Michaelis-Menten Model Research Articles

Extraction and immobilization of the urease enzyme in polyvinylpyrrolidone films: applications in biosensors

Enzymes play a fundamental role in the manufacture of biosensors, acting as biological recognition elements due to their sensitivity and selectivity in the reactions they catalyze. Among them, urease stands out as an efficient enzyme found in nature, whose function involves the hydrolysis of urea. This enzyme has been used when extracted from legume seeds, as well as various bacteria and fungi. The objective of this study was the extraction and immobilization of the urease enzyme in polyvinylpyrrolidone (PVP) films. Crude jack bean extracts were prepared at concentrations of 15, 25 and 35% in PBS/PVP, centrifuged and refrigerated. Subsequently, aliquots of each solution were applied to plates to form films, being characterized by enzymatic activity, pH, FTIR and UV-vis. The affinity with urea was evidenced by enzymatic activity, pH and UV-vis, compatible with the Michaelis-Menten model. In FTIR, the polypeptide bands characteristic of the amino acids present in the structure of the enzymes, such as those of amides I, II and III, were confirmed.

Species Differences in Ezetimibe Glucuronidation

Background: Peclinical and clinical studies have revealed that ezetimibe, an approved cholesterol-absorption inhibitor, is rapidly and extensively metabolized to a more potent metabolite, ezetimibe glucuronide. Since different species are commonly used in the pharmacokinetic and pharmacodynamic studies of ezetimibe, it is essential to determine the species difference in glucuronidation of ezetimibe in order to accurately evaluate ezetimibe’s pharmacokinetics and pharmacodynamics. The purpose of the study was to compare species differences in ezetimibe glucuronidation rates using intestinal microsomes from humans, rats, mice, monkeys, and dogs. Method: Intestinal microsomes from different species were used to assess the ezetimibe glucuronidation rates. Multiple substrate concentrations at 0.5, 2, 5, 10, 20, 30, 40, and 50 µM were tested and fitted into the Michaelis–Menten model to determine the enzyme kinetic parameters. Results: The results showed that the glucuronidation rates with these tested species were significantly different. Kinetic studies revealed that the maximum metabolic rate (Vmax) was higher in monkeys (3.87 ± 0.22 nmol/mg/min) than that in rat (2.40 ± 0.148 nmol/mg/min) and mouse (2.23 ± 0.10 nmol/mg/min), and then human (1.90 ± 0.08 nmol/mg/min) and dog (1.19 ± 0.06 nmol/mg/min). The CLint was an 8.17-fold difference among these species, following the order of mouse > dog > human > rat = monkey. Conclusions: The study revealed that the rate of ezetimibe glucuronidation in the intestine was different in different species and has an impact on ezetimibe glucuronidation in the intestine. When analyzing the pharmacodynamics, pharmacokinetics, or toxicology of ezetimibe using different models, these species differences must be taken into consideration.

Mammalian Esterase Activity: Implications for Peptide Prodrugs.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M-1 s-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s-1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

Long-term alien marsh grass in China brings high carbon uptake capacity but cannot sustain high-temperature weather

Coastal wetlands, crucial in the global carbon cycle, face increasing challenges brought by extreme climate events, particularly high temperatures above plant tolerance thresholds. These conditions often exert great impact on plant, thereby potentially reducing overall ecosystem productivity. However, it has been observed that alien species, typically exhibiting higher productivity compared to native plant. Would plant invasion offset the loss of productivity caused by high-temperature events at ecosystem scale? In this study, we utilized data from 2020 to 2023 in China's Yangtze Estuary to investigate the responses of Spartina alterniflora (alien) and Phragmites australis (native) to high-temperature stress. Our results demonstrate that though the alien vegetation exhibits higher productivity before high temperature events, it experiences significant declines during high temperatures. In average, net ecosystem productivity (NEP) and gross primary productivity (GPP) of alien plant drops by 21.03% overall, with a notable 29.59% reduction during Neap tide. In contrast, native vegetation maintains a more stable productivity profile under the same conditions. Spring tide alleviate the negative impact of high temperatures on the alien vegetation, exhibiting a distinct environmental buffering effect. Photosynthetic photon flux density emerged as a crucial factor driving productivity, yet its effectiveness was moderated by aerodynamic conductance for heat transfer (Ga_h). Through the application of the Michaelis-Menten model, we confirmed that both species maintain similar maximum light utilization efficiencies, yet native vegetation demonstrates greater resilience to thermal stress. Additionally, we observed a 33.82% overestimation in productivity by the vegetation photosynthesis model (VPM) under high temperatures, emphasizing the need to refine how Ga_h impacts are integrated, particularly when comparing the resilience of native and alien species. We emphasize necessity of incorporating canopy structure factors into ecological models and underscore the importance of maintaining tidal dynamics for coastal wetland management.

Mn3O4-FeS2/Fe2O3 composite as peroxidase-mimics for the degradation of Bisphenol A

Due to the benefits of artificial peroxidase-like enzymes, Flower-like Mn3O4-FeS2/Fe2O3 composites with multiple active sites prepared by a one-step solvothermal method.The synergistic cyclic reaction between Mn and Fe on the catalyst surface enhances the generation of active radicals, which together promote the efficient degradation of BPA. In the presence of hydrogen peroxide (H2O2), Mn3O4-FeS2/Fe2O3 could generate hydroxyl radicals (·OH) and superoxide radicals (·O2-) in buffer solution of pH=4.5 and catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidation products (ox TMB). The steady-state kinetic constants (Km) of TMB and H2O2 were obtained by Michaelis-Menten and Lineweaver-Burk models, which are 1.34 mM and 1.00 mM, respectively. This reveals the nice catalytic efficiency of Mn3O4-FeS2/Fe2O3 as enzyme mimics. Under the condition of 10 mg Mn3O4-FeS2/Fe2O3 catalyst, 100 μL H2O2 and pH = 4.5, the degradation efficiency of Bisphenol A (BPA) reaches 100 % when treating 40 mL of 50 mg L−1 BPA for 30 min at room temperature. Moreover, after 5 cycles of degradation, the BPA degradation efficiency of 85.8 % was still maintained. Therefore, the as-prepared Mn3O4-FeS2/Fe2O3 sample could potentially be applied in wastewater treatment.

Understanding Cytochrome P450 Enzyme Substrate Inhibition and Prospects for Elimination Strategies.

Cytochrome P450 (CYP450) enzymes, which are widely distributed and pivotal in various biochemical reactions, catalyze diverse processes such as hydroxylation, epoxidation, dehydrogenation, dealkylation, nitrification, and bond formation. These enzymes have been applied in drug metabolism, antibiotic production, bioremediation, and fine chemical synthesis. Recent research revealed that CYP450 catalytic kinetics deviated from the classic Michaelis-Menten model. A notable substrate inhibition phenomenon that affects the catalytic efficiency of CYP450 at high substrate concentrations was identified. However, the substrate inhibition of various reactions catalyzed by CYP450 enzymes have not been comprehensively reviewed. This review describes CYP450 substrate inhibition examples and atypical Michaelis-Menten kinetic models, and provides insight into mechanisms of these enzymes. We also reviewed 3D structure and dynamics of CYP450 with substrate binding. Outline methods for alleviating substrate inhibition in CYP450 and other enzymes, including traditional fermentation approaches and protein engineering modifications. The comprehensive analysis presented in this study lays the foundation for enhancing the catalytic efficiency of CYP450 by deregulating substrate inhibition.

Highly efficient degradation of ethanol, acetaldehyde, and ethyl acetate removal by bio-trickling filter reactors with various fillers

This study investigates the purification performance of bio-trickling filters (BTFs) using different media to treat ethanol, acetaldehyde, and ethyl acetate in kitchen waste malodorous gases. The media compared included a custom composite medium, pine bark, hollow polyhedral spheres, and ceramic particles. Over 25 days, the composite medium outperformed the traditional media, achieving removal rates of 90.13 % for ethanol, 63.89 % for acetaldehyde, and 82.56 % for ethyl acetate during the biofilm initiation phase, with the others below 60 %. Even under low empty bed residence time and high inlet concentrations, the maximum elimination capacity for ethanol, acetaldehyde, and ethyl acetate was 8.34–14.70 g/m3·h, 9.55–15.06 g/m3·h, and 6.18–10.45 g/m3·h. Kinetic analysis showed the Michaelis-Menten model fit well, indicating enhanced removal potential. High-throughput of 16S rDNA sequencing identified dominant microorganisms like Enterobacteriaceae (13.89 %), Stenotrophomonas (29.23 %), and Acinetobacter (4.09 %) in the composite medium, which thrived even at high pollutant concentrations. Principal component analysis (PCA) demonstrated differences in the microbial composition of the custom composite medium compared to traditional media under varying inlet concentrations and loads. This study provides technical support for the treatment of complex malodorous gas mixtures.

Role of Associated Water Dynamics on Protein Stability and Activity in Crowded Milieu.

Macromolecular crowding bridges in vivo and in vitro studies by simulating cellular complexities such as high viscosity and limited space while maintaining the experimental feasibility. Over the last two decades, the impact of macromolecular crowding on protein stability and activity has been a significant topic of study and discussion, though still lacking a thorough mechanistic understanding. This article investigates the role of associated water dynamics on protein stability and activity within crowded environments, using bromelain and Ficoll-70 as the model systems. Traditional crowding theory primarily attributes protein stability to entropic effects (excluded volume) and enthalpic interactions. However, our recent findings suggest that water structure modulation plays a crucial role in a crowded environment. In this report, we strengthen the conclusion of our previous study, i.e., rigid-associated water stabilizes proteins via entropy and destabilizes them via enthalpy, while flexible water has the opposite effect. In the process, we addressed previous shortcomings with a systematic concentration-dependent study using a single-domain protein and component analysis of solvation dynamics. More importantly, we analyze bromelain's hydrolytic activity using the Michaelis-Menten model to understand kinetic parameters like maximum velocity (Vmax) achieved by the system and the Michaelis-Menten coefficient (KM). Results indicate that microviscosity (not the bulk viscosity) controls the enzyme-substrate (ES) complex formation, where an increase in the microviscosity makes the ES complex formation less favorable. On the other hand, flexible associated water dynamics were found to favor the rate of product formation significantly from the ES complex, while rigid associated water hinders it. This study improves our understanding of protein stability and activity in crowded environments, highlighting the critical role of associated water dynamics.

Intrinsic Peroxidase-like Activity of Polystyrene Nanoplastics Mediates Oxidative Stress.

Nanoplastics represent a global environmental concern due to their ubiquitous presence and potential adverse impacts on public and environmental health. There is a growing need to advance the mechanistic understanding of their reactivity as they interact with biological and environmental systems. Herein, for the first time, we report that polystyrene nanoplastics (PSNPs) have intrinsic peroxidase-like activity and are able to mediate oxidative stress. The peroxidase-like activity is dependent on temperature and pH, with a maximum at pH 4.5 and 40 °C. The catalytic activity exhibits saturation kinetics, as described by the Michaelis-Menten model. The peroxidase-like activity of PSNPs is attributed to their ability to mediate electron transfer from peroxidase substrates to H2O2. Ozone-induced PSNP aging can introduce oxygen-containing groups and disrupt aromatic structures on the nanoplastic surface. While ozonation initially enhances peroxidase-like activity by increasing oxygen-containing groups without degrading many aromatic structures, extended ozonation destroys aromatic structures, significantly reducing this activity. The peroxidase-like activity of PSNPs can mediate oxidative stress, which is generally positively correlated with their aromatic structures, as suggested by the ascorbic acid assay. These results help explain the reported oxidative stress exerted by nanoplastics and provide novel insights into their environmental and public health implications.

Dual-mode optical biocompatible-sensor enabled by enzyme-like activity of UiO-66 (Zr) for ultra-sensitive ROS detection in vitro

The simple, effective and highly sensitive detection of hydrogen peroxide (H2O2), which belongs to the reactive oxygen species (ROS), at low concentrations plays an indispensable role in the field of environmental protection, biological research and safety. In this study, a dual-mode optical biosensor, UiO-66@OPD, was developed based on the inherent peroxidase mimicking activity of UiO-66 (Zr) and the optical reaction of ortho-phenylenediamine (OPD) by extending the π-system through oxidative coupling, prototropism and elimination to form OPDox, thereby exhibiting strong orangish absorbance and greenish fluorescence. The catalase-mimicking activity of UiO-66 (Zr) was demonstrated by the catalytic oxidation of methylene blue in the presence of H2O2. Moreover, the Michaelis-Menten kinetic model confirmed the intrinsic peroxidase-like activity of UiO-66@OPD as a modified MOFzyme. The synthesized UiO-66 (Zr) facilitated the oxidation of OPD to OPDox by degrading H2O2 to the hydroxyl radicals. During the oxidation process, the absorption peak at 415 nm and the fluorescence peak at 565 nm of the synthesized probe were significantly enhanced by increasing the H2O2 concentration. Moreover, a colorimetric and fluorometric ultrasensitive sensor shows a good linear relationship between the intensity enhancement and H2O2 concentration in the range of 0–600 nM for absorption and fluorescence spectra with R2 = 0.9772, and R2 = 0.9948, respectively. To demonstrate the biological performance and biocompatibility of UiO-66@OPD as a biosensor, MTT evaluation was performed for the three cell lines MCF-10 A, HEK293 and A549, indicating high biocompatibility and good cell viability for biological applications. Ultimately, this convenient, environmentally friendly, biocompatible and cost-effective catalase-mimicking-based sensor system will open a new perspective for the development of portable kite-based biosensors In vitro.

Substrate specificity of commercial lipases activated by a hydration–aggregation pretreatment in anhydrous esterification reactions

Substrate specificity in non-aqueous esterification catalyzed by commercial lipases activated by hydration–aggregation pretreatment was investigated. Four microbial lipases from Rhizopus japonicus, Burkholderia cepacia, Rhizomucor miehei, and Candida antarctica (fraction B) were used to study the effect of the carbon chain length of saturated fatty acid substrates on the esterification activity with methanol in n-hexane. Hydration–aggregation pretreatment had an activation effect on all lipases used, and different chain length dependencies of esterification activity for lipases from different origins were demonstrated. The effects of various acidic substrates with different degrees of unsaturation, aromatic rings, and alcohol substrates with different carbon chain lengths on esterification activity were examined using R. japonicus lipase, which demonstrated the most remarkable activity enhancement after hydration–aggregation pretreatment. Furthermore, in the esterification of myristic acid with methanol catalyzed by the hydrated–aggregated R. japonicus lipase, maximum reaction rate (5.43 × 10−5 mmol/(mg-biocat min)) and Michaelis constants for each substrate (48.5 mM for myristic acid, 24.7 mM for methanol) were determined by kinetic analysis based on the two-substrate Michaelis-Menten model.

Biodegradability and formulation of triclosan-degrading and plant-growth-promoting Pseudomonas sp. MS45

Abstract. Sipahutar MK. 2024. Biodegradability and formulation of triclosan-degrading and plant-growth-promoting Pseudomonas sp. MS45. Biodiversitas 25: 2980-2992. This study presents the isolation of Pseudomonas sp. MS45, a microorganism that can consume triclosan (TCS) as its only carbon and energy source, leading to its detoxification. The rarity of a toxicity study of TCS before and after biodegradation treatment is addressed in this study. Allium cepa's genotoxicity exposed TCS's cytotoxicity, generating multiple damaged chromosomes, in contrast to the insignificant consequences of its broken-down metabolites. The study also optimized the different kinetic parameters for TCS-specific biodegradation and Pseudomonas sp. MS45-specific growth. The biodegradation pattern followed the Michaelis-Menten model with a maximum specific biodegradation rate (Vmax) of 0.01 µmole h-1 mg cell protein-1 and a half saturation constant (Ks) of 21.98 µM. The growth pattern followed the Haldane model with a maximum specific growth rate (µmax) of 0.03 h-1, half saturation constant (Ks) of 4.56 µM, and TCS inhibition constant (Ki) of 32.81 µM. In addition to its TCS-degrading ability, Pseudomonas sp. MS45 also possesses various plant growth promotion traits, and it was incorporated into two solid carrier formulations: vermiculite and rice bran. The study found that vermiculite, especially with PEG, was the most consistent formulation for Pseudomonas sp. MS45, in terms of maintaining bacterial survival and TCS-degrading ability. This study marks the initial report on the biodegradation and formulation of TCS-destroying and Plant-Growth-Promoting (PGP) Pseudomonas sp. MS45, offering promising implications for environmental and agricultural applications.

Delineating acetaminophen biodegradation kinetics and metabolomics using bacterial community.

Acetaminophen [N-(4-hydroxyphenyl) acetamide, APAP]is an extensively and frequently consumed over-the-counter analgesic and antiphlogistic medication. It is being regarded as an emerging pollutant due to its continuous increment in the environment instigating inimical impacts on humans and the ecosystem. Considering its wide prevalence in the environment, there is an immense need of appropriate methods for the removal of APAP. The present study indulged screening and isolation of APAP degrading bacterial strains from pharmaceuticals-contaminated sites, followed by their molecular characterization via 16SrRNA sequencing. The phylogenetic analyses assigned the isolates to the genera Pseudomonas, Bacillus, Paracoccus, Agrobacterium, Brucella, Escherichia, and Enterobacter based on genetic relatedness. The efficacy of these strains in batch cultures tested through High-performance Liquid Chromatography (HPLC) revealed Paracoccus sp. and Enterobacter sp. as the most promising bacterial isolates degrading up to 88.96 and 85.92%, respectively of 300mg L-1 of APAP within 8days of incubation. Michaelis-Menten kinetics model parameters also elucidated the high degradation potential of these isolates. The major metabolites identified through FTIR and GC-MS analyses were 4-aminophenol, hydroquinone, and 3-hydroxy-2,4-hexadienedioic. Therefore, the outcomes of this comprehensive investigation will be of paramount significance in formulating strategies for the bioremediation of acetaminophen-contaminated sites through a natural augmentation process via native bacterial strains.

Removal Characteristics of Gas-Phase D-Limonene in Biotrickling Filter and Stoichiometric Analysis of Biological Reaction using Carbon Mass Balance

Volatile organic compounds (VOCs) pose significant risks to human health and environmental quality, prompting stringent regulations on their emissions from various industrial processes. Among VOCs, d-limonene stands out due to its low threshold and contribution to malodorous emissions. While biofiltration presents a promising approach for VOC removal, including d-limonene, a comprehensive understanding of its performance and kinetics is lacking. This study aims to comprehensively assess the performance of a lab-scale biotrickling filter in treating gas-phase d-limonene. The experimental results indicate that the biotrickling filter efficiently removed d-limonene, achieving a critical loading rate of 19.4 g m−3 h−1 and a maximum elimination capacity of 31.8 g m−3 h−1 (correspondingly, up to 85% removal) at the condition of 94.2 s of EBRT. Microbial activity played a significant role in biotrickling filter performance, with a strong linear correlation being observed between CO2 production and substrate consumption. The Michaelis–Menten model was employed to represent enzyme-catalyzed reactions, suggesting no inhibition during biotrickling filter operation.

KINETIC MODELLING OF BIOCHEMICAL REACTIONS USING MATHСAD ANALYTICAL TOOLKIT

Background. The study of the kinetics of biochemical reactions provides a better understanding of how biological processes occur in living organisms. Understanding the peculiarities of such reactions is important for the development of new technologies, in particular for the production of biologically active substances and for the synthesis of drugs. A powerful tool for solving problems in biochemical reaction kinetics is mathematical modelling, which can be carried out using computer mathematical systems, in particular the MATHCAD analytical toolkit. Aim: to substantiate the feasibility and effectiveness of using the MATHCAD analytical toolkit to solve problems of kinetic modelling of biochemical reactions in pharmaceutical research, and to review the capabilities of MATHCAD for computer modelling in pharmacy. Materials and methods. In the context of studying the rate of enzymatic reactions and developing models, such as the Michaelis-Menten model, to describe reactions in which enzymes catalyze the transformation of substrates, the use of a computer mathematical system (CMS) is considered. CMS is a software package and environment for performing mathematical computations, modelling and visualization. The possibilities of using the MATHCAD system to create mathematical models of biochemical reactions based on kinetic equations are demonstrated. This involves the creation of differential equations describing changes in reagent concentrations over time. These equations were solved using numerical methods in MATHCAD. In addition, the results obtained are visualized using 3D graphics in MATHCAD. The stages of using the MATHCAD analytical toolkit in the kinetic modelling of biochemical reactions have been determined. Results. The use of MATHCAD in the kinetic modelling of biochemical reactions is effective for the study of: (1) the kinetics of enzymatic reactions, e.g. reactions in which an enzyme catalyzes the conversion of a substrate into a product; (2) biochemical reactions that take place in reaction vessels in which reagents mix and interact; (3) modelling of reactions in reaction vessels based on the solution of differential equations of reaction kinetics; (4) the effect of inhibitors or activators on enzymatic reactions; (5) scenarios of interaction of reagents to determine changes in the kinetics of reactions that occur when different active substances are introduced; (6) kinetics of biochemical reactions in cases where reactions are accompanied by diffusion of reagents through membranes or other semi-permeable barriers; (7) modelling the effect of diffusion processes on the kinetics of biochemical reactions; (8) models describing the kinetics of decomposition of substances, for example the decomposition of biologically active compounds in the body or in the environment; (9) predicting the effect of changes in the conditions of the reaction medium (temperature, pH, concentration of reagents) on the kinetics of biochemical reactions. It is substantiated that model descriptions of the kinetics of biochemical reactions are important for forming an understanding of the functions of biological systems, including metabolism, enzymatic reactions, and other physiological phenomena. Tools have been used to visualize the modelling results in the form of three-dimensional MATHCAD graphics, which improves the understanding of the reaction mechanism and allows a more thorough analysis of its kinetics. Conclusion. MATHCAD provides an optimized environment for kinetic modelling of biochemical reactions through its ergonomic interface. Particular advantages are the ability to work with symbolic expressions and to use a wide range of built-in functions and tools for exploring mathematical models and visualizing results. The obtained results may be important both for further scientific pharmaceutical research and for implementation in the training of future Masters of Pharmacy in the discipline of "Computer Modelling in Pharmacy" in higher medical education institutions.

Evaluating the contribution of methanotrophy kinetics to uncertainty in the soil methane sink

The oxidation of atmospheric methane by soil microbes is an important natural sink for a potent greenhouse gas. However, estimates of the current and future soil methane sink are highly uncertain. Here we assessed the extent to which methanotrophy enzyme kinetics contribute to uncertainty in projections of the soil methane sink. We generated a comprehensive compilation of methanotrophy kinetic data from modern environments and assessed the patterns in kinetic parameters present in natural samples. Our compiled data enabled us to quantify the global soil methane sink through two idealized calculations comparing first-order and Michaelis–Menten models of kinetics. We show that these two kinetic models diverge only under high atmospheric CH4 scenarios, where first-order rate constants slightly overestimate the soil methane sink size, but produce similar predictions at modern atmospheric concentrations. Our compilation also shows that the kinetics of methanotrophy in natural soil samples is highly variable—both the V max (oxidation rate at saturation) and KM (half-saturation constant) in natural samples span over six orders of magnitude. However, accounting for the correlation we observe between V max and KM reduces the range of calculated uptake rates by as much as 96%. Additionally, our results indicate that variation in enzyme kinetics introduces a similar magnitude of variation in the calculated soil methane sink as temperature sensitivity. Systematic sampling of methanotroph kinetic parameters at multiple spatial scales should therefore be a key objective for closing the budget on the global soil methane sink.

Reversing the relative time courses of the peptide bond reaction with oligopeptides of different lengths and charged amino acid distributions in the ribosome exit tunnel

The kinetics of the protein elongation cycle by the ribosome depends on intertwined factors. One of these factors is the electrostatic interaction of the nascent protein with the ribosome exit tunnel. In this computational biology theoretical study, we focus on the rate of the peptide bond formation and its dependence on the ribosome exit tunnel electrostatic potential profile. We quantitatively predict how oligopeptides of variable lengths can affect the peptide bond formation rate. We applied the Michaelis-Menten model as previously extended to incorporate the mechano-biochemical effects of forces on the rate of reaction at the catalytic site of the ribosome. For a given pair of carboxy-terminal amino acid substrate at the P- and an aminoacyl-tRNA at the A-sites, the relative time courses of the peptide bond formation reaction can be reversed depending on the oligopeptide sequence embedded in the tunnel and their variable lengths from the P-site. The reversal is predicted to occur from a shift in positions of charged amino acids upstream in the oligopeptidyl-tRNA at the P-site. The position shift must be adjusted by clever design of the oligopeptide probes using the electrostatic potential profile along the exit tunnel axial path. These predicted quantitative results bring strong evidence of the importance and relative contribution of the electrostatic interaction of the ribosome exit tunnel with the nascent peptide chain during elongation.

Degradation of pretilachlor and fenclorim and effects of these compounds on bacterial communities under anaerobic condition.

Pretilachlor and safener fenclorim are the main components of herbicides widely applied to control weeds. Although some pure cultures of bacteria and fungi which degraded these compounds under aerobic conditions were isolated, no isolated pretilachlor- and fenclorim-degrading bacterial strains under anaerobic condition had been available. In this study, the degradation of these compounds and the effects of them on bacterial community structures were investigated under anaerobic conditions. The dissipation rates of pretilachlor and fenclorim in slurries were in the order: soil from paddy field ≈ sediment from river > sediment from mangrove. Moreover, three pretilachlor-degrading bacterial strains (Pseudomonas sp. Pr1, Proteiniclasticum sp. Pr2 and Paracoccus denitrificans Pr3) and two fenclorim-degrading strains (Dechloromonas sp. Fe1 and Ralstonia pickettii Fe2) isolated from a slurry of paddy soil utilized the substrates as sole carbon and energy sources under anaerobic conditions. The degradation of pure pretilachlor and fenclorim at various concentrations by corresponding mixed pure cultures followed the Michaelis-Menten model, with the maximum degradation was 3.10 ± 0.31µM/day for pretilachlor, and 2.08 ± 0.18µM/day for fenclorim. During the degradation, 2-chloro-N-(2,6-diethylphenyl) acetamide and 2,6-dimethylaniline were produced in pretilachlor degradation, and benzene was a product of fenclorim degradation. The synergistic degradation of both substrates by all isolated bacteria reduced the metabolites concentrations accumulated in media. This study provides valuable information on effects of pretilachlor and fenclorim on bacterial communities in soil and sediments, and degradation of these substrates by isolated bacteria under anaerobic condition.

Validity conditions of approximations for a target-mediated drug disposition model: A novel first-order approximation and its comparison to other approximations.

Target-mediated drug disposition (TMDD) is a phenomenon characterized by a drug's high-affinity binding to a target molecule, which significantly influences its pharmacokinetic profile within an organism. The comprehensive TMDD model delineates this interaction, yet it may become overly complex and computationally demanding in the absence of specific concentration data for the target or its complexes. Consequently, simplified TMDD models employing quasi-steady state approximations (QSSAs) have been introduced; however, the precise conditions under which these models yield accurate results require further elucidation. Here, we establish the validity of three simplified TMDD models: the Michaelis-Menten model reduced with the standard QSSA (mTMDD), the QSS model reduced with the total QSSA (qTMDD), and a first-order approximation of the total QSSA (pTMDD). Specifically, we find that mTMDD is applicable only when initial drug concentrations substantially exceed total target concentrations, while qTMDD can be used for all drug concentrations. Notably, pTMDD offers a simpler and faster alternative to qTMDD, with broader applicability than mTMDD. These findings are confirmed with antibody-drug conjugate real-world data. Our findings provide a framework for selecting appropriate simplified TMDD models while ensuring accuracy, potentially enhancing drug development and facilitating safer, more personalized treatments.

Enhancing the recovery of complex amino acids from excess sludge via low-intensity ultrasound-assisted enzymatic hydrolysis

The recovery of high-value products such as complex amino acids from activated sludge is a crucial alternative for the recycling of excess sludge, although several challenges still need to be addressed, especially in terms of extraction efficiency and product quality. Herein, a promising approach involving low-intensity ultrasound-assisted enzymatic hydrolysis (UEH) was utilized to obtain complex amino acids from sludge protein. Compared with the values obtained for single enzymatic hydrolysis (EH), after a 20-minute pre-treatment at ambient temperature using low-intensity ultrasound (0.15 W/mL), the enzyme activity increased by 28.7 %, and the degree of protein hydrolysis (DH) correspondingly rose by 93.3 %. The application of ultrasound during subsequent EH resulted in a decrease in DH, with the extent of the decrease increasing over time. This suggested that the primary cause was conformational changes of enzymes rather than mass transfer. Moreover, both the Michaelis–Menten model (R2 > 0.99) and protein hydrolysis model (R2 > 0.96) accurately simulated EH and UEH, indicating that UEH exhibits superior enzyme–substrate affinity and a higher rate of protein hydrolysis. Furthermore, after separation and purification, the total content of seven essential amino acids in the dry material exceeded that in traditional feed sources such as soybean meal. Additionally, the safety test and energy consumption analysis demonstrated that UEH has good potential for the extraction of complex amino acids from excess sludge.