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Biotechnology DOI 10.1002/biot.200600100 Biotechnol. J. 2006, 1, 1464–1470

Journal

Research Article

Production of galacto-oligosaccharides by immobilized

recombinant β-galactosidase from Aspergillus candidus

Pu Zheng1,2, Hongfeng Yu2, Zhihao Sun1,2, Ye Ni1,2, Wei Zhang3, Yunliu Fan3 and Yan Xu1,2
1Key Laboratory of Industry Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi, P. R. China
2Laboratoryof Biocatalysis, School of Biotechnology, Southern Yangtze University, Wuxi, P. R. China
3Biotechnology Research Institute, Chinese Academy of Agricultural Science, Beijing, P. R. China

The preparation of galacto-oligosaccharides (GOSs) was studied using the immobilized recombi- Received 29 June 2006
nant β-galactosidase from Aspergillus candidus CGMCC3.2919. The optimal pH and temperature Revised 18 August 2006
for the immobilized enzyme were observed at pH 6.5 and 40°C, respectively. Increasing the initial Accepted 4 September 2006
lactose concentration increased the yield of GOSs. The dilution rate was found to be a key factor
during the continuous production of GOSs. The maximum productivity, 87 g/L·h was reached
when 400 g/L lactose was fed at dilution rate of 0.8/h. The maximum GOS yield reached 37% at
dilution rate of 0.5/h. Continuous operation was maintained for 20 days in a packed-bed reactor
without apparent decrease in GOS production. The average yield of GOSs was 32%, correspon-
ding to the average productivity of 64 g/L·h, which implied that the immobilized recombinant
β-galactosidase has potential application for GOS production.

Keywords: Galacto-oligosaccharides · β-Galactosidase · Immobilized enzyme · Packed-bed reactor

1 Introduction enzymes isolated and purified from various organisms. It is

Galacto-oligosaccharides (GOSs) represent a mixture of
tri-, tetra-, penta- and hexasaccharides of galactose and
glucose with the molecular structure of (Gal)n-Glu. GOSs
constitute the major part of oligosaccharides of human
milk, and are also recognized as prebiotics because of their
non-digestibility [1, 2]. GOSs have many beneficial effects,
such as improving lactose tolerance and digestibility of
milk products; preventing pathogenic, autogenic diarrhea
and constipation; increasing absorptions of different min-
erals in the intestine; reducing toxic metabolites, undesir-
able enzymes and serum cholesterol; depressing blood
pressure, etc. [3]. Therefore, their comprehensive applica-
tions as a food additive for health purposes have led to an
increased demand for commercial GOSs.
Enzymatic synthesis employing β-galactosidase (EC
3.2.1.23) is a typical method for GOS production. β-Galac-
tosidase is one of the first few oligosaccharide-hydrolyzing
Correspondence: Professor Zhihao Sun, School of Biotechnology,
Southern Yangtze University, Wuxi 214036, P. R. China
E-mail: sunw@public1.wx.js.cn
Fax: +86-510-85808498
known to catalyze transgalactosylation as well as hydroly-
sis reactions. During the reaction, sugars other than mono-
saccharides (such as glucose and galactose) are also
formed. These di- and higher saccharides containing one
or more galactosides, which are formed by transgalactosy-
lation, are named GOSs [4]. GOSs were enzymatically syn-
thesized from lactose by Crittenden and Playne [5] using
glycosyl transfer catalyzed by the enzyme galactosidase.
Many microorganisms, such as Aspergillus niger, As-
pergillus Oryzae, Bifidobacterium bifidum, Bacillus circu-
lans, Bullera singularis, Escherichia coli, Kluyveromyces
fragilis, Kluyveromyces lactis, Saccharomyces lactis, Sac-
charopolyspora rectivigula, Sterigmatomyces elviae, and
Thermus aquaticus, have been investigated for their β-
galactosidase production [6–17]. Depending on the sources
of galactosidase, the structures and concentrations of
GOSs are quite different. This led to studies on GOS pro-
duction focused on isolation of β-galactosidase, but only a
few of them have been upscaled to commercial production
due to the high costs [18]. Since the enzymatic activity and
productivity are crucial economic factors, continuous
processes and immobilization technology were developed
to increase production efficiency [17, 19–23].

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It was reported that β-galactosidase was overex-
pressed extracellularly by recombinant Pichia pastoris
with a β-galactosidase-encoding gene from Aspergillus
candidus CGMCC3.2919 [24]. This β-galactosidase is eas-
ily and inexpensively isolated as compared with enzymes
from other sources [25]. The expression level of secreted
recombinant β-galactosidase by P. pastoris was 6 mg/mL
with enzymatic activity of 3600 U/mL in the 5-L fer-
menter, which is the highest among all kinds of recombi-
nant strains reported so far. Compared with the lactase
from Aspergillus oryzae ATCC 20423, the lactase from A.
candidus CGMCC3.2919 has preferable enzymatic prop-
erties including high thermostability, high specific activ-
ity and wide pH range for enzymatic reaction [24, 26]. In
our previous study, a novel method of immobilizing this
recombinant β-galactosidase was developed and suc-
cessfully used for the production of low-lactose milk [27].
Since β-galactosidase has two-edged activity, it can also
catalyze transgalactosylation to produce GOSs. Continu-
ous GOS production has also been investigated for indus-
trial application. In the present study, the immobilized ex-
tracellular recombinant β-galactosidase was applied for
continuous production of GOSs in a packed-bed reactor.
The operational conditions such as temperature, pH, ini-
tial lactose concentration, dilution rate of lactose solution,
and the operational stability of the reactor were investi-
gated. Under the optimal condition, long-term stability of
the immobilized enzyme was evaluated for 20 days with-
out any decrease in GOS production.
2 Materials and methods
2.1 Materials
The liquid recombinant β-galactosidase used in this
study was from recombinant Pichia pastori fermentation
broth, and had an apparent molecular mass of 130 kDa
and specific activity of 706.5 ± 2.6 U/mg, supplied by
Biotechnology Research Institute (Chinese Academy of
Agricultural Science, China).
2.2 Enzyme immobilization
The immobilized enzyme was prepared as following [27]:
57 mL liquid β-galactosidase and 100 g (wet weight) ad-
sorptive resin D113 (Cangzhou Bon Chemical Co. China.)
were mixed at 20°C with constant shaking for 1 h, then
the resin was taken out and entrapped with 3% alginate.
After submerging in 0.1 mol/L CaCl2 solution for 1 h at
4°C, the immobilized enzyme was treated with 2% glu-
taraldehyde at pH 7.0 to enhance the stabilization. Before
being stored at 4°C, the immobilized enzyme was washed
with distilled water. The activity of the immobilized en-
zyme was 964 U/g resin. One unit was defined as the
amount of enzyme that liberates 1 µmol o-nitrophenol
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
from o-nitrophenol-β-galactopyranoside/min at pH 6.5
and 30°C.
2.3 Synthesis of GOS by the immobilized β-galactosidase
Twenty grams of immobilized β-galactosidase was sus-
pended in 250-mL flasks containing 50 mL 200 g/L lactose
solution at different pHs (4.0, 5.0, 6.0, 7.0). The mixture
was incubated in water bath at different temperatures
(30–70°C) for 30 min. The reaction was stopped by heat-
ing at 100°C for 10 min, and the mixture was filtered
through a 0.45-µm membrane before HPLC analysis.
2.4 Continuous production of GOS in a packed reactor
Continuous GOS biosynthesis was performed in a
packed-bed reactor (30 × 300 mm, 60-mL effective work-
ing volume) filled with the immobilized β-galactosidase
(90 g) at 40°C. Aqueous solution containing 400 g/L lac-
tose pH 6.5–7.0 was continuously fed into the reactor from
the top of the column and the effluent was collected at the
bottom. The maximum GOS content in the produce
stream was examined by adjusting lactose feeding rate
from 20 mL/h to 88 mL/h, corresponding to dilution rate of
0.3–1.5/h. The long-term performance of the reactor was
also evaluated at a lactose flow rate of 30 mL/h.
2.5 Analytical methods
Lactose, glucose, galactose and GOSs were determined
by HPLC. Waters 1525 pump, Waters 2414 refractive in-
dex detector and Aminex HPX-87H column (300 ×
7.8 mm) were used. The mobile phase was 8 mmol/L
H2SO4. The flow rate and the injection volume were
0.5 mL/min and 5 µL, respectively. All analyses were
made in triplicate.
3 Results and discussion
3.1 Analysis of products
Figure 1 showed a typical example of the reaction mixture
separated by HPLC. Five peaks appeared, expressing
oligosaccharides, lactose, glucose and galactose in turn.
Although the peaks (1) and (2) are partially superimposed,
oligosaccharide formation is illustrated. Furthermore, the
composition of oligosaccharides was determined by LC-
MS spectrum (Waters ZMD400), as shown in Fig. 2. The
results show that the oligosaccharides comprised tri-,
tetra- and pentasaccharides with molecule weight of
539.4, 701.5 and 863.7, and further proved that the prod-
ucts of the immobilized recombinant β-galactosidase cat-
alyzed reaction were GOSs.
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Figure 1. HPLC chromatography of

the reaction mixture by immobilized
β-galactosidase with initial lactose of
400 g/L at pH 6.5 and 40°C, (1) and
(2) oligosaccharides, (3) lactose (4)
glucose, (5) galactose. Sample dilu-
tion factor: 50.

3.2 Effect of pH and temperature on GOS formation
No significant difference in pH effects on the activity be-
tween the immobilized and free β-galactosidase was ob-
served in a previous study [27]. The immobilized enzyme
has a wide operation pH ranging from pH 2.5 to 7.0. How-
ever, at lower pH the immobilization of the enzyme was
affected, which could cause β-galactosidase leakage.
Therefore, the GOS production using immobilized β-
galactosidase at a wide range of pH 4.0–7.0 was com-
pared. As shown in Table 1, all the yields of GOSs reached
about 35 g/L with initial lactose concentration of 200 g/L.
This suggested that neutral pH was feasible, and aqueous
solution of lactose could be directly used as reaction
medium.
Increasing the temperature from 25 to 40°C promoted
the GOS synthesis and lactose hydrolysis by immobilized
β-galactosidase, as shown in Fig. 3. When temperature
rose over 40°C, lactose hydrolysis declined and the GOS
content slightly increased. It might due to the product in-
hibition effect. The optimum temperature of 35–45°C,
Table 1. Effect of pH on GOS formation by the immobilized enzyme. GOS
production by immobilized β-galactosidase at various pH with initial lac-
tose of 200 g/L at 40°C. Reaction time was 30 min
Exp pH GOS formation Lactose hydrolysis
(g/L) (%)
1 4 36.7 55.9
2 5 35.1 52.8
3 6 34.4 52.6
4 7 35.1 52.9
with a production of about 33 g/L GOSs from initial lac-
tose concentration of 200 g/L, was unexpected because
the optimum temperature of the free β-galactosidase was
65°C [24]. To compare the thermal stability of the immo-
bilized and free β-galactosidase, samples were taken at
appropriate time intervals at 60°C. The half-lives of the
immobilized and free enzyme were about 87 and 62 min,
respectively (as shown in Fig. 4). Therefore, immobiliza-
tion significantly improved the thermal stability of the en-
zyme, especially under extended incubation time. After
90 min, the residual activity of immobilized β-galactosi-
dase was around 45%, which was 2.0 times of that of the
free enzyme.
3.3 Effect of initial lactose concentration on GOS formation
Initial lactose concentration was found to be the most sig-
nificant factor in GOS formation. It was clear that the pro-
duction of GOSs increased with the increase of initial lac-
tose concentration, while the lactose hydrolysis went
down slowly to a constant level (as shown in Fig. 5). This
was in agreement with results obtained by others [28]. It
was generally observed that the hydrolysis and trans-
galactosylation reaction occurred simultaneously. What
dominates the profile of the reaction is largely dependent
on lactose concentration. GOSs were produced at high
lactose concentration. When the initial lactose concentra-
tion was less than 150 g/L, no GOSs were detected. When
it was increased from 200 to 400 g/L, the GOS content in
the product increased from around 30 g/L to 87 g/L. If the
lactose concentration was further increased to over
400 g/L, crystal lactose could be separated from the solu-
tion.

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(A)
(B)
The effect of lactose concentration on initial rate of
lactose hydrolysis with the free and immobilized β-galac-
tosidase was also investigated within the concentration
range of 40–100 g/L, under which the transgalactosylation
reaction hardly occurred (data not shown). The kinetic
constants, Km and Vmax, of the free and the immobilized
lactase were 0.27 mol/L, 666 µmol/mL·min and 0.71 mol/L,
626 µmol/mL·min, respectively. The Km value of the im-
mobilized β-galactosidase was 2.7 times larger than that
of the free one. These changes in kinetic parameters may
be the consequence of either the structural changes in the
enzyme occurring upon immobilization or the lower ac-
cessibility of substrate to the active sites of the immobi-
lized enzyme [29].
3.4 Effect of dilution rate on continuous GOS formation by
the immobilized β-galactosidase in a packed-bed reactor
As β-galactosidase catalyses both hydrolysis and trans-
galactosylation reactions, lactose was hydrolyzed to glu-
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
Figure 2. (A) Mass spectra of the sample. (B) Ion current chromatogram
of the sample.
cose and galactose, and galactose was transferred to an-
other lactose molecule to produce GOSs. GOSs could also
become substrate for lactase and were slowly hydrolyzed
[3]. So the process of GOS formation may be concluded as
a balance between hydrolysis and transgalactosylation.
This phenomenon was observed in the study.
As shown in Fig. 6, lactose hydrolysis, monosaccha-
ride yield and GOS formation changed with dilution rate.
When dilution rate was decreased from 1.5/h to 0.8/h, lac-
tose hydrolysis maintained at a level of about 55%, but
monosaccharide yield decreased from 42 to 27%, while
GOS concentration increased from 52 g/L to 111 g/L. This
implied that GOSs were formed from monosaccharides in
this period. When dilution rate was decreased from 0.8/h
to 0.7/h, lactose hydrolysis increased by 5.8%, monosac-
charide yield increased by 4.3%, while GOS concentration
increased by 7.7 g/L. When the dilution rate was further
decreased from 0.7/h to 0.5/h, lactose hydrolysis kept at
similar level, monosaccharide yield were 31 and 24%, and
GOS concentration increased from 119 g/L to 148 g/L, cor-
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Figure 3. Effect of temperature on GOS production by the immobilized
β-galactosidase with initial lactose of 200 g/L at pH 6.5. Reaction time
was 30 min.
responding to GOS yield of 30–37%. This was evident that
GOS yield reached the maximum value of 37% under the
experiment condition. Further decreases in the dilution
rate led to the GOS yield decreasing dramatically and
monosaccharide yield significantly increasing. This was
likely due to the fast hydrolysis of GOSs at a low dilution
rate.
GOS productivity is, however, affected by GOS con-
centration and the dilution rate. The highest productivity
reached 87 g/L·h at dilution rate of 0.8/h. However, when
GOS yield reached its highest level of 37%, the GOS pro-
ductivity was 74 g/L·h, just 85% of the maximum. The
highest lactose hydrolysis resulted in the lowest GOS pro-
ductivity of 30 g/L·h, 66% lower than the maximum.
3.5 Operation stability of the reactor
To investigate the operational stability of the reactor, con-
tinuous operation was carried out under optimized condi-
tion. The results are shown in Fig. 7. There was no signif-
icant decaying sign during the 20 days of operation. The
average GOS yield was 32%, corresponding to average
Figure 5. Effect of initial lactose concentration on GOS production by the
immobilized β-galactosidase at pH 6.5 and 40°C. Reaction time was
30 min.
1468 © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2006, 1, 1464–1470
Figure 4. The residual enzyme activity of the immobilized and free β-galac-
tosidase at 60°C with initial lactose of 100 g/L at pH 6.5.
productivity of 64 g/L·h. This exhibits good operational
stability and commercial available.
4 Concluding remarks
We have described the operation conditions for biocon-
version of GOSs by the immobilized recombinant β-galac-
tosidase from A. candidus CGMCC3.2919 in a packed-
bed reactor. It was noted that GOS yield and GOS pro-
ductivity reached their maximum at dilution rates of 0.5/h
and 0.8/h, respectively. The maximum values of GOS yield
and GOS productivity were 37% and 87 g/L·h, respective-
ly. The GOS capacity of each gram of immobilized β-
galactosidase reached 1.1 g/day. In comparison with oth-
er immobilized enzyme such as B. circulans, B. singularis
Figure 6. Effect of dilution rate on continuous GOS production with initial
lactose concentration of 400 g/L, pH 6.5 and 40°C.
Dilution rate (/h) = flow rate/effective workking volume (60 mL);
⎛ r esidual lactose concentration ⎞
lactose hydrolysis = ⎜ 1– × 100%;
⎝ ose concentration ⎠⎟
initial lacto
glucose concentration + galactose concentration
monosaccharide yield = × 100%;
initial lactose concentration
n
GOS concentration
GOS yield = × 100%;
initial lactose concentration
GOS productivit y = GOS concentration × dilution rate (g/L·h).
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Table 2. GOS production by various immobilized β-galactosidase in continuous operation

Source of Mode of Reaction condition Max GOSa) Productivity Operation period

enzyme process (wt%) (g·L/h) (h)
Lactose Temp.
pH
conc.(g/L) (°C)
B. circulansb) CSTR 45.6 40 6.0 40.0 2.2
B. singularisb) PBR 100 45 4.8 55.0c) 4.4
A. oryzaeb) PBR 200 40 4.5 21.7 80 400
PBR 400 40 4.5 26.6 106 400
A. candidus PBR 400 40 6.5 37.1 87.1 >480
a) Max GOS is a weight percent of GOS based on the total sugars in the reaction mixture.
b) [28].
c) GOS content also includes disaccharides.

fects the population of Clostridium perfringens in the intestine of

Figure 7. Continuous production of GOSs using immobilized enzyme in
the packed bed reactor, in which 90 g immobilized β-galactosidase was
filled. Aqueous solution of 400 g/L lactose was fed at dilution rate of 0.5/h
into the reactor.
or A. oryzae (Table 2), the immobilized recombinant
β-galactosidase from A. candidus CGMCC3.2919 showed
an excellent performance in GOS production such as GOS
yield, productivity and operation period. This indicates
that the immobilized β-galactosidase is a promising alter-
native for efficient production of GOSs.
This work was supported by the State Key Basic Re-
search and Development Plan of China (No.
2003CB716008), the National Natural Science Foundation
of China (No. 20476039), and the Program for Changjiang
Scholars and Innovative Research Team in University
“PCSIRT”(IRT0532).
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