Optimization of lactulose synthesis from whey lactose by immobilized β-galactosidase and glucose isomerase

Article abstract of DOI:10.1016/j.carres.2013.01.002

In the present study, commercially available whey was used as a lactose source, and immobilized β-galactosidase and glucose isomerase were used to synthesize lactulose from whey lactose in the absence of fructose. Optimal reaction conditions, such as lactose concentration, temperature, ionic strength of the buffer, and ratio of immobilized enzymes, were determined to improve lactulose synthesis using immobilized enzymes. Lactulose synthesis using immobilized enzymes improved markedly after optimizing the reaction conditions. When the lactulose synthesis was carried out at 53.5 °C using 20% (w/v) whey lactose, 12 U/ml of immobilized β-galactosidase and 60 U/ml of immobilized glucose isomerase in 100 mM sodium phosphate buffer at pH 7.5, the lactulose concentration and specific productivity were 7.68 g/l and 0.32 mg/U h, respectively. Additionally, when the immobilized enzymes were reused for lactulose synthesis, their catalytic activity was 57.1% after 7 repeated uses.

Full text of DOI:10.1016/j.carres.2013.01.002

Carbohydrate Research 369 (2013) 1–5
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Optimization of lactulose synthesis from whey lactose
by immobilized b-galactosidase and glucose isomerase
a
a
b
a,
⇑
Yoon-Seok Song , Hee-Uk Lee , Chulhwan Park , Seung-Wook Kim
a
Department of Chemical and Biological Engineering, Korea University, 5 Ga, Anam-dong, Sungbuk-gu, Seoul 136-701, Republic of Korea
Department of Chemical Engineering, Kwangwoon University, 447-1, Wolgye-dong, Nowon-gu, Seoul 139-701, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, commercially available whey was used as a lactose source, and immobilized
b-galactosidase and glucose isomerase were used to synthesize lactulose from whey lactose in the
absence of fructose. Optimal reaction conditions, such as lactose concentration, temperature, ionic
strength of the buffer, and ratio of immobilized enzymes, were determined to improve lactulose synthe-
sis using immobilized enzymes. Lactulose synthesis using immobilized enzymes improved markedly
after optimizing the reaction conditions. When the lactulose synthesis was carried out at 53.5 °C using
Received 29 September 2012
Received in revised form 1 January 2013
Accepted 2 January 2013
Available online 10 January 2013
Keywords:
20% (w/v) whey lactose, 12 U/ml of immobilized b-galactosidase and 60 U/ml of immobilized glucose
b-Galactosidase
Glucose isomerase
Lactulose
Whey
Lactose
isomerase in 100 mM sodium phosphate buffer at pH 7.5, the lactulose concentration and specific
productivity were 7.68 g/l and 0.32 mg/U h, respectively. Additionally, when the immobilized enzymes
were reused for lactulose synthesis, their catalytic activity was 57.1% after 7 repeated uses.
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
b-galactosidase as the sole biocatalyst, and purified lactose and
1
,4,9,11
10
fructose as the substrate.
Hua et al. synthesized lactulose
Galactooligosaccharides (GOSs) have been used as functional
using b-galactosidase and glucose isomerase. However, the reac-
tion used in their study contained purified lactose and fructose.
Purified lactose is an expensive raw material for lactulose synthe-
sis. Applying milk whey, which contains 4.5–5% (w/v) lactose, is
much more cost effective. Whey is a by-product of cheese and
casein production, and hence, is an inexpensive raw material that
contains a high amount of lactose. Much of the whey produced by
the dairy industry is used as animal feed, fertilizer, and as a food
components in the food and pharmaceutical field. One compound
of industrial interest among GOSs is lactulose (non-digestible
GOS, 4-O-b-D-galactopyranosyl-D
-fructose).1 Lactulose has been
used in a wide variety of foods as a bifidus factor or as a functional
2
ingredient for intestinal regulation. Moreover, lactulose is used in
the pharmaceutical field, mainly for treating constipation, hepatic
encephalopathy, complications of liver disease, and for the mainte-
nance of blood glucose and insulin levels.3
12
additive. Despite this, about 30% of the annual world whey pro-
Methods for lactulose synthesis can be divided into chemical
and biological methods. Lactulose is chemically synthesized by
the alkaline isomerization of lactose.4 However, there are many
issues associated with the chemical synthesis of lactulose, includ-
ing product purification, lactulose degradation, side reactions, and
duction (177 million tonnes in 2006) remains underutilized.13
Whey is considered a high strength wastewater pollutant due
to its high biological (BOD) and chemical oxygen demand
1
4
(COD), and a cost-effective disposal or utilization methods have
become increasingly important to the dairy industry. So, because
of both environmental and economic concerns, whey should ide-
ally be converted into value-added products. We hypothesized
that whey could be used as an alternative substrate for the enzy-
matic synthesis of lactulose. Moreover, our group has developed
pretreatment methods that prevent the loss of b-galactosidase
and glucose isomerase activity during enzyme immobilization
1
waste management. To solve these problems, the enzymatic syn-
thesis of lactulose has been studied. b-Galactosidase is a very
important enzyme in the dairy industry, as it catalyzes the con-
version of lactose into glucose and galactose.5 It has been
broadly used for preparing lactose-hydrolyzed products to treat
lactose-intolerant or lactase-deficient patients.7 b-Galactosidases
can be used to synthesize lactulose from lactose through a trans-
galactosylation reaction, using fructose as a galactosyl accep-
,6
,8
1
5,16
through covalent bonds.
Accordingly, in this study, b-galactosidase and glucose isomer-
1
,4,9–11
tor.
In most studies, lactulose is synthesized using
ase were immobilized on silica gel according to a previously
1
5,16
reported immobilization method,
and the immobilized enzymes
⇑
Corresponding author. Tel.: +82 2 3290 3300; fax: +82 02 926 6102.
E-mail address: kimsw@korea.ac.kr (S.-W. Kim).
were used for lactulose synthesis from whey lactose without sup-
plying fructose. Immobilized glucose isomerase was used to assist
0
008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.01.002

2
Y.-S. Song et al. / Carbohydrate Research 369 (2013) 1–5
the immobilized b-galactosidase, as it can isomerize glucose pro-
duced from lactose hydrolysis to fructose. Consequently, fructose
can be continuously transformed from glucose by immobilized glu-
cose isomerase.
20 mM potassium phosphate buffer at pH 7.0, 5 mM ONPG and
immobilized b-galactosidase, and the enzyme reaction was con-
ducted at 37 °C and 150 rpm for 10 min. Two milliliters of 1 M
2 3
Na CO was added to the reaction mixture to stop the reaction. Lib-
Lactulose synthesis from whey lactose, without supplying
fructose, using immobilized b-galactosidase and glucose isomer-
ase, has not previously been reported. Reaction conditions, such
as concentration of whey lactose, temperature, ionic strength of
buffer and ratio of immobilized enzymes, were optimized to im-
prove lactulose synthesis. In addition, the reusability of the immo-
bilized enzymes was examined through repeated batch reactions.
erated 2-nitrophenol was measured at 410 nm, and its concentra-
tion was calculated using standard 2-nitrophenol solutions. One
unit of enzyme activity (U) was defined as the amount of enzyme
catalyzing the hydrolysis of 1
reaction conditions.
lmol of ONPG per minute under the
The activity of immobilized glucose isomerase was assessed by
measuring the concentration of fructose converted from glucose.
The reaction mixture contained 10 ml of 0.2 M sodium phosphate
buffer at pH 7.0, 0.2 M glucose, 20 mM MgSO
CoCl O, and immobilized glucose isomerase. The enzyme reac-
Á6H
tion was conducted at 60 °C and 100 rpm for 30 min, after which
ml of 20% (v/v) HCl was added to the reaction mixture to stop
4
Á7H
2
O, 1 mM
2
2
. Experimental
2
2
.1. Materials
2
the reaction. The fructose concentration was then measured by
the HCl-resorcinol method, in which resorcinol forms a red-colored
b-Galactosidase (EC 3.2.1.23) from Kluyveromyces lactis, whey
power, lactulose, (3-aminopropyl)triethoxysilane (3-APTES), and
o-nitrophenyl-b- -galactopyranoside (ONPG) were purchased from
complex with
5
D
-fructose, which has maximal absorbance at
20 nm. One unit of enzyme activity (U) was defined as the
amount of enzyme that catalyzed the conversion of 1 mol of glu-
D
1
9
Sigma (St. Louis, MO, USA). Glucose isomerase (EC 5.3.1.5) from
Streptomyces rubiginosus was purchased from Hampton Research
San Diego, CA, USA). Silica gels were provided by Grace Davison
Co. (Columbia, MD, USA). Lactose monohydrate and xylose were
purchased from Junsei Chemical Co. (Tokyo, Japan). Glutaraldehyde
solution was purchased from Fluka Co. (St. Gallen, Switzerland). All
other chemicals used in this study were of reagent grade.
l
cose into fructose per minute under the assay conditions described.
Activity recovery was calculated as the ratio of the activity of
immobilized enzyme to the activity of free enzyme, and was
expressed as a percentage.
(
2
.5. Preparation of whey lactose
2
.2. Pretreatment of b-galactosidase and glucose isomerase
Two hundred grams of whey power (Spray dried powder con-
before immobilization
taining lactose >65% (wt%) and protein >11% (wt%)) was added to
00 ml of sodium phosphate buffer (pH 7.5) and was mixed vigor-
ously. The mixture was centrifuged for 20 min at 13,000Âg and
5 °C, and the supernatant was adjusted to pH 7.5. The resulting
1
Lactose (30 mM) was added to 20 ml of
a 10% (v/v)
b-galactosidase solution in 0.1 M sodium phosphate buffer (pH
7
incubated at 37 °C and 150 rpm for 1 h. Glucose isomerase was
pretreated with 2.0 M xylose in 20 ml of a 10% (v/v) glucose isom-
erase solution (containing 20 mM MgSO
CoCl O in 0.2 M sodium phosphate buffer at pH 7.0) at 60 °C
and 200 rpm for 45 min. The resulting enzyme solutions were
then used for immobilization as described below.
2
.2) to pretreat the b-galactosidase.15 The mixture was then
solution was centrifuged again for 20 min at 13,000Âg and 25 °C.
The lactose concentration of the supernatant was approximately
2
0% (w/v), and the prepared lactose solution was stored at 4 °C
4 2
Á7H O
and 1 mM
20
for further use.
2
Á6H
2
16
2
.6. Lactulose synthesis
The following reaction conditions were used for lactulose syn-
2
.3. Enzyme immobilization
thesis with the immobilized enzymes: lactose (14%, 16%, 18%,
and 20%, w/v), temperature (47, 50, 53.5, 57, and 60 °C), ionic
strength (50, 100, 150, 200, 250, and 300 mM) of sodium phos-
phate buffer at pH 7.5, ratio (1:1, 1:2, 1:3, 1:4, 1:5, and 1:6) of
immobilized enzymes, agitation speed of 150 rpm, and a reaction
time of 3 h. The lactose conversion rate (defined as ([S ]À[S ])/
One gram of silica gel was silanized with 20 ml of an acetone
solution containing 0.75 M 3-APTES at 50 °C for 2 h, washed with
distilled water, and dried at 60 °C for 2 h. The dried silica gels were
added to 20 ml of 0.21 M glutaraldehyde solution in 0.1 M sodium
phosphate buffer (pH 8.0), after which it was incubated at 20 °C for
0
s
[S ] Â 100 %) and specific productivity (defined as [P]/([E] Â t),
0
1
7
2
h. The activated silica gels were then washed with distilled
mg/U h) were calculated. To test the reusability of the immobilized
water and dried at 60 °C for 2 h. The activated silica gels were
added to the pretreated b-galactosidase, or the pretreated glucose
isomerase solution, and were reacted at 20 °C for 12 h with con-
stant stirring. The immobilized enzymes were then washed with
sodium phosphate buffer and recovered by filtration.1
The amount of immobilized protein was determined according
to the difference between total protein and the amount remaining
in solution after immobilization, and the immobilization yield was
expressed as a percentage. Protein concentration was determined
using a Bio-Rad protein assay reagent according to the manufac-
turer’s protocols (Bio-Rad Laboratories, Hercules, CA, USA), and
the absorbance was measured at 750 nm.
enzymes, they were filtered and washed with 100 mM sodium
phosphate buffer (pH 7.5) between batch reactions. The relative
catalytic activity of the immobilized enzymes was calculated as
the lactulose ratio of the reused batch to the first batch, and was
expressed as a percentage. All experiments were conducted in
duplicate, and data were represented as an error bar.
5,16
2.7. Determination of sugars
Sugars were analyzed by high performance liquid chromatogra-
phy with an RI detector using a high performance carbohydrate
column (4.6 mm  250 mm, Waters Co., Milford, MA, USA) at a
9
flow rate of 1.4 ml/min at 35 °C. The mobile phase consisted of a
2
.4. Determination of immobilized enzyme activity
mixture of acetonitrile/deionized water (8:2, v/v). Concentrations
of sugars, such as lactose, lactulose, and fructose were calculated
from standard curves drawn from the standard concentrations,
respectively.
Immobilized b-galactosidase activity was assayed using ONPG
1
8
as the substrate.
The reaction mixture contained 10 ml of

Y.-S. Song et al. / Carbohydrate Research 369 (2013) 1–5
3
3
. Results and discussion
In this study, b-galactosidase and glucose isomerase were used
100
A
8
6
4
2
0
0
0
0
0
for lactulose synthesis, the balance of transgalactosylation and
isomerization of which is very important. In our previous studies
and preliminary experiments, the optimum temperature and ionic
1
5,21
strength of the two enzymes were observed to be different.
Accordingly, we focused on the optimization of the reaction condi-
tions for lactulose synthesis, such as temperature, ionic strength,
ratio of immobilized enzymes, and lactose concentration.
1
1
4%
6%
18%
2
0%
3
.1. Immobilization of b-galactosidase and glucose isomerase
We have developed pretreatment methods that prevent the loss
0
20
40
60
80
100 120 140 160 180
of b-galactosidase and glucose isomerase activity during enzyme
Time (min)
immobilization through covalent bonds.1
5,16
We suggest that the
effect of enzyme pretreatment prior to immobilization was due
to a steric effect at the enzyme active site by the substrate, and
that enzymatic regions far from the active site would react with
the solid surface. Accordingly, b-galactosidase and glucose isome-
rases pretreated with lactose or xylose were covalently immobi-
lized on activated silica gels in this study, and the immobilized
enzymes were used for lactulose synthesis. The immobilization
yield and activity recovery for b-galactosidase were 32.8% and
4
3
2
1
0
B
4
9.2%, respectively, and the activity of immobilized b-galactosidase
was 776.1 U/g matrix. The immobilization yield, activity recovery,
and activity for immobilized glucose isomerase were 52.3%, 47.1%,
and 246.7 U/g matrix, respectively.
14%
1
1
6%
8%
20%
3
.2. Effect of whey lactose concentration
0
20
40
60
80
100 120 140 160 180
Time (min)
The effect of lactose concentration on lactulose synthesis was
Figure 1. Effect of whey lactose concentration on lactose conversion rate (A) and
lactulose synthesis (B). Reactions were conducted at 47 °C using 12 U/ml immo-
bilized b-galactosidase and 12 U/ml immobilized glucose isomerase in 50 mM
sodium phosphate buffer at pH 7.5.
evaluated using different lactose concentrations (14%, 16%, 18%,
and 20%, w/v) to determine the optimum whey lactose concentra-
tion. Figure 1 shows the lactose conversion rate and lactulose con-
centration at different lactose concentrations. A higher lactose
conversion rate was observed at lower substrate concentrations
(
Fig. 1A), and lactulose synthesis was dependent on lactose concen-
1
0
8
6
4
2
0
0.20
0.15
0.10
0.05
0.00
tration (Fig. 1B). Overall, lactulose concentration increased with an
increase in lactose concentration. The highest lactulose concentra-
tion (3.54 g/l) was obtained at a lactose concentration of 20%. The
optimum concentration of whey lactose was 20% (w/v) without
supplying additional lactose. In fact, the maximum lactose concen-
tration of solution obtained from the whey was approximately 23%
Lactulose concentration
Specific productivity
(
w/v). However, lactulose synthesis by immobilized enzymes did
not occur at a lactose concentration of 23% (w/v) due to the high
viscosity of the reaction mixture. Therefore, we could not use more
than 20% (w/v) lactose concentration. This lactose concentration is
low compared with those of other studies.
to the different kinds of raw material which were utilized.
1,4,9,10
The reason is due
3
.3. Effect of temperature
46
48
50
52
54
56
58
60
o
Temperature ( C)
The effect of temperature on lactulose synthesis was evaluated
Figure 2. Effect of temperature on lactulose synthesis. Reactions were conducted
using 20% (w/v) lactose, 12 U/ml immobilized b-galactosidase, and 12 U/ml
immobilized glucose isomerase in 50 mM sodium phosphate buffer at pH 7.5.
at temperatures of 47, 50, 53.5, 57, and 60 °C. Figure 2 shows the
effect of reaction temperature on lactulose synthesis. Lactulose
concentration and specific productivity increased as temperature
increased, and maximum values were observed at 53.5 °C, above
which the values decreased sharply. This may have been due to
inactivation of b-galactosidase at elevated temperatures. The
lactulose concentration and specific productivity at 53.5 °C were
preliminary experiments showed that the optimal temperatures
for the transgalactosylation reaction by immobilized b-galactosi-
dase and isomerization by immobilized glucose isomerase were
4
2
.81 g/l and 0.16 mg/U h, respectively, after a reaction time of
.5 h. The optimum temperature values of lactulose synthesis by
2
1
47 and 60 °C, respectively. This result implies that the balance
between the transgalactosylation reaction and isomerization is
important for lactulose synthesis.
b-galactosidases and permeabilized cells of K. lactis were 30, 40,
9,10,22
and 60 °C, respectively.
Moreover, our previous study and

4
Y.-S. Song et al. / Carbohydrate Research 369 (2013) 1–5
1
0
8
6
4
2
0
0.25
1
00
Lactulose concentration
Specific productivity
0.20
0.15
0.10
0.05
0.00
8
6
4
0
0
0
20
0
50
100
150
200
250
300
0
1
2
3
4
5
6
7
Reuse
Ionic strength (mM)
Figure 5. Reusability of immobilized enzymes for lactulose synthesis. Reactions
were conducted at 53.5 °C for 2 h using 20% (w/v) lactose, 12 U/ml immobilized b-
galactosidase, and 60 U/ml immobilized glucose isomerase in 100 mM sodium
phosphate buffer at pH 7.5.
Figure 3. Effect of the buffer ionic strength on lactulose synthesis. Reactions were
conducted at 53.5 °C using 20% (w/v) lactose, 12 U/ml immobilized b-galactosidase,
and 12 U/ml immobilized glucose isomerase in sodium phosphate buffer at pH 7.5.
3
.4. Effect of ionic strength
increased to improve lactulose synthesis. Lactulose synthesis was
carried out using different ratios of immobilized b-galactosidase
and glucose isomerase (1:1, 1:2, 1:3, 1:4, 1:5, and 1:6, units of
immobilized b-galactosidase: units of immobilized glucose isomer-
ase) under the optimized conditions. Figure 4 shows the lactulose
and fructose concentrations and specific productivity at different
ratios of the immobilized enzymes. Fructose concentration
increased as the ratio of immobilized glucose isomerase increased;
the fructose concentration was 33.9 g/l at a ratio of 1:6 after a 2 h
reaction. Lactulose synthesis also increased in proportion to the
fructose concentration up to a ratio of 1:5, above which it did
not increase further. This may have been due to the mass transfer
limitation caused by increasing viscosity of the reaction mixture as
the amount of immobilized enzyme increased. A 1:5 ratio pro-
duced the highest lactulose concentration (7.68 g/l) and specific
productivity (0.32 mg/U h) among all ratios tested. This result indi-
cated that the high fructose concentration caused by the increased
concentration of immobilized glucose isomerase was closely
related to improved lactulose synthesis. Therefore, 12 U/ml of
immobilized b-galactosidase and 60 U/ml of immobilized glucose
isomerase were determined to be the most effective enzyme ratios
The effect of ionic strength (50, 100, 150, 200, 250, and 300 mM)
of the sodium phosphate buffer (pH 7.5) on lactulose synthesis was
investigated. As shown in Figure 3, lactulose synthesis was highest
at a molarity of 100 mM, in which the lactulose concentration and
specific productivity were 5.18 g/l and 0.22 mg/U h, respectively.
The lactulose concentration increased with an increase in ionic
strength up to 100 mM, above which it decreased continuously
up to 300 mM. The rate (specific productivity) of the transgalac-
tosylation reaction of K. lactis b-galactosidase was dependent on
buffer molarity. Specific productivity decreased dramatically when
the ionic strength was >100 mM. In previous studies, lactulose syn-
thesis from b-galactosidase using lactose and fructose as the sub-
,9,21,22
strate was performed at
a
molarity of 50 mM.1
This
molarity was lower than the optimum value obtained in the pres-
ent study. The reason for this difference may be due to the isomer-
ization of glucose by immobilized glucose isomerase.
3
.5. Effect of enzyme ratio
Fructose is a substrate that accepts galactoside through a trans-
1
0
galactosylation reaction of b-galactosidase from lactose hydroly-
sate. However, the fructose concentration was low during the
reaction, because of the low concentration of immobilized glucose
isomerase. Thus, the ratio of immobilized glucose isomerase was
for lactulose synthesis. Hua et al. reported lactulose synthesis
from 80% (w/v) purified lactose and 10% (w/v) fructose using
42 U/ml of b-galactosidase and 100 U/ml of glucose isomerase
(ratio of 1:2.4). However, the values in this study were different
1
1
2
0
8
6
4
2
0
40
0.4
0.3
0.2
0.1
0.0
Lactulose
Fructose
Specific productivity
30
20
10
0
1
:1
1:2
1:3
1:4
1:5
1:6
Ratio of immobilized enzymes
Figure 4. Effect of the immobilized enzyme ratios on lactulose synthesis. Reactions were conducted at 53.5 °C using 20% (w/v) lactose, immobilized b-galactosidase, and
immobilized glucose isomerase in 100 mM sodium phosphate buffer at pH 7.5. The ratio of 1:1 corresponded to 12 U/ml of immobilized b-galactosidase and 12 U/ml of
immobilized glucose isomerase.

Y.-S. Song et al. / Carbohydrate Research 369 (2013) 1–5
5
because the nature and concentrations of raw materials, as well as
the supply of fructose differed.
the Advanced Biomass R&D Center (ABC-2010-0029799) of
Korea Grant funded by the Ministry of Education, Science and
Technology.
3
.6. Reusability of immobilized enzymes
Supplementary data
The reusability of immobilized enzyme is extremely important
for industrial applications. Lactulose synthesis using immobilized
b-galactosidase has been recently reported1 , nor has the reus-
ability of immobilized b-galactosidase for lactulose synthesis.
Immobilized b-galactosidase and glucose isomerase were used
for consecutive batches of lactulose synthesis under optimum
conditions to assess reusability. The relative catalytic activity of
the immobilized enzymes for lactulose synthesis is shown in
Figure 5, and the lactulose concentration (7.54 g/l) of the first batch
was considered to be 100%. The relative catalytic activity of the
immobilized enzymes decreased with an increase in the number
of reuses. After 7 reuses, the synthesized lactulose concentration
was 4.31 g/l, and the relative catalytic activity of the immobilized
enzymes was 57.1%.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.carres.2013.
0,23
0
1.002.
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7
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This study was supported by Technology Development
Program (309016-5) for Agriculture and Forestry, Ministry for
Food, Agriculture, Forestry and Fisheries, Republic of Korea and
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