Penicillin Acylase-Catalyzed Solid-State Ampicillin Synthesis
FULL PAPERS
preparations was determined from the initial rate of the
enzymatic hydrolysis of benzylpenicillin as described earlier.[5]
The content of water in the enzyme preparations was
determined gravimetrically.
Conclusions
The possibility to perform effective penicillin acylase-
catalyzed acyl transfer to the nucleophile in the systems
at very low water content was demonstrated for the first
time. Both reaction rate and maximum ampicillin yield
were shown to be dependent on the water content,
enzyme loading, and the physicomechanical properties
of the reaction mixtures. The suggested method for the
stabilization of immobilized enzyme preparations by
inorganic salt hydrates allowed us to achieve a high
effectivity of ampicillin synthesis from the equimolar
substrate mixture and this makes the solid-state bio-
catalytic processes attractive for the further develop-
ment.
Stabilization of Penicillin Acylase Preparations by Salt
Hydrates
To study an effect of salt hydrates on the stability of PA
preparations, a weighed amount of the immobilized enzyme
was kept in a closed vessel with fixed water activity at room
temperature in the mixture with Na2SO4 ¥ 10 H2O, MgSO4 ¥
7 H2O or NaCH3COO ¥ 3 H2O (up to 30% w/w). The residual
activity of penicillin acylase preparations in the course of this
incubation was determined as described above.
Ampicillin Synthesis in the Solid-State Systems
Experimental Section
Preparation of the reaction mixture: the equimolar solution of
reagents (0.1 M of 6-APA and 0.1 M of D-PGM) in 0.01 M
phosphate buffer was adjusted to pH 6.5 usinga 1 M KOH and
dried in a closed desiccator (aw 0.4) at room temperature.
Then the mixture of reagents, containing less than 3% w/w of
water, was crushed and the obtained fine powder was used to
mix with enzyme preparation. Enzymatic synthesis was started
by mixinga 20 200 mgof immobilized PA (as a wet powder
alone or mixed with correspondingsalt hydrate), with the dry
powder of the reagents. Theonly source of water inthe reaction
mixture was water added with the moist enzyme preparation.
All reactions were performed in the desiccator at a fixed water
activity and room temperature. The content of water in the
reaction system was determined gravimetrically. Samples of
the reaction mixture were prepared by dilutingan aliquot of
the mixture in eluent to stop the enzymatic reaction, and
analyzed by HPLC.
Reagents
The Eupergit C-immobilized penicillin acylase was from
Bristol-Myers-Squibb (USA). The activity of the moist
preparation was 30 U/mg. Concentration of the active sites in
immobilized penicillin acylase, determined by titration with
phenylmethylsulfonyl fluoride (PMSF),[15] was 30 nmol per 1 g
of moist enzyme preparation. Benzylpenicillin and 6-amino-
penicillanic acid (6-APA) were from Fluka (Switzerland),
À
À
ampicillin, d-( )-phenylglycine (D-PG), and d-( )-phenyl-
glycine methyl ester (D-PGM) were from DSM (The Nether-
lands). Other chemicals and buffer components were from
Merck, Darmstadt, Germany. Organic solvents were of
analytical grade.
Analysis
Samples were analyzed by HPLC usingan Altex 110A pump, a
4.6 Â 150 mm Nucleosil C-18 column, an LKB 2138 S UV
detector at 214 nm and an LKB 2221 integrator. The eluent was
Acknowledgements
This work was financially supported by the Russian Foundation
for Basic Research (Grants 00-04-48658 to V.K.S. and 02-04-
À
1
prepared by adjustingthe pH of a 0.68 gL
solution of
œ
À
1
KH2PO4 in acetonitrile/water (30:70 v/v) containing0.68 gL
sodium dodecyl sulfate to 3.0 with phosphoric acid. The flow
06310 to M.I.Y.) and the Moscow City Government (Grant GB-
œ
18/9 to V.K.S.). We would like to thank DSM and Bristol-Myers-
À
1
rate was 1.0 mL min .
Squibb for kindly donated reagents.
Studies on Stability of Immobilized Penicillin Acylase
at the Low Water Content
References
To study the properties of immobilized penicillin acylase under
the conditions of solid-phase reactions, 100 200 mgof the
moist preparation, placed onto a slide, were kept in a closed
vessel with fixed water activity at room temperature and the
residual catalytic activity was determined in the samples taken
at intervals. The pressure of water vapor was maintained at a
fixed level usingsulfuric acid solutions of various concentra-
tions. The thermodynamic water activity (aw) was determined
as P/Ps, where P is the pressure of water vapor over the acid
solution and Ps is the pressure of saturated water vapor at a
given temperature. The residual activity of penicillin acylase
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œ
[5] M. I. Youshko, A. V. Sinev, V. K. Svedas. Biochemistry
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897