Search Results (35)

Microbial enzymes have attracted considerable attention as biocatalysts for plastic transformation, yet the experimental evidence supporting reported biodegradation varies substantially in quality and interpretation. Surface-sensitive techniques, molecular-weight analyses, identification of transformation products, microbial assimilation assays, and carbon-tracking approaches each validate different stages of polymer transformation, but they are often treated as equivalent evidence of biodegradation. This review critically examines the analytical basis of enzyme-mediated plastic and microplastic transformation and introduces the Evidence-Level Framework (ELF), which classifies studies according to the highest experimentally validated transformation endpoint, from microbial colonization (ELF 0) to polymer-derived carbon conversion (ELF 5). Systematic screening identified 102 eligible experimental studies from an initial dataset of 150 publications, all of which were classified using the ELF to provide a comprehensive assessment of the current evidence landscape. Most studies clustered within intermediate evidence levels (ELF 2–3), where analytical validation was limited to polymer chain scission or the detection of soluble transformation products. By contrast, only a small proportion demonstrated microbial assimilation or unequivocal polymer-derived carbon conversion. Hydrolysable polyesters and their associated hydrolytic enzymes consistently reached the highest ELF categories because their chemical structure, enzymatic accessibility, and analytical tractability facilitate validation of successive transformation stages. Conventional plastics, however, remain constrained by polymer recalcitrance, limited substrate accessibility, microbial metabolic capacity, and the scarcity of analytical approaches capable of tracking polymer-derived carbon through biological systems. By providing a common framework for interpreting transformation claims, the ELF establishes objective criteria for experimental design, analytical validation, and comparison across independent studies, offering a stronger foundation for more reproducible, mechanistically robust, and environmentally relevant research on microbial plastic transformation. Full article

Background/Objectives: Brassica vegetables are widely promoted as health-promoting foods, but their functional value is still commonly inferred from glucosinolate content alone. However, glucosinolates are biologically inactive precursors whose nutritional relevance depends on their conversion into bioactive hydrolysis products, particularly isothiocyanates (ITCs). This review aims to reconsider Brassica functionality through an ITC-yield-centered perspective rather than a precursor-content-based approach. Methods: This narrative review synthesizes current evidence on the biochemical, technological, gastrointestinal, and analytical determinants of glucosinolate hydrolysis and ITC formation. Particular attention is given to myrosinase activity, specifier proteins, pH, temperature, tissue disruption, food matrix effects, processing conditions, gastrointestinal transformation, inter-individual microbial variability, and the limitations of precursor-based interpretation. Results: The reviewed evidence indicates that similar glucosinolate profiles can result in markedly different levels of bioactive exposure depending on enzymatic activity, processing history, matrix context, and host-related factors. Therefore, precursor abundance alone is an unreliable surrogate for functional efficacy. Functional optimization should not aim at indiscriminate maximization of glucosinolate breakdown, but rather at selective promotion of beneficial ITC-forming pathways while limiting nitriles, epithionitriles, and goitrogenic products such as goitrin. Conversion efficiency emerges as a critical analytical bridge between glucosinolate abundance and food-level ITC formation, while subsequent bioaccessibility and host-related factors determine realized exposure. Conclusions: Brassica functionality could be more meaningfully evaluated through an ITC-yield-centered framework in which precursor abundance and conversion efficiency determine food-level ITC formation, while subsequent bioaccessibility and host-related factors shape realized exposure. This approach provides an integrative basis for breeding, food processing, product development, dietary guidance, and future evaluation of Brassica foods. Full article

Vitamin D comprises a group of fat-soluble secosteroids traditionally associated with animal physiology, calcium-phosphate homeostasis, and skeletal metabolism. However, vitamin D and related compounds have also been reported in taxonomically distinct non-animal systems, including fungi, microalgae, other algae, phytoplankton, and higher plants, although the strength of evidence differs substantially among these groups. This review synthesizes current knowledge on the occurrence, structural chemistry, UV-B-driven photochemical mechanisms, environmental determinants, analytical challenges, and biofortification potential of vitamin D formation in photosynthetic organisms and fungi. Vitamin D synthesis is initiated by UV-B radiation, primarily within the 290–315 nm range, which converts sterol precursors such as 7-dehydrocholesterol and ergosterol into previtamin D intermediates and is followed by thermal isomerization to the corresponding vitamin D forms. Continued irradiation may additionally generate lumisterol, tachysterol, and other photoproducts, thereby limiting net vitamin D accumulation. This non-enzymatic mechanism supports the interpretation that vitamin D formation can occur outside vertebrates when an appropriate 5,7-diene sterol precursor is accessible to a sufficient UV-B dose. In photosynthetic organisms and fungal matrices, net vitamin D accumulation is constrained by the spectral dose of UV-B, environmental exposure, tissue architecture, sterol localization, oxygen availability, antioxidant capacity, and ROS-mediated degradation. Studies of microalgae and phytoplankton, including reports concerning Emiliania huxleyi, suggest the occurrence or UV-B-dependent formation of both vitamin D₂ and vitamin D₃. However, these findings require evaluation according to the analytical method, use of authentic standards, experimental conditions, and confidence of compound identification. In fungi, the UV-B-induced conversion of abundant ergosterol to vitamin D₂ is well established. Microalgae represent a developing source of vitamin D₂ and vitamin D₃, whereas evidence for nutritionally relevant vitamin D accumulation in higher plants remains limited and heterogeneous. Although higher plants contain diverse phytosterols, the formation of vitamin D₄, vitamin D₅, or related analogues requires appropriate photoreactive 5,7-diene precursors and should not be inferred directly from the presence of common phytosterols such as β-sitosterol. Analytical detection remains challenging because of low concentrations, complex lipophilic matrices, and structural similarity among secosteroids and photoproducts; therefore, reliable identification requires validated analytical procedures. LC-MS/MS provides high sensitivity and selectivity but should be supported by authentic standards, preferably isotope-labelled internal standards, retention-time agreement, quantitative and qualifying ions, matrix-recovery assessment, limits of detection and quantification, and evaluation of ion suppression. Structurally similar analogues and photoproducts may additionally require orthogonal confirmation. Nutritionally, post-harvest UV-B enrichment of edible mushrooms is currently the best-validated strategy for increasing non-animal vitamin D₂ content. Microalgae constitute a developing platform for vitamin D₂ and vitamin D₃ production, whereas biofortification of higher plants remains experimental. Full article

Collagen-based biomaterials possess many advantages, such as low immunogenicity, biodegradability, biocompatibility, hydrophilicity, and ease of processability. Nevertheless, natural collagen has inherent limitations as an in vivo scaffold, including insufficient mechanical strength, low thermal stability, and low resistance to enzymatic degradation. To overcome these drawbacks, various approaches have been studied, such as mixing collagen with other biopolymers or inducing physical and chemical crosslinking. However, using non-biologically derived polymers or crosslinking agents carries the risk of persistence in the body, potentially causing cytotoxicity. Considering this, recent studies have reported that the molecular flexibility of collagen networks can be improved by activating the carboxyl groups of collagen chains using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide and then crosslinking them through amide bonding with the amino groups present in the collagen chains, or by adding free L-lysine to induce a crosslinking reaction. When the carboxyl groups of collagen are activated and form covalent bonds with amino groups, native ionic interactions (e.g., salt bridges) may be reduced, which can potentially influence the stability of its inherent higher-order structure. In this study, we proposed a selective amine-targeted cross-linking strategy designed to minimize modification of collagen carboxyl groups while enhancing mechanical properties and cellular compatibility. First, free L-glutamic acid was pre-activated to cross-link collagen chains through amide bonds with the amino groups of L-lysine residues, thereby providing a cross-linking pathway intended to reduce the involvement of collagen carboxyl groups in the reaction. By controlling the concentration of L-glutamic acid, the cross-linking rate of the collagen could be controlled within a range of 10.26% to 25.02%. All cross-linked collagen scaffolds exhibited higher tensile strength compared to non-cross-linked scaffolds. Although the scaffolds with a high cross-linking rate (25.02%) displayed excellent mechanical properties, their cellular compatibility was relatively low. Conversely, collagen scaffolds with cross-linking rates of 10.26% and 14.43% demonstrated excellent mechanical properties and very high cellular compatibility, suggesting potential applications in the fields of biomedicine and tissue engineering. The present findings are consistent with the proposed selective cross-linking strategy; however, direct experimental verification of collagen carboxyl-group preservation will require complementary analytical studies. Full article

Background/Objectives: Bisulfite-based cell-free DNA (cfDNA) methylation assays enable the detection of clinically valuable epigenetic biomarkers but often cause DNA degradation and inconsistent conversion efficiency, limiting performance in low-input liquid biopsy samples. We aimed to develop and evaluate a fully enzymatic cfDNA methylation workflow that preserves DNA integrity and supports quantitative clinical detection. Methods: The assay integrates TET2-mediated oxidation and APOBEC3A deamination with RNase H2-guided primer design, uracil-DNA glycosylase error suppression, and dual-probe detection compatible with quantitative PCR (qPCR) and digital PCR (dPCR). Performance was assessed using serial dilutions of methylated HT29 DNA, unmethylated controls, and plasma cfDNA from colorectal cancer (CRC) patients and healthy donors. Analytical sensitivity, linearity, and concordance between platforms were evaluated. Results: The 40-marker panel demonstrated higher cumulative methylation scores and more frequent methylation-positive signals in CRC cfDNA compared to controls. dPCR confirmed single-molecule resolution and clear discrimination between methylated and unmethylated templates, with occasional double-positive partitions consistent with mixed allelic methylation. Signal intensity across the dilution series followed a four-parameter logistic model, achieving detection sensitivity below 0.2% methylated DNA. qPCR and dPCR results showed strong correlation across the HT29 dilution series (R² = 0.80) and high concordance in classifying CRC and healthy samples. Conclusions: This TET2–APOBEC-based enzymatic cfDNA assay enables sensitive, quantitative, sequencing-free methylation detection under gentle conditions, supporting its application in early colorectal cancer screening and routine clinical liquid biopsy workflows. Full article

The poly(A) tail of mRNA plays a vital role in mRNA transcript stability, translational efficiency, and immunogenicity. Co-transcriptionally polyadenylated in vitro transcribed (IVT) mRNAs typically contain poly(A) tails of 50–120 nucleotide tail length due to limitations in production of template pDNA with longer poly(A) sequences. In contrast, post-transcriptional enzymatic polyadenylation of mRNA with poly(A) polymerase (PAP) presents a modular alternative to increase the tail length. However, the lack of real-time control strategies for PAP-mediated tailing has limited its broader applicability in mRNA production. Here, we develop a methodology for controlling poly(A) tail length in post-transcriptional polyadenylation of mRNA that uses adenosine triphosphate (ATP) consumption measured at-line to predict the poly(A) tail length. We establish a novel analytical method based on monolith reverse-phase chromatography to validate the poly(A) predictions. We were able to produce longer poly(A) tails and accurately determine their length in 300–700 nt range. The resulting longer poly(A) tailed reporter mRNAs outperformed the encoded and shorter poly(A) tailed mRNAs in cell-based assays. This work presents a new strategy for controlled post-transcriptional polyadenylation using ATP consumption as a process control metric, an approach which may in future be expanded to other NTP-dependent enzymatic conversions. Full article

In this study, the nonlinear and non-monotonic behavior of amperometric bienzyme biosensors employing an enzymatic trigger reaction is investigated analytically and computationally using a two-compartment model comprising an enzymatic layer and an outer diffusion layer. The trigger enzymatic reaction is coupled with a cyclic electrochemical–enzymatic conversion (CEC) process. The model is formulated as a system of reaction–diffusion equations incorporating nonlinear Michaelis–Menten kinetics and interlayer partitioning effects. Exact steady-state analytical solutions for substrate and product concentrations, as well as for the output current, are obtained for specific cases of first- and zero-order reaction kinetics. At the transition conditions, biosensor performance is further analyzed numerically using the finite difference method. The CEC biosensor exhibits the highest signal gain when the first enzyme has low activity and the second enzyme has high activity; however, under these conditions, the response time is the longest. When the first enzyme possesses a higher substrate affinity (lower Michaelis constant) than the second, the biosensor demonstrates severalfold higher current and gain compared to the reverse configuration under identical diffusion limitations. Furthermore, increasing external mass transport resistance or interfacial partitioning can enhance the apparent signal gain. Full article

Biodiesel is an alternative, sustainable, renewable, and environmentally friendly energy source, which has generated interest from the scientific community due to its low toxicity, rapid biodegradability, and zero carbon footprint. Biodiesel is a biofuel produced by the transesterification of triglycerides or the esterification of free fatty acids (FFA). Both reactions require catalysts with numerous active sites (basic, acidic, bifunctional, or enzymatic) for efficient biodiesel production. On the other hand, since the late 1990s, metal–organic frameworks (MOFs) have emerged as a new class of porous materials and have been successfully used in various fields due to their multiple properties. For this reason, MOFs have been used as heterogeneous catalysts or as a platform for designing active sites, thus improving stability and reusability. This literature review presents a comprehensive analysis of using MOFs as heterogeneous catalysts or supports for biodiesel production. The optimal parameters for transesterification/esterification are detailed, such as the alcohol/feedstock molar ratio, catalyst amount, reaction time and temperature, conversion percentage, biodiesel yield, fatty acid and water content, etc. Additionally, novel methodologies such as ultrasound and microwave irradiation for obtaining MOF-based catalysts are described. It is important to note that most studies have shown biodiesel yields >90% and multiple reuse cycles with minimal activity loss. The bibliographic analysis was conducted using the American Chemical Society (ACS) Scifinder^® database, the Elsevier B.V. Scopus^® database, and the Clarivate Analytics Web of Science^® database, under the institutional license of the Universidad Veracruzana. Keywords were searched for each section, generally limiting the document type to “reviews” and “journals,” and the language to English, and published between 2000 and 2025. Full article

Biochemical monomer upcycling of plastic waste and its conversion into value-added products is deemed necessary, as it provides a greener and more sustainable solution to plastic waste management. In the current study, the polystyrene (PS) biodegradation potential of the fungus Phanerochaete chrysosporium NA3 was evaluated using various analytical techniques, such as Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), gel permeation chromatography (GPC), and high-performance liquid chromatography (HPLC). The biodegradation capacity of the fungal strain was further evaluated using a carbon dioxide (CO₂) evolution test, which showed that the PS films treated with NA3 produced more CO₂, indicating the strain’s ability to successfully utilize PS as a carbon source. The FTIR analysis of the PS films treated with NA3 showed modifications in the polymer chemical structure, including the formation of carbonyl and hydroxyl groups, which suggests the enzymatic dissociation of the polymer and the associated biodegradation mechanism. Pretreatments were found to be effective in modifying the polymer’s properties, making it more susceptible to microbial degradation, thus further accelerating the biodegradation process. The current study strongly advocates that P. chrysosporium (NA3) can be effectively used for the biochemical monomer recovery of PS waste and could be further utilized in the upcycling of plastic waste for its conversion into value-added products under the concept of circular economy. Full article

The processing of Kappaphycus alvarezii algae to obtain carrageenan (polysaccharide) generates a residue composed mainly of glucans and galactans that can be converted to monosaccharides, making these algae a renewable feedstock that can be used to produce biofuels. This residue was subjected to batch enzyme hydrolysis with different commercial enzymatic cocktails, achieving, after 72 h of reaction time, a complete conversion of glucan to glucose for all the cocktails used. A simple mathematical model, based on a semi-empirical approach, was proposed to describe the behavior of the experimental data. The temporal profile of glucose concentration was obtained by direct analytical integration of the mathematical model, resulting in an explicit equation as a time function. Estimation of the model parameters was carried out by non-linear regression, using the least squares criterion, together with the Levenberg–Marquardt method. The quality of the model fit was evaluated by specific statistical criteria, including Fisher’s F test, the R² value, and the p-value test. The accuracy of the model was considered acceptable (p-value < 0.05 and R² ≥ 0.98), enabling its use in subsequent studies aimed at improving the enzymatic hydrolysis process under similar experimental conditions. Full article

Dopamine is a neurotransmitter which is classified as a catecholamine. It is also one of the main metabolites produced by some tumor types (such as paragangliomas and neoblastomas). As such, determining and monitoring the level of dopamine is of the utmost importance, ideally using analytical techniques that are sensitive, simple, and low in cost. Due to this, we have developed a non-enzymatic dopamine sensor that is highly sensitive, selective, and rapidly detects the presence of dopamine in the body. A hybrid material fabricated with NiO and ZnO, based on date fruit extract, was synthesized by hydrothermal methods and using NiO as a precursor material. This paper discusses the role of date fruit extracts in improving NiO’s catalytic performance with reference to ZnO and the role that they play in this process. An X-ray powder diffraction study, a scanning electron microscope study, and a Fourier transform infrared spectroscopy study were performed in order to investigate the structure of the samples. It was found that, in the composite NiO/ZnO, NiO exhibited a cubic phase and ZnO exhibited a hexagonal phase, both of which exhibited well-oriented aggregated cluster shapes in the composite. A hybrid material containing NiO and ZnO has been found to be highly electro-catalytically active in the advanced oxidation of dopamine in a phosphate buffer solution at a pH of 7.3. It has been found that this can be accomplished without the use of enzymes, and the range of oxidation used here was between 0.01 mM and 4 mM. The detection limit of non-enzymatic sensors is estimated to be 0.036 μM. Several properties of the non-enzymatic sensor presented here have been demonstrated, including its repeatability, selectivity, and reproducibility. A test was conducted on Sample 2 for the detection of banana peel and wheat grass, and the results were highly encouraging and indicated that biomass waste may be useful for the manufacture of medicines to treat chronic diseases. It is thought that date fruit extracts would prove to be valuable resources for the development of next-generation electrode materials for use in clinical settings, for energy conversion, and for energy storage. Full article

The association between microRNA (miRNA) and various diseases has been established; miRNAs have the potential to be biomarkers for these diseases. Nevertheless, the challenge of correctly quantifying an miRNA arises from its low abundance and a high degree of family homology. Therefore, in the present study, we devised a chemiluminescence (CL) detection method for miRNAs, known as the hybridization chain reaction (HCR)-CL, utilizing the enzyme-free signal amplification technology of HCR. The proposed methodology obviates the need for temperature conversion and offers a straightforward procedure owing to the absence of enzymatic participation, and the lumino-H₂O₂-mediated CL reaction occurs at a high rate. The technique successfully detected 2.5 amol of the target analyte and 50 amol of miR-146b in a 1% concentration of human serum. In summary, the method developed in this study is characterized by its ease of operation, cost-effectiveness, remarkable analytical prowess, and ability to detect miRNA without the need for total RNA extraction from serum samples. This method is expected to be widely used for biological sample testing in clinical settings. Full article

The use of non-invasive liquid biopsy-based cell-free DNA (cfDNA) analysis is an emerging method of cancer detection and intervention. Different analytical methodologies are used to investigate cfDNA characteristics, resulting in costly and long analysis processes needed for combining different data. This study investigates the possibility of using cfDNA data converted for methylation analysis for combining the cfDNA fragment size with copy number variation (CNV) in the context of early colorectal cancer detection. Specifically, we focused on comparing enzymatically and bisulfite-converted data for evaluating cfDNA fragments belonging to chromosome 18. Chromosome 18 is often reported to be deleted in colorectal cancer. We used counts of short and medium cfDNA fragments of chromosome 18 and trained a linear model (LDA) on a set of 2959 regions to predict early-stage (I–IIA) colorectal cancer on an independent test set. In total, 87.5% sensitivity and 92% specificity were obtained on the enzymatically converted libraries. Repeating the same workflow on bisulfite-converted data yielded lower accuracy results with 58.3% sensitivity, implying that enzymatic conversion preserves the cancer fragmentation footprint in whole genome data better than bisulfite conversion. These results could serve as a promising new avenue for the early detection of colorectal cancer using fragmentation and methylation approaches on the same datasets. Full article

Potato peel wastes are generated in high quantities from potato processing industries. They are pollutants to the environment, and they release greenhouse gases into the atmosphere. The present study assessed the potentiality of hydrolyzing potato wastes by amylase-producing fungi to improve biogas generation from potato peels through the anaerobic digestion process. Different fungal isolates were screened for amylase production on potato wastes, and the highest amylase producer was selected for optimizing the efficacy of producing amylases in high quantities to efficiently allow the conversion of potato organic matter into fermentable sugars that are utilized for the anaerobic digestion process. The best amylase producers were those derived from Rhizopus stolonifer (32.61 ± 0.89 U/mL). The highest cumulative methane yield from hydrolyzed potato peel was 65.23 ± 3.9 mL CH₄/g and the methane production rate was 0.39 mL CH₄/h, whereas the highest biogas yield from unhydrolyzed potato wastes was 41.32 ± 2.15 mL CH₄/g and the biogas production rate was 0.25 mL CH₄/h. Furthermore, it was found that the two combined sequential stages of anaerobic digestion (biogas production) followed by biodiesel production (enzymatic esterification) were the most effective, recording 72.36 ± 1.85 mL CH₄/g and 64.82% biodiesel of the total analytes. However, one-pot fermentation revealed that biogas yield was 22.83 ± 2.8 mL CH₄/g and the biodiesel extracted was 23.67% of the total analytes. The insights of the current paper may increase the feasibility of potato peel-based biorefinery through the biological hydrolysis strategy of potato wastes using eco-friendly enzymes. Full article

Flavonoids are biologically active natural products of great interest for their potential applications in functional foods and pharmaceuticals. A hesperetin-7-O-glucoside inclusion complex with β-cyclodextrin (HEPT7G/βCD; SunActive^® HCD) was formulated via the controlled enzymatic hydrolysis of hesperidin with naringinase enzyme. The conversion rate was nearly 98%, estimated using high-performance liquid chromatography analysis. The objective of this study was to investigate the stability, solubility, and spectroscopic features of the HEPT7G/βCD inclusion complex using Fourier-transform infrared (FTIR), Raman, ultraviolet–visible absorption (UV–vis), ¹H- and ¹³C- nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), liquid chromatography/mass spectroscopy (LC–MS), scanning electron microscopy (SEM), and powdered X-ray diffraction (PXRD) spectroscopic techniques including zeta potential, Job’s plot, and phase solubility measurements. The effects of complexation on the profiles of supramolecular interactions in analytic features, especially the chemical shifts of β-CD protons in the presence of the HEPT7G moiety, were evaluated. The stoichiometric ratio, stability, and solubility constants (binding affinity) describe the extent of complexation of a soluble complex in 1:1 stoichiometry that exhibits a greater affinity and fits better into the β-CD inner cavity. The NMR spectroscopy results identified two different configurations of the HEPT7G moiety and revealed that the HEPT7G/βCD inclusion complex has both –2S and –2R stereoisomers of hesperetin-7-O-glucoside possibly in the –2S/–2R epimeric ratio of 1/1.43 (i.e., –2S: 41.1% and –2R: 58.9%). The study indicated that encapsulation of the HEPT7G moiety in β-CD is complete inclusion, wherein both ends of HEPT7G are included in the β-CD inner hydrophobic cavity. The results showed that the water solubility and thermal stability of HEPT7G were apparently increased in the inclusion complex with β-CD. This could potentially lead to increased bioavailability of HEPT7G and enhanced health benefits of this flavonoid. Full article