Search Results (79)

We are reporting the development of a high-performance, non-enzymatic electrochemical biosensor for selective lactate detection, integrating laser-induced graphene (LIG), gold nanoparticles (AuNPs), and a molecularly imprinted polymer (MIP) synthesized from poly(3,4-ethylenedioxythiophene) (PEDOT). The LIG electrode offers a highly porous, conductive scaffold, while electrodeposited AuNPs enhance catalytic activity and signal amplification. The PEDOT-based MIP layer, electropolymerized via cyclic voltammetry, imparts molecular specificity by creating lactate-specific binding sites. Cyclic voltammetry confirmed successful molecular imprinting and enhanced interfacial electron transfer. The resulting LIG/AuNPs/MIP biosensor demonstrated a wide linear detection range from 0.1 µM to 2500 µM, with a sensitivity of 22.42 µA/log(µM) and a low limit of detection (0.035 µM). The sensor showed excellent selectivity against common electroactive interferents such as glucose and uric acid, long-term stability, and accurate recovery in artificial saliva (>95.7%), indicating strong potential for practical application. This enzyme-free platform offers a robust and scalable strategy for continuous lactate monitoring, particularly suited for wearable devices in sports performance monitoring and critical care diagnostics. Full article

Two recombinant cutinases, AfCutA and AfCutB, derived from Aspergillus fumigatus, were heterologously expressed in Pichia pastoris and systematically characterized for their biochemical properties and polyester-degrading capabilities. AfCutA demonstrated superior catalytic performance compared with AfCutB, displaying higher optimal pH (8.0–9.0 vs. 7.0–8.0), higher optimal temperature (60 °C vs. 50 °C), and greater thermostability. AfCutA exhibited increased hydrolytic activity toward p-nitrophenyl esters (C4–C16) and synthetic polyesters. Additionally, AfCutA released approximately 3.2-fold more acetic acid from polyvinyl acetate (PVAc) hydrolysis than AfCutB. Quartz crystal microbalance with dissipation monitoring (QCM-D) revealed rapid adsorption of both enzymes onto polyester films. However, their adsorption capacity on poly (ε-caprolactone) (PCL) films was significantly higher than on polybutylene succinate (PBS) films, and was influenced by pH. Comparative modeling of catalytic domains identified distinct structural differences between the two cutinases. AfCutA possesses a shallower substrate-binding cleft, fewer acidic residues, and more extensive hydrophobic regions around the active site, potentially explaining its enhanced interfacial activation and catalytic efficiency toward synthetic polyester substrates. The notably superior performance of AfCutA suggests its potential as a biocatalyst in industrial applications, particularly in polyester waste bioremediation and sustainable polymer processing. Full article

Enzymatic catalysis has gained significant attention in green chemistry due to its high specificity and efficiency under mild conditions. However, challenges related to enzyme immobilization and spatial control often limit its practical applications. In this work, we report a Marangoni flow-driven strategy to fabricate a biomimetic jellyfish-like hydrogel with tunable tentacle-like structures. The formation process occurs entirely in an aqueous system without organic solvents or post-treatment, enabling the construction of ultra-thin, free-standing hydrogels through spontaneous interfacial self-assembly. The resulting structure exhibits high surface-area geometry and excellent biocompatibility, providing a versatile platform for localized enzyme loading. This method offers a simple and scalable route for engineering soft materials with complex morphologies, and expands the design space for bioinspired hydrogel systems. Full article

Skin protection against ultraviolet (UV) radiation has long been crucial due to its role in photoaging, sunburn, and wrinkles. This study focuses on developing a bio-based sunscreen from Calendula arvensis capitula extract. Various extraction methods (maceration, sonication, and infusion) and solvents (EtOH, EtOH-H₂O, and H₂O) were explored in order to identify the most effective extract for use in a sunscreen formulation. Each extract was analyzed for its phenolic content, as well as antioxidant activities (assessed through DPPH, CAT, and FRAP assays). Enzyme inhibition assays for tyrosinase, elastase, and collagenase highlighted the low IC₅₀ values of the hydroethanolic extract. Furthermore, the in vitro sun protection factor (SPF) against UVB radiation was measured using ultraviolet spectrophotometry. A phytochemical analysis showed phenolic levels between 8 and 27 mg GAE/g, flavonoid concentrations of 7–13 mg QE/g, and tannin levels of 1.15–1.68 mg/mL, alongside moderate antioxidant activity. The ethanol maceration extract reduced the interfacial tension to 2.15 mN/m in 600 s, outperforming the conventional emulsifier polysorbate 20. The sonicated hydroethanolic extract demonstrated remarkable SPF efficacy (SPF = 193.65 ± 0.02), far exceeding that of the standard zinc oxide (SPF = 11.88 ± 0.03). The proposed formulations meet the COSMOS standards, suggesting their potential for certification as biological products. Further clinical and in vivo studies are necessary to confirm their safety and commercial viability. Full article

Phospholipase A2 (PLA2) is a superfamily of phospholipase enzymes that dock at the water/oil interface of phospholipid assemblies, hydrolyzing the ester bond at the sn-2 position. The enzymatic activity of these enzymes differs based on the nature of the substrate, its supramolecular assemblies (micelle, liposomes), and their composition, reflecting the interfacial nature of the PLA2s and requiring assays able to directly quantify this interaction of the enzyme(s) with these supramolecular assemblies. We developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye bromothymol blue (BTB), in which different POPC (3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine) self-assemblies (liposomes or mixed micelles with Triton X-100 at different molar ratios) were used to assess the enzymatic activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies and to determine the kinetic parameters of PLA2 isozymes IB and IIA for each supramolecular POPC assembly. This assay is suitable for assessing the inhibition of PLA2s with great accuracy using UV-VIS spectrophotometry, being thus amenable for screening of PLA2 enzymes and their substrates and inhibitors in conditions very similar to physiologic ones. Full article

Surfactin is widely used in the petroleum extraction, cosmetics, biopharmaceuticals and agriculture industries. It possesses antibacterial and antiviral activities and can reduce interfacial tension. Bacillus are commonly used as production chassis, but wild-type Bacillus subtilis 168 cannot synthesise surfactin. In this study, the phosphopantetheinyl transferase (PPTase) gene sfp* (with a T base removed) was overexpressed and enzyme activity was restored, enabling B. subtilis 168 to synthesise surfactin with a yield of 747.5 ± 6.5 mg/L. Knocking out ppsD and yvkC did not enhance surfactin synthesis. Overexpression of predicted surfactin transporter gene yfiS increased its titre to 1060.7 ± 89.4 mg/L, while overexpression of yerP, ycxA and ycxA-efp had little or negative effects on surfactin synthesis, suggesting YfiS is involved in surfactin efflux. By replacing the native promoter of the srfA operon encoding surfactin synthase with three promoters, surfactin synthesis was significantly reduced. However, knockout of the global transcriptional regulator gene codY enhanced the surfactin titre to 1601.8 ± 91.9 mg/L. The highest surfactin titre reached 3.89 ± 0.07 g/L, with the yield of 0.63 ± 0.02 g/g DCW, after 36 h of fed-batch fermentation in 5 L fermenter. This study provides a reference for further understanding surfactin synthesis and constructing microbial cell factories. Full article

Ionizing radiation has its unique popularity as a non-thermal decontamination technique treating with protein-rich foodstuffs to ensure the microbial and sensory quality, particularly for shell eggs. However, the changes in the functional properties of egg protein fractions such as liquid egg white (LEW) with macro/microstructural information are still controversial. Hence, this study was designed to elaborate the foaming and heat-set gelation functionality of LEW following different γ-ray irradiation dose treatments (0, 1, 3 or 5 kGy). For such, the physicochemical properties (active sulfhydryl and the hydrophobicity of protein moieties), structural characteristics (through X-ray diffraction, Fourier-transform infrared spectroscopy and differential scanning calorimetry) and interfacial activities (rheological viscosity, interfacial tension, microrheological performance) were investigated. Then, the thermal gelation of LEW in relation to the texture profile and microstructure (by means of a scanning electron microscope) was evaluated followed by the swelling potency analysis of LEW gel in enzyme-free simulated gastric juice. The results indicated that irradiation significantly increased the hydrophobicity of liquid egg white proteins (LEWPs) (p < 0.05) by exposing non-polar groups and the interfacial rearrangement from a β-sheet to linear and smaller crystal structure, leading to an enhanced foaming capacity. Microstructural analysis revealed that the higher dose irradiation (up to 5 kGy) could promote the proteins’ oxidation of LEW alongside protein aggregates formed in the amorphous region, which favored heat-set gelation. As evidenced in microrheology, ≤3 kGy irradiation provided an improved viscoelastic interface film of LEW during gelatinization. Particularly, the LEW gel treated with 1 kGy irradiation had evident swelling resistance during the times of acidification at pH 1.2. These results gave new insight into the irradiation-assisted enhancement of foaming and heat-set gelation properties of LEW. Full article

In recent years, the demand for foods without animal proteins has increased, both for health and ethical reasons. Replacing animal protein in foods can result in unappealing textures, hindering consumer acceptance. In this context, interfacial properties also play a crucial role in food systems like foam or emulsions. Therefore, the interfacial rheological behavior at the air–water interface of pea protein isolate (PPI) has been investigated to understand how affects food foam production. The PPI has been studied without modification and also through enzymatic treatment with transglutaminase (TG) to understand the interfacial properties of the modified proteins. Data obtained by static measurements have shown a surface activity of PPI comparable with other vegetable proteins, while the treatment with TG does not significantly alter the surface tension value and the interfacial adsorption rate. Differences have been found in the rearrangement rate, which decreases with TG, suggesting a possible crosslinking of the pea proteins. The PPI modified with TG, studied in dynamic conditions both in dilation and shear kinematics, are less elastic than PPI that is untreated but with a higher consistency, which may lead to poor foam stability. The lower complex interfacial modulus obtained under shear conditions also suggests a low long-time stability. Full article

The lipase from Prunus dulcis almonds has been immobilized for the first time. For this purpose, two different supports, an octadecyl methacrylate particulate support, and aminated agarose (monoaminoethyl-N-aminoethyl) have been utilized. Both immobilized biocatalysts show improved enzyme stability, but great changes in enzyme specificity were detected. The enzyme immobilized via ion exchange maintained its activity intact versus p-nitrophenyl butyrate, while the enzyme immobilized on the hydrophobic support fully lost its activity versus this substrate, which was confirmed to be due to substrate adsorption on the support. However, this biocatalyst was much more active versus triacetin (more than 10-fold), R- or S- methyl mandelate at pH 7. At pH 9, a strong effect of using phosphate or bicarbonate as reaction buffers was detected. Using bicarbonate, the interfacially immobilized enzyme presented no activity versus R-isomer, but it was very active versus the S-isomer and triacetin. Using a phosphate buffer during the reaction, all compounds were recognized as substrates. The enzyme immobilized via ion exchange was significantly more active using phosphate; in fact, using bicarbonate, the enzyme was inactive versus both methyl mandelate isomers. This paper shows for the first time a great interaction between the effects of the immobilization protocol and buffer used during reaction on the enantiospecificity of lipases. Full article

Oil body emulsions (OBEs) affect the final oil yield as an intermediate in the concurrent peanut oil and protein extraction process using an aqueous enzyme extraction (AEE) method. Roasting temperature promotes peanut cell structure breakdown, affecting OBE composition and stability and improving peanut oil and protein extraction rates. Therefore, this study aimed to investigate the effects of pretreatment at different roasting temperatures on peanut oil and protein yield extracted through AEE. The results showed that peanut oil and protein extraction rates peaked at 90 °C, 92.21%, and 77.02%, respectively. The roasting temperature did not change OBE composition but affected its stability. The OBE average particle size increased significantly with increasing temperature, while at 90 °C, the zeta potential peaked, and the interfacial protein concentration hit its lowest, indicating OBE stability was the lowest. Optical microscopy and confocal laser scanning microscopy confirmed the average particle size findings. The oil quality obtained after roasting treatment at 90 °C did not differ significantly from that at 50 °C. The protein composition remained unaffected by the roasting temperature. Conclusively, the 90 °C roasting treatment effectively improved the yield of peanut oil extracted using AEE, providing a theoretical basis for choosing a suitable pretreatment roasting temperature. Full article

Black phosphorene quantum dots (BPQDs) were prepared by ultrasonic-assisted liquid-phase exfoliation and centrifugation with morphologies proved by TEM results. Furthermore, an electrochemical enzyme sensor was prepared by co-modification of BPQDs with horseradish peroxidase (HRP) on the surface of a carbon ionic liquid electrode (CILE) for the first time. The direct electrochemical behavior of HRP was studied with a pair of well-shaped voltammetric peaks that appeared, indicating that the existence of BPQDs was beneficial to accelerate the electron transfer rate between HRP and the electrode surface. This was due to the excellent properties of BPQDs, such as small particle size, high interfacial reaction activity, fast conductivity, and good biocompatibility. The presence of BPQDs on the electrode surface provided a fast channel for direct electron transfer of HRP. Therefore, the constructed electrochemical HRP biosensor was firstly used to investigate the electrocatalytic behavior of trichloroacetic acid (TCA) and potassium bromate (KBrO₃), and the wide linear detection ranges of TCA and KBrO₃ were 4.0–600.0 mmol/L and 2.0–57.0 mmol/L, respectively. The modified electrode was applied to the actual samples detection with satisfactory results. Full article

The secreted phospholipases A2 (sPLA2s) play important roles both physiologically and pathologically, with their expression increasing significantly in diseases such as sepsis, inflammation, different cancers, glaucoma, obesity, Alzheimer’s disease and even COVID-19. The fact has led to a large-scale search for inhibitors of these enzymes. In total, several dozen promising molecules have been proposed, but not a single one has successfully passed clinical trials. The failures in clinical studies motivated in-depth fundamental studies of PLA2s. Here we review alternative ways to control sPLA2 activity, outside its catalytic site. The concept can be realized by preventing sPLA2 from attaching to the membrane surface; by binding to an external protein which blocks sPLA2 hydrolytic activity; by preventing sPLA2 from orienting properly on the membrane surface; and by preventing substrate binding to the enzyme, keeping the catalytic site unaltered. Evidence in the literature is summarized in the review with the aim to serve as a starting point for new types of sPLA2 inhibitors. Full article

This study is an extension of current research into a novel class of synthetic antihypertensive drugs referred to as “bisartans”, which are bis-alkylated imidazole derivatives bearing two symmetric anionic biphenyltetrazoles. Research to date indicates that bisartans are superior to commercially available hypertension drugs, since the former undergo stronger docking to angiotensin-converting enzyme 2 (ACE2). ACE2 is the key receptor involved in SARS-CoV-2 entry, thus initiating COVID-19 infection and in regulating levels of vasoactive peptides such as angiotensin II and beneficial heptapeptides A(1-7) and Alamandine in the renin–angiotensin system (RAS). In previous studies using in vivo rabbit-iliac arterial models, we showed that Na⁺ or K⁺ salts of selected Bisartans initiate a potent dose–response inhibition of vasoconstriction. Furthermore, computational studies revealed that bisartans undergo stable binding to the vital interfacial region between ACE2 and the SARS-CoV-2 “receptor binding domain” (i.e., the viral RBD). Thus, bisartan homologs are expected to interfere with SARS-CoV-2 infection and/or suppress disease expression in humans. The primary goal of this study was to investigate the role of tetrazole in binding and the network of amino acids of SARS-CoV-2 Spike RBD-ACE2 complex involved in interactions with sartans. This study would, furthermore, allow the expansion of the synthetic space to create a diverse suite of new bisartans in conjunction with detailed computational and in vitro antiviral studies. A critical role for tetrazole was uncovered in this study, shedding light on the vital importance of this group in the binding of sartans and bisartans to the ACE2/Spike complex. The in silico data predicting an interaction of tetrazole-containing sartans with ACE2 were experimentally validated by the results of surface plasmon resonance (SPR) analyses performed with a recombinant human ACE2 protein. Full article

Enzymatic hydrolysis of starch granules forms the fundamental basis of how nature degrades starch in plant cells, how starch is utilized as an energy resource in foods, and develops efficient, low-cost saccharification of starch, such as bioethanol and sweeteners. However, most investigations on starch hydrolysis have focused on its rates of degradation, either in its gelatinized or soluble state. These systems are inherently more well-defined, and kinetic parameters can be readily derived for different hydrolytic enzymes and starch molecular structures. Conversely, hydrolysis is notably slower for solid substrates, such as starch granules, and the kinetics are more complex. The main problems include that the surface of the substrate is multifaceted, its chemical and physical properties are ill-defined, and it also continuously changes as the hydrolysis proceeds. Hence, methods need to be developed for analyzing such heterogeneous catalytic systems. Most data on starch granule degradation are obtained on a long-term enzyme-action basis from which initial rates cannot be derived. In this review, we discuss these various aspects and future possibilities for developing experimental procedures to describe and understand interfacial enzyme hydrolysis of native starch granules more accurately. Full article

Exogenous fatty acid (eFA) activation and utilization play key roles in bacterial physiology and confer growth advantages by bypassing the need to make fatty acids for lipid synthesis. In Gram-positive bacteria, eFA activation and utilization is generally carried out by the fatty acid kinase (FakAB) two-component system that converts eFA to acyl phosphate, and the acyl-ACP:phosphate transacylase (PlsX) that catalyzes the reversible conversion of acyl phosphate to acyl–acyl carrier protein. Acyl–acyl carrier protein is a soluble format of the fatty acid that is compatible with cellular metabolic enzymes and can feed multiple processes including the fatty acid biosynthesis pathway. The combination of FakAB and PlsX enables the bacteria to channel eFA nutrients. These key enzymes are peripheral membrane interfacial proteins that associate with the membrane through amphipathic helices and hydrophobic loops. In this review, we discuss the biochemical and biophysical advances that have established the structural features that drive FakB or PlsX association with the membrane, and how these protein–lipid interactions contribute to enzyme catalysis. Full article