Surface Immobilization and Shielding of a Transaminase Enzyme for the Stereoselective Synthesis of Pharmaceutically Relevant Building Blocks.

Transaminases are enzymes capable of stereoselective reductive amination; they are of great interest in the production of chiral building blocks. However, the use of this class of enzymes in industrial processes is often hindered by their limited stability under operational conditions. Herein, we demonstrate that a transaminase enzyme from Aspergillus terreus can be immobilized at the surface of silica nanoparticles and protected in an organosilica shell of controlled thickness. The so-protected enzyme displays a high biocatalytic activity, and additionally provides the possibility to be retained in a reactor system for continuous operation and to be recycled.

A Biocatalytic Nanomaterial for the Label-Free Detection of Virus-Like Particles.

The design of nanomaterials that are capable of specific and sensitive biomolecular recognition is an on-going challenge in the chemical and biochemical sciences. A number of sophisticated artificial systems have been designed to specifically recognize a variety of targets. However, methods based on natural biomolecular detection systems using antibodies are often superior. Besides greater affinity and selectivity, antibodies can be easily coupled to enzymatic systems that act as signal amplifiers, thus permitting impressively low detection limits. The possibility to translate this concept to artificial recognition systems remains limited due to design incompatibilities. Here we describe the synthesis of a synthetic nanomaterial capable of specific biomolecular detection by using an internal biocatalytic colorimetric detection and amplification system. The design of this nanomaterial relies on the ability to accurately grow hybrid protein-organosilica layers at the surface of silica nanoparticles. The method allows for label-free detection and quantification of targets at picomolar concentrations.

Engineering Nanosized Organosilica for Molecular Recognition and Biocatalysis Applications.

A series of synthetic nanomaterials capable of molecular recognition and/or biocatalysis have been produced by exploiting the self-sorting, self-assembly and polycondensation of organosilane building blocks around protein templates. The established methodology allows for the production of thin organosilica layers of controlled thickness, down to nanometer precision. Fully synthetic virus recognition materials have been shown to specifically bind their target virus down to picomolar concentrations. The shielding of natural enzymes allowed producing nanobiocatalysts functioning under harsh operational conditions.

Enzyme Shielding in an Enzyme-thin and Soft Organosilica Layer.

The fragile nature of most enzymes is a major hindrance to their use in industrial processes. Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface of enzymes to grow an organosilica layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses.

Development and validation of a liquid chromatography-triple quadrupole mass spectrometry method for the determination of isopeptide ε-(γ-glutamyl) lysine in human urine as biomarker for transglutaminase 2 cross-linked proteins.

Determination of the levels of protein cross-linking catalysed by the activity of transglutaminase 2 in various disease states has remained a significant challenge. The ability to quantify the isopeptide ε-(γ-glutamyl) lysine, which can form as a heterogeneous bond within or between proteins has significant analytical and clinical potential as a biomarker in biofluids such as human urine. Increased transglutaminase 2 activity is associated with a number of diseases, such as fibrosis. Previously published methods have been based on classical amino acid analysis, however they require a complex multi-enzyme digestion in order to achieve complete protein digestion, whilst leaving the isopeptide cross link intact. These methods require high levels of enzymes, which contaminate the analysis and alter the dynamics of digestion. The amino acid analysis detection also lacked selectivity, especially where the levels of crosslink are expected to be low relative to the background protein levels. We have systematically addressed these challenges, by optimising the precipitation of the protein in urine, the use of innovative immobilised enzyme technology, which allows for efficient digestion without enzyme contamination and LC-MS/MS detection based on multiple reaction monitoring. This method was validated for its analytical performance characteristics, showing the method has a sensitivity of 0.1 ng/mL of ε-(γ-glutamyl) lysine in human urine with precision of less than 20% CV, and is selective as no interferences were observed that may adversely affect the analysis. As such this approach represents a significant advance in the ability to detect and quantify ε-(γ-glutamyl) lysine.

Using fluorescence-based microplate assay to assess DOM-metal binding in reactive materials for treatment of acid mine drainage.

One potential drawback of compost-based passive bioreactors, which is a promising biotechnology for acid mine drainage (AMD) treatment, is the transport of dissolved organic matter (DOM)-metal complexes in surface waters. To address this problem, the objective of this study was to assess the maximum capacity of organic substrates to release soluble DOM-metal complexes in treated water. The reactivities of DOM in maple wood chips and sawdust, composted poultry manure, and leaf compost were quantified toward Cd2+, Ni2+, Fe2+, and Cu2+ using fluorescence quenching. The DOM showed the highest reactivity toward Fe, but a limited number of available sites for sorption, whereas DOM-Cd complexes exhibited the lowest fluorescence quenching. Overall, the DOM from a mixture of wastes formed higher concentrations of DOM-metal complexes relative to sole substrates. Among DOM-metal complexes, the concentrations of DOM-Ni complexes were the highest. After reaching steady-state, low concentrations of DOM-metal complexes were released in treated water, which is in agreement with theoretical predictions based on geochemical modeling. Therefore, in addition to physicochemical characterization, fluorescence quenching technique is recommended for the substrate selection of bioreactors.

Partially shielded enzymes capable of processing large protein substrates.

We report the first method of enzyme protection enabling the production of partially shielded enzymes capable of processing substrates as large as proteins. We show that partially shielded sortase retains its transpeptidase activity and can perform bioconjugation reactions on antibodies. Moreover, a partially shielded trypsin is shown to outperform its soluble counterpart in terms of proteolytic kinetics. Remarkably, partial enzyme shielding results in a drastic increase in temporal stability of the enzyme.

Alternative spectrofluorimetric determination of short-chain volatile fatty acids in aqueous samples.

This paper presents a simple, rapid, and accurate method suitable for on-site measurement of short-chain volatile fatty acids (SCFA) in various environmental samples. This fluorimetric method involves a derivatization step of SCFA with N-(1-naphthyl)ethylenediamine (EDAN) and allows determination of acetic, butyric, propionic, valeric, lactic, succinic, and p-hydroxybenzoic acids in approximatively 10 min. To evaluate specificity and accuracy of the method, both laboratory-made waters and real samples ranging from wastewater plant and river to soils and composts have been tested. Good accuracy and correlation (r(2) = 0.9887) with HPIC determination have been obtained. The potential interference effect has been taken into account with compounds like humic substances, alcohols, amines, aldehydes, and metallic ions. This method seems thus well designed for the determination of total SCFA in waters, in the range 0.84-500 mg/L. Because this method seems well suited for following of anaerobic treatment, it has been calibrated versus acetic acid-equivalent.

Dissolved organic matter fluorescence as a water-flow tracer in the tropical wetland of Pantanal of Nhecolândia, Brazil.

The Nhecolândia is a sub-region of the Brazilian Pantanal wetland, where saline and freshwater lakes coexist in close proximity. Measurements of dissolved organic carbon (DOC) content and analysis of fluorescence excitation-emission matrices (EEM) were conducted in an effort to characterize spatial variability in concentration and source of dissolved organic matter (DOM) and to further understand the hydrochemical functioning of this complex environment. Increasing pH under the influence of evaporation resulted in an increasing DOC solubility ranging from 50 to over 300 mgC L(-1) in surface water. Spectrofluorescence characterisation indicates the presence of several families of dissolved organic matter (fulvic and humic-type and proteinaceous materials), which are related to the type of lake and its hydro-bio-geochemical functioning. Moreover, the fluorescence signatures from lake water DOM and from surrounding soil-water-extracted organic matter (WEOM) show strong similarities although some labile proteinaceous compounds disappeared during humification. Results from the characterisation of DOM and WEOM not only suggest that spectrofluorescence is a reliable technique for the tracing of water flows, but also for the marking of the origin of organic horizons in this environment.

A cyclodextrin-based polymer for sensing diclofenac in water.

An assay for the determination of diclofenac concentration, in the micromolar range in water, was developed. It is based on the use of a recently developed cyclodextrin-based polymer that possesses an inherent affinity for the target pharmaceutical. This competitive assay is exploiting the possibility to displace a fluorescent dye, adsorbed in the cyclodextrin-based polymer, by the target drug. This displacement is followed by measuring the increase in fluorescence polarization of the dye released in solution. The assay was successfully tested on a real wastewater sample with a limit of detection of 1 µM.

Virus-like particles as virus substitutes to design artificial virus-recognition nanomaterials.

Functional recognition imprints of virus-like particles, at the surface of silica particles, were generated following a strategy based on protein-templated polycondensation of organosilanes.

Modelling the dynamics of pentachlorophenol bioavailability in column experiments.

The aqueous-phase concentration of an organic pollutant found in a subsurface environment is often assumed to be its bioavailable concentration. However, the aqueous-phase concentration does not adequately reflect the dynamics of contaminant availability to microbes in flow-through systems. This paper assesses the effects of interacting processes such as sorption, biodegradation, and transport on contaminant bioavailability, and the fraction of the bioavailable contaminant that is taken up by microbes. The evolution of the bioavailable and uptake fractions is studied in two ways. Firstly, column experiments are conducted in which the introduced contaminant (pentachlorophenol, PCP) can flow through the columns, be consumed by microorganisms, or be sorbed by a solid matrix. Secondly, a phenomenological model (Flow/Sink/Reservoir model) that illustrates the dynamic nature of bioavailability and quantifies the uptake fraction is developed, based on a flow balance. Results show that after 60 h of sorption-limited bioavailability, the microorganisms induce desorption, so that the sorbed pool becomes bioavailable and bioavailability is limited by the PCP injection rate. A conclusion is drawn that the aqueous-phase concentration is a poor indicator of contaminant bioavailability to microbes.

Combining chemical sequential extractions with 3D fluorescence spectroscopy to characterize sludge organic matter.

The design and management of anaerobic digestion of sewage sludge (SS) require a relevant characterisation of the sludge organic matter (OM). Methods currently used are time-consuming and often insufficiently informative. A new method combining chemical sequential extractions (CSE) with 3D fluorescence spectroscopy was developed to provide a relevant SS characterisation to assess both OM bioaccessibility and complexity which govern SS biodegradability. CSE fractionates the sludge OM into 5 compartments of decreasing accessibility. First applied on three SS samples with different OM stability, fractionation profiles obtained were in accordance with the latter. 3D fluorescence spectroscopy revealed that the bioaccessible compartments were mainly constituted of simple and easily biodegradable OM while the unaccessible ones were largely made of complex and refractory OM. Then, primary, secondary and anaerobically digested sludge with different biodegradabilities were tested. Complexity revealed by 3D fluorescence spectroscopy was linked with biodegradability and chemical accessibility was correlated with sludge bioaccessibility.

International cross-validation of a BOD5 surrogate.

BOD5 dates back to 1912 when the Royal Commission decided to use the mean residence time of water in the rivers of England, 5 days, as a standard to measure the biochemical oxygen demand. Initially designed to protect the quality of river waters from extensive sewage discharge, the use of BOD5 has been quickly extended to waste water treatment plants (WWTPs) to monitor their efficiency on a daily basis. The measurement has been automatized but remains a tedious, time- and resource-consuming analysis. We have cross-validated a surrogate BOD5 method on two sites in France and in the USA with a total of 109 samples. This method uses a fluorescent redox indicator on a 96-well microplate to measure microbial catabolic activity for a large number of samples simultaneously. Three statistical tests were used to compare surrogate and reference methods and showed robust equivalence.

Solid-phase fluorescence spectroscopy to characterize organic wastes.

The production of solid organic waste (SOW) such as sewage sludge (SS) or municipal solid waste (MSW) has been continuously increasing in Europe since the beginning of the 1990'. Today, the European Union encourages the stabilization of these wastes using biologic processes such as anaerobic digestion and/or composting to produce bio-energy and organic fertilizers. However, the design and management of such biologic processes require knowledge about the quantity and quality of the organic matter (OM) contained in the SOW. The current methods to characterize SOW are tedious, time-consuming and often insufficiently informative. In this paper, we assess the potential of solid-phase fluorescence (SPF) spectroscopy to quickly provide a relevant characterization of SOW. First, we tested well known model compounds (tryptophan, bovine serum albumin, lignin and humic acid) and biologic matrix (Escherichia coli) in three dimensional solid-phase fluorescence (3D-SPF) spectroscopy. We recorded fluorescence spectra from proteinaceous samples but we could not record the fluorescence emitted by lignin and humic acid powders. For SOW samples, fluorescence spectra were successfully recorded for MSW and most of its sub-components (foods, cardboard) but impossible for SS, sludge compost (SC) and ligno-cellulosic wastes. Based on visual observations and additional assays, we concluded that the presence of highly light-absorptive chemical structures in such dark-colored samples was responsible for this limitation. For such samples, i.e. lignin, humic acid, SS, SC and ligno-cellulosic wastes, we show that laser induced fluorescence (LIF) spectroscopy enables the acquisition of 2D fluorescence spectra.

Organic matter as a potential complementary tool for delta(18)O data interpretation in heterogeneous aquifers.

Evaluating transit time by using delta(18)O seasonal variation is often difficult in a Mediterranean context due to the erratic rainfall signature added to the complexity of flow mixing. The present study aims to show that using organic matter can improve interpretations of the delta(18)O signal. The natural fluorescence of organic compounds and dissolved organic carbon (DOC) data were recorded in the underground low-noise laboratory, located within the catchment area of the Fontaine de Vaucluse karstic system, over a four-year period. By combining both total fluorescence and DOC, a systematic seasonal variation is observed only due to soil-water interaction. Therefore, when the recharge rate is enough at the time of the season concerned, a new specific seasonal tracer, independent of rainfall signature, is available. Besides, the DOC concentration is a tracer of rapid infiltration with short transit time associated with high DOC concentration, while long transit time waters are characterised by a low DOC concentration. Then this can also shed light on such a mixing, recent/old waters. A more sensitive interpretation of delta(18)O variations is then possible: if the rainfall amount and delta(18)O follow a seasonal trend, both tracers may be used in the same way; if the recharge is discontinuous due to discontinuous rainfall regime, delta(18)O tracing alone is usable; in case of erratic or smooth rainfall signature during the homogeneous rainy period, fluorescent organic matter tracing alone is then usable.

Discrimination of farm waste contamination by fluorescence spectroscopy coupled with multivariate analysis during a biodegradation study.

The persistence of potential tracers of dissolved organic matter (DOM) generated from farm waste-amended soil was investigated by fluorescence spectroscopy coupled with classification and regression tree (CART) and principal component analysis (PCA) during a short-term (8 days) to midterm (60 days) biodegradation study. Pig manure (PM), cow manure (CM), wheat straw (WS), and soil alone (SA) treatment inputs were used. Waste amendments were potential sources of higher DOM concentrations. PCA revealed the DOM quality differences between farm wastes and soil alone as well as a significant shift observed from the biochemical to the geochemical fluorescent fraction in SA and PM treatments. The tryptophan:Humic-Like ratio and tryptophan zone were the potential discriminators of recent and midterm pollution by farm wastes. Integral intensities of the Fulvic-Like zone and region III discriminated the PM from CM and WS during the 60 days. CART analysis showed 90 and 100% potential for farm wastes discrimination from soil during P1 and P2, respectively. The prediction successes were 72 and 57% for PM from other wastes and 60 and 100% for WS during both periods. Fluorescence spectroscopy in combination with CART analysis can be a nondestructive innovative method for monitoring susceptible farm waste contamination.