Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani.

1. A strain of the fungus Fusarium solani able to use benzonitrile as sole source of carbon and nitrogen was isolated by elective culture. 2. Respiration studies indicate that the nitrile, after degradation to benzoate, is catabolized via catechol or alternatively via p-hydroxybenzoate and 3,4-dihydroxybenzoate. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme mediating the conversion of benzonitrile into benzoate and ammonia. 4. The nitrilase enzyme was purified by DEAE-cellulose chromatography, (NH(4))(2)SO(4) precipitation and gel filtration on Sephadex G-200. The homogeneity of the purified enzyme preparation was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme showed a broad pH optimum between pH7.8 and 9.1 and a K(m) with benzonitrile as substrate of 0.039mm. The activation energy of the reaction deduced from an Arrhenius plot was 48.4kJ/mol. 6. The enzyme was susceptible to inhibition by thiol-specific reagents and certain heavy metal ions. 7. Gel filtration gave a value of 620000 for the molecular weight of the intact enzyme. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that the enzyme was composed of eight subunits of mol.wt. 76000. 8. Rates of enzymic attack on various substrates indicated that the nitrilase has a fairly broad specificity and that the fungus probably plays an important role in the biodegradation of certain nitrilic herbicides in the environment.     

A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3. Purification and characterization

A new type of nitrilase, arylacetonitrilase, has been purified from isovaleronitrile-induced cells of Alcaligenes faecalis JM3 in four steps. The purity of the enzyme was confirmed by SDS/polyacrylamide gel electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 275 kDa and consists of six subunits of identical molecular mass. The purified enzyme exhibits a pH optimum of 7.5 and a temperature optimum of 45 degrees C. The enzyme is specific for arylacetonitriles such as 2-thiophenacetonitrile, p-tolyacetonitrile, p-chlorobenzylcyanide, p-fluorobenzylcyanide and 3-pyridylacetonitrile. The enzyme stoichiometrically catalyzes the hydrolysis of arylacetonitrile to arylacetic acid and ammonia, no formation of amide occurring. However, the enzyme does not attack nitrile groups attached to aromatic and heteroaromatic rings, which are hydrolyzed preferably by the nitrilases known previously. The enzyme requires thiol compounds such as dithiothreitol and 2-mercaptoethanol to exhibit its maximum activity.     

Nitrile hydratase of Pseudomonas chlororaphis B23. Purification and characterization

Nitrile hydratase of Pseudomonas chlororaphis B23 was completely stabilized by the addition of 22 mM n-butyric acid. The enzyme was purified from extracts of methacrylamide-induced cells of P. chlororaphis B23 in eight steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular mass of about 100 kDa and consists of four subunits identical in molecular mass (approximately 25 kDa). The enzyme contained approximately 4 mol iron/mol enzyme. The concentrated solution of highly purified nitrile hydratase had a pronounced greyish green color and exhibited a broad absorption in visible range with a absorption maxima at 720 nm. A loss of enzyme activity occurred in parallel with the disappearance of the absorption in the visible range under a variety of conditions. The enzyme catalyzed stoichiometrically the hydration of nitrile to amide, and no formation of acid and ammonia were detected. The enzyme was active toward various aliphatic nitriles, particularly, nitriles with 3-6 carbon atoms, e.g. propionitrile, n-butyronitrile, acrylonitrile and cyclopropyl cyanide, served as the most suitable substrates.     

Microbial metabolism of aromatic nitriles. Enzymology of C–N cleavage by Nocardia sp. (Rhodochrous group) N.C.I.B. 11216

1. An organism utilizing benzonitrile as sole carbon and nitrogen source was isolated by the enrichment-culture technique and identified as a Nocardia sp. of the rhodochrous group. 2. Respiration studies indicate that nitrile degradation proceeds through benzoic acid and catechol. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme that catalyses the conversion of benzonitrile directly into benzoic acid without intermediate formation of benzamide. 4. This nitrilase enzyme was purified by DEAE-cellulose chromatography and gel filtration on Sephadex G-100 in the presence and absence of substrate. The purity of the enzyme was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme shows a time-dependent substrate-activation process in which the substrate catalyses the association of inactive subunits of mol.wt. 45000 to form the polymeric 12-unit active enzyme of mol.wt. 560000. The time required for complete association is highly dependent on the concentration of the enzyme, temperature and pH. 6. The associated enzyme has a pH optimum of 8.0 and K(m) with benzonitrile as substrate of 4mm. The activation energy of the reaction as deduced from the Arrhenius plot is 51.8kJ/mol. 7. Enzyme activity is inhibited by thiol-specific reagents and several metal ions. 8. Studies with different substrates indicate that the nitrilase is specific for nitrile groups directly attached to the benzene ring. Various substituents in the ring are compatible with activity, though ortho-substitution, except by fluorine, renders the nitrile invulnerable to attack. 9. The environmental implications of these findings and the possible significance of the enzyme in the regulation of metabolism are discussed.     

Molecular characterisation of a novel thermophilic nitrile hydratase

The thermophilic soil isolate, Bacillus pallidus Dac521, expresses a constitutive nitrile hydratase. The purified enzyme was found to be a 110 kDa tetramer composed of two alpha and two beta subunits with molecular masses of 27 kDa and 29 kDa, respectively. The enzyme electrophoresed as a single protein band on native PAGE but two protein bands with isoelectric points of 4.7 and 5.5 on isoelectric focusing suggested the presence of isozymes. The purified enzyme was moderately thermostable up to 55 degrees C and the enzyme activity was stable over a broad pH range. Comparisons of the N-terminal amino acid sequences of the nitrile hydratase subunits with those of other nitrile hydratases showed up to 90% identity for the beta subunit sequence but no significant identity for the alpha subunit. The enzyme hydrolysed a narrow range of aliphatic substrates and did not hydrolyse any of the cyclic, hydroxy-, di- or aromatic nitriles tested. The activity was irreversibly inhibited by the aromatic nitrile, benzonitrile. The kinetic constants for acetonitrile, acrylonitrile and propionitrile compared favourably with those of mesophilic nitrile hydratases.     

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

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

Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (alpha subunit 26 kDa, beta subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the beta subunit. However, the amino acid sequence of the alpha subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes.     

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.     

Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp. strain PCC6803 and heterologous expression and characterization of the encoded protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Nitrile hydratase of Rhodococcus sp. N-774. Purification and amino acid sequences

The nitrile hydratase of Rhodococcus sp. N-774 was purified and crystallized. The enzyme is composed of two different subunits (molecular masses: subunit alpha, 28,500 Da; subunit beta, 29,000 Da). The amino-terminal amino acid sequence of each subunit was determined. There is no sequence homology between the two subunits, suggesting that the peptides originate from different cistrons. The activity of the purified enzyme did not decrease during incubation in the dark, whereas it gradually decreased in intact cells.     

Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

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.     

Real-time monitoring of nitrile biotransformations by mid-infrared spectroscopy

In this study mid-infrared spectroscopy was used to follow the enzyme kinetics involved in nitrile biocatalysis using whole cell suspensions of the bacterium Rhodococcus rhodochrous LL100-21. The bacteria were grown on acetonitrile to induce a two-step enzymatic pathway. Acetonitrile was biotransformed to acetamide by a nitrile hydratase enzyme and subsequently to acetic acid (carboxylate ion) by an amidase enzyme. The bacteria were also grown on benzonitrile to induce a one-step enzymatic pathway. Benzonitrile was biotransformed directly to benzoic acid (carboxylate ion) by a nitrilase enzyme. These reactions were followed by React IR using a silicon probe and gave excellent quantitative and qualitative real-time data of both nitrile biocatalytic reactions. This study has shown that this novel technique has potentially useful applications in biocatalysis.     

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.     

Purification and properties of a nonheme bromoperoxidase from Streptomyces aureofaciens

The first bacterial nonheme type bromoperoxidase has been purified to homogeneity from the chlorotetracycline-producing actinomycete Streptomyces aureofaciens Tü 24. Purification was accomplished by (NH4)2SO4 precipitation, DEAE-cellulose chromatography at different pH-values, and molecular sieve chromatography. The purified enzyme has a molecular mass of 90 to 95 kDa based on ultracentrifugation and gel filtration. The enzyme is composed of three subunits of identical molecular mass (m = 31 kDa). Bromoperoxidase catalyses the bromination of monochlorodimedone, but not its chlorination, and has no peroxidase or catalase activity. The optimum pH is 4.5. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metals in equimolar amounts. Bromoperoxidase is stable in a pH range from pH 4.0 to pH 10.0 at 4 degrees C for weeks and does not loose any activity when incubated at 80 degrees C for 2 h.     

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens.

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.     

Crystallization and properties of cathepsin B from rat liver.

Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugal analysis. Its s20,w value was 2.8 S and its relative molecular mass was calculated to be 22,500 (+/- 900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with alpha-N-benzoyl-DL-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.     

Purification and properties of carnitine dehydratase from Escherichia coli--a new enzyme of carnitine metabolization

Carnitine dehydratase from Escherichia coli 044 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine. It has been purified 500-fold to electrophoretic homogeneity by chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, second phenyl-Sepharose and finally gel filtration on a Sephadex G-100 column. During the purification procedure a low-molecular-weight effector essential for enzyme activity was separated from the enzyme. The addition of this still unknown effector caused reactivation of the apoenzyme. The relative molecular mass of the apoenzyme has been estimated to be 85,000. It seems to be composed of two identical subunits with a relative molecular mass of 45,000. The purified and reactivated enzyme has been further characterized with respect to pH and temperature optimum (7.8 and 37-42 degrees C), equilibrium constant (Keq = 1.5 +/- 0.2) and substrate specifity. The enzyme is inhibited by thiol reagents. The Km value for crotonobetaine is 1.2.10(-2) M. gamma-Butyrobetaine, D(+)-carnitine and choline are competitive inhibitors of crotonobetaine hydration.     

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.