Controlled Delivery of H2O2: A Three-Enzyme Cascade Flow Reactor for Peroxidase-Catalyzed Reactions.

Peroxidases are promising catalysts for oxidation reactions, yet their practical utility has been hindered by the fact that they require hydrogen peroxide (H2O2), which at high concentrations can cause deactivation of enzymes. Practical processes involving the use of peroxidases require the frequent addition of low concentrations of H2O2. In situ generation of H2O2 can be achieved using oxidase-type enzymes. In this study, a three-enzyme cascade system comprised of a H2O2 generator (glucose oxidase (GOx)), H2O2-dependent enzymes (chloroperoxidase (CPO) or horseradish peroxidase (HRP)), and a H2O2 scavenger (catalase (CAT)) was deployed in a flow reactor. Immobilization of the enzymes on a graphite rod was achieved through electrochemically driven physical adsorption, followed by cross-linking with glutaraldehyde. Modeling studies indicated that the flow in the reactor was laminar (Reynolds number, R e < 2000) and was nearly fully developed at the midplane of the annular reactor. Immobilized CAT and GOx displayed good stability, retaining 79% and 84% of their initial activity, respectively, after three cycles of operation. Conversely, immobilized CPO exhibited a considerable reduction in activity after one use, retaining only 30% of its initial activity. The GOx-CAT-GRE system enabled controlled delivery of H2O2 in a more stable manner with a 4-fold enhancement in the oxidation of indole compared to the direct addition of H2O2. Using CPO in solution coupled with GOx-CAT-GRE yields of 90% for the oxidation of indole to 2-oxyindole and of 93% and 91% for the chlorination of thymol and carvacrol, respectively.

Combining high energy ball milling and liquid crystal templating method to prepare magnetic ordered mesoporous silica. A physico-chemical investigation.

The physico-chemical investigation of superparamagnetic MCM41 like materials prepared by the novel combination of high energy ball milling and a liquid crystal templating method is presented. Structural, morphological, textural, thermal, and preliminary magnetic characterization demonstrated the successful combination of the two synthesis techniques, avoiding the problems associated with the current methods used for the preparation of magnetic ordered mesoporous silica. MCM41 like materials with high specific surface area values (625-720 m2 g-1) and high mesopore volumes in the range 1-0.7 cm3 g-1 were obtained. The ordered mesoporous structure and accessible pores were maintained after the inclusion of increasing amounts of the magnetic component in the silica structure. All the samples showed superparamagnetic behaviour.

Electrochemical Immobilisation of Glucose Oxidase for the Controlled Production of H2O2 in a Biocatalytic Flow Reactor.

Electrochemical methods can be used to selectively modify the surfaces of electrodes, enabling the immobilisation of enzymes on defined areas on the surfaces of electrodes. Such selective immobilisation methods can be used to pattern catalysts on surfaces in a controlled manner. Using this approach, the selective patterning of the enzyme glucose oxidase on the electrodes was used to develop a flow reactor for the controlled delivery of the oxidant H2O2. GOx was immobilised on a glassy carbon electrode using polypyrrole, silica films, and diazonium linkers. The rate of production of H2O2 and the stability of the response was dependent on the immobilisation method. GOx encapsulated in polypyrrole was selected as the optimal method of immobilisation, with a rate of production of 91±11 µM h-1 for 4 hours of continuous operation. The enzyme was subsequently immobilised on carbon rod electrodes (surface area of 5.76 cm2) using a polypyrrole/Nafion® film and incorporated into a flow reactor. The rate of production of H2O2 was 602±57 µM h-1, with 100 % retention of activity after 7 h of continuous operation, demonstrating that such a system can be used to prepare H2O2 at continuous and stable rate for use in downstream oxidation reactions.

Enzyme Immobilization on Metal Organic Frameworks: the Effect of Buffer on the Stability of the Support.

Metal organic frameworks (MOFs) have been used to encapsulate an array of enzymes in a rapid and facile manner; however, the stability of MOFs as supports for enzymes has not been examined in detail. This study examines the stability of MOFs with different compositions (Fe-BTC, Co-TMA, Ni-TMA, Cu-TMA, and ZIF-zni) in buffered solutions commonly used in enzyme immobilization and biocatalysis. Stability was assessed via quantification of the release of metals by inductively coupled plasma optical emission spectroscopy. The buffers used had varied effects on different MOF supports, with incubation of all MOFs in buffers resulting in the release of metal ions to varying extents. Fe-BTC was completely dissolved in citrate, a buffer that has a profound destabilizing effect on all MOFs analyzed, precluding its use with MOFs. MOFs were more stable in acetate, potassium phosphate, and Tris HCl buffers. The results obtained provide a guide for the selection of an appropriate buffer with a particular MOF as a support for the immobilization of an enzyme. In addition, these results identify the requirement to develop methods of improving the stability of MOFs in aqueous solutions. The use of polymer coatings was evaluated with polyacrylic acid (PAA) providing an improved level of stability. Lipase was immobilized in Fe-BTC with PAA coating, resulting in a stable biocatalyst with retention of activity in comparison to the free enzyme.

Expression, Purification, and in vitro Enzyme Activity Assay of a Recombinant Aldehyde Dehydrogenase from Thermus thermophilus, Using an Escherichia coli Host.

Based on previous in-depth characterisation, aldehyde dehydrogenases (ALDH) are a diverse superfamily of enzymes, in terms of both structure and function, present in all kingdoms of life. They catalyse the oxidation of an aldehyde to carboxylic acid using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)+), and are often not substrate-specific, but rather have a broad range of associated biological functions, including detoxification and biosynthesis. We studied the structure of ALDHTt from Thermus thermophilus, as well as performed its biochemical characterisation. This allowed for insight into its potential substrates and biological roles. In this protocol, we describe ALDHTt heterologous expression in E. coli, purification, and activity assay (based on Shortall et al., 2021 ). ALDHTt was first copurified as a contaminant during caa3-type cytochrome oxidase isolation from T. thermophilus. This recombinant production system was employed for structural and biochemical analysis of wild-type and mutants, and proved efficient, yielding approximately 15-20 mg/L ALDHTt. For purification of the thermophilic his-tagged ALDHTt, heat treatment, immobilized metal affinity chromatography (IMAC), and gel filtration chromatography were used. The enzyme activity assay was performed via UV-Vis spectrophotometry, monitoring the production of reduced nicotinamide adenine dinucleotide (NADH). Graphical abstract: Flow chart outlining the steps in ALDHTt expression and purification, highlighting the approximate time required for each step.

Study of ALDH from Thermus thermophilus-Expression, Purification and Characterisation of the Non-Substrate Specific, Thermophilic Enzyme Displaying Both Dehydrogenase and Esterase Activity.

Aldehyde dehydrogenases (ALDH), found in all kingdoms of life, form a superfamily of enzymes that primarily catalyse the oxidation of aldehydes to form carboxylic acid products, while utilising the cofactor NAD(P)+. Some superfamily members can also act as esterases using p-nitrophenyl esters as substrates. The ALDHTt from Thermus thermophilus was recombinantly expressed in E. coli and purified to obtain high yields (approximately 15-20 mg/L) and purity utilising an efficient heat treatment step coupled with IMAC and gel filtration chromatography. The use of the heat treatment step proved critical, in its absence decreased yield of 40% was observed. Characterisation of the thermophilic ALDHTt led to optimum enzymatic working conditions of 50 °C, and a pH of 8. ALDHTt possesses dual enzymatic activity, with the ability to act as a dehydrogenase and an esterase. ALDHTt possesses broad substrate specificity, displaying activity for a range of aldehydes, most notably hexanal and the synthetic dialdehyde, terephthalaldehyde. Interestingly, para-substituted benzaldehydes could be processed efficiently, but ortho-substitution resulted in no catalytic activity. Similarly, ALDHTt displayed activity for two different esterase substrates, p-nitrophenyl acetate and p-nitrophenyl butyrate, but with activities of 22.9% and 8.9%, respectively, compared to the activity towards hexanal.

Enzyme immobilization on metal organic frameworks: Laccase from Aspergillus sp. is better adapted to ZIF-zni rather than Fe-BTC.

Laccase from Aspergillus sp. (LC) was immobilized within Fe-BTC and ZIF-zni metal organic frameworks through a one-pot synthesis carried out under mild conditions (room temperature and aqueous solution). The Fe-BTC, ZIF-zni MOFs, and the LC@Fe-BTC, LC@ZIF-zni immobilized LC samples were characterized by X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The kinetic parameters (KM and Vmax) and the specific activity of the free and immobilized laccase were determined. Immobilized LCs resulted in a lower specific activity compared with that of the free LC (7.7 µmol min-1 mg-1). However, LC@ZIF-zni was almost 10 times more active than LC@Fe-BTC (1.32 µmol min-1 mg-1 vs 0.17 µmol min-1 mg-1) and only 5.8 times less active than free LC. The effect of enzyme loading showed that LC@Fe-BTC had an optimal loading of 45.2 mg g-1, at higher enzyme loadings the specific activity decreased. In contrast, the specific activity of LC@ZIF-zni increased linearly over the loading range investigated. The storage stability of LC@Fe-BTC was low with a significant decrease in activity after 5 days, while LC@ZIF retained up to 50% of its original activity after 30 days storage. The difference in activity and stability between LC@Fe-BTC and LC@ZIF-zni is likely due to release of Fe3+ and the low stability of Fe-BTC MOF. Together, these results indicate that ZIF-zni is a superior support for the immobilization of laccase.

Insights into Aldehyde Dehydrogenase Enzymes: A Structural Perspective.

Aldehyde dehydrogenases engage in many cellular functions, however their dysfunction resulting in accumulation of their substrates can be cytotoxic. ALDHs are responsible for the NAD(P)-dependent oxidation of aldehydes to carboxylic acids, participating in detoxification, biosynthesis, antioxidant and regulatory functions. Severe diseases, including alcohol intolerance, cancer, cardiovascular and neurological diseases, were linked to dysfunctional ALDH enzymes, relating back to key enzyme structure. An in-depth understanding of the ALDH structure-function relationship and mechanism of action is key to the understanding of associated diseases. Principal structural features 1) cofactor binding domain, 2) active site and 3) oligomerization mechanism proved critical in maintaining ALDH normal activity. Emerging research based on the combination of structural, functional and biophysical studies of bacterial and eukaryotic ALDHs contributed to the appreciation of diversity within the superfamily. Herewith, we discuss these studies and provide our interpretation for a global understanding of ALDH structure and its purpose-including correct function and role in disease. Our analysis provides a synopsis of a common structure-function relationship to bridge the gap between the highly studied human ALDHs and lesser so prokaryotic models.

High Energy Ball Milling and Liquid Crystal Template Method: A Successful Combination for the Preparation of Magnetic Nano-Platforms.

In this study, we present the preparation of superparamagnetic ordered mesoporous silica (SOMS) for biomedical applications by the combination of high energy ball milling (HEBM) and the liquid crystal template method (LCT) to produce a material comprised of room temperature superparamagnetic Fe3O4 nanoparticles in a MCM-41 like mesostructured silica. In a typical synthesis, a mixture of Fe2O3 and silica was sealed in a stainless-steel vial with steel balls. Ball milling experiments were performed in a vibratory mill apparatus. The milling process produced nanocomposites with an average size ranging from ∼100-200 nm, where the Fe3O4 nanoparticles (4.8 nm size) are homogeneously dispersed into the amorphous SiO2 matrix. The obtained nanocomposite has been used for the preparation of the SOMS through the LCT method. Structural, morphological and textural characterization were performed using X-ray powder diffraction, transmission electron microscopy and nitrogen sorption analysis. Field dependence of magnetization was investigated and showed superparamagnetic behaviour at 300 K with a value of saturation magnetization (Ms) that is of interest for biomedical applications.

Adsorption of Malachite Green and Alizarin Red S Dyes Using Fe-BTC Metal Organic Framework as Adsorbent.

Synthetic organic dyes are widely used in various industrial sectors but are also among the most harmful water pollutants. In the last decade, significant efforts have been made to develop improved materials for the removal of dyes from water, in particular, on nanostructured adsorbent materials. Metal organic frameworks (MOFs) are an attractive class of hybrid nanostructured materials with an extremely wide range of applications including adsorption. In the present work, an iron-based Fe-BTC MOF, prepared according to a rapid, aqueous-based procedure, was used as an adsorbent for the removal of alizarin red S (ARS) and malachite green (MG) dyes from water. The synthesized material was characterized in detail, while the adsorption of the dyes was monitored by UV-Vis spectroscopy. An optimal adsorption pH of 4, likely due to the establishment of favorable interactions between dyes and Fe-BTC, was found. At this pH and at a temperature of 298 K, adsorption equilibrium was reached in less than 30 min following a pseudo-second order kinetics, with k″ of 4.29 × 10-3 and 3.98 × 10-2 g∙mg-1 min-1 for ARS and MG, respectively. The adsorption isotherm followed the Langmuir model with maximal adsorption capacities of 80 mg∙g-1 (ARS) and 177 mg∙g-1 (MG), and KL of 9.30·103 L∙mg-1 (ARS) and 51.56·103 L∙mg-1 (MG).

Antimicrobial enzymatic biofuel cells.

A compact antibiotic delivery system based on enzymatic biofuel cells was prepared, in which ampicillin was released when discharged in the presence of glucose and O2. The release of ampicillin was effective in inhibiting the growth of bacterium Escherichia coli as confirmed by ex situ and in situ release studies in culture media.

Biosensors-Recent Advances and Future Challenges in Electrode Materials.

Electrochemical biosensors benefit from the simplicity, sensitivity, and rapid response of electroanalytical devices coupled with the selectivity of biorecognition molecules. The implementation of electrochemical biosensors in a clinical analysis can provide a sensitive and rapid response for the analysis of biomarkers, with the most successful being glucose sensors for diabetes patients. This review summarizes recent work on the use of structured materials such as nanoporous metals, graphene, carbon nanotubes, and ordered mesoporous carbon for biosensing applications. We also describe the use of additive manufacturing (AM) and review recent progress and challenges for the use of AM in biosensing applications.

Enzymatic Biofuel Cells for Self-Powered, Controlled Drug Release.

Self-powered drug-delivery systems based on conductive polymers (CPs) that eliminate the need for external power sources are of significant interest for use in clinical applications. Osmium redox polymer-mediated glucose/O2 enzymatic biofuel cells (EBFCs) were prepared with an additional CP-drug layer on the cathode. On discharging the EBFCs in the presence of glucose and dioxygen, model drug compounds incorporated in the CP layer were rapidly released with negligible amounts released when the EBFCs were held at open circuit. Controlled and ex situ release of three model compounds, ibuprofen (IBU), fluorescein (FLU), and 4',6-diamidino-2-phenylindole (DAPI), was achieved with this self-powered drug-release system. DAPI released in situ in cell culture media was incorporated into retinal pigment epithelium (RPE) cells. This work demonstrates a proof-of-concept responsive drug-release system that may be used in implantable devices.

Development of graphene-based enzymatic biofuel cells: A minireview.

Enzymatic biofuel cells (EBFCs) have attracted increasing attention due to their potential to harvest energy from a wide range of fuels under mild conditions. Fabrication of effective bioelectrodes is essential for the practical application of EBFCs. Graphene possesses unique physiochemical properties making it an attractive material for the construction of EBFCs. Despite these promising properties, graphene has not been used for EBFCs as frequently as carbon nanotubes, another nanoscale carbon allotrope. This review focuses on current research progress in graphene-based electrodes, including electrodes modified with graphene derivatives and graphene composites, as well as free-standing graphene electrodes. Particular features of graphene-based electrodes such as high conductivity, mechanical flexibility and high porosity for bioelectrochemical applications are highlighted. Reports on graphene-based EBFCs from the last five years are summarized, and perspectives for graphene-based EBFCs are offered.

Specific ion effects on the enzymatic activity of alcohol dehydrogenase from Saccharomyces cerevisiae.

The enzymatic activity of alcohol dehydrogenase (ADH) in the presence of a range of electrolytes is investigated. In the presence of 150 and 200 mM cations a substantial increase in activity following the series GnCl < CsCl < KCl ∼ NaCl < LiCl was observed with a 69% increase in the presence of KCl 200 mM with respect to the salt-free solution. In the presence of 150 and 200 mM anions the increase in activity followed an ion specific trend NaF ∼ NaCl ∼ NaBr > no salt > NaClO4 > NaSCN with a peak in activity increase of 75% in the presence of NaBr. The values of the Michaelis-Menten constant (Km) did not show any significant ion specific effect, while the maximum rate (Vmax) of ethanol oxidation to acetaldehyde was strongly ion specific. The changes in specific activity and Vmax in the presence of anions likely arises from ion specific interactions with charged residues in the active site of ADH. The data indicate that the enzymatic activity of alcohol dehydrogenase can be modulated by the nature of electrolytes at physiological concentration.

Tackling the Challenges of Enzymatic (Bio)Fuel Cells.

The ever-increasing demands for clean and sustainable energy sources combined with rapid advances in biointegrated portable or implantable electronic devices have stimulated intensive research activities in enzymatic (bio)fuel cells (EFCs). The use of renewable biocatalysts, the utilization of abundant green, safe, and high energy density fuels, together with the capability of working at modest and biocompatible conditions make EFCs promising as next generation alternative power sources. However, the main challenges (low energy density, relatively low power density, poor operational stability, and limited voltage output) hinder future applications of EFCs. This review aims at exploring the underlying mechanism of EFCs and providing possible practical strategies, methodologies and insights to tackle these issues. First, this review summarizes approaches in achieving high energy densities in EFCs, particularly, employing enzyme cascades for the deep/complete oxidation of fuels. Second, strategies for increasing power densities in EFCs, including increasing enzyme activities, facilitating electron transfers, employing nanomaterials, and designing more efficient enzyme-electrode interfaces, are described. The potential of EFCs/(super)capacitor combination is discussed. Third, the review evaluates a range of strategies for improving the stability of EFCs, including the use of different enzyme immobilization approaches, tuning enzyme properties, designing protective matrixes, and using microbial surface displaying enzymes. Fourth, approaches for the improvement of the cell voltage of EFCs are highlighted. Finally, future developments and a prospective on EFCs are envisioned.

Significant Enhancement of Structural Stability of the Hyperhalophilic ADH from Haloferax volcanii via Entrapment on Metal Organic Framework Support.

The use of an in situ immobilization procedure for the immobilization of hyperhalophilic alcohol dehydrogenase in a metal organic framework material is described. The easy and rapid in situ immobilization process enables retention of activity over a broad range of pH and temperature together with a decrease in the halophilicity of the enzyme. The catalytic activity of the immobilized enzyme was studied in nonaqueous solvent mixtures with the highest retention of activity in aqueous solutions of methanol and acetonitrile. The approach demonstrates that this immobilization method can be extended to hyperhalophilic enzymes with enhancements in activity and stability.

A quasi-solid-state and self-powered biosupercapacitor based on flexible nanoporous gold electrodes.

A quasi-solid-state and flexible biofuel cell using a hydrogel electrolyte preloaded with sugar as a fuel is described. The device can function as a self-powered biosupercapacitor delivering pulses for over 600 cycles, with a power density over 10 times higher than that from the biofuel cell alone.

Nanoporous Gold-Based Biofuel Cells on Contact Lenses.

A lactate/O2 enzymatic biofuel cell (EBFC) was prepared as a potential power source for wearable microelectronic devices. Mechanically stable and flexible nanoporous gold (NPG) electrodes were prepared using an electrochemical dealloying method consisting of a pre-anodization process and a subsequent electrochemical cleaning step. Bioanodes were prepared by the electrodeposition of an Os polymer and Pediococcus sp. lactate oxidase onto the NPG electrode. The electrocatalytic response to lactate could be tuned by adjusting the deposition time. Bilirubin oxidase from Myrothecium verrucaria was covalently attached to a diazonium-modified NPG surface. A flexible EBFC was prepared by placing the electrodes between two commercially available contact lenses to avoid direct contact with the eye. When tested in air-equilibrated artificial tear solutions (3 mM lactate), a maximum power density of 1.7 ± 0.1 µW cm-2 and an open-circuit voltage of 380 ± 28 mV were obtained, values slightly lower than those obtained in phosphate buffer solution (2.4 ± 0.2 µW cm-2 and 455 ± 21 mV, respectively). The decrease was mainly attributed to interference from ascorbate. After 5.5 h of operation, the EBFC retained 20% of the initial power output.