The nitrilase from
Pseudomonas fluorescens EBC191 converted (
R,
S)-mandelonitrile with a low enantioselectivity to (
R)-mandelic acid and (
S)-mandeloamide in a ratio of about 4:1. In contrast, the same substrate was hydrolyzed by the homologous nitrilase from
Alcaligenes faecalis ATCC 8750 almost exclusively to (
R)-mandelic acid. A chimeric enzyme between both nitrilases was constructed, which represented in total 16 amino acid exchanges in the central part of the nitrilase from
P. fluorescens EBC191. The chimeric enzyme clearly resembled the nitrilase from
A. faecalis ATCC 8750 in its turnover characteristics for (
R,
S)-mandelonitrile and (
R,
S)-2-phenylpropionitrile (2-PPN) and demonstrated an even higher enantioselectivity for the formation of (
R)-mandelic acid than the nitrilase from
A. faecalis. An alanine residue (Ala165) in direct proximity to the catalytically active cysteine residue was replaced in the nitrilase from
P. fluorescens by a tryptophan residue (as found in the nitrilase from
A. faecalis ATCC 8750 and most other bacterial nitrilases) and several other amino acid residues. Those enzyme variants that possessed a larger substituent in position 165 (tryptophan, phenylalanine, tyrosine, or histidine) converted racemic mandelonitrile and 2-PPN to increased amounts of the
R enantiomers of the corresponding acids. The enzyme variant Ala165His showed a significantly increased relative activity for mandelonitrile (compared to 2-PPN), and the opposite was found for the enzyme variants carrying aromatic residues in the relevant position. The mutant forms carrying an aromatic substituent in position 165 generally formed significantly reduced amounts of mandeloamide from mandelonitrile. The important effect of the corresponding amino acid residue on the reaction specificity and enantiospecificity of arylacetonitrilases was confirmed by the construction of a Trp164Ala variant of the nitrilase from
A. faecalis ATCC 8750. This point mutation converted the highly
R-specific nitrilase into an enzyme that converted (
R,
S)-mandelonitrile preferentially to (
S)-mandeloamide.Nitrilases hydrolyze organic nitriles (R-C☰N) to the corresponding carboxylic acids and ammonia. These enzymes have been isolated from various sources, such as bacteria, fungi, and plants. Commercially, they are a very interesting group of enzymes, because nitriles are important intermediates in the chemical industry and several biotransformations have been described that utilize the chemo-, regio-, or enantioselectivity of nitrilases (
2,
6,
16,
20,
22,
29).There is an informal classification that groups nitrilases according to their substrate specificities into “benzonitrilases,” “aliphatic nitrilases,” and “arylacetonitrilases” (
17,
23). The arylacetonitrilases convert substrates, such as phenylacetonitrile and α-substituted arylacetonitriles (e.g., 2-phenylpropionitrile [2-PPN], mandelonitrile [2-hydroxyphenylacetonitrile], or phenylglycinonitrile [2-aminophenylacetonitrile]). This group of nitrilases is especially interesting for applications in biotechnology because these enzymes can enantioselectively hydrolyze α-substituted racemic nitriles to optically active carboxylic acids and thus in principle allow the production of the enantiomers of α-amino-, α-hydroxy-, and α-methylcarboxylic acids (
1,
3,
10,
34). This trait has been used for the industrial production of (substituted) (
R)-mandelic acid(s) from racemic (substituted) mandelonitrile(s) by dynamic kinetic resolution processes using different microorganisms (often strains of
Alcaligenes faecalis) (
19,
34; M. Ress-Löschke, T. Friedrich, B. Hauer, and R. Mattes, 1998, DE19848129A1, German Patent Office). An enantioselective nitrilase from
A. faecalis ATCC 8750 has been purified and characterized, and the encoding gene has been cloned (
4,
11,
26,
33).In previous work by our group, a different arylacetonitrilase was obtained from
Pseudomonas fluorescens EBC191 (
18). This enzyme converted various phenylacetonitriles (e.g., 2-PPN,
O-acetoxymandelonitrile, or mandelonitrile), and also aliphatic 2-acetoxynitriles, with moderate enantioselectivities into the corresponding α-substituted carboxylic acids. Furthermore, with some substrates, significant amounts of the corresponding amides were also formed (
5,
8,
12,
21,
27).The gene encoding the nitrilase from
P. fluorescens EBC191 was recently cloned, and it was found that the nitrilases from
P. fluorescens EBC191 and
A. faecalis ATCC 8750 are clearly homologous to each other (
12). Nevertheless, the two enzymes differ significantly in their catalytic abilities. Thus, the enzyme from
A. faecalis ATCC 8750 converts racemic mandelonitrile to (
R)-mandelic acid with a high enantioselectivity and forms almost no mandeloamide as a side product. In contrast, the enzyme from
P. fluorescens demonstrates only a low degree of enantioselectivity for the formation of (
R)-mandelic acid and forms a large amount of mandeloamide (about 16% of the totally converted mandelonitrile). We are therefore currently trying to investigate the molecular basis for these differences in order to improve the substrate specificity and enantiospecificity of nitrilases. In a previous study, we analyzed the effects of various carboxy-terminal mutations on the nitrilase of
P. fluorescens EBC191. These experiments showed that deletions of 47 to 67 amino acids from the carboxy terminus of the nitrilase resulted in variant forms that demonstrated, with mandelonitrile and 2-PPN as substrates, increased amide formation and increased formation of the
R acids associated with lower specific activities. Although these carboxy-terminal mutants showed increased enantioselectivity for the formation of (
R)-mandelic acid, the observed enantioselectivities were still much lower than those observed with the nitrilase from
A. faecalis ATCC 8750 and were also associated with increased amide formation (
13). Therefore, in the present study, additional mutants were generated in order to analyze the effects of amino acid exchanges close to the catalytic center of the nitrilase.
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