Nucleoside 2′-deoxyribosyltransferase from psychrophilic bacterium Bacillus psychrosaccharolyticus - Preparation of an immobilized biocatalyst for the enzymatic synthesis of therapeutic nucleosides

Article abstract of DOI:10.3390/molecules190811231

Nucleoside 2′-deoxyribosyltransferase (NDT) from the psychrophilic bacterium Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first time. A preliminary characterization of the recombinant protein indicates that the enzyme is an NDT type II since it catalyzes the transfer of 2′-deoxyribose between purines and pyrimidines. The enzyme (BpNDT) displays a high activity and stability in a broad range of pH and temperature. In addition, different approaches for the immobilization of BpNDT onto several supports have been studied in order to prepare a suitable biocatalyst for the one-step industrial enzymatic synthesis of different therapeutic nucleosides. Best results were obtained by adsorbing the enzyme on PEI-functionalized agarose and subsequent cross-linking with aldehyde-dextran (20 kDa and 70% oxidation degree). The immobilized enzyme could be recycled for at least 30 consecutive cycles in the synthesis of 2′-deoxyadenosine from 2′-deoxyuridine and adenine at 37 °C and pH 8.0, with a 25% loss of activity. High conversion yield of trifluridine (64.4%) was achieved in 2 h when 20 mM of 2′-deoxyuridine and 10 mM 5-trifluorothymine were employed in the transglycosylation reaction catalyzed by immobilized BpNDT at 37 °C and pH 7.5.

Full text of DOI:10.3390/molecules190811231

Molecules 2014, 19, 11231-11249; doi:10.3390/molecules190811231
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Nucleoside 2'-Deoxyribosyltransferase from Psychrophilic
Bacterium Bacillus psychrosaccharolyticus — Preparation of an
Immobilized Biocatalyst for the Enzymatic Synthesis of
Therapeutic Nucleosides
1
2
1,†
1
Alba Fresco-Taboada , Immacolata Serra , Jesús Fernández-Lucas , Carmen Acebal ,
1
2,‡,
1,‡,
Miguel Arroyo , Marco Terreni * and Isabel de la Mata
*
1
Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University
of Madrid, C/José Antonio Novais 2, 28040 Madrid, Spain; E-Mails: albaf@bbm1.ucm.es (A.F.-T.);
jesus.fernandez2@uem.es (J.F.-L.); acebal@bbm1.ucm.es (C.A.); arroyo@bbm1.ucm.es (M.A.)
Department of Drug Sciences and Italian Biocatalysis Center, Università degli Studi di Pavia,
Via Taramelli 12, 27100 Pavia, Italy; E-Mail: immacolata.serra@unipv.it
2
†
Present address: Department of Pharmacy and Biotechnology, Biomedical Sciences Faculty,
European University of Madrid, 28670, Spain.
‡
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed; E-Mails: marco.terreni@unipv.it (M.T.);
idlmata@ucm.es (I.M.); Tel.: +39-0382-98-7265 (M.T.); +34-913-944-150 (I.M.);
Fax: +39-0382-42-2975 (M.T.); +34-913-944-672 (I.M.).
Received: 11 June 2014; in revised form: 23 July 2014 / Accepted: 24 July 2014 /
Published: 31 July 2014
Abstract: Nucleoside 2'-deoxyribosyltransferase (NDT) from the psychrophilic bacterium
Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first
time. A preliminary characterization of the recombinant protein indicates that the enzyme
is an NDT type II since it catalyzes the transfer of 2'-deoxyribose between purines and
pyrimidines. The enzyme (BpNDT) displays a high activity and stability in a broad range
of pH and temperature. In addition, different approaches for the immobilization of BpNDT
onto several supports have been studied in order to prepare a suitable biocatalyst for the
one-step industrial enzymatic synthesis of different therapeutic nucleosides. Best results
were obtained by adsorbing the enzyme on PEI-functionalized agarose and subsequent
cross-linking with aldehyde-dextran (20 kDa and 70% oxidation degree). The immobilized

Molecules 2014, 19
11232
enzyme could be recycled for at least 30 consecutive cycles in the synthesis of
'-deoxyadenosine from 2'-deoxyuridine and adenine at 37 °C and pH 8.0, with a 25% loss
2
of activity. High conversion yield of trifluridine (64.4%) was achieved in 2 h when 20 mM
of 2'-deoxyuridine and 10 mM 5-trifluorothymine were employed in the transglycosylation
reaction catalyzed by immobilized BpNDT at 37 °C and pH 7.5.
Keywords: nucleoside 2'-deoxyribosyltransferase; Bacillus psychrosaccharolyticus;
enzyme immobilization; nucleoside synthesis; trifluridine
1
. Introduction
Nucleoside analogs can be recognized as physiological molecules and be incorporated into DNA
and RNA, consequently inhibiting cellular division and viral replication [1]. In this sense, some
nucleoside analogs are actually used as therapeutic agents in inhibition of cancer cell growth,
inhibition of viral replication or immunosuppression [1]. Non-natural nucleosides can be synthesized
by conventional chemical methods which include many protection and deprotection steps [2], and the
use of contaminant organic solvents. On the contrary, enzymatic synthesis of these compounds has
arisen as a friendly environmental approach, reducing time-consuming steps and increasing stereo- and
regioselectivity compared to chemical synthesis. Nucleoside phosphorylases (NPs), as well as
nucleoside 2'-deoxyribosyltransferases (NDTs), are multimeric enzymes, which can be employed in the
enzymatic synthesis of nucleosides by mediating the transfer of glycosyl residues to acceptor bases [3,4].
Nevertheless, NDTs (EC 2.4.2.6) offer many advantages in nucleoside analogs synthesis compared to
NPs, since the former ones catalyze the one-step transfer of the deoxyribosyl moiety between
2
'-deoxyribonucleosides independently of the type of base (both purines and pyrimidines) [5]. As a
matter of fact, the strict substrate specificity of NPs narrows the number of their possible applications [6].
The main microbial source of NDTs for nucleoside enzymatic synthesis comes from different
species of Lactobacillus, such as L. helveticus [7], L. leichmannii, L. fermentum [8], and L. reuteri [9].
In addition, extremophiles have also shown the presence of NDTs with applicability in nucleoside
synthesis [10,11]. In this sense, cold-adapted microbial enzymes are described to be more productive
at low temperatures than their mesophilic or thermophilic counterparts [12]. The psychrophilic
microorganism Bacillus psychrosaccharolyticus (CECT 4074, ATCC 23296, DSM 6) has been
reported to display nucleoside 2'-deoxyribosyltransferase activity [10], demonstrating that this
facultative anaerobic Gram-positive bacterium could be a NDT source. In this sense, the genome of
B. psychrosaccharolyticus has been recently published and the ndt gene that codifies a putative NDT
has been identified in its genome [13].
It should be remarked that the use of immobilized enzymes could be one of the most important
steps that should be pursued in order to move from a new enzyme towards an efficient biocatalyst.
This aspect should be particularly considered in the case of multimeric enzymes that show a very poor
stability in some of the conditions required for the development of efficient preparative processes.
Immobilization on glyoxyl-agarose might accomplish highly stabilized biocatalysts due to multipoint
covalent attachment of the enzyme (via imino-bonds formation) to the support [14]. This strategy has also

Molecules 2014, 19
11233
been successfully used for immobilization and stabilization of multimeric enzymes [14–17]. However, this
interaction could be often responsible of a remarkable loss of activity as a consequence of a strong
distortion of the 3D structure of the enzyme.
PEI-functionalized supports have been also recently employed for the immobilization of multimeric
enzymes followed by cross-linking with aldehyde-dextran in order to prevent subunit dissociation that
leads to enzyme deactivation and even product contamination [18,19]. In this case, the use of PEI-coated
carriers ensures a strong ionic interaction with the enzyme avoiding distortion of its 3D structure and other
limitations typical of covalent immobilization [20]. Similarly, the use of aldehyde macromolecules (such
as properly oxidized dextrane) allows the stabilization of the enzyme-carrier interaction by
cross-linking, preventing enzyme subunit dissociation.
In this work, cloning, production, purification and functional characterization of a novel NDT from
psychrophilic Bacillus psychrosaccharolyticus (hereafter abbreviated as BpNDT) in E. coli BL21 (DE3)
are reported. In addition, different approaches for the immobilization of BpNDT onto several carriers
have been studied in order to prepare an improved biocatalyst for the one-step enzymatic synthesis of
the therapeutic valuable trifluridine.
2
. Results and Discussion
2.1. Cloning, Expression and Purification of Nucleoside 2'-Deoxyribosyltransferase from
Bacillus psychrosaccharolyticus
The ndt gene encoding BpNDT was amplified by PCR, cloned, and overexpressed in
Escherichia coli BL21 (DE3). The recombinant BpNDT was purified from the cell extract by three
chromatographic steps consisting of an anionic-exchange chromatography, a molecular size exclusion
chromatography, and finally an isofocusing chromatography, as described in the Experimental Section.
As result of the purification process, a total of 9.8 mg of pure BpNDT per L of broth was obtained. A
single protein band with an apparent molecular mass of 16 kDa was detected by SDS-PAGE (Figure 1).
Size exclusion chromatography of pure BpNDT on Sepharose 12 10/300 GL column (GE
Healthcare) indicated that the native enzyme could be a homohexameric protein of 91.4 kDa, the same
oligomerization state as most described NDTs [7,21–25], with the exception of the tetrameric L. lactis
subsp. lactis NDT [26].
2
.2. Functional Characterization of Recombinant BpNDT
Nucleoside 2'-deoxyribosyltransferases are classified into two classes depending on their substrate
specificity: NDT type I (PDT) specific for purines (Pur ↔ Pur) and NDT type II (NDT), which
catalyzes the transfer between purines and/or pyrimidines (Pur ↔ Pur, Pur ↔ Pyr, Pyr ↔ Pyr) [7,22,27].
In order to determine which type of 2'-deoxyribosyltransferase activity was displayed by recombinant
BpNDT, different reactions with ribo- and 2'-deoxyribonucleosides were performed (dUrd + Ade, Ino
+
Ade, Urd + Ade, dUrd + Thy). As result of experiments, BpNDT should be classified as a NDT type
II (NDT) since it could catalyze the transfer of 2'-deoxyribonucleosides between both purines and
pyrimidines, however, it was not able to interchange the deoxyribosyl moiety between ribonucleosides
(
Table 1) and is free of nucleoside phosphorylases traces.

Molecules 2014, 19
11234
Figure 1. SDS-PAGE of different purification steps of BpNDT produced by E. coli BL21
DE3). Lane 1, broad range molecular weight standards from BioRad. Lane 2, 30 μg of
(
protein after sonication. Lane 3, 30 μg of protein after anionic exchange chromatography
on Bio-Scale Mini UNOsphere Q cartridge. Lane 4, 10 μg of protein after size-exclusion
chromatography on Superose 12. Lane 5, 10 μg of protein after chromatofocusing on Mono P.
kDa
1
2
3
4
5
2
1
00.0
16.3
9
6
7.4
6.2
4
5.0
3
1.0
2
1.5
1
4.4
.5
6
Table 1. Nucleoside 2'-deoxyribosiltransferase activity of BpNDT.
dUrd + Ade
Ino + Ade
Urd + Ade
dUrd + Thy
+
−
−
+
Experimental conditions: 0.40 μg of enzyme were incubated at 40 °C for 5 min with 40 μL of 10 mM
substrates in 50 mM MES buffer, pH 6.5.
The determination of the optimal conditions for activity of pure BpNDT included studies of the
influence of pH, ionic strength (I) and temperature on enzyme activity. In order to study the influence of
pH on enzyme activity, the synthesis of thymidine (Thd) from 2'-deoxyuridine (dUrd) and thymine (Thy)
was selected as the standard reaction due to pK
guarantees the correct protonated form of nucleosides and bases in solution at the pH range assayed
from 4.0 to 9.0). As shown in Figure 2, the enzyme was stable in a pH range from 6.0 to 9.0, it
a
values of thymine (9.7) and uracil (9.2), which
(
retained more than 85% activity at pH 10 and it showed optimal activity at pH 8.0.
Ionic strength (I) effect on BpNDT activity was studied by adding different concentrations of NaCl
in the reaction medium. Highest activity was maintained up to 1.0 M NaCl, whereas activity decreased
2
5% in presence of 1.5 M NaCl. As far as temperature is concerned, the BpNDT activity was studied
in the range from 4 to 80 °C, being most active at 50 °C (Figure 3). Thermal stability was analyzed by
incubating the enzyme at temperatures ranging from 4 to 90 °C during 15 min, and the results
indicated that recombinant BpNDT was stable only up to 50 °C. These results are surprising for an
enzyme from a psychrophilic microorganism. This behavior has also been reported for other enzymes
from B. psychrosaccharolyticus [28], pointing out that this microorganism should be considered a
psychrotolerant bacterium.
2
.3. Immobilization of Recombinant BpNDT
Different immobilization strategies have been attempted to prepare a robust biocatalyst of
the multimeric NDT from B. psychrosaccharolyticus, including (i) covalent attachment to

Molecules 2014, 19
11235
glyoxyl-agarose, and (ii) ionic adsorption to supports functionalized with polyethyleneimine (PEI) and
post-immobilization techniques.
Figure 2. pH dependence of BpNDT activity. Effect of pH on the BpNDT activity () and
stability () 0.4 µg of enzyme were incubated with 40 µL of 10 mM dUrd and Thy in 10 mM
citrate-phosphate buffer for 5 min, 40 °C. The ionic strength (I) at each pH was adjusted to
1
50 mM by addition of NaCl.
Figure 3. Stability in presence of 20% DMF of free BpNDT (), immobilized BpNDT on
PEI-agarose (), immobilized BpNDT on PEI-agarose followed by cross-linking (1 h)
with 50% oxidized aldehyde-dextran (), and immobilized BpNDT on PEI-agarose
followed by cross-linking (1 h) with 70% oxidized aldehyde-dextran ().

Molecules 2014, 19
.3.1. Covalent Immobilization of BpNDT on Glyoxyl-Agarose
11236
2
Covalent attachment of BpNDT to glyoxyl-agarose was carried out at two different temperatures
25 °C or 4 °C) and pH 10. Likewise, 2'-deoxyuridine (dUrd) was optionally incorporated as a
(
protective additive during the immobilization process in order to avoid possible distortions of the
enzyme active site structure upon interaction with the activated carrier (see Experimental Section). In
alkaline conditions (pH 10), immobilization takes place between aldehyde groups of the support and
ε-amino groups of lysines, mostly located on the enzyme surface. The formed imino bonds are weak
Schiff bases, which are finally reduced into stable single C-N bonds by the addition of NaBH4.
As shown in Table 2, BpNDT could be bound to glyoxyl-agarose in all cases, achieving best results
when immobilization was conducted at 25 °C in absence of dUrd as additive (55.9% of immobilization
yield). However, most of the immobilized biocatalysts were void of activity, with the exception of the
derivative that was obtained at 4 °C, but its activity was negligible after NaBH reduction (2.6%
4
recovered activity). High immobilization yields, but analogous enzyme inactivation, were also
obtained after binding of uridine phosphorylase (EC 2.4.2.3) from Bacillus subtilis (BsPyNP), (0%
recovery) [15] and thymidine phosphorylase (EC 2.4.2.4) from E. coli (EcTP) (6% recovery) [29] on
the same support. Such drastic enzymatic loss may be ascribed to the distortion of the protein structure,
or even subunit dissociation of these enzymes, caused by their multipoint attachment to highly
activated glyoxyl-agarose. In the case of tetrameric purine nucleoside phosphorylase (EC 2.4.2.1) from
Bacillus subtilis (BsPNP), inosine was added in order to stabilize this multimeric enzyme during
covalent attachment to glyoxyl-agarose, but enzyme binding was not achieved [15].
Table 2. Immobilization of BpNDT on glyoxyl (covalent) and PEI (ionic adsorption)
activated carriers.
Immobilization
Conditions
25 °C
Immobilized
Offered
Final
Recovered
Carrier
a
b
b
c
Enzyme (%)
Activity (IU)
Activity
Activity (%)
Glyoxyl-agarose
Glyoxyl-agarose
Glyoxyl-agarose
Glyoxyl-agarose
PEI-agarose
55.9
44.7
33.8
27.0
100
8.6
8.6
0
0.22
0
0
2.6
0
4 °C
d
25 °C + dUrd
8.6
d
4 °C + dUrd
8.6
0
0
25 °C
11.3
11.3
100
PEI: polyethylenimine (MM 600 Da); Covalent immobilization on glyoxyl-agarose was performed at pH 10,
a
while adsorption on PEI-carriers was performed at pH 7.5; Relative amount of bound enzyme to the support
b
with respect to the initial amount of enzyme prior to the immobilization process; IU/g wet carrier;
c
(
Activity of immobilized NDT/loaded activity) × 100; (Activity assay: synthesis of 2'-deoxyadenosine from
d
2
'-deoxyuridine and adenine; see Experimental Section); Addition of 2'-deoxyuridine as a protective
additive during immobilization.
2
.3.2. Immobilization of BpNDT on PEI-functionalized Supports
A different immobilization approach was chosen in order to keep the BpNDT quaternary structure
unaltered, in attempt to maintain the correct assembly of the oligomeric form, and thus preserving the
enzyme activity as much as possible. In this sense, ion exchange of enzymes on PEI-coated supports
was chosen as a simple non-distorting immobilization method that allows the full coverage of

Molecules 2014, 19
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multimeric enzymes by the flexible branches of polyethylenimine. In fact, PEI is a polycationic
polymer whose high density of ionized amino groups allows a strong ionic exchange with anionic
groups (carboxylic groups of both aspartate and glutamate residues) of enzyme surface located in
different subunits [30]. In our case, hydrophilic agarose functionalized with branched PEI (MM 600 Da)
were used for BpNDT immobilization at 25 °C and pH 7.5, taking into account that pI of the enzyme is
4
.6. As shown in Table 2, a complete enzyme adsorption was quickly achieved (after 1 h), and enzyme
activity was barely affected by the immobilization process. Our results indicate that immobilization of
BpNDT on PEI-coated agarose was efficiently performed without negatively affecting the oligomeric
structure of the enzyme and, therefore, preserving its catalytic activity. Similar immobilization yields
were obtained with trimeric BsUP [15], dimeric EcTP [29], and hexameric purine nucleoside
phosphorylase from Aeromonas hydrophila (AhPNPII) [17] immobilized on PEI-coated carriers,
activity recovery of all these biocatalysts was higher than 85%.
Nevertheless, immobilized biocatalysts obtained by ionic exchange of enzymes to charged supports
could be characterized by a non-optimal stability and may frequently suffer from protein leakage from
the carrier, causing loss of activity of the enzyme derivative and product contamination. The last fact is
actually an important drawback related to the industrial application of immobilized enzymes in the
production of nucleoside analogs [31,32]. In this sense, post-immobilization techniques, such as
aldehyde-dextran cross-linking of adsorbed enzymes to charged supports, have been addressed as an
efficient approach to overcome such problems [33]. In our case, immobilized BpNDT on PEI-coated
supports was further cross-linked with aldehyde-dextran in order to prevent enzyme desorption. This
approach has been successfully used in the case of BsPyNP [15] and EcTP [29], allowing the
preparation of stable biocatalysts active in enzymatic transglycosylations [34]. Aldehyde-dextran is a
polyaldehyde macromolecule, which can react with both free amino groups from the enzyme and the
PEI-coated support, affording a covalent multipoint cross-linking between the protein subunits and the
carrier. Again, the formed covalent imino bonds are weak Schiff bases, which can be reduced with
NaBH to yield single bonds [33,34]. As shown in Table 3, the influence of the oxidation degree (20%,
4
5
0% or 70%) of aldehyde-dextran (20 kDa) on the activity of the immobilized BpNDT has been
evaluated. Likewise, the cross-linking time of the post-immobilization process were also studied.
Firstly, the overall post-immobilization process generally decreased the activity of all immobilized
biocatalysts, compared to the enzyme solely adsorbed to PEI-coated supports. This effect was higher
when the cross-linking time and the oxidation degree of the dextran were increased. In such
conditions, the activity of these immobilized biocatalysts was low (≤0.5 IU and less than 6%
recovery), as a consequence of the high number of multiple bonds between the oxidized dextran and
the immobilized enzyme. In fact, a higher degree of oxidation is correlated with a higher density of
aldehyde groups, which are more likely to react at a longer cross-linking time, and the result would
presumably be a distorting effect on enzyme conformation. As expected, activity of immobilized
enzyme on PEI-agarose was increased at a shorter cross-linking time (1 h) employing aldehyde-dextran
with the lowest oxidation degree (20%).

Molecules 2014, 19
11238
Table 3. Immobilization of BpNDT on PEI-functionalized agarose and cross-linking with
aldehyde-dextran (MM 20 kDa).
Offered Activity Per
Dextran
Cross-Linking Time with
Final
Activity
2.3
Recovered
Entry
a
b
Gram of Support (IU)
Oxidation (%) Dextran (Protective Additive)
Actsivity (%)
1
2
3
4
5
6
7
6.1
6.1
6.1
6.1
8.7
8.7
8.7
20
50
50
50
70
70
70
1 h
1 h
2 h
4 h
1 h
2 h
4 h
37.7
36.1
32.8
4.9
2.2
2.0
0.3
1.8
20.7
5.7
0.5
0.3
3.4
a
IU/g wet carrier; activity assay: synthesis of 2'-deoxyadenosine from 2'-deoxyuridine and adenine (see Experimental
b
Section); (Activity of immobilized NDT/loaded activity) × 100.
2
.3.3. Stability of Immobilized BpNDT
The optimization of an enzyme-catalyzed synthesis of nucleoside analogs usually includes the
evaluation of enzyme stability in non-physiological condition. In particular, the presence of
water-miscible organic co-solvents has been evaluated, since solubility of halogenated bases and their
2
'-deoxyribonucleosides counterparts can be improved in such conditions [35]. Therefore, stability of
free and immobilized BpNDT was studied in the presence of dimethylformamide (DMF), which is one
of the most commonly used solvents for nucleoside synthesis. As depicted in Figure 3, residual activity
of the free enzyme was maintained for at least 48 h in presence of 20% DMF, whereas the derivative
obtained by adsorption on PEI coated carrier was completely unstable, losing almost all the activity
after only 2 h. This detrimental effect, which may be related to a change in enzyme microenvironment
exerted by the PEI, is progressively attenuated by increasing the oxidation degree of the aldehyde-dextran
used in the post immobilization cross-linking process. In particular, the best stability was obtained
using dextrane oxidized at 70% as probable consequence of a stabilization of the 3D structure of the
enzyme. This immobilized biocatalyst, hereafter abbreviated as PA9BpNDT, was selected for further
characterization and application in the synthesis of trifluridine.
2
.3.4. Effect of Temperature on the Activity of Immobilized BpNDT
Enzymatic activity of the best immobilized BpNDT biocatalyst (PA9BpNDT) was increased from
20 to 60 °C at pH 8.0, and then decreased very quickly as a result of the protein denaturation (Figure 4).
Optimal temperature for the free soluble enzyme in the same conditions is 10 °C lower than its
immobilized counterpart. This result could be another consequence of an enzyme rigidification
induced by the covalent crosslinking performed with oxidized-dextran. Comparing with other
immobilized NDTs, highest activity of NDT from L. reuteri (LrNDT) covalently immobilized on
epoxy-activated Sepabeads was displayed at 40 °C and pH 6.5 [36], whereas the same enzyme
covalently attached to magnetic chitosan beads showed best performance at 60 °C and pH 6.5 [37]. In
addition, these results indicate that soluble BpNDT preserves 45% of its maximal activity at 20 °C
whereas mesophilic LrNDT only maintains 20% at the same temperature [9], confirming that BpNDT
is a more suitable enzyme for synthesizing nucleosides at low temperatures than that from a

Molecules 2014, 19
11239
mesophilic microorganism. The experimental results of the effect of temperature were also used to
estimate the activation energies (see Experimental Section). In this sense, although optimal
temperature for the immobilized enzyme was 10 °C higher than that of free BpNDT, the energy of
activation of the immobilized BpNDT (E = 23.2 kJ/mol) and free enzyme (E = 23.6 kJ/mol) were
a
a
almost identical. This result indicates that the immobilization process does not affect enzyme catalysis.
Figure 4. Effect of temperature on enzyme activity of free () and immobilized ()
BpNDT (PA9BpNDT biocatalyst). Enzyme activity was assayed in 50 mM HEPES buffer
pH 8, as described in the Experimental Section.
2
.3.5. Recycling of Immobilized BpNDT
Unlike free enzymes, recycling of their immobilized counterparts offers technical and economical
advantages in industrial bioreactors. Therefore, the best immobilized BpNDT biocatalyst
PA9BpNDT) was evaluated by measuring its activity in consecutive standard assay cycles. Our
immobilized biocatalyst could be recycled for at least 30 consecutive cycles in the synthesis of
'-deoxyadenosine from 2'-deoxyuridine and adenine at 37 °C and pH 8.0, with only 25% loss of
activity (results not shown).
(
2
2
.4. Synthesis of Trifluridine Catalyzed by Immobilized BpNDT
-Trifluorothymidine (5-tFThd, trifluridine) is a nucleoside analog primarily used in ophthalmic
5
solutions for topical treatment of epithelial keratitis caused by herpes simplex virus (HSV) [38]. This
compound also exhibits potent antitumor activity since it induces double-stranded DNA breaks and
inhibits thymidylate synthase [39]. As a matter of fact, administration of the oral agent TAS-102
consisting of trifluridine in combination with a potent inhibitor of thymidine phosphorylase (which is
the enzyme that degrades trifluridine), is currently undergoing a Phase III clinical trial with patients
with refractory metastatic colorectal cancer (RECOURSE; NCT01607957) [40].
As depicted in Figure 5, synthesis of trifluridine from 2'-deoxyuridine (dUrd) and trifluorothymine
(5-tFThy) was carried out by using the best immobilized BpNDT biocatalyst (PA9BpNDT) in 10 mM

Molecules 2014, 19
11240
potassium phosphate buffer pH 7.5 at 37 °C. We analyzed the effect of substrate molar ratio, as well as
substrate concentration, on conversion at 2 h reaction time and productivity (Table 4).
Figure 5. Synthesis of 5-trifluorothymidine (trifluridine) from 2'-deoxyuridine and
5
-trifluorothymine by immobilized nucleoside 2'-deoxyribosyltransferase from Bacillus
psychrosaccharolyticus (BpNDT).
O
O
N
F3C
NH
NH
O
O
N
O
O
F3C
HO
HO
NH
O
NH
NH
O
Immobilized
+
+
BpNDT
NH
O
O
OH
OH
As observed, best conversion to trifluridine (64.4%) was achieved when 20 mM of dUrd and 10 mM
-tFThy (2:1 ratio) were employed in the transglycosylation reaction. Although conversion was
5
slightly decreased, productivity was higher (5.56 mM/h) when molar ratio was 1:1 at 20 mM substrate
concentration compared to 2:1 molar ratio (2.92 mM/h). Similar conversions have been recently
reported for the enzymatic synthesis of trifluridine catalyzed by immobilized pyrimidine nucleoside
phosphorylase from Bacillus subtilis (57%) or thymidine phosphorylase from Escherichia coli (51%)
requiring considerably longer reaction times [35], or by immobilized NDT from L. reuteri (LrNDT) on
magnetic chitosan beads (40%) at the same reaction time [37]. It is worth mentioning that immobilized
BpNDT showed a specific activity of 11.3 IU/g in trifluridine synthesis at 37 °C and pH 7.5 when
molar ratio was 1:1 at 1 mM concentration of both sugar donor (dUrd) and acceptor (5tFThy). Such
specific activity is approximately 140-fold higher than the one displayed by immobilized LrNDT on
Sepabeads in the same conditions but at 40 °C and pH 6.5 [36].
Table 4. Effect of substrate molar ratio and concentration on the enzymatic synthesis of
trifluridine (5-tFThd) catalyzed by immobilized BpNDT.
dUrd (mM)
5-tFThy (mM)
Conversion (%)
5-tFThd (mM)
0.98
Productivity (mM/h)
2
2
49.2
64.4
58.3
55.6
0.49
3.22
2.92
5.56
2
1
2
0
0
0
10
10
20
6.44
5.83
11.12
Experimental conditions: reactions were carried out by incubating the immobilized biocatalyst with 5 mL of
dUrd and 5-tFThy at different concentrations in 10 mM potassium phosphate buffer pH 7.5 at 37 °C for 2 h.
3. Experimental Section
3.1. Chemicals
2
'-Deoxyuridine (dUrd) was a gift from Pro.Bio.Sint (Varese, Italy) whereas adenine (Ade) and
2
'-deoxycytidine (dCyd) were purchased from Sigma. 5-trifluorothymine (5-tFThy) and

Molecules 2014, 19
11241
5
-trifluorothymidine (5-tFdThd) was from Carbosynth Ltd. (Berkshire, UK). Cross-linked 6% agarose
beads (Sepharose 6BCL) were from Amersham Biosciences AB (Uppsala, Sweden). Epoxy-activated

Sepabeads (EC-EP) was kindly donated by Resindion s.r.l. (Binasco, Milano, Italy). Branched
polyethylenimine (PEI) with 600 Da molecular mass, and dextran with 20 kDa molecular mass was
from Sigma-Aldrich (Milano, Italy). All other reagents and solvents (HPLC grade) were purchased
from Sigma-Aldrich (Milano, Italy).
3.2. Cloning and Expression of the ndt Gene Encoding Nucleoside 2'-Deoxyribosyltransferase from
Bacillus psychrosaccharolyticus CECT 4074
The ndt gene encoding nucleoside 2'-deoxyribosyltransferase from B. psychrosaccharolyticus was
amplified by PCR using chromosomal DNA from B. psychrosaccharolyticus CECT 4074 as a
template, obtained as described by Kieser [41]. Two synthetic primers were designed according to the
sequence of B. psychrosaccharolyticus genome [13], 5'-CCCATGGCGAAAATTTACCTAGCTTCA
CCAT-3' and 5'-GGAATTCTTATTTAACTGCTTTTAAGTACGGTTTAATAGG-3', containing the
restriction sites of NcoI and EcoRI (underlined). DNA amplification was performed under standard
conditions in a Mastercycler thermocycler (Eppendorf) using Pfu DNA polymerase. The amplified
0
.43-kb product was inserted into a pET28a(+) vector, purified with the High Pure Plasmid Isolation
Kit (Roche) and sequenced to confirm the absence of mutations by SECUGEN S.L. (Spain) according
to dideoxy chain termination method [42] with an automated sequencer, 3730 DNA Analyzer (Applied
Biosystems). This recombinant plasmid was used to transform competent E. coli BL21 (DE3) cells.
3
.3. Overproduction and Purification of Recombinant BpNDT
E. coli BL21 (DE3) cells harboring pET28Bpndt were grown on LB medium at 37 °C with
kanamycin 50 μg/mL. When the culture reached 0.6 OD600 nm, it was induced with 0.5 mM IPTG for
.5 h. After that, cells were harvested by centrifugation at 3500× g for 10 min, resuspended in 10 mM
2
potassium phosphate buffer, pH 7.0 (buffer A) and disrupted by sonication at 4 °C by a Branson digital
sonifier. The cell extract was applied onto a 5 mL Bio-Scale Mini UNOsphere Q Cartridge (BioRad)
equilibrated with buffer A at a flow rate of 0.5 mL/min using a BioLogic LP chromatographic system
(
Bio-Rad). The retained proteins were eluted with a linear gradient from 0 to 1.0 M NaCl in the same
buffer. The protein fractions containing BpNDT were pooled and concentrated with polyethylene
glycol 35,000 (Sigma) and loaded onto a 24 mL Superose 12 10/300 GL column (GE Healthcare)
equilibrated in 50 mM potassium phosphate, pH 7.0 (buffer B) at a flow rate of 0.5 mL/min using a
FPLC system (LKB-Pharmacia). The BpNDT enzyme fractions were pooled and dialyzed against
0
.025 M imidazol, pH 7.4 (buffer C) and loaded onto a 4 mL Mono P 5/200 GL column (GE
Healthcare) equilibrated with buffer C at a flow rate of 1 mL/min. Proteins were eluted with polybuffer 74,
pH 4.0 (buffer D) using a BioLogic LP chromatographic system (Bio-Rad). The protein fractions
containing BpNDT were detected by SDS-PAGE analysis. Electrophoresis was carried out on 15%
polyacrylamide slab gel with 25 mM Tris-HCl buffer, pH 8.6, 0.1% SDS [43]. Protein concentration
was determined by the Bradford method [44].

Molecules 2014, 19
.4. N-Deoxyribosyltransferase Assay for Soluble BpNDT
The standard activity assay was performed by incubating 5 μL of cell extract or 0.40 μg of pure
11242
3
enzyme with 10 mM deoxyuridine and 10 mM adenine in 50 mM HEPES buffer, pH 8.0 in a final
volume of 40 μL. The reaction mixture was incubating at 40 °C, 30 r.p.m., for 5 min. Then, activity
was stopped by addition of 40 μL of cold methanol in ice-bath and heating for 5 min at 95 °C as
described for others NDTs [9]. After centrifugation at 9000 ×g for 2 min, samples were half-diluted
with water and the nucleoside production was measured at 254 nm by HPLC (Agilent 1100 series)
with an ACE 5 μm C18-PFP column 250 mm × 46 mm (Advanced Chromatography Technologies)
equilibrated with 100% trimethyl ammonium acetate at a flow rate of 1 mL/min. Elution was carried
out by a discontinuous gradient: 0–10 min, 100% to 90% trimethyl ammonium acetate and 0% to 10%
acetonitrile, and 10–20 min, 90% to 100% trimethyl ammonium acetate and 10% to 0% acetonitrile.
Enzymatic synthesis of natural nucleosides was carried out at standard conditions described above
using 10 mM of different 2'-deoxyribonucleosides and bases. Retention times for the reference natural
compounds were as follows: uracil (Ura): 5.41 min; 2'-deoxyuridine (dUrd): 9.16 min; adenine (Ade):
1
0.14 min; 2'-deoxyadenosine (dAdo): 15.50 min; hypoxanthine (Hyp): 7.34 min; 2'-deoxyinosine
(
dIno): 10.95 min; thymine (Thy): 9.13 min; thymidine (Thd): 13.25; uric acid (UAc): 3.50 min. All
determinations were carried out by triplicate and the maximum error was below 5%. In such
conditions, one international activity unit (IU) was defined as the amount of enzyme producing
1
μmol/min of 2'-deoxyadenosine under the assay conditions.
3
.5. Effect of Temperature on Soluble and Immobilized Recombinant BpNDT
The effect of temperature on the activity of free and immobilized recombinant BpNDT was studied
at different temperatures ranging from 20 to 80 °C in 50 mM HEPES buffer pH 8. The enzymatic
activity was determined using the standard assay conditions described above but using the appropriate
temperature. The experimental results of the effect of temperature on enzyme activity were used to
estimate the activation energy by means of the equation:

E  1
a
ln(v) = −
 ⋅ + ln(A)
(
1)
R
T


where v is the specific activity of immobilized enzyme, E is the activation energy, T is the absolute
a
−1
−1
temperature in Kelvin degrees (K), R is the ideal gas constant (8.314 J·K ·mol ) and A is the
exponential factor. Activation energies were obtained from the section of negative slope of the plot of
ln(v) versus 1/T.
Thermal stability of free enzyme was determined by incubating pure BpNDT (0.08 mg/mL) at
different temperatures (from 10 to 90 °C) for 15 min. 5 μL of the enzyme were incubated on ice and
the synthesis of 2'-deoxyadenosine was measured at 40 °C as described above.
3.6. Effect of pH and Ionic Strength on Recombinant BpNDT Activity
Optimal pH of free enzyme was determined by measuring the activity of 0.4 μg of BpNDT in
10 mM potassium citrate-phosphate buffer at different pH values (from 4.0 to 9.0) using 10 mM

Molecules 2014, 19
11243
thymine and 10 mM 2'-deoxyuridine for the synthesis of thymidine under standard conditions. The
stability at different pH values was determined by incubating pure enzyme at different pH values
ranging from 4.0 to 12.0 for 15 min at 4 °C in 10 mM potassium citrate-phosphate buffer at a constant
ionic strength (I) of 150 mM. After that, enzyme samples were adjusted to pH 8 by addition of 50 mM
HEPES buffer pH 8 and BpNDT activity was measured at 40 °C, as described above. The ionic strength (I)
at each pH was adjusted to 150 mM by addition of NaCl in amounts calculated using a Visual Basic
program developed in our laboratory, which allows analysis of buffer systems with up to four
tetraprotic species. In addition, effect of ionic strength on NDT activity was studied by incubating
0.40 μg of enzyme with different concentrations of NaCl (0 to 1.5 M) in 50 mM HEPES buffer, pH 8.0
at 40 °C under standard conditions described for enzymatic assay.
3.7. Preparation of Polyethyleneimine-Functionalized Supports and Aldehyde-Dextran
Functionalization of epoxy-activated Sepabeads with PEI was performed as previously reported [15],
whereas functionalization of glyoxyl-agarose [45] with PEI was carried out as previously described [18].
On other hand, aldehyde-dextran was prepared as follows: 1.67 g of dextran (MM 20 kDa) was
4
suspended in 50 mL of distilled water and different amounts of NaIO were added to the suspension
depending on the desired percentage of oxidation [46]. The solution was stirred at 25 °C for 2 h and
finally dialyzed for 20 h.
3.8. Covalent Immobilization of BpNDT on Glyoxyl-Agarose
Five hundred milligrams of glyoxyl-agarose was added to 7.1 mL of 100 mM potassium carbonate
buffer (pH 10) solution containing 0.25 mg of BpNDT (4.7 IU) and incubated at room temperature or
°C for 3 h under mechanical stirring. In order to protect the active site, 2'-deoxyuridine (5 mM) was
optionally added during the immobilization process. Once the immobilization time was finished, Schiff
base-double bonds were reduced by NaBH (7.1 mg) for 30 min. Finally, immobilized BpNDT was
4
4
washed twice with 50 mM potassium phosphate buffer, pH 4.5, and deionized water [45].
Immobilization yield was calculated as the relative amount of bound enzyme to the support with
respect to the initial amount of enzyme prior to the immobilization process, and it was calculated as the
difference between offered protein and residual protein found in the filtrate and washing solutions
obtained after the immobilization process. The amount of protein in these enzyme solutions was
routinely determined by the Coomassie Blue method [44].
3.9. BpNDT Immobilization on PEI-Coated Sepabeads and PEI-Coated Agarose
BpNDT immobilization was carried out by ionic adsorption of the enzyme onto PEI-functionalized
supports. 500 mg of PEI-functionalized support were added to 7.1 mL of 5 mM potassium phosphate
buffer (pH 7.5) solution containing 0.25 mg of BpNDT (4.7 IU), and agitated by mechanical stirring
for 1 h at room temperature and stored at 4 °C prior to use. Immobilization yield was calculated as the
difference between offered activity and residual activity found in the filtrate and washing solutions
obtained after the immobilization process.

Molecules 2014, 19
.10. Cross-Linking of Immobilized BpNDT with Aldehyde-Dextran
11244
3
An amount of 0.71 mL of aldehyde-dextran (10% v/v) was added to the immobilization suspension
described above. After stirring for 1 h, pH was adjusted to 10 and NaBH (1 mg/mL suspension) was
4
added. After 30 min, immobilized BpNDT was washed twice with 50 mM potassium phosphate buffer
pH 4.5 and deionized water. Finally, immobilized biocatalyst was stored at 4 °C prior to use.
3.11. N-Deoxyribosiltransferase Assay for Immobilized BpNDT
Enzymatic activity of immobilized enzyme was measured using 25 mg of immobilized biocatalyst
which was added to a 1.0 mL solution containing 10 mM 2'-deoxyuridine and 10 mM adenine in
0 mM HEPES buffer pH 8.0. Reaction mixture was incubated at 37 °C for 20 min at orbital shaking.
5
After this reaction time, 300 μL aliquot of the supernatant was withdrawn using a pipette filter device.
Then, the supernatant was analyzed by HPLC to quantitatively measure the production of nucleosides
employing a Merck-Hitachi L-7100 system equipped with a UV detector L-7400 and a column oven

L-7300. Reactions were analyzed with a LiChroCART RP18 (5 μm) 250 × 4.6 mm (Merck), using as
mobile phase 10 mM potassium phosphate, methanol, water (90:9:1). The flow rate was fixed at
1
mL/min, and the UV detector was set at 260 nm. Retention times for the reference natural
compounds were as follows: uracil (Ura): 3.5 min; 2'-deoxyuridine (dUrd): 5.5 min; adenine (Ade):
.5 min; 2'-deoxyadenosine (dAdo): 16.5 min. All determinations were carried out by triplicate and the
6
maximum error was below 5%. In such conditions, one international activity unit (IU) was defined as
the amount of enzyme producing 1 μmol/min of 2'-deoxyadenosine under the assay conditions. In
order to compare with its soluble counterpart, the free enzyme was assayed in the same standard
conditions described above for the immobilized enzyme.
3.12. Stability of BpNDT in Organic Cosolvent Mixtures
Three hundred mg of immobilized BpNDT were incubated with 2 mL of 50 mM HEPES pH 8.0
containing 20% DMF for 48 h at 37 °C; 200 µL of suspension were withdrawn at different incubation
times, and activity was measured by the standard assay. For the soluble enzyme, 160 µg of BpNDT
were added to 2 mL of 50 mM HEPES pH 8.0 containing 20% DMF and incubated for 48 h at 37 °C.
At different incubation times, 0.40 μg of enzyme were withdrawn and activity was measured by the
standard assay.
3.13. Recycling of Immobilized BpNDT
Immobilized enzyme (200 mg) was evaluated for nucleoside 2'-deoxyribosyltransferase activity
after repeated performance of the same biocatalyst in the synthesis of 2'-deoxyadenosine from
2'-deoxyuridine and adenine. Immobilized BpNDT was added to a 8 mL solution containing 10 mM
2'-deoxyuridine and 10 mM adenine in 50 mM HEPES buffer pH 8.0. After enzymatic reaction for
5 min at 37 °C and 250 rpm shaking, reaction mixture was filtered and an aliquot of the supernatant
was withdrawn and half-diluted with water to be analyzed by HPLC to quantitatively measure the
reaction products as described below in the analytical methods. Then, the recovered immobilized

Molecules 2014, 19
11245
enzyme was washed three times with freshly prepared 50 mM HEPES buffer pH 8.0, and then used for
another conversion cycle.
3.14. Enzymatic Synthesis of Trifluridine Catalyzed by Immobilized BpNDT
Trifluorothymidine (trifluridine) synthesis was performed using different concentrations of
trifluorothymine and 2'-deoxyuridine (from 1 to 20 mM), which were dissolved in 5 mL of 10 mM
potassium phosphate buffer pH 7.5. The enzymatic assay was started by adding 20 mg of immobilized
biocatalyst when substrate concentration was 2 mM for both donor and acceptor, whereas 100 mg
(displaying 1.8 IU with the standard assay) of immobilized enzyme was used at higher substrate
concentrations. Reaction was performed at 37 °C for 2 h. At different reaction times, samples were
withdrawn and filtered off using a pipette filter device. Then, the supernatant was analyzed by HPLC
to quantitatively measure the reaction products employing a Merck-Hitachi L-7100 system equipped
with a UV detector L-7400, and a column oven L-7300. Reactions were analyzed with a

LiChroCART RP18 (5μm) 250 mm × 4.6 mm (Merck), using as mobile phase 10 mM potassium
phosphate, methanol, water (80:18:2). The flow rate was fixed at 1 mL/min, and the UV detector was
set at 260 nm. Retention times were: uracil (Ura): 3.1 min; 2'-deoxyuridine (dUrd): 4.2 min;
5-trifluorothymine (5-tFThy): 8.0 min; 5-trifluorothymidine (5-tFThd): 13.5 min. The produced
nucleoside was identified by comparison of its HPLC Rt with that of authentic samples [36]
.
4
. Conclusions
Here, we report, for the first time, the cloning, expression, purification and immobilization of the
nucleoside 2'-deoxyribosyltransferase Bacillus psychrosaccharolyticus (BpNDT). Best immobilized
biocatalyst was obtained by adsorbing the enzyme on PEI-functionalized agarose and subsequent
cross-linking with aldehyde-dextran (20 kDa and 70% oxidation degree). This novel biocatalyst could
be recycled for at least 30 consecutive cycles in the synthesis of 2'-deoxyadenosine from
2
'-deoxyuridine and adenine at 37 °C and pH 8.0, with a 25% loss of activity. Furthermore, the same
immobilized enzyme gave high conversion yields of trifluridine at 2 h (64.4%), starting from
'-deoxyuridine as sugar donor, and 5-trifluorothymine as base acceptor. A further detailed study on
2
scale up would be necessary to check the applicability of this novel immobilized biocatalyst in the
industrial production of these high-valuable drugs.
Acknowledgments
This work was supported by Grant S2009/PPQ-1752 from Comunidad Autónoma de Madrid
(Spain) and CTQ2009-11543 from the Spanish Ministry of Science and Innovation are also acknowledged.
Author Contributions
Conceived and designed the experiments: A.F-T., I.S., J.F-L, M.T., I.M. Performed the
experiments: A.F-T. Analyzed the data: A.F-T., I.S., J.F-L., C.A., M.A, M.T., I.M. Wrote the paper:
A.F-T., C.A., M.A., M.T., I.M.

Molecules 2014, 19
11246
Conflicts of Interest
The authors declare no conflict of interest.
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