Characterisation of the nitrile hydratase gene clusters of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> strains AJ270 and AJ300 and <Emphasis Type="Italic">Microbacterium</Emphasis> sp. AJ115 indicates horizontal gene transfer and reveals an insertion of IS1166

The nitrile metabolising strains AJ270, AJ300 and AJ115 were isolated from the same location. The strains have very similar nitrile metabolising profiles. Sequencing of the 16S rRNA gene indicates that strains AJ270 and AJ300 are novel strains of Rhodococcus erythropolis while strain AJ115 is a novel Microbacterium strain very closely related to Microbacterium oxydans and Microbacterium liquefaciens. Analysis of the structure of the nitrile hydratase/amidase gene clusters in the three strains indicates that this region is identical in these strains and that this structure is different to other nitrile hydratase/amidase gene clusters. The major difference seen is the insertion of a complete copy of the insertion sequence IS1166 in the nhr2 gene. This copy of IS1166 generates a 10 bp direct duplication at the point of insertion and has one ORF encoding a protein of 434 amino acids, with 98% homology to the transposase of IS666 from Mycobacterium avium. A gene oxd, encoding aldoxime dehydratase is found upstream of the nitrile hydratase gene cluster and an open reading frame encoding a protein with homology to GlnQ type ABC transporters is found downstream of the nitrile hydratase/amidase genes. The identity of the nitrile hydratase/amidase gene clusters in the three strains suggests horizontal gene transfer of this region. Analysis of the strains for both linear and circular plasmids indicates that both are present in the strains but hybridisation studies indicate that the nitrile hydratase/amidase gene cluster is chromosomally located. The nitrile hydratase/amidase enzymes of strain AJ270 are inducible with acetonitrile or acetamide. Interestingly although a number of Fe-type nitrile hydratases have been shown to be photosensitive, the enzyme from strain AJ270 is not.     

Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase

Rapid and direct screening of nitrile-converting enzymes is of great importance in the development of industrial biocatalytic process for pharmaceuticals and fine chemicals. In this paper, a combination of ferrous and ferric ions was used to establish a novel colorimetric screening method for nitrile hydratase and amidase with α-amino nitriles and α-amino amides as substrates, respectively. Ferrous and ferric ions reacted sequentially with the cyanide dissociated spontaneously from α-amino nitrile solution, forming a characteristic deep blue precipitate. They were also sensitive to weak basicity due to the presence of amino amide, resulting in a yellow precipitate. When amino amide was further hydrolyzed to amino acid, it gave a light yellow solution. Mechanisms of color changes were further proposed. Using this method, two isolates with nitrile hydratase activity towards 2-amino-2,3-dimethyl butyronitrile, one strain capable of hydrating 2-amino-4-(hydroxymethyl phosphiny) butyronitrile and another microbe exhibiting amidase activity against 2-amino-4-methylsulfanyl butyrlamide were obtained from soil samples and culture collections of our laboratory. Versatility of this method enabled it the first direct and inexpensive high-throughput screening system for both nitrile hydratase and amidase.     

Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for and β subunits of nitrile hydratase were cloned and sequenced.     

Control of the nitrile-hydrolyzing enzyme activity in Rhodococcus rhodochrous IFO 15564: preferential action of nitrile hydratase and amidase depending on the reaction condition factors and its application to the one-pot preparation of amides from aldehydes

The reaction conditions towards the preferential action of either nitrile hydratase or amidase in the harvested whole cells of Rhodococcus rhodochrous IFO 15564 were elaborated. The amidase showed higher heat tolerance than the nitrile hydratase and, at 45 °C the amidase worked exclusively. DMSO assisted the preferential action of nitrile hydratase, however, at more than 30% (v/v) addition of DMF, the nitrile hydratase activity was completely lost and only amidase worked. A one-pot chemo-enzymatic conversion of aldehydes to amides [(1) aq. NH₃, I₂, DMSO; (2) Na₂S₂O₃; (3) harvested cells of R. rhodochrous] was established. Under these reaction conditions, most of the amidase was lost, and the incubation of the firstly formed intermediates, nitriles in aq. NH₃ was responsible for the selective inhibition of amidase. The freezing of harvested cells in an exhaustively deionized environment provided a long-term preservable “ready to use” for the organic chemist.     

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.     

Influence of cobalt substitution on the activity of iron-type nitrile hydratase: are cobalt type nitrile hydratases regulated by carbon monoxide?

Comamonas testosteroni Ni1 nitrile hydratase is a Fe-type nitrile hydratase whose native and recombinant forms are identical. Here, the iron of Ni1 nitrile hydratase was replaced by cobalt using a chaperone based Escherichia coli expression system. Cobalt (CoNi1) and iron (FeNi1) enzymes share identical Vmax (30 nmol min(-1) mg(-1)) and Km (200 microM) toward their substrate and identical Ki values for the known competitive inhibitors of FeNi1. However, nitrophenols used as inhibitors do display a different inhibition pattern on both enzymes. Furthermore, CoNi1 and FeNi1 are also different in their sensitivity to nitric oxide and carbon monoxide, CO being selective of the cobalt enzyme. These differences are rationalized in relation to the nature of the catalytic metal center in the enzyme.     

Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats

Whole cells of Rhodococcus equi A4, a producer of nitrile hydratase and amidase activities, were immobilized in lens-shaped hydrogel particles, LentiKats^®. The immobilized biocatalyst was applied to the biotransformation of benzonitrile, 3-cyanopyridine, (R,S)-3-hydroxy-2-methylenebutanenitrile and (R,S)-3-hydroxy-2-methylene-3-phenylpropanenitrile. The stability of the nitrile hydratase during the repeated use of the biocatalyst was dependent on the type of the substrate. The enzyme was most stable during the transformation of (R,S)-3-hydroxy-2-methylenebutanenitrile. No significant loss of the amidase activity was observed within the course of the biocatalytic reaction.     

耐底物腈水合酶融合子的产酶条件优化

采用正交设计法对耐底物腈水合酶融合子的发酵条件进行优化,以发酵液起始pH,发酵周期,接种量,装料系数作为考察因素,最终确定最佳发酵条件为:起始pH8.0、发酵周期54h、接种量12％、装液系数12％.在此优化条件下融合子腈水合酶的活力达到1100万U/ml,较优化前提高了83.3％.通过响应面法对发酵培养基配方进行优化研究,采用Plackett-Burman法对8个因素进行了筛选,结果表明,葡萄糖、尿素、磷酸氢二钾、磷酸二氢钾是影响发酵液腈水合酶产量的主效应因子.用最陡爬坡试验及Central composite design设计进一步优化,利用Design-Expert软件进行二次回归分析,得到各因素的最佳浓度为:葡萄糖22.62g/L、尿素9.76g/L、K2HP04 1.22g/L、KH2PO41.268g/L.在此培养基优化配方下融合子腈水合酶的活力达到1280万U/ml,较原配方的酶活提高了16.4％.     

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.     

Efficient inducible expression of nitrile hydratase in Corynebacterium glutamicum

Nitrile hydratases are important industrial catalysts to produce valuable amides. In this study, we describe a comprehensive and systematic approach to the development of an inducible expression system for enhanced nitrile hydratase expression in Corynebacterium glutamicum. Through promoter engineering, codon optimization and design of ribosome binding site sequences, the nitrile hydratase activity toward 3-cyanopyridine was improved from 0.33 U/mg DCW to 12.03 U/mg DCW in shake-flask culture. By introduction of the novel inducible mmp expression system, the nitrile hydratase activity was further elevated to 14.97 U/mg DCW. Finally, a high nitrile hydratase yield of 1432 U/mL was achieved in a fed-batch fermentation process and used for nicotinamide production. These results provide new insights for the development of heterologous protein expression systems in C. glutamicum.     

Utilization of acetonitrile and other aliphatic nitriles by a Candida famata strain

Abstract A variant of a yeast strain identified as Candida famata isolated from gold mine effluent was able to grow on acetonitrile, acrylonitrile, butyronitrile, isobutyronitrile, methacrylnitrile, propionitrile, succinonitrile, valeronitrile, acetamide, isobutyamide, and succinamide as sole nitrogen source, after acclimatization. The yeast grew on acetonitrile and acetamide at concentrations up to 4%. The utilisation of acetonitrile and acetamide by the C. famata strain probably involves hydrolysis in a two-step reaction mediated by both inducible and intracellular nitrile hydratase and amidase.     

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.     

Optimization of Culture Conditions of Brevibacterium SP. A4 for the Production of Nitrile Hydratase

Optimum culture conditions of Brevibacterium sp. A4 for production of nitrile hydratase were determined by two mathematical methods: the Hadamard method and graphic analysis of response areas. A minimal medium was optimized and the basic roles of Fe²⁺ and Mg²⁺ were clearly shown. The influence of physico-chemical factors (pH, temperature and light conditions) on the culture and on nitrile hydratase were also studied. Various results permit the production of Brevibacterium sp. A4 cells with low protease and high nitrile hydratase contents.     

Optimization of Culture Conditions of Brevibacterium SP. A4 for the Production of Nitrile Hydratase

Optimum culture conditions of Brevibacterium sp. A4 for production of nitrile hydratase were determined by two mathematical methods: the Hadamard method and graphic analysis of response areas. A minimal medium was optimized and the basic roles of Fe²⁺ and Mg²⁺ were clearly shown. The influence of physico-chemical factors (pH, temperature and light conditions) on the culture and on nitrile hydratase were also studied. Various results permit the production of Brevibacterium sp. A4 cells with low protease and high nitrile hydratase contents.     

Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain reaction

A polymerase chain reaction (PCR) protocol was developed for the specific detection of genes coding nitrile hydratase (NHase). Primer design was based on the highly conserved sequences found in the coding region of the alpha-subunit gene corresponding to the metal-binding site. Purified genomic DNA from bacterial strains or directly from soil can serve as the target for the PCR, thus affording a simple and rapid method for screening NHase genes. The primer pairs, NHCo1/NHCo2 and NHFe1/NHFe2 yield PCR products corresponding to a partial coding sequence of cobalt and iron NHase genes, respectively. Using the PCR method, both types of iron- and cobalt-NHase-encoding genes were detected in DNA from pure cultures and soil samples. Furthermore consensus primers allowed rapid cloning and expression of novel NHases in Escherichia coli.     

Acetonitrile-hydrolysing enzymes in Rhodococcus erythropolis A10

Rhodococcus erythropolis A10 metabolizes acetonitrile by a two step process involving nitrile hydratase (NHase) and amidase. Both the enzymes were inducible and low basal levels of activities were observed in the cells grown in the absence of acetonitrile (AN). Cobalt and iron enhanced NHase, while amidase showed iron dependence. Presence of glucose or ammonium sulphate (AS) failed to affect acetonitrile utilization.     

微生物法生产丙烯酰胺的研究（Ⅰ）—腈水合酶产生菌株的培养和高活力的表达

研究了丙烯酰胺生产菌株的培养条件。通过对培养过程pH值调控、培养基补料以及诱导剂加入量的研究,使发酵液的腈水合酶的活力达到了6567u/mL菌液。这一酶活是国内外所见报道中最高的。进一步进行了丙烯腈的酶催化水合实验,产物中并没有发现副产物丙烯酸,说明在提高腈水合酶的同时,酰胺酶的活力并没有明显体现这一试验结果为工厂化生产改造以及新工艺的研究打下了基础。     

Biochemical characterization of Rhodococcus erythropolis N′4 nitrile hydratase acting on 4-chloro-3-hydroxybutyronitrile

The Rhodococcus erythropolis strain (N′4) possesses the ability to convert 4-chloro-3-hydroxybutyronitrile into the corresponding acid. This conversion was determined to be performed by its nitrile hydratase and amidase. Ammonium sulfate fractionation, DEAE ion exchange chromatography, and phenyl chromatography were used to partially purify nitrile hydratase from cell-free extract. A SDS-PAGE showed that the partially purified enzyme had two subunits and gel filtration chromatography showed that it consisted of four subunits of α₂β₂. The purified enzyme had a high specific activity of 860 U mg⁻¹ toward methacrylonitrile. The enzyme was found to have high activity at low temperature range, with a maximum activity occurring at 25 °C and be stable in the presence of organic acids at higher temperatures. The enzyme exhibited a preference for aliphatic saturated nitrile substrates over aliphatic unsaturated or aromatic ones. It was inhibited by sulfhydryl, oxidizing, and serine protease inhibitors, thus indicating that essential cysteine and serine residues can be found in the active site.The purified nitrile hydratase was able to convert 4-chloro-3-hydroxybutyronitrile into the corresponding amide at 15 °C. GC analysis showed that the initial conversion rate of the reaction was 215 mg substrate consumed min⁻¹ mg⁻¹. This demonstrated that this enzyme could be used in conjunction with a stereoselective amidase to synthesize ethyl (S)-4-chloro-3-hydroxybutyrate, an intermediate for a hypercholesterolemia drug, Atorvastatin.     

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     

Hydration of cyanopyridine to nicotinamide by whole cell nitrile hydratase

Summary 3-cyanopyridine was hydrated to nicotinamide by whole cells ofBrevibacterium R-312 containing nitrile hydratase. Cells used for kinetic studies had an initial activity of 0.30 mg nicotinamide/mg cells(dry)-min and storage half-lives (pH 8) of approximately 100 days, 10 days, 5 days and less than 1 day at 4°C, 10°C, 25°C, and 30°C respectively. Temperature and pH maxima were 35°C and 8.0, respectively. Fermentations gave a maximum total hydratase activity of 1.25 mg nicotinamide/min, but at this maximum the amidase activity was unacceptably high (25% of the hydratase activity): nicotinamide was converted too rapidly to nicotinic acid. But systematic fermentation studies (7 1) showed that harvesting at mid-log phase (18–20 h) prior to the attainment of maximum total activity gave reasonably high levels of hydratase (0.3 mg nicotinamide/mg cells-min) and acceptable levels of amidase (0.03 mg nicotinic acid/mg cells-min).