Pseudomonas marginalis: its degradative capability on organic nitriles and amides

Pseudomonas marginalis, capable of utilizing acetonitrile as the sole source of carbon and nitrogen, was isolated from an industrial waste site. P. marginalis metabolized acetonitrile into ammonia and acetate. The minimal inhibitory concentration values of different nitriles and amides for P. marginalis were in the range 5–300 mM. The bacterium was able to transform high-molecular-mass nitrile compounds and their respective amides into ammonia. The data from substrate-dependent kinetics showed that the K _m and V _max values of P. marginalis for acetonitrile were 33 mM and 67 nmol oxygen consumed min^–1 (ml cell suspension)^–1 respectively. The study with [¹⁴C]acetonitrile indicated that nearly 66% of the carbon was released as ¹⁴CO₂ and 12% was associated with the biomass. The enzyme system involved in the hydrolysis of acetonitrile was shown to be intracellular and inducible. The specific activities of the enzymes nitrile aminohydrolase and amidase were determined in the cell-free extracts of P. marginalis. Both the enzymes could hydrolyze a wide range of nitriles and amides. The present study suggests that the biodegradation of organic nitriles and the bioproduction of organic acids may be achieved with the cells of P. marginalis.     

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.     

Enzymatic degradation of nitriles by Klebsiella oxytoca

Klebsiella oxytoca, isolated from cyanide-containing wastewater, was able to utilize many nitriles as sole source of nitrogen. The major objective of this study was to explore the ability of K. oxytoca to utilize some nitriles and then further evaluate the pathways of transformation of cyanide compounds by K. oxytoca. Results from this study indicate that succinonitrile and valeronitrile were the most optimal sources of nitrogen for the growth of K. oxytoca. The biodegradation of acetonitrile proceeded with the formation of acetamide followed by acetic acid. The production of ammonia was also detected in this biodegradation experiment. Similar results were observed in the propionitrile biodegradation experiments. Collectively, this study suggests that the breakdown of acetonitrile or propionitrile by this bacterium was via a two-step enzymatic hydrolysis with amides as the intermediates and organic acids plus with ammonia as the end products.     

ESR Spectral Studies on the Free Radical Formed by the Reaction of Dehydroascorbic Acid with Amino Acid

Acrylonitrile-hydrating activity was found in various bacteria belonging to the genera Arthrobacter, Corynebacterium, Microbacterium, Nocardia, Pseudomonas and Rhodococcus. A strain, N-774, isolated by acetonitrile enrichment culture from soil showed the highest activity. Taxonomic studies indicated that strain N-774 belonged to the genus Rhodococcus. The acrylonitrile-hydrating enzyme of strain N-774 was constitutively formed in the cells. Besides acrylonitrile, many nitriles were hydrated to the corresponding amides. «-Butyronitrile, suc- cinonitrile and chloroacetonitrille served as suitable substrates. This bacterium could utilize many aliphatic nitriles and amides as a sole source of nitrogen but hardly utilized malononitrile, acrylonitrile or acrylamide. Cells having high nitrile hydratase activity (about 50units/mg of dry cells) could be easily prepared by cultivation at 30°C for 40 hr in a malt extract and yeast extract medium.     

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.     

Effects of Nitriles and Amides on the Growth and Nitrile Hydratase Activity of the Rhodococcus sp. Strain gt1

Effects of some nitriles and amides, as well as glucose and ammonium, on the growth and the nitrile hydratase (EC 4.2.1.84) activity of the Rhodococcus sp. strain gt1 isolated from soil were studied. The activity of nitrile hydratase mainly depended on the carbon and nitrogen supply to cells. The activity of nitrile hydratase was high in the presence of glucose and ammonium at medium concentrations and decreased at concentrations of glucose of more than 0.3%. Saturated unsubstituted aliphatic nitriles and amides were found to be a good source of nitrogen and carbon. However, the presence of nitriles and amides in the medium was not absolutely necessary for the expression of the activity of nitrile hydratase of the Rhodococcus sp. strain gt1.     

The Respiratory Activity of Rhodococcus rhodochrousM8 Cells Producing Nitrile-Hydrolyzing Enzymes

The respiratory activity of Rhodococcus rhodochrousM8 cells containing nitrile hydratase and amidase was studied in the presence of nitriles and amides of carbonic acids. The culturing of cells with acrylonitrile and acrylamide yielding maximum respiratory activity was studied. The optimum conditions for measurements and maintenance of respiratory activity were found. Curves for the linear concentration dependence of cell respiratory activity on 0.01–0.5 mM acrylonitrile, 0.025–1.0 mM acetonitrile, and 0.01–0.1 mM acrylamide were plotted. The selectivity of cell respiratory activity for some substrates was analyzed.     

Identification of reactive toxicants: Structure–activity relationships for amides

A diverse series of amides were evaluated for aquatic toxicity (IGC₅₀) assessed in the Tetrahymena pyriformis population growth impairment assay and for reactivity (EC₅₀) with the model soft nucleophile thiol in the form of the cysteine residue of the tripeptide glutathione. All alkylamides along with some halo-substituted amides are well predicted by the simple hydrophobicity (log K _ow)–electrophilicity (E _lumo) response-surface model [log(IGC⁻¹ ₅₀) = 0.45(log K _ow) − 0.342(E _lumo) − 1.11]. However, 2-halo amides with the halogen at the end of the molecule and α,β-unsaturated primary amides are among those derivatives identified as being more toxic than predicted by the model. Amides, which exhibit excess toxicity, were capable of forming covalent bonds through an S_N2 displacement or a Michael addition. Moreover, only those amides exhibiting excess toxicity were reactive with thiol, suggesting that the reactivity with model nucleophiles such as the thiol group may provide a means of accurately defining reactive toxicants.     

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.     

Purification and characterisation of a microbial l-carnitine amidase

A novel enzyme, l-carnitine amidase, was purified about 140-fold from a newly screened microorganism (DSM 6320) to yield a homogeneous protein. The native enzyme has a molecular mass of 125 kDa (gel filtration) and consists of two identical subunits as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Edman degradation. The pH optimum was found around pH 8.5. Out of 60 chemicals tested as substrates (amides of various aliphatic and aromatic acids, nitriles, amino acid amides and dipeptide amides) the amidase hydrolysed only l-carnitine amide. The Michaelis constant (K_m) was found to be 11.6 mm, and the pure protein had a specific activity of 328 units/mg. Complex kinetics were observed with the racemic mixture of d,l-carnitine amide as starting material during enzymatic hydrolysis. Correspondence to: M.-R. Kula     

Utilization of aliphatic nitrile by Paracoccus sp. SKG isolated from chemical waste samples

A bacterial strain Paracoccus sp. SKG capable of utilizing acetonitrile as a sole source of carbon and nitrogen was isolated from the chemical waste samples. The molecular phylogram generated using the complete sequence of 16S rDNA of the strain SKG showed close links to the bacteria grouped under Brucellaceae family, that belongs to the class alphaproteobacteria. Specifically, the 16S rDNA sequence of strain SKG has shown 99% similarity to Paracoccus sp. This bacterium has also shown impressive growth on aliphatic nitriles like acetonitrile, propionitrile, acrylonitrile, valeronitrile and their corresponding amides. The nitriles degradation has led to the accumulation of ammonia and respective carboxylic acids. The acetonitrile grown cells showed the release of ammonia that contributes to the increase in pH of the medium. However, glucose grown cells failed to produce ammonia, thus indicating the inducible nature of acetonitrile degrading enzymes in Paracoccus sp. SKG. Nitrile hydratase and amidase are the two key enzymes involved in the degradation of acetonitrile. Degradation of acetonitrile in Paracoccus sp. SKG follows the bi-enzymatic pathway. Further, this strain is capable of degrading acetonitrile in the presence of other organic solvents such as methanol, ethanol and dimethylformamide. Therefore, this strain is efficiently used for the treatment of HPLC waste stream containing acetonitrile in the presence of other organic solvents.     

Fungal nitrilases as biocatalysts: Recent developments

Of the numerous putative fungal nitrilases available from protein databases only a few enzymes were purified and characterized. The purified nitrilases from Fusarium solani, Fusarium oxysporum f. sp. melonis and Aspergillus niger share a preference for (hetero)aromatic nitriles, temperature optima between 40 and 50 °C and pH optima in the slightly alkaline region. On the other hand, they differ in their chemoselectivity, i.e. their tendency to produce amides as by-products. The production of fungal nitrilases is increased by up to three orders of magnitude on the addition of 2-cyanopyridine to the culture media. The whole-cell and subcellular biocatalysts were immobilized by various methods (LentiKats®; adsorption on hydrophobic or ion exchange resins; cross-linked enzyme aggregates). Operational stability was examined using continuous stirred membrane bioreactors. Fungal nitrilases appear promising for biocatalytic applications and biodegradation of nitrile environmental contaminants.     

Nitrilase-catalysed conversion of acrylonitrile by free and immobilized cells of <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

The biotransformation of acrylonitrile was investigated using thermophilic nitrilase produced from a new isolate Streptomyces sp. MTCC 7546 in both the free and immobilized state. Under optimal conditions, the enzyme converts nitriles to acids without the formation of amides. The whole cells of the isolate were immobilized in agar-agar and the beads so formed were evaluated for 25 cycles at 50°C. The enzyme showed a little loss of activity during reuse. Seventy-one per cent of 0.5 M acrylonitrile was converted to acid at 6 h of incubation at a very low density of immobilized cells, while 100% conversion was observed at 3 h by free cells.     

Insights into catalytic activity of industrial enzyme Co-nitrile hydratase. Docking studies of nitriles and amides

Nitrile hydratase (NHase) is an enzyme containing non-corrin Co³⁺ in the non-standard active site. NHases from Pseudonocardia thermophila JCM 3095 catalyse hydration of nitriles to corresponding amides. The efficiency of the enzyme is 100 times higher for aliphatic nitriles then aromatic ones. In order to understand better this selectivity dockings of a series of aliphatic and aromatic nitriles and related amides into a model protein based on an X-ray structure were performed. Substantial differences in binding modes were observed, showing better conformational freedom of aliphatic compounds. Distinct interactions with postranslationally modified cysteines present in the active site of the enzyme were observed. Modeling shows that water molecule activated by a metal ion may easily directly attack the docked acrylonitrile to transform this molecule into acryloamide. Thus docking studies provide support for one of the reaction mechanisms discussed in the literature. Figure Crystalographic structure of Pseudonocardia thermophila JCM 3095 nitrile hydratase (a) and the non-standard active site (b)     

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.     

Degradative versatility ofCorynebacterium pseudodiphtheriticum NCIB 10803 which uses amides as carbon source

Corynebacterium pseudodiphtheriticum NCIB 10803 was tested for aerobic growth with a large number of C sources in mineral salts medium with NH₄ ⁺ as N source. Growth was supported by some amino acids, some sugars, compounds of the Krebs’ cycle, the higher normal paraffins, normal aliphatic alcohols, fatty acids, the amides and nitriles of fatty acids, αω-alkandioic acids and some simple benzenoid compounds. Possible metabolic pathways are discussed. Degradation of catechol proceeded byortho-fision viacis-cis-muconate.     

Bacillus licheniformis protease-catalyzed peptide synthesis via the kinetically controlled approach using the carbamoylmethyl ester as an acyl donor in anhydrous acetonitrile

The couplings of N-protected amino acid esters with amino acid amides proved to be carried out in anhydrous acetonitrile in the presence of Bacillus licheniformis protease (subtilisin Carlsberg) immobilized on Celite. The maximal peptide yields were obtained with the immobilized enzyme prepared through lyophilization from a pH 10.7 buffer solution. A series of dipeptide syntheses and several segment condensations were achieved generally in high yields by the combined use of the immobilized enzyme prepared from this pH and the carbamoylmethyl ester as the acyl donor.     

Treatment of simulated wastewater containing toxic amides by immobilized <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> NHB-2 using a highly compact 5-stage plug flow reactor

Biodegradation of toxic amides by immobilized Rhodococcus rhodochrous NHB-2 has been studied to generate data for future development of reactors for the treatment of simulated wastewater containing various toxic amides. The whole resting cells were immobilized in different matrices like agar, polyacrylamide and alginate. Agar gel beads were selected for the treatment of simulated wastewater containing 100mM each acetamide, propionamide, and 10mM of acrylamide and packed in a highly compact five-stage plug flow reactor. The immobilized bacterium worked well in a broad pH range from 5 to 10, with an optimum at 8.7. The apparent K _m-value for the turnover of acetamide for the resting cells was determined to be around 40mM at pH 8.5 and 55°C, whereas the K _m-value of the purified amidase was predicted to be about 20 mM. This organism exhibited greater turnover of aliphatic amides as compared to aromatic amides. Although these cells showed maximal amide-degrading activity at 55°C, simulated wastewater treatment was carried out at 45°C, because of the greater stability of the amidase activity at that temperature. Of note, indices for overall temperature stability, based on the temperature dependence of apparent first order kinetic temperature denaturation constants, were determined to be –7.9±1.1×10^–4, and –13.7±1.3×10^–4, –14.5±0.7×10^–4, and –13.7±0.8×10^–4°Cmin, for free cells and cells immobilized in alginate, agar and polyacrylamide respectively. After 250min the reactor showed maximum degradation of acetamide, propionamide and acrylamide of about 97, 100 and 90%, respectively by using 883 enzyme activity units per reactor stage. The results of this investigation showed that R. rhodochrous NHB-2 expressing thermostable amidase could be used for the efficient treatment of wastewater containing toxic amides. Therefore, we suggest that this microbe has a very high potential for the detoxification of toxic amides from industrial effluents and other wastewaters.     

Action of Nitrile Hydratase from Rhodococcus rhodochrous IFO 15564 on Derivatives of 2,5-Anhydro-d-allononitrile

The conversion of 2,5-anhydro-d-allononitrile derivatives by a nitrile hydratase from Rhodococcus rhodochrous IFO 15564 was studied. The activity of the enzyme was strongly effected by the steric bulkiness of the substituents at the 3-position of the substrates, and the corresponding amides were obtained in high yields from the nitriles with free hydroxyl groups at the 3- and 4-positions.     

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.