Cloning and sequence analysis of the heat-stable acrylamidase from a newly isolated thermophilic bacterium, Geobacillus thermoglucosidasius AUT-01

A thermophilic bacterium capable of degrading acrylamide, AUT-01, was isolated from soil collected from a hot spring area in Montana, USA. The thermophilic strain grew with 0.2 % glucose as the sole carbon source and 1.4 mM acrylamide as the sole nitrogen source. The isolate AUT-01 was identified as Geobacillus thermoglucosidasius based on 16S rDNA sequence. An enzyme from the strain capable of transforming acrylamide to acrylic acid was purified by a series of chromatographic columns. The molecular weight of the enzyme was estimated to be 38 kDa by SDS-PAGE. The enzyme activity had pH and temperature optima of 6.2 and 70 ºC, respectively. The influence of different metals and amino acids on the ability of the purified protein to transform acrylamide to acrylic acid was evaluated. The gene from G. thermoglucosidasius encoding the acrylamidase was cloned, sequenced, and compared to aliphatic amidases from other bacterial strains. The G. thermoglucosidasius gene, amiE, encoded a 38 kDa, monomeric, heat-stable amidase that catalysed the cleavage of carbon–nitrogen bonds in acrylamide. Comparison of the amino acid sequence to other bacterial amidases revealed 99 and 82 % similarity to the amino acid sequences of Bacillus stearothermophilus and Pseudomonas aeruginosa, respectively.     

Metabolism of acrylonitrile by Klebsiella pneumoniae

A gram-negative rod-shaped bacterium capable of utilizing acrylonitrile as the sole source of nitrogen was isolated from industrial sewage and identified as Klebsiella pneumoniae. The isolate was capable of utilizing aliphatic nitriles containing 1 to 5 carbon atoms or benzonitrile as the sole source of nitrogen and either acetamide or propionamide as the sole source of both carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae was capable of hydrolyzing 6.15 mmol of acrylonitrile to 5.15 mmol of acrylamide within 24 h. The acrylamide was hydrolyzed to 1.0 mmol of acrylic acid within 72 h. Another metabolite of acrylonitrile metabolism was ammonia, which reached a maximum concentration of 3.69 mM within 48 h. Nitrile hydratase and amidase, the two hydrolytic enzymes responsible for the sequential metabolism of nitrile compounds, were induced by acrylonitrile. The optimum temperature for nitrile hydratase activity was 55°C and that for amidase was 40°C; both enzymes had pH optima of 8.0.Abbreviations PBM phosphate buffered medium - GC gas chromatography - GC/MS gas chromatography/mass spectrometry     

An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid

Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V _max and K _m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 μmol mg^?1 min^?1 and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K _i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml ^? resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.     

Nitrile-metabolizing potential of Amycolatopsis sp. IITR215

Strain Amycolatopsis sp. IITR215 was isolated from a sewage sample using polyacrylonitrile powder as the sole nitrogen source. Identification was performed by 16S rDNA analysis. The isolated strain harbored multiple nitrile-metabolizing enzymes having a wide range of substrate specificities. It metabolized nitrile and amide compounds with constitutive enzymes. Studies using an amidase inhibitor showed that hydrolysis of acrylonitrile and acrylamide occurred due to nitrile hydratase and amidase, respectively, while hydrolysis of hexanenitrile was due to the action of either nitrilase or a second nitrile hydratase/amidase system. The inhibitory effects of N-bromosuccinimide and N-ethylmaleimide on enzymes of this culture were also studied and this further indicated the involvement of either a nitrilase or a second nitrile hydratase/amidase system for hydrolysis of hexanenitrile. Interestingly, hexanenitrile hydrolysis exhibited an optimum temperature of 55 °C, whereas acrylonitrile and acrylamide hydrolysis showed an optimum temperature of 45 °C. The optimum pH was 5.8 for hexanenitrile hydrolysis and 7.0 for acrylonitrile and acrylamide hydrolysis. Hexanenitrile hydrolysis by enzymes of this strain showed better organic solvent tolerance in the presence of alcohols. The maximum enzyme activity of nitrile-metabolizing enzymes was found using media containing isobutyramide as the nitrogen source. This is the first report on constitutive multiple enzymes from the Amycolatopsis genus.     

Biotransformation of acrylonitrile to acrylamide using immobilized whole cells of Brevibacterium CH1 in a recycle fed-batch reactor

Acrylamide was produced from acrylonitrile using immobilized Brevibacterium CH1 cells that were isolated from soil and found to possess nitrile hydratase activity. The reaction conditions and stability of the enzyme activity were studied. The conversion yield was nearly 100%, including a trace amount of acrylic acid. This strain showed strong activity of nitrile hydratase toward acrylonitrile and extremely low activity of amidase toward acrylamide. A packed bed reactor was operated in a fed-batch manner for acrylamide production of high concentration. The acrylonitrile concentration was maintained below 3% and the operating temperature at 4 degrees C to minimize enzyme deactivation.     

Solvent Effects on Circular Dichroism of Colominic Acid

To produce acrylamide from acrylonitrile by use of a new enzyme, nitrile hydratase, a number of nitrile-utilizing microorganisms were screened for the enzyme activity by an intact cell system. An isobutyronitrile-utilizing bacterium, strain B23, showed the best productivity among 186 strains tested. The strain was identified taxonomically as Pseudomonas chlor or aphis. The culture and reaction conditions for the production were studied for the strain. Under the optimum conditions, 400 grams/liter of acrylamide was produced in 7.5 hr. The yield was nearly 100% with a trace amount of acrylic acid. The cell-free extract of the strain showed strong activity of nitrile hydratase toward acrylonitrile and extremely low activity of amidase toward acrylamide.     

Degradation of Acetonitrile by Pseudomonas putida

A bacterium capable of utilizing high concentrations of acetonitrile as the sole source of carbon and nitrogen was isolated from soil and identified as Pseudomonas putida. This bacterium could also utilize butyronitrile, glutaronitrile, isobutyronitrile, methacrylonitrile, propionitrile, succinonitrile, valeronitrile, and some of their corresponding amides, such as acetamide, butyramide, isobutyramide, methacrylamide, propionamide, and succinamide as growth substrates. Acetonitrile-grown cells oxidized acetonitrile with a K_m of 40.61 mM. Mass balance studies with [¹⁴C]acetonitrile indicated that nearly 66% of carbon of acetonitrile was released as ¹⁴CO₂ and 14% was associated with the biomass. Metabolites of acetonitrile in the culture medium were acetic acid and ammonia. The acetate formed in the early stages of growth completely disappeared in the later stages. Cell extracts of acetonitrile-grown cells contained activities corresponding to nitrile hydratase and amidase, which mediate the breakdown of actonitrile into acetic acid and ammonia. Both enzymes were intracellular and inducible and hydrolyzed a wide range of substrates. The specific activity of amidase was at least 150-fold higher than the activity of the enzyme nitrile hydratase.     

Nitrile Hydratase and Amidase from Rhodococcus rhodochrous Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles

Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein⁻¹) and amidase activity (38.4 nkat mg of protein⁻¹) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C.I. Basic Blue 9.     

Isolation of identical nitrilase genes from multiple bacterial strains and real-time PCR detection of the genes from soils provides evidence of horizontal gene transfer

Bacterial enzymes capable of nitrile hydrolysis have significant industrial potential. Microbacterium sp. AJ115, Rhodococcus erythropolis AJ270 and AJ300 were isolated from the same location in England and harbour identical nitrile hydratase/amidase gene clusters. Strain AJ270 has been well studied due to its nitrile hydratase and amidase activity. R. erythropolis ITCBP was isolated from Denmark and carries a very similar nitrile hydratase/amidase gene cluster. In this study, an identical nitrilase gene (nit1) was isolated from the four strains, and the nitrilase from strain AJ270 cloned and expressed in Escherichia coli. Analysis of the recombinant nitrilase has shown it to be functional with activity demonstrated towards phenylacetonitrile. A real-time PCR TaqMan^® assay was developed that allowed nit1 detection directly from soil enrichment cultures without DNA extraction, with nit1 detected in all samples tested. Real-time PCR screening of isolates from these soils resulted in the isolation of nit1 and also very similar nitrilase gene nit2 from a number of Burkholderia sp. The genes nit1 and nit2 have also been detected in many bacteria of different genera but are unstable in these isolates. It is likely that the genes were acquired by horizontal gene transfer and may be widespread in the environment.     

Microbial degradation of acrylamide monomer

Acrylamide, a neurotoxic monomer with extensive industrial applications was found to be degraded by the microorganisms present in a tropical garden soil. A bacterium capable of degrading acrylamide was isolated from this soil by enrichment. It was found to be aerobic, gram-negative, motile, short rod and identified as Pseudomonas sp. The bacterium degraded high concentrations of acrylamide (4 g/l) to acrylic acid and ammonia which were utilized as sole carbon and nitrogen source for growth. An amidase was involved in the hydrolysis of acrylamide, which could act on other short chain amides like formamide and acetamide but not on acrylamide analogues: methacrylamide and N,N-methylene bis-acrylamide. The enzyme was sensitive to catabolite repression by succinate both in presence as well as absence of nitrogen source.Abbreviations Acrylamide (ACR) High Performance Liquid Chromatography (HPLC)     

Cadmium increases catechol 2,3-dioxygenase activity in Variovorax sp. 12S, a metal-tolerant and phenol-degrading strain

A Gram-negative bacterium, designated as strain 12S, was isolated from a heavy metal-polluted soil. According to the biochemical characteristics, FAME analysis, and 16S rRNA gene sequence analysis, the isolated strain was identified as Variovorax sp. 12S. In the presence of 0.1 mM cadmium, 12S was able to completely utilize up to 1.5 mM of phenol as the sole carbon and energy source in an MSM–TRIS medium. Degradation of phenol was accompanied by a slow bacterial growth rate and an extension of the lag phase. The cells grown on phenol showed catechol 2,3-dioxygenase (C23O) activity. The activity of C23O from 12S cultivated in medium with Cd²⁺ was almost 20 % higher than in the control. Since environmental contamination with aromatic compounds is often accompanied by the presence of heavy metals, Variovorax sp. 12S and its C23O appear to be very powerful and useful tools in the biotreatment of wastewaters and soil decontamination.     

Transformation of the herbicide 2,6-dichlorobenzonitrile to the persistent metabolite 2,6-dichlorobenzamide (BAM) by soil bacteria known to harbour nitrile hydratase or nitrilase

In soil the herbicide 2,6-dichlorobenzonitrile (dichlobenil) is degraded to the persistent metabolite 2,6-dichlorobenzamide (BAM) which has been detected in 19% of samples taken from Danish groundwater. We tested if common soil bacteria harbouring nitrile-degrading enzymes, nitrile hydratases or nitrilases, were able to degrade dichlobenil in vitro. We showed that several strains degraded dichlobenil stoichiometrically to BAM in 1.5–6.0 days; formation of the amide intermediate thus showed nitrile hydratase rather than nitrilase activity, which would result in formation of 2,6-dichlorobenzoic acid. The non-halogenated␣analogue benzonitrile was also degraded, but here the benzamide intermediate accumulated only transiently showing nitrile hydratase followed by amidase activity. We conclude that a potential for dichlobenil degradation to BAM is found commonly in soil bacteria, whereas further degradation of the BAM intermediate could not be demonstrated.     

Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.     

Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli

For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli. Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of Mr 54,671. The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans. The 521-amino acid coding region was therefore expressed by use of the E. coli lac promoter in E. coli, and was found to direct a considerable amidase activity. This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide. These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase. Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner.     

Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture

Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present.The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems.Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.     

Cloning the amidase gene from Rhodococcus rhodochrous M8 and its expression in Escherichia coli

The amidase gene from Rhodococcus rhodochrous M8 was cloned by PCR amplification with primers developed by use of peptide amino acid sequences obtained after treating amidase with trypsin. Nucleotide sequence analysis of this gene revealed high homology with aliphatic amidases from R. erythropolis R312 and Pseudomonas aeruginosa. Considering the substrate specificity and the results of DNA analysis, amidase from R. rhodochrous M8 was assigned to the group of aliphatic amidases preferentially hydrolyzing short-chain aliphatic amides. The amidase gene was expressed in cells of Escherichia coli from the self promoter and from the lac promoter. To clone a fragment of R. rhodochrous M8 chromosome (approximately 9 kb), containing the entire structural gene and its flanking regions, plasmid pRY1 that can be integrated into the chromosome via homology regions was used. No sequences of the nitrile hydratase gene, the second key gene of nitrile degradation in strain R. rhodochrous M8, were detected. Thus, genes encoding amidase and nitrile hydratase in strain R. rhodochrous M8 are not organized into a single operon despite their common regulation.     

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg^?1. The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5–11.5) and temperature (40–90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K _m and V _max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min^?1 mg^?1 protein by analyzing Michaelis–Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.     

The development of a novel strategy for the microbial treatment of acrylonitrile effluents

Effluent from the manufacture of acrylonitrile is difficult to biodegrade. It contains nine major organic components: acetic acid, acrylonitrile, acrylamide, acrylic acid, acrolein, cyanopyridine, fumaronitrile, succinonitrile, and maleimide. A range of bacteria have been isolated that can grow on, or convert all of the organic components of effluent from the manufacture of acrylonitrile. These bacteria can be used as the basis of a mixed culture system to treat the effluent. The bacteria were utilised in batch and continuous cultures to degrade a synthetic wastewater containing acrylonitrile, acrylamide, acrylic acid, cyanopyridine and succinonitrile. The mixed microbial population was adapted by varying the growth rate and switching from continuous to batch and back to continuous growth, to degrade these five compounds as well as acrolein, fumaronitrile and maleimide.Abbreviations BOD Biological Oxygen Demand - COD Chemical Oxygen Demand - T _D Doubling Time - ppm parts per million - HPLC High Pressure Liquid Chromatography - GLC Gas Liquid Chromatography     

High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production

The nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase (NHase), converting acrylonitrile into acrylamide, was cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. The specific enzyme activity in recombinant C. glutamicum cells was about 13.6 μmol/min/mg dry cell weight (DCW). To overexpress the NHase, five types of plasmid variants were constructed by introducing mutations into 80 nucleotides near the translational initiation region (TIR) of nhhB. Of them, pNBM4 with seven mutations showed the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reached 53.4 g DCW/L within 18 h, and the specific and total enzyme activities were estimated to be 37.3 μmol/min/mg DCW and 1,992 μmol/min/mL, respectively. The use of recombinant Corynebacterium cells for the production of acrylamide from acrylonitrile resulted in a conversion yield of 93 % and a final acrylamide concentration of 42.5 % within 6 h when the total amount of fed acrylonitrile was 456 g.