Page 311 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

13.3 Effect of Nitrile Concentration on NHase Activity and Stability 287

reaction, E , calculated by the Arrhenius law, are comparable for the four
a,AMase
−1
−1
substrates (49.17–66.72 kJ mol ) and are all within the range (21–63 kJ mol )as
generally reported in the literature [25]. Thus each 10-degree temperature increase
caused a doubling (∼2.04 to 2.33-fold) of the kinetic constant in the investigated
temperature range.
∗
The activation energies of the inactivation process of NHase and AMase (E )
a
are completely different, AMase activity being rather stable in the temperature
◦
range 10–50 C for both aromatic substrates (benzamide and nicotinamide). Its
inactivation energy evaluated with acrylamide was lower than that generally reported
−1
in the literature (210–630 kJ mol ) [25], thus suggesting some diffusional control.
The NHase is the more fragile enzyme of the cascade system, its k -values for
d
◦
various substrates at 4 or 10 C being two orders of magnitude higher than those
◦
of the AMase at 50 C for the corresponding amides. Thus the AMase is more
thermostable than NHase, and is the only enzyme of the cascade to work at higher
temperatures. This suggests that the AMase-catalyzed step of the total conversion
of nitriles into the corresponding acids would proceed better in a separate reactor
at a high temperature [26]. Nagasawa et al. [13] also reported that in Rhodococcus
◦
◦
rhodochrous J1, the NHase is active at 10 C, while AMase is only active at 20 C
or above.
13.3
Effect of Nitrile Concentration on NHase Activity and Stability

In industrial practice, where high yields are imperative, the product concentration
achieved from the reaction should be as high as possible, to reduce the need of
subsequent downstream processing. This implies the need to use high substrate
concentrations [27]. On the other hand, the requested high activities of the enzymes
and their long-term stabilities might be negatively affected under these conditions.
These crucial points might be particularly relevant when using nitriles, as these
compounds are known to be rather detrimental to biological systems. Therefore
the enzymes and the reactor configuration must be chosen according to their
suitability to operate at high concentrations of substrate and product. Using an
extended process time, the effects of high substrate concentrations on enzymes
activity were investigated at low temperatures to exclude interference from thermal
inactivation. The data reported in Figure 13.1 were obtained for a CSMR loaded
with an activity level (U , see caption) that ensured differential conditions,
NHase
◦
and fed with an acrylonitrile buffered solution. The runs were performed at 4 C
with stirring at 250 rpm. The experiments demonstrated the ability of acrylonitrile
to depress the enzyme activity during the process. By increasing the acrylonitrile
concentration from 100 mM to 1 M, the NHase was rapidly inactivated. After
24 h of process time, the conversion obtained with a load of 107 U reached
NHase
40, 11, 2.3, and 2.3% for 300 mM, 600 mM, 800 mM, and 1 M, respectively. All
the reactors at steadystate conditions operated in differential mode, except for
the one operating at a 300 mM concentration which reached 11.9% conversion