An improved nitrilase-mediated bioprocess for synthesis of nicotinic acid from 3-cyanopyridine with hyperinduced <Emphasis Type="Italic">Nocardia globerula</Emphasis> NHB-2

Nitrilase of Nocardia globerula NHB-2 was induced by short-chain aliphatic nitriles (valeronitrile > isobutyronitrile > butyronitrile > propionitrile) and exhibited activity towards aromatic nitriles (benzonitrile > 3-cyanopyridine > 4-cyanopyridine > m-tolunitrile > p-tolunitrile). Hyperinduction of nitrilase (6.67 U mg_DCW⁻¹, 18.7 U mL⁻¹) was achieved in short incubation time (30 h, 30°C) through multiple feeding of isobutyronitrile in the growth medium. The nitrilase of this organism exhibits both substrate and product inhibition effects. In a fed batch reaction at 1 L scale using hyperinduced resting cells corresponding to 10 U mL⁻¹ nitrilase activity (1.5 mg_DCW mL⁻¹), a total of 123.11 g nicotinic acid was produced at a rate of 24 g h⁻¹ g_DCW⁻¹.     

A novel nitrilase from Ralstonia eutropha H16 and its application to nicotinic acid production

A novel aliphatic nitrilase, REH16, was found in Ralstonia eutropha H16 and overexpressed in Escherichia coli BL21(DE3), and its enzymatic properties were studied. The temperature and pH optima were 37 °C and 6.6, respectively, and the best thermostability of the nitrilase was observed at 25 °C, which preserved 95% of activity after 120 h of incubation. REH16 has a broad hydrolytic activity toward aliphatic and heterocyclic nitriles and showed high tolerance of 3-cyanopyridine; this enzyme could hydrolyze as high as 100 mM 3-cyanopyridine completely. To improve the 3-cyanopyridine conversion efficiency in an aqueous reaction system, water-miscible organic solvents were tested, and ethanol (10% v/v) was chosen as the optimal co-solvent. Finally, under optimized conditions, using the fed-batch reaction mode, total of 1050 mM 3-cyanopyridine was hydrolyzed completely in 20.8 h with eight substrate feedings, yielding 129.2 g/L production of nicotinic acid and thus showing a potential for industrial application.

     

Continuous hydrolysis of 4-cyanopyridine by nitrilases from <Emphasis Type="Italic">Fusarium solani</Emphasis> O1 and <Emphasis Type="Italic">Aspergillus niger</Emphasis> K10

The operational stabilities of nitrilases from Aspergillus niger K10 and Fusarium solani O1 were examined with 4-cyanopyridine as the substrate in continuous-stirred membrane reactors (CSMRs). The former enzyme was fairly stable at 30 °C with a deactivation constant (k _d) and enzyme half-life of 0.014 h⁻¹ and 50 h, respectively, but the latter exhibited an even higher stability characterized by k _d = 0.008 h⁻¹ and half-life of 87 h at 40 °C. Another advantage of this enzyme was its high chemoselectivity, i.e., selective transformation of nitriles into carboxylic acids, while the amide formed a high ratio of A. niger K10 nitrilase product. High conversion rates (>90%) were maintained for about 52 h using the nitrilase from F. solani O1 immobilized in cross-linked enzyme aggregates (CLEAs). The purity of isonicotinic acid was increased from 98% to >99.9% by using two CSMRs connected in series, the first one containing the F. solani O1 nitrilase and the second the amidase from Rhodococcus erythropolis A4 (both enzymes as CLEAs), the amidase hydrolyzing the by-product isonicotinamide.     

Identification and characterization of a novel nitrilase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Pf-5

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0°C and 45°C, respectively, with a specific activity of 1.7 and 1.9 μmol min⁻¹ mg protein⁻¹ for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K _m = 17.9 mM and k _cat/K _m = 0.5 mM⁻¹ s⁻¹ for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.     

Characterization and functional cloning of an aromatic nitrilase from Pseudomonas putida CGMCC3830 with high conversion efficiency toward cyanopyridine

Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due to their green characteristics and the catalytic potential in nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified to homogeneity. pI value was estimated to be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH₂ terminus was determined. Nitrilase gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The open reading frame consisted of 1113 bp encoding a protein of 370 amino acids. The predicted amino acid sequence showed the highest identity (61.6%) to nitrilase from Rhodococcus rhodochrous J1. The enzyme was highly specific toward aromatic nitriles such as 3-cyanopyridine, 4-cyanopyridine, and 2-chloro-4-cyanopyridine. It was classified as aromatic nitrilase. The nitrilase activity could reach up to 71.8 U/mg with 3-cyanopyridine as substrate, which was a prominent level among identified cyanopyridine converting enzymes. The kinetic parameters K_m and V_max for 3-cyanopyridine were 27.9 mM and 84.0 U/mg, respectively. These data would warrant it as a novel and potential candidate for creating effective nitrilases in catalytic applications of carboxylic acids synthesis through further protein engineering.     

Hyperinduction of nitrilases in filamentous fungi

2-Cyanopyridine proved to act as a powerful nitrilase inducer in Aspergillus niger K10, Fusarium solani O1, Fusarium oxysporum CCF 1414, Fusarium oxysporum CCF 483 and Penicillium multicolor CCF 2244. Valeronitrile also enhanced the nitrilase activity in most of the strains. The highest nitrilase activities were produced by fungi cultivated in a Czapek-Dox medium with both 2-cyanopyridine and valeronitrile. The specific nitrilase activities of these cultures were two to three orders of magnitude higher than those of cultures grown on other nitriles such as 3-cyanopyridine or 4-cyanopyridine.     

Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity

Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.     

Bench scale conversion of 3-cyanopyidine to nicotinamide using resting cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

The nitrile hydratase (NHase, EC 3.5.5.1) activity of Rhodococcus rhodochrous PA-34 was explored for the conversion of 3-cyanopyridine to nicotinamide. The NHase activity (∼18 U/mg dry cell weight, dcw) was observed in 0.1 M phosphate buffer, pH 8.0 containing 1M 3-cyanopyridine as substrate, and 0.75 mg of resting cells (dry cell weight) per ml reaction mixture at 40°C. However, 25°C was more suitable for prolonged batch reaction at high substrate (3-cyanopyridine) concentration. In a batch reaction (1 liter), 7M 3-cyanopyridine (729 g) was completely converted to nicotinamide (855 g) in 12h at 25°C using 9.0 g resting cells (dry cell weight) of R. rhodochrous PA-34.     

Nitrile degradation by free and immobilized cells of the thermophile Bacillus sp. UG-5B, isolated from polluted industrial waters

A moderately thermophilic Gram-positive, sporulating, rod-shaped strain of Bacillus with nitrile-degrading activity was isolated from polluted industrial waters. Whole cells and cell-free extracts from the end of exponential growth phase expressed 7.6 nkat mg⁻¹ and 2.0 nkat mg⁻¹ benzonitrile-degrading activity, respectively, after cultivation in a fermentor with complex medium containing benzonitrile as an inducer. The benzonitrile degradation took place via the nitrilase pathway directly to benzoic acid without intermediate formation of benzamide. Samples with benzonitrilase activity of 7.6 nkat mg⁻¹ converted 3 mg benzonitrile in 1 h at 45°C. The half-life of benzonitrilase activity for a whole cell suspension and for cells immobilized in 2% agar was 4.5 min and 6 min at 70°C without substrate and 3 min at 90°C with substrate, respectively. The nitrilase had a broad substrate spectrum. The active biocatalyst obtained by immobilization was used in a continuous process and total biodegradation of 14.1 mM benzonitrile and 37.2 mM 4-cyanopyridine in a column bioreactor at 50°C for 5 h was achieved.     

Biosynthesis of nicotinic acid from 3-cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150

In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0–12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50–55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid.     

Characterization of a novel nitrilase,BGC4, from <Emphasis Type="Italic">Paraburkholderia graminis</Emphasis> showing wide-spectrum substrate specificity,a potential versatile biocatalyst for the degradation of nitriles

Objective

To investigate the biodegradation of nitriles via the nitrilase-mediated pathway.

Results

A novel nitrilase, BGC4, was identified from proteobacteria Paraburkholderia graminis CD41M and its potential for use in biodegradation of toxic nitriles in industrial effluents was studied. BGC4 was overexpressed in Escherichia coli BL21 (DE3), the recombinant protein was purified and its enzymatic properties analysed. Maximum activity of BGC4 nitrilase was at 30 °C and pH 7.6. BGC4 has a broad substrate activity towards aliphatic, heterocyclic, and aromatic nitriles, as well as arylacetonitriles. Iminodiacetonitrile, an aliphatic nitrile, was the optimal substrate but comparable activities were also observed with phenylacetonitrile and indole-3-acetonitrile. BGC4-expressing cells degraded industrial nitriles, such as acrylonitrile, adiponitrile, benzonitrile, mandelonitrile, and 3-cyanopyridine, showing good tolerance and conversion rates.

Conclusion

BGC4 nitrilase has wide-spectrum substrate specificity and is suitable for efficient biodegradation of toxic nitriles.

     

Purification and characterization of a nitrilase from Aspergillus niger K10

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg^-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.     

Biosynthesis of 2-amino-2,3-dimethylbutyramide by nitrile hydratase from a newly isolated cyanide-resistant strain of <Emphasis Type="Italic">Rhodococcus qingshengii</Emphasis>

Cells of a new isolate of Rhodococcus qingshengii harboring nitrile hydratase converted 2-amino-2,3-dimethylbutyronitrile into 2-amino-2,3-dimethylbutyramide (ADBA). Cells also hydrated a broad range of substrates including saturated, unsaturated and cyclic aliphatic nitriles. The microorganism tolerated KCN and ADBA up to 5 mM and 40 g/l, respectively. At 10°C, ADBA reached 33.8 g/l (yield 84.5%) which was 2.5 times that at 30°C.     

A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization

In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V _max and K _m toward 3-cyanopyridine were 77.5 μmol min^?1 mg^?1 and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.     

Cryopreservation of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall. ex Lindl. protocorm-like bodies by encapsulation-vitrification

Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured in liquid Murashige and Skoog (MS) medium containing 0.2 mg l⁻¹ α-naphthalene acetic acid and 0.5 mg l⁻¹ 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 μmol m⁻² s⁻¹) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped in a 0.5 M CaCl₂ solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then, these were dehydrated with a plant vitrification solution 2 (PVS₂) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8, for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots, and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum.     

Dimethylformamide is a novel nitrilase inducer in Rhodococcus rhodochrous

Nitrilases are of commercial interest in the selective synthesis of carboxylic acids from nitriles. Nitrilase induction was achieved here in three bacterial strains through the incorporation of a previously unrecognised and inexpensive nitrilase inducer, dimethylformamide (DMF), during cultivation of two Rhodococcus rhodochrous strains (ATCC BAA-870 and PPPPB BD-1780), as well as a closely related organism (Pimelobacter simplex PPPPB BD-1781). Benzonitrile, a known nitrilase inducer, was ineffective in these strains. Biocatalytic product profiling, enzyme inhibition studies and protein sequencing were performed to distinguish the nitrilase activity from that of sequential nitrile hydratase-amidase activity. The expressed enzyme, a 40-kDa protein with high sequence similarity to nitrilase protein Uniprot Q-03217, hydrolyzed 3-cyanopyridine to produce nicotinic acid exclusively in strains BD-1780 and BD-1781. These strains were capable of synthesising both the vitamin nicotinic acid as well as β-amino acids, a compound class of pharmaceutical interest. The induced nitrilase demonstrated high enantioselectivity (> 99%) in the hydrolysis of 3-amino-3-phenylpropanenitrile to the corresponding carboxylic acid.

     

Regiospecific hydrolysis of dinitrile compounds by nitrilase fromRhodococcus rhodochrous J1

Summary Nitrilase fromRhodococcus rhodochrous J1 catalyses the hydrolysis of nitriles to acids without the formation of amides. TheRhodococcus nitrilase exhibited regiospecificity for dicyanobenzenes. Three- and 4-cyanobenzoic acids were synthesized from isophthalonitrile and terephthalonitrile, respectively, with conversion ratios of more than 90% using theRhodococcus nitrilase.     

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.     

Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli

An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 °C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use.