Page 313 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

13.4 Effect of Nitrile on the AMase Activity and Stability 289

0.16
0.14
Inactivation constant, k d (h −1 ) 0.10
0.12

0.08
0.06
0.04
0.02
0.00
0.0 0.2 0.4 0.6 0.8 1.0
Nitrile concentration (M)
Figure 13.2 Inactivation effect, evaluated as (50 mM Na-phosphate buffer, pH 7.0) of:
◦
−1
inactivation constant, of nitrile concentration , acrylonitrile (4 C; 1.49 U NHase ml ); ,
◦
−1
on NHase activity. k -Values from long-term benzonitrile (10 C; 0.93 U NHase ml ); ▴,
d
◦
−1
runs performed in CSMR operated in dif- 3-cyanopyridine (4 C; 2.78 U NHase ml ); ,
◦
−1
ferential mode and stirred at 250 rpm. The propionitrile (10 C; 0.32 U NHase ml ).
bioreactors were fed with buffered solution
This phenomenon was also observed with other nitriles, such as propionitrile, as
shown in Figure 13.2 where the k -values evaluated in differential CSMR-reactors
d
are reported versus the substrate concentration. These data clearly indicate that, in
a real process, a final nitrile concentration ≤300 mM is advisable to limit NHase
inactivation. Chen et al. [29] described a decrease in the efficiency of propionitrile
removal at concentrations ≥100 mM by the NHase/AMase system of Klebsiella
oxytoca. In previous studies [26], the effect of 3-cyanopyridine was investigated till
◦
300 mM and the k -values ranged from 0.004 to 0.012 h −1 at 4 C, thus indicating
d
that, apparently, the inactivating effect is rather negligible. Jin et al. [30] reported
on the inhibition effect on nitrilase activity of high 3-cyanopyridine concentration.
Benzonitrile showed a higher inactivation effect, even at low concentration
(10 mM). Also in other multienzyme systems, such as those converting α-amino
nitriles into their chiral corresponding amide derivatives [31], the NHase was
inhibited by the substrates.
On the contrary, it has to be pointed out that very high concentrations of
N-heterocyclic nitriles (up to 12 M) had been transformed to the corresponding
amides via NHase-catalyzed reactions. Moreover, the NHase activity of whole cells
of R. rhodochrous PA-34 was used for its high butyronitrile tolerance and utilized
for butyramide production [32].

13.4
Effect of Nitrile on the AMase Activity and Stability
A previous study, aiming to reach a high conversion of 3-cyanopyridine into
nicotinic acid, showed that by adopting two CSMRs arranged in series, the rate of