International Journal of Biological Macromolecules

1.Iota-carrageenan is a natural polymer with moisturizing, mucoadhesive and shear-thinning properties. In this study, we aimed to evaluate the protective effects of iota-carrageenan on ocular surface against dehydration, to demonstrate its suitability for the use in lubricant eye drops. We utilized a human epithelial corneal cell culture model to test if pre-incubation with iota-carrageenan solution could protect cells from desiccation-induced cell death, to compare its effect with other natural polymers commonly used in artificial tear products, and to determine the optimal iota-carrageenan concentration. An ex vivo porcine eye model was established to confirm the protective effect of iota-carrageenan against dehydration on ocular tissue. Pre-incubation with 1.2 mg/ml iota-carrageenan increased the survival half-life of human corneal epithelial cells upon dehydration by three-fold; the effect was in the same range as observed for large molecular weight hyaluronic acid, and superior to all other tested natural polymers. The highest tested concentration of iota-carrageenan, 1.6 mg/ml, extended the cellular survival half-life by eight-fold while maintaining healthy cellular morphology. Repeated ex vivo instillation of an iota-carrageenan-based ophthalmic formulation into porcine eyes significantly protected the ocular surface from desiccation-induced corneal damage, as shown by corneal fluorescein staining These data suggest that Iota-carrageenan effectively moisturizes and protects the ocular surface, supporting its potential as a promising novel ingredient for eye drops in the management of dry eye disease.

Brain vascular aging is increasingly recognized as a critical therapeutic target for age-related cognitive decline. Oxidative stress, bioenergetic dysfunction, and molecular damage play central roles in the progression of vascular aging, contributing to cerebrovascular dysfunction and impaired cognitive function. While naturally occurring polyphenols such as resveratrol (RSV) have demonstrated potential in mitigating aging-related pathologies, their poor bioavailability and limited brain targeting efficiency significantly constrain their therapeutic impact. As a result, high doses or advanced drug delivery strategies are necessary to achieve meaningful physiological effects. We introduce a novel nanocarrier system designed to enhance RSV delivery to the cerebral endothelium by leveraging the natural formation of an apolipoprotein E (ApoE)-enriched protein corona around fusogenic liposomes (FL) in vivo. These nanoparticles directly fuse with cytoplasmic cell membranes and thus evade endocytosis. We found that once in the circulation FL spontaneously acquire a protein corona, which is highly enriched in ApoE, a key ligand for brain endothelial low-density lipoprotein receptors (LDLR). Based on this observation, we engineered an ApoE-functionalized protein corona around FL (ApoE-FL) to systematically evaluate whether this mechanism could be exploited for targeted brain delivery. Following optimization and physicochemical characterization, the RSV-loaded liposomes were evaluated in vitro using human cerebral microvascular endothelial cells and in vivo C57BL/6 aged mice to assess their therapeutic potential. Both FL and engineered ApoE-FL liposomal delivery systems exhibited a strong affinity for endothelial cell membranes in vitro. The knockdown of the ApoE receptor, low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced liposomal docking. Microscopy analysis revealed that both ApoE-FL and non-functionalized FL directly fused with endothelial plasma membranes, thus bypassing intracellular organelles and minimizing lysosomal degradation. This suggests that the naturally formed ApoE corona in vivo may contribute to efficient cerebrovascular targeting, a property successfully replicated by the engineered ApoE corona strategy. In vivo biodistribution and kinetic studies demonstrated that especially ApoE-FL achieved enhanced brain-targeting efficiency, prolonged cerebrovascular retention, and extended targeting distance along the arteriovenous axis. This emphasizes that fusogenic liposomes effectively engage almost the entire microvascular network, including capillaries and post-capillary venules. Functionally, fusogenic liposome-delivered RSV improved blood-brain barrier (BBB) integrity, enhanced neurovascular coupling (NVC) responses, and promoted brain vascularization in aged mice. Single-cell RNA sequencing (scRNA-seq) revealed enhanced endothelial angiogenesis and barrier protective transcriptional profiles in cerebrovascular cells treated with ApoE-FL/RSV, suggesting a molecular basis for the observed vascular benefits. Liposomal RSV delivery achieved near-complete cerebrovascular and cognitive rejuvenation in aged mice applying a 2000-fold lower RSV dose than oral administration used as control sample. Thus, ApoE-FL liposomes exhibited exceptionally high delivery efficiency in deeper brain regions, further expanding their therapeutic potential. These findings underscore the importance of targeted drug delivery in optimizing therapeutic outcomes and establish ApoE-functionalized fusogenic liposomes as a promising strategy for mitigating brain vascular aging and cognitive decline. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/709925v1_ufig1.gif" ALT="Figure 1000"> View larger version (52K): org.highwire.dtl.DTLVardef@f7966dorg.highwire.dtl.DTLVardef@b4ea4corg.highwire.dtl.DTLVardef@18240a9org.highwire.dtl.DTLVardef@634f6a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

Combatting biofilm-associated methicillin-resistant Staphylococcus aureus (MRSA) infections remains a formidable challenge, largely due to bacterias heightened resistance to antibiotics and evasion of the hosts immune responses. Inhibiting biofilm formation or promoting biofilm dissolution is believed to disrupt the protective matrices and expose bacterial cells to immune clearance or antimicrobial treatments. Cross- helical amyloid fibrils of phenol-soluble modulins are the key structural components of the MRSA biofilm. In this study, we present a novel retro-inverso peptide that successfully inhibits cross- amyloid formation, induces disassembly of pre-formed fibrils of the biofilm-forming modulin peptide, and efficiently disperses MRSA biofilm biomass in a dose-dependent manner. The biophysical studies of the interaction of the designed peptide and modulins have suggested a mechanism for the biofilm disruption. The findings highlight the potential of the proteolytically resistant mirror-image peptide as a promising therapeutic agent for the treatment of biofilm-associated MRSA infections.

This study presents a multidisciplinary approach to evaluate the structure formation and digestion of lupin protein crosslinked with transglutaminase (TG). TG was applied at 0-10 U/g protein, and structural development was assessed by oscillatory rheology (G, G"), while SDS-PAGE and o-phthaldialdehyde (OPA) assays were used to evaluate protein participation and the reduction of free {varepsilon}-amino groups, respectively. Proteomics was further employed to characterise molecular features associated with crosslinking behaviour. Lupin protein showed a clear dose-dependent increase in gel strength during incubation, with G values reaching 214 {+/-} 43.9 Pa at 10 U/g TG, compared to 7.2 {+/-} 0.6 Pa in the untreated control. Across all conditions, G remained higher than G" throughout frequency sweeps, and low tan {delta} values confirmed the formation of elastic networks driven by covalent crosslinks. SDS-PAGE and OPA results consistently demonstrated efficient crosslink formation, which increased with both incubation time and TG dosage, with SDS-PAGE indicating involvement of specific protein fractions. Proteomic analysis revealed disordered structural domains in the protein are preferred regions to form crosslinks. Furthermore, TG treatment was found to slow the digestibility of the crosslinked lupin protein. Overall, this work demonstrates how integrating proteomic insights with functional measurements can guide the selection and optimisation of plant proteins for enzymatic structuring. The approach offers a rational pathway to enhance the functionality of alternative protein sources such as lupin, supporting the development of sustainable food systems, including applications in meat and dairy analogues.

Ternary composite systems formed by lactoferrin (LF), sodium alginate (Alg), and Fe(II) were designed to investigate their potential as an iron delivery platform with enhanced protein stability. The ternary LF-Alg-Fe (LAF) composites demonstrated distinct structures depending on the LF to Alg ratio and the Fe(II) concentrations. At an LF to Alg ratio of 8:2 and final Fe concentrations between 20-30 mM, the system formed complexes stabilized by electrostatic interactions. Whereas Alg-rich formulations formed hydrogels stabilized by Alg-Fe(II) egg-box cross-linking. Rheological analysis and swelling behavior indicated a higher mechanical strength in LF-rich complexes and stronger network integrity in Alg-rich hydrogels, while intermediate LF/Alg ratios showed weaker structures overall. Fourier-transform infrared spectroscopy (FTIR) spectra showed no changes in functional groups or polymer structures after composite formation, confirming composite formation via non-covalent interactions. Thermal studies indicated that these ternary systems improved LF stability, evidenced by preserved secondary structure after heating using circular dichroism (CD), and an increased denaturation temperature compared with free LF in differential scanning calorimetry (DSC). In addition, in LF-rich formulations the Fe(II) release in aqueous solution was [~]50% while in Alg-rich formulations it was much lower (< 10%). LF-Alg-Fe composites exhibit distinct structures governed by protein-polysaccharide interactions and iron-mediated cross-linking, providing a potential strategy for protein stabilization and iron fortification in food systems.

Our current understanding of carbohydrate-binding module (CBM) function is limited by the fact that most CBM research has focused on single-binding-site modules. CBM family 92 (CBM92) is a recently characterized family of predominantly trivalent proteins that bind {beta}-1,3- and {beta}-1,6-glucans with high specificity. CpCBM92A from Chitinophaga pinensis stands out as the first trivalent member of the family to be structurally determined. Multivalent CBM families are rare, and the way in which the three binding sites cooperate in ligand recognition remains unclear. Here, we use NMR spectroscopy to demonstrate how each of the proteins binding sites plays distinct roles in ligand binding. One binding site, referred to as the {beta} site, can be identified as the primary attachment point because of its higher affinity for all tested ligands, consistent with previous biochemical data suggesting it is the strongest binding site on CpCBM92A. The other two binding sites, referred to as and {gamma}, preferentially bind longer segments of {beta}-1,3- and {beta}-1,6-glucan chains, respectively. We further show that the glycosidic bond position and anomeric configuration of the binding glucosyl unit strongly affects protein affinity due to a preferred ligand pose in the binding sites. Our results provide insight into how the trivalent architecture of CBM92 might enable cross-linking of scleroglucan chains, which may guide the development of new applications for CBMs in biotechnology.

The mosquito stage of the Plasmodium falciparum life cycle is an attractive target for intervention since it is crucial for the sexual reproduction and transmission of parasites to human host. Mosquito determinants crucial for parasite infection and growth pose as lucrative targets for transmission blockers. Owing to the fact that p38 MAPK has role in immune response and vector competence, we have evaluated the potential of PROTAC molecule (NR-7h) to degrade Anopheles stephensi p38 MAPK (Asp38 MAPK), a conserved serine/threonine kinase involved in stress reactions, midgut homeostasis, and parasite survival. PROTAC-mediated degradation of Asp38 MAPK led to the disrupted development of the parasite, suggesting its crucial function in vector competence. Furthermore, NR-7h-treated mosquitoes showed higher expression of immune genes such Rel-2, TEP1, APL1, and NOS, suggesting that p38 MAPK regulates host immunity in a way that promotes parasite persistence. PROTAC-mediated degradation of target proteins, provides a more persistent and resistance-proof therapeutic effect than traditional kinase inhibitors. Our findings establish PROTACs as a novel vector-targeted strategy for the development of endectocides to limit malaria transmission.

OXA-232, an OXA-48 like carbapenemase stands amongst newly identified beta-lactamases that causes of the extensive of beta-lactam resistance. While active-site residues are well characterised, the contributions of conserved non-active-site residues in exerting enzymatic activity remain unexplored, limiting our understanding about the roles of these residues in the overall OXA-232 function. To address these gaps, the conserved residues S118, V120, L158, and D159 of OXA-232 positioned adjacent to the active-site motifs and within the omega-like loop were substituted with alanine. Substitutions of S118A and D159A rendered the expressing cells susceptible to penicillins, cephalosporins, and carbapenems, whereas the cells harbouring OXA-232V120A and OXA-232L158A proteins exhibited substrate-selective susceptibility changes. Kinetic analysis with purified proteins revealed the reduction in catalytic efficiency of all the mutants compared to wild-type protein. Though the L158A and D159A mutated proteins become deacylation-deficient, the mutations S118A and V120A exhibited selective acylation defects without trapping intermediates. It is evident from circular dichroism spectroscopy and molecular dynamics simulations that OXA-232S118A, OXA-232V120A, and OXA-232L158A nearly retained their secondary structures and compactness, except for OXA-232D159A, which presumably triggered a misfolding leading to destabilisation of the omega-loop. Interestingly, bicarbonate supplementation partially rescued the lost activities in soluble mutants, underscoring the carbamylation dependence. Taken together, these findings establish S118 and D159 as essential for core catalysis and structural integrity, with V120 and L158 modulating substrate-specific turnover and orientation. The current study reappraised the mechanistic insights of OXA-48-like carbapenemases, providing significant resources in rationally designing future therapeutics to combat carbapenem resistance.

In wheat, weak seed dormancy (SD) is related to an increased tendency for pre-harvest sprouting (PHS), which reduces yield and quality. However, the molecular mechanism underlying SD remains elusive. Here, we identified a wheat R2R3-MYB transcription factor (TaMYB83-7B) related to SD. Expression analysis showed that TaMYB83-7B was highly expressed in wheat seeds, and was more highly expressed in strong-dormancy varieties than in weak-dormancy varieties. Sequence and association analysis indicated that T/C mutations at -907 bp and -1133 bp in the TaMYB83-7B promoter were significantly associated with wheat SD, with C at both sites related to strong dormancy. Dual-luciferase reporter assays demonstrated that the transcriptional activity of the TaMYB83-7B promoter was significantly higher in strong-dormancy varieties than in weak-dormancy varieties. Further analyses indicated that TaMYB83-7B functions as a transcriptional inhibitor. Germination experiments revealed that overexpression of TaMYB83-7B significantly enhanced SD, while its loss-of-function reduced SD. Finally, TaMYB83-7B was found to regulate SD by influencing the balance between abscisic acid (ABA) and gibberellin (GA) in wheat seeds. Overall, the results of this study enhance our understanding of the complex regulatory mechanism underlying SD, and provide gene targets and molecular markers for the genetic improvement of PHS resistance in wheat.

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

TBCK-related encephalopathy (TBCKE) is a neurodevelopmental disorder associated with biallelic mutations in TBCK. Despite the increasing number of reported cases worldwide, the biochemical and biophysical properties of TBCK remain unclear, hindering molecular understanding of its role in disease. Here, we present the successful expression, purification, and biochemical characterization of full-length human TBCK produced in Spodoptera frugiperda cells. Biochemical and biophysical analyses reveal that the catalytically inactive pseudokinase domain of TBCK lacks nucleotide binding, consistent with the absence of the canonical VAIK, HRD, and DFG motifs required for catalysis. These findings support that TBCK is a class I pseudokinase and provide a foundation for future structural and functional studies to elucidate its biological role.

Mutations in MAPT, the tau gene, give rise to forms of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T), with abundant filamentous tau inclusions in brain cells. Some mutations that encode missense and deletion variants can give rise to a clinical picture of Picks disease and filaments made of three-repeat tau. Here we report the electron cryo-microscopy (cryo-EM) structures of tau filaments from individuals with MAPT mutations D252V, G272V, S320F and {Delta}G389-I392. The two-layered Pick fold was present in the individuals with mutations D252V and {Delta}G389-I392. By contrast, mutations G272V and S320F gave rise to a more open variant of the Pick fold, with residues 272-341 rotated by 20-25{degrees} with respect to the rest of the structure. These findings show that missense mutations within the filament core can modify the Pick fold, generating closely related structural variants. In addition, we were able to reconstitute the Pick fold and some of its variants using seeded assembly with recombinant 0N3R tau carrying 12 serine or threonine to aspartate substitutions (PAD12) and missense mutations D252V, G272V or S320F. This work provides a foundation for the development of structure-based diagnostic and therapeutic approaches.

The intracellular transport system is pivotal for cellular function and integrity, facilitated by cytoskeletal motor proteins such as dynein, which traverse along microtubules (MTs). The heterogeneity of the tubulin isotypes composing MTs introduces functional diversity, potentially affecting cytoskeletal motor proteins interactions with the MT. This in silico study investigated the influence of amino acid sequence variations in the C-terminal tails (CTTs) of six different Homo sapiens tubulin isotypes, TUBB2A, TUBB2B, TUBB2C, TUBB3, TUBB4A, and TUBB5, highly expressed in human brain tumors, and assessed the isotypes effect on the binding of motor protein dynein to MT. Among these isotypes, TUBB2A, TUBB2B, and TUBB2C were found to affect conformational motions of the dyneins microtubule-binding domain (MTBD) and stalk domain. The investigation highlighted the novel role of isotype-specific variations in lateral interactions between tubulin protofilaments (PFs) in determining the proximity of the {beta}-CTT of the adjacent PF to the MTBD, potentially affecting dyneins motility and suggesting how changes in isotype expression directly influence dyneins velocity and processivity and contribute to transport defects associated with neurological disorders and cancers. Isolating specific tubulin isotypes experimentally is challenging due to their high sequence similarity and complex interactions with other microtubule-associated proteins. This makes it challenging to distinguish between different tubulin isotypes and their effects, particularly in tissues where multiple isotypes are co-expressed. Additionally, these isotypes are heavily modified in vivo by post-translational modifications, which further complicate the isolation of a single, unmodified tubulin isotype. As a result, computational approaches have been necessary in this study for exploring these effects in a controlled, isotype-specific manner.

Heavy metal ATPases (HMAs) are important group of transmembrane proteins involved in homeostasis of metal ions in plant systems. In this study, a comprehensive analysis of genome assembly (VC1973A v7.1) resulted in the identification of nine HMA genes (VrHMA) and their corresponding proteins in Mungbean, an agronomically important legume crop known for its nutritional values. VrHMA proteins were also characterized based on their biomolecular features, conserved domains and motifs arrangement, transmembrane helices, pore-line helices, subcellular location and occurrence of signal peptides. Based on sequence homology, nine VrHMAs were clustered into two major substrate-specific groups: VrHMA1, VrHMA5 and VrHMA7 were categorized under the Zn/Co/Cd/Pb ATPase group, whereas the remaining six VrHMAs belong to the Cu/Ag subgroup. Gene structure analysis and promoter scanning revealed the structural divergence and presence of various stress-responsive cis-acting elements, respectively. The expression analysis of VrHMA genes in root and leaf tissues, in response to heavy metal (Zn, Cd and Cu) stress, indicates their role in the uptake, transport and sequestration of metal ions. Interestingly, VrHMA5 showed incremental upregulation in roots in response to all three heavy metal stresses, whereas its expression was only upregulated in the leaf tissues under Zn stress, which indicates its role in vascular transport in V. radiata. In addition, this study provides valuable insights into the functional roles of VrHMA genes and will lay a foundation for future genetic improvement in mung bean aimed at enhanced heavy metal stress tolerance and micronutrient homeostasis.

Xenopus is a genus of entirely aquatic frogs found in sub-Saharan Africa. Currently, the complete genomes of two species within the Xenopus genus, Xenopus laevis and Xenopus tropicalis, have been fully sequenced, annotated, and made publicly available. The two species inhabit markedly different environments: X. tropicalis lives in the hot, equatorial regions of Africa, whereas X. laevis resides in the cooler climates of southern Africa. In the present study, mutational profiling, comparative homology modeling, and computational bioinformatics were used to identify the features of adaptive evolution in Xenopus endonuclease G (EndoG) proteins. The multiple characteristics of EndoG isozymes were discovered to vary considerably between the two Xenopus species dwelling in different locations. Most notably, EndoG proteins from the psychrophilic X. laevis exhibit the increased contents of charged and polar residues, elevated pI, higher intramolecular interaction energies, B factors, molecular void volumes, and solvent accessibilities, but the decreased contents of nonpolar and aromatic amino acids, lower hydrophobicity, buried surface area, and molecular packing density compared to those from the thermophilic X. tropicalis. The observed differences strongly suggest that temperature plays a dominant role in EndoG diversification. Evaluation of intramolecular interaction energies appears to be a particularly sensitive and discriminative framework for assessing protein divergence at the structural level. Overall, this study highlights the diversification of homologous proteins in ectothermic vertebrate eukaryotes and provides mechanistic insight into protein adaptation to contrasting environments.

The role of vision in the behavior of blood-feeding mosquitoes has remained largely overlooked, particularly in species of the Anopheles genus, despite their significant impact on global health. While the importance of olfactory and thermal cues in host-seeking is well established, the contribution of visual stimuli to mating and feeding behavior remains far less understood. In particular, Anopheles mosquitoes exhibit complex swarming behavior that depends on visual input, suggesting an underexplored avenue of research with direct implications for vector control. This study introduces a genetically modified mosquito line lacking the enzyme Tan, a hydrolase involved in both dopamine and histamine metabolism, to investigate the behavioral relevance of visual cues in Anopheles. Through a combination of behavioral assays and controlled laboratory experiments, the impact of visual disruption on attraction to UV-B light, host-seeking, and blood-feeding success was assessed. The findings demonstrate a reduced light-dependent attraction in both Anopheles males and females, suggesting an impairment in visual processing or a related behavioral response. This observation has implications for reproductive success and potential adaptation to anthropogenic environments with artificial light. By leveraging this novel knockout model, the study offers new tools and perspectives to better understand how vision shapes mosquito behavior and how this knowledge could be harnessed in the development of next-generation vector control strategies.

Adenoid cystic carcinoma (ACC) of salivary gland is a "immune-cold" tumour. Annexin A3 (ANXA3) is an apoptotic protein found to be participating in immune cell infiltration in tumour microenvironment (TME) of various cancer cases. Significant low expressions of ANXA3 protein found in adenoid cystic carcinoma. We hypothesized overexpressing ANXA3 transforms ACC "cold" TME to "hot". We cultured UM-HACC-2A and UFH2 spheroids on extracellular matrix and co cultured them with peripheral blood mononuclear cells. We functionalized FDA (The Food and Drug Administration) approved Poly(lactic-co-glycolic acid) PLGA nanoparticles with anti-cMyb antibody and ANXA3 recombinant protein using streptavidin-biotin conjugation. Upon overexpressing ANXA3 in ACC spheroids in immune coculture model using functionalized nanoparticles, significant increase of tumour infiltrating lymphocytes and decrease in the size of the ACC spheroids observed. Apoptotic profiler assay further confirmed significant upregulation of apoptotic proteins, some of them participate in immune infiltration. Overall, this project exhibits promising results showing potential approach to convert ACC into an immune "hot" tumour.

This work develops and characterises a hierachichal oral drug delivery system based on the microencpasulation of drug-loaded amphiphilic nanogels within a mucoadhesive alginate/chitosan shell. Results show a more controlled release and a statistically significant oral half-life with respect to the free drug.

In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 {micro}m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions. HighlightsO_LIDevelopment of size-controlled core-shell microparticles to protect phytase C_LIO_LIPhytase-chitosan microparticles are surrounded by an alginate-{kappa}-carrageenan shell C_LIO_LIOptimization achieved 32.7% loading capacity with a uniform size of 3.2 {micro}m C_LIO_LICore-shell microparticles retained 62.7% enzyme activity after heat and digestion C_LIO_LIPhytase powder (2 mg) is required for a single maize meal C_LI