Mutant nitrilases

Figure 8.11 Relative specific activity of Alcaligenes faecalis Nitl650 nitrilase ( ) and Y296C mutant nitrilase ( ) for conversion of unsubstituted and substituted mandelonitrile mandelonitrile (MN), 2-chloromandelonitrile (2-CMN), 3-chloromandelonitrile (3-CMN), 4-chlormandelonitrile (4-CMN), 4-bromomandelonitrile (4-BMN), and 4-methylmandelonitrile (4-MeMN)...

This screening system has also been applied successfully in the directed evolution of enantioselective EHs acting as catalysts in the kinetic resolution of chiral epoxides 95,96) (Section IV.A.4). Moreover, the firm Diversa has applied the MS-based method in the desymmetrization of a prochiral dinitrile (l,3-dicyano-2-hydroxypropane) catalyzed by mutant nitrilases 46). In this industrial application, one of the nitrile moieties was labeled selectively with as in N-17, which means that the two pseiido-eaaniiovaenc products (S)- N-18 and (J )-18 differ by one mass unit. This is sufficient for the MS system to distinguish between the two products quantitatively 46). 

Recently, another interesting application of nitrilases has been demonstrated for the synthesis of pregabalin-the API of the neurophatic pain drug Lyrica. In this approach, the key step is the resolution of racemic isobutylsuccinonitrile (Scheme 10.8) [18], the process takes place with total regio- and stereoselectivity, and the (S)-acid is obtained and the (R)-substrate can be recycled under basic conditions. To improve the biocatalytic step, directed evolution was applied using the electronic polymerase chain reaction and in the first round of evolution a single C236S mutation led to a mutant with 3-fold increase in activity [19]. 

Table 8. Activity of the whole cells producing the nitrilase and its mutants...

Zhang J-E, Liu Z-Q, Zheng Y-G. Improvement of nitrilase production from a newly isolated Alcaligenes faecalis mutant for biotransformation of iminodiacefonitrile to iminodiacetic acid [J]. 7Taiwan Inst Chem Eng 2012,44 (2) 169-176. 

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