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.     

Nitrilase superfamily aryl acylamidase from the halotolerant mangrove Streptomyces sp. 211726

A novel nitrilase superfamily amidase gene, designated azl13, was cloned from Streptomyces sp. 211726. Bioinformatic and biochemical analysis indicated that Azl13 belongs to a new subfamily in branch 13 of the nitrilase superfamily. His₆-Azl13 was expressed in Escherichia coli BL21(DE3) and had the expected molecular mass of 31 kDa, and the enzymatic activity was best at 40 °C, pH 8.0. His₆-Azl13 had amidase, aryl acylamidase, and acyl transferase activities, and it displayed an unusually wide substrate spectrum. His₆-Azl13 was most active on 4-guanidinobutyramide, which is probably its natural substrate, moderately active on short-chain aliphatic amides and weakly active hydrolyzing aromatic and heterocyclic amides. His₆-Azl13 also catalyzed acyl transfer to hydroxylamine from acetamide or the herbicide propanil. The substrate spectrum differs from that of the Pseudomonas amidase RamA, probably reflecting high salinity adaptation. The broad substrate spectrum of Azl13 is potentially useful for chemical synthesis and biodegradation.     

Nitrile- and Amide-hydrolysing Activity in Kluyveromyces thermotolerans MGBY 37

Summary Forty yeast strains were screened for nitrile-hydrolysing activity. Among them Kluyveromyces thermotolerans MGBY 37 exhibited highest nitrile-hydrolysing activity (0.030 μmol/h/mg dry cell weight). This yeast contained a two-enzyme system i.e. nitrile hydratase (NHase, EC 4.2.1.84) and amidase (EC 3.5.1.4) for the hydrolysis of nitriles/amides to corresponding acids and ammonia. However, these enzymes had more affinity for N-heterocyclic aromatic and aromatic nitriles/amides rather than unsaturated and saturated aliphatic nitriles/amides. The NHase–amidase activity was constitutively produced by K. thermotolerence MGBY 37. Addition of acetonitrile in the medium enhanced the production of this activity while other nitriles and amides lowered the production of NHase–amidase activity. This organism thus exhibited two types of amidase i.e. a constitutive amidase having affinity for N-heterocyclic aromatic, unsaturated and saturated aliphatic amides and another inducible amidase with affinity for aromatic amides. Formamide proved to be the best inducer of the latter amidase activity. This is the first report on nitrile- and amide-hydrolysing activity in Kluyveromyces.     

A novel thermostable nitrilase superfamily amidase from <Emphasis Type="Italic">Geobacillus pallidus</Emphasis> showing acyl transfer activity

An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic (EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0°C, respectively. The amidase exhibited high thermal stability at 50 and 60°C, with half-lives greater than 5 h at both temperatures. At 70 and 80°C, the half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic amides, with d-selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 resulted in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability was enhanced giving half-lives of 52 min at 70°C and 30 min at 80°C. The amidase has potential for application under high temperature conditions as a biocatalyst for d-selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer.     

Nocardia globerula NHB-2: a versatile nitrile-degrading organism

A versatile nitrile-degrading bacterium was isolated by enrichment culture from the soil of a forest near Manali, Himachal Pradesh, India, and was identified as Nocardia globerula. This organism contains 3 enzymes with nitrile-degrading activity: nitrilase, nitrile hydratase, and amidase. Nocardia globerula NHB-2 cells grown on nutrient broth supplemented with 1% glucose and 0.1% yeast extract exhibited nitrile hydratase-amidase activity specific for saturated aliphatic nitriles or amide, while addition of acetonitrile in nutrient broth yielded cells with nitrile hydratase-amidase that in addition to saturated aliphatic nitriles-amide also hydrolyzed aromatic amide. Nocardia globerula NHB-2 cultivated on nutrient broth containing propionitrile exhibited nitrilase activity that hydrolyzed aromatic nitrile and unsaturated aliphatic nitrile. The versatility of this organism in the hydrolysis of various nitriles and amides makes it a potential bioresource for use in organic synthesis.     

A study of the inhibition of an amidase with a wide substrate spectrum and its consequences for the bioconversion of nitriles

Summary TheBrevibacterium sp. R 312 strain possesses a nitrile-hydratase and an amidase, both with a wide substrate spectrum. These two enzymes can be used for the bioconversion of nitriles into the corresponding organic acids: the actions of three types of compounds (nitriles, amides and acids) on the activity of the amidase are reported in the present work.     

Overexpression of a recombinant amidase in a complex auto-inducing culture: purification,biochemical characterization,and regio- and stereoselectivity

Rhodococcus erythropolis AJ270 metabolizes a wide range of nitriles via the two-step nitrile hydratase/amidase pathway. In this study, an amidase gene from R. erythropolis AJ270 was cloned and expressed in Escherichia coli BL21 (DE3). The activity reached the highest level of 22.04 U/ml in a complex auto-inducing medium using a simplified process of fermentation operation. The recombinant amidase was purified to more than 95% from the crude lysate using Ni-NTA affinity chromatography and Superose S10-300 gel filtration. The V _max and K _m values of the purified enzyme with acetamide (50 mM) were 6.89 μmol/min/mg protein and 4.12 mM, respectively, which are similar to those of the enzyme from the wild-type cell. The enzyme converted racemic α-substituted amides, O-benzylated β-hydroxy amides, and N-benzylated β-amino amides to the corresponding (S)-acids with remarkably high enantioselectivity. The ionic liquid [BMIm][PF₆] (1-butyl-3-methylimidazolium hexafluorophosphate) enhanced the activity by 1.5-fold compared with water. The adequate expression of the enzyme and excellent enantioselectivity of the recombinant amidase to a broad spectrum of amides suggest that the enzyme has prospective industrial-scale practical applications in pharmaceutical chemistry.     

A new method to prepare amides by bioconversion of corresponding nitriles

Amides of very good chemical quality have been obtained by the bioconversion of the corresponding nitriles using immobilized bacteria with nitrilase activity (nitrile hydratase). The bioreactor was polyvalent since the nitrilase used had a generalized activity and could hydrolyse a large number of nitriles. The present paper shows the polyvalence and simplicity of the process, and the quality of the amides obtained.     

Purification and characterization of a thermostable aliphatic amidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1

Amidases catalyze the hydrolysis of amides to free carboxylic acids and ammonia. Hyperthermophilic archaea are a natural reservoir of various types of thermostable enzymes. Here, we report the purification and characterization of an amidase from Pyrococcus yayanosii CH1, the first representative of a strict-piezophilic hyperthermophilic archaeon that originated from a deep-sea hydrothermal vent. An open reading frame that encoded a putative member of the nitrilase protein superfamily was identified. We cloned and overexpressed amiE in Escherichia coli C41 (DE3). The purified AmiE enzyme displayed maximal activity at 85 °C and pH 6.0 (NaH₂PO₄–Na₂HPO₄) with acetamide as the substrate and showed activity over the pH range of 4–8 and the temperature range of 4–95 °C. AmiE is a dimer and active on many aliphatic amide substrates, such as formamide, acetamide, hexanamide, acrylamide, and l-glutamine. Enzyme activity was induced by 1 mM Ca²⁺, 1 mM Al³⁺, and 1–10 mM Mg²⁺, but strongly inhibited by Zn²⁺, Cu²⁺, Ni²⁺, and Fe³⁺. The presence of acetone and ethanol significantly decreased the enzymatic activity. Neither 5 % methanol nor 5 % isopropanol had any significant effect on AmiE activity (99 and 96 % retained, respectively). AmiE displayed amidase activity although it showed high sequence homology (78 % identity) with the known nitrilase from Pyrococcus abyssi. AmiE is the most characterized archaeal thermostable amidase in the nitrilase superfamily. The thermostability and pH-stability of AmiE will attract further studies on its potential industrial applications.     

Nitrile hydrolysing activities of deep-sea and terrestrial mycolate actinomycetes

Nitrile metabolising actinomycetes previously recovered from deep-sea sediments and terrestrial soils were investigated for their nitrile transforming properties. Metabolic profiling and activity assays confirmed that all strains catalysed the hydrolysis of nitriles by a nitrile hydratase/amidase system. Acetonitrile and benzonitrile, when used as growth substrates for enzyme induction experiments, had a significant influence on the biotransformation activities towards various nitriles and amides. The specific activities of selected deep-sea and terrestrial acetonitrile-grown bacteria against a suite of nitriles and amides were higher than those of the only other reported marine nitrile-hydrolysing R. erythropolis, isolated from a shallow sediment. The increase of nitrile chain length appeared to have negative influence on the nitrile hydratase activity of acetonitrile-grown bacteria, but the same was not true for benzonitrile-grown bacteria. The nitrile hydratases and amidases were constitutive in 10 of the 16 deep-sea and terrestrial actinomycetes studied, and one strain showed an inducible hydratase and a constitutive amidase. Most of the deep-sea strains had constitutive activities and showed some of the highest activities and broadest substrate specificities of organisms included in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transformation of 2- and 4-cyanopyridines by free and immobilized cells of nitrile-hydrolyzing bacteria

The transformation dynamics of 2- and 4-cyanopyridines by cells suspended and adsorbed on inorganic carriers has been studied in the Rhodococcus ruber gt1 possessing nitrile hydratase activity and the Pseudomonas fluorescens C2 containing nitrilase. It was shown that both nitrile hydratase and nitrilase activities of immobilized cells against 2-cyanopyridine were 1.5–4 times lower compared to 4-cyanopyridine and 1.6–2 times lower than the activities of free cells against 2-cyanpopyridine. The possibility of obtaining isonicotinic acid during the combined conversion of 4-cyanopyridine by a mixed suspension of R. ruber gt1 cells with a high level of nitrile hydratase activity and R. erythropolis 11-2 cells with a pronounced activity of amidase has been shown. Immobilization of Rhodococcus cells on raw coal and Pseudomonas cells on kaolin was shown to yield a heterogeneous biocatalyst for the efficient transformation of cyanopyridines into respective amides and carboxylic acids.     

Biotransformation of aromatic and heterocyclic amides by amidase of whole cells of Rhodococcus sp. MTB5: Biocatalytic characterization and substrate specificity

In this study, an amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5 has been used for the biotransformation of aromatic, monoheterocyclic and diheterocyclic amides to corresponding carboxylic acids. Benzoic acid, nicotinic acid and pyrazinoic acid are carboxylic acids which have wide industrial applications. The amidase of this strain is found to be inducible in nature. The biocatalytic conditions for amidase present in the whole cells of MTB5 were optimized against benzamide. The enzyme exhibited optimum activity in 50?mM potassium phosphate buffer pH 7.0. The optimum temperature and substrate concentrations for this enzyme were 50?°C and 50?mM, respectively. The enzyme was quite stable for more than 6?h at 30?°C. It showed substrate specificity against different amides, including aliphatic, aromatic and heterocyclic amides. Under optimized reaction conditions, the amidase is capable of converting 50?mM each of benzamide, nicotinamide and pyrazinamide to corresponding acids within 100, 160 and 120?min, respectively, using 5?mg dry cell mass (DCM) per mL of reaction mixture. The respective percent conversion of these amides was 95.02%, 98.00% and 98.44% achieved by whole cells. The amidase in whole cells can withstand as high as 383?mM concentration of product in a reaction mixture and above which it undergoes product feedback inhibition. The results of this study suggest that Rhodococcus sp. MTB5 amidase has the potential for large-scale production of carboxylic acids of industrial value.     

Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete <Emphasis Type="Italic">Pseudonocardia thermophila</Emphasis>

A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg^–1. The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.2. The amidase is active at a broad pH range (pH 4–9) and temperature range (40–80°C) and has a half-life of 1.2 h at 70°C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The amidase has a broad substrate spectrum, including aliphatic, aromatic and amino acid amides. The presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity. Among aromatic amides with substitutions at the o-, m-, or p-position, the p-substituted amides are the preferred substrates. The highest acyl transferase activity was detected with hexanoamide, isobutyramide and propionamide. The K_m values for propionamide, methacrylamide, benzamide and 2-phenylpropionamide are 7.4, 9.2, 4.9 and 0.9 mM, respectively. The amidase is highly S-stereoselective for 2-phenylpropionamide; and the racemic amide was converted to the corresponding S-acid with an enantiomeric excess of >95% at 50% conversion of the substrate. In contrast, the d,l-tryptophanamide and d,l-methioninamide were converted to the corresponding d,l-acids at the same rate. This thermostable enzyme represents the first reported amidase from a thermophilic actinomycete.     

Transformation of the herbicide 2,6-dichlorobenzonitrile to the persistent metabolite 2,6-dichlorobenzamide (BAM) by soil bacteria known to harbour nitrile hydratase or nitrilase

In soil the herbicide 2,6-dichlorobenzonitrile (dichlobenil) is degraded to the persistent metabolite 2,6-dichlorobenzamide (BAM) which has been detected in 19% of samples taken from Danish groundwater. We tested if common soil bacteria harbouring nitrile-degrading enzymes, nitrile hydratases or nitrilases, were able to degrade dichlobenil in vitro. We showed that several strains degraded dichlobenil stoichiometrically to BAM in 1.5–6.0 days; formation of the amide intermediate thus showed nitrile hydratase rather than nitrilase activity, which would result in formation of 2,6-dichlorobenzoic acid. The non-halogenated␣analogue benzonitrile was also degraded, but here the benzamide intermediate accumulated only transiently showing nitrile hydratase followed by amidase activity. We conclude that a potential for dichlobenil degradation to BAM is found commonly in soil bacteria, whereas further degradation of the BAM intermediate could not be demonstrated.     

Immobilization of fungal nitrilase and bacterial amidase – two enzymes working in accord

A nitrilase from Aspergillus niger and an amidase from Rhodococcus erythropolis co-immobilized on a 1-mL Butyl Sepharose column were used for the hydrolysis of 4-cyanopyridine into isonicotinic acid. The former enzyme converted the nitrile into the acid:amide mixture (molar ratio ca. 3:1), while the latter enzyme hydrolyzed the amide by-product. Therefore, the ratio of amide in the total product decreased to about 5%. Sodium sulfate was used as a component of the elution buffer, as the commonly used ammonium sulfate (0.8 M) acted as an amidase inhibitor. The hydrolysis of 4-cyanopyridine by a nitrilase from F. solani gave isonicotinic acid and isonicotinamide at a molar ratio of about 98:2. When using this enzyme and the amidase immobilized on two columns operated in tandem, the percentage of isonicotinamide in total product decreased to <0.2%.     

Aliphatic Amidase from <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M8 Is Related to the Nitrilase/Cyanide Hydratase Family

A comparative study of amino acid sequence and physicochemical properties indicates the affiliation of an amidase from Rhodococcus rhodochrous M8 (EC 3.5.1.4) to the nitrilase/cyanide hydratase family. Cluster analysis and multiple alignments show that Cys166 is an active site nucleophile. The enzyme has been shown to be a typical aliphatic amidase, being the most active toward short-chain linear amides. Small polar molecules such as hydroxylamine and O-methyl hydroxylamine can serve as effective external nucleophiles in acyl transfer reactions. The kinetics of the industrially important amidase-catalyzed acrylamide hydrolysis has been studied over a wide range of substrate concentrations; inhibition during enzymatic hydrolysis by the substrate and product (acrylic acid) has been observed; an adequate kinetic scheme has been evaluated and the corresponding kinetic parameters have been determined.     

Isolation of an inducible amidase from Pseudomonas acidovorans AE1.

A bacterial strain, AEI, which hydrolysed acetanilide, was isolated from soil and identified as Pseudomonas acidovorans. Numerous amides, esters and enzyme inhibitors were tested as amidase inducers. Phenacetin was chosen as inducer for the large scale cultivation of these organisms because it was less toxic to the bacteria than acetanilide. The induction increased the enzymic activity 250-fold. In comparison, the type culture strain of P. acidovorans, ATTCCI5668, had no amidase activity which could be induced by phenacetin. Optimal growth conditions were established with respect to the concentration of carbon source and inducer so that about 10% of the extractable bacterial protein consisted of the amidase. The organisms were lysed with lysozyme in the presence of EDTA and the enzyme was isolated mainly by column chromatography procedures. A preparation form 60 g (wet wt) bacteria yielded about 100 mg highly purified amidase with a specific activity of 137 mugmol substrate hydrolysed/min/mg protien. In addition to acetanilide, the purified enzyme hydrolysed several other amides and esters. As standard substrate, p-nitroacetanilide was chosen.     

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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.     

A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles

A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C₃–C₆ dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746. Received: 26 January 1999 / Received revision: 10 June 1999 / Accepted: 27 June 1999