Page 285 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

11.3 Artificial Cascades 261

incubated in purely aqueous systems at pH 5 with benzaldehyde and cyanide
and shown to efficiently synthesize (S)-mandelic acid. It is assumed that in these
whole-cell systems, the nitrilase is at least partially protected from the low pH of
the bulk medium by the ability of E. coli to maintain a higher intracellular pH in an
acidic bulk medium [64–66]. Recently, it was demonstrated that these whole-cell
catalysts could also be used in two-phase systems in the presence of ionic liquids
for the production of synthetically relevant amounts of (S)-mandelic acid [67].
The ‘‘bienzymatic approach’’ was also used for the synthesis of α-alkyl-α-
hydroxycarboxylic acids from ketones and cyanide. The conversion of ketones
by HnLs is problematic because the reaction equilibrium is mainly on the side of
the ketones and therefore these substrates are generally not quantitatively converted
by HnLs [68, 69]. Therefore, the presence of a second enzyme, such as a nitrilase,
results in the establishment of an efficient cascade reaction. The feasibility of
this biotransformation was demonstrated for the conversion of acetophenone plus
cyanide at acidic pH-values by the recombinant whole-cell catalysts which simulta-
neously produced the nitrilase from P. fluorescens EBC191 and the MeHnL. These
cells converted acetophenone plus cyanide almost quantitatively to (S)-atrolactate
(and (S)-atrolactamide) [61].
The nitrilase from P. fluorescens EBC191 converts certain nitriles not only to
the acids but also forms significant amounts of the corresponding amides [55]
and recently several enzyme variants have been constructed that form significantly
increased amounts of amides from nitriles [70, 71]. These nitrilase variants in
combination with enantioselective HnLs also offer the possibility to synthesize
chiral 2-hydroxyamides from aldehydes (and ketones) and cyanide [66].

11.3.4
Hydroxynitrile Lyase–Nitrilase–Amidase

As mentioned above, the nitrilase from P. fluorescens EBC191 hydrolyzes (S)-
mandelonitrile into approximately 50% (S)-mandelic acid and 50% (S)-mandelic
amide [55, 60, 62], which detracts from the practical value of our bienzymatic
cascade to convert benzaldehyde into (S)-mandelic acid [62–64, 67]. An obvious
solution would be to hydrolyze the amide in situ, by including an amidase into
the biocatalyst. For this purpose, the amidase from R. erythropolis MP50 [72] was
included with the (S)-selective HnL from M. esculenta and the P. fluorescens nitrilase,
in a triple CLEA [73]. This approach proved entirely successful and (S)-mandelic acid
was obtained in nearly quantitative yield and enantiomeric excess (Figure 11.13).

11.3.5
Hydroxynitrile Lyase–Nitrile Hydratase

Another bienzymatic cascade was designed to synthesize amides instead of acids.
Aliphatic (S)-2-hydroxyamides are produced from the corresponding aldehyde and
HCN (Figure 11.14). The cascade employs MeHnL and the relatively stable NHase