Substrate Inhibition Research Articles

Bioconversion of ricinoleic acid to (R)-γ-decalactone by Clavispora lusitaniae YJ26 cells in an ionic liquid–containing biphasic fermentation system

Microbial production of chiral γ-decalactone has received considerable attention due to its high market value. However, the inhibition of substrates and products on microorganisms has significantly limited the production of chiral γ-decalactone. In this study, a green biphasic fermentation system containing 1-butyl-3-methylimidazolium tetrafluoroborate ([C4MIM]PF6) was developed to increase the synthesis of (R)-γ-decalactone from ricinoleic acid catalyzed by Clavispora lusitaniae YJ26. Under optimal conditions, the titer of γ-decalactone obtained using the biphasic system reached 2.04 g/L, which is 2.4 times that of the traditional aqueous fermentation system, and showed high enantioselectivity (e.e.> 99.0%). Fluorescence microscopy and flow cytometry results demonstrated that the addition of [C4MIM]PF6 significantly alleviated the damage of substrate and product to cell membrane and improved cell viability, thus increasing the titer of γ-decalactone. Furthermore, comparative transcriptomic analysis results suggested that the improved mechanism may be attributed to the up-regulation of genes associated with cell membrane component synthesis or cell tolerance enhancement. Overall, the experimental results provide valuable insights into the efficient microbial production of chiral decalactone in an ionic liquids–containing biphasic fermentation system.

Substrate binding and inhibition mechanism of norepinephrine transporter.

Norepinephrine transporter (NET; encoded by SLC6A2) reuptakes the majority of the released noradrenaline back to the presynaptic terminals, thereby affecting the synaptic noradrenaline level1. Genetic mutations and dysregulation of NET are associated with a spectrum of neurological conditions in humans, making NET an important therapeutic target1. However, the structure and mechanism of NET remain unclear. Here we provide cryogenic electron microscopy structures of the human NET (hNET) in three functional states-the apo state, and in states bound to the substrate meta-iodobenzylguanidine (MIBG) or the orthosteric inhibitor radafaxine. These structures were captured in an inward-facing conformation, with a tightly sealed extracellular gate and an open intracellular gate. The substrate MIBG binds at the centre of hNET. Radafaxine also occupies the substrate-binding site and might block the structural transition of hNET for inhibition. These structures provide insights into the mechanism of substrate recognition and orthosteric inhibition of hNET.

Structural basis of divergent substrate recognition and inhibition of human neurolysin

A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1–13) potently inhibits the Nln activity while dynorphin A (1–8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln.

Immobilization of Saccharomyces cerevisiae on polyhydroxyalkanoate/konjac glucan nanofiber membranes: Characterization, immobilization efficiency and cellular activity

Yeast immobilization systems can recoup yeast losses in continuous batch fermentation and relieve substrate or product inhibition. We report the use of solution blow spinning process to efficiently prepare polyhydroxyalkanoate (PHB) /konjac glucomannan (KGM) nanofiber membranes as immobilization carriers for Saccharomyces cerevisiae. The prepared PHB/KGM nanofiber membranes had fiber diameters similar to the scale of yeast cells. Incorporating KGM significantly enhanced the porosity (from 87.21 % to 91.74 %), crystallinity, and hydrophilicity (reducing water contact angle from 135.8° to 110.1°), while increasing the specific surface area (from 10.24 to 17.79 m2/g) of pure PHB nanofiber membranes. Thermal stability was maintained (degradation temperatures above 250 °C). These changes enhanced the force between the nanofiber membranes and the cells and facilitated their autoimmobilization on the nanofiber membranes. The highest yeast immobilization efficiency of 87.93 % could be achieved at a KGM addition ratio of 400:2. Yeast showed no loss of cellular activity on the immobilized carriers of natural materials and maintained or even improved fermentation kinetics during at least three consecutive alcoholic fermentations These findings indicate that PHB/KGM nanofiber membranes can serve as effective carriers for yeast immobilization, promoting the sustainable production of fermented foods.

Chemical approaches for the biomass valorisation: a comprehensive review of pretreatment strategies.

The most abundant natural renewable resource in the world, lignocellulosic biomass (LCB), has the potential to be exploited as a substitute green feedstock for the synthesis of various chemicals, materials, and biofuels. The annual global production of 13 billion tonnes of LCB offers an opportunity to cater to the increasing energy and materials requirement of process industries and also restricts the discharge of greenhouse gases. Although LCB is enriched with valuable ingredients such as cellulose, lignin, and hemicellulose, its recalcitrant nature limits its efficient utilisation. These components of LCB are strongly interlinked with each other, which resists their isolation and conversion valorisation into useful products. To disrupt the complicated structure of LCB and to isolate the lignocellulosic components in pure form, pretreatment is a crucial process in the bio-refinery, ensuring the economic feasibility of downstream processes. This review provides an outline of the structure, composition, and various sources of LCB; and the necessity of the pretreatment. Moreover, this article provides an in-depth analysis of the underlying mechanisms, advantages, and limitations of various pretreatment methods, such as physical, chemical, biological, and physicochemical. Further, the impact of chemical pretreatment techniques on the physicochemical characteristics of the material that is extracted from the biomass is also covered in detail through the rigorous evaluation of performance metrics, including substrate digestibility, sugar yield, inhibitor production, and energy requirements. This review provides a balanced and comprehensive overview of the state-of-the-art pretreatment strategies and their impact on biomass valorisation that will be useful to the scientists, engineers, and policy makers interested in biomass conversion technologies.

Multicopper Oxidase from Lactobacillus hilgardii: Mechanism of Degradation of Tyramine and Phenylethylamine in Fermented Food.

Elevated levels of biogenic amines (BAs) in fermented food can have negative effects on both the flavor and health. Mining enzymes that degrade BAs is an effective strategy for controlling their content. The study screened a strain of Lactobacillus hilgardii 1614 from fermented food system that can degrade BAs. The multiple copper oxidase genes LHMCO1614 were successfully mined after the whole genome protein sequences of homologous strains were clustered and followed by homology modeling. The enzyme molecules can interact with BAs to stabilize composite structures for catalytic degradation, as shown by molecular docking results. Ingeniously, the kinetic data showed that purified LHMCO1614 was less sensitive to the substrate inhibition of tyramine and phenylethylamine. The degradation rates of tyramine and phenylethylamine in huangjiu (18% vol) after adding LHMCO1614 were 41.35 and 40.21%, respectively. Furthermore, LHMCO1614 demonstrated universality in degrading tyramine and phenylethylamine present in other fermented foods as well. HS-SPME-GC-MS analysis revealed that, except for aldehydes, the addition of enzyme treatment did not significantly alter the levels of major flavor compounds in enzymatically treated fermented foods (p > 0.05). This study presents an enzymatic approach for regulating tyramine and phenylethylamine levels in fermented foods with potential applications both targeted and universal.

Anaerobic Digestion of Dye Wastewater and Agricultural Waste with Bio-Energy and Biochar Recovery: A Techno-Economic and Sustainable Approach

While several researchers have investigated the anaerobic digestion (AD) of textile wastewater for dye degradation, their studies suffer from lower biogas productivity due to substrate inhibition and the occurrence of secondary pollution from digestate disposal. Hence, this study focuses on using the extract of wheat straw (WS) as a co-substrate to facilitate the dye AD process, followed by recycling the digestate sludge for biochar production. In the first study, the batch digesters were operated at different dye wastewater (DW)/WS ratios (0–50% v/v), substrate-to-inoculum ratio of 0.28–0.50 g/g, pH 7.0 ± 0.2, and 37 °C. The digester operated at a DW/WS fraction of 65/35% (v/v) showed the best chemical oxygen demand (COD) removal efficiency of 68.52 ± 3.40% with bio-CH4 of 270.52 ± 19.14 mL/g CODremoved. About 52.96 ± 3.61% of the initial COD mass was converted to CH4, avoiding inhibition caused by volatile fatty acid (VFA) accumulation. In the second experiment, the dry digestate was thermally treated at 550 °C for 2 h under an oxygen-deprived condition, yielding 0.613 ± 0.031 g biochar/g. This biochar exhibited multiple functional groups, mineral contents, and high stability (O/C = 0.193). The combined digestion/pyrolysis scenario treating 35 m3/d (106.75 kg COD/d) could maintain profits from pollution reduction, biogas, biochar, and carbon trading, obtaining a 6.5-year payback period.

Drug interaction with UDP-Glucuronosyltransferase (UGT) enzymes is a predictor of drug-induced liver injury.

DILI frequently contributes to the attrition of new drug candidates and is a common cause for the withdrawal of approved drugs from the market. Although some noncytochrome P450 (non-CYP) metabolism enzymes have been implicated in DILI development, their association with DILI outcomes has not been systematically evaluated. In this study, we analyzed a large data set comprising 317 drugs and their interactions in vitro with 42 non-CYP enzymes as substrates, inducers, and/or inhibitors retrieved from historical regulatory documents using multivariate logistic regression. We examined how these in vitro drug-enzyme interactions are correlated with the drugs' potential for DILI concern, as classified in the Liver Toxicity Knowledge Base database. Our study revealed that drugs that inhibit non-CYP enzymes are significantly associated with high DILI concern. Particularly, interaction with UDP-glucuronosyltransferases (UGT) enzymes is an important predictor of DILI outcomes. Further analysis indicated that only pure UGT inhibitors and dual substrate inhibitors, but not pure UGT substrates, are significantly associated with high DILI concern. Drug interactions with UGT enzymes may independently predict DILI, and their combined use with the rule-of-two model further improves overall predictive performance. These findings could expand the currently available tools for assessing the potential for DILI in humans.

Comparative analysis of two chemostat models including substrate and biomass inhibitions

Comparative analysis of two chemostat models including substrate and biomass inhibitions

Structural Basis for Substrate Binding, Catalysis and Inhibition of Breast Cancer Target Mitochondrial Creatine Kinase by Covalent Inhibitor via Cryo-EM.

Mitochondrial creatine kinases are key players in maintaining energy homeostasis in cells by working in conjunction with cytosolic creatine kinases for energy transport from mitochondria to cytoplasm. High levels of MtCK observed in Her2+ breast cancer and inhibition of breast cancer cell growth by substrate analog, cyclocreatine, indicate dependence of cancer cells on the 'energy shuttle' for cell growth and survival. Hence, understanding the key mechanistic features of creatine kinases and their inhibition plays an important role in the development of cancer therapeutics. Herein, we present the mutational and structural investigation on understudied ubiquitous mitochondrial creatine kinase (uMtCK). Our cryo-EM structures and biochemical data on uMtCK showed closure of the loop comprising residue His61 is specific to and relies on creatine binding and the reaction mechanism of phosphoryl transfer depends on electrostatics in the active site. In addition, the previously identified covalent inhibitor CKi showed inhibition in breast cancer BT474 cells, however our biochemical and structural data indicated that CKi is not a potent inhibitor for breast cancer due to strong dependency on the covalent link formation and inability to induce conformational changes upon binding.

Strategic enrichment of ocotillol-type ginsenosides F11, RT5 and ocotillol from Panax quinquefolium

The stem and leaves of American ginseng are rich in protopanaxatriol-type, protopanaxadiol-type, and ocotillol-type ginsenosides, which have important pharmacological activities. Among them, ocotillol-type ginsenosides have emerged as promising candidates for anti-cancer therapy. However, the scarcity of ocotillol-type ginsenosides presents significant challenges in terms of cost and sustainability during the synthesis process. In this study, we utilized three key glycosidases (HaGH03, PF-bgl, and DT-bgl) as essential tools in conjunction with diverse biotransformation strategies to augment the enrichment of pseudoginsenoside F11, RT5, and ocotillol. The results demonstrate that the combination of specific macroporous resin and the whole-cell catalyst HaGH03 enables rapid separation of ginsenoside extracts from Panax quinquefolium stems and leaves (PQE), effectively enriching pseudoginsenoside F11. In addition, the engineered mutant PF-bglF327C/F223I significantly enhanced the catalytic activity and addressed substrate inhibition issues, facilitating the enrichment of pseudoginsenoside RT5. Furthermore, the synergistic utilization of PF-bglF327C/F223I and DT-bgl enabled the one-step effective enrichment of ocotillol. This study presents a novel approach for rapid extraction and conversion of ocotillol-type ginsenosides while providing a feasible combination strategy for obtaining other rare natural products in the future.

C-terminal Poly-histidine Tags Alter Escherichia coli Polyphosphate Kinase Activity and Susceptibility to Inhibition

In Escherichia coli, many environmental stressors trigger polyphosphate (polyP) synthesis by polyphosphate kinase (PPK1), including heat, nutrient restriction, toxic compounds, and osmotic imbalances. PPK1 is essential for virulence in many pathogens and has been the target of multiple screens for small molecule inhibitors that might serve as new anti-virulence drugs. However, the mechanisms by which PPK1 activity and polyP synthesis are regulated are poorly understood. Our previous attempts to uncover PPK1 regulatory elements resulted in the discovery of PPK1* mutants, which accumulate more polyP in vivo, but do not produce more in vitro. In attempting to further characterize these mutant enzymes, we discovered that the most commonly-used PPK1 purification method – Ni-affinity chromatography using a C-terminal poly-histidine tag – altered intrinsic aspects of the PPK1 enzyme, including specific activity, oligomeric state, and kinetic values. We developed an alternative purification strategy using a C-terminal C-tag which did not have these effects. Using this strategy, we were able to demonstrate major differences in the in vitro response of PPK1 to 5-aminosalicylic acid, a known PPK1 inhibitor, and observed several key differences between the wild-type and PPK1* enzymes, including changes in oligomeric distribution, increased enzymatic activity, and increased resistance to both product (ADP) and substrate (ATP) inhibition, that help to explain their in vivo effects. Importantly, our results indicate that the C-terminal poly-histidine tag is inappropriate for purification of PPK1, and that any in vitro studies or inhibitor screens performed with such tags need to be reconsidered in that light.

Biological treatment of N-methylpyrrolidone, cyclopentanone, and diethylene glycol monobutyl ether distilled residues and their effects on nitrogen removal in a full-scale wastewater treatment plant

Manufacturing processes in semiconductor and photonics industries involve the use of a significant amount of organic solvents. Recycle and reuse of these solvents produce distillate residues and require treatment before being discharged. This study aimed to evaluate the performance of the biological treatment system in a full-scale wastewater treatment plant that treats wastewater containing distillate residues from the recycling of electronic chemicals. Batch experiments were conducted to investigate the optimal operational conditions for the full-scale wastewater treatment plant. To achieve good nitrogen removal efficiency with effluent ammonia and nitrate concentrations below 20 mg N/L and 50 mg N/L, respectively, it was suggested to control the ammonia concentration and pH of the influent below 500 mg N/L and 8.0, respectively. In addition, the biodegradability of N-methylpyrrolidone, diethylene glycol monobutyl ether, and cyclopentanone distillate residues from the electronic chemicals manufacturing process were evaluated under aerobic, anoxic, and anaerobic conditions. N-methylpyrrolidone and cyclopentanone distillate residues were suggested to be treated under anoxic condition. However, substrate inhibition occurred when using cyclopentanone distillate residue as a carbon source with chemical oxygen demand (COD) levels higher than 866 mg/L and nitrate levels higher than 415 mg N/L. Under aerobic condition, the COD from both N-methylpyrrolidone and cyclopentanone distillate residues could be easily degraded. Nevertheless, a negative effect on nitrification was observed, with a prolonged lag time for ammonia oxidation as the initial COD concentration increased. The specific ammonia oxidation rate and nitrate production rate decreased under high COD concentration contributed by N-methylpyrrolidone and cyclopentanone distillate residues. Furthermore, the biodegradability of diethylene glycol monobutyl ether distillate residue was found to be low under aerobic, anoxic, and anaerobic conditions. With respect to the abundance of nitrogen removal microorganisms in the wastewater treatment plant, results showed that Comammox may have an advantage over ammonia oxidizing bacteria under high pH conditions. In addition, Comammox may have higher resistance to environmental changes. Dominance of Comammox over ammonia oxidizing bacteria under high ammonia condition was first reported in this study.

An extended bio-electrochemical model for wastewater treatment and water desalination: Insights into the performance of microbial desalination cells

This paper presents an extended mathematical model for a microbial desalination cell (MDC), integrating Haldane kinetics for substrate inhibition and proton translocation theory for substrate dissociation under dynamic flow. The model is capable of predicting exoelectrogenic and methanogenic biomass dynamics, acetate removal efficiencies, salt concentration changes, and current production. Notably, it can be used to explore the influence of operational parameters such as dilution rates, temperatures, and external resistances. Our simulations reveal the optimal conditions for maximizing exoelectrogen concentration (500 mg/L) with minimal methanogen presence (50 mg/L) in the anode chamber at an anolyte dilution rate of 0.5 d−1, a temperature of 20 °C, and an influent acetate concentration of 1500 mg/L. The model accurately predicts the salt reduction in the middle chamber (from 0.2 to 0.09 mol/L) with a salt dilution rate of 0.5 d−1 and an external resistance of 20 Ohm. The model predicts a peak power output of 2.5 mW at external resistances between 20 and 50 Ohm. The model was validated against a suite of experimental literature data, including our previously published lab-scale studies, verifying its accuracy in predicting organic and salt removal rates, current production, and desalination efficiency. This extended model is a valuable tool for understanding the complex interactions between factors that influence MDC performance and can guide the optimization of MDC design for wastewater treatment and desalination.

A dynamic kinetic resolution catalyzed by immobilized lipase coupled with a novel grinding immobilized oxovanadium：Kinetic model and performance

A dual immobilized lipase and oxovanadium co-catalyzed dynamic kinetic resolution (DKR) system was established and successfully applied to synthesize (R)-1-(3-Methoxyphenyl) acetate in batch, resulting in a key chiral intermediate of (S)-rivastigmine with highly yield (97.6 %), e.e (99.01 %) and selectivity Sel (99.4 %). Herein, oxovanadium was immobilized onto diatomite via grinding immobilization for the first time to access a novel immobilized oxovanadium (V-D) used to catalyze an in situ racemization of undesired enantiomer substrate. The racemization rate of hydrated vanadyl sulfate was found for the first time to be much higher than that of vanadyl sulfate without crystalline water because water molecules in the former may form ion clusters of solvation membranes to disperses the charge density of positive carbon ions and promote their stability. This grinding immobilization approach may become a promising alternative for the immobilization of metals, especially precious metals due to its low cost, high efficiency, easily separated and large reaction interface. The kinetic model of the kinetic resolution catalyzed by LA-MR was developed, which may obey the sequential mechanism with both substrate and product inhibition, while the in-situ racemization reaction catalyzed by V-D may comply with power law.

Identification of UDP-glucuronosyltransferase (UGT) isoforms involved in the metabolism of Chlorophenols (CPs)

Chlorophenols (CPs) are a group of pollutants that pose a great threat to the environment, they are widely used in industrial and agricultural wastes, pesticides, herbicides, textiles, pharmaceuticals and plastics. Among CPs, pentachlorophenol was listed as one of the persistent organic pollutants (POPs) by the Stockholm convention. This study aims to identify the UDP-glucosyltransferase (UGT) isoforms involved in the metabolic elimination of CPs. CPs’ mono-glucuronide was detected in the human liver microsomes (HLMs) incubation mixture with co-factor uridine-diphosphate glucuronic acid (UDPGA). HLMs-catalyzed glucuronidation metabolism reaction equations followed Michaelis-Menten or substrate inhibition type. Recombinant enzymes and chemical reagents inhibition experiments were utilized to phenotype the main UGT isoforms involved in the glucuronidation of CPs. UGT1A6 might be the major enzyme in the glucuronidation of mono-chlorophenol isomer. UGT1A1, UGT1A6, UGT1A9, UGT2B4 and UGT2B7 were the most important five UGT isoforms for metabolizing the di-chlorophenol and tri-chlorophenol isomers. UGT1A1 and UGT1A3 were the most important UGT isoforms in the catalysis of tetra-chlorophenol and pentachlorophenol isomers. Species differences were investigated using rat liver microsomes (RLMs), pig liver microsomes (PLMs), dog liver microsomes (DLMs), and monkey liver microsomes (MyLMs). All these results were helpful for elucidating the metabolic elimination and toxicity of CPs.

The Mechanism of Rh(I)-Catalyzed Coupling of Benzotriazoles and Allenes Revisited: Substrate Inhibition, Proton Shuttling, and the Role of Cationic vs Neutral Species.

Direct coupling of benzotriazole to unsaturated substrates such as allenes represents an atom-efficient method for the construction of biologically and pharmaceutically interesting functional structures. In this work, the mechanism of the N2-selective Rh complex-catalyzed coupling of benzotriazoles to allenes was investigated in depth using a combination of experimental and theoretical techniques. Substrate coordination, inhibition, and catalyst deactivation was probed in reactions of the neutral and cationic catalyst precursors [Rh(μ-Cl)(DPEPhos)]2 and [Rh(DPEPhos)(MeOH)2]+ with benzotriazole and allene, giving coordination, or coupling of the substrates. Formation of a rhodacycle, formed by unprecedented 1,2-coupling of allenes, is responsible for catalyst deactivation. Experimental and computational data suggest that cationic species, formed either by abstraction of the chloride ligand or used directly, are relevant for catalysis. Isomerization of benzotriazole and cleavage of its N-H bond are suggested to occur by counteranion-assisted proton shuttling. This contrasts with a previously proposed scenario in which oxidative N-H addition at Rh is one of the key steps. Based on the mechanistic analysis, the catalytic coupling reaction could be optimized, leading to lower reaction temperature and shorter reaction times compared to the literature.

Multiscale effects of radial mixing on mixotrophic microalgal growth and macromolecular synthesis in tubular bubble-column photobioreactors

Extremophillic microalga Chlorella sorokiniana is grown mixotrophically in a 5 L externally-illuminated tubular bubble-column photobioreactor with an external light intensity of 10,500 lx (≈195 μmol-photons.m−2.s−1) at atmospheric (0.04 %), 1 %, 2 %, 3 %, 4 %, and 5 % CO2 concentrations, with acetic acid and urea as the organic carbon and nitrogen sources, respectively, for 6–8 days. Two different setups are employed: one permits axial air flow, and another promotes radial mixing of algal particles, dissolved CO2 and nutrients along with axial sparging. We show that radial mixing increases biomass yields by reducing both photo-limitation and photo-inhibition up to CO2 concentrations <3 %, above which substrate (CO2) inhibition necessitates lower reactor mixing. Radial mixing increases the lipid yield from 1.06 and 1.61 g/L to 1.27 and 1.78 g/L at 2 % and 3 % CO2, respectively, in nitrogen-limited cases, resulting in a 10–20 % increase. We show that complete reactor mixing, facilitated by the dual effects of branched (radial) and axial sparger, is preferred only in the absence of substrate (CO2) inhibition. Subsequently, we stratify the tubular photobioreactor design strategy into two regimes based on CO2 inlet concentrations. In one regime (for inlet CO2 < 3 %), we recommend the deployment of branched sparger to facilitate both radial and axial mixing simultaneously, thereby creating a well-mixed reactor at lower CO2 concentrations. In the other regime (for inlet CO2 > 3 %), axial sparging alone is recommended since complete mixing of CO2 increases substrate inhibition by reducing the reactor pH below 7.5.

Bioremediation of phenolic compounds in coal‐contaminated environments: Bio‐kinetic studies using a mixed bacterial culture

AbstractTwo distinct bacterial strains were combined in a mixed culture to investigate the degradation of phenol. The mixed cultures consisting of species like Rhodococcus and Bacillus, were isolated from the waste effluent collected from Dankuni coal complex (DCC) in West Bengal. The experimental phenol biodegradation were performed batch wise at an optimized temperature and pH conditions of 37°C and 7.0 for a wide range of phenol concentrations initially, with saline solutions which is not supplemented with nutrients initially. Experimental results concluded a highest possible specific growth rate at 150 mg/L for phenol biodegradation of 2400 mg/L within a span of 72 h. Several significant physicochemical parameters were investigated in order to determine the ideal conditions for phenol breakdown. It was also observed that a spectroscopic study of the intermediate at 260 nm of the phenol biodegradation commenced through the formation of a cis, cis intermediate, indicating ortho‐cleavage pathway. A simulated substrate utilization and growth profiles was obtained by using substrate inhibition model equation using MATLAB R2017a were used to compare for the validation of the obtained experimental growth and substrate characterization data.

Identification of Bromophenols' glucuronidation and its induction on UDP- glucuronosyltransferases isoforms

Bromophenols (BPs) are prominent environmental pollutants extensively utilized in aquaculture, pharmaceuticals, and chemical manufacturing. This study aims to identify UDP- glucuronosyltransferases (UGTs) isoforms involved in the metabolic elimination of BPs. Mono-glucuronides of BPs were detected in human liver microsomes (HLMs) incubated with the co-factor uridine-diphosphate glucuronic acid (UDPGA). The glucuronidation metabolism reactions catalyzed by HLMs followed Michaelis-Menten or substrate inhibition kinetics. Recombinant enzymes and inhibition experiments with chemical reagents were employed to phenotype the principal UGT isoforms participating in BP glucuronidation. UGT1A6 emerged as the major enzyme in the glucuronidation of 4-Bromophenol (4-BP), while UGT1A1, UGT1A6, and UGT1A8 were identified as the most essential isoforms for metabolizing 2,4-dibromophenol (2,4-DBP). UGT1A1, UGT1A8, and UGT2B4 were deemed the most critical isoforms in the catalysis of 2,4,6-tribromophenol (2,4,6-TBP) glucuronidation. Species differences were investigated using the liver microsomes of pig (PLM), rat (RLM), monkey (MyLM), and dog (DLM). Additionally, 2,4,6-TBP effects on the expression of UGT1A1 and UGT2B7 in HepG2 cells were evaluated. The results demonstrated potential induction of UGT1A1 and UGT2B7 upon exposure to 2,4,6-TBP at a concentration of 50 μM. Collectively, these findings contribute to elucidating the metabolic elimination and toxicity of BPs.