Studies on utilization of acetonitrile by Rhodococcus erythropolis A10

A petrochemical wastewater isolate, capable of utilizing high concentrations of acetonitrile and acetamide as the sole source of carbon and nitrogen was identified as Rhodococcus erythropolis A10. Cell-free extracts of acetonitrile-grown cells exhibited activities corresponding to nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4), which mediate the two-step breakdown of acetonitrile into acetic acid and ammonia. Studies indicated that both these enzymes in R. erythropolis A10 are intracellular, inducible and capable of hydrolysing a wide range of nitriles, including simple (acetonitrile, propionitrile), branched-chain (isobutyronitrile) and dinitrile (succinonitrile). The specific activity of the amidase was found to be several-fold higher than nitrile hydratase.     

Developing an understanding of sophorolipid synthesis through application of a central composite design model

A key barrier to market penetration for sophorolipid biosurfactants is the ability to improve productivity and utilize alternative feedstocks to reduce the cost of production. To do this, a suitable screening tool is required that is able to model the interactions between media components and alter conditions to maximize productivity. In the following work, a central composite design is applied to analyse the effects of altering glucose, rapeseed oil, corn steep liquor and ammonium sulphate concentrations on sophorolipid production with Starmerella bombicola ATCC 222144 after 168 h. Sophorolipid production was analysed using standard least squares regression and the findings related to the growth (OD₆₀₀) and broth conditions (glucose, glycerol and oil concentration). An optimum media composition was found that was capable of producing 39.5 g l^–1 sophorolipid. Nitrogen and rapeseed oil sources were found to be significant, linked to their role in growth and substrate supply respectively. Glucose did not demonstrate a significant effect on production despite its importance to biosynthesis and its depletion in the broth within 96 h, instead being replaced by glycerol (via triglyceride breakdown) as the hydrophilic carbon source at the point of glucose depletion. A large dataset was obtained, and a regression model with applications towards substrate screening and process optimisation developed.     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.     

Intraspecific variation in defense against a generalist lepidopteran herbivore in populations of Eruca sativa (Mill.)

Populations of Eruca sativa (Brassicaceae) from desert and Mediterranean (Med) habitats in Israel differ in their defense against larvae of the generalist Spodoptera littoralis but not the specialist Pieris brassicae. Larvae of the generalist insect feeding on plants of the Med population gained significantly less weight than those feeding on the desert plants, and exogenous application of methyl jasmonate (MJ) on leaves of the Med plants significantly reduced the level of damage created by the generalist larvae. However, MJ treatment significantly induced resistance in plants of the desert population, whereas the generalist larvae caused similar damage to MJ‐induced and noninduced plants. Analyses of glucosinolates and expression of genes in their synthesis pathway indicated that defense in plants of the Med population against the generalist insect is governed by the accumulation of glucosinolates. In plants of the desert population, trypsin proteinase inhibitor activity was highly induced in response to herbivory by S. littoralis. Analysis of genes in the defense‐regulating signaling pathways suggested that in response to herbivory, differences between populations in the induced levels of jasmonic acid, ethylene, and salicylic acid mediate the differential defenses against the insect. In addition, expression analysis of myrosinase‐associated protein NSP2 suggested that in plants of the desert population, glucosinolates breakdown products were primarily directed to nitrile production. We suggest that proteinase inhibitors provide an effective defense in the desert plants, in which glucosinolate production is directed to the less toxic nitriles. The ecological role of nitrile production in preventing infestation by specialists is discussed.     

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.     

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     

Isolation and characterization of a bacterium possessing a novel aldoxime-dehydration activity and nitrile-degrading enzymes

A bacterial strain capable of utilizing E-pyridine-3-aldoxime as a nitrogen source was isolated from soil after a 4-month acclimation period and was identified as Rhodococcus sp. The strain contained a novel aldoxime dehydration activity that catalyzed a stoichiometric dehydration of E-pyridine-3-aldoxime to form 3-cyanopyridine. The enzyme activity was induced by various aldoximes and nitriles. The strain metabolized the aldoxime as follows: E-pyridine-3-aldoxime was dehydrated to form 3-cyanopyridine, which was converted to nicotinamide by a nitrile hydratase, and the nicotinamide was successively hydrolyzed to nicotinic acid by an amidase. Received: 21 January 1998 / Accepted: 12 May 1998     

Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1

Summary We sought the optimum conditions for production of nitrile hydratase by Rhodococcus rhodochrous J1. The addiiion of both cobalt ions and an aliphatic nitrile or amide as an inducer was indispensable for the appearance of nitrile hydratase activity in R. rhodochrous J1 cells. Crotonamide was an efficient inducer and, moreover, urea was found to be the most powerful inducer for the production of nitrile hydratase. When R. rhodochrous J1 was cultivated under optimal conditions, the enzyme activity in the culture broth and the specific activity was approximately 32,000 and 512 times higher than the initially obtained levels, respectively. The nitrile hydratase formed corresponded to more than 45% of the total soluble protein in urea-induced cells, as judged by quantitative evaluation of the gel track.Offprint requests to: T. Nagasawa     

Glucose exerts a negative effect over a peroxidase from <Emphasis Type="Italic">Trichosporon asahii</Emphasis>, with carotenoid cleaving activity

Tobacco aroma compounds were generated via lutein cleavage by the combined action of a yeast and a bacterium identified as Trichosporon asahii and Paenibacillus amylolyticus, respectively. In this study, an inverse relationship between glucose concentration and the generation of three compounds, present in the tobacco aroma profile, was observed in mixed cultures. In order to identify the organism sensitive to the sugar effect, both were grown separately. The presence of glucose suppressed β-ionone production by T. asahii grown with lutein. However, the biotransformation of the ionone into its reduced derivatives (7,8-dihydro-β-ionone and 7,8-dihydro-β-ionol) by P. amylolyticus was not affected by the sugar . This pointed to the cleavage of lutein, a step within the process necessary for the synthesis of β-ionone, as the target of the glucose effect. In vitro studies with crude extracts and concentrated cell-free medium derived from T. asahii cultures showed that the carotenoid breakdown activity was located extracellularly and only detected in supernatants from yeast cells grown in the absence of the sugar. Rather than an inhibition or a mechanism affecting the enzyme secretion, the glucose effect on lutein degradation comprised another regulatory level. Further experiments showed that the enzyme responsible for lutein breakdown and susceptible to the sugar effect exhibited a high degree of identity to fungal peroxidases, studied as well, for their involvement in carotenoid cleavage.     

Identification and characterisation of an Acinetobacter sp. capable of assimilation of a range of cyano-metal complexes,free cyanide ions and simple organic nitriles

Summary An Acinetobacter sp. strain RFB1 was shown to be capable of degrading a wide range of cyano-metal complexes, simple cyanide salts and simple organic nitrile compounds. The enzymatic activity responsible for this degradation was located in an extracellular lipid complex. This complex could not be resolved into the constitutive components under standard conditions without loss of activity. Offsprint requests to: I. Finnegan     

Utilization of acrylonitrile by bacteria isolated from petrochemical waste waters

A bacterium, utilising acrylonitrile as a sole source of carbon and nitrogen, was isolated from Indian Petrochemical Corporation Limited (IPCL) waste waters and identified as Arthrobacter sp. This strain could also utilize acetonitrile, acetamide and acrylamide individually as a source of carbon and nitrogen. The metabolic studies with the whole cells indicated the sequential conversion of the nitrile to the respective amide and then to the respective acid and ammonia. The rate of nitrile hydrolysis was slower than the corresponding amide hydrolysis. Acrylic acid, the end product of acrylonitrile breakdown, did not support the growth when provided as a carbon source.     

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.     

Nitrile biotransformations using free and immobilized cells of a thermophilic Bacillus spp

A thermophilic Bacillus spp. capable of transforming aliphatic nitriles, cyclic nitriles and dinitriles was used as a free cell suspension and immobilized in alginate beads to study the utilization of acetonitrile and acrylonitrile in a buffered biotransformation medium. The cells grew optimally at 65 degrees C and contained a nitrile hydratase-amidase enzyme system that transformed nitrile compounds stoichiometrically to the corresponding carboxylic acids. In the presence of urea or chloroacetone, amidase activity was inhibited and the amide intermediate was accumulated. Mass transfer limitation of nitrile utilization rates was observed with immobilized cells, but the alginate afforded the cells some degree of additional thermal stability and potential advantage in re-use. In vitro inhibition of the partially purified amidase was confirmed and the use of whole cells of this organism in a continuous bioreactor to generate amide products from nitrile substrates was demonstrated.     

Biodegradation of Phenanthrene by a Bacillus Species

A bacterial strain capable of utilizing phenanthrene as sole source of carbon was isolated from soil and identified as a Bacillus sp. The organism also utilized naphthalene, biphenyl, anthracene, and other aromatic compounds as growth substrates. The organism degraded phenanthrene through the intermediate formation of 1-hydroxy-2-naphthoic acid, which was further metabolized via o-phthalate by a protocatechuate pathway, as evidenced by oxygen uptake and enzymatic studies. Received: 1 December 1999 / Accepted: 5 January 2000     

Enantioselective hydrolysis of racemic 2-phenylpropionitrile and other (R,S)-2-arylpropionitriles by a new bacterial isolate,Agrobacterium tumefaciens strain d3

Bacteria were enriched from soil samples with succinate as carbon source and racemic 2-phenylpropionitrile as sole source of nitrogen. One of the isolates, strain d3, converted (R,S)-2-phenylpropionitrile with high enantioselectivity to (S)-2-phenylpropionic acid. Strain d3 was identified as Agrobacterium tumefaciens. Resting cells hydrolysed 2-phenylpropionitrile via 2-phenylpropionamide to 2-phenylpropionic acid. Racemic 2-phenylpropionitrile as well as 2-phenylpropionamide were converted to (S)-2-phenylpropionic acid with an enantiometric excess above 96%. The nitrile hydratase and the amidase were both shown to convert preferentially the S enantiomer of their respective substrate. These two enzymes were induced in the presence of (R,S)-2-phenylpropionitrile but only in the absence of ammonia. In addition to 2-phenylpropionitrile strain d3 could utilize various aliphatic and aromatic nitriles as nitrogen sources. Resting cells of strain d3 also converted (R,S)-2-phenylbutyronitrile, ibuprofen nitrile, ketoprofen nitrile and -aminophenylacetonitrile with high enantioselectivity. The nitrile- and amide-converting enzyme activities were also found in cell-free extracts.     

Screening and characterization of microorganisms capable of converting iminodiacetonitrile to iminodiacetic acid

For screening and isolation of microorganisms harboring nitrile‐hydrolyzing enzymes that mediate the hydrolysis of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA), a sensitive and specific high‐throughput screening model was established. This model integrated a solid screen‐selective culture medium plate with bromcresol purple as the pH indicator coupled to Cu‐IDA complex spectrophotometry. Four strains were selected to perform the biotransformation to IDA, which were isolated and identified as Alcaligenes faecalis, Pseudomonas chlororaphis, Pseudomonas putida and Klebsiella pneumoniae, on the basis of 16S rDNA sequence analysis in combination with physiological and biochemical characterization. Moreover, the maximum specific enzyme activity was 73.4 U/g dry cell weight obtained by A. faecalis ZJUTBX11 after optimization of the medium conditions for enzyme production. The results show that the proposed model is a suitable method for screening microorganisms with nitrile‐hydrolyzing enzymes. We suggest the A. faecalis ZJUTBX11 strain to be used for large‐scale bioconversion of IDAN to IDA, because of its excellent performance in the production of IDA.     

Aerobic mineralization of chlorobenzoates by a natural polychlorinated biphenyl-degrading mixed bacterial culture

A natural mixed aerobic bacterial culture, designated MIXE1, was found to be capable of degrading several low-chlorinated biphenyls when 4-chlorobiphenyl was used as a co-substrate. MIXE1 was capable of using all the three monochlorobenzoate (CBA) isomers tested as well as 2,5-, 3,4- and 3,5-dichlorobenzoate (dCBA) as the sole carbon and energy source. During MIXE1 growth on these substrates, a nearly stoichiometric amount of chloride was released: 0.5 g/l of each chlorobenzoate was completely mineralized by MIXE1 after 2 or 3 days of culture incubation. Two strains, namely CPE2 and CPE3, were selected from MIXE1: CPE2, referred to the Pseudomonas genus, was found to be capable of totally degrading both 2-CBA and 2,5-dCBA, whereas Alcaligenes strain CPE3 was capable of mineralizing 3-, 4-CBA and 3,4-dCBA. Substrate uptake studies carried out with whole cells of strain CPE2 suggested that 2-CBA was metabolized through catechol, while 2,5-dCBA was degraded via 4-chlorocatechol. 3-CBA, 4-CBA, and 3,4-dCBA appeared to be degraded through 3,4-dihydroxybenzoate by the CPE3 strain. MIXE1, which is capable of degrading several chlorobenzoates, should therefore be able to mineralize a number of low-chlorinated congeners of simple and complex polychlorinated biphenyl mixtures. Correspondence to: F. Fava     

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.     

Indole-3-acetonitrile production from indole glucosinolates deters oviposition by Pieris rapae

Like many crucifer-specialist herbivores, Pieris rapae uses the presence of glucosinolates as a signal for oviposition and larval feeding. Arabidopsis thaliana glucosinolate-related mutants provide a unique resource for studying the in vivo role of these compounds in affecting P. rapae oviposition. Low indole glucosinolate cyp79B2 cyp79B3 mutants received fewer eggs than wild type, confirming prior research showing that indole glucosinolates are an important oviposition cue. Transgenic plants overexpressing epithiospecifier protein, which shifts glucosinolate breakdown toward nitrile formation, are less attractive to ovipositing P. rapae females. Exogenous application of indol-3-ylmethylglucosinolate breakdown products to cyp79B2 cyp79B3 mutants showed that oviposition was increased by indole-3-carbinol and decreased by indole-3-acetonitrile (IAN). P. rapae larvae tolerate a cruciferous diet by using a gut enzyme to redirect glucosinolate breakdown toward less toxic nitriles, including IAN, rather than isothiocyanates. The presence of IAN in larval regurgitant contributes to reduced oviposition by adult females on larvae-infested plants. Therefore, production of nitriles via epithiospecifier protein in cruciferous plants, which makes the plants more sensitive to generalist herbivores, may be a counter-adaptive mechanism for reducing oviposition by P. rapae and perhaps other crucifer-specialist insects.     

Biodegradation of Carbaryl by a Micrococcus Species

A bacterium capable of utilizing carbaryl as sole source of carbon was isolated from garden soil and identified as a Micrococcus species. The organism also utilized carbofuran, naphthalene, 1-naphthol, and several other aromatic compounds as growth substrates. The organism degraded carbaryl by hydrolysis to yield 1-naphthol and methylamine. 1-Naphthol was further metabolized via salicylate by a gentisate pathway, as evidenced by oxygen uptake and enzymatic studies. Received: 27 November 2000 / Accepted: 29 December 2000