
J. M. Production of R-galactosidase by thermophilic fungus
Humicola sp. In solid-state fermentation and its application in
soymilk hydrolysis. Process Biochem. 1998, 33, 337-343.
6) Puchart, V.; Vrsansk a´ , M.; Bhat, M. K.; Biely, P. Purification
and characterization of R-galactosidase from a thermophilic
fungus Thermomyces lanuginosus. Biochem. Biophys. Acta 2000,
25) Oda, Y.; Fujisawa, T. Nucleotide sequence of alpha-galactosidase
MEL gene from Zygosaccharomyces mrakii. Curr. Microbiol.
2
000, 41, 220-222.
26) Turakainen, H.; Kristo, P.; Korhola, M. Consideration of the
evolution of the Saccharomyces cerevisiae MEL gene family
on the basis of the nucleotide sequences of the gene and their
flanking regions. Yeast 1994, 10, 1559-1568.
1
524, 27-37.
(
27) Naumov, G. I.; James, S. A.; Naumova, E. S.; Louis, E. J.;
Roberts, I. N. Three new species in the Saccharomyces sensu
stricto complex: Saccharomyces cariocanus, Saccharomyces
kudriaVzeVii and Saccharomyces mikatae. Int. J. Syst. EVol.
Microbiol. 2000, 50, 1931-1942.
7) Ishiguro, M.; Kaneko, S.; Kuno, A.; Koyama, Y.; Yoshida, S.;
Park, G. G.; Sakakibara, Y.; Kusakabe, I.; Kobayashi, H.
Purification and characterization of the recombinant Thermus
sp. strain T2 R-galactosidase expressed in Escherichia coli. Appl.
EnViron. Microbiol. 2001, 67, 1601-1606.
(
28) Wood, V.; Gwilliam, R.; Rajandream, M. A.; Lyne, M.; Lyne,
R.; Stewart, A.; Sgouros, J.; Peat, N.; Hayles, J.; Baker, S. The
genome sequence of Schizosaccharomyces pombe. Nature 2002,
(
8) Gote, M.; Umalkar, H.; Khan, I.; Khire, J. Thermostable
R-galactosidase from Bacillus stearothermophilus (NCIM 5146)
and its application in the removal of flatulence causing factors
from soymilk. Process Biochem. 2004, 39, 1723-1729.
9) Guimar a˜ es, V. M.; de Rezende, S. T.; Moreira, M. A.; Barros,
E. G.; Felix, C. R. Characterization of R-galactosidases from
germinating soybean seed and their use for hydrolysis of
oligosaccharides. Phytochemistry 2001, 58, 67-73.
4
15, 871-880.
(
29) Hart, D. O.; He, S.; Chany, C. J., II; Withers, S. G.; Sims, P. F.;
Sinnott, M. L.; Brumer, H., III. Identification of Asp-130 as the
catalytic nucleophile in the main alpha-galactosidase from
Phanerochaete chrysosporium, a family 27 glycosyl hydrolase.
J. Biochem. 2000, 39, 9826-9836.
(
(
(
(
10) Viana, S. F.; Guimar a˜ es, V. M.; Jos e´ , I. C.; Oliveira, M. G. A.;
Costa, N. M. B.; Barros, E. G.; Moreira, M. A.; Rezende, S. T.
Hydrolysis of oligosaccharides in soybean flour by soybean
R-galactosidase. Food Chem. 2005, 93, 665-670.
11) Bulpin, P. V.; Gidley, M. J.; Jeffcoat, R.; Underwood, D. J.
Development of a biotechnological process for the modification
of galactomannan polymers with plant R-galactosidase. Carbo-
hydr. Polym. 1990, 12, 155-168.
(
30) Shibuya, H.; Kobayashi, H.; Kasamo, K.; Kusakabe, I. Nucleotide
sequence of alpha-galactosidase cDNA from Mortierella Vinacea.
J. Biosci., Biotechnol., Biochem. 1995, 59, 1345-1348.
(31) Oda, Y.; Tonomura, K. R-Galactosidase from the yeast Toru-
laspora delbrueckii IFO 1255. J. Appl. Bacteriol. 1996, 80, 203-
208.
(32) Sripuan, T.; Kazuhiro, A.; Yamamoto, K.; Tongkao, D.; Kuma-
gai, H. Purification and characterization of thermostable R-ga-
lactosidase from Ganoderma lucidum. Biosci., Biotechnol.,
Biochem. 2003, 67, 1485-1491.
12) Nobre, A.; Lucas, C.; Le a˜ o, C. Transport and utilization of
hexoses and pentoses in the halotolerant yeast Debaryomyces
hansenii. Appl. EnViron. Microbiol. 1999, 65, 35494-35498.

Extracellular
R
-Galactosidase from D. hansenii UFV-1
J. Agric. Food Chem., Vol. 54, No. 6, 2006 2391
(
33) Marana, S. R.; Terra, W. R.; Ferreira, C. R. Purification and
in the active site of alpha-galactosidase from Trichoderma reesei.
J. Biochem. 1995, 308, 955-964.
(40) Cruz, R.; Park, Y. K. Production of fungal R-galactosidase and
its application to the hydrolysis of galactooligosaccharides in
soybean milk. J. Food Sci. 1982, 47, 1973-1975.
(41) Mulimani, V. H.; Ramalingam. Enzymic hydrolysis of raffinose
and stachyose in soymilk by R-galactosidase from Gibberella
fujikuroi. Biochem. Mol. Biol. Int. 1997, 59, 279-282.
(42) Prashanth, S. J.; Mulimani, V. H. Soymilk oligosaccharide
hydrolysis by Aspergillus oryzae R-galactosidase immobilized
in calcium alginate. Process Biochem. 2005, 40, 1199-1205.
(43) Dur a´ , A.; Flores, M.; Toldr a´ , F. Purification and characterization
of a glutaminase from Debaryomyces ssp. Int. J. Food Microbiol.
2002, 76, 117-126.
properties of a â-glycosidase purified from midgut cells of
Spodoptera frugiperda (Lepidoptera) larvae. Insect Biochem.
Mol. Biol. 2000, 30, 1139-1146.
(
34) Margolles-Clark, E.; Tenkanen M.; Luonteri, E.; Pentill a˜ , M.
Three R-galactosidase genes of Trichoderma reesei cloned by
expression in yeast. Eur. J. Biochem. 1996, 240, 104-111.
35) Luonteri, E.; Alatalo, E.; Siika-aho, M.; Pentill a˜ , M.; Tenkanen,
M. R-Galactosidases of Penicillium simplicissimum: Production,
purification and characterization of gene encoding AGLI. Bio-
technol. Appl. Biochem. 1998, 28, 179-188.
(
(
36) Kim, W. D.; Kobayashi, O.; Kaneko, S.; Sakakibara, Y.; Park,
G. G.; Kusakabe, I.; Tanaka, H.; Kobayashi, H. R-Galactosidase
from cultured rice (Oryza satiVa L. var. Nipponbare) cells.
Phytochemistry 2002, 61, 621-630.
(
37) Dey, P. M.; Pridham, J. B. Biochemistry of R-galactosidases.
AdV. Enzymol. 1972, 36, 114-118.
Received for review October 25, 2005. Revised manuscript received
January 5, 2006. Accepted January 26, 2006. This study was supported
by grants of the Funda c¸ a˜ o de Amparo a` Pesquisa do Estado de Minas
GeraissFAPEMIG (EDT24000, EDT268/05) and Coordena c¸ a˜ o de
Aperfei c¸ oamento de Pessoal de N ´ı vel SuperiorsCAPES, Brazil.
(
38) Varbanets, L. D.; Malanchuk, V. M.; Buglova, T. T.; Kuhlmann,
R. A. Penicillium sp.23 R-galactosidase: Purification and
substrate specificity. Carbohydr. Polym. 2001, 44, 357-363.
39) Kachurin, A. M.; Golubev, A. M.; Geison, M. M.; Veselkina,
O. S.; Isaeva-Isanova, L. S.; Neustroev, K. N. Role of methionine
(
JF0526442