Enzymatic transglycosylation of natural and modified nucleosides by immobilized thermostable nucleoside phosphorylases from Geobacillus stearothermophilus

Article abstract of DOI:10.1134/S1068162009060107

Natural and modified purine nucleosides have been synthesized using the recombinant thermostable enzymes purine nucleoside phosphorylase II (E. C. 2.4.2.1) and pyrimidine nucleoside phosphorylase (E. C. 2.4.2.2) from Geobacillus stearothermophilus B-2194. The enzymes were produced in recombinant E. coli strains and covalently immobilized on aminopropylsilochrom AP-CPG-170 after heating the cell lysates and the removal of coagulated thermolabile proteins. The resulting preparations of thermostable nucleoside phosphorylases retained a high activity after 20 reuses in nucleoside transglycosylation reactions at 70-75°C with a yield of the target products as high as 96%. Owing to the high catalytic activity, thermal stability, the ease of application, and the possibility of repeated use, the immobilized preparations of thermostable nucleoside phosphorylases are suitable for the production of pharmacologically important natural and modified nucleosides.

Full text of DOI:10.1134/S1068162009060107

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 6, pp. 739–745. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © S.A. Taran, K.N. Verevkina, S.A. Feofanov, A.I. Miroshnikov, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 6, pp. 822–829.
Enzymatic Transglycosylation of Natural and Modified
Nucleosides by Immobilized Thermostable Nucleoside
Phosphorylases from Geobacillus stearothermophilus
S. A. Tarana, b,1, K. N. Verevkinaa, S. A. Feofanova, and A. I. Miroshnikova
a Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Division, Russian Academy of Sciences, Pushchino,
Moscow oblast, 142290 Russia
b Pushchino State University, Pushchino, Moscow oblast, 142290 Russia
Received March 23, 2009; in final form, April 2, 2009
Abstract—Natural and modified purine nucleosides have been synthesized using the recombinant thermostable
enzymes purine nucleoside phosphorylase II (E. C. 2.4.2.1) and pyrimidine nucleoside phosphorylase (E. C. 2.4.2.2)
from Geobacillus stearothermophilus B-2194. The enzymes were produced in recombinant E. coli strains and
covalently immobilized on aminopropylsilochrom AP-CPG-170 after heating the cell lysates and the removal of
coagulated thermolabile proteins. The resulting preparations of thermostable nucleoside phosphorylases retained a
high activity after 20 reuses in nucleoside transglycosylation reactions at 70–75°C with a yield of the target products
as high as 96%. Owing to the high catalytic activity, thermal stability, the ease of application, and the possibility of
repeated use, the immobilized preparations of thermostable nucleoside phosphorylases are suitable for the production
of pharmacologically important natural and modified nucleosides.
Key words: purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, transglycosylation, immo-
bilized enzymes, modified nucleosides
DOI: 10.1134/S1068162009060107
INTRODUCTION
oisomers in the reaction [1, 2]. An alternative way of
obtaining natural nucleosides and their modified ana-
logues is the biosynthetic process catalyzed by N-deox-
yribosyltransferases or nucleoside phosphorylases
from microorganisms. Owing to a wide substrate spec-
ificity, nucleoside phosphorylases are more suitable
biocatalysts for industrial application [3–5].
Natural nucleosides and their modified analogues
find wide application in the production of chemothera-
peutic drugs for the treatment of viral and cancer dis-
eases.2 Also, they are used in the synthesis of, and the
search for, new compounds that are of interest in other
fields of pharmacology, as well as precursors in oligo-
nucleotide synthesis in diagnostic and therapeutic pur-
poses.
Nucleoside phosphorylases are a group of key
enzymes involved in the synthesis and exchange of
purine and pyrimidine nucleosides in living organisms,
from bacteria to mammals [6]. In the presence of phos-
phate ions, these enzymes catalyze the reversible phos-
phorolysis of ribo-, arabino-, and deoxyribonucleosides
to pentoso-1-phosphate and the corresponding bases. In
the transglycosylation reaction catalyzed by two phos-
phorylases having different substrate specificity, the
phosphate-dependent transfer of pentose between the
bases and/or nucleosides present in the mixture takes
place, which results in the formation of nucleosides dif-
ferent from the starting compounds. In particular, the
enzymes uridine phosphorylase (UP, E. C. 2.4.2.3) and
purine nucleoside phosphorylase (PNP, E. C. 2.4.2.1)
can be used together for obtaining the natural nucleo-
sides and their analogues in transglycosylation reac-
Nucleosides containing modifications in the base or
at the ribofuranose residue are obtained, as a rule, by
multistage chemical synthesis. This synthesis is very
laborious and gives a low yield of target products,
owing to the formation of a mixture of regio- and stere-
1
Corresponding author; phone: 8 (4967) 73-17-80; e-mail: corre-
sponder@rambler.ru.
Abbreviations: aAdo, 9-β-D-arabinofuranosyladenine; aUrd, 1-β-D-
2
arabinofuranosyluracil; 2ClAde, 2-chloroadenine; 2ClAdo, 2-chloro-
adenosine; 2CldAdo, 2-chloro-2'-deoxyadenosine; dThd, thymidine;
dRibP, 2-deoxyriboso-1-phosphate; 2FAdo, 2-fluoroadenosine;
2FaAdo, 9-β-D-arabinofuranosyl-2-fluoroadenine; IEP, immobilized
enzyme preparation; IPTG, isopropylthio-β-D-galactopyranoside;
PuNPII, purine nucleoside phosphorylase from G. stearothermo-
philus; PyNP, pyrimidine nucleoside phosphorylase from
G. stearothermophilus; Thy, thymine.
739

740
TARAN et al.
tions using, e.g., uridine, 2'-deoxyuridine, dThd, or trum of natural and modified purine bases as acceptors
aUrd as the donors of the sugar residue and a wide spec- [7] (see the scheme).
O
NH2
CH3
N
HN
N
O
N
H
Thy
N
H
N
Ade
NH2
O
N
CH3
N
N
N
HN
N
O
O
HO
O
OH
P
HO
HO
O
+ Pi
+ Pi
O
O
O–
PyNP
PyNPII
OH
OH
OH
dThd
dRibP
dAdo
Transglycosylation with the formation of dAdo.
The major advantage of bioconversion over chemi- 70°C is preferable for conducting the transglycosyla-
cal synthesis is the absolute regio- and stereospecificity tion, since it corresponds to the optimum of enzymatic
of processes catalyzed by nucleoside phosphorylases. activity of nucleoside phosphorylases, including those
In addition, these reactions do not require any cofactors from mesophiles [4, 12]. Also, these conditions permit
and can proceed in ecologically and technologically one to introduce into the reaction nucleosides and bases
safe water buffers. However, the limitations on the sta- at higher concentrations, which ultimately increase the
bility of biocatalysts may play an important role if spe- yield of the target product. As sources of nucleoside
cial reaction conditions are required. Thus, to provide phosphorylases, the authors of numerous papers gener-
high concentrations of weakly soluble purine nucleo- ally used bacterial strains that were either selected from
sides and their modified analogues, transglycosylation different bacterial collections by microbiological
processes must be conducted at elevated temperatures screening [12, 13] or were obtained using the classical
and pH; however, these conditions usually lead to the selection and mutagenesis methods [9]. In some inves-
destabilization of the quaternary and tertiary structures tigations, purified enzymes isolated primarily from
of the protein and in the final analysis the loss of the mesophiles were used [5]. However, these examples of
enzymatic activity. Enzymes of thermophilic bacteria transglycosylation cannot be considered as universal
stabilized by immobilization seem to be attractive as for the preparation of a wide spectrum of nucleosides
biocatalysts in the nucleotide synthesis, since they offer owing to the thermal instability of these preparations
a number of advantages over the enzymes of meso- and the nonoptimal ratio of enzymatic activities in
philic microorganisms [8] and whole bacterial cells them.
[4, 9–11] that are usually used for these purposes.
The E coli strains BL21(DE3)/pBstPNPII and
The goal of this study was to obtain IEPs of recom- BL21(DE3)/ pBstPYNP, which produce, respectively,
binant thermostable nucleoside phosphorylases PuNPII PuNPII and PyNP of G. stearothermophilus BKM-2194,
(E. C. 2.4.2.1) and PyNP (E. C. 2.4.2.2) from the ther- were created in our laboratory (data not published) for
mophilic bacterium Geobacillus stearothermophilus obtaining thermostable nucleoside phosphorylases and
B-2194 and develop a highly effective biocatalytic pro- the study of their properties with the aim of finding
cess based on the use of these preparations in transgly- approaches to the synthesis of natural and modified
cosylation reactions for the synthesis of a number of nucleosides. During the cultivation of the producers on
natural and modified nucleosides.
the rich nutrient medium TB [14], both enzymes accu-
mulated in the cytoplasm in the soluble state and the
yield of nucleoside phosphorylases upon the induction
of the culture by 0.2 mM IPTG was 40% of the total
RESULTS AND DISCUSSION
It was shown in papers devoted to the technological cellular protein, as indicated by SDS-PAGE. Owing to
production of nucleosides that a temperature of 50– the high thermal stability of recombinant G. stearother-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 6 2009

ENZYMATIC TRANSGLYCOSYLATION OF NATURAL AND MODIFIED
741
Table 1. Characteristics of preparations of G. stearothermophilus nucleoside phosphorylases isolated from recombinant
E. coli strains
Strain
Enzyme preparation Protein concentration, mg/ml carrier Specific activity, unit/mg protein
BL21(DE3)/pBstPNPII
BL21(DE3)/pBstPYNP
PuNPII
PyNP
8.3 1.2
7.8 1.4
55
4
7
112
Notes: * The activity was determined by the phosphorolysis of the corresponding substrates (inosine for PuNPII and thymidine for PyNP);
one unit of activity was defined as that amount of the enzyme that converts 1 μmol of nucleoside in 1 min at 37°C.
mophilus nucleoside phosphorylases, we were able to on the other hand, to provide a complete absence of
simplify the multistage procedure of purification of the
enzymes and separate them from the host proteins by
heating the cell lysates for 30 min to 70°C with the sub-
sequent sedimentation of thermolabile aggregates. The
characteristics of the nucleoside phosphorylase prepa-
rations obtained are given in Table 1.
Thus, the genetically engineered bacterial strains
provide a high level of production of recombinant
nucleoside phosphorylases of the thermophilic bacte-
rium G. stearothermophilus, which opens up wide pos-
sibilities for their use, together with E coli nucleoside
phosphorylases [8] and whole-cell biocatalysts [9, 11],
as suitable sources of phosphorylases for the synthesis
of natural or modified nucleosides in transglycosylation
reactions.
The preparations of recombinant nucleoside phos-
phorylases of G. stearothermophilus and E. coli were
immobilized on aminopropylated macroporous glass
AP-CPG-170. Preliminarily, the sorbent was treated
with glutaraldehyde as described in [15]. In the pres-
ence of sodium boron hydride, a covalent bond between
the aldehyde groups of glutaraldehyde and the amino
groups of the sorbent and enzymes is formed [16].
Attempts to immobilize Bacillus stearothermophilus
nucleoside phosphorylases by reversible sorption on an
anion-exchange resin were reported [17]. Despite small
losses in the nucleoside phosphorylase activity during
the immobilization, this approach, nevertheless, did not
enable one to create a biocatalyst stable enough for the
synthesis of, e.g., deoxyguanosine, which requires
extreme conditions of temperature and pH. At the same
time, there is evidence indicating that the covalent
protein desorption from the support (Table 2) upon fur-
ther use. In addition, owing to the availability of indi-
vidual enzymes isolated from producer strains, it
became possible to obtain both mono- and bienzymatic
IEPs and rapidly vary the ratio of activities of immobi-
lized enzymes during the coimmobilization of proteins
on the support (see the Experimental section).
In the course of the study, we obtained data on the
stability of IEPs of G. stearothermophilus nucleoside
phosphorylases in a wide range of pH and temperature
values. The pH optimum for IEP in the transglycosylation
reaction coincided essentially with the optimum of unim-
mobilized nucleoside phosphorylases (pH 7.5–8.0 [20]);
however, the range of values at which the enzyme
retained complete stability was wider (pH 6.5–11.5).
The temperature optimum in the synthesis of dAdo var-
ied from 80°C (at the PuNPII to PyNP activity ratio 1 : 2)
to 86°C (at the PuNPII to PyNP activity ratio 2 : 1),
Table 2. Characteristics of IEPs of G. stearothermophilus
nucleoside phosphorylases
Immobilized enzyme Protein concen- Specific activity,
preparation
tration, mg/ml
unit/mg protein
PuNPII
18.8 0.8
19.0 0.5
18.9 0.7
18.4 0.7
19.1 0.9
36 6*
74 8*
15 2**
12 2**
19 3**
PyNP
(PuNPII + PyNP) 1 : 1
immobilization of nucleoside phosphorylases stabilizes (PuNPII + PyNP) 2 : 1
the quaternary structure of enzyme complexes, which
extends the range of conditions of applying IEPs
toward higher temperatures and a wider range of pH
values [18, 19].
In this study, we optimized the conditions of the
covalent immobilization of nucleoside phosphorylases
on a solid support by adding stabilizing (inosine, dThd,
MgCl2) and adjuvant agents (Tween-20, BSA) to the
reaction. This enabled one, on one hand, to retain the
enzyme activity (about 70% of the initial activity) and,
(PuNPII + PyNP) 1 : 2
Notes: * The activity was determined by the phosphorolysis of the
corresponding substrates (see the note to Table 1).
** The activity was determined from the ability to catalyze
transglycosylation in the synthesis of dAdo under condi-
tions specified in the Experimental section; one unit of
activity in the transglycosylation reaction was defined as
that amount of the enzyme incorporated in IEP that pro-
vides the synthesis of 1 μmol of dAdo in 1 h at 75°C.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 6 2009

742
TARAN et al.
%
100
%
100
1
2
1
90
80
70
60
50
40
30
20
90
80
70
60
50
3
2
3
4
40
30
20
4
10
0
5
10
15
20
25
h
0
50
100
150
200 250
300 350
h
Fig. 1. Stability at 70°C of immobilized (1, 3) and unimmo-
bilized liquid enzyme preparations of G. stearothermophi-
lus nucleoside phosphorylases (2, 4): PuNPII (1, 2) and
PyNP (3, 4) (see the Experimental section). The maximum
degree of the conversion of the substrate by the enzyme at
the initial moment of its temperature incubation was taken
as 100%.
Fig. 2. Thermal stability of G. stearothermophilus (1, 2) and
E coli (3, 4) IEPs in the synthesis of dAdo at 60 (1, 3) and
70°C (2, 4) (see the Experimental section). The theoreti-
cally calculated yield of the target dAdo upon complete
conversion of adenine introduced to the reaction was taken
as 100%.
which is 5–10°C higher than the temperature optimum data obtained that the transglycosylase activity of the
determined for unimmobilized G. stearothermophilus
enzyme preparation upon 20 reuses decreased approxi-
mately by 0.6–0.7% per cycle at 70°C, which is, in our
opinion, a very good indicator and provides good tech-
nological prospects for these IEPs. In addition, owing
to the high specific activity of IEP, we managed to
achieve a more than 80% degree of conversion over a
period of 4–10 h with the content of the biocatalyst in
the reaction mixture being as low as 1–5% (by volume).
This enabled us to effectively use the IEPs for conduct-
ing transglycosylation reactions with a wide spectrum
of substrates, including weakly soluble substrates, such
as guanine, as well as for the synthesis of pharmaceuti-
cally significant preparations of 2CldAdo and 2FaAdo
(Table 3).
nucleoside phosphorylases.
The thermal stability of IEPs also significantly
increased compared with that of liquid preparations of
nucleoside phosphorylases (Fig. 1). The thermal stabil-
ity of IEPs of G. stearothermophilus nucleoside phos-
phorylases was examined in a series of transglycosyla-
tion reactions with the formation of dAdo at 60 and
70°C in comparison with IEPs of E. coli nucleoside
phosphorylases. For each temperature value, one and
the same sample of the biocatalyst was used, and the
total time of incubation was about 340 h (14 days). The
results obtained demonstrate a relative decrease in the
catalytic activity of IEP of G. stearothermophilus
nucleoside phosphorylases within 24 h by about 1% at
60°C and by about 9% at 70°C. At the same time, the
IEP of E. coli nucleoside phosphorylases under similar
conditions lose 8 and 45% of activity, respectively (Fig. 2).
These data confirm a higher stability of enzyme prepa-
rations of thermophilic microorganisms in nucleoside
transglycosylation reactions. This is most pronounced
in the case of pyrimidine-specific phosphorylases
whose thermal stability is a limiting factor in the bio-
conversion reaction.
Thus, we obtained preparations of G. stearothermophi-
lus nucleoside phosphorylases covalently immobilized on
aminopropylated macroporous glass AP-CPG-170. The
novel preparations of two immobilized thermostable
nucleoside phosphorylases exhibit high enzymatic
activity and stability, which makes them suitable for
repeated use as biocatalysts in transglycosylation reac-
tions. In our opinion, the application of immobiliza-
tion-stabilized nucleoside phosphorylases of thermo-
philic microorganisms, obtained from recombinant
producer strains, is the most optimal approach to the
biotechnological synthesis of nucleosides and their
analogues necessary for the needs of national medicine
By providing a high stability of IEPs, we obtained a
reproducible yield of dAdo with the repeated use (more
than 20 cycles) of one and the same sample of nucleo-
side phosphorylase IEPs. The separation of IEP from
the reaction mixture was carried out by filtration
through a glass filter; then, the IEP was transferred into
the next bioconversion reactions. It follows from the and other applications.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 6 2009

ENZYMATIC TRANSGLYCOSYLATION OF NATURAL AND MODIFIED
743
Table 3. Parameters of transglycosylation in the synthesis of natural and modified nucleosides using the IEPs of G. stearo-
thermophilus nucleoside phosphorylases*
Target IEP (activity of PuN- Pentose donor,
KH2PO4,
mM
Reaction
time, h
Base, mM
t, °C
pH
Yield**, %
product
PII : PyNP), ml
mM
dAdo
(1 : 2), 0.1
(2 : 1), 0.5
(1 : 2), 0.2
(1 : 2), 0.1
(1 : 1), 0.2
dThd, 15
dThd, 15
dThd, 15
aUrd, 15
aUrd, 15
Ade, 5
5
5
5
5
5
75
70
75
75
75
7.5
2
10
4
89
96
86
88
85
dGuo
Gua, 5
10.0***
7.5
2CldAdo
aAdo
2ClAde, 5
Ade, 5
7.5
4
2FaAdo
2FAde, 5
7.5
8
Notes: *The reaction volume is 10 ml.
** According to the data of HPLC.
*** The reaction was conducted in a 50-mM glycine-NaOH buffer (pH 10.0 at 70°C); for more detail, see the Experimental section.
EXPERIMENTAL
PII or BL21(DE3)/pBstPYNP (5 ml) was added to 100
ml of medium TB [14] containing bacto tryptone
(Difco, United States) (1.2 g), bacto yeast extract
(Difco, United States) (2.4 g), K2HPO4 (0.95 g), KH2PO4
(0.22 g), glycerol (0.8 g), and kanamycin, pH 7.0, (5
mg), and the culture was grown on a shaker (200–220
The following preparations were used: deoxyade-
nosine, deoxyguanosine, thymidine, inosine, sodium
boron hydride, and 2-mercaptoethanol (Sigma, United
States). 2ClAdo, 2ClAde, 2F-adenosine, and aUrd were
synthesized in the Laboratory of Biotechnology (Insti-
tute of Bioorganic Chemistry, RussianAcademy of Sci-
ences) by an ingenious method [21, 22]. Other chemi-
cal reagents were purchased from Promega (United
States), Serva (Germany), Fluka (Switzerland), and
Bio-Rad (United States) and were of special purity or
chemical grade. Aminopropylated glass AP-CPG-170
was used as a carrier for enzyme immobilization
(Fluka, Switzerland).
The E. coli BL21(DE3)/pBstPNPII and
BL21(DE3)/pBstPYNP producer strains have been
deposited in the collection of the Group of Technology
of Nucleic Acids and Their Components (Institute of
Bioorganic Chemistry, Russian Academy of Sciences,
Pushchino Division). Plasmids pBstPNPII and pBst-
PYNP were obtained previously in our laboratory (data
not published). They are the derivatives of plasmids of
the pET series (Novagen, United States) and contain,
respectively, the genes of PuNPII and PyNP of the ther-
mophilic bacterium G. stearothermophilus BKM-2194
under the control of the promoter of bacteriophage T7.
rpm) at 30°C for 4 h until the optical density of A550
=
1.0 was attained. Then, IPTG was added to a concentra-
tion of 0.2 mM, and the culture was incubated under the
same conditions for an additional 6 h. Optical absorp-
tion was measured at 550 nm on a UV-1800 spectro-
photometer (Shimadzu, Japan) using the software
package UV Probe. The level of expression was esti-
mated by electrophoresis in gradient (5–25%) SDS-
PAGE by monitoring the appearance of the major bands
of 27 and 46 kDa for PuNPII and PyNP, respectively.
Isolation of nucleoside phosphorylases. A cell cul-
ture was centrifuged at 4000–5000 g for 15 min. Sedi-
mented cells were suspended in two (weight/volume)
volumes of a homogenization buffer (25 mM K-phos-
phate, pH 7.5, 0.9% NaCl, 1 mM EDTA, 1 mM 2-mer-
captoethanol) and disrupted by ultrasound on a VC130
disintegrator (Cole-Parmer, United States). The homo-
genate was clarified by centrifugation for 20 min at
10000 g, and the supernatant was transferred to a glass
vessel and incubated for 30 min in a water bath at 70°C
with gentle stirring. The suspension was cooled to 4°C
and centrifuged to sediment denatured thermolabile
cellular proteins of E. coli at 10000 g for 20 min. In the
supernatant, the protein concentration was determined
by the Bradford method [24] and the specific activity of
nucleoside phosphorylases was estimated by the phos-
phorolysis of the corresponding substrates. One unit of
activity of PuNPII was defined as that amount of the
enzyme that converts 1 μmol of inosine to hypoxan-
thine in 50 mM potassium phosphate, pH 7.5, for 1 min
E. coli purine nucleoside phosphorylase and thymi-
dine phosphorylase isolated in the Laboratory of Bio-
technology (Institute of Bioorganic Chemistry, Russian
Academy of Sciences) from E. coli strains
BL21(DE3)/pERPUPH01 and BL21(DE3)/pERTPH01
[8], respectively, were used as control enzymes.
Manipulations of plasmid DNA, the preparation of
competent cells of E. coli strain BL21(DE3), and trans-
formation were carried out by standard methods [23].
Thermostable nucleoside phosphorylases of
G. stearothermophilus BKM-2194. A night inoculate at 37°C. One unit of activity of PyNP was defined as
of the recombinant E. coli strain BL21(DE3)/pBstPN- that amount of the enzyme that converts 1 μmol of thy-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 6 2009

744
TARAN et al.
midine to thymine in 50 mM potassium phosphate, 20% DMSO (v/v), IEPs of nucleoside phosphorylases
pH 7.5, for 1 min at 37°C. The resulting liquid enzyme (0.5 ml) (a PuNPII/PyNP activity ratio of 2 : 1), and
preparations were used for immobilization.
guanine (7.5 mg, 0.05 mmol) was incubated for 10 h at
70°C with gentle stirring. The yield of dGuo was 96%.
For the immobilization of nucleoside phosphorylase
preparations, aminopropylated glass AP-CPG-170 and
E. coli and G. stearothermophilus nucleoside phospho-
rylase preparations were essentially prepared as
described earlier [25]. Liquid preparations of PuNPII
and PyNP of G. stearothermophilus BKM-2194
obtained from the strains BL21(DE3)/pBstPNPII and
BL21(DE3)/pBstPYNP were diluted to a concentration
of the protein of 5 mg/ml by a buffer containing 50 mM
K-phosphate, pH 7.5, 50 mM MgCl2, 1 mM EDTA,
1 mM 2-mercaptoethanol, 0.2% Tween-20, and
0.1 mg/ml of BSA. Also, inosine and thymidine were
added to the buffer to a concentration of 10 mM for
PuNPII and PyNP, respectively. Aminopropylsilo-
chrom AP-CPG-170 prepared for immobilization and
liquid preparations of nucleoside phosphorylases were
mixed in a ratio of 20 mg of the protein of the enzyme
preparation per 1 mg of the carrier. Mixtures for immo-
bilization were prepared as monoenzymatic for each of
the nucleoside phosphorylases or multienzymatic with
different ratios of specific activities of PuNPII and
PyNP by varying the ratio of liquid enzyme prepara-
tions. The immobilization was carried out for 16 h at
room temperature under gentle stirring. Then, sodium
boron hydride was added to the reaction mixture to a
final concentration of 1 mg/ml and incubation was con-
tinued for 1 h. The preparation was washed five times
with two volumes of a 50-mM K-phosphate buffer,
pH 7.5. IEPs were stored in a 50-mM K-phosphate buffer
supplemented with sodium azide (0.2%) at 2–4°C.
2-Chloro-2'-deoxyadenosine (2ClAdo, cladribine).
A reaction mixture (10 ml) containing a 5-mM K-phos-
phate buffer (pH 7.5), 5 mM 2ClAde, 15 mM thymi-
dine, 10% DMSO (v/v), and IEPs of nucleoside phos-
phorylases (0.2 ml) (the PuNPII/PyNP activity ratio 1 : 2)
was incubated for 4 h at 75°C with gentle stirring. The
yield of 2ClAdo was 86%.
9-β-D-Arabinofuranosyladenine (aAdo). A reaction
mixture (10 ml) containing a 5-mM K-phosphate buffer
(pH 7.5), 5 mM adenine, 15 mM aUrd, 10% DMSO
(v/v), and IEPs of nucleoside phosphorylases (0.1 ml)
(a PuNPII/PyNP activity ratio of 1 : 2) was incubated
for 4 h at 75°C with gentle stirring. The yield of aAdo
was 88%.
9-β-D-Arabinofuranosyl-2-fluoroadenine (2FaAdo,
fludarabine). A reaction mixture (10 ml) containing a
5-mM K-phosphate buffer (pH 7.5), 5 mM 2FAde (or
2FAdo), 15 mM aUrd, 20% DMSO (v/v), and IEPs of
nucleoside phosphorylases (0.2 ml) (a PuNPII/PyNP
activity ratio of 1 : 1) was incubated for 8 h at 70°C with
gentle stirring. The yield of 2FaAdo was 85%.
Analysis of reaction products was performed by
TLC on Kiselgel 60-F254 plates (Merck, Germany) in
the solvent system chloroform–ethanol (4 : 1) and by
HPLC using a 254-nm detector (Beckman, United
States) in the isocratic operation mode on Separon SGX
C18 5 μl columns (3 × 150 mm). As an eluent, 5%
MeCN in 10 mM KH2PO4 was used; the elution rate
was 0.5 ml/min. After separation by TLC and HPLC,
the fractions of individual products were analyzed on a
UV-188 spectrophotometer (Shimadzu, Japan) by mea-
suring the spectra in the wavelength range from 200 to
340 nm and comparing them with standard or reference
data.
For the determination of stability at 70°C, monoen-
zymatic IEPs and liquid enzyme preparations of
G. stearothermophilus nucleoside phosphorylases
were incubated for 24 h at 70°C. At regular intervals
(every 4 h), an equal volume of an enzyme preparation
was taken and added to 1 ml of the reaction of phospho-
rolysis of inosine and thymidine for PuNPII and PyNP,
respectively. After 15 min, the residual content of
nucleoside was determined by HPLC. The maximum
degree of the conversion of the substrate by the enzyme
at the initial moment of its temperature incubation was
taken as 100%.
ACKNOWLEDGMENTS
The authors are grateful to the research workers of
the Laboratory of Biotechnology (Institute of Bioor-
ganic Chemistry, Russian Academy of Sciences),
R.S. Esipov for providing the E. coli nucleoside phos-
phorylase preparations, V.B. Berzin for the synthesis of
2Cl-adenosine and 2F-adenosine, and K.V. Antonov for
the synthesis of 1-β-D-arabinofuranosyluracil, which
were necessary for performing the experiments.
This work was supported by the Russian Foundation
for Basic Research, project nos. 08-04-12143, 08-04-
01842) and the Federal Education Agency (project of
the Thematical Plan no. 1.1.09).
2'-Deoxyadenosine (dAdo). A reaction mixture
(10 ml) containing a 5-mM K-phosphate buffer
(pH 7.5), 5 mM adenine, 15 mM thymidine, 10%
DMSO (v/v), and 0.1 ml of IEP of nucleoside phospho-
rylases (a PuNPII/PyNP activity ratio of 1 : 2) was incu-
bated for 2 h at 75°C with gentle stirring. The yield of
dAdo was 89%. For repeated use, the IEP was sepa-
rated from the reaction mixture by filtration through a
glass filter and washed on the filter with a 50-mM K-
phosphate buffer.
REFERENCES
2'-Deoxyguanosine (dGuo). A reaction mixture
(10 ml) containing a 50-mM glycine-NaOH buffer
(pH 10.0 at 70°C), 5 mM K2HPO4, 15 mM thymidine,
1. Kawakami, H., Matsushita, H., Naoi, Y., Itoh, K., and
Yoshikoshi, H., Chem. Lett., 1989, vol. 1, pp. 235–238.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 6 2009

ENZYMATIC TRANSGLYCOSYLATION OF NATURAL AND MODIFIED
745
2. Witkowski, J.T., Robins, R.K., Sidwell, R.W., and 15. Juekiewicz, M., Alegret, S., and Fabregas, E., Anal.
Simon, L.N., J. of Med. Chem, 1972, vol. 15, pp. 1150–
Chim. Acta, 1998, vol. 370, pp. 47–58.
1154.
16. Kito, M., Tawa, R., Takeshima, S., and Hirose, S.,
J. Chromatogr., 1990, vol. 528, pp. 91–99.
3. Hanrahan, J.R. and Hutchinson, D.W., J. Biotechnol.,
1992, vol. 23, pp. 193–210.
17. Hori, N., Watanabe, M., Sunagawa, K., Uehara, K., and
Mikami, Y., J. Biotechnol., 1991, vol. 114, pp. 121–131.
4. Utagawa, T., J. Molec. Catalysis B: Enzymatic, 1999,
vol. 6, pp. 215–222.
18. Zuffi, G., Ghisotti, D., Ilaria, O., Capra, E., Frascotti, G.,
Tonon, G., and Orsini, G., Biocatal. Biotransform.,
2004, vol. 22, pp. 25–33.
5. Krenitsky, T.A., Koszalka, G.W., and Tuttle, J.V., Bio-
chemistry, 1981, vol. 20, pp. 3615–3621.
19. Ubiali, D., Rocchietti, S., Scaramozzino, F., Terreni, M.,
Albertini, A., Fernandez-Lafuente, R., Guisan, J.M., and
Pregnolato, M., Adv. Synth. Catal., 2004, vol. 346,
pp. 1361–1366.
6. Pugmire, M.J. and Ealick, E., Biochem. J., 2002,
vol. 361, pp. 1–25.
7. Hatchinson, D.W., Trends Biotechnol., 1990, vol. 8,
pp. 348–353.
20. Hori, N., Watanabe, M., Yamazaki, Y., and Mikami, Y.,
8. Esipov, R.S., Gurevich, A.I., Chuvikovsky, V.,
Chupova, L.A., Muravyova, T.I., and Miroshnikov, A.I.,
Protein Express. Purif., 2002, vol. 24, pp. 56–60.
9. Barai, V.N., Zinchenko, A.I., Eroshevskaya, L.A., Kalin-
ichenko, E.N., Kulak, T.I., and Mikhailopulo, I.A, Helv.
Chim. Acta, 2002, vol. 85, pp. 1901–1908.
10. Spoldi, E., Ghisotti, D., Cali, S., Grisa, M., Orsini, G.,
Tonon, G., and Zuffi, G., Nucleosides Nucleotides, 2001,
vol. 20, pp. 977–979.
11. Taran, S.A., Verevkina, K.N., Esikova, T.Z., Feo-
fanov, S.A., and Miroshnikov, A.I., Appl. Biochem.
Microbiol., 2008, vol. 44, pp. 162–166.
12. Utagawa, T., Morisawa, H.,Yamanaka, S.,Yamazaki, A.,
Yoshinaga, F., and Hirose, Y., Agric. Biol. Chem., 1985,
vol. 49, pp. 3239–3246.
13. Yokozeki, K. and Tsuji, T., J. Molec. Catalysis B: Enzy-
matic, 2000, vol. 10, pp. 207–213.
Agric. Biol. Chem., 1989, vol. 53, pp. 3219–3224.
21. Berzin, V.B., Dorofeeva, E.V., Leonov, V.N., Luton-
ina, O.I., and Miroshnikov, A.I., RF Patent no. 2324698,
from February 5, 2007.
22. Konstantinova, I.D., Antonov, K.B., Berzin, V.B., Doro-
feeva, E.V., Fateev, I.V., Muzyka, I.S., and Miroshni-
kov, A.I., RF Patent Application no. 2008126 081 from
June 27, 2008.
23. Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular
Cloning: A Laboratory Manual, New York: Cold Spring
Harbor, Cold Spring Harbor Lab. Press, 1989.
24. Bradford, A.T., Anal. Biochem., 1976, vol. 72, pp. 248–
254.
25. Konstantinova, I.D., Leont’eva, N.A., Galegov, G.A.,
Ryzhova, O.I., Chuvikovskii, D.V., Antonov, K.V., Esi-
pov, R.S., Taran, S.A., Verevkina, K.N., Feofanov, S.A.,
and Miroshnikov, A.I., Bioorg. Khim., 2004, vol. 30,
pp. 613–620 [Russ. J. Bioorg. Chem. (Engl. Transl.),
vol. 30, pp. 553–560].
14. Tartoff, K.D. and Hobbs, C.A., Bethesda Res. Lab.
Focus, 1987, vol. 9, p. 7.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 6 2009