Escherichia coli Bl21: A useful biocatalyst for the synthesis purine nucleosides

Article abstract of DOI:10.3390/50300535

E. coli BL21 cells were able to synthesize several purine nucleosides from pyrimidine ones. Kinetics and yields of this reaction showed a strong dependence on pH, temperature, reagent concentrations and weight of wet cell paste. Yields over 90% were reached in the synthesis of adenosine.

Full text of DOI:10.3390/50300535

535
Molecules 2000, 5
Escherichia Coli Bl21: A Useful Biocatalyst for the Synthesis of
Purine Nucleosides
M.C. Rogert1, N. Martínez1, S. Porro1, E. Lewkowicz1 and A. Iribarren1,2
1Universidad Nacional de Quilmes. R. S. Peña 180, (1876) Bernal, Buenos Aires, Argentina
2INGEBI, CONICET, Vuelta de Obligado 2490, (1428) Buenos Aires, Argentina
E-mail: elewko@unq.edu.ar
Abstract: E. coli BL21 cells were able to synthesize several purine nucleosides from py-
rimidine ones. Kinetics and yields of this reaction showed a strong dependence on pH, tem-
perature, reagent concentrations and weight of wet cell paste. Yields over 90% were reached
in the synthesis of adenosine.
Introduction
Modified nucleosides are wide diffused as antiviral and antitumor drugs as well as starting materi-
als for antisense oligonucleotides. In contrast to classical chemical synthesis of these, good results as
far as yield, simplicity and regiostereospecificity are concerned can be achieved at low cost using pure
enzymes or whole cells as biocatalysts.
The methodology employed in the synthesis of purine nucleosides involves the transfer of a sugar
residue from a donor pyrimidine nucleoside to an acceptor purine base. This process requires the pres-
ence of enzymes belonging to the family of transferases, specially the purine and pyrimidine nucleo-
side phosphorylases (PNP and PyNP) which are present in most of the microorganisms [1].
In this work, several experimental variables have been studied in order to select the best conditions
necessary for the E.Coli BL21 catalyzed synthesis of purine nucleosides.
Experimental
The E. coli strain was grown in Lb medium in shaked flasks at 27ºC until saturation, centrifuged
and washed with the suitable buffer. The resultant wet cell paste was used as catalyst of the reaction [2]
after suspending in phosphate buffer (5 ml) and addition of the nucleoside and purine base. The mix-
ture was stirred and heated at constant temperature in glycerin bath and the reaction products were
analyzed by tlc and hplc.

536
Molecules 2000, 5
Results and Discussion
One of the biotransformations studied in this work is shown in Figure 1.
O
NH2
NH
N
N
N
N
O
HOH2C
HOH2C
O
HOH2C
N
O
PyNP
O
O
PNP
O P
NH2
OH
OH
OH
OH
OH
OH
N
NH
N
N
H
N
H
O
N
Figure 1. Enzymatic synthesis of adenosine.
The reagents tested were the nucleosides uridine and thymidine and the bases adenine, hypoxan-
thine and guanine. The following variables have been studied: temperature, reagent concentrations, pH
and buffer concentration, biocatalyst amount and reaction time [3]. For example, adenosine was ob-
tained with a yield higher than 90% working at 60ºC in 30 mM phosphate buffer at pH 7, with excess
of uridine during 1 hour. Longer reaction times have been observed when tymidine was used as ribose
donor.
References and Notes
1. Pal, S.; Nair, V. Biocatal. and Biotransf. 1997, 15, 147.
2. Utagawa, T.; Morisawa, H.; Yoshinaga, F.; Yamazaki, A.; Mitsugi, K.; Hirose, Y. Agric.
Biol.Chem. 1985, 49 (4), 1053.
3. Krenitsky, T.; Koszalka, G.; Tuttle, J. Biochemistry 1981, 20, 3615.