Hydroxynitrile lyase-nitrilase

HnLs and nitrilases generally convert a broad range of substrates [16, 57, 58], Therefore, a combination of enantioselective HnLs with nitrilases results 

7 7 Nitrile Converting Enzymes Involved in Natural and Synthetic Cascade Reactions 

Although the combination of HnLs and nitrilases appears at first glance to be a straightforward process, several problems had to be solved for the intended enzyme cascade. For example, synthetic HnL reactions are usually performed at pH 5 in order to suppress the uncatalyzed (and therefore nonenantioselective) hydro-cyanation reaction. Furthermore, synthetic HnL-mediated reactions are usually performed in an aqueous-organic two-phase system in order to further suppress the uncatalyzed reaction [59]. Unfortunately, nitrilases generally show only a low activity and stability under acidic conditions and are rapidly inactivated in the presence of organic solvents [1]. 

Subsequently, bienzymatic whole cell catalysts were constracted by coexpressing the (S)-HnL and nitrilase activities simultaneously in the yeast Pichia pastoris and the bacterium Escherichia coli. The recombinant E. coU cells exhibited much higher HnL and nitrilase activities compared to the P. pastoris catalysts and were therefore studied in greater detail [63, 64]. The recombinant E. coli cells were 

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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Figure 16.1 Synthetic routes to enantiomerically pure 2-hydroxycarboxylic acids, via oxynitrilase (hydroxynitrile lyase) catalysed enantioselective hydrocyanation (route A) and (R)-nitrilase (nitrilase) mediated dynamic kinetic resolution (route B).

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. 

Figure 16.3 Bienzymatic procedure for the synthesis of (S)-2-hydroxycarboxylic acids, using an (S)-specific hydroxynitrile lyase and a non-specific nitrilase in tandem.

The bienzymatic approach described above could also be advantageously applied to the synthesis of (R)-2-hydroxycarboxylic acids in cases where no satisfactorily enantioselective nitrilase is available (Figure 16.5). The best enantioselectivity in the hydrolysis of lb, for example, was 92% ee. The enantioselectivity of the hydroxynitrile lyase from ahnonds (PaHnL) in the synthesis of lb is also less then perfect [13], but we found that a combiCLEA of PaHnL and NIT-106 quantitatively converted 2b (O.IM starting concentration) into 3b with ee>99% R (reaction in 90 10 DlPE-buffer pH 5.5, as before) with very little (>3%) amide formation. 

The formation of amide by nitrilase somewhat reduces the synthetic value of our hydroxynitrile lyase-nitrUase based bienzymatic procedure for (S)-3a, as noted... 

A. (2012) Application of a recombinant Escherichia coli whole-cell catalyst expressing hydroxynitrile lyase and nitrilase activities in ionic liquids for the production of (S)-mandelic acid and (S)-mandeloamide. Adv. Synth. Catal., 354, 113-122. 

Enzymes of the hydroxynitrilase dass catalyze the addition of HCN to aldehydes, produdng cyanohydrins. Recendy, the reaction has been extended to a few ketones with modified hydroxynitrilase enzymes. In many cases, these are formed with good optical purities and such reactions are the simplest type of enzyme catalyzed carbon-carbon bond formation. By pairing hydroxynitrile lyases with nitrilases or nitrile hydratases, one-pot, multistep conversions become possible, and this also shifts the equilibrium to favor the addition products. Such concerns are particularly important when applying these catalysts to ketones where the equilibrium generally favors the starting carbonyl compound (Figure 1.17). 

The application of nitrile-converting enzymes, nitrilases, and hydroxynitrile lyases in the s)mthesis of chiral compounds and cyanohydrins is covered. 

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