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.

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.

Nanobiocatalysts with inbuilt cofactor recycling for oxidoreductase catalysis in organic solvents.

The major stumbling block in the implementation of oxidoreductase enzymes in continuous processes is their stark dependence on costly cofactors that are insoluble in organic solvents. We describe a chemical strategy that allows producing nanobiocatalysts, based on an oxidoreductase enzyme, that performs biocatalytic reactions in hydrophobic organic solvents without external cofactors. The chemical design relies on the use of a silica-based carrier nanoparticle, of which the porosity can be exploited to create an aqueous reservoir containing the cofactor. The nanoparticle core, possessing radial-centred pore channels, serves as a cofactor reservoir. It is further covered with a layer of reduced porosity. This layer serves as a support for the immobilisation of the selected enzyme yet allowing the diffusion of the cofactor from the nanoparticle core. The immobilised enzyme is, in turn, shielded by an organosilica layer of controlled thickness fully covering the enzyme. Such produced nanobiocatalysts are shown to catalyse the reduction of a series of relevant ketones into the corresponding secondary alcohols, also in a continuous flow fashion.

Transforming an esterase into an enantioselective catecholase through bioconjugation of a versatile metal-chelating inhibitor.

Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.

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.

Plasmonic photothermal activation of an organosilica shielded cold-adapted lipase co-immobilised with gold nanoparticles on silica particles.

Gold nanoparticles (AuNPs), owing to their intrinsic plasmonic properties, are widely used in applications ranging from nanotechnology and nanomedicine to catalysis and bioimaging. Capitalising on the ability of AuNPs to generate nanoscale heat upon optical excitation, we designed a nanobiocatalyst with enhanced cryophilic properties. It consists of gold nanoparticles and enzyme molecules, co-immobilised onto a silica scaffold, and shielded within a nanometre-thin organosilica layer. To produce such a hybrid system, we developed and optimized a synthetic method allowing efficient AuNP covalent immobilisation on the surface of silica particles (SPs). Our procedure allows to reach a dense and homogeneous AuNP surface coverage. After enzyme co-immobilisation, a nanometre-thin organosilica layer was grown on the surface of the SPs. This layer was designed to fulfil the dual function of protecting the enzyme from the surrounding environment and allowing the confinement, at the nanometre scale, of the heat diffusing from the AuNPs after surface plasmon resonance photothermal activation. To establish this proof of concept, we used an industrially relevant lipase enzyme, namely Lipase B from Candida Antarctica (CalB). Herein, we demonstrate the possibility to photothermally activate the so-engineered enzymes at temperatures as low as -10 °C.

Immobilisation and stabilisation of glycosylated enzymes on boronic acid-functionalised silica nanoparticles.

We report a method of glycosylated enzymes' surface immobilisation and stabilisation. The enzyme is immobilised at the surface of silica nanoparticles through the reversible covalent binding of vicinal diols of the enzyme glycans with a surface-attached boronate derivative. A soft organosilica layer of controlled thickness is grown at the silica surface, entrapping the enzyme and thus avoiding enzyme leaching. We demonstrate that this approach results not only in high and durable activity retention but also enzyme stabilisation.

Coordination-Driven Monolayer-to-Bilayer Transition in Two-Dimensional Metal-Organic Networks.

We report on monolayer-to-bilayer transitions in 2D metal-organic networks (MONs) from amphiphiles supported at the water-air interface. Functionalized calix[4]arenes are assembled through the coordination of selected transition metal ions to yield monomolecular 2D crystalline layers. In the presence of Ni(II) ions, interfacial self-assembly and coordination yields stable monolayers. Cu(II) promotes 2D coordination of a monolayer which is then diffusively reorganizing, nucleates, and grows a progressive amount of second layer islands. Atomic force microscopic data of these layers after transfer onto solid substrates reveal crystalline packing geometries with submolecular resolution as they are varying in function of the building blocks and the kinetics of the assembly. We assign this monolayer-to-bilayer transition to a diffusive reorganization of the initial monolayers owing to chemical vacancies of the predominant coordination motif formed by Cu2+ ions. Our results introduce a new dimension into the controlled monolayer-to-multilayer architecturing of 2D metal-organic networks.

Tuning the Properties of Natural Promiscuous Enzymes by Engineering Their Nano-environment.

Owing to their outstanding catalytic properties, enzymes represent powerful tools for carrying out a wide range of (bio)chemical transformations with high proficiency. In this context, enzymes with high biocatalytic promiscuity are somewhat neglected. Here, we demonstrate that a meticulous modification of a synthetic shell that surrounds an immobilized enzyme possessing broad substrate specificity allows the resulting nanobiocatalyst to be endowed with enantioselective properties while maintaining a high level of substrate promiscuity. Our results show that control of the enzyme nano-environment enables tuning of both substrate specificity and enantioselectivity. Further, we demonstrate that our strategy of enzyme supramolecular engineering allows the enzyme to be endowed with markedly enhanced stability in an organic solvent (i.e., acetonitrile). The versatility of the method was assessed with two additional substrate-promiscuous and structurally different enzymes, for which improvements in enantioselectivity and stability were confirmed. We expect this method to promote the use of supramolecularly engineered promiscuous enzymes in industrially relevant biocatalytic processes.

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.

A proteolytic nanobiocatalyst with built-in disulphide reducing properties.

We report a method to equip proteolytic nanobiocatalysts with intrinsic disulphide bond reducing properties. After immobilisation onto silica particles, selected protease enzymes are partially shielded in a nanometre-thick mercaptosilica layer acting not only as a protective system but also as a substrate reducing agent. The biocatalysts produced efficiently perform simultaneous disulphide bond reduction and protein digestion. Besides a significant simplification of the proteolysis process, this strategy allows for a drastic increase of the enzyme stability.

Hydrophobicity-responsive engineered mesoporous silica nanoparticles: application in the delivery of essential nutrients to bacteria combating oil spills.

Facile chemical modification of mesoporous silica particles allows the production of gated reservoir systems capable of hydrophobicity-triggered release. Applied to the delivery of nutrients specifically to an oil phase, the systems developed have been shown to reliably assist the bacterial degradation of hydrocarbons. The gated system developed, made of C18 hydrocarbon chains, is demonstrated to be in a closed collapsed state in an aqueous environment, yet opens up through solvation by lipophilic alkanes and releases its content on contact with the oil phase.

Supramolecular architectures of molecularly thin yet robust free-standing layers.

Stable, single-nanometer thin, and free-standing two-dimensional layers with controlled molecular architectures are desired for several applications ranging from (opto-)electronic devices to nanoparticle and single-biomolecule characterization. It is, however, challenging to construct these stable single molecular layers via self-assembly, as the cohesion of those systems is ensured only by in-plane bonds. We herein demonstrate that relatively weak noncovalent bonds of limited directionality such as dipole-dipole (-CN⋅⋅⋅NC-) interactions act in a synergistic fashion to stabilize crystalline monomolecular layers of tetrafunctional calixarenes. The monolayers produced, demonstrated to be free-standing, display a well-defined atomic structure on the single-nanometer scale and are robust under a wide range of conditions including photon and electron radiation. This work opens up new avenues for the fabrication of robust, single-component, and free-standing layers via bottom-up self-assembly.

Decoding the ocean's microbiological secrets for marine enzyme biodiscovery.

A global census of marine microbial life has been underway over the past several decades. During this period, there have been scientific breakthroughs in estimating microbial diversity and understanding microbial functioning and ecology. It is estimated that the ocean, covering 71% of the earth's surface with its estimated volume of about 2 × 1018 m3 and an average depth of 3800 m, hosts the largest population of microbes on Earth. More than 2 million eukaryotic and prokaryotic species are thought to thrive both in the ocean and on its surface. Prokaryotic cell abundances can reach densities of up to 1012 cells per millilitre, exceeding eukaryotic densities of around 106 cells per millilitre of seawater. Besides their large numbers and abundance, marine microbial assemblages and their organic catalysts (enzymes) have a largely underestimated value for their use in the development of industrial products and processes. In this perspective article, we identified critical gaps in knowledge and technology to fast-track this development. We provided a general overview of the presumptive microbial assemblages in oceans, and an estimation of what is known and the enzymes that have been currently retrieved. We also discussed recent advances made in this area by the collaborative European Horizon 2020 project 'INMARE'.

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.

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.

FMNH2-dependent monooxygenases initiate catabolism of sulfonamides in Microbacterium sp. strain BR1 subsisting on sulfonamide antibiotics.

We report a cluster of genes encoding two monooxygenases (SadA and SadB) and one FMN reductase (SadC) that enable Microbacterium sp. strain BR1 and other Actinomycetes to inactivate sulfonamide antibiotics. Our results show that SadA and SadC are responsible for the initial attack of sulfonamide molecules resulting in the release of 4-aminophenol. The latter is further transformed into 1,2,4-trihydroxybenzene by SadB and SadC prior to mineralization and concomitant production of biomass. As the degradation products lack antibiotic activity, the presence of SadA will result in an alleviated bacteriostatic effect of sulfonamides. In addition to the relief from antibiotic stress this bacterium gains access to an additional carbon source when this gene cluster is expressed. As degradation of sulfonamides was also observed when Microbacterium sp. strain BR1 was grown on artificial urine medium, colonization with such strains may impede common sulfonamide treatment during co-infections with pathogens of the urinary tract. This case of biodegradation exemplifies the evolving catabolic capacity of bacteria, given that sulfonamide bacteriostatic are purely of synthetic origin. The wide distribution of this cluster in Actinomycetes and the presence of traA encoding a relaxase in its vicinity suggest that this cluster is mobile and that is rather alarming.

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.

Enzyme Armoring by an Organosilica Layer: Synthesis and Characterization of Hybrid Organic/Inorganic Nanobiocatalysts.

The availability of highly stable and reusable enzymes is one of the main challenges in bio-based industrial processes. Enzyme immobilization and encapsulation represent promising strategies to reach this goal. In this chapter, the synthetic strategy to produce hybrid organic/inorganic nanobiocatalysts (NBC) is reported. This strategy is based on the sequential immobilization of an enzyme on the surface of silica nanoparticles followed by the growth, at the surface of the nanoparticles, of a shielding layer which serves as an armor to protect the enzyme against denaturation/degradation. This armor is produced through a thickness-controlled organosilane poly-condensation onto the nanoparticle surface around the enzyme to form a protective organosilica layer. The armored nanobiocatalysts present enhanced catalytic activity and improved stability against heat, pH, chaotropic agents, proteases, and ultrasound. The method is versatile in that it can be successfully adapted to a number of different enzymes.