Expanding the repertoire of nitrilases with broad substrate specificity and high substrate tolerance for biocatalytic applications

Enzymatic conversion of nitriles to carboxylic acids by nitrilases has gained significance in the green synthesis of several pharmaceutical precursors and fine chemicals. Although nitrilases from several sources have been characterized, there exists a scope for identifying broad spectrum nitrilases exhibiting higher substrate tolerance and better thermostability to develop industrially relevant biocatalytic processes. Through genome mining, we have identified nine novel nitrilase sequences from bacteria and evaluated their activity on a broad spectrum of 23 industrially relevant nitrile substrates. Nitrilases from Zobellia galactanivorans, Achromobacter insolitus and Cupriavidus necator were highly active on varying classes of nitriles and applied as whole cell biocatalysts in lab scale processes. Z. galactanivorans nitrilase could convert 4-cyanopyridine to achieve yields of 1.79 M isonicotinic acid within 3 h via fed-batch substrate addition. The nitrilase from A. insolitus could hydrolyze 630 mM iminodiacetonitrile at a fast rate, effecting 86 % conversion to iminodiacetic acid within 1 h. The arylaliphatic nitrilase from C. necator catalysed enantioselective hydrolysis of 740 mM mandelonitrile to (R)-mandelic acid in 4 h. Significantly high product yields suggest that these enzymes would be promising additions to the suite of nitrilases for upscale biocatalytic application.     

Characterization and functional cloning of an aromatic nitrilase from Pseudomonas putida CGMCC3830 with high conversion efficiency toward cyanopyridine

Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due to their green characteristics and the catalytic potential in nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified to homogeneity. pI value was estimated to be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH₂ terminus was determined. Nitrilase gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The open reading frame consisted of 1113 bp encoding a protein of 370 amino acids. The predicted amino acid sequence showed the highest identity (61.6%) to nitrilase from Rhodococcus rhodochrous J1. The enzyme was highly specific toward aromatic nitriles such as 3-cyanopyridine, 4-cyanopyridine, and 2-chloro-4-cyanopyridine. It was classified as aromatic nitrilase. The nitrilase activity could reach up to 71.8 U/mg with 3-cyanopyridine as substrate, which was a prominent level among identified cyanopyridine converting enzymes. The kinetic parameters K_m and V_max for 3-cyanopyridine were 27.9 mM and 84.0 U/mg, respectively. These data would warrant it as a novel and potential candidate for creating effective nitrilases in catalytic applications of carboxylic acids synthesis through further protein engineering.     

Bioconversion of Iminodiacetonitrile to Iminodiacetic acid with whole cells of <Emphasis Type="Italic">Lysinibacillus boronitolerans</Emphasis> MTCC 107614 (IICT-akl252)

Biotechnological potential of nitrilases are prompting significant interest in finding the novel microbes capable of hydrolyzing nitriles. In this view, we have screened about 450 bacterial strains for nitrilase production using bioconversion of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA) through hydrolysis and obtained six nitrilase-producing isolates. Among these six isolates, IICT-akl252 was promising which was identified as Lysinibacillus boronitolerans. This is the first report on L. boronitolerans for nitrilase activity. Optimization of various medium and reaction parameters for maximizing the nitrilase production using whole cells in shake flask was carried out for L. boronitolerans IICT-akl252. Sucrose (2 %) as a carbon source attained better nitrilase yield while IDAN appeared to be the preferable inducer (0.2 %). The maximum IDA formation was achieved with 100 mM IDAN and 150 mg/ml cells at 30 °C and pH 6.5. After optimization of the culture and reaction conditions, the activity of nitrilase was increased by 2.3-fold from 27.2 to 64.5 U. The enzyme was stable up to 1 h at 50 °C. The enzyme was able to hydrolyze aliphatic, aromatic and heterocyclic nitrile substrates.     

Exploring residues crucial for nitrilase function by site directed mutagenesis to gain better insight into sequence-function relationships

Nitrilases represent a very important class of enzymes having an array of applications. In the present scenario, where the indepth information about nitrilases is limited, the present work is an attempt to shed light on the residues crucial for the nitrilase activity. The nitrilase sequences demonstrating varying degree of identity with P. putida nitrilase were explored. A stretch of residues, fairly conserved throughout the range of higher (96%) to lower (27%) sequence identity among different nitrilases was selected and investigated for the possible functional role in nitrilase enzyme system. Subsequently, the alanine substitution mutants (T48A, W49A, L50A, P51A, G52A, Y53A and P54A) were generated. Substitution of the rationally selected conserved residues altered the substrate recognition ability, catalysis and affected the substrate specificity but had very little impact on enantioselectivity and pattern of nitrile hydrolysis.     

Bacterial Strain <Emphasis Type="Italic">Alcaligenes denitrificans</Emphasis> C-32 Containing Two Nitrilases with Different Substrate Specificities

Two genes encoding nitrilases with different properties have been found in an Alcaligenes denitrificans C-32 strain with high nitrilase activity that is currently used as a biocatalyst for commercial ammonium acrylate production. Both genes were expressed in E. coli, and the properties of the recombinant nitrilases were studied. One of these genes, which is designated as nitC1, controlled the formation of nitrilase that preferred aliphatic nitriles (acrylonitrile and butyronitrile) as best substrates. The nucleotide sequence of the gene nitC1 was almost (99%) identical to the gene sequence of an aliphatic nitrilase from Acidovorax facilis 72W (DQ4444267). In turn, nitC2 had a high level of homology (85%) with the arylacetonitrilase gene from Alcaligenes faecalis JM3 (D13419). Benzyl cyanide was shown to be the best substrate for nitC2-encoded nitrilase. In light of the results of DNA homology and differences in substrate specificity, the NitC2 and NitC1 nitrilases from Alcaligenes denitrificans C-32 were allocated to the groups of aliphatic nitrilases and arylacetonitrilases, respectively.     

Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile

A nitrilase that converts racemic mandelonitrile to R-(—)-mandelic acid was purified to apparent homogeneity from a cell extract of Alcaligenes faecalis ATCC 8750. The molecular weight of this enzyme was estimated to be 32,000±2,000 from SDS-PAGE and that of the native enzyme 460,000±30,000 from HPLC gel filtration. The enzyme preferentially hydrolyzed substituted aliphatic nitriles, in particular benzyl cyanide and its p-substituted compounds, but hydrolyzed aromatic nitriles only with difficulty. The amino-terminal amino acids were sequenced and their sequences compared with those of other nitrilases. The purified enzyme had a pH optimum of 7.5 and an optimum temperature range of 40 to 45°C. The enzyme was inhibited by various thiol reagents. It hydrolyzed racemic mandelonitrile, producing optically pure R-(—)-mandelic acid and ammonia without the concomitant production of mandelamide, evidence that this nitrilase is highly enantioselective for R-mandelonitrile.     

Evolution of nitrilases in glucosinolate-containing plants

Nitrilases, enzymes that catalyze the hydrolysis of organic cyanides, are ubiquitous in the plant kingdom. The typical plant nitrilase is a nitrilase 4 homolog which is involved in the cyanide detoxification pathway. In this pathway, nitrilase 4 converts β-cyanoalanine, the intermediate product of cyanide detoxification, into asparagine, aspartic acid and ammonia. In the Brassicaceae, a new family of nitrilases has evolved, the nitrilase 1 homologs. These enzymes are not able to use β-cyanoalanine as a substrate. Instead, they display rather broad substrate specificities and are able to hydrolyze nitriles that result from the decomposition of glucosinolates, the typical secondary metabolites of the Brassicaceae. Here we summarize and discuss data indicating that nitrilase 1 homologs have evolved to function in glucosinolate catabolism.     

Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity

Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.     

Biotransformation of β-keto nitriles to chiral (<Emphasis Type="Italic">S</Emphasis>)-β-amino acids using nitrilase and ω-transaminase

Objective

To enzymatically synthesize enantiomerically pure β-amino acids from β-keto nitriles using nitrilase and ω-transaminase.

Results

An enzyme cascade system was designed where in β-keto nitriles are initially hydrolyzed to β-keto acids using nitrilase from Bradyrhizobium japonicum and subsequently β-keto acids were converted to β-amino acids using ω-transaminases. Five different ω-transaminases were tested for this cascade reaction, To enhance the yields of β-amino acids, the concentrations of nitrilase and amino donor were optimized. Using this enzymatic reaction, enantiomerically pure (S)-β-amino acids (ee > 99%) were generated. As nitrilase is the bottleneck in this reaction, molecular docking analysis was carried out to depict the poor affinity of nitrilase towards β-keto acids.

Conclusions

A novel enzymatic route to generate enantiomerically pure aromatic (S)-β-amino acids from β-keto nitriles is demonstrated for the first time.

     

Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191

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.     

Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens, a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases. Therefore, this cysteine residue was exchanged in the nitrilase from P. fluorescens EBC191 for various amino acid residues which are present in other nitrilases at the homologous position. The influence of these mutations on the reaction specificity and enantiospecificity was analyzed with (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile as substrates. The mutants obtained demonstrated significant differences in their amide-forming capacities. The exchange of Cys163 for asparagine or glutamine residues resulted in significantly increased amounts of amides formed. In contrast, a substitution for alanine or serine residues decreased the amounts of amides formed. The newly discovered mutation was combined with previously identified mutations which also resulted in increased amide formation. Thus, variants which possessed in addition to the mutation Cys163Asn also a deletion at the C terminus of the enzyme and/or the modification Ala165Arg were constructed. These constructs demonstrated increased amide formation capacity in comparison to the mutants carrying only single mutations. The recombinant plasmids that encoded enzyme variants which formed large amounts of mandeloamide or that formed almost stoichiometric amounts of mandelic acid from mandelonitrile were used to transform Escherichia coli strains that expressed a plant-derived (S)-hydroxynitrile lyase. The whole-cell biocatalysts obtained in this way converted benzaldehyde plus cyanide either to (S)-mandeloamide or (S)-mandelic acid with high yields and enantiopurities.Nitrilases (EC 3.5.5.1) are hydrolytic enzymes found in many bacteria, fungi, and plants which convert nitriles to the corresponding carboxylic acids and ammonia. They are members of the CN hydrolase (or nitrilase) superfamily of enzymes, which also encompasses other enzymes which attack C-N bonds, such as aliphatic amidases, carbamoylases, and N-acyltransferases (1). Nitrilases possess a catalytic triad which is composed of a cysteine, a glutamate, and a lysine residue and form during the catalytic cycle a covalent adduct between the cysteine residue and the carbon atom of the nitrile group (11, 12, 29). Nitriles are important intermediates in chemical industry, and several processes which utilize the chemo-, regio-, or enantioselectivity of nitrilases for the production of commercially interesting products have been investigated (13, 16, 17, 18, 22, 26, 27, 33, 34). There is also growing biotechnological interest in nitrilases because they form (as other members of the so-called nitrilase superfamily) spiral quaternary structures which can be studied by electron microscopy and which might be useful as templates in nanotechnology (30, 31).We are currently investigating a nitrilase from Pseudomonas fluorescens EBC191 which converts various substituted phenylacetonitriles [e.g., 2-phenylpropionitrile (2-PPN), mandelonitrile (2-hydroxyphenylacetonitrile), or phenylglycinonitrile (2-aminophenylacetonitrile)] and also aliphatic 2-acetoxynitriles with moderate enantioselectivities into the corresponding α-substituted carboxylic acids. This enzyme forms with certain nitriles also significant amounts of the corresponding amides as side products (3, 5, 8, 15, 19, 24). The enzyme has recently been studied intensively in order to analyze the molecular basis for the substrate specificity, reaction specificity, and enantiospecificity of nitrilases (9, 10). In the course of these investigations, the effects of various carboxy-terminal mutations and mutations in close proximity to the catalytic active cysteine residue were analyzed. These experiments demonstrated that deletions of 47 to 67 amino acids (aa) from the carboxy terminus of the nitrilase resulted in variant forms that demonstrated increased amide formation and an increased formation of the (R)-acids (9). In addition, it was demonstrated that the size of the amino acid residue in direct proximity to the catalytic active cysteine residue (toward the C terminus) is determinative of the enantioselectivity of acid formation. Thus, it was found that only enzyme variants with large amino acid residues at this position showed a high degree of enantioselectivity for the formation of (R)-mandelic acid from racemic mandelonitrile (10). In the present study, we investigated a set of enzyme variants that carried mutations located in the amino-terminal part of the enzyme (in relation to the catalytic active cysteine residue). Thus, several mutations that resulted in changes in the enantioselectivity of the reactions and increased formation of amides were identified.     

Dimethylformamide is a novel nitrilase inducer in Rhodococcus rhodochrous

Nitrilases are of commercial interest in the selective synthesis of carboxylic acids from nitriles. Nitrilase induction was achieved here in three bacterial strains through the incorporation of a previously unrecognised and inexpensive nitrilase inducer, dimethylformamide (DMF), during cultivation of two Rhodococcus rhodochrous strains (ATCC BAA-870 and PPPPB BD-1780), as well as a closely related organism (Pimelobacter simplex PPPPB BD-1781). Benzonitrile, a known nitrilase inducer, was ineffective in these strains. Biocatalytic product profiling, enzyme inhibition studies and protein sequencing were performed to distinguish the nitrilase activity from that of sequential nitrile hydratase-amidase activity. The expressed enzyme, a 40-kDa protein with high sequence similarity to nitrilase protein Uniprot Q-03217, hydrolyzed 3-cyanopyridine to produce nicotinic acid exclusively in strains BD-1780 and BD-1781. These strains were capable of synthesising both the vitamin nicotinic acid as well as β-amino acids, a compound class of pharmaceutical interest. The induced nitrilase demonstrated high enantioselectivity (> 99%) in the hydrolysis of 3-amino-3-phenylpropanenitrile to the corresponding carboxylic acid.

     

Exploring nitrilase sequence space for enantioselective catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10(6) to 10(10) members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.     

Conversion of aliphatic nitriles by the arylacetonitrilase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> 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.     

Novel Sensitive High-Throughput Screening Strategy for Nitrilase-Producing Strains

Nitrilases have found wide use in the pharmaceutical industry for the production of fine chemicals, and it is important to have a method by which to screen libraries of isolated or engineered nitrilase variants (including bacteria and fungi). The conventional methods, such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, capillary electrophoresis, or gas chromatography, are tedious and time-consuming. Therefore, a direct and sensitive readout of the nitrilase's activity has to be considered. In this paper, we report a novel time-resolved luminescent probe: o-hydroxybenzonitrile derivatives could be applied to detect the activity of the nitrilases. By the action of nitrilases, o-hydroxybenzonitrile derivatives can be transformed to the corresponding salicylic acid derivatives, which, upon binding Tb³⁺, serve as a photon antenna and sensitize Tb³⁺ luminescence. Because of the time-resolved property of the luminescence, the background from the other proteins (especially in the fermentation system) in the assay could be reduced and, therefore, the sensitivity was increased. Moreover, because the detection was performed on a 96- or 384-well plate, the activity of the nitrilases from microorganisms could be determined quickly. Based on this strategy, the best fermentation conditions for nitrilase-producing strains were obtained.     

A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization

In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V _max and K _m toward 3-cyanopyridine were 77.5 μmol min^?1 mg^?1 and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.     

Biochemistry and biotechnology of mesophilic and thermophilic nitrile metabolizing enzymes

Mesophilic nitrile-degrading enzymes are widely dispersed in the Bacteria and lower orders of the eukaryotic kingdom. Two distinct enzyme systems, a nitrilase catalyzing the direct conversion of nitriles to carboxylic acids and separate but cotranscribed nitrile hydratase and amidase activities, are now well known. Nitrile hydratases are metalloenzymes, incorporating Fe^III or Co^II ions in thiolate ligand networks where they function as Lewis acids. In comparison, nitrilases are thiol-enzymes and the two enzyme groups have little or no apparent sequence or structural homology. The hydratases typically exist as αβ dimers or tetramers in which the α- and β-subunits are similar in size but otherwise unrelated. Nitrilases however, are usually found as homomultimers with as many as 16 subunits. Until recently, the two nitrile-degrading enzyme classes were clearly separated by functional differences, the nitrile hydratases being aliphatic substrate specific and lacking stereoselectivity, whereas the nitrilases are enantioselective and aromatic substrate specific. The recent discovery of novel enzymes in both classes (including thermophilic representatives) has blurred these functional distinctions. Purified mesophilic nitrile-degrading enzymes are typically thermolabile in buffered solution, rarely withstanding exposure to temperatures above 50°C without rapid inactivation. However, operational thermostability is often increased by addition of aliphatic acids or by use of immobilized whole cells. Low molecular stability has frequently been cited as a reason for the limited industrial application of "nitrilases"; such statements notwithstanding, these enzymes have been successfully applied for more than a decade to the kiloton production of acrylamide and more recently to the smaller-scale production of nicotinic acid, R-(−)-mandelic acid and S-(+)-ibuprofen. There is also a rapidly growing catalog of other potentially useful conversions of complex nitriles in which the regioselectivity of the enzyme coupled with the ability to achieve high conversion efficiencies without detriment to other sensitive functionalities is a distinct process advantage. Received: January 22, 1998 / Accepted: February 16, 1998     

Exploring Nitrilase Sequence Space for Enantioselective Catalysis

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 10⁶ to 10¹⁰ members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses.     

Expression,characterization of a novel nitrilase PpL19 from <Emphasis Type="Italic">Pseudomonas psychrotolerans</Emphasis> with <Emphasis Type="Italic">S</Emphasis>-selectivity toward mandelonitrile present in active inclusion bodies

Objectives

To identify a novel nitrilase with S-selectivity toward mandelonitrile that can produce (S)-mandelic acid in one step.

Results

A novel nitrilase PpL19 from Pseudomonas psychrotolerans L19 was discovered by genome mining. It showed S-selectivity with an enantiomeric excess of 52.7 % when used to hydrolyse (R, S)-mandelonitrile. No byproduct was observed. PpL19 was overexpressed in Escherichia coli BL21 (DE3) and formed inclusion bodies that were active toward mandelonitrile and stable across a broad range of temperature and pH. In addition, PpL19 hydrolysed nitriles with diverse structures; arylacetonitriles were the optimal substrates. Homology modelling and docking studies of both enantiomers of mandelonitrile in the active site of nitrilase PpL19 shed light on the enantioselectivity.

Conclusions

A novel nitrilase PpL19 from P. psychrotolerans L19 was mined and distinguished from other nitrilases as it was expressed as an active inclusion body and showed S-selectivity toward mandelonitrile.