International Journal of Food Science and Technology 2015, 50, 1625–1631 1625

Original article
Lactulose production from lactose by recombinant cellobiose
2-epimerase in permeabilised Escherichia coli cells

Mingming Wang,1,2 Ruijin Yang,1,2* Xiao Hua,2 Qiuyun Shen,2 Wenbin Zhang2 & Wei Zhao2
1 State Key Laboratory of Food Science and Technology, Jiangnan University, 214122 Wuxi, China
2 School of Food Science and Technology, Jiangnan University, 214122 Wuxi, China
(Received 8 October 2014; Accepted in revised form 20 January 2015)

Summary Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (CsCE) catalyses the single substrate lactose
into lactulose and is the most efficient enzyme ever found for the enzymatic synthesis of lactulose. Etha-
nol-permeabilised Escherichia coli cells containing CsCE were used as biocatalysts for lactulose produc-
tion. The reaction conditions for maximum lactulose production were optimised to be 600 g L 1 lactose,
pH 7.5, 80 °C and 12.5 U mL 1 of whole-cell biocatalyst. After incubated at Na2HPO4–NaH2PO4 buffer
for 2 h, approximately 390.59 g L 1 lactulose was obtained with a conversion yield of 65.1%. The lowest
production and conversion yield of epilactose were also found in Na2HPO4–NaH2PO4 buffer with a final
concentration of 11.7 g L 1 and a conversion yield <2%. The results represent a promising technology to
attain high production and conversion yield of lactulose with a high purity on industrial scale.
Keywords Cellobiose 2-epimerase, lactose, lactulose, optimum reaction conditions, permeabilised Escherichia coli cells.

and b-galactosidase (EC 3.2.1.23) in free and immobi-

Introduction
Lactose is a cheap disaccharide and several million
tons of lactose in the form of lactose-enriched cheese
whey is regarded as waste and discarded annually (Mu
et al., 2013). Hence, lactose as a raw material for the
production of lactose derivatives is probably a practi-
cal way for its efficient use (Seki & Saito, 2012). Lactu-
lose (4-O-b-D-galactopyranosyl-D-fructose), another
bioactive lactose derivative produced from the isomeri-
sation of lactose, is a nondigestible disaccharide that is
widely used in pharmaceutical, nutraceuticals and food
industries (Schumann, 2002; Aider & de Halleux, 2007;
Olano & Corzo, 2009). Lactulose has a large market
and its demand increased considerably in recent years
(Playne & Crittenden, 2009; Panesar & Kumari, 2011).
Commercially, lactose is exclusively produced by
alkaline isomerisation of lactose (Hicks & Parrish,
1980; Kozempel et al., 1995; Panesar & Kumari,
2011). As a promising alternative to this chemical
method, enzymatic production of lactulose regarding
product safety can result in clean production, and easy
purification of the product would be desirable (Panesar
& Kumari, 2011). Enzymatic synthesis of lactulose is
commonly performed by b-glycosidase (EC 3.2.1.21)
*Correspondent: Fax: +86-510-85919150;
e-mail: yrj@jiangnan.edu.cn
lised forms (Kim et al., 2006; Mayer et al., 2010; Kha-
tami et al., 2014). Low conversion yield and high costs
of enzymes are the main hurdles for the enzymatic
method. In consideration of these major obstacles,
many attempts aimed at increasing the lactulose con-
version yield and lowering the production costs have
been carried out (Bednarski & Kulikowska, 2007;
Tang et al., 2010; Song et al., 2012a,b). However, the
enzymatic synthesis of lactulose has not yet been stud-
ied on an industrial scale.
Recently, cellobiose 2-epimerase from Caldicellulosi-
ruptor saccharolyticus has been reported to be effective
for direct conversion lactose into lactulose. The highest
conversion yield (88%) and lactulose concentration
(614 g L 1) were achieved (Park et al., 2011; Kim &
Oh, 2012; Kim et al., 2013; Ojima et al., 2013). Due to
the high conversion yield of lactulose from lactose and
its thermostable property, biological synthesis of lactu-
lose with CsCE will contribute significantly to the
industry. However, the complex separation and purifi-
cation steps and activity loss of the purified enzymes
during processing will sharply increase the production
expense. The use of whole cells as biocatalysts for lac-
tulose production has advantages over purified enzymes
in many bioconversion processes because it is an eco-
nomical and easy to handle process. However, the per-
meability barrier of the cell envelope for substrates and

doi:10.1111/ijfs.12776
© 2015 Institute of Food Science and Technology
1626 Enzymatic production of lactulose M. Wang et al.
products often causes low reaction rates in whole cells
(Aider & de Halleux, 2007). To reduce the permeability
barrier, ethanol permeabilisation of cells has been dem-
onstrated to be an economical, easy, convenient and
safe process for production of lactulose (Lee et al.,
2004; Panesar et al., 2007).
This study is concerned with the preparation of
freeze-dried ethanol-permeabilised E. coli cells contain-
ing CsCE and their use as biocatalysts for lactulose
production. Reaction conditions including pH, temper-
ature, reaction time, substrate concentration, volumet-
ric activity of the biocatalyst, and molar ratio of
borate–lactose and buffer systems were optimised to
obtain maximum lactulose production.
Materials and methods
Materials
Caldicellulosiruptor saccharolyticus DSM 8903 was
generously gifted by Department of Applied Microbi-
ology, University of Lund (Sweden). Lactulose and
epilactose (HPLC grade) were purchased from Sigma-
Aldrich (Shanghai, China). Water was obtained from
a MilliQ water purification system (Millipore, Mols-
heim, France). All the other chemicals were of analyti-
cal grade except acetonitrile (HPLC grade) and
purchased from Sinopharm Chemical Reagent Co. Ltd
(Shanghai, China). All solutions were prepared with
distilled water and used without further purification.
Methods
Microorganisms, plasmid, culture conditions and enzyme
purification
Caldicellulosiruptor saccharolyticus DSM 8903, Escheri-
chia coli BL21 (DE3) and plasmid pET-28(a+) were
used as the source of genomic DNA of cellobiose
2-epimerase, host cells and the expression vector.
Recombinant E. coli cells for enzyme expression were
cultivated in 50 mL Luria-Bertani (LB) medium in 250-
mL flasks containing 10 lg mL 1 of Kanamycin at
37 °C with shaking at 200 rpm min 1. When the opti-
cal density reached 0.6 at 600 nm, lactose was added to
a final concentration of 10 g L 1 to induce cellobiose
2-epimerase expression. The culture was incubated with
shaking at 160 rpm min 1 at 25 °C for 20 h.
The purification step of the enzyme was carried out
using Q Sepharose Fast Flow and Sephadex G-75 col-
umn chromatography (Gu et al., 2014).
Permeabilisation of recombinant E. coli cells
The cultivated cells were harvested by centrifugation at
6000 g for 15 min and then resuspended in 40% (v/v)
ethanol, stirred at 4 °C for 30 min. The obtained per-
meabilised cells were washed twice with distilled water
International Journal of Food Science and Technology 2015
and then centrifugated, and after freeze-dried, the per-
meabilised E. coli cells were stored at 4 °C before being
used as a biocatalyst for lactulose production.
Enzyme assay
Unless otherwise stated, the reaction was carried out
at 80 °C in Tris–HCl buffer (50 mM, pH 7.5) contain-
ing 50 g L 1 lactose and 0.1 g L 1 purified enzyme or
freeze-dried biocatalyst for 20 min. The enzymatic
reaction was stopped by boiling for 5 min. Controls
were prepared at the same conditions with inactivated
enzyme. One unit (U) of the CsCE activity was defined
as the amount of free enzyme or permeabilised E. coli
cells required to produce 1 lmol of lactulose from lac-
tose per min at 80 °C and pH 7.5.
The quantification of lactulose was based on the
method described by Zhang et al. (2010b).
Effect of pH and temperature on enzyme activity of perme-
abilised E. coli cells
The optimal pH was investigated using two different
pH buffer systems [PIPES buffer (50 mM, pH 6.0–7.0)
and Tris–HCl buffer (50 mM, pH 7.0–8.5)]. The effect
of temperature on enzyme activity was varied from 40
to 95 °C in Tris–HCl buffer (50 mM, pH 7.5).
Optimisation of reaction conditions for lactulose production
by permeabilised E. coli cells
The various reaction conditions were optimized by
varying the respective parameters. The volumetric
activity of the biocatalyst (2.5–20 U mL-1), the sub-
strate concentration of lactose (50–700 g L-1), and the
enzymatic reaction time (0–5 h) and the borate concen-
tration (0–3 M) were optimized to obtain the maxi-
mum lactulose production. Unless otherwise stated,
the reactions were carried out at 80°C in Tris–HCl
buffer (50 mM, pH 7.5). Finally, the influence of buf-
fer systems (a. Tris–HCl buffer; b. PIPES buffer; c.
Na2HPO4–NaH2PO4 buffer; d. Deionized water; e.
Na2HPO4–Citrate buffer; f. Borate buffer solution; g.
Britton-Robinson buffer) on the production of lactu-
lose was also investigated. All the buffers were 50 mM
and adjusted to pH 7.5.
Analytical methods
Lactulose produced during the enzymatic reactions were
quantitatively monitored by HPLC analysis as described
by Hua et al. (2013).
Statistical analysis
The results expressed in various studies were reported
as mean  SD (standard deviation). Each value repre-
sents the mean for three independent experiments per-
formed in duplicate with average standard deviation
<5%. SPSS statistical software 17.0 (SPSS Inc.,
© 2015 Institute of Food Science and Technology

Chicago, IL, USA) was used to perform the statistical
analysis.
Results and discussion
Permeabilisation and biochemical properties of
permeabilised E. coli cells
To evaluate the effectiveness of ethanol as a permeabil-
ising agent, the cultivated E. coli cells were treated
with different ethanol concentration (0–80%, v/v) and
different permeabilisation time (0–75 min). Figure 1a,b
showed that the maximum activity of E. coli cells was
obtained when treated with 40% (v/v) ethanol for
30 min. In permeabilisation, the cell envelope is altered
to allow small molecules, such as lactose and lactulose
to cross freely (Panesar et al., 2007). However, the
enzyme activity decreased may be attributed to the
denaturing effect of the ethanol on the enzyme and the
leakage of the enzyme from the cells or cell lysis at
(a)
(b)
Figure 1 Ethanol permeabilisation and biochemical properties of permeabilised Escherichia coli cells and free enzyme. The activity of
untreated E. coli cells was defined as 100% in (a and b). In (c and d), the one with the highest activity was taken as 100%.
© 2015 Institute of Food Science and Technology
Enzymatic production of lactulose M. Wang et al. 1627
higher ethanol concentration and longer permeabilisa-
tion time. Thus, treated with 40% (v/v) ethanol for
30 min were chosen as the optimum conditions for
E. coli cells permeabilisation in this study.
Effects of pH and temperature on enzyme activity of
permeabilised E. coli cells and free enzyme were inves-
tigated. Both free enzyme and permeabilised E. coli
cells exhibited an optimal pH of 7.5 in 50 mM Tris–
HCl buffer, and it can be noted that under neutral pH
conditions, the relative activity was at a high level
(Fig. 1c). The activity-temperature profiles of the free
enzyme and permeabilised E. coli cells are shown in
Fig. 1d. The optimum temperature for the biocatalyst
and free enzyme was 80 °C. The free enzyme activity
decreased significantly below and above 80 °C, while
the biocatalyst exhibited some thermal stability at tem-
peratures ranging from 75 to 85 °C (Fig. 1d). This
could be attributed to the microenvironment of the
cellobiose 2-epimerase in permeabilised E. coli cells.
The environment did not change greatly and the cells
(c)
(d)
International Journal of Food Science and Technology 2015
1628 Enzymatic production of lactulose M. Wang et al.
could protect the enzyme activity conformation from
distortion or damage by heat exchange. It is more effi-
cient to produce lactulose at high temperatures
because increased temperature favours substrate solu-
bility; hence, high concentration of lactose can be used
and high concentration of lactulose can be obtained.
In addition, high temperature also resulted in a higher
reaction velocity of the enzyme, and a higher produc-
tivity of lactulose was achieved (Lee et al., 2004; Kim
& Oh, 2012). On the other hand, nonenzymatic brown-
ing (Mallard reactions) could be initiated at high tem-
peratures (Mayer et al., 2004). Future studies are
needed to find new strains that display high CsCE
activity at low temperatures or facilitate lactulose
production by improving the properties of cellobiose
2-epimerase by protein engineering. This could mini-
mise the unwanted coloured by-products generated by
nonenzymatic browning during biotransformation and
thus improve the product quality of lactulose.
Effect of the volumetric activity of the biocatalyst on
lactulose production
The effect of biocatalyst volumetric activity on the
maximum lactulose production was examined under
the given conditions with biocatalyst volumetric activ-
ity ranging from 2.5 to 20 U mL 1. As shown in
Fig. 2, it was distinctly found that the lactulose pro-
duction increased with the addition of biocatalyst, and
the maximum values were obtained at 12.5 U mL 1.
Lactulose concentration increased with the increase of
biocatalyst, due to the increased amount of CsCE
present. However, when above 12.5 U mL 1, lactulose
production was constant at approximately 275 g L 1.
This phenomenon has also been found by other
researchers. Lee et al. (2004) found that the optimal
Figure 2 Effect of the volumetric activity of the biocatalyst on
lactulose production.
International Journal of Food Science and Technology 2015
concentration of permeabilised yeast cells for lactulose
production was 10.4 g L 1. Up to this value, the pro-
ductivity of lactulose increased with the adding of b-
galactosidase in cell mass. And then the productivity
of lactulose decreased with increasing cell mass due to
steric hindrance. Thus, in this study, 12.5 U mL 1 was
chosen as the optimal volumetric activity of biocatalyst
for maximum lactulose production.
Effect of the substrate concentration of lactose on
lactulose production using permeabilised E. coli cells as
biocatalyst
As shown in Fig. 3, the production and conversion
yield of lactulose were recognised as the two indexes
to determining the optimal concentration of lactose. It
was found that lactulose production significantly
increased with addition of lactose. When lactose con-
centration was varied from 50 to 700 g L 1, the lactu-
lose almost increased linearly from 30.5 to
356.3 g L 1. When the concentration of lactose was
below 400 g L 1, the conversion yield of lactulose was
almost constant at about 60%. Above 400 g L 1, the
conversion yield of lactulose dramatically decreased to
approximately 55% at 600 g L 1 and 50.9% at
700 g L 1. Although the maximum lactulose produc-
tion was obtained at 700 g L 1 lactose, the conversion
yield was at a lower level. Taking the two key indexes
into consideration, 600 g L 1 lactose was selected as
the right substrate concentration for this study.
The concentration of lactulose increasing with
increase in lactose has also been found in other enzy-
matic syntheses of lactulose by b-galactosidase. The
transgalactosylation activity of b-galactosidase increase
with decreasing water activity, caused by the high con-
centrations of substrates (Hanssen et al., 2001; Lee
Figure 3 Effect of substrate concentration of lactose on lactulose
production.
© 2015 Institute of Food Science and Technology

et al., 2004). The decrease in conversion yield may be
caused by the high viscosity of lactose.
The time course of lactulose production using
permeabilised E. coli cells as biocatalyst
Time course of the production of lactulose from the
single substrate lactose was determined by varying the
reaction time from 0 to 6 h. As shown in Fig. 4, it
was noted that up to 2 h the lactulose concentration
significantly increased from 0 to 318 g L 1. After 2 h,
the growth curve gradually became flatted. This phe-
nomenon was also observed by other researchers, and
it may attributed to the half-life of biocatalyst (Park
et al., 2011; Kim & Oh, 2012; Kim et al., 2013). The
reaction time of lactulose production is of key impor-
tance for industrial application. Long reaction times
lead to a low productivity of lactulose. In addition,
long reaction times at high temperatures and high lac-
tose concentrations may lead to the generation of col-
oured by-products through nonenzymatic browning
(Mayer et al., 2004). Hence, in this study, 2 h was cho-
sen as the optimal reaction time for lactulose produc-
tion. After 2 h of enzymatic reaction, approximately
318.3 g L 1 lactulose was obtained.
The reaction time needed to achieve a constant lac-
tulose concentration by free cellobiose 2-epimerase
was 2 h. It was almost the same with that achieved
by permeabilised E. coli cells. This result may be due
to the reduction of the permeability barrier caused by
treating the cells with ethanol. After ethanol perme-
abilisation, the cells show a faster mass transfer of
substrate and products which may result in a more
efficient lactulose conversion similar to that of free
enzyme.
Figure 4 Effect of reaction time on lactulose production.
© 2015 Institute of Food Science and Technology
Enzymatic production of lactulose M. Wang et al. 1629
Effect of borate on lactulose production using
permeabilised E. coli cells as biocatalyst
Boric acid has successfully been used for achieving high
yields in enzymatic production of D-psicose, D-taga-
tose, L-ribulose and lactulose (Kim et al., 2008, 2013;
Zhang et al., 2010a; Salonen et al., 2013). As shown in
Fig. 5, the influence of borate on lactulose production
was determined by varying the concentration of borate
from 0 to 3 M. When the molar ratio of borate–lactose
ranged from 0 to 1, the conversion yield of lactulose
increased significantly, and the highest yield was
achieved at a 1:1 molar ratio. After this ratio, the lactu-
lose yield decreased. Compared with the biological syn-
thesis in the absence of borate, the yield of lactulose
showed an approximately 13% increase from 53% to
66% at a 1:1 molar ratio of borate–lactose. The
increased lactulose conversion yield can be explained by
the fact that the binding affinity of borate for lactulose
is 59 times higher than that for lactose (Vandenberg
et al., 1994). The addition of borate in enzymatic lactu-
lose production allows the formation of borate–lactu-
lose complex which means the newly formed lactulose
was essentially removed and allows the continuation of
lactulose formation (Kim et al., 2013).
The concentration of borate used in the reaction
solution in this study was 120 g L 1. The high con-
centration of borate may cause numerous problems
including complex desalination by ion-exchange or
nanofiltration, severe environmental pollution and
high cost. In addition, the stability of the borate–lac-
tose complex is strongly dependent on pH. In the
weak basic environment of the reaction solution, the
coordination between lactulose and borate is pro-
moted and this results in their difficult separation
Figure 5 Effect of the molar ratio of borate–lactose on lactulose
production.
International Journal of Food Science and Technology 2015
1630 Enzymatic production of lactulose M. Wang et al.
(Geffen et al., 2006). With respect to those results, it
can be concluded that it is cost-effective for lactulose
production using biological synthesis in the absent of
borate.
Effect of different buffer systems on the production of
lactulose and epilactose
CsCE not only has the ability to convert lactose into
lactulose, but can also epimerise lactose into epilactose
(Park et al., 2011). The yield of lactulose and the pro-
duction of epilactose in different buffer systems were
investigated. Figure 6a shows that the lactulose
conversion yield was markedly different in various buf-
fer systems. When the reaction was performed in
Na2HPO4–NaH2PO4 buffer (50 mM, pH 7.5), the high-
est conversion yield of lactulose was achieved with a
value of 65.1%. Interestingly, the lowest epilactose
(a)
(b)
Figure 6 Effect of different buffer systems on the production of (a)
lactulose and (b) epilactose. Unless otherwise stated, all the buffers
were adjusted to pH 7.5 in 50 mM. a: Tris-HCl buffer; b: PIPES buf-
fer; c: Na2HPO4–NaH2PO4 buffer; d: Deionized water; e: Na2HPO4-
Citrate buffer; f: Borate buffer solution; g: Britton-Robinson buffer.
International Journal of Food Science and Technology 2015
production was also found in Na2HPO4–NaH2PO4
buffer (50 mM, pH 7.5) with a final concentration of
11.7 g L 1 after 2 h (Fig. 6b). Further studies are
needed to understand why the epilactose production
changed in different buffer systems.
According to literature, the maximum lactulose
isomerisation rate was reported to be 20% when
2.1–8.6% of phosphates were added to a saturated
lactose solution at 104 °C for 20–240 min (patent of
Great Brittan No. 2031430, 1978). It has been dem-
onstrated that sulphites and phosphates have the
ability to prevent oxidation of disaccharides. For this
reason, their use as catalysts for lactose isomerisa-
tion reactions into lactulose allows the use of high
temperatures and lactose concentrations (Aider & de
Halleux, 2007). In this study, a high lactose concen-
tration and high temperature reaction system was
used. A Na2HPO4–NaH2PO4 buffer system resulted
in the highest lactulose conversion yield and the low-
est epilactose production. These are probably the
right conditions for the production of clean and safe
lactulose with higher purity.
Conclusions
This study developed a biocatalytic method for lactu-
lose production using recombinant CsCE in ethanol-
permeabilised E. coli cells. Biocatalysts in the form of
whole cells have advantages over purified enzymes
because they are simpler, easy to handle and more eco-
nomical in many industrial bioconversion processes.
The maximum lactulose yield was 390.59 g L 1 with a
conversion yield of 65.1%. Na2HPO4–NaH2PO4 buffer
can reduce epilactose yield thus increase lactulose pro-
duction and the final concentration of epilactose was
just 11.7 g L 1 with a conversion yield <2%. This
shows a great potential in the production of high pur-
ity lactulose.
Acknowledgment
The authors gratefully acknowledge the financial sup-
port provided by the National Key Technology R&D
Program in the 12th Five Year Plan of China
(2011BAD23B03), the Key Project of National Natu-
ral Science Fund (31230057) and 111 Project (B07029).
This work was also supported by the Fundamental
Research Funds for the Central Universities (JUD-
CF13001) and the Innovative Research Program for
Graduate Students of Jiangsu Province
(CXZZ13_0762). We thank Dr. (Serve) Kengen from
Wageningen UR (Holland) and Dr. Ed van Niel from
University of Lund (Sweden) for the generous dona-
tion of the genomic DNA from Caldicellulosiruptor
saccharolyticus DSM 8903 and we also thank Dr. Wu
Li for valuable suggestions.
© 2015 Institute of Food Science and Technology

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