Nitrile Hydratase Activity Research Articles

Biotransformations of nitriles mediated by in vivo nitrile hydratase of Rhodococcus erythropolis ATCC 4277 heterologously expressed in E. coli

Nitrile hydratase activity has been reported as an exciting alternative for the industrial production of a variety of compounds overwhelming its chemical counterpart; despite this, until now, a few enzymes have been thoroughly studied. Efficient expression of nitrile hydratase enzymes has been the bottleneck to explore this activity. Here, we report the cloning and expression of Rhodococcus erythropolis ATCC 4277 nitrile hydratase (α- and β-subunits) and the correspondent activator gene. Furthermore, substrate scope with whole cells of recombinant E. coli demonstrates that this Fe-type NHase could hydrate a wide range of aliphatic and aromatic nitrile with high conversion rates and moderate enantiomeric excess.

Screening and characterization of a nitrilase with significant nitrile hydratase activity.

We screened nitrilases with significant nitrile hydratase activity to exploit their potential in benzylic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on benzylic amide production by nitrilase. A sequence-based screening method using previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as "amide-formation hotspots") as molecular probes to identify putative amide-forming nitrilases. Based on the previously reported "amide-formation hotspots," we identified a nitrilase NitPG from Paraburkholderia graminis DSM 17151 that could produce a significant amount of mandelamide toward mandelonitrile and exhibited general hydration activity toward various benzylic nitriles. The time-course experiment with NitPG demonstrated that amide was also a true reaction product of nitrilase, suggesting that the nitrile catalysis by amide-forming nitrilase could be a post-transition state bifurcation-mediated enzymatic reaction. Further research demonstrated that low temperature, metal ion addition, and specific substrate structure could profoundly improve the amide formation capability of nitrilase. NitPG with broad hydration activity is a potential candidate for the enzymatic synthesis of benzylic amides for biotechnological applications. Studying the effect of nitrilase hydration activity could promote our understanding of the factors that influence amide and acid distribution.

Substrate access tunnel engineering for improving the catalytic activity of a thermophilic nitrile hydratase toward pyridine and pyrazine nitriles

Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an exothermic reaction, a NHase with high activity and stability is needed for amide production. However, the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this study, through the combination of substrate access tunnel calculation, residue conservative analysis and site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic NHase from extremophile Caldalkalibacillus thermarum TA2. A1, βLeu48, was semi-rationally identified as a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine nitriles. The specific activity of the corresponding mutant βL48H towards 3-cyanopyridine, 2-cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine carboxamides. Further structural analysis demonstrated that the βHis48 could form a long-lasting hydrogen bond with αGlu166, which contributes to the expansion of the entrance of substrate access tunnel and accelerate substrate migration.

Maturation Mechanism of Nitrile Hydratase From Streptomyces canus CGMCC 13662 and Its Structural Character

Nitrile hydratases have received significant interest both in the large-scale industrial production of acrylamide and nicotinamide, and the remediation of environmental contamination with nitrile-containing pollutants. Almost all known nitrile hydratases include an α-subunit (AnhA) and β-subunit (AnhB), and a specific activator protein is crucial for their maturation and catalytic activity. Many studies exist on nitrile hydratase characteristics and applications, but few have reported their metal insertion and post-translational maturation mechanism. In this study, we investigated the cobalt insertion and maturation mechanism of nitrile hydratase from Streptomyces canus CGMCC 13662 (ScNHase) bearing three subunits (AnhD, AnhE, and AnhA). ScNHase subunits were purified, and the cobalt content and nitrile hydratase activity of the ScNHase subunits were detected. We discovered that cobalt could insert into the cobalt-free AnhA of ScNHase in the absence of activator protein under reduction agent DL-dithiothreitol (DTT) environment. AnhD not only performed the function of AnhB of NHase, but also acted as a metal ion chaperone and self-subunit swapping chaperone, while AnhE did not act as similar performance. A cobalt direct-insertion under reduction condition coordinated self-subunit swapping mechanism is responsible for ScNHase post-translational maturation. Molecular docking of ScNHase and substrates suggested that the substrate specificity of ScNHase was correlated with its structure. ScNHase had a weak hydrophobic interaction with IAN through protein–ligand interaction analysis and, therefore, had no affinity with indole-3-acetonitrile (IAN). The post-translational maturation mechanism and structure characteristics of ScNHase could help guide research on the environmental remediation of nitrile-containing waste contamination and three-subunit nitrile hydratase.

Influence of Acrylamide on Energy Status and Survival of Bacteria of Different Systematic Groups.

We studied the effect of acrylamide on the content of intracellular ATP in the cells of bacteria of the genera Rhodococcus and Alcaligenes, the luminescence of the genetically engineered strain Escherichia coli K12 TG1 (pXen7), and the survival of bacteria of various systematic groups. According to the level of decrease in the concentration of intracellular ATP, it was found that the strain with lower amidase activity (R. erythropolis 6-21) and Gram-negative proteobacteria A. faecalis 2 were the most sensitive to acrylamide after a 20-min exposure, while the strain R. ruber gt 1 was stable, having a high nitrile hydratase activity in combination with a low amidase activity. EC50 of acrylamide for 2 h was 7.1 g/L for E. coli K12 TG1 (pXen7). Acrylamide at a concentration of 10-20 mM added to the culture medium led to a slight decrease in the number of CFUs of Rhodococcus, A. faecalis 2, and E. coli compared to the control. At an acrylamide concentration of 250 mM, from 0.016 to 0.116% of viable bacterial cells remained, and a solution of 500 mM and higher inhibited the growth of the majority of the studied strains. The results confirm that acrylamide is much less toxic to prokaryotes than to eukaryotes.

Potential environmental risks of nanopesticides: Application of Cu(OH)2 nanopesticides to soil mitigates the degradation of neonicotinoid thiacloprid

Cu(OH)2 nanopesticides and organic insecticides are continuously applied to soil at a temporal interval, while knowledge about the impact of Cu(OH)2 nanopesticides on organic insecticides degradation is currently scarce, resulting in poorly comprehensive evaluation of the potential environmental risks of Cu(OH)2 nanopesticides. Herein, a commercial Cu(OH)2 nanopesticide formulation (NPF), the active ingredient of NPF (AI-NPF), the prepared Cu(OH)2 nanotubes (NT) with comparable morphology and size to AI-NPF, and CuSO4 were respectively applied to soil at normal doses (0.5, 5 and 50 mg/kg), followed by an application of neonicotinoid thiacloprid after an interval of 21 d, showing that NPF at doses of 5 and 50 mg/kg significantly (p < 0.05) mitigated thiacloprid degradation compared to control and CuSO4. Furthermore, AI-NPF was the primary component that contributed to the mitigation effect of NPF, which was also validated by the NT. Large differences in the degradation efficiency of thiacloprid in sterilized and unsterilized soils with Cu(OH)2 nanopesticides suggested that biodegradation was the primary process responsible for thiacloprid degradation, especially as chemical degradation was negligible. Besides a decrease of thiacloprid bioavailability due to adsorption by Cu(OH)2 nanopesticides, we demonstrated that Cu(OH)2 nanopesticides changed soil microbial communities, reduced nitrile hydratase activity and down-regulated thiacloprid-degradative nth gene abundance, which thus mitigated thiacloprid biodegradation. Clearly, this study shed light on the potential environmental risks of Cu(OH)2 nanopesticide.

Biofilm Formation by Monocultures and Mixed Cultures of Alcaligenes faecalis 2 and Rhodococcus ruber gt 1

Biofilm formation by monocultures and mixed cultures of nitrile-hydrolyzing bacteria Alcaligenes faecalis 2 and Rhodococcus ruber gt 1 was studied. Biofilm formation was assessed by staining with Crystal violet, by the energy state of the cells, and by the output of the polymer matrix. A quantitative method for assessment of the output of the polymer matrix by the fluorescence of konA-tetramethylrhodamine was developed. The medium used for obtaining mixed biofilms of Al. faecalis 2 and R. ruber gt 1 contained acetamide and glucose as carbon sources. In mixed biofilms Al. faecalis 2 was shown to act as a primary colonizer, while R. ruber gt 1 was a satellite microorganism. Compared to monoculture biofilms, mixed biofilms exhibited higher output of the polymer matrix and increased biofilm formation by Al. faecalis 2 and R. ruber gt 1 on addition of the culture liquid of the second strain. Daily addition of the toxic substrate acetonitrile to planktonic and biofilm cultures resulted in bacterial adaptation, as was indicated by higher numbers of surviving cells compared to the variants with acetonitrile addition every 10 min, mainly due to nitrile hydratase activity of R. ruber gt 1, which transformed acetonitrile into nontoxic acetamide. Our results show that binary biofilms of amidase-containing Al. faecalis 2 and R. ruber gt 1, a strain with high nitrile hydratase activity, are promising as biocatalysts for acrylic acid production and as the basis for a biofilter for nitrile removal from wastewater.

Efficient inducible expression of nitrile hydratase in Corynebacterium glutamicum

Abstract Nitrile hydratases are important industrial catalysts to produce valuable amides. In this study, we describe a comprehensive and systematic approach to the development of an inducible expression system for enhanced nitrile hydratase expression in Corynebacterium glutamicum. Through promoter engineering, codon optimization and design of ribosome binding site sequences, the nitrile hydratase activity toward 3-cyanopyridine was improved from 0.33 U/mg DCW to 12.03 U/mg DCW in shake-flask culture. By introduction of the novel inducible mmp expression system, the nitrile hydratase activity was further elevated to 14.97 U/mg DCW. Finally, a high nitrile hydratase yield of 1432 U/mL was achieved in a fed-batch fermentation process and used for nicotinamide production. These results provide new insights for the development of heterologous protein expression systems in C. glutamicum.

The nitrilase PtNIT1 catabolizes herbivore-induced nitriles in Populus trichocarpa

BackgroundNitrilases are nitrile-converting enzymes commonly found within the plant kingdom that play diverse roles in nitrile detoxification, nitrogen recycling, and phytohormone biosynthesis. Although nitrilases are present in all higher plants, little is known about their function in trees. Upon herbivory, poplars produce considerable amounts of toxic nitriles such as benzyl cyanide, 2-methylbutyronitrile, and 3-methylbutyronitrile. In addition, as byproduct of the ethylene biosynthetic pathway upregulated in many plant species after herbivory, toxic β-cyanoalanine may accumulate in damaged poplar leaves. In this work, we studied the nitrilase gene family in Populus trichocarpa and investigated the potential role of the nitrilase PtNIT1 in the catabolism of herbivore-induced nitriles.ResultsA BLAST analysis revealed three putative nitrilase genes (PtNIT1, PtNIT2, PtNIT3) in the genome of P. trichocarpa. While PtNIT1 was expressed in poplar leaves and showed increased transcript accumulation after leaf herbivory, PtNIT2 and PtNIT3 appeared not to be expressed in undamaged or herbivore-damaged leaves. Recombinant PtNIT1 produced in Escherichia coli accepted biogenic nitriles such as β-cyanoalanine, benzyl cyanide, and indole-3-acetonitrile as substrates in vitro and converted them into the corresponding acids. In addition to this nitrilase activity, PtNIT1 showed nitrile hydratase activity towards β-cyanoalanine, resulting in the formation of the amino acid asparagine. The kinetic parameters of PtNIT1 suggest that the enzyme utilizes β-cyanoalanine and benzyl cyanide as substrates in vivo. Indeed, β-cyanoalanine and benzyl cyanide were found to accumulate in herbivore-damaged poplar leaves. The upregulation of ethylene biosynthesis genes after leaf herbivory indicates that herbivore-induced β-cyanoalanine accumulation is likely caused by ethylene formation.ConclusionsOur data suggest a role for PtNIT1 in the catabolism of herbivore-induced β-cyanoalanine and benzyl cyanide in poplar leaves.

Bench scale production of butyramide using free and immobilized cells of Bacillus sp. APB-6.

Butyramide is a commodity chemical having wide range of applications from material science to biological sciences including synthesis of therapeutic drugs, hydroxamic acids, and electrorheological fluids. The nitrile hydratase protein of Bacillus sp. APB-6 was explored to develop an efficient biocatalytic process for the biotransformation of butyronitrile to butyramide using free and immobilized cells. The reaction conditions for nitrile hydratase activity were not affected after immobilization of the free cells and the optimum pH and temperature for both free and immobilized cells were 8.0 and 55°C, respectively. In a 1-l batch reaction, complete conversion of 3000mM butyronitrile to butyramide was achieved using free and immobilized cells. Immobilization of the cells further enhanced their operational stability and reusability in repetitive cycles of butyramide production. This bioconversion resulted in the formation of butyramide at a rate of 522.0gh-1g-1dcw.

Biochars change the sorption and degradation of thiacloprid in soil: Insights into chemical and biological mechanisms.

One interest of using biochar as soil amendment is to reduce pesticide adverse effects. In this paper, the sorption and degradation of thiacloprid (THI) in a black soil amended by various biochars were systematically investigated, and the mechanisms therein were explored by analyzing the changes in soil physicochemical properties, degrading enzymes and genes and microorganism community. Biochar amendment increased THI sorption in soil, which was associated with an increase in organic carbon and surface area and a decrease in H/C. Amendments of 300-PT (pyrolyzing temperature) biochar promoted the biodegradation of THI by increasing the microbe abundance and improving nitrile hydratase (NHase) activity. In contrast, 500- and 700-PT biochar amendments inhibited biodegradation by reducing THI availability and changing NHase activity and THI-degradative nth gene abundance, and instead promoted chemical degradation mainly through elevated pH, active groups on mineral surface and generation of •OH and other free radicals. Furthermore, THI shifted the soil microbial community, stimulated the NHase activity and elevated nth gene abundance. Biochar amendments also changed soil bacterial community by modulating soil pH, dissolved organic matter and nitrogen and phosphorus levels, which further influenced THI biodegradation. Therefore, the impact of biochars on the fate of a pesticide in soil depends greatly on their type and properties, which should be comprehensively examined when applying biochar to soil.

Insights into the microbial degradation pathways of the ioxynil octanoate herbicide

Abstract This paper describes the biodegradation of the ioxynil octanoate herbicide by indigenous microorganisms isolated from herbicide impacted soil-enrichment cultures. Eleven positive hits out of twenty-nine microorganisms screened for nitrile hydratase, nitrilase and amidase activity were further evaluated based on their growth in microtiter plates containing liquid medium with increasing concentrations of herbicide (0.97–250 mM). Two strains were selected from this assay for biodegradation studies and were identified as Lysinibacillus boronitolerans MLH-31 and Bacillus cereus MLH-61. The bacterial degradation of ioxynil octanoate and its biodegradation products were monitored, identified and characterized by liquid chromatography tandem mass spectrometry (HPLC-MS/MS). In addition to 3,5-diiodo-4-hydroxybenzamide and 3,5-diiodo-4-hydroxybenzoic acid, which are commonly detected metabolites, two new metabolites were observed: mono-deiodinated compound 3-iodo-4-hydroxybenzoic acid and the product of Caromatic-CN cleaved 1,3-diiodophenol. The experimentally observed metabolites were correlated with the enzymatic systems involved, revealing the presence of esterases, nitrile hydratases, amidases, nitrilases, dehalogenases and carbon-carbon lyases during biodegradation. Lysinibacillus boronitolerans MLH-31 was found to degrade ioxynil octanoate at a rate of 97% over 7 days through a batch-resting cells experiment, while Bacillus cereus MLH-61 was found to do so at a rate of 75% under the same conditions.

Heterogeneous biocatalyst for nitrile and amide transformation based on cells of nitrile-hydrolyzing bacteria and multiwalled carbon nanotubes

A heterogeneous biocatalyst for the biotransformation of nitriles and amides of carboxylic acids in the form of cells of nitrile-hydrolyzing bacteria immobilized on the carrier, was created based on multiwalled carbon nanotubes (MWCNTs). It was shown that bacterial cells form aggregates in contact with powderformed purified or unpurified MWCNTs. The amount of both gram-positive and gram-negative bacteria binding with unpurified MWCNTs was significantly higher than with purified. The nitrile hydratase and amidase activity of bacterial aggregates of purified MWCNTs was preserved to a greater extent as compared to that of unpurified MWCNTs and cells adhered to the surface of the carbonized pyrosealing material with MWCNTs. Both gram-positive Rhodococcus ruber gt1 and gram-negative Alcaligenes faecalis 2 remained viable when cultured in the presence of purified or unpurified MWCNTs. The obtained heterogeneous biocatalyst can be easily separated from the medium by filtration and can be used repeatedly.

Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes.

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.

Effect of flexibility and positive charge of the C-terminal domain on the activator P14K function for nitrile hydratase inPseudomonas putida

A self-subunit swapping chaperone is crucial for cobalt incorporation into nitrile hydratase. However, further information about its structural features is not available. The flexibility and positive charge of the C-terminal domain of the self-subunit swapping chaperone (P14K) of nitrile hydratase from Pseudomonas putida NRRL-18668 play an important role in cobalt incorporation. C-terminal domain truncation, alternation of C-terminal domain flexibility through mutant P14K(G86I), and elimination of the positive charge in the C-terminal domain sharply affected nitrile hydratase cobalt content and activity. The flexible, positively charged C-terminal domain most likely carries out an external action that allows a cobalt-free nitrile hydratase to overcome an energetic barrier, resulting in a cobalt-containing nitrile hydratase.

Capturing a Sulfenic Acid with Arylboronic Acids and Benzoxaborole

Post-translational redox generation of cysteine-sulfenic acids (Cys-SOH) functions as an important reversible regulatory mechanism for many biological functions, such as signal transduction, balancing cellular redox states, catalysis, and gene transcription. Herein we show that arylboronic acids and cyclic benzoxaboroles can form adducts with sulfenic acids in aqueous medium and that these boron-based compounds can potentially be used to trap biologically significant sulfenic acids. As proof of principle we demonstrate that a benzoxaborole can inhibit the enzyme activity of an iron-containing nitrile hydratase, which requires a catalytic αCys114-SOH in the active site. The nature of the adduct and the effect of the boronic acid's pK(a)(B) on the stability constant of the adduct are discussed within.

Simultaneous purification of nitrile hydratase and amidase of Alcaligenes sp. MTCC 10674.

Alcaligenes sp. MTCC 10674 has a bienzymatic system for the hydrolysis of nitriles. The nitrile hydratase and amidase have been purified simultaneously to homogeneity using a combination of (NH)4SO4 precipitation, ion exchange chromatography and gel permeation chromatography. Nitrile hydratase and amidase have molecular weight of 47 and 114 kDa, respectively and exist as heterodimer. Optimum temperatures for maximum activity of nitrile hydratase and amidase were 15 °C (2.4 U/mg protein) and 45 °C (2.3 U/mg protein), respectively. Nitrile hydratase showed maximum 7.8 U/mg protein at 50 mM acrylonitrile and amidase has 9.2 U/mg protein at 25 mM propionamide. Nitrile hydratase has Vmax 10 μmol/min/mg and Km 40 mM, while amidase has Vmax 12.5 μmol/min/mg and Km 45.5 mM, respectively. Heavy metal ions Hg2+, Ag+, Pb2+ and Cu2+ were strong inhibitors of nitrile hydratase and amidase activity.

An aeroplysinin-1 specific nitrile hydratase isolated from the marine sponge Aplysina cavernicola.

A nitrile hydratase (NHase) that specifically accepts the nitrile aeroplysinin-1 (1) as a substrate and converts it into the dienone amide verongiaquinol (7) was isolated, partially purified and characterized from the Mediterranean sponge Aplysina cavernicola; although it is currently not known whether the enzyme is of sponge origin or produced by its symbiotic microorganisms. The formation of aeroplysinin-1 and of the corresponding dienone amide is part of the chemical defence system of A. cavernicola. The latter two compounds that show strong antibiotic activity originate from brominated isoxazoline alkaloids that are thought to protect the sponges from invasion of bacterial pathogens. The sponge was shown to contain at least two NHases as two excised protein bands from a non denaturating Blue Native gel showed nitrile hydratase activity, which was not observed for control samples. The enzymes were shown to be manganese dependent, although cobalt and nickel ions were also able to recover the activity of the nitrile hydratases. The temperature and pH optimum of the studied enzymes were found at 41 °C and pH 7.8. The enzymes showed high substrate specificity towards the physiological substrate aeroplysinin-1 (1) since none of the substrate analogues that were prepared either by partial or by total synthesis were converted in an in vitro assay. Moreover de-novo sequencing by mass spectrometry was employed to obtain information about the primary structure of the studied NHases, which did not reveal any homology to known NHases.

Random mutagenesis of the arylacetonitrilase from Pseudomonas fluorescens EBC191 and identification of variants, which form increased amounts of mandeloamide from mandelonitrile

The nitrilase from Pseudomonas fluorescens EBC191 was modified by introducing random mutations via error-prone PCR techniques in order to obtain nitrilase variants, which form increased amounts of mandeloamide from racemic mandelonitrile. A screening system was established and experimentally optimized, which allowed the screening of nitrilase variants with the intended phenotype. This system was based on the simultaneous expression of nitrilase variants and the mandeloamide converting amidase from Rhodococcus rhodochrous MP50 in recombinant Escherichia coli cells. The formation of increased amounts of mandeloamide from mandelonitrile by the nitrilase variants was detected after the addition of hydroxylamine and ferric iron ions by taking advantage of the acyltransferase activity of the amidase, which resulted in the formation of coloured iron(III)-hydroxamate complexes from mandeloamide. The system was applied for the screening of libraries of nitrilase variants and 30 enzyme variants identified, which formed increased amounts of mandeloamide from racemic mandelonitrile. The increase in amide formation was quantified by high-performance liquid chromatography and the genes encoding the relevant nitrilase variants sequenced. Thus, different types of mutations were identified. One group of mutants carried different deletions at the carboxy-terminus. The other types of variants carried amino acid exchanges in positions that had not been related previously to an increased amide formation. Finally, a nitrilase variant was created by combining two independently obtained point mutations. This enzyme variant demonstrated a true nitrile hydratase activity as it formed mandeloamide and mandelic acid in a ratio of about 19:1 from racemic mandelonitrile.

Трансформация 2- и 4-цианопиридинов свободными и иммобилизованными клетками нитрилгидролизующих бактерий

The transformation dynamics of 2- and 4-cyanopyridines by cells suspended and adsorbed on inorganic carriers has been studied in the Rhodococcus ruber gt 1 strain possessing nitrile hydratase activity and the Pseudomonas fluorescens C2 strain containing nitrilase. It was shown that both nitrile hydratase and nitrilase activities of immobilized cells against 2-cyanopyridine were 1.5-4 times lower compared to 4-cyanopyridine and 1.6-2 times lower than the activities of free cells against 2-cyanpopyridine. The possibility of obtaining isonicotinic acid during the combined conversion of 4-cyanopyridine by a mixed suspension of R. ruber gt 1 cells with a high level of nitrile hydratase activity and R. erythropolis 11-2 cells with a pronounced activity of amidase has been shown. Immobilization of Rhodococcus cells on raw coal and Pseudomonas cells on china clay was shown to yield a heterogeneous biocatalyst for the efficient transformation of cyanopyridines into respective amides and carbonic acids.