Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Duca, Daiana; Rose, David R.; Glick, Bernard R.

doi:10.1128/aem.00649-14

Cited by 41 publications

(18 citation statements)

References 66 publications

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“…Interestingly, these last three chemicals completely failed to biotransform at temperatures above 30°C (group 2b). Previous studies have suggested that bromoxynil and acetamiprid transformation to the corresponding amide is catalysed by nitrile hydratases 28 , whose activity is known to start decreasing significantly at temperatures above 20°C due to enzyme structure destabilisation 50 . Azoxystrobin, in contrast, has been shown to be primarily transformed (hydrolysis) by the action of protozoa 36 , which are unable to survive for more than few hours above 37°C 51 .…”

Section: Resultsmentioning

confidence: 99%

Understanding the Dependence of Micropollutant Biotransformation Rates on Short-Term Temperature Shifts

Meynet

Davenport

Fenner

2020

Environ. Sci. Technol.

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Temperature is a key factor that influences chemical biotransformation potential and rates, on which exposure and fate models rely to predict the environmental (micro)pollutant fate. Arrhenius-based models are currently implemented in environmental exposure assessment to adapt biotransformation rates to actual temperatures, assuming validity in the 0-30 °C range. However, evidence on how temperature shifts affect the physicochemical and microbial features in biological systems is scarce, questioning the validity of the existing modeling approaches. In this work, laboratory-scale batch assays were designed to investigate how a mixed microbial community responds to short-term temperature shifts, and how this impacts its ability to biotransform a range of structurally diverse micropollutants. Our results revealed three distinct kinetic responses at temperatures above 20 °C, mostly deviating from the classic Arrheniustype behavior. Micropollutants with similar temperature responses appeared to undergo mostly similar initial biotransformation reactions, with substitution-type reactions maintaining Arrhenius-type behavior up to higher temperatures than oxidation-type reactions. Above 20 °C, the microbial community also showed marked shifts in both composition and activity, which mostly correlated with the observed deviations from Arrhenius-type behavior, with compositional changes becoming a more relevant factor in biotransformations catalyzed by more specific enzymes (e.g., oxidation reactions). Our findings underline the need to re-examine and further develop current environmental fate models by integrating biological aspects, to improve accuracy in predicting the environmental fate of micropollutants.

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Section: Resultsmentioning

confidence: 99%

Understanding the Dependence of Micropollutant Biotransformation Rates on Short-Term Temperature Shifts

Meynet

Davenport

Fenner

2020

Environ. Sci. Technol.

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“…The molecular mass of Nit09 (36 kDa) is close to molecular masses of other known arylacetonitrilases such as the enzymes from Pseudomonas sp. UW4 (33 kDa; Duca et al 2014), Bradyrhizobium japonicum USDA110 (37 kDa; Zhu et al 2007a), or P. fluorescens EBC191 (38 kDa; Kiziak et al 2005). In contrast, the broad pH range of Nit09 activity is not that common for nitrilases of this type, including the enzymes from…”

Section: A Novel and Stable Arylacetonitrilasementioning

confidence: 99%

From sequence to function: a new workflow for nitrilase identification

Egelkamp

Friedrich

Hertel

et al. 2020

Appl Microbiol Biotechnol

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Nitrilases are industrially important biocatalysts due to their ability to degrade nitriles to carboxylic acids and ammonia. In this study, a workflow for simple and fast recovery of nitrilase candidates from metagenomes is presented. For identification of active enzymes, a NADH-coupled high-throughput assay was established. Purification of enzymes could be omitted as the assay is based on crude extract containing the expressed putative nitrilases. In addition, long incubation times were avoided by combining nitrile and NADH conversion in a single reaction. This allowed the direct measurement of nitrile degradation and provided not only insights into substrate spectrum and specificity but also in degradation efficiency. The novel assay was used for investigation of candidate nitrilase-encoding genes. Seventy putative nitrilase-encoding gene and the corresponding deduced protein sequences identified during sequence-based screens of metagenomes derived from nitrile-treated microbial communities were analyzed. Subsequently, the assay was applied to 13 selected candidate genes and proteins. Six of the generated corresponding Escherichia coli clones produced nitrilases that showed activity and one unusual nitrilase was purified and analyzed. The activity of the novel arylacetonitrilase Nit09 exhibited a broad pH range and a high long-term stability. The enzyme showed high activity for arylacetonitriles with a K M of 1.29 mM and a V max of 13.85 U/mg protein for phenylacetonitrile. In conclusion, we provided a setup for simple and rapid analysis of putative nitrilase-encoding genes from sequence to function. The suitability was demonstrated by identification, isolation, and characterization of the arylacetonitrilase. Key points • A simple and fast high-throughput nitrilase screening was developed. • A set of putative nitrilases was successfully screened with the assay. • A novel arylacetonitrilase was identified, purified, and characterized in detail.

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“…Interestingly, we found a gene encoding a nitrilase ( nit , EI613_03300), which is 74% similar that encoded by a nit that converts indole-3-acetonitrile (IAN) directly into IAA or into indol-3-acetamide (IAM) in the rhizobacterium Pseudomonas sp. UW4 [53, 54]. In the latter pathway, the conversion of IAM into IAA requires the action of IAM-hydrolases, for which we found two candidate genes, EI613_04545 e EI613_07015.…”

Section: Resultsmentioning

confidence: 99%

“…and Bradyrhizobium spp. [53, 58]. Nevertheless, the initial steps in the production of IAN in Azospirillum sp.…”

Section: Resultsmentioning

confidence: 99%

Characterization of cellular, biochemical and genomic features of the diazotrophic plant growth-promoting bacterium Azospirillum sp. UENF-412522, a novel member of the Azospirillum genus

Rodrigues

Matteoli

Gazara

et al. 2021

Preprint

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Given their remarkable beneficial effects on plant growth, several Azospirillum isolates currently integrate the formulations of various commercial inoculants. Our research group isolated a new strain, Azospirillum sp. UENF-412522, from passion fruit rhizoplane. This isolate uses carbon sources that are partially distinct from closely-related Azospirillum isolates. Scanning electron microscopy analysis and population counts demonstrate the ability of Azospirillum sp. UENF-412522 to colonize the surface of passion fruit roots. In vitro assays demonstrate the ability of Azospirillum sp. UENF-412522 to fix atmospheric nitrogen, to solubilize phosphate and to produce indole-acetic acid. Passion fruit plantlets inoculated with Azospirillum sp. UENF-41255 showed increased shoot and root fresh matter, as well as root dry matter, further highlighting its biotechnological potential for agriculture. We sequenced the genome of Azospirillum sp. UENF-412522 to investigate the genetic basis of its plant-growth promotion properties. We identified the key nif genes for nitrogen fixation, the complete PQQ operon for phosphate solubilization, the acdS gene that alleviates ethylene effects on plant growth, and the napCAB operon, which produces nitrite under anoxic conditions. We also found several genes conferring resistance to common soil antibiotics, which are critical for Azospirillum sp. UENF-412522 survival in the rhizosphere. Finally, we also assessed the Azospirillum pangenome and highlighted key genes involved in plant growth promotion. A phylogenetic reconstruction of the genus was also conducted. Our results support Azospirillum sp. UENF-412522 as a good candidate for bioinoculant formulations focused on plant growth promotion in sustainable systems.

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Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Cited by 41 publications

References 66 publications

Understanding the Dependence of Micropollutant Biotransformation Rates on Short-Term Temperature Shifts

Understanding the Dependence of Micropollutant Biotransformation Rates on Short-Term Temperature Shifts

From sequence to function: a new workflow for nitrilase identification

Characterization of cellular, biochemical and genomic features of the diazotrophic plant growth-promoting bacterium Azospirillum sp. UENF-412522, a novel member of the Azospirillum genus

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