-
PloS One 2013In 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...
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.
Topics: Acetonitriles; Alcaligenes faecalis; Aminohydrolases; Bacterial Proteins; Biotransformation; Chromatography, High Pressure Liquid; Circular Dichroism; Comamonadaceae; Escherichia coli; Hydrolysis; Imino Acids; Kinetics; Molecular Docking Simulation; Mutation; Protein Structure, Secondary; Recombinant Proteins; Rhodococcus; Sequence Alignment; Sequence Homology, Amino Acid
PubMed: 23826231
DOI: 10.1371/journal.pone.0067197 -
Journal of Molecular Microbiology and... 2012Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the...
Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacture of chelating agents, glyphosate herbicides and surfactants. In the current work, the fragment with the length of 1,110 bp encoding the Acidovorax facilis nitrilase was obtained. The recombinant nitrilase expressed in Escherichia coli BL21 (DE3) was successfully used in the production of IDA from iminodiacetonitrile. To improve the stability of operation, the recombinant cells were entrapped in polyvinyl alcohol (PVA) and sodium alginate (SA) copolymer. The maximum relative nitrilase activity with 98.1% was further observed at 1.0% SA, 8.0% PVA, 1.0% CaCl(2), and 5.0% wet cells, under conditions of 1.0% iminodiacetonitrile in distilled water and a temperature of 40°C, respectively. The entrapped cells facilitated easy separation and good recycling compared with free cells. Moreover, the immobilized cells showed good operation and storage stability. This report is the first to describe IDA preparation using immobilized recombinant E. coli harboring nitrilase.
Topics: Acetonitriles; Alginates; Aminohydrolases; Cells, Immobilized; Comamonadaceae; Gene Expression; Glucuronic Acid; Hexuronic Acids; Imino Acids; Polyvinyl Alcohol; Recombinant Proteins; Temperature
PubMed: 22441427
DOI: 10.1159/000337055 -
Biodegradation Jul 2008Focussing on the role of chlorocatechol 1,2-dioxygenase (CC12O), an oxygen-dependent key enzyme in the aerobic catabolism of chlorobenzene (CB), Pseudomonas veronii...
Focussing on the role of chlorocatechol 1,2-dioxygenase (CC12O), an oxygen-dependent key enzyme in the aerobic catabolism of chlorobenzene (CB), Pseudomonas veronii strain UFZ B549, Acidovorax facilis strain UFZ B530, and a community of indigenous groundwater bacteria were amended with CB degradation under either oxic or hypoxic conditions. All cultures readily degraded CB at high oxygen availability, but had differing abilities to completely degrade CB when exposed to oxygen limitation. For the three cultures very distinct oxygen half-saturation constants (0.3-11.7 muM) for the respective CC12Os were obtained and protein analysis showed that high affinity-type A. facilis and low affinity-type P. veronii express CC12Os, which belong to different structural clusters. From this a functional relation between CC12O type and the ability to cope with efficient ring fission under oxygen limitation is anticipated. Extremely high oxygen affinities for CC12Os support the assumption that truly oxic environments are not an essential requirement to degrade chloro(aromatic) compounds. Tiny quantities of oxygen permanently re-supplied will sufficiently maintain the growth of microaerophilic specialists with the ability to transform chloro(aromatics) via catechol intermediates.
Topics: Amino Acid Sequence; Bacteria; Bacterial Proteins; Chlorobenzenes; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Kinetics; Molecular Sequence Data; Oxygen; Sequence Homology, Amino Acid
PubMed: 17934786
DOI: 10.1007/s10532-007-9156-0 -
Biodegradation Jun 2008Limitations in the availability of oxygen restrict aerobic biodegradation of chloroaromatic compounds in groundwater ecosystems. In this context the activity of...
Limitations in the availability of oxygen restrict aerobic biodegradation of chloroaromatic compounds in groundwater ecosystems. In this context the activity of ring-cleaving chlorocatechol dioxygenases (CC12O) is crucial for effective mineralization. Previously we demonstrated that oxygen-related enzyme characteristics of CC12O can vary widely among the Proteobacteria (Balcke et al. submitted). Here, we investigated how strains with different ability to transform intermediary 3-chlorocatechol integrate into biodegradation of chlorobenzene (CB) under low or high oxygen availability. Pseudomonas veronii UFZ B549 and Acidovorax facilis UFZ B530, which had differing oxygen affinities for CC12O, were mixed together at different proportions (20:80; 80:20), and compared for degradation of chlorobenzene under oxic (215 microM O2) and hypoxic (11 microM O2) conditions. Changes in community composition in binary mixed cultures were determined and compared with an indigenous groundwater community, cultivated under comparable conditions. Community shifts were determined by FISH (fluorescent in situ hybridization) in our model system and SSCP (single stranded conformation polymorphism) fingerprinting in the groundwater community, as well as by analysis of respiratory quinones of taxonomic value. Hypoxia led to enrichment of Acidovoracae in the groundwater and binary cultures. Under hypoxic conditions cis,cis-2-chloromuconate released to the medium by A. facilis allowed for concomitant growth of P. veronii, although its low-affinity type CC12O would not imply growth on CB. Vice versa, increasing abundance of P. veronii induced intermediary 3-chlorocatechol accumulation, which was reduced by growth of A. facilis. Thus, reduced oxygen availability caused syntrophic rather than competitive interactions.
Topics: Anaerobiosis; Chlorobenzenes; Oxygen; Pseudomonas; Water
PubMed: 17882513
DOI: 10.1007/s10532-007-9149-z -
Biotechnology and Bioengineering Feb 2008A key step in a chemoenzymatic process for the production of high-purity glycolic acid (GLA) is the enzymatic conversion of glycolonitrile (GLN) to ammonium glycolate...
A key step in a chemoenzymatic process for the production of high-purity glycolic acid (GLA) is the enzymatic conversion of glycolonitrile (GLN) to ammonium glycolate using a nitrilase derived from Acidovorax facilis 72W. Protein engineering and over-expression of this nitrilase, combined with optimized fermentation of an E. coli transformant were used to increase the enzyme-specific activity up to 15-fold and the biocatalyst-specific activity up to 125-fold. These improvements enabled achievement of the desired volumetric productivity and biocatalyst productivity for the conversion of GLN to ammonium glycolate.
Topics: Acetonitriles; Aminohydrolases; Betaproteobacteria; Escherichia coli; Glycolates; Protein Engineering; Recombinant Proteins
PubMed: 17787011
DOI: 10.1002/bit.21643 -
Biodegradation Dec 2007Pseudomonas veronii strain UFZ B549, Acidovorax facilis strain UFZ B530, and a community of indigenous groundwater bacteria, adapted to oxygen limitation, were...
Pseudomonas veronii strain UFZ B549, Acidovorax facilis strain UFZ B530, and a community of indigenous groundwater bacteria, adapted to oxygen limitation, were cultivated on chlorobenzene and its metabolites 2-chloro-cis,cis-muconate and acetate/succinate under hypoxic and denitrifying conditions. Highly sensitive approaches were used to maintain defined low oxygen partial pressures in an oxygen-re-supplying headspace. With low amounts of oxygen available all cultures converted chlorobenzene, though the pure strains accumulated 3-chlorocatechol and 2-chloro-cis,cis-muconate as intermediates. Under strictly anoxic conditions no chlorobenzene transformation was observed, while 2-chloro-cis,cis-muconate, the fission product of oxidative ring cleavage, was readily degraded by the investigated chlorobenzene-degrading cultures at the expense of nitrate as terminal electron acceptor. Hence, we conclude that oxygen is an obligatory reactant for initial activation of chlorobenzene and fission of the aromatic ring, but it can be partially replaced by nitrate in respiration. The tendency to denitrify in the presence of oxygen during growth on chlorobenzene appeared to depend on the oxygen availability and the efficiency to metabolize chlorobenzene under oxygen limitation, which is largely regulated by the activity of the intradiol ring fission dioxygenase. Permanent cultivation of a groundwater consortium under reduced oxygen levels resulted in enrichment of a community almost exclusively composed of members of the beta-Proteobacteria and Bacteroidetes. Thus, it is deduced that these strains can still maintain high activities of oxygen-requiring enzymes that allow for efficient CB transformation under hypoxic conditions.
Topics: Adipates; Aerobiosis; Bacteria; Biodegradation, Environmental; Catechols; Chlorobenzenes; DNA, Ribosomal; Nitrates; Nitrogen; Oxygen; Sequence Alignment; Sorbic Acid; Time Factors
PubMed: 17279449
DOI: 10.1007/s10532-007-9104-z -
Biotechnology and Bioengineering Jul 2007Hydroxycarboxylic acid monomers can be used to prepare industrially important polymers. Enzymatic production of such hydroxycarboxylic acids is often preferred to...
Hydroxycarboxylic acid monomers can be used to prepare industrially important polymers. Enzymatic production of such hydroxycarboxylic acids is often preferred to chemical production since the reactions are run at ambient temperature, do not require strongly acidic or basic reaction conditions, and produce the desired product with high selectivity at high conversion. However, native enzymes often do not perform desired reactions with the efficiency required for commercial applications. Protein engineering was used to significantly increase the specific activity of nitrilase from Acidovorax facilis 72W for the conversion of 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid. Overexpression of engineered nitrilase enzymes in Escherichia coli, combined with immobilization of whole cells in alginate beads that can be recycled many times has facilitated the development of a commercially viable bioprocess for production of 3-hydroxyvaleric acid.
Topics: Alginates; Aminohydrolases; Bioreactors; Cells, Immobilized; Comamonadaceae; Escherichia coli; Fermentation; Industrial Microbiology; Microspheres; Mutagenesis, Site-Directed; Protein Engineering; Transformation, Genetic
PubMed: 17154311
DOI: 10.1002/bit.21289 -
Environmental Toxicology and Chemistry Jun 2005The concentration and isotopic composition of monochlorobenzene (MCB) was monitored in the plume of an anaerobic, contaminated aquifer in Bitterfeld, Germany. An...
The concentration and isotopic composition of monochlorobenzene (MCB) was monitored in the plume of an anaerobic, contaminated aquifer in Bitterfeld, Germany. An enrichment in the carbon isotopic composition of more than 4 delta units was found at the fringes of the plume relative to the center (-26.5 %), suggesting the occurrence of in situ biodegradation of MCB. A similar enrichment was measured in a detailed cross-section of the plume and in depth-specific samples obtained in a multilevel sampling well. The latter samples gave a good correlation of MCB concentrations and respective isotopic composition according to the Rayleigh equation. On the other hand, batch experiments using the aerobic MCB-degrading strains Ralstonia sp. DSM 8910, Acidovorax facilis UFZ B517, Rhodococcus erythropolis UFZ B528, and Pseudomonas veronii UFZ B547 showed that the known aerobic pathway initiated by dioxygenases does not result in a significant isotopic fractionation. Thus, a novel anaerobic pathway resulting in an isotopic fractionation appears to be the predominant process of MCB degradation in this aquifer. The study also clearly demonstrates the usefulness of isotopic fractionation analysis to prove biodegradation directly in the field, even when microcosm studies are not available and a metabolic pathway has not yet been elucidated.
Topics: Anaerobiosis; Bacteria, Aerobic; Biodegradation, Environmental; Carbon Isotopes; Chemical Fractionation; Chlorobenzenes; Fresh Water; Germany; Water Pollutants, Chemical; Water Supply
PubMed: 16117106
DOI: 10.1897/04-321r.1 -
Environmental Toxicology and Chemistry Feb 2004Five bacterial strains (Acidovorax facilis B517, Cellulomonas turbata B529, Pseudomonas veronii B547, Pseudomonas veronii B549, and Paenibacillus polymyxa B550) isolated... (Comparative Study)
Comparative Study
Five bacterial strains (Acidovorax facilis B517, Cellulomonas turbata B529, Pseudomonas veronii B547, Pseudomonas veronii B549, and Paenibacillus polymyxa B550) isolated on chlorobenzene as the sole source of carbon and energy were screened for the accumulation of the putative metabolic intermediate 3-chlorocatechol during growth on chlorobenzene under oxygen-limited conditions in the presence and absence of nitrate (1 mM). 3-Chlorocatechol accumulated in the growth media of all five strains, but accumulation was significantly less in cultures of A. facilis B517 compared to the other four strains. The presence of nitrate did not influence the biological conversion pattern. However, biologically produced nitrite reacted with 3-chlorocatechol chemically, a reaction that masked the accumulation of 3-chlorocatechol. For P. veronii B549, a clear relationship between the presence of 3-chlorocatechol in the medium and low oxygen concentrations was demonstrated. The assumption is made that accumulation of 3-chlorocatechol is due to the low enzymatic turnover of the 3-chlorocatechol cleaving enzyme, catechol-1,2-dioxygenase, at low oxygen concentrations.
Topics: Bacteria; Biodegradation, Environmental; Catechol 1,2-Dioxygenase; Catechols; Chlorobenzenes; Chromatography, Gas; Dioxygenases; Oxygen; Oxygenases
PubMed: 14982371
DOI: 10.1897/02-446 -
Applied Microbiology and Biotechnology Apr 2003A regioselective aliphatic nitrilase from Acidovorax facilis 72W was purified and characterized, and the corresponding gene was cloned and sequenced. This nitrilase gene...
A regioselective aliphatic nitrilase from Acidovorax facilis 72W was purified and characterized, and the corresponding gene was cloned and sequenced. This nitrilase gene was over-expressed in Escherichia coli, generating a microorganism that efficiently and regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic acids. The high yields obtained, mild reaction conditions used, and robustness observed make this biocatalyst suitable for industrial applications.
Topics: Amino Acid Sequence; Aminohydrolases; Base Sequence; Betaproteobacteria; Cloning, Molecular; Escherichia coli; Molecular Sequence Data; Sequence Analysis, DNA; Stereoisomerism; Substrate Specificity
PubMed: 12655453
DOI: 10.1007/s00253-002-1192-4