FMN-dependent NADH-azoreductase Research Articles

Genomic dissection of Niallia sp. for potential application in lignocellulose hydrolysis and bioremediation.

Genus Niallia has recently been separated taxonomic group from the Bacillus based on conserved signature indels in the genome. Unlike bioremediation, its role in plant biomass hydrolysis has not garnered considerable attention. The present study investigates the genomic potential of a novel Niallia sp. CRN 25 for applications in lignocellulose hydrolysis, significant enzyme production, and bioremediation. The CRN 25 strain exhibits xylosidase, cellobiosidase, α-arabinosidase, and α-D-galactosidase activity as 0.03U/ml whereas β-D-glucosidase and glucuronidase as 0.06 U/ml and 0.01U/ml, respectively. Further genome sequencing reveals nine copies of GH43 gene coding for hemicellulose-specific xylanase enzyme attached to the CBM 6 domain for increased processivity. The presence of β-glucosidase and β-galactosidase indicates the possible application of CRN 25 in facilitating the valorization of plant biomass into value-added products. Apart from this, genes of FMN-dependent NADH-azoreductase, cytochrome P450, and nitrate reductase, playing a crucial role in bioremediation processes, were annotated. Biosynthetic gene clusters (BGCs), responsible for synthesizing specialized metabolites of terpenes and lasso peptides, were also found in the genome. Conclusively genomic sketch of Niallia sp. CRN 25 reveals versatile metabolic potential for diverse environmental applications.

Metagenomic analysis of a bioreactor biofilm treating raw textile effluent and biochemical characterization of a novel azoreductase gene

Azo dyes are potent synthetic carcinogens that are widely used in the textile industries and their discharge into the surface water has detrimental consequences for human and ecosystem health. Oxidoreductases have been found to be involved in the metabolism of various xenobiotic compounds including azo dyes. Therefore, in this study shotgun metagenomic sequencing (SGMS) was used to bioprospect novel oxidoreductase genes in the biofilm of a fixed-film bioreactor treating raw textile effluent. According to the SGMS taxonomic and functional annotation, the bacteria found to be dominant in the bioreactor biofilm exhibited versatility and possessed genes that are capable of degrading azo dyes as well as other xenobiotics. A novel azoreductase gene (BM-AzoR) derived from the bioreactor biofilm metagenome was found to encode a previously uncharacterized FMN-dependent NADH azoreductase. In silico analysis of BM-AzoR gene encoded enzyme suggests that it is a thermostable and hydrophilic monomeric protein. The purified BM-AzoR is a flavin protein showing an optimal enzyme activity at pH 5 and 55 °C, respectively. The purified enzyme showed a specific azoreductase activity of 1.348 ± 0.12 U mg−1 in the presence of flavin mononucleotide (FMN) and preferred nicotinamide adenine dinucleotide (NADH) over nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. A molecular docking analysis validated the in vitro results by identifying Methyl Red as the best substrate for the BM-AzoR. The purified enzyme retained its activity in a variety of metal ions and solvents, indicating its potential application in azo dye bioremediation.

Anaerobic biodegradation of azo dye reactive black 5 by a novel strain Shewanella sp. SR1: Pathway and mechanisms

The efficiency of microbial populations in degrading refractory pollutants and the impact of adverse environmental factors often presents challenges for the biological treatment of azo dyes. In this study, the genome analysis and azo dye Reactive Black 5 (RB5) degrading capability of a newly isolated strain, Shewanella sp. SR1, were investigated. By analyzing the genome, functional genes involved in dye degradation and mechanisms for adaptation to low-temperature and high-salinity conditions were identified in SR1. The addition of co-substrates, such as glucose and yeast extract, significantly enhanced RB5 decolorization efficiency, reaching up to 87.6%. Notably, SR1 demonstrated remarkable robustness towards a wide range of NaCl concentrations (1–30 g/L) and temperatures (10–30 °C), maintaining efficient decolorization and high biomass concentration. The metabolic pathways of RB5 degradation were deduced based on the metabolites and genes detected in the genome, in which the azo bond was first cleaved by FMN-dependent NADH-azoreductase and NAD(P)H-flavin reductase, followed by deamination, desulfonation, and hydroxylation mediated by various oxidoreductases. Importantly, the degradation metabolites exhibited reduced toxicity, as revealed by toxicity analysis. These findings highlighted the great potential of Shewanella sp. SR1 for bioremediation of wastewaters contaminated with azo dyes.

Co-metabolic biodegradation of structurally discrepant dyestuffs by Klebsiella sp. KL-1: A molecular mechanism with regards to the differential responsiveness.

In this study, an attempt was made to decipher the underlying differential response mechanism of Klebsiella sp. KL-1 induced by exposure to disparate categories of dyestuffs in xylose (Xyl) co-metabolic system. Here, representative reactive black 5 (RB5), remazol brilliant blue R (RBBR) and malachite green (MG) belonging to the azo, anthraquinone and triphenylmethane categories were employed as three model dyestuffs. Klebsiella sp. KL-1 enabled nearly 98%, 80% or 97% removal of contaminants in assays Xyl+RB5, Xyl+RBBR or Xyl+MG after 48h, which was respectively 16%, 11% or 22% higher than those in the assays devoid of xylose. LC-QTOF-MS revealed an increased formation of smaller molecular weight intermediates in assay Xyl+RB5, whereas more metabolic pathways were deduced in assay Xyl+RBBR. Metaproteomics analysis displayed remarkable proteome alteration with regards to the structural difference effect of dyestuffs by Klebsiella sp. KL-1. Significant (p-value<0.05) activation of pivotal candidate NADH-quinone oxidoreductase occurred after 48h of disparate dyestuff exposure but with varying abundance. Dominant FMN-dependent NADH-azoreductase, Cytochrome d terminal oxidase or Thiol peroxidase were likewise deemed to be responsible for the catalytic cleavage of RB5, RBBR or MG, respectively. Further, the differential response mechanism towards the structurally discrepant dyestuffs was put forward. Elevated reducing force associated with the corresponding functional proteins/enzymes was transferred to the exterior of the cell to differentially decompose the target contaminants. Overall, this study was dedicated to provide in-depth insights into the molecular response mechanism of co-metabolic degradation of refractory and structurally discrepant dyestuffs by an indigenous isolated Klebsiella strain.

Eco-sustainable bioremediation of industrial azodye micropollutants by extremophilic plant growth promoting bacterium Halomonas desertis G11

Application of extremophilic plant growth promoting bacteria (PGPB) and their enzymes in bioremediation have been received increasing interest due to their eco-friendly nature and effectiveness for bio treatment of diverse industrial micro pollutants. In this work, the azo-dye decolorization potential of halophilic PGPB Halomonas desertis G11 was evaluated and optimized using central composite experimental design and response surface methodology. Interestingly, the increase of pH and NaCl concentration accelerated the dye decolorization. The model predicted a maximum removal of BEZACTIV blue S-2G dye (80%) at optimal operating conditions (dye concentration of 50 mg/L, inoculum size of 1.0%, pH of 8.2, NaCl of 5.0% and incubation time of 10 days). The experimental design model predictions are in good agreement with the experimental data, thereby providing the soundness of the developed model. The biodecolorization under pressures of high salinity and alkalinity seems to be correlated to azoreductase activity. The gene encoding FMN-dependent NADH azo-reductase from halophilic bacterium H. desertis G11 was identified and the structure and catalytic mechanism of dye decolorizing enzyme were elucidated. Results of this study provide evidence for the potential application of this azoreductase producing extremophilic bacterium as a novel candidate in the biological treatment of sediments and wastewaters contaminated by azo-dyes.

Decolorization mechanism, identification of an FMN-dependent NADH-azoreductase from a moderately halotolerant Staphylococcus sp. MEH038S, and toxicity assessment of biotransformed metabolites.

The application of halotolerant microorganisms capable of decolorizing is attractive. Decolorization mechanism, the effect of different parameters on the decolorization percentage, and toxicity analysis of Reactive Black 5 before and after decolorization were investigated in the present study. The decolorization percentage for live cells of Staphylococcus sp. strain MEH038S was more than dead cells, which demonstrated that Reactive Black 5 was decolorized through the degradation process. The results confirmed that an FMN-dependent NADH-azoreductase gene was responsible for the decolorization and then was identified as Staphylococcus sp. EFS01 azoreductase from a moderately halotolerant Staphylococcus strain for the first time. The maximal decolorization of 98.15% was observed at pH 6.5 and 35 ° C for 50mg/L of Reactive Black 5. In addition, more than 90% decolorization was achieved with 5-40g/L of NaCl. The results of Gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy showed that Reactive Black 5 was broken to the lower molecular weight compounds without any chromophoric azo groups. Phytotoxicity and fish toxicity proved that the biotransformed metabolites of Reactive Black 5degradation were more toxic than the original dye. The moderate halotolerant strain exhibited a remarkable decolorization capability and can be applied for textile wastewater treatment. PRACTITIONER POINTS: An azoreductase gene from a moderately halotolerant Staphylococcus was identified. More than 90% decolorization efficiency was observed under high-salt conditions. Biotransformed metabolites of RB5degradation were identified. Toxicity analysis of biotransformed metabolites was investigated.

Bio-Decolorization of Synthetic Dyes by a Halophilic Bacterium Salinivibrio sp.

Synthetic dyes, extensively used in various industries, act as pollutants in the aquatic environment, and pose a significant threat to living beings. In the present study, we assessed the potential of a halophilic bacterium Salinivibrio kushneri HTSP isolated from a saltpan for decolorization and bioremediation of synthetic dyes. The genomic assessment of this strain revealed the presence of genes encoding the enzymes involved in decolorization mechanisms including FMN-dependent NADH azoreductase Clade III, which cleave the azo bond of the dye, and the enzymes involved in deamination and isomerization of intermediate compounds. The dye decolorization assay was performed using this bacterial strain on three water-soluble dyes in different concentrations: Coomassie brilliant blue (CBB) G-250 (500–3,000 mg/L), Safranin, and Congo red (50–800 mg/L). Within 48 h, more than 80% of decolorization was observed in all tested concentrations of CBB G-250 and Congo red dyes. The rate of decolorization was the highest for Congo red followed by CBB G-250 and then Safranin. Using UV-Visible spectrometer and Fourier Transform Infrared (FTIR) analysis, peaks were observed in the colored and decolorized solutions. The results indicated a breakdown of dyes upon decolorization, as some peaks were shifted and lost for different vibrations of aromatic rings, aliphatic groups (–CH2, –CH3) and functional groups (–NH, –SO3H, and –SO3−) in decolorized solutions. This study has shown the potential of S. kushneri HTSP to decolorize dyes in higher concentrations at a faster pace than previously reported bacterial strains. Thus, we propose that our isolated strain can be utilized as a potential dye decolorizer and biodegradative for wastewater treatment.

Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose.

The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16–2.5 mM glucose, with a correlation coefficient, r, of 0.998.

English

The initial critical step of reduction of azo bond during the metabolism of azo dyes is catalysed by a group of NADH and FAD dependant enzyme called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis of the substrate specificity and the nature of catalysis. Azoreductase enzyme of Pseudomonas putida has a wider broad spectrum of substrate specificity and capable of degrading a wide variety of azo dyes. In the present study, the crystal structure of the enzyme from PDB and 10 azo dyes from NCBI PubChem compound were retrieved and their interactions were studied. These azo dyes were then docked with the FMN-dependent NADH-azoreductase enzyme to analyze the binding affinity of the azo dyes with the enzyme and predict the catalytic sites. Consequently, the catalytic residues of FMN-dependent and NADH dependent enzyme were then analysed in terms of properties including function, hydrogen bonding and flexibility. The results suggest that Ala-114, Phe-172 and Glu-174 play a predominant role as catalytic site residues in the enzyme. Furthermore, the approach emphasis on &nbsp;predicting the active sites of this enzyme where substrates can bind in order to give a better understanding of the biodegradation of some of the commercially important azodyes mediated by azoreductase. These results will pave way for further increase in azoreductase activity and for better understanding of the dye degradation pathway. &nbsp; Key words: Azoreductase, NADH, FMN, chemical properties, docking, active sites.&nbsp; &nbsp

Proteomic approach for identification of immunogenic proteins of Mycoplasma mycoides subsp. capri

In this study, an immunoproteomic approach was used to identify immunodominant proteins from Mycoplasma mycoides subsp. capri isolates. Membrane proteins, extracted through TX-114 phase partitioning, were separated using mono- and two-dimensional electrophoresis and detected by Western blotting with pooled sera from naturally infected goats. A total of 27 immunoreactive spots, corresponding to 13 different proteins, were identified using nanoLC–ESI-MSMS. Function annotation revealed that most of these proteins were metabolic enzymes involved in carbohydrate and energy metabolism. The immunogenic proteins identified in this study: pyruvate dehydrogenase, dihydrolipoamide acetyltransferase, dihydrolipoyl dehydrogenase, phosphate acetyltransferase, phosphopyruvate hydratase, adenine phopshoribosyltransferase, transketolase, translation elongation factor G, translation elongation factor Ts, FMN-dependent NADH-azoreductase, peptide methionine sulfoxide reductase, inorganic diphosphatase and trigger factor may be used as biomarkers for the serological diagnosis of contagious agalactia caused by M. mycoides subsp. capri.

Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus

The gene encoding an FMN-dependent NADH azoreductase, AzrG, from thermophilic Geobacillus stearothermophilus was cloned and functionally expressed in recombinant Escherichia coli. Purified recombinant AzrG is a homodimer of 23 kDa and bore FMN as a flavin cofactor. The optimal temperature of AzrG was 85 degrees C for the degradation of Methyl Red (MR). AzrG remained active for 1 h at 65 degrees C and for 1 month at 30 degrees C, demonstrating both superior thermostability and long-term stability of the enzyme. AzrG efficiently decolorized MR, Ethyl Red at 30 degrees C. Furthermore, the enzyme exhibited a wide-range of degrading activity towards several tenacious azo dyes, such as Acid Red 88, Orange I, and Congo Red. These results suggested the sustainable utilization of G. stearothermophilus as an azo-degrading strain for AzrG carrying whole-cell wastewater treatments for azo pollutants under high temperature conditions.

Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis

AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the Vmax value for p-methyl red 27-fold with a 3.8-fold increase of the Km value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The Vmax value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study.

Three-dimensional Structure of AzoR from Escherichia coli: AN OXIDEREDUCTASE CONSERVED IN MICROORGANISMS

The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2 A resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR.

Putative ACP Phosphodiesterase Gene (acpD) Encodes an Azoreductase

An FMN-dependent NADH-azoreductase of Escherichia coli was purified and analyzed for identification of the gene responsible for azo reduction by microorganisms. The N-terminal sequence of the azoreductase conformed to that of the acpD gene product, acyl carrier protein phosphodiesterase. Overexpression of the acpD gene provided the E. coli with a large amount of the 23-kDa protein and more than 800 times higher azoreductase activity. The purified gene product exhibited activity corresponding to that of the native azoreductase. The reaction followed a ping-pong mechanism requiring 2 mol of NADH to reduce 1 mol of methyl red (4'-dimethylaminoazobenzene-2-carboxylic acid) into 2-aminobenzoic acid and N,N'-dimethyl-p-phenylenediamine. On the other hand, the gene product could not convert holo-acyl carrier protein into the apo form under either in vitro or in vivo conditions. These data indicate that the acpD gene product is not acyl carrier protein phosphodiesterase but an azoreductase.