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.

Modulating Weak Protein-Protein Cross-Interactions by the Addition of Free Amino Acids at Millimolar Concentrations.

In this paper, we quantify weak protein-protein interactions in solution using cross-interaction chromatography (CIC) and surface plasmon resonance (SPR) and demonstrate that they can be modulated by the addition of millimolar concentrations of free amino acids. With CIC, we determined the second osmotic virial cross-interaction coefficient (B23) as a proxy for the interaction strength between two different proteins. We perform SPR experiments to establish the binding affinity between the same proteins. With CIC, we show that the amino acids proline, glutamine, and arginine render the protein cross-interactions more repulsive or equivalently less attractive. Specifically, we measured B23 between lysozyme (Lys) and bovine serum albumin (BSA) and between Lys and protein isolates (whey and canola). We find that B23 increases when amino acids are added to the solution even at millimolar concentrations, corresponding to protein/ligand stoichiometric ratios as low as 1:1. With SPR, we show that the binding affinity between proteins can change by 1 order of magnitude when 10 mM glutamine is added. In the case of Lys and one whey protein isolate (WPI), it changes from the mM to the M range, thus by 3 orders of magnitude. Interestingly, this efficient modulation of the protein cross-interactions does not alter the protein's secondary structure. The capacity of amino acids to modulate protein cross-interactions at mM concentrations is remarkable and may have an impact across fields in particular for specific applications in the food or pharmaceutical industries.

Disordered mesoporous silica particles: an emerging platform to deliver proteins to the lungs.

Pulmonary delivery and formulation of biologics are among the more complex and growing scientific topics in drug delivery. We herein developed a dry powder formulation using disordered mesoporous silica particles (MSP) as the sole excipient and lysozyme, the most abundant antimicrobial proteins in the airways, as model protein. The MSP had the optimal size for lung deposition (2.43 ± 0.13 µm). A maximum lysozyme loading capacity (0.35 mg/mg) was achieved in 150 mM PBS, which was seven times greater than that in water. After washing and freeze-drying, we obtained a dry powder consisting of spherical, non-aggregated particles, free from residual buffer, or unabsorbed lysozyme. The presence of lysozyme was confirmed by TGA and FT-IR, while N2 adsorption/desorption and SAXS analysis indicate that the protein is confined within the internal mesoporous structure. The dry powder exhibited excellent aerodynamic performance (fine particle fraction <5 µm of 70.32%). Lysozyme was released in simulated lung fluid in a sustained kinetics and maintaining high enzymatic activity (71-91%), whereas LYS-MSP were shown to degrade into aggregated nanoparticulate microstructures, reaching almost complete dissolution (93%) within 24 h. MSPs were nontoxic to in vitro lung epithelium. The study demonstrates disordered MSP as viable carriers to successfully deliver protein to the lungs, with high deposition and retained activity.

Advancing HIC method development: Retention-time modeling and tuning selectivity with ternary mobile-phase systems.

The use of a ternary mobile-phase system comprising ammonium sulphate, sodium chloride, and phosphate buffer was explored to tune retention and enhance selectivity in hydrophobic interaction chromatography. The accuracy of the linear solvent-strength model to predict protein retention with the ternary mobile-phase system based on isocratic scouting runs is limited, as the extrapolated retention factor at aqueous buffer conditions (k0) cannot be reliably established. The Jandera retention model utilizing a salt concentration averaged retention factor (k¯0) in aqueous buffer for ternary systems overcomes this bottleneck. Gradient retention factors were derived based on isocratic scouting runs after numerical integration of the isocratic Jandera model, leading to retention-time prediction errors below 11 % for linear gradients. Furthermore, an analytical expression was formulated to predict HIC retention for both linear and segmented linear gradients, considering the linear solvent-strength (LSS) model within ternary salt systems, relying on a fixed k0. The approach involved conducting two gradient scouting runs for each of the two binary salt systems to determine model parameters. Retention-time prediction errors for linear gradients were below 12 % for lysozyme and 3 % for trypsinogen and α-chymotrypsinogen A. Finally, the analytical expression for a ternary mobile-phase system was used in combination with a genetic algorithm to tune the HIC selectivity. With an optimized segmented ternary gradient, a critical-pair separation for a mixture of 7 proteins was achieved within 15 min with retention-time prediction errors ranging between 0.7 and 15.7 %.

Glycerol-slaved 1H-1H NMR cross-relaxation in quasi-native lysozyme.

1H-1H nuclear cross-relaxation experiments have been carried out with lysozyme in variable glycerol viscosity to study intramolecular motion, self-diffusion, and isotropic rigid-body rotational tumbling at 298 K, pH 3.8. Dynamics of intramolecular 1H-1H cross-relaxation rates, the increase in internuclear spatial distances, and lateral and rotational diffusion coefficients all show fractional viscosity dependence with a power law exponent κ in the 0.17-0.83 range. The diffusion coefficient of glycerol Ds with the bulk viscosity itself is non-Stokesian, having a fractional viscosity dependence on the medium viscosity (Ds âˆ¼ Î·-κ, κ ≈ 0.71). The concurrence and close similarity of the fractional viscosity dependence of glycerol diffusion on the one hand, and diffusion and intramolecular cross-relaxation rates of the protein on the other lead to infer that relaxation of glycerol slaves protein relaxations. Glycerol-transformed native lysozyme to a quasi-native state does not affect the conclusion that both global and internal fluctuations are slaved to glycerol relaxation.

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.

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.

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.

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.

Multimodal Affinity-Modulated Efficient Separation of Lysozyme with a Hierarchical MXene@MOF Hybrid Framework.

The development of abiotic protein affinity adsorbents remains challenging for the accurate acquisition and analysis of specific protein species. Inspired by bacterial cell walls, a hierarchical hybrid framework is fabricated through the oriented growth of an Fe-based metal organic framework (MOF) on V2C MXene for the efficient separation of lysozyme (Lys). After directed evolution of adsorptive materials, the MXene@MOF composite rich in hydroxyl groups (termed as MX@MOF-DH) is found exerting exceptional affinity for Lys. Benefiting from hydrogen-bonding, coordination, and electrostatic interaction-mediated multimodal and multivalent affinity, MX@MOF-DH reveals rapid adsorption rate (5 min), superb enrichment factor (83.1), and favorable binding capacity (609.7 mg g-1), which outperforms other latest adsorbents. Moreover, femtomolar sensitivity is achieved even in the presence of high-abundant interfering proteins, as confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis. This work not only provides an efficient approach for selective enrichment of lysozyme but also paves an avenue to construct the protein affinity reagents for specific biological medicine and analysis applications.

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.

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.

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.

Biomimetic nanoplatforms constructed from dialkylaminostyryl hetarene dyes and phospholipids exhibiting selective fluorescent response to specific proteins.

The present work explores the specificity of supramolecular assemblies comprising dialkylaminostyrylhetarene dye molecules incorporated into phosphatidylcholine (PC) or phosphatidylserine (PS) aggregates. In PS-based assemblies, the dyes demonstrate a concentration-dependent fluorescent response, distinguishing anionic proteins such as bovine serum albumin (BSA) and pepsin from lysozyme (LYZ) in aqueous solutions. Conversely, no significant response is observed when the dyes are incorporated into the well-organized bilayers of neutral PC. The fluorescent response arises from the binding of dyes to proteins, leading to the detachment of dye molecules from the assemblies, rather than from the binding of proteins to the assemblies, although the latter process is facilitated by electrostatic attraction. Thus, both the poor ordering of PS molecules and the interfacial arrangement of the dyes are prerequisites for the fluorescent response of dye-PS aggregates. The structure of the dyes significantly impacts the spectral features of dye-PS and dye-protein assemblies. An optimal dye structure has been identified for the recognition of BSA, with a limit of detection (LOD) of 10.8 nM.

Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation.

BACKGROUND: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage. RESULTS: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05). CONCLUSIONS: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

Analyzing noncovalent interactions between notoginseng saponins and lysozyme by deposition scanning intensity fading MALDI-TOF mass spectrometry.

Analysis of noncovalent interactions between natural products and proteins is important for rapid screening of active ingredients and understanding their pharmacological activities. In this work, the intensity fading MALDI-TOF mass spectrometry (IF-MALDI-MS) method with improved reproducibility was implemented to investigate the binding interactions between saponins from Panax notoginseng and lysozyme. The benchmark IF-MALDI-MS experiment was established using N,N',N″-triacetylchitotriose-lysozyme as a model system. The reproducibility of ion intensities in IF-MALDI-MS was improved by scanning the whole sample deposition with a focused laser beam. The relative standard deviation (RSD) of deposition scanning IF-MALDI-MS is 5.7%. Similar decay trends of the relative intensities of notoginseng saponins against increasing amounts of lysozyme were observed for all six notoginseng saponins. The half-maximal fading concentration (FC50) was calculated to quantitatively characterize the binding affinity of each ligand based on the decay curve. According to the FC50 values obtained, the binding affinities of the six notoginseng saponins were evaluated in the following order: notoginsenoside S > notoginsenoside Fc > ginsenoside Rb1 > ginsenoside Rd > notoginsenoside Ft1 > ginsenoside Rg1. The binding order was in accordance with molecular docking studies, which showed hydrogen bonding might play a key role in stabilizing the binding interaction. Our results demonstrated that deposition scanning IF-MALDI-MS can provide valuable information on the noncovalent interactions between ligands and proteins.

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.

Structure and slow dynamics of protein hydration water with cryopreserving DMSO and trehalose upon cooling.

We study, through molecular dynamics simulations, three aqueous solutions with one lysozyme protein and three different concentrations of trehalose and dimethyl sulfoxide (DMSO). We analyze the structural and dynamical properties of the protein hydration water upon cooling. We find that trehalose plays a major role in modifying the structure of the network of HBs between water molecules in the hydration layer of the protein. The dynamics of hydration water presents, in addition to the α-relaxation, typical of glass formers, a slower long-time relaxation process, which greatly slows down the dynamics of water, particularly in the systems with trehalose, where it becomes dominant at low temperatures. In all the solutions, we observe, from the behavior of the α-relaxation times, a shift of the Mode Coupling Theory crossover temperature and the fragile-to-strong crossover temperature toward higher values with respect to bulk water. We also observe a strong-to-strong crossover from the temperature behavior of the long-relaxation times. In the aqueous solution with only DMSO, the transition shifts to a lower temperature than in the case with only lysozyme reported in the literature. We observe that the addition of trehalose to the mixture has the opposite effect of restoring the original location of the strong-to-strong crossover. In all the solutions analyzed in this work, the observed temperature of the protein dynamical transition is slightly shifted at lower temperatures than that of the strong-to-strong crossover, but their relative order is the same, showing a correlation between the motion of the protein and that of the hydration water.

Cytokinetic engineering enhances the secretory production of recombinant human lysozyme in Komagataella phaffii.

BACKGROUND: Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast. RESULTS: Here through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ. CONCLUSIONS: Our work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.

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.