The nitrile hydratase from Rhodococcus equi A4 consisted of two kinds of subunits which slightly differed in molecular weight (both approximately 25 kDa) and showed a significant similarity in the N-terminal amino acid sequences to those of the nitrile hydratase from Rhodococcus sp. N-774. The enzyme preferentially hydrated the S-isomers of racemic 2-(2-, 4-methoxyphenyl)propionitrile, 2-(4-chlorophenyl)propionitrile and 2-(6-methoxynaphthyl)propionitrile (naproxennitrile) with E-values of 5-15. The enzyme functioned in the presence of 5-98% (v/v) of different hydrocarbons, alcohols or diisopropyl ether. The addition of 5% (v/v) of n-hexane, n-heptane, isooctane, n-hexadecane, pristane and methanol increased the E-value for the enzymatic hydration of 2-(6-methoxynaphthyl)propionitrile.

• MeSH
• dehydratasy chemie   izolace a purifikace   metabolismus   MeSH
• izoelektrický bod MeSH
• koncentrace vodíkových iontů MeSH
• methanol farmakologie   MeSH
• molekulová hmotnost MeSH
• nitrily metabolismus   MeSH
• podjednotky proteinů MeSH
• Rhodococcus equi enzymologie   MeSH
• rozpouštědla farmakologie   MeSH
• sekvence aminokyselin MeSH
• stereoizomerie MeSH
• substrátová specifita MeSH
• teplota MeSH
• uhlovodíky farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dehydratasy MeSH
• methanol MeSH
• nitrile hydratase MeSH Prohlížeč
• nitrily MeSH
• podjednotky proteinů MeSH
• rozpouštědla MeSH
• uhlovodíky MeSH

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.

• MeSH
• amidohydrolasy metabolismus   MeSH
• amidy metabolismus   toxicita   MeSH
• benzamidy metabolismus   MeSH
• biotransformace MeSH
• dehydratasy metabolismus   MeSH
• herbicidy chemie   metabolismus   MeSH
• hydrolýza MeSH
• jodbenzeny metabolismus   MeSH
• kořeny rostlin účinky léků   růst a vývoj   metabolismus   MeSH
• nitrily chemie   metabolismus   toxicita   MeSH
• Rhodococcus metabolismus   MeSH
• salát (hlávkový) účinky léků   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2,6-dichlorobenzamide MeSH Prohlížeč
• amidase MeSH Prohlížeč
• amidohydrolasy MeSH
• amidy MeSH
• benzamidy MeSH
• benzonitrile MeSH Prohlížeč
• bromoxynil MeSH Prohlížeč
• chloroxynil MeSH Prohlížeč
• dehydratasy MeSH
• dichlobanil MeSH Prohlížeč
• herbicidy MeSH
• ioxynil MeSH Prohlížeč
• jodbenzeny MeSH
• nitrile hydratase MeSH Prohlížeč
• nitrily MeSH

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.

• MeSH
• amidohydrolasy metabolismus   MeSH
• dehydratasy metabolismus   MeSH
• DNA bakterií chemie   genetika   MeSH
• genetická transkripce MeSH
• hydroxylaminy metabolismus   MeSH
• multigenová rodina MeSH
• nitrily metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• Rhodococcus enzymologie   genetika   MeSH
• sekvenční analýza DNA MeSH
• stanovení celkové genové exprese MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amidohydrolasy MeSH
• cyanohydrin MeSH Prohlížeč
• dehydratasy MeSH
• DNA bakterií MeSH
• hydroxylaminy MeSH
• nitrile hydratase MeSH Prohlížeč
• nitrily MeSH

This work critically reviews the assays of nitrile-converting and nitrile-forming enzymes (nitrilases, nitrile hydratases, amidases, aldoxime dehydratases). Most of the strains producing such enzymes were obtained by selection on media with nitriles, amides or aldoximes as nitrogen sources. Activity and enantioselectivity of the enzymes was usually assayed by time-consuming chromatographic analysis of substrates and the corresponding reaction products. Attempts at introducing faster assays resulted in several spectrophotometric methods for reaction product (ammonia, hydroxamate, methacrylamide, benzamide, etc.) determination. Recently, new methods for colorimetric and fluorimetric determination of ammonia have been developed, which appear promising for high-throughput assays. Alternatively, methods consisting in determination of NADH consumed in a coupled amination reaction or pH-responsive methods are promising for this purpose. All the above selection and screening methods establish fundamental conditions for the design of hierarchical screening projects. However, the potential of these principles, in particular spectrophotometric and fluorimetric methods, will be probably further exploited and adapted to multiwell plate and robotic systems.

• MeSH
• aminohydrolasy analýza   MeSH
• nitrily chemie   metabolismus   MeSH
• stereoizomerie MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• aminohydrolasy MeSH
• nitrilase MeSH Prohlížeč
• nitrily MeSH

Simultaneous HPLC determination of bromoxynil, ioxynil and dichlobenil, three arylnitrile herbicides, and their metabolic products in soil extracts and microbiological media is described. Limits of detection (LODs) ranged from 0.56 to 3.97 ppb. Slight modification of the mobile phase composition allowed determination of 13 other aromatic nitriles. Assay of aromatic nitrile hydratase, amidase or nitrilase activities is possible by the method developed.

• MeSH
• benzamidy analýza   metabolismus   MeSH
• herbicidy analýza   metabolismus   MeSH
• jodbenzeny analýza   metabolismus   MeSH
• kalibrace MeSH
• látky znečišťující půdu analýza   metabolismus   MeSH
• lineární modely MeSH
• nitrily analýza   metabolismus   MeSH
• Pseudomonas putida metabolismus   MeSH
• půda MeSH
• půdní mikrobiologie MeSH
• reprodukovatelnost výsledků MeSH
• Rhizobium metabolismus   MeSH
• senzitivita a specificita MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzamidy MeSH
• bromoxynil MeSH Prohlížeč
• dichlobanil MeSH Prohlížeč
• herbicidy MeSH
• ioxynil MeSH Prohlížeč
• jodbenzeny MeSH
• látky znečišťující půdu MeSH
• nitrily MeSH
• půda MeSH

In plants, aldoximes per se act as defense compounds and are precursors of complex defense compounds such as cyanogenic glucosides and glucosinolates. Bacteria rarely produce aldoximes, but some are able to transform them by aldoxime dehydratase (Oxd), followed by nitrilase (NLase) or nitrile hydratase (NHase) catalyzed transformations. Oxds are often encoded together with NLases or NHases in a single operon, forming the aldoxime-nitrile pathway. Previous reviews have largely focused on the use of Oxds and NLases or NHases in organic synthesis. In contrast, the focus of this review is on the contribution of these enzymes to plant-bacteria interactions. Therefore, we summarize the substrate specificities of the enzymes for plant compounds. We also analyze the taxonomic and ecological distribution of the enzymes. In addition, we discuss their importance in selected plant symbionts. The data show that Oxds, NLases, and NHases are abundant in Actinobacteria and Proteobacteria. The enzymes seem to be important for breaking through plant defenses and utilizing oximes or nitriles as nutrients. They may also contribute, e.g., to the synthesis of the phytohormone indole-3-acetic acid. We conclude that the bacterial and plant metabolism of aldoximes and nitriles may interfere in several ways. However, further in vitro and in vivo studies are needed to better understand this underexplored aspect of plant-bacteria interactions.

• Klíčová slova
• aldoxime dehydratase, aldoxime-nitrile pathway, indole-3-acetic acid, nitrilase, nitrile hydratase, phytohormone, plant aldoxime, plant defense, plant-bacteria interaction,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The nitrile hydratase (NHase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The resting cells (having NHase activity) (8 %; 1 mL corresponds to 22 mg dry cell mass, DCM) were immobilized in polyacrylamide gel containing 12.5 % acrylamide, 0.6 % bisacrylamide, 0.2 % diammonium persulfate and 0.4 % TEMED. The polyacrylamide entrapped cells (1.12 mg DCM/mL) completely converted acrylonitrile in 3 h at 10 °C, using 0.1 mol/L potassium phosphate buffer. In a partitioned fed batch reactor, 432 g/L acrylamide was accumulated after 1 d. The polyacrylamide discs were recycled up to 3×; 405, 210 and 170 g/L acrylamide was produced in 1st, 2nd and 3rd recycling reactions. In four cycles, a total of 1217 g acrylamide was produced by recycling the same mass of entrapped cells.

• MeSH
• akrylamid metabolismus   MeSH
• akrylonitril metabolismus   MeSH
• akrylové pryskyřice * MeSH
• bakteriální proteiny metabolismus   MeSH
• biotechnologie metody   MeSH
• dehydratasy metabolismus   MeSH
• imobilizované buňky metabolismus   MeSH
• Rhodococcus enzymologie   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylamid MeSH
• akrylonitril MeSH
• akrylové pryskyřice * MeSH
• bakteriální proteiny MeSH
• dehydratasy MeSH
• nitrile hydratase MeSH Prohlížeč
• polyacrylamide gels MeSH Prohlížeč

The purpose of this study is to summarize the current knowledge of the enzymes which are involved in the hydrolysis of cyanide, i.e., cyanide hydratases (CHTs; EC 4.2.1.66) and cyanide dihydratases (CynD; EC 3.5.5.1). CHTs are probably exclusively produced by filamentous fungi and widely occur in these organisms; in contrast, CynDs were only found in a few bacterial genera. CHTs differ from CynDs in their reaction products (formamide vs. formic acid and ammonia, respectively). Several CHTs were also found to transform nitriles but with lower relative activities compared to HCN. Mutants of CynDs and CHTs were constructed to study the structure-activity relationships in these enzymes or to improve their catalytic properties. The effect of the C-terminal part of the protein on the enzyme activity was determined by constructing the corresponding deletion mutants. CynDs are less active at alkaline pH than CHTs. To improve its bioremediation potential, CynD from Bacillus pumilus was engineered by directed evolution combined with site-directed mutagenesis, and its operation at pH 10 was thus enabled. Some of the enzymes have been tested for their potential to eliminate cyanide from cyanide-containing wastewaters. CynDs were also used to construct cyanide biosensors.

• Klíčová slova
• Cyanide biosensors, Cyanide dihydratase, Cyanide hydratase, Enzyme production, Structure-activity relationships, Wastewater bioremediation,
• MeSH
• Bacteria enzymologie   MeSH
• biosenzitivní techniky * MeSH
• biotransformace MeSH
• dehydratasy chemie   genetika   metabolismus   MeSH
• houby enzymologie   MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• hydrolýza MeSH
• koncentrace vodíkových iontů MeSH
• kyanidy analýza   metabolismus   MeSH
• látky znečišťující životní prostředí analýza   metabolismus   MeSH
• mutační analýza DNA MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• proteinové inženýrství MeSH
• stabilita enzymů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• cyanide dihydratase MeSH Prohlížeč
• cyanide hydratase MeSH Prohlížeč
• dehydratasy MeSH
• hydrolasy MeSH
• kyanidy MeSH
• látky znečišťující životní prostředí MeSH
• mutantní proteiny MeSH

The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and β-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.

• Klíčová slova
• Aldoxime–nitrile pathway, Biocatalytic applications, Database mining, Heterologous production, Metagenome mining, Nitrilase,
• MeSH
• aminohydrolasy genetika   metabolismus   MeSH
• Bacteria enzymologie   genetika   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biodegradace MeSH
• biokatalýza MeSH
• databáze proteinů MeSH
• fungální proteiny genetika   metabolismus   MeSH
• houby enzymologie   genetika   MeSH
• metagenomika MeSH
• nitrily metabolismus   MeSH
• proteinové inženýrství metody   MeSH
• rekombinantní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• aminohydrolasy MeSH
• bakteriální proteiny MeSH
• fungální proteiny MeSH
• nitrilase MeSH Prohlížeč
• nitrily MeSH
• rekombinantní proteiny MeSH

A large number of aromatic compounds and organic nitriles, the two groups of compounds covered in this review, are intermediates, products, by-products or waste products of the chemical and pharmaceutical industries, agriculture and the processing of fossil fuels. The majority of these synthetic substances (xenobiotics) are toxic and their release and accumulation in the environment pose a serious threat to living organisms. Bioremediation using various bacterial strains of the genus Rhodococcus has proved to be a promising option for the clean-up of polluted sites. The large genomes of rhodococci, their redundant and versatile catabolic pathways, their ability to uptake and metabolize hydrophobic compounds, to form biofilms, to persist in adverse conditions and the availability of recently developed tools for genetic engineering in rhodococci make them suitable industrial microorganisms for biotransformations and the biodegradation of many organic compounds. The peripheral and central catabolic pathways in rhodococci are characterized for each type of aromatics (hydrocarbons, phenols, halogenated, nitroaromatic, and heterocyclic compounds) in this review. Pathways involved in the hydrolysis of nitrile pollutants (aliphatic nitriles, benzonitrile analogues) and the corresponding enzymes (nitrilase, nitrile hydratase) are described in detail. Examples of regulatory mechanisms for the expression of the catabolic genes are given. The strains that efficiently degrade the compounds in question are highlighted and examples of their use in biodegradation processes are presented.

• MeSH
• aromatické uhlovodíky metabolismus   MeSH
• látky znečišťující životní prostředí metabolismus   MeSH
• metabolické sítě a dráhy MeSH
• nitrily metabolismus   MeSH
• průmyslový odpad * MeSH
• Rhodococcus metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• aromatické uhlovodíky MeSH
• látky znečišťující životní prostředí MeSH
• nitrily MeSH
• průmyslový odpad * MeSH