Effect of lysozyme combined with hydrothermal pretreatment on excess sludge and anaerobic digestion.

Anaerobic digestion (AD) is widely employed for sludge stabilization and waste reduction. However, the slow hydrolysis process hinders methane production and leads to prolonged sludge issues. In this study, an efficient and eco-friendly lysozyme pre-treatment method was utilized to address these challenges. By optimizing lysozyme dosage, hydrolysis and cell lysis were maximized. Furthermore, lysozyme combined with hydrothermal pretreatment enhanced overall efficiency. Results indicate that: (1) When lysozyme dosage reached 90 mg/g TS after 240 min of pretreatment, SCOD, soluble polysaccharides, and protein content reached their maxima at 855.00, 44.09, and 204.86 mg/L, respectively. This represented an increase of 85.87%, 365.58%, and 259.21% compared to the untreated sludge. Three-dimensional fluorescence spectroscopy revealed the highest fluorescence intensity in the IV region (soluble microbial product), promoting microbial metabolic activity. (2) Lysozyme combined with hydrothermal pretreatment significantly increased SCOD, soluble proteins, and polysaccharide release from sludge, reducing SCOD release time. Orthogonal experiments identified Group 3 as the most effective for SCOD and soluble polysaccharide release, while Group 9 released the most soluble proteins. The significance order of factors influencing SCOD, soluble proteins, and polysaccharide release is hydrothermal temperature > hydrothermal time > enzymatic digestion time.(3) The lysozyme-assisted hydrothermal pretreatment group exhibited the fastest release and the highest SCOD concentration of 8,135.00 mg/L during anaerobic digestion. Maximum SCOD consumption and cumulative gas production increased by 95.89% and 130.58%, respectively, compared to the control group, allowing gas production to conclude 3 days earlier.

Real-time observation of a metal complex-driven reaction intermediate using a porous protein crystal and serial femtosecond crystallography.

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.

Conjugation of Lysozyme and Epigallocatechin Gallate for Improving Antibacterial and Antioxidant Properties.

One of the main interests in the food industry is the preservation of food from spoilage by microorganisms or lipid oxidation. A novel alternative is the development of additives of natural origin with dual activity. In the present study, a chemically modified lysozyme (Lys) with epigallocatechin gallate (EGCG) was developed to obtain a conjugate (Lys-EGCG) with antibacterial/antioxidant activity to improve its properties and increase its application potential. The modification reaction was carried out using a free radical grafting method for the Lys modification reaction, using ascorbic acid and hydrogen peroxide as radical initiators in an aqueous medium. The synthesis of Lys-EGCG conjugate was confirmed by spectroscopic (FT-IR, 1H-RMN, and XPS) and calorimetry differential scanning (DSC) analyses. The EGCG binding to the Lys biomolecule was quantified by the Folin-Ciocalteu method; the antibacterial activity was evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCB) against Staphylococcus aureus and Pseudomonas fluorescens; the antioxidant activity was evaluated by ABTS, DPPH, and FRAP. The spectroscopic results showed that the Lys-EGCG conjugate was successfully obtained, and the DSC analysis revealed a 20 °C increase (P < 0.05) in the denaturation temperature of Lys due to EGCG modification. The EGCG concentration in Lys-EGCG was 97.97 ± 4.7 µmol of EGCG/g of sample. The antibacterial and antioxidant activity of the Lys-EGCG conjugate was higher (P < 0.05) than pure EGCG and Lys. The chemical modification of Lys with EGCG allows for the bioconjugate with a dual function (antibacterial/antioxidant), broadening the range of Lys and EGCG applications to different areas such as food, cosmetic, and pharmaceutical industries.

Ultrasound-Mediated Lysozyme Microbubbles Targeting NOX4 Knockdown Alleviate Cisplatin-Exposed Cochlear Hair Cell Ototoxicity.

The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.

Phospholipid-induced secondary structural changes of lysozyme polymorphic amyloid fibrils studied using vacuum-ultraviolet circular dichroism.

The hallmark of amyloidosis, such as Alzheimer's disease and Parkinson's disease, is the deposition of amyloid fibrils in various internal organs. The onset of the disease is related to the strength of cytotoxicity caused by toxic amyloid species. Furthermore, amyloid fibrils show polymorphism, where some types of fibrils are cytotoxic while others are not. It is thus essential to understand the molecular mechanism of cytotoxicity, part of which is caused by the interaction between amyloid polymorphic fibrils and cell membranes. Here, using amyloid polymorphs of hen egg white lysozyme, which is associated with hereditary systemic amyloidosis, showing different levels of cytotoxicity and liposomes of DMPC and DMPG, changes in the secondary structure of the polymorphs and the structural state of phospholipid membranes caused by the interaction were investigated using vacuum-ultraviolet circular dichroism (VUVCD) and Laurdan fluorescence measurements, respectively. Analysis has shown that the more cytotoxic polymorph increases the antiparallel ß-sheet content and causes more disorder in the membrane structure while the other less cytotoxic polymorph shows the opposite structural changes and causes less structural disorder in the membrane. These results suggest a close correlation between the structural properties of amyloid fibrils and the degree of structural disorder of phospholipid membranes, both of which are involved in the fundamental process leading to amyloid cytotoxicity.

Gas Cluster Ion Beam-Assisted Deposition for Fabricating Dry Multilayers Containing Enzyme and Substrate with On-Demand Release.

Gas cluster ion beam (GCIB)-assisted deposition is used to build multilayered protein-based structures. In this process, Ar3000-5000+ clusters bombard and sputter molecules from a reservoir (target) to a collector, an operation that can be sequentially repeated with multiple targets. The process occurs under a vacuum, making it adequate for further sample conservation in the dry state, since many proteins do not have long-term storage stability in the aqueous state. First of all, the stability in time and versatility in terms of molecule selection are demonstrated with the fabrication of peptide multilayers featuring a clear separation. Then, lysozyme and trypsin are used as protein models to show that the activity remaining on the collector after deposition is linearly proportional to the argon ion dose. The energy per atom (E/n) of the Ar clusters is a parameter that was also changed for lysozyme deposition, and its increase negatively affects activity. The intact detection of larger protein molecules by SDS-PAGE gel electrophoresis and a bioassay (trypsin at ≈25 kDa and glucose oxidase (GOx) at ≈80 kDa) is demonstrated. Finally, GOx and horseradish peroxidase, two proteins involved in the same enzymatic cascade, are successively deposited on ß-d-glucose to build an on-demand release material in which the enzymes and the substrate (ß-d-glucose) are combined in a dry trilayer, and the reaction occurs only upon reintroduction in aqueous medium.

Molecular Dynamics Simulations of Soft and Reactive Landing of Proteins Desorbed by Argon Cluster Bombardment.

Reactive molecular dynamics (MD) simulations were conducted to investigate the soft and reactive landing of hyperthermal velocity proteins transferred to a vacuum using large argon clusters. Experimentally, the interaction of argon cluster ion beams (Ar1000-5000+) with a target biofilm was previously used in such a manner to transfer lysozymes onto a collector with the retention of their bioactivity, paving the way to a new solvent-free method for complex biosurface nanofabrication. However, the experiments did not give access to a microscopic view of the interactions needed for their full understanding, which can be provided by the MD model. Our reactive force field simulations clarify the landing mechanisms of the lysozymes and their fragments on collectors with different natures (gold- and hydrogen-terminated graphite). The results highlight the conditions of soft and reactive landing on rigid surfaces, the effects of the protein structure, energy, and incidence angle before landing, and the adhesion forces with the collector substrate. Many of the obtained results can be generalized to other soft and reactive landing approaches used for biomolecules such as electrospray ionization and matrix-assisted laser desorption ionization.

Metagenomic Analysis of Rhizospheric Bacterial Community of Citrus Trees Expressing Phloem-Directed Antimicrobials.

Huanglongbing, also known as citrus greening, is currently the most devastating citrus disease with limited success in prevention and mitigation. A promising strategy for Huanglongbing control is the use of antimicrobials fused to a carrier protein (phloem protein of 16 kDa or PP16) that targets vascular tissues. This study investigated the effects of genetically modified citrus trees expressing Citrus sinensis PP16 (CsPP16) fused to human lysozyme and ß-defensin-2 on the soil microbiome diversity using 16S amplicon analysis. The results indicated that there were no significant alterations in alpha diversity, beta diversity, phylogenetic diversity, differential abundance, or functional prediction between the antimicrobial phloem-overexpressing plants and the control group, suggesting minimal impact on microbial community structure. However, microbiota diversity analysis revealed distinct bacterial assemblages between the rhizosphere soil and root environments. This study helps to understand the ecological implications of crops expressing phloem-targeted antimicrobials for vascular disease management, with minimal impact on soil microbiota.

Characterization of the structural and molecular interactions of Ferulic acid ethyl ester with human serum albumin and Lysozyme through multi-methods.

Ferulic acid ethyl ester (FAEE) is an essential raw material for the formulation of drugs for cardiovascular and cerebrovascular diseases and leukopenia. It is also used as a fixed aroma agent for food production due to its high pharmacological activity. In this study, the interaction of FAEE with Human serum albumin (HSA) and Lysozyme (LZM) was characterized by multi-spectrum and molecular dynamics simulations at four different temperatures. Additionally, the quenching mechanism of FAEE-HSA and FAEE-LZM were explored. Meanwhile, the binding constants, binding sites, thermodynamic parameters, molecular dynamics, molecular docking binding energy, and the influence of metal ions in the system were evaluated. The results of Synchronous fluorescence spectroscopy, UV-vis spectroscopy, CD, three-dimensional fluorescence spectrum, and resonance light scattering showed that the microenvironment of HSA and LZM and the protein conformation changed in the presence of FAEE. Furthermore, the effects of some common metal ions on the binding constants of FAEE-HSA and FAEE-LZM were investigated. Overall, the experimental results provide a theoretical basis for promoting the application of FAEE in the cosmetics, food, and pharmaceutical industries and significant guidance for food safety, drug design, and development.

Co-immobilization of crosslinked enzyme aggregates on lysozyme functionalized magnetic nanoparticles for enhancing stability and activity.

Surface chemistry of carriers plays a key role in enzyme loading capacity, structure rigidity, and thus catalyze activity of immobilized enzymes. In this work, the two model enzymes of horseradish peroxidase (HRP) and glucose oxidase (GOx) are co-immobilized on the lysozyme functionalized magnetic core-shell nanocomposites (LYZ@MCSNCs) to enhance their stability and activity. Briefly, the HRP and GOx aggregates are firstly formed under the crosslinker of trimesic acid, in which the loading amount and the rigidity of the enzyme can be further increased. Additionally, LYZ easily forms a robust anti-biofouling nanofilm on the surface of SiO2@Fe3O4 magnetic nanoparticles with abundant functional groups, which facilitate chemical crosslinking of HRP and GOx aggregates with minimized inactivation. The immobilized enzyme of HRP-GOx@LYZ@MCSNCs exhibited excellent recovery activity (95.6 %) higher than that of the free enzyme (HRP&GOx). Specifically, 85 % of relative activity was retained after seven cycles, while 73.5 % of initial activity was also remained after storage for 33 days at 4 °C. The thermal stability and pH adaptability of HRP-GOx@LYZ@MCSNCs were better than those of free enzyme of HRP&GOx. This study provides a mild and ecofriendly strategy for multienzyme co-immobilization based on LYZ functionalized magnetic nanoparticles using HRP and GOx as model enzymes.

Hydrophilic molecularly imprinted lysozyme-BiOBr composite with enhanced visible light utilization for selective removal of trace contaminants in water.

The development of high-efficiency molecularly imprinted photocatalysts is still challenging due to the lack of hydrophilic and suitable functional monomers. In this work, the bio-sourced lysozyme was developed as the hydrophilic functional monomer, and Cu-doped BiOBr was used as the photocatalysts, to prepare a novel hydrophilic molecularly imprinted lysozyme-BiOBr composite (BiOBr-Cu/LyzMIP) with enhanced visible light utilization. Lysozyme could form a transparent layer to mitigate the light transmission obstruction caused by the surface imprinting layer, making it an ideal functional monomer. The prepared BiOBr-Cu/LyzMIP possessed red-shifted visible-light absorption edge and minor reduction of light absorbance, indicating the enhanced utilization of visible light. Accordingly, BiOBr-Cu/LyzMIP demonstrated excellent degradation rate (99.4 % in 20 min), exceptional degradation efficiency (0.211 min-1), and superior reusability. Moreover, BiOBr-Cu/LyzMIP exhibited rapid adsorption equilibrium (20 min), good imprinting factor (2.67), and favourable degradation selectivity (>1.75), indicating the good imprinting effect resulting from abundant functional groups of lysozyme. Versatility experiments on different templates suggested that the proposed approach allowed flexibility in selecting a wide range of hazardous contaminants according to practical requirements. The present work expands the application of lysozyme-based composites in the environmental field, and provides a new one-stop pathway for efficient and sustainable treatment of contaminated water.

Induction of acute silkworm hemolymph melanization by Staphylococcus aureus treated with peptidoglycan-degrading enzymes.

Staphylococcus aureus, a Gram-positive bacterium, causes inflammatory skin diseases, such as atopic dermatitis, and serious systemic diseases, such as sepsis. In the skin and nasal environment, peptidoglycan (PGN)-degrading enzymes, including lysozyme and lysostaphin, affects S. aureus PGN. However, the effects of PGN-degrading enzymes on the acute innate immune-inducing activity of S. aureus have not yet been investigated. In this study, we demonstrated that PGN-degrading enzymes induce acute silkworm hemolymph melanization by S. aureus. Insoluble fractions of S. aureus treated with lysozyme, lysostaphin, or both enzymes, were prepared. Melanization of the silkworm hemolymph caused by the injection of these insoluble fractions was higher than that of S. aureus without enzyme treatment. These results suggest that structural changes in S. aureus PGN caused by PGN-degrading enzymes affect the acute innate immune response in silkworms.

Efficient production of a highly active lysozyme from European flat oyster Ostrea edulis.

Lysozyme, an antimicrobial agent, is extensively employed in the food and healthcare sectors to facilitate the breakdown of peptidoglycan. However, the methods to improve its catalytic activity and secretory expression still need to be studied. In the present study, twelve lysozymes from different origins were heterologously expressed using the Komagataella phaffii expression system. Among them, the lysozyme from the European flat oyster Ostrea edulis (oeLYZ) showed the highest activity. Via a semi-rational approach to reduce the structural free energy, the double mutant Y15A/S39R (oeLYZdm) with the catalytic activity 1.8-fold greater than that of the wild type was generated. Subsequently, different N-terminal fusion tags were employed to enhance oeLYZdm expression. The fusion with peptide tag 6×Glu resulted in a remarkable increase in the recombinant oeLYZdm expression, from 2.81 × 103 U mL-1 to 2.11 × 104 U mL-1 in shake flask culture, and eventually reaching 2.05 × 105 U mL-1 in a 3-L fermenter. The work produced the greatest amount of heterologous oeLYZ expression in microbial systems that are known to exist. Reducing the structural free energy and employing the N-terminal fusion tags are effective strategies to improve the catalytic activity and secretory expression of lysozyme.

Hierarchical Micro- and Mesoporous Zeolitic Imidazolate Framework-8-Based Enzyme Hybrid for Combination Antimicrobial by Lysozyme and Lactoferrin.

Pathogenic bacteria have consistently posed a formidable challenge to human health, creating the critical need for effective antibacterial solutions. In response, enzyme-metal-organic framework (MOF) composites have emerged as a promising class of antibacterial agents. This study focuses on the development of an enzyme-MOF composite based on HZIF-8, incorporating the advantages of simple synthesis, ZIF-8 antibacterial properties, lysozyme hydrolysis, and high biological safety. Through a one-pot method, core-shell nanoparticles (HZIF-8) were synthesized. This structure enables efficient immobilization of lysozyme and lactoferrin within the HZIF-8, resulting in the formation of the lysozyme-lactoferrin@HZIF-8 (LYZ-LF@HZIF-8) composite. Upon exposure to light irradiation, HZIF-8 itself possessed antibacterial properties. Lysozyme initiated the degradation of bacterial peptidoglycan and lactoferrin synergistically enhanced the antibacterial effect of lysozyme. All of the above ultimately contributed to comprehensive antibacterial activity. Antibacterial assessments demonstrated the efficacy of the LYZ-LF@HZIF-8 composite, effectively eradicating Staphylococcus aureus at a cell density of 1.5 × 106 CFU/mL with a low dosage of 200 µg/mL and completely inactivating Escherichia coli at 400 µg/mL with the same cell density. The enzyme-MOF composite exhibited significant and durable antibacterial efficacy, with no apparent cytotoxicity in vitro, thereby unveiling expansive prospects for applications in the medical and food industries.

Rapid aggregation of amyloid-like protein enhanced by mTGase to prepare functional wool fabrics for efficient and sustainable remove heavy metals from wastewater.

To counteract the increasing severity of water pollution and purify water sources, wastewater treatment materials are essential. In particular, it is necessary to improve the bonding strength between the adsorption material and the substrate in a long-term humid environment, and resist the invasion of microorganisms to prolong the service life. In this study, an amyloid-like aggregation method of lysozyme catalyzed by microbial transglutaminase (mTGase). Lysozyme self-assembles into an amyloid-like phase-transited lysozyme (PTL) in the presence of a reducing agent. Simultaneously, mTGase catalyzes acyl transfer reactions within lysozyme molecules or between lysozyme and keratin molecules, and driving PTL assembly on the wool fiber (TG-PTL@wool). This process enhances the grafting amount and fastness of PTL on the wool. Moreover, the tensile strength of wool fabric increased to 523 N. TG-PTL@wool achieves a 97.32 % removal rate of heavy metals, maintaining a removal rate of over 95 % after 5 cycles. TG-PTL@wool has excellent antibacterial property (99 %), and it remains above 90 % after 50 times of circulating washing. This study proved that mTGase can enhance the amyloid aggregation of lysozyme and enhance the bonding strength between PTL coating and substrate. Moreover, TG-PTL@wool provides a sustainable, efficient and cleaner solution for removing heavy metals from water.

Lysozyme: an endogenous antimicrobial protein with potent activity against extracellular, but not intracellular Mycobacterium tuberculosis.

Endogenous antimicrobial peptides (AMPs) play a key role in the host defense against pathogens. AMPs attack pathogens preferentially at the site of entry to prevent invasive infection. Mycobacterium tuberculosis (Mtb) enters its host via the airways. AMPs released into the airways are therefore likely candidates to contribute to the clearance of Mtb immediately after infection. Since lysozyme is detectable in airway secretions, we evaluated its antimicrobial activity against Mtb. We demonstrate that lysozyme inhibits the growth of extracellular Mtb, including isoniazid-resistant strains. Lysozyme also inhibited the growth of non-tuberculous mycobacteria. Even though lysozyme entered Mtb-infected human macrophages and co-localized with the pathogen we did not observe antimicrobial activity. This observation was unlikely related to the large size of lysozyme (14.74 kDa) because a smaller lysozyme-derived peptide also co-localized with Mtb without affecting the viability. To evaluate whether the activity of lysozyme against extracellular Mtb could be relevant in vivo, we incubated Mtb with fractions of human serum and screened for antimicrobial activity. After several rounds of sub-fractionation, we identified a highly active fraction-component as lysozyme by mass spectrometry. In summary, our results identify lysozyme as an antimycobacterial protein that is detectable as an active compound in human serum. Our results demonstrate that the activity of AMPs against extracellular bacilli does not predict efficacy against intracellular pathogens despite co-localization within the macrophage. Ongoing experiments are designed to unravel peptide modifications that occur in the intracellular space and interfere with the deleterious activity of lysozyme in the extracellular environment.

Effects of Macromolecular Cosolutes on the Kinetics of Huntingtin Aggregation Monitored by NMR Spectroscopy.

The effects of two macromolecular cosolutes, specifically the polysaccharide dextran-20 and the protein lysozyme, on the aggregation kinetics of a pathogenic huntingtin exon-1 protein (hhtex1) with a 35 polyglutamine repeat, httex1Q35, are described. A unified kinetic model that establishes a direct connection between reversible tetramerization occurring on the microsecond time scale and irreversible fibril formation on a time scale of hours/days forms the basis for quantitative analysis of httex1Q35 aggregation, monitored by measuring cross-peak intensities in a series of 2D 1H-15N NMR correlation spectra acquired during the course of aggregation. The primary effects of the two cosolutes are associated with shifts in the prenucleation tetramerization equilibrium resulting in substantial changes in concentration of "preformed" httex1Q35 tetramers. Similar effects of the two cosolutes on the tetramerization equilibrium observed for a shorter, nonaggregating huntingtin variant with a 7-glutamine repeat, httex1Q7, lend confidence to the conclusions drawn from the fits to the httex1Q35 aggregation kinetics.

Towards Enhanced Tunability of Aqueous Biphasic Systems: Furthering the Grasp of Fluorinated Ionic Liquids in the Purification of Proteins.

This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [N1112(OH)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [N1112(OH)][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs' biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C2C1Im][C4F9SO3] + 2 %wt K3PO4 and 30 %wt [C2C1Im][C4F9SO3] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.

Toward enhancement of antibody thermostability and affinity by computational design in the absence of antigen.

Over the past two decades, therapeutic antibodies have emerged as a rapidly expanding domain within the field of biologics. In silico tools that can streamline the process of antibody discovery and optimization are critical to support a pipeline that is growing more numerous and complex every year. High-quality structural information remains critical for the antibody optimization process, but antibody-antigen complex structures are often unavailable and in silico antibody docking methods are still unreliable. In this study, DeepAb, a deep learning model for predicting antibody Fv structure directly from sequence, was used in conjunction with single-point experimental deep mutational scanning (DMS) enrichment data to design 200 potentially optimized variants of an anti-hen egg lysozyme (HEL) antibody. We sought to determine whether DeepAb-designed variants containing combinations of beneficial mutations from the DMS exhibit enhanced thermostability and whether this optimization affected their developability profile. The 200 variants were produced through a robust high-throughput method and tested for thermal and colloidal stability (Tonset, Tm, Tagg), affinity (KD) relative to the parental antibody, and for developability parameters (nonspecific binding, aggregation propensity, self-association). Of the designed clones, 91% and 94% exhibited increased thermal and colloidal stability and affinity, respectively. Of these, 10% showed a significantly increased affinity for HEL (5- to 21-fold increase) and thermostability (>2.5C increase in Tm1), with most clones retaining the favorable developability profile of the parental antibody. Additional in silico tests suggest that these methods would enrich for binding affinity even without first collecting experimental DMS measurements. These data open the possibility of in silico antibody optimization without the need to predict the antibody-antigen interface, which is notoriously difficult in the absence of crystal structures.

Protonation-State Dependent Modulation of Hen Egg-White Lysozyme Fibrillation under the Influence of a Short Synthetic Peptide.

Under the influence of various conditions, misfolding of soluble proteins occurs, leading to the formation of toxic insoluble amyloids. The formation and deposition of such amyloids within the body are associated with detrimental biological consequences such as the onset of several amyloid-related diseases. Previously, we established a strategy for the rational design of peptide inhibitors against amyloid formation based on the amyloidogenic-prone region of the protein. In the current study, we have designed and identified an Asp-containing rationally designed hexapeptide (SqP4) as an excellent inhibitor of hen egg-white lysozyme (HEWL) amyloid progression in vitro. First, SqP4 showed strong affinity toward the native monomeric HEWL leading to the stabilization of the native form and restriction in the unfolding process of monomeric HEWL. Second, SqP4 was found to arrest the amyloidogenic misfolded structure of HEWL in a nonfibrillar monomer-like stage. We also observed the differential effect of the protonation state of the charged amino acid (Asp) within the peptide inhibitor on the amyloid formation of HEWL and explored the reason behind the observations. The findings of this study can be implemented in future strategies for the development of potent therapeutics against other amyloid-related diseases.