Journal of Molecular Catalysis B: Enzymatic 116 (2015) 39–44

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Journal of Molecular Catalysis B: Enzymatic

journal homepage: www.elsevier.com/locate/molcatb

Characterization of an epilactose-producing cellobiose 2-epimerase

from Thermoanaerobacterium saccharolyticum
Qiuming Chen a , Wenli Zhang a , Tao Zhang a , Bo Jiang a,b , Wanmeng Mu a,b,∗
a
State Key Laboratory of Food Science and Technology, Ministry of Education, Key Laboratory of Carbohydrate Chemistry and Biotechnology,
Jiangnan University, Wuxi, Jiangsu 214122, China
b
Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China

a r t i c l e i n f o a b s t r a c t

Article history: In this study, a recombinant cellobiose 2-epimerase (GenBank accession number, YP 006392930.1) was
Received 31 October 2014 characterized from a thermophilic bacterium, Thermoanaerobacterium saccharolyticum JW/SL-YS485. The
Received in revised form 10 March 2015 enzyme was metal independent, showed maximal epimerization activity at pH 7.0 and 60 ◦ C, and dis-
Accepted 10 March 2015
played 29.8, 15.48, and 13.5 U mg−1 for mannobiose, cellobiose, and lactose under optimum conditions,
Available online 19 March 2015
respectively. It exhibited promising thermostability under incubation below 60 ◦ C. The Km , turnover
number (kcat ), and catalytic efficiency (kcat /Km ) for lactose were 124.7 mM, 30.9 s−1 , and 0.248 mM−1 s−1 ,
Keywords:
respectively. At pH 7.0 and 60 ◦ C, 50 mM epilactose was produced from 200 mM lactose by a 0.6 ␮M of
Cellobiose 2-epimerase
Characterization
enzyme concentration after reaction for 4 h.
Epilactose © 2015 Elsevier B.V. All rights reserved.
Epimerization
Thermoanaerobacterium saccharolyticum

1. Introduction CE was first characterized from a ruminal anaerobe, Ruminococ-

Epilactose (4-O-␤-d-galactopyranosyl-d-mannose), an epimer
of lactose, is an important bioactive lactose derivative and has
recently attracted considerable attention due to its promising phys-
iological effects [1]. It is a type of non-digestible disaccharide with
good prebiotic properties [2,3], and promotes intestinal mineral
absorption [4]. Experimental results obtained in vivo indicate that
epilactose metabolism increases the level of beneficial short-chain
fatty acids and reduces the risk of arteriosclerosis [5]. Epilactose
exists in extremely small quantities in nature, and generally is
chemically formed in a small amount by heating or sterilization
of solutions of lactose [6], especially milk [7].
Recently, a great deal of attention has been focused on its
biological production. Cellobiose 2-epimerase (CE, EC 5.1.3.11)
is a potential biocatalyst for industrial production of epilac-
tose from the cheap material lactose [1]. CE mainly catalyzes
the reversible epimerization between disaccharide cellobiose
and 4-O-␤-d-glucopyranosyl-d-mannose and generally has broad
substrate specificity toward various disaccharides including man-
nobiose, lactose and epilactose [8]. In 2008, epilactose-producing
cus albus (Rual-CE) [2]. Since then, epilactose-producing CE was
identified from another five anaerobic strains, namely Eubac-
terium cellulosolvens (Euce-CE) [9], Bacteroides fragilis (Bafr-CE)
[10], Caldicellulosiruptor saccharolyticus (Casa-CE) [11], Dictyoglo-
mus turgidum (Ditu-CE) [8], and Spirochaeta thermophila (Spth-CE)
[12]. The CE from a thermohalophilic aerobe, Rhodothermus
marinus (Rhma-CE), was also proven to be able to produce
epilactose effectively [13]. In addition, Ojima et al. recently iden-
tified and characterized seven epilactose-producing CEs from
various mesophilic aerobes: Flavobacterium johnsoniae (Fljo-CE),
Pedobacter heparinus (Pehe-CE), Dyadobacter fermentans (Dyfe-CE),
Herpetosiphon aurantiacus (Heau-CE), Saccharophagus degradans
(Sade-CE), Spirosoma linguale (Spli-CE), and Teredinibacter turnerae
(Tetu-CE) [14].
In this study, a recombinant CE was characterized from a
thermophilic anaerobe, Thermoanaerobacterium saccharolyticum
JW/SL-YS485 (Thsa-CE), which showed high epimerization activ-
ity toward lactose. The epilactose production from lactose by the
enzyme was studied.

2. Materials and methods

∗ Corresponding author at: State Key Laboratory of Food Science and Technology,
2.1. Chemicals and reagents
Ministry of Education, Key Laboratory of Carbohydrate Chemistry and Biotechnol-
ogy, Jiangnan University, Wuxi, Jiangsu 214122, China. Tel.: +86 510 85919161;
fax: +86 510 85919161. Lactose and cellobiose were from Sinopharm Chemical Reagent
E-mail address: wmmu@jiangnan.edu.cn (W. Mu). (Shanghai, China). Mannobiose was from Megazyme International

http://dx.doi.org/10.1016/j.molcatb.2015.03.005
1381-1177/© 2015 Elsevier B.V. All rights reserved.
40 Q. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 39–44

Ireland Ltd. (Wicklow, Ireland). Epilactose and lactulose were
purchased from Sigma (St Louis, MO, USA). Chelating Sepharose
Fast Flow resin was from GE Healthcare (Uppsala, Sweden). Elec-
trophoresis reagents were purchased from Bio-Rad. Isopropyl
␤-d-1-thiogalactopyranoside (IPTG) and all chemicals used for
enzyme assays and characterizations in this study were of analyti-
cal grade or higher obtained from Sangon Biotech (Shanghai, China).
2.2. Gene cloning
The complete genome of the T. saccharolyticum JW/SL-YS485
chromosome was obtained from GenBank (NCBI accession number:
NC 017992). The full-length gene (locus tag: Tsac 2329), encoding
a putative protein with ID YP 006392930.1, was synthesized and
incorporated with NdeI and XhoI sites at the 5 - and 3 -termini and
then was introduced into the pET-22b(+) plasmid with the same
restriction sites to create a reconstructed plasmid, pET-Thsa-CE.
An in-frame 6 × His-tag sequence was provided at the C-terminal
sequence of the open reading frame for the simple purification of
the recombinant protein.
determined by Lineweaver–Burk plots from the Michaelis–Menten
equation.
2.6. Analytical methods
To detect the enzyme activity, lactose and epilactose were deter-
mined by an HPLC (Agilent 1200 system, Agilent technologies, CA,
USA) equipped with a refractive index detector and an Asahipak
NH2P-50-4E column (4.6 mm id × 250 mm, Shodex, Tokyo, Japan).
The column was eluted with acetonitrile/water (65:35, v/v) at room
temperature and 1 ml min−1 .
In addition, ion chromatography was used to analyze whether
the enzymatic reaction produced lactulose from lactose. The
reaction products were analyzed by Dionex ion chromatography
ICS-5000 (Sunnyvale, CA, USA) with a Dionex pulsed amperometric
detector (HPAEC-PAD) equipped with an Au electrode and a Dionex
Carbopac PA20 column (3 mm id × 150 mm, Sunnyvale, CA, USA).
The column was eluted with 1.5 mM NaOH as the mobile phase at
30 ◦ C and 0.5 ml min−1 .
3. Results and discussion

2.3. Expression and purification of recombinant Thsa-CE

3.1. Amino acid sequence alignment

The pET-Thsa-CE plasmid was transformed into host cell

The putative protein with accession No. YP 006392930, shown
Escherichia coli BL21(DE3) (Sangon Biotech, Shanghai, China). The
as N-acyl-d-glucosamine 2-epimerase (AGE) from T. saccha-
recombinant E. coli was cultivated in LB medium containing
rolyticum JW/SL-YS485 in GenBank, was identified as CE and
100 ␮g ml−1 of ampicillin with shaking (200 rpm) at 37 ◦ C until A600
renamed Thsa-CE due to the high specificity toward cellobiose.
reached 0.6. IPTG was then added to obtain a final concentration of
Similarly, many putative AGEs in GenBank have been identified as
0.5 mM, and the fermentation was continued at 28 ◦ C for 6 h. The LB
CEs without activity toward N-acetyl-d-glucosamine in previous
medium used was composed of 5 g/l yeast extract, 10 g/l tryptone,
reports [8,13,14]. By sequence analysis, Thsa-CE was determined
and 10 g/l NaCl.
to be a 392 amino acid protein with a calculated molecular mass of
The recombinant enzyme, expressed as a 6 × His-tagged fusion
46,516 Da.
protein, was purified by one-step nickel-affinity chromatography
Based on the phylogenetic tree analysis, Thsa-CE showed a much
(Novagen) according to the manufacturer’s protocol (pET His Taq
closer relationship with Casa-CE (Fig. 1). Homologous compari-
System; Novagen). The active fractions eluted were pooled and dia-
son of the amino acid sequences of various CEs was analyzed.
lyzed overnight against 50 mM sodium phosphate buffer (pH 7.0).
Thsa-CE (GenBank accession No.: YP 006392930.1) shared 52.8%
The purity and integrity of protein were checked by SDS-PAGE on
amino acid identity with Casa-CE (YP 001179132), 40–50% iden-
a 12% gels.
tity with Ditu-CE (YP 002352551), Rual-CE (BAF81108), Euce-CE
(BAG68451.1), Bafr-CE (BAH23773), and Rhma-CE (BAK61777), and
2.4. Enzyme assay less than 40% identity with other CEs (Table 1). Although more
than 10 microbial CEs were characterized, the amino acid sequence
Enzyme activity was measured by determination of the amount identity between them was interestingly very low, all less than
of produced epilactose from lactose. The reaction mixture of 50% except the identity between Thsa-CE and Casa-CE (52.8%) and
1 ml volume contained 100 mM lactose, sodium phosphate buffer the identity between Bafr-CE and Pehe-CE (50.5%) (Table S1). The
(50 mM, pH 7.0), and 1 ␮M enzyme. The reactions were carried out
at 60 ◦ C for 15 min; and were stopped by addition of HCl to the
reaction mixture at a final concentration of 500 mM. One unit (U)
of enzyme activity was defined as the amount of enzyme required
to produce 1 ␮mol of epilactose per min at pH 7.0 and 60 ◦ C.

2.5. Biochemical characterization

The characterization of the purified enzyme was detected using

lactose as a substrate. The effect of pH was determined at 60 ◦ C
using four buffer systems (50 mM), including sodium citrate buffer
(pH 5.0–6.0), sodium phosphate buffer (pH 6.0–7.5), Tris–HCl buffer
(pH 7.5–9.0), and glycine–NaOH buffer (pH 9.0–10.5). The optimum
temperature was determined by measuring the activity at different
temperatures, ranging from 30 to 80 ◦ C. The thermostability was
determined by detecting the residual activity of the enzyme that
had been pre-incubated at different temperatures.
Kinetic parameters were determined using 10–100 mM con-
Fig. 1. Phylogenetic tree of the characterized epilactose-producing CEs from various
centrations of substrate. The parameters, including the Michaelis– microorganisms. The scale bar indicates the amino acid substitutions per position.
Menten constant (Km ) and turnover number (kcat ) values, were GenBank accession numbers of the CEs are given after each species name.
 Q. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 39–44 41

Table 1
Comparison of the biochemical properties of CE from various microorganisms.

Enzyme Strain for GenBank Sequence Subunit molecular Optimum Optimum Reference
enzyme source accession no. identity (%)a mass (kDa)b temperature (◦ C) pH

Thsa-CE T. saccharolyticum YP 006392930.1 100 46.5 60 7.0 This study

Rual-CE R. albus BAF81108 48.8 45.2 30 7.5 [2]
Euce-CE E. cellulosolvens BAG68451.1 43.4 46.9 35 7.5 [9]
Bafr-CE B. fragilis BAH23773 41.6 45.7 45 7.5 [10]
Casa-CE C. saccharolyticus YP 001179132 52.8 46.5 75 7.5 [13]
Ditu-CE D. turgidum YP 002352551 49.6 46.4 70 7.0 [8]
Spth-CE S. thermophile YP 003873344 33.9 46.2 60 7.0 [12]
Rhma-CE R. marinus BAK61777 40.8 47.3 80 6.3 [17]
Fljo-CE F. johnsoniae YP 001197274 37.5 46.7 35 8.4 [14]
Pehe-CE P. heparinus YP 003094236 41.1 46.5 35 6.3 [14]
Dyfe-CE D. fermentans YP 003086363 38.3 45.2 50 7.7 [14]
Heau-CE H. aurantiacus YP 001543663 37.8 47.5 45 7.3 [14]
Sade-CE S. degradans YP 525984 37.5 47.2 35 7.7 [14]
Spli-CE S. linguale YP 003385134 36.2 46.6 45 7.7 [14]
Tetu-CE T. turnerae YP 003075376 33.4 48.0 35 8.8 [14]
a
Amino acid sequence identity with Thsa-CE.
b
Calculated based on the amino acid sequence of various CEs.

crystal structures of Rual-CE [15] and Rhma-CE [16] have recently

been identified. According to the multiple sequence alignment, two
highly conserved residues, His245 and His275, which were consid-
ered to be crucial for catalytic activity [15,16], were found in two
conserved regions (Fig. 2).

3.2. Expression and purification of Thsa-CE

E. coli overexpressing recombinant Thsa-CE by IPTG induction

exhibited a strong protein band of approximately 47 kDa on SDS-
PAGE, which was consistent with the predicted molecular mass of
Thsa-CE. The recombinant enzyme was purified to electrophoretic
homogeneity as a soluble protein from crude E. coli extract by one-
step nickel affinity chromatography, with a purification of 15-fold
and a yield of 26% (Table S2). Both SDS-PAGE (Fig. S1) and gel
filtration analysis (data not shown) were used to determine the
molecular mass of the purified enzyme, which was as approxi-
mately 47 kDa. These analyses indicated that the native Thsa-CE
existed as a 47 kDa monomer. The crystal structures of Rual-CE
[15] and Rhma-CE [16] showed that they were monomeric pro-
teins, and the Casa-CE [13], Ditu-CE [8], and Bafr-CE [9] were also
characterized as monomeric proteins according to the gel filtration
results.

3.3. HPLC analysis of the reaction products from lactose by

Thsa-CE

Lactose and epilactose could be efficiently separated and

measured by HPLC using an Asahipak NH2P-50-4E column and
refractive index detector, and the reaction products from lactose
by Thsa-CE were eluted at the retention times of lactose and epi-
lactose as shown in Fig. 3. Quantitative determination of epilactose
was carried out using HPLC based on the response value of an epi-
lactose standard. The calibration curve for epilactose was expressed
as: y = 155.65x − 163.33 (R2 = 0.985), where y denoted the peak area
and x represented the concentration of epilactose.
Fig. 2. Multiple sequence alignment of Thsa-CE, Rual-CE, and Rhma-CE. The align-
To determine whether the reaction products contained lactu- ment was performed using the ClustalW2 program (http://www.ebi.ac.uk/Tools/
lose, HPAEC-PAD was further performed using a Dionex Carbopac clustalw2/index.html). Asterisks (*), colons (:), and dots (.) indicate completely,
PA20 column (Fig. 4). The chromatographic results showed that strongly, and weakly conserved positions, respectively. The proposed catalytic
Thsa-CE produced epilactose as the only product from lactose. residues (His245 and His275) are symbolized by closed circles (䊉) according to the
crystal structures of Rual-CE [15] and Rhma-CE [16].

3.4. Effects of pH, temperature, and metal ions on Thsa-CE activity

wide pH spectrum, and more than 75% of its maximum activity
The biochemical properties of Thsa-CE were characterized using was maintained from pH 6.0 to 9.5 (Fig. S2A). The pH spectrum
lactose as substrate. The recombinant enzyme displayed maximal for activity was much broader than that of Euce-CE [9], Rhma-CE
epimerization activity at pH 7.0; however, it exhibited a relatively [17], Spth-CE [12], and Rual-CE [2]. The broad pH spectrum was
42 Q. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 39–44

Fig. 3. Chromatographic analysis of disaccharides by HPLC equipped with a refractive index detector and an Asahipak NH2P-50-4E column. Chromatograms A and B show
the standards of epilactose and lactose, respectively. C shows the HPLC profile of the enzymatic reaction products from lactose by Thsa-CE.

advantageous for the practical application of Thsa-CE under differ-
ent pH conditions, especially at slightly acidic pHs for preventing
non-enzymatic catalysis of carbohydrate substrates at high tem-
perature [1].
The optimum temperature is also an important consideration
for the potential industrial application of CE. Much attention has
recently been focused on the characterization of thermostable car-
bohydrate isomerases or epimerases. Thermophilic microbes are
important sources for isolation of thermostable enzymes. To date,
CE has been characterized from four thermostable bacteria, includ-
ing Rhma-CE [17], Casa-CE [13], Ditu-CE [8], and Spth-CE [12], and
they exhibited optimum temperatures at 80 ◦ C, 75 ◦ C, 70 ◦ C, and
60 ◦ C, respectively. Herein, CE was identified from a novel ther-
mophilic strain, T. saccharolyticum. It displayed maximal activity
at 60 ◦ C and exhibited 98% of maximal activity at 70 ◦ C (Fig. S2B).
Except for the above-mentioned CEs, other reported CEs were all
from mesophilic bacteria, and the optimum temperatures of each
were not higher than 50 ◦ C (Table 1). The effect of temperature on
enzyme stability was monitored from 50 to 65 ◦ C. Thsa-CE exhib-
ited perfect stability at 50 ◦ C, only losing 8% of its initial activity
after 48 h of incubation, and could maintain more than 50% of
its initial activity after 24 h and 6 h at 55 ◦ C and 60 ◦ C, respec-
tively (Fig. 5). However, it was easily inactivated under incubation
at 65 ◦ C.
Thsa-CE did not require metal for displaying activity and its
activity was not inhibited by EDTA, indicating that it was a
metal-independent enzyme similar to previously characterized CEs
[8–10,13,18]. The enzyme activity was investigated in the presence
of various divalent metal ions at a final concentration of 1 mM. The
Cu2+ and Zn2+ remarkably inhibited the activity, Co2+ inhibited the
activity by 35%, and all other tested metal ions slightly changed the
activity by up to 20% (Fig. S3).
3.5. Substrate specificity and enzyme kinetics
The recombinant Thsa-CE was not active for N-acyl-d-
glucosamine and d-glucose, but was active for mannobiose, cel-
lobiose, and lactose, producing 4-O-␤-mannopyranosyl-d-glucose,
4-O-␤-glucopyranosyl-d-mannose, and epilactose, respectively.
The enzyme displayed the highest activity (29.8 U mg−1 ) toward
mannobiose, and showed specific activities of 15.48 U mg−1 and
13.5 U mg−1 for cellobiose and lactose under standard conditions,
respectively (Table 2). Therefore, the Thsa-CE was, in fact, a man-
nobiose 2-epimerase. In previous works, Bafr-CE [19], Casa-CE,
Ditu-CE, Rhma-CE, and Spth-CE [12] also showed higher activity
toward mannobiose than cellobiose, and these enzymes were pro-
posed as being mannobiose 2-epimerases.
 Q. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 39–44
Fig. 4. Chromatographic analysis of disaccharides by HPAEC-PAD on a Dionex Carbopac PA20 column. Chromatogram A shows the mixed standards of lactose, epilactose,
and lactulose. B represents the enzymatic reaction products from lactose by Thsa-CE.
Fig. 5. Effect of temperature on the stability of Thsa-CE. The experiments were per-
formed by exposing the enzyme at 50 (), 55 (䊉), 60 (), and 65 ◦ C () for different
time intervals at pH 7.0. The initial enzyme activity was set as a reference value of
100%. Values are means of three replications ± standard deviation.
Thsa-CE could not convert d-glucose to d-mannose, which was
different from Casa-CE [13], Ditu-CE [8], and Spth-CE [12]. Thsa-CE
catalyzed epimerization but did not show isomerization activity
toward the substrate cellobiose and lactose (Fig. 4). It was advan-
tageous for epilactose production that the epimerization reaction
catalyzed by Thsa-CE did not produce byproducts. Ojima et al.
determined the reaction products from cellobiose or lactose by var-
ious CEs, including Fljo-CE, Pehe-CE, Dyfe-CE, Heau-CE, Sade-CE,
Spli-CE, and Tetu-CE and found that these tested CEs also produced
a sole product from each substrate [14]. However, interestingly,
Casa-CE [13] and Ditu-CE [8] catalyzed not only the epimerization
reaction but also the isomerization reaction for cellobiose and lac-
tose, and produced both epilactose and lactulose from lactose, in
which case lactulose was the main product.
The Km value for lactose (124.7 mM) was lower than that for
cellobiose (241.1 mM), indicating that Thsa-CE had higher affinity
for lactose than cellobiose. However, the enzyme showed higher
43
Fig. 6. Production of epilactose from lactose by Thsa-CE. A 200 mM concentration
of lactose was used for epilactose production by 0.6 ␮M enzyme at pH 7.0 and 60 ◦ C.
Values are means of three replications ± standard deviation.
catalysis efficiency (kcat /Km ) for cellobiose (0.417 mM−1 s−1 ) than
lactose (0.248 mM−1 s−1 ) (Table 2). In addition, the kcat , Km , and
kcat /Km values toward mannobiose were measured to be 125.6 s−1 ,
92.5 mM, and 1.358 mM−1 s−1 .
3.6. Production of epilactose from lactose by Thsa-CE
In 1 l of large-scale production reaction by purified Thsa-CE,
50 mM of epilactose could be produced from 200 mM lactose with
a conversion ratio of 25% when a 0.6 ␮M concentration of enzyme
was used for catalysis at pH 7.0 and 60 ◦ C for 4 h (Fig. 6). By compari-
son, Ditu-CE [8] and Casa-CE [11] produced epilactose from lactose
with conversion ratios of 12.8% and 15%, respectively; however,
these two enzymes produced lactulose at a high conversion ratio
(>50%).
44 Q. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 39–44

Table 2
Specific activity and kinetic parameters of various CEs for cellobiose and lactose.

Enzyme Cellobiose Lactose Reference

Specific activity kcat (s−1 ) Km (mM) kcat /Km Specific activity kcat (s−1 ) Km (mM) kcat /Km
(U mg−1 ) (mM−1 s−1 ) (U mg−1 ) (mM−1 s−1 )

Thsa-CE 15.48 ± 0.23 100.4 ± 5.6 241.1 ± 8.3 0.417 ± 0.036 13.5 ± 0.45 30.9 ± 1.8 124.7 ± 7.4 0.248 ± 0.018 This study
Rual-CE 51 63.8 13.8 4.6 28.4 52.1 33 1.6 [2]
Euce-CE 19 28.5 11.3 2.52 NRa 32.5 72 0.451 [9]
Bafr-CE 61.8 67.6 3.75 18 19.7 79.5 6.56 12.1 [10]
Casa-CE 3600b NR NR NR NR NR NR NR [13]
Ditu-CE 24.4 169.1 75.8 2.23 14.2 89.4 79.6 1.12 [8]
Spth-CE 23.4 28 4.3 6.5 7.8 NR NR NR [12]
Rhma-CE NR 80.8 27.2 2.97 87.5 111 28.8 3.85 [17]
Fljo-CE NR 39.9 53.2 0.75 NR 17.5 34.9 0.501 [14]
Pehe-CE NR 7.02 29.6 0.237 NR 5.43 24.5 0.222 [14]
Dyfe-CE NR 240 179 1.34 NR 44.9 95.7 0.469 [14]
Heau-CE NR 18.7 28.2 0.663 NR 14 51.9 0.27 [14]
Sade-CE NR 26.1 22.6 1.15 NR 7.82 29.2 0.268 [14]
Spli-CE NR 222 104 2.13 NR 92.1 206 0.447 [14]
Tetu-CE NR 165 198 0.832 NR 175 238 0.735 [14]
a
NR, not reported.
b
Remeasured to be 21.8 U mg−1 [13].

4. Conclusions [2] S. Ito, H. Taguchi, S. Hamada, S. Kawauchi, H. Ito, T. Senoura, J. Watan-

A gene encoding a putative AGE from T. saccharolyticum JW/SL-
YS485 was cloned and expressed in E. coli. The recombinant enzyme
was identified as CE by evaluating its substrate specificity. The
enzyme was metal-independent, and the optimum pH and tem-
perature were measured to be pH 7.0 and 60 ◦ C, respectively. The
enzyme could effectively produce epilactose, giving a 50 mM con-
centration of epilactose from 200 mM lactose with a conversion
ratio of 25%. It was demonstrated that Thsa-CE could be a good can-
didate for epilactose production in an industrial enzymatic process.
Acknowledgments
This work was supported by the NSFC Project (Nos. 21276001
and 31171705), the 863 Project (No. 2013AA102102), the Fun-
damental Research Funds for the Central Universities (No.
JUSRP51304A), and the Support Project of Jiangsu Province
(BK20130001).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2015.03.005.
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