Page 309 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

13.2 The Temperature Effect on the NHase–Amidase Bi-Enzymatic Cascade System 285

Table 13.1 Equations used to calculate reaction-rate in batch and continuous stirred UF-
membrane bioreactors.

Substrate Batch reactor Continuous stirred UF-membrane reactor

∗
∗
d[acid]
Amide v AMase = d[P] AMase = U AMase dt v AMase = Q [P] AMase = Q [acid]
U AMase dt
U AMase
U AMase
∗
Nitrile v = d[P] NHase = d[amide] formed v = Q [P] NHase
NHase U NHase dt U NHase dt NHase U NHase
∗
= d[amide+acid] detected = Q [amide] formed
U NHase dt U NHase
∗
= Q [amide+acid] detected
U NHase
−1
◦
Where P, U,and Q are the product concentration (mM), the units of enzyme at 20 C(μmol min ),
−1
and the flow-rate (ml min ) respectively.
NHase–AMase cascade system that naturally acts as a sequential system can be
adequately decoupled by feeding the bioreactor with the appropriate substrate. The
NHase activity was evaluated at various temperatures in the presence of acrylonitrile
[20], propionitrile [18], benzonitrile [21], and 3-cyanopyridine [22] as substrates,
while the AMase activity was separately investigated with the corresponding
amides [23]. The kinetics of both enzymes examined in an appropriate substrate
concentration range were reported to follow the Michaelis–Menten equation, as
reviewed previously [24].
Two reactor configurations (batch and CSMR) were used throughout these
studies, and the specific reaction rates were calculated from the product formed
(Table 13.1).
The operational conditions, that is, the concentration of substrate and enzyme,
the temperature range, and the reactor configuration are summarized in Table 13.2.
The activation energy of the reaction, E , was typically obtained for a batch reactor
a
and compared with that calculated for a CSMR. The data obtained in the CSMR
at steadystate enabled us, by using a semi-log plot of reaction rate versus time, to
identify a first-order mechanism of enzyme deactivation and to determine both
its first-order deactivation constant, k , and the reaction rate at time zero, r ,for
d
0
each substrate and temperature. It was thus possible to compare the effect of the
operational parameters on the activity and stability of these two enzymes. From the
Arrhenius plot of these r ,the E -values were determined for each substrate, and
0 a
were found to match the values obtained in the batch reactors.
∗
Finally, the different activation energies of the enzyme inactivation, E ,during
a
nitrile or amide reactions are also reported, and when unavailable, the inactivation
constants, k are given instead.
d
The E of the NHase appears to be dependent on the substrate used. Indeed, for
a
acrylonitrile and propionitrile, the E falls into the lower range of the values cited in
a
the literature, while for the aromatic substrates, benzonitrile and 3-cyanopyridine,
the E is higher, thus suggesting a partial effect of mass transport for the aliphatic
a
substrates. It must be also noted that the activation energies of the AMase catalyzed