S.-I. Ryu et al. / Process Biochemistry 46 (2011) 128–134
2.4. TLC, HPAEC, and HPLC analyses of reaction products
129
the side product. Sucrose synthase catalyzes the reversible cleavage
of sucrose with NDP to generate NDP-glucose (NDP-Glc) and d-
fructose. That is, this enzyme regenerates NDP-Glc from NDP using
sucrose as a cheap and renewable energy source.
The reaction products for the synthesis of trehalose or NDP-Glc were ana-
lyzed using TLC on Whatman K5F silica gel plates (Whatman, Kent, UK) with
both solvent systems of isopropyl alcohol:ethyl acetate:water (3/1/1, v/v/v) and
n-butanol:pyridine:water (7/3/1, v/v/v) [16]. Carbohydrates on the TLC plate were
visualized by dipping method as previously reported. The time-dependent change in
the composition of the reaction mixture was periodically investigated with analyti-
on a Dionex CarboPac PA10 column (0.4 cm × 25 cm, Sunnyvale, CA, USA) with an
isocratic gradient of 150 mM sodium hydroxide at a flow rate of 1.0 mL/min [20].
The standard curve for the determination of trehalose production had a slope of
15.93 (mV s/mM, peak area/trehalose concentration) with a correlation coefficient
of 0.995. The identity of the synthesized trehalose was further confirmed by hydrol-
ysis with trehalase (EC 3.2.1.28) [17]. HPLC analysis was carried out to qualitatively
and quantitatively determine the synthesis of NDP-Glc from NDP and trehalose using
a Prevail Carbohydrate ES column (250 mm × 4.6 mm, Alltech Associates, Inc., Deer-
field, IL, USA) connected to a Waters Model 510 system with 996 photodiode array
detector at 254 nm. A linear HPLC gradient was composed of 80% acetonitrile in
water (solvent A) and 0.5 M ammonium carbonate or 0.5 M NaCl in water (solvent
B). After a 20-L injection of each sample, solvent B was either increased from 0 to
30% in 20 min and then held at 30% for 5 min, followed by reduction to 0% in 5 min
for 0.5 M ammonium carbonate, or increased from 0 to 100% in 40 min and then
held at 100% for 5 min, followed by a reduction to 0% in 10 min for 0.5 M NaCl. The
analysis was performed at 25 ◦C and a flow rate of 0.8 mL/min. The standard curves
for HPLC analysis of the respective NDP-Glc were used in the range of 0–9 mM to
determine the concentration in the reaction sample, in which the slopes were 7.57
(for UDP-Glc), 7.74 (for ADP-Glc), and 7.62 (for GDP-Glc) (V s/mM, peak area/NDP-
Glc concentration) with the correlation coefficient of 0.99 for the gradient elution
of 0.5 M ammonium carbonate. The NDP-Glc synthesized was further confirmed to
be utilized as the glucosyl donor for the formation of trehalose by the enzymatic
reaction with glucose. The detailed glycosidic structure of UDP-Glc synthesized was
representatively confirmed by 13C NMR. The 13C NMR spectra of the product was
recorded with a JEOL JNM LA-400 MHz NMR spectrometer (Tokyo, Japan). The sam-
ple was dissolved in DMSO-d6 at 24 ◦C with tetramethylsilane (TMS) as the chemical
shift reference.
Recently, we have just established a novel process by effi-
ciently regenerating nucleotide sugars using another renewable
sugar, trehalose, with microbial glycosyltransferase. Previously, the
reported to synthesize trehalose or trehalose analogues with NDP-
Glc donors and monosaccharide acceptors such as glucose and
sugar-dependent glycosyltransferases is perceived to be unidirec-
tional [18]. However, trehalose glycosyltransferring synthase (TreT,
Q7LYW5) from Thermococcus litoralis has been already reported to
be reversible in the catalysis [19]. Similarly, the trehalose synthase
from P. horikoshii (hereafter abbreviated as Pyrococcus TreT) cat-
alyzed the reversible cleavage of trehalose with NDP to generate
NDP-Glc and d-glucose. In this study, the reversibility of this Pyro-
coccus TreT was described for the alternative synthesis of trehalose
and NDP-Glc in both catalytic directions. The reaction progress was
compared for various NDP-Glc and NDP substrates in the enzyme
catalysis. Regeneration yields of several NDP-Glc sugars were also
investigated. In addition, the enzymatic productions of NDP-Glcs
were evaluated in a repetitive batch mode with enzyme recycling
system.
2. Materials and methods
2.1. Materials
Nucleoside 5ꢀ-diphosphates (NDPs) such as uridine 5ꢀ-diphosphate (UDP),
adenosine 5ꢀ-diphosphate (ADP), guanosine 5ꢀ-diphosphate (GDP), and NDP-sugars
such as UDP-glucose (UDP-Glc), ADP-glucose (ADP-Glc), and GDP-glucose (GDP-
Glc) were obtained from Sigma Chemical Co (St. Louis, MO, USA). ADP was also
obtained from Amresco Inc. (Solon, OH, USA) and Genechem Inc. (Daejeon, Korea).
Cytidine 5ꢀ-diphosphate (CDP), thymidine 5ꢀ-diphosphate (TDP), CDP-glucose (CDP-
Glc), and TDP-glucose (TDP-Glc) were purchased from Genechem Inc. UDP-Glc
was also obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). ˛,˛-
Trehalose and porcine kidney trehalase were also obtained from Sigma. All other
2.5. Enzyme kinetics
The kinetic parameters for the enzyme-catalyzed synthesis of trehalose or NDP-
Glc were determined with various concentrations of donor and acceptor substrates
in 50 mM sodium acetate buffer (pH 6.0) at 60 ◦C. The donor substrates such as NDP-
Glc were employed in the range of 0.16–10.0 mM with 55.6 mM glucose acceptor
for the trehalose synthesis, and acceptor substrates such as NDP were in the range
of 4.3–340 mM with 444 mM trehalose for the synthesis of NDP-Glc. After an aliquot
(about 100 L) was taken periodically from the reaction mixture, the reaction was
immediately quenched by adding 0.1 M HCl solution and centrifuged, followed by
analysis with HPAEC or HPLC, as described above. The kinetic constants were cal-
culated by a non-linear regression program, DNRPEASY, derived by Duggleby and
Leonard [21], producing Km and kcat values.
2.2. Enzyme preparation
Pyrococcus TreT was prepared and purified as previously reported with a slight
modification [16]. Escherichia coli strain MC1061 [F− araD139 recA13 ꢀ(araABC-
leu)7696 galU galK ꢀlacX74 rpsL thi hsdR2 mcrB] was used as
a host for the
constitutive expression of the recombinant enzyme. The E. coli transformant was
cultured at 37 ◦C in Luria–Bertani (LB) medium supplemented with ampicillin
(100 g/mL). The purification of the expressed enzyme with a hexahistidine-tag
was performed by nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatogra-
phy (Qiagen, Hilden, Germany), followed by Q-Sepharose column chromatography
(Pharmacia) pre-equilibrated with 50 mM Tris–HCl buffer (pH 7.0). The enzyme was
eluted with a NaCl gradient of 0–0.5 M in the same buffer at a flow rate of 1 mL/min.
Active fractions were pooled, desalted, and concentrated by ultrafiltration.
2.6. Repetitive batch production with enzyme recycling
In order to increase overall productivities of the enzyme, the reaction was car-
ried out by repetitive batch technique with recycling of enzyme [22]. The batch
productions of respective NDP-Glc were repeated by free enzyme retained above
ultrafiltration membrane (Amicon Ultra-4 or 15 device, molecular weight cut-off
of 10 kDa, Millipore Co., Bedford, MA, USA). For one cycle of the batch reaction in
total volume of 4–10 mL, the enzyme (0.24 mg/mL) was incubated with 16% (w/v,
444 mM) trehalose as glucosyl donor and 5% (w/v, 113–124 mM) NDP (UDP, ADP,
GDP, and CDP) as acceptor at 37 ◦C for 12 h, respectively, inside the reaction reservoir
tube. Then, the solution of reaction product was collected through a centrifugation
(5000 × g) as filtrate in the membrane retention method. The centrifugal filtration
was done until an approximately 100 L of the solution inside the membrane was
remained. For the next turn of the batch reaction, the same amount of the substrate
solution was newly added into the remaining solution containing the enzyme and
the reaction was repeated at the same conditions, followed by the centrifugation.
These repetitive batch processes (2 cycles per a day) were continuously performed
for 2 or 3 weeks, depending on the substrate. The filtrate in each turn was analyzed
using high performance liquid chromatography (HPLC) with the gradient elution
of 0.5 M NaCl, pooled, and kept at 4 ◦C for the product purification. The standard
curves for the HPLC analysis of NDP-Glcs with the gradient elution of 0.5 M NaCl
were estimated to have the slopes of 5.7 (for UDP-Glc), 5.1 (for ADP-Glc), and 5.0 (for
GDP-Glc), and 5.7 (for CDP-Glc) (V s/mM, peak area/NDP-Glc concentration) in the
range of 0–16 mM with the correlation coefficient of 0.97. The NDP-Glc product was
efficiently purified from unreacted substrates such as NDP and trehalose and glu-
cose co-products by preparative HPLC on an Alltech Prevail Carbohydrate ES column
(300 mm × 20 mm) connected to a Waters Delta Prep 4000 system with the gradient
elution of 0.5 M ammonium carbonate up to 30% at a flow rate of 5.0 mL/min.
2.3. Enzyme reaction
For synthesis of trehalose, the enzyme (0.67 mg/mL) was reacted with 0.5% (w/v,
7.9–8.3 mM) NDP-Glc as a glucosyl donor (UDP-Glc, ADP-Glc, and GDP-Glc) and 5%
(w/v, 278 mM) glucose as an acceptor in 50 mM sodium acetate buffer (pH 6.0) at
37 ◦C and 60 ◦C for 24 h. Appropriate aliquots were taken periodically from the reac-
tion mixture during reaction, and the reaction was then immediately quenched
by adding 0.1 M HCl solution. The reaction mixture was centrifuged at 12,000 × g
for 10 min, and filtered using 0.45-m membrane filters for further experiments.
The formation of trehalose was confirmed by thin-layer chromatography (TLC),
and the amount of trehalose produced was determined using high performance
anionic exchange chromatography (HPAEC) as described in the section below. For
reverse synthesis of NDP-Glc, the enzyme (0.67 mg/mL) was incubated with 5% (w/v,
140 mM) trehalose as glucosyl donor and 0.5% (w/v, 11.3–12.4 mM) NDP (UDP, ADP,
GDP, CDP, and TDP) as acceptor in the same conditions for 24 h. The reaction samples
were taken periodically from the reaction mixture and the reaction was stopped by
the addition of 0.1 M HCl. After the filtration, the formation of NDP-Glc was quantita-
tively analyzed using high performance liquid chromatography (HPLC) as described
below.