Bienzymatic cascade

Mateo, C., Chmura, A., Rustler, S. et al. (2006) Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase-nitrilase bienzymatic cascade a nitrilase surprisingly shows nitrile hydratase activity. Tetrahedron Asymmetry, 17, 320-323. 

Analogously, Van Pelt et al. (2009) have realized a one-pot bienzymatic cascade combining the (5 )-selective oxynitrilase from M. esculenta and a purified nitrile hydratase from Nitriliruptor alkaliphilus for the synthesis of aliphatic S)-a-hydroxycarboxylic amides (Table 17.6) both enzymes were immobilized as CLEAs to enhance their stability (Van Pelt et al., 2009). 

Van Pelt, S., Van Rantwijk, F., and Sheldon, R.A. 2009. Synthesis of aliphatic (S)-a-hydroxycarboxyhc amides using a one-pot bienzymatic cascade of immobilized oxynit-rilase and nitrile hydratase,. Advances in Synthesis and Catalysis, 351 397 04. 

The nitrilase mediated DKR route to enantiomerically pure 2-hydroxycarboxylic acids is restricted to the (R)-enantiomers because, to our knowledge, no (S)-selec-tive nitrilases for cyanohydrin substrates are commonly available [11]. We reasoned that a fully enzymatic route to the (S)-acids should be possible by combining an (S)-selective oxynitrilase (hydroxynitrile lyase, EC 4.1.2.10, (S)-hydroxynitrile lyase) and a non-selective nitrilase in a bienzymatic cascade (see Figure 16.3). Besides being more environmentally acceptable than chemical hydrolysis, the mild reaction conditions of the combined enzymatic reaction would be compatible with a wide range of hydrolysable groups. 

As mentioned above, the nitrilase from P. Jluorescens 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 HnLfrom M. esculenta and the P.Jluorescens 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). 

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... 

The bienzymatic approach was also used for the synthesis of a-alkyl-a-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 simultaneously produced the nitrilase from P.Jluorescens EBC191 and the MeHnL. These cells converted acetophenone plus cyanide almost quantitatively to (S)-atroIactate (and (S)-atrolactamide) [61]. 

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