Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

Deciphering heterogeneous enzymatic surface reactions on xylan using surface plasmon resonance spectroscopy.

Xylans' unique properties make it attractive for a variety of industries, including paper, food, and biochemical production. While for some applications the preservation of its natural structure is crucial, for others the degradation into monosaccharides is essential. For the complete breakdown, the use of several enzymes is required, due to its structural complexity. In fact, the specificity of enzymatically-catalyzed reactions is guided by the surface, limiting or regulating accessibility and serving structurally encoded input guiding the actions of the enzymes. Here, we investigate enzymes at surfaces rich in xylan using surface plasmon resonance spectroscopy. The influence of diffusion and changes in substrate morphology is studied via enzyme surface kinetics simulations, yielding reaction rates and constants. We propose kinetic models, which can be applied to the degradation of multilayer biopolymer films. The most advanced model was verified by its successful application to the degradation of a thin film of polyhydroxybutyrate treated with a polyhydroxybutyrate-depolymerase. The herein derived models can be employed to quantify the degradation kinetics of various enzymes on biopolymers in heterogeneous environments, often prevalent in industrial processes. The identification of key factors influencing reaction rates such as inhibition will contribute to the quantification of intricate dynamics in complex systems.

The Michaelis-Menten Reaction at Low Substrate Concentrations: Pseudo-First-Order Kinetics and Conditions for Timescale Separation.

We demonstrate that the Michaelis-Menten reaction mechanism can be accurately approximated by a linear system when the initial substrate concentration is low. This leads to pseudo-first-order kinetics, simplifying mathematical calculations and experimental analysis. Our proof utilizes a monotonicity property of the system and Kamke's comparison theorem. This linear approximation yields a closed-form solution, enabling accurate modeling and estimation of reaction rate constants even without timescale separation. Building on prior work, we establish that the sufficient condition for the validity of this approximation is s 0 ≪ K , where K = k 2 / k 1 is the Van Slyke-Cullen constant. This condition is independent of the initial enzyme concentration. Further, we investigate timescale separation within the linear system, identifying necessary and sufficient conditions and deriving the corresponding reduced one-dimensional equations.

Studying protein stability in crowded environments by NMR.

Most proteins perform their functions in crowded and complex cellular environments where weak interactions are ubiquitous between biomolecules. These complex environments can modulate the protein folding energy landscape and hence affect protein stability. NMR is a nondestructive and effective method to quantify the kinetics and equilibrium thermodynamic stability of proteins at an atomic level within crowded environments and living cells. Here, we review NMR methods that can be used to measure protein stability, as well as findings of studies on protein stability in crowded environments mimicked by polymer and protein crowders and in living cells. The important effects of chemical interactions on protein stability are highlighted and compared to spatial excluded volume effects.

Monitoring CO2 kinetics as a marker of cardiopulmonary efficiency.

PURPOSE OF REVIEW: To describe current and near future developments and applications of CO2 kinetics in clinical respiratory and cardiovascular monitoring. RECENT FINDINGS: In the last years, we have witnessed a renewed interest in CO2 kinetics in relation with a better understanding of volumetric capnography and its derived parameters. This together with technological advances and improved measurement systems have expanded the monitoring potential of CO2 kinetics including breath by breath continuous end-expiratory lung volume and continuous noninvasive cardiac output. Dead space has slowly been gaining relevance in clinical monitoring and prognostic evaluation. Easy to measure dead space surrogates such as the ventilatory ratio have demonstrated a strong prognostic value in patients with acute respiratory failure. SUMMARY: The kinetics of carbon dioxide describe many relevant physiological processes. The clinical introduction of new ways of assessing respiratory and circulatory efficiency based on advanced analysis of CO2 kinetics are paving the road to a long-desired goal in clinical monitoring of critically ill patients: the integration of respiratory and circulatory monitoring during mechanical ventilation.

Discovering optimal kinetic pathways for self-assembly using automatic differentiation.

Macromolecular complexes are often composed of diverse subunits. The self-assembly of these subunits is inherently nonequilibrium and must avoid kinetic traps to achieve high yield over feasible timescales. We show how the kinetics of self-assembly benefits from diversity in subunits because it generates an expansive parameter space that naturally improves the "expressivity" of self-assembly, much like a deeper neural network. By using automatic differentiation algorithms commonly used in deep learning, we searched the parameter spaces of mass-action kinetic models to identify classes of kinetic protocols that mimic biological solutions for productive self-assembly. Our results reveal how high-yield complexes that easily become kinetically trapped in incomplete intermediates can instead be steered by internal design of rate-constants or external and active control of subunits to efficiently assemble. Internal design of a hierarchy of subunit binding rates generates self-assembly that can robustly avoid kinetic traps for all concentrations and energetics, but it places strict constraints on selection of relative rates. External control via subunit titration is more versatile, avoiding kinetic traps for any system without requiring molecular engineering of binding rates, albeit less efficiently and robustly. We derive theoretical expressions for the timescales of kinetic traps, and we demonstrate our optimization method applies not just for design but inference, extracting intersubunit binding rates from observations of yield-vs.-time for a heterotetramer. Overall, we identify optimal kinetic protocols for self-assembly as a powerful mechanism to achieve efficient and high-yield assembly in synthetic systems whether robustness or ease of "designability" is preferred.

Biosorption of arsenic (III) from aqueous solution using calcium alginate immobilized dead biomass of Acinetobacter sp. strain Sp2b.

This study presents a novel biosorbent developed by immobilizing dead Sp2b bacterial biomass into calcium alginate (CASp2b) to efficiently remove arsenic (AsIII) from contaminated water. The bacterium Sp2b was isolated from arsenic-contaminated industrial soil of Punjab, a state in India. The strain was designated Acinetobacter sp. strain Sp2b as per the 16S rDNA sequencing, GenBank accession number -OP010048.The CASp2b was used for the biosorption studies after an initial screening for the biosorption capacity of Sp2b biomass with immobilized biomass in both live and dead states. The optimum biosorption conditions were examined in batch experimentations with contact time, pH, biomass, temperature, and AsIII concentration variables. The maximum biosorption capacity (qmax = 20.1 ± 0.76 mg/g of CA Sp2b) was obtained at pH9, 35 ̊ C, 20 min contact time, and 120 rpm agitation speed. The isotherm, kinetic and thermodynamic modeling of the experimental data favored Freundlich isotherm (R2 = 0.941) and pseudo-2nd-order kinetics (R2 = 0.968) with endothermic nature (ΔH° = 27.42) and high randomness (ΔS° = 58.1).The scanning electron microscopy with energy dispersive X-ray (SEM-EDX) analysis indicated the As surface binding. The reusability study revealed the reasonable usage of beads up to 5 cycles. In conclusion, CASp2b is a promising, efficient, eco-friendly biosorbent for AsIII removal from contaminated water.

Synthesis of novel magnetic hydroxyapatite-biomass nanocomposite for arsenic and fluoride adsorption.

A magnetic nanocomposite of hydroxyapatite and biomass (HAp-CM) was synthesized through a combined ultrasonic and hydrothermal method, aiming for efficient adsorption of arsenic (As) and fluoride (F-) from drinking water in natural environments. The characterization of HAp-CM was carried out using TG, FTIR, XRD, SEM, SEM-EDS, and TEM techniques, along with the determination of pHpzc charge. FTIR analysis suggested that coordinating links are the main interactions that allow the formation of the nanocomposite. XRD data indicated that the crystalline structure of the constituent materials remained unaffected during the formation of HAp-CM. SEM-EDS analysis revelated a Ca/P molar ratio of 1.78. Adsorption assays conducted in batches demonstrated that As and F- followed a PSO kinetic model. Furthermore, As adsorption fitting well to the Langmuir model, while F- adsorption could be explained by both Langmuir and Freundlich models. The maximum adsorption capacity of HAp-CM was found to be 5.0 mg g-1 for As and 10.2 mg g-1 for F-. The influence of sorbent dosage, pH, and the presence of coexisting species on adsorption capacity was explored. The pH significantly affected the nanocomposite's efficiency in removing both pollutants. The presence of various coexisting species had different effects on F- removal efficiency, while As adsorption efficiency was generally enhanced, except in the case of PO43-. The competitive adsorption between F- and As on HAp-CM was also examined. The achieved results demonstrate that HAp-CM has great potential for use in a natural environment, particularly in groundwater remediation as a preliminary treatment for water consumption.

Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E.

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min-1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

Single-Molecule FRET Analyses of NMDA Receptors.

Single-molecule fluorescence resonance energy transfer (smFRET) enables the real-time observation of conformational changes in a single protein molecule of interest. These observations are achieved by attaching fluorophores to proteins of interest in a site-specific manner and investigating the FRET between the fluorophores. Here we describe the method wherein the FRET is studied by adhering the protein molecules to a slide using affinity-based interactions and measuring the fluorophores' fluorescence intensity from a single molecule over time. The resulting information can be used to derive distance values for a point-to-point measurement within a protein or to calculate kinetic transition rates between various conformational states of a protein. Comparing these parameters between different conditions such as the presence of protein binding partners, application of ligands, or changes in the primary sequence of the protein can provide insights into protein structural changes as well as kinetics of these changes (if in the millisecond to second timescale) that underlie functional effects. Here we describe the procedure for conducting analyses of NMDA receptor conformational changes using the above methodology and provide a discussion of various considerations that affect the design, execution, and interpretation of similar smFRET studies.

Pharmacological inhibition of α-synuclein aggregation within liquid condensates.

Aggregated forms of α-synuclein constitute the major component of Lewy bodies, the proteinaceous aggregates characteristic of Parkinson's disease. Emerging evidence suggests that α-synuclein aggregation may occur within liquid condensates formed through phase separation. This mechanism of aggregation creates new challenges and opportunities for drug discovery for Parkinson's disease, which is otherwise still incurable. Here we show that the condensation-driven aggregation pathway of α-synuclein can be inhibited using small molecules. We report that the aminosterol claramine stabilizes α-synuclein condensates and inhibits α-synuclein aggregation within the condensates both in vitro and in a Caenorhabditis elegans model of Parkinson's disease. By using a chemical kinetics approach, we show that the mechanism of action of claramine is to inhibit primary nucleation within the condensates. These results illustrate a possible therapeutic route based on the inhibition of protein aggregation within condensates, a phenomenon likely to be relevant in other neurodegenerative disorders.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant unusually preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employ static and dynamic structural methods and observe that, compared to R132H, the R132Q active site adopts a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling reveals a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis.

Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat ( Δ H ‡ = 54 kJ mol-1), significant loss in entropy ( - T Δ S ‡ = 20 kJ mol-1; 298 K) and negative activation heat capacity ( Δ C p ‡ = -0.64 kJ mol-1 K-1). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function.

Kinetics of glucose-6-phosphate dehydrogenase (G6PD) activity during Plasmodium vivax infection: implications for early radical malaria treatment.

BACKGROUND: Plasmodium vivax relapses due to dormant liver hypnozoites can be prevented with primaquine. However, the dose must be adjusted in individuals with glucose-6-phosphate-dehydrogenase (G6PD) deficiency. In French Guiana, assessment of G6PD activity is typically delayed until day (D)14 to avoid the risk if misclassification. This study assessed the kinetics of G6PD activity throughout P. vivax infection to inform the timing of treatment. METHODS: For this retrospective monocentric study, data on G6PD activity between D1 and D28 after treatment initiation with chloroquine or artemisinin-based combination therapy were collected for patients followed at Cayenne Hospital, French Guiana, between January 2018 and December 2020. Patients were divided into three groups based on the number of available G6PD activity assessments: (i) at least two measurements during the P. vivax malaria infection; (ii) two measurements: one during the current infection and one previously; (iii) only one measurement during the malaria infection. RESULTS: In total, 210 patients were included (80, 20 and 110 in groups 1, 2 and 3, respectively). Data from group 1 showed that G6PD activity remained stable in each patient over time (D1, D3, D7, D14, D21, D28). None of the patients with normal G6PD activity during the initial phase (D1-D3) of the malaria episode (n = 44) was categorized as G6PD-deficient at D14. Patients with G6PD activity < 80% at D1 or D3 showed normal activity at D14. Sex and reticulocyte count were statistically associated with G6PD activity variation. In the whole sample (n = 210), no patient had severe G6PD deficiency (< 10%) and only three between 10 and 30%, giving a G6PD deficiency prevalence of 1.4%. Among the 100 patients from group 1 and 2, 30 patients (26.5%) were lost to follow-up before primaquine initiation. CONCLUSIONS: In patients treated for P. vivax infection, G6PD activity did not vary over time. Therefore, G6PD activity on D1 instead of D14 could be used for primaquine dose-adjustment. This could allow earlier radical treatment with primaquine, that could have a public health impact by decreasing early recurrences and patients lost to follow-up before primaquine initiation. This hypothesis needs to be confirmed in larger prospective studies.

Proceedings of the 2023 Viral Clearance Symposium, Session 4: Continuous Processing.

The continuous processing session at the 2023 Viral Clearance Symposium (VCS) focused on understanding how to effectively design viral clearance operations for use in continuous processes and methods to perform viral clearance studies. In this session, an approach to directly address control considerations with operating continuous-flow reactors for low pH viral inactivation was presented. Continuous-flow low pH incubation chamber design and implications for residence time determination were discussed. Additionally, viral clearance capability between batch operation and connected operation were demonstrated to be comparable for a connected bind-elute chromatography and flow-through chromatography step. Overall, this session provided additional scientific knowledge to support viral clearance strategies when implementing a continuous manufacturing process.

Proceedings of the 2023 Viral Clearance Symposium, Session 7: Up- and Downstream Virus Retentive Filtration.

Session 7 of the 2023 Viral Clearance Symposium reviewed progresses in virus retentive filtrations applied to both upstream and downstream processing. Upstream topics included investigations and applications of media viral filtration for upstream cell culture viral risk mitigation. Downstream topics included evaluation of viral breakthrough in continuous processing using surrogate particles and demonstration of extensive viral filtration cycling with flow interruptions and long duration in connected process. Reuse of viral filters with proposed procedures was successfully demonstrated amid the supply chain challenge encountered during the pandemic. Discussions and additional considerations for the topics were also provided.

Proceedings of the 2023 Viral Clearance Symposium, Session 5: Viral Clearance Strategy and Process Understanding.

Session 5 of the 2023 Viral Clearance Symposium reviewed the strategy and process understanding of viral clearance testing. Topics included learnings from the past, leveraging surrogate-based methodologies, cleaning agents that inactivate enveloped baculoviruses, segregation, and retrovirus-like particles both in continuous process and in-use as spiking viruses. Overall, there were discussions over a wide array of viral clearance determinants.

Proceedings of the 2023 Viral Clearance Symposium, Session 1: Regulatory Updates.

At the time of the 2023 Viral Clearance Symposium in Vienna, the ongoing revision of ICH Guideline Q5A(R1) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin clearly was the dominant regulatory topic. At the symposium, the changes expected for Q5A(R2) to mirror advances of scientific knowledge, for example, the inclusion of new products, including viral-vector-derived ones, that can be subject to virus clearance, deliberations around continuous manufacturing processes, the use of prior knowledge to supplement or in part replace virus validation studies, and new molecular methods for detection of adventitious viruses, were discussed by a European and a US regulator as well as representatives from industry associations that had been involved with the drafting process.

Proceedings of the 2023 Viral Clearance Symposium: 2023 VCS Summary, Pending Questions, and Next Steps.

The 2023 Viral Clearance Symposium (VCS) was hosted by Takeda on 24 and 25 May 2023 in Vienna, Austria. The present conference extended the structure of the previous biennial symposia held between 2009 and 2019. As recapitulated in the introductory session, the genesis of the VCS, as described in the Proceedings of the 2009 VCS was "the worldwide regulatory and industry recognition that challenges, gaps, and opportunities exist, that it formally addressed could benefit the field as whole." This report provides a synopsis of the progress achieved at the conference resulting from detailed technical discussions and the pending questions that still require attention to address. The 2023 VCS was composed of nine individual sessions of short presentations followed by in-depth panel discussions from the presenters. Sessions included Regulatory Updates (with a focus on ICH Q5A(R2) efforts), including a summary of lessons learned from the 2019 VCS, and progress on these key areas mapped into 2023 VCS topics: Viral Clearance Strategy and Case Studies, New Modalities in Chromatography and Adsorptive Filters, Continuous Processing, Viral Clearance Strategy and Process Understanding, Virus Inactivation, Upstream and Downstream Virus Retentive Filtration and Cell Banks, and Advanced Technologies (advanced therapy medicinal products, next-generation sequencing).

Proceedings of the 2023 Viral Clearance Symposium, Session 3: 2023 VCS New Modalities in Chromatography and Adsorptive Filters.

The session provided an update on the application and mechanistic understanding of intensified unit operations (e.g., mixed mode depth filters, mixed mode AEX) since the last conference in 2019. One of the key gaps identified in the 2019 Viral Clearance Symposium session on the topic was for more investigation required to achieve a clear understanding of the molecular mechanisms of virus removal and the relevance of different moleculés interactions including resin, virus, and product. Further investigation into worst-case conditions for these unit operations is also warranted. One of the key outcomes from that 2019 discussion was also that multimodal anion exchangers can have robust and effective virus removal, depending on process and impurities-an observation that was recapitulated with more specific case studies and evidenced by broader application of these chromatographic resins in late-stage regulatory filings.