-
Journal of Microbiology and... May 2019Nitrilase is a valuable type of hydrolase that catalyzes nitriles into carboxylic acid and ammonia. Its applications, however, are severely restricted by the harsh...
Nitrilase is a valuable type of hydrolase that catalyzes nitriles into carboxylic acid and ammonia. Its applications, however, are severely restricted by the harsh conditions of industrial reaction processes. To solve this problem, a nitrilase from 72W was inserted into an shuttle vector for spore surface display. Western blot, enzyme activity measurements and flow cytometric analysis results all indicated a successful spore surface display of the CotB-nit fusion protein. In addition, the optimal catalytic pH value and temperature of the displayed nitrilase were determined to be 7.0 and 50°C, respectively. Moreover, results of reusability tests revealed that 64% of the initial activity of the displayed nitrilase was still retained at the 10 cycle. Furthermore, hydrolysis efficiency of upscale production of cyanocarboxylic acid was significantly higher in the displayed nitrilase-treated group than in the free group expressed by (pET-28a-nit). Generally, the display of 72W nitrilase on the spore surface of may be a useful method for immobilization of enzyme and consequent biocatalytic stabilization.
Topics: Aminohydrolases; Bacillus subtilis; Bacterial Proteins; Comamonadaceae; Enzyme Stability; Enzymes, Immobilized; Escherichia coli; Genetic Vectors; Hydrogen-Ion Concentration; Immobilization; Recombinant Fusion Proteins; Spores, Bacterial; Temperature; Time Factors
PubMed: 30955259
DOI: 10.4014/jmb.1901.01030 -
Scientific Reports Sep 2017Bacteria are essential in arsenic cycling. However, few studies have addressed 16S rRNA and arsenic-related functional gene diversity in long-term arsenic-contaminated...
Bacteria are essential in arsenic cycling. However, few studies have addressed 16S rRNA and arsenic-related functional gene diversity in long-term arsenic-contaminated tropical sediment. Here, using culture-based, metagenomic and computational approaches, we describe the diversity of bacteria, genes and enzymes involved in AsIII and AsV transformation in freshwater sediment and in anaerobic AsIII- and AsV-enrichment cultures (ECs). The taxonomic profile reveals significant differences among the communities. Arcobacter, Dechloromonas, Sedimentibacter and Clostridium thermopalmarium were exclusively found in ECs, whereas Anaerobacillus was restricted to AsV-EC. Novel taxa that are both AsV-reducers and AsIII-oxidizers were identified: Dechloromonas, Acidovorax facilis, A. delafieldii, Aquabacterium, Shewanella, C. thermopalmarium and Macellibacteroides fermentans. Phylogenic discrepancies were revealed among the aioA, arsC and arrA genes and those of other species, indicating horizontal gene transfer. ArsC and AioA have sets of amino acids that can be used to assess their functional and structural integrity and familial subgroups. The positions required for AsV reduction are conserved, suggesting strong selective pressure for maintaining the functionality of ArsC. Altogether, these findings highlight the role of freshwater sediment bacteria in arsenic mobility, and the untapped diversity of dissimilatory arsenate-reducing and arsenate-resistant bacteria, which might contribute to arsenic toxicity in aquatic environments.
Topics: Anaerobiosis; Arsenic; Bacteria; Biotransformation; Cluster Analysis; DNA, Bacterial; DNA, Ribosomal; Enzymes; Fresh Water; Genetic Variation; Geologic Sediments; Metabolic Networks and Pathways; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Water Pollutants, Chemical
PubMed: 28894204
DOI: 10.1038/s41598-017-11548-8 -
The Science of the Total Environment Aug 2023Anthropogenic activities have increased the dispersal of emerging contaminants (ECs), particularly of parabens, causing an escalation of their presence in wastewater...
Anthropogenic activities have increased the dispersal of emerging contaminants (ECs), particularly of parabens, causing an escalation of their presence in wastewater (WW). Current WW technologies do not present satisfactory efficiency or sustainability in removing these contaminants. However, bioremediation with microalgae-based systems is proving to be a relevant technology for WW polishing, and the use of microalgae-bacteria consortia can improve the efficiency of WW treatment. This work aimed to study dual cultures of selected bacteria (Raoultella ornithinolytica, Acidovorax facilis, Acinetobacter calcoaceticus, Leucobacter sp. or Rhodococcus fascians) and the microalga Chlorella vulgaris in microbial growth and WW bioremediation - removal of methylparaben (MetP) and nutrients. The association with the bacteria was antagonistic for C. vulgaris biomass productivity as a result of the decreased growth kinetics in comparison to the axenic microalga. The presence of MetP did not disturb the growth of C. vulgaris under axenic or co-cultured conditions, except when associated with R. fascians, where growth enhancement was observed. The removal of MetP by the microalga was modest (circa 30 %, with a removal rate of 0.0343 mg/L.d), but increased remarkably when the consortia were used (> 50 %, with an average removal rate > 0.0779 mg/L.d), through biodegradation and photodegradation. For nutrient removal, the consortia were found to be less effective than the axenic microalga, except for nitrogen (N) removal by C. vulgaris w/ R. fascians. The overall results propose that C. vulgaris co-cultivation with bacteria can increase MetP removal, while negatively affecting the microalga growth and the consequent reduction of sludge production, highlighting the potential of microalgae-bacteria consortia for the effective polishing of WW contaminated with parabens.
Topics: Chlorella vulgaris; Wastewater; Coculture Techniques; Parabens; Bacteria; Microalgae
PubMed: 37121314
DOI: 10.1016/j.scitotenv.2023.163746 -
Applied and Environmental Microbiology Aug 1999The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on... (Comparative Study)
Comparative Study
The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the alpha-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans(T)), and three strains isolated from freshwater ditches were affiliated with the beta-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae(T) and Acidovorax facilis(T), respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 x 10(4) dimethylmalonate-utilizing cells ml(-1), representing up to 0.4% of the total culturable nitrate-reducing population.
Topics: Anaerobiosis; Bacteria; Biodegradation, Environmental; Burkholderia; Carbon; Genes, Bacterial; Malonates; Minerals; Paracoccus; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 16S; Sewage
PubMed: 10427013
DOI: 10.1128/AEM.65.8.3319-3324.1999 -
Applied and Environmental Microbiology Feb 2001We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment...
We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4',6'-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% +/- 7.9% and 14.2% +/- 10.2% of the DAPI cell counts were detected by probes specific for alpha- and beta-Proteobacteria. These proportions increased to 12.0% +/- 3.3% and 54.0% +/- 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% +/- 1.4% and 41.1% +/- 8.4%, indicating a clear dominance of beta-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. gamma-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the alpha-Proteobacteria. In addition, with three probes highly specific for close relatives of the beta-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the beta-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of beta-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of alpha- and beta-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.
Topics: Alphaproteobacteria; Amino Acids; Betaproteobacteria; Carbohydrate Metabolism; Ecosystem; Fresh Water; Geologic Sediments
PubMed: 11157226
DOI: 10.1128/AEM.67.2.632-645.2001 -
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