Organisms maintain protein structure and function under environmental stress conditions by accumulating small organic molecules known as osmolytes. Mixtures of osmolytes can form natural deep eutectic solvents (NADESs), which have attracted attention as environmentally friendly media for biocatalysis; however, the effect of such NADESs on large, industrial enzymes and the mechanisms underlying their thermal stabilization remain unclear. In this study, we examined the effect of a NADES that mimics the osmolyte composition of the inner medulla of the rabbit kidney (RK-NADES) on the thermal stability of tetrameric l-lactate oxidase (LOX). LOX was completely inactivated at 60 °C in buffer, whereas in RK-NADES, it retained activity up to 70 °C and was inactivated above 80 °C. The apparent half-life of LOX at elevated temperatures was extended up to 15-fold compared with its individual aqueous components. Moreover, RK-NADES suppressed thermally induced changes in the tertiary structure of LOX, which indicates a structural basis for its enhanced functional stability. Molecular dynamics simulations further supported this observation, suggesting that RK-NADES components partially replace water molecules and form stabilizing hydrogen bonds with LOX. These findings provide a simple and effective approach to stabilizing enzymes under unstable conditions using NADESs designed to mimic the osmolyte composition of living cells.

Wearable enzymatic biofuel cells (EBFCs) are promising self-powered energy sources for real-time biosensing, enabling continuous physiological monitoring without external power supplies. Here, we report aqueous enzyme ink formulations incorporating water-based binders that preserve enzymatic activity and allow direct one-step screen printing of both bioanodes and biocathodes onto paper substrates. The inks consisted of MgO-templated mesoporous carbon, specific redox mediators, target enzymes (lactate oxidase, bilirubin oxidase, or flavin adenine dinucleotide-dependent glucose dehydrogenase), and polysaccharide-based thickeners, all dispersed in aqueous binder solutions. Electrochemical characterization revealed that the printed electrodes exhibited higher catalytic current densities, improved operational stability, and reduced mediator/enzyme leaching compared to conventional drop-casting methods. A lactate/oxygen EBFC assembled using the optimized inks achieved a maximum power density of 165 mu W cm-2 with an open-circuit voltage of 0.63 V, maintaining high activity over prolonged chronoamperometric operation. Long-term storage at 5 degrees C under vacuum preserved enzymatic activity most effectively. These results demonstrate that aqueous enzyme inks enable scalable, reproducible, and high-performance EBFC fabrication, offering strong potential for integration into wearable self-powered biosensing platforms.

The symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum possess a two component regulatory system comprising FixL, a histidine kinase with O2-sensor, and FixJ, a response regulator controlling the expression of nitrogen fixation-related genes. The phosphotransfer from FixL to the N-terminal receiver domain (REC) of FixJ promote the association of the C-terminal DNA-binding domain (DBD) to DNA. To understand the structural basis of the activation, solution NMR approaches were employed to FixJ in the acetyl phosphate-mediated phosphorylated and the BeF3--bound states. The backbone resonance assignments indicated the formation of symmetric homodimer in the activated states. Chemical shift changes caused by the activation were distributed on a half surface of REC as well as on a limited region in DBD, indicating that the phosphorylation propagates to DBD. Cross-saturation experiments revealed a major dimerisation interface comprising helix α4 and strand β5, which is common to the Sinorhizobium meliloti FixJ, and additional dimerisation interfaces are located on helices α3 and α5 of REC and on helices α7 and α10 on DBD. Considering that REC and DBD tumble separately both in the unphosphorylated and the phosphorylated states, the activation of FixJ can be delineated as the transition of one “inactive” ensemble structure to another “active” ensemble.

ZIF67-NQS/CNT hybrid electrodes enabling stable mediator immobilization and efficient multi-enzyme cascade reactions exhibited enhanced current output attributed to the cascade effect. Notably, the pyruvate byproduct inhibited lactate oxidase activity, while its elimination via pyruvate decarboxylase significantly improved the current response. This strategy offers both mechanistic insights and practical advantages.

Enzyme condensates are powerful tools for controlling enzymatic reactions in living cells. Recent advances in polymer science have enabled the design of artificial enzyme condensates in vitro, providing a promising approach to enhance enzymatic activity and stability for various biotechnological applications. In this review, we describe a systematic approach to engineering enzyme condensates through polymer-based strategies. First, we consider the design principles for tailoring the state of the enzyme condensates using charged polymers, including approaches that utilize enzymes as scaffolds or clients, and compare these condensates with other enzyme activation methods, highlighting the advantages and potential limitations of enzyme condensates. Second, we review the major factors that affect enzyme performance within the condensates, including size-dependent effects and local environmental changes. These data are supported by recent mechanistic studies using various enzyme systems, including oxidoreductases. Finally, we focus on possible applications and outline the key challenges in expanding the utility of enzyme condensates from single-enzyme to multienzyme systems and from solution-based to surface-bound architectures. Our comprehensive overview of enzyme condensate engineering provides a new perspective to bridge cellular organization principles and innovations in enzyme catalysis. This review highlights recent advances in engineering artificial enzyme condensates in vitro using charged polymers. Based on our recent findings, we describe strategies for designing condensates through interactions between polymers and enzymes or coenzymes. We then summarize enzyme activation mechanisms triggered by enzyme condensates, including size-dependent effects and conformational changes in enzymes. We also discuss potential applications and future directions, including multienzyme systems, integration with solid surfaces, and combination with rational enzyme design.

Sweat lactate monitoring has recently garnered increasing attention for assessing athlete performance, optimizing training, and determining fatigue levels. Thus, this study introduces a wireless dual-biosensor device for continuous monitoring of sweat lactate and pH. The pH sensor exhibits a sensitivity of-61 + 0.5 mV pH-1, while the lactate sensor displays a sensitivity of 0.0149 + 0.0004 mA cm-2 mM-1 at pH 7.5. We establish a method to precisely measure lactate within the pH range of 5.5 to 7.5, taking into account the pH-dependent sensor sensitivity. The dual-biosensor device incorporates polydimethylsiloxane microfluidics for collection of sweat, its delivery to the sensor, and disposal of used sweat, enabling continuous and precise monitoring. On-body tests demonstrate the ability of the proposed device to continuously monitor sweat pH and lactate levels in individuals during exercise.

The continuous monitoring of sweat lactate is a critical task in sports and medical applications. This paper introduces a self‐powered biosensor for the continuous monitoring of sweat lactate. A biofuel cell (BFC)is prepared using thionine as a bioanode mediator, with poly(ethylene glycol) diglycidyl ether and a chitosan–genipin membrane incorporated in the bioanode. This system achieves an open‐circuit voltage of 0.75 V and output power density of 0.163 mW cm–2 in the presence of 100 mM lactate. The self‐powered biosensor is constructed by integrating the BFC with a voltage booster and Bluetooth transmitter. An on‐body test is conducted on an exercising individual. The output signal of the self‐powered biosensor is converted into lactate concentrations through manual pH adjustment. A correlation between the lactate concentrations in sweat and blood is observed. Overall, the proposed self‐powered lactate biosensor can continuously monitor sweat lactate, with potential applications in the sports and medical domains.

Despite accumulating evidence that protein dynamics is indispensable for understanding the structural basis of biological activities, it remains challenging to visualize the spatial description of the dynamics and to associate transient conformations with their molecular functions. We have developed a new NMR protein structure determination method for the inference of multistate conformations using multiple types of NMR data, including paramagnetic NMR and residual dipolar couplings, as well as conventional NOEs. Integration of these data in the structure calculation permits delineating accurate ensemble structures of biomacromolecules. Applying the method to yeast ubiquitin hydrolase 1, we find large dynamics of its N-terminus (gating lid) and crossover loop surrounding the active site for ubiquitin-recognition and proteolysis. The N-terminus (gating lid) moves into and out of the crossover loop, suggesting their underlying functional significance. Our results, including those from biochemical analysis, show that large motion surrounding the active site contributes strongly to the efficiency of the enzymatic activity.

Structural analyses of protein filaments formed by self-assembly, such as actin, tubulin, or recombinase filaments, have suffered for decades from technical issues due to difficulties in crystallization, their large size, or the dynamic behavior inherent to their cellular function. The advent of cryo-electron microscopy has finally enabled us to obtain structures at different stages of the existence of these filaments. However, these structures correspond to frozen states, and the possibility of observations in solution is still lacking, especially for filaments characterized by a high plasticity, such as the RecA protein for homologous recombination. Here, we use a combination of SAXS measurements and integrative modeling to generate the solution structure of two known forms of the RecA nucleoprotein filament, previously characterized by electron microscopy and resolved by X-ray crystallography. The two forms differ in the cofactor bound to RecA–RecA interfaces, either ATP or ADP. Cooperative transition from one form to the other has been observed during single-molecule experiments by pulling on the filament but also in solution by modifying solvent conditions. We first compare the SAXS data against known structural information. While the crystal structure of the ATP form matches well with the SAXS data, we deduce from the SAXS profiles of the ADP-form values of the pitch (72.0 Å) and the number of monomers per turn (6.4) that differ with respect to the crystal structure (respectively, 82.7 Å and 6.0). We then monitor the transition between the two states driven by the addition of magnesium, and we show this transition occurs with 0.3 mM Mg 2+ ions with a high cooperativity.

The adaptor protein human GRB2 plays crucial roles in mediating signal transduction from cell membrane receptors to RAS and its downstream proteins by recruiting SOS1. Recent studies have revealed that GRB2 also serves as a scaffold for liquid-liquid phase separation (LLPS) with SOS1 and transmembrane receptors, which is thought to regulate the magnitude of cell signalling pathways. In this study, we employed solution NMR spectroscopy to investigate the interactions of the full-length GRB2 with proline-rich motifs (PRMs) derived from ten potential GRB2-binding sites in SOS1, as well as a peptide from a phosphorylation site of EGFR. Our findings indicate that the binding affinity of the two SH3 domains of GRB2 for PRMs differs by a factor of ten to twenty, with the N-terminal SH3 domain (NSH3) exhibiting a markedly higher affinity. The interactions of PRMs with the SH3 domains affected not only the regions surrounding the PRM binding sites on the SH3 domains but also the linker area connecting the three domains and parts of the SH2 domain. Analysis of the interaction between the phosphorylated EGFR binding site and the SH2 domain revealed chemical shift perturbations in regions distal from the known binding site of SH2. Moreover, we observed that the inter-domain interactions of the two SH3 domains with the SH2 domain of GRB2 are asymmetric. These findings suggest that the local binding of PRMs and phosphorylated EGFR to GRB2 impacts the overall structure of the GRB2 molecule, including domain orientation and dimerisation, which may contribute to LLPS formation.

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.

In contrast to intracellular gene transfer, the direct delivery of expressed proteins is a significantly challenging yet essential technique for elucidating cellular functions, including protein complex structure, liquid-liquid phase separation, therapeutic applications, and reprogramming. In this study, we developed a hybrid nanotube (HyNT) stamp system that physically inserts the HyNTs into adhesive cells, enabling the injection of target molecules through HyNT ducts. This system demonstrates the capability to deliver multiple proteins, such as lactate oxidase (LOx) and ubiquitin (UQ), to approximately 1.8 x 10(7) adhesive cells with a delivery efficiency of 89.9% and a viability of 97.1%. The delivery of LOx enzyme into HeLa cancer cells induced cell death, while enzyme-delivered healthy cells remained viable. Furthermore, our stamp system can deliver an isotope-labeled UQ into adhesive cells for detection by nuclear magnetic resonance (NMR).

Introduction Enzymes are promising catalysts with high selectivity and low environmental load. Especially, redox enzymes are attracting attentions because of their applicability to electrodes for biosensors and biofuel cells. For example, wearable lactate biosensors which can detect lactate in sweat and use the reaction to drive itself have been developed recently[1]. Various biosensors are already available today. However, most of them use single-enzyme electrodes. Compared to single-enzyme electrodes, multi-enzyme electrodes are expected to improve the performance of conventional biosensors and biofuel cells by realizing detection of multiple biomarkers and increased energy density. This study uses the multi-enzyme system involving lactate oxidase (LOx), pyruvate decarboxylase (PDC), and aldehyde dehydrogenase (ALDH) as the reaction model. In this system, four electrons are produced during one lactate molecule is converted into acetate via pyruvate, which is double the number of what are produced by an LOx single-enzyme electrode used in conventional lactate biosensors. This study aims to improve the current response of bioelectrodes by causing enzyme cascade reactions within nanoscale pores of MgO-templated carbon (MgOC). MgOC is a porous carbon material, which is believed to enhance the efficiency of cascade reactions by providing high surface area and control the enzyme activity by changing the solution environment within the nanoscale pores[2]. The prepared electrodes were evaluated electrochemically to investigate how enzyme cascade and the use of MgOC affect the electrode performance. Experimental The ink containing MgOC was dropped to glassy carbon (GC) electrodes to make MgOC electrodes. Subsequently, solution of 1,2-naphtoquinone, the mediator, was added to the MgOC surface. Finally, the mixture of certain ratios of enzymes, crosslinker, Mg-salt, and thiamine pyrophosphate (TPP) was dropped. The latter two were added to promote enzyme reactions. Using the MgOC electrode as a working electrode, three-electrode measurement system was constructed. The measurement solution was 0.1 M phosphate buffer solution (pH 7.0) containing about 15 mM of L-lactate as the substrate. Chronoamperometry (CA) was carried out with an MgOC electrode and a flat GC electrode immobilized with the same amount and ratio of enzymes to see how the use of MgOC affect the current response. Then, the enzyme cascade effect was evaluated by measuring CA of single-enzyme (LOx) electrodes and multi-enzyme (LOx+PDC+ALDH) electrodes. Furthermore, solution environment dependence of the cascade effect was measured by changing the concentration of enzymes and ammonium sulfate ((NH4)2SO4), a kosmotropic salt which is known to salt out enzymes. Results and Discussion The current density observed with the MgOC electrode immobilized with LOx, PDC, and ALDH was about 6.8 times higher than that obtained from the GC electrode also dropped with the three kinds of enzymes. This result indicates the use of MgOC as the electrode surface material is beneficial for enzyme cascade systems because of its higher surface area. We also consider that MgOC has the effect to promote product transfer in cascade systems by containing enzymes in nanoscale pores. The relationship between the pore size of MgOC and the performance of enzyme cascade electrodes will be explored in future experiments. Enzyme cascade effect was evaluated by comparing the current density of single-enzyme MgOC electrodes dropped with only LOx and multi-enzyme MgOC electrodes with LOx, PDC, and ALDH. The figure shows the results. The multi-enzyme electrodes exhibited about 1.98 times higher current density than the single-enzyme electrodes. It implies that the enzyme cascade reactions caused by LOx, PDC, and ALDH increased the number of electrons produced in the system. In this experiment, enzyme solution contained 10 mg/mL of each enzyme and 1.5 mol/L of (NH4)2SO4. When the (NH4)2SO4 concentration was raised to 3.0 mol/L, the current density increased by 2.40 times from single-enzyme to multi-enzyme. On the other hand, it rose by 2.51 times with 5 mg/mL of enzymes and 3.0 mol/L of (NH4)2SO4. Judging from the reaction scheme, the current increase should not be more than double. The increase reached around 2.5 times probably because the forward enzyme reactions were promoted to balance the equilibrium which was displaced by the addition of PDC and ALDH. The enzyme cascade effect was enhanced with increased (NH4)2SO4 concentration, but was slightly affected by the enzyme concentration. These results indicate kosmotropic salts like (NH4)2SO4 shorten the distance between enzymes by salting them out, which can positively impact the cascade reactions. The relationship between enzyme cascade effect and (NH4)2SO4 concentration will be further investigated in future studies. [1] I. Shitanda, et al., ACS Sens. 8(2023), 6, 2368-2374. [2] S. Tsujimura, K. Murata, and W. Akatsuka, J. Am. Chem. Soc. 136 (2014) 14432-14437. Figure 1

This study aimed to develop a lactate sensor with a microchannel that overcomes the issue of air bubbles interfering with the measurement of lactate levels in sweat and to evaluate its potential for continuous monitoring of lactate in sweat. To achieve continuous monitoring of lactate, a microchannel was used to supply and drain sweat from the electrodes of the lactate sensor. A lactate sensor was then developed with a microchannel that has an area specifically designed to trap air bubbles and prevent them from contacting the electrode. The sensor was evaluated by a person while exercising to test its effectiveness in monitoring lactate in sweat and its correlation with blood lactate levels. Furthermore, the lactate sensor with a microchannel in this study can be worn on the body for a long time and is expected to be used for the continuous monitoring of lactate in sweat. The developed lactate sensor with a microchannel effectively prevented air bubbles from interfering with the measurement of lactate levels in sweat. The sensor showed a concentration correlation ranging from 1 to 50 mM and demonstrated a correlation between lactate in sweat and blood. Additionally, the lactate sensor with a microchannel in this study can be worn on the body for an extended period and is expected to be useful for the continuous monitoring of lactate in sweat, particularly in the fields of medicine and sports.

Complex coacervates have received increasing attention due to their use as simple models of membrane-less organelles and microcapsule platforms. The incorporation of proteins into complex coacervates is recognized as a crucial event that enables understanding of membrane-less organelles in cells and controlling microcapsules. Here, we investigated the incorporation of proteins into complex coacervates with a focus on the progress of the incorporation process. This stands in contrast to most previous studies, which have been focused the endpoint of the incorporation process. For that purpose, client proteins, i.e., lysozyme, ovalbumin, and pyruvate oxidase, were mixed with complex coacervate scaffolds consisting of two polyelectrolytes, i.e., the positively charged poly(diallyldimethylammonium chloride) and the negatively charged carboxymethyl dextran sodium salt, and the process was studied. Spectroscopic analysis and microscopic imaging demonstrated that electrostatic factors are the primary driving force of the incorporation of the client proteins into the complex coacervate scaffolds. Moreover, we discovered the formation of multi-phase droplets when a charged protein was incorporated into a complex coacervate whose surface was charged oppositely relative to that of the protein. The droplets inside the complex coacervates were found to be the diluted phase trapped as internal vacuoles. These findings provide fundamental insight into the temporal changes at the droplet interface during the incorporation of proteins into complex coacervates. This knowledge will facilitate the understanding of biological events associated with membrane-less organelles and will contribute to the industrial development of the use of microcapsules.

Biological properties of protein molecules depend on their interaction with other molecules, and enzymes are no exception. Enzyme activities are controlled by their interaction with other molecules in living cells. Enzyme activation and their catalytic properties in the presence of different types of polymers have been studied in vitro, although these studies are restricted to only a few enzymes. In this study, we show that addition of poly-l-lysine (PLL) can increase the enzymatic activity of multiple oxidoreductases through formation of enzyme assemblies. Oxidoreductases with an overall negative charge, such as l-lactate oxidase, d-lactate dehydrogenase, pyruvate oxidase, and acetaldehyde dehydrogenase, each formed assemblies with the positively charged PLL via electrostatic interactions. The enzyme activities of these oxidoreductases in the enzyme assemblies were several-folds higher than those of the enzyme in their natural dispersed state. In the presence of PLL, the turnover number (kcat) improved for all enzymes, whereas the decrease in Michaelis constant (KM) was enzyme dependent. This type of enzyme function regulation through the formation of assemblies via simple addition of polymers has potential for diverse applications, including various industrial and research purposes.

The assembly state of enzymes is gaining interest as a mechanism for regulating the function of enzymes in living cells. One of the current topics in enzymology is the relationship between enzyme activity and the assembly state due to liquid-liquid phase separation. In this study, we demonstrated enzyme activation via the formation of enzyme assemblies using L-lactate oxidase (LOX). LOX formed hundreds of nanometer-scale assemblies with poly-L-lysine (PLL). In the presence of ammonium sulfate, the LOX-PLL clusters formed micrometer-scale liquid droplets. The enzyme activities of LOX in clusters and droplets were one order of magnitude higher than those in the dispersed state, owing to a decrease in KM and an increase in kcat. Moreover, the clusters exhibited a higher activation effect than the droplets. In addition, the conformation of LOX changed in the clusters, resulting in increased enzyme activation. Understanding enzyme activation and assembly states provides important information regarding enzyme function in living cells, in addition to biotechnology applications.

Acetaldehyde, which is an intermediate product of alcohol metabolism, is known to induce symptoms, including alcohol flushing, vomiting, and headaches in humans. Therefore, real-time monitoring of acetaldehyde levels is crucial to mitigating these health issues. However, current methods for detecting low-concentration gases necessitate the use of complex measurement equipment. In this study, we developed a low-cost, low-detection-limit, enzyme-based electrochemical biosensor for acetaldehyde gas detection that does not require sophisticated equipment. The sensor was constructed by screen-printing electrodes onto a porous polyimide film, using grafted MgO-templated carbon (GMgOC) as working electrode material, carbon for the counter electrode, and silver/ silver chloride for the reference electrode. Pyrroloquinoline-quinone-dependent aldehyde dehydrogenase was immobilized on the working electrode, and a chamber was attached to the electrode chip and filled with 1-methoxy-5-methylphenazinium methyl sulfate solution. The sensor can be used to measure acetaldehyde gas concentrations from 0.02 to 0.1 ppm, making it suitable for monitoring human skin gas. This low detection limit was achieved by delivering the analyte through the porous polyimide film on which the electrodes were printed and accumulating acetaldehyde in the mesoporous GMgOC of the working electrode. This mechanism suggests that this sensor design can be adapted to develop other low-detection limit gas sensors, such as those for screening skin gas biomarkers.