A. Petrˇícˇková et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 74–80
79
acid replacement [15,20]. These were variants of the P. fluorescens
cent to the catalytically active cysteine but toward the N-terminus
(C163N and C163Q), which produced 43 and 51% mandelamide
from (R,S)-mandelonitrile after complete conversion, respectively
[15]. NitNc carried an asparagine-residue at this position (N166),
and this may be one of the reasons for its pronounced tendency
an alanine at the respective position (A161), produced almost no
amides. The corresponding enzyme variants of the nitrilase from P.
fluorescens EBC191, which either carried an alanine or an asparagine
high e.e.-values (98 and 90%, respectively, at 35% substrate conver-
sion; Table 3). Thus for the formation of 2-phenylpropionic acid,
an inversion from R-selectivity to S-selectivity was observed, as
in the corresponding variants of NitAn (W163A; Table 3) and the
nitrilase from A. faecalis (W164A) [16]. The NitNc W168A vari-
ant was the only enzyme tested here to produce amide from
2-phenylpropionitrile (31% of the total product formed). This was
more than was found for any of the P. fluorescens nitrilase variants
previously described (the highest was 16.6% in the A165R variant,
the others were below 5%; [20]).
bulky acetophenone cyanohydrin into atrolactate (NitAn) or a mix-
ture of atrolactate and atrolactamide (NitNc).
New fungal nitrilases have significant potential in the synthesis
of enantioenriched ␣-substituted carboxylic acids or their amides
from racemic substrates. They may be also suitable for the stere-
oretentive hydrolysis or hydration of enantiopure cyanohydrins
prepared with oxynitrilases, as they are highly active at pH 4.5.
Certain amino acid residues in the proximity of the active
site have similar effects on enantioselectivity and amide produc-
tion in distantly related arylacetonitrilases (fungal enzymes and
the nitrilase from P. fluorescens or A. faecalis [20]). In principle,
this knowledge enables the creation of nitrilases with the desired
properties (enantioselectivity, amide production) and may also be
utilized in predicting the catalytic properties of putative nitrilases,
whose sequences are available from databases.
Acknowledgements
Financial support via projects P504/11/0394 and 305/09/H008
(Czech Science Foundation), IAA500200708 (Grant Agency of the
Academy of Sciences of the Czech Republic), LC06010, OC09046
(Ministry of Education of the Czech Republic), COST/ESF CM0701
(STSM fellowship COST-STSM-CM0701-6753 to A. Petrˇícˇková),
TA01021368 (Technology Agency of the Czech Republic) and
Institutional Research Concept AV0Z50200510 (Institute of Micro-
biology) is gratefully acknowledged.
3.5. Site-directed mutagenesis of the histidine residue in the
responding positions in the wild-type nitrilases from Arabidopsis
thaliana, in cyanide hydratases [28,29] and in A. niger K10 [30]. The
AtNit1 and AtNit4 isoenzymes from A. thaliana [32,33] and nitri-
Surprisingly, an increase in amide formation was only observed
for the H165N variant of NitAn (Table 2 and Fig. 3c). In contrast, the
analogous variant (H170N) of NitNc produced less mandelamide
than the parent enzyme (Table 2 and Fig. 3d).
Supplementary data associated with this article can be found, in
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