Mechanism Of Hydrolysis Research Articles

Preparation of polyclonal anti-Schistosoma mansoni cysteine protease antibodies for early diagnosis

In many parts of the tropics, schistosomiasis is a major parasitic disease second only to malaria as a cause of morbidity and mortality. Diagnostic approaches include microscopic sampling of excreta such as the Kato-Katz method, radiography, and serology. Due to their vital role in many stages of the parasitic life cycle, proteases have been under investigation as targets of immunological or chemotherapeutic anti-Schistosoma agents. Five major classes of protease have been identified on the basis of the peptide hydrolysis mechanism: serine, cysteine, aspartic, threonine, and metalloproteases. Proteases of all five catalytic classes have been identified from S. mansoni through proteomic or genetic analysis. The study aimed to produce polyclonal antibodies (pAbs) against schistosomal cysteine proteases (CP) to be used in the diagnosis of schistosomiasis. This study was conducted on S. mansoni-infected patients from highly endemic areas and from outpatients’ clinic and hospitals and other patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids). In this study, the produced polyclonal antibodies against S. mansoni cysteine protease antigens were labeled with horseradish peroxidase (HRP) conjugate and used to detect CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA. The study involved 200 S. mansoni-infected patients (diagnosed by finding characteristic eggs in the collected stool samples), 100 patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids), and 100 individuals who served as parasite-free healthy negative control. The prepared pAb succeeded in detecting CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA with a sensitivity of 98.5% and 98.0% respectively. A positive correlation was observed between S. mansoni egg counts and both stool and serum antigen concentrations. Purified 27.5 kDa CP could be introduced as a suitable candidate antigen for early immunodiagnosis using sandwich ELISA for antigen detection.Key points• Detection of cysteine protease antigens can replace parasitological examination.• Sandwich ELISA has a higher sensitivity than microscopic examination of eggs.• Identification of antigens is important for the goal of obtaining diagnostic tools.

Insight into the Role of Active Site Protonation States and Water Molecules in the Catalytic Inhibition of DPP4 by Vildagliptin.

Vildagliptin (VIL) is an antidiabetic drug that inhibits dipeptidyl peptidase-4 (DPP4) through a covalent mechanism. The molecular bases for this inhibitory process have been addressed experimentally and computationally. Nevertheless, relevant issues remain unknown such as the roles of active site protonation states and conserved water molecules nearby the catalytic center. In this work, molecular dynamics simulations were applied to examine the structures of 12 noncovalent VIL-DPP4 complexes encompassing all possible protonation states of three noncatalytic residues (His126, Asp663, Asp709) that were inconclusively predicted by different computational tools. A catalytically competent complex structure was only achieved in the system with His126 in its ε-form and nonconventional neutral states for Asp663/Asp709. This complex suggested the involvement of one water molecule in the catalytic process of His740/Ser630 activation, which was confirmed by QM/MM simulations. Our findings support the suitability of a novel water-mediated mechanism in which His740/Ser630 activation occurs concertedly with the nucleophilic attack on VIL and the imidate protonation by Tyr547. Then, the restoration of His740/ Tyr547 protonation states occurs via a two-water hydrogen bonding network in a low-barrier process, thus describing the final step of the catalytic cycle for the first time. Additionally, two hydrolytic mechanisms were proposed based on the hydrogen bonding networks formed by water molecules and the catalytic residues along the inhibitory mechanism. These findings are valuable to unveil the molecular features of the covalent inhibition of DPP4 by VIL and support the future development of novel derivatives with improved structural or mechanistic profiles.

Understanding the mechanism of the sulfur mustard hydrolysis reaction on the atomistic level from experiment and first-principles simulations

After World War II, the Baltic Sea was contaminated by dumping unused sulfur mustard (SM) and other chemical warfare agents (CWAs), which now constitutes a huge environmental hazard. We use both experimental and theoretical methods to understand the reactivity of SM in water, in particular the hydrolysis, which is one of the chemical reactions leading to the neutralization of SM. Real conditions present in the Baltic Sea are represented by performing Car-Parrinello molecular dynamics simulations for sulfur mustard in explicit water solution at finite temperature. We study the relative occurrence and stability of various SM conformers in water and analyze the solute–solvent interactions, with special focus on the formation of intra- and intermolecular hydrogen bonds and their lifetime. A missing piece toward the understanding of the sulfonium cation formation and SM hydrolysis reaction is provided by obtaining the activation energy for SM decomposition in water solution from gas chromatography–mass spectrometry (GC-MS) experiments. The complex mechanism of SM hydrolysis is also studied by umbrella sampling simulations to obtain the free energy barrier for the sulfonium cation formation and to provide an atomistic view of the reaction process.

Mechanism for dissociative hydrolysis of pyrimidine nucleoside d4N: Inversion vs retention

Employing the microhydration model that involves two to five explicit water molecules, two plausible dissociative hydrolysis pathways of 2′,3′-didehydro-2′,3′-dideoxyuridine (d4U), α-path with configuration-inversion and β-path with configuration-retention, have been investigated by M06-2X(CPCM)/6–31++G(d,p) method. Using this model, inclusion of three explicit water molecules (n = 3) is shown to be the smallest system that gives the minimal activation free energy for α-path and β-path. Our results suggest that the glycoside cleavage is the RDS, and α-path is more favorable kinetically than β-path. Whereas β-path with exergonic formation of β-dihydrofuran-like sugar with keto pyrimidine complex possesses thermodynamic preference over α-path, where the formation of α-dihydrofuran-like sugar with enol pyrimidine complex is endergonic. The free energy barriers of RDSs for d4U (25.8 kcal mol−1) and d4T (27.3 kcal mol−1) suggest that the glycosidic bond in d4T is more stable than that in d4U. The relative lower stability of d4U is probably an important factor for less antiviral activity of d4U. The small free energy barrier differences of ∼ 1 kcal mol−1 for β-path over α-path, and the reaction free energy differences of ∼ -12 kcal mol−1 for β-path lower than α-path in d4T and d4U suggest a competitive β-path in pyrimidine d4Ns. The higher free energy barriers of RDSs in ddU (27.6 kcal mol−1) and ddT (29.0 kcal mol−1) indicate that the saturated sugar moiety increases the stability of glycosidic bond in contrast to the unsaturated counter parts in d4U and d4T. NBO analysis also shows the kinetic preference of α-path over β-path. Our results provide an exploration for the less antiviral activity of d4U and the influence of saturated ribose on the glycosidic bond stability of pyrimidine d4Ns.

Biochemical analysis of SARS-CoV-2 Nsp13 helicase implicated in COVID-19 and factors that regulate its catalytic functions

Replication of the 30 kilobase genome of SARS-CoV-2, responsible for COVID-19, is a key step in the coronavirus life cycle that requires a set of virally encoded nonstructural proteins such as the highly conserved Nsp13 helicase. However, the features that contribute to catalytic properties of Nsp13 are not well established. Here, we biochemically characterized the purified recombinant SARS-CoV-2 Nsp13 helicase protein, focusing on its catalytic functions, nucleic acid substrate specificity, nucleotide/metal cofactor requirements, and displacement of proteins from RNA molecules proposed to be important for its proofreading role during coronavirus replication. We determined that Nsp13 preferentially interacts with single-stranded DNA compared to single-stranded RNA to unwind a partial duplex helicase substrate. We present evidence for functional cooperativity as a function of Nsp13 concentration, which suggests that oligomerization is important for optimal activity. In addition, under single-turnover conditions, Nsp13 unwound partial duplex RNA substrates of increasing double-stranded regions (16 - 30 base pairs) with similar efficiency, suggesting the enzyme unwinds processively in this range. We also show Nsp13-catalyzed RNA unwinding is abolished by a site-specific neutralizing linkage in the sugar-phosphate backbone, demonstrating continuity in the helicase-translocating strand is essential for unwinding the partial duplex substrate. Taken together, we demonstrate for the first time that coronavirus helicase Nsp13 disrupts a high affinity RNA-protein interaction in a uni-directional and ATP-dependent manner. Furthermore, sensitivity of Nsp13 catalytic functions to Mg2+ concentration suggests a regulatory mechanism for ATP hydrolysis, duplex unwinding, and RNA protein remodeling, processes implicated in SARS-CoV-2 replication and proofreading.

The proton transfer mechanism of the Ras GTPases, the effect of the GTPase activating protein, and oncogenic mutations.

Ras-positive cancer constitutes a major challenge for medical treatment. Ras is a central protein in the RAF/MAPK signaling pathway and mutational hot spot residues Gly12, Gly13 and Gln61 constitute the majority of oncogenic mutations which are associated with detrimental clinical prognosis. To target the aberrant signaling by directly interfering with the Ras activity, one must have a thorough understanding of the mechanism of GTP hydrolysis. We employed ab initio calculations and QM/MM enhanced sampling methods to study the possible pathways, with a particular focus on the proton transfer steps. One important requirement for a feasible mechanism is to be consistent with the activating role of the GTPase activating protein (GAP) as well as the disabling nature of key oncogenic mutations of the Ras. Our model successfully recovers the GAP activation and the decreased rate of hydrolysis in Ras mutants. The thus obtained data on the mechanism can be the foundation of a virtual screening procedure designed to find ways of modulating mutant Ras activity.

PETpyrolysis and hydrolysis mechanism in the fixed pyrolyzer

AbstractIn the conventional polyethylene terephthalate (PET) pyrolysis process, the formation of char by excessive pyrolysis is mainly due to the dehydration mechanism, so water is considered an auxiliary agent that can effectively inhibit excessive pyrolysis. The preparation of terephthalic acid (TPA) by steam‐assisted pyrolysis of PET is an effective method to achieve closed‐loop recycling of waste PET. To ensure that the reaction is mild enough to reduce excessive cracking products such as char and benzoic acid and thus increase the yield of TPA, it is critical to reduce the reaction rate while maintaining a sufficient excess steam coefficient. Under the optimal operating conditions, when the temperature rise rate was 0.5 °C min−1and the excess steam coefficient was 150, the yield of TPA was 72.5 wt.%, and the purity was 85.5%. Noticeably, the steam‐assisted pyrolysis system is a heterogeneous reaction system whose reaction mechanism is different from the conventional hydrolysis and pyrolysis reactions and has a unique reaction path. The mechanistic study indicates that, in addition to the thermal cracking of PET molecules occurring in conventional pyrolysis, hydroxyl attack and transfer, and supplementation of benzene ring hydrogen also occur between water and intermediate molecules. Meanwhile, it has also been proven that the intermolecular hydrogen transfer between intermediate molecules and water molecules is the key to reduce the intensity of the reaction and inhibit the formation of char. This discovery illustrates the mechanism of the reaction between water and PET in the steam‐assisted pyrolysis process in the fixed pyrolyzer and justifies the distinction between it and the pyrolysis and hydrolysis processes of PET. It provides a theoretical basis for optimizing the pyrolysis process of PET, which is essential for the industrialization of TPA preparation from PET steam‐assisted pyrolysis.

Progress in the Conversion of Ginsenoside Rb1 into Minor Ginsenosides Using β-Glucosidases.

In recent years, minor ginsenosides have received increasing attention due to their outstanding biological activities, yet they are of extremely low content in wild ginseng. Ginsenoside Rb1, which accounts for 20% of the total ginsenosides, is commonly used as a precursor to produce minor ginsenosides via β-glucosidases. To date, many research groups have used different approaches to obtain β-glucosidases that can hydrolyze ginsenoside Rb1. This paper provides a compilation and analysis of relevant literature published mainly in the last decade, focusing on enzymatic hydrolysis pathways, enzymatic characteristics and molecular mechanisms of ginsenoside Rb1 hydrolysis by β-glucosidases. Based on this, it can be concluded that: (1) The β-glucosidases that convert ginsenoside Rb1 are mainly derived from bacteria and fungi and are classified as glycoside hydrolase (GH) families 1 and 3, which hydrolyze ginsenoside Rb1 mainly through the six pathways. (2) Almost all of these β-glucosidases are acidic and neutral enzymes with molecular masses ranging from 44-230 kDa. Furthermore, the different enzymes vary widely in terms of their optimal temperature, degradation products and kinetics. (3) In contrast to the GH1 β-glucosidases, the GH3 β-glucosidases that convert Rb1 show close sequence-function relationships. Mutations affecting the substrate binding site might alter the catalytic efficiency of enzymes and yield different prosapogenins. Further studies should focus on elucidating molecular mechanisms and improving overall performances of β-glucosidases for better application in food and pharmaceutical industries.

Insights into the mechanism of phospholipid hydrolysis by plant non-specific phospholipase C

Non-specific phospholipase C (NPC) hydrolyzes major membrane phospholipids to release diacylglycerol (DAG), a potent lipid-derived messenger regulating cell functions. Despite extensive studies on NPCs reveal their fundamental roles in plant growth and development, the mechanistic understanding of phospholipid-hydrolyzing by NPCs, remains largely unknown. Here we report the crystal structure of Arabidopsis NPC4 at a resolution of 2.1 Å. NPC4 is divided into a phosphoesterase domain (PD) and a C-terminal domain (CTD), and is structurally distinct from other characterized phospholipases. The previously uncharacterized CTD is indispensable for the full activity of NPC4. Mechanistically, CTD contributes NPC4 activity mainly via CTDα1-PD interaction, which ultimately stabilizes the catalytic pocket in PD. Together with a series of structure-guided biochemical studies, our work elucidates the structural basis and provides molecular mechanism of phospholipid hydrolysis by NPC4, and adds new insights into the members of phospholipase family.

Rational design of poly(peptide-ester) block copolymers for enzyme-specific surface resorption.

Tissue resorption and remodeling are pivotal steps in successful healing and regeneration, and it is important to design biomaterials that are responsive to regenerative processes in native tissue. The cell types responsible for remodeling, such as macrophages in the soft tissue wound environment and osteoclasts in the bone environment, utilize a class of enzymes called proteases to degrade the organic matrix. Many hydrophobic thermoplastics used in tissue regeneration are designed to degrade and resorb passively through hydrolytic mechanisms, leaving the potential of proteolytic-guided degradation underutilized. Here, we report the design and synthesis of a tyrosol-derived peptide-polyester block copolymer where protease-mediated resorption is tuned through changing the chemistry of the base polymer backbone and protease specificity is imparted through incorporation of specific peptide sequences. Quartz crystal microbalance was used to quantify polymer surface resorption upon exposure to various enzymes. Aqueous solubility of the diacids and the thermal properties of the resulting polymer had a significant effect on enzyme-mediated polymer resorption. While peptide incorporation at 2 mol% had little effect on the final thermal and physical properties of the block copolymers, its incorporation improved polymer resorption significantly in a peptide sequence- and protease-specific manner. To our knowledge, this is the first example of a peptide-incorporated linear thermoplastic with protease-specific sensitivity reported in the literature. The product is a modular system for engineering specificity in how polyesters can resorb under physiological conditions, thus providing a potential framework for improving vascularization and integration of biomaterials used in tissue engineering.

Oxidative Degradation of Sequence-Defined Peptoid Oligomers.

Due to their N-substitution, peptoids are generally regarded as resistant to biological degradation, such as enzymatic and hydrolytic mechanisms. This stability is an especially attractive feature for therapeutic development and is a selling point of many previous biological studies. However, another key mode of degradation remains to be fully explored, namely oxidative degradation mediated by reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS are biologically relevant in numerous contexts where biomaterials may be present, thus, improving understanding of peptoid oxidative susceptibility is crucial to exploit their full potential in the biomaterials field, where an oxidatively-labile but enzymatically stable molecule can offer attractive properties. Toward this end, we demonstrate a fundamental characterization of sequence-defined peptoid chains in the presence of chemically generated ROS, as compared to ROS-susceptible peptides such as proline and lysine oligomers. Lysine oligomers showed the fastest degradation rates to ROS and the enzyme trypsin. Peptoids degraded in metal catalyzed oxidation conditions at rates on par with poly(prolines), while maintaining resistance to enzymatic degradation. Furthermore, lysine-containing peptide-peptoid hybrid molecules showed tunability in both ROS-mediated and enzyme-mediated degradation, with rates intermediate to lysine and peptoid oligomers. When lysine-mimetic side-chains were incorporated into a peptoid backbone, the rate of degradation matched that of the lysine peptide oligomers, but remained resistant to enzymatic degradation. These results expand understanding of peptoid degradation to oxidative and enzymatic mechanisms, and demonstrate the potential for peptoid incorporation into materials where selectivity towards oxidative degradation is necessary, or directed enzymatic susceptibility is desired.

Synthesis and biological evaluation of novel β-lactam-metallo β-lactamase inhibitors.

β-lactamases are enzymes that deactivate β-lactam antibiotics through a hydrolysis mechanism. There are two known types of β-lactamases: serine β-lactamases (SBLs) and metallo β-lactamases (MBLs). The two existing strategies to overcome β-lactamase-mediated resistance are (a) to develop novel β-lactam antibiotics that are not susceptible to hydrolysis by these enzymes; or (b) to develop β-lactamase inhibitors that deactivate the enzyme and thereby restore the efficacy of the co-administered antibiotics. Many commercially available SBL inhibitors are used in combination therapy with antibiotics to treat antimicrobial resistant infections; however, there are only a handful of MBL inhibitors undergoing clinical trials. In this study, we present 11 novel potential MBL inhibitors (via multi-step chemical synthesis), that have shown to completely restore the efficacy of meropenem (≤2 mg L-1) against New Delhi metallo-β-lactamase (NDM) producing Klebsiella pneumoniae in vitro. These compounds contain a cyclic amino acid zinc chelator conjugated to various commercially available β-lactam antibiotic scaffolds with the aim to improve the overall drug transport, lipophilicity, and pharmacokinetic/pharmacodynamic properties as compared to the chelator alone. Biological evaluation of compounds 24b and 24c has further highlighted the downstream application of these MBLs, since they are non-toxic at the selected doses. Time-kill assays indicate that compounds 24b and 24c exhibit sterilizing activity towards NDM producing Klebsiella pneumoniae in vitro using minimal concentrations of meropenem. Furthermore, 24b and 24c proved to be promising inhibitors of VIM-2 (Ki = 0.85 and 1.87, respectively). This study has revealed a novel series of β-lactam MBLIs that are potent, efficacious, and safe leads with the potential to develop into therapeutic MBLIs.

Peptide condensation and hydrolysis mechanisms from a proton-transfer network perspective.

Peptide bond formation is a fundamental organic chemical reaction; however, despite numerous recent reports, the computationally predicted barriers remain contradictory to experimental results. Incompleteness of the molecular mechanism for either the peptide bond formation or the reverse hydrolysis reactions is also highlighted by our lack of understanding of the seemingly equilibrium nature of the reaction that favors dipeptide formation over longer peptide chains under hydrothermal conditions. In this work, we first completed an assessment of levels of theory and evaluated chemical models that range from the neutral glycine condensation reaction in gas phase to explicitly solvated zwitterionic amino acids that are embedded in a polarizable continuum at neutral pH. Ultimately, we identified a six-step 'ping-pong' mechanism with the involvement of both zwitterions and neutral species. The carboxylate and amine end-groups of the diglycine intermediates play critical roles in the proton transfer and condensation processes. The experimental condensation barrier of 98 kJ mol-1 was approximated to be 118-129 kJ mol-1 at the MN15/def2TZVPP|SMD(water) level of theory for the rate determining step, when using the most complete model for the solvation environment. Condensed phase free energy correction employed to the rate limiting step reduced the barrier height to 106 kJ mol-1. These results have fundamental implications for understanding enzyme catalyzed peptide bond formation, peptide/protein stability, and "metabolism first" emergence of life scenarios.

Single-layer HNb3O8 with strong and nearby Lewis and Brønsted acid sites boosts amide bond hydrolysis for urease mimicking.

Urea pollution is a growing environmental concern, and its removal via catalytic hydrolysis is challenging due to the resonance-stabilized amide bonds. In nature, this reaction is catalyzed by ureases in many soil bacteria. However, the remedy of this problem with natural enzymes is not feasible as they are easily denatured and require high costs for both preparation and storage. Given this, the development of nanomaterials bearing enzyme-like activity (nanozymes) with advantages such as low production cost, simple storage, and pH/thermal stability has attracted much attention over the past decade. As inspired by the mechanism of urease-catalyzed urea hydrolysis, the co-presence of Lewis acid (LA) and Brønsted acid (BA) sites is imperative to proceed with this reaction. Herein, layered HNb3O8 samples with intrinsic BA sites were adopted for investigation. The layer reduction of this material to few-/single layers can expose Nb sites with various LA strengths depending on the degree of NbO6 distortion. Among the catalysts examined, single-layer HNb3O8 bearing strong LA and BA sites displays the best hydrolytic activity towards acetamide and urea. This sample with high thermal stability was found to outperform urease at temperatures higher than 50 °C. The acidity-activity correlation established in this study is believed to guide the future design of industrial catalysts to remediate urea pollution.

Effect of the Temperature and Molar Ratio of Water-Oil on the Enzymatic Hydrolysis Kinetics of the Soybean Oil: Experimental and Mathematical Modeling

The objective of this paper was to evaluate the kinetics of the hydrolysis of soybean oil by the action of the Lipozyme® TL IM enzyme varying the operational conditions of molar ratio water/oil (9:1-60:1) and temperature (40-64 °C). To describe the experimental data, a mathematical model based on the kinetic mechanism of Ping-Pong Bi Bi (PPBB) was proposed, in which the following steps were not considered formation of the complex enzyme/oil substrate, and formation of the acylated enzyme/oil substrate complex. The results of enzymatic hydrolysis of soybean oil indicated a yield in free fatty acids of 76% at the molar ratio of 46:1 and temperature of 52 °C. Furthermore, based on the values of the determination coefficient and root mean square error, the mathematical model based on the kinetic mechanism of PPBB showed good agreement with the experimental data in a relatively wide temperature range. Thus, it can be a useful tool for the optimization and assessment of the mechanisms of enzymatic hydrolysis of vegetable oil.

Research progress of MOF/Bismuth-based semiconductor composites in photocatalytic technology

Photocatalysis has significant potential for environmental remediation and energy conversion, with a focus on designing and developing highly efficient photocatalysts. Composite materials consisting of bismuth-based semiconductors and metal-organic frameworks (MOFs) exhibit outstanding photocatalytic activity, and have garnered significant attention from researchers as a highly sought-after material. A review is conducted on recent advances in MOF/bismuth-based semiconductor composites. On this basis, the synthesis methods of MBCs are described in detail, and then the applications of MBCs in organic pollutant degradation, Cr (VI) reduction, water (H2O) splitting, nitrogen (N2) fixation discussed. Finally, this paper highlights the current challenges in photocatalysis using MBCs and provides insights into the future development direction for MBCs photocatalysis technology. The preparation and modification methods of MBCs, the improvement of photocatalytic efficiency, the mechanism of oxidative degradation of organic matter and the mechanism of photocatalytic complete hydrolysis of water need further study.

Mechanism and kinetic model of microalgal enzymatic hydrolysis for prospective bioethanol conversion.

Tetraselmis chuii is a potential microalgae that is in consideration for producing bioethanol owing to its large content of carbohydrates. The glucose production from T. chuii through an enzymatic process with cellulase and xylanase (pretreatment process) and α-amylase and glucoamylase (saccharification process) was studied. The mechanism of the enzymatic process was developed and the kinetic models were then evaluated. For the pretreatment process, enzymes with 30% concentration reacted at 30 °C for 40 min resulted in 35.9% glucose yield. For the saccharification process, the highest glucose yield of 90.03% was obtained using simultaneous α-amylase (0.0006%) and glucoamylase (0.01%) enzymes at 55 °C and for 40 min. The kinetic models fitted well with the experimental data. The model also revealed that the saccharification process performed better than the pretreatment process with a higher kinetic constant and lower activation energy. The proposed kinetic model plays an important role in implementing processes at a larger scale.

Successful batch and continuous lyophilization of mRNA LNP formulations depend on cryoprotectants and ionizable lipids.

The limited thermostability and need for ultracold storage conditions are the major drawbacks of the currently used nucleoside-modified lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccines, which hamper the distribution of these vaccines in low-resource regions. The LNP core contains, besides mRNA and lipids, a large fraction of water. Therefore, encapsulated mRNA, or at least a part of it, is subjected to hydrolysis mechanisms similar to unformulated mRNA in an aqueous solution. It is likely that the hydrolysis of mRNA and colloidal destabilization are critical factors that decrease the biological activity of mRNA LNPs upon storage under ambient conditions. Hence, lyophilization as a drying technique is a logical and appealing method to improve the thermostability of these vaccines. In this study, we demonstrate that mRNA LNP formulations comprising a reduction-sensitive ionizable lipid can be successfully lyophilized, in the presence of 20% w/v sucrose, both by conventional batch freeze-drying and by an innovative continuous spin lyophilization process. While the chemical structure of the ionizable lipid did not affect the colloidal stability of the LNP after lyophilization and redispersion in an aqueous medium, we found that the ability of LNPs to retain the mRNA payload stably encapsulated, and mediate in vivo and in vitro mRNA translation into protein, post lyophilization strongly depended on the ionizable lipid in the LNP formulation.

Theoretical prediction on the hydrolysis rate of the new types of nerve agents: A density functional study.

Although the hydrolysis mechanism of the nerve agents, which is the main decontamination pathway, has been studied experimentally and theoretically, the reliable theoretical prediction method for the hydrolysis rate is not studied yet. Furthermore, after the CWC (Chemical Warfare Agent) list is updated, Novichok candidate structures can be more than 10,000 structures, for which it is not possible to perform the experiment for all of them for synthesizing and getting the hydrolysis rate. Therefore, developing a reliable theoretical method for hydrolysis rate prediction is crucial to prepare for the forthcoming usage of new types of nerve agents. Herein, by using DFT (Density Functional Theory), we successfully developed a new method of predicting the hydrolysis rate on nerve agents by investigating the electrophilicity index (EI) of the various A-, V-, and G-series nerve agents and found a suitable correlation with the experimental hydrolysis rate. Among the several DFT methods, wb97xD predicts the EI with the lowest % deviation of the studied nerve agents. Our results show that EI can be a good indicator to predict the hydrolysis rate of the anticipated nerve agents. Based on the result, we predicted the hydrolysis rate on another type of Novichok candidates, which could be the firm basis for developing a decontaminant and antidote with much fewer experimental efforts on new types of nerve agents.

Hydrolysis Mechanism of Carbamate Methomyl by a Novel Esterase PestE: A QM/MM Approach.

Methomyl is one of the most important carbamates that has caused potential hazardous effects on both human beings and the environment. Here, we systematically investigated the hydrolysis mechanism of methomyl catalyzed by esterase PestE using molecular dynamics simulations (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. The hydrolysis mechanism involves two elementary steps: (Ⅰ) serine-initiated nucleophilic attack and (Ⅱ) C-O bond cleavage. Our work elicits the atomic level details of the hydrolysis mechanism and free energy profiles along the reaction pathway. The Boltzmann-weighted average potential barriers are 19.1 kcal/mol and 7.5 kcal/mol for steps Ⅰ and Ⅱ, respectively. We identified serine-initiated nucleophilic attack as the rate determining-step. The deep learning-based kcat prediction model indicated that the barrier of the rate-determining step is 15.4 kcal/mol, which is in good agreement with the calculated results using Boltzmann-weighted average method. We have elucidated the importance of the protein-substrate interactions and the roles of the key active site residues during the hydrolysis process through noncovalent interactions analysis and electrostatic potential (ESP) analysis. The results provide practical value for achieving efficient degradation of carbamates by hydrolases.