Novel small multidrug resistance protein Tmt endows the Escherichia coli with triphenylmethane dyes bioremediation capability.

Dye contamination in printing and dyeing wastewater has long been a major concern due to its serious impact on both the environment and human health. In the quest for bioremediation of these hazardous dyes, biological resources such as biodegradation bacteria and enzymes have been investigated in severely polluted environments. In this context, the triphenylmethane transporter gene (tmt) was identified in six distinct clones from a metagenomic library of the printing and dyeing wastewater treatment system. Escherichia coli expressing tmt revealed 98.1% decolorization efficiency of triphenylmethane dye malachite green within 24 h under shaking culture condition. The tolerance to malachite green was improved over eightfold in the Tmt strain compared of the none-Tmt expressed strain. Similarly, the tolerance of Tmt strain to other triphenylmethane dyes like crystal violet and brilliant green, was improved by at least fourfold. Site-directed mutations, including A75G, A75S and V100G, were found to reinforce the tolerance of malachite green, and double mutations of these even further improve the tolerance. Therefore, the tmt has been demonstrated to be a specific efflux pump for triphenylmethane dyes, particularly the malachite green. By actively pumping out toxic triphenylmethane dyes, it significantly extends the cells tolerance in a triphenylmethane dye-rich environment, which may provide a promising strategy for bioremediation of triphenylmethane dye pollutants in the environments.

Treatment with senicapoc, a KCa 3.1 channel blocker, alleviates hypoxaemia in a mouse model of acute respiratory distress syndrome.

BACKGROUND AND PURPOSE: Acute respiratory distress syndrome (ARDS) is characterized by pulmonary oedema and severe hypoxaemia. We investigated whether genetic deficit or blockade of calcium-activated potassium (KCa 3.1) channels would counteract pulmonary oedema and hypoxaemia in ventilator-induced lung injury, an experimental model for ARDS. EXPERIMENTAL APPROACH: KCa 3.1 channel knockout (Kccn4-/- ) mice were exposed to ventilator-induced lung injury. Control mice exposed to ventilator-induced lung injury were treated with the KCa 3.1 channel inhibitor, senicapoc. The outcomes were oxygenation (PaO2 /FiO2 ratio), lung compliance, lung wet-to-dry weight and protein and cytokines in bronchoalveolar lavage fluid (BALF). KEY RESULTS: Ventilator-induced lung injury resulted in lung oedema, decreased lung compliance, a severe drop in PaO2 /FiO2 ratio, increased protein, neutrophils and tumour necrosis factor-alpha (TNF-α) in BALF from wild-type mice compared with Kccn4-/- mice. Pretreatment with senicapoc (10-70 mg·kg-1 ) prevented the reduction in PaO2 /FiO2 ratio, decrease in lung compliance, increased protein and TNF-α. Senicapoc (30 mg·kg-1 ) reduced histopathological lung injury score and neutrophils in BALF. After injurious ventilation, administration of 30 mg·kg-1 senicapoc also improved the PaO2 /FiO2 ratio and reduced lung injury score and neutrophils in the BALF compared with vehicle-treated mice. In human lung epithelial cells, senicapoc decreased TNF-α-induced permeability. CONCLUSIONS AND IMPLICATIONS: Genetic deficiency of KCa 3.1 channels and senicapoc improved the PaO2 /FiO2 ratio and decreased the cytokines after a ventilator-induced lung injury. Moreover, senicapoc directly affects lung epithelial cells and blocks neutrophil infiltration in the injured lung. These findings indicate that blocking KCa 3.1 channels is a potential treatment in ARDS-like disease.

Design, synthesis, and evaluation of a novel prodrug, a S-trityl-l-cysteine derivative targeting kinesin spindle protein.

Kinesin spindle protein (KSP) is expressed only in cells undergoing cell division, and hence represents an attractive target for the treatment of cancer. Several KSP inhibitors have been developed and undergone clinical trial, but their clinical use is limited by their toxicity to rapidly proliferating non-cancerous cells. To create new KSP inhibitors that are highly selective for cancer cells, we optimized the amino acid moiety of S-trityl-l-cysteine (STLC) derivative 1 using in silico modeling. Molecular docking and molecular dynamics simulation were performed to investigate the binding mode of 1 with KSP. Consistent with the structure activity relationship studies, we found that a cysteine amino moiety plays an important role in stabilizing the interaction. Based on these findings and the structure of GSH, a substrate of γ-glutamyltransferase (GGT), we designed and synthesized the prodrug N-γ-glutamylated STLC derivative 9, which could be hydrolyzed by GGT to produce 1. The KSP ATPase inhibitory activity of 9 was lower than that of 1, and LC-MS analysis indicated that 9 was converted to 1 only in the presence of GGT in vitro. In addition, the cytotoxic activity of 9 was significantly attenuated in GGT-knockdown A549 cells. Since GGT is overexpressed on the cell membrane of various cancer cells, these results suggest that compound 9 could be a promising prodrug that selectively inhibits the proliferation of GGT-expressing cancer cells.

Co-staining of KCa 3.1 Channels in NSCLC Cells with a Small-Molecule Fluorescent Probe and Antibody-Based Indirect Immunofluorescence.

The Ca2+ activated potassium channel 3.1 (KCa 3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of KCa 3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole-based BODIPY dye with a derivative of the KCa 3.1 channel inhibitor senicapoc via linkers of different length. Patch-clamp experiments revealed the inhibition of KCa 3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain KCa 3.1 channels in non-small-cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre-incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2. Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody-based indirect immunofluorescence yielded identical or very similar densities of stained KCa 3.1 channels. However, co-staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel.

Structure activity study of S-trityl-cysteamine dimethylaminopyridine derivatives as SIRT2 inhibitors: Improvement of SIRT2 binding and inhibition.

Sirtuin proteins are a highly conserved class of nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases. The pleiotropic human isoform 2 of Sirtuins (SIRT2) has been engaged in the pathogenesis of cancer in a plethora of reports around the globe. Thus, SIRT2 modulation is deemed as a promising approach for pharmaceutical intervention. Previously, we reported S-Trityl-l-Cysteine (STLC)-ornamented dimethylaminopyridine chemical entity named STC4 with a significant SIRT2 inhibitory capacity; this was separate from the conventional application of STLC scaffold as a kinesin-5 inhibitor. An interactive molecular docking study of SIRT2 and STC4 showed interaction between Asn168 of SIRT2 and the methyl ester of STC4, that appears to hinder STC4 to reach the selective pocket of the protein unlike strong SIRT2 inhibitor SirReal2. To improve its activity, herein, we utilized S-trityl cysteamine pharmacophore lacking the methyl ester. Nine compounds were synthesized and assayed affording three biopertinent SIRT2 inhibitors, and two of them, STCY1 and STCY6 showed higher inhibitory activity than STC4. These compounds have pronounced anti-proliferative activities against different cancer cell lines. A molecular docking study was executed to shed light on the supposed binding mode of the lead compound, STCY1, into the selective pocket of SIRT2 by interaction of the nitrogen of pyridine ring of the compound and Ala135 of the protein. The outcome of the study exposes that the active compounds are effective intermediates to construct more potent biological agents.

Bioremediation of malachite green by cyanobacterium Synechococcus elongatus PCC 7942 engineered with a triphenylmethane reductase gene.

Malachite green is a carcinogenic dye that has been detected in fish tissues and freshwater. Here we evaluated the malachite green decoloring ability of a photoautotrophic cyanobacterium, Synechococcus elongatus PCC 7942 (Synechococcus), that lives in freshwater. Results show that 99.5% of the dye was removed by Synechococcus through bioabsorption and bioaccumulation; however, the dye was not degraded or chemically modified. Next, we established an engineered Synechococcus strain to degrade the dye after uptake. The triphenylmethane reductase gene katmr was heterologously expressed, resulting in high production of a soluble recombinant protein. The engineered strain showed advanced decoloring abilities at a low cell density and in stressful environments. It degraded malachite green into the smaller molecules 4-methylaminobenzoic acid and 4-hydroxyl-aniline. After treatment with the engineered cyanobacterium, the growth of wheat seeds was fully recovered in the presence of malachite green. These results demonstrate the potential application of the engineered Synechococcus as a photosynthetic cell factory for the removal of malachite green from wastewater.

Construction of a Robust Cofactor Self-Sufficient Bienzyme Biocatalytic System for Dye Decolorization and its Mathematical Modeling.

A triphenylmethane reductase derived from Citrobacter sp. KCTC 18061P was coupled with a glucose 1-dehydrogenase from Bacillus sp. ZJ to construct a cofactor self-sufficient bienzyme biocatalytic system for dye decolorization. Fed-batch experiments showed that the system is robust to maintain its activity after 15 cycles without the addition of any expensive exogenous NADH. Subsequently, three different machine learning approaches, including multiple linear regression (MLR), random forest (RF), and artificial neural network (ANN), were employed to explore the response of decolorization efficiency to the variables of the bienzyme system. Statistical parameters of these models suggested that a three-layered ANN model with six hidden neurons was capable of predicting the dye decolorization efficiency with the best accuracy, compared with the models constructed by MLR and RF. Weights analysis of the ANN model showed that the ratio between two enzymes appeared to be the most influential factor, with a relative importance of 54.99% during the decolorization process. The modeling results confirmed that the neural networks could effectively reproduce experimental data and predict the behavior of the decolorization process, especially for complex systems containing multienzymes.

Citrus mangshanensis Pollen Confers a Xenia Effect on Linalool Oxide Accumulation in Pummelo Fruit by Enhancing the Expression of a Cytochrome P450 78A7 Gene CitLO1.

The aroma quality of citrus fruit is determined by volatiles that are present at extremely low levels in the citrus fruit juice sacs; it can be greatly improved by increasing volatiles. In this study, we showed that the contents of cis- and trans-linalool oxides were significantly increased in the juice sacs of three pummelos artificially pollinated with the Citrus mangshanensis (MS) pollen. A novel cytochrome P450 78A7 gene (CitLO1) was significantly upregulated in the juice sacs of Huanong Red pummelo pollinated with MS pollen in comparison to that with open pollination. Compared to wild-type tobacco Bright-Yellow2 cells, transgenic cells overexpressing CitLO1 promoted a 3- to 4-fold more conversion of (-)-linalool to cis- and trans-linalool oxides. Overall, our results suggest that MS pollen has a xenia effect on pummelo fruit aroma quality, and CitLO1 is a linalool oxide synthase gene that played an important role in the xenia effect.

Detecting Functional and Accessible Folate Receptor Expression in Cancer and Polycystic Kidneys.

Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are (i) folate receptor alpha (FRα) protein is expressed in the diseased tissues, and (ii) FRα in those tissues is accessible and functionally competent to bind systemically administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FRα binding, a multilysine containing linker that can cross-link to FRα in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FRα-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FRα protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FRα expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue, and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo.

Biodegradation of malachite green by an endophytic bacterium Klebsiella aerogenes S27 involving a novel oxidoreductase.

Endophytic microorganisms can metabolize organic contaminants and assist in plant growth, thus facilitating the phytoremediation of polluted environments. An endophytic bacterium capable of decoloring malachite green (MG) was isolated from the leaves of the wetland plant Suaeda salsa and was identified as Klebsiella aerogenes S27. Complete decolorization of MG (100 mg/l) was achieved in 8 h at 30 °C and pH 7.0. Ultraviolet-visible spectroscopy and Fourier-transform infrared spectroscopy analyses indicated the degradation of MG by the isolate. The enzymic assays of the strain showed the triphenylmethane reductase (TMR) activity. A gene encoding putative TMR-like protein (named as KaTMR) was cloned and heterologously expressed in Escherichia coli. KaTMR showed only 42.6-43.3% identities in amino acids compared with well-studied TMRs, and it phylogenetically formed a new branch in the family of TMRs. The degraded metabolites by recombinant KaTMR were detected by liquid chromatography-mass spectrometry, showing differences from the products of reported TMRs. The biotransformation pathway of MG was proposed. Phytotoxicity studies revealed the less-toxic nature of the degraded metabolites compared to the dye. This study presented the first report of an endophyte on the degradation and detoxification of triphenylmethane dye via a novel oxidoreductase, thus facilitating the study of the plant-endophyte symbiosis in the bioremediation processes.

Measurement of Proton Leak in Isolated Mitochondria.

Oxidative phosphorylation is an important energy-conserving mechanism coupling mitochondrial electron transfer to ATP synthesis. Coupling between respiration and phosphorylation is not fully efficient due to proton leaks. In this chapter, we present a method to measure proton leak activity in isolated mitochondria. The relative strength of a modular kinetic approach to probe oxidative phosphorylation is emphasized.

Efficiency of decolorization of different dyes using fungal biomass immobilized on different solid supports

Abstract Different technologies may be used for decolorization of wastewater containing dyes. Among them, biological processes are the most promising because they seem to be environmentally safe. The aim of this study was to determine the efficiency of decolorization of two dyes belonging to different classes (azo and triphenylmethane dyes) by immobilized biomass of strains of fungi (Pleurotus ostreatus - BWPH, Gleophyllum odoratum - DCa and Polyporus picipes - RWP17). Different solid supports were tested for biomass immobilization. The best growth of fungal strains was observed on the washer, brush, grid and sawdust supports. Based on the results of dye adsorption, the brush and the washer were selected for further study. These solid supports adsorbed dyes at a negligible level, while the sawdust adsorbed 82.5% of brilliant green and 19.1% of Evans blue. Immobilization of biomass improved dye removal. Almost complete decolorization of diazo dye Evans blue was reached after 24 h in samples of all strains immobilized on the washer. The process was slower when the brush was used for biomass immobilization. Comparable results were reached for brilliant green in samples with biomass of strains BWPH and RWP17. High decolorization effectiveness was reached in samples with dead fungal biomass. Intensive removal of the dyes by biomass immobilized on the washer corresponded to a significant decrease in phytotoxicity and a slight decrease in zootoxicity of the dye solutions. The best decolorization results as well as reduction in toxicity were observed for the strain P. picipes (RWP17).

Efficiency of decolorization of different dyes using fungal biomass immobilized on different solid supports.

Different technologies may be used for decolorization of wastewater containing dyes. Among them, biological processes are the most promising because they seem to be environmentally safe. The aim of this study was to determine the efficiency of decolorization of two dyes belonging to different classes (azo and triphenylmethane dyes) by immobilized biomass of strains of fungi (Pleurotus ostreatus - BWPH, Gleophyllum odoratum - DCa and Polyporus picipes - RWP17). Different solid supports were tested for biomass immobilization. The best growth of fungal strains was observed on the washer, brush, grid and sawdust supports. Based on the results of dye adsorption, the brush and the washer were selected for further study. These solid supports adsorbed dyes at a negligible level, while the sawdust adsorbed 82.5% of brilliant green and 19.1% of Evans blue. Immobilization of biomass improved dye removal. Almost complete decolorization of diazo dye Evans blue was reached after 24h in samples of all strains immobilized on the washer. The process was slower when the brush was used for biomass immobilization. Comparable results were reached for brilliant green in samples with biomass of strains BWPH and RWP17. High decolorization effectiveness was reached in samples with dead fungal biomass. Intensive removal of the dyes by biomass immobilized on the washer corresponded to a significant decrease in phytotoxicity and a slight decrease in zootoxicity of the dye solutions. The best decolorization results as well as reduction in toxicity were observed for the strain P. picipes (RWP17).

In vitro mutagenicity, NMR metabolite characterization of azo and triphenylmethanes dyes by adherents bacteria and the role of the "cna" adhesion gene in activated sludge.

Staphylococcus aureus, showing the greatest decolorization ability, was further investigated for Methyl Red (MR) Congo Red (CR), Crystal Violet (CV) and Malachite Green (MG) decolorization using response surface methodology (RSM). The chemometric methods use, based on statistical design of experiments (DOEs) such as RSM is becoming increasingly widespread in several sciences such as analytical chemistry, engineering and environmental chemistry. Stapphylococcus aureus ATCC 25923, Stapphylococcus aureus (S1) and Stapphylococcus aureus (S2), were isolated from textile wastewater plant located in KsarHellal, Tunisia and were tested for their decolorization capacity. PCR technique was utilized to identify the 3 bacterial strains and to detect the adhesin gene "cna". Biodegradation of MR, CR, CV and MG (750 ppm), were investigated under shaking condition in Mineral Salt Medium (MSM) solution at pH 7.5 and temperature 30 °C, using a 3.7 × 105 CFU/ml as inoculum size. Our results showed that Staphylococcus aureus had a high decolorization capacity. Nuclear magnetic resonance (NMR) spectroscopy analysis confirmed the biodegradation of dyes. The four dyes mutagenicity with the S9 metabolizing system decreased significantly after biodegradation and totally disappeared. Nuclear magnetic resonance (NMR) spectroscopy analysis confirmed the biodegradation of dyes.

The Comparative Study on the Rapid Decolorization of Azo, Anthraquinone and Triphenylmethane Dyes by Anaerobic Sludge.

An anaerobic sludge (AS), capable of decolorizing a variety of synthetic dyes, was acclimated and is reported here. The sludge presented a much better dye decolorizing ability than that of different individual strains. A broad spectrum of dyes could be decolorized by the sludge. Continuous decolorization tests showed that the sludge exhibited the ability to decolorize repeated additions of dye. The chemical oxygen demand (COD) removal rate of the dye wastewater reached 52% after 12 h of incubation. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) profiles revealed that the microbial community changed as a result of varying initial concentrations of dyes. Phylogenetic analysis indicated that microbial populations in the sludge belonged to the phyla Acidobacteria, Firmicutes, Bacteroidetes, Chloroflexi and Proteobacteria. The degradation products of the three types of dye were identified. For azo dyes, the anaerobic sludge converted Methyl Orange to N,N-dimethylbenzene-1,4-diamine and 4-aminobenzenesulfonic acid; for triphenylmethane dyes, after Malachite Green was decolorized, the analyzed products were found to be a mixture of N,N-dimethylbenzenamine, 3-dimethyl-aminophenol and 4-dimethylaminobenzophenone; for anthraquinone dyes, two products (acetophenone and 2-methylbenzoic acid) were observed after Reactive Blue 19 decolorization. Together, these results suggest that the anaerobic sludge has promising potential for use in the treatment of industrial wastewater containing various types of dyes.

Volatiles of Solena amplexicaulis (Lam.) Gandhi Leaves Influencing Attraction of Two Generalist Insect Herbivores.

Epilachna vigintioctopunctata Fabr. (Coleoptera: Coccinellidae) and Aulacophora foveicollis Lucas (Coleoptera: Chrysomelidae) are important pests of Solena amplexicaulis (Lam.) Gandhi (Cucurbitaceae), commonly known as creeping cucumber. The profiles of volatile organic compounds from undamaged plants, plants after 48 hr continuous feeding of adult females of either E. vigintioctopunctata or A. foveicollis, by adults of both species, and after mechanical damaging were identified and quantified by GC-MS and GC-FID analyses. Thirty two compounds were detected in volatiles of all treatments. In all plants, methyl jasmonate was the major compound. In Y-shaped glass tube olfactometer bioassays under laboratory conditions, both insect species showed a significant preference for complete volatile blends from insect damaged plants, compared to those of undamaged plants. Neither E. vigintioctopunctata nor A. foveicollis showed any preference for volatiles released by heterospecifically damaged plants vs. conspecifically damaged plants or plants attacked by both species. Epilachna vigintioctopunctata and A. foveicollis showed attraction to three different synthetic compounds, linalool oxide, nonanal, and E-2-nonenal in proportions present in volatiles of insect damaged plants. Both species were attracted by a synthetic blend of 1.64 µg linalool oxide + 3.86 µg nonanal + 2.23 µg E-2-nonenal, dissolved in 20 µl methylene chloride. This combination might be used as trapping tools in pest management strategies.

Purification and characterization of extracellular laccase produced by Ceriporiopsis subvermispora and decolorization of triphenylmethane dyes.

Laccases of white-rot fungi provide a promising future as a tool to be used in the field of biodegradation of synthetic dyes with different chemical structures. The aim of this study was production, characterization, and application of laccases from the white-rot fungus Ceriporiopsis subvermispora ATCC 90467 for decolorization of triphenylmethane dyes that could remain persistent in wastewater. Laccase was purified from a C. subvermispora culture by a four-step method resulting high specific activity of 2,571 U g-1 , 88-fold higher than crude laccase. Purified laccase (molecular weight 45 kDa) had the optimum activity at pH 2.0 and the optimum temperature 50 °C using ABTS as chromogenic substrate. Laccases efficiently decolorized triphenylmethane dyes such as Malachite Green (87.8%), Bromocresol Purple (71.6%), and Methyl Violet (68.1%) without redox mediator. However, decolorization percentage of hardly degradable triphenylmethane dyes such as Phenol Red, Bromophenol Blue, and Brilliant Blue R-250 was increased the presence of some low-molecular weight compounds (natural or synthetic redox mediators). Purified laccases were resistant to Mg2+ , Ca2+ , Ba2+ , Mn2+ , Fe2+ , Cu2+ , Zn2+ , and Sn2+ (10 mmol L-1 ). These findings suggest that laccases from C. subvermispora are able to decolorize triphenylmethane dyes without the negative influence of metal ions that can be found in wastewater.

A self-sufficient system for removal of synthetic dye by coupling of spore-displayed triphenylmethane reductase and glucose 1-dehydrogenase.

Biodegradation of triphenylmethane dyes by microorganisms is hampered by the transport barrier imposed by cell membranes. On the other hand, cell-free systems using enzyme-based biodegradation strategy are costly. Therefore, an efficient and inexpensive approach circumventing these problems is highly desirable. Here, we constructed a self-sufficient system for synthetic dye removal by coupling of spore surface-displayed triphenylmethane reductase (TMR) and glucose 1-dehydrogenase (GDH) for the first time. Display of both TMR and GDH significantly enhanced their stability under conditions of extreme pH and temperature. These engineered spores also exhibited more robust long-term stability than their purified counterparts. Furthermore, we observed that a high ratio of spore-displayed GDH is necessary for high dye degradation efficiency. These results indicate that this continuous dye removal system with cofactor regeneration offers a promising solution for dye biodegradation applications.

White-rot fungus Ganoderma sp.En3 had a strong ability to decolorize and tolerate the anthraquinone, indigo and triphenylmethane dye with high concentrations.

The ability of the white-rot fungus Ganoderma sp.En3 to decolorize different kinds of dyes widely applied in the textile and dyeing industry, including the anthraquinone dye Remazol Brilliant Blue R (RBBR), indigo dye indigo carmine and triphenylmethane dye methyl green, was evaluated in this study. Ganoderma sp.En3 had a strong capability of decolorizing high concentrations of RBBR, indigo carmine and methyl green. Obvious reduction of Chemical Oxygen Demand was observed after decolorization of different dyes. Ganoderma sp.En3 had a strong ability to tolerate RBBR, indigo carmine and methyl green with high concentrations. High concentrations of RBBR, indigo carmine and methyl green could also be efficiently decolorized by the crude enzyme of Ganoderma sp.En3. Different redox mediators such as syringaldehyde, acetosyringone and acetovanillone could enhance the decolorization capability for higher concentration of indigo carmine and methyl green. Different metal ions had little effect on the ability of the crude enzyme to decolorize indigo carmine and methyl green. Our study suggested that Ganoderma sp.En3 had a strong capability for decolorizing and tolerating high concentrations of different types of dyes such as RBBR, indigo carmine and methyl green.

CYP76C1 (Cytochrome P450)-Mediated Linalool Metabolism and the Formation of Volatile and Soluble Linalool Oxides in Arabidopsis Flowers: A Strategy for Defense against Floral Antagonists.

The acyclic monoterpene alcohol linalool is one of the most frequently encountered volatile compounds in floral scents. Various linalool oxides are usually emitted along with linalool, some of which are cyclic, such as the furanoid lilac compounds. Recent work has revealed the coexistence of two flower-expressed linalool synthases that produce the (S)- or (R)-linalool enantiomers and the involvement of two P450 enzymes in the linalool oxidation in the flowers of Arabidopsis thaliana. Partially redundant enzymes may also contribute to floral linalool metabolism. Here, we provide evidence that CYP76C1 is a multifunctional enzyme that catalyzes a cascade of oxidation reactions and is the major linalool metabolizing oxygenase in Arabidopsis flowers. Based on the activity of the recombinant enzyme and mutant analyses, we demonstrate its prominent role in the formation of most of the linalool oxides identified in vivo, both as volatiles and soluble conjugated compounds, including 8-hydroxy, 8-oxo, and 8-COOH-linalool, as well as lilac aldehydes and alcohols. Analysis of insect behavior on CYP76C1 mutants and in response to linalool and its oxygenated derivatives demonstrates that CYP76C1-dependent modulation of linalool emission and production of linalool oxides contribute to reduced floral attraction and favor protection against visitors and pests.