Nitrile Biotransformations 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.

Tailoring the Hinge Residue at the Substrate Access Tunnel Entrance Improves the Catalytic Performance of Industrialized Nitrile Hydratase Toward 3‐Cyanopyridine

AbstractNicotinamide is a high‐value chemical that has been widely applied in various aspects of daily life. Nitrile hydratase (NHase) is capable of carrying out biotransformation of nitriles to amides and has shown great potential in the green synthesis of nicotinamide. The high‐molecular mass NHase (H‐NHase) from Rhodococcus rhodochrous J1 has been applied in the industrial‐scale production of acrylamide; however, its activity is not ideal towards aromatic 3‐cyanopyridines, resulting in its substrate preference towards aliphatic nitriles. In this study, a hinge residue at the entrance of the substrate access tunnel of H‐NHase was identified through substrate access tunnel calculations, site‐saturation mutagenesis, molecular docking and molecular dynamics simulations. The corresponding mutant βW48Y showed a 5.9‐fold higher specific activity towards 3‐cyanopyridine than that of its parental enzyme, with better thermostability, proving itself to be a competitive candidate for the industrial production of nicotinamide.

Newly elected Chinese Academy of Sciences academicians in chemistry division in 2021

Newly elected Chinese Academy of Sciences academicians in chemistry division in 2021

High-throughput screening for distinguishing nitrilases from nitrile hydratases in Aspergillus and application of a Box-Behnken design for the optimization of nitrilase.

Nitrilases and nitrile hydratases/amidases hydrolyze nitriles into carboxylic acids and/or amides, which are used in industrial chemical processes. In the present study, 26 microorganisms, including yeasts and filamentous fungi, in a minimum solid mineral medium supplemented with glucose and phenylacetonitrile were screened to evaluate their biocatalytic potential. Of these microorganisms, five fungi of the genus Aspergillus were selected and subjected to colorimetry studies to evaluate the production and distinction of nitrilase and nitrile hydratase/amidase enzymes. Aspergillus parasiticus Speare 7967 and A. niger Tiegh. 8285 produced nitrilases and nitrile hydratase, respectively. Nitrilase optimization was performed using a Box-Behnken design (BBD) and fungus A. parasiticus Speare 7967 with phenylacetonitrile volume (μl), pH, and carbohydrate source (starch:glucose; g/g) as independent variables and nitrilase activity (Uml-1 ) as dependent variable. Maximum activity (2.97 × 10-3 Uml-1 ) was obtained at pH 5.5, 80μl of phenylacetonitrile, and 15g of glucose. A. parasiticus Speare 7967 showed promise in the biotransformation of nitriles to carboxylic acids.

Recent Advances and Promises in Nitrile Hydratase: From Mechanism to Industrial Applications.

Nitrile hydratase (NHase, EC 4.2.1.84) is one type of metalloenzyme participating in the biotransformation of nitriles into amides. Given its catalytic specificity in amide production and eco-friendliness, NHase has overwhelmed its chemical counterpart during the past few decades. However, unclear catalytic mechanism, low thermostablity, and narrow substrate specificity limit the further application of NHase. During the past few years, numerous studies on the theoretical and industrial aspects of NHase have advanced the development of this green catalyst. This review critically focuses on NHase research from recent years, including the natural distribution, gene types, posttranslational modifications, expression, proposed catalytic mechanism, biochemical properties, and potential applications of NHase. The developments of NHase described here are not only useful for further application of NHase, but also beneficial for the development of the fields of biocatalysis and biotransformation.

ISOLATION, IDENTIFICATION AND SUBSTRATE SPECIFICITY OF A NITRILASE PRODUCING BACTERIA, Acidovorax sp. SK1

The process of biocatalysis or biotransformation remains the core of industrial biotechnology owing to its importance in the synthesis of high-value products in a cost effective manner. Nitrile biotransformation has also achieved considerable attention in last few decades due to its widespread biotechnological and industrial applications. In the present study, a versatile, potent nitrile-degrading bacterium, Acidovorax sp. SK1 was isolated from the cracker waste dumping site of Sivakasi, Tamilnadu and characterized for its biocatalytic potential, nitrilase production and enzyme activity. A pH sensitive indicator-based assay was performed to identify the nitrile degrading ability of the isolated samples. Semi quantitative High performance thin layer chromatographic (HPTLC) method was performed for quantitative measurement of mandelic acid produced from degradation of nitrile compound, mandelonitrile. The optimization of medium and nutritional parameters were studied for the improvement of nitrilase activity, which indicated that maximum nitrilase activity was observed at an optimum pH of 7.0, agitation at 100 rpm, glucose as best carbon source (10 g/L) and yeast extract (0.1 g/L) as principal nitrogen source. Biomass is also a critical parameter in the biocatalysis of mandelonitrile to mandelic acid and at a biomass of 100 mg/L, maximum nitrilase activity of 0.026 I.U was observed. The versatility of Acidovorax sp. in the hydrolysis of mandelonitrile to pharmaceutically valuable mandelic acid makes it a potential biocatalyst in organic synthesis. The present work demonstrates the production of nitrilase and the biotransformation of mandelonitrile to mandelic acid using the newly isolated strain of Acidovorax sp. SK1 and also suggests that this strain open avenues for biotechnological application as an effective green catalyst.

Exploring a broad spectrum nitrilase from moderately halophilic bacterium Halomonas sp. IIIMB2797 isolated from saline lake.

Nitrile hydrolyzing moderate halophilic bacterium Halomonas sp. IIIMB2797 was isolated from Sambhar Lake, India. Maximum cell biomass and nitrilase production were observed at 60 g L-1 NaCl in the production media which confirms its moderate halophilic nature. Nitrilase of Halomonas sp. IIIMB2797 proved to be inducible in nature as maximum activity was observed when valeronitrile was added in the production media. Whole cells of Halomonas sp. IIIMB2797 exhibited broad substrate affinity towards aromatic and aliphatic nitriles. Optimum pH and temperature for nitrilase activity was observed at 7.0 and 45 °C, respectively. Effect of salinity on nitrilase activity was also studied and maximum activity was observed in presence of 50 g L-1 NaCl in 0.1 M phosphate buffer of pH 7.0. The interesting feature of the study is that whole cells of Halomonas sp. IIIMB2797 exhibited higher nitrilase activities in presence of organic solvents which may be useful in biotransformation of nitriles to corresponding carboxylic acids for industrial applications.

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.

Bioengineering of Nitrilases Towards Its Use as Green Catalyst: Applications and Perspectives.

Nitrilases are commercial biocatalysts used for the synthesis of plastics, paints, fibers in the chemical industries, pharmaceutical drugs and herbicides for agricultural uses. Nitrilase hydrolyses the nitriles and dinitriles to their corresponding carboxylic acids and ammonia. They have a broad range of substrate specificities as well as enantio-, regio- and chemo-selective properties which make them useful for biotransformation of nitriles to important compounds because of which they are considered as 'Green Catalysts'. Nitriles are widespread in nature and synthesized as a consequence of anthropogenic and biological activities. These are also present in certain plant species and are known to cause environmental pollution. Biotransformation using native organisms as catalysts tends to be insufficient since the enzyme of interest has very low amount in the total cellular protein, rate of reaction is slow along with the instability of enzymes. Therefore, to overcome these limitations, bioengineering offers an alternative approach to alter the properties of enzymes to enhance the applicability and stability. The present review highlights the aspects of producing the recombinant microorganisms and overexpressing the enzyme of interest for the enhanced stability at high temperatures, immobilization techniques, extremes of pH, organic solvents and hydrolysing dintriles to chiral compounds which may enhance the possibilities for creating specific enzymes for biotransformation.

Production and Characterization of a Nitrilase from Pseudomonas aeruginosa RZ44 and its Potential for Nitrile Biotransformation.

Background The conversion of nitriles into amides or carboxylic acids by nitrilase has taken its application into consideration, as the scope of its applications has recently been extended. Objectives In this study, P. aeruginosa RZ44 was isolated from sewage in the Kerman which has Nitrile-degradation activity. In order to improve the nitrilase production, several optimization were done on environmental condition. Nitrilase activity was characterized against different pHs, temperatures, ions, and substrates. Materials and Methods Enzyme activity was evaluated by determining the production of ammonia following to the modification of the phenol/hypochlorite method. Different factors that affect production of the enzyme by P. aeruginosa RZ44 were optimized and evaluated in the culture mediums. Results The results showed that degradation of the acetonitrile by P. aeruginosa RZ44 increased the pH of the growth medium from the initial pH 7.0 to 9.37. Optimizing the medium for P. aeruginosa RZ44, it was found that glucose and starch (5 g.L-1) have strongly supported nitrilase production, compared to the control. As well, urea (5 g.L-1) and yeast extract (15 g.L-1) have favored an increased biomass and nitrilase production, as the nitrogen sources. These results show that nitrilase production increases in the pH range 5.0 to 7.0 and then start decreasing. Addition of the Mg2+, Fe2+ and Na+ has supported the biomass and nitrilase production. Co2+, Mn2+ and Cu2+ were confirmed to inhibit cell growth and enzyme production. Enzyme characterization results show that, P. aeruginosa RZ44 nitrilase exhibits comparatively high activity and stability at pH 7.0 and 40°C. Nitrilase was completely inhibited by CoCl2 and CaCl2, whereas, the inhibition in the presence of MnSO4 and CuSO4 was about 60%. Time course analysis of the nitrile conversion by the resting P. aeruginosa RZ44 cells showed that nitrile substrates (i.e. acetonitrile) was hydrolyzed within 8 h. Conclusions these results indicate that P. aeruginosa RZ44 has the potential to be applied in the biotransformation of nitrile compounds.

Nitrile-metabolizing potential of Bacillus cereus strain FA12; Nitrilase production, purification, and characterization

Nitrile-hydrolyzing bacteria have the potential to perform useful biotransformations such as the production of industrially useful acids and amides. In this study, we report a nitrile-degrading bacterium with significant nitrile metabolism. Molecular characterization of 16S rDNA gene characterized this strain as Bacillus cereus. Medium optimization of B. cereus FA12 showed that biomass and nitrilase production was strongly supported by glucose (10 gL− 1) and yeast extract (10 gL− 1). Enzymatic production improved slightly in the pH range from 6.0 to 7.0. The addition of Mg+2, Fe+2, and Na+ supported biomass and nitrilase production; however, other metal ions, Co+2 and Cu+2, inhibited production. The apparent molecular mass of the purified FA12 nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was about 45 kDa. Nitrilase FA12 shows relatively high activity and stability at pH 7.0 and 40°C. Nitrilase FA12 was marginally inhibited with Ca+ 2 and Co+2, whereas inhibition in the presence of dithiothreitol or DTT was 80%. The pseudo Km (mM) values of resting cells (i.e., treating whole cells as if they were an enzyme) for acetonitrile and acetamide were determined to be 2.36 and 1.81, respectively. Under optimum situations, B. cereus FA12 resting cells produced 83 and 58 (U/mg) acetonitrile/acetamide degrading activity, respectively. Ammonia production from acetamide and acetonitrile by the B. cereus FA12 was maximum after 5 and 7 h of incubation, respectively. These results indicate that B. cereus FA12 resting cells may be used in nitrile biotransformations to produce commercially useful compounds.

Enantioselective biotransformations of nitriles in organic synthesis.

The hydration and hydrolysis of nitriles are valuable synthetic methods used to prepare carboxamides and carboxylic acids. However, chemical hydration and hydrolysis of nitriles involve harsh reaction conditions, have low selectivity, and generate large amounts of waste. Therefore, researchers have confined the scope of these reactions to simple nitrile substrates. However, biological transformations of nitriles are highly efficient, chemoselective, and environmentally benign, which has led synthetic organic chemists and biotechologists to study these reactions in detail over the last two decades. In nature, biological systems degrade nitriles via two distinct pathways: nitrilases catalyze the direct hydrolysis of nitriles to afford carboxylic acids with release of ammonia, and nitrile hydratases catalyze the conversion of nitriles into carboxamides, which then furnish carboxylic acids via hydrolysis in the presence of amidases. Researchers have subsequently developed biocatalytic methods into useful industrial processes for the manufacture of commodity chemicals, including acrylamide. Since the late 1990s, research by my group and others has led to enormous progress in the understanding and application of enantioselective biotransformations of nitriles in organic synthesis. In this Account, I summarize the important advances in enantioselective biotransformations of nitriles and amides, with a primary focus on research from my laboratory. I describe microbial whole-cell-catalyzed kinetic resolution of various functionalized nitriles, amino- and hydroxynitriles, and nitriles that contain small rings and the desymmetrization of prochiral and meso dinitriles and diamides. I also demonstrate how we can apply the biocatalytic protocol to synthesize natural products and bioactive compounds. These nitrile biotransformations offer an attractive and unique protocol for the enantioselective synthesis of polyfunctionalized organic compounds that are not readily obtainable by other methods. Nitrile substrates are readily available, and the mild reaction conditions are specific toward cyano and amido functional groups without interfering with other reactive functional groups. I anticipate that further advances in this field will lead to new and engineered nitrile-hydrolyzing enzymes or catalytic systems with improved activity and altered selectivity. These advances will broaden the scope of these transformations and their applications in organic synthesis.

Screening and Improving the Recombinant Nitrilases and Application in Biotransformation of Iminodiacetonitrile to Iminodiacetic Acid

In this study, several nitrilase genes from phylogenetically distinct organisms were expressed and purified in E. coli in order to study their ability to mediate the biotransformation of nitriles. We identified three nitrilases: Acidovorax facilis nitrilase (AcN); Alcaligenes fecalis nitrilase (AkN); and Rhodococcus rhodochrous nitrilase (RkN), which catalyzed iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA). AcN demonstrated 8.8-fold higher activity for IDAN degradation as compared to AkN and RkN. Based on homology modeling and previously described ‘hot spot’ mutations, several AcN mutants were screened for improved activity. One mutant M3 (F168V/L201N/S192F) was identified, which demonstrates a 41% enhancement in the conversion as well as a 2.4-fold higher catalytic efficiency towards IDAN as compared to wild-type AcN.

Methods of cell lysis and effect of detergents for the recovery of nitrile metabolizing enzyme from Amycolatopsis sp. IITR215

Nitrile metabolizing enzymes are of great industrial interest for the selective bio-transformation of nitriles and surface modification of synthetic polymers under mild reaction conditions. In the present work, isolated strain Amycolatopsis sp. IITR215 was cultivated in the bench top bioreactor for the recovery of maximum biomass of whole cell catalyst. Effect of different lyoprotectants was studied on nitrile metabolizing enzyme from Amycolatopsis sp. IITR215 in which sorbitol proved to be an efficient lyoprotectant . In physical and mechanical methods, only 30% activity was recovered while 85% activity was achieved in the enzymatic method using 2 g/l lysozyme. Very less activity was recovered during stationary phase when cells were grown in mineral base media containing 1 g/l yeast. Therefore, recovery of intracellular enzymes was enhanced by using different concentrations of sodium cholate and deoxycholate.

Nitrile hydratase of Rhodococcus erythropolis: characterization of the enzyme and the use of whole cells for biotransformation of nitriles

The intracellular cobalt-type nitrile hydratase was purified from the bacterium Rhodococcuserythropolis. The pure enzyme consisted of two subunits of 29 and 30 kDa. The molecular weight of the native enzyme was estimated to be 65 kDa. At 25 °C the enzyme had a half-life of 25 h. The Michaelis–Menten constants Km and vmax for the enzyme were 0.624 mM and 5.12 μmol/min/mg, respectively, using 3-cyanopyridine as the substrate. The enzyme-containing freely-suspended bacterial cells and the cells immobilized within alginate beads were evaluated for converting the various nitriles to amides. In a packed bed reactor, alginate beads (2 % alginate; 3 mm bead diameter) containing 200 mg/mL of cells, achieved a conversion of >90 % for benzonitrile and 4-cyanopyridine in 38 h (25 °C, pH 7.0) at a feed substrate concentration of 100 mM. The beads could be reused for up to six reaction cycles.

Nitrile Reductase from Geobacillus kaustophilus: A Potential Catalyst for a New Nitrile Biotransformation Reaction

AbstractThe cloning, expression and characterization of a nitrile reductase (NRed) from the thermophile Geobacillus kaustophilus is reported. The enzyme shows a 12‐fold increase in activity in response to a temperature change from 25 °C to 65 °C. The substrate scope regarding its biocatalytic applicability was investigated by testing a range of common nitriles. The narrow substrate range observed for the wild‐type enzyme prompted the rational design of GkNRed active site mutants based on a previously published homology model from Bacillus subtilis. The activities of the mutants and the wild‐type enzyme were investigated in their structure‐function relationship regarding the natural substrate 7‐cyano‐7‐deazaguanine (preQ0) as well as a range of synthesized preQ0‐like substrate structures. A distinct dependence of the wild‐type enzyme activity on specific structural modifications of the natural substrate was observed. Two non‐natural nitriles derived from preQ0 could be reduced to their corresponding amino compounds.

Biotransformation of Phenylacetonitrile to 2-Hydroxyphenylacetic Acid by Marine Fungi

Marine fungi belonging to the genera Aspergillus, Penicillium, Cladosporium, and Bionectria catalyzed the biotransformation of phenylacetonitrile to 2-hydroxyphenylacetic acid. Eight marine fungi, selected and cultured with phenylacetonitrile in liquid mineral medium, catalyzed it quantitative biotransformation to 2-hydroxyphenylacetic acid. In this study, the nitrile group was firstly hydrolysed, and then, the aromatic ring was hydroxylated, producing 2-hydroxyphenylacetic acid with 51% yield isolated. In addition, the 4-fluorophenylacetonitrile was exclusively biotransformed to 4-fluorophenylacetic acid by Aspergillus sydowii Ce19 (yield = 51%). The enzymatic biotransformation of nitriles is not trivial, and here, we describe an efficient method for production of phenylacetic acids in mild conditions.

Biotransformation of nitriles to hydroxamic acids via a nitrile hydratase–amidase cascade reaction

The transformation of benzonitrile into benzohydroxamic acid was performed by a cascade bienzymatic reaction involving nitrile hydration and acyl transfer of the intermediate benzamide onto hydroxylamine. The first step was catalyzed by either a cell-free extract from Rhodococcus erythropolis A4 (nitrile hydratase) or cell-free extracts from recombinant Escherichia coli strains expressing nitrile hydratases from Raoultella terrigena 77.1 and Klebsiella oxytoca 38.1.2; the biocatalyst in the second step was a cell-free extract from R. erythropolis A4 (amidase). When using the cell-free extract from R. erythropolis A4 in the first step, the hydrolytic amidase activity was suppressed by ammonium ions, which, however, did not inhibit the acyl transfer reaction catalyzed by the same enzyme. Aromatic and aliphatic nitriles were examined as substrates of the recombinant nitrile hydratases by using a colorimetric assay of hydroxamic acids produced in a coupled acyl transfer reaction.

Nitrile biotransformation by whole cells of Aspergillus sp. PTCC 5266

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

ChemInform Abstract: Progress of Enantioselective Nitrile Biotransformations in Organic Synthesis

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