Determination of the transgalactosylation activity of Aspergillus oryzae β-galactosidase: Effect of pH, temperature, and galactose and glucose concentrations

Article abstract of DOI:10.1016/j.carres.2011.01.030

The catalytic potential of β-galactosidase is usually determined by its hydrolytic activity over natural or synthetic substrates. However, this method poorly predicts enzyme behavior when transglycosylation instead of hydrolysis is being performed. A system for determining the transgalactosylation activity of β-galactosidase from Aspergillus oryzae was developed, and its activity was determined under conditions for the synthesis of galacto-oligosaccharides and lactulose. Transgalactosylation activity increased with temperature up to 55 °C while the effect of pH was mild in the range from pH 2.5 to 5.5, decreasing at higher values. The effect of glucose and galactose on transgalactosylation activity was also assessed both in the reactions for the synthesis of galacto-oligosaccharides and lactulose and also in the reaction of hydrolysis of o-nitrophenyl β-d-galactopiranoside. Galactose was a competitive inhibitor and its effect was stronger in the reactions of transgalactosylation than in the reaction of hydrolysis. Glucose was a mild activator of β-galactosidase in the reaction of hydrolysis, but its mechanism of action was more complex in the reactions of transgalactosylation, having this positive effect only at low concentrations while acting as an inhibitor at high concentrations. This information is relevant to properly assess the effect of monosaccharides during the reactions of the synthesis of lactose-derived oligosaccharides, such as galacto-oligosaccharides and lactulose.

Full text of DOI:10.1016/j.carres.2011.01.030

Carbohydrate Research 346 (2011) 745–752
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Determination of the transgalactosylation activity of Aspergillus oryzae
b-galactosidase: effect of pH, temperature, and galactose
and glucose concentrations
⇑
Carlos Vera, Cecilia Guerrero, Andrés Illanes
School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2147, Valparaíso, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
The catalytic potential of b-galactosidase is usually determined by its hydrolytic activity over natural or
synthetic substrates. However, this method poorly predicts enzyme behavior when transglycosylation
instead of hydrolysis is being performed. A system for determining the transgalactosylation activity of
b-galactosidase from Aspergillus oryzae was developed, and its activity was determined under conditions
for the synthesis of galacto-oligosaccharides and lactulose. Transgalactosylation activity increased with
temperature up to 55 °C while the effect of pH was mild in the range from pH 2.5 to 5.5, decreasing at
higher values. The effect of glucose and galactose on transgalactosylation activity was also assessed both
in the reactions for the synthesis of galacto-oligosaccharides and lactulose and also in the reaction of
Received 19 October 2010
Received in revised form 17 January 2011
Accepted 25 January 2011
Available online 4 February 2011
Keywords:
b-Galactosidase
Transgalatosylation
Galacto-oligosaccharides
Lactulose
hydrolysis of o-nitrophenyl b-D-galactopiranoside. Galactose was a competitive inhibitor and its effect
was stronger in the reactions of transgalactosylation than in the reaction of hydrolysis. Glucose was a
mild activator of b-galactosidase in the reaction of hydrolysis, but its mechanism of action was more
complex in the reactions of transgalactosylation, having this positive effect only at low concentrations
while acting as an inhibitor at high concentrations. This information is relevant to properly assess the
effect of monosaccharides during the reactions of the synthesis of lactose-derived oligosaccharides, such
as galacto-oligosaccharides and lactulose.
Inhibition
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
glucose unit, linked mostly by b-(1?4)- and b-(1?6)- bonds. In the
enzymatic synthesis of GOS, lactose acts both as donor and accep-
Lactose is a plentiful byproduct of cheese manufacture, whose
potential as a raw material for the production of valuable bioactive
molecules has been recently highlighted.1 The use of b-galactosi-
dase as a catalyst is attractive in performing such reactions as it
is a commodity that is widely used as a catalyst in the food indus-
try.2 b-Galactosidases can use a variety of acceptors to perform
transgalactosylation reactions; in that way valuable products like
galacto-oligosaccharides (GOS),3,4 lactulose5 and lactosucrose6
can be synthesized. The former two have been conclusively proven
as prebiotics,7 so their use in healthy foods is steadily increasing.8
During the kinetically controlled synthesis of such molecules,
b-galactosidases catalyze both the hydrolysis and transgalactosyla-
tion reactions, the latter being relevant for oligosaccharide produc-
tion. It is, therefore, necessary to assess the catalytic potential of
the enzymes in terms of their transgalactosylation activities and
not their hydrolytic activities as is customary.
tor of the galactosyl residue to yield trisaccharides (see Fig. 1)
which in turn can act as acceptors to yield tetrasaccharides and
so on. Lactulose is the non-digestible disaccharide 4-O-b-
D-galacto-
pyranosyl- -fructose. In the enzymatic synthesis of lactulose, lac-
D
tose acts as a donor of the galactose moiety, while fructose acts
as an acceptor. In this case, since lactose can also act as an acceptor,
a mixture of GOS and lactulose will be formed (see Fig. 2).
The purpose of this work was to develop an assay for determin-
ing the transgalactosylation activity of b-galactosidase to properly
assess its catalytic potential in the reactions for the synthesis of
GOS and lactulose, as well as to determine the effect of the reaction
conditions in the performance of both syntheses. The enzyme from
Aspergillus oryzae has been chosen for being one of the best indus-
trial enzymes available to perform such reactions.3,5,9
2. Experimental
2.1. Materials
GOS are non-digestible oligosaccharides composed by a variable
number of galactose units (usually from two to ten) and a terminal
b-Galactosidase from A. oryzae (EnzecoÒ Fungal Lactase) was
kindly donated by Enzyme Development Corporation, EDC (New
⇑
Corresponding author. Tel.: +56 32 2273642; fax: +56 32 2273803.
E-mail address: aillanes@ucv.cl (A. Illanes).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.01.030

746
C. Vera et al. / Carbohydrate Research 346 (2011) 745–752
Nomenclature
a
modulation factor by glucose on the affinity constant by
substrate (–)
o-NPG-E enzyme o-NPG transition complex concentration (mM)
o-NPG-E-Glu enzyme o-NPG glucose transition complex concen-
tration (mM)
v
modulation factor by glucose on the maximum reaction
rate of hydrolysis (–)
v
o-NPG reaction rate of hydrolysis of o-NPG (l )
M minꢁ1
E
free enzyme concentration (mmol kgꢁ1 or mM)
galactosyl–enzyme transition complex concentration
(mmol kgꢁ1 or mM)
vgal
specific rate of galactose release into the reaction med-
ium (mol kgꢁ1 minꢁ1 g enzꢁ1
specific rate of glucose release into the reaction medium
(mol kgꢁ1 minꢁ1 g enzꢁ1
specific rate of GOS i formation (mol kgꢁ1 minꢁ1 g enzꢁ1
specific rate of lactulose formation (mol kgꢁ1 minꢁ1
enzꢁ1
maximum specific reaction rate of glucose release into
the reaction medium (mol kgꢁ1 minꢁ1 g enzꢁ1
maximum reaction rate of o-NP release into the reaction
medium de hydrolysis de o-NPG (
M minꢁ1
vtrangal global specific reaction rate of transgalactosylated prod-
ucts formation (
mol kgꢁ1 minꢁ1 g enzꢁ1
E-Gal⁄
)
vglu
E-Gal
E-Glu
galactose–enzyme complex concentration (mmol kgꢁ1
or mM)
)
vGOS i
vlactu
)
g
glucose–enzyme complex concentration (mmol kgꢁ1 or
mM)
)
Gal
Glu
kcat
KGal
KGlu
KM
galactose concentration (mmol kgꢁ1 or mM)
glucose concentration (mmol kgꢁ1 or mM)
Vm
)
reaction rate constant of o-NPG hydrolysis (minꢁ1
)
Vmax
inhibition constant by galactose (mmol kgꢁ1 or mM)
modulation constant by glucose (mM)
Michaelis constant for lactose (mM)
l
)
l
)
KS
Lac
Michaelis constant for o-NPG (mM)
lactose concentration (mmol kgꢁ1
)
o-NPG
o-nitrophenyl b-D-galactopyranoside concentration (mM)
York, USA). The enzyme had a pH optimum between 4.5 and 5.0
and optimum temperature of 55 °C with respect to its hydrolytic
activity. The enzyme was stored at 4 °C and remained fully active
GOS-4
GOS-5
E
E
GOS-3 GOS-4
GOS-5
throughout the work.
D
-(+)-lactose monohydrate,
D-(+)-galactose,
GOS-4
GOS standards, o-nitrophenol (o-NP) and o-nitrophenyl b-
D
-galac-
GOS-4
H2O
topiranoside (o-NPG) were supplied by Sigma–Aldrich Chemical
Co. Lactulose was provided by Discovery Fine Chemicals (Wim-
borne, UK). All other reagents were of analytical grade and were
purchased from Sigma–Aldrich or E. Merck.
GOS-3
Glucose
E-Gal*
Lactose
Galactose
Lactose
Lactose
GOS-3
Galactose
2.2. Analyses
E
E
GOS-3
Lactose, fructose, and products of the reaction were determined
with a Jasco RI 2031 HPLC instrument, provided with a refractive
index detector, an isocratic pump (Jasco PU2080), and an autosam-
Galactose
E
E-Gal
pler (Jasco AS 2055), and using
(300 mm ꢀ 7.8 mm) for carbohydrate analysis (Benson Polymerics,
Reno, NV, USA). Samples were diluted and filtered through 0.22
a
BP-100 Ca2+ columns
Figure 1. Mechanism of synthesis of GOS with b-galactosidase from A. oryzae.
Modulation by glucose has not been considered.
lm
Millipore Durapore membranes prior to assay by HPLC and were
eluted with Milli-Q water at a flow-rate of 0.5 mL/min. Column
and detector temperatures were 80 and 40 °C, respectively. Reten-
tion times for substrates and products are presented on represen-
tative chromatograms as shown in Figure 3. Chromatograms were
integrated using the software ChromPass. The composition of the
samples was determined by assuming that the area of each peak
is proportional to the weight percentage of the respective sugar
on the total sugars mass.3,9–14 The accuracy of this assumption
was checked by a material balance according to Boon et al.10 Stan-
dards of fructose, galactose, glucose, galactobiose, lactose, and lac-
tulose were used to determine their retention times and check the
linear range of the measurements.
E
Lactulose
Fructose
Lactulose
Fructose
H2O
Glucose
E-Gal*
Lactose
Galactose
Lactose
GOS-3
Lactose
GOS-3
Galactose
2.3. Hydrolytic activity over o-NPG
E
E
One international unit of hydrolytic activity (IUH) was defined
as the amount of b-galactosidase that hydrolyzes 1 lmol of
Galactose
o-NPG per min at 30 mM o-NPG, pH 4.5 and 40 °C. Hydrolysis rates
were determined by measuring o-NP release during the first 5 min
of reaction. o-NP was determined by absorbance at 420 nm in a
Jenway 6715 spectrophotometer, using an extinction coefficient
E
E-Gal
Figure 2. Mechanism of synthesis of lactulose with b-galactosidase from A. oryzae.
Modulation by glucose has not been considered.

C. Vera et al. / Carbohydrate Research 346 (2011) 745–752
747
Figure 3. Typical chromatogram of galacto-oligosaccharides and lactulose synthesis using HPLC with refractive index detector and BP-100 Ca++(300 mm ꢀ 7.8 mm) column.
(a) Synthesis of GOS. (b) Synthesis of lactulose. Retention times: 8.6 (min) for GOS-5; 9.1 (min) for GOS-4; 9.9 (min) for GOS-3; 11.2 (min) for lactose; 12 (min) for lactulose;
13 (min) for glucose; 14.3 (min) for galactose and 15.4 (min) for fructose.
253.5 Mꢁ1 cmꢁ1 under the assay conditions. The assays were car-
of glucose were: 0, 100, 200, 300, and 400 mM glucose. All the
experiments were carried out in triplicate with less than 5% differ-
ence among the samples.
ried out in triplicate with less than 5% difference among samples.
2.4. The effect of galactose and glucose on the hydrolysis of
o-NPG
2.5. Transgalactosylation activity of A. oryzae b-galactosidase
The effect of galactose was analyzed by fitting the data to a
model of total competitive inhibition. The positive modulating ef-
fect of glucose on the hydrolysis of o-NPG was analyzed by fitting
the data to a model of partial mixed-type inhibition (modulation),
represented by Eq. 1, which is based on the mechanism whose
scheme is presented in Figure 4. The effect of glucose on the hydro-
lysis of o-NPG was also analyzed by fitting the data to the Michae-
lis–Menten equation and obtaining the apparent kinetic
parameters (Vap and Kap).
Experimental conditions for measuring transgalactosylation
activity in the synthesis of GOS and lactulose were selected as
those usually employed for the enzymatic synthesis of GOS with
fungal b-galactosidases.9–16
2.5.1. Transgalactosylation activity for the synthesis of GOS
For the synthesis of GOS, one international unit of b-galactosi-
dase transgalactosylation activity (IUT) was defined as the amount
a
ꢂ
v
ꢂGluꢂo-NPG
o-NPG
of enzyme that catalyzes the transglycosylation of 1 lmol of gal-
þ
þ
vo-NPG
Vmax
KS
KGluꢂKS
¼
ð1Þ
actose per min at 40% w/w initial lactose concentration, pH 4.5,
and 40 °C. The transgalactosylated galactose was determined con-
sidering the following material balance: 1 mol of galactose is trans-
galactosylated in the formation of 1 mol of GOS-3, 2 mol of
galactose are transgalactosylated in the formation of 1 mol of
GOS-4 and 3 mol of galactose are transgalactosylated in the forma-
tion of 1 mol of GOS-5 (see Fig. 1). Transgalactosylated disaccha-
rides (GOS-2) were not considered because the b-galactosidase
from A. oryzae produces insignificant amounts of GOS-2.3,9,11,12,14
Reactions were carried out in 150-mL Erlenmeyer flasks by dis-
solving 40 g of lactose in 50 g of 100 mM citrate–phosphate buffer
at the corresponding pH. Lactose was dissolved by heating the
solution at temperatures over 95 °C (no sugar degradation was ob-
served) and then, after cooling to the reaction temperature, 10 g of
properly diluted enzyme solution was added to start the reaction.
The enzyme concentration in the reaction vessel was 33 IUH/g lac-
tose in order to provide a linear range of transgalactosylation up to
15 min of reaction. Nine samples of 0.5 mL were taken at regular
intervals, and the reaction was stopped by adding 0.5 mL of
200 mM NaOH. The effect of temperature on the transgalactosyla-
tion activity was evaluated at 40, 45, 50, 55 and 60 °C while the ef-
fect of pH was evaluated at pH 2.5, 3.5, 4.5, 5.5, 6.5 and 8.5. The
assays were carried out in triplicate, with differences never
exceeding 5%.
o-NPG
KS
Glu
KGlu
aꢂGluꢂo-NPG
þ
KGluꢂKS
1 þ
Experiments were carried out at 40 °C, pH 4.5, and an enzyme con-
centration of 1 ppm in a 3-mL glass cuvette. A solution (1.8 mL)
containing a properly diluted concentration of o-NPG and glucose
or galactose dissolved on citrate–phosphate buffer 100 mM was
contacted with 0.2 mL of enzyme solution dissolved in the same
buffer. The hydrolysis rate was determined as described in Section
2.3. The concentrations of o-NPG evaluated were 0.41, 0.83, 1.66,
3.32, 6.64, 9.96, 16.61, 23.25, and 29.9 mM, the concentrations of
galactose were 0, 25, 50, 75, and 100 mM, and the concentrations
Figure 4. Mechanism of glucose modulation on the hydrolysis of o-NPG with b-
galactosidase.

748
C. Vera et al. / Carbohydrate Research 346 (2011) 745–752
2.5.2. Transgalactosylation activity for the synthesis of lactulose
For the synthesis of lactulose, one international unit of transga-
lactosylation activity (IUT) was defined as the amount of b-galacto-
The effect of galactose on the transgalactosylation activity in the
synthesis of lactulose is analyzed in the same way, considering the
reaction mechanism in Figure 2.
sidase that catalyzes the transglycosylation of 1
l
mol of galactose
The effect of galactose on transgalactosylation activity in the
synthesis of lactulose is analyzed in the same way, by considering
the reaction mechanism in Figure 2, which is represented by Eq. 5:
per min in a 50% (w/w) equimolar mixture of lactose and fructose
at pH 4.5 and 40 °C. The transgalactosylated galactose was deter-
mined considering the following material balance: 1 mol of galact-
ose is transgalactosylated in the formation of 1 mol of lactulose,
1 mol of galactose is transgalactosylated in the formation of
1 mol of GOS-3, 2 mol of galactose are transgalactosylated in the
formation of 1 mol of GOS-4, and 3 mol of galactose are transgalac-
tosylated in the formation of one mol of GOS-5 (see Fig. 2). Trans-
galactosylated disaccharides (GOS-2) were not considered because
the b-galactosidase from A. oryzae produces insignificant amounts
of GOS-2.3,9,11,12,14
Reactions were carried out in 150-mL Erlenmeyer flasks by dis-
solving 32.76 g of lactose and 17.4 g of fructose (molar ratio of 1) in
40 g of 100 mM citrate–phosphate buffer at the corresponding pH.
Substrates were dissolved by heating the solution at a temperature
over 95 °C (no degradation of sugars was observed) and then after
cooling to the reaction temperature, 10 g of a properly diluted en-
zyme solution was added to start the reaction. The enzyme concen-
tration in the reaction vessel was 64 IUH/g of lactose in order to
provide a linear range of transgalactosylation up to 15 min of reac-
tion. Nine samples of 0.5 mL were taken at regular intervals, and
the reaction was stopped by adding 0.5 mL of 200 mM NaOH.
The effect of the temperature on the transgalactosylation activity
of the b-galactosidase from A. oryzae was evaluated at 40, 45, 50,
55, and 60 °C, while the effect of pH was evaluated at pH 2.5, 3.5,
4.5, 5.5, 6.5, and 8.5. The assays were carried out in triplicate, with
differences never exceeding 5%.
n
X
vtransgal
¼
ði ꢁ 2Þ ꢂ vGOSi
þ
vlactu
¼
vglu
ꢁ
vgal
ꢃ
vglu
ð5Þ
i¼2
2.7. Parameter fitting
Kinetic and thermodynamic constants for the rate equations
were determined by nonlinear regression of experimental data to
the proposed models using the software Mathematica 7.0 (Wol-
fram Research, UK). All the experimental data for each assay were
fitted simultaneously.
2.8. Synthesis of GOS
Reactions for the synthesis of GOS were carried out at different
pH and temperatures. The purpose of these experiments was to as-
sess the transgalactosylation activity as a predictive tool for the ef-
fect of these variables on the synthesis of GOS.
Reactions were carried out at 40 °C under the same conditions
described in Section 2.5.1. The enzyme concentration on the reac-
tion medium was 33 IUH/g lactose. Samples (0.5 mL) were taken at
regular intervals during 600 min of reaction. The effect of pH was
evaluated at pH 2.5, 3.5, 4.5, 5.5, 6.5, and 8.5, and the effect of tem-
perature at 40, 45, 50, 55, and 60 °C. The experiments were carried
out in triplicate, with differences never exceeding 5%.
2.6. Effect of galactose and glucose on the transgalactosylation
activity for the synthesis of GOS and lactulose
2.9. Synthesis of lactulose
As in the case of GOS, reactions for the synthesis of lactulose
were carried out at different pHs and temperatures with the pur-
pose of also assessing the transgalactosylation activity as a predic-
tive tool of the effect of these variables on the synthesis of
lactulose.
Reactions were carried out at pH 4.5 under the same conditions
described in Section 2.5.2. The enzyme concentration on the reac-
tion medium was 320 IUH/g lactose. Samples of 0.5 mL were taken
at regular intervals during 200 min of reaction. The effect of pH
was evaluated at pH 2.5, 3.5, 4.5, 5.5, 6.5, and 8.5, and the effect
of temperature at 40, 45, 50, 55, and 60 °C. The experiments were
carried out in triplicate, with differences never exceeding 5%.
Enzyme transgalactosylation activity was determined for the
synthesis of GOS and lactulose in presence of different initial con-
centrations of galactose (0, 50, 100, 200, 400, and 600 mmol/kg)
and glucose (0, 100, 200, 400, 600, and 800 mmol/kg). The experi-
ments were carried out to pH4.5 and 40 °C as is described on Sec-
tions 2.5.1 and 2.5.2. All the experiments were carried out in
triplicate, differences never exceeding 5%.
In order to evaluate the effect of galactose, results were ana-
lyzed considering the mass balance represented by Eq. 2:
n
X
vtransgal
¼
ði ꢁ 2Þ ꢂ vGOSi
¼
vglu
ꢁ
vgal
ꢃ
vglu
ð2Þ
i¼2
which is based on the reaction mechanism shown in Figure 1 for the
synthesis of GOS.
3. Results and discussion
Under the conditions of the assay for the transgalactosylation
activity, the rate of galactose release is insignificant so that the trans-
galactosylation rate can be approximated to the rate of glucose re-
lease as indicated in Eq. 2. The competitive inhibition of galactose
on the rate of glucose release in A. oryzae b-galactosidase has been
consistently reported.17,18 Taking this into consideration, Eq. 3 is pro-
posed whose linearization, represented by Eq. 4, allows the determi-
nation of the effect of galactose on transgalactosylation activity,
provided that the Michaelis constant for lactose (KM) is known. A va-
lue of KM of 93.77 mM has been previously determined by us using
standard methodology for determining kinetic parameters.
3.1. Transgalactosylation and hydrolytic activity
Most of trangalactosylated products in the synthesis of GOS
were GOS-3 (three sugar residues). Specific trangalactosylation
activities for GOS and lactulose synthesis were 55,900 221 and
64,590 760 IUT/g of enzyme preparation, respectively. The en-
zyme has a very high transgalactosylation activity, in accordance
with high values of productivity already reported for a fungal
b-galactosidase.3,12 Hydrolytic activity for o-NPG was calculated
as 196,000 5500 IUH/g of enzyme preparation.
3.2. Effect of temperature on the transgalactosylation activity
and in the synthesis of GOS and lactulose
Vm ꢂ Lac
ꢀ
ꢂ
ꢁ
vtrangal
ꢃ
ꢃ
vglu
¼
ð3Þ
ð4Þ
Gal
KM
ꢂ
1 þ K
þ Lac
Gal
ꢂ
ꢃ
ꢂ
ꢃ
The transgalactosylation activity increased with temperature in
the range from 40 to 55 °C as shown in Figure 5a; this is reflected in
1
KM
1
KM
1 þ
þ
ꢂ Gal
vtrangal
Lac Vm
Lac ꢂ Vm ꢂ KGal

C. Vera et al. / Carbohydrate Research 346 (2011) 745–752
749
Figure 5. (a) Effect of temperature on transgalactosylation activity. } Synthesis of GOS: 40% w/w of lactose, 33 IUH/g lactose, 40 °C and pH 4.5. ꢀ Synthesis of lactulose: 50%
w/w of sugar, lactose/fructose molar ratio of 1 and 64 IUH/g lactose, 40 °C and pH 4,5. (b). Time course of synthesis of lactulose at different temperatures at 50% w/w of sugar,
lactose/fructose molar ratio of 1 and 320 IUH/g lactose, 40 °C and pH 4.5: ꢀ 40 °C, } 45 °C, j 50 °C, N 55 °C and h 60 °C.
the corresponding increase in productivity for the synthesis, both
of GOS and lactulose. Despite this, the composition of the reacting
mixture at a given lactose conversion was not affected by temper-
ature, which is in agreement with results previously pub-
lished.3,11,19 This may be the consequence of a similar effect of
temperature on the hydrolytic activity of the enzyme. In this
way, in the kinetically controlled synthesis of GOS and lactulose,
the balance between transgalactosylation and hydrolytic activity
is not altered by temperature. At temperatures higher than 55 °C
activity decreases, but this is the consequence of inactivation.
To illustrate the effect of temperature on the reactions for the
synthesis, the results are presented on the time-course for the syn-
thesis of lactulose (results are similar for the synthesis of GOS) in
Figure 5b. Maximum lactulose concentration obtained was 28 g/kg.
did not alter product distribution at a given lactose conversion in
agreement with previous reports.3,11,19
The time-course for the synthesis of GOS is used in this case to
illustrate the effect of pH (results are similar for the synthesis of
lactulose). No significant differences in the pattern of GOS synthe-
sis between pH 3.5 and 5.5 was observed, with the maximum GOS
concentration being reached at about the same time as shown in
Figure 6b. Below pH 3.5 the enzyme was rapidly inactivated. At
pHs over 5.5 the effect was different, with a decrease in conversion
as the consequence of reduced activity rather than stability. The
maximum GOS concentration was around 28% of the total sugar,
which is in agreement with previous reports.3,11
3.4. Effect of glucose and galactose on the hydrolytic and
transgalactosylation activity. Parameter fitting
3.3. Effect of pH on the transgalactosylation activity and in the
synthesis of GOS and lactulose
The effect of both monosaccharides on the transgalactosylation
activity in the synthesis of GOS and lactulose is presented in Figure
7. In both cases galactose was a strong inhibitor of transgalactosy-
lation activity. Data were fitted to the model of total competitive
inhibition by galactose represented by Eq. 3. Figure 8 shows the fit-
ting to the linearized model represented by Eq. 4. A good fitting
was obtained with a correlation coefficient of 0.99, inhibition
Catalysis with b-galactosidases is mostly acid catalysis,20 so pH
should have a strong effect on enzyme kinetics. This effect is pre-
sented in Figure 6a, both for the synthesis of GOS and lactulose.
A broad optimum range is observed between pH 2.5 and 5.5, with
the activity sharply decreasing at higher pH. In both cases, the pH
Figure 6. Effect of pH on transgalactosylation actvity. (a) } Synthesis of GOS: 40% w/w of lactose, 33 IUH/g lactose, 40 °C and pH 4.5. ꢀ Synthesis of lactulose: 50% w/w of
sugar, lactose/fructose molar ratio of 1 and 64 IUH/g lactose, 40 °C and pH 4.5. (b) Time course of synthesis of GOS at different pHs at 40% w/w of lactose, 33 IUH/g lactose and
40 °C: N pH 2.5; 4 pH 3.5; ꢀ pH 4.5; d pH 5.5; s pH 6.5; h pH 8.5.

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C. Vera et al. / Carbohydrate Research 346 (2011) 745–752
Figure 7. Effect of glucose and galactose on transgalactosylation actvity. ꢀ Galactose, N Glucose. (a) Synthesis of GOS at 40% w/w of lactose, 33 IUH/g lactose, 40 °C and pH 4.5.
(b) Synthesis of lactulose 50% w/w of sugar, lactose/fructose molar ratio of 1 and 64 IUH/g lactose, 40 °C and pH 4.5.
tosylation than for synthesis in the case of A. oryzae b-galactosi-
dase. The high affinity of this enzyme for galactose and low
affinity for lactose (KM = 93.8 mM) is a certain disadvantage with
respect to b-galactosidases from other sources. However, its very
high transgalactosylation activity is a definite advantage in terms
of specific productivity.3,12 On the other hand, the product distri-
bution is quite good with a high proportion of GOS in the form of
GOS-3 and GOS-4, which is better than the results obtained with
enzymes from other sources.9 Glucose at concentrations below
200 mmol kgꢁ1 exerted a positive effect in the transgalactosylation
activity both in the case of GOS and lactulose synthesis. At higher
concentrations inhibition was observed. Activation by glucose is
reported by its producer as a characteristic of this enzyme prepa-
ration. The effect of glucose is rather controversial, and an adverse
effect has also been reported,3,11 but no mechanism of modulation
has been proposed. To obtain a better insight on the effect of glu-
cose, its effect on the hydrolysis of o-NPG was evaluated. Results
Figure 8. Total competitive inhibition of transgalactosylation activity by galactose
at 40 °C and pH 4.5. } Synthesis of GOS; ꢀ Synthesis of lactulose.
are presented in Figure 9b, where a clear positive modulation effect
is observed both in affinity (apparent Michaelis–Menten constant,
Kap) and reactivity (apparent maximum reaction rate, Vap) at values
below 200 mmol kgꢁ1. At concentrations >200 mmol kgꢁ1 a posi-
tive effect in the maximum reaction rate is still observed, but a def-
inite decrease in affinity is produced. Results were fitted to a
mixed-type inhibition (modulation) model, obtaining a correlation
coefficient of 0.99 for the concentration of glucose between 0 and
400 mM (seeFig. 10). According to the results obtained by non- lin-
ear regression of the data presented in Table 1, a 93% increase in
the maximum rate of hydrolysis of o-NPG was obtained in the
constants being determined as 3.86 and 4.73 mmol/kg in the cases
of GOS and lactulose, respectively. Table 1 summarizes the values
of kinetic parameters, including maximum activity of transgalac-
tosylation for both syntheses. The value of the inhibition constant
by galactose is an indicative of the magnitude of the effect of gal-
actose in both cases, being somewhat stronger for the synthesis
of GOS than for lactulose. The effect of galactose on the hydrolysis
of o-NPG was also determined, as is customary (Fig. 9a). A value for
the inhibition constant of 24 mM was obtained, meaning that the
inhibition of galactose is stronger for the reactions of transgalac-
Table 1
Estimated values of kinetic parameters
Parameter
Value
Confidence interval
(95%)
Units
Experiment
Vmax
KS
KGlu
a
194.7
2.27
126.7
0.51
9.25
0.4
61.6
0.15
0.23
l
M minꢁ1
Hydrolysis of o-NPG in the presence of glucose
mM
mM
—
v
1.93
—
KGal
23.54
2.56
mM
Hydrolysis of o-NPG in the presence of galactose
Maximum transgal.
activity
KGal
59,716
2860
l
mol Gal transgal minꢁ1 gꢁ1 Transgalactosylation activity in the synthesis of GOS in the presence of
galactose
3.86
0.56
mmol kgꢁ1
Maximum transgal.
activity
KGal
68,150
1418
l
mol Gal transgal minꢁ1 gꢁ1 Transgalactosylation activity in the synthesis of lactulose in the presence
of galactose
4.73
0.3
mmol kgꢁ1

C. Vera et al. / Carbohydrate Research 346 (2011) 745–752
751
Figure 9. Effect of glucose and galactose on the hydrolysis of o-NPG at 40 °C and pH4.5. (a) ꢀ Galactose 0 mM, } Galactose 25 mM, d Galactose 50 mM, s 75 Galactose mM, N
100 mM Galactose. (b) N Apparent maximum reaction rate of o-NPG hydrolysis (Vap), d Apparent affinity constant by o-NPG (Kap) and ꢀ Vap/Kap ratio.
4. Conclusions
A methodology was developed to properly assess the potential
of b-galactosidases for catalyzing transgalactosylation reactions.
The methodology was applied to a commercial b-galactosidase
from A. oryzae, which was characterized under conditions for the
synthesis of GOS and lactulose, evaluating the effect of pH, temper-
ature and monosaccharide concentration on its transgalactosyla-
tion activity.
The results indicate the convenience of measuring actual trans-
galactosylation activity and not, as is usually done, hydrolytic
activity, since the latter poorly predicts the transgalactosylation
potential of the biocatalyst.
The results showed that the inhibitory effect of galactose is sig-
nificantly stronger for the reactions of transglycosylation than for
the reaction of the hydrolysis of lactose.
Figure 10. Model fit for glucose modulation on o-NPG hydrolysis to the experi-
mental results obtained at 40 °C and pH4.5. Initial glucose concentration: d 0 mM;
4 100 mM; j 200 mM; s 300 mM; ꢀ 400 mM. Symbols: experimental data; Solid
line: model.
Glucose, despite being an activator of the trangalactosylation
activity at moderate concentrations, is an inhibitor at concentra-
tions exceeding 200 mmol kgꢁ1, which is an important issue since
glucose release in the reaction medium is unavoidable during
transgalactosylation. This highlights the relevance of alleviating
such an effect by developing strategies for the removal of the
monosaccharides. A better insight into the mechanism of action
of glucose will allow establishing a rational approach to minimize
its effect.
presence of a saturating concentration of glucose. However, glu-
cose produced an increase in the affinity constant for o-NPG of
about 100%. The value for
constant of the modulated enzyme was Ks/
a
(see Fig. 4) was 0.53, and the affinity
, so the affinity con-
a
stant increased its value twofold. These results support the
hypothesis that glucose binds to the enzyme at a site different from
the active site. The decrease in the activity of transgalactosylation
at concentrations of glucose higher than 200 mmol kgꢁ1 is mainly
the consequence of a decrease in the affinity of the enzyme by lac-
tose, as it also occurred in the case of o-NPG. Figure 9b shows that
the Vap/Kap ratio decreased at glucose concentrations over
200 mmol kgꢁ1, showing a similar behavior to the activity of trans-
galactosylation in presence of glucose (see Fig. 7). The results ob-
tained suggest that this decrease in the activity of
Acknowledgments
This work was financially supported by Chilean Fondecyt
Grants 1100050 and 1080118, and Grant 037-112/2008 from the
Pontificia Universidad Católica de Valparaíso. The work was per-
formed within the framework of CYTED-D Enznut and NovelProbio
networks. We acknowledge the generous donations of A. oryzae
b-galactosidase by Enzyme Development Corporation (New York,
USA).
trangalactosylaction is the consequence of an increase in Kap
,
which is very significant because the Michaelis constant of lactose
is high for the b-galactosidase from A. oryzae and the solubility of
lactose is low, which is more important than the increase in Vap
produced by glucose.
The results suggest the convenience of in situ removal of the
monosaccharide from the standpoint of reaction kinetics and be-
yond the advantage in terms of product purity. Glucose and galact-
ose removal should increase conversion and productivity. Some
options have been studied like the adsorption inactivated carbon21
and nanofiltration.13 However, these strategies have still to be pro-
ven feasible under industrial process conditions.
References
1. Schaafsma, G. Int. Dairy J. 2008, 18, 458–465.
2. Husain, Q. Crit. Rev. Biotechnol. 2010, 30, 41–62.
3. Albayrak, N.; Yang, S. T. Biotechnol. Bioeng. 2002, 77, 8–19.
ˇ
ˇ
4. Pocedicová, K.; Curda, L.; Mišún, D.; Dryáková, A.; Diblíková, L. J. Food Eng.
2010, 479–484.
5. Adamczak, M. D.; Charubin, D.; Bednarsky, W. Chem. Pap. 2009, 63, 111–116.
6. Li, W.; Xiang, X.; Tang, S.; Hu, B.; Tian, L.; Sun, Y.; Ye, H.; Zeng, Y. J. Agric. Food
Chem. 2009, 57, 3927–3933.
7. Rycroft, C. E.; Jones, M. R.; Gibson, G. R.; Rastall, R. A. J. Appl. Microbiol. 2001, 91,
878–887.

752
8. Wang, Y. Res. Int. 2009, 42, 8–12.
9. Huerta, L. M.; Vera, C.; Guerrero, C.; Wilson, L.; Illanes, A. Process Biochem. 2011,
46, 245–252.
C. Vera et al. / Carbohydrate Research 346 (2011) 745–752
16. Kim, Y. S.; Park, C. S.; Oh, D. K. Enzyme Microb. Technol. 2006, 39, 903–908.
17. Illanes, A.; Zúñiga, M. E.; Chamy, R.; Marchese, P. Immobilization of Lactase and
Invertase on Crosslinked Chitin. In Bioreactor Immobilized Enzymes and Cells:
Fundamentals and Applications; Moo-Young, M., Ed.; Elsevier Applied Science:
Essex, 1988; pp 233–249.
10. Boon, M. A.; Janssen, A. E. M.; van der Padt, A. Biotechnol. Bioeng. 1999, 64, 558–
567.
11. Neri, D. F. M.; Balcão, V. M.; Costa, R. S.; Rocha, I. C. A. P.; Ferreira, E. M. F. C.;
Torres, D. P. M.; Rodriguez, L. R. M.; Carvalho, L. B., Jr.; Texeira, J. A. Food Chem.
2009, 115, 92–99.
12. Sanz Valero, J.I. Ph.D. Thesis, Ohio State University, Ohio, USA, 2009.
13. Pruksasri, S. Ph.D. Thesis, Ohio State University, Ohio, USA, 2007.
14. Cheng, C. C.; Yu, M. C.; Cheng, T. Z.; Sheu, D. H.; Duan, K. J.; Tai, W. L. Biotechnol.
Lett. 2006, 28, 793–797.
18. Portaccio, M.; Stellato, S.; Rossi, S.; Bencivenga, U.; Mohy Eldin, M. S.; Gaeta, F.
S.; Mita, D. G. Enzyme Microb. Technol. 1998, 23, 101–106.
19. Martinez-Villaluenga, C.; Cardelle-Cobas, A.; Corzo, N.; Olano, A.; Villamiel, M.
Food Chem. 2008, 107, 258–264.
20. Prenosil, J. E.; Stuker, E.; Bourne, J. R. Biotechnol. Bioeng. 1987, 30, 1019–1025.
21. Boon, M. A.; van´ t Riet, K. V.; Janssen, A. E. M. Biotechnol. Bioeng. 2000, 70, 411–
420.
15. Lee, Y. J.; Kim, C. S.; Oh, D. K. Appl. Microbiol. Biotechnol. 2004, 64, 787–793.