Microbial isobutyronitrile utilization under haloalkaline conditions

The utilization of isobutyronitrile (iBN) as a C and N source under haloalkaline conditions by microbial communities from soda lake sediments and soda soils was studied. In both cases, a consortium consisting of two different bacterial species capable of the complete degradation and utilization of iBN at pH 10 was selected. The soda lake sediment consortium consisted of a new actinobacterium and a gammaproteobacterium from the genus Marinospirillum. The former was capable of fast hydrolysis of aliphatic nitriles to the corresponding amides and much-slower further hydrolysis of the amides to carboxylic acids. Its partner cannot hydrolyze nitriles but grew rapidly on amides and carboxylic acids, thus acting as a scavenger of products released by the actinobacterium. The soda soil consortium consisted of two Bacillus species (RNA group 1). One of them initiated nitrile hydrolysis, and the other utilized the hydrolysis products isobutyroamide (iBA) and isobutyrate (iB). In contrast to the actinobacterium, the nitrile-hydrolyzing soil Bacillus grew rapidly with hydrolysis products, but it was dependent on vitamins most probably supplied by its product-utilizing partner. All four bacterial strains isolated were moderately salt-tolerant alkaliphiles with a pH range for growth from pH 7.0 to 8.5 up to 10.3 to 10.5. However, both their nitrile hydratase and amidase activities had a near-neutral pH optimum, indicating an intracellular localization of these enzymes. Despite this fact, the study demonstrated a possibility of whole-cell biocatalytic hydrolysis of various nitriles at haloalkaline conditions.     

Microbial Isobutyronitrile Utilization under Haloalkaline Conditions

The utilization of isobutyronitrile (iBN) as a C and N source under haloalkaline conditions by microbial communities from soda lake sediments and soda soils was studied. In both cases, a consortium consisting of two different bacterial species capable of the complete degradation and utilization of iBN at pH 10 was selected. The soda lake sediment consortium consisted of a new actinobacterium and a gammaproteobacterium from the genus Marinospirillum. The former was capable of fast hydrolysis of aliphatic nitriles to the corresponding amides and much-slower further hydrolysis of the amides to carboxylic acids. Its partner cannot hydrolyze nitriles but grew rapidly on amides and carboxylic acids, thus acting as a scavenger of products released by the actinobacterium. The soda soil consortium consisted of two Bacillus species (RNA group 1). One of them initiated nitrile hydrolysis, and the other utilized the hydrolysis products isobutyroamide (iBA) and isobutyrate (iB). In contrast to the actinobacterium, the nitrile-hydrolyzing soil Bacillus grew rapidly with hydrolysis products, but it was dependent on vitamins most probably supplied by its product-utilizing partner. All four bacterial strains isolated were moderately salt-tolerant alkaliphiles with a pH range for growth from pH 7.0 to 8.5 up to 10.3 to 10.5. However, both their nitrile hydratase and amidase activities had a near-neutral pH optimum, indicating an intracellular localization of these enzymes. Despite this fact, the study demonstrated a possibility of whole-cell biocatalytic hydrolysis of various nitriles at haloalkaline conditions.     

Utilization of nitriles by yeasts isolated from a Brazilian gold mine

Yeast strains from the genera Candida, Debaryomyces, Aureobasidium, Geotrichum, Pichia, Rhodotorula, Tremella, Hanseniaspora, and Cryptococcus were isolated from samples of a gold mine from liquid extraction circuit. These strains were tested for their ability to utilize acetonitrile at 12 mM as the sole nitrogen source. The yeasts that grew using acetonitrile at 12 mM were tested in the presence of acetonitrile, isobutyronitrile, methacrylnitrile, and propionitrile at concentrations of 12, 24, 48, 97, and 120 mM. One strain was selected for each nitrile and the concentration of nitrile in which the best growth occurred. Cryptococcus sp. strain UFMG-Y28 had a better growth on 120 mM propionitrile and 97 mM acetonitrile, Rhodotorula glutinis strain UFMG-Y5 on 48 mM methacrylnitrile, and Cryptococcus flavus strain UFMG-Y61 on 120 mM isobutyronitrile. The utilization of different nitriles and amides by yeast strains involves hydrolysis in a two-step reaction mediated by both inducible and intracellular nitrile hydratase and amidase.     

Chemo- and enantioselective hydrolysis of nitriles and acid amides, respectively, with resting cells of Rhodococcus sp. C3II and Rhodococcus erythropolis MP50

The two new bacterial strains, Rhodococcus sp. C3II and Rhodococcus erythropolis MP50, which have been especially selected for the enantioselective hydrolysis of pharmaceutically interesting 2-arylpropionitriles like naproxen nitrile, have been applied for the hydrolysis of various aliphatic and aromatic nitriles and acid amides. From the enantioselective hydrolysis of racemic ibuprofen amide 4, 2-phenylbutyronitrile 5a as well as the profen-related atrolactamide 8 we deduce the decisive role of both an alkyl and aryl substituent in the -position to the nitrile or amide function for high enantioselectivity of the hydrolysis. Strain C3II and MP50 differ in the activity of their nitrile hydratase–amidase enzyme systems. This is of interest for the regioselective hydrolysis of the dinitriles 10a–13a to diacids 10f–13f. While strain C3II is suitable to preferentially produce mononitrile monoamide derivatives, strain MP50 can be used especially to form mononitrile monoacid and monoamide monoacid derivatives.     

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.     

Effects of nitriles and amides on the growth and the 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 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 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 isolated from the Rhodococcus sp. strain gt1.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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)     

Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci

The aim of this work was to determine the ability of rhodococci to transform 3,5-dichloro-4-hydroxybenzonitrile (chloroxynil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), 3,5-diiodo-4-hydroxybenzonitrile (ioxynil) and 2,6-dichlorobenzonitrile (dichlobenil); to identify the products and determine their acute toxicities. Rhodococcus erythropolis A4 and Rhodococcus rhodochrous PA-34 converted benzonitrile herbicides into amides, but only the former strain was able to hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid, and produced also more of the carboxylic acids from the other herbicides compared to strain PA-34. Transformation of nitriles into amides decreased acute toxicities for chloroxynil and dichlobenil, but increased them for bromoxynil and ioxynil. The amides inhibited root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification.     

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.     

Cloning and heterologous expression of an enantioselective amidase from Rhodococcus erythropolis strain MP50

The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, alpha-chlorophenylacetamide, and alpha-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.     

Characterisation of nitrilase and nitrile hydratase biocatalytic systems

Biocatalytic transformations converting aromatic and arylaliphatic nitriles into the analogous related amide or acid were investigated. These studies included synthesis of the -substituted nitrile 3-hydroxy-3-phenylpropionitrile, subsequent enrichment and isolation on this substrate of nitrile-degrading microorganisms from the environment, and a comparative study of enzymatic reactions of nitriles by resting cell cultures and enzymes. Each biocatalyst exhibited a distinctive substrate selectivity profile, generally related to the length of the aliphatic chain of the arylaliphatic nitrile and the position of substituents on the aromatic ring or aliphatic chain. Cell-free nitrilases generally exhibited a narrower substrate range than resting whole cells of Rhodococcus strains. The Rhodococcus strains all exhibited nitrile hydratase activity and converted -hydroxy nitriles (but did not demonstrate enantioselectivity on this substrate). The biocatalysts also mediated the synthesis of a range of -hydroxy carboxylic acids or amides from aldehydes in the presence of cyanide. The use of an amidase inhibitor permits halting the nitrile hydratase/amidase reaction at the amide intermediate.