Biotransformation of halogenated 2′-deoxyribosides by immobilized lactic acid bacteria

Article abstract of DOI:10.1016/j.molcatb.2012.04.004

An efficient and green bioprocess is herein reported to obtain halogenated nucleosides by transglycosylation using immobilized lactic acid bacteria (LAB). Lactobacillus animalis ATCC 35046 showed a yield of 95% at 0.5 h to synthesize 5-fluorouracil-2′-deoxyriboside (floxuridine). Calcium alginate was the best matrix for whole-cell immobilization by entrapment. Its productivity was 87 mg/L h in a continuous bioprocess. When adsorption techniques were evaluated, DEAE-Sepharose was the support which showed higher microbial load, its productivity being 53 mg/L h. Additionally, this microorganism was able to produce 5-bromouracil-2′-deoxyriboside, 6-chloropurine-2′- deoxyriboside and 6-bromopurine-2′-deoxyriboside.

Full text of DOI:10.1016/j.molcatb.2012.04.004

Journal of Molecular Catalysis B: Enzymatic 79 (2012) 49–53
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biotransformation of halogenated 2ꢀ-deoxyribosides by immobilized lactic acid
bacteria
Claudia N. Britosa, Valeria A. Cappaa, Cintia W. Riveroa, Jorge E. Sambethb, Mario E. Lozanoa,
Jorge A. Trellesa,∗
a Laboratorio de Investigaciones en Biotecnología Sustentable (LIBioS), Universidad Nacional de Quilmes, Roque Saenz Pe˜na 352, Bernal (B1868BXD), Argentina
b Centro de Investigación y Desarrollo en Ciencias Aplicadas Dr. Jorge J. Ronco. Universidad Nacional de La Plata. 47 no. 257 – La Plata (B1904CMC), Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and green bioprocess is herein reported to obtain halogenated nucleosides by transglycosyla-
tion using immobilized lactic acid bacteria (LAB). Lactobacillus animalis ATCC 35046 showed a yield of 95%
at 0.5 h to synthesize 5-fluorouracil-2ꢀ-deoxyriboside (floxuridine). Calcium alginate was the best matrix
for whole-cell immobilization by entrapment. Its productivity was 87 mg/L h in a continuous bioprocess.
When adsorption techniques were evaluated, DEAE-Sepharose was the support which showed higher
microbial load, its productivity being 53 mg/L h. Additionally, this microorganism was able to produce 5-
bromouracil-2ꢀ-deoxyriboside, 6-chloropurine-2ꢀ-deoxyriboside and 6-bromopurine-2ꢀ-deoxyriboside.
Received 20 December 2011
Received in revised form 29 March 2012
Accepted 4 April 2012
Available online 11 April 2012
Keywords:
Lactic acid bacteria
2ꢀ-N-deoxyribosyltransferase
Whole-cell immobilization
Antitumoral compounds
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Entrapment techniques involve microorganism inclusion in a
rigid network to prevent their release into the surrounding medium
Nucleoside analogs such as 5-fluorouracil-2ꢀ-deoxyriboside
(5FUradRib), better known as floxuridine, have shown activity in
patients with colorectal, pancreatic, breast, head and neck can-
cers [1]. At present, these compounds have been synthesized by
chemical methods [2]. Additionally, nucleoside analog biotransfor-
mations can be carried out under very mild conditions, offering
environmentally clean technologies [3].
while allowing mass transfer of substrates and products [9]. In
these techniques, the matrix is produced by a physical or chemical
polymerization process [10] whereas in adsorption immobiliza-
tion techniques, microorganisms can be attached to the support
by non-specific bonds or affinity interactions [11]. In most cases,
these methodologies allow to recover the support after biocatalyst
deactivation [12].
It has been reported that some members of lactic acid bacte-
ria have an enzyme, called 2ꢀ-N-deoxyribosyltransferase (NDT, EC
2.4.2.6), which catalyzes the transglycosylation between purine or
pyrimidine bases and nucleosides in one-step reaction. NDT has
been isolated and characterized in Lactobacillus helveticus [4], Lac-
tobacillus leichmannii [5], Lactobacillus reuteri [6] and Lactococcus
lactis [7].
In this work, Lactobacillus animalis ATCC 35046 was used
for 5FUradRib biotransformation. Reaction parameters were opti-
mized in order to obtain an immobilized biocatalyst with
improved activity. Additionally, L. animalis ATCC 35046 was used
to obtain other halogenated nucleosides such as 5-bromouracil-
2ꢀ-deoxyriboside (5BrUradRib), 6-chloropurine-2ꢀ-deoxyriboside
(6ChPurdRib) and 6-bromopurine-2ꢀ-deoxyriboside (6BrPurdRib).
Microorganism immobilization is a good way to carry out
the bioprocess under preparative conditions. Entrapment and
adsorption techniques are the most widely used for whole cell
immobilization. The main advantages of this methodology are
high operational stability, easy upstream separation and bioprocess
scale-up feasibility [8].
2. Materials and methods
2.1. Materials
Nucleosides and bases were purchased from Sigma Chem.
Co. (Brazil). Culture media compounds were obtained from Bri-
tania S.A. (Argentine). Chemicals were purchased from Sigma
Chem. Co. (Brazil). Solvents used were of HPLC grade from Sintor-
gan S.A. (Argentine). Supports (DEAE-Sepharose, EDA-Sepharose,
IDA-Agarose, Q-Agarose) were purchased from Sigma–Aldrich
∗
Corresponding author. Tel.: +54 1143657100x5645; fax: +54 1143657132.
E-mail addresses: jtrelles@unq.edu.ar, jatrelles@gmail.com (J.A. Trelles).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.04.004

50
C.N. Britos et al. / Journal of Molecular Catalysis B: Enzymatic 79 (2012) 49–53
(Argentine). TLC aluminium sheets (Silica gel 60 F254) were from
Merck (Germany).
2.9. Analytical methods
Nucleoside analog biotransformations were qualitatively
evaluated by TLC in 80:20 (v/v) chloroform/methanol as mobile
phase. The quantitative analysis was performed by HPLC (Phar-
2.2. Growth conditions
macia LKB) at 254 nm using
a
Nucleodure 100C–18column
Microorganisms were grown until saturation, harvested by cen-
trifugation during 10 min at 17,500 × g, washed with 50 mM pH
7 tris(hydroxymethyl)aminomethane–HCl (Tris–HCl) buffer and
stored at 4 ◦C until use. Lactobacillus media contained 15 g/L tryp-
tone, 5 g/L soy peptone, 5 g/L NaCl pH 7. Lactococcus media: 10 g/L
tryptone, 15 g/L meat peptone, 10 g/L yeast extract, 10 g/L glucose,
1 g/L Tween® 80 pH 6.5.
(5 ␮m, 125 mm × 5 mm). Isocratic mobile phases used were
water/methanol at the following ratios: (95:5, v/v) for 5-
fluoruracil-2ꢀ-deoxyriboside (5FUradRib); (90:10, v/v) for
5-bromouracil-2ꢀ-deoxyriboside (5BrUradRib), 6-chloropurine-
2ꢀ-deoxyriboside (6ChPurdRib), 6-bromopurine-2ꢀ-deoxyriboside
(6BrPurdRib)
and
6-chloro-2-fluoropurine-2ꢀ-deoxyriboside
(6Ch2FPurdRib) and (99:1, v/v) for 5-chlorouracil-2ꢀ-deoxyriboside
(5ChUradRib) biotransformations. Product identification was per-
formed by MS–HPLC LCQ-DECAXP4 Thermo Spectrometer with
Electron Spray Ionization method (ESI). Phenomenex C18 column
(5 ␮m, 100 mm × 2 mm) and Xcalibur 1.3 software (Thermo-
Finnigan, USA) were used. Mobile phase used for 5FUradRib (t:
14.0 min, M+: 246.9) and 5BrUradRib (t: 34.4 min, M+: 308.0)
biotransformations was 95/5 (v/v) water/methanol + 0.1% acetic
acid and flow was 200 ␮L/min. Mobile phase used for 6ChPurdRib
(t: 4.2 min, M+: 271.8) and 6BrPurdRib (t: 9.4 min, M+: 315.8)
biotransformations was 15/85 (v/v) water/methanol + 0.1% acetic
acid and flow was 200 ␮L/min.
2.3. Standard reaction
5FUradRib biotransformation was carried out with 1 × 1010
colony-forming units (CFU), 6 mM thymidine (dThd) and 2 mM 5-
fluorouracil (5FUra) in 50 mM pH 7 Tris–HCl buffer at 30 ◦C and
shaking at 200 rpm. Assays were performed three times.
2.4. Lactic acid bacteria selection
Lactococcus (L. lactis subps. lactis and L. lactis subsp. cremoris)
and Lactobacillus strains (Lactobacillus acetotolerans and L. animalis)
were used to assay NDT activity at 3 h in standard conditions [13].
3. Results and discussion
3.1. Lactic acid bacteria selection
2.5. Sugar donor selection
Floxuridine biotransformation was assayed to select the best
LAB (Scheme 1). Screening was carried out with several Lactococcus
and Lactobacillus strains. L. lactis subsp. cremoris and L. acetotolerans
showed no significant activity (less than 5%) whereas L. animalis
ATCC 35046 exhibited the highest NDT activity (48% at 3 h).
Different ribosides such as thymidine (dThd), uridine (Urd), 2ꢀ-
deoxyuridine (dUrd), uracil 1-␤-d-arabinofuranoside (araUra) and
2ꢀ,3ꢀ-dideoxyuridine (ddUrd) were assayed as sugar donors. Reac-
tions were made with 1 × 1010 CFU/mL in 1 mL of 2.5 mM riboside
and 50 mM pH 7 Tris–HCl buffer, 30 ◦C at 3 h.
3.2. Sugar donor selection
2.6. Optimization of bioprocess parameters
Urd, dThd, dUrd, ddUrd and araUra were tested as starting ribo-
sides. L. animalis ATCC 35046 preferentially hydrolyzed dThd and
dUrd with yields of 38% and 35% at 3 h, respectively. Urd was
also hydrolyzed, although with lower yield (22% at 3 h). However,
when ddUrd and araUra were assayed, no hydrolytic activity was
detected. In view of these results, dThd was used as starting 2ꢀ-
deoxyriboside for 5FUradRib biotransformation.
Different reaction parameters such as different growth phases
of microorganism (stationary and exponential), buffer concentra-
tion (10, 25, 50 and 75 mM) and cation effect (1 and 10 mM Mg2+
,
K+, Zn2+, Ca2+, Cu2+, Mn2+ or Ba2+) were studied for 5FUradRib bio-
transformation in standard conditions.
Additionally, reaction time (0.25, 0.5, 1, 3 and 6 h), amount of
microorganisms (1 × 108, 1 × 109, 5 × 109 and 1 × 1010), different
reaction temperatures (20, 30, 45 and 60 ◦C), pH (6, 7 and 8) and
different 5FUra and dThd relations (2:2, 6:2 and 2:6 mM) were
evaluated.
3.3. Optimization of bioprocess parameters
5FUradRib biotransformation from dThd and 5FUra was used to
optimize bioprocess parameters at different reaction times.
2.7. Whole cells immobilization
3.3.1. Effect of microbial growth phase
L. animalis ATCC 35046 (1 × 1010 CFU) was immobilized by
entrapment in agar, agarose, polyacrylamide and sodium algi-
nate as previously described [14]. For adsorption immobilization,
200 mg of EDA-Sepharose, IDA-Agarose, DEAE-Sepharose or Q-
Agarose were incubated with 1 × 1010 CFU in 1 mL of 50 mM pH
7 Tris–HCl buffer, during 16 h at 30 ◦C and shaking at 200 rpm.
5FUradRib biotransformation was assayed using L. animalis at
exponential and stationary microbial growth phases. 5FUradRib
conversions were 48% and 71% in the exponential and stationary
phase at 1 h, respectively. This result could be due to differential
expression of NDT, an enzyme involved in the pyrimidine salvage
pathway during the stationary growth phase [15].
2.8. Biotransformation of other halogenated 2ꢀ-deoxyribosides
3.3.2. Effect of Tris–HCl concentration
Different Tris–HCl concentrations were assayed (10–75 mM)
(Fig. 1A). The highest activity was observed when the buffer con-
centration was 25 mM (43 mg/L h), while at lower (10 mM) or
higher (50 mM) Tris–HCl concentrations no significant variations
in productivity were detected. A decrease in enzyme activity was
Other purine and pyrimidine bases were tested: 5-bromouracil
(5BrUra), 5-chlorouracil (5ChUra), 6-chloropurine (6ChPur), 6-
bromopurine (6BrPur) and 6-chloro-2-fluoropurine (6Ch2FPur).
The reactions were performed at optimized conditions.

C.N. Britos et al. / Journal of Molecular Catalysis B: Enzymatic 79 (2012) 49–53
51
Scheme 1. Floxuridine biotransformation by Lactobacillus animalis ATCC 35046.
detected at 75 mM. This buffer effect has already been reported in
previous works at values close to 100 mM [16].
Finally, different cation concentrations (1 and 10 mM) were
assayed, showing no significant differences in reaction yields (data
not shown).
3.3.3. Effect of cations
Reactions were carried out with the addition of different cations
in the reaction medium and relative activity (ra) was calculated.
Results were compared with the control reaction (ra: 1).
When Mn2+ and K+ were added to the reaction medium,
5FUradRib amount decreased 10-fold (ra: 0.10) and 2-fold (ra: 0.50)
with respect to control reaction (without cations). The best rela-
tive activities (1.22 and 1.30) were achieved when Cu2+ and Zn2+
were added to the reaction medium (Fig. 1B). However, no signifi-
cant changes were observed when different alkaline earth cations
were tested (Mg2+, Ba2+, Ca2+). In lactic acid bacteria, the ABC trans-
porters activated by Zn2+ or Cu2+ cations [17] are involved in the
internalization of nucleosides and bases [15].
3.3.4. Effect of the amount of microorganisms
In order to evaluate the optimal quantity of microorganisms
for 5FUradRib biotransformation, reactions were performed with
increasing amounts of L. animalis (Fig. 1C). Biocatalytic activity was
detectable with 1 × 109 CFU, reaching a productivity of 13 mg/L h
at 0.5 h of reaction. When the amount of microorganisms was
increased 5-fold (5 × 109 CFU), the productivity was 30 mg/L h at
the same reaction time. Moreover, the best productivity (50 mg/L h)
was achieved when 1 × 1010 cells were used. Higher amounts of
microorganisms were not evaluated in microscale experiments due
to operational difficulties.
Fig. 1. Optimization of 5FUradRib biotransformation parameters. (A) buffer concentration; (B) cations (1 mM); (C) amount of microorganisms; (D) temperature.

52
C.N. Britos et al. / Journal of Molecular Catalysis B: Enzymatic 79 (2012) 49–53
Table 1
5FUradRib biotransformation with immobilized Lactobacillus animalis ATCC 35046.
Support
Yield (mg/L)
Productivity (mg/L h)
Operational
stability (h)
Alginate
DEAE-Sepharose
0.8
0.7
87
53
144
248
cases when an excess of dThd was used (3:1 ratio), leading to an
increase of 5FUradRib productivity (53 mg/L h).
Based on these results, the optimal reaction parameters for flox-
uridine biotransformation were 25 mM Tris–HCl, 1 mM Zn2+ (or
Cu2+), pH 7, 30 ◦C, 6 mM dThd and 2 mM 5FUra at 0.5 h.
3.4. Microorganism immobilization
Fig. 2. Deaminase activity of Lactobacillus animalis ATCC 35046. The activity of ade-
nine deaminase (ADA) was determinated by reaction of 2.5 mM adenine in 50 mM
Tris–HCl buffer pH 7 at different temperatures.
3.4.1. Entrapment immobilization
The selected LAB was immobilized by entrapment in thermogels
(agar, agarose), ionic gels (alginate) and synthetic polymers (poly-
acrylamide). These biocatalysts were evaluated for their 5FUradRib
biotransformation capacity.
3.3.5. Effect of temperature
In the Lactobacillus genus, NDT is responsible for transglycosy-
lation when phosphate is absent in the reaction medium and its
catalytic activity remains stable up to 50 ◦C [18]. 5FUradRib bio-
transformation was performed at different temperatures (20, 30,
45 and 60 ◦C) using dThd and 5FUra as substrates (Fig. 1D).
5FUradRib conversion values were 94 and 90% at 30 and 45 ◦C,
respectively. However, a significant decrease in 5FUradRib yield
was observed when the reaction temperature was 60 ◦C, possibly
due to adenine deaminase activity (ADA, EC 3.5.4.2) (Fig. 2) [19].
Therefore, the selected temperature for subsequent reactions was
30 ◦C.
No activity was detected in L. animalis immobilized in poly-
acrylamide, probably due to the toxic effect of acrylamide on
microorganisms [21]. Moreover, a release of L. animalis into the
surrounding medium was observed when this microorganism was
immobilized in 2 and 3% of agar or agarose. However, when 4% of
these gels were used, no microorganism release was observed but
conversion values were low.
Finally, immobilizations were made at different alginate con-
centrations (2, 3 and 4%, w/v). Calcium alginate 4% (w/v) was
the lowest matrix concentration at which there was no bacte-
rial release. This biocatalyst showed an operational stability of
44 h in batch process. To study the biocatalyst at a pre-pilot
scale, 5FUradRib biotransformation was performed in a recycled
column with a reaction volume of 12 mL (recirculation veloc-
ity: 4.2 mL/min). Under this condition, operational stability was
increased more than 3-fold (144 h) and its 5FUradRib conversion
was 0.8 mg/L.
3.3.6. Effect of pH
5FUradRib biotransformation was carried out at pH 6, 7 and 8.
A conversion rate of 51% at 0.5 h was achieved when the reaction
was evaluated under alkaline conditions and this parameter was
significantly improved under acid and neutral pH (95% at 0.5 h).
The pKa of 5FUra is 8.0 and alkaline pH values might interfere with
an adequate internalization of substrate for subsequent use.
3.4.2. Adsorption immobilization
L. animalis immobilization by adsorption was studied on dif-
ferent supports: IDA-Agarose, Q-Agarose, EDA-Sepharose and
DEAE-Sepharose.
L. animalis did not significantly attach to IDA-Agarose and
Q-Agarose, possibly due to the high negative charge density of
IDA-Agarose and the rigid surface structure of quaternary amides
in Q-Agarose [22]. However, EDA-Sepharose and DEAE-Sepharose
showed satisfactory properties as supports, displaying immobi-
lization yields of 46 and 96%, respectively. DEAE-Sepharose was
selected as support in successive batch experiments.
3.3.7. Effect of initial molar ratio (2ꢀ-deoxyriboside/base)
It has been widely reported that transglycosylation reactions
are reversible [20]. For this reason, the initial ratio of sub-
strates was analyzed (Fig. 3). When 1:3 and 1:1 dThd/5FUra
ratios were assayed, 5FUradRib productivity was 35 and 33 mg/L h,
respectively. However, the amount of 2ꢀ-deoxyribosyl-enzyme
intermediate available was significantly higher than in previous
3.4.3. Comparison of immobilized biocatalysts
5FUradRib biotransformation with L. animalis ATCC 35046
immobilized in DEAE-Sepharose and calcium alginate is shown
(Table 1). Operational stability of DEAE-Sepharose derivatives was
better (248 h) than that of calcium alginate (144 h). However, pro-
ductivity values were 53 mg/L h for DEAE-Sepharose and 87 mg/L h
for calcium alginate derivatives. Both immobilized derivatives
showed similar yields.
3.5. Biotransformation of other halogenated 2ꢀ-deoxyribosides
The possibility of using L. animalis to catalyze biotransforma-
tions with different purine and pyrimidine bases was evaluated.
The products were identified by HPLC–MS.
Fig. 3. Effect of molar ratio (2ꢀ-deoxyriboside/base) on productivity of 5FUradRib
biotransformation by Lactobacillus animalis ATCC 35046.

C.N. Britos et al. / Journal of Molecular Catalysis B: Enzymatic 79 (2012) 49–53
53
Table 2
4. Conclusions
Halogenated 2ꢀ-deoxyribosides biotransformation with Lactobacillus animalis ATCC
35046.
L. animalis ATCC 35046 was selected to produce 5FUradRib.
Reaction parameters were optimized to improve productivity and
this biocatalyst was stabilized by entrapment and adsorption
immobilization techniques. Calcium alginate and DEAE-Sepharose
derivatives showed satisfactory 5FUradRib yield and operational
stability in batch and continuous bioprocesses.
Base
Product
Yield (%)
5BrUra
5ChUra
6BrPur
6ChPur
6Ch2FPur
5BrUradRib
5ChUradRib
6BrPurdRib
6ChPurdRib
6Ch2FPurdRib
22
nd
56
44
nd
Additionally, L. animalis ATCC 35046 was able to synthesize
other halogenated nucleosides at short reaction times, and the
specificity of purine and pyrimidine bases corresponds to type II
NDT activity. These results indicate that L. animalis ATCC 35046
could be used to produce a broad spectrum of halogenated nucleo-
side analogs employing an environmentally friendly methodology.
Table 3
Green chemistry parameters of halogenated 2ꢀ-deoxyribosides biotransformation
with Lactobacillus animalis ATCC 35046.
Product
E-factora
C-efficiencyb
A-economyc
Acknowledgments
5FUradRib
5BrUradRib
6BrPurdRib
6ChPurdRib
3.0
6.1
1.3
0.9
64
64
67
67
66
71
75
73
This research was supported by Ministerio de Ciencia, Tec-
nología e Innovación Productiva de la Nación Argentina and
Universidad Nacional de Quilmes. M.E.L., J.E.S. and J.A.T. are
research members of CONICET, C.W.R. is a fellow of CONICET. We
are grateful to Carlos Manzione and Carlos Odoguardi (Technical
members of CONICET) for their collaboration in some experimental
works.
total waste mass
product mass
a
E-factor =
C-efficiency =
.
(carbon atoms in product)×100
b
.
carbons atoms in reactants
(product MW)×100
c
A-economy = ꢀ
.
(reactants MW)
5BrUradRib biotransformation yield was 22% in 4 h of reac-
tion using 5BrUra as starting base and dThd as 2ꢀ-deoxyribose
donor. Additionally, purine bases were used to evaluate the
activity and specificity of the biocatalyst. L. animalis was
able to catalyze 6ChPurdRib (44%) and 6BrPurdRib (56%) bio-
transformation in 4 h of reaction. Activity values were not
detectable when using 6Ch2FPur and 5ChUra as starting base
(Table 2).
NDTs have been classified into two classes depending on their
substrate specificity. Type I NDT is specific for purines and type
II NDT is a non-specific enzyme (purines and pyrimidines) [2].
Therefore, the results indicated the presence of NDT II activity in L.
animalis ATCC 35046.
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