Purification and characterization of a nitrilase from Aspergillus niger K10

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg^-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.     

Metabolism of acrylonitrile by Klebsiella pneumoniae

A gram-negative rod-shaped bacterium capable of utilizing acrylonitrile as the sole source of nitrogen was isolated from industrial sewage and identified as Klebsiella pneumoniae. The isolate was capable of utilizing aliphatic nitriles containing 1 to 5 carbon atoms or benzonitrile as the sole source of nitrogen and either acetamide or propionamide as the sole source of both carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae was capable of hydrolyzing 6.15 mmol of acrylonitrile to 5.15 mmol of acrylamide within 24 h. The acrylamide was hydrolyzed to 1.0 mmol of acrylic acid within 72 h. Another metabolite of acrylonitrile metabolism was ammonia, which reached a maximum concentration of 3.69 mM within 48 h. Nitrile hydratase and amidase, the two hydrolytic enzymes responsible for the sequential metabolism of nitrile compounds, were induced by acrylonitrile. The optimum temperature for nitrile hydratase activity was 55°C and that for amidase was 40°C; both enzymes had pH optima of 8.0.Abbreviations PBM phosphate buffered medium - GC gas chromatography - GC/MS gas chromatography/mass spectrometry     

Utilization of arylaliphatic nitriles by haloalkaliphilic Halomonas nitrilicus sp. nov. isolated from soda soils

An enrichment culture from saline soda soils, using acetate as carbon and energy source and 2-phenylpropionitrile as nitrogen source (PPN) at pH 10, resulted in the isolation of strain ANL-αCH3. The strain was identified as a representative of the genus Halomonas in the Gammaproteobacteria. The bacterium was capable of PPN utilization as a nitrogen source only, while phenylacetonitrile (PAN) served both as carbon, energy and nitrogen source. This capacity was not described previously for any other haloalkaliphilic bacteria. Apart from the nitriles mentioned above, resting cells of ANL-αCH3 also hydrolyzed mandelonitrile, benzonitrile, acrylonitrile, and phenylglycinonitrile, presumably using nitrilase pathway. Neither nitrile hydratase nor amidase activity was detected. The isolate showed a capacity to grow with benzoate and salicylate as carbon and energy source and demonstrated the ability to completely mineralize PAN. These clearly indicated a potential to catabolize aromatic compounds. On the basis of unique phenotype and distinct phylogeny, strain ANL-αCH3 is proposed as a novel species of the genus Halomonas—Halomonas nitrilicus sp. nov. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nitrile degradation by free and immobilized cells of the thermophile Bacillus sp. UG-5B, isolated from polluted industrial waters

A moderately thermophilic Gram-positive, sporulating, rod-shaped strain of Bacillus with nitrile-degrading activity was isolated from polluted industrial waters. Whole cells and cell-free extracts from the end of exponential growth phase expressed 7.6 nkat mg⁻¹ and 2.0 nkat mg⁻¹ benzonitrile-degrading activity, respectively, after cultivation in a fermentor with complex medium containing benzonitrile as an inducer. The benzonitrile degradation took place via the nitrilase pathway directly to benzoic acid without intermediate formation of benzamide. Samples with benzonitrilase activity of 7.6 nkat mg⁻¹ converted 3 mg benzonitrile in 1 h at 45°C. The half-life of benzonitrilase activity for a whole cell suspension and for cells immobilized in 2% agar was 4.5 min and 6 min at 70°C without substrate and 3 min at 90°C with substrate, respectively. The nitrilase had a broad substrate spectrum. The active biocatalyst obtained by immobilization was used in a continuous process and total biodegradation of 14.1 mM benzonitrile and 37.2 mM 4-cyanopyridine in a column bioreactor at 50°C for 5 h was achieved.     

Predominance of Char Sorption over Substrate Concentration and Soil pH in Influencing Biodegradation of Benzonitrile

Incomplete combustion of field crop residues results in the production of char, a material rich in charcoal-type substances. Consequently, char is an effective adsorbent of organic compounds and when incorporated into soil may adsorb soil-applied pesticides, thereby altering their susceptibility to biodegradation. We investigated the relative importance of char, soil pH and initial substrate concentration in biodegradation of pesticides in soils by measuring the biodegradation of benzonitrile in soil as a function of soil char content (0% and 1% by weight), initial benzonitrile concentration (0.1, 1.06, and 10.2 mg l⁻¹) and soil pH (5.2, 6.9 and 8.5). Preliminary experiments revealed that wheat straw char had a much greater benzonitrile sorption capacity than did soil to which the char was added. The extent of benzonitrile degradation decreased as initial benzonitrile concentration increased in both buffer solution and soil slurry. In contrast, the degradation increased as initial benzonitrile concentration increased in char-amended slurry. In un-amended soil slurry, the benzonitrile degradation was lower at pH 5.2 than at pH 6.9 or 8.5, but in char-amended soil slurry the degradation was not affected by pH, again presumably due to adsorption of benzonitrile by the char. Adsorption by soil char appears to be more important than either initial substrate concentration or soil pH in controlling benzonitrile degradation in char-amended soil slurry. The presence of crop residue-derived chars may alter pesticide degradation patterns normally observed in soils and thus significantly affect their environmental fate.     

Nitrile bioconversion by Microbacterium imperiale CBS 498-74 resting cells in batch and ultrafiltration membrane bioreactors

The biohydration of acrylonitrile, propionitrile and benzonitrile catalysed by the NHase activity contained in resting cells of Microbacterium imperiale CBS 498-74 was operated at 5, 10 and 20°C in laboratory-scale batch and membrane bioreactors. The bioreactions were conducted in buffered medium (50 mM Na₂HPO₄/NaH₂PO₄, pH 7.0) in the presence of distilled water or tap-water, to simulate a possible end-pipe biotreatment process. The integral bioreactor performances were studied with a cell loading (dry cell weight; DCW) varying from 0.1 mg_DCW per reactor to 16 mg_DCW per reactor, in order to realize near 100% bioconversion of acrylonitrile, propionitrile and benzonitrile without consistent loss of NHase activity.     

Nitrile biotransformation by whole cells of Aspergillus sp. PTCC 5266

Abstract

Aspergillus sp. PTCC 5266 exhibited nitrile-hydrating activity over a broad pH range from 6.0 to 10.0 at 26°C. It hydrated 4-nitrophenylacetonitrile, 2-chlorobenzonitrile and 3-chlorobenzonitrile to their corresponding carboxylic acids and amides, while benzyl cyanide, benzonitrile, 4-tolunitrile, cyclohexanecarbonitrile, 4-chlorobutyronitrile and isobutyronitrile gave carboxylic acids as the sole products. The maximum whole-cell nitrile-hydrating activity was observed at pH 7.0.     

Asymmetric hydrolysis of R-(−),S(+)-2-methylbutyronitrile by Rhodococcus rhodochrous NCIMB 11216

Whole cells and cell-free extracts derived from Rhodococcus rhodochrous NCIMB 11216 were shown to hydrolyse both aliphatic and aromatic nitriles, when the organism had been grown on either propionitrile or benzonitrile as the source of carbon and nitrogen. Whole cell suspensions and cell-free extracts derived from bacteria grown on either substrate were able to biotransform R-(-),S-(+)-2-methylbutyronitrile. The S-(+) enantiomer was biotransformed more rapidly than the the R-(-) enantiomer. For whole cell biotransformations at 30°C, the maximum enantiomeric excess (ee) of the remaining R-(-)-2-methylbutyronitrile was 93% when 70% of the R-(-) enantiomer had been converted to the product, 2-methylbutyric acid. For the corresponding biotransformation at 4°C, there was an ee of 93% for the residual R-(-) enantiomer of the substrate when only 60% of it had been converted to product. For biotransformations by cell-free extracts at 30°C the 2-methylbutyric acid product had an ee of 17% for the S-(+) enantiomer at the time of optimal ee for the remaining R-(-) enantiomer of the substrate. In contrast, when the reaction was carried out by whole cells, the ee for the product acid was 0.36%. This was probably due to further, non-selective metabolism of the acid, which was especially significant at the beginning of the reaction. At both temperatures, the ee for the S-(+) enantiomer of 2-methylbutyric acid was at a maximum in the early stage of the biotransformation; for example, at 4°C the maximum detectable ee was 100% when the yield was 11%.Abbreviations EDTA Ethylenediaminetetraacetic acid - ee enantiomeric excess - FID flame ionisation detector - GC gas chromatography - ¹HNMR H nuclear magnetic resonance - K _m Michaelis constant - NCIMB National Collection of Industrial and Marine Bacteria - td doubling time - V _max Maximum velocity     

Catechols of novel substrates produced using the toluene ring oxidation pathway of Pseudomonas sp. strain T-12

Summary Pseudomonas sp. strain T-12 transforms several substituted benzenes to catechols utilizing the two initial enzymes of the toluene degradative pathway, toluene-2,3-dioxygenase and toluene-2,3-dihydrodiol dehydrogenase. Several novel substrates for this catechol synthesizing system have been previously identified including cyclopropylbenzene, -methylstyrene, anisole, benzonitrile, ,,-trifluorotoluene, benzyl alcohol, 1-phenylethanol, 2-phenylethanol, p-difluorobenzene, and p-fluorobenzonitrile. The catechol products from these substrates are identified here as the 2,3-dihydroxy derivatives. Evidence is also presented which suggests that benzonitrile is metabolized like the halobenzenes and with 2,3-dihydroxybenzonitrile acting as a suicide substrate for catechol-2,3-dioxygenase. The scope and utility of Pseudomonas sp. strain T-12 catalyzed oxygenations is discussed.     

Effect of benzonitrile ester herbicides on the growth of some soil bacteria

T. CSERHÁTI, Z. ILLÉS AND S. NEMES-KÓSA. 1992. The effect of some benzonitrile ester herbicides on the growth of Bacillus megaterium, B. cereus var. mycoides, B. polymyxa, B. subtilis, Pseudomonas fluorescens and Azotobacter chrooccum was investigated in the concentration range 20–640 ppm by the agar diffusion method. The zones of inhibition, restricted growth and eventual stimulation were determined. The data matrix was evaluated by principal component analysis. Azotobacter chroococcum was the most resistant to the benzonitrile esters. The influence of benzonitrile esters on the growth of micro-organisms depended equally on the species and on the chemical structure of the herbicides. Chloro substitution considerably modified the effect whereas bromo and iodo substitution resulted in similar biological activity.     

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.     

Synergism between aromatic compounds and green leaf volatiles derived from the host plant underlies female attraction in the oriental fruit moth

Blends of volatile compounds emitted by host plants are known to mediate the attraction of gravid female herbivores to oviposition sites, but the role of individual odor components is still little understood. We characterized the olfactory response of mated female Cydia (Grapholita) molesta (Busck) (Lepidoptera: Tortricidae) to synthetic mixtures of compounds emitted by peach shoot, a key host plant of this herbivore, and investigated the role of important constituents of bioactive mixtures in moth attraction. Relative ratios of constituents of the mixtures corresponded to the natural ratio of volatile compounds collected in the plant's headspace. A significant attractant effect was found for a comparatively complex 10‐compound mixture that included four green leaf volatiles [(Z)‐3‐hexen‐1‐ol, 1‐hexanol, (E)‐2‐hexenal, and (Z)‐3‐hexen‐1‐yl acetate], five aromatics (benzaldehyde, methyl salicylate, methyl benzoate, benzonitrile, and phenylacetonitrile), and a carboxylic acid (valeric acid). Using a subtraction approach, the number of compounds was progressively decreased, resulting in a bioactive 5‐compound mixture composed of two constituents, green leaf volatiles and aromatic compounds. Further evaluations revealed that benzaldehyde and benzonitrile must be present in association with three distinct green leaf volatiles to produce an attractant effect on the female moths. This 5‐compound mixture was as attractive as natural peach shoot volatiles, which are known to comprise over 20 compounds. Results are discussed in light of the documented synergistic effect between the three general green leaf volatiles and the two specific aromatic compounds.     

Metabolism of benzonitrile and butyronitrile by Klebsiella pneumoniae.

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.     

Metabolism of benzonitrile and butyronitrile by Klebsiella pneumoniae.

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.     

Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association.

Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mM benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations.     

The nitrilases of Rhodococcus rhodochrous NCIMB 11216

Rhodococcus rhodochrous NCIMB 11216 grows on propionitrile or benzonitrile as the sole source of carbon and nitrogen. The possibility that different nitrile-hydrolyzing enzymes were produced under these two growth conditions was investigated. Nitrilase activity in whole cell suspensions from either bacteria grown on propionitrile or benzonitrile were capable of biotransforming a wide range of nitriles. The propionitrile-induced nitrile degrading activity hydrolyzed 3-cyanobenzoate and both the nitrile groups in 1,3-dicyanobenzoate. In contrast, the benzonitrile-induced activity hydrolyzed only one of the nitrile groups in 1,3-dicyanobenzoate, but did not affect 3-cyanobenzoate. Both nitrilases biotransformed -cyano-o-tolunitrile to produce 2-cyanophenylacetic acid. The nitrilases were purified by fast protein liquid chromatography and the -terminus of each enzyme sequenced. SDS-PAGE analysis identified a subunit molecular weight of 45.8 kDa for each nitrilase. The -terminal sequences showed significant similarity with other sequenced nitrilases and with the exception of a single amino acid were identical with each other. Both nitrilases had temperature and pH optima of 30°C and 8.0, respectively. The propionitrile-induced nitrilase had a K_m for benzonitrile of 20.7 m and a V_max of 12.4 μmol min⁻¹ mg⁻¹ protein whereas the benzonitrile-induced nitrilase had a K_m for benzonitrile of 8.83 m and a V_max of 0.57 μmol min⁻¹ mg⁻¹ protein.     

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.     

The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity

The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide. The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile. The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen. The recombinant enzyme shows highest nitrilase activity with benzonitrile. Site-directed mutagenesis of the F. lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity. This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same. This is the first evidence of cyanide hydratase having nitrilase activity.     

Enzymatic degradation of nitriles by a Candida guilliermondii UFMG-Y65

Candida guilliermondii UFMG-Y65, isolated from a gold mine, was able to utilize different nitriles and the corresponding amides as sole source of nitrogen, at concentrations up to 2 M. Resting cells cultivated on YCB-acetonitrile medium showed nitrile hydrolyzing enzyme activities against acrylonitrile and benzonitrile. These enzymes were inducible and intracellular; the optimum pH was 7.0-8.0, and the optimum temperature 25 degrees C-30 degrees C. Liquid chromatographic analysis indicated that C. guilliermondii UFMG-Y65 metabolized 12 mM benzonitrile to 11 mM benzoic acid and 10 mM acrylonitrile to 7.9 mM acrylic acid. The results suggest that C. guilliermondii UFMG-Y65 may be useful for the bioproduction of amides and acids, and for the bioremediation of environments contaminated with nitriles.     

Three Metabolites Produced by Valsa sp.

Two enzymes, nitrile hydratase and amidase, which participate in the conversion of trans-1,4- dicyanocyclohexane (t-DCC) to frans-4-cyanocyclohexane-l-carboxylic acid (t-MCC), a tranexamic acid intermediate, were purified and characterized. Nitrile hydratase was obtained in a homogeneous state. The molecular weight of the native enzyme was 61,400 and that of the subunit 26.900, indicating a dimer structure. Valeronitrile and butyronitrile were good substrates for the enzyme. The enzyme could also hydrate benzonitrile, p-hydroxybenzonitrile and 4-cyanobenzoic acid. t-DCC was ex-clusively hydrated to fnzws-4-cyanocycIohexane-l-car boxy amide (t-MCMA), further hydration of the nitrile group of t-MCMA and t-MCC not being observed. The presence of pyrroloquinoline quinone in the enzyme was confirmed. The presence of iron was also confirmed. The amidase of the strain was also purified. The latter enzyme could hydrate t-MCMA, yielding t-MCC. The enzyme was highly resistant to SH reagents.