January 2000, Volume 36, Issue 1, pp 4–7
Article Received: 17 August 1998

Preparative dephosphorylation of calcium salts of 2′(3″)-mononucleotides with extracellular phosphatase from Spicaria violacea
Authors: V. N. BaraiI. A. SeverinaT. A. KukharskayaA. I. Zinchenko
Institute of MicrobiologyNational Academy of SciencesBelarus

Abstract
The properties of the phosphatase present in the culture liquid ofSpicaria violacea were investigated. Based on these results, a method for preparative dephosphorylation of calcium salts of 2′(3′)-mononucleotides was proposed. A 96–98% yield was achieved at a substrate concentration of 100 mg/ml. Mild quantitative hydrolysis of RNA to nucleosides can be performed by RNA digestion to mononucleotides with Ca2+ followed by the proposed dephosphorylation procedure.

Keywords
Apply Biochemistry Nucleoside Culture Liquid Calcium Salt Chemical Hydrolysis

Preparative Dephosphorylation of Calcium Salts
of 2'(3")-Mononucleotides with Extracellular Phosphatase
from Spicaria violacea
V. N. Barai, I. A. Severina, T. A. Kukharskaya, and A. I. Zinchenko
Institute of Microbiology, National Academy of Sciences, Belarus
Received August 17, 1998
Abstract--The properties of the phosphatase present in the culture liquid of Spicaria violacea were investigated. Based on these results, a method for preparative dephosphorylation of calcium salts of 2'(3')-mononucleotides was proposed. A 96-98% yield was achieved at a substrate concentration of 100 mg/ml. Mild quantitative
hydrolysis of RNA to nucleosides can be performed by RNA digestion to mononucleotides with Ca 2§ followed
by the proposed dephosphorylation procedure.
Recently we proposed a two-step method for preparation
of natural ribonucleosides by chemical hydrolysis
of RNA with Ca 2§ ions followed by dephosphorylation
of the resulting calcium salts of mononucleotides
with phosphatase bound to the mycelium of the microscopical
fungus Spicaria violacea [1, 2]. By doing so,
we attempted to combine the advantages of chemical
hydrolysis of RNA (fast hydrolysis of RNA to mononucleotides)
and the enzymatic approach, which is characterized
by highly efficient dephosphorylation of
mononucleotides. The latter is related to the fact that
the resulting orthophosphate is precipitated by calcium
ions and does not inhibit the phosphatase any more.
In spite of these advantages, it was necessary to
increase the starting RNA concentration in the reaction
mixture from 20 to at least 100 mg/ml in order to make
the method competitive with conventional chemical
protocols [3, 4]. We had demonstrated that the phosphatase
bound to the mycelium of Spicaria violacea
can dephosphorylate calcium salts of 2'(3')-mononucleotides
with high yields at concentrations up to
100 mg/ml [5]. However, such a high concentration
requires the presence of great amounts of mycelium in
the reaction mixture. This hampers stirring the mixture
and isolating nucleosides.
We found that the culture liquid obtained during cultivation
of Spicaria violacea BM-105D in a phosphatedeficient
medium at acidic pH contained several times
more phosphatase than was bound to the mycelium.
This opened up new possibilities for the practical use of
the enzyme.
The goal of the study was to investigate the properties
of phosphatase in the culture liquid (CL) of Spicaria
violacea BM-105D and to apply it to ribonucleoside
preparation.
MATERIALS AND METHODS
RNA was isolated from baker yeast as in [6].
The mixture of calcium salts of 2'(3')-nucleoside
monophosphates was obtained by RNA hydrolysis
according to our method described in [ 1 ].
The fungus S. violacea was cultivated as in [7]. The
starting culture medium was acidified with 0.2% lactic
acid.
Phosphatase activity was assayed by the rate of
p-nitrophenyl phosphate dephosphorylation. The reaction
mixture (1 ml) containing 0.01 M p-nitrophenyl
phosphate, 0.1 M Na-acetate buffer (pH 5.0) and 20 I.tl of
the assayed solution was incubated at 60~ for 15 min.
The reaction was terminated with 2 ml of 0.4 M NaOH.
The amount of the resulting p-nitrophenol was measured
spectrophotometrically at 400 nm. The amount of
the enzyme converting 1 nmol of the substrate per
minute was taken to be unity.
The effect of pH on the phosphatase activity was
investigated in two buffers: 50 mM Na acetate buffer
(pH from 4.0 to 6.5) and Tris-HC1 buffer (pH from 7.0
to 9.0).
Stability of the enzyme at various pH values was
investigated by incubating samples of the CL filtrate
containing the above buffers at the concentration of
50 mM at 50~ for 30 min.
For investigation of thermal stability of the phosphatase,
samples of the CL filtrate containing 50 mM
Na-acetate buffer (pH 5.0) were incubated at required
temperatures for 30 min.
All numerical values reported in the study are mean
values of three or four measurements.
0003-6838/00/360t-0004525.00 9 2000 MAIK "Nauka/lnterperiodica"
PREPARATIVE DEPHOSPHORYLATION OF CALCIUM SALTS 5
RESULTS AND DISCUSSION
The experiments with the CL filtrate (phosphatase
not isolated) described below could be accomplished
owing to the fact that the filtrate had a high enzymatic
activity (4-5 U/ml) and retained this activity without
any notable loss at 40~ for several weeks. Note that
the phosphatase activity of the CL might be contributed
by several proteins.
First, we studied the effects of pH and temperature
on the phosphatase activity and stability in the CL filtrate
of S. violacea BM-105D. The results are presented
in Figure l a. They demonstrate that nearly all phosphatase
activity was observed under acidic conditions,
the pH optimum being at 5.25-5.75, and the enzyme
was stable at pH values from 4.5 to 6.5.
It is seen from Figure I b that the optimum temperature
for the phosphatase activity is 60~ Nevertheless,
15 m heating of the enzyme at temperatures above
50~ inactivated it to a marked extent. After heating at
60~ the CL filtrate retained only one-third of the
starting phosphatase activity, and heating at 65~ inactivated
the enzyme by 96%. These data may be indicative
of an effect of enzyme protection from heat inactivation
by the substrate (or reaction product). This was
especially notable at 65~ We performed experiments
to find out what stabilizes the enzyme.
The results of the experiments, shown in Table 1,
indicate that orthophosphate, a common product of
reactions catalyzed by the enzyme, exerted the greatest,
concentration-dependent, stabilizing action. It is worth
noting that the other product demonstrated no stabilizing
action on the enzyme. It was astonishing that the
substrate could only slightly protect the enzyme from
heat inactivation. Incubation of the CL filtrate with the
substrate at 65~ brought about its 50% dephosphoryTable
1. Effect of the substrate and the reaction products on
the thermal stability of phosphatase in S. violacea CL filtrate
Substrate
No
2'(3')-AMP
Adenosine
Orthophosphate
Orthophosphate
Orthophosphate
Orthophosphate
2'(Y)-AMP + orthophosphate

Heating (65~ 10 min)
Concentration
of the reaction
product, mM
0
10
10
10
5
3
1
against 5
Remanent phosphatase
activity after heating, %*
5
9
5
32
30
26
18
10
* Phosphatase activity was measured at 60~ pH 5.5.
iation, and the resulting orthophosphate was expected
to be able to protect the enzyme. This contradiction was
resolved in an experiment on the effect of a mixture
orthophosphate-substrate on the enzyme stability. It
was found that the substrate completely eliminated the
stabilizing effect of orthophosphate. In other words,
neither the substrate nor its mixture with orthophosphate
can protect the enzyme from heat inactivation.
This may result in a mismatch between the optimum
temperatures for its activity and achievement of high
yields in prolonged reactions. This is confirmed by the
results shown in Figure 2. It can be seen that during the
first 5-10 min of the reaction, the enzyme was apparA,%

100
80
60
40
20
2
I I I I T '''~
4 5 6 7 8
pH
%
100 ~-~
80 I
6O
40
20
030 4 50 60 70
t, ~
Fig. 1. Effect of pH (a) and temperature (b) on phosphatase activity and stability in S. violacea CL filtrate: (1) activity, % of the
maximum; (2) remanent activity, % of the initial value.