Development of an immobilized biocatalyst based on Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine

Article abstract of DOI:10.1016/j.cattod.2015.06.032

The immobilization of Bacillus psychrosaccharolyticus nucleoside 2′-deoxyribosyltransferase was deeply investigated and finally optimized. The best immobilization procedure resulted to be ionic adsorption on PEI 600 Da agarose followed by crosslinking with 70% oxidized dextran (20 kDa). The percentage of recovered activity was further improved (from 21% to 33%) by the addition of 20% glycerol to the immobilization mixture. The resulting biocatalyst showed a stability profile similar to that of the soluble enzyme and it was used for the preparative synthesis of trifluridine and decytabine obtaining conversions ranging from 50% to 76%.

Full text of DOI:10.1016/j.cattod.2015.06.032

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Contents lists available at ScienceDirect
Catalysis Today
journal homepage: www.elsevier.com/locate/cattod
Development of an immobilized biocatalyst based on Bacillus
psychrosaccharolyticus NDT for the preparative synthesis of
trifluridine and decytabine
Alba Fresco-Taboadaa, Immacolata Serrab,1, Miguel Arroyoa, Jesús Fernández-Lucasa,2
,
Isabel de la Mataa, Marco Terrenib,∗
a Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense University of Madrid, C/José Antonio Novais 2, 28040 Madrid, Spain
b Department of Drug Sciences and Italian Biocatalysis Center, Università degli Studi di Pavia, via Taramelli 12, 27100 Pavia, Italy
a r t i c l e i n f o
a b s t r a c t
The immobilization of Bacillus psychrosaccharolyticus nucleoside 2ꢀ-deoxyribosyltransferase was deeply
investigated and finally optimized. The best immobilization procedure resulted to be ionic adsorption
on PEI 600 Da agarose followed by crosslinking with 70% oxidized dextran (20 kDa). The percentage
of recovered activity was further improved (from 21% to 33%) by the addition of 20% glycerol to the
immobilization mixture. The resulting biocatalyst showed a stability profile similar to that of the soluble
enzyme and it was used for the preparative synthesis of trifluridine and decytabine obtaining conversions
ranging from 50% to 76%.
Article history:
Received 13 March 2015
Received in revised form 11 June 2015
Accepted 19 June 2015
Available online xxx
Keywords:
Nucleoside 2ꢀ-deoxyribosyltransferase
Biotransformations
Enzyme immobilization
Nucleoside analogues
Decytabine
© 2015 Elsevier B.V. All rights reserved.
Trifluridine
1. Introduction
reactions [5,8–10]. However, the poor ability of NPs to recog-
nize cytosine derivatives as substrates [11] and, often, the need
The use of glycosyl-transferring enzymes applied to the synthe-
sis of modified nucleosides is an established procedure endowed
with several advantages over the traditional multistep chemical
methods [1–3]. These advantages arise from the inherent proper-
ties of enzymes such as stereo-, regio-, enantiospecificity and mild
reaction conditions.
to follow a two-enzyme one-pot scheme (PyNP-PNP or UP-PNP)
to synthesize purine nucleosides, represent the main obstacle to
their broad application. In particular, this last issue is a conse-
quence of the fact that the thermodynamic equilibrium for PNP,
but not for PyNP/UP is shifted towards the nucleoside synthesis
[12].
To this aim, the use of nucleoside phosphorylases (NPs) has
been extensively described [4–7]. These enzymes are used to cat-
alyze, in presence of inorganic phosphate, the transfer of the sugar
moiety from a sugar donor nucleoside to a sugar acceptor base lead-
ing to the production of a second nucleoside through a two-step
reaction. Moreover, several NPs from different sources have been
also conveniently immobilized by following various approaches
and obtaining, in most of cases, robust biocatalysts for preparative
More recently, a second class of enzymes that mediate the
glycosyl transfer is gaining importance in the field of nucleoside
synthesis [13–15]. N-Deoxyribosyltransferases (NDTs), in fact, cat-
alyze the direct transfer of the 2ꢀ-deoxyribosyl moiety from a
2ꢀ-deoxynucleoside donor to a nucleobase acceptor through a one-
step reaction [16,17]. NDTs have been divided into two classes
depending on their substrate specificity. In particular, type I NDT
(also known as PDT) exclusively catalyzes the purine–purine trans-
fer, whereas type II NDT catalyzes the transfer between pyrimidines
and/or purines and also accepts cytosine derivatives as substrates
[12]. Taking into account the aforementioned properties, it clearly
appears that NDTs are an important completion to NPs in the
enzyme toolset aimed at the enzymatic synthesis of nucleoside ana-
logues. In fact, the use of type II NDTs allows not only to obtain
cytidine analogues but also to carry out the reaction in a one-
enzyme one-pot mode.
∗
Corresponding author.
E-mail addresses: marco.terreni@unipv.it, markt@ibiocat.eu (M. Terreni).
Present address: Department of Food, Environmental and Nutritional Sciences
1
- DeFENS, University of Milan, via L. Mangiagalli 25, I-20133 Milano, Italy.
2
Present address: Department of Pharmacy and Biotechnology, Biomedical Sci-
ences Faculty, European University of Madrid, 28670, Spain.
http://dx.doi.org/10.1016/j.cattod.2015.06.032
0920-5861/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: A. Fresco-Taboada, et al., Development of an immobilized biocatalyst based on
Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2015.06.032

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Immobilization often represents a key point to make a step for-
ward in the view of industrial application, since it provides the
biocatalyst with enhanced stability and recyclability. Differently
from NPs, immobilization of NDTs is still little studied and only
a few examples have been reported to date [15,18–20].
Both
trifluridine
and
decytabine
are
commercially
important drugs with antiviral and antitumoral activities. 5-
Trifluorothymidine, commercially known as trifluridine, is an
antiviral agent employed in ophthalmic solutions for the treat-
ment of herpes virus simple (HVS) [22]. This compound can also
induce double-strand DNA breaks and inhibit the thymidylate
synthase when it is transformed to its phosphorylated form,
displaying antitumoral activity [23]. The combination of this
compound with an inhibitor of the thymidine phosphorylase, to
inhibit degradation of trifluridine, is currently undergoing a Phase
III clinical trial with patients with refractory metastatic colorectal
cancer (NCT01607957) [24].
Recently,
a new NDT from Bacillus psychrosaccharolyticus
(BpNDT) has been cloned, produced and characterized [19]. BpNDT
can be considered a valuable enzyme since it holds several benefi-
cial properties, such as psychrophilic behavior coupled with good
thermal and pH stability.
In order to obtain an improved biocatalyst for preparative
purposes, a first attempt to immobilize this enzyme has been
also reported in the same study. Immobilization of BpNDT by
direct covalent reaction with aldehyde activated carriers caused
a complete loss of the enzyme activity. Results were slightly
improved using the approach already described for other multi-
meric enzymes: adsorption on a strong anionic exchange carrier
(agarose coated with PEI) followed by crosslinking with aldehyde-
dextran [8–10].
Additionally, 5-aza-2ꢀ-deoxycytidine, decytabine, has been
approved for use in the treatment of myelodysplastic syndromes. It
can be incorporated into DNA when it is converted to aza-dCTP and
produce the inhibition of DNA methylation, leading to enhanced
gene expression and the consequently activation of repressed genes
[25].
This strategy also allows overcoming an important drawback
associated to the immobilization of multimeric enzymes. In fact,
multimeric enzymes can undergo subunit dissociation if they are
not properly linked to the support, causing enzyme inactivation
and product contamination. Conversely, the promotion not only of
the attachment to the support but also inter-subunit bonds (with a
crosslinking agent such as dextran) allows the covalent linkage of
all the enzyme subunits onto the carrier [21].
2. Materials and methods
2.1. Chemicals
2ꢀ-Deoxyuridine (dUrd) was a gift from Pro.Bio.Sint-Euticals
(Varese, Italy) whereas adenine (Ade) was purchased from Sigma.
5-Trifluorothymine (5-tFThy), 5-trifluorothymidine (5-tFdThd), 5-
azacytosine (5-azaCyt) and 5-aza-2ꢀ-deoxycytidine (5-azaCyd)
were from Carbosynth Ltd. (Berkshire, UK). Crosslinked 6% agarose
beads (Sepharose 6BCL) were from Amersham Biosciences AB
(Uppsala, Sweden). Epoxy-activated Sepabeads® (EC-EP) were
kindly donated by Resindion s.r.l. (Binasco, Milano, Italy). Branched
polyethyleneimine (PEI) with 600 Da or 25,000 Da molecular mass,
dextran with 20,000 Da or 100,000 Da molecular mass and PEG
600 Da were from Sigma-Aldrich (Milano, Italy). All other reagents
and solvents (HPLC grade) were purchased from Sigma–Aldrich
(Milano, Italy).
Even in this case, BpNDT activity was almost lost during the
covalent crosslinking. On the other hand, when the crosslinking
degree was reduced, the enzyme preserved a good activity, in spite
of very poor stability, lower than that of the soluble enzyme [19].
The best derivative in terms of stability was achieved when the
enzyme was immobilized on 600 Da PEI-agarose and crosslinked
with dextran (20 kDa), oxidized at 70%. Nevertheless, the recovered
activity at the end of the immobilization process was too low even
when the immobilization time was reduced to one hour (about 20%
of recovered activity). This made the biocatalyst poorly exploitable
for preparative reactions. In fact, the synthesis of trifluridine was
carried out at substrate concentration not higher than 20 mM [19].
In the present work, in order to achieve an immobilized deriva-
tive with suitable activity and stability to be used in preparative
processes, immobilization of BpNDT was optimized. In particular,
the aim of this study was to modulate the process to ensure a
high crosslinking degree (that assures a suitable stability of the
enzyme preparation) but avoiding an important distortion of the
3D structure that reduces the recovered enzymatic activity after
immobilization.
2.2. Enzyme preparation
NDT from B. psychrosaccharolyticus was prepared as previ-
ously reported [19]. Briefly, the ndt gene encoding BpNDT was
amplified by PCR using chromosomal DNA from B. psychrosaccha-
rolyticus CECT 4074 as a template, cloned into the expression vector
pET28a(+), and the recombinant plasmid was used to transform
Escherichia coli BL21 (DE3) cells. After that, the overproduction of
BpNDT was carried out by the induction of the culture of E. coli BL21
(DE3) cells harboring pET28Bpndt (0.6 OD600nm) with 0.5 mM IPTG
for 2.5 h.
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
an isofocusing chromatography. The protein fractions containing
BpNDT were detected by SDS-PAGE analysis [19].
To this purpose, different factors that could influence the immo-
bilization outcome were taken into account. First, we evaluated
the effect of the reduction with NaBH4, comparing the derivative
obtained by crosslinking (before reduction of the imino bonds) with
that previously obtained with the complete procedure (crosslink-
ing and reduction).
Afterwards, we investigated the influence on the immobiliza-
tion process exerted by different variables including the nature
of the carrier, the composition of the reaction medium (i.e. addi-
tion of protective agents in order to reduce the denaturation of the
enzyme) and the size of PEI and dextran.
Finally, the best enzyme derivative (in terms of both activ-
ity and stability), obtained through the optimized immobilization
process, was used for the synthesis of 5-trifluorothymidine (trifluri-
dine). Substrate concentrations used were two-fold higher than
previously reported [19], in order to evaluate the performance of
the selected biocatalyst in similar conditions to those required
in preparative processes. In addition, the synthesis of 5-aza-2ꢀ-
deoxycytidine (decytabine) was also performed.
2.3. Preparation of polyethyleneimine-functionalized supports
and aldehyde-dextran
Functionalization of epoxy-activated Sepabeads was performed
as previously described [8]. Briefly, the support (1 g) was sus-
pended in 1 M NaCl solution pH 11 (12.6 mL) containing 10% (v/v)
of PEI (MW 600 Da or 25,000 Da). The suspension was kept under
mechanical stirring for 24 h at room temperature and then the sup-
port was filtered and washed with 1 M NaCl and deionized water.
PEI-functionalized agarose was obtained as formerly described
[21]. Briefly, aldehyde-agarose (1 g), was suspended in 1 M NaCl
solution pH 11 (12.6 mL) containing 10% (v/v) of PEI (MW 600). The
Please cite this article in press as: A. Fresco-Taboada, et al., Development of an immobilized biocatalyst based on
Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine, Catal. Today (2015),
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reaction mixture was kept under mechanical stirring for 3 h at room
temperature. NaBH4 (57 mg) was then added and the reduction
reaction was carried out for 2 h at room temperature. The activated
support was washed with phosphate buffer pH 4 and deionized
water.
Likewise, aldehyde-dextran was prepared as previously
reported [9,26,27]. In particular, 1.67 g of dextran were suspended
in 50 mL of deionized water and 3.04 g, 2.18 g, 0.87 g of NaIO4 were
added to obtain 70%, 50% and 20% oxidation degree, respectively.
The reaction was carried out for 2 h at room temperature. The
mixture was then dialyzed overnight against deionized water and
stored at −20 ◦C.
suspension were withdrawn at different incubation times, and
activity was measured by the standard assay.
On the other hand, 160 ␮g of soluble BpNDT were added to 2 mL
of 50 mM carbonate pH 10.0 and incubated for 24 h at 25 ◦C. At
different incubation times, 0.40 ␮g of enzyme were withdrawn and
activity was measured by the standard assay.
2.8. Stability of BpNDT in 20% of dimethylformamide
Stability in presence of 20% of dimethylformamide was evalu-
ated incubating 300 mg of immobilized enzyme in 2 mL of 50 mM
HEPES pH 8.0 containing 20% DMF for 48 h at 37 ◦C. At different
incubation times, 200 ␮L of suspension were withdrawn and activ-
ity was measured by the standard assay.
2.4. BpNDT immobilization on PEI-coated Sepabeads and
PEI-coated agarose
2.9. Enzymatic synthesis of trifluridine and decytabine
In a total volume of 14 mL, the activated support (1 g) was sus-
pended in 5 mM phosphate buffer at pH 7.5 containing the desired
amount of enzyme and kept under mechanical stirring for 1 h. The
immobilized preparation was filtered and washed with deionized
water. The percentage of recovered activity was calculated as fol-
lowing: (recovered activity/loaded activity) × 100.
Nucleoside analogues synthesis was performed by adding
100 mg of immobilized biocatalyst (displaying 1.8 IU with the
standard assay) to 5 mL of 10 mM potassium phosphate buffer
pH 7.5 with different concentrations of trifluorothymine and
2ꢀ-deoxyuridine (from 10 to 40 mM), for 5-trifluorothymidine syn-
thesis (trifluridine), or 5-azacytosine and 2ꢀ-deoxyuridine (from
10 to 20 mM), for 5-aza-2ꢀ-deoxycytidine synthesis (decytabine).
Reactions were performed at 37 ◦C for 3 h and, at different reac-
tion times, samples were withdrawn and filtered off using a pipette
filter device. Afterwards, the supernatant was analyzed by HPLC
to quantitatively measure the reaction products as aforemen-
tioned (section 2.6. of Materials and methods). Retention times
were: uracil (Ura): 5.41 min; 2ꢀ-deoxyuridine (dUrd): 9.16 min;
5-trifluorothymine (5-tFThy): 8.0 min; 5-trifluorothymidine (5-
tFThd): 13.5 min; 5-azacytosine (5-azaCyt): 4.5 min; 5-aza-2ꢀ-
deoxycytidine (5-azadCyd): 8.9 min. The produced nucleoside was
identified by comparison of its HPLC retention time with that of
authentic samples [10].
2.5. Crosslinking of immobilized BpNDT with aldehyde-dextran
An amount of 0.71 mL of 20 kDa or 100 kDa aldehyde-dextran
(10%, v/v) was added to the aforementioned immobilization sus-
pension (section 2.3). After stirring (1–4 h), pH was adjusted to 10
and NaBH4 was added (1 mg/mL suspension). After 30 min of reduc-
tion, 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.
2.6. N-Deoxyribosyltransferase assay for soluble and immobilized
BpNDT
Standard activity assay of immobilized enzyme was carried out
as previously described [19]. The standard activity assay using sol-
uble BpNDT was started by the addition of 4 ␮g of pure enzyme to
a 400 ␮L solution containing 10 mM 2ꢀ-deoxyuridine and 10 mM
adenine in 50 mM HEPES buffer, pH 8.0. Reaction was performed as
formerly reported [19].
3. Results and discussion
3.1. Immobilization of recombinant BpNDT on PEI-functionalized
supports
In order to immobilize recombinant BpNDT maintaining
its enzymatic activity, ionic adsorption of the enzyme to
PEI-functionalized supports was carried out. In this sense, glyoxyl-
agarose [28] and Sepabeads [29] were functionalized by two
different PEI [30] (600 or 25,000 Da molecular weight). As shown
in Table 1, when 600 Da PEI was used to coat both types of carri-
ers, the recovered activity was not affected by the immobilization
process. The immobilization yield was not even influenced by the
quantity of enzyme loaded on the activated support (Table 1). When
PEI 25,000 Da was employed to functionalize aldehyde-agarose, the
percentage of recovered activity of immobilized BpNDT diminished
to 28%.
Functionalized supports with 600 Da PEI were then chosen
for further stabilization since immobilized BpNDT obtained by
mere ionic adsorption to functionalized supports can suffer pro-
tein leakage, causing loss of activity and product contamination
[31]. This drawback can be overcome by post-immobilization tech-
niques, such as crosslinking with a bifunctional or polyfunctional
polymer [32]. In this case, BpNDT immobilized on PEI-agarose
and PEI-Sepabeads were crosslinked with aldehyde-dextran, a
polyaldehyde macromolecule obtained by oxidation with NaIO4
[33]. Different oxidation degrees (meaning a different density of
aldehyde groups) can be obtained depending on the amount of
periodate used for oxidation [9,34].
In both cases samples were analyzed by HPLC (Agilent 1100
series) to quantitatively measure the production of nucleosides
at 254 nm with a 5 ␮m C18-PFP column 250 mm × 46 mm (ACE,
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: from 0 to 10 min, the
mobile phase changed progressively from 100% trimethyl ammo-
nium acetate and 0% of acetonitrile to 90% of trimethyl ammonium
acetate and 10% of acetonitrile; and from 10 to 20 min, the mobile
phase reverted to initial conditions, from 90% to 100% trimethyl
ammonium acetate and from 10% to 0% acetonitrile. Retention
times for the reference natural compounds were as follows: uracil
(Ura): 5.41 min; 2ꢀ-deoxyuridine (dUrd): 9.16 min; adenine (Ade):
10.14 min; 2ꢀ-deoxyadenosine (dAdo): 15.50 min. All determina-
tions were carried out by triplicate and the standard deviation was
below 5%. One international activity unit (IU) was defined as the
amount of enzyme producing 1 ␮mol/min of 2ꢀ-deoxyadenosine
under assay conditions.
2.7. Stability of BpNDT at pH 10
Three hundred mg of immobilized BpNDT were incubated with
2 mL of 50 mM carbonate pH 10.0 for 24 h at 25 ◦C. 200 ␮L of
Please cite this article in press as: A. Fresco-Taboada, et al., Development of an immobilized biocatalyst based on
Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine, Catal. Today (2015),
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Table 1
Immobilization of BpNDT on PEI-functionalized carriers.
Offered protein per
gram of carrier (mg)
Offered activity per
gram of carrier (IU)
Functionalized
carrier
Molecular weight
of PEI (Da)
Final activity
(UI/g wet carrier)
Recovered
activity (%)a
0.9
0.5
0.7
1.0
11.3 [19]
6.1
9.2
PEI agarose
PEI agarose
PEI agarose
PEI Sepabeads
600
600
25,000
600
11.3
6.1
2.6
100
100
28.3
98.0
12.0
11.8
a
(Activity of immobilized BpNDT/loaded activity) × 100.
The aldehyde-dextran can react with both free amino groups of
the enzyme and PEI-coated support, resulting in a covalent multi-
point attachment between the different protein subunits and the
carrier. The number of bonds obtained during crosslinking depends
on the dextran oxidation degree as well as on the reaction time.
However, the formed imino bonds are not stable, but can be finally
reduced with NaBH4 to yield carbon-nitrogen irreversible covalent
bonds [32,35].
The recovered activity of BpNDT immobilized by ionic adsorp-
tion on 600 Da PEI-agarose and further crosslinked with 20%
oxidized 20 kDa dextran was 38%, twice the recovered activity
when 600 Da PEI-Sepabeads was used under the same crosslinking
conditions (Table 2, entries 1 and 2). For this reason, 600 Da PEI-
agarose was used for further optimization of the immobilization
process. The effect of dextran oxidation degree and crosslinking
time exerted on the immobilization process was investigated using
20 kDa dextran.
As the dextran oxidation level is increased, the number of
aldehyde groups is incremented, therefore, an increased num-
ber of covalent bonds are produced during the crosslinking step.
This confers rigidity to the protein 3D structure and allows less
conformational changes resulting, thus, in high stability of the
immobilized and crosslinked enzyme. Nevertheless, an increased
rigidity of the enzyme structure could be also responsible for a
reduced activity after immobilization.
according to a time dependent increased number of covalent bonds
[19]. However, this effect was only detected after the NaBH4 reduc-
tion step, when the reversible imino bonds were transformed into
covalent bonds (Fig. 1a) and it was much more evident when 70%
oxidized dextran was used compared with 50% oxidized dextran
(Fig. 1a). In fact, although the activity was completely retained up
to 4 h of crosslinking, the derivative prepared with 50% oxidized
dextran maintained about 40% of activity when the reduction was
carried out after 2 h of crosslinking, whereas it lost almost com-
pletely its activity when the reduction was performed after 4 h of
incubation.
Conversely, in similar conditions, the derivative prepared with
70% oxidized dextran lost almost completely its activity even when
the reduction was performed after 2 h, as a consequence of the high
number of covalent bonds produced.
These results can be confirmed following the residual activity of
the suspension during the whole crosslinking process (Fig. 1b). In
fact, the activity is completely maintained during the interaction
with both the crosslinking agents (oxidized at 50% and 70%).
Similar results were obtained using 20% oxidized dextran, but
the final residual activity (after reduction) was higher compared
with the other derivatives (Fig. 1b).
The progressive loss of activity observed depending on the time
of incubation and on the dextran oxidation degree confirms a
distortion of the enzyme 3D structure, induced by the increased
number of covalent bonds.
In order to minimize the effect of 3D structure distortion, pro-
tective agents such as 2ꢀ-deoxyuridine, glycerol and polyethylene
glycol (PEG) were added to preserve the enzyme active site during
the crosslinking process.
The use of substrates or inhibitors [36], as well as polyols
[37], has been previously reported for preserving the 3D structure
of several enzymes during immobilization, including multimeric
nucleoside phosphorylases [5]. The interaction with these additives
allows the enzyme to maintain a compact conformation and, thus,
reduces the distortion of the 3D structure occurring during covalent
immobilization or crosslinking.
In the case of BpNDT, the addition of dUrd was not effective,
but the addition of 20% glycerol to the immobilization medium
increased the final activity (expressed as % of recovered activity
In order to better understand the effect produced on the enzyme
activity by the crosslinking process, two kinds of experiments were
performed (Fig. 1):
•
Preparation of different enzyme derivatives by adsorption on PEI
carrier and incubation of the immobilized enzyme with dextran
for diverse periods of time, avoiding the final reduction.
•
Preparation of different derivatives by crosslinking of the PEI
immobilized enzyme with dextran for diverse periods of time,
followed by 30 min reduction.
As depicted in Fig. 1a, the crosslinking time appeared to be a
crucial factor for determining the effect of such crosslinking on
the enzyme activity. In fact, a progressive reduction of the recov-
ered activity was observed when incubation time was increased,
Table 2
Immobilization of BpNDT on PEI-functionalized carriers and crosslinking with aldehyde-dextran (MM 20/100 kDa).
Entry
Offered protein per
gram of carrier (mg)
Offered activity
(IU/g carrier)
Functionalized
carrier
Dextran (kDa);
oxidation degree (%)
Final (IU/g support);
recovered activity (%)
1
1.0
0.5
0.5
0.7
0.7
0.7
0.7
0.7
0.5
0.5
2.9
12.0
6.1
6.1
8.7
8.7
8.7
8.7
8.7
6.1
6.1
34.8
PEI 600 Sepabeads
PEI 600 Agarose
PEI 600 Agarose
PEI 600 Agarose
PEI 600 Agarose
PEI 600 Agarose
PEI 600 Agarose
PEI 600 Agarose
PEI 600 Agarose
PEI 600 Agarose
PEI 600 Agarose
20; 20
20; 20
20; 50
20; 70
20; 70
20; 70
20; 70
100; 20
100; 50
100; 70
20; 70
2.2; 18.7
2.3; 37.7
2.2; 36.1
1.8; 20.7
1.1; 13.3
2.8; 32.8
2.6; 30.0
4.6; 53.1
0.2; 2.9
2 [19]
3 [19]
4 [19]
5a
6b
7c
8
9
10
0.25; 4.2
19.5; 56
11b
Crosslinking time: 1 h, followed by 30 min reduction; adUrd (5 mM); bglycerol (20%); cPEG 600 Da (20%).
Please cite this article in press as: A. Fresco-Taboada, et al., Development of an immobilized biocatalyst based on
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Fig. 1. Panel (a): Immobilized BpNDT on 600 Da PEI agarose followed by crosslinking with 50% oxidized 20 kDa dextran: ᭹ after crosslinking; ᭿ after crosslinking and 30 min
reduction. Immobilized BpNDT on 600 Da PEI agarose followed by crosslinking with 70% oxidized 20 kDa dextran: ꢀ after crosslinking; ꢁ after crosslinking and 30 min
reduction. Panel (b): Crosslinking time and reduction dependence of immobilized BpNDT. ᭹ Immobilized BpNDT on 600 Da PEI agarose followed by crosslinking with 50%
oxidized 20 kDa dextran; ᭿ Immobilized BpNDT on 600 Da PEI agarose followed by crosslinking with 70% oxidized 20 kDa dextran. NaBH4 reduction was carried out in both
cases after 2 h of crosslinking.
at the end of the immobilization process) when the immobilized
enzyme was crosslinked with 70% oxidized 20 kDa dextran (Table 2,
entries 5 and 6). Similar results have been obtained including 20%
of PEG 600 in the immobilization mixture (Table 2, entry 7). Lastly,
in order to obtain a highly active biocatalyst, the enzyme load was
increased, obtaining a very good residual activity at the end of the
complete immobilization process in presence of 20% of glycerol
(56%) (Table 2, entry 11).
enzyme on PEI agarose was completely abolished, in accordance
to the results previously reported for some multimeric PyNP
and PNP [8,9]. Likewise, the immobilized biocatalyst obtained
after crosslinking with 100 kDa dextran (20% oxidation) was also
rapidly void of activity. Although highest residual activity was
achieved when 20% oxidized dextran was employed in crosslink-
ing, the obtained immobilized biocatalyst was unstable at pH 10
(Fig. 2).
A similar behavior was described for the hexameric pyrimi-
dine nucleoside phosphorylase from Clostridium perfringens (CpUP),
since uracil was added in order to stabilize this multimeric enzyme
during covalent attachment to aldehyde-agarose, but no improve-
ment in the percentage of recovered activity could be achieved
[5,8]. On the other hand, the protective effect of glycerol during
reduction was also observed in the immobilization of different PNP
and PyNP on aldehyde agarose [5].
The stability of immobilized BpNDT was progressively improved
when crosslinking was performed with an increased dextran oxi-
dation degree (Fig. 2), and was fairly similar to soluble enzyme
when BpNDT was immobilized on agarose coated with PEI (600 Da)
and crosslinked using 70% oxidized 20 kDa dextran (crosslinking
performed in presence of 20% glycerol to preserve the activity of
the absorbed enzyme). This enzyme derivative maintained 50% of
activity after 24 h of incubation (Fig. 2).
Alternatively, 100 kDa dextran was also tested: 20% oxida-
tion allowed a residual activity after crosslinking which was
much higher than that observed with 20 kDa dextran (about 50%),
although the activity loss was also dramatic with a higher oxidation
degree (Table 2, entries 8–10).
High concentrations of a large number of modified nucleosides
(g of product per liter of reaction medium) may not be soluble
even at alkaline pH, therefore, the addition of an organic solvent
is sometimes necessary. Since dimethylformamide (DMF) has been
successfully used as co-solvent in preparative synthesis of nucle-
osides [10], the effect of this co-solvent on the stability of soluble
and immobilized BpNDT was studied.
3.2. Stability of immobilized BpNDT
Once again, free BpNDT showed the highest stability in presence
of 20% DMF compared with all the immobilized derivatives (Fig. 3),
maintaining 100% of activity for at least 48 h. Furthermore, similarly
to pH studies, the immobilized enzyme by mere ionic adsorption
was completely deactivated (Fig. 3), as well as the one obtained
after crosslinking with 100 kDa dextran (20% oxidized). Likewise,
immobilized BpNDT on 600 Da PEI agarose crosslinked with 70%
oxidized dextran in presence of 20% glycerol was the most sta-
ble immobilized biocatalyst, retaining 70% of activity after 48 h in
presence of 20% DMF (Fig. 3).
The very low stability of enzymes adsorbed on ionic carriers is
surprising because it was never observed before in other enzymes,
but confirms the results obtained in the immobilization study,
where the crosslinking of the enzyme adsorbed on PEI-coated car-
riers with dextran caused a complete loss of activity. Probably,
the strong ionic microenvironment surrounding the immobilized
enzyme highly promotes, in certain conditions, the distortion of
Many unnatural nucleosides of clinical use are poorly soluble in
physiological conditions. Consequently, the use of a high pH value
[35] and/or the presence of a co-solvent [5,38] can be required to
achieve suitable product concentrations for the development of
preparative and industrial processes. However, enzyme stability
may be affected by reaction conditions different from the physio-
logical ones.
Thus, the election of an optimized enzyme biocatalyst for the
production of nucleoside analogues requires the evaluation of
enzyme stability in non-physiological conditions, such as alkaline
pH or the presence of water-miscible organic co-solvents. In that
way, a range of experimental conditions in which the immobilized
enzyme can be used without relevant loss of activity can be estab-
lished.
Surprinsingly, soluble BpNDT retained 60% of its activity after
24 h at pH 10 (Fig. 2), whereas the activity of immobilized
Please cite this article in press as: A. Fresco-Taboada, et al., Development of an immobilized biocatalyst based on
Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2015.06.032

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Fig. 2. Stability of BpNDT at pH 10. ᭹ Free BpNDT; × Immobilized BpNDT on 600 Da PEI agarose followed by crosslinking with 70% oxidized 20 kDa dextran (crosslinking
performed in presence of 20% of glycerol); ᭿ Immobilized BpNDT on 600 Da PEI agarose followed by crosslinking with 50% oxidized 20 kDa dextran; ꢁ Immobilized BpNDT
on 600 Da PEI agarose followed by crosslinking with 20% oxidized 20 kDa dextran; ꢂImmobilized BpNDT on 600 Da PEI followed by crosslinking with 20% oxidized 100 kDa
dextran; + BpNDT adsorbed on 600 Da PEI agarose.
the 3D structure (by crosslinking or by using extreme pH condi-
tions). However, further studies would be needed to confirm the
detrimental effect of PEI on the stability of BpNDT.
oxidized 20 kDa dextran in presence of 20% glycerol, since it was
the best immobilized biocatalyst in terms of activity and stability
(Scheme 1).
The enzymatic synthesis of trifluridine was thus carried out at
different concentrations of deoxyuridine (dUrd) as sugar donor and
5-trifluorothymine (5tFThy, 2a) as base acceptor. As for any equi-
librium controlled synthesis, the increase of the molar excess of one
of the substrates induces an improvement in the final conversion.
Nevertheless, we decided to study the synthesis in presence
of an equal molar amount of the sugar donor dUrd and substrate
3.3. Synthesis of trifluridine and decytabine catalyzed by
immobilized BpNDT
Preparative synthesis of 5-trifluorothymidine (trifluridine, 3a)
and 5-aza-2ꢀ-deoxycytidine (decytabine, 3b) were performed by
BpNDT adsorbed on 600 Da PEI agarose and crosslinked with 70%
Fig. 3. Stability of BpNDT in presence of 20% DMF. ᭹ Free BpNDT; × Immobilized BpNDT on PEI 600 Da agarose followed by crosslinking with 70% oxidized 20 kDa dextran
(crosslinking performed in presence of 20% of glycerol); + Immobilized BpNDT on PEI 600 Da agarose followed by crosslinking with 70% oxidized 20 kDa dextran; ᭿ Immobilized
BpNDT on PEI 600 Da agarose followed by crosslinking with 50% oxidized 20 kDa dextran; ꢁ Immobilized BpNDT on PEI 600 Da agarose followed by crosslinking with 20%
oxidized 20 kDa dextran; ꢂImmobilized BpNDT on PEI 600 Da followed by crosslinking with 20% oxidized 100 kDa dextran; ꢀ Immobilized BpNDT on PEI 600 Da agarose.
Please cite this article in press as: A. Fresco-Taboada, et al., Development of an immobilized biocatalyst based on
Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2015.06.032

G Model
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7
Scheme 1. Synthesis of trifluridine (5tFThd, 3a) and decytabine (5azadCyd, 3b) catalyzed by immobilized BpNDT (600 Da PEI agarose followed by crosslinking with 70%
oxidized 20 kDa dextran).
Table 3
Enzymatic synthesis of 5-trifluorothymidine (trifluridine, 3a) and 5-aza-2ꢀ-deoxycytidine (decytabine, 3b) catalyzed by immobilized BpNDT at different substrate
concentrations.
dUrd (1) (mM)
Base acceptor (mM)
Conversion (%)
Product (mM)
Productivity (mM/h)
10
20
20
30
40
10
20
2a (10)
2a (10)
2a (20)
2a (30)
2a (40)
2b (10)
2b (20)
58
64
56
53
50
76
51
3a (5.8)
3a (6.5)
3a (11.1)
3a (16.0)
3a (20.1)
3b (7.6)
2.92
3.22
5.56
5.34
6.71
2.53
3.36
3b (10.1)
Experimental conditions: 100 mg of biocatalyst were added to 5 mL of 1 and 2a/2b in 10 mM potassium phosphate pH 7.5 and incubated at 37 ◦C for 2–3 h.
2a, since this condition is more attractive in the view of a large
scale preparative process. Different experiments have been thus
performed increasing the concentration of both substrates up to
40 mM, corresponding to 7.2 g/L of modified base (Table 3).
As expected, the highest conversion (64%) was achieved at
20 mM dUrd and 10 mM 5tFThy (2:1 molar ratio), and it was dimin-
ished when using equimolar amounts of both substrates (Table 3).
Nevertheless, productivity was enhanced by increasing the concen-
tration of both substrates reaching the highest rate (6.71 mM/h)
when 40 mM of dUrd (1) and 5-tFThy (2a) were employed. In these
conditions, 5.9 g/L of trifluridine (3a) were obtained.
4. Conclusions
Optimization of the immobilization process of NDT from B. psy-
chrosaccharolyticus (BpNDT) has been accomplished by studying
the effect of oxidation grade and molecular mass of dextran in
crosslinking of the absorbed enzyme on PEI functionalized sup-
ports. It has been observed that the activity loss was likely due to the
distortion of the 3D structure of the enzyme caused by the reduc-
tion of imino bonds towards irreversible C-N covalent bonds. This
detrimental effect was higher as larger was the number of covalent
bonds obtained during the crosslinking (by increasing the reaction
time and/or the oxidation degree of the crosslinking agent). This
evidence allowed the optimization of the immobilization process
in order to obtain a highly active BpNDT derivative that maintained
the good stability induced by the formation of several covalent
bonds during the immobilization.
A stabilized and active biocatalyst was obtained on 600 Da
PEI-agarose followed by crosslinking with 70% oxidized dextran
(20 kDa) in presence of 20% glycerol or PEG. These additives behave
as protective agents during the final reduction step of the immo-
bilization process, reducing the distortion of the 3D structure of
the protein. In particular, by using 20% glycerol, a biocatalyst
with a recovered activity (>50%) much higher than that previously
reported (about 20%) was obtained [19], also maintaining good sta-
bility in presence of 20% DMF as well as at pH 10.
The performance of the immobilized BpNDT was exactly the
same compared with the soluble enzyme, providing about 64% of
conversion of 2a into product 3a, using 2:1 of molar excess of dUrd
(1) (data not shown).
Syntheses of 3a by immobilized BpNDT lead to higher produc-
tivities than those transglycosylations performed by immobilized
UP from Bacillus subtilis (BsUP) or TP from Escherichia coli (EcTP)
in the same reaction conditions [9,10]. In fact, immobilized BpNDT
was able to achieve 58% conversion yield after 2 h in presence of 1:1
substrate molar ratio (productivity: 1.6 mM/h/IU), whereas immo-
bilized BsUP and EcTP afforded approximately the same conversion
(57% and 51%, respectively) to compound 3a using a 2:1 substrate
molar ratio, in 10 h and 3 h respectively (productivity: 0.2 mM/h/IU)
[9,10].
In addition, immobilized BpNDT was also able to catalyze
the synthesis of decytabine (3b) with similar conversion yields
(Table 3). In this case, it is worth mentioning that EcTP or BsUP
are not able to catalyze the synthesis of 3b using 5-azacytosine
(2b) as substrate (results not shown). This fact can be explained
considering that UP and TP are inactive toward cytidine, according
to previous literature data [11].
The optimized biocatalyst was successfully used to carry out
the synthesis of two interesting therapeutic nucleoside analogues:
trifluridine and decytabine. These syntheses were performed in
presence of high substrate concentration and, in both cases, higher
conversion yields and productivities were achieved in compari-
son to immobilized nucleoside phosphorylases (BsUP and EcTP),
demonstrating the biotechnological interest of this NDT.
Please cite this article in press as: A. Fresco-Taboada, et al., Development of an immobilized biocatalyst based on
Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2015.06.032

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The results now achieved in the immobilization of this enzyme
complete the set of immobilized enzymes that can be used for
the synthesis of antiviral and antitumoral nucleosides, allowing
the selection of the most adequate biocatalyst depending on the
desired product. In particular, the immobilized BpNDT could be
the first choice for the one-pot, one-enzyme synthesis of modified
pyrimidine or purine nucleosides including decytabine and other
cytosine derivatives.
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Please cite this article in press as: A. Fresco-Taboada, et al., Development of an immobilized biocatalyst based on
Bacillus psychrosaccharolyticus NDT for the preparative synthesis of trifluridine and decytabine, Catal. Today (2015),
http://dx.doi.org/10.1016/j.cattod.2015.06.032