Biotransformation of 5′-O-β-D-galactosyl-floxuridine by immobilized β-galactosidase from Kocuria rhizophila

Article abstract of DOI:10.1016/j.jfluchem.2018.07.012

5-fluorouracil-2’-deoxyriboside (FUdR), an antimetabolite known as floxuridine, is a halogenated nucleoside extensively used in the clinical treatment of colon carcinoma and hepatic metastases. This drug presents low bioavailability, thus requiring large doses and frequent administration, which leads to long-lasting and severe side effects in clinical practice. In order to overcome this problem, galactosylated nucleoside analogues were obtained using immobilized β-galactosidase in Ca-alginate with yields of 80% at only 7 h. Additionally, the obtained biocatalyst was stable for 6 months in storage conditions (4 °C) and could be reused at least 16 times without loss of its activity at 30 °C. This work describes for the first time an efficient, eco-compatible and simple bioprocess for obtaining 5′-O-β-D-galactosyl-floxuridine using an immobilized biocatalyst.

Full text of DOI:10.1016/j.jfluchem.2018.07.012

JournalofFluorineChemistry214(2018)58–62
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Biotransformation of 5´-O-β-D-galactosyl-floxuridine by immobilized β-
galactosidase from Kocuria rhizophila
⁎
Andrea Sarquiza,b, Cintia W. Riveroa,b, Claudia N. Britosa, Jorge A. Trellesa,b,
a
Laboratory of Sustainable Biotechnology (LIBioS National University of Quilmes), Bernal, Argentina
b
National Scientific and Technical Research Council (CONICET) Ciudad Autónoma de Buenos Aires (CABA), Argentina
A R T I C L E I N F O
A B S T R A C T
Keywords:
Enzyme stabilization
Floxuridine
Galactosylation
Biocatalysis
5-fluorouracil-2’-deoxyriboside (FUdR), an antimetabolite known as floxuridine, is a halogenated nucleoside
extensively used in the clinical treatment of colon carcinoma and hepatic metastases. This drug presents low
bioavailability, thus requiring large doses and frequent administration, which leads to long-lasting and severe
side effects in clinical practice. In order to overcome this problem, galactosylated nucleoside analogues were
obtained using immobilized β-galactosidase in Ca-alginate with yields of 80% at only 7 h. Additionally, the
obtained biocatalyst was stable for 6 months in storage conditions (4 °C) and could be reused at least 16 times
without loss of its activity at 30 °C.
This work describes for the first time an efficient, eco-compatible and simple bioprocess for obtaining 5´-O-β-
D-galactosyl-floxuridine using an immobilized biocatalyst.
1. Introduction
conditions and display chemical enantio- and regioselectivity [1].
However, there are few publications on enzymatic glycosylation of
Nucleoside analogues are synthetic compounds that have been de-
veloped to mimic the physiological functions of their natural versions in
order to interfere with the cellular metabolism and subsequently be
incorporated into the DNA and RNA to inhibit cell division or/and viral
replication [1]. These compounds have played an important role in
anticancer and antiviral therapies for the last ten years. 5-fluorouracil-
2’-deoxyriboside (FUdR), a halogenated pyrimidinic nucleoside, has
been extensively used in the clinical treatment of colon carcinoma and
hepatic metastases, and has also shown antitumoral activity in breast,
head and neck cancers [2,3]. However, FUdR has some disadvantages
such as low oral bioavailability and selectivity that lead to severe side
effects in clinical practice [4]. It has been reported that the glycosyla-
tion of nucleosides masks their toxicity and improves their cell ab-
sorption [5]. For instance, Abraham et al. have shown that FUdR is a
hundred times more toxic to bone marrow cells in Balb/c mice than 5´-
O-β-D-galactosyl-5-fluorouridine (5-Gal-FUdR) [6]. Nucleoside drugs
and their derivatives are mainly synthesized by chemical methods that
involve arduous and tedious protection/deprotection steps causing
unwanted accumulation of racemic mixtures that affect further pur-
ification; for these cases, biosynthesis is a promising alternative [7].
Biocatalysis has several advantages over chemical synthesis due to the
general properties of enzymes, which are active under mild reaction
nucleosides and in all these cases soluble enzymes are used, which
hinders the product purification and does not allow its reuse [8,9].
Enzyme immobilization has received considerable attention in recent
decades due to the fact that enzyme activity and stability can be im-
proved by this technique [10]. Entrapment methods are widely used for
enzyme immobilization; these techniques are based on the inclusion of
the enzyme within a rigid network to prevent their release into the
surrounding medium, while still allowing mass transfer [11]. The most
commonly used supports for this methodology are agar, agarose, algi-
nate, β-carrageenan, and polyacrylamide [12–16]. Alginate is a naturally
occurring anionic polymer typically obtained from brown seaweed that
has been extensively used in biotechnology, medicine and pharmaceu-
tical industry due to its biocompatibility, low toxicity, and low cost [17].
The aim of this work was to biosynthesize, for the first time, 5-Gal-
FUdR using a stabilized biocatalyst based on an immobilized β-ga-
lactosidase (3.2.1.23) from Kocuria rhizophila (Scheme 1).
2. Material and methods
2.1. Screening for β-galactosidase activity
Seventy bacterial strains from LIBioS collection were screened for
⁎
Corresponding author.
E-mail address: jtrelles@unq.edu.ar (J.A. Trelles).
https://doi.org/10.1016/j.jfluchem.2018.07.012
Received 24 April 2018; Received in revised form 28 July 2018; Accepted 28 July 2018
Availableonline30July2018
0022-1139/©2018ElsevierB.V.Allrightsreserved.

A. Sarquiz et al.
JournalofFluorineChemistry214(2018)58–62
Scheme 1. Enzymatic galactosylation
of FUdR.
extracellular β-galactosidase activity. Bacterial cultures were grown for
16 h at optimum conditions for each genus. β-galactosidase hydrolytic
activity was determined by measuring of o-nitrophenol release from
ortho-nitrophenyl-β-galactoside (ONPG) at 415 nm. The reaction con-
ditions were ONPG 1 mM, NaCl 1 mM, and tris(hydroxymethyl)ami-
nomethane-HCl (Tris-HCl) 25 mM, pH 7 at 30 °C. One unit of enzyme
activity (U) was defined as the quantity of enzyme that will liberate
1 μmol o-nitrophenol per min under assay conditions.
detector, automatic injector and fraction collector Thermo Scientific
Dionex Ultimate 3000 Series (ThermoScientific®). Product identifica-
tion was conducted by MS-HPLC under the above-mentioned conditions
(5-Gal-FUdR; M+: 407) using
a LCQ-DECAXP4 Thermo/Finnigan
spectrometer with the electron spray ionization method (ESI) and one
ion trap detector.
2.7. Statistical analysis
2.2. β-galactosidase purification
All experiments were performed in triplicate. One-way analysis of
variance (ANOVA) was used to determine significant differences among
variables. Differences with a probability value of p < 0.05 were con-
Kocuria rhizophila was grown in an optimized culture medium, until
saturation, and the supernatant was ultrafiltered up to 100× volume
concentration. The enzyme was characterized by electrophoresis.
Native gradient gel electrophoresis according to the modified method of
Laemmli was used to estimate the molecular weight of the protein by
the detection of the enzyme activity in the gel [18].
sidered significant, and all data were reported as mean
SD.
Statgraphics Centurion XV program (version 15.1.02) was used.
3. Results
3.1. Selection of strains with β-galactosidase activity
2.3. Enzyme stabilization
Seventy strains from several bacterial genera such as
Thermonospora, Brevibacterium, Chromobacterium, Citrobacter,
According to Tanash et al. [19], different concentrations of β-ga-
lactosidase (50, 100, 200, 300, 600 μg/mL) solution were mixed with
sodium alginate to final concentrations of 2%. The entrapment was
carried out by dropping alginate solution in 0.1 M CaCl2. Afterwards,
the same assay was performed with different sodium alginate con-
centrations with the purpose of optimizing β-galactosidase retention in
the matrix.
Lactobacillus and Kocuria were screened for β-galactosidase activity
using batch fermentation. Among all evaluated strains, 14 strains that
were able to hydrolyze ONPG were selected (Table 1). Kocuria rhizo-
phila showed higher activity (0.15 U/mL) than all the other strains and
was chosen for subsequent assays. The culture medium was optimized
and β-galactosidase expression and activity were improved sig-
nificantly, leading to an increase of 50% in enzyme activity.
2.4. Optimization of FUdR galactosylation
Different reaction parameters such as pH (4, 5, 6, 7 and 8), tem-
perature (20, 30, 45, 60 °C) and cation addition (Mg2+ and Na+) were
analyzed. Besides, the ONPG/FUdR molar ratio (1:1, 1:2 and 2:1 mM)
was studied for FUdR galactosylation in 1 mL of reaction medium for
12 h. Finally, a kinetic of the reaction was evaluated at different times
(1, 2, 3, 4, 5, 7 and 12 h).
3.2. β-galactosidase molecular weight
Enzyme characterization was carried out by electrophoretic tech-
niques. By using a stained native electrophoresis, it could be confirmed
that it was a lactase with demonstrated activity and their molecular
weight was around 310 kDa (Fig. 1).
2.5. Biocatalyst stability
Table 1
Screening of strains for β-galactosidase activity.
Storage stability was tested at two temperatures (4 °C and 30 °C) and
was defined as the relative activity of FUdR galactosylation between the
first and the successive reactions using the same biocatalyst. Reusability
of immobilized enzyme in Ca-alginate was evaluated using 5’-Gal-FUdR
synthesis as standard reaction. The used beads were washed three times
using buffer Tris-HCl pH 7 at the end of each cycle.
Negative
Positivea
Genus
Strains
tested
Genus
Strains
tested
Aeromonas
Arthrobacter
Bacillus
Enterobacter
Enterococcus
Erwinia
Klebsiella
Lactobacillus
Proteus
Pseudomonas
Serratia
Streptomyces
Xanthomonas
4
2
9
3
1
3
3
7
3
3
2
15
1
Bacillus
3
1
1
3
1
2
1
2
Brevibacterium
Chromobacterium
Citrobacter
2.6. Analytical methods
Kocuria
Lactobacillus
Thermonospora
Xanthomonas
FUdR galactosylation quantitative analysis was performed by HPLC
(Gilson) at 254 nm (Detector UV/Vis 156, Gilson) with an Agilent
Zorbax Eclipse XDB C-18 column (5 μm, 150 mm × 5 mm). The mobile
phase consisted of a gradient elution (1.4 mL/min) with water/me-
thanol (96/4, v/v) from 0 to 6.5 min, and then water/methanol (80/20,
v/v) from 6.5 min was used. The retention times for 5-Gal-FUdR, FUdR
and ONPG were 2.0, 5.4, and 12.0 min, respectively. The 5-Gal-FUdR
was separated and purified by using a UHPLC equipped with UV–vis
a
The positive response is expressed regarding ONPG hydrolysis.
59

A. Sarquiz et al.
JournalofFluorineChemistry214(2018)58–62
3% (w/v), maintaining the CaCl2 concentration at 100 mM, the op-
timum concentration that present the highest activity (3.20 U/mg
0.14) was 2%. Lower and higher concentration of alginate presented
undesirable protein release into the reaction, and diffusion restrictions
respectively [20].
3.4. Biosynthesis of 5´-O-β-d-galactosyl-floxuridine
In order to evaluate the effect of pH and temperature different
conditions were tested. Fig. 3a shows that the optimal pH range to
which the maximum yield has been obtained is 6–8, which matches
with the optimum pH of this enzyme. As shown in Fig. 3b, the product
yield is altered marginally when the temperature increased up to 30 °C.
Further increase of temperature resulted in sharp decrement of the
yield, possibly due to the denaturation of the biocatalyst, because the
bacteria that produce this enzyme (K. rhizophila) is a mesophilic mi-
croorganism.
Fig. 1. Electrophoresis on a nondenaturing gradient gel. Arrow indicates β-
galactosidase. Lane 1: Molecular weight marker; lane 2: supernatant of K. rhi-
zophila culture stained with Coomassie brilliant blue G250; lane 3: supernatant
of K. rhizophila culture stained with ONPG. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this
article).
Since Na+ and Mg2+ are required for maximal activity of β-ga-
lactosidase [21], biotransformations were carried out by adding these
cations. When Na+ and Mg2+ were added to the reaction medium, the
yields of the reaction were enhanced by 50% and 30% when compared
to the reaction without cation addition. This difference in the activity
could be explain by a crystallography reported by Matthews that shown
how the Na+ interacts with the enzyme active site and intermediates of
the transgalactosylation reaction. Even though Mg2+ is not necessary
for structure, has been reported that this cation is important for mod-
ulating the chemistry and mediating the interactions between the active
site components [22].
Originally, lactose and galactose were tested as glycosyl donors.
Unfortunately, no galactosylated derivative was formed. Thus the ac-
tive glycosyl donor ONPG was selected in this work. The reaction
conditions were buffer Tris-HCl 25 mM pH 7.0 containing 2 mM FUdR,
1 mM ONPG, 1 mM NaCl and 0.3 U of β-galactosidase and substrate
ratio FUdR/ONPG was evaluated at 12 h, achieving a maximum yield
when the molar ratio was 2:1 (Fig. 4a). This could be due because there
is a competitive hydrolysis of ONPG in which water acts as a nucleo-
philic agent, so the reaction can be controlled kinetically, therefore a
high concentration of the substrate FUdR will displace the reaction
toward synthetic direction. To gain a deeper insight into the enzymatic
process, the time courses of the synthesis of O-galactosylated derivative
were followed (Fig. 4b) reaching a yield of 80% at only 7 h. Thus a
significant decrease of reaction times previously reported was achieved,
which allowed increasing the process productivity (50 μg/h).
Fig. 2. Optimization of β-galactosidase immobilization conditions. Protein
concentration. No significant differences among treatments were named with
the same letter (LSD test, p-value < 0.05).
Table 2 compares the transglycosylation efficiencies of this study
with the previously reported. The yields obtained with the β-galacto-
sidase from K. rhizophila and reactions times were significantly im-
proved compared to those reported with other glycosidases for 5-Gal-
FUdR biotransformation. Additionally, in this work the enzyme was
immobilized which increase the protein stability and facilitate their
3.3. Enzyme stabilization
Protein concentration positively affected the activity, which in-
creased until an initial enzyme concentration of 300 μg/L (Fig. 2). On
the other hand, when alginate concentration was increased from 1% to
Fig. 3. Effect of buffer pH (a) and temperature (b) on enzymatic galactosylation of FUdR. Reaction conditions: 1 mM FUdR, 1 mM ONPG, 1 mM NaCl and 0.3 U of β-
galactosidase. When pH was evaluated the temperature was 30 °C and when temperature was assayed pH was 7.
60

A. Sarquiz et al.
JournalofFluorineChemistry214(2018)58–62
Fig. 4. Optimization of FUdR galacto-
sylation biosynthesis.
(a) Substrate molar ratio. (b) FUdR
galactosylation catalyzed by β-galacto-
sidase at different times. Reaction
conditions: Buffer Tris-HCl 25 mM pH
7.0 containing 2 mM FUdR, 1 mM
ONPG, 1 mM NaCl and 0.3 U of β-ga-
lactosidase. No significant differences
among treatments were named with
the same letter (LSD test, p-value <
0.05).
able to biosynthesize galactosylated floxuridine at short reaction times
with high yields. These results indicate that the use of immobilized
enzyme is an alternative to produce a broad spectrum of galactosylated
nucleosides employing an environmentally friendly methodology.
Table 2
Transglycosylation efficiencies of different β –galactosidase.
Source of enzyme
Yield Reaction Type of
Reuses Reference
(%)
Time (h) biocatalyst
Bovine liver (comercial)
Bovine liver
75
72
60
11
Free enzyme
Free enzyme
1
1
[8]
[9]
Acknowledgments
(crude extract)
Bovine liver
(purified enzyme)
Bovine liver
(crude extract)
Bacteria
This research was supported by National Agency for Scientific and
Technological Promotion (PICT 2013-2658 and PICT 2014-3438),
National Council of Scientific and Technical Research (PIP 2014-KA5-
00805) and National University of Quilmes (PUNQ 1409/15).
50
68
80
24
11
7
Free enzyme
Free enzyme
1
[23]
1
[24]
Immobilize
enzyme
16
This study
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