Biological Catalysts Research Articles

Two fluorinases prioritized from protein families of fluorinase, SAM-dependent chlorinase and hydroxide adenosyltransferase.

Fluorinases represent the only known biological catalysts capable of forming carbon-fluorine bonds, but their slow catalytic rate limits their broader application. In this study, two fluorinases, FlASbac and FlAPbac, were identified from a pool of 12 718 nonredundant proteins using a genome-mining approach, with FlASbac showing high catalytic activity. Both newly identified fluorinases contain a Phe50 residue in place of the Trp50 typically found in fluorinases. Structural and mutagenesis studies revealed that the Trp50 or Phe50 residue at this position is crucial for fluorinase activity. This work highlights the utility of genomic enzymology in expanding the repertoire of biocatalysts for fluorination chemistry.

Exploring the conformational space of the mobile flap in Sporosarcina pasteurii urease by cryo-electron microscopy.

To fully understand enzymatic dynamics, it is essential to explore the complete conformational space of a biological catalyst. The catalytic mechanism of the nickel-dependent urease, the most efficient enzyme known, holds significant relevance for medical, pharmaceutical, and agro-environmental applications. A critical aspect of urease function is the conformational change of a helix-turn-helix motif that covers the active site cavity, known as the mobile flap. This motif has been observed in either an open or a closed conformation through X-ray crystallography studies and has been proposed to stabilize the coordination of a urea molecule to the essential dinuclear Ni(II) cluster in the active site, a requisite for subsequent substrate hydrolysis. This study employs cryo-electron microscopy (cryo-EM) to investigate the transient states within the conformational space of the mobile flap, devoid of the possible constraints of crystallization conditions and solid-state effects. By comparing two cryo-EM structures of Sporosarcina pasteurii urease, one in its native form and the other inhibited by N-(n-butyl) phosphoric triamide (NBPTO), we have unprecedently identified an intermediate state between the open and the catalytically efficient closed conformation of the helix-turn-helix motif, suggesting a role of its tip region in this transition between the two states.

Highly specific colorimetric detection of sarcosine using surface molecular imprinted Zn/Ce-ZIF

Despite significant progress in nanozyme research and the advancement of analytical techniques, the inherent lack of specificity for target analytes often limits their utility in analysis. Integrating specific recognition capabilities into inorganic nanomaterials, independent of biological catalysts or adaptors, represents a crucial breakthrough in the field. Detecting Sarcosine (Sar) in human urine has recently emerged as a non-invasive biomarker for prostate cancer (PCa), presenting a valuable diagnostic tool. This study introduces a novel method for embedding molecular imprinting sites directly onto the surface of a Zn/Ce-based zeolitic imidazolate framework (Zn/Ce-ZIF) nanozyme, facilitating the development of a highly specific colorimetric assay for precise Sar measurement. By utilizing the lanthanide metal cerium as the catalytic element and ZIF-8 as the structural scaffold, we synthesized spherical Zn/Ce-ZIF nanozymes with exceptional oxidase-like catalytic efficiency. The efficiency of molecular imprinting experiments and the ability of molecularly imprinted polymers (MIPs) to identify target molecules were significantly enhanced by using theortical calculations to screen suitable functional monomers. The molecularly imprinted nanozyme (Zn/Ce-ZIF@MIP) initiates a colorimetric oxidation reaction of 3,3′,5,5′-tetramethylbenzidine (TMB), wherein the presence of Sar facilitates selective recognition and capture by the MIP shell, modulating the colorimetric response by hindering TMB’s access to the catalytic site. An intelligent color extraction detection device has been developed for the rapid perception of Sar. This colorimetric sensing platform has been validated through the detection of Sar in simulated urine samples. Overall, the application of surface molecular imprinting enhances the functionality of nanozymes in analytical fields.

Bioelectrocatalytic carbon dioxide reduction by an engineered formate dehydrogenase from Thermoanaerobacter kivui

Electrocatalytic carbon dioxide (CO2) reduction by CO2 reductases is a promising approach for biomanufacturing. Among all known biological or chemical catalysts, hydrogen-dependent carbon dioxide reductase from Thermoanaerobacter kivui (TkHDCR) possesses the highest activity toward CO2 reduction. Herein, we engineer TkHDCR to generate an electro-responsive carbon dioxide reductase considering the safety and convenience. To achieve this purpose, a recombinant Escherichia coli TkHDCR overexpression system is established. The formate dehydrogenase is obtained via subunit truncation and rational design, which enables direct electron transfer (DET)-type bioelectrocatalysis with a near-zero overpotential. By applying a constant voltage of −500 mV (vs. SHE) to a mediated electrolytic cell, 22.8 ± 1.6 mM formate is synthesized in 16 h with an average production rate of 7.1 ± 0.5 μmol h−1cm−2, a Faradaic efficiency of 98.9% and a half-cell energy efficiency of 94.4%. This study provides an enzyme candidate for high efficient CO2 reduction and opens up a way to develop paradigm for CO2-based bio-manufacturing.

Electric Field-Induced Sequential Prototropic Tautomerism in Enzyme-like Nanopocket Created by Single Molecular Break Junction.

Mastering the control of external stimuli-induced chemical transformations with detailed insights into the mechanistic pathway is the key for developing efficient synthetic strategies and designing functional molecular systems. Enzymes, the most potent biological catalysts, efficiently utilize their built-in electric field to catalyze and control complex chemical reactions within the active site. Herein, we have demonstrated the interfacial electric field-induced prototropic tautomerization reaction in acylhydrazone entities by creating an enzymatic-like nanopocket within the atomically sharp gold electrodes using a mechanically controlled break junction (MCBJ) technique. In addition to that, the molecular system used here contains two coupled acylhydrazone reaction centers, hence demonstrating a cooperative stepwise electric field-induced reaction realized at the single molecular level. Furthermore, the mechanistic studies revealed a proton relay-assisted tautomerization showing the importance of external factors such as solvent in such electric field-driven reactions. Finally, single-molecule charge transport and energetics calculations of different molecular species at various applied electric fields using a polarizable continuum solvent model confirm and support our experimental findings. Thus, this study demonstrates that mimicking an enzymatic pocket using a single molecular junction's interfacial electric field as a trigger for chemical reactions can open new avenues to the field of synthetic chemistry.

Водорозчинні вітаміни, їхнє значення в обмінних процесах, рості та розвитку дітей різного віку

Vitamins are highly biologically active low-molecular compounds of different chemical nature, with different compositions, structures, and physicochemical properties. In contrast to proteins, fats, and carbohydrates, which are macronutrients, vitamins belong to micronutrients, which have high biological activity, although they do not function as an energy source or plastic material. Aim - to raise awareness of the biological and clinical effects of water-soluble vitamins, their importance in metabolic processes, growth and development of children. Vitamins are classified according to their physical and chemical characteristics as water-soluble and fat-soluble. Water-soluble vitamins include C, H, P, B12, B9, B6, B5, B3, B2, B1. Water-soluble vitamins perform the function of biological catalysts of various metabolic processes, growth and development of children, regulate most of the vital functions of the child's body, necessary for the balanced work of all organs and systems. Water-soluble vitamins do not accumulate in the body, so their concentration in tissues depends on consumption with diet. Water-soluble vitamins are extremely necessary for the growth and development, health of children of all ages. Water-soluble vitamins are extremely necessary for maintaining healthy skin and mucous membranes, ensuring adequate hematopoiesis, and the normal functioning of the cardiovascular, nervous, endocrine, and digestive systems. Conclusions. Water-soluble vitamins are essential exogenous alimentary factors that should be steadily supplied to the child's body with food in doses in accordance with physiological needs. All vital processes in the child's body take place with the direct participation of water-soluble vitamins. Water-soluble vitamins regulate the function of various tissues, organs and systems, activating a large number of biological ones, and contribute to the body's resistance to external factors. Screening for disorders of the metabolism of water-soluble vitamins and their correction in children can improve health and increase the quality of life.

Mechanism of Action of Formate Dehydrogenases.

The molybdenum- and tungsten-containing formate dehydrogenases from a variety of microorganisms catalyze the reversible interconversion of formate and CO2; several, in fact, function as CO2 reductases in the reverse direction under physiological conditions. CO2 reduction catalyzed by these enzymes occurs under mild temperature and pressure rather than the elevated conditions required for current industrial processes. Given the contemporary importance of remediation of atmospheric CO2 to address global warming, there has been considerable interest in the application of these enzymes in bioreactors. Equally important, understanding the detailed means by which these biological catalysts convert CO2 to formate, a useful and easily transported feedstock chemical, might also inspire the development of a new generation of highly efficient, biomimetic synthetic catalysts. Here we have examined the ability of the FdsDABG formate dehydrogenase from Cupriavidus necator to catalyze the exchange of solvent oxygen into product CO2 during the course of formate oxidation under single-turnover conditions. Negligible incorporation of 18O is observed when the experiment is performed in H218O, indicating that bicarbonate cannot be the immediate product of the enzyme-catalyzed reaction, as previously concluded. These results, in conjunction with the observation that the reductive half-reaction exhibits mildly acid-catalyzed rather than base-catalyzed chemistry, are consistent with a reaction mechanism involving direct hydride transfer from formate to the enzyme's molybdenum center, directly yielding CO2. Our results are inconsistent with any mechanism in which the initial product formed on oxidation of formate is bicarbonate.

ОСОБЛИВОСТІ ВИКОРИСТАННЯ ФЕРМЕНТІВ У ХАРЧОВИХ ТЕХНОЛОГІЯХ ДЛЯ ПІДВИЩЕННЯ ЕФЕКТИВНОСТІ ВИРОБНИЦТВА

In the modern food industry, various food additives are actively used to improve the quality of the finished product. Enzymes are extremely effective biological catalysts of protein nature, which, acting in a strictly defined sequence, increase the speed of chemical reactions hundreds of times. The use of enzymes has now become the most important industrial principle of food technology improvement. The use of enzymes in food technologies is at the stage of active development, their range and the functions they can perform in food systems are expanding. The effectiveness of the introduction of enzymes as technological additives-improvers is beyond doubt. The article is devoted to highlighting the peculiarities of the introduction of enzymes into food products of the bakery, confectionery, sugar, meat, alcohol, starch and molasses industries, etc. It is shown that the use of enzymes makes it possible to intensify technological processes, increase the yield and quality of products. The main functions of enzymes in food technology are acceleration of biochemical processes, improvement of organoleptic properties of products, increase of yield of beverages, meat, bread and confectionery products, increase of extractability of beverages, intensification of deep hydrolysis of starch and proteins and milk sugar - lactose, prevention of oxidation processes and development aerobic microorganisms, enrichment of valuable nitrogenous nutrition of yeast wort; combating non-biological clouding of beer, adjusting the technological characteristics of flour, strengthening the gluten framework and improving plasticity in frozen dough, ensuring the rheological characteristics of puff pastry and the necessary consistency at high sugar concentrations in fondant candies, caramel, praline, etc. It was concluded that the achievement of high product quality, high product yield and intensification of technological processes, which directly affects the production efficiency, are practically impossible without the use of technological additives - enzymes.

Biological Waste-Derived Dual-Site Catalyst Empowers Electro-Fenton Systems to Sustainably Decontaminate Livestock Wastewater

Biological Waste-Derived Dual-Site Catalyst Empowers Electro-Fenton Systems to Sustainably Decontaminate Livestock Wastewater

Homogeneous HER electrocatalysis using monothiolate ligand-based {FeS} complexes: A review

Renewable energy sources are considered as efficient, low cost and sustainable energy systems with the capability of achieving a decarbonized world. Of particular interest has been the use of hydrogen as an affordable and clean energy for achieving a sustainable (renewable) energy economy. The hydrogenases discovered almost 90 years ago and found in green algae and anaerobic bacteria, are known to be highly efficient biological catalysts for hydrogen evolution in nature. Out of the three types of hydrogenases ([FeFe], [NiFe] and [Fe] only) the [FeFe] hydrogenases have drawn particular attention due to their higher activity/efficiency and their ability to reversibly reduce protons to dihydrogen. Several models of the [FeFe] hydrogenase enzymes have been reported over the last three decades. This review article mainly describes in details the characteristics of mono- and di-nuclear {FeS} complexes with monothiolates (-SR) as the bridging ligands that have been reported as electrocatalysts for the hydrogen evolution reaction (HER).

Production and characterization of acidophilic xylanase from wood degrading white rot fungus by solid-state fermentation of wheat straw

Xylanases (EC 3.2.1.8) catalyze the breakdown of xylan, which is the second most abundant polysaccharide in plant cell walls. Biological catalysts have gained greater global attention than chemical catalysts in different industrial processes because they are highly selective, easy to control and have a negligible environmental impact. The aim of this study was to investigate the xylanolytic potential of white-rot fungi, optimize their physicochemical conditions and characterize the resulting xylanase. Sixty-eight white-rot fungus (WRF) isolates were screened for their xylanolytic potential and growth conditions for maximal xylanase production using cheap agricultural residue (wheat straw) as the sole carbon source. Five WRF isolates with high xylanase yields (73.63 ± 0.0283–63.6 ± 0.01247 U/ml) were selected by qualitative and quantitative screening methods. The optimum xylanase production occurred at pH 5.0 and 28 °C. Solid-state fermentation (SSF) yielded a high amount of xylanase. The highest xylanase activity (80.9–61.274 U/mL) was recorded in the pH range of 5.0–6.5 and at 50 °C. The metal ions Mg2+, Ca2+ and Mn2+ enhanced the activity of xylanase (127.28–110.06 %), while Cu2+, Fe2+ and K+ inhibited the activity with 43.4–17 % losses. The km and Vmax were 0.32–0.545 mg/mL and 86.95–113.63 μmol/min/mg, respectively. This finding indicates that wheat straw can be used for large-scale xylanase production under SSF conditions. The pH and temperature profiles and stabilities indicate that the xylanase produced in the present study can be applied in food and animal feed industries.

Screening for the economic production of hydrolytic enzymes from locally-isolated fungi

Background Enzymes are complex proteins serving as biological catalysts to facilitate reactions in mild and environment-friendly conditions. Saprophytic fungi have long been harnessed for the efficient production of several industrially-significant enzymes whose market is still growing to cope with the increase in demand and natural resources’ depletion. Objective This investigation was performed with respect to the economic viewpoint of terrestrial fungi utilization and their hydrolytic enzymes’ biosynthetic potential. Materials and methods Several terrestrial fungi were isolated, cultivated on cheap agricultural wastes, and evaluated for industrial relevance. Solid-state fermentation was conducted to further boost the economic value and sustainability. The enzymatic productivity was estimated through solid-phase radial diffusion correlating the zones’ diameters to the enzymatic activity. Results and conclusion Six soil fungi were isolated, five belonging to the order Eurotiales and one to Mucorales. The molds belonged to four different genera; Aspergillus sydowii, Aspergillus versicolor, Aspergillus ustus, Fennelia flavipes (anamorph: Aspergillus flavipes), Cunninghamella elegans and Paecilomyces lilacinus. Many of the tested agricultural wastes were able to support the biosynthesis of the explored constitutive enzymes, recording better activity than the standard synthetic medium. Under the test conditions, L-asparaginase and protease were the most frequently detected enzymes while banana and mandarine peels led to the highest enzymes’ activity. In light of the global direction towards sustainability, enzymes can have immense prospects to sustain the industrial sectors innocuously. The cost-effectiveness of the manufacturing processes can be enhanced by accommodating the fiscal challenges for operating conditions. Using agrarian residues as raw material, highly productive enzyme producers, and cheaper solid-state fermentation processes are factors that may contribute to the efficacy, efficiency and economic feasibility of the enzyme-based processes.

Identifying Oncogenic Missense Single Nucleotide Polymorphisms in Human SAT1 Gene Using Computational Algorithms and Molecular Dynamics Tools.

The human SAT1 gene encodes spermidine/spermine N1-acetyltransferase 1 (SSAT1), a regulatory biological catalyst of polyamine catabolism. Numerous essential biological processes, such as cellular proliferation, differentiation, and survival, depend on polyamines like spermidine and spermine. Thus, SSAT1 is involved in key cellular activities such as proliferation and survival of cells and mediates various diseases including cancer. A plethora of studies established the involvement of missense single nucleotide polymorphisms (SNPs) in numerous pathological conditions due to their ability to adversely affect the structure and subsequent function of the protein. To date, an in silico study to identify the pathogenic missense SNPs of the human SAT1 gene has not been accomplished yet. This study aimed to filter the missense SNPs that were functionally detrimental and pathogenic. The rs757435207 (I21N) was ascertained to be the most deleterious and pathogenic by all algorithmic tools. Stability and evolutionary conservation analysis tools also stated that I21N variant decreased the stability and was located in the highly conserved residue. Molecular dynamics simulation revealed that I21N caused substantial alterations in the conformational stability and dynamics of the SSAT1 protein. Consequently, the I21N variant could disrupt the native functional roles of the SSAT1 enzyme. Therefore, the I21N variant was identified and concluded to be an oncogenic missense variant of the human SAT1 gene. Overall, the findings of this study would be a great directory of future experimental research to develop personalized medicine.

Pickering Emulsions Biocatalysis: Recent Developments and Emerging Trends.

Biocatalysis within biphasic systems is gaining significant attention in the field of synthetic chemistry, primarily for its ability to solve the problem of incompatible solubilities between biocatalysts and organic compounds. By forming an emulsion from these two-phase systems, a larger surface area is created, which greatly improves the mass transfer of substrates to the biocatalysts. Among the various types of emulsions, Pickering emulsions stand out due to their excellent stability, compatibility with biological substances, and the ease with which they can be formed and separated. This makes them ideal for reusing both the emulsifiers and the biocatalysts. This review explores the latest developments in biocatalysis using Pickering emulsions. It covers the structural features, methods of creation, innovations in flow biocatalysis, and the role of interfaces in these processes. Additionally, the challenges and future directions are discussed in combining chemical and biological catalysts within Pickering emulsion frameworks to advance synthetic methodologies.

Selection of a promiscuous minimalist cAMP phosphodiesterase from a library of de novo designed proteins

The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To probe how unevolved sequences can develop new functions, we use ultrahigh-throughput droplet microfluidics to screen for phosphoesterase activity amidst a library of more than one million sequences based on a de novo designed 4-helix bundle. Characterization of hits revealed that acquisition of function involved a large jump in sequence space enriching for truncations that removed >40% of the protein chain. Biophysical characterization of a catalytically active truncated protein revealed that it dimerizes into an α-helical structure, with the gain of function accompanied by increased structural dynamics. The identified phosphodiesterase is a manganese-dependent metalloenzyme that hydrolyses a range of phosphodiesters. It is most active towards cyclic AMP, with a rate acceleration of ~109 and a catalytic proficiency of >1014 M−1, comparable to larger enzymes shaped by billions of years of evolution.

Eutectogels as Promising Materials in Biocatalysis

AbstractThe entrapment of deep eutectic solvents (DES) and eutectic systems into porous scaffolds renders a new class of soft and nonvolatile materials called eutectogels that have recently stepped into the spotlight in different areas ranging from electronics to drug delivery. Recent progress in the use of DES in biocatalysis, where they have been demonstrated to improve substrate supply, conversion, and enzyme stability, has opened an unparalleled opportunity to exploit the merits of eutectogels for immobilizing biological catalysts. The resulting functional materials could outperform traditional hydrogels and ionic liquid gels, offering fresh perspectives to broaden the application scope of many enzymes. In this perspective, we go into the potential of eutectogels as innovative scaffolds that support biocatalytic reactions and discuss different applications where these systems could show plain benefits compared to traditional materials. Future directions for this newly developed technology are highlighted.

Monitoring Enzyme Activity Using Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

Enzymes serve as pivotal biological catalysts that accelerate essential chemical reactions, thereby influencing a variety of physiological processes. Consequently, the monitoring of enzyme activity and inhibition not only yields crucial insights into health and disease conditions but also forms the basis of research in drug discovery, toxicology, and the understanding of disease mechanisms. In this context, near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) have emerged as effective tools for tracking enzyme activity and inhibition through diverse strategies. This perspective explores the physicochemical attributes of SWCNTs that render them well-suited for such monitoring. Additionally, we delve into the various strategies developed so far for successfully monitoring enzyme activity and inhibition, emphasizing the distinctive features of each principle. Furthermore, we contrast the benefits of SWCNT-based NIR probes with conventional gold standards in monitoring enzyme activity. Lastly, we highlight the current challenges faced in this field and suggest potential solutions to propel it forward. This perspective aims to contribute to the ongoing progress in biodiagnostics and seeks to engage the wider community in developing and applying enzymatic assays using SWCNTs.

The Rhodium Analogue of Coenzyme B12 as an Anti‐Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors

AbstractCoenzyme B12 (AdoCbl; 5′‐deoxy‐5′‐adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light‐responsive cobalt‐corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA‐binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl‐bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non‐natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound “base‐off/His‐on” to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti‐photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl‐based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non‐radical syn‐1,2‐elimination mechanism, an aspect not previously considered for this photoreceptor.

The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

A Potentially Versatile Enzyme Sensor Platform: Enzyme-Loaded, Tagged, Porous Polymeric Nanocapsules.

Enzymes are essential to life and indispensable in a wide range of industries (food, pharmaceutical, medical, biosensing, etc.); however, a significant shortcoming of these fragile biological catalysts is their poor stability. To address this challenge, a variety of immobilization methods have been described to enhance the enzyme's stability. These immobilization methods generally are specific to an individual enzyme or optimal for a particular application. The aim of this study is to explore the utility of porous, indicator moiety-tagged, polymeric nanocapsules (NCs) for the encapsulation of enzymes and measurement of the enzyme's substrate. As a model enzyme, glucose oxidase (GOx) is used. The GOx enzyme-loaded, fluorophore-tagged NCs were synthesized by using self-assembled surfactant vesicle templates. To show that the biological activity of GOx is preserved during entrapment, the rate of the GOx enzyme catalyzed reaction was measured. To evaluate the protective features of the porous NCs, the encapsulated GOx enzyme activity was followed in the presence of hydrolytic enzymes. During the encapsulation of GOx and the purification of the GOx-loaded NCs, the GOx activity decayed less than 10%, and up to 30% of the encapsulated GOx activity could be retained for 3-5 days in the presence of hydrolytic enzymes. In support of the potentially unique advantages of the enzyme-loaded NCs, as a proof-of-concept example, the fluorophore-tagged, GOx-loaded NCs were used for the determination of glucose in the concentration range between 18 and 162 mg/dL and for imaging the distribution of glucose concentration in imaging experiments.