Enzymes of aldoxime–nitrile pathway for organic synthesis

Aldoxime–nitrile pathway is one of the important routes of carbon and nitrogen metabolism in many life forms and a key interface for plant–microbe interactions. This pathway starts with transformation of amino acids to aldoximes, which are converted to nitriles and the later are ultimately hydrolyzed to acids and ammonia. Understanding and engineering of the enzymes involved in this pathway viz. cytochrome P450/CYP79, aldoxime dehydratase, nitrilase, nitrile hydratase, amidase and hydroxynitrile lyase, presents unprecedented opportunities in biocatalysis and green chemistry. Co-expressing these enzymes in prokaryotic and eukaryotic microbial hosts and tailoring their properties i.e. activity, specificity, stability and enantioselectivity may lead to develop sustainable bioprocesses for the synthesis of industrially important nitriles, amides and acids.     

Nocardia globerula NHB-2: a versatile nitrile-degrading organism

A versatile nitrile-degrading bacterium was isolated by enrichment culture from the soil of a forest near Manali, Himachal Pradesh, India, and was identified as Nocardia globerula. This organism contains 3 enzymes with nitrile-degrading activity: nitrilase, nitrile hydratase, and amidase. Nocardia globerula NHB-2 cells grown on nutrient broth supplemented with 1% glucose and 0.1% yeast extract exhibited nitrile hydratase-amidase activity specific for saturated aliphatic nitriles or amide, while addition of acetonitrile in nutrient broth yielded cells with nitrile hydratase-amidase that in addition to saturated aliphatic nitriles-amide also hydrolyzed aromatic amide. Nocardia globerula NHB-2 cultivated on nutrient broth containing propionitrile exhibited nitrilase activity that hydrolyzed aromatic nitrile and unsaturated aliphatic nitrile. The versatility of this organism in the hydrolysis of various nitriles and amides makes it a potential bioresource for use in organic synthesis.     

Effects of nitriles and amides on the growth and the nitrile hydratase activity of the Rhodococcus sp. strain gt1

Effects of some nitriles and amides, as well as glucose and ammonium, on the growth and the nitrile hydratase (EC 4.2.1.84) activity of the Rhodococcus sp. strain gt1 isolated from soil were studied. The activity of nitrile hydratase mainly depended on carbon and nitrogen supply to cells. The activity of nitrile hydratase was high in the presence of glucose and ammonium at medium concentrations and decreased at concentrations of glucose more than 0.3%. Saturated unsubstituted aliphatic nitriles and amides were found to be a good source of nitrogen and carbon. However, the presence of nitriles and amides in the medium was not absolutely necessary for the expression of the activity of nitrile hydratase isolated from the Rhodococcus sp. strain gt1.     

Effects of Nitriles and Amides on the Growth and Nitrile Hydratase Activity of the Rhodococcus sp. Strain gt1

Effects of some nitriles and amides, as well as glucose and ammonium, on the growth and the nitrile hydratase (EC 4.2.1.84) activity of the Rhodococcus sp. strain gt1 isolated from soil were studied. The activity of nitrile hydratase mainly depended on the carbon and nitrogen supply to cells. The activity of nitrile hydratase was high in the presence of glucose and ammonium at medium concentrations and decreased at concentrations of glucose of more than 0.3%. Saturated unsubstituted aliphatic nitriles and amides were found to be a good source of nitrogen and carbon. However, the presence of nitriles and amides in the medium was not absolutely necessary for the expression of the activity of nitrile hydratase of the Rhodococcus sp. strain gt1.     

Isolation,identification and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> ZJB-063, a versatile nitrile-converting bacterium

Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of ɛ-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of ɛ-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.     

Nitrile hydrolysing activities of deep-sea and terrestrial mycolate actinomycetes

Nitrile metabolising actinomycetes previously recovered from deep-sea sediments and terrestrial soils were investigated for their nitrile transforming properties. Metabolic profiling and activity assays confirmed that all strains catalysed the hydrolysis of nitriles by a nitrile hydratase/amidase system. Acetonitrile and benzonitrile, when used as growth substrates for enzyme induction experiments, had a significant influence on the biotransformation activities towards various nitriles and amides. The specific activities of selected deep-sea and terrestrial acetonitrile-grown bacteria against a suite of nitriles and amides were higher than those of the only other reported marine nitrile-hydrolysing R. erythropolis, isolated from a shallow sediment. The increase of nitrile chain length appeared to have negative influence on the nitrile hydratase activity of acetonitrile-grown bacteria, but the same was not true for benzonitrile-grown bacteria. The nitrile hydratases and amidases were constitutive in 10 of the 16 deep-sea and terrestrial actinomycetes studied, and one strain showed an inducible hydratase and a constitutive amidase. Most of the deep-sea strains had constitutive activities and showed some of the highest activities and broadest substrate specificities of organisms included in this study. This revised version was published online in August 2006 with corrections to the Cover Date.     

Constitutive acetonitrile hydrolysing activity of Nocardia globerula NHB-2: Optimization of production and reaction conditions

Nocardia globerula NHB-2 exhibited an intracellular acetonitrile hydrolysing activity (AHA) when cultivated in nutrient broth supplemented with glucose (10.0 g/l) and yeast extract (1.0 g/l), at pH 8.0, 30 degrees C for 21 hr. Maximum AHA was recorded in the culture containing 0.1 M of sodium phosphate buffer, (pH 8.8) at 45 degrees C for 15 min with 600 micromol of acetonitrile and resting cells of N. globerula NHB-2 equivalent to 1.0 ml culture broth. This activity was stable up to 40 degrees C and was completely inactivated at or above 60 degrees C. About five-fold increase in AHA was observed after optimization of culture and reaction conditions. Under the optimized conditions, this organism hydrolyzed various nitriles and amides such as propionitrile, benzonitrile. acetamide, and acrylamide to corresponding acids. This nitrile/amide hydrolysing activity of N. globerula NHB-2 has potential applications in enzymatic synthesis of organic acids and bioremediation of nitriles and amides contaminated soil and water system.     

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

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

Nitrile- and Amide-hydrolysing Activity in Kluyveromyces thermotolerans MGBY 37

Summary Forty yeast strains were screened for nitrile-hydrolysing activity. Among them Kluyveromyces thermotolerans MGBY 37 exhibited highest nitrile-hydrolysing activity (0.030 μmol/h/mg dry cell weight). This yeast contained a two-enzyme system i.e. nitrile hydratase (NHase, EC 4.2.1.84) and amidase (EC 3.5.1.4) for the hydrolysis of nitriles/amides to corresponding acids and ammonia. However, these enzymes had more affinity for N-heterocyclic aromatic and aromatic nitriles/amides rather than unsaturated and saturated aliphatic nitriles/amides. The NHase–amidase activity was constitutively produced by K. thermotolerence MGBY 37. Addition of acetonitrile in the medium enhanced the production of this activity while other nitriles and amides lowered the production of NHase–amidase activity. This organism thus exhibited two types of amidase i.e. a constitutive amidase having affinity for N-heterocyclic aromatic, unsaturated and saturated aliphatic amides and another inducible amidase with affinity for aromatic amides. Formamide proved to be the best inducer of the latter amidase activity. This is the first report on nitrile- and amide-hydrolysing activity in Kluyveromyces.     

Biotransformation of nitriles by rhodococci

Rhodococci have been shown to be capable of a very wide range of biotransformations. Of these, the conversion of nitriles into amides or carboxylic acids has been studied in great detail because of the biotechnological potential of such activities. Initial investigations used relatively simple aliphatic nitriles. These studies were quickly followed by the examination of the regio- and stereoselective properties of the enzymes involved, which has revealed the potential synthetic utility of rhodococcal nitrile biotransforming enzymes. Physiological studies on rhodococci have shown the importance of growth medium design and bioreactor operation for the maximal conversion of nitriles. This in turn has resulted in some truly remarkable biotransformation activities being obtained, which have been successfully exploited for commercial organic syntheses (e.g. acrylamide production from acrylonitrile).The two main types of enzyme involved in nitrile biotransformations by rhodococci are nitrile hydratases (amide synthesis) and nitrilases (carboxylic acid synthesis with no amide intermediate released). It is becoming clear that many rhodococci contain both activities and multiple forms of each enzyme, often induced in a complex way by nitrogen containing molecules. The genes for many nitrile-hydrolysing enzymes have been identified and sequenced. The crystal structure of one nitrile hydratase is now available and has revealed many interesting aspects of the enzyme structure in relationship to its catalytic activity and substrate selectivity.     

Action of Nitrile Hydratase from Rhodococcus rhodochrous IFO 15564 on Derivatives of 2,5-Anhydro-d-allononitrile

The conversion of 2,5-anhydro-d-allononitrile derivatives by a nitrile hydratase from Rhodococcus rhodochrous IFO 15564 was studied. The activity of the enzyme was strongly effected by the steric bulkiness of the substituents at the 3-position of the substrates, and the corresponding amides were obtained in high yields from the nitriles with free hydroxyl groups at the 3- and 4-positions.     

Nitrile hydratases (NHases): At the interface of academia and industry

Nitrile hydratase (NHase, EC 4.2.1.84) is one of the key enzymes of nitrile metabolism in a large number of microbes that catalyses the hydration of nitriles to corresponding amides, and has been successfully adopted in chemical industry for production of acrylamide, nicotinamide and 5-cyanovaleramide. However, NHase is still under active consideration of enzymologists to expand its potential for synthesis of various amides. Most of the NHases have been reported for their limited substrates acceptability, low enantioselectivity and thermostability and therefore a considerable improvement is required for developing as robust biocatalyst for synthesis of a range of organic amides. Studies on biochemical properties, gene configuration, active-site chemical models and site-directed mutagenesis have given the insight into the structural and functional characteristics of NHase. Keeping in view, the present review critically describes the available information on natural sources (based on activity and phylogenetic analysis), biochemical properties, catalysis–structure relationship, molecular expression and potential applications of this enzyme.     

Characterisation of nitrilase and nitrile hydratase biocatalytic systems

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

Auxin-like activity of 1,2-benzisothiazole derivatives

The 1,2-benzisothiazol-3-yl-acetic and -3-yl-butyric acids and their ethyl esters, amides and nitriles are generally active in the split pea stem test, induce an increase in both length and fresh weight of pea internodes, inhibit the development of pea roots, and, with some exceptions (1,2-benzisothiazol-3-yl-butyric amide and nitrile), induce the production of ethylene by pea segments. Moreover they stimulate cell multiplication and raise the degree of hydration of Helianthus tuberosus explants grown in vitro. These activities are often similar or sometimes higher than those of IAA. By contrast, the 1,2-benzisothiazole derivatives having a side chain with an odd number of carbon atoms (-3-yl-carboxylic and propionic acids, amides, ethyl esters and nitriles) are inactive or show a far lower activity.     

Exploratory studies to investigate a linked prebiotic origin of RNA and coded peptides. 2nd communication. Derivation and reactivity of xylose phosphates

The potentially prebiotic synthesis of ribo-nucleotides by stepwise pyrimidine nucleobase assembly on arabinose-3-phosphate derivatives has been demonstrated in previous work. Consideration of the provenance of pentose phosphates, by aldolisation or sugar phosphorylation, suggested that 2-phosphate derivatives might be generated more easily than 3-phosphate derivatives. In the 2-phosphate series, nucleobase-assembly chemistry to give ribo-nucleotides/nucleic acid can be envisaged from xylo-configured starting materials. In this paper, the derivation of xylose-2-phosphate derivatives by aldol chemistry and attempts to demonstrate subsequent pyrimidine nucleobase assembly are reported.     

A comparative study of warheads for design of cysteine protease inhibitors

The effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were explored. The oxime was almost an order of magnitude more potent than the corresponding nitrile and has the potential to provide access to the prime side of the catalytic site. Dipeptide aldehydes and azadipeptide nitriles were found to be two orders of magnitude more potent cruzain inhibitors than the corresponding dipeptide nitriles although potency differences were modulated by substitution at P1 and P3. Replacement of the α methylene of a dipeptide aldehyde with cyclopropane led to a loss of potency of almost three orders of magnitude. The vinyl esters and amides that were characterized as reversible inhibitors were less potent than the corresponding nitrile by between one and two orders of magnitude.     

A new method to prepare amides by bioconversion of corresponding nitriles

Amides of very good chemical quality have been obtained by the bioconversion of the corresponding nitriles using immobilized bacteria with nitrilase activity (nitrile hydratase). The bioreactor was polyvalent since the nitrilase used had a generalized activity and could hydrolyse a large number of nitriles. The present paper shows the polyvalence and simplicity of the process, and the quality of the amides obtained.     

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.     

Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis: recent insights and promises

Nitrile-converting enzymes, including nitrilase and nitrile hydratase (NHase), have received increasing attention from researchers of industrial biocatalysis because of their critical role as a tool in organic synthesis of carboxylic acids and amides from nitriles. To date, these bioconversion approaches are considered as one of the most potential industrial processes using resting cells or purified enzymes as catalysts for production of food additives, pharmaceutical, and agrochemical precursors. This review focuses on the distribution and catalytic mechanism research of nitrile-converting enzymes in recent years. Molecular biology aspects to improve the biocatalytic performance of microbial nitrilase and NHase are demonstrated. The process developments of microbial nitrilase and NHase for organic synthesis are also discussed.     

Microbial Isobutyronitrile Utilization under Haloalkaline Conditions

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