The catalytic and ROS-scavenging activities of green synthesized, antiferromagneticα-Fe2O3nanoparticle with a prismatic octahedron morphology from pomegranate rind extract.

Phenolic compounds (like 4-nitrophenol) and dyes (like methyl orange) are common by-products discharged by many industries as wastes; they are toxic and may induce discomfort and irritation in humans when ingested. Most of these compounds can be made less toxic through catalytic degradation. Metal oxide nanoparticles are found to have high catalytic activity and can degrade toxic phenolic compounds and dyes. In the current study, pomegranate rind extract was used for the green synthesis of iron oxide nanoparticles that exhibited an octahedron morphology revealed by scanning electron microscopy analysis. Energy dispersive x-ray analysis showed 47.96% content of Fe (by weight); high resolution-transmission electron microscopy analysis confirmed that the nanoparticles had a particle size of 22.54 ± 4.13 nm. The particles were further characterized by x-ray diffraction, fourier transform-infrared spectroscopy, vibrating sample magnetometer, and thermogravimetric analysis. The nanoparticle proved to be efficient in reducing 4-nitrophenol and methyl orange. It was also found to be non-toxic towards murine macrophages, RAW 264.7 with good ROS-scavenging potential compared to control.

Sea lamprey cardiac mitochondrial bioenergetics after exposure to TFM and its metabolites.

Population control of invasive sea lamprey relies heavily on lampricide treatment of infested streams. The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is thought to impair mitochondrial ATP production through uncoupling oxidative phosphorylation. However, the effect of TFM on the entire electron transport chain (complexes I to V) in the mitochondria is not clear. In addition, TFM is reduced in phase I metabolism by sea lamprey at higher levels than in other fish species. The effects of these TFM reductive metabolites on mitochondria have not been explored. In this study, we sought to examine the effects of TFM and its reductive metabolite amino-TFM (TFMa) on cardiac mitochondrial oxygen consumption and membrane potential to delineate potential mechanisms for toxicity. To determine if molecules with similar structure also exhibit similar effects on mitochondria, we used 4-nitro-3-methylphenol (NMP) and its reductive metabolites 4-amino-3-methylphenol (NMPa) and 4-nitroso-3-methylphenol (NMPn) for comparisons. We found that mitochondrial bioenergetics was heavily affected with increasing concentrations of TFM, NMP, and NMPa when complexes I and II of the electron transport chain were examined, indicating that the toxic action of these compounds was exerted not only by uncoupling complex V, but also affecting complexes I and II.

Phytosynthesis of Cu/rGO using Euphorbia cheiradenia Boiss extract and study of its ability in the reduction of organic dyes and 4-nitrophenol in aqueous medium.

For the first time, copper nanoparticles (Cu NPs) superficially deposited on reduced graphene oxide (rGO) using Euphorbia cheiradenia Boiss leaf aqueous media. A beneficial series of analytical methods was used to characterise E. cheiradenia Boiss leaf extract and involved nanostructures. The Cu/rGO nanocomposite (NC) obtained from the conversion of Cu2+ ions to Cu NPs and GO to rGO undergoes the plant extract and used as a heterogeneous and reusable nanocatalyst for the destruction of 4-nitrophenol, rhodamine B, methylene blue, methyl orange and congo red using sodium borohydride at ambient temperature. In addition, Cu/rGO NC has reusability for many times in the reduction reactions with no decreasing of its catalytic capability.

Water-soluble MoS2 quantum dots for facile and sensitive fluorescence sensing of alkaline phosphatase activity in serum and live cells based on the inner filter effect.

We report a facile and sensitive method for the detection of alkaline phosphatase (ALP) activity in serum and live cells using molybdenum disulfide quantum dots (MoS2 QDs) based on the Inner Filter Effect (IFE). In the present work, water soluble MoS2 QDs with bright green fluorescence were synthesized through direct ultrasonic exfoliation of MoS2 powder in 85 vol% aqueous ethanol solution. p-Nitrophenylphosphate (PNPP) was employed to act as an ALP substrate, and its enzyme catalytic product (p-nitrophenol (PNP)) functioned as a powerful absorber in the IFE to influence the excitation of MoS2 QDs. PNPP was transformed into PNP in the presence of ALP, leading to the transition of the absorption peak from 310 nm to 405 nm and therefore resulted in a complementary overlap between the absorption of PNP and the excitation of MoS2 QDs. The fluorescence of MoS2 QDs was quenched due to the significant weakening of the excitation of MoS2 QDs by competitive absorption between QDs and PNP. A good linear relationship was obtained from 0 to 5 U L-1 (R2 = 0.9919) using the present IFE based sensing strategy with the lowest detection activity of 0.1 U L-1. The proposed sensing approach was successfully applied to ALP sensing in serum samples and ALP inhibitor investigation, as well as in ALP cell imaging.

Biosynthesis of waste pistachio shell supported silver nanoparticles for the catalytic reduction processes.

Silver nanoparticles (NPs) are immobilised on pistachio shell surface by Cichorium intybus L. leaves extract as an antioxidant media. The Fourier transform infrared spectra, X-ray diffraction, field-emission scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy, and transmission electron microscope analyses confirmed the support of silver NPs on the pistachio shell (Ag NPs/pistachio shell). Ag NPs on the pistachio shell had a diameter basically in the 10-15 nm range. Reduction reactions of 4-nitrophenol (4-NP), and organic dyes at ambient condition were used in the investigation of the catalytic performance of the prepared catalyst. Through this research, the Ag NPs/pistachio shell shows a high activity and recyclability, and reusability without loss of its catalytic activity.

Bioaugmented soil aquifer treatment for P-nitrophenol removal in wastewater unique for cold regions.

P-nitrophenol (PNP) is a toxic and recalcitrant organic pollutant and a usual intermediate in the production of fine chemicals, which has posed a significant threat to subsurface environment safety. Soil aquifer treatment (SAT) is a promising method to remove and remediate contamination in vadose zone with low cost and high efficiency. However, there are still research gaps for the treatment of recalcitrant contaminants by SAT in cold regions, such as un-robust indigenous microbes and low temperature constraint in vadose zone. The bioaugmentation technology was first introduced into SAT in order to enhance the removal ability of PNP by SAT operated in cold regions in this study. A high-efficiency PNP-degrading bacterium was successfully isolated, which can efficiently degrade PNP below 200 mg L-1 with a degradation rate above 99% at 15 °C close to the real subsurface temperature in cold regions, and added into SAT for bioaugmentation. The feasibility of bioaugmented SAT and associated PNP removal process were investigated by laboratory sand columns, along with effects of the SAT operative parameters (namely PNP loading concentration, flow rate and soil saturation level of SAT). Within the range of PNP loading stresses tested (1-200 mg L-1), PNP removal efficiency was optimal at constant flow rate of 219 mL d-1 in unsaturated operating condition of SAT under 15 °C among all the investigated experimental conditions. Longer hydraulic residence time increased the PNP removal rate, although the accumulated mass removed reduced and the removal efficiencies remained constant in unsaturated operating condition of SAT. It is found from the comparison between the PNP removals via both unsaturated and saturated columns that slight difference only in the removal rate of PNP was observed and the highly efficient bioaugmented SAT can completely degrade PNP of 10 mg L-1 within 5 wetting/drying cycles under both scenarios.

From laboratory to environmental conditions: a new approach for chemical's biodegradability assessment.

With thousands of organic chemicals released every day into our environment, Europe and other continents are confronted with increased risk of health and environmental problems. Even if a strict regulation such as REgistration, Authorization and restriction of CHemicals (REACH) is imposed and followed by industry to ensure that they prove the harmlessness of their substances, not all testing procedures are designed to cope with the complexity of the environment. This is especially true for the evaluation of persistence through biodegradability assessment guidelines. Our new approach has been to adapt "in the lab" biodegradability assessment to the environmental conditions and model the probability for a biodegradation test to be positive in the form of a logistic function of both the temperature and the viable cell density. Here, a proof of this new concept is proposed with the establishment of tri-dimensional biodegradability profiles of six chemicals (sodium benzoate, 4-nitrophenol, diethylene glycol, 2,4,5-trichlorophenol, atrazine, and glyphosate) between 4 to 30 °C and 10(4) to 10(8) cells ml(-1) as can be found in environmental compartments in time and space. The results show a significant increase of the predictive power of existing screening lab-scale tests designed for soluble substances. This strategy can be complementary to those current testing strategies with the creation of new indicators to quantify environmental persistence using lab-scale tests.

Kinetic characterization of a novel acid ectophosphatase from Enterobacter asburiae.

Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10(8) cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4 °C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows "Michaelis-Menten" kinetics with V = 61.2 U/mg and K0.5 = 60 µM, while ATP hydrolysis showed V = 19.7 U/mg, K0.5 = 110 µM, and nH = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K0.5 = 84 µM, and nH = 2.3. Arsenate and phosphate were competitive inhibitors with K i = 0.6 mM and K i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K0.5 = 2.2 mM and nH = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.

A Chromogenic Assay Suitable for High-Throughput Determination of Limit Dextrinase Activity in Barley Malt Extracts.

Twenty-four malt samples were assayed for limit dextrinase activity using a chromogenic assay developed recently in our group. The assay utilizes a small soluble chromogenic substrate which is hydrolyzed selectively by limit dextrinase in a coupled assay to release the chromophore 2-chloro-4-nitrophenol. The release of the chromophore, corresponding to the activity of limit dextrinase, can be followed by measuring the UV absorption at 405 nm. The 24 malt samples represented a wide variation of limit dextrinase activities, and these activities could be clearly differentiated by the assay. The results obtained were comparable with the results obtained from a commercially available assay, Limit-Dextrizyme from Megazyme International Ireland. Furthermore, the improved assay uses a soluble substrate. That makes it well suited for high-throughput screening as it can be handled in a 96-well plate format.

Effect of saikosaponins and extracts of vinegar-baked Bupleuri Radix on the activity of ß-glucuronidase.

In Traditional Chinese Medicine, liver targeting is usually achieved by coadministration with Vinegar-baked Radix Bupleuri (VBRB), but the mechanism is unclear. In this paper, the influence of VBRB on the activity of ß-glucuronidase was investigated and compared with that of saikosaponins. The activity of ß-glucuronidase was measured by microplate reader using a 4-nitrophenyl-ß-d-glucuronide substrate. The change of 4-nitrophenol content was used to characterize the activity of ß-glucuronidase. Bupleurum chinenes were found to be the inhibitor of ß-glucuronidase. The inhibition rate of Bupleurum chinenes extracts BC1 (high molecular weight polysaccharides), BC2 (ethanol soluble/water insoluble component), BC3 (extracted by n-butanol, soluble in water), and BC4 (low molecular weight water soluble parts) on the activity of ß-glucuronidase was found to be 45.15%, 33.94%, 24.94%, and 34.54%, respectively, after 1 h incubation, with BC1 showing the highest inhibition rate. In contrast, the saikosaponins were demonstrated to be the promoter of ß-glucuronidase, with promotion rates of 333.56%, 217.04%, 247.87%, 149.75%, and 92.50% for saikosaponin standard samples A, B, B2, C, and D, respectively, (p<0.05). In conclusion, inhibiting the activity of ß-glucuronidase might be one of the reasons why VBRB could influence drug distribution upon its coadministration with other drugs. Since saikosaponins and VBRB extracts have opposite effect, more attention should be paid to the content of saikosaponins in the extracts upon its application.

Spatial distribution and expression of intracellular and extracellular acid phosphatases of cluster roots at different developmental stages in white lupin.

Acid phosphatases (APases) play a key role in phosphorus (P) acquisition and recycling in plants. White lupin (Lupinus albus L.) forms cluster roots (CRs) and produces large amounts of APases under P deficiency. However, the relationships between the activity of intracellular and extracellular APases (EC 3.1.3.2) and CR development are not fully understood. Here, comparative studies were conducted to examine the spatial variation pattern of APase activity during CR development using the enzyme-labelled fluorescence-97 (ELF-97) and the p-nitrophenyl phosphate methods. The activity of intracellular and extracellular APases was significantly enhanced under P deficiency in the non-CRs and CRs at different developmental stages. These two APases exhibited different spatial distribution patterns during CR development, and these distribution patterns were highly modified by P deficiency. The activity of extracellular APase increased steadily with CR development from meristematic, juvenile, mature to senescent stages under P deficiency. In comparison, P deficiency-induced increase in the activity of intracellular APase remained relatively constant during CR development. Increased activity of intracellular and extracellular APases was associated with enhanced expression of LaSAP1 encoding intracellular APase and LaSAP2 encoding extracellular APase. The expression levels of these two genes were significantly higher at transcriptional level in both mature and senescent CRs. Taken together, these findings demonstrate that both activity and gene expression of intracellular or extracellular APases exhibit a differential response pattern during CR development, depending on root types, CR developmental stages and P supply. Simultaneous in situ determination of intracellular and extracellular APase activity has proved to be an effective approach for studying spatial variation of APases during CR development.

Generation of assays and antibodies to facilitate the study of human 5'-tyrosyl DNA phosphodiesterase.

Topoisomerases regulate DNA topology by the transient cleavage and religation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5' DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5'-tyrosyl DNA phosphodiesterase that repairs this topoisomerase-mediated DNA damage, thereby playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in increased susceptibility and sensitivity to Topo II-induced DNA double-strand breaks, thereby revealing Tdp2 as a potentially attractive anticancer target. No drug-like inhibitors of Tdp2 have been identified to date, and assays suitable for high-throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogeneous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. In addition, a new and specific Tdp2 antibody is described. Together, these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.

Effect of monocytes/macrophages on the early osteogenic differentiation of hBMSCs.

Heterotypic cell interactions are essential for the homeostasis of bone tissue, in particular the widely studied interaction between osteoblasts and osteoclasts. Closely related with osteoclasts are monocytes/macrophages. These have been shown to produce osteogenic factors, e.g. BMP-2, which plays a key role in bone metabolism. However, the mechanisms through which monocytes/macrophages interact with osteoblasts are still elusive. The aim of this work was to assess the influence of human peripheral blood monocytes/macrophages over the early osteogenic differentiation of human bone marrow stromal cells (hBMSCs) in the presence of dexamethasone-supplemented medium. The co-cultures were performed using porous transwells that allowed the interaction between both cell types through the production of paracrine factors. The potential effect of BMP-2 produced by monocytes/macrophages was addressed by adding an anti-BMP-2 antibody to the co-cultures. hBMSCs cultured in the presence of monocytes/macrophages had a higher proliferation rate than hBMSCs monocultures. The quantification of early osteogenic marker alkaline phosphatase (ALP) revealed higher activity of this enzyme in cells in the co-culture throughout the time of culture. Both of these effects were inhibited by adding an anti-BMP-2 antibody to the cultures. Moreover, qRT-PCR for osteocalcin and osteopontin transcripts showed overexpression of both markers. Once again, the effect of monocytes/macrophages over hBMSC osteogenic differentiation was completely inhibited in the co-cultures by blocking BMP-2. The present report confirmed that monocytes/macrophages produce BMP-2, which promotes osteogenic differentiation and proliferation of hBMSCs cumulatively to dexamethasone-supplemented medium. This potentially implies that monocyte/macrophages play a stronger role in bone homeostasis than so far supposed.

Efficiency of biological activator formulated material (BAFM) for volatile organic compounds removal--preliminary batch culture tests with activated sludge.

During biological degradation, such as biofiltration of air loaded with volatile organic compounds, the pollutant is passed through a bed packed with a solid medium acting as a biofilm support. To improve microorganism nutritional equilibrium and hence to enhance the purification capacities, a Biological Activator Formulated Material (BAFM) was developed, which is a mixture of solid nutrients dissolving slowly in a liquid phase. This solid was previously validated on mineral pollutants: ammonia and hydrogen sulphide. To evaluate the efficiency of such a material for biodegradation of some organic compounds, a simple experiment using an activated sludge batch reactor was carried out. The pollutants (sodium benzoate, phenol, p-nitrophenol and 2-4-dichlorophenol) were in the concentration range 100 to 1200 mg L(-1). The positive impact of the formulated material was shown. The improvement of the degradation rates was in the range 10-30%. This was the consequence of the low dissolution of the nutrients incorporated during material formulation, followed by their consumption by the biomass, as shown for urea used as a nitrogen source. Owing to its twofold interest (mechanical resistance and nutritional supplementation), the Biological Activator Formulated Material seems to be a promising material. Its addition to organic or inorganic supports should be investigated to confirm its relevance for implementation in biofilters.

The role of phosphorus in the ectendomycorrhiza continuum of desert truffle mycorrhizal plants.

The influence of inorganic and organic phosphorus (P) and the absence of P in the culture medium on the type of mycorrhizal colonization formed (ecto-, ectendo-, or endomycorrhiza) during Helianthemum almeriense x Terfezia claveryi symbiosis in in vitro conditions was analyzed. This is the first time that the relative proportions of the different mycorrhizal types in mycorrhizal roots of H. almeriense have been quantified and statistically analyzed. The relative proportions of the mycorrhizal types depended on the P source in the medium, suggesting that it is the organic P form that induces the formation of intracellular colonization. The above association should be considered as a continuum between intra- and intercellular colonizations, the most appropriate term for defining it being ectendomycorrhiza. The influence of the endogenous concentration of P on plant growth was also analyzed. P translocation was observed from shoot to roots, especially in mycorrhizal plants because mycorrhizal roots showed higher growth than non-mycorrhizal roots and/or because of an extra P demand from mycelium inside the roots. Soluble and cell wall acid phosphatases activities from H. almeriense roots were kinetically characterized at optimum pH (5.0), using p-nitrophenyl phosphate as substrate, with K (m) values of 3.4 and 1.8 mM, respectively. Moreover, the plant acid phosphatase and fungal alkaline phosphatases activities were histochemically localised in mycorrhizal H. almeriense roots by fluorescence with enzyme-labelled fluorescence substrate.

Vitamin E decreases bone mass by stimulating osteoclast fusion.

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts are multinucleated cells that are formed by mononuclear preosteoclast fusion. Fat-soluble vitamins such as vitamin D are pivotal in maintaining skeletal integrity. However, the role of vitamin E in bone remodeling is unknown. Here, we show that mice deficient in α-tocopherol transfer protein (Ttpa(-/-) mice), a mouse model of genetic vitamin E deficiency, have high bone mass as a result of a decrease in bone resorption. Cell-based assays indicated that α-tocopherol stimulated osteoclast fusion, independent of its antioxidant capacity, by inducing the expression of dendritic-cell-specific transmembrane protein, an essential molecule for osteoclast fusion, through activation of mitogen-activated protein kinase 14 (p38) and microphthalmia-associated transcription factor, as well as its direct recruitment to the Tm7sf4 (a gene encoding DC-STAMP) promoter. Indeed, the bone abnormality seen in Ttpa(-/-) mice was rescued by a Tm7sf4 transgene. Moreover, wild-type mice or rats fed an α-tocopherol-supplemented diet, which contains a comparable amount of α-tocopherol to supplements consumed by many people, lost bone mass. These results show that serum vitamin E is a determinant of bone mass through its regulation of osteoclast fusion.

Differential induction, purification and characterization of cold active lipase from Yarrowia lipolytica NCIM 3639.

The production, purification and characterization of cold active lipases by Yarrowia lipolytica NCIM 3639 is described. The study presents a new finding of production of cell bound and extracellular lipase activities depending upon the substrate used for growth. The strain produced cell bound and extracellular lipase activity when grown on olive oil and Tween 80, respectively. The organism grew profusely at 20 °C and at initial pH of 5.5, producing maximum extracellular lipase. The purified lipase has a molecular mass of 400 kDa having 20 subunits forming a multimeric native protein. Further the enzyme displayed an optimum pH of 5.0 and optimum temperature of 25 °C. Peptide mass finger printing reveled that some peptides showed homologues sequence (42%) to Yarrowia lipolytica LIP8p. The studies on hydrolysis of racemic lavandulyl acetate revealed that extracellular and cell bound lipases show preference over the opposite antipodes of irregular monoterpene, lavandulyl acetate.

Effects of brucine combined with glycyrrhetinic acid or liquiritin on rat hepatic cytochrome P450 activities in vivo.

Abstract: The activities of four CYP450 enzymes (CYP3A, 1A2, 2El and 2C) and the mRNA expression levels of CYP1A2, 2El, 2Cll and 3A1 in rat liver were determined after Wistar rats were orally administered with brucine (BR) at three dosage levels (3, 15 and 60 mg.kg-1 per day) and the high dose of BR combined with glycyrrhetinic acid (GA, 25 mg.kg-1 per day) or liquiritin (LQ, 20 mg.kg-1 per day) for 7 consecutive days. Compared with the control, brucine caused 24.5% and 34.6% decrease of CYP3A-associated testosterone 6beta-hydroxylation (6betaTesto-OH) and CYP2C-associated tolbutamide hydroxylation (Tol-OH), respectively, and 146.1% increase of CYP2El-associated para-nitrophenol hydroxylation (PNP-OH) at the high dose level. On the other hand, (BR+GA) caused 51.4% and 33.5% decrease, respectively, of CYP2El-associated PNP-OH and CYP1A2-associated ethoxyresorufin-O-de-ethylation (EROD) as compared with the high dose of BR group. Meanwhile, (BR+LQ) caused 41.1% decrease of CYP2El-associated PNP-OH and 37.7% increase of CYP2C-associated Tol-OH. The results indicated that the co-administration of BR with GA or LQ had effect on mRNA expression and activities of the CYP450 enzymes mentioned above to some extent, and the in vivo antagonism of LQ on BR-induced CYPs adverse effects and the in vivo inhibitory action of GA on CYP2E1 and 1A2 might play an important role in the detoxification of Radix Glycyrrhizae against Strychnos nux-vomica L.

Mineralization of mono-nitrophenols by Bjerkandera adusta and Lentinus squarrosulus and their extracellular ligninolytic enzymes.

Nitroaromatic compounds constitute a major class of widely distributed environmental contaminants. Fifty fungal strains were screened for their potential to tolerance with 2-nitrophenol, 3-nitrophenol and 4-nitrophenol on solid medium supplemented with 2% malt extract (MEA). Growth rate (mm/day) was determined at three concentrations (0.25, 0.5 and 1 mM) of all the three nitrophenols. From the fifty fungal strains only Bjerkandera adusta and Lentinus squarrosulus were able to tolerate all the three nitrophenols (NPs). These white-rot fungi (WRF) were chosen for liquid medium studies for the mineralization of mono-nitrophenols and ligninolytic enzyme activity at 0.25 mM concentration. Both varieties completely removed 2-NP and 3-NP while 4-NP was hard to mineralize. AAO (Aryl Alcohol Oxidase) is the main oxidase enzyme in B. adusta while laccase plays important role in L. squarrosulus. MnP (Manganese peroxidase) is the main peroxidase enzyme in both varieties. These fungal strains were capable to degrade nitrophenols and could be used for bioremediation applications on large scale.

Activity of immobilised rat hepatic microsomal CYP2E1 using alumina membrane as a support.

Porous alumina membranes are attractive materials for the construction of biosensors and also have utility for the production of immobilised enzyme bioreactors. Microsomes from rat liver were adsorbed onto alumina membrane activated by silane. Microsomal membranes were pumped through the channels where they became immobilised by binding to amine groups on the surface of the alumina membrane. In an effort to gain a quantitative understanding of the effects of microsomal film growth on enzyme activity, we compared the para-nitrophenol (pNP) hydroxylase activity of the microsomes by varying the amount of microsomes fixed in alumina microchannels. The alumina membrane was placed in a fluidic device at a fast flow that afforded short residence time (seconds) to obtain transformation of pNP to 4-nitrocatechol (pNC), which was detected by LC-MS/MS. This enabled the use of this bioreactor where CYP2E1 activity is low and tissue sources are limiting. The microsomes, successfully immobilised on the alumina membranes, were used to produce stable biocatalytic reactors that can be used repeatedly over a period of 2 months.