Page 308 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

284 13 Key-Study on NHase/AMase System

as K , V , inhibition constant with specific inhibitors, and optimal temperature
m max
are available in the literature. However, very often there is a lack of information
on the stability of these enzymes under operational process conditions, despite the
fact that it is known that a bioprocess is feasible as long as the enzyme maintains
its activity within a defined percentage according to the process economics. The
classical ways of speeding up the reaction by increasing the temperature and/or the
driving force (high substrate concentration) might not be appropriate because of a
dramatic loss of activity. From this point of view, whole cells, where the enzyme
might be protected from the external environment, and the use of immobilized
enzymes might be more advantageous than employing soluble enzymes. These
options can be economically attractive for some large-scale processes. However,
drawbacks such as the low substrate or the low product permeability of whole cells
or of the immobilized enzymes are still limiting factors.
In the past decade, the in situ NHase–AMase bienzymatic system of Microbac-
terium imperiale CBS 498-74 resting cells has been investigated with various
substrates. Interesting findings on the kinetic behavior of each enzyme involved
in this cascade process were obtained, suggesting the possibility of using whole
cells instead of purified enzymes. The extensive use of continuous stirred ultrafil-
tration (UF)-membrane bioreactors (CSMRs) enables us to explain the effects of
key parameters such as temperature, substrate concentration, and cell loading on
the reaction rate [18, 19]. In this reactor configuration, after a transient period of
product accumulation, steadystate conditions are reached, in which the substrate
and product concentrations in the outflow stream are equal to those inside the
reactor. Appropriate mixing ensures the homogeneity of the reactor content, thus
avoiding biocatalyst segregation. A suitable choice of residence time and of biocat-
alyst concentration enables the reactor to be operated at differential conditions (low
substrate conversion), allowing to elucidate bioreactor performance and enzyme
kinetics. On the other hand, by a long-term, continuous monitoring of the enzyme
activity and stability under the reaction conditions required by the manufactur-
ing process (high temperature, high substrate, and product concentrations), it is
possible to verify the feasibility of process scale-up and/or its applicability at the
industrial level.
This chapter focuses on how the kinetics of the in situ bi-sequential NHase/AMase
cascade system in resting cells is affected by temperature and nitrile concentration.
The effects of temperature on the activity and stability of each enzyme of the
cascade system involved in the bioconversion of selected nitriles and amides have
been evaluated in terms of activation energy. In addition to this, the effect of high
substrate concentration of various nitriles on the activity and stability of both the
NHase and AMase is also documented in long-term experiments.

13.2
The Temperature Effect on the NHase–Amidase Bi-Enzymatic Cascade System
The effect of temperature on each enzyme of the in situ bi-enzymatic cascade
system was investigated using whole resting cells of M. imperiale CBS 498-74. The