Pseudomonas Fluorescens EBC191 Research Articles

Chemoenzymatic enantioselective synthesis of phenylglycine and phenylglycine amide by direct coupling of the Strecker synthesis with a nitrilase reaction

The conversion of rac-phenylglycinonitrile by different variants of the nitrilase from Pseudomonas fluorescens EBC191 (EC 3.5.5.1) was studied and the amounts and chiral composition of the formed phenylglycine and phenylglycine amide compared. Muteins that converted rac-phenylglycinonitrile to extraordinarily high amounts of phenylglycine or phenylglycine amide were tested for the chemoenzymatic enantioselective one-pot synthesis of (R)- and (S)-phenylglycine and (R)- and (S)-phenylglycine amide. The chemoenzymatic synthesis combined the initial step in the traditional chemical Strecker synthesis which results in the formation of rac-phenylglycinonitrile from benzaldehyde, cyanide, and ammonia with the enzymatic conversion of the formed nitrile by the nitrilase variants. The aminonitrile synthesis was optimized in order to obtain conditions which allowed under mildly alkaline conditions (pH 9.5) maximal yields of phenylglycinonitrile and the in-situ racemization of the compound. The racemic phenylglycinonitrile was directly converted under the alkaline conditions without any interposed purification step by cells of Escherichia coli overexpressing recombinant nitrilase variants. The application of a mutant of E. coli defect in a (S)-phenylglycine amide hydrolysing peptidase (E. coli JM109ΔpepA) expressing a highly reaction- and (R)-specific nitrilase variant allowed the synthesis of (R)-phenylglycine with ee-values ≥ 95% in yields up to 81% in relation to the initially added benzaldehyde. These yields indicated a dynamic kinetic resolution which involved the racemization of (S)- to (R)-phenylglycinonitrile under the used alkaline conditions with the concurrent hydrolysis of (R)-phenylglycinonitrile to (R)-phenylglycine. The addition of resting cells of E. coli JM109ΔpepA synthesizing an amide forming nitrilase variant to the final product of the Strecker synthesis and/or using E. coli strains with an intact aminopeptidase gene resulted in the preferred formation of (S)-phenylglycine amide, (R)-phenylglycine amide or (S)-phenylglycine.

Synthesis of (R)-mandelic acid and (R)-mandelic acid amide by recombinant E. coli strains expressing a (R)-specific oxynitrilase and an arylacetonitrilase

ObjectivesChiral 2-hydroxycarboxylic acids and 2-hydroxycarboxamides are valuable synthons for the chemical industry.ResultsThe biocatalytic syntheses of (R)-mandelic acid and (R)-mandelic acid amide by recombinant Escherichia coli clones were studied. Strains were constructed which simultaneously expressed a (R)-specific oxynitrilase (hydroxynitrile lyase) from the plant Arabidopsis thaliana together with the arylacetonitrilase from the bacterium Pseudomonas fluorescens EBC191. In addition, recombinant strains were constructed which expressed a previously described acid tolerant variant of the oxynitrilase and an amide forming variant of the nitrilase. The whole cell catalysts which simultaneously expressed the (R)-specific oxynitrilase and the wild-type nitrilase transformed in slightly acidic buffer systems benzaldehyde plus cyanide preferentially to (R)-mandelic acid with ee-values > 95%. The combination of the (R)-specific oxynitrilase with the amide forming nitrilase variant gave whole cell catalysts which converted at pH-values ≤ pH 5 benzaldehyde plus cyanide with a high degree of enantioselectivity (ee > 90%) to (R)-mandelic acid amide. The acid and the amide forming catalysts also converted chlorinated benzaldehydes with cyanide to chlorinated mandelic acid or chlorinated mandelic acid amides.ConclusionsEfficient systems for the biocatalytic production of (R)-2-hydroxycarboxylic acids and (R)-2-hydroxycarboxamides were generated.

Comparative Analysis of the Conversion of Mandelonitrile and 2-Phenylpropionitrile by a Large Set of Variants Generated from a Nitrilase Originating from Pseudomonas fluorescens EBC191

The arylacetonitrilase from the bacterium Pseudomonas fluorescens EBC191 has been intensively studied as a model to understand the molecular basis for the substrate-, reaction-, and enantioselectivity of nitrilases. The nitrilase converts various aromatic and aliphatic nitriles to the corresponding acids and varying amounts of the corresponding amides. The enzyme has been analysed by site-specific mutagenesis and more than 50 different variants have been generated and analysed for the conversion of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile. These comparative analyses demonstrated that single point mutations are sufficient to generate enzyme variants which hydrolyse (R,S)-mandelonitrile to (R)-mandelic acid with an enantiomeric excess (ee) of 91% or to (S)-mandelic acid with an ee-value of 47%. The conversion of (R,S)-2-phenylpropionitrile by different nitrilase variants resulted in the formation of either (S)- or (R)-2-phenylpropionic acid with ee-values up to about 80%. Furthermore, the amounts of amides that are produced from (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile could be changed by single point mutations between 2%–94% and <0.2%–73%, respectively. The present study attempted to collect and compare the results obtained during our previous work, and to obtain additional general information about the relationship of the amide forming capacity of nitrilases and the enantiomeric composition of the products.

Conversion of phenylglycinonitrile by recombinant Escherichia coli cells synthesizing variants of the arylacetonitrilase from Pseudomonas fluorescens EBC191

The conversion of phenylglycinonitrile (2-aminophenylacetonitrile) by Escherichia coli strains was studied, which recombinantly expressed the arylacetonitrilase (NitA) from Pseudomonas fluorescens EBC191 and different nitrilase variants with altered reaction specificities. The whole-cell catalysts which formed the wild-type nitrilase converted (R,S)-phenylglycinonitrile preferentially to (S)-phenylglycine with a low degree of enantioselectivity. A recombinant strain which formed a variant of NitA produced mainly (S)-phenylglycine amide from (R,S)-phenylglycinonitrile and a second variant showed an almost complete enantioconversion and produced (R)-phenylglycine and left (S)-phenylglycinonitrile. The microbial-produced (S)-phenylglycinonitrile was used to study the chemical racemisation of (S)-phenylglycinonitrile at alkaline pH values in order to establish a dynamic kinetic resolution of the substrate. Subsequently, the conversion of (R,S)-phenylglycinonitrile by the whole-cell catalysts was studied at a pH of 10.8 which allowed a sufficient racemisation rate of phenylglycinonitrile. Surprisingly, under these conditions, strongly increased amounts of (S)-phenylglycine were formed by the recombinant E. coli cells expressing the amide-forming nitrilase variant. The aminopeptidase PepA from E. coli was identified by the construction of a deletion mutant and subsequent complementation as responsible amidase activity, which converted (S)-phenylglycine amide to (S)-phenylglycine.

Conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191.

The conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 (NitA) was analyzed. The nitrilase hydrolysed a wide range of aliphatic mono- and dinitriles and showed a preference for unsaturated aliphatic substrates containing 5-6 carbon atoms. In addition, increased reaction rates were also found for aliphatic nitriles carrying electron withdrawing substituents (e.g. chloro- or hydroxy-groups) close to the nitrile group. Aliphatic dinitriles were attacked only at one of the nitrile groups and with most of the tested dinitriles the monocarboxylates were detected as major products. In contrast, fumarodinitrile was converted to the monocarboxylate and the monocarboxamide in a ratio of about 65:35. Significantly different relative amounts of the two products were observed with two nitrilase variants with altered reaction specifities. NitA converted some aliphatic substrates with higher rates than 2-phenylpropionitrile, which is one of the standard substrates for arylacetonitrilases. This indicated that the traditional classification of nitrilases as "arylacetonitrilases", "aromatic" or "aliphatic" nitrilases might require some corrections. This was also suggested by the construction of some variants of NitA which were modified in an amino acid residue which was previously suggested to be essential for the conversion of aliphatic substrates by a homologous nitrilase.

Improvement of the amides forming capacity of the arylacetonitrilase from Pseudomonas fluorescens EBC191 by site-directed mutagenesis.

The influence of different amino acid substitutions in the nitrilase from Pseudomonas fluorescens EBC191 (NitA) on the catalytical activity and the ability to form amides was investigated. The enzyme variant Glu137Ala was constructed because glutamate residues homologous to Glu137 are highly conserved among different members of the nitrilase superfamily and it has been suggested that these residues are indispensable for the hydrolysis of amides by enzymes belonging to the nitrilase superfamily. The enzyme variant Glu137Ala demonstrated less than 1 % of the wild-type activity but was still enzymatically competent to convert mandelonitrile to mandelic acid and mandeloamide. The tryptophan residue at position 188, which was previously identified as important for the amide forming capacity of the nitrilase, was exchanged by saturation mutagenesis for all other proteinogenic amino acids. Surprisingly, 18 of these 19 exchanges resulted in an increased formation of mandeloamide from (R,S)-mandelonitrile and three of these variants converted (R,S)-mandelonitrile to more than 90 % of mandeloamide. Furthermore, these modifications also resulted in a reversal of stereoselectivity and these variants formed in contrast to the wild-type enzyme and almost all other known nitrilases preferentially (S)-mandelic acid. The synthetic potential of one of these variants was demonstrated by the construction of recombinant E. coli clones which simultaneously expressed the nitrilase variant and the (S)-hydroxynitrile lyase (oxynitrilase) from the cassava plant (Manihot esculenta). These "bienzymatic catalysts" converted benzaldehyde plus cyanide almost exclusively to (S)-mandeloamide and did not show any inhibition in the presence of cyanide in concentrations up to 200 mM.

Random mutagenesis of the arylacetonitrilase from Pseudomonas fluorescens EBC191 and identification of variants, which form increased amounts of mandeloamide from mandelonitrile

The nitrilase from Pseudomonas fluorescens EBC191 was modified by introducing random mutations via error-prone PCR techniques in order to obtain nitrilase variants, which form increased amounts of mandeloamide from racemic mandelonitrile. A screening system was established and experimentally optimized, which allowed the screening of nitrilase variants with the intended phenotype. This system was based on the simultaneous expression of nitrilase variants and the mandeloamide converting amidase from Rhodococcus rhodochrous MP50 in recombinant Escherichia coli cells. The formation of increased amounts of mandeloamide from mandelonitrile by the nitrilase variants was detected after the addition of hydroxylamine and ferric iron ions by taking advantage of the acyltransferase activity of the amidase, which resulted in the formation of coloured iron(III)-hydroxamate complexes from mandeloamide. The system was applied for the screening of libraries of nitrilase variants and 30 enzyme variants identified, which formed increased amounts of mandeloamide from racemic mandelonitrile. The increase in amide formation was quantified by high-performance liquid chromatography and the genes encoding the relevant nitrilase variants sequenced. Thus, different types of mutations were identified. One group of mutants carried different deletions at the carboxy-terminus. The other types of variants carried amino acid exchanges in positions that had not been related previously to an increased amide formation. Finally, a nitrilase variant was created by combining two independently obtained point mutations. This enzyme variant demonstrated a true nitrile hydratase activity as it formed mandeloamide and mandelic acid in a ratio of about 19:1 from racemic mandelonitrile.

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

AbstractThe conversion of benzaldehyde and cyanide into mandelic acid and mandeloamide by a recombinant Escherichia coli strain which simultaneously expressed an (S)‐hydroxynitrile lyase (oxynitrilase) from cassava (Manihot esculenta) and an arylacetonitrilase from Pseudomonas fluorescens EBC191 was studied. Benzaldehyde exhibited a pronounced inhibitory effect on the nitrilase activity in concentrations ≥25 mM. Therefore, it was tested if two‐phase systems consisting of a buffered aqueous phase and the ionic liquid 1‐butyl‐1‐pyrrolidinium bis(trifluoromethanesulfonyl)imide (BMpl NTf2) or 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMim PF6) could be used for the intended biotransformation. The distribution coefficients of the substrates, intermediates and products of the reaction were determined and it was found that BMpl NTf2 and BMim PF6 were highly efficient as substrate reservoirs for benzaldehyde. The recombinant E. coli strain was active in the presence of BMpl NTf2 or BMim PF6 phases and converted benzaldehyde and cyanide into mandelic acid and mandeloamide. The two‐phase systems allowed the conversion of benzaldehyde dissolved in the ionic liquids to a concentration of 700 mM with product yields (=sum of mandelic acid and mandeloamide) of 87–100%. The cells were slightly more effective in the presence of BMpl NTf2 than in the presence of BMim PF6. In both two‐phase systems benzaldehyde and cyanide were converted into (S)‐mandeloamide and (S)‐mandelic acid with enantiomeric excesses ≥94%. The recombinant E. coli cells formed, in the two‐phase systems with ionic liquids and increased substrate concentrations, higher relative amounts of mandeloamide than in a purely aqueous system with lower substrate concentrations.

Conversion of sterically demanding α,α-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191.

The nitrilase from Pseudomonas fluorescens EBC191 converted 2-methyl-2-phenylpropionitrile, which contains a quaternary carbon atom in the α-position toward the nitrile group, and also similar sterically demanding substrates, such as 2-hydroxy-2-phenylpropionitrile (acetophenone cyanohydrin) or 2-acetyloxy-2-methylphenylacetonitrile. 2-Methyl-2-phenylpropionitrile was hydrolyzed to almost stoichiometric amounts of the corresponding acid. Acetophenone cyanohydrin was transformed to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide were formed preferentially from acetophenone cyanohydrin. A homology model of the nitrilase suggested that steric hindrance with amino acid residue Tyr54 could impair the binding or conversion of sterically demanding substrates. Therefore, several enzyme variants that carried mutations in the respective residues were generated and subsequently analyzed for the substrate specificity and enantioselectivity of the reactions. Enzyme variants that demonstrated increased relative activities for the conversion of acetophenone cyanohydrin were identified. The chiral analysis of these reactions demonstrated peculiar reaction kinetics, which suggested that the enzyme variants converted the nonpreferred (S)-enantiomer of acetophenone cyanohydrin with a higher reaction rate than that of the (preferred) (R)-enantiomer. Recombinant whole-cell catalysts that simultaneously produced the nitrilase from P. fluorescens EBC191 and a plant-derived (S)-oxynitrilase from cassava (Manihot esculenta) converted acetophenone plus cyanide at pH 4.5 to (S)-atrolactate and (S)-atrolactamide. These recombinant cells are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones.

Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase fromPseudomonas fluorescensEBC191

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.

Construction of Recombinant Escherichia coli Catalysts which Simultaneously Express an (S)‐Oxynitrilase and Different Nitrilase Variants for the Synthesis of (S)‐Mandelic Acid and (S)‐Mandelic Amide from Benzaldehyde and Cyanide

AbstractRecombinant Escherichia coli strains were constructed which simultaneously expressed the genes encoding the (S)‐oxynitrilase from cassava (Manihot esculenta) together with the wild‐type or a mutant variant of the arylacetonitrilase from Pseudomonas fluorescens EBC191 in a single organism under the control of a rhamnose‐inducible promoter. The whole cell catalysts obtained converted benzaldehyde and potassium cyanide in aqueous media at pH 5.2 mainly to (S)‐mandelic acid and/or (S)‐mandelic amide and synthesized only low amounts of the corresponding (R)‐enantiomers. The conversion of benzaldehyde and potassium cyanide (KCN) by a whole‐cell catalyst simultaneously expressing the (S)‐oxynitrilase and the wild‐type nitrilase resulted in a ratio of (S)‐mandelic acid to (S)‐mandelic amide of about 4:3. This could be explained by the strong nitrile hydratase activity of the wild‐type nitrilase with (S)‐mandelonitrile as substrate. The relative proportion of (S)‐mandelic amide formed in this system was significantly increased by coexpressing the (S)‐oxynitrilase with a carboxy‐terminally truncated variant of the nitrilase. This whole‐cell catalyst converted benzaldehyde and KCN to mandelic amide and mandelic acid in a ratio of about 9:1. The ee of the (S)‐mandelic amide formed was calculated to be &gt;95%.

Conversion of mandelonitrile and phenylglycinenitrile by recombinant E. coli cells synthesizing a nitrilase from Pseudomonas fluorescens EBC191

The enzymatic conversion of (chiral) α-amino- and α-hydroxynitriles is hampered by the low stability of these compounds under neutral conditions. In contrast, these substrates are much more stable at acidic pH-values. Therefore, the spontaneous decomposition of the model compounds mandelonitrile and phenylglycinenitrile was analysed in acid aqueous buffers at different pH-values. Both compounds showed at 4 °C half-lives of more than 24 h in Na-citrate buffer with pH-values of 5 and lower. Phenylglycinenitrile demonstrated a higher stability at all pH-values tested than mandelonitrile. The enzymatic conversion of mandelontrile was analysed at pH-values of 4–7 using a recombinantly produced nitrilase from Pseudomonas fluorescens EBC191. These experiments demonstrated that cell extracts and resting cells of the recombinant organism ( E. coli JM109(pIK9)) showed at pH 5 the highest conversion rates for mandelonitrile. This was explained by the higher bioavailability of mandelonitrile in combination with significant residual enzyme activities at this pH. The resting cell system was superior to the cell-free system in respect to conversion rates, acid stability, and tolerance against high substrate concentrations. The enantioconservative conversion of the pure enantiomers of mandelonitrile to the respective mandelic acid enantiomers was achieved with resting cells at pH 5, but was not possible at pH 7. Significantly more mandeloamide as side-product was formed from ( S)-mandelonitrile than from ( R)-mandelonitrile by resting cells. The higher stability of phenylglycinenitrile in combination with the high enzyme activities allowed an efficient conversion of this substrate already at neutral pH-values.

Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme

The gene encoding an enantioselective arylacetonitrilase was identified on a 3.8 kb DNA fragment from the genomic DNA of Pseudomonas fluorescens EBC191. The gene was isolated, sequenced and cloned into the L-rhamnose-inducible expression vector pJOE2775. The nitrilase was produced in large quantities and purified as a histidine-tagged enzyme from crude extracts of L-rhamnose-induced cells of Escherichia coli JM109. The purified nitrilase was significantly stabilized during storage by the addition of 1 M ammonium sulfate. The temperature optimum (50 degrees C), pH optimum (pH 6.5), and specific activity of the recombinant nitrilase were similar to those of the native enzyme from P. fluorescens EBC191. The enzyme hydrolysed various phenylacetonitriles with different substituents in the 2-position and also heterocyclic and bicyclic arylacetonitriles to the corresponding carboxylic acids. The conversion of most arylacetonitriles was accompanied by the formation of different amounts of amides as by-products. The relative amounts of amides formed from different nitriles increased with an increasing negative inductive effect of the substituent in the 2-position. The acids and amides that were formed from chiral nitriles demonstrated in most cases opposite enantiomeric excesses. Thus mandelonitrile was converted by the nitrilase preferentially to R-mandelic acid and S-mandelic acid amide. The nitrilase gene is physically linked in the genome of P. fluorescens with genes encoding the degradative pathway for mandelic acid. This might suggest a natural function of the nitrilase in the degradation of mandelonitrile or similar naturally occurring hydroxynitriles.