Isolation and characterization of a bacterium possessing a novel aldoxime-dehydration activity and nitrile-degrading enzymes

A bacterial strain capable of utilizing E-pyridine-3-aldoxime as a nitrogen source was isolated from soil after a 4-month acclimation period and was identified as Rhodococcus sp. The strain contained a novel aldoxime dehydration activity that catalyzed a stoichiometric dehydration of E-pyridine-3-aldoxime to form 3-cyanopyridine. The enzyme activity was induced by various aldoximes and nitriles. The strain metabolized the aldoxime as follows: E-pyridine-3-aldoxime was dehydrated to form 3-cyanopyridine, which was converted to nicotinamide by a nitrile hydratase, and the nicotinamide was successively hydrolyzed to nicotinic acid by an amidase. Received: 21 January 1998 / Accepted: 12 May 1998     

Biosynthesis of nicotinic acid from 3-cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150

In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0–12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50–55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid.     

Nitrile hydratase involved in aldoxime metabolism from Rhodococcus sp. strain YH3-3 purification and characterization.

Nitrile hydratase responsible for aldoxime metabolism from the E-pyridine-3-aldoxime degrading bacterium, Rhodococcus sp. strain YH3-3 was purified and characterized. Addition of cobalt ion was necessary for the formation of enzyme. The enzyme activity was highly induced not only by nitriles and amides but also by several aldoxime compounds. The enzyme was purified approximately 108-fold with a 16% yield from the cell-free extract of the strain. The native enzyme had a Mr of approximately 130 000 and consisted of two subunits (alpha-subunit, 27 100; beta-subunit, 34 500). The enzyme contained approximately 2 mol cobalt per mol enzyme; it showed a maximum activity at 60 degrees C and at 40 degrees C under the rate assay and end-point assay conditions, respectively, and was stable over a wide range of pH (pH 2.5-11.0). The enzyme had a wide substrate specificity: it acted on aliphatic saturated and unsaturated as well as aromatic nitriles. The N-terminus of the beta-subunit showed good sequence similarities with those of other nitrile hydratases. Nitrile hydratase is part of the metabolic pathway for aldoximes in microorganisms.     

Nitrile hydratase-catalyzed production of isonicotinamide,picolinamide and pyrazinamide from 4-cyanopyridine, 2-cyanopyridine and cyanopyrazine in Rhodococcus rhodochrous J1

Nitrile hydratase, which occurs abundantly in cells of Rhodococcus rhodochrous J1, catalyzes the hydration of 4- and 2-cyanopyridine and cyanopyrazine to isonicotinamide, picolinamide and pyrazinamide, respectively. Using resting cells, the reaction conditions for the production of isonicotinamide, picolinamide and pyrazinamide were optimized. Under the optimum reaction conditions, 100% of the added 9 M 4-cyanopyridine, 8 M 2-cyanopyridine and 8 M cyanopyrazine was converted to isonicotinamide, picolinamide and pyrazinamide, respectively, without the formation of the corresponding acids. The highest yields achieved corresponded to 1099 g of isonicotinamide, 977 g of picolinamide and 985 g of pyrazinamide per litre of reaction mixture containing resting cells (1.17 g dry weight). The isonicotinamide, picolinamide and pyrazinamide were crystallized and then identified physicochemically. The substrate specificity of the Rhodococcus nitrile hydratase for various aromatic nitriles was also examined.     

A propionitrile-induced nitrilase of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. NDB 1165 and its application in nicotinic acid synthesis

Rhodococcus sp. NDB 1165, a nitrile-transforming organism was isolated from temperate forest soil of Himalayas. The nitrilase (EC 3.5.5.2) activity of this organism had higher substrate specificity toward aromatic nitriles (benzonitrile, 3-cyanopyridine and 4-cyanopyridine) and unsaturated aliphatic nitrile (acrylonitrile) in comparison to saturated aliphatic nitriles (acetonitrile, propionitrile, butyronitrile and isobutyronitrile) nitrile and arylacetonitrile (phenylacetonitrile and indole-3-acetonitrile). The nitrilase of Rhodococcus sp. NDB 1165 was inducible in nature and propionitrile proved to be an efficient inducer. However, the salts of ferrous and cobalt ions had an inhibitory effect. Under optimized reaction conditions (pH 8.0 and temperature 45°C) the nitrilase activity of this organism was 2.39 ± 0.07 U/mg dry cell mass (dcm). The half-life of this enzyme was 150 min and 40 min at 45°C and 50°C respectively. However, it was quite stable at 40°C and around 58 % activity was retained even after 6 h at this temperature. The V _max and K _m value of this nitrilase were 1.67 μmol/ml min and 0.1 M respectively using 3-cyanopyridine as substrate. However, the decrease in V _max and K _m values (0.56 μmol/ml min and 0.02 M, respectively) were ␣observed at >0.05 M 3-cyanopyridine which revealed that this enzyme experienced uncompetitive inhibition at higher substrate concentrations. Under optimized reaction conditions, 1.6 M 3-cyanopyridine was successfully converted in to nicotinic acid using 2.0 mg resting cells (dcm)/ml reaction mixture in 11 h. This is the highest production of nicotinic acid i.e. 8.95 mg/mg resting cells (dcm)/h as compared to nitrilase systems reported hitherto.     

Distribution of aldoxime dehydratase in microorganisms

The distribution of phenylacetaldoxime-degrading and pyridine-3-aldoxime-degrading ability was examined with intact cells of 975 microorganisms, including 45 genera of bacteria, 11 genera of actinomyces, 22 genera of yeasts, and 37 genera of fungi, by monitoring the decrease of the aldoximes by high-pressure liquid chromatography. The abilities were found to be widely distributed in bacteria, actinomyces, fungi, and some yeasts: 98 and 107 strains degraded phenylacetaldoxime and pyridine-3-aldoxime, respectively. All of the active strains exhibited not only the aldoxime-dehydration activity to form nitrile but also nitrile-hydrolyzing activity. On the other hand, all of 19 nitrile-degrading microorganisms (13 species, 7 genera) were found to exhibit aldoxime dehydration activity. It is shown that aldoxime dehydratase and nitrile-hydrolyzing activities are widely distributed among 188 aldoxime and 19 nitrile degraders and that the enzymes were induced by aldoximes or nitriles.     

Distribution of Aldoxime Dehydratase in Microorganisms

The distribution of phenylacetaldoxime-degrading and pyridine-3-aldoxime-degrading ability was examined with intact cells of 975 microorganisms, including 45 genera of bacteria, 11 genera of actinomyces, 22 genera of yeasts, and 37 genera of fungi, by monitoring the decrease of the aldoximes by high-pressure liquid chromatography. The abilities were found to be widely distributed in bacteria, actinomyces, fungi, and some yeasts: 98 and 107 strains degraded phenylacetaldoxime and pyridine-3-aldoxime, respectively. All of the active strains exhibited not only the aldoxime-dehydration activity to form nitrile but also nitrile-hydrolyzing activity. On the other hand, all of 19 nitrile-degrading microorganisms (13 species, 7 genera) were found to exhibit aldoxime dehydration activity. It is shown that aldoxime dehydratase and nitrile-hydrolyzing activities are widely distributed among 188 aldoxime and 19 nitrile degraders and that the enzymes were induced by aldoximes or nitriles.     

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.     

Characterization of a novel nitrilase,BGC4, from <Emphasis Type="Italic">Paraburkholderia graminis</Emphasis> showing wide-spectrum substrate specificity,a potential versatile biocatalyst for the degradation of nitriles

Objective

To investigate the biodegradation of nitriles via the nitrilase-mediated pathway.

Results

A novel nitrilase, BGC4, was identified from proteobacteria Paraburkholderia graminis CD41M and its potential for use in biodegradation of toxic nitriles in industrial effluents was studied. BGC4 was overexpressed in Escherichia coli BL21 (DE3), the recombinant protein was purified and its enzymatic properties analysed. Maximum activity of BGC4 nitrilase was at 30 °C and pH 7.6. BGC4 has a broad substrate activity towards aliphatic, heterocyclic, and aromatic nitriles, as well as arylacetonitriles. Iminodiacetonitrile, an aliphatic nitrile, was the optimal substrate but comparable activities were also observed with phenylacetonitrile and indole-3-acetonitrile. BGC4-expressing cells degraded industrial nitriles, such as acrylonitrile, adiponitrile, benzonitrile, mandelonitrile, and 3-cyanopyridine, showing good tolerance and conversion rates.

Conclusion

BGC4 nitrilase has wide-spectrum substrate specificity and is suitable for efficient biodegradation of toxic nitriles.

     

Enantioselective hydration of 2-arylpropionitriles by a nitrile hydratase from Agrobacterium tumefaciens strain d3

The enantioselective nitrile hydratase from the bacterium Agrobacterium tumefaciens d3 was purified and completely separated from the amidase activity that is also present in cell extracts prepared from this strain. The nitrile hydratase had an activity optimum at pH 7.0 and a temperature optimum of 40 °C. The holoenzyme had a molecular mass of 69 kDa, the subunits a molecular mass of 27 kDa. The enzyme hydrated various 2-arylpropionitriles and other aromatic and heterocyclic nitriles. With racemic 2-phenylpropionitrile, 2-phenylbutyronitrile, 2-(4-chlorophenyl)propionitrile, 2-(4-methoxy)propionitrile or ketoprofen nitrile the corresponding (S)-amides were formed enantioselectively. The highest enantiomeric excesses (ee >90% until about 30% of the respective substrates were converted) were found for the amides formed from 2-phenylpropionitrile, 2-phenylbutyronitrile and ketoprofen nitrile. For the reaction of the purified nitrile hydratase, higher ee values were found than when whole cells were used in the presence of an inhibitor of the amidase activity. The enantioselectivity of the whole-cell reaction was enhanced by increasing the reaction temperature. Received: 20 June 1997 / Received revision: 28 August 1997 / Accepted: 29 August 1997     

Herbivore‐induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalist caterpillar

Numerous plant species emit volatile nitriles upon herbivory, but the biosynthesis as well as the relevance of these nitrogenous compounds in plant–insect interactions remains unknown. Populus trichocarpa has been shown to produce a complex blend of nitrogenous volatiles, including aldoximes and nitriles, after herbivore attack. The aldoximes were previously reported to be derived from amino acids by the action of cytochrome P450 enzymes of the CYP79 family. Here we show that nitriles are derived from aldoximes by another type of P450 enzyme in P. trichocarpa. First, feeding of deuterium‐labeled phenylacetaldoxime to poplar leaves resulted in incorporation of the label into benzyl cyanide, demonstrating that poplar volatile nitriles are derived from aldoximes. Then two P450 enzymes, CYP71B40v3 and CYP71B41v2, were characterized that produce aliphatic and aromatic nitriles from their respective aldoxime precursors. Both possess typical P450 sequence motifs but do not require added NADPH or cytochrome P450 reductase for catalysis. Since both enzymes are expressed after feeding by gypsy moth caterpillars, they are likely to be involved in herbivore‐induced volatile nitrile emission in P. trichocarpa. Olfactometer experiments showed that these volatile nitriles have a strong repellent activity against gypsy moth caterpillars, suggesting they play a role in induced direct defense against poplar herbivores.     

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.     

Biosynthetic Pathway for the Cyanide-Free Production of Phenylacetonitrile in Escherichia coli by Utilizing Plant Cytochrome P450 79A2 and Bacterial Aldoxime Dehydratase

The biosynthetic pathway for the production of phenylacetonitrile (PAN), which has a wide variety of uses in chemical and pharmaceutical industries, was constructed in Escherichia coli utilizing enzymes from the plant glucosinolate-biosynthetic and bacterial aldoxime-nitrile pathways. First, the single-step reaction to produce E,Z-phenylacetaldoxime (PAOx) from l-Phe was constructed in E. coli by introducing the genes encoding cytochrome P450 (CYP) 79A2 and CYP reductase from Arabidopsis thaliana, yielding the E,Z-PAOx-producing transformant. Second, this step was expanded to the production of PAN by further introducing the aldoxime dehydratase (Oxd) gene from Bacillus sp. strain OxB-1, yielding the PAN-producing transformant. The E,Z-PAOx-producing transformant also produced phenethyl alcohol and PAN as by-products, which were suggested to be the metabolites of E,Z-PAOx produced by E. coli enzymes, while the PAN-producing transformant accumulated only PAN in the culture broth, which suggested that the CYP79A2 reaction (the conversion of l-Phe to E,Z-PAOx) was a potential bottleneck in the PAN production pathway. Expression of active CYP79A2 and concentration of biomass were improved by the combination of the autoinduction method, coexpression of groE, encoding the heat shock protein GroEL/GroES, N-terminal truncation of CYP79A2, and optimization of the culture conditions, yielding a >60-fold concentration of E,Z-PAOx (up to 2.9 mM). The concentration of PAN was 4.9 mM under the optimized conditions. These achievements show the potential of this bioprocess to produce nitriles and nitrile derivatives in the absence of toxic chemicals.     

Isolation,identification and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> ZJB-063, a versatile nitrile-converting bacterium

Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of ɛ-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of ɛ-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.     

Z-phenylacetaldoxime degradation by a novel aldoxime dehydratase from Bacillus sp. strain OxB-1

Z-phenylacetaldoxime (Z-PAOx) degrading bacterium, identified as Bacillus sp. strain OxB-1, was isolated from soil after 2 months acclimation. The enzyme involved in the degradation of Z-PAOx was induced by the aldoxime and required FMN for its activity. The enzyme was partially purified from the cell-free extract of the strain and shown to catalyze the stoichiometric dehydration reaction of Z-PAOx to form phenylacetonitrile (PAN). Activities of nitrilase and amidase acting on PAN and phenylacetamide (PAAm), respectively, to form phenylacetate (PAA) were found in the strain grown on Z-PAOx. This is the first report of aldoxime dehydratase co-existing with nitrile degrading enzymes in bacteria.     

X-ray Crystal Structure of Michaelis Complex of Aldoxime Dehydratase

Aldoxime dehydratase (Oxd) catalyzes the dehydration of aldoximes (R–CH=N–OH) to their corresponding nitrile (R–CN). Oxd is a heme-containing enzyme that catalyzes the dehydration reaction as its physiological function. We have determined the first two structures of Oxd: the substrate-free OxdRE at 1.8 Å resolution and the n-butyraldoxime- and propionaldoxime-bound OxdREs at 1.8 and 1.6 Å resolutions, respectively. Unlike other heme enzymes, the organic substrate is directly bound to the heme iron in OxdRE. We determined the structure of the Michaelis complex of OxdRE by using the unique substrate binding and activity regulation properties of Oxd. The Michaelis complex was prepared by x-ray cryoradiolytic reduction of the ferric dead-end complex in which Oxd contains a Fe³⁺ heme form. The crystal structures reveal the mechanism of substrate recognition and the catalysis of OxdRE.     

Enantioselective hydrolysis of racemic 2-phenylpropionitrile and other (R,S)-2-arylpropionitriles by a new bacterial isolate,Agrobacterium tumefaciens strain d3

Bacteria were enriched from soil samples with succinate as carbon source and racemic 2-phenylpropionitrile as sole source of nitrogen. One of the isolates, strain d3, converted (R,S)-2-phenylpropionitrile with high enantioselectivity to (S)-2-phenylpropionic acid. Strain d3 was identified as Agrobacterium tumefaciens. Resting cells hydrolysed 2-phenylpropionitrile via 2-phenylpropionamide to 2-phenylpropionic acid. Racemic 2-phenylpropionitrile as well as 2-phenylpropionamide were converted to (S)-2-phenylpropionic acid with an enantiometric excess above 96%. The nitrile hydratase and the amidase were both shown to convert preferentially the S enantiomer of their respective substrate. These two enzymes were induced in the presence of (R,S)-2-phenylpropionitrile but only in the absence of ammonia. In addition to 2-phenylpropionitrile strain d3 could utilize various aliphatic and aromatic nitriles as nitrogen sources. Resting cells of strain d3 also converted (R,S)-2-phenylbutyronitrile, ibuprofen nitrile, ketoprofen nitrile and -aminophenylacetonitrile with high enantioselectivity. The nitrile- and amide-converting enzyme activities were also found in cell-free extracts.     

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.     

Bench scale conversion of 3-cyanopyidine to nicotinamide using resting cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

The nitrile hydratase (NHase, EC 3.5.5.1) activity of Rhodococcus rhodochrous PA-34 was explored for the conversion of 3-cyanopyridine to nicotinamide. The NHase activity (∼18 U/mg dry cell weight, dcw) was observed in 0.1 M phosphate buffer, pH 8.0 containing 1M 3-cyanopyridine as substrate, and 0.75 mg of resting cells (dry cell weight) per ml reaction mixture at 40°C. However, 25°C was more suitable for prolonged batch reaction at high substrate (3-cyanopyridine) concentration. In a batch reaction (1 liter), 7M 3-cyanopyridine (729 g) was completely converted to nicotinamide (855 g) in 12h at 25°C using 9.0 g resting cells (dry cell weight) of R. rhodochrous PA-34.