Enzymatic Reaction Research Articles

Surface-modified CMOS biosensors

Biosensors translate biological events into electronic signals that quantify biological processes. They are increasingly used in in vitro diagnostics applications that leverage their ability to process small sample volumes. One recent trend has been to integrate biosensors with complementary metal-oxide-semiconductor (CMOS) chips to provide enhanced miniaturization, parallel sensing, and low power consumption at a low cost. CMOS-enabled biosensors are used in monitoring DNA hybridization, enzymatic reactions, and cell proliferation, to name a few applications. This paper explores the materials and processes used in emerging CMOS biosensors. We discuss subtractive and additive processes for creating electrodes for electrochemical sensing applications. We discuss functionalization techniques for creating bioelectronic interfaces that allow molecular events to be transduced into the electrical domain using a plurality of modalities that are readily provided by CMOS chips. Example modalities featured are optical sensing, electrochemical detection, electrical detection, magnetic sensing, and mechanical sensing.

A Urease‐Based pH Photoswitch: A General Route to Light‐to‐pH Transduction

AbstractNew approaches for the integration of chemical and physical stimuli to control the dynamics of artificial enzymatic reaction networks (ERNs) are needed. Here, we present a general approach to convert a light stimulus into a time‐programmed pH response. We developed and characterized a panel of photoswitchable inhibitors of urease. Urease activity, now regulated by light via the photoinhibitors, leads to an increase in pH upon hydrolysis of urea into ammonia. Careful choice of characteristics of light, and concentrations of enzyme, substrate, and photoinhibitor, allowed us to control the timing of the pH transition. Furthermore, as all enzymes have an activity–pH profile, the urease photoinhibitor system can be used to regulate the activities of other enzymes in small reaction networks.

Molecular Photoswitching Unlocks Glucose Oxidase for Synergistically Reinforcing Fenton Reactions for Antitumor Chemodynamic Therapy.

We have developed a new type of nanoparticles with potent antitumor activity photoactivatable via the combination of molecular photoswitching of spiropyran (SP) and enzymatic reaction of glucose oxidase (GOx). As two key processes involved therein, Fe(III)-to-Fe(II) photoreduction in Fe(III) metal-organic frameworks (MOFs) brings about the release of free Fe2+/Fe3+ while the photoswitching of SP to merocyanine (MC) unlocks the enzymatic activity of GOx that was pre-passivated by SP. The release of free Fe3+ boosts its hydrolysis and therefore enables the acidification of microenvironment, which is further reinforced by one of the products of the GOx-mediated glucose oxidation reaction, gluconic acid (GlcA). Based on the generation of Fe2+ and acidic milieu together with another product of the oxidation reaction, hydrogen peroxide (H2O2), these two processes jointly present triple enabling factors for generating lethal hydroxyl radicals (⋅OH) species via Fenton reactions and therefore oxidative stress capable of inhibiting tumor. The antitumor potency of such nanoparticle is verified in tumor-bearing model mice in vivo, proclaiming its potential as a potent and safe agent based on the unique mechanism of optically manipulating enzyme activity for synergistic antitumor therapeutics with high spatial precision, enhanced efficacy and minimized side effects.

MRNA for Body Fluid and Individual Identification.

Biological stains are one of the most important pieces of evidence, playing a multifaceted role in forensic investigations. An integral facet of forensic practice involves the identification of body fluids, typically achieved through chemical and enzymatic reactions. In recent decades, the introduction of mRNA markers has been posited as a pivotal advancement to augment the capabilities of body fluid identification (BFID). The mRNA coding region single-nucleotide polymorphisms (cSNPs) also present notable advantages, particularly in the task of individual identification. Here, we review the specificity and stability of mRNA markers in the context of BFID and the prowess of mRNA polymorphism in individual identification. Additionally, innovative methods for mRNA detection are discussed.

Degradation mechanism of difructose dianhydride III in Blautia species.

Di-fructofuranose 1,2':2,3' dianhydride (DFA-III) is a cyclic fructo-disaccharide, which is produced by the condensation of two fructose molecules via the caramelization or enzymatic reaction of inulin fructotransferase. A strain of Blautia producta was known to utilize DFA-III as a carbohydrate source; however, the mechanisms remain unclear. In this study, we characterized the glycoside hydrolase (GH) family 91 DFA-III hydrolase (DFA-IIIase) from B. parvula NBRC 113351. Recombinant BpDFA-IIIase catalyzed the reversible conversion of DFA-III to inulobiose, which is further degraded to fructose by the cooperative action of DFA-IIIase and GH32 β-D-fructofuranosidase. DFA-III was utilized in several Blautia species with a gene cluster for DFA-III degradation (e.g., B. parvula NBRC 113351, B. hydrogenotrophica JCM 14656, and B. wexlerae JCM 35486), but not by B. wexlerae JCM 31267, which does not possess the gene cluster. Furthermore, B. hansenii JCM 14655, which cannot metabolize fructose, could not utilize DFA-III; however, it could degrade DFA-III to fructose in the presence of DFA-III-degrading enzymes. Fecal fermentation tests showed that Blautia species are important gut microbe for degrading DFA-III. KEY POINTS: • BpDFA-IIIase is the first characterized DFA-IIIase in intestinal non-pathogenic bacteria. • DFA-IIIase is widely conserved in Blautia species. • DFA-III is degraded to d-fructose through inulobiose by the cooperative action of DFA-IIIase and β-d-fructofuranosidase.

Biostimulant-Based Molecular Priming Improves Crop Quality and Enhances Yield of Raspberry and Strawberry Fruits

Background/Objectives: The biostimulant SuperFifty, produced from the brown algae Ascophyllum nodosum, can improve crop quality and yield and mitigate stress tolerance in model and crop plants such as Arabidopsis thaliana, pepper, and tomato. However, the effect of SuperFifty on raspberries and strawberries has not been well studied, especially in terms of nutritional properties and yield. The aim of this study was to investigate the effect of SuperFifty on the quality and quantity of raspberry and strawberry fruits, with a focus on metabolic composition and essential elements, which together determine the nutritional properties and total yield of these two crops. Methods: Metabolome analysis was performed by liquid chromatography–mass spectrometry analysis (LC-MS), and essential elements analysis was performed by inductively coupled plasma-mass spectrometry (ICP-MS). Results: Here, we demonstrate that SuperFifty increases the fruit size of both raspberries and strawberries and enhances the yield in these two berry crops by 42.1% (raspberry) and 33.9% (strawberry) while preserving the nutritional properties of the fruits. Metabolome analysis of 100 metabolites revealed that antioxidants, essential amino acids, organic acids, sugars, and vitamins, such as glutathione, alanine, asparagine, histidine, threonine, serine, tryptophan, sucrose, citric acid, pantothenic acid (vitamin B5), as well as other primary metabolites, remain the same in the SuperFifty-primed fruits. Secondary metabolites, such as caffeic acid, p-coumaric acid, kaempferol, and quercetin, also maintained their levels in the SuperFifty-primed fruits. Analysis of essential elements demonstrated that elements important for human health, such as Zn, Mn, Fe, B, Cu, K, and Ca, maintain the same levels in the raspberry and strawberry fruits obtained from the biostimulant-primed plants. Magnesium, an important element known as a co-factor in many enzymatic reactions related to both plant physiology and human health, increased in both raspberry and strawberry fruits primed with SuperFifty. Finally, we discuss the potential financial and health benefits of the SuperFifty-induced priming for both growers and consumers. Conclusions: We demonstrate that SuperFifty significantly enhances the yield of both raspberries and strawberries, improves the marketable grade of the fruits (larger and heavier fruits), and enhances the nutritional properties by elevating Mg content in the fruits. Altogether, this biostimulant-induced molecular priming offers an environmentally friendly, efficient, and sustainable way to enhance the yield and quality of berry crops, with clear benefits to both berry producers and customers.

Membrane-dependent reactions of blood coagulation: classical view and state-of-the-art concepts

The complex mechanism called hemostasis evolved in living organisms to prevent blood loss when a blood vessel is damaged. In this process, two closely interconnected systems are distinguished: platelet-vascular and plasmatic hemostasis. Plasmatic hemostasis is a system of proteolytic reactions, in which blood plasma proteins called coagulation factors are involved. A key feature of this system is the localization of enzymatic reactions on the surface of phospholipid membranes, which increases their rate by up to 5 orders of magnitude. This review describes the basic mechanisms of coagulation factors binding to phospholipid membranes, pathways for complex assembly and activation reactions, and discusses the role of membranes in this process, their composition and sources. The binding of coagulation factors to procoagulant membranes leads not only to the acceleration of coagulation reactions, but also to their selective localization in restricted areas and protection from being washed away by the flow. The efficiency of coagulation reactions is regulated by the composition of the outer layer of the membrane, primarily through a special mechanism of mitochondria-dependent necrotic platelet death.

Molecular Evolution and Adaptation Strategies in Marine Ciliates: An Inspiration for Cold-Adapted Enzyme Engineering and Drug Binding Analysis

In the present review, we summarize genome mining of genomic data obtained from the psychrophilic Antarctic marine ciliate Euplotes focardii and its evolutionary-close mesophilic cosmopolitan counterpart E. crassus. This analysis highlights adaptation strategies that are unique to the Antarctic ciliate, including antioxidant gene duplication and distinctive substitutions that may play roles in increased drug binding affinity and enzyme reaction rate in cold environments. Enzymes from psychrophiles are usually characterized by high activities and reaction rates at low temperatures compared with their counterparts from mesophiles and thermophiles. As a rule, catalyst cold activity derives from an increased structural flexibility that may lead to protein denaturation in response to temperature fluctuation. Molecular thermolability has been a major drawback of using macromolecules from psychrophiles in industrial applications. Here, we report a case study in which the role of peculiar amino acid substitution in cold adaptation is demonstrated by site-directed mutagenesis. Combined with a rational design approach, these substitutions can be used for site-directed mutagenesis to obtain cold-active catalysts that are structurally stable. Furthermore, molecular docking analysis of β-tubulin isotypes extrapolated from E. focardii and E. crassus genomes allowed us to obtain additional insight on the taxol binding site and drug affinity. E. focardii genome mining and the comparison with the mesophilic sibling counterpart can be used as an inspiration for molecular engineering for medical and industrial applications.

Synergetic enzymatic reactive crystallization of cefaclor catalyzed by PGA

In the context of the severe global carbon emission crisis, green and sustainable enzymatic reactions are garnering widespread attention in the industry. However, these processes still face numerous challenges, such as limitations imposed by reaction equilibrium, product decomposition, and inhibition. Product removal via crystallization has the potential to address these issues. More importantly, crystal product properties can be directly manipulated during enzymatic reactive crystallization (ERC), such as polymorphic purity, size, and morphology in a single batch. In the synthesis of cefaclor catalyzed by penicillin G acylase (PGA), primary and secondary hydrolysis greatly limited the efficiency of the reaction. Thus, ERC was performed to combine the advantages of green enzymatic reactions and controllable direct crystallization. Initially, a supersaturated solution of the substrate was established, and cefaclor crystals were seeded to the enzymatic reaction system at a proper time. Subsequently, operating parameters were adjusted to facilitate selective crystallization of cefaclor. It is noteworthy that both immobilized and free enzymes were examined in our research. Ultimately, cefaclor crystals with purity exceeding 99% were obtained directly. The yield of cefaclor reached as high as 88%. It is believed that this method held significant promise for the green and efficient production of high-purity cefaclor and demonstrated considerable potential for application in more similar pharmaceutical scenarios.

Multifrequency-STD NMR unveils the first Michaelis complex of an intramolecular trans-sialidase from Ruminococcus gnavus

RgNanH is an intramolecular trans-sialidase expressed by the human gut symbiont Ruminococcus gnavus, to utilise intestinal sialylated mucin glycan epitopes. Its catalytic domain, belonging to glycoside hydrolase GH33 family, cleaves off terminal sialic acid residues from mucins, releasing 2,7-anhydro-Neu5Ac which is then used as metabolic substrate by R. gnavus to proliferate in the mucosal environment. RgNanH is one of the three intramolecular trans-sialidases (IT-sialidases) characterised to date, and the first from a gut commensal organism. Here, saturation transfer difference NMR (STD NMR) in combination with computational techniques (molecular docking and CORCEMA-ST) were used to elucidate the specificity, kinetics and relative affinity of RgNanH for sialoglycans and 2,7-anhydro-Neu5Ac. We propose the first 3D model for the Michaelis complex of an IT-sialidase. This confirms the sialic acid to be the main recognition element for the interaction in the enzymatic cleft and highlights the crucial role of Trp698 to make CH-π stacking with the galactose residue of the substrate 3′-sialyllactose. The same contact is shown not to be possible for 6′-sialyllactose, due to geometrical constrains of the α-2,6 linkage. Indeed 6′-sialyllactose is not a substrate, even though it is shown to bind to RgNanH by STD NMR. These findings corroborate the role of Trp698 for the α-2,3 specificity of trans-sialidases. In this structural study, the use of Differential Epitope Mapping STD NMR (DEEP-STD NMR) approach allowed the validation of the proposed 3D models in solution. These structural approaches are shown to be instrumental in shedding light on the molecular mechanisms underpinning enzymatic reactions in the absence of enzyme-substrate X-ray structures.

Structural dynamics of the Ca2+-regulated cutinase towards structure-based improvement of PET degradation activity

We previously revealed the structural basis of Ca2+ dependent regulation of a polyethylene terephthalate (PET)-degrading enzyme, Cut190, and proposed a unique reaction cycle in which the enzyme repeatedly binds and releases Ca2+. Here, we report crystal structures of Cut190 mutants with high thermal stability complexed with PET-like ligands that contain aromatic rings. The structural information has allowed us to perform further computational analyses using a PET-trimer bound model. Our multicanonical molecular dynamics simulations and subsequent analyses of the free energy landscapes revealed a novel intermediate form that occurs during the enzymatic reaction cycle. Furthermore, the computational analyses were used to investigate the effect of the point mutations F77L and F81L in the Ca2+-binding site, which showed that the former stabilizes the engaged and open forms to improve transition between the open and active forms, while the latter extremely increases the open form. Subsequent experiments showed that the F77L mutation increased the activity, while the F81L mutation decreased the activity. Our computational analysis has enabled us to explore the dynamics of Cut190 on a completely new level, providing key insights into how the balance between the various conformations influences the reaction cycle and ultimately how to improve the reaction cycle.

Kinetics of galactooligosaccharides (GOS) production with two β-galactosidases: Metallic ion effect and mathematical model

Galactooligosaccharides (GOS) are functional carbohydrates produced from different lactose sources like cheese whey. They are relevant products in the dairy industry. However, its enzymatic production implies a challenge due to its inhibition by metallic ions presence. Enzymatic production of GOS from lactose using β-galactosidase from Kluyveromyces lactis and Aspergillus oryzae were studied in the presence of metallic ions (calcium, potassium, sodium, and magnesium). Also, a kinetic enzymatic model was proposed and correlated to explain the metallic ion effect in the GOS production. For both enzymes, stronger inhibition effects were observed when calcium was present, followed by potassium, sodium, and magnesium. Consequently, additional efforts must be made for the calcium remotion before the enzymatic reaction for industrial applications. Moreover, the second-order kinetic model proposed in this work describes the GOS production using both enzymes with metallic ions presence. It was stablished that for GOS production using β-gal from K. lactis and A. oryzae with various salts, calcium showed the strongest inhibition for both enzymes, followed by potassium and sodium. A second-order kinetic model accurately described GOS production, better reflecting hydrolysis inhibition than transgalactosylation inhibition

One-ElectronNO to N2O Pathways via Heme Models and Lewis Acid: Metal Effectsand Differences from the Enzymatic Reaction.

Some pathogens use heme-containing nitric oxide reductases (NORs) to reduce NO to N2O as their defense mechanism to detoxify NO and reduce nitrosative stress. This reduction is also significant in the global N cycle. Our previous experimental work showed that Fe and Co porphyrin NO complexes can couple with external NO to form N2O when activated by the Lewis acid BF3. A key difference from conventional two-electron enzymatic reaction is that one electron is sufficient. However, a complete understanding of the entire reaction pathways and the more favorable reactivity for Fe remains unknown. Here, we present a quantum chemical study to provide such information. Our results confirmed Fe's higher experimental reactivity, showing advantages in all steps of the reaction pathway: easier metal oxidation for NO reduction and N-O cleavage as well as a larger size to expedite the N/O coordination mode transition. The Co system, with a similar product energy as the enzyme, shows potential for further development in catalytic NO coupling. This work also offers the first evidence that this new one-electron NO reduction is both kinetically competitive and thermodynamically more favorable than the native pathway, supporting future initiatives in optimizing NO reduction agents in biology, environment, and industry.

Xylose Acetals - a New Class of Sustainable Solvents and Their Application in Enzymatic Polycondensation.

The use of organic solvents in academic research and industry applications is facing increasing regulatory pressure due to environmental and health concerns. Consequently, there is a growing demand for sustainable solvents, particularly in the enzymatic synthesis and processing of polyesters. Biocatalysts offer a sustainable method for producing these materials; however, achieving high molecular weights often necessitates use of solvents. In this work, we introduce a new class of alternative aprotic solvents with medium polarity produced directly from agricultural waste biomass in up to 83 mol% yield (on xylan basis). The new solvents have a largely unmodified xylose core and acetal functionality, yet they show no peroxide formation and provide reduced flammability risk. We also demonstrate their successful application in enzymatic polycondensation reactions with Candida antarctica lipase B (CaLB). In particular, the solvent dibutylxylose (DBX) outperformed the hazardous solvent diphenyl ether and facilitated polycondensation of the lignin-derived diester pyridine-2,4-dicarboxylate, yielding polyesters with a Mn of >15 kDa. Computational modelling studies provided further insight into the molecular structure and dynamics of CaLB in the presence of new solvents. Lastly, up to 98 wt% of the new xylose acetals were successfully recovered and recycled, further contributing to the sustainability of the overall process.

The role of cuproptosis in gastric cancer

As a biologically essential transition metal, copper is widely involved in various enzymatic reactions and crucial biological processes in the body. It plays an increasingly important role in maintaining normal cellular metabolism and supporting the growth and development of the human body. As a trace element, copper maintains the dynamic balance of its concentration in body fluids through active homeostatic mechanisms. Both excess and deficiency of copper ions can impair cell function, ultimately leading to cell damage and death. Cuproptosis is a novel form of cell death where copper ions cause cell death by directly binding to the lipoylated components of the citric acid cycle (CAC) in mitochondrial respiration and interfering with the levels of iron-sulfur cluster (Fe-S cluster) proteins, ultimately causing protein toxic stress. Its primary characteristics are Cu2+ concentration dependence and high expression in mitochondrial respiratory cells. Recent research has revealed that, compared to other forms of programmed cell death such as apoptosis, necrosis, and autophagy, cuproptosis has unique morphological and biochemical features. Cuproptosis is associated with the occurrence and development of various diseases, including cancer, neurodegenerative diseases, and cardiovascular diseases. This article focuses on a review of the relevance of cuproptosis in gastric cancer (GC).

CRISPR/Cas9-Mediated Genome Editing of T4 Bacteriophage for High-Throughput Antimicrobial Susceptibility Testing.

The accurate and effective determination of antimicrobial resistance is essential to limiting the spread of infectious diseases and ensuring human health. Herein, a simple, accurate, and high-throughput phage-based colorimetric sensing strategy was developed for antimicrobial susceptibility testing (AST). Taking advantage of the CRISPR/Cas9 system, the genome of the T4 phage was modularly engineered to carry lacZ-α (lacZa), a marker gene encoding the α-fragment of β-galactosidase (β-gal). T4lacZa phages were identified by blue-white selection and then used for a biosensing application. In this strategy, the bacterial solution is exposed to the T4lacZa phage, causing target bacteria to overexpress β-gal. Upon the addition of a colorimetric substrate, the β-gal initiates an enzymatic reaction, resulting in a solution color change from yellow to red. This sensing strategy offers a visual way to monitor bacterial growth in the presence of antibiotics, enabling the determination of bacterial antimicrobial susceptibility. As a proof of concept, our developed sensing strategy was successfully applied to identify 9 different multidrug-resistant Escherichia coli (E. coli) in urine samples with 100% specificity. Compared with conventional disk diffusion susceptibility tests, the engineered phage-based sensing strategy can shorten the detection time by at least half without losing detection sensitivity, providing an alternative high-throughput method for AST in clinical diagnosis.

Construction of Liposome‐Based Extracellular Artificial Organelles on Individual Living Cells

AbstractIntegration of living cells with extrinsic functional entities gives rise to bioaugmented nanobiohybrids, which hold tremendous potential across diverse fields such as cell therapy, biocatalysis, and cell robotics. This study presents a biocompatible method for incorporating multilayered functional liposomes onto the cell surface, creating extracellular artificial organelles or exorganelles. The introduction of various extrinsic functionalities to cells is achieved without comprising their viabilities. The integration of extrinsic enzymatic reactions is exemplified through the cascade reaction involving glucose oxidase and horseradish peroxidase. Furthermore, our protocol offers the design flexibility to customize liposome compositions, thereby providing effective cell modification. The versatility of the liposome‐based exorganelle approach establishes an advanced chemical tool, empowering cells with novel functionalities that surpass or are complementary to their innate capabilities.

Membrane Interactions of GET1 and GET2 Facilitate Fiber Cell Initiation through the Guided Entry of the TA Protein Pathway in Cotton.

The guided entry of TA proteins (GET) pathway, which is responsible for the post-translational targeting and insertion of the tail-anchored (TA) protein into the endoplasmic reticulum (ER), plays an important role in physiological processes such as protein sorting, vesicle trafficking, cell apoptosis, and enzymatic reactions in which the GET1/2 complex is indispensable. However, a comprehensive study of the GET1 and GET2 genes and the GET pathway in cotton has not yet been carried out. Here, 12 GET1 and 21 GET2 genes were identified in nine representative plant species, and the phylogenetic relationships, gene structures, protein motifs, cis-regulatory elements (CREs), and temporal and spatial expression profiles were analyzed thoroughly. Our study indicated that GhGET1s and GhGET2s might be localized on ER membranes. According to expression profiling and CREs analysis, GhGET2-A02 was identified as a promising candidate for fiber cell development, interacting with two GhGET1s in the membrane, with a binding bias toward GhGET1-A06. Silencing of GhGET1-A06 or GhGET2-A02 reduced fiber initiation and elongation. In summary, our research provides important evidence for understanding the gene families and functions of GET1 and GET2 in cotton and provides clues for molecular breeding of high-quality cotton fiber varieties.

Cover Feature: Tutorial Review on the Set‐Up and Running of Quantum Mechanical Cluster Models for Enzymatic Reaction Mechanisms (Chem. Eur. J. 60/2024)

Cover Feature: Tutorial Review on the Set‐Up and Running of Quantum Mechanical Cluster Models for Enzymatic Reaction Mechanisms (Chem. Eur. J. 60/2024)

Colorimetric aptasensing of microcystin-LR using DNA-conjugated polydiacetylene.

Polydiacetylene (PDA) holds promise as a versatile material for biosensing applications due to its unique optical properties and self-assembly capabilities. In this study, we developed a colorimetric detection biosensor system utilizing PDA and aptamer for the detection of microcystin-LR (MC-LR), a potent hepatotoxin found in cyanobacteria-contaminated environments. The biosensor was constructed by immobilizing MC-LR-specific aptamer on magnetic beads, where the aptamer was hybridized with a urease-labelled complementary DNA (cDNA-urease). Upon binding MC-LR, the aptamer undergoes a conformational change to release cDNA-urease. The released cDNA-urease is subsequently captured by PDA bearing a single-stranded DNA (ssDNA). The enzymatic reaction triggers a distinctive color transition of PDA from blue to red. The results demonstrate exceptional sensitivity, with a linear detection range of 5-100ng/mL and a limit of detection as low as 1ng/mL. The practicability of the colorimetric method was demonstrated by detecting different levels of MC-LR in spiked water samples. The recoveries ranged from 77.3 to 102% and the color change, visible to the naked eye, underscores the practical utility for on-site applications. Selectivity for MC-LR over other microcystin variants (MC-RR and MC-YR) was confirmed. The colorimetric detection platform capitalizes on the properties of PDA and nucleic acid, offering a robust method for detecting small molecules with potential applications in environmental monitoring and public health.