Enzymatic Degradation Research Articles

Multifunctional DNA-Collagen Biomaterials: Developmental Advances and Biomedical Applications.

The complexation of nucleic acids and collagen forms a platform biomaterial greater than the sum of its parts. This union of biomacromolecules merges the extracellular matrix functionality of collagen with the designable bioactivity of nucleic acids, enabling advances in regenerative medicine, tissue engineering, gene delivery, and targeted therapy. This review traces the historical foundations and critical applications of DNA-collagen complexes and highlights their capabilities, demonstrating them as biocompatible, bioactive, and tunable platform materials. These complexes form structures across length scales, including nanoparticles, microfibers, and hydrogels, a process controlled by the relative amount of each component and the type of nucleic acid and collagen. The broad distribution of different types of collagen within the body contributes to the extensive biological relevance of DNA-collagen complexes. Functional nucleic acids can form these complexes, such as siRNA, antisense oligonucleotides, DNA origami nanostructures, and, in particular, single-stranded DNA aptamers, often distinguished by their rapid self-assembly at room temperature and formation without external stimuli and modifications. The simple and seamless integration of nucleic acids within collagenous matrices enhances biomimicry and targeted bioactivity, and provides stability against enzymatic degradation, positioning DNA-collagen complexes as an advanced biomaterial system for many applications including angiogenesis, bone tissue regeneration, wound healing, and more.

Enzymatic Degradation of Ochratoxin A: The Role of Ultra-Pure Water

Ochratoxin A (OTA) is a toxic mycotoxin, making its removal from food essential for public health. This study examines OTA degradation by porcine pancreatic lipase (PPL) in ultra-pure water versus buffer systems through in vitro assays and molecular modeling. The results show that PPL fully degrades OTA in ultra-pure water within 7 h at 44 °C, whereas only partial degradation occurs in phosphate buffer. After 4 h, PPL in water degrades 91% of OTA, compared to only 12% in buffer. The enzyme’s half-life is longer in water (~4 h 4 min) than in phosphate buffer (~2 h 30 min), suggesting better stability in water. Other buffers, including acetate, citrate, and borate, confirmed higher degradation efficiency in low-conductivity, acidic environments similar to ultra-pure water. Additionally, using the model compound p-nitrophenyl octanoate (p-NPO), it was found that p-NPO degrades faster in buffer, likely due to a salting-out effect. Molecular modeling and circular dichroism analysis indicate that PPL’s secondary structure in water promotes an ideal conformation for OTA binding. This study suggests ultra-pure water as a greener, sustainable option for reducing mycotoxins in food, with broad industrial applications.

Enhancing Probiotic Viability in Yogurt: The Role of Apple Fibers in Supporting Lacticaseibacillus casei ATCC 393 During Storage and Gastrointestinal Transit

Probiotics are widely recognized for their health benefits, but their viability during food processing and digestion poses significant challenges. The present study evaluated the impact of incorporating apple fibers into yogurt on the viability of the probiotic strain Lacticaseibacillus casei ATCC 393 during production, storage, and simulated gastrointestinal digestion. Apple fibers, a by-product of apple processing, were used as a prebiotic ingredient due to their functional and technological benefits. The incorporation of apple fibers increased probiotic viability during 28 days of refrigerated storage, improving it from 90.4% in the control yogurt to 93.9%. Under simulated gastrointestinal conditions, yogurt alone acted as a protective matrix, preserving probiotic viability, during gastric (71.0% at pH 2 after 3 h) and intestinal digestion (73.3% at 0.3% bile salts after 6 h). The inclusion of apple fibers further enhanced this protection, reducing probiotic viability loss in both gastric (81.9% at pH 2 after 3 h) and intestinal (79.0% at 0.3% bile salts after 6 h) environments. Similar results were obtained using the INFOGEST 2.0 static protocol. After the completion of the protocol (oral, gastric and intestinal phase) a viability of 71.1% (6.61 logCFU/g) was observed in the yogurt with apple fibers compared to 64.5% (6.10 logCFU/g) in the control yogurt. This enhanced protection could be attributed to the potential prebiotic properties of apple fibers, including their pectin and cellulose content, which may shield probiotics from acidic and enzymatic degradation. These findings highlight the potential of apple fiber-enriched yogurt as a functional food that supports probiotic viability during storage and throughout gastrointestinal transit. These insights may open the way for developing new food products with enhanced health benefits, aligning with growing consumer demand for functional foods.

Pathway Elucidation and Key Enzymatic Processes in the Biodegradation of Difenoconazole by Pseudomonas putida A-3.

The extensive agricultural use of the fungicide difenoconazole (DIF) and its associated toxicity increasingly damage ecosystems and human health. Thus, an urgent need is to develop environmentally friendly technological approaches capable of effectively removing DIF residues. In this study, strain Pseudomonas putida A-3 was isolated for the first time which can degrade DIF efficiently. After optimization of the degradation conditions, the degradation rate reached 75.98%. Moreover, a new DIF degradation pathway, including hydroxylation, hydrolysis, dechlorination, and ether bond breaking. The acute and chronic toxicity of DIF degradation products assessed using ECOSAR software showed lower toxicity than the parent compound. Furthermore, strain A-3 remarkably accelerated the degradation of DIF in contaminated water-sediment systems. We successfully predicted six potential key enzymes for DIF degradation based on the results of whole genome sequencing, RT-qPCR, and molecular docking. Overall, the results revealed novel pathways for DIF biodegradation and provide a strong candidate for bioremediation of DIF residue-polluted environments.

Enzymatic degradation of polybutylene succinate by recombinant cutinase cloned from Paraphoma chrysanthemicola.

Polybutylene succinate (PBS), a biodegradable plastic, can be used as an alternative to traditional plastics to effectively solve the growing plastic pollution. Although PBS is theoretically completely biodegradable, slow degradation remains a problem in practical applications, leading to the possibility of environmental pollution. In this study, after the PBS degradation ability of the fungus Paraphoma chrysanthemicola was determined, a P. chrysanthemicola cutinase (PCC) gene was cloned and expressed in Pichia pastoris and its PBS degradation ability was further characterized. With a molecular weight of approximately 20kDa, PCC showed good PBS degradation activity at pH 6.0-8.0 and 20-40°C. Metal ions have different effects on PCC activity. Specifically, Ca2+, Zn2+, and Co2+ promoted enzyme activity, whereas Cu2+, Fe2+, and Ni2+ inhibited enzyme activity. The weight loss of the PBS films was greater than 50% after 60h of PCC treatment, and scanning electron microscopy revealed the appearance of cracks on the surface of the PBS films during the degradation process, which deepened with the progression of degradation time. This PBS degradation by PCC occurs via surface erosion, with the resulting degradation products being mainly 1,4-succinic acid and succinic acid butanediyl ester. This study provides a preliminary elucidation of the enzymatic mechanisms involved in PBS degradation by PCC and offers insights into the development of more effective biotechnological approaches to address the environmental challenges associated with plastic waste.

Effect of Glycosylation on the Enzymatic Degradation of D-Amino Acid-Containing Peptides

Effect of Glycosylation on the Enzymatic Degradation of D-Amino Acid-Containing Peptides

Platelet-rich plasma alleviates skin photoaging by activating autophagy and inhibiting inflammasome formation.

Platelet-rich plasma (PRP) holds promising prospects for the treatment of skin photoaging. This study aims to unravel the mechanism underlying PRP's anti-photoaging properties. Partial skin of rats was irradiated with ultraviolet (UV) and injected with PRP, and the skin appearance, pathological state, and aging conditions were determined. Apoptosis, reactive oxygen species (ROS), and collagen levels in skin tissues were detected. HaCaT cells were stimulated with UVB, and the effects of PRP on cells and collagen degradation enzymes were evaluated. Furthermore, the mechanism of the autophagy-NLRP3 inflammasome pathway was explored by treating cells with the autophagy inhibitor 3-MA. Erythema, ulceration, and wrinkles appeared on the skin of rats after being irradiated by UV. PRP could enhance skin tenderness and improve skin pathology and aging. PRP inhibited cell apoptosis, ROS generation, and collagen degradation in skin tissue. PRP elevated UVB-stimulated HaCaT cell activity, reduced oxidative stress, senescence, and MMP-1. Furthermore, 3-MA treatment reversed the inhibition of NLRP3 inflammasome by PRP, suggesting that autophagy mediated the regulation of PRP. To summarize, this study elucidates the regulatory mechanism of PRP on the autophagy-NLRP3 inflammasome pathway in the photoaging. These findings may provide a novel theoretical foundation for the clinical application of PRP.

Hyperelastic constitutive modeling of healthy and enzymatically mediated degraded articular cartilage.

This research demonstrates a systematic curve fitting approach for acquiring parametric values of hyperelastic constitutive models for both healthy and enzymatically mediated degenerated cartilage to facilitate finite element modeling of cartilage. Several widely used phenomenological hyperelastic constitutive models were tested to adequately capture the changes in cartilage mechanics that vary with the differential/unequal abundance of matrix metalloproteinases (MMPs). Trauma and physiological conditions result in an increased production of collagenases (MMP-1) and gelatinases (MMP-9), which impacts the load-bearing ability of cartilage by significantly deteriorating its extracellular matrix (ECM). The material parameters in the constitutive equation of each hyperelastic model are significant for developing a comprehensive computational interpretation of MMP mediated degenerated cartilage. Stress-strain responses obtained from indentation test were fitted with selected Ogden, polynomial, reduced polynomial, and van der Waals hyperelastic constitutive models by optimizing their adjustable parameters (material constants). The goodness of fit of the 2nd order reduced polynomial and van der Waals model exhibited the closest data fitting with the experimental stress-strain distributions of healthy and degraded articular cartilage. The coefficient of the shear modulus for the 2nd order reduced polynomial decreased gradually by 21.9% to 80.1% with more enzymatic degradation of collagen fibril due to the relative abundance of MMP-1 (collagenases), and 28.5% to 69.2% for the van der Waals model. Our findings showed that the major materials coefficients of the models were reduced in the degenerated cartilages, and the reduction varied differentially with the relative abundance of MMPs-1 and 9, correlating the severity of degeneration. This work advances the understanding of cartilage mechanics and offers insights into the impact of biochemical (enzymatic) effects on cartilage degradation.

Release Profile and Antibacterial Activity of Thymus sibthorpii Essential Oil-Incorporated, Optimally Stabilized Type I Collagen Hydrogels.

Antimicrobial resistance is one of the drastically increasing major global health threats due to the misuse and overuse of antibiotics as traditional antimicrobial agents, which render urgent the need for alternative and safer antimicrobial agents, such as essential oils (EOs). Although the strong antimicrobial activity of various EOs has already been studied and revealed, their characteristic high sensitivity and volatility drives the need towards a more efficient drug administration method via a biomaterial system. Herein, the potential of Thymus sibthorpii EO incorporated in functionalized antibacterial collagen hydrogels was investigated. At first, the optimally stabilized type I collagen hydrogels via six different multi-arm poly (ethylene glycol) succinimidyl glutarate (starPEG) crosslinkers were determined by assessing the free amine content and the resistance to enzymatic degradation. Subsequently, 0.5, 1, and 2% v/v of EO were incorporated into optimized collagen hydrogels, and the release profile, as well as release kinetics, were studied. Finally, biomaterial cytocompatibility tests were performed. Thymus sibthorpii EO was released from the hydrogel matrix via Fickian diffusion and showed sustained release and 0.5% v/v EO-loaded hydrogels showed adequate antibacterial activity against Staphylococcus aureus and did not show any statistically significant difference compared to penicillin (p < 0.05). Moreover, none of the fabricated composite antibacterial scaffolds displayed any cytotoxicity on NIH-3T3 fibroblasts. In conclusion, this work presents an innovative antibacterial biomaterial system for tissue engineering applications, which could serve as a promising alternative to antibiotics, contributing to coping with the issue of antimicrobial resistance.

Recent Advances in Peptide-Loaded PLGA Nanocarriers for Drug Delivery and Regenerative Medicine.

Peptide-loaded poly(lactide-co-glycolide) (PLGA) nanocarriers represent a transformative approach to addressing the challenges of peptide-based therapies. These systems offer solutions to peptide instability, enzymatic degradation, and limited bioavailability by providing controlled release, targeted delivery, and improved stability. The versatility of PLGA nanocarriers extends across therapeutic domains, including cancer therapy, neurodegenerative diseases, vaccine development, and regenerative medicine. Innovations in polymer chemistry, surface functionalization, and advanced manufacturing techniques, such as microfluidics and electrospraying, have further enhanced the efficacy and scalability of these systems. This review highlights the key physicochemical properties, preparation strategies, and proven benefits of peptide-loaded PLGA systems, emphasizing their role in sustained drug release, immune activation, and tissue regeneration. Despite remarkable progress, challenges such as production scalability, cost, and regulatory hurdles remain.

Degradation mechanism of PGNa by the immobilized degrading enzymes from magnetic fermentation residue biochar.

The development of a method to efficiently remove high concentrations of penicillin G sodium (PGNa) from the environment is important for human and animal health and safety. In this study, the degradative enzymes were immobilized by adsorption using biochar from penicillin fermentation waste residue, which could efficiently remove PGNa (900mg/L) from an aqueous solution, with a removal rate of 99.84% within 20min. The successful immobilization of the PGNa-degrading enzymes was verified by scanning electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric analyzer. The adsorption mechanism of SAMB on the enzymes was also analyzed by adsorption kinetics and molecular docking calculations, which were mainly chemisorption and physisorption. By comparing with the free enzymes, the immobilized enzymes were found to have better thermal stability, pH adaptability, reusability, and storage stability. In addition, combined with the LC-MS analysis of the removal products, three removal pathways were postulated, including the reactions of β-lactam ring cleavage, decarboxylation, demethylation, deamidation, and oxidation. Finally, the toxicity of the removal products was evaluated using ECOSAR software, and the results showed that the intermediate products had low toxicity. This study is the first to use penicillin waste residue immobilized degradative enzymes to remove PGNa, and it provides a new low-cost option for remediation of PGNa contamination in the environment. It is important for the efficient degradation of residual PGNa in the environment.

A Novel Process for Oleacein Production from Olive Leaves Using Freeze Drying Methodology.

The abundant yet underutilized olive leaves, a renewable by-product of olive cultivation, offer untapped potential for producing high-value bioactive compounds, notably oleacein. Existing extraction methods are often inefficient, yielding low quantities of oleacein due to enzymatic degradation of its precursor, oleuropein, during conventional processing and storage. This study aimed to overcome these limitations by exploring a novel methodology based on freeze-drying, to facilitate the in situ enzymatic biotransformation of oleuropein into oleacein directly within the plant matrix. Olive leaves were subjected to three drying methods-ambient air drying, microwave drying, and freeze-drying-and their phenolic profiles were analyzed. The findings demonstrated that freeze drying uniquely promotes the selective activation of β-glucosidase and esterase enzymes while simultaneously inhibiting oxidative enzymes, such as polyphenol oxidase and peroxidase, resulting in significantly enriched oleacein content. This process eliminates the need for extensive post-extraction transformations, providing a cost-effective, scalable, and sustainable approach to oleacein production. The proposed methodology aligns with circular economy principles and holds substantial potential for applications in pharmaceuticals, nutraceuticals, and functional food industries.

Laccase immobilized on reduced graphene oxide sponges for simultaneous adsorption and enzymatic degradation of endocrine disrupting chemicals.

The presence of endocrine disrupting chemicals (EDCs) in water can impart detrimental effects on public health by mimicking the behaviors of natural hormones and their associated receptors in human body. Studies have demonstrated that ligninolytic enzymes such as laccase can degrade various phenolic compounds, including a broad range of EDCs. In this study, the technique of covalent immobilization of laccase through carbodiimide coupling chemistry on highly adsorptive reduced graphene oxide (rGO) sponges was utilized to effectively remove two representative EDCs; namely, bisphenol A (BPA) and triclosan (TCS) from water. The bio-functionalized adsorbent (rGO-LA) showed a significant improvement in removing BPA (87 % after 24 h) compared to pristine rGO sponge, (~40 % after 24 h). The removal efficiency of both adsorbents for TCS was as high as 84 %, with faster kinetics being observed for rGO-LA. Further investigation using gas-chromatography-mass spectroscopy revealed that the bio-functionalization not only improved the removal efficiency of the adsorbent, but also facilitated the adsorption of the metabolites generated during the biodegradation of BPA and TCS. When temperature was increased to 40 °C, the removal efficiency and kinetics of rGO-LA sponges were improved significantly for BPA (83 % removal in 4 h) and TCS (73 % removal after 4 h). The study highlights the synergy of enzymatic degradation and adsorption, with enhanced performance observed at elevated temperatures, offering a promising solution for effective EDCs mitigation in water treatment, while also ensuring comprehensive contaminant removal by adsorbing the generated metabolites.

Fibroin-Hybrid Systems: Current Advances in Biomedical Applications.

Fibroin, a protein extracted from silk, offers advantageous properties such as non-immunogenicity, biocompatibility, and ease of surface modification, which have been widely utilized for a variety of biomedical applications. However, in vivo studies have revealed critical challenges, including rapid enzymatic degradation and limited stability. To widen the scope of this natural biomacromolecule, the grafting of polymers onto the protein surface has been advanced as a platform to enhance protein stability and develop smart conjugates. This review article brings into focus applications of fibroin-hybrid systems prepared using chemical modification of the protein with polymers and inorganic compounds. A selection of recent preclinical evaluations of these hybrids is included to highlight the significance of this approach.

Impact of Enzymatic Degradation Treatment on Physicochemical Properties, Antioxidant Capacity, and Prebiotic Activity of Lilium Polysaccharides.

In order to overcome the bioavailability limitation of Lilium polysaccharide (LPS) caused by its high molecular weight and complex structure, two low-molecular-weight degraded polysaccharides, namely G-LPS(8) and G-LPS(16), were prepared through enzymatic degradation. The molecular weight of LPS was significantly reduced by enzymolysis, leading to increased exposure of internal functional groups and altering the molar ratio of its constituent monosaccharides. The results of antioxidant experiments showed that enzymatic hydrolysis had the potential to enhance the antioxidant performance of LPS. In vitro fermentation experiments revealed that LPS and its derivatives exerted different prebiotic effects on intestinal microbial communities. Specifically, LPS mainly inhibited the growth of harmful bacteria such as Fusobacterium, while G-LPS(8) and G-LPS(16) tended to promote the growth of beneficial bacteria like Megamonas, Bacteroides, and Parabacteroides. Metabolomic analysis revealed that LPSs with varying molecular weights exerted comparable promoting effects on multiple amino acid and carbohydrate metabolic pathways. Importantly, with the reduction in molecular weight, G-LPS(16) also particularly stimulated sphingolipid metabolism, nucleotide metabolism, as well as ascorbic acid and uronic acid metabolism, leading to the significant increase in specific metabolites such as sphingosine. Therefore, this study suggests that properly degraded LPS components have greater potential as a prebiotic for improving gut health.

The Evolution of Antimicrobial Resistance in Acinetobacter baumannii and New Strategies to Fight It.

Acinetobacter baumannii is considered one of the prioritized ESKAPE microorganisms for the research and development of novel treatments by the World Health Organization, especially because of its remarkable persistence and drug resistance. In this review, we describe how this can be acquired by the enzymatic degradation of antibiotics, target site modification, altered membrane permeability, multidrug efflux pumps, and their ability to form biofilms. Also, the evolution of drug resistance in A. baumannii, which is mainly driven by mobile genetic elements, is reported, with particular reference to plasmid-associated resistance, resistance islands, and insertion sequences. Finally, an overview of existing, new, and alternative therapies is provided.

Cryo-SEM and large volume FIB-SEM of Arabidopsis cotyledons: Degradation of lipid bodies, biogenesis of glyoxysomes and reorganisation of organelles during germination.

Until recently, the lack of three-dimensional visualisation of whole cells at the electron microscopic (EM) level has led to a significant gap in our understanding of the interaction of cellular organelles and their interconnection. This is particularly true with regard to the role of the endoplasmic reticulum (ER). In this study, we perform three-dimensional reconstructions of serial FIB/SEM stacks and anaglyphs derived from volume rendering, cryo-scanning electron microscopy (cryo-SEM) and state-of-the-art electron microscopy immobilisation and imaging techniques. The results show that glyoxysomes are formed de novo in large numbers and in characteristic clusters on the ER upon germination in mesophyll cells of Arabidopsis cotyledons. The degradation of lipid bodies during germination occurs not only via the ER, which enlarges by taking up polar lipids resulting from enzymatic degradation by lipases, but also via glyoxysomes, which engulf lipid bodies. Dictyosomal (Golgi-derived) vesicles, which fuse with glyoxysomes or their precursors, also appear to be involved in the differentiation of glyoxysomes from segments of the ER. The formation of the central vacuole is the result of the fusion of protein storage vacuoles (protein bodies), which become complex three-dimensional structures during germination. Our observations also suggest that the vacuole plays a role in the degradation of glyoxysomes. The evidence provided in three dimensions shows that the endoplasmic reticulum plays a central role in the biogenesis and degradation of lipid bodies, the ontogeny of glyoxysomes and the development of plastids in the mesophyll cells of Arabidopsis cotyledons.

Identification and Characterization of a New Thermophilic κ-Carrageenan Sulfatase.

Carrageenans are sulfated polysaccharides found in the cell wall of certain red seaweeds. They are widely used in the food industry for their gelling and stabilizing properties. In nature, carrageenans undergo enzymatic modification and degradation by marine organisms. Characterizing these enzymes is crucial for understanding carrageenan utilization and may eventually enable the development of targeted processes to modify carrageenans for industrial applications. In our study, we characterized a κ-carrageenan sulfatase, AMOR_S1_16A, belonging to the sulfatase S1_16 subfamily, which selectively desulfates the nonreducing end galactoses of κ-carrageenan oligomers in an exomode. Notably, AMOR_S1_16A represents the first κ-carrageenan sulfatase within the S1_16 subfamily and exhibits a novel enzymatic activity. This study provides further understanding of the substrate specificity and characteristics of the S1_16 subfamily. Moreover, this research highlights that many processes and enzymes remain to be discovered to fully understand carrageenan utilization pathways and to develop enzymatic processes for carrageenan modification and processing.

Alpha-1 Antitrypsin as a Regulatory Protease Inhibitor Modulating Inflammation and Shaping the Tumor Microenvironment in Cancer.

Alpha-1 antitrypsin (AAT) is a key serine protease inhibitor for regulating proteases such as neutrophil elastase. AAT restrains the pulmonary matrix from enzymatic degradation, and a deficiency in AAT leads to inflammatory tissue damage in the lungs, resulting in chronic obstructive pulmonary disease. Due to the crucial biological function of AAT, the emerging research interest in this protein has shifted to its role in cancer-associated inflammation and the dynamics of the tumor microenvironment. However, the lack of comprehensive reviews in this field hinders our understanding of AAT as an essential immune modulator with great potential in cancer immunotherapy. Therefore, in this review, we have elucidated the pivotal roles of AAT in inflammation and the tumor microenvironment, including the structure and molecular properties of AAT, its molecular functions in the regulation of the inflammatory response and tumor microenvironment, and its clinical implications in cancer including its diagnosis, prognosis, and therapeutic intervention. This review seeks to bridge the gap in the understanding of AAT between inflammatory diseases and cancer, and to foster deeper investigations into its translational potential in cancer immunotherapy in the future.

A screening method for polyester films-degrading microorganisms and enzymes.

Enzymatic degradation of plastic pollution offers a promising environmentally friendly waste management strategy, however, suitable biocatalysts must be screened and developed. Traditional screening methods using soluble or solubilised polymers do not necessarily identify enzymes that are effective against solid or crystalline polymers. This study presents a simple, time-saving and cost-effective method for identifying microorganisms and enzymes capable of degrading polymeric films. The method was tested on polycaprolactone (PCL), polyethylene terephthalate (PET), polylactate (PLA) and polyhydroxybutyrate/polyhydroxyvalerate (PHB/PHV) films. It involves two steps: first, screening for PCL diol (PCLD)-degrading microorganisms on agar plates, and second, testing these microorganisms on polyester films. Using this screening method, over 100 PCLD-degrading microorganisms and 27 E. coli clones carrying genomic or metagenomic DNA fragments have been isolated. In addition, recombinant cutinases from Streptomyces scabiei and Thermobifida fusca have been tested. Approximately 66 % of the microorganisms forming halos on PCLD agar plates hydrolysed PCL and 6 % - the biaxially oriented PET film. In addition, five PLA- and four PHB/PHV-degrading esterases have been identified. The proposed method is effective for detecting both wild-type and recombinant microorganisms, as well as recombinant enzymes from in vitro transcription-translation reactions. Screening for thermostable and thermophilic enzymes, including those resistant to organic solvents or environmental inhibitors, is also easily implemented.