-
Applied and Environmental Microbiology Aug 2020The enzymatic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF) has gained interest in recent years, as FDCA is a renewable precursor of...
The enzymatic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF) has gained interest in recent years, as FDCA is a renewable precursor of poly(ethylene-2,5-furandicarboxylate) (PEF). 5-Hydroxymethylfurfural oxidases (HMFOs) form a flavoenzyme family with genes annotated in a dozen bacterial species but only one enzyme purified and characterized to date (after heterologous expression of a sp. HMFO gene). This oxidase acts on both furfuryl alcohols and aldehydes and, therefore, is able to catalyze the conversion of HMF into FDCA through 2,5-diformylfuran (DFF) and 2,5-formylfurancarboxylic acid (FFCA), with only the need of oxygen as a cosubstrate. To enlarge the repertoire of HMFO enzymes available, genetic databases were screened for putative HMFO genes, followed by heterologous expression in After unsuccessful trials with other bacterial HMFO genes, HMFOs from two species were produced as active soluble enzymes, purified, and characterized. The sp. enzyme was also produced and purified in parallel for comparison. Enzyme stability against temperature, pH, and hydrogen peroxide, three key aspects for application, were evaluated (together with optimal conditions for activity), revealing differences between the three HMFOs. Also, the kinetic parameters for HMF, DFF, and FFCA oxidation were determined, the new HMFOs having higher efficiencies for the oxidation of FFCA, which constitutes the bottleneck in the enzymatic route for FDCA production. These results were used to set up the best conditions for FDCA production by each enzyme, attaining a compromise between optimal activity and half-life under different conditions of operation. HMFO is the only enzyme described to date that can catalyze by itself the three consecutive oxidation steps to produce FDCA from HMF. Unfortunately, only one HMFO enzyme is currently available for biotechnological application. This availability is enlarged here by the identification, heterologous production, purification, and characterization of two new HMFOs, one from and one from an unidentified species. Compared to the previously known HMFO, the new enzyme from exhibits better performance for FDCA production in wider pH and temperature ranges, with higher tolerance for the hydrogen peroxide formed, longer half-life during oxidation, and higher yield and total turnover numbers in long-term conversions under optimized conditions. All these features are relevant properties for the industrial production of FDCA. In summary, gene screening and heterologous expression can facilitate the selection and improvement of HMFO enzymes as biocatalysts for the enzymatic synthesis of renewable building blocks in the production of bioplastics.
Topics: Bacterial Proteins; Dicarboxylic Acids; Escherichia coli; Furaldehyde; Furans; Methylophilaceae; Microorganisms, Genetically-Modified; Oxidoreductases; Pseudomonas
PubMed: 32503910
DOI: 10.1128/AEM.00842-20 -
Ecotoxicology and Environmental Safety Sep 2020Microorganisms' role in pesticide degradation has been studied widely. Insitu treatments of effluents containing pesticides such as biological beds (biobeds) are...
Microorganisms' role in pesticide degradation has been studied widely. Insitu treatments of effluents containing pesticides such as biological beds (biobeds) are efficient biological systems where biomixture (mixture of substrates) and microorganisms are the keys in pesticide treatment; however, microbial activity has been studied poorly, and its potential beyond biobeds has not been widely explored. In this study, the capacity of microbial consortium and bacteria-pure strains isolated from a biomixture (soil-straw; 1:1, v/v) used to treat agricultural effluents under real conditions were evaluated during a bioremediation process of five pesticides commonly used Yucatan Mexico. Atrazine, carbofuran, and glyphosate had the highest degradations (>90%) using the microbial consortium; 2,4-D and diazinon were the most persistent (DT = 8.64 and 6.63 days). From the 21 identified bacteria species in the microbial consortium, Pseudomonas nitroreducens was the most abundant (52%) according to identified sequences. For the pure strains evaluation 2,4-D (DT = 9.87 days), carbofuran (DT = 8.27 days), diazinon (DT = 8.80 days) and glyphosate (DT = 8.59 days) were less persistent in the presence of the mixed consortium (Ochrobactrum sp. DGG-1-3, Ochrobactrum sp. Ge-14, Ochrobactrum sp. B18 and Pseudomonas citronellolis strain ADA-23B). Time, pesticide, and strain type were significant (P < 0.05) in pesticide degradation, so this process is multifactorial. Microbial consortium and pure strains can be used to increase the biobed efficiency by inoculation, even in the remediation of soil contaminated by pesticides in agricultural areas.
Topics: 2,4-Dichlorophenoxyacetic Acid; Agriculture; Atrazine; Bacteria; Biodegradation, Environmental; Carbofuran; Diazinon; Glycine; Microbial Consortia; Pesticides; Pseudomonas; Soil; Soil Pollutants; Glyphosate
PubMed: 32464440
DOI: 10.1016/j.ecoenv.2020.110734 -
3 Biotech Apr 20205-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the primary target for the broad-spectrum herbicide, glyphosate. Improvement of gene for high level of glyphosate...
5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the primary target for the broad-spectrum herbicide, glyphosate. Improvement of gene for high level of glyphosate tolerance is important to generate glyphosate-tolerant crops. In this study, we report the isolation and characterization of genes of glyphosate-tolerant strains FY43 and FY47. Both strains FY43 and FY47, which showed glyphosate tolerance up to 8.768% (518.4 mM, 32 × higher than field application), were isolated from soil samples collected from oil palm plantation with a long history of glyphosate application. The glyphosate tolerance property of genes of strains FY43 and FY47 was functionally characterized by expressing the genes in strain BL21(DE3). Error-prone PCR was performed to mutagenize native gene of strains FY43 and FY47. Ten mutagenized EPSPS with amino acid changes (R21C, N265S, A329T, P71L, T258A, L184F, G292C, G292S, L35F and A242V) were generated through error-prone PCR. Both native and mutated genes of strains FY43 and FY47 were introduced into strain BL21(DE3) and transformants were selected on basal salt medium supplemented with 8.768% (518.4 mM) glyphosate. Mutants with mutations (R21C, N265S, A329T, P71L, T258A, L35F, A242V, L184F and G292C) showed sensitivity to 8.768% glyphosate, whereas glyphosate tolerance for mutant with G292S mutation was not affected by the mutation.
PubMed: 32257739
DOI: 10.1007/s13205-020-02176-7 -
Analytical Chemistry Jan 2020Humic substances (HSs) are important electron acceptors and donors in soils and aquifers. The coupling of anoxic nitrogen (N) cycling to the function of HSs as a redox...
Humic substances (HSs) are important electron acceptors and donors in soils and aquifers. The coupling of anoxic nitrogen (N) cycling to the function of HSs as a redox battery, however, remains poorly understood. Mediated electrochemical analysis is an emerging tool to determine the redox properties (i.e., electron donating capacity (EDC), electron accepting capacity (EAC), and redox state) of HS. However, the presence of nitrite (NO), a central intermediate of the N-cycle, interferes with the electrochemical determination of the EAC. To eliminate this interference, we developed a bioassay to remove nitrite in HS samples using the denitrifying bacterium . Cell suspensions of completely removed NO at various concentrations (1, 2, and 5 mM) from humic acid samples (1 g HA/L) of different redox states. As is not able to exchange electrons with dissolved humic acids, the procedure allows an accurate and reliable determination of the EAC of humic acid samples. The proposed method thus opens new perspectives in biogeochemistry to study interactions between HSs and N cycling.
Topics: Electrochemical Techniques; Electrons; Humic Substances; Nitrites; Pseudomonas
PubMed: 31751112
DOI: 10.1021/acs.analchem.9b03683 -
Microbiology (Reading, England) Jan 2020Azelaic acid is a dicarboxylic acid that has recently been shown to play a role in plant-bacteria signalling and also occurs naturally in several cereals. Several...
Azelaic acid is a dicarboxylic acid that has recently been shown to play a role in plant-bacteria signalling and also occurs naturally in several cereals. Several bacteria have been reported to be able to utilize azelaic acid as a unique source of carbon and energy, including . In this study, we utilize as a model organism to study bacterial degradation of and response to azelaic acid. We report genetic evidence of azelaic acid degradation and the identification of a transcriptional regulator that responds to azelaic acid in DSM 9128. Three mutants possessing transposons in genes of an acyl-CoA ligase, an acyl-CoA dehydrogenase and an isocitrate lyase display a deficient ability in growing in azelaic acid. Studies on transcriptional regulation of these genes resulted in the identification of an IclR family repressor that we designated as AzeR, which specifically responds to azelaic acid. A bioinformatics survey reveals that AzeR is confined to a few proteobacterial genera that are likely to be able to degrade and utilize azelaic acid as the sole source of carbon and energy.
Topics: Bacteria; Bacterial Proteins; Dicarboxylic Acids; Gene Expression Regulation, Bacterial; Molecular Structure; Mutation; Phylogeny; Promoter Regions, Genetic; Pseudomonas; Repressor Proteins; Transcription Factors
PubMed: 31621557
DOI: 10.1099/mic.0.000865 -
Bioresource Technology Nov 2019The indiscriminate use of pesticides leads to serious food safety and toxicity issues and threatens the environment and biodiversity. Pseudomonas nitroreducens AR-3...
The indiscriminate use of pesticides leads to serious food safety and toxicity issues and threatens the environment and biodiversity. Pseudomonas nitroreducens AR-3 isolated from pesticide contaminated agricultural soil removed 97% of chlorpyrifos (CP) in just 8 h, in a mineral salt medium (MSM) containing glucose (1.0 g/L) and yeast extract (0.5 g/L) at 30 °C and 2% (v/v) inoculum when challenged with 100 mg/L CP. 3, 5, 6-trichloro 2-pyridinol (TCP), the degradation product of CP was detected only in low levels, indicating its further degradation. Organophosphate hydrolase (OPH), the enzyme considered responsible for CP degradation, had an intracellular localization. Crude OPH (1 mg/ml) removed 42% of 100 mg/L chlorpyrifos in just 2 h, indicating a rapid rate of degradation. Ultra-fast degradation of chlorpyrifos with an inducible OPH marks the potential of P. nitroreducens AR-3 for bioremediation of organophosphates. The strain AR-3 has the fastest rate of organophosphate degradation reported till date among Pseudomonads.
Topics: Biodegradation, Environmental; Chlorpyrifos; Pesticides; Pseudomonas
PubMed: 31466023
DOI: 10.1016/j.biortech.2019.122025 -
Scientific Reports May 2019Bacteria in the environment play a major role in the degradation of widely used man-made recalcitrant organic compounds. Pseudomonas nitroreducens TX1 is of special...
Bacteria in the environment play a major role in the degradation of widely used man-made recalcitrant organic compounds. Pseudomonas nitroreducens TX1 is of special interest because of its high efficiency to remove nonionic ethoxylated surfactants. In this study, a novel approach was demonstrated by a bacterial enzyme involved in the formation of radicals to attack ethoxylated surfactants. The dihydrolipoamide dehydrogenase was purified from the crude extract of strain TX1 by using octylphenol polyethoxylate (OPEO) as substrate. The extent of removal of OPEOs during the degradation process was conducted by purified recombinant enzyme from E. coli BL21 (DE3) in the presence of the excess of metal mixtures (Mn, Mg, Zn, and Cu). The metabolites and the degradation rates were analyzed and determined by liquid chromatography-mass spectrometry. The enzyme was demonstrated to form Fenton reagent in the presence of an excess of metals. Under this in vitro condition, it was shown to be able to shorten the ethoxylate chains of OPEO. After 2 hours of reaction, the products obtained from the degradation experiment revealed a prominent ion peak at m/z = 493.3, namely the ethoxylate chain unit is 6 (OPEO) compared to OPEO (m/z = 625.3), the main undegraded surfactant in the no enzyme control. It revealed that the concentration of OPEO and OPEO decreased by 90% and 40% after 4 hours, respectively. The disappearance rates for the OPEO homologs correlated to the length of the exothylate chains, suggesting it is not a specific enzymatic reaction which cleaves one unit by unit from the end of the ethoxylate chain. The results indicate the diverse and novel strategy by bacteria to catabolize organic compounds by using existing housekeeping enzyme(s).
Topics: Bacterial Proteins; Chromatography, Liquid; Copper; Dihydrolipoamide Dehydrogenase; Escherichia coli; Magnesium; Manganese; Mass Spectrometry; Phenols; Pseudomonas; Surface-Active Agents; Zinc
PubMed: 31048711
DOI: 10.1038/s41598-019-43266-8 -
AoB PLANTS Apr 2019Gametophytes of the fern develop into either hermaphrodites or males. As hermaphrodites develop, they secrete antheridiogen, or A, into the environment, inducing male...
Gametophytes of the fern develop into either hermaphrodites or males. As hermaphrodites develop, they secrete antheridiogen, or A, into the environment, inducing male development in undifferentiated gametophytes. Hermaphrodites are composed of archegonia, antheridia, rhizoids and a notch meristem, while males consist of antheridia and rhizoids. Much of the research on sexual and morphological development concerns gametophytes grown in sterile environments. Using biochemical and molecular techniques we identify a soil bacterium and explore its effects on sexual and rhizoid development. Hermaphrodite and male gametophytes were exposed to this bacterium and the effects on sexual development, rhizoid length and rhizoid number were explored. The bacterium was identified as a pseudomonad, . Gametophytes grown in the presence of the pseudomonad were more likely to develop into hermaphrodites across all gametophyte densities. Across all gametophyte sizes, hermaphrodites had rhizoids that were 2.95× longer in the presence of the pseudomonad while males had rhizoids that were 2.72× longer in the presence of the pseudomonad. Both hermaphrodite and male gametophytes developed fewer rhizoids in the presence of the pseudomonad. Control hermaphrodites produced 1.23× more rhizoids across all gametophyte sizes. For male gametophytes grown in the absence of the pseudomonad, the rate of increase in the number of rhizoids was greater with increasing size in the control than the rate of increase in males grown in the presence of the pseudomonad. The pseudomonad may be acting on gametophyte sexual development via several potential mechanisms: degradation of A, changes in nutrient availability or phytohormone production. The pseudomonad may also increase rhizoid number through production of phytohormones or changes in nutrient availability.
PubMed: 31019671
DOI: 10.1093/aobpla/plz012 -
Bioscience, Biotechnology, and... Feb 2019Theanine (γ-glutamylethylamide) is an amino acid analog that reduces blood pressure and improves immune responses. The ϒ-glutamyltranspeptidase (GGT) from Pseudomonas...
Theanine (γ-glutamylethylamide) is an amino acid analog that reduces blood pressure and improves immune responses. The ϒ-glutamyltranspeptidase (GGT) from Pseudomonas nitroreducens IFO12694 (PnGGT) has a unique preference for primary amines as ϒ-glutamyl acceptors over standard L-amino acids and peptides. This characteristic is useful for the synthesis of theanine. We used X-ray crystallographic analysis to understand the structural basis of PnGGT's hydrolysis and transpeptidation reactions and to characterize its previously unidentified acceptor site. Structural studies of PnGGT have shown that key interactions between three residues (Trp385, Phe417, and Trp525) distinguish PnGGT from other GGTs. We studied the roles of these residues in the distinct biochemical properties of PnGGT using site-directed mutagenesis. All mutants showed a significant decrease in hydrolysis activity and an increase in transpeptidase activity, suggesting that the aromatic side chains of Trp385, Phe417, and Trp525 were involved in the recognition of acceptor substrates. Abbreviations: ϒ-glutamyl peptide, theanine, X-ray crystallography.
Topics: Amino Acid Sequence; Amino Acids; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Hydrolysis; Mutagenesis, Site-Directed; Protein Conformation; Pseudomonas; Sequence Homology, Amino Acid; Substrate Specificity; gamma-Glutamyltransferase
PubMed: 30507352
DOI: 10.1080/09168451.2018.1547104 -
Bioresource Technology Feb 2019Synthetic oily bilge water (OBW) treatment and subsequent production of biopolymer were studied by using a sequential batch reactor (SBR). The effect of various...
Synthetic oily bilge water (OBW) treatment and subsequent production of biopolymer were studied by using a sequential batch reactor (SBR). The effect of various influencing parameters such as solids retention time (SRT), cycle time (CT), substrate concentration, pH level on the organic removal and synthesis of polyhydroxyalkanoates (PHA) was examined by novel soil bacteria isolated from hydrocarbon contaminated site near Karaikal port, India. The isolates were identified as Pseudomonas tuomuerensis and Pseudomonas nitroreducens using 16S rRNA. Sudan Black B staining was performed to visualize the presence of PHA. The experimental results showed that a decrease in substrate concentration to 5000 mg/L of soluble COD (CODs) showed maximum organic removal (81%) and maximum PHA yields of its cell dry mass (81%). The PHA yield was maximum at SRT of 5 d, pH = 7 and CT of 24 h. The produced PHA was characterized by using FTIR, XRD and SEM analysis.
Topics: Biopolymers; Hydrocarbons; India; Oils; Polyhydroxyalkanoates; Pseudomonas; RNA, Ribosomal, 16S; Wastewater
PubMed: 30445269
DOI: 10.1016/j.biortech.2018.11.003