Bariloche protein symposium argentine society for biochemistry and molecular biology

84
BIOCELL, 27 (Suppl. I), 2003
BT-P3.
SYNTHESIS OF NUCLEOSIDES ANALOGUES USING
IMMOBILIZED WHOLE CELLS
Jorge Trelles
1
, Leticia Bentancor
1
, Mariano Grasselli
1
, Elizabeth
Lewkowicz
1
, and Adolfo Iribarren
1,2
1
Universidad Nacional de Quilmes. R.S. Peña 180, (1876) Bernal,
Buenos Aires, Argentina. 
2
INGEBI, CONICET, Vuelta de Obligado
2490, (1428) Buenos Aires, Argentina. E-mail: [e-mail]
Whole immobilized cells have been used as efficient biocatalysts
in the production of many pharmaceutical compounds.
Sugar and/or base modified nucleosides have been extensively
employed in cancer and antiviral therapies and, more recently, for
antisense strategies. Traditionally, nucleosides are prepared by
chemical methods that often involve difficult, inefficient and time-
consuming multistage processes. Microbial preparations of both
natural and unnatural nucleosides are an alternative methodology,
which offer regio and stereoselectivity and simple experimental
conditions.
Recently, we have demonstrated the high efficiency of immobilized
E. coli BL21 for the synthesis of adenosine. The results showed
that this biocatalyst maintained high activity for long periods both
at 4ºC and 37ºC and previde good reuse capacity and high
productivity. In this work the results obtened using different
immovilized biocatalysts for the synthesis of nucleosides analogues
with optimal yield are shown.
BT-P4.
CYCLODEXTRINS SYRUP OBTAINED FROM STARCH
BY ENZYMATIC CONVERSION
Szerman, Natalia; Rosso, Adriana M.; Rossi, Ana L.; Ferrarotti,
Susana A. and Krymkiewicz, Norberto.
Departamento de Ciencias Básicas, Universidad Nacional de
Luján, cc 221, 6700 Luján, Buenos Aires, Argentina. E-mail:
[e-mail]
Cyclodextrins (CD) are cyclic oligosaccharides containing six (
α),
seven (
β) or eight (γ) glucose units α-1,4 linked made from starch
by cyclodextrin glucanotransferase (cgtase).They are able to form
inclusion complexes with many hydrophobic molecules, changing
their physical and chemical properties. for this reason, CD are
used in food industry for stabilizing flavors, masking odors,
emulsifying oils and protecting substances from decomposition
induced by light, temperature and air.
In this work, an enzymatic process was developed for the
production of CD syrup from starch. the enzyme was produced by
bacillus circulans df 9r, isolated in our laboratory, purified by starch
adsorption and preserved by lyophilization.
The production of CD was optimized using experimental designs.
the fixed conditions were 5% (w/v) cassava starch, 20 ue/g of
starch, ph 6.4, temperature 56°c and 90 minutes of reaction under
continuous shaking (100 rpm). the conversion of cassava starch to
CD achieved was 22.8% (w/w).
The product was concentrated under reduced pressure. the syrup
obtained was analyzed by hplc and the ratio of 
α: β: γ -CD was
0.65 : 1.00 : 0.18.
BT-P5.
MILK CLOTTING AND PROTEOLYTIC ACTIVITIES OF
PLANT PROTEASES - A COMPARISON TO CHYMOSIN
Pardo, Marcelo F.
1
; Brutti, Cristina, Jesica Rodríguez; Fernández
Gener, Martín B.; La Blunda, Julia; Natalucci, Claudia L.
2
 and
Caffini, Néstor O.
2
LIPROVE, Depto. Cs. Biológicas, Fac. Cs. Exactas, UNLP,
Argentina. E-mail: [e-mail]
The milk clotting and proteolytic activities of a new plant protease,
named onopordosin, isolated from Onopordon achantium L.
(Asteraceae), have been determined and compared to those of
chymosin using cow and goat milk. Fresh flowers were grinded
under liquid nitrogen, suspended in 0.1 M citrate buffer at pH 5.5
and centrifuged at 10.000g for 5 min (crude extract). Clotting
activity was measured on both bovine and caprine milk at 30ºC
and pH 6.5. The substrate (10 ml) was mixed with 0.2 ml of crude
enzyme extract. Aliquots (100 
µl) were taken at different times,
added to 700 
µl of 6M urea and 3 % β-mercaptoethanol, and then
mixed with an equal volume of denaturing electrophoresis buffer.
Tricine-SDS gels were used, as they are suitable to resolve mixtures
of small peptides. Characteristic and differential proteolytic
patterns on the main casein components (
α
s1
, 
α
s2 
and 
β fractions)
were obtained for each substrate. After 10 min minimum
degradation rate was observed for 
α
s2
, 
α
s1 
and 
β caseins for both
substrates. New peptides appeared in all cases after 60 min (20
kDa, 16 kDa and 14 kDa). The patterns could be correlated with
the modification of functional properties and the production of
bitter peptides.
1
UNLP fellow. 
2
CIC Researcher Career. Supported by grants from
ANPCyT, CONICET, UNLP and UNLu
BT-P6.
HIERONYMAIN II, A NEW PEPTIDASE FROM FRUITS
OF BROMELIA HIERONYMI MEZ (BROMELIACEAE)
Bruno, Mariela A.; Stabile, Mª de las Mercedes; Mercerat, Julio
R.; Caffini, Néstor O.
1
 and López, Laura M.I.
2
LIPROVE, Depto. de Cs. Biológicas, Fac. Cs. Exactas, UNLP, La
Plata. E-mail: [e-mail]
The best known family of cysteine peptidases is the papain family,
which contains endopeptidases with broad specifity (such as
papain), or a narrow specifity (such as glycyl endopeptidase),
aminopeptidases, and peptidases with both endo- and exopeptidase
activities. Most plant cysteine peptidases belong to the papain
family, including those of Bromeliaceae. From unripe fruits of
Bromelia hieronymi Mez (Bromeliaceae) a new peptidase, named
hieronymain II, was purified to homogeneity by acetone
fractionation followed by anionic exchange chromatography
(FPLC) on Q- Sepharose HP and rechromatographied employing
the same exchanger but different saline gradient. Homogeneity of
the enzyme was confirmed by isoelectric focusing-zymogram.
Hieronymain II is a basic peptidase (pI 8.3) and its molecular mass
was 25 kDa (SDS-PAGE). Maximum proteolytic activity on casein
(more than 90% of maximum activity) was achieved at pH 7.5-
9.0. The enzyme was completely inhibited by E-64 and iodoacetic
and activated by the addition of cysteine; these results strongly
suggest that the isolated protease should be included within the
cysteine group.
1 
CIC and 
2
CONICET Researcher Career. Supported by grants from
ANPCyT, CONICET, CIC, UNLP and CYTED.

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