This chapter describes three complementary in vitro assays designed to evaluate the antimicrobial potential and cytotoxic safety of endolysins. The Kirby-Bauer disk diffusion method enables rapid screening of endolysin activity based on inhibition zones against bacterial lawns. A serial dilution assay quantifies bactericidal efficacy through colony-forming unit (CFU) reduction, allowing dose-dependent assessment of killing activity. To assess host safety, a hemolytic assay using human red blood cells measures potential cytotoxicity by monitoring membrane lysis. Together, these methods offer a practical and reproducible framework for early-stage functional screening of candidate endolysins under controlled laboratory conditions.

Background/Objectives: Cryptosporidiosis, caused by Cryptosporidium parvum, is a significant cause of diarrheal disease, particularly affecting young children and immunocompromised individuals. With current treatments offering limited efficacy, there is an urgent need for novel therapeutic targets. Methods: In this study, we report the cloning, expression, and functional characterization of a glutathione transferase (GST) from C. parvum (CpGST). Results: Biocomputing analysis revealed a single gene encoding a cytosolic enzyme with distinct structural features, compared to human cytosolic homologs. Structural modeling indicated a non-canonical thioredoxin fold and a truncated C-terminal domain, suggesting functional divergence. CpGST was expressed in Escherichia coli, and its enzymatic properties were characterized. Although the enzyme displayed a narrow substrate spectrum, it showed a distinct substrate preference, retaining catalytic activity toward the standard GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and cumene hydroperoxide (CuOOH). Steady-state kinetic analysis revealed limited affinity for both reduced glutathione (GSH) and CDNB. Inhibition analysis identified several polyphenols and synthetic curcumin analogues as potent inhibitors, with IC50 values in the low micromolar range. Kinetic analysis with the most potent inhibitor revealed a mixed-type inhibition mechanism. Conclusions: These findings support the classification of CpGST as a structurally and functionally distinct member of the GST family, likely adapted to the parasite’s physiology and metabolism. The enzyme’s divergence from human GSTs, along with its favorable druggability profile, underscores its potential as a target for anti-cryptosporidial drug development, particularly in strategies aimed at disrupting stress response and detoxification pathways.

This chapter presents a set of protocols for evaluating endolysin activity through nephelometric turbidity assays. Nephelometric assays measure the decrease in optical density over time using either heat-inactivated bacterial cells or purified peptidoglycan (PG) as substrates, offering a rapid and quantitative approach to assess lytic enzyme function. Complementary methods for PG extraction, purification, and dye-labeling are also included. Together, these protocols support consistent and reproducible assessment of endolysin activity across diverse experimental contexts.

Microalgae display remarkable resilience to harsh environments, partly through the biosynthesis of diverse secondary metabolites. Cyanobacteria and red algae are well known to produce mycosporine-like amino acids (MAAs)-low-molecular-weight, water-soluble UV-absorbing compounds with anti-inflammatory, anticancer, and antimicrobial activities. By contrast, green microalgae typically lack detectable MAAs under standard conditions, and their responses under abiotic stress remain poorly characterized. Here, we investigated the freshwater green microalga Jaagichlorella luteoviridis grown under three stressors (salinity, heat, and UV) and assessed MAA induction. High-performance liquid chromatography (HPLC) revealed that stressed cultures accumulated multiple MAAs, whereas untreated controls showed no such accumulation. All stress treatments (UV, salinity, and heat) produced a substantial increase in peak intensity at 323-350 nm, whereas the control samples showed significantly lower absorption in this region. We also optimized an MAA extraction protocol suitable for "green" downstream applications in the pharmaceutical, nutraceutical, and cosmeceutical sectors and formulated an emulsion showing preliminary positive results and exhibiting an increased SPF index from 3.60 (control) to 3.78 when 0.2% MAA extract was added. Transcriptomic profiling against a reference genome revealed stress-specific differential gene expression and overexpression of specific genes of the MAA pathway, like ArioC and AroM/Aro1 SAM methyltransferases, thus identifying candidate targets for engineering enhanced MAA production. Given market demand for environmentally friendly and safe bioactives, microalgae represent a promising source of these valuable molecules.

The emergence of multidrug-resistant (MDR) bacterial pathogens, particularly Acinetobacter baumannii, has necessitated the development of novel antimicrobial strategies. Endolysins, which are bacteriophage-derived peptidoglycan hydrolases, have shown promise as alternative therapeutics against antibiotic-resistant bacteria. The endolysin AbLys1 from A. baumannii phage TZA1 belongs to the glycoside hydrolase family 24 (GH24) and displays high selectivity and bacteriolytic activity against the Gram-negative A. baumannii. In this study, an engineered form of AbLys1 was obtained by fusion with an antimicrobial α-helical decapeptide. Compared to the wild-type enzyme, the engineered enzyme exhibited enhanced lytic activity against A. baumannii and showed lytic activity against a broader panel of bacterial species, including Enterococcus and Staphylococcus, as well as Salmonella sp., Klebsiella oxytoca and Escherichia coli, with activity levels varying among strains. In addition, the enzyme exhibited activity against plant pathogens, such as Pseudomonas syringae and several Xanthomonas strains. The enzyme was encapsulated in an alginate gel matrix (art-AbLys1-Alg), and its storage stability, bacteriolytic, and antibiofilm activities were investigated. Thermostability analysis revealed that the encapsulated enzyme exhibited a dramatic increase in stability, retaining >75% of its activity after 120 days at 4 °C. In addition, art-AbLys1-Alg showed an increased biofilm reduction ability for A. baumannii biofilm compared to the free enzymes. The findings of this study demonstrate that the combined strategy of antimicrobial peptide (AMP) fusion and alginate encapsulation significantly enhances the functionality and practical applicability of AbLys1 in diverse biomedical and environmental contexts.

Industrialization and urbanization have caused serious contamination of water bodies, and the removal of chemical contaminants has become a major challenge. Chlorite is a harmful anthropogenic compound with a serious environmental impact and has been detected in groundwater, drinking water, and soil. Enzymes are considered sustainable tools for bioremediation, with chlorite dismutase (Cld) being a notable example. This enzyme has unique properties owing to the rare dioxygen bond formation that it catalyzes. In the present study, we report the cloning, biochemical, and structural characterization of the dimeric Cld from Pseudomonas aeruginosa ( Pa Cld). Pa Cld is a heme b oxidoreductase that can decompose chlorite ( or OClO − ) into harmless chloride (Cl − ) and dioxygen (O 2 ) with high turnover rates. The structure of Pa Cld was determined at atomic (0.99 Å) resolution using X‐ray crystallography. Additionally, steady‐state kinetics and stability studies provided valuable insights into the catalytic mechanism of dimeric Clds. Apart from chlorite bioremediation of water, Clds can also be used in biomedical and synthetic biology as well as in enzymatic cascades with O 2 ‐utilizing enzymes.

Deoxynivalenol (DON) is a toxicologically relevant trichothecene mycotoxin frequently found in cereal products. It is a virulence factor produced by the plant pathogen Fusarium graminearum during cereal crop infections. Investigating plant defense mechanisms is crucial for understanding plant resistance to F. graminearum and identifying new biocatalysts for DON detoxification. Previous studies identified DON-thiol adducts in cereal samples, indicating partial DON detoxification by glutathione transferases (GSTs). DON possesses two electrophilic centers for thiol conjugation, resulting in either epoxide opening at C13 or Michael addition at C10. At present, information on plant GSTs that catalyze these reactions is limited. In this study, Fusarium-inducible wheat GSTs were identified by analyzing the transcriptome of Fusarium-infected wheat heads. Twelve highly induced genes of the tau and phi GST classes were heterologously expressed and purified, biochemically characterized with model substrates, and assayed for activity with DON. Use of LC-MS showed that four of the selected tau class GSTs conjugated DON to GSH by epoxide opening (DON-13-GSH) and/or the reversible Michael addition reaction (DON-10-GSH). The crystal structure of a wheat GST (herein designated "TaGST-10") in complex with DON-13-GSH was solved at a resolution of 2.3 Å and provided insights into the binding of DON at the active site of tau class GSTs. Our results corroborate the hypothesis that enzyme-catalyzed, GSH-mediated DON detoxification may be involved in plant response to Fusarium infection.

Microalgae offer a sustainable and versatile source of bioactive compounds. Their rapid growth, efficient CO2 utilization, and adaptability make them a promising alternative to traditional production methods. Key compounds, such as proteins, polyunsaturated fatty acids (PUFAs), polyphenols, phytosterols, pigments, and mycosporine-like amino acids (MAAs), hold significant commercial value and are widely utilized in food, nutraceuticals, cosmetics, and pharmaceuticals, driving innovation across multiple industries. Their antiviral and enzyme-producing capabilities further enhance industrial and medical applications. Additionally, microalgae-based biostimulants and plant elicitor peptides (PEPs) contribute to sustainable agriculture by enhancing plant growth and resilience to environmental stressors. The GRAS status of several species facilitates market integration, but challenges in scaling and cost reduction remain. Advances in biotechnology and metabolic engineering will optimize production, driving growth in the global microalgae industry. With increasing consumer demand for natural, eco-friendly products, microalgae will play a vital role in health, food security, and environmental sustainability.

The 16S microbial community profiling of a metagenomics library from geothermal spring at Lisvori (Lesvos island, Greece) enabled the identification of a putative sequence exhibiting 95% identity to the γ‐type carbonic anhydrase (γ‐CA) from Caloramator australicus (γ‐ Ca CA). The sequence of γ‐ Ca CA was amplified by PCR, cloned, and expressed in E. coli . Activity assays showed that γ‐ Ca CA possesses very low, but detectable, anhydrase activity, while exhibiting no measurable esterase activity. Differential scanning fluorimetry (DSF) revealed that the enzyme shows high thermal stability with a melting temperature ( T m ) approximately 65–75°C in the pH range between 5.5 and 9.0. The structure of γ ‐Ca CA was determined by X‐ray crystallography at 1.11 Å resolution, the highest resolution reported so far for a γ ‐ CA. The enzyme was crystallized as a trimer in the crystallographic asymmetric unit and contains three zinc‐binding sites, one at each interface of neighboring subunits of the trimer. Structure‐based rational design enabled the design and creation of a mutant enzyme (γ ‐Ca CAmut) which possessed a heptapeptide insertion at the active‐site loop and two‐point mutations. Kinetic analysis demonstrated that γ‐ Ca CAmut was successfully converted into a catalytically active esterase indicating successful activity gain through structure‐guided engineering. The thermostability of γ‐ Ca CAmut was significantly increased, aligning with the thermostability typically observed in hyperthermostable enzymes. X‐ray crystallographic analysis of the γ‐ Ca CAmut structure at 2.1 Å resolution, provided detailed structural insights into how the mutations impact the overall enzyme structure, function, and thermostability. These findings provide valuable structural and functional insights into γ‐CAs and demonstrate a strategy for converting an inactive enzyme into a catalytically active form through rational design.

Xyloglucan is a complex, highly substituted plant biomass polysaccharide, which is largely overlooked in the design of enzyme cocktails for lignocellulose saccharification, due to its presence in specific plant tissues only, and its low content. Thus, the microbial mechanisms for its degradation have not been thoroughly studied. However, in the frame of the biorefinery concept, xyloglucan monomers also have to be utilized for the design of efficient bioprocesses. Moreover, in plant tissues, xyloglucan often covers cellulose fibrils, impeding the access of cellulases. In order to shed light on the enzymatic degradation of xyloglucan, a novel GH12 family xyloglucanase was studied, from the basidiomycete Abortiporus biennis. The enzyme was heterologously produced in Pichia pastoris, purified and characterized. AbiXeg12a is a 28 kDa glycoprotein, with relatively strict substrate specificity, since it is only active in xyloglucan and β-glucan. The main hydrolysis products are the oligomers XXXG, XLXG/XXLG, XLLG and the optimum activity conditions are pH 4.5 and 55 °C. The enzyme contributes to the saccharification of corn bran and apple pulp by a commercial cellulase preparation, increasing the release of reducing sugars by up to 39 % and 18 %, respectively, while the addition of AbiXeg12a can minimize the enzyme load of the reaction, at least for apple pulp, without loss in reducing sugar yield. Overall, the importance of xyloglucanases on the saccharification of xyloglucan-containing substrates was demonstrated in this study. The results could contribute to the design of more efficient, tailor-made enzyme cocktails for the saccharification and subsequent valorization of lignocellulose.

Tau class glutathione transferases (GSTUs) play essential roles in plant defense by facilitating the nucleophilic attack of glutathione (GSH) to a wide range of electrophilic xenobiotics. In addition to their conjugating activity, these enzymes possess hydroperoxidase function, enabling the detoxification of harmful organic hydroperoxides into less reactive alcohols. In this study, we identified three closely related GST isoenzymes (96-98 % sequence identity) from Cicer arietinum (CaGSTUs) through computational homology screening. Full-length cDNAs encoding these GSTs were cloned, recombinantly produced in E. coli, and purified for functional characterization. Enzyme kinetics were evaluated using model substrates, cumene hydroperoxide (CuOOH) and 1-chloro-2,4-dinitrobenzene (CDNB), revealing that CaGSTU1-1 displayed superior hydroperoxidase activity and thermal stability. Based on these properties, CaGSTU1-1 was selected as the parental scaffold for directed evolution via DNA shuffling, using the homologous Glycine max isoenzyme GmGSTU4-4. Screening of the generated chimeric library resulted in the identification of a new variant, CaGmGSTU, which demonstrated a fourfold enhancement in catalytic turnover and efficiency toward both substrates. Additionally, CaGmGSTU exhibited altered ligand-binding characteristics, including increased affinity for selected pesticides. Structural modeling and viscosity-dependence kinetics indicated that these enhancements were primarily driven by changes in enzyme flexibility. Given the widespread toxicity of hydroperoxides and related pollutants, CaGmGSTU represents a promising tool for detoxification applications in environmental and agricultural biotechnology.

The unique metabolic capabilities and fast growth rates of microalgae render them promising candidates for various industrial applications, such as biofuel production, pharmaceuticals, nutraceuticals, and wastewater treatment. Metabolic engineering is a powerful approach used to enhance the sustainable production of high-value compounds in microalgae, improve their stress tolerance, growth characteristics and suitability for large-scale cultivation. This review provides a snapshot of the current state of knowledge on omics and metabolic engineering research to further enhance our understanding on microalgal metabolism and enable the development of optimized strains with improved productivity and functionality. More specifically, it focuses on the recent breakthroughs in microalgal omics, driven by advancements in genomics technologies, such as improved sequencing platforms and bioinformatics tools, that have enabled the functional characterization of key genes, identification of metabolic pathways, and elucidation of microalgae cell physiology. Conventional and state-of-the-art genetic engineering approaches used in the last decades to manipulate the metabolic pathways of microalgae in a targeted manner, are highlighted in the scope of microalgal optimization. In this review, the different applications of genetic engineering and their impact on microalgae industry are also discussed. Integrating pan-omics data in future research is crucial for predicting novel functional interactions and identifying aspects of metabolic flux, towards enhancing algal strain-engineering techniques.

The comparative analysis of homologous enzymes is a valuable approach for elucidating enzymes’ structure–function relationships. Glutathione transferases (GSTs, EC. 2.5.1.18) are crucial enzymes in maintaining the homeostatic stability of plant cells by performing various metabolic, regulatory, and detoxifying functions. They are promiscuous enzymes that catalyze a broad range of reactions that involve the nucleophilic attack of the activated thiolate of glutathione (GSH) to electrophilic compounds. In the present work, three highly homologous (96–98%) GSTs from ryegrass Lolium perenne (LpGSTs) were identified by in silico homology searches and their full-length cDNAs were isolated, cloned, and expressed in E. coli cells. The recombinant enzymes were purified by affinity chromatography and their substrate specificity and kinetic parameters were determined. LpGSTs belong to the tau class of the GST superfamily, and despite their high sequence homology, their substrate specificity displays remarkable differences. High catalytic activity was determined towards hydroxyperoxides and alkenals, suggesting a detoxification role towards oxidative stress metabolites. The prediction of the structure of the most active LpGST by molecular modeling allowed the identification of a non-conserved residue (Phe215) with key structural and functional roles. Site-saturation mutagenesis at position 215 and the characterization of eight mutant enzymes revealed that this site plays pleiotropic roles, affecting the affinity of the enzyme for the substrates, catalytic constant, and structural stability. The results of the work have improved our understanding of the GST family in L. perenne, a significant threat to agriculture, sustainable food production, and safety worldwide.

Verticillium dahliae is a xylem-invading fungal pathogen that causes vascular wilt in a wide range of angiosperms. The pathogen uses a variety of virulence factors to invade and colonize its hosts. Here, we report that VdNEP, an NLP (Necrosis and ethylene inducing peptide 1-Like Protein), functions as one such factor in multiple hosts. Eggplant leaves treated with VdNEP developed necrotic symptoms. Overexpression of VdNEP by incorporating extra copies of the VdNEP gene increased virulence to cotton, eggplant and tomato plants, suggesting its role as a virulence factor in diverse plants. Increased expression of VdNEP among the transformants did not correlate with the number of VdNEP inserts, suggesting that its expression was affected by the genomic context of the insertion sites. Interestingly, a transformant derived from a defoliating strain with high VdNEP transcript levels caused disease symptoms in tomato plants, whereas the corresponding wild-type strain did not cause visible symptoms. The amount of V. dahliae DNA in plants infected with this VdNEP-overexpressing transformant was 22 times higher than that in plants infected with the wild-type isolate, further supporting the critical role of VdNEP in infection. A VdNEP-EGFP fusion was constructed to follow its localization in fungal cells and during infection.

Multidrug resistance (MDR) mechanisms in cancer cells are greatly influenced by glutathione transferase P1-1 (hGSTP1-1). The use of synthetic or natural compounds as hGSTP1-1 inhibitors is considered an effective approach to overcome MDR. Nine compounds consisting of coumarin-6-sulfonamide linked to chalcone derivatives were synthesized and evaluated for their ability to inhibit hGSTP1-1. Among the synthetic derivatives, compounds 5g, 5f, and 5a displayed the most potent inhibitory effect, with IC50 values of 12.2 ± 0.5 μΜ, 12.7 ± 0.7 and 16.3 ± 0.6, respectively. Kinetic inhibition analysis of the most potent molecule, 5g, showed that it behaves as a mixed-type inhibitor of the target enzyme. An in vitro cytotoxicity assessment of 5a, 5f, and 5g against the human prostate cancer cell lines DU-145 and PC3, as well as the breast cancer cell line MCF-7, demonstrated that compound 5g exhibited the most pronounced cytotoxic effect on all tested cell lines. Molecular docking studies were performed to predict the structural and molecular determinants of 5g, 5f, and 5a binding to hGSTP1-1. In agreement with the experimental data, the results revealed that 5g exhibited the lowest docking score among the three studied inhibitors as a consequence of shape complementarity, governed by van der Waals, hydrogen bonds and a π-π stacking interaction. These findings suggest that coumarin-chalcone hybrids offer new perspectives for the development of safe and efficient natural product-based sensitizers that can target hGSTP1-1 for anticancer purposes.

The misuse and overuse of antibiotics have contributed to a rapid emergence of antibiotic-resistant bacterial pathogens. This global health threat underlines the urgent need for innovative and novel antimicrobials. Endolysins derived from bacteriophages or prophages constitute promising new antimicrobials (so-called enzybiotics), exhibiting the ability to break down bacterial peptidoglycan (PG). In the present work, metagenomic analysis of soil samples, collected from thermal springs, allowed the identification of a prophage-derived endolysin that belongs to the N -acetylmuramoyl- L -alanine amidase type 2 (NALAA-2) family and possesses a LysM (lysin motif) region as a cell wall binding domain (CWBD). The enzyme (Ami1) was cloned and expressed in Escherichia coli , and its bactericidal and lytic activity was characterized. The results indicate that Ami1 exhibits strong bactericidal and antimicrobial activity against a broad range of bacterial pathogens, as well as against isolated peptidoglycan (PG). Among the examined bacterial pathogens, Ami1 showed highest bactericidal activity against Staphylococcus aureus s and Staphylococcus epidermidis cells. Thermostability analysis revealed a melting temperature of 64.2 ± 0.6 °C. Overall, these findings support the potential that Ami1, as a broad spectrum antimicrobial agent, could be further assessed as enzybiotic for the effective treatment of bacterial infections. Key points • Metagenomic analysis allowed the identification of a novel prophage endolysin • The endolysin belongs to type 2 amidase family with lysin motif region • The endolysin displays high thermostability and broad bactericidal spectrum

Human glutathione transferase A4-4 (hGSTA4-4) displays high catalytic efficiency towards 4-hydroxyalkenals and other cytotoxic and mutagenic products of radical reactions and lipid peroxidation. Its role as a target for the chemosensitization of cancer cells has not been investigated so far. In this study, the inhibitory potency of twelve selected natural products and ten monocarbonyl curcumin derivatives against hGSTA4-4 was studied. Among natural products, ellagic acid turned out to be the strongest inhibitor with an IC50 value of 0.44 ± 0.01 μM. Kinetic analysis using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as variable substrates showed that ellagic acid behaved as a competitive inhibitor towards both GSH and CDNB, with Ki values of 0.39 ± 0.02 and 0.63 ± 0.03 μM, respectively. Among the curcumin derivatives studied, three proved to be the most potent inhibitors, in the order DM151 > DM101 > DM100, with IC50 values of 2.4 ± 0.1 μM, 12.7 ± 1.1 μΜ and 16.9 ± 0.4 μΜ, respectively. Further kinetic inhibition analysis of the most active derivative, DM151, demonstrated that this compound is a mixed inhibitor towards CDNB with inhibition constants of Ki = 4.1 ± 0.5 μM and Ki’ = 0.536 ± 0.034 μM, while it is a competitive inhibitor towards GSH with a Ki = 0.98 ± 0.11 μM. Molecular docking studies were performed to interpret the differences in binding of ellagic acid and curcumin derivatives to hGSTA4-4. The in silico measured docking scores were consistent with the obtained experimental data. Hydrogen bonds appear to be the main contributors to the specific binding of monocarbonyl curcumin derivatives, while π-π stacking interactions play a key role in the enzyme–ellagic acid interaction. In vitro cytotoxicity assessment of the worst (DM148) and the best (DM151) inhibitors was performed against glioblastoma cell lines U-251 MG and U-87 MG. The results revealed that DM151 displays considerably higher cytotoxicity against both glioblastoma cell lines, while the glioblastoma cytotoxicity of DM148 was very limited. Furthermore, low and non-toxic doses of DM151 sensitized U-251 MG cells to the first-line glioblastoma chemotherapeutic temozolomide (TMZ), allowing us to propose for the first time that hGSTA4-4 inhibitors may be attractive therapeutic partners for TMZ to optimize its clinical effect in glioblastoma chemotherapy.

The unique metabolic capabilities and fast growth rates of microalgae render them promising candidates for various industrial applications, such as biofuel production, pharmaceuticals, nutraceuticals, and wastewater treatment. Metabolic engineering is a powerful approach used to enhance the sustainable production of highvalue compounds in microalgae, improve their stress tolerance, growth characteristics and suitability for large-scale cultivation. This review provides a snapshot of the current state of knowledge on omics and metabolic engineering research to further enhance our understanding on microalgal metabolism and enable the development of optimized strains with improved productivity and functionality. More specifically, it focuses on the recent breakthroughs in microalgal omics, driven by advancements in genomics technologies, such as improved sequencing platforms and bioinformatics tools, that have enabled the functional characterization of key genes, identification of metabolic pathways, and elucidation of microalgae cell physiology. Conventional and state-ofthe-art genetic engineering approaches used in the last decades to manipulate the metabolic pathways of microalgae in a targeted manner, are highlighted in the scope of microalgal optimization. In this review, the different applications of genetic engineering and their impact on microalgae industry are also discussed. Integrating pan-omics data in future research is crucial for predicting novel functional interactions and identifying aspects of metabolic flux, towards enhancing algal strain-engineering techniques.