Design of a Biocatalytic Flow Reactor Based on Hierarchically Structured Monolithic Silica for Producing Galactooligosaccharides (GOSs).

Climate change mitigation requires the development of greener chemical processes. In this context, biocatalysis is a pivotal key enabling technology. The advantages of biocatalysis include lower energy consumption levels, reduced hazardous waste production and safer processes. The possibility to carry out biocatalytic reactions under flow conditions provides the additional advantage to retain the biocatalyst and to reduce costly downstream processes. Herein, we report a method to produce galactooligosaccharides (GOSs) from a largely available feedstock (i.e. lactose from dairy production) using a flow reactor based on hierarchically structured monolithic silica. This reactor allows for fast and efficient biotransformation reaction in flow conditions.

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 Two-Dimensional Polymer Synthesized at the Air/Water Interface.

A trifunctional, partially fluorinated anthracene-substituted triptycene monomer was spread at an air/water interface into a monolayer, which was transformed into a long-range-ordered 2D polymer by irradiation with a standard UV lamp. The polymer was analyzed by Brewster angle microscopy, scanning tunneling microscopy measurements, and non-contact atomic force microscopy, which confirmed the generation of a network structure with lattice parameters that are virtually identical to a structural model network based on X-ray diffractometry of a closely related 2D polymer. The nc-AFM images highlight the long-range order over areas of at least 300×300 nm2 . As required for a 2D polymer, the pore sizes are monodisperse, except for the regions where the network is somewhat stretched because it spans over protrusions. Together with a previous report on the nature of the cross-links in this network, the structural information provided herein leaves no doubt that a 2D polymer has been synthesized under ambient conditions at an air/water interface.

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.

Sequential lignin depolymerization by combination of biocatalytic and formic acid/formate treatment steps.

Lignin, a complex three-dimensional amorphous polymer, is considered to be a potential natural renewable resource for the production of low-molecular-weight aromatic compounds. In the present study, a novel sequential lignin treatment method consisting of a biocatalytic oxidation step followed by a formic acid-induced lignin depolymerization step was developed and optimized using response surface methodology. The biocatalytic step employed a laccase mediator system using the redox mediator 1-hydroxybenzotriazole. Laccases were immobilized on superparamagnetic nanoparticles using a sorption-assisted surface conjugation method allowing easy separation and reuse of the biocatalysts after treatment. Under optimized conditions, as much as 45 wt% of lignin could be solubilized either in aqueous solution after the first treatment or in ethyl acetate after the second (chemical) treatment. The solubilized products were found to be mainly low-molecular-weight aromatic monomers and oligomers. The process might be used for the production of low-molecular-weight soluble aromatic products that can be purified and/or upgraded applying further downstream processes.

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.

Two-Dimensional Calix[4]arene-based Metal-Organic Coordination Networks of Tunable Crystallinity.

A flexible and versatile method to fabricate two-dimensional metal-organic coordination networks (MOCNs) by bottom-up self-assembly is described. 2D crystalline layers were formed at the air-water interface, coordinated by ions from the liquid phase, and transferred onto a solid substrate with their crystallinity preserved. By using an inherently three-dimensional amphiphile, namely 25,26,27,28-tetrapropoxycalix[4]arene-5,11,17,23-tetracarboxylic acid, and a copper metal node, large and monocrystalline dendritic MOCN domains were formed. The method described allows for the fabrication of monolayers of tunable crystallinity on liquid and solid substrates. It can be applied to a large range of differently functionalized organic building blocks, also beyond macrocycles, which can be interconnected by diverse metal nodes.

Production of superparamagnetic nanobiocatalysts for green chemistry applications.

Immobilization of enzymes on solid supports is a convenient method for increasing enzymatic stability and enabling enzyme reuse. In the present work, a sorption-assisted surface conjugation method was developed and optimized to immobilize enzymes on the surface of superparamagnetic nanoparticles. An oxidative enzyme, i.e., laccase from Trametes versicolor was used as model enzyme. The immobilization method consists of the production of superparamagnetic nanoparticles by co-precipitation of FeCl2 and FeCl3. Subsequently, the particle surface is modified with an organosilane containing an amino group. Next, the enzymes are adsorbed on the particle surface before a cross-linking agent, i.e., glutaraldehyde is added which links the amino groups on the particle surface with the amino groups of the enzymes and leads to internal cross-linking of the enzymes as well. The method was optimized using response surface methodology regarding optimal enzyme and glutaraldehyde amounts, pH, and reaction times. Results allowed formulation of biocatalysts having high specific enzymatic activity and improved stability. The biocatalysts showed considerably higher stability compared with the dissolved enzymes over a pH range from 3 to 9 and in the presence of several chemical denaturants. To demonstrate the reusability of the immobilized enzymes, they were applied as catalysts for the production of a phenoxazinone dye. Virtually, 100 % of the precursor was transformed to the dye in each of the ten conducted reaction cycles while on average 84.5 % of the enzymatic activity present at the beginning of a reaction cycle was retained after each cycle highlighting the considerable potential of superparamagnetic biocatalysts for application in industrial processes.

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.

Interfacial binding of divalent cations to calixarene-based Langmuir monolayers.

The interactions of Langmuir monolayers produced through the self-assembly of an amphiphilic p-carboxycalix[4]arene (1) with a series of divalent, fourth-period transition metals, at the air-water interface, were investigated. Changes in the interfacial behavior of 1 in response to the presence of CuCl2, CoCl2, MnCl2, and NiCl2 were studied by means of Langmuir compression isotherms and Brewster angle microscopy (BAM). The measurements revealed that the self-assembly properties of 1 are significantly affected by Cu(2+) ions. The interactions of 1-based monolayers with Co(2+) and Cu(2+) ions were further investigated by means of synchrotron radiation-based X-ray reflectivity (XRR), X-ray near-total-reflection fluorescence (XNTRF), and grazing incidence X-ray diffraction (GIXD). XNTRF and XRR analyses revealed that the monolayer of 1 binds more strongly to Cu(2+) than Co(2+) ions. In the presence of relatively high concentrations of Cu(2+) ions in the subphase (1.4 × 10(-3) M), XNTRF exhibited anomalous depth profile behavior and GIXD measurements showed considerably strong diffuse scattering. Both measurements suggest the formation of Cu(2+) clusters contiguous to the monolayer of 1.

Reversible Supramolecular Surface Attachment of Enzyme-Polymer Conjugates for the Design of Biocatalytic Filtration Membranes.

To be used successfully in continuous reactor systems, enzymes must either be retained using filtration membranes or immobilized on a solid component of the reactor. Whereas the first approach requires large amounts of energy, the second approach is limited by the low temporal stability of enzymes under operational conditions. To circumvent these major stumbling blocks, we have developed a strategy that enables the reversible supramolecular immobilization of bioactive enzyme-polymer conjugates at the surface of filtration membranes. The polymer is produced through a reversible addition-fragmentation transfer method; it contains multiple adamantyl moieties that are used to bind the resulting conjugate at the surface of the membrane which has surface-immobilized cyclodextrin macrocycles. This supramolecular modification is stable under operational conditions and allows for efficient biocatalysis, and can be reversed by competitive host-guest interactions.

Sequence-specific DNA interactions with calixarene-based langmuir monolayers.

The interactions of an amphiphilic calixarene, namely p-guanidino-dodecyloxy-calix[4]arene, 1, self-assembled as Langmuir monolayers, with short double stranded DNA, were investigated by surface pressure-area (π-A) isotherms, surface ellipsometry and Brewster angle microscopy (BAM). Three DNA 30mers were used as models, poly(AT), poly(GC) and a random DNA sequence with 50% of G:C base pairs. The interactions of these model DNA duplexes with 1-based Langmuir monolayers were studied by measuring compression isotherms using increasing DNA concentrations (10(-6), 10(-5), 10(-4), and 5 × 10(-4) g L(-1)) in the aqueous subphase. The isotherms of 1 showed an expansion of the monolayer with, interestingly, significant differences depending on the duplex DNA sequence studied. Indeed, the interactions of 1-based monolayers with poly(AT) led to an expansion of the monolayer that was significantly more pronounced that for monolayers on subphases of poly(GC) and the random DNA sequence. The structure and thickness of 1-based Langmuir monolayers were investigated by BAM and surface ellipsometry that showed differences in thickness and structure between a monolayer formed on pure water or on a DNA subphase, with here again relevant dissimilarities depending on the DNA composition.

Laccases to take on the challenge of emerging organic contaminants in wastewater.

The removal of emerging organic contaminants from municipal wastewater poses a major challenge unsatisfactorily addressed by present wastewater treatment processes. Enzyme-catalyzed transformation of emerging organic contaminants (EOC) has been proposed as a possible solution to this major environmental issue more than a decade ago. Especially, laccases gained interest in this context in recent years due to their broad substrate range and since they only need molecular oxygen as a cosubstrate. In order to ensure the stability of the enzymes and allow their retention and reuse, either immobilization or insolubilization of the biocatalysts seems to be the prerequisite for continuous wastewater treatment applications. The present review summarizes the research conducted on EOC transformation with laccases and presents an overview of the possible immobilization techniques. The goal is to assess the state of the art and identify the next necessary steps that have to be undertaken in order to implement laccases as a tertiary wastewater treatment process in sewage treatment plants.

Advanced enzymatic elimination of phenolic contaminants in wastewater: a nano approach at field scale.

The removal of recalcitrant chemicals in wastewater treatment systems is an increasingly relevant issue in industrialized countries. The elimination of persistent xenobiotics such as endocrine-disrupting chemicals (EDCs) emitted by municipal and industrial sewage treatment plants remains an unsolved challenge. The existing efficacious physico-chemical methods, such as advanced oxidation processes, are resource-intensive technologies. In this work, we investigated the possibility to remove phenolic EDCs [i.e., bisphenol A (BPA)] by means of a less energy and chemical consuming technology. To that end, cheap and resistant oxidative enzymes, i.e., laccases, were immobilized onto silica nanoparticles. The resulting nanobiocatalyst produced at kilogram scale was demonstrated to possess a broad substrate spectrum regarding the degradation of recalcitrant pollutants. This nanobiocatalyst was applied in a membrane reactor at technical scale for tertiary wastewater treatment. The system efficiently removed BPA and the results of long-term field tests illustrated the potential of fumed silica nanoparticles/laccase composites for advanced biological wastewater treatment.

Surface chemistry at Swiss Universities of Applied Sciences.

In the Swiss Universities of Applied Sciences, a number of research groups are involved in surface science, with different methodological approaches and a broad range of sophisticated characterization techniques. A snapshot of the current research going on in different groups from the University of Applied Sciences and Arts Western Switzerland (HES-SO), the Zurich University of Applied Sciences (ZHAW) and the University of Applied Sciences and Arts Northwestern Switzerland (FHNW) is given.

Exploiting cyclodextrins as artificial chaperones to enhance enzyme protection through supramolecular engineering.

We report a method of enzyme stabilisation exploiting the artificial protein chaperone properties of ß-cyclodextrin (ß-CD) covalently embedded in an ultrathin organosilica layer. Putative interaction points of this artificial chaperone system with the surface of the selected enzyme were studied in silico using a protein energy landscape exploration simulation algorithm. We show that this enzyme shielding method allows for drastic enhancement of enzyme stability under thermal and chemical stress conditions, along with broadening the optimal temperature range of the biocatalyst. The presence of the ß-CD macrocycle within the protective layer supports protein refolding after treatment with a surfactant.

Ipso-hydroxylation and subsequent fragmentation: a novel microbial strategy to eliminate sulfonamide antibiotics.

Sulfonamide antibiotics have a wide application range in human and veterinary medicine. Because they tend to persist in the environment, they pose potential problems with regard to the propagation of antibiotic resistance. Here, we identified metabolites formed during the degradation of sulfamethoxazole and other sulfonamides in Microbacterium sp. strain BR1. Our experiments showed that the degradation proceeded along an unusual pathway initiated by ipso-hydroxylation with subsequent fragmentation of the parent compound. The NADH-dependent hydroxylation of the carbon atom attached to the sulfonyl group resulted in the release of sulfite, 3-amino-5-methylisoxazole, and benzoquinone-imine. The latter was concomitantly transformed to 4-aminophenol. Sulfadiazine, sulfamethizole, sulfamethazine, sulfadimethoxine, 4-amino-N-phenylbenzenesulfonamide, and N-(4-aminophenyl)sulfonylcarbamic acid methyl ester (asulam) were transformed accordingly. Therefore, ipso-hydroxylation with subsequent fragmentation must be considered the underlying mechanism; this could also occur in the same or in a similar way in other studies, where biotransformation of sulfonamides bearing an amino group in the para-position to the sulfonyl substituent was observed to yield products corresponding to the stable metabolites observed by us.

Solid lipid nanoparticle-based calix[n]arenes and calix-resorcinarenes as building blocks: synthesis, formulation and characterization.

Solid lipid nanoparticles (SLNs) have attracted increasing attention during recent years. This paper presents an overview about the use of calix[n]arenes and calix-resorcinarenes in the formulation of SLNs. Because of their specific inclusion capability both in the intraparticle spaces and in the host cavities as well as their capacity for functionalization, these colloidal nanostructures represent excellent tools for the encapsulation of different active pharmaceutical ingredients (APIs) in the area of drug targeting, cosmetic additives, contrast agents, etc. Various synthetic routes to the supramolecular structures will be given. These various routes lead to the formulation of the corresponding SLNs. Characterization, properties, toxicological considerations as well as numerous corresponding experimental studies and analytical methods will be also exposed and discussed.

Cyclodextrin-based combinatorial polymers: efficient binders of pharmaceuticals in water.

Cyclodextrins are cyclic oligomers of glucose; they are widely used in a large range of industrial applications because of their molecular inclusion properties. We used cyclodextrins to prepare different libraries of polymers and tested their ability to selectively recognize pharmaceuticals in water. It was demonstrated that the chemical composition of the polymer strongly influences its binding properties. The developed strategy can be used to produce selective sorbent nanomaterials of pharmaceuticals.