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Fosfato sódico
----- 1 M
___ 100 mM
...... 10 mM
a) ¿Qué efecto tiene la presencia del fosfato sobre la inmovilización? Propón una posible explicación
mobilization courses of PCL (A) and TLL (B) on octyl agarose beads at different buffer concentrations. Experiments were performed as described in Section 2. Pointed
natants at 10 mMb) Calcula
sodium YE para
phosphate, solidla inmovilización
line: supernatants at 100sobre ambos
mM sodium soportes
phosphate, a las
dashed diferentes
line: supernatantsconcentraciones de fosfato
at 1 M sodium phosphate.
c) ¿cuál fue el valor de YP? ¿Cuál es la densidad de la enzima inmovilizada?
Supón que no existe desactivación enzimática durante el proceso de inmovilización.
t of experimental conditions on the irreversible resistant to irreversible inactivation. The small effect on the inhi-
n by D-pNPP using lipases immobilized on octyl and CNBr bition rate of the octyl-TLL of the ionic strength should be related
eads to the fact that the enzymes maintain their open form even under
2) Una ß-galactosidasa es inmovilizada sobrethe unhighest
soporte de agarosa heterofuncional. El rendimiento
ionic strength used. Only the interaction with the sup-
de inmovilización en proteína,
shows the inactivation of TLL immobilized on octyl or Y P=95%, y en actividad Y E=85%.
port may be stronger atEl protocolo
high de inmovilización
ionic strength and this may produce
consiste
rose beads by the action en poner en
of D-pNPP contacto
under 10 gcon-
different de soporte
some(densidad=0,7 g mL
minor diffusional -1 ) con 9 to
limitation mLthedeentry
una of
preparación
the inhibitor. The
he covalent preparation is inhibited slower
de ß-galactosidasa solublethan
y 93the
mLoctyl use of
de tampón 0.01% of detergent
bicarbonato, (CTAB)durante
en agitación, produced a significant
3 h. Al finalizarincrease
el
ion even at low proceso,
ionic strength
la actividad del catalizador inmovilizado es 286 U g , la actividad remanente en solución form
suggesting that the octyl in the inhibition rate
-1 of CNBr-TLL, suggesting that the open
ion has a higher exposition
es 2,6 U mL of the
-1, ycatalytic Ser. The
la proteína increase en solución
remanente of the enzyme
es 0,18may
mg be mLnow stabilized (as it has been reported in
-1. Determina:
strength has a small impact on the inactivation rate of many studies [40–45]) thus exposing the catalytic Ser to the action
a) La actividad perdida durante la inmovilización
-TLL preparation while this impact is much higher using of D-pNPP.
b) La carga de proteína en el soporte (masa deUsing
lent preparation. The results agree with the hypothesis
proteína inmovilizada por unidad de masa del
PCL (Fig. 4), results are similar. Octyl-PCL is inhibited more
ume that the octylsoporte)
support immobilized and stabilized rapidly than CNBr-PCL and the increase in ionic strength produced
c) La(that
form of the lipase actividad
permitsespecífica de la solución
a rapid inactivation of deaß-galactosidasa contactada
much more significant inicialmente
slowdown en U mg-1rate
of the inhibition . on the
me) while the CNBr immobilized
RESPUESTAS: the enzyme keeping the covalent preparation. The addition of 0.01% of Triton X-100 to the
sed-form equilibrium. Upon
a) 239,5 U increasing the ionic strength CNBr-PCL increased the inhibition rate.
nhibition, the open formmg
b) 34,8 of g
CNBr-TLL
-1 is less favored due Thus, these results also agree with the hypothesis that the octyl
ge hydrophobic pocket exposed to the medium, the equi- preparation has stabilized the open form of the lipase that cannot
c) 9,16 U mg -1
s shifted toward the closed form, making the lipase more close even in the presence of high concentrations of buffer.